Extracellular vesicle-derived miRNAs improve stem cell-based therapeutic approaches in muscle wasting conditions.

Skeletal muscle holds an intrinsic capability of growth and regeneration both in physiological conditions and in case of injury. Chronic muscle illnesses, generally caused by genetic and acquired factors, lead to deconditioning of the skeletal muscle structure and function, and are associated with a significant loss in muscle mass. At the same time, progressive muscle wasting is a hallmark of aging. Given the paracrine properties of myogenic stem cells, extracellular vesicle-derived signals have been studied for their potential implication in both the pathogenesis of degenerative neuromuscular diseases and as a possible therapeutic target. In this study, we screened the content of extracellular vesicles from animal models of muscle hypertrophy and muscle wasting associated with chronic disease and aging. Analysis of the transcriptome, protein cargo, and microRNAs (miRNAs) allowed us to identify a hypertrophic miRNA signature amenable for targeting muscle wasting, consisting of miR-1 and miR-208a. We tested this signature among others in vitro on mesoangioblasts (MABs), vessel-associated adult stem cells, and we observed an increase in the efficiency of myogenic differentiation. Furthermore, injections of miRNA-treated MABs in aged mice resulted in an improvement in skeletal muscle features, such as muscle weight, strength, cross-sectional area, and fibrosis compared to controls. Overall, we provide evidence that the extracellular vesicle-derived miRNA signature we identified enhances the myogenic potential of myogenic stem cells.

Interstitial Cell Remodeling Promotes Aberrant Adipogenesis in Dystrophic Muscles.

Fibrosis and fat replacement in skeletal muscle are major complications that lead to a loss of mobility in chronic muscle disorders, such as muscular dystrophy. However, the in vivo properties of adipogenic stem and precursor cells remain unclear, mainly due to the high cell heterogeneity in skeletal muscles. Here, we use single-cell RNA sequencing to decomplexify interstitial cell populations in healthy and dystrophic skeletal muscles. We identify an interstitial CD142-positive cell population in mice and humans that is responsible for the inhibition of adipogenesis through GDF10 secretion. Furthermore, we show that the interstitial cell composition is completely altered in muscular dystrophy, with a near absence of CD142-positive cells. The identification of these adipo-regulatory cells in the skeletal muscle aids our understanding of the aberrant fat deposition in muscular dystrophy, paving the way for treatments that could counteract degeneration in patients with muscular dystrophy.

Growth Factor Screening in Dystrophic Muscles Reveals PDGFB/PDGFRB-Mediated Migration of Interstitial Stem Cells.

Progressive muscle degeneration followed by dilated cardiomyopathy is a hallmark of muscular dystrophy. Stem cell therapy is suggested to replace diseased myofibers by healthy myofibers, although so far, we are faced by low efficiencies of migration and engraftment of stem cells. Chemokines are signalling proteins guiding cell migration and have been shown to tightly regulate muscle tissue repair. We sought to determine which chemokines are expressed in dystrophic muscles undergoing tissue remodelling. Therefore, we analysed the expression of chemokines and chemokine receptors in skeletal and cardiac muscles from Sarcoglycan-α null, Sarcoglycan-ß null and immunodeficient Sgcß-null mice. We found that several chemokines are dysregulated in dystrophic muscles. We further show that one of these, platelet-derived growth factor-B, promotes interstitial stem cell migration. This finding provides perspective to an approachable mechanism for improving stem cell homing towards dystrophic muscles.

Aging affects the in vivo regenerative potential of human mesoangioblasts.

Sarcopenia is the age-related loss of muscle mass, strength, and function. Although the role of human satellite cells (SCs) as adult skeletal muscle stem cells has been deeply investigated, little is known about the impact of aging on muscle interstitial stem cells. Here, we isolated the non-SC CD56- fraction from human muscle biopsies of young and elderly subjects. The elderly interstitial cell population contained a higher number of CD15+ and PDGFRα+ cells when compared to young samples. In addition, we found that the CD56- /ALP+ cells were well represented as a multipotent stem cell population inside the CD56- fraction. CD56- /ALP+ /CD15- cells were clonogenic, and since they were myogenic and expressed NG2, α-SMA and PDGFRß can be considered mesoangioblasts (MABs). Interestingly, elderly MABs displayed a dramatic impairment in the myogenic differentiation ability in vitro and when transplanted in dystrophic immunodeficient Sgcb-null Rag2-null γc-null mice. In addition, elderly MABs proliferated less, but yet retained other multilineage capabilities. Overall, our results indicate that aging negatively impacted on the regenerative potential of MABs and this should be carefully considered for potential therapeutic applications of MABs.

Folic Acid Exposure Rescues Spina Bifida Aperta Phenotypes in Human Induced Pluripotent Stem Cell Model.

Neural tube defects (NTDs) are severe congenital abnormalities, caused by failed closure of neural tube during early embryonic development. Periconceptional folic acid (FA) supplementation greatly reduces the risk of NTDs. However, the molecular mechanisms behind NTDs and the preventive role of FA remain unclear. Here, we use human induced pluripotent stem cells (iPSCs) derived from fetuses with spina bifida aperta (SBA) to study the pathophysiology of NTDs and explore the effects of FA exposure. We report that FA exposure in SBA model is necessary for the proper formation and maturation of neural tube structures and robust differentiation of mesodermal derivatives. Additionally, we show that the folate antagonist methotrexate dramatically affects the formation of neural tube structures and FA partially reverts this aberrant phenotype. In conclusion, we present a novel model for human NTDs and provide evidence that it is a powerful tool to investigate the molecular mechanisms underlying NTDs, test drugs for therapeutic approaches.

Morphological and functional analyses of skeletal muscles from an immunodeficient animal model of limb-girdle muscular dystrophy type 2E.

