Hypothesis / aims of study
Interstitial cystitis/bladder pain syndrome (IC/BPS) is characterized by urothelial barrier disruption and impaired epithelial regeneration, with limited recovery from current therapies. We hypothesized that bladder organoids (BLOs) can enhance epithelial repair in injured bladders through structural anchoring and activation of Wnt1–β-catenin signaling. This study aimed to evaluate the regenerative potential and mechanisms of proliferative and differentiated BLOs (P-BLO and D-BLO) in a preclinical bladder injury model.
Study design, materials and methods
Rat proliferative (P-BLO) and differentiated (D-BLO) bladder organoids were generated in 3D matrix gel cultures (Reference 1). In a cyclophosphamide (CYP)-induced rat bladder injury model, hyaluronic acid (HA), P-BLO, or D-BLO were instilled intravesically twice at a 48-hour interval. Bladder repair was assessed 24 hours after instillation via tissue histology, pain thresholds, and urinary spot counting. Intravesical retention of instillates was evaluated via in vivo imaging 60 minutes post-instillation. Single-cell RNA sequencing (scRNA-seq) was performed on normal, CYP-injured bladders, and organoids to dissect the cellular and molecular mechanisms underlying epithelial regeneration.
Results
P-BLO and HA instillation increased pain thresholds, reduced urinary spots, thickened the urothelium, increased the proportion of E-cadherin+ cells, and decreased IL-1β+ cells, whereas D-BLO had no significant effects. After the second instillation, P-BLO outperformed HA in pain reduction, epithelial marker restoration, and suppression of inflammation. In vivo imaging revealed superior intravesical retention of P-BLO compared with HA or D-BLO. scRNA-seq revealed that P-BLO upregulated Spp1–Itga9 and Cd44–Lama2 interactions with injured urothelium, and activated Wnt1–β-catenin signaling, counteracting CYP-induced suppression of Ctnnb1.
Interpretation of results
P-BLO promotes epithelial regeneration and repair via dual anchoring structures (Spp1–Itga9 and Cd44–Lama2) and reactivation of Wnt1–β-catenin signaling. Superior retention and functional integration of P-BLO relative to HA contribute to enhanced epithelial restoration and pain alleviation.