资金已被提供由美国国立卫生研究院拨款5T32CA09216 - 26（对JSB），和K01 AR055619 - 01A1和3 K01 AR055619 - 03S1（对于DML），以及在Alex的柠檬水架基金会，在哈佛大学干细胞研究所和美国癌症学会。

1。斑马鱼胚胎的DNA显微注射法<ol><li>线性化RAG2:11 GFP的 DNA结构:cMyc和RAG2:在37℃过夜消化每个NotI质粒10μg。 </li><li>酚:氯仿提取物和沉淀物的质粒，并重新悬浮20μLH 2 O </li><li>确定在1％琼脂糖凝胶运行20μL，10μL和高范围的DNA阶梯5μL1:1，1:5和1:10稀释的每个消化质粒DNA浓度。这种类型的定量一般比光谱仪读数更准确。 </li><li> CG1应变（或其他同系株）稀释成的DNA注射的60ng/μL终浓度为1M氯化钾0.5 X TE斑马鱼的胚胎，使用30ng/μLRAG2:cMyc +30ng/μLRAG2:绿色荧光蛋白。 </li><li>注射应执行先前展示的12，13 。简言之，男性和女性的斑马鱼交配室，注射前的晚上。在当天注射，DNA是加载到注射针，压力调整，使一个50微米直径的泡沫是被驱逐（30pg的DNA），这是一个阶段千分尺测量。然后，注入单细胞阶段的胚胎，用的DNA直接注射到细胞中，入蛋黄不。注入CG1的胚胎和幼虫的生存是普遍较差，只有5％的鱼类转基因成功整合，因此，应注射&gt; 600胚胎，以确保一些鱼会发展成白血病。 </li><li>商店在28.5 ° C注射的胚胎，死胚后24小时删除。动物，然后在5天的生活转移到大型坦克，并提出了前面描述的 14 。 </li></ol> 2。主T的所有在斑马鱼幼虫的筛选<ol><li>注射后大约28天，麻醉幼虫的斑马鱼，鱼制水25ML加入200μL4ng / M Tricane - S在培养皿。 </li><li>检查T - ALL中通过使用20X放大倍率的epifluorescence显微镜，使用二十〇分之四百八十五激发波长和二十五分之五百三十○排放过滤器检测到绿色荧光（图1）荧光标记的发展幼虫。 </li><li>单独的肿瘤阳性幼虫从那些是否定的。负幼虫可在稍后的时间点检查，一旦肿瘤可能已越来越大，以确保没有T一切积极鱼被错过。 </li><li>显示器的T一切积极的鱼，每周至少一次使用epifluorescence显微镜追踪肿瘤的生长。 </li></ol> 3。荧光标记的原代白血病细胞的分离纯化<ol><li>当GFP阳性的白血病细胞已超过50％的动物，牺牲在培养皿含9毫升鱼系统水盘4ng/mL Tricane - S的加入1ML的鱼。 </li><li>放置在一个新的培养皿菜含有5％胎牛血清1ML 0.9倍PBS缓冲细胞的鱼。浸渍用刀片，吸管混合分解大细胞团块的鱼，然后通过细胞到一个50毫升管的40微米滤网。这是没有必要分解成单个细胞的所有组织团块。 </li><li> 4ML聚苯乙烯管吸取500μL的细胞。加入1μL1mg/ml的碘化丙啶（PI），将标签的死细胞。涡，然后继续在冰上的细胞。 </li><li>牺牲CG1鱼，野生型，浸渍，并以同样的方式，应变上述收集transplant.Add 4ML 5％FBS在0.9倍PBS期间的正常细胞，肿瘤细胞的载体作用，总的50毫升管量各5ml。 </li><li>计数在5％胎牛血清的正常细胞，在0.9倍PBS稀释至3X10 5细胞/ mL，然后吸管100μL/well一个96孔板。 </li><li>到96孔板包含以下剂量的血细胞中使用荧光激活细胞分选（FACS），排序GFP阳性，有价证券负肿瘤细胞（图2A，B）的10个细胞/孔48孔，100个细胞/孔到24孔，000个细胞/孔12井，10000个细胞/成6口井。此外，万细胞应成井排序不正常的血细胞，重新分析使用流式细胞仪评估排序细胞的纯度和可行性（图2C）。 </li></ol> 4。限制稀释移植到成年斑马鱼<ol><li>颗粒细胞在2000xg离心10分钟，96孔板。 </li><li>取出上清液95μL，细胞重悬在余下的5μL。 </li><li>举行成人（&gt; 60天）同基因斑马鱼腹侧，并注入腹腔细胞悬液，用一个26G / 2"微量注射器。斑马鱼没有被移植前的麻醉。通常情况下，45鱼10白血病细胞移植，20 100个细胞移植，7移植1000细胞和5个鱼与10,000的T - ALL细胞移植。 </li></ol> 5。分析，以确定白血病起始细胞频率<ol><li>移植后约28天，检查epifluoresence显微镜使用的二十分之四百八十五过滤器检测到绿色荧光的激发波长和25分之530排放移植鱼。记录每注入鱼总数量的积极的鱼的数量。 </li><li>重新审视鱼长达90天，每14天，并记录积极鱼类的数量。当一个肿瘤阳性的鱼变得死气沉沉的，牺牲Tricane - S过量的动物。 </li><li>结果输入到一个表，图3d所示。上传数据到基于Web的ELDA（至尊有限稀释分析）在统计软件<a href="http://bioinf.wehi.edu.au/software/elda/index.html">http://bioinf.wehi.edu.au/software/elda/index.html</a> 15，其中使用频率肿瘤的正面和负面的动物每个移植的剂量，以确定在主T - ALL的白血病细胞自我更新的频率。 </li></ol> 6。代表性的成果: 从同源鱼的胚胎显微注射DNA导入结果往往在注射胚胎的成活率低，&lt;60％的胚胎存活24小时。此外，注入同源鱼的生存是在幼虫发育普遍较差，与通常&lt;20％存活28天。因此重要的是注入大量的胚胎，以创造转基因鱼，将开发的T - ALL。 作为一个例子，CG1应变胚胎注入RAG2::cMyc + RAG2::GFP。合作的转基因在胚胎发育分隔，使每一个细胞表达绿色荧光蛋白表达cMyc。小学后28天（图1）T - ALL的幼虫进行了筛选。先前的研究表明，大约有5％的注射鱼将其胸腺内GFP阳性T细胞（图1），这些鱼的100％将发展成白血病T 细胞成为转化11。虽然小斑马鱼的比例可能有更先进的白血病是很容易检测到28天，最多只会有GFP阳性（图1）胸腺，使幼虫应进行检查高放大倍率下单独，以确保一切积极鱼被选中。 GFP阳性的T - ALL细胞将超过50％的45-70天之间的动物。 在提供的例子中，斑马鱼生活在65天处死，和白血病细胞中分离和FACS限制稀释移植排序。单细胞，碘化丙啶消极和GFP阳性（图2）整理成一个96孔板。再分析10,000排序细胞（理想情况下，以评估可行性（最好&gt; 95％）和纯度&gt; 92％，图2）。移植的T -承滴盘一般28天前出现的，但某些肿瘤的可能需要60天以上的发展。在这个例子中，在第28天，早期肿瘤被视为GFP阳性细胞注射部位（图3B）附近的区域，而一些T承滴盘更多的进展，扩大填补腹腔（图3C） 。限制从三个不同的主T型承滴盘的稀释细胞移植的实验数据显示在图3D。对于这些实验中，超过220个的动物移植，可以很容易地由一个人在几个小时内完成。使用ELDA软件的数据分析表明，平均135的T - ALL细胞自我更新的白血病传播细胞（图3E）。 <img src="/files/ftp_upload/2790/2790fig1.jpg" alt="图1"/> 图1斑马鱼幼虫注射用抹布一细胞阶段::- cMyc +抹布:- EGFP原代白血病的增长进行了筛选，注射后28天。鱼（*）胸腺内GFP阳性T细胞，这种鱼将开发T - ALL的T细胞随着时间的推移转变。在第28天，这个阶段是很常见的的。一条鱼（**）有先进的T - ALL中，已蔓延到软组织。剩下的两个鱼是负数，为肿瘤的生长。采取了16倍的放大倍率的图像。 <img src="/files/ftp_upload/2790/2790fig2.jpg" alt="图2"/> 图2。荧光标记的T - ALL细胞排序从患病动物和限制稀释细胞移植实验中使用。首先，门被吸引到选择单细胞（左上面板），然后碘化丙啶阴性细胞（中间左侧面板）。最后，门被吸引到选择的GFP阳性排序白血病细胞（左侧面板较低）。排序细胞应重新分析，以评估可行性和移植前的纯度（右图）。 <img src="/files/ftp_upload/2790/2790fig3.jpg" alt="图3"/> 图3。斑马鱼是研究白血病移植后28天增长。鱼是要么肿瘤NEGative（一），在注射部位（二）越来越多，有一个小的肿瘤，或有进展性白血病（三）。 7X放大倍率的图像。白血病阳性％，每个稀释度移植鱼类总数的鱼的总人数记录，（四）。数据输入到基于Web的ELDA方案计算自我更新白血病细胞和上下95％可信区间（五）。

