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

Journal name:
Nature
Volume:
516,
Pages:
400–404
Date published:
DOI:
doi:10.1038/nature13863
Received
Accepted
Published online

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.

At a glance

Figures

  1. Generation of three-dimensional posterior foregut spheroids.
    Figure 1: Generation of three-dimensional posterior foregut spheroids.

    a, Sox2 marks foregut endoderm and Cdx2 marks mid/hindgut endoderm in E8.5 (14-somite stage) mouse embryo. b, c, Quantitative PCR (qPCR) analysis (b) and wholemount immunostaining (c) for patterning markers at day 6 in human pluripotent stem-cell definitive endoderm cultures exposed to 3 days in media alone (control) or with the indicated growth factors/antagonists. WNT3A (WNT) and FGF4 (FGF) induced CDX2 expression whereas the BMP antagonist NOG repressed CDX2 and induced high levels of the foregut marker SOX2. Results are normalized to expression in control endoderm (stage-matched, no-growth-factor-treated). *P < 0.05 compared to control; **P < 0.005 compared to WNT/FGF; two-tailed Student’s t-test; n = 3 biological replicates per condition, data representative of 6 independent experiments. d, Quantitation of SOX2- and CDX2-expressing cells in day 6 spheroids generated in hindgut (WNT/FGF4) and foregut (WNT/FGF4/NOG) patterning conditions. Data are expressed as the percentage of cells expressing indicated markers, normalized to the total number of cells in the spheroids. *P < 1.0 × 10−6; two-tailed Student’s t-test; n = 5 biological replicates per condition, data representative of 3 independent experiments. e, The posterior foregut in the E8.5 mouse embryo expressed both Sox2 and Hnf1β. f, g, Exposing cultures to retinoic acid (RA) on the final day (day 5–6) of the spheroid generation step induced expression of HNF1β in SOX2-expressing epithelium, measured by both qPCR (f) and wholemount immunofluorescent staining (g) at day 6, indicating the formation of posterior foregut spheroids. *P < 0.005; two-tailed Student’s t-test; n = 3 biological replicates per condition, data representative of 3 independent experiments. Scale bars, 100 μm (a and e) and 50 µm (c and g). Error bars represent s.d.

  2. Specification and growth of human antral gastric organoids.
    Figure 2: Specification and growth of human antral gastric organoids.

    a, Schematic representation of the in vitro culture system used to direct the differentiation of pluripotent stem cells into three-dimensional gastric organoids. b, Defining molecular domains of the posterior foregut in E10.5 mouse embryos with Sox2, Pdx1 and Cdx2; Sox2/Pdx1, antrum (a); Sox2, fundus (f); Pdx1, dorsal and ventral pancreas (dp and vp); Pdx1/Cdx2, duodenum (d). c, Posterior foregut spheroids exposed for three days to retinoic acid (2 μM) exhibited >100-fold induction of PDX1 compared to control spheroids, measured by qPCR at day 9. *P < 0.05; two-tailed Student’s t-test; n = 3 biological replicates per condition, data representative of 4 independent experiments. d, Time course qPCR analysis of antral differentiation (according to protocol detailed in Fig. 2a) demonstrated sequential activation of SOX2 at day 6 (posterior foregut (FG) endoderm), followed by induction of PDX1 at day 9 (presumptive antrum). Day-9 antral spheroids had a 500-fold increase in SOX2 and a 10,000-fold increase in PDX1 relative to day-3 definitive endoderm (DE). *P < 0.05; two-tailed Student’s t-test; n = 3 biological replicates per time point, data representative of 2 independent experiments. The pancreatic marker PTF1A was not significantly increased. e, Stereomicrographs showing morphological changes during growth of gastric organoids. By 4 weeks, the epithelium of hGOs exhibited a complex folded and glandular architecture (arrows). D, day. f, Comparison of mouse stomach at E18.5 and day-34 hGOs. Pdx1 was highly expressed in the mouse antrum but excluded from the fundus. Human gastric organoids expressed PDX1 throughout the epithelium and exhibited morphology similar to the late gestational mouse antrum (arrows). Scale bars, 100 μm (b and f) and 250 µm (e). Error bars represent s.d.

