在本协议中所述的工作成为可能，通过与美国加州再生医学研究所（CIRM）的协作联盟的一部分，由维多利亚州政府资助。

所有过程都在无菌的层流罩中进行。所有介质和溶液平衡至37℃，除非另有规定。 1，扩展人类的产品分成合同（人类胚胎干细胞和iPS细胞，WA-09本议定书中所使用）的转注意：这是没有必要扩大对基质胶或Geltrex人类胚胎干细胞。人类胚胎干细胞展开对小鼠胚胎成纤维细胞（MEF中），可用于电或核转遵循MEF去除步骤（见第2.8节）。 <ol><li>制备1×100毫米培养皿和DR4的MEF中的2.0×10 4个细胞每cm 2在MEF媒体1×75平方厘米烧瓶中（ 见表1）上的菜肴预先涂有0.1％w / v的明胶。 注意：CF1，可以使用BLK6或MF1的MEF，但是，它建议抗生素有抗药性的MEF线被使用， 例如，DR410。 </li><li>第二天，通过胚胎干细胞到100毫米的菜预镀在1.1节准备的MEF。要做到这一点ASPI从培养的菜肴速率的hESC介质，洗的hESC与Ca2 + / Mg2 +的-free Hanks&#39;平衡盐溶液（HBSS）中，于37℃/ 5％CO 2在潮湿的培养箱中添加分散酶（ 见表2为适当体积）和地点。继3-5分钟孵化，吸分散酶，轻轻地洗1 - 2次的HBSS删除的MEF。添加人类胚胎干细胞介质（ 见表3），用细胞刮刀，或5毫升吸管轻轻刮落掉的培养皿的表面。 </li><li>收集悬浮在培养皿中的菌落片段入15ml离心管中。冲洗培养皿用人类胚胎干细胞的媒体和转移到15ml试管中。小心不要打破团块成单个细胞或小片段。 </li><li>离心5分钟，在160 XG以沉淀细胞集落的碎片。 </li><li>吸出上清液。挖掘15ml试管底部用手指拧松落碎片颗粒。根据分流比在人类胚胎干细胞介质轻轻悬浮细胞集落的碎片（normall4和1：8为宜）雅间1分流比。要格外小心，不要使团块成单个细胞或小片段，因为这会影响replating效率。 </li><li>此前replating人类胚胎干细胞碎片上的MEF用HBSS洗1X。慢慢地将适量含于人类胚胎干细胞介质的hESC片段的细胞悬浮液（ 见表2）添加到菜预镀的MEF。 </li><li>将培养皿架子上的孵化器，慢慢地前后移动和左右摇晃均匀分布的殖民地片段。 </li><li>每天更换人类胚胎干细胞介质。 </li><li>为了准备由MEF（MEF-CM）条件培养基洗75 平方厘米瓶预镀的MEF 1个用HBSS。人类胚胎干细胞中添加媒体。以下24个小时，收集条件培养基，换上新鲜的人类胚胎干细胞介质。每天最多12天重复。存储的MEF-CM在4℃下直到需要。 </li></ol> 2，准备人类胚胎干细胞的转染<ol><li>观察人类胚胎干细胞的生长每天在显微镜下。当菌落变成40-50％汇合时，板3×100毫米培养皿中，每平方厘米2.0×10 5的MEF。 </li><li>当菌落变成60 - 70％汇合，他们准备被转染。 &#39;新郎&#39;殖民地去除分化的殖民地，殖民地的核心。 注意：由于我们相信更高的转染和当细胞处于对数期生长靶向效率发生，故建议电穿孔进行之前的hESC达到≥70％汇合。 </li><li>用HBSS洗涤人类胚胎干细胞克隆，并添加的Accutase。 注意：这是没有必要预先治疗人类胚胎干细胞与ROCK抑制剂Y-27632时，在步骤2.7以Y-27632孵育会发生如足以抑制</li><li>孵育10 - 20分钟，在37℃/ 5％CO 2，直至菌落呈现单个细胞在显微镜下的观察。磨碎细胞以1毫升吸管。 </li><li>添加人类胚胎干细胞介质和过滤到15毫升锥形管使用40微米的细胞straineR以除去细胞团块。 </li><li>离心机在160×g离心5分钟。重悬细胞沉淀在新鲜胚胎干细胞介质，并重复以除去任何残留的Accutase溶液。 </li><li>重悬的细胞在胚胎干细胞介质含有10μM的Y-27632，总细胞悬浮液转移到100mm培养皿预先涂有明胶。 </li><li>在37°C / 5％CO 2的至少30分钟。 </li><li>在此期间的MEF将坚持以明胶涂层菜。收集使用1毫升吸管非粘附的hESC成新鲜15ml离心管中，并确定使用血球细胞密度。 </li></ol> 3，电穿孔胚胎干细胞<ol><li>使用0.22微米过滤器，并加入10纳克/ ml重组人成纤维细胞生长因子2（rhFGF2）过滤器CM-MEF媒体。 </li><li>传输距离2.9节7.5×106个胚胎干细胞到一个新的15毫升锥形管和离心160×g离心5分钟。 </li><li>在此期间，成立了电穿孔：选择程序的指数ELEctroporation并设置以下参数，电压：250伏，容量：500μF，和阻力：∞-。 </li><li>吸出上清液，并于0.8ml冰冷却的0.22微米过滤的HBSS中重悬细胞沉淀。将溶液转移到无菌的0.4cm电比色皿。 </li><li>添加电视hPITX3进靶向构建（30微克DNA），并用200微升移液器轻轻混匀。在冰上孵育5分钟。在这段时间删除PITX3 ZFN表达的一个分装解冻冰。当电比色皿解冻转移7.5微升ZFN基因与DNA /细胞混合物。用200微升移液器轻轻混匀。 注意：作为目标的轨迹是有助于靶向效率的主要因素。基因座编码沉默基因，不表达于胚胎干细胞的基因，如PITX3是难以用常规的同源重组6进行定位。因此，该协议利用定制的ZFN为人类PITX3基因诱导的DNA双链break。 </li><li>通过电穿孔以下的电穿孔仪屏幕上的说明进行操作。 </li><li>用200微升吸管从电比色皿的内容传送到一个​​15毫升的试管含有10ml人类胚胎干细胞介质。与每个时间，并转移到15ml试管中加入200μl人类胚胎干细胞介质的两次冲洗比色皿。会出现“粘液状”碎片组成的DNA和蛋白从裂解的细胞将细胞存活产生不利影响的。离心机在160×g离心5分钟以除去这些碎屑。 </li><li>吸出上清液，并轻轻拍打细胞沉淀松动。重悬在30毫升补充的MEF-CM含有10微米Y-27632和replate上预镀用的MEF 3×100毫米的菜肴。 </li><li>每天更换介质。在第2天，Y-27632可抽出并生长的细胞中只有人类胚胎干细胞的介质。 </li><li>后2-3天的人类胚胎干细胞应该是50-70％汇合。在这一点上开始抗生素选择（ 例如，0.5微克/毫升嘌呤霉素）。 </li><li>保持SEL挠度通过每天增加新鲜胚胎干细胞/嘌呤霉素媒体。后〜7天小菌落应该是可见的。如果不使用抗生素有抗药性的MEF，MEF中通过每平方厘米增加一个额外的1.0×10 4个细胞的补充。 </li></ol> 4，采摘转基因人类胚胎干细胞克隆<ol><li>大约14 - 22天内选择（当殖民地已经达到约1毫米直径）准备板的扩展和筛选。通过解冻基底膜或Geltrex的等分在4℃下至少5小时的开始。稀释按制造商的指示，并添加75微升至3.0×96孔板各孔中。通过在2.0×10 4个细胞每cm 2电镀的MEF制备3×48孔板中。孵育两个板在o / n 37℃/ 5％的CO 2。 </li><li>用在2毫升吸管的端部的21G针头切网格次日解剖菌落成16-25件。 </li><li>吸出基质胶从96孔板中，并替换为supplemented CM-MEF媒体。洗的MEF在48孔板1个用HBSS，并添加人类胚胎干细胞的介质。 </li><li>用200微升吸管轻轻转移解剖殖民地的三分之一到48孔板进行扩展。传送解剖菌落的其他三分之二到含有补充CM-MEF媒体对应的96孔板中。 </li><li>通过每日更换介质保持两板（补充CM-MEF培养基的96孔基质胶包被的平板和人类胚胎干细胞培养基包含的MEF 48孔板;都补充有0.5微克/毫升嘌呤霉素）。 </li><li>扩大直到96孔板100％汇合，然后开始通过PCR筛选。 </li></ol> 5，筛选和扩展阳性克隆<ol><li>要准备的基因组DNA（基于11法）由反相删除板所有媒体，然后涂抹到纸巾。 </li><li>冲洗各孔两次，用RT PBS（100μl/孔，每次洗涤），转化和印迹，作为步骤5.1。地方板在-80°C 2 F或1 - 2小时，细胞裂解援助（板可以无限期地保存这样的）。 </li><li>而细胞在-80℃下使烷基肌氨酸钠裂解缓冲液（ 见表4）（50μl/孔）。裂解细胞，暖板5分钟，在RT再吸移管的裂解缓冲液到每个孔中。封含有湿润纸巾湿度创建一个可密封的容器中的每个盘磁带，地方的边缘，并培育在o / n 60℃。 </li><li>孵育后加入150μl5M氯化钠10毫升-20℃的无水乙醇，混合均匀，并加入100微升，每孔（注：酒精会去阴天时NaCl的加入）。轻轻拍打板混合（不要吸管），并留下了至少30分钟，在室温。在此期间，溶液会清楚的DNA会沉淀。 </li><li>除去溶液如在步骤5.1，然后加入150微升70％乙醇中的每个孔中。轻轻翻转板和以前一样吸干。重复此步骤，进一步的2倍。 注：由于沉淀的DNA自然阿德水库于组织培养塑料它不会成为在这些洗涤步骤被撞出。） </li><li>最后一次洗涤后，短暂离心平板以最高速度来浓缩DNA到各孔然后空气干燥的在室温下（即37℃）下至少30分钟。 </li><li>添加40 - 50微升T0.1E液（pH8.0）（ 见表5）或TE（pH为8.0），向每个孔中并置于平板在65℃下进行1小时或在4℃CO / N使用用于PCR之前。通过轻轻敲击板的侧面重悬DNA。使用1-2微升的再悬浮的DNA / 10微升的PCR反应。 </li><li>使用Expand长模板PCR系统与hPITX3Ł臂根。 F和hPITX3Ļ臂GFPŕ引物进行克隆的初步筛选，在58℃退火，45秒延伸，35个循环。 </li><li>与Hyperladder 1KB在1％琼脂糖凝胶Electrophorese样本含有Gel​​Red在100伏45分钟，并通过紫外线照射检测阳性克隆。 </li><li>验证PCR阳性克隆，通过消化，从EXPA基因组DNAnded克隆（见下）用HindIII和杂交探针的5&#39;和3&#39;的载体同源臂，以Southern印迹（如先前所述 ）。通过PCR使用引物对hPITX3 5&#39;探针F和R，并hPITX3 3&#39;探针F和R，用EcoRI消化，并连接到一个通用的克隆载体中产生的探针。 </li><li>根据阳性克隆的数目，通过预镀用的MEF（2.0×10 4个细胞每cm 2）的制备2孔6孔板。 </li><li>第二天预处理阳性克隆在48孔板为10μm的Y-27632最少1小时的。 </li><li>用HBSS洗阳性克隆，并加入300μl的Accutase的。 </li><li>当菌落呈现单细胞，加入1 ml人类胚胎干细胞介质和收集到15毫升管。 </li><li>每天更换介质。在第2天，Y-27632可抽出并生长的细胞在人类胚胎干细胞的介质。 </li><li>当殖民地的70 - 80％融合，传代培养和冷冻的结肠其余纽约片段。 </li></ol>

<ol>
	<li>Nefzger, C. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lmx1a+Allows+Context-Specific+Isolation+of+Progenitors+of+GABAergic+or+Dopaminergic+Neurons+During+Neural+Differentiation+of+Embryonic+Stem+Cells.">Lmx1a Allows Context-Specific Isolation of Progenitors of GABAergic or Dopaminergic Neurons During Neural Differentiation of Embryonic Stem Cells.</a> Stem Cells. 30 (7), 1349-1361 (2012).</li><li>Zeng, W. R., Fabb, S. R. A., Haynes, J. M., Pouton, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extended+periods+of+neural+induction+and+propagation+of+embryonic+stem+cell-derived+neural+progenitors+with+EGF+and+FGF2+enhances+Lmx1a+expression+and+neurogenic+potential.">Extended periods of neural induction and propagation of embryonic stem cell-derived neural progenitors with EGF and FGF2 enhances Lmx1a expression and neurogenic potential.</a> Neurochemistry International. 59 (3), 394-403 (2011).</li><li>Collin, J., Lako, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Concise+review:+putting+a+finger+on+stem+cell+biology:+zinc+finger+nuclease-driven+targeted+genetic+editing+in+human+embryonic+stem+cells.">Concise review: putting a finger on stem cell biology: zinc finger nuclease-driven targeted genetic editing in human embryonic stem cells.</a> Stem Cells. 29 (7), 1021-1033 (2011).</li><li>Carroll, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progress+and+prospects:+zinc-finger+nucleases+as+gene+therapy+agents.">Progress and prospects: zinc-finger nucleases as gene therapy agents.</a> Gene Therapy. 15 (22), 1463-1468 (2008).</li><li>Urnov, F. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+efficient+endogenous+human+gene+correction+using+designed+zinc-finger+nucleases.">Highly efficient endogenous human gene correction using designed zinc-finger nucleases.</a> Nature. 435 (7042), 646-651 (2005).</li><li>Hockemeyer, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+targeting+of+expressed+and+silent+genes+in+human+ESCs+and+iESCs+using+zinc-finger+nucleases.">Efficient targeting of expressed and silent genes in human ESCs and iESCs using zinc-finger nucleases.</a> Nature Biotechnology. 27 (9), 851-857 (2009).</li><li>Lombardo, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+editing+in+human+stem+cells+using+zinc+finger+nucleases+and+integrase-defective+lentiviral+vector+delivery.">Gene editing in human stem cells using zinc finger nucleases and integrase-defective lentiviral vector delivery.</a> Nature Biotechnology. 25 (11), (2007).</li><li>Boch, J., Bonas, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Xanthomonas+AvrBs3+family-type+III+effectors:+discovery+and+function.">Xanthomonas AvrBs3 family-type III effectors: discovery and function.</a> Annual Review of Phytopathology. 48, 419-436 (2010).</li><li>Hockemeyer, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+engineering+of+human+embryonic+cells+using+TALE+nucleases.">Genetic engineering of human embryonic cells using TALE nucleases.</a> Nature Biotechnology. 29 (8), 731-734 (2011).</li><li>Tucker, K. L., Wang, Y., Dausman, J., Jaenisch, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+transgenic+mouse+strain+expressing+four+drug-selectable+marker+genes.">A transgenic mouse strain expressing four drug-selectable marker genes.</a> Nucleic Acids Research. 25 (18), 3745-3746 (1997).</li><li>Ram&#237;rez-Solis, R., Rivera-P&#233;rez, J., Wallace, J. D., Wims, M., Zheng, H., Bradley, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genomic+DNA+microextraction:+a+method+to+screen+numerous+samples.">Genomic DNA microextraction: a method to screen numerous samples.</a> Analytical Biochemistry. 201 (2), 331-335 (1992).</li><li>Kawasaki, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+midbrain+dopaminergic+neurons+from+ES+cells+by+stromal+cell-derived+inducing+activity.">Induction of midbrain dopaminergic neurons from ES cells by stromal cell-derived inducing activity.</a> Neuron. 28 (1), 31-40 (2000).</li><li>Denham, M., Thompson, L. H., Leung, J., P&#233;bay, A., Bj&#246;rklund, A., Dottori, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gli1+is+an+inducing+factor+in+generating+floor+plate+progenitor+cells+from+human+embryonic+stem+cells.">Gli1 is an inducing factor in generating floor plate progenitor cells from human embryonic stem cells.</a> Stem Cells. 28 (10), 1805-1815 (2010).</li><li>Smidt, M. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+homeodomain+gene+Ptx3+has+highly+restricted+brain+expression+in+mesencephalic+dopaminergic+neurons.">A homeodomain gene Ptx3 has highly restricted brain expression in mesencephalic dopaminergic neurons.</a> Proceedings of the National Academy of Sciences U.S.A. 94, 13305-13310 (1997).</li></ol>

作者什么都没有透露。

对记者的细胞系的产生提供了强有力的手段来跟踪，可视化和从人类胚胎干细胞来源的异质群体隔离感兴趣的细胞。然而，基因通过常规的同源重组靶向已被证明是非常低效的人类胚胎干细胞3。在这个协议中，我们描述了一个相对简单的技术采用广泛使用的目标向量的ZFN一起引入到RP的外显子的PITX3基因之一，在人类胚胎干细胞。我们手中的基因靶向系统是高效的，用含有正确的目...

当前的胚胎干细胞（ESC）分化的协议导致异质细胞群，特别是相对于特定的神经元表型的推导。因此，虽然文化包含所需的细胞表型，其他的神经元，非神经和未分化的细胞类型是经常存在1。这些特点限制了ESC键的应用程序源细胞来源的细胞替代疗法的使用和体外疾病模型。 遗传报告基因的细胞系提供了可视化，跟踪和隔离所关注的细胞的装置，只要该报告蛋白（RP）中的表达再现的内源性表达。使用的启动子紧接基因编码区上游的可用于推导粗遗传记者但这样的构建缺乏控制内源基因表达的精确调控元件。与此相反，同源重组提供了机会，确保安置计划的高保真表达。在过去，针对设计用于同源重组在感兴趣的特定基因的载体已被用于使用电穿孔作为DNA递送1,2的手段对目标在小鼠胚胎干细胞（mESCs分化）的RP。但是通过传统的同源重组报告细胞系的生成是非常低效的人类胚胎干细胞（胚胎干细胞），因此只被记录在极少数情况下（在3审查）。通过使用含有融合的霍 1核酸酶与特定地点的锌指结构，统称为锌指核酸酶（ZFN）的工程嵌合蛋白，DNA双链断裂可以在预先确定的基因组位点引入。当用同源性的DNA载体的DNA双链断裂的两侧添加，基因组位点可通过同源重组得到修复，从而使该DNA供体序列的掺入。该技术已被证明非常有用˚F或在两个人原代细胞4,5和6,7人类胚胎干细胞的基因组编辑。更近期的工作已经利用转录激活因子样效应物核酸酶（TALENS），所使用的植物转录因子的病原体如图8所示 ，位点特异性核酸酶9来帮助设计。 在下面的协议我们展示了人类胚胎干细胞报道细胞系的产生，含有同源靶向载体6用的ZFN对人垂体同源3（PITX3）轨迹在一起的EGFP的电穿孔。下列抗生素选择为2-3周，胚胎干细胞具有正确整合的DNA可以手动拾取，膨胀并通过PCR筛选最初，并随后通过Southern杂交验证。

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			<td style="width:207px;">GeneWorks, Australia</td>
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</table>

zinc-finger nuclease enhanced gene targeting in human embryonic stem cells

与当前的人类胚胎干细胞（ESC）的分化方案的一个主要限制是异质的细胞群的产生。这些培养物含有感兴趣的细胞，而且还污染了未分化的胚胎干细胞，非神经衍生物和其他神经元亚型。这限制了它们的使用在体外和体内应用中，如在体外模型用于药物开发的或细胞替代疗法。为了帮助解决这个问题，记者的细胞系，它提供可视化，跟踪和隔离感兴趣的细胞的方法，可以设计。然而，要实现这个在人胚胎干细胞通过常规的同源重组是极其低效的。这个协议描述了一种具有靶向垂体同源使用定制设计的锌指核酸酶，其引入位点特异性双链DNA断裂3（PITX3）位点在人胚胎干细胞，一起PITX3 - 绿色荧光蛋白特异性的DNA供体载体。以下的PITX3报道细胞系的产生，其然后可以使用发表的方案为在研究中使用的分化，例如在体外帕金森氏病模型或细胞替代疗法。

以下共电穿孔的定制设计PITX3锌指一对连同PITX3 - EGFP特异性DNA供体载体中，并随后嘌呤选择，阳性胚胎干细胞克隆，通过基因组PCR筛选（ 图1），首先检测到的。这些PCR阳性克隆与5&#39;和3&#39;特异性探针的Southern印迹杂交确认正确的定位外显子1的PITX3轨迹（ 图2），具有19％的效率。在图3所示的免疫荧光图像表明EGFP表达由PITX3子使用?...

Watch this Scientific Journal Video about Zinc-finger Nuclease Enhanced Gene Targeting in Human Embryonic Stem Cells at JoVE.com

Zinc-finger Nuclease Enhanced Gene Targeting in Human Embryonic Stem Cells

记者的细胞系提供了可视化，跟踪和隔离，从不同的人群感兴趣的细胞的方法。然而，基因靶向用常规的同源重组在人胚胎干细胞是非常低效的。在这里，我们将介绍使用锌指核酸酶增强同源重组靶向中枢神经系统中脑特异性转录因子PITX3基因与绿色荧光蛋白。

锌指核酸酶增强基因打靶的人胚胎干细胞

One major limitation with current human embryonic stem cell (ESC) differentiation protocols is the generation of heterogeneous cell ...

zinc-finger-nuclease-enhanced-gene-targeting-human-embryonic-stem

Research

JoVE Journal

Biology

10.7K 次观看.  Monash University. 记者的细胞系提供了可视化，跟踪和隔离，从不同的人群感兴趣的细胞的方法。然而，基因靶向用常规的同源重组在人胚胎干细胞是非常低效的。在这里，我们将介绍使用锌指核酸酶增强同源重组靶向中枢神经系统中脑特异性转录因子PITX3基因与绿色荧光蛋白。 

视频: Zinc-finger Nuclease Enhanced Gene Targeting in Human Embryonic Stem Cells

Propagation of Human Embryonic Stem (ES) Cells

Propagation of Human Embryonic Stem ES Cells

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 ...

Probing for Mitochondrial Complex Activity in Human Embryonic Stem Cells

Mitochondria are cytoplasmic organelles that have a primary role in cellular metabolism and homeostasis, regulation of the cell signaling network, and programmed cell death. Mitochondria produce ATP, regulate the cytoplasmic redox state and Ca2+ balance, catabolize fatty acids, synthesize heme, nucleotides, steroid hormones, amino acids, and help assemble iron-sulfur clusters in proteins. Mitochondria also have an essential role in heat production. Mutations of the mitochondrial genome cause several types of human disorder. The accumulation of mtDNA mutations correlates with aging and is suspected to have an important role in the development of cancer. Due to their vitally important role in all cell types, the function of mitochondria must also be critical for stem cells. Key advances have been made in our understanding of stem cell viability, proliferation, and differentiation capacity. But the functional activity of stem cells, in particular their energy status, was not yet been studied in detail. Almost nothing is known about the mitochondrial properties of human embryonic stem cells (hESCs) and their differentiated precursor progeny. One way to understand and evaluate the role of mitochondria in hESC function and developmental potential is to directly measure the activity of mitochondrial respiratory complexes. Here, we describe high resolution clear native gel electrophoresis and subsequent in gel activity visualization as a method for analyzing the five respiratory chain complexes of hESCs.

In this video, we will show you how the mitochondrial respiratory chain complexes of human embryonic stem cells can be analyzed using in gel activity assays.

在人类胚胎干细胞的线粒体复杂的活动初探

Mitochondria are cytoplasmic organelles that have a primary role in cellular metabolism and homeostasis, regulation of the cell signaling network, and ...

In vitro and in vivo Bioluminescence Reporter Gene Imaging of Human Embryonic Stem Cells

The discovery of human embryonic stem cells (hESCs) has dramatically increased the tools available to medical scientists interested in regenerative medicine. However, direct injection of hESCs, and cells differentiated from hESCs, into living organisms has thus far been hampered by significant cell death, teratoma formation, and host immune rejection. Understanding the in vivo hESC behavior after transplantation requires novel imaging techniques to longitudinally monitor hESC localization, proliferation, and viability. Molecular imaging has given investigators a high-throughput, inexpensive, and sensitive means for tracking in vivo cell proliferation over days, weeks, and even months. This advancement has significantly increased the understanding of the spatio-temporal kinetics of hESC engraftment, proliferation, and teratoma-formation in living subjects.

A major advance in molecular imaging has been the extension of noninvasive reporter gene assays from molecular and cellular biology into in vivo multi-modality imaging platforms. These reporter genes, under control of engineered promoters and enhancers that take advantage of the host cell s transcriptional machinery, are introduced into cells using a variety of vector and non-vector methods. Once in the cell, reporter genes can be transcribed either constitutively or only under specific biological or cellular conditions, depending on the type of promoter used. Transcription and translation of reporter genes into bioactive proteins is then detected with sensitive, noninvasive instrumentation (e.g., CCD cameras) using signal-generating probes such as D-luciferin.

To avoid the need for excitatory light to track stem cells in vivo as is required for fluorescence imaging, bioluminescence reporter gene imaging systems require only an exogenously administered probe to induce light emission. Firefly luciferase, derived from the firefly Photinus pyralis, encodes an enzyme that catalyzes D-luciferin to the optically active metabolite, oxyluciferin. Optical activity can then be monitored with an external CCD camera. Stably transduced cells that carry the reporter construct within their chromosomal DNA will pass the reporter construct DNA to daughter cells, allowing for longitudinal monitoring of hESC survival and proliferation in vivo. Furthermore, because expression of the reporter gene product is required for signal generation, only viable parent and daughter cells will create bioluminescence signal; apoptotic or dead cells will not. 

In this video, the specific materials and methods needed for tracking stem cell proliferation and teratoma formation with bioluminescence imaging will be described.

With the growing interest in stem cell therapies, molecular imaging techniques are ideal for monitoring stem cell behavior after transplantation. Luciferase reporter genes have enabled non-invasive, repetitive assessment of cell survival, location, and proliferation in vivo. This video will demonstrate how to track hESC proliferation in a living mouse.

在体外和体内生物发光记者人类胚胎干细胞的基因显像

The discovery of human embryonic stem cells (hESCs) has dramatically increased the tools available to medical scientists interested in regenerative ...

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 ...

In vitro Labeling of Human Embryonic Stem Cells for Magnetic Resonance Imaging

Human embryonic stem cells (hESC) have demonstrated the ability to restore the injured myocardium. Magnetic resonance imaging (MRI) has emerged as one of the predominant imaging modalities to assess the restoration of the injured myocardium. Furthermore, ex-vivo labeling agents, such as iron-oxide nanoparticles, have been employed to track and localize the transplanted stem cells. However, this method does not monitor a fundamental cellular biology property regarding the viability of transplanted cells. It has been known that manganese chloride (MnCl2) enters the cells via voltage-gated calcium (Ca2+) channels when the cells are biologically active, and accumulates intracellularly to generate T1 shortening effect. Therefore, we suggest that manganese-guided MRI can be useful to monitor cell viability after the transplantation of hESC into the myocardium.
In this video, we will show how to label hESC with MnCl2 and how those cells can be clearly seen by using MRI in vitro. At the same time, biological activity of Ca2+-channels will be modulated utilizing both Ca2+-channel agonist and antagonist to evaluate concomitant signal changes.

In this video, we are showing how to label human embryonic stem cells (hESC) with manganese chloride (MnCl2) which can enter cells via voltage-gated calcium channels when the cells are biologically active. Additionally, we show the use of MnCl2 as cellular MRI contrast agent to determine the in vitro viability of hESC.

在人类胚胎干细胞体外培养的标签用于磁共振成像

Human embryonic stem cells (hESC) have demonstrated the ability to restore the injured myocardium. Magnetic resonance imaging (MRI) has emerged as one of ...

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 ...

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 ...

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.

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.