The different expression of key markers on urothelial holoclonal, meroclonal, and paraclonal cells in in vitro culture

Abstract

Urothelial cell populations which differ in morphology and proliferation capacities can be isolated from the urinary bladder. The goal of this study was to analyze a clonal, proliferative, and self-renewing potential of porcine urothelial cells and to compare expression of selected adhesion and tight junction molecules, urothelial and stem cell markers for the urothelial clone types. Urothelial cells were isolated from 10 porcine urinary bladders. Three different clone types: holoclone-, meroclone-and paraclonelike colonies were identified based on their morphology. To characterize and compare the urothelial clones the immunofluorescent stains were performed. Expression of pancytokeratin (PanCK), Ki-67 and p63 was higher for holoclone-like cells compared to meroclone-and paraclone-like cells (P < 0.05). Meroclone-like cells expressed higher levels of p63 compared to paraclone-like cells (P < 0.05). The level of Ki-67 and PanCK for meroclone-and paraclone-like cells was comparable (P > 0.05). b1 and b4 integrins were not expressed. Expression of zonula occludens-1 (ZO-1) in cell-cell junctions for paraclone-, meroclone-and holoclone-like cells was 17.6 AE 0.6, 14.7 AE 0.5, and 16.1 AE 0.4, respectively. The results of actin filaments (F-actin) expression were 253,634 AE 6,920 for meroclone-like cells, 198,512 AE 7,977 for paraclone-like cells and 133,544 AE 3,169 for holoclone-like cells. Three urothelial cell types with differing features can be isolated from the bladder. Holoclone-like cells are the richest in stem cells and should be used in further studies for construction of neo-bladder or neo-conduit using tissue engineering methods. Supporting InformationAdditional supporting information may be found in the online version of this article at the publisher's web-site. Figure S1. Representative fluorescence signals of integrins b4 (a, d, g), b1 (b e, h) and Merge (c, f, i) in the holoclonelike (a-c), meroclone-like (d-f) and paraclone-like (g-i) cells. Laser scanning confocal microscopy; bar 25 mm.The porcine urothelial cell populations M. Buhl et al.