Immunocytochemical characterisation of cultures of human bladder mucosal cells

Abstract

BackgroundThe functional role of the bladder urothelium has been the focus of much recent research. The bladder mucosa contains two significant cell types: urothelial cells that line the bladder lumen and suburothelial interstitial cells or myofibroblasts. The aims of this study were to culture these cell populations from human bladder biopsies and to perform immunocytochemical characterisation.MethodsPrimary cell cultures were established from human bladder biopsies (n = 10). Individual populations of urothelial and myofibroblast-like cells were isolated using magnetic activated cell separation (MACS). Cells were slow growing, needing 3 to 5 weeks to attain confluence.ResultsCytokeratin 20 positive cells (umbrella cells) were isolated at primary culture and also from patients' bladder washings but these did not proliferate. In primary culture, proliferating cells demonstrated positive immunocytochemical staining to cytokeratin markers (AE1/AE3 and A0575) as well fibroblasts (5B5) and smooth muscle (αSMA) markers. An unexpected finding was that populations of presumptive urothelial and myofibroblast-like cells, isolated using the MACS beads, stained for similar markers. In contrast, staining for cytokeratins and fibroblast or smooth muscle markers was not co-localised in full thickness bladder sections.ConclusionsOur results suggest that, in culture, bladder mucosal cells may undergo differentiation into a myoepithelial cell phenotype indicating that urothelial cells have the capacity to respond to environmental changes. This may be important pathologically but also suggests that studies of the physiological function of these cells in culture may not give a reliable indicator of human physiology.

Defining protein expression in the urothelium: a problem of more than transitional interest

“…The urothelium is stratified and therefore has a complex multilayered phenotype. While some features of the urothelium are certainly retained in culture, the expression of differentiation markers like cytokeratins and uroplakins does not reproduce that seen in vivo (46,58,70). Depending on the culture conditions and the way in which cultures are established and maintained, urothelial cells can exist in a proliferative state or in a quiescent, differentiated state.…”

“…These may be pharmacological or mechanical, for example. However, it would be best to regard most urothelial culture systems as having regenerative or myoepithelial cell properties (70) and to be circumspect when drawing conclusions about expression.…”

“…FACS or MACS offers a way to isolate a specific population or subpopulation of urothelial cells (70). Utilizing epithelialspecific cell surface markers such as uroplakin, epithelial cell adhesion molecule (EpCAM), or E-cadherin, it is possible to obtain a highly enriched fraction of urothelial cells (23).…”

Evaluation of Silk Biomaterials in Combination with Extracellular Matrix Coatings for Bladder Tissue Engineering with Primary and Pluripotent Cells

“…To verify ECM protein coatings on non-seeded scaffolds, the following primary antibodies were used: anti-collagen type I (Abcam, Cambridge, MA, cat.# ab34710, 1∶200 dilution), anti-collagen type IV (Abcam, cat.# ab6586, 1∶200 dilution), and anti-fibronectin (Millipore, cat.# AB2047, 1∶200 dilution). For differentiation analyses, contractile smooth muscle markers such as α-actin and SM22α [38] as well as urothelial-associated proteins, uroplakins (UP) [39] and cytokeratins (CK) [40], were detected using the following primary antibodies: anti-α-actin (Sigma-Aldrich, cat.# A2457, 1∶200 dilution), anti-SM22α (Abcam, Cambridge, MA, cat.# ab14106, 1∶200 dilution), anti-pan-UP (rabbit antisera raised against total bovine UP extracts, 1∶100 dilution), anti-pan-CK (Dako, Carpinteria, CA, cat.# M3515, 1∶200 dilution). Specimens were incubated with respective species-matched Cy3-conjugated secondary antibodies (Millipore) to ascertain primary antibody localization.…”

microRNA response elements-regulated TRAIL expression shows specific survival-suppressing activity on bladder cancer

“…The previously described procedures that have been approved by Ethical Review Board in General Hospital of Chengdu Military Area Command of Chinese PLA (Chengdu, China) was followed to establish the primary BMC culture [47]. The BMCs were immortalized using adenoviral vector, Adeno-SV40 (Applied Biological Materials Inc., Canada), according to the manufacturer’s instructions.…”