ABSTRACT
In Duchenne muscular dystrophy (DMD), sarcolemma fragility and myofiber necrosis produce cellular debris that attract inflammatory cells. Macrophages and T-lymphocytes infiltrate muscles in response to damage-associated molecular pattern signalling and the release of TNF-α, TGF-ß and interleukins prevent skeletal muscle improvement from the inflammation. This immunological scenario was extended by the discovery of a specific response to muscle antigens and a role for regulatory T cells (Tregs) in muscle regeneration. Normally, autoimmunity is avoided by autoreactive T-lymphocyte deletion within thymus, while in the periphery Tregs monitor effector T-cells escaping from central regulatory control. Here, we report impairment of thymus architecture of mdx mice together with decreased expression of ghrelin, autophagy dysfunction and AIRE down-regulation. Transplantation of dystrophic thymus in recipient nude mice determine the up-regulation of inflammatory/fibrotic markers, marked metabolic breakdown that leads to muscle atrophy and loss of force. These results indicate that involution of dystrophic thymus exacerbates muscular dystrophy by altering central immune tolerance.
Subject(s)
Immune Tolerance/immunology , Muscle, Skeletal/pathology , Muscular Atrophy/pathology , Muscular Dystrophy, Animal/pathology , Thymus Gland/pathology , Animals , Autophagy/physiology , Ghrelin/biosynthesis , Macrophages/immunology , Male , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Inbred mdx , Mice, Nude , Muscular Dystrophy, Duchenne/pathology , T-Lymphocytes/transplantation , T-Lymphocytes, Regulatory/immunology , Thymus Gland/transplantation , Transcription Factors/biosynthesis , AIRE ProteinABSTRACT
The immune infiltrate present in acutely injured or dystrophic skeletal muscle has been shown to play an important role in the process of muscle regeneration. Our work has described, for the first time, muscle regulatory T cells (Tregs), a unique population in phenotype and function capable of promoting skeletal muscle repair. Herein, we describe the methods we have optimized to study muscle Tregs, including their isolation from injured muscle, immuno-labeling for analysis/separation by flow cytometry, and measurement of their proliferation status.
Subject(s)
Cell Separation/methods , Muscle, Skeletal/cytology , Regeneration/immunology , T-Lymphocytes, Regulatory/cytology , Humans , Muscular Dystrophy, Duchenne/immunology , Muscular Dystrophy, Duchenne/pathology , Phenotype , T-Lymphocytes, Regulatory/immunologyABSTRACT
Parabiosis experiments indicate that impaired regeneration in aged mice is reversible by exposure to a young circulation, suggesting that young blood contains humoral "rejuvenating" factors that can restore regenerative function. Here, we demonstrate that the circulating protein growth differentiation factor 11 (GDF11) is a rejuvenating factor for skeletal muscle. Supplementation of systemic GDF11 levels, which normally decline with age, by heterochronic parabiosis or systemic delivery of recombinant protein, reversed functional impairments and restored genomic integrity in aged muscle stem cells (satellite cells). Increased GDF11 levels in aged mice also improved muscle structural and functional features and increased strength and endurance exercise capacity. These data indicate that GDF11 systemically regulates muscle aging and may be therapeutically useful for reversing age-related skeletal muscle and stem cell dysfunction.
Subject(s)
Aging/physiology , Bone Morphogenetic Proteins/physiology , Growth Differentiation Factors/physiology , Muscle, Skeletal/blood supply , Muscle, Skeletal/physiology , Myoblasts, Skeletal/physiology , Regeneration , Rejuvenation , Age Factors , Aging/blood , Aging/drug effects , Animals , Bone Morphogenetic Proteins/administration & dosage , Bone Morphogenetic Proteins/blood , Growth Differentiation Factors/administration & dosage , Growth Differentiation Factors/blood , Male , Mice , Mice, Inbred C57BL , Muscle, Skeletal/drug effects , Myoblasts, Skeletal/drug effects , ParabiosisABSTRACT
Long recognized to be potent suppressors of immune responses, Foxp3(+)CD4(+) regulatory T (Treg) cells are being rediscovered as regulators of nonimmunological processes. We describe a phenotypically and functionally distinct population of Treg cells that rapidly accumulated in the acutely injured skeletal muscle of mice, just as invading myeloid-lineage cells switched from a proinflammatory to a proregenerative state. A Treg population of similar phenotype accumulated in muscles of genetically dystrophic mice. Punctual depletion of Treg cells during the repair process prolonged the proinflammatory infiltrate and impaired muscle repair, while treatments that increased or decreased Treg activities diminished or enhanced (respectively) muscle damage in a dystrophy model. Muscle Treg cells expressed the growth factor Amphiregulin, which acted directly on muscle satellite cells in vitro and improved muscle repair in vivo. Thus, Treg cells and their products may provide new therapeutic opportunities for wound repair and muscular dystrophies.