INTRODUCTION: Limb-girdle muscular dystrophy type 2E (LGMD2E) is caused by mutations in the ß-sarcoglycan gene, which is expressed in skeletal, cardiac, and smooth muscles. ß-Sarcoglycan-deficient (Sgcb-null) mice develop severe muscular dystrophy and cardiomyopathy with focal areas of necrosis. METHODS: In this study we performed morphological (histological and cellular characterization) and functional (isometric tetanic force and fatigue) analyses in dystrophic mice. Comparison studies were carried out in 1-month-old (clinical onset of the disease) and 7-month-old control mice (C57Bl/6J, Rag2/γc-null) and immunocompetent and immunodeficient dystrophic mice (Sgcb-null and Sgcb/Rag2/γc-null, respectively). RESULTS: We found that the lack of an immunological system resulted in an increase of calcification in striated muscles without impairing extensor digitorum longus muscle performance. Sgcb/Rag2/γc-null muscles showed a significant reduction of alkaline phosphate-positive mesoangioblasts. DISCUSSION: The immunological system counteracts skeletal muscle degeneration in the murine model of LGMD2E. Muscle Nerve, 2018.

MicroRNAs promote skeletal muscle differentiation of mesodermal iPSC-derived progenitors.

Muscular dystrophies (MDs) are often characterized by impairment of both skeletal and cardiac muscle. Regenerative strategies for both compartments therefore constitute a therapeutic avenue. Mesodermal iPSC-derived progenitors (MiPs) can regenerate both striated muscle types simultaneously in mice. Importantly, MiP myogenic propensity is influenced by somatic lineage retention. However, it is still unknown whether human MiPs have in vivo potential. Furthermore, methods to enhance the intrinsic myogenic properties of MiPs are likely needed, given the scope and need to correct large amounts of muscle in the MDs. Here, we document that human MiPs can successfully engraft into the skeletal muscle and hearts of dystrophic mice. Utilizing non-invasive live imaging and selectively induced apoptosis, we report evidence of striated muscle regeneration in vivo in mice by human MiPs. Finally, combining RNA-seq and miRNA-seq data, we define miRNA cocktails that promote the myogenic potential of human MiPs.

Exploring Wound-Healing Genomic Machinery with a Network-Based Approach.

The molecular mechanisms underlying tissue regeneration and wound healing are still poorly understood despite their importance. In this paper we develop a bioinformatics approach, combining biology and network theory to drive experiments for better understanding the genetic underpinnings of wound healing mechanisms and for selecting potential drug targets. We start by selecting literature-relevant genes in murine wound healing, and inferring from them a Protein-Protein Interaction (PPI) network. Then, we analyze the network to rank wound healing-related genes according to their topological properties. Lastly, we perform a procedure for in-silico simulation of a treatment action in a biological pathway. The findings obtained by applying the developed pipeline, including gene expression analysis, confirms how a network-based bioinformatics method is able to prioritize candidate genes for in vitro analysis, thus speeding up the understanding of molecular mechanisms and supporting the discovery of potential drug targets.

Mesodermal iPSC-derived progenitor cells functionally regenerate cardiac and skeletal muscle.

Conditions such as muscular dystrophies (MDs) that affect both cardiac and skeletal muscles would benefit from therapeutic strategies that enable regeneration of both of these striated muscle types. Protocols have been developed to promote induced pluripotent stem cells (iPSCs) to differentiate toward cardiac or skeletal muscle; however, there are currently no strategies to simultaneously target both muscle types. Tissues exhibit specific epigenetic alterations; therefore, source-related lineage biases have the potential to improve iPSC-driven multilineage differentiation. Here, we determined that differential myogenic propensity influences the commitment of isogenic iPSCs and a specifically isolated pool of mesodermal iPSC-derived progenitors (MiPs) toward the striated muscle lineages. Differential myogenic propensity did not influence pluripotency, but did selectively enhance chimerism of MiP-derived tissue in both fetal and adult skeletal muscle. When injected into dystrophic mice, MiPs engrafted and repaired both skeletal and cardiac muscle, reducing functional defects. Similarly, engraftment into dystrophic mice of canine MiPs from dystrophic dogs that had undergone TALEN-mediated correction of the MD-associated mutation also resulted in functional striatal muscle regeneration. Moreover, human MiPs exhibited the same capacity for the dual differentiation observed in murine and canine MiPs. The findings of this study suggest that MiPs should be further explored for combined therapy of cardiac and skeletal muscles.

Myogenic induction of adult and pluripotent stem cells using recombinant proteins.

Met Activating Genetically Improved Chimeric Factor 1 (Magic-F1) is a human recombinant protein, derived from dimerization of the receptor-binding domain of hepatocyte growth factor. Previous experiments demonstrate that in transgenic mice, the skeletal muscle specific expression of Magic-F1 can induce a constitutive muscular hypertrophy, improving running performance and accelerating muscle regeneration after injury. In order to evaluate the therapeutic potential of Magic-F1, we tested its effect on multipotent and pluripotent stem cells. In murine mesoangioblasts (adult vessel-associated stem cells), the presence of Magic-F1 did not alter their osteogenic, adipogenic or smooth muscle differentiation ability. However, when analyzing their myogenic potential, mesoangioblasts expressing Magic-F1 differentiated spontaneously into myotubes. Finally, Magic-F1 inducible cassette was inserted into a murine embryonic stem cell line by homologous recombination. When embryonic stem cells were subjected to myogenic differentiation, the presence of Magic-F1 resulted in the upregulation of Pax3 and Pax7 that enhanced the myogenic commitment of transgenic pluripotent stem cells. Taken together our results candidate Magic-F1 as a potent myogenic stimulator, able to enhance muscular differentiation from both adult and pluripotent stem cells.