特别感谢博士鲍里斯Greber的设立激活素A酶联免疫吸附试验协议在Greber 等2007年发表的。我们特别感谢莫妮卡Shevack夫人准备的图形概览。我们非常感谢所有帮助和拍摄之前和期间的宝贵建议Heiko福克斯博士。我们要感谢所有成员保持稳定供应MEFs和CM的的Adjaye实验室，尤其是伊丽莎白·索查。我们也承认我们的同事，在他们的永久支持MPIMG动物设施。这项工作是由马克斯 - 普朗克协会和[BMBF的部分资金;授权号0315717A]，伙伴的ERASysBio + ERA-NET的加在第七框架计划下，欧盟支持的倡议。

1。分离的小鼠胚胎成纤维细胞（MEFs） 非无菌条件下进行以下两个步骤。 <ol><li>颈椎脱位，牺牲在13或14 DPC（一天后coitum）怀孕鼠标（CF1的，哈伦，美国）。 </li><li>剖析了子宫角，简单地在70％（V / V）乙醇和地方成猎鹰管无钙+镁 PBS冲洗+（GIBCO，Invitrogen公司）。 </li></ol>下面的步骤进行了组织文化罩在无菌条件下使用无菌文书。 <ol start="3"><li>培养皿放入子宫角分开，从每个胚胎的胎盘和胚胎囊。 </li><li>解剖头部和红色机关，在PBS清洗，并放置在一个干净的培养皿的胚胎。细剁碎的组织，用消毒的刀片，直到它变成可能吸取。 </li><li>加入1毫升的0.05％胰蛋白酶/ EDTA（GIBCO，Invitrogen公司），包裹体丁100 Kunitz型DNA酶I（USB）的单位，每胚胎。 </li><li>转入50毫升猎鹰管组织和孵育15分钟，在37°C。孵化后每5分钟，移液器上下彻底分离的细胞。 </li><li>加入新鲜制备的MEF的介质的体积约1灭活胰蛋白酶。 MEF培养基（组件使媒体的500毫升，混合所有的元件和过滤器）: 450毫升培养液，胎牛血清（50毫升10％（V / V）），200毫米L-谷氨酰胺（1/100（V / V））5毫升，5毫升的青霉素，链霉素（1/100（V / V））。 </li><li>离心细胞与低速（300 XG），5分钟，小心地取出上清，重悬细胞沉淀在温暖的MEF的介质。 </li><li>板约一个细胞的数量，这相当于在每个T150大掌柜（TPP）的烧瓶3-4涂有0.2％明胶（明胶从牛皮肤，B型，西格玛），2小时的胚胎。成纤维细胞（P0，通道0），是唯一有能力重视明胶涂层烧瓶细胞。 </l我&gt; <li>理想的情况下，细胞是80-90％汇合后24小时，在这个阶段的P0细胞的重要组成部分，是为将来使用冻结。 </li><li>展开余下的T150大掌柜的P0到P3或P4细胞（瓶），然后灭活和使用馈线replate胚胎干细胞或生产条件培养基（CM）。 </li></ol> 2。灭活和电镀MEFs细胞（饲养层细胞的制备） 所有步骤都是在无菌条件下进行组织培养引擎盖。 <ol><li>涂T150瓶明胶0.2％，在室温下孵育至少2小时。 </li><li>在PBS稀释丝裂霉素C（1毫克/毫升）和过滤器。 </li><li>吸MEFs中的媒体，用PBS洗的Ca 2 + Mg 2 +的 。 </li><li>将20毫升含10微克/毫升丝裂霉素ÇMEFs中的介质。 </li><li>在37℃孵育2小时Ç与丝裂霉素C含中，然后用PBS，trypsinize，离心（300 XG 5分钟），并在温暖的培养基重悬细胞两次。 </li><li>计数细胞和板密度56.000细胞/在T150瓶厘米2厘米以下6天的生产和使用。 </li></ol> 3。条件培养液（CM）的制备所有步骤都是在无菌条件下进行组织培养引擎盖。 <ol><li>在56.000细胞/厘米2的密度灭活MEFs中电镀取代胚胎干细胞培养基（澳，无条件中等）（0.5毫升/厘米2）的MEF介质后的第二天，补充新鲜的FGF2的4纳克/毫升。 </li><li>收集培养24 h后，从饲养瓶CM和补充新鲜胚胎干细胞中4纳克/毫升FGF2的进料器。 </li><li>在接下来的6天里重复此过程。每天商店收集于-20°C CM </li><li>经过6天的混合介质和过滤器（康宁，0.22微米，PAS）的所有等分。 50毫升分装并储存于-80°C。 </li><li> &lt;补充基底膜上种植的胚胎干细胞，然后加入额外的4纳克/毫升，FGF2的CM。/ LI&gt; </ol> 4。测量条件培养基一个激活素（ELISA）15 <ol><li>将所有样品和试剂室温。 </li><li>稀释捕获抗体（人力资源\鼠标\大鼠激活素A单克隆抗体，R＆D系统）1％BSA的PBS中，加入微孔板（100μL/孔）孵育在室温下过夜。 </li><li> 24小时后用PBST（PBS 0.05％吐温20）（300μL/孔）洗井三次，然后在室温下1小时块（1％的BSA / PBS，300μL/孔）。 </li><li>在这段时间内，准备激活素A（R＆D系统）的标准曲线，其中包括7稀释（浓度低于30毫微克/毫升）和空白样品。线性工作范围的激活素A是介于0.25和32毫微克/毫升。 </li><li>井（100μL/孔）的标准和样品的重复和孵化在室温下为2小时。 </li><li>洗井的3倍（300μLPBST）。 </li><li>新增二级（生物素）抗体（人/鼠/鼠生物素化单克隆抗体，激活素A R＆D系统）（0。25微克/毫升在1％的BSA / PBS）和孵化在室温下为2小时。 </li><li>洗井的3倍（300μLPBST），加链霉亲和素-HRP（稀释1％BSA / PBS，研发系统）和孵化在室温为20分钟。 </li><li>洗井的3倍（300μLPBST），加入100微升底物溶液（Quantikine，R＆D系统），在室温30分钟，在黑暗中孵育。 </li><li>每孔加入100微升一站式解决方案（Quantikine，R＆D系统），轻轻混匀。 </li><li>集酶标仪（分子器件的光谱最大250，全球医疗器械公司，明尼苏达州）至450纳米，在540或570 nm波长校正，确定每个光密度。 </li></ol> 5。代表结果在图1的隔离程序的总体方案。人类胚胎干细胞和培养hiPSCs不同条件下的典型形态呈现在图2。形态<html> 图3给出MEFs和灭活馈线细胞用来准备厘米。在一般情况下，细胞应该合流隔离后24小时，并随时可以冻结或扩大。但是，有时可能需要2-3天才能获得融合文化。细胞在第4代，而不是后来的CM应准备。这是至关重要的，因为主细胞只能被扩大为4-5代衰老来临前。 细胞因子，激活素A，被认为是作为饲养层细胞分泌的支持14多能干细胞分化生长的最关键因素。厘米的激活素水平测量（ 图4）是一个非常方便的定量分析，以监测MEFs中的质量。 <img alt="图1" src="/files/ftp_upload/3854/3854fig1.jpg" /> 图1。MEFs细胞分离过程示意图。 内容】"&gt; <img alt="图2" src="/files/ftp_upload/3854/3854fig2.jpg" /> 图2。典型的未分化的胚胎干细胞培养（一）饲养层细胞（B）条件培养基（三）定义介质的存在形态。 hiPSCs中存在（D和E）饲养层细胞培养的典型形态。 <img alt="图3" src="/files/ftp_upload/3854/3854fig3.jpg" /> 图3典型形态的小鼠胚胎成纤维细胞（MEFs）（A）通过电镀/隔离两天后，（B）在灭活密度56.000细胞/厘米，饲养层。2 0（P0） <img alt="图4" src="/files/ftp_upload/3854/3854fig4.jpg" /> 酶联免疫吸附试验（ELISA）为基础的条件培养基（CM）公关一个激活素浓度的测量图4。epared与来自CF1的小鼠品系的小鼠胚胎成纤维细胞。公分为6天，然后汇总。厘米"1"和CM"2"是指不同的媒体和对无条件媒体的澳批次。由于功能的调节过程MEFs细胞，激活一个分泌到培养基激活素A是在澳大几乎无法察觉。

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没有利益冲突的声明。

这里的MEFs细胞分离过程，使建立一个标准化的培养条件下对人类胚胎干细胞和hiPSCs。此外，基于ELISA的系统，用于评估饲养层细胞的细胞因子生产的MEF派生的空调媒体质量的一个有用的指标。提供配套成纤维细胞的小鼠品系（CF1的）的日常维护是必要的，以避免批次到批次的培养基的变化。此外，建议隔离从几个小鼠胚胎细胞的同时获得一致的质量。当然新鲜分离MEFs细胞可以保持冻结在P0和P1。也灭活MEFs中可以保持在适当数额取决于细胞培养的要求等分冻结。一般应约250.000细胞接种在6孔板培养胚胎干细胞和iPS细胞的单井。建立和优化的方法可以经常使用，以尽量减少复合驱之间的变异租实验。

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td> DMEM培养液（高糖） </td><td> Gibco公司，Invitrogen公司</td><td> 41966-052 </td><td></td></tr><tr><td>胎牛血清</td><td> biochrom </td><td> S0115 </td><td></td></tr><tr><td> L-谷氨酰胺</td><td> Gibco公司，Invitrogen公司</td><td> 25030-024 </td><td></td></tr><tr><td>青霉素链霉素</td><td> Gibco公司，Invitrogen公司</td><td> 15140-122 </td><td></td></tr><tr><td>真空过滤系统</td><td>康宁</td><td> 431097 </td><td> 500毫升，0.22微米，首席助理秘书长</td></tr><tr><td>二甲基亚砜</td><td>西格玛</td><td> D2650 </td><td></td></tr><tr><td>淘汰赛DMEM培养液</td><td> Gibco公司，Invitrogen公司</td><td> 10829-018 </td><td></td></tr><tr><td>淘汰赛辑嗯更换</td><td> Gibco公司，Invitrogen公司</td><td> 10828-028 </td><td></td></tr><tr><td>非必需氨基酸</td><td> Gibco公司，Invitrogen公司</td><td> 11140-035 </td><td></td></tr><tr><td> β-巯基乙醇</td><td>西格玛</td><td> M7522 </td><td></td></tr><tr><td> PBS </td><td> Gibco公司，Invitrogen公司</td><td> 14190-169 </td><td></td></tr><tr><td> DNA酶I </td><td>西格玛</td><td> D4527 </td><td></td></tr><tr><td>丝裂霉素C </td><td>罗氏公司</td><td> 10107409001 </td><td></td></tr><tr><td>碱性成纤维细胞生长因子</td><td> PeproTech公司</td><td> 100-18B </td><td></td></tr><tr><td>生物素Anti-human/mouse/rat激活素A抗体</td><td> R＆D系统</td><td> BAM3381 </td><td></td></tr><tr><td>明胶</td><td>西格玛</td><td> G9391 </td><td></td></tr><tr><td>人/鼠/大鼠激活素一个单克隆抗体</td><td> R＆D系统</td><td> MAB3381 </td><td></td></tr><tr><td> 0.05％胰蛋白酶EDTA </td><td> Gibco公司，Invitrogen公司</td><td> 25300-054 </td><td></td></tr><tr><td>超薄光圈剪刀的</td><td>福斯特</td><td> 14088-10 </td><td></td></tr><tr><td>超等格雷夫钳</td><td>福斯特</td><td> 11150-10 </td><td></td></tr><tr><td> pierse固定钳</td><td>福斯特</td><td> 18155-13 </td><td></td></tr></tbody></table>

preparation of mouse embryonic fibroblast cells suitable for culturing human embryonic and induced pluripotent stem cells

在一般情况下，人类胚胎干细胞（胚胎干细胞）和人类诱导多能干细胞（hiPSCs 1）可变量的条件下培养。然而，它是不容易的建立一个有效的制度，为培养这些细胞。由于文化条件可以影响基因表达，在胚胎干细胞和hiPSCs赋予全能性，培养方法的优化和标准化是至关重要的。 人类胚胎干细胞系的建立首次使用MEFs细胞作为饲养层细胞和胎牛血清（FBS），含培养基2。接下来，胎牛血清被替换Knockout血清替代品（KSR）和FGF2的，从而提高3胚胎干细胞的增殖。最后，馈线自由文化系统培养细胞使基底膜涂层板KSR-含条件培养基（MEFs细胞条件培养基）4。随后，胚胎干细胞培养条件化学defin馈线自由文化走向编辑条件5-7。此外，异种组件由病原体和动物蛋白培养方法，以避免潜在的污染已建立了8个 。 获得改善条件与人类细胞系（如胎儿肌肉和皮肤细胞9日，10成人皮肤细胞，包皮成纤维11-12，13羊膜间质细胞），小鼠饲养层细胞已被替换。然而，使用人包皮成纤维细胞源性饲养层保持未分化的胚胎干细胞的效率是不是只要从小鼠饲养细胞分泌激活素A 14的较低水平，由于高。显然，是在老鼠和人类饲养层细胞生长因子生产的明显差异。 老鼠和人类饲养层细胞的转录组的分析结果显示支持和不支持细胞之间的显着性差异。外源性FGF2的关键是maintai宁胚胎干细胞和hiPSCs自我更新，并已被确定为一个关键调节因子TGFβ1，激活素A和小鬼（骨形态发生蛋白受体拮抗剂）在饲养层细胞的表达。激活素A诱导的OCT4，SOX2和NANOG的在15-16人胚胎干细胞的表达已被证明。 长期培养，胚胎干细胞和hiPSCs可以生长分裂灭活MEFs细胞或基底膜涂层板在MEF-CM（MEF的条件培养基）馈线的条件下，以维持其未分化状态。两种培养条件下的成功完全取决于饲养层细胞的质量，因为它们直接影响了人类胚胎干细胞的生长。 在这里，我们提出了一个优化的分离和培养小鼠胚胎成纤维细胞（MEFs）的方法，制备条件培养基（CM）和酶联免疫吸附试验（ELISA）评估各级激活素在媒体。

Watch this Scientific Journal Video about Preparation of Mouse Embryonic Fibroblast Cells Suitable for Culturing Human Embryonic and Induced Pluripotent Stem Cells at JoVE.com

Preparation of Mouse Embryonic Fibroblast Cells Suitable for Culturing Human Embryonic and Induced Pluripotent Stem Cells

小鼠胚胎成纤维细胞（MEFs）的质量取决于鼠标CF-1如正确的应变。全能性支持MEFs和条件培养基（厘米），从这些应该包含激活素A，小鬼和TGFβ1的最佳浓度为激活素/ Nodal和FGF通路所需的合作保持自我更新和全能性。

适用于培养人类胚胎和诱导多能干细胞的小鼠胚胎成纤维细胞的制备

In general, human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs)1 can be cultured under variable conditions. However, it ...

preparation-mouse-embryonic-fibroblast-cells-suitable-for-culturing

Research

JoVE Journal

Biology

90.6K 次观看.  Max Planck Institute for Molecular Genetics. 小鼠胚胎成纤维细胞（MEFs）的质量取决于鼠标CF-1如正确的应变。全能性支持MEFs和条件培养基（厘米），从这些应该包含激活素A，小鬼和TGFβ1的最佳浓度为激活素/ Nodal和FGF通路所需的合作保持自我更新和全能性。

视频: Preparation of Mouse Embryonic Fibroblast Cells Suitable for Culturing Human Embryonic and Induced Pluripotent Stem Cells

Propagation of Human Embryonic Stem (ES) Cells

Propagation of Human Embryonic Stem ES Cells

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

人类胚胎干细胞（ES细胞）的繁殖

科研

生物学

Derivation of Human Embryonic Stem Cells by Immunosurgery

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


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

人类胚胎干细胞的推导Immunosurgery

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

Chromatin Immunoprecipitation from Human Embryonic Stem Cells

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

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

从人类胚胎干细胞的染色质免疫沉淀

The functional and structural complexity of the myriad of cells in metazoan organisms arises from a small number of stem cells. Stem cells are ...

Culture and Maintenance of Human Embryonic Stem Cells  

Human embryonic stem (hES) cells must be monitored and cared for in order to maintain healthy, undifferentiated cultures. At minimum, the cultures must be  fed  every day by performing a complete medium change to replenish lost nutrients and to keep the cultures free of unwanted differentiation factors. Although a small amount of differentiation is normal and expected in stem cell cultures, the culture should be routinely cleaned up by manually removing, or "picking" differentiated areas. Identifying and removing excess differentiation from hES cell cultures are essential techniques in the maintenance of a healthy population of cells.

Culture and Maintenance of Human Embryonic Stem Cells

This protocol demonstrates how to maintain healthy, undifferentiated human embryonic stem (ES) cells.

文化和维护人类胚胎干细胞

Human embryonic stem (hES) cells must be monitored and cared for in order to maintain healthy, undifferentiated cultures. At minimum, the cultures must be ...

Chromosomal Spread Preparation of Human Embryonic Stem Cells for Karyotyping

Although human embryonic stem cells (hESC) have been shown to present a stable diploid karyotype 1, many studies have reported that depending on culture conditions they become prone to acquire chromosomal anomalies such as addition of whole or parts of chromosomes. Indeed, during long-term culture, karyotypic alterations are observed when enzymatic or chemical dissociation are used 2,3,4, while manual dissection of colonies for passaging retains a stable karyotype 5. Besides, changes in the environment such as the removal of feeder cells also seem to compromise the genetic integrity of hESC 3,6. Once chromosomal alterations could affect cellular physiology, the characterization of the genetic integrity of hESC in vitro is crucial considering hESC as an essential tool in embryogenesis studies and drug testing. Furthermore, for future therapeutic purposes chromosomal changes are a real concern as it is frequently associated to carcinogenesis.
Here we show a simple and useful method to obtain high quality chromosome spreads for subsequent analysis of chromosome set by G-banding, FISH, SKY or CGH techniques 7,8. We recommend checking the chromosomal status routinely with intervals of 5 passages in order to monitor the appearance of translocations and aneuploidies
Priscila Britto and Rafaela Sartore contributed equally to the paper.

Karyotyping is a simple and useful technique widely used for detecting genetic alterations. Here we describe a step by step protocol for chromosome spread preparation of human embryonic stem cells for monitoring the chromosomal status of these cells maintained in culture.

为核型分析人类胚胎干细胞的染色体蔓延制备

Although human embryonic stem cells (hESC) have been shown to present a stable diploid karyotype 1, many studies have reported that depending on culture ...

Freezing and Thawing Human Embryonic Stem Cells

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells from a frozen stock have become important procedures performed in routine hES cell culture. Since hES cells are very sensitive to the stresses of freezing and thawing, special care must taken. Here we demonstrate the proper technique for rapidly thawing hES cells from liquid nitrogen stocks, plating them on mouse embryonic feeder cells, and slowly freezing them for long-term storage.

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells from a frozen stock have become important procedures performed in routine hES cell culture. Since hES cells are very sensitive to the stresses of freezing and thawing, special care must taken. Here we demonstrate the proper technique for rapidly thawing hES cells from liquid nitrogen stocks, plating them on mouse embryonic feeder cells, and slowly freezing them for long-term storage.

人类胚胎干细胞冷冻和解冻

Since James Thomson et al developed a technique in 1998 to isolate and grow hES in culture, freezing cells for later use and thawing and expanding cells ...

Isolation and Derivation of Mouse Embryonic Germinal Cells

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum (dpc) mouse embryos.  Developing gonadal ridges from mouse embryos (C57BL6J) were dissociated by mechanical disruption with collagenase, then plated in a mouse embryo fibroblast feeder layer (MEF-CF1) that was previously mitotically inactivated with mitomycin C in the presence of knockout media and supplemented with Leukemia Inhibitor Factor (LIF), basic Fibroblast Growth Factor (bFGF), and Stem Cell Factor (SCF).  Using these optimized methods for PCG identification, isolation, and establishment of culture conditions permits long term cultures of EG cells for more than 40 days.  The embryonic germinal cell lines showed embryonic phenotype and expression of common used markers of the pluripotent state.  Isolation and derivation of germinal cells in culture provide a tool to understand their development in vitro and offer the opportunity to monitor cumulative damage at genetic and epigenetic levels after exposure to oxidative stress.

The ability of embryonic germinal cells to differentiate into primordial germinal cells during early development stages is a perfect model to address our hypothesis about cancer and infertility. This protocol shows how to isolate primordial germinal cells from developing gonads in 10.5-11.5 days post coitum mouse embryos.

小鼠胚胎生殖细胞的分离和推导

The ability of embryonic germinal cells (EG) to differentiate into primordial germinal cells (PGCs) and later into gametes during early developmental ...

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 ...

Fate Mapping of Human Embryonic Stem Cells by Teratoma Formation 

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

Fate Mapping of Human Embryonic Stem Cells by Teratoma Formation

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

人类胚胎干细胞畸胎瘤形成命运的映射

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

Transfecting and Nucleofecting Human Induced Pluripotent Stem Cells

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

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

人类诱导多能干细胞的转染和Nucleofecting

Genetic modification is continuing to be an essential tool in studying stem cell biology and in setting forth potential clinical applications of human ...