Primary Oligodendrocyte Precursor Cell (OPC) Cultures.

OPC cultures were developed from the cortices of neonatal Sprague-Dawley rat pups (P0–2; Harlan), as previously described [15]. Cortices were denuded of meninges and hippocampi and were then mechanically minced and manually homogenized with a flame polished Pasteur pipet into a single cell suspension in culture medium (DMEM/F12, Gibco) containing 10% fetal bovine serum (FBS; Denville Scientific), and 1% penicillin/streptomycin (Fisher Scientific). The cell suspension was spun for 10 min (1800 rpm, RT) and the pellet was resuspended in culture media for plating into flasks coated with poly-L-lysine (PLL 0.3 mg/mL; Sigma Aldrich). Confluent cell monolayers were then shaken for 1 hour (55 rpm, 37˚C), media exchanged with fresh culture media to remove any cellular debris and microglia and then returned to the incubator for 2–4 hours (37°C). Cultures were then shaken overnight (255 rpm) to lift OPCs. Supernatant from shaken flasks was spun down (1800 rpm, 10 min), the pellet resuspended in media, and then incubated (37°C), for 3–4 hrs to limit astrocyte contamination. Media was collected, spun down (1800 rpm, 10 min), resuspended in fresh OPC media, and cells plated onto poly-ornithine coated coverslips. For poly-ornithine coating: glass coverslips were washed in sterile 1N HCl (30 min, RT), washed with sterile water (3 times) and then coated with poly-L-ornithine (0.05 mg/mL; Sigma) for 3 hrs (37°C). Once plated onto coverglass, media was exchanged for differentiation media for up to 4 days and then OL maturation was assessed. Differentiation media: Neurobasal media (Gibco) with 2% B27 serum-free supplement (Gibco), 2mM Lglutamine (Gibco), and 10 ng/mL T3 (Sigma). For experimental treatments, OPCs were treated with specific peptides or inhibitors daily for 4 consecutive days (see below) and their effect on OL differentiation assessed by immunocytochemistry using markers to identify each stage of OL differentiation.