Abstract

Mouse embryonic stem cells derived from the epiblast1 contribute to the somatic lineages and the germline but are excluded from the extra-embryonic tissues that are derived from the trophectoderm and the primitive endoderm2 upon reintroduction to the blastocyst. Here we report that cultures of expanded potential stem cells can be established from individual eight-cell blastomeres, and by direct conversion of mouse embryonic stem cells and induced pluripotent stem cells. Remarkably, a single expanded potential stem cell can contribute both to the embryo proper and to the trophectoderm lineages in a chimaera assay. Bona fide trophoblast stem cell lines and extra-embryonic endoderm stem cells can be directly derived from expanded potential stem cells in vitro. Molecular analyses of the epigenome and single-cell transcriptome reveal enrichment for blastomere-specific signature and a dynamic DNA methylome in expanded potential stem cells. The generation of mouse expanded potential stem cells highlights the feasibility of establishing expanded potential stem cells for other mammalian species.

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Acknowledgements

We thank colleagues of the Research Support Facility (B. Doe, S. Newman, E. Grau and others), Y. Hooks, Sequencing (N. Smerdon) and FACS core facilities (B. L. Ng and J. Graham) at the Sanger Institute, the animal facility at the Cancer Research UK Cambridge Institute, and P. Humphreys of the University of Cambridge, for technical support; S. Gerety for the fluorescence stereo microscope, J. K. Kim for informatics advice, S. Rice for help on DNA bisulfite sequencing analysis; J. Nichols, A. Martinez Arias, K. McDole and Y. Zheng for reagents; J. Thomson, E. Robertson and A. Ang for comments. We acknowledge the following funding and support: Wellcome Trust Clinical PhD Fellowship for Academic Clinicians (D.J.R.); PhD fellowship (Portuguese Foundation for Science and Technology, FCT (SFRH/BD/84964/2012)) (L.A.); Japan Society for the Promotion of Science fellowship (Y.T.); National Institutes of Health (RP-PG-0310-10002) (A.C.W.); European Molecular Biology Organization (ALTF938-2014) and Marie Sklodowska-Curie Individual Fellowship (M.A.E.-M.); Biotechnology and Biological Sciences Research Council (BB/K010867/1) and Wellcome Trust (095645/Z/11/Z) (W.R.); Bloodwise (12029), Cancer Research UK (C1163/A12765 and C1163/A21762) and Wellcome Trust core funding (SCI 097922/Z/11/Z) (B.G.); Leading Advanced Projects for Medical Innovation, Japan Agency for Medical Research and Development (H.N. and H.M.); National Health and Medical Research Council Senior Principal Research Fellowship (1110751) (P.P.L.T.); National Natural Science Foundation of China (81671579, 31370904, 30972691) and The National Key Research and Development Program (2017YFA0104500) (L.L.). P.L. thanks M. Stratton, A. Bradley, N. Copeland, N. Jenkins and J. Lupski for their encouragement in these experiments. P.L. is an affiliate faculty member of the Wellcome Trust-MRC Stem Cell Institute, University of Cambridge. The P.L. laboratory is supported by the Wellcome Trust (grant numbers 098051 and 206194).

Author information

Author notes

    • Jian Yang
    • , David J. Ryan
    • , Wei Wang
    • , Jason Cheuk-Ho Tsang
    • , Guocheng Lan
    •  & Hideki Masaki

    These authors contributed equally to this work.

Affiliations

  1. Wellcome Trust Sanger Institute, Hinxton, Cambridge CB10 1HH, UK

    • Jian Yang
    • , David J. Ryan
    • , Wei Wang
    • , Jason Cheuk-Ho Tsang
    • , Xuefei Gao
    • , Liliana Antunes
    • , Yong Yu
    • , Zhexin Zhu
    • , Juexuan Wang
    • , Aleksandra A. Kolodziejczyk
    • , Lia S. Campos
    • , Cui Wang
    • , Fengtang Yang
    • , Beiyuan Fu
    • , Michael Woods
    • , Xi Chen
    • , James Bussell
    • , Jacqui White
    • , Ramiro Ramirez-Solis
    • , Wolf Reik
    • , Sarah A. Teichmann
    • , Liming Lu
    •  & Pentao Liu
  2. Cancer Research UK Cambridge Institute, University of Cambridge, Li Ka Shing Centre, Cambridge CB2 0RE, UK

    • Guocheng Lan
    •  & Xiangang Zou
  3. Division of Stem Cell Therapy, Institute of Medical Science, University of Tokyo, Tokyo 108-8639, Japan

    • Hideki Masaki
    •  & Hiromitsu Nakauchi
  4. European Molecular Biology Laboratory, European Bioinformatics Institute, Hinxton, Cambridge CB10 1SD, UK

