PROJECT SUMMARY / ABSTRACT Diabetes affects nearly 10% of the adult population (30 million), and these numbers are expected to double by the year 2050. The pathophysiology of diabetes profoundly impairs all tissue reparative processes, leading to chronic non-healing wounds in affected patients. Diabetic foot ulcers affect between 15-30% of all diabetic individuals and represent the leading cause of lower limb amputations in the United States. Conventional methods to treat diabetes, such as with insulin or oral hypoglycemic agents, can control the disease but do not prevent diabetic complications, as demonstrated by continued progressive organ dysfunction even decades after medical optimization. This highlights a clear need for new therapeutic approaches. Over the past 15 years of NIH funding, our laboratory has made important contributions to our understanding of the critical molecular and cellular pathways in normal and diabetic tissue repair. We have identified hyperglycemia-related impairments in both the local microenvironment and progenitor cell homing and cytokine production that contribute to the pathogenesis of diabetic complications. We have demonstrated that diabetes results in depletion of critical cell subpopulations, resulting in decreased neovascularization and impaired tissue healing. To understand the effects of diabetes on cell population dynamics with greater precision, we have developed novel single cell “-omics” techniques to identify critical perturbations in cell subpopulations at the single cell level. It is our fundamental hypothesis that diabetes alters the “cellular ecology” of heterogeneous cell populations involved in tissue repair and that normalization of those cell subpopulations can treat or reverse diabetic complications. In this proposal, we will integrate emerging multimodal -omics technologies to definitively characterize the behavior of cell subpopulations in diabetic complications, including wound healing. We will extend this work therapeutically by using cell-based approaches to normalize these defects to treat and prevent diabetic complications. To begin, we will employ a novel multiplex approach for high-throughput single cell sequencing to definitively characterize the behavior of resident tissue and progenitor cell subpopulations in human diabetic and non-diabetic wounds (Specific Aim 1). We will then confirm these human observations in animal models and define these changes with spatial resolution by integrating single cell sequencing with next-generation spatial transcriptomic and proteomic technologies and precisely delineate where in the three-dimensional wound environment these differences exist (Specific Aim 2). Finally, we will use this information to optimize the systemic delivery of cell-based therapeutics in order to prevent or reverse diabetes-induced defects in relevant cell populations and thereby correct diabetic complications (Specific Aim 3). Taken together, this novel approach for identi...