scholarly journals Menstrual Blood-derived Cells Confer Human Dystrophin Expression in the Murine Model of Duchenne Muscular Dystrophy via Cell Fusion and Myogenic Transdifferentiation

2007 ◽  
Vol 18 (5) ◽  
pp. 1586-1594 ◽  
Author(s):  
Chang-Hao Cui ◽  
Taro Uyama ◽  
Kenji Miyado ◽  
Masanori Terai ◽  
Satoru Kyo ◽  
...  
Keyword(s):  
Duchenne Muscular Dystrophy ◽  
Muscular Dystrophy ◽  
Progenitor Cells ◽  
Cell Fusion ◽  
Menstrual Blood ◽  
Mdx Mice ◽  
Dystrophin Expression ◽  
Human Dystrophin

Duchenne muscular dystrophy (DMD), the most common lethal genetic disorder in children, is an X-linked recessive muscle disease characterized by the absence of dystrophin at the sarcolemma of muscle fibers. We examined a putative endometrial progenitor obtained from endometrial tissue samples to determine whether these cells repair muscular degeneration in a murine mdx model of DMD. Implanted cells conferred human dystrophin in degenerated muscle of immunodeficient mdx mice. We then examined menstrual blood–derived cells to determine whether primarily cultured nontransformed cells also repair dystrophied muscle. In vivo transfer of menstrual blood–derived cells into dystrophic muscles of immunodeficient mdx mice restored sarcolemmal expression of dystrophin. Labeling of implanted cells with enhanced green fluorescent protein and differential staining of human and murine nuclei suggest that human dystrophin expression is due to cell fusion between host myocytes and implanted cells. In vitro analysis revealed that endometrial progenitor cells and menstrual blood–derived cells can efficiently transdifferentiate into myoblasts/myocytes, fuse to C2C12 murine myoblasts by in vitro coculturing, and start to express dystrophin after fusion. These results demonstrate that the endometrial progenitor cells and menstrual blood–derived cells can transfer dystrophin into dystrophied myocytes through cell fusion and transdifferentiation in vitro and in vivo.

scholarly journals Targeting fibrosis in the Duchenne Muscular Dystrophy mice model: an uphill battle

2021 ◽  
Author(s):  
Marine Theret ◽  
Marcela Low ◽  
Lucas Rempel ◽  
Fang Fang Li ◽  
Lin Wei Tung ◽  
...  
Keyword(s):  
Duchenne Muscular Dystrophy ◽  
Muscular Dystrophy ◽  
Large Scale ◽  
Drug Screen ◽  
Mdx Mice ◽  
Mice Model ◽  
Positive Effects ◽  
In Vitro Drug Screening

AbstractAimFibrosis is the most common complication from chronic diseases, and yet no therapy capable of mitigating its effects is available. Our goal is to unveil specific signallings regulating the fibrogenic process and to identify potential small molecule candidates that block fibrogenic differentiation of fibro/adipogenic progenitors.MethodWe performed a large-scale drug screen using muscle-resident fibro/adipogenic progenitors from a mouse model expressing EGFP under the Collagen1a1 promotor. We first confirmed that the EGFP was expressed in response to TGFβ1 stimulation in vitro. Then we treated cells with TGFβ1 alone or with drugs from two libraries of known compounds. The drugs ability to block the fibrogenic differentiation was quantified by imaging and flow cytometry. From a two-rounds screening, positive hits were tested in vivo in the mice model for the Duchenne muscular dystrophy (mdx mice). The histopathology of the muscles was assessed with picrosirius red (fibrosis) and laminin staining (myofiber size).Key findingsFrom the in vitro drug screening, we identified 21 drugs and tested 3 in vivo on the mdx mice. None of the three drugs significantly improved muscle histopathology.SignificanceThe in vitro drug screen identified various efficient compounds, none of them strongly inhibited fibrosis in skeletal muscle of mdx mice. To explain these observations, we hypothesize that in Duchenne Muscular Dystrophy, in which fibrosis is a secondary event due to chronic degeneration and inflammation, the drugs tested could have adverse effect on regeneration or inflammation, balancing off any positive effects and leading to the absence of significant results.

