This work was supported by the Ministry of Science and Technology of China (2015CB964902, 2013CB966902 and 2012CB966601), the National Natural Science Foundation of China (81500148, 81570164 and 81421002), the Loma Linda University School of Medicine GCAT grant (2015), and Telemedicine and Advanced Technology Research Center (W81XWH-08-1-0697).

人类所有的PB样品均从当地研究伦理委员会批准可用的识别信息，从天津血液中心的匿名捐赠者的成人获得。 1.远藤无质粒制备<ol><li>使用商业质粒纯化Maxi试剂盒根据制造商的协议从大肠杆菌中提取附加型载体。对于最后一步，用不含内毒素的无菌水代替TE缓冲以溶解DNA沉淀。 </li><li>使用商业紫外/可见光分光光度计测量DNA浓度。的浓度通常为大于1微克/微升，用A260 / A280和A260 / A230比值比分别为1.8和2.0，越大。 </li></ol> 2.文化传媒<ol><li>制备红细胞介质:造血干细胞扩增培养基补充有100纳克/毫升的人类干细胞因子（SCF），10纳克/毫升白细胞介素3（IL3），2 U / ml的促红细胞生成素（EPO），20纳克/毫升的胰岛素生长因子-1（IGF1），1μM地塞米松和0.2mM的1-硫代甘油。过滤用0.22μm注射器过滤消毒。红细胞介质可以储存在4℃最多一个月。 </li><li>制备的iPSC培养基:DMEM / F12培养基补充有1X L-谷氨酰胺，1×青霉素/链霉素，1×非必需氨基酸溶液，50纳克/毫升成纤维细胞生长因子2（FGF2（Dulbecco氏改良的Eagle培养基/营养混合物F-12） ），1×胰岛素 - 转铁蛋白 - 亚硒酸盐补充物（ITS），和50毫克/毫升抗坏血酸。过滤用0.22μm注射器过滤消毒。 IPSC的介质可以存储在4℃最多一个月。 </li><li>制备MEF培养基:Dulbecco改良的Eagle培养基（DMEM;高葡萄糖）补充有1X青霉素/链霉素和10％胎牛血清（FBS）。过滤用0.22μm注射器过滤消毒。 MEF培养基可以储存于4℃多达一个月或使用前新鲜加入1倍的L-谷氨酰胺。 </li><li> Prepare IPSC的冷冻保存介质（2×）:将5-克D-海藻糖在30毫升无菌水中在37℃的水浴中。使温度至4℃，然后加入10毫升胎牛血清和10ml二甲亚砜（DMSO）中。过滤用0.22μm注射器过滤消毒。储存在4℃长达3个月24。 </li></ol> 3.外周血单个核细胞的分离（PB跨国公司） <ol><li>结合10毫升新鲜PB样品和10毫升PBS缓冲液到50 mL管拌匀。 PBS带来到室温，或用37℃之前。 注:约1 - 3×10 6跨国公司（新鲜或冷冻）可以在1毫升的PB来获得。培养6天后，期望有0.5 - 1×10 6，由于成熟细胞的死亡总细胞培养过程中。约1×10 7个细胞可以从10毫升新鲜的PB来获得。 </li><li>使用连接到一个长针10毫升注射器加入10毫升的Ficoll到管的底部。这种亲塞斯应缓慢进行，以确保聚蔗糖层不与PB混合。 </li><li>在400×g离心30分钟，离心以低的加速和减速速率。离心后，该PB跨国公司是在位于该PB等离子体和聚蔗糖之间的白色层（血沉棕黄层）。 </li><li>慢慢吸〜10毫升的PB等离子体，而不会干扰血沉棕黄层。小心收获用1mL移液管尖，以一个新的50毫升管中的白色层。收集的细胞从白色层的总体积为3之间 - 毫升6。 </li><li>添加PBS，使总体积至30ml，并用10毫升吸管混合均匀。离心在400×g离心10分钟。 </li><li>取出20毫升培养液（ 即 Iscove改进Dulbecco培养基，IMDM）上清，悬浮细胞沉淀，拌匀。离心机在400×g离心10分钟，以除去大多数的血小板。 </li><li>去除上清，重悬细胞沉淀在1 - 2毫升IMDM。细胞可以使用˚F或直接文化，还是以备后用冷冻了下来。对于冷冻，加入冻存培养基等量。等分试样的细胞在冷冻管（0.5 - 每瓶1mL）和转让冷冻管至-80℃，立即C冰箱。 PB跨国公司可以存储在-80℃冰箱中几个星期或转移到液氮罐用于长期贮存。 注意:当解冻冷冻的细胞，虽然我们的冷冻保存介质可以维持细胞活力，细胞数目可能会由于在解冻过程减少细胞死亡。建议在1 - 10×10 7个细胞将在每个小瓶中冷冻。 </li></ol> 4.在扩张中红系PB跨国公司<ol><li>在准备15毫升管5毫升IMDM培养基。迅速解冻冷冻的PB跨国公司在37℃水浴中，然后将它们与IMDM培养基转移到管中。 