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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

Immunohistochemistry of cultured bladder mucosal cells. At primary culture (A, B, C) cells were counter stained to demonstrate morphology (A). A small number of cells were large with a spreading morphology, these stained for CK20 (B). Other cells were more elongated and branching and stained for AO575 (C). Cells were also stained after treatment with MACS beads. Cells that bound to the beads (D, E, F, myofibroblast-like cells) were stained for 5B5 (D), AE1/AE3 (E) and αSMA (F). Cells that eluted from the beads (G, H, I, urothelial cells) were stained for 5B5 (G), A0575 (H) and αSMA (I). (Bar = 100 μm)
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Figure 1: Immunohistochemistry of cultured bladder mucosal cells. At primary culture (A, B, C) cells were counter stained to demonstrate morphology (A). A small number of cells were large with a spreading morphology, these stained for CK20 (B). Other cells were more elongated and branching and stained for AO575 (C). Cells were also stained after treatment with MACS beads. Cells that bound to the beads (D, E, F, myofibroblast-like cells) were stained for 5B5 (D), AE1/AE3 (E) and αSMA (F). Cells that eluted from the beads (G, H, I, urothelial cells) were stained for 5B5 (G), A0575 (H) and αSMA (I). (Bar = 100 μm)

Mentions: In preliminary immunocytochemical studies (n = 2, Figure 1), cells in primary culture were found to display diverse morphologies (Figure 1A). Large spreading umbrella-like cells were isolated early in culture, but these cells did not proliferate. Immunostaining of cultures containing these large spreading cells demonstrated that they expressed CK20 (Figure 1B).


Immunocytochemical characterisation of cultures of human bladder mucosal cells.

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

Immunohistochemistry of cultured bladder mucosal cells. At primary culture (A, B, C) cells were counter stained to demonstrate morphology (A). A small number of cells were large with a spreading morphology, these stained for CK20 (B). Other cells were more elongated and branching and stained for AO575 (C). Cells were also stained after treatment with MACS beads. Cells that bound to the beads (D, E, F, myofibroblast-like cells) were stained for 5B5 (D), AE1/AE3 (E) and αSMA (F). Cells that eluted from the beads (G, H, I, urothelial cells) were stained for 5B5 (G), A0575 (H) and αSMA (I). (Bar = 100 μm)
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Immunohistochemistry of cultured bladder mucosal cells. At primary culture (A, B, C) cells were counter stained to demonstrate morphology (A). A small number of cells were large with a spreading morphology, these stained for CK20 (B). Other cells were more elongated and branching and stained for AO575 (C). Cells were also stained after treatment with MACS beads. Cells that bound to the beads (D, E, F, myofibroblast-like cells) were stained for 5B5 (D), AE1/AE3 (E) and αSMA (F). Cells that eluted from the beads (G, H, I, urothelial cells) were stained for 5B5 (G), A0575 (H) and αSMA (I). (Bar = 100 μm)
Mentions: In preliminary immunocytochemical studies (n = 2, Figure 1), cells in primary culture were found to display diverse morphologies (Figure 1A). Large spreading umbrella-like cells were isolated early in culture, but these cells did not proliferate. Immunostaining of cultures containing these large spreading cells demonstrated that they expressed CK20 (Figure 1B).

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