Myoblast Transplantation Research Articles

Expanding Donor Muscle‐Derived Cells for Transplantation

Studies in mice showed tremendous promise for the eventual clinical utility of myoblast transplantation to treat human muscular dystrophies. Initial attempts to translate the murine studies to humans, however, were not successful, due in part to limited engraftability of expanded donor myoblasts. Conventionally, muscle cells have been cultured on collagen-coated tissue culture-treated polystyrene. However, this promotes lineage progression and differentiation of cells, which limits engraftment potential. This unit describes the isolation of canine muscle-derived cells, ex vivo expansion of cells on plates coated with a modified Notch ligand, and the xenotransplant method used to evaluate engraftment potential. Activation of Notch signaling in freshly isolated canine muscle-derived cells with Delta-1(ext)-IgG inhibits myogenic differentiation, and maintains cells earlier in myogenic lineage progression. Delta-1(ext)-IgG-expanded cells engraft into the regenerating muscle of NOD/SCID mice more effectively than control cells expanded on human IgG, as evidenced by a significant increase in the number of muscle fibers expressing canine dystrophin in recipient murine muscle. Therefore, this protocol provides the basis for further developing culture conditions for ex vivo expansion of donor muscle cells for transplant.

191st ENMC International Workshop: Recent advances in oculopharyngeal muscular dystrophy research: From bench to bedside 8-10 June 2012, Naarden, The Netherlands

This workshop hosted a group of 19 clinicians and basic scientist from 5 countries (France, USA, Canada, Denmark and The Netherlands). Recent research developments and possible collaboration in advancing therapies for oculopharyngeal muscular dystrophy (OPMD), a late onset, rare and progressive neurodegeneration disorder were discussed in this meeting. The disease is caused by alanine expansion mutations in the gene encoding for poly(A)-binding protein nuclear 1 (PABPN1), resulting in accumulation of mutant PABPN1 in the cell nucleus and the formation of insoluble aggregates. Therefore, OPMD is categorized together with neurodegenerative disorders that are caused by single amino acid repeats. While the genetic cause for those disorders is often recognized, the molecular basis for symptoms is still obscure. PABPN1 is ubiquitously expressed and it is not known why symptoms develop only after midlife and initially only in subsets of skeletal muscles [1,2]. While the genetic cause for OPMD was reported in 1998 and good surgical treatments exist for major early symptoms, there are currently no medical options for the more general skeletal muscle involvement, no validated biomarkers and no clinical severity scale.

Transplantation of novel vascular endothelial growth factor gene delivery system manipulated skeletal myoblasts promote myocardial repair

Skeletal myoblast (SkM) transplantation combined with vascular endothelial growth factor (VEGF) gene delivery has been proposed as a promising therapy for cardiac repair. Nevertheless, the defective gene vectors and unregulable VEGF expression in vivo hinder its application. Therefore, the search for an economical, effective, controllable gene delivery system is quite necessary. In our study, hyperbranched polyamidoamine (h-PAMAM) dendrimer was synthesized as a novel gene delivery vector using a modified method. And hypoxia-regulated human VEGF-165 plasmids (pHRE-hVEGF165) were constructed for controllable VEGF gene expression. The efficiency and feasibility of h-PAMAM-HRE-hVEGF165 gene delivery system manipulated SkM transplantation for cardiac repair were investigated in myocardial infarction models. The h-PAMAM encapsulated pHRE-hVEGF165 could resist nuclease digestion for over 120 min. In primary SkMs, h-PAMAM-pHRE-hVEGF165 gene delivery system showed high transfection efficiency (43.47 ± 2.22%) and minor cytotoxicity (cell viability = 91.38 ± 0.48%). And the transfected SkMs could express hVEGF165 for 18 days under hypoxia in vitro. For myocardial infarction models, intramyocardial transplantation of the transfected SkMs could result in reduction of apoptotic myocardiocytes, improvement of grafted cell survival, decrease of infarct size and interstitial fibrosis, and increase of blood vessel density, which inhibited left ventricle remodeling and improved heart function at the late phase following infarction. These results indicate that h-PAMAM based pHRE-hVEGF165 gene delivery into SkMs is feasible and effective, and may serve as a novel and promising gene therapy strategy in ischemic heart disease.

Promotion of muscle regeneration by myoblast transplantation combined with the controlled and sustained release of bFGFcpr

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.

