Production and cryopreservation of definitive endoderm from human pluripotent stem cells under defined and scalable culture conditions

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.