Project Abstract Maintenance and regeneration of various organs is dependent upon specialized stem cell populations. These populations require highly specific environments and external cues to maintain their multipotent capabilities or specifically trigger expansion and differentiation resulting in growth and development or repairing of the organ after damage. The advent of stem cell derived enteroid and organoid culture models has given insight into the signaling requirements of many stem cell populations and allowed for intensive study of the necessary niche signals that direct behavior. Previous data has suggested a role for sub-epithelial cells (SECs) in maintaining the intestinal stem cell niche. Our lab recently described an SEC population in the developing human intestine defined by the unique expression of F3 and PDGFRA (F3+/PDGFRAhi). This population, which lines the entire crypt-villus axis, can be further divided by NPY expression into villus localized (NPY+) and crypt localized (NPY-) populations using both single cell RNA sequencing (scRNAseq) and fluorescent in situ hybridization (FiSH). My preliminary data has compared single cell RNA-sequencing (scRNA-seq) data and identified differentially expressed genes in villus-SECs and crypt-SECs, including secreted proteins and receptors, identifying putative novel intestinal niche factors. One of the putative niche factors that my analysis identified was HGF (Hepatocyte Growth Factor), which was enriched in the crypt-base SECs. The HGF receptor c-MET was found to be localized to the intestinal epithelium in both mouse and human scRNA-seq datasets during fetal development. HGF is well known for its roles in development, wound healing/regeneration, and cancer in many tissues including the intestinal epithelium. My proposal is designed to test the hypothesis that the crypt-based SECs have intestinal niche supporting function through HGF signaling we will 1) characterize the distinct SEC sup-populations and 2) determine if HGF is necessary and sufficient to support human intestinal stem cell growth and development in induced pluripotent stem cell-derived human intestinal organoids (iPSC-HIOs). After HIO survival in media supplemented with HGF is confirmed, the HIO cultures both before and after transplantation beneath a mouse kidney capsule will be analyzed to define any differences between the EGF-HIOs and HGF-HIOs.