Immortalization eliminates a roadblock during cellular reprogramming into iPS cells (original) (raw)

Nature volume 460, pages 1145–1148 (2009)Cite this article

Abstract

The overexpression of defined transcription factors in somatic cells results in their reprogramming into induced pluripotent stem (iPS) cells1,2,3. The extremely low efficiency and slow kinetics of in vitro reprogramming suggest that further rare events are required to generate iPS cells. The nature and identity of these events, however, remain elusive. We noticed that the reprogramming potential of primary murine fibroblasts into iPS cells decreases after serial passaging and the concomitant onset of senescence. Consistent with the notion that loss of replicative potential provides a barrier for reprogramming, here we show that cells with low endogenous p19Arf (encoded by the Ink4a/Arf locus, also known as Cdkn2a locus) protein levels and immortal fibroblasts deficient in components of the Arf–Trp53 pathway yield iPS cell colonies with up to threefold faster kinetics and at a significantly higher efficiency than wild-type cells, endowing almost every somatic cell with the potential to form iPS cells. Notably, the acute genetic ablation of Trp53 (also known as p53) in cellular subpopulations that normally fail to reprogram rescues their ability to produce iPS cells. Our results show that the acquisition of immortality is a crucial and rate-limiting step towards the establishment of a pluripotent state in somatic cells and underscore the similarities between induced pluripotency and tumorigenesis.

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References

  1. Hochedlinger, K. & Plath, K. Epigenetic reprogramming and induced pluripotency. Development 136, 509–523 (2009)
    Article CAS Google Scholar
  2. Takahashi, K. et al. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131, 861–872 (2007)
    Article CAS Google Scholar
  3. Takahashi, K. & Yamanaka, S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126, 663–676 (2006)
    Article CAS Google Scholar
  4. Maherali, N. et al. Directly reprogrammed fibroblasts show global epigenetic remodeling and widespread tissue contribution. Cell Stem Cell 1, 55–70 (2007)
    Article CAS Google Scholar
  5. Okita, K., Ichisaka, T. & Yamanaka, S. Generation of germline-competent induced pluripotent stem cells. Nature 448, 313–317 (2007)
    Article ADS CAS Google Scholar
  6. Wernig, M. et al. In vitro reprogramming of fibroblasts into a pluripotent ES-cell-like state. Nature 448, 318–324 (2007)
    Article ADS CAS Google Scholar
  7. Hockemeyer, D. et al. A drug-inducible system for direct reprogramming of human somatic cells to pluripotency. Cell Stem Cell 3, 346–353 (2008)
    Article CAS Google Scholar
  8. Maherali, N. et al. A high-efficiency system for the generation and study of human induced pluripotent stem cells. Cell Stem Cell 3, 340–345 (2008)
    Article CAS Google Scholar
  9. Wernig, M. et al. A drug-inducible transgenic system for direct reprogramming of multiple somatic cell types. Nature Biotechnol. 26, 916–924 (2008)
    Article CAS Google Scholar
  10. Collado, M., Blasco, M. A. & Serrano, M. Cellular senescence in cancer and aging. Cell 130, 223–233 (2007)
    Article CAS Google Scholar
  11. Parrinello, S. et al. Oxygen sensitivity severely limits the replicative lifespan of murine fibroblasts. Nature Cell Biol. 5, 741–747 (2003)
    Article CAS Google Scholar
  12. Zindy, F. et al. Arf tumor suppressor promoter monitors latent oncogenic signals in vivo . Proc. Natl Acad. Sci. USA 100, 15930–15935 (2003)
    Article ADS CAS Google Scholar
  13. Stadtfeld, M., Maherali, N., Breault, D. T. & Hochedlinger, K. Defining molecular cornerstones during fibroblast to iPS cell reprogramming in mouse. Cell Stem Cell 2, 230–240 (2008)
    Article CAS Google Scholar
  14. Brambrink, T. et al. Sequential expression of pluripotency markers during direct reprogramming of mouse somatic cells. Cell Stem Cell 2, 151–159 (2008)
    Article CAS Google Scholar
  15. Sharpless, N. E. et al. Loss of p16Ink4a with retention of p19Arf predisposes mice to tumorigenesis. Nature 413, 86–91 (2001)
    Article ADS CAS Google Scholar
  16. Serrano, M. et al. Role of the INK4a locus in tumor suppression and cell mortality. Cell 85, 27–37 (1996)
    Article CAS Google Scholar
  17. Bennett, D. C., Cooper, P. J. & Hart, I. R. A line of non-tumorigenic mouse melanocytes, syngeneic with the B16 melanoma and requiring a tumour promoter for growth. Int. J. Cancer 39, 414–418 (1987)
    Article CAS Google Scholar
  18. Kamijo, T. et al. Tumor suppression at the mouse INK4a locus mediated by the alternative reading frame product p19ARF. Cell 91, 649–659 (1997)
    Article CAS Google Scholar
  19. Hanna, J. et al. Direct reprogramming of terminally differentiated mature B lymphocytes to pluripotency. Cell 133, 250–264 (2008)
    Article CAS Google Scholar
  20. Ventura, A. et al. Cre-lox-regulated conditional RNA interference from transgenes. Proc. Natl Acad. Sci. USA 101, 10380–10385 (2004)
    Article ADS CAS Google Scholar
  21. Dickson, M. A. et al. Human keratinocytes that express hTERT and also bypass a p16(INK4a)-enforced mechanism that limits life span become immortal yet retain normal growth and differentiation characteristics. Mol. Cell. Biol. 20, 1436–1447 (2000)
    Article CAS Google Scholar
  22. Mali, P. et al. Improved efficiency and pace of generating induced pluripotent stem cells from human adult and fetal fibroblasts. Stem Cells 26, 1998–2005 (2008)
    Article CAS Google Scholar
  23. Zhao, Y. et al. Two supporting factors greatly improve the efficiency of human iPSC generation. Cell Stem Cell 3, 475–479 (2008)
    Article CAS Google Scholar
  24. Molofsky, A. V. et al. Increasing p16INK4a expression decreases forebrain progenitors and neurogenesis during ageing. Nature 443, 448–452 (2006)
    Article ADS CAS Google Scholar
  25. Krishnamurthy, J. et al. p16INK4a induces an age-dependent decline in islet regenerative potential. Nature 443, 453–457 (2006)
    Article ADS CAS Google Scholar
  26. Janzen, V. et al. Stem-cell ageing modified by the cyclin-dependent kinase inhibitor p16INK4a. Nature 443, 421–426 (2006)
    Article ADS CAS Google Scholar
  27. Eminli, S. et al. Differentiation stage determines reprogramming potential of hematopoietic cells into iPS cells. Nature Genet (in the press)
  28. Sommer, C. A. et al. iPS cell generation using a single lentiviral stem cell cassette. Stem Cells 27, 543–549 (2008)
    Article Google Scholar
  29. Ventura, A. et al. Restoration of p53 function leads to tumour regression in vivo . Nature 445, 661–665 (2007)
    Article CAS Google Scholar