注:时间在水浴的长度取决于该冷冻保存的细胞的体积和冷冻管ü上SED。通常，冷冻的细胞会在1分钟内解冻。 </li><li>离心在400×g离心5 - 10分钟。除去上清，悬浮红细胞中的介质中的细胞沉淀。加10μl台盼蓝溶液10μL细胞悬液，拌匀。台盼蓝染色1中的死细胞 - 3分钟。算使用血球在显微镜下的细胞。 </li><li>在非组织培养培养的PB跨国公司（非TC）处理的6孔平板，每孔2毫升介质，在细胞密度〜5×10 6个细胞/ mL，在37℃下在5％CO 2的培养箱。 </li><li> 1毫升新鲜红细胞直接培养基添加到每个孔中，而不为D 3和5改变介质。 </li><li>在d 6，收获的核转的PB跨国公司。 </li></ol> 5. PB MNC核转和再编程<ol><li>核转染的前一天，预涂层TC处理的6孔板有0.1％明胶在37℃下20分钟。解冻灭活的小鼠胚胎纤维原稻瘟病（MEF）饲养细胞在37℃水浴中，并立即将它们转移到含有5毫升MEF培养基的15毫升管中。 </li><li>离心在400×g离心5分钟，去除上清。从6孔板除去明胶，重悬在MEF培养基的细胞沉淀，然后将它们转移到明胶预处理6孔板中。在每孔，种子2 -悬浮在2毫升MEF培养基4×10 5灭活MEF细胞。 </li><li>培养在5％CO 2的加湿培养箱中在MEF饲养细胞在37℃。 </li><li>关于核转染的当天，吸出的MEF培养基，并用1毫升红培养基更换。在一个5％CO 2的加湿培养箱中30分钟，在37℃ -预平衡10培养板。 </li><li>添加2微克在pEV-OCT4-2A-SOX2，1微克在pEV-MYC，1微克在pEV-KLF4和0.5微克在pEV-BCL-XL在一个1.5毫升的无菌Eppendorf管中。在50℃下加热该管5分钟，以防止污染，并冷却到室温。加57μL核转缓冲区和13微升补充。 </li><li>收获2×10 6培养的PB跨国公司到5毫升管在200 xg离心离心7分钟。除去上清液和质粒和核转染缓冲混合物添加到细胞沉淀。用手指轻弹管拌匀。 注:低至2×10 5细胞可用于核转染，但较低的重编程效率应该预期的那样好。 </li><li>的DNA和细胞悬浮液转移到在试剂盒中提供的反应杯和盖的反应杯中。在核转染设备上选择程序U-008。将试管插入支架，然后按OK应用U-008计划。 </li><li>立即在各反应杯传递细胞预热1毫升红细胞介质，并以预平衡的MEF板 - 取试管进出该支座，0.5添加。由于高重编程效率和捐助者的变异，种子细胞范围的不同数量的1-10×10 5细胞p呃也强烈鼓励。 </li><li>板转移到低氧室，并且该腔室用的92％的N 2，5％CO 2和3％O 2组成的混合气体冲洗，为1 -以20升/分钟的速率2分钟。在37℃下密封所述腔室，培养的细胞后。 </li><li>在D 2后核转染，直接添加2毫升的iPSC介质到每个孔中。 </li><li>在d 4后核转，取出3毫升培养基，并加入2毫升新鲜的iPSC网上平台。为D 4后核转染，大多数活细胞将已附着于饲养层。 </li><li> 18 - ð6后核转染之后，通过使500微升废培养基并添加补充有0.25毫丁酸钠直到14天2毫升新鲜E8介质改变介质每2天。 注:其他饲养细胞可以在每当观察到饲养细胞已被分离的培养孔进行添加。解冻的MEF（参见5.1和5.2）后，重悬细胞沉淀在E8中的一个小的体积（ 即 〜0。2毫升为一个6孔板的一个孔中），然后添加的MEF到培养孔中。大约可以添加2％FBS的增加细胞附着。可替代地，MEF条件培养基也可以使用，但它是更费力。 </li></ol> 6.扩展的iPS细胞注:在大多数情况下，大量的iPSC菌落出现在D 8 - 10后核转。 14天之后，大的iPSC菌落通常都愿意采摘。 <ol><li>采摘菌落之前，添加补充有ROCK抑制剂，Y27632（10μM）500μLE8介质，在进料器或基质胶包被的24孔平板的一个孔中。用10或20微升吸管划伤，并挑选在显微镜下的殖民地。传输各菌落的件含有培养基的孔中。 注:基质胶的浓度不同于一个批次到另一个。通常我们用1:100稀释基质胶。 <ol><li>为了避免交叉污染，挑殖民地，它们大，从他人一道很好地分开。在37℃下在5％CO 2的湿润培养箱中培养的iPSC。 注意:应该注意的是，当有数十或数百个菌落在一个6孔板的每个孔中的iPSC群体可以是多克隆由于细胞迁移。 </li></ol></li><li>不要为第2天更改网上平台。 ROCK抑制剂可促进小菌落25的存活率。 </li><li>从D 2起，中每天被移除用过的培养基并在每个加入500μL新鲜E8中也发生改变。 </li><li>约1周后，当菌落足够大，取出培养基，并在37℃下处理300微升细胞剥离溶液（0.5毫摩尔EDTA的PBS）细胞1 - 3分钟。取出细胞脱离解决方案，并添加含ROCK抑制剂（10μM）暂停iPS细胞E8网上平台。