Subject(s)
Muscle, Skeletal/cytology , Muscle, Skeletal/physiology , Regeneration , T-Lymphocytes, Regulatory/physiology , Amphiregulin , Animals , EGF Family of Proteins , Glycoproteins/metabolism , Intercellular Signaling Peptides and Proteins/metabolism , Lymphoid Tissue/cytology , Mice , Mice, Inbred C57BL , Muscle, Skeletal/immunology , Muscle, Skeletal/injuries , Muscular Dystrophies/pathology , Muscular Dystrophies/physiopathology , Muscular Dystrophies/therapy , Receptors, Antigen, T-Cell/metabolism , T-Lymphocytes, Regulatory/cytology , T-Lymphocytes, Regulatory/immunology , TranscriptomeABSTRACT
Mutations in the Survival of Motor Neuron (SMN) gene underlie the development of spinal muscular atrophy (SMA), which currently represents the leading genetic cause of mortality in infants and toddlers. SMA is characterized by degeneration of spinal cord motor neurons and muscle atrophy. Although SMA is often considered to be a motor neuron disease, accumulating evidence suggests that muscle cells themselves may be affected by low levels of SMN. Here, we examine satellite cells, tissue-resident stem cells that play an essential role in the growth and repair of skeletal muscle, isolated from a severe SMA mouse model (Smn(-/-); SMN2(+/+)). We found similar numbers of satellite cells in the muscles of SMA and wild-type (Smn(+/+); SMN2(+/+)) mice at postnatal day 2 (P2), and, when isolated from skeletal muscle using cell surface marker expression, these cells showed comparable survival and proliferative potential. However, SMA satellite cells differentiate abnormally, revealed by the premature expression of muscle differentiation markers, and, especially, by a reduced efficiency in forming myotubes. These phenotypes suggest a critical role of SMN protein in the intrinsic regulation of muscle differentiation and suggest that abnormal muscle development contributes to the manifestation of SMA symptoms.
Subject(s)
Muscular Atrophy, Spinal/metabolism , Satellite Cells, Skeletal Muscle/metabolism , Survival of Motor Neuron 1 Protein/metabolism , Animals , Animals, Newborn , Apoptosis , Cell Differentiation , Cell Proliferation , Cell Survival , Cells, Cultured , Disease Models, Animal , Flow Cytometry , Humans , Immunohistochemistry , Mice , Mice, Knockout , Mice, Transgenic , Microscopy, Fluorescence , Muscle Fibers, Skeletal/cytology , Muscle Fibers, Skeletal/metabolism , Muscular Atrophy, Spinal/genetics , PAX7 Transcription Factor/metabolism , Survival of Motor Neuron 1 Protein/genetics , Survival of Motor Neuron 2 Protein/genetics , Survival of Motor Neuron 2 Protein/metabolism , Syndecans/metabolism , Time FactorsABSTRACT
Calorie restriction (CR) extends life span and ameliorates age-related pathologies in most species studied, yet the mechanisms underlying these effects remain unclear. Using mouse skeletal muscle as a model, we show that CR acts in part by enhancing the function of tissue-specific stem cells. Even short-term CR significantly enhanced stem cell availability and activity in the muscle of young and old animals, in concert with an increase in mitochondrial abundance and induction of conserved metabolic and longevity regulators. Moreover, CR enhanced endogenous muscle repair and CR initiated in either donor or recipient animals improved the contribution of donor cells to regenerating muscle after transplant. These studies indicate that metabolic factors play a critical role in regulating stem cell function and that this regulation can influence the efficacy of recovery from injury and the engraftment of transplanted cells.
