Identification of Quiescent LGR5⁺ Stem Cells in the Human Colon

Background & aims: In the mouse intestinal epithelium, Lgr5⁺ stem cells are vulnerable to injury, owing to their predominantly cycling nature, and their progenies de-differentiate to replenish the stem cell pool. However, how human colonic stem cells behave in homeostasis and during regeneration remains unknown.

Methods: Transcriptional heterogeneity among colonic epithelial cells was analyzed by means of single-cell RNA sequencing analysis of human and mouse colonic epithelial cells. To trace the fate of human colonic stem or differentiated cells, we generated LGR5-tdTomato, LGR5-iCasase9-tdTomato, LGR5-split-Cre, and KRT20-ERCreER knock-in human colon organoids via genome engineering. p27⁺ dormant cells were further visualized with the p27-mVenus reporter. To analyze the dynamics of human colonic stem cells in vivo, we orthotopically xenotransplanted fluorescence-labeled human colon organoids into immune-deficient mice. The cell cycle dynamics in xenograft cells were evaluated using 5-ethynyl-2'-deoxyuridine pulse-chase analysis. The clonogenic capacity of slow-cycling human stem cells or differentiated cells was analyzed in the context of homeostasis, LGR5 ablation, and 5-fluorouracil-induced mucosal injury.

Results: Single-cell RNA sequencing analysis illuminated the presence of nondividing LGR5⁺ stem cells in the human colon. Visualization and lineage tracing of slow-cycling LGR5⁺p27⁺ cells and orthotopic xenotransplantation validated their homeostatic lineage-forming capability in vivo, which was augmented by 5-FU-induced mucosal damage. Transforming growth factor-β signaling regulated the quiescent state of LGR5⁺ cells. Despite the plasticity of differentiated KRT20⁺ cells, they did not display clonal growth after 5-FU-induced injury, suggesting that occupation of the niche environment by LGR5⁺p27⁺ cells prevented neighboring differentiated cells from de-differentiating.

Conclusions: Our results highlight the quiescent nature of human LGR5⁺ colonic stem cells and their contribution to post-injury regeneration.