Romito Elena, Battistella Ingrid, Plakhova Vera, Paplekaj Arteda, Forastieri Chiara, Toffolo Emanuela, Musio Carlo, Conti Luciano, Battaglioli Elena, Rusconi Francesco
Department of Medical Biotechnology and Translational Medicine, Università degli Studi di Milano, Via Fratelli Cervi, 93, Segrate, Milan 20054, Italy.
Department of Cellular, Computational and Integrative Biology - CIBIO, Università degli Studi di Trento, Via Sommarive, 9, Trento 38123, Italy.
J Neurosci Methods. 2024 Oct;410:110225. doi: 10.1016/j.jneumeth.2024.110225. Epub 2024 Jul 23.
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.
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.
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.
Our protocol provides clear instructions for generating reliable human neuronal cultures with defined electrophysiological properties to investigate neuronal differentiation and model diseases in vitro.
对神经元的研究是解开神经系统复杂性的基础。长期以来，来自啮齿动物的原代神经元培养一直是实验研究的基石，但与它们的非人类性质和伦理问题相关的局限性促使了替代方法的发展。近年来，从人诱导多能干细胞（hiPSC）中衍生神经元已成为一种强大的选择，为各种应用提供了可扩展的细胞来源。源自hiPSC的神经祖细胞（NPC）可以有效地分化为功能性神经元，提供了一个在体外研究人类神经生理学和病理学的平台。然而，在不同实验环境中实现一致且可重复的结果仍然存在挑战。
我们的目标是提供一个逐步的方法学方案，通过额外的说明和参数来补充现有程序，以指导研究人员获得可重复的结果。
我们概述了将hiPSC衍生的NPC分化为具有电活性神经元的程序，包括初始细胞密度、形态、维持和分化。我们还描述了用于评估神经元表型的特定标志物的分析，以及用于评估神经元兴奋性生物物理特性的电生理分析。此外，我们对三种不同的化学方法——氯化钾（KCl）、N-甲基-D-天冬氨酸（NMDA）和荷包牡丹碱——进行了比较分析，以诱导神经元去极化，并评估它们对诱导快速和慢速翻译后、转录和转录后反应的影响。
我们的方案为生成具有明确电生理特性的可靠人类神经元培养物提供了清晰的说明，以在体外研究神经元分化和模拟疾病。
