P86. Identification of urothelial stem cells for tissue engineering

Abstract

Introduction Stem cells (SCs) are able to self-renew and to differentiate into various cell types. During life, SCs are involved in organogenesis, but also in maintenance and repair of adult tissue. In reconstructive medicine, the availability of transferable tissue often is limited. For reconstructive tissue engineering, the combination of SCs with biomatrices may be a viable alternative approach. Urothelium, a layer of epithelial cells lining the bladder wall, is composed of basal, intermediate, and superficial cells. It has been hypothesized that the SCs are located in the basal cell layer. Since isolating urothelial SCs to combine with biomatrices may generate a basic construct for bladder tissue engineering, the identification and selection of urothelial SCs are crucial issues that need to be investigated. The aim of this study is to identify SCs from urothelium by identifying slow-cycling BrdU-retaining cells together with candidate SCs markers. Method: Tissue from newborn (5 days postnatal), pubertal (1 month), and adult (6 months) mice harvested from BrdU pulsed pregnant mice was examined by immunohistochemistry. Additionally, harvested tissue was examined for cells with a potential SC phenotype, including Cytokeratin 7 (CK7), Cytokeratin 14 (CK14), Cytokeratin 20 (CK20), and β-catenin. Result: The potential SCs in bladder were identified by the BrdU-retained cell population in 1 month old mice of ~8% and 6-month-old mice of ~1%. Most of BrdU retained cells were localized in basal part of epithelium as detected by co-localization of BrdU with CK 7, the latter staining all basal cells. CK14 was differentially expressed in newborn and adult mice, with only rare cells in the adult basal cell layer. In a minority population of BrdU (less than 0.1%) retained cells in 6-month-old mice were located in the umbrella cells (CK 20 positive). β-catenin showed ubiquitous staining of basal urothelium. Conclusion BrdU retained cells were reduced over time in adult life, suggesting a SC pool in the adult bladder. The markers being used showed differential localization and cell lineage of BrdU retained cells, where the basal cell layer contained the lion share of BrdU retained cells. Identification of membrane markers in the BrdU pool may enable cell sorting and isolation of bladder SCs. Ultimately isolated cells can then be expanded and cultured on biomatrices to generate a basic construct for bladder tissue engineering, and to repair and restore bladder function.