Molecular Imaging to Target Transplanted Muscle Progenitor Cells

Duchenne muscular dystrophy (DMD) is a severe genetic neuromuscular disorder that affects 1 in 3,500 boys, and is characterized by progressive muscle degeneration. In patients, the ability of resident muscle satellite cells (SCs) to regenerate damaged myofibers becomes increasingly inefficient. Therefore, transplantation of muscle progenitor cells (MPCs)/myoblasts from healthy subjects is a promising therapeutic approach to DMD. A major limitation to the use of stem cell therapy, however, is a lack of reliable imaging technologies for long-term monitoring of implanted cells, and for evaluating its effectiveness. Here, we describe a non-invasive, real-time approach to evaluate the success of myoblast transplantation. This method takes advantage of a unified fusion reporter gene composed of genes (firefly luciferase [fluc], monomeric red fluorescent protein [mrfp] and sr39 thymidine kinase [sr39tk]) whose expression can be imaged with different imaging modalities. A variety of imaging modalities, including positron emission tomography (PET), single-photon emission computed tomography (SPECT), magnetic resonance imaging (MRI), optical imaging, and high frequency 3D-ultrasound are now available, each with unique advantages and limitations. Bioluminescence imaging (BLI) studies, for example, have the advantage of being relatively low cost and high-throughput. It is for this reason that, in this study, we make use of the firefly luciferase (fluc) reporter gene sequence contained within the fusion gene and bioluminescence imaging (BLI) for the short-term localization of viable C2C12 myoblasts following implantation into a mouse model of DMD (muscular dystrophy on the X chromosome [mdx] mouse). Importantly, BLI provides us with a means to examine the kinetics of labeled MPCs post-implantation, and will be useful to track cells repeatedly over time and following migration. Our reporter gene approach further allows us to merge multiple imaging modalities in a single living subject; given the tomographic nature, fine spatial resolution and ability to scale up to larger animals and humans, PET will form the basis of future work that we suggest may facilitate rapid translation of methods developed in cells to preclinical models and to clinical applications.

Modification of experimental vein grafts by homologous transplantation of skeletal myoblasts

Modification of experimental vein grafts by homologous transplantation of skeletal myoblasts

Impact of Hepatocyte Growth Factor on Skeletal Myoblast Transplantation Late after Myocardial Infarction

In clinical studies, skeletal myoblast (SKMB) transplantation late after myocardial infarction (MI) has minimal impact on left ventricular (LV) function. This may be related to our previous observation that the extent of SKMB engraftment is minimal in chronic MI when compared to acute MI, which correlates with decreased hepatocyte growth factor (HGF) expression, an important regulator of SKMB function. Here, we investigated delivery of exogenous HGF as a strategy for augmenting SKMB engraftment late after MI. Rats underwent SKMB transplantation 4 weeks after coronary ligation. HGF or vehicle control was delivered intravenously during the subsequent 2 weeks. LV function was assessed by MRI before and 2 weeks after SKMB transplantation. We evaluated HGF delivery, SKMB engraftment, and expression of genes associated with post-MI remodeling. Serum HGF was 6.2 ± 2.4 ng/mL after 2 weeks of HGF infusion (n = 7), but undetectable in controls (n = 7). LV end-diastolic volume and ejection fraction did not improve with HGF treatment (321 ± 27 mm3, 42% ± 2% vs. 285 ± 33 mm3, 43% ± 2%, HGF vs. control). MIs were larger in HGF-treated animals (50 ± 7 vs. 30 ± 6 mm3, P = 0.046), but the volume of engrafted SKMBs or percentage of MIs occupied by SKMBs did not increase with HGF (1.7 ± 0.3 mm3, 4.7% ± 1.9% vs. 1.4 ± 0.4 mm3, 5.3% ± 1.6%, HGF vs. control). Expression of genes associated with post-infarction remodeling was not altered by HGF. Delivery of exogenous HGF failed to augment SKMB engraftment and functional recovery in chronic MI. Expression of genes associated with LV remodeling was not altered by HGF. Alternative strategies to enhance engraftment of SKMB must be explored to optimize the clinical efficacy of SKMB transplantation.

Evaluation of macroporous blood and plasma scaffolds for skeletal muscle tissue engineering.

