PLP是桑福德儿童健康研究中心的助理研究员。 R01AR056712，R01AR052779并从关节炎，肌肉骨骼的健康/国立研究所和皮肤病（NIAMS），MDA和桑福德儿童健康研究奖P30 AR061303：该作品已被以下补助工党的支持。这项工作在一定程度得益于科研经费从该项目中的欧盟第七框架计划FP7 -健康- 2009 ENDOSTEM 241440（ 血管相关的干细胞和肌肉干细胞的修复和维护肌肉组织中的激活）股份有限公司是由支持。 CIRM奖学金。

1，感染人类胚胎干细胞此协议需要高滴度的慢病毒编码BAF60C和MyoD的13。这些结构可根据要求提供。 <ol><li>在开始之前，建议<ol><li>为了确保感染，板的hESC感染上无饲养条件下（ 图2A）的病毒摄取过程，以消除蜂窝竞争对手高效率。事实上，MEF中是出了名更容易感染相比，人类胚胎干细胞，并能胜任MyoD调控的转换。因此，从人类胚胎干细胞培养消除的MEF增加了感染率。 </li><li>建议在有高滴度的慢病毒，以确保感染的高效率。选项，以提高感染效率一节“ 检查感染”的讨论。 </li><li>加入1 ml 1毫克/毫升的基质胶的各孔中制备的基质胶包被的板并离开在室温至少1小时（或O / N密封在4℃下如果前一天准备）。 </li></ol></li><li>感染过程<ol><li>一天前感染<ol><li>加胚胎干细胞克隆和恢复补充培养基中于1,000倍稀释（最终浓度为2mM）。 </li></ol></li><li>每天0 - 感染BAF60C 注：执行与BAF60C人类胚胎干细胞首先由感染MyoD的感染。 <ol><li>分解一个6孔板的hESC的在37℃下生长的菌落14在单细胞悬浮液温育1毫升的TrypLE的5分钟的阱</li><li>停止过户到一个15毫升的试管中，加入9毫升人类胚胎干细胞中，离心5分钟1200转的。 </li><li>在离心，加入到1ml mTeSR1的，聚凝胺（6毫克/毫升）和胚胎干细胞克隆和恢复补编（2毫米）准备感染混合在一个干净的试管中。 </li><li>重悬细胞沉淀（约5×10 5 -1×10 6细胞从一个C人类胚胎干细胞的onfluent孔）用1ml的混合感染和板到6 - 孔低安装板，从而防止细胞粘附到塑料。 </li><li>添加BAF60C-IRES-GFP的病毒在10 8 MOI，并培育3小时的孵化器</li><li>收集含病毒的细胞悬浮液，并在2的基质胶包被的孔等分。然后加入2毫升mTeSR1 +胚胎干细胞克隆与恢复补充，每孔留下的O / N的孵化器。 </li></ol></li><li>第1天<ol><li>小心地取出含有病毒的媒介和替换用2毫升mTeSR1and胚胎干细胞克隆和恢复补充。细胞将开始结块， 如图2B所示 。 </li></ol></li><li>第2天 - 感染MyoD的<ol><li>在继续之前检查在荧光显微镜下的细胞的荧光状态，以确保有感染的高效率。如果没有，请参阅“ 检查侵染离子“。 </li><li>如果细胞已经达到至少80％汇合，进行如下操作;否则等待一个额外的前一天诉讼。 </li><li>取出介质，并加入1 ml的TrypLE试剂解离的细胞。孵育5分钟，在37°C。 </li><li>重复描述from1.2.2.2 - 1.2.2.6添加MyoD的病毒在10 8 MOI。 </li></ol></li><li> 3天<ol><li>小心地取出含有病毒的媒介和替换用2毫升mTeSR1and胚胎干细胞克隆和恢复补充。 </li><li>在基质胶板的MEF涂层板在2.5×10 5 / cm 2的第二天，让被感染的细胞的繁殖。 </li></ol></li><li> 4天<ol><li>取出介质，并加入1 ml的TrypLE试剂解离的细胞。孵育5分钟，在37°C。 </li><li>细胞转移中有15毫升猎鹰管，5分钟在1200转加9毫升人类胚胎干细胞培养基和离心机。 </li><li> Ř的eMove上清，重新悬浮于6ml人类胚胎干细胞培养基。 </li><li>从含的MEF-6孔板和板的hESC 1:2分流比移除介质，配药3毫升每个MEFs中含有良好。 提示！ 当殖民地达到汇合，一些MyoD/BAF60C-infected细胞的井可以在液氮中冷冻的储备量。冷冻培养基包括90％KOSR和10％DMSO加2mM的胚胎干细胞克隆和恢复补充。其余的井可以再暴露在分化协议-见下面的说明。如果需要更多的细胞产生更多myospheres，受感染的人类胚胎干细胞可以被进一步传播如下： </li><li>除去培养基并用1ml的1毫克/毫升胶原酶IV为5分钟，在37℃下</li><li>除去胶原酶IV和加入1 ml人类胚胎干细胞培养基。 </li><li>在解剖镜下用刮10ml的尖殖民地。 </li><li>收集科洛尼的块ES在一个15毫升管中，并允许以沉淀3分钟。然后用1:4去除上清，重悬在人类胚胎干细胞培养基和板块上的MEF板。 </li></ol></li></ol></li><li>检查感染<ol><li>而传播的单元格，则建议在板的受感染细胞在小规模收获用于RNA分析，并通过免疫荧光，以检查生物标志物，反映在每个阶段正确的生物学状态进行蛋白质表达分析（ 见代表性结果 ）。对于高滴度慢病毒预期受感染的细胞的百分比应至少为80％。 </li><li>以保证高效率的慢病毒载体可以被修改，引入一个荧光标记物的选择和/或抗生素抗性。我们的慢病毒表达质粒BAF60C还带有GFP荧光。 </li><li>人类胚胎干细胞分选的荧光标记物。中的感染效率低的情况下，建议使用荧光ctivated细胞分选（FACS分选），以丰富的细胞表达BAF60C病毒再感染的细胞具有高滴度的MyoD病毒。 <ol><li>加胚胎干细胞克隆和恢复补充培养基中的排序的前一天。 </li><li>分拣的当天，通过用TrypLE分离的细胞（如在第0天描述），重悬细胞，用FACS-KO管更换血清加人胚胎干细胞克隆和恢复补充。 </li><li>在排序结束后，收集MEF中板增加胚胎干细胞克隆与恢复补充的细胞KO血清替代和中厚板。 </li></ol></li></ol></li></ol> 2，EB-样诱导和分化<ol><li>诱导分化过程<ol><li>每天0 - EB感应<ol><li>确保从BAF60C/Myod-infected人类胚胎干细胞分化开始之前，有在井（ 图2C）大量殖民地。 </li><li>每口井Ř的eMove培养基，洗净，用PBS加1毫升1毫克/毫升胶原酶IV。孵育5分钟，在37℃下</li><li>除去胶原酶和加入1 ml EB培养基。 </li><li>在解剖镜下机械迅速用10毫升尖刮离解成菌落400-800细胞聚集。 </li><li>收集菌落的组块中有15毫升管中，并允许以沉淀3分钟。除去上清液，重新悬浮在3ml的EB培养基中，将细胞悬液转移至6孔板的低安装板的一个孔中。 </li></ol></li><li>第1天<ol><li>在继续操作之前检查菌落的大小和细胞死亡。如果大块大块的存在，打破他们轻轻吹打向上和向下与5毫升吸管，分3-4次。如果细胞死亡是非常高的（很多漂浮细胞和碎片）通过收集聚集在一个15毫升管通过重力更换培养基并用3毫升新鲜的EB中厚板它们，否则请执行以下步骤。 </li><li>添加1.5毫升新鲜的EB培养基中的孔</li></ol></li><li> 2天<ol><li>通过收集从各孔中有15毫升管中的聚集体改变介质。让他们安顿下来2分钟。 </li><li>除去上清液，更换用3毫升新鲜EB培养基中，并重新分配在同一个井。 </li></ol></li><li> 3天<ol><li>加入1.5毫升新鲜EB培养基中的井。 </li></ol></li><li> 4天<ol><li>请按“2天”中所述。 </li></ol></li><li>第5天-的EB-成簇状（ 图2D）肌分化。 <ol><li>收集的聚集体如2.1.3.1中所述，用2毫升的PBS洗涤一次;允许聚合去滓。 </li><li>除去PBS中，加3毫升DM介质;板在同一井。 </li></ol></li><li>第6天 - 第20天<ol><li>从D6至D20用新鲜培养基更换培养基，每3天。代表myospheres示F中IGURE 2E。 </li></ol></li></ol></li><li>检查myosphere分化<ol><li>在进一步继续之前，建议通过使包埋在OCT化合物中myospheres的部分，如在先前的朱庇特的协议12说明检查myospheres内肌分化的效率。肌源性标记物，如myognenin（克隆F5D，DSHB）和肌球蛋白重链（克隆MF20，DSHB）（ 见代表性成果 ）进行免疫荧光染色。 提示！要成功与染色为肌细胞生成素上的EB部分通过治疗进行抗原修复用十二烷基硫酸钠（SDS），0.5％5分钟，这是至关重要的。此外，也能够使用该协议用于使用F5D和MF20抗体的双染色。 </li></ol></li></ol> 3。媒体食谱<ol><li> ESC培养基 DMEM-F12 1 2mM L-谷氨酰胺 20％淘汰赛辑嗯更换培养基（KOSR） 0.1mM非必需氨基酸（NEAA） 50 U / ml青霉素/链霉素（PEN /链球菌） 0.1 mM的β-巯基乙醇 8纳克/毫升碱性成纤维细胞生长因子（bFGF） </li><li> EB培养基中 DMEM F12 1 2mM L-谷氨酰胺 15％FBS的 5％马血清</li><li> DM媒体 DMEF F12 1X液态培养基添加剂 1X N-2，补充</li></ol>

