The human stomach features distinct, regionalized functionalities along the anterior–posterior axis1,2. Historically, studies on stomach patterning have used animal models to identify the underlying principles3–7. Recently, human pluripotent stem (hPS)-cell-based gastric organoids for modelling domain-specific development of the fundic and antral epithelium are emerging8–10. However, recapitulating self-organized fundic–antral patterning in early stomach organogenesis remains challenging, presenting a considerable barrier for advancing knowledge of stomach organogenesis. Here we report human gastroids—a self-organized multilineage gastric organoid derived from hPS cells—to model gastric fundic–antral patterning in vitro. Through multi-germ-layer co-development, we generate gastroids that feature an epithelial chamber with bipolar fundic–antral patterning, annexed with neural populations near the fundic domain while enveloped by mesenchymal cells, therefore showing molecular, cellular, structural and anatomical similarity to stomach development in vivo. Non-endodermal cells, especially neural populations, function as a critical signalling centre to instruct fundic–antral patterning in gastroids through WNT-mediated crosstalk. Single-cell transcriptomic profiling and genetic silencing further reveal NR2F2 as a key mediator of fundic–antral patterning in gastroid development. This study reveals a principle for instructing gastric patterning and provides a higher-fidelity platform for advancing knowledge of stomach organogenesis and gastric organoid development.
