Myoblast Transplantation Research Articles

Transcription Factor Rational Design Improves Directed Differentiation of Human Mesenchymal Stem Cells Into Skeletal Myocytes

There is great interest in transdifferentiating cells from one lineage into those of another and in dedifferentiating mature cells back into a stem/progenitor cell state by deploying naturally occurring transcription factors (TFs). Often, however, steering cellular differentiation pathways in a predictable and efficient manner remains challenging. Here, we investigated the principle of combining domains from different lineage-specific TFs to improve directed cellular differentiation. As proof-of-concept, we engineered the whole-human TF MyoDCD, which has the NH(2)-terminal transcription activation domain (TAD) and adjacent DNA-binding motif of MyoD COOH-terminally fused to the TAD of myocardin (MyoCD). We found via reporter gene and marker protein assays as well as by a cell fusion readout system that, targeting the TAD of MyoCD to genes normally responsive to the skeletal muscle-specific TF MyoD enforces more robust myogenic reprogramming of nonmuscle cells than that achieved by the parental, prototypic master TF, MyoD. Human mesenchymal stem cells (hMSCs) transduced with a codon-optimized microdystrophin gene linked to a synthetic striated muscle-specific promoter and/or with MyoD or MyoDCD were evaluated for complementing the genetic defect in Duchenne muscular dystrophy (DMD) myocytes through heterotypic cell fusion. Cotransduction of hMSCs with MyoDCD and microdystrophin led to chimeric myotubes containing the highest dystrophin levels.

The Survival of Myoblasts After Intramuscular Transplantation Is Improved When Fewer Cells Are Injected

Myoblast transplantation has long been studied as a potential therapy for Duchenne muscular dystrophy as the incorporation of donor myoblasts into host muscle allows the production of functional dystrophin protein. However, the clinical feasibility of this approach is limited by the poor survival of the donor cells in the weeks after transplantation. It has recently been determined that the intramuscular transplantation of large numbers of cells can lead to the formation of ischemic necrosis in the center of these cell masses. For this reason, the relationship between donor cell survival and the number of cells transplanted was investigated. Myoblasts were prepared from the hind limb muscles of male C57BL/10Sn mice and transplanted into the tibialis anterior muscles of female mdx mice at one of the following amounts: 10, 10, 10, or 10 cells. The survival of the transplanted cells was analyzed using a Y chromosome-specific qPCR. It was found that donor cell survival was improved 1 week after transplantation when fewer myoblasts were transplanted, including the observation of donor cell proliferation after the transplantation of 10 myoblasts. However, concentration effects and long-term survival complicate the interpretation of these results. These results indicate that early donor myoblast survival was dependent on the number of cells transplanted and the volume of liquid used to deliver them into the muscle. We believe that this finding has implications for the design and interpretation of future experimentation relating to intramuscular cell therapies.

Allogenic Skeletal Myoblast Transplantation in Acute Myocardial Infarction Model Rats

The limitations of syngenic cell therapy include patient safety and quality control of the source cells. Therefore, it is important to develop and assess procedures using allogenic cells. We investigated the impact of allogenic skeletal myoblast (SMB) transplantation on acute myocardial infarction with respect to immune response, donor cell survival, and therapeutic efficacy. Female Lewis rats underwent proximal left anterior descending coronary artery ligation. Fifteen minutes later, they underwent major histocompatibility (MHC)-matched Lewis SMB transplantation (group S) and MHC-mismatched ACI SMB transplantation (group A), or treated with buffer injection as a control (group C). Flow cytometry showed that the SMBs expressed MHC antigens and B7 signal molecules in vitro. In group A, transcription levels of interleukin-2 receptor and interferon-γ were significantly increased 7 days after transplantation, and the area surrounding the donor SMBs was intensely infiltrated with CD4- and CD8-positive cells. Estimation of the number of donor cells in the recipient left ventricular chamber revealed that except for day 0, group A had fewer donor SMBs, which disappeared faster, compared with group S. Echocardiography demonstrated that the ejection fraction (EF) of group A was lower than that of group S. MHC-mismatched allogenic SMB transplantation in infarcted myocardium induces the immune response and acceleration of donor cell clearance, decreasing the therapeutic effect. Donor cell survival and inflammation may play important roles in the therapeutic mechanism of SMB transplantation therapy for acute myocardial infarction.

Final results of a phase IIa, randomised, open-label trial to evaluate the percutaneous intramyocardial transplantation of autologous skeletal myoblasts in congestive heart failure patients: the SEISMIC trial

The SEISMIC study was an open-label, prospective, randomised study to assess the safety and feasibility of percutaneous myoblast implantation in heart failure patients with implanted cardioverter-defibrillators (ICD). Patients were randomised 2:1 to autologous skeletal myoblast therapy vs. optimal medical treatment. The primary safety end-point was defined as the incidence of procedural and device related serious adverse events, whereas the efficacy endpoints were defined as the change in global LVEF by MUGA scan, change in NYHA classification of heart failure and in the distance achieved during a six-minute walk test (6MW) at 6-month follow-up. Forty subjects were randomised to the treatment arm (n=26), or to the control arm (n=14). There were 12 sustained arrhythmic events and one death after episodes of ventricular tachycardia (VT) in the treatment group and 14 events in the control group (P=ns). At 6-month follow-up, 6MW distance improved by 60.3±54.1?meters in the treated group as compared to no improvement in the control group (0.4±185.7?meters; P=ns). In the control group, 28.6% experienced worsening of heart failure status (4/14), while 14.3% experienced an improvement in NYHA classification (2/14). In the myoblast-treatment arm, one patient experienced a deterioration in NYHA classification (8.0%), whereas five patients improved one or two classes (20.0%; P=0.06). However, therapy did not improve global LVEF measured by MUGA at 6-month follow-up. These data indicate that implantation of myoblasts in patients with HF is feasible, appears to be safe and may provide symptomatic relief, though no significant effect was detected on global LVEF.

Blocking the Myostatin Signal With a Dominant Negative Receptor Improves the Success of Human Myoblast Transplantation in Dystrophic Mice

Duchenne muscular dystrophy (DMD) is a recessive disease caused by a dystrophin gene mutation. Myoblast transplantation permits to introduce the dystrophin gene in dystrophic muscle fibers. However, the success of this approach is reduced by the short duration of the regeneration following the transplantation, which reduces the number of hybrid fibers. Myostatin (MSTN) is a negative regulator of skeletal muscle development and responsible for limiting regeneration. It binds with high affinity to the activin type IIB receptor (ActRIIB). Our aim was to verify whether the success of the myoblast transplantation is enhanced by blocking the MSTN signal with expression of a dominant negative mutant of ActRIIB (dnActRIIB). In vitro, blocking MSTN activity with a lentivirus carrying dnActRIIB increased proliferation and fusion of human myoblasts because MSTN regulates the expression of several myogenic regulatory factors. In vivo, myoblasts infected with the dnActRIIB lentivirus were transplanted in immunodeficient dystrophic mice. Dystrophin immunostaining of tibialis anterior (TA) cross-sections of these mice 1 month post-transplantation revealed more human dystrophin-positive myofibers following the transplantation of dnActRIIB myoblasts than of control myoblasts. Thus, blocking the MSTN signal with dnActRIIB improved the success of myoblast transplantation by increasing the myoblast proliferation and fusion and changed the expression of myogenic regulatory factors.

Laminin-111: A Potential Therapeutic Agent for Duchenne Muscular Dystrophy

Duchenne muscular dystrophy (DMD) still needs effective treatments, and myoblast transplantation (MT) is considered as an approach to repair damaged skeletal muscles. DMD is due to the complete loss of dystrophin from muscles. The lack of link between the contracting apparatus and the extracellular matrix leads to frequent damage to the sarcolemma triggering muscle fiber necrosis. Laminins are major proteins in the extracellular matrix. Laminin-111 is normally present in skeletal and cardiac muscles in mice and humans but only during embryonic development. In this study, we showed that intramuscular injection of laminin-111 increased muscle strength and resistance in mdx mice. We also used laminin-111 as a coadjuvant in MT, and we showed this protein decreased considerably the repetitive cycles of degeneration, inflammatory reaction, and regeneration. Moreover, MT is significantly improved. To explain the improvement, we confirmed with the same myoblast cell batch that laminin-111 improves proliferation and drastically increases migration in vitro. These results are extremely important because DMD could be treated only by the injection of a recombinant protein, a simple and safe therapy to prevent loss of muscle function. Moreover, the improvement in MT would be significant to treat the muscles of DMD patients who are already weak.

