• KRT5high TP63-expressing basal cells are identified as key drivers of urothelial regeneration, capable of differentiating into intermediate and umbrella cells.
• A focal mucosal resection model and single-cell RNA sequencing reveal the stemness signature of these basal cells, providing a cellular basis for bladder tissue engineering.
• Transplantation of cultured basal cell sheets onto a pre-set capsule vascular bed successfully reconstructs a physiologically functional urothelium with barrier integrity in vivo.
• This bioengineered urothelium offers a promising strategy for bladder reconstruction and a platform for drug screening and disease modeling.