Despite the global prevalence of gastric disease, there are few adequate models in which to study the fundus epithelium of the human stomach. We differentiated human pluripotent stem cells (hPSCs) 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 signalling in mouse embryos led to conversion of fundic to antral epithelium, and that 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 signalling pathways in epithelial morphogenesis and differentiation of fundic cell types, including chief cells and functional parietal cells. hFGOs are a powerful model for studying the development of the human fundus and the molecular bases of human gastric physiology and pathophysiology, and also represent a new platform for drug discovery.
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We thank J. Whitsett and D. Sinner for Axin2-LacZ animals, and Y. Zavros, A. Zorn and members of the Wells and Zorn laboratories for reagents and feedback. This work was supported by National Institutes of Health grants R01DK092456, U19 AI116491, U18EB021780 (J.M.W.), R01DK102551 (M.H.M.), and NIGMS Medical Scientist Training Program T32 GM063483. We acknowledge core support from the Cincinnati Digestive Disease Center Award (P30 DK0789392), CCHMC Confocal Imaging Core, CCHMC Pluripotent Stem Cell Facility, and CCHMC Pathology Core.
The authors declare no competing financial interests.
Nature thanks J. Mills, B.-K. Koo and the other anonymous reviewer(s) for their contribution to the peer review of this work.
Extended data figures and tables
a, Analysis of Sox2, Pdx1, and Gata4 in the embryonic mouse stomach (E14.5) showed that the fundus (f) was Sox2+Gata4+Pdx1−, whereas the antrum (a) was Sox2+Gata4+Pdx1+. The forestomach (fs) expressed Sox2 but neither Gata4 nor Pdx1. b, Brightfield stereomicrograph showing dissected regions of the E14.5 mouse stomach that were analysed by qPCR. fs, forestomach; f, fundus; a, antrum; d, duodenum. c, Dissected regions in b were analysed by qPCR for known regionally expressed markers (Sox2, P63, Gata4, Pdx1, and Cdx2) to validate the accuracy of micro-dissection. qPCR analysis of the dissected E14.5 stomach regions showed that putative fundus markers Irx1, Irx2, Irx3, Irx5, and Pitx1 were enriched in the fundus compared to the antrum. n = 4 biological replicates per dissected region. Scale bar, 500 μm. Error bars represent s.d.
a, By E12.4 and E14.5, ectopic Pdx1 expression was observed throughout the dorsal gastric epithelium and in the most proximal gastric epithelium of cKO embryos. b, qPCR analysis of dissected regions (as shown in Extended Data Fig. 1b) of E14.5 cKO foregut showed significant upregulation of Pdx1 in the fundus and forestomach domains. Conversely, expression of Irx2, Irx3, and Irx5 was markedly reduced in these proximal regions. *P < 0.05; two-tailed Student’s t-test, n = 3 biological replicates per dissected region for each genotype. c, Stereomicrographs of E18.5 dissected viscera demonstrated that cKO embryos exhibited lung agenesis as previously reported. The GI tract, particularly the stomach, was greatly reduced in size. d, Immunofluorescent staining at E18.5 revealed mosaic deletion pattern of Ctnnb1. Boxed regions are shown in Fig. 1e. e, In the E18.5 cKO stomach, recombined glands lacking Ctnnb1 staining did not contain parietal cells whereas robust parietal cell differentiation was observed in Ctnnb1-positive glands. Scale bars, 200 μm (a), 500 μm (d) and 50 μm (e). Error bars represent s.d.
a, We investigated how long CHIR treatment was necessary to establish fundus identity. Brief CHIR treatment (days 6–9) and subsequent growth of organoids in control growth medium until day 34 resulted in fundic organoids expressing the antral marker PDX1, suggesting that short CHIR treatment did not produce a stable fundic fate. We then tested whether longer exposures to CHIR were required to retain fundic fate and found that only continuous treatment through at least day 29 could maintain low expression of the antral marker PDX1. *P < 0.05 compared to control antral hGOs; two-tailed Student’s t-test. n = 3 biological replicates, data representative of two independent experiments. b, c, Over the course of the protocol, PDX1 remained low in CHIR-treated organoids, whereas IRX5 expression was persistently elevated. *P < 0.05; two-tailed Student’s t-test; n = 3 biological replicates per time point. d, Conversion of day 6 posterior foregut spheroids to early stage gastric organoids (day 20) is greater than 80% efficient in both the hAGO and hFGO protocols. e, At d20, hFGO epithelium is ~90% GATA4+SOX2+PDX1− whereas hAGO epithelium is ~90% GATA4+SOX2+PDX1+. **P < 0.001, two-tailed Student’s t-test, n = 4 biological replicates per experiment, two individual experiments shown. Scale bars, 100 μm (c) and 200 μm (d).
Extended Data Figure 4 BMP-dependence of induction of intestinal fate from foregut progenitors by Wnt/β-catenin activation.
a, The intestine-specific transcription factor CDX2 was not significantly induced in CHIR-treated hGOs at either day 9 or day 20. b, Neither fundic nor antral hGOs expressed genes associated with intestinal cell types, including MUC2, CCK, and SCT, when compared to human intestinal organoids (hIOs). *P < 0.05 compared to hIO; two-tailed Student’s t-test. n = 3 biological replicates. c, Anterior–posterior fate is coordinately controlled by WNT and BMP activity. In the presence of the BMP inhibitor Noggin, all organoids maintained foregut (SOX2+) regardless of Wnt/β-catenin pathway activity; however in the presence of BMP4, all organoids were posteriorized (CDX2+). Activation of Wnt (CHIR) in a BMP inhibited state resulted in fundus pattern (SOX2+PDX1−CDX2−) whereas activation of WNT (CHIR) and addition of BMP4 resulted in an intestinal fate (CDX2+). *P < 0.05 compared to analogous Noggin-treated condition; two-tailed Student’s t-test. n = 3 biological replicates. d, Immunofluorescent staining of human tissues revealed that CLDN18 was a gastric-specific epithelial marker that is not found in the intestine. Scale bar, 200 μm. Error bars represent s.e.m.
