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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European Nucleotide Archive
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).
Extended data figures
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.
About this article
Nature Methods (2017)