Forced cell cycle exit and modulation of GABAA, CREB, and GSK3β signaling promote functional maturation of induced pluripotent stem cell-derived neurons
Forced cell cycle exit and modulation of GABAA, CREB, and GSK3β signaling promote functional maturation of induced pluripotent stem cell-derived neurons
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DOI：
10.1152/ajpcell.00166.2015
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发表时间：
2016-04-01
影响因子：
5.5
通讯作者：
Kemp, Paul J.
中科院分区：
文献类型：
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作者：
Telezhkin, Vsevolod;Schnell, Christian;Kemp, Paul J.
尽管已经开发了许多从各种多能干细胞中分化出神经元的方案，但大多数方案都集中在能够有效地指定合适的神经元亚型，很少有人被设计成增强或加速功能成熟。其中，大多数使用的功能成熟过程相当漫长，没有一个完全表征神经元功能的所有方面，从自发动作电位的产生到突触后受体的成熟。在这里，我们描述了一个简单的方案，它只使用两个补充的介质的顺序添加，这两个补充介质已经被配方来分离神经分化的两个关键阶段，神经发生和突触形成，每个阶段都具有不同的信号要求。利用这些介质，这个新的方案同步了神经发生，并提高了多能干细胞来源的神经前体的成熟率。用该方法分化的神经元表现出较大的细胞电容和相对超极化的静息膜电位；此外，它们还表现出增强的：1)自发电活动；2)再生诱导动作电位串活动；3)钠电流可获得性；4)突触电流。这是通过快速和均匀地发展成熟的、抑制性的GABA(A)受体表型来实现的，这一表型由钙成像和GABA(A)受体阻滞剂在多电极阵列记录中唤起致痫网络活动的能力所证明。此外，由于该协议可以利用扩大和冻结的预构图神经前体细胞在21天内交付成熟神经元，因此它既可以扩展，也可以转移到高通量平台，用于功能筛选。
Although numerous protocols have been developed for differentiation of neurons from a variety of pluripotent stem cells, most have concentrated on being able to specify effectively appropriate neuronal subtypes and few have been designed to enhance or accelerate functional maturity. Of those that have, most employ time courses of functional maturation that are rather protracted, and none have fully characterized all aspects of neuronal function, from spontaneous action potential generation through to postsynaptic receptor maturation. Here, we describe a simple protocol that employs the sequential addition of just two supplemented media that have been formulated to separate the two key phases of neural differentiation, the neurogenesis and synaptogenesis, each characterized by different signaling requirements. Employing these media, this new protocol synchronized neurogenesis and enhanced the rate of maturation of pluripotent stem cell-derived neural precursors. Neurons differentiated using this protocol exhibited large cell capacitance with relatively hyperpolarized resting membrane potentials; moreover, they exhibited augmented: 1) spontaneous electrical activity; 2) regenerative induced action potential train activity; 3) Na+ current availability, and 4) synaptic currents. This was accomplished by rapid and uniform development of a mature, inhibitory GABA(A) receptor phenotype that was demonstrated by Ca2+ imaging and the ability of GABA(A) receptor blockers to evoke seizurogenic network activity in multielectrode array recordings. Furthermore, since this protocol can exploit expanded and frozen prepatterned neural progenitors to deliver mature neurons within 21 days, it is both scalable and transferable to high-throughput platforms for the use in functional screens.