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.

  • Article
  • Published:

Direct cell reprogramming is a stochastic process amenable to acceleration

Nature volume 462, pages 595–601 (2009)Cite this article

Abstract

Direct reprogramming of somatic cells into induced pluripotent stem (iPS) cells can be achieved by overexpression of Oct4, Sox2, Klf4 and c-Myc transcription factors, but only a minority of donor somatic cells can be reprogrammed to pluripotency. Here we demonstrate that reprogramming by these transcription factors is a continuous stochastic process where almost all mouse donor cells eventually give rise to iPS cells on continued growth and transcription factor expression. Additional inhibition of the p53/p21 pathway or overexpression of Lin28 increased the cell division rate and resulted in an accelerated kinetics of iPS cell formation that was directly proportional to the increase in cell proliferation. In contrast, Nanog overexpression accelerated reprogramming in a predominantly cell-division-rate-independent manner. Quantitative analyses define distinct cell-division-rate-dependent and -independent modes for accelerating the stochastic course of reprogramming, and suggest that the number of cell divisions is a key parameter driving epigenetic reprogramming to pluripotency.

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

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

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

Figure 1: Models of progressing to a pluripotent state during direct reprogramming.
Figure 2: Long-term analysis of reprogramming monoclonal populations.
Figure 3: Cell-division-rate-dependent and -independent acceleration of reprogramming.
Figure 4: Quantitative analysis of cell reprogramming.
Figure 5: Distinct modes for accelerating reprogramming to pluripotency.

Similar content being viewed by others

References

  1. Hanna, J. et al. Treatment of sickle cell anemia mouse model with iPS cells generated from autologous skin. Science 318, 1920–1923 (2007)

    Article  CAS  ADS  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. Wernig, M. et al. In vitro reprogramming of fibroblasts into a pluripotent ES-cell-like state. Nature 448, 318–324 (2007)

    Article  CAS  ADS  Google Scholar 

  5. Yu, J. et al. Induced pluripotent stem cell lines derived from human somatic cells. Science 318, 1917–1920 (2007)

    Article  CAS  ADS  Google Scholar 

  6. Jaenisch, R. & Young, R. Stem cells, the molecular circuitry of pluripotency and nuclear reprogramming. Cell 132, 567–582 (2008)

    Article  CAS  Google Scholar 

  7. Yamanaka, S. Elite and stochastic models for induced pluripotent stem cell generation. Nature 460, 49–52 (2009)

    Article  CAS  ADS  Google Scholar 

  8. Hanna, J., Carey, B. W. & Jaenisch, R. Reprogramming of somatic cell identity. Cold Spring Harb. Symp. Quant. Biol. 10.1101/sqb.2008.73.025 (2008)

  9. Hanna, J. et al. Direct reprogramming of terminally differentiated mature B lymphocytes to pluripotency. Cell 133, 250–264 (2008)

    Article  CAS  Google Scholar 

  10. 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 

  11. Markoulaki, S. et al. Transgenic mice with defined combinations of drug-inducible reprogramming factors. Nature Biotechnol. 27, 169–171 (2009)

    Article  CAS  Google Scholar 

  12. Hanna, J. et al. Metastable pluripotent states in NOD-mouse-derived ESCs. Cell Stem Cell 4, 513–524 (2009)

    Article  CAS  Google Scholar 

  13. Eminli, S. et al. Differentiation stage determines potential of hematopoietic cells for reprogramming into induced pluripotent stem cells. Nature Genet. 41, 968–976 (2009)

    Article  CAS  Google Scholar 

  14. Sridharan, R. et al. Role of the murine reprogramming factors in the induction of pluripotency. Cell 136, 364–377 (2009)

    Article  CAS  Google Scholar 

  15. Mikkelsen, T. S. et al. Dissecting direct reprogramming through integrative genomic analysis. Nature 454, 49–55 (2008)

    Article  CAS  ADS  Google Scholar 

  16. Utikal, J. et al. Immortalization eliminates a roadblock during cellular reprogramming into iPS cells. Nature 460, 1145–1148 (2009)

    Article  CAS  ADS  Google Scholar 

  17. Huangfu, D. et al. Induction of pluripotent stem cells by defined factors is greatly improved by small-molecule compounds. Nature Biotechnol. 26, 795–797 (2008)

