Rapid Neuronal Differentiation of Induced Pluripotent Stem Cells for Measuring Network Activity on Micro-electrode Arrays
Rapid Neuronal Differentiation of Induced Pluripotent Stem Cells for Measuring Network Activity on Micro-electrode Arrays
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DOI：
10.3791/54900
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发表时间：
2017-01-01
影响因子：
1.2
通讯作者：
Kasri, Nael Nadif
中科院分区：
文献类型：
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作者：
Frega, Monica;van Gestel, Sebastianus H. C.;Kasri, Nael Nadif
源自人类诱导多能干细胞 (hiPSC) 的神经元为研究神经系统疾病提供了一种有前景的新工具。在过去的十年中，已经开发了许多将 hiPSC 分化为神经元的方案。然而，这些协议通常速度缓慢、变异性高、重现性低且效率低。此外，通过这些协议获得的神经元通常不成熟，并且在单细胞和网络水平上缺乏足够的功能活动，除非神经元培养数月。部分由于这些限制，hiPSC 衍生的神经元网络的功能特性仍然没有得到很好的表征。在这里，我们采用了最近发布的协议，该协议描述了通过转录因子 Neurogenin-212 的强制表达从 hiPSC 产生人类神经元。该方案快速（3 周内产生成熟神经元）且高效，转导细胞的转化效率接近 100%（> 95% 的 DAPI 阳性细胞为 MAP2 阳性）。此外，该协议产生了同质的兴奋性神经元群体，这将允许研究细胞类型对神经系统疾病的特定贡献。我们通过生成稳定转导的 hiPSC 细胞修改了原始方案，使我们能够明确控制神经元总数。然后使用这些细胞在微电极阵列上生成 hiPSC 衍生的神经元网络。通过这种方式，可以测量和表征 hiPSC 衍生的神经元网络的自发电生理活动，同时保持细胞密度方面的实验间一致性。所提出的协议广泛适用，特别是对于人类神经元网络的机械和药理学研究。
Neurons derived from human induced Pluripotent Stem Cells (hiPSCs) provide a promising new tool for studying neurological disorders. In the past decade, many protocols for differentiating hiPSCs into neurons have been developed. However, these protocols are often slow with high variability, low reproducibility, and low efficiency. In addition, the neurons obtained with these protocols are often immature and lack adequate functional activity both at the single-cell and network levels unless the neurons are cultured for several months. Partially due to these limitations, the functional properties of hiPSC-derived neuronal networks are still not well characterized. Here, we adapt a recently published protocol that describes production of human neurons from hiPSCs by forced expression of the transcription factor neurogenin-212. This protocol is rapid (yielding mature neurons within 3 weeks) and efficient, with nearly 100% conversion efficiency of transduced cells (>95% of DAPI-positive cells are MAP2 positive). Furthermore, the protocol yields a homogeneous population of excitatory neurons that would allow the investigation of cell-type specific contributions to neurological disorders. We modified the original protocol by generating stably transduced hiPSC cells, giving us explicit control over the total number of neurons. These cells are then used to generate hiPSC-derived neuronal networks on micro-electrode arrays. In this way, the spontaneous electrophysiological activity of hiPSC-derived neuronal networks can be measured and characterized, while retaining interexperimental consistency in terms of cell density. The presented protocol is broadly applicable, especially for mechanistic and pharmacological studies on human neuronal networks.