scholarly journals Differential Effects of Halofuginone Enantiomers on Muscle Fibrosis and Histopathology in Duchenne Muscular Dystrophy

2021 ◽  
Vol 22 (13) ◽  
pp. 7063
Author(s):  
Sharon Mordechay ◽  
Shaun Smullen ◽  
Paul Evans ◽  
Olga Genin ◽  
Mark Pines ◽  
...  
Keyword(s):  
Duchenne Muscular Dystrophy ◽  
Muscular Dystrophy ◽  
Motor Coordination ◽  
Mdx Mice ◽  
Cell Viability Assay ◽  
Muscle Fibrosis ◽  
Antifibrotic Therapy ◽  
Racemic Form ◽  
Viability Assay

Progressive loss of muscle and muscle function is associated with significant fibrosis in Duchenne muscular dystrophy (DMD) patients. Halofuginone, an analog of febrifugine, prevents fibrosis in various animal models, including those of muscular dystrophies. Effects of (+)/(−)-halofuginone enantiomers on motor coordination and diaphragm histopathology in mdx mice, the mouse model for DMD, were examined. Four-week-old male mice were treated with racemic halofuginone, or its separate enantiomers, for 10 weeks. Controls were treated with saline. Racemic halofuginone-treated mice demonstrated better motor coordination and balance than controls. However, (+)-halofuginone surpassed the racemic form’s effect. No effect was observed for (−)-halofuginone, which behaved like the control. A significant reduction in collagen content and degenerative areas, and an increase in utrophin levels were observed in diaphragms of mice treated with racemic halofuginone. Again, (+)-halofuginone was more effective than the racemic form, whereas (−)-halofuginone had no effect. Both racemic and (+)-halofuginone increased diaphragm myofiber diameters, with no effect for (−)-halofuginone. No effects were observed for any of the compounds tested in an in-vitro cell viability assay. These results, demonstrating a differential effect of the halofuginone enantiomers and superiority of (+)-halofuginone, are of great importance for future use of (+)-halofuginone as a DMD antifibrotic therapy.

scholarly journals Applications of CRISPR/Cas9 for the Treatment of Duchenne Muscular Dystrophy

2018 ◽  
Vol 8 (4) ◽  
pp. 38 ◽  
Author(s):  
Kenji Lim ◽  
Chantal Yoon ◽  
Toshifumi Yokota
Keyword(s):  
Duchenne Muscular Dystrophy ◽  
Muscular Dystrophy ◽  
Wide Spectrum ◽  
Membrane Integrity ◽  
Dystrophin Gene ◽  
Muscle Membrane ◽  
Reading Frame ◽  
Long Term Treatment

Duchenne muscular dystrophy (DMD) is a fatal X-linked recessive neuromuscular disease prevalent in 1 in 3500 to 5000 males worldwide. As a result of mutations that interrupt the reading frame of the dystrophin gene (DMD), DMD is characterized by a loss of dystrophin protein that leads to decreased muscle membrane integrity, which increases susceptibility to degeneration. CRISPR/Cas9 technology has garnered interest as an avenue for DMD therapy due to its potential for permanent exon skipping, which can restore the disrupted DMD reading frame in DMD and lead to dystrophin restoration. An RNA-guided DNA endonuclease system, CRISPR/Cas9 allows for the targeted editing of specific sequences in the genome. The efficacy and safety of CRISPR/Cas9 as a therapy for DMD has been evaluated by numerous studies in vitro and in vivo, with varying rates of success. Despite the potential of CRISPR/Cas9-mediated gene editing for the long-term treatment of DMD, its translation into the clinic is currently challenged by issues such as off-targeting, immune response activation, and sub-optimal in vivo delivery. Its nature as being mostly a personalized form of therapy also limits applicability to DMD patients, who exhibit a wide spectrum of mutations. This review summarizes the various CRISPR/Cas9 strategies that have been tested in vitro and in vivo for the treatment of DMD. Perspectives on the approach will be provided, and the challenges faced by CRISPR/Cas9 in its road to the clinic will be briefly discussed.

