我们要感谢来自莫纳什拉金斯计划，以及从NHMRC民防部队和NHMRC项目补助资金支持。此外，我们要感谢苏美廉为她建设性的意见，埃德温娜McGlinn对她的支持和莫纳什Flowcore团队（尤其是亚当Dinsdale）为他们生产的视频帮助。

1，仪器设置/试剂准备/基因分型<ol><li>制备iPS细胞培养基:补充500毫升敲除DMEM培养基用75毫升胎牛血清（FBS）中，加入5ml L-谷氨酰胺，将5ml的非必需氨基酸，500微升β-巯基乙醇，5×10 5单位白血病抑制因子（ LIF）。请参阅材料清单用于产品和购买信息用于在该原稿的上下文中使用的试剂。 </li><li>制备MEF培养基:补充500毫升DMEM培养基用50ml的FBS，加入5ml L-谷氨酰胺，将5ml的非必需氨基酸，加入5ml丙酮酸钠，500微升β-巯基乙醇。 </li><li>准备冻存液:对于10毫升，结合9毫升FBS与1毫升DMSO中。 </li><li>制备明胶涂覆的菜肴加入3-5毫升0.1％猪明胶溶液至T75烧瓶中，在室温下孵育至少10分钟，并吸出物使用前右。 </li><li>准备FACS标签介质:对于10毫升，结合9.9毫升的PBS用0.1ml胎牛血清。 </li><li>执行基因分型PCR检测的ROSA26 m2rtTA轨迹:与Taq DNA聚合酶，根据制造商的说明进行反应。使用修改过的氯化镁为3.5毫米和下面的3个引物-浓度:oIMR8052:5&#39;GCG AAG AGT TTG TCC TCA ACC 3&#39;，oIMR8545:5&#39;AAA GTC GCT CTG AGT TGT达3&#39;，oIMR8546:5&#39;GGA GCG GGA GAA ATG GAT ATG 3&#39;。循环条件为:94℃，3分钟; 35个循环（94℃，30秒，65℃1分钟，72℃1分钟）; 72℃，2分钟;保持在12℃。预计产品尺寸:650 bp的野生型等位基因和340个基点的突变等位基因。 </li><li>进行基因分型PCR检测胶原StemCa / OKSM所在地:进行反应，与Taq DNA聚合酶按照说明进行操作。使用修改过的氯化镁 2.5毫米及以下3个引物-浓度:彩色/ FRT-B:5&#39;CCCTCCATGTGTGACCAAGG 3&#39;，COL / frtA1:5&#39;GCACAGCATTGCGGACATGC 3&#39;，COL / frtC1:5&#39;GCAGAAGCGCGGCCGTCTGG 3&#39;。循环条件为:95℃1分钟2个循环（94℃30秒，70℃45秒），6个循环（94℃30秒，68℃45秒）和30个循环的（94℃，20秒，60℃，1分钟）; 72℃，5分钟;保持在12℃。预计产品尺寸:331 bp的野生型等位基因和551个基点的突变等位基因。 </li><li>进行所有离心步骤在400 XG为3分钟，在4℃。 </li></ol> 2代小鼠胚胎成纤维细胞<ol><li>与m2rtTA应变的异性成员进行OKSM应变的动物之间的交配时机。在13.5天的胚胎剔除由母亲和去除子宫角收获胚胎。去除之前的子宫角，喷腹部用80％的乙醇溶液中，以避免微生物的污染。 </li><li>转移子宫角成含有10毫升无菌磷酸盐缓冲盐水的10cm的组织培养皿（PBS）。用消毒的手术等级剪刀剪开子宫角到含有一个胚胎件（ 见图3A，B）。 </li><li>使用解剖显微镜（最好放置在组织培养罩内），灭菌镊子小心取出子宫包膜及周围每个胚胎的胚外膜。每个胚胎转移到一个单独的10cm皿用10毫升的PBS。 </li><li>通过移除胚胎的头，四肢，尾部和内脏器官（心脏，肝，肠等 ）用钳子进行（参见图3C）。转移胚胎的头到1.5毫升试管中，并冻结下来，因此它可以被用于基因分型，如果有必要的。 </li><li>转移胚胎的躯干到空10厘米板及使用两个手术刀剁碎的胚胎，持续2分钟。加入200μl的胰蛋白酶/ EDTA溶液对胚胎切碎的顶部，并培育在室温下3-5分钟。继续切碎另外2分钟，加入2mL的MEF介质中<html>激活胰蛋白酶，然后转移到一个15毫升的试管中。 </li><li>用1000微升吸管，进一步分解机械轻柔吹打组织。转移到明胶包被的10cm皿和MEF媒体增加一个额外的10毫升注意:无论是常氧和缺氧的孵化器，可用于文化，是指讨论的更多的信息。 </li><li>经过24-48小时的板应密布的MEF。 </li><li>可选地，所述细胞可以被进一步传播或冷冻下来。对于细胞的冷冻，取出培养基，冲洗培养皿，用10ml PBS以除去介质和覆盖痕迹用3ml胰蛋白酶/ EDTA溶液。孵育3-5分钟，在37℃以下，加入5ml的MEF媒体和转移到15ml离心管中失活胰蛋白酶消化。降速和悬浮颗粒在3毫升冷冻介质。转移到3离心管，并在单元格中冷冻集装箱冷冻下来。 </li></ol> 3，重新编程的MEF ove_content"&gt;注:离心沉淀细胞在200 XG 5分钟，在4℃下，用氧正常组织培养孵化器重新编程它可以是有益的派生和扩大缺氧条件下（5％的氧气在MEF中，更多信息见讨论。 ）。 <ol><li>快速解冻低传离心管（P0-P1，2-3神达细胞）在水浴（37℃），其内容用10ml预热的MEF媒体转移到管。颗粒细胞，重新悬浮于12ml新鲜MEF的媒体，调成明胶包被的T75培养瓶中，让细胞恢复在继续操作之前24-48小时。注意:MEF中也可以导出后直接使用。 </li><li>以下去除培养基，用胰蛋白酶/ EDTA溶液（3ml 3-5分钟，在37℃）重叠洗MEFs细胞用PBS和cellularize。通过加入5ml的MEF介质的淬火胰蛋白酶和通过温和混合进一步解离的细胞，用10毫升吸管。确定使用血球细胞的数量。 </li><li>对于reprogramming实验中，种子的MEF到明胶包被的T75烧瓶中，在6.7×10 3个细胞/ cm 2的密度（〜每瓶0.5宇细胞）在含有2微克/毫升的强力霉素的iPSC媒体。请参考表1关于播种的建议，得到大约2宇重编程的中间体，用于每个时间点。注:重新编程开始与另外强力霉素（2微克/毫升）的补充培养基</li><li>对于第6天更换介质隔日新鲜强力霉素补充的iPSC媒体（12毫升每T75瓶中媒体）。超过此点更新媒体，每天如果有高的细胞数。双倍的培养体积，如果媒体的变化只能每隔一天进行。 </li><li>收获的重编程的中间体在所需的时间点（建议:天3，6，9，12），通过除去介质，漂洗用PBS适当体积（10毫升的T75烧瓶）中，用胰蛋白酶-EDTA溶液覆盖（3毫升的T75瓶）。孵育为在37℃下3-5分钟，并通过加入5ml的iPSC媒体接着温和移液急冷。 </li><li>计数与血球细胞悬浮液（见上文），粒料通过离心，吸出上清液，重悬的细胞在10ml的PBS中，以除去胰蛋白酶的任何痕迹。再次沉淀细胞，吸出上清液，并继续执行步骤4.1隔离重编程的中间体。注:细胞进行重编程可冷冻保存，并在以后阶段解冻用于FACS分离。 </li><li>要建立完全重编程的iPS培养，细胞生长在DOX无IPSC的媒体再4-7天。注:成功重编程的文化包含了大量由12天菌落（ 见图4）。在此期间，异常iPS细胞仍然需要OKSM表达会消失。 </li><li>或者，传播剩余完全重新编程的iPS培养:起初，种子照射的MEF为15×10 3个细胞/ cm 2的密度</s达&gt;于12ml的iPSC媒体上涂的T75明胶烧瓶一天或6小时前实验。 </li><li>接着，cellularize iPS细胞培养物通过除去介质，漂洗的T75烧瓶中，用10ml的PBS中，重叠用3ml胰蛋白酶-EDTA溶液，并在37℃温育3-5分钟。猝灭胰蛋白酶通过加入5ml的iPSC媒体接着温和的混合，转移到15毫升锥形管中，短暂离心，然后在10毫升的iPS媒体再悬浮。 </li><li>转移500μl的该细胞悬浮液到被照射的MEF（T75瓶），并允许培养物中生长4-5天。注:建立的iPS培养物可以是冷冻保存或用于实验。克隆细胞系可以通过解剖显微镜22下选择单独的iPSC克隆得到。 </li><li>冷冻保存汇合iPSC的培养在2.8描述的MEF。 </li></ol> 4，抗体标记<ol><li>重新编程的文化重悬细胞沉淀，在第3节准备中FACS标签介质补充有抗体（抗Thy-1.2太平洋蓝1:400，抗SSEA-1的生物素1:400和抗EpCAM的FITC 1:400）。用200微升每5万个细胞补充标签组合和在冰上孵育。 </li><li> 10分钟后，轻轻拍打管使细胞重新悬浮，并培育在冰上另外的10分钟。加入10毫升冷PBS中，每管和离心。 </li><li>对于每个管被染色的制备200μl的标签介质的补充有1微升链霉亲和PeCy7（1:200）每5百万个细胞。当细胞已沉淀，小心地吸去上清，重悬在补充有链亲和素-PeCy7标签介质的适当体积。 </li><li>细胞保持在冰上10分钟，点击重悬，孵育冰上再过10分钟，每管加冷PBS 10毫升，降速和吸出上清液。 </li><li>在标签介质重悬细胞沉淀物在约1×10 7个细胞/ ml补充有碘化丙啶（PI）（2微克/毫升）。通过将悬浮液通过70微米过滤去除细胞团块。转移细胞到一个适当的FACS管并保持在冰上。 </li><li>准备与各个抗体单独样品（抗Thy-1.2，抗-SSEA-1和抗EpCAM的）。请注意:这些是必需的，以执行在分析中使用的三个通道的色彩补偿。此外，还需要未标记细胞来设置电压和栅极进行排序。理想的情况是iPS细胞用于补偿:对保持在照射的MEF 0.5-1×10 6个iPS细胞种群被存储在液氮中冻存管。 MEFs细胞的存在是必不可少的，以获得在THY-1.2信道的信号。 </li><li>解冻iPS细​​胞的离心管所描述的的MEF（第3节）。离心，悬浮细胞在800微升标记缓冲液和200μL该样品被划为无标签的控制。 </li><li>利用剩余的细胞作为补偿控制。 3 15ml试管，加入抗体之间的鸿沟细胞如下:太平洋蓝补偿控制:添加抗Thy-1.2太平洋蓝抗体的0.5微升。 FITC补偿管:添加抗-EpCAM-FITC抗体的0.5微升。 PE-Cy7的补偿控制:0.5μL抗SSEA-1-生物素的抗体。 </li><li>保持细胞 - 抗体样品在冰上10分钟后，通过敲击混合并孵育在冰上10分钟。 </li><li>洗涤样品，用10ml冷PBS以除去未结合的抗体，向下自旋细胞，吸出上清液。重悬细胞沉淀物中加入200μl的FACS标签介质。 </li><li>抗SSEA-1-生物素标记的样品，加入链霉亲和PeCy7共轭（每200微升细胞1微升）。标签单元所描述的第一抗体（SSEA-1-生物素）。 </li><li>通过所有样本，包括未标记的控制通过一个70微米的过滤器，并添加PI（2微克/毫升）。细胞转移到FACS管并保持在冰上，直到需要。 </li></ol>中间体5，流式细胞仪分离注意:细胞用荧光激活细胞分选仪用405纳米，488纳米和560纳米激发激光器和一个100微米的喷嘴排列。 <ol><li>对于前向和侧向散射设电压，使得细胞群被正确显示。 如图5A所示，以排除杂物细胞周围画一个门。注:电压对细胞分选正确的设置可能会很复杂，需要一个有经验的流式细胞仪操作员。 </li><li>使用前向散射（FSC）的高度相对于FSC区域排除聚集体（ 图5B）。 </li><li>在可视化的PI信道与FSC到栅极上的PI阴性的活细胞（ 图5C）。 </li><li>使用未标记细胞调整为PB，FITC / GFP，PE-Cy7的的荧光通道的电压。在各通道的下端理想地定位在细胞群。 </li><li>置栅极与未标记的对照细胞中， 如图6A，B到子级分的重编程崇拜URES进入第3天，6和9群:SSEA-1 / THY-1.2 +细胞; SSEA-1 / THY-1.2〜细胞;和重新编程中间体SSEA-1 + / THY-1.2 - 细胞。 </li><li>进一步细分的第9天SSEA-1 + / THY-1.2〜部分采用PE-Cy7的抗FITC印迹;周围画SSE-A1 + / EPCAM +细胞及SSEA-1 + / Epcam-细胞的大门。置栅极与未标记的对照细胞中， 如图6C所示，D。 </li><li>排序所需的亚群（参见图7为用于MEF中代表性的FACS型材，3天/第6天/第9天/ 12天培养物和iPS细胞）之前预先填入与iPS细胞介质（1-2毫升）中相应的收集管。 </li><li>流式细胞仪分选，根据制造商的协议执行。 </li><li>经过流式细胞仪分离提交的细胞分子分析（RNA /蛋白提取，染色质免疫沉淀的DNA甲基化分析等）。可替代地，各种细胞组分可以是培养的。 </li></ol>

