Name: analyzing mitochondrial transport and morphology in human induced pluripotent stem cell-derived neurons in hereditary spastic paraplegia
Uploaded: 2020-02-09T13:00:00
Description: Watch this Scientific Journal Video about Analyzing Mitochondrial Transport and Morphology in Human Induced Pluripotent Stem Cell-Derived Neurons in Hereditary Spastic Paraplegia at JoVE.com

这项工作得到了痉挛性截瘫基金会、布雷泽基金会和NIH（R21NS109837）的支持。

1. 从iPSC生成端脑谷氨酸神经元
注：用于维护iPSC及其分化为端脑谷氨酸神经元的详细协议与前面描述的18相似。在这里，介绍并强调了人类多能干细胞分化过程中的关键过程。
<ol><li>人类胚胎干细胞（hESC）培养基细胞（MEF）的小鼠胚胎成纤维细胞（MEF）分供器的培养iPSC，辅以成纤维细胞生长因子（bFGF，4纳克/mL）。</li>
	<li>用消炎（1mg/mL）孵育3分钟后，将i...

<ol>
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本文介绍了一种利用红色荧光染料和ImageJ软件分析神经元斧子线粒体迁移和形态的方法，为神经退行性疾病的斧子退化和线粒体形态学研究提供了独特的平台。该协议中有几个关键步骤，包括线粒体的染色、活细胞成像和分析图像。在这种方法中，使用荧光染料染色线粒体。由于人类iPSC衍生的神经元很容易从培养皿中分离，因此在培养皿中保留一些溶液并轻轻添加神经基质介质非常重要。洗涤可...

