Project Summary The radiation-induced gastrointestinal syndrome (RIGS) results from dose-dependent, cytocidal effects of radiation on intestinal crypt cells. There are currently no approved medical countermeasures to alleviate the RIGS. While active Lgr5+ stem cells are efficiently ablated, regeneration emerges from reserve, quiescent progenitor/stem cells that are radio-resistant. The exact relationship between these two progenitor populations (radio-resistant and radio-sensitive) remains undefined, and more information is needed to develop therapies for RIGS and other disorders. An unbiased approach is critically needed to fully characterize the diversity of cells in normal and regenerating intestine, independent of prior knowledge or purification assays. Work from our lab and the ISCC has demonstrated a key role for the stromal mesenchyme and other niche cells in modulating ISC homeostasis and regeneration, but a comprehensive regulatory model does not exist. Matrix proteins are also part of the niche, and we have been able to grow both enteroids and human intestinal organoids in tissue-specific hydrogels. Our investigators and collaborators have developed novel R-spondin and Wnt agonists for in vivo delivery but the optimal combinations for treatment of RIGS have not been defined. Thus, a full understanding of the cross-talk signals between ISCs and niche cells, and models for stimulating or replacing intestinal ISCs, is needed for advances in therapy for RIGS. In collaboration with other members of the ISCC, we are proposing two specific aims. Aim 1. How does the heterogeneity of intestinal crypt cells and adjacent stromal cells change after radiation? We will analyze highly pure crypt epithelial cells and stromal cells using a novel multiplex scRNA-seq platform before and after radiation, and apply computational tools (metaVIPER) to develop a hierarchical structure and regulatory model. Aim 2. Can therapy with niche factors or stem cells be used to mitigate radiation or inflammatory injury to the intestine? We will study novel combinations of factors to mitigate RIGS, and then use tissue-specific hydrogels and novel factors and cells to grow in vitro and transplant in vivo human intestinal organoids to treat colitis and radiation proctitis. Overall, this proposal will use novel technology developed at Columbia/Einstein to advance collaboratively the ISCC mission to regenerate the human intestine.