Mammalian male germ-cell development consists of three distinct phases: primordial germ cell (PGC) development, male germ-cell specification for spermatogonium development, and ensuing spermatogenesis. Here, we show an in vitro reconstitution of whole male germ-cell development by pluripotent stem cells (PSCs). Mouse embryonic stem cells (mESCs) are induced into PGC-like cells (mPGCLCs), which are expanded for epigenetic reprogramming. In reconstituted testes under an optimized condition, such mPGCLCs differentiate into spermatogonium-like cells with proper developmental transitions, gene expression, and cell-cycle dynamics and are expanded robustly as germline stem cell-like cells (GSCLCs) with an appropriate androgenetic epigenome. Importantly, GSCLCs show vigorous spermatogenesis, not only upon transplantation into testes in vivo but also under an in vitro culture of testis transplants, and the resultant spermatids contribute to fertile offspring. By uniting faithful recapitulations of the three phases of male germ-cell development, our study creates a paradigm for the in vitro male gametogenesis by PSCs.

哺乳动物雄性生殖细胞发育包括三个不同的阶段：原始生殖细胞 (PGC) 发育、精原细胞发育的雄性生殖细胞规范和随后的精子发生。在这里，我们展示了体外多能干细胞 (PSC) 重建整个雄性生殖细胞发育。小鼠胚胎干细胞 (mESCs) 被诱导为 PGC 样细胞 (mPGCLCs)，这些细胞被扩展用于表观遗传重编程。在优化条件下的重组睾丸中，此类 mPGCLC 分化为具有适当发育转变、基因表达和细胞周期动力学的精原细胞样细胞，并作为具有适当雄激素表观基因组的生殖干细胞样细胞 (GSCLC) 稳健扩增。重要的是，GSCLCs 不仅在体内移植到睾丸后，而且在体外移植后都表现出旺盛的精子发生睾丸移植物的培养，以及由此产生的精子细胞有助于生育后代。通过结合对雄性生殖细胞发育三个阶段的忠实再现，我们的研究为PSC 的体外雄性配子发生创造了一个范例。