Figure 4

Differentiation of neural progenitor cells. HiPSC cell lines were directed to neural progenitor cells (A,B,E,F) and subsequently differentiated into neuronal cells. Neural progenitor cells expressed typical markers like Nestin (A,E, shown in red) or Sox2 (B,F shown in red). Differentiation of neural progenitor cells was induced by withdrawal of growth factors and resulted in cells demonstrating a neuronal morphology, expressing neuronal markers like MAP2 (C,G shown in green) and Tuj1 (D,H shown in red). Inset in C and D shows an example of a higher magnification to demonstrate the dense network of processes built up by the cells during differentiation. Patch clamp experiments were performed to analyze the maturation of the cells into functional neuronal cells. Voltage steps elicited inward and outward directed Na⁺ and K⁺ currents (I), where Na⁺ currents could be blocked by TTX (J) and possessed I/ V relationship typical for voltage gated Na⁺ channels (K). The example shows recordings of a mutNPC1 cell, differentiated for four weeks. After 7–9 weeks of differentiation, we observed spontaneous action potentials (L) as well as spontaneous postsynaptic currents (M). (N) shows a superimposed average of spontaneous postsynaptic currents with fast decay kinetics (red trace) and slow decay kinetics (black trace), indicating input of different synaptic sites. The example shows recordings of a mutNPC1 cell, differentiated for 7 weeks.