Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan, Israel.
J Neurosci Methods. 2013 Mar 30;214(1):9-14. doi: 10.1016/j.jneumeth.2012.12.026. Epub 2013 Jan 8.
Human embryonic stem cells (hESC) are potentially an unlimited source of neurons for study and therapy for human disease. Directed differentiation of hESC has been performed using many different methods, often via neural precursor intermediates generated from aggregates of hESC. We describe here a protocol based on commercially available reusable silicone micromolds and two small molecule growth factor inhibitors to simply and reproducibly generate human neurons from hESC. Hundreds of neurospheres were generated with a single pipettation of hESC into agarose multiwell plates made with the micromolds. This was followed by suspension culture with two medium changes, and plating of clumps cut from the neurospheres on laminin-coated coverslips. After two weeks of terminal differentiation, 90%+ of cells expressed neuronal proteins, and many of the neurons expressed markers of peripheral sensory neurons. The neurons made with this method underwent productive infection with the human-specific pathogenic virus varicella zoster, demonstrating the utility of the neurons for addressing clinically relevant research questions. This simple method should allow laboratories experienced in growing human pluripotent cells to easily generate neurons for studies of nerve cell biology and pathology.
人类胚胎干细胞（hESC）是研究和治疗人类疾病的神经元的潜在无限来源。已经使用许多不同的方法来定向分化 hESC，通常是通过从 hESC 聚集物中产生的神经前体细胞中间产物。我们在这里描述了一种基于商业上可重复使用的可重复使用的硅胶微模具和两种小分子生长因子抑制剂的方案，可简单且可重复地从 hESC 中生成人类神经元。通过将 hESC 单次吸入琼脂糖多微孔板中，可生成数百个神经球。然后，通过两次培养基更换进行悬浮培养，并将从神经球中切下的团块铺在层粘连蛋白包被的载玻片上。经过两周的终末分化，90%+的细胞表达神经元蛋白，并且许多神经元表达周围感觉神经元的标志物。用这种方法产生的神经元可被人类特异性致病病毒水痘带状疱疹有效地感染，证明了这些神经元用于解决临床相关研究问题的实用性。这种简单的方法应该允许有经验的培养人类多能细胞的实验室能够轻松地生成神经元，用于研究神经细胞生物学和病理学。
