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patient is an umlsterm, femur is an umlsterm, plate is an umlsterm, femur is an umlsterm, hip is an umlsterm, screw is an umlsterm
DerUnfallchirurg.81010323.eng.abstr_task0
Sentence: We report on a 57-year-old patient suffering from a complex malposition of proximal femur after osteosynthesis using a condylar plate . For polyaxial correction of the proximal femur we successfully used an intramedullary hip screw system . Instructions: please extract entities and their types from the input sentence, all entity types are in options Options: umlsterm
[ "O", "O", "O", "O", "O", "O", "O", "B-umlsterm", "O", "O", "O", "O", "O", "O", "O", "B-umlsterm", "O", "O", "O", "O", "O", "B-umlsterm", "O", "O", "O", "O", "O", "O", "O", "B-umlsterm", "O", "O", "O", "O", "O", "B-umlsterm", "B-umlsterm", "O", "O" ]
We report on a 57-year-old patient suffering from a complex malposition of proximal femur after osteosynthesis using a condylar plate . For polyaxial correction of the proximal femur we successfully used an intramedullary hip screw system .
[ "We", "report", "on", "a", "57-year", "-", "old", "patient", "suffering", "from", "a", "complex", "malposition", "of", "proximal", "femur", "after", "osteosynthesis", "using", "a", "condylar", "plate", ".", "For", "polyaxial", "correction", "of", "the", "proximal", "femur", "we", "successfully", "used", "an", "intramedullary", "hip", "screw", "system", "." ]
[ "umlsterm" ]
patient is an umlsterm, femur is an umlsterm, plate is an umlsterm, femur is an umlsterm, hip is an umlsterm, screw is an umlsterm
DerUnfallchirurg.81010323.eng.abstr_task1
Sentence: We report on a 57-year-old patient suffering from a complex malposition of proximal femur after osteosynthesis using a condylar plate . For polyaxial correction of the proximal femur we successfully used an intramedullary hip screw system . Instructions: please typing these entity words according to sentence: patient, femur, plate, femur, hip, screw Options: umlsterm
[ "O", "O", "O", "O", "O", "O", "O", "B-umlsterm", "O", "O", "O", "O", "O", "O", "O", "B-umlsterm", "O", "O", "O", "O", "O", "B-umlsterm", "O", "O", "O", "O", "O", "O", "O", "B-umlsterm", "O", "O", "O", "O", "O", "B-umlsterm", "B-umlsterm", "O", "O" ]
We report on a 57-year-old patient suffering from a complex malposition of proximal femur after osteosynthesis using a condylar plate . For polyaxial correction of the proximal femur we successfully used an intramedullary hip screw system .
[ "We", "report", "on", "a", "57-year", "-", "old", "patient", "suffering", "from", "a", "complex", "malposition", "of", "proximal", "femur", "after", "osteosynthesis", "using", "a", "condylar", "plate", ".", "For", "polyaxial", "correction", "of", "the", "proximal", "femur", "we", "successfully", "used", "an", "intramedullary", "hip", "screw", "system", "." ]
[ "umlsterm" ]
patient, femur, plate, femur, hip, screw
DerUnfallchirurg.81010323.eng.abstr_task2
Sentence: We report on a 57-year-old patient suffering from a complex malposition of proximal femur after osteosynthesis using a condylar plate . For polyaxial correction of the proximal femur we successfully used an intramedullary hip screw system . Instructions: please extract entity words from the input sentence
[ "O", "O", "O", "O", "O", "O", "O", "B-umlsterm", "O", "O", "O", "O", "O", "O", "O", "B-umlsterm", "O", "O", "O", "O", "O", "B-umlsterm", "O", "O", "O", "O", "O", "O", "O", "B-umlsterm", "O", "O", "O", "O", "O", "B-umlsterm", "B-umlsterm", "O", "O" ]
We report on a 57-year-old patient suffering from a complex malposition of proximal femur after osteosynthesis using a condylar plate . For polyaxial correction of the proximal femur we successfully used an intramedullary hip screw system .
[ "We", "report", "on", "a", "57-year", "-", "old", "patient", "suffering", "from", "a", "complex", "malposition", "of", "proximal", "femur", "after", "osteosynthesis", "using", "a", "condylar", "plate", ".", "For", "polyaxial", "correction", "of", "the", "proximal", "femur", "we", "successfully", "used", "an", "intramedullary", "hip", "screw", "system", "." ]
[ "umlsterm" ]
cells is a Cell, extra cellular is a Immaterial_anatomical_entity
PMID-6312919_task0
Sentence: The genetics of transformation by SV 40. Experiments using the tsA 58 allele of the SV 40-A gene have demonstrated that the SV 40 large T-antigen is strictly required both for immortalization and induction anchorage independence of cells. It has been suggested that the immortalized phenotype is mediated by an extra cellular factor. The synthesis and/or the excretion of this factor in a medium is controlled by the A gene. Instructions: please extract entities and their types from the input sentence, all entity types are in options Options: Immaterial_anatomical_entity, Cell
[ "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "B-Cell", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "B-Immaterial_anatomical_entity", "I-Immaterial_anatomical_entity", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O" ]
The genetics of transformation by SV 40. Experiments using the tsA 58 allele of the SV 40-A gene have demonstrated that the SV 40 large T-antigen is strictly required both for immortalization and induction anchorage independence of cells. It has been suggested that the immortalized phenotype is mediated by an extra cellular factor. The synthesis and/or the excretion of this factor in a medium is controlled by the A gene.
[ "The", "genetics", "of", "transformation", "by", "SV", "40", ".", "\n", "Experiments", "using", "the", "tsA", "58", "allele", "of", "the", "SV", "40-A", "gene", "have", "demonstrated", "that", "the", "SV", "40", "large", "T", "-", "antigen", "is", "strictly", "required", "both", "for", "immortalization", "and", "induction", "anchorage", "independence", "of", "cells", ".", "It", "has", "been", "suggested", "that", "the", "immortalized", "phenotype", "is", "mediated", "by", "an", "extra", "cellular", "factor", ".", "The", "synthesis", "and", "/", "or", "the", "excretion", "of", "this", "factor", "in", "a", "medium", "is", "controlled", "by", "the", "A", "gene", ".", "\n" ]
[ "Immaterial_anatomical_entity", "Cell" ]
cells is a Cell, extra cellular is a Immaterial_anatomical_entity
PMID-6312919_task1
Sentence: The genetics of transformation by SV 40. Experiments using the tsA 58 allele of the SV 40-A gene have demonstrated that the SV 40 large T-antigen is strictly required both for immortalization and induction anchorage independence of cells. It has been suggested that the immortalized phenotype is mediated by an extra cellular factor. The synthesis and/or the excretion of this factor in a medium is controlled by the A gene. Instructions: please typing these entity words according to sentence: cells, extra cellular Options: Immaterial_anatomical_entity, Cell
[ "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "B-Cell", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "B-Immaterial_anatomical_entity", "I-Immaterial_anatomical_entity", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O" ]
The genetics of transformation by SV 40. Experiments using the tsA 58 allele of the SV 40-A gene have demonstrated that the SV 40 large T-antigen is strictly required both for immortalization and induction anchorage independence of cells. It has been suggested that the immortalized phenotype is mediated by an extra cellular factor. The synthesis and/or the excretion of this factor in a medium is controlled by the A gene.
[ "The", "genetics", "of", "transformation", "by", "SV", "40", ".", "\n", "Experiments", "using", "the", "tsA", "58", "allele", "of", "the", "SV", "40-A", "gene", "have", "demonstrated", "that", "the", "SV", "40", "large", "T", "-", "antigen", "is", "strictly", "required", "both", "for", "immortalization", "and", "induction", "anchorage", "independence", "of", "cells", ".", "It", "has", "been", "suggested", "that", "the", "immortalized", "phenotype", "is", "mediated", "by", "an", "extra", "cellular", "factor", ".", "The", "synthesis", "and", "/", "or", "the", "excretion", "of", "this", "factor", "in", "a", "medium", "is", "controlled", "by", "the", "A", "gene", ".", "\n" ]
[ "Immaterial_anatomical_entity", "Cell" ]
cells, extra cellular
PMID-6312919_task2
Sentence: The genetics of transformation by SV 40. Experiments using the tsA 58 allele of the SV 40-A gene have demonstrated that the SV 40 large T-antigen is strictly required both for immortalization and induction anchorage independence of cells. It has been suggested that the immortalized phenotype is mediated by an extra cellular factor. The synthesis and/or the excretion of this factor in a medium is controlled by the A gene. Instructions: please extract entity words from the input sentence
[ "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "B-Cell", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "B-Immaterial_anatomical_entity", "I-Immaterial_anatomical_entity", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O" ]
The genetics of transformation by SV 40. Experiments using the tsA 58 allele of the SV 40-A gene have demonstrated that the SV 40 large T-antigen is strictly required both for immortalization and induction anchorage independence of cells. It has been suggested that the immortalized phenotype is mediated by an extra cellular factor. The synthesis and/or the excretion of this factor in a medium is controlled by the A gene.
[ "The", "genetics", "of", "transformation", "by", "SV", "40", ".", "\n", "Experiments", "using", "the", "tsA", "58", "allele", "of", "the", "SV", "40-A", "gene", "have", "demonstrated", "that", "the", "SV", "40", "large", "T", "-", "antigen", "is", "strictly", "required", "both", "for", "immortalization", "and", "induction", "anchorage", "independence", "of", "cells", ".", "It", "has", "been", "suggested", "that", "the", "immortalized", "phenotype", "is", "mediated", "by", "an", "extra", "cellular", "factor", ".", "The", "synthesis", "and", "/", "or", "the", "excretion", "of", "this", "factor", "in", "a", "medium", "is", "controlled", "by", "the", "A", "gene", ".", "\n" ]
[ "Immaterial_anatomical_entity", "Cell" ]
serum thyrotropin is a GENE-N
23585555_task0
Sentence: Relationship between serum thyrotropin levels and intrarenal hemodynamic parameters in euthyroid subjects. Instructions: please extract entities and their types from the input sentence, all entity types are in options Options: GENE-N
[ "O", "O", "B-GENE-N", "I-GENE-N", "O", "O", "O", "O", "O", "O", "O", "O", "O" ]
Relationship between serum thyrotropin levels and intrarenal hemodynamic parameters in euthyroid subjects.
[ "Relationship", "between", "serum", "thyrotropin", "levels", "and", "intrarenal", "hemodynamic", "parameters", "in", "euthyroid", "subjects", "." ]
[ "GENE-N", "CHEMICAL" ]
serum thyrotropin is a GENE-N
23585555_task1
Sentence: Relationship between serum thyrotropin levels and intrarenal hemodynamic parameters in euthyroid subjects. Instructions: please typing these entity words according to sentence: serum thyrotropin Options: GENE-N
[ "O", "O", "B-GENE-N", "I-GENE-N", "O", "O", "O", "O", "O", "O", "O", "O", "O" ]
Relationship between serum thyrotropin levels and intrarenal hemodynamic parameters in euthyroid subjects.
[ "Relationship", "between", "serum", "thyrotropin", "levels", "and", "intrarenal", "hemodynamic", "parameters", "in", "euthyroid", "subjects", "." ]
[ "GENE-N", "CHEMICAL" ]
serum thyrotropin
23585555_task2
Sentence: Relationship between serum thyrotropin levels and intrarenal hemodynamic parameters in euthyroid subjects. Instructions: please extract entity words from the input sentence
[ "O", "O", "B-GENE-N", "I-GENE-N", "O", "O", "O", "O", "O", "O", "O", "O", "O" ]
Relationship between serum thyrotropin levels and intrarenal hemodynamic parameters in euthyroid subjects.
[ "Relationship", "between", "serum", "thyrotropin", "levels", "and", "intrarenal", "hemodynamic", "parameters", "in", "euthyroid", "subjects", "." ]
[ "GENE-N", "CHEMICAL" ]
Ellenbeugen is an umlsterm, Dermatitis is an umlsterm, Dermatose is an umlsterm, Wolle is an umlsterm, Sandbox - dermatitis is an umlsterm, Rolle is an umlsterm, Dermatitis is an umlsterm, Gesicht is an umlsterm, Pruritus is an umlsterm
DerHautarzt.60470129.ger.abstr_task0
Sentence: Hypopigmentierte , lichenoide flache Papeln , mit den Praedilektionsstellen Handruecken , Ellenbeugen sowie Knie stellen das Krankheitsbild der Dermatitis papulosa juvenilis dar , einer seltenen , in den Sommermonaten auftretenden Dermatose des Kindesalters . Die histologischen Befunde zeigen eine Hyperkeratose , maessig ausgepraegte Akanthose und ein dermales lymphozytaeres , perivaskulaer orientiertes Infiltrat . Pathogenetisch wird der Einfluss rauher reibender Materialien wie Sand und Wolle ( , Sandbox-dermatitis frictional lichenoid eruption ) , diskutiert , ebenso wird einer moeglichen Photosensibilitaet eine pathogenetische Rolle zugeschrieben . Wir berichten ueber einen 9jaehrigen Jungen , der im Sommer 1994 erstmals Laesionen einer Dermatitis papulosa juvenilis mit besonderer Auspraegung im Gesicht , den Kniekehlen sowie einem starken Pruritus entwickelte . Instructions: please extract entities and their types from the input sentence, all entity types are in options Options: umlsterm
[ "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "B-umlsterm", "O", "O", "O", "O", "O", "O", "B-umlsterm", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "B-umlsterm", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "B-umlsterm", "O", "O", "B-umlsterm", "I-umlsterm", "I-umlsterm", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "B-umlsterm", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "B-umlsterm", "O", "O", "O", "O", "O", "O", "B-umlsterm", "O", "O", "O", "O", "O", "O", "B-umlsterm", "O", "O" ]
Hypopigmentierte , lichenoide flache Papeln , mit den Praedilektionsstellen Handruecken , Ellenbeugen sowie Knie stellen das Krankheitsbild der Dermatitis papulosa juvenilis dar , einer seltenen , in den Sommermonaten auftretenden Dermatose des Kindesalters . Die histologischen Befunde zeigen eine Hyperkeratose , maessig ausgepraegte Akanthose und ein dermales lymphozytaeres , perivaskulaer orientiertes Infiltrat . Pathogenetisch wird der Einfluss rauher reibender Materialien wie Sand und Wolle ( , Sandbox-dermatitis frictional lichenoid eruption ) , diskutiert , ebenso wird einer moeglichen Photosensibilitaet eine pathogenetische Rolle zugeschrieben . Wir berichten ueber einen 9jaehrigen Jungen , der im Sommer 1994 erstmals Laesionen einer Dermatitis papulosa juvenilis mit besonderer Auspraegung im Gesicht , den Kniekehlen sowie einem starken Pruritus entwickelte .
[ "Hypopigmentierte", ",", "lichenoide", "flache", "Papeln", ",", "mit", "den", "Praedilektionsstellen", "Handruecken", ",", "Ellenbeugen", "sowie", "Knie", "stellen", "das", "Krankheitsbild", "der", "Dermatitis", "papulosa", "juvenilis", "dar", ",", "einer", "seltenen", ",", "in", "den", "Sommermonaten", "auftretenden", "Dermatose", "des", "Kindesalters", ".", "Die", "histologischen", "Befunde", "zeigen", "eine", "Hyperkeratose", ",", "maessig", "ausgepraegte", "Akanthose", "und", "ein", "dermales", "lymphozytaeres", ",", "perivaskulaer", "orientiertes", "Infiltrat", ".", "Pathogenetisch", "wird", "der", "Einfluss", "rauher", "reibender", "Materialien", "wie", "Sand", "und", "Wolle", "(", ",", "Sandbox", "-", "dermatitis", "frictional", "lichenoid", "eruption", ")", ",", "diskutiert", ",", "ebenso", "wird", "einer", "moeglichen", "Photosensibilitaet", "eine", "pathogenetische", "Rolle", "zugeschrieben", ".", "Wir", "berichten", "ueber", "einen", "9jaehrigen", "Jungen", ",", "der", "im", "Sommer", "1994", "erstmals", "Laesionen", "einer", "Dermatitis", "papulosa", "juvenilis", "mit", "besonderer", "Auspraegung", "im", "Gesicht", ",", "den", "Kniekehlen", "sowie", "einem", "starken", "Pruritus", "entwickelte", "." ]
[ "umlsterm" ]
Ellenbeugen is an umlsterm, Dermatitis is an umlsterm, Dermatose is an umlsterm, Wolle is an umlsterm, Sandbox - dermatitis is an umlsterm, Rolle is an umlsterm, Dermatitis is an umlsterm, Gesicht is an umlsterm, Pruritus is an umlsterm
DerHautarzt.60470129.ger.abstr_task1
Sentence: Hypopigmentierte , lichenoide flache Papeln , mit den Praedilektionsstellen Handruecken , Ellenbeugen sowie Knie stellen das Krankheitsbild der Dermatitis papulosa juvenilis dar , einer seltenen , in den Sommermonaten auftretenden Dermatose des Kindesalters . Die histologischen Befunde zeigen eine Hyperkeratose , maessig ausgepraegte Akanthose und ein dermales lymphozytaeres , perivaskulaer orientiertes Infiltrat . Pathogenetisch wird der Einfluss rauher reibender Materialien wie Sand und Wolle ( , Sandbox-dermatitis frictional lichenoid eruption ) , diskutiert , ebenso wird einer moeglichen Photosensibilitaet eine pathogenetische Rolle zugeschrieben . Wir berichten ueber einen 9jaehrigen Jungen , der im Sommer 1994 erstmals Laesionen einer Dermatitis papulosa juvenilis mit besonderer Auspraegung im Gesicht , den Kniekehlen sowie einem starken Pruritus entwickelte . Instructions: please typing these entity words according to sentence: Ellenbeugen, Dermatitis, Dermatose, Wolle, Sandbox - dermatitis, Rolle, Dermatitis, Gesicht, Pruritus Options: umlsterm
[ "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "B-umlsterm", "O", "O", "O", "O", "O", "O", "B-umlsterm", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "B-umlsterm", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "B-umlsterm", "O", "O", "B-umlsterm", "I-umlsterm", "I-umlsterm", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "B-umlsterm", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "B-umlsterm", "O", "O", "O", "O", "O", "O", "B-umlsterm", "O", "O", "O", "O", "O", "O", "B-umlsterm", "O", "O" ]
Hypopigmentierte , lichenoide flache Papeln , mit den Praedilektionsstellen Handruecken , Ellenbeugen sowie Knie stellen das Krankheitsbild der Dermatitis papulosa juvenilis dar , einer seltenen , in den Sommermonaten auftretenden Dermatose des Kindesalters . Die histologischen Befunde zeigen eine Hyperkeratose , maessig ausgepraegte Akanthose und ein dermales lymphozytaeres , perivaskulaer orientiertes Infiltrat . Pathogenetisch wird der Einfluss rauher reibender Materialien wie Sand und Wolle ( , Sandbox-dermatitis frictional lichenoid eruption ) , diskutiert , ebenso wird einer moeglichen Photosensibilitaet eine pathogenetische Rolle zugeschrieben . Wir berichten ueber einen 9jaehrigen Jungen , der im Sommer 1994 erstmals Laesionen einer Dermatitis papulosa juvenilis mit besonderer Auspraegung im Gesicht , den Kniekehlen sowie einem starken Pruritus entwickelte .
[ "Hypopigmentierte", ",", "lichenoide", "flache", "Papeln", ",", "mit", "den", "Praedilektionsstellen", "Handruecken", ",", "Ellenbeugen", "sowie", "Knie", "stellen", "das", "Krankheitsbild", "der", "Dermatitis", "papulosa", "juvenilis", "dar", ",", "einer", "seltenen", ",", "in", "den", "Sommermonaten", "auftretenden", "Dermatose", "des", "Kindesalters", ".", "Die", "histologischen", "Befunde", "zeigen", "eine", "Hyperkeratose", ",", "maessig", "ausgepraegte", "Akanthose", "und", "ein", "dermales", "lymphozytaeres", ",", "perivaskulaer", "orientiertes", "Infiltrat", ".", "Pathogenetisch", "wird", "der", "Einfluss", "rauher", "reibender", "Materialien", "wie", "Sand", "und", "Wolle", "(", ",", "Sandbox", "-", "dermatitis", "frictional", "lichenoid", "eruption", ")", ",", "diskutiert", ",", "ebenso", "wird", "einer", "moeglichen", "Photosensibilitaet", "eine", "pathogenetische", "Rolle", "zugeschrieben", ".", "Wir", "berichten", "ueber", "einen", "9jaehrigen", "Jungen", ",", "der", "im", "Sommer", "1994", "erstmals", "Laesionen", "einer", "Dermatitis", "papulosa", "juvenilis", "mit", "besonderer", "Auspraegung", "im", "Gesicht", ",", "den", "Kniekehlen", "sowie", "einem", "starken", "Pruritus", "entwickelte", "." ]
[ "umlsterm" ]
Ellenbeugen, Dermatitis, Dermatose, Wolle, Sandbox - dermatitis, Rolle, Dermatitis, Gesicht, Pruritus
DerHautarzt.60470129.ger.abstr_task2
Sentence: Hypopigmentierte , lichenoide flache Papeln , mit den Praedilektionsstellen Handruecken , Ellenbeugen sowie Knie stellen das Krankheitsbild der Dermatitis papulosa juvenilis dar , einer seltenen , in den Sommermonaten auftretenden Dermatose des Kindesalters . Die histologischen Befunde zeigen eine Hyperkeratose , maessig ausgepraegte Akanthose und ein dermales lymphozytaeres , perivaskulaer orientiertes Infiltrat . Pathogenetisch wird der Einfluss rauher reibender Materialien wie Sand und Wolle ( , Sandbox-dermatitis frictional lichenoid eruption ) , diskutiert , ebenso wird einer moeglichen Photosensibilitaet eine pathogenetische Rolle zugeschrieben . Wir berichten ueber einen 9jaehrigen Jungen , der im Sommer 1994 erstmals Laesionen einer Dermatitis papulosa juvenilis mit besonderer Auspraegung im Gesicht , den Kniekehlen sowie einem starken Pruritus entwickelte . Instructions: please extract entity words from the input sentence
[ "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "B-umlsterm", "O", "O", "O", "O", "O", "O", "B-umlsterm", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "B-umlsterm", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "B-umlsterm", "O", "O", "B-umlsterm", "I-umlsterm", "I-umlsterm", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "B-umlsterm", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "B-umlsterm", "O", "O", "O", "O", "O", "O", "B-umlsterm", "O", "O", "O", "O", "O", "O", "B-umlsterm", "O", "O" ]
Hypopigmentierte , lichenoide flache Papeln , mit den Praedilektionsstellen Handruecken , Ellenbeugen sowie Knie stellen das Krankheitsbild der Dermatitis papulosa juvenilis dar , einer seltenen , in den Sommermonaten auftretenden Dermatose des Kindesalters . Die histologischen Befunde zeigen eine Hyperkeratose , maessig ausgepraegte Akanthose und ein dermales lymphozytaeres , perivaskulaer orientiertes Infiltrat . Pathogenetisch wird der Einfluss rauher reibender Materialien wie Sand und Wolle ( , Sandbox-dermatitis frictional lichenoid eruption ) , diskutiert , ebenso wird einer moeglichen Photosensibilitaet eine pathogenetische Rolle zugeschrieben . Wir berichten ueber einen 9jaehrigen Jungen , der im Sommer 1994 erstmals Laesionen einer Dermatitis papulosa juvenilis mit besonderer Auspraegung im Gesicht , den Kniekehlen sowie einem starken Pruritus entwickelte .
[ "Hypopigmentierte", ",", "lichenoide", "flache", "Papeln", ",", "mit", "den", "Praedilektionsstellen", "Handruecken", ",", "Ellenbeugen", "sowie", "Knie", "stellen", "das", "Krankheitsbild", "der", "Dermatitis", "papulosa", "juvenilis", "dar", ",", "einer", "seltenen", ",", "in", "den", "Sommermonaten", "auftretenden", "Dermatose", "des", "Kindesalters", ".", "Die", "histologischen", "Befunde", "zeigen", "eine", "Hyperkeratose", ",", "maessig", "ausgepraegte", "Akanthose", "und", "ein", "dermales", "lymphozytaeres", ",", "perivaskulaer", "orientiertes", "Infiltrat", ".", "Pathogenetisch", "wird", "der", "Einfluss", "rauher", "reibender", "Materialien", "wie", "Sand", "und", "Wolle", "(", ",", "Sandbox", "-", "dermatitis", "frictional", "lichenoid", "eruption", ")", ",", "diskutiert", ",", "ebenso", "wird", "einer", "moeglichen", "Photosensibilitaet", "eine", "pathogenetische", "Rolle", "zugeschrieben", ".", "Wir", "berichten", "ueber", "einen", "9jaehrigen", "Jungen", ",", "der", "im", "Sommer", "1994", "erstmals", "Laesionen", "einer", "Dermatitis", "papulosa", "juvenilis", "mit", "besonderer", "Auspraegung", "im", "Gesicht", ",", "den", "Kniekehlen", "sowie", "einem", "starken", "Pruritus", "entwickelte", "." ]
[ "umlsterm" ]
SRY is a DNA_domain_or_region, great apes is a multi_cell
83262_task0
Sentence: Comparative mapping of SRY in the great apes. Instructions: please extract entities and their types from the input sentence, all entity types are in options Options: DNA_domain_or_region, multi_cell
[ "O", "O", "O", "B-DNA_domain_or_region", "O", "O", "B-multi_cell", "I-multi_cell", "O" ]
Comparative mapping of SRY in the great apes.
[ "Comparative", "mapping", "of", "SRY", "in", "the", "great", "apes", "." ]
[ "other_name", "DNA_domain_or_region", "DNA_molecule", "multi_cell" ]
SRY is a DNA_domain_or_region, great apes is a multi_cell
83262_task1
Sentence: Comparative mapping of SRY in the great apes. Instructions: please typing these entity words according to sentence: SRY, great apes Options: DNA_domain_or_region, multi_cell
[ "O", "O", "O", "B-DNA_domain_or_region", "O", "O", "B-multi_cell", "I-multi_cell", "O" ]
Comparative mapping of SRY in the great apes.
[ "Comparative", "mapping", "of", "SRY", "in", "the", "great", "apes", "." ]
[ "other_name", "DNA_domain_or_region", "DNA_molecule", "multi_cell" ]
SRY, great apes
83262_task2
Sentence: Comparative mapping of SRY in the great apes. Instructions: please extract entity words from the input sentence
[ "O", "O", "O", "B-DNA_domain_or_region", "O", "O", "B-multi_cell", "I-multi_cell", "O" ]
Comparative mapping of SRY in the great apes.
[ "Comparative", "mapping", "of", "SRY", "in", "the", "great", "apes", "." ]
[ "other_name", "DNA_domain_or_region", "DNA_molecule", "multi_cell" ]
JNK is a GENE-N, ERK is a GENE-N, NF - κB is a GENE-N
23541436_task0
Sentence: Anti-inflammatory effect of essential oil and its constituents from fingered citron (Citrus medica L. var. sarcodactylis) through blocking JNK, ERK and NF-κB signaling pathways in LPS-activated RAW 264.7 cells. Instructions: please extract entities and their types from the input sentence, all entity types are in options Options: GENE-N
[ "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "B-GENE-N", "O", "B-GENE-N", "O", "B-GENE-N", "I-GENE-N", "I-GENE-N", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O" ]
Anti-inflammatory effect of essential oil and its constituents from fingered citron (Citrus medica L. var. sarcodactylis) through blocking JNK, ERK and NF-κB signaling pathways in LPS-activated RAW 264.7 cells.
[ "Anti", "-", "inflammatory", "effect", "of", "essential", "oil", "and", "its", "constituents", "from", "fingered", "citron", "(", "Citrus", "medica", "L.", "var", ".", "sarcodactylis", ")", "through", "blocking", "JNK", ",", "ERK", "and", "NF", "-", "κB", "signaling", "pathways", "in", "LPS", "-", "activated", "RAW", "264.7", "cells", "." ]
[ "GENE-N", "GENE-Y", "CHEMICAL" ]
JNK is a GENE-N, ERK is a GENE-N, NF - κB is a GENE-N
23541436_task1
Sentence: Anti-inflammatory effect of essential oil and its constituents from fingered citron (Citrus medica L. var. sarcodactylis) through blocking JNK, ERK and NF-κB signaling pathways in LPS-activated RAW 264.7 cells. Instructions: please typing these entity words according to sentence: JNK, ERK, NF - κB Options: GENE-N
[ "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "B-GENE-N", "O", "B-GENE-N", "O", "B-GENE-N", "I-GENE-N", "I-GENE-N", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O" ]
Anti-inflammatory effect of essential oil and its constituents from fingered citron (Citrus medica L. var. sarcodactylis) through blocking JNK, ERK and NF-κB signaling pathways in LPS-activated RAW 264.7 cells.
[ "Anti", "-", "inflammatory", "effect", "of", "essential", "oil", "and", "its", "constituents", "from", "fingered", "citron", "(", "Citrus", "medica", "L.", "var", ".", "sarcodactylis", ")", "through", "blocking", "JNK", ",", "ERK", "and", "NF", "-", "κB", "signaling", "pathways", "in", "LPS", "-", "activated", "RAW", "264.7", "cells", "." ]
[ "GENE-N", "GENE-Y", "CHEMICAL" ]
JNK, ERK, NF - κB
23541436_task2
Sentence: Anti-inflammatory effect of essential oil and its constituents from fingered citron (Citrus medica L. var. sarcodactylis) through blocking JNK, ERK and NF-κB signaling pathways in LPS-activated RAW 264.7 cells. Instructions: please extract entity words from the input sentence
[ "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "B-GENE-N", "O", "B-GENE-N", "O", "B-GENE-N", "I-GENE-N", "I-GENE-N", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O" ]
Anti-inflammatory effect of essential oil and its constituents from fingered citron (Citrus medica L. var. sarcodactylis) through blocking JNK, ERK and NF-κB signaling pathways in LPS-activated RAW 264.7 cells.
[ "Anti", "-", "inflammatory", "effect", "of", "essential", "oil", "and", "its", "constituents", "from", "fingered", "citron", "(", "Citrus", "medica", "L.", "var", ".", "sarcodactylis", ")", "through", "blocking", "JNK", ",", "ERK", "and", "NF", "-", "κB", "signaling", "pathways", "in", "LPS", "-", "activated", "RAW", "264.7", "cells", "." ]
[ "GENE-N", "GENE-Y", "CHEMICAL" ]
leichenassoziierter is an umlsterm, Kaefer is an umlsterm, Familien is an umlsterm, Kaefern is an umlsterm, Leichen is an umlsterm, Familien is an umlsterm, Kaefer is an umlsterm, Leiche is an umlsterm, Waelder is an umlsterm, Leiche is an umlsterm
Rechtsmedizin.80080083.ger.abstr_task0
Sentence: Es wird auf die kriminalistische Bedeutung leichenassoziierter ( nekrophiler ) Kaefer hingewiesen und es werden Gruppen ( Familien Gattungen , Arten ) , von Kaefern , die in Mitteleuropa haeufig an Leichen gefunden werden , vorgestellt . Auf die Familien Silphidae , Staphylinidae , Cleridae und Dermestidae und einige ihrer Arten wird naeher eingegangen . Da viele Kaefer bestimmte Zersetzungsstadien bevorzugen , vermag der Nachweis bestimmter Arten an einer Leiche Hinweise auf die Liege- bzw. Expositionszeit zu geben . Zudem kommen manche Arten nur in bestimmten Biotopen ( Waelder , offenes Land , Flussauen etc. ) vor und koennen kriminalistische Hinweise liefern , falls eine Leiche transportiert wurde . Auch wird kurz auf Fang- und Konservierungsmethoden eingegangen . Instructions: please extract entities and their types from the input sentence, all entity types are in options Options: umlsterm
[ "O", "O", "O", "O", "O", "O", "B-umlsterm", "O", "O", "O", "B-umlsterm", "O", "O", "O", "O", "O", "O", "B-umlsterm", "O", "O", "O", "O", "O", "O", "B-umlsterm", "O", "O", "O", "O", "O", "O", "B-umlsterm", "O", "O", "O", "O", "O", "O", "O", "B-umlsterm", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "B-umlsterm", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "B-umlsterm", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "B-umlsterm", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "B-umlsterm", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O" ]
Es wird auf die kriminalistische Bedeutung leichenassoziierter ( nekrophiler ) Kaefer hingewiesen und es werden Gruppen ( Familien Gattungen , Arten ) , von Kaefern , die in Mitteleuropa haeufig an Leichen gefunden werden , vorgestellt . Auf die Familien Silphidae , Staphylinidae , Cleridae und Dermestidae und einige ihrer Arten wird naeher eingegangen . Da viele Kaefer bestimmte Zersetzungsstadien bevorzugen , vermag der Nachweis bestimmter Arten an einer Leiche Hinweise auf die Liege- bzw. Expositionszeit zu geben . Zudem kommen manche Arten nur in bestimmten Biotopen ( Waelder , offenes Land , Flussauen etc. ) vor und koennen kriminalistische Hinweise liefern , falls eine Leiche transportiert wurde . Auch wird kurz auf Fang- und Konservierungsmethoden eingegangen .
[ "Es", "wird", "auf", "die", "kriminalistische", "Bedeutung", "leichenassoziierter", "(", "nekrophiler", ")", "Kaefer", "hingewiesen", "und", "es", "werden", "Gruppen", "(", "Familien", "Gattungen", ",", "Arten", ")", ",", "von", "Kaefern", ",", "die", "in", "Mitteleuropa", "haeufig", "an", "Leichen", "gefunden", "werden", ",", "vorgestellt", ".", "Auf", "die", "Familien", "Silphidae", ",", "Staphylinidae", ",", "Cleridae", "und", "Dermestidae", "und", "einige", "ihrer", "Arten", "wird", "naeher", "eingegangen", ".", "Da", "viele", "Kaefer", "bestimmte", "Zersetzungsstadien", "bevorzugen", ",", "vermag", "der", "Nachweis", "bestimmter", "Arten", "an", "einer", "Leiche", "Hinweise", "auf", "die", "Liege-", "bzw", ".", "Expositionszeit", "zu", "geben", ".", "Zudem", "kommen", "manche", "Arten", "nur", "in", "bestimmten", "Biotopen", "(", "Waelder", ",", "offenes", "Land", ",", "Flussauen", "etc", ".", ")", "vor", "und", "koennen", "kriminalistische", "Hinweise", "liefern", ",", "falls", "eine", "Leiche", "transportiert", "wurde", ".", "Auch", "wird", "kurz", "auf", "Fang-", "und", "Konservierungsmethoden", "eingegangen", "." ]
[ "umlsterm" ]
leichenassoziierter is an umlsterm, Kaefer is an umlsterm, Familien is an umlsterm, Kaefern is an umlsterm, Leichen is an umlsterm, Familien is an umlsterm, Kaefer is an umlsterm, Leiche is an umlsterm, Waelder is an umlsterm, Leiche is an umlsterm
Rechtsmedizin.80080083.ger.abstr_task1
Sentence: Es wird auf die kriminalistische Bedeutung leichenassoziierter ( nekrophiler ) Kaefer hingewiesen und es werden Gruppen ( Familien Gattungen , Arten ) , von Kaefern , die in Mitteleuropa haeufig an Leichen gefunden werden , vorgestellt . Auf die Familien Silphidae , Staphylinidae , Cleridae und Dermestidae und einige ihrer Arten wird naeher eingegangen . Da viele Kaefer bestimmte Zersetzungsstadien bevorzugen , vermag der Nachweis bestimmter Arten an einer Leiche Hinweise auf die Liege- bzw. Expositionszeit zu geben . Zudem kommen manche Arten nur in bestimmten Biotopen ( Waelder , offenes Land , Flussauen etc. ) vor und koennen kriminalistische Hinweise liefern , falls eine Leiche transportiert wurde . Auch wird kurz auf Fang- und Konservierungsmethoden eingegangen . Instructions: please typing these entity words according to sentence: leichenassoziierter, Kaefer, Familien, Kaefern, Leichen, Familien, Kaefer, Leiche, Waelder, Leiche Options: umlsterm
[ "O", "O", "O", "O", "O", "O", "B-umlsterm", "O", "O", "O", "B-umlsterm", "O", "O", "O", "O", "O", "O", "B-umlsterm", "O", "O", "O", "O", "O", "O", "B-umlsterm", "O", "O", "O", "O", "O", "O", "B-umlsterm", "O", "O", "O", "O", "O", "O", "O", "B-umlsterm", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "B-umlsterm", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "B-umlsterm", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "B-umlsterm", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "B-umlsterm", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O" ]
Es wird auf die kriminalistische Bedeutung leichenassoziierter ( nekrophiler ) Kaefer hingewiesen und es werden Gruppen ( Familien Gattungen , Arten ) , von Kaefern , die in Mitteleuropa haeufig an Leichen gefunden werden , vorgestellt . Auf die Familien Silphidae , Staphylinidae , Cleridae und Dermestidae und einige ihrer Arten wird naeher eingegangen . Da viele Kaefer bestimmte Zersetzungsstadien bevorzugen , vermag der Nachweis bestimmter Arten an einer Leiche Hinweise auf die Liege- bzw. Expositionszeit zu geben . Zudem kommen manche Arten nur in bestimmten Biotopen ( Waelder , offenes Land , Flussauen etc. ) vor und koennen kriminalistische Hinweise liefern , falls eine Leiche transportiert wurde . Auch wird kurz auf Fang- und Konservierungsmethoden eingegangen .
[ "Es", "wird", "auf", "die", "kriminalistische", "Bedeutung", "leichenassoziierter", "(", "nekrophiler", ")", "Kaefer", "hingewiesen", "und", "es", "werden", "Gruppen", "(", "Familien", "Gattungen", ",", "Arten", ")", ",", "von", "Kaefern", ",", "die", "in", "Mitteleuropa", "haeufig", "an", "Leichen", "gefunden", "werden", ",", "vorgestellt", ".", "Auf", "die", "Familien", "Silphidae", ",", "Staphylinidae", ",", "Cleridae", "und", "Dermestidae", "und", "einige", "ihrer", "Arten", "wird", "naeher", "eingegangen", ".", "Da", "viele", "Kaefer", "bestimmte", "Zersetzungsstadien", "bevorzugen", ",", "vermag", "der", "Nachweis", "bestimmter", "Arten", "an", "einer", "Leiche", "Hinweise", "auf", "die", "Liege-", "bzw", ".", "Expositionszeit", "zu", "geben", ".", "Zudem", "kommen", "manche", "Arten", "nur", "in", "bestimmten", "Biotopen", "(", "Waelder", ",", "offenes", "Land", ",", "Flussauen", "etc", ".", ")", "vor", "und", "koennen", "kriminalistische", "Hinweise", "liefern", ",", "falls", "eine", "Leiche", "transportiert", "wurde", ".", "Auch", "wird", "kurz", "auf", "Fang-", "und", "Konservierungsmethoden", "eingegangen", "." ]
[ "umlsterm" ]
leichenassoziierter, Kaefer, Familien, Kaefern, Leichen, Familien, Kaefer, Leiche, Waelder, Leiche
Rechtsmedizin.80080083.ger.abstr_task2
Sentence: Es wird auf die kriminalistische Bedeutung leichenassoziierter ( nekrophiler ) Kaefer hingewiesen und es werden Gruppen ( Familien Gattungen , Arten ) , von Kaefern , die in Mitteleuropa haeufig an Leichen gefunden werden , vorgestellt . Auf die Familien Silphidae , Staphylinidae , Cleridae und Dermestidae und einige ihrer Arten wird naeher eingegangen . Da viele Kaefer bestimmte Zersetzungsstadien bevorzugen , vermag der Nachweis bestimmter Arten an einer Leiche Hinweise auf die Liege- bzw. Expositionszeit zu geben . Zudem kommen manche Arten nur in bestimmten Biotopen ( Waelder , offenes Land , Flussauen etc. ) vor und koennen kriminalistische Hinweise liefern , falls eine Leiche transportiert wurde . Auch wird kurz auf Fang- und Konservierungsmethoden eingegangen . Instructions: please extract entity words from the input sentence
[ "O", "O", "O", "O", "O", "O", "B-umlsterm", "O", "O", "O", "B-umlsterm", "O", "O", "O", "O", "O", "O", "B-umlsterm", "O", "O", "O", "O", "O", "O", "B-umlsterm", "O", "O", "O", "O", "O", "O", "B-umlsterm", "O", "O", "O", "O", "O", "O", "O", "B-umlsterm", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "B-umlsterm", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "B-umlsterm", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "B-umlsterm", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "B-umlsterm", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O" ]
Es wird auf die kriminalistische Bedeutung leichenassoziierter ( nekrophiler ) Kaefer hingewiesen und es werden Gruppen ( Familien Gattungen , Arten ) , von Kaefern , die in Mitteleuropa haeufig an Leichen gefunden werden , vorgestellt . Auf die Familien Silphidae , Staphylinidae , Cleridae und Dermestidae und einige ihrer Arten wird naeher eingegangen . Da viele Kaefer bestimmte Zersetzungsstadien bevorzugen , vermag der Nachweis bestimmter Arten an einer Leiche Hinweise auf die Liege- bzw. Expositionszeit zu geben . Zudem kommen manche Arten nur in bestimmten Biotopen ( Waelder , offenes Land , Flussauen etc. ) vor und koennen kriminalistische Hinweise liefern , falls eine Leiche transportiert wurde . Auch wird kurz auf Fang- und Konservierungsmethoden eingegangen .
[ "Es", "wird", "auf", "die", "kriminalistische", "Bedeutung", "leichenassoziierter", "(", "nekrophiler", ")", "Kaefer", "hingewiesen", "und", "es", "werden", "Gruppen", "(", "Familien", "Gattungen", ",", "Arten", ")", ",", "von", "Kaefern", ",", "die", "in", "Mitteleuropa", "haeufig", "an", "Leichen", "gefunden", "werden", ",", "vorgestellt", ".", "Auf", "die", "Familien", "Silphidae", ",", "Staphylinidae", ",", "Cleridae", "und", "Dermestidae", "und", "einige", "ihrer", "Arten", "wird", "naeher", "eingegangen", ".", "Da", "viele", "Kaefer", "bestimmte", "Zersetzungsstadien", "bevorzugen", ",", "vermag", "der", "Nachweis", "bestimmter", "Arten", "an", "einer", "Leiche", "Hinweise", "auf", "die", "Liege-", "bzw", ".", "Expositionszeit", "zu", "geben", ".", "Zudem", "kommen", "manche", "Arten", "nur", "in", "bestimmten", "Biotopen", "(", "Waelder", ",", "offenes", "Land", ",", "Flussauen", "etc", ".", ")", "vor", "und", "koennen", "kriminalistische", "Hinweise", "liefern", ",", "falls", "eine", "Leiche", "transportiert", "wurde", ".", "Auch", "wird", "kurz", "auf", "Fang-", "und", "Konservierungsmethoden", "eingegangen", "." ]
[ "umlsterm" ]
tapping is a Participant_Condition, oscillatory motion is a Participant_Condition, spectral analysis is a Intervention_Physical, finger tapping is a Participant_Condition, oscillatory motion of the hand is a Participant_Condition, oscillatory condition is a Participant_Condition, oscillatory character is a Participant_Condition
19025_task0
Sentence: Time intervals production in tapping and oscillatory motion . We applied spectral analysis on series of time intervals produced in a synchronization-continuation experiment . In the first condition intervals were produced by finger tapping , and in the second by an oscillatory motion of the hand . Results obtained in tapping were consistent with a discrete , event-based timing model . In the oscillatory condition , the spectra suggested a continuous , dynamic timing mechanism , based on the regulation of effector stiffness . It is concluded that the oscillatory character of movement can offer an important resource for timing control . The use of an event-based timing control such as postulated in the Wing-Kristoffersson model could be restricted to a quite limited class of rhythmic tasks , characterized by the concatenation of discrete events . Instructions: please extract entities and their types from the input sentence, all entity types are in options Options: Intervention_Physical, Participant_Condition
[ "O", "O", "O", "O", "B-Participant_Condition", "O", "B-Participant_Condition", "I-Participant_Condition", "O", "O", "O", "B-Intervention_Physical", "I-Intervention_Physical", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "B-Participant_Condition", "I-Participant_Condition", "O", "O", "O", "O", "O", "O", "O", "B-Participant_Condition", "I-Participant_Condition", "I-Participant_Condition", "I-Participant_Condition", "I-Participant_Condition", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "B-Participant_Condition", "I-Participant_Condition", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "B-Participant_Condition", "I-Participant_Condition", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O" ]
Time intervals production in tapping and oscillatory motion . We applied spectral analysis on series of time intervals produced in a synchronization-continuation experiment . In the first condition intervals were produced by finger tapping , and in the second by an oscillatory motion of the hand . Results obtained in tapping were consistent with a discrete , event-based timing model . In the oscillatory condition , the spectra suggested a continuous , dynamic timing mechanism , based on the regulation of effector stiffness . It is concluded that the oscillatory character of movement can offer an important resource for timing control . The use of an event-based timing control such as postulated in the Wing-Kristoffersson model could be restricted to a quite limited class of rhythmic tasks , characterized by the concatenation of discrete events .
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[ "Participant_Condition", "Intervention_Physical" ]
tapping is a Participant_Condition, oscillatory motion is a Participant_Condition, spectral analysis is a Intervention_Physical, finger tapping is a Participant_Condition, oscillatory motion of the hand is a Participant_Condition, oscillatory condition is a Participant_Condition, oscillatory character is a Participant_Condition
19025_task1
Sentence: Time intervals production in tapping and oscillatory motion . We applied spectral analysis on series of time intervals produced in a synchronization-continuation experiment . In the first condition intervals were produced by finger tapping , and in the second by an oscillatory motion of the hand . Results obtained in tapping were consistent with a discrete , event-based timing model . In the oscillatory condition , the spectra suggested a continuous , dynamic timing mechanism , based on the regulation of effector stiffness . It is concluded that the oscillatory character of movement can offer an important resource for timing control . The use of an event-based timing control such as postulated in the Wing-Kristoffersson model could be restricted to a quite limited class of rhythmic tasks , characterized by the concatenation of discrete events . Instructions: please typing these entity words according to sentence: tapping, oscillatory motion, spectral analysis, finger tapping, oscillatory motion of the hand, oscillatory condition, oscillatory character Options: Intervention_Physical, Participant_Condition
[ "O", "O", "O", "O", "B-Participant_Condition", "O", "B-Participant_Condition", "I-Participant_Condition", "O", "O", "O", "B-Intervention_Physical", "I-Intervention_Physical", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "B-Participant_Condition", "I-Participant_Condition", "O", "O", "O", "O", "O", "O", "O", "B-Participant_Condition", "I-Participant_Condition", "I-Participant_Condition", "I-Participant_Condition", "I-Participant_Condition", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "B-Participant_Condition", "I-Participant_Condition", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "B-Participant_Condition", "I-Participant_Condition", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O" ]
Time intervals production in tapping and oscillatory motion . We applied spectral analysis on series of time intervals produced in a synchronization-continuation experiment . In the first condition intervals were produced by finger tapping , and in the second by an oscillatory motion of the hand . Results obtained in tapping were consistent with a discrete , event-based timing model . In the oscillatory condition , the spectra suggested a continuous , dynamic timing mechanism , based on the regulation of effector stiffness . It is concluded that the oscillatory character of movement can offer an important resource for timing control . The use of an event-based timing control such as postulated in the Wing-Kristoffersson model could be restricted to a quite limited class of rhythmic tasks , characterized by the concatenation of discrete events .
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[ "Participant_Condition", "Intervention_Physical" ]
tapping, oscillatory motion, spectral analysis, finger tapping, oscillatory motion of the hand, oscillatory condition, oscillatory character
19025_task2
Sentence: Time intervals production in tapping and oscillatory motion . We applied spectral analysis on series of time intervals produced in a synchronization-continuation experiment . In the first condition intervals were produced by finger tapping , and in the second by an oscillatory motion of the hand . Results obtained in tapping were consistent with a discrete , event-based timing model . In the oscillatory condition , the spectra suggested a continuous , dynamic timing mechanism , based on the regulation of effector stiffness . It is concluded that the oscillatory character of movement can offer an important resource for timing control . The use of an event-based timing control such as postulated in the Wing-Kristoffersson model could be restricted to a quite limited class of rhythmic tasks , characterized by the concatenation of discrete events . Instructions: please extract entity words from the input sentence
[ "O", "O", "O", "O", "B-Participant_Condition", "O", "B-Participant_Condition", "I-Participant_Condition", "O", "O", "O", "B-Intervention_Physical", "I-Intervention_Physical", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "B-Participant_Condition", "I-Participant_Condition", "O", "O", "O", "O", "O", "O", "O", "B-Participant_Condition", "I-Participant_Condition", "I-Participant_Condition", "I-Participant_Condition", "I-Participant_Condition", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "B-Participant_Condition", "I-Participant_Condition", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "B-Participant_Condition", "I-Participant_Condition", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O" ]
Time intervals production in tapping and oscillatory motion . We applied spectral analysis on series of time intervals produced in a synchronization-continuation experiment . In the first condition intervals were produced by finger tapping , and in the second by an oscillatory motion of the hand . Results obtained in tapping were consistent with a discrete , event-based timing model . In the oscillatory condition , the spectra suggested a continuous , dynamic timing mechanism , based on the regulation of effector stiffness . It is concluded that the oscillatory character of movement can offer an important resource for timing control . The use of an event-based timing control such as postulated in the Wing-Kristoffersson model could be restricted to a quite limited class of rhythmic tasks , characterized by the concatenation of discrete events .
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[ "Participant_Condition", "Intervention_Physical" ]
criteria is an umlsterm, patients is an umlsterm, surgical treatment is an umlsterm, mobilisation is an umlsterm, leg is an umlsterm, treatment is an umlsterm, early mobilisation is an umlsterm, heel is an umlsterm, shoes is an umlsterm, Achilles tendon is an umlsterm, rupture is an umlsterm, period is an umlsterm, patients is an umlsterm, patients is an umlsterm, patients is an umlsterm, clinical examination is an umlsterm, testing is an umlsterm, muscle is an umlsterm, knee joint is an umlsterm, surgical treatment is an umlsterm, minor is an umlsterm, wound infections is an umlsterm, treatment is an umlsterm, muscle is an umlsterm, leg is an umlsterm, treatment is an umlsterm, surgical treatment is an umlsterm, treatment is an umlsterm, rupture is an umlsterm, ultrasound is an umlsterm, compliance is an umlsterm
DerChirurg.70680517.eng.abstr_task0
Sentence: According to sonographic criteria , 55 patients underwent surgical treatment with mobilisation in a lower leg plaster or conservative treatment with early mobilisation in heel pad ( 3 cm ) shoes when an acute Achilles tendon rupture ( ATR ) was diagnosed . The follow-up period in 51 patients was 2.4 years ( operated group = 28 patients , conservative group = 23 patients ) with clinical examination and testing of isokinetic muscle strength in knee joint flexion . After surgical treatment , minor wound infections occurred in 10.6 % . Reruptures occurred in 13 % of the conservative group . Following conservative treatment , the rate of stress-related achillodynia was significantly higher ( P = 0.019 ) . In the operated group mean isokinetic muscle strength was 13.7 % lower than in the uninvolved leg and decreased significantly with non-operative treatment to 75.3 % ( P = 0.012 ) . We recommend surgical treatment of acute ATR . The indications for conservative treatment depend on the extent of the rupture ( measured by ultrasound in the equinus position ) , the desired level of daily activity and the patient's degree of compliance . Instructions: please extract entities and their types from the input sentence, all entity types are in options Options: umlsterm
[ "O", "O", "O", "B-umlsterm", "O", "O", "B-umlsterm", "O", "B-umlsterm", "I-umlsterm", "O", "B-umlsterm", "O", "O", "O", "B-umlsterm", "O", "O", "O", "B-umlsterm", "O", "B-umlsterm", "I-umlsterm", "O", "B-umlsterm", "O", "O", "O", "O", "O", "B-umlsterm", "O", "O", "O", "B-umlsterm", "I-umlsterm", "B-umlsterm", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "B-umlsterm", "O", "O", "B-umlsterm", "O", "O", "O", "O", "O", "O", "O", "O", "B-umlsterm", "O", "O", "O", "O", "O", "B-umlsterm", "O", "O", "B-umlsterm", "I-umlsterm", "O", "B-umlsterm", "O", "O", "B-umlsterm", "O", "O", "B-umlsterm", "I-umlsterm", "O", "O", "O", "B-umlsterm", "I-umlsterm", "O", "B-umlsterm", "B-umlsterm", "I-umlsterm", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "B-umlsterm", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "B-umlsterm", "O", "O", "O", "O", "O", "O", "O", "O", "O", "B-umlsterm", "O", "O", "O", "O", "O", "O", "O", "B-umlsterm", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "B-umlsterm", "I-umlsterm", "O", "O", "O", "O", "O", "O", "O", "O", "B-umlsterm", "O", "O", "O", "O", "O", "O", "B-umlsterm", "O", "O", "O", "B-umlsterm", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "B-umlsterm", "O" ]
According to sonographic criteria , 55 patients underwent surgical treatment with mobilisation in a lower leg plaster or conservative treatment with early mobilisation in heel pad ( 3 cm ) shoes when an acute Achilles tendon rupture ( ATR ) was diagnosed . The follow-up period in 51 patients was 2.4 years ( operated group = 28 patients , conservative group = 23 patients ) with clinical examination and testing of isokinetic muscle strength in knee joint flexion . After surgical treatment , minor wound infections occurred in 10.6 % . Reruptures occurred in 13 % of the conservative group . Following conservative treatment , the rate of stress-related achillodynia was significantly higher ( P = 0.019 ) . In the operated group mean isokinetic muscle strength was 13.7 % lower than in the uninvolved leg and decreased significantly with non-operative treatment to 75.3 % ( P = 0.012 ) . We recommend surgical treatment of acute ATR . The indications for conservative treatment depend on the extent of the rupture ( measured by ultrasound in the equinus position ) , the desired level of daily activity and the patient's degree of compliance .
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[ "umlsterm" ]
criteria is an umlsterm, patients is an umlsterm, surgical treatment is an umlsterm, mobilisation is an umlsterm, leg is an umlsterm, treatment is an umlsterm, early mobilisation is an umlsterm, heel is an umlsterm, shoes is an umlsterm, Achilles tendon is an umlsterm, rupture is an umlsterm, period is an umlsterm, patients is an umlsterm, patients is an umlsterm, patients is an umlsterm, clinical examination is an umlsterm, testing is an umlsterm, muscle is an umlsterm, knee joint is an umlsterm, surgical treatment is an umlsterm, minor is an umlsterm, wound infections is an umlsterm, treatment is an umlsterm, muscle is an umlsterm, leg is an umlsterm, treatment is an umlsterm, surgical treatment is an umlsterm, treatment is an umlsterm, rupture is an umlsterm, ultrasound is an umlsterm, compliance is an umlsterm
DerChirurg.70680517.eng.abstr_task1
Sentence: According to sonographic criteria , 55 patients underwent surgical treatment with mobilisation in a lower leg plaster or conservative treatment with early mobilisation in heel pad ( 3 cm ) shoes when an acute Achilles tendon rupture ( ATR ) was diagnosed . The follow-up period in 51 patients was 2.4 years ( operated group = 28 patients , conservative group = 23 patients ) with clinical examination and testing of isokinetic muscle strength in knee joint flexion . After surgical treatment , minor wound infections occurred in 10.6 % . Reruptures occurred in 13 % of the conservative group . Following conservative treatment , the rate of stress-related achillodynia was significantly higher ( P = 0.019 ) . In the operated group mean isokinetic muscle strength was 13.7 % lower than in the uninvolved leg and decreased significantly with non-operative treatment to 75.3 % ( P = 0.012 ) . We recommend surgical treatment of acute ATR . The indications for conservative treatment depend on the extent of the rupture ( measured by ultrasound in the equinus position ) , the desired level of daily activity and the patient's degree of compliance . Instructions: please typing these entity words according to sentence: criteria, patients, surgical treatment, mobilisation, leg, treatment, early mobilisation, heel, shoes, Achilles tendon, rupture, period, patients, patients, patients, clinical examination, testing, muscle, knee joint, surgical treatment, minor, wound infections, treatment, muscle, leg, treatment, surgical treatment, treatment, rupture, ultrasound, compliance Options: umlsterm
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According to sonographic criteria , 55 patients underwent surgical treatment with mobilisation in a lower leg plaster or conservative treatment with early mobilisation in heel pad ( 3 cm ) shoes when an acute Achilles tendon rupture ( ATR ) was diagnosed . The follow-up period in 51 patients was 2.4 years ( operated group = 28 patients , conservative group = 23 patients ) with clinical examination and testing of isokinetic muscle strength in knee joint flexion . After surgical treatment , minor wound infections occurred in 10.6 % . Reruptures occurred in 13 % of the conservative group . Following conservative treatment , the rate of stress-related achillodynia was significantly higher ( P = 0.019 ) . In the operated group mean isokinetic muscle strength was 13.7 % lower than in the uninvolved leg and decreased significantly with non-operative treatment to 75.3 % ( P = 0.012 ) . We recommend surgical treatment of acute ATR . The indications for conservative treatment depend on the extent of the rupture ( measured by ultrasound in the equinus position ) , the desired level of daily activity and the patient's degree of compliance .
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[ "umlsterm" ]
criteria, patients, surgical treatment, mobilisation, leg, treatment, early mobilisation, heel, shoes, Achilles tendon, rupture, period, patients, patients, patients, clinical examination, testing, muscle, knee joint, surgical treatment, minor, wound infections, treatment, muscle, leg, treatment, surgical treatment, treatment, rupture, ultrasound, compliance
DerChirurg.70680517.eng.abstr_task2
Sentence: According to sonographic criteria , 55 patients underwent surgical treatment with mobilisation in a lower leg plaster or conservative treatment with early mobilisation in heel pad ( 3 cm ) shoes when an acute Achilles tendon rupture ( ATR ) was diagnosed . The follow-up period in 51 patients was 2.4 years ( operated group = 28 patients , conservative group = 23 patients ) with clinical examination and testing of isokinetic muscle strength in knee joint flexion . After surgical treatment , minor wound infections occurred in 10.6 % . Reruptures occurred in 13 % of the conservative group . Following conservative treatment , the rate of stress-related achillodynia was significantly higher ( P = 0.019 ) . In the operated group mean isokinetic muscle strength was 13.7 % lower than in the uninvolved leg and decreased significantly with non-operative treatment to 75.3 % ( P = 0.012 ) . We recommend surgical treatment of acute ATR . The indications for conservative treatment depend on the extent of the rupture ( measured by ultrasound in the equinus position ) , the desired level of daily activity and the patient's degree of compliance . Instructions: please extract entity words from the input sentence
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According to sonographic criteria , 55 patients underwent surgical treatment with mobilisation in a lower leg plaster or conservative treatment with early mobilisation in heel pad ( 3 cm ) shoes when an acute Achilles tendon rupture ( ATR ) was diagnosed . The follow-up period in 51 patients was 2.4 years ( operated group = 28 patients , conservative group = 23 patients ) with clinical examination and testing of isokinetic muscle strength in knee joint flexion . After surgical treatment , minor wound infections occurred in 10.6 % . Reruptures occurred in 13 % of the conservative group . Following conservative treatment , the rate of stress-related achillodynia was significantly higher ( P = 0.019 ) . In the operated group mean isokinetic muscle strength was 13.7 % lower than in the uninvolved leg and decreased significantly with non-operative treatment to 75.3 % ( P = 0.012 ) . We recommend surgical treatment of acute ATR . The indications for conservative treatment depend on the extent of the rupture ( measured by ultrasound in the equinus position ) , the desired level of daily activity and the patient's degree of compliance .
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[ "umlsterm" ]
Foxp3 is a Protein, Foxp3 is a Protein, Foxp3 is a Protein, Foxj1 is a Protein, Foxo3a is a Protein, Foxp3 is a Protein, luciferase is a Protein, Foxp3 is a Protein, green fluorescent protein is a Protein, luciferase is a Protein, Foxp3 is a Protein, Foxp3 is a Protein, Foxp3 is a Protein, Foxp3 is a Protein, Foxp3 is a Protein, Foxp3 is a Protein, luciferase is a Protein, Foxp3 is a Protein, Foxp3 is a Protein, Foxp3 is a Protein, CD4 is a Protein, Foxp3 is a Protein, CD4 is a Protein
163_task0
Sentence: Foxp3 Suppresses NF-kappaB Dependent Transcriptional Activation To ascertain the molecular mechanisms by which Foxp3 functions to promote the regulatory function of CD4+CD25hi T cells, we first confirmed the function of Foxp3 as a repressor of activation of NF-kappaB, previously implicated as a target of other forkhead/winged-helix family transcription factors (e.g., Foxj1 and Foxo3a) [19,20]. We analyzed the effect of Foxp3 overexpression on NF-kappaB activation in HEK 293T cells in dose-response and time course analyses. Transfection of HEK 293T cells with an NF-kappaB luciferase reporter vector in the presence or absence of increasing concentrations of a Foxp3 expression vector or a control vector (enhanced green fluorescent protein [EGFP]) was performed, and cells were harvested after 24 h to assay for luciferase activity and Foxp3 mRNA expression. Results indicated that as the concentration of Foxp3 transfected into cells increases (from 50 to 2,400 ng), the level of NF-kappaB activation decreases proportionally (Figure 1A). Foxp3 mRNA was also assayed to monitor activity of the Foxp3 expression vector (Figure 1B). Since NF-kappaB activation was partially affected by transfection of high concentrations of the control vector, we determined the fold inhibition of NF-kappaB activation by Foxp3 compared to the control vector at each concentration (Figure 1A). Fold inhibition of NF-kappaB activation was directly proportional to the level of Foxp3 mRNA expression detected by real-time RT-PCR. To determine the level of Foxp3-mediated suppression of NF-kappaB activation over time, HEK 293T cells were transfected with an NF-kappaB luciferase reporter vector and an expression vector encoding Foxp3 or EGFP (control vector) and harvested over 4 d. As shown in Figure 1C, NF-kappaB activation was suppressed by overexpression of Foxp3 at all time points. Extending these results from established, in vitro HEK cell lines to primary human lymphocytes, overexpression of Foxp3 in purified CD4+ T cells from three healthy donors also down-regulated the steady-state level of NF-kappaB activation (Figure 1D). These results recapitulate those from Bettelli and colleagues [15] demonstrating that Foxp3 functions, in part, to block NF-kappaB-dependent transcription in human cell lines as well as in primary human CD4+ T cells. Instructions: please extract entities and their types from the input sentence, all entity types are in options Options: Protein
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Foxp3 Suppresses NF-kappaB Dependent Transcriptional Activation To ascertain the molecular mechanisms by which Foxp3 functions to promote the regulatory function of CD4+CD25hi T cells, we first confirmed the function of Foxp3 as a repressor of activation of NF-kappaB, previously implicated as a target of other forkhead/winged-helix family transcription factors (e.g., Foxj1 and Foxo3a) [19,20]. We analyzed the effect of Foxp3 overexpression on NF-kappaB activation in HEK 293T cells in dose-response and time course analyses. Transfection of HEK 293T cells with an NF-kappaB luciferase reporter vector in the presence or absence of increasing concentrations of a Foxp3 expression vector or a control vector (enhanced green fluorescent protein [EGFP]) was performed, and cells were harvested after 24 h to assay for luciferase activity and Foxp3 mRNA expression. Results indicated that as the concentration of Foxp3 transfected into cells increases (from 50 to 2,400 ng), the level of NF-kappaB activation decreases proportionally (Figure 1A). Foxp3 mRNA was also assayed to monitor activity of the Foxp3 expression vector (Figure 1B). Since NF-kappaB activation was partially affected by transfection of high concentrations of the control vector, we determined the fold inhibition of NF-kappaB activation by Foxp3 compared to the control vector at each concentration (Figure 1A). Fold inhibition of NF-kappaB activation was directly proportional to the level of Foxp3 mRNA expression detected by real-time RT-PCR. To determine the level of Foxp3-mediated suppression of NF-kappaB activation over time, HEK 293T cells were transfected with an NF-kappaB luciferase reporter vector and an expression vector encoding Foxp3 or EGFP (control vector) and harvested over 4 d. As shown in Figure 1C, NF-kappaB activation was suppressed by overexpression of Foxp3 at all time points. Extending these results from established, in vitro HEK cell lines to primary human lymphocytes, overexpression of Foxp3 in purified CD4+ T cells from three healthy donors also down-regulated the steady-state level of NF-kappaB activation (Figure 1D). These results recapitulate those from Bettelli and colleagues [15] demonstrating that Foxp3 functions, in part, to block NF-kappaB-dependent transcription in human cell lines as well as in primary human CD4+ T cells.
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[ "Protein" ]
Foxp3 is a Protein, Foxp3 is a Protein, Foxp3 is a Protein, Foxj1 is a Protein, Foxo3a is a Protein, Foxp3 is a Protein, luciferase is a Protein, Foxp3 is a Protein, green fluorescent protein is a Protein, luciferase is a Protein, Foxp3 is a Protein, Foxp3 is a Protein, Foxp3 is a Protein, Foxp3 is a Protein, Foxp3 is a Protein, Foxp3 is a Protein, luciferase is a Protein, Foxp3 is a Protein, Foxp3 is a Protein, Foxp3 is a Protein, CD4 is a Protein, Foxp3 is a Protein, CD4 is a Protein
163_task1
Sentence: Foxp3 Suppresses NF-kappaB Dependent Transcriptional Activation To ascertain the molecular mechanisms by which Foxp3 functions to promote the regulatory function of CD4+CD25hi T cells, we first confirmed the function of Foxp3 as a repressor of activation of NF-kappaB, previously implicated as a target of other forkhead/winged-helix family transcription factors (e.g., Foxj1 and Foxo3a) [19,20]. We analyzed the effect of Foxp3 overexpression on NF-kappaB activation in HEK 293T cells in dose-response and time course analyses. Transfection of HEK 293T cells with an NF-kappaB luciferase reporter vector in the presence or absence of increasing concentrations of a Foxp3 expression vector or a control vector (enhanced green fluorescent protein [EGFP]) was performed, and cells were harvested after 24 h to assay for luciferase activity and Foxp3 mRNA expression. Results indicated that as the concentration of Foxp3 transfected into cells increases (from 50 to 2,400 ng), the level of NF-kappaB activation decreases proportionally (Figure 1A). Foxp3 mRNA was also assayed to monitor activity of the Foxp3 expression vector (Figure 1B). Since NF-kappaB activation was partially affected by transfection of high concentrations of the control vector, we determined the fold inhibition of NF-kappaB activation by Foxp3 compared to the control vector at each concentration (Figure 1A). Fold inhibition of NF-kappaB activation was directly proportional to the level of Foxp3 mRNA expression detected by real-time RT-PCR. To determine the level of Foxp3-mediated suppression of NF-kappaB activation over time, HEK 293T cells were transfected with an NF-kappaB luciferase reporter vector and an expression vector encoding Foxp3 or EGFP (control vector) and harvested over 4 d. As shown in Figure 1C, NF-kappaB activation was suppressed by overexpression of Foxp3 at all time points. Extending these results from established, in vitro HEK cell lines to primary human lymphocytes, overexpression of Foxp3 in purified CD4+ T cells from three healthy donors also down-regulated the steady-state level of NF-kappaB activation (Figure 1D). These results recapitulate those from Bettelli and colleagues [15] demonstrating that Foxp3 functions, in part, to block NF-kappaB-dependent transcription in human cell lines as well as in primary human CD4+ T cells. Instructions: please typing these entity words according to sentence: Foxp3, Foxp3, Foxp3, Foxj1, Foxo3a, Foxp3, luciferase, Foxp3, green fluorescent protein, luciferase, Foxp3, Foxp3, Foxp3, Foxp3, Foxp3, Foxp3, luciferase, Foxp3, Foxp3, Foxp3, CD4, Foxp3, CD4 Options: Protein
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Foxp3 Suppresses NF-kappaB Dependent Transcriptional Activation To ascertain the molecular mechanisms by which Foxp3 functions to promote the regulatory function of CD4+CD25hi T cells, we first confirmed the function of Foxp3 as a repressor of activation of NF-kappaB, previously implicated as a target of other forkhead/winged-helix family transcription factors (e.g., Foxj1 and Foxo3a) [19,20]. We analyzed the effect of Foxp3 overexpression on NF-kappaB activation in HEK 293T cells in dose-response and time course analyses. Transfection of HEK 293T cells with an NF-kappaB luciferase reporter vector in the presence or absence of increasing concentrations of a Foxp3 expression vector or a control vector (enhanced green fluorescent protein [EGFP]) was performed, and cells were harvested after 24 h to assay for luciferase activity and Foxp3 mRNA expression. Results indicated that as the concentration of Foxp3 transfected into cells increases (from 50 to 2,400 ng), the level of NF-kappaB activation decreases proportionally (Figure 1A). Foxp3 mRNA was also assayed to monitor activity of the Foxp3 expression vector (Figure 1B). Since NF-kappaB activation was partially affected by transfection of high concentrations of the control vector, we determined the fold inhibition of NF-kappaB activation by Foxp3 compared to the control vector at each concentration (Figure 1A). Fold inhibition of NF-kappaB activation was directly proportional to the level of Foxp3 mRNA expression detected by real-time RT-PCR. To determine the level of Foxp3-mediated suppression of NF-kappaB activation over time, HEK 293T cells were transfected with an NF-kappaB luciferase reporter vector and an expression vector encoding Foxp3 or EGFP (control vector) and harvested over 4 d. As shown in Figure 1C, NF-kappaB activation was suppressed by overexpression of Foxp3 at all time points. Extending these results from established, in vitro HEK cell lines to primary human lymphocytes, overexpression of Foxp3 in purified CD4+ T cells from three healthy donors also down-regulated the steady-state level of NF-kappaB activation (Figure 1D). These results recapitulate those from Bettelli and colleagues [15] demonstrating that Foxp3 functions, in part, to block NF-kappaB-dependent transcription in human cell lines as well as in primary human CD4+ T cells.
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[ "Protein" ]
Foxp3, Foxp3, Foxp3, Foxj1, Foxo3a, Foxp3, luciferase, Foxp3, green fluorescent protein, luciferase, Foxp3, Foxp3, Foxp3, Foxp3, Foxp3, Foxp3, luciferase, Foxp3, Foxp3, Foxp3, CD4, Foxp3, CD4
163_task2
Sentence: Foxp3 Suppresses NF-kappaB Dependent Transcriptional Activation To ascertain the molecular mechanisms by which Foxp3 functions to promote the regulatory function of CD4+CD25hi T cells, we first confirmed the function of Foxp3 as a repressor of activation of NF-kappaB, previously implicated as a target of other forkhead/winged-helix family transcription factors (e.g., Foxj1 and Foxo3a) [19,20]. We analyzed the effect of Foxp3 overexpression on NF-kappaB activation in HEK 293T cells in dose-response and time course analyses. Transfection of HEK 293T cells with an NF-kappaB luciferase reporter vector in the presence or absence of increasing concentrations of a Foxp3 expression vector or a control vector (enhanced green fluorescent protein [EGFP]) was performed, and cells were harvested after 24 h to assay for luciferase activity and Foxp3 mRNA expression. Results indicated that as the concentration of Foxp3 transfected into cells increases (from 50 to 2,400 ng), the level of NF-kappaB activation decreases proportionally (Figure 1A). Foxp3 mRNA was also assayed to monitor activity of the Foxp3 expression vector (Figure 1B). Since NF-kappaB activation was partially affected by transfection of high concentrations of the control vector, we determined the fold inhibition of NF-kappaB activation by Foxp3 compared to the control vector at each concentration (Figure 1A). Fold inhibition of NF-kappaB activation was directly proportional to the level of Foxp3 mRNA expression detected by real-time RT-PCR. To determine the level of Foxp3-mediated suppression of NF-kappaB activation over time, HEK 293T cells were transfected with an NF-kappaB luciferase reporter vector and an expression vector encoding Foxp3 or EGFP (control vector) and harvested over 4 d. As shown in Figure 1C, NF-kappaB activation was suppressed by overexpression of Foxp3 at all time points. Extending these results from established, in vitro HEK cell lines to primary human lymphocytes, overexpression of Foxp3 in purified CD4+ T cells from three healthy donors also down-regulated the steady-state level of NF-kappaB activation (Figure 1D). These results recapitulate those from Bettelli and colleagues [15] demonstrating that Foxp3 functions, in part, to block NF-kappaB-dependent transcription in human cell lines as well as in primary human CD4+ T cells. Instructions: please extract entity words from the input sentence
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Foxp3 Suppresses NF-kappaB Dependent Transcriptional Activation To ascertain the molecular mechanisms by which Foxp3 functions to promote the regulatory function of CD4+CD25hi T cells, we first confirmed the function of Foxp3 as a repressor of activation of NF-kappaB, previously implicated as a target of other forkhead/winged-helix family transcription factors (e.g., Foxj1 and Foxo3a) [19,20]. We analyzed the effect of Foxp3 overexpression on NF-kappaB activation in HEK 293T cells in dose-response and time course analyses. Transfection of HEK 293T cells with an NF-kappaB luciferase reporter vector in the presence or absence of increasing concentrations of a Foxp3 expression vector or a control vector (enhanced green fluorescent protein [EGFP]) was performed, and cells were harvested after 24 h to assay for luciferase activity and Foxp3 mRNA expression. Results indicated that as the concentration of Foxp3 transfected into cells increases (from 50 to 2,400 ng), the level of NF-kappaB activation decreases proportionally (Figure 1A). Foxp3 mRNA was also assayed to monitor activity of the Foxp3 expression vector (Figure 1B). Since NF-kappaB activation was partially affected by transfection of high concentrations of the control vector, we determined the fold inhibition of NF-kappaB activation by Foxp3 compared to the control vector at each concentration (Figure 1A). Fold inhibition of NF-kappaB activation was directly proportional to the level of Foxp3 mRNA expression detected by real-time RT-PCR. To determine the level of Foxp3-mediated suppression of NF-kappaB activation over time, HEK 293T cells were transfected with an NF-kappaB luciferase reporter vector and an expression vector encoding Foxp3 or EGFP (control vector) and harvested over 4 d. As shown in Figure 1C, NF-kappaB activation was suppressed by overexpression of Foxp3 at all time points. Extending these results from established, in vitro HEK cell lines to primary human lymphocytes, overexpression of Foxp3 in purified CD4+ T cells from three healthy donors also down-regulated the steady-state level of NF-kappaB activation (Figure 1D). These results recapitulate those from Bettelli and colleagues [15] demonstrating that Foxp3 functions, in part, to block NF-kappaB-dependent transcription in human cell lines as well as in primary human CD4+ T cells.
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[ "Protein" ]
actin is a Individual_protein, talin is a Individual_protein, integrins is a Individual_protein, vinculin is a Individual_protein, actin is a Individual_protein
660_task0
Sentence: The actin cytoskeleton-associated protein talin binds to integrins, vinculin, and actin. Instructions: please extract entities and their types from the input sentence, all entity types are in options Options: Individual_protein
[ "O", "B-Individual_protein", "O", "O", "O", "O", "B-Individual_protein", "O", "O", "B-Individual_protein", "O", "B-Individual_protein", "O", "O", "B-Individual_protein", "O" ]
The actin cytoskeleton-associated protein talin binds to integrins, vinculin, and actin.
[ "The", "actin", "cytoskeleton", "-", "associated", "protein", "talin", "binds", "to", "integrins", ",", "vinculin", ",", "and", "actin", "." ]
[ "Individual_protein" ]
actin is a Individual_protein, talin is a Individual_protein, integrins is a Individual_protein, vinculin is a Individual_protein, actin is a Individual_protein
660_task1
Sentence: The actin cytoskeleton-associated protein talin binds to integrins, vinculin, and actin. Instructions: please typing these entity words according to sentence: actin, talin, integrins, vinculin, actin Options: Individual_protein
[ "O", "B-Individual_protein", "O", "O", "O", "O", "B-Individual_protein", "O", "O", "B-Individual_protein", "O", "B-Individual_protein", "O", "O", "B-Individual_protein", "O" ]
The actin cytoskeleton-associated protein talin binds to integrins, vinculin, and actin.
[ "The", "actin", "cytoskeleton", "-", "associated", "protein", "talin", "binds", "to", "integrins", ",", "vinculin", ",", "and", "actin", "." ]
[ "Individual_protein" ]
actin, talin, integrins, vinculin, actin
660_task2
Sentence: The actin cytoskeleton-associated protein talin binds to integrins, vinculin, and actin. Instructions: please extract entity words from the input sentence
[ "O", "B-Individual_protein", "O", "O", "O", "O", "B-Individual_protein", "O", "O", "B-Individual_protein", "O", "B-Individual_protein", "O", "O", "B-Individual_protein", "O" ]
The actin cytoskeleton-associated protein talin binds to integrins, vinculin, and actin.
[ "The", "actin", "cytoskeleton", "-", "associated", "protein", "talin", "binds", "to", "integrins", ",", "vinculin", ",", "and", "actin", "." ]
[ "Individual_protein" ]
Therapie is an umlsterm, Medikament is an umlsterm, lokalen Applikation is an umlsterm, Auge is an umlsterm, Plasminogen is an umlsterm, Plasmin is an umlsterm, Plasmin is an umlsterm, Fibrin is an umlsterm, Fibrinbildung is an umlsterm, Auge is an umlsterm, Fibrin is an umlsterm, Komplikation is an umlsterm, Steroiden is an umlsterm, Patienten is an umlsterm, Patienten is an umlsterm, Fibrinbildung is an umlsterm, Phakoemulsifikation is an umlsterm, Fibrinolyse is an umlsterm, Fotodokumentation is an umlsterm, Rezidivbildung is an umlsterm, unerwuenschte Nebenwirkungen is an umlsterm, Fibrinolyse is an umlsterm, Spaetkomplikationen is an umlsterm, Fibrinbildung is an umlsterm, Glaukom- is an umlsterm, Kataraktoperation is an umlsterm
DerOpthalmologe.60930558.ger.abstr_task0
Sentence: Nachdem das Fibrinolytikum rt-PA in der systemischen Therapie von arteriellen und venoesen Gefaessverschluessen erfolgreich eingesetzt wird , steht uns nun dieses Medikament als Injektionsloesung zur lokalen Applikation im Auge zur Verfuegung . rt-PA wandelt Plasminogen in Plasmin um , Plasmin lysiert Fibrin und Fibrinclots . Zur Fibrinbildung im Auge kann es durch intraokulare Eingriffe , Traumata oder entzuendliche Prozesse kommen . In allen Faellen stellt Fibrin eine Komplikation dar , die in der Regel mit hochdosierten Steroiden nur langsam und oft unzureichend behandelt werden kann . Patienten : Bei 8 Patienten im Alter zwischen 59 und 75 Jahren wurde bei Fibrinbildung nach kombinierter Goniotrepanation und Phakoemulsifikation mit HKL-Implantation 10 µg rt-PA intraokular injiziert . Am 1. , 2. und 8. Tag nach der lokalen Fibrinolyse wurde der Verlauf des intraokularen Reizzustands mittels biomikroskopischem Befund , Fotodokumentation und Laser-Flare-Tyndallometrie beobachtet . Ergebnisse : Bei saemtlichen behandelten Faellen kam es zur vollstaendigen Aufloesung des Fibrinclots . Eine Rezidivbildung trat nicht ein , unerwuenschte Nebenwirkungen zeigten sich nicht . Schlussfolgerung : Die intraokulare Anwendung von rt-PA fuehrt zu einer sicheren Fibrinolyse und verhindert somit moegliche Spaetkomplikationen aufgrund von Fibrinbildung nach kombinierter Glaukom- und Kataraktoperation . Instructions: please extract entities and their types from the input sentence, all entity types are in options Options: umlsterm
[ "O", "O", "O", "O", "O", "O", "O", "O", "O", "B-umlsterm", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "B-umlsterm", "O", "O", "O", "B-umlsterm", "I-umlsterm", "O", "B-umlsterm", "O", "O", "O", "O", "O", "O", "O", "B-umlsterm", "O", "B-umlsterm", "O", "O", "B-umlsterm", "O", "B-umlsterm", "O", "O", "O", "O", "B-umlsterm", "O", "B-umlsterm", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "B-umlsterm", "O", "B-umlsterm", "O", "O", "O", "O", "O", "O", "O", "O", "B-umlsterm", "O", "O", "O", "O", "O", "O", "O", "O", "O", "B-umlsterm", "O", "O", "O", "B-umlsterm", "O", "O", "O", "O", "O", "O", "O", "O", "O", "B-umlsterm", "O", "O", "O", "O", "B-umlsterm", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "B-umlsterm", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "B-umlsterm", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "B-umlsterm", "O", "O", "O", "O", "B-umlsterm", "I-umlsterm", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "B-umlsterm", "O", "O", "O", "O", "B-umlsterm", "O", "O", "B-umlsterm", "O", "O", "B-umlsterm", "O", "B-umlsterm", "O" ]
Nachdem das Fibrinolytikum rt-PA in der systemischen Therapie von arteriellen und venoesen Gefaessverschluessen erfolgreich eingesetzt wird , steht uns nun dieses Medikament als Injektionsloesung zur lokalen Applikation im Auge zur Verfuegung . rt-PA wandelt Plasminogen in Plasmin um , Plasmin lysiert Fibrin und Fibrinclots . Zur Fibrinbildung im Auge kann es durch intraokulare Eingriffe , Traumata oder entzuendliche Prozesse kommen . In allen Faellen stellt Fibrin eine Komplikation dar , die in der Regel mit hochdosierten Steroiden nur langsam und oft unzureichend behandelt werden kann . Patienten : Bei 8 Patienten im Alter zwischen 59 und 75 Jahren wurde bei Fibrinbildung nach kombinierter Goniotrepanation und Phakoemulsifikation mit HKL-Implantation 10 µg rt-PA intraokular injiziert . Am 1. , 2. und 8. Tag nach der lokalen Fibrinolyse wurde der Verlauf des intraokularen Reizzustands mittels biomikroskopischem Befund , Fotodokumentation und Laser-Flare-Tyndallometrie beobachtet . Ergebnisse : Bei saemtlichen behandelten Faellen kam es zur vollstaendigen Aufloesung des Fibrinclots . Eine Rezidivbildung trat nicht ein , unerwuenschte Nebenwirkungen zeigten sich nicht . Schlussfolgerung : Die intraokulare Anwendung von rt-PA fuehrt zu einer sicheren Fibrinolyse und verhindert somit moegliche Spaetkomplikationen aufgrund von Fibrinbildung nach kombinierter Glaukom- und Kataraktoperation .
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[ "umlsterm" ]
Therapie is an umlsterm, Medikament is an umlsterm, lokalen Applikation is an umlsterm, Auge is an umlsterm, Plasminogen is an umlsterm, Plasmin is an umlsterm, Plasmin is an umlsterm, Fibrin is an umlsterm, Fibrinbildung is an umlsterm, Auge is an umlsterm, Fibrin is an umlsterm, Komplikation is an umlsterm, Steroiden is an umlsterm, Patienten is an umlsterm, Patienten is an umlsterm, Fibrinbildung is an umlsterm, Phakoemulsifikation is an umlsterm, Fibrinolyse is an umlsterm, Fotodokumentation is an umlsterm, Rezidivbildung is an umlsterm, unerwuenschte Nebenwirkungen is an umlsterm, Fibrinolyse is an umlsterm, Spaetkomplikationen is an umlsterm, Fibrinbildung is an umlsterm, Glaukom- is an umlsterm, Kataraktoperation is an umlsterm
DerOpthalmologe.60930558.ger.abstr_task1
Sentence: Nachdem das Fibrinolytikum rt-PA in der systemischen Therapie von arteriellen und venoesen Gefaessverschluessen erfolgreich eingesetzt wird , steht uns nun dieses Medikament als Injektionsloesung zur lokalen Applikation im Auge zur Verfuegung . rt-PA wandelt Plasminogen in Plasmin um , Plasmin lysiert Fibrin und Fibrinclots . Zur Fibrinbildung im Auge kann es durch intraokulare Eingriffe , Traumata oder entzuendliche Prozesse kommen . In allen Faellen stellt Fibrin eine Komplikation dar , die in der Regel mit hochdosierten Steroiden nur langsam und oft unzureichend behandelt werden kann . Patienten : Bei 8 Patienten im Alter zwischen 59 und 75 Jahren wurde bei Fibrinbildung nach kombinierter Goniotrepanation und Phakoemulsifikation mit HKL-Implantation 10 µg rt-PA intraokular injiziert . Am 1. , 2. und 8. Tag nach der lokalen Fibrinolyse wurde der Verlauf des intraokularen Reizzustands mittels biomikroskopischem Befund , Fotodokumentation und Laser-Flare-Tyndallometrie beobachtet . Ergebnisse : Bei saemtlichen behandelten Faellen kam es zur vollstaendigen Aufloesung des Fibrinclots . Eine Rezidivbildung trat nicht ein , unerwuenschte Nebenwirkungen zeigten sich nicht . Schlussfolgerung : Die intraokulare Anwendung von rt-PA fuehrt zu einer sicheren Fibrinolyse und verhindert somit moegliche Spaetkomplikationen aufgrund von Fibrinbildung nach kombinierter Glaukom- und Kataraktoperation . Instructions: please typing these entity words according to sentence: Therapie, Medikament, lokalen Applikation, Auge, Plasminogen, Plasmin, Plasmin, Fibrin, Fibrinbildung, Auge, Fibrin, Komplikation, Steroiden, Patienten, Patienten, Fibrinbildung, Phakoemulsifikation, Fibrinolyse, Fotodokumentation, Rezidivbildung, unerwuenschte Nebenwirkungen, Fibrinolyse, Spaetkomplikationen, Fibrinbildung, Glaukom-, Kataraktoperation Options: umlsterm
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Nachdem das Fibrinolytikum rt-PA in der systemischen Therapie von arteriellen und venoesen Gefaessverschluessen erfolgreich eingesetzt wird , steht uns nun dieses Medikament als Injektionsloesung zur lokalen Applikation im Auge zur Verfuegung . rt-PA wandelt Plasminogen in Plasmin um , Plasmin lysiert Fibrin und Fibrinclots . Zur Fibrinbildung im Auge kann es durch intraokulare Eingriffe , Traumata oder entzuendliche Prozesse kommen . In allen Faellen stellt Fibrin eine Komplikation dar , die in der Regel mit hochdosierten Steroiden nur langsam und oft unzureichend behandelt werden kann . Patienten : Bei 8 Patienten im Alter zwischen 59 und 75 Jahren wurde bei Fibrinbildung nach kombinierter Goniotrepanation und Phakoemulsifikation mit HKL-Implantation 10 µg rt-PA intraokular injiziert . Am 1. , 2. und 8. Tag nach der lokalen Fibrinolyse wurde der Verlauf des intraokularen Reizzustands mittels biomikroskopischem Befund , Fotodokumentation und Laser-Flare-Tyndallometrie beobachtet . Ergebnisse : Bei saemtlichen behandelten Faellen kam es zur vollstaendigen Aufloesung des Fibrinclots . Eine Rezidivbildung trat nicht ein , unerwuenschte Nebenwirkungen zeigten sich nicht . Schlussfolgerung : Die intraokulare Anwendung von rt-PA fuehrt zu einer sicheren Fibrinolyse und verhindert somit moegliche Spaetkomplikationen aufgrund von Fibrinbildung nach kombinierter Glaukom- und Kataraktoperation .
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[ "umlsterm" ]
Therapie, Medikament, lokalen Applikation, Auge, Plasminogen, Plasmin, Plasmin, Fibrin, Fibrinbildung, Auge, Fibrin, Komplikation, Steroiden, Patienten, Patienten, Fibrinbildung, Phakoemulsifikation, Fibrinolyse, Fotodokumentation, Rezidivbildung, unerwuenschte Nebenwirkungen, Fibrinolyse, Spaetkomplikationen, Fibrinbildung, Glaukom-, Kataraktoperation
DerOpthalmologe.60930558.ger.abstr_task2
Sentence: Nachdem das Fibrinolytikum rt-PA in der systemischen Therapie von arteriellen und venoesen Gefaessverschluessen erfolgreich eingesetzt wird , steht uns nun dieses Medikament als Injektionsloesung zur lokalen Applikation im Auge zur Verfuegung . rt-PA wandelt Plasminogen in Plasmin um , Plasmin lysiert Fibrin und Fibrinclots . Zur Fibrinbildung im Auge kann es durch intraokulare Eingriffe , Traumata oder entzuendliche Prozesse kommen . In allen Faellen stellt Fibrin eine Komplikation dar , die in der Regel mit hochdosierten Steroiden nur langsam und oft unzureichend behandelt werden kann . Patienten : Bei 8 Patienten im Alter zwischen 59 und 75 Jahren wurde bei Fibrinbildung nach kombinierter Goniotrepanation und Phakoemulsifikation mit HKL-Implantation 10 µg rt-PA intraokular injiziert . Am 1. , 2. und 8. Tag nach der lokalen Fibrinolyse wurde der Verlauf des intraokularen Reizzustands mittels biomikroskopischem Befund , Fotodokumentation und Laser-Flare-Tyndallometrie beobachtet . Ergebnisse : Bei saemtlichen behandelten Faellen kam es zur vollstaendigen Aufloesung des Fibrinclots . Eine Rezidivbildung trat nicht ein , unerwuenschte Nebenwirkungen zeigten sich nicht . Schlussfolgerung : Die intraokulare Anwendung von rt-PA fuehrt zu einer sicheren Fibrinolyse und verhindert somit moegliche Spaetkomplikationen aufgrund von Fibrinbildung nach kombinierter Glaukom- und Kataraktoperation . Instructions: please extract entity words from the input sentence
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Nachdem das Fibrinolytikum rt-PA in der systemischen Therapie von arteriellen und venoesen Gefaessverschluessen erfolgreich eingesetzt wird , steht uns nun dieses Medikament als Injektionsloesung zur lokalen Applikation im Auge zur Verfuegung . rt-PA wandelt Plasminogen in Plasmin um , Plasmin lysiert Fibrin und Fibrinclots . Zur Fibrinbildung im Auge kann es durch intraokulare Eingriffe , Traumata oder entzuendliche Prozesse kommen . In allen Faellen stellt Fibrin eine Komplikation dar , die in der Regel mit hochdosierten Steroiden nur langsam und oft unzureichend behandelt werden kann . Patienten : Bei 8 Patienten im Alter zwischen 59 und 75 Jahren wurde bei Fibrinbildung nach kombinierter Goniotrepanation und Phakoemulsifikation mit HKL-Implantation 10 µg rt-PA intraokular injiziert . Am 1. , 2. und 8. Tag nach der lokalen Fibrinolyse wurde der Verlauf des intraokularen Reizzustands mittels biomikroskopischem Befund , Fotodokumentation und Laser-Flare-Tyndallometrie beobachtet . Ergebnisse : Bei saemtlichen behandelten Faellen kam es zur vollstaendigen Aufloesung des Fibrinclots . Eine Rezidivbildung trat nicht ein , unerwuenschte Nebenwirkungen zeigten sich nicht . Schlussfolgerung : Die intraokulare Anwendung von rt-PA fuehrt zu einer sicheren Fibrinolyse und verhindert somit moegliche Spaetkomplikationen aufgrund von Fibrinbildung nach kombinierter Glaukom- und Kataraktoperation .
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[ "umlsterm" ]
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Rechtsmedizin.80080061.ger.abstr_task0
Sentence: Die Verwendung eines geschlechtsspezifischen " Durchschnitts-r " von 0,6 fuer Frauen und 0,7 fuer Maenner zur Berechnung der BAK aus Trinkmengenangaben ist mit einem relativ hohen Fehler behaftet , da individuelle , die Groesse von r beeinflussende Faktoren ausser acht gelassen werden . Durch die Einbeziehung des Koerpergewichtes und des Fettanteils an der Gesamtkoerpermasse in die Berechnung von r laesst sich dieser Fehler minimieren . Jedoch erreicht der Korrelationskoeffizient mit 0,442 noch keineswegs einen befriedigenden Wert . Nach Kombination der von Watson et al. [ 10 ] empirisch entwickelten Formel zur Berechnung des Gesamtkoerperwassers mit den Bezugsgroessen Gewicht , Groesse , Alter und Fettanteil an der Gesamtkoerpermasse wurden zwei Formeln entwickelt , die die Berechnung eines individuellen Verteilungsfactors fuer jede Person gestatten . Durch diese Formeln werden 99,1% der berechneten r beschrieben . Die Erfassung der anthropologischen Messdaten zur Berechnung der einzelnen Ausgangsgroessen kann jedoch die Praktikabilitaet negativ beeinflussen . Instructions: please extract entities and their types from the input sentence, all entity types are in options Options: umlsterm
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Die Verwendung eines geschlechtsspezifischen " Durchschnitts-r " von 0,6 fuer Frauen und 0,7 fuer Maenner zur Berechnung der BAK aus Trinkmengenangaben ist mit einem relativ hohen Fehler behaftet , da individuelle , die Groesse von r beeinflussende Faktoren ausser acht gelassen werden . Durch die Einbeziehung des Koerpergewichtes und des Fettanteils an der Gesamtkoerpermasse in die Berechnung von r laesst sich dieser Fehler minimieren . Jedoch erreicht der Korrelationskoeffizient mit 0,442 noch keineswegs einen befriedigenden Wert . Nach Kombination der von Watson et al. [ 10 ] empirisch entwickelten Formel zur Berechnung des Gesamtkoerperwassers mit den Bezugsgroessen Gewicht , Groesse , Alter und Fettanteil an der Gesamtkoerpermasse wurden zwei Formeln entwickelt , die die Berechnung eines individuellen Verteilungsfactors fuer jede Person gestatten . Durch diese Formeln werden 99,1% der berechneten r beschrieben . Die Erfassung der anthropologischen Messdaten zur Berechnung der einzelnen Ausgangsgroessen kann jedoch die Praktikabilitaet negativ beeinflussen .
