PROJECT SUMMARY / ABSTRACTDiabetes affects nearly 10% of the adult population (30 million) and these numbers are expected to double bythe year 2050. The pathophysiology of diabetes profoundly impairs all tissue reparative processes leading tochronic non-healing wounds in affected patients. Diabetic foot ulcers affect between 15-30% of all diabeticindividuals and represent the leading cause of lower limb amputations in the United States. Conventionalmethods to treat diabetes such as with insulin or oral hypoglycemic agents can control the disease but do notprevent diabetic complications as demonstrated by continued progressive organ dysfunction even decades aftermedical optimization. This highlights a clear need for new therapeutic approaches. Over the past 15 years ofNIH funding our laboratory has made important contributions to our understanding of the critical molecular andcellular pathways in normal and diabetic tissue repair. We have identified hyperglycemia-related impairments inboth the local microenvironment and progenitor cell homing and cytokine production that contribute to thepathogenesis of diabetic complications. We have demonstrated that diabetes results in depletion of critical cellsubpopulations resulting in decreased neovascularization and impaired tissue healing. To understand theeffects 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 ourfundamental hypothesis that diabetes alters the cellular ecology of heterogeneous cell populations involvedin 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 thebehavior of cell subpopulations in diabetic complications including wound healing. We will extend this worktherapeutically by using cell-based approaches to normalize these defects to treat and prevent diabeticcomplications. To begin we will employ a novel multiplex approach for high-throughput single cell sequencingto definitively characterize the behavior of resident tissue and progenitor cell subpopulations in human diabeticand non-diabetic wounds (Specific Aim 1). We will then confirm these human observations in animal models anddefine these changes with spatial resolution by integrating single cell sequencing with next-generation spatialtranscriptomic and proteomic technologies and precisely delineate where in the three-dimensional woundenvironment these differences exist (Specific Aim 2). Finally we will use this information to optimize the systemicdelivery of cell-based therapeutics in order to prevent or reverse diabetes-induced defects in relevant cellpopulations and thereby correct diabetic complications (Specific Aim 3). Taken together this novel approach foridentifying spatially characterizing and correcting subpopulation deficits in diabetic complications will providenew insights into diabetic pathophysiology and inform novel strategies to prevent and treat these complications.