Myogenic Cell Transplantation Research Articles

Challenges and Considerations of Preclinical Development for iPSC-Based Myogenic Cell Therapy.

Cell therapies derived from induced pluripotent stem cells (iPSCs) offer a promising avenue in the field of regenerative medicine due to iPSCs' expandability, immune compatibility, and pluripotent potential. An increasing number of preclinical and clinical trials have been carried out, exploring the application of iPSC-based therapies for challenging diseases, such as muscular dystrophies. The unique syncytial nature of skeletal muscle allows stem/progenitor cells to integrate, forming new myonuclei and restoring the expression of genes affected by myopathies. This characteristic makes genome-editing techniques especially attractive in these therapies. With genetic modification and iPSC lineage specification methodologies, immune-compatible healthy iPSC-derived muscle cells can be manufactured to reverse the progression of muscle diseases or facilitate tissue regeneration. Despite this exciting advancement, much of the development of iPSC-based therapies for muscle diseases and tissue regeneration is limited to academic settings, with no successful clinical translation reported. The unknown differentiation process in vivo, potential tumorigenicity, and epigenetic abnormality of transplanted cells are preventing their clinical application. In this review, we give an overview on preclinical development of iPSC-derived myogenic cell transplantation therapies including processes related to iPSC-derived myogenic cells such as differentiation, scaling-up, delivery, and cGMP compliance. And we discuss the potential challenges of each step of clinical translation. Additionally, preclinical model systems for testing myogenic cells intended for clinical applications are described.

Human pluripotent stem cell-derived myogenic progenitors undergo maturation to quiescent satellite cells upon engraftment.

Human pluripotent stem cell (hPSC)-derived myogenic progenitor cell (MPC) transplantation is a promising therapeutic approach for a variety of degenerative muscle disorders. Here, using an MPC-specific fluorescent reporter system (PAX7::GFP), we demonstrate that hPSC-derived MPCs can contribute to the regeneration of myofibers in mice following local injury and in mice deficient of dystrophin (mdx). We also demonstrate that a subset of PAX7::GFP MPCs engraft within the basal lamina of regenerated myofibers, adopt a quiescent state, and contribute to regeneration upon reinjury and in mdx mouse models. This subset of PAX7::GFP MPCs undergo a maturation process and remodel their molecular characteristics to resemble those of late-stage fetal MPCs/adult satellite cells following invivo engraftment. These in-vivo-matured PAX7::GFP MPCs retain a cell-autonomous ability to regenerate and can repopulate in the niche of secondary recipient mice, providing a proof of principle for future hPSC-based cell therapy for muscle disorders.

Myogenic Cell Transplantation in Genetic and Acquired Diseases of Skeletal Muscle.

This article will review myogenic cell transplantation for congenital and acquired diseases of skeletal muscle. There are already a number of excellent reviews on this topic, but they are mostly focused on a specific disease, muscular dystrophies and in particular Duchenne Muscular Dystrophy. There are also recent reviews on cell transplantation for inflammatory myopathies, volumetric muscle loss (VML) (this usually with biomaterials), sarcopenia and sphincter incontinence, mainly urinary but also fecal. We believe it would be useful at this stage, to compare the same strategy as adopted in all these different diseases, in order to outline similarities and differences in cell source, pre-clinical models, administration route, and outcome measures. This in turn may help to understand which common or disease-specific problems have so far limited clinical success of cell transplantation in this area, especially when compared to other fields, such as epithelial cell transplantation. We also hope that this may be useful to people outside the field to get a comprehensive view in a single review. As for any cell transplantation procedure, the choice between autologous and heterologous cells is dictated by a number of criteria, such as cell availability, possibility of in vitro expansion to reach the number required, need for genetic correction for many but not necessarily all muscular dystrophies, and immune reaction, mainly to a heterologous, even if HLA-matched cells and, to a minor extent, to the therapeutic gene product, a possible antigen for the patient. Finally, induced pluripotent stem cell derivatives, that have entered clinical experimentation for other diseases, may in the future offer a bank of immune-privileged cells, available for all patients and after a genetic correction for muscular dystrophies and other myopathies.

