Co-culture of PSC-derived OPCs with rat primary Dorsal Root Ganglion (DRG) neurons in vitro

The care and experimental procedures of all animals were in accordance with a protocol approved by the Japanese National Research Institute for Child Health and Development Animal Care Committee [Permit Number: 2002–005, 2002–006 and 2005–005]. Pregnant rats were sacrificed with overdose of sodium pentobarbital (100 mg/kg body weight). DRG neurons were isolated from E15 SD rat spinal cord regions as previously described [16] and then dissociated and plated onto collagen type I-coated coverslips (IWAKI). Non-neuronal cells were eliminated by cycling them three times with medium containing 5-fluorodeoxyuridine and uridine. Myelinating co-cultures were established by seeding approximately 2 × 10⁵ differentiated monkey ESCs or human iPSCs onto purified DRG neurons as previously described [17]. The cryopreserved monkey ESCs or human iPSCs-derived OPCs that were stored for 131 or 60 days were used. Co-cultures were maintained and medium was replaced every 3 days.

Co-cultures were fixed first with 4% paraformaldehyde and then with 100% cold methanol [18]. The fixed cells were permeabilized with PBS containing 0.1% Tween-20 and blocked using the Blocking One kit (Nacalai Tesque). The cells were incubated first with primary antibodies and then with appropriate Alexa Fluor-conjugated secondary antibodies. The coverslips or dishes were mounted with Vectashield reagent containing 4′,6-diamidino-2-phenylindole (DAPI; Vector Laboratories, Burlingame, CA). Primary antibodies were as follows: mouse monoclonal anti-MBP (1:100; BioLegend, SMI-94R) and rabbit polyclonal anti-neurofilament 200 (1:100; Sigma-Aldrich, N4142) antibody. The secondary antibodies were as follows: Alexa Fluor 488 goat anti-rabbit IgG (H+L), and Alexa Fluor 594 donkey anti-mouse IgG (H+L). The fluorescence images were captured with a fluorescence microscopy system (DMI4000B; Leica, DeltaVision Elite Imaging System; GE Healthcare) and analyzed with AF6000 software (Leica).