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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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.
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.
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