Extended Data Fig. 8: MRISCs require MAP3K2 to promote intestinal organoid growth via R-spondin 1.

a, Bright-field images of organoids cultured for 0, 3, or 5 days alone (no stromal cells), or co-cultured with MRISCs or with non-MRISC stromal cells in the absence (−) or presence (+) of R-spondin 1 or anti-R-spondin 1 antibody (Ab). Results are representative of three independent experiments. Scale bars, 100 μm. b, Average circumferences of colon organoids in a at day 5. Each symbol represents a single organoid (n = 15 organoids over three independent experiments). c, Dark-field images of organoids co-cultured with MRISCs or CD138⁺ cluster 2 stromal cells with an anti-R-spondin 1 antibody (Ab) or a control antibody (isotype). Bottom panels show magnified images of boxed areas. Results are representative of three independent experiments. Scale bars, 500 μm (top); 200 μm (bottom). d, Average circumferences of colon organoids in c at day 4. Each symbol represents a single organoid (n = 15 organoids over three independent experiments). e, Images of tdTomato⁺ cells in the colons of wild-type mice orthotopically injected with PBS alone or with tdTomato⁺ colon stromal cells in PBS (Extended Data Fig. 3k). White dashed lines indicated baseline of colon crypt. Results are representative of two independent experiments. Scale bars, 200 μm. f, qRT–PCR of tdTomato expression in the colons of wild-type mice orthotopically injected with PBS or tdTomato⁺ colon stromal cells (n = 4 colon segments over two independent experiments) (Extended Data Fig. 3k). g, qRT–PCR of Axin2, Lgr5, Rspo1, and tdTomato expression in colons from DSS-exposed wild-type mice (n = 4 colon segments over two independent experiments) orthotopically injected with PBS or with wild-type or Map3k2^−/− MRISCs from colons of wild-type or Map3k2^−/−R26-mTmG mice (Extended Data Fig. 3k). Data presented as mean ± s.e.m. Numbers in plots are P values calculated by two-tailed unpaired Student’s t-test.

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