Fernandopulle Michael S, Prestil Ryan, Grunseich Christopher, Wang Chao, Gan Li, Ward Michael E
National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland.
Gladstone Institute of Neurological Disease, Gladstone Institutes, San Francisco, California.
Curr Protoc Cell Biol. 2018 Jun;79(1):e51. doi: 10.1002/cpcb.51. Epub 2018 May 18.
Accurate modeling of human neuronal cell biology has been a long-standing challenge. However, methods to differentiate human induced pluripotent stem cells (iPSCs) to neurons have recently provided experimentally tractable cell models. Numerous methods that use small molecules to direct iPSCs into neuronal lineages have arisen in recent years. Unfortunately, these methods entail numerous challenges, including poor efficiency, variable cell type heterogeneity, and lengthy, expensive differentiation procedures. We recently developed a new method to generate stable transgenic lines of human iPSCs with doxycycline-inducible transcription factors at safe-harbor loci. Using a simple two-step protocol, these lines can be inducibly differentiated into either cortical (i Neurons) or lower motor neurons (i LMN) in a rapid, efficient, and scalable manner (Wang et al., 2017). In this manuscript, we describe a set of protocols to assist investigators in the culture and genetic engineering of iPSC lines to enable transcription factor-mediated differentiation of iPSCs into i Neurons or i LMNs, and we present neuronal culture conditions for various experimental applications. © 2018 by John Wiley & Sons, Inc.
对人类神经元细胞生物学进行精确建模一直是一项长期挑战。然而，将人类诱导多能干细胞（iPSC）分化为神经元的方法最近提供了易于实验操作的细胞模型。近年来出现了许多使用小分子将iPSC引导至神经元谱系的方法。不幸的是，这些方法面临诸多挑战，包括效率低下、细胞类型异质性可变以及分化过程冗长且昂贵。我们最近开发了一种新方法，可在安全港位点生成具有强力霉素诱导型转录因子的人类iPSC稳定转基因系。使用简单的两步方案，这些细胞系能够以快速、高效且可扩展的方式被诱导分化为皮质神经元（iNeurons）或低位运动神经元（iLMN）（Wang等人，2017年）。在本手稿中，我们描述了一套方案，以协助研究人员对iPSC系进行培养和基因工程，从而实现转录因子介导的iPSC向iNeurons或iLMN的分化，并且我们展示了适用于各种实验应用的神经元培养条件。© 2018约翰威立国际出版公司
