A bioengineered novel human functional neuromuscular junction platform for drug testing and diabetes studies

Abd Al Samid, Marwah (2020) A bioengineered novel human functional neuromuscular junction platform for drug testing and diabetes studies. Doctoral thesis (PhD), Manchester Metropolitan University.

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Abstract

Background: The neuromuscular junction (NMJ) is a unique chemical synaptic connection between muscle fibre and motor neurons. NMJ is a complex structure that serves to efficiently communicate the electrical impulse from the motor neuron to the skeletal muscle to signal contraction making it difficult to isolate and dissect to enable the understanding of the underlying mechanisms and factors affecting neurodegeneration and muscle wasting associated with ageing and diseases (i.e. cancer and diabetes). Despite several decades of NMJs research, the prospect of in vivo NMJ studies is limited and these studies are challenging to implement. Thus, new sophisticated models are required to more efficiently trial novel drugs and compounds designed to enhance muscle growth and regeneration. Objective: The aim of this project is to establish a novel functional human NMJs platform, which is serum and neural complex media/neural growth factor-free, using human immortalised myoblasts and human embryonic stem cells (hESCs)-derived neural progenitor cells (NPCs) which could be used as disease model to study diseases associated with NMJ dysfunction. Methods: immobilised human myoblasts were co-cultured with hESCs for 7 days in serum and neural growth factors free differentiation media. In this co-culture model, functional NMJs form, myotubes exhibit advanced differentiation into muscle tissue and they undergo nerve-evoked contractions. The model fully characterised using different antibodies against specific markers for NMJ formation and for motor neurons and myoblast differentiation. The functionality of the NMJ was assessed using different pharmacological drugs. Finally, the model was evaluated as diabetic specific model using advanced glycation end products and cross-talk between muscle and motor neurons and endogenously secreted neural growth factors were investigated. Results: It was confirmed that the NPCs had matured into cholinergic motor neurons using choline acetyltransferase and βIII-tubulin immunostaining. Multiple NMJ innervation sites were formed from neuronal axon sprouting branched along the myotubes resulting in extensive, spontaneous contractile activity shown in the myotubes. Postsynaptic site of NMJs was further characterised by staining dihydropyridine receptors, ryanodine receptors, and acetylcholine receptors by α-bungarotoxin (α-BTX). The functional assessments using different agonists and antagonists pharmacological drugs (L-glutamic acid, α-BTX and Tubocurarine) showed that this system behaved physiologically and muscle contraction was motor neurons-driven. The model was successfully applied as diabetic platform in which the bi-directional communications between myotubes and motor neurons were impaired and consequently essential neural growth factor levels were detrimentally affected. Conclusion: A functional entirely human motor unit serum and neural growth factors free platform was successfully established and characterised for in vitro investigations and validated as a diabetic platform that replicate the diabetes in human.