Adult skeletal muscle harbours a population of muscle stem cells (MuSCs) that are required for repair after tissue injury. In youth, MuSCs return to a reversible state of cell-cycle arrest termed 'quiescence' after injury resolution. Conversely, some MuSCs in aged muscle remain semi-activated, causing a premature response to injuries that results in incomplete repair and eventual stem cell depletion. Regulating this balance between MuSC quiescence and activation may hold the key to restoring tissue homeostasis with age, but is incompletely understood. To fill this gap, we developed a simple and tractable in vitro method, to rapidly inactivate MuSCs freshly isolated from young murine skeletal muscle, and return them to a quiescent-like state for at least 1-week, which we name mini-IDLE (Inactivation and Dormancy LEveraged in vitro). This was achieved by introducing MuSCs into a 3D bioartificial niche comprised of a thin sheet of mouse myotubes, which we demonstrate provides the minimal cues necessary to induce quiescence. With different starting numbers of MuSCs, the assay revealed cellular heterogeneity and population-level adaptations that converged on a common niche repopulation density; behaviours previously observed only in vivo. Quiescence-associated hallmarks included a Pax7+CalcR+DDX6+MyoD-c-FOS- signature, quiescent-like morphologies, and polarized niche markers. Leveraging high-content bioimaging pipelines, we demonstrate a relationship between morphology and cell fate signatures for possible real-time morphology-based screening. When using MuSCs from aged muscle, they displayed aberrant proliferative activities and delayed inactivation kinetics, among other quiescence-associated defects that we show are partially rescued by wortmannin treatment. Thus, the assay offers an unprecedented opportunity to systematically investigate long-standing queries in areas such as regulation of pool size and functional heterogeneity within the MuSC population, and to uncover quiescence regulators in youth and age.
Keywords: aging; in vitro assay; inactivation; mouse; muscle stem cell; niche; quiescence; regenerative medicine; skeletal muscle; stem cells.
When our muscles are injured, stem cells in the tissue are activated to start the repair process. However, when there is no damage, these cells tend to stay in a protective, dormant state known as quiescence. If quiescence is not maintained, the stem cells cannot properly repair when the muscle is damaged. This happens in old age, when a proportion of the cells remain semi-activated, and become depleted. However, researchers still do not fully understand how quiescence is regulated. This is partly because in order to study quiescence, live animals must be used, because muscle stem cells immediately come out of quiescence when they are removed from muscle tissue. To overcome this experimental limitation, Jacques et al. developed a new method to study muscle stem cells by transferring them from mice into three-dimensional engineered muscle tissue grown in the lab. This tissue is made by infiltrating the pores of teabag paper with muscle progenitor cells, which then fuse with one another to make a thin muscle that contains three layers of contractile muscle cells. Introducing muscle stem cells from young healthy animals into this engineered muscle tissue allowed them to return to a quiescent-like state and to remain in that state for at least a week. Cells from older animals could also be returned to dormancy if they were chemically treated after placing them in the engineered muscle tissue. The approach works in a miniaturized fashion, with each engineered tissue requiring less than one per cent of the muscle stem cells collected from each mouse. This allows 100 times as many experiments compared to the current methods using live animals. This system could help researchers to study the genetic and chemical influences on muscle stem cell quiescence. Further understanding in this area could lead to treatments that restore healing abilities in older muscle tissue.
© 2022, Jacques et al.