Abstract
Skeletal muscle is increasingly considered an endocrine organ secreting myokines and extracellular vesicles (exosomes and microvesicles), which can affect physiological changes with an impact on different pathological conditions, including regenerative processes, aging, and myopathies. Primary human myoblasts are an essential tool to study the muscle vesicle secretome. Since their differentiation in conditioned media does not induce any signs of cell death or cell stress, artefactual effects from those processes are unlikely. However, adult human primary myoblasts senesce in long-term tissue culture, so a major technical challenge is posed by the need to avoid artefactual effects resulting from pre-senescent changes. Since these cells should be studied within a strictly controlled pre-senescent division count (<21 divisions), and yields of myoblasts per muscle biopsy are low, it is difficult or impossible to amplify sufficiently large cell numbers (some 250 × 106 myoblasts) to obtain sufficient conditioned medium for the standard ultracentrifugation approach to exosome isolation.Thus, an optimized strategy to extract and study secretory muscle vesicles is needed. In this study, conditions are optimized for the in vitro cultivation of human myoblasts, and the quality and yield of exosomes extracted using an ultracentrifugation protocol are compared with a modified polymer-based precipitation strategy combined with extra washing steps. Both vesicle extraction methods successfully enriched exosomes, as vesicles were positive for CD63, CD82, CD81, floated at identical density (1.15-1.27 g.ml−1), and exhibited similar size and cup-shape using electron microscopy and NanoSight tracking. However, the modified polymer-based precipitation was a more efficient strategy to extract exosomes, allowing their extraction in sufficient quantities to explore their content or to isolate a specific subpopulation, while requiring >30 times fewer differentiated myoblasts than what is required for the ultracentrifugation method. In addition, exosomes could still be integrated into recipient cells such as human myotubes or iPSC-derived motor neurons.Modified polymer-based precipitation combined with extra washing steps optimizes exosome yield from a lower number of differentiated myoblasts and less conditioned medium, avoiding senescence and allowing the execution of multiple experiments without exhausting the proliferative capacity of the myoblasts.
Highlights
In addition to its classical role in locomotion, skeletal muscle is increasingly recognized to have a role in signaling via its secretory functions
The maximum number of divisions is not age-dependent, which is consistent with our previous study showing that the myoblasts extracted from subjects have the same proliferative capacity as myoblasts extracted from young adults [41] (Fig. 1b)
In order to avoid potential artifacts arising from cells that are nearing, or have reached senescence, we suggest that myoblasts under 21 divisions should be used to study the muscle secretome (Fig. 1), and we, sampled cells within this window for all subsequent experiments
Summary
In addition to its classical role in locomotion, skeletal muscle is increasingly recognized to have a role in signaling via its secretory functions. Interleukin-6 (IL-6) [1] and musculin [2] have been identified to originate and be secreted from skeletal muscle in vivo, and the secretomic profiles of muscle cells in vitro, such as C2C12 myotubes [3, 4], human myotubes [5], and rat muscle explants [6] include growth factors (e.g., follistatin-like protein 1, IGF2, TGF), cytokines, and inhibitors of collagenase (e.g., TIMP2). In addition to proteins exiting the cell by classical secretory pathways, muscle cells release proteinassociated vesicles [5] These extracellular vesicles (EVs) are widely studied in different physiological and pathological contexts, and are known to play a key role in tissue homeostasis [8], embryogenesis and development [9], cell survival [10], inflammatory and metabolic diseases [11, 12], cancer metastasis [13]. Since primary human myoblasts should be studied within a strictly controlled pre-senescent division count (
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