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

Abstract

BackgroundDuchenne 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.MethodsA comparative study of miRNAs expression levels and cellular localization was performed on 9-month-old 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.ResultsWe 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.ConclusionWe 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12891-016-1060-5) contains supplementary material, which is available to authorized users.

Human serum and platelet lysate are appropriate xeno-free alternatives for clinical-grade production of human MuStem cell batches

“…Over the past 6 years, several studies have demonstrated the clinical efficacy of MuStem cell treatment to repair muscle tissue, highlighting the potential of this muscle-derived stem cell population in cell-based therapies [ 53 – 56 ]. hMuStem cells have been previously isolated, expanded, and characterized using FBS-supplemented growth culture media [ 57 ].…”

“…Based on their initial delayed adhesion properties, we isolated and characterized a population of adult stem cells (named MuStem cells) from healthy dog skeletal muscle, and demonstrated their efficacy following vascular delivery into the clinically relevant dog model of Duchenne muscular dystrophy (DMD), in which we observed persistent clinical stabilization and significant muscle repair [ 53 – 56 ]. More recently, we isolated and extensively characterized the human counterparts of these cells (hMuStem cells), and demonstrated their regenerative ability when delivered into injured muscle [ 57 ].…”

Human MuStem Cell Grafting into Infarcted Rat Heart Attenuates Adverse Tissue Remodeling and Preserves Cardiac Function

“…Through signaling mediated by several biological pathways, MuStem cell implantation limits the progression of muscle damage and stabilizes the clinical status of recipient dogs. 29 , 30 , 31 More recently, we isolated the human counterpart of MuStem cells (hMuStem cells), for which we have demonstrated comparably robust muscular-regeneration capacity after delivery into injured skeletal muscle in immunodeficient mice. 32 These compelling findings point to hMuStem cells as attractive candidates for the treatment of skeletal muscle diseases.…”

>b<Avaliação de Pericitos no modelo GRMD (>i<Golden Retriever Muscular Dystrophy>/i<)>/b<

“…No homem, os sintomas clínicos são demonstrados de forma dinâmica, afetando o músculo estriado esquelético, músculo liso e músculo cardíaco (BROLIO et al, 2014;MARKHAM et al, 2017). Por se tratar de uma doença de caráter congênito, as manifestações clínicas da doença aparecem desde o início da infância e surgem com maior evidência entre três e cinco anos de idade (ARAUJO et al, 2017 (ROBRIQUET et al, 2016;SCHNEIDER et al, 2018).…”

“…observada melhora clínica nos cães afetados(ROBRIQUET et al, 2016; COENEN-STASS; WOOD; ROBERTS, 2017).Outro estudo com terapia gênica demonstrou que cães GRMD com quatro dias de idade que receberam a injeção intravenosa de um vetor associado ao adenovírus (AAV9) carreando um códon humano com o mini-gene da distrofina sob influência do citomegalovírus (CMV), exibiram expressão muscular generalizada do transgene posteriormente a 16 semanas de tratamento, entretanto sem implicações clínicas satisfatórias(FRAYSSE et al, 2017; KORNEGAY, 2017;LE GUINER et al, 2017; VOIT et al, 2017).…”