Generation of Functional Neuromuscular Junctions from Human Pluripotent Stem Cell Lines.
Generation of Functional Neuromuscular Junctions from Human Pluripotent Stem Cell Lines.
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DOI：
10.3389/fncel.2015.00473
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发表时间：
2015
影响因子：
5.3
通讯作者：
Koistinaho J
中科院分区：
文献类型：
--
作者：
Puttonen KA;Ruponen M;Naumenko N;Hovatta OH;Tavi P;Koistinaho J
几种神经肌肉疾病涉及神经肌肉接头（NMJ）的功能障碍，但目前还没有针对NMJ电生理特征的患者特异性人体模型。我们接种细胞的神经诱导的胚状体样球来自诱导多能干细胞（iPSC）或胚胎干细胞（ESC）线作为单层无碱性成纤维细胞因子（bFGF）和观察分化的神经元以及自发收缩，多核骨骼肌肌管。肌管显示横纹，肌球蛋白重链的免疫反应，肌动蛋白束典型的肌定向细胞，并产生自发和诱发的动作电位（AP）。成肌分化与MyoD 1、myogenin和I型ryanodine受体的表达有关。神经元形成终板样结构，与α-银环蛇毒素（一种在NMJ突触后膜中高度表达的烟碱乙酰胆碱受体标记物）强结合，并表达运动神经元标记物SMI-32以及突触标记物SV 2。胆碱能受体的药理刺激导致肌管膜的强烈去极化和肌浆中的Ca 2+浓度升高，而电刺激诱发肌管中的Ca 2+瞬变。用N-甲基-D-天冬氨酸刺激运动神经元导致肌管和终板中可再现的AP，显示出典型的mEPP和约10 mV的去极化肌管的强直活性。我们的结论是，同时分化的神经元和肌管从患者特异性iPSC或ESC的结果也在功能性NMJ的发展。我们的人类NMJ模型可能作为一个重要的工具，研究正常的发展，疾病的机制和新的药物靶点涉及NMJ功能障碍和变性。
Several neuromuscular diseases involve dysfunction of neuromuscular junctions (NMJs), yet there are no patient-specific human models for electrophysiological characterization of NMJ. We seeded cells of neurally-induced embryoid body-like spheres derived from induced pluripotent stem cell (iPSC) or embryonic stem cell (ESC) lines as monolayers without basic fibroblast factor (bFGF) and observed differentiation of neuronal as well as spontaneously contracting, multinucleated skeletal myotubes. The myotubes showed striation, immunoreactivity for myosin heavy chain, actin bundles typical for myo-oriented cells, and generated spontaneous and evoked action potentials (APs). The myogenic differentiation was associated with expression of MyoD1, myogenin and type I ryanodine receptor. Neurons formed end plate like structures with strong binding of α-bungarotoxin, a marker of nicotinic acetylcholine receptors highly expressed in the postsynaptic membrane of NMJs, and expressed SMI-32, a motoneuron marker, as well as SV2, a marker for synapses. Pharmacological stimulation of cholinergic receptors resulted in strong depolarization of myotube membrane and raised Ca2+ concentration in sarcoplasm, while electrical stimulation evoked Ca2+ transients in myotubes. Stimulation of motoneurons with N-Methyl-D-aspartate resulted in reproducible APs in myotubes and end plates displayed typical mEPPs and tonic activity depolarizing myotubes of about 10 mV. We conclude that simultaneous differentiation of neurons and myotubes from patient-specific iPSCs or ESCs results also in the development of functional NMJs. Our human model of NMJ may serve as an important tool to investigate normal development, mechanisms of diseases and novel drug targets involving NMJ dysfunction and degeneration.