In Vivo Assessment of the Electrophysiological Integration and Arrhythmogenic Risk of Myocardial Cell Transplantation Strategies

Cell replacement strategies are promising interventions aiming to improve myocardial performance. Yet, the electrophysiological impact of these approaches has not been elucidated. We assessed the electrophysiological consequences of grafting of two candidate cell types, that is, skeletal myoblasts and human embryonic stem cell-derived cardiomyocytes (hESC-CMs). The fluorescently labeled (DiO) candidate cells were grafted into the rat's left ventricular myocardium. Two weeks later, optical mapping was performed using the Langendorff-perfused rat heart preparation. Images were obtained with appropriate filters to delineate the heart's anatomy, to identify the DiO-labeled cells, and to associate this information with the voltage-mapping data (using the voltage-sensitive dye PGH-I). Histological examination revealed the lack of gap junctions between grafted skeletal myotubes and host cardiomyocytes. In contrast, positive Cx43 immunostaining was observed between donor and host cardiomyocytes in the hESC-CMs-transplanted hearts. Optical mapping demonstrated either normal conduction (four of six) or minimal conduction slowing (two of six) at the hESC-CMs engraftment sites. In contrast, marked slowing of conduction or conduction block was seen (seven of eight) at the myoblast transplantation sites. Ventricular arrhythmias could not be induced in the hESC-CM hearts following programmed electrical stimulation but were inducible in 50% of the myoblast-engrafted hearts. In summary, a unique method for assessment of the electrophysiological impact of myocardial cell therapy is presented. Our results demonstrate the ability of hESC-CMs to functionally integrate with host tissue. In contrast, transplantation of cells that do not form gap junctions (skeletal myoblats) led to localized conduction disturbances and to the generation of a proarrhythmogenic substrate.

Myoblast transplantation: A possible surgical treatment for a severe pediatric disease

Duchenne muscular dystrophy (DMD) is a genetic X-linked recessive orphan disease that affects approximately 1 in 3 500 male births. Boys with DMD have progressive and predictable muscle destruction due to the absence of dystrophin, a protein present under the muscle fiber membrane. This absence induces contraction-related membrane damage and activation of inflammatory necrosis and fibrosis, leading to cardiac/diaphragmatic failure and death. The authors support the therapeutic role of myoblast transplantation in DMD, and describe the history and rationale for such an approach.

Past, present and future of myoblast transplantation in the treatment of Duchenne muscular dystrophy

DMD is a genetic X-linked recessive disease that affects approximately one in 3500 male births. Boys with DMD have progressive and predictable muscle destruction because of the absence of Dys, a protein present under the muscle fiber membrane. Dys deficiency induces contraction-related membrane damages, activation of inflammatory-necrosis-fibrosis up to the cardiac-diaphragmatic failure and death. This review supports the therapeutic role of MT associated with immunosuppression in DMD patients, describing the history and the rationale of such approach. The authors underline the importance to evaluate a protocol of myoblast intradermal multi-injection to apply in young DMD patients

