Abstract
Satellite cells (SC) are the stem cells of skeletal muscles. They are quiescent in adult animals but resume proliferation to allow muscle hypertrophy or regeneration after injury. The mechanisms balancing quiescence, self-renewal, and differentiation of SC are difficult to analyze in vivo owing to their complexity and in vitro because the staminal character of SC is lost when they are removed from the niche and is not adequately reproduced in the culture models currently available. To overcome these difficulties, we set up a culture model of the myogenic C2C12 cell line in suspension. When C2C12 cells are cultured in suspension, they enter a state of quiescence and form three-dimensional aggregates (myospheres) that produce the extracellular matrix and express markers of quiescent SC. In the initial phase of culture, a portion of the cells fuses in syncytia and abandons the myospheres. The remaining cells are mononucleated and quiescent but resume proliferation and differentiation when plated in a monolayer. The notch pathway controls the quiescent state of the cells as shown by the fact that its inhibition leads to the resumption of differentiation. Within this context, notch3 appears to play a central role in the activity of this pathway since the expression of notch1 declines soon after aggregation. In summary, the culture model of C2C12 in suspension may be used to study the cellular interactions of muscle stem cells and the pathways controlling SC quiescence entrance and maintenance.
Highlights
Satellite cells (SC) lie between the basal lamina and the sarcolemma of skeletal muscle fibers
Mouse C2C12 myoblasts seeded in nonadhesive conditions form aggregates that appear within 3-4 h
Our work complements that published in other studies that used myosphere cultures maintained within a chemically defined media reach of growth factors
Summary
Satellite cells (SC) lie between the basal lamina and the sarcolemma of skeletal muscle fibers. In adult muscle, they are quiescent until physical exercise or muscle damage induces their activation. A central question in adult stem cell biology regards the elucidation of the molecular mechanisms that preserve the regenerative potential of the tissues by maintaining a population of reversibly quiescent stem cells. Several findings suggest that reversible quiescence is not a state of cell inactivity but is the result of specific molecular programs [2, 3]. It is not possible to maintain the quiescent state of SC in vitro because any isolation procedure triggers their activation and converts them into cycling myoblasts that undergo differentiation. SC lose their staminality because their return to quiescence is precluded in the monolayer culture by the lack of an appropriate niche
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