Fig. 1. Characterization of cellular heterogeneity and marker expression in intestinal organoids.

(A) Illustration of mouse intestinal organoid phenotypes derived from distinct stages. Epithelial cells isolated from the fetus at E16.5 give rise to FEnS in three-dimensional (3D) culture, while cells isolated from the adult intestine give rise to budding aOrgs. (B) Uniform Manifold Approximation and Projection (UMAP) plot of cells from FEnS or aOrgs following scRNA-seq. Left: FEnS (pink) and aOrg (blue). Right: Annotation of cell types based on canonical marker expression (see fig. S1A). Arrows indicate adult-like clusters of cells derived from FEnS. (C) PAGA trajectory of clusters from (B). The size of the nodes is proportional to the number of cells in each cluster, and the thickness of the edges represents confidence in transcriptional connection. Arrows indicate adult-like fetal clusters I and II. (D) Volcano plot of DE surface marker genes between the fetal progenitor cluster (pink) and adult secretory clusters (blue). x axis: Log₂ fold change (LFC); y axis: Log₁₀ P value. Kit (adult marker) and Ly6a (fetal marker) are highlighted. Outliers with 15 < LFC < −15 and high P values were removed. (E) UMAP plot showing expression levels of Kit (left) and Ly6a (right). Arrows indicate the adult-like fetal clusters I and II. (F) Violin plots of Ly6a and Kit expression across cell type clusters of fetal and adult cells. (G) Immunofluorescence images of CD117 expression (top, scale bars, 100 μm) and SCA1 expression (bottom, scale bars, 50 μm) in 3D FEnS and aOrgs. Arrows = CD117⁺ cells; asterisks = debris. (H) Flow cytometry–based contour plots of CD117 (Kit) and SCA1 (Ly6a) expression in FEnS and aOrgs. Quantification of indicated populations is summarized in the bar plot to the right (n = 3). Error bars indicate SD, unpaired Student’s t test. ***P < 0.001 and ****P < 0.0001. DAPI, 4′,6-diamidino-2-phenylindole; FSC, forward scatter.