Bcl-2 Improves Myoblast Sheet Therapy in Rat Chronic Heart Failure

Myoblast transplantation therapy for chronic heart failure (HF) is impaired by early donor cell death and reduced graft viability. Although epicardial implantation of cell sheets can prevent the initial loss of transplanted cells, limited vascularization subjects the sheets to apoptotic stress. We studied the efficacy of antiapoptotic bcl2 in myoblast sheet therapy for rat chronic HF. Myocardial infarction was induced by left anterior descending coronary artery ligation and HF was allowed to develop for 4 weeks. Thereafter, wild type (L6-WT; n=16) or Bcl-2-expressing (L6-Bcl2; n=19) myoblast sheets were transplanted epicardially. Control rats (n=21) underwent left anterior descending coronary artery ligation and re-thoracotomy. Five rats were sham-operated in both surgeries. Four weeks after transplantation, only the L6-Bcl2 rats showed improved left ventricular ejection fraction. Their vascular density in the damaged myocardium was greater, and they had more proliferating cells. The L6-Bcl2 group had an increased amount of myocytes in the infarct area. Soluble factors from L6-Bcl2 sheets induced a 2.9-fold increase in endothelial cell proliferation, and enhanced endothelial wound healing as compared to the L6-WT sheets. These effects were inhibited by SU5416 and were thus dependent on Flt1/Flk1 signaling. In conclusion, bcl2 improves efficacy of myoblast sheet transplantation and promotes proangiogenic paracrine signaling.

Intramuscular Transplantation of Human Postnatal Myoblasts Generates Functional Donor-Derived Satellite Cells

Myogenic cell transplantation is an experimental approach for the treatment of myopathies. In this approach, transplanted cells need to fuse with pre-existing myofibers, form new myofibers, and generate new muscle precursor cells (MPCs). The last property was fully reported following myoblast transplantation in mice but remains poorly studied with human myoblasts. In this study, we provide evidence that the intramuscular transplantation of postnatal human myoblasts in immunodeficient mice generates donor-derived MPCs and specifically donor-derived satellite cells. In a first experiment, cells isolated from mouse muscles 1 month after the transplantation of human myoblasts proliferated in vitro as human myoblasts. These cells were retransplanted in mice and formed myofibers expressing human dystrophin. In a second experiment, we observed that inducing muscle regeneration 2 months following transplantation of human myoblasts led to myofiber regeneration by human-derived MPCs. In a third experiment, we detected by immunohistochemistry abundant human-derived satellite cells in mouse muscles 1 month after transplantation of postnatal human myoblasts. These human-derived satellite cells may correspond totally or partially to the human-derived MPCs evidenced in the first two experiments. Finally, we present evidence that donor-derived satellite cells may be produced in patients that received myoblast transplantation.

Clinical Use of Immunosuppressants in Duchenne Muscular Dystrophy

Duchenne muscular dystrophy (DMD) is a degenerative disease primarily affecting voluntary muscles with secondary consequences on heart and breathing muscles. DMD is an X-linked recessive disease that results in the loss of dystrophin, a key muscle protein. Inflammation can play different roles in DMD; it can be a secondary response to muscle degeneration, a primary cause of degeneration, or can contribute to the disease progression. Several immunosuppressants have been used with the aim to reduce the inflammation associated with DMD. Most recently, myoblast transplantation has shown the possibility to restore the dystrophin lack in the DMD patient's muscle fibers and this evidence has emphasized the importance of the use of immunosuppressants and the necessity of studying them and their secondary effects. The aim of this review is to analyze the main immunosuppressants drugs starting from the mdx mice experiments and concluding with the most recent human clinical studies.

In vivo Fluorescence Imaging of Muscle Cell Regeneration by Transplanted EGFP-labeled Myoblasts

In vivo fluorescence imaging (FLI) enables monitoring fluorescent protein (FP)-labeled cells and proteins in living organisms noninvasively. Here, we examined whether this modality could reach a sufficient sensitivity to allow evaluation of the regeneration process of enhanced green fluorescent protein (eGFP)-labeled muscle precursors (myoblasts). Using a basic FLI station, we were able to detect clear fluorescence signals generated by 40,000 labeled cells injected into a tibialis anterior (TA) muscle of mouse. We observed that the signal declined to approximately 25% on the 48 hours of cell injection followed by a recovery starting at the second day and reached a peak of approximately 45% of the original signal by the 7th day, suggesting that the survived population underwent a limited run of proliferation before differentiation. To assess whether transplanted myoblasts could form satellite cells, we injured the transplanted muscles repeatedly with cardiotoxin. We observed a recovery of fluorescence signal following a disappearance of the signal after each cardiotoxin injection. Histology results showed donor-derived cells located underneath basal membrane and expressing Pax7, confirming that the regeneration observed by imaging was indeed mediated by donor-derived satellite cells. Our results show that FLI is a powerful tool that can extend our ability to unveil complicated biological processes such as stem cell-mediated regeneration.

