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Concomitant activation of the Wnt pathway and suppression of Mapk signalling by two small molecule inhibitors (2i) in the presence of leukaemia inhibitory factor (LIF) (hereafter termed 2i/L) induces a naive state in mouse embryonic stem (ES) cells that resembles the inner cell mass (ICM) of the pre-implantation embryo1. Since the ICM exists only transiently in vivo, it remains unclear how sustained propagation of naive ES cells in vitro affects their stability and functionality. Here we show that prolonged culture of male mouse ES cells in 2i/L results in irreversible epigenetic and genomic changes that impair their developmental potential. Furthermore, we find that female ES cells cultured in conventional serum plus LIF medium phenocopy male ES cells cultured in 2i/L. Mechanistically, we demonstrate that the inhibition of Mek1/2 is predominantly responsible for these effects, in part through the downregulation of DNA methyltransferases and their cofactors. Finally, we show that replacement of the Mek1/2 inhibitor with a Src inhibitor preserves the epigenetic and genomic integrity as well as the developmental potential of ES cells. Taken together, our data suggest that, although short-term suppression of Mek1/2 in ES cells helps to maintain an ICM-like epigenetic state, prolonged suppression results in irreversible changes that compromise their developmental potential.

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  • 09 August 2017

    The name of the PKC inhibitor in the main text and Methods was corrected.


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We thank members of the Hochedlinger laboratory for suggestions as well as L. Barrett for critical reading of the manuscript. Additionally, we thank A. Galvin, M. Gesner and M. Handley at the Massachusetts General Hospital flow cytometry core. We also thank A. L. Hawkins, S. Wang, A. Aggarwal and C. C. Morton at Brigham and Woman’s Hospital CytoGenomics Core Laboratory for karyotyping analysis. We are grateful to K. Shioda from the Shioda laboratory for technical support for genome sequencing. We are also grateful to W. Yu at The Sidney Kimmel Cancer Center Microarray Core Facility at Johns Hopkins University for aCGH analysis, who was supported by NIH grant P30 CA006973 entitled Regional Oncology Research Center. We thank T. Theunissen and R. Jaenisch for sharing human OCT4–eGFP ES cell lines and T. Graf for providing the Rex1–GFP ES cells. A.J.H. is supported by an American Cancer Society–New England Division–Ellison Foundation Postdoctoral Fellowship (PF-15-130-01-DDC). B.D.S. is supported by an EMBO long-term Fellowship (ALTF 1143-2015). T.S. is supported by NIH grant R21ES024861. A.Me. is a New York Stem Cell Foundation Robertson Investigator and supported by NIH grant 1P50HG006193 and the New York Stem Cell Foundation. K.H. was supported by funds from the MGH, HHMI, NIH (R01 HD058013-06) and the Gerald and Darlene Jordan Chair in Regenerative Medicine.

Author information

Author notes

    • Jiho Choi
    •  & Aaron J. Huebner

    These authors contributed equally to this work.


  1. Massachusetts General Hospital Department of Molecular Biology, Boston, Massachusetts 02114, USA

    • Jiho Choi
    • , Aaron J. Huebner
    • , Ryan M. Walsh
    • , Andrej Savol
    • , Bruno Di Stefano
    • , Justin Brumbaugh
    • , Ruslan I. Sadreyev
    •  & Konrad Hochedlinger
  2. Massachusetts General Hospital Cancer Center and Center for Regenerative Medicine, Boston, Massachusetts 02114, USA

    • Jiho Choi
    • , Aaron J. Huebner
    • , Ryan M. Walsh
    • , Bruno Di Stefano
    • , Justin Brumbaugh
    • , Junko Odajima
    • , Toshi Shioda
    •  & Konrad Hochedlinger
  3. Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, Massachusetts 02138, USA

    • Jiho Choi
    • , Aaron J. Huebner
    • , Kendell Clement
    • , Ryan M. Walsh
    • , Bruno Di Stefano
    • , Justin Brumbaugh
    • , Alexander Meissner
    •  & Konrad Hochedlinger
  4. Harvard Stem Cell Institute, 1350 Massachusetts Avenue, Cambridge, Massachusetts 02138, USA

    • Jiho Choi
    • , Aaron J. Huebner
    • , Kendell Clement
    • , Ryan M. Walsh
    • , Bruno Di Stefano
    • , Justin Brumbaugh
    • , Alexander Meissner
    •  & Konrad Hochedlinger
  5. Broad Institute of MIT and Harvard, Cambridge, Massachusetts 02142, USA

    • Kendell Clement
    • , Hongcang Gu
    • , Arman Mohammad
    • , Andreas Gnirke
    •  & Alexander Meissner
  6. Department of Genetics, Yale University School of Medicine, 10 Amistad Street, New Haven, Connecticut 06519, USA

    • Kaixuan Lin
    •  & Andrew Xiao
  7. New York University Langone Medical Center, New York 10016, USA

    • Sang-Yong Kim
  8. Center for Integrative Medical Sciences, RIKEN National Research and Development Agency, 1-7-22 Suehiuro-cho, Tsurumi-ku, Yokohama-shi, Kanagawa-ken 230-0045, Japan

    • Jafar Sharif
    •  & Haruhiko Koseki
  9. Department of Cell Biology, Harvard Medical School, 240 Longwood Avenue, Boston, Massachusetts 02115, USA

    • Christopher M. Rose
    •  & Steven Gygi
  10. Centre de recherche sur le cancer de l’Université Laval, CRCHU de Québec, L’Hôtel-Dieu de Québec, 9, rue McMahon, Quebec G1R 2J6, Canada

    • Jean Charron


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J. Choi and A.J.H. performed the bulk of the cell culture, teratoma assays, dot blot and flow cytometry analyses; J. Choi, A.J.H. and S.-Y.K. performed the blastocyst injections; J. Choi, A.J.H. and J. Charron generated the Mek1/2 double-knockout iPS cell lines; R.M.W. generated the XGXT ES cell line; J.S. and H.K. generated the Dnmt triple-knockout ES cell line; J. Choi, A.J.H. and J.B. conducted the western blot analysis; J.B., C.M.R. and S.G. performed the proteomics analysis; B.D.S. performed experiments related to human ES cell culture; J.O. and T.S. performed the genome sequencing analyses and analysed the RNA-seq data of interspecies iPS cell lines; K.C., H.G., A.Mo., A.G. and A.Me. performed the RRBS analyses; A.S. and R.I.S. performed the RNA-seq analyses; K.L. and A.X. conducted the H2A.X ChIP–seq analysis.; J. Choi, A.J.H. and K.H. designed the experiments, interpreted the results and wrote the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Konrad Hochedlinger.

Reviewer Information Nature thanks T. Zwaka and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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