Modelling functional human neuromuscular junctions in a differentially-perturbable microfluidic environment, validated through recombinant monosynaptic pseudotyped ΔG-rabies virus tracing

Abstract

Sweden Graphical abstractFunctional neuromuscular junction in a microfluidic chip (a) Overview of microfluidic chip. Human iPS cell-derived motor neuron aggregates (spheroids indicated by black arrows) are seeded in the three lateral compartments of the chip, while human myotubes (white arrows) are seeded in the middle compartment. (b) Directed connectivity and retrograde virus tracing.Outgrowing axons (yellow arrow) from the motor neuron aggregate enter the directional axon tunnels (grey rectangles) and form connections with the myotubes (white arrow) within the opposite compartment. Addition of a designer monosynaptic pseudotyped ΔG-rabies virus to the myotube compartment, infects the myotubes (green) expressing an exogenous receptor (TVA) and rabies glycoprotein (G), subsequently making infectious viruses that are retrogradely transported through the motor neuron axons (green arrow) back to the neuronal cell bodies within the aggregate, validating neuromuscular junction functionality. AbstractCompartmentalized microfluidic culture systems provide new perspectives in in vitro disease modelling as they enable co-culture of different relevant cell types in interconnected but fluidically isolated microenvironments. Such systems are thus particularly interesting in the context of in vitro modelling of mechanistic aspects of neurodegenerative diseases such as amyotrophic lateral sclerosis, which progressively affect the function of neuromuscular junctions, as they enable the co-culture of motor neurons and muscle cells in separate, but interconnected compartments. In combination with cell reprogramming technologies for the generation of human (including patient-specific) motor neurons, microfluidic platforms can thus become important research tools in preclinical studies. In this study, we present the application of a microfluidic chip with a differentially-perturbable microenvironment as a platform for establishing functional neuromuscular junctions using human induced pluripotent stem cell derived motor neurons and human myotubes. As a novel approach, we demonstrate the functionality of the platform using a designer pseudotyped ΔG-rabies virus for retrograde monosynaptic tracing.

Advances in In Vitro Models of Neuromuscular Junction: Focusing on Organ‐on‐a‐Chip, Organoids, and Biohybrid Robotics

“…The results showed that iPS cell‐derived MN formed a functional coordinated network before in vitro NMJ model analysis. [ 82 ] Duc et al. combined microfluidics, soft lithography, and MEA to create an NMJ on specific electrode patterns to stimulate presynaptic axons and record postsynaptic muscle activity.…”

“…The results showed that iPS cell-derived MN formed a functional coordinated network before in vitro NMJ model analysis. [82] Duc et al combined microfluidics, soft lithography, and MEA to create an NMJ on specific electrode patterns to stimulate presynaptic axons and record postsynaptic muscle activity. Owing the combination of MEA and microfluidic technology, the authors formed a fully functional human NMJ and recorded the muscle action potentials triggered by induced neuronal electrical stimulation.…”

“…As the transferred genes contained fluorescent markers, the pathway of viral infection in neurons can be evaluated by fluorescence analysis, which validates the function of NMJ. [ 82 ]…”

Organ on a Chip: A Novel in vitro Biomimetic Strategy in Amyotrophic Lateral Sclerosis (ALS) Modeling

“…In addition to monosynaptic retrograde tracing, alpha-bungarotoxin, as the inhibitor of postsynaptic nicotinic acetylcholine receptors has been applied which has a crucial role in determining the contractile activity of myotubes. Furthermore, the use of immunostaining assay has also been examined as an effective approach to confirm the connection between myotubes and MNs in the suggested model ( 119 ).…”

Human neuromuscular junction on micro-structured microfluidic devices implemented with a custom micro electrode array (MEA)

“…In the last three years, there has been a considerable increase in the number of papers presenting several models of hNMJs. For example, a functional NMJ platform has been validated by combining microfluidic chips and pseudotyped ΔG-rabies virus for retrograde monosynaptic tracing 27 or chips coupled with MEA for spontaneous activity recording. 28 Most of the time, muscle activity is measured by evaluating contraction after electrical stimulation using patch-clamp 29 or optogenetic neuronal stimulation.…”