    Article  CAS  Google Scholar 

  18. Signer, R. A., Montecino-Rodriguez, E., Witte, O. N. & Dorshkind, K. Aging and cancer resistance in lymphoid progenitors are linked processes conferred by p16Ink4a and Arf. Genes Dev. 22, 3115–3120 (2008)

    Article  CAS  Google Scholar 

  19. Chen, J., Lansford, R., Stewart, V., Young, F. & Alt, F. W. RAG-2-deficient blastocyst complementation: an assay of gene function in lymphocyte development. Proc. Natl Acad. Sci. USA 90, 4528–4532 (1993)

    Article  CAS  ADS  Google Scholar 

  20. Okita, K., Ichisaka, T. & Yamanaka, S. Generation of germline-competent induced pluripotent stem cells. Nature 448, 313–317 (2007)

    Article  CAS  ADS  Google Scholar 

  21. 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 

  22. 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 

  23. Banito, A. et al. Senescence impairs successful reprogramming to pluripotent stem cells. Genes Dev. 23, 2134–2139 (2009)

    Article  CAS  Google Scholar 

  24. Hong, H. et al. Suppression of induced pluripotent stem cell generation by the p53-p21 pathway. Nature 460, 1132–1135 (2009)

    Article  CAS  ADS  Google Scholar 

  25. Kawamura, T. et al. Linking the p53 tumour suppressor pathway to somatic cell reprogramming. Nature 460, 1140–1144 (2009)

    Article  CAS  ADS  Google Scholar 

  26. Li, H. et al. The Ink4/Arf locus is a barrier for iPS cell reprogramming. Nature 460, 1136–1139 (2009)

    Article  CAS  ADS  Google Scholar 

  27. Marión, R. M. et al. A p53-mediated DNA damage response limits reprogramming to ensure iPS cell genomic integrity. Nature 460, 1149–1153 (2009)

    Article  ADS  Google Scholar 

  28. Ventura, A. et al. Cre-lox-regulated conditional RNA interference from transgenes. Proc. Natl Acad. Sci. USA 101, 10380–10385 (2004)

    Article  CAS  ADS  Google Scholar 

  29. Rowland, B. D., Bernards, R. & Peeper, D. S. The KLF4 tumour suppressor is a transcriptional repressor of p53 that acts as a context-dependent oncogene. Nature Cell Biol. 7, 1074–1082 (2005)

    Article  CAS  Google Scholar 

  30. Xu, B., Zhang, K. & Huang, Y. Lin28 modulates cell growth and associates with a subset of cell cycle regulator mRNAs in mouse embryonic stem cells. RNA 15, 357–361 (2009)

    Article  CAS  Google Scholar 

  31. Silva, J. et al. Nanog is the gateway to the pluripotent ground state. Cell 138, 722–737 (2009)

    Article  CAS  Google Scholar 

  32. Chambers, I. et al. Nanog safeguards pluripotency and mediates germline development. Nature 450, 1230–1234 (2007)

    Article  CAS  ADS  Google Scholar 

  33. Mitsui, K. et al. The homeoprotein Nanog is required for maintenance of pluripotency in mouse epiblast and ES cells. Cell 113, 631–642 (2003)

    Article  CAS  Google Scholar 

  34. Navarro, P. et al. Molecular coupling of Xist regulation and pluripotency. Science 321, 1693–1695 (2008)

    Article  CAS  ADS  Google Scholar 

  35. Silva, J., Chambers, I., Pollard, S. & Smith, A. Nanog promotes transfer of pluripotency after cell fusion. Nature 441, 997–1001 (2006)

    Article  CAS  ADS  Google Scholar 

  36. Darr, H., Mayshar, Y. & Benvenisty, N. Overexpression of NANOG in human ES cells enables feeder-free growth while inducing primitive ectoderm features. Development 133, 1193–1201 (2006)

    Article  CAS  Google Scholar 

  37. Niwa, H., Ogawa, K., Shimosato, D. & Adachi, K. A parallel circuit of LIF signalling pathways maintains pluripotency of mouse ES cells. Nature 460, 118–122 (2009)

    Article  CAS  ADS  Google Scholar 

  38. Huang, S. Reprogramming cell fates: reconciling rarity with robustness. Bioessays 31, 546–560 (2009)

    Article  CAS  Google Scholar 

  39. Kærn, M., Elston, T. C., Blake, W. J. & Collins, J. J. Stochasticity in gene expression: from theories to phenotypes. Nature Rev. Genet. 6, 451–464 (2005)

