Mesenchymal stem cells are used for different therapeutic approaches, e.g. for osteoarthritis, lesions of the tendon as well as for bone defects. Current research on the mechanism of stem cells on the repair of damaged tissue suggest an important role of a cell-to-cell communication through secreted extracellular vesicles, mainly represented by exosomes. To enhance the scarce knowledge on the functional role of exosomes we compared as a first step different techniques to isolate and identify exosomes from the supernatant of equine adipose derived mesenchymal stem cells for further characterization and usage in functional assays. It was possible to obtain exosomes secreted from equine adipose derived mesenchymal stem cells with three common techniques: a stepwise ultracentrifugation at 100.000 g, an ultrafiltration with 3 kDa exclusion membranes and a charge-based precipitation method. The mean sizes and amounts of exosomes isolated with the different techniques were measured by the nanoparticle tracking analysis. The diameter ranged between 116.2 nm (ultracentrifugation), 453.1 nm (precipitation) and 178.7 nm (ultrafiltration), the counts of particles / ml ranged between 9.6 × 108 (ultracentrifugation), 2.02 × 109 (precipitation) and 52.5 × 109 (ultrafiltration). Relevant marker for exosomes, tetraspanins CD9, CD63 and CD81 were detectable by immunofluorescence staining of the investigated exosomes secreting mesenchymal stem cells. In addition, transmission electron microscopy and immunogold labeling with CD9 and CD90 was performed to display the morphological shape of exosomes and existence of marker relevant for exosomes (CD9) and mesenchymal stem cells (CD90). Western blot analysis of CD9 and CD90 of exosomes ensured the specificity of the rare available respectively cross reacting antibodies against equine antigens. Exosomes generated by equine mesenchymal stem cells can be obtained by ultrafiltration and ultracentrifugation in an equal quality for in vitro experiments. Especially for later therapeutic usage we recommend ultrafiltration due to a higher concentration without aggregation of extracellular vesicles in comparison to exosomes obtained by ultracentrifugation.

Myostatin regulates both muscle mass and muscle metabolism. The myostatin null (MSTN^−/−) mouse has a hypermuscular phenotype owing to both hypertrophy and hyperplasia of the myofibres. The enlarged muscles display a reliance on glycolysis for energy production; however, enlarged muscles that develop in the absence of myostatin have compromised force‐generating capacity. Recent evidence has suggested that endurance exercise training increases the oxidative properties of muscle. Here, we aimed to identify key changes in the muscle phenotype of MSTN^−/− mice that can be induced by training. To this end, we subjected MSTN^−/− mice to two different forms of training, namely voluntary wheel running and swimming, and compared the response at the morphological, myocellular and molecular levels. We found that both regimes normalized changes of myostatin deficiency and restored muscle function. We showed that both exercise training regimes increased muscle capillary density and the expression of Ucp3, Cpt1α, Pdk4 and Errγ, key markers for oxidative metabolism. Cross‐sectional area of hypertrophic myofibres from MSTN^−/− mice decreased towards wild‐type values in response to exercise and, in this context, Bnip3, a key autophagy‐related gene, was upregulated. This reduction in myofibre size caused an increase of the nuclear‐to‐cytoplasmic ratio towards wild‐type values. Importantly, both training regimes increased muscle force in MSTN^−/− mice. We conclude that impaired skeletal muscle function in myostatin‐deficient mice can be improved through endurance exercise‐mediated remodelling of muscle fibre size and metabolic profile.

Insulin resistance (IR) in skeletal muscle is a prerequisite for type 2 diabetes and is often associated with obesity. IR also develops alongside muscle atrophy in older individuals in sarcopenic obesity. The molecular defects that underpin this syndrome are not well characterized, and there is no licensed treatment. Deletion of the transforming growth factor-β family member myostatin, or sequestration of the active peptide by overexpression of the myostatin propeptide/latency-associated peptide (ProMyo) results in both muscle hypertrophy and reduced obesity and IR. We aimed to establish whether local myostatin inhibition would have a paracrine/autocrine effect to enhance glucose disposal beyond that simply generated by increased muscle mass, and the mechanisms involved. We directly injected adeno-associated virus expressing ProMyo in right tibialis cranialis/extensor digitorum longus muscles of rats and saline in left muscles and compared the effects after 17 days. Both test muscles were increased in size (by 7 and 11%) and showed increased radiolabeled 2-deoxyglucose uptake (26 and 47%) and glycogen storage (28 and 41%) per unit mass during an intraperitoneal glucose tolerance test. This was likely mediated through increased membrane protein levels of GLUT1 (19% higher) and GLUT4 (63% higher). Interestingly, phosphorylation of phosphoinositol 3-kinase signaling intermediates and AMP-activated kinase was slightly decreased, possibly because of reduced expression of insulin-like growth factor-I in these muscles. Thus, myostatin inhibition has direct effects to enhance glucose disposal in muscle beyond that expected of hypertrophy alone, and this approach may offer potential for the therapy of IR syndromes.

