Publications by authors named "Ornella Cappellari"

20 Publications

  • Page 1 of 1

BB-301: a silence and replace AAV-based vector for the treatment of oculopharyngeal muscular dystrophy.

Mol Ther Nucleic Acids 2021 Jun 18;24:67-78. Epub 2021 Feb 18.

Benitec Biopharma, Inc., Hayward, CA 94545, USA.

Oculopharyngeal muscular dystrophy (OPMD) is a rare autosomal dominant disease that results from an alanine expansion in the N-terminal domain of Poly-A Binding Protein Nuclear-1 (PABPN1). We have recently demonstrated that a two-vector gene therapy strategy significantly ameliorated the pathology in a mouse model of OPMD. This approach entailed intramuscular injection of two recombinant adeno-associated viruses (AAVs), one expressing three short hairpin RNAs (shRNAs) to silence both mutant and wild-type PABPN1 and one expressing a codon-optimized version of PABPN1 that is insensitive to RNA interference. Here we report the continued development of this therapeutic strategy by delivering "silence and replace" sequences in a single AAV vector named BB-301. This construct is composed of a modified AAV serotype 9 (AAV9) capsid that expresses a unique single bifunctional construct under the control of the muscle-specific Spc5-12 promoter for the co-expression of both the codon-optimized PABPN1 protein and two small inhibitory RNAs (siRNAs) against PABPN1 modeled into microRNA (miRNA) backbones. A single intramuscular injection of BB-301 results in robust inhibition of mutant PABPN1 and concomitant replacement of the codon-optimized PABPN1 protein. The treatment restores muscle strength and muscle weight to wild-type levels as well as improving other physiological hallmarks of the disease in a mouse model of OPMD.
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http://dx.doi.org/10.1016/j.omtn.2021.02.017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7940701PMC
June 2021

Gain-of-Function STIM1 L96V Mutation Causes Myogenesis Alteration in Muscle Cells From a Patient Affected by Tubular Aggregate Myopathy.

Front Cell Dev Biol 2021 26;9:635063. Epub 2021 Feb 26.

Department of Pharmacy-Drug Sciences, University of Bari, Bari, Italy.

Tubular Aggregate Myopathy (TAM) is a hereditary ultra-rare muscle disorder characterized by muscle weakness and cramps or myasthenic features. Biopsies from TAM patients show the presence of tubular aggregates originated from sarcoplasmic reticulum due to altered Ca homeostasis. TAM is caused by gain-of-function mutations in STIM1 or ORAI1, proteins responsible for Store-Operated-Calcium-Entry (SOCE), a pivotal mechanism in Ca signaling. So far there is no cure for TAM and the mechanisms through which or gene mutation lead to muscle dysfunction remain to be clarified. It has been established that post-natal myogenesis critically relies on Ca influx through SOCE. To explore how Ca homeostasis dysregulation associated with TAM impacts on muscle differentiation cascade, we here performed a functional characterization of myoblasts and myotubes deriving from patients carrying STIM1 L96V mutation by using fura-2 cytofluorimetry, high content imaging and real-time PCR. We demonstrated a higher resting Ca concentration and an increased SOCE in STIM1 mutant compared with control, together with a compensatory down-regulation of genes involved in Ca handling (. Differentiating STIM1 L96V myoblasts persisted in a mononuclear state and the fewer multinucleated myotubes had distinct morphology and geometry of mitochondrial network compared to controls, indicating a defect in the late differentiation phase. The alteration in myogenic pathway was confirmed by gene expression analysis regarding early () and late () differentiation markers together with mitochondrial markers (. We provided evidences of mechanisms responsible for a defective myogenesis associated to TAM mutant and validated a reliable cellular model usefull for TAM preclinical studies.
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http://dx.doi.org/10.3389/fcell.2021.635063DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7952532PMC
February 2021

Ultrasonography validation for early alteration of diaphragm echodensity and function in the mdx mouse model of Duchenne muscular dystrophy.

PLoS One 2021 12;16(1):e0245397. Epub 2021 Jan 12.

Section of Pharmacology, Department of Pharmacy-Drug Sciences, University of Bari "Aldo Moro", Bari, Italy.

