Abstract
IntroductionThe 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.MethodsClonal 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.ResultsWe 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 Ca2+ channels and spontaneous calcium oscillations, and (c) stably engraft into lesioned rat spinal cord without tumorogenicity.ConclusionsWe 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.
Highlights
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
We demonstrated that on removal of growth factors and 4-hydroxy tamoxifen (4-OHT), these cells differentiate into V2 interneurons and motoneurons, consistent with the expression of p2 and progenitor subdomain (pMN) domain markers in the progenitor cells
We demonstrated that a subset of these neurons exhibit spontaneous calcium oscillations typically observed in dissociated embryonic rat motoneurons cultures [23]
Summary
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. Stem cells derived from a range of different tissue sources and developmental stages have been studied for their capacity to elicit functional recovery in animal models of SCI [3,4] One such approach has been to generate immortalized neural stem cell lines from postmortem human fetal spinal cord tissue for transplantation [5,6,7,8]. We generated three clonal neural stem cell lines from human fetal spinal cord, designated SPC-01, SPC-04, and SPC-06, conditionally immortalized with 4-hydroxy tamoxifen (4-OHT)-inducible cMyc (cMycERTAM) [9] This technology involves transducing primary dissociated cells with a retrovirus containing the gene cMyc fused to a mutated form of the estrogen receptor. Removal of 4-OHT from the media results in inactivation of cMycER and terminal cellular differentiation [11]
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