Letter | Published:

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

Nature volume 516, pages 400404 (18 December 2014) | Download Citation

Abstract

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.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Accessions

Primary accessions

ArrayExpress

Data deposits

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

References

  1. 1.

    & Helicobacter pylori virulence factors in gastric carcinogenesis. Cancer Lett. 282, 1–8 (2009)

  2. 2.

    , & Peptic ulcer disease today. Nature Clin. Pract. Gastroenterol. Hepatol. 3, 80–89 (2006)

  3. 3.

    The global health burden of infection-associated cancers in the year 2002. Int. J. Cancer 118, 3030–3044 (2006)

  4. 4.

    Helicobacter pylori infection and disease: from humans to animal models. Dis. Model. Mech. 1, 50–55 (2008)

  5. 5.

    & Gastric epithelial stem cells. Gastroenterology 140, 412–424 (2011)

  6. 6.

    et al. Directed differentiation of human pluripotent stem cells into intestinal tissue in vitro. Nature 470, 105–109 (2011)

  7. 7.

    et al. Highly efficient generation of human hepatocyte-like cells from induced pluripotent stem cells. Hepatology 51, 297–305 (2010)

  8. 8.

    et al. Production of pancreatic hormone-expressing endocrine cells from human embryonic stem cells. Nature Biotechnol. 24, 1392–1401 (2006)

  9. 9.

    et al. Efficient differentiation of human embryonic stem cells to definitive endoderm. Nature Biotechnol. 23, 1534–1541 (2005)

  10. 10.

    , , & Generating human intestinal tissue from pluripotent stem cells in vitro. Nature Protocols 6, 1920–1928 (2011)

  11. 11.

    , , & Signals from lateral plate mesoderm instruct endoderm toward a pancreatic fate. Dev. Biol. 259, 109–122 (2003)

  12. 12.

    , , , & BMP signalling regulates anteroposterior endoderm patterning in zebrafish. Mech. Dev. 118, 29–37 (2002)

  13. 13.

    et al. Generation of anterior foregut endoderm from human embryonic and induced pluripotent stem cells. Nature Biotechnol. 29, 267–272 (2011)

  14. 14.

    , , & Retinoic acid regulates morphogenesis and patterning of posterior foregut derivatives. Dev. Biol. 297, 433–445 (2006)

  15. 15.

    et al. Dorsal pancreas agenesis in retinoic acid-deficient Raldh2 mutant mice. Dev. Biol. 284, 399–411 (2005)

  16. 16.

    , & Retinoic acid generated by Raldh2 in mesoderm is required for mouse dorsal endodermal pancreas development. Dev. Dyn. 232, 950–957 (2005)

  17. 17.

    et al. The role of the transcriptional regulator Ptf1a in converting intestinal to pancreatic progenitors. Nature Genet. 32, 128–134 (2002)

  18. 18.

    & Stimulation of rat oxyntic gland mucosal growth by epidermal growth factor. Am. J. Physiol. 238, G45–G49 (1980)

  19. 19.

    Postnatal undernutrition: effect of epidermal growth factor on growth and function of the gastrointestinal tract in rats. J. Pediatr. Gastroenterol. Nutr. 3, 618–625 (1984)

  20. 20.

    et al. Cell dynamics in fetal intestinal epithelium: implications for intestinal growth and morphogenesis. Development 138, 4423–4432 (2011)

  21. 21.

    et al. Role of the homeodomain transcription factor Bapx1 in mouse distal stomach development. Gastroenterology 136, 1701–1710 (2009)

  22. 22.

    et al. Cell lineage distribution atlas of the human stomach reveals heterogeneous gland populations in the gastric antrum. Gut (2014)

  23. 23.

    et al. Lgr5+ve stem cells drive self-renewal in the stomach and build long-lived gastric units in vitro. Cell Stem Cell 6, 25–36 (2010)

  24. 24.

    et al. Neurogenin3 is differentially required for endocrine cell fate specification in the intestinal and gastric epithelium. EMBO J. 21, 6338–6347 (2002)

  25. 25.

    , , & Neurogenin 3 is essential for the proper specification of gastric enteroendocrine cells and the maintenance of gastric epithelial cell identity. Genes Dev. 16, 1488–1497 (2002)

  26. 26.

    , , & A mechanism by which Helicobacter pylori infection of the antrum contributes to the development of duodenal ulcer. Gastroenterology 110, 1386–1394 (1996)

  27. 27.

    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, 114–121 (2000)

  28. 28.

    et al. Helicobacter pylori CagA protein targets the c-Met receptor and enhances the motogenic response. J. Cell Biol. 161, 249–255 (2003)

  29. 29.

    et al. Helicobacter pylori cagA+ strains and dissociation of gastric epithelial cell proliferation from apoptosis. J. Natl. Cancer Inst. 89, 863–868 (1997)

  30. 30.

    et al. Activin and BMP4 synergistically promote formation of definitive endoderm in human embryonic stem cells. Stem Cells 30, 631–642 (2012)

  31. 31.

    et al. The pINDUCER lentiviral toolkit for inducible RNA interference in vitro and in vivo. Proc. Natl Acad. Sci. USA 108, 3665–3670 (2011)

  32. 32.

    , , , & Simple and highly efficient BAC recombineering using galK selection. Nucleic Acids Res. 33, e36 (2005)

  33. 33.

    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, 458–466 (2013)

  34. 34.

    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, 5791–5795 (1993)

  35. 35.

    , , & Helicobacter pylori enter and survive within multivesicular vacuoles of epithelial cells. Cell. Microbiol. 4, 677–690 (2002)

  36. 36.

    et al. Gastric Sonic Hedgehog acts as a macrophage chemoattractant during the immune response to Helicobacter pylori. Gastroenterology 142, 1150–1159 (2012)

  37. 37.

    et al. Analysis of the human tissue-specific expression by genome-wide integration of transcriptomics and antibody-based proteomics. Mol. Cell. Proteomics 13, 397–406 (2014)

  38. 38.

    et al. The NIH roadmap epigenomics mapping consortium. Nature Biotechnol. 28, 1045–1048 (2010)

  39. 39.

    , , & Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol. 10, R25 (2009)

  40. 40.

    , & TopHat: discovering splice junctions with RNA-Seq. Bioinformatics 25, 1105–1111 (2009)

  41. 41.

    et al. Differential analysis of gene regulation at transcript resolution with RNA-seq. Nature Biotechnol. 31, 46–53 (2013)

  42. 42.

    et al. Dynamic chromatin remodeling mediated by polycomb proteins orchestrates pancreatic differentiation of human embryonic stem cells. Stem Cells 12, 224–237 (2013)

Download references

Acknowledgements

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

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

Authors

  1. Search for Kyle W. McCracken in:

  2. Search for Emily M. Catá in:

  3. Search for Calyn M. Crawford in:

  4. Search for Katie L. Sinagoga in:

  5. Search for Michael Schumacher in:

  6. Search for Briana E. Rockich in:

  7. Search for Yu-Hwai Tsai in:

  8. Search for Christopher N. Mayhew in:

  9. Search for Jason R. Spence in:

  10. Search for Yana Zavros in:

  11. Search for James M. Wells in:

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 interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Yana Zavros or James M. Wells.

Extended data

About this article

Publication history

Received

Accepted

Published

DOI

https://doi.org/10.1038/nature13863

Further reading

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.