A Defined Strategy for Multi-Lineage Differentiation of Gastric Organoids

In the gastric glands, adult stem cells differentiate into multiple epithelial lineages, including pit mucous cells, acid-secreting parietal cells, and enzyme-producing chief cells. Efficient and controllable lineage specification from adult gastric stem cells is essential for modeling gastric homeostasis and disease in vitro. Organoids are a good model for investigating cell differentiation and organ formation. However, methods to achieve appropriate differentiation of multiple gastric lineages are inefficient. Here, we provide a detailed stepwise protocol for the differentiation of functional cells in human or mouse gastric organoids through targeted modulation of key signaling pathways. The workflow enables reproducible generation of distinct gastric epithelial cell types and provides a tractable platform to dissect signaling requirements. This platform facilitates downstream mechanistic exploration and enhances the translational utility of gastric organoids for disease modeling and therapeutic development.