Gastric diseases, including peptic ulcer disease and gastric cancer, affect 10% of the world’s population and are largely due to chronic H. pylori infection1–3. Species differences in embryonic development and architecture of the adult stomach make animal models suboptimal for studying human stomach organogenesis and pathogenesis4, and there is no experimental model of normal human gastric mucosa. Here we report the de novo generation of three-dimensional human gastric tissue in vitro through the directed differentiation of human pluripotent stem cells (hPSCs). We identified that temporal manipulation of the FGF, WNT, BMP, retinoic acid and EGF signaling pathways and three-dimensional growth are sufficient to generate human gastric organoids (hGOs). Developing hGOs progressed through molecular and morphogenetic stages that were nearly identical to the developing antrum of the mouse stomach. Organoids formed primitive gastric gland- and pit-like domains, proliferative zones containing LGR5-expressing cells, surface and antral mucous cells, and a diversity of gastric endocrine cells. We used hGO cultures to identify novel signaling mechanisms that regulate early endoderm patterning and gastric endocrine cell differentiation upstream of the transcription factor NEUROG3. Using hGOs to model pathogenesis of human disease, we found that H. pylori infection resulted in rapid association of the virulence factor CagA with the c-Met receptor, activation of signaling and induction of epithelial proliferation. Together, these studies describe a novel and robust in vitro system for elucidating the mechanisms underlying human stomach development and disease.

We describe a protocol to generate 3-dimensional human intestinal tissue (called organoids) in vitro from human pluripotent stem cells. To generate intestinal organoids, pluripotent stem cells are first differentiated into FOXA2+/SOX17+ endoderm by treating the cells with ActivinA for 3 days. Following endoderm induction, the pluripotent stem cells are patterned into CDX2+ mid/hindgut tissue using FGF4 and WNT3a. During this patterning step, 3-dimensional mid/hindgut spheroids bud from the monolayer epithelium attached to the tissue culture dish. The 3-dimensional spheroids are further cultured in matrigel along with pro-intestinal growth factors, and proliferate and expand over 1–3 months to give rise to intestinal tissue, complete with intestinal mesenchyme and epithelium consisting of all of the major intestinal cell types. To date, this is the only method to efficiently direct differentiation of human pluripotent stem cells into 3-dimensional human intestinal tissue in vitro.

Despite the global prevalence of gastric disease, there are few adequate models to study the fundus epithelium of the human stomach. We differentiated human pluripotent stem cells (PSCs) into gastric organoids containing fundic epithelium by first identifying and then recapitulating key events in embryonic fundus development. We found that disruption of Wnt/β-catenin signaling in mouse embryos led to conversion of fundic to antral epithelium, while β-catenin activation in hPSC-derived foregut progenitors promoted the development of human fundic-type gastric organoids (hFGOs). We then used hFGOs to identify temporally distinct roles for multiple signaling pathways in epithelial morphogenesis and differentiation of fundic cell types, including chief cells and functional parietal cells. While hFGOs are a powerful new model for studying the development of the human fundus and its lineages, they also represent a critical new model system to study the molecular basis of human gastric physiology, pathophysiology, and drug discovery.

Organogenesis of the trachea and lungs requires a complex series of mesoderm-endoderm interactions mediated by WNT, BMP, retinoic acid (RA) and hedgehog (Hh), but how these pathways interact in a gene regulatory network is less clear. Using Xenopus embryology, mouse genetics, and human ES cell cultures we identified a conserved signaling cascade that initiates respiratory lineage specification. We show that RA has multiple roles; first RA pre-patterns the lateral plate mesoderm and then it promotes Hh ligand expression in the foregut endoderm. Hh subsequently signals back to the pre-patterned mesoderm to promote expression of the lung-inducing ligands Wnt2/2b and Bmp4. Finally, RA regulates the competence of the endoderm to activate the Nkx2-1+ respiratory program in response to these mesodermal WNT and BMP signals. These data provide insights into early lung development and a paradigm for how mesenchymal signals are coordinated with epithelial competence during organogenesis.