(502b) Photo-Responsive Hydrogel System to Study Mechano-Transduction during Intestinal Tissue Homeostasis

The epithelial lining of our gut acquires a crypt-villi architecture and serves many crucial functions including digestion, nutrient absorption, and maintenance of barrier. Due to the presence of high shear and other stressors in the intestinal lumen, the epithelium experiences very rapid cellular turnover and regenerates continuously from multipotent leucine-rich repeat-containing G-protein-coupled receptor-5-positive (Lgr5+) intestinal stem cells (ISCs) present at the crypt base. During homeostasis, the ISC niche exists together with differentiated cells in the crypt, like Paneth cells in the small intestine. Anomalies in this homeostatic regulation can cause pathological conditions like inflammatory bowel disease (IBD) and colorectal cancer (CRC) which annually affects 3.1 million and 149,000 people respectively in the USA alone. While soluble biochemical cues secreted by the surrounding mesenchymal cells constitute important role in guiding cell fate specification and organization, recently the role of mechanical cue in the process of ISC differentiation has come to light. However, the spatiotemporal role of mechano-transduction in the crypt formation and differentiation process is not fully understood. More importantly, what role the cell nuclei play in integrating the extracellular mechanical cues and subsequent genetic transformation during the differentiation process is unknown. To investigate this, we employed an interdisciplinary approach integrating murine ISC derived primary organoids, novel photo-responsive hydrogels and sophisticated microscopic techniques. The photo-responsive poly(ethylene glycol) (PEG) hydrogel system is composed of PEG chains functionalized with nitrobenzyl azide and DBCO. Upon mixing of the two PEG monomers, the azide reacts with DBCO via a strain promoted alkyne-azide cycloaddition (SPAAC) bio-click reaction that can be used to encapsulate ISCs and grow organoids. Uniquely, the ortho-nitrobenzyl (oNB) crosslinks can be cleaved with a 405 nm confocal laser, softening the hydrogel at predefined regions adjacent to the organoid, thus directing crypt formation at specified points in space and time. We demonstrated formation of intestinal crypts of controlled dimensions at high fidelity. Using our controllable crypt growth approach, we also revealed significant differences in the nuclear envelope architecture and composition as stem cells differentiate into Paneth cells. This is accompanied by changes in chromatin condensation states of the cells due to altered histone methylation levels. Additionally, this presentation will discuss the role of nuclear mechano-transduction in CRC using both publicly available datasets and patient derived tissue micro array samples. Cumulatively, we showcase the design and utilization of a novel photo-responsive hydrogel platform for studying intestinal crypt homeostasis and pathology. Our research will also reveal what role nuclear mechano-transduction plays during tissue morpho dynamics and pathophysiology.