We next determined the functional characteristics of the neurons using electrophysiology and calcium imaging. After 4 weeks of differentiation on hippocampal astrocytes, whole-cell patch-clamp recording revealed that the PROX1+ cells showed characteristics of functional neurons, such as normal levels of transient sodium inward currents and sustained potassium outward currents induced by voltage step depolarization (Figure 3A); the cells were able to fire action potentials following somatic current injection (Figure 3C). Importantly, a majority of the neurons (eight out of ten) were functionally active, with spontaneous bursts of action potentials (Figure 3B) as well as spontaneous postsynaptic currents (Figure 3D–3F), indicating formation of functional neural networks. Because spontaneous neuronal activity leads to increases in intracellular calcium levels and activation of signaling pathways that are important for the regulation of neuronal processes (Spitzer et al., 2004), we next turned to calcium imaging to evaluate the maturation of the hESC-derived hippocampal neural networks at 3 weeks and 6 weeks postdifferentiation. We observed a significantly increased percentage of active neurons as well as a higher frequency of calcium transients at 6 weeks versus 3
