The protocol for directed differentiation of gastric organoids was adapted from our previous protocol7 (link), and Supplementary Table 1 contains the complete list of media and growth factors for each stage. For differentiation, hPSCs were dissociated into single cells using Accutase (Stem Cell Technologies) and plated into 24-well plates at a density of roughly 200,000 cells per well in mTesR1 with Y-27632 (10 μM; Stemgent). The following day, cells were differentiated into definitive endoderm (DE) by adding Activin A (100 ng/ml; Cell Guidance Systems) in RPMI 1640 media (Invitrogen) for three days. Media was also supplemented with NEAA (1×; Gibco) and defined FBS (dFBS; Invitrogen) at 0%, 0.2%, and 2.0% on days 1, 2, and 3, respectively. Additionally, BMP4 (50 ng/ml; R&D Systems) was added on the first day. Subsequently, DE was differentiated to posterior foregut endoderm by exposing cells to CHIR99021 (2 μM; Stemgent), FGF4 (500 ng/ml; R&D Systems), and Noggin (200 ng/ml; R&D systems) for three days in RPMI 1640 supplemented with NEAA and 2.0% dFBS. Retinoic acid (2 μM; Sigma Aldrich) was added for the final day. Media was changed every day. This process resulted in the spontaneous formation of three-dimensional posterior foregut spheroids.