Simplified in vitro engineering of functional mammalian neuromuscular junctions between embryonic rat motor neurons and immortalised human skeletal muscle cells

Saini, Jasdeep (2019) Simplified in vitro engineering of functional mammalian neuromuscular junctions between embryonic rat motor neurons and immortalised human skeletal muscle cells. Doctoral thesis (PhD), Manchester Metropolitan University.

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Abstract

Neuromuscular junction (NMJ) research is vital to advance the understanding of neuromuscular (NM) pathologies and development of novel therapies for diseases associated with NM dysfunction and deterioration. Several in vivo animal models manifest phenotypes observed in NM diseases. Unfortunately, in vivo NMJ research with animal models present many challenges due to inaccurate reproduction of human disease. For example, the most widely used animal model for Duchenne muscular dystrophy, the mdx mouse, is a good genetic and biochemical model, presenting total deficiency of the protein dystrophin in the muscle. However, this in vivo model is not useful for clinical trials due to the very mild phenotype expressed. Therefore, in vitro models were established, yet limitations exist. For example, inclusion of serum influences translation of animal data into human trials, inclusion of complex neurotrophic/growth factors can interfere with drug discovery, the initiation of skeletal muscle (SkM) contractions requiring electric pulse or chemical stimuli, and time consuming culture methods to induce spontaneous SkM contractions. Therefore, the aim of this thesis was to establish and characterise a simplified co-culture system that allows in vitro research of functional NMJs, representative of in vivo conditions. Immortalised human SkM stem cells were co-cultured with motor neurons (MNs) from rat embryo spinal cord explants, using for the first time a culture media formulation free from serum and neurotrophic or growth factors. This co-culture resulted in NMJ formation and contractile SkM cells. The de novo formation of NMJs was validated via characterisation of pre- and post-synaptic structures of the junctional apparatus. Interactions between the specialised membranes of presynaptic MN terminals with postsynaptic motor end plates (MEPs) located on SkM cells, along with supporting neuroglia, permitted chemical transmission of acetylcholine from MNs across structural bridges to bind with receptors on the MEPs. These interactions were associated with contractile activity and advanced differentiation of innervated SkM fibres. Functionality of NMJs was verified through the application of known agonists and antagonists to the co-culture system and confirmed that the contractile activity observed in the innervated SkM fibres were driven via NMJs. An ELISA-based microarray identified the presence of trophic factors required for MN, SkM, and NMJ development. Ultimately, engineering of this novel in vitro NMJ system represents an accessible platform to investigate NMJ formation and function, as well as providing a breakthrough assay via the system’s ability to respond to drug interventions through measurable output, initiate spontaneous SkM cell contractions, and induce advanced differentiation of SkM Cells. Therefore, this novel system provides a tool to screen pharmacological or genetic therapies for diseased linked with SkM, MNs, and NMJs.