OBJECTIVE: To examine the kinetics of growth, differentiation and senescence of normal human urothelium in an organoid-like culture model. MATERIALS AND METHODS: Micro-dissected normal human urothelium explants were grown on porous membranes pretreated with various matrix components. Between 5 and 30 days of culture, cell proliferation was assessed by BrdU incorporation. Differentiation was evaluated on the basis of cytokeratin (Ck) and uroplakin (UP) expression. Epidermal growth factor family mRNA expression was monitored during explant outgrowth. Senescence was assessed by measuring endogenous beta-galactosidase activity and p16(INK4a) mRNA expression. RESULTS: Collagen IV was the most efficient matrix component for urothelial cell expansion. BrdU incorporation by urothelial cells was 5% between 15 and 30 days, corresponding to steady-state urothelium in vivo. Heparin-binding EGF (HB-EGF), Amphiregulin (AR) and Transforming Growth Factor alpha (TGF alpha) expression correlated with increased cell proliferation. UPII expression was stable throughout culture. P16(INK4a) mRNA expression and beta-galactosidase activity increased on day 25, giving signs of senescence. CONCLUSIONS: This model retains many characteristics of the urothelium in vivo. It can be used for pharmacological studies between 15 to 25 days and to study mechanisms such as wound healing, proliferation and senescence.
OBJECTIVE: To examine the kinetics of growth, differentiation and senescence of normal human urothelium in an organoid-like culture model. MATERIALS AND METHODS: Micro-dissected normal human urothelium explants were grown on porous membranes pretreated with various matrix components. Between 5 and 30 days of culture, cell proliferation was assessed by BrdU incorporation. Differentiation was evaluated on the basis of cytokeratin (Ck) and uroplakin (UP) expression. Epidermal growth factor family mRNA expression was monitored during explant outgrowth. Senescence was assessed by measuring endogenous beta-galactosidase activity and p16(INK4a) mRNA expression. RESULTS: Collagen IV was the most efficient matrix component for urothelial cell expansion. BrdU incorporation by urothelial cells was 5% between 15 and 30 days, corresponding to steady-state urothelium in vivo. Heparin-binding EGF (HB-EGF), Amphiregulin (AR) and Transforming Growth Factor alpha (TGF alpha) expression correlated with increased cell proliferation. UPII expression was stable throughout culture. P16(INK4a) mRNA expression and beta-galactosidase activity increased on day 25, giving signs of senescence. CONCLUSIONS: This model retains many characteristics of the urothelium in vivo. It can be used for pharmacological studies between 15 to 25 days and to study mechanisms such as wound healing, proliferation and senescence.
Authors: Benjamin O Murray; Carlos Flores; Corin Williams; Deborah A Flusberg; Elizabeth E Marr; Karolina M Kwiatkowska; Joseph L Charest; Brett C Isenberg; Jennifer L Rohn Journal: Front Cell Infect Microbiol Date: 2021-05-26 Impact factor: 5.293