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Manufacturing human iPSC-based neuromuscular circuits in compartmented devices


Neuromuscular disorders encompass a range of conditions from muscular dystrophies such as Duchenne muscular dystrophy (DMD) to some neurodegenerative disorders such as amyotrophic lateral sclerosis (ALS). Despite the diversity in clinical presentation, all of these disorders lack suitable treatments.  Furthermore, in many of these conditions, axonal and neuromuscular synapse dysfunction have been implicated by research as significant pathological factors. The need for treatments and the known pathological factors highlight the pressing need for in vitro models to accurately recapitulate these aspects of human neuromuscular physiology.

In a new publication, out now in Bio-protocol, Peter Harley and Ivo Lieberam, along with colleagues from QMUL and NUS, Singapore, describe a new model of neuromuscular circuits. In their protocol, Harley et al. report their findings from co-culturing neural spheroids, composed of human pluripotent stem cell (PSC)-derived motor neurons and astrocytes, and human PSC-derived myofibers. The authors co-cultured these PSC-derived motor neurons, astrocytes and myofibers in 3D compartmentalised microdevices with the aim of generating functional human neuromuscular circuits in vitro.

Motor axons were observed to project from a CNS-like compartment along microchannels in their microphysiological model. This in turn innervated skeletal myofibers plated in a separate muscle compartment, recapitulating the spatial organisation of neuromuscular circuits in vivo. Harley et al. used optogenetics, particle image velocimetry (PIV) analysis and immunohistochemistry to monitor functional neurotransmission, axonal growth and neuromuscular synapse number and morphology in their model. This tripartite approach was employed to look at DMD- and ALS-specific phenotypes, incorporating patient-derived, and CRISPR-corrected, human PSC-derived motor neurons and skeletal myogenic progenitors into the model. Their approach also allowed the authors to test candidate drugs for their capacity to rescue pathological phenotypes. Harley et al.’s straightforward protocol is amenable to high power imaging and generates in vivo-like anatomical separation between the CNS and peripheral muscle. In addition to the applications the authors report, their approach opens up the possibility to carry out live axonal transport and synaptic imaging assays in future studies.

Harley et al. describe a method for assembling circuits from stem cell-derived motor neurons, astrocytes and myofibers in open microdevice arrays. The authors show how this approach can be used to measure neuromuscular synapse formation/maintenance and optogenetically induced myofiber contractions in disease models of Duchenne muscular dystrophy and amyotrophic lateral sclerosis.