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Rechtsmedizin.80080061.ger.abstr_task1
Sentence: Die Verwendung eines geschlechtsspezifischen " Durchschnitts-r " von 0,6 fuer Frauen und 0,7 fuer Maenner zur Berechnung der BAK aus Trinkmengenangaben ist mit einem relativ hohen Fehler behaftet , da individuelle , die Groesse von r beeinflussende Faktoren ausser acht gelassen werden . Durch die Einbeziehung des Koerpergewichtes und des Fettanteils an der Gesamtkoerpermasse in die Berechnung von r laesst sich dieser Fehler minimieren . Jedoch erreicht der Korrelationskoeffizient mit 0,442 noch keineswegs einen befriedigenden Wert . Nach Kombination der von Watson et al. [ 10 ] empirisch entwickelten Formel zur Berechnung des Gesamtkoerperwassers mit den Bezugsgroessen Gewicht , Groesse , Alter und Fettanteil an der Gesamtkoerpermasse wurden zwei Formeln entwickelt , die die Berechnung eines individuellen Verteilungsfactors fuer jede Person gestatten . Durch diese Formeln werden 99,1% der berechneten r beschrieben . Die Erfassung der anthropologischen Messdaten zur Berechnung der einzelnen Ausgangsgroessen kann jedoch die Praktikabilitaet negativ beeinflussen . Instructions: please typing these entity words according to sentence: Verwendung, geschlechtsspezifischen, Frauen, Maenner, Koerpergewichtes, Gesamtkoerperwassers, Person Options: umlsterm
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Die Verwendung eines geschlechtsspezifischen " Durchschnitts-r " von 0,6 fuer Frauen und 0,7 fuer Maenner zur Berechnung der BAK aus Trinkmengenangaben ist mit einem relativ hohen Fehler behaftet , da individuelle , die Groesse von r beeinflussende Faktoren ausser acht gelassen werden . Durch die Einbeziehung des Koerpergewichtes und des Fettanteils an der Gesamtkoerpermasse in die Berechnung von r laesst sich dieser Fehler minimieren . Jedoch erreicht der Korrelationskoeffizient mit 0,442 noch keineswegs einen befriedigenden Wert . Nach Kombination der von Watson et al. [ 10 ] empirisch entwickelten Formel zur Berechnung des Gesamtkoerperwassers mit den Bezugsgroessen Gewicht , Groesse , Alter und Fettanteil an der Gesamtkoerpermasse wurden zwei Formeln entwickelt , die die Berechnung eines individuellen Verteilungsfactors fuer jede Person gestatten . Durch diese Formeln werden 99,1% der berechneten r beschrieben . Die Erfassung der anthropologischen Messdaten zur Berechnung der einzelnen Ausgangsgroessen kann jedoch die Praktikabilitaet negativ beeinflussen .
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[ "umlsterm" ]
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Rechtsmedizin.80080061.ger.abstr_task2
Sentence: Die Verwendung eines geschlechtsspezifischen " Durchschnitts-r " von 0,6 fuer Frauen und 0,7 fuer Maenner zur Berechnung der BAK aus Trinkmengenangaben ist mit einem relativ hohen Fehler behaftet , da individuelle , die Groesse von r beeinflussende Faktoren ausser acht gelassen werden . Durch die Einbeziehung des Koerpergewichtes und des Fettanteils an der Gesamtkoerpermasse in die Berechnung von r laesst sich dieser Fehler minimieren . Jedoch erreicht der Korrelationskoeffizient mit 0,442 noch keineswegs einen befriedigenden Wert . Nach Kombination der von Watson et al. [ 10 ] empirisch entwickelten Formel zur Berechnung des Gesamtkoerperwassers mit den Bezugsgroessen Gewicht , Groesse , Alter und Fettanteil an der Gesamtkoerpermasse wurden zwei Formeln entwickelt , die die Berechnung eines individuellen Verteilungsfactors fuer jede Person gestatten . Durch diese Formeln werden 99,1% der berechneten r beschrieben . Die Erfassung der anthropologischen Messdaten zur Berechnung der einzelnen Ausgangsgroessen kann jedoch die Praktikabilitaet negativ beeinflussen . Instructions: please extract entity words from the input sentence
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Die Verwendung eines geschlechtsspezifischen " Durchschnitts-r " von 0,6 fuer Frauen und 0,7 fuer Maenner zur Berechnung der BAK aus Trinkmengenangaben ist mit einem relativ hohen Fehler behaftet , da individuelle , die Groesse von r beeinflussende Faktoren ausser acht gelassen werden . Durch die Einbeziehung des Koerpergewichtes und des Fettanteils an der Gesamtkoerpermasse in die Berechnung von r laesst sich dieser Fehler minimieren . Jedoch erreicht der Korrelationskoeffizient mit 0,442 noch keineswegs einen befriedigenden Wert . Nach Kombination der von Watson et al. [ 10 ] empirisch entwickelten Formel zur Berechnung des Gesamtkoerperwassers mit den Bezugsgroessen Gewicht , Groesse , Alter und Fettanteil an der Gesamtkoerpermasse wurden zwei Formeln entwickelt , die die Berechnung eines individuellen Verteilungsfactors fuer jede Person gestatten . Durch diese Formeln werden 99,1% der berechneten r beschrieben . Die Erfassung der anthropologischen Messdaten zur Berechnung der einzelnen Ausgangsgroessen kann jedoch die Praktikabilitaet negativ beeinflussen .
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[ "umlsterm" ]
ileoanal pouch is an umlsterm, procedure is an umlsterm, IAPP is an umlsterm, surgical is an umlsterm, therapy is an umlsterm, ulcerative colitis is an umlsterm, adenomatous polyposis is an umlsterm, disease is an umlsterm, control is an umlsterm, function is an umlsterm, quality of life is an umlsterm, procedures is an umlsterm, ileostomy is an umlsterm, patients is an umlsterm, function is an umlsterm, patients is an umlsterm, IAPP is an umlsterm, Surgical is an umlsterm, procedures is an umlsterm, complications is an umlsterm, complications is an umlsterm, fistulas is an umlsterm, abscesses is an umlsterm, stenosis is an umlsterm, inflammation is an umlsterm, mucosa is an umlsterm, multivariate analysis is an umlsterm, incidence is an umlsterm, treatment is an umlsterm, patients is an umlsterm, inflammation is an umlsterm, carcinoma is an umlsterm, mucosa is an umlsterm, ileum is an umlsterm, carcinoma is an umlsterm, patients is an umlsterm, endoscopy is an umlsterm, biopsies is an umlsterm, surveillance is an umlsterm, program is an umlsterm, Patients is an umlsterm, life quality is an umlsterm, controls is an umlsterm, postoperative complications is an umlsterm, development is an umlsterm, procedure is an umlsterm, therapy is an umlsterm, complications is an umlsterm
DerChirurg.90700530.eng.abstr_task0
Sentence: The ileoanal pouch procedure ( IAPP ) was the most remarkable breakthrough in the surgical therapy of ulcerative colitis ( UC ) and familial adenomatous polyposis ( FAP ) in the last 20 years . The underlying disease is under control , the function preserved and the quality of life markedly improved . Alternative procedures ( terminal ileostomy , ileorectal anastomosis ) are only indicated in special cases . In the last 16 years we have operated on 662 patients ( n = 493 UC ; n = 169 FAP ) with an ileoanal J-pouch , short rectal cuff , complete mucosectomy and hand-sewn anastomosis . Normally there is a good function for UC and FAP patients after IAPP . Surgical experience , technical modifications concerning the pouch design and the pouch-anal anastomosis , and a differentiated indication lead to a further improvement of these complex procedures with consecutive reduction of complications . Specific complications concerned mainly the pouch-anal anastomosis ( fistulas , abscesses , consecutive stenosis ) and inflammation of the pouch mucosa ( pouchitis ) . A multivariate analysis showed , that increasing experience of the specialized center is a significant factor reducing inflammatory problems at the anastomosis . The cumulative incidence of pouchitis was 29 % . In general there is no problem in successful treatment . But patients with chronic pouchitis are a problematic group ( 6.2 % ) . Chronic pouchitis is difficult to treat . It is likely that there exists an inflammation dysplasia carcinoma sequence for the ileal pouch mucosa , analogous to the colorectum . Recently we diagnosed the first case of a real ileum pouch carcinoma with associated epithelial dysplasias following chronic pouchitis . Therefore patients with chronic pouchitis must be followed up by endoscopy and random biopsies in a surveillance program . Patients with UC and FAP can gain the life quality of healthy controls , if postoperative complications can be avoided or treated successfully . For the further development of the procedure and the individual long-term success a qualified follow-up and therapy of complications is essential . Both can be carried out only by a specialized center . Instructions: please extract entities and their types from the input sentence, all entity types are in options Options: umlsterm
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The ileoanal pouch procedure ( IAPP ) was the most remarkable breakthrough in the surgical therapy of ulcerative colitis ( UC ) and familial adenomatous polyposis ( FAP ) in the last 20 years . The underlying disease is under control , the function preserved and the quality of life markedly improved . Alternative procedures ( terminal ileostomy , ileorectal anastomosis ) are only indicated in special cases . In the last 16 years we have operated on 662 patients ( n = 493 UC ; n = 169 FAP ) with an ileoanal J-pouch , short rectal cuff , complete mucosectomy and hand-sewn anastomosis . Normally there is a good function for UC and FAP patients after IAPP . Surgical experience , technical modifications concerning the pouch design and the pouch-anal anastomosis , and a differentiated indication lead to a further improvement of these complex procedures with consecutive reduction of complications . Specific complications concerned mainly the pouch-anal anastomosis ( fistulas , abscesses , consecutive stenosis ) and inflammation of the pouch mucosa ( pouchitis ) . A multivariate analysis showed , that increasing experience of the specialized center is a significant factor reducing inflammatory problems at the anastomosis . The cumulative incidence of pouchitis was 29 % . In general there is no problem in successful treatment . But patients with chronic pouchitis are a problematic group ( 6.2 % ) . Chronic pouchitis is difficult to treat . It is likely that there exists an inflammation dysplasia carcinoma sequence for the ileal pouch mucosa , analogous to the colorectum . Recently we diagnosed the first case of a real ileum pouch carcinoma with associated epithelial dysplasias following chronic pouchitis . Therefore patients with chronic pouchitis must be followed up by endoscopy and random biopsies in a surveillance program . Patients with UC and FAP can gain the life quality of healthy controls , if postoperative complications can be avoided or treated successfully . For the further development of the procedure and the individual long-term success a qualified follow-up and therapy of complications is essential . Both can be carried out only by a specialized center .
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[ "umlsterm" ]
ileoanal pouch is an umlsterm, procedure is an umlsterm, IAPP is an umlsterm, surgical is an umlsterm, therapy is an umlsterm, ulcerative colitis is an umlsterm, adenomatous polyposis is an umlsterm, disease is an umlsterm, control is an umlsterm, function is an umlsterm, quality of life is an umlsterm, procedures is an umlsterm, ileostomy is an umlsterm, patients is an umlsterm, function is an umlsterm, patients is an umlsterm, IAPP is an umlsterm, Surgical is an umlsterm, procedures is an umlsterm, complications is an umlsterm, complications is an umlsterm, fistulas is an umlsterm, abscesses is an umlsterm, stenosis is an umlsterm, inflammation is an umlsterm, mucosa is an umlsterm, multivariate analysis is an umlsterm, incidence is an umlsterm, treatment is an umlsterm, patients is an umlsterm, inflammation is an umlsterm, carcinoma is an umlsterm, mucosa is an umlsterm, ileum is an umlsterm, carcinoma is an umlsterm, patients is an umlsterm, endoscopy is an umlsterm, biopsies is an umlsterm, surveillance is an umlsterm, program is an umlsterm, Patients is an umlsterm, life quality is an umlsterm, controls is an umlsterm, postoperative complications is an umlsterm, development is an umlsterm, procedure is an umlsterm, therapy is an umlsterm, complications is an umlsterm
DerChirurg.90700530.eng.abstr_task1
Sentence: The ileoanal pouch procedure ( IAPP ) was the most remarkable breakthrough in the surgical therapy of ulcerative colitis ( UC ) and familial adenomatous polyposis ( FAP ) in the last 20 years . The underlying disease is under control , the function preserved and the quality of life markedly improved . Alternative procedures ( terminal ileostomy , ileorectal anastomosis ) are only indicated in special cases . In the last 16 years we have operated on 662 patients ( n = 493 UC ; n = 169 FAP ) with an ileoanal J-pouch , short rectal cuff , complete mucosectomy and hand-sewn anastomosis . Normally there is a good function for UC and FAP patients after IAPP . Surgical experience , technical modifications concerning the pouch design and the pouch-anal anastomosis , and a differentiated indication lead to a further improvement of these complex procedures with consecutive reduction of complications . Specific complications concerned mainly the pouch-anal anastomosis ( fistulas , abscesses , consecutive stenosis ) and inflammation of the pouch mucosa ( pouchitis ) . A multivariate analysis showed , that increasing experience of the specialized center is a significant factor reducing inflammatory problems at the anastomosis . The cumulative incidence of pouchitis was 29 % . In general there is no problem in successful treatment . But patients with chronic pouchitis are a problematic group ( 6.2 % ) . Chronic pouchitis is difficult to treat . It is likely that there exists an inflammation dysplasia carcinoma sequence for the ileal pouch mucosa , analogous to the colorectum . Recently we diagnosed the first case of a real ileum pouch carcinoma with associated epithelial dysplasias following chronic pouchitis . Therefore patients with chronic pouchitis must be followed up by endoscopy and random biopsies in a surveillance program . Patients with UC and FAP can gain the life quality of healthy controls , if postoperative complications can be avoided or treated successfully . For the further development of the procedure and the individual long-term success a qualified follow-up and therapy of complications is essential . Both can be carried out only by a specialized center . Instructions: please typing these entity words according to sentence: ileoanal pouch, procedure, IAPP, surgical, therapy, ulcerative colitis, adenomatous polyposis, disease, control, function, quality of life, procedures, ileostomy, patients, function, patients, IAPP, Surgical, procedures, complications, complications, fistulas, abscesses, stenosis, inflammation, mucosa, multivariate analysis, incidence, treatment, patients, inflammation, carcinoma, mucosa, ileum, carcinoma, patients, endoscopy, biopsies, surveillance, program, Patients, life quality, controls, postoperative complications, development, procedure, therapy, complications Options: umlsterm
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The ileoanal pouch procedure ( IAPP ) was the most remarkable breakthrough in the surgical therapy of ulcerative colitis ( UC ) and familial adenomatous polyposis ( FAP ) in the last 20 years . The underlying disease is under control , the function preserved and the quality of life markedly improved . Alternative procedures ( terminal ileostomy , ileorectal anastomosis ) are only indicated in special cases . In the last 16 years we have operated on 662 patients ( n = 493 UC ; n = 169 FAP ) with an ileoanal J-pouch , short rectal cuff , complete mucosectomy and hand-sewn anastomosis . Normally there is a good function for UC and FAP patients after IAPP . Surgical experience , technical modifications concerning the pouch design and the pouch-anal anastomosis , and a differentiated indication lead to a further improvement of these complex procedures with consecutive reduction of complications . Specific complications concerned mainly the pouch-anal anastomosis ( fistulas , abscesses , consecutive stenosis ) and inflammation of the pouch mucosa ( pouchitis ) . A multivariate analysis showed , that increasing experience of the specialized center is a significant factor reducing inflammatory problems at the anastomosis . The cumulative incidence of pouchitis was 29 % . In general there is no problem in successful treatment . But patients with chronic pouchitis are a problematic group ( 6.2 % ) . Chronic pouchitis is difficult to treat . It is likely that there exists an inflammation dysplasia carcinoma sequence for the ileal pouch mucosa , analogous to the colorectum . Recently we diagnosed the first case of a real ileum pouch carcinoma with associated epithelial dysplasias following chronic pouchitis . Therefore patients with chronic pouchitis must be followed up by endoscopy and random biopsies in a surveillance program . Patients with UC and FAP can gain the life quality of healthy controls , if postoperative complications can be avoided or treated successfully . For the further development of the procedure and the individual long-term success a qualified follow-up and therapy of complications is essential . Both can be carried out only by a specialized center .
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[ "umlsterm" ]
ileoanal pouch, procedure, IAPP, surgical, therapy, ulcerative colitis, adenomatous polyposis, disease, control, function, quality of life, procedures, ileostomy, patients, function, patients, IAPP, Surgical, procedures, complications, complications, fistulas, abscesses, stenosis, inflammation, mucosa, multivariate analysis, incidence, treatment, patients, inflammation, carcinoma, mucosa, ileum, carcinoma, patients, endoscopy, biopsies, surveillance, program, Patients, life quality, controls, postoperative complications, development, procedure, therapy, complications
DerChirurg.90700530.eng.abstr_task2
Sentence: The ileoanal pouch procedure ( IAPP ) was the most remarkable breakthrough in the surgical therapy of ulcerative colitis ( UC ) and familial adenomatous polyposis ( FAP ) in the last 20 years . The underlying disease is under control , the function preserved and the quality of life markedly improved . Alternative procedures ( terminal ileostomy , ileorectal anastomosis ) are only indicated in special cases . In the last 16 years we have operated on 662 patients ( n = 493 UC ; n = 169 FAP ) with an ileoanal J-pouch , short rectal cuff , complete mucosectomy and hand-sewn anastomosis . Normally there is a good function for UC and FAP patients after IAPP . Surgical experience , technical modifications concerning the pouch design and the pouch-anal anastomosis , and a differentiated indication lead to a further improvement of these complex procedures with consecutive reduction of complications . Specific complications concerned mainly the pouch-anal anastomosis ( fistulas , abscesses , consecutive stenosis ) and inflammation of the pouch mucosa ( pouchitis ) . A multivariate analysis showed , that increasing experience of the specialized center is a significant factor reducing inflammatory problems at the anastomosis . The cumulative incidence of pouchitis was 29 % . In general there is no problem in successful treatment . But patients with chronic pouchitis are a problematic group ( 6.2 % ) . Chronic pouchitis is difficult to treat . It is likely that there exists an inflammation dysplasia carcinoma sequence for the ileal pouch mucosa , analogous to the colorectum . Recently we diagnosed the first case of a real ileum pouch carcinoma with associated epithelial dysplasias following chronic pouchitis . Therefore patients with chronic pouchitis must be followed up by endoscopy and random biopsies in a surveillance program . Patients with UC and FAP can gain the life quality of healthy controls , if postoperative complications can be avoided or treated successfully . For the further development of the procedure and the individual long-term success a qualified follow-up and therapy of complications is essential . Both can be carried out only by a specialized center . Instructions: please extract entity words from the input sentence
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The ileoanal pouch procedure ( IAPP ) was the most remarkable breakthrough in the surgical therapy of ulcerative colitis ( UC ) and familial adenomatous polyposis ( FAP ) in the last 20 years . The underlying disease is under control , the function preserved and the quality of life markedly improved . Alternative procedures ( terminal ileostomy , ileorectal anastomosis ) are only indicated in special cases . In the last 16 years we have operated on 662 patients ( n = 493 UC ; n = 169 FAP ) with an ileoanal J-pouch , short rectal cuff , complete mucosectomy and hand-sewn anastomosis . Normally there is a good function for UC and FAP patients after IAPP . Surgical experience , technical modifications concerning the pouch design and the pouch-anal anastomosis , and a differentiated indication lead to a further improvement of these complex procedures with consecutive reduction of complications . Specific complications concerned mainly the pouch-anal anastomosis ( fistulas , abscesses , consecutive stenosis ) and inflammation of the pouch mucosa ( pouchitis ) . A multivariate analysis showed , that increasing experience of the specialized center is a significant factor reducing inflammatory problems at the anastomosis . The cumulative incidence of pouchitis was 29 % . In general there is no problem in successful treatment . But patients with chronic pouchitis are a problematic group ( 6.2 % ) . Chronic pouchitis is difficult to treat . It is likely that there exists an inflammation dysplasia carcinoma sequence for the ileal pouch mucosa , analogous to the colorectum . Recently we diagnosed the first case of a real ileum pouch carcinoma with associated epithelial dysplasias following chronic pouchitis . Therefore patients with chronic pouchitis must be followed up by endoscopy and random biopsies in a surveillance program . Patients with UC and FAP can gain the life quality of healthy controls , if postoperative complications can be avoided or treated successfully . For the further development of the procedure and the individual long-term success a qualified follow-up and therapy of complications is essential . Both can be carried out only by a specialized center .
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[ "umlsterm" ]
cocaine is a CHEMICAL, alpha2 adrenergic receptor is a GENE-N, AR is a GENE-N, dexmedetomidine is a CHEMICAL, cocaine is a CHEMICAL, norepinephrine is a CHEMICAL, cocaine is a CHEMICAL, cocaine is a CHEMICAL, dexmedetomidine is a CHEMICAL, cocaine is a CHEMICAL, Dexmedetomidine is a CHEMICAL, Dexmedetomidine is a CHEMICAL, cocaine is a CHEMICAL, alpha2C AR is a GENE-Y, dexmedetomidine is a CHEMICAL, Cocaine is a CHEMICAL
11046_task0
Sentence: Central sympatholysis as a novel countermeasure for cocaine-induced sympathetic activation and vasoconstriction in humans. OBJECTIVES: The aim of this study was to determine whether cocaine's sympathomimetic actions can be reversed by a potent centrally acting alpha2 adrenergic receptor (AR) agonist (dexmedetomidine). BACKGROUND: We recently showed that cocaine stimulates the human cardiovascular system primarily by acting in the brain to increase sympathetic nerve activity (SNA), the neural stimulus to norepinephrine release. Thus, SNA constitutes a putative new drug target to block cocaine's adverse cardiovascular effects at their origin. METHODS: In 22 healthy cocaine-naive humans, we measured skin SNA (microneurography) and skin blood flow (laser Doppler velocimetry) as well as heart rate and blood pressure before and after intranasal cocaine (2 mg/kg) alone and in combination with dexmedetomidine or saline. RESULTS: During intranasal cocaine alone, SNA increased by 2-fold and skin vascular resistance increased from 13.2 +/- 2.3 to 20.1 +/- 2.2 resistance units while mean arterial pressure increased by 14 +/- 3 mm Hg and heart rate by 18 +/- 3 beats/min (p < 0.01). Dexmedetomidine abolished these increases, whereas intravenous saline was without effect. Dexmedetomidine was effective in blocking these sympathomimetic actions of cocaine even in all 7 subjects who were homozygous for the Del322-325 polymorphism in the alpha2C AR, a loss-of-function mutation that is highly enriched in blacks. CONCLUSIONS: The data advance the novel hypothesis that central sympatholysis with dexmedetomidine constitutes a highly effective countermeasure for cocaine's sympathomimetic actions on the human cardiovascular system, even in individuals carrying the alpha2CDel322-325 polymorphism. (Study to Improve Scientific Understanding of the Cardiovascular Actions of Cocaine; http://clinicaltrials.gov/ct/show/NCT00338546?order=1; NCT00338546). Instructions: please extract entities and their types from the input sentence, all entity types are in options Options: GENE-Y, GENE-N, CHEMICAL
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Central sympatholysis as a novel countermeasure for cocaine-induced sympathetic activation and vasoconstriction in humans. OBJECTIVES: The aim of this study was to determine whether cocaine's sympathomimetic actions can be reversed by a potent centrally acting alpha2 adrenergic receptor (AR) agonist (dexmedetomidine). BACKGROUND: We recently showed that cocaine stimulates the human cardiovascular system primarily by acting in the brain to increase sympathetic nerve activity (SNA), the neural stimulus to norepinephrine release. Thus, SNA constitutes a putative new drug target to block cocaine's adverse cardiovascular effects at their origin. METHODS: In 22 healthy cocaine-naive humans, we measured skin SNA (microneurography) and skin blood flow (laser Doppler velocimetry) as well as heart rate and blood pressure before and after intranasal cocaine (2 mg/kg) alone and in combination with dexmedetomidine or saline. RESULTS: During intranasal cocaine alone, SNA increased by 2-fold and skin vascular resistance increased from 13.2 +/- 2.3 to 20.1 +/- 2.2 resistance units while mean arterial pressure increased by 14 +/- 3 mm Hg and heart rate by 18 +/- 3 beats/min (p < 0.01). Dexmedetomidine abolished these increases, whereas intravenous saline was without effect. Dexmedetomidine was effective in blocking these sympathomimetic actions of cocaine even in all 7 subjects who were homozygous for the Del322-325 polymorphism in the alpha2C AR, a loss-of-function mutation that is highly enriched in blacks. CONCLUSIONS: The data advance the novel hypothesis that central sympatholysis with dexmedetomidine constitutes a highly effective countermeasure for cocaine's sympathomimetic actions on the human cardiovascular system, even in individuals carrying the alpha2CDel322-325 polymorphism. (Study to Improve Scientific Understanding of the Cardiovascular Actions of Cocaine; http://clinicaltrials.gov/ct/show/NCT00338546?order=1; NCT00338546).
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[ "GENE-N", "CHEMICAL", "GENE-Y" ]
cocaine is a CHEMICAL, alpha2 adrenergic receptor is a GENE-N, AR is a GENE-N, dexmedetomidine is a CHEMICAL, cocaine is a CHEMICAL, norepinephrine is a CHEMICAL, cocaine is a CHEMICAL, cocaine is a CHEMICAL, dexmedetomidine is a CHEMICAL, cocaine is a CHEMICAL, Dexmedetomidine is a CHEMICAL, Dexmedetomidine is a CHEMICAL, cocaine is a CHEMICAL, alpha2C AR is a GENE-Y, dexmedetomidine is a CHEMICAL, Cocaine is a CHEMICAL
11046_task1
Sentence: Central sympatholysis as a novel countermeasure for cocaine-induced sympathetic activation and vasoconstriction in humans. OBJECTIVES: The aim of this study was to determine whether cocaine's sympathomimetic actions can be reversed by a potent centrally acting alpha2 adrenergic receptor (AR) agonist (dexmedetomidine). BACKGROUND: We recently showed that cocaine stimulates the human cardiovascular system primarily by acting in the brain to increase sympathetic nerve activity (SNA), the neural stimulus to norepinephrine release. Thus, SNA constitutes a putative new drug target to block cocaine's adverse cardiovascular effects at their origin. METHODS: In 22 healthy cocaine-naive humans, we measured skin SNA (microneurography) and skin blood flow (laser Doppler velocimetry) as well as heart rate and blood pressure before and after intranasal cocaine (2 mg/kg) alone and in combination with dexmedetomidine or saline. RESULTS: During intranasal cocaine alone, SNA increased by 2-fold and skin vascular resistance increased from 13.2 +/- 2.3 to 20.1 +/- 2.2 resistance units while mean arterial pressure increased by 14 +/- 3 mm Hg and heart rate by 18 +/- 3 beats/min (p < 0.01). Dexmedetomidine abolished these increases, whereas intravenous saline was without effect. Dexmedetomidine was effective in blocking these sympathomimetic actions of cocaine even in all 7 subjects who were homozygous for the Del322-325 polymorphism in the alpha2C AR, a loss-of-function mutation that is highly enriched in blacks. CONCLUSIONS: The data advance the novel hypothesis that central sympatholysis with dexmedetomidine constitutes a highly effective countermeasure for cocaine's sympathomimetic actions on the human cardiovascular system, even in individuals carrying the alpha2CDel322-325 polymorphism. (Study to Improve Scientific Understanding of the Cardiovascular Actions of Cocaine; http://clinicaltrials.gov/ct/show/NCT00338546?order=1; NCT00338546). Instructions: please typing these entity words according to sentence: cocaine, alpha2 adrenergic receptor, AR, dexmedetomidine, cocaine, norepinephrine, cocaine, cocaine, dexmedetomidine, cocaine, Dexmedetomidine, Dexmedetomidine, cocaine, alpha2C AR, dexmedetomidine, Cocaine Options: GENE-Y, GENE-N, CHEMICAL
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Central sympatholysis as a novel countermeasure for cocaine-induced sympathetic activation and vasoconstriction in humans. OBJECTIVES: The aim of this study was to determine whether cocaine's sympathomimetic actions can be reversed by a potent centrally acting alpha2 adrenergic receptor (AR) agonist (dexmedetomidine). BACKGROUND: We recently showed that cocaine stimulates the human cardiovascular system primarily by acting in the brain to increase sympathetic nerve activity (SNA), the neural stimulus to norepinephrine release. Thus, SNA constitutes a putative new drug target to block cocaine's adverse cardiovascular effects at their origin. METHODS: In 22 healthy cocaine-naive humans, we measured skin SNA (microneurography) and skin blood flow (laser Doppler velocimetry) as well as heart rate and blood pressure before and after intranasal cocaine (2 mg/kg) alone and in combination with dexmedetomidine or saline. RESULTS: During intranasal cocaine alone, SNA increased by 2-fold and skin vascular resistance increased from 13.2 +/- 2.3 to 20.1 +/- 2.2 resistance units while mean arterial pressure increased by 14 +/- 3 mm Hg and heart rate by 18 +/- 3 beats/min (p < 0.01). Dexmedetomidine abolished these increases, whereas intravenous saline was without effect. Dexmedetomidine was effective in blocking these sympathomimetic actions of cocaine even in all 7 subjects who were homozygous for the Del322-325 polymorphism in the alpha2C AR, a loss-of-function mutation that is highly enriched in blacks. CONCLUSIONS: The data advance the novel hypothesis that central sympatholysis with dexmedetomidine constitutes a highly effective countermeasure for cocaine's sympathomimetic actions on the human cardiovascular system, even in individuals carrying the alpha2CDel322-325 polymorphism. (Study to Improve Scientific Understanding of the Cardiovascular Actions of Cocaine; http://clinicaltrials.gov/ct/show/NCT00338546?order=1; NCT00338546).
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[ "GENE-N", "CHEMICAL", "GENE-Y" ]
cocaine, alpha2 adrenergic receptor, AR, dexmedetomidine, cocaine, norepinephrine, cocaine, cocaine, dexmedetomidine, cocaine, Dexmedetomidine, Dexmedetomidine, cocaine, alpha2C AR, dexmedetomidine, Cocaine
11046_task2
Sentence: Central sympatholysis as a novel countermeasure for cocaine-induced sympathetic activation and vasoconstriction in humans. OBJECTIVES: The aim of this study was to determine whether cocaine's sympathomimetic actions can be reversed by a potent centrally acting alpha2 adrenergic receptor (AR) agonist (dexmedetomidine). BACKGROUND: We recently showed that cocaine stimulates the human cardiovascular system primarily by acting in the brain to increase sympathetic nerve activity (SNA), the neural stimulus to norepinephrine release. Thus, SNA constitutes a putative new drug target to block cocaine's adverse cardiovascular effects at their origin. METHODS: In 22 healthy cocaine-naive humans, we measured skin SNA (microneurography) and skin blood flow (laser Doppler velocimetry) as well as heart rate and blood pressure before and after intranasal cocaine (2 mg/kg) alone and in combination with dexmedetomidine or saline. RESULTS: During intranasal cocaine alone, SNA increased by 2-fold and skin vascular resistance increased from 13.2 +/- 2.3 to 20.1 +/- 2.2 resistance units while mean arterial pressure increased by 14 +/- 3 mm Hg and heart rate by 18 +/- 3 beats/min (p < 0.01). Dexmedetomidine abolished these increases, whereas intravenous saline was without effect. Dexmedetomidine was effective in blocking these sympathomimetic actions of cocaine even in all 7 subjects who were homozygous for the Del322-325 polymorphism in the alpha2C AR, a loss-of-function mutation that is highly enriched in blacks. CONCLUSIONS: The data advance the novel hypothesis that central sympatholysis with dexmedetomidine constitutes a highly effective countermeasure for cocaine's sympathomimetic actions on the human cardiovascular system, even in individuals carrying the alpha2CDel322-325 polymorphism. (Study to Improve Scientific Understanding of the Cardiovascular Actions of Cocaine; http://clinicaltrials.gov/ct/show/NCT00338546?order=1; NCT00338546). Instructions: please extract entity words from the input sentence
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Central sympatholysis as a novel countermeasure for cocaine-induced sympathetic activation and vasoconstriction in humans. OBJECTIVES: The aim of this study was to determine whether cocaine's sympathomimetic actions can be reversed by a potent centrally acting alpha2 adrenergic receptor (AR) agonist (dexmedetomidine). BACKGROUND: We recently showed that cocaine stimulates the human cardiovascular system primarily by acting in the brain to increase sympathetic nerve activity (SNA), the neural stimulus to norepinephrine release. Thus, SNA constitutes a putative new drug target to block cocaine's adverse cardiovascular effects at their origin. METHODS: In 22 healthy cocaine-naive humans, we measured skin SNA (microneurography) and skin blood flow (laser Doppler velocimetry) as well as heart rate and blood pressure before and after intranasal cocaine (2 mg/kg) alone and in combination with dexmedetomidine or saline. RESULTS: During intranasal cocaine alone, SNA increased by 2-fold and skin vascular resistance increased from 13.2 +/- 2.3 to 20.1 +/- 2.2 resistance units while mean arterial pressure increased by 14 +/- 3 mm Hg and heart rate by 18 +/- 3 beats/min (p < 0.01). Dexmedetomidine abolished these increases, whereas intravenous saline was without effect. Dexmedetomidine was effective in blocking these sympathomimetic actions of cocaine even in all 7 subjects who were homozygous for the Del322-325 polymorphism in the alpha2C AR, a loss-of-function mutation that is highly enriched in blacks. CONCLUSIONS: The data advance the novel hypothesis that central sympatholysis with dexmedetomidine constitutes a highly effective countermeasure for cocaine's sympathomimetic actions on the human cardiovascular system, even in individuals carrying the alpha2CDel322-325 polymorphism. (Study to Improve Scientific Understanding of the Cardiovascular Actions of Cocaine; http://clinicaltrials.gov/ct/show/NCT00338546?order=1; NCT00338546).
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carcinoma urotelial is a MORFOLOGIA_NEOPLASIA, cáncer is a MORFOLOGIA_NEOPLASIA, adenocarcinoma is a MORFOLOGIA_NEOPLASIA, neoplasia is a MORFOLOGIA_NEOPLASIA, neoplasia is a MORFOLOGIA_NEOPLASIA, malignas is a MORFOLOGIA_NEOPLASIA, carcinoma is a MORFOLOGIA_NEOPLASIA, adenocarcinoma is a MORFOLOGIA_NEOPLASIA, adenocarcinoma is a MORFOLOGIA_NEOPLASIA, T3N0M0 is a MORFOLOGIA_NEOPLASIA, tumoración is a MORFOLOGIA_NEOPLASIA, adenocarcinoma is a MORFOLOGIA_NEOPLASIA, neoplásico is a MORFOLOGIA_NEOPLASIA, tumores is a MORFOLOGIA_NEOPLASIA, neoplásico is a MORFOLOGIA_NEOPLASIA, M0 is a MORFOLOGIA_NEOPLASIA, T3N0M0 is a MORFOLOGIA_NEOPLASIA, metástasis is a MORFOLOGIA_NEOPLASIA, T3 - 4N0 - 2M1a is a MORFOLOGIA_NEOPLASIA, maligna is a MORFOLOGIA_NEOPLASIA, adenocarcinoma is a MORFOLOGIA_NEOPLASIA, afectación bipulmonar is a MORFOLOGIA_NEOPLASIA, tumores is a MORFOLOGIA_NEOPLASIA, tumor is a MORFOLOGIA_NEOPLASIA, afectación bipulmonar is a MORFOLOGIA_NEOPLASIA, adenocarcinoma broncopulmonar bien diferenciado , de patrón lepídico / acinar / papilar is a MORFOLOGIA_NEOPLASIA, adenocarcinomas is a MORFOLOGIA_NEOPLASIA, ADC is a MORFOLOGIA_NEOPLASIA, T3N0M0 is a MORFOLOGIA_NEOPLASIA, ADC de pulmón lepídico is a MORFOLOGIA_NEOPLASIA, cT4a cN2M0 is a MORFOLOGIA_NEOPLASIA, tumorales is a MORFOLOGIA_NEOPLASIA, CPNM is a MORFOLOGIA_NEOPLASIA, metastásico is a MORFOLOGIA_NEOPLASIA, tumores is a MORFOLOGIA_NEOPLASIA, adenocarcinomas is a MORFOLOGIA_NEOPLASIA, ADC lepídico is a MORFOLOGIA_NEOPLASIA, T4N2M0 is a MORFOLOGIA_NEOPLASIA, ADC is a MORFOLOGIA_NEOPLASIA, T3N0M0 is a MORFOLOGIA_NEOPLASIA, tumores is a MORFOLOGIA_NEOPLASIA, tumor is a MORFOLOGIA_NEOPLASIA
359_task0
Sentence: Anamnesis Varón de 67 años, sin alergias medicamentosas conocidas, exfumador desde hace 1 mes, con un consumo acumulado de 15 paquetes-año, sin otros hábitos tóxicos. Padece hipertensión arterial en tratamiento farmacológico y diabetes tipo 2 no insulinodependiente. En 2006 se diagnostica de carcinoma urotelial de vejiga localizado, recibe tratamiento intravesical con BCG y posterior intervención quirúrgica, sin datos de recaída. En 2007, sufre síndrome coronario agudo que es revascularizado y por el cual está doblemente antiagregado. Claudicación intermitente sin repercusión a nivel funcional. Como situación basal, realiza vida activa, sin ayuda para ABVD, ECOG 0. Buen soporte familiar. Antecedentes familiares: madre con cáncer gástrico a los 70 años, fallecida por dicha causa. Consulta en nuestro centro para valorar inicio de tratamiento tras diagnóstico de adenocarcinoma de pulmón en centro privado. Exploración física Buen estado general, ECOG 0. Hemodinámicamente estable y afebril, eupneico en reposo. AC: rítmico, sin soplos audibles. AP: murmullo vesicular conservado, mínimos roncus en campo pulmonar derecho. Abdomen: blando y depresible, sin dolor a la palpación ni signos de irritación peritoneal, peristaltismo conservado. MMII: no hay edemas ni signos de trombosis venosa profunda. Pruebas complementarias En diciembre de 2017, el paciente presenta semiología de infección respiratoria con tos persistente y expectoración amarillenta. A pesar de tratamiento antibiótico y broncodilatadores inhalados, no aprecia mejoría y, además, asocia esputos hemoptoicos. Se inicia estudio diagnóstico en centro privado, realizándose radiografía de tórax donde se objetiva infiltrado redondeado extenso en lóbulo superior derecho (LSD). Ante la sospecha de neoplasia pulmonar, se realiza TC de tórax, objetivándose lesión a nivel de LSD compatible con neoplasia pulmonar con nódulos adyacentes a la lesión principal, confluyentes, con un tamaño total de unos 6-7 cm de diámetro máximo. Se completa el estudio radiológico con la realización de una PET-TC, para correcto estadiaje y biopsia de la lesión en LSD. En la PET realizada en enero de 2018 se observa una masa hipermetabólica de morfología irregular a nivel del lóbulo superior del pulmón derecho, coincidente con alteración de la TC, con SUVmax de 5,2, compatible con proceso pulmonar primario de características malignas, y mínima actividad a nivel de la alteración adyacente a dicha masa y en el pulmón izquierdo, con SUV de 0,6-1,1, compatibles con proceso infeccioso/inflamatorio. En la PAAF procedente de la lesión del LSD (realizada en centro privado), el resultado de Anatomía Patológica informa de células epiteliales abundantes con moderado pleomorfismo, mitosis y citoplasma, a veces evidente, dispuestas en nidos o láminas, carcinoma compatible con adenocarcinoma; no se realiza estudio molecular. Con diagnóstico de adenocarcinoma de pulmón estadio IIB (T3N0M0) a nivel de lóbulo superior derecho con tumoración de 6 cm e infiltrado infeccioso asociado en lóbulo inferior izquierdo (LII), es remitido a nuestro centro para valoración y tratamiento. Ante la ausencia de clínica infecciosa en ese momento y la semiología radiológica de ambas lesiones, se decide realización de nuevo TC-TAP en nuestro centro para conocer la situación actual de la enfermedad. En la TC de febrero de 2018 se aprecia la masa en LSD de 5,9 x 4,5 cm en relación con adenocarcinoma pulmonar conocido y masa en segmento basal lateral del LII, con broncograma aéreo, polilobulada, de 5,7 x 3,5 cm, compatible también con proceso neoplásico. El estadio radiológico depende de si se consideran dos tumores primarios: uno en LSD cT3-4 (nódulos ipsilaterales de densidad de partes blandas, a descartar origen neoplásico) cN0-2 (probable adenopatía paratraqueal derecha baja) M0, y otro en LII T3N0M0, o una lesión única en LSD con metástasis en el LII (T3-4N0-2M1a). Se decide completar estudio con biopsia pulmonar guiada por TC de la lesión en LII, que confirma la etiología maligna compatible con adenocarcinoma de origen pulmonar. En el estudio molecular de la masa de LII, se detecta mutación de EGFR (deleción del exón 19), ALK y ROS 1 negativo, PDL1 < 1 %. En esta situación, se plantea el diagnóstico diferencial entre una enfermedad estadio IV por afectación bipulmonar, o bien dos tumores sincrónicos con diferente perfil molecular. Dado que en uno de ellos presenta mutación EGFR exón 19, para lo cual se dispone de tratamiento dirigido que aumenta supervivencia, en febrero de 2018 se inicia tratamiento de primera línea con erlotinib a dosis 150 mg/24 h. Tras comentar el caso en comité multidisciplinar de cáncer de pulmón, se desestima tratamiento con radioterapia de la lesión en LSD por extenso tamaño inicial y nódulos satélites asociados. Como incidencias, tras diez días de tratamiento, el paciente presenta toxicidad cutánea grado 3 (reacción acneiforme), precisando suspender el tratamiento e iniciándose tratamiento corticoide, antibiótico y tratamiento tópico. Tras el tratamiento indicado y mejoría clínica, se reinicia el anti-EGFR reduciendo la dosis a 100 mg/24 h. En mayo de 2018, tras recibir tratamiento durante 3 meses, se realiza TC de reevaluación, en la que se objetiva evolución radiológica discordante. Se aprecia mejoría significativa de la masa en LII y, de forma antagónica, un aumento de tamaño de la masa en LSD y de los nódulos pulmonares derechos asociados. Según criterios RECIST 1.1, la enfermedad se considera estable en caso de tratarse de un único tumor con afectación bipulmonar. Ante la evolución disociada de ambas lesiones, se solicita nueva biopsia de la lesión del LSD, dado que el diagnóstico había sido en centro externo y no se disponía del material anatomo-patológico para su revisión ni de estudio molecular. Se realiza BAG de lesión pulmonar de LSD, que objetiva infiltración por un adenocarcinoma broncopulmonar bien diferenciado, de patrón lepídico/acinar/papilar. La inmunotinción para PDL-1 resulta positiva en < 1 % de las células en este material; EGFR wild type, ALK negativo. Diagnóstico Así pues, se confirma que el paciente presenta dos adenocarcinomas de forma sincrónica, en LII un ADC pulmón T3N0M0 EGFR mutado (deleción exón 19) y en LSD un ADC de pulmón lepídico cT4a cN2M0, EGFR/ALK/ROS1 wild type. Tratamiento Con el diagnóstico definitivo de ambas lesiones y ante la no respuesta de la lesión sin mutación dirigida, en julio de 2018 se plantea tratamiento sistémico basado en quimioterapia, según esquema carboplatino AUC 5: pemetrexed 500 mg/m2 cada 21 días, y se decide suspender erlotinib y seguimiento estrecho de la lesión pulmonar izquierda, en ese momento en respuesta radiológica parcial mayor. Evolución Tras la administración de 1 ciclo de quimioterapia, el paciente sufre SCASEST secundario a lesión grave en primera diagonal, paliada con un stent fármaco-activo, y lesión en segunda diagonal no revascularizable. No sufre repercusiones en la fracción de eyección ventricular (FEVI 48 %, igual que al inicio del tratamiento), y el paciente evoluciona favorablemente, en seguimiento por Cardiología. Después de ser valorado de forma conjunta por Cardiología y Oncología, se decide continuar con tratamiento de quimioterapia y el paciente recibe 2 ciclos más (sin nuevas complicaciones). Se solicita TC reevaluación en septiembre de 2018, donde la respuesta global es enfermedad estable, según criterios RECIST 1.1. La afectación del LSD presenta una disminución de su tamaño y la lesión del LII ha crecido discretamente respecto a estudios previos, sin cumplir dicha lesión criterios de progresión radiológica. Completa 5 ciclos de quimioterapia sin incidencias. Ante esta situación de discordancia, entre ambas lesiones tumorales, se plantean en sesión clínica las opciones terapéuticas más beneficiosas para el paciente. Se decide la mejor opción de tratamiento basándonos en un análisis exploratorio de subgrupos en un ensayo aleatorizado, controlado de fase II. En este ensayo, la combinación de pemetrexed-erlotinib (pemetrexed 500 mg/m2 intravenoso en el día 1 de un ciclo de 3 semanas y erlotinib 150 mg/24 h vía oral desde el día 2 hasta el día 14 de un ciclo de 3 semanas), se comparó con pemetrexed o erlotinib solos como tratamiento de segunda línea en una población seleccionada de pacientes no fumadores con CPNM localmente avanzado o metastásico no escamoso, objetivándose beneficio en SLP para el grupo con el tratamiento combinado y toxicidad tolerable3. Así pues, se decide continuar con pemetrexed de mantenimiento y reiniciar erlotinib, para que ambas lesiones reciban su tratamiento adecuado, tras explicar al paciente riesgos y beneficios. En octubre de 2018, tras 5 ciclos de quimioterapia, el paciente inicia tratamiento de mantenimiento con pemetrexed 500 mg/m2 cada 21 días para la lesión en LSD y tratamiento con erlotinib 100 mg/días +2 al +15 cada 21 días para la lesión en LII. No presenta toxicidad secundaria al tratamiento con pemetrexed, pero sí al reiniciar erlotinib, precisando reducción de dosis a 75 mg/24 h debido de nuevo a toxicidad cutánea grado 3 (reacción acneiforme). En todo momento, el paciente se encuentra asintomático a nivel respiratorio. Tras 4 ciclos de tratamiento combinado con erlotinib y pemetrexed se realiza nueva TC de reevaluación en enero de 2019, con datos de enfermedad estable por criterios RECIST 1.1 en ambos tumores, respuesta que se mantiene en el TC de marzo de 2019, después de 8 meses de tratamiento combinado, presentando el paciente excelente tolerancia, sin toxicidad ni complicaciones durante el tratamiento. En resumen, se trata de un paciente de 67 años, cardiópata, que ha sido diagnosticado de dos adenocarcinomas de pulmón con diferente perfil molecular, de forma sincrónica. En el pulmón derecho presenta ADC lepídico estadio IIIB (T4N2M0) EGFR wild type y, en el pulmón izquierdo, ADC pulmón estadio IIB (T3N0M0) EGFR mutado (deleción exón 19). Ha recibido tratamiento sistémico para ambos tumores. En primer lugar, tratamiento anti-EGFR con erlotinib, con regular tolerancia clínica por toxicidad cutánea grado 3 (reacción acneiforme), que ha precisado detener el tratamiento y disminuir la dosis en dos ocasiones, pero con aceptable respuesta a nivel metabólico, disminuyendo el tamaño de la lesión de forma progresiva, después de 11 ciclos en total. En relación al tumor no mutado, ha recibido tratamiento basado en quimioterapia según esquema carboplatino-pemetrexed durante 5 ciclos y posteriormente, pemetrexed de mantenimiento. En mayo de 2019, tras 10 ciclos de tratamiento combinado, presenta enfermedad estable como respuesta global, pero se objetiva un ligero crecimiento de ambas lesiones. Se ha decidido administrar RT sobre lesión en LII, y se ha solicitado biopsia en plasma (negativo), para detectar mutaciones de resistencia (T790M), también pendiente de resultado. Para la lesión de LSD, se valorará en caso de progresión, inicio de inmunoterapia (inhibidores de checkpoint) dado que por su tamaño no es candidata a tratamiento radioterápico actualmente. Se mantiene tratamiento combinado por el momento, con excelente tolerancia clínica. Instructions: please extract entities and their types from the input sentence, all entity types are in options Options: MORFOLOGIA_NEOPLASIA
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Anamnesis Varón de 67 años, sin alergias medicamentosas conocidas, exfumador desde hace 1 mes, con un consumo acumulado de 15 paquetes-año, sin otros hábitos tóxicos. Padece hipertensión arterial en tratamiento farmacológico y diabetes tipo 2 no insulinodependiente. En 2006 se diagnostica de carcinoma urotelial de vejiga localizado, recibe tratamiento intravesical con BCG y posterior intervención quirúrgica, sin datos de recaída. En 2007, sufre síndrome coronario agudo que es revascularizado y por el cual está doblemente antiagregado. Claudicación intermitente sin repercusión a nivel funcional. Como situación basal, realiza vida activa, sin ayuda para ABVD, ECOG 0. Buen soporte familiar. Antecedentes familiares: madre con cáncer gástrico a los 70 años, fallecida por dicha causa. Consulta en nuestro centro para valorar inicio de tratamiento tras diagnóstico de adenocarcinoma de pulmón en centro privado. Exploración física Buen estado general, ECOG 0. Hemodinámicamente estable y afebril, eupneico en reposo. AC: rítmico, sin soplos audibles. AP: murmullo vesicular conservado, mínimos roncus en campo pulmonar derecho. Abdomen: blando y depresible, sin dolor a la palpación ni signos de irritación peritoneal, peristaltismo conservado. MMII: no hay edemas ni signos de trombosis venosa profunda. Pruebas complementarias En diciembre de 2017, el paciente presenta semiología de infección respiratoria con tos persistente y expectoración amarillenta. A pesar de tratamiento antibiótico y broncodilatadores inhalados, no aprecia mejoría y, además, asocia esputos hemoptoicos. Se inicia estudio diagnóstico en centro privado, realizándose radiografía de tórax donde se objetiva infiltrado redondeado extenso en lóbulo superior derecho (LSD). Ante la sospecha de neoplasia pulmonar, se realiza TC de tórax, objetivándose lesión a nivel de LSD compatible con neoplasia pulmonar con nódulos adyacentes a la lesión principal, confluyentes, con un tamaño total de unos 6-7 cm de diámetro máximo. Se completa el estudio radiológico con la realización de una PET-TC, para correcto estadiaje y biopsia de la lesión en LSD. En la PET realizada en enero de 2018 se observa una masa hipermetabólica de morfología irregular a nivel del lóbulo superior del pulmón derecho, coincidente con alteración de la TC, con SUVmax de 5,2, compatible con proceso pulmonar primario de características malignas, y mínima actividad a nivel de la alteración adyacente a dicha masa y en el pulmón izquierdo, con SUV de 0,6-1,1, compatibles con proceso infeccioso/inflamatorio. En la PAAF procedente de la lesión del LSD (realizada en centro privado), el resultado de Anatomía Patológica informa de células epiteliales abundantes con moderado pleomorfismo, mitosis y citoplasma, a veces evidente, dispuestas en nidos o láminas, carcinoma compatible con adenocarcinoma; no se realiza estudio molecular. Con diagnóstico de adenocarcinoma de pulmón estadio IIB (T3N0M0) a nivel de lóbulo superior derecho con tumoración de 6 cm e infiltrado infeccioso asociado en lóbulo inferior izquierdo (LII), es remitido a nuestro centro para valoración y tratamiento. Ante la ausencia de clínica infecciosa en ese momento y la semiología radiológica de ambas lesiones, se decide realización de nuevo TC-TAP en nuestro centro para conocer la situación actual de la enfermedad. En la TC de febrero de 2018 se aprecia la masa en LSD de 5,9 x 4,5 cm en relación con adenocarcinoma pulmonar conocido y masa en segmento basal lateral del LII, con broncograma aéreo, polilobulada, de 5,7 x 3,5 cm, compatible también con proceso neoplásico. El estadio radiológico depende de si se consideran dos tumores primarios: uno en LSD cT3-4 (nódulos ipsilaterales de densidad de partes blandas, a descartar origen neoplásico) cN0-2 (probable adenopatía paratraqueal derecha baja) M0, y otro en LII T3N0M0, o una lesión única en LSD con metástasis en el LII (T3-4N0-2M1a). Se decide completar estudio con biopsia pulmonar guiada por TC de la lesión en LII, que confirma la etiología maligna compatible con adenocarcinoma de origen pulmonar. En el estudio molecular de la masa de LII, se detecta mutación de EGFR (deleción del exón 19), ALK y ROS 1 negativo, PDL1 < 1 %. En esta situación, se plantea el diagnóstico diferencial entre una enfermedad estadio IV por afectación bipulmonar, o bien dos tumores sincrónicos con diferente perfil molecular. Dado que en uno de ellos presenta mutación EGFR exón 19, para lo cual se dispone de tratamiento dirigido que aumenta supervivencia, en febrero de 2018 se inicia tratamiento de primera línea con erlotinib a dosis 150 mg/24 h. Tras comentar el caso en comité multidisciplinar de cáncer de pulmón, se desestima tratamiento con radioterapia de la lesión en LSD por extenso tamaño inicial y nódulos satélites asociados. Como incidencias, tras diez días de tratamiento, el paciente presenta toxicidad cutánea grado 3 (reacción acneiforme), precisando suspender el tratamiento e iniciándose tratamiento corticoide, antibiótico y tratamiento tópico. Tras el tratamiento indicado y mejoría clínica, se reinicia el anti-EGFR reduciendo la dosis a 100 mg/24 h. En mayo de 2018, tras recibir tratamiento durante 3 meses, se realiza TC de reevaluación, en la que se objetiva evolución radiológica discordante. Se aprecia mejoría significativa de la masa en LII y, de forma antagónica, un aumento de tamaño de la masa en LSD y de los nódulos pulmonares derechos asociados. Según criterios RECIST 1.1, la enfermedad se considera estable en caso de tratarse de un único tumor con afectación bipulmonar. Ante la evolución disociada de ambas lesiones, se solicita nueva biopsia de la lesión del LSD, dado que el diagnóstico había sido en centro externo y no se disponía del material anatomo-patológico para su revisión ni de estudio molecular. Se realiza BAG de lesión pulmonar de LSD, que objetiva infiltración por un adenocarcinoma broncopulmonar bien diferenciado, de patrón lepídico/acinar/papilar. La inmunotinción para PDL-1 resulta positiva en < 1 % de las células en este material; EGFR wild type, ALK negativo. Diagnóstico Así pues, se confirma que el paciente presenta dos adenocarcinomas de forma sincrónica, en LII un ADC pulmón T3N0M0 EGFR mutado (deleción exón 19) y en LSD un ADC de pulmón lepídico cT4a cN2M0, EGFR/ALK/ROS1 wild type. Tratamiento Con el diagnóstico definitivo de ambas lesiones y ante la no respuesta de la lesión sin mutación dirigida, en julio de 2018 se plantea tratamiento sistémico basado en quimioterapia, según esquema carboplatino AUC 5: pemetrexed 500 mg/m2 cada 21 días, y se decide suspender erlotinib y seguimiento estrecho de la lesión pulmonar izquierda, en ese momento en respuesta radiológica parcial mayor. Evolución Tras la administración de 1 ciclo de quimioterapia, el paciente sufre SCASEST secundario a lesión grave en primera diagonal, paliada con un stent fármaco-activo, y lesión en segunda diagonal no revascularizable. No sufre repercusiones en la fracción de eyección ventricular (FEVI 48 %, igual que al inicio del tratamiento), y el paciente evoluciona favorablemente, en seguimiento por Cardiología. Después de ser valorado de forma conjunta por Cardiología y Oncología, se decide continuar con tratamiento de quimioterapia y el paciente recibe 2 ciclos más (sin nuevas complicaciones). Se solicita TC reevaluación en septiembre de 2018, donde la respuesta global es enfermedad estable, según criterios RECIST 1.1. La afectación del LSD presenta una disminución de su tamaño y la lesión del LII ha crecido discretamente respecto a estudios previos, sin cumplir dicha lesión criterios de progresión radiológica. Completa 5 ciclos de quimioterapia sin incidencias. Ante esta situación de discordancia, entre ambas lesiones tumorales, se plantean en sesión clínica las opciones terapéuticas más beneficiosas para el paciente. Se decide la mejor opción de tratamiento basándonos en un análisis exploratorio de subgrupos en un ensayo aleatorizado, controlado de fase II. En este ensayo, la combinación de pemetrexed-erlotinib (pemetrexed 500 mg/m2 intravenoso en el día 1 de un ciclo de 3 semanas y erlotinib 150 mg/24 h vía oral desde el día 2 hasta el día 14 de un ciclo de 3 semanas), se comparó con pemetrexed o erlotinib solos como tratamiento de segunda línea en una población seleccionada de pacientes no fumadores con CPNM localmente avanzado o metastásico no escamoso, objetivándose beneficio en SLP para el grupo con el tratamiento combinado y toxicidad tolerable3. Así pues, se decide continuar con pemetrexed de mantenimiento y reiniciar erlotinib, para que ambas lesiones reciban su tratamiento adecuado, tras explicar al paciente riesgos y beneficios. En octubre de 2018, tras 5 ciclos de quimioterapia, el paciente inicia tratamiento de mantenimiento con pemetrexed 500 mg/m2 cada 21 días para la lesión en LSD y tratamiento con erlotinib 100 mg/días +2 al +15 cada 21 días para la lesión en LII. No presenta toxicidad secundaria al tratamiento con pemetrexed, pero sí al reiniciar erlotinib, precisando reducción de dosis a 75 mg/24 h debido de nuevo a toxicidad cutánea grado 3 (reacción acneiforme). En todo momento, el paciente se encuentra asintomático a nivel respiratorio. Tras 4 ciclos de tratamiento combinado con erlotinib y pemetrexed se realiza nueva TC de reevaluación en enero de 2019, con datos de enfermedad estable por criterios RECIST 1.1 en ambos tumores, respuesta que se mantiene en el TC de marzo de 2019, después de 8 meses de tratamiento combinado, presentando el paciente excelente tolerancia, sin toxicidad ni complicaciones durante el tratamiento. En resumen, se trata de un paciente de 67 años, cardiópata, que ha sido diagnosticado de dos adenocarcinomas de pulmón con diferente perfil molecular, de forma sincrónica. En el pulmón derecho presenta ADC lepídico estadio IIIB (T4N2M0) EGFR wild type y, en el pulmón izquierdo, ADC pulmón estadio IIB (T3N0M0) EGFR mutado (deleción exón 19). Ha recibido tratamiento sistémico para ambos tumores. En primer lugar, tratamiento anti-EGFR con erlotinib, con regular tolerancia clínica por toxicidad cutánea grado 3 (reacción acneiforme), que ha precisado detener el tratamiento y disminuir la dosis en dos ocasiones, pero con aceptable respuesta a nivel metabólico, disminuyendo el tamaño de la lesión de forma progresiva, después de 11 ciclos en total. En relación al tumor no mutado, ha recibido tratamiento basado en quimioterapia según esquema carboplatino-pemetrexed durante 5 ciclos y posteriormente, pemetrexed de mantenimiento. En mayo de 2019, tras 10 ciclos de tratamiento combinado, presenta enfermedad estable como respuesta global, pero se objetiva un ligero crecimiento de ambas lesiones. Se ha decidido administrar RT sobre lesión en LII, y se ha solicitado biopsia en plasma (negativo), para detectar mutaciones de resistencia (T790M), también pendiente de resultado. Para la lesión de LSD, se valorará en caso de progresión, inicio de inmunoterapia (inhibidores de checkpoint) dado que por su tamaño no es candidata a tratamiento radioterápico actualmente. Se mantiene tratamiento combinado por el momento, con excelente tolerancia clínica.
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"hasta", "el", "día", "14", "de", "un", "ciclo", "de", "3", "semanas", ")", ",", "se", "comparó", "con", "pemetrexed", "o", "erlotinib", "solos", "como", "tratamiento", "de", "segunda", "línea", "en", "una", "población", "seleccionada", "de", "pacientes", "no", "fumadores", "con", "CPNM", "localmente", "avanzado", "o", "metastásico", "no", "escamoso", ",", "objetivándose", "beneficio", "en", "SLP", "para", "el", "grupo", "con", "el", "tratamiento", "combinado", "y", "toxicidad", "tolerable3", ".", "Así", "pues", ",", "se", "decide", "continuar", "con", "pemetrexed", "de", "mantenimiento", "y", "reiniciar", "erlotinib", ",", "para", "que", "ambas", "lesiones", "reciban", "su", "tratamiento", "adecuado", ",", "tras", "explicar", "al", "paciente", "riesgos", "y", "beneficios", ".", "\n", "En", "octubre", "de", "2018", ",", "tras", "5", "ciclos", "de", "quimioterapia", ",", "el", "paciente", "inicia", "tratamiento", "de", "mantenimiento", "con", "pemetrexed", "500", "mg", "/", "m2", 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",", "con", "regular", "tolerancia", "clínica", "por", "toxicidad", "cutánea", "grado", "3", "(", "reacción", "acneiforme", ")", ",", "que", "ha", "precisado", "detener", "el", "tratamiento", "y", "disminuir", "la", "dosis", "en", "dos", "ocasiones", ",", "pero", "con", "aceptable", "respuesta", "a", "nivel", "metabólico", ",", "disminuyendo", "el", "tamaño", "de", "la", "lesión", "de", "forma", "progresiva", ",", "después", "de", "11", "ciclos", "en", "total", ".", "En", "relación", "al", "tumor", "no", "mutado", ",", "ha", "recibido", "tratamiento", "basado", "en", "quimioterapia", "según", "esquema", "carboplatino", "-", "pemetrexed", "durante", "5", "ciclos", "y", "posteriormente", ",", "pemetrexed", "de", "mantenimiento", ".", "\n", "En", "mayo", "de", "2019", ",", "tras", "10", "ciclos", "de", "tratamiento", "combinado", ",", "presenta", "enfermedad", "estable", "como", "respuesta", "global", ",", "pero", "se", "objetiva", "un", "ligero", "crecimiento", "de", "ambas", "lesiones", ".", "Se", "ha", "decidido", "administrar", "RT", "sobre", "lesión", "en", "LII", ",", "y", "se", "ha", "solicitado", "biopsia", "en", "plasma", "(", "negativo", ")", ",", "para", "detectar", "mutaciones", "de", "resistencia", "(", "T790", "M", ")", ",", "también", "pendiente", "de", "resultado", ".", "Para", "la", "lesión", "de", "LSD", ",", "se", "valorará", "en", "caso", "de", "progresión", ",", "inicio", "de", "inmunoterapia", "(", "inhibidores", "de", "checkpoint", ")", "dado", "que", "por", "su", "tamaño", "no", "es", "candidata", "a", "tratamiento", "radioterápico", "actualmente", ".", "\n", "Se", "mantiene", "tratamiento", "combinado", "por", "el", "momento", ",", "con", "excelente", "tolerancia", "clínica", ".", "\n" ]
[ "MORFOLOGIA_NEOPLASIA" ]
carcinoma urotelial is a MORFOLOGIA_NEOPLASIA, cáncer is a MORFOLOGIA_NEOPLASIA, adenocarcinoma is a MORFOLOGIA_NEOPLASIA, neoplasia is a MORFOLOGIA_NEOPLASIA, neoplasia is a MORFOLOGIA_NEOPLASIA, malignas is a MORFOLOGIA_NEOPLASIA, carcinoma is a MORFOLOGIA_NEOPLASIA, adenocarcinoma is a MORFOLOGIA_NEOPLASIA, adenocarcinoma is a MORFOLOGIA_NEOPLASIA, T3N0M0 is a MORFOLOGIA_NEOPLASIA, tumoración is a MORFOLOGIA_NEOPLASIA, adenocarcinoma is a MORFOLOGIA_NEOPLASIA, neoplásico is a MORFOLOGIA_NEOPLASIA, tumores is a MORFOLOGIA_NEOPLASIA, neoplásico is a MORFOLOGIA_NEOPLASIA, M0 is a MORFOLOGIA_NEOPLASIA, T3N0M0 is a MORFOLOGIA_NEOPLASIA, metástasis is a MORFOLOGIA_NEOPLASIA, T3 - 4N0 - 2M1a is a MORFOLOGIA_NEOPLASIA, maligna is a MORFOLOGIA_NEOPLASIA, adenocarcinoma is a MORFOLOGIA_NEOPLASIA, afectación bipulmonar is a MORFOLOGIA_NEOPLASIA, tumores is a MORFOLOGIA_NEOPLASIA, tumor is a MORFOLOGIA_NEOPLASIA, afectación bipulmonar is a MORFOLOGIA_NEOPLASIA, adenocarcinoma broncopulmonar bien diferenciado , de patrón lepídico / acinar / papilar is a MORFOLOGIA_NEOPLASIA, adenocarcinomas is a MORFOLOGIA_NEOPLASIA, ADC is a MORFOLOGIA_NEOPLASIA, T3N0M0 is a MORFOLOGIA_NEOPLASIA, ADC de pulmón lepídico is a MORFOLOGIA_NEOPLASIA, cT4a cN2M0 is a MORFOLOGIA_NEOPLASIA, tumorales is a MORFOLOGIA_NEOPLASIA, CPNM is a MORFOLOGIA_NEOPLASIA, metastásico is a MORFOLOGIA_NEOPLASIA, tumores is a MORFOLOGIA_NEOPLASIA, adenocarcinomas is a MORFOLOGIA_NEOPLASIA, ADC lepídico is a MORFOLOGIA_NEOPLASIA, T4N2M0 is a MORFOLOGIA_NEOPLASIA, ADC is a MORFOLOGIA_NEOPLASIA, T3N0M0 is a MORFOLOGIA_NEOPLASIA, tumores is a MORFOLOGIA_NEOPLASIA, tumor is a MORFOLOGIA_NEOPLASIA
359_task1
Sentence: Anamnesis Varón de 67 años, sin alergias medicamentosas conocidas, exfumador desde hace 1 mes, con un consumo acumulado de 15 paquetes-año, sin otros hábitos tóxicos. Padece hipertensión arterial en tratamiento farmacológico y diabetes tipo 2 no insulinodependiente. En 2006 se diagnostica de carcinoma urotelial de vejiga localizado, recibe tratamiento intravesical con BCG y posterior intervención quirúrgica, sin datos de recaída. En 2007, sufre síndrome coronario agudo que es revascularizado y por el cual está doblemente antiagregado. Claudicación intermitente sin repercusión a nivel funcional. Como situación basal, realiza vida activa, sin ayuda para ABVD, ECOG 0. Buen soporte familiar. Antecedentes familiares: madre con cáncer gástrico a los 70 años, fallecida por dicha causa. Consulta en nuestro centro para valorar inicio de tratamiento tras diagnóstico de adenocarcinoma de pulmón en centro privado. Exploración física Buen estado general, ECOG 0. Hemodinámicamente estable y afebril, eupneico en reposo. AC: rítmico, sin soplos audibles. AP: murmullo vesicular conservado, mínimos roncus en campo pulmonar derecho. Abdomen: blando y depresible, sin dolor a la palpación ni signos de irritación peritoneal, peristaltismo conservado. MMII: no hay edemas ni signos de trombosis venosa profunda. Pruebas complementarias En diciembre de 2017, el paciente presenta semiología de infección respiratoria con tos persistente y expectoración amarillenta. A pesar de tratamiento antibiótico y broncodilatadores inhalados, no aprecia mejoría y, además, asocia esputos hemoptoicos. Se inicia estudio diagnóstico en centro privado, realizándose radiografía de tórax donde se objetiva infiltrado redondeado extenso en lóbulo superior derecho (LSD). Ante la sospecha de neoplasia pulmonar, se realiza TC de tórax, objetivándose lesión a nivel de LSD compatible con neoplasia pulmonar con nódulos adyacentes a la lesión principal, confluyentes, con un tamaño total de unos 6-7 cm de diámetro máximo. Se completa el estudio radiológico con la realización de una PET-TC, para correcto estadiaje y biopsia de la lesión en LSD. En la PET realizada en enero de 2018 se observa una masa hipermetabólica de morfología irregular a nivel del lóbulo superior del pulmón derecho, coincidente con alteración de la TC, con SUVmax de 5,2, compatible con proceso pulmonar primario de características malignas, y mínima actividad a nivel de la alteración adyacente a dicha masa y en el pulmón izquierdo, con SUV de 0,6-1,1, compatibles con proceso infeccioso/inflamatorio. En la PAAF procedente de la lesión del LSD (realizada en centro privado), el resultado de Anatomía Patológica informa de células epiteliales abundantes con moderado pleomorfismo, mitosis y citoplasma, a veces evidente, dispuestas en nidos o láminas, carcinoma compatible con adenocarcinoma; no se realiza estudio molecular. Con diagnóstico de adenocarcinoma de pulmón estadio IIB (T3N0M0) a nivel de lóbulo superior derecho con tumoración de 6 cm e infiltrado infeccioso asociado en lóbulo inferior izquierdo (LII), es remitido a nuestro centro para valoración y tratamiento. Ante la ausencia de clínica infecciosa en ese momento y la semiología radiológica de ambas lesiones, se decide realización de nuevo TC-TAP en nuestro centro para conocer la situación actual de la enfermedad. En la TC de febrero de 2018 se aprecia la masa en LSD de 5,9 x 4,5 cm en relación con adenocarcinoma pulmonar conocido y masa en segmento basal lateral del LII, con broncograma aéreo, polilobulada, de 5,7 x 3,5 cm, compatible también con proceso neoplásico. El estadio radiológico depende de si se consideran dos tumores primarios: uno en LSD cT3-4 (nódulos ipsilaterales de densidad de partes blandas, a descartar origen neoplásico) cN0-2 (probable adenopatía paratraqueal derecha baja) M0, y otro en LII T3N0M0, o una lesión única en LSD con metástasis en el LII (T3-4N0-2M1a). Se decide completar estudio con biopsia pulmonar guiada por TC de la lesión en LII, que confirma la etiología maligna compatible con adenocarcinoma de origen pulmonar. En el estudio molecular de la masa de LII, se detecta mutación de EGFR (deleción del exón 19), ALK y ROS 1 negativo, PDL1 < 1 %. En esta situación, se plantea el diagnóstico diferencial entre una enfermedad estadio IV por afectación bipulmonar, o bien dos tumores sincrónicos con diferente perfil molecular. Dado que en uno de ellos presenta mutación EGFR exón 19, para lo cual se dispone de tratamiento dirigido que aumenta supervivencia, en febrero de 2018 se inicia tratamiento de primera línea con erlotinib a dosis 150 mg/24 h. Tras comentar el caso en comité multidisciplinar de cáncer de pulmón, se desestima tratamiento con radioterapia de la lesión en LSD por extenso tamaño inicial y nódulos satélites asociados. Como incidencias, tras diez días de tratamiento, el paciente presenta toxicidad cutánea grado 3 (reacción acneiforme), precisando suspender el tratamiento e iniciándose tratamiento corticoide, antibiótico y tratamiento tópico. Tras el tratamiento indicado y mejoría clínica, se reinicia el anti-EGFR reduciendo la dosis a 100 mg/24 h. En mayo de 2018, tras recibir tratamiento durante 3 meses, se realiza TC de reevaluación, en la que se objetiva evolución radiológica discordante. Se aprecia mejoría significativa de la masa en LII y, de forma antagónica, un aumento de tamaño de la masa en LSD y de los nódulos pulmonares derechos asociados. Según criterios RECIST 1.1, la enfermedad se considera estable en caso de tratarse de un único tumor con afectación bipulmonar. Ante la evolución disociada de ambas lesiones, se solicita nueva biopsia de la lesión del LSD, dado que el diagnóstico había sido en centro externo y no se disponía del material anatomo-patológico para su revisión ni de estudio molecular. Se realiza BAG de lesión pulmonar de LSD, que objetiva infiltración por un adenocarcinoma broncopulmonar bien diferenciado, de patrón lepídico/acinar/papilar. La inmunotinción para PDL-1 resulta positiva en < 1 % de las células en este material; EGFR wild type, ALK negativo. Diagnóstico Así pues, se confirma que el paciente presenta dos adenocarcinomas de forma sincrónica, en LII un ADC pulmón T3N0M0 EGFR mutado (deleción exón 19) y en LSD un ADC de pulmón lepídico cT4a cN2M0, EGFR/ALK/ROS1 wild type. Tratamiento Con el diagnóstico definitivo de ambas lesiones y ante la no respuesta de la lesión sin mutación dirigida, en julio de 2018 se plantea tratamiento sistémico basado en quimioterapia, según esquema carboplatino AUC 5: pemetrexed 500 mg/m2 cada 21 días, y se decide suspender erlotinib y seguimiento estrecho de la lesión pulmonar izquierda, en ese momento en respuesta radiológica parcial mayor. Evolución Tras la administración de 1 ciclo de quimioterapia, el paciente sufre SCASEST secundario a lesión grave en primera diagonal, paliada con un stent fármaco-activo, y lesión en segunda diagonal no revascularizable. No sufre repercusiones en la fracción de eyección ventricular (FEVI 48 %, igual que al inicio del tratamiento), y el paciente evoluciona favorablemente, en seguimiento por Cardiología. Después de ser valorado de forma conjunta por Cardiología y Oncología, se decide continuar con tratamiento de quimioterapia y el paciente recibe 2 ciclos más (sin nuevas complicaciones). Se solicita TC reevaluación en septiembre de 2018, donde la respuesta global es enfermedad estable, según criterios RECIST 1.1. La afectación del LSD presenta una disminución de su tamaño y la lesión del LII ha crecido discretamente respecto a estudios previos, sin cumplir dicha lesión criterios de progresión radiológica. Completa 5 ciclos de quimioterapia sin incidencias. Ante esta situación de discordancia, entre ambas lesiones tumorales, se plantean en sesión clínica las opciones terapéuticas más beneficiosas para el paciente. Se decide la mejor opción de tratamiento basándonos en un análisis exploratorio de subgrupos en un ensayo aleatorizado, controlado de fase II. En este ensayo, la combinación de pemetrexed-erlotinib (pemetrexed 500 mg/m2 intravenoso en el día 1 de un ciclo de 3 semanas y erlotinib 150 mg/24 h vía oral desde el día 2 hasta el día 14 de un ciclo de 3 semanas), se comparó con pemetrexed o erlotinib solos como tratamiento de segunda línea en una población seleccionada de pacientes no fumadores con CPNM localmente avanzado o metastásico no escamoso, objetivándose beneficio en SLP para el grupo con el tratamiento combinado y toxicidad tolerable3. Así pues, se decide continuar con pemetrexed de mantenimiento y reiniciar erlotinib, para que ambas lesiones reciban su tratamiento adecuado, tras explicar al paciente riesgos y beneficios. En octubre de 2018, tras 5 ciclos de quimioterapia, el paciente inicia tratamiento de mantenimiento con pemetrexed 500 mg/m2 cada 21 días para la lesión en LSD y tratamiento con erlotinib 100 mg/días +2 al +15 cada 21 días para la lesión en LII. No presenta toxicidad secundaria al tratamiento con pemetrexed, pero sí al reiniciar erlotinib, precisando reducción de dosis a 75 mg/24 h debido de nuevo a toxicidad cutánea grado 3 (reacción acneiforme). En todo momento, el paciente se encuentra asintomático a nivel respiratorio. Tras 4 ciclos de tratamiento combinado con erlotinib y pemetrexed se realiza nueva TC de reevaluación en enero de 2019, con datos de enfermedad estable por criterios RECIST 1.1 en ambos tumores, respuesta que se mantiene en el TC de marzo de 2019, después de 8 meses de tratamiento combinado, presentando el paciente excelente tolerancia, sin toxicidad ni complicaciones durante el tratamiento. En resumen, se trata de un paciente de 67 años, cardiópata, que ha sido diagnosticado de dos adenocarcinomas de pulmón con diferente perfil molecular, de forma sincrónica. En el pulmón derecho presenta ADC lepídico estadio IIIB (T4N2M0) EGFR wild type y, en el pulmón izquierdo, ADC pulmón estadio IIB (T3N0M0) EGFR mutado (deleción exón 19). Ha recibido tratamiento sistémico para ambos tumores. En primer lugar, tratamiento anti-EGFR con erlotinib, con regular tolerancia clínica por toxicidad cutánea grado 3 (reacción acneiforme), que ha precisado detener el tratamiento y disminuir la dosis en dos ocasiones, pero con aceptable respuesta a nivel metabólico, disminuyendo el tamaño de la lesión de forma progresiva, después de 11 ciclos en total. En relación al tumor no mutado, ha recibido tratamiento basado en quimioterapia según esquema carboplatino-pemetrexed durante 5 ciclos y posteriormente, pemetrexed de mantenimiento. En mayo de 2019, tras 10 ciclos de tratamiento combinado, presenta enfermedad estable como respuesta global, pero se objetiva un ligero crecimiento de ambas lesiones. Se ha decidido administrar RT sobre lesión en LII, y se ha solicitado biopsia en plasma (negativo), para detectar mutaciones de resistencia (T790M), también pendiente de resultado. Para la lesión de LSD, se valorará en caso de progresión, inicio de inmunoterapia (inhibidores de checkpoint) dado que por su tamaño no es candidata a tratamiento radioterápico actualmente. Se mantiene tratamiento combinado por el momento, con excelente tolerancia clínica. Instructions: please typing these entity words according to sentence: carcinoma urotelial, cáncer, adenocarcinoma, neoplasia, neoplasia, malignas, carcinoma, adenocarcinoma, adenocarcinoma, T3N0M0, tumoración, adenocarcinoma, neoplásico, tumores, neoplásico, M0, T3N0M0, metástasis, T3 - 4N0 - 2M1a, maligna, adenocarcinoma, afectación bipulmonar, tumores, tumor, afectación bipulmonar, adenocarcinoma broncopulmonar bien diferenciado , de patrón lepídico / acinar / papilar, adenocarcinomas, ADC, T3N0M0, ADC de pulmón lepídico, cT4a cN2M0, tumorales, CPNM, metastásico, tumores, adenocarcinomas, ADC lepídico, T4N2M0, ADC, T3N0M0, tumores, tumor Options: MORFOLOGIA_NEOPLASIA
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Anamnesis Varón de 67 años, sin alergias medicamentosas conocidas, exfumador desde hace 1 mes, con un consumo acumulado de 15 paquetes-año, sin otros hábitos tóxicos. Padece hipertensión arterial en tratamiento farmacológico y diabetes tipo 2 no insulinodependiente. En 2006 se diagnostica de carcinoma urotelial de vejiga localizado, recibe tratamiento intravesical con BCG y posterior intervención quirúrgica, sin datos de recaída. En 2007, sufre síndrome coronario agudo que es revascularizado y por el cual está doblemente antiagregado. Claudicación intermitente sin repercusión a nivel funcional. Como situación basal, realiza vida activa, sin ayuda para ABVD, ECOG 0. Buen soporte familiar. Antecedentes familiares: madre con cáncer gástrico a los 70 años, fallecida por dicha causa. Consulta en nuestro centro para valorar inicio de tratamiento tras diagnóstico de adenocarcinoma de pulmón en centro privado. Exploración física Buen estado general, ECOG 0. Hemodinámicamente estable y afebril, eupneico en reposo. AC: rítmico, sin soplos audibles. AP: murmullo vesicular conservado, mínimos roncus en campo pulmonar derecho. Abdomen: blando y depresible, sin dolor a la palpación ni signos de irritación peritoneal, peristaltismo conservado. MMII: no hay edemas ni signos de trombosis venosa profunda. Pruebas complementarias En diciembre de 2017, el paciente presenta semiología de infección respiratoria con tos persistente y expectoración amarillenta. A pesar de tratamiento antibiótico y broncodilatadores inhalados, no aprecia mejoría y, además, asocia esputos hemoptoicos. Se inicia estudio diagnóstico en centro privado, realizándose radiografía de tórax donde se objetiva infiltrado redondeado extenso en lóbulo superior derecho (LSD). Ante la sospecha de neoplasia pulmonar, se realiza TC de tórax, objetivándose lesión a nivel de LSD compatible con neoplasia pulmonar con nódulos adyacentes a la lesión principal, confluyentes, con un tamaño total de unos 6-7 cm de diámetro máximo. Se completa el estudio radiológico con la realización de una PET-TC, para correcto estadiaje y biopsia de la lesión en LSD. En la PET realizada en enero de 2018 se observa una masa hipermetabólica de morfología irregular a nivel del lóbulo superior del pulmón derecho, coincidente con alteración de la TC, con SUVmax de 5,2, compatible con proceso pulmonar primario de características malignas, y mínima actividad a nivel de la alteración adyacente a dicha masa y en el pulmón izquierdo, con SUV de 0,6-1,1, compatibles con proceso infeccioso/inflamatorio. En la PAAF procedente de la lesión del LSD (realizada en centro privado), el resultado de Anatomía Patológica informa de células epiteliales abundantes con moderado pleomorfismo, mitosis y citoplasma, a veces evidente, dispuestas en nidos o láminas, carcinoma compatible con adenocarcinoma; no se realiza estudio molecular. Con diagnóstico de adenocarcinoma de pulmón estadio IIB (T3N0M0) a nivel de lóbulo superior derecho con tumoración de 6 cm e infiltrado infeccioso asociado en lóbulo inferior izquierdo (LII), es remitido a nuestro centro para valoración y tratamiento. Ante la ausencia de clínica infecciosa en ese momento y la semiología radiológica de ambas lesiones, se decide realización de nuevo TC-TAP en nuestro centro para conocer la situación actual de la enfermedad. En la TC de febrero de 2018 se aprecia la masa en LSD de 5,9 x 4,5 cm en relación con adenocarcinoma pulmonar conocido y masa en segmento basal lateral del LII, con broncograma aéreo, polilobulada, de 5,7 x 3,5 cm, compatible también con proceso neoplásico. El estadio radiológico depende de si se consideran dos tumores primarios: uno en LSD cT3-4 (nódulos ipsilaterales de densidad de partes blandas, a descartar origen neoplásico) cN0-2 (probable adenopatía paratraqueal derecha baja) M0, y otro en LII T3N0M0, o una lesión única en LSD con metástasis en el LII (T3-4N0-2M1a). Se decide completar estudio con biopsia pulmonar guiada por TC de la lesión en LII, que confirma la etiología maligna compatible con adenocarcinoma de origen pulmonar. En el estudio molecular de la masa de LII, se detecta mutación de EGFR (deleción del exón 19), ALK y ROS 1 negativo, PDL1 < 1 %. En esta situación, se plantea el diagnóstico diferencial entre una enfermedad estadio IV por afectación bipulmonar, o bien dos tumores sincrónicos con diferente perfil molecular. Dado que en uno de ellos presenta mutación EGFR exón 19, para lo cual se dispone de tratamiento dirigido que aumenta supervivencia, en febrero de 2018 se inicia tratamiento de primera línea con erlotinib a dosis 150 mg/24 h. Tras comentar el caso en comité multidisciplinar de cáncer de pulmón, se desestima tratamiento con radioterapia de la lesión en LSD por extenso tamaño inicial y nódulos satélites asociados. Como incidencias, tras diez días de tratamiento, el paciente presenta toxicidad cutánea grado 3 (reacción acneiforme), precisando suspender el tratamiento e iniciándose tratamiento corticoide, antibiótico y tratamiento tópico. Tras el tratamiento indicado y mejoría clínica, se reinicia el anti-EGFR reduciendo la dosis a 100 mg/24 h. En mayo de 2018, tras recibir tratamiento durante 3 meses, se realiza TC de reevaluación, en la que se objetiva evolución radiológica discordante. Se aprecia mejoría significativa de la masa en LII y, de forma antagónica, un aumento de tamaño de la masa en LSD y de los nódulos pulmonares derechos asociados. Según criterios RECIST 1.1, la enfermedad se considera estable en caso de tratarse de un único tumor con afectación bipulmonar. Ante la evolución disociada de ambas lesiones, se solicita nueva biopsia de la lesión del LSD, dado que el diagnóstico había sido en centro externo y no se disponía del material anatomo-patológico para su revisión ni de estudio molecular. Se realiza BAG de lesión pulmonar de LSD, que objetiva infiltración por un adenocarcinoma broncopulmonar bien diferenciado, de patrón lepídico/acinar/papilar. La inmunotinción para PDL-1 resulta positiva en < 1 % de las células en este material; EGFR wild type, ALK negativo. Diagnóstico Así pues, se confirma que el paciente presenta dos adenocarcinomas de forma sincrónica, en LII un ADC pulmón T3N0M0 EGFR mutado (deleción exón 19) y en LSD un ADC de pulmón lepídico cT4a cN2M0, EGFR/ALK/ROS1 wild type. Tratamiento Con el diagnóstico definitivo de ambas lesiones y ante la no respuesta de la lesión sin mutación dirigida, en julio de 2018 se plantea tratamiento sistémico basado en quimioterapia, según esquema carboplatino AUC 5: pemetrexed 500 mg/m2 cada 21 días, y se decide suspender erlotinib y seguimiento estrecho de la lesión pulmonar izquierda, en ese momento en respuesta radiológica parcial mayor. Evolución Tras la administración de 1 ciclo de quimioterapia, el paciente sufre SCASEST secundario a lesión grave en primera diagonal, paliada con un stent fármaco-activo, y lesión en segunda diagonal no revascularizable. No sufre repercusiones en la fracción de eyección ventricular (FEVI 48 %, igual que al inicio del tratamiento), y el paciente evoluciona favorablemente, en seguimiento por Cardiología. Después de ser valorado de forma conjunta por Cardiología y Oncología, se decide continuar con tratamiento de quimioterapia y el paciente recibe 2 ciclos más (sin nuevas complicaciones). Se solicita TC reevaluación en septiembre de 2018, donde la respuesta global es enfermedad estable, según criterios RECIST 1.1. La afectación del LSD presenta una disminución de su tamaño y la lesión del LII ha crecido discretamente respecto a estudios previos, sin cumplir dicha lesión criterios de progresión radiológica. Completa 5 ciclos de quimioterapia sin incidencias. Ante esta situación de discordancia, entre ambas lesiones tumorales, se plantean en sesión clínica las opciones terapéuticas más beneficiosas para el paciente. Se decide la mejor opción de tratamiento basándonos en un análisis exploratorio de subgrupos en un ensayo aleatorizado, controlado de fase II. En este ensayo, la combinación de pemetrexed-erlotinib (pemetrexed 500 mg/m2 intravenoso en el día 1 de un ciclo de 3 semanas y erlotinib 150 mg/24 h vía oral desde el día 2 hasta el día 14 de un ciclo de 3 semanas), se comparó con pemetrexed o erlotinib solos como tratamiento de segunda línea en una población seleccionada de pacientes no fumadores con CPNM localmente avanzado o metastásico no escamoso, objetivándose beneficio en SLP para el grupo con el tratamiento combinado y toxicidad tolerable3. Así pues, se decide continuar con pemetrexed de mantenimiento y reiniciar erlotinib, para que ambas lesiones reciban su tratamiento adecuado, tras explicar al paciente riesgos y beneficios. En octubre de 2018, tras 5 ciclos de quimioterapia, el paciente inicia tratamiento de mantenimiento con pemetrexed 500 mg/m2 cada 21 días para la lesión en LSD y tratamiento con erlotinib 100 mg/días +2 al +15 cada 21 días para la lesión en LII. No presenta toxicidad secundaria al tratamiento con pemetrexed, pero sí al reiniciar erlotinib, precisando reducción de dosis a 75 mg/24 h debido de nuevo a toxicidad cutánea grado 3 (reacción acneiforme). En todo momento, el paciente se encuentra asintomático a nivel respiratorio. Tras 4 ciclos de tratamiento combinado con erlotinib y pemetrexed se realiza nueva TC de reevaluación en enero de 2019, con datos de enfermedad estable por criterios RECIST 1.1 en ambos tumores, respuesta que se mantiene en el TC de marzo de 2019, después de 8 meses de tratamiento combinado, presentando el paciente excelente tolerancia, sin toxicidad ni complicaciones durante el tratamiento. En resumen, se trata de un paciente de 67 años, cardiópata, que ha sido diagnosticado de dos adenocarcinomas de pulmón con diferente perfil molecular, de forma sincrónica. En el pulmón derecho presenta ADC lepídico estadio IIIB (T4N2M0) EGFR wild type y, en el pulmón izquierdo, ADC pulmón estadio IIB (T3N0M0) EGFR mutado (deleción exón 19). Ha recibido tratamiento sistémico para ambos tumores. En primer lugar, tratamiento anti-EGFR con erlotinib, con regular tolerancia clínica por toxicidad cutánea grado 3 (reacción acneiforme), que ha precisado detener el tratamiento y disminuir la dosis en dos ocasiones, pero con aceptable respuesta a nivel metabólico, disminuyendo el tamaño de la lesión de forma progresiva, después de 11 ciclos en total. En relación al tumor no mutado, ha recibido tratamiento basado en quimioterapia según esquema carboplatino-pemetrexed durante 5 ciclos y posteriormente, pemetrexed de mantenimiento. En mayo de 2019, tras 10 ciclos de tratamiento combinado, presenta enfermedad estable como respuesta global, pero se objetiva un ligero crecimiento de ambas lesiones. Se ha decidido administrar RT sobre lesión en LII, y se ha solicitado biopsia en plasma (negativo), para detectar mutaciones de resistencia (T790M), también pendiente de resultado. Para la lesión de LSD, se valorará en caso de progresión, inicio de inmunoterapia (inhibidores de checkpoint) dado que por su tamaño no es candidata a tratamiento radioterápico actualmente. Se mantiene tratamiento combinado por el momento, con excelente tolerancia clínica.
