Preparation and Culture of Myogenic Precursor Cells/Primary Myoblasts from Skeletal Muscle of Adult and Aged Humans

Abstract

Skeletal muscle homeostasis depends on muscle growth (hypertrophy), atrophy and regeneration. During ageing and in several diseases, muscle wasting occurs. Loss of muscle mass and function is associated with muscle fiber type atrophy, fiber type switching, defective muscle regeneration associated with dysfunction of satellite cells, muscle stem cells, and other pathophysiological processes. These changes are associated with changes in intracellular as well as local and systemic niches. In addition to most commonly used rodent models of muscle ageing, there is a need to study muscle homeostasis and wasting using human models, which due to ethical implications, consist predominantly of in vitro cultures. Despite the wide use of human Myogenic Progenitor Cells (MPCs) and primary myoblasts in myogenesis, there is limited data on using human primary myoblast and myotube cultures to study molecular mechanisms regulating different aspects of age-associated muscle wasting, aiding in the validation of mechanisms of ageing proposed in rodent muscle. The use of human MPCs, primary myoblasts and myotubes isolated from adult and aged people, provides a physiologically relevant model of molecular mechanisms of processes associated with muscle growth, atrophy and regeneration. Here we describe in detail a robust, inexpensive, reproducible and efficient protocol for the isolation and maintenance of human MPCs and their progeny — myoblasts and myotubes from human muscle samples using enzymatic digestion. Furthermore, we have determined the passage number at which primary myoblasts from adult and aged people undergo senescence in an in vitro culture. Finally, we show the ability to transfect these myoblasts and the ability to characterize their proliferative and differentiation capacity and propose their suitability for performing functional studies of molecular mechanisms of myogenesis and muscle wasting in vitro.

Efficient and high yield isolation of myoblasts from skeletal muscle

“…S2B) (Ocalan et al, 1988). In addition, recent studies showed that laminin promotes proliferation of human myoblasts (Chowdhury et al, 2015; Soriano-Arroquia et al, 2017) and maintains their myogenic phenotype over long-term culture (Penton et al, 2016), suggesting that this isolation protocol may also be applicable to human skeletal muscle myoblasts. The high serum medium supplemented with CEE and bFGF facilitated the migration and expansion of myoblasts on matrigel.…”

Inflamma-miR-21 Negatively Regulates Myogenesis during Ageing

“…Primary myoblasts from adult (6 months old) and old (24 months old) mice were prepared from EDL muscles following single fibre isolation as previously described [12,39]. Briefly, EDL muscle from both legs was digested with 1.5 U/mL collagenase D, 2.4 U/mL dispase II and 2.5 mM CaCl 2 .…”

“…Digested muscle was filtered and spun to remove undigested tissue and plated on surfaces covered with 10 µg/mL laminin and incubated with DMEM media with 20% FBS, 10% horse serum, 1% l-glutamine and 1% penicillin/streptomycin at 37 • C and 5% CO 2 . Primary myoblasts were grown in DMEM media supplemented with 10% FBS, 1% l-glutamine and 1% penicillin/streptomycin [39].…”

“…Primary myoblasts were cultured as described previously [39]. Myogenic differentiation was induced by placing 90% confluent cells in DMEM supplemented with 2% horse serum and 1% penicillin/streptomycin (differentiation media; DM).…”

The root cause of Duchenne muscular dystrophy is the lack of dystrophin in smooth muscle of blood vessels rather than in skeletal muscle per se

“…At the neuromuscular synapse, the DAPC is also formed with utrophin, also a 427 kDa protein, instead of dystrophin. The phosphorylation of α-DB1 through NRG/ERBB signaling stabilized acetylcholine receptors (AChRs) at the neuromuscular synapse 1,2 . Duchenne and Becker muscular dystrophy (D/BMD) patients have mutation(s) in the dystrophin gene, resulting in the expression of a truncated dystrophin protein [3][4][5][6] . The main body of research on DMD argues for the lack of dystrophin in skeletal muscle as the cause for DMD.…”