Letter | Published:

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

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


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.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.


Primary accessions

Gene Expression Omnibus


  1. 1.

    & Naive and primed pluripotent states. Cell Stem Cell 4, 487–492 (2009)

  2. 2.

    & Regulatory principles of pluripotency: from the ground state up. Cell Stem Cell 15, 416–430 (2014)

  3. 3.

    et al. A unique regulatory phase of DNA methylation in the early mammalian embryo. Nature 484, 339–344 (2012)

  4. 4.

    et al. The ground state of embryonic stem cell self-renewal. Nature 453, 519–523 (2008)

  5. 5.

    et al. FGF signaling inhibition in ES cells drives rapid genome-wide demethylation to the epigenetic ground state of pluripotency. Cell Stem Cell 13, 351–359 (2013)

  6. 6.

    et al. Whole-genome bisulfite sequencing of two distinct interconvertible DNA methylomes of mouse embryonic stem cells. Cell Stem Cell 13, 360–369 (2013)

  7. 7.

    et al. Synergistic mechanisms of DNA demethylation during transition to ground-state pluripotency. Stem Cell Reports 1, 518–531 (2013)

  8. 8.

    et al. Naive pluripotency is associated with global DNA hypomethylation. Nat. Struct. Mol. Biol. 20, 311–316 (2013)

  9. 9.

    et al. The transcriptional and epigenomic foundations of ground state pluripotency. Cell 149, 590–604 (2012)

  10. 10.

    et al. Capture of authentic embryonic stem cells from rat blastocysts. Cell 135, 1287–1298 (2008)

  11. 11.

    et al. Derivation and characterization of mouse embryonic stem cells from permissive and nonpermissive strains. Nat. Protocols 9, 559–574 (2014)

  12. 12.

    et al. Naive pluripotent stem cells derived directly from isolated cells of the human inner cell mass. Stem Cell Reports 6, 437–446 (2016)

  13. 13.

    et al. The ancestor of extant Japanese fancy mice contributed to the mosaic genomes of classical inbred strains. Genome Res. 23, 1329–1338 (2013)

  14. 14.

    et al. Global hypomethylation of the genome in XX embryonic stem cells. Nat. Genet. 37, 1274–1279 (2005)

  15. 15.

    et al. DUSP9 modulates DNA hypomethylation in female mouse pluripotent stem cells. Cell Stem Cell 20, 706–719.e7 (2017)

  16. 16.

    , & Targeted mutation of the DNA methyltransferase gene results in embryonic lethality. Cell 69, 915–926 (1992)

  17. 17.

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

  18. 18.

    et al. Germ-line passage is required for establishment of methylation and expression patterns of imprinted but not of nonimprinted genes. Genes Dev. 10, 1008–1020 (1996)

  19. 19.

    et al. Molecular criteria for defining the naive human pluripotent state. Cell Stem Cell 19, 502–515 (2016)

  20. 20.

    et al. Developmental potential of mouse primordial germ cells. Development 126, 1823–1832 (1999)

  21. 21.

    et al. Premature termination of reprogramming in vivo leads to cancer development through altered epigenetic regulation. Cell 156, 663–677 (2014)

  22. 22.

    et al. Aberrant silencing of imprinted genes on chromosome 12qF1 in mouse induced pluripotent stem cells. Nature 465, 175–181 (2010)

  23. 23.

    , , , & Full-term development of mice from enucleated oocytes injected with cumulus cell nuclei. Nature 394, 369–374 (1998)

  24. 24.

    et al. Maternal DNA methylation regulates early trophoblast development. Dev. Cell 36, 152–163 (2016)

  25. 25.

