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In vitro establishment of expanded-potential stem cells from mouse pre-implantation embryos or embryonic stem cells

Nature Protocolsvolume 14pages350378 (2019) | Download Citation

Abstract

Molecular and embryology studies have demonstrated that mouse pre-implantation embryo development is a process of progressive cell fate determination. At the time of implantation, three cell lineages are present in the developing blastocyst: the trophectoderm (TE), the epiblast (Epi) and the primitive endoderm (PrE). From these early embryo cells, trophoblast stem (TS) cells, embryonic stem (ES) cells and extra-embryonic endoderm stem (XEN) cells can be derived. Recently, we derived stem cells with blastomere-like features from mouse cleavage-stage embryos, which we named expanded-potential stem cells (EPSCs). Here, we provide detailed protocols that describe how to establish EPSCs from single eight-cell-stage blastomeres or whole eight-cell pre-implantation mouse embryos, or by conversion of mouse ES cells or induced pluripotent stem (iPS) cells reprogrammed from fibroblasts. It takes 2–3 weeks to derive EPSCs from each cell source. The EPSCs derived from these protocols can differentiate into all embryonic and extra-embryonic lineages when implanted into chimeras. Furthermore, bona fide TS and XEN cell lines can be derived from EPSCs in vitro.

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Data availability

The data that support the findings of this study are available from the corresponding author upon request. Source data for Fig. 10 are available online.

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

Key reference using this protocol

Yang, J. et al. Nature 550, 393–397 (2017) https://doi.org/10.1038/nature24052

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Acknowledgements

We thank our colleagues from the research support facility (M. Woods, C. Sinclair, E. Brown, B. Doe, S. Newman, E. Grau and others) at the Sanger Institute and the animal facility at CRUK-CI. The P.L. lab was supported by the Wellcome Trust (grant numbers: 098051 and 206194) and internal funding from the University of Hong Kong.

Author information

Author notes

  1. These authors contributed equally: Jian Yang, David J. Ryan, Guocheng Lan.

Affiliations

  1. Wellcome Trust Sanger Institute, Hinxton, UK

    • Jian Yang
    • , David J. Ryan
    •  & Pentao Liu
  2. Cancer Research UK Cambridge Institute, Li Ka Shing Centre, University of Cambridge, Cambridge, UK

    • Guocheng Lan
    •  & Xiangang Zou
  3. Li Ka Shing Faculty of Medicine, Stem Cell and Regenerative Medicine Consortium, School of Biomedical Sciences, University of Hong Kong, Hong Kong, China

    • Pentao Liu

Authors

  1. Search for Jian Yang in:

  2. Search for David J. Ryan in:

  3. Search for Guocheng Lan in:

  4. Search for Xiangang Zou in:

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Contributions

J.Y. and D.J.R. drafted the protocol. J.Y., D.J.R. and P.L. wrote the protocol with inputs from G.L. and X.Z. The initial observations of pre-implantation embryos in EPSCM were made by D.J.R. Most experiments presented in this protocol were performed by J.Y. All chimeric blastocysts were generated by G.L. and X.Z.

Competing interests

Genome Research Limited has filed a provisional patent application that covers the derivation and maintenance of EPSCs (WO2016079146). P.L., D.J.R. and J.Y. are listed as inventors. The other authors declare no competing interests.

Corresponding authors

Correspondence to Jian Yang or Pentao Liu.

Integrated supplementary information

  1. Supplementary Figure 1 Diagrams of constructs.

    These constructs include episomal vectors used in MEF reprogramming experiment and PB vectors used in EPSCs transfection. CAG: CMV early enhancer/chick β-actin; SV40/pA: SV40 polyA; Orip: origin of plasmid replication; EBNA-1: EBV nuclear antigen 1; T2A, E2A: 2A peptides; bpA: bovine growth hormone polyA.

  2. Supplementary Figure 2 Gating strategy.

    Non-transfected EPSCs (DR25) were used as negative control. In all sorting experiments, cell population was initially identified on a FSC/SSC plot, using a polygon gate to separate distinct cell population from debris and feeder (over 85%). The boundary between positive and negative was determined by negative control. And negative population in control was over 99.5%. Using these gates, the sample, DR25-EPSCs transfected with an mCherry fluorescence reporter construct was sorted (76.5% mCherry positive) and plated. a. Negative control, untransfected DR25-EPSCs. b. Sample, DR25-EPSCs transfected with PBCAGmCherry.

Supplementary information

  1. Supplementary Text and Figures

    Supplementary Figures 1 and 2, and Supplementary Table 1

  2. Reporting Summary

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DOI

https://doi.org/10.1038/s41596-018-0096-4

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