Tissue intrinsic properties of regeneration in homeostasis and injury in the mammalian small intestine

Abstract

Epithelial tissues must undergo regeneration to maintain homeostasis, as these tissues are subjected to some of the harshest environments the organism faces. The intestinal epithelium is one such tissue which has been used for decades to understand the process of regeneration in homeostasis and disease. The intestinal epithelium is particularly amenable to experimentation, given its clear zonation of the functional units (villi) and the stem cell niche (crypts). The tissue resident stem cells are clearly identifiable by their ability to give rise to the rest of the cells of the intestinal epithelium. Single stem cells have the potential to give rise to self-organized epithelial structures called intestinal organoids, allowing for the study of the tissue intrinsic properties of the intestinal epithelium. In Chapter 2, I provide a method to culture intestinal organoids and detail improvements in protocols to generate data from imaging experiments and single-cell RNA sequencing. In Chapter 3, I interrogate the crypt fission mechanism underlying inflation-collapse events of stem cell zones (organoid crypts) in intestinal organoids, which are not under intrinsic size control. I find that cystic fibrosis transmembrane receptor (CFTR) and mechanosensitive ion channel Piezo1 are necessary, but not sufficient to explain inflation-collapse in intestinal organoids, and that a Clusterin-expressing state is correlated with inflation. Finally in Chapter 4, I interrogate the Clusterin-expressing cell state which has been dubbed the ‘revival’ stem cell – a cell type which can regenerate the intestinal epithelium after injury. I find no evidence suggesting that Clusterin-expressing cells are a unique cell type, and I find that this gene signature is associated with both cell death and a lack of recovery of the epithelial barrier when it is induced in Lgr5+ cells and their recent progeny following irradiation.