Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Protocol
  • Published:

Retrovirally mediated telomerase immortalization of neural progenitor cells

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.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

Figure 1: hTERT transduction of progenitor cells can yield phenotypically restricted lines.
Figure 2: Immortalized lines sustain high levels of telomerase and long telomeres.
Figure 3: hSC11V-TERT cells retain mitotic and karyotypic stability.

Similar content being viewed by others

References

  1. Wichterle, H., Lieberam, I., Porter, J.A. & Jessell, T.M. Directed differentiation of embryonic stem cells into motor neurons 110, 385–397 (2002).

  2. Roy, N.S. et al. Functional engraftment of human ES cell-derived dopaminergic neurons enriched by co-culture with telomerase-immortalized midbrain astrocytes. Nat. Med. 12, 1259–1268 (2006).

    Article  CAS  Google Scholar 

  3. Roy, N. et al. Enhancer-specified GFP-based FACS purification of human spinal motor neurons from embryonic stem cells. Exp. Neurol. 196, 224–234 (2005).

    Article  CAS  Google Scholar 

  4. Windrem, M.S. et al. Fetal and adult human oligodendrocyte progenitor cell isolates myelinate the congenitally dysmyelinated brain. Nat. Med. 10, 93–97 (2004).

    Article  CAS  Google Scholar 

  5. Dietrich, J., Noble, M. & Mayer-Proschel, M. Characterization of A2B5+ glial precursor cells from cryopreserved human fetal brain progenitor cells. Glia 40, 65–77 (2002).

    Article  Google Scholar 

  6. Roy, N.S. et al. In vitro neurogenesis by progenitor cells isolated from the adult human hippocampus. Nat. Med. 6, 271–277 (2000).

    Article  CAS  Google Scholar 

  7. Roy, N.S. et al. Identification, isolation, and promoter-defined separation of mitotic oligodendrocyte progenitor cells from the adult human subcortical white matter. J. Neurosci. 19, 9986–9995 (1999).

    Article  CAS  Google Scholar 

  8. Goldman, S.A. Stem and progenitor cell-based therapy of the human central nervous system. Nat. Biotechnol. 23, 862–871 (2005).

    Article  CAS  Google Scholar 

  9. Wright, W., Piatyszek, M., Rainey, W., Byrd, W. & Shay, J. Telomerase activity in human germline and embryonic tissues and cells. Dev. Genet. 18, 173–179 (1996).

    Article  CAS  Google Scholar 

  10. Harley, C.B., Futcher, A.B. & Greider, C.W. Telomeres shorten during ageing of human fibroblasts. Nature 345, 458–460 (1990).

    Article  CAS  Google Scholar 

  11. Allsopp, R. et al. Telomere length predicts replicative capacity of human fibroblasts. Proc. Natl. Acad. Sci. USA 89, 10114–10118 (1992).

    Article  CAS  Google Scholar 

  12. Ulaner, G. & Giudice, L. Developmental regulation of telomerase activity in human fetal tissues during gestation. Mol. Hum. Reprod. 3, 769–773 (1997).

    Article  CAS  Google Scholar 

  13. Ostenfeld, T. et al. Human neural precursor cells express low levels of telomerase in vitro and show diminishing cell proliferation with extensive axonal outgrowth following transplantation. Exp. Neurol. 164, 215–226 (2000).

    Article  CAS  Google Scholar 

  14. Bodnar, A.G. et al. Extension of life-span by introduction of telomerase into normal human cells. Science 279, 349–352 (1998).

    CAS  Google Scholar 

  15. Morales, C. et al. Absence of cancer-associated changes in human fibroblasts immortalized with telomerase. Nat. Genet. 21, 115–118 (1999).

    Article  CAS  Google Scholar 

  16. Forsyth, N.R., Evans, A.P., Shay, J.W. & Wright, W.E. Developmental differences in the immortalization of lung fibroblasts by telomerase. Aging Cell 2, 235–243 (2003).

    Article  CAS  Google Scholar 

  17. Yang, J. et al. Human endothelial cell life extension by telomerase expression. J. Biol. Chem. 274, 26141–26148 (1999).

