Laboratory of Clinical and Experimental Toxicology, and Pavia Poison Centre-National Toxicology Information Centre, Toxicology Unit, Istituti Clinici Scientifici Maugeri IRCCS, Pavia, Italy.
Department of Obstetrics and Gynecology, Fondazione IRCCS Policlinico San Matteo, University of Pavia, Pavia, Italy.
Curr Protoc. 2022 Apr;2(4):e423. doi: 10.1002/cpz1.423.
Neurotoxicity (NT) testing for regulatory purposes is based on in vivo animal testing. There is general consensus, however, about the need for the development of alternative methodologies to allow researchers to more rapidly and cost effectively screen large numbers of chemicals for their potential to cause NT, or to investigate their mode of action. In vitro assays are considered an important source of information for making regulatory decisions, and human cell-based systems are recommended as one of the most relevant models in toxicity testing, to reduce uncertainty in the extrapolation of results from animal-based models. Human neuronal models range from various neuroblastoma cell lines to stem cell-derived systems, including those derived from mesenchymal stem/stromal cells (hMSC). hMSCs exhibit numerous advantages, including the fact that they can be obtained in high yield from healthy human adult tissues, can be cultured with a minimal laboratory setup and without genetic manipulations, are able of continuous and repeated self-renewal, are nontumorigenic, and can form large populations of stably differentiated cells representative of different tissues, including neuronal cells. hMSCs derived from human umbilical cord (hUC) in particular possess several prominent advantages, including a painless, non-invasive, and ethically acceptable collection procedure, simple and convenient preparation, and high proliferation capacity. In addition, hMSCs can be efficiently differentiated into neuron-like cells (hNLCs), which can then be used for the assessment of neuronal toxicity of potential neurotoxic compounds in humans. Here, we describe a step-by-step procedure to use hMSCs from the umbilical cord for in vitro neurotoxicity testing. First, we describe how to isolate, amplify, and store hMSCs derived from the umbilical cord. We then outline the steps to transdifferentiate these cells into hNLCs, and then use the hNLCs for neurotoxicity testing by employing multiple common cytotoxicity assays after treatment with test compounds. The approach follows the most updated guidance on using human cell-based systems. These protocols will allow investigators to implement an alternative system for obtaining primary NLCs of human origin, and support advancement in neurotoxicity research. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Isolation and maintenance of human mesenchymal stem/stromal cells (hMSCs) obtained from the umbilical cord lining membrane Basic Protocol 2: Transdifferentiation of hMSCs into neuron-like cells (hNLCs) and basic neurotoxicity assessment.
神经毒性 (NT) 测试是为了监管目的而进行的，基于体内动物测试。然而，人们普遍认为，需要开发替代方法，以便研究人员能够更快速、更经济有效地筛选大量化学物质，以评估它们是否具有引起 NT 的潜力，或研究它们的作用模式。体外测定被认为是做出监管决策的重要信息来源，而基于人类细胞的系统被推荐为毒性测试中最相关的模型之一，以减少从基于动物的模型推断结果的不确定性。人类神经细胞模型范围从各种神经母细胞瘤细胞系到干细胞衍生系统，包括源自间充质干细胞/基质细胞 (hMSC) 的系统。hMSCs 具有许多优点，包括可以从健康的成人组织中以高产量获得、可以在最小的实验室设置和无需遗传操作的情况下培养、能够连续和重复自我更新、非致瘤性以及能够形成大量代表不同组织的稳定分化细胞，包括神经元细胞。特别是来源于人脐带的 hMSC 具有几个突出的优点，包括无痛、非侵入性和符合伦理的采集程序、简单方便的制备方法和高增殖能力。此外，hMSCs 可以有效地分化为类神经元细胞 (hNLCs)，然后可以用于评估潜在神经毒性化合物对人类神经元的毒性。在这里，我们描述了使用脐带间充质干细胞进行体外神经毒性测试的逐步程序。首先，我们描述了如何分离、扩增和储存源自脐带的 hMSC。然后，我们概述了将这些细胞转分化为 hNLC 的步骤，然后在使用测试化合物处理后，通过采用多种常见的细胞毒性测定法来使用 hNLC 进行神经毒性测试。该方法遵循使用基于人类细胞的系统的最新指南。这些方案将允许研究人员实施一种替代系统来获得源自人类的原代 NLC，并支持神经毒性研究的进展。© 2022 作者。Wiley 期刊出版公司出版的《当代协议》。基础方案 1：从脐带衬里膜中分离和维持人间充质干细胞 (hMSC)基础方案 2：hMSC 向类神经元细胞 (hNLC)的转分化和基本神经毒性评估。
