Extended Data Fig. 1: Small intestine ALI organoids possess different mesenchymal and epithelial cell types.

a, IF whole-mount staining of small intestine organoids at day 14 showing SMA⁺ or PDGFRA⁺ fibroblasts, CD31⁺ endothelial cells and PGP9.5⁺ neurons (green), ECAD⁺ epithelium (white) and DAPI (blue) (representative images from n = 3 biological replicates). b, IF whole-mount staining of small intestine organoids at day 14 showing MUC2⁺ goblet cells, CHGA⁺ enteroendocrine cells, LYZ1⁺ Paneth cells (green), ECAD⁺ epithelium (white) and DAPI (blue) (representative images from n = 3 biological replicates). c, Enlargement of organoid cup-shaped MUC2⁺ goblet cells (representative image from n = 3 biological replicates). d, Violin plots of CD14 and CD68 mRNA expression from CeD organoid scRNA-seq and a scatter plot of CD14 and CD68 mRNA co-expression in the myeloid compartment. e, Whole-mount IF staining of small intestine ALI organoid CD4⁺ (green) and CD8⁺ (red) T cells, showing enrichment of CD8⁺ T cells within the EPCAM⁺ epithelial compartment (white). DAPI (blue). In contrast, CD4⁺ T cells localize to non-epithelial lamina propria-like areas (representative image from n = 3 biological replicates). All scale bars are 100 µm, except (c) in which the scale bar is 50 µm.