Project Summary Skeletal muscle is one of the most regenerative tissues in the human body. Skeletal muscle progenitor cells (SMPCs) contribute to developmental myogenesis, and skeletal muscle stem cells (satellite cells, SCs) contribute to postnatal muscle homeostasis and regeneration. In Duchenne Muscular Dystrophy (DMD), a prevalent but fatal X-linked muscle wasting disease that affects 1:5000 live male births, a loss-of-function mutation in the DMD gene results in the absence of functional dystrophin protein to stabilize muscle fibers. This leads to continuous damage from repeated muscle degeneration and regeneration and compromises the regenerative ability of SCs. There is currently no cure for DMD. Differentiating human pluripotent stem cells (hPSCs) into SCs is a valuable resource for developing cell replacement therapies for DMD. However, current hPSC directed myogenic differentiation protocols result in embryonic/fetal-like SMPCs that cannot be maintained over multiple passages (i.e. self-renew) in culture. SMPCs also do not engraft as efficiently as postnatal SCs, and it is not known how to mature SMPCs to SCs because it is unclear how they molecularly differ. Metabolism plays a key role in regulating cell state and function in stem cells across development. We performed single cell RNA sequencing analysis that demonstrated that expression of most genes in glycolysis, tricarboxylic cycle, and oxidative phosphorylation decrease across human myogenic development. However, how metabolic activity supports SMPCs or can be used to transition SMPCs to SCs in humans has not been investigated beyond the transcriptional level. By more closely evaluating the metabolic profiles of tissue-derived SCs and hPSC-derived myogenic subpopulations including hPSC-derived SMPCs (hPSC-SMPCs), specific metabolic enzymes and metabolites in the aforementioned pathways will be targeted as candidates to support hPSC-SMPC self-renewal or promote hPSC-SMPC maturation to SCs in culture. Tightly connected to metabolism is the epigenome which has a direct role in transitioning between cell states, particularly through modulating chromatin accessibility at cis-regulatory regions to mediate transcription factor (TF) binding and control gene expression. Evaluating differences in chromatin accessibility of TF binding motifs between the same myogenic populations identifies TFs that support and regulate SMPC and SC states. Several TF candidates have been identified as potential regulators of SMPC-to-SC maturation. Expression of TF candidates will be modulated to support hPSC-SMPCs or promote their maturation toward SC fate. This work will for the first time shed light on the metabolic and epigenetic roles in maintaining and transitioning between human muscle progenitor and stem cell states. These findings will enhance the regenerative potential of hPSC-derived muscle cells and enable the development of improved myogenic cell therapies for DMD. This study will be performed at ...