The endodermal germ layer gives rise to respiratory epithelium, hepatocytes, pancreatic cells and intestinal lineages, among other cell types. These lineages can be differentiated from human pluripotent stem cells (hPSCs) via a common definitive endoderm (DE) intermediate that is characterized by the co-expression of the cell surface markers CXCR4, c-KIT and EPCAM and the transcription factors SOX17 and FOXA2. Here we provide a detailed protocol for mass production of DE from hPSCs in scalable and easy-to-handle suspension culture using a rotating Erlenmeyer flask or a sophisticated, fully controllable, 150-ml stirred tank bioreactor. This protocol uses two different media formulations that are chemically defined and xeno free and therefore good manufacturing practice ready. Our protocol allows for efficient hPSC-derived DE specification in multicellular aggregates within 3 days and generates up to 1 × 10⁸ DE cells with >92% purity in one differentiation batch when using the bioreactor. The hPSC-derived DE cells that are generated can be cryopreserved for later downstream differentiation into various endodermal lineages. This protocol should facilitate the flexible production of mature DE derivatives for physiologically relevant disease models, high-throughput drug screening, toxicology testing and cellular therapies.

内胚层胚层产生呼吸道上皮细胞、肝细胞、胰腺细胞和肠谱系，以及其他细胞类型。这些谱系可以通过共同的定形内胚层 (DE) 中间体从人类多能干细胞 (hPSC) 中分化出来，该中间体的特征是细胞表面标志物 CXCR4、c-KIT 和 EPCAM 以及转录因子 SOX17 和 FOXA2 的共表达。在这里，我们提供了一个详细的协议，用于使用旋转锥形瓶或复杂、完全可控的 150-ml 搅拌罐生物反应器在可扩展且易于处理的悬浮培养中从 hPSC 大规模生产 DE。该协议使用两种不同的培养基配方，它们是化学定义的且不含异种物质，因此准备好良好的生产实践。^{使用生物反应器时，一个分化批次中有8}个纯度 > 92% 的 DE 细胞。产生的 hPSC 衍生的 DE 细胞可以冷冻保存，以便以后在下游分化成各种内胚层谱系。该协议应有助于灵活生产用于生理相关疾病模型、高通量药物筛选、毒理学测试和细胞疗法的成熟 DE 衍生物。