PROJECT SUMMARY Human pluripotent stem cells (hPSCs) are a promising renewable source of differentiated insulin-producing islets for diabetes cell replacement therapy. Within the islets of diabetic patients, beta cells are dead or dys- functional, causing loss of blood glucose control. There is no cure for diabetes, and current treatments are insufficient in controlling the disease for many patients. Transplantation of insulin-secreting cells could be an effective treatment for diabetes, and a small number of patients have been implanted with cadaveric donor islets, remaining insulin independent for years. Unfortunately, many factors limit this approach, particularly the scarcity and variability of isolated human islets, with patients often requiring islets from multiple donors to achieve normal blood sugar. The lack of mature replacement tissues is a critical barrier to cellular therapy. We have previously published a strategy for the scalable generation of hPSC-derived islets (SC-islets) in vitro that are capable of secreting insulin and restoring normoglycemia in diabetic mice. However, a current challenge with these in vitro-derived cells is the incomplete and uncontrolled differentiation to fully mature SC-islets that are equal to primary cells. Our goal is to understand how modulating the microenvironment affects hPSC cell fate decisions to and the subsequent maturation of SC-islets and to use this to innovate SC-islet generation strategies for use in cellular therapy. To this end, we will study patterning of definitive endoderm and other intermediate cell types produced by microenvironmental perturbation. Subsequent generation of the SC-islets will be investigated, including characterizing in vitro and in vivo function and marker expression. The outcome of this proposal will have a positive impact by filling gaps in our understanding of what controls SC-islet cell fate specification to individual cell types, as well as how these cells mature. Successful completion of our studies will inform strategies to improve SC-islet differentiation protocols for diabetes cell replacement therapy and disease modeling.