Discussion
Prevalence of motor neurone diseases such as amyotrophic lateral sclerosis (ALS) has driven the need for in vitro platforms to provide physiologically relevant muscle-motor neuron modules for investigating disease mechanisms, new drug targets and screening of compounds to treat ALS.
We aim to develop a model of the Neuromuscular Junction in vitro that closely mimics the relationship between human muscle & motor neurons, using human iPSC-generated cell types.
Validation of cellular innervation and synapse development was achieved using microfluidic chambers. Isolated Co-culture of iPSC progenitor cells to mature skeletal and motor neuron cell types was performed and immunostaining was conducted using commercially available antibodies. IPSC derived Motor neurons and Skeletal muscle were stained at Day 18 post plating with neuronal and muscle markers (HB9, CHAT, Islet-1, Actin, Titin, Myosin).
Extracellular field potentials were acquired on day 15 post plating using a MEA system, utilizing 16 electrodes per well across a 48 well plate. A 3D scaffold is also used with 48 well MEA plates and both Motor neuron and Skeletal muscle progenitors were seeded and differentiated in situ. Within 10 days neurons display synchronized multiple train burst firing responses indicating a mature neuron response and drive Skeletal muscle contractility.
Within 10 days in both 2D and 3D, the Motor neuron cellular neuraxis was found to be highly developed, crossing the fluidic barrier and innervating with mature Skeletal muscles. Motor neurons were observed overlapping the postsynaptic acetyl choline receptors and muscle by ICC.
Results demonstrate the development of isolated 2D and 3D culture environments offers unique methodologies for measuring cell-to-cell interactions and NMJ development and the potential for generating human models of the NMJ for drug discovery and research.
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