Fig. 1
CD49f labels an organoid-forming urothelial cell population with stem cell features. a Urothelial cyto-organization highlighting the three cell layers: basal (CD49f and KRT5), intermediate (KRT5), and luminal (WGA-binding) markers. U, urothelium; LP, lamina propria (scale bar, 50 μm). Color code for the scheme: brown, basal; pink, basal-proliferative; blue, intermediate; green, umbrella. b Representative images of organoids from low-passage urothelial cell suspensions embedded in Matrigel in complete medium (upper left). The remaining panels correspond to the leave-one-out experiments (see panel c) (scale bar, 500 μm). c Quantification of organoid growth in leave-one-out experiments (WR condition: WNT3A and RSPO1 were omitted). Number of organoids normalized to complete medium; error bars indicate SEM. d, e FACS plots depicting the analysis and isolation of fresh epithelial cells according to cell surface markers (EpCAM, CD49f, CD44, and WGA) and size (n = 2) (scale bar, 500 μm). f Quantification of the organoid-forming capacity of freshly isolated and sorted urothelial cells (100 cells/Matrigel drop) compared to the unsorted population (Urothelium); results from a representative experiment (n = 2); error bars indicate SEM. g Clonal growth capacity of freshly isolated urothelial cells FACS-sorted according to CD49f expression (CD49fhigh vs. CD49flow) and seeded at 1, 10, and 100 cells/Matrigel drop (n = 3). The proportion of Matrigel drops showing outgrowth (bars, Y-axis) and the number of organoids/drop (1, 2, 3, and 4; according to the color code) are shown. h Monoclonal origin of organoids established starting from a 1:1 mixture of EGFP- and mTomato-expressing cells (n = 2) (scale bar, 500 μm). ANOVA and Mann–Whitney tests were applied; *p ≤ 0.05, **p ≤ 0.01; ***p ≤ 0.001. Source data are provided as a Source Data file