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一个在癌症研究中使用斑马鱼的主要优势在于可以在相对较低的成本使用大量的动物。这是特别重要的限制稀释细胞移植实验，开发移植动物肿瘤移植总数的比例用于确定肿瘤启动细胞频率。在这里介绍的方法，用于移植的动物超过70％检测，提供了一个传播肿瘤细胞数量的准确估计。使用动物的数量和移植的肿瘤细胞的剂量应为一个给定的癌症模型优化;例如，如果初步实验表明，肿瘤细胞，是罕见的，有用的移植剂量可10万元，50000，10000和1000细胞每移植手术。 斑马鱼的同源菌株有限稀释分析是有用的。然而，这些菌株尚未常用在所有的实验室，和一些斑马鱼的癌症模型中只存在异体鱼。有限稀释细胞移植可以在野生型菌株发生免疫消融，虽然细胞的自我更新频率可能比如果斑马鱼的同源共使用8高出10-100倍计算。重要的是要注意，应移植到非免疫匹配，照射收件人使用更大剂量的细胞，某些肿瘤细胞就无法生存移植到外国的微环境。此外，许多肿瘤会开始倒退21天以后，当收件人动物的免疫系统恢复正常，因此应评估肿瘤生长移植后每7天的动物。 这里介绍的方法适用于使用任何斑马鱼癌症模型，迄今被用来确定肿瘤启动细胞的频率，在T细胞急性淋巴细胞白血病 8，和实体瘤模型，横纹肌肉瘤 16 。这些肿瘤合作与癌基因和荧光标记的胚胎注射荧光标记，因为DNA的合作隔离，任何表达的原癌基因的细胞也将表达荧光蛋白17。虽然，因为它有助于追踪肿瘤的生长，而不需要牺牲动物，并提供了一​​个直接的方法，分离肿瘤细胞，流式细胞仪，荧光建议，它是可能的肿瘤细胞与抗肿瘤特异性厂商的抗体标志，以方便其净化的，但在斑马鱼的抗体染色，可能需要优化。 最后，一​​旦传播肿瘤细胞的发病率已经被确定为一个给定的的肿瘤类型，它有可能使用这些检测，以确定的机制，执政的细胞频率。例如，原发肿瘤细胞可以被视为与途径的特异性抑制剂，限制稀释移植，或移植鱼本身可能与药物剂量。此外，肿瘤本身的基因可能会操纵一个感兴趣的基因注入一个细胞阶段鱼，使产生的原发性肿瘤过度表达的基因。在这些实验中，影响细胞自我更新频率，将在限制稀释实验观察。

