scholarly journals MON-710 ACVR1 Activation in Primary and iPS-Derived Human Skeletal Muscle Stem Cells Impairs Myogenic Transcriptional Signature and Function

2020 ◽  
Vol 4 (Supplement_1) ◽  
Author(s):  
Emilie Barruet ◽  
Steven Garcia ◽  
Stanley Tamaki ◽  
Blanca M Morales ◽  
Jake Wu ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Stem Cells ◽  
Heterotopic Ossification ◽  
Satellite Cells ◽  
High Efficiency ◽  
Skeletal Muscle Regeneration ◽  
Type I ◽  
Muscle Repair ◽  
Mouse Muscle ◽  
Muscle Stem Cells

Abstract Developing optimal strategies for skeletal muscle regeneration and repair requires a detailed understanding of how these processes are regulated. The number of primary human satellite cells that can be obtained is usually extremely low, and may be impaired in disease of impaired skeletal muscle repair. One such condition is fibrodysplasia ossificans progressiva (FOP), a progressive disease characterized by massive heterotopic ossification in skeletal muscles and aberrant skeletal muscle repair after injury. FOP patients have activating mutations in the Activin A Type I receptor (ACVR1), a bone morphogenetic protein (BMP) receptor. Our overall hypothesis is that activated ACVR1 signaling caused by the ACVR1 R206H mutation incites inappropriate activation of human muscle stem cells (satellite cells, PAX7 expressing cells), causing loss of muscle cell fate and aberrant muscle repair. Since human satellite cells are difficult to obtain from live tissue donors, and injury can trigger heterotopic ossification, we created human induced pluripotent stem cell (iPSC)-derived muscle stem cells (iMuSCs) from FOP and control iPSC lines. We found that control and FOP iPSCs can differentiate into PAX7+ cells with high efficiency. Control and FOP iMuSCs can regenerate injured mouse muscle and form new human fibers, but both showed few PAX7 cells after transplant. Single cell RNA sequencing showed cell heterogeneity, and specific subsets of PAX7+ cells. FOP iMuSCs showed a chondrogenic/osteogenic signature (e.g COL1A1, DCN, OGN) with higher p38 pathway signaling activity. Skeletal muscle samples from autopsies of patients with FOP also showed increased expression of COL1A1. Additionally, we found that primary human FOP satellite cells can engraft and regenerate injured muscle, but with lower efficiency than control satellite cells. These studies used a novel iMuSC strategy to elucidate how increased ACVR1 activity affects human satellite cells function, and compare these iMuSCs to primary human satellite cells. These approaches will be useful to identify new therapeutic targets for conditions affecting skeletal muscle, and will improve our understanding of how muscle and bone interact in development and disease pathophysiology.

scholarly journals Pro-myogenic small molecules revealed by a chemical screen on primary muscle stem cells

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Sean M. Buchanan ◽  
Feodor D. Price ◽  
Alessandra Castiglioni ◽  
Amanda Wagner Gee ◽  
Joel Schneider ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Stem Cells ◽  
Stem Cell ◽  
Small Molecules ◽  
Satellite Cell ◽  
Satellite Cells ◽  
Muscle Repair ◽  
Muscle Stem Cells

Abstract Satellite cells are the canonical muscle stem cells that regenerate damaged skeletal muscle. Loss of function of these cells has been linked to reduced muscle repair capacity and compromised muscle health in acute muscle injury and congenital neuromuscular diseases. To identify new pathways that can prevent loss of skeletal muscle function or enhance regenerative potential, we established an imaging-based screen capable of identifying small molecules that promote the expansion of freshly isolated satellite cells. We found several classes of receptor tyrosine kinase (RTK) inhibitors that increased freshly isolated satellite cell numbers in vitro. Further exploration of one of these compounds, the RTK inhibitor CEP-701 (also known as lestaurtinib), revealed potent activity on mouse satellite cells both in vitro and in vivo. This expansion potential was not seen upon exposure of proliferating committed myoblasts or non-myogenic fibroblasts to CEP-701. When delivered subcutaneously to acutely injured animals, CEP-701 increased both the total number of satellite cells and the rate of muscle repair, as revealed by an increased cross-sectional area of regenerating fibers. Moreover, freshly isolated satellite cells expanded ex vivo in the presence of CEP-701 displayed enhanced muscle engraftment potential upon in vivo transplantation. We provide compelling evidence that certain RTKs, and in particular RET, regulate satellite cell expansion during muscle regeneration. This study demonstrates the power of small molecule screens of even rare adult stem cell populations for identifying stem cell-targeting compounds with therapeutic potential.

