A biomimetic tissue from cultured normal human urothelial cells: analysis of physiological function

Abstract

The urinary bladder and associated tract is lined by the urothelium. Once considered as just an impermeable epithelium, it is becoming evident that the urothelium not only functions as a volume-accommodating urinary barrier but has additional roles, including sensory signaling. Lack of access to normal human urothelium has hampered physiological investigation, and although cell culture systems have been developed, there has been a failure to demonstrate that normal human urothelial (NHU) cells grown in vitro retain the capacity to form a functional differentiated urothelium. The aim of this study was to develop a biomimetic human urothelium from NHU cell cultures. Urothelial cells isolated from normal human urothelium and serially propagated as monolayers in serum-free culture were homogeneous and adopted a proliferative, nondifferentiated phenotype. In the presence of serum and physiological concentrations of calcium, these cells could be reproducibly induced to form stratified urothelia consisting of basal, intermediate, and superficial cells, with differential expression of cytokeratins and superficial tight junctions. Functionally, the neotissues showed characteristics of native urothelium, including high transepithelial electrical resistance of >3,000 Omega.cm(2), apical membrane-restricted amiloride-sensitive sodium ion channels, basal expression of Na(+)-K(+)-ATPase, and low diffusive permeability to urea, water, and dextran. This model represents major progress in developing a biomimetic human urothelial culture model to explore molecular and functional relationships in normal and dysfunctional bladder physiology.

Characterization of tight junction proteins in cultured human urothelial cells

“…ZO-1 has previously been reported in mammalian bladder and cultured human urothelial cells (Acharya et al 2004, Cross et al 2005. We have confirmed the presence of ZO-1 in TEU-2 cultures and further shown the presence of ZO-2 and ZO-3.…”

“…Claudins 1, 4, occludin and and ZO-1 in cultured human urothelial cells and a differentiation-associated profile of claudins 3, 4, 5, 7, ZO-1 and occludin for normal utereric urothelium in situ has been reported (Cross et al 2005, Varley et al 2006). These investigators employed human primary and subcoultured cells which have a finite lifespan.…”

Immortalisation of Normal Human Urothelial Cells Compromises Differentiation Capacity

“…Urothelial cells were grown in KSFMc containing 5% (v/v) adult bovine serum (ABS) before subculture onto Snapwell™ membranes. TER measurements were made as described [13].…”

“…Sub-culture of NHU cells in medium containing serum and 2mM calcium stimulates them to stratify and develop functional barrier properties equivalent to normal urothelia [13]. We examined the ability of para-malignant cell lines to form a tight barrier, as assessed by measurement of trans-epithelial resistance (TER).…”

“…In low-calcium, serum-free medium, NHU cells adopt a rapidlyproliferating, non-differentiated regenerative phenotype [10] driven by autocrine EGFR signalling [11]. In vitro-propagated NHU cell lines retain differentiation capacity and can be induced to stratify [10], express genes associated with late/terminal urothelial cytodifferentiation [12] and form a functional, barrier-epithelium [13]. The genetic manipulation of NHU cells with retroviruses has enabled the generation of "paramalignant" [14] human urothelial sub-lines carrying defined genetic alterations, such as inactivated p53 or p16 functions [15][16][17].…”