The mdx mouse model of Duchenne muscular dystrophy is characterized by functional and structural alterations of the diaphragm since early stages of pathology, closely resembling patients' condition. In recent years, ultrasonography has been proposed as a useful longitudinal non-invasive technique to assess mdx diaphragm dysfunction and evaluate drug efficacy over time. To date, only a few preclinical studies have been conducted. Therefore, an independent validation of this method by different laboratories is needed to increase results reliability and reduce biases. Here, we performed diaphragm ultrasonography in 3- and 6-month-old mdx mice, the preferred age-window for pharmacology studies. The alteration of diaphragm function over time was measured as diaphragm ultrasound movement amplitude. At the same time points, a first-time assessment of diaphragm echodensity was performed, as an experimental index of progressive loss of contractile tissue. A parallel evaluation of other in vivo and ex vivo dystrophy-relevant readouts was carried out. Both 3- and 6-month-old mdx mice showed a significant decrease in diaphragm amplitude compared to wild type (wt) mice. This index was well-correlated either with in vivo running performance or ex vivo isometric tetanic force of isolated diaphragm. In addition, diaphragms from 6-month-old dystrophic mice were also highly susceptible to eccentric contraction ex vivo. Importantly, we disclosed an age-dependent increase in echodensity in mdx mice not observed in wt animals, which was independent from abdominal wall thickness. This was accompanied by a notable increase of pro-fibrotic TGF-β1 levels in the mdx diaphragm and of non-muscle tissue amount in diaphragm sections stained by hematoxylin & eosin. Our findings corroborate the usefulness of diaphragm ultrasonography in preclinical drug studies as a powerful tool to monitor mdx pathology progression since early stages.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0245397PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7802948PMC
June 2021

Simvastatin Treatment Does Not Ameliorate Muscle Pathophysiology in a Mouse Model for Duchenne Muscular Dystrophy.

J Neuromuscul Dis 2020 Oct 5. Epub 2020 Oct 5.

Department of Comparative Biomedical Sciences, Neuromuscular Diseases Group, Royal Veterinary College, London, United Kingdom.

Duchenne muscular dystrophy is an X-linked, recessive muscular dystrophy in which the absence of the dystrophin protein leads to fibrosis, inflammation and oxidative stress, resulting in loss of muscle tissue. Drug repurposing, i.e. using drugs already approved for other disorders, is attractive as it decreases development time. Recent studies suggested that simvastatin, a cholesterol lowering drug used for cardiovascular diseases, has beneficial effects on several parameters in mdx mice. To validate properly the effectiveness of simvastatin, two independent labs tested the effects of 12-week simvastatin treatment in either young (starting at 4 weeks of age) or adult (starting at 12 weeks of age) mdx mice. In neither study were benefits of simvastatin treatment observed on muscle function, histology or expression of genes involved in fibrosis, regeneration, oxidative stress and autophagy. Unexpectedly, although the treatment protocol was similar, simvastatin plasma levels were found to be much lower than observed in a previous study. In conclusion, in two laboratories, simvastatin did not ameliorate disease pathology in mdx mice, which could either be due to the ineffectiveness of simvastatin itself or due to the low simvastatin plasma levels following oral administration via the food.
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http://dx.doi.org/10.3233/JND-200524DOI Listing
October 2020

Ergogenic Effect of BCAAs and L-Alanine Supplementation: Proof-of-Concept Study in a Murine Model of Physiological Exercise.

Nutrients 2020 Jul 30;12(8). Epub 2020 Jul 30.

Section of Pharmacology, Department of Pharmacy-Drug Sciences, University of Bari "Aldo Moro", Orabona 4-Campus, 70125 Bari, Italy.

Background: Branched-chain amino acids (BCAAs: leucine, isoleucine, valine) account for 35% of skeletal muscle essential amino acids (AAs). As such, they must be provided in the diet to support peptide synthesis and inhibit protein breakdown. Although substantial evidence has been collected about the potential usefulness of BCAAs in supporting muscle function and structure, dietary supplements containing BCAAs alone may not be effective in controlling muscle protein turnover, due to the rate-limiting bioavailability of other AAs involved in BCAAs metabolism.

Methods: We aimed to evaluate the in vivo/ex vivo effects of a 4-week treatment with an oral formulation containing BCAAs alone (2:1:1) on muscle function, structure, and metabolism in a murine model of physiological exercise, which was compared to three modified formulations combining BCAAs with increasing concentrations of L-Alanine (ALA), an AA controlling BCAAs catabolism.

