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BACKGROUND: In vitro generation of human urothelium from stem cells would be a major advancement in the regenerative medicine field, scientifically and clinically. In addition, deciphering the mechanisms of urothelial differentiation would facilitate investigation of deviated differentiation of normal progenitors into urothelial cancer stem cells, and could point to areas of intervention for improved treatments. While the induction of murine embryonic stem cells (ESCs) into urothelium has been evaluated, the ability to induce human ESCs has not been investigated. Our goal was to develop an in vitro protocol for induction of human ESCs into definitive endoderm (DE) and then urothelium, free of matrices and cell contact. Using conditioned medium and non-contact experiments, we also sought to evaluate the impact of adult epithelial and mesenchymal cells upon induction. Lastly, we sought to determine the temporal association of transcription regulators, known to be involved in urothelial differentiation, with uroplakin (UP) expression in this in vitro system.
METHODS: HESCs (H9) were maintained undifferentiated on irradiated MEFs. Colonies were induced into DE in RPMI with Activin A and graduated FBS concentrations for 9 days. The ability to induce DE into urothelium was evaluated with these conditions in a 21-day culture system: Uromedia with and without all-trans retinoic acid (RA), non-contact co-culture with adult urothelium, with and without urothelial conditioned medium, and with and without bladder smooth muscle conditioned medium. To induce terminal differentiation, troglitasone (a PPARγ activator) was added and EGF was removed on day 18. Induction from two other HESC lines (H1, HuES8) and two IPSC lines (IMR-90-4, WT-2) was also tested. Differentiation markers Oct4, Nanog, Tra160, FoxA2, Sox17 and uroplakins (UPs) were analyzed by PCR, immunocytochemistry (ICC) and flow cytometry at different times during the induction process. The temporal expression of transcription factors IRF-1, GATA-4 and Grainyhead factor/Get1 with UP expression were analyzed by flow cytometry.
RESULTS: Analyses of HESC-derived cultures by flow cytometry and ICC demonstrate that up to 60% of cells could be induced into urothelium. Neither RA, co-culture with adult cells or conditioned medium increased urothelial induction over base medium. Uroplakin expression was seen within a few days of induction from DE, concurrently with the expected decrease in Sox17. Additional HESC and IPSC lines could also be induced to urothelium. Flow cytometry- based temporal evaluation of transcription factor expression during early differentiation suggested an association of IRF-1, GATA-4 and Get1 with UP expression. ICC also showed cellular co-localization of these transcription factors with UP-expressing cells.
CONCLUSIONS: Human embryonic stem cells and IPSCs can be induced into definitive endoderm and then urothelium with controlled media conditions in vitro. Unlike murine cells, supplemental RA was not required for urothelial induction. Unlike other cell types, stimulation from adult cells or medium conditioned with adult cells was not necessary. Transcription factors IRF-1, GATA-4 and Get1 are expressed at the same time as the definitive endoderm loses its differentiation markers and begins expressing uroplakins. These findings will facilitate further study of abnormal differentiation and investigation of induced pluripotent stem cells for tissue engineering applications.