Myogenic progenitor cell transplantation for muscle regeneration following hindlimb ischemia and reperfusion

BackgroundMuscle is severely affected by ischemia/reperfusion injury (IRI). Quiescent satellite cells differentiating into myogenic progenitor cells (MPC) possess a remarkable regenerative potential. We herein established a model of local application of MPC in murine hindlimb ischemia/reperfusion to study cell engraftment and differentiation required for muscle regeneration.MethodsA clamping model of murine (C57b/6 J) hindlimb ischemia was established to induce IRI in skeletal muscle. After 2 h (h) warm ischemic time (WIT) and reperfusion, reporter protein expressing MPC (TdTomato or Luci-GFP, 1 × 106 cells) obtained from isolated satellite cells were injected intramuscularly. Surface marker expression and differentiation potential of MPC were analyzed in vitro by flow cytometry and differentiation assay. In vivo bioluminescence imaging and histopathologic evaluation of biopsies were performed to quantify cell fate, engraftment and regeneration.Results2h WIT induced severe IRI on muscle, and muscle fiber regeneration as per histopathology within 14 days after injury. Bioluminescence in vivo imaging demonstrated reporter protein signals of MPC in 2h WIT animals and controls over the study period (75 days). Bioluminescence signals were detected at the injection site and increased over time. TdTomato expressing MPC and myofibers were visible in host tissue on postoperative days 2 and 14, respectively, suggesting that injected MPC differentiated into muscle fibers. Higher reporter protein signals were found after 2h WIT compared to controls without ischemia, indicative for enhanced growth and/or engraftment of MPC injected into IRI-affected muscle antagonizing muscle damage caused by IRI.ConclusionWIT-induced IRI in muscle requests increased numbers of injected MPC to engraft and persist, suggesting a possible rational for cell therapy to antagonize IRI. Further investigations are needed to evaluate the regenerative capacity and therapeutic advantage of MPC in the setting of ischemic limb injury.

Therapeutic Strategies for Duchenne Muscular Dystrophy: An Update.

Neuromuscular disorders encompass a heterogeneous group of conditions that impair the function of muscles, motor neurons, peripheral nerves, and neuromuscular junctions. Being the most common and most severe type of muscular dystrophy, Duchenne muscular dystrophy (DMD), is caused by mutations in the X-linked dystrophin gene. Loss of dystrophin protein leads to recurrent myofiber damage, chronic inflammation, progressive fibrosis, and dysfunction of muscle stem cells. Over the last few years, there has been considerable development of diagnosis and therapeutics for DMD, but current treatments do not cure the disease. Here, we review the current status of DMD pathogenesis and therapy, focusing on mutational spectrum, diagnosis tools, clinical trials, and therapeutic approaches including dystrophin restoration, gene therapy, and myogenic cell transplantation. Furthermore, we present the clinical potential of advanced strategies combining gene editing, cell-based therapy with tissue engineering for the treatment of muscular dystrophy.

Potential Therapies Using Myogenic Stem Cells Combined with Bio-Engineering Approaches for Treatment of Muscular Dystrophies.

Muscular dystrophies (MDs) are a group of heterogeneous genetic disorders caused by mutations in the genes encoding the structural components of myofibres. The current state-of-the-art treatment is oligonucleotide-based gene therapy that restores disease-related protein. However, this therapeutic approach has limited efficacy and is unlikely to be curative. While the number of studies focused on cell transplantation therapy has increased in the recent years, this approach remains challenging due to multiple issues related to the efficacy of engrafted cells, source of myogenic cells, and systemic injections. Technical innovation has contributed to overcoming cell source challenges, and in recent studies, a combination of muscle resident stem cells and gene editing has shown promise as a novel approach. Furthermore, improvement of the muscular environment both in cultured donor cells and in recipient MD muscles may potentially facilitate cell engraftment. Artificial skeletal muscle generated by myogenic cells and muscle resident cells is an alternate approach that may enable the replacement of damaged tissues. Here, we review the current status of myogenic stem cell transplantation therapy, describe recent advances, and discuss the remaining obstacles that exist in the search for a cure for MD patients.

