An injury-responsive Rac-to-Rho GTPase switch drives activation of muscle stem cells through rapid cytoskeletal remodeling

Kann, Allison P.; Hung, Margaret; Wang, Wei; Nguyen, Jo; Gilbert, Penney M.; Wu, Zhuhao; Krauss, Robert M.

doi:10.1016/j.stem.2022.04.016

Cited by 53 publications

(112 citation statements)

References 86 publications

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“…It was recently discovered that quiescent MuSCs localize the N-cadherin adhesion molecule to the tips of elaborated cytoplasmic projections (coined ‘quiescent projections’) 48 , a feature we also observed within our culture assay at 7 DPE ( Figure 6A ). While examples such as these were relatively uncommon occurrences, in each case the Pax7 + donor cell morphology was characterized by a long oval-shaped nucleus and very long, elaborated cytoplasmic projections.…”

Section: Resultssupporting

confidence: 76%

“…Qualitatively, the engrafted MuSCs in our cultures adopted an elongated morphology over time ( Figure 2B-C, Figure 3A, Figure 5A ), reminiscent of quiescent MuSCs in vivo , and contrasting against with morphologies observed in 2D cultures ( Supplementary Figure 7 ). 12,47,48 Therefore, we next overcame a significant data analysis bottleneck by establishing and validating a CellProfiler™-based image analysis pipeline in order to segment and evaluate donor MuSCs in our phenotypic datasets (see Methods and Supplementary Figure 8 ). 49 The cytoplasmic elongation of mononucleated Pax7 + donor cell bodies was captured by applying a ratio of max/min feret diameter to segmented images of tissues immunostained for YFP, Pax7, and DAPI.…”

Section: Resultsmentioning

confidence: 99%

“…The rice-like nuclear morphology and elaborated cytoplasmic projections of the Pax7 + donor cells at 7 DPE resembled quiescent features of MuSCs in vivo that were shown to be induced and maintained by tipping the Rho family GTPase balance to favour cytoskeletal remodelling events caused by Rac signaling. 48…”

Section: Resultsmentioning

confidence: 99%

“…MuSCs in situ display long cytoplasmic projections that were initially described from electron microscopy analyses 65 , and more recently evaluated using tissue clearing and intravital imaging methodologies 12,47,48 . The elaborate MuSC morphologies arising in our cultures offer a new opportunity to explore the cellular and molecular mechanisms driving the acquisition of quiescent-like morphologies, but also the relevance of this phenotypic feature on MuSC behavior and fate.…”

Section: Discussionmentioning

confidence: 99%

“…The elaborate MuSC morphologies arising in our cultures offer a new opportunity to explore the cellular and molecular mechanisms driving the acquisition of quiescent-like morphologies, but also the relevance of this phenotypic feature on MuSC behavior and fate. Indeed, long elaborated cytoplasmic projections have been associated with a deeper quiescent state 48 , and have been ruled out as a migratory apparatus 47 , favoring instead a role in ‘niche sensing’, though that remains to be determined. 47,48 Quite surprisingly, we found that acquisition of quiescent-like morphologies and anatomical hallmarks was dependent on interactions between MuSCs and their immediate niche, occurring in the absence of other resident muscle cell types.…”

Section: Discussionmentioning

confidence: 99%

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Rescue of aged muscle stem cell quiescence defects by AKT inhibition revealed with a 3D biomimetic culture assay

Jacques

Kuang

Kann

et al. 2022

Preprint

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Adult skeletal muscle harbors a population of muscle stem cells (MuSCs) that are required to repair or reform multinucleated myofibers after a tissue injury. In youth, a portion of MuSCs return to a reversible state of cell cycle arrest, termed quiescence, after injury resolution. By contrast, a proportion of aged MuSCs exist in a semi-activated state under homeostatic conditions, and prematurely respond to subsequent injury cues, thereby failing to return the tissue to its pre-injury state. The heterogeneity of MuSC function is linked to quiescence depth, but regulation of the balance between MuSC quiescence and activation in youth and in age is incompletely understood. This is due in part to the paucity of scalable methods that support MuSC quiescence in culture, and in turn necessitates reliance on low-throughput in vivo studies. To fill this gap, we developed a simple, 96-well format method to inactivate MuSCs isolated from skeletal muscle tissue, and return them to a quiescent-like state for at least one-week by culturing them within a three-dimensional engineered sheet of myotubes. Seeding the myotube sheets with different numbers of MuSCs elicited population-level adaptation activities that converged on a common steady-state niche repopulation density. By evaluating MuSC engraftment over time in culture, we observed reversible cell cycle exit that required both myotubes and a 3D culture environment. Additional quiescence-associated hallmarks were identified including a Pax7+CalcR+MyoD-c-FOS- molecular signature, quiescent-like morphology including oval-shaped nuclei and long cytoplasmic projections with N-cadherin+ tips, as well as the acquisition of polarized niche markers. We further demonstrate a relationship between morphology and cell fate signature using high-content imaging and CellProfilerTM-based image analysis pipelines. MuSC functional heterogeneity during engraftment was observed across all metrics tested, suggesting in vivo-like subpopulation activities are reflected in the assay. Notably, aged MuSCs introduced into young 3D myotube cultures displayed aberrant proliferative activities, delayed inactivation kinetics, and activation-associated morphologies that we show are rescued by wortmannin treatment. Thus, this miniaturized, biomimetic culture assay offers an unprecedented opportunity to uncover regulators of quiescence in youth and in age.

