作者要感谢来自美国国立卫生研究院的资助下支持这项研究:K08DK082783（AR），P30DK56465（BTS），U01HL099993（BTS），T32HL00715036（SKS）和K12HL0806406（SKS）;而JP麦卡锡基金会（AR）。

签署知情同意书，批准由弗雷德·哈钦森癌症研究中心的机构审查委员会和儿童"费城医院，从患者采集外周血标本之前获得所有机构的指导方针的变化。所有的动物实验，包括MEF生成和畸胎瘤的形成被批准的机构动物护理和使用委员会。 外周血单个核细胞和红细胞扩张的1。聚蔗糖分离 - 0天<ol><li>稀释外周血1:1的Dulbecco磷酸盐缓冲盐水（DPBS）。在一个15毫升的圆底聚苯乙烯试管，小心7层毫升稀释血置于3mL室温的Ficoll-Hypaque上的。 </li><li>离心样品以400×g离心30分钟。 </li><li>的外周血单个核细胞将落户在等离子和聚蔗糖 - 泛影葡胺，看作是一个白色混浊层之间的接口。用无菌移液管，收集PB管委会为一体的15毫升锥形管。使体积达到用DPBS 10毫升</li><li>使用血球计数器，计数外周血单个核细胞。 </li><li>移液管2×10 6个PBMC中到一个单独的15毫升锥形管中，离心分离机，在300×g离心5分钟。降速剩余细胞并冷冻，以2×10 6个PBMC中/ ml，在90％胎牛血清（FBS）/ 10％二甲亚砜（DMSO）的浓度。 </li><li>制备膨胀介质:QBSF-60无血清培养基（避光）+青霉素/链霉素（1％）+抗坏血酸（50毫微克/毫升）+干细胞因子（SCF，50纳克/毫升）+。白细胞介素3（IL- 3，10纳克/毫升）+促红细胞生成素（EPO，2单位/毫升）+胰岛素样生长因子-1（IGF-1，40纳克/毫升）+地塞米松（1μM;从光保护;解冻新鲜等分试样的每个媒体改变）。 </li><li>重悬2×10 6个PBMC中的2ml新鲜配制的扩展培养基，并放入12孔组织培养板的一个孔中，并孵育在37℃的潮湿培养箱具有5气氛％ 的 O 2，5％的CO 2和90％N 2。 </li><li>在3日和6日，更换介质。 <ol><li>收集细胞并用2ml QBSF-60的洗井以收集任何残留的细胞。 </li><li>离心细胞悬浮液，在300×g离心5分钟，吸出上清液。 </li><li>悬浮细胞在2毫升的新拓展中，并将其返回到相同的12孔板。 </li></ol></li></ol> 2.重编程细胞的核转 - 9日<ol><li>吸管的重新编程质粒进入无菌1.5毫升Eppendorf管中（ 表1）。 </li><li>拌上补充（由制造商提供）和吸管核转解到无菌1.5毫升Eppendorf管中。 </li><li>吸管2毫升扩展中等入12孔板和地点的一个孔，在湿润的37℃培养箱（5％O 2，5％CO 2和90％N 2），用于平衡加入细胞之前的。 </li><li>收集培养的细胞和洗井用2ml QBSF-60，以收集剩余的细胞。 </li><li>离心将细胞在300×g离心5分钟，吸出上清液。 </li><li>通过重新悬浮于5ml的DPBS中，并在300×g离心离心5分钟清洗细胞。 </li><li>吸出上清液。 </li><li>悬浮于100μl核转染溶液+补充细胞沉淀，同时注意避免产生气泡。 </li><li>吸取细胞悬浮液到含有重编程的质粒，移液器上下一旦管，样品吸取到核转染反应杯的底部。 </li><li>将试管放入核转染机和运行程序的T-019。 </li><li>转移100微升的预热膨胀介质（从平衡化板在步骤2.3）进入核转试管。 </li><li>立即收集全部样品和沉积成的预热膨胀介质的井。避免收集白色，浮尸细胞碎片。 </li&gt; <li>将板在湿润的37℃培养箱（5％O 2，5％CO 2和90％N 2）的生长。 </li></ol> 3.电镀饲养细胞 - 10日或11 <ol><li>在第10天，板式给料机的细胞。 <ol><li>涂层1 6孔组织培养板用明胶。 <ol><li>吸管0.1％，1明胶中Hank氏缓冲盐溶液（HBSS）插入所述板的每个孔中。 </li><li>将板在湿润的37℃培养箱中培养至少5分钟。 </li><li>在即将使用前，吸从板的明胶溶液。 </li></ol></li><li>解冻的照射在3000 cGy的在10ml预热的MEF培养基（Dulbecco改良的Eagle培养基（DMEM）+胎牛血清（10％）+青霉素/链霉素（1％））的2×10 6的小鼠胚胎成纤维细胞的一个小瓶。 </li><li>离心将细胞在300×g离心5分钟，吸出上清液。重悬细胞于12ml MEF媒体和吸管2毫升我n要在明胶包被的6孔板的每个孔中。将板在湿润的37℃培养箱（5％O 2，5％CO 2和90％N 2），直到第12天。 </li></ol></li></ol> 4.在电镀饲养细胞和细胞介质变化 - 12日<ol><li>收集nucleofected细胞成一体的15毫升锥形管中，并通过洗涤以及用2ml QBSF-60的收集任何残留的细胞。离心样品以300×g离心5分钟。 </li><li>准备重编程培养基:Iscove氏改良的Dulbecco氏培养基（IMDM）+ FBS（10％）+非动物L-谷氨酰胺（1毫米）+非必需氨基酸（NEAA，1％）+青霉素/链霉素（1％）+β β-巯基乙醇（0.1毫摩尔）+碱性成纤维细胞生长因子（bFGF，加入新鲜的在馈送，10纳克/毫升）+抗坏血酸（加入新鲜的进料，50微克/毫升）。 </li><li>吸出上清液，重悬细胞于12ml重编程中的。吸管2毫升％的细胞悬浮液以及成在6孔板饲养细胞（如准备在第3节）。离心反应板在50×g离心30分钟，在室温下进行。 </li><li>将板在湿润的37℃培养箱（5％O 2，5％CO 2和90％N 2）的生长。改变重编程中的每一天，直到IPSC的殖民地开始出现（1-2周）。 </li><li>制备人类胚胎干细胞培养基:DMEM / F12 1:1 +敲除血清替代品（20％）+青霉素/链霉素（1％）+ NEAA（1％）+非动物L-谷氨酰胺（1毫米）+丙酮酸钠（1％） +碳酸氢钠（0.12％）+β巯基乙醇（0.1毫摩尔）+ bFGF的（补充新鲜的进料，10纳克/毫升）的</li><li>准备HDAC抑制剂:丁酸钠（在喂养补充新鲜的，100微米），suberanilohydroxamic酸（SAHA，加入新鲜的饲养，200纳米）。一旦IPSC的菌落出现，开始饲养细胞，人类胚胎干细胞中+ HDAC抑制剂，每天换液。 注:丁酸钠是一种小分子被吸附到塑料管中，叔herefore其存储在硼硅玻璃管在4℃下。 </li></ol> 5.隔离的iPSC克隆<ol><li>一次的iPSC集落有足够大的分离（20-25个细胞），选择其中一个，P20移液管。 </li><li>准备HDAC抑制剂:丁酸钠（加入新鲜的饲养，100微米），萨哈（补充新鲜的饲养，200纳米） </li><li>将分离出的菌落到含有人类胚胎干细胞培养基+ HDAC抑制剂+克隆与恢复补充个人35毫米菜肴的iMEFs。 </li><li>当天采摘的殖民地后，改变介质为人类胚胎干细胞中+ HDAC抑制剂。 </li><li>每天更换培养基，直到IPSC的殖民地准备好传代。 </li><li>从介质中删除HDAC抑制剂一次菌落传代2-3稳定。 </li></ol>

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作者宣称，他们有没有竞争的财务权益。

对于成功的iPSC生成使用此协议的时候，有一些应该考虑几个重要的注意事项。在红细胞膨胀阶段，媒体改变调度必须严格遵守，因为偏差可能导致目标的祖细胞群体的低效刺激和iPSC的产生效率较低。重要的是，使用新鲜地塞米松与各介质变化的新的扩展介质;和QBSF-60基础培养基和地塞米松在存储期间应避光。在核转染，所述的DPBS洗涤是关键，除去从细胞悬浮液过量的溶质可能导致电流从nucleofecto...

iPS细胞是从体组织经由一组的多能性基因的最小的异位表达而得。该技术最初是由人成纤维细胞与OCT4，SOX2，KLF4和cMYC的 ，其高度表达在多能状态1的逆转录病毒转导的展示。这些瞬时表达"重编程因子"改变靶细胞的表观遗传学景观和基因表达谱类似于人类胚胎干细胞2。一旦创建了iPS细胞有可能分化成任何类型的组织作进一步调查。因此，它们有望用于再生医学，疾病建模和基因治疗的应用中使用。然而，破坏与整合的病毒基因组有改变内源基因的表达，影响细胞表型的潜力，最终偏置的科学成果。此外，随机的病毒整合可能会导致有害细胞éffects，包括恶性转化3或重新表达致癌基因的转基因4的可能性。未来的临床应用需要非整合的iPSC生成。 附加体，它是染色体外的环状DNA分子，提供了一项战略，以产生成本效益，整合无iPS细胞5。在表1所示的附加 ​​型载体的组合表达重编程因子OCT4，SOX2，KLF4，MYCL和LIN28A。该pCXLE_hOCT3 / 4- shp53-F质粒还含有p53基因的shRNA对TP53的临时抑制，增强细胞的初始化6。复制缺陷型pCXWB-EBNA1载体通过提供核抗原表达7一过性升高促进重编程因子的放大和提高重编程效率。该pCXLE_EGFP质粒可以被添加到核转染混合物为射探测器的目的rmining转染效率或用于细胞分选的应用程序。除P CXWB-EBNA1的，在该协议中使用的附加 ​​型质粒含有的Epstein-Barr病毒起源的病毒复制和EBNA1基因，其宿主细胞8的分裂过程介导的复制和附加体的分区。该附加体都不约而同的用连续的iPSC扩张7丢失。亚克隆和iPS细胞与附加型载体的损失，这可以从eGFP的表达缺失推断的特性，可能会导致完全集成免费的iPSC供将来的临床应用。 固有的iPSC生成的过程是抑制的细胞系特异性基因和多能性相关基因的重新激活。基因表达的调节发生在细胞核内的多个级别，包括修饰的DNA和染色质，以允许转录因子，调节DNA元件，和RNA聚合酶的访问目标基因。通过全球性染色质修饰的后生景观改造是重新表达的一个关键组成部分的多能性的遗传程序。一个具体的染色质修饰是在基因表达的调控是重要的乙酰化的组蛋白在特定的赖氨酸残基，其允许访问通过组蛋白的DNA线圈的张力下降到目标基因。 HDAC抑制剂是小分子已经显示出增强的iPSC重编程和人类胚胎干细胞的自我更新9,10，可能是由于支承乙酰化状态11。下面描述的协议中，适于从使用整合的慢病毒12的现有出版物，提供了从外周血使用附加体和HDAC抑制剂的步骤的分步方法优化的iPSC产生。这里所用的HDAC抑制剂浓度是半那些由洁具所描述的， 等。 如图9所示 ，并经常导致了2倍的增加完全重新编程的iPSC集落，标准游离重编程协议，而不HDAC抑制剂。重新编程的这个水平看齐与我们观察到与慢病毒方法的效率。使用此协议，我们已经有效地产生iPSC的个人老87岁。

<table><tbody><tr><td>Ficoll-Paque PLUS</td><td>Fisher Scientific</td><td>45-001-750</td><td>Store at 4 &amp;deg;C. Warm to room temperature before use.</td></tr><tr><td>DPBS</td><td>Life Technologies</td><td>14190-250</td><td>Store at 4 &amp;deg;C.</td></tr><tr><td>RPMI 1640</td><td>Life Technologies</td><td>11875-093</td><td>Store at 4 &amp;deg;C.</td></tr><tr><td>fetal bovine serum (FBS)</td><td>Life Technologies</td><td>10437028</td><td>Store at -20 &amp;deg;C until needed. Thaw, aliquot, store at 4 &amp;deg;C.</td></tr><tr><td>dimethyl sulfoxide (DMSO)</td><td>Sigma-Aldrich</td><td>154938</td><td>Store at room temperature.</td></tr><tr><td>QBSF-60 serum-free medium</td><td>Fisher Scientific</td><td>50-983-234</td><td>Store at 4 &amp;deg;C.</td></tr><tr><td>penicillin/streptomycin</td><td>Life Technologies</td><td>15140122</td><td>Store at 4 &amp;deg;C.</td></tr><tr><td>Cell Line Nucleofector Kit V</td><td>Lonza</td><td>VCA-1003</td><td>Store at 4 &amp;deg;C.</td></tr><tr><td>2% gelatin solution</td><td>Sigma-Aldrich</td><td>G1393</td><td>Store at 4 &amp;deg;C, liquify in 37 &amp;deg;C water bath before use.</td></tr><tr><td>Hank&#039;s Balanced Saline Solution (HBSS)</td><td>Life Technologies</td><td>14175-103</td><td>Store at 4 &amp;deg;C.</td></tr><tr><td>Dulbecco&#039;s Modified Eagle Medium (DMEM)</td><td>Life Technologies</td><td>11965-092</td><td>Store at 4 &amp;deg;C.</td></tr><tr><td>Iscove&#039;s Modified Dulbecco&#039;s Medium (IMDM)</td><td>Life Technologies</td><td>12440-061</td><td>Store at 4 &amp;deg;C.</td></tr><tr><td>L-glutamine, 200 mM</td><td>Life Technologies</td><td>25030-081</td><td>Aliquot, freeze at -20 &amp;deg;C.