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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.

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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.


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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).

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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.

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Correspondence to James M. Wells.

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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.

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

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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).

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