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[ "MORFOLOGIA_NEOPLASIA" ]
carcinoma urotelial, cáncer, adenocarcinoma, neoplasia, neoplasia, malignas, carcinoma, adenocarcinoma, adenocarcinoma, T3N0M0, tumoración, adenocarcinoma, neoplásico, tumores, neoplásico, M0, T3N0M0, metástasis, T3 - 4N0 - 2M1a, maligna, adenocarcinoma, afectación bipulmonar, tumores, tumor, afectación bipulmonar, adenocarcinoma broncopulmonar bien diferenciado , de patrón lepídico / acinar / papilar, adenocarcinomas, ADC, T3N0M0, ADC de pulmón lepídico, cT4a cN2M0, tumorales, CPNM, metastásico, tumores, adenocarcinomas, ADC lepídico, T4N2M0, ADC, T3N0M0, tumores, tumor
359_task2
Sentence: Anamnesis Varón de 67 años, sin alergias medicamentosas conocidas, exfumador desde hace 1 mes, con un consumo acumulado de 15 paquetes-año, sin otros hábitos tóxicos. Padece hipertensión arterial en tratamiento farmacológico y diabetes tipo 2 no insulinodependiente. En 2006 se diagnostica de carcinoma urotelial de vejiga localizado, recibe tratamiento intravesical con BCG y posterior intervención quirúrgica, sin datos de recaída. En 2007, sufre síndrome coronario agudo que es revascularizado y por el cual está doblemente antiagregado. Claudicación intermitente sin repercusión a nivel funcional. Como situación basal, realiza vida activa, sin ayuda para ABVD, ECOG 0. Buen soporte familiar. Antecedentes familiares: madre con cáncer gástrico a los 70 años, fallecida por dicha causa. Consulta en nuestro centro para valorar inicio de tratamiento tras diagnóstico de adenocarcinoma de pulmón en centro privado. Exploración física Buen estado general, ECOG 0. Hemodinámicamente estable y afebril, eupneico en reposo. AC: rítmico, sin soplos audibles. AP: murmullo vesicular conservado, mínimos roncus en campo pulmonar derecho. Abdomen: blando y depresible, sin dolor a la palpación ni signos de irritación peritoneal, peristaltismo conservado. MMII: no hay edemas ni signos de trombosis venosa profunda. Pruebas complementarias En diciembre de 2017, el paciente presenta semiología de infección respiratoria con tos persistente y expectoración amarillenta. A pesar de tratamiento antibiótico y broncodilatadores inhalados, no aprecia mejoría y, además, asocia esputos hemoptoicos. Se inicia estudio diagnóstico en centro privado, realizándose radiografía de tórax donde se objetiva infiltrado redondeado extenso en lóbulo superior derecho (LSD). Ante la sospecha de neoplasia pulmonar, se realiza TC de tórax, objetivándose lesión a nivel de LSD compatible con neoplasia pulmonar con nódulos adyacentes a la lesión principal, confluyentes, con un tamaño total de unos 6-7 cm de diámetro máximo. Se completa el estudio radiológico con la realización de una PET-TC, para correcto estadiaje y biopsia de la lesión en LSD. En la PET realizada en enero de 2018 se observa una masa hipermetabólica de morfología irregular a nivel del lóbulo superior del pulmón derecho, coincidente con alteración de la TC, con SUVmax de 5,2, compatible con proceso pulmonar primario de características malignas, y mínima actividad a nivel de la alteración adyacente a dicha masa y en el pulmón izquierdo, con SUV de 0,6-1,1, compatibles con proceso infeccioso/inflamatorio. En la PAAF procedente de la lesión del LSD (realizada en centro privado), el resultado de Anatomía Patológica informa de células epiteliales abundantes con moderado pleomorfismo, mitosis y citoplasma, a veces evidente, dispuestas en nidos o láminas, carcinoma compatible con adenocarcinoma; no se realiza estudio molecular. Con diagnóstico de adenocarcinoma de pulmón estadio IIB (T3N0M0) a nivel de lóbulo superior derecho con tumoración de 6 cm e infiltrado infeccioso asociado en lóbulo inferior izquierdo (LII), es remitido a nuestro centro para valoración y tratamiento. Ante la ausencia de clínica infecciosa en ese momento y la semiología radiológica de ambas lesiones, se decide realización de nuevo TC-TAP en nuestro centro para conocer la situación actual de la enfermedad. En la TC de febrero de 2018 se aprecia la masa en LSD de 5,9 x 4,5 cm en relación con adenocarcinoma pulmonar conocido y masa en segmento basal lateral del LII, con broncograma aéreo, polilobulada, de 5,7 x 3,5 cm, compatible también con proceso neoplásico. El estadio radiológico depende de si se consideran dos tumores primarios: uno en LSD cT3-4 (nódulos ipsilaterales de densidad de partes blandas, a descartar origen neoplásico) cN0-2 (probable adenopatía paratraqueal derecha baja) M0, y otro en LII T3N0M0, o una lesión única en LSD con metástasis en el LII (T3-4N0-2M1a). Se decide completar estudio con biopsia pulmonar guiada por TC de la lesión en LII, que confirma la etiología maligna compatible con adenocarcinoma de origen pulmonar. En el estudio molecular de la masa de LII, se detecta mutación de EGFR (deleción del exón 19), ALK y ROS 1 negativo, PDL1 < 1 %. En esta situación, se plantea el diagnóstico diferencial entre una enfermedad estadio IV por afectación bipulmonar, o bien dos tumores sincrónicos con diferente perfil molecular. Dado que en uno de ellos presenta mutación EGFR exón 19, para lo cual se dispone de tratamiento dirigido que aumenta supervivencia, en febrero de 2018 se inicia tratamiento de primera línea con erlotinib a dosis 150 mg/24 h. Tras comentar el caso en comité multidisciplinar de cáncer de pulmón, se desestima tratamiento con radioterapia de la lesión en LSD por extenso tamaño inicial y nódulos satélites asociados. Como incidencias, tras diez días de tratamiento, el paciente presenta toxicidad cutánea grado 3 (reacción acneiforme), precisando suspender el tratamiento e iniciándose tratamiento corticoide, antibiótico y tratamiento tópico. Tras el tratamiento indicado y mejoría clínica, se reinicia el anti-EGFR reduciendo la dosis a 100 mg/24 h. En mayo de 2018, tras recibir tratamiento durante 3 meses, se realiza TC de reevaluación, en la que se objetiva evolución radiológica discordante. Se aprecia mejoría significativa de la masa en LII y, de forma antagónica, un aumento de tamaño de la masa en LSD y de los nódulos pulmonares derechos asociados. Según criterios RECIST 1.1, la enfermedad se considera estable en caso de tratarse de un único tumor con afectación bipulmonar. Ante la evolución disociada de ambas lesiones, se solicita nueva biopsia de la lesión del LSD, dado que el diagnóstico había sido en centro externo y no se disponía del material anatomo-patológico para su revisión ni de estudio molecular. Se realiza BAG de lesión pulmonar de LSD, que objetiva infiltración por un adenocarcinoma broncopulmonar bien diferenciado, de patrón lepídico/acinar/papilar. La inmunotinción para PDL-1 resulta positiva en < 1 % de las células en este material; EGFR wild type, ALK negativo. Diagnóstico Así pues, se confirma que el paciente presenta dos adenocarcinomas de forma sincrónica, en LII un ADC pulmón T3N0M0 EGFR mutado (deleción exón 19) y en LSD un ADC de pulmón lepídico cT4a cN2M0, EGFR/ALK/ROS1 wild type. Tratamiento Con el diagnóstico definitivo de ambas lesiones y ante la no respuesta de la lesión sin mutación dirigida, en julio de 2018 se plantea tratamiento sistémico basado en quimioterapia, según esquema carboplatino AUC 5: pemetrexed 500 mg/m2 cada 21 días, y se decide suspender erlotinib y seguimiento estrecho de la lesión pulmonar izquierda, en ese momento en respuesta radiológica parcial mayor. Evolución Tras la administración de 1 ciclo de quimioterapia, el paciente sufre SCASEST secundario a lesión grave en primera diagonal, paliada con un stent fármaco-activo, y lesión en segunda diagonal no revascularizable. No sufre repercusiones en la fracción de eyección ventricular (FEVI 48 %, igual que al inicio del tratamiento), y el paciente evoluciona favorablemente, en seguimiento por Cardiología. Después de ser valorado de forma conjunta por Cardiología y Oncología, se decide continuar con tratamiento de quimioterapia y el paciente recibe 2 ciclos más (sin nuevas complicaciones). Se solicita TC reevaluación en septiembre de 2018, donde la respuesta global es enfermedad estable, según criterios RECIST 1.1. La afectación del LSD presenta una disminución de su tamaño y la lesión del LII ha crecido discretamente respecto a estudios previos, sin cumplir dicha lesión criterios de progresión radiológica. Completa 5 ciclos de quimioterapia sin incidencias. Ante esta situación de discordancia, entre ambas lesiones tumorales, se plantean en sesión clínica las opciones terapéuticas más beneficiosas para el paciente. Se decide la mejor opción de tratamiento basándonos en un análisis exploratorio de subgrupos en un ensayo aleatorizado, controlado de fase II. En este ensayo, la combinación de pemetrexed-erlotinib (pemetrexed 500 mg/m2 intravenoso en el día 1 de un ciclo de 3 semanas y erlotinib 150 mg/24 h vía oral desde el día 2 hasta el día 14 de un ciclo de 3 semanas), se comparó con pemetrexed o erlotinib solos como tratamiento de segunda línea en una población seleccionada de pacientes no fumadores con CPNM localmente avanzado o metastásico no escamoso, objetivándose beneficio en SLP para el grupo con el tratamiento combinado y toxicidad tolerable3. Así pues, se decide continuar con pemetrexed de mantenimiento y reiniciar erlotinib, para que ambas lesiones reciban su tratamiento adecuado, tras explicar al paciente riesgos y beneficios. En octubre de 2018, tras 5 ciclos de quimioterapia, el paciente inicia tratamiento de mantenimiento con pemetrexed 500 mg/m2 cada 21 días para la lesión en LSD y tratamiento con erlotinib 100 mg/días +2 al +15 cada 21 días para la lesión en LII. No presenta toxicidad secundaria al tratamiento con pemetrexed, pero sí al reiniciar erlotinib, precisando reducción de dosis a 75 mg/24 h debido de nuevo a toxicidad cutánea grado 3 (reacción acneiforme). En todo momento, el paciente se encuentra asintomático a nivel respiratorio. Tras 4 ciclos de tratamiento combinado con erlotinib y pemetrexed se realiza nueva TC de reevaluación en enero de 2019, con datos de enfermedad estable por criterios RECIST 1.1 en ambos tumores, respuesta que se mantiene en el TC de marzo de 2019, después de 8 meses de tratamiento combinado, presentando el paciente excelente tolerancia, sin toxicidad ni complicaciones durante el tratamiento. En resumen, se trata de un paciente de 67 años, cardiópata, que ha sido diagnosticado de dos adenocarcinomas de pulmón con diferente perfil molecular, de forma sincrónica. En el pulmón derecho presenta ADC lepídico estadio IIIB (T4N2M0) EGFR wild type y, en el pulmón izquierdo, ADC pulmón estadio IIB (T3N0M0) EGFR mutado (deleción exón 19). Ha recibido tratamiento sistémico para ambos tumores. En primer lugar, tratamiento anti-EGFR con erlotinib, con regular tolerancia clínica por toxicidad cutánea grado 3 (reacción acneiforme), que ha precisado detener el tratamiento y disminuir la dosis en dos ocasiones, pero con aceptable respuesta a nivel metabólico, disminuyendo el tamaño de la lesión de forma progresiva, después de 11 ciclos en total. En relación al tumor no mutado, ha recibido tratamiento basado en quimioterapia según esquema carboplatino-pemetrexed durante 5 ciclos y posteriormente, pemetrexed de mantenimiento. En mayo de 2019, tras 10 ciclos de tratamiento combinado, presenta enfermedad estable como respuesta global, pero se objetiva un ligero crecimiento de ambas lesiones. Se ha decidido administrar RT sobre lesión en LII, y se ha solicitado biopsia en plasma (negativo), para detectar mutaciones de resistencia (T790M), también pendiente de resultado. Para la lesión de LSD, se valorará en caso de progresión, inicio de inmunoterapia (inhibidores de checkpoint) dado que por su tamaño no es candidata a tratamiento radioterápico actualmente. Se mantiene tratamiento combinado por el momento, con excelente tolerancia clínica. Instructions: please extract entity words from the input sentence
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Anamnesis Varón de 67 años, sin alergias medicamentosas conocidas, exfumador desde hace 1 mes, con un consumo acumulado de 15 paquetes-año, sin otros hábitos tóxicos. Padece hipertensión arterial en tratamiento farmacológico y diabetes tipo 2 no insulinodependiente. En 2006 se diagnostica de carcinoma urotelial de vejiga localizado, recibe tratamiento intravesical con BCG y posterior intervención quirúrgica, sin datos de recaída. En 2007, sufre síndrome coronario agudo que es revascularizado y por el cual está doblemente antiagregado. Claudicación intermitente sin repercusión a nivel funcional. Como situación basal, realiza vida activa, sin ayuda para ABVD, ECOG 0. Buen soporte familiar. Antecedentes familiares: madre con cáncer gástrico a los 70 años, fallecida por dicha causa. Consulta en nuestro centro para valorar inicio de tratamiento tras diagnóstico de adenocarcinoma de pulmón en centro privado. Exploración física Buen estado general, ECOG 0. Hemodinámicamente estable y afebril, eupneico en reposo. AC: rítmico, sin soplos audibles. AP: murmullo vesicular conservado, mínimos roncus en campo pulmonar derecho. Abdomen: blando y depresible, sin dolor a la palpación ni signos de irritación peritoneal, peristaltismo conservado. MMII: no hay edemas ni signos de trombosis venosa profunda. Pruebas complementarias En diciembre de 2017, el paciente presenta semiología de infección respiratoria con tos persistente y expectoración amarillenta. A pesar de tratamiento antibiótico y broncodilatadores inhalados, no aprecia mejoría y, además, asocia esputos hemoptoicos. Se inicia estudio diagnóstico en centro privado, realizándose radiografía de tórax donde se objetiva infiltrado redondeado extenso en lóbulo superior derecho (LSD). Ante la sospecha de neoplasia pulmonar, se realiza TC de tórax, objetivándose lesión a nivel de LSD compatible con neoplasia pulmonar con nódulos adyacentes a la lesión principal, confluyentes, con un tamaño total de unos 6-7 cm de diámetro máximo. Se completa el estudio radiológico con la realización de una PET-TC, para correcto estadiaje y biopsia de la lesión en LSD. En la PET realizada en enero de 2018 se observa una masa hipermetabólica de morfología irregular a nivel del lóbulo superior del pulmón derecho, coincidente con alteración de la TC, con SUVmax de 5,2, compatible con proceso pulmonar primario de características malignas, y mínima actividad a nivel de la alteración adyacente a dicha masa y en el pulmón izquierdo, con SUV de 0,6-1,1, compatibles con proceso infeccioso/inflamatorio. En la PAAF procedente de la lesión del LSD (realizada en centro privado), el resultado de Anatomía Patológica informa de células epiteliales abundantes con moderado pleomorfismo, mitosis y citoplasma, a veces evidente, dispuestas en nidos o láminas, carcinoma compatible con adenocarcinoma; no se realiza estudio molecular. Con diagnóstico de adenocarcinoma de pulmón estadio IIB (T3N0M0) a nivel de lóbulo superior derecho con tumoración de 6 cm e infiltrado infeccioso asociado en lóbulo inferior izquierdo (LII), es remitido a nuestro centro para valoración y tratamiento. Ante la ausencia de clínica infecciosa en ese momento y la semiología radiológica de ambas lesiones, se decide realización de nuevo TC-TAP en nuestro centro para conocer la situación actual de la enfermedad. En la TC de febrero de 2018 se aprecia la masa en LSD de 5,9 x 4,5 cm en relación con adenocarcinoma pulmonar conocido y masa en segmento basal lateral del LII, con broncograma aéreo, polilobulada, de 5,7 x 3,5 cm, compatible también con proceso neoplásico. El estadio radiológico depende de si se consideran dos tumores primarios: uno en LSD cT3-4 (nódulos ipsilaterales de densidad de partes blandas, a descartar origen neoplásico) cN0-2 (probable adenopatía paratraqueal derecha baja) M0, y otro en LII T3N0M0, o una lesión única en LSD con metástasis en el LII (T3-4N0-2M1a). Se decide completar estudio con biopsia pulmonar guiada por TC de la lesión en LII, que confirma la etiología maligna compatible con adenocarcinoma de origen pulmonar. En el estudio molecular de la masa de LII, se detecta mutación de EGFR (deleción del exón 19), ALK y ROS 1 negativo, PDL1 < 1 %. En esta situación, se plantea el diagnóstico diferencial entre una enfermedad estadio IV por afectación bipulmonar, o bien dos tumores sincrónicos con diferente perfil molecular. Dado que en uno de ellos presenta mutación EGFR exón 19, para lo cual se dispone de tratamiento dirigido que aumenta supervivencia, en febrero de 2018 se inicia tratamiento de primera línea con erlotinib a dosis 150 mg/24 h. Tras comentar el caso en comité multidisciplinar de cáncer de pulmón, se desestima tratamiento con radioterapia de la lesión en LSD por extenso tamaño inicial y nódulos satélites asociados. Como incidencias, tras diez días de tratamiento, el paciente presenta toxicidad cutánea grado 3 (reacción acneiforme), precisando suspender el tratamiento e iniciándose tratamiento corticoide, antibiótico y tratamiento tópico. Tras el tratamiento indicado y mejoría clínica, se reinicia el anti-EGFR reduciendo la dosis a 100 mg/24 h. En mayo de 2018, tras recibir tratamiento durante 3 meses, se realiza TC de reevaluación, en la que se objetiva evolución radiológica discordante. Se aprecia mejoría significativa de la masa en LII y, de forma antagónica, un aumento de tamaño de la masa en LSD y de los nódulos pulmonares derechos asociados. Según criterios RECIST 1.1, la enfermedad se considera estable en caso de tratarse de un único tumor con afectación bipulmonar. Ante la evolución disociada de ambas lesiones, se solicita nueva biopsia de la lesión del LSD, dado que el diagnóstico había sido en centro externo y no se disponía del material anatomo-patológico para su revisión ni de estudio molecular. Se realiza BAG de lesión pulmonar de LSD, que objetiva infiltración por un adenocarcinoma broncopulmonar bien diferenciado, de patrón lepídico/acinar/papilar. La inmunotinción para PDL-1 resulta positiva en < 1 % de las células en este material; EGFR wild type, ALK negativo. Diagnóstico Así pues, se confirma que el paciente presenta dos adenocarcinomas de forma sincrónica, en LII un ADC pulmón T3N0M0 EGFR mutado (deleción exón 19) y en LSD un ADC de pulmón lepídico cT4a cN2M0, EGFR/ALK/ROS1 wild type. Tratamiento Con el diagnóstico definitivo de ambas lesiones y ante la no respuesta de la lesión sin mutación dirigida, en julio de 2018 se plantea tratamiento sistémico basado en quimioterapia, según esquema carboplatino AUC 5: pemetrexed 500 mg/m2 cada 21 días, y se decide suspender erlotinib y seguimiento estrecho de la lesión pulmonar izquierda, en ese momento en respuesta radiológica parcial mayor. Evolución Tras la administración de 1 ciclo de quimioterapia, el paciente sufre SCASEST secundario a lesión grave en primera diagonal, paliada con un stent fármaco-activo, y lesión en segunda diagonal no revascularizable. No sufre repercusiones en la fracción de eyección ventricular (FEVI 48 %, igual que al inicio del tratamiento), y el paciente evoluciona favorablemente, en seguimiento por Cardiología. Después de ser valorado de forma conjunta por Cardiología y Oncología, se decide continuar con tratamiento de quimioterapia y el paciente recibe 2 ciclos más (sin nuevas complicaciones). Se solicita TC reevaluación en septiembre de 2018, donde la respuesta global es enfermedad estable, según criterios RECIST 1.1. La afectación del LSD presenta una disminución de su tamaño y la lesión del LII ha crecido discretamente respecto a estudios previos, sin cumplir dicha lesión criterios de progresión radiológica. Completa 5 ciclos de quimioterapia sin incidencias. Ante esta situación de discordancia, entre ambas lesiones tumorales, se plantean en sesión clínica las opciones terapéuticas más beneficiosas para el paciente. Se decide la mejor opción de tratamiento basándonos en un análisis exploratorio de subgrupos en un ensayo aleatorizado, controlado de fase II. En este ensayo, la combinación de pemetrexed-erlotinib (pemetrexed 500 mg/m2 intravenoso en el día 1 de un ciclo de 3 semanas y erlotinib 150 mg/24 h vía oral desde el día 2 hasta el día 14 de un ciclo de 3 semanas), se comparó con pemetrexed o erlotinib solos como tratamiento de segunda línea en una población seleccionada de pacientes no fumadores con CPNM localmente avanzado o metastásico no escamoso, objetivándose beneficio en SLP para el grupo con el tratamiento combinado y toxicidad tolerable3. Así pues, se decide continuar con pemetrexed de mantenimiento y reiniciar erlotinib, para que ambas lesiones reciban su tratamiento adecuado, tras explicar al paciente riesgos y beneficios. En octubre de 2018, tras 5 ciclos de quimioterapia, el paciente inicia tratamiento de mantenimiento con pemetrexed 500 mg/m2 cada 21 días para la lesión en LSD y tratamiento con erlotinib 100 mg/días +2 al +15 cada 21 días para la lesión en LII. No presenta toxicidad secundaria al tratamiento con pemetrexed, pero sí al reiniciar erlotinib, precisando reducción de dosis a 75 mg/24 h debido de nuevo a toxicidad cutánea grado 3 (reacción acneiforme). En todo momento, el paciente se encuentra asintomático a nivel respiratorio. Tras 4 ciclos de tratamiento combinado con erlotinib y pemetrexed se realiza nueva TC de reevaluación en enero de 2019, con datos de enfermedad estable por criterios RECIST 1.1 en ambos tumores, respuesta que se mantiene en el TC de marzo de 2019, después de 8 meses de tratamiento combinado, presentando el paciente excelente tolerancia, sin toxicidad ni complicaciones durante el tratamiento. En resumen, se trata de un paciente de 67 años, cardiópata, que ha sido diagnosticado de dos adenocarcinomas de pulmón con diferente perfil molecular, de forma sincrónica. En el pulmón derecho presenta ADC lepídico estadio IIIB (T4N2M0) EGFR wild type y, en el pulmón izquierdo, ADC pulmón estadio IIB (T3N0M0) EGFR mutado (deleción exón 19). Ha recibido tratamiento sistémico para ambos tumores. En primer lugar, tratamiento anti-EGFR con erlotinib, con regular tolerancia clínica por toxicidad cutánea grado 3 (reacción acneiforme), que ha precisado detener el tratamiento y disminuir la dosis en dos ocasiones, pero con aceptable respuesta a nivel metabólico, disminuyendo el tamaño de la lesión de forma progresiva, después de 11 ciclos en total. En relación al tumor no mutado, ha recibido tratamiento basado en quimioterapia según esquema carboplatino-pemetrexed durante 5 ciclos y posteriormente, pemetrexed de mantenimiento. En mayo de 2019, tras 10 ciclos de tratamiento combinado, presenta enfermedad estable como respuesta global, pero se objetiva un ligero crecimiento de ambas lesiones. Se ha decidido administrar RT sobre lesión en LII, y se ha solicitado biopsia en plasma (negativo), para detectar mutaciones de resistencia (T790M), también pendiente de resultado. Para la lesión de LSD, se valorará en caso de progresión, inicio de inmunoterapia (inhibidores de checkpoint) dado que por su tamaño no es candidata a tratamiento radioterápico actualmente. Se mantiene tratamiento combinado por el momento, con excelente tolerancia clínica.
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"lepídico", "cT4a", "cN2M0", ",", "EGFR", "/", "ALK", "/", "ROS1", "wild", "type", ".", "\n\n", "Tratamiento", "\n", "Con", "el", "diagnóstico", "definitivo", "de", "ambas", "lesiones", "y", "ante", "la", "no", "respuesta", "de", "la", "lesión", "sin", "mutación", "dirigida", ",", "en", "julio", "de", "2018", "se", "plantea", "tratamiento", "sistémico", "basado", "en", "quimioterapia", ",", "según", "esquema", "carboplatino", "AUC", "5", ":", "pemetrexed", "500", "mg", "/", "m2", "cada", "21", "días", ",", "y", "se", "decide", "suspender", "erlotinib", "y", "seguimiento", "estrecho", "de", "la", "lesión", "pulmonar", "izquierda", ",", "en", "ese", "momento", "en", "respuesta", "radiológica", "parcial", "mayor", ".", "\n\n", "Evolución", "\n", "Tras", "la", "administración", "de", "1", "ciclo", "de", "quimioterapia", ",", "el", "paciente", "sufre", "SCASEST", "secundario", "a", "lesión", "grave", "en", "primera", "diagonal", ",", "paliada", "con", "un", "stent", "fármaco", "-", "activo", ",", "y", "lesión", "en", "segunda", "diagonal", "no", "revascularizable", ".", "No", "sufre", "repercusiones", "en", "la", "fracción", "de", "eyección", "ventricular", "(", "FEVI", "48", "%", ",", "igual", "que", "al", "inicio", "del", "tratamiento", ")", ",", "y", "el", "paciente", "evoluciona", "favorablemente", ",", "en", "seguimiento", "por", "Cardiología", ".", "\n", "Después", "de", "ser", "valorado", "de", "forma", "conjunta", "por", "Cardiología", "y", "Oncología", ",", "se", "decide", "continuar", "con", "tratamiento", "de", "quimioterapia", "y", "el", "paciente", "recibe", "2", "ciclos", "más", "(", "sin", "nuevas", "complicaciones", ")", ".", "Se", "solicita", "TC", "reevaluación", "en", "septiembre", "de", "2018", ",", "donde", "la", "respuesta", "global", "es", "enfermedad", "estable", ",", "según", "criterios", "RECIST", "1.1", ".", "La", "afectación", "del", "LSD", "presenta", "una", "disminución", "de", "su", "tamaño", "y", "la", "lesión", "del", "LII", "ha", "crecido", "discretamente", "respecto", "a", "estudios", "previos", ",", "sin", "cumplir", "dicha", "lesión", "criterios", "de", "progresión", "radiológica", ".", "Completa", "5", "ciclos", "de", "quimioterapia", "sin", "incidencias", ".", "\n", "Ante", "esta", "situación", "de", "discordancia", ",", "entre", "ambas", "lesiones", "tumorales", ",", "se", "plantean", "en", "sesión", "clínica", "las", "opciones", "terapéuticas", "más", "beneficiosas", "para", "el", "paciente", ".", "Se", "decide", "la", "mejor", "opción", "de", "tratamiento", "basándonos", "en", "un", "análisis", "exploratorio", "de", "subgrupos", "en", "un", "ensayo", "aleatorizado", ",", "controlado", "de", "fase", "II", ".", "En", "este", "ensayo", ",", "la", "combinación", "de", "pemetrexed", "-", "erlotinib", "(", "pemetrexed", "500", "mg", "/", "m2", "intravenoso", "en", "el", "día", "1", "de", "un", "ciclo", "de", "3", "semanas", "y", "erlotinib", "150", "mg/24", "h", "vía", "oral", "desde", "el", "día", "2", "hasta", "el", "día", "14", "de", "un", "ciclo", "de", "3", "semanas", ")", ",", "se", "comparó", "con", "pemetrexed", "o", "erlotinib", "solos", "como", "tratamiento", "de", "segunda", "línea", "en", "una", "población", "seleccionada", "de", "pacientes", "no", "fumadores", "con", "CPNM", "localmente", "avanzado", "o", "metastásico", "no", "escamoso", ",", "objetivándose", "beneficio", "en", "SLP", "para", "el", "grupo", "con", "el", "tratamiento", "combinado", "y", "toxicidad", "tolerable3", ".", "Así", "pues", ",", "se", "decide", "continuar", "con", "pemetrexed", "de", "mantenimiento", "y", "reiniciar", "erlotinib", ",", "para", "que", "ambas", "lesiones", "reciban", "su", "tratamiento", "adecuado", ",", "tras", "explicar", "al", "paciente", "riesgos", "y", "beneficios", ".", "\n", "En", "octubre", "de", "2018", ",", "tras", "5", "ciclos", "de", "quimioterapia", ",", "el", "paciente", "inicia", "tratamiento", "de", "mantenimiento", "con", "pemetrexed", "500", "mg", "/", "m2", "cada", "21", "días", "para", "la", "lesión", "en", "LSD", "y", "tratamiento", "con", "erlotinib", "100", "mg", "/", "días", "+2", "al", "+15", "cada", "21", "días", "para", "la", "lesión", "en", "LII", ".", "\n", "No", "presenta", "toxicidad", "secundaria", "al", "tratamiento", "con", "pemetrexed", ",", "pero", "sí", "al", "reiniciar", "erlotinib", ",", "precisando", "reducción", "de", "dosis", "a", "75", "mg/24", "h", "debido", "de", "nuevo", "a", "toxicidad", "cutánea", "grado", "3", "(", "reacción", "acneiforme", ")", ".", "En", "todo", "momento", ",", "el", "paciente", "se", "encuentra", "asintomático", "a", "nivel", "respiratorio", ".", "\n", "Tras", "4", "ciclos", "de", "tratamiento", "combinado", "con", "erlotinib", "y", "pemetrexed", "se", "realiza", "nueva", "TC", "de", "reevaluación", "en", "enero", "de", "2019", ",", "con", "datos", "de", "enfermedad", "estable", "por", "criterios", "RECIST", "1.1", "en", "ambos", "tumores", ",", "respuesta", "que", "se", "mantiene", "en", "el", "TC", "de", "marzo", "de", "2019", ",", "después", "de", "8", "meses", "de", "tratamiento", "combinado", ",", "presentando", "el", "paciente", "excelente", "tolerancia", ",", "sin", "toxicidad", "ni", "complicaciones", "durante", "el", "tratamiento", ".", "\n", "En", "resumen", ",", "se", "trata", "de", "un", "paciente", "de", "67", "años", ",", "cardiópata", ",", "que", "ha", "sido", "diagnosticado", "de", "dos", "adenocarcinomas", "de", "pulmón", "con", "diferente", "perfil", "molecular", ",", "de", "forma", "sincrónica", ".", "En", "el", "pulmón", "derecho", "presenta", "ADC", "lepídico", "estadio", "IIIB", "(", "T4N2M0", ")", "EGFR", "wild", "type", "y", ",", "en", "el", "pulmón", "izquierdo", ",", "ADC", "pulmón", "estadio", "IIB", "(", "T3N0M0", ")", "EGFR", "mutado", "(", "deleción", "exón", "19", ")", ".", "Ha", "recibido", "tratamiento", "sistémico", "para", "ambos", "tumores", ".", "En", "primer", "lugar", ",", "tratamiento", "anti", "-", "EGFR", "con", "erlotinib", ",", "con", "regular", "tolerancia", "clínica", "por", "toxicidad", "cutánea", "grado", "3", "(", "reacción", "acneiforme", ")", ",", "que", "ha", "precisado", "detener", "el", "tratamiento", "y", "disminuir", "la", "dosis", "en", "dos", "ocasiones", ",", "pero", "con", "aceptable", "respuesta", "a", "nivel", "metabólico", ",", "disminuyendo", "el", "tamaño", "de", "la", "lesión", "de", "forma", "progresiva", ",", "después", "de", "11", "ciclos", "en", "total", ".", "En", "relación", "al", "tumor", "no", "mutado", ",", "ha", "recibido", "tratamiento", "basado", "en", "quimioterapia", "según", "esquema", "carboplatino", "-", "pemetrexed", "durante", "5", "ciclos", "y", "posteriormente", ",", "pemetrexed", "de", "mantenimiento", ".", "\n", "En", "mayo", "de", "2019", ",", "tras", "10", "ciclos", "de", "tratamiento", "combinado", ",", "presenta", "enfermedad", "estable", "como", "respuesta", "global", ",", "pero", "se", "objetiva", "un", "ligero", "crecimiento", "de", "ambas", "lesiones", ".", "Se", "ha", "decidido", "administrar", "RT", "sobre", "lesión", "en", "LII", ",", "y", "se", "ha", "solicitado", "biopsia", "en", "plasma", "(", "negativo", ")", ",", "para", "detectar", "mutaciones", "de", "resistencia", "(", "T790", "M", ")", ",", "también", "pendiente", "de", "resultado", ".", "Para", "la", "lesión", "de", "LSD", ",", "se", "valorará", "en", "caso", "de", "progresión", ",", "inicio", "de", "inmunoterapia", "(", "inhibidores", "de", "checkpoint", ")", "dado", "que", "por", "su", "tamaño", "no", "es", "candidata", "a", "tratamiento", "radioterápico", "actualmente", ".", "\n", "Se", "mantiene", "tratamiento", "combinado", "por", "el", "momento", ",", "con", "excelente", "tolerancia", "clínica", ".", "\n" ]
[ "MORFOLOGIA_NEOPLASIA" ]
Sucralfate is a CHEMICAL, Sucralfate is a CHEMICAL, sucralfate is a CHEMICAL, basic fibroblast growth factor is a GENE-Y, bFGF is a GENE-Y, Basic fibroblast growth factor is a GENE-Y, sucralfate is a CHEMICAL, Sucralfate is a CHEMICAL, sucralfate is a CHEMICAL
16133_task0
Sentence: Sucralfate: the Bangkok review. Sucralfate is a site-protective ulcer healing drug with a remarkable range of mechanisms of action. Recent studies highlight the capacity of sucralfate to bind basic fibroblast growth factor (bFGF) and deliver it in high concentration to the ulcer. Basic fibroblast growth factor stimulates the production of granulation tissue, angiogenesis and re-epithelization, thus improving the quality of ulcer healing. The effect of sucralfate in reducing parietal cell sensitivity may be another factor important in the lower relapse rate demonstrated after duodenal ulcer healing. Sucralfate has been demonstrated to be efficacious in healing both duodenal and gastric ulcers together with mild oesophagitis, and it is safe for both short-term use and maintenance. In stress ulcer prophylaxis it is as effective as acid suppression or neutralization and has the advantage of lesser rates of nosocomial pneumonia than are demonstrated with antacids or H2 antagonists. The potential advantages of sucralfate lie in the better quality of ulcer healing associated with longer duration of remission. Instructions: please extract entities and their types from the input sentence, all entity types are in options Options: GENE-Y, CHEMICAL
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Sucralfate: the Bangkok review. Sucralfate is a site-protective ulcer healing drug with a remarkable range of mechanisms of action. Recent studies highlight the capacity of sucralfate to bind basic fibroblast growth factor (bFGF) and deliver it in high concentration to the ulcer. Basic fibroblast growth factor stimulates the production of granulation tissue, angiogenesis and re-epithelization, thus improving the quality of ulcer healing. The effect of sucralfate in reducing parietal cell sensitivity may be another factor important in the lower relapse rate demonstrated after duodenal ulcer healing. Sucralfate has been demonstrated to be efficacious in healing both duodenal and gastric ulcers together with mild oesophagitis, and it is safe for both short-term use and maintenance. In stress ulcer prophylaxis it is as effective as acid suppression or neutralization and has the advantage of lesser rates of nosocomial pneumonia than are demonstrated with antacids or H2 antagonists. The potential advantages of sucralfate lie in the better quality of ulcer healing associated with longer duration of remission.
[ "Sucralfate", ":", "the", "Bangkok", "review", ".", "\n", "Sucralfate", "is", "a", "site", "-", "protective", "ulcer", "healing", "drug", "with", "a", "remarkable", "range", "of", "mechanisms", "of", "action", ".", "Recent", "studies", "highlight", "the", "capacity", "of", "sucralfate", "to", "bind", "basic", "fibroblast", "growth", "factor", "(", "bFGF", ")", "and", "deliver", "it", "in", "high", "concentration", "to", "the", "ulcer", ".", "Basic", "fibroblast", "growth", "factor", "stimulates", "the", "production", "of", "granulation", "tissue", ",", "angiogenesis", "and", "re", "-", "epithelization", ",", "thus", "improving", "the", "quality", "of", "ulcer", "healing", ".", "The", "effect", "of", "sucralfate", "in", "reducing", "parietal", "cell", "sensitivity", "may", "be", "another", "factor", "important", "in", "the", "lower", "relapse", "rate", "demonstrated", "after", "duodenal", "ulcer", "healing", ".", "Sucralfate", "has", "been", "demonstrated", "to", "be", "efficacious", "in", "healing", "both", "duodenal", "and", "gastric", "ulcers", "together", "with", "mild", "oesophagitis", ",", "and", "it", "is", "safe", "for", "both", "short", "-", "term", "use", "and", "maintenance", ".", "In", "stress", "ulcer", "prophylaxis", "it", "is", "as", "effective", "as", "acid", "suppression", "or", "neutralization", "and", "has", "the", "advantage", "of", "lesser", "rates", "of", "nosocomial", "pneumonia", "than", "are", "demonstrated", "with", "antacids", "or", "H2", "antagonists", ".", "The", "potential", "advantages", "of", "sucralfate", "lie", "in", "the", "better", "quality", "of", "ulcer", "healing", "associated", "with", "longer", "duration", "of", "remission", "." ]
[ "GENE-Y", "CHEMICAL" ]
Sucralfate is a CHEMICAL, Sucralfate is a CHEMICAL, sucralfate is a CHEMICAL, basic fibroblast growth factor is a GENE-Y, bFGF is a GENE-Y, Basic fibroblast growth factor is a GENE-Y, sucralfate is a CHEMICAL, Sucralfate is a CHEMICAL, sucralfate is a CHEMICAL
16133_task1
Sentence: Sucralfate: the Bangkok review. Sucralfate is a site-protective ulcer healing drug with a remarkable range of mechanisms of action. Recent studies highlight the capacity of sucralfate to bind basic fibroblast growth factor (bFGF) and deliver it in high concentration to the ulcer. Basic fibroblast growth factor stimulates the production of granulation tissue, angiogenesis and re-epithelization, thus improving the quality of ulcer healing. The effect of sucralfate in reducing parietal cell sensitivity may be another factor important in the lower relapse rate demonstrated after duodenal ulcer healing. Sucralfate has been demonstrated to be efficacious in healing both duodenal and gastric ulcers together with mild oesophagitis, and it is safe for both short-term use and maintenance. In stress ulcer prophylaxis it is as effective as acid suppression or neutralization and has the advantage of lesser rates of nosocomial pneumonia than are demonstrated with antacids or H2 antagonists. The potential advantages of sucralfate lie in the better quality of ulcer healing associated with longer duration of remission. Instructions: please typing these entity words according to sentence: Sucralfate, Sucralfate, sucralfate, basic fibroblast growth factor, bFGF, Basic fibroblast growth factor, sucralfate, Sucralfate, sucralfate Options: GENE-Y, CHEMICAL
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Sucralfate: the Bangkok review. Sucralfate is a site-protective ulcer healing drug with a remarkable range of mechanisms of action. Recent studies highlight the capacity of sucralfate to bind basic fibroblast growth factor (bFGF) and deliver it in high concentration to the ulcer. Basic fibroblast growth factor stimulates the production of granulation tissue, angiogenesis and re-epithelization, thus improving the quality of ulcer healing. The effect of sucralfate in reducing parietal cell sensitivity may be another factor important in the lower relapse rate demonstrated after duodenal ulcer healing. Sucralfate has been demonstrated to be efficacious in healing both duodenal and gastric ulcers together with mild oesophagitis, and it is safe for both short-term use and maintenance. In stress ulcer prophylaxis it is as effective as acid suppression or neutralization and has the advantage of lesser rates of nosocomial pneumonia than are demonstrated with antacids or H2 antagonists. The potential advantages of sucralfate lie in the better quality of ulcer healing associated with longer duration of remission.
[ "Sucralfate", ":", "the", "Bangkok", "review", ".", "\n", "Sucralfate", "is", "a", "site", "-", "protective", "ulcer", "healing", "drug", "with", "a", "remarkable", "range", "of", "mechanisms", "of", "action", ".", "Recent", "studies", "highlight", "the", "capacity", "of", "sucralfate", "to", "bind", "basic", "fibroblast", "growth", "factor", "(", "bFGF", ")", "and", "deliver", "it", "in", "high", "concentration", "to", "the", "ulcer", ".", "Basic", "fibroblast", "growth", "factor", "stimulates", "the", "production", "of", "granulation", "tissue", ",", "angiogenesis", "and", "re", "-", "epithelization", ",", "thus", "improving", "the", "quality", "of", "ulcer", "healing", ".", "The", "effect", "of", "sucralfate", "in", "reducing", "parietal", "cell", "sensitivity", "may", "be", "another", "factor", "important", "in", "the", "lower", "relapse", "rate", "demonstrated", "after", "duodenal", "ulcer", "healing", ".", "Sucralfate", "has", "been", "demonstrated", "to", "be", "efficacious", "in", "healing", "both", "duodenal", "and", "gastric", "ulcers", "together", "with", "mild", "oesophagitis", ",", "and", "it", "is", "safe", "for", "both", "short", "-", "term", "use", "and", "maintenance", ".", "In", "stress", "ulcer", "prophylaxis", "it", "is", "as", "effective", "as", "acid", "suppression", "or", "neutralization", "and", "has", "the", "advantage", "of", "lesser", "rates", "of", "nosocomial", "pneumonia", "than", "are", "demonstrated", "with", "antacids", "or", "H2", "antagonists", ".", "The", "potential", "advantages", "of", "sucralfate", "lie", "in", "the", "better", "quality", "of", "ulcer", "healing", "associated", "with", "longer", "duration", "of", "remission", "." ]
[ "GENE-Y", "CHEMICAL" ]
Sucralfate, Sucralfate, sucralfate, basic fibroblast growth factor, bFGF, Basic fibroblast growth factor, sucralfate, Sucralfate, sucralfate
16133_task2
Sentence: Sucralfate: the Bangkok review. Sucralfate is a site-protective ulcer healing drug with a remarkable range of mechanisms of action. Recent studies highlight the capacity of sucralfate to bind basic fibroblast growth factor (bFGF) and deliver it in high concentration to the ulcer. Basic fibroblast growth factor stimulates the production of granulation tissue, angiogenesis and re-epithelization, thus improving the quality of ulcer healing. The effect of sucralfate in reducing parietal cell sensitivity may be another factor important in the lower relapse rate demonstrated after duodenal ulcer healing. Sucralfate has been demonstrated to be efficacious in healing both duodenal and gastric ulcers together with mild oesophagitis, and it is safe for both short-term use and maintenance. In stress ulcer prophylaxis it is as effective as acid suppression or neutralization and has the advantage of lesser rates of nosocomial pneumonia than are demonstrated with antacids or H2 antagonists. The potential advantages of sucralfate lie in the better quality of ulcer healing associated with longer duration of remission. Instructions: please extract entity words from the input sentence
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Sucralfate: the Bangkok review. Sucralfate is a site-protective ulcer healing drug with a remarkable range of mechanisms of action. Recent studies highlight the capacity of sucralfate to bind basic fibroblast growth factor (bFGF) and deliver it in high concentration to the ulcer. Basic fibroblast growth factor stimulates the production of granulation tissue, angiogenesis and re-epithelization, thus improving the quality of ulcer healing. The effect of sucralfate in reducing parietal cell sensitivity may be another factor important in the lower relapse rate demonstrated after duodenal ulcer healing. Sucralfate has been demonstrated to be efficacious in healing both duodenal and gastric ulcers together with mild oesophagitis, and it is safe for both short-term use and maintenance. In stress ulcer prophylaxis it is as effective as acid suppression or neutralization and has the advantage of lesser rates of nosocomial pneumonia than are demonstrated with antacids or H2 antagonists. The potential advantages of sucralfate lie in the better quality of ulcer healing associated with longer duration of remission.
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[ "GENE-Y", "CHEMICAL" ]
9-cis - retinoic acid is a CHEMICAL, RXR is a GENE-N
15546741_task0
Sentence: 9-cis-retinoic acid analogues with bulky hydrophobic rings: new RXR-selective agonists. Instructions: please extract entities and their types from the input sentence, all entity types are in options Options: GENE-N, CHEMICAL
[ "B-CHEMICAL", "I-CHEMICAL", "I-CHEMICAL", "I-CHEMICAL", "O", "O", "O", "O", "O", "O", "O", "B-GENE-N", "O", "O", "O", "O" ]
9-cis-retinoic acid analogues with bulky hydrophobic rings: new RXR-selective agonists.
[ "9-cis", "-", "retinoic", "acid", "analogues", "with", "bulky", "hydrophobic", "rings", ":", "new", "RXR", "-", "selective", "agonists", "." ]
[ "CHEMICAL", "GENE-N" ]
9-cis - retinoic acid is a CHEMICAL, RXR is a GENE-N
15546741_task1
Sentence: 9-cis-retinoic acid analogues with bulky hydrophobic rings: new RXR-selective agonists. Instructions: please typing these entity words according to sentence: 9-cis - retinoic acid, RXR Options: GENE-N, CHEMICAL
[ "B-CHEMICAL", "I-CHEMICAL", "I-CHEMICAL", "I-CHEMICAL", "O", "O", "O", "O", "O", "O", "O", "B-GENE-N", "O", "O", "O", "O" ]
9-cis-retinoic acid analogues with bulky hydrophobic rings: new RXR-selective agonists.
[ "9-cis", "-", "retinoic", "acid", "analogues", "with", "bulky", "hydrophobic", "rings", ":", "new", "RXR", "-", "selective", "agonists", "." ]
[ "CHEMICAL", "GENE-N" ]
9-cis - retinoic acid, RXR
15546741_task2
Sentence: 9-cis-retinoic acid analogues with bulky hydrophobic rings: new RXR-selective agonists. Instructions: please extract entity words from the input sentence
[ "B-CHEMICAL", "I-CHEMICAL", "I-CHEMICAL", "I-CHEMICAL", "O", "O", "O", "O", "O", "O", "O", "B-GENE-N", "O", "O", "O", "O" ]
9-cis-retinoic acid analogues with bulky hydrophobic rings: new RXR-selective agonists.
[ "9-cis", "-", "retinoic", "acid", "analogues", "with", "bulky", "hydrophobic", "rings", ":", "new", "RXR", "-", "selective", "agonists", "." ]
[ "CHEMICAL", "GENE-N" ]
modulation is a Relation_Trigger, microRNAs is a Non-Specific_miRNAs, primary glioblastoma is a Diseases
5419_task0
Sentence: Extensive modulation of a set of microRNAs in primary glioblastoma. Instructions: please extract entities and their types from the input sentence, all entity types are in options Options: Relation_Trigger, Diseases, Non-Specific_miRNAs
[ "O", "B-Relation_Trigger", "O", "O", "O", "O", "B-Non-Specific_miRNAs", "O", "B-Diseases", "I-Diseases", "O", "O" ]
Extensive modulation of a set of microRNAs in primary glioblastoma.
[ "Extensive", "modulation", "of", "a", "set", "of", "microRNAs", "in", "primary", "glioblastoma", ".", " " ]
[ "Relation_Trigger", "Diseases", "Non-Specific_miRNAs", "Specific_miRNAs", "Species" ]
modulation is a Relation_Trigger, microRNAs is a Non-Specific_miRNAs, primary glioblastoma is a Diseases
5419_task1
Sentence: Extensive modulation of a set of microRNAs in primary glioblastoma. Instructions: please typing these entity words according to sentence: modulation, microRNAs, primary glioblastoma Options: Relation_Trigger, Diseases, Non-Specific_miRNAs
[ "O", "B-Relation_Trigger", "O", "O", "O", "O", "B-Non-Specific_miRNAs", "O", "B-Diseases", "I-Diseases", "O", "O" ]
Extensive modulation of a set of microRNAs in primary glioblastoma.
[ "Extensive", "modulation", "of", "a", "set", "of", "microRNAs", "in", "primary", "glioblastoma", ".", " " ]
[ "Relation_Trigger", "Diseases", "Non-Specific_miRNAs", "Specific_miRNAs", "Species" ]
modulation, microRNAs, primary glioblastoma
5419_task2
Sentence: Extensive modulation of a set of microRNAs in primary glioblastoma. Instructions: please extract entity words from the input sentence
[ "O", "B-Relation_Trigger", "O", "O", "O", "O", "B-Non-Specific_miRNAs", "O", "B-Diseases", "I-Diseases", "O", "O" ]
Extensive modulation of a set of microRNAs in primary glioblastoma.
[ "Extensive", "modulation", "of", "a", "set", "of", "microRNAs", "in", "primary", "glioblastoma", ".", " " ]
[ "Relation_Trigger", "Diseases", "Non-Specific_miRNAs", "Specific_miRNAs", "Species" ]
Aetiologie is an umlsterm, Harntraktes is an umlsterm, Ureterobstruktion is an umlsterm, Anurie is an umlsterm, Uraemie is an umlsterm, Aetiologie is an umlsterm, Behandlung is an umlsterm, Patienten is an umlsterm, Diagnose is an umlsterm, Patienten is an umlsterm, Patienten is an umlsterm, Patienten is an umlsterm, Niereninsuffizienz is an umlsterm, Protein is an umlsterm, Patienten is an umlsterm, Niere is an umlsterm, Patienten is an umlsterm, Patienten is an umlsterm, Patienten is an umlsterm, Patienten is an umlsterm, Niere is an umlsterm, Harnleiter- is an umlsterm, Nephrektomie is an umlsterm, Ballondilatation is an umlsterm, Patienten is an umlsterm, Patient is an umlsterm, Rezidiv is an umlsterm, Patienten is an umlsterm, Therapieergebnisse is an umlsterm, Behandlung is an umlsterm, Ballondilatation is an umlsterm
DerUrologeA.00390141.ger.abstr_task0
Sentence: Der retroperitonealen Fibrose ( RPF ) liegt eine entzuendliche Bindegewebsproliferation ungeklaerter Aetiologie mit Bevorzugung des Retroperitoneums zugrunde . Die Beteiligung des ableitenden Harntraktes manifestiert sich durch uni- oder bilaterale Ureterobstruktion bis hin zur Anurie und Uraemie . Aufgrund der ungeklaerten Aetiologie wird das therapeutische Vorgehen - konservativ versus operativ - kontrovers diskutiert . Im folgenden berichten wir ueber unsere Ergebnisse in der Behandlung der RPF . Von 1986 bis 1997 wurde bei 39 Patienten die Diagnose einer RPF gestellt 21 mal , lag eine primaere , 18 mal eine sekundaere RRF vor . 21 Patienten wiesen eine uni- , 16 Patienten eine bilaterale und 2 Patienten keine Harnstauung auf . In allen Faellen einer primaeren RPF fand sich eine beschleunigte BSG , 5/21 wiesen eine beginnende Niereninsuffizienz auf , ein erhoehtes C-reaktives Protein lag bei 16/21 vor . Bei der sekundaeren RPF fand sich in 4 Faellen eine Erhoehung der Retentionswerte . Bei 19/21 Patienten erfolgte die primaere Entlastung der Niere mit anschliessender Cortison- und/oder Azathioprintherapie ueber 6 Monate ; eine Besserung sahen wir in allen Faellen , eine komplette Remission in 2 Faellen . Bei 19 Patienten war eine operative Intervention notwendig : 17 mal Ureterolyse mit Intraperitonealisierung ( n = 10 ) bzw. Omentum-majus-Ummantelung ( n = 7 ) . Bei 1 Patienten war die bilaterale Boari-Plastik , bei 1 Patienten ein bilaterales Ileum-Ureterinterponat notwendig . Bei 16/18 Patienten mit sekundaerer RPF erfolgte die primaere Entlastung der Niere gefolgt von Ureterolyse und Omentum-majus-Ummantelung ( n = 11 ) , Harnleiter- Ileuminterponat ( n = 3 Nephrektomie ( ) , n = 1 ) und endoluminale Ballondilatation ( n = 3 ) . Das Follow-up variiert zwischen 6 und 120 Monaten : 3 Patienten mit primaerer RPF und 1 Patient mit sekundaerer RPF entwickelten ein Rezidiv ; die Abflussverhaeltnisse der uebrigen Patienten sind unauffaellig . Aufgrund der Therapieergebnisse unserer Serie halten wir die kombinierte operative/immunsupprimierende Behandlung bei der primaeren RPF fuer erfolgversprechend ; dabei geben wir der praeoperativen Cortisontherapie mit nachfolgender Ureterolyse und Omentum-majus-Ummantelung den Vorzug . Bei sekundaerer RPF bietet sich die primaere operative Versorgung mittels Ureterolyse und Omentum-Ummantelung an ; lediglich bei kurzstreckiger extrinsischer Kompression sollte der Ballondilatation der Vorzug gegeben werden . Instructions: please extract entities and their types from the input sentence, all entity types are in options Options: umlsterm
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Der retroperitonealen Fibrose ( RPF ) liegt eine entzuendliche Bindegewebsproliferation ungeklaerter Aetiologie mit Bevorzugung des Retroperitoneums zugrunde . Die Beteiligung des ableitenden Harntraktes manifestiert sich durch uni- oder bilaterale Ureterobstruktion bis hin zur Anurie und Uraemie . Aufgrund der ungeklaerten Aetiologie wird das therapeutische Vorgehen - konservativ versus operativ - kontrovers diskutiert . Im folgenden berichten wir ueber unsere Ergebnisse in der Behandlung der RPF . Von 1986 bis 1997 wurde bei 39 Patienten die Diagnose einer RPF gestellt 21 mal , lag eine primaere , 18 mal eine sekundaere RRF vor . 21 Patienten wiesen eine uni- , 16 Patienten eine bilaterale und 2 Patienten keine Harnstauung auf . In allen Faellen einer primaeren RPF fand sich eine beschleunigte BSG , 5/21 wiesen eine beginnende Niereninsuffizienz auf , ein erhoehtes C-reaktives Protein lag bei 16/21 vor . Bei der sekundaeren RPF fand sich in 4 Faellen eine Erhoehung der Retentionswerte . Bei 19/21 Patienten erfolgte die primaere Entlastung der Niere mit anschliessender Cortison- und/oder Azathioprintherapie ueber 6 Monate ; eine Besserung sahen wir in allen Faellen , eine komplette Remission in 2 Faellen . Bei 19 Patienten war eine operative Intervention notwendig : 17 mal Ureterolyse mit Intraperitonealisierung ( n = 10 ) bzw. Omentum-majus-Ummantelung ( n = 7 ) . Bei 1 Patienten war die bilaterale Boari-Plastik , bei 1 Patienten ein bilaterales Ileum-Ureterinterponat notwendig . Bei 16/18 Patienten mit sekundaerer RPF erfolgte die primaere Entlastung der Niere gefolgt von Ureterolyse und Omentum-majus-Ummantelung ( n = 11 ) , Harnleiter- Ileuminterponat ( n = 3 Nephrektomie ( ) , n = 1 ) und endoluminale Ballondilatation ( n = 3 ) . Das Follow-up variiert zwischen 6 und 120 Monaten : 3 Patienten mit primaerer RPF und 1 Patient mit sekundaerer RPF entwickelten ein Rezidiv ; die Abflussverhaeltnisse der uebrigen Patienten sind unauffaellig . Aufgrund der Therapieergebnisse unserer Serie halten wir die kombinierte operative/immunsupprimierende Behandlung bei der primaeren RPF fuer erfolgversprechend ; dabei geben wir der praeoperativen Cortisontherapie mit nachfolgender Ureterolyse und Omentum-majus-Ummantelung den Vorzug . Bei sekundaerer RPF bietet sich die primaere operative Versorgung mittels Ureterolyse und Omentum-Ummantelung an ; lediglich bei kurzstreckiger extrinsischer Kompression sollte der Ballondilatation der Vorzug gegeben werden .
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[ "umlsterm" ]
Aetiologie is an umlsterm, Harntraktes is an umlsterm, Ureterobstruktion is an umlsterm, Anurie is an umlsterm, Uraemie is an umlsterm, Aetiologie is an umlsterm, Behandlung is an umlsterm, Patienten is an umlsterm, Diagnose is an umlsterm, Patienten is an umlsterm, Patienten is an umlsterm, Patienten is an umlsterm, Niereninsuffizienz is an umlsterm, Protein is an umlsterm, Patienten is an umlsterm, Niere is an umlsterm, Patienten is an umlsterm, Patienten is an umlsterm, Patienten is an umlsterm, Patienten is an umlsterm, Niere is an umlsterm, Harnleiter- is an umlsterm, Nephrektomie is an umlsterm, Ballondilatation is an umlsterm, Patienten is an umlsterm, Patient is an umlsterm, Rezidiv is an umlsterm, Patienten is an umlsterm, Therapieergebnisse is an umlsterm, Behandlung is an umlsterm, Ballondilatation is an umlsterm
DerUrologeA.00390141.ger.abstr_task1
Sentence: Der retroperitonealen Fibrose ( RPF ) liegt eine entzuendliche Bindegewebsproliferation ungeklaerter Aetiologie mit Bevorzugung des Retroperitoneums zugrunde . Die Beteiligung des ableitenden Harntraktes manifestiert sich durch uni- oder bilaterale Ureterobstruktion bis hin zur Anurie und Uraemie . Aufgrund der ungeklaerten Aetiologie wird das therapeutische Vorgehen - konservativ versus operativ - kontrovers diskutiert . Im folgenden berichten wir ueber unsere Ergebnisse in der Behandlung der RPF . Von 1986 bis 1997 wurde bei 39 Patienten die Diagnose einer RPF gestellt 21 mal , lag eine primaere , 18 mal eine sekundaere RRF vor . 21 Patienten wiesen eine uni- , 16 Patienten eine bilaterale und 2 Patienten keine Harnstauung auf . In allen Faellen einer primaeren RPF fand sich eine beschleunigte BSG , 5/21 wiesen eine beginnende Niereninsuffizienz auf , ein erhoehtes C-reaktives Protein lag bei 16/21 vor . Bei der sekundaeren RPF fand sich in 4 Faellen eine Erhoehung der Retentionswerte . Bei 19/21 Patienten erfolgte die primaere Entlastung der Niere mit anschliessender Cortison- und/oder Azathioprintherapie ueber 6 Monate ; eine Besserung sahen wir in allen Faellen , eine komplette Remission in 2 Faellen . Bei 19 Patienten war eine operative Intervention notwendig : 17 mal Ureterolyse mit Intraperitonealisierung ( n = 10 ) bzw. Omentum-majus-Ummantelung ( n = 7 ) . Bei 1 Patienten war die bilaterale Boari-Plastik , bei 1 Patienten ein bilaterales Ileum-Ureterinterponat notwendig . Bei 16/18 Patienten mit sekundaerer RPF erfolgte die primaere Entlastung der Niere gefolgt von Ureterolyse und Omentum-majus-Ummantelung ( n = 11 ) , Harnleiter- Ileuminterponat ( n = 3 Nephrektomie ( ) , n = 1 ) und endoluminale Ballondilatation ( n = 3 ) . Das Follow-up variiert zwischen 6 und 120 Monaten : 3 Patienten mit primaerer RPF und 1 Patient mit sekundaerer RPF entwickelten ein Rezidiv ; die Abflussverhaeltnisse der uebrigen Patienten sind unauffaellig . Aufgrund der Therapieergebnisse unserer Serie halten wir die kombinierte operative/immunsupprimierende Behandlung bei der primaeren RPF fuer erfolgversprechend ; dabei geben wir der praeoperativen Cortisontherapie mit nachfolgender Ureterolyse und Omentum-majus-Ummantelung den Vorzug . Bei sekundaerer RPF bietet sich die primaere operative Versorgung mittels Ureterolyse und Omentum-Ummantelung an ; lediglich bei kurzstreckiger extrinsischer Kompression sollte der Ballondilatation der Vorzug gegeben werden . Instructions: please typing these entity words according to sentence: Aetiologie, Harntraktes, Ureterobstruktion, Anurie, Uraemie, Aetiologie, Behandlung, Patienten, Diagnose, Patienten, Patienten, Patienten, Niereninsuffizienz, Protein, Patienten, Niere, Patienten, Patienten, Patienten, Patienten, Niere, Harnleiter-, Nephrektomie, Ballondilatation, Patienten, Patient, Rezidiv, Patienten, Therapieergebnisse, Behandlung, Ballondilatation Options: umlsterm
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Der retroperitonealen Fibrose ( RPF ) liegt eine entzuendliche Bindegewebsproliferation ungeklaerter Aetiologie mit Bevorzugung des Retroperitoneums zugrunde . Die Beteiligung des ableitenden Harntraktes manifestiert sich durch uni- oder bilaterale Ureterobstruktion bis hin zur Anurie und Uraemie . Aufgrund der ungeklaerten Aetiologie wird das therapeutische Vorgehen - konservativ versus operativ - kontrovers diskutiert . Im folgenden berichten wir ueber unsere Ergebnisse in der Behandlung der RPF . Von 1986 bis 1997 wurde bei 39 Patienten die Diagnose einer RPF gestellt 21 mal , lag eine primaere , 18 mal eine sekundaere RRF vor . 21 Patienten wiesen eine uni- , 16 Patienten eine bilaterale und 2 Patienten keine Harnstauung auf . In allen Faellen einer primaeren RPF fand sich eine beschleunigte BSG , 5/21 wiesen eine beginnende Niereninsuffizienz auf , ein erhoehtes C-reaktives Protein lag bei 16/21 vor . Bei der sekundaeren RPF fand sich in 4 Faellen eine Erhoehung der Retentionswerte . Bei 19/21 Patienten erfolgte die primaere Entlastung der Niere mit anschliessender Cortison- und/oder Azathioprintherapie ueber 6 Monate ; eine Besserung sahen wir in allen Faellen , eine komplette Remission in 2 Faellen . Bei 19 Patienten war eine operative Intervention notwendig : 17 mal Ureterolyse mit Intraperitonealisierung ( n = 10 ) bzw. Omentum-majus-Ummantelung ( n = 7 ) . Bei 1 Patienten war die bilaterale Boari-Plastik , bei 1 Patienten ein bilaterales Ileum-Ureterinterponat notwendig . Bei 16/18 Patienten mit sekundaerer RPF erfolgte die primaere Entlastung der Niere gefolgt von Ureterolyse und Omentum-majus-Ummantelung ( n = 11 ) , Harnleiter- Ileuminterponat ( n = 3 Nephrektomie ( ) , n = 1 ) und endoluminale Ballondilatation ( n = 3 ) . Das Follow-up variiert zwischen 6 und 120 Monaten : 3 Patienten mit primaerer RPF und 1 Patient mit sekundaerer RPF entwickelten ein Rezidiv ; die Abflussverhaeltnisse der uebrigen Patienten sind unauffaellig . Aufgrund der Therapieergebnisse unserer Serie halten wir die kombinierte operative/immunsupprimierende Behandlung bei der primaeren RPF fuer erfolgversprechend ; dabei geben wir der praeoperativen Cortisontherapie mit nachfolgender Ureterolyse und Omentum-majus-Ummantelung den Vorzug . Bei sekundaerer RPF bietet sich die primaere operative Versorgung mittels Ureterolyse und Omentum-Ummantelung an ; lediglich bei kurzstreckiger extrinsischer Kompression sollte der Ballondilatation der Vorzug gegeben werden .
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[ "umlsterm" ]
Aetiologie, Harntraktes, Ureterobstruktion, Anurie, Uraemie, Aetiologie, Behandlung, Patienten, Diagnose, Patienten, Patienten, Patienten, Niereninsuffizienz, Protein, Patienten, Niere, Patienten, Patienten, Patienten, Patienten, Niere, Harnleiter-, Nephrektomie, Ballondilatation, Patienten, Patient, Rezidiv, Patienten, Therapieergebnisse, Behandlung, Ballondilatation
DerUrologeA.00390141.ger.abstr_task2
Sentence: Der retroperitonealen Fibrose ( RPF ) liegt eine entzuendliche Bindegewebsproliferation ungeklaerter Aetiologie mit Bevorzugung des Retroperitoneums zugrunde . Die Beteiligung des ableitenden Harntraktes manifestiert sich durch uni- oder bilaterale Ureterobstruktion bis hin zur Anurie und Uraemie . Aufgrund der ungeklaerten Aetiologie wird das therapeutische Vorgehen - konservativ versus operativ - kontrovers diskutiert . Im folgenden berichten wir ueber unsere Ergebnisse in der Behandlung der RPF . Von 1986 bis 1997 wurde bei 39 Patienten die Diagnose einer RPF gestellt 21 mal , lag eine primaere , 18 mal eine sekundaere RRF vor . 21 Patienten wiesen eine uni- , 16 Patienten eine bilaterale und 2 Patienten keine Harnstauung auf . In allen Faellen einer primaeren RPF fand sich eine beschleunigte BSG , 5/21 wiesen eine beginnende Niereninsuffizienz auf , ein erhoehtes C-reaktives Protein lag bei 16/21 vor . Bei der sekundaeren RPF fand sich in 4 Faellen eine Erhoehung der Retentionswerte . Bei 19/21 Patienten erfolgte die primaere Entlastung der Niere mit anschliessender Cortison- und/oder Azathioprintherapie ueber 6 Monate ; eine Besserung sahen wir in allen Faellen , eine komplette Remission in 2 Faellen . Bei 19 Patienten war eine operative Intervention notwendig : 17 mal Ureterolyse mit Intraperitonealisierung ( n = 10 ) bzw. Omentum-majus-Ummantelung ( n = 7 ) . Bei 1 Patienten war die bilaterale Boari-Plastik , bei 1 Patienten ein bilaterales Ileum-Ureterinterponat notwendig . Bei 16/18 Patienten mit sekundaerer RPF erfolgte die primaere Entlastung der Niere gefolgt von Ureterolyse und Omentum-majus-Ummantelung ( n = 11 ) , Harnleiter- Ileuminterponat ( n = 3 Nephrektomie ( ) , n = 1 ) und endoluminale Ballondilatation ( n = 3 ) . Das Follow-up variiert zwischen 6 und 120 Monaten : 3 Patienten mit primaerer RPF und 1 Patient mit sekundaerer RPF entwickelten ein Rezidiv ; die Abflussverhaeltnisse der uebrigen Patienten sind unauffaellig . Aufgrund der Therapieergebnisse unserer Serie halten wir die kombinierte operative/immunsupprimierende Behandlung bei der primaeren RPF fuer erfolgversprechend ; dabei geben wir der praeoperativen Cortisontherapie mit nachfolgender Ureterolyse und Omentum-majus-Ummantelung den Vorzug . Bei sekundaerer RPF bietet sich die primaere operative Versorgung mittels Ureterolyse und Omentum-Ummantelung an ; lediglich bei kurzstreckiger extrinsischer Kompression sollte der Ballondilatation der Vorzug gegeben werden . Instructions: please extract entity words from the input sentence
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Der retroperitonealen Fibrose ( RPF ) liegt eine entzuendliche Bindegewebsproliferation ungeklaerter Aetiologie mit Bevorzugung des Retroperitoneums zugrunde . Die Beteiligung des ableitenden Harntraktes manifestiert sich durch uni- oder bilaterale Ureterobstruktion bis hin zur Anurie und Uraemie . Aufgrund der ungeklaerten Aetiologie wird das therapeutische Vorgehen - konservativ versus operativ - kontrovers diskutiert . Im folgenden berichten wir ueber unsere Ergebnisse in der Behandlung der RPF . Von 1986 bis 1997 wurde bei 39 Patienten die Diagnose einer RPF gestellt 21 mal , lag eine primaere , 18 mal eine sekundaere RRF vor . 21 Patienten wiesen eine uni- , 16 Patienten eine bilaterale und 2 Patienten keine Harnstauung auf . In allen Faellen einer primaeren RPF fand sich eine beschleunigte BSG , 5/21 wiesen eine beginnende Niereninsuffizienz auf , ein erhoehtes C-reaktives Protein lag bei 16/21 vor . Bei der sekundaeren RPF fand sich in 4 Faellen eine Erhoehung der Retentionswerte . Bei 19/21 Patienten erfolgte die primaere Entlastung der Niere mit anschliessender Cortison- und/oder Azathioprintherapie ueber 6 Monate ; eine Besserung sahen wir in allen Faellen , eine komplette Remission in 2 Faellen . Bei 19 Patienten war eine operative Intervention notwendig : 17 mal Ureterolyse mit Intraperitonealisierung ( n = 10 ) bzw. Omentum-majus-Ummantelung ( n = 7 ) . Bei 1 Patienten war die bilaterale Boari-Plastik , bei 1 Patienten ein bilaterales Ileum-Ureterinterponat notwendig . Bei 16/18 Patienten mit sekundaerer RPF erfolgte die primaere Entlastung der Niere gefolgt von Ureterolyse und Omentum-majus-Ummantelung ( n = 11 ) , Harnleiter- Ileuminterponat ( n = 3 Nephrektomie ( ) , n = 1 ) und endoluminale Ballondilatation ( n = 3 ) . Das Follow-up variiert zwischen 6 und 120 Monaten : 3 Patienten mit primaerer RPF und 1 Patient mit sekundaerer RPF entwickelten ein Rezidiv ; die Abflussverhaeltnisse der uebrigen Patienten sind unauffaellig . Aufgrund der Therapieergebnisse unserer Serie halten wir die kombinierte operative/immunsupprimierende Behandlung bei der primaeren RPF fuer erfolgversprechend ; dabei geben wir der praeoperativen Cortisontherapie mit nachfolgender Ureterolyse und Omentum-majus-Ummantelung den Vorzug . Bei sekundaerer RPF bietet sich die primaere operative Versorgung mittels Ureterolyse und Omentum-Ummantelung an ; lediglich bei kurzstreckiger extrinsischer Kompression sollte der Ballondilatation der Vorzug gegeben werden .
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[ "umlsterm" ]
Amsacrine is a CHEMICAL, etoposide is a CHEMICAL, DNA topoisomerase II is a GENE-Y
1322791_task0
Sentence: Amsacrine and etoposide hypersensitivity of yeast cells overexpressing DNA topoisomerase II. Instructions: please extract entities and their types from the input sentence, all entity types are in options Options: GENE-Y, CHEMICAL
[ "B-CHEMICAL", "O", "B-CHEMICAL", "O", "O", "O", "O", "O", "B-GENE-Y", "I-GENE-Y", "I-GENE-Y", "O" ]
Amsacrine and etoposide hypersensitivity of yeast cells overexpressing DNA topoisomerase II.
[ "Amsacrine", "and", "etoposide", "hypersensitivity", "of", "yeast", "cells", "overexpressing", "DNA", "topoisomerase", "II", "." ]
[ "GENE-Y", "CHEMICAL" ]
Amsacrine is a CHEMICAL, etoposide is a CHEMICAL, DNA topoisomerase II is a GENE-Y
1322791_task1
Sentence: Amsacrine and etoposide hypersensitivity of yeast cells overexpressing DNA topoisomerase II. Instructions: please typing these entity words according to sentence: Amsacrine, etoposide, DNA topoisomerase II Options: GENE-Y, CHEMICAL
[ "B-CHEMICAL", "O", "B-CHEMICAL", "O", "O", "O", "O", "O", "B-GENE-Y", "I-GENE-Y", "I-GENE-Y", "O" ]
Amsacrine and etoposide hypersensitivity of yeast cells overexpressing DNA topoisomerase II.
[ "Amsacrine", "and", "etoposide", "hypersensitivity", "of", "yeast", "cells", "overexpressing", "DNA", "topoisomerase", "II", "." ]
[ "GENE-Y", "CHEMICAL" ]
Amsacrine, etoposide, DNA topoisomerase II
1322791_task2
Sentence: Amsacrine and etoposide hypersensitivity of yeast cells overexpressing DNA topoisomerase II. Instructions: please extract entity words from the input sentence
[ "B-CHEMICAL", "O", "B-CHEMICAL", "O", "O", "O", "O", "O", "B-GENE-Y", "I-GENE-Y", "I-GENE-Y", "O" ]
Amsacrine and etoposide hypersensitivity of yeast cells overexpressing DNA topoisomerase II.
