神经石生长分析与神经毒性评估与人类神经原生细胞衍生神经元
1Department of Molecular Genetics, Faculty of Biological Sciences,Tarbiat Modares University, 2Center for Therapeutic Innovation and Department of Psychiatry & Behavioral Sciences,University of Miami Miller School of Medicine, 3Department of Biochemistry and Molecular Biology,University of Miami Miller School of Medicine, 4Persian BayanGene Research and Training Center
Summary
提出的方案描述了一种小分子化合物的神经质生长测定和神经毒性评估方法。
Abstract
神经土虫生长测定和神经毒性评估是可以使用本文所述方法进行的两项主要研究。该协议提供可靠的神经元形态分析，以及神经石长度和突触蛋白定位和在小分子化合物处理时对富足的修改进行定量测量。除了在神经性生长研究中应用介绍的方法外，还可以进行神经毒性评估，根据其潜在的发育神经毒性效应评估、区分和排名商业化合物。
尽管细胞系现在广泛用于神经科学的复合筛选分析，但它们在基因和表型上往往与组织起源不同。另一方面，原细胞维持体内观察到的重要标记和功能。因此，由于这些细胞的转化潜力和生理相关性，可以提供神经素生长测定和神经毒性评估，可以大大受益于使用人类神经祖细胞（hNPCs）作为主要的人类细胞模型。
本文所介绍的方法可以利用人类神经祖细胞衍生神经元（一种密切代表人类生物学的细胞模型）来筛选化合物诱导神经性生长和神经毒性的能力。
Introduction
神经素生长是一个基础性神经网络和神经再生1，2,的过程。受伤后，神经衰弱在神经系统的再生中起着关键作用。神经外生长也是诱导神经元再生活动细胞外信号的一个重要元素，可增强神经退行性疾病和神经元损伤的结果3，4，5，6。4,5,63
通过保持其分化潜力，在产生各种神经血统，人类神经祖细胞（hNPCs）可以提供一个模型系统的研究中枢神经系统（CNS）的功能和发展7,7，8，9。,9hNPCs作为初级人体细胞模型的高转化潜力和生理相关性，在神经素生长相关的药物发现筛查中提供了相当大的优势。然而，对高通量测定的原细胞模型的维护和缩放可能非常耗时和劳动密集型 10、11、12、13。10,11,12,13
除了在神经素生长研究中应用介绍的方法外，神经毒性评估是使用 hNPC 衍生神经元的另一种应用。有成千上万的商业化合物，要么没有检查，要么神经毒性潜力不灵。因此，更可靠和有效的筛选实验，以评估，区分和排名化合物的基础上，其潜力，以引起发育神经毒性是高需求14。神经紊乱的流行率和发病率的增加，以及环境中大量未经测试的化合物，需要开发更可信和更有效的实验，以识别可能构成神经毒性15的危险环境化合物。
本文所介绍的方法可以利用人类神经祖细胞衍生神经元（一种密切代表人类生物学的细胞模型）来筛选化合物诱导神经素生长和神经毒性的能力。
Protocol
伦理声明：通过国家卫生研究院（NIH）支持的组织分配计划，从西雅图华盛顿大学出生缺陷研究实验室接收胎儿标本。出生缺陷研究实验室获得父母的适当书面知情同意，组织采购由华盛顿大学机构审查委员会监测。所有工作均经迈阿密大学人类学科研究办公室批准。 1. 人类神经祖细胞的分离与培养 将脑组织放在 100 mm 培养皿中，然后用钳子小心地?…
Representative Results
手稿中提出的协议已成功地用于最近发表的两篇论文22、23。,23图3演示了利用NPPC衍生神经元来研究HDAC抑制剂作为表观遗传化合物对中性粒细胞延伸的影响，作为小分子化合物的神经质生成和随后的神经原能的标记。 此外，在图4中，还通过同时区分384孔板中的hNPC来评估测试化合物的神经…
Discussion
该协议是少数发表论文之一，描述使用试验化合物治疗时神经石长度的测试。此外，我们描述了如何使用hNPCs进行神经性生长测定和神经毒性评估。通过利用这种神经性生长测定和神经毒性评估对NPPC衍生神经元，一类表观遗传小分子化合物，HDAC抑制剂的神经原能，在诱导神经素叶生长中表现出22。此外，在Sartor等人提出的另一篇论文中，该协议用于在用几种表观遗传修饰剂化合?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项研究由NIMAD研究补助金（940714）资助给MAF。
Materials
| 4-well Glass Chamber Slides | Sigma | PEZGS0816 | |
| Alexa Fluor 488 | Invitrogen | A-11001 | |
| Alexa Fluor 594 | Invitrogen | R37117 | |
| Antibiotic-Antimycotic | Gibco | 15240062 | |
| Anti-β-Tubulin III | Thermo | MA1-118X | |
| B27 | Thermo | 17504001 | |
| B27 – minus vitamin A | Thermo | 12587010 | |
| BDNF | PeproTech | 450-02 | |
| BSA | Sigma | A8531 | |
| CellTiter-Glo | Promega | G7572 | |
| CoolCell | Corning | 432000 | Cell freezing containers ensuring standardized controlled-rate -1℃/minute cell freezing in a -80℃ freezer |
| CryoStor CS10 | StemCell Technologies | 7930 | Cryopreservation medium containing 10% DMSO |
| DAPI | Thermo | D1306 | |
| DMEM/F12 | Gibco | 11320033 | |
| DMSO | Sigma | 34869-100ML | |
| EGF | Gibco | PHG0311 | |
| FGF | Gibco | PHG6015 | |
| Formaldehyde | Thermo | FB002 | |
| GDNF | PeproTech | 450-10 | |
| Glutamax | Gibco | 35050061 | L-alanyl-L-glutamine supplement |
| Goat Serum | Thermo | 50062Z | |
| Heparin | Calbiochem | 375095 | |
| Laminin | Sigma | L2020-1MG | |
| L-Ascorbic Acid | Sigma | A92902-25G | |
| L-lysine | Sigma | L5501 | |
| MEM non-essential amino acids | Gibco | 11140050 | |
| mFreSR | StemCell Technologies | 5854 | Serum-free cryopreservation medium designed for the cryopreservation of human embryonic and induced pluripotent stem cells |
| N2 | Gibco | 17502048 | |
| NaCl | Sigma | 71376 | |
| Neurobasal Medium | Gibco | 21103049 | |
| Nunc 384-Well Polystyrene White Microplates | Thermo | 164610 | |
| PBS | Thermo | 10010-049 | |
| Poly‐L‐lysine | Sigma | P5899-5MG | |
| ProLong Gold Antifade Mountant | Thermo | P10144 | |
| Retinoic Acid | Sigma | R2625 | |
| Sodium Azide | Sigma | S2002 | |
| StemPro Accutase | Gibco | A1110501 | Cell dissociation reagent containing proteolytic and collagenolytic enzymes |
| Synaptophysin | Thermo | MA5-14532 | |
| Tris Base | Sigma | 10708976001 | |
| Triton X-100 | Sigma | X100-100ML |
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Cite This Article
Bagheri, A., Razavipour, S. F., Wahlestedt, C., Mowla, S. J., Faghihi, M. A. A Neurite Outgrowth Assay and Neurotoxicity Assessment with Human Neural Progenitor Cell-Derived Neurons. J. Vis. Exp. (162), e60955, doi:10.3791/60955 (2020).