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.

原始人类多能干细胞 (hPSC) 可用于在小鼠-人类嵌合胚胎中生成所有三个胚层的成熟人类细胞。在这里，我们描述了一种通过注射从启动状态转换而来的幼稚 hPSC 来生成鼠-人嵌合胚胎的方案。引物 hPSCs 用哺乳动物雷帕霉素抑制剂 (Torin1) 靶标处理 3 小时，然后分离成单个细胞，然后将其接种在 2iLI 培养基中的小鼠胚胎成纤维细胞上，这种条件与培养小鼠胚胎干细胞的条件基本相同。3–4 天后，具有小鼠胚胎干细胞形态的明亮圆顶形集落在 2iLI 培养基中传代。已建立的幼稚 hPSC 被注射到小鼠囊胚中，产生含有 0.1-4.0% 人类细胞的 E17.5 小鼠胚胎，通过 18S 核糖体 DNA 扩增子的下一代测序进行量化。