Figure 4.

CD PDOs retain evidence of altered cell composition, high oxidative stress, and turnover

(A) Schematic summarizing the relative expression of type, as determined by gene expression (markers used for each cell type in parentheses). Up-arrow, upregulation. Down-arrow, downregulation. P, penetrating CD. See Figures S9A–S9D for violin plots displayed as both clinical and molecular subtypes of CD.

(B) Violin plots show the ratio of LYZ and MUC2 transcripts in CD PDOs vs. healthy controls (B [left], all CD subtypes combined; B [right], separated into molecular subtypes of CD). Statistical significance was assessed by Mann-Whitney. Only significant p values are displayed (n = 6–15 subjects in each group). See Figure S9E for the same data, displayed as clinical subtypes of CD.

(C) FFPE of CD PDOs of the IDICD subtypes were analyzed for goblet (MUC2; green) and Paneth (lysozyme; red) cells by confocal immunofluorescence. Representative images are shown. Scale bar, 150 μm. Boxed regions (numbered 1, 2 in upper panels) are magnified below. See Figure S9F for quantification of images and Figure S9G for summary.

(D) Electron micrographs of Paneth cells in healthy and CD PDOs of IDICD subtypes are shown. The boxed region on the left is magnified on the right. BM, basement membranes; AV, apical villi; SG, secretory granules. Scale bar, 1 μm in top and middle panels and 2 μm in the bottom panel.

(E) Violin plots show the extent of DNA damage, as determined by flow cytometry analysis of γH2AX in CD PDOs vs. healthy controls (E [left], all CD subtypes combined; E [right], separated into molecular subtypes of CD). Statistical significance was assessed by Mann-Whitney (left) or one-way ANOVA (right). Only significant p values are displayed.

(F) Violin plots show the extent of oxidative DNA/RNA damage in CD PDOs vs. healthy controls. Statistical significance was assessed by Mann-Whitney. Only significant p values are displayed. See Figures S10A and S10B for the display of the findings based on clinical subtypes of CD.

(G) Violin plots show the extent of BrDU incorporation over 24 h on four-day-old CD PDOs grown in 96-well plates prior to assessment by ELISA. Statistical significance was assessed by Mann-Whitney (left) and one-way ANOVA (right). Only significant p values are displayed (n = 5–8 subjects in each group; 2–3 repeats in each PDO). See Figures S10C and S10D for the display of the findings based on clinical subtypes of CD.

(H) Violin plots show % cells with Ki67-positive nuclei in CD PDOs vs. healthy controls. Statistical significance was assessed by Mann-Whitney. Only significant p values are displayed (n = 2–5 subjects in each group; 2–3 repeats in each PDO). See Figures S10E and S10F for the display of the findings based on clinical subtypes of CD. See Table S2 for subjects analyzed in each assay. See also Figure S11 for cellular apoptosis in CD PDOs at baseline and upon challenge with TNF-α.