Deciphering cell motility and spatial sensing of intestinal cell types using an ex vivo intestinal model

Abstract

The intestine is a highly organized tissue with two distinct regions: the crypt and the villus. When stem cells divide at the crypt bottom, half of their progeny migrates upwards towards the villus, where they differentiate into various cell types, including the abundant absorptive enterocytes. However, the precise mechanisms governing this migration and tissue organization remain poorly understood. In this thesis, novel methodologies, such as long-term intravital imaging and decellularization of mouse intestine, are used to study cell type-specific motility within the tissue architecture. Moreover, work in this thesis probes the mechanisms mediating intestinal regeneration and aging, and the clonal competition during tumor development.

In paper 1, we employ long-term intravital imaging to identify a greater number of longterm functioning intestinal stem cells (ISCs) in the small intestine compared to the colon. We further investigate this phenomenon by combining intravital imaging and the novel ex vivo live cell imaging assay to discover that stem cells in the small intestine display downward motility directed by Wnt-ligands.

In Paper 2, the ex vivo live cell imaging assay was utilized to investigate active cell migration in several cell types. Our findings reveal that both ISCs and paneth cells possess an intrinsic ability to perceive positional cues embedded in the extracellular matrix (ECM), which guides them to their native location, the crypt. In contrast, enterocytes, lack this capability. Finally, we discovered that during aging ECM loses the signals guiding crypt homing of ISCs, and that the tumor-causing mutations render cells insensitive to ECM signals resulting in loss of crypt homing.

In Paper 3, we introduce an optimized intestinal decellularization protocol and demonstrate its capacity to regenerate the intestinal epithelium from single-seeded stem cells, freshly isolated crypts, or organoids. During regeneration following damage, we discovered mesenchymally produced Asporin, which promotes Tgfβ-signaling and induces fetal-like reprogramming in intestinal tissue. Additionally, we observed that chronic upregulation of Asporin in the aged intestinal tissue hampers tissue repair.

In Paper 4, we elucidate how Apc-mutant ISCs gain a clonal advantage over wild-type ISCs. We reveal that Apc-mutant ISCs secrete the Wnt-inhibitor Notum, which reduces the stemness and competitiveness of wild-type ISCs. Inhibition of Notum reverted the clonal advantage of Apc-mutant cells and reduced tumor burden.

In conclusion, this thesis focused on highlighting the interplay between intestinal epithelial cells and the ECM, particularly the ability of ISCs and paneth cells to sense positional cues embedded in the ECM, guiding them to their native location. Additionally, key mechanisms disrupted during aging and in intestinal cancer are elucidated.