Fig. 4. Increased number of differentiated pit cells leads to higher H. pylori adhesion.

a Scheme of the directed differentiation setup. b Western blot analysis of pit markers (MUC5AC, GKN1, and GKN2) and neck markers (MUC6) in 2D monolayers upon differentiation according to the scheme in (a). c Scheme of the experimental setup. Differentiated monolayers were infected with a H. pylori strain expressing GFP at a MOI of 1 for 6 h. d–f Quantification of adhered bacteria to 2D monolayer cells by (d) flow cytometry, (e) CFU assay and (f) qRT-PCR of CagA expression (fold over host GAPDH; f). Results are normalized against standard media (ENRWFGTi_, grey symbols). g Confocal images of differentiated gastric 2D monolayers infected with GFP-expressing H. pylori. Nuclei were counterstained with Hoechst 33342. Scale bar: 25 µm. h Western blot analysis of phosphorylated CagA (P-Tyr) in H. pylori-infected 2D monolayers. i and j Quantification of bacterial attachment (CagA total protein over host alpha-tubulin; i and CagA translocation (P-Tyr over CagA; j) by western blot. k Scheme of the experimental setup. Differentiated 3D organoids were microinjected with H. pylori (MOI 1 for 6 h). l Quantification of PSCA-positive cells in 3D organoids measured by flow cytometry. m Quantification of adhered bacteria to 3D organoid cells by flow cytometry. Data from organoid lines derived from individual donors are shown as symbols (patient #1-circle, #10-diamond, #71-triangle, and #72-square), with mean values with horizontal lines. Data in panel b, d–j are representative of or show data of organoid lines from 4 donors, l and m of 3 donors, and 1 (b, f, i and j), 3 (g, h, l, m), 4 (d) or 5 (e) independent experiments. Statistical analysis of data in panels d–f, i, j and l, m was performed using one-way ANOVA with Tukey’s multiple comparisons post-hoc test. Source data are provided as a Source Data file.