miRNA-Based Rapid Differentiation of Purified Neurons from hPSCs Advancestowards Quick Screening for Neuronal Disease Phenotypes In Vitro
miRNA-Based Rapid Differentiation of Purified Neurons from hPSCs Advancestowards Quick Screening for Neuronal Disease Phenotypes In Vitro
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DOI：
10.3390/cells9030532
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发表时间：
2020-03-01
期刊：
影响因子：
6
通讯作者：
Okano, Hideyuki
中科院分区：
文献类型：
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作者：
Ishikawa, Mitsuru;Aoyama, Takeshi;Okano, Hideyuki
通过高效、简单、快速的分化方法获得具有高生理功能的分化细胞对于利用人多能干细胞（hPSC）体外模拟神经元疾病至关重要。目前，涉及一种或几种转录因子的瞬时表达的方法已经被建立为用于在快速的单个步骤中诱导神经元分化的技术。也有报道称，microRNA可以作为重编程效应物，直接将人皮肤成纤维细胞重编程为神经元。在这项研究中，我们测试了添加神经元microRNA、miRNA-9/9* 和miR-124（miR-9/9*-124）对于使用Tet-On驱动的神经生成素2基因（Ngn 2）（一种前神经因子）表达的hPSC神经元诱导方法的影响。虽然已经确定Ngn 2由于其神经原性作用可以促进从多能干细胞分化成具有高纯度的神经元，但是单独使用Ngn 2错误表达的神经元成熟需要很长或无限期的时间。使用本发明的方法，细胞保持高神经元分化率，同时表现出神经元成熟标志物的基因表达增加、自发钙振荡和具有网络爆发的高电活性，如通过多点电极系统评估的。此外，当将这种方法应用于患有早老素-1（PS1）或早老素-2（PS2）突变的阿尔茨海默病（AD）患者的iPSC时，观察到细胞表型，例如淀粉样蛋白β 42细胞外分泌量增加、异常耗氧量和增加。细胞中的活性氧自由基在比之前报道的更短的培养时间内观察到。因此，强烈预期结合Ngn 2和miR-9/9*-124的诱导方法将能够更快速和简单地筛选各种类型的神经元疾病表型并促进药物发现。
Obtaining differentiated cells with high physiological functions by an efficient, but simple and rapid differentiation method is crucial for modeling neuronal diseases in vitro using human pluripotent stem cells (hPSCs). Currently, methods involving the transient expression of one or a couple of transcription factors have been established as techniques for inducing neuronal differentiation in a rapid, single step. It has also been reported that microRNAs can function as reprogramming effectors for directly reprogramming human dermal fibroblasts to neurons. In this study, we tested the effect of adding neuronal microRNAs, miRNA-9/9*, and miR-124 (miR-9/9*-124), for the neuronal induction method of hPSCs using Tet-On-driven expression of the Neurogenin2 gene (Ngn2), a proneural factor. While it has been established that Ngn2 can facilitate differentiation from pluripotent stem cells into neurons with high purity due to its neurogenic effect, a long or indefinite time is required for neuronal maturation with Ngn2 misexpression alone. With the present method, the cells maintained a high neuronal differentiation rate while exhibiting increased gene expression of neuronal maturation markers, spontaneous calcium oscillation, and high electrical activity with network bursts as assessed by a multipoint electrode system. Moreover, when applying this method to iPSCs from Alzheimer's disease (AD) patients with presenilin-1 (PS1) or presenilin-2 (PS2) mutations, cellular phenotypes such as increased amount of extracellular secretion of amyloid beta 42, abnormal oxygen consumption, and increased reactive oxygen species in the cells were observed in a shorter culture period than those previously reported. Therefore, it is strongly anticipated that the induction method combining Ngn2 and miR-9/9*-124 will enable more rapid and simple screening for various types of neuronal disease phenotypes and promote drug discovery.