Results: A preliminary pharmacokinetic study confirmed the ability of ALA to boost up BCAAs bioavailability. After 4 weeks, (BCAAs + 2ALA) had the best protective effect on mice force and fatigability, as well as on muscle morphology and metabolic indices.

Conclusion: Our study corroborates the use of BCAAs + ALA to support muscle health during physiological exercise, underlining how the relative BCAAs/ALA ratio is important to control BCAAs distribution.
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http://dx.doi.org/10.3390/nu12082295DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7468919PMC
July 2020

"The Social Network" and Muscular Dystrophies: The Lesson Learnt about the Niche Environment as a Target for Therapeutic Strategies.

Cells 2020 07 9;9(7). Epub 2020 Jul 9.

Section of Pharmacology, Department of Pharmacy-Drug Sciences, University of Bari "Aldo Moro", via Orabona 4-Campus, 70125 Bari, Italy.

The muscle stem cells niche is essential in neuromuscular disorders. Muscle injury and myofiber death are the main triggers of muscle regeneration via satellite cell activation. However, in degenerative diseases such as muscular dystrophy, regeneration still keep elusive. In these pathologies, stem cell loss occurs over time, and missing signals limiting damaged tissue from activating the regenerative process can be envisaged. It is unclear what comes first: the lack of regeneration due to satellite cell defects, their pool exhaustion for degeneration/regeneration cycles, or the inhibitory mechanisms caused by muscle damage and fibrosis mediators. Herein, Duchenne muscular dystrophy has been taken as a paradigm, as several drugs have been tested at the preclinical and clinical levels, targeting secondary events in the complex pathogenesis derived from lack of dystrophin. We focused on the crucial roles that pro-inflammatory and pro-fibrotic cytokines play in triggering muscle necrosis after damage and stimulating satellite cell activation and self-renewal, along with growth and mechanical factors. These processes contribute to regeneration and niche maintenance. We review the main effects of drugs on regeneration biomarkers to assess whether targeting pathogenic events can help to protect niche homeostasis and enhance regeneration efficiency other than protecting newly formed fibers from further damage.
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http://dx.doi.org/10.3390/cells9071659DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7407800PMC
July 2020

Safety issues and harmful pharmacological interactions of nutritional supplements in Duchenne muscular dystrophy: considerations for Standard of Care and emerging virus outbreaks.

Pharmacol Res 2020 08 30;158:104917. Epub 2020 May 30.

Unit of Pharmacology, Department of Pharmacy and Drug Sciences, University of Bari Aldo Moro, Bari, Italy. Electronic address:

At the moment, little treatment options are available for Duchenne muscular dystrophy (DMD). The absence of the dystrophin protein leads to a complex cascade of pathogenic events in myofibres, including chronic inflammation and oxidative stress as well as altered metabolism. The attention towards dietary supplements in DMD is rapidly increasing, with the aim to counteract pathology-related alteration in nutrient intake, the consequences of catabolic distress or to enhance the immunological response of patients as nowadays for the COVID-19 pandemic emergency. By definition, supplements do not exert therapeutic actions, although a great confusion may arise in daily life by the improper distinction between supplements and therapeutic compounds. For most supplements, little research has been done and little evidence is available concerning their effects in DMD as well as their preventing actions against infections. Often these are not prescribed by clinicians and patients/caregivers do not discuss the use with their clinical team. Then, little is known about the real extent of supplement use in DMD patients. It is mistakenly assumed that, since compounds are of natural origin, if a supplement is not effective, it will also do no harm. However, supplements can have serious side effects and also have harmful interactions, in terms of reducing efficacy or leading to toxicity, with other therapies. It is therefore pivotal to shed light on this unclear scenario for the sake of patients. This review discusses the supplements mostly used by DMD patients, focusing on their potential toxicity, due to a variety of mechanisms including pharmacodynamic or pharmacokinetic interactions and contaminations, as well as on reports of adverse events. This overview underlines the need for caution in uncontrolled use of dietary supplements in fragile populations such as DMD patients. A culture of appropriate use has to be implemented between clinicians and patients' groups.
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http://dx.doi.org/10.1016/j.phrs.2020.104917DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7261230PMC
August 2020

Cisplatin-Induced Skeletal Muscle Dysfunction: Mechanisms and Counteracting Therapeutic Strategies.

