AUTOLOGOUS TRANSPLANTATION OF UROTHELIUM INTO DEMUCOSALIZED GASTROINTESTINAL SEGMENTS - EVIDENCE FOR EPITHELIALIZATION AND DIFFERENTIATION OF IN-VITRO EXPANDED AND TRANSPLANTED UROTHELIAL CELLS

Purpose: Our study established a technique for in vitro expansion and subsequent transplantation of autologous urothelial cells into vascula rized seromuscular segments from stomach and colon in sheep. The proof of proliferation and differentiation of the transplanted urothelium i n the absence of resident urothelium is considered to be a prerequisit e for use of this technique in bladder augmentation. Materials and Met hods: Autologous sheep urothelial cells were expanded in vitro and gro wn on collagen membranes for sheet grafting. Using a vital stain, viab ility and confluency status of the urothelial graft were determined be fore transplantation into demucosalized segments isolated from the she ep stomach and colon gastrointestinal pouches. The gastrointestinal se gments were sewn up and remained in the abdomen as small pouches stich ed to the abdominal wall. Take and differentiation of transplanted cel ls within the pouch were assessed two and three weeks later using hist ological and immunohistological means. Results: Urothelial cells grew well on collagen membranes, A confluency status >40% and co-culturing with 3T3 feeder cells favored successful transplantation. Two weeks af ter transplantation a multilayered urothelial-like epithelium was foun d to line the lumen of the pouch. The epithelium was characterized by a distinct urothelium-typical distribution of basal and luminal kerati ns and the expression of the umbrella cell-specific marker uroplakin I II, Moreover, the epithelium had an underlying basal lamina which foca lly contained collagen type IV. Conclusions: The data indicate that in vitro expanded urothelial cells are capable of epithelializing demuco salized gastrointestinal segments forming a genuine, differentiated '' neo''urothelium.