Purified OPCs were placed on coverslips coated with poly-l -lysine and laminin (20,000 cells/coverslip), and FGF2 (20 ng/mL; R&D Systems, Minneapolis, MN, USA) or total protein from CT CHO cells or A1-expressing CHO cells (0.1 mg/mL, see above) were added in differentiation medium (BME:F12 media (Gibco | Thermo Fisher Scientific, Waltham, MA USA) [1:1] supplemented with 100 μg/mL transferrin, 20 μg/mL putrescine, 12.8 ng/mL progesterone, 10.4 ng/mL selenium, 25 μg/mL insulin, 0.8 μg/mL thyroxine, 0.6% glucose, and 6.6 mM glutamine, as reported previously [37 (link),41 (link),44 (link)]. AraC (5 µM; Sigma-Aldrich, San Luis, AZ, USA) was added to the cultures to inhibit proliferation and after 5–7 days in vitro (DIV), the cells were fixed for immunocytochemical detection of CNPase (1:200; Covance, Princeton, NJ, USA) and Olig-2 (1:200; Millipore, Burlington, NJ, USA). Finally, 10 random fields per coverslip were photographed under a Leica microscope using a20X objective and the proportion of CNPase+ cells was assessed relative to the respective controls (±s.e.m.).
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