不要打破殖民地成单个细胞，这可能会导致过度的细胞死亡。的细胞团块与5 - 50细胞s为适合的iPSC通道。 </li><li>转移50 - 细胞悬浮液的100％到已预涂馈线或基质胶12或6孔板的每个孔中。 </li><li>对于基质胶涂层板长期培养（见注6.1），每天换的媒介。当细胞汇合达到40 - 60％，治疗用1mL细胞剥离溶液（0.5毫摩尔EDTA的PBS）将细胞在37℃〜3分钟。当殖民地开始蜷缩，除去细胞分离液，并添加E8中含有ROCK抑制剂（10μM）暂停iPS细胞。传代细胞为4的分裂因素 - 8. ROCK抑制剂应该传代细胞时提高生存进行添加，删除和1天之后，以防止分化。 </li><li>根据制造商的协议5分钟 - 冻结向下iPSCs的，治疗与细胞剥离溶液细胞在37℃，3。当殖民地的iPSC开始蜷缩，吸出缓冲，并添加0.5 - 1毫升E8网上平台。 </li> &lt;李&gt;添加冷冻介质的细胞悬液等量拌匀。冷冻的iPSCs可以存储在-80℃冰箱数周或在长期贮存液氮中。 </li></ol> 7.选择的iPS细胞无残留附加型质粒<ol><li>第5代，收获后的iPS细胞，提取基因组DNA。 注意:通常培养5代，剩余的游离质粒是检测不到的。因此，几乎所有在高于5（ 即通道10）的通道的的iPSC集落的缺乏残余游离质粒。 </li><li>从转染的PB跨国公司使用基因组DNA作为阴性对照。添加1.6皮克在pEV-OCT4-2A-SOX2质粒到1微克阴性对照DNA模仿细胞每个细胞在pEV质粒的一个副本。 </li><li>添加9微升PCR级水，包括100纳克基因组DNA和1μL的特异性引物（各10μM的正向和反向引物）到10μL的高保真PCR主混合物。规范化上午通过DNA GAPDH的&#39;mount。使用PCR引物如下:EBNA1-F TTTAATACGATTGAGGGCGTCT，EBNA1-R GGTTTTGAAGGATGCGATTAAG，WPRE-F GGTTTAAACGCGTCGACAAT，WPRE-R GTTGCGTCAGCAAACACAGT，GAPDH-F GAGTCCACTGGCGTCTTC，GAPDH-R GACTGTGGTCATGAGTCCTTC。 </li><li>孵育在98℃下将反应混合物进行60秒;接着98℃下10秒，60℃30秒，72℃30秒。后30个循环;在72℃延伸反应5分钟。 </li><li>负载5微升PCR产物在1％琼脂糖凝胶的每一孔中。在100 30分钟五，选择与EBNA1和WPRE进一步培养和下游分析检测不到频段的iPSC克隆 - 运行20凝胶。 </li></ol> 8.流式细胞仪<ol><li>收获的iPSC通过在37℃用的Accutase处理它们为3 - 5分钟以获得单细胞悬浮液。加入1微升的PE缀合的抗TRA-1-60或eFluor 570缀合的抗SSEA4或同种型抗体到100微升细胞悬浮液（1 - 5×10 5个细胞）在5mL管中。孵育在黑暗的位置的管子在RT 20分钟。 </li><li>在RT染色20分钟后，加入2毫升的PBS和离心机在400×g离心5分钟。悬浮细胞在300微升PBS中荧光激活细胞分选（FACS）分析利用流式细胞仪细胞分析仪的流动。 23,26 </li></ol> 9.共焦成像<ol><li>种子在iPS细胞基质胶涂层玻片。 </li><li>后3 - 培养4天，取出生长培养基，并用4％多聚甲醛（PFA）在室温30分钟固定。用PBS洗涤细胞两次。 注:PFA是有毒有害的。 </li><li>治疗与在RT 0.1％的Triton X-100的PBS（PBS-T）的细胞30分钟。用PBS洗涤细胞两次。 </li><li>方框用封闭缓冲液（在PBS中的5％山羊血清，V:V）的所述细胞在RT 1小时。 </li><li>在封闭步骤，稀释第一抗体（100倍）在封闭缓冲液中。 注:该抗体信息是在材料的表列/设备。 </li><li>孵育稀释的抗体细胞在4°CO / N。 </li><li>用PBS-T用PBS洗两次15分钟，接着两次15分钟。 </li><li>孵育在室温下进行2小时的稀释的荧光团共轭次级抗体的细胞。 </li><li>用PBS-T用PBS洗涤细胞两次进行15分钟，接着两次15分钟。 </li><li>染色在RT用DAPI（1微克/毫升）的PBS细胞核10分钟。 </li><li>用PBS洗细胞两次15分钟。 </li><li>捕捉图像用共聚焦显微镜。 23,27 </li></ol> 10.畸胎瘤分析收获1×10 6的iPSC与细胞分离溶液（0.5毫摩尔EDTA在PBS中），并悬浮细胞在200μL的DMEM / F12稀释（1:1）的基质胶。 <ol><li>注入皮下细胞到NOD / SCID免疫缺陷小鼠的后臀尖。 </li><li>在8 - 的iPSC注射后12周时，解剖和修复10％福尔马林畸胎瘤。 28 </li><li>经过显微切片和行HE（H＆E）染色，分析样本。 29 </li></ol>

