(J-373) Craniofacial muscle stem cells for regenerative medicine applications

Introduction:: Skeletal muscle possesses a robust ability to regenerate following injury due to the presence of a dedicated stem cell called muscle satellite cell (MuSC). However, with aging and muscular dystrophies, MuSCs lose their myogenic capacity, which contributes to muscle wasting. Interestingly, previous research has shown that certain craniofacial muscles, such as extraocular muscles, are resistant to aging, Duchenne muscular dystrophy, and Amyotrophic Lateral Sclerosis. Furthermore, recent evidence suggests that, due to the heterogeneity of the muscle stem cell niche, craniofacial muscle stem cells have distinct myogenesis and remodeling characteristics that contribute to a more dynamic MuSC microenvironment. Although there is some reports on cellular and tissue-related phenotypes, the exact mechanisms of MuSC niche regulation and the therapeutic potential of craniofacial MuSC have not been explored. We hypothesized that craniofacial MuSCs exhibit enhanced regenerative capacity due to their high concentration of mitochondria, resulting in more efficient myogenesis following injury.

Materials and Methods:: To characterize myogenic functions of craniofacial MuSC, proliferation and differentiation were assessed with myoblasts. For mitochondrial function, myoblasts were isolated and underwent Seahorse XF flux analyses to assess mitochondrial bioenergetics. MuSCs from craniofacial and hindlimb muscles of C57BL6 mice were stained with mitoBrilliant and compared to assess differences in mitochondrial morphologies using confocal microscopy. Whole tissue of neuron-specific yellow fluorescence expressing Thy1-YFP mice were fixed and stained for immunofluorescence imaging.

Results, Conclusions, and Discussions:: The craniofacial muscle, masseter displayed a higher number of neuromuscular junctions, as evidenced by a higher number of a-bungarotoxin staining compared to TA tissue. Aged tibialis anterior muscle shows greater fragmentation of neuromuscular junctions in comparison to the masseter muscle. Differences in mitochondrial volume were also observed in the masseter muscle. The oxygen consumption rate of masseter mitochondria was lower than that of limb mitochondria. Masseter muscle myoblasts exhibit a general increase in proliferation followed by delayed differentiation compared to limb myoblasts when cultured in the same conditions. Overall, these data demonstrate that craniofacial MuSCs possess a slowed aging behavior although the mitochondrial function seems less efficient than hindlimb muscle.