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In vitro reprogramming of fibroblasts into a pluripotent ES-cell-like state

Nature volume 448, pages 318–324 (2007)Cite this article

Abstract

Nuclear transplantation can reprogramme a somatic genome back into an embryonic epigenetic state, and the reprogrammed nucleus can create a cloned animal or produce pluripotent embryonic stem cells. One potential use of the nuclear cloning approach is the derivation of ‘customized’ embryonic stem (ES) cells for patient-specific cell treatment, but technical and ethical considerations impede the therapeutic application of this technology. Reprogramming of fibroblasts to a pluripotent state can be induced in vitro through ectopic expression of the four transcription factors Oct4 (also called Oct3/4 or Pou5f1), Sox2, c-Myc and Klf4. Here we show that DNA methylation, gene expression and chromatin state of such induced reprogrammed stem cells are similar to those of ES cells. Notably, the cells—derived from mouse fibroblasts—can form viable chimaeras, can contribute to the germ line and can generate live late-term embryos when injected into tetraploid blastocysts. Our results show that the biological potency and epigenetic state of in-vitro-reprogrammed induced pluripotent stem cells are indistinguishable from those of ES cells.

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Figure 1: Generation of Oct4- and Nanog-selected iPS cells.
Figure 2: Expression and promoter methylation analysis of iPS cells.
Figure 3: Reprogrammed MEFs acquire an ES-cell-like epigenetic state.
Figure 4: Efficient silencing of retroviral transcripts in induced pluripotent cells.
Figure 5: Developmental pluripotency of reprogrammed fibroblasts.

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References

  1. Hochedlinger, K. & Jaenisch, R. Nuclear transplantation, embryonic stem cells, and the potential for cell therapy. N. Engl. J. Med. 349, 275–286 (2003)

    Article  CAS  Google Scholar 

  2. Yang, X. et al. Nuclear reprogramming of cloned embryos and its implications for therapeutic cloning. Nature Genet. 39, 295–302 (2007)

    Article  CAS  Google Scholar 

  3. Hochedlinger, K. & Jaenisch, R. Nuclear reprogramming and pluripotency. Nature 441, 1061–1067 (2006)

    Article  ADS  CAS  Google Scholar 

  4. Tada, M., Takahama, Y., Abe, K., Nakatsuji, N. & Tada, T. Nuclear reprogramming of somatic cells by in vitro hybridization with ES cells. Curr. Biol. 11, 1553–1558 (2001)

    Article  CAS  Google Scholar 

  5. Cowan, C. A., Atienza, J., Melton, D. A. & Eggan, K. Nuclear reprogramming of somatic cells after fusion with human embryonic stem cells. Science 309, 1369–1373 (2005)

    Article  ADS  CAS  Google Scholar 

  6. Jaenisch, R. Human cloning—the science and ethics of nuclear transplantation. N. Engl. J. Med. 351, 2787–2791 (2004)

    Article  CAS  Google Scholar 

  7. 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 

  8. Labosky, P. A., Barlow, D. P. & Hogan, B. L. Mouse embryonic germ (EG) cell lines: transmission through the germline and differences in the methylation imprint of insulin-like growth factor 2 receptor (Igf2r) gene compared with embryonic stem (ES) cell lines. Development 120, 3197–3204 (1994)

    CAS  PubMed  Google Scholar 

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

    Article  CAS  Google Scholar 

  10. Loh, Y. H. et al. The Oct4 and Nanog transcription network regulates pluripotency in mouse embryonic stem cells. Nature Genet. 38, 431–440 (2006)

    Article  CAS  Google Scholar 

  11. Lee, T. I. et al. Control of developmental regulators by Polycomb in human embryonic stem cells. Cell 125, 301–313 (2006)

    Article  CAS  Google Scholar 

  12. Boyer, L. A. et al. Polycomb complexes repress developmental regulators in murine embryonic stem cells. Nature 441, 349–353 (2006)

    Article  ADS  CAS  Google Scholar 

  13. Bernstein, B. E. et al. A bivalent chromatin structure marks key developmental genes in embryonic stem cells. Cell 125, 315–326 (2006)

