3 research outputs found

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

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    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

    A functional human motor unit platform engineered from human embryonic stem cells and immortalized skeletal myoblasts.

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    Although considerable research on neuromuscular junctions (NMJs) has been conducted, the prospect of in vivo NMJ studies is limited and these studies are challenging to implement. Therefore, there is a clear unmet need to develop a feasible, robust, and physiologically relevant in vitro NMJ model. We aimed to establish a novel functional human NMJs platform, which is serum and neural complex media/neural growth factor-free, using human immortalized myoblasts and human embryonic stem cells (hESCs)-derived neural progenitor cells (NPCs) that can be used to understand the mechanisms of NMJ development and degeneration. Immortalized human myoblasts were co-cultured with hESCs derived committed NPCs. Over the course of the 7 days myoblasts differentiated into myotubes and NPCs differentiated into motor neurons. Neuronal axon sprouting branched to form multiple NMJ innervation sites along the myotubes and the myotubes showed extensive, spontaneous contractile activity. Choline acetyltransferase and βIII-tubulin immunostaining confirmed that the NPCs had matured into cholinergic motor neurons. Postsynaptic site of NMJs was further characterized by staining dihydropyridine receptors, ryanodine receptors, and acetylcholine receptors by α-bungarotoxin. We established a functional human motor unit platform for in vitro investigations. Thus, this co-culture system can be used as a novel platform for 1) drug discovery in the treatment of neuromuscular disorders, 2) deciphering vital features of NMJ formation, regulation, maintenance, and repair, and 3) exploring neuromuscular diseases, age-associated degeneration of the NMJ, muscle aging, and diabetic neuropathy and myopathy

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

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    International audienceBackground: Neuromuscular junctions (NMJs) consist of the presynaptic cholinergic moto-neuron 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
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