Hu Bao-Yang, Zhang Su-Chun
Department of Anatomy, Waisman Center, School of Medicine and Public Health, the WiCell Institute, University of Wisconsin-Madison, Madison, WI, USA.
Methods Mol Biol. 2010;636:123-37. doi: 10.1007/978-1-60761-691-7_8.
We describe a chemically defined protocol for efficient differentiation of human embryonic stem cells (hESCs) to neural epithelial cells and then to functional spinal motor neurons. This protocol comprises four major steps. Human ESCs are differentiated without morphogens into neuroepithelial cells that form neural tube-like rosettes in the first 2 weeks. The neuroepithelial cells are then specified to OLIG2-expressing motoneuron progenitors in the presence of retinoic acid (RA) and sonic hedgehog (SHH) in the following 2 weeks. These OLIG2 progenitors generate postmitotic, HB9 expressing motoneurons at the fifth week and mature to functional motor neurons thereafter. The protein factor SHH can be replaced by a small molecule purmorphamine in the entire process, which may facilitate potential clinical applications. This protocol has been shown equally effective in generating motor neurons from human induced pluropotent stem (iPS) cells.
我们描述了一种化学定义的方案，可将人类胚胎干细胞（hESCs）高效分化为神经上皮细胞，进而分化为功能性脊髓运动神经元。该方案包括四个主要步骤。在无形态发生素的情况下，人类胚胎干细胞在前两周分化为形成神经管样玫瑰花结的神经上皮细胞。在接下来的两周里，神经上皮细胞在视黄酸（RA）和音猬因子（SHH）的作用下分化为表达OLIG2的运动神经元祖细胞。这些OLIG2祖细胞在第五周产生有丝分裂后的、表达HB9的运动神经元，并在此后成熟为功能性运动神经元。在整个过程中，蛋白质因子SHH可用小分子嘌呤吗啡替代，这可能有助于潜在的临床应用。该方案已被证明在从人类诱导多能干细胞（iPS细胞）生成运动神经元方面同样有效。
