Name: in vivo clonal tracking of hematopoietic stem and progenitor cells marked by five fluorescent proteins using confocal and multiphoton microscopy
Uploaded: 2014-08-06T16:00:00.000+00:00
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Description: Watch this Scientific Journal Video about In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy at JoVE.com

This work was supported by the Intramural Research Program of the National Heart, Lung, Blood Institute of the National Institutes of Health. We thank Boris Fehse (University Medical Center Hamburg-Eppendorf, Hamburg, Germany) for providing the five LeGO vector plasmids; Christian A. Combs and Neal S. Young (NHLBI, NIH) for discussions, support and encouragement throughout this study, and Andre LaRochelle (NHLBI, NIH) for assistance with tail vein injections.

所有小鼠饲养，并按照指南的护理和使用美国国立卫生研究院实验动物的处理，并参加了一个NHLBI动物护理和使用委员会批准的协议。女B6.SJL-PTPRC（D）Pep3（B）/ BoyJ（B6.SJL）和C57BL / 6小鼠，6〜12周龄，分别作为供体和受体，分别为。 在表1中提供的材料，试剂和设备的列表。 鼠标HSPCs 1。乐高传导和骨髓移植在执行经过认证的生物安全2级（BSL-2）橱柜（组织培养罩）下面介绍的程序。 <ol><li>生产LEGO向量编码5FPS的<ol><li>使用慢病毒"基因本体论"（LEGO）的载体质粒，分别来自强组成SFFV启动，包括乐高，Cer2（蔚蓝），LEGO-G2（EGFP），LEGO-V2（金星），LEGO-T2的编码不同的FP （TdTomato），以及LEGO-C2（mCherry）鲍里斯Fehse博士（大学医学中心汉堡Eppendorf公司，德国汉堡）友情提供;现在也可以从Addgene 5-7。 </li><li>以产生LEGO矢量，种子5×10 6个293T细胞以10 cm的培养皿（使用12个培养皿的每个向量）24小时在转染前在8ml I10介质（IMDM补充有10％FCS和谷氨酰胺/青霉素/链霉素）中。 </li><li>转用稍微修改CAPHOS包建议的细胞磷酸钙: <ol><li>对于每块板组合15微克LEGO载体，12微克pCDNA3.HIVgag / pol.4xCTE，4微克上一页达，和1微克pMD2.G-VSV-G质粒中存在的50微升2.5M的氯化钙2，加水至产生500微升总体积。 </li><li>沉淀的DNA的质粒用500μl2×HEBS（HEPES缓冲盐水）中，并添加到293T细胞中的各板前孵育20分钟，在室温下进行。 </li><li>孵育细胞6小时，在37℃的5％CO 2培养箱;删除媒体和补充新鲜培养基6小时后转染。 </li></ol></li><li>收集培养物上清液含有慢病毒颗粒从各板，每天，持续3天。每一天，下面收集上清液加入新鲜I10媒体每块板。 <ol><li>通过0.22微米的微孔，游泳池过滤并在71,900 XG和4℃下浓缩上清液通过超速离心上的SW28转子2小时。 </li><li>悬浮上清液1毫升StemSpan媒体。分装慢股200微升，并储存在-80℃。 </li></ol></li></ol></li><li>慢病毒颗粒的滴定 通过利用转导NIH3T3细胞单位/毫升的定量测定LEGO载体颗粒的收集上清液的效价。滴度允许需要导致感染复数（MOI）的所需的相对多重每个矢量的上清液用于实验对象的体积的计算细胞。 <ol><li>板5×10 4个NIH3T3细胞在2ml I10媒体的前一天滴定的每孔（12孔板）的细胞。 </li><li>制备载体上清液的5倍连续稀释液在添加有硫酸鱼精蛋白I10媒体（4毫克/毫升为1000倍）。 </li><li>从细胞中取出介质，并更换500微升含2，0.8，0.32和0.128微升浓病毒相当于上清液稀释。 </li><li>确定细胞每孔存在于转导的时间数（否），计算一个控制良好。 </li><li>转导后6小时后，加入2mL I10媒体的孵育3天，在37℃，5％CO 2。 </li><li>从用胰蛋白酶-EDTA的每个孔收获细胞，并测定荧光细胞（％FP）经由流的百分比术。 </li><li>计算滴度如下，平均每孔（稀释）得到的滴度产生介于1和50％的％FP:效价（载体颗粒/毫升）=否矢量上清液（毫升）的x％的FP /音量。平均得到的各稀释液，如果％FP是介于1和50％的效价。 </li></ol></li><li>小鼠细胞收集，净化，转导，并移植（ 图1A示意性地显示从马利德等 4改编）。 <ol><li>经批准的程序牺牲供体小鼠。收获骨髓前后肢:解剖肱骨，股骨和胫骨，彻底去除所有肌肉，韧带和多余的组织从骨骼，贯穿各骨尽可能接近到接头的端部，并冲洗骨髓出用27-0 16G针含I10媒体注射器的骨头。 </li><li>沉淀全骨髓细胞10分钟，在500 XG，4℃，裂解红细胞两次用50ml ACK缓冲液中。将细胞再悬浮于10ml I10媒体。 </li><li>根据修改后的试剂盒protoco净化谱系阴性（Lin-的）祖细胞的MACS谱系耗尽试剂盒L: <ol><li>使用30微升抗生物素微珠代替10微升/ 10 7个细胞;完成清洗步骤与I2介质（IMDM补充有2％FCS）代替缓冲液。 </li><li>将培养与生物素标记抗体鸡尾酒和抗生物素微珠保温之间的一个洗涤步骤;和平衡色谱柱与I10，而不是缓冲区。 </li></ol></li><li>培养Lin-的细胞48小时，以5×10 5细胞/ ml，在StemSpan媒体的起始密度补充有10纳克/毫升鼠IL-3，100毫微克/毫升鼠IL-11，100纳克/毫升人FLT-3配位体，和50毫微克/毫升小鼠干细胞因子。 </li><li>转让1至4×10 5个细胞于12孔板中包被Retronectin和转导与LEGO上清，每在感染复数为6-7（MOI），以实现对每个FP 50％的表达效率，在StemSpan的存在介质和细胞因子的上面详述和4微克/毫升的硫酸鱼精蛋白。 </li><li>转导细胞每个LEGO分别为矢量描述的步骤1.3.5及以下转导的细胞混合（称为混合）或同时共转导细胞与所有5 LEGO载体上清液（称为钴）。 24小时后取出电池，用StemSpan媒体洗一次无细胞因子和它们悬浮在StemSpan媒体无细胞因子进行移植，或共聚焦显微镜前，将其转移至新鲜培养基与细胞因子和培养更多的96小时和流式细胞仪。 <ol><li>通过注入导Lin-的HSPCs经尾静脉注射入11 Gy的单剂量预照射的收件人（前6-18小时，从而移植）的收件人进行骨髓移植，在100μl的体积，在每只小鼠的细胞的剂量对应于1 -4×10 5个Lin-的细胞最初镀转导。在每个实验中，移植动物的整个队列与转导的供体骨髓细胞的相同的人口。 </li><li>对于转导ħ 体外表征的SPC，培养的细胞在StemSpan额外4-5天补充有细胞因子和通过流式细胞仪对转染效率进行分析，并通过显微镜进行颜色的多样性。 <ol><li>牺牲个别小鼠和检索的组织和器官的成像，在不同的时间点达120天移植后。 <ol><li>用乙醇喷雾鼠标的皮毛和切割的腹侧避免头发散发肌肤。 </li><li>根据标准程序，脾，腘淋巴结，肺，心脏，皮肤，脂肪组织，骨骼肌，肾脏，肝脏，以及头部，以获得颅盖骨解剖和切除。 </li><li>另外，使用颅骨或建立骨窗实时成像重复随着时间的推移2,8,9或图象表面的器官，如在活的动物的皮肤和淋巴结。 </li></ol></li><li>将各个器官的35毫米数0玻璃盖培养皿中50-10056，L PBS成像它们完好无如步骤2.5中所述切片，固定或进一步加工。 </li><li>对于容积成像，保持器官的冷PBS备用。对于时间推移成像立即着手，含20毫米的HEPES 10％胎牛血清，37℃成像器官的DMEM </li></ol></li></ol></li></ol></li></ol> 2，激光共聚焦和双光子显微镜和图像分析<ol><li>执行使用装有多线氩，二极管561纳米，氦氖594纳米，氦氖633纳米可见激光徕卡SP5的五个通道共聚焦和多系统显微成像。使用双光子模式下，脉冲飞秒钛:蓝宝石（TI-SA）激光器，可调谐的激励，从680到1,080 nm处色散校正。使用在涉及红移的FP（tdTomato和mCherry），光学参量振荡器（OPO）激光器的实验来尽量延长输出波长范围为1,030-1,300纳米，依次或同时用叔他的Ti-萨激光。 </li><li>使用HCX-IRAPO-L 25X / 0.95 NA水浸渍目标（WD = 2.5 mm）表示时HC-PLAPO-CS 20X / 0.70 NA干物镜（WD = 0.6 MM），或HC-PL-IRAPO 40X图像的各种组织/1.1 NA水浸物镜（WD = 0.6毫米）。 </li><li>得到收集发射光的λ栈（xyλ）共焦荧光光谱中为5nm-带宽从BM细胞转导的单个FP LEGO矢量以及控制的可见光谱中的，未移植BM。 <ol><li>图像处理与Leica LAS-AF V2.4.1软件创建的所有5张参考光谱，以及自发荧光（ 图2A）。使用个别的FP光谱和荧光亮度的差（表示为％的EGFP）大仓（79％），金星（156％），tdTomato（283％）和mCherry（47％）的设定5序列成像通道，如下所示:蔚蓝（458 / 468-482纳米），绿色荧光蛋白（488 / 496-514纳米），金星（514 / 523-558纳米），tdTomato（561 / 579-597纳米）和mCherry（594 / 618-670纳米） （ <str翁&gt;图2B）。 </li><li>验证准确的FP分离，如转导的细胞与每个FP仅在相应的信道可见和不存在来自其他地方（ 图2C）。另外，该定义的多通道方法创造了一个独特的5频道"COLORPRINT"，使后续的颜色分析和克隆跟踪，并具有减少采集时间相比，光谱成像的优点。 </li></ol></li><li>单独的荧光发射，在2光子模式中，通过高效率的自定义分色镜和收集具有四个通道的非退扫描检测器（NDD）如下:二向色镜在568 nm的后跟一个分色镜在465nm处，接着à四十五分之四百五十二nm发射滤波器，倍频或自发荧光和蔚蓝，对于绿色荧光蛋白或金星在tdTomato或mCherryà四十○分之五百二十五nm发射滤波器和648 nm的分色镜和六十零分之六百二nm发射滤波器。 <ol><li>通过双光子显微镜我揭示的结构信息ntrinsic对比成像（如上文所述）的组织自发荧光的由弹性蛋白，NADH，（激发在780nm处）和二次谐波产生的从骨和纤维状胶原信号（SHG）（激发在收集在背散射920纳米或860纳米方向）10,11。 </li></ol></li><li>对于3D体绘制，沿z轴收集一系列的xyz的图像（通常为1×1×4微米的3体素尺寸）在5微米的间隔的范围内的深度（150-300微米）在整个骨髓和其他组织中，在使用软件的瓦片函数大的区域，以自动生成缝合卷包含整个胸骨窝，约2.5×1.2 平方毫米（XY）和300微米（Z）（ 图2D-2E）及电影1。 <ol><li>颅骨图像直接通过颅骨骨髓切片没有2,8。 </li><li>沿矢状平面（平分），然后部分胸骨在横向通过切割面3,4,12 2胸骨福斯和图像之间的联合（概述图1B改编自高久等[3]）。 </li></ol></li><li>四维时间推移成像，将新鲜切除腘窝淋巴结，脾，肺，或颅骨骨样品未​​切割到35 mm数0盖玻片的培养皿，在50〜100μLDMEM，10％FBS的20毫​​米肝素钠在37℃并利用双光子钛萨激发（860 nm）的结合激发焦（458纳米，488纳米，514纳米，561纳米，594纳米）和徕卡共振扫描仪（8000赫兹/秒），以Z-堆栈（〜为80μm），在20秒的间隔长达1小时。 <ol><li>对于同时三种颜色2P成像采用TI萨调整在860 nm处同时与OPO激光（调谐在1,130 nm波长tdTomato;或1140纳米mCherry）（ 电影2）。 </li></ol></li><li>三维体积和四维时间序列的重建与分析<ol><li>使用了Imaris 64软件是版本7.6，或替代的软件包来转换平铺电池堆的原始图像到容积重建（3D）的骨髓和不同的组织，并将其输出为3D旋转电影内的荧光细胞的数据，如前面所描述的3,4 （ 电影1）一步一步的例子: <ol><li>在了Imaris，打开Z堆栈的图像;为了显示3D体积可视化选择"超越"。 </li><li>使用"浏览"，打开音量360°。 </li><li>选择"动画"制作动画;使用关键帧添加所选的意见，变焦，音量的旋转;预览影片，根据需要进行编辑和保存在文件中所需的影片格式（如:AVI，MOV）。 </li></ol></li><li>对于三维细胞和克隆频率，位置，大小和分布进行定量评估，进一步的处理使用了Imaris XT模块的Z堆栈的集成进行距离MEAS MATLAB应用程序urements（使用距离变换算法）和簇分析，如先前所描述的3,4。变换的图像栈的序列随时间成体积渲染四维（4D）电影了Imaris 64的软件，并使用自回归运动算法在三维空间中使用的点和面分析为细胞运动的半自动跟踪。 </li><li>如下进行细胞的多谱分析:提取多色加入所有5个通道通过了Imaris XT通道运算到一个新的通道标记点;确定所有点（细胞）中的每一个的5分量信道的平均强度的荧光。在导出的Excel软件中的值绘制的图表显示了独特的"COLORPRINT"，即每个FP个体细胞（图2E图）的相对％。纠正组织漂移的数据集，并计算细胞的速度和位移随时间推移，轨迹长度，路径和距离;在Excel中SOF出口值tware绘制图形。组装与Adobe Photoshop的CSS 5软件的复合数字。 </li></ol></li></ol>

