Figure 3. Generation of spiking mESC-OPCs from nonspiking mESC-OPCs.

(A) Representative gel image showing the absence of Na_V α subunits and the presence of β1 subunit (SCN1B) in the purified D30 GFP⁺ cells. Some of the α subunits were detected in the D10 differentiated cells. In day 10 cells, Olig2 but not NG2 was detectable. In day 30 purified cells, both Olig2 and NG2 were detected. (B) Representative gel image showing that the expression of SCN2A gene transcript was detected one day after infected with baculovirus carrying SCN2A gene, and decreased over time in culture. At 8 days after viral infection, the expression was undetectable. (C) Representative images showing that the mESC-OPCs infected with the baculovirus were positive for Na_V1.2 and remained expressing the OPC marker A2B5. Scale bar: 50 µm. Blue, DAPI-stained nuclei. (D) Representative traces showing the inward TTX-sensitive Na⁺ current (indicated by arrows) and outward K⁺ current recorded from infected mESC-OPCs. The boxed traces of I_Na on the right were enlarged. (E) I-V relationship of I_Na recorded from the infected mESC-OPCs (n = 7). (F) Left, current-clamp recording showing an example of the transduced mESC-OPC with graded spikes that had greater amplitudes at stronger depolarization (inset), when the membrane potential was held at −60 mV. Right, an example of the transduced mESC-OPC firing all-or-none action potentials when the membrane potential was held at −100 mV (current injection, 300 ms, 20 pA steps from −20 to 160 pA). (G) Representative traces showing the TTX-sensitive Na⁺ current (indicated by arrows) and outward potassium current recorded from the mESC-OPCs cultured in the presence of laminin. (H) Current-clamp recording showing the slight spikes recorded from the mESC-OPCs cultured in the presence of laminin, when the membrane potential was held at −60 mV (current injection, 300 ms, 20 pA steps from −20 to 80 pA). Abbreviations: mESCs, mouse embryonic stem cells; OPCs, oligodendrocyte progenitor cells; Na_V, voltage-gated sodium channel; DAPI, 4´, 6-diamindino-2-phenylindole; TTX, tetrodotoxin.