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Derivation of extraembryonic endoderm stem (XEN) cells from mouse embryos and embryonic stem cells

Nature Protocols volume 8, pages 1028–1041 (2013)Cite this article

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Abstract

At the time of implantation in the maternal uterus, the mouse blastocyst possesses an inner cell mass comprising two lineages: epiblast (Epi) and primitive endoderm (PrE). Representative stem cells derived from these two cell lineages can be expanded and maintained indefinitely in vitro as either embryonic stem (ES) or XEN cells, respectively. Here we describe protocols that can be used to establish XEN cell lines. These include the establishment of XEN cells from blastocyst-stage embryos in either standard embryonic or trophoblast stem (TS) cell culture conditions. We also describe protocols for establishing XEN cells directly from ES cells by either retinoic acid and activin-based conversion or by overexpression of the GATA transcription factor Gata6. XEN cells are a useful model of PrE cells, with which they share gene expression, differentiation potential and lineage restriction. The robust protocols for deriving XEN cells described here can be completed within 2–3 weeks.

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Figure 1: Overview of early embryonic development.
Figure 2: Stem cell types that can be derived and propagated in culture representing the three blastocyst lineages.
Figure 3: Recovery of blastocyst-stage embryos from uteri of adult female mice.
Figure 4: Timeline for XEN cell derivation from mouse blastocysts.
Figure 5: Timeline for cXEN cell derivation from mouse ES cells.

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Acknowledgements

We thank A. Foley, M. Kang and S. Wamaitha for comments on this protocol. Work in our laboratories is supported by the Human Frontier Science Program, US National Institutes of Health (NIH) grants RO1-HD052115 and RO1-DK084391 to A.-K.H.; the New York State Department of Health NYSTEM IDEA grant C024318 to A.-K.H.; and the March of Dimes Foundation Research Grant 1-FY11-436 to K.K.N. L.T.Y.C. is supported by a Medical Research Council (MRC) capacity-building studentship. K.K.N. is supported by a Centre for Trophoblast Research Next Generation Fellowship.

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Author notes

  1. Kathy K Niakan

    Present address: Present address: Division of Stem Cell Biology and Developmental Genetics, Medical Research Council National Institute for Medical Research, London, UK.,

Authors and Affiliations

  1. The Anne McLaren Laboratory for Regenerative Medicine, University of Cambridge, Cambridge, UK

    Kathy K Niakan & Lily T Y Cho

  2. Department of Physiology, Centre for Trophoblast Research, Development and Neuroscience, University of Cambridge, Cambridge, UK

    Kathy K Niakan

  3. Developmental Biology Program, Sloan-Kettering Institute, New York, New York, USA

    Nadine Schrode & Anna-Katerina Hadjantonakis

  4. Department of Surgery, University of Cambridge, Cambridge, UK

    Lily T Y Cho

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  1. Kathy K Niakan
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Contributions

K.K.N. and A.-K.H. outlined the protocol. K.K.N., N.S. and A.-K.H. wrote the protocol manuscript with the help of L.T.Y.C.

Corresponding authors

Correspondence to Kathy K Niakan or Anna-Katerina Hadjantonakis.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Figure 1

Molecular characteristics of XEN cell lines. Immunofluorescence analysis of mouse ES cells (mESCs), embryo-derived XEN cells, cXEN cells and iXEN cells generated by Gata6 - overexpression (OE) in mESCs after 6 days. Cell lines were analyzed for the expression of Oct4 (sc-5279, Santa Cruz Biotech, 1:500), Dab2 (SC-13982, Santa Cruz Biotech, 1:500), Gata4 (SC-9053, Santa Cruz Biotech, 1:500) or Sox7 (MAB2766, R&D, 1:500) (red) with DAPI (blue) merge using the method described in reference 14. Scale bars: 100 μm. (PDF 431 kb)

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Niakan, K., Schrode, N., Cho, L. et al. Derivation of extraembryonic endoderm stem (XEN) cells from mouse embryos and embryonic stem cells. Nat Protoc 8, 1028–1041 (2013). https://doi.org/10.1038/nprot.2013.049

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