The Differentiation of Patient-derived Prostate Organoids and the Influence of Vitamin D

Organoid culture is an in vitro method to expand epithelial cells into small structures that mimic organ morphology and function. For the prostate, they are the only benign model to exhibit androgen signaling and fully differentiated luminal cells. In order to characterize and improve upon this model, prostate epithelial organoids were grown from patient tissue and their cell content was interrogated using single cell RNA sequencing and immunostaining. Results indicate that organoids consist of a resident KRT13+ stem cell, downstream KRT6A progenitors, dividing cells along the basal and luminal hierarchy, and polarized cell types that have high expression of integrin. Once the model was fully characterized, it was used to test the hypothesis that vitamin D promotes differentiation in prostate epithelium. Vitamin D (1,25D) is an essential hormone that regulates systemic calcium homeostasis and cell fate decisions. The prostate is a hormonally regulated gland that requires steroids for the differentiation of terminal luminal cells. To explore the role of vitamin D in this context, we examined the influence of 1,25D over human prostate epithelial differentiation utilizing the primary cell organoid model and determined targets by single cell RNA sequencing. 1,25D promoted differentiation by inhibiting canonical Wnt activity via upregulation of DKK1, and promoted epithelial growth through suppression of DKK3. This action was observed in lineage-committed cells, demonstrating that vitamin D selectively regulates cells that are on the path to differentiation. Overall this thesis describes the cells cultivated in prostate organoids, and discusses the effect of the steroid 1,25D their differentiation.