3D Compartmentalised Human Pluripotent Stem Cell–derived Neuromuscular Co-cultures

Abstract

Human neuromuscular diseases represent a diverse group of disorders with unmet clinical need, ranging from muscular dystrophies, such as Duchenne muscular dystrophy (DMD), to neurodegenerative disorders, such as amyotrophic lateral sclerosis (ALS). In many of these conditions, axonal and neuromuscular synapse dysfunction have been implicated as crucial pathological events, highlighting the need for in vitro disease models that accurately recapitulate these aspects of human neuromuscular physiology. The protocol reported here describes the co -culture of neural spheroids composed of human pluripotent stem cell (PSC)-derived motor neurons and astrocytes, and human PSC-derived myofibers in 3D compartmentalised microdevices to generate functional human neuromuscular circuits in vitro. In this microphysiological model, motor axons project from a central nervous system (CNS)-like compartment along microchannels to innervate skeletal myofibers plated in a separate muscle compartment. This mimics the spatial organization of neuromuscular circuits in vivo. Optogenetics, particle image velocimetry (PIV) analysis, and immunocytochemistry are used to control, record, and quantify functional neuromuscular transmission, axonal outgrowth, and neuromuscular synapse number and morphology. This approach has been applied to study diseasespecific phenotypes for DMD and ALS by incorporating patient-derived and CRISPR-corrected human PSC-derived motor neurons and skeletal myogenic progenitors into the model, as well as testing candidate drugs for rescuing pathological phenotypes. The main advantages of this approach are: i) its simple design; ii) the in vivo-like anatomical separation between CNS and peripheral muscle; and iii) the amenability of the approach to high power imaging. This opens up the possibility for carrying out live axonal transport and synaptic imaging assays in future studies, in addition to the applications reported in this study.

Engineered human myogenic cells in hydrogels generate functional myofibers within dystrophic mouse muscle

“…Healthy human skeletal muscle-derived CD133+ cells (hCD133+) were described 11 and obtained from Medical Research Council Centre for Neuromuscular Diseases Biobank (ID: 8206). CORR-R3381X and CORR-K2957fs human MPCs were generated using a transgene-free myogenic differentiation protocol 58 from two precisely CRISPR-corrected DMD patient-derived PSC lines, respectively 33,34,59 . hCD133+ cells were maintained in Megacell Skeletal Muscle Growth Medium (For details, see Supplementary Table S3), whereas CORR-R3381X and CORR-K2957fs MPCs were maintained in Promocell Skeletal Muscle Growth Medium (Promocell, C-23060).…”

Leveraging Biomaterial Platforms to Study Aging-Related Neural and Muscular Degeneration

“…A challenge encountered in the generation of stem-cell-derived neuromuscular models is the detachment of muscle fibers from their culture substrate as they contract. To circumvent this issue, Kamm and colleagues created a culture system based on the support of myofibers between micropillar cantilevers that deflected upon contraction [305,307,308].…”

Spinal Cord Organoids to Study Motor Neuron Development and Disease

“…Recent studies used these devices to generate gradients of drugs, such as Riluzole [ 232 ] or Rapamycin [ 122 ], and demonstrated their suitability for optimizing drug concentration and efficacy. Organoids cultured in combination with microfluidic devices are often termed organ-on-a-chip, and they have been recently been applied in the MND field to generate structures where MN somas and muscle fibers are contained in separate compartments and connected by motor axons confined into nerve bundles [ 118 , 120 , 233 ]. This procedure enabled the reliable measurement of muscle contractions that could be easily manipulated by chemical or genetic approaches, and created advanced platforms to test drugs with potential impact on axon fascicles and NMJ functionality.…”