Identification of a cKit⁺ colonic crypt base secretory cell that supports Lgr5⁺ stem cells in mice

Background & Aims: Paneth cells contribute to the small intestinal niche of Lgr5⁺ stem cells. Although the colon also contains Lgr5 ⁺ stem cells, it does not contain Paneth cells. We investigated the existence of colonic Paneth-like cells that have a distinct transcriptional signature and support Lgr5⁺ stem cells. Methods: We used multicolor fluorescence-activated cell sorting to isolate different subregions of colon crypts, based on known markers, from dissociated colonic epithelium of mice. We performed multiplexed single-cell gene expression analysis with quantitative reverse transcriptase polymerase chain reaction followed by hierarchical clustering analysis to characterize distinct cell types. We used immunostaining and fluorescence-activated cell sorting analyses with in vivo administration of a Notch inhibitor and in vitro organoid cultures to characterize different cell types. Results: Multicolor fluorescence-activated cell sorting could isolate distinct regions of colonic crypts. Four major epithelial subtypes or transcriptional states were revealed by gene expression analysis of selected populations of single cells. One of these, the goblet cells, contained a distinct cKit/CD117⁺ crypt base subpopulation that expressed Dll1, Dll4, and epidermal growth factor, similar to Paneth cells, which were also marked by cKit. In the colon, cKit⁺ goblet cells were interdigitated with Lgr5⁺ stem cells. In vivo, this colonic cKit⁺ population was regulated by Notch signaling; administration of a γ-secretase inhibitor to mice increased the number of cKit⁺ cells. When isolated from mouse colon, cKit⁺ cells promoted formation of organoids from Lgr5⁺ stem cells, which expressed Kitl/stem cell factor, the ligand for cKit. When organoids were depleted of cKit⁺ cells using a toxin-conjugated antibody, organoid formation decreased. Conclusions: cKit marks small intestinal Paneth cells and a subset of colonic goblet cells that are regulated by Notch signaling and support Lgr5⁺ stem cells.