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作者什么都没有透露。

为了成功地重新编程的MEF成iPS细胞和纯化的重编程的中间体在高量，必须注意的是对整体效率产生影响的因素。在特定批次的FBS的用于补充的iPS媒体可具有有害的影响。一般而言积极的经验与胚胎干（ES）细胞的合格的FBS，但血清来自多家供应商可能会确定一个更便宜的选择批量测试发了言。 另一个因素是在重编程效率的影响是MEF中15,20的基因型。虽然从杂合子小鼠（HET），同时为OKSM和m2rtTA轨迹做改编，homozygousity在一个或更高的重编程效率，这两个区域的结果计算出来的MEF。的确，从OKSM和m2rtTA杂交后代衍生的MEF显示顺序如下（OKSM / m2rtTA）增加重编程效率:HET / HET &lt;均相/ HET &lt;HET /同系物&lt;均相/均聚物（请注意，在表1中给出的数字是为HET / HET动物）。在极少数情况下男性的HOMO /同源动物识别，后宫多m2rtTA雌性交配可以设置。所有的这些杂交后代将HET / HOMO的OKSM / m2rtTA位点，分别为。此外，胎的快速基因分型被推荐为较大同窝仔使用年轻小鼠进行繁殖（6-15周龄）时获得的。 此外，使用低传代MEFs细胞重编程被强调23。如果MEF中推导并扩大在含氧量正常的组织培养箱，我们强烈建议您只使用这些细胞到通道2。然而，如果实验者访问低氧培养箱（5％氧气）的MEF中的推导和扩展，通道数高达3仍然会产生良好的效果23。 万一实验者遇到与重新编程相关的问题，<html>如所概述的，最有可能的解释是在DOX另外，小鼠的不正确基因型或使用FBS的是不利于对iPS细胞产生时高传代数。然而，在罕见的情况下，我们观察到的MEF没能即使在理想的条件下重新编程。在这种情况下，自发从头删除m2rtTA的开放阅读框之内被认定为的根本原因。 该方法被成功地适应了通过提取磁性细胞分离（MACS），SSEA-1 +的中间体。有一个明显的时间优势，在使用MACS，然而，SSEA-1 +细胞群的"纯度降低到80-90％，而不是95％以上，这取决于实验的细胞是目的地为可能构成的类型一个问题。因此，一个选择是使用MACS富集SSEA-1 +细胞随后通过FACS来提高纯度和/或分数成SSEA-1 + / +的EpCAM和SSEA-1 + / Epcam-细胞。 "&gt;当由所概述的协议和小鼠模型中产生的iPS细胞已被证明是多能性和有效地向嵌合动物15,20的所有组织中，以产生胚完全由通过四倍体互补这些iPS细胞的还原能力已证明24。异常甲基化模式在DLK-DIO3基因簇已被确定为潜在的病因24，然而，添加抗坏血酸时的50微克/毫升到媒体重编程过程中的浓度会产生四倍体互补主管iPS细胞24。请注意，另一种可重复编程的小鼠模型改造以表达重编程因子在不同的化学计量比可以甚至在不存在的抗坏血酸处理25产生四倍体主管iPS细胞，然而，在我们手中的细胞从该菌株中重新编程，在相当低的频率进行比较在本文中使用鼠标模式升。 在这个手稿中描述的方法允许重新编程的中间体的分离从人口的耐火散装和应该被看作是非常有用的工具，解剖和理解重编程过程中，除去不必使用未分离的人口为仿形的上限制（ 例如，从耐火细胞高信号噪声）。本文所描述的抗体结合，使从小鼠细胞中以高纯度的中间体的分离，但它是可能的额外的细胞表面标记将在未来，这将允许在更高的纯度，得到的中间体被发现。请注意，SSEA-1的表达是不是人类诱导多能干细胞的标志，因此该协议不能再编程人来源的细胞中。总之，一旦分离所描述的方法重编程的中间体，可用于分子分析，包括表达profilin的克，染色质免疫沉淀法，甲基化分析和蛋白质分析。

