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. 2024 Jul 18;19(8):1172–1188. doi: 10.1016/j.stemcr.2024.06.006

Figure 2.

Figure 2

Altered cell fate patterns in the ulcerated epithelium samples

(A) Immunostaining for CEACAM1 (green), PCNA (red), and β-catenin (yellow) human tissues. Scale bar: 100 μm.

(B) UMAP depicting 4,351 colonic epithelial cells from patients with UC following cell type annotation as in (1B).

(C and D) Trajectory graph based on over-clustering and RNA velocity analysis of epithelial cells from ulcerated samples. Arrows (D) point to velocity vectors going from differentiated cell clusters toward progenitor clusters.

(E) (Left) representative image of immunohistochemistry staining for KRT20 and KI67 in healthy and ulcerated human colonic samples. Arrows indicate KRT20/KI67 double-positive cells. (Right) bar plots showing percentage of KI67 single-positive and KRT20/KI67 double-positive cells in the indicate areas. Each dot represents an individual donor (average of at least 5 crypts each). H, healthy controls; UC, ulcerated samples. ∗ for p < 0.05 by Student’s t test. Scale bar: 100 μm.

(F) Density plots showing of KRT20 expression (y axis) and proliferation score (x axis) in the healthy (left) and ulcerated (right) datasets.

(G) Representative flow cytometry analysis of KRT20-tdTomato human organoid reporter line. A wild-type line was used as a control for tdTomato levels. Schematic of the reporter gene is shown above.

(H) Quantitative reverse-transcription PCR (RT-qPCR) analysis of the indicated genes in tdTomato− and tdTomato+ sorted from KRT20-tdTomato reporter line. Expression levels of genes of interest were normalized to GAPDH. Bars show fold change relative to KRT20-tdTomato- cells ±SEM; each dot represents an independent experiment. ∗∗∗ for p < 0.005 by ordinary 2-way AVOVA with Sidak’s correction for multiple comparison.

(I) (Left) representative phase-contrast images of human colon organoids formed from sorted tdTomato− and tdTomato+ (bottom) cells 10 days after seeding. Scale bars: 275 μm. (Right) quantification of organoid-forming efficiency and size of organoids formed from purified KRT20-tdTomato- and tdTomato+ cells. For efficiency, plot shows mean ± SEM; each dot represents an independent experiment (average of four replica per experiment). For size, plot was generated using the Tukey method. The number of organoids analyzed within one representative experiment is shown below the bars.