Fig 2: The protein expression pattern of the ZEB1-KO cells was weakly altered in comparison to wildtype cells. Shown are representative Western blot data of at least three independent experiments and three consecutive passages (P1, P2, and P3). Note that some blots, such as CDH2 and CK5 of the HS578T-Hyg cells, CDH1 and CK5 of M13HS-2 and -8 cells were double stained and thus have identical β-actin housekeeping bands. EIF4E was used as a housekeeping gene for WNT5A expression of the M13HS-8 and M13HS-8 ZEB1-KO cells. Blots for the HS578T-Hyg/HS578T ZEB1-KO and M13HS-2/M13HS-2 ZEB1-KO cells were derived from one blot. Here, bands from other samples were cut off (indicated by a dashed line). The arrow marks the active form of WNT5A. P1, P2, and P3 indicate three consecutive passage numbers. Original Western blot data are shown in Supplementary Figure S1.

Fig 3: Generation of AR and FOXA1 double-positive UGS cells. (A) The immunofluorescence results of AR after differentiation with different conditions. Scale bar = 50 μm (CTRL: control). (B) Statistical results of AR-positive cells in (A) (n = 3). (C) The immunofluorescence results of FOXA1 after differentiation with different conditions. Scale bar = 50 μm. (D) Statistical results of FOXA1-positive cells in (C) (n = 3). (E) Changes in the mRNA levels of the prostatic markers SOX9, P63 (TP63), and CK5 (KRT5) after differentiation with different conditions (n = 4). (F-G) The immunofluorescence results (F) and mRNA levels (G, n = 4) of NKX3-1 after differentiation. Scale bar = 50 μm. ns, not significant (p > 0.05), *p < 0.05, **p < 0.01, ***p < 0.001

Fig 4: Differentiation of NKX3-1, AR, and FOXA1 triple-positive UGS cells. (A) Schematic diagram of the UGS differentiation from hindgut (HG: hindgut cell, UGS: Urogenital sinus cell, Doxy: Doxycycline). (B-C) Western blot results (B) (Full-length blots/gels are presented in Supplementary Fig. 4A) and immunofluorescence images (C) of AR protein in cells treated with DHT, R1881, and without androgen induction as control during UGS differentiation. The molecular weight of the protein was labeled at right. Scale bar, 50 μm (CTRL: control). (D-F) Western blot results (D) (Full-length blots/gels are presented in Supplementary Fig. 4B) and immunofluorescence images (E-F) of AR, FOXA1 and NKX3-1 proteins in cells treated with (E) or without (F) Doxy during UGS differentiation. The molecular weight of the protein was labeled at right. Scale bar, 50 μm. (G) Statistical analysis of the percentages of positive cells for NKX3-1, AR, and FOXA1 with or without Doxy induction during UGS differentiation (n = 3). (H) Relative mRNA expression levels of NKX3-1, AR, and FOXA1 with or without Doxy induction during UGS differentiation (n = 3). (I) Changes in relative mRNA expression levels of markers at various stages throughout the differentiation process from ESCs to UGS (n = 3) (ES: human embryonic stem cell, DE: definitive endoderm cell). (J) Immunofluorescence results of CK5, CK8/18, and P63 in UGS cells. Scale bar, 50 μm. (K-L) Immunofluorescence images (K) and relative mRNA expression level (L) of NKX3-1 after adding Doxy induction for 6 days, for 3 days’ treatment followed by 3 days’ withdrawal, and without Doxy induction for 6 days during UGS differentiation (n = 3). Scale bar, 50 μm. ns, not significant (p > 0.05), *p < 0.05, **p < 0.01, ***p < 0.001

Fig 5: The formation of structural prostate organoids through 3D culture of ESC-derived UGS cells. (A) Illustration of the generation of prostatic organoids from human ESCs. (B) Brightfield images of organoids at week 1, 2, 3, and 5. Scale bar, 50 μm. (C) Diameter statistical graph of organoids after 3D cultivation for the first 20 days. (D) DAPI immunofluorescence results of organoids on week 3 and 5. Scale bar, 50 μm. (E) Immunofluorescence results of NKX3-1, KI67, FOXA1, and AR on week 3 of organoids cultivation. Scale bar, 50 μm. (F-I) H&E staining images (F), immunofluorescence results of E-Cadherin (G), P63 and CK18 (H), and CK5 and CK18 (I) of organoids at week 4. Scale bar, 50 μm. (J) Flow cytometry results of CK18 of organoids. 3D cultured organoids for 9 days were used (ISO: isotype control)