这项工作是支持由Helse OG Rehabilitering（JLB），挪威研究理事会（GH和JCG）和奥斯陆大学（NK细胞和JCG）的补助金。

1。分离和培养成胚胎的人类干细胞注射的准备<ol><li>作为在本议定书中的例子（脑，脂肪，骨髓）的人类干细胞分离在不同的方式从不同的组织。例如，成体神经干细胞分离的脑室壁内镜神经外科，然后分离单个细胞在体外培养的过程中收集的组织块。自发形成的神经干细胞神经球，漂浮在培养液中，而其他类型的细胞坚持底部的菜 7 。脂肪间充质干细胞是从吸脂材料，是消化酶和紧张，以消除纤维组织，然后离心除去成熟的脂肪细胞。由此产生的细胞悬浮于培养基中血清补充，以促进干细胞的增殖，超过其他类型的细胞 8造血干细胞和祖细胞可以从骨髓抽吸隔离使用特异性抗体标记的磁性粒子（Myltenyi生物技术，德国的积极选择或DYNAL - Invitrogen公司，美国）和磁性细胞分离和/或使用抗体的共轭荧光基团和荧光激活细胞分选（FACS）9。 </li><li>为了便于以后识别，标签上的干细胞，用荧光标记的鸡胚胎植入前。干细胞可以被标记的亲脂性荧光染料，如使用DII（Invitrogen公司，美国）或PKH26供应商推荐的协议（美国Sigma公司）植入前不久，或为一个永久性标签，基本上没有污染宿主细胞的危险，他们可以像绿色荧光蛋白的荧光蛋白基因转导，利用慢病毒载体，而仍然在文化10。 </li><li>不同的文化和传播人类干细胞的协议。文献中可以找到详细的协议。简言之，球造血干细胞通常在烧瓶中培养，而贴壁干细胞通常在培养皿中培养（方便trituration分裂时，见下文），在合适的介质。分裂或收割，颗粒球形成细胞在1200RPM和重悬5分钟不超过5分钟在37摄氏度预热胰蛋白酶/ EDTA溶液的离心。对于贴壁细胞的胰蛋白酶/ EDTA溶液的培养皿中，并在5分钟后磨碎。终止加入的Ca 2 +含中等，有时补充白蛋白的胰蛋白酶。要去除胰蛋白酶/ EDTA，离心机的细胞在1200RPM（5分钟），取出上清液，悬浮在寒冷的注射车辆（通常是中等或磷酸盐缓冲液），再离心（5分）在1200RPM。删除这最上清，轻轻悬浮细胞，以创建一个高度集中的细胞悬液。 </li><li>保存在一个小的，加盖的瓶或离心管放在冰上几个小时在注射之前，人类干细胞的悬浮液。在这种状态下的生存可能会依赖细胞类型。根据细胞的属性注射车辆必须进行优化，包括他们的附着性（如低的Ca 2 +车辆可能有助于防止结块）和抗缺氧和pH值的变化。 </li></ol> 2。卵孵化和准备<ol><li>商店受精鸡蛋在冷却室（例如：Termaks，卑尔根，挪威），在14-15 ° C之前孵化。在此温度下的发展被逮捕开始，直到孵化。超过10天左右或在较低温度下储存的受精卵率较低的后续发展和生存。 </li><li>重新初始化发展的鸡蛋放在一个加湿，强制通风孵化器（例如：粘结剂，纽约，美国）在38-39 ° C，并孵化，直到达到所需的发展阶段（为分期，见文献11。 ）。 </li><li>打开鸡蛋前，通过蛋壳（不太深，以避免刺入蛋黄）中插入一个18针的注射器针头和疏散4毫升的白蛋白创建一个以上的胚胎的空气袋（图A）。这将创建一个压降，在几分钟内的空气通过多孔的外壳进入扳平。由于空气的进入，它收集在壳体内的最高点，因此，从外壳的内表面分离胚胎。 </li><li>与传统的塑料胶带密封注射器针头创建孔。 </li></ol> 3。拉玻璃微量<ol><li>拉从高硼硅玻璃玻璃微量（例如，1.2毫米外径× 0.94 mm内径，传统的电极拉马（例如，美国哈佛大学器械），模型P - 2000，美国），萨特仪器。调整拉拔器设置，以获得具有高输入电阻时，microel使用的微量ectrodes。提示应约1-1.5厘米长。轴可以被标记在1 mm间隔计算卷弹出。 </li><li>打破弯曲用钳，他们或推动对固体表面，在显微镜下观察时，一个可行的大小微量提示。突破应尽量均匀左右的玻璃的周边和由此产生的尖端内径应适应不堵塞细胞的大小。 20-30微米内径通常效果很好。 </li></ol> 4。快速绿色染料溶液（对比试剂的制备） <ol><li>准备的快速绿色染料（Sigma - Aldrich公司，美国）的0.1％（W / V）溶液溶解在鸡生理盐水（137毫米氯化钠，氯化钾5毫米，2毫米氯化钙 2，1毫米氯化镁 2，1毫米娜磷酸，5毫米HEPES，11毫米葡萄糖，pH值7.4）。 </li><li>通过0.22微米Millex GP过滤器（Millipore公司有限公司，百福，美国）的过滤器快速的绿色解决方案。 </li></ol> 5。开放的鸡蛋和对比胚胎<ol><li>广场上的鸡蛋的顶部两片磁带，涵盖空气口袋的尺寸。保持鸡蛋为主，所以，录音的面积（气囊）最重要的是，切一个小窗口内的录音使用一个坚固的解剖剪刀面积边界。磁带增加了支持，并有助于防止蛋壳碎片坠落到胚胎。与气囊相关的气泡可以使用塑料枪头或其他钝探头后端弹出。 </li><li>绘制成一个用25号针头的1 ml注射器一些快速的绿色解决方案。取下注射器和针头在任何气泡。弯曲的针，一个45度角，渗透在胚胎板外的点（确定由血管circumscribe环;穿出内的胚胎板增加死亡率）和精心指导下胚胎的绒绣。注入的染料，直到达到所需的对比度。胚胎，这通常是透明的，现在应该站出来作为一个对深绿色（图B）白色的幽灵。许多研究者使用墨汁（稀释到10％V / V），而不是快速绿色。在这种情况下，重要的是，使用印度墨水是无毒的，如伯利坎润版液印度墨水，。 </li></ol> 6。制作后脑和脊髓神经管病变<ol><li>生产削尖的钨针，火焰短（2-3厘米）件的直径为100微米的钨棒（目录号719000，A＆R的系统，西雅图，华盛顿），蚀刻与乙炔/氧气的火炬。这样做是很简单的杆端插入火炬的火焰，直到一个火花看到，它代表的钨外层的爆炸性的侵蚀，留下锋利的尖。 </li><li>通过使用一个削尖的钨针，片和偏转卵黄膜覆盖面积显微。 </li><li>使用第二个削尖的钨针（往往是破坏后的第一针的尖端通过卵黄膜切片，不适合随后的显微外科），仔细切割通过上皮覆神经管和偏转，然后切周围和下一块神经管被删除，使用短，见效快向上招（图C）。横向削减和纵向削减通常是最成功的。切不要过深或穿透底层的血管，这通常是致命的的危险。 </li><li>轻轻翻转或切除的组织块拖到远离病变的部位使用的钨针。如果这被证明是困难的，它也可以被删除，使用软管连接到一个玻璃微吸管吸口。 </li></ol> 7。人类干细胞注射 A.注入病变神经管绘制成的提示使用塑料管的玻璃微吸管口吸少量的细胞。被注入的细胞类型的细胞悬液浓度必须优化。安装在一个显微的微管，并把它连接到一个微量泵（例如：PV830气动PicoPump，WPI的，华盛顿，美国）。在解剖镜下使用的视觉控制，引导到病灶的微量的提示，并仔细驱逐增加空气压力（恒压脉冲或逐渐增加的压力）（图C）的细胞。当沉积在病变部位所需的细胞量，仔细撤回的微量。 B.神经管的管腔注入在某些情况下，它可能是可取的细胞注入到发育中的大脑脑室，例如，如果细胞侵入神经组织病变的情况下获得的访问。细胞注射到任何BR流明AIN地区需要使用管腔提供一个足够大的插座，很容易进入的发展阶段; HH阶段12日至18日在这方面有利。的病变，是没有必要的。只需皮尔斯的神经管与玻璃微管细胞注射前的墙上。这种方法的成功的关键要素是微量渗透唐突的清晰度;太慢了一个运动和微量提示可能只有酒窝神经管内壁无穿透。 C.全身注射全身注射液可通过多余的胚胎血管（HH阶段15起，当这些船只发达）。这是不寻常的陪同一些出血，因为有很多的小动脉和少数较大静脉，动脉注射一般都更安全，当涉及到胚胎的生存。另一方面，静脉注射到心脏细胞提供更快的访问，从而可能更均匀分布的细胞。一个成功的注入需要一个尖锐的微量直径小于目标船只。方法沿其轴线的水平（约30度）的一个小角度的船只。直到船只开始闭塞，对船只的微量推。然后推进一步坚定，突然运动渗透。慢慢地缩回，直到血液被看作是通过毛细作用绘制成的微尖。采用恒压，然后进行细胞注射后，微管是一个快速移动的缩回。 8。密封鸡蛋<ol><li>注射后，我们仍然允许注射的细胞来解决，坚持站在鸡蛋几分钟。以避免脱水（它可以很快发生，并能证明致命的），奠定了一块沾在窗口，在此期间的休息时间组织或滤纸。 </li><li>几分钟后，细胞纷纷落户，干在窗口的外壳，并与传统的塑料胶带密封的窗口。确保这种密封很紧，没有白蛋白可以通过开放或与壳体之间的磁带（这常常被证明是致命的，在随后的培养）的泄漏。 </li><li>更换蛋被迫草案孵化器的进一步发展。观察可以在窗口中删除磁带的时间间隔。 </li></ol> 9。胚胎夹层<ol><li>一旦胚胎已经达到了预期的阶段，打开鸡蛋，一碗成一个冰冷的生理盐水或磷酸盐缓冲液（磁带覆盖窗口应削减首次允许分开的两半蛋壳）。冰冷的生理盐水，低温麻醉胚胎。心应迅速减缓，并在几分钟内停止。 </li><li>从胚外膜分离胚胎阶段，使用ref。 11。 </li><li>杀头的胚胎，其传输到一个清扫菜，硅橡胶地板，并包含含氧生理盐水辅以葡萄糖（137毫米氯化钠，氯化钾5毫米，2毫米氯化钙 2，1毫米氯化镁 2，1毫米的钠磷酸盐， 5 mM的HEPES，11毫米葡萄糖，pH值7.4）和针快腹侧。取下腹部和胸内脏。使用角度的剪刀，使纵切口，沿脊柱腹外每个方面。特别是在后期阶段（后发展6天），最初的切口是最好的腰骶椎骨和脊髓之间的空间是最大的地区。删除，脊柱揭示脊髓腹侧。确保生理盐水仍然由纯医用氧气在约10分钟一班冒泡含氧。 </li><li>小心剪去两边的新生的背，腹根，脊髓样所需的水平，并轻轻椎管。脊髓在这种状态下存活多小时，可承受12和生理解剖13,14实验。 </li></ol><img src="/files/ftp_upload/2071/2071fig1.jpg" alt="图1" /> 图1）疏散白蛋白适当的做法。请注意胚胎的位置和针的位置。二）对比的胚胎。胚胎光盘外，注意针的切入点。 c）过程的示意图表示单方面脊髓神经管病变细胞移植。

<ol>
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鸡胚为异种器官移植实验的方便和灵活的动物模型。胚胎和胎儿发育期间缺乏免疫系统功能，允许从容忍任何种类的孵化温度的细胞的生存和发展。鸡胚胎已被用于测试各类干细胞（文献15）。的再生潜力。哺乳动物干细胞能够融入鸡胚胎组织，包括中枢神经系统，在那里他们可以引起神经元的轴突预测，突触连接和电生理特性，可以在功能神经回路1,2的背景下进行评估。 由于手术过程中是比较容易的，注射是根据视觉，而不是立体控制，可以很容易地增加胚胎数量，以适应​​不同的实验条件，在一个单一的实验。通常情况下，细胞可以注入20-30胚胎在4小时的实验。注射后的胚胎死亡率是主要限制因素。必须采取预防措施，以避免出血和脱水。要知道，有某些关键的发展阶段自然死亡率增加。补充几毫升的温暖，鸡消毒振铃以下注射和关键发育阶段的前一天的胚胎，以及确保，鸡蛋中的窗口，是良好的密封和导向性，以避免泄漏，提高生存率。如果通过胶带密封泄漏发生，请取出磁带，干燥的表面，并重新磁带。一些研究者用蜡和玻璃或塑料盖玻片密封的窗口。死亡率不应与假手术胚胎注入大大不同。 虽然我们一直致力于人类干细胞注射到大脑和脊髓的原基的干细胞也可以被注入到其他器官原基（见4）例如文献。。各器官对自身的挑战。例如，注射入心可以是具有挑战性的，因为高的机械性能，吸管渗透和，因为运动，另一方面可以减缓冷却至室温，在注射之前，胚胎。机关缺乏流明可能无法容纳足够数量的注射，因此可能需要手术的病变，以创建一个插座。在我们的经验，后病变组织的再生是一个优势，因为它可以促进组织的人类干细胞的融合。如分散somitic间质的方式来实现纳入到间质的衍生工具，包括神经嵴衍生的外围神经节 16,17或迁移的神经嵴的胚胎间质细胞也被注入。

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<td>Fluorescent microscope</td>
<td>Microscope</td>
<td>Olympus IX-81</td>
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<td>Stereo Investigator</td>
<td>Program</td>
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<td>MIP-GFP mice</td>
<td>Mice</td>
<td>Jackson Laboratory</td>
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<td>Mathematica</td>
<td>Program</td>
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xenotransplantation of human stem cells into the chicken embryo

鸡胚是研究胚胎和胎儿的正常发展，为异种器官移植实验的经典动物模型来研究细胞的行为，在一个标准化的<em&gt;在体内</em&gt;环境。鸡胚的主要优点包括成本低，可访问性高，便于手术操作，缺乏一个全面发展的免疫系统。异种移植到鸡胚可以提供有价值的信息，对细胞增殖，分化和行为，定义的胚胎组织龛信号细胞的反应，并致瘤潜力。移植到鸡胚细胞，也可以对其他动物模型移植实验的一个步骤。最近鸡胚已经用于评估的人类干细胞和祖细胞源性的潜力，植入到神经原基<sup&gt; 1-6</sup&gt;。我们在这个视频文件移植到发育中的鸡胚胎的神经系统的人类干细胞的整个过程。受精卵孵化的过程开始，直到胚胎所需的年龄开发。然后打开蛋壳和胚胎注射胚胎和蛋黄之间的染料对比。微小病变采用显微神经管，建立一个后续整合到宿主组织细胞沉积，促进再生的网站。我们证明人类干细胞的成神经管形成的后脑和脊髓，和成的部分，形成大脑的神经管的管腔的一部分的这种病变的注射。作为一种替代方式，提供吻合血管的组织，包括中枢神经系统的细胞也表现出全身注射到胚外静脉和动脉。最后，我们将展示如何从鸡蛋中删除的胚胎，经过数天的进一步发展，以及如何免费为随后的生理，病理或生化分析解剖脊髓。

Watch this Scientific Journal Video about Xenotransplantation of Human Stem Cells into the Chicken Embryo at JoVE.com

Xenotransplantation of Human Stem Cells into the Chicken Embryo

在本文中我们提出一个人类干细胞移植到鸡胚中央神经系统的各地区的方法。这提供了一个<em&gt;在体内</em&gt;评估各类胚胎组织环境中的人类干细胞的增殖和分化的模型。

人类干细胞异种移植到鸡胚

The chicken embryo is a classical animal model for studying normal embryonic and fetal development and for xenotransplantation experiments to study the ...

xenotransplantation-of-human-stem-cells-into-the-chicken-embryo

Research

JoVE Journal

Biology

19.2K 次观看.  University of Oslo. 在本文中我们提出一个人类干细胞移植到鸡胚中央神经系统的各地区的方法。这提供了一个在体内评估各类胚胎组织环境中的人类干细胞的增殖和分化的模型。

视频: Xenotransplantation of Human Stem Cells into the Chicken Embryo

Propagation of Human Embryonic Stem (ES) Cells

Propagation of Human Embryonic Stem ES Cells

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

人类胚胎干细胞（ES细胞）的繁殖

科研

生物学

Transfecting Human Neural Stem Cells with the Amaxa Nucleofector

Transfection of primary mammalian neural cells, such as human neural stem/precursor cells (hNSPCs), with commonly used cationic lipid transfection reagents has often resulted in poor cell viability and low transfection efficiency.  Other mechanical methods of introducing a gene of interest, such as a &#8220;gene gun&#8221; or microinjection, are also limited by poor cell viability and low numbers of transfected cells.  The strategy of using viral constructs to introduce an exogenous gene into primary cells has been constrained by both the amount of time and labor required to create viral vectors and potential safety concerns.  We describe here a step-by-step protocol for transfecting hNSPCs using Amaxa's Nucleofector device and technology with electrical current parameters and buffer solutions specifically optimized for transfecting neural stem cells.  Using this protocol, we have achieved initial transfection efficiencies of ~35% and ~70% after stable transfection.  The protocol entails combining a high number of hNSPCs with the DNA to be transfected in the appropriate buffer followed by electroporation with the Nucleofector device.

Introducing a gene of interest into a cell is a powerful method for elucidating its function in vivo. This protocol describes an efficient method of transfecting a culture of human neural stem/precursor cells (hNSPCs) using the Nucleofector electroporation apparatus made by Amaxa.

人类神经干细胞转染与Amaxa Nucleofector

Transfection of primary mammalian neural cells, such as human neural stem/precursor cells (hNSPCs), with commonly used cationic lipid transfection ...

Counting Human Neural Stem Cells

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

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

人类神经干细胞计数

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

Passaging Human Neural Stem Cells

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

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

传代人类神经干细胞

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

Immunocytochemistry: Human Neural Stem Cells

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

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

免疫组化：人类神经干细胞

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

In Ovo Electroporations of HH Stage 10 Chicken Embryos

Large size and external development of the chicken embryo have long made it a valuable tool in the study of developmental biology.  With the advent of molecular biological techniques, the chick has become a useful system in which to study gene regulation and function.  By electroporating DNA or RNA constructs into the developing chicken embryo, genes can be expressed or knocked down in order to analyze in vivo gene function.  Similarly, reporter constructs can be used for fate mapping or to examine putative gene regulatory elements.  Compared to similar experiments in mouse, chick electroporation has the advantages of being quick, easy and inexpensive.  This video demonstrates first how to make a window in the eggshell to manipulate the embryo.  Next, the embryo is visualized with a dilute solution of India ink injected below the embryo.  A glass needle and pipette are used to inject DNA and Fast Green dye into the developing neural tube, then platinum electrodes are placed parallel to the embryo and short electrical pulses are administered with a pulse generator.  Finally, the egg is sealed with tape and placed back into an incubator for further development.  Additionally, the video shows proper egg storage and handling and discusses possible causes of embryo loss following electroporation.

Chick in ovo electroporation is a technique which allows genetic manipulation of the avian embryo. Common applications of this technique include functional analysis of genes and putative enhancer elements. This video demonstrates neural tube electroporation in HH 10 chick embryos. Injection technique and proper egg handling are discussed.

在OVO的HH阶段10个鸡胚Electroporations

Large size and external development of the chicken embryo have long made it a valuable tool in the study of developmental biology.  With the advent of ...

Differentiation of Embryonic Stem Cells into Oligodendrocyte Precursors

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

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

胚胎干细胞分化成少突胶质前体

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

Lentiviral-mediated Knockdown During Ex Vivo Erythropoiesis of Human Hematopoietic Stem Cells

Erythropoiesis is a commonly used model system to study cell differentiation. During erythropoiesis, pluripotent adult human hematopoietic stem cells (HSCs) differentiate into oligopotent progenitors, committed precursors and mature red blood cells 1. This process is regulated for a large part at the level of gene expression, whereby specific transcription factors activate lineage-specific genes while concomitantly repressing genes that are specific to other cell types 2. Studies on transcription factors regulating erythropoiesis are often performed using human and murine cell lines that represent, to some extent, erythroid cells at given stages of differentiation 3-5. However transformed cell lines can only partially mimic erythroid cells and most importantly they do not allow one to comprehensibly study the dynamic changes that occur as cells progress through many stages towards their final erythroid fate. Therefore, a current challenge remains the development of a protocol to obtain relatively homogenous populations of primary HSCs and erythroid cells at various stages of differentiation in quantities that are sufficient to perform genomics and proteomics experiments.
Here we describe an ex vivo cell culture protocol to induce erythroid differentiation from human hematopoietic stem/progenitor cells that have been isolated from either cord blood, bone marrow, or adult peripheral blood mobilized with G-CSF (leukapheresis). This culture system, initially developed by the Douay laboratory 6, uses cytokines and co-culture on mesenchymal cells to mimic the bone marrow microenvironment. Using this ex vivo differentiation protocol, we observe a strong amplification of erythroid progenitors, an induction of differentiation exclusively towards the erythroid lineage and a complete maturation to the stage of enucleated red blood cells. Thus, this system provides an opportunity to study the molecular mechanism of transcriptional regulation as hematopoietic stem cells progress along the erythroid lineage. 
Studying erythropoiesis at the transcriptional level also requires the ability to over-express or knockdown specific factors in primary erythroid cells. For this purpose, we use a lentivirus-mediated gene delivery system that allows for the efficient infection of both dividing and non-dividing cells 7. Here we show that we are able to efficiently knockdown the transcription factor TAL1 in primary human erythroid cells. In addition, GFP expression demonstrates an efficiency of lentiviral infection close to 90%. Thus, our protocol provides a highly useful system for characterization of the regulatory network of transcription factors that control erythropoiesis.

Lentiviral-mediated Knockdown During Ex Vivo Erythropoiesis of Human Hematopoietic Stem Cells

An ex vivo protocol to generate mature human red blood cells from hematopoietic stem/progenitors is described. Additionally we describe an efficient lentiviral-delivery method to knockdown the transcription factor TAL1 in primary erythroid cells. The efficiency of lentivirus mediated gene delivery is demonstrated using GFP expressing viruses.

慢病毒介导的击倒在体外</em人造血干细胞>红细胞

Erythropoiesis is a commonly used model system to study cell differentiation. During erythropoiesis, pluripotent adult human hematopoietic stem cells ...

Reprogramming Human Somatic Cells into Induced Pluripotent Stem Cells (iPSCs) Using Retroviral Vector with GFP

Human embryonic stem cells (hESCs) are pluripotent and an invaluable cellular sources for in vitro disease modeling and regenerative medicine1. It has been previously shown that human somatic cells can be reprogrammed to pluripotency by ectopic expression of four transcription factors (Oct4, Sox2, Klf4 and Myc) and become induced pluripotent stem cells (iPSCs)2-4 . Like hESCs, human iPSCs are pluripotent and a potential source for autologous cells. Here we describe the protocol to reprogram human fibroblast cells with the four reprogramming factors cloned into GFP-containing retroviral backbone4. Using the following protocol, we generate human iPSCs in 3-4 weeks under human ESC culture condition. Human iPSC colonies closely resemble hESCs in morphology and display the loss of GFP fluorescence as a result of retroviral transgene silencing. iPSC colonies isolated mechanically under a fluorescence microscope behave in a similar fashion as hESCs. In these cells, we detect the expression of multiple pluripotency genes and surface markers.

Reprogramming Human Somatic Cells into Induced Pluripotent Stem Cells iPSCs Using Retroviral Vector with GFP

A method to generate human induced pluripotent stem cells (iPSCs) via retrovirus-mediated ectopic expression of OCT4, SOX2, KLF4 and MYC is described. A practical way to identify human iPSC colonies based on GFP expression is also discussed.

人类体细胞重编程为诱导多能干细胞（iPS细胞）用绿色荧光蛋白逆转录病毒载体

Human embryonic stem cells (hESCs) are pluripotent and an invaluable cellular sources for in vitro disease modeling and regenerative medicine1. It has ...

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

诱导上皮和分析间质转化

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...