Skip to main content

Thank you for visiting You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Generation of human antral and fundic gastric organoids from pluripotent stem cells


The human stomach contains two primary domains: the corpus, which contains the fundic epithelium, and the antrum. Each of these domains has distinct cell types and functions, and therefore each presents with unique disease pathologies. Here, we detail two protocols to differentiate human pluripotent stem cells (hPSCs) into human gastric organoids (hGOs) that recapitulate both domains. Both protocols begin with the differentiation of hPSCs into definitive endoderm (DE) using activin A, followed by the generation of free-floating 3D posterior foregut spheroids using FGF4, Wnt pathway agonist CHIR99021 (CHIR), BMP pathway antagonist Noggin, and retinoic acid. Embedding spheroids in Matrigel and continuing 3D growth in epidermal growth factor (EGF)-containing medium for 4 weeks results in antral hGOs (hAGOs). To obtain fundic hGOs (hFGOs), spheroids are additionally treated with CHIR and FGF10. Induced differentiation of acid-secreting parietal cells in hFGOs requires temporal treatment of BMP4 and the MEK inhibitor PD0325901 for 48 h on protocol day 30. In total, it takes ~34 d to generate hGOs from hPSCs. To date, this is the only approach that generates functional human differentiated gastric cells de novo from hPSCs.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Fig. 1: Overview of the protocol.
Fig. 2: Identifying appropriate confluence of target hPSCs to ensure optimal differentiation to DE and robust generation of posterior foregut spheroids.
Fig. 3: Analysis of differentiation efficiency of day 3 DE and day 6 posterior foregut monolayer cultures by immunofluorescence.
Fig. 4: Morphological changes during protocol days 4–20 of culture associated with posterior foregut spheroid generation, and outgrowth of hAGOs and hFGOs in 3D Matrigel suspension.
Fig. 5: Analysis of differentiation efficiency of day 20 hAGOs and hFGOs by immunofluorescence.
Fig. 6: Formation and differentiation of antral and fundic epithelium in hGOs grown in 3D Matrigel suspension after reducing density.


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

    CAS  Article  Google Scholar 

  2. McCracken, K. W. et al. Wnt/beta-catenin promotes gastric fundus specification in mice and humans. Nature 541, 182–187 (2017).

    CAS  Article  Google Scholar 

  3. Bartfeld, S. et al. In vitro expansion of human gastric epithelial stem cells and their responses to bacterial infection. Gastroenterology 148, 126–136.e6 (2015).

    Article  Google Scholar 

  4. Bertaux-Skeirik, N. et al. CD44 plays a functional role in Helicobacter pylori-induced epithelial cell proliferation. PLoS Pathog. 11, e1004663 (2015).

    Article  Google Scholar 

  5. Dedhia, P. H., Bertaux-Skeirik, N., Zavros, Y. & Spence, J. R. Organoid models of human gastrointestinal development and disease. Gastroenterology 150, 1098–1112 (2016).

    Article  Google Scholar 

  6. Schlaermann, P. et al. A novel human gastric primary cell culture system for modelling Helicobacter pylori infection in vitro. Gut 65, 202–213 (2016).

    CAS  Article  Google Scholar 

  7. Willet, S. G. & Mills, J. C. Stomach organ and cell lineage differentiation: from embryogenesis to adult homeostasis. Cell. Mol. Gastroenterol. Hepatol. 2, 546–559 (2016).

    Article  Google Scholar 

  8. Zorn, A. M. & Wells, J. M. Molecular basis of vertebrate endoderm development. Int. Rev. Cytol. 259, 49–111 (2007).

    CAS  Article  Google Scholar 

  9. Munera, J. O. et al. Differentiation of human pluripotent stem cells into colonic organoids via transient activation of BMP signaling. Cell Stem Cell 21, 51–64.e6 (2017).

    CAS  Article  Google Scholar 

  10. Rankin, S. A. et al. Timing is everything: reiterative Wnt, BMP and RA signaling regulate developmental competence during endoderm organogenesis. Dev. Biol. 434, 121–132 (2018).

    CAS  Article  Google Scholar 

  11. Stevens, M. L. et al. Genomic integration of Wnt/beta-catenin and BMP/Smad1 signaling coordinates foregut and hindgut transcriptional programs. Development 144, 1283–1295 (2017).

    CAS  Article  Google Scholar 

  12. Roberts, D. J. et al. Sonic hedgehog is an endodermal signal inducing Bmp-4 and Hox genes during induction and regionalization of the chick hindgut. Development 121, 3163–3174 (1995).

