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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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Related in: MedlinePlus

Dual labeling of MACS treated cell populations. Cells that bound to the MACS anti-fibroblast beads (myofibroblast-like cells, A, B, C) or that were eluted from the MACS beads (urothelial cells, D, E, F) were stained with (A, D) A0575 (brown) and 5B5 (Red) (B, E) with A0575 (brown) and αSMA (Red) and (C, F) αSMA (brown) and 5B5 (Red). (Bar = 100 μm)
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Figure 2: Dual labeling of MACS treated cell populations. Cells that bound to the MACS anti-fibroblast beads (myofibroblast-like cells, A, B, C) or that were eluted from the MACS beads (urothelial cells, D, E, F) were stained with (A, D) A0575 (brown) and 5B5 (Red) (B, E) with A0575 (brown) and αSMA (Red) and (C, F) αSMA (brown) and 5B5 (Red). (Bar = 100 μm)

Mentions: Because MACS treated cultures showed similarities in immunocytochemical staining, dual labeling was performed on both bound and eluted populations (Table 2). The anti-fibroblast positive cells (myofibroblast-like cells) that were bound to the beads demonstrated dual labelling for cytokeratin markers together with 5B5 and αSMA (Figure 2A, B, C). Similarly, cells that were anti-fibroblast negative (urothelial cells) that were eluted from the columns also displayed the same pattern of dual labelling for cytokeratins together with 5B5 and αSMA (Figure 2D, E, F).


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 labeling of MACS treated cell populations. Cells that bound to the MACS anti-fibroblast beads (myofibroblast-like cells, A, B, C) or that were eluted from the MACS beads (urothelial cells, D, E, F) were stained with (A, D) A0575 (brown) and 5B5 (Red) (B, E) with A0575 (brown) and αSMA (Red) and (C, F) αSMA (brown) and 5B5 (Red). (Bar = 100 μm)
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Dual labeling of MACS treated cell populations. Cells that bound to the MACS anti-fibroblast beads (myofibroblast-like cells, A, B, C) or that were eluted from the MACS beads (urothelial cells, D, E, F) were stained with (A, D) A0575 (brown) and 5B5 (Red) (B, E) with A0575 (brown) and αSMA (Red) and (C, F) αSMA (brown) and 5B5 (Red). (Bar = 100 μm)
Mentions: Because MACS treated cultures showed similarities in immunocytochemical staining, dual labeling was performed on both bound and eluted populations (Table 2). The anti-fibroblast positive cells (myofibroblast-like cells) that were bound to the beads demonstrated dual labelling for cytokeratin markers together with 5B5 and αSMA (Figure 2A, B, C). Similarly, cells that were anti-fibroblast negative (urothelial cells) that were eluted from the columns also displayed the same pattern of dual labelling for cytokeratins together with 5B5 and αSMA (Figure 2D, E, F).

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