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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.

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.

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. [ABSTRACT FROM AUTHOR]

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 uman 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. [ABSTRACT FROM AUTHOR]

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-Dysfprmd Prkdcscid/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.

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. [ABSTRACT FROM AUTHOR]

Intracardiac transplantation of undifferentiated skeletal muscle cells (myoblasts) has emerged as a promising therapy for myocardial infarct repair and is already undergoing clinical trials. The fact that cells originating from skeletal muscle have different electrophysiological properties than cardiomyocytes, however, may considerably limit the success of this therapy and, in addition, cause side effects. Indeed, a major problem observed after myoblast transplantation is the occurrence of ventricular arrhythmias. The most often transient nature of these arrhythmias may suggest that, once transplanted into cardiac tissue, skeletal muscle cells adopt more cardiac-like electrophysiological properties. To test whether a cardiac cell environment can indeed modify electrophysiological parameters of skeletal muscle cells, we treated mouse C2Cl2 myocytes with medium preconditioned by primary cardiocytes and compared their functional sodium current properties with those of control cells. We found this treatment to significantly alter the activation and inactivation properties of sodium currents from "skeletal muscle" to more "cardiac"-like ones. Sodium currents of cardiac-conditioned cells showed a reduced sensitivity to block by tetrodotoxin. These findings and reverse transcription PCR experiments suggest that an upregulation of the expression of the cardiac sodium channel isoform Nav1.5 versus the skeletal muscle isoform Nav1.4 is responsible for the observed changes in sodium current function. We conclude that cardiomyocytes alter sodium channel isoform expression of skeletal muscle cells via a paracrine mechanism. Thereby, skeletal muscle cells with more cardiac-like sodium current properties are generated. [ABSTRACT FROM AUTHOR]

Abstract: Transplantation of normal muscle precursor cells is a potential approach to restore dystrophin expression within dystrophin [deficient] mdx mice, a model of Duchenne Muscular Dystrophy. This study aims to evaluate whether exercise could improve graft success and hybrid fiber distribution within mdx muscle. eGFP+ Muscle precursor cells were transplanted into tibialis anterior muscles of mdx mice using a single injection trajectory. During the following weeks, muscle fiber breaks were induced by making mdx mice swim. To evaluate fiber damage, Evans blue solution was injected intraperitoneally to mice 16h before their sacrifice. Tibialis anterior muscles were then harvested and eGFP, dystrophin and Evans blue labeling were analyzed by fluorescent microscopy. Twenty minutes of exercise (i.e., swimming) were used to induce damage in about 30% of TA muscle fibers. Graft success, evaluated as the percentage of hybrid fibers which are eGFP+, was improved by 1.9-fold after swimming 3 times per week during 4 weeks and by 1.8-fold after daily swimming. Hybrid muscle fiber transversal and longitudinal distribution were also increased after repeated physical efforts. Exercise induced fiber breaks, which improved MPC recruitment and fusion and increased long-term graft success and also transverse and longitudinal distribution of hybrid fibers. [Copyright &y& Elsevier]

Abstract: Human skeletal muscle has been considered as an ideal target for cell-mediated therapy. However, the positive results obtained in dystrophic animal models using the resident precursor satellite cell population have been followed by discouraging evidences obtained in the clinical trials involving Duchenne muscular dystrophy patients. This text reviews the recent advances that many groups have achieved to identify from the stem cell compartment putative candidates for cell therapy. We focused our attention on stem cells with myogenic potential which might be able to improve transplantation efficiency and therefore could be used as a therapeutic tool for neuromuscular diseases. [Copyright &y& Elsevier]

The regenerative capacity of skeletal muscle will depend on the number of available satellite cells and their proliferative capacity. We have measured both parameters in ageing, and have shown that although the proliferative capacity of satellite cells is decreasing during muscle growth, it then stabilizes in the adult, whereas the number of satellite cells decreases during ageing. We have also developed a model to evaluate the regenerative capacity of human satellite cells by implantation into regenerating muscles of immunodeficient mice. Using telomere measurements, we have shown that the proliferative capacity of satellite cells is dramatically decreased in muscle dystrophies, thus hampering the possibilities of autologous cell therapy. Immortalization by telomerase was unsuccessful, and we currently investigate the factors involved in cell cycle exits in human myoblasts. We have also observed that insulin-like growth factor-1 (IGF-1), a factor known to provoke hypertrophy, does not increase the proliferative potential of satellite cells, which suggests that hypertrophy is provoked by increasing the number of satellite cells engaged in differentiation, thus possibly decreasing the compartment of reserve cells. We conclude that autologous cell therapy can be applied to specific targets when there is a source of satellite cells which is not yet exhausted. This is the case of Oculo–Pharyngeal Muscular Dystrophy (OPMD), a late onset muscular dystrophy, and we participate to a clinical trial using autologous satellite cells isolated from muscles spared by the disease. [ABSTRACT FROM AUTHOR]

