19 FUNCTIONAL UROTHELIAL DIFFERENTIATION OF URINE-DERIVED STEM CELLS FOR POTENTIAL USE IN URETHRAL TISSUE REPAIR

Abstract

You have accessJournal of UrologyTrauma/Reconstruction: Traum & Reconstructive Surgery (1)1 Apr 201319 FUNCTIONAL UROTHELIAL DIFFERENTIATION OF URINE-DERIVED STEM CELLS FOR POTENTIAL USE IN URETHRAL TISSUE REPAIR Guihua Liu, Shantaram Bharadwaj, Anthony Atala, and Yuanyuan Zhang Guihua LiuGuihua Liu Winston Salem, NC More articles by this author , Shantaram BharadwajShantaram Bharadwaj Winston Salem, NC More articles by this author , Anthony AtalaAnthony Atala Winston Salem, NC More articles by this author , and Yuanyuan ZhangYuanyuan Zhang Winston Salem, NC More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2013.02.1393AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Urothelial cells play important and contradictory roles in the function of the urinary system. They act as a permeability barrier and protect underlying muscle tissue from the caustic effects of urine, which prevent formation of scarring in urethral tissue repair. It is a challenge to induce stem cells to differentiate into urothelial cells with barrier function. Our previous studies demonstrated that urine-derived stem cells (USC) possess self-renewal and multipotent capacity, and can give rise to cells expressing urothelial markers. The goal of this study was to determine whether USC can differentiate into urothelial cells with barrier function in vitro, for potential use in urethral tissue engineering. METHODS Urine-derived cells were harvested from 6 healthy individuals and then induced to differentiate into urothelial cells using epithelial differentiation medium containing epidermal growth factor (EGF) for up to 2 weeks. These differentiated cells were compared to non-differentiated USC. Induced cells were assessed for morphologic changes and expression of urothelial markers via RT-PCR, Western blotting, and immunofluorescent staining. Functional tight junction markers were also assessed by real-time PCR, immunofluorescent stain, and transmission electron microscopy (TEM). Finally, differentiated (4.5×105 cells) and non-induced USC (4.5×105 cells) were seeded on collagen-coated inserts for barrier function assessmenton day 7 and 14. RESULTS Morphologically, urothelial differentiated USC formed cobblestone-like cells. These differentiated USCs expressed transcripts and proteins of urothelial-specific (Uroplakin-III, -Ia) and general epithelial cell markers (CK7, CK13, CK20 and AE1/AE3) in a time-dependent manner. Expression of these markers also depended on the amount of EGF in the media. In an assay of barrier function, urothelial differentiated USC expressed tight junction gene and protein markers (ZO-1 and E-cadherin). TEM demonstrated the ultrastructural aspects of urothelial differentiated USC, including tight junction formation between neighboring cells. Using a fluorescent tracer on differentiated cells cultured on inserts, at least a 50% decrease in leakage of the tracer across the insert occurred after 3h in vitro, indicating that these cells could perform a barrier function similar to the normal urothelial cells. CONCLUSIONS USC can be efficiently differentiated into functional urothelial cells with barrier microstructures, and these cells may be a potential cell source for tissue-engineered urethras. © 2013 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 189Issue 4SApril 2013Page: e7-e8 Advertisement Copyright & Permissions© 2013 by American Urological Association Education and Research, Inc.MetricsAuthor Information Guihua Liu Winston Salem, NC More articles by this author Shantaram Bharadwaj Winston Salem, NC More articles by this author Anthony Atala Winston Salem, NC More articles by this author Yuanyuan Zhang Winston Salem, NC More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...