Skeletal Myoblast Transplantation Research Articles

Retraction: Transplantation of HGF gene-engineered skeletal myoblasts improve infarction recovery in a rat myocardial ischemia model.

Retraction: Transplantation of HGF gene-engineered skeletal myoblasts improve infarction recovery in a rat myocardial ischemia model.

Correction: Transplantation of HGF gene-engineered skeletal myoblasts improve infarction recovery in a rat myocardial ischemia model

[This corrects the article DOI: 10.1371/journal.pone.0175807.].

Potential use of superparamagnetic iron oxide nanoparticles for in vitro and in vivo bioimaging of human myoblasts

Myocardial infarction (MI) is one of the most frequent causes of death in industrialized countries. Stem cells therapy seems to be very promising for regenerative medicine. Skeletal myoblasts transplantation into postinfarction scar has been shown to be effective in the failing heart but shows limitations such, e.g. cell retention and survival. We synthesized and investigated superparamagnetic iron oxide nanoparticles (SPIONs) as an agent for direct cell labeling, which can be used for stem cells imaging. High quality, monodisperse and biocompatible DMSA-coated SPIONs were obtained with thermal decomposition and subsequent ligand exchange reaction. SPIONs’ presence within myoblasts was confirmed by Prussian Blue staining and inductively coupled plasma mass spectrometry (ICP-MS). SPIONs’ influence on tested cells was studied by their proliferation, ageing, differentiation potential and ROS production. Cytotoxicity of obtained nanoparticles and myoblast associated apoptosis were also tested, as well as iron-related and coating-related genes expression. We examined SPIONs’ impact on overexpression of two pro-angiogenic factors introduced via myoblast electroporation method. Proposed SPION-labeling was sufficient to visualize firefly luciferase-modified and SPION-labeled cells with magnetic resonance imaging (MRI) combined with bioluminescence imaging (BLI) in vivo. The obtained results demonstrated a limited SPIONs’ influence on treated skeletal myoblasts, not interfering with basic cell functions.

Transplantation of HGF gene-engineered skeletal myoblasts improve infarction recovery in a rat myocardial ischemia model.

BackgroundSkeletal myoblast transplantation seems a promising approach for the repair of myocardial infarction (MI). However, the low engraftment efficacy and impaired angiogenic ability limit the clinical efficiency of the myoblasts. Gene engineering with angiogenic growth factors promotes angiogenesis and enhances engraftment of transplanted skeletal myoblasts, leading to improved infarction recovery in myocardial ischemia. The present study evaluated the therapeutic effects of hepatocyte growth factor (HGF) gene-engineered skeletal myoblasts on tissue regeneration and restoration of heart function in a rat MI model.Methods and resultsThe skeletal myoblasts were isolated, expanded, and transduced with adenovirus carrying the HGF gene (Ad-HGF). Male SD rats underwent ligation of the left anterior descending coronary artery. After 2 weeks, the surviving rats were randomized into four groups and treated with skeletal myoblasts by direct injection into the myocardium. The survival and engraftment of skeletal myoblasts were determined by real-time PCR and in situ hybridization. The cardiac function with hemodynamic index and left ventricular architecture were monitored; The adenovirus-mediated-HGF gene transfection increases the HGF expression and promotes the proliferation of skeletal myoblasts in vitro. Transplantation of HGF-engineered skeletal myoblasts results in reduced infarct size and collagen deposition, increased vessel density, and improved cardiac function in a rat MI model. HGF gene modification also increases the myocardial levels of HGF, VEGF, and Bcl-2 and enhances the survival and engraftment of skeletal myoblasts.ConclusionsHGF engineering improves the regenerative effect of skeletal myoblasts on MI by enhancing their survival and engraftment ability.

A New In Vitro Co-Culture Model Using Magnetic Force-Based Nanotechnology.

Skeletal myoblast (SkMB) transplantation has been conducted as a therapeutic strategy for severe heart failure. However, arrhythmogenicity following transplantation remains unsolved. We developed an in vitro model of myoblast transplantation with "patterned" or "randomly-mixed" co-culture of SkMBs and cardiomyocytes enabling subsequent electrophysiological, and arrhythmogenic evaluation. SkMBs were magnetically labeled with magnetite nanoparticles and co-cultured with neonatal rat ventricular myocytes (NRVMs) on multi-electrode arrays. SkMBs were patterned by a magnet beneath the arrays. Excitation synchronicity was evaluated by Ca(2+) imaging using a gene-encoded Ca(2+) indicator, G-CaMP2. In the monoculture of NRVMs (control), conduction was well-organized. In the randomly-mixed co-culture of NRVMs and SkMBs (random group), there was inhomogeneous conduction from multiple origins. In the "patterned" co-culture where an en bloc SKMB-layer was inserted into the NRVM-layer, excitation homogenously propagated although conduction was distorted by the SkMB-area. The 4-mm distance conduction time (CT) in the random group was significantly longer (197 ± 126 ms) than in control (17 ± 3 ms). In the patterned group, CT through NRVM-area did not change (25 ± 3 ms), although CT through the SkMB-area was significantly longer (132 ± 77 ms). The intervals between spontaneous excitation varied beat-to-beat in the random group, while regular beating was recorded in the control and patterned groups. Synchronized Ca(2+) transients of NRVMs were observed in the patterned group, whereas those in the random group were asynchronous. Patterned alignment of SkMBs is feasible with magnetic nanoparticles. Using the novel in vitro model mimicking cell transplantation, it may become possible to predict arrhythmogenicity due to heterogenous cell transplantation. J. Cell. Physiol. 231: 2249-2256, 2016. © 2016 Wiley Periodicals, Inc.

骨・軟骨疾患の再生医療

Cardiovascular regenerative therapy has the potential to improve the treatment of peripheral artery disease (PAD), ischemic heart disease (IHD) and severe heart failure. Autologous implantation of bone marrow-mononuclear cells into ischemic limbs was reported as an effective application by the secretion of various angiogenic factors or cytokines. On the other hand, bone marrow cells and skeletal myoblasts transplantation for cardiac angiogenic therapy, injected directry into cardiac ischemic lesion, was generally insufficient to repair severe myocardial damage. To improve the efficacy of cardiac cell therapy, cell sheet technology was developed to reduce the damage of the transplanted cells. The discovery of iPS cells and cardiac master genes have changed the field of cardiac regenerative medicine. Because cardiomyocytes are terminally differentiated cells with limited regenerative capacity in adult heart, implantation of cardiomyocyte cell-sheet differentiated from iPS cells or the other pluripotent cell types, is expected as a new strategy of cardiac cell therapy. Moreover, direct cardiac reprograming which means cardiac fibroblasts induced converting directry into cardiomyocytes in damaged cardiac tissue, was discovered recentry. Here, this review summarizes recent advances in cardiovascular regenerative therapy and discusses the perspectives and challenges of new technology in basic biology and clinical applications.

The Paracrine Effect of Skeletal Myoblasts Is Cardioprotective Against Oxidative Stress and Involves EGFR-ErbB4 Signaling, Cystathionase, and the Unfolded Protein Response.

Therapeutic effects of skeletal myoblast transplantation into the myocardium are mediated via paracrine factors. We investigated the ability of myoblast-derived soluble mediators to protect cardiomyocytes from oxidative stress. Fetal rat cardiac cells were treated with conditioned medium from cultures of myoblasts or cardiac fibroblasts, and oxidative stress was induced with H2O2. Myoblast-derived factors effectively prevented oxidative stress-induced cardiac cell death and loss of mitochondrial membrane potential. This protective effect was mediated via epidermal growth factor (EGF) receptor and c-Met signaling, and mimicked by neuregulin 1 but not EGF. Microarray analysis of cardiac cells treated with myoblast versus cardiac fibroblast-derived mediators revealed differential regulation of genes associated with antioxidative effects: cystathionine-γ-lyase (cst), xanthine oxidase, and thioredoxin-interacting protein as well as tribbles homolog 3 (trib3). Cardiac cell pretreatment with tunicamycin, an inducer of trib3, also protected them against H2O2-induced cell death. Epicardial transplantation of myoblast sheets in a rat model of acute myocardial infarction was used to evaluate the expression of CST and trib3 as markers of myoblasts' paracrine effect in vivo. Myoblast sheets induced expression of the CST as well as trib3 in infarcted myocardium. CST localized around blood vessels, suggesting smooth muscle cell localization. Our results provide a deeper molecular insight into the therapeutic mechanisms of myoblast-derived paracrine signaling in cardiac cells and suggest that myoblast transplantation therapy may prevent oxidative stress-induced cardiac deterioration and progression of heart failure.

Human myoblast transplantation in mice infarcted heart alters the expression profile of cardiac genes associated with left ventricle remodeling

BackgroundMyocardial infarction (MI) and left ventricle remodeling (LVR) are two of the most challenging disease entities in developed societies. Since conventional treatment cannot fully restore heart function new approaches were attempted to develop new strategies and technologies that could be used for myocardial regeneration. One of these strategies pursued was a cell therapy — particularly applying skeletal muscle stem cells (SkMCs). Methods and resultsUsing NOD-SCID murine model of MI and human skeletal myoblast transplantation we were able to show that SkMC administration significantly affected gene expression profile (p<0.05) (NPPB, CTGF, GATA4, SERCA2a, PLB) of the heart ventricular tissue and this change was beneficial for the heart function. We have also shown, that the level of heart biomarker, NT-proBNP, decreased in animals receiving implanted cells and that the NT-proBNP level negatively correlated with left ventricle area fraction change (LVFAC) index which makes NT-proBNP an attractive tool in assessing the efficacy of cell therapy both in the animal model and prospectively in clinical trials. ConclusionsThe results obtained suggest that transplanted SkMCs exerted beneficial effect on heart regeneration and were able to inhibit LVR which was confirmed on the molecular level, giving hope for new ways of monitoring novel cellular therapies for MI.

Extending Flaps Lifts an Infarcted Heart Toward Repair

Normal hearts comprise 2 × 109 myocytes, but this number may be doubled in excessively hypertrophied hearts. After a heart attack caused by blockage of one of the arteries that supply the heart, approximately 20% or more of cardiomyocytes are acutely and irreversibly lost in the ischemic area. However, the damage is not limited to the acute interruption of blood flow. Reperfusion of the ischemic territory can cause additional damage due to a burst of toxic reactive oxygen species. Moreover, additional cardiomyocytes are lost in the peri-infarct area during the subacute phase, leading to extension of the infarcted area. The ensuing remodeling response, which consists of cardiomyocyte hypertrophy and collagen accumulation, is insufficient to compensate for the massive cell loss, thus leading to scar expansion, ventricular chamber dilation, and failure.

Cardiac Analysis of Autologous Transplantation of Cocultured Skeletal Myoblasts and Mesenchymal Cells in a Rat Model Doxorubicin-Induced Cardiotoxicity: Histopathological and Functional Studies

Objective: Investigate the functional and histopathological effects of combined transplantation of mesenchymal stem cells (MSCs) and skeletal myoblasts (SM) in rats submitted to doxorubicin (DOX) induced cardiomyopathy. Methods: Myocardiopathy was induced through intraperitoneal applications of 3.75 mg/kg/day DOX once a week for 4 weeks in all experimental animals. Echocardiography examination was conducted to evaluate heart function. Myocardial cell apoptosis was examined morphologically by optical microscopy. The expression of both, von Willebrand factor and MyoD proteins was analysed using immunohistochemical staining. Results: Our results showed that all the experimental animals developed cardio-toxicity and exhibited decrease in heart function 4 weeks after the administration of DOX (P<0.05). The ventricular function significantly improved in rats that received sub-epicardial injection of co-culture of SM and MSC (CO group) when compared to rats that received only saline sub-epicardial injection (CG group) (P<0.05). Conclusion: The results of this study provide evidences that the myocardial co-culture transplantation of SM and MSCs contributed to the treatment the DOX-induced cardiotoxicity.

On-chip monitoring of skeletal myoblast transplantation for the treatment of hypoxia-induced myocardial injury.

