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Immunocytochemical characterisation of cultures of human bladder mucosal cells.

Woodman JR, Mansfield KJ, Lazzaro VA, Lynch W, Burcher E, Moore KH - BMC Urol (2011)

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Detrusor Muscle Laboratory, The St George Hospital, University of New South Wales, Sydney, NSW 2052, Australia.

ABSTRACT

Background: The 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.

Methods: Primary 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.

Results: Cytokeratin 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.

Conclusions: Our 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.

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Dual labelling of urothelim (Uro) and lamina propria (lp) of human bladder. Full thickness segments of human bladder were stained with (A) the cytokeratin marker A0575 (brown) and αSMA (red) or (B) AE1/AE3 (brown) and αSMA (red). (C) with CK20 (brown) and αSMA (red). D. Full thickness segments were stained with 5B5 (red). Sections were counterstained with haematoxylin. (Bar = 50 μm)
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Figure 4: Dual labelling of urothelim (Uro) and lamina propria (lp) of human bladder. Full thickness segments of human bladder were stained with (A) the cytokeratin marker A0575 (brown) and αSMA (red) or (B) AE1/AE3 (brown) and αSMA (red). (C) with CK20 (brown) and αSMA (red). D. Full thickness segments were stained with 5B5 (red). Sections were counterstained with haematoxylin. (Bar = 50 μm)

Mentions: To confirm the localization of the antibodies used in this study immunohistochemical staining was also performed in full thickness bladder segments (Figure 4). The urothelium demonstrated strong immunoreactivity for the cytokeratin markers (A0575, Figure 4A and AE1/AE3, Figure 4B). Although many of the superficial umbrella cells were lost during processing, the remaining superficial cells demonstrated immunoreactivity to CK20 (Figure 4C). None of the urothelial cells stained positively for αSMA (Figure 4A, B and 4C).


Immunocytochemical characterisation of cultures of human bladder mucosal cells.

Woodman JR, Mansfield KJ, Lazzaro VA, Lynch W, Burcher E, Moore KH - BMC Urol (2011)

Dual labelling of urothelim (Uro) and lamina propria (lp) of human bladder. Full thickness segments of human bladder were stained with (A) the cytokeratin marker A0575 (brown) and αSMA (red) or (B) AE1/AE3 (brown) and αSMA (red). (C) with CK20 (brown) and αSMA (red). D. Full thickness segments were stained with 5B5 (red). Sections were counterstained with haematoxylin. (Bar = 50 μm)
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3104367&req=5

Figure 4: Dual labelling of urothelim (Uro) and lamina propria (lp) of human bladder. Full thickness segments of human bladder were stained with (A) the cytokeratin marker A0575 (brown) and αSMA (red) or (B) AE1/AE3 (brown) and αSMA (red). (C) with CK20 (brown) and αSMA (red). D. Full thickness segments were stained with 5B5 (red). Sections were counterstained with haematoxylin. (Bar = 50 μm)
Mentions: To confirm the localization of the antibodies used in this study immunohistochemical staining was also performed in full thickness bladder segments (Figure 4). The urothelium demonstrated strong immunoreactivity for the cytokeratin markers (A0575, Figure 4A and AE1/AE3, Figure 4B). Although many of the superficial umbrella cells were lost during processing, the remaining superficial cells demonstrated immunoreactivity to CK20 (Figure 4C). None of the urothelial cells stained positively for αSMA (Figure 4A, B and 4C).

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Detrusor Muscle Laboratory, The St George Hospital, University of New South Wales, Sydney, NSW 2052, Australia.

ABSTRACT

Background: The 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.

Methods: Primary 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.

Results: Cytokeratin 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.

Conclusions: Our 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.

Show MeSH
Related in: MedlinePlus