Induction of neural progenitor cells, oligodendrocyte progenitor cells, and oligodendrocytes by stem cell differentiation using landmark transcription factors

A method for inducing differentiation of stem cell into NPCs, OPCs, oligodendrocytes sequentially or directly said method comprising:

• a) plating iPSCs and/or ESCs on a coated culturing vessel, wherein the iPSCs and/ESCs are seeded using about 70,000 cells to about 14,000,000 cells per culture flask or culture well-plate in medium comprising containing minimal media-alpha (MEMa), or Dulbecco's Modified Eagle Medium:
• Nutrient Mixture F-12 (DMEM/F12), or DMEM with B27 supplement all with knock out replacement serum (KSR);
• b) transfecting the cells of step a) with mRNA encoding cell fate factors selected from the group consisting of one or more of Ngn2, Pax6, Sox2, Brn2 and FoxO family factors as individual mRNA or combined mRNA transfection;
• c) culturing said iPSCs and/or ESCs of step b) until the cells differentiate to NPCs.
• d) isolating the NPCs from step c):
• e) plating NPCs on coated cell culture vessels, wherein the NPCs are seeded using about 70,000 cells to about 14,000,000 cells per culture flask or culture well-plate in medium comprising MEMa, or DMEM/F12, or DMEM, all with KSR and one or more of the medium supplements selected from the group consisting of ascorbic acid, SAG, PDGF, HGF, IGF1, T3, insulin, RA, B27, CAMP and biotin;
• f) transfecting the cells of step e) with one or more of the group consisting of Oligo2, NKX2.2, SOX10, and Oligo1 mRNA individually or in combination using transfection reagents;
• g) repeating the transfection of step e) at least two more times;
• h) culturing the cells of step g) until the morphology of the cells change from the morphology of an NPC cell to an OPC cell;
• i) isolating the OPCs from step h);
• j) plating the isolated OPCs from step j) onto coated cell culture vessels, wherein the OPCs are seeded using about 70,000 cells to about 14,000,000 cells per culture flask or culture well-plate in medium comprising MEMa, or DMEM/F12, or DMEM, all with KSR and one or more of the medium supplements selected from the group consisting of ascorbic acid, N2, B27, biotin, Cam, T3, and insulin;
• and
• K) culturing the cells for at least about 1 week until oligodendrocytes cells appear with tree branch-looking processes and/or produce myelin basic protein (MBP).