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

Peter Harley; Amaia Paredes-Redondo; Gianluca Grenci; Virgile Viasnoff; Yung-Yao Lin; Ivo Lieberam

doi:10.21769/BioProtoc.4624

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

Bio Protoc. 2023 Mar 5;13(5):e4624. doi: 10.21769/BioProtoc.4624.

Authors

Peter Harley^{1

2}, Amaia Paredes-Redondo^{3

4}, Gianluca Grenci⁵, Virgile Viasnoff⁵, Yung-Yao Lin^{3

4}, Ivo Lieberam^{1

2}

Affiliations

¹ Centre for Gene Therapy & Regenerative Medicine, Kings College London, London SE1 9RT, UK.
² Centre for Developmental Neurobiology and MRC Centre for Neurodevelopmental Disorders, Institute of Psychiatry, Psychology and Neuroscience, Kings College London, London SE1 1UL, UK.
³ Centre for Genomics and Child Health, Blizard Institute, Barts and the London School of Medicine and Dentistry, Queen Mary University of London, 4 Newark Street, London E1 2AT, UK.
⁴ Centre for Predictive in vitro Model, Queen Mary University of London, Mile End Road, London E1 4NS, UK.
⁵ Mechanobiology Institute, National University of Singapore, 5a Engineering Drive 1, 117411 Singapore.

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 disease-specific 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. Graphical abstract Graphical abstract abbreviations: Channelrhodopsin-2 (CHR2+), pluripotent stem cell (PSC), motor neurons (MNs), myofibers (MFs), neuromuscular junction (NMJ).

Keywords: ALS; Compartmentalised microdevice; DMD; Human pluripotent stem cells; Motor neuron; Myofiber; Neuromuscular co-culture; Optogenetics; Tissue engineering.

Abstract

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