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Modelling human development and disease in pluripotent stem-cell-derived gastric organoids


Gastric diseases, including peptic ulcer disease and gastric cancer, affect 10% of the world’s population and are largely due to chronic Helicobacter pylori infection1,2,3. 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. We show that temporal manipulation of the FGF, WNT, BMP, retinoic acid and EGF signalling 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 signalling 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 signalling and induction of epithelial proliferation. Together, these studies describe a new and robust in vitro system for elucidating the mechanisms underlying human stomach development and disease.

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Figure 1: Generation of three-dimensional posterior foregut spheroids.
Figure 2: Specification and growth of human antral gastric organoids.
Figure 3: hGOs contain differentiated antral cell types.
Figure 4: hGOs exhibit acute responses to H. pylori infection.

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The RNAseq data from hGOs have been deposited in ArrayExpress with accession number E-MTAB-2885.


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We thank A. Zorn, J. Whitsett, N. Shroyer, the Pluripotent Stem Cell Facility and members of the Wells and Zorn laboratories for reagents and feedback. We also thank M. Kofron for assistance with confocal imaging and T. Westbrook for providing the pInducer20 vector. We thank R. Peek for assistance with analysing electron micrograph images. This work was supported by National Institutes of Health grants R01DK080823, R01DK092456 and K01DK091415, NIGMS Medical Scientist Training Program T32 GM063483, and the American Gastroenterological Association: Robert and Sally Funderburg Research Award in Gastric Cancer. We also acknowledge core support from the Cincinnati Digestive Disease Center Award (P30 DK0789392), Clinical Translational Science Award (U54 RR025216), the Michigan Gastrointestinal Peptide Research Center (MGPRC; NIDDK 5P30DK034933), and technical support from CCHMC Confocal Imaging Core, CCHMC Pathology Core, and CCHMC Viral Vector Core.

Author information




K.W.M. and J.M.W. conceived the study and experimental design, performed and analysed experiments and co-wrote the manuscript. Y.Z. designed, performed and helped analyse H. pylori experiments. E.M.C., C.M.C., K.L.S. and M.S. performed experiments. C.N.M. generated and characterized the iPS cell line. B.E.R., Y.-H.T. and J.R.S. designed, generated and characterized the LGR5-eGFP reporter hES cell line and performed RNA-seq experiments and analysis. All authors contributed to the writing or editing of the manuscript.

Corresponding authors

Correspondence to Yana Zavros or James M. Wells.

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Competing interests

The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 BMP signalling is required in parallel with activation of WNT and FGF to promote a posterior fate.

a, Activation of WNT signalling with WNT3A or the GSK3β inhibitor CHIR99021 (CHIR; 2 μM) induced a posterior fate and this was blocked by BMP inhibition. n = 3 biological replicates per condition. b, Activation of WNT signalling with WNT3A (not shown) or CHIR induced gut tube morphogenesis and spheroid production. c, Immunofluorescent staining of monolayer cultures confirmed the high efficiency of CDX2 induction by CHIR/FGF treatment, and that NOG blocked posterior CDX2 expression and induced expression of the foregut marker SOX2. d, qPCR analysis of BMP target genes MSX1/2 indicated that BMP activity is not increased in response to WNT/FGF, but target genes are suppressed in response to NOG, suggesting that NOG acts on endogenous BMP signalling. n = 3 biological replicates per condition. e, Addition of BMP2 (100 ng ml−1) did not substitute for or augment the ability of WNT/FGF to posteriorize endoderm. These data indicate that the posteriorizing effect of WNT/FGF is not mediated by upregulation of BMP signalling but does require endogenous BMP activity. n = 3 biological replicates per condition. Scale bars, 1 mm (b) and 100 μm (c). Error bars represent s.d.

Extended Data Figure 2 Gastric organoid differentiation is efficient in multiple pluripotent stem cell lines.

a, Table comparing spheroid formation and characteristics between two human ES cell lines (H1 and H9) and one iPS cell line (72.3). Spheroid number was averaged from n = 8 wells per cell line; total cells per spheroid and epithelial composition were determined from whole mount staining (DAPI for total cell number and FOXA2 for epithelial cells) and quantification from n = 6 spheroids per cell line. Error bars represent s.d. b, Immunofluorescent staining of day-34 hGOs derived from ES cell line H1 and iPS cell line 72.3. iPS-cell-derived organoids exhibit the same morphological and molecular features of those derived from ES cells. c, Organ epithelial cell type quantification in day-34 hGOs. Greater than 90% of the epithelium is antral, indicated by PDX1 expression and lack of PTF1A expression, whereas less than 5% express markers associated with other organs derived from endoderm including CDX2 (intestine), albumin (liver) and p63 (squamous epithelium). Data shown are averages from n = 6 hGOs. dg, Characterization of iPS cell line 72.3 used in a. d, e, iPS cell line 72.3 exhibited normal morphological characteristics of pluripotent stem-cell colonies, as compared to the H1 hESC line (d) and had a normal 46;XY karyotype (e). f, g, iPS cell line 72.3 expressed pluripotent markers OCT3/4 and NANOG (f), and demonstrated pluripotency by differentiation into endoderm, mesoderm, and ectoderm lineages in an in vivo teratoma assay (g). h, Human pluripotent stem-cell scorecard assay results demonstrating that ES cell line H1 and iPS cell line 72.3 have similar pluripotency and differentiation potential, and that iPS cell line 72.3 does not have a lineage bias. EB, differentiated as embryoid bodies for 14 days; UD, undifferentiated. Scale bars, 100 μm. Error bars represent s.d.

