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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 ( I nactivation and D ormancy LE veraged 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.
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This Methods paper seeks procedures evaluating the balance between muscle cell quiescence and activation. These could well permit investigations of long-standing questions in key areas of muscle function. The latter include the regulation of adult stem cell pool size and functional heterogeneities in this, as well as regulators of muscle quiescence.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 agreed to share their name with the authors.)
Reviewer #1 (Public Review):
This Methods paper explores methods of assaying the balance between muscle cell quiescence and activation. If successful, it offers a miniaturized assay that will permit systematic investigations of long-standing queries in key areas of muscle function such as regulation of adult stem cell pool size and functional heterogeneity. It could also be used to discover regulators of quiescence.
Reviewer #2 (Public Review):
This is interesting and timely work that seeks to recapitulate aspects of the in vivo muscle stem cell (MuSC) niche in an in vitro format. The authors have innovated a three dimensional bioartificial niche composed of thin sheets of multinucleated myotubes in a 96-well plate format. By seeding fresh isolated MuSCs onto myotube sheets, the authors show that 'donor' MuSCs maintain features of MuSC quiescence for a period up to one week. Hallmarks of in vivo quiescent MuSCs from young adult mice, include Pax7 and CalcR expression, oval shaped nuclei, cytoplasmic projections and, to a limited extent niche interactions, were recapitulated in the in vitro system. In contrast, quiescence associated features could not be recapitulated when aged MuSCs were the 'donor' cells. The authors provide some evidence that …
Reviewer #2 (Public Review):
This is interesting and timely work that seeks to recapitulate aspects of the in vivo muscle stem cell (MuSC) niche in an in vitro format. The authors have innovated a three dimensional bioartificial niche composed of thin sheets of multinucleated myotubes in a 96-well plate format. By seeding fresh isolated MuSCs onto myotube sheets, the authors show that 'donor' MuSCs maintain features of MuSC quiescence for a period up to one week. Hallmarks of in vivo quiescent MuSCs from young adult mice, include Pax7 and CalcR expression, oval shaped nuclei, cytoplasmic projections and, to a limited extent niche interactions, were recapitulated in the in vitro system. In contrast, quiescence associated features could not be recapitulated when aged MuSCs were the 'donor' cells. The authors provide some evidence that age-associated quiescence defects could be rescued by Akt inhibition (wortmannin), providing some proof-of-concept that the novel 3D biomimetic niche may provide a new tool to investigate the regulation and deregulation of stem cell quiescence.
This is a timely contribution that demonstrates the utility of a myotube sheet biomimetic niche, in a 96 well format, to recapture some biological aspects of MuSC quiescence ex vivo. The format should provide a novel biological context within which features of MuSC quiescence can be studied.
The simplicity of the 3D niche in a 96 well plate format is very attractive and will be a significant resource to the community.
The authors use multiple lines of evidence, to show aspects of MuSC quiescence are achieved in vitro, including Pax7 expression, CalcR expression, lack of cell proliferation (Ki67 immunolabeling, EdU incorporation), lack of MyoD and c-Fos expression, along with morphological features including oval shaped nuclei and cytoplasmic projections.
The authors provide good data that aged donor MuSCs are defective to enter quiescence in the in vitro system, mimicking some of their known properties in vivo.
The presented data does not fully capture the innovative 3D bioengineered myotube substrate, nor does it capture the advance that has been made, beyond more traditional 2D culture techniques. (The work is innovative, but the innovation is not captured by the presented data).
The title of the manuscript does not reflect the balance of the article. Although the authors have presented some statistically significant data showing Akt inhibition (Wortmannin) rescues aged MuSC defects in their assay, I would expect more lines of evidence, including rescue experiments in vivo, to support the authors conclusion that their assay revealed Akt inhibition as a targetable pathway to rescue aged MuSC quiescence.
To summarize: the authors have presented a novel 3D bioengineered myotube substrate in a 96 well plate format, and have shown it's utility to maintain quiescence properties of young adult MuSCs in vitro. The simplicity of the method, shown in a 96 well plate format, will be an important new tool to study MuSC stem cell properties. The manuscript would benefit from a high resolution, high magnification of the 3D bioengineered myotube substrates compared to more conventional myotubes. The authors continue by showing that when aged MuSCs are introduced to the in vitro system, they remain partially defective to maintain quiescence but can be rescued by Akt inhibition (wortmannin). However, the authors conclusion that Akt inhibition rescues quiescence properties of aged MuSCs is only partially supported by the presented data.