a, Transmission electron micrographs show that hFGO glands exhibited organized architecture with narrow apical membranes. b, Both hFGOs and hAGOs contained a supporting layer of FOXF1+VIM+ undifferentiated fibroblasts. Scale bars, 5 μm (a) and 100 μm (b).
a, Antral and fundic hGOs exhibited comparable expression of the mucous cell markers MUC5AC and MUC6. b, As shown in a transmission electron micrograph, hFGOs contained abundant cells exhibiting granule pattern consistent with mucous neck cells, the precursors to differentiated chief cells. c, Exogenous expression of NEUROG3 in hGOs derived from a NEUROG3-deficient hESC line induced robust differentiation of SYP-positive endocrine cells. While both hAGOs and hFGOs formed GHRL+ and SST+ endocrine cells, specification of GAST+ G-cells was observed only in hAGOs. d, Expression comparison of cell lineage markers in hGOs and human gastric biopsy tissue. qPCR analyses demonstrated that hGOs exhibited comparable expression levels of several lineage markers (MUC5AC, ATP4B), while other markers were expressed at much lower levels (ATP4A, PGA5, and PGC) than found in the fully differentiated, mature human stomach. Scale bars, 5 μm (b) and 100 μm (c). Error bars represent s.d. (a) and s.e.m. (b).
a, Unlike parietal cells, which expressed functional markers (Atp4b) as early as late embryonic stages, chief cell gene products were not detectable until much later stages of development. In the embryonic (E18.5) and juvenile (P12) stomach, Gif and Pgc were not yet expressed, indicating that chief cells mature much later in development than other lineages in the gastric epithelium. b, Despite the absence of Pgc, the P12 mouse stomach did contain abundant glandular cells expressing nuclear Mist1, a chief cell-specific marker. Thus, chief cells were specified earlier but took several weeks to develop robust expression of terminal differentiation markers. Scale bars, 100 μm (a) and 200 μm (b).
Extended Data Figure 8 Screen for pathways that promote differentiation of parietal cells in fundic hGOs.
a, To test for growth factors or small molecules capable of inducing parietal cell differentiation, hFGOs were exposed for two days (30–32) to the indicated agonist or antagonist and then analysed at day 34. In a screening experiment of different pathways, only MEK inhibition with PD03 was found to robustly induce expression of ATP4A/B. b, Reduction or removal of EGF from the culture medium was not sufficient to reproduce the effect of MEK inhibition. c, The ability of PD03/BMP4 to induce parietal cell development was exclusive to fundic hGOs, as antral hGOs did not express fundic markers in response to PD03/BMP4. d, Exposure to PD03/BMP4 rapidly increased expression of ATP4A and ATP4B in fundic hGOs. e, Induction of parietal cell generation with PD03/BMP4 did not significantly alter the differentiation of chief cells (PGA5 and PGC) and endocrine cells (CHGA). f, The manipulations at each stage of the hFGO differentiation protocol were required for robust parietal cell differentiation, as removal of any single step led to loss of ATP4A/B expression. Error bars represent s.d. (a–c) and s.e.m. (d–f).
a, The dye SNAFR5F exhibits responsiveness over pH range of 5–8, which makes it well suited to detect physiological changes in response to parietal cell-mediated acid secretion. b, Media and luminal pH measurements recorded before (closed circles) and 60 min after addition of histamine (open circles). Antral hGOs did not respond, whereas the fundic hGO luminal pH decreased in response to histamine. The acidification was inhibited by pre-treatment of organoids with famotidine or omeprazole. Furthermore, omeprazole was sufficient to raise the pH in fundic organoids before histamine exposure, suggesting baseline acid secretion in the fundic organoids. Media pH did not change in any organoids. ***P < 0.001 compared to before histamine; $$$P < 0.001 compared to luminal pH without histamine; ###P < 0.001 compared to luminal pH with histamine; 2-tailed Student’s t-test. c, hFGOs contained parietal cell-dense glands in which acridine orange (AO) accumulated in nearly all of the cells lining the lumen of the gland. d, AO accumulation was observed in a canalicular-type pattern in parietal cells in hFGOs. Scale bars, 10 μm. Error bars represent s.d.
a, Schematic representation of experiments to determine the presence of gastric stem cells in hGOs. b, When fragments were grown in culture medium containing only EGF, they did not grow or expand to form new organoids. However, addition of CHIR and FGF10 to the culture medium was sufficient to support the growth of individual fragments into newly formed organoids. c, Following two passages, hFGOs still expressed genes consistent with a gastric phenotype, including PGC, MUC6, MUC5AC, and GHRL. This ability to undergo serial passaging with maintenance of gastric identity supports the conclusion that hFGOs contain cells with properties analogous to those of adult gastric stem cells. d, Although passaged hFGOs expressed markers associated with several differentiated gastric cell types, they did not express genes associated with parietal cells, such as ATP4B. Furthermore, differentiation of parietal cells could not be induced through MEK inhibition as they could before passaging. Error bars represent s.d.
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McCracken, K., Aihara, E., Martin, B. et al. Wnt/β-catenin promotes gastric fundus specification in mice and humans. Nature 541, 182–187 (2017). https://doi.org/10.1038/nature21021
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