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The authors have no competing or conflicting interests to disclose.

健康人或病人采集血样方便，无创，使之成为基础研究和临床细胞治疗一个有吸引力的细胞来源。在这里，我们描述了高效代从外周血免费集成为iPS细胞的协议。这种可重复和经济实惠的方法应该受益的iPSC领域。 我们已经报道有负责高效PB重新编程30两个关键因素。一个是OCT4的表达等摩尔和Sox2用2A连接器31。为了实现这一目标，在pEV-OCT4-2A-SOX2在该协议中使用?...

几个转录因子的强制表达后 （ 即 OCT4，SOX2，MYC和KLF4），体细胞可以被重新编程诱导多能干细胞（iPS细胞），其保持在再生医学和细胞替代疗法1-3应用大有希望。迄今为止，多种方法已被开发，以提高重编程4-7的成功率。病毒载体诱导重新编程被广泛地用于有效地产生iPSCs的，因为病毒整合导致高电平时，重新编程因子的稳定表达。然而，该载体DNA导入细胞基因组的永久整合可能诱发插入诱变5。此外，重新编程因子失活不足可能扰乱的iPSC分化8。这样，不重编程因子整合使用的iPSC的是必要的，尤其是对在细胞治疗应用中使用。 9的质粒。的oriP元件促进在哺乳动物细胞中的质粒复制，而EBNA1元件系绳含oriP-质粒DNA的染色体DNA，其允许所述附加体的宿主细胞的分裂过程中的分区。相较于其他自由整合的办法，包括仙台病毒（SV）和RNA转染，电动汽车具备多种优势5,6,10。作为质粒DNA，电动车可以很容易地生产并在内部修改，使它们非常实惠。此外，电动车进行重新编程是较少的劳动密集的过程，因为与电动汽车的单个转染足以的iPSC产生，而几的RNA转染所必需的成功重新编程。 ðermal成纤维细胞已在许多重编程研究中使用。然而，皮肤活检不仅是一种侵入性和痛苦的过程，而且还费时扩大细胞足够的数量进行重新编程。更令人关注的，成年供体的皮肤细胞经常被暴露于长期紫外光辐射，这可能会导致与肿瘤有关，从而限制了从皮肤成纤维细胞11,12衍生的iPSC应用突变。最近，已经报道，人正常皮肤细胞积累的体细胞突变与多种癌症的基因，包括大多数皮肤鳞状细胞癌的主要驱动力，是在强正选择13。 在对比皮肤成纤维细胞，外周血（PB）的细胞是细胞重新编程的一个优选来源，因为1）血细胞可以通过微创方法来容易地得到，2）的外周血细胞是造血干细胞的后代居住在骨髓，从而防止有害的辐射。外周血单核细胞（PB跨国公司）可以在从血沉棕黄层层以下用Ficoll-Hypaque上（1.077克/ mL）的一个简单的梯度离心一小时进行收集。得到的PB跨国公司是由淋巴细胞，单核细胞和少数造血祖细胞（高性能计算机）14。虽然人类T淋巴细胞是在PB中的主要细胞类型中的一种，成熟T细胞中含有的T细胞受体（TCR）基因重排和缺一个完整的基因组中从而限制其应用程序15,16的潜力。然而，通过iPSC的生成T细胞的复兴可能在嵌合抗原受体（CAR）T细胞疗法17-19潜在的应用。相比较而言，高性能计算机有一个完整的基因组，并很容易重新编程的。虽然只有0.01 - 0.1％，外周循环细胞是高性能计算机，这些细胞可被体外扩增在红细胞中有利于erythr的增殖OID祖细胞14。 在我们以前的研究中，我们所用的因子的BCL-XL在除山因子（OCT4，SOX2，MYC和KLF4），这导致在PB中重编程efficency 20 10倍的增加。 BCL-X L，也称为BCL2L1，是细胞死亡的有效抑制剂，通过抑制胱天蛋白酶21,22的激活。但是，BCL-X L也可以在维持多潜能21,22发挥着重要作用。最近，我们已进一步通过分别表达MYC和KLF4与两个向量，这导致在重新编程的效率23的大致100倍增加优化了EV重新编程系统。使用该方法，重编程效率，由通过在转染起始细胞数除以细胞集落数来定义，是0.2 - 来自健康供体的0.5％。如下，我们描述了从PB生成自由整合，iPSCs的详细的实验步骤。

