Fig 2: Retinoic acid promotes lumen formation in SMG organoids. (A) SMG organoids were cultured with different doses of RA and treated with 200 nM VIP on day 4. Microscopic images of lumen formation were obtained on day 6. Scale bars indicate 100 µm. (B) The proportion of organoids containing internal lumen were calculated, followed by statistical analysis (n = 3). Results are expressed as the mean ± SD. ***p < 0.001. (C–E) SMG organoids were cultured with or without RA in combination with 1 nM bFGF and 5 nM FGF10 throughout the culturing period. At day 9, organoids were either unstimulated or stimulated with VIP for 1 day. (C) Branching ducts were observed with brightfield microscopy. Black arrows indicate formation of lumens near end buds. Scale bars indicate 100 µm. (D) Harvested organoid were subjected to H&E staining to observe lumen formation in branching ducts (red arrows). Scale bars indicate 100 µm. (E) Organoids were subjected to immunofluorescence staining for KRT5 (green) and KRT7 (red). Scale bars indicate 50 µm. Nuclei were counterstained with DAPI (blue). All experiments were performed three times independently.

Fig 3: Identification of OLFM4-expressing cells in normal human adult prostate epithelium. (a) Uniform Manifold Approximation and Projection (UMAP) plots of integrated data from single-cell RNA sequencing of the GSE117403 dataset. Left panel shows 19 clusters of total prostate cells; right panel shows OLFM4-expressing cells (purple color) in clusters 7 and 12. (b) Representative triple-color immunofluorescent staining of normal prostatic urethra tubular epithelium. OLFM4 and CK13 (green); CD44 (red); CK5 (cyan); DAPI (blue). Scale bar: 20 µm. (c) Representative double-color immunofluorescent staining of normal prostatic tissues. OLFM4 (green); SCGB1A1 (red); DAPI (blue). Left panel shows low-magnification image. Scale bar: 100 µm. Right panel shows high-magnification image. Scale bar: 20 µm. Arrow indicates OLFM4+/SCGB1A1+ cells.

Fig 4: Identification of OLFM4-expressing RWPE1 cells. (a) Uniform Manifold Approximation and Projection (UMAP) plots of integrated data from single-cell RNA sequencing of RWPE1 cells. Left panel shows 13 clusters of total RWPE1 cells; right panel shows OLFM4-expressing cells (purple color) in clusters 3 and 7. (b) Heat map illustrates OLFM4 co-expression with stem/progenitor-cell marker genes, cytokeratins, and others in 37 OLFM4-expressing RWPE1 cells. Sq., squamous. P, progenitor. *Indicates lines for stem/progenitor-cell marker genes; arrow indicates line for the OLFM4 gene. (c) Representative triple-color immunofluorescent staining of RWPE1 cells. OLFM4 (green); CK13 and CD44 (red); CK5 (cyan); DAPI (blue). Scale bar: 20 µm.

Fig 5: Lineage-Tracing Analyses of Urothelial Subpopulations during Regeneration Focal Mucosal Defects(A) Sites of mucosal resection and NRC regions in Tm-induced Krt5CreERT2;mTmG mice at 3 weeks post-op revealing GFP expression in de novo BC-1, IC-1, and subsets of S cells in neotissues as well as GFP+ host BC-1 cells in NRC.(B) Sites of mucosal resection and NRC areas in Tm-induced Upk2iCreERT2;mTmG mice at 3 weeks post-op revealing GFP expression in de novo IC-1, and S cell fractions in neotissues as well as GFP+ host IC-1 and S in NRC.(C) Quantitation of GFP+ S cells in neotissues present in resected bladders of Tm-induced Krt5CreERT2;mTmG and Upk2iCreERT2;mTmG mice following 3 weeks post-op. Means ± SD.For (A) and (B), respective marker expression following IHC evaluations is displayed in red, green, or pink (Cy3, FITC, Cy5 labeling), and blue denotes DAPI nuclear counterstain. In some cases, Cy5 labeling was false colored to white. Yellow arrows denote IC-1 cells (KRT5-P63+UP+KRT20-) and white arrows denote S cells (KRT5-P63-UP+KRT20+). For all panels, data were acquired from n = 3 animals per experimental group with n = 3 sections per animal analyzed for neotissue and NRC evaluations. Scale bars represent 200 µm (A and B).