<table border="1"><tbody><tr><th>试剂名称</th><th>公司</th><th>产品编号</th></tr><tr><td> NotI限制酶</td><td> New England Biolabs公司</td><td> R0189 </td></tr><tr><td>酚:氯仿</td><td> EMD的生化</td><td> 6805 - OP </td></tr><tr><td> 5M氯化钾</td><td> Sigma - Aldrich公司</td><td> P9541 </td></tr><tr><td> 100X的Tris - EDTA </td><td> Sigma - Aldrich公司</td><td> T9285 </td></tr><tr><td>高范围的DNA阶梯</td><td> Fermentas公司</td><td> R0621 </td></tr><tr><td>同基因斑马鱼</td><td></td><td>参考文献6-8 </td></tr><tr><td>胎牛血清</td><td> Gibco公司</td><td> 16000-036 </td></tr><tr><td>碘化丙啶</td><td> Sigma - Aldrich公司</td><td> P4864 </td></tr><tr><td> 26G / 2"Microsyringe </td><td>汉密尔顿</td><td> 80366 </td></tr><tr><td> 40微米网状细胞过滤器</td><td>屋宇署猎鹰</td><td> 352340 </td></tr></tbody></table>

quantifying the frequency of tumor-propagating cells using limiting dilution cell transplantation in syngeneic zebrafish

自我更新的癌细胞内肿瘤的细胞类型有无限的能力，促进肿瘤的生长，从而传播肿瘤细胞或肿瘤细胞。它被认为是针对销毁这些细胞自我更新将阻止肿瘤的进展和停止复发，大大提高了患者的预后1。最常用的方法来确定的频率自我更新在肿瘤细胞，是一个限制稀释细胞移植实验，其中肿瘤细胞移植到受体动物剂量的增加;开发肿瘤的动物的比重是用于计算数量自我更新的细胞内原发肿瘤样本2，3。理想的情况下，大量的动物将被用来在每个有限稀释实验，以准确地确定传播肿瘤细胞的频率。然而，大规模的实验，涉及小鼠是昂贵的，最重要的限制稀释检测只用10-15％的实验小鼠。 斑马鱼已获得了作为癌症模型中的突出地位，在很大程度上是由于其易于遗传操作和可以进行大规模实验的经济。此外，在斑马鱼为蓝本的癌症类型，已发现密切模仿其对应的人类疾病4。虽然有可能从一条鱼的肿瘤细胞移植亚致死照射收件人动物的免疫系统在21天以后的再生，往往导致肿瘤缩小5。同基因斑马鱼的最近成立大大促进了肿瘤移植研究 6-8 。由于这些动物的基因完全相同，移植的肿瘤细胞嫁接到收件人的鱼，和肿瘤的生长健壮，可长时间监测。同基因斑马鱼是理想的限制稀释移植，肿瘤细胞检测，没有适应生长在国外的微环境，这可能低估了细胞的自我更新频率 9 ，10。此外，一个细胞移植已成功完成8斑马鱼的同源和数百个动物，可以很容易地和经济移植在同一时间，这两个服务提供一个更准确的估计，自我更新的细胞频率。 在这里，一种方法是建立小学，荧光标记的T细胞急性淋巴细胞性白血病（T - ALL的）在同基因斑马鱼，移栽在这些肿瘤的限制稀释到成年的鱼，以确定细胞自我更新频率。虽然白血病为例，该协议是合适的，以确定肿瘤细胞传播，使用任何癌症模型中的斑马鱼的频率。

Watch this Scientific Journal Video about Quantifying the Frequency of Tumor-propagating Cells Using Limiting Dilution Cell Transplantation in Syngeneic Zebrafish at JoVE.com

Quantifying the Frequency of Tumor-propagating Cells Using Limiting Dilution Cell Transplantation in Syngeneic Zebrafish

