Letter | Published:

Derivation of ground-state female ES cells maintaining gamete-derived DNA methylation

Nature volume 548, pages 224227 (10 August 2017) | Download Citation

Abstract

Inhibitors of Mek1/2 and Gsk3β, known as 2i, enhance the derivation of embryonic stem (ES) cells and promote ground-state pluripotency in rodents1,2. Here we show that the derivation of female mouse ES cells in the presence of 2i and leukaemia inhibitory factor (2i/L ES cells) results in a widespread loss of DNA methylation, including a massive erasure of genomic imprints. Despite this global loss of DNA methylation, early-passage 2i/L ES cells efficiently differentiate into somatic cells, and this process requires genome-wide de novo DNA methylation. However, the majority of imprinting control regions (ICRs) remain unmethylated in 2i/L-ES-cell-derived differentiated cells. Consistently, 2i/L ES cells exhibit impaired autonomous embryonic and placental development by tetraploid embryo complementation or nuclear transplantation. We identified the derivation conditions of female ES cells that display 2i/L-ES-cell-like transcriptional signatures while preserving gamete-derived DNA methylation and autonomous developmental potential. Upon prolonged culture, however, female ES cells exhibited ICR demethylation regardless of culture conditions. Our results provide insights into the derivation of female ES cells reminiscent of the inner cell mass of preimplantation embryos.

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Acknowledgements

We are grateful to T. Shiroishi, M. Saitou and S. Yokobayashi for helpful suggestions, K. Woltjen for providing piggyBac vectors, P. Karagiannis for critical reading of this manuscript, and T. Ukai, M. Kabata, S. Sakurai, D. Seki and T. Sato for technical assistance. Y.Y. was supported in part by P-CREATE, SICORP, Japan Agency for Medical Research and Development (AMED); JSPS KAKENHI 15H04721; the Princess Takamatsu Cancer Research Fund; the Takeda Science Foundation; and the Naito Foundation. Y.Y. and T.Y. were supported by Core Center for iPS Cell Research, Research Center Network for Realization of Regenerative Medicine, AMED. T.Y. was supported by AMED-CREST; JSPS KAKENHI 15H01352; and iPS Cell Research Fund. M.Y. was supported by JSPS KAKENHI 15J05792.

Author information

Affiliations

  1. Department of Life Science Frontiers, Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto 606-8507, Japan

    • Masaki Yagi
    • , Akito Tanaka
    • , Katsunori Semi
    • , Takuya Yamamoto
    •  & Yasuhiro Yamada
  2. Faculty of Life and Environmental Sciences, University of Yamanashi, Kofu, Yamanashi 400-8510, Japan

    • Satoshi Kishigami
    • , Eiji Mizutani
    • , Sayaka Wakayama
    •  & Teruhiko Wakayama
  3. Advanced Biotechnology Center, University of Yamanashi, Kofu, Yamanashi 400-8510, Japan

    • Eiji Mizutani
    • , Sayaka Wakayama
    •  & Teruhiko Wakayama
  4. AMED-CREST, AMED 1-7-1 Otemachi, Chiyodaku, Tokyo 100-0004, Japan

    • Takuya Yamamoto

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Contributions

M.Y. and Y.Y. designed and conceived the study and wrote the manuscript. M.Y. generated cell lines, performed experiments, analysed microarray data and generated WGBS and methyl-seq libraries. S.K., E.M., S.W. and T.W. performed nuclear transfer. K.S. provided technical instructions. A.T., S.K., S.W. and T.W. performed 2n and 4n blastocyst injections. T.Y. analysed all the WGBS, methyl-seq and RNA-seq data.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Takuya Yamamoto or Yasuhiro Yamada.

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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    Supplementary Data

    This file contains uncropped gel image data from figures 4g and extended data figures 4c, 6h, 8c, 8k, 9c, 9d, 9g, 10b and 10c.

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DOI

https://doi.org/10.1038/nature23286

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