Lgr5(+) intestinal stem cells reside in an unlicensed G(1) phase

During late mitosis and the early G(1) 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 G(1) 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 G(1) entry. We propose that the unlicensed G(1) phase of intestinal stem cells creates a temporal window when proliferative fate decisions can be made.