Int J Mol Sci 2020 Feb 13;21(4). Epub 2020 Feb 13.

Department of Pharmacy-Drug Sciences, University of Bari, 70125 Bari, Italy.

Among the severe side effects induced by cisplatin chemotherapy, muscle wasting is the most relevant one. This effect is a major cause for a clinical decline of cancer patients, since it is a negative predictor of treatment outcome and associated to increased mortality. However, despite its toxicity even at low doses, cisplatin remains the first-line therapy for several types of solid tumors. Thus, effective pharmacological treatments counteracting or minimizing cisplatin-induced muscle wasting are urgently needed. The dissection of the molecular pathways responsible for cisplatin-induced muscle dysfunction gives the possibility to identify novel promising therapeutic targets. In this context, the use of animal model of cisplatin-induced cachexia is very useful. Here, we report an update of the most relevant researches on the mechanisms underlying cisplatin-induced muscle wasting and on the most promising potential therapeutic options to preserve muscle mass and function.
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http://dx.doi.org/10.3390/ijms21041242DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7072891PMC
February 2020

Established PABPN1 intranuclear inclusions in OPMD muscle can be efficiently reversed by AAV-mediated knockdown and replacement of mutant expanded PABPN1.

Hum Mol Genet 2019 10;28(19):3301-3308

Centres of Gene and Cell Therapy and Biomedical Sciences, School of Biological Sciences, Royal Holloway, University of London, Egham TW20 0EX, Surrey, UK.

Oculopharyngeal muscular dystrophy (OPMD) is a rare autosomal dominant late-onset muscular dystrophy affecting approximately 1:100 000 individuals in Europe. OPMD is mainly characterized by progressive eyelid drooping (ptosis) and dysphagia although muscles of the limbs can also be affected late in life. This muscle disease is due to a trinucleotide repeat expansion in the polyA-binding protein nuclear-1 gene. Patients express a protein with an 11-18 alanine tract that is misfolded and prone to form intranuclear inclusions, which are the hallmark of the disease. Other features of OPMD include muscle fibrosis and atrophy in affected muscles. Currently, no pharmacological treatments are available, and OPMD patients can only be referred to surgeons for cricopharyngeal myotomy or corrective surgery of extraocular muscles to ease ptosis. We recently tested a two-AAV `silence' and `replace' vector-based gene therapy treatment in a mouse model of OPMD. We demonstrate here that this gene therapy approach can revert already established insoluble aggregates and partially rescues the muscle from atrophy, which are both crucially important since in most cases OPMD patients already have an established disease when diagnosed. This strategy also prevents the formation of muscle fibrosis and stabilizes the muscle strength to the level of healthy muscles. Furthermore, we show here that similar results can be obtained using a single AAV vector incorporating both the `silence' and `replace' cassettes. These results further support the application of a gene therapy approach as a novel treatment for OPMD in humans.
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http://dx.doi.org/10.1093/hmg/ddz167DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7343048PMC
October 2019

Inhibition of myostatin improves muscle atrophy in oculopharyngeal muscular dystrophy (OPMD).

J Cachexia Sarcopenia Muscle 2019 10 7;10(5):1016-1026. Epub 2019 May 7.

Centres of Gene and Cell Therapy and Biomedical Sciences, School of Biological Sciences, Royal Holloway-University of London, Surrey, UK.

Background: Oculopharyngeal muscular dystrophy (OPMD) is a late-onset muscle disease affecting one per 80 000 of the general population characterized by profound dysphagia and ptosis, and limb weakness at later stages. Affected muscles are characterized by increased fibrosis and atrophy. Myostatin is a negative regulator of muscle mass, and inhibition of myostatin has been demonstrated to ameliorate symptoms in dystrophic muscles.

Methods: In this study, we performed a systemic delivery of a monoclonal antibody to immunologically block myostatin in the A17 mouse model of OPMD. The mice were administered a weekly dose of 10 mg/kg RK35 intraperitonially for 10 weeks, following which histological analyses were performed on the samples.

