Quantitative proteome profiling of dystrophic dog skeletal muscle reveals a stabilized muscular architecture and protection against oxidative stress after systemic delivery of MuStem cells.

International audienceProteomic profiling plays a decisive role in the elucidation of molecular signatures representative of a specific clinical context. MuStem cell-based therapy represents a promising approach for clinical applications to cure Duchenne Muscular Dystrophy (DMD). To expand our previous studies collected in the clinically relevant DMD animal model, we decided to investigate the skeletal muscle proteome four months after systemic delivery of allogenic MuStem cells. Quantitative proteomics with isotope-coded protein labelling (ICPL) was used to compile quantitative changes in the protein expression profiles of muscle in transplanted Golden Retriever Muscular Dystrophy (GRMD) dogs as compared to GRMD dogs. A total of 492 proteins were quantified, including 25 that were overrepresented and 46 that were underrepresented after MuStem cell transplantation. Interestingly, this study demonstrates that somatic stem cell therapy impacts on the structural integrity of the muscle fascicle by acting on fibres and its connections with the extracellular matrix. We also show that cell infusion promotes protective mechanisms against oxidative stress and favours the initial phase of muscle repair. This study allows us to identify putative candidates for tissue markers that might be of great value in objectively exploring the clinical benefits resulting from our cell-based therapy for DMD. This article is protected by copyright. All rights reserved

Isolation and Characterization of MuStem cell-derived extracellular vesicles: potential roles in regenerative medicine?

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Identification in GRMD dog muscle of critical miRNAs involved in pathophysiology and effects associated with MuStem cell transplantation

Background: Duchenne muscular dystrophy (DMD) is an X-linked muscle disease that leads to fibre necrosis and progressive paralysis. At present, DMD remains a lethal disease without any effective treatment, requiring a better understanding of the pathophysiological processes and comprehensive assessment of the newly identified therapeutic strategies. MicroRNAs including members of the muscle-specific myomiR family have been identified as being deregulated in muscle of DMD patients and in mdx mice used as a model for DMD. In recent years, the Golden Retriever muscular dystrophy (GRMD) dog has appeared as the crucial animal model for objectively assessing the potential of new innovative approaches. Here, we first aim at establishing the muscle expression pattern of five selected miRNAs in this clinically relevant model to determine if they are similarly affected compared with other DMD contexts. Second, we attempt to show whether these miRNAs could be impacted by the systemic delivery of a promising stem cell candidate (referred to as MuStem cells) to implement our knowledge on its mode of action and/or identify markers associated with cell therapy efficacy. Methods: A comparative study of miRNAs expression levels and cellular localization was performed on 9-monthold healthy dogs, as well as on three sub-sets of GRMD dog (without immunosuppression or cell transplantation, with continuous immunosuppressive regimen and with MuStem cell transplantation under immunosuppression), using RT-qPCR and in situ hybridization. Results: We find that miR-222 expression is markedly up-regulated in GRMD dog muscle compared to healthy dog, while miR-486 tends to be down-expressed. Intriguingly, the expression of miR-1, miR-133a and miR-206 does not change. In situ hybridization exploration reveals, for the first time, that miR-486 and miR-206 are mainly localized in newly regenerated fibres in GRMD dog muscle. In addition, we show that cyclosporine-based immunosuppression, classically used in allogeneic cell transplantation, exclusively impacts the miR-206 expression. Finally, we demonstrate that intra-arterial administration of MuStem cells results in up-regulation of miR-133a and miR-222 concomitantly with a down-expression of two sarcomeric proteins corresponding to miR-222 targets. Conclusion: We point out a differential muscle expression of miR-222 and miR-486 associated with the pathophysiology of the clinically relevant GRMD dog model with a tissue localization focused on regenerated fibres. We also establish a modified expression of miR-133a and miR-222 subsequent to MuStem cell infusion

Exploration of muscle from GRMD dogs tranplanted with MuStem cells using “omics” approaches

