Figure 1.

Hopx⁺ CARSCs Promote Colitis-Associated Regeneration In Vivo

(A–D) Sections of colonic mucosa from wild-type (WT) mice treated with vehicle, 7 days of DSS, or 7 days of DSS with a 14-day “washout” period were stained with H&E (A, top panels), Epcam (green), Ki67 (red) (A, bottom panels), or in situ probes against Lgr5 (D, top panels) and Hopx mRNAs (D, bottom panels). Arrows and arrowheads denote crypt bases. White dashed lines indicate crypt/lamina propria boundaries. The asterisk denotes an ulcer. Percentage of atrophic (yellow) and hypertrophic (green) crypts within the distal-most colon (1 cm) under various conditions of DSS-induced colitis were plotted as mean ± SD (B) (A, atrophic crypts; H, hypertrophic crypts). The percentage of Ki67⁺ crypt epithelial cells was plotted as mean ± SD for homeostatic, atrophic, and hypertrophic crypts (C). n = 3–4 mice/group.

(E and F) Transiently lineage-labeled cells (red) from Hopx^CreERT2/Rosa^Td or Lgr5^CreERT2/Rosa^Td mice were co-stained with Tacstd2 (green) (E). The percentage of Tacstd2⁺ crypts in the mid and distal colon that were co-labeled with tdTomato from the two CreERT2 lines was plotted as mean ± SD (F). n = 3 mice/group.

(G) Single Hopx⁺ cells at the regenerative stage of DSS-induced colitis were sorted and cultured in Matrigel with 50% L-WRN media (left panel). Light and tdTomato fluorescent images of spheroids on day 6 after plating (right panels).

(H) Experimental scheme for lineage tracing assays of Hopx⁺ CARSCs from Hopx^CreERT2/Rosa^Td mice at the regenerative stage of DSS-induced colitis (top panel). TdTomato⁺ traced clones in the distal colon were co-stained with Muc2 (goblet cells), Chga (enteroendocrine cells), and Slc26a3 (colonocytes).

(I–K) Experimental scheme for Hopx⁺ CARSCs ablation (I, top panel). Crypt morphology was defined by H&E stained colonic sections from Hopx^CreER/Rosa^DTR mice (arrows in I) and their WT littermate controls subjected to the same procedure. The number of atrophic/degenerating crypts per mid + distal colon section was plotted as mean ± SD (J). n = 4–6 mice/group. Colon length was plotted as a box-whisker plot (K). n = 7–9 mice/group.

Two-tailed Student’s t test in (C), (F), and (J): ^∗∗p < 0.01. Two-tailed Mann-Whitney U test in (K): ^∗∗p < 0.01. Bars: (G) 500 μm; (A, D, E, H, and I) 100 μm; (insets of A, D, and H) 25 μm. Histological, in situ hybridization and immuno-fluorescent images are representative of at least 3 mice examined.

See also Figure S1.