    Article  CAS  Google Scholar 

  18. Jiang, X.R. et al. Telomerase expression in human somatic cells does not induce changes associated with a transformed phenotype. Nat. Genet. 21, 111–114 (1999).

    Article  CAS  Google Scholar 

  19. Wright, W.E. & Shay, J.W. Telomere biology in aging and cancer. J. Am. Geriatr. Soc. 53, S292–S294 (2005).

    Article  Google Scholar 

  20. Wang, J., Hannon, G.J. & Beach, D.H. Risky immortalization by telomerase. Nature 405, 755–756 (2000).

    Article  CAS  Google Scholar 

  21. Zhu, C.-H. et al. Cellular senescence in human myoblasts is overcome by hTERT and cyclin-dependent kinase 4: consequences in aging muscle and therapeutic strategies for muscular dystrophies. Aging Cell 6, 515–523 (2007).

    Article  CAS  Google Scholar 

  22. Yang, G. et al. Knockdown of p53 combined with expression of the catalytic subunit of telomerase is sufficient to immortalize primary human ovarian surface epithelial cells. Carcinogenesis 28, 174–182 (2007).

    Article  CAS  Google Scholar 

  23. Haga, K. et al. Efficient immortalization of primary human cells by p16INK4a-specific short hairpin RNA or Bmi-1, combined with introduction of hTERT. Cancer Sci. 98, 147–154 (2007).

    Article  CAS  Google Scholar 

  24. Yang, G., Rosen, D.G., Colacino, J.A., Mercado-Uribe, I. & Liu, J. Disruption of the retinoblastoma pathway by small interfering RNA and ectopic expression of the catalytic subunit of telomerase lead to immortalization of human ovarian surface epithelial cells. Oncogene 26, 1492–1498 (2007).

    Article  CAS  Google Scholar 

  25. Goldman, S. & Sim, F. Neural progenitor cells of the adult brain. in Stem Cells: Nuclear Programming and Therapeutic Applications. Novartis Foundation Symposium no. 265 (ed. Gearhart, J.) 66–92 (John Wiley, London, 2005).

    Google Scholar 

  26. Goldman, S. Glia as neural progenitor cells. Trends Neurosci. 26, 590–596 (2003).

    Article  CAS  Google Scholar 

  27. Roy, N.S. et al. Telomerase immortalization of neuronally restricted progenitor cells derived from the human fetal spinal cord. Nat. Biotechnol. 22, 297–305 (2004).

    Article  CAS  Google Scholar 

  28. Sherr, C. & DePinho, R. Cellular senescence: mitotic clock or culture shock? Cell 18, 407–410 (2000).

    Article  Google Scholar 

  29. Todaro, G., Habel, K. & Green, H. Antigenic and cultural properties of cells doubly transformed by polyoma virus and SV40. Virology 27, 179–185 (1965).

    Article  CAS  Google Scholar 

  30. Wright, W.E., Shay, J.W. & Piatyszek, M.A. Modifications of a telomeric repeat amplification protocol (TRAP) result in increased reliability, linearity and sensitivity. Nucleic Acids Res. 23, 3794–3795 (1995).

    Article  CAS  Google Scholar 

  31. Herbert, B.-S., Hiochreiter, A., Wright, W. & Shay, J. Nonradioactive detection of telomerase activity using the telomeric repeat amplification protocol. Nat. Protoc. 1, 1583–1590 (2006).

    Article  CAS  Google Scholar 

  32. Watson, J., Chambers, S. & Smith, P. A pragmatic approach to the analysis of DNA histograms with a definable G1 peak. Cytometry 8, 1–8 (1987).

    Article  CAS  Google Scholar 

  33. Terry, N. & White, R. Flow cytometry after bromodeoxyuridine labeling to measure S and G2+M phase durations plus doubling times in vitro and in vivo. Nat. Protoc. 1, 859–869 (2006).

    Article  CAS  Google Scholar 

Download references

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.

Author information

Authors and Affiliations

Authors

Corresponding authors

Correspondence to Neeta S Roy or Steven A Goldman.

Ethics declarations

Competing interests

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.

Rights and permissions

Reprints and permissions

About this article

Cite this article

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

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nprot.2007.402

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing