Search

Stem cell-based therapies are a promising approach for the treatment of degenerative muscular diseases; however, clinical trials have shown inconclusive and even disappointing results so far. Noninvasive cell monitoring by medicine imaging could improve the understanding of the survival and biodistribution of cells following injection. In this study, we assessed the canine sodium iodide symporter (cNIS) reporter gene as an imaging tool to track by single-photon emission computed tomography (SPECT/CT) transduced canine myoblasts after intramuscular (IM) administrations in dogs. cNIS-expressing cells kept their myogenic capacities and showed strong 99 mTc-pertechnetate (99 mTcO4−) uptake efficiency both in vitro and in vivo. cNIS expression allowed visualization of cells by SPECT/CT along time: 4 h, 48 h, 7 days, and 30 days after IM injection; biopsies collected 30 days post administration showed myofiber’s membranes expressing cNIS. This study demonstrates that NIS can be used as a reporter to track cells in vivo in the skeletal muscle of large animals. Our results set a proof of concept of the benefits NIS-tracking tool may bring to the already challenging cell-based therapies arena in myopathies and pave the way to a more efficient translation to the clinical setting from more accurate pre-clinical results., Graphical Abstract

Background The hepatocyte growth factor (HGF) is required for the activation of muscle progenitor cells called satellite cells (SC), plays a role in the migration of proliferating SC (myoblasts), and is present as a soluble factor during muscle regeneration, along with extracellular matrix (ECM) molecules. In this study, we aimed at determining whether HGF is able to interact with ECM proteins, particularly laminin 111 and fibronectin, and to modulate human myoblast migration. Methods We evaluated the expression of the HGF-receptor c-Met, laminin, and fibronectin receptors by immunoblotting, flow cytometry, or immunofluorescence and used Transwell assays to analyze myoblast migration on laminin 111 and fibronectin in the absence or presence of HGF. Zymography was used to check whether HGF could modulate the production of matrix metalloproteinases by human myoblasts, and the activation of MAPK/ERK pathways was evaluated by immunoblotting. Results We demonstrated that human myoblasts express c-Met, together with laminin and fibronectin receptors. We observed that human laminin 111 and fibronectin have a chemotactic effect on myoblast migration, and this was synergistically increased when low doses of HGF were added. We detected an increase in MMP-2 activity in myoblasts treated with HGF. Conversely, MMP-2 inhibition decreased the HGF-associated stimulation of cell migration triggered by laminin or fibronectin. HGF treatment also induced in human myoblasts activation of MAPK/ERK pathways, whose specific inhibition decreased the HGF-associated stimulus of cell migration triggered by laminin 111 or fibronectin. Conclusions We demonstrate that HGF induces ERK phosphorylation and MMP production, thus stimulating human myoblast migration on ECM molecules. Conceptually, these data state that the mechanisms involved in the migration of human myoblasts comprise both soluble and insoluble moieties. This should be taken into account to optimize the design of therapeutic cell transplantation strategies by improving the migration of donor cells within the host tissue, a main issue regarding this approach. Electronic supplementary material The online version of this article (10.1186/s13395-017-0138-6) contains supplementary material, which is available to authorized users.

Summary Satellite cells are responsible for skeletal muscle regeneration. Upon activation, they proliferate as transient amplifying myoblasts, most of which fuse into regenerating myofibers. Despite their remarkable differentiation potential, these cells have limited migration capacity, which curtails clinical use for widespread forms of muscular dystrophy. Conversely, skeletal muscle perivascular cells have less myogenic potential but better migration capacity than satellite cells. Here we show that modulation of Notch and PDGF pathways, involved in developmental specification of pericytes, induces perivascular cell features in adult mouse and human satellite cell-derived myoblasts. DLL4 and PDGF-BB-treated cells express markers of perivascular cells and associate with endothelial networks while also upregulating markers of satellite cell self-renewal. Moreover, treated cells acquire trans-endothelial migration ability while remaining capable of engrafting skeletal muscle upon intramuscular transplantation. These results extend our understanding of muscle stem cell fate plasticity and provide a druggable pathway with clinical relevance for muscle cell therapy., Graphical Abstract, Highlights • DLL4 and PDGF-BB change satellite cell morphology, proliferation, and differentiation • DLL4 and PDGF-BB induce both perivascular and satellite cell gene expression • Treated satellite cells acquire perivascular cell properties and improved migration • Human satellite cell-derived myoblasts respond to DLL4 and PDGF-BB treatment, Gerli and Moyle and colleagues show that treatment with molecules involved in developmental specification of pericytes (DLL4 and PDGF-BB) alters satellite cell fate and provides them with features potentially relevant for novel cell therapy protocols.

