Extended Data Fig. 10: Testing intestinal epithelial cells for equipotency.
a, Detection of LGR5–eGFP (green) and CD44 (red) in the E16.5 small intestine isolated from Lgr5-eGFP-ires-creERT2 E16.5 mice. Tissue was counterstained with DAPI (blue). Samples from three mice were analysed and a representative image is shown. Scale bars, 50 μm. b, Cartoon showing the positions used to quantify the pattern of CD44 and LGR5 expression. Quantification of the localization of CD44 and LGR5 positive cells. A total of 14 intervillus regions (x axis) were quantified up to position ±10 (y axis). c, d, Representative FACS dot plot illustrating the gating strategy used to quantify CD44. Dots represent the quantification in individual mice (n = 4). e, Spheroids forming from cells isolated based on DAPI−EpCAM+LGR5–eGFP− and DAPI−EpCAM+LGR5–eGFP− from the proximal half of the small intestine from mice at E16.5. Representative images of n = 3 biologically independent samples. f, Quantification of spheroid seeding efficiency following isolation based on either CD44 or LGR5 (n = 3 mice per condition). g–i, Gating strategy for purification of villus and intervillus cells for transplantation. The gating hierarchy of the panels is numbered i–vi (g), an example of purity is indicated (h) and an mT/mG-derived organoid is shown (i). Spheroids were generated from a pool of n = 6 biologically independent samples. j, k, Outline for transplantation experiment. In brief, experimental colitis was induced in RAG2−/− mice by administration of DSS in the drinking water. Organoids from the different cultures were subsequently infused into lumen of the mice (j) and engrafted regions were immunostained for lineage and stem-cell markers (k). l, Scheme summarizing the main findings of this work.