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Section: Resultssupporting

confidence: 76%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

See 3 more Smart Citations

Rescue of aged muscle stem cell quiescence defects by AKT inhibition revealed with a 3D biomimetic culture assay

Jacques

Kuang

Kann

et al. 2022

Preprint

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Granular Hydrogels Improve Myogenic Invasion and Repair after Volumetric Muscle Loss

Tanner,

Schiltz,

Narra

et al. 2024

Adv Healthcare Materials

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Skeletal muscle injuries including volumetric muscle loss (VML) lead to excessive tissue scarring and permanent functional disability. Despite high prevalence, there is currently no effective treatment for VML. Bioengineering interventions such as biomaterials that fill the VML defect to support cell and tissue growth are a promising therapeutic strategy. However, traditional biomaterials developed for this purpose lack the pore features needed to support cell infiltration. The present study investigates for the first time, the impact of granular hydrogels on muscle repair – hypothesizing that their flowability will permit conformable filling of the defect site and their inherent porosity will support the invasion of native myogenic cells, leading to effective muscle repair. We prepared small and large microparticle fragments from photocurable hyaluronic acid polymer via extrusion fragmentation and facile size sorting. In assembled granular hydrogels, particle size and degree of packing significantly influenced pore features, rheological behavior, and injectability. Using a mouse model of volumetric muscle loss (VML), we demonstrate that, compared to bulk hydrogels, granular hydrogels support early‐stage (satellite cell invasion) and late‐stage (myofiber regeneration) muscle repair processes. Together, these results highlight the promising potential of injectable and porous granular hydrogels in supporting endogenous repair after severe muscle injury.This article is protected by copyright. All rights reserved

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Biomaterials‐Based Technologies in Skeletal Muscle Tissue Engineering

Luo,

Zhang,

Wan

et al. 2024

Adv Healthcare Materials

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For many clinically prevalent severe injuries, the inherent regenerative capacity of skeletal muscle remains inadequate. Skeletal muscle tissue engineering (SMTE) seeks to meet this clinical demand. With continuous progress in biomedicine and related technologies including micro/nanotechnology and 3D printing, numerous studies have uncovered various intrinsic mechanisms regulating skeletal muscle regeneration and developed tailored biomaterial systems based on these understandings. Here, we discussed the skeletal muscle structure and regeneration process and explored in detail the diverse biomaterial systems derived from various technologies. Biomaterials serve not merely as local niches for cell growth, but also as scaffolds endowed with structural or physicochemical properties that provide tissue regenerative cues such as topographical, electrical, and mechanical signals. They can also act as delivery systems for stem cells and bioactive molecules that have been shown as key participants in endogenous repair cascades. To achieve bench‐to‐bedside translation, we have also summarized the typical effect enabled by biomaterial systems and the potential underlying molecular mechanisms. We hope to provide insights into the roles of biomaterials in SMTE from cellular and molecular perspectives. Finally, we provided perspectives on the advancement of SMTE, for which gene therapy, exosomes, and hybrid biomaterials may hold promise to make important contributions.This article is protected by copyright. All rights reserved

show abstract

An injury-responsive Rac-to-Rho GTPase switch drives activation of muscle stem cells through rapid cytoskeletal remodeling

Cited by 53 publications

References 86 publications

Rescue of aged muscle stem cell quiescence defects by AKT inhibition revealed with a 3D biomimetic culture assay

Rescue of aged muscle stem cell quiescence defects by AKT inhibition revealed with a 3D biomimetic culture assay

Granular Hydrogels Improve Myogenic Invasion and Repair after Volumetric Muscle Loss

Biomaterials‐Based Technologies in Skeletal Muscle Tissue Engineering

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