Fig 2: (A) Representative phase contrast image of HIO-derived epithelial cells seeded into the Chip. Cells were exposed to continuous media flow at 30 µL/h and imaged after 0, 3, 5, and 7 days. Static Chip was imaged after 7 days. Scale bar = 200 µm. (B) Stitched phase contrast image of HIO-epithelial cells that were exposed to continual media flow at 30 µL/h for 5 days. Scale bar = 1 mm. (C) Representative brightfield image of cross-section of Chip that was exposed to continual media flow at 30 µL/h for 14 days. Scale bar = 250 µm. Representative fluorescent images showing (D) E-cadherin/CDH1 (red), ZO-1 (green), DAPI (blue), and (E) E-cadherin (blue), CDX2 (red), and Villin (green), in cross-section of Chip under conditions similar to C. Both scale bars = 50 µm. (F) Representative fluorescent images showing E-cadherin/CDH1 (red), and MUC2, lysozyme, FABP2, and chromogranin A (all green), counterstained with DAPI (blue), in cross-section of Chips that were exposed to continual media flow at 30 µL/h for 7 days. Scale bar = 10 µm. (G) Representative images of in situ hybridization for LGR5+ and WDR43+ (white arrows) in conditions similar to F. Scale bar = 10 µm. (H) Representative fluorescent image showing E-cadherin/CDH1 (blue), Ki67 (green), and DAPI (blue) in conditions similar to F. Scale bar = 100 µm. Graph shows percent Ki67+ nuclei per section.

Fig 3: Tight junction expression occurs in colonic mucosa from control mice with claudin-2 antibody; FFPE tissues. (A) Representative images of colonic mucosa from wild-type mice (n = 3) stained with E-cadherin (E-cad) antibody to identify lateral cell membranes, claudin-2 (Cldn2) antibody to identify tight junctions, and Hoechst 33342 (HOE) to identify nuclei. As has been determined before in mice,32 in our hands the 12H2 antibody to claudin-2 localized to cells in the crypt base in colon in paraffin sections. (B and C) In the crypt base, claudin-2 co-localized with the tight junction protein ZO-1 (white signal in B, arrows in C) and was also found along the basolateral membrane (arrowheads). Claudin-2 was excluded from the base of cells in colon from FFPE tissues (asterisk). Scale bars: (A) 100 µm; (B) 20 µm; (C) 10 µm.

Fig 4: BRCA1mut-FTE organoids exhibit histopathological features of STIC(A) Representative H&E stain of different histopathology for organoids at 4 months in culture, compared to 80i-CTR line. Type I obtained from the 08i-BRCA line and Type II obtained from the 79i-BRCA line, with images stitched using Stereo Investigator. Scale bar, 50 µm.(B) Table for total organoids analyzed in each cell line at 4 months (n = 5 different biological replicates used per cell line).(C) FTE organoid thickness distribution by individual lines.(D) Cellular outgrowth measured as arithmetic average roughness by individual lines.(E and F) Nuclei and cell surface distribution based on DAPI and CDH1 staining and corresponding quantification. Scale bars, 10 µM (E) 50 and 25 µm (F). (n = 3 different biological replicates used per organoid). Significance calculated using the Student’s t test and Welch ANOVA;*p < 0.05, ***p < 0.001, and ****p < 0.0001.

Fig 5: The overexpression of CDX2 in induced gastric organoids(A) A schematic representation of the protocol of gastric organoid differentiation from CDX2-iPSC. The administration of DOX (1 µM) started between days 35 and 40.(B) Immunofluorescence analyses of the MUC5AC and E-cadherin (E-Cad) expression in differentiated gastric organoids at day 37 without DOX treatment. Scale bar, 40 µm.(C) Morphologies of gastric organoids derived from CDX2-iPSC after 1 µM DOX treatment for 10 days from Day 36. The representative data of three independent experiments are shown. Scale bars, 200 µm.(D) An RT-PCR analysis showed the mRNA expression of CDX2 at Day45 with or without DOX treatment for 9 days. The expression of CDX2 was increased in DOX(+) organoids. GAPDH was used as an endogenous control.(E) Immunofluorescence analyses of CDX2 and mCherry in gastric organoids derived from CDX2-iPSC at Day45 with or without DOX treatment for 10 days. Scale bars, 20 µm.