Photoresponsive materials to study matricellular signaling dynamics during crypt formation and fission

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ABSTRACT The small intestine is lined with a single layer of epithelial cells that is organized into crypt-villus units. These epithelial cells perform multiple functions, such as aiding in digestion, nutrient absorbance, and serving as a barrier, but these processes also damage the epithelial layer. As a result, intestinal stem cell (ISCs) located near the base of crypts are responsible for the constant renewal and rapid replenishment of all intestinal cell types, but the crypts themselves also require maintenance through a process called fission. Crypt fission occurs when a single intestinal crypt divides and generates a new daughter crypt, which is essential for maintaining overall crypt density and intestinal health. Despite the important role of crypt fission in injury-induced crypt regeneration and its mis-regulation in intestinal diseases (e.g., inflammatory bowel disease), little is known about the mechanisms of how crypt fission is initiated, progresses through crypt bifurcation, and is regulated under different pathophysiological conditions. Thus, the proposed research aims to use photoresponsive hydrogels for the culture of intestinal organoids and then develop a robust and predictable in vitro model of crypt fission events. Our innovative materials and reductionist approach will allow us to precisely tune the properties of the ISC niche and understand how epithelial cells and ECM signaling contribute to crypt formation and fission. We will test hypotheses related to the role of crypt cells and matricellular signaling and its effects on organoid symmetry breaking, crypt formation, crypt fission, and the potential compensatory cellular response to local tissue damage. Specifically, we aim to: 1. Investigate the role of spatiotemporally varying cell-matrix on real time crypt formation in intestinal organoids. 2. Iteratively pattern intestinal organoid-laden hydrogels to study the role of the ECM interactions in guiding crypt fission. and 3. Spatiotemporally regulate crypt cell composition to investigate its role in crypt fission.