背景:对神经元的研究是揭示神经系统复杂性的基础。来自啮齿动物的原代神经元培养物长期以来一直是实验研究的基石,然而,与非人性和伦理问题相关的限制促使了替代方案的发展。近年来,从人类诱导的多能干细胞(hiPSCs)中衍生神经元已成为一种强大的选择,为各种应用提供可扩展的细胞来源。来自hiPSCs的神经祖细胞(NPCs)可以有效地分化为功能性神经元,提供了体外研究人类神经生理和病理的平台。然而,在整个实验环境中,在实现一致和可重复的结果方面仍然存在挑战.
方法:我们的目标是提供一个循序渐进的方法协议,用额外的指令和参数扩充现有的程序,指导研究人员取得可重复的结果。
结果:我们概述了hiPSC衍生的NPCs分化为有电能力的神经元的程序,包括初始细胞密度,形态学,维护,和差异化。我们还描述了用于评估神经元表型的特定标记的分析,与电生理分析一起评估神经元兴奋性的生物物理特性。此外,我们对三种不同的化学方法进行了比较分析——氯化钾,N-甲基-D-天冬氨酸(NMDA),和双核蛋白-诱导神经元去极化,并评估它们对快速和慢速翻译后诱导的影响,转录,和转录后反应。
结论:我们的方案为产生具有确定的电生理特性的可靠的人类神经元培养物提供了明确的指导,以研究体外神经元分化和模型疾病。
BACKGROUND: The study of neurons is fundamental to unraveling the complexities of the nervous system. Primary neuronal cultures from rodents have long been a cornerstone of experimental studies, yet limitations related to their non-human nature and ethical concerns have prompted the development of alternatives. In recent years, the derivation of neurons from human-induced pluripotent stem cells (hiPSCs) has emerged as a powerful option, offering a scalable source of cells for diverse applications. Neural progenitor cells (NPCs) derived from hiPSCs can be efficiently differentiated into functional neurons, providing a platform to study human neural physiology and pathology in vitro. However, challenges persist in achieving consistent and reproducible outcomes across experimental settings.
METHODS: Our aim is to provide a step-by-step methodological protocol, augmenting existing procedures with additional instructions and parameters, to guide researchers in achieving reproducible results.
RESULTS: We outline procedures for the differentiation of hiPSC-derived NPCs into electrically competent neurons, encompassing initial cell density, morphology, maintenance, and differentiation. We also describe the analysis of specific markers for assessing neuronal phenotype, along with electrophysiological analysis to evaluate biophysical properties of neuronal excitability. Additionally, we conduct a comparative analysis of three different chemical methods-KCl, N-methyl-D-aspartate (NMDA), and bicuculline-to induce neuronal depolarization and assess their effects on the induction of both fast and slow post-translational, transcriptional, and post-transcriptional responses.
CONCLUSIONS: Our protocol provides clear instructions for generating reliable human neuronal cultures with defined electrophysiological properties to investigate neuronal differentiation and model diseases in vitro.