MicroRNAs (miRNAs) are small, non-coding RNA molecules that are mainly involved in translational repression by binding to specific messenger RNAs. Recently, miRNAs have emerged as biomarkers, relevant for a multitude of pathophysiological conditions, and cells can selectively sort miRNAs into extracellular vesicles for paracrine and endocrine effects. In the overall context of muscle-wasting conditions, a multitude of miRNAs has been implied as being responsible for the typical dysregulation of anabolic and catabolic pathways. In general, chronic muscle disorders are associated with the main characteristic of a substantial loss in muscle mass. Muscular dystrophies (MDs) are a group of genetic diseases that cause muscle weakness and degeneration. Typically, MDs are caused by mutations in those genes responsible for upholding the integrity of muscle structure and function. Recently, the dysregulation of miRNA levels in such pathological conditions has been reported. This revelation is imperative for both MDs and other muscle-wasting conditions, such as sarcopenia and cancer cachexia. The expression levels of miRNAs have immense potential for use as potential diagnostic, prognostic and therapeutic biomarkers. Understanding the role of miRNAs in muscle-wasting conditions may lead to the development of novel strategies for the improvement of patient management.

Muscular dystrophies are debilitating neuromuscular disorders for which no cure exists. As this disorder affects both cardiac and skeletal muscle, patients would benefit from a cellular therapy that can simultaneously regenerate both tissues. The current protocol to derive bipotent mesodermal progenitors which can differentiate into cardiac and skeletal muscle relies on the spontaneous formation of embryoid bodies, thereby hampering further clinical translation. Additionally, as skeletal muscle is the largest organ in the human body, a high myogenic potential is necessary for successful regeneration. Here, we have optimized a protocol to generate chemically defined human induced pluripotent stem cell-derived mesodermal progenitors (cdMiPs). We demonstrate that these cells contribute to myotube formation and differentiate into cardiomyocytes, both in vitro and in vivo. Furthermore, the addition of valproic acid, a clinically approved small molecule, increases the potential of the cdMiPs to contribute to myotube formation that can be prevented by NOTCH signaling inhibitors. Moreover, valproic acid pre-treated cdMiPs injected in dystrophic muscles increase physical strength and ameliorate the functional performances of transplanted mice. Taken together, these results constitute a novel approach to generate mesodermal progenitors with enhanced myogenic potential using clinically approved reagents.

Significant loss of muscle mass may occur in cachexia and sarcopenia, which are major causes of mortality and disability. Cachexia represents a complex multi-organ syndrome associated with cancer and chronic diseases. It is often characterized by body weight loss, inflammation, and muscle and adipose wasting. Progressive muscle loss is also a hallmark of healthy aging, which is emerging worldwide as a main demographic trend. A great challenge for the health care systems is the age-related decline in functionality which threatens the independence and quality of life of elderly people. This biological decline can also be associated with functional muscle loss, known as sarcopenia. Previous studies have shown that microRNAs (miRNAs) play pivotal roles in the development and progression of muscle wasting in both cachexia and sarcopenia. These small non-coding RNAs, often carried in extracellular vesicles, inhibit translation by targeting messenger RNAs, therefore representing potent epigenetic modulators. The molecular mechanisms behind cachexia and sarcopenia, including the expression of specific miRNAs, share common and distinctive trends. The aim of the present review is to compile recent evidence about shared and divergent epigenetic mechanisms, particularly focusing on miRNAs, between cachexia and sarcopenia to understand a facet in the underlying muscle wasting associated with these morbidities and disclose potential therapeutic interventions.

