Derivation of pluripotent epiblast stem cells from mammalian embryos

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Abstract

Although the first mouse embryonic stem (ES) cell lines were derived 25 years ago1,2 using feeder-layer-based blastocyst cultures, subsequent efforts to extend the approach to other mammals, including both laboratory and domestic species, have been relatively unsuccessful. The most notable exceptions were the derivation of non-human primate ES cell lines3 followed shortly thereafter by their derivation of human ES cells4. Despite the apparent common origin and the similar pluripotency of mouse and human embryonic stem cells, recent studies have revealed that they use different signalling pathways to maintain their pluripotent status. Mouse ES cells depend on leukaemia inhibitory factor and bone morphogenetic protein, whereas their human counterparts rely on activin (INHBA)/nodal (NODAL) and fibroblast growth factor (FGF). Here we show that pluripotent stem cells can be derived from the late epiblast layer of post-implantation mouse and rat embryos using chemically defined, activin-containing culture medium that is sufficient for long-term maintenance of human embryonic stem cells. Our results demonstrate that activin/Nodal signalling has an evolutionarily conserved role in the derivation and the maintenance of pluripotency in these novel stem cells. Epiblast stem cells provide a valuable experimental system for determining whether distinctions between mouse and human embryonic stem cells reflect species differences or diverse temporal origins.

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Figure 1: Derivation of pluripotent epiblast stem cells (EpiSCs) from the late epiblast layer of embryos at post-implantation stages.
Figure 2: Embryonic identity of EpiSCs.
Figure 3: EpiSCs are capable of differentiating into the three primary germ layers in vitro and in vivo.

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Acknowledgements

We thank A. McLaren for her support. We thank A. Smith for CGR8 cells, A. Nagy for R1 cells, P. Andrew for the SSEA-1 antibody, L. Wicker for access to NOD mice, and S. Thiru for advice in the teratoma analysis. This work was supported by an MRC International Appointments Initiative grant (R.A.P), MRC/Juvenile Diabetes Research Foundation Centre funding (R.A.P., I.G.M.B.), Remedi (I.G.M.B.), the Wellcome Trust Functional Genomics Initiative on Stem Cells (L.E.S, M.W.B.T.), the Addenbrookes National Institute for Health Research Biomedical Research Centre, and a Diabetes UK Career Development fellowship (L.V.). We dedicate this paper to the memory of our colleague Isabelle Bouhon.

Author Contributions L.V. conceived the experiment and did the molecular analysis; I.G.M.B. derived and cultured the EpiSCs; R.A.P. performed the epiblast dissections and together with I.G.M.B. carried out the aggregation, chimaera and clonal assays; L.E.S. and M.T. obtained the microarray data; P.R-G. performed the epigenetic analysis; B.S. performed the blastocyst immunosurgery and ICM cultures; S.M.C.d.S.L. did the Stella–GFP dissection and Blimp1 staining; S.K.H. provided PCR analysis of chimaeras; A.C. did karyotyping of human ES cell lines; L.A-R. carried out the teratoma work; L.V., R.A.P. and I.G.M.B. analysed the data and co-wrote the paper.

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Correspondence to Ludovic Vallier.

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