Abstract Duchenne Muscular Dystrophy (DMD) is caused by mutations in the gene coding for dystrophin, which functions to maintain muscle fiber structure and function in the whole body by preventing it from being damaged during muscle contraction. Presently, there is no definitive treatment for DMD patients, and current therapies focus on prolonging survival and improving quality of life. Definitive treatment will require that functional dystrophin protein is restored in all affected muscle groups. Possible approaches to restoring dystrophin expression in muscle fibers include cell therapy.We hope that transplantation of a particular type of cell capable of regenerating muscle will help us to develop new therapeutic approaches to this disease. Muscle stem cells, termed satellite cells, isolated from healthy donors or patients should be able to provide dystrophin and repair muscle damage in DMD patients. For efficient DMD therapy, satellite cells which maintain the ability to self-renew are also necessary. However, satellite cells are rare (2-7% of all muscle nuclei) and often difficult to isolate. In addition, efficient myogenic differentiation and the scale-up of myogenic differentiation remain elusive and must be developed further in order to generate effective cell-based therapy for chronic muscle diseases. Patient-derived induced pluripotent stem cells (iPSCs) are the ideal cell source to obtain an unlimited number of myogenic cells that escape immune rejection after engraftment. However, the failure of systemic delivery of the injected cells has hindered practical application in patients. Our long-term goals are to develop an effective supply of satellite cells for cell therapy, to find an efficient systemic delivery method that can treat all affected muscles, and to enable the injected cells to self-renew and reach an effective mass. The specific aims of this application: 1. To determine possible interactions between CD24-expressing satellite cells and capillary endothelial cells via real-time imaging and 3- D imaging after our recently established whole muscle tissue clearing protocol. 2. To develop systemic delivery methods for satellite cells and hiPSC-derived myogenic progenitor cells in combination with genetic modification, including transduction of an extravasation factor, CD24, and Mannitol, an agent for enhancing vascular permeabilization. 3. To develop systemic delivery methods for satellite cells via Extracellular Vesicle (EV)- mediated CD24 transfer. In combination with gene and protein transduction, this concerted approach will help us to make satellite cells that can be systemically transplanted into patients for a definitive cure of DMD. The proposed specific aims will test whether systemically-injected satellite cells can be delivered to a target injured muscle and contribute to muscle regeneration as well as to the satellite cell pool. The anticipated outcome of the proposed specific aims will provide valuable insights for sa...