In Vivo Magnetic Resonance Tracking of Transplanted Myoblasts Labeled with Magnetic Iron Oxide Nanoparticle in a Rabbit Model of Fecal Incontinence

Purpose Prior to the application of myoblast transplantation technique for regeneration of anal sphincter to patients, noninvasive methods for tracking the temporal and spatial homings of these cells are essential. Magnetic resonance imaging (MRI) may emerge as an ideal imaging modality to monitor stem cell therapy with high spatial resolution. We investigated homing of transplanted superparamagnetic iron oxide (SPIO) nanoparticle-labeled muscle progenitor cells using MRI in an experimental rabbit model of fecal incontinence to determine the transplanted cell's engraftment efficiency and functional capability. Material and Methods The anal sphincters of sixteen male rabbits were damaged by sphincterotomy and were randomized to two groups. In group I myoblasts were isolated from quadriceps myofiber explants and labeled with SPIO nanoparticles, and injected into the injured sphincter. In group II, saline buffer was injected. Cell labeling efficiency was assessed using viability, proliferation, Prussian-blue staining, and MRI in-vitro. T2-weighted MR examinations were performed immediately before, 1 hour, 7, 14, 28, and 60 days after transplantation and findings were correlated with histomorphological, EMG, and manometric study. Results Labeling did not influence cellular parameters. Distribution and migration of SPIO-labeled cells was successfully documented with in-vivo MR imaging. Although, hypointense signals on T2-weighted images decreased significantly in the sphincter at the intervals, myoblasts could be tracked until 60 days after implantation. Histochemical analyses confirmed the presence of Prussian-blue-stain positive cells in the regenerating sphincter mainly at the injured site. The concurrent functional study revealed improvement in the mean resting anal canal pressure and sphincteric EMG activity. Conclusions Our study illustrates the potential of noninvasive MRI techniques for in-vivo monitoring of magnetically labeled myoblasts for anal sphincter myoplasty. The presence of the labeled cell corresponding with functional improvement confirmed their capability for recapitulation of a myogenic program in a deficient anal sphincter.

Engraftment Is Optimal When Myoblasts Are Transplanted Early: The Role of Hepatocyte Growth Factor

In a recent clinical trial, skeletal myoblast (SKMB) transplantation performed late after myocardial infarction (MI) did not improve left ventricular function. We hypothesized that (1) delaying SKMB transplantation until a chronic infarct scar has developed reduces engraftment, and (2) hepatocyte growth factor (HGF), a main regulator of SKMBs, is present in acute but not chronic MI, potentially influencing engraftment. Rats underwent coronary artery ligation followed by SKMB transplantation immediately (n = 12) or delayed by 5 weeks (n = 11). The volume of engrafted SKMBs was quantified 6 weeks later. Hepatocyte growth factor was evaluated by computerized analysis of immunohistochemical labeling of rat heart sections 48 hours, 1 week, 2 weeks, and 5 weeks after coronary artery ligation. The impact of HGF on SKMB proliferation and its ability to protect against oxidative stress and hypoxia was evaluated in vitro. Skeletal myoblast transplantation immediately after MI resulted in an engraftment volume of 29.1 +/- 2.9 mm(3). However, delaying SKMB transplantation 5 weeks caused a 95% drop in engraftment (1.4 +/- 0.3 mm(3); p < 0.001). Hepatocyte growth factor labeling in MIs 48 hours after coronary artery ligation was similar to control myocardium (18.0 +/- 2.0 versus 16.8 +/- 1.3 units). However, HGF declined progressively at 1, 2, and 5 weeks after MI (9.1 +/- 1.4, 4.2 +/- 0.4, and 3.1 +/- 0.6 units, respectively; p < 0.05 versus 48 hours). Hepatocyte growth factor caused a dose-dependent increase in SKMB proliferation in vitro and protected against oxidative stress and hypoxia. These results demonstrate that engraftment of SKMBs is impaired when transplantation is delayed until a chronic infarct has developed. Hepatocyte growth factor in MI declines with time and may enhance engraftment of SKMBs transplanted early after MI. Delivery of exogenous HGF to enhance SKMB engraftment in chronic infarcts warrants further investigation.