[ "Amsacrine", "and", "etoposide", "hypersensitivity", "of", "yeast", "cells", "overexpressing", "DNA", "topoisomerase", "II", "." ]
[ "GENE-Y", "CHEMICAL" ]
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Sentence: BackgroundHuman embryonic stem cells (hESCs) offer a virtually unlimited source of neural cells for structural repair in neurological disorders, such as stroke. Neural cells can be derived from hESCs either by direct enrichment, or by isolating specific growth factor-responsive and expandable populations of human neural stem cells (hNSCs). Studies have indicated that the direct enrichment method generates a heterogeneous population of cells that may contain residual undifferentiated stem cells that could lead to tumor formation in vivo.Methods/Principal FindingsWe isolated an expandable and homogenous population of hNSCs (named SD56) from hESCs using a defined media supplemented with epidermal growth factor (EGF), basic fibroblast growth factor (bFGF) and leukemia inhibitory growth factor (LIF). These hNSCs grew as an adherent monolayer culture. They were fully neuralized and uniformly expressed molecular features of NSCs, including nestin, vimentin and radial glial markers. These hNSCs did not express the pluripotency markers Oct4 or Nanog, nor did they express markers for the mesoderm or endoderm lineages. The self-renewal property of the hNSCs was characterized by a predominant symmetrical mode of cell division. The SD56 hNSCs differentiated into neurons, astrocytes and oligodendrocytes throughout multiple passages in vitro, as well as after transplantation. Together, these criteria confirm the definitive NSC identity of the SD56 cell line. Importantly, they exhibited no chromosome abnormalities and did not form tumors after implantation into rat ischemic brains and into naïve nude rat brains and flanks. Furthermore, hNSCs isolated under these conditions migrated toward the ischemia-injured adult brain parenchyma and improved the independent use of the stroke-impaired forelimb two months post-transplantation.Conclusions/SignificanceThe SD56 human neural stem cells derived under the reported conditions are stable, do not form tumors in vivo and enable functional recovery after stroke. These properties indicate that this hNSC line may offer a renewable, homogenous source of neural cells that will be valuable for basic and translational research. To date there have been no effective treatments for improving residual structural and functional deficits resulting from stroke. Current therapeutic approaches, such as the use of thrombolytics, benefit only 1 to 4% of patients [1]. Consequently, the majority of stroke patients experience progression of ischemia associated with debilitating neurological deficits. Recent evidence has suggested that the transplantation of cells derived from cord blood, bone marrow or brain tissue (fetal and adult) enhances sensorimotor function in experimental models of stroke [2], [3]. However, the normal human-derived somatic stem cells used in these studies have a limited capacity to differentiate into the diverse neural cell types optimal for structural and physiological tissue repair and are not amenable for large-scale cell production. Unlike other sources of stem cells, hESC lines possess a nearly unlimited self-renewal capacity and the developmental potential to differentiate into virtually any cell type of the organism. As such, they constitute an ideal source of cells for regenerative medicine. The successful derivation of hESC lines from the inner cell mass of preimplantation embryos and their long-term maintenance in vitro over multiple passages has been demonstrated [4] and standardized. Differentiation and enrichment processes that direct hESCs towards a neural lineage have also been achieved. To promote neuralization, ESCs were cultured in a defined media supplemented with morphogens or growth factors [5], [6], [7] or cultured under conditions that promote “rosettes”, structures morphologically similar to the developing neural tube [8], [9]. This neuralization process has proven invaluable in understanding the specification of hESC-derived neural tissue [10], [11], [12]. However, the enriched neural progeny derived displayed overgrowth and limited migration after grafting into normal newborn mice [13] and lesioned adult rat striatum [12], [14], [15], [16]. The inner cores of these grafts contained tumorigenic precursor cells (reviewed in [17]). These findings suggest that neural cells generated by acute exposure to growth factors and/or morphogens may still be heterogeneous and potentially tumorigenic. We report an alternative method for the isolation and the perpetuation of a multipotent hNSC line from the hESCs with a primitive mode of self-renewal. We also demonstrate their long-term expansion, non-tumorigenic properties and functional engraftability in an experimental model of stroke. 1. In vitro isolation, growth and differentiation of hESC-derived hNSCsThe hESCs were maintained and expanded on mouse feeder layer in media supplemented with bFGF (Figure 1A). After cell dissociation, a portion of the hESCs was cultured in serum free medium containing EGF, bFGF and LIF. These factors are known to stimulate the proliferation of human fetal-derived NSCs [18], [19]. After 3 days in vitro (DIV), there was selective survival and growth of cells that aggregated in clusters or spheres (Figure 1B). These primary spheres were harvested and replated in fresh media. During the following week, the spheres attached to the flask and a fibroblast-like cell population began to migrate out (Figure 1C). Secondary spheres (2° spheres) were generated from these cultures and lifted off by the end of the week leaving a hollow in the middle of the attached cells (Figure 1D). The floating 2° spheres were collected and replated in fresh growth medium for 2 weeks. The cultures were then passaged by collagenase cell dissociation every 7 DIV for an additional 4 passages (Figure S1). At the 5th and 6th passages, spheres were dissociated into a single-cell suspension using trypsin-EDTA. At this stage there was a change in the hNSCs' adherent properties, and the cells began to grow as a monolayer with multiple foci of cells throughout the culture (Figure 1F). The adherent hNSC culture stained uniformly for nestin (Figure 1K), vimentin (Figure 1L) and with the radial glial marker 3CB2 (Figure 1M) indicating their homogeneity and NSC identity. Under these culture conditions, it is noteworthy that we did not observe the formation of rosettes which has been previously reported to occur under certain conditions during neuralization of hESCs [8], [20], [21]. RT-PCR analysis confirmed that these hNSCs did not express the pluripotency transcripts Oct-4 and Nanog (Figure 1I). Furthermore, the hNSCs did not express transcripts for brachyury and foxa2, marker genes for mesoderm and endoderm respectively (negative result, data not shown).10.1371/journal.pone.0001644.g001Figure 1Isolation and purification of hNSCs from the hESCs.The hESCs were grown on a mouse feeder layer (A). Primary neurospheres (B) were isolated and replated to eliminate other non-neural cells. The selectively harvested secondary neurospheres (arrow in C), left behind hollow cores in the surface area (star in D) where they attached earlier. They were perpetuated for an additional 5 passages (E). These 2° spheres were then passaged using a single cell dissociation protocol (F). Arrow in F shows an example of a focus of proliferating cells. (G, H) The hNSCs were passaged every 5–7 days, as described in the Methods section. Starting from an initial population of 1 million cells, the cumulative cell number was calculated at each passage as the fold of increase×the total cell number and plotted as logarithm with base 2 in function of time (G). The cell perpetuation (G) and population doubling (H) analysis demonstrated the continuous and stable growth of the hNSCs. (I) RT-PCR analysis showing the down regulation of the pluripotency transcripts Oct4 and Nanog in secondary neurospheres and in expanded hNSCs at passage 8 (P8). (J) Cytogenetic evaluation of the SD56 hNSCs line at passage 12 by standard G-banding was performed. Twenty metaphase cells were analyzed and showed a normal female chromosome complement (46,XX). Isolated and expanded hNSCs expressed the neural precursor cell markers nestin (K), Vimentin (L) and the radial glial cell marker 3CB2 (M) in virtually all the progeny. (N-P) Clonal self-renewal ability of the isolated hNSCs through symmetrical cell division. Single (N), two-cell stage (O) and four-cell stage (P) of a hNSC proliferating over a 3-day culture period. Note the symmetrical segregation of BrdU and nestin in the progeny. Bars: (A, B, C, D) 200 µm; (E, F) 100 µm; (K–M) 20 µm; (N–P) 10 µm.To ascertain self-renewal ability under clonal conditions, a single cell suspension was plated at clonal density (1–2 cell/10 µl). To determine if the hNSCs divide symmetrically, we pulsed cultures with the thymidine analog, bromodeoxyuridine (BrdU), after plating and looked for the expression of nestin in the progeny. Cultures were fixed after 1, 2 or 3 DIV (Figure 1N–P). After 2 days, plated single cells first underwent a symmetric cell division and gave rise to daughter cells that were both positive for BrdU and nestin. The clone of cells continued to grow over the 3 DIV and all the progeny expressed nestin. BrdU labeling demonstrated that it was rare for only one daughter cell to inherit the BrdU and thus had undergone asymmetric segregation of the chromatids (Figure S2). G-band karyotyping of these hNSCs confirmed the normal, non-transformed nature of cells after 12 passages (Figure 1J). We named the derived hNSCs SD56 (intermittently referred to as SD56 hNSCs or hNSCs).Under these defined growth conditions, the hNSCs showed stable growth with a 2.7±0.2 fold increase every 5 to 7 days (Figure 1G). The population doubling at each passage averaged at 1.4±0.1 (Figure 1H). The viability of hNSCs at each passage was consistent at the approximate value of 98%. The projected cumulative cell numbers demonstrated that trillions of cells could be generated over a period of 5 months (Figure 1G). We expanded the isolated hNSCs lines for over 20 passages with a stable phenotype. An initial cell bank of 75 vials containing 2 to 5 million cells each was generated and cryopreserved.Upon removal of the mitogenic factors and plating on a coverslip pre-coated with poly-L-ornithine (PLO) substrate, the hNSCs spontaneously differentiated into neurons, astrocytes and oligodendrocytes, a property that is consistent with normal multipotent hNSCs (Figure 2). After 2 DIV, hNSCs expressed transcripts for the neural-specific genes nestin, Notch1 and neural cell adhesion molecule (N-CAM) (Figure 2A) and for the lineage specific markers β-tubulin class III, medium-size neurofilament (NF-M) and microtubule-associated protein 2 (MAP-2) for neurons, GFAP for astrocytes and myelin basic protein (MBP) for oligodendrocytes (Figure 2A). Transcripts for the GABAergic cell marker glutamic acid decarboxylase (GAD) were expressed, but transcripts for the tyrosine hydroxylase (TH), a marker for dopaminergic neurons, were undetectable. Immunocytochemical analysis (Figure 2B–F) of 10 day-old cultures demonstrated that the proportion of nestin+ cells was 36.6±2.7%, 62.5±2.8% expressed the neuronal marker TuJ1, 1.9±0.3% expressed the astrocytic marker GFAP and 7.1±0.4% were oligodendrocytes and expressed galactocerebrocide (GC) (Figure 2F). A subset (9.8±1.6%) of the GFAP+ astrocytes co-expressed nestin.10.1371/journal.pone.0001644.g002Figure 2hESC-derived hNSCs spontaneously differentiated into the 3 principal central nervous system cell types.Dissociated hNSCs were washed free of growth factors and plated on poly-L-onithine coated glass coverslips. Differentiated cultures were either harvested after 2 DIV for total RNA extraction and RT-PCR analysis or fixed after 10 DIV and processed for indirect immunocytochemistry. (A) Differentiated hNSCs expressed the neural-specific transcripts nestin and Notch1 as well as transcripts: for neurons (β-tubulin class III, MAP-2, NCAM and medium-size neurofilament, NF-M), for astrocytes (GFAP) and for oligodendrocytes (MBP). The hNSCs expressed transcripts for GAD, but not for TH. B, C & D, morphology of NSC-derived progeny differentiated into GFAP+ astrocytes (B), GC-expressing oligodendrocytes (C) and TuJ1+ neurons (D), DAPI (blue) show life cell nuclei. (E) Photo showing cultures double-immunostained for TuJ1 (green) and nestin (red) (DAPI, blue). (F) Quantitative analysis of immunostained 10 day-old cultures for the 3 neural cell types. Results are mean±s.e.m. of three independent experiments, each performed in duplicate. Bars: (c) 20 µm; (d, e) 10 µm. 2. The isolated hNSCs are normal and do not form tumors in normal nude ratsThe self-renewal and pluripotent abilities of the hESCs are also associated with tumorigenic properties. Therefore, the first critical step toward developing therapeutic hNSCs is to verify that they are non-tumorigenic. The monolayer culture of SD56 hNSCs clearly demonstrated contact inhibition of growth, a normal karyotype and did not express the pluripotency transcripts Oct-4 and Nanog. Removal of mitogenic factors and continued culture on plastic resulted in cell senescence that is characteristic of non-transformed cells. To determine whether SD56 hNSCs form tumors in vivo, we transplanted them at high density (see Methods) into the forebrain and subcutaneously into the flank of nude rats. The animals were kept for a two-month post-transplant survival period. To label mitotically active cells in vivo during S-phase, the rats were injected IP with BrdU (50 mg/kg) every 8 hours during the last 24 hours before euthanasia. The transit amplifying endogenous precursors located in the subventricular zone (SVZ) were labeled (Figure S3); however, we were unable to detect grafted SD56 hNSCs co-localizing the human-specific nuclear marker hNuc and BrdU (Figure S3). No surviving SD56 hNSCs were detected in the flank of the transplanted animals suggesting that the grafted cells are not tumorigenic. 3. Transplanted cells survived, migrated toward and engrafted into the stroke-damaged host tissueTo investigate the survival and functional engraftment in an injury environment, hNSCs (4×105) were transplanted into the ischemic boundary zone in the rat striatum one week after the middle cerebral artery occlusion (MCAO) was performed. Animals were euthanized two months later and the brains processed for histo-pathology and immunocytochemistry. Grafted SD56 hNSCs, identified with hNuc, demonstrated a 37.0±15.8% survival rate and a remarkable dispersion toward the stroke-damaged tissue with no sign of overgrowth or tumorigenesis. The majority of grafted cells (61.2±4.7%) migrated at least 200 µm away from the injection site and penetrated an average distance of 806.4±49.3 µm into the stroke-damaged tissue (Figure 3A–C). Immunostaining with the blood vessel marker, GluT1, revealed dilated vessels in the infarcted striatum and a close association between vessels and the grafted hNSCs (Figure 3B, 3C). The grafted cells rarely expressed the proliferation marker Ki67 (Figure 3D), 29.8±4.4% expressed nestin (Figure 3E), 6.5±0.9% expressed doublecortin (DCX) and 60.8±8.1% were TuJ1+ (Figure 3F, G). Grafted cells rarely co-expressed the astroglial marker GFAP (Figure 3H) or differentiated into CNPase-expressing oligodendrocytes (Figure 3I). Immunostaining for GAD demonstrated that 25.1±2.3% of grafted hNSCs differentiated into GABAergic neurons while less than 2% were positive for glutamate (Figure 3J, K).10.1371/journal.pone.0001644.g003Figure 3Dispersion, engraftment and differentiation of the hNSCs in stroke-lesioned animals.(A) Schematic drawing of a frontal section through the striatum illustrating the dispersion of grafted hNSCs in the focal ischemia-lesioned parenchyma (shaded area). (B, C) Photos show frontal sections through the graft in the striatum immunostained with the human specific antibodies: anti-hNuc (green in B & C) and anti-GluT1 (red, B & C) showing blood vessels and dispersed hNSCs in the graft zone. C: higher magnification of the inset in B. (D–I) Photos taken from frontal sections through the graft in the striatum double immunoprocessed for cell proliferation and neural lineage markers. (D) Note the endogenous Ki67+ cells (red cells, arrow) in the stroke damaged area and the hNuc+ (green)/Ki67- grafted hNSCs (arrowheads). (E) Examples of grafted SD56 hNSCs showing co-expression of hNuc (green) and nestin (red). (F) Confocal 3D reconstructed orthogonal images of the hNuc+(green)/DCX+(red) NSCs (arrowheads) viewed in the x-z plan on the top and y-z plan on the right. (G) Examples show the majority of grafted NSC progeny co-expressing hNuc (red) and the neuronal marker TuJ1 (green). Grafted NSCs rarely differentiate into GFAP+ astrocytes (H). In I, rare example of grafted NSC progeny becoming an oligodendrocyte identified by the expression of CNPase (green). Grafted NSCs expressed the GABAergic marker GAD65/67 (J) and rarely expressed glutamate (K). (Abbreviations: Cx: cortex, Str: striatum). Bars: (B, C) 100 µm; (D, F) 20 µm; (E, G–K) 10 µm. 4. Transplanted cells improve sensorimotor function of the stroke-disabled forelimbWe asked whether transplanted SD56 hNSCs could enhance the recovery of sensorimotor function that is compromised in the stroke-injured rats. We used the cylinder test to measure the sensorimotor asymmetry in forelimb use during spontaneous exploration [22]. To establish the baseline of the stroke-induced sensorimotor deficit, spontaneous behavior of rats in a transparent cylinder was videotaped one week after stroke (pre-transplant, Figure 4). Tests were then conducted 4 and 8 weeks after vehicle and SD56 hNSCs transplantation. Stable asymmetry in forelimb use was observed 7 days post-stroke (pre-transplant, Figure 4). Ischemic rats used their impaired forelimbs (contralateral to lesion) during lateral exploration less than they did before stroke. Transplantation of SD56 hNSCs significantly enhanced the independent use of the impaired contralateral forelimb 4 weeks post transplantation (P<0.05 vs pre-transplant). Eight weeks after transplantation the improvement in the use of the impaired forelimb was stable and significant when compared to the pre-transplant group and significantly improved in comparison to vehicle treated group at 8 weeks (Figure 4). In the vehicle treated group, the independent use of the contralateral forelimb remained impaired 4 and 8 weeks post-injection. In an independent study and using the same MCAO rat animal model, we found that transplantation of dermal fibroblasts did not improve the stroke-induced motor deficits (unpublished data).10.1371/journal.pone.0001644.g004Figure 4Transplantation of NSCs improves sensorimotor function of the stroke-disabled forelimb.Forelimb use during spontaneous lateral exploration was measured in the cylinder test (see Method and Results sections for details). Groups of vehicle injected (n = 7) and hNSCs (n = 10) transplanted are represented. The animals were tested as described in Method section. Note the significant increase in the independent use of the impaired contralateral forelimb at 4 and 8 weeks post transplantation (P<0.05 vs pre-transplant group). The contralateral forelimb remained impaired in the vehicle treated group at 4 and 8 weeks post-injection. Bars represent percentages±s.e.m. of steps taken by the ipsilateral, contralateral and both forelimbs simultaneously. *P<0.05 vs pre-transplant group; #P<0.05 vs vehicle groups. Our results indicate that a self-renewable and homogenous population of hNSCs, SD56, was derived from hESCs using defined media supplemented with a specific combination of growth factors. The SD56 hNSCs grew as an adherent monolayer culture, uniformly expressed molecular features of hNSCs including nestin, vimentin and the radial glial marker 3CB2, and did not express the pluripotency markers Oct4 or Nanog. The self-renewal property of the hNSCs was characterized by a predominant symmetrical mode of cell division. They exhibited no chromosomal abnormalities and demonstrated non-tumorigenic properties after implantation into ischemic brains and into naïve nude rat brains and flanks. Furthermore, the transplanted SD56 hNSCs migrated toward the stroke-damaged adult brain parenchyma, engrafted and improved the independent use of the stroke-impaired forelimb. Maintenance of stem cells requires symmetrical and asymmetrical cell divisions to both expand and to give rise to specialized progeny of a specific tissue (reviewed in [23]). In vivo, a complex cellular micro-environment or niche ensures the self-maintenance property of NSCs [24], [25], [26], [27]. In vitro, defined growth factors and extracellular matrices support stem cell self-renewal [28], [29]. The embryonic stem cells can propagate in a predominantly proliferative symmetrical mode, leading to homogeneous cell cultures growing relatively quickly with minimal cell differentiation [30], [31], [32], [33], [34]. Tissue specific stem cells, however, self-renew in a predominant asymmetric mode to maintain them selves and compensate for the loss of differentiated cells due to disease or injury. Thus, NSCs isolated from developing or adult brain grow as a mixture of undifferentiated and differentiated cells due the predominant asymmetrical mode of cell division [35], [36], [37], [38], [39], [40], [41]. A recent study has reported that a murine ESC-derived NSC line (LC1) is propagated as an adherent homogenous culture with a dominant mode of symmetrical self-renewal [21]. A combination of EGF and FGF2 was sufficient to propagate these NSCs as an adherent monolayer. However, the SD56 hNSC line described here required the combination of EGF, bFGF and LIF for self-maintenance. Although there are morphological and molecular similarities between our hNSCs and the NSCs previously described [21], the methods of isolation and growth are different. In addition to the different combination of growth factors used, the hNSC line we have isolated did not go through the rosette-structure stage. The in vitro analysis of BrdU incorporation and nestin expression indicated that our hNSCs divide predominantly symmetrically. This type of growth pattern is characteristic of primitive normal stem cells undergoing mostly symmetric cell division to increase the stem cell pool at the early stage of development or during tissue regeneration after injury [23]. RT-PCR and immunocytochemistry analysis demonstrated that these undifferentiated SD56 cells did not express any pluripotency, endodermal or mesodermal markers. Furthermore, the SD56 hNSCs described here exhibited the multipotential characteristic to differentiate into neurons, astrocytes and oligodendrocytes both in vitro and upon transplantation. Together these findings suggest that the hNSC line we isolated are appropriately programmed and share similar characteristics with the definitive NSCs of the developing brain. The SD56 hNSCs demonstrated a remarkable ability to migrate toward the stroke-damaged parenchyma of the adult rat brain. This directed migration by the majority of the grafted cells could be due to their uniform cellular composition, which results in an equal response to the host microenvironment. In previous studies, subpopulations of transplanted hESCs that were enriched in neural cells migrated in host microenvironments conducive to cell migration, such as the developing brain or in structures such as the rostral migratory stream [13], [20]. In the adult lesioned brain, the grafted hESC-derived neural cells proliferated and formed rosettes [14], teratomas [12], [15] or a cellular mass that induced a gliotic host response whereby local astrocytes demarcated the grafts [16]. Enriched neural cultures derived from mouse [42] and monkey ESCs [43] have produced behavioral improvements when transplanted into animal models of stroke and brain injury. However, in these cases, the transplanted non-human ESCs also formed a mass with signs of overgrowth in the core, as well as deformations [44], [45], [46]. ESCs plated at low density acquire neural identity within few hours after plating [47]. Interestingly, nearly all viable cells expressed nestin, the early neural fate marker Sox1, and the pluripotency marker Oct4. Together, these studies are seminal and suggest that complete neuralization may not be achieved through certain enrichment processes, consequently the neural cells could revert to a pluripotent stage [17]. The dispersion of the grafted hNSCs within host parenchyma may allow for more graft-host interactions that could stabilize differentiation, inhibit growth and prevent gliotic host response. In the present study, SD56 hNSC-transplanted animals demonstrated a stable improvement in the sensorimotor function when evaluated for spontaneous exploratory activity in the cylinder test that detects long-lasting deficits in forelimb use in the experimental models of stroke [22]. The transplantation of hNSCs significantly enhanced the independent use of the impaired contralateral forelimb 8 weeks post transplantation. This is the first report demonstrating that the transplantation of hNSCs derived from hESCs can improve neurologic behavior after experimental stroke. Together, these findings are encouraging and suggest that these cells are promising for future development. In addition to their therapeutic application, the hNSCs isolated under the reported conditions offer a means to interrogate host environments and unravel mechanistic features of self-renewal, non-tumorigenicity and functional engraftability in animal models of neurological disorders. hESC and NSC CulturesThe hESC line H9 (WiCell Research Institute) was propagated every 5 to 7 days on irradiated mouse embryonic fibroblasts. The cell culture media consisted of a 1∶1 mixture of Dulbecco's modified Eagle's medium (DMEM) and F12 nutrient, 20% serum replacement (Invitrogen), 0.1 mM β-mercaptoethanol, 2 µg/ml heparin and 4 ng/ml bFGF (R&D Systems). To generate the NSCs, dissociated hESCs were cultured in a chemically defined medium composed of DMEM/F12 (1∶1) including glucose (0.6%), glutamine (2 mM), sodium bicarbonate (3 mM), and HEPES buffer (5 mM) [all from Sigma except glutamine (Invitrogen)]. A defined hormone mix and salt mixture (Sigma), including insulin (25 mg/ml), transferrin (100 mg/ml), progesterone (20 nM), putrescine (60 mM), and selenium chloride (30 nM) was used in place of serum. The medium was supplemented with EGF (20 ng/ml), bFGF (10 ng/ml) and LIF (10 ng/ml). Dissociated hNSCs were plated at a density of 100,000 cell/ml in Corning T75 (Invitrogen) culture flasks in the defined media together with the growth factors. After 5–7 DIV, the adherent culture was incubated in 0.025%trypsin/0.01% EDTA (w/v) for 1 min followed by the addition of trypsin inhibitor (Invitrogen) then gently triturated to achieve single cell suspension. The cells were then washed twice with fresh medium and reseeded in fresh growth factor-containing media at 100,000 cells/ml. This procedure was performed for 21 passages and the fold of increase and population doubling were calculated at each passage. For clonal analysis, single spheres or confluent hNSC cultures were single cell dissociated and serially diluted to yield 1–2 cell/10 µl. A 10-µl-cell suspension was then added to each of 96 or 24 well plates containing 200–300 µl of growth media. Wells containing one viable cell were marked the next day and re-scored 5 to 7 days later for cell proliferation. The differentiation of the hNSCs was performed as previously described [48]. Dissociated hNSCs were plated at a density of 106 cells/ml in control (media/hormone mix) medium devoid of any growth factors and supplemented with 1% fetal bovine serum (FBS) on poly-L-ornithine-coated (15 mg/ml; Sigma) glass coverslips in 24-well Nunclon culture dishes with 0.5 ml/well. After 2, 7–15 DIV cultures were fixed and processed for immunocytochemistry or used for RT-PCR analysis. Karyotype analysisLong-term cultures of hNSCs were incubated at 37°C and harvested for metaphase chromosomes when the cultures were 75% confluent. Metaphase chromosomes were obtained by standard chromosome harvest methods by exposure to Colcemid at 0.1 µg/ml for 1 hour at 37°C, a 2-minute exposure to trypsin/EDTA, hypotonized with 0.057 M KCl and fixed with 3∶1 methanol:acetic acid. Slide preparations were made by dropping the fixed cell pellet onto cold, wet slides and air-dried. After incubating the slides at 90°C for 30 minutes, chromosomes were trypsin banded and then Wright/Giemsa stained for G-banding analysis. Twenty metaphase cells were completely analyzed and a normal female chromosome complement was found (46,XX). Tumorigenicity in nude ratsAll animal experiments were conducted according to the National Institute of Health (NIH) guidelines and approved by the IACUC. Normal adult NIH-Nude rats (n = 5, 8 week-old, Taconic, Germantown, New York, United States) were used to test the tumorigenic potential of the SD56 hNSCs. Undifferentiated hNSCs from passage 9 were single cell dissociated using trypsin-EDTA and suspended at the concentration of 125,000 cell/µl in preparation for cell transplantation. Two µl of the cell suspension were stereotaxically transplanted into 4 sites within the striatum at the following coordinates: AP: +1.0 mm, ML: +3.2 mm, DV: −5.0; AP: +0.5 mm, ML: +3.0 mm, DV: −5.0; AP: −0.5 mm, ML: +3.0 mm, DV: −5.0; AP: −1.0 mm, ML: +3.5 mm, DV: −5.0 mm with the incisor bar set at 3.4 mm. The injection rate was 1 µl/min, and the cannula was left in place for an additional 5 min before retraction. For the flank tumor assay, 2×106 cells (125,000 cell/µl) were injected subcutaneously to the side of the adult nude rats. To label mitotically active cells in vivo during S-phase, the rats were injected IP with the BrdU (50 mg/kg, Sigma) every 8 hours during the last 24 hours before euthanasia. After 2-month survival time, rats were euthanized and a postmortem examination for tumor formation was performed. Induction of Focal Ischemia and Cell TransplantationAll animal experimentations were conducted according to the National Institute of Health (NIH) guidelines and approved by the IACUC. Sprague Dawley adult male rats (n = 17, 275g–310g, Charles River Laboratories, Wilmington, Massachusetts, United States) were subjected to one and a half hour suture occlusion of the middle cerebral artery (MCAO), as previously described [49] and immunosuppressed 2 days before cell transplantation and daily thereafter for one week with i.p. injections of cyclosporine A (20 mg/ml, Sandimmune, Novartis Pharmaceuticals). Thereafter oral cyclosporine was used at 210 µg/ml in drinking water until euthanasia. Undifferentiated SD56 hNSCs from passages between P9 and P13 were single cell dissociated using trypsin-EDTA in preparation for cell transplantation. One week after the stroke lesion, 2 µl of the hNSCs, at a concentration of 50,000 cell/µl, were stereotaxically transplanted into 4 sites within the lesioned striatum (n = 10) at the following coordinates: AP: +1.0 mm, ML: +3.2 mm, DV: −5.0; AP: +0.5 mm, ML: +3.0 mm, DV: −5.0; AP: −0.5 mm, ML: +3.0 mm, DV: −5.0; AP: −1.0 mm, ML: +3.5 mm, DV: −5.0 mm with the incisor bar set at 3.4 mm. The injection rate was 1 µl/min, and the cannula was left in place for an additional 5 min before retraction. As a control group, we used rats subjected to ischemia and injected with the vehicle (n = 7). All animals underwent baseline motor behavioral assessment before and after the ischemic lesion, and 4 & 8 weeks after cell transplantation. The animals were killed after 2-month survival time by transcardial perfusion with phosphate buffered saline (PBS) followed by 4% paraformaldehyde. The brains were cryoprotected in an increasing gradient of 10, 20 and 30% sucrose solution and cryostat sectioned at 40 µm and processed for immunocytochemistry. ImmunocytochemistryCultures were fixed with 4% paraformaldehyde for 15 min. Both cells and brain sections were rinsed in PBS for 3×5 min then incubated for 2 hrs (cultures) or overnight (brain sections) with the appropriate primary antibodies for multiple labeling. Secondary antibodies raised in the appropriate hosts and conjugated to FITC, RITC, AMCA, CY3 or CY5 chromogenes (Jackson ImmunoResearch) were used. Cells and sections were counterstained with the nuclear marker 4′,6-diamidine-2′-phenylindole dihydrochloride (DAPI). Positive and negative controls were included in each run. Immunostained sections were coverslipped using fluorsave (Calbiochem) as the mounting medium. The following antibodies were used: Anti-human Nuclei (hNuc, monoclonal 1∶100, Chemicon), Anti-TuJ1 (monoclonal 1∶100, Covance; Polyclonal 1∶200, Aves Labs); anti-GAD65/67 (polyclonal 1∶1000, Chemicon); Anti-glial fibrillary acidic protein (GFAP, monoclonal 1∶1000, Chemicon; polyclonal 1∶200, Aves Labs); Anti-galactocerebrocide (GC, monoclonal 1∶250, Chemicon); Anti-CNPase (polyclonal 1∶200, Aves Labs); Anti-Glucose Transporter type 1 (Glut-1 polyclonal, 1∶500, Chemicon); Anti-Nestin (polyclonal 1∶1000, Chemicon); Anti-vimentin (monoclonal 1∶500, Calbiochem); Anti-3CB2 (monoclonal 1∶500, Developmental Studies Hybridoma Bank); Anti-doublecortin (DCX, polyclonal 1∶250, SantaCruz Biotechnology); Anti-Ki67 (polyclonal 1∶250, SantaCruz Biotechnology). Fluorescence was detected, analyzed and photographed with a Zeiss LSM550 laser scanning confocal photomicroscope. For each animal, quantitative estimates of the total number of grafted cells were stereologically determined using the optical fractionator procedure [50]. A computer-assisted image analysis system was performed using Stereo Investigator software (MicroBrightField, Inc.). The rostral and caudal limits of the reference volume were determined by first and last frontal sections containing grafted cells. The striatum and cortex were accurately outlined at low magnification (2.5× objective). The optical fractionator probe was selected to perform systematic sampling of the immunoreactive cell population distributed within the serial sections to estimate the population number in the volume of tissue. The counting frame of the optical fractionator was defined at 50×50 µm squares and the systematic sampling was performed by translating a grid with 200×200 µm squares onto the sections of interest using the Stereo Investigator software. The sample sites were systematically and automatically generated by the computer and examined using a 60× objective of a Nikon Eclipse TE 300 microscope. The counting frame displayed inclusion and exclusion lines and only immunoreactive cell bodies falling within the counting frame with no contact with the exclusion lines were counted. The cell dispersion was measured by counting the number of cells within 200 µm distance from the graft site. The number and distance in µm of cells dispersed beyond 200 µm was also measured. An average of 2,000 cells was counted per animal. Double labeling was determined using the confocal laser scanning microscope by random sampling of 100 or more cells per marker for each animal, scoring first for hNuc+, followed by DAPI+ nuclei and then the marker of choice. The double labeling was always confirmed in x-z and y-z cross-sections produced by the orthogonal projections of z-series. Reverse Transcription-Polymerase Chain Reaction (RT-PCR) analysisTotal RNA was extracted from cultured cells using RNAeasy kit (Quiagen). Aliquots (1 µg) of total RNA from the cells were reverse transcribed in the presence of 50 mM Tris-HCl, pH 8.3, 75 mM KCl, 3 mM MgCl2, 10 mM DTT, 0.5 µM dNTPs, and 0.5 µg oligo-dT(12–18) with 200 U Superscript RNase H-Reverse Transcriptase (Invitrogen). PCR amplification was performed using standard procedure with Taq Polymerase. Aliquots of cDNA equivalent to 50 ng of total RNA were amplified in 25 µl reactions containing 10 mM Tris-HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl2 , 50 pmol of each primer, 400 µM dNTPs, and 0.5 U AmpliTaq DNA polymerase (Perkin-Elmer). PCR was performed using the following thermal profile: 4 min at 94°C; 1 min at 94°C, 1 min at 60°C, 1.5 min at 72°C, for 30–40 cycles; 7 min at 72°C, and finally a soak at 4°C overnight. The following day, 10 µl aliquots of the amplified products were run on a 2% agarose Tris–acetate gel containing 0.5 mg/ml ethidium bromide. The products were visualized through a UV transilluminator, captured in a digital format using Quantify One Gel Analysis software (Bio-Rad Laboratories) on a Macintosh G4 computer.The PCR primers specific to each transcript were as follows: GFAP, forward (F), 5′-TCATCGCTCAGGAGGTCCTT–3′ Reverse (R), 5′-CTG TTGCCAGAGATGGAGGTT–3′; MAP2 (F) 5′-GAAGACTCGCATCCGAATGG–3′, (R) 5′-CGCAGGATAGGAGGAAGAGACT–3′; MBP (F) 5′-TTAGCTGAATTC GCGTGTGG–3′, (R) 5′-GAGGAAGTGAATGAGCCGGTTA-3′ were deigned using the Primer Designer software, Version 2.0 (Scientific and Educational Software) [48]. 18S, β-tubulin class III, N-CAM, Nestin, NF-M, Notch-1 primers [51]. Oct4, Nanog primers [11]. FOXa2 (HNF3B), Brachyury primers [52]. Behavioral testsThe cylinder test was used to assess the spontaneous forelimb use during lateral exploration movement [22]. Rats were placed in a transparent acrylic cylinder (20 cm diameter) for 5 minutes. The cylinder encourages use of the forelimbs for vertical exploration. A mirror was placed behind the cylinder so that the forelimbs could be viewed at all times. Testing sessions were videotaped and forelimb use was scored by a blinded operator. Movements scored were the independent use of the left or right forelimb or simultaneous use of both the left and right forelimb to contact the wall of the cylinder during a full rear, to initiate a weight-shifting movement, or to regain center of gravity while moving laterally in a vertical posture along the wall. Animals were tested for their baselines after stroke and 4 and 8 weeks after cell transplantation. Statistical analysisOutcome measurement for each experiment was reported as mean±SEM. All data were analyzed using SPSS 11 for Mac OS X (SPSS Inc.). Significance of inter-group differences was performed by applying Student's t-test where appropriate. The One-Way ANOVA analysis was used to compare group differences for the forelimb use as the dependant variable and groups as the single independent factor variable. Differences between the groups were determined using Bonferroni's post hoc test. A P-value of less than 0.05 was considered to be statistically significant. Instructions: please extract entities and their types from the input sentence, all entity types are in options Options: GeneProtein, Species, Anatomy, CellLine, CellComponent, CellType
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BackgroundHuman embryonic stem cells (hESCs) offer a virtually unlimited source of neural cells for structural repair in neurological disorders, such as stroke. Neural cells can be derived from hESCs either by direct enrichment, or by isolating specific growth factor-responsive and expandable populations of human neural stem cells (hNSCs). Studies have indicated that the direct enrichment method generates a heterogeneous population of cells that may contain residual undifferentiated stem cells that could lead to tumor formation in vivo.Methods/Principal FindingsWe isolated an expandable and homogenous population of hNSCs (named SD56) from hESCs using a defined media supplemented with epidermal growth factor (EGF), basic fibroblast growth factor (bFGF) and leukemia inhibitory growth factor (LIF). These hNSCs grew as an adherent monolayer culture. They were fully neuralized and uniformly expressed molecular features of NSCs, including nestin, vimentin and radial glial markers. These hNSCs did not express the pluripotency markers Oct4 or Nanog, nor did they express markers for the mesoderm or endoderm lineages. The self-renewal property of the hNSCs was characterized by a predominant symmetrical mode of cell division. The SD56 hNSCs differentiated into neurons, astrocytes and oligodendrocytes throughout multiple passages in vitro, as well as after transplantation. Together, these criteria confirm the definitive NSC identity of the SD56 cell line. Importantly, they exhibited no chromosome abnormalities and did not form tumors after implantation into rat ischemic brains and into naïve nude rat brains and flanks. Furthermore, hNSCs isolated under these conditions migrated toward the ischemia-injured adult brain parenchyma and improved the independent use of the stroke-impaired forelimb two months post-transplantation.Conclusions/SignificanceThe SD56 human neural stem cells derived under the reported conditions are stable, do not form tumors in vivo and enable functional recovery after stroke. These properties indicate that this hNSC line may offer a renewable, homogenous source of neural cells that will be valuable for basic and translational research. To date there have been no effective treatments for improving residual structural and functional deficits resulting from stroke. Current therapeutic approaches, such as the use of thrombolytics, benefit only 1 to 4% of patients [1]. Consequently, the majority of stroke patients experience progression of ischemia associated with debilitating neurological deficits. Recent evidence has suggested that the transplantation of cells derived from cord blood, bone marrow or brain tissue (fetal and adult) enhances sensorimotor function in experimental models of stroke [2], [3]. However, the normal human-derived somatic stem cells used in these studies have a limited capacity to differentiate into the diverse neural cell types optimal for structural and physiological tissue repair and are not amenable for large-scale cell production. Unlike other sources of stem cells, hESC lines possess a nearly unlimited self-renewal capacity and the developmental potential to differentiate into virtually any cell type of the organism. As such, they constitute an ideal source of cells for regenerative medicine. The successful derivation of hESC lines from the inner cell mass of preimplantation embryos and their long-term maintenance in vitro over multiple passages has been demonstrated [4] and standardized. Differentiation and enrichment processes that direct hESCs towards a neural lineage have also been achieved. To promote neuralization, ESCs were cultured in a defined media supplemented with morphogens or growth factors [5], [6], [7] or cultured under conditions that promote “rosettes”, structures morphologically similar to the developing neural tube [8], [9]. This neuralization process has proven invaluable in understanding the specification of hESC-derived neural tissue [10], [11], [12]. However, the enriched neural progeny derived displayed overgrowth and limited migration after grafting into normal newborn mice [13] and lesioned adult rat striatum [12], [14], [15], [16]. The inner cores of these grafts contained tumorigenic precursor cells (reviewed in [17]). These findings suggest that neural cells generated by acute exposure to growth factors and/or morphogens may still be heterogeneous and potentially tumorigenic. We report an alternative method for the isolation and the perpetuation of a multipotent hNSC line from the hESCs with a primitive mode of self-renewal. We also demonstrate their long-term expansion, non-tumorigenic properties and functional engraftability in an experimental model of stroke. 1. In vitro isolation, growth and differentiation of hESC-derived hNSCsThe hESCs were maintained and expanded on mouse feeder layer in media supplemented with bFGF (Figure 1A). After cell dissociation, a portion of the hESCs was cultured in serum free medium containing EGF, bFGF and LIF. These factors are known to stimulate the proliferation of human fetal-derived NSCs [18], [19]. After 3 days in vitro (DIV), there was selective survival and growth of cells that aggregated in clusters or spheres (Figure 1B). These primary spheres were harvested and replated in fresh media. During the following week, the spheres attached to the flask and a fibroblast-like cell population began to migrate out (Figure 1C). Secondary spheres (2° spheres) were generated from these cultures and lifted off by the end of the week leaving a hollow in the middle of the attached cells (Figure 1D). The floating 2° spheres were collected and replated in fresh growth medium for 2 weeks. The cultures were then passaged by collagenase cell dissociation every 7 DIV for an additional 4 passages (Figure S1). At the 5th and 6th passages, spheres were dissociated into a single-cell suspension using trypsin-EDTA. At this stage there was a change in the hNSCs' adherent properties, and the cells began to grow as a monolayer with multiple foci of cells throughout the culture (Figure 1F). The adherent hNSC culture stained uniformly for nestin (Figure 1K), vimentin (Figure 1L) and with the radial glial marker 3CB2 (Figure 1M) indicating their homogeneity and NSC identity. Under these culture conditions, it is noteworthy that we did not observe the formation of rosettes which has been previously reported to occur under certain conditions during neuralization of hESCs [8], [20], [21]. RT-PCR analysis confirmed that these hNSCs did not express the pluripotency transcripts Oct-4 and Nanog (Figure 1I). Furthermore, the hNSCs did not express transcripts for brachyury and foxa2, marker genes for mesoderm and endoderm respectively (negative result, data not shown).10.1371/journal.pone.0001644.g001Figure 1Isolation and purification of hNSCs from the hESCs.The hESCs were grown on a mouse feeder layer (A). Primary neurospheres (B) were isolated and replated to eliminate other non-neural cells. The selectively harvested secondary neurospheres (arrow in C), left behind hollow cores in the surface area (star in D) where they attached earlier. They were perpetuated for an additional 5 passages (E). These 2° spheres were then passaged using a single cell dissociation protocol (F). Arrow in F shows an example of a focus of proliferating cells. (G, H) The hNSCs were passaged every 5–7 days, as described in the Methods section. Starting from an initial population of 1 million cells, the cumulative cell number was calculated at each passage as the fold of increase×the total cell number and plotted as logarithm with base 2 in function of time (G). The cell perpetuation (G) and population doubling (H) analysis demonstrated the continuous and stable growth of the hNSCs. (I) RT-PCR analysis showing the down regulation of the pluripotency transcripts Oct4 and Nanog in secondary neurospheres and in expanded hNSCs at passage 8 (P8). (J) Cytogenetic evaluation of the SD56 hNSCs line at passage 12 by standard G-banding was performed. Twenty metaphase cells were analyzed and showed a normal female chromosome complement (46,XX). Isolated and expanded hNSCs expressed the neural precursor cell markers nestin (K), Vimentin (L) and the radial glial cell marker 3CB2 (M) in virtually all the progeny. (N-P) Clonal self-renewal ability of the isolated hNSCs through symmetrical cell division. Single (N), two-cell stage (O) and four-cell stage (P) of a hNSC proliferating over a 3-day culture period. Note the symmetrical segregation of BrdU and nestin in the progeny. Bars: (A, B, C, D) 200 µm; (E, F) 100 µm; (K–M) 20 µm; (N–P) 10 µm.To ascertain self-renewal ability under clonal conditions, a single cell suspension was plated at clonal density (1–2 cell/10 µl). To determine if the hNSCs divide symmetrically, we pulsed cultures with the thymidine analog, bromodeoxyuridine (BrdU), after plating and looked for the expression of nestin in the progeny. Cultures were fixed after 1, 2 or 3 DIV (Figure 1N–P). After 2 days, plated single cells first underwent a symmetric cell division and gave rise to daughter cells that were both positive for BrdU and nestin. The clone of cells continued to grow over the 3 DIV and all the progeny expressed nestin. BrdU labeling demonstrated that it was rare for only one daughter cell to inherit the BrdU and thus had undergone asymmetric segregation of the chromatids (Figure S2). G-band karyotyping of these hNSCs confirmed the normal, non-transformed nature of cells after 12 passages (Figure 1J). We named the derived hNSCs SD56 (intermittently referred to as SD56 hNSCs or hNSCs).Under these defined growth conditions, the hNSCs showed stable growth with a 2.7±0.2 fold increase every 5 to 7 days (Figure 1G). The population doubling at each passage averaged at 1.4±0.1 (Figure 1H). The viability of hNSCs at each passage was consistent at the approximate value of 98%. The projected cumulative cell numbers demonstrated that trillions of cells could be generated over a period of 5 months (Figure 1G). We expanded the isolated hNSCs lines for over 20 passages with a stable phenotype. An initial cell bank of 75 vials containing 2 to 5 million cells each was generated and cryopreserved.Upon removal of the mitogenic factors and plating on a coverslip pre-coated with poly-L-ornithine (PLO) substrate, the hNSCs spontaneously differentiated into neurons, astrocytes and oligodendrocytes, a property that is consistent with normal multipotent hNSCs (Figure 2). After 2 DIV, hNSCs expressed transcripts for the neural-specific genes nestin, Notch1 and neural cell adhesion molecule (N-CAM) (Figure 2A) and for the lineage specific markers β-tubulin class III, medium-size neurofilament (NF-M) and microtubule-associated protein 2 (MAP-2) for neurons, GFAP for astrocytes and myelin basic protein (MBP) for oligodendrocytes (Figure 2A). Transcripts for the GABAergic cell marker glutamic acid decarboxylase (GAD) were expressed, but transcripts for the tyrosine hydroxylase (TH), a marker for dopaminergic neurons, were undetectable. Immunocytochemical analysis (Figure 2B–F) of 10 day-old cultures demonstrated that the proportion of nestin+ cells was 36.6±2.7%, 62.5±2.8% expressed the neuronal marker TuJ1, 1.9±0.3% expressed the astrocytic marker GFAP and 7.1±0.4% were oligodendrocytes and expressed galactocerebrocide (GC) (Figure 2F). A subset (9.8±1.6%) of the GFAP+ astrocytes co-expressed nestin.10.1371/journal.pone.0001644.g002Figure 2hESC-derived hNSCs spontaneously differentiated into the 3 principal central nervous system cell types.Dissociated hNSCs were washed free of growth factors and plated on poly-L-onithine coated glass coverslips. Differentiated cultures were either harvested after 2 DIV for total RNA extraction and RT-PCR analysis or fixed after 10 DIV and processed for indirect immunocytochemistry. (A) Differentiated hNSCs expressed the neural-specific transcripts nestin and Notch1 as well as transcripts: for neurons (β-tubulin class III, MAP-2, NCAM and medium-size neurofilament, NF-M), for astrocytes (GFAP) and for oligodendrocytes (MBP). The hNSCs expressed transcripts for GAD, but not for TH. B, C & D, morphology of NSC-derived progeny differentiated into GFAP+ astrocytes (B), GC-expressing oligodendrocytes (C) and TuJ1+ neurons (D), DAPI (blue) show life cell nuclei. (E) Photo showing cultures double-immunostained for TuJ1 (green) and nestin (red) (DAPI, blue). (F) Quantitative analysis of immunostained 10 day-old cultures for the 3 neural cell types. Results are mean±s.e.m. of three independent experiments, each performed in duplicate. Bars: (c) 20 µm; (d, e) 10 µm. 2. The isolated hNSCs are normal and do not form tumors in normal nude ratsThe self-renewal and pluripotent abilities of the hESCs are also associated with tumorigenic properties. Therefore, the first critical step toward developing therapeutic hNSCs is to verify that they are non-tumorigenic. The monolayer culture of SD56 hNSCs clearly demonstrated contact inhibition of growth, a normal karyotype and did not express the pluripotency transcripts Oct-4 and Nanog. Removal of mitogenic factors and continued culture on plastic resulted in cell senescence that is characteristic of non-transformed cells. To determine whether SD56 hNSCs form tumors in vivo, we transplanted them at high density (see Methods) into the forebrain and subcutaneously into the flank of nude rats. The animals were kept for a two-month post-transplant survival period. To label mitotically active cells in vivo during S-phase, the rats were injected IP with BrdU (50 mg/kg) every 8 hours during the last 24 hours before euthanasia. The transit amplifying endogenous precursors located in the subventricular zone (SVZ) were labeled (Figure S3); however, we were unable to detect grafted SD56 hNSCs co-localizing the human-specific nuclear marker hNuc and BrdU (Figure S3). No surviving SD56 hNSCs were detected in the flank of the transplanted animals suggesting that the grafted cells are not tumorigenic. 3. Transplanted cells survived, migrated toward and engrafted into the stroke-damaged host tissueTo investigate the survival and functional engraftment in an injury environment, hNSCs (4×105) were transplanted into the ischemic boundary zone in the rat striatum one week after the middle cerebral artery occlusion (MCAO) was performed. Animals were euthanized two months later and the brains processed for histo-pathology and immunocytochemistry. Grafted SD56 hNSCs, identified with hNuc, demonstrated a 37.0±15.8% survival rate and a remarkable dispersion toward the stroke-damaged tissue with no sign of overgrowth or tumorigenesis. The majority of grafted cells (61.2±4.7%) migrated at least 200 µm away from the injection site and penetrated an average distance of 806.4±49.3 µm into the stroke-damaged tissue (Figure 3A–C). Immunostaining with the blood vessel marker, GluT1, revealed dilated vessels in the infarcted striatum and a close association between vessels and the grafted hNSCs (Figure 3B, 3C). The grafted cells rarely expressed the proliferation marker Ki67 (Figure 3D), 29.8±4.4% expressed nestin (Figure 3E), 6.5±0.9% expressed doublecortin (DCX) and 60.8±8.1% were TuJ1+ (Figure 3F, G). Grafted cells rarely co-expressed the astroglial marker GFAP (Figure 3H) or differentiated into CNPase-expressing oligodendrocytes (Figure 3I). Immunostaining for GAD demonstrated that 25.1±2.3% of grafted hNSCs differentiated into GABAergic neurons while less than 2% were positive for glutamate (Figure 3J, K).10.1371/journal.pone.0001644.g003Figure 3Dispersion, engraftment and differentiation of the hNSCs in stroke-lesioned animals.(A) Schematic drawing of a frontal section through the striatum illustrating the dispersion of grafted hNSCs in the focal ischemia-lesioned parenchyma (shaded area). (B, C) Photos show frontal sections through the graft in the striatum immunostained with the human specific antibodies: anti-hNuc (green in B & C) and anti-GluT1 (red, B & C) showing blood vessels and dispersed hNSCs in the graft zone. C: higher magnification of the inset in B. (D–I) Photos taken from frontal sections through the graft in the striatum double immunoprocessed for cell proliferation and neural lineage markers. (D) Note the endogenous Ki67+ cells (red cells, arrow) in the stroke damaged area and the hNuc+ (green)/Ki67- grafted hNSCs (arrowheads). (E) Examples of grafted SD56 hNSCs showing co-expression of hNuc (green) and nestin (red). (F) Confocal 3D reconstructed orthogonal images of the hNuc+(green)/DCX+(red) NSCs (arrowheads) viewed in the x-z plan on the top and y-z plan on the right. (G) Examples show the majority of grafted NSC progeny co-expressing hNuc (red) and the neuronal marker TuJ1 (green). Grafted NSCs rarely differentiate into GFAP+ astrocytes (H). In I, rare example of grafted NSC progeny becoming an oligodendrocyte identified by the expression of CNPase (green). Grafted NSCs expressed the GABAergic marker GAD65/67 (J) and rarely expressed glutamate (K). (Abbreviations: Cx: cortex, Str: striatum). Bars: (B, C) 100 µm; (D, F) 20 µm; (E, G–K) 10 µm. 4. Transplanted cells improve sensorimotor function of the stroke-disabled forelimbWe asked whether transplanted SD56 hNSCs could enhance the recovery of sensorimotor function that is compromised in the stroke-injured rats. We used the cylinder test to measure the sensorimotor asymmetry in forelimb use during spontaneous exploration [22]. To establish the baseline of the stroke-induced sensorimotor deficit, spontaneous behavior of rats in a transparent cylinder was videotaped one week after stroke (pre-transplant, Figure 4). Tests were then conducted 4 and 8 weeks after vehicle and SD56 hNSCs transplantation. Stable asymmetry in forelimb use was observed 7 days post-stroke (pre-transplant, Figure 4). Ischemic rats used their impaired forelimbs (contralateral to lesion) during lateral exploration less than they did before stroke. Transplantation of SD56 hNSCs significantly enhanced the independent use of the impaired contralateral forelimb 4 weeks post transplantation (P<0.05 vs pre-transplant). Eight weeks after transplantation the improvement in the use of the impaired forelimb was stable and significant when compared to the pre-transplant group and significantly improved in comparison to vehicle treated group at 8 weeks (Figure 4). In the vehicle treated group, the independent use of the contralateral forelimb remained impaired 4 and 8 weeks post-injection. In an independent study and using the same MCAO rat animal model, we found that transplantation of dermal fibroblasts did not improve the stroke-induced motor deficits (unpublished data).10.1371/journal.pone.0001644.g004Figure 4Transplantation of NSCs improves sensorimotor function of the stroke-disabled forelimb.Forelimb use during spontaneous lateral exploration was measured in the cylinder test (see Method and Results sections for details). Groups of vehicle injected (n = 7) and hNSCs (n = 10) transplanted are represented. The animals were tested as described in Method section. Note the significant increase in the independent use of the impaired contralateral forelimb at 4 and 8 weeks post transplantation (P<0.05 vs pre-transplant group). The contralateral forelimb remained impaired in the vehicle treated group at 4 and 8 weeks post-injection. Bars represent percentages±s.e.m. of steps taken by the ipsilateral, contralateral and both forelimbs simultaneously. *P<0.05 vs pre-transplant group; #P<0.05 vs vehicle groups. Our results indicate that a self-renewable and homogenous population of hNSCs, SD56, was derived from hESCs using defined media supplemented with a specific combination of growth factors. The SD56 hNSCs grew as an adherent monolayer culture, uniformly expressed molecular features of hNSCs including nestin, vimentin and the radial glial marker 3CB2, and did not express the pluripotency markers Oct4 or Nanog. The self-renewal property of the hNSCs was characterized by a predominant symmetrical mode of cell division. They exhibited no chromosomal abnormalities and demonstrated non-tumorigenic properties after implantation into ischemic brains and into naïve nude rat brains and flanks. Furthermore, the transplanted SD56 hNSCs migrated toward the stroke-damaged adult brain parenchyma, engrafted and improved the independent use of the stroke-impaired forelimb. Maintenance of stem cells requires symmetrical and asymmetrical cell divisions to both expand and to give rise to specialized progeny of a specific tissue (reviewed in [23]). In vivo, a complex cellular micro-environment or niche ensures the self-maintenance property of NSCs [24], [25], [26], [27]. In vitro, defined growth factors and extracellular matrices support stem cell self-renewal [28], [29]. The embryonic stem cells can propagate in a predominantly proliferative symmetrical mode, leading to homogeneous cell cultures growing relatively quickly with minimal cell differentiation [30], [31], [32], [33], [34]. Tissue specific stem cells, however, self-renew in a predominant asymmetric mode to maintain them selves and compensate for the loss of differentiated cells due to disease or injury. Thus, NSCs isolated from developing or adult brain grow as a mixture of undifferentiated and differentiated cells due the predominant asymmetrical mode of cell division [35], [36], [37], [38], [39], [40], [41]. A recent study has reported that a murine ESC-derived NSC line (LC1) is propagated as an adherent homogenous culture with a dominant mode of symmetrical self-renewal [21]. A combination of EGF and FGF2 was sufficient to propagate these NSCs as an adherent monolayer. However, the SD56 hNSC line described here required the combination of EGF, bFGF and LIF for self-maintenance. Although there are morphological and molecular similarities between our hNSCs and the NSCs previously described [21], the methods of isolation and growth are different. In addition to the different combination of growth factors used, the hNSC line we have isolated did not go through the rosette-structure stage. The in vitro analysis of BrdU incorporation and nestin expression indicated that our hNSCs divide predominantly symmetrically. This type of growth pattern is characteristic of primitive normal stem cells undergoing mostly symmetric cell division to increase the stem cell pool at the early stage of development or during tissue regeneration after injury [23]. RT-PCR and immunocytochemistry analysis demonstrated that these undifferentiated SD56 cells did not express any pluripotency, endodermal or mesodermal markers. Furthermore, the SD56 hNSCs described here exhibited the multipotential characteristic to differentiate into neurons, astrocytes and oligodendrocytes both in vitro and upon transplantation. Together these findings suggest that the hNSC line we isolated are appropriately programmed and share similar characteristics with the definitive NSCs of the developing brain. The SD56 hNSCs demonstrated a remarkable ability to migrate toward the stroke-damaged parenchyma of the adult rat brain. This directed migration by the majority of the grafted cells could be due to their uniform cellular composition, which results in an equal response to the host microenvironment. In previous studies, subpopulations of transplanted hESCs that were enriched in neural cells migrated in host microenvironments conducive to cell migration, such as the developing brain or in structures such as the rostral migratory stream [13], [20]. In the adult lesioned brain, the grafted hESC-derived neural cells proliferated and formed rosettes [14], teratomas [12], [15] or a cellular mass that induced a gliotic host response whereby local astrocytes demarcated the grafts [16]. Enriched neural cultures derived from mouse [42] and monkey ESCs [43] have produced behavioral improvements when transplanted into animal models of stroke and brain injury. However, in these cases, the transplanted non-human ESCs also formed a mass with signs of overgrowth in the core, as well as deformations [44], [45], [46]. ESCs plated at low density acquire neural identity within few hours after plating [47]. Interestingly, nearly all viable cells expressed nestin, the early neural fate marker Sox1, and the pluripotency marker Oct4. Together, these studies are seminal and suggest that complete neuralization may not be achieved through certain enrichment processes, consequently the neural cells could revert to a pluripotent stage [17]. The dispersion of the grafted hNSCs within host parenchyma may allow for more graft-host interactions that could stabilize differentiation, inhibit growth and prevent gliotic host response. In the present study, SD56 hNSC-transplanted animals demonstrated a stable improvement in the sensorimotor function when evaluated for spontaneous exploratory activity in the cylinder test that detects long-lasting deficits in forelimb use in the experimental models of stroke [22]. The transplantation of hNSCs significantly enhanced the independent use of the impaired contralateral forelimb 8 weeks post transplantation. This is the first report demonstrating that the transplantation of hNSCs derived from hESCs can improve neurologic behavior after experimental stroke. Together, these findings are encouraging and suggest that these cells are promising for future development. In addition to their therapeutic application, the hNSCs isolated under the reported conditions offer a means to interrogate host environments and unravel mechanistic features of self-renewal, non-tumorigenicity and functional engraftability in animal models of neurological disorders. hESC and NSC CulturesThe hESC line H9 (WiCell Research Institute) was propagated every 5 to 7 days on irradiated mouse embryonic fibroblasts. The cell culture media consisted of a 1∶1 mixture of Dulbecco's modified Eagle's medium (DMEM) and F12 nutrient, 20% serum replacement (Invitrogen), 0.1 mM β-mercaptoethanol, 2 µg/ml heparin and 4 ng/ml bFGF (R&D Systems). To generate the NSCs, dissociated hESCs were cultured in a chemically defined medium composed of DMEM/F12 (1∶1) including glucose (0.6%), glutamine (2 mM), sodium bicarbonate (3 mM), and HEPES buffer (5 mM) [all from Sigma except glutamine (Invitrogen)]. A defined hormone mix and salt mixture (Sigma), including insulin (25 mg/ml), transferrin (100 mg/ml), progesterone (20 nM), putrescine (60 mM), and selenium chloride (30 nM) was used in place of serum. The medium was supplemented with EGF (20 ng/ml), bFGF (10 ng/ml) and LIF (10 ng/ml). Dissociated hNSCs were plated at a density of 100,000 cell/ml in Corning T75 (Invitrogen) culture flasks in the defined media together with the growth factors. After 5–7 DIV, the adherent culture was incubated in 0.025%trypsin/0.01% EDTA (w/v) for 1 min followed by the addition of trypsin inhibitor (Invitrogen) then gently triturated to achieve single cell suspension. The cells were then washed twice with fresh medium and reseeded in fresh growth factor-containing media at 100,000 cells/ml. This procedure was performed for 21 passages and the fold of increase and population doubling were calculated at each passage. For clonal analysis, single spheres or confluent hNSC cultures were single cell dissociated and serially diluted to yield 1–2 cell/10 µl. A 10-µl-cell suspension was then added to each of 96 or 24 well plates containing 200–300 µl of growth media. Wells containing one viable cell were marked the next day and re-scored 5 to 7 days later for cell proliferation. The differentiation of the hNSCs was performed as previously described [48]. Dissociated hNSCs were plated at a density of 106 cells/ml in control (media/hormone mix) medium devoid of any growth factors and supplemented with 1% fetal bovine serum (FBS) on poly-L-ornithine-coated (15 mg/ml; Sigma) glass coverslips in 24-well Nunclon culture dishes with 0.5 ml/well. After 2, 7–15 DIV cultures were fixed and processed for immunocytochemistry or used for RT-PCR analysis. Karyotype analysisLong-term cultures of hNSCs were incubated at 37°C and harvested for metaphase chromosomes when the cultures were 75% confluent. Metaphase chromosomes were obtained by standard chromosome harvest methods by exposure to Colcemid at 0.1 µg/ml for 1 hour at 37°C, a 2-minute exposure to trypsin/EDTA, hypotonized with 0.057 M KCl and fixed with 3∶1 methanol:acetic acid. Slide preparations were made by dropping the fixed cell pellet onto cold, wet slides and air-dried. After incubating the slides at 90°C for 30 minutes, chromosomes were trypsin banded and then Wright/Giemsa stained for G-banding analysis. Twenty metaphase cells were completely analyzed and a normal female chromosome complement was found (46,XX). Tumorigenicity in nude ratsAll animal experiments were conducted according to the National Institute of Health (NIH) guidelines and approved by the IACUC. Normal adult NIH-Nude rats (n = 5, 8 week-old, Taconic, Germantown, New York, United States) were used to test the tumorigenic potential of the SD56 hNSCs. Undifferentiated hNSCs from passage 9 were single cell dissociated using trypsin-EDTA and suspended at the concentration of 125,000 cell/µl in preparation for cell transplantation. Two µl of the cell suspension were stereotaxically transplanted into 4 sites within the striatum at the following coordinates: AP: +1.0 mm, ML: +3.2 mm, DV: −5.0; AP: +0.5 mm, ML: +3.0 mm, DV: −5.0; AP: −0.5 mm, ML: +3.0 mm, DV: −5.0; AP: −1.0 mm, ML: +3.5 mm, DV: −5.0 mm with the incisor bar set at 3.4 mm. The injection rate was 1 µl/min, and the cannula was left in place for an additional 5 min before retraction. For the flank tumor assay, 2×106 cells (125,000 cell/µl) were injected subcutaneously to the side of the adult nude rats. To label mitotically active cells in vivo during S-phase, the rats were injected IP with the BrdU (50 mg/kg, Sigma) every 8 hours during the last 24 hours before euthanasia. After 2-month survival time, rats were euthanized and a postmortem examination for tumor formation was performed. Induction of Focal Ischemia and Cell TransplantationAll animal experimentations were conducted according to the National Institute of Health (NIH) guidelines and approved by the IACUC. Sprague Dawley adult male rats (n = 17, 275g–310g, Charles River Laboratories, Wilmington, Massachusetts, United States) were subjected to one and a half hour suture occlusion of the middle cerebral artery (MCAO), as previously described [49] and immunosuppressed 2 days before cell transplantation and daily thereafter for one week with i.p. injections of cyclosporine A (20 mg/ml, Sandimmune, Novartis Pharmaceuticals). Thereafter oral cyclosporine was used at 210 µg/ml in drinking water until euthanasia. Undifferentiated SD56 hNSCs from passages between P9 and P13 were single cell dissociated using trypsin-EDTA in preparation for cell transplantation. One week after the stroke lesion, 2 µl of the hNSCs, at a concentration of 50,000 cell/µl, were stereotaxically transplanted into 4 sites within the lesioned striatum (n = 10) at the following coordinates: AP: +1.0 mm, ML: +3.2 mm, DV: −5.0; AP: +0.5 mm, ML: +3.0 mm, DV: −5.0; AP: −0.5 mm, ML: +3.0 mm, DV: −5.0; AP: −1.0 mm, ML: +3.5 mm, DV: −5.0 mm with the incisor bar set at 3.4 mm. The injection rate was 1 µl/min, and the cannula was left in place for an additional 5 min before retraction. As a control group, we used rats subjected to ischemia and injected with the vehicle (n = 7). All animals underwent baseline motor behavioral assessment before and after the ischemic lesion, and 4 & 8 weeks after cell transplantation. The animals were killed after 2-month survival time by transcardial perfusion with phosphate buffered saline (PBS) followed by 4% paraformaldehyde. The brains were cryoprotected in an increasing gradient of 10, 20 and 30% sucrose solution and cryostat sectioned at 40 µm and processed for immunocytochemistry. ImmunocytochemistryCultures were fixed with 4% paraformaldehyde for 15 min. Both cells and brain sections were rinsed in PBS for 3×5 min then incubated for 2 hrs (cultures) or overnight (brain sections) with the appropriate primary antibodies for multiple labeling. Secondary antibodies raised in the appropriate hosts and conjugated to FITC, RITC, AMCA, CY3 or CY5 chromogenes (Jackson ImmunoResearch) were used. Cells and sections were counterstained with the nuclear marker 4′,6-diamidine-2′-phenylindole dihydrochloride (DAPI). Positive and negative controls were included in each run. Immunostained sections were coverslipped using fluorsave (Calbiochem) as the mounting medium. The following antibodies were used: Anti-human Nuclei (hNuc, monoclonal 1∶100, Chemicon), Anti-TuJ1 (monoclonal 1∶100, Covance; Polyclonal 1∶200, Aves Labs); anti-GAD65/67 (polyclonal 1∶1000, Chemicon); Anti-glial fibrillary acidic protein (GFAP, monoclonal 1∶1000, Chemicon; polyclonal 1∶200, Aves Labs); Anti-galactocerebrocide (GC, monoclonal 1∶250, Chemicon); Anti-CNPase (polyclonal 1∶200, Aves Labs); Anti-Glucose Transporter type 1 (Glut-1 polyclonal, 1∶500, Chemicon); Anti-Nestin (polyclonal 1∶1000, Chemicon); Anti-vimentin (monoclonal 1∶500, Calbiochem); Anti-3CB2 (monoclonal 1∶500, Developmental Studies Hybridoma Bank); Anti-doublecortin (DCX, polyclonal 1∶250, SantaCruz Biotechnology); Anti-Ki67 (polyclonal 1∶250, SantaCruz Biotechnology). Fluorescence was detected, analyzed and photographed with a Zeiss LSM550 laser scanning confocal photomicroscope. For each animal, quantitative estimates of the total number of grafted cells were stereologically determined using the optical fractionator procedure [50]. A computer-assisted image analysis system was performed using Stereo Investigator software (MicroBrightField, Inc.). The rostral and caudal limits of the reference volume were determined by first and last frontal sections containing grafted cells. The striatum and cortex were accurately outlined at low magnification (2.5× objective). The optical fractionator probe was selected to perform systematic sampling of the immunoreactive cell population distributed within the serial sections to estimate the population number in the volume of tissue. The counting frame of the optical fractionator was defined at 50×50 µm squares and the systematic sampling was performed by translating a grid with 200×200 µm squares onto the sections of interest using the Stereo Investigator software. The sample sites were systematically and automatically generated by the computer and examined using a 60× objective of a Nikon Eclipse TE 300 microscope. The counting frame displayed inclusion and exclusion lines and only immunoreactive cell bodies falling within the counting frame with no contact with the exclusion lines were counted. The cell dispersion was measured by counting the number of cells within 200 µm distance from the graft site. The number and distance in µm of cells dispersed beyond 200 µm was also measured. An average of 2,000 cells was counted per animal. Double labeling was determined using the confocal laser scanning microscope by random sampling of 100 or more cells per marker for each animal, scoring first for hNuc+, followed by DAPI+ nuclei and then the marker of choice. The double labeling was always confirmed in x-z and y-z cross-sections produced by the orthogonal projections of z-series. Reverse Transcription-Polymerase Chain Reaction (RT-PCR) analysisTotal RNA was extracted from cultured cells using RNAeasy kit (Quiagen). Aliquots (1 µg) of total RNA from the cells were reverse transcribed in the presence of 50 mM Tris-HCl, pH 8.3, 75 mM KCl, 3 mM MgCl2, 10 mM DTT, 0.5 µM dNTPs, and 0.5 µg oligo-dT(12–18) with 200 U Superscript RNase H-Reverse Transcriptase (Invitrogen). PCR amplification was performed using standard procedure with Taq Polymerase. Aliquots of cDNA equivalent to 50 ng of total RNA were amplified in 25 µl reactions containing 10 mM Tris-HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl2 , 50 pmol of each primer, 400 µM dNTPs, and 0.5 U AmpliTaq DNA polymerase (Perkin-Elmer). PCR was performed using the following thermal profile: 4 min at 94°C; 1 min at 94°C, 1 min at 60°C, 1.5 min at 72°C, for 30–40 cycles; 7 min at 72°C, and finally a soak at 4°C overnight. The following day, 10 µl aliquots of the amplified products were run on a 2% agarose Tris–acetate gel containing 0.5 mg/ml ethidium bromide. The products were visualized through a UV transilluminator, captured in a digital format using Quantify One Gel Analysis software (Bio-Rad Laboratories) on a Macintosh G4 computer.The PCR primers specific to each transcript were as follows: GFAP, forward (F), 5′-TCATCGCTCAGGAGGTCCTT–3′ Reverse (R), 5′-CTG TTGCCAGAGATGGAGGTT–3′; MAP2 (F) 5′-GAAGACTCGCATCCGAATGG–3′, (R) 5′-CGCAGGATAGGAGGAAGAGACT–3′; MBP (F) 5′-TTAGCTGAATTC GCGTGTGG–3′, (R) 5′-GAGGAAGTGAATGAGCCGGTTA-3′ were deigned using the Primer Designer software, Version 2.0 (Scientific and Educational Software) [48]. 18S, β-tubulin class III, N-CAM, Nestin, NF-M, Notch-1 primers [51]. Oct4, Nanog primers [11]. FOXa2 (HNF3B), Brachyury primers [52]. Behavioral testsThe cylinder test was used to assess the spontaneous forelimb use during lateral exploration movement [22]. Rats were placed in a transparent acrylic cylinder (20 cm diameter) for 5 minutes. The cylinder encourages use of the forelimbs for vertical exploration. A mirror was placed behind the cylinder so that the forelimbs could be viewed at all times. Testing sessions were videotaped and forelimb use was scored by a blinded operator. Movements scored were the independent use of the left or right forelimb or simultaneous use of both the left and right forelimb to contact the wall of the cylinder during a full rear, to initiate a weight-shifting movement, or to regain center of gravity while moving laterally in a vertical posture along the wall. Animals were tested for their baselines after stroke and 4 and 8 weeks after cell transplantation. Statistical analysisOutcome measurement for each experiment was reported as mean±SEM. All data were analyzed using SPSS 11 for Mac OS X (SPSS Inc.). Significance of inter-group differences was performed by applying Student's t-test where appropriate. The One-Way ANOVA analysis was used to compare group differences for the forelimb use as the dependant variable and groups as the single independent factor variable. Differences between the groups were determined using Bonferroni's post hoc test. A P-value of less than 0.05 was considered to be statistically significant.
[ "BackgroundHuman", "embryonic", "stem", "cells", "(", "hESCs", ")", "offer", "a", "virtually", "unlimited", "source", "of", "neural", "cells", "for", "structural", "repair", "in", "neurological", "disorders", ",", "such", "as", "stroke", ".", "Neural", "cells", "can", "be", "derived", "from", "hESCs", "either", "by", "direct", "enrichment", ",", "or", "by", "isolating", "specific", "growth", "factor", "-", "responsive", "and", "expandable", "populations", "of", "human", "neural", "stem", "cells", "(", "hNSCs", ")", ".", "Studies", "have", "indicated", "that", "the", "direct", "enrichment", "method", "generates", "a", "heterogeneous", "population", "of", "cells", "that", "may", "contain", "residual", "undifferentiated", "stem", "cells", "that", "could", "lead", "to", "tumor", "formation", "in", "vivo", ".", "Methods", "/", "Principal", "FindingsWe", "isolated", "an", "expandable", "and", "homogenous", "population", "of", "hNSCs", "(", "named", "SD56", ")", "from", "hESCs", "using", "a", "defined", "media", "supplemented", "with", "epidermal", "growth", "factor", "(", "EGF", ")", ",", "basic", "fibroblast", "growth", "factor", "(", "bFGF", ")", "and", "leukemia", "inhibitory", "growth", "factor", "(", "LIF", ")", ".", "These", "hNSCs", "grew", "as", "an", "adherent", "monolayer", "culture", ".", "They", "were", "fully", "neuralized", "and", "uniformly", "expressed", "molecular", "features", "of", "NSCs", ",", "including", "nestin", ",", "vimentin", "and", "radial", "glial", "markers", ".", "These", "hNSCs", "did", "not", "express", "the", "pluripotency", "markers", "Oct4", "or", "Nanog", ",", "nor", "did", "they", "express", "markers", "for", "the", "mesoderm", "or", "endoderm", "lineages", ".", "The", "self", "-", "renewal", "property", "of", "the", "hNSCs", "was", "characterized", "by", "a", "predominant", "symmetrical", "mode", "of", "cell", "division", ".", "The", "SD56", "hNSCs", "differentiated", "into", "neurons", ",", "astrocytes", "and", "oligodendrocytes", "throughout", "multiple", "passages", "in", "vitro", ",", "as", "well", "as", "after", "transplantation", ".", "Together", ",", "these", "criteria", "confirm", "the", "definitive", "NSC", "identity", "of", "the", "SD56", "cell", "line", ".", "Importantly", ",", "they", "exhibited", "no", "chromosome", "abnormalities", "and", "did", "not", "form", "tumors", "after", "implantation", "into", "rat", "ischemic", "brains", "and", "into", "naïve", "nude", "rat", "brains", "and", "flanks", ".", "Furthermore", ",", "hNSCs", "isolated", "under", "these", "conditions", "migrated", "toward", "the", "ischemia", "-", "injured", "adult", "brain", "parenchyma", "and", "improved", "the", "independent", "use", "of", "the", "stroke", "-", "impaired", "forelimb", "two", "months", "post", "-", "transplantation", ".", "Conclusions", "/", "SignificanceThe", "SD56", "human", "neural", "stem", "cells", "derived", "under", "the", "reported", "conditions", "are", "stable", ",", "do", "not", "form", "tumors", "in", "vivo", "and", "enable", "functional", "recovery", "after", "stroke", ".", "These", "properties", "indicate", "that", "this", "hNSC", "line", "may", "offer", "a", "renewable", ",", "homogenous", "source", "of", "neural", "cells", "that", "will", "be", "valuable", "for", "basic", "and", "translational", "research", ".", "\n", "To", "date", "there", "have", "been", "no", "effective", "treatments", "for", "improving", "residual", "structural", "and", "functional", "deficits", "resulting", "from", "stroke", ".", "Current", "therapeutic", "approaches", ",", "such", "as", "the", "use", "of", "thrombolytics", ",", "benefit", "only", "1", "to", "4", "%", "of", "patients", "[", "1", "]", ".", "Consequently", ",", "the", "majority", "of", "stroke", "patients", "experience", "progression", "of", "ischemia", "associated", "with", "debilitating", "neurological", "deficits", ".", "Recent", "evidence", "has", "suggested", "that", "the", "transplantation", "of", "cells", "derived", "from", "cord", "blood", ",", "bone", "marrow", "or", "brain", "tissue", "(", "fetal", "and", "adult", ")", "enhances", "sensorimotor", "function", "in", "experimental", "models", "of", "stroke", "[", "2", "]", ",", "[", "3", "]", ".", "However", ",", "the", "normal", "human", "-", "derived", "somatic", "stem", "cells", "used", "in", "these", "studies", "have", "a", "limited", "capacity", "to", "differentiate", "into", "the", "diverse", "neural", "cell", "types", "optimal", "for", "structural", "and", "physiological", "tissue", "repair", "and", "are", "not", "amenable", "for", "large", "-", "scale", "cell", "production", ".", "Unlike", "other", "sources", "of", "stem", "cells", ",", "hESC", "lines", "possess", "a", "nearly", "unlimited", "self", "-", "renewal", "capacity", "and", "the", "developmental", "potential", "to", "differentiate", "into", "virtually", "any", "cell", "type", "of", "the", "organism", ".", "As", "such", ",", "they", "constitute", "an", "ideal", "source", "of", "cells", "for", "regenerative", "medicine", ".", "The", "successful", "derivation", "of", "hESC", "lines", "from", "the", "inner", "cell", "mass", "of", "preimplantation", "embryos", "and", "their", "long", "-", "term", "maintenance", "in", "vitro", "over", "multiple", "passages", "has", "been", "demonstrated", "[", "4", "]", "and", "standardized", ".", "Differentiation", "and", "enrichment", "processes", "that", "direct", "hESCs", "towards", "a", "neural", "lineage", "have", "also", "been", "achieved", ".", "To", "promote", "neuralization", ",", "ESCs", "were", "cultured", "in", "a", "defined", "media", "supplemented", "with", "morphogens", "or", "growth", "factors", "[", "5", "]", ",", "[", "6", "]", ",", "[", "7", "]", "or", "cultured", "under", "conditions", "that", "promote", "“", "rosettes", "”", ",", "structures", "morphologically", "similar", "to", "the", "developing", "neural", "tube", "[", "8", "]", ",", "[", "9", "]", ".", "This", "neuralization", "process", "has", "proven", "invaluable", "in", "understanding", "the", "specification", "of", "hESC", "-", "derived", "neural", "tissue", "[", "10", "]", ",", "[", "11", "]", ",", "[", "12", "]", ".", "However", ",", "the", "enriched", "neural", "progeny", "derived", "displayed", "overgrowth", "and", "limited", "migration", "after", "grafting", "into", "normal", "newborn", "mice", "[", "13", "]", "and", "lesioned", "adult", "rat", "striatum", "[", "12", "]", ",", "[", "14", "]", ",", "[", "15", "]", ",", "[", "16", "]", ".", "The", "inner", "cores", "of", "these", "grafts", "contained", "tumorigenic", "precursor", "cells", "(", "reviewed", "in", "[", "17", "]", ")", ".", "These", "findings", "suggest", "that", "neural", "cells", "generated", "by", "acute", "exposure", "to", "growth", "factors", "and", "/", "or", "morphogens", "may", "still", "be", "heterogeneous", "and", "potentially", "tumorigenic", ".", "We", "report", "an", "alternative", "method", "for", "the", "isolation", "and", "the", "perpetuation", "of", "a", "multipotent", "hNSC", "line", "from", "the", "hESCs", "with", "a", "primitive", "mode", "of", "self", "-", "renewal", ".", "We", "also", "demonstrate", "their", "long", "-", "term", "expansion", ",", "non", "-", "tumorigenic", "properties", "and", "functional", "engraftability", "in", "an", "experimental", "model", "of", "stroke", ".", "\n", "1", ".", "In", "vitro", "isolation", ",", "growth", "and", "differentiation", "of", "hESC", "-", "derived", "hNSCsThe", "hESCs", "were", "maintained", "and", "expanded", "on", "mouse", "feeder", "layer", "in", "media", "supplemented", "with", "bFGF", "(", "Figure", "1A", ")", ".", "After", "cell", "dissociation", ",", "a", "portion", "of", "the", "hESCs", "was", "cultured", "in", "serum", "free", "medium", "containing", "EGF", ",", "bFGF", "and", "LIF", ".", "These", "factors", "are", "known", "to", "stimulate", "the", "proliferation", "of", "human", "fetal", "-", "derived", "NSCs", "[", "18", "]", ",", "[", "19", "]", ".", "After", "3", "days", "in", "vitro", "(", "DIV", ")", ",", "there", "was", "selective", "survival", "and", "growth", "of", "cells", "that", "aggregated", "in", "clusters", "or", "spheres", "(", "Figure", "1B", ")", ".", "These", "primary", "spheres", "were", "harvested", "and", "replated", "in", "fresh", "media", ".", "During", "the", "following", "week", ",", "the", "spheres", "attached", "to", "the", "flask", "and", "a", "fibroblast", "-", "like", "cell", "population", "began", "to", "migrate", "out", "(", "Figure", "1C", ")", ".", "Secondary", "spheres", "(", "2", "°", "spheres", ")", "were", "generated", "from", "these", "cultures", "and", "lifted", "off", "by", "the", "end", "of", "the", "week", "leaving", "a", "hollow", "in", "the", "middle", "of", "the", "attached", "cells", "(", "Figure", "1D", ")", ".", "The", "floating", "2", "°", "spheres", "were", "collected", "and", "replated", "in", "fresh", "growth", "medium", "for", "2", "weeks", ".", "The", "cultures", "were", "then", "passaged", "by", "collagenase", "cell", "dissociation", "every", "7", "DIV", "for", "an", "additional", "4", "passages", "(", "Figure", "S1", ")", ".", "At", "the", "5th", "and", "6th", "passages", ",", "spheres", "were", "dissociated", "into", "a", "single", "-", "cell", "suspension", "using", "trypsin", "-", "EDTA", ".", "At", "this", "stage", "there", "was", "a", "change", "in", "the", "hNSCs", "'", "adherent", "properties", ",", "and", "the", "cells", "began", "to", "grow", "as", "a", "monolayer", "with", "multiple", "foci", "of", "cells", "throughout", "the", "culture", "(", "Figure", "1F", ")", ".", "The", "adherent", "hNSC", "culture", "stained", "uniformly", "for", "nestin", "(", "Figure", "1", "K", ")", ",", "vimentin", "(", "Figure", "1L", ")", "and", "with", "the", "radial", "glial", "marker", "3CB2", "(", "Figure", "1", "M", ")", "indicating", "their", "homogeneity", "and", "NSC", "identity", ".", "Under", "these", "culture", "conditions", ",", "it", "is", "noteworthy", "that", "we", "did", "not", "observe", "the", "formation", "of", "rosettes", "which", "has", "been", "previously", "reported", "to", "occur", "under", "certain", "conditions", "during", "neuralization", "of", "hESCs", "[", "8", "]", ",", "[", "20", "]", ",", "[", "21", "]", ".", "RT", "-", "PCR", "analysis", "confirmed", "that", "these", "hNSCs", "did", "not", "express", "the", "pluripotency", "transcripts", "Oct-4", "and", "Nanog", "(", "Figure", "1I", ")", ".", "Furthermore", ",", "the", "hNSCs", "did", "not", "express", "transcripts", "for", "brachyury", "and", "foxa2", ",", "marker", "genes", "for", "mesoderm", "and", "endoderm", "respectively", "(", "negative", "result", ",", "data", "not", "shown).10.1371", "/", "journal.pone.0001644.g001Figure", "1Isolation", "and", "purification", "of", "hNSCs", "from", "the", "hESCs", ".", "The", "hESCs", "were", "grown", "on", "a", "mouse", "feeder", "layer", "(", "A", ")", ".", "Primary", "neurospheres", "(", "B", ")", "were", "isolated", "and", "replated", "to", "eliminate", "other", "non", "-", "neural", "cells", ".", "The", "selectively", "harvested", "secondary", "neurospheres", "(", "arrow", "in", "C", ")", ",", "left", "behind", "hollow", "cores", "in", "the", "surface", "area", "(", "star", "in", "D", ")", "where", "they", "attached", "earlier", ".", "They", "were", "perpetuated", "for", "an", "additional", "5", "passages", "(", "E", ")", ".", "These", "2", "°", "spheres", "were", "then", "passaged", "using", "a", "single", "cell", "dissociation", "protocol", "(", "F", ")", ".", "Arrow", "in", "F", "shows", "an", "example", "of", "a", "focus", "of", "proliferating", "cells", ".", "(", "G", ",", "H", ")", "The", "hNSCs", "were", "passaged", "every", "5–7", "days", ",", "as", "described", "in", "the", "Methods", "section", ".", "Starting", "from", "an", "initial", "population", "of", "1", "million", "cells", ",", "the", "cumulative", "cell", "number", "was", "calculated", "at", "each", "passage", "as", "the", "fold", "of", "increase×the", "total", "cell", "number", "and", "plotted", "as", "logarithm", "with", "base", "2", "in", "function", "of", "time", "(", "G", ")", ".", "The", "cell", "perpetuation", "(", "G", ")", "and", "population", "doubling", "(", "H", ")", "analysis", "demonstrated", "the", "continuous", "and", "stable", "growth", "of", "the", "hNSCs", ".", "(", "I", ")", "RT", "-", "PCR", "analysis", "showing", "the", "down", "regulation", "of", "the", "pluripotency", "transcripts", "Oct4", "and", "Nanog", "in", "secondary", "neurospheres", "and", "in", "expanded", "hNSCs", "at", "passage", "8", "(", "P8", ")", ".", "(", "J", ")", "Cytogenetic", "evaluation", "of", "the", "SD56", "hNSCs", "line", "at", "passage", "12", "by", "standard", "G", "-", "banding", "was", "performed", ".", "Twenty", "metaphase", "cells", "were", "analyzed", "and", "showed", "a", "normal", "female", "chromosome", "complement", "(", "46,XX", ")", ".", "Isolated", "and", "expanded", "hNSCs", "expressed", "the", "neural", "precursor", "cell", "markers", "nestin", "(", "K", ")", ",", "Vimentin", "(", "L", ")", "and", "the", "radial", "glial", "cell", "marker", "3CB2", "(", "M", ")", "in", "virtually", "all", "the", "progeny", ".", "(", "N", "-", "P", ")", "Clonal", "self", "-", "renewal", "ability", "of", "the", "isolated", "hNSCs", "through", "symmetrical", "cell", "division", ".", "Single", "(", "N", ")", ",", "two", "-", "cell", "stage", "(", "O", ")", "and", "four", "-", "cell", "stage", "(", "P", ")", "of", "a", "hNSC", "proliferating", "over", "a", "3-day", "culture", "period", ".", "Note", "the", "symmetrical", "segregation", "of", "BrdU", "and", "nestin", "in", "the", "progeny", ".", "Bars", ":", "(", "A", ",", "B", ",", "C", ",", "D", ")", "200", "µm", ";", "(", "E", ",", "F", ")", "100", "µm", ";", "(", "K", "–", "M", ")", "20", "µm", ";", "(", "N", "–", "P", ")", "10", "µm", ".", "To", "ascertain", "self", "-", "renewal", "ability", "under", "clonal", "conditions", ",", "a", "single", "cell", "suspension", "was", "plated", "at", "clonal", "density", "(", "1–2", "cell/10", "µl", ")", ".", "To", "determine", "if", "the", "hNSCs", "divide", "symmetrically", ",", "we", "pulsed", "cultures", "with", "the", "thymidine", "analog", ",", "bromodeoxyuridine", "(", "BrdU", ")", ",", "after", "plating", "and", "looked", "for", "the", "expression", "of", "nestin", "in", "the", "progeny", ".", "Cultures", "were", "fixed", "after", "1", ",", "2", "or", "3", "DIV", "(", "Figure", "1N", "–", "P", ")", ".", "After", "2", "days", ",", "plated", "single", "cells", "first", "underwent", "a", "symmetric", "cell", "division", "and", "gave", "rise", "to", "daughter", "cells", "that", "were", "both", "positive", "for", "BrdU", "and", "nestin", ".", "The", "clone", "of", "cells", "continued", "to", "grow", "over", "the", "3", "DIV", "and", "all", "the", "progeny", "expressed", "nestin", ".", "BrdU", "labeling", "demonstrated", "that", "it", "was", "rare", "for", "only", "one", "daughter", "cell", "to", "inherit", "the", "BrdU", "and", "thus", "had", "undergone", "asymmetric", "segregation", "of", "the", "chromatids", "(", "Figure", "S2", ")", ".", "G", "-", "band", "karyotyping", "of", "these", "hNSCs", "confirmed", "the", "normal", ",", "non", "-", "transformed", "nature", "of", "cells", "after", "12", "passages", "(", "Figure", "1J", ")", ".", "We", "named", "the", "derived", "hNSCs", "SD56", "(", "intermittently", "referred", "to", "as", "SD56", "hNSCs", "or", "hNSCs).Under", "these", "defined", "growth", "conditions", ",", "the", "hNSCs", "showed", "stable", "growth", "with", "a", "2.7±0.2", "fold", "increase", "every", "5", "to", "7", "days", "(", "Figure", "1", "G", ")", ".", "The", "population", "doubling", "at", "each", "passage", "averaged", "at", "1.4±0.1", "(", "Figure", "1H", ")", ".", "The", "viability", "of", "hNSCs", "at", "each", "passage", "was", "consistent", "at", "the", "approximate", "value", "of", "98", "%", ".", "The", "projected", "cumulative", "cell", "numbers", "demonstrated", "that", "trillions", "of", "cells", "could", "be", "generated", "over", "a", "period", "of", "5", "months", "(", "Figure", "1", "G", ")", ".", "We", "expanded", "the", "isolated", "hNSCs", "lines", "for", "over", "20", "passages", "with", "a", "stable", "phenotype", ".", "An", "initial", "cell", "bank", "of", "75", "vials", "containing", "2", "to", "5", "million", "cells", "each", "was", "generated", "and", "cryopreserved", ".", "Upon", "removal", "of", "the", "mitogenic", "factors", "and", "plating", "on", "a", "coverslip", "pre", "-", "coated", "with", "poly", "-", "L", "-", "ornithine", "(", "PLO", ")", "substrate", ",", "the", "hNSCs", "spontaneously", "differentiated", "into", "neurons", ",", "astrocytes", "and", "oligodendrocytes", ",", "a", "property", "that", "is", "consistent", "with", "normal", "multipotent", "hNSCs", "(", "Figure", "2", ")", ".", "After", "2", "DIV", ",", "hNSCs", "expressed", "transcripts", "for", "the", "neural", "-", "specific", "genes", "nestin", ",", "Notch1", "and", "neural", "cell", "adhesion", "molecule", "(", "N", "-", "CAM", ")", "(", "Figure", "2A", ")", "and", "for", "the", "lineage", "specific", "markers", "β", "-", "tubulin", "class", "III", ",", "medium", "-", "size", "neurofilament", "(", "NF", "-", "M", ")", "and", "microtubule", "-", "associated", "protein", "2", "(", "MAP-2", ")", "for", "neurons", ",", "GFAP", "for", "astrocytes", "and", "myelin", "basic", "protein", "(", "MBP", ")", "for", "oligodendrocytes", "(", "Figure", "2A", ")", ".", "Transcripts", "for", "the", "GABAergic", "cell", "marker", "glutamic", "acid", "decarboxylase", "(", "GAD", ")", "were", "expressed", ",", "but", "transcripts", "for", "the", "tyrosine", "hydroxylase", "(", "TH", ")", ",", "a", "marker", "for", "dopaminergic", "neurons", ",", "were", "undetectable", ".", "Immunocytochemical", "analysis", "(", "Figure", "2B", "–", "F", ")", "of", "10", "day", "-", "old", "cultures", "demonstrated", "that", "the", "proportion", "of", "nestin+", "cells", "was", "36.6±2.7", "%", ",", "62.5±2.8", "%", "expressed", "the", "neuronal", "marker", "TuJ1", ",", "1.9±0.3", "%", "expressed", "the", "astrocytic", "marker", "GFAP", "and", "7.1±0.4", "%", "were", "oligodendrocytes", "and", "expressed", "galactocerebrocide", "(", "GC", ")", "(", "Figure", "2F", ")", ".", "A", "subset", "(", "9.8±1.6", "%", ")", "of", "the", "GFAP+", "astrocytes", "co", "-", "expressed", "nestin.10.1371", "/", "journal.pone.0001644.g002Figure", "2hESC", "-", "derived", "hNSCs", "spontaneously", "differentiated", "into", "the", "3", "principal", "central", "nervous", "system", "cell", "types", ".", "Dissociated", "hNSCs", "were", "washed", "free", "of", "growth", "factors", "and", "plated", "on", "poly", "-", "L", "-", "onithine", "coated", "glass", "coverslips", ".", "Differentiated", "cultures", "were", "either", "harvested", "after", "2", "DIV", "for", "total", "RNA", "extraction", "and", "RT", "-", "PCR", "analysis", "or", "fixed", "after", "10", "DIV", "and", "processed", "for", "indirect", "immunocytochemistry", ".", "(", "A", ")", "Differentiated", "hNSCs", "expressed", "the", "neural", "-", "specific", "transcripts", "nestin", "and", "Notch1", "as", "well", "as", "transcripts", ":", "for", "neurons", "(", "β", "-", "tubulin", "class", "III", ",", "MAP-2", ",", "NCAM", "and", "medium", "-", "size", "neurofilament", ",", "NF", "-", "M", ")", ",", "for", "astrocytes", "(", "GFAP", ")", "and", "for", "oligodendrocytes", "(", "MBP", ")", ".", "The", "hNSCs", "expressed", "transcripts", "for", "GAD", ",", "but", "not", "for", "TH", ".", "B", ",", "C", "&", "D", ",", "morphology", "of", "NSC", "-", "derived", "progeny", "differentiated", "into", "GFAP+", "astrocytes", "(", "B", ")", ",", "GC", "-", "expressing", "oligodendrocytes", "(", "C", ")", "and", "TuJ1", "+", "neurons", "(", "D", ")", ",", "DAPI", "(", "blue", ")", "show", "life", "cell", "nuclei", ".", "(", "E", ")", "Photo", "showing", "cultures", "double", "-", "immunostained", "for", "TuJ1", "(", "green", ")", "and", "nestin", "(", "red", ")", "(", "DAPI", ",", "blue", ")", ".", "(", "F", ")", "Quantitative", "analysis", "of", "immunostained", "10", "day", "-", "old", "cultures", "for", "the", "3", "neural", "cell", "types", ".", "Results", "are", "mean±s.e.m", ".", "of", "three", "independent", "experiments", ",", "each", "performed", "in", "duplicate", ".", "Bars", ":", "(", "c", ")", "20", "µm", ";", "(", "d", ",", "e", ")", "10", "µm", ".", "2", ".", "The", "isolated", "hNSCs", "are", "normal", "and", "do", "not", "form", "tumors", "in", "normal", "nude", "ratsThe", "self", "-", "renewal", "and", "pluripotent", "abilities", "of", "the", "hESCs", "are", "also", "associated", "with", "tumorigenic", "properties", ".", "Therefore", ",", "the", "first", "critical", "step", "toward", "developing", "therapeutic", "hNSCs", "is", "to", "verify", "that", "they", "are", "non", "-", "tumorigenic", ".", "The", "monolayer", "culture", "of", "SD56", "hNSCs", "clearly", "demonstrated", "contact", "inhibition", "of", "growth", ",", "a", "normal", "karyotype", "and", "did", "not", "express", "the", "pluripotency", "transcripts", "Oct-4", "and", "Nanog", ".", "Removal", "of", "mitogenic", "factors", "and", "continued", "culture", "on", "plastic", "resulted", "in", "cell", "senescence", "that", "is", "characteristic", "of", "non", "-", "transformed", "cells", ".", "To", "determine", "whether", "SD56", "hNSCs", "form", "tumors", "in", "vivo", ",", "we", "transplanted", "them", "at", "high", "density", "(", "see", "Methods", ")", "into", "the", "forebrain", "and", "subcutaneously", "into", "the", "flank", "of", "nude", "rats", ".", "The", "animals", "were", "kept", "for", "a", "two", "-", "month", "post", "-", "transplant", "survival", "period", ".", "To", "label", "mitotically", "active", "cells", "in", "vivo", "during", "S", "-", "phase", ",", "the", "rats", "were", "injected", "IP", "with", "BrdU", "(", "50", "mg", "/", "kg", ")", "every", "8", "hours", "during", "the", "last", "24", "hours", "before", "euthanasia", ".", "The", "transit", "amplifying", "endogenous", "precursors", "located", "in", "the", "subventricular", "zone", "(", "SVZ", ")", "were", "labeled", "(", "Figure", "S3", ")", ";", "however", ",", "we", "were", "unable", "to", "detect", "grafted", "SD56", "hNSCs", "co", "-", "localizing", "the", "human", "-", "specific", "nuclear", "marker", "hNuc", "and", "BrdU", "(", "Figure", "S3", ")", ".", "No", "surviving", "SD56", "hNSCs", "were", "detected", "in", "the", "flank", "of", "the", "transplanted", "animals", "suggesting", "that", "the", "grafted", "cells", "are", "not", "tumorigenic", ".", "3", ".", "Transplanted", "cells", "survived", ",", "migrated", "toward", "and", "engrafted", "into", "the", "stroke", "-", "damaged", "host", "tissueTo", "investigate", "the", "survival", "and", "functional", "engraftment", "in", "an", "injury", "environment", ",", "hNSCs", "(", "4×105", ")", "were", "transplanted", "into", "the", "ischemic", "boundary", "zone", "in", "the", "rat", "striatum", "one", "week", "after", "the", "middle", "cerebral", "artery", "occlusion", "(", "MCAO", ")", "was", "performed", ".", "Animals", "were", "euthanized", "two", "months", "later", "and", "the", "brains", "processed", "for", "histo", "-", "pathology", "and", "immunocytochemistry", ".", "Grafted", "SD56", "hNSCs", ",", "identified", "with", "hNuc", ",", "demonstrated", "a", "37.0±15.8", "%", "survival", "rate", "and", "a", "remarkable", "dispersion", "toward", "the", "stroke", "-", "damaged", "tissue", "with", "no", "sign", "of", "overgrowth", "or", "tumorigenesis", ".", "The", "majority", "of", "grafted", "cells", "(", "61.2±4.7", "%", ")", "migrated", "at", "least", "200", "µm", "away", "from", "the", "injection", "site", "and", "penetrated", "an", "average", "distance", "of", "806.4±49.3", "µm", "into", "the", "stroke", "-", "damaged", "tissue", "(", "Figure", "3A", "–", "C", ")", ".", "Immunostaining", "with", "the", "blood", "vessel", "marker", ",", "GluT1", ",", "revealed", "dilated", "vessels", "in", "the", "infarcted", "striatum", "and", "a", "close", "association", "between", "vessels", "and", "the", "grafted", "hNSCs", "(", "Figure", "3B", ",", "3C", ")", ".", "The", "grafted", "cells", "rarely", "expressed", "the", "proliferation", "marker", "Ki67", "(", "Figure", "3D", ")", ",", "29.8±4.4", "%", "expressed", "nestin", "(", "Figure", "3E", ")", ",", "6.5±0.9", "%", "expressed", "doublecortin", "(", "DCX", ")", "and", "60.8±8.1", "%", "were", "TuJ1", "+", "(", "Figure", "3F", ",", "G", ")", ".", "Grafted", "cells", "rarely", "co", "-", "expressed", "the", "astroglial", "marker", "GFAP", "(", "Figure", "3H", ")", "or", "differentiated", "into", "CNPase", "-", "expressing", "oligodendrocytes", "(", "Figure", "3I", ")", ".", "Immunostaining", "for", "GAD", "demonstrated", "that", "25.1±2.3", "%", "of", "grafted", "hNSCs", "differentiated", "into", "GABAergic", "neurons", "while", "less", "than", "2", "%", "were", "positive", "for", "glutamate", "(", "Figure", "3J", ",", "K).10.1371", "/", "journal.pone.0001644.g003Figure", "3Dispersion", ",", "engraftment", "and", "differentiation", "of", "the", "hNSCs", "in", "stroke", "-", "lesioned", "animals.