UCSF

The intestinal epithelium is a single layer of cells that plays a critical role in digestion, absorbs nutrients from food, and coordinates the delicate interplay between microbes in the gut lumen and the immune system. Epithelial homeostasis is crucial for maintaining health; disruption of homeostasis results in disorders including inflammatory bowel disease and cancer. The advent of 3D intestinal epithelial organoids has greatly advanced our understanding of the molecular underpinnings of epithelial homeostasis and disease. Recently, we developed an intestinal monolayer culture system that recapitulates important features of 3D organoids and the in vivo intestinal epithelium such as tissue renewal, representation of diverse epithelial cell types, self-organization, and apical-basolateral polarization. Intestinal monolayers are cultured in microtiter plates, enabling high-throughput experiments. Furthermore, their 2D nature makes it easier to distinguish individual cells by fluorescent microscopy, enabling quantitative analysis of single cell behaviors within the epithelial tissue.In Chapter 1, I describe experimental methods for generating intestinal monolayers and computational methods for analyzing immunofluorescence images of intestinal monolayers. We outline experimental methods for generating intestinal monolayers from freshly isolated intestinal crypts, frozen intestinal crypts, and 3D organoids. Fresh crypts are easily obtained from murine or human intestinal samples, and the ability to derive intestinal monolayers from both frozen crypts and 3D organoids enables genetic modification and/or biobanking of patient samples for future studies. We outline computational methods for identifying distinct epithelial cell types in immunofluorescence images of intestinal monolayers. Together, these methods enable detailed studies of epithelial homeostasis and drug-induced gastrointestinal toxicity. In Chapter 2, I investigate the responses of the intestinal epithelium to individual and paired perturbations across eight epithelial signaling pathways to better understand how complex milieus of microenvironmental signals are interpreted to coordinate tissue cell-type composition. Renewing tissues have the remarkable ability to continually produce both proliferative progenitor and specialized differentiated cell types. Using a high-throughput approach that combines intestinal monolayers and quantitative imaging, we identified conditions that enrich for specific cell types as well as interactions between pathways. Importantly, we found that modulation of transit-amplifying cell proliferation changes the ratio of differentiated secretory to absorptive cell types. These observations highlight an underappreciated role for transit-amplifying cells in the tuning of differentiated cell-type composition. Finally, in Chapter 3, I establish the ability to use murine small and large intestine-derived monolayers to screen drugs for toxicity. Gastrointestinal toxicity is a major concern in the development of drugs. As a proof-of-concept, we applied this system to assess gastrointestinal toxicity of ~50 clinically used oncology drugs, encompassing diverse mechanisms of action. Nearly all tested drugs had a deleterious effect on the gut, with increased sensitivity in the small intestine. The identification of differential toxicity between the small and large intestine enabled us to pinpoint differences in drug uptake, drug metabolism and cell signaling across the gut. These results highlight an under-appreciated distinction between small and large intestine toxicity and suggest distinct tissue properties important for modulating drug-induced gastrointestinal toxicity. The ability to accurately predict where and how drugs affect the murine gut will accelerate preclinical drug development.