The Satchi-Fainaro research laboratory is partially supported by The Association for International Cancer Research (AICR), German-Israel Foundation (GIF), THE ISRAEL SCIENCE FOUNDATION (Grant No. 1309/10), Swiss Bridge Award, and by grants from the Israeli National Nanotechnology Initiative (INNI), Focal Technology Area (FTA) program: Nanomedicine for Personalized Theranostics, and by The Leona M. and Harry B. Helmsley Nanotechnology Research Fund.

注：所有动物的程序获得批准，并符合医学实验动物管理和使用委员会的特拉维夫大学萨克勒学校（IACUC）的标准执行。 1.空调媒体准备<ol><li>种子2×10 6的U-87 MG细胞在10cm的培养皿中的体积8毫升Dulbecco改进的Eagle培养基（DMEM），补充有10％胎牛血清（FBS）和1％青霉素/链霉素/制霉菌素溶液。 </li><li>孵育细胞在37 °℃; 5％CO 2，直到48小时实现全面融合。 </li><li>使用10毫升注射器，撤回所有介质从板和过滤通过0.45微米的过滤入15ml管中。 </li><li>未经洗涤细胞板，加入1 ml胰蛋白酶溶液（0.25％），并培育。 </li><li>当所有的细胞分离，使用至少5毫升的DMEM补充有10％FBS的灭活胰蛋白酶和预型体细胞计数。 </li><li<html> &gt;计数使用血球或其他细胞计数装置细胞的数量，并根据所使用的计数方法计算细胞的数目。 注：将在后面以正常化的CM的浓度被使用。 </li><li>所述CM转移到玻璃圆底烧瓶中并浓缩，在高真空旋转蒸发。为了加快该过程，增加浴温至37℃，如果需要的话。继续下去，直到CM达到糊状的质地。 </li><li>悬浮从每个培养皿中的400微升生理盐水CM。当使用一个以上的板，可使用等效的体积。例如，对于2培养板中，使用800微升。用每块板的4只小鼠。 </li><li>为了比较不同的细胞系或治疗，根据在步骤1.6接收到的细胞的比率调整音量。例如，如果一个板包含1.5倍以上的细胞，添加200μl的盐水，以预先存在的400微升。这将是调节的CM </li></ol>标题“&gt; 2。ECM-模仿硅胶塞接种到小鼠<ol><li>接种前的ECM-模仿凝胶到小鼠体内，准备以下材料： <ol><li>生长因子减少苯酚的无ECM-模拟物凝胶（解冻过夜，在实验前一天，在4℃下在冰上）。 </li><li>电动剃须刀。 </li><li>氯胺酮和甲苯噻嗪以麻醉小鼠。 </li><li>冰冷的1000微升无菌针头。 </li><li>冰冷1ml注射器和27G的针头。 </li></ol></li><li>准备每只小鼠含有80微升调节的CM 1.5ml的Eppendorf管中，并保持在冰上。 </li><li>向每个管中，加入600微升冰冷的生长因子被解冻过夜，在4℃，在冰上减少苯酚的无ECM-模拟凝胶。对ECM-模拟凝胶是液体在4℃，并固化在体温;为了防止对ECM-模仿凝胶从固化，用冰冷1000微升的提示。混合管仔细不会形成气泡，并保持在冰上。 </li><li>麻醉6周老B使用氯胺酮（100毫克/千克）和赛拉嗪（12毫克/千克）ALB / c小鼠。根据在开始实验前的机构动物护理和使用委员会， 即确认适当麻醉。动物已经达到麻醉自己正确的飞机时，他们不再以坚定的脚趾捏响应。 </li><li>剃鼠的腹部和消毒使用外科擦洗/醇。 </li><li>使用冷1ml注射器和27G的针来注入Eppendorf管皮下的内容。进行注射非常缓慢，以防止泡沫的形成。一旦所有卷管理，不弹出针几分钟，直至凝固发生。 </li></ol> 3.评估ECM-模仿硅胶塞血管化利用超声注：这部分协议要求操作超声成像系统，或经授权的技术人员的协助下以前的知识。 <ol><li>在开始之前，必须在Following材料和工具准备： <ol><li>电动剃须刀。 </li><li>脱毛霜。 </li><li>氯胺酮和甲苯噻嗪以麻醉小鼠。 </li><li>非针对性的微泡。 </li><li>激活设备。 </li><li>美国成像系统，其包括一个55兆赫708探针和3D获取显微。 </li></ol></li><li>插头后三周接种内测量血管： 注：在U-87 MG型动物模型，3周被发现代表了成像的理想时间点。根据不同的设置，校准从接种后塞几天开始血管动力学。 <ol><li>麻醉用氯胺酮（100毫克/千克）和赛拉嗪（12毫克/公斤）小鼠。根据在开始实验前的机构动物护理和使用委员会， 即确认适当麻醉。动物已经达到麻醉自己正确的飞机时，他们不再以坚定的脚趾捏响应。 </li><li>剃小鼠的腹部区域，并用脱毛霜。 </li><li>固定的小鼠仰卧在微操纵器的阶段。 </li><li>使用含生理盐水1ml注射器和一个地下30针，将针尾静脉内，固定针头牢固，松动的注射器。 </li><li>建立起来用于对比度3D模式的图像采集会话中： <ol><li>按“3D”初始化3D马达阶段，并将换能器在所需的扫描区域的中间。 </li><li>按“3D”，并输入扫描距离和步长，然后按“扫描”。 </li><li>确保整个插头被扫描中显示的3D图像数据。如果不是这样，调整振子，以使整个插头是在扫描和重复步骤3.5.2可见。 </li><li>按“对比度”，推动了时间增益补偿（TGC）控制到左边使图像变暗。 </li></ol></li><li>激活使用小瓶混合机是DESI微泡gnated对微泡的活化为45秒。 </li><li>混合在1ml注射器将50μl的微泡用50μl生理盐水。 </li><li>更换含生理盐水松动注射器含有活性微泡的注射器。注入稀释微泡入尾静脉，小鼠的，等待几秒钟的气泡，以达到稳定状态，然后获得插头的3D造影增强电影回放，使用3D采集电机： <ol><li>按“3D”，然后选择“扫描”，以获得3D对比度增强图像，然后按“电影商店”保存图像数据。 </li><li>按“3D”，然后单击“消灭3D”，以销毁的微泡。 </li><li>按“3D”再选择“扫描”收购后破坏电影圈，然后按“电影商店”。 </li></ol></li><li>生成的叠加图像帧之前和之后的破坏微泡： <ol><li>在＆＃8220;参考设置“在屏幕的左侧点击”创建引用“。 </li><li>按“学习管理”，打开获得的预破坏电影圈。 </li><li>单击“进程电影”生成绿色的对比叠加，然后单击“加载到3D”。 </li></ol></li><li>使用美国成像软件，以定量的插头内的血液量得到的三维图像： <ol><li>按“扫描/冻结”，然后按“模式设置”。 </li><li>单击“音量”，然后选择“并行”。 </li><li>通过分割一系列轮廓创建目标组织的三维体积。 </li><li>点击“F​​inish”完成分割。 注：在造影剂的量将在屏幕的左下侧显示。 </li></ol></li><li>在程序结束时，小鼠放置在温暖，干净，干燥，安静的环境，远离其他动物。使用市售的手术加热垫或白炽灯（50-75瓦），12-14英寸的距离放置啮齿动物提供温暖。连续监测小鼠，直到保持直立的姿势和行走正常。 </li></ol></li></ol> 4.评价ECM-模仿硅胶塞脉管形态使用纤维状的共聚焦显微内镜注：这部分协议的执行紧随美国成像，它需要操作纤维质的共聚焦显微内镜成像系统，或经授权的技术人员的协助下以前的知识。 <ol><li>开始前，有下列材料和工具准备： <ol><li>手术工具，如镊子，剪刀和可吸收缝合线。 </li><li>的FITC缀合的葡聚糖（70 kDa）的。 </li><li>校准溶液（3试管各含有过氧化氢，水或荧光溶液）。 </li><li>所述成像系统和一个探头适于血管根据制造商的成像。 </li></ol></li><li>麻醉用氯胺酮（100毫克/千克）和赛拉嗪（12毫克/公斤）小鼠。 </li><li>辖四，经尾静脉，200微升10毫克/毫升的FITC-标记的葡聚糖。 </li><li>使用外科擦洗/酒精消毒腹部区域，并从插头施加一个小的切口大约1厘米。 </li><li>将显微内镜探针上的插头的顶部轻轻同时保持插头完好无损。 </li><li>收购： <ol><li>一旦探针被设置在插头上时，按使用脚踏板“开始激光”。荧光应该显示在屏幕上。 </li><li>按“记录”在屏幕上或&#39;选择&#39;使用的脚踏板获得短片（最长30秒）。 </li><li>电影之间，通过将尖端的含管水冲洗探头。 </li><li>完成后，取下探头，并用干净的棉签尖端。 </li></ol></li><li>分析平均值已经SSEL直径： <ol><li>点击“检测微血管”图标在上面板以打开容器检测模块窗口。 </li><li>点击“首选项”，并标注在“统计的利益”和“显示直方图条”中的“显示模式”，“平均血管直径”。 </li><li>点击“检测”，以显示选定的测量。 </li></ol></li><li>为了分析在邻近血管的区域的荧光信号强度： <ol><li>请点击左侧面板中的“创建圈子的投资回报率”，创造在感兴趣的领域了一圈。 </li><li>点击“范围内的投资回报率计算直方图”计算与本区域的值。 </li></ol></li></ol>小鼠5.后处理程序<ol><li>在每个成像过程的结束，将小鼠在一个温暖，干净，干燥，安静的环境，远离其他动物。使用市售-一个提供温暖vailable手术加热垫或白炽灯（50-75 W），放置在12-14远离啮齿动物。连续监测小鼠，直到保持直立的姿势和行走正常。 </li><li>对于塞“血管额外的时间点进一步分析，应用以下条件恢复： <ol><li>使用缝合吸收缝合线的小切口。 </li><li>给予小鼠镇痛如丁丙诺啡（1毫克/千克）经皮下，并返回到单独的笼子中。 </li><li>遵守所有的动物，以确定是否进一步止痛治疗是必要的和重新评估的日常监测和评估遇险。 </li></ol></li><li>在终点的实验中，小鼠安乐死通过首先施加异氟烷，随后通过颈脱位。 </li><li>如果额外的影像将在初始成像步骤周进行，所有的程序应保持无菌越好。应进行外科手术的皮肤擦洗，消毒娃之三和无菌棉尖端应当用来清洁显微内镜探针，并且探针应该使用无菌水各动物之间进行消毒。 </li></ol>

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The authors have nothing to disclose.

结合互补的成像方法允许在不同的血管生成组件的微血管密度，功能和形态的信息的获取。在ECM-模拟凝胶塞装载的CM产生一个肿瘤微环境，这是从其他细胞群中分离，如内皮细胞，该被招募并渗出到插头。 通过执行，并行地，微泡造影美国成像和纤维状共聚焦显微内镜成像，我们可以得出结论，一池由快速生长的血管生成的肿瘤产生的细胞分泌的因素，而不癌细胞本身，不?...

基质胶塞血管生成测定首先由Kibbey 等人于1992年，在那里它被用来由肽SIKVAV（丝氨酸-异亮氨酸-赖氨酸-缬氨酸-丙氨酸-缬氨酸）1来评估血管生成的刺激说明。相对于其他的体内血管发生测定法，像鼠标角膜血管发生或鸡胚尿囊膜测定法，该测定法是比较容易进行2。注入的ECM-模拟凝胶可包含细胞，促血管生成或抗血管生成的化合物和/或因素。当评估抗血管生成化合物的活性，所述插头通常包含的血管内皮生长因子（VEGF）和成纤维细胞生长因子（FGF）的混合物中，并且所述抗血管生成物质可直接施用到插头或全身三4。 对ECM-模拟凝胶皮下注射那里分钟内固化。血管招聘中产生的插头评估。典型ý通过以下注射荧光素异硫氰酸（FITC）测定血红蛋白水平内塞，通过荧光信号的测量进行标记的葡聚糖，通过组织学切片内皮特异性标记物的免疫染色或通过荧光激活细胞分选（FACS）2,5， 6。但是，此评价只允许终点分析，缺乏有关的血管的功能和形态信息。此外，血浆量的测量结果，无论是由血红蛋白水平或通过使用葡聚糖-FITC是一个指标，可能会产生误导，因为血液含量是受血管的大小和血停滞池的程度。这是特别重要的，当招募血管的特征在于增强的渗透性和保留（EPR）效果7。 我们在此通过结合两个互补成像方式提出了一种新的，更精确的，方法的招募血管可视化。 HIGH-分辨率微泡超声造影（美国）联合活fibred共焦显微内镜不仅能够提供有关血管密度，同时也对自己的形态和功能的信息。此外，这种分析可以在几个时间点，从而使监控血管发生动力学来执行。美国是一种广泛使用的成像模态而具有以下静脉内（IV）注射仍然存在只在血管隔室8-11微泡的高空间分辨率的血管成像。微泡是产生一个强回声信号时与美国脉冲激发，因此可作为一个良好的造影剂气体填充的微泡。使用下列微泡破坏美国成像系统软件中获取的插头的3D图像。所产生的图像是由前，后破坏微泡的图像帧组成的，并反映在彩色视频强度差。这些叠加图像由软件自动显示。因此，插头内的功能血管的百分比可以量化。纤维状共聚焦显微内镜高分辨率作为通过血管形态报告的补充成像方式。在这种微创方法，图像采集的FITC标记的右旋糖酐12静脉给药后。荧光聚合物染料的血液，从而作为“造影剂”的血管形态和血流。此外，由于所注入的聚合物是在70 kDa的大小，它只能交叉渗漏血管如肿瘤血管中找到。这将导致从血管外的FITC-标记的葡聚糖的高背景。因此，纤维状的共聚焦显微内镜可用于可视化的EPR效应典型特征在肿瘤neovasculature-放大，泄漏的，高度缠结的船只，具有平端。 该系统模型descri床的U-87 MG人脑胶质瘤和休眠克隆13的血管生成潜力的比较。插头内血管进行评价后三周的ECM-模拟凝胶接种。结果表明，含有的CM从插头内的血管的U-87 MG快速增长的数量，密度和功能性，方面当与含有的CM从休眠肿瘤产生的细胞内塞血管相比肿瘤产生细胞显著增加。因此，研究者们能够得出这样的结论CM从隔离U-87 MG快速增长的肿瘤细胞产生含有较高水平的促血管生成因子，从休眠的肿瘤细胞产生的CM相比。这些因素刺激功能血管的形成通过积极地影响所有步骤的血管发生级联（ 即 ，增殖，出芽，迁移，最后，形成内皮细胞的管状结构）。

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	<tbody>
		<tr height="40">
			<td dir="LTR" height="40" style="height:40px;width:255px;">Name of the Material/Equipment</td>
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			<td dir="LTR" style="width:179px;">Catalog number</td>
			<td dir="LTR" style="width:251px;">Comments/Description&#160;</td>
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			<td dir="LTR" height="40" style="height:40px;width:255px;">Rotary Vacuum Evaporator</td>
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			<td dir="LTR" style="width:179px;">-</td>
			<td style="width:251px;">-</td>
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			<td dir="LTR" height="40" style="height:40px;width:255px;">Growth-factors reduced phenol-free Matrigel</td>
			<td dir="LTR" style="width:179px;">BD Bioscience</td>
			<td dir="LTR" style="width:179px;">FAL356231</td>
			<td style="width:251px;">Thaw overnight, at 40C on ice&#160;</td>
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			<td dir="LTR" height="40" style="height:40px;width:255px;">Depilatory cream&#160;</td>
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			<td dir="LTR" height="40" style="height:40px;width:255px;">Vevo2100 US imaging system</td>
			<td dir="LTR" style="width:179px;">VisualSonics Inc.&#160;</td>
			<td dir="LTR" style="width:179px;">-</td>
			<td dir="LTR" style="width:251px;">Requires previous knowledge in operating or the assistance of an authorized technician</td>
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			<td dir="LTR" height="40" style="height:40px;width:255px;">55 MHz 708 probe</td>
			<td dir="LTR" style="width:179px;">VisualSonics Inc.</td>
			<td dir="LTR" style="width:179px;">-</td>
			<td dir="LTR" style="width:251px;">-</td>
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			<td dir="LTR" height="40" style="height:40px;width:255px;">Vevo2100 imaging software</td>
			<td dir="LTR" style="width:179px;">VisualSonics Inc.</td>
			<td dir="LTR" style="width:179px;">-</td>
			<td dir="LTR" style="width:251px;">-</td>
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			<td dir="LTR" height="40" style="height:40px;width:255px;">VialMix&#160;</td>
			<td dir="LTR" style="width:179px;">DEFINITY Imaging</td>
			<td dir="LTR" style="width:179px;">VMIX</td>
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			<td dir="LTR" height="40" style="height:40px;width:255px;">DEFINITY microbubbles</td>
			<td dir="LTR" style="width:179px;">DEFINITY Imaging</td>
			<td dir="LTR" style="width:179px;">DE4</td>
			<td dir="LTR" style="width:251px;">Activate for 45 seconds using Vialmix before use</td>
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		<tr height="40">
			<td height="40" style="height:40px;width:255px;">CellVizio (Fibered confocal endomicroscope)</td>
			<td dir="LTR" style="width:179px;">Mauna Kea Technologies</td>
			<td dir="LTR" style="width:179px;">-</td>
			<td dir="LTR" style="width:251px;">Requires previous knowledge in operating or the assistance of an authorized technician</td>
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			<td dir="LTR" height="40" style="height:40px;width:255px;">ProFlex MiniO/30 probe</td>
			<td dir="LTR" style="width:179px;">Mauna Kea Technologies</td>
			<td dir="LTR" style="width:179px;">-</td>
			<td dir="LTR" style="width:251px;">-</td>
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			<td dir="LTR" height="40" style="height:40px;width:255px;">70 kD FITC-labeled Dextran</td>
			<td dir="LTR" style="width:179px;">Sigma-Aldrich</td>
			<td dir="LTR" style="width:179px;">46945</td>
			<td dir="LTR" style="width:251px;">-</td>
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		<tr height="40">
			<td dir="LTR" height="40" style="height:40px;width:255px;">ImageCell software</td>
			<td dir="LTR" style="width:179px;">Mauna Kea Technologies</td>
			<td dir="LTR" style="width:179px;">-</td>
			<td dir="LTR" style="width:251px;">-</td>
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		<tr height="40">
			<td dir="LTR" height="40" style="height:40px;width:255px;">Calibration Kit</td>
			<td dir="LTR" style="width:179px;">Mauna Kea Technologies</td>
			<td dir="LTR" style="width:179px;">-</td>
			<td dir="LTR" style="width:251px;">-</td>
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		<tr height="40">
			<td align="left" height="40" style="height:40px;">Dulbecco&#8217;s modified Eagle&#8217;s medium (DMEM)</td>
			<td align="left">Gibco Life Tecchnologies</td>
			<td dir="LTR" style="width:179px;">41965-039</td>
			<td></td>
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			<td align="left" height="37" style="height:37px;">Fetal bovine serum (FBS)</td>
			<td align="left">Biological Industries&#160;</td>
			<td align="left">04-007-1A</td>
			<td></td>
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			<td align="left" height="39" style="height:39px;">Penicillin-Streptomycin-Nystatin Solution</td>
			<td align="left">Biological Industries&#160;</td>
			<td align="left">03-032-1B</td>
			<td></td>
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monitoring functionality and morphology of vasculature recruited by factors secreted by fast-growing tumor-generating cells

在小鼠中的皮下基质胶栓测定是用于亲和抗血管生成因子在体内的评价所选择的方法。在该方法中，期望的因素被引入到冷液体的ECM-模拟凝胶其中，皮下注射后，固化形成模仿癌环境的环境。这个矩阵允许的宿主细胞，如内皮细胞，和血管因此，形成的渗透。 这里我们提出了一种新的改性的基质胶塞测定法，其可被利用来说明由癌细胞分泌的因子池的血管生成潜力，而不是一个特定的因子（ 例如，bFGF及VEGF）或代理。含ECM-模拟凝胶插头用来引进主机（ 即鼠标）与分泌，以快速增长的肿瘤生成胶质母细胞瘤细 ​​胞的CM因素池。我们前面所述的血管生成潜在的广泛的比较的U-87 MG人类成胶质细胞瘤及其蛰伏衍生克隆，在该系统模型，示出在U-87 MG亲代细胞诱导的血管生成。所述CM通过过滤从任一细胞系的汇合组织培养板下面48小时培养收集介质制备。因此，它仅包含由细胞分泌的因素，而不细胞本身。这里描述的是两个成像模态的组合，微泡超声造影成像和活体纤维质共焦显微内镜，为准确，实时的程度，形态表征插头内新形成的血管和功能。

植入ECM-模拟胶栓从快速增长的血管生成肿瘤产生的细胞系U-87 MG，导致广泛的血管生成CM。这种脉管可以通过使用两个互补的成像方法进行了广泛探索。 微泡造影剂增强成像美国由表示内的U-87 MG的CM加载插头，而不是检测不到血流含有从休眠肿瘤产生细胞的CM插头内（ 图1）广泛的血流量显示的功能性血管进入插头广泛招募。 这些新形成的血?...

Watch this Scientific Journal Video about Monitoring Functionality and Morphology of Vasculature Recruited by Factors Secreted by Fast-growing Tumor-generating Cells at JoVE.com

Monitoring Functionality and Morphology of Vasculature Recruited by Factors Secreted by Fast-growing Tumor-generating Cells

We describe a matrigel plug assay to illustrate angiogenic potential of a pool of factors secreted by cancer cells using two complementary imaging modalities, ultrasound and endomicroscopy. The matrigel, an extracellular matrix (ECM)-mimic gel, is utilized to introduce the host (mouse) with angiogenic factors secreted to the conditioned media (C.M.).&#160;

监控功能和血管形态由快速增长的肿瘤产生细胞分泌的因素招募

The subcutaneous matrigel plug assay in mice is a method of choice for the in vivo evaluation of pro- and anti-angiogenic factors. In this method, desired ...

monitoring-functionality-morphology-vasculature-recruited-factors

Research

JoVE Journal

Biology

9.7K 次观看.  Tel Aviv University. We describe a matrigel plug assay to illustrate angiogenic potential of a pool of factors secreted by cancer cells using two complementary imaging modalities, ultrasound and endomicroscopy. The matrigel, an extracellular matrix (ECM)-mimic gel, is utilized to introduce the host (mouse) with angiogenic factors secreted to the conditioned media (C.M.). 

视频: Monitoring Functionality and Morphology of Vasculature Recruited by Factors Secreted by Fast-growing Tumor-generating Cells

Derivation of Human Embryonic Stem Cells by Immunosurgery

The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both medical applications and as a research 
tool for addressing fundamental questions in development and disease. Here, we provide a 
concise, step-by-step protocol for the derivation of human embryonic stem cells from embryos 
by immunosurgical isolation of the inner cell mass. 


The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both medical applications and as a research 
tool for addressing fundamental questions in development and disease. Here, we provide a 
concise, step-by-step protocol for the derivation of human embryonic stem cells from embryos 
by immunosurgical isolation of the inner cell mass.

人类胚胎干细胞的推导Immunosurgery

The ability of human embryonic stem cells to self-renew and differentiate into all cell types of 
the body suggests that they hold great promise for both ...

科研

生物学

Monitoring Acupuncture Effects on Human Brain by fMRI

Functional MRI is used to study the effects of acupuncture on the BOLD response and the functional connectivity of the human brain.  Results demonstrate that acupuncture mobilizes a limbic-paralimbic-neocortical network and its anti-correlated sensorimotor/paralimbic network at multiple levels of the brain and that the hemodynamic response is influenced by the psychophysical response.  Physiological monitoring may be performed to explore the peripheral response of the autonomic nerve function. This video describes the studies performed at LI4 (hegu), ST36 (zusanli) and LV3 (taichong), classical acupoints that are commonly used for modulatory and pain-reducing actions. Some issues that require attention in the applications of fMRI to acupuncture investigation are noted.

FMRI and physiological monitoring is used to study the effects of Acupuncture on the central and peripheral nervous systems. Acupuncture mobilizes a limbic-paralimbic-neocortical network, with great overlap with the default mode network, to modulate neurological activity, possibly related to its autonomic effect in the peripheral nervous system.

通过功能磁共振成像监测针灸对人脑的影响

Functional MRI is used to study the effects of acupuncture on the BOLD response and the functional connectivity of the human brain.  Results demonstrate ...

Fate Mapping of Human Embryonic Stem Cells by Teratoma Formation

Human embryonic stem cells (hESCs) have an unlimited capacity for self-renewal, and the ability to differentiate into cells derived from all three embryonic germ layers (1). Directed differentiation of hESCs into specific cell types has generated much interest in the field of regenerative medicine (e.g., (2-5)), and methods for determining the in vivo fate of selected or manipulated hESCs are essential to this endeavor. We have adapted a highly efficient teratoma formation assay for this purpose. A small number of specifically selected hESCs is mixed with undifferentiated wild type hESCs and Phaseolus vulgaris lectin to form a cell pellet. This is grafted beneath the kidney capsule in an immunodeficient mouse. As few as 2.5 x 105 hESCs are needed to form a 16 cm3 teratoma within 8-12 weeks. The fate of the originally selected hESCs can then be determined by immunohistochemistry. This method provides a valuable tool for characterizing tissue-specific reagents for cell-based therapy.

Directed differentiation of hESCs into specific cells has generated much interest in regenerative medicine. We provide a concise, step-by-step protocol for determining the in vivo fate of selected hESCs that provides a valuable tool for characterizing tissue-specific reagents for cell-based therapy.

人类胚胎干细胞畸胎瘤形成命运的映射

Human embryonic stem cells (hESCs) have an unlimited capacity for self-renewal, and the ability to differentiate into cells derived from all three ...

In vivo Liver Endocytosis Followed by Purification of Liver Cells by Liver Perfusion

The liver is the metabolic center of the mammalian body and serves as a filter for the blood. The basic architecture of the liver is illustrated in figure 1 in which more than 85% of the liver mass is composed of hepatocytes and the remaining 15% of the cellular mass is composed of Kupffer cells (KCs), stellate cells (HSCs), and sinusoidal endothelial cells (SECs). SECs form the blood vessel walls within the liver and contain specialized morphology called fenestrae within in the cytoplasm. Fenestration of the cytoplasm is the appearance of holes (&tilde;100 &mu;m) within the cells so that the SECs act as a sieve in which most chylomicrons, chylomicron remnants and macromolecules, but not cells, pass through to the hepatocytes and HSCs 1 (Fig. 1). Due to the lack of a basement membrane, the gap between the SECs and hepatocytes form the Space of Disse. HSCs occupy this space and play a prominent role in regulation and response to injury, storage of retinoic acid and immunoregulation of the liver 2.
SECs are among the most endocytically active cells of the body displaying an array of scavenger receptors on their cell surface 3. These include SR-A, Stabilin-1 and Stabilin-2. Generally, small colloidal particles less than 230 nm and macromolecules in buffer phase are taken up by SECs, whereas, large particles and cellular debris is endocytosed (phagocytosed) by KCs 4. Thus, the bulk clearance of extracellular material such as the glycosaminoglycans from blood is largely dependent on the health and endocytic functions of SECs 5,6. For example, an increase in blood hyaluronan levels is indicative of liver disease ranging from mild to more severe forms 7.
With the exception of one report 8, there are no immortalized SEC cell lines in existence. Even this immortalized cell line is de-differentiated in that it does not express scavenger receptors that are present on primary SECs (our data, not shown). All cell biological studies must be performed on primary cells obtained freshly from the animal. Unfortunately, SECs dedifferentiate under standard culture conditions and must be used within 1 or 2 days upon isolation from the animal. Differentiation of SECs is marked by the expression of Stabilin-2 or HARE receptor 9 , CD31, and the presence of cytoplasmic fenestration 1. Differentiation of SECs can be extended by the addition of VEGF in culture media or by culturing cells in hepatocyte conditioned medium 10,11.
In this report, we will demonstrate the endocytic activity of SECs in the intact organ using radio-labeled heparin for hyaluronan for the SEC-specific Stabilin-2 receptor. We will then purify hepatocytes and SECs from the perfused liver to measure endocytosis.

In vivo Liver Endocytosis Followed by Purification of Liver Cells by Liver Perfusion

The study of liver sinusoidal endothelial cells (SECs) must be performed with primary cells obtained from the animal as no cell lines exist. This method relies on liver digestion and differential centrifugation for SEC purification for subsequent culturing and experimentation.

在体内肝细胞的分离纯化的肝细胞内吞其次是肝脏灌注

The liver is the metabolic center of the mammalian body and serves as a filter for the blood.  The basic architecture of the liver is illustrated in ...

Monitoring Equilibrium Changes in RNA Structure by 'Peroxidative' and 'Oxidative' Hydroxyl Radical Footprinting

RNA molecules play an essential role in biology. In addition to transmitting genetic information, RNA can fold into unique tertiary structures fulfilling a specific biologic role as regulator, binder or catalyst. Information about tertiary contact formation is essential to understand the function of RNA molecules. Hydroxyl radicals (&bull;OH) are unique probes of the structure of nucleic acids due to their high reactivity and small size.1 When used as a footprinting probe, hydroxyl radicals map the solvent accessible surface of the phosphodiester backbone of DNA1 and RNA2 with as fine as single nucleotide resolution. Hydroxyl radical footprinting can be used to identify the nucleotides within an intermolecular contact surface, e.g. in DNA-protein1 and RNA-protein complexes. Equilibrium3 and kinetic4 transitions can be determined by conducting hydroxyl radical footprinting as a function of a solution variable or time, respectively. A key feature of footprinting is that limited exposure to the probe (e.g., 'single-hit kinetics') results in the uniform sampling of each nucleotide of the polymer.5
In this video article, we use the P4-P6 domain of the Tetrahymena ribozyme to illustrate RNA sample preparation and the determination of a Mg(II)-mediated folding isotherms. We describe the use of the well known hydroxyl radical footprinting protocol that requires H2O2 (we call this the 'peroxidative' protocol) and a valuable, but not widely known, alternative that uses naturally dissolved O2 (we call this the 'oxidative' protocol). An overview of the data reduction, transformation and analysis procedures is presented.

Monitoring Equilibrium Changes in RNA Structure by &#039;Peroxidative&#039; and &#039;Oxidative&#039; Hydroxyl Radical Footprinting

This protocol describes how to quantify the Mg(II)-dependent formation of RNA tertiary structure by two methods of hydroxyl radical footprinting.

监测RNA结构平衡的变动情况“过氧化”和“氧化”羟基自由基足

RNA molecules play an essential role in biology. In addition to transmitting genetic information, RNA can fold into unique tertiary structures fulfilling ...

Monitoring Kinase and Phosphatase Activities Through the Cell Cycle by Ratiometric FRET

F&ouml;rster resonance energy transfer (FRET)-based reporters1 allow the assessment of endogenous kinase and phosphatase activities in living cells. Such probes typically consist of variants of CFP and YFP, intervened by a phosphorylatable sequence and a phospho-binding domain. Upon phosphorylation, the probe changes conformation, which results in a change of the distance or orientation between CFP and YFP, leading to a change in FRET efficiency (Fig 1). Several probes have been published during the last decade, monitoring the activity balance of multiple kinases and phosphatases, including reporters of PKA2, PKB3, PKC4, PKD5, ERK6, JNK7, Cdk18, Aurora B9 and Plk19. Given the modular design, additional probes are likely to emerge in the near future10. 
Progression through the cell cycle is affected by stress signaling pathways 11. Notably, the cell cycle is regulated differently during unperturbed growth compared to when cells are recovering from stress12.Time-lapse imaging of cells through the cell cycle therefore requires particular caution. This becomes a problem particularly when employing ratiometric imaging, since two images with a high signal to noise ratio are required to correctly interpret the results. Ratiometric FRET imaging of cell cycle dependent changes in kinase and phosphatase activities has predominately been restricted to sub-sections of the cell cycle8,9,13,14.
Here, we discuss a method to monitor FRET-based probes using ratiometric imaging throughout the human cell cycle. The method relies on equipment that is available to many researchers in life sciences and does not require expert knowledge of microscopy or image processing.

FRET-based reporters are increasingly used to monitor kinase and phosphatase activities in live cells. Here we describe a method on how to use FRET-based reporters to assess cell cycle-dependent changes in target phosphorylation.

通过细胞周期激酶和磷酸酶活性成比例的FRET监测

F&ouml;rster resonance energy transfer (FRET)-based reporters1 allow the assessment of endogenous kinase and phosphatase activities in living cells. Such ...

Monitoring Plasmid Replication in Live Mammalian Cells over Multiple Generations by Fluorescence Microscopy

Few naturally-occurring plasmids are maintained in mammalian cells. Among these are genomes of gamma-herpesviruses, including Epstein-Barr virus (EBV) and Kaposi's Sarcoma-associated herpesvirus (KSHV), which cause multiple human malignancies 1-3. These two genomes are replicated in a licensed manner, each using a single viral protein and cellular replication machinery, and are passed to daughter cells during cell division despite their lacking traditional centromeres 4-8.
Much work has been done to characterize the replications of these plasmid genomes using methods such as Southern blotting and fluorescence in situ hybridization (FISH). These methods are limited, though. Quantitative PCR and Southern blots provide information about the average number of plasmids per cell in a population of cells. FISH is a single-cell assay that reveals both the average number and the distribution of plasmids per cell in the population of cells but is static, allowing no information about the parent or progeny of the examined cell.
Here, we describe a method for visualizing plasmids in live cells. This method is based on the binding of a fluorescently tagged lactose repressor protein to multiple sites in the plasmid of interest 9. The DNA of interest is engineered to include approximately 250 tandem repeats of the lactose operator (LacO) sequence. LacO is specifically bound by the lactose repressor protein (LacI), which can be fused to a fluorescent protein. The fusion protein can either be expressed from the engineered plasmid or introduced by a retroviral vector. In this way, the DNA molecules are fluorescently tagged and therefore become visible via fluorescence microscopy. The fusion protein is blocked from binding the plasmid DNA by culturing cells in the presence of IPTG until the plasmids are ready to be viewed.
This system allows the plasmids to be monitored in living cells through several generations, revealing properties of their synthesis and partitioning to daughter cells. Ideal cells are adherent, easily transfected, and have large nuclei. This technique has been used to determine that 84% of EBV-derived plasmids are synthesized each generation and 88% of the newly synthesized plasmids partition faithfully to daughter cells in HeLa cells. Pairs of these EBV plasmids were seen to be tethered to or associated with sister chromatids after their synthesis in S-phase until they were seen to separate as the sister chromatids separated in Anaphase10. The method is currently being used to study replication of KSHV genomes in HeLa cells and SLK cells. HeLa cells are immortalized human epithelial cells, and SLK cells are immortalized human endothelial cells. Though SLK cells were originally derived from a KSHV lesion, neither the HeLa nor SLK cell line naturally harbors KSHV genomes11. In addition to studying viral replication, this visualization technique can be used to investigate the effects of the addition, removal, or mutation of various DNA sequence elements on synthesis, localization, and partitioning of other recombinant plasmid DNAs.

A method of observing individual DNA molecules in live cells is described. The technique is based on the binding of a fluorescently tagged lac repressor protein to binding sites engineered into the DNA of interest. This method can be adapted to follow many recombinant DNAs in live cells over time.

在Live哺乳动物细胞中质粒复制多代荧光显微镜监测

Few naturally-occurring plasmids are maintained in mammalian cells.  Among these are genomes of gamma-herpesviruses, including Epstein-Barr virus  (EBV) ...

In vivo Reprogramming of Adult Somatic Cells to Pluripotency by Overexpression of Yamanaka Factors

Induced pluripotent stem (iPS) cells that result from the reprogramming of somatic cells to a pluripotent state by forced expression of defined factors are offering new opportunities for regenerative medicine. Such clinical applications of iPS cells have been limited so far, mainly due to the poor efficiency of the existing reprogramming methodologies and the risk of the generated iPS cells to form tumors upon implantation.
We hypothesized that the reprogramming of somatic cells towards pluripotency could be achieved in vivo by gene transfer of reprogramming factors. In order to efficiently reprogram cells in vivo, high levels of the Yamanaka (OKSM) transcription factors need to be expressed at the target tissue. This can be achieved by using different viral or nonviral gene vectors depending on the target tissue. In this particular study, hydrodynamic tail-vein (HTV) injection of plasmid DNA was used to deliver the OKSM factors to mouse hepatocytes. This provided proof-of-evidence of in vivo reprogramming of adult, somatic cells towards a pluripotent state with high efficiency and fast kinetics. Furthermore no tumor or teratoma formation was observed in situ.
It can be concluded that reprogramming somatic cells in vivo may offer a potential approach to induce enhanced pluripotency rapidly, efficiently, and safely compared to in vitro performed protocols and can be applied to different tissue types in the future.

In vivo Reprogramming of Adult Somatic Cells to Pluripotency by Overexpression of Yamanaka Factors

This study demonstrates the reprogramming of somatic cells towards pluripotency in vivo without the generation of teratomas. We used hydrodynamic tail vein injection of plasmid DNA encoding the Yamanka factors to induce the in vivo reprogramming of adult hepatocytes into cells of enhanced pluripotency.

Induced pluripotent stem (iPS) cells that result from the reprogramming of somatic cells to a pluripotent state by forced expression of defined factors ...

Monitoring Intraspecies Competition in a Bacterial Cell Population by Cocultivation of Fluorescently Labelled Strains

Many microorganisms such as bacteria proliferate extremely fast and the populations may reach high cell densities. Small fractions of cells in a population always have accumulated mutations that are either detrimental or beneficial for the cell. If the fitness effect of a mutation provides the subpopulation with a strong selective growth advantage, the individuals of this subpopulation may rapidly outcompete and even completely eliminate their immediate fellows. Thus, small genetic changes and selection-driven accumulation of cells that have acquired beneficial mutations may lead to a complete shift of the genotype of a cell population. Here we present a procedure to monitor the rapid clonal expansion and elimination of beneficial and detrimental mutations, respectively, in a bacterial cell population over time by cocultivation of fluorescently labeled individuals of the Gram-positive model bacterium Bacillus subtilis. The method is easy to perform and very illustrative to display intraspecies competition among the individuals in a bacterial cell population.

Bacteria may accumulate either detrimental or beneficial mutations during their lifetime. In a population of cells individuals that have accumulated beneficial mutations may rapidly outcompete their fellows. Here we present a simple procedure to visualize intraspecies competition in a bacterial cell population over time using fluorescently labeled individuals.&#160;

通过共生荧光标记菌株监测细菌细胞群中的物种内部竞争

Many microorganisms such as bacteria proliferate extremely fast and the populations may reach high cell densities. Small fractions of cells in a ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

在造血干细胞和祖细胞的成年小鼠颅骨骨髓体内四维跟踪

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...