(A", ")", "Schematic", "drawing", "of", "a", "frontal", "section", "through", "the", "striatum", "illustrating", "the", "dispersion", "of", "grafted", "hNSCs", "in", "the", "focal", "ischemia", "-", "lesioned", "parenchyma", "(", "shaded", "area", ")", ".", "(", "B", ",", "C", ")", "Photos", "show", "frontal", "sections", "through", "the", "graft", "in", "the", "striatum", "immunostained", "with", "the", "human", "specific", "antibodies", ":", "anti", "-", "hNuc", "(", "green", "in", "B", "&", "C", ")", "and", "anti", "-", "GluT1", "(", "red", ",", "B", "&", "C", ")", "showing", "blood", "vessels", "and", "dispersed", "hNSCs", "in", "the", "graft", "zone", ".", "C", ":", "higher", "magnification", "of", "the", "inset", "in", "B.", "(", "D", "–", "I", ")", "Photos", "taken", "from", "frontal", "sections", "through", "the", "graft", "in", "the", "striatum", "double", "immunoprocessed", "for", "cell", "proliferation", "and", "neural", "lineage", "markers", ".", "(", "D", ")", "Note", "the", "endogenous", "Ki67", "+", "cells", "(", "red", "cells", ",", "arrow", ")", "in", "the", "stroke", "damaged", "area", "and", "the", "hNuc+", "(", "green)/Ki67-", "grafted", "hNSCs", "(", "arrowheads", ")", ".", "(", "E", ")", "Examples", "of", "grafted", "SD56", "hNSCs", "showing", "co", "-", "expression", "of", "hNuc", "(", "green", ")", "and", "nestin", "(", "red", ")", ".", "(", "F", ")", "Confocal", "3D", "reconstructed", "orthogonal", "images", "of", "the", "hNuc+(green)/DCX+(red", ")", "NSCs", "(", "arrowheads", ")", "viewed", "in", "the", "x", "-", "z", "plan", "on", "the", "top", "and", "y", "-", "z", "plan", "on", "the", "right", ".", "(", "G", ")", "Examples", "show", "the", "majority", "of", "grafted", "NSC", "progeny", "co", "-", "expressing", "hNuc", "(", "red", ")", "and", "the", "neuronal", "marker", "TuJ1", "(", "green", ")", ".", "Grafted", "NSCs", "rarely", "differentiate", "into", "GFAP+", "astrocytes", "(", "H", ")", ".", "In", "I", ",", "rare", "example", "of", "grafted", "NSC", "progeny", "becoming", "an", "oligodendrocyte", "identified", "by", "the", "expression", "of", "CNPase", "(", "green", ")", ".", "Grafted", "NSCs", "expressed", "the", "GABAergic", "marker", "GAD65/67", "(", "J", ")", "and", "rarely", "expressed", "glutamate", "(", "K", ")", ".", "(", "Abbreviations", ":", "Cx", ":", "cortex", ",", "Str", ":", "striatum", ")", ".", "Bars", ":", "(", "B", ",", "C", ")", "100", "µm", ";", "(", "D", ",", "F", ")", "20", "µm", ";", "(", "E", ",", "G", "–", "K", ")", "10", "µm", ".", "4", ".", "Transplanted", "cells", "improve", "sensorimotor", "function", "of", "the", "stroke", "-", "disabled", "forelimbWe", "asked", "whether", "transplanted", "SD56", "hNSCs", "could", "enhance", "the", "recovery", "of", "sensorimotor", "function", "that", "is", "compromised", "in", "the", "stroke", "-", "injured", "rats", ".", "We", "used", "the", "cylinder", "test", "to", "measure", "the", "sensorimotor", "asymmetry", "in", "forelimb", "use", "during", "spontaneous", "exploration", "[", "22", "]", ".", "To", "establish", "the", "baseline", "of", "the", "stroke", "-", "induced", "sensorimotor", "deficit", ",", "spontaneous", "behavior", "of", "rats", "in", "a", "transparent", "cylinder", "was", "videotaped", "one", "week", "after", "stroke", "(", "pre", "-", "transplant", ",", "Figure", "4", ")", ".", "Tests", "were", "then", "conducted", "4", "and", "8", "weeks", "after", "vehicle", "and", "SD56", "hNSCs", "transplantation", ".", "Stable", "asymmetry", "in", "forelimb", "use", "was", "observed", "7", "days", "post", "-", "stroke", "(", "pre", "-", "transplant", ",", "Figure", "4", ")", ".", "Ischemic", "rats", "used", "their", "impaired", "forelimbs", "(", "contralateral", "to", "lesion", ")", "during", "lateral", "exploration", "less", "than", "they", "did", "before", "stroke", ".", "Transplantation", "of", "SD56", "hNSCs", "significantly", "enhanced", "the", "independent", "use", "of", "the", "impaired", "contralateral", "forelimb", "4", "weeks", "post", "transplantation", "(", "P<0.05", "vs", "pre", "-", "transplant", ")", ".", "Eight", "weeks", "after", "transplantation", "the", "improvement", "in", "the", "use", "of", "the", "impaired", "forelimb", "was", "stable", "and", "significant", "when", "compared", "to", "the", "pre", "-", "transplant", "group", "and", "significantly", "improved", "in", "comparison", "to", "vehicle", "treated", "group", "at", "8", "weeks", "(", "Figure", "4", ")", ".", "In", "the", "vehicle", "treated", "group", ",", "the", "independent", "use", "of", "the", "contralateral", "forelimb", "remained", "impaired", "4", "and", "8", "weeks", "post", "-", "injection", ".", "In", "an", "independent", "study", "and", "using", "the", "same", "MCAO", "rat", "animal", "model", ",", "we", "found", "that", "transplantation", "of", "dermal", "fibroblasts", "did", "not", "improve", "the", "stroke", "-", "induced", "motor", "deficits", "(", "unpublished", "data).10.1371", "/", "journal.pone.0001644.g004Figure", "4Transplantation", "of", "NSCs", "improves", "sensorimotor", "function", "of", "the", "stroke", "-", "disabled", "forelimb", ".", "Forelimb", "use", "during", "spontaneous", "lateral", "exploration", "was", "measured", "in", "the", "cylinder", "test", "(", "see", "Method", "and", "Results", "sections", "for", "details", ")", ".", "Groups", "of", "vehicle", "injected", "(", "n", " ", "=", " ", "7", ")", "and", "hNSCs", "(", "n", " ", "=", " ", "10", ")", "transplanted", "are", "represented", ".", "The", "animals", "were", "tested", "as", "described", "in", "Method", "section", ".", "Note", "the", "significant", "increase", "in", "the", "independent", "use", "of", "the", "impaired", "contralateral", "forelimb", "at", "4", "and", "8", "weeks", "post", "transplantation", "(", "P<0.05", "vs", "pre", "-", "transplant", "group", ")", ".", "The", "contralateral", "forelimb", "remained", "impaired", "in", "the", "vehicle", "treated", "group", "at", "4", "and", "8", "weeks", "post", "-", "injection", ".", "Bars", "represent", "percentages±s.e.m", ".", "of", "steps", "taken", "by", "the", "ipsilateral", ",", "contralateral", "and", "both", "forelimbs", "simultaneously", ".", "*", "P<0.05", "vs", "pre", "-", "transplant", "group", ";", "#", "P<0.05", "vs", "vehicle", "groups", ".", "\n", "Our", "results", "indicate", "that", "a", "self", "-", "renewable", "and", "homogenous", "population", "of", "hNSCs", ",", "SD56", ",", "was", "derived", "from", "hESCs", "using", "defined", "media", "supplemented", "with", "a", "specific", "combination", "of", "growth", "factors", ".", "The", "SD56", "hNSCs", "grew", "as", "an", "adherent", "monolayer", "culture", ",", "uniformly", "expressed", "molecular", "features", "of", "hNSCs", "including", "nestin", ",", "vimentin", "and", "the", "radial", "glial", "marker", "3CB2", ",", "and", "did", "not", "express", "the", "pluripotency", "markers", "Oct4", "or", "Nanog", ".", "The", "self", "-", "renewal", "property", "of", "the", "hNSCs", "was", "characterized", "by", "a", "predominant", "symmetrical", "mode", "of", "cell", "division", ".", "They", "exhibited", "no", "chromosomal", "abnormalities", "and", "demonstrated", "non", "-", "tumorigenic", "properties", "after", "implantation", "into", "ischemic", "brains", "and", "into", "naïve", "nude", "rat", "brains", "and", "flanks", ".", "Furthermore", ",", "the", "transplanted", "SD56", "hNSCs", "migrated", "toward", "the", "stroke", "-", "damaged", "adult", "brain", "parenchyma", ",", "engrafted", "and", "improved", "the", "independent", "use", "of", "the", "stroke", "-", "impaired", "forelimb", ".", "Maintenance", "of", "stem", "cells", "requires", "symmetrical", "and", "asymmetrical", "cell", "divisions", "to", "both", "expand", "and", "to", "give", "rise", "to", "specialized", "progeny", "of", "a", "specific", "tissue", "(", "reviewed", "in", "[", "23", "]", ")", ".", "In", "vivo", ",", "a", "complex", "cellular", "micro", "-", "environment", "or", "niche", "ensures", "the", "self", "-", "maintenance", "property", "of", "NSCs", "[", "24", "]", ",", "[", "25", "]", ",", "[", "26", "]", ",", "[", "27", "]", ".", "In", "vitro", ",", "defined", "growth", "factors", "and", "extracellular", "matrices", "support", "stem", "cell", "self", "-", "renewal", "[", "28", "]", ",", "[", "29", "]", ".", "The", "embryonic", "stem", "cells", "can", "propagate", "in", "a", "predominantly", "proliferative", "symmetrical", "mode", ",", "leading", "to", "homogeneous", "cell", "cultures", "growing", "relatively", "quickly", "with", "minimal", "cell", "differentiation", "[", "30", "]", ",", "[", "31", "]", ",", "[", "32", "]", ",", "[", "33", "]", ",", "[", "34", "]", ".", "Tissue", "specific", "stem", "cells", ",", "however", ",", "self", "-", "renew", "in", "a", "predominant", "asymmetric", "mode", "to", "maintain", "them", "selves", "and", "compensate", "for", "the", "loss", "of", "differentiated", "cells", "due", "to", "disease", "or", "injury", ".", "Thus", ",", "NSCs", "isolated", "from", "developing", "or", "adult", "brain", "grow", "as", "a", "mixture", "of", "undifferentiated", "and", "differentiated", "cells", "due", "the", "predominant", "asymmetrical", "mode", "of", "cell", "division", "[", "35", "]", ",", "[", "36", "]", ",", "[", "37", "]", ",", "[", "38", "]", ",", "[", "39", "]", ",", "[", "40", "]", ",", "[", "41", "]", ".", "A", "recent", "study", "has", "reported", "that", "a", "murine", "ESC", "-", "derived", "NSC", "line", "(", "LC1", ")", "is", "propagated", "as", "an", "adherent", "homogenous", "culture", "with", "a", "dominant", "mode", "of", "symmetrical", "self", "-", "renewal", "[", "21", "]", ".", "A", "combination", "of", "EGF", "and", "FGF2", "was", "sufficient", "to", "propagate", "these", "NSCs", "as", "an", "adherent", "monolayer", ".", "However", ",", "the", "SD56", "hNSC", "line", "described", "here", "required", "the", "combination", "of", "EGF", ",", "bFGF", "and", "LIF", "for", "self", "-", "maintenance", ".", "Although", "there", "are", "morphological", "and", "molecular", "similarities", "between", "our", "hNSCs", "and", "the", "NSCs", "previously", "described", "[", "21", "]", ",", "the", "methods", "of", "isolation", "and", "growth", "are", "different", ".", "In", "addition", "to", "the", "different", "combination", "of", "growth", "factors", "used", ",", "the", "hNSC", "line", "we", "have", "isolated", "did", "not", "go", "through", "the", "rosette", "-", "structure", "stage", ".", "The", "in", "vitro", "analysis", "of", "BrdU", "incorporation", "and", "nestin", "expression", "indicated", "that", "our", "hNSCs", "divide", "predominantly", "symmetrically", ".", "This", "type", "of", "growth", "pattern", "is", "characteristic", "of", "primitive", "normal", "stem", "cells", "undergoing", "mostly", "symmetric", "cell", "division", "to", "increase", "the", "stem", "cell", "pool", "at", "the", "early", "stage", "of", "development", "or", "during", "tissue", "regeneration", "after", "injury", "[", "23", "]", ".", "RT", "-", "PCR", "and", "immunocytochemistry", "analysis", "demonstrated", "that", "these", "undifferentiated", "SD56", "cells", "did", "not", "express", "any", "pluripotency", ",", "endodermal", "or", "mesodermal", "markers", ".", "Furthermore", ",", "the", "SD56", "hNSCs", "described", "here", "exhibited", "the", "multipotential", "characteristic", "to", "differentiate", "into", "neurons", ",", "astrocytes", "and", "oligodendrocytes", "both", "in", "vitro", "and", "upon", "transplantation", ".", "Together", "these", "findings", "suggest", "that", "the", "hNSC", "line", "we", "isolated", "are", "appropriately", "programmed", "and", "share", "similar", "characteristics", "with", "the", "definitive", "NSCs", "of", "the", "developing", "brain", ".", "The", "SD56", "hNSCs", "demonstrated", "a", "remarkable", "ability", "to", "migrate", "toward", "the", "stroke", "-", "damaged", "parenchyma", "of", "the", "adult", "rat", "brain", ".", "This", "directed", "migration", "by", "the", "majority", "of", "the", "grafted", "cells", "could", "be", "due", "to", "their", "uniform", "cellular", "composition", ",", "which", "results", "in", "an", "equal", "response", "to", "the", "host", "microenvironment", ".", "In", "previous", "studies", ",", "subpopulations", "of", "transplanted", "hESCs", "that", "were", "enriched", "in", "neural", "cells", "migrated", "in", "host", "microenvironments", "conducive", "to", "cell", "migration", ",", "such", "as", "the", "developing", "brain", "or", "in", "structures", "such", "as", "the", "rostral", "migratory", "stream", "[", "13", "]", ",", "[", "20", "]", ".", "In", "the", "adult", "lesioned", "brain", ",", "the", "grafted", "hESC", "-", "derived", "neural", "cells", "proliferated", "and", "formed", "rosettes", "[", "14", "]", ",", "teratomas", "[", "12", "]", ",", "[", "15", "]", "or", "a", "cellular", "mass", "that", "induced", "a", "gliotic", "host", "response", "whereby", "local", "astrocytes", "demarcated", "the", "grafts", "[", "16", "]", ".", "Enriched", "neural", "cultures", "derived", "from", "mouse", "[", "42", "]", "and", "monkey", "ESCs", "[", "43", "]", "have", "produced", "behavioral", "improvements", "when", "transplanted", "into", "animal", "models", "of", "stroke", "and", "brain", "injury", ".", "However", ",", "in", "these", "cases", ",", "the", "transplanted", "non", "-", "human", "ESCs", "also", "formed", "a", "mass", "with", "signs", "of", "overgrowth", "in", "the", "core", ",", "as", "well", "as", "deformations", "[", "44", "]", ",", "[", "45", "]", ",", "[", "46", "]", ".", "ESCs", "plated", "at", "low", "density", "acquire", "neural", "identity", "within", "few", "hours", "after", "plating", "[", "47", "]", ".", "Interestingly", ",", "nearly", "all", "viable", "cells", "expressed", "nestin", ",", "the", "early", "neural", "fate", "marker", "Sox1", ",", "and", "the", "pluripotency", "marker", "Oct4", ".", "Together", ",", "these", "studies", "are", "seminal", "and", "suggest", "that", "complete", "neuralization", "may", "not", "be", "achieved", "through", "certain", "enrichment", "processes", ",", "consequently", "the", "neural", "cells", "could", "revert", "to", "a", "pluripotent", "stage", "[", "17", "]", ".", "The", "dispersion", "of", "the", "grafted", "hNSCs", "within", "host", "parenchyma", "may", "allow", "for", "more", "graft", "-", "host", "interactions", "that", "could", "stabilize", "differentiation", ",", "inhibit", "growth", "and", "prevent", "gliotic", "host", "response", ".", "In", "the", "present", "study", ",", "SD56", "hNSC", "-", "transplanted", "animals", "demonstrated", "a", "stable", "improvement", "in", "the", "sensorimotor", "function", "when", "evaluated", "for", "spontaneous", "exploratory", "activity", "in", "the", "cylinder", "test", "that", "detects", "long", "-", "lasting", "deficits", "in", "forelimb", "use", "in", "the", "experimental", "models", "of", "stroke", "[", "22", "]", ".", "The", "transplantation", "of", "hNSCs", "significantly", "enhanced", "the", "independent", "use", "of", "the", "impaired", "contralateral", "forelimb", "8", "weeks", "post", "transplantation", ".", "This", "is", "the", "first", "report", "demonstrating", "that", "the", "transplantation", "of", "hNSCs", "derived", "from", "hESCs", "can", "improve", "neurologic", "behavior", "after", "experimental", "stroke", ".", "Together", ",", "these", "findings", "are", "encouraging", "and", "suggest", "that", "these", "cells", "are", "promising", "for", "future", "development", ".", "In", "addition", "to", "their", "therapeutic", "application", ",", "the", "hNSCs", "isolated", "under", "the", "reported", "conditions", "offer", "a", "means", "to", "interrogate", "host", "environments", "and", "unravel", "mechanistic", "features", "of", "self", "-", "renewal", ",", "non", "-", "tumorigenicity", "and", "functional", "engraftability", "in", "animal", "models", "of", "neurological", "disorders", ".", "\n", "hESC", "and", "NSC", "CulturesThe", "hESC", "line", "H9", "(", "WiCell", "Research", "Institute", ")", "was", "propagated", "every", "5", "to", "7", "days", "on", "irradiated", "mouse", "embryonic", "fibroblasts", ".", "The", "cell", "culture", "media", "consisted", "of", "a", "1∶1", "mixture", "of", "Dulbecco", "'s", "modified", "Eagle", "'s", "medium", "(", "DMEM", ")", "and", "F12", "nutrient", ",", "20", "%", "serum", "replacement", "(", "Invitrogen", ")", ",", "0.1", "mM", "β", "-", "mercaptoethanol", ",", "2", "µg", "/", "ml", "heparin", "and", "4", "ng", "/", "ml", "bFGF", "(", "R&D", "Systems", ")", ".", "To", "generate", "the", "NSCs", ",", "dissociated", "hESCs", "were", "cultured", "in", "a", "chemically", "defined", "medium", "composed", "of", "DMEM", "/", "F12", "(", "1∶1", ")", "including", "glucose", "(", "0.6", "%", ")", ",", "glutamine", "(", "2", "mM", ")", ",", "sodium", "bicarbonate", "(", "3", "mM", ")", ",", "and", "HEPES", "buffer", "(", "5", "mM", ")", "[", "all", "from", "Sigma", "except", "glutamine", "(", "Invitrogen", ")", "]", ".", "A", "defined", "hormone", "mix", "and", "salt", "mixture", "(", "Sigma", ")", ",", "including", "insulin", "(", "25", "mg", "/", "ml", ")", ",", "transferrin", "(", "100", "mg", "/", "ml", ")", ",", "progesterone", "(", "20", "nM", ")", ",", "putrescine", "(", "60", "mM", ")", ",", "and", "selenium", "chloride", "(", "30", "nM", ")", "was", "used", "in", "place", "of", "serum", ".", "The", "medium", "was", "supplemented", "with", "EGF", "(", "20", "ng", "/", "ml", ")", ",", "bFGF", "(", "10", "ng", "/", "ml", ")", "and", "LIF", "(", "10", "ng", "/", "ml", ")", ".", "Dissociated", "hNSCs", "were", "plated", "at", "a", "density", "of", "100,000", "cell", "/", "ml", "in", "Corning", "T75", "(", "Invitrogen", ")", "culture", "flasks", "in", "the", "defined", "media", "together", "with", "the", "growth", "factors", ".", "After", "5–7", "DIV", ",", "the", "adherent", "culture", "was", "incubated", "in", "0.025%trypsin/0.01", "%", "EDTA", "(", "w", "/", "v", ")", "for", "1", "min", "followed", "by", "the", "addition", "of", "trypsin", "inhibitor", "(", "Invitrogen", ")", "then", "gently", "triturated", "to", "achieve", "single", "cell", "suspension", ".", "The", "cells", "were", "then", "washed", "twice", "with", "fresh", "medium", "and", "reseeded", "in", "fresh", "growth", "factor", "-", "containing", "media", "at", "100,000", "cells", "/", "ml", ".", "This", "procedure", "was", "performed", "for", "21", "passages", "and", "the", "fold", "of", "increase", "and", "population", "doubling", "were", "calculated", "at", "each", "passage", ".", "For", "clonal", "analysis", ",", "single", "spheres", "or", "confluent", "hNSC", "cultures", "were", "single", "cell", "dissociated", "and", "serially", "diluted", "to", "yield", "1–2", "cell/10", "µl", ".", "A", "10-µl", "-", "cell", "suspension", "was", "then", "added", "to", "each", "of", "96", "or", "24", "well", "plates", "containing", "200–300", "µl", "of", "growth", "media", ".", "Wells", "containing", "one", "viable", "cell", "were", "marked", "the", "next", "day", "and", "re", "-", "scored", "5", "to", "7", "days", "later", "for", "cell", "proliferation", ".", "The", "differentiation", "of", "the", "hNSCs", "was", "performed", "as", "previously", "described", "[", "48", "]", ".", "Dissociated", "hNSCs", "were", "plated", "at", "a", "density", "of", "106", "cells", "/", "ml", "in", "control", "(", "media", "/", "hormone", "mix", ")", "medium", "devoid", "of", "any", "growth", "factors", "and", "supplemented", "with", "1", "%", "fetal", "bovine", "serum", "(", "FBS", ")", "on", "poly", "-", "L", "-", "ornithine", "-", "coated", "(", "15", "mg", "/", "ml", ";", "Sigma", ")", "glass", "coverslips", "in", "24-well", "Nunclon", "culture", "dishes", "with", "0.5", "ml", "/", "well", ".", "After", "2", ",", "7–15", "DIV", "cultures", "were", "fixed", "and", "processed", "for", "immunocytochemistry", "or", "used", "for", "RT", "-", "PCR", "analysis", ".", "Karyotype", "analysisLong", "-", "term", "cultures", "of", "hNSCs", "were", "incubated", "at", "37", "°", "C", "and", "harvested", "for", "metaphase", "chromosomes", "when", "the", "cultures", "were", "75", "%", "confluent", ".", "Metaphase", "chromosomes", "were", "obtained", "by", "standard", "chromosome", "harvest", "methods", "by", "exposure", "to", "Colcemid", "at", "0.1", "µg", "/", "ml", "for", "1", "hour", "at", "37", "°", "C", ",", "a", "2-minute", "exposure", "to", "trypsin", "/", "EDTA", ",", "hypotonized", "with", "0.057", "M", "KCl", "and", "fixed", "with", "3∶1", "methanol", ":", "acetic", "acid", ".", "Slide", "preparations", "were", "made", "by", "dropping", "the", "fixed", "cell", "pellet", "onto", "cold", ",", "wet", "slides", "and", "air", "-", "dried", ".", "After", "incubating", "the", "slides", "at", "90", "°", "C", "for", "30", "minutes", ",", "chromosomes", "were", "trypsin", "banded", "and", "then", "Wright", "/", "Giemsa", "stained", "for", "G", "-", "banding", "analysis", ".", "Twenty", "metaphase", "cells", "were", "completely", "analyzed", "and", "a", "normal", "female", "chromosome", "complement", "was", "found", "(", "46,XX", ")", ".", "Tumorigenicity", "in", "nude", "ratsAll", "animal", "experiments", "were", "conducted", "according", "to", "the", "National", "Institute", "of", "Health", "(", "NIH", ")", "guidelines", "and", "approved", "by", "the", "IACUC", ".", "Normal", "adult", "NIH", "-", "Nude", "rats", "(", "n", " ", "=", " ", "5", ",", "8", "week", "-", "old", ",", "Taconic", ",", "Germantown", ",", "New", "York", ",", "United", "States", ")", "were", "used", "to", "test", "the", "tumorigenic", "potential", "of", "the", "SD56", "hNSCs", ".", "Undifferentiated", "hNSCs", "from", "passage", "9", "were", "single", "cell", "dissociated", "using", "trypsin", "-", "EDTA", "and", "suspended", "at", "the", "concentration", "of", "125,000", "cell/µl", "in", "preparation", "for", "cell", "transplantation", ".", "Two", "µl", "of", "the", "cell", "suspension", "were", "stereotaxically", "transplanted", "into", "4", "sites", "within", "the", "striatum", "at", "the", "following", "coordinates", ":", "AP", ":", "+1.0", "mm", ",", "ML", ":", "+3.2", "mm", ",", "DV", ":", "−5.0", ";", "AP", ":", "+0.5", "mm", ",", "ML", ":", "+3.0", "mm", ",", "DV", ":", "−5.0", ";", "AP", ":", "−0.5", "mm", ",", "ML", ":", "+3.0", "mm", ",", "DV", ":", "−5.0", ";", "AP", ":", "−1.0", "mm", ",", "ML", ":", "+3.5", "mm", ",", "DV", ":", "−5.0", "mm", "with", "the", "incisor", "bar", "set", "at", "3.4", "mm", ".", "The", "injection", "rate", "was", "1", "µl", "/", "min", ",", "and", "the", "cannula", "was", "left", "in", "place", "for", "an", "additional", "5", "min", "before", "retraction", ".", "For", "the", "flank", "tumor", "assay", ",", "2×106", "cells", "(", "125,000", "cell/µl", ")", "were", "injected", "subcutaneously", "to", "the", "side", "of", "the", "adult", "nude", "rats", ".", "To", "label", "mitotically", "active", "cells", "in", "vivo", "during", "S", "-", "phase", ",", "the", "rats", "were", "injected", "IP", "with", "the", "BrdU", "(", "50", "mg", "/", "kg", ",", "Sigma", ")", "every", "8", "hours", "during", "the", "last", "24", "hours", "before", "euthanasia", ".", "After", "2-month", "survival", "time", ",", "rats", "were", "euthanized", "and", "a", "postmortem", "examination", "for", "tumor", "formation", "was", "performed", ".", "Induction", "of", "Focal", "Ischemia", "and", "Cell", "TransplantationAll", "animal", "experimentations", "were", "conducted", "according", "to", "the", "National", "Institute", "of", "Health", "(", "NIH", ")", "guidelines", "and", "approved", "by", "the", "IACUC", ".", "Sprague", "Dawley", "adult", "male", "rats", "(", "n", " ", "=", " ", "17", ",", "275g–310", "g", ",", "Charles", "River", "Laboratories", ",", "Wilmington", ",", "Massachusetts", ",", "United", "States", ")", "were", "subjected", "to", "one", "and", "a", "half", "hour", "suture", "occlusion", "of", "the", "middle", "cerebral", "artery", "(", "MCAO", ")", ",", "as", "previously", "described", "[", "49", "]", "and", "immunosuppressed", "2", "days", "before", "cell", "transplantation", "and", "daily", "thereafter", "for", "one", "week", "with", "i.p", ".", "injections", "of", "cyclosporine", "A", "(", "20", "mg", "/", "ml", ",", "Sandimmune", ",", "Novartis", "Pharmaceuticals", ")", ".", "Thereafter", "oral", "cyclosporine", "was", "used", "at", "210", "µg", "/", "ml", "in", "drinking", "water", "until", "euthanasia", ".", "Undifferentiated", "SD56", "hNSCs", "from", "passages", "between", "P9", "and", "P13", "were", "single", "cell", "dissociated", "using", "trypsin", "-", "EDTA", "in", "preparation", "for", "cell", "transplantation", ".", "One", "week", "after", "the", "stroke", "lesion", ",", "2", "µl", "of", "the", "hNSCs", ",", "at", "a", "concentration", "of", "50,000", "cell/µl", ",", "were", "stereotaxically", "transplanted", "into", "4", "sites", "within", "the", "lesioned", "striatum", "(", "n", " ", "=", " ", "10", ")", "at", "the", "following", "coordinates", ":", "AP", ":", "+1.0", "mm", ",", "ML", ":", "+3.2", "mm", ",", "DV", ":", "−5.0", ";", "AP", ":", "+0.5", "mm", ",", "ML", ":", "+3.0", "mm", ",", "DV", ":", "−5.0", ";", "AP", ":", "−0.5", "mm", ",", "ML", ":", "+3.0", "mm", ",", "DV", ":", "−5.0", ";", "AP", ":", "−1.0", "mm", ",", "ML", ":", "+3.5", "mm", ",", "DV", ":", "−5.0", "mm", "with", "the", "incisor", "bar", "set", "at", "3.4", "mm", ".", "The", "injection", "rate", "was", "1", "µl", "/", "min", ",", "and", "the", "cannula", "was", "left", "in", "place", "for", "an", "additional", "5", "min", "before", "retraction", ".", "As", "a", "control", "group", ",", "we", "used", "rats", "subjected", "to", "ischemia", "and", "injected", "with", "the", "vehicle", "(", "n", " ", "=", " ", "7", ")", ".", "All", "animals", "underwent", "baseline", "motor", "behavioral", "assessment", "before", "and", "after", "the", "ischemic", "lesion", ",", "and", "4", "&", "8", "weeks", "after", "cell", "transplantation", ".", "The", "animals", "were", "killed", "after", "2-month", "survival", "time", "by", "transcardial", "perfusion", "with", "phosphate", "buffered", "saline", "(", "PBS", ")", "followed", "by", "4", "%", "paraformaldehyde", ".", "The", "brains", "were", "cryoprotected", "in", "an", "increasing", "gradient", "of", "10", ",", "20", "and", "30", "%", "sucrose", "solution", "and", "cryostat", "sectioned", "at", "40", "µm", "and", "processed", "for", "immunocytochemistry", ".", "ImmunocytochemistryCultures", "were", "fixed", "with", "4", "%", "paraformaldehyde", "for", "15", "min", ".", "Both", "cells", "and", "brain", "sections", "were", "rinsed", "in", "PBS", "for", "3×5", "min", "then", "incubated", "for", "2", "hrs", "(", "cultures", ")", "or", "overnight", "(", "brain", "sections", ")", "with", "the", "appropriate", "primary", "antibodies", "for", "multiple", "labeling", ".", "Secondary", "antibodies", "raised", "in", "the", "appropriate", "hosts", "and", "conjugated", "to", "FITC", ",", "RITC", ",", "AMCA", ",", "CY3", "or", "CY5", "chromogenes", "(", "Jackson", "ImmunoResearch", ")", "were", "used", ".", "Cells", "and", "sections", "were", "counterstained", "with", "the", "nuclear", "marker", "4′,6-diamidine-2′-phenylindole", "dihydrochloride", "(", "DAPI", ")", ".", "Positive", "and", "negative", "controls", "were", "included", "in", "each", "run", ".", "Immunostained", "sections", "were", "coverslipped", "using", "fluorsave", "(", "Calbiochem", ")", "as", "the", "mounting", "medium", ".", "The", "following", "antibodies", "were", "used", ":", "Anti", "-", "human", "Nuclei", "(", "hNuc", ",", "monoclonal", "1∶100", ",", "Chemicon", ")", ",", "Anti", "-", "TuJ1", "(", "monoclonal", "1∶100", ",", "Covance", ";", "Polyclonal", "1∶200", ",", "Aves", "Labs", ")", ";", "anti", "-", "GAD65/67", "(", "polyclonal", "1∶1000", ",", "Chemicon", ")", ";", "Anti", "-", "glial", "fibrillary", "acidic", "protein", "(", "GFAP", ",", "monoclonal", "1∶1000", ",", "Chemicon", ";", "polyclonal", "1∶200", ",", "Aves", "Labs", ")", ";", "Anti", "-", "galactocerebrocide", "(", "GC", ",", "monoclonal", "1∶250", ",", "Chemicon", ")", ";", "Anti", "-", "CNPase", "(", "polyclonal", "1∶200", ",", "Aves", "Labs", ")", ";", "Anti", "-", "Glucose", "Transporter", "type", "1", "(", "Glut-1", "polyclonal", ",", "1∶500", ",", "Chemicon", ")", ";", "Anti", "-", "Nestin", "(", "polyclonal", "1∶1000", ",", "Chemicon", ")", ";", "Anti", "-", "vimentin", "(", "monoclonal", "1∶500", ",", "Calbiochem", ")", ";", "Anti-3CB2", "(", "monoclonal", "1∶500", ",", "Developmental", "Studies", "Hybridoma", "Bank", ")", ";", "Anti", "-", "doublecortin", "(", "DCX", ",", "polyclonal", "1∶250", ",", "SantaCruz", "Biotechnology", ")", ";", "Anti", "-", "Ki67", "(", "polyclonal", "1∶250", ",", "SantaCruz", "Biotechnology", ")", ".", "Fluorescence", "was", "detected", ",", "analyzed", "and", "photographed", "with", "a", "Zeiss", "LSM550", "laser", "scanning", "confocal", "photomicroscope", ".", "For", "each", "animal", ",", "quantitative", "estimates", "of", "the", "total", "number", "of", "grafted", "cells", "were", "stereologically", "determined", "using", "the", "optical", "fractionator", "procedure", "[", "50", "]", ".", "A", "computer", "-", "assisted", "image", "analysis", "system", "was", "performed", "using", "Stereo", "Investigator", "software", "(", "MicroBrightField", ",", "Inc", ".", ")", ".", "The", "rostral", "and", "caudal", "limits", "of", "the", "reference", "volume", "were", "determined", "by", "first", "and", "last", "frontal", "sections", "containing", "grafted", "cells", ".", "The", "striatum", "and", "cortex", "were", "accurately", "outlined", "at", "low", "magnification", "(", "2.5×", "objective", ")", ".", "The", "optical", "fractionator", "probe", "was", "selected", "to", "perform", "systematic", "sampling", "of", "the", "immunoreactive", "cell", "population", "distributed", "within", "the", "serial", "sections", "to", "estimate", "the", "population", "number", "in", "the", "volume", "of", "tissue", ".", "The", "counting", "frame", "of", "the", "optical", "fractionator", "was", "defined", "at", "50×50", "µm", "squares", "and", "the", "systematic", "sampling", "was", "performed", "by", "translating", "a", "grid", "with", "200×200", "µm", "squares", "onto", "the", "sections", "of", "interest", "using", "the", "Stereo", "Investigator", "software", ".", "The", "sample", "sites", "were", "systematically", "and", "automatically", "generated", "by", "the", "computer", "and", "examined", "using", "a", "60×", "objective", "of", "a", "Nikon", "Eclipse", "TE", "300", "microscope", ".", "The", "counting", "frame", "displayed", "inclusion", "and", "exclusion", "lines", "and", "only", "immunoreactive", "cell", "bodies", "falling", "within", "the", "counting", "frame", "with", "no", "contact", "with", "the", "exclusion", "lines", "were", "counted", ".", "The", "cell", "dispersion", "was", "measured", "by", "counting", "the", "number", "of", "cells", "within", "200", "µm", "distance", "from", "the", "graft", "site", ".", "The", "number", "and", "distance", "in", "µm", "of", "cells", "dispersed", "beyond", "200", "µm", "was", "also", "measured", ".", "An", "average", "of", "2,000", "cells", "was", "counted", "per", "animal", ".", "Double", "labeling", "was", "determined", "using", "the", "confocal", "laser", "scanning", "microscope", "by", "random", "sampling", "of", "100", "or", "more", "cells", "per", "marker", "for", "each", "animal", ",", "scoring", "first", 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human Nuclei is a GeneProtein, hNuc is a GeneProtein, Anti - TuJ1 is a GeneProtein, anti - GAD65/67 is a GeneProtein, Anti - glial fibrillary acidic protein is a GeneProtein, GFAP is a GeneProtein, Anti - galactocerebrocide is a GeneProtein, GC is a GeneProtein, Anti - CNPase is a GeneProtein, Anti - Glucose Transporter type 1 is a GeneProtein, Glut-1 is a GeneProtein, Anti - Nestin is a GeneProtein, Anti - vimentin is a GeneProtein, Anti-3CB2 is a GeneProtein, Anti - doublecortin is a GeneProtein, DCX is a GeneProtein, Anti - Ki67 is a GeneProtein, rostral is a Anatomy, caudal is a Anatomy, striatum is a Anatomy, cortex is a Anatomy, nuclei is a CellComponent, Polymerase is a GeneProtein, RNase is a GeneProtein, Transcriptase is a GeneProtein, polymerase is a GeneProtein, GFAP is a GeneProtein, MAP2 is a GeneProtein, MBP is a GeneProtein, β - tubulin class III is a GeneProtein, N - CAM is a GeneProtein, Nestin is a GeneProtein, NF - M is a GeneProtein, Notch-1 is a GeneProtein, Oct4 is a GeneProtein, Nanog is a GeneProtein, FOXa2 is a GeneProtein, HNF3B is a GeneProtein, Brachyury is a GeneProtein, Rats is a Species, forelimbs is a Anatomy, forelimbs is a Anatomy
18286199_task1
Sentence: BackgroundHuman embryonic stem cells (hESCs) offer a virtually unlimited source of neural cells for structural repair in neurological disorders, such as stroke. Neural cells can be derived from hESCs either by direct enrichment, or by isolating specific growth factor-responsive and expandable populations of human neural stem cells (hNSCs). Studies have indicated that the direct enrichment method generates a heterogeneous population of cells that may contain residual undifferentiated stem cells that could lead to tumor formation in vivo.Methods/Principal FindingsWe isolated an expandable and homogenous population of hNSCs (named SD56) from hESCs using a defined media supplemented with epidermal growth factor (EGF), basic fibroblast growth factor (bFGF) and leukemia inhibitory growth factor (LIF). These hNSCs grew as an adherent monolayer culture. They were fully neuralized and uniformly expressed molecular features of NSCs, including nestin, vimentin and radial glial markers. These hNSCs did not express the pluripotency markers Oct4 or Nanog, nor did they express markers for the mesoderm or endoderm lineages. The self-renewal property of the hNSCs was characterized by a predominant symmetrical mode of cell division. The SD56 hNSCs differentiated into neurons, astrocytes and oligodendrocytes throughout multiple passages in vitro, as well as after transplantation. Together, these criteria confirm the definitive NSC identity of the SD56 cell line. Importantly, they exhibited no chromosome abnormalities and did not form tumors after implantation into rat ischemic brains and into naïve nude rat brains and flanks. Furthermore, hNSCs isolated under these conditions migrated toward the ischemia-injured adult brain parenchyma and improved the independent use of the stroke-impaired forelimb two months post-transplantation.Conclusions/SignificanceThe SD56 human neural stem cells derived under the reported conditions are stable, do not form tumors in vivo and enable functional recovery after stroke. These properties indicate that this hNSC line may offer a renewable, homogenous source of neural cells that will be valuable for basic and translational research. To date there have been no effective treatments for improving residual structural and functional deficits resulting from stroke. Current therapeutic approaches, such as the use of thrombolytics, benefit only 1 to 4% of patients [1]. Consequently, the majority of stroke patients experience progression of ischemia associated with debilitating neurological deficits. Recent evidence has suggested that the transplantation of cells derived from cord blood, bone marrow or brain tissue (fetal and adult) enhances sensorimotor function in experimental models of stroke [2], [3]. However, the normal human-derived somatic stem cells used in these studies have a limited capacity to differentiate into the diverse neural cell types optimal for structural and physiological tissue repair and are not amenable for large-scale cell production. Unlike other sources of stem cells, hESC lines possess a nearly unlimited self-renewal capacity and the developmental potential to differentiate into virtually any cell type of the organism. As such, they constitute an ideal source of cells for regenerative medicine. The successful derivation of hESC lines from the inner cell mass of preimplantation embryos and their long-term maintenance in vitro over multiple passages has been demonstrated [4] and standardized. Differentiation and enrichment processes that direct hESCs towards a neural lineage have also been achieved. To promote neuralization, ESCs were cultured in a defined media supplemented with morphogens or growth factors [5], [6], [7] or cultured under conditions that promote “rosettes”, structures morphologically similar to the developing neural tube [8], [9]. This neuralization process has proven invaluable in understanding the specification of hESC-derived neural tissue [10], [11], [12]. However, the enriched neural progeny derived displayed overgrowth and limited migration after grafting into normal newborn mice [13] and lesioned adult rat striatum [12], [14], [15], [16]. The inner cores of these grafts contained tumorigenic precursor cells (reviewed in [17]). These findings suggest that neural cells generated by acute exposure to growth factors and/or morphogens may still be heterogeneous and potentially tumorigenic. We report an alternative method for the isolation and the perpetuation of a multipotent hNSC line from the hESCs with a primitive mode of self-renewal. We also demonstrate their long-term expansion, non-tumorigenic properties and functional engraftability in an experimental model of stroke. 1. In vitro isolation, growth and differentiation of hESC-derived hNSCsThe hESCs were maintained and expanded on mouse feeder layer in media supplemented with bFGF (Figure 1A). After cell dissociation, a portion of the hESCs was cultured in serum free medium containing EGF, bFGF and LIF. These factors are known to stimulate the proliferation of human fetal-derived NSCs [18], [19]. After 3 days in vitro (DIV), there was selective survival and growth of cells that aggregated in clusters or spheres (Figure 1B). These primary spheres were harvested and replated in fresh media. During the following week, the spheres attached to the flask and a fibroblast-like cell population began to migrate out (Figure 1C). Secondary spheres (2° spheres) were generated from these cultures and lifted off by the end of the week leaving a hollow in the middle of the attached cells (Figure 1D). The floating 2° spheres were collected and replated in fresh growth medium for 2 weeks. The cultures were then passaged by collagenase cell dissociation every 7 DIV for an additional 4 passages (Figure S1). At the 5th and 6th passages, spheres were dissociated into a single-cell suspension using trypsin-EDTA. At this stage there was a change in the hNSCs' adherent properties, and the cells began to grow as a monolayer with multiple foci of cells throughout the culture (Figure 1F). The adherent hNSC culture stained uniformly for nestin (Figure 1K), vimentin (Figure 1L) and with the radial glial marker 3CB2 (Figure 1M) indicating their homogeneity and NSC identity. Under these culture conditions, it is noteworthy that we did not observe the formation of rosettes which has been previously reported to occur under certain conditions during neuralization of hESCs [8], [20], [21]. RT-PCR analysis confirmed that these hNSCs did not express the pluripotency transcripts Oct-4 and Nanog (Figure 1I). Furthermore, the hNSCs did not express transcripts for brachyury and foxa2, marker genes for mesoderm and endoderm respectively (negative result, data not shown).10.1371/journal.pone.0001644.g001Figure 1Isolation and purification of hNSCs from the hESCs.The hESCs were grown on a mouse feeder layer (A). Primary neurospheres (B) were isolated and replated to eliminate other non-neural cells. The selectively harvested secondary neurospheres (arrow in C), left behind hollow cores in the surface area (star in D) where they attached earlier. They were perpetuated for an additional 5 passages (E). These 2° spheres were then passaged using a single cell dissociation protocol (F). Arrow in F shows an example of a focus of proliferating cells. (G, H) The hNSCs were passaged every 5–7 days, as described in the Methods section. Starting from an initial population of 1 million cells, the cumulative cell number was calculated at each passage as the fold of increase×the total cell number and plotted as logarithm with base 2 in function of time (G). The cell perpetuation (G) and population doubling (H) analysis demonstrated the continuous and stable growth of the hNSCs. (I) RT-PCR analysis showing the down regulation of the pluripotency transcripts Oct4 and Nanog in secondary neurospheres and in expanded hNSCs at passage 8 (P8). (J) Cytogenetic evaluation of the SD56 hNSCs line at passage 12 by standard G-banding was performed. Twenty metaphase cells were analyzed and showed a normal female chromosome complement (46,XX). Isolated and expanded hNSCs expressed the neural precursor cell markers nestin (K), Vimentin (L) and the radial glial cell marker 3CB2 (M) in virtually all the progeny. (N-P) Clonal self-renewal ability of the isolated hNSCs through symmetrical cell division. Single (N), two-cell stage (O) and four-cell stage (P) of a hNSC proliferating over a 3-day culture period. Note the symmetrical segregation of BrdU and nestin in the progeny. Bars: (A, B, C, D) 200 µm; (E, F) 100 µm; (K–M) 20 µm; (N–P) 10 µm.To ascertain self-renewal ability under clonal conditions, a single cell suspension was plated at clonal density (1–2 cell/10 µl). To determine if the hNSCs divide symmetrically, we pulsed cultures with the thymidine analog, bromodeoxyuridine (BrdU), after plating and looked for the expression of nestin in the progeny. Cultures were fixed after 1, 2 or 3 DIV (Figure 1N–P). After 2 days, plated single cells first underwent a symmetric cell division and gave rise to daughter cells that were both positive for BrdU and nestin. The clone of cells continued to grow over the 3 DIV and all the progeny expressed nestin. BrdU labeling demonstrated that it was rare for only one daughter cell to inherit the BrdU and thus had undergone asymmetric segregation of the chromatids (Figure S2). G-band karyotyping of these hNSCs confirmed the normal, non-transformed nature of cells after 12 passages (Figure 1J). We named the derived hNSCs SD56 (intermittently referred to as SD56 hNSCs or hNSCs).Under these defined growth conditions, the hNSCs showed stable growth with a 2.7±0.2 fold increase every 5 to 7 days (Figure 1G). The population doubling at each passage averaged at 1.4±0.1 (Figure 1H). The viability of hNSCs at each passage was consistent at the approximate value of 98%. The projected cumulative cell numbers demonstrated that trillions of cells could be generated over a period of 5 months (Figure 1G). We expanded the isolated hNSCs lines for over 20 passages with a stable phenotype. An initial cell bank of 75 vials containing 2 to 5 million cells each was generated and cryopreserved.Upon removal of the mitogenic factors and plating on a coverslip pre-coated with poly-L-ornithine (PLO) substrate, the hNSCs spontaneously differentiated into neurons, astrocytes and oligodendrocytes, a property that is consistent with normal multipotent hNSCs (Figure 2). After 2 DIV, hNSCs expressed transcripts for the neural-specific genes nestin, Notch1 and neural cell adhesion molecule (N-CAM) (Figure 2A) and for the lineage specific markers β-tubulin class III, medium-size neurofilament (NF-M) and microtubule-associated protein 2 (MAP-2) for neurons, GFAP for astrocytes and myelin basic protein (MBP) for oligodendrocytes (Figure 2A). Transcripts for the GABAergic cell marker glutamic acid decarboxylase (GAD) were expressed, but transcripts for the tyrosine hydroxylase (TH), a marker for dopaminergic neurons, were undetectable. Immunocytochemical analysis (Figure 2B–F) of 10 day-old cultures demonstrated that the proportion of nestin+ cells was 36.6±2.7%, 62.5±2.8% expressed the neuronal marker TuJ1, 1.9±0.3% expressed the astrocytic marker GFAP and 7.1±0.4% were oligodendrocytes and expressed galactocerebrocide (GC) (Figure 2F). A subset (9.8±1.6%) of the GFAP+ astrocytes co-expressed nestin.10.1371/journal.pone.0001644.g002Figure 2hESC-derived hNSCs spontaneously differentiated into the 3 principal central nervous system cell types.Dissociated hNSCs were washed free of growth factors and plated on poly-L-onithine coated glass coverslips. Differentiated cultures were either harvested after 2 DIV for total RNA extraction and RT-PCR analysis or fixed after 10 DIV and processed for indirect immunocytochemistry. (A) Differentiated hNSCs expressed the neural-specific transcripts nestin and Notch1 as well as transcripts: for neurons (β-tubulin class III, MAP-2, NCAM and medium-size neurofilament, NF-M), for astrocytes (GFAP) and for oligodendrocytes (MBP). The hNSCs expressed transcripts for GAD, but not for TH. B, C & D, morphology of NSC-derived progeny differentiated into GFAP+ astrocytes (B), GC-expressing oligodendrocytes (C) and TuJ1+ neurons (D), DAPI (blue) show life cell nuclei. (E) Photo showing cultures double-immunostained for TuJ1 (green) and nestin (red) (DAPI, blue). (F) Quantitative analysis of immunostained 10 day-old cultures for the 3 neural cell types. Results are mean±s.e.m. of three independent experiments, each performed in duplicate. Bars: (c) 20 µm; (d, e) 10 µm. 2. The isolated hNSCs are normal and do not form tumors in normal nude ratsThe self-renewal and pluripotent abilities of the hESCs are also associated with tumorigenic properties. Therefore, the first critical step toward developing therapeutic hNSCs is to verify that they are non-tumorigenic. The monolayer culture of SD56 hNSCs clearly demonstrated contact inhibition of growth, a normal karyotype and did not express the pluripotency transcripts Oct-4 and Nanog. Removal of mitogenic factors and continued culture on plastic resulted in cell senescence that is characteristic of non-transformed cells. To determine whether SD56 hNSCs form tumors in vivo, we transplanted them at high density (see Methods) into the forebrain and subcutaneously into the flank of nude rats. The animals were kept for a two-month post-transplant survival period. To label mitotically active cells in vivo during S-phase, the rats were injected IP with BrdU (50 mg/kg) every 8 hours during the last 24 hours before euthanasia. The transit amplifying endogenous precursors located in the subventricular zone (SVZ) were labeled (Figure S3); however, we were unable to detect grafted SD56 hNSCs co-localizing the human-specific nuclear marker hNuc and BrdU (Figure S3). No surviving SD56 hNSCs were detected in the flank of the transplanted animals suggesting that the grafted cells are not tumorigenic. 3. Transplanted cells survived, migrated toward and engrafted into the stroke-damaged host tissueTo investigate the survival and functional engraftment in an injury environment, hNSCs (4×105) were transplanted into the ischemic boundary zone in the rat striatum one week after the middle cerebral artery occlusion (MCAO) was performed. Animals were euthanized two months later and the brains processed for histo-pathology and immunocytochemistry. Grafted SD56 hNSCs, identified with hNuc, demonstrated a 37.0±15.8% survival rate and a remarkable dispersion toward the stroke-damaged tissue with no sign of overgrowth or tumorigenesis. The majority of grafted cells (61.2±4.7%) migrated at least 200 µm away from the injection site and penetrated an average distance of 806.4±49.3 µm into the stroke-damaged tissue (Figure 3A–C). Immunostaining with the blood vessel marker, GluT1, revealed dilated vessels in the infarcted striatum and a close association between vessels and the grafted hNSCs (Figure 3B, 3C). The grafted cells rarely expressed the proliferation marker Ki67 (Figure 3D), 29.8±4.4% expressed nestin (Figure 3E), 6.5±0.9% expressed doublecortin (DCX) and 60.8±8.1% were TuJ1+ (Figure 3F, G). Grafted cells rarely co-expressed the astroglial marker GFAP (Figure 3H) or differentiated into CNPase-expressing oligodendrocytes (Figure 3I). Immunostaining for GAD demonstrated that 25.1±2.3% of grafted hNSCs differentiated into GABAergic neurons while less than 2% were positive for glutamate (Figure 3J, K).10.1371/journal.pone.0001644.g003Figure 3Dispersion, engraftment and differentiation of the hNSCs in stroke-lesioned animals.(A) Schematic drawing of a frontal section through the striatum illustrating the dispersion of grafted hNSCs in the focal ischemia-lesioned parenchyma (shaded area). (B, C) Photos show frontal sections through the graft in the striatum immunostained with the human specific antibodies: anti-hNuc (green in B & C) and anti-GluT1 (red, B & C) showing blood vessels and dispersed hNSCs in the graft zone. C: higher magnification of the inset in B. (D–I) Photos taken from frontal sections through the graft in the striatum double immunoprocessed for cell proliferation and neural lineage markers. (D) Note the endogenous Ki67+ cells (red cells, arrow) in the stroke damaged area and the hNuc+ (green)/Ki67- grafted hNSCs (arrowheads). (E) Examples of grafted SD56 hNSCs showing co-expression of hNuc (green) and nestin (red). (F) Confocal 3D reconstructed orthogonal images of the hNuc+(green)/DCX+(red) NSCs (arrowheads) viewed in the x-z plan on the top and y-z plan on the right. (G) Examples show the majority of grafted NSC progeny co-expressing hNuc (red) and the neuronal marker TuJ1 (green). Grafted NSCs rarely differentiate into GFAP+ astrocytes (H). In I, rare example of grafted NSC progeny becoming an oligodendrocyte identified by the expression of CNPase (green). Grafted NSCs expressed the GABAergic marker GAD65/67 (J) and rarely expressed glutamate (K). (Abbreviations: Cx: cortex, Str: striatum). Bars: (B, C) 100 µm; (D, F) 20 µm; (E, G–K) 10 µm. 4. Transplanted cells improve sensorimotor function of the stroke-disabled forelimbWe asked whether transplanted SD56 hNSCs could enhance the recovery of sensorimotor function that is compromised in the stroke-injured rats. We used the cylinder test to measure the sensorimotor asymmetry in forelimb use during spontaneous exploration [22]. To establish the baseline of the stroke-induced sensorimotor deficit, spontaneous behavior of rats in a transparent cylinder was videotaped one week after stroke (pre-transplant, Figure 4). Tests were then conducted 4 and 8 weeks after vehicle and SD56 hNSCs transplantation. Stable asymmetry in forelimb use was observed 7 days post-stroke (pre-transplant, Figure 4). Ischemic rats used their impaired forelimbs (contralateral to lesion) during lateral exploration less than they did before stroke. Transplantation of SD56 hNSCs significantly enhanced the independent use of the impaired contralateral forelimb 4 weeks post transplantation (P<0.05 vs pre-transplant). Eight weeks after transplantation the improvement in the use of the impaired forelimb was stable and significant when compared to the pre-transplant group and significantly improved in comparison to vehicle treated group at 8 weeks (Figure 4). In the vehicle treated group, the independent use of the contralateral forelimb remained impaired 4 and 8 weeks post-injection. In an independent study and using the same MCAO rat animal model, we found that transplantation of dermal fibroblasts did not improve the stroke-induced motor deficits (unpublished data).10.1371/journal.pone.0001644.g004Figure 4Transplantation of NSCs improves sensorimotor function of the stroke-disabled forelimb.Forelimb use during spontaneous lateral exploration was measured in the cylinder test (see Method and Results sections for details). Groups of vehicle injected (n = 7) and hNSCs (n = 10) transplanted are represented. The animals were tested as described in Method section. Note the significant increase in the independent use of the impaired contralateral forelimb at 4 and 8 weeks post transplantation (P<0.05 vs pre-transplant group). The contralateral forelimb remained impaired in the vehicle treated group at 4 and 8 weeks post-injection. Bars represent percentages±s.e.m. of steps taken by the ipsilateral, contralateral and both forelimbs simultaneously. *P<0.05 vs pre-transplant group; #P<0.05 vs vehicle groups. Our results indicate that a self-renewable and homogenous population of hNSCs, SD56, was derived from hESCs using defined media supplemented with a specific combination of growth factors. The SD56 hNSCs grew as an adherent monolayer culture, uniformly expressed molecular features of hNSCs including nestin, vimentin and the radial glial marker 3CB2, and did not express the pluripotency markers Oct4 or Nanog. The self-renewal property of the hNSCs was characterized by a predominant symmetrical mode of cell division. They exhibited no chromosomal abnormalities and demonstrated non-tumorigenic properties after implantation into ischemic brains and into naïve nude rat brains and flanks. Furthermore, the transplanted SD56 hNSCs migrated toward the stroke-damaged adult brain parenchyma, engrafted and improved the independent use of the stroke-impaired forelimb. Maintenance of stem cells requires symmetrical and asymmetrical cell divisions to both expand and to give rise to specialized progeny of a specific tissue (reviewed in [23]). In vivo, a complex cellular micro-environment or niche ensures the self-maintenance property of NSCs [24], [25], [26], [27]. In vitro, defined growth factors and extracellular matrices support stem cell self-renewal [28], [29]. The embryonic stem cells can propagate in a predominantly proliferative symmetrical mode, leading to homogeneous cell cultures growing relatively quickly with minimal cell differentiation [30], [31], [32], [33], [34]. Tissue specific stem cells, however, self-renew in a predominant asymmetric mode to maintain them selves and compensate for the loss of differentiated cells due to disease or injury. Thus, NSCs isolated from developing or adult brain grow as a mixture of undifferentiated and differentiated cells due the predominant asymmetrical mode of cell division [35], [36], [37], [38], [39], [40], [41]. A recent study has reported that a murine ESC-derived NSC line (LC1) is propagated as an adherent homogenous culture with a dominant mode of symmetrical self-renewal [21]. A combination of EGF and FGF2 was sufficient to propagate these NSCs as an adherent monolayer. However, the SD56 hNSC line described here required the combination of EGF, bFGF and LIF for self-maintenance. Although there are morphological and molecular similarities between our hNSCs and the NSCs previously described [21], the methods of isolation and growth are different. In addition to the different combination of growth factors used, the hNSC line we have isolated did not go through the rosette-structure stage. The in vitro analysis of BrdU incorporation and nestin expression indicated that our hNSCs divide predominantly symmetrically. This type of growth pattern is characteristic of primitive normal stem cells undergoing mostly symmetric cell division to increase the stem cell pool at the early stage of development or during tissue regeneration after injury [23]. RT-PCR and immunocytochemistry analysis demonstrated that these undifferentiated SD56 cells did not express any pluripotency, endodermal or mesodermal markers. Furthermore, the SD56 hNSCs described here exhibited the multipotential characteristic to differentiate into neurons, astrocytes and oligodendrocytes both in vitro and upon transplantation. Together these findings suggest that the hNSC line we isolated are appropriately programmed and share similar characteristics with the definitive NSCs of the developing brain. The SD56 hNSCs demonstrated a remarkable ability to migrate toward the stroke-damaged parenchyma of the adult rat brain. This directed migration by the majority of the grafted cells could be due to their uniform cellular composition, which results in an equal response to the host microenvironment. In previous studies, subpopulations of transplanted hESCs that were enriched in neural cells migrated in host microenvironments conducive to cell migration, such as the developing brain or in structures such as the rostral migratory stream [13], [20]. In the adult lesioned brain, the grafted hESC-derived neural cells proliferated and formed rosettes [14], teratomas [12], [15] or a cellular mass that induced a gliotic host response whereby local astrocytes demarcated the grafts [16]. Enriched neural cultures derived from mouse [42] and monkey ESCs [43] have produced behavioral improvements when transplanted into animal models of stroke and brain injury. However, in these cases, the transplanted non-human ESCs also formed a mass with signs of overgrowth in the core, as well as deformations [44], [45], [46]. ESCs plated at low density acquire neural identity within few hours after plating [47]. Interestingly, nearly all viable cells expressed nestin, the early neural fate marker Sox1, and the pluripotency marker Oct4. Together, these studies are seminal and suggest that complete neuralization may not be achieved through certain enrichment processes, consequently the neural cells could revert to a pluripotent stage [17]. The dispersion of the grafted hNSCs within host parenchyma may allow for more graft-host interactions that could stabilize differentiation, inhibit growth and prevent gliotic host response. In the present study, SD56 hNSC-transplanted animals demonstrated a stable improvement in the sensorimotor function when evaluated for spontaneous exploratory activity in the cylinder test that detects long-lasting deficits in forelimb use in the experimental models of stroke [22]. The transplantation of hNSCs significantly enhanced the independent use of the impaired contralateral forelimb 8 weeks post transplantation. This is the first report demonstrating that the transplantation of hNSCs derived from hESCs can improve neurologic behavior after experimental stroke. Together, these findings are encouraging and suggest that these cells are promising for future development. In addition to their therapeutic application, the hNSCs isolated under the reported conditions offer a means to interrogate host environments and unravel mechanistic features of self-renewal, non-tumorigenicity and functional engraftability in animal models of neurological disorders. hESC and NSC CulturesThe hESC line H9 (WiCell Research Institute) was propagated every 5 to 7 days on irradiated mouse embryonic fibroblasts. The cell culture media consisted of a 1∶1 mixture of Dulbecco's modified Eagle's medium (DMEM) and F12 nutrient, 20% serum replacement (Invitrogen), 0.1 mM β-mercaptoethanol, 2 µg/ml heparin and 4 ng/ml bFGF (R&D Systems). To generate the NSCs, dissociated hESCs were cultured in a chemically defined medium composed of DMEM/F12 (1∶1) including glucose (0.6%), glutamine (2 mM), sodium bicarbonate (3 mM), and HEPES buffer (5 mM) [all from Sigma except glutamine (Invitrogen)]. A defined hormone mix and salt mixture (Sigma), including insulin (25 mg/ml), transferrin (100 mg/ml), progesterone (20 nM), putrescine (60 mM), and selenium chloride (30 nM) was used in place of serum. The medium was supplemented with EGF (20 ng/ml), bFGF (10 ng/ml) and LIF (10 ng/ml). Dissociated hNSCs were plated at a density of 100,000 cell/ml in Corning T75 (Invitrogen) culture flasks in the defined media together with the growth factors. After 5–7 DIV, the adherent culture was incubated in 0.025%trypsin/0.01% EDTA (w/v) for 1 min followed by the addition of trypsin inhibitor (Invitrogen) then gently triturated to achieve single cell suspension. The cells were then washed twice with fresh medium and reseeded in fresh growth factor-containing media at 100,000 cells/ml. This procedure was performed for 21 passages and the fold of increase and population doubling were calculated at each passage. For clonal analysis, single spheres or confluent hNSC cultures were single cell dissociated and serially diluted to yield 1–2 cell/10 µl. A 10-µl-cell suspension was then added to each of 96 or 24 well plates containing 200–300 µl of growth media. Wells containing one viable cell were marked the next day and re-scored 5 to 7 days later for cell proliferation. The differentiation of the hNSCs was performed as previously described [48]. Dissociated hNSCs were plated at a density of 106 cells/ml in control (media/hormone mix) medium devoid of any growth factors and supplemented with 1% fetal bovine serum (FBS) on poly-L-ornithine-coated (15 mg/ml; Sigma) glass coverslips in 24-well Nunclon culture dishes with 0.5 ml/well. After 2, 7–15 DIV cultures were fixed and processed for immunocytochemistry or used for RT-PCR analysis. Karyotype analysisLong-term cultures of hNSCs were incubated at 37°C and harvested for metaphase chromosomes when the cultures were 75% confluent. Metaphase chromosomes were obtained by standard chromosome harvest methods by exposure to Colcemid at 0.1 µg/ml for 1 hour at 37°C, a 2-minute exposure to trypsin/EDTA, hypotonized with 0.057 M KCl and fixed with 3∶1 methanol:acetic acid. Slide preparations were made by dropping the fixed cell pellet onto cold, wet slides and air-dried. After incubating the slides at 90°C for 30 minutes, chromosomes were trypsin banded and then Wright/Giemsa stained for G-banding analysis. Twenty metaphase cells were completely analyzed and a normal female chromosome complement was found (46,XX). Tumorigenicity in nude ratsAll animal experiments were conducted according to the National Institute of Health (NIH) guidelines and approved by the IACUC. Normal adult NIH-Nude rats (n = 5, 8 week-old, Taconic, Germantown, New York, United States) were used to test the tumorigenic potential of the SD56 hNSCs. Undifferentiated hNSCs from passage 9 were single cell dissociated using trypsin-EDTA and suspended at the concentration of 125,000 cell/µl in preparation for cell transplantation. Two µl of the cell suspension were stereotaxically transplanted into 4 sites within the striatum at the following coordinates: AP: +1.0 mm, ML: +3.2 mm, DV: −5.0; AP: +0.5 mm, ML: +3.0 mm, DV: −5.0; AP: −0.5 mm, ML: +3.0 mm, DV: −5.0; AP: −1.0 mm, ML: +3.5 mm, DV: −5.0 mm with the incisor bar set at 3.4 mm. The injection rate was 1 µl/min, and the cannula was left in place for an additional 5 min before retraction. For the flank tumor assay, 2×106 cells (125,000 cell/µl) were injected subcutaneously to the side of the adult nude rats. To label mitotically active cells in vivo during S-phase, the rats were injected IP with the BrdU (50 mg/kg, Sigma) every 8 hours during the last 24 hours before euthanasia. After 2-month survival time, rats were euthanized and a postmortem examination for tumor formation was performed. Induction of Focal Ischemia and Cell TransplantationAll animal experimentations were conducted according to the National Institute of Health (NIH) guidelines and approved by the IACUC. Sprague Dawley adult male rats (n = 17, 275g–310g, Charles River Laboratories, Wilmington, Massachusetts, United States) were subjected to one and a half hour suture occlusion of the middle cerebral artery (MCAO), as previously described [49] and immunosuppressed 2 days before cell transplantation and daily thereafter for one week with i.p. injections of cyclosporine A (20 mg/ml, Sandimmune, Novartis Pharmaceuticals). Thereafter oral cyclosporine was used at 210 µg/ml in drinking water until euthanasia. Undifferentiated SD56 hNSCs from passages between P9 and P13 were single cell dissociated using trypsin-EDTA in preparation for cell transplantation. One week after the stroke lesion, 2 µl of the hNSCs, at a concentration of 50,000 cell/µl, were stereotaxically transplanted into 4 sites within the lesioned striatum (n = 10) at the following coordinates: AP: +1.0 mm, ML: +3.2 mm, DV: −5.0; AP: +0.5 mm, ML: +3.0 mm, DV: −5.0; AP: −0.5 mm, ML: +3.0 mm, DV: −5.0; AP: −1.0 mm, ML: +3.5 mm, DV: −5.0 mm with the incisor bar set at 3.4 mm. The injection rate was 1 µl/min, and the cannula was left in place for an additional 5 min before retraction. As a control group, we used rats subjected to ischemia and injected with the vehicle (n = 7). All animals underwent baseline motor behavioral assessment before and after the ischemic lesion, and 4 & 8 weeks after cell transplantation. The animals were killed after 2-month survival time by transcardial perfusion with phosphate buffered saline (PBS) followed by 4% paraformaldehyde. The brains were cryoprotected in an increasing gradient of 10, 20 and 30% sucrose solution and cryostat sectioned at 40 µm and processed for immunocytochemistry. ImmunocytochemistryCultures were fixed with 4% paraformaldehyde for 15 min. Both cells and brain sections were rinsed in PBS for 3×5 min then incubated for 2 hrs (cultures) or overnight (brain sections) with the appropriate primary antibodies for multiple labeling. Secondary antibodies raised in the appropriate hosts and conjugated to FITC, RITC, AMCA, CY3 or CY5 chromogenes (Jackson ImmunoResearch) were used. Cells and sections were counterstained with the nuclear marker 4′,6-diamidine-2′-phenylindole dihydrochloride (DAPI). Positive and negative controls were included in each run. Immunostained sections were coverslipped using fluorsave (Calbiochem) as the mounting medium. The following antibodies were used: Anti-human Nuclei (hNuc, monoclonal 1∶100, Chemicon), Anti-TuJ1 (monoclonal 1∶100, Covance; Polyclonal 1∶200, Aves Labs); anti-GAD65/67 (polyclonal 1∶1000, Chemicon); Anti-glial fibrillary acidic protein (GFAP, monoclonal 1∶1000, Chemicon; polyclonal 1∶200, Aves Labs); Anti-galactocerebrocide (GC, monoclonal 1∶250, Chemicon); Anti-CNPase (polyclonal 1∶200, Aves Labs); Anti-Glucose Transporter type 1 (Glut-1 polyclonal, 1∶500, Chemicon); Anti-Nestin (polyclonal 1∶1000, Chemicon); Anti-vimentin (monoclonal 1∶500, Calbiochem); Anti-3CB2 (monoclonal 1∶500, Developmental Studies Hybridoma Bank); Anti-doublecortin (DCX, polyclonal 1∶250, SantaCruz Biotechnology); Anti-Ki67 (polyclonal 1∶250, SantaCruz Biotechnology). Fluorescence was detected, analyzed and photographed with a Zeiss LSM550 laser scanning confocal photomicroscope. For each animal, quantitative estimates of the total number of grafted cells were stereologically determined using the optical fractionator procedure [50]. A computer-assisted image analysis system was performed using Stereo Investigator software (MicroBrightField, Inc.). The rostral and caudal limits of the reference volume were determined by first and last frontal sections containing grafted cells. The striatum and cortex were accurately outlined at low magnification (2.5× objective). The optical fractionator probe was selected to perform systematic sampling of the immunoreactive cell population distributed within the serial sections to estimate the population number in the volume of tissue. The counting frame of the optical fractionator was defined at 50×50 µm squares and the systematic sampling was performed by translating a grid with 200×200 µm squares onto the sections of interest using the Stereo Investigator software. The sample sites were systematically and automatically generated by the computer and examined using a 60× objective of a Nikon Eclipse TE 300 microscope. The counting frame displayed inclusion and exclusion lines and only immunoreactive cell bodies falling within the counting frame with no contact with the exclusion lines were counted. The cell dispersion was measured by counting the number of cells within 200 µm distance from the graft site. The number and distance in µm of cells dispersed beyond 200 µm was also measured. An average of 2,000 cells was counted per animal. Double labeling was determined using the confocal laser scanning microscope by random sampling of 100 or more cells per marker for each animal, scoring first for hNuc+, followed by DAPI+ nuclei and then the marker of choice. The double labeling was always confirmed in x-z and y-z cross-sections produced by the orthogonal projections of z-series. Reverse Transcription-Polymerase Chain Reaction (RT-PCR) analysisTotal RNA was extracted from cultured cells using RNAeasy kit (Quiagen). Aliquots (1 µg) of total RNA from the cells were reverse transcribed in the presence of 50 mM Tris-HCl, pH 8.3, 75 mM KCl, 3 mM MgCl2, 10 mM DTT, 0.5 µM dNTPs, and 0.5 µg oligo-dT(12–18) with 200 U Superscript RNase H-Reverse Transcriptase (Invitrogen). PCR amplification was performed using standard procedure with Taq Polymerase. Aliquots of cDNA equivalent to 50 ng of total RNA were amplified in 25 µl reactions containing 10 mM Tris-HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl2 , 50 pmol of each primer, 400 µM dNTPs, and 0.5 U AmpliTaq DNA polymerase (Perkin-Elmer). PCR was performed using the following thermal profile: 4 min at 94°C; 1 min at 94°C, 1 min at 60°C, 1.5 min at 72°C, for 30–40 cycles; 7 min at 72°C, and finally a soak at 4°C overnight. The following day, 10 µl aliquots of the amplified products were run on a 2% agarose Tris–acetate gel containing 0.5 mg/ml ethidium bromide. The products were visualized through a UV transilluminator, captured in a digital format using Quantify One Gel Analysis software (Bio-Rad Laboratories) on a Macintosh G4 computer.The PCR primers specific to each transcript were as follows: GFAP, forward (F), 5′-TCATCGCTCAGGAGGTCCTT–3′ Reverse (R), 5′-CTG TTGCCAGAGATGGAGGTT–3′; MAP2 (F) 5′-GAAGACTCGCATCCGAATGG–3′, (R) 5′-CGCAGGATAGGAGGAAGAGACT–3′; MBP (F) 5′-TTAGCTGAATTC GCGTGTGG–3′, (R) 5′-GAGGAAGTGAATGAGCCGGTTA-3′ were deigned using the Primer Designer software, Version 2.0 (Scientific and Educational Software) [48]. 18S, β-tubulin class III, N-CAM, Nestin, NF-M, Notch-1 primers [51]. Oct4, Nanog primers [11]. FOXa2 (HNF3B), Brachyury primers [52]. Behavioral testsThe cylinder test was used to assess the spontaneous forelimb use during lateral exploration movement [22]. Rats were placed in a transparent acrylic cylinder (20 cm diameter) for 5 minutes. The cylinder encourages use of the forelimbs for vertical exploration. A mirror was placed behind the cylinder so that the forelimbs could be viewed at all times. Testing sessions were videotaped and forelimb use was scored by a blinded operator. Movements scored were the independent use of the left or right forelimb or simultaneous use of both the left and right forelimb to contact the wall of the cylinder during a full rear, to initiate a weight-shifting movement, or to regain center of gravity while moving laterally in a vertical posture along the wall. Animals were tested for their baselines after stroke and 4 and 8 weeks after cell transplantation. Statistical analysisOutcome measurement for each experiment was reported as mean±SEM. All data were analyzed using SPSS 11 for Mac OS X (SPSS Inc.). Significance of inter-group differences was performed by applying Student's t-test where appropriate. The One-Way ANOVA analysis was used to compare group differences for the forelimb use as the dependant variable and groups as the single independent factor variable. Differences between the groups were determined using Bonferroni's post hoc test. A P-value of less than 0.05 was considered to be statistically significant. 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BackgroundHuman embryonic stem cells (hESCs) offer a virtually unlimited source of neural cells for structural repair in neurological disorders, such as stroke. Neural cells can be derived from hESCs either by direct enrichment, or by isolating specific growth factor-responsive and expandable populations of human neural stem cells (hNSCs). Studies have indicated that the direct enrichment method generates a heterogeneous population of cells that may contain residual undifferentiated stem cells that could lead to tumor formation in vivo.Methods/Principal FindingsWe isolated an expandable and homogenous population of hNSCs (named SD56) from hESCs using a defined media supplemented with epidermal growth factor (EGF), basic fibroblast growth factor (bFGF) and leukemia inhibitory growth factor (LIF). These hNSCs grew as an adherent monolayer culture. They were fully neuralized and uniformly expressed molecular features of NSCs, including nestin, vimentin and radial glial markers. These hNSCs did not express the pluripotency markers Oct4 or Nanog, nor did they express markers for the mesoderm or endoderm lineages. The self-renewal property of the hNSCs was characterized by a predominant symmetrical mode of cell division. The SD56 hNSCs differentiated into neurons, astrocytes and oligodendrocytes throughout multiple passages in vitro, as well as after transplantation. Together, these criteria confirm the definitive NSC identity of the SD56 cell line. Importantly, they exhibited no chromosome abnormalities and did not form tumors after implantation into rat ischemic brains and into naïve nude rat brains and flanks. Furthermore, hNSCs isolated under these conditions migrated toward the ischemia-injured adult brain parenchyma and improved the independent use of the stroke-impaired forelimb two months post-transplantation.Conclusions/SignificanceThe SD56 human neural stem cells derived under the reported conditions are stable, do not form tumors in vivo and enable functional recovery after stroke. These properties indicate that this hNSC line may offer a renewable, homogenous source of neural cells that will be valuable for basic and translational research. To date there have been no effective treatments for improving residual structural and functional deficits resulting from stroke. Current therapeutic approaches, such as the use of thrombolytics, benefit only 1 to 4% of patients [1]. Consequently, the majority of stroke patients experience progression of ischemia associated with debilitating neurological deficits. Recent evidence has suggested that the transplantation of cells derived from cord blood, bone marrow or brain tissue (fetal and adult) enhances sensorimotor function in experimental models of stroke [2], [3]. However, the normal human-derived somatic stem cells used in these studies have a limited capacity to differentiate into the diverse neural cell types optimal for structural and physiological tissue repair and are not amenable for large-scale cell production. Unlike other sources of stem cells, hESC lines possess a nearly unlimited self-renewal capacity and the developmental potential to differentiate into virtually any cell type of the organism. As such, they constitute an ideal source of cells for regenerative medicine. The successful derivation of hESC lines from the inner cell mass of preimplantation embryos and their long-term maintenance in vitro over multiple passages has been demonstrated [4] and standardized. Differentiation and enrichment processes that direct hESCs towards a neural lineage have also been achieved. To promote neuralization, ESCs were cultured in a defined media supplemented with morphogens or growth factors [5], [6], [7] or cultured under conditions that promote “rosettes”, structures morphologically similar to the developing neural tube [8], [9]. This neuralization process has proven invaluable in understanding the specification of hESC-derived neural tissue [10], [11], [12]. However, the enriched neural progeny derived displayed overgrowth and limited migration after grafting into normal newborn mice [13] and lesioned adult rat striatum [12], [14], [15], [16]. The inner cores of these grafts contained tumorigenic precursor cells (reviewed in [17]). These findings suggest that neural cells generated by acute exposure to growth factors and/or morphogens may still be heterogeneous and potentially tumorigenic. We report an alternative method for the isolation and the perpetuation of a multipotent hNSC line from the hESCs with a primitive mode of self-renewal. We also demonstrate their long-term expansion, non-tumorigenic properties and functional engraftability in an experimental model of stroke. 1. In vitro isolation, growth and differentiation of hESC-derived hNSCsThe hESCs were maintained and expanded on mouse feeder layer in media supplemented with bFGF (Figure 1A). After cell dissociation, a portion of the hESCs was cultured in serum free medium containing EGF, bFGF and LIF. These factors are known to stimulate the proliferation of human fetal-derived NSCs [18], [19]. After 3 days in vitro (DIV), there was selective survival and growth of cells that aggregated in clusters or spheres (Figure 1B). These primary spheres were harvested and replated in fresh media. During the following week, the spheres attached to the flask and a fibroblast-like cell population began to migrate out (Figure 1C). Secondary spheres (2° spheres) were generated from these cultures and lifted off by the end of the week leaving a hollow in the middle of the attached cells (Figure 1D). The floating 2° spheres were collected and replated in fresh growth medium for 2 weeks. The cultures were then passaged by collagenase cell dissociation every 7 DIV for an additional 4 passages (Figure S1). At the 5th and 6th passages, spheres were dissociated into a single-cell suspension using trypsin-EDTA. At this stage there was a change in the hNSCs' adherent properties, and the cells began to grow as a monolayer with multiple foci of cells throughout the culture (Figure 1F). The adherent hNSC culture stained uniformly for nestin (Figure 1K), vimentin (Figure 1L) and with the radial glial marker 3CB2 (Figure 1M) indicating their homogeneity and NSC identity. Under these culture conditions, it is noteworthy that we did not observe the formation of rosettes which has been previously reported to occur under certain conditions during neuralization of hESCs [8], [20], [21]. RT-PCR analysis confirmed that these hNSCs did not express the pluripotency transcripts Oct-4 and Nanog (Figure 1I). Furthermore, the hNSCs did not express transcripts for brachyury and foxa2, marker genes for mesoderm and endoderm respectively (negative result, data not shown).10.1371/journal.pone.0001644.g001Figure 1Isolation and purification of hNSCs from the hESCs.The hESCs were grown on a mouse feeder layer (A). Primary neurospheres (B) were isolated and replated to eliminate other non-neural cells. The selectively harvested secondary neurospheres (arrow in C), left behind hollow cores in the surface area (star in D) where they attached earlier. They were perpetuated for an additional 5 passages (E). These 2° spheres were then passaged using a single cell dissociation protocol (F). Arrow in F shows an example of a focus of proliferating cells. (G, H) The hNSCs were passaged every 5–7 days, as described in the Methods section. Starting from an initial population of 1 million cells, the cumulative cell number was calculated at each passage as the fold of increase×the total cell number and plotted as logarithm with base 2 in function of time (G). The cell perpetuation (G) and population doubling (H) analysis demonstrated the continuous and stable growth of the hNSCs. (I) RT-PCR analysis showing the down regulation of the pluripotency transcripts Oct4 and Nanog in secondary neurospheres and in expanded hNSCs at passage 8 (P8). (J) Cytogenetic evaluation of the SD56 hNSCs line at passage 12 by standard G-banding was performed. Twenty metaphase cells were analyzed and showed a normal female chromosome complement (46,XX). Isolated and expanded hNSCs expressed the neural precursor cell markers nestin (K), Vimentin (L) and the radial glial cell marker 3CB2 (M) in virtually all the progeny. (N-P) Clonal self-renewal ability of the isolated hNSCs through symmetrical cell division. Single (N), two-cell stage (O) and four-cell stage (P) of a hNSC proliferating over a 3-day culture period. Note the symmetrical segregation of BrdU and nestin in the progeny. Bars: (A, B, C, D) 200 µm; (E, F) 100 µm; (K–M) 20 µm; (N–P) 10 µm.To ascertain self-renewal ability under clonal conditions, a single cell suspension was plated at clonal density (1–2 cell/10 µl). To determine if the hNSCs divide symmetrically, we pulsed cultures with the thymidine analog, bromodeoxyuridine (BrdU), after plating and looked for the expression of nestin in the progeny. Cultures were fixed after 1, 2 or 3 DIV (Figure 1N–P). After 2 days, plated single cells first underwent a symmetric cell division and gave rise to daughter cells that were both positive for BrdU and nestin. The clone of cells continued to grow over the 3 DIV and all the progeny expressed nestin. BrdU labeling demonstrated that it was rare for only one daughter cell to inherit the BrdU and thus had undergone asymmetric segregation of the chromatids (Figure S2). G-band karyotyping of these hNSCs confirmed the normal, non-transformed nature of cells after 12 passages (Figure 1J). We named the derived hNSCs SD56 (intermittently referred to as SD56 hNSCs or hNSCs).Under these defined growth conditions, the hNSCs showed stable growth with a 2.7±0.2 fold increase every 5 to 7 days (Figure 1G). The population doubling at each passage averaged at 1.4±0.1 (Figure 1H). The viability of hNSCs at each passage was consistent at the approximate value of 98%. The projected cumulative cell numbers demonstrated that trillions of cells could be generated over a period of 5 months (Figure 1G). We expanded the isolated hNSCs lines for over 20 passages with a stable phenotype. An initial cell bank of 75 vials containing 2 to 5 million cells each was generated and cryopreserved.Upon removal of the mitogenic factors and plating on a coverslip pre-coated with poly-L-ornithine (PLO) substrate, the hNSCs spontaneously differentiated into neurons, astrocytes and oligodendrocytes, a property that is consistent with normal multipotent hNSCs (Figure 2). After 2 DIV, hNSCs expressed transcripts for the neural-specific genes nestin, Notch1 and neural cell adhesion molecule (N-CAM) (Figure 2A) and for the lineage specific markers β-tubulin class III, medium-size neurofilament (NF-M) and microtubule-associated protein 2 (MAP-2) for neurons, GFAP for astrocytes and myelin basic protein (MBP) for oligodendrocytes (Figure 2A). Transcripts for the GABAergic cell marker glutamic acid decarboxylase (GAD) were expressed, but transcripts for the tyrosine hydroxylase (TH), a marker for dopaminergic neurons, were undetectable. Immunocytochemical analysis (Figure 2B–F) of 10 day-old cultures demonstrated that the proportion of nestin+ cells was 36.6±2.7%, 62.5±2.8% expressed the neuronal marker TuJ1, 1.9±0.3% expressed the astrocytic marker GFAP and 7.1±0.4% were oligodendrocytes and expressed galactocerebrocide (GC) (Figure 2F). A subset (9.8±1.6%) of the GFAP+ astrocytes co-expressed nestin.10.1371/journal.pone.0001644.g002Figure 2hESC-derived hNSCs spontaneously differentiated into the 3 principal central nervous system cell types.Dissociated hNSCs were washed free of growth factors and plated on poly-L-onithine coated glass coverslips. Differentiated cultures were either harvested after 2 DIV for total RNA extraction and RT-PCR analysis or fixed after 10 DIV and processed for indirect immunocytochemistry. (A) Differentiated hNSCs expressed the neural-specific transcripts nestin and Notch1 as well as transcripts: for neurons (β-tubulin class III, MAP-2, NCAM and medium-size neurofilament, NF-M), for astrocytes (GFAP) and for oligodendrocytes (MBP). The hNSCs expressed transcripts for GAD, but not for TH. B, C & D, morphology of NSC-derived progeny differentiated into GFAP+ astrocytes (B), GC-expressing oligodendrocytes (C) and TuJ1+ neurons (D), DAPI (blue) show life cell nuclei. (E) Photo showing cultures double-immunostained for TuJ1 (green) and nestin (red) (DAPI, blue). (F) Quantitative analysis of immunostained 10 day-old cultures for the 3 neural cell types. Results are mean±s.e.m. of three independent experiments, each performed in duplicate. Bars: (c) 20 µm; (d, e) 10 µm. 2. The isolated hNSCs are normal and do not form tumors in normal nude ratsThe self-renewal and pluripotent abilities of the hESCs are also associated with tumorigenic properties. Therefore, the first critical step toward developing therapeutic hNSCs is to verify that they are non-tumorigenic. The monolayer culture of SD56 hNSCs clearly demonstrated contact inhibition of growth, a normal karyotype and did not express the pluripotency transcripts Oct-4 and Nanog. Removal of mitogenic factors and continued culture on plastic resulted in cell senescence that is characteristic of non-transformed cells. To determine whether SD56 hNSCs form tumors in vivo, we transplanted them at high density (see Methods) into the forebrain and subcutaneously into the flank of nude rats. The animals were kept for a two-month post-transplant survival period. To label mitotically active cells in vivo during S-phase, the rats were injected IP with BrdU (50 mg/kg) every 8 hours during the last 24 hours before euthanasia. The transit amplifying endogenous precursors located in the subventricular zone (SVZ) were labeled (Figure S3); however, we were unable to detect grafted SD56 hNSCs co-localizing the human-specific nuclear marker hNuc and BrdU (Figure S3). No surviving SD56 hNSCs were detected in the flank of the transplanted animals suggesting that the grafted cells are not tumorigenic. 3. Transplanted cells survived, migrated toward and engrafted into the stroke-damaged host tissueTo investigate the survival and functional engraftment in an injury environment, hNSCs (4×105) were transplanted into the ischemic boundary zone in the rat striatum one week after the middle cerebral artery occlusion (MCAO) was performed. Animals were euthanized two months later and the brains processed for histo-pathology and immunocytochemistry. Grafted SD56 hNSCs, identified with hNuc, demonstrated a 37.0±15.8% survival rate and a remarkable dispersion toward the stroke-damaged tissue with no sign of overgrowth or tumorigenesis. The majority of grafted cells (61.2±4.7%) migrated at least 200 µm away from the injection site and penetrated an average distance of 806.4±49.3 µm into the stroke-damaged tissue (Figure 3A–C). Immunostaining with the blood vessel marker, GluT1, revealed dilated vessels in the infarcted striatum and a close association between vessels and the grafted hNSCs (Figure 3B, 3C). The grafted cells rarely expressed the proliferation marker Ki67 (Figure 3D), 29.8±4.4% expressed nestin (Figure 3E), 6.5±0.9% expressed doublecortin (DCX) and 60.8±8.1% were TuJ1+ (Figure 3F, G). Grafted cells rarely co-expressed the astroglial marker GFAP (Figure 3H) or differentiated into CNPase-expressing oligodendrocytes (Figure 3I). Immunostaining for GAD demonstrated that 25.1±2.3% of grafted hNSCs differentiated into GABAergic neurons while less than 2% were positive for glutamate (Figure 3J, K).10.1371/journal.pone.0001644.g003Figure 3Dispersion, engraftment and differentiation of the hNSCs in stroke-lesioned animals.(A) Schematic drawing of a frontal section through the striatum illustrating the dispersion of grafted hNSCs in the focal ischemia-lesioned parenchyma (shaded area). (B, C) Photos show frontal sections through the graft in the striatum immunostained with the human specific antibodies: anti-hNuc (green in B & C) and anti-GluT1 (red, B & C) showing blood vessels and dispersed hNSCs in the graft zone. C: higher magnification of the inset in B. (D–I) Photos taken from frontal sections through the graft in the striatum double immunoprocessed for cell proliferation and neural lineage markers. (D) Note the endogenous Ki67+ cells (red cells, arrow) in the stroke damaged area and the hNuc+ (green)/Ki67- grafted hNSCs (arrowheads). (E) Examples of grafted SD56 hNSCs showing co-expression of hNuc (green) and nestin (red). (F) Confocal 3D reconstructed orthogonal images of the hNuc+(green)/DCX+(red) NSCs (arrowheads) viewed in the x-z plan on the top and y-z plan on the right. (G) Examples show the majority of grafted NSC progeny co-expressing hNuc (red) and the neuronal marker TuJ1 (green). Grafted NSCs rarely differentiate into GFAP+ astrocytes (H). In I, rare example of grafted NSC progeny becoming an oligodendrocyte identified by the expression of CNPase (green). Grafted NSCs expressed the GABAergic marker GAD65/67 (J) and rarely expressed glutamate (K). (Abbreviations: Cx: cortex, Str: striatum). Bars: (B, C) 100 µm; (D, F) 20 µm; (E, G–K) 10 µm. 4. Transplanted cells improve sensorimotor function of the stroke-disabled forelimbWe asked whether transplanted SD56 hNSCs could enhance the recovery of sensorimotor function that is compromised in the stroke-injured rats. We used the cylinder test to measure the sensorimotor asymmetry in forelimb use during spontaneous exploration [22]. To establish the baseline of the stroke-induced sensorimotor deficit, spontaneous behavior of rats in a transparent cylinder was videotaped one week after stroke (pre-transplant, Figure 4). Tests were then conducted 4 and 8 weeks after vehicle and SD56 hNSCs transplantation. Stable asymmetry in forelimb use was observed 7 days post-stroke (pre-transplant, Figure 4). Ischemic rats used their impaired forelimbs (contralateral to lesion) during lateral exploration less than they did before stroke. Transplantation of SD56 hNSCs significantly enhanced the independent use of the impaired contralateral forelimb 4 weeks post transplantation (P<0.05 vs pre-transplant). Eight weeks after transplantation the improvement in the use of the impaired forelimb was stable and significant when compared to the pre-transplant group and significantly improved in comparison to vehicle treated group at 8 weeks (Figure 4). In the vehicle treated group, the independent use of the contralateral forelimb remained impaired 4 and 8 weeks post-injection. In an independent study and using the same MCAO rat animal model, we found that transplantation of dermal fibroblasts did not improve the stroke-induced motor deficits (unpublished data).10.1371/journal.pone.0001644.g004Figure 4Transplantation of NSCs improves sensorimotor function of the stroke-disabled forelimb.Forelimb use during spontaneous lateral exploration was measured in the cylinder test (see Method and Results sections for details). Groups of vehicle injected (n = 7) and hNSCs (n = 10) transplanted are represented. The animals were tested as described in Method section. Note the significant increase in the independent use of the impaired contralateral forelimb at 4 and 8 weeks post transplantation (P<0.05 vs pre-transplant group). The contralateral forelimb remained impaired in the vehicle treated group at 4 and 8 weeks post-injection. Bars represent percentages±s.e.m. of steps taken by the ipsilateral, contralateral and both forelimbs simultaneously. *P<0.05 vs pre-transplant group; #P<0.05 vs vehicle groups. Our results indicate that a self-renewable and homogenous population of hNSCs, SD56, was derived from hESCs using defined media supplemented with a specific combination of growth factors. The SD56 hNSCs grew as an adherent monolayer culture, uniformly expressed molecular features of hNSCs including nestin, vimentin and the radial glial marker 3CB2, and did not express the pluripotency markers Oct4 or Nanog. The self-renewal property of the hNSCs was characterized by a predominant symmetrical mode of cell division. They exhibited no chromosomal abnormalities and demonstrated non-tumorigenic properties after implantation into ischemic brains and into naïve nude rat brains and flanks. Furthermore, the transplanted SD56 hNSCs migrated toward the stroke-damaged adult brain parenchyma, engrafted and improved the independent use of the stroke-impaired forelimb. Maintenance of stem cells requires symmetrical and asymmetrical cell divisions to both expand and to give rise to specialized progeny of a specific tissue (reviewed in [23]). In vivo, a complex cellular micro-environment or niche ensures the self-maintenance property of NSCs [24], [25], [26], [27]. In vitro, defined growth factors and extracellular matrices support stem cell self-renewal [28], [29]. The embryonic stem cells can propagate in a predominantly proliferative symmetrical mode, leading to homogeneous cell cultures growing relatively quickly with minimal cell differentiation [30], [31], [32], [33], [34]. Tissue specific stem cells, however, self-renew in a predominant asymmetric mode to maintain them selves and compensate for the loss of differentiated cells due to disease or injury. Thus, NSCs isolated from developing or adult brain grow as a mixture of undifferentiated and differentiated cells due the predominant asymmetrical mode of cell division [35], [36], [37], [38], [39], [40], [41]. A recent study has reported that a murine ESC-derived NSC line (LC1) is propagated as an adherent homogenous culture with a dominant mode of symmetrical self-renewal [21]. A combination of EGF and FGF2 was sufficient to propagate these NSCs as an adherent monolayer. However, the SD56 hNSC line described here required the combination of EGF, bFGF and LIF for self-maintenance. Although there are morphological and molecular similarities between our hNSCs and the NSCs previously described [21], the methods of isolation and growth are different. In addition to the different combination of growth factors used, the hNSC line we have isolated did not go through the rosette-structure stage. The in vitro analysis of BrdU incorporation and nestin expression indicated that our hNSCs divide predominantly symmetrically. This type of growth pattern is characteristic of primitive normal stem cells undergoing mostly symmetric cell division to increase the stem cell pool at the early stage of development or during tissue regeneration after injury [23]. RT-PCR and immunocytochemistry analysis demonstrated that these undifferentiated SD56 cells did not express any pluripotency, endodermal or mesodermal markers. Furthermore, the SD56 hNSCs described here exhibited the multipotential characteristic to differentiate into neurons, astrocytes and oligodendrocytes both in vitro and upon transplantation. Together these findings suggest that the hNSC line we isolated are appropriately programmed and share similar characteristics with the definitive NSCs of the developing brain. The SD56 hNSCs demonstrated a remarkable ability to migrate toward the stroke-damaged parenchyma of the adult rat brain. This directed migration by the majority of the grafted cells could be due to their uniform cellular composition, which results in an equal response to the host microenvironment. In previous studies, subpopulations of transplanted hESCs that were enriched in neural cells migrated in host microenvironments conducive to cell migration, such as the developing brain or in structures such as the rostral migratory stream [13], [20]. In the adult lesioned brain, the grafted hESC-derived neural cells proliferated and formed rosettes [14], teratomas [12], [15] or a cellular mass that induced a gliotic host response whereby local astrocytes demarcated the grafts [16]. Enriched neural cultures derived from mouse [42] and monkey ESCs [43] have produced behavioral improvements when transplanted into animal models of stroke and brain injury. However, in these cases, the transplanted non-human ESCs also formed a mass with signs of overgrowth in the core, as well as deformations [44], [45], [46]. ESCs plated at low density acquire neural identity within few hours after plating [47]. Interestingly, nearly all viable cells expressed nestin, the early neural fate marker Sox1, and the pluripotency marker Oct4. Together, these studies are seminal and suggest that complete neuralization may not be achieved through certain enrichment processes, consequently the neural cells could revert to a pluripotent stage [17]. The dispersion of the grafted hNSCs within host parenchyma may allow for more graft-host interactions that could stabilize differentiation, inhibit growth and prevent gliotic host response. In the present study, SD56 hNSC-transplanted animals demonstrated a stable improvement in the sensorimotor function when evaluated for spontaneous exploratory activity in the cylinder test that detects long-lasting deficits in forelimb use in the experimental models of stroke [22]. The transplantation of hNSCs significantly enhanced the independent use of the impaired contralateral forelimb 8 weeks post transplantation. This is the first report demonstrating that the transplantation of hNSCs derived from hESCs can improve neurologic behavior after experimental stroke. Together, these findings are encouraging and suggest that these cells are promising for future development. In addition to their therapeutic application, the hNSCs isolated under the reported conditions offer a means to interrogate host environments and unravel mechanistic features of self-renewal, non-tumorigenicity and functional engraftability in animal models of neurological disorders. hESC and NSC CulturesThe hESC line H9 (WiCell Research Institute) was propagated every 5 to 7 days on irradiated mouse embryonic fibroblasts. The cell culture media consisted of a 1∶1 mixture of Dulbecco's modified Eagle's medium (DMEM) and F12 nutrient, 20% serum replacement (Invitrogen), 0.1 mM β-mercaptoethanol, 2 µg/ml heparin and 4 ng/ml bFGF (R&D Systems). To generate the NSCs, dissociated hESCs were cultured in a chemically defined medium composed of DMEM/F12 (1∶1) including glucose (0.6%), glutamine (2 mM), sodium bicarbonate (3 mM), and HEPES buffer (5 mM) [all from Sigma except glutamine (Invitrogen)]. A defined hormone mix and salt mixture (Sigma), including insulin (25 mg/ml), transferrin (100 mg/ml), progesterone (20 nM), putrescine (60 mM), and selenium chloride (30 nM) was used in place of serum. The medium was supplemented with EGF (20 ng/ml), bFGF (10 ng/ml) and LIF (10 ng/ml). Dissociated hNSCs were plated at a density of 100,000 cell/ml in Corning T75 (Invitrogen) culture flasks in the defined media together with the growth factors. After 5–7 DIV, the adherent culture was incubated in 0.025%trypsin/0.01% EDTA (w/v) for 1 min followed by the addition of trypsin inhibitor (Invitrogen) then gently triturated to achieve single cell suspension. The cells were then washed twice with fresh medium and reseeded in fresh growth factor-containing media at 100,000 cells/ml. This procedure was performed for 21 passages and the fold of increase and population doubling were calculated at each passage. For clonal analysis, single spheres or confluent hNSC cultures were single cell dissociated and serially diluted to yield 1–2 cell/10 µl. A 10-µl-cell suspension was then added to each of 96 or 24 well plates containing 200–300 µl of growth media. Wells containing one viable cell were marked the next day and re-scored 5 to 7 days later for cell proliferation. The differentiation of the hNSCs was performed as previously described [48]. Dissociated hNSCs were plated at a density of 106 cells/ml in control (media/hormone mix) medium devoid of any growth factors and supplemented with 1% fetal bovine serum (FBS) on poly-L-ornithine-coated (15 mg/ml; Sigma) glass coverslips in 24-well Nunclon culture dishes with 0.5 ml/well. After 2, 7–15 DIV cultures were fixed and processed for immunocytochemistry or used for RT-PCR analysis. Karyotype analysisLong-term cultures of hNSCs were incubated at 37°C and harvested for metaphase chromosomes when the cultures were 75% confluent. Metaphase chromosomes were obtained by standard chromosome harvest methods by exposure to Colcemid at 0.1 µg/ml for 1 hour at 37°C, a 2-minute exposure to trypsin/EDTA, hypotonized with 0.057 M KCl and fixed with 3∶1 methanol:acetic acid. Slide preparations were made by dropping the fixed cell pellet onto cold, wet slides and air-dried. After incubating the slides at 90°C for 30 minutes, chromosomes were trypsin banded and then Wright/Giemsa stained for G-banding analysis. Twenty metaphase cells were completely analyzed and a normal female chromosome complement was found (46,XX). Tumorigenicity in nude ratsAll animal experiments were conducted according to the National Institute of Health (NIH) guidelines and approved by the IACUC. Normal adult NIH-Nude rats (n = 5, 8 week-old, Taconic, Germantown, New York, United States) were used to test the tumorigenic potential of the SD56 hNSCs. Undifferentiated hNSCs from passage 9 were single cell dissociated using trypsin-EDTA and suspended at the concentration of 125,000 cell/µl in preparation for cell transplantation. Two µl of the cell suspension were stereotaxically transplanted into 4 sites within the striatum at the following coordinates: AP: +1.0 mm, ML: +3.2 mm, DV: −5.0; AP: +0.5 mm, ML: +3.0 mm, DV: −5.0; AP: −0.5 mm, ML: +3.0 mm, DV: −5.0; AP: −1.0 mm, ML: +3.5 mm, DV: −5.0 mm with the incisor bar set at 3.4 mm. The injection rate was 1 µl/min, and the cannula was left in place for an additional 5 min before retraction. For the flank tumor assay, 2×106 cells (125,000 cell/µl) were injected subcutaneously to the side of the adult nude rats. To label mitotically active cells in vivo during S-phase, the rats were injected IP with the BrdU (50 mg/kg, Sigma) every 8 hours during the last 24 hours before euthanasia. After 2-month survival time, rats were euthanized and a postmortem examination for tumor formation was performed. Induction of Focal Ischemia and Cell TransplantationAll animal experimentations were conducted according to the National Institute of Health (NIH) guidelines and approved by the IACUC. Sprague Dawley adult male rats (n = 17, 275g–310g, Charles River Laboratories, Wilmington, Massachusetts, United States) were subjected to one and a half hour suture occlusion of the middle cerebral artery (MCAO), as previously described [49] and immunosuppressed 2 days before cell transplantation and daily thereafter for one week with i.p. injections of cyclosporine A (20 mg/ml, Sandimmune, Novartis Pharmaceuticals). Thereafter oral cyclosporine was used at 210 µg/ml in drinking water until euthanasia. Undifferentiated SD56 hNSCs from passages between P9 and P13 were single cell dissociated using trypsin-EDTA in preparation for cell transplantation. One week after the stroke lesion, 2 µl of the hNSCs, at a concentration of 50,000 cell/µl, were stereotaxically transplanted into 4 sites within the lesioned striatum (n = 10) at the following coordinates: AP: +1.0 mm, ML: +3.2 mm, DV: −5.0; AP: +0.5 mm, ML: +3.0 mm, DV: −5.0; AP: −0.5 mm, ML: +3.0 mm, DV: −5.0; AP: −1.0 mm, ML: +3.5 mm, DV: −5.0 mm with the incisor bar set at 3.4 mm. The injection rate was 1 µl/min, and the cannula was left in place for an additional 5 min before retraction. As a control group, we used rats subjected to ischemia and injected with the vehicle (n = 7). All animals underwent baseline motor behavioral assessment before and after the ischemic lesion, and 4 & 8 weeks after cell transplantation. The animals were killed after 2-month survival time by transcardial perfusion with phosphate buffered saline (PBS) followed by 4% paraformaldehyde. The brains were cryoprotected in an increasing gradient of 10, 20 and 30% sucrose solution and cryostat sectioned at 40 µm and processed for immunocytochemistry. ImmunocytochemistryCultures were fixed with 4% paraformaldehyde for 15 min. Both cells and brain sections were rinsed in PBS for 3×5 min then incubated for 2 hrs (cultures) or overnight (brain sections) with the appropriate primary antibodies for multiple labeling. Secondary antibodies raised in the appropriate hosts and conjugated to FITC, RITC, AMCA, CY3 or CY5 chromogenes (Jackson ImmunoResearch) were used. Cells and sections were counterstained with the nuclear marker 4′,6-diamidine-2′-phenylindole dihydrochloride (DAPI). Positive and negative controls were included in each run. Immunostained sections were coverslipped using fluorsave (Calbiochem) as the mounting medium. The following antibodies were used: Anti-human Nuclei (hNuc, monoclonal 1∶100, Chemicon), Anti-TuJ1 (monoclonal 1∶100, Covance; Polyclonal 1∶200, Aves Labs); anti-GAD65/67 (polyclonal 1∶1000, Chemicon); Anti-glial fibrillary acidic protein (GFAP, monoclonal 1∶1000, Chemicon; polyclonal 1∶200, Aves Labs); Anti-galactocerebrocide (GC, monoclonal 1∶250, Chemicon); Anti-CNPase (polyclonal 1∶200, Aves Labs); Anti-Glucose Transporter type 1 (Glut-1 polyclonal, 1∶500, Chemicon); Anti-Nestin (polyclonal 1∶1000, Chemicon); Anti-vimentin (monoclonal 1∶500, Calbiochem); Anti-3CB2 (monoclonal 1∶500, Developmental Studies Hybridoma Bank); Anti-doublecortin (DCX, polyclonal 1∶250, SantaCruz Biotechnology); Anti-Ki67 (polyclonal 1∶250, SantaCruz Biotechnology). Fluorescence was detected, analyzed and photographed with a Zeiss LSM550 laser scanning confocal photomicroscope. For each animal, quantitative estimates of the total number of grafted cells were stereologically determined using the optical fractionator procedure [50]. A computer-assisted image analysis system was performed using Stereo Investigator software (MicroBrightField, Inc.). The rostral and caudal limits of the reference volume were determined by first and last frontal sections containing grafted cells. The striatum and cortex were accurately outlined at low magnification (2.5× objective). The optical fractionator probe was selected to perform systematic sampling of the immunoreactive cell population distributed within the serial sections to estimate the population number in the volume of tissue. The counting frame of the optical fractionator was defined at 50×50 µm squares and the systematic sampling was performed by translating a grid with 200×200 µm squares onto the sections of interest using the Stereo Investigator software. The sample sites were systematically and automatically generated by the computer and examined using a 60× objective of a Nikon Eclipse TE 300 microscope. The counting frame displayed inclusion and exclusion lines and only immunoreactive cell bodies falling within the counting frame with no contact with the exclusion lines were counted. The cell dispersion was measured by counting the number of cells within 200 µm distance from the graft site. The number and distance in µm of cells dispersed beyond 200 µm was also measured. An average of 2,000 cells was counted per animal. Double labeling was determined using the confocal laser scanning microscope by random sampling of 100 or more cells per marker for each animal, scoring first for hNuc+, followed by DAPI+ nuclei and then the marker of choice. The double labeling was always confirmed in x-z and y-z cross-sections produced by the orthogonal projections of z-series. Reverse Transcription-Polymerase Chain Reaction (RT-PCR) analysisTotal RNA was extracted from cultured cells using RNAeasy kit (Quiagen). Aliquots (1 µg) of total RNA from the cells were reverse transcribed in the presence of 50 mM Tris-HCl, pH 8.3, 75 mM KCl, 3 mM MgCl2, 10 mM DTT, 0.5 µM dNTPs, and 0.5 µg oligo-dT(12–18) with 200 U Superscript RNase H-Reverse Transcriptase (Invitrogen). PCR amplification was performed using standard procedure with Taq Polymerase. Aliquots of cDNA equivalent to 50 ng of total RNA were amplified in 25 µl reactions containing 10 mM Tris-HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl2 , 50 pmol of each primer, 400 µM dNTPs, and 0.5 U AmpliTaq DNA polymerase (Perkin-Elmer). PCR was performed using the following thermal profile: 4 min at 94°C; 1 min at 94°C, 1 min at 60°C, 1.5 min at 72°C, for 30–40 cycles; 7 min at 72°C, and finally a soak at 4°C overnight. The following day, 10 µl aliquots of the amplified products were run on a 2% agarose Tris–acetate gel containing 0.5 mg/ml ethidium bromide. The products were visualized through a UV transilluminator, captured in a digital format using Quantify One Gel Analysis software (Bio-Rad Laboratories) on a Macintosh G4 computer.The PCR primers specific to each transcript were as follows: GFAP, forward (F), 5′-TCATCGCTCAGGAGGTCCTT–3′ Reverse (R), 5′-CTG TTGCCAGAGATGGAGGTT–3′; MAP2 (F) 5′-GAAGACTCGCATCCGAATGG–3′, (R) 5′-CGCAGGATAGGAGGAAGAGACT–3′; MBP (F) 5′-TTAGCTGAATTC GCGTGTGG–3′, (R) 5′-GAGGAAGTGAATGAGCCGGTTA-3′ were deigned using the Primer Designer software, Version 2.0 (Scientific and Educational Software) [48]. 18S, β-tubulin class III, N-CAM, Nestin, NF-M, Notch-1 primers [51]. Oct4, Nanog primers [11]. FOXa2 (HNF3B), Brachyury primers [52]. Behavioral testsThe cylinder test was used to assess the spontaneous forelimb use during lateral exploration movement [22]. Rats were placed in a transparent acrylic cylinder (20 cm diameter) for 5 minutes. The cylinder encourages use of the forelimbs for vertical exploration. A mirror was placed behind the cylinder so that the forelimbs could be viewed at all times. Testing sessions were videotaped and forelimb use was scored by a blinded operator. Movements scored were the independent use of the left or right forelimb or simultaneous use of both the left and right forelimb to contact the wall of the cylinder during a full rear, to initiate a weight-shifting movement, or to regain center of gravity while moving laterally in a vertical posture along the wall. Animals were tested for their baselines after stroke and 4 and 8 weeks after cell transplantation. Statistical analysisOutcome measurement for each experiment was reported as mean±SEM. All data were analyzed using SPSS 11 for Mac OS X (SPSS Inc.). Significance of inter-group differences was performed by applying Student's t-test where appropriate. The One-Way ANOVA analysis was used to compare group differences for the forelimb use as the dependant variable and groups as the single independent factor variable. Differences between the groups were determined using Bonferroni's post hoc test. A P-value of less than 0.05 was considered to be statistically significant.