Background: Adipose tissue-derived mesenchymal stem cells (ASCs) offer a promising cell source for therapeutic applications in musculoskeletal disorders. The appropriate selection of ASCs from various fat depots for cell-based therapy is challenging. The present study aims to compare stemness and multipotency of ASCs derived from retroperitoneal (RP), subcutaneous (SC), and lipoma (LP) fat to assess their usefulness for clinical application.Methods: Equine ASCs from the three fat tissue sources were isolated and characterized. The cell viability, proliferation, and self-renewal were evaluated using MTT, sulforhodamine B, and colony forming unit (CFU) assays. Stem cell relative marker CD44, CD90, and CD105 and tumor marker CA9 and osteopontin (OPN) expression were quantified using RT-qPCR. Multipotency of ASCs for adipogenic, osteogenic, and chondrogenic differentiation was examined by quantifying Oil Red O and Alizarin Red S staining, alkaline phosphatase activity (ALP), and expression of differentiation relative markers. All data were statistically analyzed using ANOVA.Results: RP fat-derived ASCs showed a higher cell proliferation rate compared to SC and LP derived cells. In contrast, ASCs from lipoma displayed a lower proliferation rate and impaired CFU capacities. The expression of CD44, CD90, and CD105 was upregulated in RP and SC derived cells but not in LP cells. RP fat-derived cells displayed a higher adipogenic potential compared to SC and LP cells. Although ASCs from all fat sources showed enhanced ALP activity following osteogenic differentiation, SC fat-derived cells revealed upregulated ALP and bone morphogenetic protein-2 expression together with a higher calcium deposition. We found an enhanced chondrogenic potency of RP and SC fat-derived cells as shown by Alcian blue staining and upregulation of aggrecan (Aggre), cartilage oligomeric matrix protein precursor (COMP), and collagen 2a1 (Col2a1) expression compared to LP. The expression of OPN and CA9 was exclusively upregulated in the ASCs of LP.Conclusions: The results provide evidence of variation in ASC performance not only between normal fat depots but also compared to LP cells which suggest a different molecular regulation controlling the cell fate. These data provided are useful when considering a source for cell replacement therapy in equine veterinary medicine.

Background: Skeletal muscle-derived stem cells (SC) have become a promising approach for investigating myogenic differentiation and optimizing tissue regeneration. Muscle regeneration is performed by SC, a self-renewal cell population underlying the basal lamina of muscle fibers. Here, we examined the impact of hypoxia condition on the regenerative capacity of SC either in their native microenvironment or via isolation in a monolayer culture using ectopic differentiation inductions. Furthermore, the effect of low oxygen tension on myogenic differentiation protocols of the myoblasts cell line C2C12 was examined. Methods: Hind limb muscles of wild type mice were processed for both SC/fiber isolation and myoblast extraction using magnetic beads. SC were induced for myogenic, adipogenic and osteogenic commitments under normoxic (21% O₂) and hypoxic (3% O₂) conditions. SC proliferation and differentiation were evaluated using histological staining, immunohistochemistry, morphometric analysis and RT-qPCR. The data were statistically analyzed using ANOVA. Results: The data revealed enhanced SC proliferation and motility following differentiation induction after 48 h under hypoxia. Following myogenic induction, the number of undifferentiated cells positive for Pax7 were increased at 72 h under hypoxia. Hypoxia upregulated MyoD and downregulated Myogenin expression at day-7 post-myogenic induction. Hypoxia promoted both SC adipogenesis and osteogenesis under respective induction as shown by using Oil Red O and Alizarin Red S staining. The expression of adipogenic markers; peroxisome proliferator activated receptor gamma (PPARγ) and fatty acid-binding protein 4 (FABP4) were upregulated under hypoxia up to day 14 compared to normoxic condition. Enhanced osteogenic differentiation was detected under hypoxic condition via upregulation of osteocalcin and osteopontin expression up to day 14 as well as, increased calcium deposition at day 21. Hypoxia exposure increases the number of adipocytes and the size of fat vacuoles per adipocyte compared to normoxic culture. Combining the differentiation medium with dexamethasone under hypoxia improves the efficiency of the myogenic differentiation protocol of C2C12 by increasing the length of the myotubes. Conclusions: Hypoxia exposure increases cell resources for clinical applications and promotes SC multipotency and thus beneficial for tissue regeneration.