Nintedanib as a new therapeutic agent for Duchenne muscular dystrophy: preclinical in vitro and in vivo studies

2017 ◽  
Vol 27 ◽  
pp. S191
Author(s):  
P. Piñol ◽  
E. Fernández-Simón ◽  
X. Suárez ◽  
N. de Luna ◽  
A. Molins ◽  
...  
Keyword(s):  
Duchenne Muscular Dystrophy ◽  
Muscular Dystrophy ◽  
Therapeutic Agent ◽  
In Vivo Studies

scholarly journals Soluble guanylate cyclase stimulation mitigates skeletal and cardiac muscle dysfunction in a mdx model of Duchenne muscular dystrophy

2021 ◽  
Author(s):  
Ling Zhang ◽  
Yuanyuan Xu ◽  
Keyvan Yousefi ◽  
Camila I. Irion ◽  
Roger A. Alvarez ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Duchenne Muscular Dystrophy ◽  
Muscular Dystrophy ◽  
Guanylate Cyclase ◽  
Soluble Guanylate Cyclase ◽  
Signal Propagation ◽  
Guanosine Monophosphate ◽  
Muscle Pathology ◽  
Mdx Mice

AbstractThe impairment of neuronal nitric oxide synthase (nNOS) signaling contributes to disease pathology in the muscle wasting disorder Duchenne muscular dystrophy (DMD). nNOS signal propagation occurs through nitric oxide sensitive soluble guanylate cyclase (sGC), a critical source of cyclic guanosine monophosphate (cGMP) in muscle. Although both nNOS and sGC activity are impaired in DMD patients, little is known about sGC as a therapeutic target. In this study, we tested the hypothesis that stimulating sGC activity with the allosteric agonist BAY41-8543 mitigates striated muscle pathology in the mdx4cv mouse model of DMD. In contrast to DMD patients, mdx mice exhibited greater basal sGC activity than wild type controls with preservation of cGMP levels due partly to upregulation of sGC in some muscles. Stimulating sGC activity in mdx mice with BAY41-8543 substantially reduced skeletal muscle damage, macrophage densities and inflammation and significantly increased resistance to contraction-induced fatigue. BAY41-8543 also enhanced in vivo diaphragm function while reducing breathing irregularities suggesting improved respiratory function. BAY41-8543 attenuated cardiac hypertrophic remodeling, fibrosis and diastolic dysfunction including left atrium enlargement in aged mdx mice. Overall, sGC stimulation significantly mitigated skeletal and cardio-respiratory dysfunction in mdx4cv mice. Importantly, this study provides compelling pre-clinical evidence supporting sGC as a novel target in DMD and the repurposing of FDA-approved sGC stimulators, such as riociguat and veraciguat, as a novel therapeutic approach for DMD.

scholarly journals Role of proteoglycans and glycosaminoglycans in Duchenne muscular dystrophy

Glycobiology ◽  
2018 ◽  
Vol 29 (2) ◽  
pp. 110-123 ◽  
Author(s):  
Laurino Carmen ◽  
Vadala’ Maria ◽  
Julio Cesar Morales-Medina ◽  
Annamaria Vallelunga ◽  
Beniamino Palmieri ◽  
...  
Keyword(s):  
Duchenne Muscular Dystrophy ◽  
Muscular Dystrophy ◽  
Mammalian Cells ◽  
Muscle Development ◽  
Glycoprotein Complex ◽  
Experimental Therapies ◽  
Dystrophin Protein

