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.

中文翻译：

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用于药理效力测试的电刺激神经肌肉接头生物反应器模型

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、突触维持和发病机制的重要工具。之前已经使用多种骨骼肌和运动神经元细胞来源开发了许多模型。这些模型可以通过整合 NMJ 本地发育环境的有益特征来推进。我们通过在电刺激下生物反应器培养转分化的肌细胞和干细胞衍生的运动神经元，创建了 NMJ 的功能性体外模型。与共培养介质结合，电刺激导致运动神经元和肌细胞的成熟和功能得到改善，这通过成熟的细胞结构、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（AChR）的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，我们记录了影响 NMJ 信号转导过程中关键途径的药物的效力：(i) 乙酰胆碱 (ACh) 合成，(ii) ACh 囊泡储存，(iii) ACh 突触释放， (iv) 突触间隙中的 AChR 激活和 (v) ACh 失活。该模型以浓度依赖性方式对药物做出正确反应。因此，我们建议这种NMJ体外模型可以用作神经肌肉疾病药理学筛选和对照研究的平台。