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[Advanced Science]Organoid-Based Human Stomach Micro-Physiological System to Recapitulate the Dynamic Mucosal Defense Mechanism

2023-12-22

https://onlinelibrary.wiley.com/doi/10.1002/advs.202300164

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Organoid-Based Human Stomach Micro-Physiological System to Recapitulate the Dynamic Mucosal Defense Mechanism

Hye-Jin Jeong, Ji-Hyeon Park, Joo H. Kang, Jonathan Sabaté del Río, Seong-Ho Kong, Tae-Eun Park



Figure 1

Reconstitution of the human stomach in an MPS device. a) Schematic illustration of the human gastric mucosa (left) and the human stomach MPS (hsMPS) with a singularized human gastric antral organoid (hAOs) cultured on the apical luminal channel interfaced with primary gastric stromal cells (gMSCs) on the basal abluminal channel (center). At the top right, a bright field image of hAOs (bar, 250 µm) used to generate gastric epithelium, and at the bottom right, an immunofluorescence micrograph of the gMSCs labeled with Vimentin (bar, 100 µm) are shown. b) Photograph of the hsMPS (bar, 1 cm). c) Timeline for the reconstitution of the human stomach in an MPS device. d) Immunofluorescence micrographs of the gastric epithelium in the hsMPS at 6 days labeled with E-cadherin, mucin 5AC, mucin 6, trefoil factor 1, and gastrin (bar, 20 µm).

 

Figure 2

Sustainable gastric stem cell activity in the hsMPS. a) Immunofluorescence images of Ki-67 in gastric epithelial cells (bar, 20 µm) (left) in the hsMPS in monocultures (M) and co-cultures (C) under static (top) and flow conditions (bottom). Percentage of Ki-67-positive epithelial cells analyzed using ImageJ under static and flow conditions (right). b) Organoid forming efficacy assay in the hsMPS in monocultures (M) and co-cultures (C) under flow conditions. c,d) Flow cytometry analysis of LGR5- (left), CCK2R- (right) positive gastric epithelial cells representing gastric stem cells; and SOX2-positive gastric progenitor cells in the hsMPS in monocultures (M) and co-cultures (C) under flow conditions compared to hAOs. e) Schematic diagram illustrating the orchestrated gastric epithelial proliferation and differentiation in the hsMPS by mesenchymal niche factors derived from gMSCs under fluid flow. The results are presented as the mean ± SEM. For statistical analysis, a one-way ANOVA and Tukey's test was performed (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001). ns: non-significant.

Figure 3

Fluid flow-enhanced cellular niche function of gMSC and telocyte-like morphogenesis. a) Schematic illustration of fluid flow-enhanced niche function of gMSC. b) The mRNA expression ratios of genes encoding FOXL1 of gMSCs in flow versus static conditions. c) Immunofluorescence micrographs of the gMSCs in the hsMPS labeled with acetylated tubulin marker (AcTub, green) and telocyte-specific marker (FOXL1, red) (bar, 100 µm) (left). d) Quantification of Foxl1-positive gMSCs using a flow cytometer under static and flow conditions. e) Quantification of human Wnt2b, Egf, and Fgf10 levels using ELISA in the media collected from hsMPS (C) under static and flow conditions. f) Percentage of Ki-67-positive gMSCs analyzed using ImageJ under static and flow conditions. g) mRNA expression ratio of Notch receptors (NOTCH1, NOTCH2, NOTCH3, and NOTCH4), Notch ligands (DLL1, DLL3, DLL4, JAG1, and JAG2). h) The mRNA expression ratio of transcription factor HES1 initiated by the Notch signaling pathway in gMSCs under static and flow conditions. i) Percentage of Ki-67-positive gMSCs analyzed using ImageJ when treated with a Notch inhibitor, LY3039478 (100 nm). The results are presented as the mean ± SEM. For statistical analysis, a Student's t-test was performed (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001).

Figure 7

Immune pathogenesis in H. pylori-infected hsMPSs co-cultivated with PBMC. a) Schematic illustration of the H. pylori-infected human gastric mucosa (left) and H. pylori-infected hsMPSs co-cultured with peripheral blood mononuclear cells (PBMCs). b) Representative fluorescence images of PBMCs adhering to the basal side of the abluminal microchannel membrane of a non-infected (upper, left) and H. pylori-infected hsMPS at an MOI of 10 (lower, left) stained with CellTracker Green (bar, 250 µm). Relative number of PBMCs adhering to the basal side of the membrane of H. pylori-infected hsMPS at MOI 10 compared to non-infected control (right). For statistical analysis, a Student's t-test was performed (*p < 0.05). The mRNA expression ratios of NF-κB-mediated inflammatory cytokines c) TNFa, d) IL8, e) IL1B, f) LTB, g) CXCL3, and h) CCL20 in gastric epithelial cells when infected with H. pylori at an MOI of 10 in the presence and absence of PBMCs in the abluminal microchannel relative to the non-infected control (NI). The results are presented as the mean ± SEM, n  =  2 for the independent hsMPS experiments. For statistical analysis, one-way ANOVA with Tukey's multiple comparisons test was performed (*p < 0.05; **p < 0.01; ***p <0.001; ****p < 0.0001). TNFa, tumor necrosis factor alpha; IL, interleukin; LTB, lymphotoxin beta; CCL/ CXCL, chemokine (C-C/C-X-C motif) ligand; −, without PBMC; +, with PBMC.