Results: This treatment significantly (P < 0.01) improved body mass (11%) and muscle mass (for the tibialis anterior and extensor digitorum longus by 19% and 41%) in the A17 mice treated with RK35 when compared to saline controls. Similarly, a significantly (P < 0.01) increased muscle strength (18% increase in maximal tetanic force) and myofibre diameter (17% and 44% for the tibialis anterior and extensor digitorum longus), and reduced expression of markers of muscle fibrosis (40% reduction in area of expression), was also observed. No change in the density of intranuclear inclusions (a hallmark of disease progression of OPMD) was however observed.

Conclusions: Our study supports the clinical translation of such antibody-mediated inhibition of myostatin as a treatment of OPMD. This strategy has implications to be used as adjuvant therapies with gene therapy based approaches, or to stabilize the muscle prior to myoblast transplantation.
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http://dx.doi.org/10.1002/jcsm.12438DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6818462PMC
October 2019

Pharmacological modulation of the ER stress response ameliorates oculopharyngeal muscular dystrophy.

Hum Mol Genet 2019 05;28(10):1694-1708

Sorbonne Université, INSERM, Association Institut de Myologie, Centre de Recherche en Myologie, UMRS974, 47 bd de l'Hôpital, Paris, France.

Oculopharyngeal muscular dystrophy (OPMD) is a rare late onset genetic disease leading to ptosis, dysphagia and proximal limb muscles at later stages. A short abnormal (GCN) triplet expansion in the polyA-binding protein nuclear 1 (PABPN1) gene leads to PABPN1-containing aggregates in the muscles of OPMD patients. Here we demonstrate that treating mice with guanabenz acetate (GA), an FDA-approved antihypertensive drug, reduces the size and number of nuclear aggregates, improves muscle force, protects myofibers from the pathology-derived turnover and decreases fibrosis. GA targets various cell processes, including the unfolded protein response (UPR), which acts to attenuate endoplasmic reticulum (ER) stress. We demonstrate that GA increases both the phosphorylation of the eukaryotic translation initiation factor 2α subunit and the splicing of Xbp1, key components of the UPR. Altogether these data show that modulation of protein folding regulation is beneficial for OPMD and promote the further development of GA or its derivatives for treatment of OPMD in humans. Furthermore, they support the recent evidences that treating ER stress could be therapeutically relevant in other more common proteinopathies.
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http://dx.doi.org/10.1093/hmg/ddz007DOI Listing
May 2019

A Dynamic Simulation of Musculoskeletal Function in the Mouse Hindlimb During Trotting Locomotion.

Front Bioeng Biotechnol 2018 16;6:61. Epub 2018 May 16.

Structure and Motion Lab, Department of Comparative Biomedical Sciences, Royal Veterinary College, Hatfield, United Kingdom.

Mice are often used as animal models of various human neuromuscular diseases, and analysis of these models often requires detailed gait analysis. However, little is known of the dynamics of the mouse musculoskeletal system during locomotion. In this study, we used computer optimization procedures to create a simulation of trotting in a mouse, using a previously developed mouse hindlimb musculoskeletal model in conjunction with new experimental data, allowing muscle forces, activation patterns, and levels of mechanical work to be estimated. Analyzing musculotendon unit (MTU) mechanical work throughout the stride allowed a deeper understanding of their respective functions, with the rectus femoris MTU dominating the generation of positive and negative mechanical work during the swing and stance phases. This analysis also tested previous functional inferences of the mouse hindlimb made from anatomical data alone, such as the existence of a proximo-distal gradient of muscle function, thought to reflect adaptations for energy-efficient locomotion. The results do not strongly support the presence of this gradient within the mouse musculoskeletal system, particularly given relatively high negative net work output from the ankle plantarflexor MTUs, although more detailed simulations could test this further. This modeling analysis lays a foundation for future studies of the control of vertebrate movement through the development of neuromechanical simulations.
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http://dx.doi.org/10.3389/fbioe.2018.00061DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5964171PMC
May 2018

Functional Rescue of Dystrophin Deficiency in Mice Caused by Frameshift Mutations Using Campylobacter jejuni Cas9.

Mol Ther 2018 06 30;26(6):1529-1538. Epub 2018 Mar 30.

