Production and isolation of NG2+ oligodendrocyte precursors from human embryonic stem cells in defined serum-free medium

Human embryonic stem cells (hESCs) are a promising source of oligodendrocyte precursor cells (OPCs) and oligodendrocytes. These cells can be used to repair myelin in central nervous system deficits such as multiple sclerosis or traumas such as spinal cord injury. Here, we introduce a novel differentiation method for the production of OPCs from hESCs. OPCs were differentiated as spheres in defined serum-free medium supplemented with recombinant human growth factors. A broad gene expression analysis revealed that this OPC population expressed Olig1/2, Sox10, PDGFR, Nkx2.2, Nkx6.2, oligodendrocyte-myelin glycoprotein, myelin basic protein (MBP), and proteolipid protein (PLP). According to quantitative RT-PCR analyses addition of ciliary neurotrophic factor (CNTF) upregulated the Olig2 mRNA levels in the OPC population. According to the flow cytometry analyses the OPC population was > 90% NG2-positive, > 80% PDGFR-positive, and > 60% CD44-positive, and further matured into O4- (45%) and GalC- (80%) positive oligodendrocyte populations when cultured on top of human extracellular matrix proteins, which were used instead of Matrigel. In addition, OPCs matured into myelin-forming cells when cocultured with neuronal cells. The multilayered myelin sheet formation around axons was detected with transmission electron microscopy in cocultures. Further, the OPC populations could be purified with sorting of NG2+ cells. These NG2+ cells reformed spheres that remained stable during prolonged culturing (7 weeks), and matured into GalC-positive oligodendrocytes. Importantly, these NG2+ spheres were free of pluripotent Tra1-81, Oct-4, and CD326-positive hESCs. Thus, this method is suitable for the efficient production of OPCs and in the future for therapeutic graft production.