Subject(s)
Caloric Restriction , Mitochondria/physiology , Muscle, Skeletal/physiology , Satellite Cells, Skeletal Muscle/physiology , Aging/physiology , Animals , Cells, Cultured , Colony-Forming Units Assay , Galactose/pharmacology , Mice , Mice, Inbred C57BL , Mitochondria/drug effects , Muscle Development/drug effects , Organ Specificity , Oxidative Phosphorylation/drug effectsABSTRACT
Suboptimal nutrition during prenatal and early postnatal development is associated with increased risk for type 2 diabetes during adult life. A hallmark of such diabetes risk is altered body composition, including reduced lean mass and increased adiposity. Since stem cell number and activity are important determinants of muscle mass, modulation of perinatal nutrition could alter stem cell number/function, potentially mediating developmentally programmed reductions in muscle mass. Skeletal muscle precursors (SMP) were purified from muscle of mice subjected to prenatal undernutrition and/or early postnatal high-fat diet (HFD)--experimental models that are both associated with obesity and diabetes risk. SMP number was determined by flow cytometry, proliferative capacity measured in vitro, and regenerative capacity of these cells determined in vivo after muscle freeze injury. Prenatally undernutrition (UN) mice showed significantly reduced SMP frequencies [Control (C) 4.8% ± 0.3% (% live cells) vs. UN 3.2% ± 0.4%, P=0.015] at 6 weeks; proliferative capacity was unaltered. Reduced SMP in UN was associated with 32% decrease in regeneration after injury (C 16% ± 3% of injured area vs. UN 11% ± 2%; P<0.0001). SMP frequency was also reduced in HFD-fed mice (chow 6.4% ± 0.6% vs. HFD 4.7% ± 0.4%, P=0.03), and associated with 44% decreased regeneration (chow 16% ± 2.7% vs. HFD 9% ± 2.2%; P<0.0001). Prenatal undernutrition was additive with postnatal HFD. Thus, both prenatal undernutrition and postnatal overnutrition reduce myogenic stem cell frequency and function, indicating that developmentally established differences in muscle-resident stem cell populations may provoke reductions in muscle mass and repair and contribute to diabetes risk.
Subject(s)
Maternal Nutritional Physiological Phenomena , Muscle, Skeletal/pathology , Stem Cells/pathology , Wound Healing , Absorptiometry, Photon , Animals , Animals, Newborn , Birth Weight/drug effects , Body Composition/drug effects , Cell Count , Dietary Fats/pharmacology , Feeding Behavior/drug effects , Female , Male , Maternal Nutritional Physiological Phenomena/drug effects , Mice , Muscle Development/drug effects , Muscle Fibers, Skeletal/drug effects , Muscle Fibers, Skeletal/pathology , Muscle, Skeletal/drug effects , Muscle, Skeletal/physiopathology , Nutritional Status , Organ Size , Regeneration/drug effects , Stem Cells/drug effects , Wound Healing/drug effectsABSTRACT
Brown fat is specialized for energy expenditure and has therefore been proposed to function as a defense against obesity. Despite recent advances in delineating the transcriptional regulation of brown adipocyte differentiation, cellular lineage specification and developmental cues specifying brown-fat cell fate remain poorly understood. In this study, we identify and isolate a subpopulation of adipogenic progenitors (Sca-1(+)/CD45(-)/Mac1(-); referred to as Sca-1(+) progenitor cells, ScaPCs) residing in murine brown fat, white fat, and skeletal muscle. ScaPCs derived from different tissues possess unique molecular expression signatures and adipogenic capacities. Importantly, although the ScaPCs from interscapular brown adipose tissue (BAT) are constitutively committed brown-fat progenitors, Sca-1(+) cells from skeletal muscle and subcutaneous white fat are highly inducible to differentiate into brown-like adipocytes upon stimulation with bone morphogenetic protein 7 (BMP7). Consistent with these findings, human preadipocytes isolated from subcutaneous white fat also exhibit the greatest inducible capacity to become brown adipocytes compared with cells isolated from mesenteric or omental white fat. When muscle-resident ScaPCs are re-engrafted into skeletal muscle of syngeneic mice, BMP7-treated ScaPCs efficiently develop into adipose tissue with brown fat-specific characteristics. Importantly, ScaPCs from obesity-resistant mice exhibit markedly higher thermogenic capacity compared with cells isolated from obesity-prone mice. These data establish the molecular characteristics of tissue-resident adipose progenitors and demonstrate a dynamic interplay between these progenitors and inductive signals that act in concert to specify brown adipocyte development.
