The mini-IDLE 3D biomimetic culture assay enables interrogation of mechanisms governing muscle stem cell quiescence and niche repopulation

  1. Erik Jacques
  2. Yinni Kuang
  3. Allison P Kann
  4. Fabien Le Grand
  5. Robert S Krauss
  6. Penney M Gilbert  Is a corresponding author
  1. University of Toronto, Canada
  2. Icahn School of Medicine at Mount Sinai, United States
  3. INSERM - CNRS - Université Pierre et Marie Curie, France

Abstract

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.

Data availability

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.

Article and author information

Author details

  1. Erik Jacques

    Institute of Biomedical Engineering, University of Toronto, Toronto, Canada
    Competing interests
    The authors declare that no competing interests exist.
  2. Yinni Kuang

    Donnelly Centre, University of Toronto, Toronto, Canada
    Competing interests
    The authors declare that no competing interests exist.
  3. Allison P Kann

    Department of Cell, Developmental, and Regenerative Biology, Icahn School of Medicine at Mount Sinai, New York, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-0111-9081
  4. Fabien Le Grand

    INSERM - CNRS - Université Pierre et Marie Curie, Paris, France
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-7843-3899
  5. Robert S Krauss

    Department of Cell, Developmental, and Regenerative Biology, Icahn School of Medicine at Mount Sinai, New York, United States
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-7661-3335
  6. Penney M Gilbert

    Institute of Biomedical Engineering, University of Toronto, Toronto, Canada
    For correspondence
    Penney.Gilbert@utoronto.ca
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-5509-9616

Funding

Canadian Institutes of Health Research (CGS-D Scholarship)

  • Erik Jacques

Ontario Ministry of Research and Innovation (Ontario Graduate Scholarship)

  • Erik Jacques

Mitacs (Globalink Research Award)

  • Erik Jacques

Canadian Institutes of Health Research (Michael Smith Foreign Study Supplement)

  • Erik Jacques

Canada First Research Excellence Fund (Medicine by Design (MbDC2-2019-02))

  • Penney M Gilbert

Natural Sciences and Engineering Research Council of Canada (Canada Research Chair in Endogenous Repair)

  • Penney M Gilbert

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Ethics

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.

Reviewing Editor

  1. Christopher L-H Huang, University of Cambridge, United Kingdom

Publication history

  1. Received: July 10, 2022
  2. Accepted: December 19, 2022
  3. Accepted Manuscript published: December 20, 2022 (version 1)

Copyright

© 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.

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  1. Erik Jacques
  2. Yinni Kuang
  3. Allison P Kann
  4. Fabien Le Grand
  5. Robert S Krauss
  6. Penney M Gilbert
(2022)
The mini-IDLE 3D biomimetic culture assay enables interrogation of mechanisms governing muscle stem cell quiescence and niche repopulation
eLife 11:e81738.
https://doi.org/10.7554/eLife.81738
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