小鼠星形胶质细胞的分离和直接神经元重编程
Summary
在这里，我们描述了一个详细的方案，以产生来自产后小鼠中枢神经系统不同区域的星形胶质细胞的高度富集培养物，并通过强迫转录因子将其转化为功能性神经元。
Abstract
直接神经元重编程是一种强大的方法，可以从不同的起始细胞群中产生功能性神经元，而无需通过多能中间体。这项技术不仅在疾病建模领域有很大的希望，因为它允许将例如患有神经退行性疾病的患者的成纤维细胞转化为神经元，而且还代表了基于细胞的替代疗法的有希望的替代方案。在这种情况下，一个重大的科学突破是证明中枢神经系统内分化的非神经细胞，如星形胶质细胞，可以在 体外转化为功能性神经元。从那时起， 在体外 直接将星形胶质细胞重编程为神经元提供了对强制身份转换背后的分子机制以及阻止有效重编程的障碍的实质性见解。然而，由于用于分离，培养和重编程星形胶质细胞的方法不同，因此很难比较在不同实验室进行的 体外实验的结果。在这里，我们描述了一个详细的方案，通过磁细胞分选 从 出生后小鼠中枢神经系统的不同区域可靠地分离和培养具有高纯度的星形胶质细胞。此外，我们还提供 通过 病毒转导或DNA转染将培养的星形胶质细胞重新编程为神经元的方案。这种简化和标准化的方案可用于研究细胞身份维持的分子机制，新神经元身份的建立，以及特定神经元亚型及其功能特性的产生。
Introduction
哺乳动物中枢神经系统（CNS）高度复杂，由数百种不同的细胞类型组成，包括大量不同的神经元亚型1，2，3，4，5，6。与其他器官或组织7，8，9不同，哺乳动物CNS的再生能力非常有限;创伤性脑损伤或神经变性后的神经元丢失是不可逆的，通常会导致运动和认知缺陷10.为了挽救大脑功能，替换丢失神经元的不同策略正在进行密集研究11.其中，将体细胞直接重编程为功能性神经元正在成为一种有前途的治疗方法12。直接重编程或转分化是将一种分化的细胞类型转化为新身份而不通过中间增殖或多能状态13，14，15，16的过程。该方法通过鉴定MyoD1作为足以将成纤维细胞转化为肌肉细胞17，18的因子而开创，该方法已成功应用于将几种细胞类型重新编程为功能神经元19，20，21。
星形胶质细胞是CNS22，23中最丰富的大胶质细胞，由于多种原因，它是一种特别有希望的直接神经元重编程的细胞类型。首先，它们广泛而均匀地分布在CNS中，为新神经元提供了丰富的 定位 源。其次，星形胶质细胞和神经元在发育上密切相关，因为它们在胚胎发育过程中共享一个共同的祖先，即桡骨神经胶质细胞24。与来自不同胚层的细胞重编程相比，两种细胞类型的共同胚胎起源似乎有助于神经元转换19，21。此外，星形胶质细胞通过其桡神经胶质细胞起源遗传的模式信息也保留在成年星形胶质细胞25，26，27中，并且似乎有助于产生区域上合适的神经元亚型28，29，30。因此，研究和了解星形胶质细胞转化为神经元是实现该技术在基于细胞的替换策略中的全部潜力的重要组成部分。
体外培养的星形胶质细胞转化为神经元在直接神经元重编程领域取得了几项突破，包括：i）鉴定足以从星形胶质细胞中产生神经元的转录因子15，19，31，ii）在同一细胞背景下由不同重编程因子触发的分子机制的解开32，iii）强调星形胶质细胞的发育起源对诱导不同神经元亚型的影响28，29，33。此外，星形胶质细胞的体外直接转化解开了限制神经元直接重编程34，35的几个主要障碍，例如活性氧（ROS）产生增加34以及星形胶质细胞的线粒体蛋白质组和神经元35之间的差异。因此，这些观察结果强烈支持使用星形胶质细胞的原代培养物作为直接神经元重编程的模型，以研究生物学12中的几个基本问题，这些问题与细胞身份维持，防止细胞命运变化的障碍以及代谢在重编程中的作用有关。
在这里，我们提出了一个详细的方案，以非常高的纯度从出生后（P）年龄的小鼠中分离星形胶质细胞，如从小鼠脊髓29中分离星形胶质细胞所证明的那样。我们还提供 通过 病毒转导或DNA质粒转染将星形胶质细胞重新编程为神经元的方案。重编程的细胞可以在转导后7天（7 DPT）进行分析，以评估各个方面，例如重编程效率和神经元形态，或者可以在培养物中维持数周，以评估其随时间推移的成熟度。重要的是，该方案不是脊髓星形胶质细胞特异性的，可以很容易地应用于从其他各种大脑区域分离星形胶质细胞，包括皮质灰质，中脑和小脑。
Protocol
以下程序遵循慕尼黑亥姆霍兹中心关于保护用于科学目的的动物的指令2010/63 / EU的动物护理指南。请确保遵守进行解剖的机构的动物护理指南。 1. 解剖、解离和培养材料的准备 注意：在生物安全柜内准备所有培养试剂，并仅使用高压灭菌或无菌设备工作。解剖和解离试剂可以在生物安全柜外制备。 通过在H 2 O中用聚-D-赖氨酸（储备1mg…
Representative Results
星形胶质细胞的原代培养通常在MAC分选和接种后7至10天内达到80%-90%的汇合度（图1B）。通常，单个T25培养瓶产生约1-1.5×106个细胞，当以每孔5-5.5×10个细胞的密度接种细胞时，这足以容纳20-30个盖玻片。接种后的第二天，细胞通常覆盖盖玻片表面的50%-60%（图1C）。在这个阶段，培养几乎完全由星形胶质细胞组成，而其他细胞类型，如神经…
Discussion
小鼠星形胶质细胞的原代培养是研究直接神经元重编程的卓越体外模型系统。事实上，尽管在出生后阶段分离出，但细胞表达典型的星形胶质细胞标志物29，保留模式基因的表达28，29，并保持增殖能力，类似于38岁时的体内星形胶质细胞。在MACS介导的分离后，细胞首先粘附在烧瓶上，然后开始增殖，产?…
Declarações
The authors have nothing to disclose.