Subject(s)
Adipocytes, Brown/physiology , Adipose Tissue, White/cytology , Antigens, Ly/metabolism , Cell Differentiation/physiology , Membrane Proteins/metabolism , Muscle, Skeletal/cytology , Stem Cells/physiology , Adipocytes, Brown/cytology , Animals , Blotting, Western , Bone Morphogenetic Protein 7/metabolism , Flow Cytometry , Fluorescent Antibody Technique , Humans , Immunohistochemistry , Mice , Thermogenesis/physiologyABSTRACT
Stem cells are rare and unique precursor cells that participate in the building and rebuilding of tissues and organs during embryogenesis, postnatal growth, and injury repair. Stem cells are distinctively endowed with the ability to both self-renew and differentiate, such that they can replenish the stem cell pool while continuing to produce the differentiated daughter cells that are essential for tissue function. Stem cell self-renewal/differentiation decisions must be carefully controlled during organogenesis, tissue homeostasis, and regeneration, as failure in stem cell maintenance or activation can lead to progressive tissue wasting, while unchecked self-renewal is a hallmark of many cancers. Here, we review evidence implicating the Notch signaling pathway, an evolutionarily conserved cell fate determinant with widespread roles in a variety of tissues and organisms, as a crucial regulator of stem cell behavior. As discussed below, this pathway plays varied and critical roles at multiple stages of organismal development, in lineage-specific differentiation of pluripotent embryonic stem cells, and in controlling stem cell numbers and activity in the context of age-related tissue degeneration, injury-induced tissue repair, and malignancy.
Subject(s)
Cell Differentiation , Receptors, Notch/metabolism , Regeneration , Signal Transduction , Stem Cells/cytology , Stem Cells/physiology , Animals , HumansABSTRACT
We describe the immunostaining methods we commonly use to detect the more robust cell markers identifying the various cell populations obtained by the enzymatic and or mechanical dissociation of muscle satellite cells: CD34, m-Cadherin, and Pax7, self-renewing muscle stem cells expressing CXCR4 and beta1-integrin, populations of proliferative myogenic progenitor cells expressing, Pax3, Pax7, Myf5, MyoD1, and desmin, differentiating myoblasts expressing myogenin and eMHC, and the CD45 expressing leukocyte lineage cells that infiltrate injured and regenerating skeletal muscle.
Subject(s)
Flow Cytometry/methods , Immunoassay/methods , Muscle Fibers, Skeletal/cytology , Stem Cells/cytology , Stem Cells/immunology , Animals , Antigens/analysis , Antigens/immunology , Cell Adhesion , Clone Cells , Mice , Mice, Transgenic , Staining and LabelingABSTRACT
Satellite cells reside beneath the basal lamina of skeletal muscle fibers and include cells that act as precursors for muscle growth and repair. Although they share a common anatomical localization and typically are considered a homogeneous population, satellite cells actually exhibit substantial heterogeneity. We used cell-surface marker expression to purify from the satellite cell pool a distinct population of skeletal muscle precursors (SMPs) that function as muscle stem cells. When engrafted into muscle of dystrophin-deficient mdx mice, purified SMPs contributed to up to 94% of myofibers, restoring dystrophin expression and significantly improving muscle histology and contractile function. Transplanted SMPs also entered the satellite cell compartment, renewing the endogenous stem cell pool and participating in subsequent rounds of injury repair. Together, these studies indicate the presence in adult skeletal muscle of prospectively isolatable muscle-forming stem cells and directly demonstrate the efficacy of myogenic stem cell transplant for treating muscle degenerative disease.