Download references

Acknowledgements

We thank M. Roussel and C. Sherr for providing us with _Arf_–GFP cells, D. C. Bennett and E. Sviderskaya for sharing Melan A cells, and A. Ventura and T. Jacks for tail biopsies of conditional _Trp53_-mutant mice. We also thank A. Tzatsos and N. Bardeesy for suggestions, for critical reading of the manuscript and for providing _Ink4a/Arf_-/- MEFs. We are grateful to P. Follett for blastocyst injections and L. Prickett and K. Folz-Donahue for assistance with FACS. J.U. was supported by a postdoctoral fellowship from the Mildred Scheel Foundation, J.M.P. by an ECOR fellowship, and M.S. by a fellowship from the Schering Foundation. J.G.R. was supported by an NIH Skin Disease Research Center Grant. N.M. was supported by a graduated scholarship from the Natural Sciences and Engineering Council of Canada. Support to K.H. came from the NIH Director’s Innovator Award, the Harvard Stem Cell Institute, the Kimmel Foundation and the V Foundation.

Author Contributions J.U., J.M.P. and K.H. conceived the study, interpreted results and wrote the manuscript, J.U. and J.M.P. performed most of the experiments with help from W.K., R.M.W. and A.K. M.S., N.M. and J.G.R. provided essential study material and helped with interpretation of results.

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Author notes

  1. Jochen Utikal and Jose M. Polo: These authors contributed equally to this work.

Authors and Affiliations

  1. Massachusetts General Hospital Cancer Center and Center for Regenerative Medicine, Harvard Stem Cell Institute, 185 Cambridge Street, Boston, Massachusetts 02114, USA ,
    Jochen Utikal, Jose M. Polo, Matthias Stadtfeld, Nimet Maherali, Warakorn Kulalert, Ryan M. Walsh, Adam Khalil & Konrad Hochedlinger
  2. Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, Massachusetts 02138, USA,
    Jochen Utikal, Jose M. Polo, Matthias Stadtfeld, Nimet Maherali, Warakorn Kulalert, Ryan M. Walsh, Adam Khalil & Konrad Hochedlinger
  3. Department of Dermatology, Venereology and Allergology, University Medical Center Mannheim, Ruprecht-Karl-University of Heidelberg, Theodor-Kutzer-Ufer 1-3, 68135 Mannheim, Germany,
    Jochen Utikal
  4. Department of Molecular and Cellular Biology, Harvard University, 7 Divinity Avenue, Cambridge, Massachusetts 02138, USA,
    Nimet Maherali
  5. Department of Dermatology, Brigham and Women’s Hospital and Harvard Skin Disease Research Center, 77 Avenue Louis Pasteur, Boston, Massachusetts 02115, USA,
    James G. Rheinwald

Authors

  1. Jochen Utikal
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  2. Jose M. Polo
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  3. Matthias Stadtfeld
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  4. Nimet Maherali
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  5. Warakorn Kulalert
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  6. Ryan M. Walsh
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  7. Adam Khalil
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  8. James G. Rheinwald
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  9. Konrad Hochedlinger
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Corresponding author

Correspondence toKonrad Hochedlinger.

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Utikal, J., Polo, J., Stadtfeld, M. et al. Immortalization eliminates a roadblock during cellular reprogramming into iPS cells.Nature 460, 1145–1148 (2009). https://doi.org/10.1038/nature08285

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Editorial Summary

On iPS cells and p53: removing the roadblock

Pluripotency can be induced in somatic cells by overexpression of a set of transcription factors. The process has extremely low efficiency and slow kinetics. Here Utikal et al. show that cells with low endogenous p19Arf levels and immortal fibroblasts deficient for components of the Ink4a/Arf/p53 pathway yield iPS colonies with a threefold faster kinetics and at a significantly higher efficiency compared with wild-type cells, reaching frequencies of up to 100%. Genetic deletion of p53 in cellular subpopulations that normally fail to reprogram rescues their ability to produce iPS cells.

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