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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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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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.
Integrated supplementary information
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.
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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Nature Cell Biology (2019)