    Article  CAS  Google Scholar 

  14. Azuara, V. et al. Chromatin signatures of pluripotent cell lines. Nature Cell Biol. 8, 532–538 (2006)

    Article  CAS  Google Scholar 

  15. Jackson-Grusby, L. et al. Loss of genomic methylation causes p53-dependent apoptosis and epigenetic deregulation. Nature Genet. 27, 31–39 (2001)

    Article  CAS  Google Scholar 

  16. Li, E., Bestor, T. H. & Jaenisch, R. Targeted mutation of the DNA methyltransferase gene results in embryonic lethality. Cell 69, 915–926 (1992)

    Article  CAS  Google Scholar 

  17. Meissner, A. et al. Reduced representation bisulfite sequencing for comparative high-resolution DNA methylation analysis. Nucleic Acids Res. 33, 5868–5877 (2005)

    Article  CAS  Google Scholar 

  18. 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 

  19. Holm, T. M. et al. Global loss of imprinting leads to widespread tumorigenesis in adult mice. Cancer Cell 8, 275–285 (2005)

    Article  CAS  Google Scholar 

  20. Okano, M., Bell, D. W., Haber, D. A. & Li, E. DNA methyltransferases Dnmt3a and Dnmt3b are essential for de novo methylation and mammalian development. Cell 99, 247–257 (1999)

    Article  CAS  Google Scholar 

  21. Stewart, C. L., Stuhlmann, H., Jähner, D. & Jaenisch, R. De novo methylation, expression, and infectivity of retroviral genomes introduced into embryonal carcinoma cells. Proc. Natl Acad. Sci. USA 79, 4098–4102 (1982)

    Article  ADS  CAS  Google Scholar 

  22. Jähner, D. et al. De novo methylation and expression of retroviral genomes during mouse embryogenesis. Nature 298, 623–628 (1982)

    Article  ADS  Google Scholar 

  23. Maherali, N. et al. Global epigenetic remodeling in directly reprogrammed fibroblasts. Cell Stem Cells (in the press)

  24. 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 

  25. Chapman, V., Forrester, L., Sanford, J., Hastie, N. & Rossant, J. Cell lineage specific undermethylation of mouse repetitive DNA. Nature 307, 284–286 (1984)

    Article  ADS  CAS  Google Scholar 

  26. Walsh, C. P., Chaillet, J. R. & Bestor, T. H. Transcription of IAP endogenous retroviruses is constrained by cytosine methylation. Nature Genet. 20, 116–117 (1998)

    Article  CAS  Google Scholar 

  27. Blelloch, R. et al. Reprogramming efficiency following somatic cell nuclear transfer is influenced by the differentiation and methylation state of the donor nucleus. Stem Cells 24, 2007–2013 (2006)

    Article  CAS  Google Scholar 

  28. Lucifero, D., Mertineit, C., Clarke, H. J., Bestor, T. H. & Trasler, J. M. Methylation dynamics of imprinted genes in mouse germ cells. Genomics 79, 530–538 (2002)

    Article  CAS  Google Scholar 

  29. Hochedlinger, K., Yamada, Y., Beard, C. & Jaenisch, R. Ectopic expression of Oct-4 blocks progenitor-cell differentiation and causes dysplasia in epithelial tissues. Cell 121, 465–477 (2005)

    Article  CAS  Google Scholar 

  30. Wernig, M. et al. Functional integration of embryonic stem cell-derived neurons in vivo. J. Neurosci. 24, 5258–5268 (2004)

    Article  CAS  Google Scholar 

  31. Brambrink, T., Hochedlinger, K., Bell, G. & Jaenisch, R. ES cells derived from cloned and fertilized blastocysts are transcriptionally and functionally indistinguishable. Proc. Natl Acad. Sci. USA 103, 933–938 (2006)

    Article  ADS  CAS  Google Scholar 

  32. Eads, C. A. & Laird, P. W. Combined bisulfite restriction analysis (COBRA). Methods Mol. Biol. 200, 71–85 (2002)

    CAS  PubMed  Google Scholar 

  33. Peitz, M., Pfannkuche, K., Rajewsky, K. & Edenhofer, F. Ability of the hydrophobic FGF and basic TAT peptides to promote cellular uptake of recombinant Cre recombinase: a tool for efficient genetic engineering of mammalian genomes. Proc. Natl Acad. Sci. USA 99, 4489–4494 (2002)