The field of tissue engineering has a growing need for suitable scaffold materials to become attractive as a clinical therapy. To use a completely autologous construct to repair a damaged or diseased tissue is an appealing thought. As a model system, two types of scaffolds were prepared from biological fluids: blood and plasma. The prepared scaffolds formed a macroporous structure with elastic mechanical properties that were further evaluated with myoblast cell line (C2C12) cultivation and transplantation into mouse skeletal muscle. The cells were found to attach, proliferate, and migrate through all the different scaffolds. Moreover, the cells underwent myogenic differentiation, showing typical cell morphology aligned in a parallel fashion. An increased level of myogenin mRNA was found with the time of culture. Furthermore, myogenic markers MyoD1, desmin, myogenin and myosin, as well as β-dystroglycan and the laminin α2 chain, were found to be expressed. In vivo data indicated that the scaffolds degraded and were replaced with regenerated muscle fibres. We conclude that the two types of macroporous scaffolds based on blood or plasma have potential in the field of skeletal muscle tissue engineering.

Nine-year follow-up of local implantation of autologous skeletal myoblasts in a patient with coronary heart disease

Patient:gender – Male, age – 63 year-oldPrimary Diagnosis:Acute myocardial infarctionCo-existing Diseases:HypertensionMedication:Aspirin • beta-blocker • captoprilClinical Procedure:CABG • autologous skeletal myoblast transplantation • PCISpecialty:CardiologyObjective:Unusual or unexpected effect of treatment.Background:Cell transplantation has been viewed as a promising strategy for end-stage heart failure, but long-term follow-up results are lacking.Case Report:In December 2002 we began transplanting autologous skeletal myoblasts in one patient because of serious coronary heart disease. Here, we present the 9-year follow-up results of this patient. No ventricular tachyarrhythmias were detected after treatment. The patient had another myocardial infarction in April 2012 and was treated successful with PCI.Conclusions:Autologous skeletal myoblast transplantation with bypass surgery is associated with improvement in cardiac function and lack of adverse effects in long-term follow-up, making it a promising therapy for patients with heart failure.

Cutaneous Pigmentation in Health and Disease: Novel and Well-Established Players

Cutaneous pigment formation and aberration in disease are addressed. Dynoodt et al. (this issue) present data on a specific micro RNA that downregulates proteins involved in melanogenesis and melanosome movement. Kim et al. (this issue) present data showing that Wnt signaling is involved in the pathogenesis of melasma. Both articles enhance our understanding of cutaneous pigmentation and point to targets in the development of novel therapeutic modalities.

Targeted Gene Addition of Microdystrophin in Mice Skeletal Muscle via Human Myoblast Transplantation

Zinc finger nucleases (ZFN) can facilitate targeted gene addition to the genome while minimizing the risks of insertional mutagenesis. Here, we used a previously characterized ZFN pair targeting the chemokine (C-C motif) receptor 5 (CCR5) locus to introduce, as a proof of concept, the enhanced green fluorescent protein (eGFP) or the microdystrophin genes into human myoblasts. Using integrase-defective lentiviral vectors (IDLVs) and chimeric adenoviral vectors to transiently deliver template DNA and ZFN respectively, we achieved up to 40% targeted gene addition in human myoblasts. When the O6-methylguanine-DNA methyltransferaseP140K gene was co-introduced with eGFP, the frequency of cells with targeted integration could be increased to over 90% after drug selection. Importantly, gene-targeted myoblasts retained their mitogenic activity and potential to form myotubes both in vitro and in vivo when injected into the tibialis anterior of immune-deficient mice. Altogether, our results could lead to the development of improved cell therapy transplantation protocols for muscular diseases.

Low Molecular Weight Dextran Sulfate Binds to Human Myoblasts and Improves their Survival after Transplantation in Mice

Introduction: Myoblasts are skeletal muscle progenitors cells responsible for skeletal muscle growth and regeneration. As such and because myoblasts faces the complex challenges of clinical application, myoblast transplantation appears to be a promising approach for treatment of extensive skeletal muscle destruction occurring in severe myopathies such as Duchenne muscular dystrophy. Nevertheless, even in autologous combination, such approaches are impaired by the rapid death, limited migration and rejection of transplanted myoblasts by the host. Low molecular weight dextran sulfate (DXS), a sulfated polysaccharide, has been reported to act as a cytoprotectant in vitro and in vivo. These beneficial effects of DXS in modulating innate immunity make this substance a possible candidate to improve the success of myoblast transplantation. Methods: Clonal cultures of human myoblasts were prepared from human muscle biopsies obtained during corrective orthopedic surgery. Myoblasts were 100% positives for the myogenic markers CD56 and desmin as assessed by flow cytometry. For in vivo experiments, human myoblasts were transduced with a lentiviral vector encoding the renilla luciferase bioluminescence marker gene (Rluc). Non-invasive bioluminescence imaging was used to quantify survival of engrafted human myoblasts after transplantation in immunodeficient mice. All research with human samples and mice was performed in compliance with the institutional guidelines, national as well as international guidelines. Results: In vitro, DXS binds dose dependently to human myoblasts and significantly inhibits staurosporine mediated apoptosis and necrosis. DXS pretreatment also protects human myoblasts from natural killer cell-mediated cytotoxicity in a dose dependent manner. At a multiplicity of infection of 1, 80% of the cells expressed the renilla luciferase transgene after lentiviral infection. Rluc transduced myoblasts kept their myogenic capacity to proliferate and to differentiate in vitro as assessed by fusion indexes. In vivo, we observed a two-fold improvement in survival of human myoblasts that were pretreated with DXS (10 mg/ml) as compared to untreated cells, at 1 day and at 3 days after transplantation (77.9 ± 10.1% vs 39.4 ± 4.9%; p = 0.0009 and 38.1 ± 8.5% vs 15.1 ± 3.4%; p = 0.01, respectively). No significant difference was observed at 7 days (8.0 ± 2.1 % vs 3.6 ± 0.7%; p = 0.06) and at 14 days post injection with values that were indistinguishable from background levels. Conclusion: Taken together, we provide evidence that DXS act as a myoblast protectant in vitro and in vivo, able locally to prevent early cell death. This protective effect of DXS was efficient up to 72h after pretreatment. DXS might represent a useful therapeutic reagent to improve the success of myoblast cell transplantation. More experiments are needed to evaluate if combination of DXS pretreatment of human myoblasts with systemic DXS administration of the recipient may be envisaged to enhance myoblast transplantation success.

Experimental study on the therapy of acute myocardial infarction with the transplantation of skeletal myoblasts transfected with vascular endothelial growth factor gene regulated by hypoxic response elements

Objective To investigate the therapy of acute myocardial infarction with the transplantation of skeletal myoblasts transfected with pcDNA3.1/vascular endothelial growth factor (VEGF) vector and pEGFP-C3-9HRE-CMV-VEGF vector.Methods The rat models of acute myocardial infarction were established.Serum-free medium (group A),control skeletal myoblasts (group B),skeletal myoblasts transfected withpcDNA3.1/VEGF vector (group C) and skeletal myoblasts transfected with pEGFP-C3-9HRE-CMV-VEGF vector (group D) were injected into the border zone surrounding the infarct respectively.The mortality,cardiac function,infarct size,grafted myoblasts,expression of VEGF and angiogenesis were observed 4 weeks later to demonstrate the therapy of acute myocardial infarction with transfected skeletal myoblasts.Results The mortality rates were better in groups C (10.0)％ and D (13.3)％ than in groups B (26.7) ％.The cardiac function were better in groups C (32.9 ± 1.4) ％ and D (33.1 ± 1.4) ％than in groups A(44.5 ± 1.7) ％ and B (38.2 ± 1.5) ％.The expression of VEGF and angiogenesis were increased in groups C and D than in groups A and B.Conclusion This combined strategy of skeletal myoblasts transplantation with gene therapy could be of importance for the treatment of acute myocardial infarction. Key words: Hypoxic response elements; Vascular endothelial growth factor; Gene therapy; Cell transplantation; Acute myocardial infarction

The use of cell-sheet technique eliminates arrhythmogenicity of skeletal myoblast-based therapy to the heart with enhanced therapeutic effects

BackgroundClinical application of skeletal myoblast transplantation has been curtailed due to arrhythmogenicity and inconsistent therapeutic benefits observed in previous studies. However, these issues may be solved by the use of a new cell-delivery mode. It is now possible to generate “cell-sheets” using temperature-responsive dishes without artificial scaffolds. This study aimed to validate the safety and efficacy of epicardial placement of myoblast-sheets (myoblast-sheet therapy) in treating heart failure. Methods and resultsAfter coronary artery ligation in rats, the same numbers of syngeneic myoblasts were transplanted by intramyocardial injection or cell-sheet placement. Continuous radio-telemetry monitoring detected increased ventricular arrhythmias, including ventricular tachycardia, after intramyocardial injection compared to the sham-control, while these were abolished in myoblast-sheet therapy. This effect was conjunct with avoidance of islet-like cell-cluster formation that disrupts electrical conduction, and with prevention of increased arrhythmogenic substrates due to exaggerated inflammation. Persistent ectopic donor cells were found in the lung only after intramyocardial injection, strengthening the improved safety of myoblast-sheet therapy. In addition, myoblast-sheet therapy enhanced cardiac function, corresponding to a 9.2-fold increase in donor cell survival, compared to intramyocardial injection. Both methods achieved reduced infarct size, decreased fibrosis, attenuated cardiomyocyte hypertrophy, and increased neovascular formation, in association with myocardial upregulation of a group of relevant molecules. The pattern of these beneficial changes was similar between two methods, but the degree was more substantial after myoblast-sheet therapy. ConclusionThe cell-sheet technique enhanced safety and therapeutic efficacy of myoblast-based therapy, compared to the current method, thereby paving the way for clinical application.

Macrophages Improve Survival, Proliferation and Migration of Engrafted Myogenic Precursor Cells into MDX Skeletal Muscle

Transplantation of muscle precursor cells is of therapeutic interest for focal skeletal muscular diseases. However, major limitations of cell transplantation are the poor survival, expansion and migration of the injected cells. The massive and early death of transplanted myoblasts is not fully understood although several mechanisms have been suggested. Various attempts have been made to improve their survival or migration. Taking into account that muscle regeneration is associated with the presence of macrophages, which are helpful in repairing the muscle by both cleansing the debris and deliver trophic cues to myoblasts in a sequential way, we attempted in the present work to improve myoblast transplantation by coinjecting macrophages. The present data showed that in the 5 days following the transplantation, macrophages efficiently improved: i) myoblast survival by limiting their massive death, ii) myoblast expansion within the tissue and iii) myoblast migration in the dystrophic muscle. This was confirmed by in vitro analyses showing that macrophages stimulated myoblast adhesion and migration. As a result, myoblast contribution to regenerating host myofibres was increased by macrophages one month after transplantation. Altogether, these data demonstrate that macrophages are beneficial during the early steps of myoblast transplantation into skeletal muscle, showing that coinjecting these stromal cells may be used as a helper to improve the efficiency of parenchymal cell engraftment.

VEGF165 attenuates the Th17/Treg imbalance that exists when transplanting allogeneic skeletal myoblasts to treat acute myocardial infarction

To investigate whether Th17/Treg imbalance exists, and whether VEGF(165) attenuates the imbalance in allogeneic skeletal myoblast transplantation (allo-SMT) for acute myocardial infarction (AMI). On days 1, 2, 4, and 7 after allo-SMT, the percentages and ratios of Th17 and Treg cells were analyzed by flow cytometry in three groups-the AMI group, the AMI-S group (allo-SMT) and the AMI-V group (with VEGF(165) treatment). Subsequently, related proinflammatory and regulatory cytokines and key transcription factors, ROR-γt mRNA and Foxp3 mRNA expression, were examined by Bio-plex and real-time polymerase chain reaction, respectively. On days 1, 2, 4, and 7, the percentage of Tregs, related cytokine concentrations and transcript factor Foxp3 mRNA in the AMI-S group were lower than those in the AMI group, while those in the AMI-V group were higher than those in the AMI group. However, the percentage of Th17 cells, related cytokine concentrations and ROR-γt mRNA in the AMI-S group were higher than those in the AMI group; those in the AMI-V group were lower than those in the AMI group. Compared with the AMI group, the ratios of Th17/Treg cells significantly increased in the AMI-S group and decreased in the AMI-V group. Th17/Treg imbalance participated in the formation and development of the inflammatory and immune response after allo-SMT. However, transfected VEGF(165) was able to relieve the severity of the Th17/Treg imbalance.

Role of autologous myoblast transplantation in endplate regeneration and neuromuscular function restoration after direct nerve implantation in rats

Objective To observe the effects of autologous myoblast transplantation on endplate regeneration and neuromuscular function restoration following direct nerve implantation （DNI） in rats so as to offer experimental basis for the use of myoblasts in neural regeneration area and further lay founda- tion for the research using myohlasts as transgenic carriers. Methods A total of 20 male SD rats were randomly divided into experimental group and control group, with 10 rats in each group. Models of DNI in rat gastrocnemius were established. The experimental group was injected with primarily cultured autolo- gous myoblasts to the DNI location, while the control group was injected with isometric medium without autologous myoblasts. The effects of myoblasts on the neuromuscular function recovery following DNI were studied by detecting the tibial functional index （FFI）, neuro-electrophysiology and pathohistology. Results The experimental group displayed faster tibial nerve function recovery than the control group （P 〈 0.01 ）. The peak to peak value （PPV） of gastrocnemius neuro-electrophysiology, area under the curve and regenerated endplate number of the experimental group had statistical significances as compared with the control group （P 〈 0.01 ）. Conclusion Myoblast autotransplantation accelerates the recovery of neuromuscular function after rat DNI and increases the number of the regenerated endplates. Key words: Myoblasts ; Transplantation; Neuromuscular function repairation

Myostatin: genetic variants, therapy and gene doping

Since its discovery, myostatin (MSTN) has been at the forefront of muscle therapy research because intrinsic mutations or inhibition of this protein, by either pharmacological or genetic means, result in muscle hypertrophy and hyperplasia. In addition to muscle growth, MSTN inhibition potentially disturbs connective tissue, leads to strength modulation, facilitates myoblast transplantation, promotes tissue regeneration, induces adipose tissue thermogenesis and increases muscle oxidative phenotype. It is also known that current advances in gene therapy have an impact on sports because of the illicit use of such methods. However, the adverse effects of these methods, their impact on athletic performance in humans and the means of detecting gene doping are as yet unknown. The aim of the present review is to discuss biosynthesis, genetic variants, pharmacological/genetic manipulation, doping and athletic performance in relation to the MSTN pathway. As will be concluded from the manuscript, MSTN emerges as a promising molecule for combating muscle wasting diseases and for triggering wide-ranging discussion in view of its possible use in gene doping.

Mechano growth factor (MGF) promotes proliferation and inhibits differentiation of porcine satellite cells (PSCs) by down-regulation of key myogenic transcriptional factors

Porcine satellite cells represent an ideal model system for studying the cellular and molecular basis regulating myogenic stem cell proliferation and differentiation and for exploring the experimental conditions for myoblast transplantation. Here, we investigated the effects of mechano growth factor (MGF), a spliced variant of the IGF-1 gene, on porcine satellite cells. We show that MGF potently stimulated proliferation while inhibited differentiation of porcine satellite cells. MGF-treatment acutely down-regulates the expression of myogenic determination factor (MyoD) and the cyclin-dependent kinase inhibitor p21. MGF-treatment also markedly reduced the overall expression of cyclin B1 and key factors of the myogenic regulatory and myocyte enhancer families, including Myogenein and MEF2A. Taken together, the gene expression data from MGF-treated porcine satellite cells are in favor of a molecular model in which MGF inhibits porcine satellite cell differentiation by down-regulating either the activity or expression of MyoD, which, in turn, suppresses the expression of key genes required for cell cycle progression and differentiation, such as p21, Myogenin, and MEF2. Overall, our findings are in support of the previous suggestion that MGF may be used in vivo and in vitro to promote proliferation of myogenic stem cells to prevent and treat age-related muscle degenerative diseases.

Increased angiogenesis and improved left ventricular function after transplantation of myoblasts lacking the MyoD gene into infarcted myocardium.

Skeletal myoblast transplantation has therapeutic potential for repairing damaged heart. However, the optimal conditions for this transplantation are still unclear. Recently, we demonstrated that satellite cell-derived myoblasts lacking the MyoD gene (MyoD−/−), a master transcription factor for skeletal muscle myogenesis, display increased survival and engraftment compared to wild-type controls following transplantation into murine skeletal muscle. In this study, we compare cell survival between wild-type and MyoD−/− myoblasts after transplantation into infarcted heart. We demonstrate that MyoD−/− myoblasts display greater resistance to hypoxia, engraft with higher efficacy, and show a larger improvement in ejection fraction than wild-type controls. Following transplantation, the majority of MyoD−/− and wild-type myoblasts form skeletal muscle fibers while cardiomyocytes do not. Importantly, the transplantation of MyoD−/− myoblasts induces a high degree of angiogenesis in the area of injury. DNA microarray data demonstrate that paracrine angiogenic factors, such as stromal cell-derived factor-1 (SDF-1) and placental growth factor (PlGF), are up-regulated in MyoD−/− myoblasts. In addition, over-expression and gene knockdown experiments demonstrate that MyoD negatively regulates gene expression of these angiogenic factors. These results indicate that MyoD−/− myoblasts impart beneficial effects after transplantation into an infarcted heart, potentially due to the secretion of paracrine angiogenic factors and enhanced angiogenesis in the area of injury. Therefore, our data provide evidence that a genetically engineered myoblast cell type with suppressed MyoD function is useful for therapeutic stem cell transplantation.