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

Exploration of muscle from GRMD dogs transplanted with MuStem cells using "omics" approaches

International audienc

Additional file 2: Figure S2. of Identification in GRMD dog muscle of critical miRNAs involved in pathophysiology and effects associated with MuStem cell transplantation

Clinical follow-up. Clinical scores of mock GRMD dogs (---) and MuStem cell-injected dogs (—) are represented as mean ± SD. The clinical score of each GRMD dog was assessed weekly and expressed as a percentage of a theoretical healthy dog score. Limits of the MuStem cell delivery window are indicated (dashed lines). (PDF 32 kb

Differential Gene Expression Profiling of Dystrophic Dog Muscle after MuStem Cell Transplantation.

Several adult stem cell populations exhibit myogenic regenerative potential, thus representing attractive candidates for therapeutic approaches of neuromuscular diseases such as Duchenne Muscular Dystrophy (DMD). We have recently shown that systemic delivery of MuStem cells, skeletal muscle-resident stem cells isolated in healthy dog, generates the remodelling of muscle tissue and gives rise to striking clinical benefits in Golden Retriever Muscular Dystrophy (GRMD) dog. This global effect, which is observed in the clinically relevant DMD animal model, leads us to question here the molecular pathways that are impacted by MuStem cell transplantation. To address this issue, we compare the global gene expression profile between healthy, GRMD and MuStem cell treated GRMD dog muscle, four months after allogenic MuStem cell transplantation.In the dystrophic context of the GRMD dog, disease-related deregulation is observed in the case of 282 genes related to various processes such as inflammatory response, regeneration, calcium ion binding, extracellular matrix organization, metabolism and apoptosis regulation. Importantly, we reveal the impact of MuStem cell transplantation on several molecular and cellular pathways based on a selection of 31 genes displaying signals specifically modulated by the treatment. Concomitant with a diffuse dystrophin expression, a histological remodelling and a stabilization of GRMD dog clinical status, we show that cell delivery is associated with an up-regulation of genes reflecting a sustained enhancement of muscle regeneration. We also identify a decreased mRNA expression of a set of genes having metabolic functions associated with lipid homeostasis and energy. Interestingly, ubiquitin-mediated protein degradation is highly enhanced in GRMD dog muscle after systemic delivery of MuStem cells.Overall, our results provide the first high-throughput characterization of GRMD dog muscle and throw new light on the complex molecular/cellular effects associated with muscle repair and the clinical efficacy of MuStem cell-based therapy

Update on hypoxia-inducible factors and hydroxylases in oxygen regulatory pathways:from physiology to therapeutics

Abstract The “Hypoxia Nantes 2016” organized its second conference dedicated to the field of hypoxia research. This conference focused on “the role of hypoxia under physiological conditions as well as in cancer” and took place in Nantes, France, in October 6–7, 2016. The main objective of this conference was to bring together a large group of scientists from different spheres of hypoxia. Recent advances were presented and discussed around different topics: genomics, physiology, musculoskeletal, stem cells, microenvironment and cancer, and oxidative stress. This review summarizes the major highlights of the meeting

Clinical follow-up.

<p>(A) Mean±SD clinical scores of mock GRMD dogs and MuStem cell-injected dogs (GRMD^MuStem). The clinical score of each GRMD dog was assessed weekly and expressed as a percentage of a theoretical healthy dog score. Limits of the MuStem cell delivery window are indicated (dashed lines). (B) Right lateral view of a GRMD^MuStem dog, #7G^Mu. (C) Right lateral view of mock GRMD dog, #4G. Note the anterior weight transfer and plantigrady.</p

Flow chart of the statistical filtration of microarray data.

<p>Procedure leading to classification for each of the three pairwise comparisons of healthy, GRMD and GRMD^MuStem dog muscle samples.</p

Skeletal Muscle Regenerative Potential of Human MuStem Cells following Transplantation into Injured Mice Muscle.

After intra-arterial delivery in the dystrophic dog, allogeneic muscle-derived stem cells, termed MuStem cells, contribute to long-term stabilization of the clinical status and preservation of the muscle regenerative process. However, it remains unknown whether the human counterpart could be identified, considering recent demonstrations of divergent features between species for several somatic stem cells. Here, we report that MuStem cells reside in human skeletal muscle and display a long-term ability to proliferate, allowing generation of a clinically relevant amount of cells. Cultured human MuStem (hMuStem) cells do not express hematopoietic, endothelial, or myo-endothelial cell markers and reproducibly correspond to a population of early myogenic-committed progenitors with a perivascular/mesenchymal phenotypic signature, revealing a blood vessel wall origin. Importantly, they exhibit both myogenesis in vitro and skeletal muscle regeneration after intramuscular delivery into immunodeficient host mice. Together, our findings provide new insights supporting the notion that hMuStem cells could represent an interesting therapeutic candidate for dystrophic patients