Mapping early fate determination in Lgr5+ crypt stem cells using a novel Ki67-RFP allele

Cycling Lgr5(+) stem cells fuel the rapid turnover of the adult intestinal epithelium. The existence of quiescent Lgr5(+) cells has been reported, while an alternative quiescent stem cell population is believed to reside at crypt position +4. Here, we generated a novel Ki67(RFP) knock-in allele that identifies dividing cells. Using Lgr5-GFP; Ki67(RFP) mice, we isolated crypt stem and progenitor cells with distinct Wnt signaling levels and cell cycle features and generated their molecular signature using microarrays. Stem cell potential of these populations was further characterized using the intestinal organoid culture. We found that Lgr5(high) stem cells are continuously in cell cycle, while a fraction of Lgr5(low) progenitors that reside predominantly at +4 position exit the cell cycle. Unlike fast dividing CBCs, Lgr5(low) Ki67(-) cells have lost their ability to initiate organoid cultures, are enriched in secretory differentiation factors, and resemble the Dll1 secretory precursors and the label-retaining cells of Winton and colleagues. Our findings support the cycling stem cell hypothesis and highlight the cell cycle heterogeneity of early progenitors during lineage commitment.