A molecular roadmap for the emergence of early-embryonic-like cells in culture

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Unlike pluripotent cells, which generate only embryonic tissues, totipotent cells can generate a full organism, including extra-embryonic tissues. A rare population of cells resembling 2-cell-stage embryos arises in pluripotent embryonic stem (ES) cell cultures. These 2-cell-like cells display molecular features of totipotency and broader developmental plasticity. However, their specific nature and the process through which they arise remain outstanding questions. Here we identified intermediate cellular states and molecular determinants during the emergence of 2-cell-like cells. By deploying a quantitative single-cell expression approach, we identified an intermediate population characterized by expression of the transcription factor ZSCAN4 as a precursor of 2-cell-like cells. By using a small interfering RNA (siRNA) screen, we identified epigenetic regulators of 2-cell-like cell emergence, including the non-canonical PRC1 complex PRC1.6 and the EP400–TIP60 complex. Our data shed light on the mechanisms that underlie exit from the ES cell state toward the formation of early-embryonic-like cells in culture and identify key epigenetic pathways that promote this transition.

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We thank A. Smith (Wellcome Trust/MRC Stem Cell Institute) for providing the knock-in REX1 reporter cell line, M. Ko (Keio University) for the Zscan4c promoter plasmid, R. Enriquez-Gasca for providing a classification of MERVLs before publication, D. Reinberg (New York University Langone School of Medicine) for the rabbit antibody to PRDM14, A. Ettinger for time-lapse analysis, C. Ebel, D. Pich, T. Hofer and W. Hammerschmidt for help and access to FACS, the INGESTEM infrastructure for access to the IGBMC high-throughput high-content screening workstation, C. Thibault, F. Recillas-Targa and M. Zurita-Ortega for helpful discussions and A. Burton for critical reading of the manuscript. M.-E.T.-P. acknowledges funding from EpiGeneSys NoE, ERC-Stg ‘NuclearPotency’ (280840), the EMBO Young Investigator Programme, the Fondation Schlumberger pour l’Education et la Recherche (2016-Torres-Padilla) and the Helmholtz Association. J.M.V. acknowledges funding from the Max Planck Society and Epigenesys NoE. T.I. was a recipient of postdoctoral fellowships from the Uehara Memorial Foundation and the Human Frontier Science Programme (LT000015/2012-l). D.R.-T. was partially supported by a DGECI fellowship (2890/2014) from the National University of Mexico.

Author information

Author notes

    • Takashi Ishiuchi

    Present address: Division of Epigenetics and Development, Medical Institute of Bioregulation, Kyushu University, Fukuoka, Japan

  1. Diego Rodriguez-Terrones and Xavier Gaume contributed equally to this work.


  1. Institute of Epigenetics and Stem Cells (IES), Helmholtz Zentrum München, Munich, Germany

    • Diego Rodriguez-Terrones
    • , Xavier Gaume
    • , Takashi Ishiuchi
    • , Audrey Penning
    •  & Maria-Elena Torres-Padilla
  2. Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS–INSERM, U964, Strasbourg, France

    • Diego Rodriguez-Terrones
    • , Xavier Gaume
    • , Amélie Weiss
    • , Arnaud Kopp
    •  & Laurent Brino
  3. Max Planck Institute for Molecular Biomedicine, Münster, Germany

    • Kai Kruse
    •  & Juan M. Vaquerizas
  4. Faculty of Biology, Ludwig Maximilians Universität, Munich, Germany

    • Maria-Elena Torres-Padilla


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D.R.-T., X.G. and T.I. designed, performed and analyzed experiments. D.R.-T. performed most of the computational analyses. A.W. performed the screen together with X.G., under the supervision of L.B. A.K. implemented the screening analysis pipeline with L.B. K.K. performed bioinformatics analysis under the supervision of J.M.V. A.P. performed experiments for screen validation. M.-E.T.-P. designed and supervised the study. All authors contributed to manuscript preparation and read, commented on and approved the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Maria-Elena Torres-Padilla.

Integrated supplementary information

Supplementary information

  1. Supplementary Text and Figures

    Supplementary Figures 1–12, Supplementary Table 10 and Supplementary Note

  2. Life Sciences Reporting Summary

  3. Supplementary Table 1

    List of TaqMan assays.

  4. Supplementary Table 2

    Raw data from the Biomark expression analysis.

  5. Supplementary Table 3

    Significantly differentially expressed genes between transitional states, based on the Biomark expression data.

  6. Supplementary Table 4

    List of siRNA targets used in the library.

  7. Supplementary Table 5

    List of siRNAs used for validation and subsequent experiments.

  8. Supplementary Table 6

    List of all primers used in this study.

  9. Supplementary Table 7

    Results from primary screening.

  10. Supplementary Table 8

    Results from secondary screening.

  11. Supplementary Table 9

    Differentially expressed genes across each transitional state.

  12. Supplementary Video 1

    Embryonic stem cells transitioning to the 2-cell-like state, through an intermediate Zscan4 + state—example 1. Example video for the time-lapse experiments shown in Fig. 2. The destabilized 2C::tbGFP reporter is shown in green, the destabilized ZSCAN4::mCherry reporter is shown in red and the constitutively expressed H2B-iRFP marking all nuclei is shown in cyan.

  13. Supplementary Video 2

    Embryonic stem cells transitioning to the 2-cell-like state, through an intermediate Zscan4 + state—example 2. Example video for the time-lapse experiments shown in Fig. 2. The destabilized 2C::tbGFP reporter is shown in green, the destabilized ZSCAN4::mCherry reporter is shown in red and the constitutively expressed H2B-iRFP marking all nuclei is shown in cyan.

  14. Supplementary Video 3

    Embryonic stem cells transitioning to the 2-cell-like state, through an intermediate Zscan4 + state—example 3. Example video for the time-lapse experiments shown in Fig. 2. The destabilized 2C::tbGFP reporter is shown in green, the destabilized ZSCAN4::mCherry reporter is shown in red and the constitutively expressed H2B-iRFP marking all nuclei is shown in cyan.