线粒体运动和分布在满足极化神经元的可变和专门能量需求方面发挥着至关重要的作用。神经元可以延长极长的斧子，通过形成突触与目标连接，而突触对Ca2+缓冲和电电流需要高水平的能量。线粒体从索马到斧子的传输对于支持神经元的斧突和突触功能至关重要。空间和时空动态线粒体运动是通过快速的辅助体传输以每秒几微米的速度进行。
具体来说，运动或适配器蛋白，如激酶和dynein，参与沿着微管的快速细胞器传输，以控制线粒体2，3的运动。正常的神经元活动需要将新组装的线粒体从神经元索马正确传输到远端斧子（异端等向等同体传输），并将线粒体从远端斧子反向运输回细胞体（逆向传输）。最近的研究表明，不正确的线粒体分配与神经元缺陷和运动神经元退行性疾病4，5密切相关。因此，要剖析线粒体在神经退化中的作用，必须建立研究活文化中沿斧突运动的线粒体运动的方法。
在检查和分析线粒体的跟踪时，有两个主要挑战：（1） 从每个帧的背景识别线粒体，（2） 分析和生成每个帧之间的连接。在解决第一个挑战时，广泛使用荧光标记方法区分线粒体与背景，如MitoTracker染料或荧光融合线粒体靶向蛋白（例如mito-GFP）6、7、8的转染。为了分析帧之间的关联，在前面的研究9中描述了几种算法和软件工具。在最近的一篇论文中，研究人员比较了四种不同的自动化工具（例如，沃洛蒂、伊马里斯、wrMTrck和差异跟踪器），以量化线粒体传输。结果表明，尽管轨道长度、线粒体位移、运动持续时间和速度存在差异，但这些自动化工具适用于治疗后的运输差异。除了这些工具，一个集成插件"宏"为ImageJ（由Rietdorf和Seitz编写）已广泛用于分析线粒体传输11。此方法生成可用于分析线粒体运动（包括逆向和逆行方向的速度）的图形。
线粒体是高度动态的细胞器，在数量和形态上不断变化，以响应生理和病理条件。线粒体裂变和融合严格调节线粒体形态和平衡。线粒体裂变和融合之间的不平衡可诱发极短或长线粒体网络，从而损害线粒体功能，导致神经元异常活动和神经退化。受损的线粒体传输和形态涉及各种神经退行性疾病，如阿尔茨海默病、帕金森病、亨廷顿舞蹈症和遗传性痉挛性截瘫（HSP）12、13、14、15。HSP是一个异质的遗传性神经系统疾病组，其特征是皮质脊柱退化，随后未能控制下肢肌肉16、17。在这项研究中，iPSC衍生前脑神经元用于评估HSP中的线粒体迁移和形态。该方法为检查活培养体中神经元斧子的线粒体动力学提供了独特的范例学范式。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Accutase Cell Detachment Solution</td>
			<td>Innovative Cell Technologies</td>
			<td>AT104</td>
			<td></td>
		</tr>
		<tr>
			<td>Biosafety hood</td>
			<td>Thermo Scientific</td>
			<td>1300 SERIES A2</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin (BSA)</td>
			<td>Sigma</td>
			<td>A-7906</td>
			<td></td>
		</tr>
		<tr>
			<td>Brain derived neurotrophic factor (BDNF)</td>
			<td>Peprotech</td>
			<td>450-02</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Thermo Scientific</td>
			<td>Sorvall Legend X1R/ 75004261</td>
			<td></td>
		</tr>
		<tr>
			<td>Coverslips</td>
			<td>Chemiglass Life Sciences</td>
			<td>1760-012</td>
			<td></td>
		</tr>
		<tr>
			<td>Cyclic AMP (cAMP)</td>
			<td>Sigma-Aldrich</td>
			<td>D0627</td>
			<td></td>
		</tr>
		<tr>
			<td>Dispase</td>
			<td>Gibco</td>
			<td>17105-041</td>
			<td></td>
		</tr>
		<tr>
			<td>Dorsomorphin</td>
			<td>Selleckchem</td>
			<td>S7146</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s modified eagle medium with F12 nutrient mixture (DMEM/F12)</td>
			<td>Corning</td>
			<td>10-092-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Gibco</td>
			<td>16141-002</td>
			<td></td>
		</tr>
		<tr>
			<td>Fibroblast growth factor 2 (FGF2, bFGF)</td>
			<td>Peprotech</td>
			<td>100-18B</td>
			<td></td>
		</tr>
		<tr>
			<td>Geltrex LDEV-Free Reduced Growth Factor Basement Membrane Matrix</td>
			<td>Gibco</td>
			<td>A1413201</td>
			<td></td>
		</tr>
		<tr>
			<td>Gem21 NeuroPlex Serum-Free Supplement</td>
			<td>Gemini</td>
			<td>400-160</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Bottom Dishes</td>
			<td>MatTek</td>
			<td>P35G-0.170-14-C</td>
			<td></td>
		</tr>
		<tr>
			<td>9&#39;&#39; glass pipetes</td>
			<td>VWR</td>
			<td>14673-043</td>
			<td></td>
		</tr>
		<tr>
			<td>Glial derived neurotrophic factor (BDNF)</td>
			<td>Sigma-Aldrich</td>
			<td>D0627</td>
			<td></td>
		</tr>
		<tr>
			<td>GlutaMAX-I</td>
			<td>Gibco</td>
			<td>35050-061</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>Sigma</td>
			<td>H3149</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin growth factor 1 (IGF1)</td>
			<td>Invitrogen</td>
			<td>M7512</td>
			<td></td>
		</tr>
		<tr>
			<td>Knockout Serum Replacer</td>
			<td>Gibco</td>
			<td>A31815</td>
			<td></td>
		</tr>
		<tr>
			<td>Laminin</td>
			<td>Sigma</td>
			<td>L-6274</td>
			<td></td>
		</tr>
		<tr>
			<td>2-Mercaptoethanol</td>
			<td>Sigma</td>
			<td>M3148-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>MitoTracker CMXRos</td>
			<td>Invitrogen</td>
			<td>M7512</td>
			<td></td>
		</tr>
		<tr>
			<td>Neurobasal medium</td>
			<td>Gibco</td>
			<td>21103-049</td>
			<td></td>
		</tr>
		<tr>
			<td>Non Essential Amino Acids</td>
			<td>Gibco</td>
			<td>11140-050</td>
			<td></td>
		</tr>
		<tr>
			<td>N2 NeuroPle Serum-Free Supplement</td>
			<td>Gemini</td>
			<td>400-163</td>
			<td></td>
		</tr>
		<tr>
			<td>Olympus microscope IX83</td>
			<td>Olympus</td>
			<td>IX83-ZDC2</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Corning</td>
			<td>21-031-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Phase contrast microscope</td>
			<td>Olympus</td>
			<td>CKX41/ IX2-SLP</td>
			<td></td>
		</tr>
		<tr>
			<td>6 well plates</td>
			<td>Corning</td>
			<td>353046</td>
			<td></td>
		</tr>
		<tr>
			<td>24 well plates</td>
			<td>Corning</td>
			<td>353047</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-L-ornithine hydrobromide (polyornithine))</td>
			<td>Sigma-Aldrich</td>
			<td>P3655</td>
			<td></td>
		</tr>
		<tr>
			<td>SB431542</td>
			<td>Stemgent</td>
			<td>04-0010</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile 50ml Disposable Vacuum Filtration System 0.22 &#956;m Millipore Express&#174; Plus Membrane</td>
			<td>Millipore</td>
			<td>SCGP00525</td>
			<td></td>
		</tr>
		<tr>
			<td>Stericup 500/1000 ml Durapore 0.22 &#956;M PVDF</td>
			<td>Millipore</td>
			<td>SCGVU10RE</td>
			<td></td>
		</tr>
		<tr>
			<td>Tbr1 antibody (1:2000)</td>
			<td>Chemicon</td>
			<td>AB9616</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin inhibitor</td>
			<td>Gibco</td>
			<td>17075029</td>
			<td></td>
		</tr>
		<tr>
			<td>50 ml tubes</td>
			<td>Phenix</td>
			<td>SS-PH50R</td>
			<td></td>
		</tr>
		<tr>
			<td>15 ml tubes</td>
			<td>Phenix</td>
			<td>SS-PH15R</td>
			<td></td>
		</tr>
		<tr>
			<td>T25 flasks (untreated)</td>
			<td>VWR</td>
			<td>10861-572</td>
			<td></td>
		</tr>
		<tr>
			<td>Plugins for softwares</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bio-formats Package</td>
			<td><a href="http://downloads.openmicroscopy.org/bio-formats/5.1.0/" target="_blank">http://downloads.openmicroscopy.org/bio-formats/5.1.0/</a></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Fiji software</td>
			<td><a href="https://fiji.sc/" target="_blank">https://fiji.sc/</a></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Kymograph Plugin</td>
			<td><a href="https://www.embl.de/eamnet/html/body_kymograph.html" target="_blank">https://www.embl.de/eamnet/html/body_kymograph.html</a></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MultipleKymograph.class</td>
			<td><a href="https://www.embl.de/eamnet/html/body_kymograph.html" target="_blank">https://www.embl.de/eamnet/html/body_kymograph.html</a></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>MultipleOverlay.class</td>
			<td><a href="https://www.embl.de/eamnet/html/body_kymograph.html" target="_blank">https://www.embl.de/eamnet/html/body_kymograph.html</a></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>WalkingAverage.class</td>
			<td><a href="https://www.embl.de/eamnet/html/body_kymograph.html" target="_blank">https://www.embl.de/eamnet/html/body_kymograph.html</a></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>StackDifference.class</td>
			<td><a href="https://www.embl.de/eamnet/html/body_kymograph.html" target="_blank">https://www.embl.de/eamnet/html/body_kymograph.html</a></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Straighten_.jar</td>
			<td><a href="https://imagej.nih.gov/ij/plugins/straighten.html" target="_blank">https://imagej.nih.gov/ij/plugins/straighten.html</a></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>tsp050706.txt</td>
			<td><a href="https://www.embl.de/eamnet/html/body_kymograph.html" target="_blank">https://www.embl.de/eamnet/html/body_kymograph.html</a></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

analyzing mitochondrial transport and morphology in human induced pluripotent stem cell-derived neurons in hereditary spastic paraplegia

神经元对高能量有强烈的需求，以支持其功能。在人类神经元中观察到沿着斧子的线粒体传输受损，这可能导致各种疾病状态的神经退化。虽然研究活人类神经中的线粒体动力学具有挑战性，但这种模式对于研究线粒体在神经退化中的作用至关重要。本文介绍一个协议，用于分析前脑神经元的线粒体迁移和线粒体形态学，该词从人类诱导多能干细胞（iPSCs）中提取。iPSCs 使用成熟的方法分化为端脑谷氨酸神经元。神经元的线粒体被MitoTracker CMXRos染色，并且使用装有细胞培养箱的活细胞成像显微镜捕获斧子内的线粒体运动。使用带有"多Kymograph"、"生物格式导入器"和"宏"插件的软件对延时图像进行分析。生成线粒体传输的线粒体图，并从测速仪中读取反逆向线粒体的平均线粒体速度。在线粒体形态分析方面，利用ImageJ获得线粒体长度、面积和纵横比。总之，该协议允许沿斧子对线粒体贩运进行表征，并分析其形态，以促进神经退行性疾病的研究。

在这里，人类iPSCs被分化为端脑谷氨酸神经元，其特征是用Tbr1和βIII图布林标记进行免疫染色（图1A）。为了检查线粒体的同线体传输，这些细胞被红色荧光染料染色，并进行了延时成像。由于ImageJ是现成的，更容易获得，线粒体传输被进一步分析与"多Kymograph"和"宏"图像J插件，如图1所示。
线粒体运动有三种各自的状?...