Center for Genome Engineering, Institute for Basic Science (IBS), Seoul 08826, Republic of Korea; Department of Basic Science, University of Science and Technology, Daejeon 34113, Republic of Korea; Department of Chemistry, Seoul National University, Seoul 08826, Republic of Korea. Electronic address:

Duchenne muscular dystrophy (DMD) is a fatal, X-linked muscle-wasting disease caused by mutations in the DMD gene. In 51% of DMD cases, a reading frame is disrupted because of deletion of several exons. Here, we show that CjCas9 derived from Campylobacter jejuni can be used as a gene-editing tool to correct an out-of-frame Dmd exon in Dmd knockout mice. Herein, we used Cas9 derived from S. pyogenes to generate Dmd knockout mice with a frameshift mutation in Dmd gene. Then, we expressed CjCas9, its single-guide RNA, and the EGFP gene in the tibialis anterior muscle of the Dmd knockout mice using an all-in-one adeno-associated virus (AAV) vector. CjCas9 cleaved the target site in the Dmd gene efficiently in vivo and induced small insertions or deletions at the target site. This treatment resulted in conversion of the disrupted Dmd reading frame from out of frame to in frame, leading to the expression of dystrophin in the sarcolemma. Importantly, muscle strength was enhanced in the CjCas9-treated muscles, without off-target mutations, indicating high efficiency and specificity of CjCas9. This work suggests that in vivo DMD frame correction, mediated by CjCas9, has great potential for the treatment of DMD and other neuromuscular diseases.
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http://dx.doi.org/10.1016/j.ymthe.2018.03.018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5986736PMC
June 2018

Three-Dimensional Human iPSC-Derived Artificial Skeletal Muscles Model Muscular Dystrophies and Enable Multilineage Tissue Engineering.

Cell Rep 2018 Apr;23(3):899-908

Department of Cell and Developmental Biology, University College London, London WC1E 6DE, UK. Electronic address:

Generating human skeletal muscle models is instrumental for investigating muscle pathology and therapy. Here, we report the generation of three-dimensional (3D) artificial skeletal muscle tissue from human pluripotent stem cells, including induced pluripotent stem cells (iPSCs) from patients with Duchenne, limb-girdle, and congenital muscular dystrophies. 3D skeletal myogenic differentiation of pluripotent cells was induced within hydrogels under tension to provide myofiber alignment. Artificial muscles recapitulated characteristics of human skeletal muscle tissue and could be implanted into immunodeficient mice. Pathological cellular hallmarks of incurable forms of severe muscular dystrophy could be modeled with high fidelity using this 3D platform. Finally, we show generation of fully human iPSC-derived, complex, multilineage muscle models containing key isogenic cellular constituents of skeletal muscle, including vascular endothelial cells, pericytes, and motor neurons. These results lay the foundation for a human skeletal muscle organoid-like platform for disease modeling, regenerative medicine, and therapy development.
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http://dx.doi.org/10.1016/j.celrep.2018.03.091DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5917451PMC
April 2018

Muscle moment arms and sensitivity analysis of a mouse hindlimb musculoskeletal model.

J Anat 2016 10 12;229(4):514-35. Epub 2016 May 12.

Structure and Motion Lab, Comparative Biomedical Sciences, Royal Veterinary College, Hatfield, UK.

Musculoskeletal modelling has become a valuable tool with which to understand how neural, muscular, skeletal and other tissues are integrated to produce movement. Most musculoskeletal modelling work has to date focused on humans or their close relatives, with few examples of quadrupedal animal limb models. A musculoskeletal model of the mouse hindlimb could have broad utility for questions in medicine, genetics, locomotion and neuroscience. This is due to this species' position as a premier model of human disease, having an array of genetic tools for manipulation of the animal in vivo, and being a small quadruped, a category for which few models exist. Here, the methods used to develop the first three-dimensional (3D) model of a mouse hindlimb and pelvis are described. The model, which represents bones, joints and 39 musculotendon units, was created through a combination of previously gathered muscle architecture data from microdissections, contrast-enhanced micro-computed tomography (CT) scanning and digital segmentation. The model allowed muscle moment arms as well as muscle forces to be estimated for each musculotendon unit throughout a range of joint rotations. Moment arm analysis supported the reliability of musculotendon unit placement within the model, and comparison to a previously published rat hindlimb model further supported the model's reliability. A sensitivity analysis performed on both the force-generating parameters and muscle's attachment points of the model indicated that the maximal isometric muscle moment is generally most sensitive to changes in either tendon slack length or the coordinates of insertion, although the degree to which the moment is affected depends on several factors. This model represents the first step in the creation of a fully dynamic 3D computer model of the mouse hindlimb and pelvis that has application to neuromuscular disease, comparative biomechanics and the neuromechanical basis of movement. Capturing the morphology and dynamics of the limb, it enables future dissection of the complex interactions between the nervous and musculoskeletal systems as well as the environment.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5013061PMC
http://dx.doi.org/10.1111/joa.12461DOI Listing
October 2016

Musculoskeletal Geometry, Muscle Architecture and Functional Specialisations of the Mouse Hindlimb.