  3. hGOs contain differentiated antral cell types.
    Figure 3: hGOs contain differentiated antral cell types.

    a, Schematic representation of a typical antral gland showing normal cell types and associated molecular markers. bg, Immunofluorescent staining demonstrated that day-34 hGOs consisted of normal cell types found in the antrum, but not the fundus. The hGO epithelium contained surface mucous cells that express MUC5AC (b, left), similar to the P12 mouse antrum (b, right), but not ATP4B-expressing parietal cells (c, left) that characterize the fundus (c, right). SOX9+ cells were found at the base of the hGO epithelium (d, left), similar to the progenitor cells found in the P12 antrum (d, right). Furthermore, hGOs contained MUC6+ antral gland cells (e) and LGR5-expressing cells (yellow arrow) (f). Boxed regions in bf are shown as high magnification images below (b, c, d) or to the right (e, f) of the original. g, Day-34 hGOs also contained endocrine cells (SYP) that expressed the gastric hormones GAST, SST, GHRL and serotonin (5-HT). Scale bars, 100 μm (original images in bf) and 20 μm (magnified images in bf and g). Marker expression data are representative from a minimum of 10 independent experiments, except LGR5-eGFP data, which is a representative example from two separate experiments. DAPI, 4′,6-diamidine-2-phenylindole.

  4. hGOs exhibit acute responses to H. pylori infection.
    Figure 4: hGOs exhibit acute responses to H. pylori infection.

    a, Day-34 hGOs contained a zone of MKI67+ proliferative cells similar to the embryonic (E18.5) and postnatal (P12) mouse antrum. b, Using hGOs to model human-specific disease processes of H. pylori infection. Pathogenic (G27) and attenuated (ΔCagA) bacteria were microinjected into the lumen of hGOs and after 24 h, bacteria (both G27 and ΔCagA strains) were tightly associated with the apical surface of the hGO epithelium. c, Immunoprecipitation (IP) for the oncogene c-Met demonstrates that H. pylori induced a robust activation (tyrosine phosphorylation (pTyr)) of c-Met, and this is a CagA-dependent process. Furthermore, CagA immunoprecipitated with c-Met, suggesting that these proteins interact in hGO epithelial cells. Phosphorylated c-Met (phos. c-MET) and CagA control lysates were not immunoprecipitated but used to confirm molecular masses. The molecular mass markers are indicated (130 and 170 kilodaltons (kDa)) and shown in Extended Data Fig. 9c. IB, immunoblotting. d, Within 24 h, H. pylori infection caused a CagA-dependent twofold increase in the number of proliferating cells in the hGO epithelium, measured by 5-ethynyl-2′-deoxyuridine (EdU) incorporation. *P < 0.05; two-tailed Student’s t-test; n = 3 biological replicates per condition, data representative of 4 independent experiments. Scale bars, 100 μm (a) and 20 μm (b). Error bars represent s.e.m.

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

  6. Gastric organoid differentiation is efficient in multiple pluripotent stem cell lines.
    Extended Data Fig. 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.

  7. Retinoic acid posteriorizes foregut endoderm.
    Extended Data Fig. 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.

  8. hGOs recapitulate normal antrum development of mouse embryos.
    Extended Data Fig. 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).

  9. Mesenchymal differentiation in gastric organoids.
    Extended Data Fig. 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.

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

  11. Characterization of LGR5-eGFP BAC transgenic reporter ES cell line.
    Extended Data Fig. 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.

  12. NEUROG3 expression and endocrine differentiation are reduced in a high EGF environment.
    Extended Data Fig. 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.

  13. H. pylori infection of hGOs.
    Extended Data Fig. 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).

  14. Summary of methods for the directed differentiation of gastric organoids.
    Extended Data Fig. 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.

