Oligodendrocyte generation from mouse stem cells:
Mouse D3 ESCs were grown on MEF plates by feeding with mouse ESC Medium supplemented with 1,000 U/ml mouse Leukemia inhibitory factor (LIF; Millipore) every day. Mouse ESC Medium consisted of, KnockOut DMEM (Life Technologies), 15% KnockOut serum replacement (Life Technologies), GlutaMAX (Life Technologies), 1x non-essential amino acids (Gibco), and 55 µM 2-mercaptoethanol (Gibco).
NPCs were generated and differentiated as described below that was modified from a previous study (Marchetto et al., 2008). ESCs were grown in suspension in mouse ESC Medium without LIF for the first day and in N2/B27 Medium supplemented with 500 ng/ml Noggin (PeproTech) for four more days. N2/B27 Medium: DMEM/F12-Glutamax Medium (Gibco), 1x N2 supplement (Gibco), 1x B27 supplement (Gibco). Next, embryoid bodies were dissociated, plated on and maintained on laminin- (Life Technologies) coated dishes in N2/B27 Medium supplemented with 20 ng/ml Epidermal growth factor (EGF;PeproTech), 20 ng/ml Fibroblast growth factor-basic (bFGF; Stemgent).
On D1, OPC differentiation started by switching NPCs into Differentiation Medium: N2/B27 Medium, 10 ng/ml Insulin-like growth factor 1 (IGF-1; R&D Systems), 10 ng/ml Platelet-derived grow factor-α (PDGF-α; R&D Systems). On D8, OPCs were switched to Maturation Medium: N2/B27 Medium, 10 ng/ml Ciliary neurotrophic factor (CNTF; Sigma-Aldrich), 5 ng/ml Neurotrophin-3 (NT3; Sigma-Aldrich) + 40 ng/ml Triiodothyronine (T3; Sigma-Aldrich). From D1, cells were fed every other day.
Please cite: Kerman, BE., Kim HJ., Padmanabhan, K., Mei, A., Georges, S., Joens, MS., Fitzpatrick, JAJ., Japelli, R., Chandross, K., August, P., and Gage, FH. (2015) In vitro myelin formation using embryonic stem cells. Development 142(12):2213-25.