Summary

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-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 biased their differentiation towards a colorectal fate. WNT activation biased differentiation towards a urothelial fate, giving rise to human urothelial organoids (HUOs). HUOs contained cell types present in human urothelial tissue. Based on our results, we propose a mechanism whereby WNT signaling patterns the ventral cloaca, prior to cloacal septation, to give rise to the urogenital sinus.