<p>Simplified in vitro engineering of neuromuscular junctions between rat embryonic motoneurons and immortalized human skeletal muscle cells</p>

Abstract

Background Neuromuscular junctions (NMJs) consist of the presynaptic cholinergic motoneuron terminals and the corresponding postsynaptic motor endplates on skeletal muscle fibers. At the NMJ the action potential of the neuron leads, via release of acetylcholine, to muscle membrane depolarization that in turn is translated into muscle contraction and physical movement. Despite the fact that substantial NMJ research has been performed, the potential of in vivo NMJ investigations is inadequate and difficult to employ. A simple and reproducible in vitro NMJ model may provide a robust means to study the impact of neurotrophic factors, growth factors, and hormones on NMJ formation, structure, and function. Methods This report characterizes a novel in vitro NMJ model utilizing immortalized human skeletal muscle stem cells seeded on 35 mm glass-bottom dishes, cocultured and innervated with spinal cord explants from rat embryos at ED 13.5. The cocultures were fixed and stained on day 14 for analysis and assessment of NMJ formation and development. Results This unique serum- and trophic factor-free system permits the growth of cholinergic motoneurons, the formation of mature NMJs, and the development of highly differentiated contractile myotubes, which exhibit appropriate configuration of transversal triads, representative of in vivo conditions. Conclusion This coculture system provides a tool to study vital features of NMJ formation, regulation, maintenance, and repair, as well as a model platform to explore neuromuscular diseases and disorders affecting NMJs.

A Novel Bioengineered Functional Motor Unit Platform to Study Neuromuscular Interaction

“…The first spontaneously contracting myotubes were observed ~72 h after co-culture, while no contractions were observed in aneurally cultured myotubes which confirm our previous observation [ 27 ], as myotubes matured in the co-culture, the number of contracting myotubes and their contraction frequency increased. After seven days in co-culture, the myotubes were contracting continuously in a systematic pattern as a unified network (see Video S1 ), behaving as an individual motor unit prompted by motor neuron stimulation.…”

“…In order to provide new avenues that facilitate the study of NMJ in development, disease progression to therapeutic intervention, appropriate NMJ experimental models are therefore needed. In the present study, we extended our recent work [ 27 ] to fully characterise the NMJ in a rat spinal cord segment and primary human muscle cells co-culture and assess its contractile functionality. The advantages of this platform are (1) the presence of supporting Schwann and glial cells may possibly enhance NMJ robustness and improve function of motor neurons in vitro and (2) the formation of functional NMJs that are associated with highly differentiated and motor neuron-driven contracting myotubes.…”

“…Furthermore, it has also been demonstrated that denervated myotubes within a nerve-muscle co-culture system fail to exhibit contractile function or characteristics of mature development; they eventually deteriorate, despite the elevated concentration of nerve-derived secretions in the culture environment [ 50 ]. Interestingly, the myotubes in our co-culture model initiated spontaneous contractions as early as 72 h post co-culture [ 27 ], suggesting the initial emergence of NMJ formation, due to the myotube requirement for nervous input from the motor neurons to induce contraction [ 51 ]. When compared to aneural in vitro SkMC cultures, the benefits of our co-culture system are apparent, as co-cultured myotubes exhibit advanced stages of differentiation that monocultures of SkMCs fail to achieve.…”

“…All animal work undertaken was approved by the Home Office and carried out at the University of Manchester, in accordance with the Animal Scientific Procedures Act 1986 [ 27 ]. Time-mated Sprague Dawley rats obtained from Charles River Laboratories (Oxford, UK) were sacrificed with CO 2 when embryos were roughly embryonic development day (ED) 13.5.…”

“…This further complicates drug discovery and toxicology studies due to possible cross-communication of the novel compound with factors contained within the added media, possibly explaining why many promising therapies do not translate to clinics [ 26 ]. Recently, we have developed a co-culture of rat embryonic spinal cord explants with primary human myoblasts resulted in NMJ formation and enhanced differentiation of myotubes that was stable for at least 2 weeks without the need of serum or neural growth factors [ 27 ].…”