The recent advances, offered by cell therapy in the regenerative medicine field, offer a revolutionary potential for the development of innovative cures to restore compromised physiological functions or organs. Adult myogenic precursors, such as myoblasts or satellite cells, possess a marked regenerative capacity, but the exploitation of this potential still encounters significant challenges in clinical application, due to low rate of proliferation in vitro, as well as a reduced self-renewal capacity. In this scenario, induced pluripotent stem cells (iPSCs) can offer not only an inexhaustible source of cells for regenerative therapeutic approaches, but also a valuable alternative for in vitro modeling of patient-specific diseases. In this study we established a reliable protocol to induce the myogenic differentiation of iPSCs, generated from pericytes and fibroblasts, exploiting skeletal muscle-derived extracellular vesicles (EVs), in combination with chemically defined factors. This genetic integration-free approach generates functional skeletal myotubes maintaining the engraftment ability in vivo. Our results demonstrate evidence that EVs can act as biological “shuttles” to deliver specific bioactive molecules for a successful transgene-free differentiation offering new opportunities for disease modeling and regenerative approaches.

Aims/hypothesis: Treatment of vascular complications of diabetes remains inadequate. We reported that muscle pericytes (MPs) from limb muscles of vascular patients with diabetes mellitus display elevated levels of oxidative stress causing a dysfunctional phenotype. Here, we investigated whether treatment with dimethyl-2-oxoglutarate (DM-2OG), a tricarboxylic acid cycle metabolite with antioxidant properties, can restore a healthy metabolic and functional phenotype. Methods: MPs were isolated from limb muscles of diabetes patients with vascular disease (D-MPs) and from non-diabetic control participants (ND-MPs). Metabolic status was assessed in untreated and DM-2OG-treated (1 mmol/l) cells using an extracellular flux analyser and anion-exchange chromatography–mass spectrometry (IC-MS/MS). Redox status was measured using commercial kits and IC-MS/MS, with antioxidant and metabolic enzyme expression assessed by quantitative RT-PCR and western blotting. Myogenic differentiation and proliferation and pericyte–endothelial interaction were assessed as functional readouts. Results: D-MPs showed mitochondrial dysfunction, suppressed glycolytic activity and reduced reactive oxygen species-buffering capacity, but no suppression of antioxidant systems when compared with ND-MP controls. DM-2OG supplementation improved redox balance and mitochondrial function, without affecting glycolysis or antioxidant systems. Nonetheless, this was not enough for treated D-MPs to regain the level of proliferation and myogenic differentiation of ND-MPs. Interestingly, DM-2OG exerted a positive effect on pericyte–endothelial cell interaction in the co-culture angiogenesis assay, independent of the diabetic status. Conclusions/interpretation: These novel findings support the concept of using DM-2OG supplementation to improve pericyte redox balance and mitochondrial function, while concurrently allowing for enhanced pericyte–endothelial crosstalk. Such effects may help to prevent or slow down vasculopathy in skeletal muscles of people with diabetes.

The absence of a native extracellular matrix and the use of xenogeneic sera are often associated with rapid tenocyte function losses during in vitro culture. Herein, we assessed the influence of different sera (equine serum and foetal bovine serum) on equine tenocyte morphology, viability, metabolic activity, proliferation and protein synthesis as a function of tissue-specific extracellular matrix deposition (induced via macromolecular crowding), aging (passages 3, 6, 9) and time in culture (days 3, 5, 7). In comparison to cells at passage 3, at day 3, in foetal bovine serum and without macromolecular crowding (traditional equine tenocyte culture), the highest number of significantly decreased readouts were observed for cells in foetal bovine serum, at passage 3, at day 5 and day 7 and without macromolecular crowding. Again, in comparison to traditional equine tenocyte culture, the highest number of significantly increased readouts were observed for cells in equine serum, at passage 3 and passage 6, at day 7 and with macromolecular crowding. Our data advocate the use of an allogeneic serum and tissue-specific extracellular matrix for effective expansion of equine tenocytes.

Atherosclerosis is the main cause of mortality globally, being at the basis of most cardiovascular diseases. It is a multifactorial disease, arising from complex interactions comprising changes in lipid metabolism, inflammation and oxidative stress. These factors contribute to endothelial damage and dysfunction, the accumulation of immune cells and smooth muscle cells in the intima, ultimately leading to the formation of atherosclerotic plaques, which restricts blood flow through the vessels. Much progress has been made in the last decades in debunking the underlying mechanisms of atherosclerosis development, especially concerning the evaluation and prediction of plaque stability and the understanding of the roles played by each of the involved cell types. As yet, mechanisms that drive plaque development toward specific 'vulnerable' phenotypes remain undiscovered. Based on recent advancements in RNA therapeutics, this review aims to illustrate a comprehensive overview of miRNAs relevant to various aspects of atherosclerosis and emphasizes their theranostic potential, highlighting their dual role as both drug targets and biomarkers.