Differentiation of G-Olig2 embryonic stem cells (ESCs) into oligodendrocytes. (A) Differentiation procedures and cell morphology of each step: (upper left frame) G-Olig2 ESC colony, (upper right) embryoid bodies (EBs), (lower left) neural lineage cells (arrow), and (lower right) oligodendrocyte-like cells (arrow). (B) RT-PCR results. Nestin, MAP2, and β-tublin III are markers for neurons. GFAP for astrocytes. NG2, Olig1, Olig2, MAG, myelin basic protein (MBP), and PLP are oligodendrocyte markers. Minus sign (−) indicates spontaneously differentiated cells; plus sign (+) indicates induced cells. Step 1 (EBs), step 2 (NPCs), step 3 (OPCs), and step 4 (oligodendrocytes) are shown. In lane 1, nestin and β-tublin III are expressed in undifferentiated G-Olig2. In lanes 2 and 3 (EBs), nestin and β-tublin III are expressed. In lanes 4 and 5 (NPCs), NG2, MAP2, Olig1, Olig2, and MBP transcription factors are induced. In lanes 6 and 7 (OPCs), nestin, β-tublin III, NG2, MAP2, Olig1, Olig2, and MBP are expressed. In lanes 8 and 9 (oligodendrocyte), oligodendrocyte-specific genes MAG, MBP, and PLP are expressed at the terminal stage. Nestin, β-tublin III, NG2, MAP2, Olig1, Olig2, and GFAP genes are also expressed. (C) eGFP expression-positive cells in each step was detected by fluorescence microscopy, and the percentage was analyzed through fluorescence-activated cell sorting. Step 1 (EBs): only sporadic eGFP-positive cells in the EBs. Step 2 (NPCs): 52.59% ± 6.58% of NPCs was eGFP-positive. Step3 (OPCs) and Step4 (oligodendrocytes): 76% ± 5.76% of OPCs and 78.4% ± 5.95% of oligodendrocytes were eGFP-positive. (D) Immunostaining of terminal differentiated cells: (a) (NeuN)-positive cells, (b) β-tublin III-positive cells, (c) GFAP-positive cells, (d) A2B5-positive cells, (e) NG2-positive cells, (f) O4-positive cells, (g) GALC-positive cells, and (h) the percentage of positive cells. The secondary antibodies are labeled with rhodamine (red). nuclei were stained with DAPI (blue).