Watch this Scientific Journal Video about Analyzing Mitochondrial Transport and Morphology in Human Induced Pluripotent Stem Cell-Derived Neurons in Hereditary Spastic Paraplegia at JoVE.com

Analyzing Mitochondrial Transport and Morphology in Human Induced Pluripotent Stem Cell-Derived Neurons in Hereditary Spastic Paraplegia

受损的线粒体迁移和形态学涉及各种神经退行性疾病。提出的协议使用诱导多能干细胞衍生前脑神经元来评估线粒体迁移和遗传性痉挛性截瘫的形态。该协议允许沿斧子对线粒体贩运进行表征，并分析其形态，这将有助于神经退行性疾病的研究。

遗传性痉挛性截瘫中人类诱导多能干细胞衍生神经元的线粒体迁移与形态分析

Neurons have intense demands for high energy in order to support their functions. Impaired mitochondrial transport along axons has been observed in human ...

线粒体功能障碍是许多神经退行性疾病的基础。我们的协议为检查轴突中的线粒体动力学提供了一个重要工具，有助于研究涉及轴突退化的神经系统疾病。通过结合线粒体标记、活细胞成像和诱导多能干细胞技术，我们的协议可用于特征线粒体随用轴线体贩运，并分析其形态。在干细胞培养和神经退行性疾病的动物模型中可以观察到线粒体运输和形态受损，为治疗这些疾病提供了潜在的治疗目标。经过第35天的培养，分离出与人类诱导多能干细胞区别的神经球，以1mg/ml的细胞分离溶液在37摄氏度下两分钟。在孵化结束时，通过离心收集细胞簇，并在NDM的一毫升中重新塞粒。板约五簇细胞在一百微升中每多余物氨酸和乳糖脱氢酶提升无病毒降低生长因子地下室膜基质涂覆35毫米玻璃底盘。然后添加一毫升NDM，辅以B27、环状腺苷单磷酸盐、胰岛素样生长因子、人脑衍生神经营养因子和胶质细胞衍生神经营养因子。要沿前脑神经元轴突的线粒体可视化，首先在NDM中用50纳米摩尔红色荧光染料染色神经元，在37摄氏度下进行三分钟，然后用温暖的NDM洗涤两次。接下来，将培养力置于荧光显微镜的舞台上，并选择 40 倍目标。在相场下，根据轴的形态特征识别轴子。要区分线粒体沿轴突运动的方向，可以清楚地识别神经元的细胞体。区分细胞体和轴突后，调整轴突中线粒体的暴露时间和焦点，每五秒钟捕获轴突内的线粒体传输，共五分钟。要分析 ImageJ 中线粒体传输打开斐济，并选择生物格式插件。使用生物格式导入器导入 tiff 系列的延时图像，并选择标准 ImageJ 和打开所有系列。检查自动缩放分割通道，然后单击"确定"以像素为单位注意图像的帧数和大小。调整所有 60 帧的亮度和对比度后，使用分段线绘制一条线，从单元格主体到端轴。要生成 Kymograph，请选择多个 Kymograph 插件并选择线宽。测量移动线粒体打开 tsp050607 的距离、时间值和速度。txt 在宏插件中。并在线粒体从上位到劣等区域的线粒体运动轨迹上画一条分割线。绘制行后打开从宏插件中的 tsp 读取速度，以读取与线对应的分段速度。接下来打开原始图像，并使用线条工具沿比例图条绘制一条线。选择"分析"可测量线的长度，并将 dx 现在和距离单位从像素转换为微米。然后将时间从像素更改为秒。要分析 ImageJ 中轴突内的线粒体长度和面积，请安装拉直下划线 DOT JAR 插件，并打开感兴趣的轴突图像。将 32 位图像转换为 8 位，并使用分段线工具跟踪轴孔。选择"直划下划线 JAR"插件，将灯丝宽线的宽度设置为 50 像素。再次跟踪轴孔以生成拉直的轴孔并调整图像阈值。选择分析集测量周长拟合椭圆和形状描述符以设置测量值并使用线函数测量原始图像中的比例条，如所示。在"分析"下设置比例输入以像素为单位的距离和长度单位以设置比例。然后选择全局，将比例设置为所有图像。最后选择分析粒子以确定面积，并选择显示结果以查看生成的测量结果。分化成电脑谷胱甘肽神经元后，细胞可以由其T-B-R一和β三管状蛋白标记表达来描述。使用红色荧光染料染色和延时成像可以评估线粒体的斧头运输。单个线粒体可以保持静态或在轴突内逆行或逆行方向移动。线粒体沿轴线体运动的速度可以量化，如图所示。例如，使用市售分析软件，与野生型神经元相比，S-P-G-3-A神经元中运动线粒体的百分比显著降低。要分析线粒体面积长度和纵横比轴突可以在图像分析软件中拉直，并通过调整阈值进行选择。线粒体区域周长宽度和纵横比可以从拉直的轴突获得。例如，线粒体长度和纵横比在S-P-G 15神经元轴突中显著减少，而控制野生型轴突。加热培养介质和显微镜培养箱以轻轻清洗细胞，使细胞在成像前稳定20分钟是十分重要的。活细胞成像后，培养物可以固定并进行免疫污染，以检查其他感兴趣的蛋白质的表达。研究活人神经中的线粒体动力学是具有挑战性的。该技术为研究神经系统疾病的线粒体动力学和神经退化提供了独特的工具。

