Figure 1

Characterization of in vitro neurogenesis of iPS cells and neural differentiation of iPS pNPCs. (A) Time scale diagram for neural differentiation of iPS cells and pNPC expansion as neurospheres. Left panel shows typical alkaline phosphatase positive iPS cell colonies (red). The rest of the panels depict overlays of phase contrast images and the GFP signal of Oct-4-driven GFP. iPS cells were positive for GFP; pNPCs, neurospheres and expanded pNPCs were negative for GFP (B) FACS quantification of the Oct-4-GFP positive cells (P1) at d 0 and d 5 of differentiation for clonal line 1 (C1) and 2 (C2). Data are represented as mean ± SD, n = 3. (C) Expression analysis of different pluripotency and neural genes by RT-PCR in iPS cells and pNPCs. Mouse embryonic stem cell line (ESC wtB1), wtB1-derived pNPCs, neural stem cells (mNSC) and mouse embryonic fibroblasts (MEF) were used as controls. (D) Percentage of cells from C1 and C2 expressing markers for neurons (MAP-2), oligodendrocytes (PDGFR) and astrocytes (GFAP) at d 10 of differentiation. Data is represented as mean ± SD, n = 3. (E) Representative images of marker expression during pNPC differentiation. At d 0 of differentiation cells expressed the neural stem cell marker nestin and the neuronal progenitor marker Pax6. After 10 d in culture pNPCs expressed the neuronal markers MAP-2 and TAU, the astrocytic marker GFAP and the oligodendrocytic marker PDGFR. Staining for the oligodendrocyte marker O4 became visible after 14 d of differentiation. After 22 d, cells expressed the presynaptic marker synapsin, the postsynaptic marker homer1 and myelin basic protein, a marker for mature oligodendrocytes. (F) Immunochemical labeling of active synapses. Fluorescent picture showing staining for synapsin (green), and synaptotagmin I (red) and the mask for analyzing the frequency of colocalized dots (yellow, arrowhead). Graph shows the quantification of the mean percentage of double stained dots. Data represent mean ± SD, n = 3. (G) Electrophysiological characterization of pNPCs at d 13/14. I/V curves of VC-stimulation in pNPC C1 neuronlike cells before and after treatment with ttx (tetrodotoxin, sodium channel blocker) or TEA (tetraethylamonium, potassium channel blocker). Stimulation potential (mV) is plotted against the maximum measured inward or outward current (current was normalized to cell size (pA/pF), data represent mean ± SD; n = 6, *p < 0.05, **p < 0.01 (paired t test for normal and Wilcoxon test for not normal distributed samples). A representative trace of membrane potential responding to step depolarization by current injection (depolarizations: black lines; hyperpolarizations: gray lines) and a representative trace of whole cell currents in voltage clamp mode responding to step depolarization by current injection. Current injections (−50 pA, +10 pA) into pNPC-derived neurons in current clamp (CC)-mode. Stimulation via stepwise increase of membrane potential (−80 mV to +55 mV, step size 15 mV) in VC-mode. Scale bars (A): first and fourth subpanels from left, 100 µm; second, third and fifth sub-panels from left, 50 µm; (E): top subpanels and second through fourth bottom subpanels, 25 µm; first and fifth bottom subpanels, 10 µm.