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

Nature Protocols volume 14, pages 350–378 (2019)Cite this article

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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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Fig. 1: Dynamic of OCT4 and CDX2 expression in embryos cultured in EPSCM.
Fig. 2: Proliferation and apoptosis in embryos cultured in EPSCM.
Fig. 3: Colony formation assay of single cells from filled blastocyst.
Fig. 4: Embryo collection and blastomere preparation.
Fig. 5: Derivation of EPSCs from eight-cell embryos on SNL76/7 feeders.
Fig. 6: Derivation of feeder-free EPSCs from eight-cell embryos.
Fig. 7: Derivation of EPSCs from single blastomeres.
Fig. 8: Conversion of ES cells to EPSCs.
Fig. 9: MEF reprogramming and conversion of iPS cells to EPSCs.
Fig. 10: RT–qPCR analysis of Axin2 and H19 expression.
Fig. 11: Examples of unsuccessful EPSC chimera formation.

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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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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.

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Author notes

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

Authors and 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

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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.

Corresponding authors

Correspondence to Jian Yang or Pentao Liu.

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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.

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Key reference using this protocol

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

Integrated supplementary information

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.

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.

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Yang, J., Ryan, D.J., Lan, G. et al. In vitro establishment of expanded-potential stem cells from mouse pre-implantation embryos or embryonic stem cells. Nat Protoc 14, 350–378 (2019). https://doi.org/10.1038/s41596-018-0096-4

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