PROJECT SUMMARY Inflammatory Bowel Diseases (IBD) currently affect >1.6 million Americans. IBD is characterized by disruption to the intestinal epithelium and a high inflammatory environment. Available treatments target the inflammation through biological agents, however, fewer efforts have focused on epithelium healing and there are no broadly applicable therapies to repair intestinal epithelium. Human intestinal organoids (HIOs) are three-dimensional (3D) multicellular structures, derived from either adult intestinal stem cells or human pluripotent stem cells (hPSCs), that recapitulate human intestinal tissue architecture. HIOs are a promising cell source for intestinal epithelium repair, disease modeling, and drug screening. Previous work has demonstrated that HIOs engraft to the injured intestinal wall in vivo, however, these approaches are significantly limited by the lack of an appropriate delivery vehicle to drive HIO engraftment. HIO generation from hPSCs is multi-stage directed differentiation process comprising three stages: (I) differentiation into a definitive endoderm monolayer, (II) hindgut and primitive tube differentiation into free-floating, self-organized 3D aggregates (human intestinal spheroids, HIS), and (III) intestinal specification into HIOs within a 3D extracellular matrix. This in vitro culture process spans a 2D growth substrate (stage I and II) to a 3D matrix (stage III). Stage III requires culture within Matrigel, a murine tumor- derived basement membrane extract with ill-defined composition, lot-to-lot variability, and limited clinical translation potential. Another roadblock to HIO technologies is the low yield and consistency of HIS differentiation in HIOs. The objectives of this project are to (1) engineer a synthetic hydrogel platform with independent control of the biochemical and biophysical cues guiding the entire in vitro differentiation of hPSCs into HIOs, and (2) deliver HIOs in a synthetic coating to intestinal injuries in vivo. The central hypothesis is that an engineered synthetic matrix with appropriate biophysical and biochemical cues will support the HIO self-organization, growth, and differentiation process and enhance HIO engraftment and healing of intestinal wounds. Aim 1: Engineer a 2D synthetic matrix promoting spheroid generation from hPSCs. Aim 2: Evaluate the maturation of HIOs from the generated spheroids within synthetic niches. Aim 3: Engineer a clinically translatable therapeutic delivery material for HIOs to injured intestinal tissue. The results of this study will increase the clinical relevance the generated HIOs and will provide a scalable and translatable synthetic material for the differentiation and delivery of HIOs.

PROJECT NARRATIVE Human intestinal organoids (HIOs) represent an excellent in vitro model of the human intestine and a promising source for intestinal tissue repair therapies. However, the HIO in vitro differentiation process is limited by low yield of organoids, while the therapeutic translatability is hampered by a lack of clinically-relevant delivery materials. This project will result in a well-defined engineered synthetic matrix to guide the HIO in vitro differentiation process and in vivo delivery, increasing the clinical relevance of the HIO transformative therapeutic potential.