我们感谢帮助编辑的马里亚尼实验室的协议。这项工作是由一个摆围博士后培训津贴（JLF），的CIRM的BRIDGES实习（TZTT），罗伯特·E.五月R.莱特基金会（FVM），美国南加州大学（FVM）。

1。 LysoTracker染小鼠胚胎<ol><li>生成的小鼠胚胎放置年轻女性到男性螺柱笼（5-6周龄）。监视以下早晨指示，交配阴道栓的外观发生。如果女性怀孕了，中午在这一天被称为0.5 DPC（天交配后）。这里介绍的是该程序的胚胎7-13 DPC的理想选择。 </li><li>在胚胎的利益，根据经批准的协议和安乐死的女性，切除子宫。从一个10厘米的培养皿中，用Hanks，BSS（无酚红）的蜕膜中取出胚胎。删除多余的胚胎外膜，至少做汉克斯BSS冲洗，以去除多余的组织和血液中。 </li><li>准备LysoTracker胚胎染色解决方案（·汉克斯的5毫米LysoTracker在BSS）。 1-2胎是一个方便的音量5毫升。轻轻地混合均匀地发放染料。 </li><li>添加的LysoTracker胚胎染色Solut的的胚胎在微量离心管中的离子或小瓶中，在37℃下孵育45分钟。 </li><li>冲洗轻轻地汉克斯BSS 4次，每次洗5分钟。 </li><li>固定在4％多聚甲醛过夜。 </li><li>汉克斯BSS，10分钟一次冲洗，删除​​该修补程序。 </li><li>脱水通过甲醇系列（50％，75％，80％，100％，5分钟每个步骤），以消除背景染色。这是很重要的，在成像过程中，为实现良好的信号 - 噪声。 </li><li>在这一点上，你可以无限期储存的胚胎样品中甲醇在-20°C，避光。 </li></ol> 注意:可以采取组织样本在任何步骤，用于DNA分型，然而，它可以是最方便的，只是在储存之前采取的样品，使多达此步骤中所有的样品可以是分批处理。收集组织样本，并把所有的尾巴，四肢或头，从每个胚胎成单独的管。补充水分的组织样本，并准备genoty平。 2。 ES文化的差异LysoTracker染色<ol><li>准备通过悬滴法15的胚状体与起始密度为500个细胞每20μl下拉。 </li><li> 3天之后，悬浮培养的胚状体转移到10厘米非粘附培养皿（ 即那些用于细菌的工作）。改变介质每2天5天，培养。 </li><li>第7天，胚体转移到的糊化8以及玻片（加入0.1％明胶的腔室，等待5分钟，删除和添加媒体）。传输每室1-2胚体。 </li><li>文化10天，媒体每隔一天。成为移液管小心地放置在拐角处的腔室滑动，以便不扰动胚体附着到玻璃表面。此外，当加入新鲜的培养基中也放置在移液管的拐角处的腔室和慢慢释放介质。 </li><li>经过10天的邪教组织余吕杏茜作为阳性对照，诱导细胞凋亡，治疗一些孔与0.1 mm和1.0 mm H 2 O 2（H 2 O 2，以新媒体，混合，然后加入到细胞）为60分钟。冲洗两次，D-PBS在正规媒体和培养过夜。 </li><li>轻轻吸现有媒体和添加LysoTracker细胞染色溶液（500的纳米LysoTracker（最终浓度）在媒体，每个腔室的300微升）。 </li><li>在37℃下孵育15分钟。 </li><li>冲洗两次，用D-PBS 2次，每次5 min。 </li><li>固定在4％多聚甲醛在室温15分钟。 </li><li>冲洗两次，用D-PBS，5分钟。 </li></ol> 3。 ，查看LysoTracker被弄脏的胚胎或ES文化<ol><li>准备用于显微镜的细胞样品，通过抽吸的D-PBS;除去腔室关闭的幻灯片;空气干燥约5分钟，然后加入水溶液安装介质（Vectashield与DAPI染色或类似）。在而dryin将陪衬的障碍g至降低的荧光漂白。安装了深刻的抑郁症玻璃盘或小培养皿在Vectashield或类似的胚胎。 </li><li>可视化解剖或复合式显微镜罗丹明或德克萨斯州的红色过滤器（配备LysoTracker RED DND-99，激发/发射:577/590纳米）。染色通常是相当明亮，所以通常没有必要长时间曝光。 </li></ol> 4。使用成像技术定量分析结果<ol><li>在手动模式下相同的曝光条件下对样品的数码照片。 </li><li>打开Adobe Photoshop或类似的成像程序中的图像。要确定的染色的LysoTracker量，选择的红色通道和阈值，使得红染色现在是由白色像素表示的图像（的图像转换为黑色和白色的）。选择白色的像素，并使用直方图功能，得到的总数。 </li><li>使用蓝色通道数数量的核中的字段。它可以是有用的，以记录的计数标注的图像。 </li><li>记录的值，然后计算平均每个细胞的染色水平。重复此分析相同的阈值设置为你想品尝的所有图像。 </li></ol> 5。代表性的成果有一个正常的胚胎发育过程中的PCD模式时，可以改变一个生长因子基因被删除，如刺猬（嘘），16,17。由于LysoTracker是一个小分子染料是高度扩散性，全胚胎可以是可视化评估PCD甚至深范围内的间充质12,13领域。 图1显示了LysoTracker染色模式为正常和 Shh - / -的突变小鼠胚胎10.5dpc。在这个阶段中，PCD 嘘 - / -突变胚胎中大大增加，特别是在四肢和体节的区域选择性纳秒。成像部分的胚胎可以全部安装在更高的放大倍率（1B）评估的总体格局。解剖的部分也可以用振动切片机（1C）或低温恒温器（DF），更详细地揭示的PCD区域截面。 TUNEL分析最好是薄的部分，末端转移酶的渗透问题较少。 LysoTracker染色和TUNEL检测可以做到在同一组织（ 图1D-G）。这些结果表明，虽然目前还没有一个严格的1:1之间的相关性LysoTracker和TUNEL染色，一般地区的PCD重叠相当。在一个较高的倍率，是有可能看到泪点TUNEL检测与染色阳性，但不与在LysoTracker染料，泪点仅与LysoTracker染料，染色，和泪点有重叠染色。 当使用EB的明胶涂覆的表面上镀覆，分化细胞将migratË向外。经过10天的培养后，细胞会分化成多种类型的细胞，包括神经细胞，肌细胞，心肌。 图2中所示的培养包含混合种群的这些类型的细胞，但是人们可以适应我们的协议来研究PCD的一个特定的细胞类型的兴趣。例如，可以处理的EB与维甲酸增强分化的神经元19，然后的LysoTracker方法可用于在不同的条件下，以评估神经元细胞死亡。在分化的ES细胞的常规培养，也有少数的细胞经受PCD和这些LysoTracker和/或TUNEL法测定（ 图2A），可以检测到与。 PCD可以由许多不同的细胞毒性剂，如过氧化氢 ​​（H 2 O 2）诱导的氧化损伤，并启动凋亡通路20,21诱导。低剂量的H 2 O 2同时增加LysoTracker和TUNEL染色不进行治疗，而高剂量细胞的收缩及的广泛LysoTracker和TUNEL阳性结果。在胚胎的结果相似，它是可以看到泪点TUNEL检测与染色阳性，但不与在LysoTracker染料，泪点仅与LysoTracker染料，染色，和泪点有重叠染色。血清饥饿诱导自噬（ 图2D）的条件相比，诱导apopotosis细胞毒性剂的应用的后果。已经历了一段血清饥饿（1小时）的细胞表现出不同的模式，LysoTracker和TUNEL染色不高度相关。处理培养物相比，大大提高LysoTracker染色，但是TUNEL阳性是在正常水平。 采用的标准工具在Adobe Photoshop中，可以量化LysoTracker染色用一个简单的阈值方法，允许一个计算细胞群的平均染色水平。 图3A-B示出了如何在量化过程中的样本图像看起来。开始LysoTracker在蓝色通道（面板图3A）在红色通道和DAPI染色的核染色与一个基准图像，该图像的红色通道的阈值将所述图像转换为黑色和白色的白色像素然后选择和计数（ 图3A）。选择一个阈值水平"代表LysoTracker染色模式。任何量化的方法，使用图像，重要的是要避免过度曝光的照片，因为这有可能导致过分强调差异。然后，可以在该领域的原子核的数目计数看着只是蓝色通道（ 图3B）和标记的细胞核计数时（ 图3B）的使用的绘制工具如画笔。一个可以表达日通过计算每个细胞的平均水平的染色的染色（像素/细胞核计数数）的E级。如果一个人希望使用这种算法的高通量筛选，筛选包可以测量亮度在一个特定的通道，也有一个对象标识符功能，将计算核的一个领域，但最典型的应用手工的方法是快速和简单。举一个例子，一个样品区，从图2A-C进行定量的结果证实，增加浓度的H 2 O 2的结果，在一个较高的水平，LysoTracker染色（ 图3C）。 <img src="/files/ftp_upload/4254/4254fig1.jpg" alt="图1"/> 图1。在正常和Shh - / -胚胎LysoTracker染色。 A，A&#39;。正常和 Shh - / -小鼠胚胎10.5 DPC，与LysoTracker红染色，安装在Vectashield的，并拍摄下解剖显微镜的epiflourescence。括号表示的前肢的位置B，B&#39;，前肢正常和 Shh - / -胚胎在更高的放大倍率成像。请注意正常域的细胞死亡包括&#39;后路坏死区"（标有星号）和在近端的肢芽的间充质细胞（用箭头标记）的区域。 的Shh文献null四肢表现出更强的染色在内侧和远侧部C，C&#39;内侧的部分，通过前肢正常和空肢芽芽。相同的部分（12微米，冷冻注意近端间充质细胞（箭头）和AER（箭头）的正常肢芽和强染色在背，腹和远端间充质细胞内的空肢芽中存在的染色。DF。一节）被拍摄到的LysoTracker和TUNEL染色（罗氏1684795）。该节通过的嘘空mesenc的远侧部分hyme。箭头指向的AER。然而注意如何整体图案是非常类似的，使用这两种方法，染色不完全重合，G，G&#39;，人们可以看到，在较高的放大倍数的AER与间充质的存在，会泪点是LysoTracker或TUNEL阳性仅作为以及泪点有重叠染色。 <img src="/files/ftp_upload/4254/4254fig2.jpg" alt="图2"/> 图2。 LysoTracker染色分化的ES细胞中，分化细胞铺板在8室玻片在没有的LIF。为了显示，如何LysoTracker和TUNEL染色比较，2种不同剂量的H 2 O 2诱导的细胞凋亡途径，而使用1小时的血清饥饿诱导的自噬途径。这些图像A-A":未处理的文化与一个蔡司LSM5的共聚焦显微镜显示一些正常的细胞凋亡的LysoTracker和TUNEL染色。使用这两种技术大多是重叠（白色箭头）的图案，然而，人们可以看到几个泪点，只LysoTracker阳性（红色箭头） 的B-B"CC"的:治疗与增加量的H 2 O 2刺激细胞凋亡较低的的剂量显示LysoTracker和未经处理的细胞相比，的TUNEL积极泪点。高剂量诱导更LysoTracker和TUNEL阳性泪点。白色箭头指向到重叠的染色，而红色箭头指向泪小点，积极LysoTracker只。它也可以看到泪点只阳性TUNEL染色（绿色箭头）。DD":血清饥饿可诱导自噬作用，它可刺激材料在溶酶体和autophagolysosomes的一个积累。注意多少LysoTracker泪点是可见的，未经处理的文化。 TUNEL阳性泪小点（其中大部分有重叠LysoTracker的染色ING，白箭头），但是，数字是不是大大增加相比，未经处理的条件。此外，在血清饥饿条件下，LysoTracker只有积极的泪点是非常普遍的（红色箭头）。 <img src="/files/ftp_upload/4254/4254fig3.jpg" alt="图3"/> 图3。量化方法进行评估水平的人口使用Adobe Photoshop的细胞染色。 A:选择图2A的部分的LysoTracker（红色通道）和DAPI染色（细胞核，蓝色通道）在此面板中，A&#39;:图3A阈值，将其转换为黑色和红色通道白图像。白色像素被选择和计算使用直方图功能，B-B&#39;蓝色通道的细胞核和计数时，图像可以进行注释，如画笔绘图功能。C:这是一个前面板一个样品从图2A-C充足的量化。数据表示为每核的像素。 <a href="http://www.jove.com/files/ftp_upload/4254/4254fig3large.jpg" target="_blank">点击此处查看大图</a> 。

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B., Orrenius, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dual+regulation+of+caspase+activity+by+hydrogen+peroxide:+implications+for+apoptosis.">Dual regulation of caspase activity by hydrogen peroxide: implications for apoptosis.</a> FEBS Lett. 414, 552-556 (1997).</li><li>Wood, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+cells+engulf+and+clear+apoptotic+footplate+cells+in+macrophageless+PU.1+null+mouse+embryos.">Mesenchymal cells engulf and clear apoptotic footplate cells in macrophageless PU.1 null mouse embryos.</a> Development. 127, 5245-5252 (2000).</li><li>Scott, R. C., Juhasz, G., Neufeld, T. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+induction+of+autophagy+by+Atg1+inhibits+cell+growth+and+induces+apoptotic+cell+death.">Direct induction of autophagy by Atg1 inhibits cell growth and induces apoptotic cell death.</a> Curr. Biol. 17, 1-11 (2007).</li><li>Rodriguez-Enriquez, S., Kim, I., Currin, R. T., Lemasters, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tracker+dyes+to+probe+mitochondrial+autophagy+(mitophagy)+in+rat+hepatocytes.">Tracker dyes to probe mitochondrial autophagy (mitophagy) in rat hepatocytes.</a> Autophagy. 2, 39-46 (2006).</li><li>Bampton, E. T., Goemans, C. G., Niranjan, D., Mizushima, N., Tolkovsky, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+dynamics+of+autophagy+visualized+in+live+cells:+from+autophagosome+formation+to+fusion+with+endo/lysosomes.">The dynamics of autophagy visualized in live cells: from autophagosome formation to fusion with endo/lysosomes.</a> Autophagy. 1, 23-36 (2005).</li><li>Boya, P., Mellen, M. A., de la Rosa, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+autophagy+is+related+to+programmed+cell+death+during+the+development+of+the+nervous+system.">How autophagy is related to programmed cell death during the development of the nervous system.</a> Biochem. Soc. Trans. 36, 813-817 (2008).</li><li>Galluzzi, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Guidelines+for+the+use+and+interpretation+of+assays+for+monitoring+cell+death+in+higher+eukaryotes.">Guidelines for the use and interpretation of assays for monitoring cell death in higher eukaryotes.</a> Cell Death Differ. 16, 1093-1107 (2009).</li><li>Klionsky, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Guidelines+for+the+use+and+interpretation+of+assays+for+monitoring+autophagy+in+higher+eukaryotes.">Guidelines for the use and interpretation of assays for monitoring autophagy in higher eukaryotes.</a> Autophagy. 4, 151-175 (2008).</li></ol>

没有利益冲突的声明。

细胞凋亡的重要标志包括与TUNEL检测的DNA损伤的检测，沿与caspase活性的检测（检测的单克隆抗体或结合分子如ZVAD-芬兰马克），观察细胞的变化，和介绍的磷脂酰丝氨酸（PS ）在膜表面上（Annexin V结合）中检测到的。与每个这些检测方法也有一些缺点。例如，TUNEL法检测中使用的末端转移不穿透超越几个细胞层。膜联蛋白V可以标记细胞不进行细胞凋亡，如那些具有的膜破坏，（膜联蛋白V得到在细?...

<table border="1"><tbody><tr><td> 的试剂的名称 </td><td> 公司 </td><td></td><td> 目录编号 </td></tr><tr><td> DND-99 LysoTracker红， </td><td> Invitrogen公司</td><td> ＃L-7528 </td><td></td></tr><tr><td>汉克斯BSS </td><td> Invitrogen公司</td><td> 14025-076 </td><td></td></tr><tr><td>多聚甲醛</td><td> EMD </td><td> EM-PX0055-3 </td><td></td></tr><tr><td> Vectashield </td><td> VECTOR </td><td> H-1200 </td><td></td></tr><tr><td> DMEM </td><td> Cellgro </td><td> 10-013-CV </td><td></td></tr><tr><td>非必需氨基酸</td><td> Cellgro </td><td> 25-025-CI </td><td></td></tr><tr><td>丙酮酸钠</td><td> Cellgro </td><td> 25-000-CI </td><td></td></tr><tr><td> FBS </td><td> Hyclone公司</td><td> SH30071.02 </td><td></td></tr><tr><td>笔链球菌</td><td> Invitrogen公司</td><td> 15140-122 </td><td></td></tr><tr><td> β-巯基乙醇，50mM的</td><td> Invitrogen公司</td><td> 21985-023 </td><td></td></tr><tr><td> LabTek-II玻片 （8孔） </td><td> Nalge Nunc公司国际</td><td> 154534 </td><td></td></tr><tr><td> 0.1％明胶</td><td>密理博</td><td> ES-006-B </td><td></td></tr><tr><td>的Dulbecco PBS（D-PBS） </td><td> Cellgro </td><td> 21-031-CV </td><td></td></tr><tr><td></td><td></td><td></td><td>解决方案食谱 4％多聚甲醛 100毫升: <ol><li>混合4克多聚甲醛，90毫升H 2 O，和NaOH（2N NaOH中的下降）。多聚甲醛不会进入溶液中，直到添加了一些NaOH溶液的pH值增加。 </li><li>搅拌并加热，在60℃，直到所有的粉末是在溶液中（约10-20分钟）。 不要过热。 </li><li>加入〜10毫升的10倍的PBS中达到最终体积为100ml。 </li><li>方便的（〜10毫升〜40毫升）的等分试样在-20℃下储存。 </li></ol> 警告:在&#39;做样子&#39;形式（压缩的小颗粒）的多聚甲醛减粉末，因此，可以测量以外的机罩。但是你还是应该戴防尘口罩（N95至少）在处理过程中。 EB文化传媒对于500毫升EB媒体: <table border="1"><tbody><tr><td> DMEM: </td><td> 404.5毫升 </td></tr><tr><td> FBS: </td><td> 75.0毫升</td></tr><tr><td> L-谷氨酰胺: </td><td> 5.0毫升</td></tr><tr><td>青霉素/链霉素: </td><td> 5.0毫升</td></tr><tr><td>非必需氨基酸: </td><td> 5.0毫升</td></tr><tr><td>丙酮酸钠: </td><td> 5.0毫升</td></tr><tr><td> β-巯基乙醇: </td><td> 500微升</td></tr></tbody></table></td></tr></tbody></table>

use of lysotracker to detect programmed cell death in embryos and differentiating embryonic stem cells

程序性细胞死亡（PCD）发生于成人，以维持正常组织稳态和胚胎发育过程中形成组织和器官1,2,6,7。在开发过程中，有毒化学物质或基因的改变可以导致PCD增加或改变PCD的模式，导致发育异常和出生缺陷3-5。要了解这些缺陷的病因学，胚胎的研究，使用区分胚胎干细胞（ES细胞） 在体外实验中，可以补充。 细胞凋亡是一种形式的PCD研究，既包括内在的和外在的信号，激活半胱天冬酶的酶级联。细胞特性的变化包括膜皱缩，核收缩，且DNA碎片。不涉及其他形式的PCD半胱天冬酶的激活，最终的结果可能是长期自噬。无论PCD的途径，死亡的细胞需要被删除。在成年人中，免疫细胞PERFORM功能，而在胚胎的免疫系统尚未开发，拆除产生的一种替代机制。这种机制涉及到邻近的细胞（称为"非专业吞噬细胞"），吞噬作用，他们认识到表面上的死细胞和吞噬8-10"吃我"的信号。吞噬后，碎片带来的溶酶体降解。因此无论PCD机制，溶酶体活性的增加可以增加细胞死亡相关。 要学习PCD，一个简单的分析，可视化厚的组织和多层区分文化的溶酶体中是有用的。 LysoTracker染料是一种高度可溶性小分子，被保持在如酸性亚细胞溶酶体11-13。的染料是采取了通过扩散和通过循环。由于是不是一个障碍，可视化的PCD在厚厚的组织和多层次的文化渗透是可能的12,13 </支持&gt;。与此相反，TUNEL（末端脱氧核苷酸转移酶缺口末端标记法）分析14，仅限于小样本，组织切片和单层培养，因为此过程需要进入/渗透终端转移。 相反，扩散和溶解的溶剂苯胺蓝，DND-99 LysoTracker红，是可以解决的，明亮的，稳定的。染色可以可视化与标准荧光或共聚焦显微镜在整个贴装或部分，使用水性或溶剂型的安装媒体12,13。在这里，我们描述了使用此染料看，PCD在正常和 Sonic Hedgehog null小鼠胚胎的协议。此外，我们表现出ES细胞培养鉴别分析PCD，并提出一个简单的量化方法。总之，LysoTracker染色检测PCD的其他方法可以是一个很好的补充。

Watch this Scientific Journal Video about Use of LysoTracker to Detect Programmed Cell Death in Embryos and Differentiating Embryonic Stem Cells at JoVE.com

Use of LysoTracker to Detect Programmed Cell Death in Embryos and Differentiating Embryonic Stem Cells

我们提出了一个简单的协议，可视化地区的小鼠胚胎中的细胞程序性死亡（PCD）和鉴别胚胎干（ES）细胞培养，使用高度水溶性染料LysoTracker。

使用LysoTracker检测及鉴别胚胎干细胞在胚胎细胞程序性死亡

Programmed cell death (PCD) occurs in adults to maintain normal tissue homeostasis and during embryological development to shape tissues and ...

use-lysotracker-to-detect-programmed-cell-death-embryos

Research

Education

JoVE Journal

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Cell Biology

Biology

Unit 1

Cell Death

SCIENTISTS IN ACTION

23.1K 次观看.  University of Southern California. 我们提出了一个简单的协议，可视化地区的小鼠胚胎中的细胞程序性死亡（PCD）和鉴别胚胎干（ES）细胞培养，使用高度水溶性染料LysoTracker。

视频: Use of LysoTracker to Detect Programmed Cell Death in Embryos and Differentiating Embryonic Stem Cells

Dissection of 6.5 dpc Mouse Embryos

Analysis of gene expression patterns during early stages of mammalian embryonic development can provide important clues about gene function, cell-cell interaction and signaling mechanisms that guide embryonic patterning. However, dissection of the mouse embryo from the decidua shortly after implantation can be a challenging procedure, and detailed step-by-step documentation of this process is lacking.

Here we demonstrate how post-implantation (6.5 dpc) embryos are isolated by first dissecting the uterus of a pregnant mouse (detection of the vaginal plug was designated day 0.5 poist coitum) and subsequently dissecting the embryo from maternal decidua. The dissection of Reichert's membrane is described as well as the removal of the ectoplacental cone.

Isolation of postimplantation-stage embryos allows one to study gene patterning and analyze cell-lineage decision making processes during embryonic development, but proper dissection of the early embryo can be challenging. This protocol describes a method for isolating early primitive-streak-stage embryos (~6.5 days post coitum [dpc]).

6.5 DPC小鼠胚胎剖析

Analysis of gene expression patterns during early stages of mammalian embryonic development can provide important clues about gene function, cell-cell ...

科研

生物学

Derivation of Hematopoietic Stem Cells from Murine Embryonic Stem Cells

A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny.  Embryonic stem cells (ESC) are derived from the blastocyst of the early embryo and are pluripotent in differentiative ability.  Their vast differentiative potential has made them the focus of much research centered on deducing how to coax them to generate clinically useful cell types.  The successful derivation of hematopoietic stem cells (HSC) from mouse ESC has recently been accomplished and can be visualized in this video protocol.  HSC, arguably the most clinically exploited cell population, are used to treat a myriad of hematopoietic malignancies and disorders.  However, many patients that might benefit from HSC therapy lack access to suitable donors.  ESC could provide an alternative source of HSC for these patients.  The following protocol establishes a baseline from which ESC-HSC can be studied and inform efforts to isolate HSC from human ESC.  In this protocol, ESC are differentiated as embryoid bodies (EBs) for 6 days in commercially available serum pre-screened for optimal hematopoietic differentiation.  EBs are then dissociated and infected with retroviral HoxB4.  Infected EB-derived cells are plated on OP9 stroma, a bone marrow stromal cell line derived from the calvaria of  M-CSF-/- mice, and co-cultured in the presence of hematopoiesis promoting cytokines for ten days.  During this co-culture, the infected cells expand greatly, resulting in the generation a heterogeneous pool of 100s of millions of cells.  These cells can then be used to rescue and reconstitute lethally irradiated mice.

This protocol details the derivation of transplantable hematopoietic stem cells from mouse embryonic stem cells (ESC) and their subsequent injection into lethally irradiated recipient mice. Briefly, ESC are differentiated as embryoid bodies, which are then infected with retroviral HoxB4 and co-cultured with OP9 stromal cells and hematopoietic cytokines.

从小鼠胚胎干细胞的造血干细胞的推导

A stem cell is defined as a cell with the capacity to both self-renew and generate multiple differentiated progeny.  Embryonic stem cells (ESC) are ...

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 ...

Preparation of embryos for Electron Microscopy of the Drosophila embryonic heart tube

The morphogenesis of the Drosophila embryonic heart tube has emerged as a valuable model system for studying cell migration, cell-cell adhesion and cell shape changes during embryonic development. One of the challenges faced in studying this structure is that the lumen of the heart tube, as well as the membrane features that are crucial to heart tube formation, are difficult to visualize in whole mount embryos, due to the small size of the heart tube and intra-lumenal space relative to the embryo. The use of transmission electron microscopy allows for higher magnification of these structures and gives the advantage of examining the embryos in cross section, which easily reveals the size and shape of the lumen. In this video, we detail the process for reliable fixation, embedding, and sectioning of late stage Drosophila embryos in order to visualize the heart tube lumen as well as important cellular structures including cell-cell junctions and the basement membrane.

Preparation of embryos for Electron Microscopy of the Drosophila embryonic heart tube

We describe a process for fixation, embedding, sectioning, and imaging of late stage Drosophila embryos for Trasmission Electron Microscopy of the embryonic heart tube. This technique allows for the visualization of the heart tube lumen as well as the basement membrane, which lines the lumen of the heart.

电子显微镜对胚胎的制备果蝇胚胎心管

The morphogenesis of the Drosophila  embryonic heart tube has emerged as a valuable model system for studying cell migration, cell-cell adhesion and cell ...

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 ...

Video Bioinformatics Analysis of Human Embryonic Stem Cell Colony Growth

Because video data are complex and are comprised of many images, mining information from video material is difficult to do without the aid of computer software.  Video bioinformatics is a powerful quantitative approach for extracting spatio-temporal data from video images using computer software to perform dating mining and analysis. In this article, we introduce a video bioinformatics method for quantifying the growth of human embryonic stem cells (hESC) by analyzing time-lapse videos collected in a Nikon BioStation CT incubator equipped with a camera for video imaging. In our experiments, hESC colonies that were attached to Matrigel were filmed for 48 hours in the BioStation CT.  To determine the rate of growth of these colonies, recipes were developed using CL-Quant software which enables users to extract various types of data from video images.  To accurately evaluate colony growth, three recipes were created. The first segmented the image into the colony and background, the second enhanced the image to define colonies throughout the video sequence accurately, and the third measured the number of pixels in the colony over time.  The three recipes were run in sequence on video data collected in a BioStation CT to analyze the rate of growth of individual hESC colonies over 48 hours. To verify the truthfulness of the CL-Quant recipes, the same data were analyzed manually using Adobe Photoshop software. When the data obtained using the CL-Quant recipes and Photoshop were compared, results were virtually identical, indicating the CL-Quant recipes were truthful. The method described here could be applied to any video data to measure growth rates of hESC or other cells that grow in colonies. In addition, other video bioinformatics recipes can be developed in the future for other cell processes such as migration, apoptosis, and cell adhesion.  

Video bioinformatics is the automated processing, analysis, understanding, and data mining of biological spatio-temporal data extracted from microscopic videos. The purpose of this article is to demonstrate a method for measuring human embryonic stem cell colony growth using a video bioinformatics method.

视频人类胚胎干细胞克隆生长的生物信息学分析

Because video data are complex and are comprised of many images, mining information from video material is difficult to do without the aid of computer ...

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.

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 ...

Whole-mount Immunohistochemical Analysis for Embryonic Limb Skin Vasculature: a Model System to Study Vascular Branching Morphogenesis in Embryo

Whole-mount immunohistochemical analysis for imaging the entire vasculature is pivotal for understanding the cellular mechanisms of branching morphogenesis. We have developed the limb skin vasculature model to study vascular development in which a pre-existing primitive capillary plexus is reorganized into a hierarchically branched vascular network. Whole-mount confocal microscopy with multiple labelling allows for robust imaging of intact blood vessels as well as their cellular components including endothelial cells, pericytes and smooth muscle cells, using specific fluorescent markers. Advances in this limb skin vasculature model with genetic studies have improved understanding molecular mechanisms of vascular development and patterning. The limb skin vasculature model has been used to study how peripheral nerves provide a spatial template for the differentiation and patterning of arteries. This video article describes a simple and robust protocol to stain intact blood vessels with vascular specific antibodies and fluorescent secondary antibodies, which is applicable for vascularized embryonic organs where we are able to follow the process of vascular development.

We introduce a whole-mount immunohistochemistry and laser scanning confocal microscopy with multiple labelling for analyzing intricate vascular network formation in mouse embryonic limb skin.

全安装胚胎肢体皮肤血管的免疫组织化学分析:一个模型系统，研究在胚胎血管分支形态发生

Whole-mount immunohistochemical analysis for imaging the entire vasculature is pivotal for understanding the cellular mechanisms of branching ...

Efficient Derivation of Human Cardiac Precursors and Cardiomyocytes from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based transplantation or by cardiac tissue engineering1-3. Cardiomyocytes become terminally-differentiated soon after birth and lose their ability to proliferate. There is no evidence that stem/progenitor cells derived from other sources, such as the bone marrow or the cord blood, are able to give rise to the contractile heart muscle cells following transplantation into the heart1-3. The need to regenerate or repair the damaged heart muscle has not been met by adult stem cell therapy, either endogenous or via cell delivery1-3. The genetically stable human embryonic stem cells (hESCs) have unlimited expansion ability and unrestricted plasticity, proffering a pluripotent reservoir for in vitro derivation of large supplies of human somatic cells that are restricted to the lineage in need of repair and regeneration4,5. Due to the prevalence of cardiovascular disease worldwide and acute shortage of donor organs, there is intense interest in developing hESC-based therapies as an alternative approach. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity6-8 (see a schematic in Fig. 1A). In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic9-11. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules12 (see a schematic in Fig. 1B). After screening a variety of small molecules and growth factors, we found that such defined conditions rendered nicotinamide (NAM) sufficient to induce the specification of cardiomesoderm direct from pluripotent hESCs that further progressed to cardioblasts that generated human beating cardiomyocytes with high efficiency (Fig. 2). We defined conditions for induction of cardioblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human cardiac cells across the spectrum of developmental stages for cell-based therapeutics.

We have established a protocol for induction of cardioblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human cardiac progenitors and functional cardiomyocytes for cardiovascular repair.