Conditions under which skeletal myoblasts are cultured in vitro are critical to growth and differentiation of these cells into mature skeletal myofibers. We examined several culture conditions that promoted human skeletal myoblast (HSkM) culture and examined the effect of microRNAs and mechanical stimulation on differentiation. Culture conditions for HSkM are different from those that enable rapid C2C12 myoblast differentiation. Culture on a growth factor-reduced Matrigel (GFR-MG)-coated surface in 2% equine serum-supplemented differentiation medium to promote HSkM differentiation under static conditions was compared with culture conditions used for C2C12 cell differentiation. Such conditions led to a Ͼ20-fold increase in myogenic miR-1, miR-133a, and miR-206 expression, a Ͼ2-fold increase in myogenic transcription factor Mef-2C expression, and an increase in sarcomeric ␣-actinin protein. Imposing Ϯ10% cyclic stretch at 0.5 Hz for 1 h followed by 5 h of rest over 2 wk produced a Ͼ20% increase in miR-1, miR-133a, and miR-206 expression in 8% equine serum and a Ͼ35% decrease in 2% equine serum relative to static conditions. HSkM differentiation was accelerated in vitro by inhibition of proliferationpromoting miR-133a: immunofluorescence for sarcomeric ␣-actinin exhibited accelerated development of striations compared with the corresponding negative control, and Western blotting showed 30% more ␣-actinin at day 6 postdifferentiation. This study showed that 100 g/ml GFR-MG coating and 2% equine serum-supplemented differentiation medium enhanced HSkM differentiation and myogenic miR expression and that addition of antisense miR-133a alone can accelerate primary human skeletal muscle differentiation in vitro. skeletal muscle; microRNA; myogenesis; differentiation; tissue engineering THE DYNAMICS OF PRIMARY HUMAN skeletal myoblast (HSkM) growth and differentiation into mature myofibers are critical to their use for applications in regenerative medicine, such as repair of severe muscle injuries, congenital defects, and muscular dystrophy. Most studies have focused on rodent myoblasts, and the bulk of the research has been carried out with the widely used immortalized murine C2C12 myoblast line, which is used for ease of culture, differentiation potential, and accessibility (7). Culture conditions for these cells have been optimized to ensure rapid growth and effective differentiation in vitro. These cells do not require protein-coated substrates and differentiate well in a range of equine serum-supplemented differentiation media (DM) (14,24,28,29,32). While C2C12 cells have provided invaluable information about key mechanistic steps in differentiation (31), extensive in vitro cultivation may lead to deviations from normal biological processes that are important for differentiation of myoblasts in a normal in vivo environment. Less is known about the dynamics of key differentiation factors in primary human myoblasts and how culture conditions affect the changes in these factors (3).In vitro, rodent myoblast differentiati...