Accession codes

Primary accessions

ArrayExpress

References

  1. Wen, S. & Moss, S. F. Helicobacter pylori virulence factors in gastric carcinogenesis. Cancer Lett. 282, 18 (2009)
  2. Yuan, Y., Padol, I. T. & Hunt, R. H. Peptic ulcer disease today. Nature Clin. Pract. Gastroenterol. Hepatol. 3, 8089 (2006)
  3. Parkin, D. M. The global health burden of infection-associated cancers in the year 2002. Int. J. Cancer 118, 30303044 (2006)
  4. Peek, R. M. Helicobacter pylori infection and disease: from humans to animal models. Dis. Model. Mech. 1, 5055 (2008)
  5. Mills, J. C. & Shivdasani, R. A. Gastric epithelial stem cells. Gastroenterology 140, 412424 (2011)
  6. Spence, J. R. et al. Directed differentiation of human pluripotent stem cells into intestinal tissue in vitro. Nature 470, 105109 (2011)
  7. Si-Tayeb, K. et al. Highly efficient generation of human hepatocyte-like cells from induced pluripotent stem cells. Hepatology 51, 297305 (2010)
  8. D’Amour, K. A. et al. Production of pancreatic hormone-expressing endocrine cells from human embryonic stem cells. Nature Biotechnol. 24, 13921401 (2006)
  9. D’Amour, K. A. et al. Efficient differentiation of human embryonic stem cells to definitive endoderm. Nature Biotechnol. 23, 15341541 (2005)
  10. McCracken, K. W., Howell, J. C., Spence, J. R. & Wells, J. M. Generating human intestinal tissue from pluripotent stem cells in vitro. Nature Protocols 6, 19201928 (2011)
  11. Kumar, M., Jordan, N., Melton, D. & Grapin-Botton, A. Signals from lateral plate mesoderm instruct endoderm toward a pancreatic fate. Dev. Biol. 259, 109122 (2003)
  12. Tiso, N., Filippi, A., Pauls, S., Bortolussi, M. & Argenton, F. BMP signalling regulates anteroposterior endoderm patterning in zebrafish. Mech. Dev. 118, 2937 (2002)
  13. Green, M. D. et al. Generation of anterior foregut endoderm from human embryonic and induced pluripotent stem cells. Nature Biotechnol. 29, 267272 (2011)
  14. Wang, Z., Dollé, P., Cardoso, W. V. & Niederreither, K. Retinoic acid regulates morphogenesis and patterning of posterior foregut derivatives. Dev. Biol. 297, 433445 (2006)
  15. Martín, M. et al. Dorsal pancreas agenesis in retinoic acid-deficient Raldh2 mutant mice. Dev. Biol. 284, 399411 (2005)
  16. Molotkov, A., Molotkova, N. & Duester, G. Retinoic acid generated by Raldh2 in mesoderm is required for mouse dorsal endodermal pancreas development. Dev. Dyn. 232, 950957 (2005)
  17. Kawaguchi, Y. et al. The role of the transcriptional regulator Ptf1a in converting intestinal to pancreatic progenitors. Nature Genet. 32, 128134 (2002)
  18. Johnson, L. R. & Guthrie, P. D. Stimulation of rat oxyntic gland mucosal growth by epidermal growth factor. Am. J. Physiol. 238, G45G49 (1980)
  19. Majumdar, A. P. Postnatal undernutrition: effect of epidermal growth factor on growth and function of the gastrointestinal tract in rats. J. Pediatr. Gastroenterol. Nutr. 3, 618625 (1984)
  20. Grosse, A. S. et al. Cell dynamics in fetal intestinal epithelium: implications for intestinal growth and morphogenesis. Development 138, 44234432 (2011)
  21. Verzi, M. P. et al. Role of the homeodomain transcription factor Bapx1 in mouse distal stomach development. Gastroenterology 136, 17011710 (2009)
  22. Choi, E. et al. Cell lineage distribution atlas of the human stomach reveals heterogeneous gland populations in the gastric antrum. Gut (2014)
  23. Barker, N. et al. Lgr5+ve stem cells drive self-renewal in the stomach and build long-lived gastric units in vitro. Cell Stem Cell 6, 2536 (2010)
  24. Jenny, M. et al. Neurogenin3 is differentially required for endocrine cell fate specification in the intestinal and gastric epithelium. EMBO J. 21, 63386347 (2002)
  25. Lee, C. S., Perreault, N., Brestelli, J. E. & Kaestner, K. H. Neurogenin 3 is essential for the proper specification of gastric enteroendocrine cells and the maintenance of gastric epithelial cell identity. Genes Dev. 16, 14881497 (2002)
  26. Olbe, L., Hamlet, A., Dalenbäck, J. & Fändriks, L. A mechanism by which Helicobacter pylori infection of the antrum contributes to the development of duodenal ulcer. Gastroenterology 110, 13861394 (1996)
  27. Xia, H. H. et al. Antral-type mucosa in the gastric incisura, body, and fundus (antralization): a link between Helicobacter pylori infection and intestinal metaplasia? Am. J. Gastroenterol. 95, 114121 (2000)
  28. Churin, Y. et al. Helicobacter pylori CagA protein targets the c-Met receptor and enhances the motogenic response. J. Cell Biol. 161, 249255 (2003)
  29. Peek, R. M. et al. Helicobacter pylori cagA+ strains and dissociation of gastric epithelial cell proliferation from apoptosis. J. Natl. Cancer Inst. 89, 863868 (1997)
  30. Teo, A. K. K. et al. Activin and BMP4 synergistically promote formation of definitive endoderm in human embryonic stem cells. Stem Cells 30, 631642 (2012)
  31. Meerbrey, K. L. et al. The pINDUCER lentiviral toolkit for inducible RNA interference in vitro and in vivo. Proc. Natl Acad. Sci. USA 108, 36653670 (2011)
  32. Warming, S., Costantino, N., Court, D. L., Jenkins, N. A. & Copeland, N. G. Simple and highly efficient BAC recombineering using galK selection. Nucleic Acids Res. 33, e36 (2005)
  33. Okita, K. et al. An efficient nonviral method to generate integration-free human-induced pluripotent stem cells from cord blood and peripheral blood cells. Stem Cells 31, 458466 (2013)
  34. Covacci, A. et al. Molecular characterization of the 128-kDa immunodominant antigen of Helicobacter pylori associated with cytotoxicity and duodenal ulcer. Proc. Natl Acad. Sci. USA 90, 57915795 (1993)
  35. Amieva, M. R., Salama, N. R., Tompkins, L. S. & Falkow, S. Helicobacter pylori enter and survive within multivesicular vacuoles of epithelial cells. Cell. Microbiol. 4, 677690 (2002)
  36. Schumacher, M. A. et al. Gastric Sonic Hedgehog acts as a macrophage chemoattractant during the immune response to Helicobacter pylori. Gastroenterology 142, 11501159 (2012)
  37. Fagerberg, L. et al. Analysis of the human tissue-specific expression by genome-wide integration of transcriptomics and antibody-based proteomics. Mol. Cell. Proteomics 13, 397406 (2014)
  38. Bernstein, B. E. et al. The NIH roadmap epigenomics mapping consortium. Nature Biotechnol. 28, 10451048 (2010)
  39. Langmead, B., Trapnell, C., Pop, M. & Salzberg, S. L. Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol. 10, R25 (2009)
  40. Trapnell, C., Pachter, L. & Salzberg, S. L. TopHat: discovering splice junctions with RNA-Seq. Bioinformatics 25, 11051111 (2009)
  41. Trapnell, C. et al. Differential analysis of gene regulation at transcript resolution with RNA-seq. Nature Biotechnol. 31, 4653 (2013)
  42. Xie, R. et al. Dynamic chromatin remodeling mediated by polycomb proteins orchestrates pancreatic differentiation of human embryonic stem cells. Stem Cells 12, 224237 (2013)

