Currently an in vitro model that fully recapitulates the human embryonic gonad is lacking. Here we describe a fully defined feeder-free protocol to generate early testis-like cells with the ability to be cultured as an organoid, from human induced pluripotent stem cells. This stepwise approach uses small molecules to mimic embryonic development, with upregulation of bipotential gonad markers (LHX9, EMX2, GATA4, and WT1) at day 10 of culture, followed by induction of testis Sertoli cell markers (SOX9, WT1, and AMH) by day 15. Aggregation into 3D structures and extended culture on Transwell filters yielded organoids with defined tissue structures and distinct Sertoli cell marker expression. These studies provide insight into human gonadal development, suggesting that a population of precursor cells may originate from a more lateral region of the mesoderm. Our protocol represents a significant advance toward generating a much-needed human gonad organoid for studying disorders/differences of sex development.

目前缺乏完全概括人类胚胎性腺的体外模型。在这里，我们描述了一个完全定义的无饲养层协议，以从人类诱导的多能干细胞中生成具有作为类器官培养能力的早期睾丸样细胞。这种逐步方法使用小分子来模拟胚胎发育，在培养的第 10 天上调双潜能性腺标记物（LHX9、EMX2、GATA4和WT1），然后诱导睾丸支持细胞标记物（SOX9、WT1和AMH）) 到第 15 天。在 Transwell 过滤器上聚集成 3D 结构和扩展培养产生具有明确组织结构和不同支持细胞标记表达的类器官。这些研究提供了对人类性腺发育的深入了解，表明前体细胞群可能起源于中胚层的更外侧区域。我们的协议代表了在生成用于研究性发育障碍/差异的急需的人类性腺类器官方面取得的重大进展。