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