Abstract
Skeletal muscle stem cells, satellite cells, are normally quiescent but become activated upon muscle injury. Recruitment of resident satellite cells may be a useful strategy for treatment of muscle disorders, but little is known about gene expression in quiescent human satellite cells or the mechanisms involved in their early activation. We have developed a method to induce quiescence in purified primary human myoblasts isolated from healthy individuals. Analysis of the resting state showed absence of BrdU incorporation and lack of KI67 expression, as well as the extended kinetics during synchronous reactivation into the cell cycle, confirming arrest in the G0 phase. Reactivation studies showed that the majority (>95%) of the G0 arrested cells were able to re-enter the cell cycle, confirming reversibility of arrest. Furthermore, a panel of important myogenic factors showed expression patterns similar to those reported for mouse satellite cells in G0, reactivated and differentiated cultures, supporting the applicability of the human model. In addition, gene expression profiling showed that a large number of genes (4598) were differentially expressed in cells activated from G0 compared to long term exponentially proliferating cultures normally used for in vitro studies. Human myoblasts cultured through many passages inevitably consist of a mixture of proliferating and non-proliferating cells, while cells activated from G0 are in a synchronously proliferating phase, and therefore may be a better model for in vivo proliferating satellite cells. Furthermore, the temporal propagation of proliferation in these synchronized cultures resembles the pattern seen in vivo during regeneration. We therefore present this culture model as a useful and novel condition for molecular analysis of quiescence and reactivation of human myoblasts.
Highlights
IntroductionIn bone marrow and epithelia, the stem cell population is continuously active and maintains the homeostasis of the tissues [1,2,3,4], while in skeletal muscle, the tissue specific stem cells (satellite cells) are normally quiescent but can be recruited after an injury
Tissue specific stem cells are present in many adult tissues
We have previously characterized the cell population isolated according to the described procedure and found that 95% of the cells were positive for the human myoblast marker NCAM [37], confirming that the isolation and culturing methods result in a highly purified satellite cell population
Summary
In bone marrow and epithelia, the stem cell population is continuously active and maintains the homeostasis of the tissues [1,2,3,4], while in skeletal muscle, the tissue specific stem cells (satellite cells) are normally quiescent but can be recruited after an injury. Most of the resulting myoblasts continue into differentiation, fuse and form new muscle fibers, but a small minority returns to G0 and restore the resting SC compartment [5,6,7,8]. Even the more common form of muscle weakness seen in sarcopenia, inactivity and prolonged bed rest due to surgery or illness, especially in elderly, might be treatment targets as these conditions involves muscular atrophy resulting in loss of muscle mass and strength [11,12,13,14]
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