Extended Data Figure 4. Human gastric organoids recapitulate normal antrum development of mouse embryos.

a, Comparison of transcription factor expression between hGO development and in vivo stomach organogenesis. Four embryonic stages (E12.5, E14.5, E16.5 and E18.5) and one postnatal stage (P12) of in vivo antrum development were analyzed for expression of the following transcription factors: Sox2, Pdx1, Gata4, Klf5, and FoxF1. The same markers were analyzed at two stages (day 13 and day 34) of in vitro hGO development and revealed that organoid development parallels what occurs in vivo. At early stages of antrum development the epithelial marker Sox2 was expressed ubiquitously but at later stages it is down-regulated, while other epithelial transcription factors, Pdx1, Gata4 and Klf5, exhibit persistent expression in the epithelium throughout development. Both early and late stage hGOs contain FoxF1-positive mesenchymal cells surrounding the epithelium, similar to the in vivo antrum. b, Early stage human gastric organoids exhibit stereotypic epithelial architecture and nuclear behavior. At 13 days, hGOs contained pseudostratified epithelia that display apicobasal polarity marked by the apical marker aPKC and the basolateral marker E-Cadherin, similar to the E12.5 mouse antrum. Further, extensions of apical membrane (white arrows) were seen within deeper portions of the organoid epithelium. Both the E12.5 mouse antrum and day 7 hGOs appeared to undergo interkinetic nuclear migration, indicated by the presence of mitotic nuclei, pHH3, in only the apical portions of cells. c, EGF is required for morphogenesis in gastric organoids. Bright field images demonstrate the requirement for EGF in epithelial morphogenesis including folding and gland formation at late stages of hGO differentiation. When EGF is removed from the growth medium at day 27, prior to glandular morphogenesis, the hGO epithelium retains a simple, cuboidal structure that fails to form glands. Scale bars, 100 μm in a, 50 μm in b, 2 mm in c.