Muscle is the site or the target of immunologic injury in several diseases. Whereas under physiologic conditions muscle fibers are negative for major histocompatibility complex (MHC) class I antigens, these are upregulated under pathologic conditions, thus rendering muscle a possible target for the recognition by cytotoxic CD8 T cells. Cultured muscle cells are capable of presenting antigens to CD4 and CD8 T cells, further indicating that muscle fibers in vivo are critically involved in the initiating or perpetuating steps of inflammatory responses. The finding that muscle fibers in autoimmune inflammatory myopathies in vivo and cultured muscle cells in vitro express the nonclassical major histocompatibility complex molecule HLA-G raises several hypothesis concerning its possible pathophysiologic role. We review present knowledge on the functional consequences of muscle-related HLA-G and provide concepts of its relevance under pathologic conditions. We further speculate on the potential therapeutic implications of HLA-G that relate to special approaches such as myoblast transplantation or strategies against inflammatory aggression in general. [Copyright &y& Elsevier]

Nonhuman primates were used to define myoblast transplantation strategies applicable to humans. Nevertheless, previous experiments were based on the use of myotoxins concomitant with the myoblast injections. Since myotoxins must be avoided for clinical applications, we analyzed the efficacy of simple myoblast injections (i.e., myoblasts resuspended only in saline) into monkey muscles. We also evaluated different FK506 dosages (in combination or not with mycophenolate mofetil) for immunosuppression. Allogeneic myoblasts transduced with the β-galactosidase (β-Gal) gene were implanted in the muscles of 19 monkeys by injections placed 1 to 2 mm from each other. A biopsy was performed at the implanted sites 1 month later, and histologically studied for demonstration of β-Gal+ myofibers, lymphocyte infiltration, and CD8+ cells. The presence of antibodies against the donor myoblasts and the blood levels of FK506 were analyzed. Our results show that: (1) If myoblast injections are sufficiently close to each other, high percentages of hybrid myofibers can be obtained following myoblast transplantation in primates (25 to 67% with an interinjection distance of 1 mm). (2) Efficient immunosuppression can be reached by increasing FK506 dosages, but also by combining this drug with mycophenolate mofetil, a combination that reduces toxic effects. The present results represent a step towards a better designing of myoblast transplantation strategies in humans. [Copyright &y& Elsevier]

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Although myoblast transplantation is an attractive method for muscle regeneration, its efficiency remains limited. The efficacy of myoblast transplantation in combination with the controlled and sustained delivery of basic fibroblast growth factor (bFGF) was investigated. Defects of thigh muscle in Sprague-Dawley (SD) rats were created, and GFP-positive myoblasts were subsequently transplanted. The rats were divided into three groups. In control group 1 (C1) only myoblasts were transplanted, while in control group 2 (C2) myoblasts were introduced along with empty gelatin hydrogel microspheres. In the experimental group (Ex), myoblasts were transplanted along with bFGF incorporated into gelatin hydrogel microspheres. Four weeks after transplantation, GFP-positive myoblasts were found to be integrated into the recipient muscle and to contribute to muscle fibre regeneration in all groups. A significantly higher expression level of GFP in the Ex group demonstrated that the survival rate of transplanted myoblasts in Ex was remarkably improved compared with that in C1 and C2. Furthermore, myofibre regeneration, characterized by centralization of the nuclei, was markedly accelerated in Ex. The expression level of CD31 in Ex was higher than that in both C1 and C2, but the differences were not statistically significant. A significantly higher expression level of Myogenin and a lower expression level of MyoD1 were both observed in Ex after 4 weeks, suggesting the promotion of differentiation to myotubes. Our findings suggest that the controlled and sustained release of bFGF from gelatin hydrogel microspheres improves the survival rate of transplanted myoblasts and promotes muscle regeneration by facilitating myogenesis rather than angiogenesis., (Copyright © 2013 John Wiley & Sons, Ltd.)

Myoblast transplantation (MT) is a method to introduce healthy genes into abnormal skeletal muscle. It has been considered as a therapeutic modality in the last few decades for diseases such as Duchenne Muscular Dystrophy (DMD). However, challenges including cell death and poor graft engraftment have limited its application. The current experiment utilizes MMP1 gene transfer to improve the efficacy of myoblast transplantation into the diseased dystrophic skeletal muscle of mdx mice. Our results indicated that MMP1 expression can promote myogenic differentiation and fusion capacities, increase migration of MMP1 expressing myoblasts in vitro, as well as improve engraftment of dystrophin positive myofibers in vivo. Taken together, our observation suggests that the addition of MMP1 can overcome limitations in MT and improve its clinical efficacy.

