Sundberg Maria, Skottman Heli, Suuronen Riitta, Narkilahti Susanna
Regea-Institute for Regenerative Medicine, University of Tampere and Tampere University Hospital, Tampere, Finland.
Stem Cell Res. 2010 Sep;5(2):91-103. doi: 10.1016/j.scr.2010.04.005. Epub 2010 Jun 9.
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 (7weeks), 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.
人类胚胎干细胞（hESCs）是少突胶质前体细胞（OPCs）和少突胶质细胞的一个有前景的来源。这些细胞可用于修复中枢神经系统缺陷（如多发性硬化症）或创伤（如脊髓损伤）中的髓鞘。在此，我们介绍一种从hESCs生产OPCs的新型分化方法。OPCs在添加重组人类生长因子的特定无血清培养基中分化为球体。广泛的基因表达分析表明，该OPC群体表达Olig1/2、Sox10、血小板衍生生长因子受体（PDGFR）、Nkx2.2、Nkx6.2、少突胶质细胞髓磷脂糖蛋白、髓磷脂碱性蛋白（MBP）和蛋白脂质蛋白（PLP）。根据定量逆转录聚合酶链反应（RT-PCR）分析，添加睫状神经营养因子（CNTF）可上调OPC群体中Olig2 mRNA水平。根据流式细胞术分析，OPC群体中>90%为神经胶质抗原2（NG2）阳性、>80%为PDGFR阳性、>60%为CD44阳性，当在人细胞外基质蛋白上培养时，可进一步成熟为O4阳性（45%）和半乳糖脑苷脂（GalC）阳性（80%）的少突胶质细胞群体，这里用人细胞外基质蛋白代替基质胶。此外，OPCs与神经元细胞共培养时可成熟为形成髓鞘的细胞。在共培养物中通过透射电子显微镜检测到轴突周围多层髓鞘片的形成。此外，OPC群体可通过分选NG2(+)细胞进行纯化。这些NG2(+)细胞重新形成球体，在长时间培养（7周）期间保持稳定，并成熟为GalC阳性的少突胶质细胞。重要的是，这些NG2(+)球体不含多能性的Tra1-81、八聚体结合转录因子4（Oct-4）和上皮细胞黏附分子（CD326）阳性的hESCs。因此，该方法适用于高效生产OPCs，未来也适用于治疗性移植物的生产。