Invited Commentary

Invited Commentary

Invited Commentary

Invited Commentary

Clinical trials update from the Heart Failure Society of America Meeting 2009: FAST, IMPROVE‐HF, COACH galectin‐3 substudy, HF‐ACTION nuclear substudy, DAD‐HF, and MARVEL‐1

This article presents findings and a commentary on late-breaking trials presented during the meeting of the Heart Failure Society of America in September 2009. Unpublished reports should be considered as preliminary, since analyses may change in the final publication. The FAST trial showed somewhat better performance of intrathoracic impedance for prediction of deterioration in patients with heart failure (HF) when compared with daily weighing. The IMPROVE-HF study reported the benefits of education on the management of patients with systolic HF. Galectin-3 appeared a useful method for improving risk stratification of patients with chronic HF in a substudy of the COACH trial. A nuclear substudy of the HF-ACTION trial failed to demonstrate that resting myocardial perfusion imaging, a measure of myocardial scar and viability, was clinically useful. A small randomized controlled trial (DAD-HF) suggested that the use of low-dose dopamine in patients with acutely decompensated HF was associated with less deterioration in renal function and less hypokalaemia. The MARVEL-1 trial raises further concerns about the safety of myoblast transplantation in ischaemic HF.

Electrophysiological Challenges of Cell-Based Myocardial Repair

Cardiovascular diseases remain the major cause of death in the Western world.1 Stem and progenitor cell (SPC)–based therapies in animal models and human trials in recent years have suggested promising therapeutic potential and drawn intense public interest. Possible beneficial mechanisms of cell-based therapies include generation of new and mature ventricular cardiomyocytes (cardiomyogenesis), recruitment of endogenous SPCs for cardiomyogenesis, and salvage of native myocardium through paracrine, angiogenic, and antiapoptotic effects.2,3 We refer readers to several excellent reviews for an overview of cell-based therapies for cardiac diseases.2–5 Here we discuss potential electrophysiological challenges and arrhythmic risks posed by cell therapies designed to replace damaged myocardium. Although adverse outcomes after cell therapies, such as lethal arrhythmias, occurred in early clinical trials of skeletal myoblast transplantation,5–8 recent clinical trials using various sources of SPCs, novel modes of cell delivery, better cell selection, and different timing of cell transplantation did not show any significant increase in arrhythmic incidence. It is tempting to conclude on this basis that cell-based therapies are “safe” and nonarrhythmogenic (reviewed in References 2 and 4); however, the negligible production of new cardiomyocytes documented for many SPCs employed makes it difficult to extrapolate their safety records toward future trials in which substantial numbers of new cardiomyocytes might be generated or introduced. Clinical trials to test cardiomyocyte replacement (cardiomyogenesis), long a goal of SPC-based therapy, are bound to take place in the near future, thanks to many investigators who are developing technologies for efficient generation of cardiomyocytes from SPCs and for increasing their retention after transplantation. In weighing the benefits of replacement therapies, we should not overlook the fact that SPC-derived cardiomyocytes exhibit variable electrophysiological properties and are typically immature compared with adult cardiomyocytes (see below). This immaturity of primitive SPC-derived cardiomyocytes remains a major hurdle toward developing safe …

La greffe de myoblastes dans le muscle squelettique améliore la tolérance au glucose dans un modèle de souris diabétique

Aims/hypothesis We aimed to demonstrate the feasibility and efficacy of intra-muscular transplantation of human skeletal myoblasts (hSkMs) for attenuation of hyperglycaemia and improvement of insulin sensitivity using a mouse model of type 2 diabetes mellitus.