Introduction: Since myoblasts have been limited by poor cell survival after cellular myoplasty, the major goal of the current study was to determine whether improving myoblast survival with an antioxidant could improve cardiac function after the transplantation of the myoblasts into an acute myocardial infarction., Background: We previously demonstrated that early myogenic progenitors such as muscle-derived stem cells (MDSCs) exhibited superior cell survival and improved cardiac repair after transplantation into infarcted hearts compared to myoblasts, which we partially attributed to MDSC's higher antioxidant levels., Aim: To determine if antioxidant treatment could increase myoblast survival, subsequently improving cardiac function after myoblast transplantation into infarcted hearts., Materials and Methods: Myoblasts were pre-treated with the antioxidant N-acetylcysteine (NAC) or the glutathione depleter, diethyl maleate (DEM), and injected into infarcted murine hearts. Regenerative potential was monitored by cell survival and cardiac function., Results: At early time points, hearts injected with NAC-treated myoblasts exhibited increased donor cell survival, greater cell proliferation, and decreased cellular apoptosis, compared to untreated myoblasts. NAC-treated myoblasts significantly improved cardiac contractility, reduced fibrosis, and increased vascular density compared to DEM-treated myoblasts, but compared to untreated myoblasts, no difference was noted., Discussion: While early survival of myoblasts transplanted into infarcted hearts was augmented by NAC pre-treatment, cardiac function remained unchanged compared to non-treated myoblasts., Conclusion: Despite improving cell survival with NAC treated myoblast transplantation in a MI heart, cardiac function remained similar to untreated myoblasts. These results suggest that the reduced cardiac regenerative potential of myoblasts, when compared to MDSCs, is not only attributable to cell survival but is probably also related to the secretion of paracrine factors by the MDSCs., Competing Interests: Conflict of Interests Johnny Huard received consulting fees and royalty payments from Cook MyoSite, Inc. during the period of time in which this project was performed.

The effect of pretreatments of host muscles with metalloproteinases (MMPs) or with notexin on the migration of transplanted myoblasts was investigated. Transgenic TnILacZ mice in which the β-galactosidase gene is under the control of a quail fast skeletal troponin I gene promoter were used as donors. A polyethylene microtube with four perforations was inserted in the tibialis anterior (TA) of CD1 mice. Both pretreatment substances and cells were slowly injected through that microtube. Muscles were pretreated 2 days before myoblast injection either with a mixture of collagenase, matrilysin, and notexin or with only collagenase and matrilysin or only notexin. As control for our experiments, TnILacZ and C2C12 myoblasts were also injected in TA muscles not pretreated. Comparison of short and long-term myoblast radial migration was performed using a dye (PKH26) and X-gal staining, respectively. The recipient mice were immunosuppressed with FK506. Two days after myoblast transplantation, the cell movement in muscles pretreated with collagenase, matrilysin, and notexin was slightly greater than in muscles pretreated only with collagenase and matrilysin but was about twice that observed in muscles treated with notexin alone. Almost no radial migration of TnILacZ myoblasts was observed in untreated muscles. The C2C12 myoblasts showed a four-to fivefold higher migration capacity than TnILacZ myoblasts. At 15 days after TnILacZ myoblast transplantation, the farthest positive β-gal muscle fibers show a two- to threefold extension of the initial migration observed at 2 days, demonstrating the ability of myoblasts to continue the migration following all pretreatments and even in the untreated muscles. In addition, more muscle fibers expressed the β-gal reporter gene in muscles pretreated only with MMPs. Our results clearly demonstrate that muscle pretreatments with MMPs increase myoblast migration and fusion with host muscle fibers after transplantation and that the C2C12 cell line producing MMPs has a higher migratory capacity.

Myogenic precursors in adult skeletal muscle (satellite cells) are mitotically quiescent but can proliferate in response to a variety of stresses including muscle injury. To gain further understanding of adult myoblasts, we are analyzing myogenesis of satellite cells on fibers isolated from adult rat muscle. In this culture model, satellite cells are maintained in their in situ position underneath the fiber basement membrane. Employing two different approaches to monitor proliferation of satellite cells on isolated fibers (autoradiography following 3 H-thymidine incorporation and immunofluorescence of cells positive for proliferating cell nuclear antigen (PCNA)), we show in the present study that satellite cells initiate cell proliferation at 12 to 24 hours following fiber culture establishment and that cell proliferation is reduced to minimal levels by 60 to 72 hours in culture. Maximal number of proliferating cells is seen at 36 to 48 hours in culture. These PCNA+ satellite cells transit into the differentiated, myogenin+ state following about 24 hours in the proliferative state. Continuous exposure of the fiber culture to FGF2 (basic FGF; added at the time of culture establishment) leads to a 2 fold increase in the number of PCNA+ cells by 48 hours in culture but the overall schedule of proliferation and transition into the myogenin+ state is not affected. Delaying the addition of FGF2 until 15 to 18 hours following the initiation of the fiber culture does not reduce its effect. However, the addition of FGF2 at 24 hours or later results in a progressive reduction in the number of proliferating satellite cells. Exposure of fiber cultures to transforming growth factor β (TGFβ1) leads to a reduction in the number of proliferating cells in both the absence or presence of FGF2. We propose that FGF2 enhances the number of proliferating cells by facilitating the recruitment of additional satellite cells from the quiescent state. However, satellite cells on isolated fibers conform to a highly coordinated program and rapidly transit from proliferation to differentiation regardless of the presence of FGF. The identification of agents that can prolong the proliferative state of satellite cells when the cells undergo myogenesis in their native position by the intact myofiber might be useful in improving myoblast transplantation into skeletal muscle for cell-mediated gene therapy.