The impact of myogenic cell transplantation on collateral capillary arteriogenesis

Current treatments for peripheral arterial occlusive disease (PAOD), including angioplasty and bypass grafting fail in up to 25% of patients, or are not indicated in up to 50%, so there is a need to develop additional treatment options. Patients with native collaterals have a better prognosis, so a treatment such as cell transplantation that promotes collateral arteriogenesis is a potential PAOD treatment. Unfortunately, bone marrow derived stem cells, the main cell type that has been investigated, have limited clinical effectiveness in enhancing pre‐existing collateral growth. Therefore, delivering a stem cell type native to the collateral‐containing tissue, such as myogenic stem cells, could have improved outcomes. Additionally, about 20% of patients never develop collaterals, and no one, to our knowledge, has evaluated the effect of cell therapy on collateral capillary arteriogenesis (CCA), which can reperfuse ischemic tissue in animals without native arteriolar collaterals and therefore represents a potential therapeutic target in patients who lack native collaterals. To test the hypothesis that myogenic cells enhance CCA, we ligated the spinotrapezius feed artery in Balb/C mice to induce CCA, and delivered myogenic cells, thrombin (positive control), or vehicle to the muscle. Seven days post ligation, ‐‐‐‐arterialized collateral capillary (ACC) number and diameter were determined. As expected, thrombin increased ACC number, but myogenic cells had no effect, 9.3 ± 0.5 vs 7.1 ± 0.4, respectively. However, myogenic cells increased ACC diameter as compared to untreated (12.1 ± 0.8 μm vs 10.3 ± 0.3 μm, respectively) and both myogenic cells and thrombin increased the maximum ACC diameter (29.9 ± 3.4 μm and 27.9 ± 2.0 μm vs 18.8 ± 1.1 μm, respectively). These results are consistent with previous studies indicating that thrombin enhances arteriogenesis and suggest that myogenic cells have a similar effect, but likely without the deleterious pro‐inflammatory effects of thrombin. Because myogenic cells increase arteriogenesis and macrophages are an essential part of arteriogenesis, we also tested the hypothesis that myogenic cells increase macrophage content by performing CD68 staining. Although macrophage number increased following ligation, neither myoblasts nor thrombin further increased macrophage number. The lack of difference in macrophage number despite enhanced CCA could be because the day‐7 timepoint was too late, as macrophages peak around day‐3, or because a shift towards either classically‐activated macrophages or alternatively‐activated macrophages, which have a greater effect on arteriogenesis, would not be detected by the CD68 stain. Therefore, future studies may include a day‐3 timepoint and a stain for alternatively‐activated macrophages.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

De Novo Circulating Antidonor's Cell Antibodies During Induced Acute Rejection of Allogeneic Myofibers in Myogenic Cell Transplantation: A Study in Nonhuman Primates

BackgroundTransplantation of myogenic cells has potential applications in the treatment of muscle pathologies. Excluding purely autologous cell transplantation, graft viability depends on an adequate control of acute rejection (AR). To contribute in understanding AR in this context, we analyzed whether de novo circulating antibodies against donor’s cells are detected during induced AR of graft-derived myofibers in nonhuman primates.MethodsWe allotransplanted satellite cell-derived myoblasts in macaques immunosuppressed with tacrolimus. To induce AR of graft-derived myofibers, we administered tacrolimus for 4 weeks to allow complete myofiber formation, and then we stopped tacrolimus administration. Cell-grafted sites were biopsied at tacrolimus withdrawal and then every 2 weeks and analyzed by histology until AR completion. Blood samples were taken before immunosuppression, at tacrolimus withdrawal and then every 2 weeks to detect antibodies against the donor’s cells by flow cytometry.ResultsThere was an increase of antibodies against the donor’s cells related to AR in all monkeys. This increase was variable in intensity, and preceded, coincided or followed the histological evidence of AR (focal accumulations of lymphocytes) and/or the loss of myofibers of donor origin, and remained until the end of the follow-up (up to 8 weeks after tacrolimus withdrawal).ConclusionsFlow cytometry detection of de novo circulating antibodies against the donor’s cells was consistently associated with AR. A clear increase in this antibody detection indicated current or recent AR. Smaller increases in comparison to the preimmunosuppression values were not associated with AR.

Wnt7a stimulates myogenic stem cell motility and engraftment resulting in improved muscle strength

Wnt7a/Fzd7 signaling stimulates skeletal muscle growth and repair by inducing the symmetric expansion of satellite stem cells through the planar cell polarity pathway and by activating the Akt/mTOR growth pathway in muscle fibers. Here we describe a third level of activity where Wnt7a/Fzd7 increases the polarity and directional migration of mouse satellite cells and human myogenic progenitors through activation of Dvl2 and the small GTPase Rac1. Importantly, these effects can be exploited to potentiate the outcome of myogenic cell transplantation into dystrophic muscles. We observed that a short Wnt7a treatment markedly stimulated tissue dispersal and engraftment, leading to significantly improved muscle function. Moreover, myofibers at distal sites that fused with Wnt7a-treated cells were hypertrophic, suggesting that the transplanted cells deliver activated Wnt7a/Fzd7 signaling complexes to recipient myofibers. Taken together, we describe a viable and effective ex vivo cell modulation process that profoundly enhances the efficacy of stem cell therapy for skeletal muscle.

Safety Assessment of Myogenic Stem Cell Transplantation and Resulting Tumor Formation

To assess for stem cell migration to liver and lung after transplantation in injured rat anal sphincters. To evaluate histological findings of unanticipated ectopic foci of growth. This is a prospective study involving 33 female virginal Sprague-Dawley rats. Anal sphincters were transected and repaired under sterile technique. Animals received injections of 5.0 × 10 myogenic stem cells (24 rats) or sham control (9 rats) and were killed on day 30. Liver and lung samples were obtained. Upon encountering abnormal foci of growth, further staining protocols were employed. Enzyme-linked immunosorbent assay studies evaluated stem cell media for in vitro growth factor secretion. No evidence of cell migration to liver or lung was found at the time of euthanasia in any study animal. Ectopic foci of growth were noted in 2 transplant rats. Further histological evaluations of these growths were consistent with benign tumors: no nuclear abnormalities and no evidence of proliferation at day 30. Enzyme-linked immunosorbent assay studies demonstrated positive secretion of vascular endothelial growth factor and insulin growth factor into the media of cultured rat myogenic stem cells. Whereas distant migration was not encountered in the liver or lung, 2 transplanted rats developed abnormal foci of growth, that is, tumors, from the external anal sphincter-raising further safety questions. Additional evaluation of these foci seemed benign. Possible explanations include cell trapping, stem cell overgrowth, and/or paracrine factors. The lack of cell migration supports that future investigation of safety parameters could focus locally.

Acute Rejection of Myofibers in Nonhuman Primates: Key Histopathologic Features

The aim of this study was to define the histologic features of acute rejection of myofibers, particularly in the context of therapeutic myogenic cell transplantation. Myoblasts expressing or not expressing β-galactosidase were transplanted into 13 macaques that were divided into 3 protocols: withdrawal of immunosuppression, low immunosuppression, and progressive reduction of immunosuppression. The biopsy samples were obtained from cell-grafted sites at different intervals, and cryostat sections of biopsies were analyzed. The grafts were lost in all the monkeys at different periods after transplantation depending on the protocol and in association with low blood levels of tacrolimus. In all cases, graft loss was associated with the presence of dense focal accumulations of CD8-positive and CD4-positive lymphocytes and a component of macrophages. The lymphocyte accumulations totally or partially surrounded some myofibers and often invaded them; they were mainly endomysial. These histopathologic patterns in nonhuman primates and their similarity with preliminary observations in humans may facilitate the translation of these results to the histologic diagnosis of acute rejection of myofibers in human clinical trials of myogenic cell transplantation and probably gene therapy.

