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Generation of mouse–human chimeric embryos

Abstract

Naive human pluripotent stem cells (hPSCs) can be used to generate mature human cells of all three germ layers in mouse–human chimeric embryos. Here, we describe a protocol for generating mouse–human chimeric embryos by injecting naive hPSCs converted from the primed state. Primed hPSCs are treated with a mammalian target of rapamycin inhibitor (Torin1) for 3 h and dissociated to single cells, which are plated on mouse embryonic fibroblasts in 2iLI medium, a condition essentially the same for culturing mouse embryonic stem cells. After 3–4 d, bright, dome-shaped colonies with mouse embryonic stem cell morphology are passaged in 2iLI medium. Established naive hPSCs are injected into mouse blastocysts, which produce E17.5 mouse embryos containing 0.1–4.0% human cells as quantified by next-generation sequencing of 18S ribosomal DNA amplicons. The protocol is suitable for studying the development of hPSCs in mouse embryos and may facilitate the generation of human cells, tissues and organs in animals.

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Fig. 1: Overview of the protocol.
Fig. 2: Growth of mESC-like colonies after hPSCs are treated with Torin1.
Fig. 3: Two methods to passage naive hPSC colonies.
Fig. 4: Continuous passaging of naive hPSCs in 2iLI.
Fig. 5: Injecting naive hPSCs into mouse blastocysts.
Fig. 6: Equipment and tools needed to perform embryo transfers.
Fig. 7: NGS quantification of human DNA in mouse–human chimeric embryos.

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Acknowledgements

We thank D. Barnas at the Gene Targeting and Transgenic Resource of Roswell Park Comprehensive Cancer Center for injections and transfers of mouse embryos. The work was supported by NYSTEM contracts C028129 (J.F.), C029556 (J.F.) and C30290GG (fellowship for H.L.) and the Buffalo Blue Sky Initiative (J.F.). We thank E.C. Fisher and M.A. Juncker for reading the manuscript.

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Authors

Contributions

B.Z. and H.L. contributed equally to the study. B.Z. developed the conversion protocol based on the initial method of Z.H. H.L. developed the method to analyze chimeric embryos by NGS. H.J. contributed to the analyses of naive hPSCs and chimeric embryos. A.B.S. performed blastocyst injections and embryo transfers. B.J.M. and D.A.Y. performed bioinformatics analysis of NGS data. J.F. conceived and supervised the study. All authors contributed to writing the paper.

Corresponding author

Correspondence to Jian Feng.

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Competing interests

J.F. is a cofounder of Vitropy, LLC and ASDDR, LLC, and has a patent application regarding this work (no. 16/346534). All other authors declare no competing interests.

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Peer review information Nature Protocols thanks Jacob Hanna and the other, anonymous reviewer(s) for their contribution to the peer review of this work.

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Key reference using this protocol

Hu, Z. et al. Sci Adv 6, eaaz0298 (2020): https://doi.org/10.1126/sciadv.aaz0298

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Zhang, B., Li, H., Hu, Z. et al. Generation of mouse–human chimeric embryos. Nat Protoc 16, 3954–3980 (2021). https://doi.org/10.1038/s41596-021-00565-7

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