Muscle satellite cell proliferation and association: new insights from myofiber time-lapse imaging

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.

Eph/ephrin interactions modulate muscle satellite cell motility and patterning

“…However, a role for signaling between activated satellite cells and damaged or undamaged myofibers is suggested by, not only the expression of individual Ephs and ephrins on each cell type, but our in vitro motility assays; future work will focus on demonstrating an in vivo role as well. The expression not only of Eph receptors but also of ephrin ligands by activated satellite cells provides the possibility for Eph-mediated signaling between activated satellite cells also: our recent observations that cell-cell affinity between daughter cells is heterogeneous based on the plane of cell division with respect to the myofiber (Siegel et al, 2011) might be the result of such signaling. Finally, the continued expression of a subset of Ephs and ephrins after myogenic differentiation, as well as the more ordered…”

Isolation and Culture of Individual Myofibers and their Satellite Cells from Adult Skeletal Muscle

“…In 1995, Rosenblattmodified the Bischoff protocol such that myofibers are singly picked and handled separately after collagenase digestion instead of being isolated by gravity sedimentation 2,3 . The Rosenblatt or Bischoff protocol has since been adapted to different muscles, age or conditions [3][4][5][6] . The single myofiber isolation technique is an indispensable tool due its unique advantages.…”

G-CSF supports long-term muscle regeneration in mouse models of muscular dystrophy

“…We then examined the direct role of G-CSF on SCs from wild-type mice. At 72 h, the subpopulation of activated SCs had lost MYOD expression in vivo and on cultured myofibres [21][22][23] . Surprisingly, G-CSF significantly increased the population of SCs throughout the differentiation stages, including self-renewal SCs, as shown by the PAX7 þ /MYOD À cells at 72 h in wild-type mice; in addition, csf3r À / À mice showed significantly decreased numbers of SCs throughout the differentiation stages ( Fig.…”

“…3a,b). At 24 h, most PAX7 þ cells express MYOD, are activated and thereafter proliferate and commit to differentiation [21][22][23] . In the csf3r À / À mice, the number of MYOD þ /PAX7 þ cells was significantly decreased in PAX7 þ cells activated for 24 h (Fig.…”