Fig. 4

Generation and characterization of diPSC-NPCs. a diPSCs were coaxed into NPCs (diPSC-NPCs) via EB formation in the presence of dorsomorphin (DM) and SB431542 (SB). EBs (top panel) and neural rosettes (middle and bottom panels) are shown. b The NPCs selected and expanded from neural rosettes were immunostained with neural precursor markers Sox2, Nestin, Sox1, and Pax6. Bottom panels are higher-magnification images of top panels. c RT-PCR analysis showed the expression of markers for both NPCs (Sox1 and Pax6) and neural rosettes (PLZF, DACH1, NR2F, and PLAGL1) in NPCs derived from hESCs, diPSC1, and diPSC2. d A differential interference contrast image of neurons differentiated from diPSC-NPCs. e, f TUJ1⁺ neurons at low and high magnifications, respectively. g, h MAP2⁺ neurons at low and high magnifications, respectively. (i and j) NCAM⁺ neurons at low and high magnifications, respectively. k Dense NF⁺/MAP2⁺ axon fibers. l MAP2⁺/NeuN⁺ neurons. m TUJ1⁺/TH⁺ dopaminergic neuron at 21 days of differentiation. n, o vGlut2⁺/MAP2⁺ glutamatergic neuron at 21 days of differentiation. The image in (o) was a high magnification of the white box in (n). p Some diPSC-NPCs differentiated into Olig2-positive oligodendrocytes. q Na⁺ channel-mediated current was examined to prove the functionality of the differentiated neurons. Na⁺ currents were produced in neurons. Voltage steps from −60 mV to +50 mV were applied to the patched cells, and several positive currents were found (left, n = 3 cells/total 15 cells). By applying 0.5 μM TTX, a Na⁺ channel blocker, currents were totally abolished (right). Boxes were amplified to show Na⁺ currents and their disappearance precisely. r Action potentials were also generated in differentiated neurons. Narrow spikes were recorded in the cells which showed Na⁺ currents (left). Protocol involved sequential steps in stimulation (right, top). Detected action potential spikes were also blocked by TTX, just as Na⁺ current in (q) (right, bottom). diPSC disc cell-derived induced pluripotent stem cell, EB embryoid body, hESC human embryonic stem cell, MAP2 microtubule-associated protein 2, NCAM neural cell adhesion molecule, NF neurofilament, NPC neural precursor cell, Olig2 oligodendrocyte lineage transcription factor 2, RT-PCR reverse transcription-polymerase chain reaction, Sox2 SRY (sex determining region)-box 2, TH tyrosine hydroxylase, TTX tetrodotoxin, TUJ1 beta-III tubulin, vGlut2 vesicular glutamate transporter 2.