Spatial Transcriptomic Mapping Reveals Wnt/β-catenin Gradient-Dependent Intestinal Stem Cell Niche Heterogeneity and Regeneration Dynamics

Abstract

Intestinal stem cells (ISCs) reside at the crypt base and maintain tissue homeostasis through continuous self-renewal and differentiation. While the Wnt/β-catenin signaling pathway is known to regulate ISC fate, the spatial organization and functional heterogeneity within the stem cell niche remain incompletely understood. Here, we employed spatial transcriptomics combined with high-resolution confocal imaging to generate a comprehensive molecular atlas of the intestinal crypt. Our analysis revealed distinct ISC subpopulations distributed along a Wnt gradient: high Wnt activity at the crypt base promotes active cycling stem cells (aISCs) expressing Lgr5 and Olfm4, while the +4 position harbors reserve stem cells (rISCs) with lower Wnt activity and expression of Bmi1 and Hopx. Single-molecule fluorescence in situ hybridization (smFISH) confirmed this spatial patterning. Following radiation-induced injury, we discovered that rISCs rapidly upregulate β-catenin signaling and transition to an aISC state, demonstrating remarkable plasticity. Organoid culture experiments with GSK-3β inhibitor treatment recapitulated this transition. Furthermore, lineage tracing using inducible Cre-loxP systems revealed that Paneth cells serve as a critical niche component by secreting Wnt3 and Dll4 ligands, forming a localized signaling hub. Depletion of Paneth cells disrupted ISC maintenance and impaired regeneration capacity. These findings provide unprecedented spatial resolution of Wnt-dependent ISC heterogeneity and identify potential therapeutic targets for intestinal regenerative medicine.