Allogeneic islets transplanted into the liver have shown promise clinically for treatment of T1D, yet their supply is limited. Recent reports on the generation of insulin producing cells from human embryonic stem cells (hESC) has demonstrated their potential as a cell source. The hESC are cultured in vitro to generate insulin positive cells, and are transplanted to complete their maturation toward ?-cells. Importantly, these publications note that not all diabetic recipients of these cells become normoglycemic, and indicates the need for systems that consistently and efficiently promote differentiation in vitro and in vivo to achieve mature ?-cells capable of restoring normoglycemia. Recent advances in 3D culture of organoids have demonstrated the production of complex tissues through maintenance of tissue structure and cellular organization. We propose that microporous scaffolds can provide cues that promotes differentiation and supports the cellular organization into islet-like structures. PI Dr. Shea has developed the scaffolds for the transplantation of primary islets into mice at a clinically translatable site that allows for efficient engraftment and function, and the reversal of hyperglycemia with a minimal islet mass. co-PI Dr. Spence is a developmental biologist with expertise in organoid culture that is collaborating on the scaffold design and analysis of in vivo maturation. Aim 1 will test the hypothesis that the differentiation of hES-derived pancreatic progenitors on 3D microporous scaffolds can increase the efficiency for forming immature ?-cells in vitro. Scaffolds will be created with controlled structures. Aim 2 focuses on facilitating the transition from immature ?-cells to mature ?-cells, a step that can architecture and modified with ECM proteins to facilitate organization and differentiation of cells into islet like only be achieved through transplantation and the in vivo environment, at a clinically translatable site. Importantly, the scaffold/cell constructs can be directly transplanted, which is anticipated to enhance engraftment and function by avoiding the disruption of cell-matrix and cell-cell contacts that occurs during the manipulation involved with preparing 2D cultured cells for transplant. The scaffolds will be augmented prior to transplantation to deliver VEGF and thereby recruit vasculature that can provide nutrients and distribute insulin, as well provide endothelial cells interactions that are critical for endocrine differentiation in vivo. Traditional assays involving PCR, IHC, and flow cytometry will be employed to characterize the tissue properties. However, we apply a transcription factor (TF) reporter system to quantify activity throughout differentiation, and thus provide unprecedented insight to the developmental processes as a function of the biomaterial design. Furthermore, we will employ human Pro-insulin promoter-C-peptide- Super Folded Green Florescent Protein (hPro-CpepSfGFP) to assess the insulin storage and secretion by individual cells in vitro and in vivo. for cell differentiation to mature ?-cells, with the potential to reverse hyperglycemia. Successful completion of the studies would identify scaffold designs to enhance the consistency and efficiency