The study was supported by the Ministry of Health and Department of Educational Assistance, University and Research of the Autonomous Province of Bolzano and the South Tyrolean Sparkasse Foundation.

外周血单个核细胞（外周血单个核细胞）后，从签署知情同意书和南蒂罗尔省的伦理委员会批准献血者健康人外周血标本中分离。实验是按照在表达赫尔辛基宣言的原则进行。所有的数据收集和分析了匿名。 1.分离外周血单个核细胞（外周血单个核细胞） <ol><li>从全血中离心后的血沉棕黄层获得外周血单个核细胞，没有密度梯度分离。 <ol><li>收集8毫升静脉外周血成柠檬酸钠缓冲的塑料管，并储存在室温（25℃）。在采集后12小时过程血。 </li><li>离心管中，在2000×g离心15分钟，在4℃下，在吊桶式转子，开关关闭离心机刹车。 </li><li>收集阴天棕黄层（上层血浆相和下之间的层相含有大部分的红细胞），将含有富集的PBMNC级分（500微升）到离心管筒。 </li><li>悬浮500μl的棕黄层用500μl含有90％FBS和20％DMSO的2×冷冻介质中，在为了得到1毫升，用10％的DMSO浓度的总体积。 </li><li>冻结在受控速率冷冻容器小瓶在-80℃下。商店细胞在液氮中更长时间。 </li></ol></li><li>密度梯度分离后获得外周血单个核细胞<ol><li>收集12ml含静脉外周血成柠檬酸钠缓冲的塑料管，并储存在室温。在采集后12小时过程血。 </li><li>稀释血液用无菌PBS以35 ml的终体积。 </li><li>小心缓慢，层35毫升稀释血液15毫升多聚蔗糖溶液（用于密度梯度分离）（p值= 1.077）在50ml锥形管（比率3:1）。 </li><li>离心管中，在800×g离心在室温30分钟在吊桶式转子，开关关闭离心机刹车。 </li><li>吸上，含血浆黄相并丢弃。仔细，含有外周血单个核细胞的不透明白色相间层转移到新的50ml锥形管中。 </li><li>将总体积至30ml用无菌PBS和离心机在300×g离心在室温10分钟。 </li><li>弃上清，重悬细胞用30ml PBS中。计数活细胞的数目，根据台盼蓝排除25。 </li><li>离心机外周血单个核细胞在300×g离心10分钟，在室温，吸出上清液，并丢弃它。 </li><li>重悬细胞沉淀在含有90％FBS和10％DMSO的冷冻介质的一个合适的量，以获得5×10 6个细胞/ ml的浓度。 </li><li>等分试样1毫升每小瓶（约5×10 6个细胞/ ml）并冷冻小瓶在受控速率冷冻集装箱- 80℃。商店细胞在液氮中更长时间。 </li></o升&gt; </li></ol> 2.解冻和外周血单个核细胞的电镀（0天） <ol><li>从全血中离心后的血沉棕黄层获得外周血单个核细胞，没有密度梯度分离。 <ol><li>开始使用从冷冻血沉棕黄层外周血单个核细胞的协议，解冻1小瓶细胞在水浴中在37℃和稀释的血沉棕黄层用无菌PBS至2ml的最终体积。 </li><li>传送稀释的血沉棕黄层到50毫升锥形管，加入10毫升红细胞裂解缓冲液，孵育在室温10分钟。 </li><li>而制成500毫升红细胞裂解缓冲液，加入0.5克碳酸氢钾，4.145克氯化铵，100微升的0.5M EDTA溶液至500ml的超纯水，pH值7.2-7.4。 </li><li>将总体积至50ml用无菌PBS和离心机在300×g离心在室温10分钟。 </li><li>弃去上清液，并用50ml无菌PBS洗涤沉淀，离心血沉棕黄层，在300×g离心在室温10分钟。 </li><li>悬浮细胞中1毫升PBMNC介质，如前所述22，组成：IMDM和火腿的F-12（比例为1：1），胰岛素-转铁-硒-乙醇胺（ITS-X），经化学为1％的1％定义脂质浓缩（CDL），1％青霉素/链霉素，L-抗坏血酸酸0.005％，牛血清白蛋白（BSA）的0.5％，1-硫代甘油（终浓度都是200μM），干细胞因子SCF（100毫微克/毫升），白细胞介素-3，IL-3（10毫微克/毫升），促红细胞生成素的EPO（2单位/毫升），胰岛素样生长因子IGF-1（40毫微克/毫升），地塞米松（1μM）和全息转铁蛋白（100μg/ ml的）。 </li><li>他们转移到一个12孔板用标准组织培养处理过的表面的一个孔，无涂层，以2×10 6个细胞/ ml的密度。孵育细胞在37℃，5％CO 2的加湿培养箱中48小时，直到变换媒体的步骤（见第3节）。 </li></ol></li><li>密度梯度分离后获得外周血单个核细胞<ol><li>要使用冷冻的外周血单个核细胞后启动协议密度梯度分离，解冻1小瓶细胞在水浴中在37℃下，转移细胞到5毫升PBMNC培养基。离心在300×g离心在室温10分钟。 </li><li>重悬细胞于1ml PBMNC培养基中，并将它们转移到一个12孔板的一个孔，以2×10 6个细胞/ ml的密度。孵育细胞在37℃，5％CO 2的加湿培养箱中48小时，直到变换媒体的步骤（见第3节）。 </li></ol></li></ol> 3.文化与扩张外周血单个核细胞的（2-13天） <ol><li>收集外周血单个核细胞在悬浮培养中，用移液管以1毫升尖，在15ml锥形管中并离心，在300×g离心在室温10分钟。 </li><li>弃去上清液并将细胞重新悬浮在1ml新鲜的外周血单个核细胞培养基。 </li><li>转移细胞进入1井的12孔板用标准组织培养处理过的表面，没有涂布，并孵育它们在37℃，5％CO 2的加湿培养箱。 </li><li&gt;重复这些步骤，每两天，和分裂细胞的比例1：当他们到达汇合适当（80％左右）2。 </li></ol> 4.转染外周血单个核细胞与附加型质粒（第14天） 注意：执行四个商业intergation无游离型质粒的分离，使用商业试剂盒进行质粒纯化携带的多能性基因和评估使用琼脂糖凝胶电泳分析纯化的质粒的质量，根据制造商的说明。 <ol><li>收集在15ml锥形管中外周血单个核细胞并计数活细胞的数量25（台盼蓝排斥的基础上）。 </li><li>离心机2×10 6个细胞，在300×g离心在室温10分钟。 </li><li>重悬2×10 6个细胞在含有100微升的再悬浮缓冲液T和各1μg质粒DNA转染混合物（一个质粒携带OCT3 / 4和shRNA抗p53，一个质粒携带SOX2和KLF4，人们质粒携带的L- MYC和LIN28和一种质粒携带绿色荧光蛋白，以检查转染效率）。 </li><li>吸含有细胞的转染混合物加入100微升尖端和插入与样品垂直插入管中，填充用3ml电解缓冲液的E2，置于电穿孔机的吸移管站的移液管，按制造商的说明。 </li><li>使用以下程序有效电穿孔细胞：1650伏，10毫秒，3个脉冲。 </li><li>重悬电穿孔的细胞（2×10 6个细胞）于2ml预热PBMNC培养基中，加入0.25毫丁酸钠（NAB）和计数存活细胞的电穿孔规程后的数字25（台盼蓝排斥的基础上）。 </li><li>转移细胞到6孔板的一个孔中无涂层，轻轻摇动平板孵育所述细胞在37℃，5％CO 2的加湿培养箱。 </li><li>电穿孔，COUN后的第二天吨GFP阳性细胞的荧光显微镜下从8-10随机字段的数量，以评估转染效率。 </li><li>维持转染的细胞不分裂为3天，直到镀它们的MEF（参见第6节）。 </li><li>代替加入2ml新鲜外周血单个核细胞培养基，再加0.25毫酸钠每两天。 </li></ol> 5.准备菜肴馈线细胞共培养（16天） <ol><li>外套一个6孔板用1ml基底膜基质的15分钟的，在37℃和板小鼠胚胎成纤维细胞（MEFs）以2×10 5个细胞的密度/孔用2毫升10％FBS的含有孔DMEM（MEF中）1天传代电外周血单个核细胞到MEF饲养细胞之前。 </li></ol> 6.镀染外周血单个核细胞上的MEF（17-19天） <ol><li>收集悬浮培养外周血单个核细胞，用移液管与1ml尖，在15ml锥形管中，并离心转染外周血单个核细胞在300×g离心10分钟在RT。 </li><li>弃上清，重悬沉淀在2ml新鲜PBMNC介质，加上0.25mM的丁酸钠。 </li><li>板上的细胞上的MEF涂覆的6孔板孵育所述细胞在37℃，5％CO 2的加湿培养箱。 </li><li>两天后，吸出培养基并用2ml的iPSC介质的敲除DMEM（KO-DMEM），20％KO-血清替代（KOSR），1mM的NEAAs，1％青霉素/链霉素，20mM的组成取代的L-谷氨酰胺，0.1mM的β巯基乙醇，10纳克/毫升的FGF（基本的bFGF）和0.25毫酸钠。 </li><li>更换每天上午2毫升新鲜的iPSC中，每天检查的细胞。 注：在大约22-25天，iPS细胞的第一殖民地应该是可见的。 </li></ol> 7.采摘和扩张诱导多能干细胞的（iPS细胞）克隆（30-35天） 注：在转染后约30-35天，菌落的iPSC应准备好采摘和replating。重编程效率应ESTI配合作为的iPSC集落/电穿孔的细胞的总数量的数量的百分比，如先前报道26。 <ol><li>传代的iPSC集落的前一天，制备MEF饲养在12孔板（1.25×10 5个细胞/孔）。 </li><li>吸的MEF培养基，改变用1mL的iPSC培养基用10ng / ml的bFGF和10μM的Y-27632。手动用针一个菌落，以获得栅格解剖在采摘机罩的解剖显微镜下，每次，通过移液收集较小的块和每个单独的转移成的12孔板涂覆的MEF一个阱。 </li><li>通道和扩展每个12孔板到6孔板中的比率为1：2，然后每6孔板中的比率1：进一步传播4。解剖和手动展开的iPSC用于第一2-3代（如在步骤7.2中全面描述），然后用一种酶分解过程（从步骤7.4启动）。 </li><li>对于酶分解协议，干净的iPSC科洛尼ES通过吸差异部分，在采摘机罩的解剖显微镜下。 </li><li>孵育的iPSC用1mlⅣ型胶原酶（1毫克/毫升）10分钟，在37℃。 </li><li>吸酶溶液冲洗细胞用1ml KO-DMEM中并收集菌落在15ml锥形管中。离心，在100×g离心在室温2分钟。 </li><li>吸出上清，重悬在2ml的iPSC介质的菌落用10ng / ml的bFGF和10μM的Y-27632和板他们在MEF涂覆的6孔板（比率1:4）。 </li></ol> iPS细胞的表征8.（5-15之间代） <ol><li>碱性磷酸酶染色<ol><li>培养中的iPSC介质3-5天的的iPSC集落用10ng / ml的bFGF。 </li><li>用于开始碱性磷酸酶染色方案，冲洗的iPSC一次用PBS，然后修复它们用1ml 4％PFA的20分钟，室温。 </li><li>用PBS洗细胞三次，以去除残留的PFA。 </li><li>使用染色固定的细胞根据制造商的说明商用碱性磷酸酶染色试剂盒。 </li></ol></li><li>免疫荧光<ol><li>培养中的iPSC介质3-5天的的iPSC集落用10ng / ml的bFGF。 </li><li>启动免疫荧光测定法中，用PBS洗涤一次的iPSC，然后修复它们用1ml 4％PFA的20分钟，在室温。 </li><li>用PBS洗细胞三次，以去除残留的PFA。 </li><li>对待固定的iPSC用1ml透化/阻挡含有4％山羊血清，0.1％BSA，0.1％的Triton X-100和0.05％叠氮化钠溶液（NaN 3的）处理1小时，在室温。 </li><li>吸出封闭溶液并孵育固定的细胞用3％BSA的第一抗体和0.05％NaN 3的溶液O / N在4℃（检查一次抗体列表的材料表和建议的抗体稀释因子）。 </li><li>第二天，洗涤的iPSC三次，用PBS，然后孵育细胞的Alexa Fluor 488山羊抗小鼠和Alexa FLUOR 555山羊抗兔抗体，稀释1：1000中的3％BSA和0.05％NaN 3的溶液，1小时，在37℃。 </li><li>染色用DAPI（1微克/毫升）的原子核在室温10分钟在黑暗中，接着是三个时间用PBS清洗。 </li></ol></li><li>在胚体iPS细胞分化（EBS） <ol><li>培养中的iPSC介质5-6天的iPSC集落用10ng / ml的bFGF。 </li><li>通过抽吸去除的iPSC菌落差异的部分，在采摘机罩的解剖显微镜下。 </li><li>孵育的iPSC用1mlⅣ型胶原酶（1毫克/毫升）10分钟，在37℃。 </li><li>吸酶溶液冲洗细胞用1ml KO-DMEM中并收集菌落在15ml锥形管中。离心，在100×g离心在室温2分钟。 </li><li>吸出上清，重悬菌落在2ml KO-DMEM中组成的EB培养基中，20％的定义胎牛血清（FBS），1毫NEAAs，1％青霉素/链霉素，20mM的L-谷氨酰胺，0.1毫46;巯基乙醇。 </li><li>板中的悬浮液中6天的iPSC集落到超低附件6孔板中，为了使球状三维胚体（EB）的形成。变化2毫升新鲜EB培养基每两天。 </li><li>在第7天，收集EB中和板他们到0.1％明胶包被的6孔板的2ml的EB培养基中，并在分化维持胚20天。 </li><li>变化2毫升新鲜EB培养基每两天。 </li></ol></li><li>定量RT-PCR基因表达分析<ol><li>培养中的iPSC介质5-6天的iPSC集落用10ng / ml的bFGF。 </li><li>提取外周血单个核细胞，iPS细胞或胚使用按照制造商的说明1毫升商业试剂总RNA。 </li><li>评估RNA的浓度和纯度通过合适的方法，如使用分光光度计（高品质的RNA用A 260 / A 280和A 260 / A比值230&gt; 1.8）27。 </li><li>检查使用根据制造商的协议（高品质的RNA RQI&gt; 9.5）28商业试剂盒的RNA完整性。 </li><li>执行使用根据制造商的说明商用逆转录试剂盒1微克总RNA进行逆转录反应。 </li><li>制备含5毫微克cDNA模板，7.5微升SYBR Green超，2微升5μM引物的qPCR的混合物（前锋：1微升和反向：1微升）和6微升RNA酶/ DNA酶的水每个反应29。 </li><li>使用以下程序进行的qRT-PCR：起始变性步骤，在95℃进行10分钟，然后是40个循环分成一个变性步骤在95℃下15秒，引物退火和延伸步骤在60℃进行45秒29。 </li><li>正常化使用GAPDH作为管家基因28的其它基因的表达（ 见表1中所列的引物）。 </li></ol></li><li>定量PCR对于转基因排除<ol><li>使用将1ml含有50mM Tris，100毫摩尔EDTA，100毫摩尔NaCl，裂解缓冲液外周血单个核细胞或iPS细胞提取的DNA的1％十二烷基硫酸钠（SDS）和20微克/ ml的蛋白酶K. </li><li>加100％的异丙醇等量的（1毫升）中，以允许沉淀DNA，离心在10,000rpm×g离心5分钟，在室温颠倒混合三次。 </li><li>弃去上清液，用1毫升70％乙醇和离心机以10,000×g离心洗DNA沉淀进行5分钟，在室温。吸弃上清，重悬它核糖核酸酶/ DNA酶的水。 </li><li>评估DNA浓度和纯度通过合适的方法，如使用分光光度计（高品质的RNA用A 260 / A 280和A 260 / A比值230&gt; 1.8）27。 </li><li>制备含5毫微克的DNA模板，7.5微升SYBR Green超，2微升5μM引物的qPCR的混合物（前锋：1微升和反向：1微升）和6＃181;微升RNA酶/ DNA酶的水用于每个反应29。 </li><li>正常化具体游离质粒EBNA-1基因的使用FBX-15作为持家基因29（参照表1中所列的引物）的表达。 </li></ol></li><li>核型分析<ol><li>文化的iPSC菌落5-6天到无饲养基底膜基质涂板定义的商业，无饲养维修中的iPS细胞，按照生产商的说明。 </li><li>要启动的核型分析协议，分离的iPSC集落用1ml细胞脱附溶液中5分钟，在37℃，直到获得单细胞解离。 </li><li>收集细胞，离心，在400×g离心5分钟，在RT。 </li><li>重悬的iPSC在2ml培养基中专门为进行产前诊断技术培养细胞的发展。板单细胞到iPSCs的基底膜基质镀 ​​膜玻璃的菜肴，并培育他们在37℃，5％CO 2 &lt;/ SUB&gt;孵化器。 </li><li>后2-4天，直接添加10微升/毫升秋水仙碱到培养并在培养箱孵育3小时，在37℃，5％的CO 2。 </li><li>吸出培养基并孵育的iPSC在1ml的低渗溶液（0.6％的柠檬酸钠和0.13％氯化钾），用于在室温下10分钟。 </li><li>冲洗细胞用1ml 5％乙酸溶液和修复的iPSC用甲醇/乙酸溶液（比率3:1）中的机器具有受控温度和湿度（28℃，42％RH）。 </li><li>浸泡和染色固定的细胞15-20分钟在黑暗阿的溶液在室温（获得Q-带）30。 </li><li>在获得放大100倍29在荧光显微镜下的细胞分裂中期。 </li></ol></li></ol>

