Center for Precision Genome Editing and Genetic Technologies for Biomedicine, Pirogov Russian National Research Medical University, Ostrovitianov Street, Moscow, Russia, 117997.
Faculty of Biology, Lomonosov Moscow State University, GSP-1, Leninskie Gory, Moscow, Russia, 119991.
Neurochem Res. 2022 Apr;47(4):952-966. doi: 10.1007/s11064-021-03497-6. Epub 2021 Dec 2.
The study of human neurons and their interaction with neurochemicals is difficult due to the inability to collect primary biomaterial. However, recent advances in the cultivation of human stem cells, methods for their neuronal differentiation and chimeric fluorescent calcium indicators have allowed the creation of model systems in vitro. In this paper we report on the development of a method to obtain human neurons with the GCaMP6s calcium indicator, based on a human iPSC line with the TetON-NGN2 transgene complex. The protocol we developed allows us quickly, conveniently and efficiently obtain significant amounts of human neurons suitable for the study of various neurochemicals and their effects on specific neurophysiological activity, which can be easily registered using fluorescence microscopy. In the neurons we obtained, glutamate (Glu) induces rises in [Ca] which are caused by ionotropic receptors for Glu, predominantly of the NMDA-type. Taken together, these facts allow us to consider the model we have created to be a useful and successful development of this technology.
由于无法收集主要的生物材料，研究人类神经元及其与神经化学物质的相互作用是困难的。然而，最近在培养人类干细胞、神经元分化方法以及嵌合荧光钙指示剂方面的进展，使得体外模型系统的创建成为可能。在本文中，我们报告了一种基于带有 TetON-NGN2 转基因复合物的人 iPSC 系，用 GCaMP6s 钙指示剂获得人神经元的方法的开发。我们开发的方案使我们能够快速、方便、有效地获得大量适合研究各种神经化学物质及其对特定神经生理活动影响的人神经元，这些可以使用荧光显微镜轻松记录。在我们获得的神经元中，谷氨酸 (Glu) 诱导 [Ca] 的升高，这是由 Glu 的离子型受体引起的，主要是 NMDA 型。综上所述，这些事实使我们认为我们创建的模型是这项技术的一个有用且成功的发展。
