Name: mitoception: transferring isolated human msc mitochondria to glioblastoma stem cells
Uploaded: 2017-02-22T15:00:00
Description: Watch this Scientific Journal Video about MitoCeption: Transferring Isolated Human MSC Mitochondria to Glioblastoma Stem Cells at JoVE.com

我们感谢安德烈Parmeggiani（L2C和DIMNP，蒙彼利埃），伯努瓦夏洛（IES，蒙彼利埃），以及实验室的有益讨论的成员，克里斯托夫Duperray与流式细胞仪分析，蒙彼利埃RIO成像设备（MRI）提供帮助对于FACS和共聚焦显微镜充足的环境。国行是从LabEx Numev（ANR公约-10-的LabX-20）一个研究生奖学金的支持。 AB是由来自华沙和欧洲联盟的大学本科生奖学金支持（N°POKL.04.01.02-00-221 / 12）。 MLV是从国家科学研究中心（CNRS）工作的科学家。

1天 1.间充质干细胞的标记（MSC）线粒体（可选） <ol><li>线粒体制备前两天，种子人MSC在100毫米培养皿中，在10毫升αMEM/ FBS 10％，以便具有在第1天在培养4×10 5个干细胞。 </li><li>冲洗的MSC用PBS（4毫升）中，并添加4毫升αMEM/ FBS 1％（预热至37℃）。 </li><li>添加所需的线粒体活体染料的量，并培育细胞30分钟，在37℃培养箱。 </li><li>除去线粒体染料溶液，用预热4毫升（37?...

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越来越多的研究表明，细胞可以交换线粒体，这些线粒体对靶细胞的代谢和功能产生深远影响。因此，重要的是掌握工具来定量从供体细胞中这些靶细胞转移线粒体使它们的生物效应的精确研究。 这里所描述的协议最初是摸索出从人间充质干细胞中分离出的线粒体转移到粘附癌细胞系MDA-MB-231 33。如这里所示，它可适于该成长为体外神经球癌症干细胞?...

线粒体是在他们中的养分吸收，以及在能量和代谢物生产中发挥中心作用的真核细胞中发现的细胞器。这些细胞器包含圆形线粒体DNA（mtDNA的），16.6 kb的长，编码电子传递链复合物，tRNA和个rRNA 1的蛋白质。这些细胞器的功能是对细胞稳态和几个病状已与线粒体功能障碍1，2，3相关联的关键。线粒体状态已经例如被链接到炎症，感染性疾病和癌症，与用于转移和电阻后果治疗4，5，6，7后面这种情况。 线粒体显示的非凡能力，<html> “供体”和“目标”细胞间的转移得到。这导致在靶细胞的能量代谢的变化，以及在其它功能上的修改，例如组织修复和对化疗药物抗性，如最近由不同实验室8，9，10，11，12，13，14，15中所示16。人类间充质干细胞（MSCs）显示这个能力到线粒体转移到各种各样的靶细胞，包括心肌细胞，血管内皮细胞，肺泡上皮细胞，肾小管细胞和癌细胞，导致这些细胞的功能特性的修改8，&gt; 9，10，12，17，18。 线粒体交换现在显示为一种广泛使用的机制，允许多个不同的细胞类型中与彼此通信并修改他们的生物特性。这种线粒体交换可以通过隧道纳米管（TNT）的形成发生，涉及连接蛋白含有43缝隙连接8或M-SEC / TNFaip2和exocyst复杂的19。可替代地，也被示出的线粒体转移到通过抑制蛋白结构域的蛋白1介导的微泡（ARMMs）20介导的。有趣的是，线粒体转移的功效是有联系的所述的Rho GTP酶1的表达率MIRO1 21，用于说明在iPSC-之间线粒体转移功效的差别的关键因素干细胞与成人骨髓间充质干22。 尽管这种财富有关的细胞 - 细胞线粒体交换数据的，相对知之甚少此线粒体转移的代谢和生物结果。因此，它完全值得设置适当的工具充分评估这种转移的生物效应。多年来，若干技术方法从供体转移到线粒体受体细胞已被提出。这包括直接注射的线粒体成卵母细胞23，24，25，细胞融合，以产生transmitochondrial胞质杂种26,27 并且，最近，利用光热分离的线粒体的转移nanoblades 28。 我们和其他人以前证明孤立的和线粒体的能力河口到由活细胞内化，因为无论是在体外和体内观察到的 29，30，31，通过机制，提出了涉及巨吞32。我们进一步开发出一种方法，叫做MitoCeption，以定量地转移（从干细胞）分离线粒体靶细胞，与（贴壁）中列举的MDA-MB-231乳腺癌细胞系31。该协议在这里被改编为分离人MSC线粒体的转移胶质母细胞瘤干细胞（GSCS）。 胶质母细胞瘤是大脑中迅速成为处理有抗性，主要是由于胶质母细胞瘤干细胞（GSC）的肿瘤33内本侵略性恶性肿瘤。这些GSCS成长为体外神经球并产生异种移植模型的肿瘤。胶质母细胞瘤中的肿瘤细胞具有容量，使细胞与细胞间的连接，如最近所示为星形细胞脑肿瘤细胞通过扩展的微管，配线，通过该线粒体（以及钙和细胞核）可以迁移，导致耐放疗星形网络34。胶质母细胞瘤可以招募肿瘤微环境内的许多不同的细胞，包括干细胞35，36。我们表明，干细胞可以与在共培养GSCS细胞 - 细胞连接，并传送其线粒体（数据未显示），这是预期到修改GSC功能特性。本协议说明了MitoCeption技术如何可用于从人MSC与确定它们的功能的生物结果的目的传送线粒体，分离的预先，对人类GSCS。的多能和高致瘤性GB4 GSC线37在本研究中使用。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Mitochondria Isolation Kit for Tissue&#160;</td>
			<td>Fisher Scientific&#160;</td>
			<td>10579663</td>
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			<td>Fisher Scientific&#160;</td>
			<td>11520536</td>
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			<td>BE12-169F</td>
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			<td>DMEM/F-12 without glutamine,&#160;</td>
			<td>Fisher Scientific&#160;</td>
			<td>11540566</td>
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			<td>Invitrogen&#160;</td>
			<td>25030-024&#160;</td>
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			<td>Sigma&#160;</td>
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			<td>R&#38;D Systems</td>
			<td>233-FB-025</td>
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			<td>Human EGF&#160;</td>
			<td>Peprotech&#160;</td>
			<td>AF-100-15&#160;</td>
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			<td>Heparin</td>
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			<td>H3149&#160;</td>
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			<td>MERCK</td>
			<td>2382</td>
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			<td>Trypsine Inhibitor&#160;</td>
			<td>Sigma&#160;</td>
			<td>T9003</td>
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			<td>SIGMA&#160;</td>
			<td>10104159001</td>
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			<td>Invitrogen&#160;</td>
			<td>25200056</td>
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			<td>Trypsin</td>
			<td>Gibco&#160;</td>
			<td>15090-046</td>
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			<td>Protease inhibitors EDTA free</td>
			<td>Sigma</td>
			<td>4693159001</td>
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			<td>Ciprofloxacine&#160;</td>
			<td>Sigma</td>
			<td>17850-5G-F</td>
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			<td>Fungine&#160;</td>
			<td>Invivogen</td>
			<td>ant-fn-1</td>
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			<td>Fungizone&#160;</td>
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			<td>15290018</td>
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			<td>EU0410</td>
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			<td>Molecular Probes</td>
			<td>M7514</td>
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			<td>&#160;MitoTracker Red CMXRos</td>
			<td>Molecular Probes</td>
			<td>&#160;M7512</td>
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			<td>MitoTracker Deep Red FM</td>
			<td>Molecular Probes</td>
			<td>&#160;M22426&#160;</td>
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			<td>&#160;CellTracker Green CMFDA</td>
			<td>Molecular Probes</td>
			<td>C7025</td>
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			<td>&#160;CellTracker Blue CMF2HC</td>
			<td>Molecular Probes</td>
			<td>C12881</td>
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			<td>RIPA</td>
			<td>Santa Cruz</td>
			<td>sc-24948</td>
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			<td>FluoroDish Sterile Culture Dish</td>
			<td>World Precision Instruments</td>
			<td>FD35-100</td>
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			<td>Hemacytometer</td>
			<td>Fisher Scientific</td>
			<td>267110</td>
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			<td>FACS tubes</td>
			<td>Beckman Coulter</td>
			<td>2,523,749</td>
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			<td>FACS apparatus</td>
			<td>Gallios&#160;</td>
			<td>&#160;3L 10C</td>
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			<td>LC FAST START DNA MASTER PLUS&#160;</td>
			<td>Roche</td>
			<td>3515885001</td>
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mitoception: transferring isolated human msc mitochondria to glioblastoma stem cells

线粒体发挥细胞代谢，能源生产和细胞凋亡的控制中心作用。不足的线粒体功能已经发现负责非常多样的疾病，从病症神经系统癌症。有趣的是，线粒体最近已显示，以显示有能力的细胞类型之间从人间充质干细胞（MSC）的共培养条件转移，特别是癌症细胞，代谢和功能的后果对线粒体受体细胞，进一步增强了当前兴趣这些细胞器的生物学性质。 评估在靶细胞的转移的MSC线粒体的效果是最重要的，以了解这样的细胞 - 细胞相互作用的生物学结果。这里所描述的MitoCeption协议允许从供体细胞中预先分离到靶细胞的线粒体的转移，使用MSC线粒体和胶质母细胞瘤干细胞（GSC）作为模型系统。该协议之前已经用于从干细胞转移线粒体，分离的，以贴壁的MDA-MB-231癌细胞。此线粒体传输协议此处适于呈递如体外神经球生长的具体特殊性GSCS。分离的线粒体的转移可以使用线粒体活体染料后跟荧光激活细胞分选（FACS）和共聚焦成像。使用线粒体供体和靶细胞具有不同的单倍型（单核苷酸多态性）的也允许基于其圆线粒体DNA（mtDNA的）的靶细胞的浓度被转印线粒体的检测。一旦该协议已经被验证这些标准，窝藏转移的线粒体的细胞可以被进一步分析，以确定对生物性能如细胞代谢，可塑性，增殖和对治疗的反应的外源线粒体的作用。

概述从间质干细胞（MSC）和由MitoCeption其转移到靶向胶质母细胞瘤干细胞（GSC）线粒体的分离的过程步骤示于图1。 GSCS生长神经球，以维护自己的干细胞特性的肿瘤干细胞。用于协议，GSCS接种为单细胞几小时线粒体（步骤3），以允许更高的线粒体转印效率的转印之前（见的FACS数据图3B）。以下第5（第3天），这些细胞被再次观察到作为形成神经球...

Watch this Scientific Journal Video about MitoCeption: Transferring Isolated Human MSC Mitochondria to Glioblastoma Stem Cells at JoVE.com

MitoCeption: Transferring Isolated Human MSC Mitochondria to Glioblastoma Stem Cells

这里，协议（MitoCeption）呈现给传送线粒体，从人类间质干细胞（MSC）隔离，胶质母细胞瘤干细胞（GSC），具有研究它们对GSC代谢和功能的生物效应的目的。类似的协议可以适于其它类型的细胞之间转移线粒体。

MitoCeption：转让人离MSC线粒体脑胶质瘤干细胞

Mitochondria play a central role for cell metabolism, energy production and control of apoptosis. Inadequate mitochondrial function has been found ...

Research

JoVE Journal

Biology

Propagation of Human Embryonic Stem (ES) Cells

Propagation of Human Embryonic Stem ES Cells

人类胚胎干细胞（ES细胞）的繁殖

科研

生物学

Counting Human Neural Stem Cells

人类神经干细胞计数

Knowledge of the exact number of viable cells in a given volume of a cell suspension is required for many routine tissue culture manipulations, such as ...

Passaging Human Neural Stem Cells

传代人类神经干细胞

The ability to manipulate human neural stem/precursor cells (hNSPCs) in vitro provides a means to investigate their utility as cell transplants for ...

Immunocytochemistry: Human Neural Stem Cells

免疫组化：人类神经干细胞

Immunocytochemistry is a very powerful and fairly straightforward method for determining the presence, subcellular localization, and relative abundance of ...

Chromatin Immunoprecipitation from Human Embryonic Stem Cells

从人类胚胎干细胞的染色质免疫沉淀

The functional and structural complexity of the myriad of cells in metazoan organisms arises from a small number of stem cells. Stem cells are ...

Purification of Mitochondria from Yeast Cells

从酵母细胞线粒体的纯化

Mitochondria are the main site of ATP production during aerobic metabolism in eukaryotic non-photosynthetic cells1. These complex organelles also play ...

Freezing and Thawing Human Embryonic Stem Cells

人类胚胎干细胞冷冻和解冻

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells ...

Differentiation of Embryonic Stem Cells into Oligodendrocyte Precursors

胚胎干细胞分化成少突胶质前体

Oligodendrocytes are the myelinating cells of the central nervous system. For regenerative cell therapy in demyelinating diseases, there is significant ...

Transfecting and Nucleofecting Human Induced Pluripotent Stem Cells

人类诱导多能干细胞的转染和Nucleofecting

Genetic modification is continuing to be an essential tool in studying stem cell biology and in setting forth potential clinical applications of human ...

Multi-parameter Measurement of the Permeability Transition Pore Opening in Isolated Mouse Heart Mitochondria

小鼠离体心肌线粒体通透性转换孔开放中的多参数测量。

The mitochondrial permeability transition pore (mtPTP) is a non specific channel that forms in the inner mitochondrial membrane to transport solutes with ...