title: Conditionally immortalized stem cell lines from human spinal cord retain regional identity and generate functional V2a interneurons and motorneurons.
abstract: BACKGROUND
  The use of immortalized neural stem cells either as models of neural development in vitro or as cellular therapies in central nervous system (CNS) disorders has been controversial. This controversy has centered on the capacity of immortalized cells to retain characteristic features of the progenitor cells resident in the tissue of origin from which they were derived, and the potential for tumorogenicity as a result of immortalization. Here, we report the generation of conditionally immortalized neural stem cell lines from human fetal spinal cord tissue, which addresses these issues.
  
  
  METHODS
  Clonal neural stem cell lines were derived from 10-week-old human fetal spinal cord and conditionally immortalized with an inducible form of cMyc. The derived lines were karyotyped, transcriptionally profiled by microarray, and assessed against a panel of spinal cord progenitor markers with immunocytochemistry. In addition, the lines were differentiated and assessed for the presence of neuronal fate markers and functional calcium channels. Finally, a clonal line expressing eGFP was grafted into lesioned rat spinal cord and assessed for survival, differentiation characteristics, and tumorogenicity.
  
  
  RESULTS
  We demonstrate that these clonal lines (a) retain a clear transcriptional signature of ventral spinal cord progenitors and a normal karyotype after extensive propagation in vitro, (b) differentiate into relevant ventral neuronal subtypes with functional T-, L-, N-, and P/Q-type Ca(2+) channels and spontaneous calcium oscillations, and (c) stably engraft into lesioned rat spinal cord without tumorogenicity.
  
  
  CONCLUSIONS
  We propose that these cells represent a useful tool both for the in vitro study of differentiation into ventral spinal cord neuronal subtypes, and for examining the potential of conditionally immortalized neural stem cells to facilitate functional recovery after spinal cord injury or disease.
authors.forename: Graham
authors.initials: G
authors.lastname: Cocks
authors.forename: Nataliya
authors.initials: N
authors.lastname: Romanyuk
authors.forename: Takashi
authors.initials: T
authors.lastname: Amemori
authors.forename: Pavla
authors.initials: P
authors.lastname: Jendelova
authors.forename: Oksana
authors.initials: O
authors.lastname: Forostyak
authors.forename: Aaron R
authors.initials: AR
authors.lastname: Jeffries
authors.forename: Leo
authors.initials: L
authors.lastname: Perfect
authors.forename: Sandrine
authors.initials: S
authors.lastname: Thuret
authors.forename: Govindan
authors.initials: G
authors.lastname: Dayanithi
authors.forename: Eva
authors.initials: E
authors.lastname: Sykova
authors.forename: Jack
authors.initials: J
authors.lastname: Price
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism / Q000494:pharmacology; D002454:Cell Differentiation / Q000187:drug effects; D002478:Cells, Cultured; D004151:Dipeptides / Q000494:pharmacology; D005333:Fetus / Q000166:cytology; D006801:Humans; D007395:Interneurons / Q000166:cytology* / Q000378:metabolism; D007621:Karyotyping; D008297:Male; D009046:Motor Neurons / Q000166:cytology* / Q000378:metabolism; D058953:Neural Stem Cells / Q000166:cytology* / Q000187:drug effects / Q000637:transplantation; D051381:Rats; D017208:Rats, Wistar; D051880:Receptors, Notch / Q000037:antagonists & inhibitors / Q000378:metabolism; D013116:Spinal Cord / Q000166:cytology*; D013119:Spinal Cord Injuries / Q000628:therapy; D014183:Transplantation, Heterologous
chemical_list: D004151:Dipeptides; C419410:N-(N-(3,5-difluorophenacetyl)alanyl)phenylglycine tert-butyl ester; D051880:Receptors, Notch; D002118:Calcium