Adult skeletal muscle harbors a population of muscle stem cells (MuSCs) that are required to repair or reform multinucleated myofibers after tissue injury. In youth, MuSCs return to a reversible state of cell cycle arrest termed 'quiescence' after injury resolution. By contrast, a proportion of MuSCs in aged muscle remain in a semi-activated state, causing a premature response to subsequent injury cues that results in incomplete tissue repair and eventual stem cell depletion. Regulation of the balance between MuSC quiescence and activation in youth and in age 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, with a 96-well footprint, to rapidly inactivate MuSCs freshly isolated from young murine skeletal muscle tissue, and return them to a quiescent-like state for at least one-week, which we name mini-IDLE (Inactivation and Dormancy LEveraged in vitro). This was achieved by introducing MuSCs into a three-dimensional (3D) bioartificial niche comprised of a thin sheet of multinucleated mouse myotubes, which we iterate, and analyze temporally, to show that these in vivo niche features provide the minimal cues necessary to inactivate MuSCs and induce quiescence. By seeding the 3D myotube sheets with different starting numbers of MuSCs, the assay revealed cellular heterogeneity and population-level adaptation activities that converged on a common steady-state niche repopulation density; behaviors previously observed only in vivo. Quiescence-associated hallmarks included a Pax7+CalcR+DDX6+MyoD-c-FOS- molecular signature, in vivo quiescent-like morphologies including oval-shaped nuclei and long cytoplasmic projections with N-cadherin+ tips, as well as the acquisition of polarized niche markers. Leveraging high-content imaging and bespoke CellProfilerTM-based image analysis pipelines, we demonstrate a relationship between morphology and cell fate signatures opening up the possibility of real-time morphology-based screening. When MuSCs from aged muscle were introduced into the assay, they displayed aberrant proliferative activities, delayed inactivation kinetics, persistence of activation-associated morphologies, and population depletion; quiescence-associated defects that we show are rescued by wortmannin treatment. Thus, the miniaturized assay offers an unprecedented opportunity to systematically investigate long-standing queries in areas such as regulation of adult stem cell pool size and functional heterogeneity within the MuSC population, and to uncover regulators of quiescence in youth and in age.
All data generated and analysed during this study are included in the manuscript files. In addition, a Source Data file containing all of the numerical data used to generate each of the figures has been provided.
- Erik Jacques
- Erik Jacques
- Erik Jacques
- Erik Jacques
- Penney M Gilbert
- Penney M Gilbert
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Animal experimentation: All animal use protocols were reviewed and approved by the local Animal Care Committee (ACC) within the Division of Comparative Medicine (DCM) at the University of Toronto. All methods in this study were conducted as described in the approved animal use protocols (#20012838) and more broadly in accordance with the guidelines and regulations of the DCM ACC and the Canadian Council on Animal Care.
- Christopher L-H Huang, University of Cambridge, United Kingdom
© 2022, Jacques et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
The acute traumatic or surgical loss of skeletal muscle, known as volumetric muscle loss (VML), is a devastating type of injury that results in exacerbated and persistent inflammation followed by fibrosis. The mechanisms that mediate the magnitude and duration of the inflammatory response and ensuing fibrosis after VML remain understudied, and as such, the development of regenerative therapies has been limited. To address this need, we profiled how lipid mediators, which are potent regulators of the immune response after injury, varied with VML injuries that heal or result in fibrosis. We observed that non-healing VML injuries displayed increased pro-inflammatory eicosanoids and a lack of pro-resolving lipid mediators. Treatment of VML with a pro-resolving lipid mediator synthesized from docosahexaenoic acid, called Maresin 1, ameliorated fibrosis through reduction of neutrophils and macrophages and enhanced recovery of muscle strength. These results expand our knowledge of the dysregulated immune response that develops after VML and identify a novel immuno-regenerative therapeutic modality in Maresin 1.
Breakdown of neuromuscular junctions (NMJs) is an early pathological hallmark of amyotrophic lateral sclerosis (ALS) that blocks neuromuscular transmission, leading to muscle weakness, paralysis and, ultimately, premature death. Currently, no therapies exist that can prevent progressive motor neuron degeneration, muscle denervation, or paralysis in ALS. Here, we report important advances in the development of an optogenetic, neural replacement strategy that can effectively restore innervation of severely affected skeletal muscles in the aggressive SOD1G93A mouse model of ALS, thus providing an interface to selectively control the function of targeted muscles using optical stimulation. We also identify a specific approach to confer complete survival of allogeneic replacement motor neurons. Furthermore, we demonstrate that an optical stimulation training paradigm can prevent atrophy of reinnervated muscle fibers and results in a tenfold increase in optically evoked contractile force. Together, these advances pave the way for an assistive therapy that could benefit all ALS patients.