Project Summary The human intestine is a remarkable organ which stores and secretes a variety of hormones from enteroendocrine (EEC) cells. These hormones play critical roles in regulating human feeding behavior and satiety, and their dysregulation leads to overeating and a host of other metabolic disorders. For these reasons, there is a need in the therapeutics marketplace for in vitro intestinal EEC cell platform that precisely recapitulates the physiology of in vivo intestines. To meet this need, Altis Biosystems Inc., an early stage biotechnology company, has collaborated with scientists at academic laboratories to develop a novel, primary-stem-cells- based, in vitro intestinal model (termed RepliGut). We have finished the SBIR Phase I program by optimizing the RepliGut platform to enrich enterochromaffin (EC) cells, a subtype of EEC cells, and increase barrier integrity. We have developed a simple but efficient method that significantly increases the formation of EEC cells compared with the starting culture conditions. We have investigated signaling molecules for forced differentiation towards EEC lineage allocation, quantified assays for serotonin, and investigated passage and donor variation. We validated the platform with a small-scale compound screen for serotonin secretion from EC cells. All proposed milestones in the Phase I SBIR were accomplished, thus providing a solid foundation for this Phase II SBIR application. The focus of this Phase II proposal is to continue the optimization of EEC formation to meet the market needs for high-throughput screening assays. Besides EC cells, we will extend our research to the other important subtype, enteroendocrine L-cells, which secrete GLP-1 and PYY in response to the ingestion of food. Additional characterizations will be focused on the uniformity of cell behaviors within a 96-well format. Low well-to-well and plate-to-plate variation will be confirmed before use as a cellular assay platform. Potential regional-, sex- and age-based variations will be further investigated by testing stem cells derived from 5 donors and all 6 sections of intestine. The platform will be validated by screening a large library of metabolic compounds. Performance characteristics of the platform will then be evaluated in comparisons of our in-house assays vs. kits shipped to collaborating laboratories.

Project Narrative The novel enteroendocrine cell-rich RepliGut platform will have broad applications in screening and validating therapeutics to manipulate feeding behavior and satiety which will revolutionize the treatment of many metabolic disorders in humans.