有限稀释细胞移植试验是用来确定传播肿瘤细胞的频率。本协议描述了一个产生，发展荧光标记的白血病和细节，如何限制稀释到成年斑马鱼的腹腔这些白血病细胞分离和移植的同源斑马鱼的方法。

量化传播肿瘤细胞的频率使用斑马鱼的同源有限稀释细胞移植

Self-renewing cancer cells are the only cell types within a tumor that have an unlimited ability to promote tumor growth, and are thus known as ...

quantifying-frequency-tumor-propagating-cells-using-limiting-dilution

Nanyang Technological University

Research

JoVE Journal

Biology

19.1K 次观看.  Harvard Medical School. 有限稀释细胞移植试验是用来确定传播肿瘤细胞的频率。本协议描述了一个产生，发展荧光标记的白血病和细节，如何限制稀释到成年斑马鱼的腹腔这些白血病细胞分离和移植的同源斑马鱼的方法。

视频: Quantifying the Frequency of Tumor-propagating Cells Using Limiting Dilution Cell Transplantation in Syngeneic Zebrafish

Isolation and Transplantation of Hematopoietic Stem Cells (HSCs)

Isolation and Transplantation of Hematopoietic Stem Cells HSCs

I am Christina Ceso. I am a postdoc in David's Den lab and I work on the hematopoietic stem cells. Today we are going to purify long-term recuperating hematopoietic stem cells and we'll transplant them into a mouse.We start by spraying the mice with alcohol so that first of all, the mind starts sterilized and also the hair doesn't interfere with the dissection. We use PBS with 2%serum for the dissection. I cut the back skin and I pull the skin apart in order to expose the tissue.In order to isolate the tibia, I hold the tibia with tweezers. I cut the tendon on top of the knee and I cut the muscles below the knee and after that I cut through the knee so that the TBI is not connected to the rest of the limb anymore and I pull in order to isolate it. I then hold the TB upside down and with the scissors I separate the foot from the tibia In order to obtain the femur.I cut the muscles around the femur and then I dislocate the femur from the hip. In order to isolate the hip, I cut the muscle around the hip bone on the side of the tail of the mouse and then I pull the hip away from the body of the mouth In order to isolate the spine. I cut through the tail as close as I can to the beginning of the back and then I cut on the two sides of the spine and below the spine, trying to stay as close as possible to the spine so that the peritoneum is not cut and the intestine is left untouched.A first round of cleaning of the bones is done with a scalpel, which is used to scrape the bone, the muscles away from the bone. The scalpel is good to clean the femurs, the hip and the spine. In order to clean the tibia, I hold them with forceps and I use the forceps to remove as much tissue as possible.A second round of cleaning is done using clean wipe to completely clean the bones outta the muscle, and I use the F wipe to clean the tibia, the femurs and the hips. So this is how bones look. After cleaning, I prefer 50 amount tubes with filters on top and I transfer the bones into the mortar.I add the section media and I crush the bones ask of the first round of crashing of the bones. I piped the mixture in order to release as many styles as possible into solution and reduce clumps. I transferred the mixture on the tubes through the filters and this is what the bone fragments look like after the first round of crushing.So I add more dissection medium to the bone fragments and I flash them again. I transferred the seline mixture to the tubes through the filters and at this point the remaining bone fragments are white and there is no bone marrow left. The tubes are filled with PBS 2%serum and in order to have them all with the same weight so that they're balanced in the centrifuge, but also in order to wash the filters so that we obtain as many cells as possible, I then centrifuge the cell mixture and after centrifugation, the palt includes the bone marrow cells.I aspirate the medium and I move back and forward the tube on the rack so that the pelle is becomes loose and fewer clamps are formed. When I add medium, at this point I add one ml of medium per tube and I ics since I'm sorting hematopoietic stem cells. Later on, I'm going to store an ALI output of total bone marrow sizes at this point, which I will use later for the fact single color control.I add lineage antibody cocktail. I ordered the samples to mix them well and I invaded four degrees for 15 minutes. In the meantime, I start preparing the columns for the lineage division.I put the columns on the magnet and I prepare Ian tubes under the columns. I also prepared the gas PBS using aster flip filter and attaching it to the vacuum so that all the bubbles of air move in the top part of the filter. I then add three ml of the gas DBS to each column in order for the column to become wet.After the incubation with the antibodies, I add medium to the cell samples and I centrifuge them in order to remove the excess of antibodies. After the centrifugation, the stain cells are in the pallet. I aspirate the medium and I suspend the pallet in one mile of the gas PBSI Add the strip tab beans, I mix the cells and I incubate for 15 minutes at four degrees.After the incubation, I put new elucian tubes under the columns. I add two ml of PBS to each tube with the cells and I transferred the cell mixture to the column through a 40 micron filter. I wash the tubes with more medium in order to collect all the cells remaining and I apply the remaining medium through the filters to the columns.The progenitor cells lineage low are alluded into the collection views. I pulled the luted samples and I sent tofu them. So after the centrifugation, the depleted cells are in a white pate.I aspirate the medium and re suspend the cells. I store an adequate of the depleted cells and I add the antibodies to the cell mixture for the sorting and also to all the single color controls. The antibodies are incubated in that with the size for 15 minutes in the fridge.After the ation, I add medium to the cells and and centrifuge them to remove the ex excess of antibodies. After the centrifugation, the cells are in the pt, I aspirate the media that I suspend the cells and I transfer the cells into fox tubes with a blue filter. At this point, the cells are ready to be sorted.I place, I place the mouse under a heating lamp for a few minutes so that the veins dilate and they are more easily seen. When preparing the syringe, you have to be careful to eliminate all the bubbles from the, the mixture that is in the syringe and then you bend the needle at 90 degrees. I put the mouse under a glass cup with a weight on top so that it can run away.I sterilize the tail with the alcohol pad so that the site of injection is sterile, but also it's easier to identify the vein. If you look at the tail of the mouse from the top in the middle and top of the tail, there is an artery while on the two sides there are the two veins and we inject the cells in one of the two veins. I insert the needle into the vein and I inject following injection.It is to wait for a couple of seconds before removing the needle, the needle, and after that I clean the side of injection. This is a representation of the syringe with the ben needle bent at 90 degrees with the knee, the at this ankle compared to the tail, which makes the injection the easiest. When you injecting the tail of the mouse, you have to keep in mind that the tail vein is extremely superficial, so really this is the orientation of the needle compared to the tail.And it looks like you will just keep sliding on top of the tail, but actually you will get into the tail then. I am Christina Ceso. I am a post in David Kaden lab, and I work on the hematopoietic stem cells.I usually use for animals to start with, and I had more variability at the beginning, but once you become consistent with your methods, I consistently get about 50, 000 to 60, 000 stem cells out for mice. I did my PhD in London with Dr.Fiona Wat working on derma stem cells. After that, I just wanted to have a wider knowledge of adult stem cells in various tissues, and so I decided to move to David Kaden lab to work on hematopoietic stem cells, mainly because the things that are known about hematopoietic stem cells are the things that are not known for the epidermic stem cells and a number of assays that can be done with epi.Epidermal stem cells are not really done yet with the hematopoietic stem cells, and so at this point I'm trying to put together the, the knowledge from the two different fields and ultimately I'm interested in the study of the mechanism to regulate adult tissue sensors in general, so not just the epidermis or the stem cells, but also different tissues because there are, there are similar mechanism that is, it's always the same genes that are involved in the regulation that just interact with each other in different ways, and it's just interesting to understand what makes each stem cell be a stem, but then ultimately a stem cell in a different tissue. So giving rise to a different protein.

分离和造血干细胞移植（造血干细胞）

科研

生物学

Transplantation of Whole Kidney Marrow in Adult Zebrafish

Hematopoietic stem cells (HSC) are a rare population of pluripotent cells that maintain all the differentiated blood lineages throughout the life of an organism. The functional definition of a HSC is a transplanted cell that has the ability to reconstitute all the blood lineages of an irradiated recipient long term. This designation was established by decades of seminal work in mammalian systems. Using hematopoietic cell transplantation (HCT) and reverse genetic manipulations in the mouse, the underlying regulatory factors of HSC biology are beginning to be unveiled, but are still largely under-explored. Recently, the zebrafish has emerged as a powerful genetic model to study vertebrate hematopoiesis. Establishing HCT in zebrafish will allow scientists to utilize the large-scale genetic and chemical screening methodologies available in zebrafish to reveal novel mechanisms underlying HSC regulation. In this article, we demonstrate a method to perform HCT in adult zebrafish. We show the dissection and preparation of zebrafish whole kidney marrow, the site of adult hematopoiesis in the zebrafish, and the introduction of these donor cells into the circulation of irradiated recipient fish via intracardiac injection. Additionally, we describe the post-transplant care of fish in an "ICU" to increase their long-term health. In general, gentle care of the fish before, during, and after the transplant is critical to increase the number of fish that will survive more than one month following the procedure, which is essential for assessment of long term (&lt;3 month) engraftment. The experimental data used to establish this protocol will be published elsewhere. The establishment of this protocol will allow for the merger of large-scale zebrafish genetics and transplant biology.

In this article, we demonstrate a method to perform HCT in adult zebrafish.

在成年斑马鱼的全肾骨髓移植

Hematopoietic stem cells (HSC) are a rare population of pluripotent cells that maintain all the differentiated blood lineages throughout the life of an ...

Tracheotomy: A Method for Transplantation of Stem Cells to the Lung

Lung disease is a leading cause of death and likely to become an epidemic given increases in pollution and smoking worldwide. Advances in stem cell therapy may alleviate many of the symptoms associated with lung disease and induce alveolar repair in adults. Concurrent with the ongoing search for stem cells applicable for human treatment, precise delivery and homing (to the site of disease) must be reassured for successful therapy. Here, I report that stem cells can safely be instilled via the trachea opening a non-stop route to the lung. This method involves a skin incision, caudal insertion of a cannula into and along the tracheal lumen, and injection of a stem cell vehicle mixture into airways of the lung. A broad range of media solutions and stabilizers can be instilled via tracheotomy, resulting in the ability to deliver a wider range of cell types. With alveolar epithelium confining these cells to the lumen, lung expansion and negative pressure during inhalation may also assist in stem cell integration. Tracheal delivery of stem cells, with a quick uptake and the ability to handle a large range of treatments, could accelerate the development of cell-based therapies, opening new avenues for treatment of lung disease.

Significant breakthroughs in stem cell identification are continuously being made. To translate these discoveries, however, novel methods for cellular delivery must be devised. Here I report that the airways provide a safe route for stem cell transplantation to the lungs.

气管切开术：干细胞移植肺的方法

Lung disease is a leading cause of death and likely to become an epidemic given increases in pollution and smoking worldwide. Advances in stem cell ...

Lens Transplantation in Zebrafish and its Application in the Analysis of Eye Mutants

The lens plays an important role in the development of the optic cup[1,2]. Using the zebrafish as a model organism, questions regarding lens development can be addressed. The zebrafish is useful for genetic studies due to several advantageous characteristics, including small size, high fecundity, short lifecycle, and ease of care. Lens development occurs rapidly in zebrafish. By 72 hpf, the zebrafish lens is functionally mature [3]. Abundant genetic and molecular resources are available to support research in zebrafish. In addition, the similarity of the zebrafish eye to those of other vertebrates provides basis for its use as an excellent animal model of human defects[4-7]. Several zebrafish mutants exhibit lens abnormalities, including high levels of cell death, which in some cases leads to a complete degeneration of lens tissues [8]. To determine whether lens abnormalities are due to intrinsic causes or to defective interactions with the surrounding tissues, transplantation of a mutant lens into a wild-type eye is performed. Using fire-polished metal needles, mutant or wild-type lenses are carefully dissected from the donor animal, and transferred into the host. To distinguish wild-type and mutant tissues, a transgenic line is used as the donor. This line expresses membrane-bound GFP in all tissues, including the lens. This transplantation technique is an essential tool in the studies of zebrafish lens mutants.

Lens development involves interactions with other tissues. Several zebrafish eye mutants are characterized by an abnormally small lens size. Here we demonstrate a lens transplantation experiment to determine whether this phenotype is due to intrinsic causes or defective interactions with tissues that surround the lens.

在斑马鱼和镜头移植在眼突变分析中的应用

The lens plays an important role in the development of the optic cup[1,2]. Using the zebrafish as a model organism, questions regarding lens development ...

Generation of Human CD40-activated B cells

CD40-activated B cells (CD40-B cells) have been identified as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cancer immunotherapy 1-3. Compared to Dendritic cells (DCs), the best characterized APC, CD40-B cells have several distinct biological and technical properties. Similar to DCs, B cells show an increased expression of MHC and co-stimulatory molecules (Fig.1b), exhibit a strong migratory capacity and present antigen presentation efficiently to T cells, after stimulation with interleukin-4 and CD40 ligand (CD40L). However, in contrast to immature or mature DCs, CD40-B cells express the full lymph node homing triad consisting of CD62L, CCR7/CXCR4, and leukocyte function antigen-1 (LFA1, CD11a/CD18), necessary for homing to secondary lymphoid organs (Fig.1a) 3. CD40-B cells can be generated without difficulties from very small amounts of peripheral blood which can be further expanded in vitro to very large amounts of highly-pure CD40-B cells (&gt;109 cells per patient) from healthy donors as well as cancer patients (Fig.1c,d) 1,4. 
In this protocol we demonstrate how to obtain fully activated CD40-B cells from human PBMC. Key molecules for the cell culture are CD40 ligand, interleukin-4 (IL-4) and cyclosporin A (CsA), which are replenished in a 3-4 day culture cycle. For laboratory purposes CD40-stimulation is provided by NIH/3T3 cells expressing recombinant human CD40 ligand (tCD40L NIH/3T3) 5. To avoid contamination with non-transfected cells, expression of the human CD40 ligand on the transfectants has to be checked regularly (Fig.2). 
After 14 days CD40-B cell cultures consist of more than 95% pure B cells and an expansion of CD40-B cells over 65 days is frequently possible without any loss of function 1, 4. CD40-B cells efficiently take up, process and present antigens to T cells 6. They do not only prime na&#970;ve, but also expand memory T cells 7,8. CD40-activated B cells can be used to study B-cell activation, differentiation and function. Moreover, they represent a promising tool for therapeutic or preventive vaccination against tumors 9.

In this video we present the ex vivo generation and expansion of human CD40-activated B cells (CD40-B) from peripheral blood mononuclear cells (PBMC) by stimulation with CD40 ligand and interleukin-4.

人CD40激活的B细胞的生成

CD40-activated B cells (CD40-B cells) have been identified as an alternative source of immuno-stimulatory antigen-presenting cells (APC) for cancer ...

Tissue Targeted Embryonic Chimeras: Zebrafish Gastrula Cell Transplantation

Certain fundamental questions in the field of developmental biology can only be answered when cells are placed in novel environments or when small groups of cells in a larger context are altered.  Watching how one cell interacts with and behaves in a unique environment is essential to characterizing cell functions.  Determining how the localized misexpression of a specific protein influences surrounding cells provides insightful information on the roles that protein plays in a variety of developmental processes.  Our lab uses the zebrafish model system to uniquely combine genetic approaches with classical transplantation techniques to generate genotypic or phenotypic chimeras.  We study neuron-glial cell interactions during the formation of forebrain commissures in zebrafish. This video describes a method that allows our lab to investigate the role of astroglial populations in the diencephalon and the roles of specific guidance cues that influence projecting axons as they cross the midline. Due to their transparency zebrafish embryos are ideal models for this type of ectopic cell placement or localized gene misexpression. Tracking transplanted cells can be accomplished using a vital dye or a transgenic fish line expressing a fluorescent protein. We demonstrate here how to prepare donor embryos with a vital dye tracer for transplantation, as well as how to extract and transplant cells from one gastrula staged embryo to another.  We present data showing ectopic GFP+ transgenic cells within the forebrain of zebrafish embryos and characterize the location of these cells with respect to forebrain commissures.  In addition, we show laser scanning confocal timelapse microscopy of Alexa 594 labeled cells transplanted into a GFP+ transgenic host embryo.  These data provide evidence that gastrula staged transplantation enables the targeted positioning of ectopic cells to address a variety of questions in Developmental Biology.

Zebrafish cell transplantation enables the combination of genetics and embryology to generate tissue specific chimeras. This video demonstrates gastrula staged cell transplantations that have allowed our lab to investigate the roles of astroglial populations and specific guidance cues during commissure formation in the forebrain.

组织有针对性的胚胎嵌合体：斑马鱼原肠胚细胞移植

Certain fundamental questions in the field of developmental biology can only be answered when cells are placed in novel environments or when small groups ...

Transplantation of Cells Directly into the Kidney of Adult Zebrafish

Regenerative medicine based on the transplantation of stem or progenitor cells into damaged tissues has the potential to treat a wide range of chronic diseases1. However, most organs are not easily accessible, necessitating the need to develop surgical methods to gain access to these structures. In this video article, we describe a method for transplanting cells directly into the kidney of adult zebrafish, a popular model to study regeneration and disease2. Recipient fish are pre-conditioned by irradiation to suppress the immune rejection of the injected cells3. We demonstrate how the head kidney can be exposed by a lateral incision in the flank of the fish, followed by the injection of cells directly in to the organ. Using fluorescently labeled whole kidney marrow cells comprising a mixed population of renal and hematopoietic precursors, we show that nephron progenitors can engraft and differentiate into new renal tissue - the gold standard of any cell-based regenerative therapy. This technique can be adapted to deliver purified stem or progenitor cells and/or small molecules to the kidney as well as other internal organs and further enhances the zebrafish as a versatile model to study regenerative medicine.

Cell transplantation is an essential technique for studying tissue regeneration and for developing cell-based therapies of disease. We demonstrate here a microsurgical technique that permits the transplantation of genetically labeled cells directly into the kidney of adult zebrafish fish.

直接进入成年斑马鱼的肾细胞移植

Regenerative medicine based on the transplantation of stem or progenitor cells into damaged tissues has the potential to treat a wide range of chronic ...

In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina

Many devastating inherited eye diseases result in progressive and irreversible blindness because humans cannot regenerate dying or diseased retinal neurons. In contrast, the adult zebrafish retina possesses the robust ability to spontaneously regenerate any neuronal class that is lost in a variety of different retinal damage models, including retinal puncture, chemical ablation, concentrated high temperature, and intense light treatment 1-8. Our lab extensively characterized regeneration of photoreceptors following constant intense light treatment and inner retinal neurons after intravitreal ouabain injection 2, 5, 9. In all cases, resident M&uuml;ller glia re-enter the cell cycle to produce neuronal progenitors, which continue to proliferate and migrate to the proper retinal layer, where they differentiate into the deficient neurons. 
	We characterized five different stages during regeneration of the light-damaged retina that were highlighted by specific cellular responses. We identified several differentially expressed genes at each stage of retinal regeneration by mRNA microarray analysis 10. Many of these genes are also critical for ocular development. To test the role of each candidate gene/protein during retinal regeneration, we needed to develop a method to conditionally limit the expression of a candidate protein only at times during regeneration of the adult retina. 
	Morpholino oligos are widely used to study loss of function of specific proteins during the development of zebrafish, Xenopus, chick, mouse, and tumors in human xenografts 11-14. These modified oligos basepair with complementary RNA sequence to either block the splicing or translation of the target RNA. Morpholinos are stable in the cell and can eliminate or "knockdown" protein expression for three to five days 12. 
	Here, we describe a method to efficiently knockdown target protein expression in the adult zebrafish retina. This method employs lissamine-tagged antisense morpholinos that are injected into the vitreous of the adult zebrafish eye. Using electrode forceps, the morpholino is then electroporated into all the cell types of the dorsal and central retina. Lissamine provides the charge on the morpholino for electroporation and can be visualized to assess the presence of the morpholino in the retinal cells. 
	Conditional knockdown in the retina can be used to examine the role of specific proteins at different times during regeneration. Additionally, this approach can be used to study the role of specific proteins in the undamaged retina, in such processes as visual transduction and visual processing in second order neurons.

In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina

A method to conditionally knockdown a target protein&#x2019;s expression in the adult zebrafish retina is described, which involves intravitreally injecting antisense morpholinos and electroporating them into the retina. The resulting protein is knocked down for several days, which allows testing the protein&#x2019;s role in the regenerating or intact retina.

在体内电穿孔Morpholinos到成年斑马鱼视网膜

Many devastating inherited eye diseases result in progressive and irreversible blindness because humans cannot regenerate dying or diseased retinal ...

Studying Proteolysis of Cyclin B at the Single Cell Level in Whole Cell Populations

Equal distribution of chromosomes between the two daughter cells during cell division is a prerequisite for guaranteeing genetic stability 1. Inaccuracies during chromosome separation are a hallmark of malignancy and associated with progressive disease 2-4. The spindle assembly checkpoint (SAC) is a mitotic surveillance mechanism that holds back cells at metaphase until every single chromosome has established a stable bipolar attachment to the mitotic spindle1. The SAC exerts its function by interference with the activating APC/C subunit Cdc20 to block proteolysis of securin and cyclin B and thus chromosome separation and mitotic exit. Improper attachment of chromosomes prevents silencing of SAC signaling and causes continued inhibition of APC/CCdc20 until the problem is solved to avoid chromosome missegregation, aneuploidy and malignant growths1.
Most studies that addressed the influence of improper chromosomal attachment on APC/C-dependent proteolysis took advantage of spindle disruption using depolymerizing or microtubule-stabilizing drugs to interfere with chromosomal attachment to microtubules. Since interference with microtubule kinetics can affect the transport and localization of critical regulators, these procedures bear a risk of inducing artificial effects 5.
To study how the SAC interferes with APC/C-dependent proteolysis of cyclin B during mitosis in unperturbed cell populations, we established a histone H2-GFP-based system which allowed the simultaneous monitoring of metaphase alignment of mitotic chromosomes and proteolysis of cyclin B 6.
To depict proteolytic profiles, we generated a chimeric cyclin B reporter molecule with a C-terminal SNAP moiety 6 (Figure 1). In a self-labeling reaction, the SNAP-moiety is able to form covalent bonds with alkylguanine-carriers (SNAP substrate) 7,8 (Figure 1). SNAP substrate molecules are readily available and carry a broad spectrum of different fluorochromes. Chimeric cyclin B-SNAP molecules become labeled upon addition of the membrane-permeable SNAP substrate to the growth medium 7 (Figure 1). Following the labeling reaction, the cyclin B-SNAP fluorescence intensity drops in a pulse-chase reaction-like manner and fluorescence intensities reflect levels of cyclin B degradation 6 (Figure 1). Our system facilitates the monitoring of mitotic APC/C-dependent proteolysis in large numbers of cells (or several cell populations) in parallel. Thereby, the system may be a valuable tool to identify agents/small molecules that are able to interfere with proteolytic activity at the metaphase to anaphase transition. Moreover, as synthesis of cyclin B during mitosis has recently been suggested as an important mechanism in fostering a mitotic block in mice and humans by keeping cyclin B expression levels stable 9,10, this system enabled us to analyze cyclin B proteolysis as one element of a balanced equilibrium 6.

Metaphase to anaphase transition is triggered through anaphase-promoting complex (APC/C)-dependent ubiquitination and subsequent destruction of cyclin B. Here, we established a system which, following pulse-chase labeling, allows monitoring cyclin B proteolysis in entire cell populations and facilitates the detection of interference by the mitotic checkpoint.

在整个细胞群在单细胞水平上研究细胞周期蛋白B的蛋白水解

Equal distribution of chromosomes between the two daughter cells during cell division is a prerequisite for guaranteeing genetic stability 1. Inaccuracies ...

Analyzing Craniofacial Morphogenesis in Zebrafish Using 4D Confocal Microscopy

Time-lapse imaging is a technique that allows for the direct observation of the process of morphogenesis, or the generation of shape. Due to their optical clarity and amenability to genetic manipulation, the zebrafish embryo has become a popular model organism with which to perform time-lapse analysis of morphogenesis in living embryos. Confocal imaging of a live zebrafish embryo requires that a tissue of interest is persistently labeled with a fluorescent marker, such as a transgene or injected dye. The process demands that the embryo is anesthetized and held in place in such a way that healthy development proceeds normally. Parameters for imaging must be set to account for three-dimensional growth and to balance the demands of resolving individual cells while getting quick snapshots of development. Our results demonstrate the ability to perform long-term in vivo imaging of fluorescence-labeled zebrafish embryos and to detect varied tissue behaviors in the cranial neural crest that cause craniofacial abnormalities. Developmental delays caused by anesthesia and mounting are minimal, and embryos are unharmed by the process. Time-lapse imaged embryos can be returned to liquid medium and subsequently imaged or fixed at later points in development. With an increasing abundance of transgenic zebrafish lines and well-characterized fate mapping and transplantation techniques, imaging any desired tissue is possible. As such, time-lapse in vivo imaging combines powerfully with zebrafish genetic methods, including analyses of mutant and microinjected embryos.

Time-lapse confocal imaging is a powerful technique useful for characterizing embryonic development. Here, we describe the methodology and characterize craniofacial morphogenesis in wild-type, as well as pdgfra, smad5, and smo mutant embryos.

使用4D共聚焦显微镜分析颅面形态的斑马鱼

Time-lapse imaging is a technique that allows for the direct observation of the process of morphogenesis, or the generation of shape. Due to their optical ...