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

Compartmentalized 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 {Delta}G-rabies virus for retrograde monosynaptic tracing.\n\nGraphical abstract\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=186 SRC=\"FIGDIR/small/745513v1_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (91K):\norg.highwire.dtl.DTLVardef@d34487org.highwire.dtl.DTLVardef@3804b8org.highwire.dtl.DTLVardef@1be0389org.highwire.dtl.DTLVardef@1da414c_HPS_FORMAT_FIGEXP M_FIG Functional neuromuscular junction in a microfluidic chip\n\n(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.\n\n(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 {Delta}G-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.\n\nC_FIG