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Biologia dello sviluppo

人脑神经前体细胞神经发育表型的快速检测 (npc)

Published: March 02, 2018
doi:
10.3791/56628
, , , , , , , , ,
1Department of Neuroscience and Cell Biology,Rutgers Robert Wood Johnson Medical School, 2Center for Advanced Biotechnology and Medicine, Department of Neuroscience and Cell Biology,Rutgers Robert Wood Johnson Medical School, 3The Child Health Institute of NJ, Department of Obstetrics, Gynecology, and Reproductive Services,Rutgers Robert Wood Johnson Medical School, 4The Child Health Institute of NJ, Department of Neuroscience and Cell Biology,Rutgers Robert Wood Johnson Medical School, 5Department of Genetics,Rutgers University

Summary

神经发育过程, 如增殖, 迁移和突起的生长, 往往是不安的精神疾病。因此, 我们提出的协议, 快速和重现性评估这些神经发育过程中的人类 iPSC 派生的 npc。这些协议还允许评估相关生长因子和疗法对 NPC 发展的影响。

Abstract

人类大脑发育通过一系列精确编排的过程进行, 早期的阶段以增殖、迁移和突起为特色;后阶段的特征是轴突/枝晶生长和突触形成。在神经发育紊乱中, 通常有一个或多个过程被打乱, 导致大脑形成和功能异常。随着人类诱导的多潜能干细胞 (hiPSC) 技术的出现, 研究人员现在有大量的人类细胞供应, 可以被区分成几乎任何细胞类型, 包括神经元。这些细胞可以用来研究正常的大脑发育和疾病发病机制。一些使用 hiPSCs 模型神经精神疾病的协议使用晚期分化的神经元或使用3D 的文化系统称为 organoids。这些方法在研究人类疾病发病机制方面已证明是宝贵的, 但也存在一些弊端。hiPSCs 分化为神经元和 organoids 的产生是冗长而昂贵的过程, 可能会影响可以评估的实验和变量的数量。此外, 在有丝分裂后的神经元和 organoids 允许研究与疾病有关的过程, 包括枝晶生长和 synaptogenesis, 它们排除了对早期的过程, 如扩散和迁移的研究。在神经发育紊乱, 如自闭症, 丰富的遗传和验尸证据表明早期发育过程中的缺陷。神经前体细胞 (npc), 一个高度增生的细胞群体, 可能是一个合适的模型, 其中提出问题的发育过程和疾病的启动。我们现在扩展了从研究老鼠和大鼠皮质文化的发展到人类 npc 的方法学。使用 npc 可以让我们调查与疾病有关的表型, 并定义不同的变量 (例如, 生长因子, 药物) 如何影响发展过程, 包括增殖, 迁移, 并在短短几天的分化。最终, 此工具集可用于重现性和高通量的方式, 以确定神经发育紊乱疾病的特定机制和表型。

Introduction

使用简单的有机体和老鼠模型阐明了基本的大脑发展和疾病发病机制。尽管有这些进展, 许多精神疾病的病因仍然难以捉摸, 因为并非所有的研究结果都与人类疾病的复杂方面直接相关。此外, 人脑的更大复杂性往往使人的发育和动物疾病的建模变得困难。随着人类诱导多潜能干细胞 (hiPSCs) 技术的发展和发展, 体细胞可以重新编程成干细胞, 然后分化为神经细胞来研究人类疾病。hiPSCs 和 “涨价可能” 技术的进步 (基因组学、转录组学、蛋白质组学、新陈代谢) 有望彻底改变人类大脑发育的认识。这些技术现在可以在个案的基础上对神经精神疾病的特征进行 “精确医学” 处理。

目前在 hiPSC 疾病建模领域的主要内容是将细胞分化成单层的特定神经元亚型, 或者使用一个叫做 organoid 的3D 培养系统来重述大脑发育的各个方面1,2, 3。这些系统在研究和揭露人类发展和疾病的独特方面非常有价值4,5,6,7。然而, 在他们准备学习之前, 神经元文化和 organoids 通常需要从几周到几个月的文化。这些协议的耗时性和维护这些文化系统所需的资源量常常限制可以进行的实验数量和可测试的变量 (如生长因子或药物) 的数量。此外, 许多利用有丝分裂后神经元和 organoids 的研究都集中在后来的发展过程, 如枝晶突起或突触形成。虽然这些过程牵涉到发育紊乱的病理学, 如自闭症和精神分裂症, 早期的发展事件发生之前, 明确的神经元分化也重要的疾病发病机制8 ,9,10,11,12,13。事实上, 最近的基因组研究表明, 由增殖、过程和迁移组成的中胎儿期在自闭症发病机制中尤为重要11,14。因此, 研究神经干细胞和祖细胞群, 以更好地了解这些早期的过程是很重要的。Organoid 系统由于其3D 性质和组织结构而被认为可以更好地概括人脑发育, 但它包含了一个前体池, 用于研究这些早期事件。然而, organoids 的祖群往往稀疏, 更像是径向胶质细胞, 而不是神经干细胞或祖细胞5,15。因此, 有一个高吞吐量的方法来研究神经发育在积极增殖细胞的早期阶段是有益的。

在实验室中, 我们创建了一个使用 hiPSC 神经前体细胞 (npc) 的协议, 一个神经干细胞和祖细胞的混合种群, 高度增生, 以研究神经发育过程, 如增殖、细胞迁移和初始过程 (突起) 扩展。这些化验是从我们实验室使用的技术中开发的, 数十年来成功地研究了老鼠和老鼠皮层培养的神经发育 16, 17, 18, 19, 20, 21,22,23。重要的是, 它还表明, 在鼠和小鼠培养系统中定义的表型和调控信号是高度预测的机制, 是活跃的在体内, 表明这些技术的价值16, 17,18,19,24。在 hiPSCs 与 npc 的初步分化之后, 这些方法使我们能够在几天内研究重要的发育过程。这些方法有许多优点: (1) 它们需要很少的复杂设备, 易于实现, (2) 可以在短时间内进行大量的实验复制, 从而快速确认结果的重现性, (3)培养变量, 如涂层基质, 生长因子的影响, 药物的活性, 可以快速和经济高效地测试。此外, 我们利用细胞外生长因子作为不同发育过程的关键调控者的良好作用。npc 被暴露在选择发展信号, 直接刺激诸如扩散, 突起和细胞迁移等事件, 并发现它们增强了识别在控制条件下不明显的缺陷的能力19,25,26,27,28. 同样, 评估药物的容易性为采用精确的医学技术来测试各种治疗干预措施的功效提供了有力的途径。因此, 本议定书促进了高通量、重现性和直截了当的方法, 研究早期大脑发育、疾病发病机制以及生长因子和药物对神经发育表型的潜在有益影响。

Protocol

1. 安全程序和生物安全柜维护 生物安全 Level-2 (BSL-2) 安全程序 遵循该机构的指导方针, 使用 BSL-2 材料。根据机构的做法处理 BSL-2 材料。标明用于 BSL-2 材料的房间和设备。佩戴所有个人防护用品 (PPE), 包括实验室外套和手套。 安全柜维护 使用 BSL-2 级材料认证的生物安全柜。 在生物安全柜中打开 UV 光, 至少15分钟, 然后用10…

Representative Results

这些研究的目的之一是确定 npc 的增殖活动, 即细胞数量的增加。这是通过评估 DNA 合成的总细胞总数, 一个高通量的方法, 测量将放射性示踪剂氚胸苷纳入细胞提取物, 并反映所有从事 s-阶段的细胞, 无论是合成5分钟或整整两个小时。此外, 这些化验可以确定进入 S 相和总细胞数的细胞比例, 一种更劳动密集型的单细胞检测。细胞在 s-阶段合成 DNA, 这是有丝分裂和细胞分裂之…

Discussion

这里提出的协议说明了快速和简单的方法来研究基本神经发育过程和测试生长因子和药物使用 hiPSC 衍生神经前体细胞。hiPSC 技术已经革命性的研究神经发育疾病的发病机制, 为我们提供了前所未有的机会, 从受影响的人的活体神经细胞细胞。事实上, 已经有许多 hiPSC 研究的神经发育紊乱, 包括 Rett 综合征, 蒂莫西综合征, 脆性 x 综合征, 精神分裂症, 发现了疾病特异的畸变在树突, 突触和神经元函数<s…

Divulgazioni

The authors have nothing to disclose.

Acknowledgements

这项工作得到了新泽西州州长自闭症医学研究和治疗委员会 (CAUT13APS010) 的支持;CAUT14APL031;CAUT15APL041), 南茜. Mindworks 标志着家庭基金会, 慈善领导信任, 以及更大 MetroWest 新泽西州的犹太社区基金会。

Materials

PSC Neural Induction Medium:
Protocol Link: https://goo.gl/euub7a 		 ThermoFischer Scientific A1647801 This is a kit that consists of Neurobasal (NB) medium and a 50x Neural Induction Supplement (NIS). The NIS is used to make 1X Neural Induction Medium and 100% Expansion Medium
Advanced DMEM/F12 Medium ThermoFischer Scientific 12634-010 Component of 100% Expansion Medium
Neurobasal Medium ThermoFischer Scientific 21103049 Component of both NIM and 100% Expansion Medium 
hESC-qualified Matrigel Corning 354277 hESC-qualified extracellular matrix-mimic gel (ECM-mimic gel) 
Y-27632 (2HCl), 1 mg Stem Cell Technologies 72302 ROCK inhibitor
6 well plates Corning COR-3506 Polystyrene plates used for NPC maintenance and for Neurosphere Migration Assay 
24 well plates ThermoFischer Scientific 2021-05 Polystyrene plates: Used for NPC DNA Synthesis Assay
35 mm dishes ThermoFischer Scientific 2021-01 Polystyrene plates: Used for NPC S-Phase Entry and Neurite Assay
Natural Mouse Laminin Invitrogen 23017-015 Substrate for coating plates: Used for NPC DNA Synthesis, S-Phase Entry, and Cell Number Assays
Fibronectin Sigma F1141 Substrate for coating plates: Used for Neurite Assay 
Poly-D-Lysine Sigma P0899 Substrate for coating plates
Penicillin/Streptomycin ThermoFischer Scientific 15140122 Antibiotic, component of NIM, 100% Expansion and 30% Expansion Media 
StemPro Accutase Gibco A11105-01 1X Cell Detachment Solution 
2.5% Trypsin (10X) Gibco 15090-046 10X enzymatic solution
0.5 M EDTA ThermoFischer Scientific AM9261 used in trypsin solution for lifting cells for DNA synthesis assay
tritiated [3H]-thymidine PerkinElmer NET027E001 Radioactive tritium, thymidine
Fisherbrand 7 mL HDPE Scintillation Vials Fisherbrand 03-337-1 Vials for liquid scintillation counting
EcoLite(+) MP Biomedicals 0188247501  Liquid scintillation cocktail
LS 6500 multi-purpose liquid scintillation counter Beckman Coulter 8043-30-1194 Liquid Scintillation Counter
Skatron Semi-automactic Cell Harvester Type 11019 Molecular Devices & Skatron Instruments, Inc. Semi-automatic cell harvester
Click-iT EdU Alexa Fluor® 488 Imaging Kit ThermoFisher Scientific C10337 EdU and staining kit for S-Phase Entry Assay
Trypan Blue Solution, 0.4% ThermoFisher Scientific 15250061 Assessing viability of cells
Grade GF/C filter paper GE Healthcare Life Sciences, Whatman 1822-849 Glass fiber filter paper
Human Basic FGF-2 Peprotech 100-18B growth factor
Pituitary Adenylate Cyclase Activating Polypeptide (PACAP-38) BACHEM H-8430 neuropeptide
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Tags

Neural Precursor CellsNeurodevelopmental PhenotypesNeural DevelopmentCell CharacterizationCell CultureCell DetachmentCell HarvestingTritiated ThymidineCell ProliferationNeurodevelopmental DisordersNeuropsychiatric DisordersRapid And Reproducible Method

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Williams, M., Prem, S., Zhou, X., Matteson, P., Yeung, P. L., Lu, C., Pang, Z., Brzustowicz, L., Millonig, J. H., Dicicco-Bloom, E. Rapid Detection of Neurodevelopmental Phenotypes in Human Neural Precursor Cells (NPCs). J. Vis. Exp. (133), e56628, doi:10.3791/56628 (2018).
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