scholarly journals PPARγ Controls Ectopic Adipogenesis and Cross-Talks with Myogenesis During Skeletal Muscle Regeneration

2018 ◽  
Vol 19 (7) ◽  
pp. 2044 ◽  
Author(s):  
Gabriele Dammone ◽  
Sonia Karaz ◽  
Laura Lukjanenko ◽  
Carine Winkler ◽  
Federico Sizzano ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Stem Cells ◽  
Adipose Tissue ◽  
Muscle Regeneration ◽  
Ex Vivo ◽  
Skeletal Muscle Regeneration ◽  
Whole Body ◽  
Muscle Repair ◽  
Muscle Stem Cells ◽  
Muscle Fat Infiltration

Skeletal muscle is a regenerative tissue which can repair damaged myofibers through the activation of tissue-resident muscle stem cells (MuSCs). Many muscle diseases with impaired regeneration cause excessive adipose tissue accumulation in muscle, alter the myogenic fate of MuSCs, and deregulate the cross-talk between MuSCs and fibro/adipogenic progenitors (FAPs), a bi-potent cell population which supports myogenesis and controls intra-muscular fibrosis and adipocyte formation. In order to better characterize the interaction between adipogenesis and myogenesis, we studied muscle regeneration and MuSC function in whole body Pparg null mice generated by epiblast-specific Cre/lox deletion (PpargΔ/Δ). We demonstrate that deletion of PPARγ completely abolishes ectopic muscle adipogenesis during regeneration and impairs MuSC expansion and myogenesis after injury. Ex vivo assays revealed that perturbed myogenesis in PpargΔ/Δ mice does not primarily result from intrinsic defects of MuSCs or from perturbed myogenic support from FAPs. The immune transition from a pro- to anti-inflammatory MuSC niche during regeneration is perturbed in PpargΔ/Δ mice and suggests that PPARγ signaling in macrophages can interact with ectopic adipogenesis and influence muscle regeneration. Altogether, our study demonstrates that a PPARγ-dependent adipogenic response regulates muscle fat infiltration during regeneration and that PPARγ is required for MuSC function and efficient muscle repair.

scholarly journals Progressive and Coordinated Mobilization of the Skeletal Muscle Niche throughout Tissue Repair Revealed by Single-Cell Proteomic Analysis

Cells ◽  
2021 ◽  
Vol 10 (4) ◽  
pp. 744
Author(s):  
Matthew Borok ◽  
Nathalie Didier ◽  
Francesca Gattazzo ◽  
Teoman Ozturk ◽  
Aurelien Corneau ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Stem Cells ◽  
Stem Cell ◽  
Muscle Regeneration ◽  
Cell Types ◽  
Skeletal Muscle Regeneration ◽  
Muscle Repair ◽  
Muscle Stem Cell ◽  
Muscle Stem Cells ◽  
Cell Lineages

Background: Skeletal muscle is one of the only mammalian tissues capable of rapid and efficient regeneration after trauma or in pathological conditions. Skeletal muscle regeneration is driven by the muscle satellite cells, the stem cell population in interaction with their niche. Upon injury, muscle fibers undergo necrosis and muscle stem cells activate, proliferate and fuse to form new myofibers. In addition to myogenic cell populations, interaction with other cell types such as inflammatory cells, mesenchymal (fibroadipogenic progenitors—FAPs, pericytes) and vascular (endothelial) lineages are important for efficient muscle repair. While the role of the distinct populations involved in skeletal muscle regeneration is well characterized, the quantitative changes in the muscle stem cell and niche during the regeneration process remain poorly characterized. Methods: We have used mass cytometry to follow the main muscle cell types (muscle stem cells, vascular, mesenchymal and immune cell lineages) during early activation and over the course of muscle regeneration at D0, D2, D5 and D7 compared with uninjured muscles. Results: Early activation induces a number of rapid changes in the proteome of multiple cell types. Following the induction of damage, we observe a drastic loss of myogenic, vascular and mesenchymal cell lineages while immune cells invade the damaged tissue to clear debris and promote muscle repair. Immune cells constitute up to 80% of the mononuclear cells 5 days post-injury. We show that muscle stem cells are quickly activated in order to form new myofibers and reconstitute the quiescent muscle stem cell pool. In addition, our study provides a quantitative analysis of the various myogenic populations during muscle repair. Conclusions: We have developed a mass cytometry panel to investigate the dynamic nature of muscle regeneration at a single-cell level. Using our panel, we have identified early changes in the proteome of stressed satellite and niche cells. We have also quantified changes in the major cell types of skeletal muscle during regeneration and analyzed myogenic transcription factor expression in satellite cells throughout this process. Our results highlight the progressive dynamic shifts in cell populations and the distinct states of muscle stem cells adopted during skeletal muscle regeneration. Our findings give a deeper understanding of the cellular and molecular aspects of muscle regeneration.

