Stem Cells and Cloning: Advances and Applications (Feb 2019)
Simplified in vitro engineering of neuromuscular junctions between rat embryonic motoneurons and immortalized human skeletal muscle cells
Abstract
Jasdeep Saini,1 Alessandro Faroni,2,3 Marwah Abd Al Samid,1 Adam J Reid,2,3 Adam P Lightfoot,1 Kamel Mamchaoui,4 Vincent Mouly,4 Gillian Butler-Browne,4 Jamie S McPhee,5 Hans Degens,1,6,7 Nasser Al-Shanti1 1Musculoskeletal Science & Sports Medicine Research Centre, School of Healthcare Science, Manchester Metropolitan University, Manchester, UK; 2Blond McIndoe Laboratories, Division of Cell Matrix Biology and Regenerative Medicine, School of Biological Sciences, Faculty of Biology Medicine and Health, University of Manchester, Manchester Academic Health Science Centre, Manchester, UK; 3Department of Plastic Surgery & Burns, University Hospitals of South Manchester, Manchester Academic Health Science Centre, Manchester, UK; 4Center for Research in Myology, Sorbonne Université–INSERM, Paris, France; 5Department of Sport and Exercise Science, Manchester Metropolitan University, Manchester, UK; 6Institute of Sport Science and Innovations, Lithuanian Sports University, Kaunas, Lithuania; 7University of Medicine and Pharmacy of Targu Mures, Targu Mures, Romania 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. Keywords: neuromuscular junction, NMJ, coculture, myoblast, myotube, motor neuron, motoneuron
