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

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 LE>veraged 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.

A self-renewing biomimetic skeletal muscle construct engineered using induced myogenic progenitor cells

“…In contrast, iSMCs appear highly proliferative, mirroring an ongoing regeneration response in vitro, most likely due to the presence of the F/R/C small molecule cocktail. It will be of interest to assess if a fraction of the Pax7 + cells in iSMCs exit the cell cycle and return to quiescence, as previously reported for SCs seeded on myofibers [56][57][58] . Additionally, it will be of interest to assess whether serum withdrawal or modulation of additional signaling pathways might confer quiescence rather than activation on the Pax7 + cell population in iSMCs, rendering them more similar to muscle tissue during homeostasis 57,59,60 .…”

mini-MEndR: A 96-well mixed species culture assay to co-evaluate human and mouse Pax7+cell-mediated skeletal muscle repair

“…The copyright holder for this preprint this version posted January 21, 2023. ; https://doi.org/10.1101/2023.01.20.524941 doi: bioRxiv preprint populations in culture and then 12 hrs later, enumerated the viable GFP positive and negative cells in each well 17 . This uncovered a dramatic loss of the GFP-negative population from the MACSenriched cell population, resulting in an enrichment for GFP-positive cells that matched values obtained using FACS-enrichment.…”

Muscle stem cells get a new look: Dynamic cellular projections as sensors of the stem cell niche

“…[ 20 ] A second example of phenotypic reversibility is provided by Jacques et al., who recently reported that seeding freshly isolated MuSCs (which have entered the activation process and are fully rounded cells) onto a 3D, biomimetic culture of myotubes restored expression of quiescence markers and features of quiescent morphology, including long cellular projections. [ 72 ] Furthermore, treatment of these cultures with Y‐27632 hastened this process. [ 72 ] Therefore, myotubes in a bioengineered in vitro niche provide information sufficient to push activated MuSCs back towards full quiescence.…”

“…[ 72 ] Furthermore, treatment of these cultures with Y‐27632 hastened this process. [ 72 ] Therefore, myotubes in a bioengineered in vitro niche provide information sufficient to push activated MuSCs back towards full quiescence.…”