Name: isolation and direct neuronal reprogramming of mouse astrocytes
Uploaded: 2022-07-07T15:00:00
Description: Watch this Scientific Journal Video about Isolation and Direct Neuronal Reprogramming of Mouse Astrocytes at JoVE.com

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。

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。
<ol><li>通过在H 2 O中用聚-D-赖氨酸（储备1mg...

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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?...

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。

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	<tbody>
		<tr>
			<td>0.05% Trypsin/EDTA</td>
			<td>Life Technologies</td>
			<td>25300054</td>
			<td></td>
		</tr>
		<tr>
			<td>4&#39;, 6-Diamidino-2-phenyindole, dilactate (DAPI)</td>
			<td>Sigma-Aldrich</td>
			<td>D9564</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-mouse IgG1 Alexa 647</td>
			<td>Thermo Fisher</td>
			<td>A21240</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-Mouse IgG1 Biotin</td>
			<td>Southernbiotech</td>
			<td>Cat# 1070-08; RRID: AB_2794413</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-mouse IgG2b Alexa 488</td>
			<td>Thermo Fisher</td>
			<td>A21121</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-rabbit Alexa 546</td>
			<td>Thermo Fisher</td>
			<td>A11010</td>
			<td></td>
		</tr>
		<tr>
			<td>Aqua Poly/Mount</td>
			<td>Polysciences</td>
			<td>Cat# 18606-20</td>
			<td></td>
		</tr>
		<tr>
			<td>B27 Supplement</td>
			<td>Life Technologies</td>
			<td>17504044</td>
			<td></td>
		</tr>
		<tr>
			<td>BDNF</td>
			<td>Peprotech</td>
			<td>450-02</td>
			<td></td>
		</tr>
		<tr>
			<td>bFGF</td>
			<td>Life Technologies</td>
			<td>13256029</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumine (BSA)</td>
			<td>Sigma-Aldrich</td>
			<td>Cat# A9418</td>
			<td></td>
		</tr>
		<tr>
			<td>cAMP</td>
			<td>Sigma Aldrich</td>
			<td>D0260</td>
			<td></td>
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		<tr>
			<td>C-Tubes</td>
			<td>Miltenyi Biotec</td>
			<td>130-093-237</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F12</td>
			<td>Life Technologies</td>
			<td>21331020</td>
			<td></td>
		</tr>
		<tr>
			<td>Dorsomorphin</td>
			<td>Sigma Aldrich</td>
			<td>P5499</td>
			<td></td>
		</tr>
		<tr>
			<td>EGF</td>
			<td>Life Technologies</td>
			<td>PHG0311</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>PAN Biotech</td>
			<td>P30-3302</td>
			<td></td>
		</tr>
		<tr>
			<td>Forskolin</td>
			<td>Sigma Aldrich</td>
			<td>F6886</td>
			<td></td>
		</tr>
		<tr>
			<td>GDNF</td>
			<td>Peprotech</td>
			<td>450-10</td>
			<td></td>
		</tr>
		<tr>
			<td>gentleMACS Octo Dissociator</td>
			<td>Miltenyi Biotec</td>
			<td>130-096-427</td>
			<td></td>
		</tr>
		<tr>
			<td>GFAP</td>
			<td>Dako</td>
			<td>Cat# Z0334; RRID: AB_100013482</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma Aldrich</td>
			<td>G8769</td>
			<td></td>
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		<tr>
			<td>GlutaMax</td>
			<td>Life Technologies</td>
			<td>35050038</td>
			<td></td>
		</tr>
		<tr>
			<td>HBSS</td>
			<td>Life Technologies</td>
			<td>14025050</td>
			<td></td>
		</tr>
		<tr>
			<td>Hepes</td>
			<td>Life Technologies</td>
			<td>15630056</td>
			<td></td>
		</tr>
		<tr>
			<td>Lipofectamine 2000 (Transfection reagent)</td>
			<td>Thermo Fisher</td>
			<td>Cat# 11668019</td>
			<td></td>
		</tr>
		<tr>
			<td>MACS SmartStrainer 70&#181;m</td>
			<td>Miltenyi Biotec</td>
			<td>130-098-462</td>
			<td></td>
		</tr>
		<tr>
			<td>MiniMACS Seperator</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-102</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse anti-ACSA-2 MicroBeat Kit&#160;</td>
			<td>Miltenyi Biotec</td>
			<td>130-097-678</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse IgG1 anti-Synaptophysin 1</td>
			<td>Synaptic Systems</td>
			<td>Cat# 101 011 RRID:AB_887824)</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse IgG2b anti-Tuj-1 (&#946;III-tub)</td>
			<td>Sigma Aldrich</td>
			<td>T8660</td>
			<td></td>
		</tr>
		<tr>
			<td>MS columns</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-201</td>
			<td></td>
		</tr>
		<tr>
			<td>N2 Supplement</td>
			<td>Life Technologies</td>
			<td>17502048</td>
			<td></td>
		</tr>
		<tr>
			<td>Neural Tissue Dissociation Kit&#160;</td>
			<td>Miltenyi Biotec</td>
			<td>130-092-628</td>
			<td></td>
		</tr>
		<tr>
			<td>NT3</td>
			<td>Peprotech</td>
			<td>450-03</td>
			<td></td>
		</tr>
		<tr>
			<td>octoMACS Separator</td>
			<td>Miltenyi Biotec</td>
			<td>130-042-109</td>
			<td></td>
		</tr>
		<tr>
			<td>OptiMEM &#8211; GlutaMAX (serum-reduced medium)</td>
			<td>Thermo Fisher</td>
			<td>Cat# 51985-026</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Life Technologies</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-D-Lysine</td>
			<td>Sigma Aldrich</td>
			<td>P1149</td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit anti-RFP</td>
			<td>Rockland</td>
			<td>Cat# 600-401-379; RRID:AB_2209751</td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit anti-Sox9</td>
			<td>Sigma-Aldrich</td>
			<td>Cat# AB5535; RRID:AB_2239761</td>
			<td></td>
		</tr>
		<tr>
			<td>Streptavidin Alexa 405</td>
			<td>Thermo Fisher</td>
			<td>Cat# S32351</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma-Aldrich</td>
			<td>Cat# T9284</td>
			<td></td>
		</tr>
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isolation and direct neuronal reprogramming of mouse astrocytes

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。

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...

Watch this Scientific Journal Video about Isolation and Direct Neuronal Reprogramming of Mouse Astrocytes at JoVE.com

Isolation and Direct Neuronal Reprogramming of Mouse Astrocytes

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。

小鼠星形胶质细胞的分离和直接神经元重编程

Direct neuronal reprogramming is a powerful approach to generate functional neurons from different starter cell populations without passing through ...

星形胶质细胞是一种有趣的自体细胞群，可靶向直接神经元编程。该协议提供了一种可靠的技术来分离来自不同区域或中枢神经系统的高纯度培养星形胶质细胞。该协议的设计和优化旨在研究星形胶质细胞重新编程为功能性神经元的能力，而不会混淆因素，例如不同启动群体的星形胶质细胞纯度差异。演示该程序将由实验室的博士后Bob Hersbach和我们实验室的技术援助Tatiana Simon完成。首先，将安乐死小鼠的躯干放入35毫米培养皿中并将其放在冰上。用剪刀打开皮肤，用小剪刀去除椎骨。然后提取脊髓并将其置于冰上的解剖缓冲液中。在具有两倍放大倍数的立体定向解剖显微镜下，使用镊子从分离的脊髓中取出脑膜，并将解剖和清洁的脊髓组织转移到C管中。从神经组织解离试剂盒中加入50微升酶P和30微升先前制备的酶混合物到每个C管中。倒置C管并将它们放在加热的解离物上，确保所有组织都收集在管的盖子中。通过从分离器中取出色谱柱，加入800微升星形胶质细胞培养基，并用随附的柱塞将细胞推出色谱柱来躲避细胞。通过加入4.2毫升星形胶质细胞培养基，每毫升补充10纳克EGF和bFGF，将细胞平板在先前制备的培养瓶中。没有必要在培养瓶中涂覆从其他大脑区域分离的星形胶质细胞，但它为从脊髓分离的星形胶质细胞提供了更好的底物。在37摄氏度和5%二氧化碳下培养细胞。在安全等级为一级的生物安全柜下进行星形胶质细胞座位，并按照文本手稿中所述使用聚-D-赖氨酸涂层玻璃盖玻片准备 24 孔板。对于电镀，从P2小鼠中分离出六条脊髓，产生约100万个细胞。从含有培养的星形胶质细胞的T-12.5培养瓶中吸出培养基，并用1x PBS洗涤一次。通过加入0.5毫升0.05%胰蛋白酶EDTA从培养瓶中分离星形胶质细胞，并在37摄氏度下孵育5分钟。轻轻敲击烧瓶的侧面以从培养瓶表面释放细胞，并在明场显微镜下使用10倍放大镜检查脱落。用2.5毫升星形胶质细胞培养基停止胰蛋白酶化，并在15毫升管中收集细胞悬液。在 300 RCF 下在 4 摄氏度下离心五分钟。吸出上清液并在一毫升星形胶质细胞培养基中重新发送细胞。使用血细胞计数器或自动细胞计数系统计算细胞浓度。根据细胞数量，用补充有EGF和bFGF的新鲜星形胶质细胞培养基以每因子10纳克/毫升稀释细胞悬液。在37摄氏度和5%二氧化碳下向先前制备的24孔板和培养细胞的每个孔中加入500微升细胞悬液。通过将一毫升B27补充剂添加到49毫升碱性培养基中来制备神经元分化培养基。24至48小时后，根据细胞是否已转导或转染，每孔用1毫升神经元分化培养基替换星形胶质细胞培养基。用9%二氧化碳在37摄氏度下培养细胞。为了提高重编程效率，当用分化培养基替换星形胶质细胞培养基时，用佛司可林和背吗啡补充神经元分化培养基，最终浓度分别为30微摩尔和1微摩尔。当选择用佛司可林和背吗啡治疗细胞时，在初始治疗后两天提供第二剂背吗啡。将第二种处理直接添加到培养基中。星形胶质细胞的原代培养物在磁辅助细胞分选和铺板后7至10天内达到80%至90%汇合度。电镀后的第二天，细胞通常覆盖50%至60%的盖玻片表面。在这个阶段，培养物主要由星形胶质细胞组成，而其他细胞类型如神经原始细胞几乎不存在。在转导或转染后7天，诱导的神经元细胞与星形胶质细胞明显可区分。神经元细胞体小于未重编程的星形胶质细胞具有较长的过程，神经元标志物，β三微管蛋白呈阳性，星形胶质细胞标志物GFAP呈阴性。在转导或转染后21天，许多诱导的神经元能够发射动作电位，并且对成熟的神经元标志物NeuN和泛突触蛋白Synaptophysin呈阳性。正确剖析感兴趣的区域至关重要。避免周围组织的污染。必须注意从孤立的脊髓中去除脑膜和背根神经节。该方法可用于从中枢神经系统的各个区域分离星形胶质细胞，包括大脑皮层、背侧中脑和小脑。在未来，这项技术将使我们能够扩展我们对区域星形胶质细胞及其重新编程为功能性神经元的潜力的了解。