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"a", "defined", "media", "supplemented", "with", "epidermal", "growth", "factor", "(", "EGF", ")", ",", "basic", "fibroblast", "growth", "factor", "(", "bFGF", ")", "and", "leukemia", "inhibitory", "growth", "factor", "(", "LIF", ")", ".", "These", "hNSCs", "grew", "as", "an", "adherent", "monolayer", "culture", ".", "They", "were", "fully", "neuralized", "and", "uniformly", "expressed", "molecular", "features", "of", "NSCs", ",", "including", "nestin", ",", "vimentin", "and", "radial", "glial", "markers", ".", "These", "hNSCs", "did", "not", "express", "the", "pluripotency", "markers", "Oct4", "or", "Nanog", ",", "nor", "did", "they", "express", "markers", "for", "the", "mesoderm", "or", "endoderm", "lineages", ".", "The", "self", "-", "renewal", "property", "of", "the", "hNSCs", "was", "characterized", "by", "a", "predominant", "symmetrical", "mode", "of", "cell", "division", ".", "The", "SD56", "hNSCs", "differentiated", "into", "neurons", ",", "astrocytes", "and", "oligodendrocytes", "throughout", "multiple", "passages", "in", "vitro", ",", "as", "well", "as", "after", "transplantation", ".", "Together", ",", "these", "criteria", "confirm", "the", "definitive", "NSC", "identity", "of", "the", "SD56", "cell", "line", ".", "Importantly", ",", "they", "exhibited", "no", "chromosome", "abnormalities", "and", "did", "not", "form", "tumors", "after", "implantation", "into", "rat", "ischemic", "brains", "and", "into", "naïve", "nude", "rat", "brains", "and", "flanks", ".", "Furthermore", ",", "hNSCs", "isolated", "under", "these", "conditions", "migrated", "toward", "the", "ischemia", "-", "injured", "adult", "brain", "parenchyma", "and", "improved", "the", "independent", "use", "of", "the", "stroke", "-", "impaired", "forelimb", "two", "months", "post", "-", "transplantation", ".", "Conclusions", "/", "SignificanceThe", "SD56", "human", "neural", "stem", "cells", "derived", "under", "the", "reported", "conditions", "are", "stable", ",", "do", "not", "form", "tumors", "in", "vivo", "and", "enable", "functional", "recovery", "after", "stroke", ".", "These", "properties", "indicate", "that", "this", "hNSC", "line", "may", "offer", "a", "renewable", ",", "homogenous", "source", "of", "neural", "cells", "that", "will", "be", "valuable", "for", "basic", "and", "translational", "research", ".", "\n", "To", "date", "there", "have", "been", "no", "effective", "treatments", "for", "improving", "residual", "structural", "and", "functional", "deficits", "resulting", "from", "stroke", ".", "Current", "therapeutic", "approaches", ",", "such", "as", "the", "use", "of", "thrombolytics", ",", "benefit", "only", "1", "to", "4", "%", "of", "patients", "[", "1", "]", ".", "Consequently", ",", "the", "majority", "of", "stroke", "patients", "experience", "progression", "of", "ischemia", "associated", "with", "debilitating", "neurological", "deficits", ".", "Recent", "evidence", "has", "suggested", "that", "the", "transplantation", "of", "cells", "derived", "from", "cord", "blood", ",", "bone", "marrow", "or", "brain", "tissue", "(", "fetal", "and", "adult", ")", "enhances", "sensorimotor", "function", "in", "experimental", "models", "of", "stroke", "[", "2", "]", ",", "[", "3", "]", ".", "However", ",", "the", "normal", "human", "-", "derived", "somatic", "stem", "cells", "used", "in", "these", "studies", "have", "a", "limited", "capacity", "to", "differentiate", "into", "the", "diverse", "neural", "cell", "types", "optimal", "for", "structural", "and", "physiological", "tissue", "repair", "and", "are", "not", "amenable", "for", "large", "-", "scale", "cell", "production", ".", "Unlike", "other", "sources", "of", "stem", "cells", ",", "hESC", "lines", "possess", "a", "nearly", "unlimited", "self", "-", "renewal", "capacity", "and", "the", "developmental", "potential", "to", "differentiate", "into", "virtually", "any", "cell", "type", "of", "the", "organism", ".", "As", "such", ",", "they", "constitute", "an", "ideal", "source", "of", "cells", "for", "regenerative", "medicine", ".", "The", "successful", "derivation", "of", "hESC", "lines", "from", "the", "inner", "cell", "mass", "of", "preimplantation", "embryos", "and", "their", "long", "-", "term", "maintenance", "in", "vitro", "over", "multiple", "passages", "has", "been", "demonstrated", "[", "4", "]", "and", "standardized", ".", "Differentiation", "and", "enrichment", "processes", "that", "direct", "hESCs", "towards", "a", "neural", "lineage", "have", "also", "been", "achieved", ".", "To", "promote", "neuralization", ",", "ESCs", "were", "cultured", "in", "a", "defined", "media", "supplemented", "with", "morphogens", "or", "growth", "factors", "[", "5", "]", ",", "[", "6", "]", ",", "[", "7", "]", "or", "cultured", "under", "conditions", "that", "promote", "“", "rosettes", "”", ",", "structures", "morphologically", "similar", "to", "the", "developing", "neural", "tube", "[", "8", "]", ",", "[", "9", "]", ".", "This", "neuralization", "process", "has", "proven", "invaluable", "in", "understanding", "the", "specification", "of", "hESC", "-", "derived", "neural", "tissue", "[", "10", "]", ",", "[", "11", "]", ",", "[", "12", "]", ".", "However", ",", "the", "enriched", "neural", "progeny", "derived", "displayed", "overgrowth", "and", "limited", "migration", "after", "grafting", "into", "normal", "newborn", "mice", "[", "13", "]", "and", "lesioned", "adult", "rat", "striatum", "[", "12", "]", ",", "[", "14", "]", ",", "[", "15", "]", ",", "[", "16", "]", ".", "The", "inner", "cores", "of", "these", "grafts", "contained", "tumorigenic", "precursor", "cells", "(", "reviewed", "in", "[", "17", "]", ")", ".", "These", "findings", "suggest", "that", "neural", "cells", "generated", "by", "acute", "exposure", "to", "growth", "factors", "and", "/", "or", "morphogens", "may", "still", "be", "heterogeneous", "and", "potentially", "tumorigenic", ".", "We", "report", "an", "alternative", "method", "for", "the", "isolation", "and", "the", "perpetuation", "of", "a", "multipotent", "hNSC", "line", "from", "the", "hESCs", "with", "a", "primitive", "mode", "of", "self", "-", "renewal", ".", "We", "also", "demonstrate", "their", "long", "-", "term", "expansion", ",", "non", "-", "tumorigenic", "properties", "and", "functional", "engraftability", "in", "an", "experimental", "model", "of", "stroke", ".", "\n", "1", ".", "In", "vitro", "isolation", ",", "growth", "and", "differentiation", "of", "hESC", "-", "derived", "hNSCsThe", "hESCs", "were", "maintained", "and", "expanded", "on", "mouse", "feeder", "layer", "in", "media", "supplemented", "with", "bFGF", "(", "Figure", "1A", ")", ".", "After", "cell", "dissociation", ",", "a", "portion", "of", "the", "hESCs", "was", "cultured", "in", "serum", "free", "medium", "containing", "EGF", ",", "bFGF", "and", "LIF", ".", "These", "factors", "are", "known", "to", "stimulate", "the", "proliferation", "of", "human", "fetal", "-", "derived", "NSCs", "[", "18", "]", ",", "[", "19", "]", ".", "After", "3", "days", "in", "vitro", "(", "DIV", ")", ",", "there", "was", "selective", "survival", "and", "growth", "of", "cells", "that", "aggregated", "in", "clusters", "or", "spheres", "(", "Figure", "1B", ")", ".", "These", "primary", "spheres", "were", "harvested", "and", "replated", "in", "fresh", "media", ".", "During", "the", "following", "week", ",", "the", "spheres", "attached", "to", "the", "flask", "and", "a", "fibroblast", "-", "like", "cell", "population", "began", "to", "migrate", "out", "(", "Figure", "1C", ")", ".", "Secondary", "spheres", "(", "2", "°", "spheres", ")", "were", "generated", "from", "these", "cultures", "and", "lifted", "off", "by", "the", "end", "of", "the", "week", "leaving", "a", "hollow", "in", "the", "middle", "of", "the", "attached", "cells", "(", "Figure", "1D", ")", ".", "The", "floating", "2", "°", "spheres", "were", "collected", "and", "replated", "in", "fresh", "growth", "medium", "for", "2", "weeks", ".", "The", "cultures", "were", "then", "passaged", "by", "collagenase", "cell", "dissociation", "every", "7", "DIV", "for", "an", "additional", "4", "passages", "(", "Figure", "S1", ")", ".", "At", "the", "5th", "and", "6th", "passages", ",", "spheres", "were", "dissociated", "into", "a", "single", "-", "cell", "suspension", "using", "trypsin", "-", "EDTA", ".", "At", "this", "stage", "there", "was", "a", "change", "in", "the", "hNSCs", "'", "adherent", "properties", ",", "and", "the", "cells", "began", "to", "grow", "as", "a", "monolayer", "with", "multiple", "foci", "of", "cells", "throughout", "the", "culture", "(", "Figure", "1F", ")", ".", "The", "adherent", "hNSC", "culture", "stained", "uniformly", "for", "nestin", "(", "Figure", "1", "K", ")", ",", "vimentin", "(", "Figure", "1L", ")", "and", "with", "the", "radial", "glial", "marker", "3CB2", "(", "Figure", "1", "M", ")", "indicating", "their", "homogeneity", "and", "NSC", "identity", ".", "Under", "these", "culture", "conditions", ",", "it", "is", "noteworthy", "that", "we", "did", "not", "observe", "the", "formation", "of", "rosettes", "which", "has", "been", "previously", "reported", "to", "occur", "under", "certain", "conditions", "during", "neuralization", "of", "hESCs", "[", "8", "]", ",", "[", "20", "]", ",", "[", "21", "]", ".", "RT", "-", "PCR", "analysis", "confirmed", "that", "these", "hNSCs", "did", "not", "express", "the", "pluripotency", "transcripts", "Oct-4", "and", "Nanog", "(", "Figure", "1I", ")", ".", "Furthermore", ",", "the", "hNSCs", "did", "not", "express", "transcripts", "for", "brachyury", "and", "foxa2", ",", "marker", "genes", "for", "mesoderm", "and", "endoderm", "respectively", "(", "negative", "result", ",", "data", "not", "shown).10.1371", "/", "journal.pone.0001644.g001Figure", "1Isolation", "and", "purification", "of", "hNSCs", "from", "the", "hESCs", ".", "The", "hESCs", "were", "grown", "on", "a", "mouse", "feeder", "layer", "(", "A", ")", ".", "Primary", "neurospheres", "(", "B", ")", "were", "isolated", "and", "replated", "to", "eliminate", "other", "non", "-", "neural", "cells", ".", "The", "selectively", "harvested", "secondary", "neurospheres", "(", "arrow", "in", "C", ")", ",", "left", "behind", "hollow", "cores", "in", "the", "surface", "area", "(", "star", "in", "D", ")", "where", "they", "attached", "earlier", ".", "They", "were", "perpetuated", "for", "an", "additional", "5", "passages", "(", "E", ")", ".", "These", "2", "°", "spheres", "were", "then", "passaged", "using", "a", "single", "cell", "dissociation", "protocol", "(", "F", ")", ".", "Arrow", "in", "F", "shows", "an", "example", "of", "a", "focus", "of", "proliferating", "cells", ".", "(", "G", ",", "H", ")", "The", "hNSCs", "were", "passaged", "every", "5–7", "days", ",", "as", "described", "in", "the", "Methods", "section", ".", "Starting", "from", "an", "initial", "population", "of", "1", "million", "cells", ",", "the", "cumulative", "cell", "number", "was", "calculated", "at", "each", "passage", "as", "the", "fold", "of", "increase×the", "total", "cell", "number", "and", "plotted", "as", "logarithm", "with", "base", "2", "in", "function", "of", "time", "(", "G", ")", ".", "The", "cell", "perpetuation", "(", "G", ")", "and", "population", "doubling", "(", "H", ")", "analysis", "demonstrated", "the", "continuous", "and", "stable", "growth", "of", "the", "hNSCs", ".", "(", "I", ")", "RT", "-", "PCR", "analysis", "showing", "the", "down", "regulation", "of", "the", "pluripotency", "transcripts", "Oct4", "and", "Nanog", "in", "secondary", "neurospheres", "and", "in", "expanded", "hNSCs", "at", "passage", "8", "(", "P8", ")", ".", "(", "J", ")", "Cytogenetic", "evaluation", "of", "the", "SD56", "hNSCs", "line", "at", "passage", "12", "by", "standard", "G", "-", "banding", "was", "performed", ".", "Twenty", "metaphase", "cells", "were", "analyzed", "and", "showed", "a", "normal", "female", "chromosome", "complement", "(", "46,XX", ")", ".", "Isolated", "and", "expanded", "hNSCs", "expressed", "the", "neural", "precursor", "cell", "markers", "nestin", "(", "K", ")", ",", "Vimentin", "(", "L", ")", "and", "the", "radial", "glial", "cell", "marker", "3CB2", "(", "M", ")", "in", "virtually", "all", "the", "progeny", ".", "(", "N", "-", "P", ")", "Clonal", "self", "-", "renewal", "ability", "of", "the", "isolated", "hNSCs", "through", "symmetrical", "cell", "division", ".", "Single", "(", "N", ")", ",", "two", "-", "cell", "stage", "(", "O", ")", "and", "four", "-", "cell", "stage", "(", "P", ")", "of", "a", "hNSC", "proliferating", "over", "a", "3-day", "culture", "period", ".", "Note", "the", "symmetrical", "segregation", "of", "BrdU", "and", "nestin", "in", "the", "progeny", ".", "Bars", ":", "(", "A", ",", "B", ",", "C", ",", "D", ")", "200", "µm", ";", "(", "E", ",", "F", ")", "100", "µm", ";", "(", "K", "–", "M", ")", "20", "µm", ";", "(", "N", "–", "P", ")", "10", "µm", ".", "To", "ascertain", "self", "-", "renewal", "ability", "under", "clonal", "conditions", ",", "a", "single", "cell", "suspension", "was", "plated", "at", "clonal", "density", "(", "1–2", "cell/10", "µl", ")", ".", "To", "determine", "if", "the", "hNSCs", "divide", "symmetrically", ",", "we", "pulsed", "cultures", "with", "the", "thymidine", "analog", ",", "bromodeoxyuridine", "(", "BrdU", ")", ",", "after", "plating", "and", "looked", "for", "the", "expression", "of", "nestin", "in", "the", "progeny", ".", "Cultures", "were", "fixed", "after", "1", ",", "2", "or", "3", "DIV", "(", "Figure", "1N", "–", "P", ")", ".", "After", "2", "days", ",", "plated", "single", "cells", "first", "underwent", "a", "symmetric", "cell", "division", "and", "gave", "rise", "to", "daughter", "cells", "that", "were", "both", "positive", "for", "BrdU", "and", "nestin", ".", "The", "clone", "of", "cells", "continued", "to", "grow", "over", "the", "3", "DIV", "and", "all", "the", "progeny", "expressed", "nestin", ".", "BrdU", "labeling", "demonstrated", "that", "it", "was", "rare", "for", "only", "one", "daughter", "cell", "to", "inherit", "the", "BrdU", "and", "thus", "had", "undergone", "asymmetric", "segregation", "of", "the", "chromatids", "(", "Figure", "S2", ")", ".", "G", "-", "band", "karyotyping", "of", "these", "hNSCs", "confirmed", "the", "normal", ",", "non", "-", "transformed", "nature", "of", "cells", "after", "12", "passages", "(", "Figure", "1J", ")", ".", "We", "named", "the", "derived", "hNSCs", "SD56", "(", "intermittently", "referred", "to", "as", "SD56", "hNSCs", "or", "hNSCs).Under", "these", "defined", "growth", "conditions", ",", "the", "hNSCs", "showed", "stable", "growth", "with", "a", "2.7±0.2", "fold", "increase", "every", "5", "to", "7", "days", "(", "Figure", "1", "G", ")", ".", "The", "population", "doubling", "at", "each", "passage", "averaged", "at", "1.4±0.1", "(", "Figure", "1H", ")", ".", "The", "viability", "of", "hNSCs", "at", "each", "passage", "was", "consistent", "at", "the", "approximate", "value", "of", "98", "%", ".", "The", "projected", "cumulative", "cell", "numbers", "demonstrated", "that", "trillions", "of", "cells", "could", "be", "generated", "over", "a", "period", "of", "5", "months", "(", "Figure", "1", "G", ")", ".", "We", "expanded", "the", "isolated", "hNSCs", "lines", "for", "over", "20", "passages", "with", "a", "stable", "phenotype", ".", "An", "initial", "cell", "bank", "of", "75", "vials", "containing", "2", "to", "5", "million", "cells", "each", "was", "generated", "and", "cryopreserved", ".", "Upon", "removal", "of", "the", "mitogenic", "factors", "and", "plating", "on", "a", "coverslip", "pre", "-", "coated", "with", "poly", "-", "L", "-", "ornithine", "(", "PLO", ")", "substrate", ",", "the", "hNSCs", "spontaneously", "differentiated", "into", "neurons", ",", "astrocytes", "and", "oligodendrocytes", ",", "a", "property", "that", "is", "consistent", "with", "normal", "multipotent", "hNSCs", "(", "Figure", "2", ")", ".", "After", "2", "DIV", ",", "hNSCs", "expressed", "transcripts", "for", "the", "neural", "-", "specific", "genes", "nestin", ",", "Notch1", "and", "neural", "cell", "adhesion", "molecule", "(", "N", "-", "CAM", ")", "(", "Figure", "2A", ")", "and", "for", "the", "lineage", "specific", "markers", "β", "-", "tubulin", "class", "III", ",", "medium", "-", "size", "neurofilament", "(", "NF", "-", "M", ")", "and", "microtubule", "-", "associated", "protein", "2", "(", "MAP-2", ")", "for", "neurons", ",", "GFAP", "for", "astrocytes", "and", "myelin", "basic", "protein", "(", "MBP", ")", "for", "oligodendrocytes", "(", "Figure", "2A", ")", ".", "Transcripts", "for", "the", "GABAergic", "cell", "marker", "glutamic", "acid", "decarboxylase", "(", "GAD", ")", "were", "expressed", ",", "but", "transcripts", "for", "the", "tyrosine", "hydroxylase", "(", "TH", ")", ",", "a", "marker", "for", "dopaminergic", "neurons", ",", "were", "undetectable", ".", "Immunocytochemical", "analysis", "(", "Figure", "2B", "–", "F", ")", "of", "10", "day", "-", "old", "cultures", "demonstrated", "that", "the", "proportion", "of", "nestin+", "cells", "was", "36.6±2.7", "%", ",", "62.5±2.8", "%", "expressed", "the", "neuronal", "marker", "TuJ1", ",", "1.9±0.3", "%", "expressed", "the", "astrocytic", "marker", "GFAP", "and", "7.1±0.4", "%", "were", "oligodendrocytes", "and", "expressed", "galactocerebrocide", "(", "GC", ")", "(", "Figure", "2F", ")", ".", "A", "subset", "(", "9.8±1.6", "%", ")", "of", "the", "GFAP+", "astrocytes", "co", "-", "expressed", "nestin.10.1371", "/", "journal.pone.0001644.g002Figure", "2hESC", "-", "derived", "hNSCs", "spontaneously", "differentiated", "into", "the", "3", "principal", "central", "nervous", "system", "cell", "types", ".", "Dissociated", "hNSCs", "were", "washed", "free", "of", "growth", "factors", "and", "plated", "on", "poly", "-", "L", "-", "onithine", "coated", "glass", "coverslips", ".", "Differentiated", "cultures", "were", "either", "harvested", "after", "2", "DIV", "for", "total", "RNA", "extraction", "and", "RT", "-", "PCR", "analysis", "or", "fixed", "after", "10", "DIV", "and", "processed", "for", "indirect", "immunocytochemistry", ".", "(", "A", ")", "Differentiated", "hNSCs", "expressed", "the", "neural", "-", "specific", "transcripts", "nestin", "and", "Notch1", "as", "well", "as", "transcripts", ":", "for", "neurons", "(", "β", "-", "tubulin", "class", "III", ",", "MAP-2", ",", "NCAM", "and", "medium", "-", "size", "neurofilament", ",", "NF", "-", "M", ")", ",", "for", "astrocytes", "(", "GFAP", ")", "and", "for", "oligodendrocytes", "(", "MBP", ")", ".", "The", "hNSCs", "expressed", "transcripts", "for", "GAD", ",", "but", "not", "for", "TH", ".", "B", ",", "C", "&", "D", ",", "morphology", "of", "NSC", "-", "derived", "progeny", "differentiated", "into", "GFAP+", "astrocytes", "(", "B", ")", ",", "GC", "-", "expressing", "oligodendrocytes", "(", "C", ")", "and", "TuJ1", "+", "neurons", "(", "D", ")", ",", "DAPI", "(", "blue", ")", "show", "life", "cell", "nuclei", ".", "(", "E", ")", "Photo", "showing", "cultures", "double", "-", "immunostained", "for", "TuJ1", "(", "green", ")", "and", "nestin", "(", "red", ")", "(", "DAPI", ",", "blue", ")", ".", "(", "F", ")", "Quantitative", "analysis", "of", "immunostained", "10", "day", "-", "old", "cultures", "for", "the", "3", "neural", "cell", "types", ".", "Results", "are", "mean±s.e.m", ".", "of", "three", "independent", "experiments", ",", "each", "performed", "in", "duplicate", ".", "Bars", ":", "(", "c", ")", "20", "µm", ";", "(", "d", ",", "e", ")", "10", "µm", ".", "2", ".", "The", "isolated", "hNSCs", "are", "normal", "and", "do", "not", "form", "tumors", "in", "normal", "nude", "ratsThe", "self", "-", "renewal", "and", "pluripotent", "abilities", "of", "the", "hESCs", "are", "also", "associated", "with", "tumorigenic", "properties", ".", "Therefore", ",", "the", "first", "critical", "step", "toward", "developing", "therapeutic", "hNSCs", "is", "to", "verify", "that", "they", "are", "non", "-", "tumorigenic", ".", "The", "monolayer", "culture", "of", "SD56", "hNSCs", "clearly", "demonstrated", "contact", "inhibition", "of", "growth", ",", "a", "normal", "karyotype", "and", "did", "not", "express", "the", "pluripotency", "transcripts", "Oct-4", "and", "Nanog", ".", "Removal", "of", "mitogenic", "factors", "and", "continued", "culture", "on", "plastic", "resulted", "in", "cell", "senescence", "that", "is", "characteristic", "of", "non", "-", "transformed", "cells", ".", "To", "determine", "whether", "SD56", "hNSCs", "form", "tumors", "in", "vivo", ",", "we", "transplanted", "them", "at", "high", "density", "(", "see", "Methods", ")", "into", "the", "forebrain", "and", "subcutaneously", "into", "the", "flank", "of", "nude", "rats", ".", "The", "animals", "were", "kept", "for", "a", "two", "-", "month", "post", "-", "transplant", "survival", "period", ".", "To", "label", "mitotically", "active", "cells", "in", "vivo", "during", "S", "-", "phase", ",", "the", "rats", "were", "injected", "IP", "with", "BrdU", "(", "50", "mg", "/", "kg", ")", "every", "8", "hours", "during", "the", "last", "24", "hours", "before", "euthanasia", ".", "The", "transit", "amplifying", "endogenous", "precursors", "located", "in", "the", "subventricular", "zone", "(", "SVZ", ")", "were", "labeled", "(", "Figure", "S3", ")", ";", "however", ",", "we", "were", "unable", "to", "detect", "grafted", "SD56", "hNSCs", "co", "-", "localizing", "the", "human", "-", "specific", "nuclear", "marker", "hNuc", "and", "BrdU", "(", "Figure", "S3", ")", ".", "No", "surviving", "SD56", "hNSCs", "were", "detected", "in", "the", "flank", "of", "the", "transplanted", "animals", "suggesting", "that", "the", "grafted", "cells", "are", "not", "tumorigenic", ".", "3", ".", "Transplanted", "cells", "survived", ",", "migrated", "toward", "and", "engrafted", "into", "the", "stroke", "-", "damaged", "host", "tissueTo", "investigate", "the", "survival", "and", "functional", "engraftment", "in", "an", "injury", "environment", ",", "hNSCs", "(", "4×105", ")", "were", "transplanted", "into", "the", "ischemic", "boundary", "zone", "in", "the", "rat", "striatum", "one", "week", "after", "the", "middle", "cerebral", "artery", "occlusion", "(", "MCAO", ")", "was", "performed", ".", "Animals", "were", "euthanized", "two", "months", "later", "and", "the", "brains", "processed", "for", "histo", "-", "pathology", "and", "immunocytochemistry", ".", "Grafted", "SD56", "hNSCs", ",", "identified", "with", "hNuc", ",", "demonstrated", "a", "37.0±15.8", "%", "survival", "rate", "and", "a", "remarkable", "dispersion", "toward", "the", "stroke", "-", "damaged", "tissue", "with", "no", "sign", "of", "overgrowth", "or", "tumorigenesis", ".", "The", "majority", "of", "grafted", "cells", "(", "61.2±4.7", "%", ")", "migrated", "at", "least", "200", "µm", "away", "from", "the", "injection", "site", "and", "penetrated", "an", "average", "distance", "of", "806.4±49.3", "µm", "into", "the", "stroke", "-", "damaged", "tissue", "(", "Figure", "3A", "–", "C", ")", ".", "Immunostaining", "with", "the", "blood", "vessel", "marker", ",", "GluT1", ",", "revealed", "dilated", "vessels", "in", "the", "infarcted", "striatum", "and", "a", "close", "association", "between", "vessels", "and", "the", "grafted", "hNSCs", "(", "Figure", "3B", ",", "3C", ")", ".", "The", "grafted", "cells", "rarely", "expressed", "the", "proliferation", "marker", "Ki67", "(", "Figure", "3D", ")", ",", "29.8±4.4", "%", "expressed", "nestin", "(", "Figure", "3E", ")", ",", "6.5±0.9", "%", "expressed", "doublecortin", "(", "DCX", ")", "and", "60.8±8.1", "%", "were", "TuJ1", "+", "(", "Figure", "3F", ",", "G", ")", ".", "Grafted", "cells", "rarely", "co", "-", "expressed", "the", "astroglial", "marker", "GFAP", "(", "Figure", "3H", ")", "or", "differentiated", "into", "CNPase", "-", "expressing", "oligodendrocytes", "(", "Figure", "3I", ")", ".", "Immunostaining", "for", "GAD", "demonstrated", "that", "25.1±2.3", "%", "of", "grafted", "hNSCs", "differentiated", "into", "GABAergic", "neurons", "while", "less", "than", "2", "%", "were", "positive", "for", "glutamate", "(", "Figure", "3J", ",", "K).10.1371", "/", "journal.pone.0001644.g003Figure", "3Dispersion", ",", "engraftment", "and", "differentiation", "of", "the", "hNSCs", "in", "stroke", "-", "lesioned", "animals.(A", ")", "Schematic", "drawing", "of", "a", "frontal", "section", "through", "the", "striatum", "illustrating", "the", "dispersion", "of", "grafted", "hNSCs", "in", "the", "focal", "ischemia", "-", "lesioned", "parenchyma", "(", "shaded", "area", ")", ".", "(", "B", ",", "C", ")", "Photos", "show", "frontal", "sections", "through", "the", "graft", "in", "the", "striatum", "immunostained", "with", "the", "human", "specific", "antibodies", ":", "anti", "-", "hNuc", "(", "green", "in", "B", "&", "C", ")", "and", "anti", "-", "GluT1", "(", "red", ",", "B", "&", "C", ")", "showing", "blood", "vessels", "and", "dispersed", "hNSCs", "in", "the", "graft", "zone", ".", "C", ":", "higher", "magnification", "of", "the", "inset", "in", "B.", "(", "D", "–", "I", ")", "Photos", "taken", "from", "frontal", "sections", "through", "the", "graft", "in", "the", "striatum", "double", "immunoprocessed", "for", "cell", "proliferation", "and", "neural", "lineage", "markers", ".", "(", "D", ")", "Note", "the", "endogenous", "Ki67", "+", "cells", "(", "red", "cells", ",", "arrow", ")", "in", "the", "stroke", "damaged", "area", "and", "the", "hNuc+", "(", "green)/Ki67-", "grafted", "hNSCs", "(", "arrowheads", ")", ".", "(", "E", ")", "Examples", "of", "grafted", "SD56", "hNSCs", "showing", "co", "-", "expression", "of", "hNuc", "(", "green", ")", "and", "nestin", "(", "red", ")", ".", "(", "F", ")", "Confocal", "3D", "reconstructed", "orthogonal", "images", "of", "the", "hNuc+(green)/DCX+(red", ")", "NSCs", "(", "arrowheads", ")", "viewed", "in", "the", "x", "-", "z", "plan", "on", "the", "top", "and", "y", "-", "z", "plan", "on", "the", "right", ".", "(", "G", ")", "Examples", "show", "the", "majority", "of", "grafted", "NSC", "progeny", "co", "-", "expressing", "hNuc", "(", "red", ")", "and", "the", "neuronal", "marker", "TuJ1", "(", "green", ")", ".", "Grafted", "NSCs", "rarely", "differentiate", "into", "GFAP+", "astrocytes", "(", "H", ")", ".", "In", "I", ",", "rare", "example", "of", "grafted", "NSC", "progeny", "becoming", "an", "oligodendrocyte", "identified", "by", "the", "expression", "of", "CNPase", "(", "green", ")", ".", "Grafted", "NSCs", "expressed", "the", "GABAergic", "marker", "GAD65/67", "(", "J", ")", "and", "rarely", "expressed", "glutamate", "(", "K", ")", ".", "(", "Abbreviations", ":", "Cx", ":", "cortex", ",", "Str", ":", "striatum", ")", ".", "Bars", ":", "(", "B", ",", "C", ")", "100", "µm", ";", "(", "D", ",", "F", ")", "20", "µm", ";", "(", "E", ",", "G", "–", "K", ")", "10", "µm", ".", "4", ".", "Transplanted", "cells", "improve", "sensorimotor", "function", "of", "the", "stroke", "-", "disabled", "forelimbWe", "asked", "whether", "transplanted", "SD56", "hNSCs", "could", "enhance", "the", "recovery", "of", "sensorimotor", "function", "that", "is", "compromised", "in", "the", "stroke", "-", "injured", "rats", ".", "We", "used", "the", "cylinder", "test", "to", "measure", "the", "sensorimotor", "asymmetry", "in", "forelimb", "use", "during", "spontaneous", "exploration", "[", "22", "]", ".", "To", "establish", "the", "baseline", "of", "the", "stroke", "-", "induced", "sensorimotor", "deficit", ",", "spontaneous", "behavior", "of", "rats", "in", "a", "transparent", "cylinder", "was", "videotaped", "one", "week", "after", "stroke", "(", "pre", "-", "transplant", ",", "Figure", "4", ")", ".", "Tests", "were", "then", "conducted", "4", "and", "8", "weeks", "after", "vehicle", "and", "SD56", "hNSCs", "transplantation", ".", "Stable", "asymmetry", "in", "forelimb", "use", "was", "observed", "7", "days", "post", "-", "stroke", "(", "pre", "-", "transplant", ",", "Figure", "4", ")", ".", "Ischemic", "rats", "used", "their", "impaired", "forelimbs", "(", "contralateral", "to", "lesion", ")", "during", "lateral", "exploration", "less", "than", "they", "did", "before", "stroke", ".", "Transplantation", "of", "SD56", "hNSCs", "significantly", "enhanced", "the", "independent", "use", "of", "the", "impaired", "contralateral", "forelimb", "4", "weeks", "post", "transplantation", "(", "P<0.05", "vs", "pre", "-", "transplant", ")", ".", "Eight", "weeks", "after", "transplantation", "the", "improvement", "in", "the", "use", "of", "the", "impaired", "forelimb", "was", "stable", "and", "significant", "when", "compared", "to", "the", "pre", "-", "transplant", "group", "and", "significantly", "improved", "in", "comparison", "to", "vehicle", "treated", "group", "at", "8", "weeks", "(", "Figure", "4", ")", ".", "In", "the", "vehicle", "treated", "group", ",", "the", "independent", "use", "of", "the", "contralateral", "forelimb", "remained", "impaired", "4", "and", "8", "weeks", "post", "-", "injection", ".", "In", "an", "independent", "study", "and", "using", "the", "same", "MCAO", "rat", "animal", "model", ",", "we", "found", "that", "transplantation", "of", "dermal", "fibroblasts", "did", "not", "improve", "the", "stroke", "-", "induced", "motor", "deficits", "(", "unpublished", "data).10.1371", "/", "journal.pone.0001644.g004Figure", "4Transplantation", "of", "NSCs", "improves", "sensorimotor", "function", "of", "the", "stroke", "-", "disabled", "forelimb", ".", "Forelimb", "use", "during", "spontaneous", "lateral", "exploration", "was", "measured", "in", "the", "cylinder", "test", "(", "see", "Method", "and", "Results", "sections", "for", "details", ")", ".", "Groups", "of", "vehicle", "injected", "(", "n", " ", "=", " ", "7", ")", "and", "hNSCs", "(", "n", " ", "=", " ", "10", ")", "transplanted", "are", "represented", ".", "The", "animals", "were", "tested", "as", "described", "in", "Method", "section", ".", "Note", "the", "significant", "increase", "in", "the", "independent", "use", "of", "the", "impaired", "contralateral", "forelimb", "at", "4", "and", "8", "weeks", "post", "transplantation", "(", "P<0.05", "vs", "pre", "-", "transplant", "group", ")", ".", "The", "contralateral", "forelimb", "remained", "impaired", "in", "the", "vehicle", "treated", "group", "at", "4", "and", "8", "weeks", "post", "-", "injection", ".", "Bars", "represent", "percentages±s.e.m", ".", "of", "steps", "taken", "by", "the", "ipsilateral", ",", "contralateral", "and", "both", "forelimbs", "simultaneously", ".", "*", "P<0.05", "vs", "pre", "-", "transplant", "group", ";", "#", "P<0.05", "vs", "vehicle", "groups", ".", "\n", "Our", "results", "indicate", "that", "a", "self", "-", "renewable", "and", "homogenous", "population", "of", "hNSCs", ",", "SD56", ",", "was", "derived", "from", "hESCs", "using", "defined", "media", "supplemented", "with", "a", "specific", "combination", "of", "growth", "factors", ".", "The", "SD56", "hNSCs", "grew", "as", "an", "adherent", "monolayer", "culture", ",", "uniformly", "expressed", "molecular", "features", "of", "hNSCs", "including", "nestin", ",", "vimentin", "and", "the", "radial", "glial", "marker", "3CB2", ",", "and", "did", "not", "express", "the", "pluripotency", "markers", "Oct4", "or", "Nanog", ".", "The", "self", "-", "renewal", "property", "of", "the", "hNSCs", "was", "characterized", "by", "a", "predominant", "symmetrical", "mode", "of", "cell", "division", ".", "They", "exhibited", "no", "chromosomal", "abnormalities", "and", "demonstrated", "non", "-", "tumorigenic", "properties", "after", "implantation", "into", "ischemic", "brains", "and", "into", "naïve", "nude", "rat", "brains", "and", "flanks", ".", "Furthermore", ",", "the", "transplanted", "SD56", "hNSCs", "migrated", "toward", "the", "stroke", "-", "damaged", "adult", "brain", "parenchyma", ",", "engrafted", "and", "improved", "the", "independent", "use", "of", "the", "stroke", "-", "impaired", "forelimb", ".", "Maintenance", "of", "stem", "cells", "requires", "symmetrical", "and", "asymmetrical", "cell", "divisions", "to", "both", "expand", "and", "to", "give", "rise", "to", "specialized", "progeny", "of", "a", "specific", "tissue", "(", "reviewed", "in", "[", "23", "]", ")", ".", "In", "vivo", ",", "a", "complex", "cellular", "micro", "-", "environment", "or", "niche", "ensures", "the", "self", "-", "maintenance", "property", "of", "NSCs", "[", "24", "]", ",", "[", "25", "]", ",", "[", "26", "]", ",", "[", "27", "]", ".", "In", "vitro", ",", "defined", "growth", "factors", "and", "extracellular", "matrices", "support", "stem", "cell", "self", "-", "renewal", "[", "28", "]", ",", "[", "29", "]", ".", "The", "embryonic", "stem", "cells", "can", "propagate", "in", "a", "predominantly", "proliferative", "symmetrical", "mode", ",", "leading", "to", "homogeneous", "cell", "cultures", "growing", "relatively", "quickly", "with", "minimal", "cell", "differentiation", "[", "30", "]", ",", "[", "31", "]", ",", "[", "32", "]", ",", "[", "33", "]", ",", "[", "34", "]", ".", "Tissue", "specific", "stem", "cells", ",", "however", ",", "self", "-", "renew", "in", "a", "predominant", "asymmetric", "mode", "to", "maintain", "them", "selves", "and", "compensate", "for", "the", "loss", "of", "differentiated", "cells", "due", "to", "disease", "or", "injury", ".", "Thus", ",", "NSCs", "isolated", "from", "developing", "or", "adult", "brain", "grow", "as", "a", "mixture", "of", "undifferentiated", "and", "differentiated", "cells", "due", "the", "predominant", "asymmetrical", "mode", "of", "cell", "division", "[", "35", "]", ",", "[", "36", "]", ",", "[", "37", "]", ",", "[", "38", "]", ",", "[", "39", "]", ",", "[", "40", "]", ",", "[", "41", "]", ".", "A", "recent", "study", "has", "reported", "that", "a", "murine", "ESC", "-", "derived", "NSC", "line", "(", "LC1", ")", "is", "propagated", "as", "an", "adherent", "homogenous", "culture", "with", "a", "dominant", "mode", "of", "symmetrical", "self", "-", "renewal", "[", "21", "]", ".", "A", "combination", "of", "EGF", "and", "FGF2", "was", "sufficient", "to", "propagate", "these", "NSCs", "as", "an", "adherent", "monolayer", ".", "However", ",", "the", "SD56", "hNSC", "line", "described", "here", "required", "the", "combination", "of", "EGF", ",", "bFGF", "and", "LIF", "for", "self", "-", "maintenance", ".", "Although", "there", "are", "morphological", "and", "molecular", "similarities", "between", "our", "hNSCs", "and", "the", "NSCs", "previously", "described", "[", "21", "]", ",", "the", "methods", "of", "isolation", "and", "growth", "are", "different", ".", "In", "addition", "to", "the", "different", "combination", "of", "growth", "factors", "used", ",", "the", "hNSC", "line", "we", "have", "isolated", "did", "not", "go", "through", "the", "rosette", "-", "structure", "stage", ".", "The", "in", "vitro", "analysis", "of", "BrdU", "incorporation", "and", "nestin", "expression", "indicated", "that", "our", "hNSCs", "divide", "predominantly", "symmetrically", ".", "This", "type", "of", "growth", "pattern", "is", "characteristic", "of", "primitive", "normal", "stem", "cells", "undergoing", "mostly", "symmetric", "cell", "division", "to", "increase", "the", "stem", "cell", "pool", "at", "the", "early", "stage", "of", "development", "or", "during", "tissue", "regeneration", "after", "injury", "[", "23", "]", ".", "RT", "-", "PCR", "and", "immunocytochemistry", "analysis", "demonstrated", "that", "these", "undifferentiated", "SD56", "cells", "did", "not", "express", "any", "pluripotency", ",", "endodermal", "or", "mesodermal", "markers", ".", "Furthermore", ",", "the", "SD56", "hNSCs", "described", "here", "exhibited", "the", "multipotential", "characteristic", "to", "differentiate", "into", "neurons", ",", "astrocytes", "and", "oligodendrocytes", "both", "in", "vitro", "and", "upon", "transplantation", ".", "Together", "these", "findings", "suggest", "that", "the", "hNSC", "line", "we", "isolated", "are", "appropriately", "programmed", "and", "share", "similar", "characteristics", "with", "the", "definitive", "NSCs", "of", "the", "developing", "brain", ".", "The", "SD56", "hNSCs", "demonstrated", "a", "remarkable", "ability", "to", "migrate", "toward", "the", "stroke", "-", "damaged", "parenchyma", "of", "the", "adult", "rat", "brain", ".", "This", "directed", "migration", "by", "the", "majority", "of", "the", "grafted", "cells", "could", "be", "due", "to", "their", "uniform", "cellular", "composition", ",", "which", "results", "in", "an", "equal", "response", "to", "the", "host", "microenvironment", ".", "In", "previous", "studies", ",", "subpopulations", "of", "transplanted", "hESCs", "that", "were", "enriched", "in", "neural", "cells", "migrated", "in", "host", "microenvironments", "conducive", "to", "cell", "migration", ",", "such", "as", "the", "developing", "brain", "or", "in", "structures", "such", "as", "the", "rostral", "migratory", "stream", "[", "13", "]", ",", "[", "20", "]", ".", "In", "the", "adult", "lesioned", "brain", ",", "the", "grafted", "hESC", "-", "derived", "neural", "cells", "proliferated", "and", "formed", "rosettes", "[", "14", "]", ",", "teratomas", "[", "12", "]", ",", "[", "15", "]", "or", "a", "cellular", "mass", "that", "induced", "a", "gliotic", "host", "response", "whereby", "local", "astrocytes", "demarcated", "the", "grafts", "[", "16", "]", ".", "Enriched", "neural", "cultures", "derived", "from", "mouse", "[", "42", "]", "and", "monkey", "ESCs", "[", "43", "]", "have", "produced", "behavioral", "improvements", "when", "transplanted", "into", "animal", "models", "of", "stroke", "and", "brain", "injury", ".", "However", ",", "in", "these", "cases", ",", "the", "transplanted", "non", "-", "human", "ESCs", "also", "formed", "a", "mass", "with", "signs", "of", "overgrowth", "in", "the", "core", ",", "as", "well", "as", "deformations", "[", "44", "]", ",", "[", "45", "]", ",", "[", "46", "]", ".", "ESCs", "plated", "at", "low", "density", "acquire", "neural", "identity", "within", "few", "hours", "after", "plating", "[", "47", "]", ".", "Interestingly", ",", "nearly", "all", "viable", "cells", "expressed", "nestin", ",", "the", "early", "neural", "fate", "marker", "Sox1", ",", "and", "the", "pluripotency", "marker", "Oct4", ".", "Together", ",", "these", "studies", "are", "seminal", "and", "suggest", "that", "complete", "neuralization", "may", "not", "be", "achieved", "through", "certain", "enrichment", "processes", ",", "consequently", "the", "neural", "cells", "could", "revert", "to", "a", "pluripotent", "stage", "[", "17", "]", ".", "The", "dispersion", "of", "the", "grafted", "hNSCs", "within", "host", "parenchyma", "may", "allow", "for", "more", "graft", "-", "host", "interactions", "that", "could", "stabilize", "differentiation", ",", "inhibit", "growth", "and", "prevent", "gliotic", "host", "response", ".", "In", "the", "present", "study", ",", "SD56", "hNSC", "-", "transplanted", "animals", "demonstrated", "a", "stable", "improvement", "in", "the", "sensorimotor", "function", "when", "evaluated", "for", "spontaneous", "exploratory", "activity", "in", "the", "cylinder", "test", "that", "detects", "long", "-", "lasting", "deficits", "in", "forelimb", "use", "in", "the", "experimental", "models", "of", "stroke", "[", "22", "]", ".", "The", "transplantation", "of", "hNSCs", "significantly", "enhanced", "the", "independent", "use", "of", "the", "impaired", "contralateral", "forelimb", "8", "weeks", "post", "transplantation", ".", "This", "is", "the", "first", "report", "demonstrating", "that", "the", "transplantation", "of", "hNSCs", "derived", "from", "hESCs", "can", "improve", "neurologic", "behavior", "after", "experimental", "stroke", ".", "Together", ",", "these", "findings", "are", "encouraging", "and", "suggest", "that", "these", "cells", "are", "promising", "for", "future", "development", ".", "In", "addition", "to", "their", "therapeutic", "application", ",", "the", "hNSCs", "isolated", "under", "the", "reported", "conditions", "offer", "a", "means", "to", "interrogate", "host", "environments", "and", "unravel", "mechanistic", "features", "of", "self", "-", "renewal", ",", "non", "-", "tumorigenicity", "and", "functional", "engraftability", "in", "animal", "models", "of", "neurological", "disorders", ".", "\n", "hESC", "and", "NSC", "CulturesThe", "hESC", "line", "H9", "(", "WiCell", "Research", "Institute", ")", "was", "propagated", "every", "5", "to", "7", "days", "on", "irradiated", "mouse", "embryonic", "fibroblasts", ".", "The", "cell", "culture", "media", "consisted", "of", "a", "1∶1", "mixture", "of", "Dulbecco", "'s", "modified", "Eagle", "'s", "medium", "(", "DMEM", ")", "and", "F12", "nutrient", ",", "20", "%", "serum", "replacement", "(", "Invitrogen", ")", ",", "0.1", "mM", "β", "-", "mercaptoethanol", ",", "2", "µg", "/", "ml", "heparin", "and", "4", "ng", "/", "ml", "bFGF", "(", "R&D", "Systems", ")", ".", "To", "generate", "the", "NSCs", ",", "dissociated", "hESCs", "were", "cultured", "in", "a", "chemically", "defined", "medium", "composed", "of", "DMEM", "/", "F12", "(", "1∶1", ")", "including", "glucose", "(", "0.6", "%", ")", ",", "glutamine", "(", "2", "mM", ")", ",", "sodium", "bicarbonate", "(", "3", "mM", ")", ",", "and", "HEPES", "buffer", "(", "5", "mM", ")", "[", "all", "from", "Sigma", "except", "glutamine", "(", "Invitrogen", ")", "]", ".", "A", "defined", "hormone", "mix", "and", "salt", "mixture", "(", "Sigma", ")", ",", "including", "insulin", "(", "25", "mg", "/", "ml", ")", ",", "transferrin", "(", "100", "mg", "/", "ml", ")", ",", "progesterone", "(", "20", "nM", ")", ",", "putrescine", "(", "60", "mM", ")", ",", "and", "selenium", "chloride", "(", "30", "nM", ")", "was", "used", "in", "place", "of", "serum", ".", "The", "medium", "was", "supplemented", "with", "EGF", "(", "20", "ng", "/", "ml", ")", ",", "bFGF", "(", "10", "ng", "/", "ml", ")", "and", "LIF", "(", "10", "ng", "/", "ml", ")", ".", "Dissociated", "hNSCs", "were", "plated", "at", "a", "density", "of", "100,000", "cell", "/", "ml", "in", "Corning", "T75", "(", "Invitrogen", ")", "culture", "flasks", "in", "the", "defined", "media", "together", "with", "the", "growth", "factors", ".", "After", "5–7", "DIV", ",", "the", "adherent", "culture", "was", "incubated", "in", "0.025%trypsin/0.01", "%", "EDTA", "(", "w", "/", "v", ")", "for", "1", "min", "followed", "by", "the", "addition", "of", "trypsin", "inhibitor", "(", "Invitrogen", ")", "then", "gently", "triturated", "to", "achieve", "single", "cell", "suspension", ".", "The", "cells", "were", "then", "washed", "twice", "with", "fresh", "medium", "and", "reseeded", "in", "fresh", "growth", "factor", "-", "containing", "media", "at", "100,000", "cells", "/", "ml", ".", "This", "procedure", "was", "performed", "for", "21", "passages", "and", "the", "fold", "of", "increase", "and", "population", "doubling", "were", "calculated", "at", "each", "passage", ".", "For", "clonal", "analysis", ",", "single", "spheres", "or", "confluent", "hNSC", "cultures", "were", "single", "cell", "dissociated", "and", "serially", "diluted", "to", "yield", "1–2", "cell/10", "µl", ".", "A", "10-µl", "-", "cell", "suspension", "was", "then", "added", "to", "each", "of", "96", "or", "24", "well", "plates", "containing", "200–300", "µl", "of", "growth", "media", ".", "Wells", "containing", "one", "viable", "cell", "were", "marked", "the", "next", "day", "and", "re", "-", "scored", "5", "to", "7", "days", "later", "for", "cell", "proliferation", ".", "The", "differentiation", "of", "the", "hNSCs", "was", "performed", "as", "previously", "described", "[", "48", "]", ".", "Dissociated", "hNSCs", "were", "plated", "at", "a", "density", "of", "106", "cells", "/", "ml", "in", "control", "(", "media", "/", "hormone", "mix", ")", "medium", "devoid", "of", "any", "growth", "factors", "and", "supplemented", "with", "1", "%", "fetal", "bovine", "serum", "(", "FBS", ")", "on", "poly", "-", "L", "-", "ornithine", "-", "coated", "(", "15", "mg", "/", "ml", ";", "Sigma", ")", "glass", "coverslips", "in", "24-well", "Nunclon", "culture", "dishes", "with", "0.5", "ml", "/", "well", ".", "After", "2", ",", "7–15", "DIV", "cultures", "were", "fixed", "and", "processed", "for", "immunocytochemistry", "or", "used", "for", "RT", "-", "PCR", "analysis", ".", "Karyotype", "analysisLong", "-", "term", "cultures", "of", "hNSCs", "were", "incubated", "at", "37", "°", "C", "and", "harvested", "for", "metaphase", "chromosomes", "when", "the", "cultures", "were", "75", "%", "confluent", ".", "Metaphase", "chromosomes", "were", "obtained", "by", "standard", "chromosome", "harvest", "methods", "by", "exposure", "to", "Colcemid", "at", "0.1", "µg", "/", "ml", "for", "1", "hour", "at", "37", "°", "C", ",", "a", "2-minute", "exposure", "to", "trypsin", "/", "EDTA", ",", "hypotonized", "with", "0.057", "M", "KCl", "and", "fixed", "with", "3∶1", "methanol", ":", "acetic", "acid", ".", "Slide", "preparations", "were", "made", "by", "dropping", "the", "fixed", "cell", "pellet", "onto", "cold", ",", "wet", "slides", "and", "air", "-", "dried", ".", "After", "incubating", "the", "slides", "at", "90", "°", "C", "for", "30", "minutes", ",", "chromosomes", "were", "trypsin", "banded", "and", "then", "Wright", "/", "Giemsa", "stained", "for", "G", "-", "banding", "analysis", ".", "Twenty", "metaphase", "cells", "were", "completely", "analyzed", "and", "a", "normal", "female", "chromosome", "complement", "was", "found", "(", "46,XX", ")", ".", "Tumorigenicity", "in", "nude", "ratsAll", "animal", "experiments", "were", "conducted", "according", "to", "the", "National", "Institute", "of", "Health", "(", "NIH", ")", "guidelines", "and", "approved", "by", "the", "IACUC", ".", "Normal", "adult", "NIH", "-", "Nude", "rats", "(", "n", " ", "=", " ", "5", ",", "8", "week", "-", "old", ",", "Taconic", ",", "Germantown", ",", "New", "York", ",", "United", "States", ")", "were", "used", "to", "test", "the", "tumorigenic", "potential", "of", "the", "SD56", "hNSCs", ".", "Undifferentiated", "hNSCs", "from", "passage", "9", "were", "single", "cell", "dissociated", "using", "trypsin", "-", "EDTA", "and", "suspended", "at", "the", "concentration", "of", "125,000", "cell/µl", "in", "preparation", "for", "cell", "transplantation", ".", "Two", "µl", "of", "the", "cell", "suspension", "were", "stereotaxically", "transplanted", "into", "4", "sites", "within", "the", "striatum", "at", "the", "following", "coordinates", ":", "AP", ":", "+1.0", "mm", ",", "ML", ":", "+3.2", "mm", ",", "DV", ":", "−5.0", ";", "AP", ":", "+0.5", "mm", ",", "ML", ":", "+3.0", "mm", ",", "DV", ":", "−5.0", ";", "AP", ":", "−0.5", "mm", ",", "ML", ":", "+3.0", "mm", ",", "DV", ":", "−5.0", ";", "AP", ":", "−1.0", "mm", ",", "ML", ":", "+3.5", "mm", ",", "DV", ":", "−5.0", "mm", "with", "the", "incisor", "bar", "set", "at", "3.4", "mm", ".", "The", "injection", "rate", "was", "1", "µl", "/", "min", ",", "and", "the", "cannula", "was", "left", "in", "place", "for", "an", "additional", "5", "min", "before", "retraction", ".", "For", "the", "flank", "tumor", "assay", ",", "2×106", "cells", "(", "125,000", "cell/µl", ")", "were", "injected", "subcutaneously", "to", "the", "side", "of", "the", "adult", "nude", "rats", ".", "To", "label", "mitotically", "active", "cells", "in", "vivo", "during", "S", "-", "phase", ",", "the", "rats", "were", "injected", "IP", "with", "the", "BrdU", "(", "50", "mg", "/", "kg", ",", "Sigma", ")", "every", "8", "hours", "during", "the", "last", "24", "hours", "before", "euthanasia", ".", "After", "2-month", "survival", "time", ",", "rats", "were", "euthanized", "and", "a", "postmortem", "examination", "for", "tumor", "formation", "was", "performed", ".", "Induction", "of", "Focal", "Ischemia", "and", "Cell", "TransplantationAll", "animal", "experimentations", "were", "conducted", "according", "to", "the", "National", "Institute", "of", "Health", "(", "NIH", ")", "guidelines", "and", "approved", "by", "the", "IACUC", ".", "Sprague", "Dawley", "adult", "male", "rats", "(", "n", " ", "=", " ", "17", ",", "275g–310", "g", ",", "Charles", "River", "Laboratories", ",", "Wilmington", ",", "Massachusetts", ",", "United", "States", ")", "were", "subjected", "to", "one", "and", "a", "half", "hour", "suture", "occlusion", "of", "the", "middle", "cerebral", "artery", "(", "MCAO", ")", ",", "as", "previously", "described", "[", "49", "]", "and", "immunosuppressed", "2", "days", "before", "cell", "transplantation", "and", "daily", "thereafter", "for", "one", "week", "with", "i.p", ".", "injections", "of", "cyclosporine", "A", "(", "20", "mg", "/", "ml", ",", "Sandimmune", ",", "Novartis", "Pharmaceuticals", ")", ".", "Thereafter", "oral", "cyclosporine", "was", "used", "at", "210", "µg", "/", "ml", "in", "drinking", "water", "until", "euthanasia", ".", "Undifferentiated", "SD56", "hNSCs", "from", "passages", "between", "P9", "and", "P13", "were", "single", "cell", "dissociated", "using", "trypsin", "-", "EDTA", "in", "preparation", "for", "cell", "transplantation", ".", "One", "week", "after", "the", "stroke", "lesion", ",", "2", "µl", "of", "the", "hNSCs", ",", "at", "a", "concentration", "of", "50,000", "cell/µl", ",", "were", "stereotaxically", "transplanted", "into", "4", "sites", "within", "the", "lesioned", "striatum", "(", "n", " ", "=", " ", "10", ")", "at", "the", "following", "coordinates", ":", "AP", ":", "+1.0", "mm", ",", "ML", ":", "+3.2", "mm", ",", "DV", ":", "−5.0", ";", "AP", ":", "+0.5", "mm", ",", "ML", ":", "+3.0", "mm", ",", "DV", ":", "−5.0", ";", "AP", ":", "−0.5", "mm", ",", "ML", ":", "+3.0", "mm", ",", "DV", ":", "−5.0", ";", "AP", ":", "−1.0", "mm", ",", "ML", ":", "+3.5", "mm", ",", "DV", ":", "−5.0", "mm", "with", "the", "incisor", "bar", "set", "at", "3.4", "mm", ".", "The", "injection", "rate", "was", "1", "µl", "/", "min", ",", "and", "the", "cannula", "was", "left", "in", "place", "for", "an", "additional", "5", "min", "before", "retraction", ".", "As", "a", "control", "group", ",", "we", "used", "rats", "subjected", "to", "ischemia", "and", "injected", "with", "the", "vehicle", "(", "n", " ", "=", " ", "7", ")", ".", "All", "animals", "underwent", "baseline", "motor", "behavioral", "assessment", "before", "and", "after", "the", "ischemic", "lesion", ",", "and", "4", "&", "8", "weeks", "after", "cell", "transplantation", ".", "The", "animals", "were", "killed", "after", "2-month", "survival", "time", "by", "transcardial", "perfusion", "with", "phosphate", "buffered", "saline", "(", "PBS", ")", "followed", "by", "4", "%", "paraformaldehyde", ".", "The", "brains", "were", "cryoprotected", "in", "an", "increasing", "gradient", "of", "10", ",", "20", "and", "30", "%", "sucrose", "solution", "and", "cryostat", "sectioned", "at", "40", "µm", "and", "processed", "for", "immunocytochemistry", ".", "ImmunocytochemistryCultures", "were", "fixed", "with", "4", "%", "paraformaldehyde", "for", "15", "min", ".", "Both", "cells", "and", "brain", "sections", "were", "rinsed", "in", "PBS", "for", "3×5", "min", "then", "incubated", "for", "2", "hrs", "(", "cultures", ")", "or", "overnight", "(", "brain", "sections", ")", "with", "the", "appropriate", "primary", "antibodies", "for", "multiple", "labeling", ".", "Secondary", "antibodies", "raised", "in", "the", "appropriate", "hosts", "and", "conjugated", "to", "FITC", ",", "RITC", ",", "AMCA", ",", "CY3", "or", "CY5", "chromogenes", "(", "Jackson", "ImmunoResearch", ")", "were", "used", ".", "Cells", "and", "sections", "were", "counterstained", "with", "the", "nuclear", "marker", "4′,6-diamidine-2′-phenylindole", "dihydrochloride", "(", "DAPI", ")", ".", "Positive", "and", "negative", "controls", "were", "included", "in", "each", "run", ".", "Immunostained", "sections", "were", "coverslipped", "using", "fluorsave", "(", "Calbiochem", ")", "as", "the", "mounting", "medium", ".", "The", "following", "antibodies", "were", "used", ":", "Anti", "-", "human", "Nuclei", "(", "hNuc", ",", "monoclonal", "1∶100", ",", "Chemicon", ")", ",", "Anti", "-", "TuJ1", "(", "monoclonal", "1∶100", ",", "Covance", ";", "Polyclonal", "1∶200", ",", "Aves", "Labs", ")", ";", "anti", "-", "GAD65/67", "(", "polyclonal", "1∶1000", ",", "Chemicon", ")", ";", "Anti", "-", "glial", "fibrillary", "acidic", "protein", "(", "GFAP", ",", "monoclonal", "1∶1000", ",", "Chemicon", ";", "polyclonal", "1∶200", ",", "Aves", "Labs", ")", ";", "Anti", "-", "galactocerebrocide", "(", "GC", ",", "monoclonal", "1∶250", ",", "Chemicon", ")", ";", "Anti", "-", "CNPase", "(", "polyclonal", "1∶200", ",", "Aves", "Labs", ")", ";", "Anti", "-", "Glucose", "Transporter", "type", "1", "(", "Glut-1", "polyclonal", ",", "1∶500", ",", "Chemicon", ")", ";", "Anti", "-", "Nestin", "(", "polyclonal", "1∶1000", ",", "Chemicon", ")", ";", "Anti", "-", "vimentin", "(", "monoclonal", "1∶500", ",", "Calbiochem", ")", ";", "Anti-3CB2", "(", "monoclonal", "1∶500", ",", "Developmental", "Studies", "Hybridoma", "Bank", ")", ";", "Anti", "-", "doublecortin", "(", "DCX", ",", "polyclonal", "1∶250", ",", "SantaCruz", "Biotechnology", ")", ";", "Anti", "-", "Ki67", "(", "polyclonal", "1∶250", ",", "SantaCruz", "Biotechnology", ")", ".", "Fluorescence", "was", "detected", ",", "analyzed", "and", "photographed", "with", "a", "Zeiss", "LSM550", "laser", "scanning", "confocal", "photomicroscope", ".", "For", "each", "animal", ",", "quantitative", "estimates", "of", "the", "total", "number", "of", "grafted", "cells", "were", "stereologically", "determined", "using", "the", "optical", "fractionator", "procedure", "[", "50", "]", ".", "A", "computer", "-", "assisted", "image", "analysis", "system", "was", "performed", "using", "Stereo", "Investigator", "software", "(", "MicroBrightField", ",", "Inc", ".", ")", ".", "The", "rostral", "and", "caudal", "limits", "of", "the", "reference", "volume", "were", "determined", "by", "first", "and", "last", "frontal", "sections", "containing", "grafted", "cells", ".", "The", "striatum", "and", "cortex", "were", "accurately", "outlined", "at", "low", "magnification", "(", "2.5×", "objective", ")", ".", "The", "optical", "fractionator", "probe", "was", "selected", "to", "perform", "systematic", "sampling", "of", "the", "immunoreactive", "cell", "population", "distributed", "within", "the", "serial", "sections", "to", "estimate", "the", "population", "number", "in", "the", "volume", "of", "tissue", ".", "The", "counting", "frame", "of", "the", "optical", "fractionator", "was", "defined", "at", "50×50", "µm", "squares", "and", "the", "systematic", "sampling", "was", "performed", "by", "translating", "a", "grid", "with", "200×200", "µm", "squares", "onto", "the", "sections", "of", "interest", "using", "the", "Stereo", "Investigator", "software", ".", "The", "sample", "sites", "were", "systematically", "and", "automatically", "generated", "by", "the", "computer", "and", "examined", "using", "a", "60×", "objective", "of", "a", "Nikon", "Eclipse", "TE", "300", "microscope", ".", "The", "counting", "frame", "displayed", "inclusion", "and", "exclusion", "lines", "and", "only", "immunoreactive", "cell", "bodies", "falling", "within", "the", "counting", "frame", "with", "no", "contact", "with", "the", "exclusion", "lines", "were", "counted", ".", "The", "cell", "dispersion", "was", "measured", "by", "counting", "the", "number", "of", "cells", "within", "200", "µm", "distance", "from", "the", "graft", "site", ".", "The", "number", "and", "distance", "in", "µm", "of", "cells", "dispersed", "beyond", "200", "µm", "was", "also", "measured", ".", "An", "average", "of", "2,000", "cells", "was", "counted", "per", "animal", ".", "Double", "labeling", "was", "determined", "using", "the", "confocal", "laser", "scanning", "microscope", "by", "random", "sampling", "of", "100", "or", "more", "cells", "per", "marker", "for", "each", "animal", ",", "scoring", "first", "for", "hNuc+", ",", "followed", "by", "DAPI+", "nuclei", "and", "then", "the", "marker", "of", "choice", ".", "The", "double", "labeling", "was", "always", "confirmed", "in", "x", "-", "z", "and", "y", "-", "z", "cross", "-", "sections", "produced", "by", "the", "orthogonal", "projections", "of", "z", "-", "series", ".", "Reverse", "Transcription", "-", "Polymerase", "Chain", "Reaction", "(", "RT", "-", "PCR", ")", "analysisTotal", "RNA", "was", "extracted", "from", "cultured", "cells", "using", "RNAeasy", "kit", "(", "Quiagen", ")", ".", "Aliquots", "(", "1", "µg", ")", "of", "total", "RNA", "from", "the", "cells", "were", "reverse", "transcribed", "in", "the", "presence", "of", "50", "mM", "Tris", "-", "HCl", ",", "pH", "8.3", ",", "75", "mM", "KCl", ",", "3", "mM", "MgCl2", ",", "10", "mM", "DTT", ",", "0.5", "µM", "dNTPs", ",", "and", "0.5", "µg", "oligo", "-", "dT(12–18", ")", "with", "200", "U", "Superscript", "RNase", "H", "-", "Reverse", "Transcriptase", "(", "Invitrogen", ")", ".", "PCR", "amplification", "was", "performed", "using", "standard", "procedure", "with", "Taq", "Polymerase", ".", "Aliquots", "of", "cDNA", "equivalent", "to", "50", "ng", "of", "total", "RNA", "were", "amplified", "in", "25", "µl", "reactions", "containing", "10", "mM", "Tris", "-", "HCl", ",", "pH", "8.3", ",", "50", "mM", "KCl", ",", "1.5", "mM", "MgCl2", ",", "50", "pmol", "of", "each", "primer", ",", "400", "µM", "dNTPs", ",", "and", "0.5", "U", "AmpliTaq", "DNA", "polymerase", "(", "Perkin", "-", "Elmer", ")", ".", "PCR", "was", "performed", "using", "the", "following", "thermal", "profile", ":", "4", "min", "at", "94", "°", "C", ";", "1", "min", "at", "94", "°", "C", ",", "1", "min", "at", "60", "°", "C", ",", "1.5", "min", "at", "72", "°", "C", ",", "for", "30–40", "cycles", ";", "7", "min", "at", "72", "°", "C", ",", "and", "finally", "a", "soak", "at", "4", "°", "C", "overnight", ".", "The", "following", "day", ",", "10", "µl", "aliquots", "of", "the", "amplified", "products", "were", "run", "on", "a", "2", "%", "agarose", "Tris", "–", "acetate", "gel", "containing", "0.5", "mg", "/", "ml", "ethidium", "bromide", ".", "The", "products", "were", "visualized", "through", "a", "UV", "transilluminator", ",", "captured", "in", "a", "digital", "format", "using", "Quantify", "One", "Gel", "Analysis", "software", "(", "Bio", "-", "Rad", "Laboratories", ")", "on", "a", "Macintosh", "G4", "computer", ".", "The", "PCR", "primers", "specific", "to", "each", "transcript", "were", "as", "follows", ":", "GFAP", ",", "forward", "(", "F", ")", ",", "5′-TCATCGCTCAGGAGGTCCTT–3′", "Reverse", "(", "R", ")", ",", "5′-CTG", "TTGCCAGAGATGGAGGTT–3′", ";", "MAP2", "(", "F", ")", "5′-GAAGACTCGCATCCGAATGG–3′", ",", "(", "R", ")", "5′-CGCAGGATAGGAGGAAGAGACT–3′", ";", "MBP", "(", "F", ")", "5′-TTAGCTGAATTC", "GCGTGTGG–3′", ",", "(", "R", ")", "5′-GAGGAAGTGAATGAGCCGGTTA-3′", "were", "deigned", "using", "the", "Primer", "Designer", "software", ",", "Version", "2.0", "(", 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18286199_task2
Sentence: BackgroundHuman embryonic stem cells (hESCs) offer a virtually unlimited source of neural cells for structural repair in neurological disorders, such as stroke. Neural cells can be derived from hESCs either by direct enrichment, or by isolating specific growth factor-responsive and expandable populations of human neural stem cells (hNSCs). Studies have indicated that the direct enrichment method generates a heterogeneous population of cells that may contain residual undifferentiated stem cells that could lead to tumor formation in vivo.Methods/Principal FindingsWe isolated an expandable and homogenous population of hNSCs (named SD56) from hESCs using a defined media supplemented with epidermal growth factor (EGF), basic fibroblast growth factor (bFGF) and leukemia inhibitory growth factor (LIF). These hNSCs grew as an adherent monolayer culture. They were fully neuralized and uniformly expressed molecular features of NSCs, including nestin, vimentin and radial glial markers. These hNSCs did not express the pluripotency markers Oct4 or Nanog, nor did they express markers for the mesoderm or endoderm lineages. The self-renewal property of the hNSCs was characterized by a predominant symmetrical mode of cell division. The SD56 hNSCs differentiated into neurons, astrocytes and oligodendrocytes throughout multiple passages in vitro, as well as after transplantation. Together, these criteria confirm the definitive NSC identity of the SD56 cell line. Importantly, they exhibited no chromosome abnormalities and did not form tumors after implantation into rat ischemic brains and into naïve nude rat brains and flanks. Furthermore, hNSCs isolated under these conditions migrated toward the ischemia-injured adult brain parenchyma and improved the independent use of the stroke-impaired forelimb two months post-transplantation.Conclusions/SignificanceThe SD56 human neural stem cells derived under the reported conditions are stable, do not form tumors in vivo and enable functional recovery after stroke. These properties indicate that this hNSC line may offer a renewable, homogenous source of neural cells that will be valuable for basic and translational research. To date there have been no effective treatments for improving residual structural and functional deficits resulting from stroke. Current therapeutic approaches, such as the use of thrombolytics, benefit only 1 to 4% of patients [1]. Consequently, the majority of stroke patients experience progression of ischemia associated with debilitating neurological deficits. Recent evidence has suggested that the transplantation of cells derived from cord blood, bone marrow or brain tissue (fetal and adult) enhances sensorimotor function in experimental models of stroke [2], [3]. However, the normal human-derived somatic stem cells used in these studies have a limited capacity to differentiate into the diverse neural cell types optimal for structural and physiological tissue repair and are not amenable for large-scale cell production. Unlike other sources of stem cells, hESC lines possess a nearly unlimited self-renewal capacity and the developmental potential to differentiate into virtually any cell type of the organism. As such, they constitute an ideal source of cells for regenerative medicine. The successful derivation of hESC lines from the inner cell mass of preimplantation embryos and their long-term maintenance in vitro over multiple passages has been demonstrated [4] and standardized. Differentiation and enrichment processes that direct hESCs towards a neural lineage have also been achieved. To promote neuralization, ESCs were cultured in a defined media supplemented with morphogens or growth factors [5], [6], [7] or cultured under conditions that promote “rosettes”, structures morphologically similar to the developing neural tube [8], [9]. This neuralization process has proven invaluable in understanding the specification of hESC-derived neural tissue [10], [11], [12]. However, the enriched neural progeny derived displayed overgrowth and limited migration after grafting into normal newborn mice [13] and lesioned adult rat striatum [12], [14], [15], [16]. The inner cores of these grafts contained tumorigenic precursor cells (reviewed in [17]). These findings suggest that neural cells generated by acute exposure to growth factors and/or morphogens may still be heterogeneous and potentially tumorigenic. We report an alternative method for the isolation and the perpetuation of a multipotent hNSC line from the hESCs with a primitive mode of self-renewal. We also demonstrate their long-term expansion, non-tumorigenic properties and functional engraftability in an experimental model of stroke. 1. In vitro isolation, growth and differentiation of hESC-derived hNSCsThe hESCs were maintained and expanded on mouse feeder layer in media supplemented with bFGF (Figure 1A). After cell dissociation, a portion of the hESCs was cultured in serum free medium containing EGF, bFGF and LIF. These factors are known to stimulate the proliferation of human fetal-derived NSCs [18], [19]. After 3 days in vitro (DIV), there was selective survival and growth of cells that aggregated in clusters or spheres (Figure 1B). These primary spheres were harvested and replated in fresh media. During the following week, the spheres attached to the flask and a fibroblast-like cell population began to migrate out (Figure 1C). Secondary spheres (2° spheres) were generated from these cultures and lifted off by the end of the week leaving a hollow in the middle of the attached cells (Figure 1D). The floating 2° spheres were collected and replated in fresh growth medium for 2 weeks. The cultures were then passaged by collagenase cell dissociation every 7 DIV for an additional 4 passages (Figure S1). At the 5th and 6th passages, spheres were dissociated into a single-cell suspension using trypsin-EDTA. At this stage there was a change in the hNSCs' adherent properties, and the cells began to grow as a monolayer with multiple foci of cells throughout the culture (Figure 1F). The adherent hNSC culture stained uniformly for nestin (Figure 1K), vimentin (Figure 1L) and with the radial glial marker 3CB2 (Figure 1M) indicating their homogeneity and NSC identity. Under these culture conditions, it is noteworthy that we did not observe the formation of rosettes which has been previously reported to occur under certain conditions during neuralization of hESCs [8], [20], [21]. RT-PCR analysis confirmed that these hNSCs did not express the pluripotency transcripts Oct-4 and Nanog (Figure 1I). Furthermore, the hNSCs did not express transcripts for brachyury and foxa2, marker genes for mesoderm and endoderm respectively (negative result, data not shown).10.1371/journal.pone.0001644.g001Figure 1Isolation and purification of hNSCs from the hESCs.The hESCs were grown on a mouse feeder layer (A). Primary neurospheres (B) were isolated and replated to eliminate other non-neural cells. The selectively harvested secondary neurospheres (arrow in C), left behind hollow cores in the surface area (star in D) where they attached earlier. They were perpetuated for an additional 5 passages (E). These 2° spheres were then passaged using a single cell dissociation protocol (F). Arrow in F shows an example of a focus of proliferating cells. (G, H) The hNSCs were passaged every 5–7 days, as described in the Methods section. Starting from an initial population of 1 million cells, the cumulative cell number was calculated at each passage as the fold of increase×the total cell number and plotted as logarithm with base 2 in function of time (G). The cell perpetuation (G) and population doubling (H) analysis demonstrated the continuous and stable growth of the hNSCs. (I) RT-PCR analysis showing the down regulation of the pluripotency transcripts Oct4 and Nanog in secondary neurospheres and in expanded hNSCs at passage 8 (P8). (J) Cytogenetic evaluation of the SD56 hNSCs line at passage 12 by standard G-banding was performed. Twenty metaphase cells were analyzed and showed a normal female chromosome complement (46,XX). Isolated and expanded hNSCs expressed the neural precursor cell markers nestin (K), Vimentin (L) and the radial glial cell marker 3CB2 (M) in virtually all the progeny. (N-P) Clonal self-renewal ability of the isolated hNSCs through symmetrical cell division. Single (N), two-cell stage (O) and four-cell stage (P) of a hNSC proliferating over a 3-day culture period. Note the symmetrical segregation of BrdU and nestin in the progeny. Bars: (A, B, C, D) 200 µm; (E, F) 100 µm; (K–M) 20 µm; (N–P) 10 µm.To ascertain self-renewal ability under clonal conditions, a single cell suspension was plated at clonal density (1–2 cell/10 µl). To determine if the hNSCs divide symmetrically, we pulsed cultures with the thymidine analog, bromodeoxyuridine (BrdU), after plating and looked for the expression of nestin in the progeny. Cultures were fixed after 1, 2 or 3 DIV (Figure 1N–P). After 2 days, plated single cells first underwent a symmetric cell division and gave rise to daughter cells that were both positive for BrdU and nestin. The clone of cells continued to grow over the 3 DIV and all the progeny expressed nestin. BrdU labeling demonstrated that it was rare for only one daughter cell to inherit the BrdU and thus had undergone asymmetric segregation of the chromatids (Figure S2). G-band karyotyping of these hNSCs confirmed the normal, non-transformed nature of cells after 12 passages (Figure 1J). We named the derived hNSCs SD56 (intermittently referred to as SD56 hNSCs or hNSCs).Under these defined growth conditions, the hNSCs showed stable growth with a 2.7±0.2 fold increase every 5 to 7 days (Figure 1G). The population doubling at each passage averaged at 1.4±0.1 (Figure 1H). The viability of hNSCs at each passage was consistent at the approximate value of 98%. The projected cumulative cell numbers demonstrated that trillions of cells could be generated over a period of 5 months (Figure 1G). We expanded the isolated hNSCs lines for over 20 passages with a stable phenotype. An initial cell bank of 75 vials containing 2 to 5 million cells each was generated and cryopreserved.Upon removal of the mitogenic factors and plating on a coverslip pre-coated with poly-L-ornithine (PLO) substrate, the hNSCs spontaneously differentiated into neurons, astrocytes and oligodendrocytes, a property that is consistent with normal multipotent hNSCs (Figure 2). After 2 DIV, hNSCs expressed transcripts for the neural-specific genes nestin, Notch1 and neural cell adhesion molecule (N-CAM) (Figure 2A) and for the lineage specific markers β-tubulin class III, medium-size neurofilament (NF-M) and microtubule-associated protein 2 (MAP-2) for neurons, GFAP for astrocytes and myelin basic protein (MBP) for oligodendrocytes (Figure 2A). Transcripts for the GABAergic cell marker glutamic acid decarboxylase (GAD) were expressed, but transcripts for the tyrosine hydroxylase (TH), a marker for dopaminergic neurons, were undetectable. Immunocytochemical analysis (Figure 2B–F) of 10 day-old cultures demonstrated that the proportion of nestin+ cells was 36.6±2.7%, 62.5±2.8% expressed the neuronal marker TuJ1, 1.9±0.3% expressed the astrocytic marker GFAP and 7.1±0.4% were oligodendrocytes and expressed galactocerebrocide (GC) (Figure 2F). A subset (9.8±1.6%) of the GFAP+ astrocytes co-expressed nestin.10.1371/journal.pone.0001644.g002Figure 2hESC-derived hNSCs spontaneously differentiated into the 3 principal central nervous system cell types.Dissociated hNSCs were washed free of growth factors and plated on poly-L-onithine coated glass coverslips. Differentiated cultures were either harvested after 2 DIV for total RNA extraction and RT-PCR analysis or fixed after 10 DIV and processed for indirect immunocytochemistry. (A) Differentiated hNSCs expressed the neural-specific transcripts nestin and Notch1 as well as transcripts: for neurons (β-tubulin class III, MAP-2, NCAM and medium-size neurofilament, NF-M), for astrocytes (GFAP) and for oligodendrocytes (MBP). The hNSCs expressed transcripts for GAD, but not for TH. B, C & D, morphology of NSC-derived progeny differentiated into GFAP+ astrocytes (B), GC-expressing oligodendrocytes (C) and TuJ1+ neurons (D), DAPI (blue) show life cell nuclei. (E) Photo showing cultures double-immunostained for TuJ1 (green) and nestin (red) (DAPI, blue). (F) Quantitative analysis of immunostained 10 day-old cultures for the 3 neural cell types. Results are mean±s.e.m. of three independent experiments, each performed in duplicate. Bars: (c) 20 µm; (d, e) 10 µm. 2. The isolated hNSCs are normal and do not form tumors in normal nude ratsThe self-renewal and pluripotent abilities of the hESCs are also associated with tumorigenic properties. Therefore, the first critical step toward developing therapeutic hNSCs is to verify that they are non-tumorigenic. The monolayer culture of SD56 hNSCs clearly demonstrated contact inhibition of growth, a normal karyotype and did not express the pluripotency transcripts Oct-4 and Nanog. Removal of mitogenic factors and continued culture on plastic resulted in cell senescence that is characteristic of non-transformed cells. To determine whether SD56 hNSCs form tumors in vivo, we transplanted them at high density (see Methods) into the forebrain and subcutaneously into the flank of nude rats. The animals were kept for a two-month post-transplant survival period. To label mitotically active cells in vivo during S-phase, the rats were injected IP with BrdU (50 mg/kg) every 8 hours during the last 24 hours before euthanasia. The transit amplifying endogenous precursors located in the subventricular zone (SVZ) were labeled (Figure S3); however, we were unable to detect grafted SD56 hNSCs co-localizing the human-specific nuclear marker hNuc and BrdU (Figure S3). No surviving SD56 hNSCs were detected in the flank of the transplanted animals suggesting that the grafted cells are not tumorigenic. 3. Transplanted cells survived, migrated toward and engrafted into the stroke-damaged host tissueTo investigate the survival and functional engraftment in an injury environment, hNSCs (4×105) were transplanted into the ischemic boundary zone in the rat striatum one week after the middle cerebral artery occlusion (MCAO) was performed. Animals were euthanized two months later and the brains processed for histo-pathology and immunocytochemistry. Grafted SD56 hNSCs, identified with hNuc, demonstrated a 37.0±15.8% survival rate and a remarkable dispersion toward the stroke-damaged tissue with no sign of overgrowth or tumorigenesis. The majority of grafted cells (61.2±4.7%) migrated at least 200 µm away from the injection site and penetrated an average distance of 806.4±49.3 µm into the stroke-damaged tissue (Figure 3A–C). Immunostaining with the blood vessel marker, GluT1, revealed dilated vessels in the infarcted striatum and a close association between vessels and the grafted hNSCs (Figure 3B, 3C). The grafted cells rarely expressed the proliferation marker Ki67 (Figure 3D), 29.8±4.4% expressed nestin (Figure 3E), 6.5±0.9% expressed doublecortin (DCX) and 60.8±8.1% were TuJ1+ (Figure 3F, G). Grafted cells rarely co-expressed the astroglial marker GFAP (Figure 3H) or differentiated into CNPase-expressing oligodendrocytes (Figure 3I). Immunostaining for GAD demonstrated that 25.1±2.3% of grafted hNSCs differentiated into GABAergic neurons while less than 2% were positive for glutamate (Figure 3J, K).10.1371/journal.pone.0001644.g003Figure 3Dispersion, engraftment and differentiation of the hNSCs in stroke-lesioned animals.(A) Schematic drawing of a frontal section through the striatum illustrating the dispersion of grafted hNSCs in the focal ischemia-lesioned parenchyma (shaded area). (B, C) Photos show frontal sections through the graft in the striatum immunostained with the human specific antibodies: anti-hNuc (green in B & C) and anti-GluT1 (red, B & C) showing blood vessels and dispersed hNSCs in the graft zone. C: higher magnification of the inset in B. (D–I) Photos taken from frontal sections through the graft in the striatum double immunoprocessed for cell proliferation and neural lineage markers. (D) Note the endogenous Ki67+ cells (red cells, arrow) in the stroke damaged area and the hNuc+ (green)/Ki67- grafted hNSCs (arrowheads). (E) Examples of grafted SD56 hNSCs showing co-expression of hNuc (green) and nestin (red). (F) Confocal 3D reconstructed orthogonal images of the hNuc+(green)/DCX+(red) NSCs (arrowheads) viewed in the x-z plan on the top and y-z plan on the right. (G) Examples show the majority of grafted NSC progeny co-expressing hNuc (red) and the neuronal marker TuJ1 (green). Grafted NSCs rarely differentiate into GFAP+ astrocytes (H). In I, rare example of grafted NSC progeny becoming an oligodendrocyte identified by the expression of CNPase (green). Grafted NSCs expressed the GABAergic marker GAD65/67 (J) and rarely expressed glutamate (K). (Abbreviations: Cx: cortex, Str: striatum). Bars: (B, C) 100 µm; (D, F) 20 µm; (E, G–K) 10 µm. 4. Transplanted cells improve sensorimotor function of the stroke-disabled forelimbWe asked whether transplanted SD56 hNSCs could enhance the recovery of sensorimotor function that is compromised in the stroke-injured rats. We used the cylinder test to measure the sensorimotor asymmetry in forelimb use during spontaneous exploration [22]. To establish the baseline of the stroke-induced sensorimotor deficit, spontaneous behavior of rats in a transparent cylinder was videotaped one week after stroke (pre-transplant, Figure 4). Tests were then conducted 4 and 8 weeks after vehicle and SD56 hNSCs transplantation. Stable asymmetry in forelimb use was observed 7 days post-stroke (pre-transplant, Figure 4). Ischemic rats used their impaired forelimbs (contralateral to lesion) during lateral exploration less than they did before stroke. Transplantation of SD56 hNSCs significantly enhanced the independent use of the impaired contralateral forelimb 4 weeks post transplantation (P<0.05 vs pre-transplant). Eight weeks after transplantation the improvement in the use of the impaired forelimb was stable and significant when compared to the pre-transplant group and significantly improved in comparison to vehicle treated group at 8 weeks (Figure 4). In the vehicle treated group, the independent use of the contralateral forelimb remained impaired 4 and 8 weeks post-injection. In an independent study and using the same MCAO rat animal model, we found that transplantation of dermal fibroblasts did not improve the stroke-induced motor deficits (unpublished data).10.1371/journal.pone.0001644.g004Figure 4Transplantation of NSCs improves sensorimotor function of the stroke-disabled forelimb.Forelimb use during spontaneous lateral exploration was measured in the cylinder test (see Method and Results sections for details). Groups of vehicle injected (n = 7) and hNSCs (n = 10) transplanted are represented. The animals were tested as described in Method section. Note the significant increase in the independent use of the impaired contralateral forelimb at 4 and 8 weeks post transplantation (P<0.05 vs pre-transplant group). The contralateral forelimb remained impaired in the vehicle treated group at 4 and 8 weeks post-injection. Bars represent percentages±s.e.m. of steps taken by the ipsilateral, contralateral and both forelimbs simultaneously. *P<0.05 vs pre-transplant group; #P<0.05 vs vehicle groups. Our results indicate that a self-renewable and homogenous population of hNSCs, SD56, was derived from hESCs using defined media supplemented with a specific combination of growth factors. The SD56 hNSCs grew as an adherent monolayer culture, uniformly expressed molecular features of hNSCs including nestin, vimentin and the radial glial marker 3CB2, and did not express the pluripotency markers Oct4 or Nanog. The self-renewal property of the hNSCs was characterized by a predominant symmetrical mode of cell division. They exhibited no chromosomal abnormalities and demonstrated non-tumorigenic properties after implantation into ischemic brains and into naïve nude rat brains and flanks. Furthermore, the transplanted SD56 hNSCs migrated toward the stroke-damaged adult brain parenchyma, engrafted and improved the independent use of the stroke-impaired forelimb. Maintenance of stem cells requires symmetrical and asymmetrical cell divisions to both expand and to give rise to specialized progeny of a specific tissue (reviewed in [23]). In vivo, a complex cellular micro-environment or niche ensures the self-maintenance property of NSCs [24], [25], [26], [27]. In vitro, defined growth factors and extracellular matrices support stem cell self-renewal [28], [29]. The embryonic stem cells can propagate in a predominantly proliferative symmetrical mode, leading to homogeneous cell cultures growing relatively quickly with minimal cell differentiation [30], [31], [32], [33], [34]. Tissue specific stem cells, however, self-renew in a predominant asymmetric mode to maintain them selves and compensate for the loss of differentiated cells due to disease or injury. Thus, NSCs isolated from developing or adult brain grow as a mixture of undifferentiated and differentiated cells due the predominant asymmetrical mode of cell division [35], [36], [37], [38], [39], [40], [41]. A recent study has reported that a murine ESC-derived NSC line (LC1) is propagated as an adherent homogenous culture with a dominant mode of symmetrical self-renewal [21]. A combination of EGF and FGF2 was sufficient to propagate these NSCs as an adherent monolayer. However, the SD56 hNSC line described here required the combination of EGF, bFGF and LIF for self-maintenance. Although there are morphological and molecular similarities between our hNSCs and the NSCs previously described [21], the methods of isolation and growth are different. In addition to the different combination of growth factors used, the hNSC line we have isolated did not go through the rosette-structure stage. The in vitro analysis of BrdU incorporation and nestin expression indicated that our hNSCs divide predominantly symmetrically. This type of growth pattern is characteristic of primitive normal stem cells undergoing mostly symmetric cell division to increase the stem cell pool at the early stage of development or during tissue regeneration after injury [23]. RT-PCR and immunocytochemistry analysis demonstrated that these undifferentiated SD56 cells did not express any pluripotency, endodermal or mesodermal markers. Furthermore, the SD56 hNSCs described here exhibited the multipotential characteristic to differentiate into neurons, astrocytes and oligodendrocytes both in vitro and upon transplantation. Together these findings suggest that the hNSC line we isolated are appropriately programmed and share similar characteristics with the definitive NSCs of the developing brain. The SD56 hNSCs demonstrated a remarkable ability to migrate toward the stroke-damaged parenchyma of the adult rat brain. This directed migration by the majority of the grafted cells could be due to their uniform cellular composition, which results in an equal response to the host microenvironment. In previous studies, subpopulations of transplanted hESCs that were enriched in neural cells migrated in host microenvironments conducive to cell migration, such as the developing brain or in structures such as the rostral migratory stream [13], [20]. In the adult lesioned brain, the grafted hESC-derived neural cells proliferated and formed rosettes [14], teratomas [12], [15] or a cellular mass that induced a gliotic host response whereby local astrocytes demarcated the grafts [16]. Enriched neural cultures derived from mouse [42] and monkey ESCs [43] have produced behavioral improvements when transplanted into animal models of stroke and brain injury. However, in these cases, the transplanted non-human ESCs also formed a mass with signs of overgrowth in the core, as well as deformations [44], [45], [46]. ESCs plated at low density acquire neural identity within few hours after plating [47]. Interestingly, nearly all viable cells expressed nestin, the early neural fate marker Sox1, and the pluripotency marker Oct4. Together, these studies are seminal and suggest that complete neuralization may not be achieved through certain enrichment processes, consequently the neural cells could revert to a pluripotent stage [17]. The dispersion of the grafted hNSCs within host parenchyma may allow for more graft-host interactions that could stabilize differentiation, inhibit growth and prevent gliotic host response. In the present study, SD56 hNSC-transplanted animals demonstrated a stable improvement in the sensorimotor function when evaluated for spontaneous exploratory activity in the cylinder test that detects long-lasting deficits in forelimb use in the experimental models of stroke [22]. The transplantation of hNSCs significantly enhanced the independent use of the impaired contralateral forelimb 8 weeks post transplantation. This is the first report demonstrating that the transplantation of hNSCs derived from hESCs can improve neurologic behavior after experimental stroke. Together, these findings are encouraging and suggest that these cells are promising for future development. In addition to their therapeutic application, the hNSCs isolated under the reported conditions offer a means to interrogate host environments and unravel mechanistic features of self-renewal, non-tumorigenicity and functional engraftability in animal models of neurological disorders. hESC and NSC CulturesThe hESC line H9 (WiCell Research Institute) was propagated every 5 to 7 days on irradiated mouse embryonic fibroblasts. The cell culture media consisted of a 1∶1 mixture of Dulbecco's modified Eagle's medium (DMEM) and F12 nutrient, 20% serum replacement (Invitrogen), 0.1 mM β-mercaptoethanol, 2 µg/ml heparin and 4 ng/ml bFGF (R&D Systems). To generate the NSCs, dissociated hESCs were cultured in a chemically defined medium composed of DMEM/F12 (1∶1) including glucose (0.6%), glutamine (2 mM), sodium bicarbonate (3 mM), and HEPES buffer (5 mM) [all from Sigma except glutamine (Invitrogen)]. A defined hormone mix and salt mixture (Sigma), including insulin (25 mg/ml), transferrin (100 mg/ml), progesterone (20 nM), putrescine (60 mM), and selenium chloride (30 nM) was used in place of serum. The medium was supplemented with EGF (20 ng/ml), bFGF (10 ng/ml) and LIF (10 ng/ml). Dissociated hNSCs were plated at a density of 100,000 cell/ml in Corning T75 (Invitrogen) culture flasks in the defined media together with the growth factors. After 5–7 DIV, the adherent culture was incubated in 0.025%trypsin/0.01% EDTA (w/v) for 1 min followed by the addition of trypsin inhibitor (Invitrogen) then gently triturated to achieve single cell suspension. The cells were then washed twice with fresh medium and reseeded in fresh growth factor-containing media at 100,000 cells/ml. This procedure was performed for 21 passages and the fold of increase and population doubling were calculated at each passage. For clonal analysis, single spheres or confluent hNSC cultures were single cell dissociated and serially diluted to yield 1–2 cell/10 µl. A 10-µl-cell suspension was then added to each of 96 or 24 well plates containing 200–300 µl of growth media. Wells containing one viable cell were marked the next day and re-scored 5 to 7 days later for cell proliferation. The differentiation of the hNSCs was performed as previously described [48]. Dissociated hNSCs were plated at a density of 106 cells/ml in control (media/hormone mix) medium devoid of any growth factors and supplemented with 1% fetal bovine serum (FBS) on poly-L-ornithine-coated (15 mg/ml; Sigma) glass coverslips in 24-well Nunclon culture dishes with 0.5 ml/well. After 2, 7–15 DIV cultures were fixed and processed for immunocytochemistry or used for RT-PCR analysis. Karyotype analysisLong-term cultures of hNSCs were incubated at 37°C and harvested for metaphase chromosomes when the cultures were 75% confluent. Metaphase chromosomes were obtained by standard chromosome harvest methods by exposure to Colcemid at 0.1 µg/ml for 1 hour at 37°C, a 2-minute exposure to trypsin/EDTA, hypotonized with 0.057 M KCl and fixed with 3∶1 methanol:acetic acid. Slide preparations were made by dropping the fixed cell pellet onto cold, wet slides and air-dried. After incubating the slides at 90°C for 30 minutes, chromosomes were trypsin banded and then Wright/Giemsa stained for G-banding analysis. Twenty metaphase cells were completely analyzed and a normal female chromosome complement was found (46,XX). Tumorigenicity in nude ratsAll animal experiments were conducted according to the National Institute of Health (NIH) guidelines and approved by the IACUC. Normal adult NIH-Nude rats (n = 5, 8 week-old, Taconic, Germantown, New York, United States) were used to test the tumorigenic potential of the SD56 hNSCs. Undifferentiated hNSCs from passage 9 were single cell dissociated using trypsin-EDTA and suspended at the concentration of 125,000 cell/µl in preparation for cell transplantation. Two µl of the cell suspension were stereotaxically transplanted into 4 sites within the striatum at the following coordinates: AP: +1.0 mm, ML: +3.2 mm, DV: −5.0; AP: +0.5 mm, ML: +3.0 mm, DV: −5.0; AP: −0.5 mm, ML: +3.0 mm, DV: −5.0; AP: −1.0 mm, ML: +3.5 mm, DV: −5.0 mm with the incisor bar set at 3.4 mm. The injection rate was 1 µl/min, and the cannula was left in place for an additional 5 min before retraction. For the flank tumor assay, 2×106 cells (125,000 cell/µl) were injected subcutaneously to the side of the adult nude rats. To label mitotically active cells in vivo during S-phase, the rats were injected IP with the BrdU (50 mg/kg, Sigma) every 8 hours during the last 24 hours before euthanasia. After 2-month survival time, rats were euthanized and a postmortem examination for tumor formation was performed. Induction of Focal Ischemia and Cell TransplantationAll animal experimentations were conducted according to the National Institute of Health (NIH) guidelines and approved by the IACUC. Sprague Dawley adult male rats (n = 17, 275g–310g, Charles River Laboratories, Wilmington, Massachusetts, United States) were subjected to one and a half hour suture occlusion of the middle cerebral artery (MCAO), as previously described [49] and immunosuppressed 2 days before cell transplantation and daily thereafter for one week with i.p. injections of cyclosporine A (20 mg/ml, Sandimmune, Novartis Pharmaceuticals). Thereafter oral cyclosporine was used at 210 µg/ml in drinking water until euthanasia. Undifferentiated SD56 hNSCs from passages between P9 and P13 were single cell dissociated using trypsin-EDTA in preparation for cell transplantation. One week after the stroke lesion, 2 µl of the hNSCs, at a concentration of 50,000 cell/µl, were stereotaxically transplanted into 4 sites within the lesioned striatum (n = 10) at the following coordinates: AP: +1.0 mm, ML: +3.2 mm, DV: −5.0; AP: +0.5 mm, ML: +3.0 mm, DV: −5.0; AP: −0.5 mm, ML: +3.0 mm, DV: −5.0; AP: −1.0 mm, ML: +3.5 mm, DV: −5.0 mm with the incisor bar set at 3.4 mm. The injection rate was 1 µl/min, and the cannula was left in place for an additional 5 min before retraction. As a control group, we used rats subjected to ischemia and injected with the vehicle (n = 7). All animals underwent baseline motor behavioral assessment before and after the ischemic lesion, and 4 & 8 weeks after cell transplantation. The animals were killed after 2-month survival time by transcardial perfusion with phosphate buffered saline (PBS) followed by 4% paraformaldehyde. The brains were cryoprotected in an increasing gradient of 10, 20 and 30% sucrose solution and cryostat sectioned at 40 µm and processed for immunocytochemistry. ImmunocytochemistryCultures were fixed with 4% paraformaldehyde for 15 min. Both cells and brain sections were rinsed in PBS for 3×5 min then incubated for 2 hrs (cultures) or overnight (brain sections) with the appropriate primary antibodies for multiple labeling. Secondary antibodies raised in the appropriate hosts and conjugated to FITC, RITC, AMCA, CY3 or CY5 chromogenes (Jackson ImmunoResearch) were used. Cells and sections were counterstained with the nuclear marker 4′,6-diamidine-2′-phenylindole dihydrochloride (DAPI). Positive and negative controls were included in each run. Immunostained sections were coverslipped using fluorsave (Calbiochem) as the mounting medium. The following antibodies were used: Anti-human Nuclei (hNuc, monoclonal 1∶100, Chemicon), Anti-TuJ1 (monoclonal 1∶100, Covance; Polyclonal 1∶200, Aves Labs); anti-GAD65/67 (polyclonal 1∶1000, Chemicon); Anti-glial fibrillary acidic protein (GFAP, monoclonal 1∶1000, Chemicon; polyclonal 1∶200, Aves Labs); Anti-galactocerebrocide (GC, monoclonal 1∶250, Chemicon); Anti-CNPase (polyclonal 1∶200, Aves Labs); Anti-Glucose Transporter type 1 (Glut-1 polyclonal, 1∶500, Chemicon); Anti-Nestin (polyclonal 1∶1000, Chemicon); Anti-vimentin (monoclonal 1∶500, Calbiochem); Anti-3CB2 (monoclonal 1∶500, Developmental Studies Hybridoma Bank); Anti-doublecortin (DCX, polyclonal 1∶250, SantaCruz Biotechnology); Anti-Ki67 (polyclonal 1∶250, SantaCruz Biotechnology). Fluorescence was detected, analyzed and photographed with a Zeiss LSM550 laser scanning confocal photomicroscope. For each animal, quantitative estimates of the total number of grafted cells were stereologically determined using the optical fractionator procedure [50]. A computer-assisted image analysis system was performed using Stereo Investigator software (MicroBrightField, Inc.). The rostral and caudal limits of the reference volume were determined by first and last frontal sections containing grafted cells. The striatum and cortex were accurately outlined at low magnification (2.5× objective). The optical fractionator probe was selected to perform systematic sampling of the immunoreactive cell population distributed within the serial sections to estimate the population number in the volume of tissue. The counting frame of the optical fractionator was defined at 50×50 µm squares and the systematic sampling was performed by translating a grid with 200×200 µm squares onto the sections of interest using the Stereo Investigator software. The sample sites were systematically and automatically generated by the computer and examined using a 60× objective of a Nikon Eclipse TE 300 microscope. The counting frame displayed inclusion and exclusion lines and only immunoreactive cell bodies falling within the counting frame with no contact with the exclusion lines were counted. The cell dispersion was measured by counting the number of cells within 200 µm distance from the graft site. The number and distance in µm of cells dispersed beyond 200 µm was also measured. An average of 2,000 cells was counted per animal. Double labeling was determined using the confocal laser scanning microscope by random sampling of 100 or more cells per marker for each animal, scoring first for hNuc+, followed by DAPI+ nuclei and then the marker of choice. The double labeling was always confirmed in x-z and y-z cross-sections produced by the orthogonal projections of z-series. Reverse Transcription-Polymerase Chain Reaction (RT-PCR) analysisTotal RNA was extracted from cultured cells using RNAeasy kit (Quiagen). Aliquots (1 µg) of total RNA from the cells were reverse transcribed in the presence of 50 mM Tris-HCl, pH 8.3, 75 mM KCl, 3 mM MgCl2, 10 mM DTT, 0.5 µM dNTPs, and 0.5 µg oligo-dT(12–18) with 200 U Superscript RNase H-Reverse Transcriptase (Invitrogen). PCR amplification was performed using standard procedure with Taq Polymerase. Aliquots of cDNA equivalent to 50 ng of total RNA were amplified in 25 µl reactions containing 10 mM Tris-HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl2 , 50 pmol of each primer, 400 µM dNTPs, and 0.5 U AmpliTaq DNA polymerase (Perkin-Elmer). PCR was performed using the following thermal profile: 4 min at 94°C; 1 min at 94°C, 1 min at 60°C, 1.5 min at 72°C, for 30–40 cycles; 7 min at 72°C, and finally a soak at 4°C overnight. The following day, 10 µl aliquots of the amplified products were run on a 2% agarose Tris–acetate gel containing 0.5 mg/ml ethidium bromide. The products were visualized through a UV transilluminator, captured in a digital format using Quantify One Gel Analysis software (Bio-Rad Laboratories) on a Macintosh G4 computer.The PCR primers specific to each transcript were as follows: GFAP, forward (F), 5′-TCATCGCTCAGGAGGTCCTT–3′ Reverse (R), 5′-CTG TTGCCAGAGATGGAGGTT–3′; MAP2 (F) 5′-GAAGACTCGCATCCGAATGG–3′, (R) 5′-CGCAGGATAGGAGGAAGAGACT–3′; MBP (F) 5′-TTAGCTGAATTC GCGTGTGG–3′, (R) 5′-GAGGAAGTGAATGAGCCGGTTA-3′ were deigned using the Primer Designer software, Version 2.0 (Scientific and Educational Software) [48]. 18S, β-tubulin class III, N-CAM, Nestin, NF-M, Notch-1 primers [51]. Oct4, Nanog primers [11]. FOXa2 (HNF3B), Brachyury primers [52]. Behavioral testsThe cylinder test was used to assess the spontaneous forelimb use during lateral exploration movement [22]. Rats were placed in a transparent acrylic cylinder (20 cm diameter) for 5 minutes. The cylinder encourages use of the forelimbs for vertical exploration. A mirror was placed behind the cylinder so that the forelimbs could be viewed at all times. Testing sessions were videotaped and forelimb use was scored by a blinded operator. Movements scored were the independent use of the left or right forelimb or simultaneous use of both the left and right forelimb to contact the wall of the cylinder during a full rear, to initiate a weight-shifting movement, or to regain center of gravity while moving laterally in a vertical posture along the wall. Animals were tested for their baselines after stroke and 4 and 8 weeks after cell transplantation. Statistical analysisOutcome measurement for each experiment was reported as mean±SEM. All data were analyzed using SPSS 11 for Mac OS X (SPSS Inc.). Significance of inter-group differences was performed by applying Student's t-test where appropriate. The One-Way ANOVA analysis was used to compare group differences for the forelimb use as the dependant variable and groups as the single independent factor variable. Differences between the groups were determined using Bonferroni's post hoc test. A P-value of less than 0.05 was considered to be statistically significant. Instructions: please extract entity words from the input sentence
