Therapeutic potential of stem cell-exosomes in counteracting NMJ degeneration during muscle atrophy: a novel in vitro microfluidic system

Osteosarcopenia is a widespread geriatric condition resulting from the coexistence of osteoporosis and sarcopenia, where the bone-muscle crosstalk is, in part, involved. Given the close, reciprocal influence between muscle and nerve it is not surprising that aging is associated with parallel biochemical and morphological changes at the neuromuscular junction (NMJ). Human amniotic fluid stem cells (hAFSC), have been introduced as an attractive and potent stem cell source for clinical applications due to their collection procedures, which minimize ethical issues. Extracellular vesicles (EVs) derived from hAFSC exhibit antioxidant properties that may underlie their anti-aging and cytoprotective effects, making them a promising potential treatment for age-related diseases. To better clarify this aspect, protein and miRNA expression profiles of EVs secreted by hAFSCs were evaluated. To study cell interactions under both healthy and pathological conditions occurring in musculo-skeletal apparatus, first we developed a three-culture system exploiting the use of well-known transwell supports. This system enables physical and biochemical interactions among myotubes and neurons — optionally treated with extracellular vesicles (EVs) — and osteoblasts induced toward an osteoporotic phenotype. Collectively, this method allowed us to understand how the modifications induced in osteoblasts during bone disorders trigger a cascade of detrimental effects in the muscle and neuron parts. Moreover, we demonstrated the efficacy of hAFSC-EVs in preventing NMJ dysfunction, muscle atrophy, and osteoblast impairment. Therefore, we fabricated a novel PDMS device using soft lithography to create a multicompartment neuro-muscular-bone (NMB) culturing method that, unlike the transwell system, allowed us to gain live-cell microscopy, functionality experiments, fluidically isolated channels, and time-dependent communication between compartments. In conclusion, in this work we validated two different systems to study NMB alterations associated to osteo-muscular diseases and we demonstrated that hAFSC-EVs represent a potential therapeutic cell-free strategy for the treatment of these age-related degenerative pathologies.