A comprehensive elucidation of the unexpected adverse events that occur in skeletal myoblast transplantation is fundamental for the optimization of myocardial therapeutic effects. However, a well-defined method to study the interactions between skeletal myoblasts and cardiomyocytes during the healing process is out of reach. Here, we describe a microfluidic method for monitoring the interactions between skeletal myoblasts and hypoxia-injured cardiomyocytes in a spatiotemporally-controlled manner, mimicking the in vivo cell transplantation process. A myocardial hypoxia environment was created using an oxygen consumption blocking reagent, carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone. Meanwhile, the interactions between the skeletal L6 myoblasts and hypoxia-injured myocardium H9c2 cells were investigated, and the effects of a L6 conditional medium on H9c2 cells were comparatively analyzed by quantitatively measuring the morphological and pathophysiological dynamics of H9c2 cells. The results showed that skeletal myoblasts could repair hypoxia-injured H9c2 cells mainly through direct cell-to-cell interactions. This simple on-chip assay for investigating myocardial repair processes may provide avenues for the in vitro screening of drug-induced cardiotoxicity.

Long-term follow-up after autologous skeletal myoblast transplantation in ischaemic heart disease

Short-term follow-up after autologous skeletal myoblasts (ASM) transplantation (Tx) (Myoblast Autologous Grafting in Ischaemic Cardiomyopathy (MAGIC) Phase II Study) for the treatment of ischaemic cardiomyopathy revealed improved left ventricular (LV) remodelling. Our study reports the longest long-term worldwide follow-up of a single-centre cohort, focusing on the safety and efficacy of ASM-Tx. The multicentre MAGIC Phase II Study involved 120 patients and was conducted between 2004 and 2006. Out of the 120 patients involved in the entire study, the cohort treated at our institution contained 7 patients only. These 7 patients received ASM-Tx (injection volume: 400 million cells, n = 2 low dosage; 800 million cells, n = 2 high dosage) or placebo (n = 3) injections, in addition to coronary artery bypass grafting (CABG). After closure of the MAGIC registry, we conducted a long-term follow-up for our 7-patient cohort. The mean follow-up was 72.0 ± 5.3 months. The follow-up was complete for echo data, implanted cardioverter defibrillator (ICD) report, clinical investigation and New York Heart Association (NYHA) class. At final follow-up, all the patients were alive, and 5 were in NYHA class 1 or 2. There were 6 hospitalizations for congestive heart failure during the follow-up (1 patient from each group). One patient (placebo group) was treated twice for ventricular fibrillation by the ICD. The LV ejection fraction remained stable in all the three groups (31.1 ± 3.9% preoperative vs 29.4 ± 4.4% at final follow-up). The LV volumes were reduced in the high-dosage group, remained unchanged in the low-dosage group and deteriorated in the placebo group. Our long-term data confirm the findings of the MAGIC study. The LV function did not improve, but the long-term LV volumes in the high-dosage group were reduced. During the follow-up, there were also no additional arrhythmogenic incidences. Our data could imply that CABG in combination with ASM-Tx is safe and has beneficial therapeutic effects in the long-term. However, due to the small patient number, the clinical impact is limited.

Skeletal myoblast transplantation on gene expression profiles of insulin signaling pathway and mitochondrial biogenesis and function in skeletal muscle

AimThe study aims to investigate the gene expression profiling of insulin signaling pathway and mitochondrial biogenesis and function in the skeletal muscle of KK mice. MethodsKK mice were divided into the following groups: KK control group, basal medium (M199) only; KK fibroblast group, with human fibroblast transplantation; KK myoblast group, with human skeletal myoblast transplantation. C57BL mice received hSkM transplantation as a normal control. Cells were transplanted into mice hind limb skeletal muscle. All animals were treated with cyclosporine for 6 weeks only. The mice were sacrificed in a fasting state at 12 weeks after treatment. Hind limb skeletal muscle was harvested and used for study of gene expression profiling. ResultshSkMs survived extensively in mice skeletal muscle at 12 weeks after cell transplantation. Glucose tolerance test showed a significant decrease of blood glucose in the mice of KK myoblast group compared to the KK control and fibroblast groups. Transcriptional patterns of insulin signaling pathway showed alterations in KK myoblast as compared with KK control group (23 genes), KK fibroblast group (7 genes), and C57BL group (8 genes). Transcriptional patterns of mitochondrial biogenesis and function also had alterations in KK myoblast as compared with KK control group (27 genes), KK fibroblast group (9 genes), and C57BL group (6 genes). ConclusionsThese data demonstrated for the first time that hSKM transplantation resulted in a change of gene transcript in multiple genes involved in insulin signaling pathway and mitochondrial biogenesis and function.

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.

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.

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.

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.