In vitro models of the neuromuscular junction (NMJ) are emerging as a valuable tool to study synaptogenesis, synaptic maintenance, and pathogenesis of neurodegenerative diseases. Many models have previously been developed using a variety of cell sources for skeletal muscle and motoneurons. These models can advanced by integrating beneficial features of the native developmental milieu of the NMJ. We created a functional in vitro model of NMJ by bioreactor cultivation of transdifferentiated myocytes and stem cell-derived motoneurons, in the presence of electrical stimulation. In conjunction with a coculture medium, electrical stimulation resulted in improved maturation and function of motoneurons and myocytes, as evidenced by mature cellular structures, increased expression of neuronal and muscular genes, clusterization of acetylcholine receptors (AChRs) in the vicinity of motoneurons, and the response to glutamate stimulation. To validate the model and demonstrate its utility for pharmacological testing, we documented the potency of drugs that affect key pathways during NMJ signal transduction: (i) acetylcholine (ACh) synthesis, (ii) ACh vesicular storage, (iii) ACh synaptic release, (iv) AChR activation, and (v) ACh inactivation in the synaptic cleft. The model properly responded to the drugs in a concentration-dependent manner. We thus propose that this in vitro model of NMJ could be used as a platform in pharmacological screening and controlled studies of neuromuscular diseases.

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神经肌肉接头与电刺激的生物反应器模型，用于药理效能测试

神经肌肉接头（NMJ）的体外模型正在成为研究神经退行性疾病的突触发生，突触维持和发病机理的一种有价值的工具。以前已经使用骨骼肌和运动神经元的各种细胞来源开发了许多模型。通过整合NMJ本地开发环境的有益功能，可以改进这些模型。我们创建了一个功能体外在电刺激下，通过生物反应器培养转分化的心肌细胞和干细胞衍生的运动神经元，建立NMJ模型。结合共培养基，电刺激可改善运动神经元和肌细胞的成熟度和功能，如成熟的细胞结构，神经元和肌肉基因的表达增加，运动神经元附近的乙酰胆碱受体（AChRs）聚集以及对谷氨酸刺激的反应。为了验证该模型并证明其在药理学测试中的实用性，我们记录了在NMJ信号转导过程中影响关键途径的药物的效力：（i）乙酰胆碱（ACh）合成，（ii）ACh囊泡贮藏，（iii）ACh突触释放， （iv）AChR激活，和（v）突触间隙中的ACh失活。该模型以浓度依赖的方式适当地响应药物。因此，我们建议NMJ的体外模型可以用作神经肌肉疾病的药理筛选和对照研究的平台。