    Article  Google Scholar 

  40. Raj, A. & van Oudenaarden, A. Nature, nurture, or chance: stochastic gene expression and its consequences. Cell 135, 216–226 (2008)

    Article  CAS  Google Scholar 

  41. Boyer, L. A. et al. Core transcriptional regulatory circuitry in human embryonic stem cells. Cell 122, 947–956 (2005)

    Article  CAS  Google Scholar 

  42. Egli, D., Birkhoff, G. & Eggan, K. Mediators of reprogramming: transcription factors and transitions through mitosis. Nature Rev. Mol. Cell Biol. 9, 505–516 (2008)

    Article  CAS  Google Scholar 

  43. Kim, J. B. et al. Oct4-induced pluripotency in adult neural stem cells. Cell 136, 411–419 (2009)

    Article  CAS  Google Scholar 

  44. Shi, Y. et al. Induction of pluripotent stem cells from mouse embryonic fibroblasts by Oct4 and Klf4 with small-molecule compounds. Cell Stem Cell 3, 568–574 (2008)

    Article  CAS  Google Scholar 

  45. Boiani, M. et al. Variable reprogramming of the pluripotent stem cell marker Oct4 in mouse clones: distinct developmental potentials in different culture environments. Stem Cells 23, 1089–1104 (2005)

    Article  CAS  Google Scholar 

  46. Egli, D., Rosains, J., Birkhoff, G. & Eggan, K. Developmental reprogramming after chromosome transfer into mitotic mouse zygotes. Nature 447, 679–685 (2007)

    Article  CAS  ADS  Google Scholar 

Download references

Acknowledgements

We would like to thank N. Maheshri, K. Wood, Y. Bugnanim, B. Carey, J. Cassady and F. Soldner for commenting on the manuscript; J. Dausmann, S. Markoulaki, P. Schorderet and M. Pawlak for conducting blastocysts injections; and members of the Jaenisch laboratory for discussions. R.J. is supported by grants from the NIH (RO1-HDO45022, R37-CA084198, RO1-CA087869). J.H. is supported by a postdoctoral fellowship from Helen Hay Whitney Foundation. K.S. is supported by the Society in Science: The Brano-Weiss fellowship. J.v.Z. is supported by the Human Frontiers Science Program. A.v.O. is supported by a National Institutes of Health (NIH) Director’s Pioneer award and an award from the National Cancer Institute (U54CA143874).

Author Contributions J.H., K.S. and R.J. conceived the ideas for this study and designed the experiments. J.H. conducted the reprogramming experiments and data collection. M.P.C. and C.J.L. assisted in construct preparation, transgene induction and blastocyst injections. K.S., B.P., J.v.Z, J.H., and A.v.O. performed statistical and numerical analyses, and also constructed the stochastic models. J.H., K.S. and R.J. wrote the manuscript with contributions from all other authors.

Author information

Author notes

  1. Jacob Hanna and Krishanu Saha: These authors contributed equally to this work.

Authors and Affiliations

  1. The Whitehead Institute for Biomedical Research,,

    Jacob Hanna, Krishanu Saha, Christopher J. Lengner, Menno P. Creyghton & Rudolf Jaenisch

  2. Department of Physics,,

    Bernardo Pando, Jeroen van Zon & Alexander van Oudenaarden

  3. Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02142, USA,

    Jeroen van Zon, Alexander van Oudenaarden & Rudolf Jaenisch

Authors
  1. Jacob Hanna
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Krishanu Saha
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Bernardo Pando
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Jeroen van Zon
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Christopher J. Lengner
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Menno P. Creyghton
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Alexander van Oudenaarden
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Rudolf Jaenisch
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Jacob Hanna or Rudolf Jaenisch.

Ethics declarations

Competing interests

R.J. is an advisor to Stemgent and a cofounder of Fate Therapeutics.

Supplementary information

Supplementary Figures

This file contains Supplementary Figures 1 - 19 with Legends. (PDF 3164 kb)

PowerPoint slides

Rights and permissions

Reprints and permissions

About this article

Cite this article

Hanna, J., Saha, K., Pando, B. et al. Direct cell reprogramming is a stochastic process amenable to acceleration . Nature 462, 595–601 (2009). https://doi.org/10.1038/nature08592

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nature08592

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

Advanced 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