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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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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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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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