<table><tbody><tr><td>Hematopoietic Stem Cell Expansion Medium</td><td>Sigma</td><td>S0192</td><td>Store at 4 &amp;deg;C</td></tr><tr><td>Human Stem Cell Factor (SCF)</td><td>Peprotech</td><td>300-07</td><td>Store at -20 or -80 &amp;deg;C</td></tr><tr><td>Interleukin-3 (IL-3)</td><td>Peprotech</td><td>AF-200-03</td><td>Store at -20 or -80 &amp;deg;C</td></tr><tr><td>Erythropoietin (EPO)</td><td>Peprotech</td><td>100-64</td><td>Store at -20 or -80 &amp;deg;C</td></tr><tr><td>Insulin Growth Factor-1 (IGF-1)</td><td>Peprotech</td><td>100-11</td><td>Store at -20 or -80 &amp;deg;C</td></tr><tr><td>Dexamethasone</td><td>Sigma</td><td>D4902</td><td>Store at -20 or -80 &amp;deg;C</td></tr><tr><td>1-thioglycerol (MTG)</td><td>Sigma</td><td>M6145</td><td>Store at -20 or -80 &amp;deg;C</td></tr><tr><td>DMEM/F12 medium</td><td>Gibco</td><td>112660-012</td><td>Store at 4 &amp;deg;C</td></tr><tr><td>L-glutamine (100x)</td><td>Gibco</td><td>25030-081</td><td>Store at -20 &amp;deg;C</td></tr><tr><td>Penicillin/Streptomycin (100x)</td><td>Gibco</td><td>15140-122</td><td>Store at -20 &amp;deg;C</td></tr><tr><td>Non-essential Amino Acids solution (100x)</td><td>Gibco</td><td>11140-050</td><td>Store at 4 &amp;deg;C</td></tr><tr><td>Fibroblast Growth Factor 2 (FGF2)</td><td>Peprotech</td><td>100-18B</td><td>Store at -20 or -80 &amp;deg;C</td></tr><tr><td>Insulin-Transferrin-Selenium (ITS, 100x)</td><td>Gibco</td><td>41400-045</td><td>Store at 4 &amp;deg;C</td></tr><tr><td>Ascorbic acid</td><td>Sigma</td><td>49752</td><td>Store at -20 &amp;deg;C</td></tr><tr><td>DMEM (high glucose) medium</td><td>Thermo</td><td>SH30243.01B</td><td>Store at 4 &amp;deg;C</td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Hyclone</td><td>SV30087.01</td><td>Store at -20 &amp;deg;C</td></tr><tr><td>Ficoll</td><td>GE Healthcare, SIGMA</td><td>17-5442-02</td><td>Store at RT</td></tr><tr><td>Trehalose</td><td>Sigma</td><td>T9531</td><td>Store at 4 &amp;deg;C</td></tr><tr><td>Dimethylsulfoxide (DMSO)</td><td>Sigma</td><td>D2650</td><td>Store at RT, protect from light</td></tr><tr><td>Endofree Plasmid Maxi Kit (10)</td><td>Qiagen</td><td>12362</td><td>Store at RT</td></tr><tr><td>IMDM</td><td>Gibco</td><td>21056-023</td><td>Store at 4 &amp;deg;C</td></tr><tr><td>Human CD34+ Cell Nucleofection Kit</td><td>Lonza</td><td>VPA-1003</td><td>Store at RT, nucleofection buffer and supplement should be stored at 4 &amp;deg;C</td></tr><tr><td>Sodium Butyrate</td><td>Sigma</td><td>B5887</td><td>Store at -20 or -80 &amp;deg;C</td></tr><tr><td>ROCK inhibitor - Y27632</td><td>STEMGENT</td><td>04-0012-10</td><td>Store at -20 &amp;deg;C</td></tr><tr><td>Essential 8 basal medium (E8)</td><td>Gibco</td><td>A15169-01</td><td>Store at 4 &amp;deg;C, the supplement should be stored at -20 or -80 &amp;deg;C</td></tr><tr><td>Matrigel</td><td>BD</td><td>354277</td><td>Store at -20 or -80 &amp;deg;C</td></tr><tr><td>2x EasyTaq PCR SuperMix (+dye)</td><td>TransGen Biotech</td><td>AS111</td><td>Store at -20 &amp;deg;C</td></tr><tr><td>Cell detachment solution</td><td>STEMGENT</td><td>01-0006</td><td>Store at -20 &amp;deg;C, Accutase as a cell detachment solution to obtain a single-cell suspension</td></tr><tr><td>DAPI</td><td>Sigma</td><td>D9542-1MG</td><td>Store at 4 or -20 &amp;deg;C</td></tr><tr><td>Anti-Nanog AF488</td><td>BD</td><td>560791</td><td>Store at 4 &amp;deg;C, primary antibody used for Immunofluorescence, dilute 1/100 when used</td></tr><tr><td>Anti-OCT4</td><td>abcam</td><td>ab19857</td><td>Store at 4 &amp;deg;C, primary antibody used for Immunofluorescence, dilute 1/100 when used</td></tr><tr><td>AF488 donkey anti-mouse IgG</td><td>Invitrogen</td><td>A21202</td><td>Store at 4 &amp;deg;C, secondary antibody used for Immunofluorescence,&amp;nbsp; dilute 1/500 when used</td></tr><tr><td>PE anti-human TRA-1-60-R antibody</td><td>Biolegend</td><td>330610</td><td>Store at 4 &amp;deg;C, antibody used for flow cytometry</td></tr><tr><td>eFluor 570-conjugated anti-SSEA4</td><td>eBioscience</td><td>41-8843</td><td>Store at 4 &amp;deg;C, antibody used for flow cytometry</td></tr><tr><td>Isotype antibody</td><td>eBioscience</td><td>11-4011</td><td>Store at 4 &amp;deg;C, antibody used for flow cytometry</td></tr><tr><td>Alkaline Phosphatase Detection Kit</td><td>SiDanSai</td><td>1102-100</td><td>Store at 4 &amp;deg;C</td></tr><tr><td>Genomic DNA Extraction Kit</td><td>TIANGEN</td><td>DP304-02</td><td>Store at RT</td></tr><tr><td>Trypan Blue solution</td><td>Sigma</td><td>T8154</td><td>Store at RT</td></tr><tr><td>Flow cytometry cell analyzer</td><td>BD</td><td>LSRII</td><td></td></tr><tr><td>Spinning Disk Confocal microscope (SDC)</td><td>PerkinElmer</td><td>UltraVIEW VOX</td><td>for confocal imaging</td></tr><tr><td>Nucleofection device</td><td>Lonza</td><td>Nucleofector 2b</td><td>for the nucleofection of PB MNC</td></tr><tr><td>ImageQuant LAS-4010</td><td>GE</td><td></td><td>to take photos of AP staining in bulk</td></tr></tbody></table>

generation of integration-free induced pluripotent stem cells from human peripheral blood mononuclear cells using episomal vectors

