Department of Anatomy and Neurology, School of Medicine and Public Health, Waisman Center, WiCell Institute, University of Wisconsin-Madison, Madison, Wisconsin, USA.
Nat Protoc. 2009;4(11):1614-22. doi: 10.1038/nprot.2009.186. Epub 2009 Oct 15.
We have developed a four-part protocol to differentiate human embryonic stem cells (hESCs) to oligodendrocyte progenitor cells (OPCs) according to developmental principles. In the first 2 weeks, hESCs are induced to differentiate into neuroepithelial cells, which form neural tube-like rosettes. In the following 10 d, these neuroepithelial cells are specified to OLIG2-expressing progenitors in the presence of retinoic acid (RA) and sonic hedgehog (SHH). Upon treatment with fibroblast growth factor 2 (FGF2) for another 10 d, these progenitors convert to OLIG2 and NKX2.2-expressing pre-OPCs. Finally, the pre-OPCs take 8-9 weeks to differentiate into OPCs, which express additional markers of oligodendrocytes, such as SOX10, platelet-derived growth factor receptor alpha (PDGFRalpha) and NG2. The unique aspects of the protocol are the use of FGF2 to promote the differentiation of gliogenic pre-OPCs in the third part and the removal of FGF2 during the transition of pre-OPCs to OPCs. This 3-month differentiation protocol consistently yields OPCs of high purity capable of producing myelin sheaths in vivo.
我们开发了一个四步方案，根据发育原则将人类胚胎干细胞（hESC）分化为少突胶质前体细胞（OPC）。在前 2 周，hESC 被诱导分化为神经上皮细胞，形成神经管样玫瑰花结。在接下来的 10 天中，在视黄酸（RA）和 sonic hedgehog（SHH）存在的情况下，这些神经上皮细胞被特化为 OLIG2 表达的祖细胞。在用成纤维细胞生长因子 2（FGF2）处理 10 天后，这些祖细胞转化为 OLIG2 和 NKX2.2 表达的前 OPC。最后，前 OPC 需要 8-9 周分化为 OPC，其表达少突胶质细胞的其他标志物，如 SOX10、血小板衍生生长因子受体 alpha（PDGFRalpha）和 NG2。该方案的独特之处在于在第三部分中使用 FGF2 促进神经发生前 OPC 的分化，以及在过渡到 OPC 期间去除 FGF2。这个 3 个月的分化方案始终产生高纯度的 OPC，能够在体内产生髓鞘。
