Fig. 2.

Growth factor-depleted organoids recapitulate the urothelial differentiation program. a Experimental design applied to induce urothelial organoid differentiation: organoids cultured until day 7 in complete medium were maintained for seven additional days in differentiation medium. b Image of organoids displaying the features quantified in panel c: d, diameter; l, lumen; t, thickness of the epithelial layer. The signal distribution was measured across the organoids as indicated by the arrow in both cases (scale bar, 100 μm). c Signal distribution (in microns) acquired by confocal microscopy displaying the quantification of organoid features (diameter) of individual proliferative (P) (n = 57) and differentiated (D) (n = 71) organoids; color code indicates the intensity of the signal: green, low; yellow, intermediate; red, high. d Violin plots representing organoid features. e RT–qPCR analysis of expression of genes regulated during differentiation. Data are normalized to Hprt expression (Mann–Whitney test, error bars indicate SD). f Western blot (WB) analysis showing expression of TP63 (basal marker), UPK3a, and UPK1b (luminal markers) in P and D organoids in three independent experiments. Urothelial bladder cancer cell lines (ScaBER, RT112, VMCUB1, and RT4) were used as controls. g Immunofluorescence analysis of urothelial markers in P and D organoids. Normal urothelium is shown for comparison. DAPI staining is shown in blue (scale bar, 1000 μm). Source data are provided as a Source Data file