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BackgroundHuman embryonic stem cells (hESCs) offer a virtually unlimited source of neural cells for structural repair in neurological disorders, such as stroke. Neural cells can be derived from hESCs either by direct enrichment, or by isolating specific growth factor-responsive and expandable populations of human neural stem cells (hNSCs). Studies have indicated that the direct enrichment method generates a heterogeneous population of cells that may contain residual undifferentiated stem cells that could lead to tumor formation in vivo.Methods/Principal FindingsWe isolated an expandable and homogenous population of hNSCs (named SD56) from hESCs using a defined media supplemented with epidermal growth factor (EGF), basic fibroblast growth factor (bFGF) and leukemia inhibitory growth factor (LIF). These hNSCs grew as an adherent monolayer culture. They were fully neuralized and uniformly expressed molecular features of NSCs, including nestin, vimentin and radial glial markers. These hNSCs did not express the pluripotency markers Oct4 or Nanog, nor did they express markers for the mesoderm or endoderm lineages. The self-renewal property of the hNSCs was characterized by a predominant symmetrical mode of cell division. The SD56 hNSCs differentiated into neurons, astrocytes and oligodendrocytes throughout multiple passages in vitro, as well as after transplantation. Together, these criteria confirm the definitive NSC identity of the SD56 cell line. Importantly, they exhibited no chromosome abnormalities and did not form tumors after implantation into rat ischemic brains and into naïve nude rat brains and flanks. Furthermore, hNSCs isolated under these conditions migrated toward the ischemia-injured adult brain parenchyma and improved the independent use of the stroke-impaired forelimb two months post-transplantation.Conclusions/SignificanceThe SD56 human neural stem cells derived under the reported conditions are stable, do not form tumors in vivo and enable functional recovery after stroke. These properties indicate that this hNSC line may offer a renewable, homogenous source of neural cells that will be valuable for basic and translational research. To date there have been no effective treatments for improving residual structural and functional deficits resulting from stroke. Current therapeutic approaches, such as the use of thrombolytics, benefit only 1 to 4% of patients [1]. Consequently, the majority of stroke patients experience progression of ischemia associated with debilitating neurological deficits. Recent evidence has suggested that the transplantation of cells derived from cord blood, bone marrow or brain tissue (fetal and adult) enhances sensorimotor function in experimental models of stroke [2], [3]. However, the normal human-derived somatic stem cells used in these studies have a limited capacity to differentiate into the diverse neural cell types optimal for structural and physiological tissue repair and are not amenable for large-scale cell production. Unlike other sources of stem cells, hESC lines possess a nearly unlimited self-renewal capacity and the developmental potential to differentiate into virtually any cell type of the organism. As such, they constitute an ideal source of cells for regenerative medicine. The successful derivation of hESC lines from the inner cell mass of preimplantation embryos and their long-term maintenance in vitro over multiple passages has been demonstrated [4] and standardized. Differentiation and enrichment processes that direct hESCs towards a neural lineage have also been achieved. To promote neuralization, ESCs were cultured in a defined media supplemented with morphogens or growth factors [5], [6], [7] or cultured under conditions that promote “rosettes”, structures morphologically similar to the developing neural tube [8], [9]. This neuralization process has proven invaluable in understanding the specification of hESC-derived neural tissue [10], [11], [12]. However, the enriched neural progeny derived displayed overgrowth and limited migration after grafting into normal newborn mice [13] and lesioned adult rat striatum [12], [14], [15], [16]. The inner cores of these grafts contained tumorigenic precursor cells (reviewed in [17]). These findings suggest that neural cells generated by acute exposure to growth factors and/or morphogens may still be heterogeneous and potentially tumorigenic. We report an alternative method for the isolation and the perpetuation of a multipotent hNSC line from the hESCs with a primitive mode of self-renewal. We also demonstrate their long-term expansion, non-tumorigenic properties and functional engraftability in an experimental model of stroke. 1. In vitro isolation, growth and differentiation of hESC-derived hNSCsThe hESCs were maintained and expanded on mouse feeder layer in media supplemented with bFGF (Figure 1A). After cell dissociation, a portion of the hESCs was cultured in serum free medium containing EGF, bFGF and LIF. These factors are known to stimulate the proliferation of human fetal-derived NSCs [18], [19]. After 3 days in vitro (DIV), there was selective survival and growth of cells that aggregated in clusters or spheres (Figure 1B). These primary spheres were harvested and replated in fresh media. During the following week, the spheres attached to the flask and a fibroblast-like cell population began to migrate out (Figure 1C). Secondary spheres (2° spheres) were generated from these cultures and lifted off by the end of the week leaving a hollow in the middle of the attached cells (Figure 1D). The floating 2° spheres were collected and replated in fresh growth medium for 2 weeks. The cultures were then passaged by collagenase cell dissociation every 7 DIV for an additional 4 passages (Figure S1). At the 5th and 6th passages, spheres were dissociated into a single-cell suspension using trypsin-EDTA. At this stage there was a change in the hNSCs' adherent properties, and the cells began to grow as a monolayer with multiple foci of cells throughout the culture (Figure 1F). The adherent hNSC culture stained uniformly for nestin (Figure 1K), vimentin (Figure 1L) and with the radial glial marker 3CB2 (Figure 1M) indicating their homogeneity and NSC identity. Under these culture conditions, it is noteworthy that we did not observe the formation of rosettes which has been previously reported to occur under certain conditions during neuralization of hESCs [8], [20], [21]. RT-PCR analysis confirmed that these hNSCs did not express the pluripotency transcripts Oct-4 and Nanog (Figure 1I). Furthermore, the hNSCs did not express transcripts for brachyury and foxa2, marker genes for mesoderm and endoderm respectively (negative result, data not shown).10.1371/journal.pone.0001644.g001Figure 1Isolation and purification of hNSCs from the hESCs.The hESCs were grown on a mouse feeder layer (A). Primary neurospheres (B) were isolated and replated to eliminate other non-neural cells. The selectively harvested secondary neurospheres (arrow in C), left behind hollow cores in the surface area (star in D) where they attached earlier. They were perpetuated for an additional 5 passages (E). These 2° spheres were then passaged using a single cell dissociation protocol (F). Arrow in F shows an example of a focus of proliferating cells. (G, H) The hNSCs were passaged every 5–7 days, as described in the Methods section. Starting from an initial population of 1 million cells, the cumulative cell number was calculated at each passage as the fold of increase×the total cell number and plotted as logarithm with base 2 in function of time (G). The cell perpetuation (G) and population doubling (H) analysis demonstrated the continuous and stable growth of the hNSCs. (I) RT-PCR analysis showing the down regulation of the pluripotency transcripts Oct4 and Nanog in secondary neurospheres and in expanded hNSCs at passage 8 (P8). (J) Cytogenetic evaluation of the SD56 hNSCs line at passage 12 by standard G-banding was performed. Twenty metaphase cells were analyzed and showed a normal female chromosome complement (46,XX). Isolated and expanded hNSCs expressed the neural precursor cell markers nestin (K), Vimentin (L) and the radial glial cell marker 3CB2 (M) in virtually all the progeny. (N-P) Clonal self-renewal ability of the isolated hNSCs through symmetrical cell division. Single (N), two-cell stage (O) and four-cell stage (P) of a hNSC proliferating over a 3-day culture period. Note the symmetrical segregation of BrdU and nestin in the progeny. Bars: (A, B, C, D) 200 µm; (E, F) 100 µm; (K–M) 20 µm; (N–P) 10 µm.To ascertain self-renewal ability under clonal conditions, a single cell suspension was plated at clonal density (1–2 cell/10 µl). To determine if the hNSCs divide symmetrically, we pulsed cultures with the thymidine analog, bromodeoxyuridine (BrdU), after plating and looked for the expression of nestin in the progeny. Cultures were fixed after 1, 2 or 3 DIV (Figure 1N–P). After 2 days, plated single cells first underwent a symmetric cell division and gave rise to daughter cells that were both positive for BrdU and nestin. The clone of cells continued to grow over the 3 DIV and all the progeny expressed nestin. BrdU labeling demonstrated that it was rare for only one daughter cell to inherit the BrdU and thus had undergone asymmetric segregation of the chromatids (Figure S2). G-band karyotyping of these hNSCs confirmed the normal, non-transformed nature of cells after 12 passages (Figure 1J). We named the derived hNSCs SD56 (intermittently referred to as SD56 hNSCs or hNSCs).Under these defined growth conditions, the hNSCs showed stable growth with a 2.7±0.2 fold increase every 5 to 7 days (Figure 1G). The population doubling at each passage averaged at 1.4±0.1 (Figure 1H). The viability of hNSCs at each passage was consistent at the approximate value of 98%. The projected cumulative cell numbers demonstrated that trillions of cells could be generated over a period of 5 months (Figure 1G). We expanded the isolated hNSCs lines for over 20 passages with a stable phenotype. An initial cell bank of 75 vials containing 2 to 5 million cells each was generated and cryopreserved.Upon removal of the mitogenic factors and plating on a coverslip pre-coated with poly-L-ornithine (PLO) substrate, the hNSCs spontaneously differentiated into neurons, astrocytes and oligodendrocytes, a property that is consistent with normal multipotent hNSCs (Figure 2). After 2 DIV, hNSCs expressed transcripts for the neural-specific genes nestin, Notch1 and neural cell adhesion molecule (N-CAM) (Figure 2A) and for the lineage specific markers β-tubulin class III, medium-size neurofilament (NF-M) and microtubule-associated protein 2 (MAP-2) for neurons, GFAP for astrocytes and myelin basic protein (MBP) for oligodendrocytes (Figure 2A). Transcripts for the GABAergic cell marker glutamic acid decarboxylase (GAD) were expressed, but transcripts for the tyrosine hydroxylase (TH), a marker for dopaminergic neurons, were undetectable. Immunocytochemical analysis (Figure 2B–F) of 10 day-old cultures demonstrated that the proportion of nestin+ cells was 36.6±2.7%, 62.5±2.8% expressed the neuronal marker TuJ1, 1.9±0.3% expressed the astrocytic marker GFAP and 7.1±0.4% were oligodendrocytes and expressed galactocerebrocide (GC) (Figure 2F). A subset (9.8±1.6%) of the GFAP+ astrocytes co-expressed nestin.10.1371/journal.pone.0001644.g002Figure 2hESC-derived hNSCs spontaneously differentiated into the 3 principal central nervous system cell types.Dissociated hNSCs were washed free of growth factors and plated on poly-L-onithine coated glass coverslips. Differentiated cultures were either harvested after 2 DIV for total RNA extraction and RT-PCR analysis or fixed after 10 DIV and processed for indirect immunocytochemistry. (A) Differentiated hNSCs expressed the neural-specific transcripts nestin and Notch1 as well as transcripts: for neurons (β-tubulin class III, MAP-2, NCAM and medium-size neurofilament, NF-M), for astrocytes (GFAP) and for oligodendrocytes (MBP). The hNSCs expressed transcripts for GAD, but not for TH. B, C & D, morphology of NSC-derived progeny differentiated into GFAP+ astrocytes (B), GC-expressing oligodendrocytes (C) and TuJ1+ neurons (D), DAPI (blue) show life cell nuclei. (E) Photo showing cultures double-immunostained for TuJ1 (green) and nestin (red) (DAPI, blue). (F) Quantitative analysis of immunostained 10 day-old cultures for the 3 neural cell types. Results are mean±s.e.m. of three independent experiments, each performed in duplicate. Bars: (c) 20 µm; (d, e) 10 µm. 2. The isolated hNSCs are normal and do not form tumors in normal nude ratsThe self-renewal and pluripotent abilities of the hESCs are also associated with tumorigenic properties. Therefore, the first critical step toward developing therapeutic hNSCs is to verify that they are non-tumorigenic. The monolayer culture of SD56 hNSCs clearly demonstrated contact inhibition of growth, a normal karyotype and did not express the pluripotency transcripts Oct-4 and Nanog. Removal of mitogenic factors and continued culture on plastic resulted in cell senescence that is characteristic of non-transformed cells. To determine whether SD56 hNSCs form tumors in vivo, we transplanted them at high density (see Methods) into the forebrain and subcutaneously into the flank of nude rats. The animals were kept for a two-month post-transplant survival period. To label mitotically active cells in vivo during S-phase, the rats were injected IP with BrdU (50 mg/kg) every 8 hours during the last 24 hours before euthanasia. The transit amplifying endogenous precursors located in the subventricular zone (SVZ) were labeled (Figure S3); however, we were unable to detect grafted SD56 hNSCs co-localizing the human-specific nuclear marker hNuc and BrdU (Figure S3). No surviving SD56 hNSCs were detected in the flank of the transplanted animals suggesting that the grafted cells are not tumorigenic. 3. Transplanted cells survived, migrated toward and engrafted into the stroke-damaged host tissueTo investigate the survival and functional engraftment in an injury environment, hNSCs (4×105) were transplanted into the ischemic boundary zone in the rat striatum one week after the middle cerebral artery occlusion (MCAO) was performed. Animals were euthanized two months later and the brains processed for histo-pathology and immunocytochemistry. Grafted SD56 hNSCs, identified with hNuc, demonstrated a 37.0±15.8% survival rate and a remarkable dispersion toward the stroke-damaged tissue with no sign of overgrowth or tumorigenesis. The majority of grafted cells (61.2±4.7%) migrated at least 200 µm away from the injection site and penetrated an average distance of 806.4±49.3 µm into the stroke-damaged tissue (Figure 3A–C). Immunostaining with the blood vessel marker, GluT1, revealed dilated vessels in the infarcted striatum and a close association between vessels and the grafted hNSCs (Figure 3B, 3C). The grafted cells rarely expressed the proliferation marker Ki67 (Figure 3D), 29.8±4.4% expressed nestin (Figure 3E), 6.5±0.9% expressed doublecortin (DCX) and 60.8±8.1% were TuJ1+ (Figure 3F, G). Grafted cells rarely co-expressed the astroglial marker GFAP (Figure 3H) or differentiated into CNPase-expressing oligodendrocytes (Figure 3I). Immunostaining for GAD demonstrated that 25.1±2.3% of grafted hNSCs differentiated into GABAergic neurons while less than 2% were positive for glutamate (Figure 3J, K).10.1371/journal.pone.0001644.g003Figure 3Dispersion, engraftment and differentiation of the hNSCs in stroke-lesioned animals.(A) Schematic drawing of a frontal section through the striatum illustrating the dispersion of grafted hNSCs in the focal ischemia-lesioned parenchyma (shaded area). (B, C) Photos show frontal sections through the graft in the striatum immunostained with the human specific antibodies: anti-hNuc (green in B & C) and anti-GluT1 (red, B & C) showing blood vessels and dispersed hNSCs in the graft zone. C: higher magnification of the inset in B. (D–I) Photos taken from frontal sections through the graft in the striatum double immunoprocessed for cell proliferation and neural lineage markers. (D) Note the endogenous Ki67+ cells (red cells, arrow) in the stroke damaged area and the hNuc+ (green)/Ki67- grafted hNSCs (arrowheads). (E) Examples of grafted SD56 hNSCs showing co-expression of hNuc (green) and nestin (red). (F) Confocal 3D reconstructed orthogonal images of the hNuc+(green)/DCX+(red) NSCs (arrowheads) viewed in the x-z plan on the top and y-z plan on the right. (G) Examples show the majority of grafted NSC progeny co-expressing hNuc (red) and the neuronal marker TuJ1 (green). Grafted NSCs rarely differentiate into GFAP+ astrocytes (H). In I, rare example of grafted NSC progeny becoming an oligodendrocyte identified by the expression of CNPase (green). Grafted NSCs expressed the GABAergic marker GAD65/67 (J) and rarely expressed glutamate (K). (Abbreviations: Cx: cortex, Str: striatum). Bars: (B, C) 100 µm; (D, F) 20 µm; (E, G–K) 10 µm. 4. Transplanted cells improve sensorimotor function of the stroke-disabled forelimbWe asked whether transplanted SD56 hNSCs could enhance the recovery of sensorimotor function that is compromised in the stroke-injured rats. We used the cylinder test to measure the sensorimotor asymmetry in forelimb use during spontaneous exploration [22]. To establish the baseline of the stroke-induced sensorimotor deficit, spontaneous behavior of rats in a transparent cylinder was videotaped one week after stroke (pre-transplant, Figure 4). Tests were then conducted 4 and 8 weeks after vehicle and SD56 hNSCs transplantation. Stable asymmetry in forelimb use was observed 7 days post-stroke (pre-transplant, Figure 4). Ischemic rats used their impaired forelimbs (contralateral to lesion) during lateral exploration less than they did before stroke. Transplantation of SD56 hNSCs significantly enhanced the independent use of the impaired contralateral forelimb 4 weeks post transplantation (P<0.05 vs pre-transplant). Eight weeks after transplantation the improvement in the use of the impaired forelimb was stable and significant when compared to the pre-transplant group and significantly improved in comparison to vehicle treated group at 8 weeks (Figure 4). In the vehicle treated group, the independent use of the contralateral forelimb remained impaired 4 and 8 weeks post-injection. In an independent study and using the same MCAO rat animal model, we found that transplantation of dermal fibroblasts did not improve the stroke-induced motor deficits (unpublished data).10.1371/journal.pone.0001644.g004Figure 4Transplantation of NSCs improves sensorimotor function of the stroke-disabled forelimb.Forelimb use during spontaneous lateral exploration was measured in the cylinder test (see Method and Results sections for details). Groups of vehicle injected (n = 7) and hNSCs (n = 10) transplanted are represented. The animals were tested as described in Method section. Note the significant increase in the independent use of the impaired contralateral forelimb at 4 and 8 weeks post transplantation (P<0.05 vs pre-transplant group). The contralateral forelimb remained impaired in the vehicle treated group at 4 and 8 weeks post-injection. Bars represent percentages±s.e.m. of steps taken by the ipsilateral, contralateral and both forelimbs simultaneously. *P<0.05 vs pre-transplant group; #P<0.05 vs vehicle groups. Our results indicate that a self-renewable and homogenous population of hNSCs, SD56, was derived from hESCs using defined media supplemented with a specific combination of growth factors. The SD56 hNSCs grew as an adherent monolayer culture, uniformly expressed molecular features of hNSCs including nestin, vimentin and the radial glial marker 3CB2, and did not express the pluripotency markers Oct4 or Nanog. The self-renewal property of the hNSCs was characterized by a predominant symmetrical mode of cell division. They exhibited no chromosomal abnormalities and demonstrated non-tumorigenic properties after implantation into ischemic brains and into naïve nude rat brains and flanks. Furthermore, the transplanted SD56 hNSCs migrated toward the stroke-damaged adult brain parenchyma, engrafted and improved the independent use of the stroke-impaired forelimb. Maintenance of stem cells requires symmetrical and asymmetrical cell divisions to both expand and to give rise to specialized progeny of a specific tissue (reviewed in [23]). In vivo, a complex cellular micro-environment or niche ensures the self-maintenance property of NSCs [24], [25], [26], [27]. In vitro, defined growth factors and extracellular matrices support stem cell self-renewal [28], [29]. The embryonic stem cells can propagate in a predominantly proliferative symmetrical mode, leading to homogeneous cell cultures growing relatively quickly with minimal cell differentiation [30], [31], [32], [33], [34]. Tissue specific stem cells, however, self-renew in a predominant asymmetric mode to maintain them selves and compensate for the loss of differentiated cells due to disease or injury. Thus, NSCs isolated from developing or adult brain grow as a mixture of undifferentiated and differentiated cells due the predominant asymmetrical mode of cell division [35], [36], [37], [38], [39], [40], [41]. A recent study has reported that a murine ESC-derived NSC line (LC1) is propagated as an adherent homogenous culture with a dominant mode of symmetrical self-renewal [21]. A combination of EGF and FGF2 was sufficient to propagate these NSCs as an adherent monolayer. However, the SD56 hNSC line described here required the combination of EGF, bFGF and LIF for self-maintenance. Although there are morphological and molecular similarities between our hNSCs and the NSCs previously described [21], the methods of isolation and growth are different. In addition to the different combination of growth factors used, the hNSC line we have isolated did not go through the rosette-structure stage. The in vitro analysis of BrdU incorporation and nestin expression indicated that our hNSCs divide predominantly symmetrically. This type of growth pattern is characteristic of primitive normal stem cells undergoing mostly symmetric cell division to increase the stem cell pool at the early stage of development or during tissue regeneration after injury [23]. RT-PCR and immunocytochemistry analysis demonstrated that these undifferentiated SD56 cells did not express any pluripotency, endodermal or mesodermal markers. Furthermore, the SD56 hNSCs described here exhibited the multipotential characteristic to differentiate into neurons, astrocytes and oligodendrocytes both in vitro and upon transplantation. Together these findings suggest that the hNSC line we isolated are appropriately programmed and share similar characteristics with the definitive NSCs of the developing brain. The SD56 hNSCs demonstrated a remarkable ability to migrate toward the stroke-damaged parenchyma of the adult rat brain. This directed migration by the majority of the grafted cells could be due to their uniform cellular composition, which results in an equal response to the host microenvironment. In previous studies, subpopulations of transplanted hESCs that were enriched in neural cells migrated in host microenvironments conducive to cell migration, such as the developing brain or in structures such as the rostral migratory stream [13], [20]. In the adult lesioned brain, the grafted hESC-derived neural cells proliferated and formed rosettes [14], teratomas [12], [15] or a cellular mass that induced a gliotic host response whereby local astrocytes demarcated the grafts [16]. Enriched neural cultures derived from mouse [42] and monkey ESCs [43] have produced behavioral improvements when transplanted into animal models of stroke and brain injury. However, in these cases, the transplanted non-human ESCs also formed a mass with signs of overgrowth in the core, as well as deformations [44], [45], [46]. ESCs plated at low density acquire neural identity within few hours after plating [47]. Interestingly, nearly all viable cells expressed nestin, the early neural fate marker Sox1, and the pluripotency marker Oct4. Together, these studies are seminal and suggest that complete neuralization may not be achieved through certain enrichment processes, consequently the neural cells could revert to a pluripotent stage [17]. The dispersion of the grafted hNSCs within host parenchyma may allow for more graft-host interactions that could stabilize differentiation, inhibit growth and prevent gliotic host response. In the present study, SD56 hNSC-transplanted animals demonstrated a stable improvement in the sensorimotor function when evaluated for spontaneous exploratory activity in the cylinder test that detects long-lasting deficits in forelimb use in the experimental models of stroke [22]. The transplantation of hNSCs significantly enhanced the independent use of the impaired contralateral forelimb 8 weeks post transplantation. This is the first report demonstrating that the transplantation of hNSCs derived from hESCs can improve neurologic behavior after experimental stroke. Together, these findings are encouraging and suggest that these cells are promising for future development. In addition to their therapeutic application, the hNSCs isolated under the reported conditions offer a means to interrogate host environments and unravel mechanistic features of self-renewal, non-tumorigenicity and functional engraftability in animal models of neurological disorders. hESC and NSC CulturesThe hESC line H9 (WiCell Research Institute) was propagated every 5 to 7 days on irradiated mouse embryonic fibroblasts. The cell culture media consisted of a 1∶1 mixture of Dulbecco's modified Eagle's medium (DMEM) and F12 nutrient, 20% serum replacement (Invitrogen), 0.1 mM β-mercaptoethanol, 2 µg/ml heparin and 4 ng/ml bFGF (R&D Systems). To generate the NSCs, dissociated hESCs were cultured in a chemically defined medium composed of DMEM/F12 (1∶1) including glucose (0.6%), glutamine (2 mM), sodium bicarbonate (3 mM), and HEPES buffer (5 mM) [all from Sigma except glutamine (Invitrogen)]. A defined hormone mix and salt mixture (Sigma), including insulin (25 mg/ml), transferrin (100 mg/ml), progesterone (20 nM), putrescine (60 mM), and selenium chloride (30 nM) was used in place of serum. The medium was supplemented with EGF (20 ng/ml), bFGF (10 ng/ml) and LIF (10 ng/ml). Dissociated hNSCs were plated at a density of 100,000 cell/ml in Corning T75 (Invitrogen) culture flasks in the defined media together with the growth factors. After 5–7 DIV, the adherent culture was incubated in 0.025%trypsin/0.01% EDTA (w/v) for 1 min followed by the addition of trypsin inhibitor (Invitrogen) then gently triturated to achieve single cell suspension. The cells were then washed twice with fresh medium and reseeded in fresh growth factor-containing media at 100,000 cells/ml. This procedure was performed for 21 passages and the fold of increase and population doubling were calculated at each passage. For clonal analysis, single spheres or confluent hNSC cultures were single cell dissociated and serially diluted to yield 1–2 cell/10 µl. A 10-µl-cell suspension was then added to each of 96 or 24 well plates containing 200–300 µl of growth media. Wells containing one viable cell were marked the next day and re-scored 5 to 7 days later for cell proliferation. The differentiation of the hNSCs was performed as previously described [48]. Dissociated hNSCs were plated at a density of 106 cells/ml in control (media/hormone mix) medium devoid of any growth factors and supplemented with 1% fetal bovine serum (FBS) on poly-L-ornithine-coated (15 mg/ml; Sigma) glass coverslips in 24-well Nunclon culture dishes with 0.5 ml/well. After 2, 7–15 DIV cultures were fixed and processed for immunocytochemistry or used for RT-PCR analysis. Karyotype analysisLong-term cultures of hNSCs were incubated at 37°C and harvested for metaphase chromosomes when the cultures were 75% confluent. Metaphase chromosomes were obtained by standard chromosome harvest methods by exposure to Colcemid at 0.1 µg/ml for 1 hour at 37°C, a 2-minute exposure to trypsin/EDTA, hypotonized with 0.057 M KCl and fixed with 3∶1 methanol:acetic acid. Slide preparations were made by dropping the fixed cell pellet onto cold, wet slides and air-dried. After incubating the slides at 90°C for 30 minutes, chromosomes were trypsin banded and then Wright/Giemsa stained for G-banding analysis. Twenty metaphase cells were completely analyzed and a normal female chromosome complement was found (46,XX). Tumorigenicity in nude ratsAll animal experiments were conducted according to the National Institute of Health (NIH) guidelines and approved by the IACUC. Normal adult NIH-Nude rats (n = 5, 8 week-old, Taconic, Germantown, New York, United States) were used to test the tumorigenic potential of the SD56 hNSCs. Undifferentiated hNSCs from passage 9 were single cell dissociated using trypsin-EDTA and suspended at the concentration of 125,000 cell/µl in preparation for cell transplantation. Two µl of the cell suspension were stereotaxically transplanted into 4 sites within the striatum at the following coordinates: AP: +1.0 mm, ML: +3.2 mm, DV: −5.0; AP: +0.5 mm, ML: +3.0 mm, DV: −5.0; AP: −0.5 mm, ML: +3.0 mm, DV: −5.0; AP: −1.0 mm, ML: +3.5 mm, DV: −5.0 mm with the incisor bar set at 3.4 mm. The injection rate was 1 µl/min, and the cannula was left in place for an additional 5 min before retraction. For the flank tumor assay, 2×106 cells (125,000 cell/µl) were injected subcutaneously to the side of the adult nude rats. To label mitotically active cells in vivo during S-phase, the rats were injected IP with the BrdU (50 mg/kg, Sigma) every 8 hours during the last 24 hours before euthanasia. After 2-month survival time, rats were euthanized and a postmortem examination for tumor formation was performed. Induction of Focal Ischemia and Cell TransplantationAll animal experimentations were conducted according to the National Institute of Health (NIH) guidelines and approved by the IACUC. Sprague Dawley adult male rats (n = 17, 275g–310g, Charles River Laboratories, Wilmington, Massachusetts, United States) were subjected to one and a half hour suture occlusion of the middle cerebral artery (MCAO), as previously described [49] and immunosuppressed 2 days before cell transplantation and daily thereafter for one week with i.p. injections of cyclosporine A (20 mg/ml, Sandimmune, Novartis Pharmaceuticals). Thereafter oral cyclosporine was used at 210 µg/ml in drinking water until euthanasia. Undifferentiated SD56 hNSCs from passages between P9 and P13 were single cell dissociated using trypsin-EDTA in preparation for cell transplantation. One week after the stroke lesion, 2 µl of the hNSCs, at a concentration of 50,000 cell/µl, were stereotaxically transplanted into 4 sites within the lesioned striatum (n = 10) at the following coordinates: AP: +1.0 mm, ML: +3.2 mm, DV: −5.0; AP: +0.5 mm, ML: +3.0 mm, DV: −5.0; AP: −0.5 mm, ML: +3.0 mm, DV: −5.0; AP: −1.0 mm, ML: +3.5 mm, DV: −5.0 mm with the incisor bar set at 3.4 mm. The injection rate was 1 µl/min, and the cannula was left in place for an additional 5 min before retraction. As a control group, we used rats subjected to ischemia and injected with the vehicle (n = 7). All animals underwent baseline motor behavioral assessment before and after the ischemic lesion, and 4 & 8 weeks after cell transplantation. The animals were killed after 2-month survival time by transcardial perfusion with phosphate buffered saline (PBS) followed by 4% paraformaldehyde. The brains were cryoprotected in an increasing gradient of 10, 20 and 30% sucrose solution and cryostat sectioned at 40 µm and processed for immunocytochemistry. ImmunocytochemistryCultures were fixed with 4% paraformaldehyde for 15 min. Both cells and brain sections were rinsed in PBS for 3×5 min then incubated for 2 hrs (cultures) or overnight (brain sections) with the appropriate primary antibodies for multiple labeling. Secondary antibodies raised in the appropriate hosts and conjugated to FITC, RITC, AMCA, CY3 or CY5 chromogenes (Jackson ImmunoResearch) were used. Cells and sections were counterstained with the nuclear marker 4′,6-diamidine-2′-phenylindole dihydrochloride (DAPI). Positive and negative controls were included in each run. Immunostained sections were coverslipped using fluorsave (Calbiochem) as the mounting medium. The following antibodies were used: Anti-human Nuclei (hNuc, monoclonal 1∶100, Chemicon), Anti-TuJ1 (monoclonal 1∶100, Covance; Polyclonal 1∶200, Aves Labs); anti-GAD65/67 (polyclonal 1∶1000, Chemicon); Anti-glial fibrillary acidic protein (GFAP, monoclonal 1∶1000, Chemicon; polyclonal 1∶200, Aves Labs); Anti-galactocerebrocide (GC, monoclonal 1∶250, Chemicon); Anti-CNPase (polyclonal 1∶200, Aves Labs); Anti-Glucose Transporter type 1 (Glut-1 polyclonal, 1∶500, Chemicon); Anti-Nestin (polyclonal 1∶1000, Chemicon); Anti-vimentin (monoclonal 1∶500, Calbiochem); Anti-3CB2 (monoclonal 1∶500, Developmental Studies Hybridoma Bank); Anti-doublecortin (DCX, polyclonal 1∶250, SantaCruz Biotechnology); Anti-Ki67 (polyclonal 1∶250, SantaCruz Biotechnology). Fluorescence was detected, analyzed and photographed with a Zeiss LSM550 laser scanning confocal photomicroscope. For each animal, quantitative estimates of the total number of grafted cells were stereologically determined using the optical fractionator procedure [50]. A computer-assisted image analysis system was performed using Stereo Investigator software (MicroBrightField, Inc.). The rostral and caudal limits of the reference volume were determined by first and last frontal sections containing grafted cells. The striatum and cortex were accurately outlined at low magnification (2.5× objective). The optical fractionator probe was selected to perform systematic sampling of the immunoreactive cell population distributed within the serial sections to estimate the population number in the volume of tissue. The counting frame of the optical fractionator was defined at 50×50 µm squares and the systematic sampling was performed by translating a grid with 200×200 µm squares onto the sections of interest using the Stereo Investigator software. The sample sites were systematically and automatically generated by the computer and examined using a 60× objective of a Nikon Eclipse TE 300 microscope. The counting frame displayed inclusion and exclusion lines and only immunoreactive cell bodies falling within the counting frame with no contact with the exclusion lines were counted. The cell dispersion was measured by counting the number of cells within 200 µm distance from the graft site. The number and distance in µm of cells dispersed beyond 200 µm was also measured. An average of 2,000 cells was counted per animal. Double labeling was determined using the confocal laser scanning microscope by random sampling of 100 or more cells per marker for each animal, scoring first for hNuc+, followed by DAPI+ nuclei and then the marker of choice. The double labeling was always confirmed in x-z and y-z cross-sections produced by the orthogonal projections of z-series. Reverse Transcription-Polymerase Chain Reaction (RT-PCR) analysisTotal RNA was extracted from cultured cells using RNAeasy kit (Quiagen). Aliquots (1 µg) of total RNA from the cells were reverse transcribed in the presence of 50 mM Tris-HCl, pH 8.3, 75 mM KCl, 3 mM MgCl2, 10 mM DTT, 0.5 µM dNTPs, and 0.5 µg oligo-dT(12–18) with 200 U Superscript RNase H-Reverse Transcriptase (Invitrogen). PCR amplification was performed using standard procedure with Taq Polymerase. Aliquots of cDNA equivalent to 50 ng of total RNA were amplified in 25 µl reactions containing 10 mM Tris-HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl2 , 50 pmol of each primer, 400 µM dNTPs, and 0.5 U AmpliTaq DNA polymerase (Perkin-Elmer). PCR was performed using the following thermal profile: 4 min at 94°C; 1 min at 94°C, 1 min at 60°C, 1.5 min at 72°C, for 30–40 cycles; 7 min at 72°C, and finally a soak at 4°C overnight. The following day, 10 µl aliquots of the amplified products were run on a 2% agarose Tris–acetate gel containing 0.5 mg/ml ethidium bromide. The products were visualized through a UV transilluminator, captured in a digital format using Quantify One Gel Analysis software (Bio-Rad Laboratories) on a Macintosh G4 computer.The PCR primers specific to each transcript were as follows: GFAP, forward (F), 5′-TCATCGCTCAGGAGGTCCTT–3′ Reverse (R), 5′-CTG TTGCCAGAGATGGAGGTT–3′; MAP2 (F) 5′-GAAGACTCGCATCCGAATGG–3′, (R) 5′-CGCAGGATAGGAGGAAGAGACT–3′; MBP (F) 5′-TTAGCTGAATTC GCGTGTGG–3′, (R) 5′-GAGGAAGTGAATGAGCCGGTTA-3′ were deigned using the Primer Designer software, Version 2.0 (Scientific and Educational Software) [48]. 18S, β-tubulin class III, N-CAM, Nestin, NF-M, Notch-1 primers [51]. Oct4, Nanog primers [11]. FOXa2 (HNF3B), Brachyury primers [52]. Behavioral testsThe cylinder test was used to assess the spontaneous forelimb use during lateral exploration movement [22]. Rats were placed in a transparent acrylic cylinder (20 cm diameter) for 5 minutes. The cylinder encourages use of the forelimbs for vertical exploration. A mirror was placed behind the cylinder so that the forelimbs could be viewed at all times. Testing sessions were videotaped and forelimb use was scored by a blinded operator. Movements scored were the independent use of the left or right forelimb or simultaneous use of both the left and right forelimb to contact the wall of the cylinder during a full rear, to initiate a weight-shifting movement, or to regain center of gravity while moving laterally in a vertical posture along the wall. Animals were tested for their baselines after stroke and 4 and 8 weeks after cell transplantation. Statistical analysisOutcome measurement for each experiment was reported as mean±SEM. All data were analyzed using SPSS 11 for Mac OS X (SPSS Inc.). Significance of inter-group differences was performed by applying Student's t-test where appropriate. The One-Way ANOVA analysis was used to compare group differences for the forelimb use as the dependant variable and groups as the single independent factor variable. Differences between the groups were determined using Bonferroni's post hoc test. A P-value of less than 0.05 was considered to be statistically significant.
[ "BackgroundHuman", "embryonic", "stem", "cells", "(", "hESCs", ")", "offer", "a", "virtually", "unlimited", "source", "of", "neural", "cells", "for", "structural", "repair", "in", "neurological", "disorders", ",", "such", "as", "stroke", ".", "Neural", "cells", "can", "be", "derived", "from", "hESCs", "either", "by", "direct", "enrichment", ",", "or", "by", "isolating", "specific", "growth", "factor", "-", "responsive", "and", "expandable", "populations", "of", "human", "neural", "stem", "cells", "(", "hNSCs", ")", ".", "Studies", "have", "indicated", "that", "the", "direct", "enrichment", "method", "generates", "a", "heterogeneous", "population", "of", "cells", "that", "may", "contain", "residual", "undifferentiated", "stem", "cells", "that", "could", "lead", "to", "tumor", "formation", "in", "vivo", ".", "Methods", "/", "Principal", "FindingsWe", "isolated", "an", "expandable", "and", "homogenous", "population", "of", "hNSCs", "(", "named", "SD56", ")", "from", "hESCs", "using", "a", "defined", "media", "supplemented", "with", "epidermal", "growth", "factor", "(", "EGF", ")", ",", "basic", "fibroblast", "growth", "factor", "(", "bFGF", ")", "and", "leukemia", "inhibitory", "growth", "factor", "(", "LIF", ")", ".", "These", "hNSCs", "grew", "as", "an", "adherent", "monolayer", "culture", ".", "They", "were", "fully", "neuralized", "and", "uniformly", "expressed", "molecular", "features", "of", "NSCs", ",", "including", "nestin", ",", "vimentin", "and", "radial", "glial", "markers", ".", "These", "hNSCs", "did", "not", "express", "the", "pluripotency", "markers", "Oct4", "or", "Nanog", ",", "nor", "did", "they", "express", "markers", "for", "the", "mesoderm", "or", "endoderm", "lineages", ".", "The", "self", "-", "renewal", "property", "of", "the", "hNSCs", "was", "characterized", "by", "a", "predominant", "symmetrical", "mode", "of", "cell", "division", ".", "The", "SD56", "hNSCs", "differentiated", "into", "neurons", ",", "astrocytes", "and", "oligodendrocytes", "throughout", "multiple", "passages", "in", "vitro", ",", "as", "well", "as", "after", "transplantation", ".", "Together", ",", "these", "criteria", "confirm", "the", "definitive", "NSC", "identity", "of", "the", "SD56", "cell", "line", ".", "Importantly", ",", "they", "exhibited", "no", "chromosome", "abnormalities", "and", "did", "not", "form", "tumors", "after", "implantation", "into", "rat", "ischemic", "brains", "and", "into", "naïve", "nude", "rat", "brains", "and", "flanks", ".", "Furthermore", ",", "hNSCs", "isolated", "under", "these", "conditions", "migrated", "toward", "the", "ischemia", "-", "injured", "adult", "brain", "parenchyma", "and", "improved", "the", "independent", "use", "of", "the", "stroke", "-", "impaired", "forelimb", "two", "months", "post", "-", "transplantation", ".", "Conclusions", "/", "SignificanceThe", "SD56", "human", "neural", "stem", "cells", "derived", "under", "the", "reported", "conditions", "are", "stable", ",", "do", "not", "form", "tumors", "in", "vivo", "and", "enable", "functional", "recovery", "after", "stroke", ".", "These", "properties", "indicate", "that", "this", "hNSC", "line", "may", "offer", "a", "renewable", ",", "homogenous", "source", "of", "neural", "cells", "that", "will", "be", "valuable", "for", "basic", "and", "translational", "research", ".", "\n", "To", "date", "there", "have", "been", "no", "effective", "treatments", "for", "improving", "residual", "structural", "and", "functional", "deficits", "resulting", "from", "stroke", ".", "Current", "therapeutic", "approaches", ",", "such", "as", "the", "use", "of", "thrombolytics", ",", "benefit", "only", "1", "to", "4", "%", "of", "patients", "[", "1", "]", ".", "Consequently", ",", "the", "majority", "of", "stroke", "patients", "experience", "progression", "of", "ischemia", "associated", "with", "debilitating", "neurological", "deficits", ".", "Recent", "evidence", "has", "suggested", "that", "the", "transplantation", "of", "cells", "derived", "from", "cord", "blood", ",", "bone", "marrow", "or", "brain", "tissue", "(", "fetal", "and", "adult", ")", "enhances", "sensorimotor", "function", "in", "experimental", "models", "of", "stroke", "[", "2", "]", ",", "[", "3", "]", ".", "However", ",", "the", "normal", "human", "-", "derived", "somatic", "stem", "cells", "used", "in", "these", "studies", "have", "a", "limited", "capacity", "to", "differentiate", "into", "the", "diverse", "neural", "cell", "types", "optimal", "for", "structural", "and", "physiological", "tissue", "repair", "and", "are", "not", "amenable", "for", "large", "-", "scale", "cell", "production", ".", "Unlike", "other", "sources", "of", "stem", "cells", ",", "hESC", "lines", "possess", "a", "nearly", "unlimited", "self", "-", "renewal", "capacity", "and", "the", "developmental", "potential", "to", "differentiate", "into", "virtually", "any", "cell", "type", "of", "the", "organism", ".", "As", "such", ",", "they", "constitute", "an", "ideal", "source", "of", "cells", "for", "regenerative", "medicine", ".", "The", "successful", "derivation", "of", "hESC", "lines", "from", "the", "inner", "cell", "mass", "of", "preimplantation", "embryos", "and", "their", "long", "-", "term", "maintenance", "in", "vitro", "over", "multiple", "passages", "has", "been", "demonstrated", "[", "4", "]", "and", "standardized", ".", "Differentiation", "and", "enrichment", "processes", "that", "direct", "hESCs", "towards", "a", "neural", "lineage", "have", "also", "been", "achieved", ".", "To", "promote", "neuralization", ",", "ESCs", "were", "cultured", "in", "a", "defined", "media", "supplemented", "with", "morphogens", "or", "growth", "factors", "[", "5", "]", ",", "[", "6", "]", ",", "[", "7", "]", "or", "cultured", "under", "conditions", "that", "promote", "“", "rosettes", "”", ",", "structures", "morphologically", "similar", "to", "the", "developing", "neural", "tube", "[", "8", "]", ",", "[", "9", "]", ".", "This", "neuralization", "process", "has", "proven", "invaluable", "in", "understanding", "the", "specification", "of", "hESC", "-", "derived", "neural", "tissue", "[", "10", "]", ",", "[", "11", "]", ",", "[", "12", "]", ".", "However", ",", "the", "enriched", "neural", "progeny", "derived", "displayed", "overgrowth", "and", "limited", "migration", "after", "grafting", "into", "normal", "newborn", "mice", "[", "13", "]", "and", "lesioned", "adult", "rat", "striatum", "[", "12", "]", ",", "[", "14", "]", ",", "[", "15", "]", ",", "[", "16", "]", ".", "The", "inner", "cores", "of", "these", "grafts", "contained", "tumorigenic", "precursor", "cells", "(", "reviewed", "in", "[", "17", "]", ")", ".", "These", "findings", "suggest", "that", "neural", "cells", "generated", "by", "acute", "exposure", "to", "growth", "factors", "and", "/", "or", "morphogens", "may", "still", "be", "heterogeneous", "and", "potentially", "tumorigenic", ".", "We", "report", "an", "alternative", "method", "for", "the", "isolation", "and", "the", "perpetuation", "of", "a", "multipotent", "hNSC", "line", "from", "the", "hESCs", "with", "a", "primitive", "mode", "of", "self", "-", "renewal", ".", "We", "also", "demonstrate", "their", "long", "-", "term", "expansion", ",", "non", "-", "tumorigenic", "properties", "and", "functional", "engraftability", "in", "an", "experimental", "model", "of", "stroke", ".", "\n", "1", ".", "In", "vitro", "isolation", ",", "growth", "and", "differentiation", "of", "hESC", "-", "derived", "hNSCsThe", "hESCs", "were", "maintained", "and", "expanded", "on", "mouse", "feeder", "layer", "in", "media", "supplemented", "with", "bFGF", "(", "Figure", "1A", ")", ".", "After", "cell", "dissociation", ",", "a", "portion", "of", "the", "hESCs", "was", "cultured", "in", "serum", "free", "medium", "containing", "EGF", ",", "bFGF", "and", "LIF", ".", "These", "factors", "are", "known", "to", "stimulate", "the", "proliferation", "of", "human", "fetal", "-", "derived", "NSCs", "[", "18", "]", ",", "[", "19", "]", ".", "After", "3", "days", "in", "vitro", "(", "DIV", ")", ",", "there", "was", "selective", "survival", "and", "growth", "of", "cells", "that", "aggregated", "in", "clusters", "or", "spheres", "(", "Figure", "1B", ")", ".", "These", "primary", "spheres", "were", "harvested", "and", "replated", "in", "fresh", "media", ".", "During", "the", "following", "week", ",", "the", "spheres", "attached", "to", "the", "flask", "and", "a", "fibroblast", "-", "like", "cell", "population", "began", "to", "migrate", "out", "(", "Figure", "1C", ")", ".", "Secondary", "spheres", "(", "2", "°", "spheres", ")", "were", "generated", "from", "these", "cultures", "and", "lifted", "off", "by", "the", "end", "of", "the", "week", "leaving", "a", "hollow", "in", "the", "middle", "of", "the", "attached", "cells", "(", "Figure", "1D", ")", ".", "The", "floating", "2", "°", "spheres", "were", "collected", "and", "replated", "in", "fresh", "growth", "medium", "for", "2", "weeks", ".", "The", "cultures", "were", "then", "passaged", "by", "collagenase", "cell", "dissociation", "every", "7", "DIV", "for", "an", "additional", "4", "passages", "(", "Figure", "S1", ")", ".", "At", "the", "5th", "and", "6th", "passages", ",", "spheres", "were", "dissociated", "into", "a", "single", "-", "cell", "suspension", "using", "trypsin", "-", "EDTA", ".", "At", "this", "stage", "there", "was", "a", "change", "in", "the", "hNSCs", "'", "adherent", "properties", ",", "and", "the", "cells", "began", "to", "grow", "as", "a", "monolayer", "with", "multiple", "foci", "of", "cells", "throughout", "the", "culture", "(", "Figure", "1F", ")", ".", "The", "adherent", "hNSC", "culture", "stained", "uniformly", "for", "nestin", "(", "Figure", "1", "K", ")", ",", "vimentin", "(", "Figure", "1L", ")", "and", "with", "the", "radial", "glial", "marker", "3CB2", "(", "Figure", "1", "M", ")", "indicating", "their", "homogeneity", "and", "NSC", "identity", ".", "Under", "these", "culture", "conditions", ",", "it", "is", "noteworthy", "that", "we", "did", "not", "observe", "the", "formation", "of", "rosettes", "which", "has", "been", "previously", "reported", "to", "occur", "under", "certain", "conditions", "during", "neuralization", "of", "hESCs", "[", "8", "]", ",", "[", "20", "]", ",", "[", "21", "]", ".", "RT", "-", "PCR", "analysis", "confirmed", "that", "these", "hNSCs", "did", "not", "express", "the", "pluripotency", "transcripts", "Oct-4", "and", "Nanog", "(", "Figure", "1I", ")", ".", "Furthermore", ",", "the", "hNSCs", "did", "not", "express", "transcripts", "for", "brachyury", "and", "foxa2", ",", "marker", "genes", "for", "mesoderm", "and", "endoderm", "respectively", "(", "negative", "result", ",", "data", "not", "shown).10.1371", "/", "journal.pone.0001644.g001Figure", "1Isolation", "and", "purification", "of", "hNSCs", "from", "the", "hESCs", ".", "The", "hESCs", "were", "grown", "on", "a", "mouse", "feeder", "layer", "(", "A", ")", ".", "Primary", "neurospheres", "(", "B", ")", "were", "isolated", "and", "replated", "to", "eliminate", "other", "non", "-", "neural", "cells", ".", "The", "selectively", "harvested", "secondary", "neurospheres", "(", "arrow", "in", "C", ")", ",", "left", "behind", "hollow", "cores", "in", "the", "surface", "area", "(", "star", "in", "D", ")", "where", "they", "attached", "earlier", ".", "They", "were", "perpetuated", "for", "an", "additional", "5", "passages", "(", "E", ")", ".", "These", "2", "°", "spheres", "were", "then", "passaged", "using", "a", "single", "cell", "dissociation", "protocol", "(", "F", ")", ".", "Arrow", "in", "F", "shows", "an", "example", "of", "a", "focus", "of", "proliferating", "cells", ".", "(", "G", ",", "H", ")", "The", "hNSCs", "were", "passaged", "every", "5–7", "days", ",", "as", "described", "in", "the", "Methods", "section", ".", "Starting", "from", "an", "initial", "population", "of", "1", "million", "cells", ",", "the", "cumulative", "cell", "number", "was", "calculated", "at", "each", "passage", "as", "the", "fold", "of", "increase×the", "total", "cell", "number", "and", "plotted", "as", "logarithm", "with", "base", "2", "in", "function", "of", "time", "(", "G", ")", ".", "The", "cell", "perpetuation", "(", "G", ")", "and", "population", "doubling", "(", "H", ")", "analysis", "demonstrated", "the", "continuous", "and", "stable", "growth", "of", "the", "hNSCs", ".", "(", "I", ")", "RT", "-", "PCR", "analysis", "showing", "the", "down", "regulation", "of", "the", "pluripotency", "transcripts", "Oct4", "and", "Nanog", "in", "secondary", "neurospheres", "and", "in", "expanded", "hNSCs", "at", "passage", "8", "(", "P8", ")", ".", "(", "J", ")", "Cytogenetic", "evaluation", "of", "the", "SD56", "hNSCs", "line", "at", "passage", "12", "by", "standard", "G", "-", "banding", "was", "performed", ".", "Twenty", "metaphase", "cells", "were", "analyzed", "and", "showed", "a", "normal", "female", "chromosome", "complement", "(", "46,XX", ")", ".", "Isolated", "and", "expanded", "hNSCs", "expressed", "the", "neural", "precursor", "cell", "markers", "nestin", "(", "K", ")", ",", "Vimentin", "(", "L", ")", "and", "the", "radial", "glial", "cell", "marker", "3CB2", "(", "M", ")", "in", "virtually", "all", "the", "progeny", ".", "(", "N", "-", "P", ")", "Clonal", "self", "-", "renewal", "ability", "of", "the", "isolated", "hNSCs", "through", "symmetrical", "cell", "division", ".", "Single", "(", "N", ")", ",", "two", "-", "cell", "stage", "(", "O", ")", "and", "four", "-", "cell", "stage", "(", "P", ")", "of", "a", "hNSC", "proliferating", "over", "a", "3-day", "culture", "period", ".", "Note", "the", "symmetrical", "segregation", "of", "BrdU", "and", "nestin", "in", "the", "progeny", ".", "Bars", ":", "(", "A", ",", "B", ",", "C", ",", "D", ")", "200", "µm", ";", "(", "E", ",", "F", ")", "100", "µm", ";", "(", "K", "–", "M", ")", "20", "µm", ";", "(", "N", "–", "P", ")", "10", "µm", ".", "To", "ascertain", "self", "-", "renewal", "ability", "under", "clonal", "conditions", ",", "a", "single", "cell", "suspension", "was", "plated", "at", "clonal", "density", "(", "1–2", "cell/10", "µl", ")", ".", "To", "determine", "if", "the", "hNSCs", "divide", "symmetrically", ",", "we", "pulsed", "cultures", "with", "the", "thymidine", "analog", ",", "bromodeoxyuridine", "(", "BrdU", ")", ",", "after", "plating", "and", "looked", "for", "the", "expression", "of", "nestin", "in", "the", "progeny", ".", "Cultures", "were", "fixed", "after", "1", ",", "2", "or", "3", "DIV", "(", "Figure", "1N", "–", "P", ")", ".", "After", "2", "days", ",", "plated", "single", "cells", "first", "underwent", "a", "symmetric", "cell", "division", "and", "gave", "rise", "to", "daughter", "cells", "that", "were", "both", "positive", "for", "BrdU", "and", "nestin", ".", "The", "clone", "of", "cells", "continued", "to", "grow", "over", "the", "3", "DIV", "and", "all", "the", "progeny", "expressed", "nestin", ".", "BrdU", "labeling", "demonstrated", "that", "it", "was", "rare", "for", "only", "one", "daughter", "cell", "to", "inherit", "the", "BrdU", "and", "thus", "had", "undergone", "asymmetric", "segregation", "of", "the", "chromatids", "(", "Figure", "S2", ")", ".", "G", "-", "band", "karyotyping", "of", "these", "hNSCs", "confirmed", "the", "normal", ",", "non", "-", "transformed", "nature", "of", "cells", "after", "12", "passages", "(", "Figure", "1J", ")", ".", "We", "named", "the", "derived", "hNSCs", "SD56", "(", "intermittently", "referred", "to", "as", "SD56", "hNSCs", "or", "hNSCs).Under", "these", "defined", "growth", "conditions", ",", "the", "hNSCs", "showed", "stable", "growth", "with", "a", "2.7±0.2", "fold", "increase", "every", "5", "to", "7", "days", "(", "Figure", "1", "G", ")", ".", "The", "population", "doubling", "at", "each", "passage", "averaged", "at", "1.4±0.1", "(", "Figure", "1H", ")", ".", "The", "viability", "of", "hNSCs", "at", "each", "passage", "was", "consistent", "at", "the", "approximate", "value", "of", "98", "%", ".", "The", "projected", "cumulative", "cell", "numbers", "demonstrated", "that", "trillions", "of", "cells", "could", "be", "generated", "over", "a", "period", "of", "5", "months", "(", "Figure", "1", "G", ")", ".", "We", "expanded", "the", "isolated", "hNSCs", "lines", "for", "over", "20", "passages", "with", "a", "stable", "phenotype", ".", "An", "initial", "cell", "bank", "of", "75", "vials", "containing", "2", "to", "5", "million", "cells", "each", "was", "generated", "and", "cryopreserved", ".", "Upon", "removal", "of", "the", "mitogenic", "factors", "and", "plating", "on", "a", "coverslip", "pre", "-", "coated", "with", "poly", "-", "L", "-", "ornithine", "(", "PLO", ")", "substrate", ",", "the", "hNSCs", "spontaneously", "differentiated", "into", "neurons", ",", "astrocytes", "and", "oligodendrocytes", ",", "a", "property", "that", "is", "consistent", "with", "normal", "multipotent", "hNSCs", "(", "Figure", "2", ")", ".", "After", "2", "DIV", ",", "hNSCs", "expressed", "transcripts", "for", "the", "neural", "-", "specific", "genes", "nestin", ",", "Notch1", "and", "neural", "cell", "adhesion", "molecule", "(", "N", "-", "CAM", ")", "(", "Figure", "2A", ")", "and", "for", "the", "lineage", "specific", "markers", "β", "-", "tubulin", "class", "III", ",", "medium", "-", "size", "neurofilament", "(", "NF", "-", "M", ")", "and", "microtubule", "-", "associated", "protein", "2", "(", "MAP-2", ")", "for", "neurons", ",", "GFAP", "for", "astrocytes", "and", "myelin", "basic", "protein", "(", "MBP", ")", "for", "oligodendrocytes", "(", "Figure", "2A", ")", ".", "Transcripts", "for", "the", "GABAergic", "cell", "marker", "glutamic", "acid", "decarboxylase", "(", "GAD", ")", "were", "expressed", ",", "but", "transcripts", "for", "the", "tyrosine", "hydroxylase", "(", "TH", ")", ",", "a", "marker", "for", "dopaminergic", "neurons", ",", "were", "undetectable", ".", "Immunocytochemical", "analysis", "(", "Figure", "2B", "–", "F", ")", "of", "10", "day", "-", "old", "cultures", "demonstrated", "that", "the", "proportion", "of", "nestin+", "cells", "was", "36.6±2.7", "%", ",", "62.5±2.8", "%", "expressed", "the", "neuronal", "marker", "TuJ1", ",", "1.9±0.3", "%", "expressed", "the", "astrocytic", "marker", "GFAP", "and", "7.1±0.4", "%", "were", "oligodendrocytes", "and", "expressed", "galactocerebrocide", "(", "GC", ")", "(", "Figure", "2F", ")", ".", "A", "subset", "(", "9.8±1.6", "%", ")", "of", "the", "GFAP+", "astrocytes", "co", "-", "expressed", "nestin.10.1371", "/", "journal.pone.0001644.g002Figure", "2hESC", "-", "derived", "hNSCs", "spontaneously", "differentiated", "into", "the", "3", "principal", "central", "nervous", "system", "cell", "types", ".", "Dissociated", "hNSCs", "were", "washed", "free", "of", "growth", "factors", "and", "plated", "on", "poly", "-", "L", "-", "onithine", "coated", "glass", "coverslips", ".", "Differentiated", "cultures", "were", "either", "harvested", "after", "2", "DIV", "for", "total", "RNA", "extraction", "and", "RT", "-", "PCR", "analysis", "or", "fixed", "after", "10", "DIV", "and", "processed", "for", "indirect", "immunocytochemistry", ".", "(", "A", ")", "Differentiated", "hNSCs", "expressed", "the", "neural", "-", "specific", "transcripts", "nestin", "and", "Notch1", "as", "well", "as", "transcripts", ":", "for", "neurons", "(", "β", "-", "tubulin", "class", "III", ",", "MAP-2", ",", "NCAM", "and", "medium", "-", "size", "neurofilament", ",", "NF", "-", "M", ")", ",", "for", "astrocytes", "(", "GFAP", ")", "and", "for", "oligodendrocytes", "(", "MBP", ")", ".", "The", "hNSCs", "expressed", "transcripts", "for", "GAD", ",", "but", "not", "for", "TH", ".", "B", ",", "C", "&", "D", ",", "morphology", "of", "NSC", "-", "derived", "progeny", "differentiated", "into", "GFAP+", "astrocytes", "(", "B", ")", ",", "GC", "-", "expressing", "oligodendrocytes", "(", "C", ")", "and", "TuJ1", "+", "neurons", "(", "D", ")", ",", "DAPI", "(", "blue", ")", "show", "life", "cell", "nuclei", ".", "(", "E", ")", "Photo", "showing", "cultures", "double", "-", "immunostained", "for", "TuJ1", "(", "green", ")", "and", "nestin", "(", "red", ")", "(", "DAPI", ",", "blue", ")", ".", "(", "F", ")", "Quantitative", "analysis", "of", "immunostained", "10", "day", "-", "old", "cultures", "for", "the", "3", "neural", "cell", "types", ".", "Results", "are", "mean±s.e.m", ".", "of", "three", "independent", "experiments", ",", "each", "performed", "in", "duplicate", ".", "Bars", ":", "(", "c", ")", "20", "µm", ";", "(", "d", ",", "e", ")", "10", "µm", ".", "2", ".", "The", "isolated", "hNSCs", "are", "normal", "and", "do", "not", "form", "tumors", "in", "normal", "nude", "ratsThe", "self", "-", "renewal", "and", "pluripotent", "abilities", "of", "the", "hESCs", "are", "also", "associated", "with", "tumorigenic", "properties", ".", "Therefore", ",", "the", "first", "critical", "step", "toward", "developing", "therapeutic", "hNSCs", "is", "to", "verify", "that", "they", "are", "non", "-", "tumorigenic", ".", "The", "monolayer", "culture", "of", "SD56", "hNSCs", "clearly", "demonstrated", "contact", "inhibition", "of", "growth", ",", "a", "normal", "karyotype", "and", "did", "not", "express", "the", "pluripotency", "transcripts", "Oct-4", "and", "Nanog", ".", "Removal", "of", "mitogenic", "factors", "and", "continued", "culture", "on", "plastic", "resulted", "in", "cell", "senescence", "that", "is", "characteristic", "of", "non", "-", "transformed", "cells", ".", "To", "determine", "whether", "SD56", "hNSCs", "form", "tumors", "in", "vivo", ",", "we", "transplanted", "them", "at", "high", "density", "(", "see", "Methods", ")", "into", "the", "forebrain", "and", "subcutaneously", "into", "the", "flank", "of", "nude", "rats", ".", "The", "animals", "were", "kept", "for", "a", "two", "-", "month", "post", "-", "transplant", "survival", "period", ".", "To", "label", "mitotically", "active", "cells", "in", "vivo", "during", "S", "-", "phase", ",", "the", "rats", "were", "injected", "IP", "with", "BrdU", "(", "50", "mg", "/", "kg", ")", "every", "8", "hours", "during", "the", "last", "24", "hours", "before", "euthanasia", ".", "The", "transit", "amplifying", "endogenous", "precursors", "located", "in", "the", "subventricular", "zone", "(", "SVZ", ")", "were", "labeled", "(", "Figure", "S3", ")", ";", "however", ",", "we", "were", "unable", "to", "detect", "grafted", "SD56", "hNSCs", "co", "-", "localizing", "the", "human", "-", "specific", "nuclear", "marker", "hNuc", "and", "BrdU", "(", "Figure", "S3", ")", ".", "No", "surviving", "SD56", "hNSCs", "were", "detected", "in", "the", "flank", "of", "the", "transplanted", "animals", "suggesting", "that", "the", "grafted", "cells", "are", "not", "tumorigenic", ".", "3", ".", "Transplanted", "cells", "survived", ",", "migrated", "toward", "and", "engrafted", "into", "the", "stroke", "-", "damaged", "host", "tissueTo", "investigate", "the", "survival", "and", "functional", "engraftment", "in", "an", "injury", "environment", ",", "hNSCs", "(", "4×105", ")", "were", "transplanted", "into", "the", "ischemic", "boundary", "zone", "in", "the", "rat", "striatum", "one", "week", "after", "the", "middle", "cerebral", "artery", "occlusion", "(", "MCAO", ")", "was", "performed", ".", "Animals", "were", "euthanized", "two", "months", "later", "and", "the", "brains", "processed", "for", "histo", "-", "pathology", "and", "immunocytochemistry", ".", "Grafted", "SD56", "hNSCs", ",", "identified", "with", "hNuc", ",", "demonstrated", "a", "37.0±15.8", "%", "survival", "rate", "and", "a", "remarkable", "dispersion", "toward", "the", "stroke", "-", "damaged", "tissue", "with", "no", "sign", "of", "overgrowth", "or", "tumorigenesis", ".", "The", "majority", "of", "grafted", "cells", "(", "61.2±4.7", "%", ")", "migrated", "at", "least", "200", "µm", "away", "from", "the", "injection", "site", "and", "penetrated", "an", "average", "distance", "of", "806.4±49.3", "µm", "into", "the", "stroke", "-", "damaged", "tissue", "(", "Figure", "3A", "–", "C", ")", ".", "Immunostaining", "with", "the", "blood", "vessel", "marker", ",", "GluT1", ",", "revealed", "dilated", "vessels", "in", "the", "infarcted", "striatum", "and", "a", "close", "association", "between", "vessels", "and", "the", "grafted", "hNSCs", "(", "Figure", "3B", ",", "3C", ")", ".", "The", "grafted", "cells", "rarely", "expressed", "the", "proliferation", "marker", "Ki67", "(", "Figure", "3D", ")", ",", "29.8±4.4", "%", "expressed", "nestin", "(", "Figure", "3E", ")", ",", "6.5±0.9", "%", "expressed", "doublecortin", "(", "DCX", ")", "and", "60.8±8.1", "%", "were", "TuJ1", "+", "(", "Figure", "3F", ",", "G", ")", ".", "Grafted", "cells", "rarely", "co", "-", "expressed", "the", "astroglial", "marker", "GFAP", "(", "Figure", "3H", ")", "or", "differentiated", "into", "CNPase", "-", "expressing", "oligodendrocytes", "(", "Figure", "3I", ")", ".", "Immunostaining", "for", "GAD", "demonstrated", "that", "25.1±2.3", "%", "of", "grafted", "hNSCs", "differentiated", "into", "GABAergic", "neurons", "while", "less", "than", "2", "%", "were", "positive", "for", "glutamate", "(", "Figure", "3J", ",", "K).10.1371", "/", "journal.pone.0001644.g003Figure", "3Dispersion", ",", "engraftment", "and", "differentiation", "of", "the", "hNSCs", "in", "stroke", "-", "lesioned", "animals.(A", ")", "Schematic", "drawing", "of", "a", "frontal", "section", "through", "the", "striatum", "illustrating", "the", "dispersion", "of", "grafted", "hNSCs", "in", "the", "focal", "ischemia", "-", "lesioned", "parenchyma", "(", "shaded", "area", ")", ".", "(", "B", ",", "C", ")", "Photos", "show", "frontal", "sections", "through", "the", "graft", "in", "the", "striatum", "immunostained", "with", "the", "human", "specific", "antibodies", ":", "anti", "-", "hNuc", "(", "green", "in", "B", "&", "C", ")", "and", "anti", "-", "GluT1", "(", "red", ",", "B", "&", "C", ")", "showing", "blood", "vessels", "and", "dispersed", "hNSCs", "in", "the", "graft", "zone", ".", "C", ":", "higher", "magnification", "of", "the", "inset", "in", "B.", "(", "D", "–", "I", ")", "Photos", "taken", "from", "frontal", "sections", "through", "the", "graft", "in", "the", "striatum", "double", "immunoprocessed", "for", "cell", "proliferation", "and", "neural", "lineage", "markers", ".", "(", "D", ")", "Note", "the", "endogenous", "Ki67", "+", "cells", "(", "red", "cells", ",", "arrow", ")", "in", "the", "stroke", "damaged", "area", "and", "the", "hNuc+", "(", "green)/Ki67-", "grafted", "hNSCs", "(", "arrowheads", ")", ".", "(", "E", ")", "Examples", "of", "grafted", "SD56", "hNSCs", "showing", "co", "-", "expression", "of", "hNuc", "(", "green", ")", "and", "nestin", "(", "red", ")", ".", "(", "F", ")", "Confocal", "3D", "reconstructed", "orthogonal", "images", "of", "the", "hNuc+(green)/DCX+(red", ")", "NSCs", "(", "arrowheads", ")", "viewed", "in", "the", "x", "-", "z", "plan", "on", "the", "top", "and", "y", "-", "z", "plan", "on", "the", "right", ".", "(", "G", ")", "Examples", "show", "the", "majority", "of", "grafted", "NSC", "progeny", "co", "-", "expressing", "hNuc", "(", "red", ")", "and", "the", "neuronal", "marker", "TuJ1", "(", "green", ")", ".", "Grafted", "NSCs", "rarely", "differentiate", "into", "GFAP+", "astrocytes", "(", "H", ")", ".", "In", "I", ",", "rare", "example", "of", "grafted", "NSC", "progeny", "becoming", "an", "oligodendrocyte", "identified", "by", "the", "expression", "of", "CNPase", "(", "green", ")", ".", "Grafted", "NSCs", "expressed", "the", "GABAergic", "marker", "GAD65/67", "(", "J", ")", "and", "rarely", "expressed", "glutamate", "(", "K", ")", ".", "(", "Abbreviations", ":", "Cx", ":", "cortex", ",", "Str", ":", "striatum", ")", ".", "Bars", ":", "(", "B", ",", "C", ")", "100", "µm", ";", "(", "D", ",", "F", ")", "20", "µm", ";", "(", "E", ",", "G", "–", "K", ")", "10", "µm", ".", "4", ".", "Transplanted", "cells", "improve", "sensorimotor", "function", "of", "the", "stroke", "-", "disabled", "forelimbWe", "asked", "whether", "transplanted", "SD56", "hNSCs", "could", "enhance", "the", "recovery", "of", "sensorimotor", "function", "that", "is", "compromised", "in", "the", "stroke", "-", "injured", "rats", ".", "We", "used", "the", "cylinder", "test", "to", "measure", "the", "sensorimotor", "asymmetry", "in", "forelimb", "use", "during", "spontaneous", "exploration", "[", "22", "]", ".", "To", "establish", "the", "baseline", "of", "the", "stroke", "-", "induced", "sensorimotor", "deficit", ",", "spontaneous", "behavior", "of", "rats", "in", "a", "transparent", "cylinder", "was", "videotaped", "one", "week", "after", "stroke", "(", "pre", "-", "transplant", ",", "Figure", "4", ")", ".", "Tests", "were", "then", "conducted", "4", "and", "8", "weeks", "after", "vehicle", "and", "SD56", "hNSCs", "transplantation", ".", "Stable", "asymmetry", "in", "forelimb", "use", "was", "observed", "7", "days", "post", "-", "stroke", "(", "pre", "-", "transplant", ",", "Figure", "4", ")", ".", "Ischemic", "rats", "used", "their", "impaired", "forelimbs", "(", "contralateral", "to", "lesion", ")", "during", "lateral", "exploration", "less", "than", "they", "did", "before", "stroke", ".", "Transplantation", "of", "SD56", "hNSCs", "significantly", "enhanced", "the", "independent", "use", "of", "the", "impaired", "contralateral", "forelimb", "4", "weeks", "post", "transplantation", "(", "P<0.05", "vs", "pre", "-", "transplant", ")", ".", "Eight", "weeks", "after", "transplantation", "the", "improvement", "in", "the", "use", "of", "the", "impaired", "forelimb", "was", "stable", "and", "significant", "when", "compared", "to", "the", "pre", "-", "transplant", "group", "and", "significantly", "improved", "in", "comparison", "to", "vehicle", "treated", "group", "at", "8", "weeks", "(", "Figure", "4", ")", ".", "In", "the", "vehicle", "treated", "group", ",", "the", "independent", "use", "of", "the", "contralateral", "forelimb", "remained", "impaired", "4", "and", "8", "weeks", "post", "-", "injection", ".", "In", "an", "independent", "study", "and", "using", "the", "same", "MCAO", "rat", "animal", "model", ",", "we", "found", "that", "transplantation", "of", "dermal", "fibroblasts", "did", "not", "improve", "the", "stroke", "-", "induced", "motor", "deficits", "(", "unpublished", "data).10.1371", "/", "journal.pone.0001644.g004Figure", "4Transplantation", "of", "NSCs", "improves", "sensorimotor", "function", "of", "the", "stroke", "-", "disabled", "forelimb", ".", "Forelimb", "use", "during", "spontaneous", "lateral", "exploration", "was", "measured", "in", "the", "cylinder", "test", "(", "see", "Method", "and", "Results", "sections", "for", "details", ")", ".", "Groups", "of", "vehicle", "injected", "(", "n", " ", "=", " ", "7", ")", "and", "hNSCs", "(", "n", " ", "=", " ", "10", ")", "transplanted", "are", "represented", ".", "The", "animals", "were", "tested", "as", "described", "in", "Method", "section", ".", "Note", "the", "significant", "increase", "in", "the", "independent", "use", "of", "the", "impaired", "contralateral", "forelimb", "at", "4", "and", "8", "weeks", "post", "transplantation", "(", "P<0.05", "vs", "pre", "-", "transplant", "group", ")", ".", "The", "contralateral", "forelimb", "remained", "impaired", "in", "the", "vehicle", "treated", "group", "at", "4", "and", "8", "weeks", "post", "-", "injection", ".", "Bars", "represent", "percentages±s.e.m", ".", "of", "steps", "taken", "by", "the", "ipsilateral", ",", "contralateral", "and", "both", "forelimbs", "simultaneously", ".", "*", "P<0.05", "vs", "pre", "-", "transplant", "group", ";", "#", "P<0.05", "vs", "vehicle", "groups", ".", "\n", "Our", "results", "indicate", "that", "a", "self", "-", "renewable", "and", "homogenous", "population", "of", "hNSCs", ",", "SD56", ",", "was", "derived", "from", "hESCs", "using", "defined", "media", "supplemented", "with", "a", "specific", "combination", "of", "growth", "factors", ".", "The", "SD56", "hNSCs", "grew", "as", "an", "adherent", "monolayer", "culture", ",", "uniformly", "expressed", "molecular", "features", "of", "hNSCs", "including", "nestin", ",", "vimentin", "and", "the", "radial", "glial", "marker", "3CB2", ",", "and", "did", "not", "express", "the", "pluripotency", "markers", "Oct4", "or", "Nanog", ".", "The", "self", "-", "renewal", "property", "of", "the", "hNSCs", "was", "characterized", "by", "a", "predominant", "symmetrical", "mode", "of", "cell", "division", ".", "They", "exhibited", "no", "chromosomal", "abnormalities", "and", "demonstrated", "non", "-", "tumorigenic", "properties", "after", "implantation", "into", "ischemic", "brains", "and", "into", "naïve", "nude", "rat", "brains", "and", "flanks", ".", "Furthermore", ",", "the", "transplanted", "SD56", "hNSCs", "migrated", "toward", "the", "stroke", "-", "damaged", "adult", "brain", "parenchyma", ",", "engrafted", "and", "improved", "the", "independent", "use", "of", "the", "stroke", "-", "impaired", "forelimb", ".", "Maintenance", "of", "stem", "cells", "requires", "symmetrical", "and", "asymmetrical", "cell", "divisions", "to", "both", "expand", "and", "to", "give", "rise", "to", "specialized", "progeny", "of", "a", "specific", "tissue", "(", "reviewed", "in", "[", "23", "]", ")", ".", "In", "vivo", ",", "a", "complex", "cellular", "micro", "-", "environment", "or", "niche", "ensures", "the", "self", "-", "maintenance", "property", "of", "NSCs", "[", "24", "]", ",", "[", "25", "]", ",", "[", "26", "]", ",", "[", "27", "]", ".", "In", "vitro", ",", "defined", "growth", "factors", "and", "extracellular", "matrices", "support", "stem", "cell", "self", "-", "renewal", "[", "28", "]", ",", "[", "29", "]", ".", "The", "embryonic", "stem", "cells", "can", "propagate", "in", "a", "predominantly", "proliferative", "symmetrical", "mode", ",", "leading", "to", "homogeneous", "cell", "cultures", "growing", "relatively", "quickly", "with", "minimal", "cell", "differentiation", "[", "30", "]", ",", "[", "31", "]", ",", "[", "32", "]", ",", "[", "33", "]", ",", "[", "34", "]", ".", "Tissue", "specific", "stem", "cells", ",", "however", ",", "self", "-", "renew", "in", "a", "predominant", "asymmetric", "mode", "to", "maintain", "them", "selves", "and", "compensate", "for", "the", "loss", "of", "differentiated", "cells", "due", "to", "disease", "or", "injury", ".", "Thus", ",", "NSCs", "isolated", "from", "developing", "or", "adult", "brain", "grow", "as", "a", "mixture", "of", "undifferentiated", "and", "differentiated", "cells", "due", "the", "predominant", "asymmetrical", "mode", "of", "cell", "division", "[", "35", "]", ",", "[", "36", "]", ",", "[", "37", "]", ",", "[", "38", "]", ",", "[", "39", "]", ",", "[", "40", "]", ",", "[", "41", "]", ".", "A", "recent", "study", "has", "reported", "that", "a", "murine", "ESC", "-", "derived", "NSC", "line", "(", "LC1", ")", "is", "propagated", "as", "an", "adherent", "homogenous", "culture", "with", "a", "dominant", "mode", "of", "symmetrical", "self", "-", "renewal", "[", "21", "]", ".", "A", "combination", "of", "EGF", "and", "FGF2", "was", "sufficient", "to", "propagate", "these", "NSCs", "as", "an", "adherent", "monolayer", ".", "However", ",", "the", "SD56", "hNSC", "line", "described", "here", "required", "the", "combination", "of", "EGF", ",", "bFGF", "and", "LIF", "for", "self", "-", "maintenance", ".", "Although", "there", "are", "morphological", "and", "molecular", "similarities", "between", "our", "hNSCs", "and", "the", "NSCs", "previously", "described", "[", "21", "]", ",", "the", "methods", "of", "isolation", "and", "growth", "are", "different", ".", "In", "addition", "to", "the", "different", "combination", "of", "growth", "factors", "used", ",", "the", "hNSC", "line", "we", "have", "isolated", "did", "not", "go", "through", "the", "rosette", "-", "structure", "stage", ".", "The", "in", "vitro", "analysis", "of", "BrdU", "incorporation", "and", "nestin", "expression", "indicated", "that", "our", "hNSCs", "divide", "predominantly", "symmetrically", ".", "This", "type", "of", "growth", "pattern", "is", "characteristic", "of", "primitive", "normal", "stem", "cells", "undergoing", "mostly", "symmetric", "cell", "division", "to", "increase", "the", "stem", "cell", "pool", "at", "the", "early", "stage", "of", "development", "or", "during", "tissue", "regeneration", "after", "injury", "[", "23", "]", ".", "RT", "-", "PCR", "and", "immunocytochemistry", "analysis", "demonstrated", "that", "these", "undifferentiated", "SD56", "cells", "did", "not", "express", "any", "pluripotency", ",", "endodermal", "or", "mesodermal", "markers", ".", "Furthermore", ",", "the", "SD56", "hNSCs", "described", "here", "exhibited", "the", "multipotential", "characteristic", "to", "differentiate", "into", "neurons", ",", "astrocytes", "and", "oligodendrocytes", "both", "in", "vitro", "and", "upon", "transplantation", ".", "Together", "these", "findings", "suggest", "that", "the", "hNSC", "line", "we", "isolated", "are", "appropriately", "programmed", "and", "share", "similar", "characteristics", "with", "the", "definitive", "NSCs", "of", "the", "developing", "brain", ".", "The", "SD56", "hNSCs", "demonstrated", "a", "remarkable", "ability", "to", "migrate", "toward", "the", "stroke", "-", "damaged", "parenchyma", "of", "the", "adult", "rat", "brain", ".", "This", "directed", "migration", "by", "the", "majority", "of", "the", "grafted", "cells", "could", "be", "due", "to", "their", "uniform", "cellular", "composition", ",", "which", "results", "in", "an", "equal", "response", "to", "the", "host", "microenvironment", ".", "In", "previous", "studies", ",", "subpopulations", "of", "transplanted", "hESCs", "that", "were", "enriched", "in", "neural", "cells", "migrated", "in", "host", "microenvironments", "conducive", "to", "cell", "migration", ",", "such", "as", "the", "developing", "brain", "or", "in", "structures", "such", "as", "the", "rostral", "migratory", "stream", "[", "13", "]", ",", "[", "20", "]", ".", "In", "the", "adult", "lesioned", "brain", ",", "the", "grafted", "hESC", "-", "derived", "neural", "cells", "proliferated", "and", "formed", "rosettes", "[", "14", "]", ",", "teratomas", "[", "12", "]", ",", "[", "15", "]", "or", "a", "cellular", "mass", "that", "induced", "a", "gliotic", "host", "response", "whereby", "local", "astrocytes", "demarcated", "the", "grafts", "[", "16", "]", ".", "Enriched", "neural", "cultures", "derived", "from", "mouse", "[", "42", "]", "and", "monkey", "ESCs", "[", "43", "]", "have", "produced", "behavioral", "improvements", "when", "transplanted", "into", "animal", "models", "of", "stroke", "and", "brain", "injury", ".", "However", ",", "in", "these", "cases", ",", "the", "transplanted", "non", "-", "human", "ESCs", "also", "formed", "a", "mass", "with", "signs", "of", "overgrowth", "in", "the", "core", ",", "as", "well", "as", "deformations", "[", "44", "]", ",", "[", "45", "]", ",", "[", "46", "]", ".", "ESCs", "plated", "at", "low", "density", "acquire", "neural", "identity", "within", "few", "hours", "after", "plating", "[", "47", "]", ".", "Interestingly", ",", "nearly", "all", "viable", "cells", "expressed", "nestin", ",", "the", "early", "neural", "fate", "marker", "Sox1", ",", "and", "the", "pluripotency", "marker", "Oct4", ".", "Together", ",", "these", "studies", "are", "seminal", "and", "suggest", "that", "complete", "neuralization", "may", "not", "be", "achieved", "through", "certain", "enrichment", "processes", ",", "consequently", "the", "neural", "cells", "could", "revert", "to", "a", "pluripotent", "stage", "[", "17", "]", ".", "The", "dispersion", "of", "the", "grafted", "hNSCs", "within", "host", "parenchyma", "may", "allow", "for", "more", "graft", "-", "host", "interactions", "that", "could", "stabilize", "differentiation", ",", "inhibit", "growth", "and", "prevent", "gliotic", "host", "response", ".", "In", "the", "present", "study", ",", "SD56", "hNSC", "-", "transplanted", "animals", "demonstrated", "a", "stable", "improvement", "in", "the", "sensorimotor", "function", "when", "evaluated", "for", "spontaneous", "exploratory", "activity", "in", "the", "cylinder", "test", "that", "detects", "long", "-", "lasting", "deficits", "in", "forelimb", "use", "in", "the", "experimental", "models", "of", "stroke", "[", "22", "]", ".", "The", "transplantation", "of", "hNSCs", "significantly", "enhanced", "the", "independent", "use", "of", "the", "impaired", "contralateral", "forelimb", "8", "weeks", "post", "transplantation", ".", "This", "is", "the", "first", "report", "demonstrating", "that", "the", "transplantation", "of", "hNSCs", "derived", "from", "hESCs", "can", "improve", "neurologic", "behavior", "after", "experimental", "stroke", ".", "Together", ",", "these", "findings", "are", "encouraging", "and", "suggest", "that", "these", "cells", "are", "promising", "for", "future", "development", ".", "In", "addition", "to", "their", "therapeutic", "application", ",", "the", "hNSCs", "isolated", "under", "the", "reported", "conditions", "offer", "a", "means", "to", "interrogate", "host", "environments", "and", "unravel", "mechanistic", "features", "of", "self", "-", "renewal", ",", "non", "-", "tumorigenicity", "and", "functional", "engraftability", "in", "animal", "models", "of", "neurological", "disorders", ".", "\n", "hESC", "and", "NSC", "CulturesThe", "hESC", "line", "H9", "(", "WiCell", "Research", "Institute", ")", "was", "propagated", "every", "5", "to", "7", "days", "on", "irradiated", "mouse", "embryonic", "fibroblasts", ".", "The", "cell", "culture", "media", "consisted", "of", "a", "1∶1", "mixture", "of", "Dulbecco", "'s", "modified", "Eagle", "'s", "medium", "(", "DMEM", ")", "and", "F12", "nutrient", ",", "20", "%", "serum", "replacement", "(", "Invitrogen", ")", ",", "0.1", "mM", "β", "-", "mercaptoethanol", ",", "2", "µg", "/", "ml", "heparin", "and", "4", "ng", "/", "ml", "bFGF", "(", "R&D", "Systems", ")", ".", "To", "generate", "the", "NSCs", ",", "dissociated", "hESCs", "were", "cultured", "in", "a", "chemically", "defined", "medium", "composed", "of", "DMEM", "/", "F12", "(", "1∶1", ")", "including", "glucose", "(", "0.6", "%", ")", ",", "glutamine", "(", "2", "mM", ")", ",", "sodium", "bicarbonate", "(", "3", "mM", ")", ",", "and", "HEPES", "buffer", "(", "5", "mM", ")", "[", "all", "from", "Sigma", "except", "glutamine", "(", "Invitrogen", ")", "]", ".", "A", "defined", "hormone", "mix", "and", "salt", "mixture", "(", "Sigma", ")", ",", "including", "insulin", "(", "25", "mg", "/", "ml", ")", ",", "transferrin", "(", "100", "mg", "/", "ml", ")", ",", "progesterone", "(", "20", "nM", ")", ",", "putrescine", "(", "60", "mM", ")", ",", "and", "selenium", "chloride", "(", "30", "nM", ")", "was", "used", "in", "place", "of", "serum", ".", "The", "medium", "was", "supplemented", "with", "EGF", "(", "20", "ng", "/", "ml", ")", ",", "bFGF", "(", "10", "ng", "/", "ml", ")", "and", "LIF", "(", "10", "ng", "/", "ml", ")", ".", "Dissociated", "hNSCs", "were", "plated", "at", "a", "density", "of", "100,000", "cell", "/", "ml", "in", "Corning", "T75", "(", "Invitrogen", ")", "culture", "flasks", "in", "the", "defined", "media", "together", "with", "the", "growth", "factors", ".", "After", "5–7", "DIV", ",", "the", "adherent", "culture", "was", "incubated", "in", "0.025%trypsin/0.01", "%", "EDTA", "(", "w", "/", "v", ")", "for", "1", "min", "followed", "by", "the", "addition", "of", "trypsin", "inhibitor", "(", "Invitrogen", ")", "then", "gently", "triturated", "to", "achieve", "single", "cell", "suspension", ".", "The", "cells", "were", "then", "washed", "twice", "with", "fresh", "medium", "and", "reseeded", "in", "fresh", "growth", "factor", "-", "containing", "media", "at", "100,000", "cells", "/", "ml", ".", "This", "procedure", "was", "performed", "for", "21", "passages", "and", "the", "fold", "of", "increase", "and", "population", "doubling", "were", "calculated", "at", "each", "passage", ".", "For", "clonal", "analysis", ",", "single", "spheres", "or", "confluent", "hNSC", "cultures", "were", "single", "cell", "dissociated", "and", "serially", "diluted", "to", "yield", "1–2", "cell/10", "µl", ".", "A", "10-µl", "-", "cell", "suspension", "was", "then", "added", "to", "each", "of", "96", "or", "24", "well", "plates", "containing", "200–300", "µl", "of", "growth", "media", ".", "Wells", "containing", "one", "viable", "cell", "were", "marked", "the", "next", "day", "and", "re", "-", "scored", "5", "to", "7", "days", "later", "for", "cell", "proliferation", ".", "The", "differentiation", "of", "the", "hNSCs", "was", "performed", "as", "previously", "described", "[", "48", "]", ".", "Dissociated", "hNSCs", "were", "plated", "at", "a", "density", "of", "106", "cells", "/", "ml", "in", "control", "(", "media", "/", "hormone", "mix", ")", "medium", "devoid", "of", "any", "growth", "factors", "and", "supplemented", "with", "1", "%", "fetal", "bovine", "serum", "(", "FBS", ")", "on", "poly", "-", "L", "-", "ornithine", "-", "coated", "(", "15", "mg", "/", "ml", ";", "Sigma", ")", "glass", "coverslips", "in", "24-well", "Nunclon", "culture", "dishes", "with", "0.5", "ml", "/", "well", ".", "After", "2", ",", "7–15", "DIV", "cultures", "were", "fixed", "and", "processed", "for", "immunocytochemistry", "or", "used", "for", "RT", "-", "PCR", "analysis", ".", "Karyotype", "analysisLong", "-", "term", "cultures", "of", "hNSCs", "were", "incubated", "at", "37", "°", "C", "and", "harvested", "for", "metaphase", "chromosomes", "when", "the", "cultures", "were", "75", "%", "confluent", ".", "Metaphase", "chromosomes", "were", "obtained", "by", "standard", "chromosome", "harvest", "methods", "by", "exposure", "to", "Colcemid", "at", "0.1", "µg", "/", "ml", "for", "1", "hour", "at", "37", "°", "C", ",", "a", "2-minute", "exposure", "to", "trypsin", "/", "EDTA", ",", "hypotonized", "with", "0.057", "M", "KCl", "and", "fixed", "with", "3∶1", "methanol", ":", "acetic", "acid", ".", "Slide", "preparations", "were", "made", "by", "dropping", "the", "fixed", "cell", "pellet", "onto", "cold", ",", "wet", "slides", "and", "air", "-", "dried", ".", "After", "incubating", "the", "slides", "at", "90", "°", "C", "for", "30", "minutes", ",", "chromosomes", "were", "trypsin", "banded", "and", "then", "Wright", "/", "Giemsa", "stained", "for", "G", "-", "banding", "analysis", ".", "Twenty", "metaphase", "cells", "were", "completely", "analyzed", "and", "a", "normal", "female", "chromosome", "complement", "was", "found", "(", "46,XX", ")", ".", "Tumorigenicity", "in", "nude", "ratsAll", "animal", "experiments", "were", "conducted", "according", "to", "the", "National", "Institute", "of", "Health", "(", "NIH", ")", "guidelines", "and", "approved", "by", "the", "IACUC", ".", "Normal", "adult", "NIH", "-", "Nude", "rats", "(", "n", " ", "=", " ", "5", ",", "8", "week", "-", "old", ",", "Taconic", ",", "Germantown", ",", "New", "York", ",", "United", "States", ")", "were", "used", "to", "test", "the", "tumorigenic", "potential", "of", "the", "SD56", "hNSCs", ".", "Undifferentiated", "hNSCs", "from", "passage", "9", "were", "single", "cell", "dissociated", "using", "trypsin", "-", "EDTA", "and", "suspended", "at", "the", "concentration", "of", "125,000", "cell/µl", "in", "preparation", "for", "cell", "transplantation", ".", "Two", "µl", "of", "the", "cell", "suspension", "were", "stereotaxically", "transplanted", "into", "4", "sites", "within", "the", "striatum", "at", "the", "following", "coordinates", ":", "AP", ":", "+1.0", "mm", ",", "ML", ":", "+3.2", "mm", ",", "DV", ":", "−5.0", ";", "AP", ":", "+0.5", "mm", ",", "ML", ":", "+3.0", "mm", ",", "DV", ":", "−5.0", ";", "AP", ":", "−0.5", "mm", ",", "ML", ":", "+3.0", "mm", ",", "DV", ":", "−5.0", ";", "AP", ":", "−1.0", "mm", ",", "ML", ":", "+3.5", "mm", ",", "DV", ":", "−5.0", "mm", "with", "the", "incisor", "bar", "set", "at", "3.4", "mm", ".", "The", "injection", "rate", "was", "1", "µl", "/", "min", ",", "and", "the", "cannula", "was", "left", "in", "place", "for", "an", "additional", "5", "min", "before", "retraction", ".", "For", "the", "flank", "tumor", "assay", ",", "2×106", "cells", "(", "125,000", "cell/µl", ")", "were", "injected", "subcutaneously", "to", "the", "side", "of", "the", "adult", "nude", "rats", ".", "To", "label", "mitotically", "active", "cells", "in", "vivo", "during", "S", "-", "phase", ",", "the", "rats", "were", "injected", "IP", "with", "the", "BrdU", "(", "50", "mg", "/", "kg", ",", "Sigma", ")", "every", "8", "hours", "during", "the", "last", "24", "hours", "before", "euthanasia", ".", "After", "2-month", "survival", "time", ",", "rats", "were", "euthanized", "and", "a", "postmortem", "examination", "for", "tumor", "formation", "was", "performed", ".", "Induction", "of", "Focal", "Ischemia", "and", "Cell", "TransplantationAll", "animal", "experimentations", "were", "conducted", "according", "to", "the", "National", "Institute", "of", "Health", "(", "NIH", ")", "guidelines", "and", "approved", "by", "the", "IACUC", ".", "Sprague", "Dawley", "adult", "male", "rats", "(", "n", " ", "=", " ", "17", ",", "275g–310", "g", ",", "Charles", "River", "Laboratories", ",", "Wilmington", ",", "Massachusetts", ",", "United", "States", ")", "were", "subjected", "to", "one", "and", "a", "half", "hour", "suture", "occlusion", "of", "the", "middle", "cerebral", "artery", "(", "MCAO", ")", ",", "as", "previously", "described", "[", "49", "]", "and", "immunosuppressed", "2", "days", "before", "cell", "transplantation", "and", "daily", "thereafter", "for", "one", "week", "with", "i.p", ".", "injections", "of", "cyclosporine", "A", "(", "20", "mg", "/", "ml", ",", "Sandimmune", ",", "Novartis", "Pharmaceuticals", ")", ".", "Thereafter", "oral", "cyclosporine", "was", "used", "at", "210", "µg", "/", "ml", "in", "drinking", "water", "until", "euthanasia", ".", "Undifferentiated", "SD56", "hNSCs", "from", "passages", "between", "P9", "and", "P13", "were", "single", "cell", "dissociated", "using", "trypsin", "-", "EDTA", "in", "preparation", "for", "cell", "transplantation", ".", "One", "week", "after", "the", "stroke", "lesion", ",", "2", "µl", "of", "the", "hNSCs", ",", "at", "a", "concentration", "of", "50,000", "cell/µl", ",", "were", "stereotaxically", "transplanted", "into", "4", "sites", "within", "the", "lesioned", "striatum", "(", "n", " ", "=", " ", "10", ")", "at", "the", "following", "coordinates", ":", "AP", ":", "+1.0", "mm", ",", "ML", ":", "+3.2", "mm", ",", "DV", ":", "−5.0", ";", "AP", ":", "+0.5", "mm", ",", "ML", ":", "+3.0", "mm", ",", "DV", ":", "−5.0", ";", "AP", ":", "−0.5", "mm", ",", "ML", ":", "+3.0", "mm", ",", "DV", ":", "−5.0", ";", "AP", ":", "−1.0", "mm", ",", "ML", ":", "+3.5", "mm", ",", "DV", ":", "−5.0", "mm", "with", "the", "incisor", "bar", "set", "at", "3.4", "mm", ".", "The", "injection", "rate", "was", "1", "µl", "/", "min", ",", "and", "the", "cannula", "was", "left", "in", "place", "for", "an", "additional", "5", "min", "before", "retraction", ".", "As", "a", "control", "group", ",", "we", "used", "rats", "subjected", "to", "ischemia", "and", "injected", "with", "the", "vehicle", "(", "n", " ", "=", " ", "7", ")", ".", "All", "animals", "underwent", "baseline", "motor", "behavioral", "assessment", "before", "and", "after", "the", "ischemic", "lesion", ",", "and", "4", "&", "8", "weeks", "after", "cell", "transplantation", ".", "The", "animals", "were", "killed", "after", "2-month", "survival", "time", "by", "transcardial", "perfusion", "with", "phosphate", "buffered", "saline", "(", "PBS", ")", "followed", "by", "4", "%", "paraformaldehyde", ".", "The", "brains", "were", "cryoprotected", "in", "an", "increasing", "gradient", "of", "10", ",", "20", "and", "30", "%", "sucrose", "solution", "and", "cryostat", "sectioned", "at", "40", "µm", "and", "processed", "for", "immunocytochemistry", ".", "ImmunocytochemistryCultures", "were", "fixed", "with", "4", "%", "paraformaldehyde", "for", "15", "min", ".", "Both", "cells", "and", "brain", "sections", "were", "rinsed", "in", "PBS", "for", "3×5", "min", "then", "incubated", "for", "2", "hrs", "(", "cultures", ")", "or", "overnight", "(", "brain", "sections", ")", "with", "the", "appropriate", "primary", "antibodies", "for", "multiple", "labeling", ".", "Secondary", "antibodies", "raised", "in", "the", "appropriate", "hosts", "and", "conjugated", "to", "FITC", ",", "RITC", ",", "AMCA", ",", "CY3", "or", "CY5", "chromogenes", "(", "Jackson", "ImmunoResearch", ")", "were", "used", ".", "Cells", "and", "sections", "were", "counterstained", "with", "the", "nuclear", "marker", "4′,6-diamidine-2′-phenylindole", "dihydrochloride", "(", "DAPI", ")", ".", "Positive", "and", "negative", "controls", "were", "included", "in", "each", "run", ".", "Immunostained", "sections", "were", "coverslipped", "using", "fluorsave", "(", "Calbiochem", ")", "as", "the", "mounting", "medium", ".", "The", "following", "antibodies", "were", "used", ":", "Anti", "-", "human", "Nuclei", "(", "hNuc", ",", "monoclonal", "1∶100", ",", "Chemicon", ")", ",", "Anti", "-", "TuJ1", "(", "monoclonal", "1∶100", ",", "Covance", ";", "Polyclonal", "1∶200", ",", "Aves", "Labs", ")", ";", "anti", "-", "GAD65/67", "(", "polyclonal", "1∶1000", ",", "Chemicon", ")", ";", "Anti", "-", "glial", "fibrillary", "acidic", "protein", "(", "GFAP", ",", "monoclonal", "1∶1000", ",", "Chemicon", ";", "polyclonal", "1∶200", ",", "Aves", "Labs", ")", ";", "Anti", "-", "galactocerebrocide", "(", "GC", ",", "monoclonal", "1∶250", ",", "Chemicon", ")", ";", "Anti", "-", "CNPase", "(", "polyclonal", "1∶200", ",", "Aves", "Labs", ")", ";", "Anti", "-", "Glucose", "Transporter", "type", "1", "(", "Glut-1", "polyclonal", ",", "1∶500", ",", "Chemicon", ")", ";", "Anti", "-", "Nestin", "(", "polyclonal", "1∶1000", ",", "Chemicon", ")", ";", "Anti", "-", "vimentin", "(", "monoclonal", "1∶500", ",", "Calbiochem", ")", ";", "Anti-3CB2", "(", "monoclonal", "1∶500", ",", "Developmental", "Studies", "Hybridoma", "Bank", ")", ";", "Anti", "-", "doublecortin", "(", "DCX", ",", "polyclonal", "1∶250", ",", "SantaCruz", "Biotechnology", ")", ";", "Anti", "-", "Ki67", "(", "polyclonal", "1∶250", ",", "SantaCruz", "Biotechnology", ")", ".", "Fluorescence", "was", "detected", ",", "analyzed", "and", "photographed", "with", "a", "Zeiss", "LSM550", "laser", "scanning", "confocal", "photomicroscope", ".", "For", "each", "animal", ",", "quantitative", "estimates", "of", "the", "total", "number", "of", "grafted", "cells", "were", "stereologically", "determined", "using", "the", "optical", "fractionator", "procedure", "[", "50", "]", ".", "A", "computer", "-", "assisted", "image", "analysis", "system", "was", "performed", "using", "Stereo", "Investigator", "software", "(", "MicroBrightField", ",", "Inc", ".", ")", ".", "The", "rostral", "and", "caudal", "limits", "of", "the", "reference", "volume", "were", "determined", "by", "first", "and", "last", "frontal", "sections", "containing", "grafted", "cells", ".", "The", "striatum", "and", "cortex", "were", "accurately", "outlined", "at", "low", "magnification", "(", "2.5×", "objective", ")", ".", "The", "optical", "fractionator", "probe", "was", "selected", "to", "perform", "systematic", "sampling", "of", "the", "immunoreactive", "cell", "population", "distributed", "within", "the", "serial", "sections", "to", "estimate", "the", "population", "number", "in", "the", "volume", "of", "tissue", ".", "The", "counting", "frame", "of", "the", "optical", "fractionator", "was", "defined", "at", "50×50", "µm", "squares", "and", "the", "systematic", "sampling", "was", "performed", "by", "translating", "a", "grid", "with", "200×200", "µm", "squares", "onto", "the", "sections", "of", "interest", "using", "the", "Stereo", "Investigator", "software", ".", "The", "sample", "sites", "were", "systematically", "and", "automatically", "generated", "by", "the", "computer", "and", "examined", "using", "a", "60×", "objective", "of", "a", "Nikon", "Eclipse", "TE", "300", "microscope", ".", "The", "counting", "frame", "displayed", "inclusion", "and", "exclusion", "lines", "and", "only", "immunoreactive", "cell", "bodies", "falling", "within", "the", "counting", "frame", "with", "no", "contact", "with", "the", "exclusion", "lines", "were", "counted", ".", "The", "cell", "dispersion", "was", "measured", "by", "counting", "the", "number", "of", "cells", "within", "200", "µm", "distance", "from", "the", "graft", "site", ".", "The", "number", "and", "distance", "in", "µm", "of", "cells", "dispersed", "beyond", "200", "µm", "was", "also", "measured", ".", "An", "average", "of", "2,000", "cells", "was", "counted", "per", "animal", ".", "Double", "labeling", "was", "determined", "using", "the", "confocal", "laser", "scanning", "microscope", "by", "random", "sampling", "of", "100", "or", "more", "cells", "per", "marker", "for", "each", "animal", ",", "scoring", "first", "for", "hNuc+", ",", "followed", "by", "DAPI+", "nuclei", "and", "then", "the", "marker", "of", "choice", ".", "The", "double", "labeling", "was", "always", "confirmed", "in", "x", "-", "z", "and", "y", "-", "z", "cross", "-", "sections", "produced", "by", "the", "orthogonal", "projections", "of", "z", "-", "series", ".", "Reverse", "Transcription", "-", "Polymerase", "Chain", "Reaction", "(", "RT", "-", "PCR", ")", "analysisTotal", "RNA", "was", "extracted", "from", "cultured", "cells", "using", "RNAeasy", "kit", "(", "Quiagen", ")", ".", "Aliquots", "(", "1", "µg", ")", "of", "total", "RNA", "from", "the", "cells", "were", "reverse", "transcribed", "in", "the", "presence", "of", "50", "mM", "Tris", "-", "HCl", ",", "pH", "8.3", ",", "75", "mM", "KCl", ",", "3", "mM", "MgCl2", ",", "10", "mM", "DTT", ",", "0.5", "µM", "dNTPs", ",", "and", "0.5", "µg", "oligo", "-", "dT(12–18", ")", "with", "200", "U", "Superscript", "RNase", "H", "-", "Reverse", "Transcriptase", "(", "Invitrogen", ")", ".", "PCR", "amplification", "was", "performed", "using", "standard", "procedure", "with", "Taq", "Polymerase", ".", "Aliquots", "of", "cDNA", "equivalent", "to", "50", "ng", "of", "total", "RNA", "were", "amplified", "in", "25", "µl", "reactions", "containing", "10", "mM", "Tris", "-", "HCl", ",", "pH", "8.3", ",", "50", "mM", "KCl", ",", "1.5", "mM", "MgCl2", ",", "50", "pmol", "of", "each", "primer", ",", "400", "µM", "dNTPs", ",", "and", "0.5", "U", "AmpliTaq", "DNA", "polymerase", "(", "Perkin", "-", "Elmer", ")", ".", "PCR", "was", "performed", "using", "the", "following", "thermal", "profile", ":", "4", "min", "at", "94", "°", "C", ";", "1", "min", "at", "94", "°", "C", ",", "1", "min", "at", "60", "°", "C", ",", "1.5", "min", "at", "72", "°", "C", ",", "for", "30–40", "cycles", ";", "7", "min", "at", "72", "°", "C", ",", "and", "finally", "a", "soak", "at", "4", "°", "C", "overnight", ".", "The", "following", "day", ",", "10", "µl", "aliquots", "of", "the", "amplified", "products", "were", "run", "on", "a", "2", "%", "agarose", "Tris", "–", "acetate", "gel", "containing", "0.5", "mg", "/", "ml", "ethidium", "bromide", ".", "The", "products", "were", "visualized", "through", "a", "UV", "transilluminator", ",", "captured", "in", "a", "digital", "format", "using", "Quantify", "One", "Gel", "Analysis", "software", "(", "Bio", "-", "Rad", "Laboratories", ")", "on", "a", "Macintosh", "G4", "computer", ".", "The", "PCR", "primers", "specific", "to", "each", "transcript", "were", "as", "follows", ":", "GFAP", ",", "forward", "(", "F", ")", ",", "5′-TCATCGCTCAGGAGGTCCTT–3′", "Reverse", "(", "R", ")", ",", "5′-CTG", "TTGCCAGAGATGGAGGTT–3′", ";", "MAP2", "(", "F", ")", "5′-GAAGACTCGCATCCGAATGG–3′", ",", "(", "R", ")", "5′-CGCAGGATAGGAGGAAGAGACT–3′", ";", "MBP", "(", "F", ")", "5′-TTAGCTGAATTC", "GCGTGTGG–3′", ",", "(", "R", ")", "5′-GAGGAAGTGAATGAGCCGGTTA-3′", "were", "deigned", "using", "the", "Primer", "Designer", "software", ",", "Version", "2.0", "(", "Scientific", "and", "Educational", "Software", ")", "[", "48", "]", ".", "18S", ",", "β", "-", "tubulin", "class", "III", ",", "N", "-", "CAM", ",", "Nestin", ",", "NF", "-", "M", ",", "Notch-1", "primers", "[", "51", "]", ".", "Oct4", ",", "Nanog", "primers", "[", "11", "]", ".", "FOXa2", "(", "HNF3B", ")", ",", "Brachyury", "primers", "[", "52", "]", ".", "Behavioral", "testsThe", "cylinder", "test", "was", "used", "to", "assess", "the", "spontaneous", "forelimb", "use", "during", "lateral", "exploration", "movement", "[", "22", "]", ".", "Rats", "were", "placed", "in", "a", "transparent", "acrylic", "cylinder", "(", "20", "cm", "diameter", ")", "for", "5", "minutes", ".", "The", "cylinder", "encourages", "use", "of", "the", "forelimbs", "for", "vertical", "exploration", ".", "A", "mirror", "was", "placed", "behind", "the", "cylinder", "so", "that", "the", "forelimbs", "could", "be", "viewed", "at", "all", "times", ".", "Testing", "sessions", "were", "videotaped", "and", "forelimb", "use", "was", "scored", "by", "a", "blinded", "operator", ".", "Movements", "scored", "were", "the", "independent", "use", "of", "the", "left", "or", "right", "forelimb", "or", "simultaneous", "use", "of", "both", "the", "left", "and", "right", "forelimb", "to", "contact", "the", "wall", "of", "the", "cylinder", "during", "a", "full", "rear", ",", "to", "initiate", "a", "weight", "-", "shifting", "movement", ",", "or", "to", "regain", "center", "of", "gravity", "while", "moving", "laterally", "in", "a", "vertical", "posture", "along", "the", "wall", ".", "Animals", "were", "tested", "for", "their", "baselines", "after", "stroke", "and", "4", "and", "8", "weeks", "after", "cell", "transplantation", ".", "Statistical", "analysisOutcome", "measurement", "for", "each", "experiment", "was", "reported", "as", "mean±SEM", ".", "All", "data", "were", "analyzed", "using", "SPSS", "11", "for", "Mac", "OS", "X", "(", "SPSS", "Inc", ".", ")", ".", "Significance", "of", "inter", "-", "group", "differences", "was", "performed", "by", "applying", "Student", "'s", "t", "-", "test", "where", "appropriate", ".", "The", "One", "-", "Way", "ANOVA", "analysis", "was", "used", "to", "compare", "group", "differences", "for", "the", "forelimb", "use", "as", "the", "dependant", "variable", "and", "groups", "as", "the", "single", "independent", "factor", "variable", ".", "Differences", "between", "the", "groups", "were", "determined", "using", "Bonferroni", "'s", "post", "hoc", "test", ".", "A", "P", "-", "value", "of", "less", "than", "0.05", "was", "considered", "to", "be", "statistically", "significant", ".", "\n" ]
[ "CellType", "GeneProtein", "Species", "Anatomy", "CellComponent", "CellLine" ]
alkaline phosphatase is a GENE-N, perylene is a CHEMICAL
23144001_task0
Sentence: Real-time fluorescence turn-on detection of alkaline phosphatase activity with a novel perylene probe. Instructions: please extract entities and their types from the input sentence, all entity types are in options Options: GENE-N, CHEMICAL
[ "O", "O", "O", "O", "O", "O", "O", "O", "O", "B-GENE-N", "I-GENE-N", "O", "O", "O", "O", "B-CHEMICAL", "O", "O" ]
Real-time fluorescence turn-on detection of alkaline phosphatase activity with a novel perylene probe.