Aim: To perform an early cost-effectiveness analysis of in vitro expanded myoblasts (IVM) and minced myofibers versus midurethral slings (MUS) for surgical treatment of female stress urinary incontinence. Methods: Cost-effectiveness and sensitivity analyses were performed using a decision tree comprising previously published data and expert opinions. Results & conclusion: In the base case scenario, MUS was the cost-effective strategy with a negative incremental cost-effectiveness ratio compared with IVM and a positive incremental cost-effectiveness ratio compared with minced myofibers. However, the sensitivity analysis indicates that IVM may become an alternative providing greater effect at a higher cost. With the possibility of becoming more effective, IVM treatment would be advantageous over MUS given its reduced invasiveness and lower risks of complications.

Dysferlin-deficient muscular dystrophy is a progressive disease characterized by muscle weakness and wasting for which there is no treatment. It is caused by mutations in DYSF, a large, multiexonic gene that forms a coding sequence of 6.2 kb. Sleeping Beauty (SB) transposon is a nonviral gene transfer vector, already used in clinical trials. The hyperactive SB system consists of a transposon DNA sequence and a transposase protein, SB100X, that can integrate DNA over 10 kb into the target genome. We constructed an SB transposon-based vector to deliver full-length human DYSF cDNA into dysferlin-deficient H2K A/J myoblasts. We demonstrate proper dysferlin expression as well as highly efficient engraftment (>1,100 donor-derived fibers) of the engineered myoblasts in the skeletal muscle of dysferlin- and immunodeficient B6.Cg-Dysf(prmd) Prkdc(scid)/J (Scid/BLA/J) mice. Nonviral gene delivery of full-length human dysferlin into muscle cells, along with a successful and efficient transplantation into skeletal muscle are important advances towards successful gene therapy of dysferlin-deficient muscular dystrophy.