Extended Data Figure 3 Retinoic acid posteriorizes foregut endoderm.

a, Lineage diagram that summarizes the patterning effects of noggin and retinoic acid in the formation of both anterior and posterior foregut endoderm (aFG and pFG, respectively). b, Bright-field images show that retinoic acid increased the number of spheroids that are produced from foregut monolayer cultures. c, A lower power image of Fig. 1d showing immunofluorescent image of an E8.5, 14-somite stage embryo with Hnf1β protein localized to the posterior portion of the foregut. Boxed region of embryo is shown in Fig. 1d. d, qPCR analysis of gene expression in foregut spheroids treated with retinoic acid. Posterior foregut markers HNF1B and HNF6 were robustly induced by 24-h exposure to retinoic acid. Although retinoic acid induced posterior foregut gene expression it did not induce expression of the posterior marker CDX2. *P < 0.05; Student’s t-test; n = 3 biological replicates per condition, data representative of 3 independent experiments. Scale bars, 1 mm (b) and 100 μm (c). Error bars represent s.d.

Extended Data Figure 4 hGOs 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 analysed for expression of the following transcription factors: Sox2, Pdx1, Gata4, Klf5 and FoxF1. The same markers were analysed at two stages (days 13 and 34) of in vitro hGO development and revealed that organoid development parallels that which occurs in vivo. At early stages of antrum development the epithelial marker Sox2 was expressed ubiquitously but at later stages it is downregulated, 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+ mesenchymal cells surrounding the epithelium, similar to the in vivo antrum. b, Early-stage hGOs exhibit stereotypic epithelial architecture and nuclear behaviour. At day 13, hGOs contained pseudo-stratified epithelia that display apicobasal polarity marked by the apical marker aPKC and the basolateral marker E-cadherin, similar to the E12.5 mouse antrum. Furthermore, 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, phosphohistone H3 (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, before glandular morphogenesis, the hGO epithelium retains a simple, cuboidal structure that fails to form glands. Scale bars, 100 µm (a), 50 μm (b) and 2 mm (c).

Extended Data Figure 5 Mesenchymal differentiation in gastric organoids.

a, Temporal expression analysis of the antral mesenchyme transcription factor BAPX1. Similar to its known embryonic expression pattern, BAPX1 is upregulated during the earlier stages of hGO differentiation and then downregulated coincident with functional cell type marker expression. n = 3 biological replicates per time point. b, Staining for mesenchymal cell type markers revealed that day-34 hGOs contain FOXF1/VIM-positive submucosal fibroblasts and a small number of VIM/ACTA2-expressing subepithelial fibroblasts. hGOs lack a robust smooth muscle layer, indicated by ACTA2/desmin-positive cells in the in vivo antrum. Scale bars, 100 μm. Error bars represent s.d.

Extended Data Figure 6 Induction of genes during development of hGOs that mark specific differentiated antral cell types.

a, qPCR analyses of cell lineage differentiation marker expression at several stages throughout the gastric organoid differentiation protocol (days 0, 3, 6, 9, 20, 27 and 34) and day-34 human intestinal organoids (hIO). Beginning at day 27, hGOs robustly induced genes expressed in differentiated cell types including surface mucous cells (MUC5AC, TFF1, TFF3 and GKN1) and antral gland cells (TFF2). hGOs do not upregulate the expression of markers found in fundic lineages such as parietal cells (ATP4A and ATP4B) and chief cells (MIST1) or intestinal goblet cells (MUC2). Expression levels are normalized to day-3 definitive endoderm cultures. n = 3 biological replicates per time point. b, Muc5AC-expressing surface mucous cells in the late fetal (E18.5) mouse antrum are not yet confined to a pit region and are more broadly distributed through the antral epithelium. Furthermore, these pit cells exhibit high amounts of intracellular mucin staining, similar to day-34 hGOs. c, Global gene expression profiling of day-34 hGOs was performed using RNA-seq, and data were compared to published RNA-seq data sets from human tissues. Hierarchical clustering revealed that hGOs closely resemble human fetal stomach tissue but not human fetal intestine. Error bars represent s.d.