诱导多能干细胞（iPS细胞）持有疾病建模和再生疗法大有希望。我们以前报道的使用游离型载体（EV）来产生从外周血单核细胞（PB跨国公司）自由集成-iPS细胞。所使用的附加型载体是与来自爱泼斯坦-巴尔（EB）病毒oriP和EBNA1元件，其分别允许在哺乳动物细胞中质粒的复制和长期retainment，掺入的DNA的质粒。随着进一步的优化，数以千计的iPSC集落可以在1毫升的外周血中获得。为实现高的重编程效率的两个关键因素是:1）利用一个2A的"自切割"肽连结OCT4和SOX2，从而实现这两个因素的等摩尔的表达; 2）使用两个向量来表达MYC和KLF4个别。在这里，我们描述了一个一步一步的协议，用于生成免费的集成-IP成人外周血标本旺。生成的iPS细胞是整合无残留游离质粒后5代检测不到。虽然重编程效率比得上仙台病毒（SV）矢量，EV质粒是比市售的SV矢量相当更经济。这个经济实惠的EV重新编程系统适用于在再生医学临床应用潜力，并为PB跨国公司的自由集成-间质干细胞，神经干细胞等的直接重编程的方法。

使用此协议中，我们可以从1×10 5 nucleofected PB跨国公司获得数百菌落（ 图1A和1B）。重编程效率为约0.2 - 0.5％和菌落表达多潜能标志物（ 图1C和1D）。使用所描述的协议产生的iPSC是积分-自由和具有形成畸胎瘤构成3胚层（ 图1E和1F）的能力。 <img...

Watch this Scientific Journal Video about Generation of Integration-free Induced Pluripotent Stem Cells from Human Peripheral Blood Mononuclear Cells Using Episomal Vectors at JoVE.com

Generation of Integration-free Induced Pluripotent Stem Cells from Human Peripheral Blood Mononuclear Cells Using Episomal Vectors

This protocol describes a detailed method for efficient generation of integration-free iPSCs from human adult peripheral blood cells. With the use of four oriP/EBNA-based episomal vectors to express the reprogramming factors, KLF4, MYC, BCL-XL, or OCT4 and SOX2, thousands of iPSC colonies can be obtained from 1 mL of peripheral blood.

从人体外周血单个核细胞融合，无诱导多能干细胞的生成方法游离型载体

Induced Pluripotent Stem Cells (iPSCs) hold great promise for disease modeling and regenerative therapies. We previously reported the use of Episomal ...

generation-integration-free-induced-pluripotent-stem-cells-from-human

Research

JoVE Journal

Developmental Biology

10.6K 次观看.  Chinese Academy of Medical Sciences and Peking Union Medical College. This protocol describes a detailed method for efficient generation of integration-free iPSCs from human adult peripheral blood cells. With the use of four oriP/EBNA-based episomal vectors to express the reprogramming factors, KLF4, MYC, BCL-XL, or OCT4 and SOX2, thousands of iPSC colonies can be obtained from 1 mL of peripheral blood. 

视频: Generation of Integration-free Induced Pluripotent Stem Cells from Human Peripheral Blood Mononuclear Cells Using Episomal Vectors

Direct Induction of Human Neural Stem Cells from Peripheral Blood Hematopoietic Progenitor Cells

Human disease specific neuronal cultures are essential for generating in vitro models for human neurological diseases. However, the lack of access to primary human adult neural cultures raises unique challenges. Recent developments in induced pluripotent stem cells (iPSC) provides an alternative approach to derive neural cultures from skin fibroblasts through patient specific iPSC, but this process is labor intensive, requires special expertise and large amounts of resources, and can take several months. This prevents the wide application of this technology to the study of neurological diseases. To overcome some of these issues, we have developed a method to derive neural stem cells directly from human adult peripheral blood, bypassing the iPSC derivation process. Hematopoietic progenitor cells enriched from human adult peripheral blood were cultured in vitro and transfected with Sendai virus vectors containing transcriptional factors Sox2, Oct3/4, Klf4, and c-Myc. The transfection results in morphological changes in the cells which are further selected by using human neural progenitor medium containing basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF). The resulting cells are characterized by the expression for neural stem cell markers, such as nestin and SOX2. These neural stem cells could be further differentiated to neurons, astroglia and oligodendrocytes in specified differentiation media. Using easily accessible human peripheral blood samples, this method could be used to derive neural stem cells for further differentiation to neural cells for in vitro modeling of neurological disorders and may advance studies related to the pathogenesis and treatment of those diseases.

A method was developed to directly derive human neural stem cells from hematopoietic progenitor cells enriched from peripheral blood cells.

人类神经干细胞从外周血造血祖细胞直接诱导

Human disease specific neuronal cultures are essential for generating in vitro models for human neurological diseases. However, the lack of access to ...

