One major goal of regenerative medicine is to produce insulin secreting ?-cells for transplantation therapy to treat Type 1 diabetes (T1D). Differentiation of embryonic stem cells is a powerful technology to generate functional insulin+ cells. However, teratoma formation is a serious concern with this approach. Alternatively, adult tissues could be converted into insulin+ cells in a process termed reprogramming. Reprogramming adult cells carries little risk for teratoma, but the reprogramming efficiency tends to be low and the resulting insulin+ cells have limited functionality. There is a critical ned to define the best tissue source and reprogramming method for this approach. My laboratory pioneered a reprogramming method based on a cocktail of defined genetic factors (Ngn3, Pdx1, Mafa, referred to as NPM factors). NPM factors are sufficient to convert pancreatic acinar cells to stable and functional insulin+ cells in animal models. In a comprehensive screen of adult tissues, we discovered that cells residing in the adult gastric mucosa also respond rapidly to NPM factors and generate functional insulin+ cells. Significantly, gastric insulin+ cells become glucose responsive faster than acinar-derived insulin+ cells. Thus, gastric tissue is a highly promising adult tissue source to produce functional insulin+ cells. Importantly, human gastric cells can be cultured and propagated as organoids in large numbers from cadaveric sources. Delivery of NPM factors led to formation of c-peptide+ cells in the human organoids, raising the exciting possibility of generating functional human insulin+ cells with this approach. One major aim of the proposal is to gain deeper understanding of the reprogramming process and the resulting gastric insulin+ cells. Specifically, we will determine the molecular and functional similarity of the induced gastric insulin+ cells with native islet beta-cells. We will investigate stability of the induced cells and test the hypothesis that islet structure formation will lead to heir long-term stability. We will also define the individual function of NPM factors in the reprogramming process. The second major aim of the proposal is to develop methods for efficient induction of functional insulin+ cells from human gastric tissues with combined treatment of genetic factors and signaling pathway modulators. Together, these studies will provide the necessary foundation for developing a novel technology to produce functional human insulin+ cells for therapeutic transplantation.