Abstract
BackgroundAs the resident stem cells of skeletal muscle, satellite cells are activated by extracellular cues associated with local damage. Once activated, satellite cells will re-enter the cell cycle to proliferate and supply a population of myoblasts, which will repair or replace damaged myofibers by differentiating and fusing either with an existing myofiber or with each other. There is also evidence that the orientation of cell division with respect to the myofiber may indicate or convey asymmetry in the two daughter cells. Our recent studies with time-lapse imaging of myofiber-associated satellite cells in vitro have yielded new data on the timing and orientation of satellite cell divisions, and revealed persistent differences in the behavior of daughter cells from planar versus vertical divisions.ResultsWe analyzed 244 individual fiber-associated satellite cells in time-lapse video from 24 to 48 hours after myofiber harvest. We found that initial cell division in fiber culture is not synchronous, although presumably all cells were activated by the initial trauma of harvest; that cell cycling time is significantly shorter than previously thought (as short as 4.8 hours, averaging 10 hours between the first and second divisions and eight hours between the second and third); and that timing of subsequent divisions is not strongly correlated with timing of the initial division. Approximately 65% of first and 80% of second cell divisions occur parallel to the axis of the myofiber, whereas the remainder occur outside the plane of the fiber surface (vertical division). We previously demonstrated that daughter cells frequently remain associated with each other after division or reassociate after a brief separation, and that unrelated cells may also associate for significant periods of time. We show in this paper that daughter cells resulting from a vertical division remain associated with one another several times longer than do daughters from a horizontal division. However, the total average time of association between sister cells is not significantly different from the total average time of association between unrelated cells.ConclusionsThese longitudinal characterizations of satellite cell behavior shortly after activation provide new insights into cell proliferation and association as a function of relatedness, and indicate significant and consistent heterogeneity within the population based on these metrics.
Highlights
As the resident stem cells of skeletal muscle, satellite cells are activated by extracellular cues associated with local damage
Satellite cells are the resident stem cells of skeletal muscle; they are considered to be self-renewing, and serve to generate a population of differentiation-competent myoblasts that will participate as needed in muscle growth, repair and regeneration [1,2]
We have recently described a method of following fiber-associated satellite cells longitudinally over extended periods of time in vitro, using time-lapse microscopy [11]. This provides an advantage in characterizing satellite cell activity because we can directly visualize and follow individual satellite cells through multiple phases of activity, including exit from the basal lamina, proliferation, and movement along the myofiber
Summary
As the resident stem cells of skeletal muscle, satellite cells are activated by extracellular cues associated with local damage. Our previous work focused primarily on cell motility and the cellular and environmental factors required for efficient movement on the myofiber, a number of other activities were noted, including a much higher than expected degree of asynchrony in the timing of satellite cell divisions, and a surprising tendency for cells to both remain as cell doublets for extended periods of time after cell division and to associate as apparent doublets with unrelated cells These behaviors would have a significant effect on interpretation and analysis of fixed and stained cell preparations, so we set out to tabulate and quantify these aspects of satellite cell activity after activation
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