Neuromuscular junctions (NMJs) are specialized synapses, crucial for the communication between spinal motor neurons (MNs) and skeletal muscle. NMJs become vulnerable in degenerative diseases, such as muscle atrophy, where the crosstalk between the different cell populations fails, and the regenerative ability of the entire tissue is hampered. How skeletal muscle sends retrograde signals to MNs through NMJs represents an intriguing field of research, and the role of oxidative stress and its sources remain poorly understood. Recent works demonstrate the myofiber regeneration potential of stem cells, including amniotic fluid stem cells (AFSC), and secreted extracellular vesicles (EVs) as cell-free therapy. To study NMJ perturbations during muscle atrophy, we generated an MN/myotube co-culture system through Xona^TM microfluidic devices, and muscle atrophy was induced in vitro by Dexamethasone (Dexa). After atrophy induction, we treated muscle and MN compartments with AFSC-derived EVs (AFSC-EVs) to investigate their regenerative and anti-oxidative potential in counteracting NMJ alterations. We found that the presence of EVs reduced morphological and functional in vitro defects induced by Dexa. Interestingly, oxidative stress, occurring in atrophic myotubes and thus involving neurites as well, was prevented by EV treatment. Here, we provided and validated a fluidically isolated system represented by microfluidic devices for studying human MN and myotube interactions in healthy and Dexa-induced atrophic conditions—allowing the isolation of subcellular compartments for region-specific analyses—and demonstrated the efficacy of AFSC-EVs in counteracting NMJ perturbations.

Muscular dystrophies are debilitating neuromuscular disorders for which no cure exists. As this disorder affects both cardiac and skeletal muscle, patients would benefit from a cellular therapy that can simultaneously regenerate both tissues. The current protocol to derive bipotent mesodermal progenitors which can differentiate into cardiac and skeletal muscle relies on the spontaneous formation of embryoid bodies, thereby hampering further clinical translation. Additionally, as skeletal muscle is the largest organ in the human body, a high myogenic potential is necessary for successful regeneration. Here, we have optimized a protocol to generate chemically defined induced pluripotent stem cell-derived mesodermal progenitors (cdMiPs). We demonstrate that these cells contribute to myotube formation and differentiate into cardiomyocytes, both in vitro and in vivo. Furthermore, the addition of valproic acid, a clinically approved small molecule, increases the potential of the cdMiPs to contribute to myotube formation without compromising their ability to differentiate towards cardiomyocytes. This effect is mediated through the activation of the Notch signaling pathway. Taken together, these results constitute a novel approach to generate mesodermal progenitors with enhanced myogenic potential using clinically approved reagents, which opens the door to new therapeutic solutions in the treatment of muscular dystrophy.

Objective. Human induced pluripotent stem cells (hiPSCs) represent a clinically relevant cell source for skeletal muscle tissue engineering, yet transgene-free maturation remains challenging. Moreover, the potential of spheroids as building blocks for muscle engineering remains poorly defined. This study evaluated whether hiPSC-derived myogenic spheroids enhance biofabrication outcomes in an in vitro bio-artificial muscle (BAM) system compared to a conventional single cell approach. Methods. CD56+ hiPSC-derived myoblasts were generated using a transgene-free protocol and differentiated toward 2D myotubes. In 3D, myoblasts were aggregated into 140-200 µm spheroids. Spheroids or single cells were encapsulated in a fibrin-based BAM, kept under tension using metal pins. Non-encapsulated spheroids and BAMs were cultured up to 15 days and evaluated for myosin heavy chain (MYH)+ organization, transcriptional profile, and contractile function. Results. In 2D, MYH+ myotubes showing multinucleation and, occasionally, sarcomere striation were formed. Spheroids were successfully generated, increased in size and circularity, and showed an enhancing MYH positivity, particularly during the proliferation phase, and displayed a fetal/perinatal myogenic state. In BAMs, spheroids promoted MYH positivity and established larger continuous MYH+ structures. Transcriptional profiles of spheroid and single cell BAMs were broadly similar. Tetanic forces were generally higher in spheroid BAMs; however, this difference did not reach statistical significance. Conclusion. hiPSC-derived myogenic spheroids advance MYH expression, but terminal maturation is constrained, likely due to their intrinsically isotropic nature. Within BAMs, spheroids enhance MYH+ myotube formation, but do not overcome limitations in myoblast fusion. Incomplete spheroid outgrowth and poor control over their spatial distribution are important challenges for their integration. Future work should focus on improving hiPSC differentiation efficiency, implementing spheroids in earlier differentiation stages, and optimizing spheroid outgrowth and positioning, alongside incorporating remaining muscle components as fibroblasts and vascular cells. This will refine whether spheroid-based assembly can add value in specific biofabrication contexts.