Conditions that promote primary human skeletal myoblast culture and muscle differentiation in vitro.

TitleConditions that promote primary human skeletal myoblast culture and muscle differentiation in vitro.
Publication TypeJournal Article
Year of Publication2014
AuthorsCheng, CS, El-Abd, Y, Bui, K, Hyun, Y-E, Hughes, RH, Kraus, WE, and Truskey, GA
JournalAmerican journal of physiology. Cell physiology
Volume306
Issue4
Start PageC385
PaginationC385 - C395
Date Published02/2014
Abstract

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 >20-fold increase in myogenic miR-1, miR-133a, and miR-206 expression, a >2-fold increase in myogenic transcription factor Mef-2C expression, and an increase in sarcomeric α-actinin protein. Imposing ±10% cyclic stretch at 0.5 Hz for 1 h followed by 5 h of rest over 2 wk produced a >20% increase in miR-1, miR-133a, and miR-206 expression in 8% equine serum and a >35% decrease in 2% equine serum relative to static conditions. HSkM differentiation was accelerated in vitro by inhibition of proliferation-promoting miR-133a: immunofluorescence for sarcomeric α-actinin exhibited accelerated development of striations compared with the corresponding negative control, and Western blotting showed 30% more α-actinin at day 6 postdifferentiation. This study showed that 100 μg/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.

URLhttp://dx.doi.org/10.1152/ajpcell.00179.2013
DOI10.1152/ajpcell.00179.2013
Short TitleAmerican journal of physiology. Cell physiology