PLoS One 2016 26;11(4):e0147669. Epub 2016 Apr 26.

Neuromuscular Diseases Group, Comparative Biomedical Sciences, Royal Veterinary College, 4 Royal College Street, London, NW1 0TU, United Kingdom.

Mice are one of the most commonly used laboratory animals, with an extensive array of disease models in existence, including for many neuromuscular diseases. The hindlimb is of particular interest due to several close muscle analogues/homologues to humans and other species. A detailed anatomical study describing the adult morphology is lacking, however. This study describes in detail the musculoskeletal geometry and skeletal muscle architecture of the mouse hindlimb and pelvis, determining the extent to which the muscles are adapted for their function, as inferred from their architecture. Using I2KI enhanced microCT scanning and digital segmentation, it was possible to identify 39 distinct muscles of the hindlimb and pelvis belonging to nine functional groups. The architecture of each of these muscles was determined through microdissections, revealing strong architectural specialisations between the functional groups. The hip extensors and hip adductors showed significantly stronger adaptations towards high contraction velocities and joint control relative to the distal functional groups, which exhibited larger physiological cross sectional areas and longer tendons, adaptations for high force output and elastic energy savings. These results suggest that a proximo-distal gradient in muscle architecture exists in the mouse hindlimb. Such a gradient has been purported to function in aiding locomotor stability and efficiency. The data presented here will be especially valuable to any research with a focus on the architecture or gross anatomy of the mouse hindlimb and pelvis musculature, but also of use to anyone interested in the functional significance of muscle design in relation to quadrupedal locomotion.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0147669PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4846001PMC
February 2017

Pericytes in development and pathology of skeletal muscle.

Circ Res 2013 Jul;113(3):341-7

Department of Cell and Developmental Biology, University College London, United Kingdom.

Increasing attention is currently devoted to the multiple roles that pericytes (also defined as mural, Rouget, or perivascular cells) may play during angiogenesis, vascular homeostasis, and pathology. Many recent excellent reviews thoroughly address these topics (see below); hence, we will not discuss them in detail here. However, not much is known about origin, heterogeneity, gene expression, and developmental potential of pericytes during fetal and postnatal development. This is likely because of the paucity of markers expressed by pericytes and the absence of truly unique ones. Thus, in vivo identification and ex perspective isolation are challenging and explain the relative little data available in comparison with neighbor but far more characterized cells such as the endothelium. Despite this preliminary knowledge, we will propose that contribution to growing mesoderm tissues may be an important role for pericytes. Thus, their ability to contribute to tissue regeneration may be a consequence of their role in tissue growth. However, in a severely damaged or diseased tissue, acute or chronic inflammation likely results in the production of signaling molecules that are different from those present in developing tissues, thus explaining why pericytes are easily diverted from a regenerative to a fibrotic fate.
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http://dx.doi.org/10.1161/CIRCRESAHA.113.300203DOI Listing
July 2013

Dll4 and PDGF-BB convert committed skeletal myoblasts to pericytes without erasing their myogenic memory.

Dev Cell 2013 Mar 7;24(6):586-99. Epub 2013 Mar 7.

Department of Cell and Developmental Biology and Centre for Stem Cells and Regenerative Medicine, University College London, WC1E 6DE London, UK.

