We next evaluated whether control hiPSC-astrocytes exhibit spontaneous calcium transients, as has been described for astrocytes (Volterra et al., 2014). We used the calcium indicator Fluo-4AM to monitor calcium signaling under basal conditions and in response to a pulse of extracellular glutamate (3 μM) (Zhang et al., 2016). A single pulse of glutamate produced a slow calcium response in both hiPSC-astrocytes and primary human fetal astrocytes (Figure 5). In addition, some cells exhibited spontaneous calcium spikes, suggesting the presence of a network of connected astrocytes (Scemes and Giaume, 2006) (Figures 5A–5D). To quantify these responses, we measured the frequency of spontaneous activity, the number of spontaneous spikes per time, and the amplitude of the calcium spike (Figures 5E–5G). There was no statistical difference in the frequency and number of spikes between hiPSC-astrocytes and primary astrocytes. Interestingly, the amplitude of the spontaneous calcium spike was significantly higher in hiPSC-astrocytes compared with primary astrocytes. Taken together, the excitability of hiPSC-astrocytes was largely indistinguishable from that of primary human astrocytes.
