Limb girdle muscular dystrophy 2B (LGMD2B) is a late-onset progressive muscular dystrophy resulting from mutations in the dysferlin gene. Dysferlin is a membrane-associated protein, highly expressed in skeletal and cardiac muscle fibers where it orchestrates membrane repair in response to various injuries. Currently, there are no ongoing clinical trials or therapies to slow disease progression or cure LGMD2B. While useful for in vivo mechanistic studies, dysferlin-deficient (BLAJ) mice, a model of LGDM2B, exhibit a mild disease phenotype compared to humans, limiting mouse utility for translational studies. Developing a high-fidelity in vitro model of human LGMD2B muscle would complement mouse studies and allow patient-specific disease modeling and drug discovery. Thus, the overarching goal of this project is to engineer a novel 3D human skeletal muscle tissue model (“myobundle”) that replicates the main structural, functional, and metabolic features of LGMD2B. Specifically, we will utilize human iPSC lines from three healthy and three LGMD2B donors to engineer LGMD2B myobundles that exhibit reproducible deficits in muscle contractile function, calcium homeostasis, and lipid handling, while showing drug responses consistent with studies in BLAJ mice and LGMD2B patients. Importantly, a defining feature of LGMD2B muscle is the ectopic fat formation suggested to occur due to adipogenic differentiation of muscle interstitial cells (MICs). We will thus develop a novel tissue-engineered model of intramuscular adipose tissue (IMAT) accumulation in LGMD2B muscle by co-culturing MICs isolated from LGMD2B human muscle biopsies and iPSC-derived muscle progenitor cells. In this novel co-culture system, we will identify pro-adipogenic factors secreted from LGMD2B muscle and study their ability to induce ectopic fat formation. Since immune cell infiltration and biased macrophage polarization are additional defining features of LGMD2B muscle, we will engineer co- and tri-cultured muscle-macrophage myobundles to further characterize roles of heterocellular interactions and inflammatory milieu in injury response and fat accumulation in LGMD2B. Finally, our preliminary studies suggest that the cholesterol metabolism in LGMD2B muscle is impaired and contributes to the disease, which we will further study pharmacologically, biochemically, and histologically in LGMD2B myobundles and BLAJ mice. Overall, we expect that the novel tissue-engineered model of human LGMD2B muscle developed in this project will enable new mechanistic and pharmacological studies, eventually leading to first clinical trials for LGMD2B.