PROJECT ABSTRACT Intestinal Stem Cells (ISCs) are responsible for maintenance and regeneration of the intestinal epithelium, and reside in a domain called the crypt, supported by physical and biochemical cues that make up the stem cell niche. Despite the vital role ISCs play in human intestinal development, homeostasis and disease, the signaling cues that make up the human ISC niche are poorly understood. By analyzing single cell RNA sequencing data from human duodenal samples spanning approximately 7-to-21-week post conception, we determined that Epidermal Growth Factor (EGF), which is commonly used to create an artificial in vitro niche for growing intestinal organoids, was expressed on the differentiated cells of the villus, far from the stem cell domain. We also identified another EGF family member, Epiregulin (EREG), which was highly enriched in the stem cell domain and spatial location confirmed to the crypt. Functional experiments suggest that culturing isolated intestinal epithelium (known as enteroids) in EREG or EGF lead to dramatically different cellular outcomes and spatial organization. Based on this data, the overarching goal of this proposal is to determine the functional role of EREG as a novel intestinal stem cell niche cue. We will test the hypothesis that EREG, and not EGF, is a critical physiologically relevant growth factor for intestinal stem cell homeostasis and plays a key role in human intestinal development. We will test this hypothesis through the following specific aims: 1) Elucidate the mechanism by which EGF and EREG act on human ISCs; 2) Assess the role of EREG in a complex model of the developing human intestine in vitro and following xenotransplantation.