IPSC-Derived Human Neurons with GCaMP6s Expression Allow In Vitro Study of Neurophysiological Responses to Neurochemicals

Abstract

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.

Keywords: GCaMP6s; Glutamate; IPSC; Neurochemicals; Neurons; TetON–NGN2.

Fig. 1

A Experimental design. B Phase contrast and fluorescence images of GCaMP6s illustrating the differentiation of transgenic iPSCs into neurons. By the 2d DD (day of differentiation), we observed the transformation of iPSCs into neuron-like cells, and by the 8th DD they already had long neurites. By 21 DD, the neurons formed a network with numerous neurites that formed bizarre patterns on the substrate. Scale bar 200 µm

Fig. 2

Immunocytochemical staining of neural culture at 21 DD for major neuronal marker proteins (beta-III-tubulin (Tuj1), Synaptophysin (SYN), Synapsin (SYP), NeuN, hNCAM, Neuron-specific enolase (NSE). A Fluorescence microscopy; Scale bar 200 µm. B Gene expression analysis by quantitative RT-PCR of the neural cell gene-markers (TUBB3, TH, MAP2, NES, MAPT, PAX6, ASCL1, BRN2, SOX1). Two-group averaging (iPSC and Neurons) of two independent biological replicates in each, significance level (p-value) in all cases less than 0.05. Normalization expression level using the home-keeping genes PSMB4 and ECM7 for all samples, logarithmic representation

Fig. 3

Images of neurons at 23 DD. A Bright field. B Fluorescence images of cells expressing the protein Ca²⁺ sensor GCaMP6s. C Fluorescence images of cells expressing the protein Ca²⁺ sensor GCaMP6s and loaded with the synthetic Ca²⁺ indicator Fura-2. Scale bars correspond to 250 µm

Fig. 4

Changes in [Ca²⁺]_i induced by glutamate (Glu) alone and in the presence of inhibitors of ionotropic Glu receptors in neural culture. Only the graphs for those cells that responded to Glu by an increase in [Ca²⁺]_i, are depicted. Glu was added at a concentration of 100 μM in the presence of 10 μM glycine (in Mg²⁺-free buffer). Changes in [Ca²⁺]_i are presented as the ratios of the Fura-2 signals upon fluorescence excitation at 340 and 380 nm (A, E, C, G) and as the GCaMP6s fluorescence signals (B, F, D, H). The Fura-2 fluorescence ratio (F340/F380) was considered to be 0 in resting cells and 1 at a saturating Ca²⁺ concentration in the presence of the Ca²⁺ ionophore Ionomycin (Iono, 2 μM, 5 mM Ca^2 + in the buffer). GCaMP6s fluorescence signals (f) are normalized relative to the basal level in resting cells (F/F_o). To quantitatively compare the Ca²⁺ responses of the cells to Glu alone and in the presence of inhibitors, the areas under the curves of the [Ca²⁺]_i changes (Aria Under Curves, AUC, rel.un.) were calculated for CNQX (C, D) and MK-801 (G, H). GCaMP6s fluorescence was excited at 442 nm. The fluorescence signals of Fura-2 and GCaMP6s were recorded at 544 nm (see methods for details). To calibrate the maximum signals of Fura-2 and GCaMP6s, the Ca²⁺ ionophore ionomycin (2 μM in the presence of 5 mM CaCl₂) was added at the end of experiments. Measurements were made in cultures at 23 DD

Fig. 5

Changes in [Ca²⁺]_i and [Na⁺]_i induced in neural culture by Glu alone and in the presence of a cocktail of inhibitors of ionotropic glutamate receptors. Presented changes in [Ca²⁺]_i (A, B) and [Na⁺]_i (C, D) are only for those cells that increased GCaMP6s fluorescence in response to Glu. Changes in [Na⁺]_i are represented as the ratio of SBFI fluorescence signals excited at 340 and 380 nm (F340/F380). SBFI and GCaMP6s fluorescence signals were monitored at 544 nm. To quantitatively compare the [Ca²⁺]_i and [Na⁺]_i responses of cells to Glu alone and in the presence of the cocktail of ionotropic Glu receptor inhibitors MK-801 and CNQX (9 μM each), the areas under the curves of [Ca²⁺]_i and [Na⁺]_i changes were used (see also legend to Fig. 4). The area under the curve (AUC) was calculated as the area between the [Ca²⁺]_i or [Na⁺]_i curve and the horizontal lines corresponding the values that [Ca²⁺]_i or [Na⁺]_i of each cell had just before the Glu addition. Measurements were performed in cultures at 32 DD. The conditions for recording and presentation of the plots are the same as in Fig. 4

Fig. 6

Changes of [Ca²⁺]_i induced by plasma membrane depolarization and the purinergic P2-receptor agonist ATP. Fura-2 (A) and GCaMP6s (B) fluorescence signals in response to [Ca²⁺]_i changes induced by plasma membrane depolarization with 50 mM KCl and P2-receptor stimulation with ATP (100 μM) in neurons obtained by differentiation of iPSCs