Extended Data Figure 7 Characterization of LGR5-eGFP BAC transgenic reporter ES cell line.

a, H9 LGR5-eGFP ES cell line did not show eGFP fluorescence in undifferentiated, pluripotent stem cells. b, Upon differentiation to definitive endoderm, robust eGFP expression was observed, consistent with published microarray and RNA-sequencing analyses that show LGR5 as a highly enriched endoderm transcript6,42. Top, DAPI and eGFP staining; bottom, eGFP co-localization with endoderm markers SOX17 and FOXA2. c, FACS was used to sort LGR5-eGFPLO and LGR5-eGFPHI from 3-day activin-A-treated definitive endoderm cultures. d, qPCR was used to measure LGR5, FOXA2 and SOX17 expression levels in undifferentiated H9 LGR5-eGFP cells (blue bars, stem cell) and in FACS-purified H9 LGR5-eGFP endoderm (red bars, LGR5-eGFPLO; green bars, LGR5-eGFPHI). As expected, LGR5, FOXA2 and SOX17 were all highly enriched in both LGR5-eGFPLO and LGR5-eGFPHI endoderm populations compared to undifferentiated controls, and the LGR5-eGFPHI cells showed significant enrichment of LGR5 mRNA, but not FOXA2 or SOX17, compared to the LGR5-eGFPLO population. n = 3 biological replicates for each group and error bars represent s.e.m. *P < 0.05 using two-tailed Student’s t-test. This analysis suggests that the LGR5-eGFP BAC construct drives eGFP expression in endoderm cells with the highest levels of LGR5 expression. e, H9 LGR5-eGFP ES cells were differentiated into antral gastric organoids. Bright-field and eGFP stereomicrographs of day-30 hGOs showed that the organoid epithelium developed regionally-restricted areas of LGR5-eGFP expression, suggesting that LGR5+ stem-cell populations formed during the differentiation of the organoids. Scale bars, 100 µm.

Extended Data Figure 8 NEUROG3 expression and endocrine differentiation are reduced in a high EGF environment.

a, Endocrine cell differentiation in the antrum is first evident at E18.5 and highly robust at postnatal stages (P12 shown). As early as E18.5, all expected gastric endocrine subtype hormones are present, including gastrin, ghrelin, somatostatin and serotonin (5-HT). b, High levels of EGF (100 ng ml−1) repressed NEUROG3 expression, however a reduction in EGF concentration (10 ng ml−1) at day 30 resulted in a significant increase in NEUROG3 expression measured at day 34 by qPCR. *P < 0.05; Student’s t-test; n = 5 biological replicates, data representative of 3 independent experiments. c, hGOs maintained in high concentrations of EGF (100 ng ml−1) had very few endocrine cells at day 34, shown by staining for the pan-endocrine marker CHGA. However, a reduction of EGF concentration (to 10 ng ml−1) at day 30 resulted in more physiological numbers of endocrine cells in the gastric epithelium. d, Schematic indicating the effects of EGF at different stages of hGO growth, morphogenesis, and cell type specification. High levels of EGF were required at early developmental stages for growth and morphogenesis, however, it repressed endocrine differentiation at late stages of development; thus, the EGF concentration was reduced at day 30 to allow for endocrine cell development. e, To test whether EGF repression of endocrine differentiation occurs upstream of NEUROG3, hGOs were generated from an ES cell line stably transfected with a dox-inducible NEUROG3-overexpressing transgene. hGOs were maintained in high EGF (100 ng ml−1), then at day 30 were treated with doxycycline (1 μg ml−1) for 24 h and then analysed at day 34. f, g, Dox-treated hGOs show robust activation of endocrine markers CHGA, GAST, GHRL and SST (f), and they contain CHGA-, GHRL- and SST- expressing cells with endocrine morphology (g). *P < 0.05; Student’s t-test; n = 3 biological replicates per condition, data representative of 2 independent experiments. Therefore, NEUROG3 overexpression was sufficient to induce gastric endocrine cell fate in a high-EGF environment. Scale bars, 100 μm. Error bars represent s.d.

Extended Data Figure 9 H. pylori infection of hGOs.

a, hGOs were used to model human-specific disease processes of H. pylori infection. Bacteria were microinjected into the lumen of hGOs and bacteria were detected in the lumen 24 h after injection by bright-field microscopy (black arrow). b, Electron micrograph illustrating the attachment of an H. pylori bacterium to an hGO epithelial cell 24 h after injection. Scale bar, 500 nm. c, Western blots from Fig. 4 that show the molecular mass markers in the first lane. The darker exposure for the CagA western blot (CagA dark) was included to show the molecular mass markers (170 and 130 kDa).

Extended Data Figure 10 Summary of methods for the directed differentiation of gastric organoids.

Each step in the differentiation process is indicated, along with representative stereomicrographs. EGF-100 and EGF-10 represent EGF at 100 ng ml−1 and 10 ng ml−1, respectively.

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McCracken, K., Catá, E., Crawford, C. et al. Modelling human development and disease in pluripotent stem-cell-derived gastric organoids. Nature 516, 400–404 (2014).

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