胚胎干（ES）细胞衍生自胚泡期胚胎1的内细胞团。在适当的培养条件下，他们的自我更新和多能性维持。在2006年山和同事证明成熟细胞因子可被重新编程为所谓的诱导多能干细胞（iPS）细胞的转录的强制表达的Oct-4，KLF-4，SOX-2，c-Myc的（OKSM）2。 iPS细胞，如胚胎干细胞，可以产生身体的所有细胞类型中，但是，它们是免费的周围ES细胞3的产生的伦理约束。此外，iPS细胞进行个性化的再生医学的承诺和保持巨大的潜力样疾病建模应用及体外药物筛选4,5。为了重新编程技术来实现这种潜力，需要核重新编程的基本机制，可以充分地理解。然而，着力剖析了重新编程拍hway受到阻碍的事实，只有极少数的细胞重新编程（0.1-1％）。成功地重新编程成纤维细胞已报道经过不同的一系列事件，包括间质向上皮过渡6-10，在重新编程，激活的内源性核的多能性网络11-14的最后阶段。我们和其他人12,13,15-17最近已鉴定了一组细胞表面标记物，其允许稀有中间体从耐火散装种群的分离。重编程小鼠胚胎成纤维细胞（MEF中）发生在2个星期之久的重编程过程15 THY-1.2，SSEA1和EpCAM的（等等）的表达变化。早期重编程的MEF的一个子集时下调成纤维细胞的身份标志（THY-1.2）的表达式，然后开始表达多能性相关的标志物SSEA-1 12。在重新编程SSEA1阳性细胞的最后阶段reactiv吃了内源性多能性基因，如10月4 10-13,15。这最后一个转换标记在由EpCAM的可检测的表达（参见图1），或在稍后的阶段PECAM 15细胞表面。最近，奥马利等人报道了用CD44和ICAM1的作为替代或补充，THY-1.2和SSEA-1的重编程的中间体的鉴定。我们先前的FACS提取从第0天，第3天，第6天，第9天与第12天重新编程文化重新编程的中间体，以及从建立的iPS细胞系基于这些细胞表面标记15,18。对于下面描述的重编程系统和条件下，我们已经表明，在单细胞水平上，虽然人群是安静均匀的，也有一定程度的异质性，在确定中间人口。但是应当注意的是，细胞仅这些人群中的一个子集能够进展到相应的下一个站重编程过程的锗和产生iPS细胞集落，在不同的效率，这已被广泛先前15,19特征。此外，这些人群的重编程效率将取决于以及上再电镀和培养条件。为了增加实验的重复性，我们使用已设计，以表达控制住ROSA26轨迹和下强力霉素敏感子20,21的控制多顺反子OKSM磁带转录反式激活（m2rtTA）可重新编程的小鼠品系。使用这种小鼠模型规避的iPS细胞的产生， 即异构出发人口与细胞间的变化在数量和病毒插入的整合位点的位置，传统的病毒方法的不必要的副作用。两种转基因小鼠品系（OKSM，m2rtTA），可作为纯合创始人动物的杰克逊实验室，已经对以建立reprogra划线mmable小鼠模型（参见图2）。在该原稿，我们将详细描述如何导出的MEF，产生iPS细胞，并分离出重编程的中间体在用FACS转换过程的不同阶段。

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			<td dir="LTR" height="21" style="height:21px;width:212px;">70 &#181;m Cell strainer&#160;</td>
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			<td dir="LTR" style="width:171px;">352340</td>
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			<td dir="LTR" height="21" style="height:21px;width:212px;">Taq DNA Polymerase&#160;</td>
			<td dir="LTR" style="width:191px;">Life Technologies&#160;</td>
			<td dir="LTR" style="width:171px;">10342-020&#160;</td>
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cell surface marker mediated purification of ips cell intermediates from a reprogrammable mouse model

Mature cells can be reprogrammed to a pluripotent state. These so called induced pluripotent stem (iPS) cells are able to give rise to all cell types of the body and consequently have vast potential for regenerative medicine applications. Traditionally iPS cells are generated by viral introduction of transcription factors Oct-4, Klf-4, Sox-2, and c-Myc (OKSM) into fibroblasts. However, reprogramming is an inefficient process with only 0.1-1% of cells reverting towards a pluripotent state, making it difficult to study the reprogramming mechanism. A proven methodology that has allowed the study of the reprogramming process is to separate the rare intermediates of the reaction from the refractory bulk population. In the case of mouse embryonic fibroblasts (MEFs), we and others have previously shown that reprogramming cells undergo a distinct series of changes in the expression profile of cell surface markers which can be used for the separation of these cells. During the early stages of OKSM expression successfully reprogramming cells lose fibroblast identity marker Thy-1.2 and up-regulate pluripotency associated marker Ssea-1. The final transition of a subset of Ssea-1 positive cells towards the pluripotent state is marked by the expression of Epcam during the late stages of reprogramming. Here we provide a detailed description of the methodology used to isolate reprogramming intermediates from cultures of reprogramming MEFs. In order to increase experimental reproducibility we use a reprogrammable mouse strain that has been engineered to express a transcriptional transactivator (m2rtTA) under control of the Rosa26 locus and OKSM under control of a doxycycline responsive promoter. Cells isolated from these mice are isogenic and express OKSM homogenously upon addition of doxycycline. We describe in detail the establishment of the reprogrammable mice, the derivation of MEFs, and the subsequent isolation of intermediates during reprogramming into iPS cells via fluorescent activated cells sorting (FACS).

一个E13.5小鼠胚胎的解剖，分解和电镀后，一块10厘米的菜预计将在约1至2天到达汇合。在这个阶段，这是正常的文化包含了一些贴个组织尚未cellularized正常。传代后，这些就会消失。 经强力霉素诱导，重新编程的MEF发生明显的形态变化。约6天，早聚集状斑块应该开始出现（ 图4C）。这些将继续在进一步培养大小增长（参见图4D，E）。良好的重编程实验应导致&gt; 500菌落每T75，最初接种5×10 5个细胞（ 图4E）。建立的iPS培养具有特色的圆顶状的菌落，应该大多是缺乏分化的细胞（ 图4F）的。有时候，额外的iPS丘尔的传代清晰度可能需要除去未分化的/部分地重编程的细胞;细胞融合瓶可以按1:10的比例进行拆分到饲养层照射的MEF。 如上面所提到的，重新编程期间，形态学和分子变化是由对THY-1.2，SSEA-1和最终的EpCAM（参见图7A-F）的变化在FACS型材反射。正如前面12,15的报道，而MEF中主要是正面的THY-1.2和消极的其他标志物，第3天的文化已经看起来明显不同。细胞中的大部分已经开始下调THY-1.2和表达这些THY-1.2阴性细胞的非常小的子集，已成为阳性的SSEA-1，实际的重编程中间这个时间点。重编程的中间体，可以从日提取3培养物的数目是非常难以预测的百分比SSEA-1 + / THY-1.2 - 通常位于一个范围之间viabl的1-10％的E细胞。关于天6,9 SSEA-1 +细胞的增加的百分比，通常为10％，远高于可被检测到。大约12天SSEA-1阳性细胞的一个子集，可以检测该标记也为阳性的EpCAM。第12天培养物，如3天培养，表示在中间体的纯化的瓶颈，如SSEA-1.2 + / EPCAM +细胞的百分比是可变的，通常落在只有2-4％的所有活细胞的范围内。在表1中重新编程中间体的预期数量仅作为一个粗略的近似。建立真正的iPS细胞培养将是强阳性的SSEA-1和EpCAM的。 <img alt="图1" fo:content-width="5in" src="/files/ftp_upload/51728/51728fig1highres.jpg" width="500" /> 重新规划路径时图1表面标志的变化:下调成纤维细胞的身份标记THY-1.2之后是上调SSEA-1，SSEA-1阳性细胞向多能状态的一个子集的转换是通过收购EpCAM的大约12天的指示。 <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/51728/51728fig1large.jpg" target="_blank">请点击这里查看该图的放大版本。</a> <img alt="图2" fo:content-width="5in" src="/files/ftp_upload/51728/51728fig2highres.jpg" width="500" /> 图2中的可重新编程的小鼠模型的示意图。m2rtTA表达在遍在有源ROSA26轨迹的控制在多西环素存在下（DOX）的m2rtTA蛋白结合至四环素依赖的启动子（tetOP）在胶原1A1（COL1A1）轨迹产生的四要素盒的表达。两个双顺反子盒是由内部核糖体进入位点（IRES）连接的。对于Oct-4的/ KLF-4和Sox-2 / c-Myc的开放阅读框是复<html>通过自我裂解F2A和E2A序列的sed，分别<a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/51728/51728fig2large.jpg" target="_blank">请点击这里查看该图的放大版本。</a> <img alt="图3" fo:content-width="5in" src="/files/ftp_upload/51728/51728fig3highres.jpg" width="500" /> 图3胚解剖:（A）传输的子宫角成10cm皿填充用10ml PBS中，（B）中切成含有一个胚件。 （ 三）使用镊子摆脱多余的胚胎组织的胚胎并取出头部，四肢，尾部和内脏。 <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/51728/51728fig3large.jpg" target="_blank">请点击这里查看该图的放大版本。</a> LES / ftp_upload / 51728 / 51728fig4highres.jpg"WIDTH ="500"/&gt; 图重编程过程中4形态的变化。蚁群般的补丁成为从第6天起培养明显。真正的iPS细胞的特点是圆顶形形态。 （ 一）第0天/ MEF中，（B）3日，（C）6日（四）9日（五 ）第12天，（F）的 iPS细胞培养。比例尺:200微米<a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/51728/51728fig4large.jpg" target="_blank">，请点击这里查看该图的放大版本。</a> <img alt="图5" fo:content-width="5in" src="/files/ftp_upload/51728/51728fig5highres.jpg" width="500" /> 图5流式细胞仪的基本设置。（一）排除杂物侧面散射与前向散射面积印迹。 （B）中通过门控单细胞群与前向散射面积与前向散射高级印迹排除非碎屑聚集。 （ 三）通过门控PI的低事件用点燃式通道与前向散射面积污点。排除从单细胞群的细胞死亡<a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/51728/51728fig5large.jpg" target="_blank">，请点击这里查看该图的放大版本。</a> <img alt="图6" fo:content-width="5in" src="/files/ftp_upload/51728/51728fig6highres.jpg" width="500" /> 图6门（A，B）使用设置了门的THY-1.2 + / SSEA-1细胞，THY-1.2〜/ SSEA-1细胞和未标记的对照组细胞THY-1.2〜/ SSEA-1 +细胞。 （三）使用未标记的控制单元格，设置闸门的EpCAM的正面和负面的EpCAM细胞。 （D）SSEA-1 + / THY-1.2 -细胞可以分离到EpCAM的正面和成EpCAM的人口负。 <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/51728/51728fig6large.jpg" target="_blank">请点击这里查看该图的放大版本。</a> <img alt="图7" fo:content-width="5in" src="/files/ftp_upload/51728/51728fig7highres.jpg" width="500" /> 图7 THY-1.2与SSEA-1流式细胞仪印迹在重编程过程的各个阶段。（一）日0 / MEF中，（B）3日，（C）6日（四）9日（五 ）日12，（F）的iPS细胞培养物。 表1建议的接种密度，瓶数和预期的结果，导致胚胎杂OKSM和m2rtTA P1的MEF中。 <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/51728/51728fig7large.jpg" target="_blank">请点击ħ</a>ERE查看这个数字的放大版本。 <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td>日</td><td>接种在第0天T75瓶的数目</td><td>流式细胞仪后SSEA1 +细胞总数</td><td>流式细胞仪后SSEA1 + / EPCAM +细胞总数</td></tr><tr><td> 3 </td><td> 8 </td><td> 2000000 </td><td></td></tr><tr><td> 6 </td><td> 4 </td><td> 2000000 </td><td></td></tr><tr><td> 9 </td><td> 4 </td><td> 2000000 </td><td></td></tr><tr><td> 12 </td><td> 10 </td><td>千万</td><td> 2000000 </td></tr></tbody></table>

Watch this Scientific Journal Video about Cell Surface Marker Mediated Purification of iPS Cell Intermediates from a Reprogrammable Mouse Model at JoVE.com

Cell Surface Marker Mediated Purification of iPS Cell Intermediates from a Reprogrammable Mouse Model

Mouse embryonic fibroblast can be reprogrammed into induced pluripotent stem cells at low efficiency by the forced expression of transcription factors Oct-4, Sox-2, Klf-4, c-Myc. The rare intermediates of the reprogramming reaction are FACS isolated via labeling with antibodies against cell surface makers Thy-1.2, Ssea-1, and Epcam.

的iPS细胞从中间体可重编程小鼠模型的细胞表面标志物介导的净化

Mature cells can be reprogrammed to a pluripotent state. These so called induced pluripotent stem (iPS) cells are able to give rise to all cell types of ...

cell-surface-marker-mediated-purification-ips-cell-intermediates-from

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

12.2K 次观看.  Monash University. Mouse embryonic fibroblast can be reprogrammed into induced pluripotent stem cells at low efficiency by the forced expression of transcription factors Oct-4, Sox-2, Klf-4, c-Myc. The rare intermediates of the reprogramming reaction are FACS isolated via labeling with antibodies against cell surface makers Thy-1.2, Ssea-1, and Epcam. 

视频: Cell Surface Marker Mediated Purification of iPS Cell Intermediates from a Reprogrammable Mouse Model

Chromatographic Purification of Highly Active Yeast Ribosomes

Eukaryotic ribosomes are much more labile as compared to their eubacterial and archael counterparts, thus posing a significant challenge to researchers. Particularly troublesome is the fact that lysis of cells releases a large number of proteases and nucleases which can degrade ribosomes. Thus, it is important to separate ribosomes from these enzymes as quickly as possible. Unfortunately, conventional differential ultracentrifugation methods leaves ribosomes exposed to these enzymes for unacceptably long periods of time, impacting their structural integrity and functionality. To address this problem, we utilize a chromatographic method using a cysteine charged Sulfolink resin. This simple and rapid application significantly reduces co-purifying proteolytic and nucleolytic activities, producing high yields of intact, highly biochemically active yeast ribosomes. We suggest that this method should also be applicable to mammalian ribosomes. The simplicity of the method, and the enhanced purity and activity of chromatographically purified ribosome represents a significant technical advancement for the study of eukaryotic ribosomes.

Contamination of preparations of eukaryotic ribosomes purified by traditional methods by co-purifying nucleases and proteases negatively impacts on downstream biochemical and structural analyses. A rapid and simple chromatographic purification method is used to solve this problem using yeast ribosomes as a model system.

高活性酵母核糖体的色谱分离纯化

Eukaryotic ribosomes are much more labile as compared to their eubacterial and archael counterparts, thus posing a significant challenge to researchers.  ...

科研

生物学

Isolation and Purification of Kinesin from Drosophila Embryos

Motor proteins move cargos along microtubules, and transport them to specific sub-cellular locations. Because altered transport is suggested to underlie a variety of neurodegenerative diseases, understanding microtubule based motor transport and its regulation will likely ultimately lead to improved therapeutic approaches. Kinesin-1 is a eukaryotic motor protein which moves in an anterograde (plus-end) direction along microtubules (MTs), powered by ATP hydrolysis. Here we report a detailed purification protocol to isolate active full length kinesin from Drosophila embryos, thus allowing the combination of Drosophila genetics with single-molecule biophysical studies. Starting with approximately 50 laying cups, with approximately 1000 females per cup, we carried out overnight collections. This provided approximately 10 ml of packed embryos. The embryos were bleach dechorionated (yielding approximately 9 grams of embryos), and then homogenized. After disruption, the homogenate was clarified using a low speed spin followed by a high speed centrifugation. The clarified supernatant was treated with GTP and taxol to polymerize MTs. Kinesin was immobilized on polymerized MTs by adding the ATP analog, 5'-adenylyl imidodiphosphate at room temperature. After kinesin binding, microtubules were sedimented via high speed centrifugation through a sucrose cushion. The microtubule pellet was then re-suspended, and this process was repeated. Finally, ATP was added to release the kinesin from the MTs. High speed centrifugation then spun down the MTs, leaving the kinesin in the supernatant. This kinesin was subjected to a centrifugal filtration using a 100 KD cut off filter for further purification, aliquoted, snap frozen in liquid nitrogen, and stored at -80 &deg;C. SDS gel electrophoresis and western blotting was performed using the purified sample. The motor activity of purified samples before and after the final centrifugal filtration step was evaluated using an in vitro single molecule microtubule assay. The kinesin fractions before and after the centrifugal filtration showed processivity as previously reported in literature. Further experiments are underway to evaluate the interaction between kinesin and other transport related proteins.

Isolation and Purification of Kinesin from Drosophila Embryos

This is a protocol to isolate active full length Kinesin from Drosophila embryos for single-molecule biophysical studies. We show how to collect embryos, make the embryo lysate, and then polymerize microtubules (MTs). Kinesin is purified by immobilizing it on the MTs, spinning down the Kinesin-MT complexes, and then releasing the kinesin from the MTs via ATP addition.

kinesin的分离和纯化果蝇胚胎

Motor proteins move cargos along microtubules, and transport them to specific sub-cellular locations. Because altered transport is suggested to underlie a ...

Avidity-based Extracellular Interaction Screening (AVEXIS) for the Scalable Detection of Low-affinity Extracellular Receptor-Ligand Interactions

Extracellular protein:protein interactions between secreted or membrane-tethered proteins are critical for both initiating intercellular communication and ensuring cohesion within multicellular organisms. Proteins predicted to form extracellular interactions are encoded by approximately a quarter of human genes1, but despite their importance and abundance, the majority of these proteins have no documented binding partner. Primarily, this is due to their biochemical intractability: membrane-embedded proteins are difficult to solubilise in their native conformation and contain structurally-important posttranslational modifications. Also, the interaction affinities between receptor proteins are often characterised by extremely low interaction strengths (half-lives &lt; 1 second) precluding their detection with many commonly-used high throughput methods2. 
Here, we describe an assay, AVEXIS (AVidity-based EXtracellular Interaction Screen) that overcomes these technical challenges enabling the detection of very weak protein interactions (t1/2 &le; 0.1 sec) with a low false positive rate3. The assay is usually implemented in a high throughput format to enable the systematic screening of many thousands of interactions in a convenient microtitre plate format (Fig. 1). It relies on the production of soluble recombinant protein libraries that contain the ectodomain fragments of cell surface receptors or secreted proteins within which to screen for interactions; therefore, this approach is suitable for type I, type II, GPI-linked cell surface receptors and secreted proteins but not for multipass membrane proteins such as ion channels or transporters.
The recombinant protein libraries are produced using a convenient and high-level mammalian expression system4, to ensure that important posttranslational modifications such as glycosylation and disulphide bonds are added. Expressed recombinant proteins are secreted into the medium and produced in two forms: a biotinylated bait which can be captured on a streptavidin-coated solid phase suitable for screening, and a pentamerised enzyme-tagged (&beta;-lactamase) prey. The bait and prey proteins are presented to each other in a binary fashion to detect direct interactions between them, similar to a conventional ELISA (Fig. 1). The pentamerisation of the proteins in the prey is achieved through a peptide sequence from the cartilage oligomeric matrix protein (COMP) and increases the local concentration of the ectodomains thereby providing significant avidity gains to enable even very transient interactions to be detected. By normalising the activities of both the bait and prey to predetermined levels prior to screening, we have shown that interactions having monomeric half-lives of 0.1 sec can be detected with low false positive rates3.

Avidity-based Extracellular Interaction Screening AVEXIS for the Scalable Detection of Low-affinity Extracellular Receptor-Ligand Interactions

AVEXIS is a high throughput protein interaction assay developed to systematically screen for novel extracellular receptor-ligand pairs involved in cellular recognition processes. It is specifically designed to detect transient protein interactions that are difficult to identify using other high throughput approaches.

基于亲和力，外的相互作用筛选（AVEXIS）外低亲和力受体 - 配体相互作用的可扩展检测

Extracellular protein:protein interactions between secreted or membrane-tethered proteins are critical for both initiating intercellular communication and ...

Generation of Mice Derived from Induced Pluripotent Stem Cells

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also produce a source of patient-matched cells for regenerative therapies. iPSCs may be generated using multiple protocols and derived from multiple cell sources. Once generated, iPSCs are tested using a variety of assays including immunostaining for pluripotency markers, generation of three germ layers in embryoid bodies and teratomas, comparisons of gene expression with embryonic stem cells (ESCs) and production of chimeric mice with or without germline contribution2. Importantly, iPSC lines that pass these tests still vary in their capacity to produce different differentiated cell types2. This has made it difficult to establish which iPSC derivation protocols, donor cell sources or selection methods are most useful for different applications.
The most stringent test of whether a stem cell line has sufficient developmental potential to generate all tissues required for survival of an organism (termed full pluripotency) is tetraploid embryo complementation (TEC)3-5. Technically, TEC involves electrofusion of two-cell embryos to generate tetraploid (4n) one-cell embryos that can be cultured in vitro to the blastocyst stage6. Diploid (2n) pluripotent stem cells (e.g. ESCs or iPSCs) are then injected into the blastocoel cavity of the tetraploid blastocyst and transferred to a recipient female for gestation (see Figure 1). The tetraploid component of the complemented embryo contributes almost exclusively to the extraembryonic tissues (placenta, yolk sac), whereas the diploid cells constitute the embryo proper, resulting in a fetus derived entirely from the injected stem cell line.
Recently, we reported the derivation of iPSC lines that reproducibly generate adult mice via TEC1. These iPSC lines give rise to viable pups with efficiencies of 5-13%, which is comparable to ESCs3,4,7 and higher than that reported for most other iPSC lines8-12. These reports show that direct reprogramming can produce fully pluripotent iPSCs that match ESCs in their developmental potential and efficiency of generating pups in TEC tests. At present, it is not clear what distinguishes between fully pluripotent iPSCs and less potent lines13-15. Nor is it clear which reprogramming methods will produce these lines with the highest efficiency. Here we describe one method that produces fully pluripotent iPSCs and "all- iPSC" mice, which may be helpful for investigators wishing to compare the pluripotency of iPSC lines or establish the equivalence of different reprogramming methods.

Generating induced pluripotent stem cell (iPSC) lines produces lines of differing developmental potential even when they pass standard tests for pluripotency. Here we describe a protocol to produce mice derived entirely from iPSCs, which defines the iPSC lines as possessing full pluripotency1.

诱导多能干细胞来源于小鼠的生成

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also ...

Glycopeptide Capture for Cell Surface Proteomics

Cell surface proteins, including extracellular matrix proteins, participate in all major cellular processes and functions, such as growth, differentiation, and proliferation. A comprehensive characterization of these proteins provides rich information for biomarker discovery, cell-type identification, and drug-target selection, as well as helping to advance our understanding of cellular biology and physiology. Surface proteins, however, pose significant analytical challenges, because of their inherently low abundance, high hydrophobicity, and heavy post-translational modifications. Taking advantage of the prevalent glycosylation on surface proteins, we introduce here a high-throughput glycopeptide-capture approach that integrates the advantages of several existing N-glycoproteomics means. Our method can enrich the glycopeptides derived from surface proteins and remove their glycans for facile proteomics using LC-MS. The resolved N-glycoproteome comprises the information of protein identity and quantity as well as their sites of glycosylation. This method has been applied to a series of studies in areas including cancer, stem cells, and drug toxicity. The limitation of the method lies in the low abundance of surface membrane proteins, such that a relatively large quantity of samples is required for this analysis compared to studies centered on cytosolic proteins.

Cell surface proteins are biologically important and widely glycosylated. We introduce here a glycopeptide-capture approach to solubilize, enrich, and deglycosylate these proteins for facile LC-MS based proteomic analyses.

糖肽捕获的细胞表面蛋白组学

Cell surface proteins, including extracellular matrix proteins, participate in all major cellular processes and functions, such as growth, ...

MicroRNA Expression Profiles of Human iPS Cells, Retinal Pigment Epithelium Derived From iPS, and Fetal Retinal Pigment Epithelium

The objective of this report is to describe the protocols for comparing the microRNA (miRNA) profiles of human induced-pluripotent stem (iPS) cells, retinal pigment epithelium (RPE) derived from human iPS cells (iPS-RPE), and fetal RPE. The protocols include collection of RNA for analysis by microarray, and the analysis of microarray data to identify miRNAs that are differentially expressed among three cell types. The methods for culture of iPS cells and fetal RPE are explained. The protocol used for differentiation of RPE from human iPS is also described. The RNA extraction technique we describe was selected to allow maximal recovery of very small RNA for use in a miRNA microarray. Finally, cellular pathway and network analysis of microarray data is explained. These techniques will facilitate the comparison of the miRNA profiles of three different cell types.

The microRNA (miRNA) profiles of human induced-pluripotent stem (iPS) cells, retinal pigment epithelium (RPE) derived from human induced-pluripotent stem (iPS) cells (iPS-RPE), and fetal RPE, were compared.

人类iPS细胞的微小RNA表达谱，视网膜色素上皮衍生自IPS和胎儿视网膜色素上皮

The objective of this report is to describe the protocols for comparing the microRNA (miRNA) profiles of human induced-pluripotent stem (iPS) cells, ...

Efficient iPS Cell Generation from Blood Using Episomes and HDAC Inhibitors

This manuscript illustrates a protocol for efficiently creating integration-free human induced pluripotent stem cells (iPSCs) from peripheral blood using episomal plasmids and histone deacetylase (HDAC) inhibitors. The advantages of this approach include: (1) the use of a minimal amount of peripheral blood as a source material; (2) nonintegrating reprogramming vectors; (3) a cost effective method for generating vector free iPSCs; (4) a single transfection; and (5) the use of small molecules to facilitate epigenetic reprogramming. Briefly, peripheral blood mononuclear cells (PBMCs) are isolated from routine phlebotomy samples and then cultured in defined growth factors to yield a highly proliferative erythrocyte progenitor cell population that is remarkably amenable to reprogramming. Nonintegrating, nontransmissible episomal plasmids expressing OCT4, SOX2, KLF4, MYCL, LIN28A, and a p53 short hairpin (sh)RNA are introduced into the derived erythroblasts via a single nucleofection. Cotransfection of an episome that expresses enhanced green fluorescent protein (eGFP) allows for easy identification of transfected cells. A separate replication-deficient plasmid expressing Epstein-Barr nuclear antigen 1 (EBNA1) is also added to the reaction mixture for increased expression of episomal proteins. Transfected cells are then plated onto a layer of irradiated mouse embryonic fibroblasts (iMEFs) for continued reprogramming. As soon as iPSC-like colonies appear at about twelve days after nucleofection, HDAC inhibitors are added to the medium to facilitate epigenetic remodeling. We have found that the inclusion of HDAC inhibitors routinely increases the generation of fully reprogrammed iPSC colonies by 2 fold. Once iPSC colonies exhibit typical human embryonic stem cell (hESC) morphology, they are gently transferred to individual iMEF-coated tissue culture plates for continued growth and expansion.

Here we describe a protocol for generating human induced pluripotent stem cells from peripheral blood using an episome based reprogramming strategy and histone deacetylase inhibitors.

从血液高效的iPS细胞生成使用附加体和HDAC抑制剂

This manuscript illustrates a protocol for efficiently creating integration-free human induced pluripotent stem cells (iPSCs) from peripheral blood using ...

Light Sheet Fluorescence Microscopy of Plant Roots Growing on the Surface of a Gel

One of the key questions in understanding plant development is how single cells behave in a larger context of the tissue. Therefore, it requires the observation of the whole organ with a high spatial- as well as temporal resolution over prolonged periods of time, which may cause photo-toxic effects. This protocol shows a plant sample preparation method for light-sheet microscopy, which is characterized by mounting the plant vertically on the surface of a gel. The plant is mounted in such a way that the roots are submerged in a liquid medium while the leaves remain in the air. In order to ensure photosynthetic activity of the plant, a custom-made lighting system illuminates the leaves. To keep the roots in darkness the water surface is covered with sheets of black plastic foil. This method allows long-term imaging of plant organ development in standardized conditions.

This protocol shows a plant sample preparation method for light-sheet microscopy. The setup is characterized by mounting the plant vertically on the surface of a gel and letting it grow in controlled bright conditions. This allows long-term observation of plant organ development in standardized conditions.

植物根系的轻质板材荧光显微镜生长在一个凝胶表面

One of the key questions in understanding plant development is how single cells behave in a larger context of the tissue. Therefore, it requires the ...

Trans-inner Cell Mass Injection of Embryonic Stem Cells Leads to Higher Chimerism Rates

In an effort to increase efficiency in the creation of genetically modified mice via ES Cell methodologies, we present an adaptation to the current blastocyst injection protocol. Here we report that a simple rotation of the embryo, and injection through Trans-Inner cell mass (TICM) increased the percentage of chimeric mice from 31% to 50%, with no additional equipment or further specialized training. 26 different inbred clones, and 35 total clones were injected over a period of 9 months. There was no significant difference in either pregnancy rate or recovery rate of embryos between traditional injection techniques and TICM. Therefore, without any major alteration in the injection process and a simple positioning of the blastocyst and injecting through the ICM, releasing the ES cells into the blastocoel cavity can potentially improve the quantity of chimeric production and subsequent germline transmission.

Here we present a protocol to increase chimera production without the use of new equipment. A simple orientation change of the embryo for injection can increase the number of embryos produced, and potentially reduce the timeline to germline transmission.

胚胎干细胞的跨内细胞质量注射导致嵌合体率增高

In an effort to increase efficiency in the creation of genetically modified mice via ES Cell methodologies, we present an adaptation to the current ...

Primary Cell Cultures from the Mouse Retinal Pigment Epithelium

The retinal pigment epithelium (RPE) is a highly polarized multi-functional epithelium that is located between the neural retina and the choroid of the eye. It is a single sheet of pigmented cells that are hexagonally packed and connected by tight junctions. The main functions of the RPE include absorption of light, phagocytosis of the shed photoreceptor outer segments, spatial buffering of ions, transport of nutrients, ions and water as well as active involvement in the visual cycle. With such important and diverse functions, it is critically important to study the biology of RPE cells. A number of RPE cell lines have been established; however, passaged and immortalized cells are known to quickly lose some of the morphological and physiological characteristics of natural RPE cells. Thus, primary cells are more suitable for studying different aspects of RPE cell biology and function. Mouse primary RPE cell culture is very useful to researchers since mouse models are widely used in biological studies, however collecting RPE cells from mouse is also very challenging due to their small size. Here, we present a protocol for establishing primary mouse RPE cell cultures which includes enucleation and dissection of the eyes and isolation of the RPE sheets to yield the cells for culturing. This method enables efficient cell recovery. The RPE cells obtained from two mice&#160;can reach confluency on one 12 mm polyester membrane insert pre-loaded in culture plate after one week of culture and display some of the original properties of bona fide RPE cells such as hexagonal shape and pigmentation after two weeks of culture.

The retinal pigment epithelium (RPE) is a multi-functional epithelium of the eye. Here we present a protocol to establish primary cell cultures derived from the murine RPE.

小鼠视网膜色素上皮细胞的原代培养

The retinal pigment epithelium (RPE) is a highly polarized multi-functional epithelium that is located between the neural retina and the choroid of the ...