PROJECT SUMMARY The increasing global prevalence of obesity and its association with diabetes motivate a need for mechanistic understanding of pathogenesis. Obesity is a complex, multifactorial disease with contributions from the control circuitry of appetite and energy expenditure in the brain and critical endocrine and metabolic signals from peripheral organs including fat tissue. At the cellular level, dramatic changes in fat cell structure and function are defining features of obesity, and link obesity to insulin resistance through mediators including leptin, adiponectin, and proinflammatory lipids and cytokines. While appetite reduction has appropriately received a great deal of attention in obesity research resulting in the recent approval of multiple GLP1 drugs for obesity, mechanistic understanding of the contributions of fat tissue and therapeutic progress toward increasing energy expenditure has had limited success. A major reason for this lack of therapeutic progress is that the pharmaceutical industry lacks scalable drug discovery tools for testing drugs or determining mechanisms of action in mature human fat cells. The overarching goal of the current R43 Phase I application is to demonstrate how MelliCell’s proprietary M3 platform can generate normal and obese-like fat cells that closely mimic ex vivo mature fat cells relative to immature fat cells derived from the conventional two-dimensional (2D) method. To test this, we collaborate with Brigham and Women’s Hospital and propose two specific milestones. In Milestone 1, we will compare the differentiation efficiency, lipid droplet size, insulin sensitivity, cytokine and hormone secretion in ex vivo vs. M3 and 2D vs. M3 fat cells, obtained from the same donor. The results from this aim will quantitatively evaluate the robustness of M3 technology by displaying phenotypic features and functional readouts like those exhibited by mature ex vivo fat cells. In Milestone 2, considering the variety of fat-cell derived biomarkers for obesity, we will use state-of-the-art multiomics to demonstrate the ability of M3 technology to differentiate donors with versus without obesity. Parameters for testing will include multi-feature imaging, mRNA/protein quantification, endocrine function, and lipolysis and glucose uptake assays. Common disease associated genetic variants known to be highly prevalent (for example, CAV1 single nucleotide variants or SNVs) will also be analyzed to further classify phenotypes in M3 adipocytes. Completion of this Phase I study will establish the generation of a novel fat cell engineering technology with direct comparison to in vivo tissue for modeling human pathology. Progression to Phase II will facilitate expanded testing of obesity-linked SNPs to capture donor specific responses to disease relevant stimuli, enabling the development of therapeutics with potential to reverse the metabolic dysfunction in fat cells in obesity that leads to insulin resistance and diabetes.