Summary

Does fibrotic gut stiffening caused by inflammatory bowel diseases (IBD) direct the fate of intestinal stem cells (ISCs)? To address this question we first developed a novel long-term culture of quasi-3D gut organoids plated on hydrogel matrix of varying stiffness. Stiffening from 0.6kPa to 9.6kPa significantly reduces Lgr5 high ISCs and Ki67 + progenitor cells while promoting their differentiation towards goblet cells. These stiffness-driven events are attributable to YAP nuclear translocation. Matrix stiffening also extends the expression of the stemness marker Olfactomedin 4 (Olfm4) into villus-like regions, mediated by cytoplasmic YAP. We next used single-cell RNA sequencing to generate for the first time the stiffness-regulated transcriptional signatures of ISCs and their differentiated counterparts. These signatures confirm the impact of stiffening on ISC fate and additionally suggest a stiffening-induced switch in metabolic phenotype, from oxidative phosphorylation to glycolysis. Finally, we used colon samples from IBD patients as well as chronic colitis murine models to confirm the in vivo stiffening-induced epithelial deterioration similar to that observed in vitro . Together, these results demonstrate stiffness-dependent ISC reprograming wherein YAP nuclear translocation diminishes ISCs and Ki67 + progenitors and drives their differentiation towards goblet cells, suggesting stiffening as potential target to mitigate gut epithelial deterioration during IBD.