Miyoshi myopathy (MM), limb girdle muscular dystrophy 2B (LGMD2B) and distal anterior compartment myopathy (DACM) are rare autosomal recessive muscle disorders caused by mutations in dysferlin. They are encompassed by the term dysferlinopathy and affect the proximal and/or distal muscles of the limbs. Patients usually become wheelchair bound within 15 years of disease onset due to progressive muscle degeneration, weakness and atrophy. No treatment is available. The dysferlin gene spans over 150 kb of genomic DNA in chromosome 2p13 and comprises 55 exons that form a coding sequence of 6.2 kb. Dysferlin is a 237 kDa protein of the ferlin family. It is expressed in many tissues, but more strongly in muscle, where it is found in mature myofibers and to some extent also in satellite cells and myoblasts. It has important roles in membrane repair and in maintaining the transverse tubule structure in myofibers. Besides alterations of these two functions, defects in dysferlin cause increased inflammatory attack to muscle fibers thereby enhancing the muscle pathology. Muscle regeneration relies on muscle stem cells, called satellite cells, and their progeny of muscle precursors, the myoblasts. Upon muscle injury caused by severe trauma or muscle disorders, they are capable of extensive proliferation to repair the damaged tissue. Satellite cells are marked by the expression of the transcription factor Pax7, they can be isolated based on specific surface markers and have a robust engraftment potential when transplanted into regenerating muscles. Therefore, autologous or allogeneic satellite cell and myoblast transplantation are envisioned as promising therapeutic alternatives. Given that a single gene is causative for dysferlinopathy, gene therapy also holds great promise. However, the large size of the full-length dysferlin coding sequence represents a challenge for gene transfer approaches because most viral vectors used in gene therapy have a lower cargo capacity. Sleeping Beauty transposon is a non-viral bi-component vector system consisting of a transposon DNA sequence and a transposase protein. The transposase excises the transposon from a donor plasmid and integrates it into the target genome. Since the transposon can be engineered to carry any sequence of interest, Sleeping Beauty is very valuable as a genetic tool for stable gene transfer and has been widely used in gene therapy. Sleeping Beauty system can be used to integrate transgenes up to ~8 kb with high efficiency. It is therefore well-suited for delivery of full- length dysferlin. In this work, I developed a non-viral cell-based gene therapy approach for dysferlinopathy. Using Sleeping Beauty transposon, I transferred the full-length human dysferlin cDNA into dysferlin deficient H2K A/J mouse myoblasts, leading to restoration of dysferlin protein expression in vitro. I transplanted the corrected myoblasts into dysferlin deficient Scid/blAJ mice following local irradiation of the recipient muscles. This step was necessary to ablate the endogenous satellite cells and promote engraftment of the transplanted cells. I found up to ~90 dysferlin expressing myofibers 3 and 6 weeks post-transplantation. Inducing regeneration in the recipient muscles simultaneously to cell transplantation by injection of cardiotoxin, a snake venom toxin that causes acute damage to myofibers, further enhanced engraftment of the donor cells. In muscles treated with cardiotoxin in addition to irradiation, I found up to ~1000 donor-derived myofibers 3 and 6 weeks after grafting, which were present almost along the entire length of the muscles. Moreover, I found numerous Pax7+ cells in the vicinity of donor- derived myofibers in the grafted muscles. Given that recipient muscles were irradiated prior to grafting, this suggests that donor myoblasts were able to repopulate the host satellite cell compartment upon transplantation. Engraftment as satellite cells would ensure long-term contribution of donor- derived cells to muscle regeneration. These results show the feasibility of long-term full-length dysferlin reconstitution in skeletal muscle through ex vivo gene transfer with Sleeping Beauty., Miyoshi Myopathie (MM), Gliedergürteldystrophie Typ 2B (engl. limb girdle muscular dystrophy 2B, LGMD2B) und die DACM (distal anterior compartment myopathy) sind seltene, autosomal rezessiv vererbte Krankheiten, die durch Mutationen im Dysferlin-Gen verursacht werden. Diese muskulären Erkrankungen werden unter dem Begriff Dysferlinopathie zusammengefasst und betreffen die proximale und/oder die distale Muskulatur der Extremitäten. Durch eine zunehmende Schwächung und Atrophie der Muskeln sind Patienten normalerweise bereits 15 Jahre nach Krankheitsbeginn an den Rollstuhl gebunden. Behandlungsmöglichkeiten gibt es hier keine. Das Dysferlin-Gen umfasst einen 150 kb-Bereich der genomischen DNA auf Chromosom 2p13 und beinhaltet 55 Exons mit einer kodierenden Sequenz von 6,2 kb. Dysferlin ist ein 237 kDa Protein der Ferlinfamilie. Es ist in vielen Geweben vorhanden, vor allem im Muskelgewebe, wo es in ausgereiften Muskelfasern, aber auch in Satellitenzellen und Myoblasten exprimiert wird. Dysferlin spielt eine wichtige Rolle bei der Membranreparatur und der Aufrechterhaltung der T-Tubuli Struktur in den Muskelfasern. Auβer dem erhöht sich durch ein defektes Dysferlinprotein die Entzündungsanfälligkeit der Muskelfasern. Die Regenerationsfähigkeit von Muskeln beruht auf den Muskelstammzellen (Satellitenzellen) und ihren Abkömmlingen, den Muskelvorläuferzellen (Myoblasten). Bei Muskelverletzungen, z.B. durch schwere Verwundungen oder durch Muskelerkrankungen, vermehren sich diese Zellen extensiv um das geschädigte Gewebe zu reparieren. Satellitenzellen sind durch die Expression des Transkriptionsfaktors Pax7 gekennzeichnet. Ihre Isolation basiert auf spezifischen Oberflächenmarkern. Zudem haben sie ein hohes Anwachspotential, wenn sie in sich regenerierendes Muskelgewebe tranplantiert werden. Deshalb gilt die autologe oder allogene Transplantation von Satellitenzellen oder Myoblasten als vielversprechende therapeutische Alternative. Da Dysferlinopathien durch ein einzelnes Gen verursacht werden, ist zudem die Gentherapie hier sehr erfolgversprechend. Allerdings werden Gentransferversuche durch die Gröβe der kodierenden Dysferlinsequenz erschwert, da die meisten viralen Vektoren, welche für Gentherapiezwecke benutzt werden, eine zu geringe Ladungskapazität besitzen. Das Sleeping Beauty Transposon ist ein nicht-virales 2-Komponenten Vektorsystem, welches aus der DNA Sequenz des Transposons und einem Transposase Protein besteht. Die Transposase schneidet das Transposon aus dem Donorplasmid heraus und integriert es in das Zielgenom. Da das Transposon so verändert werden kann, dass es jede Zielsequenz beinhalten kann, ist Sleeping Beauty ein sehr wichtiges, nicht-virales, genetisches Werkzeug, das einen stabilen Gentransfer ermöglicht und deshalb häufig in der Gentherapie verwendet wird. Durch das Sleeping Beauty System kann ein bis zu 8 kb groβes Transgen effizient integriert werden. Damit ist es auch für die Integration des vollständigen Dysferlin-Gens sehr gut geeignet. Im Rahmen dieser Arbeit habe ich eine Methode entwickelt, welche einen nicht-viralen zellbasierten Ansatz zur Gentherapie von Dysferlinopathien ermöglicht. Durch Nutzung des Sleeping Beauty Transposons ist es mir gelungen, die vollständige humane Dysferlin cDNA-Sequenz in Dysferlin-defiziente H2K A/J Myoblasten der Maus zu transferieren und die Dysferlinexpression in vitro wieder herzustellen. Die so korrigierten Myoblasten habe ich in Dysferlin-defiziente Scid/blAJ Mäuse transplantiert, in denen der Empfängermuskel zuvor lokal bestrahlt wurde. Dieser Schritt war notwendig, um die Anzahl der endogenen Satellitenzellen zu verringern und das Anwachsen der transplantierten Zellen zu fördern. 3-6 Wochen nach der Transplantation konnte ich eine Dysferlinexpression in bis zu 90 Muskelfasern nachweisen. Eine gleichzeitig zur Transplantation stattfindende Injektion des Zellgiftes Cardiotoxin führte zu einem wesentlich verbesserten Anwachsen der Donorzellen. Muskeln, welche zusätzlich zur Bestrahlung noch mit Cardiotoxin geschädigt wurden, wiesen 3 bis 6 Wochen nach der Transplantation bis zu 1000 Dysferlin exprimierende Muskelfasern auf. Zudem konnte ich in den transplantierten Muskeln eine Vielzahl von Pax7 positiven Zellen in der Umgebung der von den Spenderzellen abgeleiteten Myofibrillen identifizieren. Da der Empfängermuskel vor der Transplantation bestrahlt wurde, deutet dieses darauf hin, dass das Satellitenzellkompartiment des Empfängermuskels nach der Transplantation wieder neu besiedelt wurde. Das Anwachsen der Myoblasten als Satellitenzellen würde daher einen dauerhaften Beitrag der Spenderzellen zur Regeneration des Muskels belegen. Diese Ergebnisse zeigen, dass eine Rekonstruktion des vollständigen Dysferlin Proteins in Skelettmuskeln durch Sleeping Beauty vermittelten Gentransfer möglich ist.

