Fig 2: Conserved numbers but impaired functionality of stem cells in Cd44?ie mice.a IHC staining of OLFM4. Scale bar: 50 µm. b Average number of OLFM4-positive stem cells per crypt. Error bars, ±SE. N = number of mice, n = number of crypts. c SI organoids observed 5 days after Cd44 deletion and controls. Scale bar: 200 µm. d Budded vs. round organoid numbers after deletion of Cd44 and passaging (P) every 5 days. n = number of organoids. Error bars, ±SE. e Representative images of f. Scale bar: 200 µm. f Relative organoid number along passages in Cd44fl/fl;VillinCreERT2 organoid cultured with or without 4-OHT treatment. N = 3 mice. Scale bar: 500 µm. g Organoid vs. cystic organoid numbers after deletion of Cd44 and treatment with Wnt3a CM, control CM or CHIR. N = 3 experiments. Error bars, ±SE. h Representative images of g CHIR and Wnt3a conditions. Scale bar: 200 µm. Student’s t test; *p < 0.05, **p < 0.01, ns = not significant. 4-OHT 4-hydroxytamoxifen. The dotted line in g indicates that the CHIR and Wnt3a conditions are two independent but related experiments.

Fig 3: Stretching induced stemness promotion. a and b showed the imaging and quantification of IHC staining for Olfm4. c Venn diagram showing the overlap among ISC signature genes (green), up-regulated genes (red), and down-regulated genes (purple). The significance was evaluated by Fisher’s exact test. d GSEA plot of static and stretching treated organoid sample compared with a gene list containing the top 384 ISC signature genes. NES, normalized enrichment score. e Quantification of the expression level of Lgr5, Ascl2, Olfm4, Msi1, Alpi, and Chga using qRT-PCR. f Images of organoids expressing Lgr5-eGFP at day1 post passaging. The right images were for stretching and the left for static. Red arrows marked out the round transparent ISC sphere complexes. g Quantification of ISC sphere ratio. Student’s t-test: ***P < 0.001. **P < 0.01. *P < 0.05. Scale bars, 50 μm in a, 100 μm in f

Fig 4: Stem cell formation and cellular proliferation in the small intestine of PCdel mice.(A, B) Representative images of immunofluorescence staining of ileal tissue from WT and PCdel mice for Olfm4 (A, intestinal stem cell marker) and Ki67 (B, transient amplifying cells). Scale bar, 100 μm. (C, D) Quantification of Olfm4 (C) positive area per crypt (150 crypts were used for analysis) and Ki67 (D) positive area (n = 24 crypts per group). No statistically significant difference was noted (unpaired t test).

Fig 5: Stem cell maintenance and differentiation in PCdel mice.(A, B, C, D) mRNA expression of stem cell expressed genes Lgr5 (A), Olfm4 (B), Ascl2 (C), and Bmi1 (D) was determined by qRT-PCR analysis in ileal tissue from WT and PCdel mice (n = 6 per each genotype). No statistically significant difference was noted (unpaired t test). (E, F, G) Representative images of Alcian blue staining (E, Goblet cells), Dclk-1 immunofluorescence staining (F, Tuft cells), and Cga immunofluorescence staining (G, enteroendocrine cells) of ileal tissue from WT and PCdel mice. Scale bar, 100 μm. (H, I, J) Corresponding quantification of Goblet cells (H), Tuft cells (I), and enteroendocrine cells (J) numbers (each dot representing a crypt, n = 3 mice per genotype). No statistically significant differences seen (unpaired t test).