A. A. Galiakberova,corresponding author1,2 A. M. Surin,3,4 Z. V. Bakaeva,3,5 R. R. Sharipov,4 Dongxing Zhang,6 D. A. Dorovskoy,6 K. M. Shakirova,1 A. P. Fisenko,3 and E. B. Dashinimaev1,6,7
"1Center for Precision Genome Editing and Genetic Technologies for Biomedicine, Pirogov Russian National Research Medical University, Ostrovitianov Street, Moscow, Russia 117997
2Faculty of Biology, Lomonosov Moscow State University, GSP-1, Leninskie Gory, Moscow, Russia 119991
3Laboratory of Neurobiology, “National Medical Research Center of Children’s Health”, Russian Ministry of Health, Lomonosov Avenue, Moscow, Russia 119991
4Laboratory of Pathology of Ion Transport and Intracellular Signaling, Institute of General Pathology and Pathophysiology, Baltiyskaya St., Moscow, Russia 125315
5Department of General Biology and Physiology, Gorodovikov Kalmyk State University, Pushkin St., Elista, Russia 358000
6Moscow Institute of Physics and Technology (State University), Institutskiy per., 141701 Dolgoprudny, Russia
7Koltzov Institute of Developmental Biology, Russian Academy of Sciences, Vavilov St., Moscow, Russia 119334
A. A. Galiakberova, Email: [email protected]
corresponding authorCorresponding author."
Calcium Imaging, Neuroscience
"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 [Ca2+]i 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.
The online version contains supplementary material available at 10.1007/s11064-021-03497-6.
Keywords: IPSC, Neurons, TetON–NGN2, GCaMP6s, Neurochemicals, Glutamate
DOI: https://dx.doi.org/ 10.1007/s11064-021-03497-6
Measurements of changes in [Ca2+]i and [Na+]i were performed using an image analysis system based on a Nikon Ti2 microscope (Japan), an LED-based illumination system (PE-340-fura, CoolLED, USA), a triple-band beam-splitter mirror DM468/526/596, a set of excitation filters 340 ± 13 and 387 ± 6 nm for Fura-2 and SBFI, and 442 ± 21 nm for GCaMP6s.
Product Associated Features
The 340 nm and 380 nm LED illumination system provides the optimum excitation wavelengths for Fura-2-based calcium imaging, allowing high-precision, stable, high-throughput imaging with video-rate time resolution.
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