Abstract
The interaction between embryonic and extraembryonic tissues is critical in natural mouse embryogenesis. Here, to enable such interaction in vitro, we describe a protocol to assemble a complete mouse embryo model using mouse embryonic stem cells and induced embryonic stem cells to express Cdx2 (or trophoblast stem cells) and Gata4 to reconstitute the epiblast, extraembryonic ectoderm and visceral endoderm lineages, respectively. The resulting complete embryo models recapitulate development from embryonic day 5.0 to 8.5, generating advanced embryonic and extraembryonic tissues that develop through gastrulation to initiate organogenesis to form a head and a beating heart structure as well as a yolk sac and chorion. Once the required stem cell lines are stably maintained in culture, the protocol requires 1 day to assemble complete embryo models and a further 8 days to culture them until headfold stages, although structures can be collected at earlier developmental stages as required. This protocol can be easily performed by researchers with experience in mouse stem cell culture, although they will benefit from knowledge of natural mouse embryos at early postimplantation stages.
Key points
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This protocol is for the assembly of complete mouse embryo models from embryonic and induced stem cells. These embryo models develop to generate advanced embryonic and extraembryonic tissues.
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While there are many existing in vitro models of mouse embryogenesis, these complete induced embryo models are among the only models reported to recapitulate development from gastrulation to headfold stages.
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Acknowledgements
The authors thank S. Raj for providing the images for Fig. 3, K. Ivanovitch for advice on the handling and storage of rat serum and M.Z.-G. laboratory members for their helpful comments. This work was supported by National Institutes of Health Pioneer Award (DP1 HD104575-01), the Allen Discovery Center for Lineage Tracing; European Research Council (669198), the Wellcome Trust (207415/Z/17/Z), Open Philanthropy/Silicon Valley Community Foundation and Weston Havens Foundation. K.Y.C.L. was supported by the Croucher Foundation and the Cambridge Trust.
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K.Y.C.L. performed the experiments. K.Y.C.L. and G.A. developed the protocol with the guidance from M.Z.-G. M.Z.-G. conceived and supervised the project. K.Y.C.L., G.A. and M.Z.-G. wrote the manuscript.
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A patent ‘Generation of synthetic embryos from multiple stem cell types’ was filed by California Institute of Technology and the University of Cambridge under CIT file number: CIT-8826-P and serial number: 63/344,251.
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Nature Protocols thanks Miki Ebisuya and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
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Key references using this protocol
Amadei, G. et al. Nature 610, 143–153 (2022): https://doi.org/10.1038/s41586-022-05246-3
Amadei, G. et al. Dev. Cell 56, 366–382.e9 (2021): https://doi.org/10.1016/j.devcel.2020.12.004
Lau, K. Y. C. et al. Cell Stem Cell 29, 1445–1458.e8 (2022): https://doi.org/10.1016/j.stem.2022.08.013
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Lau, K.Y.C., Amadei, G. & Zernicka-Goetz, M. Assembly of complete mouse embryo models from embryonic and induced stem cell types in vitro. Nat Protoc 18, 3662–3689 (2023). https://doi.org/10.1038/s41596-023-00891-y
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DOI: https://doi.org/10.1038/s41596-023-00891-y
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