Abstract Duchenne muscular dystrophy (DMD) is an inherited fatal X-linked myogenic disorder with a prevalence of 1 in 3500 male live births. It affects voluntary muscles, and heart and breathing muscles. DMD is characterized by continuous degeneration and regeneration cycles resulting in extensive fibrosis and a progressive reduction in muscle mass. Since the identification of a reduction in dystrophin protein as the cause of this disorder, numerous innovative and experimental therapies, focusing on increasing the levels of dystrophin, have been proposed, but the clinical improvement has been unsatisfactory. Dystrophin forms the dystrophin-associated glycoprotein complex and its proteins have been studied as a promising novel therapeutic target to treat DMD. Among these proteins, cell surface glycosaminoglycans (GAGs) are found almost ubiquitously on the surface and in the extracellular matrix (ECM) of mammalian cells. These macromolecules interact with numerous ligands, including ECM constituents, adhesion molecules and growth factors that play a crucial role in muscle development and maintenance. In this article, we have reviewed in vitro, in vivo and clinical studies focused on the functional role of GAGs in the pathophysiology of DMD with the final aim of summarizing the state of the art of GAG dysregulation within the ECM in DMD and discussing future therapeutic perspectives.

scholarly journals Enhanced CRISPR-Cas9 correction of Duchenne muscular dystrophy in mice by a self-complementary AAV delivery system

2020 ◽  
Vol 6 (8) ◽  
pp. eaay6812 ◽  
Author(s):  
Yu Zhang ◽  
Hui Li ◽  
Yi-Li Min ◽  
Efrain Sanchez-Ortiz ◽  
Jian Huang ◽  
...  
Keyword(s):  
Duchenne Muscular Dystrophy ◽  
Muscular Dystrophy ◽  
Genome Editing ◽  
Dystrophin Gene ◽  
Adeno Associated Virus ◽  
Guide Rnas ◽  
Dystrophin Expression ◽  
Cas9 Nuclease ◽  
High Doses

Duchenne muscular dystrophy (DMD) is a lethal neuromuscular disease caused by mutations in the dystrophin gene (DMD). Previously, we applied CRISPR-Cas9–mediated “single-cut” genome editing to correct diverse genetic mutations in animal models of DMD. However, high doses of adeno-associated virus (AAV) are required for efficient in vivo genome editing, posing challenges for clinical application. In this study, we packaged Cas9 nuclease in single-stranded AAV (ssAAV) and CRISPR single guide RNAs in self-complementary AAV (scAAV) and delivered this dual AAV system into a mouse model of DMD. The dose of scAAV required for efficient genome editing were at least 20-fold lower than with ssAAV. Mice receiving systemic treatment showed restoration of dystrophin expression and improved muscle contractility. These findings show that the efficiency of CRISPR-Cas9–mediated genome editing can be substantially improved by using the scAAV system. This represents an important advancement toward therapeutic translation of genome editing for DMD.

In vitro assessment of anti-fibrotic activity does not predict their in vivo efficacy in murine models of Duchenne muscular dystrophy

Life Sciences ◽  
2021 ◽  
pp. 119482
Author(s):  
Marine Theret ◽  
Marcela Low ◽  
Lucas Rempel ◽  
Fang Fang Li ◽  
Lin Wei Tung ◽  
...  
Keyword(s):  
Duchenne Muscular Dystrophy ◽  
Muscular Dystrophy ◽  
Murine Models ◽  
In Vivo Efficacy ◽  
In Vitro Assessment

scholarly journals CD13 is a Critical Regulator of Cell-cell Fusion in Osteoclastogenesis

2020 ◽  
Author(s):  
Mallika Ghosh ◽  
Ivo Kalajzic ◽  
Hector Leonardo Aguila ◽  
Linda H Shapiro
Keyword(s):  
Bone Marrow ◽  
Bone Formation ◽  
Progenitor Cells ◽  
Cell Fusion ◽  
Formation Rate ◽  
Giant Cells ◽  
Bone Formation Rate ◽  
Cell Cell