It takes all kinds: heterogeneity among satellite cells and fibro-adipogenic progenitors during skeletal muscle regeneration

Development ◽  
2021 ◽  
Vol 148 (21) ◽  
Author(s):  
Brittany C. Collins ◽  
Gabrielle Kardon
Keyword(s):  
Skeletal Muscle ◽  
Satellite Cells ◽  
Regenerative Process ◽  
Skeletal Muscle Regeneration ◽  
Anatomical Location ◽  
Muscle Stem Cells ◽  
Functional Consequences ◽  
Transplantation Experiments ◽  
Complete Regeneration ◽  
Vertebrate Skeletal Muscle

ABSTRACT Vertebrate skeletal muscle is composed of multinucleate myofibers that are surrounded by muscle connective tissue. Following injury, muscle is able to robustly regenerate because of tissue-resident muscle stem cells, called satellite cells. In addition, efficient and complete regeneration depends on other cells resident in muscle – including fibro-adipogenic progenitors (FAPs). Increasing evidence from single-cell analyses and genetic and transplantation experiments suggests that satellite cells and FAPs are heterogeneous cell populations. Here, we review our current understanding of the heterogeneity of satellite cells, their myogenic derivatives and FAPs in terms of gene expression, anatomical location, age and timing during the regenerative process – each of which have potentially important functional consequences.

scholarly journals The role of Pitx2 and Pitx3 in muscle stem cells gives new insights into P38α MAP kinase and redox regulation of muscle regeneration

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Aurore L'honoré ◽  
Pierre-Henri Commère ◽  
Elisa Negroni ◽  
Giorgia Pallafacchina ◽  
Bertrand Friguet ◽  
...  
Keyword(s):  
Stem Cells ◽  
Map Kinase ◽  
Satellite Cells ◽  
Muscle Regeneration ◽  
Redox Regulation ◽  
Primary Cultures ◽  
Rapid Expansion ◽  
Skeletal Muscle Regeneration ◽  
Muscle Stem Cells ◽  
P38α Map Kinase

Skeletal muscle regeneration depends on satellite cells. After injury these muscle stem cells exit quiescence, proliferate and differentiate to regenerate damaged fibres. We show that this progression is accompanied by metabolic changes leading to increased production of reactive oxygen species (ROS). Using Pitx2/3 single and double mutant mice that provide genetic models of deregulated redox states, we demonstrate that moderate overproduction of ROS results in premature differentiation of satellite cells while high levels lead to their senescence and regenerative failure. Using the ROS scavenger, N-Acetyl-Cysteine (NAC), in primary cultures we show that a physiological increase in ROS is required for satellite cells to exit the cell cycle and initiate differentiation through the redox activation of p38α MAP kinase. Subjecting cultured satellite cells to transient inhibition of P38α MAP kinase in conjunction with NAC treatment leads to their rapid expansion, with striking improvement of their regenerative potential in grafting experiments.

Single-fiber isolation and maintenance of satellite cell quiescence

2005 ◽  
Vol 83 (5) ◽  
pp. 674-676 ◽  
Author(s):  
Ashley C Wozniak ◽  
Judy E Anderson
Keyword(s):  
Skeletal Muscle ◽  
Satellite Cell ◽  
Satellite Cells ◽  
Muscle Fibers ◽  
Single Fiber ◽  
Skeletal Muscle Regeneration ◽  
Mouse Muscle ◽  
Collagenase Digestion ◽  
Cell Quiescence ◽  
Gentle Agitation