[ "Real", "-", "time", "fluorescence", "turn", "-", "on", "detection", "of", "alkaline", "phosphatase", "activity", "with", "a", "novel", "perylene", "probe", "." ]
[ "GENE-N", "CHEMICAL" ]
alkaline phosphatase is a GENE-N, perylene is a CHEMICAL
23144001_task1
Sentence: Real-time fluorescence turn-on detection of alkaline phosphatase activity with a novel perylene probe. Instructions: please typing these entity words according to sentence: alkaline phosphatase, perylene Options: GENE-N, CHEMICAL
[ "O", "O", "O", "O", "O", "O", "O", "O", "O", "B-GENE-N", "I-GENE-N", "O", "O", "O", "O", "B-CHEMICAL", "O", "O" ]
Real-time fluorescence turn-on detection of alkaline phosphatase activity with a novel perylene probe.
[ "Real", "-", "time", "fluorescence", "turn", "-", "on", "detection", "of", "alkaline", "phosphatase", "activity", "with", "a", "novel", "perylene", "probe", "." ]
[ "GENE-N", "CHEMICAL" ]
alkaline phosphatase, perylene
23144001_task2
Sentence: Real-time fluorescence turn-on detection of alkaline phosphatase activity with a novel perylene probe. Instructions: please extract entity words from the input sentence
[ "O", "O", "O", "O", "O", "O", "O", "O", "O", "B-GENE-N", "I-GENE-N", "O", "O", "O", "O", "B-CHEMICAL", "O", "O" ]
Real-time fluorescence turn-on detection of alkaline phosphatase activity with a novel perylene probe.
[ "Real", "-", "time", "fluorescence", "turn", "-", "on", "detection", "of", "alkaline", "phosphatase", "activity", "with", "a", "novel", "perylene", "probe", "." ]
[ "GENE-N", "CHEMICAL" ]
Hybridisierung is an umlsterm, Analyse is an umlsterm, Tumorgenoms is an umlsterm, Software is an umlsterm, farbkodierte is an umlsterm, Chromosomen is an umlsterm, Genotyp is an umlsterm, Phaenotyp is an umlsterm, Tumoren is an umlsterm
DerPathologe.60170342.ger.abstr_task0
Sentence: Die komparative genomische Hybridisierung ( " comparative genomic hybridization " , CGH ) ist ein molekularzytogenetisches Verfahren , welches die umfassende Analyse eines Tumorgenoms auf Ueber- und Unterrepraesentationen von DNA-Abschnitten ermoeglicht . Um quantitative und reproduzierbare Aussagen ueber die genetischen Aberrationen machen zu koennen , wurde eine Software entwickelt , welche die objektive Erfassung von Ort und Art der Veraenderungen ermoeglicht . Das Ergebnis wird in Form eines CGH-Summenkaryogramms dargestellt , welches die genetischen Veraenderungen als farbkodierte Chromosomen dokumentiert . Ziel dieser Untersuchungen ist es , auf der Grundlage von Summen-Karyogrammen eine Korrelation zwischen Genotyp und Phaenotyp herzustellen und damit eine genetische Charakterisierung von Tumoren zu erreichen , die die morphologische Beschreibung ergaenzt . Instructions: please extract entities and their types from the input sentence, all entity types are in options Options: umlsterm
[ "O", "O", "O", "B-umlsterm", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "B-umlsterm", "O", "B-umlsterm", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "B-umlsterm", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "B-umlsterm", "B-umlsterm", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "B-umlsterm", "O", "B-umlsterm", "O", "O", "O", "O", "O", "O", "O", "B-umlsterm", "O", "O", "O", "O", "O", "O", "O", "O", "O" ]
Die komparative genomische Hybridisierung ( " comparative genomic hybridization " , CGH ) ist ein molekularzytogenetisches Verfahren , welches die umfassende Analyse eines Tumorgenoms auf Ueber- und Unterrepraesentationen von DNA-Abschnitten ermoeglicht . Um quantitative und reproduzierbare Aussagen ueber die genetischen Aberrationen machen zu koennen , wurde eine Software entwickelt , welche die objektive Erfassung von Ort und Art der Veraenderungen ermoeglicht . Das Ergebnis wird in Form eines CGH-Summenkaryogramms dargestellt , welches die genetischen Veraenderungen als farbkodierte Chromosomen dokumentiert . Ziel dieser Untersuchungen ist es , auf der Grundlage von Summen-Karyogrammen eine Korrelation zwischen Genotyp und Phaenotyp herzustellen und damit eine genetische Charakterisierung von Tumoren zu erreichen , die die morphologische Beschreibung ergaenzt .
[ "Die", "komparative", "genomische", "Hybridisierung", "(", "\"", "comparative", "genomic", "hybridization", "\"", ",", "CGH", ")", "ist", "ein", "molekularzytogenetisches", "Verfahren", ",", "welches", "die", "umfassende", "Analyse", "eines", "Tumorgenoms", "auf", "Ueber-", "und", "Unterrepraesentationen", "von", "DNA", "-", "Abschnitten", "ermoeglicht", ".", "Um", "quantitative", "und", "reproduzierbare", "Aussagen", "ueber", "die", "genetischen", "Aberrationen", "machen", "zu", "koennen", ",", "wurde", "eine", "Software", "entwickelt", ",", "welche", "die", "objektive", "Erfassung", "von", "Ort", "und", "Art", "der", "Veraenderungen", "ermoeglicht", ".", "Das", "Ergebnis", "wird", "in", "Form", "eines", "CGH", "-", "Summenkaryogramms", "dargestellt", ",", "welches", "die", "genetischen", "Veraenderungen", "als", "farbkodierte", "Chromosomen", "dokumentiert", ".", "Ziel", "dieser", "Untersuchungen", "ist", "es", ",", "auf", "der", "Grundlage", "von", "Summen", "-", "Karyogrammen", "eine", "Korrelation", "zwischen", "Genotyp", "und", "Phaenotyp", "herzustellen", "und", "damit", "eine", "genetische", "Charakterisierung", "von", "Tumoren", "zu", "erreichen", ",", "die", "die", "morphologische", "Beschreibung", "ergaenzt", "." ]
[ "umlsterm" ]
Hybridisierung is an umlsterm, Analyse is an umlsterm, Tumorgenoms is an umlsterm, Software is an umlsterm, farbkodierte is an umlsterm, Chromosomen is an umlsterm, Genotyp is an umlsterm, Phaenotyp is an umlsterm, Tumoren is an umlsterm
DerPathologe.60170342.ger.abstr_task1
Sentence: Die komparative genomische Hybridisierung ( " comparative genomic hybridization " , CGH ) ist ein molekularzytogenetisches Verfahren , welches die umfassende Analyse eines Tumorgenoms auf Ueber- und Unterrepraesentationen von DNA-Abschnitten ermoeglicht . Um quantitative und reproduzierbare Aussagen ueber die genetischen Aberrationen machen zu koennen , wurde eine Software entwickelt , welche die objektive Erfassung von Ort und Art der Veraenderungen ermoeglicht . Das Ergebnis wird in Form eines CGH-Summenkaryogramms dargestellt , welches die genetischen Veraenderungen als farbkodierte Chromosomen dokumentiert . Ziel dieser Untersuchungen ist es , auf der Grundlage von Summen-Karyogrammen eine Korrelation zwischen Genotyp und Phaenotyp herzustellen und damit eine genetische Charakterisierung von Tumoren zu erreichen , die die morphologische Beschreibung ergaenzt . Instructions: please typing these entity words according to sentence: Hybridisierung, Analyse, Tumorgenoms, Software, farbkodierte, Chromosomen, Genotyp, Phaenotyp, Tumoren Options: umlsterm
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Die komparative genomische Hybridisierung ( " comparative genomic hybridization " , CGH ) ist ein molekularzytogenetisches Verfahren , welches die umfassende Analyse eines Tumorgenoms auf Ueber- und Unterrepraesentationen von DNA-Abschnitten ermoeglicht . Um quantitative und reproduzierbare Aussagen ueber die genetischen Aberrationen machen zu koennen , wurde eine Software entwickelt , welche die objektive Erfassung von Ort und Art der Veraenderungen ermoeglicht . Das Ergebnis wird in Form eines CGH-Summenkaryogramms dargestellt , welches die genetischen Veraenderungen als farbkodierte Chromosomen dokumentiert . Ziel dieser Untersuchungen ist es , auf der Grundlage von Summen-Karyogrammen eine Korrelation zwischen Genotyp und Phaenotyp herzustellen und damit eine genetische Charakterisierung von Tumoren zu erreichen , die die morphologische Beschreibung ergaenzt .
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[ "umlsterm" ]
Hybridisierung, Analyse, Tumorgenoms, Software, farbkodierte, Chromosomen, Genotyp, Phaenotyp, Tumoren
DerPathologe.60170342.ger.abstr_task2
Sentence: Die komparative genomische Hybridisierung ( " comparative genomic hybridization " , CGH ) ist ein molekularzytogenetisches Verfahren , welches die umfassende Analyse eines Tumorgenoms auf Ueber- und Unterrepraesentationen von DNA-Abschnitten ermoeglicht . Um quantitative und reproduzierbare Aussagen ueber die genetischen Aberrationen machen zu koennen , wurde eine Software entwickelt , welche die objektive Erfassung von Ort und Art der Veraenderungen ermoeglicht . Das Ergebnis wird in Form eines CGH-Summenkaryogramms dargestellt , welches die genetischen Veraenderungen als farbkodierte Chromosomen dokumentiert . Ziel dieser Untersuchungen ist es , auf der Grundlage von Summen-Karyogrammen eine Korrelation zwischen Genotyp und Phaenotyp herzustellen und damit eine genetische Charakterisierung von Tumoren zu erreichen , die die morphologische Beschreibung ergaenzt . Instructions: please extract entity words from the input sentence
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Die komparative genomische Hybridisierung ( " comparative genomic hybridization " , CGH ) ist ein molekularzytogenetisches Verfahren , welches die umfassende Analyse eines Tumorgenoms auf Ueber- und Unterrepraesentationen von DNA-Abschnitten ermoeglicht . Um quantitative und reproduzierbare Aussagen ueber die genetischen Aberrationen machen zu koennen , wurde eine Software entwickelt , welche die objektive Erfassung von Ort und Art der Veraenderungen ermoeglicht . Das Ergebnis wird in Form eines CGH-Summenkaryogramms dargestellt , welches die genetischen Veraenderungen als farbkodierte Chromosomen dokumentiert . Ziel dieser Untersuchungen ist es , auf der Grundlage von Summen-Karyogrammen eine Korrelation zwischen Genotyp und Phaenotyp herzustellen und damit eine genetische Charakterisierung von Tumoren zu erreichen , die die morphologische Beschreibung ergaenzt .
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[ "umlsterm" ]
synthetic appeasing pheromone is a Intervention_Pharmacological, synthetic , dog - appeasing pheromone ( sDAP ) is a Intervention_Pharmacological, 46 dogs is a Participant_Sample-size, sDAP solution is a Intervention_Physical, sham treated with the carrier is a Intervention_Control, sham treatment is a Intervention_Control, behavioral , neuroendocrine , immune , and acute - phase responses is a Outcome_Mental, Behavioral response variables is a Outcome_Mental, serum prolactin concentration is a Outcome_Physical, alertness and visual exploration behaviors is a Outcome_Mental, serum prolactin concentrations is a Outcome_Physical, behavioral and neuroendocrine perioperative stress responses is a Outcome_Mental, recovery is a Outcome_Other
47468_task0
Sentence: Effect of a synthetic appeasing pheromone on behavioral , neuroendocrine , immune , and acute-phase perioperative stress responses in dogs . OBJECTIVE To study the effects of a synthetic , dog-appeasing pheromone ( sDAP ) on the behavioral , neuroendocrine , immune , and acute-phase perioperative stress responses in dogs undergoing elective orchiectomy or ovariohysterectomy . DESIGN Randomized , controlled clinical trial . ANIMALS 46 dogs housed in animal shelters and undergoing elective orchiectomy or ovariohysterectomy . PROCEDURES Intensive care unit cages were sprayed with sDAP solution or sham treated with the carrier used in the solution 20 minutes prior to use . Dogs ( n = 24 and 22 in the sDAP and sham treatment exposure groups , respectively ) were placed in treated cages for 30 minutes before and after surgery . Indicators of stress ( ie , alterations in behavioral , neuroendocrine , immune , and acute-phase responses ) were evaluated perioperatively . Behavioral response variables , salivary cortisol concentration , WBC count , and serum concentrations of glucose , prolactin , haptoglobin , and C-reactive protein were analyzed . RESULTS Behavioral response variables and serum prolactin concentration were influenced by sDAP exposure . Dogs exposed to sDAP were more likely to have alertness and visual exploration behaviors after surgery than were dogs exposed to sham treatment . Decreases in serum prolactin concentrations in response to perioperative stress were significantly smaller in dogs exposed to sDAP , compared with findings in dogs exposed to the sham treatment . Variables examined to evaluate the hypothalamic-pituitary-adrenal axis , immune system , and acute-phase responses were unaffected by treatment . CONCLUSIONS AND CLINICAL RELEVANCE sDAP appeared to affect behavioral and neuroendocrine perioperative stress responses by modification of lactotropic axis activity . Use of sDAP in a clinical setting may improve the recovery and welfare of dogs undergoing surgery . Instructions: please extract entities and their types from the input sentence, all entity types are in options Options: Intervention_Pharmacological, Intervention_Physical, Intervention_Control, Outcome_Physical, Participant_Sample-size, Outcome_Other, Outcome_Mental
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Effect of a synthetic appeasing pheromone on behavioral , neuroendocrine , immune , and acute-phase perioperative stress responses in dogs . OBJECTIVE To study the effects of a synthetic , dog-appeasing pheromone ( sDAP ) on the behavioral , neuroendocrine , immune , and acute-phase perioperative stress responses in dogs undergoing elective orchiectomy or ovariohysterectomy . DESIGN Randomized , controlled clinical trial . ANIMALS 46 dogs housed in animal shelters and undergoing elective orchiectomy or ovariohysterectomy . PROCEDURES Intensive care unit cages were sprayed with sDAP solution or sham treated with the carrier used in the solution 20 minutes prior to use . Dogs ( n = 24 and 22 in the sDAP and sham treatment exposure groups , respectively ) were placed in treated cages for 30 minutes before and after surgery . Indicators of stress ( ie , alterations in behavioral , neuroendocrine , immune , and acute-phase responses ) were evaluated perioperatively . Behavioral response variables , salivary cortisol concentration , WBC count , and serum concentrations of glucose , prolactin , haptoglobin , and C-reactive protein were analyzed . RESULTS Behavioral response variables and serum prolactin concentration were influenced by sDAP exposure . Dogs exposed to sDAP were more likely to have alertness and visual exploration behaviors after surgery than were dogs exposed to sham treatment . Decreases in serum prolactin concentrations in response to perioperative stress were significantly smaller in dogs exposed to sDAP , compared with findings in dogs exposed to the sham treatment . Variables examined to evaluate the hypothalamic-pituitary-adrenal axis , immune system , and acute-phase responses were unaffected by treatment . CONCLUSIONS AND CLINICAL RELEVANCE sDAP appeared to affect behavioral and neuroendocrine perioperative stress responses by modification of lactotropic axis activity . Use of sDAP in a clinical setting may improve the recovery and welfare of dogs undergoing surgery .
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[ "Outcome_Mental", "Intervention_Pharmacological", "Outcome_Physical", "Intervention_Control", "Intervention_Physical", "Outcome_Other", "Participant_Sample-size" ]
synthetic appeasing pheromone is a Intervention_Pharmacological, synthetic , dog - appeasing pheromone ( sDAP ) is a Intervention_Pharmacological, 46 dogs is a Participant_Sample-size, sDAP solution is a Intervention_Physical, sham treated with the carrier is a Intervention_Control, sham treatment is a Intervention_Control, behavioral , neuroendocrine , immune , and acute - phase responses is a Outcome_Mental, Behavioral response variables is a Outcome_Mental, serum prolactin concentration is a Outcome_Physical, alertness and visual exploration behaviors is a Outcome_Mental, serum prolactin concentrations is a Outcome_Physical, behavioral and neuroendocrine perioperative stress responses is a Outcome_Mental, recovery is a Outcome_Other
47468_task1
Sentence: Effect of a synthetic appeasing pheromone on behavioral , neuroendocrine , immune , and acute-phase perioperative stress responses in dogs . OBJECTIVE To study the effects of a synthetic , dog-appeasing pheromone ( sDAP ) on the behavioral , neuroendocrine , immune , and acute-phase perioperative stress responses in dogs undergoing elective orchiectomy or ovariohysterectomy . DESIGN Randomized , controlled clinical trial . ANIMALS 46 dogs housed in animal shelters and undergoing elective orchiectomy or ovariohysterectomy . PROCEDURES Intensive care unit cages were sprayed with sDAP solution or sham treated with the carrier used in the solution 20 minutes prior to use . Dogs ( n = 24 and 22 in the sDAP and sham treatment exposure groups , respectively ) were placed in treated cages for 30 minutes before and after surgery . Indicators of stress ( ie , alterations in behavioral , neuroendocrine , immune , and acute-phase responses ) were evaluated perioperatively . Behavioral response variables , salivary cortisol concentration , WBC count , and serum concentrations of glucose , prolactin , haptoglobin , and C-reactive protein were analyzed . RESULTS Behavioral response variables and serum prolactin concentration were influenced by sDAP exposure . Dogs exposed to sDAP were more likely to have alertness and visual exploration behaviors after surgery than were dogs exposed to sham treatment . Decreases in serum prolactin concentrations in response to perioperative stress were significantly smaller in dogs exposed to sDAP , compared with findings in dogs exposed to the sham treatment . Variables examined to evaluate the hypothalamic-pituitary-adrenal axis , immune system , and acute-phase responses were unaffected by treatment . CONCLUSIONS AND CLINICAL RELEVANCE sDAP appeared to affect behavioral and neuroendocrine perioperative stress responses by modification of lactotropic axis activity . Use of sDAP in a clinical setting may improve the recovery and welfare of dogs undergoing surgery . Instructions: please typing these entity words according to sentence: synthetic appeasing pheromone, synthetic , dog - appeasing pheromone ( sDAP ), 46 dogs, sDAP solution, sham treated with the carrier, sham treatment, behavioral , neuroendocrine , immune , and acute - phase responses, Behavioral response variables, serum prolactin concentration, alertness and visual exploration behaviors, serum prolactin concentrations, behavioral and neuroendocrine perioperative stress responses, recovery Options: Intervention_Pharmacological, Intervention_Physical, Intervention_Control, Outcome_Physical, Participant_Sample-size, Outcome_Other, Outcome_Mental
[ "O", "O", "O", "B-Intervention_Pharmacological", "I-Intervention_Pharmacological", "I-Intervention_Pharmacological", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "B-Intervention_Pharmacological", "I-Intervention_Pharmacological", "I-Intervention_Pharmacological", "I-Intervention_Pharmacological", "I-Intervention_Pharmacological", "I-Intervention_Pharmacological", "I-Intervention_Pharmacological", "I-Intervention_Pharmacological", "I-Intervention_Pharmacological", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "B-Participant_Sample-size", "I-Participant_Sample-size", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "B-Intervention_Physical", "I-Intervention_Physical", "O", "B-Intervention_Control", "I-Intervention_Control", "I-Intervention_Control", "I-Intervention_Control", "I-Intervention_Control", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "B-Intervention_Control", "I-Intervention_Control", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "B-Outcome_Mental", "I-Outcome_Mental", "I-Outcome_Mental", "I-Outcome_Mental", "I-Outcome_Mental", "I-Outcome_Mental", "I-Outcome_Mental", "I-Outcome_Mental", "I-Outcome_Mental", "I-Outcome_Mental", "I-Outcome_Mental", "O", "O", "O", "O", "O", "B-Outcome_Mental", "I-Outcome_Mental", "I-Outcome_Mental", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "B-Outcome_Physical", "I-Outcome_Physical", "I-Outcome_Physical", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "B-Outcome_Mental", "I-Outcome_Mental", "I-Outcome_Mental", "I-Outcome_Mental", "I-Outcome_Mental", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "B-Outcome_Physical", "I-Outcome_Physical", "I-Outcome_Physical", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "B-Outcome_Mental", "I-Outcome_Mental", "I-Outcome_Mental", "I-Outcome_Mental", "I-Outcome_Mental", "I-Outcome_Mental", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "B-Outcome_Other", "O", "O", "O", "O", "O", "O", "O" ]
Effect of a synthetic appeasing pheromone on behavioral , neuroendocrine , immune , and acute-phase perioperative stress responses in dogs . OBJECTIVE To study the effects of a synthetic , dog-appeasing pheromone ( sDAP ) on the behavioral , neuroendocrine , immune , and acute-phase perioperative stress responses in dogs undergoing elective orchiectomy or ovariohysterectomy . DESIGN Randomized , controlled clinical trial . ANIMALS 46 dogs housed in animal shelters and undergoing elective orchiectomy or ovariohysterectomy . PROCEDURES Intensive care unit cages were sprayed with sDAP solution or sham treated with the carrier used in the solution 20 minutes prior to use . Dogs ( n = 24 and 22 in the sDAP and sham treatment exposure groups , respectively ) were placed in treated cages for 30 minutes before and after surgery . Indicators of stress ( ie , alterations in behavioral , neuroendocrine , immune , and acute-phase responses ) were evaluated perioperatively . Behavioral response variables , salivary cortisol concentration , WBC count , and serum concentrations of glucose , prolactin , haptoglobin , and C-reactive protein were analyzed . RESULTS Behavioral response variables and serum prolactin concentration were influenced by sDAP exposure . Dogs exposed to sDAP were more likely to have alertness and visual exploration behaviors after surgery than were dogs exposed to sham treatment . Decreases in serum prolactin concentrations in response to perioperative stress were significantly smaller in dogs exposed to sDAP , compared with findings in dogs exposed to the sham treatment . Variables examined to evaluate the hypothalamic-pituitary-adrenal axis , immune system , and acute-phase responses were unaffected by treatment . CONCLUSIONS AND CLINICAL RELEVANCE sDAP appeared to affect behavioral and neuroendocrine perioperative stress responses by modification of lactotropic axis activity . Use of sDAP in a clinical setting may improve the recovery and welfare of dogs undergoing surgery .
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[ "Outcome_Mental", "Intervention_Pharmacological", "Outcome_Physical", "Intervention_Control", "Intervention_Physical", "Outcome_Other", "Participant_Sample-size" ]
synthetic appeasing pheromone, synthetic , dog - appeasing pheromone ( sDAP ), 46 dogs, sDAP solution, sham treated with the carrier, sham treatment, behavioral , neuroendocrine , immune , and acute - phase responses, Behavioral response variables, serum prolactin concentration, alertness and visual exploration behaviors, serum prolactin concentrations, behavioral and neuroendocrine perioperative stress responses, recovery
47468_task2
Sentence: Effect of a synthetic appeasing pheromone on behavioral , neuroendocrine , immune , and acute-phase perioperative stress responses in dogs . OBJECTIVE To study the effects of a synthetic , dog-appeasing pheromone ( sDAP ) on the behavioral , neuroendocrine , immune , and acute-phase perioperative stress responses in dogs undergoing elective orchiectomy or ovariohysterectomy . DESIGN Randomized , controlled clinical trial . ANIMALS 46 dogs housed in animal shelters and undergoing elective orchiectomy or ovariohysterectomy . PROCEDURES Intensive care unit cages were sprayed with sDAP solution or sham treated with the carrier used in the solution 20 minutes prior to use . Dogs ( n = 24 and 22 in the sDAP and sham treatment exposure groups , respectively ) were placed in treated cages for 30 minutes before and after surgery . Indicators of stress ( ie , alterations in behavioral , neuroendocrine , immune , and acute-phase responses ) were evaluated perioperatively . Behavioral response variables , salivary cortisol concentration , WBC count , and serum concentrations of glucose , prolactin , haptoglobin , and C-reactive protein were analyzed . RESULTS Behavioral response variables and serum prolactin concentration were influenced by sDAP exposure . Dogs exposed to sDAP were more likely to have alertness and visual exploration behaviors after surgery than were dogs exposed to sham treatment . Decreases in serum prolactin concentrations in response to perioperative stress were significantly smaller in dogs exposed to sDAP , compared with findings in dogs exposed to the sham treatment . Variables examined to evaluate the hypothalamic-pituitary-adrenal axis , immune system , and acute-phase responses were unaffected by treatment . CONCLUSIONS AND CLINICAL RELEVANCE sDAP appeared to affect behavioral and neuroendocrine perioperative stress responses by modification of lactotropic axis activity . Use of sDAP in a clinical setting may improve the recovery and welfare of dogs undergoing surgery . Instructions: please extract entity words from the input sentence
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Effect of a synthetic appeasing pheromone on behavioral , neuroendocrine , immune , and acute-phase perioperative stress responses in dogs . OBJECTIVE To study the effects of a synthetic , dog-appeasing pheromone ( sDAP ) on the behavioral , neuroendocrine , immune , and acute-phase perioperative stress responses in dogs undergoing elective orchiectomy or ovariohysterectomy . DESIGN Randomized , controlled clinical trial . ANIMALS 46 dogs housed in animal shelters and undergoing elective orchiectomy or ovariohysterectomy . PROCEDURES Intensive care unit cages were sprayed with sDAP solution or sham treated with the carrier used in the solution 20 minutes prior to use . Dogs ( n = 24 and 22 in the sDAP and sham treatment exposure groups , respectively ) were placed in treated cages for 30 minutes before and after surgery . Indicators of stress ( ie , alterations in behavioral , neuroendocrine , immune , and acute-phase responses ) were evaluated perioperatively . Behavioral response variables , salivary cortisol concentration , WBC count , and serum concentrations of glucose , prolactin , haptoglobin , and C-reactive protein were analyzed . RESULTS Behavioral response variables and serum prolactin concentration were influenced by sDAP exposure . Dogs exposed to sDAP were more likely to have alertness and visual exploration behaviors after surgery than were dogs exposed to sham treatment . Decreases in serum prolactin concentrations in response to perioperative stress were significantly smaller in dogs exposed to sDAP , compared with findings in dogs exposed to the sham treatment . Variables examined to evaluate the hypothalamic-pituitary-adrenal axis , immune system , and acute-phase responses were unaffected by treatment . CONCLUSIONS AND CLINICAL RELEVANCE sDAP appeared to affect behavioral and neuroendocrine perioperative stress responses by modification of lactotropic axis activity . Use of sDAP in a clinical setting may improve the recovery and welfare of dogs undergoing surgery .
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[ "Outcome_Mental", "Intervention_Pharmacological", "Outcome_Physical", "Intervention_Control", "Intervention_Physical", "Outcome_Other", "Participant_Sample-size" ]
Linsen is an umlsterm
DerOpthalmologe.70940678.ger.abstr_task0
Sentence: Fragestellung : Lassen sich die Wundstabilitaet und Astigmatismusinduktion einer linearen kornealen Tunnelinzision zur Implantation faltbarer Linsen durch Radialisierung einer Schnittkomponente verbessern ? Instructions: please extract entities and their types from the input sentence, all entity types are in options Options: umlsterm
[ "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "B-umlsterm", "O", "O", "O", "O", "O", "O" ]
Fragestellung : Lassen sich die Wundstabilitaet und Astigmatismusinduktion einer linearen kornealen Tunnelinzision zur Implantation faltbarer Linsen durch Radialisierung einer Schnittkomponente verbessern ?
[ "Fragestellung", ":", "Lassen", "sich", "die", "Wundstabilitaet", "und", "Astigmatismusinduktion", "einer", "linearen", "kornealen", "Tunnelinzision", "zur", "Implantation", "faltbarer", "Linsen", "durch", "Radialisierung", "einer", "Schnittkomponente", "verbessern", "?" ]
[ "umlsterm" ]
Linsen is an umlsterm
DerOpthalmologe.70940678.ger.abstr_task1
Sentence: Fragestellung : Lassen sich die Wundstabilitaet und Astigmatismusinduktion einer linearen kornealen Tunnelinzision zur Implantation faltbarer Linsen durch Radialisierung einer Schnittkomponente verbessern ? Instructions: please typing these entity words according to sentence: Linsen Options: umlsterm
[ "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "B-umlsterm", "O", "O", "O", "O", "O", "O" ]
Fragestellung : Lassen sich die Wundstabilitaet und Astigmatismusinduktion einer linearen kornealen Tunnelinzision zur Implantation faltbarer Linsen durch Radialisierung einer Schnittkomponente verbessern ?
[ "Fragestellung", ":", "Lassen", "sich", "die", "Wundstabilitaet", "und", "Astigmatismusinduktion", "einer", "linearen", "kornealen", "Tunnelinzision", "zur", "Implantation", "faltbarer", "Linsen", "durch", "Radialisierung", "einer", "Schnittkomponente", "verbessern", "?" ]
[ "umlsterm" ]
Linsen
DerOpthalmologe.70940678.ger.abstr_task2
Sentence: Fragestellung : Lassen sich die Wundstabilitaet und Astigmatismusinduktion einer linearen kornealen Tunnelinzision zur Implantation faltbarer Linsen durch Radialisierung einer Schnittkomponente verbessern ? Instructions: please extract entity words from the input sentence
[ "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "B-umlsterm", "O", "O", "O", "O", "O", "O" ]
Fragestellung : Lassen sich die Wundstabilitaet und Astigmatismusinduktion einer linearen kornealen Tunnelinzision zur Implantation faltbarer Linsen durch Radialisierung einer Schnittkomponente verbessern ?
[ "Fragestellung", ":", "Lassen", "sich", "die", "Wundstabilitaet", "und", "Astigmatismusinduktion", "einer", "linearen", "kornealen", "Tunnelinzision", "zur", "Implantation", "faltbarer", "Linsen", "durch", "Radialisierung", "einer", "Schnittkomponente", "verbessern", "?" ]
[ "umlsterm" ]
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HNO.00480583.eng.abstr_task0
Sentence: Fine needle aspiration biopsy ( FNAB ) under ultrasound control is an established diagnostic procedure for the head and neck region . Because of the disintegration of tissues , the diagnostic value of the method is limited resulting in only moderate specificity . In a prospective study , we performed a new , semi-automatic biopsy method in patients who had been diagnosed with sonographically confirmed pathologic masses in the head-neck region . This biopsy is carried out with a spring-loaded biopsy pistol which uses a disposable 20-gauge , specially designed cutting needle . Because this method combines the low invasiveness of FNAB with the high specificity of an excisional biopsy , a high tissue quality is obtained . Comparing these bioptic results with those of subsequent excisional biopsies proves that this new method yields a sensitivity of close to 100% for the detection of lymph node metastases of squamous cell carcinomas ( SCC ) . The tissue cylinders have a reproducible size and allow ultrastructural investigations in the transmission electron microscope ( TEM ) on the ultrastructural level . Due to the excellent tissue preservation in the biopsy cylinders , ultrastructural studies , using transmission electron microscopy , may be carried out with the biopsy material . Furthermore , following paraffin embedding of biopsy cylinders , serial sections may be obtained for special staining techniques , and immunohistological investigations are possible which may serve as an adjunct in the diagnosis of , e.g. , lymphoproliferative lesions with a sensitivity of 96% . Summarizing , the new semi-automatic biopsy technique obtains tissue probes of high quality with low invasiveness which enables highly sensitive diagnosis of head and neck lesions . Instructions: please extract entities and their types from the input sentence, all entity types are in options Options: umlsterm
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Fine needle aspiration biopsy ( FNAB ) under ultrasound control is an established diagnostic procedure for the head and neck region . Because of the disintegration of tissues , the diagnostic value of the method is limited resulting in only moderate specificity . In a prospective study , we performed a new , semi-automatic biopsy method in patients who had been diagnosed with sonographically confirmed pathologic masses in the head-neck region . This biopsy is carried out with a spring-loaded biopsy pistol which uses a disposable 20-gauge , specially designed cutting needle . Because this method combines the low invasiveness of FNAB with the high specificity of an excisional biopsy , a high tissue quality is obtained . Comparing these bioptic results with those of subsequent excisional biopsies proves that this new method yields a sensitivity of close to 100% for the detection of lymph node metastases of squamous cell carcinomas ( SCC ) . The tissue cylinders have a reproducible size and allow ultrastructural investigations in the transmission electron microscope ( TEM ) on the ultrastructural level . Due to the excellent tissue preservation in the biopsy cylinders , ultrastructural studies , using transmission electron microscopy , may be carried out with the biopsy material . Furthermore , following paraffin embedding of biopsy cylinders , serial sections may be obtained for special staining techniques , and immunohistological investigations are possible which may serve as an adjunct in the diagnosis of , e.g. , lymphoproliferative lesions with a sensitivity of 96% . Summarizing , the new semi-automatic biopsy technique obtains tissue probes of high quality with low invasiveness which enables highly sensitive diagnosis of head and neck lesions .
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[ "umlsterm" ]
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HNO.00480583.eng.abstr_task1
Sentence: Fine needle aspiration biopsy ( FNAB ) under ultrasound control is an established diagnostic procedure for the head and neck region . Because of the disintegration of tissues , the diagnostic value of the method is limited resulting in only moderate specificity . In a prospective study , we performed a new , semi-automatic biopsy method in patients who had been diagnosed with sonographically confirmed pathologic masses in the head-neck region . This biopsy is carried out with a spring-loaded biopsy pistol which uses a disposable 20-gauge , specially designed cutting needle . Because this method combines the low invasiveness of FNAB with the high specificity of an excisional biopsy , a high tissue quality is obtained . Comparing these bioptic results with those of subsequent excisional biopsies proves that this new method yields a sensitivity of close to 100% for the detection of lymph node metastases of squamous cell carcinomas ( SCC ) . The tissue cylinders have a reproducible size and allow ultrastructural investigations in the transmission electron microscope ( TEM ) on the ultrastructural level . Due to the excellent tissue preservation in the biopsy cylinders , ultrastructural studies , using transmission electron microscopy , may be carried out with the biopsy material . Furthermore , following paraffin embedding of biopsy cylinders , serial sections may be obtained for special staining techniques , and immunohistological investigations are possible which may serve as an adjunct in the diagnosis of , e.g. , lymphoproliferative lesions with a sensitivity of 96% . Summarizing , the new semi-automatic biopsy technique obtains tissue probes of high quality with low invasiveness which enables highly sensitive diagnosis of head and neck lesions . Instructions: please typing these entity words according to sentence: needle, aspiration biopsy, ultrasound, control, diagnostic, procedure, head, neck, tissues, diagnostic, value, method, specificity, prospective study, biopsy, method, patients, pathologic, head - neck, biopsy, biopsy, needle, method, specificity, biopsy, tissue, biopsies, method, sensitivity, lymph node, metastases, squamous cell carcinomas, SCC, tissue, transmission, electron, TEM, tissue preservation, biopsy, transmission electron microscopy, biopsy, paraffin embedding, biopsy, serial, techniques, diagnosis, sensitivity, biopsy, technique, tissue, probes, diagnosis, head, neck Options: umlsterm
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Fine needle aspiration biopsy ( FNAB ) under ultrasound control is an established diagnostic procedure for the head and neck region . Because of the disintegration of tissues , the diagnostic value of the method is limited resulting in only moderate specificity . In a prospective study , we performed a new , semi-automatic biopsy method in patients who had been diagnosed with sonographically confirmed pathologic masses in the head-neck region . This biopsy is carried out with a spring-loaded biopsy pistol which uses a disposable 20-gauge , specially designed cutting needle . Because this method combines the low invasiveness of FNAB with the high specificity of an excisional biopsy , a high tissue quality is obtained . Comparing these bioptic results with those of subsequent excisional biopsies proves that this new method yields a sensitivity of close to 100% for the detection of lymph node metastases of squamous cell carcinomas ( SCC ) . The tissue cylinders have a reproducible size and allow ultrastructural investigations in the transmission electron microscope ( TEM ) on the ultrastructural level . Due to the excellent tissue preservation in the biopsy cylinders , ultrastructural studies , using transmission electron microscopy , may be carried out with the biopsy material . Furthermore , following paraffin embedding of biopsy cylinders , serial sections may be obtained for special staining techniques , and immunohistological investigations are possible which may serve as an adjunct in the diagnosis of , e.g. , lymphoproliferative lesions with a sensitivity of 96% . Summarizing , the new semi-automatic biopsy technique obtains tissue probes of high quality with low invasiveness which enables highly sensitive diagnosis of head and neck lesions .
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[ "umlsterm" ]
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HNO.00480583.eng.abstr_task2
Sentence: Fine needle aspiration biopsy ( FNAB ) under ultrasound control is an established diagnostic procedure for the head and neck region . Because of the disintegration of tissues , the diagnostic value of the method is limited resulting in only moderate specificity . In a prospective study , we performed a new , semi-automatic biopsy method in patients who had been diagnosed with sonographically confirmed pathologic masses in the head-neck region . This biopsy is carried out with a spring-loaded biopsy pistol which uses a disposable 20-gauge , specially designed cutting needle . Because this method combines the low invasiveness of FNAB with the high specificity of an excisional biopsy , a high tissue quality is obtained . Comparing these bioptic results with those of subsequent excisional biopsies proves that this new method yields a sensitivity of close to 100% for the detection of lymph node metastases of squamous cell carcinomas ( SCC ) . The tissue cylinders have a reproducible size and allow ultrastructural investigations in the transmission electron microscope ( TEM ) on the ultrastructural level . Due to the excellent tissue preservation in the biopsy cylinders , ultrastructural studies , using transmission electron microscopy , may be carried out with the biopsy material . Furthermore , following paraffin embedding of biopsy cylinders , serial sections may be obtained for special staining techniques , and immunohistological investigations are possible which may serve as an adjunct in the diagnosis of , e.g. , lymphoproliferative lesions with a sensitivity of 96% . Summarizing , the new semi-automatic biopsy technique obtains tissue probes of high quality with low invasiveness which enables highly sensitive diagnosis of head and neck lesions . Instructions: please extract entity words from the input sentence
[ "O", "B-umlsterm", "B-umlsterm", "I-umlsterm", "O", "O", "O", "O", "B-umlsterm", "B-umlsterm", "O", "O", "O", "B-umlsterm", "B-umlsterm", "O", "O", "B-umlsterm", "O", "B-umlsterm", "O", "O", "O", "O", "O", "O", "O", "B-umlsterm", "O", "O", "B-umlsterm", "B-umlsterm", "O", "O", "B-umlsterm", "O", "O", "O", "O", "O", "O", "B-umlsterm", "O", "O", "O", "B-umlsterm", "I-umlsterm", "O", "O", "O", "O", "O", "O", "O", "O", "O", "B-umlsterm", "B-umlsterm", "O", "B-umlsterm", "O", "O", "O", "O", "O", "O", "O", "B-umlsterm", "O", "O", "O", "B-umlsterm", "I-umlsterm", "I-umlsterm", "O", "O", "O", "B-umlsterm", "O", "O", "O", "O", "O", "O", "O", "O", "B-umlsterm", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "B-umlsterm", "O", "O", "O", "B-umlsterm", "O", "O", "O", "O", "O", "O", "O", "O", "O", "B-umlsterm", "O", "O", "O", "B-umlsterm", "O", "O", "O", "B-umlsterm", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "B-umlsterm", "O", "O", "O", "O", "B-umlsterm", "O", "O", "B-umlsterm", "O", "O", "O", "O", "O", "O", "O", "O", "O", "B-umlsterm", "I-umlsterm", "B-umlsterm", "O", "B-umlsterm", "I-umlsterm", "I-umlsterm", "O", "B-umlsterm", "O", "O", "O", "B-umlsterm", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "B-umlsterm", "B-umlsterm", "O", "O", "B-umlsterm", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "B-umlsterm", "I-umlsterm", "O", "O", "B-umlsterm", "O", "O", "O", "O", "O", "O", "B-umlsterm", "I-umlsterm", "I-umlsterm", "O", "O", "O", "O", "O", "O", "O", "B-umlsterm", "O", "O", "O", "O", "O", "B-umlsterm", "I-umlsterm", "O", "B-umlsterm", "O", "O", "B-umlsterm", "O", "O", "O", "O", "O", "O", "O", "B-umlsterm", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "B-umlsterm", "O", "O", "O", "O", "O", "O", "O", "O", "O", "B-umlsterm", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "B-umlsterm", "B-umlsterm", "O", "B-umlsterm", "B-umlsterm", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "B-umlsterm", "O", "B-umlsterm", "O", "B-umlsterm", "O", "O" ]
Fine needle aspiration biopsy ( FNAB ) under ultrasound control is an established diagnostic procedure for the head and neck region . Because of the disintegration of tissues , the diagnostic value of the method is limited resulting in only moderate specificity . In a prospective study , we performed a new , semi-automatic biopsy method in patients who had been diagnosed with sonographically confirmed pathologic masses in the head-neck region . This biopsy is carried out with a spring-loaded biopsy pistol which uses a disposable 20-gauge , specially designed cutting needle . Because this method combines the low invasiveness of FNAB with the high specificity of an excisional biopsy , a high tissue quality is obtained . Comparing these bioptic results with those of subsequent excisional biopsies proves that this new method yields a sensitivity of close to 100% for the detection of lymph node metastases of squamous cell carcinomas ( SCC ) . The tissue cylinders have a reproducible size and allow ultrastructural investigations in the transmission electron microscope ( TEM ) on the ultrastructural level . Due to the excellent tissue preservation in the biopsy cylinders , ultrastructural studies , using transmission electron microscopy , may be carried out with the biopsy material . Furthermore , following paraffin embedding of biopsy cylinders , serial sections may be obtained for special staining techniques , and immunohistological investigations are possible which may serve as an adjunct in the diagnosis of , e.g. , lymphoproliferative lesions with a sensitivity of 96% . Summarizing , the new semi-automatic biopsy technique obtains tissue probes of high quality with low invasiveness which enables highly sensitive diagnosis of head and neck lesions .
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[ "umlsterm" ]
Three Component Model ( 3CM ) is an intervention, usual care is an intervention, PTSD is a participant
285_task0
Sentence: To compare a collaborative approach , the Three Component Model ( 3CM ) , with usual care for treating PTSD in primary care . Instructions: please extract entities and their types from the input sentence, all entity types are in options Options: intervention, participant
[ "O", "O", "O", "O", "O", "O", "O", "B-intervention", "I-intervention", "I-intervention", "I-intervention", "I-intervention", "I-intervention", "O", "O", "B-intervention", "I-intervention", "O", "O", "B-participant", "O", "O", "O", "O" ]
To compare a collaborative approach , the Three Component Model ( 3CM ) , with usual care for treating PTSD in primary care .
[ "To", "compare", "a", "collaborative", "approach", ",", "the", "Three", "Component", "Model", "(", "3CM", ")", ",", "with", "usual", "care", "for", "treating", "PTSD", "in", "primary", "care", "." ]
[ "intervention", "participant" ]
Three Component Model ( 3CM ) is an intervention, usual care is an intervention, PTSD is a participant
285_task1
Sentence: To compare a collaborative approach , the Three Component Model ( 3CM ) , with usual care for treating PTSD in primary care . Instructions: please typing these entity words according to sentence: Three Component Model ( 3CM ), usual care, PTSD Options: intervention, participant
[ "O", "O", "O", "O", "O", "O", "O", "B-intervention", "I-intervention", "I-intervention", "I-intervention", "I-intervention", "I-intervention", "O", "O", "B-intervention", "I-intervention", "O", "O", "B-participant", "O", "O", "O", "O" ]
To compare a collaborative approach , the Three Component Model ( 3CM ) , with usual care for treating PTSD in primary care .
[ "To", "compare", "a", "collaborative", "approach", ",", "the", "Three", "Component", "Model", "(", "3CM", ")", ",", "with", "usual", "care", "for", "treating", "PTSD", "in", "primary", "care", "." ]
[ "intervention", "participant" ]
Three Component Model ( 3CM ), usual care, PTSD
285_task2
Sentence: To compare a collaborative approach , the Three Component Model ( 3CM ) , with usual care for treating PTSD in primary care . Instructions: please extract entity words from the input sentence
[ "O", "O", "O", "O", "O", "O", "O", "B-intervention", "I-intervention", "I-intervention", "I-intervention", "I-intervention", "I-intervention", "O", "O", "B-intervention", "I-intervention", "O", "O", "B-participant", "O", "O", "O", "O" ]
To compare a collaborative approach , the Three Component Model ( 3CM ) , with usual care for treating PTSD in primary care .
[ "To", "compare", "a", "collaborative", "approach", ",", "the", "Three", "Component", "Model", "(", "3CM", ")", ",", "with", "usual", "care", "for", "treating", "PTSD", "in", "primary", "care", "." ]
[ "intervention", "participant" ]
rosuvastatin is a CHEMICAL, HMG - CoA reductase is a GENE-Y
11501230_task0
Sentence: Clinical rationale for rosuvastatin, a potent new HMG-CoA reductase inhibitor. Instructions: please extract entities and their types from the input sentence, all entity types are in options Options: GENE-Y, CHEMICAL
[ "O", "O", "O", "B-CHEMICAL", "O", "O", "O", "O", "B-GENE-Y", "I-GENE-Y", "I-GENE-Y", "I-GENE-Y", "O", "O" ]
Clinical rationale for rosuvastatin, a potent new HMG-CoA reductase inhibitor.
[ "Clinical", "rationale", "for", "rosuvastatin", ",", "a", "potent", "new", "HMG", "-", "CoA", "reductase", "inhibitor", "." ]
[ "GENE-Y", "CHEMICAL", "GENE-N" ]
rosuvastatin is a CHEMICAL, HMG - CoA reductase is a GENE-Y
11501230_task1
Sentence: Clinical rationale for rosuvastatin, a potent new HMG-CoA reductase inhibitor. Instructions: please typing these entity words according to sentence: rosuvastatin, HMG - CoA reductase Options: GENE-Y, CHEMICAL
[ "O", "O", "O", "B-CHEMICAL", "O", "O", "O", "O", "B-GENE-Y", "I-GENE-Y", "I-GENE-Y", "I-GENE-Y", "O", "O" ]
Clinical rationale for rosuvastatin, a potent new HMG-CoA reductase inhibitor.
[ "Clinical", "rationale", "for", "rosuvastatin", ",", "a", "potent", "new", "HMG", "-", "CoA", "reductase", "inhibitor", "." ]
[ "GENE-Y", "CHEMICAL", "GENE-N" ]
rosuvastatin, HMG - CoA reductase
11501230_task2
Sentence: Clinical rationale for rosuvastatin, a potent new HMG-CoA reductase inhibitor. Instructions: please extract entity words from the input sentence
[ "O", "O", "O", "B-CHEMICAL", "O", "O", "O", "O", "B-GENE-Y", "I-GENE-Y", "I-GENE-Y", "I-GENE-Y", "O", "O" ]
Clinical rationale for rosuvastatin, a potent new HMG-CoA reductase inhibitor.
[ "Clinical", "rationale", "for", "rosuvastatin", ",", "a", "potent", "new", "HMG", "-", "CoA", "reductase", "inhibitor", "." ]
[ "GENE-Y", "CHEMICAL", "GENE-N" ]
prostate - specific membrane antigen is a GENE-Y, phenylalkylphosphonamidates is a CHEMICAL, PSMA is a GENE-Y, phenylalkylphosphonamidate is a CHEMICAL, glutamic acid is a CHEMICAL, PSMA is a GENE-Y, phenyl- and benzylphosphonamidates is a CHEMICAL, phenethyl is a CHEMICAL, alkyl is a CHEMICAL, phosphorus is a CHEMICAL, alkyl is a CHEMICAL, phenylalkyl is a CHEMICAL, PSMA is a GENE-Y
15846_task0
Sentence: Probing for a hydrophobic a binding register in prostate-specific membrane antigen with phenylalkylphosphonamidates. To explore for the existence of an auxiliary hydrophobic binding register remote from the active site of PSMA a series of phenylalkylphosphonamidate derivatives of glutamic acid were synthesized and evaluated for their inhibitory potencies against PSMA. Both the phenyl- and benzylphosphonamidates (1a and 1b) exhibited only modest inhibitory potency against. The phenethyl analog 1c was intermediate in inhibitory potency while inhibitors possessing a longer alkyl tether from the phenyl ring, resulted in markedly improved K(i) values. The greatest inhibitory potency was obtained for the inhibitors in which the phenyl ring was extended furthest from the central phosphorus (1f, n=5 and 1g, n=6). The slightly serrated pattern that emerged as the alkyl tether increased from three to six methylene units suggests that inhibitory potency is not simply correlated to increased hydrophobicity imparted by the phenylalkyl chain, but rather that one or more hydrophobic binding registers may exist remote from the substrate recognition architecture in the active site of PSMA. Instructions: please extract entities and their types from the input sentence, all entity types are in options Options: GENE-Y, CHEMICAL
[ "O", "O", "O", "O", "O", "O", "O", "O", "B-GENE-Y", "I-GENE-Y", "I-GENE-Y", "I-GENE-Y", "I-GENE-Y", "O", "B-CHEMICAL", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "B-GENE-Y", "O", "O", "O", "B-CHEMICAL", "O", "O", "B-CHEMICAL", "I-CHEMICAL", "O", "O", "O", "O", "O", "O", "O", "O", "O", "B-GENE-Y", "O", "O", "O", "B-CHEMICAL", "I-CHEMICAL", "I-CHEMICAL", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "B-CHEMICAL", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "B-CHEMICAL", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "B-CHEMICAL", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "B-CHEMICAL", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "B-CHEMICAL", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "B-GENE-Y", "O" ]
Probing for a hydrophobic a binding register in prostate-specific membrane antigen with phenylalkylphosphonamidates. To explore for the existence of an auxiliary hydrophobic binding register remote from the active site of PSMA a series of phenylalkylphosphonamidate derivatives of glutamic acid were synthesized and evaluated for their inhibitory potencies against PSMA. Both the phenyl- and benzylphosphonamidates (1a and 1b) exhibited only modest inhibitory potency against. The phenethyl analog 1c was intermediate in inhibitory potency while inhibitors possessing a longer alkyl tether from the phenyl ring, resulted in markedly improved K(i) values. The greatest inhibitory potency was obtained for the inhibitors in which the phenyl ring was extended furthest from the central phosphorus (1f, n=5 and 1g, n=6). The slightly serrated pattern that emerged as the alkyl tether increased from three to six methylene units suggests that inhibitory potency is not simply correlated to increased hydrophobicity imparted by the phenylalkyl chain, but rather that one or more hydrophobic binding registers may exist remote from the substrate recognition architecture in the active site of PSMA.
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[ "CHEMICAL", "GENE-Y" ]
prostate - specific membrane antigen is a GENE-Y, phenylalkylphosphonamidates is a CHEMICAL, PSMA is a GENE-Y, phenylalkylphosphonamidate is a CHEMICAL, glutamic acid is a CHEMICAL, PSMA is a GENE-Y, phenyl- and benzylphosphonamidates is a CHEMICAL, phenethyl is a CHEMICAL, alkyl is a CHEMICAL, phosphorus is a CHEMICAL, alkyl is a CHEMICAL, phenylalkyl is a CHEMICAL, PSMA is a GENE-Y
15846_task1
Sentence: Probing for a hydrophobic a binding register in prostate-specific membrane antigen with phenylalkylphosphonamidates. To explore for the existence of an auxiliary hydrophobic binding register remote from the active site of PSMA a series of phenylalkylphosphonamidate derivatives of glutamic acid were synthesized and evaluated for their inhibitory potencies against PSMA. Both the phenyl- and benzylphosphonamidates (1a and 1b) exhibited only modest inhibitory potency against. The phenethyl analog 1c was intermediate in inhibitory potency while inhibitors possessing a longer alkyl tether from the phenyl ring, resulted in markedly improved K(i) values. The greatest inhibitory potency was obtained for the inhibitors in which the phenyl ring was extended furthest from the central phosphorus (1f, n=5 and 1g, n=6). The slightly serrated pattern that emerged as the alkyl tether increased from three to six methylene units suggests that inhibitory potency is not simply correlated to increased hydrophobicity imparted by the phenylalkyl chain, but rather that one or more hydrophobic binding registers may exist remote from the substrate recognition architecture in the active site of PSMA. Instructions: please typing these entity words according to sentence: prostate - specific membrane antigen, phenylalkylphosphonamidates, PSMA, phenylalkylphosphonamidate, glutamic acid, PSMA, phenyl- and benzylphosphonamidates, phenethyl, alkyl, phosphorus, alkyl, phenylalkyl, PSMA Options: GENE-Y, CHEMICAL
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Probing for a hydrophobic a binding register in prostate-specific membrane antigen with phenylalkylphosphonamidates. To explore for the existence of an auxiliary hydrophobic binding register remote from the active site of PSMA a series of phenylalkylphosphonamidate derivatives of glutamic acid were synthesized and evaluated for their inhibitory potencies against PSMA. Both the phenyl- and benzylphosphonamidates (1a and 1b) exhibited only modest inhibitory potency against. The phenethyl analog 1c was intermediate in inhibitory potency while inhibitors possessing a longer alkyl tether from the phenyl ring, resulted in markedly improved K(i) values. The greatest inhibitory potency was obtained for the inhibitors in which the phenyl ring was extended furthest from the central phosphorus (1f, n=5 and 1g, n=6). The slightly serrated pattern that emerged as the alkyl tether increased from three to six methylene units suggests that inhibitory potency is not simply correlated to increased hydrophobicity imparted by the phenylalkyl chain, but rather that one or more hydrophobic binding registers may exist remote from the substrate recognition architecture in the active site of PSMA.
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[ "CHEMICAL", "GENE-Y" ]
prostate - specific membrane antigen, phenylalkylphosphonamidates, PSMA, phenylalkylphosphonamidate, glutamic acid, PSMA, phenyl- and benzylphosphonamidates, phenethyl, alkyl, phosphorus, alkyl, phenylalkyl, PSMA
15846_task2
Sentence: Probing for a hydrophobic a binding register in prostate-specific membrane antigen with phenylalkylphosphonamidates. To explore for the existence of an auxiliary hydrophobic binding register remote from the active site of PSMA a series of phenylalkylphosphonamidate derivatives of glutamic acid were synthesized and evaluated for their inhibitory potencies against PSMA. Both the phenyl- and benzylphosphonamidates (1a and 1b) exhibited only modest inhibitory potency against. The phenethyl analog 1c was intermediate in inhibitory potency while inhibitors possessing a longer alkyl tether from the phenyl ring, resulted in markedly improved K(i) values. The greatest inhibitory potency was obtained for the inhibitors in which the phenyl ring was extended furthest from the central phosphorus (1f, n=5 and 1g, n=6). The slightly serrated pattern that emerged as the alkyl tether increased from three to six methylene units suggests that inhibitory potency is not simply correlated to increased hydrophobicity imparted by the phenylalkyl chain, but rather that one or more hydrophobic binding registers may exist remote from the substrate recognition architecture in the active site of PSMA. Instructions: please extract entity words from the input sentence
[ "O", "O", "O", "O", "O", "O", "O", "O", "B-GENE-Y", "I-GENE-Y", "I-GENE-Y", "I-GENE-Y", "I-GENE-Y", "O", "B-CHEMICAL", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "B-GENE-Y", "O", "O", "O", "B-CHEMICAL", "O", "O", "B-CHEMICAL", "I-CHEMICAL", "O", "O", "O", "O", "O", "O", "O", "O", "O", "B-GENE-Y", "O", "O", "O", "B-CHEMICAL", "I-CHEMICAL", "I-CHEMICAL", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "B-CHEMICAL", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "B-CHEMICAL", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "B-CHEMICAL", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "B-CHEMICAL", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "B-CHEMICAL", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "B-GENE-Y", "O" ]
Probing for a hydrophobic a binding register in prostate-specific membrane antigen with phenylalkylphosphonamidates. To explore for the existence of an auxiliary hydrophobic binding register remote from the active site of PSMA a series of phenylalkylphosphonamidate derivatives of glutamic acid were synthesized and evaluated for their inhibitory potencies against PSMA. Both the phenyl- and benzylphosphonamidates (1a and 1b) exhibited only modest inhibitory potency against. The phenethyl analog 1c was intermediate in inhibitory potency while inhibitors possessing a longer alkyl tether from the phenyl ring, resulted in markedly improved K(i) values. The greatest inhibitory potency was obtained for the inhibitors in which the phenyl ring was extended furthest from the central phosphorus (1f, n=5 and 1g, n=6). The slightly serrated pattern that emerged as the alkyl tether increased from three to six methylene units suggests that inhibitory potency is not simply correlated to increased hydrophobicity imparted by the phenylalkyl chain, but rather that one or more hydrophobic binding registers may exist remote from the substrate recognition architecture in the active site of PSMA.
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[ "CHEMICAL", "GENE-Y" ]
Alpha1-adrenoceptors is a GENE-N
17603545_task0
Sentence: Alpha1-adrenoceptors are required for normal male sexual function. Instructions: please extract entities and their types from the input sentence, all entity types are in options Options: GENE-N
[ "B-GENE-N", "O", "O", "O", "O", "O", "O", "O", "O" ]
Alpha1-adrenoceptors are required for normal male sexual function.
[ "Alpha1-adrenoceptors", "are", "required", "for", "normal", "male", "sexual", "function", "." ]
[ "GENE-N", "GENE-Y", "CHEMICAL" ]
Alpha1-adrenoceptors is a GENE-N
17603545_task1
Sentence: Alpha1-adrenoceptors are required for normal male sexual function. Instructions: please typing these entity words according to sentence: Alpha1-adrenoceptors Options: GENE-N
[ "B-GENE-N", "O", "O", "O", "O", "O", "O", "O", "O" ]
Alpha1-adrenoceptors are required for normal male sexual function.
[ "Alpha1-adrenoceptors", "are", "required", "for", "normal", "male", "sexual", "function", "." ]
[ "GENE-N", "GENE-Y", "CHEMICAL" ]
Alpha1-adrenoceptors
17603545_task2
Sentence: Alpha1-adrenoceptors are required for normal male sexual function. Instructions: please extract entity words from the input sentence
[ "B-GENE-N", "O", "O", "O", "O", "O", "O", "O", "O" ]
Alpha1-adrenoceptors are required for normal male sexual function.
[ "Alpha1-adrenoceptors", "are", "required", "for", "normal", "male", "sexual", "function", "." ]
[ "GENE-N", "GENE-Y", "CHEMICAL" ]
Joints is an umlsterm, nerve is an umlsterm, nociceptors is an umlsterm, pain is an umlsterm, humans is an umlsterm, nociceptors is an umlsterm, tissue is an umlsterm, nociceptors is an umlsterm, pain is an umlsterm, mediators is an umlsterm, bradykinin is an umlsterm, prostaglandins is an umlsterm, function is an umlsterm, nociceptors is an umlsterm, function is an umlsterm, neurogenic inflammation is an umlsterm, release is an umlsterm, neuropeptides is an umlsterm, substance P is an umlsterm, calcitonin gene - related peptide is an umlsterm, tissue is an umlsterm, function is an umlsterm, nociceptors is an umlsterm, generation is an umlsterm, tissue is an umlsterm
ManuelleMedizin.70350077.eng.abstr_task0
Sentence: Joints are innervated by nociceptive nerve fibres ( nociceptors ) , which are only activated by tissue-damaging stimuli . This activation causes pain in conscious humans . The sensory terminals of many nociceptors in the tissue are sensitized by inflammatory processes . Under these conditions , normally innocuous and non-painful stimuli are sufficient to activate nociceptors and to elicit pain . The effect of inflammatory mediators such as bradykinin and prostaglandins is important for the process of sensitization . In addition to their sensory function , many nociceptors have an efferent function . They evoke a neurogenic inflammation by the release of neuropeptides such as substance P and calcitonin gene-related peptide into the tissue . The efferent function of nociceptors contributes to the generation of inflammatory lesions of the tissue . Instructions: please extract entities and their types from the input sentence, all entity types are in options Options: umlsterm
[ "B-umlsterm", "O", "O", "O", "O", "B-umlsterm", "O", "O", "B-umlsterm", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "B-umlsterm", "O", "O", "B-umlsterm", "O", "O", "O", "O", "O", "O", "B-umlsterm", "O", "O", "B-umlsterm", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "B-umlsterm", "O", "O", "O", "B-umlsterm", "O", "O", "O", "O", "O", "B-umlsterm", "O", "O", "B-umlsterm", "O", "B-umlsterm", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "O", "B-umlsterm", "O", "O", "B-umlsterm", "O", "O", "O", "B-umlsterm", "O", "O", "O", "O", "B-umlsterm", "I-umlsterm", "O", "O", "B-umlsterm", "O", "B-umlsterm", "O", "O", "B-umlsterm", "I-umlsterm", "O", "B-umlsterm", "I-umlsterm", "I-umlsterm", "I-umlsterm", "I-umlsterm", "O", "O", "B-umlsterm", "O", "O", "O", "B-umlsterm", "O", "B-umlsterm", "O", "O", "O", "B-umlsterm", "O", "O", "O", "O", "O", "B-umlsterm", "O" ]
Joints are innervated by nociceptive nerve fibres ( nociceptors ) , which are only activated by tissue-damaging stimuli . This activation causes pain in conscious humans . The sensory terminals of many nociceptors in the tissue are sensitized by inflammatory processes . Under these conditions , normally innocuous and non-painful stimuli are sufficient to activate nociceptors and to elicit pain . The effect of inflammatory mediators such as bradykinin and prostaglandins is important for the process of sensitization . In addition to their sensory function , many nociceptors have an efferent function . They evoke a neurogenic inflammation by the release of neuropeptides such as substance P and calcitonin gene-related peptide into the tissue . The efferent function of nociceptors contributes to the generation of inflammatory lesions of the tissue .
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[ "umlsterm" ]
Joints is an umlsterm, nerve is an umlsterm, nociceptors is an umlsterm, pain is an umlsterm, humans is an umlsterm, nociceptors is an umlsterm, tissue is an umlsterm, nociceptors is an umlsterm, pain is an umlsterm, mediators is an umlsterm, bradykinin is an umlsterm, prostaglandins is an umlsterm, function is an umlsterm, nociceptors is an umlsterm, function is an umlsterm, neurogenic inflammation is an umlsterm, release is an umlsterm, neuropeptides is an umlsterm, substance P is an umlsterm, calcitonin gene - related peptide is an umlsterm, tissue is an umlsterm, function is an umlsterm, nociceptors is an umlsterm, generation is an umlsterm, tissue is an umlsterm
ManuelleMedizin.70350077.eng.abstr_task1
Sentence: Joints are innervated by nociceptive nerve fibres ( nociceptors ) , which are only activated by tissue-damaging stimuli . This activation causes pain in conscious humans . The sensory terminals of many nociceptors in the tissue are sensitized by inflammatory processes . Under these conditions , normally innocuous and non-painful stimuli are sufficient to activate nociceptors and to elicit pain . The effect of inflammatory mediators such as bradykinin and prostaglandins is important for the process of sensitization . In addition to their sensory function , many nociceptors have an efferent function . They evoke a neurogenic inflammation by the release of neuropeptides such as substance P and calcitonin gene-related peptide into the tissue . The efferent function of nociceptors contributes to the generation of inflammatory lesions of the tissue . Instructions: please typing these entity words according to sentence: Joints, nerve, nociceptors, pain, humans, nociceptors, tissue, nociceptors, pain, mediators, bradykinin, prostaglandins, function, nociceptors, function, neurogenic inflammation, release, neuropeptides, substance P, calcitonin gene - related peptide, tissue, function, nociceptors, generation, tissue Options: umlsterm
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Joints are innervated by nociceptive nerve fibres ( nociceptors ) , which are only activated by tissue-damaging stimuli . This activation causes pain in conscious humans . The sensory terminals of many nociceptors in the tissue are sensitized by inflammatory processes . Under these conditions , normally innocuous and non-painful stimuli are sufficient to activate nociceptors and to elicit pain . The effect of inflammatory mediators such as bradykinin and prostaglandins is important for the process of sensitization . In addition to their sensory function , many nociceptors have an efferent function . They evoke a neurogenic inflammation by the release of neuropeptides such as substance P and calcitonin gene-related peptide into the tissue . The efferent function of nociceptors contributes to the generation of inflammatory lesions of the tissue .
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[ "umlsterm" ]
Joints, nerve, nociceptors, pain, humans, nociceptors, tissue, nociceptors, pain, mediators, bradykinin, prostaglandins, function, nociceptors, function, neurogenic inflammation, release, neuropeptides, substance P, calcitonin gene - related peptide, tissue, function, nociceptors, generation, tissue
ManuelleMedizin.70350077.eng.abstr_task2
Sentence: Joints are innervated by nociceptive nerve fibres ( nociceptors ) , which are only activated by tissue-damaging stimuli . This activation causes pain in conscious humans . The sensory terminals of many nociceptors in the tissue are sensitized by inflammatory processes . Under these conditions , normally innocuous and non-painful stimuli are sufficient to activate nociceptors and to elicit pain . The effect of inflammatory mediators such as bradykinin and prostaglandins is important for the process of sensitization . In addition to their sensory function , many nociceptors have an efferent function . They evoke a neurogenic inflammation by the release of neuropeptides such as substance P and calcitonin gene-related peptide into the tissue . The efferent function of nociceptors contributes to the generation of inflammatory lesions of the tissue . Instructions: please extract entity words from the input sentence
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Joints are innervated by nociceptive nerve fibres ( nociceptors ) , which are only activated by tissue-damaging stimuli . This activation causes pain in conscious humans . The sensory terminals of many nociceptors in the tissue are sensitized by inflammatory processes . Under these conditions , normally innocuous and non-painful stimuli are sufficient to activate nociceptors and to elicit pain . The effect of inflammatory mediators such as bradykinin and prostaglandins is important for the process of sensitization . In addition to their sensory function , many nociceptors have an efferent function . They evoke a neurogenic inflammation by the release of neuropeptides such as substance P and calcitonin gene-related peptide into the tissue . The efferent function of nociceptors contributes to the generation of inflammatory lesions of the tissue .
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[ "umlsterm" ]
DPP-4 is a GENE-Y, sitagliptin is a CHEMICAL, insulin is a GENE-Y, sitagliptin is a CHEMICAL, insulin is a GENE-Y, insulin is a GENE-N, insulin is a GENE-Y, insulin is a GENE-Y, insulin is a GENE-Y, insulin is a GENE-Y, sulfonylureas is a CHEMICAL, α - glucosidase is a GENE-N, metformin is a CHEMICAL, hemoglobin A1c is a GENE-Y, HbA1c is a GENE-Y, sitagliptin is a CHEMICAL, insulin is a GENE-Y, HbA1c is a GENE-Y, glycated albumin is a GENE-Y, sitagliptin is a CHEMICAL, sitagliptin is a CHEMICAL, insulin is a GENE-Y, sitagliptin is a CHEMICAL, insulin is a GENE-Y, sitagliptin is a CHEMICAL, insulin is a GENE-Y
33593_task0
Sentence: Add-on therapy with the DPP-4 inhibitor sitagliptin improves glycemic control in insulin-treated Japanese patients with type 2 diabetes mellitus. The effect of add-on therapy with sitagliptin on glycemic control was prospectively investigated in patients with type 2 diabetes mellitus (T2DM) receiving insulin alone or insulin combined with oral antidiabetic drugs. Seventy-one patients were evaluated (38 men and 33 women aged 63.9±10.2 years). They were divided into three groups, which were 45 patients receiving premixed insulin twice daily, 15 patients receiving multiple daily insulin injections, and 11 patients receiving basal insulin with oral antidiabetic drugs (basal insulin therapy). Concomitant oral drugs included sulfonylureas, α-glucosidase inhibitors and metformin. The hemoglobin A1c (HbA1c) of all patients improved significantly from 8.1±1.2% to 7.6±1.1% after 12 weeks of add-on therapy with sitagliptin (p<0.01), and the insulin dosage was reduced from 27.3±15.8 U/day to 24.5±16.5 U/day (p<0.001). Body weight did not change after the start of concomitant therapy and severe hypoglycemia was not observed. The baseline HbA1c and glycated albumin levels were identified as factors that predicted the response to add-on therapy with sitagliptin. These findings suggest that add-on therapy with sitagliptin can be expected to achieve improvement of poor glycemic control irrespective of a patient's demographic profile. Stratified analysis based on the insulin regimen revealed a stronger antidiabetic effect and a high efficacy of sitagliptin when it was added to basal insulin therapy. The results of this investigation confirmed that add-on therapy with sitagliptin to various insulin regimens could improve glycemic control without severe hypoglycemia and/or weight gain. Instructions: please extract entities and their types from the input sentence, all entity types are in options Options: GENE-N, GENE-Y, CHEMICAL
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Add-on therapy with the DPP-4 inhibitor sitagliptin improves glycemic control in insulin-treated Japanese patients with type 2 diabetes mellitus. The effect of add-on therapy with sitagliptin on glycemic control was prospectively investigated in patients with type 2 diabetes mellitus (T2DM) receiving insulin alone or insulin combined with oral antidiabetic drugs. Seventy-one patients were evaluated (38 men and 33 women aged 63.9±10.2 years). They were divided into three groups, which were 45 patients receiving premixed insulin twice daily, 15 patients receiving multiple daily insulin injections, and 11 patients receiving basal insulin with oral antidiabetic drugs (basal insulin therapy). Concomitant oral drugs included sulfonylureas, α-glucosidase inhibitors and metformin. The hemoglobin A1c (HbA1c) of all patients improved significantly from 8.1±1.2% to 7.6±1.1% after 12 weeks of add-on therapy with sitagliptin (p<0.01), and the insulin dosage was reduced from 27.3±15.8 U/day to 24.5±16.5 U/day (p<0.001). Body weight did not change after the start of concomitant therapy and severe hypoglycemia was not observed. The baseline HbA1c and glycated albumin levels were identified as factors that predicted the response to add-on therapy with sitagliptin. These findings suggest that add-on therapy with sitagliptin can be expected to achieve improvement of poor glycemic control irrespective of a patient's demographic profile. Stratified analysis based on the insulin regimen revealed a stronger antidiabetic effect and a high efficacy of sitagliptin when it was added to basal insulin therapy. The results of this investigation confirmed that add-on therapy with sitagliptin to various insulin regimens could improve glycemic control without severe hypoglycemia and/or weight gain.
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[ "GENE-Y", "CHEMICAL", "GENE-N" ]
DPP-4 is a GENE-Y, sitagliptin is a CHEMICAL, insulin is a GENE-Y, sitagliptin is a CHEMICAL, insulin is a GENE-Y, insulin is a GENE-N, insulin is a GENE-Y, insulin is a GENE-Y, insulin is a GENE-Y, insulin is a GENE-Y, sulfonylureas is a CHEMICAL, α - glucosidase is a GENE-N, metformin is a CHEMICAL, hemoglobin A1c is a GENE-Y, HbA1c is a GENE-Y, sitagliptin is a CHEMICAL, insulin is a GENE-Y, HbA1c is a GENE-Y, glycated albumin is a GENE-Y, sitagliptin is a CHEMICAL, sitagliptin is a CHEMICAL, insulin is a GENE-Y, sitagliptin is a CHEMICAL, insulin is a GENE-Y, sitagliptin is a CHEMICAL, insulin is a GENE-Y
33593_task1
Sentence: Add-on therapy with the DPP-4 inhibitor sitagliptin improves glycemic control in insulin-treated Japanese patients with type 2 diabetes mellitus. The effect of add-on therapy with sitagliptin on glycemic control was prospectively investigated in patients with type 2 diabetes mellitus (T2DM) receiving insulin alone or insulin combined with oral antidiabetic drugs. Seventy-one patients were evaluated (38 men and 33 women aged 63.9±10.2 years). They were divided into three groups, which were 45 patients receiving premixed insulin twice daily, 15 patients receiving multiple daily insulin injections, and 11 patients receiving basal insulin with oral antidiabetic drugs (basal insulin therapy). Concomitant oral drugs included sulfonylureas, α-glucosidase inhibitors and metformin. The hemoglobin A1c (HbA1c) of all patients improved significantly from 8.1±1.2% to 7.6±1.1% after 12 weeks of add-on therapy with sitagliptin (p<0.01), and the insulin dosage was reduced from 27.3±15.8 U/day to 24.5±16.5 U/day (p<0.001). Body weight did not change after the start of concomitant therapy and severe hypoglycemia was not observed. The baseline HbA1c and glycated albumin levels were identified as factors that predicted the response to add-on therapy with sitagliptin. These findings suggest that add-on therapy with sitagliptin can be expected to achieve improvement of poor glycemic control irrespective of a patient's demographic profile. Stratified analysis based on the insulin regimen revealed a stronger antidiabetic effect and a high efficacy of sitagliptin when it was added to basal insulin therapy. The results of this investigation confirmed that add-on therapy with sitagliptin to various insulin regimens could improve glycemic control without severe hypoglycemia and/or weight gain. Instructions: please typing these entity words according to sentence: DPP-4, sitagliptin, insulin, sitagliptin, insulin, insulin, insulin, insulin, insulin, insulin, sulfonylureas, α - glucosidase, metformin, hemoglobin A1c, HbA1c, sitagliptin, insulin, HbA1c, glycated albumin, sitagliptin, sitagliptin, insulin, sitagliptin, insulin, sitagliptin, insulin Options: GENE-N, GENE-Y, CHEMICAL
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Add-on therapy with the DPP-4 inhibitor sitagliptin improves glycemic control in insulin-treated Japanese patients with type 2 diabetes mellitus. The effect of add-on therapy with sitagliptin on glycemic control was prospectively investigated in patients with type 2 diabetes mellitus (T2DM) receiving insulin alone or insulin combined with oral antidiabetic drugs. Seventy-one patients were evaluated (38 men and 33 women aged 63.9±10.2 years). They were divided into three groups, which were 45 patients receiving premixed insulin twice daily, 15 patients receiving multiple daily insulin injections, and 11 patients receiving basal insulin with oral antidiabetic drugs (basal insulin therapy). Concomitant oral drugs included sulfonylureas, α-glucosidase inhibitors and metformin. The hemoglobin A1c (HbA1c) of all patients improved significantly from 8.1±1.2% to 7.6±1.1% after 12 weeks of add-on therapy with sitagliptin (p<0.01), and the insulin dosage was reduced from 27.3±15.8 U/day to 24.5±16.5 U/day (p<0.001). Body weight did not change after the start of concomitant therapy and severe hypoglycemia was not observed. The baseline HbA1c and glycated albumin levels were identified as factors that predicted the response to add-on therapy with sitagliptin. These findings suggest that add-on therapy with sitagliptin can be expected to achieve improvement of poor glycemic control irrespective of a patient's demographic profile. Stratified analysis based on the insulin regimen revealed a stronger antidiabetic effect and a high efficacy of sitagliptin when it was added to basal insulin therapy. The results of this investigation confirmed that add-on therapy with sitagliptin to various insulin regimens could improve glycemic control without severe hypoglycemia and/or weight gain.
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[ "GENE-Y", "CHEMICAL", "GENE-N" ]
DPP-4, sitagliptin, insulin, sitagliptin, insulin, insulin, insulin, insulin, insulin, insulin, sulfonylureas, α - glucosidase, metformin, hemoglobin A1c, HbA1c, sitagliptin, insulin, HbA1c, glycated albumin, sitagliptin, sitagliptin, insulin, sitagliptin, insulin, sitagliptin, insulin
33593_task2
Sentence: Add-on therapy with the DPP-4 inhibitor sitagliptin improves glycemic control in insulin-treated Japanese patients with type 2 diabetes mellitus. The effect of add-on therapy with sitagliptin on glycemic control was prospectively investigated in patients with type 2 diabetes mellitus (T2DM) receiving insulin alone or insulin combined with oral antidiabetic drugs. Seventy-one patients were evaluated (38 men and 33 women aged 63.9±10.2 years). They were divided into three groups, which were 45 patients receiving premixed insulin twice daily, 15 patients receiving multiple daily insulin injections, and 11 patients receiving basal insulin with oral antidiabetic drugs (basal insulin therapy). Concomitant oral drugs included sulfonylureas, α-glucosidase inhibitors and metformin. The hemoglobin A1c (HbA1c) of all patients improved significantly from 8.1±1.2% to 7.6±1.1% after 12 weeks of add-on therapy with sitagliptin (p<0.01), and the insulin dosage was reduced from 27.3±15.8 U/day to 24.5±16.5 U/day (p<0.001). Body weight did not change after the start of concomitant therapy and severe hypoglycemia was not observed. The baseline HbA1c and glycated albumin levels were identified as factors that predicted the response to add-on therapy with sitagliptin. These findings suggest that add-on therapy with sitagliptin can be expected to achieve improvement of poor glycemic control irrespective of a patient's demographic profile. Stratified analysis based on the insulin regimen revealed a stronger antidiabetic effect and a high efficacy of sitagliptin when it was added to basal insulin therapy. The results of this investigation confirmed that add-on therapy with sitagliptin to various insulin regimens could improve glycemic control without severe hypoglycemia and/or weight gain. Instructions: please extract entity words from the input sentence
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Add-on therapy with the DPP-4 inhibitor sitagliptin improves glycemic control in insulin-treated Japanese patients with type 2 diabetes mellitus. The effect of add-on therapy with sitagliptin on glycemic control was prospectively investigated in patients with type 2 diabetes mellitus (T2DM) receiving insulin alone or insulin combined with oral antidiabetic drugs. Seventy-one patients were evaluated (38 men and 33 women aged 63.9±10.2 years). They were divided into three groups, which were 45 patients receiving premixed insulin twice daily, 15 patients receiving multiple daily insulin injections, and 11 patients receiving basal insulin with oral antidiabetic drugs (basal insulin therapy). Concomitant oral drugs included sulfonylureas, α-glucosidase inhibitors and metformin. The hemoglobin A1c (HbA1c) of all patients improved significantly from 8.1±1.2% to 7.6±1.1% after 12 weeks of add-on therapy with sitagliptin (p<0.01), and the insulin dosage was reduced from 27.3±15.8 U/day to 24.5±16.5 U/day (p<0.001). Body weight did not change after the start of concomitant therapy and severe hypoglycemia was not observed. The baseline HbA1c and glycated albumin levels were identified as factors that predicted the response to add-on therapy with sitagliptin. These findings suggest that add-on therapy with sitagliptin can be expected to achieve improvement of poor glycemic control irrespective of a patient's demographic profile. Stratified analysis based on the insulin regimen revealed a stronger antidiabetic effect and a high efficacy of sitagliptin when it was added to basal insulin therapy. The results of this investigation confirmed that add-on therapy with sitagliptin to various insulin regimens could improve glycemic control without severe hypoglycemia and/or weight gain.
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[ "GENE-Y", "CHEMICAL", "GENE-N" ]
prophylaxis is an umlsterm, therapy is an umlsterm, ointment is an umlsterm, glass is an umlsterm, glass is an umlsterm, patients is an umlsterm, feeling is an umlsterm, physical handicap is an umlsterm, therapeutic is an umlsterm, gravity is an umlsterm, gold is an umlsterm, implantation is an umlsterm, Methods is an umlsterm, patients is an umlsterm, lead is an umlsterm, weights is an umlsterm, patients is an umlsterm, adhesive is an umlsterm, skin is an umlsterm, method is an umlsterm, persons is an umlsterm, patients is an umlsterm, method is an umlsterm, patients is an umlsterm, procedure is an umlsterm, visual function is an umlsterm, cosmetics is an umlsterm, glass is an umlsterm, lead is an umlsterm, weights is an umlsterm, adhesive is an umlsterm, method is an umlsterm, gold is an umlsterm, implantation is an umlsterm
DerNervenarzt.60670667.eng.abstr_task0
Sentence: Background : For prophylaxis or therapy of lagophthalmic keratopathy ointment application or hour glass dressing is indicated . The hour glass dressing has the disadvantage of a continuous visual impairment by steaming up of the moisture chamber . The main problem for the patients is the strong subjective feeling of physical handicap . A therapeutic alternative is the gravity dependent lidloading , which is known as gold implantation in cases of irreversible lagophthalmos . Methods and patients : By lead weights of 0,8 to 2,0 g , which are fixed to the upper lid by a foil glueing on both sides ( Tesafix ) or in allergic patients by an adhesive layer , well tolerated by the skin ( Combihesive ) , the lidclosure can become restored without impairment of lidopening . In a controlled study the described method was first tested on 10 normal persons and than applied to 22 patients with lagophthalmos . Meanwhile the new method was applied to 32 further patients . Results : The " liddynamic " procedure is effective and well tolerated ; it is more accepted , especially during the day , because of better visual function and better cosmetics . The hour glass dressing , is still of importance in serious cases of kerathopathy during the night . Conclusion : " lidloading " with lead weights , which are glued by an adhesive layer to the upper lid , can be recommended as a new method in cases of reversible lagophthalmos or as a preparing step before a gold implantation . Instructions: please extract entities and their types from the input sentence, all entity types are in options Options: umlsterm
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Background : For prophylaxis or therapy of lagophthalmic keratopathy ointment application or hour glass dressing is indicated . The hour glass dressing has the disadvantage of a continuous visual impairment by steaming up of the moisture chamber . The main problem for the patients is the strong subjective feeling of physical handicap . A therapeutic alternative is the gravity dependent lidloading , which is known as gold implantation in cases of irreversible lagophthalmos . Methods and patients : By lead weights of 0,8 to 2,0 g , which are fixed to the upper lid by a foil glueing on both sides ( Tesafix ) or in allergic patients by an adhesive layer , well tolerated by the skin ( Combihesive ) , the lidclosure can become restored without impairment of lidopening . In a controlled study the described method was first tested on 10 normal persons and than applied to 22 patients with lagophthalmos . Meanwhile the new method was applied to 32 further patients . Results : The " liddynamic " procedure is effective and well tolerated ; it is more accepted , especially during the day , because of better visual function and better cosmetics . The hour glass dressing , is still of importance in serious cases of kerathopathy during the night . Conclusion : " lidloading " with lead weights , which are glued by an adhesive layer to the upper lid , can be recommended as a new method in cases of reversible lagophthalmos or as a preparing step before a gold implantation .
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[ "umlsterm" ]