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我们什么都没有透露。

这里提出的协议描述了如何直接从人类胚胎干细胞产生收缩​​肌纤维（myospheres）的三维集群。提出的策略具有前所未有的非凡潜力以生产微型肌肉悬吊，可适合作为“病在一盘”的模式为筛选试验和发育研究。此外，为了从胚胎干细胞产生myospheres该方法是简单的，并且不需要在分化过程中的任何FACS分选工序中，其典型地具有在细胞中回收的产率产生不利影响。此外，分化过程中的分拣过程会干扰聚合，因为它意味着在EB解离成单细胞。 所提出的方法是基于人类胚胎干细胞的表观遗传学reprograming与特定的因素，MyoD的和BaAF60C，它们不表达多能性胚胎干细胞。 MyoD的和BAF60C提供的“核心”蛋白复合物第在标记的基因组位点从该转录进入激活骨骼肌肌原程序。这两个因素的慢病毒感染的手段被传递到细胞中，因此，关键是要获得具有高感染效率这两个因素中的所有单元格。这可以通过使用高滴度的病毒或病毒赋予了选择标记物来实现。培养条件在优化肌分化的程度也起着重要的作用。我们提出了一种基于血清的分化方案为MyoD/BAF60C的表达胚胎干细胞成簇生肌前体的分化，其次是（含有胰岛素转铁蛋白 - ITS）孵育在无血清的确定成分培养基，以实现转换生肌前体的成骨骼肌肌管。然而，它可能是其他定义的媒体可以实现相同或更好的肌分化。该协议的一个电流限制是，它依赖于使用的胎牛血清，其中，除了含有ING许多动物蛋白和物质，显示很多到很多的变化。这可能会导致myospheres内低效率或异质生肌转换。值得注意的是，并非所有的BAF60/MyoD-expressing胚胎干细胞衍生的EB-样结构是myospheres;即，有些是EB状集合体完全由肌纤维。 myospheres通常在胚胎干细胞表达BAF60C和MyoD的派生数可以从30到60％的估计，在免疫染色在协议结束时进行的EB段（见结果）而有所不同。丰富培养皿仅供myospheres一个潜在的方法是使用一个生肌记者。这将有利于丢弃部分或完全没有区别的EB-成簇状并仅选择了myospheres。 最后，更好地了解相关的介绍的因素时间要求的机制是提高交付的方法是有用的。例如，如果BAF60C和MyoD的需要只为sh的以“踢”的细胞在正确的方向，基于改进的基因传递方法可能适用ORT时间。相反，如果需要一个较长的时间之前将细胞利用其内源性蛋白的因素，附加型的方法将被推荐给消除变异和由于整合在基因组中不加控制的效果。最后，我们注意到，它是强制性的，以表达BAF60C，以达到人类胚胎干细胞转化为骨骼肌之前或在同一时间MyoD的（从未之后）。对于BAF60C前表达的要求想必依靠其作用，通过基因组6,15预先设定的后生景观进行适当的MyoD染色质分布。因此，未来的协议可能由化合物或促进人类胚胎干细胞BAF60C表达其他操作得到改善。

由于其独特的能力，自我更新，同时保持多能性人类胚胎干细胞（胚胎干细胞）被认为在再生医学的宝贵资源。茎病变肌肉和人类胚胎干细胞或诱导多能干细胞的细胞介导的再增殖（IPSC）衍生代骨骼肌体外疾病模型是研究目的是确定治疗方法和阐明许多神经肌肉疾病的发病机制主要目标。然而，这些研究已经由人类胚胎干细胞，以转化成骨骼肌细胞的电阻和通过的关于向骨骼肌成肌的hESC承诺的分子调控信息的缺乏的挑战。事实上，以前曾试图从人类胚胎干细胞生成骨骼肌祖细胞显示，只有胚体（EB）衍生的后代或间质细胞有能力来激活骨骼肌成肌下面的转录激活因子的异常表达（ 如PAX3或MYF5）1-3或暴露于特定的培养条件4-5。 之前我们已经表明，SWI / SNF成分，BAF60C（由SMARCD3编码），是转录机制，使肌肉特异性基因座6 MyoD调控活化的重要组成部分。我们最近发现，选择性缺席BAF60C的赋予到胚胎干细胞向骨骼肌成肌7是在BAF60C另有观察到表达的体细胞8-10中MyoD调控活化的阻力，这个过程通常被称为生肌转换。 BAF60C的强制表达MyoD的能够直接激活骨骼肌肉发育中的胚胎干细胞，在特定的培养条件下，与BAF60C和MyoD的强加一个后生签字，承诺对人类胚胎干细胞的肌源性谱系7。值得注意的是，以前的蛋白质组学分析揭示了选择性缺席BAF6的0℃时，SWI / SNF组件中，在胚胎干细胞11。我们用这些知识来强加人类胚胎干细胞的表观遗传承诺到生肌血统，导致可能被整合，形成立体的收缩骨骼肌成肌细胞的同质群体的产生结构（myospheres）在功能上模仿小型化骨骼肌7。事实上，我们从人类胚胎干细胞产生骨骼肌祖细胞的方法依赖于人类胚胎干细胞的肌源性谱系，这是表型潜直至细胞暴露于分化的信号，如细 ​​胞的表观遗传学承诺聚集和培养在分化培养基（具体见协议）。该策略允许人类胚胎干细胞的同质人口膨胀所表观遗传学致力于骨骼肌谱系，并适用于形成了重述组织和骨骼肌功能特性的三维收缩myospheres肌肉。该myospheres提供小型化的肌肉可开发为一种“病在培养皿中”肌肉疾病模型的第一个证据。当从来自患者的iPS细胞产生的，这些myospheres所澄清的长期发展问题的潜力和罕见疾病的发病机制，此外还提供了巨大的潜力作为治疗化合物的高通量筛选的工具。我们还注意到，myosphere分析的一个直接读出可以通过免疫组化可提供的部分，在由戈麦斯等人 12朱庇特的协议描述

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			<td height="21" style="height:21px;width:259px;">6 well plate</td>
			<td style="width:259px;">Corning</td>
			<td style="width:259px;">3506</td>
			<td style="width:64px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">6 well low attachment plate</td>
			<td style="width:259px;">Corning</td>
			<td style="width:259px;">3471</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">GFR Matrigel</td>
			<td style="width:259px;">BD Biosciencies</td>
			<td style="width:259px;">356231</td>
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			<td height="25" style="height:25px;width:259px;">MEFs (growth arrested)14</td>
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			<td height="21" style="height:21px;width:259px;">Collagenase IV</td>
			<td style="width:259px;">Invitrogen</td>
			<td style="width:259px;">17104-019</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:259px;">DMEM-F12</td>
			<td style="width:259px;">Invitrogen</td>
			<td style="width:259px;">15-090-CV</td>
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			<td height="21" style="height:21px;width:259px;">KOSR</td>
			<td style="width:259px;">Invitrogen</td>
			<td style="width:259px;">10828-028</td>
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			<td height="21" style="height:21px;width:259px;">1mM L-glutamine (100X)</td>
			<td style="width:259px;">Invitrogen</td>
			<td style="width:259px;">35050-61</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Pen/Strep (100X)</td>
			<td style="width:259px;">Invitrogen</td>
			<td style="width:259px;">15070-063</td>
			<td></td>
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			<td height="21" style="height:21px;width:259px;">2-Mercaptoethanol</td>
			<td style="width:259px;">Invitrogen</td>
			<td style="width:259px;">21985-023</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:259px;">NEAA (100X)</td>
			<td style="width:259px;">Invitrogen</td>
			<td style="width:259px;">11140-050</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Polybrene</td>
			<td style="width:259px;">Millipore</td>
			<td style="width:259px;">TR-1003-G</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:259px;">ITS Liquid Media Supplement</td>
			<td style="width:259px;">Sigma</td>
			<td style="width:259px;">I3146</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">N2-supplement&#160;</td>
			<td style="width:259px;">Invitrogen</td>
			<td style="width:259px;">17502-048</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:259px;">TrypLE express</td>
			<td style="width:259px;">Invitrogen</td>
			<td style="width:259px;">12605-010</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:259px;">FBS</td>
			<td style="width:259px;">HyClone</td>
			<td style="width:259px;">SH30396.03</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:259px;">HS</td>
			<td style="width:259px;">Invitrogen</td>
			<td style="width:259px;">16050114</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:259px;"><a href="https://www.stemgent.com/products/193">hES Cell Cloning &#38; Recovery Supplement</a></td>
			<td style="width:259px;">Stemgent</td>
			<td style="width:259px;">01-0014-100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">bfgf</td>
			<td style="width:259px;">Sigma</td>
			<td style="width:259px;">4114-TC</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">mTeSR1</td>
			<td style="width:259px;">Stemcell Technologies</td>
			<td style="width:259px;">5850</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Anti-MyoD</td>
			<td style="width:259px;">BD Biosciences</td>
			<td style="width:259px;">554130</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Anti-MyHC</td>
			<td style="width:259px;">DSHB</td>
			<td style="width:259px;">MF20</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:259px;">Anti-myogenin</td>
			<td style="width:259px;">DSHB</td>
			<td style="width:259px;">F5D</td>
			<td></td>
		</tr>
	</tbody>
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generation of myospheres from hescs by epigenetic reprogramming

从人胚胎干细胞（胚胎干细胞）的骨骼肌细胞的均匀和丰富人口的产生是基于细胞的疗法和一个“病在培养皿中的”人神经肌肉疾病的模型的要求。主要的障碍，如低丰度和兴趣的人群，以及缺乏对三维收缩结构的形成协议的异质性，限制了干细胞的应用神经肌肉疾病。我们设计了一个协议，它通过引入异位定义因素克服了这些限制在人类胚胎干细胞 - 肌判定因素的MyoD和SWI / SNF染色质重塑复合成分BAF60C - 那些能够胚胎干细胞重新编程为骨骼肌细胞。在这里，我们描述了协议的成立，以产生人类胚胎干细胞来源的成肌细胞，促进其聚集成三维微型结构（myospheres）在功能上模仿小型化骨骼肌7 </sup&gt;。

图1A示出了协议的示意建议从胚胎干细胞产生myospheres。的关键步骤，以控制（由箭头表示）是感染的人类胚胎干细胞的效率和myospheres内生肌转换。 在我们的实验中，我们采取的慢病毒载体编码BAF60C携带GFP荧光的优点。我们通常感染细胞BAF60C和FACS排序72小时感染后，以丰富的表达BAF60C人口。当细胞达到约70-80％汇合，我们与感染MyoD的慢病毒。第一次感染后一般需要两三天的细胞，达到了汇合。 图1B示出了外源基因的诱导相对于未感染的人类胚胎干细胞倍的表达水平。 图1C示出了在引入的因子蛋白质水平的表达和分布。 BAF60C<html>表情显示为绿色荧光，而MyoD的表达是通过免疫荧光法检测与MyoD的抗体。我们认为0日一天，我们开始感染人类胚胎干细胞分化为EB状集合体。后在DM介质15天，myospheres的一部分（通常是一个完整的孔）被包埋在OCT化合物12和切片进行免疫荧光为生肌标记来检查生肌转换效率。 图1D示出了代表性的染色为肌细胞生成蛋白和肌球蛋白重链中最佳的肌分化。 Myospheres可以有不同的或不寻常的形状，或许作为融合较小myospheres的结果。从10日开始，就可以观察到在myospheres零星收缩（ 参见电影1和2）。 43/51243fig1.jpg“/&gt; 图1 A）示意图用于从人胚胎干细胞。 乙myospheres）在感染BAF60C和MyoD的（H9-BM）通过qRT-PCR监测，并表示为倍数诱导胚胎干细胞的外源基因的mRNA水平的产生的协议，如相比于模拟感染的hESC表示由于在上myosphere部分染色肌细胞生成素D）免疫荧光进行的矢量的IRES-GFP的元件（MYOG MyoD的染色（红色）和BAF60C荧光（绿色）（H9-CTR）。C）的免疫荧光图像），肌球蛋白重链（MYHC）和counterstained的DAPI（见免疫详情）。比例尺为100μm。 <img alt="图2" fo:content-width="5in" fo:src="/files/ftp_upload/51243/51243fig2highres.jpg" src="/files/ftp_upload/51243/51243fig2.jpg" /> 图2。细胞的外观在不同阶段的代表性图片协议从感染（第一部分）的分化（第二部分）。比例尺为100μm。而EB-样结构从D1到D5大多是圆形的（D），增长了根据图1A该计划提出了一个不可预知的形状，是异质文化15天myospheres;看到在E和 F两个例子。

Watch this Scientific Journal Video about Generation of Myospheres From hESCs by Epigenetic Reprogramming at JoVE.com

Generation of Myospheres From hESCs by Epigenetic Reprogramming

在这里，我们描述了基于人类胚胎干细胞（胚胎干细胞）的产生向着那宽容的培养条件下形成的收缩肌纤维（myospheres）三维簇骨骼肌祖细胞，它概括人类的生物学特性均匀人口表观遗传重编程的协议骨骼肌。

Myospheres的产生从人类胚胎干细胞通过表观遗传修饰

Generation of a homogeneous and abundant population of skeletal muscle cells from human embryonic stem cells (hESCs) is a requirement for cell-based ...

generation-of-myospheres-from-hescs-by-epigenetic-reprogramming

Research

JoVE Journal

Biology

7.8K 次观看.  Sanford-Burnham Institute for Medical Research. 在这里，我们描述了基于人类胚胎干细胞（胚胎干细胞）的产生向着那宽容的培养条件下形成的收缩肌纤维（myospheres）三维簇骨骼肌祖细胞，它概括人类的生物学特性均匀人口表观遗传重编程的协议骨骼肌。 

视频: Generation of Myospheres From hESCs by Epigenetic Reprogramming

From MEFs to Matrigel I: Passaging hESCs in the Presence of MEFs

This video demonstrates how to grow human embryonic stem cells (hESCs) on mouse embryonic fibroblast (MEF) feeder cells.


This video demonstrates how to grow human embryonic stem cells (hESCs) on mouse embryonic fibroblast (MEF) feeder cells. Part 1 of 3.

从MEFs基质胶我的MEFs传代的胚胎干细胞

This video demonstrates how to grow human embryonic stem cells (hESCs) on mouse embryonic fibroblast (MEF) feeder cells.
...

科研

生物学

From MEFs to Matrigel 2: Splitting hESCs from MEFs onto Matrigel

This video demonstrates how to grow human embryonic stem cells (hESCs) on mouse embryonic fibroblast (MEF) feeder cells, how to passage hESCs from MEF plates to feeder cell-free Matrigel plates.


This video demonstrates how to maintain the growth of human embryonic stem cells (hESCs) in feeder cell-free conditions and how to continuously passage hESCs in feeder cell-free conditions. Confirmation of hESC pluripotency grown in feeder cell-free conditions by immunofluorescence microscopy is also demonstrated. Part 2 of 3.

从MEFs基质胶2：到基质胶从MEFs分裂的胚胎干细胞

This video demonstrates how to grow human embryonic stem cells (hESCs) on mouse embryonic fibroblast (MEF) feeder cells, how to passage hESCs from MEF ...

From MEFs to Matrigel 3: Passaging hESCs from Matrigel onto Matrigel

This video demonstrates how to maintain the growth of human embryonic stem cells (hESCs) in feeder cell-free conditions and how to continuously passage hESCs in feeder cell-free conditions. Confirmation of hESC pluripotency grown in feeder cell-free conditions by immunofluorescence microscopy is also demonstrated.

This video demonstrates how to maintain the growth of human embryonic stem cells (hESCs) in feeder cell-free conditions and how to continuously passage hESCs in feeder cell-free conditions. Confirmation of hESC pluripotency grown in feeder cell-free conditions by immunofluorescence microscopy is also demonstrated. Part 3 of 3.

从MEFs基质胶3：传代的胚胎干细胞从基质胶到基质胶

This video demonstrates how to maintain the growth of human embryonic stem cells (hESCs) in feeder cell-free conditions and how to continuously passage ...

Generation of Single-Cell Suspensions from Mouse Neural Tissue

Within the nervous system, hundreds of neuronal and glial cell types have been described. Each specific cell type in the brain or spinal cord has a repertoire of cell surface molecules, or molecular determinants, through which it can be identified and characterized. Currently, robust cell identification and separation technologies require single-cell preparations to be generated while simultaneously limiting cell death and destruction of characteristic surface protein. The gentleMACS Dissociator, when used in combination with trypsin or papain-based dissociation kits, can effectively and gently dissociate brain tissue while preserving antigen epitopes and limiting cell loss. Standardized preparation of single-cell suspensions is achieved using C Tubes and optimized, preset gentleMACS Programs. Once generated, single-cell suspensions can be treated with monoclonal conjugates like Anti-Prominin-1 MicroBeads, which identify neural progenitors, or purified further using Myelin Removal Beads.

Dissociating cells from specific tissue types requires specific parameters for tissue aggitation to obtain a high volume of viable, culturable cells. The Miltenyi gentleMACS Dissociator optimizes this task with a simple, practical protocol. In this publication the use of this apparatus on nerual tissue is explained.

从小鼠神经组织产生单细胞悬浮液

Within the nervous system, hundreds of neuronal and glial cell types have been described. Each specific cell type in the brain or spinal cord has a ...

Generation of Induced Pluripotent Stem Cells by Reprogramming Mouse Embryonic Fibroblasts with a Four Transcription Factor, Doxycycline Inducible Lentiviral Transduction System

Using a defined set of transcription factors and cell culture conditions, Yamanaka and colleagues demonstrated that retrovirus-mediated delivery and expression of Oct4, Sox2, c-Myc, and Klf4 is capable of inducing pluripotency in mouse fibroblasts.1 Subsequent reports have demonstrated the utility of the doxycycline (DOX) inducible lentiviral delivery system for the generation of both primary and secondary iPS cells from a variety of other adult mouse somatic cell types.2,3
Induced pluripotent stem (iPS) cells are similar to embryonic stem (ES) cells in morphology, proliferation and ability to induce teratoma formation. Both types of cell can be used as the pluripotent starting material for the generation of differentiated cells or tissues in regenerative medicine.4-6 iPS cells also have a distinct advantage over ES cells as they exhibit key properties of ES cells without the ethical dilemma of embryo destruction.
Here we demonstrate the protocol for reprogramming mouse embryonic fibroblast (MEF) cells with the Stemgent DOX Inducible Mouse TF Lentivirus Set. We also demonstrate that the Stemgent DOX Inducible Mouse TF Lentivirus Set is capable of expressing each of the four transcription factors upon transduction into MEFs thereby inducing a pluripotent stem cell state that displays the pluripotency markers characteristic of ES cells.

The Stemgent Dox Inducible Mouse TF Lentivirus Set can reprogram mouse embryonic fibroblasts (MEFs) to induced pluripotent stem (iPS) cells. Here we demonstrate the protocol for DOX-inducible expression of mouse reprogramming transcription factors Oct4, Sox2, Klf4 and c-Myc to generate iPS colonies that express common mES pluripotency markers.

重新用四个转录因子的小鼠胚胎成纤维细胞诱导多能干细胞的产生，强力霉素诱导慢病毒转导系统

Using a defined set of transcription factors and cell culture conditions, Yamanaka and colleagues demonstrated that retrovirus-mediated delivery and ...

Generation of Induced Pluripotent Stem Cells by Reprogramming Human Fibroblasts with the Stemgent Human TF Lentivirus Set

In 2006, Yamanaka and colleagues first demonstrated that retrovirus-mediated delivery and expression of Oct4, Sox2, c-Myc and Klf4 is capable of inducing the pluripotent state in mouse fibroblasts.1 The same group also reported the successful reprogramming of human somatic cells into induced pluripotent stem (iPS) cells using human versions of the same transcription factors delivered by retroviral vectors.2 Additionally, James Thomson et al. reported that the lentivirus-mediated co-expression of another set of factors (Oct4, Sox2, Nanog and Lin28) was capable of reprogramming human somatic cells into iPS cells.3
iPS cells are similar to ES cells in morphology, proliferation and the ability to differentiate into all tissue types of the body. Human iPS cells have a distinct advantage over ES cells as they exhibit key properties of ES cells without the ethical dilemma of embryo destruction. The generation of patient-specific iPS cells circumvents an important roadblock to personalized regenerative medicine therapies by eliminating the potential for immune rejection of non-autologous transplanted cells.
Here we demonstrate the protocol for reprogramming human fibroblast cells using the Stemgent Human TF Lentivirus Set. We also show that cells reprogrammed with this set begin to show iPS morphology four days post-transduction. Using the Stemolecule Y27632, we selected for iPS cells and observed correct morphology after three sequential rounds of colony picking and passaging. We also demonstrate that after reprogramming cells displayed the pluripotency marker AP, surface markers TRA-1-81, TRA-1-60, SSEA-4, and SSEA-3, and nuclear markers Oct4, Sox2 and Nanog.

We demonstrate the protocol for the generation of induced pluripotent stem cells from human somatic cells using lentivirus-mediated delivery of the human factors Oct4, Sox2, Nanog, and Lin28. Pluripotency was confirmed by morphology and the presence of embryonic stem (ES) cell-specific markers.

由Stemgent人类TF慢病毒重新编程人类成纤维细胞生成诱导多能干细胞

In 2006, Yamanaka and colleagues first demonstrated that retrovirus-mediated delivery and expression of Oct4, Sox2, c-Myc and Klf4 is capable of inducing ...

Generation of Transgenic C. elegans by Biolistic Transformation

The number of laboratories using the free living nematode C. elegans is rapidly growing. The popularity of this biological model is attributed to a rapid generation time and short life span, easy and inexpensive maintenance, fully sequenced genome, and array of RNAi resources and mutant animals. Additionally, analysis of the C. elegans genome revealed a great similarity between worms and higher vertebrates, which suggests that research in worms could be an important adjunct to studies performed in whole mice or cultured cells. A powerful and important part of worm research is the ability to use transgenic animals to study gene localization and function. Transgenic animals can be created either via microinjection of the worm germline or through the use of biolistic bombardment. Bombardment is a newer technique and is less familiar to a number of labs. Here we describe a simple protocol to generate transgenic worms by biolistic bombardment with gold particles using the Bio-Rad PDS-1000 system. Compared with DNA microinjection into hermaphrodite germline, this protocol has the advantage of not requiring special skills from the operator with regards to identifying worm anatomy or performing microinjection. Further multiple transgenic lines are usually obtained from a single bombardment. Also in contrast to microinjection, biolistic bombardment produces transgenic animals with both extrachromosomal arrays and integrated transgenes. The ability to obtain integrated transgenic lines can avoid the use of mutagenic protocols to integrate foreign DNA. In conclusion, biolistic bombardment can be an attractive method for the generation of transgenic animals, especially for investigators not interested in investing the time and effort needed to become skilled at microinjection.

Generation of Transgenic C. elegans by Biolistic Transformation

Transgenic worms are commonly used in C. elegans research. Described is a simple, yet effective, protocol to introduce transgenes into worms using biolistic bombardment with DNA-coated gold particles. The effort involved and results of bombardment compare favorably with microinjection for the generation of transgenic animals.

生成的基因 C。线虫通过基因枪转化

The number of laboratories using the free living nematode C. elegans is rapidly growing. The popularity of this biological model is attributed to a rapid ...

DNA Extraction from Paraffin Embedded Material for Genetic and Epigenetic Analyses

Disease development and progression are characterized by frequent genetic and epigenetic aberrations including chromosomal rearrangements, copy number gains and losses and DNA methylation. Advances in high-throughput, genome-wide profiling technologies, such as microarrays, have significantly improved our ability to identify and detect these specific alterations. However as technology continues to improve, a limiting factor remains sample quality and availability. Furthermore, follow-up clinical information and disease outcome are often collected years after the initial specimen collection. Specimens, typically formalin-fixed and paraffin embedded (FFPE), are stored in hospital archives for years to decades. DNA can be efficiently and effectively recovered from paraffin-embedded specimens if the appropriate method of extraction is applied. High quality DNA extracted from properly preserved and stored specimens can support quantitative assays for comparisons of normal and diseased tissues and generation of genetic and epigenetic signatures 1. To extract DNA from paraffin-embedded samples, tissue cores or microdissected tissue are subjected to xylene treatment, which dissolves the paraffin from the tissue, and then rehydrated using a series of ethanol washes. Proteins and harmful enzymes such as nucleases are subsequently digested by proteinase K. The addition of lysis buffer, which contains denaturing agents such as sodium dodecyl sulfate (SDS), facilitates digestion 2. Nucleic acids are purified from the tissue lysate using buffer-saturated phenol and high speed centrifugation which generates a biphasic solution. DNA and RNA remain in the upper aqueous phase, while proteins, lipids and polysaccharides are sequestered in the inter- and organic-phases respectively. Retention of the aqueous phase and repeated phenol extractions generates a clean sample. Following phenol extractions, RNase A is added to eliminate contaminating RNA. Additional phenol extractions following incubation with RNase A are used to remove any remaining enzyme. The addition of sodium acetate and isopropanol precipitates DNA, and high speed centrifugation is used to pellet the DNA and facilitate isopropanol removal. Excess salts carried over from precipitation can interfere with subsequent enzymatic assays, but can be removed from the DNA by washing with 70% ethanol, followed by centrifugation to re-pellet the DNA 3. DNA is re-suspended in distilled water or the buffer of choice, quantified and stored at -20&deg;C. Purified DNA can subsequently be used in downstream applications which include, but are not limited to, PCR, array comparative genomic hybridization 4 (array CGH), methylated DNA Immunoprecipitation (MeDIP) and sequencing, allowing for an integrative analysis of tissue/tumor samples.

This video demonstrates the protocol for DNA extraction from formalin-fixed paraffin-embedded material. This is a multi-day procedure in which tissue sections are deparaffinized with xylene, rehydrated with ethanol and treated with proteinase K to purify and isolate DNA for subsequent gene-specific or genome-wide analysis.

从石蜡包埋材料的遗传和表观遗传分析的DNA提取

Disease development and progression are characterized by frequent genetic and epigenetic aberrations including chromosomal rearrangements, copy number ...

In vivo Reprogramming of Adult Somatic Cells to Pluripotency by Overexpression of Yamanaka Factors

Induced pluripotent stem (iPS) cells that result from the reprogramming of somatic cells to a pluripotent state by forced expression of defined factors are offering new opportunities for regenerative medicine. Such clinical applications of iPS cells have been limited so far, mainly due to the poor efficiency of the existing reprogramming methodologies and the risk of the generated iPS cells to form tumors upon implantation.
We hypothesized that the reprogramming of somatic cells towards pluripotency could be achieved in vivo by gene transfer of reprogramming factors. In order to efficiently reprogram cells in vivo, high levels of the Yamanaka (OKSM) transcription factors need to be expressed at the target tissue. This can be achieved by using different viral or nonviral gene vectors depending on the target tissue. In this particular study, hydrodynamic tail-vein (HTV) injection of plasmid DNA was used to deliver the OKSM factors to mouse hepatocytes. This provided proof-of-evidence of in vivo reprogramming of adult, somatic cells towards a pluripotent state with high efficiency and fast kinetics. Furthermore no tumor or teratoma formation was observed in situ.
It can be concluded that reprogramming somatic cells in vivo may offer a potential approach to induce enhanced pluripotency rapidly, efficiently, and safely compared to in vitro performed protocols and can be applied to different tissue types in the future.

In vivo Reprogramming of Adult Somatic Cells to Pluripotency by Overexpression of Yamanaka Factors

This study demonstrates the reprogramming of somatic cells towards pluripotency in vivo without the generation of teratomas. We used hydrodynamic tail vein injection of plasmid DNA encoding the Yamanka factors to induce the in vivo reprogramming of adult hepatocytes into cells of enhanced pluripotency.

Induced pluripotent stem (iPS) cells that result from the reprogramming of somatic cells to a pluripotent state by forced expression of defined factors ...

Generation of Transgenic Hydra by Embryo Microinjection

As a member of the phylum Cnidaria, the sister group to all bilaterians, Hydra can shed light on fundamental biological processes shared among multicellular animals. Hydra is used as a model for the study of regeneration, pattern formation, and stem cells. However, research efforts have been hampered by lack of a reliable method for gene perturbations to study molecular function. The development of transgenic methods has revitalized the study of Hydra biology1. Transgenic Hydra allow for the tracking of live cells, sorting to yield pure cell populations for biochemical analysis, manipulation of gene function by knockdown and over-expression, and analysis of promoter function. Plasmid DNA injected into early stage embryos randomly integrates into the genome early in development. This results in hatchlings that express transgenes in patches of tissue in one or more of the three lineages (ectodermal epithelial, endodermal epithelial, or interstitial). The success rate of obtaining a hatchling with transgenic tissue is between 10% and 20%. Asexual propagation of the transgenic hatchling is used to establish a uniformly transgenic line in a particular lineage. Generating transgenic Hydra is surprisingly simple and robust, and here we describe a protocol that can be easily implemented at low cost.

Generation of Transgenic Hydra by Embryo Microinjection

Stably transgenic Hydra are made by microinjection of plasmid DNA into embryos followed by random genomic integration and asexual propagation to establish a uniform line. Transgenic Hydra are used to track cell movements, overexpress genes, study promoter function, or knock down gene expression using RNAi.