Protective effect of sodium ascorbate on efficacy of intramuscular transplantation of autologous muscle‐derived cells

The possible reason for elimination of myogenic cells after transplantation is inflammation at the injection site associated with oxidative stress. The aim of this study was to determine whether preconditioning of muscle-derived cells with an antioxidant, sodium ascorbate, can influence the fate of transplanted cells. Autologous transplantation of muscle-derived cells was performed in rabbits. Isolated cells were identified, lipofected with β-galactosidase, preincubated or not with sodium ascorbate, and injected intramuscularly. Two weeks after autologous transplantation in the edge of a previous muscle defect, donor cells formed multinucleated young myotubes. Pretreatment of cells with sodium ascorbate before injection resulted in a significant increase of donor cells at the injection site 2 weeks after transfer. These results show that: (1) preincubation with antioxidant can increase the efficacy of myogenic cell transplantation; and (2) oxidative stress may play a role in elimination of cells after autologous transplantation.

Intramuscular cell transplantation as a potential treatment of myopathies: clinical and preclinical relevant data

Introduction: Myopathies produce deficits in skeletal muscle function and, in some cases, literally progressive loss of skeletal muscles. The transplantation of cells able to differentiate into myofibers is an experimental strategy for the potential treatment of some of these diseases.Areas covered: Among the two routes used to deliver cells to skeletal muscles, that is intramuscular and intravascular, this paper focuses on the intramuscular route due to our expertise and because it is the most used in animal experiments and the only tested so far in humans. Given the absence of recent reviews about clinical observations and the profusion based on mouse results, this review prioritizes observations made in humans and non-human primates. The review provides a vision of cell transplantation in myology centered on what can be learned from clinical trials and from preclinical studies in non-human primates and leading mouse studies.Expert opinion: Experiments on myogenic cell transplantation in mice are essential to quickly identify potential treatments, but studies showing the possibility to scale up the methods in large mammals are indispensable to determine their applicability in humans and to design clinical protocols.

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.

Growth Factor Coinjection Improves the Migration Potential of Monkey Myogenic Precursors without Affecting Cell Transplantation Success

Duchenne muscular dystrophy (DMD) is an inherited disease and a main target of myogenic cell transplantation (MT). After the failure of the first clinical trials with DMD patients, the poor migration of transplanted cells has been suspected to be a major problem for a more effective clinical application of MT. Previous investigations suggested that the quantity and dispersion of myofibers containing donor cell nuclei might be improved by increasing the migration of the transplanted cells outside the injection sites. Because the coinjection of motogenic factors with human myoblasts enhanced their intramuscular migration following MT in SCID mice, the present study aimed to investigate whether this approach was appropriate to increase MT success in muscles of nonhuman primates. In vitro studies indicated that IGF-1 or bFGF increased components of proteolytic systems involved in myoblast migration. In vitro and in vivo experiments also demonstrated that coinjection of bFGF or IGF-1 was able to improve monkey myogenic cell migration and invasion. Sixty hours after MT in skeletal muscle tissue, the migration distances reached by monkey myoblasts increased by nearly twofold when one of the growth factors was coinjected with the cells. However, long-term observations in adult monkeys suggest that promigratory treatments are not intrinsically sufficient to improve the success of MT. Even if short-term observations reveal that grafted cells are not always trapped inside the injection site and in spite of the fact that both factors enhanced transplanted cell migration, myofibers including grafted cell nuclei were still restrained to the injection trajectory without notable difference in their amount or their dispersion. The incapacity of transplanted cells to fuse with undamaged myofibers, which are located outside the injection sites, is a priority problem to solve in order to improve transplantation success and reduce the number of injections required for the treatment of DMD patients.