Download references

Author information

Affiliations

  1. Division of Developmental Biology, Cincinnati Children’s Hospital Medical Center, 3333 Burnet Avenue, Cincinnati, Ohio 45229-3039, USA

    • Kyle W. McCracken,
    • Emily M. Catá,
    • Calyn M. Crawford,
    • Katie L. Sinagoga,
    • Christopher N. Mayhew &
    • James M. Wells
  2. Department of Molecular and Cellular Physiology, University of Cincinnati, Cincinnati, Ohio 45267, USA

    • Michael Schumacher &
    • Yana Zavros
  3. Department of Cell and Developmental Biology, University of Michigan Medical School, Ann Arbor, Michigan 48109-2200, USA

    • Briana E. Rockich &
    • Jason R. Spence
  4. Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, Michigan 48109-2200, USA

    • Yu-Hwai Tsai &
    • Jason R. Spence
  5. Division of Endocrinology, Cincinnati Children’s Hospital Medical Center, 3333 Burnet Avenue, Cincinnati, Ohio 45229-3039, USA

    • James M. Wells

Contributions

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.

Competing financial interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to:

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

Author details

Extended data figures and tables

Extended Data Figures

  1. Extended Data Figure 1: BMP signalling is required in parallel with activation of WNT and FGF to promote a posterior fate. (537 KB)

    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.

  2. Extended Data Figure 2: Gastric organoid differentiation is efficient in multiple pluripotent stem cell lines. (541 KB)

    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.

  3. Extended Data Figure 3: Retinoic acid posteriorizes foregut endoderm. (279 KB)

    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.

  4. Extended Data Figure 4: hGOs recapitulate normal antrum development of mouse embryos. (1,383 KB)

    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).

  5. Extended Data Figure 5: Mesenchymal differentiation in gastric organoids. (559 KB)

    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.

  6. Extended Data Figure 6: Induction of genes during development of hGOs that mark specific differentiated antral cell types. (457 KB)

    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.

  7. Extended Data Figure 7: Characterization of LGR5-eGFP BAC transgenic reporter ES cell line. (932 KB)

    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.

  8. Extended Data Figure 8: NEUROG3 expression and endocrine differentiation are reduced in a high EGF environment. (922 KB)

    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.

  9. Extended Data Figure 9: H. pylori infection of hGOs. (266 KB)

    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).

  10. Extended Data Figure 10: Summary of methods for the directed differentiation of gastric organoids. (268 KB)

    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.

Additional data