Abstract
Despite a variety of therapeutic options for urothelial carcinoma of the bladder (UCB) in the early stages, the efficacy of treatment options in metastatic disease remains modest. Investigating the influence of different mutational patterns during the complex process of carcinogenesis may enable the development of innovative therapeutic agents. The differentiation of human pluripotent stem cells (hPSCs) into mature bladder urothelial cells offers a distinct platform to study embryonic development and disease modelling in the human urothelium. Given the precisely defined genetic background of those cells, it becomes evident that hPSCs-derived urothelium provides a valuable tool to study the processes during early carcinogenesis upon defined oncogenic stimuli. Differentiation of hPSCs into urothelial cells was performed by mimicking different stages of embryonic development with the induction of definitive endoderm, a hindgut differentiation step and finally, the induction of mature urothelium. To study the influence of different genes on the carcinogenesis of UCB, knockouts of TP53, BRCA2 and ATM were generated by CRISPR-Cas9 mediated gene editing. A protocol for the differentiation of urothelial cells from hPSCs was established by reaching different milestones of embryonic development (1. definitive endoderm, 2. hindgut, 3. urothelium). The induction of urothelium was confirmed by upregulation of uroplakins (UPK2, UPK3a) in the differentiated cells. Functional relevance of the knockouts was confirmed by reduced gene and protein expression (BRCA2, TP53), the presence of truncated proteins (ATM and BRCA2) and enhanced sensitivity to genotoxic stress. An efficient generation of urothelial cells from hPSCs is feasible. The functional relevance of different gene knockouts underlines the validity of the gene-editing strategy. In future studies, the significance of exogenous and endogenous carcinogenic stimuli in the context of distinct mutational signatures during early carcinogenesis of UCB can be investigated in this defined genetic background.
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