Subject(s)
Adult Stem Cells/cytology , Muscle, Skeletal/cytology , Satellite Cells, Skeletal Muscle/cytology , Adult Stem Cells/chemistry , Animals , Cell Separation , Dystrophin/genetics , Dystrophin/metabolism , Humans , Membrane Proteins/analysis , Mice , Mice, Inbred C57BL , Muscle Contraction , Muscle, Skeletal/physiology , Muscular Dystrophy, Animal/therapy , Satellite Cells, Skeletal Muscle/chemistry , Stem Cell TransplantationABSTRACT
Myoblast fusion is a highly regulated process that is important during muscle development and myofiber repair and is also likely to play a key role in the incorporation of donor cells in myofibers for cell-based therapy. Although several proteins involved in muscle cell fusion in Drosophila are known, less information is available on the regulation of this process in vertebrates, including humans. To identify proteins that are regulated during fusion of human myoblasts, microarray studies were performed on samples obtained from human fetal skeletal muscle of seven individuals. Primary muscle cells were isolated, expanded, induced to fuse in vitro, and gene expression comparisons were performed between myoblasts and early or late myotubes. Among the regulated genes, melanoma cell adhesion molecule (M-CAM) was found to be significantly downregulated during human fetal muscle cell fusion. M-CAM expression was confirmed on activated myoblasts, both in vitro and in vivo, and on myoendothelial cells (M-CAM(+) CD31(+)), which were positive for the myogenic markers desmin and MyoD. Lastly, in vitro functional studies using M-CAM RNA knockdown demonstrated that inhibition of M-CAM expression enhances myoblast fusion. These studies identify M-CAM as a novel marker for myogenic progenitors in human fetal muscle and confirm that downregulation of this protein promotes myoblast fusion.
Subject(s)
Biomarkers/metabolism , CD146 Antigen/metabolism , Cell Fusion , Fetus/anatomy & histology , Muscle, Skeletal , Myoblasts/physiology , Adult , Animals , CD146 Antigen/genetics , Cell Fractionation , Cells, Cultured , Endothelial Cells/metabolism , Female , Gene Expression Profiling , Gestational Age , Humans , Muscle, Skeletal/cytology , Muscle, Skeletal/embryology , Muscle, Skeletal/physiology , Myoblasts/cytology , Oligonucleotide Array Sequence Analysis , Pregnancy , RNA InterferenceABSTRACT
The process of muscle regeneration in normal and dystrophic muscle depends on locally produced cytokines and growth factors and requires the activity of the urokinase plasminogen activator/urokinase plasminogen activator receptor/plasminogen activator inhibitor-1 system. In this study we tested the effect of basic fibroblast growth factor (bFGF), hepatocyte growth factor (HGF) and transforming growth factor-beta (TGFbeta) on the fibrinolytic pattern of normal and dystrophic satellite cells, their mitogenic and motogenic activities and the dependence of such activities on the cell-associated fibrinolytic system. We have observed that the urokinase plasminogen activator (u-PA) receptor is weakly upregulated by bFGF in normal satellite cells, while it is strongly up-regulated by TGFbeta, mainly in dystrophic myoblasts. bFGF up-regulated u-PA in both normal and dystrophic myoblasts grown in primary culture, while a striking down-regulation was observed with TGFbeta. TGFbeta was the only growth factor able to exceptionally up-regulate plasminogen activator inhibitor-1 (PAI-1), mainly in dystrophic satellite cells. HGF did not show any activity on the fibrinolytic system. Proliferation and invasion into Matrigel matrices of normal and dystrophic cells occurred regardless of the growth factor-dependent regulation of the fibrinolytic system. Nevertheless, each growth factor required the efficiency of the constitutive cell-associated fibrinolytic system to operate, as shown by impairment of growth factor activity with antagonists of u-PA and of its receptor. Noteworthy, TGFbeta induced a dose-dependent increase of Matrigel invasion only in dystrophic myoblasts. Since TGFbeta-challenged dystrophic myoblasts undergo an exceptional up-regulation of the receptor and of PAI-1, we propose the possibility that the TGFbeta-induced fibrinolytic pattern (low urokinase plasminogen activator, high receptor and high PAI-1) may be exploited to promote survival and spreading of transplanted engineered myoblasts in Duchenne muscular dystrophy.