Research

JoVE Journal

Neuroscience

Isolation and Culture of Mouse Cortical Astrocytes

小鼠脑皮层星形胶质细胞的分离和培养

Astrocytes  are an abundant cell type in the mammalian brain, yet much remains to be  learned about their molecular and functional characteristics. In ...

科研

神经科学

Sex Differences in Mouse Hippocampal Astrocytes after In-Vitro Ischemia

Sex Differences in Mouse Hippocampal Astrocytes after In-Vitro Ischemia

性别差异在小鼠海马星形胶质细胞<em&gt;在体外</em&gt;缺血

Astrogliosis following hypoxia/ischemia (HI)-related brain injury plays a role in increased morbidity and mortality in neonates. Recent clinical studies ...

Modeling Neuronal Death and Degeneration in Mouse Primary Cerebellar Granule Neurons

小鼠小脑颗粒神经元神经元死亡和变性的模型研究

Cerebellar granule neurons (CGNs) are a commonly used neuronal model, forming an abundant homogeneous population in the cerebellum. In light of their ...

Chronic Transcranial Electrical Stimulation and Intracortical Recording in Rats

大鼠慢性经颅电刺激及 Intracortical 记录

Transcranial electrical stimulation (TES) is a powerful and relatively simple approach to diffusely influence brain activity either randomly or in a ...

Analyzing the Size, Shape, and Directionality of Networks of Coupled Astrocytes

耦合星形胶质细胞网络的大小、形状和方向性分析

It has become increasingly clear that astrocytes modulate neuronal function not only at the synaptic and single-cell levels, but also at the network ...

In Vivo Direct Reprogramming of Resident Glial Cells into Interneurons by Intracerebral Injection of Viral Vectors

在Vivo直接重新编程居民胶质细胞到内神经通过病毒载体的脑内注射

Converting resident glia in the brain into functional and subtype-specific neurons in vivo provides a step forward towards the development of alternative ...

Visualizing and Analyzing Intracellular Transport of Organelles and Other Cargos in Astrocytes

可视化和分析星形细胞中细胞内器官和其他货物的细胞内传输

Astrocytes are among the most abundant cell types in the adult brain, where they play key roles in a multiplicity of functions. As a central player in ...

In Utero Electroporation of Multiaddressable Genome-Integrating Color (MAGIC) Markers to Individualize Cortical Mouse Astrocytes

In Utero Electroporation of Multiaddressable Genome-Integrating Color MAGIC Markers to Individualize Cortical Mouse Astrocytes

在多地址基因组集成颜色 （MAGIC） 标记的 Utero 电冲中， 以个性化皮质小鼠星形细胞

Protoplasmic astrocytes (PrA) located in the mouse cerebral cortex are tightly juxtaposed, forming an apparently continuous three-dimensional matrix at ...

Neuronal Differentiation from Mouse Embryonic Stem Cells In vitro

小鼠胚胎干细胞体外神经元分化

The neural differentiation of mouse embryonic stem cells (mESCs) is a potential tool for elucidating the key mechanisms involved in neurogenesis and ...

In vitro Modeling for Neurological Diseases using Direct Conversion from Fibroblasts to Neuronal Progenitor Cells and Differentiation into Astrocytes

使用从纤维细胞直接转换为神经元祖先细胞和分化为星形细胞的神经系统疾病体外建模

Research on neurological disorders focuses primarily on the impact of neurons on disease mechanisms. Limited availability of animal models severely ...