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The authors have nothing to disclose.

在过去，只有这样才能获得携带特定遗传变异的人类多能干细胞是招募家长进行胚胎植入前遗传学诊断，并产生从他们丢弃的囊胚31,32胚胎干细胞。使用方法重新编程，研究人员现在可以生成携带几乎所有的基因型患者iPS细胞。的可能性，以从患者的特定细胞系就易于访问的源是非常重要的，因为它允许病症的病理生理学和随后鉴定的新的治疗方法的研究。 在本研究?...

在2006年，该基山中伸弥1展示首次从成年小鼠和人类体细胞可以通过四种重编程因子（Oct-4的，SOX-2，KLF-4，和异位表达被转换成多能状态c-Myc的），产生了所谓的诱导多能干细胞（iPS细胞）2。患者特异性的iPSC中的形态，增殖和分化成三胚细胞类型（中胚层，内胚层和外胚层）的能力方面非常类似于人类胚胎干细胞（胚胎干细胞），而缺乏相关的使用人类胚胎干细胞和旁路的伦理问题可能的免疫排斥反应3。因此，iPS细胞表现为患者特异性的细胞为基础研究，药物筛选，疾病建模，毒性的评价，和再生医学的目的4的最重要来源之一。 几种方法已用于iPSC的生成：病毒整合载体（逆转录病毒5，慢病毒6），病毒非整合载体（腺病毒7），仙台病毒8，BAC座子9，附加型载体10，蛋白质11或RNA递送12。虽然使用病毒介导的方法可导致高效率的重编程，病毒载体整合到宿主细胞，因此潜在随机插入 ​​突变的基因组中，基因表达，和沉默的转基因的再活化的分化过程中的永久改变，不能排除13。 使iPSCs的安全再生医学，已经作出努力来推导的iPSC未经外源DNA整合到细胞基因组中。虽然应纳税病毒载体和转座子被开发出来，它仍然是不清楚短载体序列，这不可避免地留在切除后的转导的细胞，和转座表达，是否可以诱导改变在细胞ular功能13。尽管其高重编程效率，仙台病毒代表一种昂贵的方法，并达到通授权的关注与开发的这个系统必须限制其应用在翻译研究的潜力公司。此外，需要对直接引入蛋白质和RNA的需要多个递送重新编程分子的固有技术局限性此介绍的，和总重编程效率是非常低的14。值得注意的是，具有成本效益的基础上，利用附加型质粒的病毒-自由和非整合的方法已被成功地报道了皮肤成纤维细胞15的再编程。具体而言，在本工作中，我们决定使用市售集成无附加型质粒，如先前报道10,15。 迄今为止，皮肤成纤维细胞代表了最流行 ​​施主小区类型5。然而，其他细胞来源已经更迭sfully重编程为iPS细胞包括角质形成细胞16，骨髓间充质干细胞17，脂肪基质细胞18，毛囊19与牙髓细胞20。这些细胞的分离，需要外科手术，并以建立原代细胞培养，需要在体外细胞扩增几个星期。 有鉴于此，起始细胞类型的选择是至关重要的，但同样重要的是能够从方便和较少侵入性的组织，例如血液产生的iPSC。两个脐带血单核细胞（CBMNCs）21,22和外周血单核细胞（外周血单个核细胞）14,22-24表示细胞为iPS细胞的推导合适来源。 虽然成人PBMNC重编程的效率比CBMNCs 22的低20-50倍，它们仍然是最方便的细胞类型为取样目的。在事实上，PBMNC取样有被微创的优点，此外，这些细胞不需要重新编程实验之前在体外广泛扩展。迄今为止，不同的协议已经报道，密度梯度分离后外周血单个核细胞可冷冻后冷冻和解冻天至数月并重新编程成iPS细胞22,23之前膨胀为几天。然而，就我们所知没有报道描述从冷冻的血沉棕黄层外周血单个核细胞的重编程。重要的是，没有密度梯度分离收集冷冻棕黄色大衣代表存储在从人口研究大型生物库，从而代表材料的生产的iPSC以避免进一步的样品采集的方便池中最常见的血液样本。 此处我们报告首次病毒无iPSCs的产生来自人类冷冻血沉棕黄层，根据先前描述的方案22。在此外，从密度梯度分离之后得到的冷冻外周血单个核细胞生成的iPSCs，对于非密度梯度纯化PBMNC结果的控制协议。

<table border="0" cellpadding="0" cellspacing="0">
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			<td>Iscove&#39;s modified Dulbecco&#39;s medium (IMDM)&#160;</td>
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			<td>21056-023</td>
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			<td>PeproTech</td>
			<td>200-03</td>
			<td>10 ng/ml (Stock: 10 &#181;g/ml)</td>
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			<td>Recombinant Human Insulin-like Growth Factor (IGF-1)</td>
			<td>PeproTech</td>
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			<td>25030-024</td>
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			<td>MEM Non-Essential Amino Acids Solution (100X)</td>
			<td>Gibco</td>
			<td>11140-050</td>
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			<td>2-Mercaptoethanol&#160;</td>
			<td>Gibco</td>
			<td>31350-010</td>
			<td>0.1 mM (Stock: 50 mM)</td>
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			<td>Sigma-Aldrich</td>
			<td>B5887</td>
			<td>0.25 mM (Stock: 0.5 M)</td>
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			<td>Recombinant Human FGF basic, 145 aa&#160;</td>
			<td>R&#38;D Systems&#160;</td>
			<td>4114-TC</td>
			<td>10 ng/ml (Stock: 10 &#181;g/ml)</td>
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			<td>Y-27632 dihydrochloride</td>
			<td>Sigma-Aldrich</td>
			<td>Y0503&#160;</td>
			<td>10 &#181;M (Stock: 10 mM)</td>
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			<td>Fetal Defined Bovine Serum</td>
			<td>Hyclone</td>
			<td>SH 30070.03</td>
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			<td>EmbryoMax 0.1% Gelatin Solution</td>
			<td>Merck-Millipore</td>
			<td>ES-006-B</td>
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			<td>Matrigel Basement Membrane Matrix Growth Factor Reduced</td>
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			<td>Collagenase, Type IV</td>
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			<td>Accutase</td>
			<td>PAA Laboratories GmbH</td>
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			<td>Plasmid pCXLE-hOCT3/4-shp53-F</td>
			<td>Addgene&#160;</td>
			<td>27077</td>
			<td>1 &#181;g (Stock: 1 &#181;g/&#181;l)</td>
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			<td>Addgene&#160;</td>
			<td>27078</td>
			<td>1 &#181;g (Stock: 1 &#181;g/&#181;l)</td>
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			<td>Plasmid pCXLE-hUL</td>
			<td>Addgene&#160;</td>
			<td>27080</td>
			<td>1 &#181;g (Stock: 1 &#181;g/&#181;l)</td>
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		<tr>
			<td>Plasmid pCXLE-EGFP</td>
			<td>Addgene&#160;</td>
			<td>27082</td>
			<td>1 &#181;g (Stock: 1 &#181;g/&#181;l)</td>
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			<td>Alkaline Phosphatase Staining Kit</td>
			<td>Stemgent</td>
			<td>00-0009</td>
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		<tr>
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			<td>Merck-Millipore</td>
			<td>MAB4360</td>
			<td>1/250</td>
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		<tr>
			<td>Anti-TRA-1-81 Antibody</td>
			<td>Merck-Millipore</td>
			<td>MAB4381</td>
			<td>1/250</td>
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			<td>Anti-Oct-3/4 Antibody</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-9081</td>
			<td>1/500</td>
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		<tr>
			<td>Anti-Nanog Antibody</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-33759</td>
			<td>1/500</td>
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		<tr>
			<td>Anti-Troponin I Antibody</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-15368</td>
			<td>1/500</td>
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		<tr>
			<td>Anti-&#945;-Actinin (Sarcomeric) Antibody</td>
			<td>Sigma-Aldrich</td>
			<td>A7732</td>
			<td>1/250</td>
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		<tr>
			<td>Neuronal Class III &#223;-Tubulin (TUJ1) Antibody</td>
			<td>Covance Research Products Inc&#160;</td>
			<td>MMS-435P-100</td>
			<td>1/500</td>
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		<tr>
			<td>Anti-Tyrosine Hydroxylase (TH) Antibody</td>
			<td>Calbiochem</td>
			<td>657012</td>
			<td>1/200</td>
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		<tr>
			<td>Alexa Fluor 488 Goat Anti-Mouse&#160;</td>
			<td>Molecular Probes</td>
			<td>A-11029</td>
			<td>1/1000</td>
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		<tr>
			<td>Alexa Fluor 555 Goat Anti-Rabbit</td>
			<td>Molecular Probes</td>
			<td>A-21429</td>
			<td>1/1000</td>
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		<tr>
			<td>Ultra-Low Attachment Cell Culture 6-well plate</td>
			<td>Corning</td>
			<td>3471</td>
			<td></td>
		</tr>
		<tr>
			<td>Trizol Reagent</td>
			<td>Ambion</td>
			<td>15596-018&#160;</td>
			<td>reagent for RNA extraction</td>
		</tr>
		<tr>
			<td>SuperScript VILO cDNA Synthesis Kit</td>
			<td>Invitrogen</td>
			<td>11754050</td>
			<td>Reverse transcriptase kit</td>
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		<tr>
			<td>iTaq Universal SYBR Green Supermix</td>
			<td>Bio-Rad</td>
			<td>172-5124</td>
			<td></td>
		</tr>
		<tr>
			<td>CFX96 Real-Time PCR Detection System</td>
			<td>Bio-Rad</td>
			<td>185-5195</td>
			<td></td>
		</tr>
		<tr>
			<td>Experion Automated Electrophoresis System</td>
			<td>Bio-Rad</td>
			<td>700-7000</td>
			<td>Instrument to check RNA integrity</td>
		</tr>
		<tr>
			<td>Experion RNA Highsense Analysis kit</td>
			<td>Bio-Rad</td>
			<td>7007105</td>
			<td>Reagent kit to check RNA integrity</td>
		</tr>
		<tr>
			<td>Dissecting microscope (SteREO Discovery V12 )</td>
			<td>Zeiss</td>
			<td>495007</td>
			<td></td>
		</tr>
		<tr>
			<td>NeonTransfection System 100 &#181;L Kit</td>
			<td>Invitrogen</td>
			<td>MPK10025</td>
			<td>Reagent kit for electroporation</td>
		</tr>
		<tr>
			<td>Neon Transfection System</td>
			<td>Invitrogen</td>
			<td>MPK5000</td>
			<td>Instrument used for electroporation</td>
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		<tr>
			<td>NanoDrop UV/Vis Spectrophotometer</td>
			<td>Thermo Scientific</td>
			<td>ND-2000</td>
			<td>Instrument for DNA/RNA quantification</td>
		</tr>
		<tr>
			<td>EndoFree Plasmid Maxi Kit</td>
			<td>Qiagen</td>
			<td>12362</td>
			<td>Plasmid purification kit</td>
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</table>

generation of induced pluripotent stem cells from frozen buffy coats using non-integrating episomal plasmids

体细胞可以通过迫使四个转录因子（Oct-4的，SOX-2，KLF-4，和c-Myc）的表达重编程为诱导性多能干细胞（iPS细胞），通常由人类胚胎干细胞中表达（胚胎干细胞） 。由于它们的相似性与人类胚胎干细胞，iPS细胞已成为潜在的患者特异性再生医学的重要工具，避免了与人类胚胎干细胞相关的伦理问题。为了获得适合于临床应用的细胞，转基因的无iPSCs的需要产生，以避免转基因激活，改变的基因表达和误导分化。此外，高效率的和廉价的重编程方法是必要的，以获得足够的iPSC用于治疗目的。鉴于这种需求，一个高效的非整合附加型质粒的方法是对的iPSC衍生的优选的选择。隔离，其中当前用于重编程的目的是最常见的细胞类型是成纤维细胞，需要组织活检，一个我nvasive外科手术的病人。因此，人外周血代表的iPSC一代最容易和最不侵入性组织。 在这项研究中，使用非整合附加型质粒具有成本效益和病毒 - 自由协议报从后全血离心分离，没有密度梯度分离从冷冻血沉棕黄层获得的人外周血单核细胞（外周血单个核细胞）的iPSCs的产生。

在这项研究中的简单而有效的协议，用于病毒无iPSCs的由外周血单个核细胞从全血中离心分离和密度梯度分离报道后获得外周血单个核细胞的重新编程，比较后冷冻的血沉棕黄层中分离的重编程的产生。 图1A示出的示意图详细的协议。解冻后，分离的外周血单个核细胞，显示出典型的圆形形状，在特定的血液培养基中14天（ 图1B）展开，然后转染了附加型质粒。经过10-15天...

Watch this Scientific Journal Video about Generation of Induced Pluripotent Stem Cells from Frozen Buffy Coats using Non-integrating Episomal Plasmids at JoVE.com

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

诱导多能干细胞（iPS细胞）代表的患者特异性组织为临床应用和基础研究的来源。在这里，我们提出了一个详细的协议来重新编程从冷冻棕黄色大衣将获得使用非整合游离病毒质粒无iPSCs的人外周血单个核细胞（外周血单个核细胞）。

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, ...

generation-induced-pluripotent-stem-cells-from-frozen-buffy-coats

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JoVE Journal

Developmental Biology

12.8K 次观看.  European Academy Bozen/Bolzano (EURAC). 诱导多能干细胞（iPS细胞）代表的患者特异性组织为临床应用和基础研究的来源。在这里，我们提出了一个详细的协议来重新编程从冷冻棕黄色大衣将获得使用非整合游离病毒质粒无iPSCs的人外周血单个核细胞（外周血单个核细胞）。 

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

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 ...

科研

发育生物学

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 ...

Generation of Induced-pluripotent Stem Cells Using Fibroblast-like Synoviocytes Isolated from Joints of Rheumatoid Arthritis Patients

Mature somatic cells can be reversed into a pluripotent stem cell-like state using a defined set of reprogramming factors. Numerous studies have generated induced-Pluripotent Stem Cells (iPSCs) from various somatic cell types by transducing four Yamanaka transcription factors: Oct4, Sox2, Klf4 and c-Myc. The study of iPSCs remains at the cutting edge of biological and clinical research. In particular, patient-specific iPSCs can be used as a pioneering tool for the study of disease pathobiology, since iPSCs can be induced from the tissue of any individual. Rheumatoid arthritis (RA) is a chronic inflammatory disease, classified by the destruction of cartilage and bone structure in the joint. Synovial hyperplasia is one of the major reasons or symptoms that lead to these results in RA. Fibroblast-like Synoviocytes (FLSs) are the main component cells in the hyperplastic synovium. FLSs in the joint limitlessly proliferate, eventually invading the adjacent cartilage and bone. Currently, the hyperplastic synovium can be removed only by a surgical procedure. The removed synovium is used for RA research as a material that reflects the inflammatory condition of the joint. As a major player in the pathogenesis of RA, FLSs can be used as a material to generate and investigate the iPSCs of RA patients. In this study, we used the FLSs of a RA patient to generate iPSCs. Using a lentiviral system, we discovered that FLSs can generate RA patient-specific iPSC. The iPSCs generated from FLSs can be further used as a tool to study the pathophysiology of RA in the future.

Here we describe a protocol for generating human induced-pluripotent stem cells from patient-derived fibroblast-like synoviocytes, using a lentiviral system without feeder cells.

诱导多能干细胞使用成纤维样滑膜生成类风湿关节炎患者的关节分离

Mature somatic cells can be reversed into a pluripotent stem cell-like state using a defined set of reprogramming factors. Numerous studies have generated ...

Generation of Induced Pluripotent Stem Cells from Human Melanoma Tumor-infiltrating Lymphocytes

Adoptive transfer of ex vivo expanded autologous tumor-infiltrating lymphocytes (TILs) can mediate durable and complete responses in significant subsets of patients with metastatic melanoma. Major obstacles of this approach are the reduced viability of transferred T cells, caused by telomere shortening, and the limited number of TILs obtained from patients. Less-differentiated T cells with long telomeres would be an ideal T cell subset for adoptive T cell therapy;however, generating large numbers of these less-differentiated T cells is problematic. This limitation of adoptive T cell therapy can be theoretically overcome by using induced pluripotent stem cells (iPSCs) that self-renew, maintain pluripotency, have elongated telomeres, and provide an unlimited source of autologous T cells for immunotherapy. Here, we present a protocol to generate iPSCs using Sendai virus vectors for the transduction of reprogramming factors into TILs. This protocol generates fully reprogrammed, vector-free clones. These TIL-derived iPSCs might be able to generate less-differentiated patient- and tumor-specific T cells for adoptive T cell therapy.

The goal of this protocol is to show the protocol for reprogramming melanoma tumor-infiltrating lymphocytes into induced pluripotent stem cells.

从人黑色素瘤肿瘤浸润淋巴细胞诱导多能干细胞的产生

Adoptive transfer of ex vivo expanded autologous tumor-infiltrating lymphocytes (TILs) can mediate durable and complete responses in significant subsets ...

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 ...

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

Induced Pluripotent Stem Cells (iPSCs) hold great promise for disease modeling and regenerative therapies. We previously reported the use of Episomal Vectors (EV) to generate integration-free iPSCs from peripheral blood mononuclear cells (PB MNCs). The episomal vectors used are DNA plasmids incorporated with oriP and EBNA1 elements from the Epstein-Barr (EB) virus, which&#160;allow for replication and long-term retainment of plasmids in mammalian cells, respectively. With further optimization, thousands of iPSC colonies can be obtained from 1 mL of peripheral blood. Two critical factors for achieving high reprogramming efficiencies&#160;are: 1) the use of a 2A &#34;self-cleavage&#34; peptide to link OCT4 and SOX2, thus achieving equimolar expression of the two factors; 2) the use of two vectors to express MYC and KLF4 individually. Here we describe a step-by-step protocol for generating integration-free iPSCs from adult peripheral blood samples. The generated iPSCs are integration-free as residual episomal plasmids are undetectable after five passages. Although the reprogramming efficiency is comparable to that of Sendai Virus (SV) vectors, EV plasmids are considerably more economical than the commercially available SV vectors. This affordable EV reprogramming system holds potential for clinical applications in regenerative medicine and provides an approach for the direct reprogramming of PB MNCs to integration-free mesenchymal stem cells, neural stem cells, etc.

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 ...

Defined and Scalable Generation of Hepatocyte-like Cells from Human Pluripotent Stem Cells

Human pluripotent stem cells (hPSCs) possess great value for biomedical research. hPSCs can be scaled and differentiated to all cell types found in the human body. The differentiation of hPSCs to human hepatocyte-like cells (HLCs) has been extensively studied, and efficient differentiation protocols have been established. The combination of extracellular matrix and biological stimuli, including growth factors, cytokines, and small molecules, have made it possible to generate HLCs that resemble primary human hepatocytes. However, the majority of procedures still employ undefined components, giving rise to batch-to-batch variation. This serves as a significant barrier to the application of the technology. To tackle this issue, we developed a defined system for hepatocyte differentiation using human recombinant laminins as extracellular matrices in combination with a serum-free differentiation process. Highly efficient hepatocyte specification was achieved, with demonstrated improvements in both HLC function and phenotype. Importantly, this system is easy to scale up using research and GMP-grade hPSC lines&#160;promising advances in cell-based modelling and therapies.

The method presented here describes a scalable and good manufacturing practice (GMP)-ready differentiation system to generate human hepatocyte-like cells from pluripotent stem cells. It serves as a cost-effective and standardized system to generate human hepatocyte-like cells for basic and applied human liver research.

界定和由人多能干细胞的肝细胞样细胞的可扩展代

Human pluripotent stem cells (hPSCs) possess great value for biomedical research. hPSCs can be scaled and differentiated to all cell types found in the ...

Generation of Standardized and Reproducible Forebrain-type Cerebral Organoids from Human Induced Pluripotent Stem Cells

The human cortex is highly expanded and exhibits a complex structure with specific functional areas, providing higher brain function, such as cognition. Efforts to study human cerebral cortex development have been limited by the availability of model systems. Translating results from rodent studies to the human system is restricted by species differences and studies on human primary tissues are hampered by a lack of tissue availability as well as ethical concerns. Recent development in human pluripotent stem cell (PSC) technology include the generation of three-dimensional (3D) self-organizing organotypic culture systems, which mimic to a certain extent human-specific brain development in vitro. Currently, various protocols are available for the generation of either whole brain or brain-region specific organoids. The method for the generation of homogeneous and reproducible forebrain-type organoids from induced PSC (iPSC), which we previously established and describe here, combines the intrinsic ability of PSC to self-organize with guided differentiation towards the anterior neuroectodermal lineage and matrix embedding to support the formation of a continuous neuroepithelium. More specifically, this protocol involves: (1) the generation of iPSC aggregates, including the conversion of iPSC colonies to a confluent monolayer culture; (2) the induction of anterior neuroectoderm; (3) the embedding of neuroectodermal aggregates in a matrix scaffold; (4) the generation of forebrain-type organoids from neuroectodermal aggregates; and (5) the fixation and validation of forebrain-type organoids. As such, this protocol provides an easily applicable system for the generation of standardized and reproducible iPSC-derived cortical tissue structures in vitro.

Cerebral organoids represent a new model system to investigate early human brain development in vitro. This article provides the detailed methodology to efficiently generate homogeneous dorsal forebrain-type organoids from human induced pluripotent stem cells including critical characterization and validation steps.

人诱导多能干细胞的前脑型脑 Organoids 标准化和可再生性的生成

The human cortex is highly expanded and exhibits a complex structure with specific functional areas, providing higher brain function, such as cognition. ...

Generation of 3D Skin Organoid from Cord Blood-derived Induced Pluripotent Stem Cells

The skin is the body&#8217;s largest organ and has many functions. The skin acts as a physical barrier and protector of the body and regulates bodily functions. Biomimetics is the imitation of the models, systems, and elements of nature for the purpose of solving complex human problems1. Skin biomimetics is a useful tool for in vitro disease research and in vivo regenerative medicine. Human induced pluripotent stem cells (iPSCs) have the characteristic of unlimited proliferation and the ability of differentiation to three germ layers. Human iPSCs are generated from various primary cells, such as blood cells, keratinocytes, fibroblasts, and more. Among them, cord blood mononuclear cells (CBMCs) have emerged as an alternative cell source from the perspective of allogeneic regenerative medicine. CBMCs are useful in regenerative medicine because human leukocyte antigen (HLA) typing is essential to the cell banking system. We provide a method for the differentiation of CBMC-iPSCs into keratinocytes and fibroblasts and for generation of a 3D skin organoid. CBMC-iPSC-derived keratinocytes and fibroblasts have characteristics similar to a primary cell line. The 3D skin organoids are generated by overlaying an epidermal layer onto a dermal layer. By transplanting this 3D skin organoid, a humanized mice model is generated. This study shows that a 3D human iPSC-derived skin organoid may be a novel, alternative tool for dermatologic research in vitro and in vivo.

We propose a protocol that shows how to differentiate induced pluripotent stem cell-derived keratinocytes and fibroblasts and generate a 3D skin organoid, using these keratinocytes and fibroblasts. This protocol contains an additional step of generating a humanized mice model. The technique presented here will improve dermatologic research.

从脐带血源性多能干细胞中提取三维皮肤有机细胞

The skin is the body&#8217;s largest organ and has many functions. The skin acts as a physical barrier and protector of the body and regulates bodily ...

In Vitro Generation of Somite Derivatives from Human Induced Pluripotent Stem Cells

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give rise to multiple cell types, including the myotome (MYO), sclerotome (SCL), dermatome (D), and syndetome (SYN), which in turn develop into skeletal muscle, axial skeleton, dorsal dermis, and axial tendon/ligament, respectively. Therefore, the generation of SMs and their derivatives from human induced pluripotent stem cells (iPSCs) is critical to obtain pluripotent stem cells (PSCs) for application in regenerative medicine and for disease research in the field of orthopedic surgery. Although the induction protocols for MYO and SCL from PSCs have been previously reported by several researchers, no study has yet demonstrated the induction of SYN and D from iPSCs. Therefore, efficient induction of fully competent SMs remains a major challenge. Here, we recapitulate human SM patterning with human iPSCs in vitro by mimicking the signaling environment during chick/mouse SM development, and report on methods of systematic induction of SM derivatives (MYO, SCL, D, and SYN) from human iPSCs under chemically defined conditions through the presomitic mesoderm (PSM) and SM states. Knowledge regarding chick/mouse&#160;SM development was successfully applied to the induction of SMs with human iPSCs. This method could be a novel tool for studying human somitogenesis and patterning without the use of embryos and for cell-based therapy and disease modeling.

We present here a protocol for the differentiation of human induced pluripotent stem cells into each somite derivative (myotome, sclerotome, dermatome, and syndetome) in chemically defined conditions, which has applications in future disease modeling and cell-based therapies in orthopedic surgery.

人诱导多能干细胞体外生成体细胞衍生物的研究

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give ...