Skeletal muscle satellite cells are important for muscle repairing and muscle mass growth. For a successful muscle regenerative process, satellite cells have to sequentially undergoing different stages of myogenic process, i.e. proliferative state and differentiation state. To support this process, the presence of different circulating factors, such as immune cells, cytokines, and hormones, at the appropriate time course is critical. Among these factors, hormones, such as testosterone, cortisol, and IGF-1, have shown to play an important role in satellite cell proliferation and differentiation. Studies investigated the effect of testosterone on satellite cell using a supraphysiological dose in human or in cell culture demonstrated that testosterone is critical in satellite cell myogenic process. Due to the anabolic effect of testosterone on muscle, studies had been focused on the physiological means to increase the circulating testosterone concentration in the body to maximize the muscle mass growth from resistance exercise. The acute and transient increase in testosterone has shown to be beneficial to muscle mass growth and strength gain; however, this change in physiological testosterone concentration on satellite cell myogenesis is not known. Therefore the purpose of this dissertation is to first determine the effect of acute change in exercise-induced hormones on satellite cell myogenic state, then to determine if testosterone promotes satellite cell proliferation.

Muscle aging is associated with a decrease in the number of satellite cells and their progeny, muscle progenitor cells (MPCs) that are available for muscle repair and regeneration. However, there is an increase in non-immuno-hematopoietic cells (CD45 negative) in regenerating muscle from aged mice characterized by high stem cell antigen -1(Sca-1) expression. In aged regenerating muscle, 14.2% of cells are CD45neg Sca-1pos while 7.2% of cells are CD45neg Sca-1pos in young adult muscle. In vitro, CD45neg Sca-1pos cells over express genes associated with fibrosis, potentially controlled by Wnt2. These cells are proliferative, non-myogenic and non-adipogenic, and arise in clonally-derived MPCs cultures from aged mice. Both in vitro and in vivo studies suggest that CD45neg Sca-1pos cells from aged muscle are more susceptible to apoptosis than their MPCs, which may contribute to depletion of the satellite cell pool. Therefore, with age, a subset of MPCs takes on an altered phenotype, which is marked by high Sca-1 expression. This altered phenotype prevents these cells from participating in muscle regeneration or replenishing the satellite cell pool, and instead may contribute to fibrosis in aged muscle.

Duchenne Muscular Dystrophy (DMD) is a disorder caused by loss-of-function mutations in dystrophin, a critical protein that maintains muscle fiber integrity. Our lab discovered that dystrophin-deficient skeletal muscle stem cells, also known as satellite cells, cannot generate enough myogenic progenitors for proper muscle regeneration. Previously, we demonstrated that WNT7A, a protein expressed during muscle regeneration, stimulates symmetric division of satellite cells, and gives rise to two daughter satellite cells. Conversely, epidermal growth factor (EGF) induces asymmetric division, which generates one daughter satellite cell and one committed precursor cell. We aimed to investigate these satellite cell division mechanisms following WNT7A or EGF treatment in a human model using healthy and DMD-patient derived hiPSCs differentiated into the myogenic lineage. The presence of satellite-like cells was confirmed in both lines by their characteristic expression of PAX7 and other myogenic markers. Intriguingly, DMD-patient hiPSCs precociously differentiated compared to healthy control human induced pluripotent stem cells (hiPSCs). More notably, WNT7A treatment had a potent effect on the DMD differentiated cells. High content analysis revealed an expansion of the satellite-like cell pool as observed by a higher number of PAX7+ cells within the total population and gene expression analysis demonstrated a significant increase in global PAX7 expression. In contrast, EGF treatment reduced the number of PAX7+ cells and increased the proportion of MYOG+ cells within the myogenic population, indicating an increase in myogenic progenitors. Taken together, WNT7A and EGF can alter the myogenic differentiation program of healthy and DMD-patient derived hiPSCs by modulating the satellite-like cell division dynamics.