LGR5⁺ Stem Cells Maintain Apex Position in Regenerative Hierarchy of the Intestinal Epithelium During Homeostasis and Injury

The cellular origin of intestinal epithelial regeneration has been a subject of continued debate, with recent models challenging the primacy of WNT-dependent LGR5⁺ crypt base columnar (CBC) cells as the central regenerative population. Here, we revisit this question through quantitative integration of single-cell transcriptomic, chromatin accessibility, spatial, and lineage-tracing analyses across the proximal-to-distal axis of the small intestinal epithelium. Our data show that under homeostatic conditions, LGR5⁺ cells exclusively sustain epithelial self-renewal in nearly all crypt–villus units along the entire length of the small intestine. Following irradiation or chemotoxic injury, surviving LGR5⁺ CBC cells and their immediate progeny undergo transcriptional and epigenetic reprogramming into transient fetal-like cell states that initiate epithelial repair. Crucially, successful regeneration depends on the reactivation of canonical WNT/β-catenin signaling, as evidenced by increased TCF motif accessibility and upregulation of WNT target genes, including Lgr5. Accordingly, pharmacological inhibition of WNT signaling blocks the reconstitution of LGR5⁺ cells and crypt regeneration, leading to epithelial collapse. These findings reconcile prior controversies by demonstrating that epithelial regeneration throughout the small intestine, even following injury, is ultimately driven by LGR5⁺ CBC cells in a WNT-dependent manner.