Differentiation of human pluripotent stem cells into urothelial organoids via transient activation of WNT signaling.

The cloaca is a transient structure that forms in the terminal hindgut giving rise to the rectum dorsally and the urogenital sinus ventrally. Similarly, human hindgut cultures derived from human pluripotent stem cells generate human colonic organoids (HCOs) which also contain co-developing urothelial tissue. In this study, our goal was to identify pathways involved in cloacal patterning and apply this to human hindgut cultures. RNA sequencing (RNA-seq) data comparing dorsal versus ventral cloaca in e10.5 mice revealed that WNT signaling was elevated in the ventral versus dorsal cloaca. Inhibition of WNT signaling in hindgut cultures maintained their differentiation toward colonic organoids. WNT activation promoted differentiation toward human urothelial organoids (HUOs). HUOs contained developmental stage specific cell types present in mammalian urothelial tissue including co-developing mesenchyme. Therefore, HUOs offer a powerful in vitro model for dissecting the regulatory pathways that control the dynamic emergence of stage specific cell types within the human urothelium.