Extended Data Fig. 4: PGE₂-driven spheroids contain more functional stem cells.

a, Crypts isolated from the small intestine of wild-type mice were grown into organoids by 3D culture with OGM or OGM that was supplemented daily with 0.1 μM 16,16-dimethyl PGE₂ (dmPGE₂). Indicative images and quantification of the absolute numbers of organoids and spheroids grown per 3D structure are shown. n = 6 3D cultures were evaluated per condition. Scale bar, 100 μm. b, Assessment of stem-cell activity in organoids or PGE₂-driven spheroids grown as in a by dissociation into single cells and 3D culture in OGM. Growth of crypts and organoids from the same initial number of cells was quantified on day 14. The results are indicative of five independent experiments starting from independent crypt isolations. c, Normal crypts were grown into organoids with OGM in a 3D co-culture with primary mouse intestinal fibroblasts with or without 10 μM ONO-AE3-208 (Ptger4/EP4 inhibitor). Indicative images and quantification of the absolute numbers of organoids and spheroids grown per 3D structure are shown. n = 6 3D co-cultures were evaluated per condition. Scale bar, 200 μm. d, Separation of n = 2,192 fibroblasts and epithelial cells in single-cell RNA-seq data from fibroblast–crypt organotypic cultures on the basis of the expression of key marker genes. Expression of intestinal epithelial marker genes (Epcam, Atp1b1 and Krt8) and fibroblast marker genes (Sparc, Col1a1 and Col3a1) in single cells from Ptger4-ON and Ptger4–OFF fibroblast–crypt co-cultures is shown projected onto t-SNE plots. e–j, Single-cell data from Ptger4-ON and Ptger4–OFF fibroblast–crypt co-cultures as shown in Fig. 3d, visualized on the respective t-SNE plot depicting n = 1,585 epithelial cells. e, Expression of epithelial population-specific signatures (metagenes) per single epithelial cell. Population signatures were calculated on the basis of single-cell profiling of the mouse intestinal epithelium¹⁵. f, Cell cycle analysis of single epithelial cells projected onto the t-SNE plot. g–j, Expression levels of metagenes for the signatures or transcriptional programs of RSC¹⁶ (g), β-catenin (h), Yap (i) and early (non-tumour) Apc^Min/+ tumorigenesis (j) per single epithelial cell projected onto t-SNE plots. k, Data from n = 1,585 single epithelial cells, visualized in violin plots for each co-culture condition (Ptger4-ON or Ptger4-OFF). The entire range of metagene expression levels per single epithelial cell for the signatures or transcriptional programs of RSCs, β-catenin, Yap and early Apc^Min/+ tumorigenesis is displayed. In a, c, two-way ANOVA. Data are mean ± s.e.m. ****P < 0.0001.

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