Lgr5⁺ intestinal stem cells reside in an unlicensed G₁ phase

During late mitosis and the early G₁ phase, the origins of replication are licensed by binding to double hexamers of MCM2-7. In this study, we investigated how licensing and proliferative commitment are coupled in the epithelium of the small intestine. We developed a method for identifying cells in intact tissue containing DNA-bound MCM2-7. Interphase cells above the transit-amplifying compartment had no DNA-bound MCM2-7, but still expressed the MCM2-7 protein, suggesting that licensing is inhibited immediately upon differentiation. Strikingly, we found most proliferative Lgr5⁺ stem cells are in an unlicensed state. This suggests that the elongated cell-cycle of intestinal stem cells is caused by an increased G₁ length, characterized by dormant periods with unlicensed origins. Significantly, the unlicensed state is lost in Apc-mutant epithelium, which lacks a functional restriction point, causing licensing immediately upon G₁ entry. We propose that the unlicensed G₁ phase of intestinal stem cells creates a temporal window when proliferative fate decisions can be made.