Supplementary information files for: Bioengineered model of the human motor unit with physiologically functional neuromuscular junctions

Supplementary files for article: Bioengineered model of the human motor unit with physiologically functional neuromuscular junctions.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.<br>

本数据集为论文《具备生理功能性神经肌肉接头的人类运动单元生物工程模型》的补充材料。长期以来，针对人类神经肌肉接头（neuromuscular junction, NMJ）的研究多采用实验动物、模式生物或单层细胞培养体系，但此类模型无法复现突触的生理复杂性。因此，目前关于人类NMJ发育的相关数据仍较为匮乏，且缺乏可用于运动单元研究的标准化实验系统。本研究针对这一需求，提供了构建人类运动单元三维生物工程模型的完整实验方法。与单层培养体系相比，诱导多能干细胞（induced pluripotent stem cell, iPSC）来源的运动神经元前体经球体培养后，OLIG2、ISLET1与SMI32这三种运动神经元相关标志物的mRNA转录水平分别提升约400倍、150倍与200倍。贴壁球体的轴突投射在单层培养环境中可超过1000 μm，将其接种至三维细胞外基质后，SMI32与VACHT的mRNA转录水平可进一步上调，且上调幅度与I型胶原蛋白的浓度呈剂量依赖性关系。生物工程构建的骨骼肌可产生功能性强直收缩与单收缩曲线，同时表现出乙酰胆碱受体（acetylcholine receptor, AChR）聚集增强的特征，且MUSK与LRP4的mRNA转录水平显著升高，提示突触后膜的组织结构得到优化。随后本研究确定了功能性支配三维肌肉组织所需的运动神经元球体数量（即运动池规模），成功构建出具备生理功能的人类NMJ，该模型可观测到突触前膜、突触后膜与运动神经轴突的共定位现象。三维运动单元中的自发放电活动显著升高，通过乙酰胆碱受体拮抗抑制实验证实，该放电活动由运动神经驱动。功能分析结果显示，受支配的运动单元的收缩峰时间与半松弛时间均显著缩短，提示兴奋-收缩耦联的生理功能得到增强。本研究提供了可分别优化iPSC来源运动神经元与原代人骨骼肌成熟度的实验方案，通过使用细胞类型特异性细胞外基质与标准化发育时序，成功构建出可用于神经肌肉接头生理学研究的人类运动单元生物工程模型。