FIG 1.

Neuronal differentiation of human iPSCs (hiPSCs) in 2D cultures. (A to F) hiPSCs (A) are differentiated into columnar epithelial cells, forming neural rosettes (B). (C) hiPSC-derived neural rosettes are expanded as monolayer cultures of neural stem cells/neural progenitor cells (collectively referred as neural precursor cells [NPCs] in this study). (D) NPCs are further differentiated into neurons, illustrated using Tuj1 immunofluorescence (red) with Hoechst 33342 counterstaining of nuclei (blue). (E) These cells express the glutamate receptors GluRB, GluR5, and GluR6. Lanes M, molecular size markers. (F) Coimmunostaining of hiPSC-derived neurons with PSD-95 (green) and MAP2 (red) revealed PSD-95-labeled dendritic protrusions resembling a spine. (A to C) Phase-contrast microscopy; (D, F) confocal fluorescence microscopy. Bars, 50 μm (A and B), 100 μm (C), 75 μm (D), 5 μm (F). (G to J) Electrophysiological recordings of hiPSC-derived neurons. In voltage clamp experiments on cells with a resting potential equal to or more negative than −40 mV, when the membrane potential was depolarized from −100 mV to 20 mV starting from a holding potential of −85 mV, two notable currents were evoked: a fast inward Na⁺ component (evoked starting from −30 mV until 20 mV, with a maximum value of current at −30 mV of −2,263 ± 329.4 pA; mean ± SEM, n = 4 cells) and a slower outward K⁺ component (evoked starting from −30 mV until 20 mV, with a maximum value of current at 20 mV of 1,020 ± 179.5 pA; mean ± SEM, n = 4 cells). In current clamp mode, depolarizing steps of current from −100 pA to 80 pA were used to detect the ability of the cells to generate action potentials. hiPSC-derived neurons showed repetitive evoked action potentials that were in line with the recorded currents. (G) Voltage clamp recording. The currents recorded consist of an inward Na⁺ component and of an outward K⁺ component. (H) Currents were recorded using a voltage clamp protocol consisting of depolarizing steps from −100 mV to 20 mV starting from a holding potential of −85 mV. (I and J) A representative trace of evoked action potentials obtained in current clamp mode (I) injecting depolarizing steps of current from −100 pA to 80 pA (J).