Bioengineered model of the human motor unit with physiologically functional neuromuscular junctions

Abstract Investigations of the human neuromuscular junction (NMJ) have predominately utilised experimental animals, model organisms, or monolayer cell cultures that fail to represent the physiological complexity of the synapse. Consequently, there remains a paucity of data regarding the development...

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Autores principales:	Rowan P. Rimington, Jacob W. Fleming, Andrew J. Capel, Patrick C. Wheeler, Mark P. Lewis
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2021
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/4e4e49a1611143bc96fb271b7731c6bd
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spelling	oai:doaj.org-article:4e4e49a1611143bc96fb271b7731c6bd2021-12-02T18:25:03ZBioengineered model of the human motor unit with physiologically functional neuromuscular junctions10.1038/s41598-021-91203-52045-2322https://doaj.org/article/4e4e49a1611143bc96fb271b7731c6bd2021-06-01T00:00:00Zhttps://doi.org/10.1038/s41598-021-91203-5https://doaj.org/toc/2045-2322Abstract Investigations of the human neuromuscular junction (NMJ) have predominately utilised experimental animals, model organisms, or monolayer cell cultures that fail to represent the physiological complexity of the synapse. Consequently, there remains a paucity of data regarding the development of the human NMJ and a lack of systems that enable investigation of the motor unit. This work addresses this need, providing the methodologies to bioengineer 3D models of the human motor unit. Spheroid culture of iPSC derived motor neuron progenitors augmented the transcription of OLIG2, ISLET1 and SMI32 motor neuron mRNAs ~ 400, ~ 150 and ~ 200-fold respectively compared to monolayer equivalents. Axon projections of adhered spheroids exceeded 1000 μm in monolayer, with transcription of SMI32 and VACHT mRNAs further enhanced by addition to 3D extracellular matrices in a type I collagen concentration dependent manner. Bioengineered skeletal muscles produced functional tetanic and twitch profiles, demonstrated increased acetylcholine receptor (AChR) clustering and transcription of MUSK and LRP4 mRNAs, indicating enhanced organisation of the post-synaptic membrane. The number of motor neuron spheroids, or motor pool, required to functionally innervate 3D muscle tissues was then determined, generating functional human NMJs that evidence pre- and post-synaptic membrane and motor nerve axon 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. Functional analysis outlined decreased time to peak twitch and half relaxation times, indicating enhanced physiology of excitation contraction coupling in innervated motor units. Our findings provide the methods to maximise the maturity of both iPSC motor neurons and primary human skeletal muscle, utilising cell type specific extracellular matrices and developmental timelines to bioengineer the human motor unit for the study of neuromuscular junction physiology.Rowan P. RimingtonJacob W. FlemingAndrew J. CapelPatrick C. WheelerMark P. LewisNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 11, Iss 1, Pp 1-15 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Rowan P. Rimington Jacob W. Fleming Andrew J. Capel Patrick C. Wheeler Mark P. Lewis Bioengineered model of the human motor unit with physiologically functional neuromuscular junctions
description	Abstract Investigations of the human neuromuscular junction (NMJ) have predominately utilised experimental animals, model organisms, or monolayer cell cultures that fail to represent the physiological complexity of the synapse. Consequently, there remains a paucity of data regarding the development of the human NMJ and a lack of systems that enable investigation of the motor unit. This work addresses this need, providing the methodologies to bioengineer 3D models of the human motor unit. Spheroid culture of iPSC derived motor neuron progenitors augmented the transcription of OLIG2, ISLET1 and SMI32 motor neuron mRNAs ~ 400, ~ 150 and ~ 200-fold respectively compared to monolayer equivalents. Axon projections of adhered spheroids exceeded 1000 μm in monolayer, with transcription of SMI32 and VACHT mRNAs further enhanced by addition to 3D extracellular matrices in a type I collagen concentration dependent manner. Bioengineered skeletal muscles produced functional tetanic and twitch profiles, demonstrated increased acetylcholine receptor (AChR) clustering and transcription of MUSK and LRP4 mRNAs, indicating enhanced organisation of the post-synaptic membrane. The number of motor neuron spheroids, or motor pool, required to functionally innervate 3D muscle tissues was then determined, generating functional human NMJs that evidence pre- and post-synaptic membrane and motor nerve axon 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. Functional analysis outlined decreased time to peak twitch and half relaxation times, indicating enhanced physiology of excitation contraction coupling in innervated motor units. Our findings provide the methods to maximise the maturity of both iPSC motor neurons and primary human skeletal muscle, utilising cell type specific extracellular matrices and developmental timelines to bioengineer the human motor unit for the study of neuromuscular junction physiology.
format	article
author	Rowan P. Rimington Jacob W. Fleming Andrew J. Capel Patrick C. Wheeler Mark P. Lewis
author_facet	Rowan P. Rimington Jacob W. Fleming Andrew J. Capel Patrick C. Wheeler Mark P. Lewis
author_sort	Rowan P. Rimington
title	Bioengineered model of the human motor unit with physiologically functional neuromuscular junctions
title_short	Bioengineered model of the human motor unit with physiologically functional neuromuscular junctions
title_full	Bioengineered model of the human motor unit with physiologically functional neuromuscular junctions
title_fullStr	Bioengineered model of the human motor unit with physiologically functional neuromuscular junctions
title_full_unstemmed	Bioengineered model of the human motor unit with physiologically functional neuromuscular junctions
title_sort	bioengineered model of the human motor unit with physiologically functional neuromuscular junctions
publisher	Nature Portfolio
publishDate	2021
url	https://doaj.org/article/4e4e49a1611143bc96fb271b7731c6bd
work_keys_str_mv	AT rowanprimington bioengineeredmodelofthehumanmotorunitwithphysiologicallyfunctionalneuromuscularjunctions AT jacobwfleming bioengineeredmodelofthehumanmotorunitwithphysiologicallyfunctionalneuromuscularjunctions AT andrewjcapel bioengineeredmodelofthehumanmotorunitwithphysiologicallyfunctionalneuromuscularjunctions AT patrickcwheeler bioengineeredmodelofthehumanmotorunitwithphysiologicallyfunctionalneuromuscularjunctions AT markplewis bioengineeredmodelofthehumanmotorunitwithphysiologicallyfunctionalneuromuscularjunctions
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Bioengineered model of the human motor unit with physiologically functional neuromuscular junctions

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