Coupling Computational Analysis and Primary Human Stem Cell Models to Understand the Human Intestine in Health and Disease

The Planar Cell Polarity (PCP) complex, which coordinates the front-back asymmetry of cells within a tissue, plays an essential role in many diverse biological contexts. While recent literature has elucidated some of these roles, whether PCP regulates cell migration in the intestinal epithelium is still unknown. Furthermore, while the key protein components underlying PCP function have been identified, a mechanistic understanding of how these components function together as a system to drive multicellular behavior is still lacking. In this thesis, I seek to answer these questions in tandem by combining novel primary human intestinal stem cell models with computational modeling. First, I develop new transgenesis and gene editing techniques, building on previous advances in primary human intestinal stem cell (hISC) culture to develop a new hISC gene editing pipeline that achieves up to 80% transfection efficiency and allows for high-throughput cloning and expansion of transgenic lines in 3–4 weeks. Second, I develop a comprehensive multicellular mathematical model of Planar Cell Polarity, centered around key PCP phosphorylation events, to directly simulate the protein interactions that drive PCP. My model confirms key PCP phenotypes, predicts unique roles for the two known positive feedback mechanisms, and predicts that VANGL-mediated DVL phosphorylation may be an underappreciated PCP regulatory mechanism. To test model predictions, I utilize my new gene editing methods to develop a novel VANGL2 inducible overexpression hISC cell line. Using this cell line, I demonstrate a significant role for the PCP complex in mediating collective migration in the intestinal epithelium and provide additional support for the existence of a VANGL-mediated negative feedback mechanism in the PCP reaction network. Together, my model provides novel insights into the mechanisms that regulate PCP, while my experimental results highlight an unappreciated role for PCP in intestinal biology.