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Abstract

An estimated 400 million people worldwide suffer from bladder diseases. However, little is known at the cellular level as to what causes or initiates many of these diseases. Therefore, this thesis has focused on understanding one cell type within the bladder wall, the urothelial cell. Urothelial cells are capable of keeping urine from entering the blood stream, which can be only a few microns away. Until now, an urothelial stem cell, or holoclone, with the capacity to self-renew and differentiate into fully mature urothelial cells has not been isolated. Therefore, we have tried to find a new adult epithelial stem cell population in vitro that could be useful for regenerative medicine applications. Cells were isolated from both human and porcine tissues from various locations within the urinary tract. We found that a porcine bladder urothelial holoclone had the capacity to self-renew extensively and to differentiate into fully differentiated urothelial cells. In addition, from single cell analysis, we also found a human urothelial holoclone but could not yet show that this human urothelial holoclone could differentiate. Thus, we believe the potential exists to isolate urothelial holoclones and use them in engineered tissue constructs. Furthermore, work was also focused on evaluating a molecular biotechnology tool to sort live cells based on mRNA expression as a novel means to isolate holoclones. Molecular beacons are hairpin structures that can sense mRNA present within a cell, and this approach to sort live cells has not been investigated in great detail. We designed and found a molecular beacon specific to Sox2 mRNA that could sort live Sox2-positive cells from a mixed cell population. This sorted Sox2-positive population showed specific phenotypic behavior that has only previously been shown with sorted cells based on protein surface markers. We hope that this work can aid in the design of tissue-engineered constructs that incorporate urothelial holoclones for patients that need bladder replacements. Thus, a molecular beacon sorting strategy could be used to isolate urothelial holoclones that could be embedded in engineered urotheliums. In addition, a molecular beacon strategy to analyze the number of urothelial holoclones that survive in an implanted urothelium could potentially enhance the success rate of implanted tissues for regenerative medicine applications in the bladder.

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