Bioengineered model of the human motor unit with physiologically functional neuromuscular junctions
Investigations of the human neuromuscular junction (NMJ) have predominately utilised experimental animals and model organisms. Consequently, there remains a paucity of data regarding the development of the human NMJ and a lack of systems that enable temporal investigation of the motor unit. This work addresses this need, providing the methodologies to bioengineer 3D models of the human motor unit. Separate maturation of primary human skeletal muscle and iPSC derived motor neurons seeks to accurately represent neuromuscular development via controlled addition of motor axons following primary myogenesis. Spheroid cultures of motor neuron progenitors augmented the transcription of OLIG2, ISLET1 and SMI32 motor neuron mRNAs [~]400, [~]150 and [~]200-fold respectively. Axon projections of adhered motor neuron spheroids exceeded 1000m in monolayer, with transcription of SMI32 and VACHT mRNAs further enhanced in a concentration dependent manner within optimised 3D type I collagen extracellular matrices. Bioengineered skeletal muscles produce functional forces, demonstrate increased acetylcholine receptor (AChR) clustering, and transcription of MUSK and LRP4 mRNAs indicating enhanced organisation of the post-synaptic membrane. Dosed integration of motor neuron spheroids outlined the motor pool required to functionally innervate muscle tissues in 3D, generating physiologically functional human NMJs that evidence pre- and post-synaptic membrane and motor nerve terminal co-localisation. Spontaneous firing was significantly elevated in 3D motor units, confirmed to be driven by the motor nerve via antagonistic inhibition of the AChR. Finally, functional analyses outlined decreased time to peak twitch and half relaxation times, indicating enhanced physiology of excitation contraction coupling of NMJs within innervated motor units.