    , , , & Derivation of completely cell culture-derived mice from early-passage embryonic stem cells. Proc. Natl Acad. Sci. USA 90, 8424–8428 (1993)

  26. 26.

    et al. Interaction between Oct3/4 and Cdx2 determines trophectoderm differentiation. Cell 123, 917–929 (2005)

  27. 27.

    et al. The two active X chromosomes in female ES cells block exit from the pluripotent state by modulating the ES cell signaling network. Cell Stem Cell 14, 203–216 (2014)

  28. 28.

    et al. Global landscape and regulatory principles of DNA methylation reprogramming for germ cell specification by mouse pluripotent stem cells. Dev. Cell 39, 87–103 (2016)

  29. 29.

    et al. Dual inhibition of Src and GSK3 maintains mouse embryonic stem cells, whose differentiation is mechanically regulated by Src signaling. Stem Cells 30, 1394–1404 (2012)

  30. 30.

    et al. Naive human pluripotent cells feature a methylation landscape devoid of blastocyst or germline memory. Cell Stem Cell 18, 323–329 (2016)

  31. 31.

    , , , & NIG_MoG: a mouse genome navigator for exploring intersubspecific genetic polymorphisms. Mamm. Genome 26, 331–337 (2015)

  32. 32.

    et al. Maintenance of self-renewal ability of mouse embryonic stem cells in the absence of DNA methyltransferases Dnmt1, Dnmt3a and Dnmt3b. Genes Cells 11, 805–814 (2006)

  33. 33.

    et al. Inducible transgene expression in human iPS cells using versatile all-in-one piggyBac transposons. Methods Mol. Biol. 1357, 111–131 (2016)

  34. 34.

    Cutadapt removes adapter sequences from high-throughput sequencing reads. EMBnet.journal 17, 10–12 (2011)

  35. 35.

    & Bismark: a flexible aligner and methylation caller for Bisulfite-Seq applications. Bioinformatics 27, 1571–1572 (2011)

  36. 36.

    & Fast gapped-read alignment with Bowtie 2. Nat. Methods 9, 357–359 (2012)

  37. 37.

    et al. Integrative genomics viewer. Nat. Biotechnol. 29, 24–26 (2011)

  38. 38.

    et al. The UCSC Genome Browser database: 2015 update. Nucleic Acids Res. 43, D670–D681 (2015)

  39. 39.

    et al. Orphan CpG islands identify numerous conserved promoters in the mammalian genome. PLoS Genet. 6, e1001134 (2010)

  40. 40.

    et al. Contribution of intragenic DNA methylation in mouse gametic DNA methylomes to establish oocyte-specific heritable marks. PLoS Genet. 8, e1002440 (2012)

  41. 41.

    et al. Dynamic stage-specific changes in imprinted differentially methylated regions during early mammalian development and prevalence of non-CpG methylation in oocytes. Development 138, 811–820 (2011)

  42. 42.

    et al. TopHat2: accurate alignment of transcriptomes in the presence of insertions, deletions and gene fusions. Genome Biol. 14, R36 (2013)

  43. 43.

    et al. Transcript assembly and quantification by RNA-seq reveals unannotated transcripts and isoform switching during cell differentiation. Nat. Biotechnol. 28, 511–515 (2010)

  44. 44.

    et al. Multiplex genome engineering using CRISPR/Cas systems. Science 339, 819–823 (2013)

  45. 45.

    , , , & Mice cloned from embryonic stem cells. Proc. Natl Acad. Sci. USA 96, 14984–14989 (1999)

  46. 46.

    et al. Latrunculin A can improve the birth rate of cloned mice and simplify the nuclear transfer protocol by gently inhibiting actin polymerization. Biol. Reprod. 86, 180 (2012)

  47. 47.

    et al. Latrunculin A treatment prevents abnormal chromosome segregation for successful development of cloned embryos. PLoS One 8, e78380 (2013)

Download references


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


  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


  1. Search for Masaki Yagi in:

  2. Search for Satoshi Kishigami in:

  3. Search for Akito Tanaka in:

  4. Search for Katsunori Semi in:

  5. Search for Eiji Mizutani in:

  6. Search for Sayaka Wakayama in:

  7. Search for Teruhiko Wakayama in:

  8. Search for Takuya Yamamoto in:

  9. Search for Yasuhiro Yamada in:


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.

Extended data

Supplementary information

PDF files

  1. 1.

    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.

About this article

Publication history






Further reading


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.

Newsletter Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing