Intestinal homeostasis and regeneration after injury are controlled by 2 different types of cells: slow cycling, injury-resistant reserve intestinal stem cells (ISCs) and actively proliferative ISCs. Putative reserve ISCs have been identified using a variety of methods, including CreER insertions at Hopx or Bmi1 loci in mice and DNA label retention. Label-retaining cells (LRCs) include dormant stem cells in several tissues; in the intestine, LRCs appear to share some properties with reserve ISCs, which can be marked by reporter alleles. We investigated the relationships between these populations.

Studies were performed in Lgr5−EGFP-IRESCreERT2, Bmi1-CreERT2, Hopx-CreERT2, and TRE-H2BGFP::Hopx-CreERT2::lox-stop-lox-tdTomato mice. Intestinal epithelial cell populations were purified; we compared reporter allele-marked reserve ISCs and several LRC populations (marked by H2B-GFP retention) using histologic flow cytometry and functional and single-cell gene expression assays.

LRCs were dynamic and their cellular composition changed with time. Short-term LRCs had properties of secretory progenitor cells undergoing commitment to the Paneth or enteroendocrine lineages, while retaining some stem cell activity. Long-term LRCs lost stem cell activity and were a homogenous population of terminally differentiated Paneth cells. Reserve ISCs marked with HopxCreER were primarily quiescent (in G0), with inactive Wnt signaling and robust stem cell activity. In contrast, most LRCs were in G1 arrest and expressed genes that are regulated by the Wnt pathway or are in the secretory lineage.

LRCs are molecularly and functionally distinct from reporter-marked reserve ISCs. This information provides an important basis for future studies of relationships among ISC populations.