Myoblasts, the precursors of skeletal muscle fibers, can be induced to withdraw from the cell cycle and differentiate in vitro. Recent studies have also identified undifferentiated subpopulations that can self-renew and generate myogenic cells (Baroffio, A., M. Hamann, L. Bernheim, M.-L. Bochaton-Pillat, G. Gabbiani, and C.R. Bader. 1996. Differentiation. 60:47–57; Yoshida, N., S. Yoshida, K. Koishi, K. Masuda, and Y. Nabeshima. 1998. J. Cell Sci. 111:769–779). Cultured myoblasts can also differentiate and contribute to repair and new muscle formation in vivo, a capacity exploited in attempts to develop myoblast transplantation (MT) for genetic modification of adult muscle. Our studies of the dynamics of MT demonstrate that cultures of myoblasts contain distinct subpopulations defined by their behavior in vitro and divergent responses to grafting. By comparing a genomic and a semiconserved marker, we have followed the fate of myoblasts transplanted into muscles of dystrophic mice, finding that the majority of the grafted cells quickly die and only a minority are responsible for new muscle formation. This minority is behaviorally distinct, slowly dividing in tissue culture, but rapidly proliferative after grafting, suggesting a subpopulation with stem cell–like characteristics.

Myoblast transplantation has been extensively studied as a gene complementation approach for genetic diseases such as Duchenne Muscular Dystrophy. This approach has been found capable of delivering dystrophin, the product missing in Duchenne Muscular Dystrophy muscle, and leading to an increase of strength in the dystrophic muscle. This approach, however, has been hindered by numerous limitations, including immunological problems, and low spread and poor survival of the injected myoblasts. We have investigated whether antiinflammatory treatment and use of different populations of skeletal muscle–derived cells may circumvent the poor survival of the injected myoblasts after implantation. We have observed that different populations of muscle-derived cells can be isolated from skeletal muscle based on their desmin immunoreactivity and differentiation capacity. Moreover, these cells acted differently when injected into muscle: 95% of the injected cells in some populations died within 48 h, while others richer in desmin-positive cells survived entirely. Since pure myoblasts obtained from isolated myofibers and myoblast cell lines also displayed a poor survival rate of the injected cells, we have concluded that the differential survival of the populations of muscle-derived cells is not only attributable to their content in desmin-positive cells. We have observed that the origin of the myogenic cells may influence their survival in the injected muscle. Finally, we have observed that myoblasts genetically engineered to express an inhibitor of the inflammatory cytokine, IL-1, can improve the survival rate of the injected myoblasts. Our results suggest that selection of specific muscle-derived cell populations or the control of inflammation can be used as an approach to improve cell survival after both myoblast transplantation and the myoblast-mediated ex vivo gene transfer approach.