Enhancement of Myogenic and Muscle Repair Capacities of Human Adipose–derived Stem Cells With Forced Expression of MyoD

Muscle disorders such as Duchenne muscular dystrophy (DMD) still need effective treatments, and mesenchymal stem cells (MSCs) may constitute an attractive cell therapy alternative because they are multipotent and accessible in adult tissues. We have previously shown that human multipotent adipose-derived stem (hMADS) cells were able to restore dystrophin expression in the mdx mouse. The goal of this work was to improve the myogenic potential of hMADS cells and assess the impact on muscle repair. Forced expression of MyoD in vitro strongly induced myogenic differentiation while the adipogenic differentiation was inhibited. Moreover, MyoD-expressing hMADS cells had the capacity to fuse with DMD myoblasts and to restore dystrophin expression. Importantly, transplantation of these modified hMADS cells into injured muscles of immunodepressed Rag2(-/-)gammaC(-/-) mice resulted in a substantial increase in the number of hMADS cell-derived fibers. Our approach combined the easy access of MSCs from adipose tissue, the highly efficient lentiviral transduction of these cells, and the specific improvement of myogenic differentiation through the forced expression of MyoD. Altogether our results highlight the capacity of modified hMADS cells to contribute to muscle repair and their potential to deliver a repairing gene to dystrophic muscles.

Association of electrostimulation with cell transplantation in ischemic heart disease

Until now, cell therapy has constituted a passive therapeutic approach; the only effects seem to be related to the reduction of the myocardial fibrosis and the limitation of the adverse ventricular remodeling. Cardiac resynchronization therapy is indicated in patients with heart failure to correct conduction disorders associated with chronic systolic and diastolic dysfunction. The association of electrostimulation with cellular cardiomyoplasty could be a way to transform passive cell therapy into "dynamic cellular support." Electrostimulation of ventricles following skeletal myoblast implantation should induce the contraction of the transplanted cells and a higher expression of slow myosin, which is better adapted for chronic ventricular assistance. The purpose of this study is to evaluate myogenic cell transplantation in an ischemic heart model associated with cardiac resynchronization therapy. Twenty two sheep were included. All animals underwent myocardial infarction by ligation of 2 coronary artery branches (distal left anterior descending artery and D2). After 4 weeks, autologous cultured myoblasts were injected in the infarcted areas with or without pacemaker implantation. Atrial synchronized biventricular pacing was performed using epicardial electrodes. Echocardiography was performed at 4 weeks (baseline) and 12 weeks after infarction. Echocardiography showed a significant improvement in ejection fraction and limitation of left ventricular dilatation in cell therapy with cardiac resynchronization therapy as compared with the other groups. Viable cells were identified in the infarcted areas. Differentiation of myoblasts into myotubes and enhanced expression of slow myosin heavy chain was observed in the electrostimulated group. Transplantation of cells with cardiac resynchronization therapy caused an increase in diastolic wall thickening in the infarcted zone relative to cells-only group and cardiac resynchronization therapy-only group. Biventricular pacing seems to induce synchronous contraction of transplanted myoblasts and the host myocardium, thus improving ventricular function. Electrostimulation was related with enhanced expression of slow myosin and the organization of myoblasts in myotubes, which are better adapted at performing cardiac work. Patients with heart failure presenting myocardial infarct scars and indication for cardiac resynchronization therapy might benefit from simultaneous cardiac pacing and cell therapy.

