Supplementary Figure 6: TGFA overexpression induced a Ménétrier disease-like state in e-ST in vitro cultures.

(a) Phase-contrast and fluorescence images of ES cells with or without Tet. The scale bar represents 100 μm. (b) qPCR of human TGFA in ES cells and e-ST cultured with or without Tet. Expression was normalised to Gapdh. Grey bar: Knock-in ES cells, Black bar: e-ST differentiated from Knock-in ES cells at day 42. N.D., not detected. n = 3; biological independent repeats. Error bars are s.e.m. (c) Differentiation scheme of TGFA-inducible e-ST. (d,e) Immunofluorescence staining of the epithelium and mesenchymal layer of e-ST. Immunofluorescence staining of EpCAM and Desmin in e-ST on day 54 with or without Tet at lower magnification (d). Scale bar represents 200 μm. Arrowheads indicate the hypertrophic epithelia. Higher magnification of the white outlined area is shown in (e). Immunofluorescence staining of EpCAM, Venus: TGFA, and Muc5ac in e-ST on day 54 without Tet (e). Scale bar represents 30 μm. (f–h) Immunofluorescence staining of marker proteins of pit cells, parietal cells, and proliferative cells in e-ST cultured with or without Tet. Muc5ac⁺ pit cells (Tet⁻ day 54, Tet⁺ day 42) (g), H⁺/K⁺ ATPase⁺ parietal cells at day 42 (h), and Ki67+proliferative cells on day 42 (i) in e-ST cultured with or without Tet. Scale bar are 30 μm. (i–k) Quantification of EpCAM⁺ epithelial layers in Tet⁺ and Tet⁻ e-ST. Muc5ac⁺ pit cells (j), H⁺/K⁺ ATPase⁺ parietal cell % (k), and Ki67⁺ cell % (l). Statistical analyses were performed by comparing Tet+ with Tet−. Significant differences between Tet+ and Tet− are shown; t-test, ^∗, p < 0.05,^∗∗, p < 0.01, Pit cells; p = 0.01655, Parietal cells; p = 0.00427, Ki67⁺ cells; p = 0.01582, n = 3; biologically independent samples. Error bars are s.e.m. (l) Measurement of acid secretion from Tet⁺ and Tet⁻ e-ST cultures at day 54. The pH change measured every hour (0, 1, 2, 3 h). His (Histamine). n = 3; biologically independent samples. Error bars are s.e.m. For Supplementary Fig. 4a, d–h are representative images. Details of the reproducibility and statistical analyses are summarised in Supplementary Table 3 and Methods section.