科研

发育生物学

Generation of Induced Pluripotent Stem Cells from Frozen Buffy Coats using Non-integrating Episomal Plasmids

Somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs) by forcing the expression of four transcription factors (Oct-4, Sox-2, Klf-4, and c-Myc), typically expressed by human embryonic stem cells (hESCs). Due to their similarity with hESCs, iPSCs have become an important tool for potential patient-specific regenerative medicine, avoiding ethical issues associated with hESCs. In order to obtain cells suitable for clinical application, transgene-free iPSCs need to be generated to avoid transgene reactivation, altered gene expression and misguided differentiation. Moreover, a highly efficient and inexpensive reprogramming method is necessary to derive sufficient iPSCs for therapeutic purposes. Given this need, an efficient non-integrating episomal plasmid approach is the preferable choice for iPSC derivation. Currently the most common cell type used for reprogramming purposes are fibroblasts, the isolation of which requires tissue biopsy, an invasive surgical procedure for the patient. Therefore, human peripheral blood represents the most accessible and least invasive tissue for iPSC generation.
In this study, a cost-effective and viral-free protocol using non-integrating episomal plasmids is reported for the generation of iPSCs from human peripheral blood mononuclear cells (PBMNCs) obtained from frozen buffy coats after whole blood centrifugation and without density gradient separation.

Induced pluripotent stem cells (iPSCs) represent a source of patient-specific tissues for clinical applications and basic research. Here, we present a detailed protocol to reprogram human peripheral blood mononuclear cells (PBMNCs) obtained from frozen buffy coats into viral-free iPSCs using non-integrating episomal plasmids.

使用非整合附加型质粒从冷冻血沉棕黄层诱导多能干细胞的产生

Somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs) by forcing the expression of four transcription factors (Oct-4, Sox-2, ...

Direct Induction of Hemogenic Endothelium and Blood by Overexpression of Transcription Factors in Human Pluripotent Stem Cells

During development, hematopoietic cells arise from a specialized subset of endothelial cells, hemogenic endothelium (HE). Modeling HE development in vitro is essential for mechanistic studies of the endothelial-hematopoietic transition and hematopoietic specification. Here, we describe a method for the efficient induction of HE from human pluripotent stem cells (hPSCs) by way of overexpression of different sets of transcription factors. The combination of ETV2 and GATA1 or GATA2 TFs is used to induce HE with pan-myeloid potential, while a combination of GATA2 and TAL1 transcription factors allows for the production of HE with erythroid and megakaryocytic potential. The addition of LMO2 to GATA2 and TAL1 combination substantially accelerates differentiation and increases erythroid and megakaryocytic cells production. This method provides an efficient and rapid means of HE induction from hPSCs and allows for the observation of the endothelial-hematopoietic transition in a culture dish. The protocol includes hPSCs transduction procedures and post-transduction analysis of HE and blood progenitors.

This protocol describes the efficient induction of hemogenic endothelium and multipotential hematopoietic progenitors from human pluripotent stem cells via the forced expression of transcription factors.

Hemogenic内皮细胞直接诱导和血液中转录因子在人多能干细胞表达

During development, hematopoietic cells arise from a specialized subset of endothelial cells, hemogenic endothelium (HE). Modeling HE development in vitro ...

Generation of Integration-free Human Induced Pluripotent Stem Cells Using Hair-derived Keratinocytes

Recent advances in reprogramming allow us to turn somatic cells into human induced pluripotent stem cells (hiPSCs). Disease modeling using patient-specific hiPSCs allows the study of the underlying mechanism for pathogenesis, also providing a platform for the development of in vitro drug screening and gene therapy to improve treatment options. The promising potential of hiPSCs for regenerative medicine is also evident from the increasing number of publications (>7000) on iPSCs in recent years. Various cell types from distinct lineages have been successfully used for hiPSC generation, including skin fibroblasts, hematopoietic cells and epidermal keratinocytes. While skin biopsies and blood collection are routinely performed in many labs as a source of somatic cells for the generation of hiPSCs, the collection and subsequent derivation of hair keratinocytes are less commonly used. Hair-derived keratinocytes represent a non-invasive approach to obtain cell samples from patients. Here we outline a simple non-invasive method for the derivation of keratinocytes from plucked hair. We also provide instructions for maintenance of keratinocytes and subsequent reprogramming to generate integration-free hiPSC using episomal vectors.

This manuscript provides a step-by-step procedure for the derivation and maintenance of human keratinocytes from plucked hair and subsequent generation of integration-free human induced pluripotent stem cells (hiPSCs) by episomal vectors.

集成的无人类诱导多能干细胞的生成方法头发衍生角质形成细胞

Recent advances in reprogramming allow us to turn somatic cells into human induced pluripotent stem cells (hiPSCs). Disease modeling using ...

Generation of Induced Pluripotent Stem Cells from Human Peripheral T Cells Using Sendai Virus in Feeder-free Conditions

Recently, iPSCs have attracted attention as a new source of cells for regenerative therapies. Although the initial method for generating iPSCs relied on dermal fibroblasts obtained by invasive biopsy and retroviral genomic insertion of transgenes, there have been many efforts to avoid these disadvantages. Human peripheral T cells are a unique cell source for generating iPSCs. iPSCs derived from T cells contain rearrangements of the T cell receptor (TCR) genes and are a source of antigen-specific T cells. Additionally, T cell receptor rearrangement in the genome has the potential to label individual cell lines and distinguish between transplanted and donor cells. For safe clinical application of iPSCs, it is important to minimize the risk of exposing newly generated iPSCs to harmful agents. Although fetal bovine serum and feeder cells have been essential for pluripotent stem cell culture, it is preferable to remove them from the culture system to reduce the risk of unpredictable pathogenicity. To address this, we have established a protocol for generating iPSCs from human peripheral T cells using Sendai virus to reduce the risk of exposing iPSCs to undefined pathogens. Although handling Sendai virus requires equipment with the appropriate biosafety level, Sendai virus infects activated T cells without genome insertion, yet with high efficiency. In this protocol, we demonstrate the generation of iPSCs from human peripheral T cells in feeder-free conditions using a combination of activated T cell culture and Sendai virus.