Advances in myogenic cell transplantation and skeletal muscle tissue engineering

Curative treatments are currently not available for people suffering from one of the many prevalent muscle myopathies. One approach to ameliorate these conditions relies on the cell-based transplantation of potential myogenic precursors, or more optimistically, the transfer of engineered skeletal muscle tissue. To date, clinical trials with myogenic stem cell transplantation have met with only modest success while the transplantation of engineered muscle tissue is at the earliest stages of development. Despite the slow progress, these studies have provided insights and avenues that will eventually lead to a powerful therapeutic tool.

Characterization of the paracrine effects of human skeletal myoblasts transplanted in infarcted myocardium

The discrepancy between the functional improvements yielded experimentally by skeletal myoblasts (SM) transplanted in infarcted myocardium and the paucity of their long-term engraftment has raised the hypothesis of cell-mediated paracrine mechanisms. We analyzed gene expression and growth factors released by undifferentiated human SM (CD56(+)), myotubes (SM cultured until confluence) and fibroblasts-like cells (CD56(-)). Gene expression revealed up-regulation of pro-angiogenic (PGF), anti-apoptotics (BAG-1, BCL-2), heart development (TNNT2, TNNC1) and extracellular matrix remodelling (MMP-2, MMP-7) genes in SM. In line with the gene expression profile, the analysis of culture supernatants of SM by ELISA identified the release of growth factors involved in angiogenesis (VEGF, PIGF, angiogenin, angiopoietin, HGF and PDGF-BB) as well as proteases involved in matrix remodelling (MMP2, MMP9 and MMP10) and their inhibitors (TIMPs). Culture of smooth muscle cells (SMC), cardiomyocytes (HL-1) and human umbilical vein endothelial cells (HUVECs) with SM-released conditioned media demonstrated an increased proliferation of HUVEC, SMC and cardiomyocytes (p<0.05) and a decrease in apoptosis of cardiomyocytes (p<0.05). Analysis of nude rats transplanted with human SM demonstrated expression of human-specific MMP-2, TNNI3, CNN3, PGF, TNNT2, PAX7, TGF-beta, and IGF-1 1 month after transplant. Our data support the paracrine hypothesis whereby myoblast-secreted factors may contribute to the beneficial effects of myogenic cell transplantation in infarcted myocardium.

Central Tolerance to Myogenic Cell Transplants Does Not Include Muscle Neoantigens

Duchenne muscular dystrophy is a fatal genetic disease caused by lack of dystrophin. Myogenic cell transplantation (MT), a potential therapy for Duchenne muscular dystrophy, can restore dystrophin expression in muscles. Because allogeneic MT is highly resistant to peripheral tolerance, we proposed to induce central tolerance. However, given its immunogenicity, we asked whether central tolerance to donor major histocompatibility complex would allow long-term expression of dystrophin, a tissue-specific neoantigen in dystrophic recipients. Central tolerance was induced in C57BL/10J mdx (dystrophic) mice by allogeneic bone marrow transplantation (BMT) after conditioning with either lethal total body irradiation (TBI) or an established nonmyeloablative protocol (anti-CD154, anti-CD8 mAbs, and low-dose TBI). Recipients subsequently received donor-strain MT or skin grafts. Long-term hematopoietic chimeras generated using either lethal TBI or the nonmyeloablative regimen were tolerant to donor skin grafts and both primary and secondary donor MT (>90 days). Myogenic cell transplantation survival was decreased when chimerism was transient, which was most common with nonmyeloablative conditioning and fully rather than haplo-mismatched donors. Interestingly, regardless of conditioning, MT was associated with localized muscle infiltration with Foxp3CD4, CD25CD4, and PerforinCD8 cells, whereas skin grafts lacked infiltration. Central tolerance achieved using regimens that eliminate nearly all endogenous peripheral lymphocytes (i.e., lethal irradiation) or a nonmyeloablative protocol that depleted peripheral CD8 cells, results in lymphocytic infiltration in muscles that received MT but not in skin allografts. This suggests that muscle-specific infiltration may result from lack of negative selection for peripheral neoantigens in the thymus after BMT and that tolerance after MT may rely on peripheral regulatory mechanisms.