Skeletal muscle transplantation strategies for muscle repair or gene therapy involve either the injection of proliferating myoblasts followed by fusion with host myofibers or implantation of ex vivo differentiated myofibers; however, both implant procedures are associated with significant cell loss. Biodegradable porous, gas-foamed poly-lactide-co-glycolide (PLG) scaffolds have desirable characteristics for cell transfer and were used to study attachment, growth, differentiation and survival of human myogenic cells. Primary human myoblasts suspended in clinical grade extracellular matrixes (ECMs) and adhered to PLG scaffolds differentiated in vitro into high-density tropomyosin positive myofibers. An immunodeficient non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mouse implant model was used to study the transfer and in vivo survival of differentiated human myofibers on these scaffolds. Scaffold rigidity allowed the myofibers to be maintained under tension in vitro and following subcutaneous transplantation in vivo. Following implantation, myofiber density on the PLG scaffolds decreased linearly by 78% over a 4-week period. ECM composed of either Tisseel((R)) fibrin or Zyderm((R)) collagen type I did not significantly affect in vivo cell viability over the 4-week period. Varying PLG scaffold microsphere content (10-100%) also had little effect on cell survival in vivo. In contrast, when the residual NK cell population in the immunodeficient NOD/SCID mouse model was depleted with anti-asialo GM1 (ASGM1) antiscrum, in vivo cell survival significantly increased from 22% to 34% after 4 weeks. With further improvements in cell survival, PLG scaffolds may prove useful for the implantation of primary human myofibers in future clinical applications. (c) 2007 Elsevier Ltd. All rights reserved. ispartof: Biomaterials vol:29 issue:1 pages:75-84 ispartof: location:Netherlands status: published

In this work we set out to determine if the murine macrophage J774 cell line can be used to produce myogenic growth factors. Activated J774 macrophages were grown in serum-free conditions. The macrophage-conditioned medium (MCM) was then used to treat cultures of primary myoblasts and regenerating muscle tissue, in vitro and in vivo respectively. MCM activity in vitro was tested by analyzing the expression of muscle-specific transcription factors, in parallel with the proliferation and differentiation rates of the cells. The macrophage-secreted factors greatly enhanced the proliferative potential of both rat and human primary myoblasts and were found to be highly muscle-specific. In vivo, MCM administration markedly enhanced the regenerative processes in damaged muscles. The ability to produce large amounts of macrophage-secreted myogenic factor(s) in the absence of serum holds great promise for its biochemical characterization and successive application in therapeutic protocols, both for ex vivo gene therapy and for muscle repair.

Myoblast transfer therapy has been extensively studied for a wide range of clinical applications, such as tissue engineering for muscular loss, cardiac surgery or Duchenne Muscular Dystrophy treatment. However, this approach has been hindered by numerous limitations, including early myoblast death after injection and specific immune response after transplantation with allogenic cells. Different cell sources have been analyzed to overcome some of these limitations. The object of our study was to investigate the growth potential, characterization and integration in vivo of human primary fetal skeletal muscle cells. These data together show the potential for the creation of a cell bank to be used as a cell source for muscle cell therapy and tissue engineering. For this purpose, we developed primary muscular cell cultures from biopsies of human male thigh muscle from a 16-week-old fetus and from donors of 13 and 30 years old. We show that fetal myogenic cells can be successfully isolated and expanded in vitro from human fetal muscle biopsies, and that fetal cells have higher growth capacities when compared to young and adult cells. We confirm lineage specificity by comparing fetal muscle cells to fetal skin and bone cells in vitro by immunohistochemistry with desmin and 5.1 H11 antibodies. For the feasibility of the cell bank, we ensured that fetal muscle cells retained intrinsic characteristics after 5 years cryopreservation. Finally, human fetal muscle cells marked with PKH26 were injected in normal C57BL/6 mice and were found to be present up to 4 days. In conclusion we estimate that a human fetal skeletal muscle cell bank can be created for potential muscle cell therapy and tissue engineering.