Abstract
Traditional methods of generating immortalized lines of both somatic cells and their progenitors have relied on the use of oncogenes. However, the resulting lines are typically anaplastic in vitro and tumorigenic in vivo, and hence of limited utility. The overexpression of telomerase, as mediated by the induced overexpression of human telomerase reverse transcriptase (hTERT), has permitted the generation of stable, non-oncogenic lines of a variety of cell types. This strategy for immortalization has found special utility in the central nervous system, as few stable lines are available for the study of either human neural progenitor cells, or of neurons or glia of restricted phenotype. We describe the use of retroviral hTERT overexpression for generating lines of immortalized human neural progenitor cells, whose neuronal progeny are phenotypically restricted, post-mitotic and functionally competent. Although we focus here on telomerase immortalization of spinal neural progenitors, this is a broadly applicable protocol for using hTERT to immortalize human fetal neural progenitors of any pre-selected phenotype and for characterizing the cell lines thereby generated.
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Acknowledgements
This work was supported by NINDS grants R01NS39559 and R01NS33106, the Mathers Charitable Foundation, the National Multiple Sclerosis Society, the Michael J Fox Foundation, the NY State Spinal Cord Injury Research Program, the High Q Foundation and the Adelson Medical Research Foundation.
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Roy and Goldman hold US Patent 7,150,989, covering the use of hTERT over-expression for immortalizing CNS stem and progenitor cells. Dr. Goldman also holds US Patents 6,245,564 and 6,692,957, which cover the use of promoter-based cell sorting for isolating phenotypically-restricted progenitor cells, prior to immortalization. All patents are assigned to the Cornell Research Foundation.
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Roy, N., Chandler-Militello, D., Lu, G. et al. Retrovirally mediated telomerase immortalization of neural progenitor cells. Nat Protoc 2, 2815–2825 (2007). https://doi.org/10.1038/nprot.2007.402
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DOI: https://doi.org/10.1038/nprot.2007.402
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