The activity of satellite cells during myogenesis, development, or skeletal muscle regeneration is strongly modelled using cultures of single muscle fibers. However, there are variations in reported features of gene or protein expression as examined with single-fiber cultures. Here, we examined the potential differences in activation of satellite cells on normal mouse muscle fibers produced during a standard isolation protocol, with or without agitation during collagenase digestion. Activation was detected in satellite cells on fibers after 24 and 48 h of culture in basal growth medium using immunodetection of the incorporation of bromodeoxyuridine (BrdU) into DNA and quantification of the number of BrdU-positive cells per fiber. After 24 and 48 h in culture under nonactivating conditions, the number of activated (BrdU+) satellite cells was greater on fibers that had received gentle agitation during collagenase digestion than on those that were subject to digestion without agitation during isolation. The findings are interpreted to mean that at least some of the variation among published reports may derive from the application of various methods of fiber isolation. The information should be useful for maintaining satellite cell quiescence during studies of the regulatory steps that lead to satellite cell activation.Key words: activation, skeletal muscle, proliferation, single-fiber culture, myogenesis.

scholarly journals Stem cells for skeletal muscle repair

2011 ◽  
Vol 366 (1575) ◽  
pp. 2297-2306 ◽  
Author(s):  
Jennifer L. Shadrach ◽  
Amy J. Wagers
Keyword(s):  
Skeletal Muscle ◽  
Stem Cells ◽  
Satellite Cell ◽  
Satellite Cells ◽  
Cell Function ◽  
Therapeutic Potential ◽  
Muscle Disease ◽  
Muscle Fibres ◽  
Muscle Repair ◽  
Muscle Satellite Cells

Skeletal muscle is a highly specialized tissue composed of non-dividing, multi-nucleated muscle fibres that contract to generate force in a controlled and directed manner. Skeletal muscle is formed during embryogenesis from a subset of muscle precursor cells, which generate both differentiated muscle fibres and specialized muscle-forming stem cells known as satellite cells. Satellite cells remain associated with muscle fibres after birth and are responsible for muscle growth and repair throughout life. Failure in satellite cell function can lead to delayed, impaired or failed recovery after muscle injury, and such failures become increasingly prominent in cases of progressive muscle disease and in old age. Recent progress in the isolation of muscle satellite cells and elucidation of the cellular and molecular mediators controlling their activity indicate that these cells represent promising therapeutic targets. Such satellite cell-based therapies may involve either direct cell replacement or development of drugs that enhance endogenous muscle repair mechanisms. Here, we discuss recent breakthroughs in understanding both the cell intrinsic and extrinsic regulators that determine the formation and function of muscle satellite cells, as well as promising paths forward to realizing their full therapeutic potential.

scholarly journals Assessing Autophagy in Muscle Stem Cells

2021 ◽  
Vol 8 ◽  
Author(s):  
Silvia Campanario ◽  
Ignacio Ramírez-Pardo ◽  
Xiaotong Hong ◽  
Joan Isern ◽  
Pura Muñoz-Cánoves
Keyword(s):  
Skeletal Muscle ◽  
Stem Cells ◽  
Satellite Cell ◽  
Satellite Cells ◽  
Rapid Assessment ◽  
Muscle Stem Cells ◽  
Reporter Mice ◽  
Ability To Regenerate ◽  
Resident Stem Cells ◽  
Autophagic Activity

The skeletal muscle tissue in the adult is relatively stable under normal conditions but retains a striking ability to regenerate by its resident stem cells (satellite cells). Satellite cells exist in a quiescent (G0) state; however, in response to an injury, they reenter the cell cycle and start proliferating to provide sufficient progeny to form new myofibers or undergo self-renewal and returning to quiescence. Maintenance of satellite cell quiescence and entry of satellite cells into the activation state requires autophagy, a fundamental degradative and recycling process that preserves cellular proteostasis. With aging, satellite cell regenerative capacity declines, correlating with loss of autophagy. Enhancing autophagy in aged satellite cells restores their regenerative functions, underscoring this proteostatic activity’s relevance for tissue regeneration. Here we describe two strategies for assessing autophagic activity in satellite cells from GFP-LC3 reporter mice, which allows direct autophagosome labeling, or from non-transgenic (wild-type) mice, where autophagosomes can be immunostained. Treatment of GFP-LC3 or WT satellite cells with compounds that interfere with autophagosome-lysosome fusion enables measurement of autophagic activity by flow cytometry and immunofluorescence. Thus, the methods presented permit a relatively rapid assessment of autophagy in stem cells from skeletal muscle in homeostasis and in different pathological scenarios such as regeneration, aging or disease.

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