Next we tested the ability of the cells in hCSs to differentiate into electrically active mature neurons. We dissociated ~130-day-old hCSs and cultured cells in monolayer for 2 weeks before imaging them using the ratiometric calcium dye Fura-2. We observed that neurons displayed abundant spontaneous calcium spikes (Fig. 4a,b). We also directly loaded hCSs with the calcium indicator Fluo-4, sectioned hCSs and acutely imaged them with a confocal microscope (n = 3 hCSs). Again, we observed spontaneous calcium spikes (Supplementary Video 2). We then used patch clamping to record from human cortical neurons. All recorded neurons (n = 28 cells from hCSs differentiated from two hiPSC clones) produced a transient inward current after depolarization beyond −30 mV (Fig. 4c) that was blocked by tetrodotoxin (TTX) (Fig. 4d). This inward voltage-gated Na+ current was followed by activation of a more sustained K+ current. Importantly, depolarizing current injection revealed that all of the recorded neurons reliably produced action potentials (n = 9 cells from hCSs differentiated from two hiPSC clones) (Fig. 4e).
