PROJECT SUMMARY: Congenital or acquired disruption of the enteric nervous system (ENS) leads to neurointestinal diseases, including Hirschsprung disease, esophageal achalasia, gastroparesis, chronic intestinal pseudo-obstruction, and others. Normal development of a functional ENS relies on the coordinated interaction between enteric neural crest-derived cells and their microenvironment, including the extracellular matrix (ECM), as the enteric neural crest-derived cells migrate rostrocaudally along the embryonic gut mesenchyme.1 These interactions ensure proper cell migration, proliferation, and differentiation into the enteric neurons and glial cells that regulate numerous gastrointestinal processes, including motor and sensory function. Numerous ECM and ECM binding proteins have been shown to promote enteric neural crest-derived cell migration, while others (including agrin and chondroitin sulfate proteoglycans) have been shown to inhibit migration, demonstrating a critical role of the ECM in ENS formation. Despite an improving understanding of normal ENS development, current therapies for neurointestinal diseases are lacking, as they target symptoms rather than the underlying pathology of an absent or abnormal ENS. Our lab and others are investigating the potential of enteric neuronal stem cell transplantation as a novel approach to restoring innervation and gut function in these patients, but studies have been hampered by limited engraftment, migration, and proliferation of transplanted cells.1 Given the reliance of ENS development on the local environment, we believe that successful development of enteric neuronal stem cell transplantation relies on a permissive local ECM to support the transplanted cells. We hypothesize that inhibitory ECM proteins (agrin and chondroitin sulfate proteoglycans) are over-expressed following enteric neuronal stem cell transplantation, thwarting the migration of stem cells. In this grant we therefore aim to identify ECM gene expression differences between the aganglionic and normal colon, characterize ECM protein expression following enteric neuronal stem cell transplantation, and to leverage our understanding of normal ENS development to modify the microenvironment after enteric neuronal stem cell transplantation by knocking down ECM proteins that inhibit cell migration in order to enhance the migration of transplanted cells.