Duchenne Muscular Dystrophy (DMD), the most common form of inherited neuromuscular disorder, is caused by mutations in the dystrophin gene leading to the protein lack. Membrane disorganization and subsequent alterations in signaling pathways and energy metabolism play important roles in muscle fibre necrosis. Systemic delivery of MuStem cells, skeletal muscle-resident stem cells isolated in healthy dog, generate a remodeling of muscle tissue and gives rise to striking clinical benefits in Golden Retriever Muscular Dystrophy (GRMD) dog. To pursue investigation of the consequences on the skeletal muscle tissue 6 months after cell transplantation with undedicated approach, we used here a combined analysis of transcriptomics (gene expression microarrays) and quantitative proteomics (ICPL/LC-MS/MS) (Robriquet et al., 2015). At molecular level, we determined that MuStem cell administration enhances muscle regeneration, promotes ubiquitin-mediated protein degradation in parallel with a decrease expression of genes associated with lipid homeostasis and energy metabolism. Furthermore, the proteomic approach confirmed a main impact of MuStem cell delivery on muscle regeneration, metabolism as well as homeostasis pathways. In addition, we establish that the analysis of a limited set of miRNAs in skeletal muscle clearly discriminates between immunosuppression context and MuStem cell therapyrelated effects on GRMD dogs. Overall, the combination of transcriptomics, proteomics and miRNA approaches allowed to pave the way to the understanding of MuStem cell action modalities as stimulation of muscle fibre formation. This strategy has a great potential to considerably contribute to the identification of therapeutic biomarkers of MuStem cell transplantation and thus represents an interesting tool to monitor therapeutic effects during DMD-dedicated preclinical studies

Differential gene and miRNA profiling in dystrophic dog and impact of MuStem cell-based therapy

Differential gene and miRNA profiling in dystrophic dog and impact of MuStem cell-based therapy. Annual Congress of the European Society of Gene and Cell Therapy (ESGCT

Myocardial infarct repair with human adult muscle-derived stem cells “MuStem”

Myocardial infarction is a leading cause of morbidity and mortality worldwide. Although medical and surgical treatments can significantly improve patient outcomes, no treatment currently available is able to generate new contractile tissue or reverse ischemic myocardium. Driven by the recent understanding that regenerative processes do exist in the myocardium, the use of stem cells has emerged as a promising therapeutic approach with high expectations. The literature describes the use of cells from various sources. Many publications show the promising use of these cells to regenerate damaged myocardium in both animal models and Human; however, more studies are needed to directly compare cells of various origins in efforts to draw conclusions on the most appropriate source in order to positively impact on the heart tissue remodeling and function

Human MuStem cell grafting into infarcted rat heart attenuates adverse tissue remodeling and preserves cardiac function hMuStem cells preserve function of infarcted heart

International audienceMyocardial infarction is one of the leading causes of mortality and morbidity worldwide. Whereas transplantation of several cell types into the infarcted heart has produced promising preclinical results, clinical studies using analogous human cells have shown limited structural and functional benefits. In dogs and humans, we have described a type of muscle-derived stem cells termed MuStem cells that efficiently promoted repair of injured skeletal muscle. Enhanced survival rate, long-term engraftment, and participation in muscle fiber formation were reported, leading to persistent tissue remodeling and clinical benefits. With the consideration of these features that are restricted or absent in cells tested so far for myocardial infarction, we wanted to investigate the capacity of human MuStem cells to repair infarcted hearts. Their local administration in immunodeficient rats 1 week after induced infarction resulted in reduced fibrosis and increased angiogenesis 3 weeks post-transplantation. Importantly, foci of human fibers were detected in the infarct site. Treated rats also showed attenuated left-ventricle dilation and preservation of contractile function. Interestingly, no spontaneous arrhythmias were observed. Our findings support the potential of MuStem cells, which have already been proposed as therapeutic candidates for dystrophic patients, to treat myocardial infarction and position them as an attractive tool for muscle-regenerative medicine