Objective: To investigate the immunoreactivity of p63 in monolayered and stratified human urothelial cell cultures and in normal urothelial tissues to assess the differentiation status of in vitro stratified urothelial constructs.
Methods: p63 expression was detected immunohistochemically in native normal human bladder, ureter, and renal pelvis tissues and immunocytochemically in monolayered urothelial cell cultures and urothelial constructs stratified in vitro. Additionally, expression of pancytokeratin, cytokeratin 20 (CK20), uroplakin III, and fibroblast surface antigen was investigated.
Results: In native tissues, urothelial cell layers showed the most intensive p63 staining in the basal cells; the superficial umbrella cells were predominantly negative. Monolayered urothelial cell cultures revealed reduced p63 expression with ongoing culture passages. In vitro stratified urothelial constructs exhibited p63 expression similar to that of native urothelium. CK20-reactive cells were absent in the monolayered cultures but present in the stratified cell cultures and in the urothelial constructs. In native urothelium, only superficial cells stained positive for CK20. Uroplakin III was not present in either monolayered urothelial cell cultures or stratified urothelial constructs. Cultured cells were always positive for pancytokeratin and negative for fibroblast surface antigen.
Conclusions: p63 is a new biomarker for differentiation and stratification of urothelium created in vitro. For proposed clinical applications of in vitro stratified urothelium in reconstructive urology, urothelial constructs should exhibit expression of significant marker proteins similar to that of native urothelium. Our results show such similarity of expression for pancytokeratin, p63, and CK20, an encouraging possibility for confirming the functionality of tissue-engineered urothelia after clinical application.