This protocol describes how to generate induced pluripotent stem cells (iPSCs) from human peripheral T cells in feeder-free conditions using a combination of matrigel and Sendai virus vectors containing reprogramming factors.

从人外周T细胞无饲养条件下，使用仙台病毒诱导多能干细胞的生成

Recently, iPSCs have attracted attention as a new source of cells for regenerative therapies. Although the initial method for generating iPSCs relied on ...

Induced Pluripotent Stem Cell Generation from Blood Cells Using Sendai Virus and Centrifugation

The recent development of human induced pluripotent stem cells (hiPSCs) proved that mature somatic cells can return to an undifferentiated, pluripotent state. Now, reprogramming is done with various types of adult somatic cells: keratinocytes, urine cells, fibroblasts, etc. Early experiments were usually done with dermal fibroblasts. However, this required an invasive surgical procedure to obtain fibroblasts from the patients. Therefore, suspension cells, such as blood and urine cells, were considered ideal for reprogramming because of the convenience of obtaining the primary cells. Here, we report an efficient protocol for iPSC generation from peripheral blood mononuclear cells (PBMCs). By plating the transduced PBMCs serially to a new, matrix-coated plate using centrifugation, this protocol can easily provide iPSC colonies. This method is also applicable to umbilical cord blood mononuclear cells (CBMCs). This study presents a simple and efficient protocol for the reprogramming of PBMCs and CBMCs.

We propose a protocol for reprogramming peripheral blood mononuclear cells (PBMCs) into induced pluripotent stem cells (iPSCs). By plating the transduced blood cells onto matrix-coated plates with centrifugation, iPSCs are successfully induced from floating cells. This technique suggests a simple and effective reprogramming protocol for cells such as PBMCs and CBMCs.

从血细胞使用仙台病毒和离心诱导多能干细胞代

The recent development of human induced pluripotent stem cells (hiPSCs) proved that mature somatic cells can return to an undifferentiated, pluripotent ...

Integration Free Derivation of Human Induced Pluripotent Stem Cells Using Laminin 521 Matrix

Xeno-free and fully defined conditions are key parameters for robust and reproducible generation of homogenous human induced pluripotent stem (hiPS) cells. Maintenance of hiPS cells on feeder cells or undefined matrices are susceptible to batch variances, pathogenic contamination and risk of immunogenicity. Utilizing the defined recombinant human laminin 521 (LN-521) matrix in combination with xeno-free and defined media formulations reduces variability and allows for the consistent generation of hiPS cells. The Sendai virus (SeV) vector is a non-integrating RNA-based system, thus circumventing concerns associated with the potential disruptive effect on genome integrity integrating vectors can have. Furthermore, these vectors have demonstrated relatively high efficiency in the reprogramming of dermal fibroblasts. In addition, enzymatic single cell passaging of cells facilitates homogeneous maintenance of hiPS cells without substantial prior experience of stem cell culture. Here we describe a protocol that has been extensively tested and developed with a focus on reproducibility and ease of use, providing a robust and practical way to generate defined and xeno-free human hiPS cells from fibroblasts.

Robust derivation of human induced pluripotent stem (hiPS) cells was achieved by using non-integrating Sendai virus (SeV) vector mediated reprogramming of dermal fibroblasts. hiPS cell maintenance and clonal expansion was performed using xeno-free and chemically defined culture conditions with recombinant human laminin 521 (LN-521) matrix and Essential E8 (E8) Medium.

使用层粘连蛋白521矩阵整合人诱导多能干细胞的自由衍生

Xeno-free and fully defined conditions are key parameters for robust and reproducible generation of homogenous human induced pluripotent stem (hiPS) ...

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, ...

生物学

GM-Free Generation of Blood-Derived Neuronal Cells

Many human&#160;neurological disorders are caused by degeneration of neurons and glial cells in the brain. Due to limitations in pharmacological and other therapeutic strategies, there is currently no cure available for the injured or diseased brain. Cell replacement appears as a promising therapeutic strategy for neurodegenerative conditions. To this day, neural stem cells (NSCs) have been successfully generated from fetal tissues, human embryonic cells (ES) or induced pluripotent stem cells (iPSC). A process of dedifferentiation was initiated by activation of the novel human GPI-linked glycoprotein, which leads to generation of pluripotent stem cells. These blood-derived pluripotent stem cells (BD-PSCs) differentiate in vitro into cells with a neural phenotype as shown by brightfield and immunofluorescence microscopy. Ultrastructural analysis of these cells by means of electron microscopy confirms their primitive structure as well as neuronal-like morphology and subcellular characteristics.

We present a genetically modified-free (GM-free) method to obtain cells with a neuronal phenotype from reprogrammed peripheral blood cells. Activation of a signaling pathway linked to novel human GPI-linked protein reveals an efficient GM-free method for obtaining human pluripotent stem cells.

无转基因生成血液衍生神经元细胞

Many human&#160;neurological disorders are caused by degeneration of neurons and glial cells in the brain. Due to limitations in pharmacological and other ...

神经科学

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 ...