    • Aleksandra A. Kolodziejczyk
    •  & Sarah A. Teichmann
  5. Medical Research Council Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge CB2 0QH, UK

    • Zhen Zhong
  6. Epigenetics Programme, The Babraham Institute, Cambridge CB22 3AT, UK

    • Melanie A. Eckersley-Maslin
    •  & Wolf Reik
  7. Cambridge Institute for Medical Research, University of Cambridge, Cambridge CB2 0XY, UK

    • Yosuke Tanaka
    • , Adam C. Wilkinson
    •  & Berthold Göttgens
  8. Wellcome Trust-MRC Cambridge Stem Cell Institute, University of Cambridge, Cambridge CB2 0XY, UK

    • Yosuke Tanaka
    • , Adam C. Wilkinson
    •  & Berthold Göttgens
  9. Division of Cellular Therapy, Institute of Medical Science, University of Tokyo, Tokyo 108-8639, Japan

    • Yosuke Tanaka
  10. Embryology Unit, Children’s Medical Research Institute, University of Sydney, Westmead, New South Wales 2145, Australia

    • Patrick P. L. Tam
  11. School of Medical Sciences, Sydney Medical School, University of Sydney, Westmead, New South Wales 2145, Australia

    • Patrick P. L. Tam
  12. Institute for Stem Cell Biology and Regenerative Medicine, Department of Genetics, Stanford University School of Medicine, Stanford, California 94305-5461, USA

    • Hiromitsu Nakauchi
  13. Shanghai Institute of Immunology, Shanghai Jiaotong University School of Medicine, Shanghai 200025, China.

    • Liming Lu

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Contributions

D.J.R. and W.W. developed EPSCM and derived mouse EPSC lines. J.Y. performed most of the experiments, made the final figures and edited the manuscript. J.C.-H.T performed and analysed the RNA-seq and chromatin immunoprecipitation followed by sequencing (ChIP–seq) experiments, produced the genomics figures and wrote the manuscript. G.L. performed most of the injections. H.M. performed independent EPSC injection experiments at Nakauchi laboratory. X.G., L.A., Y.Y., Z. Zhu, J.W., A.A.K. and C.W. performed genotyping, additional line derivation, FACS, sequencing and chimaera experiments. L.S.C. interpreted the histology data. F.Y. and B.F. karyotyped EPSC and ES cell lines. Z.Zho. performed confocal imaging and interpretation. M.A.E.-M. and W.R. provided DNA methylation data. M.W., Y.T. and A.C.W. performed additional experiments. X.C. analysed TSC RNA-seq data. J.B., J.W., R.R.-S., W.R., B.G., S.A.T., H.N. and X.Z. provided microinjection resources, sequencing and other support. L.L. and P.P.L.T. contributed intellectually and assisted with the revision of the manuscript. P.L. devised the concept, supervised the overall research project and prepared the manuscript for publication.

Competing interests

The Genome Research Limited has filed a provisional patent application that covers the derivation and maintenance of expanded potential stem cells (European patent application number 15797300.9-1402). P.L., D.J.R., J.Y., W.W. and X.G. are listed as inventors.

Corresponding author

Correspondence to Pentao Liu.

Reviewer Information Nature thanks A.-K. Hadjantonakis 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 Information

    This file contains Supplementary Figure 1 (source gels) and the gating strategy of mCherry+ placenta cells. The mCherry+ placenta cells were analysed using LSR II Fortessa cytometry (Bector Dickinson) analyser, and the data were analysed using FlowJo. Because of the various sizes of trophoblasts in placenta, the majority of starting cells were included in FSC/SSC gates. The boundary between positive and negative is defined according the negative control. We also used a non-overlapping channel (GFP) to gate out the false positive cells due to autoflurescence in placenta cells.

  2. 2.

    Reporting Summary

Excel files

  1. 1.

    Supplementary Data

    This file contains Bed files of signal peaks identified in EPSCs by histone modifications ChIP-seq (H3K4me3, H3K27me3 and H3K27Ac super enhancers).

  2. 2.

    Supplementary Data

    This file contains a list of ChIP-seq and RNA-seq sequencing data deposited.

Zip files

  1. 1.

    Supplementary Data

    This file contains Supplementary Tables 1-7 and a Supplementary Table guide.

Videos

  1. 1.

    Preimplantation embryo in EPSCM

    Transgenic Oct4 EGFP 4C-8C embryos were cultured in a gelatinized 96 well plate in EPSCM. At 80 hours in EPSCM, the embryos were imaged on Leica AF6000 fluorescence microscope. The microscope stage and objectives were enclosed by a cage incubator and maintained at 37°C, 5% CO2. A 20X objective was used for all positions. Time-lapses were acquired using GFP filter-set and transmitted light images were sequentially captured. Images were acquired at 30 minutes interval for 55 hours in total.

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DOI

https://doi.org/10.1038/nature24052

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