Reserve intestinal stem cells (rISCs) are quiescent/slowly cycling under homeostatic conditions, allowing for their identification with label-retention assays. rISCs mediate epithelial regeneration after tissue damage by converting to actively proliferating stem cells (aISCs) that self renew and demonstrate multipotency, which are defining properties of stem cells. Little is known about the genetic mechanisms that regulate the production and maintenance of rISCs. High expression levels of the transcription factor Sox9 (Sox9^high) are associated with rISCs. This study investigates the role of SOX9 in regulating the rISC state.

We used fluorescence-activated cell sorting to isolate cells defined as aISCs (Lgr5^high) and rISCs (Sox9^high) from Lgr5^EGFP and Sox9^EGFP reporter mice. Expression of additional markers associated with active and reserve ISCs were assessed in Lgr5^high and Sox9^high populations by single-cell gene expression analyses. We used label-retention assays to identify whether Sox9^high cells were label-retatining cells (LRCs). Lineage-tracing experiments were performed in Sox9-CreERT2 mice to measure the stem cell capacities and radioresistance of Sox9-expressing cells. Conditional SOX9 knockout mice and inducible-conditional SOX9 knockout mice were used to determine whether SOX9 was required to maintain LRCs and rISC function.

Lgr5^high and a subset of crypt-based Sox9^high cells co-express markers of aISC and rISC (Lgr5, Bmi1, Lrig1, and Hopx). LRCs express high levels of Sox9 and are lost in SOX9-knockout mice. SOX9 is required for epithelial regeneration after high-dose irradiation. Crypts from SOX9-knockout mice have increased sensitivity to radiation, compared with control mice, which could not be attributed to impaired cell-cycle arrest or DNA repair.

SOX9 limits proliferation in LRCs and imparts radiation resistance to rISCs in mice.