Co-Culturing DRG Neurons and IFRS1 Cells

Co-culturing of DRG neurons and IFRS1 cells was conducted as previously described [22 (link)], with slight modifications (Figure 5a). Briefly, DRG neurons were seeded on type I collagen-coated chamber slides (Matsunami Glass Ind., LTD, Osaka, Japan) and Aclar fluorocarbon coverslips (Nissin EM Co., Tokyo, Japan) at an approximate density of 2 × 10³/cm², and were maintained for 7 days in DMEM/F12 with N2 supplement (Thermo Fisher), 10 ng/mL NGF (R&D Systems, Inc., Minneapolis, MN, USA), 10 ng/mL glial cell line-derived neurotrophic factor (R&D Systems), and 10 ng/mL ciliary neurotrophic factor (Peprotech, Rocky Hill, NJ, USA). After confirming the neurite elongation from the neuronal cell bodies under a phase-contrast microscope, IFRS1 cells are added to the neurons at an approximate density of 2 × 10⁴/cm²; the co-cultured cells were incubated for 2 days in DMEM/F12 containing 5% FBS, and subsequently maintained for 14 days in DMEM/F12/B27 with 50 μg/mL ascorbic acid (Wako) to induce myelination. The cells were then incubated for 2 days in the serum-free medium in the presence or absence of 100 μM zonisamide and for 7 days in the presence of 10 μM OHP.