Multi-level characterization of balanced inhibitory-excitatory cortical neuron network derived from human pluripotent stem cells.

hNES cell-derived neuronal culture protocol.hiPSC-derived NES cells were generated and characterized by (A-E) immunocytochemistry for PLZF, PAX6, SOX2, (F) morphology as well as (G) RT-PCR for SOX2, Nestin, PAX6, PLZF, DACH1, ZNF312 and HES5, and later differentiated into low-density neuronal cultures. (H) Schematic representation of the differentiation protocol. (I-L) Representative images of differentiating neurons at day 4, 8, 20 and direct contact culture mode day 49 are shown. (M, N) Cartoon representation of direct and indirect contact co-culture model. Scale bars are 25 μm. See also S1 and S2 Figs for hiPSC characterization and hESC-derived hNES cell characterization.

RNA and immunocytochemical analysis of hNES cell-derived neuronal cultures in direct contact.RNA was isolated at different stages of differentiation (day 5 (n = 3), day 8 (n = 2), day 18 (n = 3) and day 49 (n = 3)) altogether from Line B and C. RNA expression profiles of (A) caudal (CoupTF2, PROX1), (B) medial (SATB1, LHX6) and (C) lateral (MEIS2, GSX2) ganglionic eminence markers. (D) Expression of sub-pallium (ASCL1), and pre-synaptic markers (VGAT, VGLUT1) overtime during differentiation. (E) Cortical progenitors (BF1, TBR2) and pallium marker (PAX6); (F) cortical upper layers (CUX1, BRN2, SATB2); (G) deep cortical layers (TBR1, CTIP2) and (H) rosette and neural stem cell (HES5, DACH1, PLZF, SOX2) markers were also assessed in the RNA samples. All the primers data was normalized to the expression of housekeeping gene EIF4G2. Error bars represent standard mean error (SEM) per time point from n = 3 in day 5, 18, 49 and n = 2 in day 8. One-way ANOVA was performed and with significant differences (p<0.05) in some gene expressions, a post hoc test represents significant differences (in asterisks) among the time points with a p<0.05. Immunocytochemical analysis at day 49 (n = 2) for Glutamatergic lineage markers CTIP2 (I), SATB2 (J), CGE marker PROX1 (K), LGE marker MEIS2 (L) and Calbindin (M) with MAP2. (N) Only the neuronal population is human-derived as shown by immunocytochemistry for human nucleus (HN), GFAP (rat astrocytes) and MAP2. Scale bars are 25 μm.

Immunocytochemical analysis of glutamatergic and GABAergic pre/post-synaptic markers in direct contact co-cultures.Immunocytochemical analysis was performed at day 49 from Line B. hPSC-derived neurons were analyzed for the expression of (A-C) MAP2, GAD65/67, SMI-312, (F-H) VGAT and synaptophysin1, (K-M) VGLUT1 and HOMER1. Ratio of (D) GAD65/MAP2-positive cells (n = 2), (E) Synaptophysin1 puncta per MAP2 soma (n = 3) (I) VGAT/synaptophysin1-positive puncta (n = 3), (J) VGAT puncta per MAP2 soma (n = 3) (N) VGLUT1/synaptophysin1-positive (n = 3) puncta and (O) VGLUT1 puncta per MAP2 soma (n = 3) (10 FOV’s per experiment) were quantified. Error bars represent SEM. Insets represent closer magnifications. One-way ANOVA was performed, no significant difference was found between experiments for GAD65 and VGAT analysis. Significant differences with p<0.05 was found between experiments B1, B2 and B3 for VGLUT1 analysis. FOV–fields of view; Scale bars are 5 μm.

Functional analysis of differentiated neurons in direct contact co-cultures.At day 49 we recorded Line A-derived (n = 2) and Line B-derived (n = 2) neurons, which show (A) fast inward Na+ currents followed by long-lasting outward K+ currents; current was evoked by voltage steps ranging from -90 mV to 50 mV with 10 mV increments. Inset shows fast Na+ currents in absence or presence of Na+ channel antagonist TTX (1 μM). (B) Quantification of peak Na+ and K+ currents evoked in (A) is shown. (C) We recorded action potentials evoked by a current injection of 100 pA and also (D) spontaneously generated action potentials in current clamp mode. (E) Spontaneous post-synaptic events from a single neuron (holding potential at -70 mV in voltage clamp mode) recorded in presence of 1μM TTX, TTX + 40 μM Bicuculline and TTX + 40 μM Bicuculline + 50 μM AP5 + 10 μM DNQX are shown. (F) Postsynaptic current evoked by local field stimulation (2 mA, 1 ms). We recorded calcium traces at day 49 of differentiation; (G, H) hiPSC-derived neurons loaded with Fluo-5 AM ester. (I) Raster plot showing onset and duration of intracellular calcium events from ROIs represented on G; upon TTX addition and 20 minutes after TTX was washed away (J) or traces represented in H upon bicuculline, bicuculline + AP5 + CNQX addition and 20 minutes after drugs were washed away (representative calcium traces for ROI 2 (G, I) and ROI 9 (H, J)) are shown. (K) We then analyzed activity dependent intracellular calcium traces (red dotted bars indicate beginning of electrical field stimulation and number of pulses applied). Scale bars are 10 μm. Data are represented as mean ± SEM. See also S4 Fig for hESC-derived neurons calcium imaging experiments.

Characterization of the differentiated neurons in indirect contact co-cultures.In indirect co-cultures we differentiated the human neurons in close proximity of rat astrocyte layers (Line B1 and B2). (A, B) Bright field images of indirect cultures during differentiation day 26 and 56 are shown. (C-F) We observed the expression of MAP2, VGAT, synaptophysin1 and (G-J) VGLUT1 and HOMER1 co-localizing puncta using immunocytochemistry at day 56. We then analyzed (K) spontaneous mEPSCs and mIPSCs (holding potential at -70mV in voltage clamp mode) recorded in 1 μM TTX supplemented with 40 μM Bicuculline or 50 μM AP5 + 10 μM DNQX respectively. (L) Quantification of amplitude, area, time to decay and frequency of mEPSCs and mIPSCs was also analyzed (n = 2). We then recorded calcium traces of (M, N) neurons loaded with Fluo-5 AM ester from n = 2. (O) Raster plot showing onset and duration of intracellular calcium events from ROIs represented on M and N respectively; upon TTX addition and 20 minutes after TTX was washed away (P) or upon bicuculline, bicuculline + AP5 + CNQX addition and 20 minutes after drugs were washed away (representative calcium traces for ROI 1(M, O) and ROI 8 (N, P)) are shown. (Q) Analysis of activity dependent intracellular calcium traces (red dotted bars indicate beginning of electrical field stimulation and number of pulses applied). Scale bars are 10 μm. Data are represented as mean ± SEM. See also S5 Fig for indirect contact plate preparation method.

Proteomic analysis of indirect contact co-culture neurons representing reproducible cultures.(A) Hierarchical clustering of protein groups based on expression intensity present in the day 56 differentiated neurons. Columns represent triplicates from 2 experiments (B1 and B2) normalized for loading and appear similar in highly expressed proteins (color key values > 4). (B) Average of the coefficient of variation normalized to loading for all the proteins. (C) Average and standard deviations of intensity based absolute quantification (iBAQ) values for pre-, post-synaptic and growth cone proteins in day 56 neuronal cultures.