从多能干细胞生成人类神经元和少突胶质细胞，用于模拟神经元-少突胶质细胞相互作用
Summary
由于工具和方法不足，神经退行性变中的神经元-神经胶质相互作用尚未得到很好的理解。在这里，我们描述了从人类多能干细胞中获得诱导神经元，少突胶质细胞前体细胞和少突胶质细胞的优化方案，并提供这些方法在理解阿尔茨海默病细胞类型特异性贡献方面的价值示例。
Abstract
在阿尔茨海默病（AD）和其他神经退行性疾病中，少突胶质细胞衰竭是一种常见的早期病理特征，但它如何促进疾病的发展和进展，特别是在大脑的灰质中，仍然在很大程度上是未知的。少突胶质细胞谱系细胞的功能障碍以髓鞘形成缺陷和少突胶质细胞前体细胞（OPCs）自我更新受损为特征。这两种缺陷至少部分是由神经元和少突胶质细胞之间沿着病理学积累的相互作用的破坏引起的。OPC在中枢神经系统发育过程中产生髓鞘少突胶质细胞。在成熟的大脑皮层中，OPC是主要的增殖细胞（占总脑细胞的~5%），并以神经活动依赖性的方式控制新的髓磷脂形成。由于缺乏适当的工具，这种神经元到少突胶质细胞的通讯研究明显不足，特别是在AD等神经退行性疾病的背景下。近年来，我们的小组和其他人在改进目前可用的方案方面取得了重大进展，以从人类多能干细胞中单独生成功能性神经元和少突胶质细胞。在这篇手稿中，我们描述了我们的优化程序，包括建立一个共培养系统来模拟神经元 – 少突胶质细胞连接。我们的说明性结果表明，OPCs/少突胶质细胞对脑淀粉样变性和突触完整性做出了意想不到的贡献，并强调了该方法在AD研究中的实用性。这种还原论方法是从大脑内部固有的复杂性中剖析特定异细胞相互作用的有力工具。我们在这里描述的方案有望促进未来对神经变性发病机制中少突胶质细胞缺陷的研究。
Introduction
少突胶质细胞谱系细胞（包括少突胶质细胞前体细胞 （OPC）、髓鞘少突胶质细胞和介于两者之间的过渡型细胞）构成了人类脑细胞的主要群体1，它们积极参与许多关键功能，在整个神经发育和衰老过程中正常运作和维护我们的中枢神经系统2，3，4.虽然少突胶质细胞以产生髓磷脂以促进神经元活动传递并支持白质中的轴突健康而闻名，但OPC在髓鞘形成稀缺的灰质中含量丰富（~5%），并执行活动依赖性信号传导功能以控制学习行为和记忆形成5，6，7，8.少突胶质细胞在阿尔茨海默病（AD）和其他年龄相关神经退行性疾病的发病机制中的功能和功能障碍的研究不足9。适当的模型系统的不足和指导实验路径前进的一般知识的不足是造成这一差距的主要原因。
鉴于从多能干细胞（包括胚胎干（ES）和诱导多能干细胞（iPS）细胞）中提取人脑细胞的最新突破，这种细胞模型与现代基因编辑工具相结合已成为处理大脑中细胞相互作用的复杂联系的强大工具，并且能够证明人类特有的疾病表现10，11.考虑到单个脑细胞类型在面对相同的AD促进条件时可以表现出不同甚至相互矛盾的效果12，13，这种干细胞方法独特地提供了以前使用已建立的体内或体外模型错过的细胞类型特异性信息，这些模型仅提供来自脑细胞类型集合的汇总读数。在过去的十年中，已经开发了大量可靠的协议，以通过ES / iPS细胞的转分化或从其他终末分化细胞类型（例如成纤维细胞）的直接转化中产生人类神经元14，15。特别是，将关键的神经源性转录因子（例如，神经原蛋白2，Ngn2）16应用于人多能干细胞，可以为纯培养物产生表征良好的神经元细胞类型的均质群体，而无需与神经胶质细胞共培养12，17，18。对于诱导的人少突胶质细胞，有一些已发布的方案可以产生与其主要对应物高度相似的功能细胞，具有广泛的效率和时间和资源需求19，20，21，22，23，24，25，26，27，28.迄今为止，这些方案均未应用于研究少突胶质细胞如何响应和影响AD发病机制。
在这里，我们描述了我们改进的人类诱导神经元（iNs）和OPC /少突胶质细胞（iOPC / iOLs）的单一和混合培养方案。这里描述的iN协议基于广泛使用的Ngn2方法16，并且具有无胶质细胞的附加特征。所得iN是同质的，与皮质层2/3兴奋性神经元高度相似，具有特征性的锥体形态、基因表达模式和电生理特征17，18（图1）。为了克服多能干细胞定向分化的一些基本障碍，我们开发了一种简单有效的低剂量二甲基亚砜（DMSO）预处理方法29，30，并报告了人ES / iPS细胞转分化为iOPC和iOLs31的增强倾向，基于Douvaras和Fossati32广泛适应的方案.我们进一步简化了方案，并掺入了一种强大的分化促进化合物克立姆定7，33，34，以加速少突胶质细胞成熟过程。结果（图2），iOPCs可以在2周内产生（标记物O4的~95%阳性），iOLs可以在四周内产生（表达成熟的标志物MBP和PLP1）。有趣的是，我们发现单独iOPCs分泌大量淀粉样蛋白-β（Aβ），与独立的转录组学数据一致，数据显示淀粉样蛋白前体蛋白（APP）和加工蛋白酶β分泌酶（BACE1）在少突胶质细胞谱系细胞中的丰富表达35，36。此外，我们的iN-iOPC共培养系统通过MBP阳性iOL过程促进轴突的鞘化，并为突触形成提供重要支持（图3）。因此，我们在下面详细介绍的方案与先前编目的神经元-少突胶质共培养方法相比具有技术和生物学优势，并有望更好地模拟AD中的神经变性。
