Abstract
BackgroundDuring the process of muscle regeneration, activated stem cells termed satellite cells proliferate, and then differentiate to form new myofibers that restore the injured area. Yet not all satellite cells contribute to muscle repair. Some continue to proliferate, others die, and others become quiescent and are available for regeneration following subsequent injury. The mechanisms that regulate the adoption of different cell fates in a muscle cell precursor population remain unclear.MethodsWe have used live cell imaging and lineage tracing to study cell fate in the C2 myoblast line.ResultsAnalyzing the behavior of individual myoblasts revealed marked variability in both cell cycle duration and viability, but similarities between cells derived from the same parental lineage. As a consequence, lineage sizes and outcomes differed dramatically, and individual lineages made uneven contributions toward the terminally differentiated population. Thus, the cohort of myoblasts undergoing differentiation at the end of an experiment differed dramatically from the lineages present at the beginning. Treatment with IGF-I increased myoblast number by maintaining viability and by stimulating a fraction of cells to complete one additional cell cycle in differentiation medium, and as a consequence reduced the variability of the terminal population compared with controls.ConclusionOur results reveal that heterogeneity of responses to external cues is an intrinsic property of cultured myoblasts that may be explained in part by parental lineage, and demonstrate the power of live cell imaging for understanding how muscle differentiation is regulated.
Highlights
During the process of muscle regeneration, activated stem cells termed satellite cells proliferate, and differentiate to form new myofibers that restore the injured area
We plated a mixture of unmarked myoblasts with myoblasts expressing enhanced green fluorescent protein (EGFP) under control of the constitutively active EF-1α promoter, and tracked EGFP-positive cells every 15 min using an automated cell counting algorithm (Figure 2A)
Treatment with IGF-I increased myoblast number by maintaining viability and by stimulating a fraction of cells to complete one additional cell cycle in differentiation medium (DM), and as a consequence reduced the variability of the terminal population compared with controls
Summary
During the process of muscle regeneration, activated stem cells termed satellite cells proliferate, and differentiate to form new myofibers that restore the injured area. Others die, and others become quiescent and are available for regeneration following subsequent injury. Muscle regeneration following injury occurs through stimulation of muscle stem cells, termed satellite cells [1]. A similar series of steps occurs during muscle differentiation in culture. In both situations not all cells exposed to the same milieu have the same outcome. Because proliferation and death can occur simultaneously within a population, and can skew the fraction of cells that differentiate, it has been challenging to determine why some cells adopt one fate rather than another
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