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℃）αMEM/ FBS 1％冲洗细胞两次，并加回4毫升αMEM/ FBS 10％。孵育在37℃的细胞。 </li><li> 30分钟后，改变培养基（10毫升αMEM/ FBS 10％）和，另一次，2小时后。 </li></ol> 1天 2.标签胶质母细胞瘤干细胞（GSC）（可选，见讨论部分） <table border="1" fo:keep-together.within页="1"FO:保持与 - next.within页="总是"&gt; <tbody><tr><td> 细胞培养基 </td><td> 组成 </td></tr><tr><td rowspan="5"> GSC基础培养基</td><td> DMEM / F-12补充有</td></tr><tr><td>胰岛素20毫克/毫升</td></tr><tr><td> N2补充1X </td></tr><tr><td>葡萄糖3g / L的</td></tr><tr><td> L-谷氨酰胺2mM的</td></tr><tr><td rowspan="9"> GSC增殖培养基</td><td>添加到基础培养基: </td></tr><tr><td> B27的补充1X </td></tr><tr><td> EGF 10毫微克/毫升</td></tr><tr><td> bFGF的10毫微克/毫升</td></tr><tr><td>为Fungine 10毫克/毫升</td></tr><tr><td>两性霉素B 0.25毫克/毫升</td></tr><tr><td>肝素2mg / ml的</td></tr><tr><td>环丙沙星2微克/毫升</td></tr><tr><td>庆大霉素2微克/毫升</td></tr><tr><td rowspan="4"> MSC增殖培养基</td><td> ＆＃945; MEM补充有</td></tr><tr><td> L-谷氨酰胺2mM的</td></tr><tr><td> 10％FBS的</td></tr><tr><td> bFGF的2毫微克/毫升</td></tr></tbody></table> 表1:文化传媒。 <ol><li>分离生长为在聚HEMA包被的细胞培养烧瓶神经球GSCS（GB4细胞线32（10×10 6个细胞）（参见步骤3.1至3.12）。 </li><li>在10 5个细胞在48孔板种子GSCS /孔在GSC增殖培养基（500微升）（ 见表1）。 </li><li>离心板在270×g离心20℃5分钟。 </li><li>添加所需的细胞活体染料的量，并在37℃下孵育30分钟。 </li><li>加入500μlGSC基础培养基（ 表1）每48孔板的孔。 </li><li>离心在270 xg离心该板在20℃下5分钟。吸出上清液。 </li><li>重复步骤2.5至2.6。 </li><li>加入500μlGSC基础培养基中，并在37℃孵育30分钟。 </li><li>离心在270 xg离心该板在20℃下5分钟。吸出上清液。 </li><li>加入500μlGSC增殖培养基的每孔的48孔板，并在37℃培养箱孵育。 注意:GSCS的量表示（10×10 6个细胞）允许执行不同的剂量-反应实验和FACS对照。一旦实验条件更精确地定义此量可被缩减。 </li></ol>第2天 3.胶质母细胞瘤干细胞接种<ol><li>在50ml管中收集的GSC神经球（10×10 6个细胞）通过离心在270×g离心5分钟，在20℃。 </li><li>在20℃下洗涤细胞用5毫升的HBSS，和离心机在270×g离心5分钟。 </li><li>吸出上清液。 </li><li>轻轻重悬在100μl胰蛋白酶-EDTA（0.25％）（在GSC粒料每10×10 <s达&gt; 6个细胞） </li><li>孵育在37℃下3分钟。 </li><li>添加10微升的CaCl 2（20毫米）和2μlDNA酶I（10毫克/毫升）。 </li><li>通过与P200吸管轻轻吹打向上和向下（30-50x）游离神经球。避免气泡。检查所有GSCS解离的显微镜下。 </li><li>添加10微升胰蛋白酶抑制剂（5％）和10毫升的HBSS。 </li><li>离心机GSCS在在20℃下270×g离心7分钟。 </li><li>弃上清，加入10 mL GSC基础培养基（ 表1）。 </li><li>算GSCS用托马计数室，然后离心细胞以270×g离心7分钟，在20℃。 </li><li>添加GSC增殖培养基（ 表1）的适当的体积达到10 6 GSCS / ml的细胞浓度。 </li><li>种子10 5 GSCS每一个96孔板的孔（100微升细胞悬浮液）。 </li><li>离心在270×g离心7分钟将板在20℃在机器人以获得GSCS井汤姆。 </li><li>在37℃下的96孔板直到第5节。 </li></ol>第2天 4. MSC线粒体隔离<ol><li>调整微管离心机温度至4℃。 </li><li>准备2个1.5毫升管的"A"和"C"含有线粒体萃取（200微升试剂A和400μl的试剂C，二者均含有的不含EDTA的蛋白酶抑制剂）的试剂。准备其他两管，标有"地中海"和"美图"。置于冰上所有的管子。还准备为管线粒体连续稀释。 </li><li>洗的MSC与预热10毫升（37℃）的PBS。 </li><li>洗的MSC用2ml胰蛋白酶（无EDTA）中10秒，并加入1 ml的胰蛋白酶（无EDTA）中。孵育细胞5-10分钟，在37℃。 </li><li>通过加入10mlαMEM/ FBS 10％，转移到50ml管中收回的MSC。 </li><li>离心细胞于在20℃下270×g离心5分钟。 </li><li>弃去上清液，加入10毫升αMEM/ FBS 10％至细胞沉淀。 </li><li>计数和计时Malassez室中的干细胞。 </li><li>离心机的MSCs（4-5×10 5）在20℃270×g离心5分钟。 </li><li>弃去上清液，加入1米冰冷αMEM/ FBS 10％到细胞沉淀，将细胞转移到"MSC"标记的管（在步骤4.2中制备）。保持管冰。 </li><li>离心机包含的MSC在900×g离心5分钟的管中，在4℃下。 </li><li>取下管所有剩余的网上平台。 </li><li>加入200μl线粒体提取试剂A（含不含EDTA的蛋白酶抑制剂）的。旋涡中速，持续5秒，离开冰管整整2分钟。 </li><li>以最大的速度添加2.5微升线粒体分离试剂B涡流的10秒，然后离开冰管中。重复每30秒5分钟。 </li><li>加入200μl线粒体提取试剂C（含不含EDTA的蛋白酶抑制剂）的。通过倾斜混合管（ROughly 30次，不要旋涡）。离心管中，在700×g离心在4℃下10分钟。 </li><li>转移上清液（含有MSC线粒体）至"三刀"管（在步骤4.2中制备）。 </li><li>离心机在3000 XG，在4℃下15分钟，得到线粒体沉淀。弃去上清液。沉淀包含分离线粒体。 </li><li>冲洗用200μl试剂C的。然后，离心分离的线粒体沉淀以12,000 xg离心，于4℃下5分钟，以获得线粒体沉淀。 </li></ol> 5.转让隔离MSC线粒体到GSCS（MitoCeption） <ol><li>添加200微升预冷的（0℃）的GSC增殖培养基由干细胞（4×10 5）中分离出的线粒体沉淀。 </li><li>稀释线粒体制剂（在GSC增殖培养基），以20微升的线粒体悬浮液的一贯添加到GSCS。 </li><li>添加分离线粒体的体积到96-W的孔中ELL板含有GSCS（从步骤3.15），在所需的浓度（0.1至10微克）。添加线粒体慢慢靠近井的底部，覆盖了整个表面至少一次。 </li><li>用于控制MSC线粒体活体染料渗漏，添加相同量的线粒体制剂（0.1至10微克）的含有GSC增殖培养基只（无GSCS）（参见用于活体染料泄漏检测部6.2）的96孔平板的孔中。 </li><li>离心机含有与MSC线粒体（步骤5.3）和控制板（步骤5.4）的线粒体收件人GSCS在1500 xg离心在4℃下15分钟的96孔板中。 </li><li>放置在37℃细胞培养箱培养板离心后立即。 注意:线粒体传输协议依赖于在足够的离心力的培养细胞的线粒体悬浮液的离心分离，具有一批离心，可以根据不同的SYST调整线粒体供体/受体细胞的他们。 </li></ol>第3天 6.流式细胞仪和共焦成像的线粒体传递的分析<ol><li> GSC样品的制备用于FACS分析 注意:如果MSC线粒体用活体染料预先（第1）标记的，效率可通过FACS线粒体转移后24小时监视。 <ol><li>离心机的96孔板用在270×g离心5分钟MitoCepted GSCS在20℃。 </li><li>弃去上清液。 </li><li>在每个孔中加入100μl胰蛋白酶（无EDTA）中。孵育在37℃下3分钟。吸管上下解离形成在24小时时间段的神经球。 </li><li>加入100微升GSC基础培养基。 </li><li>离心板在270×g离心5分钟，在20℃，并丢弃上清液。 </li><li>悬浮在GSCS 300微升GSC基础培养基，并转移到FACS适应管。 </li><li>执行的FACSalysis。 </li></ol></li><li> 从分离线粒体线粒体活体染料泄漏控制（FACS） 注意:该步骤的目的是确定本底的FACS信号，不会反映真正的MSC线粒体转移，而是从用于MitoCeption（步骤5.3）标记的MSC线粒体活体染料的光泄漏。 <ol><li> GSC种子细胞在步骤3.1所描述的3.15。 </li><li>孵育所述细胞在37℃下1小时。 </li><li>离心机仅在1500 xg离心含有线粒体（来自步骤5.4到5.6）中5分钟，在20℃的96孔板中。 </li><li>从GSC 96孔板（步骤6.2.2）吸出培养基，并通过与线粒体只（上清液从步骤6.2.3）孵育培养基更换。 </li><li>孵育含有GSCS的96孔板在37℃下2小时。 </li><li>请按步骤6.1.1 6.1.7 FACS分析。 </li></ol></li><li> GSC SAMP的制备LES的共焦成像 注意:如果MSC线粒体转移到GSCS是通过荧光成像分析，无论是MSC和GSCS需要预先分别与线粒体（步骤1）和小区（步骤2）活体染料标记。 <ol><li>准备GSCS如步骤6.1.1到6.1.5。 </li><li>悬浮GSCS在2毫升含2％FBS和种子35毫米培养皿（玻璃底）GSC增殖培养基。 </li><li>用转让荧光MSC线粒体24小时后的GSCS执行共焦成像。 </li></ol></li></ol>

<ol>
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A., Ma, Y., Wong, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+effects+of+cancer+mitochondria+on+energy+metabolism+and+tumorigenesis:+utility+of+transmitochondrial+cybrids.">Functional effects of cancer mitochondria on energy metabolism and tumorigenesis: utility of transmitochondrial cybrids.</a> Ann N Y Acad Sci. 1201, 137-146 (2010).</li><li>Wu, T. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+Transfer+by+Photothermal+Nanoblade+Restores+Metabolite+Profile+in+Mammalian+Cells.">Mitochondrial Transfer by Photothermal Nanoblade Restores Metabolite Profile in Mammalian Cells.</a> Cell Metab. 23 (5), 921-929 (2016).</li><li>Masuzawa, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transplantation+of+autologously+derived+mitochondria+protects+the+heart+from+ischemia-reperfusion+injury.">Transplantation of autologously derived mitochondria protects the heart from ischemia-reperfusion injury.</a> Am J Physiol Heart Circ Physiol. 304 (7), H966-H982 (2013).</li><li>Kitani, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+human+mitochondrial+transfer:+a+novel+concept+based+on+the+endosymbiotic+theory.">Direct human mitochondrial transfer: a novel concept based on the endosymbiotic theory.</a> Transplant Proc. 46 (4), 1233-1236 (2014).</li><li>Caicedo, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MitoCeption+as+a+new+tool+to+assess+the+effects+of+mesenchymal+stem/stromal+cell+mitochondria+on+cancer+cell+metabolism+and+function.">MitoCeption as a new tool to assess the effects of mesenchymal stem/stromal cell mitochondria on cancer cell metabolism and function.</a> Sci Rep. 5, 9073 (2015).</li><li>Kesner, E. 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INSERM（国家研究所德拉桑特等德拉RECHERCHE MEDICALE），与CJ和ML.V.挂靠，对MitoCeption技术（EP14306154.7）申请了专利。

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>
		</tr>
		<tr>
			<td>N-2 Supplement (100X)</td>
			<td>Fisher Scientific&#160;</td>
			<td>11520536</td>
		</tr>
		<tr>
			<td>B-27 Supplement W/O VIT A (50X)</td>
			<td>Fisher Scientific&#160;</td>
			<td>11500446</td>
		</tr>
		<tr>
			<td>HBSS w/o Ca2+ w/o Mg2+</td>
			<td>&#160;Sigma</td>
			<td>H4385</td>
		</tr>
		<tr>
			<td>poly Heme&#160;</td>
			<td>Sigma&#160;</td>
			<td>P3932</td>
		</tr>
		<tr>
			<td>aMEM w/o glutamine</td>
			<td>Ozyme</td>
			<td>BE12-169F</td>
		</tr>
		<tr>
			<td>DMEM/F-12 without glutamine,&#160;</td>
			<td>Fisher Scientific&#160;</td>
			<td>11540566</td>
		</tr>
		<tr>
			<td>L-Glutamine&#160;</td>
			<td>Invitrogen&#160;</td>
			<td>25030-024&#160;</td>
		</tr>
		<tr>
			<td>Glucose&#160;</td>
			<td>Sigma&#160;</td>
			<td>G7021</td>
		</tr>
		<tr>
			<td>Insuline&#160;</td>
			<td>Sigma&#160;</td>
			<td>I 1882&#160;</td>
		</tr>
		<tr>
			<td>&#160;Human&#160;bFGF&#160;</td>
			<td>R&#38;D Systems</td>
			<td>233-FB-025</td>
		</tr>
		<tr>
			<td>Human EGF&#160;</td>
			<td>Peprotech&#160;</td>
			<td>AF-100-15&#160;</td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>Sigma</td>
			<td>H3149&#160;</td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>MERCK</td>
			<td>2382</td>
		</tr>
		<tr>
			<td>Trypsine Inhibitor&#160;</td>
			<td>Sigma&#160;</td>
			<td>T9003</td>
		</tr>
		<tr>
			<td>DNase I</td>
			<td>SIGMA&#160;</td>
			<td>10104159001</td>
		</tr>
		<tr>
			<td>Trypsine 0.25% /EDTA 1 mM</td>
			<td>Invitrogen&#160;</td>
			<td>25200056</td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Gibco&#160;</td>
			<td>15090-046</td>
		</tr>
		<tr>
			<td>Protease inhibitors EDTA free</td>
			<td>Sigma</td>
			<td>4693159001</td>
		</tr>
		<tr>
			<td>Ciprofloxacine&#160;</td>
			<td>Sigma</td>
			<td>17850-5G-F</td>
		</tr>
		<tr>
			<td>Fungine&#160;</td>
			<td>Invivogen</td>
			<td>ant-fn-1</td>
		</tr>
		<tr>
			<td>Fungizone&#160;</td>
			<td>Thermofisher</td>
			<td>15290018</td>
		</tr>
		<tr>
			<td>Gentamycin</td>
			<td>Euromedex</td>
			<td>EU0410</td>
		</tr>
		<tr>
			<td>&#160;MitoTracker Green FM</td>
			<td>Molecular Probes</td>
			<td>M7514</td>
		</tr>
		<tr>
			<td>&#160;MitoTracker Red CMXRos</td>
			<td>Molecular Probes</td>
			<td>&#160;M7512</td>
		</tr>
		<tr>
			<td>MitoTracker Deep Red FM</td>
			<td>Molecular Probes</td>
			<td>&#160;M22426&#160;</td>
		</tr>
		<tr>
			<td>&#160;CellTracker Green CMFDA</td>
			<td>Molecular Probes</td>
			<td>C7025</td>
		</tr>
		<tr>
			<td>&#160;CellTracker Blue CMF2HC</td>
			<td>Molecular Probes</td>
			<td>C12881</td>
		</tr>
		<tr>
			<td>RIPA</td>
			<td>Santa Cruz</td>
			<td>sc-24948</td>
		</tr>
		<tr>
			<td>FluoroDish Sterile Culture Dish</td>
			<td>World Precision Instruments</td>
			<td>FD35-100</td>
		</tr>
		<tr>
			<td>Hemacytometer</td>
			<td>Fisher Scientific</td>
			<td>267110</td>
		</tr>
		<tr>
			<td>FACS tubes</td>
			<td>Beckman Coulter</td>
			<td>2,523,749</td>
		</tr>
		<tr>
			<td>FACS apparatus</td>
			<td>Gallios&#160;</td>
			<td>&#160;3L 10C</td>
		</tr>
		<tr>
			<td>LC FAST START DNA MASTER PLUS&#160;</td>
			<td>Roche</td>
			<td>3515885001</td>
		</tr>
	</tbody>
</table>

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 ...

mitoception-transferring-isolated-human-msc-mitochondria-to

Research

JoVE Journal

Biology

12.7K 次观看.  Montpellier University. 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。 

视频: MitoCeption: Transferring Isolated Human MSC Mitochondria to Glioblastoma Stem Cells

Propagation of Human Embryonic Stem (ES) Cells

Propagation of Human Embryonic Stem ES Cells

Yeah, so I'm Lauren Deon. I'm in charge of the Human Amgen stem cell co facility at MGH. Yeah, so I'm Lauren, I'm in charge of the Human AM stem cell co facility at MGH.So I've been working with human Amgen stem cells for years now since I was a postdoc in George De Lab before, from four, four years. And so right now we have two cell lines, H one and H nine, which are both from Y cell. And so the way we are expanding or maintaining these cells in culture is by, at least the way we are splitting these cells is by using collagenous.So what I usually do is for the first few passage after throwing the cells, I use a technical, the picking colonies. So in this case, I can check under the microscope which colonies are good and differentiate and nice shape, and I will collect only these colonies and I will cut them in few pieces and put them back in, in a fresh flat. And the, the colonies that look kind of differentiate it are just left them alone.The other way to pass the cells is by using trypsin. So I don't use this technique here. I think collagenase is a little bit safer because human being stem cells don't like to be individualized or they don't like to be alone.So if you do trypsin, if you use trypsin for a little bit too long, if you use it for like three to four minutes, which is the way we use strips in for other type of cells, human stem cells will be individualized and most of them will die or just differentiate or will not attach to the myth. So the idea is you have to keep the cells as clumps. You don't want them to be alone in the in, in the flask.So using collagenase, the advantage is to keep cells as plants. Using chipsy, you just select for cells that will survive this very, very harsh process of g opsonization. So these cells are usually very highly proliferative.So you get, you can adapt your cells to this tripsin process. The problem is that you have cells that they behave differently, they go much faster. And one risk is that you can get some commercial abnormality to, to this, to this very high division process.So you say that ization can cause selection of cells in culture for most proliferative cells.Right. More ative and probably also higher survival rates than other cells. So after proliferation, basically culture after proliferation in tripsin, after several passages become a different culture, these different properties.So it's, it's hard to figure out if they are exactly identical. If you do the test, the usual test, let's say you check markers, regular markers of end differentiated cells like OC four, nano one 60 SC four C, C3, all this molecule might still be expressed. If you do TER formation, this cells might still differentiate and be formed like al like normal cells.If you look at the karyotype, you might have some abnormality, but you might still have a normal karyotype. The, the, the selective advantage might be due to just a mutation that you cannot see by doing a karyotype. So the fact that they grow much faster is by itself I think a little bit worser.And if we want to use these cells for transplantation at some point, I would definitely not use this type of cells.Okay. So would you like to tell me what are the main technical problems with using this technique for, for passages of cells? So using S, yes.It's just a little bit longer and also it's, since the cells are not going as fast, it's just much longer to get a high number of cells to do some experiment. So it's just less convenient I would say. Would you like to tell me about few words about the goals of your experiment?How do you use CS cells? Okay, so my main project here is to get endothelial cells from human AB stem cells. And the idea is to be able to make blood.And we are working a lot with tissue bioengineering labs and one of their goal is to make tissue in vitro. But one of the limitation to this is the lack of blood vessels going through this tissues. So if we can bring endothelial cells or put endothelial cells in this tissue, we would have better growth of this tissue.So the idea few sources of cells are known for endothelial cells like cold blood or pressure blood, but it would be interesting to have another source of cells like human years. And the advantage is since human years can be expanded and most infinitely, you could modify these cells, make some inducible system and be able to use it. Then in your final product, your material cells, in this case.

人类胚胎干细胞（ES细胞）的繁殖

科研

生物学

Counting Human Neural Stem Cells

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 plating cells for immunocytochemistry or for cell transfections. This protocol describes a straightforward and fast method for differentiating between live and dead cells and quantifying the cell concentration and total cell number using a hemacytometer. This procedure first requires detaching cells from a growth surface and resuspending them in media. Next, the cells are diluted in a solution of Trypan blue (ideally to a concentration that will give 20-50 cells per quadrant) and placed in the hemacytometer. Finally, averaging the counts of viable cells in several randomly selected quadrants, dividing the average by the volume of one 1 mm2 quadrant (0.1 &mu;l) and multiplying by the dilution factor gives the number of cells per l. Multiplying this cell concentration by the total volume in &mu;l gives the total cell number. This protocol describes counting human neural stem/precursor cells (hNSPCs), but can also be used for many other cell types.

Knowledge of the exact number of viable cells is required for many tissue culture manipulations. This protocol describes how to differentiate between live and dead cells and quantify cells using a hemacytometer. Although it describes counting human neural stem/precursor cells (hNSPCs), it can be used for other cell types.

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

The ability to manipulate human neural stem/precursor cells (hNSPCs) in vitro provides a means to investigate their utility as cell transplants for therapeutic purposes as well as to explore many fundamental processes of human neural development and pathology.  This protocol presents a simple method of culturing and passaging hNSPCs in hopes of standardizing this technique and increasing reproducibility of human stem cell research.  The hNSPCs we use were isolated from cadaveric postnatal brain cortices by the National Human Neural Stem Cell Resource and grown as adherent cultures on flasks coated with fibronectin (Palmer et al., 2001; Schwartz et al., 2003).  We culture our hNSPCs in a DMEM:F12 serum-free media supplemented with EGF, FGF, and PDGF and passage them 1:2 approximately every seven days.  Using these conditions, the majority of the cells in the culture maintain a bipolar morphology and express markers of undifferentiated neural stem cells (such as nestin and sox2).

The ability to manipulate human neural stem/precursor cells (hNSPCs) in vitro allows to investigate their utility as cell transplants for therapeutic purposes and to explore human neural development. This protocol presents a method of culturing and passaging hNSPCs in hopes of increasing reproducibility of human stem cell research.

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

Immunocytochemistry is a very powerful and fairly straightforward method for determining the presence, subcellular localization, and relative abundance of an antigen of interest, most commonly a protein, in cultured cells. This protocol presents an easy-to-follow series of steps that will enable researchers to conserve primary and secondary antibodies while getting high quality, reproducible qualitative and quantitative data out of their staining. There are two aspects of this protocol that help to conserve the volume of antibody necessary for staining. For one, the cells are grown on small, circular coverslips that are placed in wells of a tissue culture plate. After fixation, the cells on coverslips can be removed from the wells of the plate. For antibody staining, the coverslip with cells is inverted onto a small drop of antibody solution on parafilm and is covered with a second piece of parafilm to prevent drying. Using this method, only ~25 &mu;l of antibody solution is needed for each coverslip (or sample) to be stained. This protocol describes immunostaining of human neural stem/precursor cells (hNSPCs), but can be used for many other cell types.

Immunocytochemistry is a powerful method to determine the presence, subcellular localization, and relative abundance of an antigen of interest in cultured cells. This protocol presents an easy-to-follow series of steps that will enable one to conserve antibodies and get the most out of one's staining.

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

The functional and structural complexity of the myriad of cells in metazoan organisms arises from a small number of stem cells. Stem cells are characterized by two fundamental properties: self-renewal and multipotency that allows a stem cell to differentiate into virtually any cell type 1. The progression stem cell to differentiated cell is characterized by loss of multipotency, structural and morphological changes and the hierarchic activity of transcription factors and signaling molecules, whose activities establish and maintain cell-type specific gene expression patterns. At the molecular level, cell differentiation involves dynamic changes of the structure and composition of chromatin and the detection of those dynamic changes can provide valuable insights into the functional features of stem cells and the cell differentiation process 2,3. Chromatin is a highly compacted DNA-protein complex that forms when cells package chromosomal DNA with proteins, mainly histones 4. Stemcellness and cell differentiation has been correlated with the presence of specific arrays of regulatory proteins such as epigenetic factors, histone variants, and transcription factors 2,3,5.
 Chromatin immunoprecipitation (ChIP) provides a valuable method to monitor the presence of RNA, proteins, and protein modifications in chromatin 6,7. The comparison of chromatin from different cell types can elucidate dynamic changes in protein-chromatin associations that occur during cell differentiation.
Chromatin immunoprecipitation involves the purification of in vivo cross-linked chromatin. The isolated chromatin is reduced to smaller fragments by enzymatic digestion or mechanical force. Chromatin fragments are precipitated using specific antibodies to target proteins or protein and DNA modifications. The precipitated DNA or RNA is purified and used as a template for PCR or DNA microarray based assays. Prerequisites for a successful ChIP are high quality antibodies to the desired antigen and the availability of chromatin from control cells that do not express the target molecule. ChIP can correlate the presence of proteins, protein and RNA modifications, and RNA with specific target DNA, and depending on the choice of outread tool, detects the association of target molecules at specific target genes or in the context of an entire genome. The comparison of the distribution of proteins in the chromatin of differentiating cells can elucidate the dynamic changes of chromatin composition that coincide with the progression of cells along a cell lineage.

The differentiation of ESC coincides with cell-type specific changes in the structure and composition of chromatin. The detection of those changes provides valuable insights into the mechanisms that define stemcellness and cell differentiation. Chromatin immunoprecipitation (ChIP) represents a valuable method to dissect the molecular mechanisms underlying stem cell differentiation.

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

Mitochondria are the main site of ATP production during aerobic metabolism in eukaryotic non-photosynthetic cells1. These complex organelles also play essential roles in apoptotic cell death2, cell survival3, mammalian development4, neuronal development and function4, intracellular signalling5, and longevity regulation6. Our understanding of these complex biological processes controlled by mitochondria relies on robust methods for assessing their morphology, their protein and lipid composition, the integrity of their DNA, and their numerous vital functions. The budding yeast Saccharomyces cerevisiae, a genetically and biochemically manipulable unicellular eukaryote with annotated genome and well-defined proteome, is a valuable model for studying the molecular and cellular mechanisms underlying essential biological functions of mitochondria. For these types of studies, it is crucial to have highly pure mitochondria. Here we present a detailed description of a rapid and effective method for purification of yeast mitochondria. This method enables the isolation of highly pure mitochondria that are essentially free of contamination by other organelles and retain their structural and functional integrity after their purification. Mitochondria purified by this method are suitable for cell-free reconstitution of essential mitochondrial processes and can be used for the analysis of mitochondrial structure and functions, mitochondrial proteome and lipidome, and mitochondrial DNA.

We describe a rapid and effective method for purification of mitochondria from the yeast Saccharomyces cerevisiae. This method enables the high-yield isolation of pure mitochondria that are essentially free of contamination by other organelles and retain their structural and functional integrity after their purification.

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

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 from a frozen stock have become important procedures performed in routine hES cell culture. Since hES cells are very sensitive to the stresses of freezing and thawing, special care must taken. Here we demonstrate the proper technique for rapidly thawing hES cells from liquid nitrogen stocks, plating them on mouse embryonic feeder cells, and slowly freezing them for long-term storage.

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 from a frozen stock have become important procedures performed in routine hES cell culture. Since hES cells are very sensitive to the stresses of freezing and thawing, special care must taken. Here we demonstrate the proper technique for rapidly thawing hES cells from liquid nitrogen stocks, plating them on mouse embryonic feeder cells, and slowly freezing them for long-term storage.

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

Oligodendrocytes are the myelinating cells of the central nervous system. For regenerative cell therapy in demyelinating diseases, there is significant interest in deriving a pure population of lineage-committed oligodendrocyte precursor cells (OPCs) for transplantation. OPCs are characterized by the activity of the transcription factor Olig2 and surface expression of a proteoglycan NG2. Using the GFP-Olig2 (G-Olig2) mouse embryonic stem cell (mESC) reporter line, we optimized conditions for the differentiation of mESCs into GFP+Olig2+NG2+ OPCs. In our protocol, we first describe the generation of embryoid bodies (EBs) from mESCs. Second, we describe treatment of mESC-derived EBs with small molecules: (1) retinoic acid (RA) and (2) a sonic hedgehog (Shh) agonist purmorphamine (Pur) under defined culture conditions to direct EB differentiation into the oligodendroglial lineage. By this approach, OPCs can be obtained with high efficiency (&gt;80%) in a time period of 30 days. Cells derived from mESCs in this protocol are phenotypically similar to OPCs derived from primary tissue culture. The mESC-derived OPCs do not show the spiking property described for a subpopulation of brain OPCs in situ. To study this electrophysiological property, we describe the generation of spiking mESC-derived OPCs by ectopically expressing NaV1.2 subunit. The spiking and nonspiking cells obtained from this protocol will help advance functional studies on the two subpopulations of OPCs.

We describe a small molecule-based protocol for differentiation of mouse embryonic stem cells into oligodendrocyte precursor cells (OPCs). This protocol generates Olig2+NG2+ OPCs with high efficiency by 30 days of differentiation. We also describe a method to generate "spiking" OPCs that can fire action potentials.

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

Genetic modification is continuing to be an essential tool in studying stem cell biology and in setting forth potential clinical applications of human embryonic stem cells (HESCs)1. While improvements in several gene delivery methods have been described2-9, transfection remains a capricious process for HESCs, and has not yet been reported in human induced pluripotent stem cells (iPSCs). In this video, we demonstrate how our lab routinely transfects and nucleofects human iPSCs using plasmid with an enhanced green fluorescence protein (eGFP) reporter. Human iPSCs are adapted and maintained as feeder-free cultures to eliminate the possibility of feeder cell transfection and to allow efficient selection of stable transgenic iPSC clones following transfection. For nucleofection, human iPSCs are pre-treated with ROCK inhibitor11, trypsinized into small clumps of cells, nucleofected and replated on feeders in feeder cell-conditioned medium to enhance cell recovery. Transgene-expressing human iPSCs can be obtained after 6 hours. Antibiotic selection is applied after 24 hours and stable transgenic lines appear within 1 week. Our protocol is robust and reproducible for human iPSC lines without altering pluripotency of these cells.

Despite recent advancements in genetic modification, transfection of human embryonic stem cells (HESCs) remains a capricious process. To our knowledge, systematic and efficient methods to transfect human induced pluripotent stem cells (iPSCs) have not been reported. Here, we describe robust protocols to efficiently transfect and nucleofect human iPSCs.

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

The mitochondrial permeability transition pore (mtPTP) is a non specific channel that forms in the inner mitochondrial membrane to transport solutes with a molecular mass smaller than 1.5 kDa. Although the definitive molecular identity of the pore is still under debate, proteins such as cyclophilin D, VDAC and ANT contribute to mtPTP formation. While the involvement of mtPTP opening in cell death is well established1, accumulating evidence indicates that the mtPTP serves a physiologic role during mitochondrial Ca2+ homeostasis2, bioenergetics and redox signaling 3.
mtPTP opening is triggered by matrix Ca2+ but its activity can be modulated by several other factors such as oxidative stress, adenine nucleotide depletion, high concentrations of Pi, mitochondrial membrane depolarization or uncoupling, and long chain fatty acids4. In vitro, mtPTP opening can be achieved by increasing Ca2+ concentration inside the mitochondrial matrix through exogenous additions of Ca2+ (calcium retention capacity). When Ca2+ levels inside mitochondria reach a certain threshold, the mtPTP opens and facilitates Ca2+ release, dissipation of the proton motive force, membrane potential collapse and an increase in mitochondrial matrix volume (swelling) that ultimately leads to the rupture of the outer mitochondrial membrane and irreversible loss of organelle function.
Here we describe a fluorometric assay that allows for a comprehensive characterization of mtPTP opening in isolated mouse heart mitochondria. The assay involves the simultaneous measurement of 3 mitochondrial parameters that are altered when mtPTP opening occurs: mitochondrial Ca2+ handling (uptake and release, as measured by Ca2+ concentration in the assay medium), mitochondrial membrane potential, and mitochondrial volume. The dyes employed for Ca2+ measurement in the assay medium and mitochondrial membrane potential are Fura FF, a membrane impermeant, ratiometric indicator which undergoes a shift in the excitation wavelength in the presence of Ca2+, and JC-1, a cationic, ratiometric indicator which forms green monomers or red aggregates at low and high membrane potential, respectively. Changes in mitochondrial volume are measured by recording light scattering by the mitochondrial suspension. Since high-quality, functional mitochondria are required for the mtPTP opening assay, we also describe the steps necessary to obtain intact, highly coupled and functional isolated heart mitochondria.

A spectrofluorometric protocol for the measurement of the mitochondrial permeability transition pore opening in isolated mouse heart mitochondria is presented here. The assay involves the simultaneous measurement of mitochondria Ca2+ handling, mitochondrial membrane potential and mitochondrial volume. The procedure for obtaining high-quality and functional heart mitochondria is also described.

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 ...