Protocol
Representative Results
Discussion
除了通过髓鞘形成稳定突触结构和促进盐信号传导的物理和代谢支持外，少突胶质细胞谱系细胞还可以通过与神经元的快速和动态串扰来塑造神经元活动模式5，6，7。虽然在AD病理学中，少突胶质反应最初被认为仅继发于炎症和氧化应激，但现在有有希望的证据表明，髓鞘完整性受损是Aβ聚集和tau过度磷酸化出现<…
Divulgations
The authors have nothing to disclose.
Acknowledgements
这项工作得到了美国国立卫生研究院（R00 AG054616 至 YAH，T32 GM136566 至 K.C.）、斯坦福大学医学院和 Siebel 奖学金（授予 SC）的资助。YAH是布朗转化科学研究所转化神经科学中心的GFL转化教授。
Materials
| Accutase | STEMCELL Technologies | 7920 | |
| B27 supplement | ThermoFisher | 17504044 | |
| bFGF | ThermoFisher | PHG 0266 | |
| cAMP | MilliporeSigma | A9501 | |
| Clemastine | MilliporeSigma | SML0445 | |
| DMEM/F12 medium | STEMCELL Technologies | 36254 | |
| DMSO | ThermoFisher | D12345 | |
| Doxycycline | MilliporeSigma | D3072 | |
| Fetal Bovine Serum | ScienCell | 10 | |
| H1 human ES cells | WiCell | WA01 | |
| Matrigel | Corning | 354234 | |
| mTeSR plus | STEMCELL Technologies | 5825 | |
| N2 supplement | ThermoFisher | 17502001 | |
| Neurobasal A medium | ThermoFisher | 10888-022 | |
| Non Essential Amino Acids | ThermoFisher | 11140-050 | |
| PDGF-AA | R&D Systems | 221-AA-010 | |
| PEI | VWR | 71002-812 | |
| pMDLg/pRRE | Addgene | 12251 | |
| Polybrene | MilliporeSigma | TR-1003-G | |
| pRSV-REV | Addgene | 12253 | |
| Puromycin | ThermoFisher | A1113803 | |
| ROCK Inhibitor Y-27632 | STEMCELL Technologies | 72302 | |
| SAG | Tocris | 4366 | |
| STEMdiff Neural Progenitor Freezing Media | STEMCELL Technologies | 5838 | |
| STEMdiff SMADi Neural Induction Kit | STEMCELL Technologies | 8581 | |
| T3 triiodothyronine | MilliporeSigma | T6397 | |
| Tempo-iOlogo: Human iPSC-derived OPCs | Tempo BioScience | SKU102 | |
| TetO-Ng2-Puro | Addgene | 52047 | |
| VSV-G | Addgene | 12259 |
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