Research

JoVE Journal

Neuroscience

Neuromodulation and Mitochondrial Transport: Live Imaging in Hippocampal Neurons over Long Durations

神经调节和线粒体运输：在较长的时间里，海马神经元的实时成像

To understand the relationship between mitochondrial transport and neuronal function, it is critical to observe mitochondrial behavior in live cultured ...

科研

神经科学

Efficient Derivation of Human Neuronal Progenitors and Neurons from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

人类神经祖细胞和神经元小分子诱导人类胚胎干细胞的多能干高效的推导

There is a large unfulfilled need for a clinically-suitable human neuronal cell source for repair or regeneration of the damaged central nervous system ...

The Specification of Telencephalic Glutamatergic Neurons from Human Pluripotent Stem Cells

从人类多能干细胞的规范在前脑谷氨酸能神经元

Here, a stepwise procedure for efficiently generating telencephalic glutamatergic neurons from human pluripotent stem cells (PSCs) has been described. The ...

Analyzing Dendritic Morphology in Columns and Layers

分析树突状形态的列和图层

In many regions of the central nervous systems, such as the fly optic lobes and the vertebrate cortex, synaptic circuits are organized in layers and ...

Modelling Zika Virus Infection of the Developing Human Brain In Vitro Using Stem Cell Derived Cerebral Organoids

Modelling Zika Virus Infection of the Developing Human Brain In Vitro Using Stem Cell Derived Cerebral Organoids

利用干细胞衍生脑 Organoids体外培养人脑组织 Zika 病毒感染的模拟研究

The recent emergence of Zika virus (ZIKV) in susceptible populations has led to an abrupt increase in microcephaly and other neurodevelopmental conditions ...

Method for High Speed Stretch Injury of Human Induced Pluripotent Stem Cell-derived Neurons in a 96-well Format

96井格式人诱导多潜能干细胞源神经元高速拉伸损伤的方法

Traumatic brain injury (TBI) is a major clinical challenge with high morbidity and mortality. Despite decades of pre-clinical research, no proven ...

Compartmentalization of Human Stem Cell-Derived Neurons within Pre-Assembled Plastic Microfluidic Chips

预组装塑料微流体芯片中人类干细胞衍生神经元的分段化

Use of microfluidic devices to compartmentalize cultured neurons has become a standard method in neuroscience. This protocol shows how to use a ...

Post-differentiation Replating of Human Pluripotent Stem Cell-derived Neurons for High-content Screening of Neuritogenesis and Synapse Maturation

分化后再蛋白的人类多能干细胞衍生神经元，用于高含量的内尿成生和突合性成熟筛查

Neurons differentiated in two-dimensional culture from human pluripotent stem-cell-derived neural progenitor cells (NPCs) represent a powerful model ...

Conversion of Human Induced Pluripotent Stem Cells (iPSCs) into Functional Spinal and Cranial Motor Neurons Using PiggyBac Vectors

Conversion of Human Induced Pluripotent Stem Cells iPSCs into Functional Spinal and Cranial Motor Neurons Using PiggyBac Vectors

利用 PiggyBac 载体将人诱导的多能干细胞 (Ipsc) 转化为功能性脊柱和颅内运动神经元

We describe here a method to obtain functional spinal and cranial motor neurons from human induced pluripotent stem cells (iPSCs). Direct conversion into ...