AbstractIn vertebrates, bone formation is dynamically controlled by the activity of two specialized cell types: the bone-generating osteoblasts and bone-degrading osteoclasts. Osteoblasts produce the soluble receptor activator of NFκB ligand (RANKL) that binds to its receptor RANK on the surface of osteoclast precursor cells to promote osteoclastogenesis, a process that involves cell-cell fusion and assembly of molecular machinery to ultimately degrade the bone. CD13 is a transmembrane aminopeptidase that is highly expressed in cells of myeloid lineage has been shown to regulate dynamin-dependent receptor endocytosis and recycling and is a necessary component of actin cytoskeletal organization. In the present study, we show that CD13-deficient mice display a normal distribution of osteoclast progenitor populations in the bone marrow, but present a low bone density phenotype. Further, the endosteal bone formation rate is similar between genotypes, indicating a defect in osteoclast-specific function in vivo. Loss of CD13 led to exaggerated in vitro osteoclastogenesis as indicated by significantly enhanced fusion of bone marrow-derived multinucleated osteoclasts in the presence of M-CSF and RANKL, resulting in abnormally large cells with remarkably high numbers of nuclei with a concomitant increase in bone resorption activity. Similarly, we also observed increased formation of multinucleated giant cells (MGC) in CD13KO bone marrow progenitor cells stimulated with IL-4 and IL-13, suggesting that CD13 may regulate cell-cell fusion events via a common pathway, independent of RANKL signaling. Mechanistically, while expression levels of the fusion-regulatory proteins dynamin and DC-STAMP are normally downregulated as fusion progresses in fusion-competent mononucleated progenitor cells, in the absence of CD13 they are uniformly sustained at high levels, even in mature multi-nucleated osteoclasts. Taken together, we conclude that CD13 may regulate cell-cell fusion by controlling expression and localization of key fusion proteins that are critical for both osteoclast and MGC fusion.

Contractile efficiency of dystrophic mdx mouse muscle: in vivo and ex vivo assessment of adaptation to exercise of functional end points

2017 ◽  
Vol 122 (4) ◽  
pp. 828-843 ◽  
Author(s):  
Roberta Francesca Capogrosso ◽  
Paola Mantuano ◽  
Anna Cozzoli ◽  
Francesca Sanarica ◽  
Ada Maria Massari ◽  
...  
Keyword(s):  
Duchenne Muscular Dystrophy ◽  
Muscular Dystrophy ◽  
Muscle Damage ◽  
Functional Adaptation ◽  
Mdx Mouse ◽  
Mdx Mice ◽  
Chronic Exercise ◽  
Exercise Protocol ◽  
Dystrophic Muscle

Progressive weakness is a typical feature of Duchenne muscular dystrophy (DMD) patients and is exacerbated in the benign mdx mouse model by in vivo treadmill exercise. We hypothesized a different threshold for functional adaptation of mdx muscles in response to the duration of the exercise protocol. In vivo weakness was confirmed by grip strength after 4, 8, and 12 wk of exercise in mdx mice. Torque measurements revealed that exercise-related weakness in mdx mice correlated with the duration of the protocol, while wild-type (WT) mice were stronger. Twitch and tetanic forces of isolated diaphragm and extensor digitorum longus (EDL) muscles were lower in mdx compared with WT mice. In mdx, both muscle types exhibited greater weakness after a single exercise bout, but only in EDL after a long exercise protocol. As opposite to WT muscles, mdx EDL ones did not show any exercise-induced adaptations against eccentric contraction force drop. qRT-PCR analysis confirmed the maladaptation of genes involved in metabolic and structural remodeling, while damage-related genes remained significantly upregulated and angiogenesis impaired. Phosphorylated AMP kinase level increased only in exercised WT muscle. The severe histopathology and the high levels of muscular TGF-β1 and of plasma matrix metalloproteinase-9 confirmed the persistence of muscle damage in mdx mice. Therefore, dystrophic muscles showed a partial degree of functional adaptation to chronic exercise, although not sufficient to overcome weakness nor signs of damage. The improved understanding of the complex mechanisms underlying maladaptation of dystrophic muscle paves the way to a better managment of DMD patients. NEW & NOTEWORTHY We focused on the adaptation/maladaptation of dystrophic mdx mouse muscles to a standard protocol of exercise to provide guidance in the development of more effective drug and physical therapies in Duchenne muscular dystrophy. The mdx muscles showed a modest functional adaptation to chronic exercise, but it was not sufficient to overcome the progressive in vivo weakness, nor to counter signs of muscle damage. Therefore, a complex involvement of multiple systems underlies the maladaptive response of dystrophic muscle.

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