## 1 Introduction
A study demonstrated changes in cell proliferation, migration, differentiation capacities, and cell death under hypoxia [27; 28; 29]. Meanwhile, short-term hypoxia has been shown to have a positive effect on wound healing, including the activation of MDSCs, angiogenesis, and phagocytic infiltration [30]. However, long-term hypoxic conditions can result in muscle wasting, as seen in chronic obstructive pulmonary disease [31; 32]. Under chronic hypoxia conditions, the oxidative capacity of muscle fibers is reduced, leading to muscle atrophy [33; 34] due to promoted catabolic activities and impaired regeneration [35]. Likewise, cultivating neural crest cells under hypoxic condition resulted in increased cell proliferation and differentiation, as well as reduced cell death [36]. A study found that a 3-6% O\({}_{2}\) concentration not only improved the proliferation and myogenic differentiation of MDSCs, but also it reduced the apoptotic activities and inhibited the accumulation of reactive oxygen species [28; 37; 38]. Previous studies have reported the effect of hypoxia on cell division. One study found that hypoxia condition promotes cell cycle progression and inactivates cell cycle inhibitors [37]. However, hypoxia can also inhibit differentiation, as evidenced by the downregulation of MRFs and the reduction in myotube formation in muscle cells [39; 40]. In addition, hypoxia has been found to inhibit the final stage of myogenic differentiation by reducing MyoD expression [39; 41]. In the same context, studies have found that hypoxia inhibits protein synthesis by interfering with Akt/mTOR signaling [41; 42; 43]. Conversely, hypoxia stimulates the quiescence and self-renewal of primary myoblasts by activating the Notch signaling pathway [44; 45]. Studies have found that primary satellite cells and myoblasts have an increased multipotency under hypoxia, leading to adipogenic and osteogenic fate [40; 46]. Hypoxia-inducible factor-1 alpha (HIF-1\(\alpha\)) is a well-established master regulator of oxygen homeostasis that activates angiogenesis by upregulating vascular endothelial growth factor (VEGF) expression [29; 47]. This improves the regeneration of ischemic lesions [48]. Neuronal nitric oxide synthase (nNOS/NOS1) is mainly expressed in skeletal muscle and appears to play an important role under hypoxia [49; 50]. To determine the optimal microenvironment for myogenic differentiation of MDSCs, the present study evaluates the effects of various models of mechanical fluid shear stress, including cyclic and periodic stress, as well as modulated oxygen concentration, on the physiological parameters and differentiation capacity of muscle stem cells. To achieve our goal, we evaluated cell viability, cell number, and differentiation parameters using standard biological assays, immunohistochemical analysis, morphometric measurement, and the quantification of the relative gene expression. The results provide evidence of enhanced myogenic differentiation capacity under mechanical FSS, suggesting that optimized mechanical FSS stress could improve cell performance and their regenerative potential for clinical applications. Although enhanced angiogenesis and NO production occur under hypoxia, the data suggest that the myogenic differentiation of MDSCs is impaired under hypoxia. This highlights the importance of suitable oxygen concentrations for muscle differentiation.