Transcription factor regulation in urothelial-type differentiation and reprogramming

For bladder tissue-engineering, there is a need for a surrogate epithelial cell source for patients whose own bladder epithelial cells (urothelial cells) are compromised by disease or cancer. Current work in the field has focused on attempting to differentiate various types of stem cells into urothelial cells using conditioned medium, co-culture or specific factors. These approaches have lacked clear focus, making it obvious that a better understanding of the transcription factors underlying urothelial lineage, development, and cell differentiation is necessary. The aim of the work presented in this thesis was to acquire specific knowledge regarding transcription factors involved in urothelial cell differentiation, which could be applied to an in vitro transdifferentiation approach of cell reprogramming. Since buccal mucosa is readily available, and already used as a grafted tissue for urology surgical applications, buccal epithelial cells were identified as a potential starting cell type. An in vitro comparison of human buccal epithelial cells to urothelial cells revealed that buccal epithelial cells had weak transcript and protein expression of four transcription factors that play key roles in urothelial cell differentiation: ELF3, FOXA1, GATA3 and PPARγ. Since ELF3 and FOXA1 have been previously shown to act downstream of PPARγ in urothelial cells, PPARγ and GATA3 were chosen as key upstream transcription factors to overexpress in buccal epithelial cells. Investigation into PPARγ expression in human urothelial cells revealed that of the two main PPARγ protein isoforms, PPARγ1 was most critical for urothelial cell differentiation. Knockdown of GATA3 expression in urothelial cells using specific siRNA revealed an important role for GATA3 in maintaining the differentiated state of urothelium. Individual overexpression of GATA3 and PPARγ1 in human buccal epithelial cells was unable to cause complete transdifferentiation to urothelial cells, but each resulted in the upregulation of downstream genes that play critical roles in urothelial cell differentiation and barrier formation. This work revealed key differences in transcription factor expression between buccal epithelial cells and urothelial cells, allowing for a better understanding of transcription factors involved in urothelial cell differentiation. The work provides a starting point for future urothelial-type cell reprogramming efforts which will likely require combined overexpression of several of these identified transcription factors to achieve complete transdifferentiation.