Crosstalk among neurons, muscle and bone on a chip: impact of stem cell derived extracellular vesicles on osteosarcopenia

Aging is associated with gradual degeneration, in mass and function, of the neuro-musculo-skeletal system. Neuromuscular junctions (NMJs) are specialized synapses, which are crucial for the communication between spinal motor neurons (MNs) and skeletal muscle. NMJs become vulnerable in degenerative diseases, such as muscle atrophy, where the impairment of NMJs results in muscle weakness. During aging this muscle-wasting condition occurs and is called sarcopenia. Indeed, osteoporosis and sarcopenia (osteosarcopenia (OS)) are twin-aging diseases. In this dramatic context, the crosstalk between the different cell populations fails, and the regeneration ability of the entire system is hampered. The molecular mechanisms that cause both muscle and bone loss are still unclear, and how skeletal muscle and NMJ send retrograde signals to MNs represents an intriguing field of research. However, the clinical data suggest that therapeutic approaches targeting either sarcopenia or osteoporosis alone may not be sufficient to effectively prevent fracture and disability. Novel approaches for treatments are advised to target the whole system simultaneously in the future. In order to study the perturbation in NMJs occurring in muscle atrophy, beside bone cells, an ideal model would contain MNs, myotubes and osteoblasts to recapitulate the human disease etiology or pathology. Therefore, the first aim of this project is to develop a personalized homemade microfluidic device to generate and study compartmentalized human NMJs in an in vitro muscle model connected to the bone side. A selective treatment in bone compartment with dexamethasone (DEX), a synthetic glucocorticoid (GC), will be used to induce osteoporosis: in this way, we could investigate if this pathological condition could be the driver to have a OS linked to NMJ perturbations. Recent works demonstrate paracrine actions of stem cells, including human amniotic fluid stem cells (hAFSC) that stimulate adult myogenesis and osteogenesis. In particular, extracellular vesicles (EVs), represent promising candidates for hAFSC-mediated tissue regeneration. Studies on the regulation of the whole neuro-musculo-skeletal system by EVs are still lacking, therefore, as a second aim of the project, we will treat the pathological condition with hAFSC-derived EVs (hAFSCEVs). For this project, each unit will be mainly devoted to develop specific aspects of NMJ/muscle/bone interaction with the common goal to set-up a new optimized in vitro model to investigate the complex interplay between tissues occurring in OS or other neuro-musculo-skeletal disorders. In conclusion, we will provide a validated fluidically isolated system for studying cell interactions in healthy and pathological conditions, allowing the isolation of cellular compartments for region-specific analyses, which will also be applicable to identify prospective targets for future interventions, such as EVs produced by stem cells.