Figure 4.

Recapitulating Cycles of Colonic Epithelial Injury-Regeneration In Vitro

(A) Scheme of mature ALI culture re-submersion and re-exposure to air.

(B–M) ALI monolayers prior to re-submersion (ALId21; B–D), 24 h (Re-Sub 24h; E–G), and 7 days (Re-Sub 7d; H–J) after re-submersion, as well as 14 days after re-exposure to ALI (Re-ALId14; K–M) were examined by H&E staining (B, E, H, and K), immunostaining for UEA1 (C, F, I, and L) and Ki67 (D, G, J, and M).

(N) Heatmap of regenerative epithelial markers at 0 h, 8 h, 24 h, and 7 days after re-submersion. Data are row normalized. n = 3 replicates/time point.

(O and P) GSEA-based analysis comparing the mRNA profiles of re-submerge d7 cells to the regenerating epithelium of DSS-treated mice and fetal spheroid epithelium (Mustata et al., 2013, Yui et al., 2018). NES = 2.01 (O) and 2.65 (P). FDR < 0.001. See also Tables S5 and S6.

(Q–S) Whole mount images of ALI cultures derived from Hopx^CreER/Rosa^Td mice. The presence of Hopx-expressing cells (red) at indicated time points (Q, ALId21; R, Re-sub d2; S, Re-ALI d2) was examined by transiently labeling monolayers with 4-OH tamoxifen 24 h before imaging.

(T–V) Sections of mouse colons from Hopx^CreER/Rosa^Td mice treated with no DSS, DSS for 7 days (DSS d7), and DSS for 7 days with a 14-day recovery phase (DSS d7 + d14). Hopx⁺ cells (red) were transiently labeled with tamoxifen within homeostatic (T), atrophic (U), and hypertrophic crypts (V) (arrowheads in T–V, respectively). White dashed lines mark the crypt/stroma boundary.

Bars: (K–M) 25 μm; (S and V) 50 μm. Histological and immuno-stained images are representative of at least 3 Transwell samples or animals examined.

See also Figure S4.