    CAS  PubMed  Google Scholar 

  13. Bayha, E., Jorgensen, M. C., Serup, P. & Grapin-Botton, A. Retinoic acid signaling organizes endodermal organ specification along the entire antero-posterior axis. PLoS ONE 4, e5845 (2009).

    Article  Google Scholar 

  14. Molotkov, A., Molotkova, N. & Duester, G. Retinoic acid generated by Raldh2 in mesoderm is required for mouse dorsal endodermal pancreas development. Dev. Dyn. 232, 950–957 (2005).

    CAS  Article  Google Scholar 

  15. Wang, Z., Dolle, P., Cardoso, W. V. & Niederreither, K. Retinoic acid regulates morphogenesis and patterning of posterior foregut derivatives. Dev. Biol. 297, 433–445 (2006).

    CAS  Article  Google Scholar 

  16. Engevik, K. A., Matthis, A. L., Montrose, M. H. & Aihara, E. Organoids as a model to study infectious disease. Methods Mol. Biol. 1734, 71–81 (2018).

    CAS  Article  Google Scholar 

  17. Takebe, T., Wells, J. M., Helmrath, M. A. & Zorn, A. M. Organoid center strategies for accelerating clinical translation. Cell Stem Cell 22, 806–809 (2018).

    CAS  Article  Google Scholar 

  18. Finkbeiner, S. R. et al. Transcriptome-wide analysis reveals hallmarks of human intestine development and maturation in vitro and in vivo. Stem Cell Rep. 4, 1140–1155 (2015).

    CAS  Article  Google Scholar 

  19. Kroon, E. et al. Pancreatic endoderm derived from human embryonic stem cells generates glucose-responsive insulin-secreting cells in vivo. Nat. Biotechnol. 26, 443–452 (2008).

    CAS  Article  Google Scholar 

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

    CAS  Article  Google Scholar 

  21. Schumacher, M. A. et al. Helicobacter pylori-induced sonic hedgehog expression is regulated by NFκB pathway activation: the use of a novel in vitro model to study epithelial response to infection. Helicobacter 20, 19–28 (2015).

    CAS  Article  Google Scholar 

  22. Wroblewski, L. E. et al. Helicobacter pylori targets cancer-associated apical-junctional constituents in gastroids and gastric epithelial cells. Gut 64, 720–730 (2015).

    CAS  Article  Google Scholar 

  23. McCracken, K. W., Howell, J. C., Wells, J. M. & Spence, J. R. Generating human intestinal tissue from pluripotent stem cells in vitro. Nat. Protoc. 6, 1920–1928 (2011).

    CAS  Article  Google Scholar 

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

    Article  Google Scholar 

  25. Workman, M. J. et al. Engineered human pluripotent-stem-cell-derived intestinal tissues with a functional enteric nervous system. Nat. Med. 23, 49–59 (2017).

    CAS  Article  Google Scholar 

  26. Sato, T. et al. Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche. Nature 459, 262–265 (2009).

    CAS  Article  Google Scholar 

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

    CAS  Article  Google Scholar 

Download references


We thank D. Kechele for comments on the manuscript. This work was supported by grants from the National Institutes of Health (R01DK092456, U19AI116491, and P01HD093363 to J.M.W.). We also acknowledge core support from the Pluripotent Stem Cell Facility of Cincinnati Children’s Hospital Medical Center. We acknowledge core support from a Cincinnati Digestive Disease Center award (P30DK0789392).

Author information

Authors and Affiliations



J.M.W., K.W.M., and T.R.B. conceived the study and experimental design. T.R.B. and J.M.W. co-wrote the manuscript. T.R.B. produced all images for the figures, and J.M.W. produced the protocol schematic. K.W.M. and T.R.B. analyzed the data and performed experiments.

Corresponding author

Correspondence to James M. Wells.

Ethics declarations

Competing interests

J.M.W. and K.W.M. are listed on the following patent applications: PCT/US2015/032626, ‘Methods and systems for converting precursor cells into gastric tissues through directed differentiation’, and PCT/US2017/031309, ‘Methods for the in vitro manufacture of gastric fundus tissue and compositions related to same’. T.R.B. declares no competing interests.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Related links

Key references using this protocol

McCracken, K. W. et al. Nature 516, 400–404 (2014):

McCracken, K. W. et al. Nature 541, 182–187 (2017):

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Broda, T.R., McCracken, K.W. & Wells, J.M. Generation of human antral and fundic gastric organoids from pluripotent stem cells. Nat Protoc 14, 28–50 (2019).

Download citation

  • Published:

  • Issue Date:

  • DOI:

This article is cited by


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.


Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing