Name: cell surface marker mediated purification of ips cell intermediates from a reprogrammable mouse model
Uploaded: 2014-09-06T15:00:00.000+00:00
Duration: 10 min 32 s
Description: Watch this Scientific Journal Video about Cell Surface Marker Mediated Purification of iPS Cell Intermediates from a Reprogrammable Mouse Model at JoVE.com

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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W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surface+marker+antigens+in+the+characterization+of+human+embryonic+stem+cells.">Surface marker antigens in the characterization of human embryonic stem cells.</a> Stem Cell Research. 3, 3-11 (2009).</li><li>O'Malley, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-resolution+analysis+with+novel+cell-surface+markers+identifies+routes+to+iPS+cells.">High-resolution analysis with novel cell-surface markers identifies routes to iPS cells.</a> Nature. , (2013).</li><li>Hansson, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+coordinated+proteome+dynamics+during+reprogramming+of+somatic+cells+to+pluripotency.">Highly coordinated proteome dynamics during reprogramming of somatic cells to pluripotency.</a> Cell Reports. 2, 1579-1592 (2012).</li><li>Stadtfeld, M., Maherali, N., Breault, D. T., Hochedlinger, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defining+molecular+cornerstones+during+fibroblast+to+iPS+cell+reprogramming+in+mouse.">Defining molecular cornerstones during fibroblast to iPS cell reprogramming in mouse.</a> Cell Stem Cell. 2, 230-240 (2008).</li><li>Stadtfeld, M., Maherali, N., Borkent, M., Hochedlinger, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+reprogrammable+mouse+strain+from+gene-targeted+embryonic+stem+cells.">A reprogrammable mouse strain from gene-targeted embryonic stem cells.</a> Nature Methods. 7, 53-55 (2009).</li><li>Sommer, C. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induced+pluripotent+stem+cell+generation+using+a+single+lentiviral+stem+cell+cassette.">Induced pluripotent stem cell generation using a single lentiviral stem cell cassette.</a> Stem Cells. 27, 543-549 (2009).</li><li>Takahashi, K., Okita, K., Nakagawa, M., Yamanaka, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+pluripotent+stem+cells+from+fibroblast+cultures.">Induction of pluripotent stem cells from fibroblast cultures.</a> Nature Protocols. 2, 3081-3089 (2007).</li><li>Utikal, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immortalization+eliminates+a+roadblock+during+cellular+reprogramming+into+iPS+cells.">Immortalization eliminates a roadblock during cellular reprogramming into iPS cells.</a> Nature. 460, 1145-1148 (2009).</li><li>Stadtfeld, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ascorbic+acid+prevents+loss+of+Dlk1-Dio3+imprinting+and+facilitates+generation+of+all-iPS+cell+mice+from+terminally+differentiated+B+cells.">Ascorbic acid prevents loss of Dlk1-Dio3 imprinting and facilitates generation of all-iPS cell mice from terminally differentiated B cells.</a> Nature Genetics. 44, 398-405 (2012).</li><li>Carey, B. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reprogramming+factor+stoichiometry+influences+the+epigenetic+state+and+biological+properties+of+induced+pluripotent+stem+cells.">Reprogramming factor stoichiometry influences the epigenetic state and biological properties of induced pluripotent stem cells.</a> Cell Stem Cell. 9, 588-598 (2011).</li></ol>

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。

<table><tbody><tr><td>Anti-mouse CD90.2 (Thy1.2) Pacific Blue&amp;nbsp;</td><td>eBioscience&amp;nbsp;</td><td>48-0902-82&amp;nbsp;</td><td></td></tr><tr><td>Anti-Human/Mouse SSEA-1 Biotin&amp;nbsp;</td><td>eBioscience&amp;nbsp;</td><td>13-8813&amp;nbsp;</td><td></td></tr><tr><td>Streptavidin PE-Cy 7&amp;nbsp;</td><td>eBioscience&amp;nbsp;</td><td>25-4317-82&amp;nbsp;</td><td></td></tr><tr><td>Anti-Mouse CD326(Epcam) Fitc&amp;nbsp;</td><td>eBioscience&amp;nbsp;</td><td>48-5791-82&amp;nbsp;</td><td></td></tr><tr><td>Sodium pyruvate&amp;nbsp;</td><td>Life Technologies&amp;nbsp;</td><td>11360-070&amp;nbsp;</td><td></td></tr><tr><td>MEM Non-Essential Amino Acids (NEAA)&amp;nbsp;</td><td>Life Technologies&amp;nbsp;</td><td>11140-050&amp;nbsp;</td><td></td></tr><tr><td>Embryonic Stem Cell qualified Foetal Bovine Serum (FBS)&amp;nbsp;</td><td>Life Technologies&amp;nbsp;</td><td>10439024</td><td></td></tr><tr><td>Trypsin-EDTA (0.25%)&amp;nbsp;</td><td>Life Technologies&amp;nbsp;</td><td>25200-056&amp;nbsp;</td><td></td></tr><tr><td>GlutaMAX</td><td>Life Technologies&amp;nbsp;</td><td>35050-070&amp;nbsp;</td><td></td></tr><tr><td>&amp;beta;-Mercaptoethanol&amp;nbsp;</td><td>Life Technologies&amp;nbsp;</td><td>21985-023&amp;nbsp;</td><td></td></tr><tr><td>DMSO (Dimethyl Sulfoxide)&amp;nbsp;</td><td>Sigma-Aldrich&amp;nbsp;</td><td>D8418&amp;nbsp;</td><td></td></tr><tr><td>Penicillin/streptomycin&amp;nbsp;</td><td>Life Technologies&amp;nbsp;</td><td>15140</td><td></td></tr><tr><td>Propidium Iodide solution (PI)&amp;nbsp;</td><td>Sigma-Aldrich&amp;nbsp;</td><td>P4864-10ML&amp;nbsp;</td><td></td></tr><tr><td>Gelatine from porcine skin&amp;nbsp;</td><td>Sigma-Aldrich&amp;nbsp;</td><td>G1890&amp;nbsp;</td><td></td></tr><tr><td>Doxycyclin hyclate&amp;nbsp;</td><td>Sigma-Aldrich&amp;nbsp;</td><td>33429-100MG-R&amp;nbsp;</td><td></td></tr><tr><td>Leukemia Inhibitory Factor (LIF)&amp;nbsp;</td><td>Millipore&amp;nbsp;</td><td>ESG1107&amp;nbsp;</td><td></td></tr><tr><td>DMEM High Glucose&amp;nbsp;</td><td>Life Technologies&amp;nbsp;</td><td>11960-044&amp;nbsp;</td><td></td></tr><tr><td>DPBS (Dulbecco&#039;s phosphate buffer saline)&amp;nbsp;</td><td>Life Technologies&amp;nbsp;</td><td>14190-144&amp;nbsp;</td><td></td></tr><tr><td>KnockOut DMEM&amp;nbsp;</td><td>Life Technologies&amp;nbsp;</td><td>10829-018&amp;nbsp;</td><td></td></tr><tr><td>Irradiated MEFs&amp;nbsp;</td><td>Life Technologies&amp;nbsp;</td><td>S1520-100&amp;nbsp;</td><td></td></tr><tr><td>75-cm&lt;sup&gt;2&lt;/sup&gt; Tissue culture flasks&amp;nbsp;</td><td>Corning/BD Bioscience&amp;nbsp;</td><td>430641</td><td></td></tr><tr><td>15-ml centrifuge tubes&amp;nbsp;</td><td>Corning/BD Bioscience&amp;nbsp;</td><td>430791</td><td></td></tr><tr><td>50-ml centrifuge tubes&amp;nbsp;</td><td>Corning/BD Bioscience&amp;nbsp;</td><td>430829</td><td></td></tr><tr><td>Disposable surgical blades&amp;nbsp;</td><td>Clifford Hallam</td><td>SM0501</td><td></td></tr><tr><td>Cryogenic vials&amp;nbsp;</td><td>Corning/BD Bioscience&amp;nbsp;</td><td>430487</td><td></td></tr><tr><td>70 &amp;micro;m Cell strainer&amp;nbsp;</td><td>BD Bioscience&amp;nbsp;</td><td>352340</td><td></td></tr><tr><td>Taq DNA Polymerase&amp;nbsp;</td><td>Life Technologies&amp;nbsp;</td><td>10342-020&amp;nbsp;</td><td></td></tr></tbody></table>

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://www-jove-com.bdigitaluss.remotexs.co/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://www-jove-com.bdigitaluss.remotexs.co/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://www-jove-com.bdigitaluss.remotexs.co/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://www-jove-com.bdigitaluss.remotexs.co/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://www-jove-com.bdigitaluss.remotexs.co/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://www-jove-com.bdigitaluss.remotexs.co/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://www-jove-com.bdigitaluss.remotexs.co/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 San Sebastian

Research

JoVE Journal

Biology

12.3K 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

Generating iPS Cells from MEFS through Forced Expression of Sox-2, Oct-4, c-Myc, and Klf4

Pluripotency can be induced in differentiated murine by viral transduction of Oct4, Sox2, Klf4, and c-Myc (Takahashi and Yamanaka, 2006; Wernig, et al., 2007; Okita, et al., 2007; Maherali, et al., 2007). We have devised a reprogramming strategy in which these four transcription factors are expressed from doxycycline (dox)-inducible lentiviral vectors (Brambrink et al., 2008). Using these inducible constructs, we can derive induced pluripotent stem (iPS) cells from mouse embryonic fibroblasts (MEFs). In this video, we demonstrate the procedure for the generation of inducible lentiviruses that express the four transcription factors and show how to infect MEFs with these viruses in order to produce iPS cells. By using inducible lentiviruses, the expression of the four factors in controlled by the addition of doxycyline to the culture medium. The advantage of this system over the traditional retroviral infection is the ability to turn the genes on and off so that the kinetics of reprogramming and gene expression requirements can be analyzed in detail.

This video shows the procedure for generating induced pluripotent stem cells using inducible lentivirus that express Oct4, Sox2, c-Myc and Klf4.

从MEFS iPS细胞生成通过强制SOX - 2的表达，10月4，c - Myc基因和Klf4

Pluripotency can be induced in differentiated murine by viral transduction of Oct4, Sox2, Klf4, and c-Myc (Takahashi and Yamanaka, 2006;  Wernig, et al., ...

科研

生物学

Directed Differentiation of Induced Pluripotent Stem Cells towards T Lymphocytes

Adoptive cell transfer (ACT) of antigen-specific CD8+ cytotoxic T lymphocytes (CTLs) is a promising treatment for a variety of malignancies 1. CTLs can recognize malignant cells by interacting tumor antigens with the T cell receptors (TCR), and release cytotoxins as well as cytokines to kill malignant cells. It is known that less-differentiated and central-memory-like (termed highly reactive) CTLs are the optimal population for ACT-based immunotherapy, because these CTLs have a high proliferative potential, are less prone to apoptosis than more differentiated cells and have a higher ability to respond to homeostatic cytokines 2-7. However, due to difficulties in obtaining a high number of such CTLs from patients, there is an urgent need to find a new approach to generate highly reactive Ag-specific CTLs for successful ACT-based therapies.
TCR transduction of the self-renewable stem cells for immune reconstitution has a therapeutic potential for the treatment of diseases 8-10. However, the approach to obtain embryonic stem cells (ESCs) from patients is not feasible. Although the use of hematopoietic stem cells (HSCs) for therapeutic purposes has been widely applied in clinic 11-13, HSCs have reduced differentiation and proliferative capacities, and HSCs are difficult to expand in in vitro cell culture 14-16. Recent iPS cell technology and the development of an in vitro system for gene delivery are capable of generating iPS cells from patients without any surgical approach. In addition, like ESCs, iPS cells possess indefinite proliferative capacity in vitro, and have been shown to differentiate into hematopoietic cells. Thus, iPS cells have greater potential to be used in ACT-based immunotherapy compared to ESCs or HSCs.
Here, we present methods for the generation of T lymphocytes from iPS cells in vitro, and in vivo programming of antigen-specific CTLs from iPS cells for promoting cancer immune surveillance. Stimulation in vitro with a Notch ligand drives T cell differentiation from iPS cells, and TCR gene transduction results in iPS cells differentiating into antigen-specific T cells in vivo, which prevents tumor growth. Thus, we demonstrate antigen-specific T cell differentiation from iPS cells. Our studies provide a potentially more efficient approach for generating antigen-specific CTLs for ACT-based therapies and facilitate the development of therapeutic strategies for diseases.

Generation of T lymphocytes from induced pluripotent stem (iPS) cells gives an alternative approach of using embryonic stem cells for T cell-based immunotherapy. The method shows that by utilizing either in vitro or in vivo induction system, iPS cells are able to differentiate into both conventional and antigen-specific T lymphocytes.

定向分化诱导性多潜能干细胞向T淋巴细胞

Adoptive cell transfer (ACT) of antigen-specific CD8+  cytotoxic T lymphocytes (CTLs) is a promising treatment for a variety of  malignancies 1. CTLs can  ...

免疫与感染

Subretinal Transplantation of MACS Purified Photoreceptor Precursor Cells into the Adult Mouse Retina

Vision impairment and blindness due to the loss of the light-sensing cells of the retina, i.e. photoreceptors, represents the main reason for disability in industrialized countries. Replacement of degenerated photoreceptors by cell transplantation represents a possible treatment option in future clinical applications. Indeed, recent preclinical studies demonstrated that immature photoreceptors, isolated from the neonatal mouse retina at postnatal day 4, have the potential to integrate into the adult mouse retina following subretinal transplantation. Donor cells generated a mature photoreceptor morphology including inner and outer segments, a round cell body located at the outer nuclear layer, and synaptic terminals in close proximity to endogenous bipolar cells. Indeed, recent reports demonstrated that donor photoreceptors functionally integrate into the neural circuitry of host mice. For a future clinical application of such cell replacement approach, purified suspensions of the cells of choice have to be generated and placed at the correct position for proper integration into the eye. For the enrichment of photoreceptor precursors, sorting should be based on specific cell surface antigens to avoid genetic reporter modification of donor cells. Here we show magnetic-associated cell sorting (MACS) - enrichment of transplantable rod photoreceptor precursors isolated from the neonatal retina of photoreceptor-specific reporter mice based on the cell surface marker CD73. Incubation with anti-CD73 antibodies followed by micro-bead conjugated secondary antibodies allowed the enrichment of rod photoreceptor precursors by MACS to approximately 90%. In comparison to flow cytometry, MACS has the advantage that it can be easier applied to GMP standards and that high amounts of cells can be sorted in relative short time periods. Injection of enriched cell suspensions into the subretinal space of adult wild-type mice resulted in a 3-fold higher integration rate compared to unsorted cell suspensions.

Cell transplantation represents a strategy for the treatment of retinal degeneration characterized by photoreceptor loss. Here we describe a method for enrichment of transplantable photoreceptors and their subretinal grafting into adult mice.

MACS纯化光感受器前体细胞的亚视移植到成年小鼠视网膜中

Vision impairment and blindness due to the loss of the light-sensing cells of the retina, i.e. photoreceptors, represents the main reason for disability ...

医学

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 ...

Rapid and Refined CD11b Magnetic Isolation of Primary Microglia with Enhanced Purity and Versatility

Microglia are the primary responders to central nervous system insults; however, much remains unknown about their role in regulating neuroinflammation. Microglia are mesodermal cells that function similarly to macrophages in surveying inflammatory stress. The classical (M1-type) and alternative (M2-type) activations of macrophages have also been extended to microglia in an effort to better understand the underlying interplay these phenotypes have in neuroinflammatory conditions such as Parkinson&#39;s, Alzheimer&#39;s, and Huntington&#39;s Diseases. In vitro experimentation utilizing primary microglia offers rapid and reliable results that may be extended to the in vivo environment. Although this is a clear advantage over in vivo experimentation, isolating microglia while achieving adequate yields of optimal purity has been a challenge. Common methods currently in use either suffer from low recovery, low purity, or both. Herein, we demonstrate a refinement of the column-free CD11b magnetic separation method that achieves a high cell recovery and enhanced purity in half the amount of time. We propose this optimized method as a highly useful model of primary microglial isolation for the purposes of studying neuroinflammation and neurodegeneration.

Here, we present a protocol to isolate microglia from postnatal mouse pups (day 1) for in vitro experimentation. This improvised method of isolation generates both high yield and purity, a significant advantage over alternate methods that allows broad range experimentation for the purposes of elucidating microglial biology.

原代小胶质的快速和精细的CD11b电磁隔离带增强的纯度和多功能性

Microglia are the primary responders to central nervous system insults; however, much remains unknown about their role in regulating neuroinflammation. ...

神经科学

Selecting and Isolating Colonies of Human Induced Pluripotent Stem Cells Reprogrammed from Adult Fibroblasts

Herein we present a protocol of reprogramming human adult fibroblasts into human induced pluripotent stem cells (hiPSC) using retroviral vectors encoding Oct3/4, Sox2, Klf4 and c-myc (OSKM) in the presence of sodium butyrate 1-3. We used this method to reprogram late passage (&gt;p10) human adult fibroblasts derived from Friedreich's ataxia patient (GM03665, Coriell Repository). The reprogramming approach includes highly efficient transduction protocol using repetitive centrifugation of fibroblasts in the presence of virus-containing media. The reprogrammed hiPSC colonies were identified using live immunostaining for Tra-1-81, a surface marker of pluripotent cells, separated from non-reprogrammed fibroblasts and manually passaged 4,5. These hiPSC were then transferred to Matrigel plates and grown in feeder-free conditions, directly from the reprogramming plate. Starting from the first passage, hiPSC colonies demonstrate characteristic hES-like morphology. Using this protocol more than 70% of selected colonies can be successfully expanded and established into cell lines. The established hiPSC lines displayed characteristic pluripotency markers including surface markers TRA-1-60 and SSEA-4, as well as nuclear markers Oct3/4, Sox2 and Nanog. 
The protocol presented here has been established and tested using adult fibroblasts obtained from Friedreich's ataxia patients and control individuals 6, human newborn fibroblasts, as well as human keratinocytes.

We present a protocol for efficient reprogramming of human somatic cells into human induced pluripotent stem cells (hiPSC) using retroviral vectors encoding Oct3/4, Sox2, Klf4 and c-myc (OSKM) and identification of correctly reprogrammed hiPSC by live staining with Tra-1-81 antibody.

选择和隔离殖民地的人类诱导多能干细胞重新编程成年成纤维细胞

Herein we present a protocol of reprogramming human adult fibroblasts into human induced pluripotent stem cells (hiPSC) using retroviral vectors encoding ...

Reprogramming Human Somatic Cells into Induced Pluripotent Stem Cells (iPSCs) Using Retroviral Vector with GFP

Human embryonic stem cells (hESCs) are pluripotent and an invaluable cellular sources for in vitro disease modeling and regenerative medicine1. It has been previously shown that human somatic cells can be reprogrammed to pluripotency by ectopic expression of four transcription factors (Oct4, Sox2, Klf4 and Myc) and become induced pluripotent stem cells (iPSCs)2-4 . Like hESCs, human iPSCs are pluripotent and a potential source for autologous cells. Here we describe the protocol to reprogram human fibroblast cells with the four reprogramming factors cloned into GFP-containing retroviral backbone4. Using the following protocol, we generate human iPSCs in 3-4 weeks under human ESC culture condition. Human iPSC colonies closely resemble hESCs in morphology and display the loss of GFP fluorescence as a result of retroviral transgene silencing. iPSC colonies isolated mechanically under a fluorescence microscope behave in a similar fashion as hESCs. In these cells, we detect the expression of multiple pluripotency genes and surface markers.

Reprogramming Human Somatic Cells into Induced Pluripotent Stem Cells iPSCs Using Retroviral Vector with GFP

A method to generate human induced pluripotent stem cells (iPSCs) via retrovirus-mediated ectopic expression of OCT4, SOX2, KLF4 and MYC is described. A practical way to identify human iPSC colonies based on GFP expression is also discussed.

人类体细胞重编程为诱导多能干细胞（iPS细胞）用绿色荧光蛋白逆转录病毒载体

Human embryonic stem cells (hESCs) are pluripotent and an invaluable cellular sources for in vitro disease modeling and regenerative medicine1. It has ...

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 ...

Generation of Human Induced Pluripotent Stem Cells from Peripheral Blood Using the STEMCCA Lentiviral Vector

Through the ectopic expression of four transcription factors, Oct4, Klf4, Sox2 and cMyc, human somatic cells can be converted to a pluripotent state, generating so-called induced pluripotent stem cells (iPSCs)1-4. Patient-specific iPSCs lack the ethical concerns that surround embryonic stem cells (ESCs) and would bypass possible immune rejection. Thus, iPSCs have attracted considerable attention for disease modeling studies, the screening of pharmacological compounds, and regenerative therapies5.
We have shown the generation of transgene-free human iPSCs from patients with different lung diseases using a single excisable polycistronic lentiviral Stem Cell Cassette (STEMCCA) encoding the Yamanaka factors6. These iPSC lines were generated from skin fibroblasts, the most common cell type used for reprogramming. Normally, obtaining fibroblasts requires a skin punch biopsy followed by expansion of the cells in culture for a few passages. Importantly, a number of groups have reported the reprogramming of human peripheral blood cells into iPSCs7-9. In one study, a Tet inducible version of the STEMCCA vector was employed9, which required the blood cells to be simultaneously infected with a constitutively active lentivirus encoding the reverse tetracycline transactivator. In contrast to fibroblasts, peripheral blood cells can be collected via minimally invasive procedures, greatly reducing the discomfort and distress of the patient. A simple and effective protocol for reprogramming blood cells using a constitutive single excisable vector may accelerate the application of iPSC technology by making it accessible to a broader research community. Furthermore, reprogramming of peripheral blood cells allows for the generation of iPSCs from individuals in which skin biopsies should be avoided (i.e. aberrant scarring) or due to pre-existing disease conditions preventing access to punch biopsies.
Here we demonstrate a protocol for the generation of human iPSCs from peripheral blood mononuclear cells (PBMCs) using a single floxed-excisable lentiviral vector constitutively expressing the 4 factors. Freshly collected or thawed PBMCs are expanded for 9 days as described10,11 in medium containing ascorbic acid, SCF, IGF-1, IL-3 and EPO before being transduced with the STEMCCA lentivirus. Cells are then plated onto MEFs and ESC-like colonies can be visualized two weeks after infection. Finally, selected clones are expanded and tested for the expression of the pluripotency markers SSEA-4, Tra-1-60 and Tra-1-81. This protocol is simple, robust and highly consistent, providing a reliable methodology for the generation of human iPSCs from readily accessible 4 ml of blood.

Here we show a simple and effective protocol for the generation of human iPSCs from 3-4 ml of peripheral blood using a single lentiviral reprogramming vector. Reprogramming of readily available blood cells promises to accelerate the utilization of iPSC technology by making it accessible to a broader research community.

代人类诱导多能干细胞从外周血中使用STEMCCA慢病毒载体

Through the ectopic expression of four transcription factors, Oct4, Klf4, Sox2 and cMyc, human somatic cells can be converted to a pluripotent state, ...

Efficient Generation Human Induced Pluripotent Stem Cells from Human Somatic Cells with Sendai-virus

A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs include modeling pathogenesis of human genetic diseases, autologous cell therapy after gene correction, and personalized drug screening by providing a source of patient-specific and symptom relevant cells. However, there are several hurdles to overcome, such as eliminating the remaining reprogramming factor transgene expression after human iPSCs production. More importantly, residual transgene expression in undifferentiated human iPSCs could hamper proper differentiations and misguide the interpretation of disease-relevant in vitro phenotypes. With this reason, integration-free and/or transgene-free human iPSCs have been developed using several methods, such as adenovirus, the piggyBac system, minicircle vector, episomal vectors, direct protein delivery and synthesized mRNA. However, efficiency of reprogramming using integration-free methods is quite low in most cases.
Here, we present a method to isolate human iPSCs by using Sendai-virus (RNA virus) based reprogramming system. This reprogramming method shows consistent results and high efficiency in cost-effective manner.

Here, we present our established method to reprogram human somatic cells into transgene-free human iPSCs with Sendai virus, which shows consistent outcome and enhanced efficiency.

从人类体细胞与仙台病毒高效的新一代人类诱导多能干细胞

A few years ago, the establishment of human induced pluripotent stem cells (iPSCs) ushered in a new era in biomedicine. Potential uses of human iPSCs ...