Acknowledgements
我们要感谢伊内斯·米尔哈恩克隆了用于重编程的构建体，感谢宝琳娜·克莱比克克隆病毒，感谢马格达莱娜·格茨和朱迪思·费舍尔-斯特恩贾克对手稿的评论。
Materials
| 0.05% Trypsin/EDTA | Life Technologies | 25300054 | |
| 4', 6-Diamidino-2-phenyindole, dilactate (DAPI) | Sigma-Aldrich | D9564 | |
| anti-mouse IgG1 Alexa 647 | Thermo Fisher | A21240 | |
| anti-Mouse IgG1 Biotin | Southernbiotech | Cat# 1070-08; RRID: AB_2794413 | |
| anti-mouse IgG2b Alexa 488 | Thermo Fisher | A21121 | |
| anti-rabbit Alexa 546 | Thermo Fisher | A11010 | |
| Aqua Poly/Mount | Polysciences | Cat# 18606-20 | |
| B27 Supplement | Life Technologies | 17504044 | |
| BDNF | Peprotech | 450-02 | |
| bFGF | Life Technologies | 13256029 | |
| Bovine Serum Albumine (BSA) | Sigma-Aldrich | Cat# A9418 | |
| cAMP | Sigma Aldrich | D0260 | |
| C-Tubes | Miltenyi Biotec | 130-093-237 | |
| DMEM/F12 | Life Technologies | 21331020 | |
| Dorsomorphin | Sigma Aldrich | P5499 | |
| EGF | Life Technologies | PHG0311 | |
| Fetal Bovine Serum | PAN Biotech | P30-3302 | |
| Forskolin | Sigma Aldrich | F6886 | |
| GDNF | Peprotech | 450-10 | |
| gentleMACS Octo Dissociator | Miltenyi Biotec | 130-096-427 | |
| GFAP | Dako | Cat# Z0334; RRID: AB_100013482 | |
| Glucose | Sigma Aldrich | G8769 | |
| GlutaMax | Life Technologies | 35050038 | |
| HBSS | Life Technologies | 14025050 | |
| Hepes | Life Technologies | 15630056 | |
| Lipofectamine 2000 (Transfection reagent) | Thermo Fisher | Cat# 11668019 | |
| MACS SmartStrainer 70µm | Miltenyi Biotec | 130-098-462 | |
| MiniMACS Seperator | Miltenyi Biotec | 130-042-102 | |
| Mouse anti-ACSA-2 MicroBeat Kit | Miltenyi Biotec | 130-097-678 | |
| Mouse IgG1 anti-Synaptophysin 1 | Synaptic Systems | Cat# 101 011 RRID:AB_887824) | |
| Mouse IgG2b anti-Tuj-1 (βIII-tub) | Sigma Aldrich | T8660 | |
| MS columns | Miltenyi Biotec | 130-042-201 | |
| N2 Supplement | Life Technologies | 17502048 | |
| Neural Tissue Dissociation Kit | Miltenyi Biotec | 130-092-628 | |
| NT3 | Peprotech | 450-03 | |
| octoMACS Separator | Miltenyi Biotec | 130-042-109 | |
| OptiMEM – GlutaMAX (serum-reduced medium) | Thermo Fisher | Cat# 51985-026 | |
| Penicillin/Streptomycin | Life Technologies | 15140122 | |
| Poly-D-Lysine | Sigma Aldrich | P1149 | |
| Rabbit anti-RFP | Rockland | Cat# 600-401-379; RRID:AB_2209751 | |
| Rabbit anti-Sox9 | Sigma-Aldrich | Cat# AB5535; RRID:AB_2239761 | |
| Streptavidin Alexa 405 | Thermo Fisher | Cat# S32351 | |
| Triton X-100 | Sigma-Aldrich | Cat# T9284 |
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Citar este artigo
Hersbach, B. A., Simon, T., Masserdotti, G. Isolation and Direct Neuronal Reprogramming of Mouse Astrocytes. J. Vis. Exp. (185), e64175, doi:10.3791/64175 (2022).