Lin Chuang-Yu, Yoshida Michiko, Li Li-Tzu, Saito Megumu K
Department of Clinical Application, Center for iPS Cell Research and Application (CiRA), Kyoto University;
Department of Clinical Application, Center for iPS Cell Research and Application (CiRA), Kyoto University; Department of Pediatrics, Kyoto Prefectural University of Medicine.
J Vis Exp. 2020 Dec 3(166). doi: 10.3791/61396.
The neuromuscular junction (NMJ) is a specialized synapse that transmits action potentials from the motor neuron to skeletal muscle for mechanical movement. The architecture of the NMJ structure influences the functions of the neuron, the muscle and the mutual interaction. Previous studies have reported many strategies by co-culturing the motor neurons and myotubes to generate NMJ in vitro with complex induction process and long culture period but have struggled to recapitulate mature NMJ morphology and function. Our in vitro NMJ induction system is constructed by differentiating human iPSC in a single culture dish. By switching the myogenic and neurogenic induction medium for induction, the resulting NMJ contained pre- and post- synaptic components, including motor neurons, skeletal muscle and Schwann cells in the one month culture. The functional assay of NMJ also showed that the myotubes contraction can be triggered by Ca then inhibited by curare, an acetylcholine receptor (AChR) inhibitor, in which the stimulating signal is transmitted through NMJ. This simple and robust approach successfully derived the complex structure of NMJ with functional connectivity. This in vitro human NMJ, with its integrated structures and function, has promising potential for studying pathological mechanisms and compound screening.
神经肌肉接头（NMJ）是一种特殊的突触，它将动作电位从运动神经元传递到骨骼肌以实现机械运动。NMJ结构的架构会影响神经元、肌肉的功能以及它们之间的相互作用。先前的研究报道了许多通过共培养运动神经元和肌管在体外生成NMJ的策略，这些策略诱导过程复杂且培养周期长，但难以重现成熟NMJ的形态和功能。我们的体外NMJ诱导系统是通过在单个培养皿中分化人诱导多能干细胞（iPSC）构建而成的。通过切换成肌和成神经诱导培养基进行诱导，在一个月的培养过程中，所产生的NMJ包含突触前和突触后成分，包括运动神经元、骨骼肌和施万细胞。NMJ的功能测定还表明，肌管收缩可由钙离子触发，然后被箭毒（一种乙酰胆碱受体（AChR）抑制剂）抑制，其中刺激信号通过NMJ传递。这种简单而可靠的方法成功地衍生出了具有功能连接性的复杂NMJ结构。这种具有完整结构和功能的体外人NMJ在研究病理机制和化合物筛选方面具有广阔的应用前景。