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作者宣称没有竞争的经济利益。

在这里，我们描述了一个功能强大的方法，最近发明了克隆细胞跟踪的细节，用相结合的组合与5FP编码LEGO载体和成像串联共聚焦和双光子显微镜标记来实现体积和动态成像活组织中产生大量的多样性。这延长了使用的FP为主色调，通过记录共焦光谱身份5"独特"的8位通道标识。因此蔚蓝，绿色荧光蛋白，金星，tdTomato，mCherry的每一个细胞内的相对比例创造了独特的"COLORPRINT"鉴定克隆后代。由5fps的?...

A correction was made to <a href="/video/51669">In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy</a>. At the time of publication, there was a typo in the discussion, which displayed an incorrect depth.
This was corrected in the discussion from:
This approach combines the benefits of single-cell resolved high resolution imaging together with optical sectioning via confocal microscopy. Very large x - y (mm2) regions of the intact dense tissue volume can be examined by generating tiled-images. These high resolution images from optical sections can be used to computationally reconstruct (automatically and &#8220;on-the-fly&#8221;) complete 3D volumes of great complexity to depths of ~ 30 &#956;m, comprising ~ 20-30 layers of cells, vascular, bone and collagen structures. 3D reconstructions can be used for morphometric non-invasive quantitative analyses of biologic interest. One important caveat to point out is that large volume/high resolution imaging is time-consuming, for instance 1 hr per ~1 mm3 of tissue (one fossae of the sternum), therefore we recommend imaging no more than 1 mouse per experiment per day when bone marrow as well as different tissues need to be examined in detail.
to:
This approach combines the benefits of single-cell resolved high resolution imaging together with optical sectioning via confocal microscopy. Very large x - y (mm2) regions of the intact dense tissue volume can be examined by generating tiled-images. These high resolution images from optical sections can be used to computationally reconstruct (automatically and &#8220;on-the-fly&#8221;) complete 3D volumes of great complexity to depths of ~ 300 &#956;m, comprising ~ 20-30 layers of cells, vascular, bone and collagen structures. 3D reconstructions can be used for morphometric non-invasive quantitative analyses of biologic interest. One important caveat to point out is that large volume/high resolution imaging is time-consuming, for instance 1 hr per ~1 mm3 of tissue (one fossae of the sternum), therefore we recommend imaging no more than 1 mouse per experiment per day when bone marrow as well as different tissues need to be examined in detail.

A correction was made to <a href="/video/51669">In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy</a>. At the time of publication, there was a typo in the discussion, which displayed an incorrect depth.

Erratum: In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy

血细胞，称为造血的生产，是由一个小群体的造血干细胞和祖细胞（HSPCs）的维护。这些细胞位于骨髓（BM）中的一个复杂的微环境的生态位选自成骨细胞，基质细胞，脂肪组织，和血管结构之内，所有牵连的自我更新和分化1,2的控制。由于完整的BM向来人迹罕至的直接观察，HSPC及其微环境之间的相互作用仍然在体内主要是未鉴定。以前，我们建立了一个方法，用共聚焦荧光和反射镜3形象化完整的BM的3D架构。我们的特点扩大了BM绿色荧光蛋白标记的HSPCs，但只使用一个单一的FP排除再生分析，在克隆的水平。就在最近，我们又利用慢病毒基因本体论（LEGO）的载体组成型表达FLuorescent蛋白（fps）的有效标记细胞4,5。共转导HSPC与3或5向量生成的组合色彩多样的调色板，允许多个独立的HSPC克隆跟踪。 标记HSPC结合允许跟踪各个HSPC归巢和植入辐射小鼠的骨髓新的成像技术。 LEGO载体用于3或5个不同的帧率（从蔚蓝，绿色荧光蛋白，金星，tdTomato和mCherry）标记HSPC，并按照他们的植入时间的推移，在骨髓，激光共聚焦和双光子显微镜，让清晰的骨骼和其他可视化矩阵结构和HSPC克隆到其微环境的部件的关系。 HSPC纯化来自C57BL / 6小鼠骨髓谱系阴性细胞，转导的LEGO矢量，并通过尾静脉注射回输入髓烧蚀同类系小鼠。通过监测胸骨骨髓组织中大量的可视化，我们在年初发生的骨内植入骨髓再生。细胞簇与不同颜色的光谱出现最初在靠近骨和进展集中随着时间的推移。有趣的是，经过3个多星期的骨髓由宏观克隆簇，提示造血个别HSPCs衍生的连续传播​​的精髓，而不是HSPCs通过血液广泛传播。有较少的颜色多样性在晚的时间点，这表明难以传感长期再植细胞与多个矢量。 此外，HSPCs在脾脏形成集群，器官还负责造血小鼠。 HPSC衍生的单个细胞可在胸腺中得到解决，淋巴结，脾，肝，肺，心脏，皮肤，骨骼肌，脂肪组织和肾脏以及。 3D图像可以定性和定量地进行评估，以欣赏细胞与组织的最小扰动的分布。最后，我们说明在四维实时动态研究ð结合共振扫描多光子显微镜和共聚焦时间推移成像的可行性。这种方法使频谱标记的细胞群体的非侵入性的高解析度，多层面的细胞命运追踪其完整的3D架构，提供组织再生和病理学研究的有力工具。

<table><tbody><tr><td colspan="4">&lt;strong&gt;Production of Viruses&lt;/strong&gt;</td></tr><tr><td>LeGO-Cer2</td><td>Addgene</td><td>27338</td><td>Expression of Cerulean</td></tr><tr><td>LeGO-G2</td><td>Addgene</td><td>25917</td><td>Expression of eGFP</td></tr><tr><td>LeGO-V2</td><td>Addgene</td><td>27340</td><td>Expression of Venus</td></tr><tr><td>LeGO-T2</td><td>Addgene</td><td>27342</td><td>Expression of tdTomato</td></tr><tr><td>LeGO-C2</td><td>Addgene</td><td>27339</td><td>Expression of mCherry</td></tr><tr><td>Calciumm Phosphate Transfection Kit</td><td>Sigma</td><td>CAPHOS-1KT</td><td></td></tr><tr><td>Tissue culture dish</td><td>BD Falcon</td><td>353003</td><td></td></tr><tr><td>IMDM</td><td>Gibco, Life Technology</td><td>12440-053</td><td></td></tr><tr><td>Fetal Bovine Serum Heat Inactivated</td><td>Sigma</td><td>F4135-500ML</td><td></td></tr><tr><td>Pen Strep Glutamine</td><td>Gibco, Life Technology</td><td>10378-016</td><td></td></tr><tr><td>Centrifuge Tubes</td><td>Beckman Coulter</td><td>326823</td><td></td></tr><tr><td>SW28 Ultracentrifuge</td><td>Beckman Coulter</td><td>L60</td><td></td></tr><tr><td>Millex Syringe Driven Filter Unite (0.22 &amp;micro;m)</td><td>Millipore</td><td>SLGS033SS</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;Mouse Cell Collection, Purification, Transduction, and Transplantation&lt;/strong&gt;</td></tr><tr><td>ACK lysing buffer</td><td>Quality Biologicals Inc.</td><td>118-156-101</td><td></td></tr><tr><td>Lineage Cell Depletion Kit (mouse)</td><td>Miltenyi Biotec Inc. USA</td><td>130-090-858</td><td></td></tr><tr><td>LS columns + tubes</td><td>Miltenyi Biotec Inc. USA</td><td>130-041-306</td><td></td></tr><tr><td>Pre-Separation Filters (30 &amp;micro;m)</td><td>Miltenyi Biotec Inc. USA</td><td>130-041-407</td><td></td></tr><tr><td>StemSpan SFEM serum-free medium for culture and expansion of hematopoietic cells (500 ml)</td><td>StemCell Technologies Inc&amp;nbsp;</td><td>9650</td><td></td></tr><tr><td>Murine IL-11 CF</td><td>R &amp;amp; D Systems Inc</td><td>418-ML-025/CF</td><td>100 &amp;micro;g/ml is 1,000x</td></tr><tr><td>Recombinant murine SCF</td><td>RDI Division of Fitzgerald Industries Intl&amp;nbsp;</td><td>RDI-2503</td><td>100 &amp;micro;g/ml is 2,000x</td></tr><tr><td>Recombinant murine IL-3</td><td>R &amp;amp; D Systems Inc</td><td>403-ML-050</td><td>20 &amp;micro;g/ml is 2,000x</td></tr><tr><td>FLT-3 Ligand</td><td>Miltenyi Biotec Inc. USA</td><td>130-096-480</td><td>100 &amp;micro;g/ml is 1,000x</td></tr><tr><td>12-well plates, Costar</td><td>Corning Inc.</td><td>3527</td><td></td></tr><tr><td>Retronectin</td><td>Takara Bio Inc</td><td>T100A/B</td><td>Resuspend at 50 &amp;micro;g/ml</td></tr><tr><td>Protamine Sulfate</td><td>Sigma</td><td>P-4020</td><td>4 &amp;micro;g/ml is 1,000x</td></tr><tr><td colspan="4">&lt;strong&gt;Confocal and Two-photon Microscopy&lt;/strong&gt;</td></tr><tr><td>DMEM</td><td>Lonza</td><td>12-614F</td><td></td></tr><tr><td>1 M Hepes</td><td>Cellgro, Mediatech Inc.</td><td>25-060-CI</td><td></td></tr><tr><td>Glass bottom culture dish P35G-0-20-C</td><td>MatTek Corporation</td><td>P35G-0-20-C</td><td></td></tr><tr><td>Glass bottom culture dish P35G-0-14-C</td><td>MatTek Corporation</td><td>P35G-0-14-C</td><td></td></tr><tr><td>Nunc Lab-Tek Chambered Coverglass #1 Borosilicate coverglass; 4-well</td><td>Thermo Scientific</td><td>155383</td><td></td></tr><tr><td>Coverslips 22 mm No 2</td><td>Thomas Scientific</td><td>6662-Q55</td><td></td></tr><tr><td>Leica TCS SP5 AOBS five channels confocal and multi-photon microscope</td><td>Leica Microsystems</td><td></td><td></td></tr><tr><td>Chameleon Vision II -TiSaph laser range 680-1,080 nm</td><td>Coherent</td><td></td><td></td></tr><tr><td>Chameleon Compact OPO laser range 1,030-1,350 nm</td><td>Coherent</td><td></td><td></td></tr><tr><td>HCX-IRAPO-L 25X/0.95 NA water dipping objective (WD=2.5 mm)</td><td>Leica Microsystems</td><td></td><td></td></tr><tr><td>HC-PLAPO-CS 20X/0.70 NA dry objective (WD=0.6 mm)</td><td>Leica Microsystems</td><td></td><td></td></tr><tr><td>HC-PL-IRAPO 40X/1.1 NA water immersion objective (WD=0.6 mm)</td><td>Leica Microsystems</td><td></td><td></td></tr><tr><td>Imaris software version 7.6</td><td>Bitplane</td><td></td><td></td></tr></tbody></table>

in vivo clonal tracking of hematopoietic stem and progenitor cells marked by five fluorescent proteins using confocal and multiphoton microscopy

我们开发并验证了一种荧光标记的方法对造血干细胞和祖细胞（HSPCs）以高空间和时间分辨率使用小鼠骨髓移植实验模型体内造血研究的克隆跟踪。遗传组合使用的编码荧光蛋白（FPS）的慢病毒载体标记通过先进的显微成像使细胞命运的映射。矢量编码五种不同的FPS:蔚蓝，绿色荧光蛋白，金星，tdTomato和mCherry被用来同时转导HSPCs，创建色彩标记的细胞多样的调色板。使用共聚焦/双光子混合显微成像能够唯一地标记细胞的同时高分辨率评估和其与组织的结构部件结合的后代。体积分析在大面积显示，频谱编码HSPC来源的细胞，可以检测非侵入性地在各个完整的组织，包括骨髓（BM），对于时间移植后广泛周期。现场研究相结合的视频速率多光子激光扫描共聚焦时间推移成像的4D演示动态的细胞和克隆跟踪以定量的方式的可能性。

全安装3D共焦胸骨骨髓时程/ 2 - 光子显微镜的重建揭示了移植和扩展移植联合5FPS的具有显着特征的模式:克隆出现了明确界定，均匀标有颜色的调色板宽，初期进展随着时间的推移优先包含多一种颜色的细胞。共焦显微镜的设置和成像5fps的标记HSPC在胸骨骨髓代表性实例示于图2和视频1和2。 完整组织的整个贴三维共聚焦/双光子显微镜重建...

Watch this Scientific Journal Video about In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy at JoVE.com

In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy

组合5的荧光蛋白的造血干细胞和祖细胞的标记允许在通过共聚焦和双光子显微镜体内克隆跟踪，再生期间提供见解骨髓造血体系结构。此方法允许频谱编码HSPCs来源的细胞在完整组织的时间移植后广泛周期的非侵入性的命运映射。

在造血干细胞和祖细胞利用共聚焦和多光子显微镜五荧光蛋白标记的活体克隆跟踪

We developed and validated a fluorescent marking methodology for clonal tracking of hematopoietic stem and progenitor cells (HSPCs) with high spatial and ...

in-vivo-clonal-tracking-hematopoietic-stem-progenitor-cells-marked

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy

13.1K 次观看.  NHLBI/NIH. 组合5的荧光蛋白的造血干细胞和祖细胞的标记允许在通过共聚焦和双光子显微镜体内克隆跟踪，再生期间提供见解骨髓造血体系结构。此方法允许频谱编码HSPCs来源的细胞在完整组织的时间移植后广泛周期的非侵入性的命运映射。 

视频: In vivo Clonal Tracking of Hematopoietic Stem and Progenitor Cells Marked by Five Fluorescent Proteins using Confocal and Multiphoton Microscopy

Visualizing Cell-to-cell Transfer of HIV using Fluorescent Clones of HIV and Live Confocal Microscopy

By fusing the green fluorescent protein to their favorite proteins, biologists now have the ability to study living complex cellular processes using fluorescence video microscopy. To track the movements of the human immunodeficiency virus core protein during cell-to-cell transmission of human immunodeficiency virus, we have GFP-tagged the Gag protein in the context of an infectious molecular clone of HIV, called HIV Gag-iGFP. We study this viral clone using video confocal microscopy. In the following visualized experiment, we transfect a human T cell line with HIV Gag-iGFP, and we use fluorescently labeled uninfected CD4+ T cells to serve as target cells for the virus. Using the different fluorescent labels we can readily follow viral production and transport across intercellular structures called virological synapses. Simple gas permeable imaging chambers allow us to observe synapses with live confocal microscopy from minutes to days. These approaches can be used to track viral proteins as they move in from one cell to the next.

This visualized experiment is a guide for utilizing a fluorescent molecular clone of HIV for live confocal imaging experiments.

可视化的细胞与细胞转移的艾滋病毒，艾滋病毒和现场共聚焦显微镜的荧光克隆

By fusing the green fluorescent protein to their favorite proteins, biologists now have the ability to study living complex cellular processes using ...

科研

免疫与感染

A Method for Lineage Tracing of Corneal Cells Using Multi-color Fluorescent Reporter Mice

Lineage tracing experiments define the origin, fate and behavior of cells in a specific tissue or organism. This technique has been successfully applied for many decades, revealing seminal findings in developmental biology. More recently, it was adopted by stem cell biologists to identify and track different stem cell populations with minimal experimental intervention. The recent developments in mouse genetics, the availability of a large number of mouse strains, and the advancements in fluorescent microscopy allow the straightforward design of powerful lineage tracing systems for various tissues with basic expertise, using commercially available tools. We have recently taken advantage of this powerful methodology to explore the origin and fate of stem cells at the ocular surface using R26R-Confetti mouse. This model offers a multi-color genetic system, for the expression of 4 fluorescent genes in a random manner. Here we describe the principles of this methodology and provide an adaptable protocol for designing lineage tracing experiments; specifically for the corneal epithelium as well as for other tissues.

In this article we describe the principles for designing and performing a multi-color lineage tracing experiment using R26R-Confetti mice. We provide a specific protocol for tracking corneal epithelial cells which can be modified for other tissues of interest.

方法对角膜细胞的谱系追踪使用多色荧光记者小鼠

Lineage tracing experiments define the origin, fate and behavior of cells in a specific tissue or organism. This technique has been successfully applied ...

发育生物学

Combining Intravital Fluorescent Microscopy (IVFM) with Genetic Models to Study Engraftment Dynamics of Hematopoietic Cells to Bone Marrow Niches

Increasing evidence indicates that normal hematopoiesis is regulated by distinct microenvironmental cues in the BM, which include specialized cellular niches modulating critical hematopoietic stem cell (HSC) functions1,2. Indeed, a more detailed picture of the hematopoietic microenvironment is now emerging, in which the endosteal and the endothelial niches form functional units for the regulation of normal HSC and their progeny3,4,5. New studies have revealed the importance of perivascular cells, adipocytes and neuronal cells in maintaining and regulating HSC function6,7,8. Furthermore, there is evidence that cells from different lineages, i.e. myeloid and lymphoid cells, home and reside in specific niches within the BM microenvironment. However, a complete mapping of the BM microenvironment and its occupants is still in progress.
Transgenic mouse strains expressing lineage specific fluorescent markers or mice genetically engineered to lack selected molecules in specific cells of the BM niche are now available. Knock-out and lineage tracking models, in combination with transplantation approaches, provide the opportunity to refine the knowledge on the role of specific &#34;niche&#34; cells for defined hematopoietic populations, such as HSC, B-cells, T-cells, myeloid cells and erythroid cells. This strategy can be further potentiated by merging the use of two-photon microscopy of the calvarium. By providing in vivo high resolution imaging and 3-D rendering of the BM calvarium, we can now determine precisely the location where specific hematopoietic subsets home in the BM and evaluate the kinetics of their expansion over time. Here, Lys-GFP transgenic mice (marking myeloid cells)9 and RBPJ knock-out mice (lacking canonical Notch signaling)10 are used in combination with IVFM to determine the engraftment of myeloid cells to a Notch defective BM microenvironment.

Combining Intravital Fluorescent Microscopy IVFM with Genetic Models to Study Engraftment Dynamics of Hematopoietic Cells to Bone Marrow Niches

Intravital fluorescence microscopy (IVFM) of the calvarium is applied in combination with genetic animal models to study the homing and engraftment of hematopoietic cells into bone marrow (BM) niches.

结合活体荧光显微镜 (IVFM) 和遗传模型研究造血细胞对骨髓生态位的植入动力学

Increasing evidence indicates that normal hematopoiesis is regulated by distinct microenvironmental cues in the BM, which include specialized cellular ...

Labeling Stem Cells with Fluorescent Dyes for non-invasive Detection with Optical Imaging

Optical imaging (OI) is an easy, fast and inexpensive tool for in vivo monitoring of new stem cell based therapies. The technique is based on ex vivo labeling of stem cells with a fluorescent dye, subsequent intravenous injection of the labeled cells and visualization of their accumulation in specific target organs or pathologies. The presented technique demonstrates how we label human mesenchymal stem cells (hMSC) by simple incubation with the lipophilic fluorescent dye DiD (C67H103CIN2O3S) and how we label human embryonic stem cells (hESC) with the FDA approved fluorescent dye Indocyanine Green (ICG). The uptake mechanism is via adherence and diffusion of the lypophilic dye across the phospholipid cell membrane bilayer. The labeling efficiency is usually improved if the cells are incubated with the dye in serum-free media as opposed to incubation in serum-containing media. Furthermore, the addition of the transfection agent Protamine Sulfate significantly improves contrast agent uptake.

This video shows techniques for labeling of human embryonic stem cells and mesenchymal stem cells with fluorescent dyes. This technique can be used for an in vivo tracking of transplanted stem cells with optical imaging and for histopathological correlations with fluorescence microscopy.

与非侵入性检测荧光染料标记的干细胞与光学成像

Optical imaging (OI) is an easy, fast and inexpensive tool for in vivo monitoring of new stem cell based therapies. The technique is based on ex vivo ...

生物学

Development, Expansion, and In vivo Monitoring of Human NK Cells from Human Embryonic Stem Cells (hESCs) and Induced Pluripotent Stem Cells (iPSCs)

We present a method for deriving natural killer (NK) cells from undifferentiated hESCs and iPSCs using a feeder-free approach. This method gives rise to high levels of NK cells after 4 weeks culture and can undergo further 2-log expansion with artificial antigen presenting cells. hESC- and iPSC-derived NK cells developed in this system have a mature phenotype and function. The production of large numbers of genetically modifiable NK cells is applicable for both basic mechanistic as well as anti-tumor studies. Expression of firefly luciferase in hESC-derived NK cells allows a non-invasive approach to follow NK cell engraftment, distribution, and function. We also describe a dual-imaging scheme that allows separate monitoring of two different cell populations to more distinctly characterize their interactions in vivo. This method of derivation, expansion, and dual in vivo imaging provides a reliable approach for producing NK cells and their evaluation which is necessary to improve current NK cell adoptive therapies.

Development, Expansion, and In vivo Monitoring of Human NK Cells from Human Embryonic Stem Cells hESCs and Induced Pluripotent Stem Cells iPSCs

This protocol describes the development, expansion, and in vivo imaging of NK cells derived from hESCs and iPSCs.

开发，扩展，<em&gt;在体内</em&gt;从人类胚胎干细胞（hESCs）监测人类NK细胞和诱导多能干细胞（iPS细胞）

We present a method for deriving natural killer (NK) cells from undifferentiated hESCs and iPSCs using a feeder-free approach. This method gives rise to ...

生物工程

Sequential In vivo Imaging of Osteogenic Stem/Progenitor Cells During Fracture Repair

Bone turns over continuously and is highly regenerative following injury. Osteogenic stem/progenitor cells have long been hypothesized to exist, but in vivo demonstration of such cells has only recently been attained. Here, in vivo imaging techniques to investigate the role of endogenous osteogenic stem/progenitor cells (OSPCs) and their progeny in bone repair are provided. Using osteo-lineage cell tracing models and intravital imaging of induced microfractures in calvarial bone, OSPCs can be directly observed during the first few days after injury, in which critical events in the early repair process occur. Injury sites can be sequentially imaged revealing that OSPCs relocate to the injury, increase in number and differentiate into bone forming osteoblasts. These methods offer a means of investigating the role of stem cell-intrinsic and extrinsic molecular regulators for bone regeneration and repair.

Sequential In vivo Imaging of Osteogenic Stem/Progenitor Cells During Fracture Repair

Quantitative measurement of bone progenitor function in fracture healing requires high resolution serial imaging technology. Here, protocols are provided for using intravital microscopy and osteo-lineage tracking to sequentially image and quantify the migration, proliferation and differentiation of endogenous osteogenic stem/progenitor cells in the process of repairing bone fracture.

顺序<em&gt;在体内</em&gt;成骨干/祖细胞在骨折修复的影像学

Bone turns over continuously and is highly regenerative following injury. Osteogenic stem/progenitor cells have long been hypothesized to exist, but in ...

医学

Tracking Mouse Bone Marrow Monocytes In Vivo

Real time multiphoton imaging provides a great opportunity to study cell trafficking and cell-to-cell interactions in their physiological 3-dimensionnal environment. Biological activities of immune cells mainly rely on their motility capacities. Blood monocytes have short half-life in the bloodstream; they originate in the bone marrow and are constitutively released from it. In inflammatory condition, this process is enhanced, leading to blood monocytosis and subsequent infiltration of the peripheral inflammatory tissues. Identifying the biomechanical events controlling monocyte trafficking from the bone marrow towards the vascular network is an important step to understand monocyte physiopathological relevance. We performed in vivo time-lapse imaging by two-photon microscopy of the skull bone marrow of the Csf1r-Gal4VP16/UAS-ECFP (MacBlue) mouse. The MacBlue mouse expresses the fluorescent reporters enhanced cyan fluorescent protein (ECFP) under the control of a myeloid specific promoter 1, in combination with vascular network labelling. We describe how this approach enables the tracking of individual medullar monocytes in real time to further quantify the migratory behaviour within the bone marrow parenchyma and the vasculature, as well as cell-to-cell interactions. This approach provides novel insights into the biology of the bone marrow monocyte subsets and allows to further address how these cells can be influenced in specific pathological conditions.

Tracking Mouse Bone Marrow Monocytes In Vivo

Monocytes are key regulators of innate immunity and play a critical role in the renewal of the peripheral mononuclear phagocytic system and in case of inflammation. This manuscript describes the procedure of real time imaging of the mouse calvaria bone marrow to study the monocyte mobilisation mechanism.

跟踪小鼠骨髓单核细胞<em&gt;在体内</em

Real time multiphoton imaging provides a great opportunity to study cell trafficking and cell-to-cell interactions in their physiological 3-dimensionnal ...

Clonal Genetic Tracing using the Confetti Mouse to Study Mineralized Tissues

Labeling an individual cell in the body to monitor which cell types it can give rise to and track its migration through the organism or determine its longevity can be a powerful way to reveal mechanisms of tissue development and maintenance. One of the most important tools currently available to monitor cells in vivo is the Confetti mouse model. The Confetti model can be used to genetically label individual cells in living mice with various fluorescent proteins in a cell type-specific manner and monitor their fate, as well as the fate of their progeny over time, in a process called clonal genetic tracing or clonal lineage tracing. This model was generated almost a decade ago and has contributed to an improved understanding of many biological processes, particularly related to stem cell biology, development, and renewal of adult tissues. However, preserving the fluorescent signal until image collection and simultaneous capturing of various fluorescent signals is technically challenging, particularly for mineralized tissue. This publication describes a step-by-step protocol for using the Confetti model to analyze growth plate cartilage that can be applied to any mineralized or nonmineralized tissue.

This method describes the use of the R26R-Confetti (Confetti) mouse model to study mineralized tissues, covering all steps from the breeding strategy to the image acquirements. Included is a general protocol that can be applied to all soft tissues and a modified protocol that can be applied to mineralized tissues.

使用纸屑小鼠研究矿化组织的克隆遗传追踪

Labeling an individual cell in the body to monitor which cell types it can give rise to and track its migration through the organism or determine its ...

Live Imaging Followed by Single Cell Tracking to Monitor Cell Biology and the Lineage Progression of Multiple Neural Populations

Understanding the mechanisms that control critical biological events of neural cell populations, such as proliferation, differentiation, or cell fate decisions, will be crucial to design therapeutic strategies for many diseases affecting the nervous system. Current methods to track cell populations rely on their final outcomes in still images and they generally fail to provide sufficient temporal resolution to identify behavioral features in single cells. Moreover, variations in cell death, behavioral heterogeneity within a cell population, dilution, spreading, or the low efficiency of the markers used to analyze cells are all important handicaps that will lead to incomplete or incorrect read-outs of the results. Conversely, performing live imaging and single cell tracking under appropriate conditions represents a powerful tool to monitor each of these events. Here, a time-lapse video-microscopy protocol, followed by post-processing, is described to track neural populations with single cell resolution, employing specific software. The methods described enable researchers to address essential questions regarding the cell biology and lineage progression of distinct neural populations.

A robust protocol to monitor neural populations by time-lapse video-microscopy followed by software-based post-processing is described. This method represents a powerful tool to identify biological events in a selected population during live imaging experiments.

活体影像随访单细胞追踪监测细胞生物学及多神经种群的谱系进展

Understanding the mechanisms that control critical biological events of neural cell populations, such as proliferation, differentiation, or cell fate ...

神经科学

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) ...

在造血干细胞和祖细胞利用共聚焦和多光子显微镜五荧光蛋白标记的活体克隆跟踪

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