Pericytes are endothelial-associated cells that contribute to vessel wall. Here, we report that pericytes may derive from direct conversion of committed skeletal myoblasts. When exposed to Dll4 and PDGF-BB, but not Dll1, skeletal myoblasts downregulate myogenic genes, except Myf5, and upregulate pericyte markers, whereas inhibition of Notch signaling restores myogenesis. Moreover, when cocultured with endothelial cells, skeletal myoblasts, previously treated with Dll4 and PDGF-BB, adopt a perithelial position stabilizing newly formed vessel-like networks in vitro and in vivo. In a transgenic mouse model in which cells expressing MyoD activate Notch, skeletal myogenesis is abolished and pericyte genes are activated. Even if overexpressed, Myf5 does not trigger myogenesis because Notch induces Id3, partially sequestering Myf5 and inhibiting MEF2 expression. Myf5-expressing cells adopt a perithelial position, as occasionally also observed in wild-type (WT) embryos. These data indicate that endothelium, via Dll4 and PDGF-BB, induces a fate switch in adjacent skeletal myoblasts.
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http://dx.doi.org/10.1016/j.devcel.2013.01.022DOI Listing
March 2013

Transplantation of genetically corrected human iPSC-derived progenitors in mice with limb-girdle muscular dystrophy.

Sci Transl Med 2012 Jun;4(140):140ra89

Department of Cell and Developmental Biology and Centre for Stem Cells and Regenerative Medicine, University College London, London, UK.

Mesoangioblasts are stem/progenitor cells derived from a subset of pericytes found in muscle that express alkaline phosphatase. They have been shown to ameliorate the disease phenotypes of different animal models of muscular dystrophy and are now undergoing clinical testing in children affected by Duchenne's muscular dystrophy. Here, we show that patients with a related disease, limb-girdle muscular dystrophy 2D (LGMD2D), which is caused by mutations in the gene encoding α-sarcoglycan, have reduced numbers of this pericyte subset and thus produce too few mesoangioblasts for use in autologous cell therapy. Hence, we reprogrammed fibroblasts and myoblasts from LGMD2D patients to generate human induced pluripotent stem cells (iPSCs) and developed a protocol for the derivation of mesoangioblast-like cells from these iPSCs. The iPSC-derived mesoangioblasts were expanded and genetically corrected in vitro with a lentiviral vector carrying the gene encoding human α-sarcoglycan and a promoter that would ensure expression only in striated muscle. When these genetically corrected human iPSC-derived mesoangioblasts were transplanted into α-sarcoglycan-null immunodeficient mice, they generated muscle fibers that expressed α-sarcoglycan. Finally, transplantation of mouse iPSC-derived mesoangioblasts into α-sarcoglycan-null immunodeficient mice resulted in functional amelioration of the dystrophic phenotype and restoration of the depleted progenitors. These findings suggest that transplantation of genetically corrected mesoangioblast-like cells generated from iPSCs from LGMD2D patients may be useful for treating this type of muscular dystrophy and perhaps other forms of muscular dystrophy as well.
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http://dx.doi.org/10.1126/scitranslmed.3003541DOI Listing
June 2012

Noggin recruits mesoderm progenitors from the dorsal aorta to a skeletal myogenic fate.

Dev Biol 2012 May 18;365(1):91-100. Epub 2012 Feb 18.

Division of Regenerative Medicine, San Raffaele Scientific Institute, Milan.

Embryonic mesoangioblasts are the in vitro counterpart of vessel-associated progenitors, able to differentiate into different mesoderm cell types. To investigate signals recruiting these progenitors to a skeletal myogenic fate, we developed an in vitro assay, based upon co-culture of E11.5 dorsal aorta (from MLC3F-nLacZ transgenic embryos, expressing nuclear beta galactosidase only in striated muscle) with differentiating C2C12 or primary myoblasts. Under these conditions muscle differentiation from cells originating from the vessel can be quantified by counting the number of beta gal+nuclei. Results indicated that Noggin (but not Follistatin, Chordin or Gremlin) stimulates while BMP2/4 inhibits myogenesis from dorsal aorta progenitors; neutralizing antibodies and shRNA greatly reduce these effects. In contrast, TGF-β1, VEGF, Wnt7A, Wnt3A, bFGF, PDGF-BB and IGF1 have no effect. Sorting experiments indicated that the majority of these myogenic progenitors express the pericyte marker NG2. Moreover they are abundant in the thoracic segment at E10.5 and in the iliac bifurcation at E11.5 suggesting the occurrence of a cranio-caudal wave of competent cells along the aorta. BMP2 is expressed in the dorsal aorta and Noggin in newly formed muscle fibers suggesting that these two tissues compete to recruit mesoderm cells to a myogenic or to a perithelial fate in the developing fetal muscle.
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http://dx.doi.org/10.1016/j.ydbio.2012.02.015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3327875PMC
May 2012