    Article  ADS  CAS  Google Scholar 

  34. Eggan, K. et al. Hybrid vigor, fetal overgrowth, and viability of mice derived by nuclear cloning and tetraploid embryo complementation. Proc. Natl Acad. Sci. USA 98, 6209–6214 (2001)

    Article  ADS  CAS  Google Scholar 

  35. Naviaux, R. K., Costanzi, E., Haas, M. & Verma, I. M. The pCL vector system: rapid production of helper-free, high-titer, recombinant retroviruses. J. Virol. 70, 5701–5705 (1996)

    CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

We thank H. Suh, D. Fu and J. Dausman for technical assistance; J. Love for help with the microarray analysis; S. Markoulaki for help with blastocyst injections; F. Edenhofer for a gift of Tat-Cre; and S. Yamanaka for the Nanog-neo construct. We acknowledge L. Zagachin in the MGH Nucleic Acid Quantitation core for assistance with real-time PCR. We also thank C. Lengner, C. Beard and M. Creyghton for constructive criticism. M.W. was supported in part by fellowships from the Human Frontiers Science Organization Program and the Ellison Foundation; B.B. by grants from the Burroughs Wellcome Fund, the Harvard Stem Cell Institute and the NIH; and R.J. by grants from the NIH. All microarray data from this study are available from Array Express at the EBI (http://www.ebi.ac.uk/arrayexpress) under the accession number E-MEXP-1037.

Author Contributions M.W., A.M. and R.J. conceived and designed the experiments and wrote the manuscript; M.W. derived all iPS lines; M.W. and A.M. performed the in vitro and in vivo characterization of the iPS lines (teratoma, 2N and 4N injections and IHC) and the conditional Dnmt1 experiment; A.M. investigated the promoter and imprinting methylation; M.K. and B.B. performed and analysed the real-time PCRs and ChIP experiments; R.F. and K.H. generated the selectable MEFs and TTFs; R.F. performed western blot and PCR analyses; and T.B. performed the microarray analysis and the proviral integration Southern blots.

Author information

Author notes

  1. Konrad Hochedlinger

    Present address: Present address: Center for Regenerative Medicine and Cancer Center, Massachusetts General Hospital, Harvard Medical School and Harvard Stem Cell Institute, Boston, Massachusetts 02414, USA.,

  2. Marius Wernig, Alexander Meissner, Ruth Foreman, Tobias Brambrink and Manching Ku: These authors contributed equally to this work.

Authors and Affiliations

  1. Whitehead Institute for Biomedical Research and,,

    Marius Wernig, Alexander Meissner, Ruth Foreman, Tobias Brambrink, Konrad Hochedlinger & Rudolf Jaenisch

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

    Ruth Foreman & Rudolf Jaenisch

  3. Molecular Pathology Unit and Center for Cancer Research, Massachusetts General Hospital, Charlestown, Massachusetts 02129, USA,

    Manching Ku & Bradley E. Bernstein

  4. Broad Institute of Harvard and MIT, Cambridge, Massachusetts 02142, USA,

    Bradley E. Bernstein

  5. Department of Pathology, Harvard Medical School, Boston, Massachusetts 02115, USA,

    Bradley E. Bernstein

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Correspondence to Rudolf Jaenisch.

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All microarray data from this study are available from Array Express at the EBI (http://www.ebi.ac.uk/arrayexpress) under the accession number E-MEXP-1037. Reprints and permissions information is available at www.nature.com/reprints. The authors declare no competing financial interests.

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This file contains Supplementary Figures S1-S5 with Legends and Supplementary Table S1. (PDF 4803 kb)

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Wernig, M., Meissner, A., Foreman, R. et al. In vitro reprogramming of fibroblasts into a pluripotent ES-cell-like state. Nature 448, 318–324 (2007). https://doi.org/10.1038/nature05944

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