</td></tr><tr><td>non-essential amino acids (NEAA), 100X</td><td>Life Technologies</td><td>11140-050</td><td>Store at 4 &amp;deg;C, away from light.</td></tr><tr><td>DMEM/Ham&#039;s F12, 1:1</td><td>Fisher Scientific</td><td>SH30023.02</td><td>Store at 4 &amp;deg;C.</td></tr><tr><td>KnockOut Serum Replacement</td><td>Life Technologies</td><td>10828-028</td><td>Aliquot, freeze at -20 &amp;deg;C.</td></tr><tr><td>sodium pyruvate, 100 mM</td><td>Life Technologies</td><td>11360-070</td><td>Store at 4 &amp;deg;C.</td></tr><tr><td>sodium bicarbonate, 7.5%</td><td>Life Technologies</td><td>25080094</td><td>Store at 4 &amp;deg;C.</td></tr><tr><td>basic fibroblast growth factor (bFGF)</td><td>Life Technologies</td><td>PHG0263</td><td>Make 10 mg/ml stock in DPBS, aliquot, freeze at -20 &amp;deg;C. Once thawed, store at 4 &amp;deg;C.</td></tr><tr><td>sodium butyrate&amp;nbsp;</td><td>Sigma-Aldrich</td><td>B5887-1G</td><td>Make 2000x stock (400 mM) in DPBS, aliquot, freeze at -20 &amp;deg;C. Once thawed, store at 4 &amp;deg;C.</td></tr><tr><td>hES Cell Cloning &amp;amp; Recovery Supplement</td><td>Stemgent</td><td>01-0014-500</td><td>Store at -20 &amp;deg;C until needed. Once thawed, store at 4 &amp;deg;C.</td></tr><tr><td>ESC-qualified BD matrigel</td><td>BD Biosciences</td><td>35-4277</td><td>Thaw overnight on ice at 4 &amp;deg;C, aliquot into pre-chilled tubes using pre-chilled pipette tips. Store at -20 &amp;deg;C until needed. Thaw at 4 &amp;deg;C, use immediately.</td></tr><tr><td>StemSpan SFEM&amp;nbsp;</td><td>STEMCELL Technologies</td><td>9650</td><td>Aliquot, freeze at -20 &amp;deg;C.</td></tr><tr><td>ascorbic acid, powdered</td><td>Sigma-Aldrich</td><td>A4403-100MG</td><td>Make 5 mg/ml stock in DPBS, sterile filter, store at 4 &amp;deg;C.</td></tr><tr><td>recombinant human stem cell factor (SCF)</td><td>R &amp;amp; D Systems</td><td>255-SC-010</td><td>Make 100 &amp;mu;g/m stock in SFEM, aliquot, freeze at -20 &amp;deg;C. Once thawed, store at 4 &amp;deg;C.</td></tr><tr><td>recombinant human interleukin 3 (IL-3)</td><td>R &amp;amp; D Systems</td><td>203-IL-010</td><td>Make 100 &amp;mu;g/ml stock in SFEM, aliquot, freeze at -20 &amp;deg;C. Once thawed, store at 4 &amp;deg;C.</td></tr><tr><td>erythropoietin (EPO)</td><td>R &amp;amp; D Systems</td><td>287-TC-500</td><td>Make 1000 U/ml stock in SFEM, aliquot, freeze at -20 &amp;deg;C. Once thawed, store at 4 &amp;deg;C.</td></tr><tr><td>recombinant human insulin-like growth factor 1 (IGF-1)</td><td>R &amp;amp; D Systems</td><td>291-G1-200</td><td>Make 100 &amp;mu;g/ml stock in SFEM, aliquot, freeze at -20 &amp;deg;C. Once thawed, store at 4 &amp;deg;C.</td></tr><tr><td>&amp;beta;-mercaptoethanol</td><td>Sigma-Aldrich</td><td>M7522</td><td>Make 1000x stock (100 mM) in DPBS.</td></tr><tr><td>dexamethasone</td><td>Sigma-Aldrich</td><td>D4902-25MG</td><td>Make 50x stock (50 &amp;mu;M) in DPBS, sterile filter, store at 4 &amp;deg;C.</td></tr><tr><td>SAHA (vorinostat)</td><td>Cayman Chemical</td><td>149647-78-9</td><td>Make 2,000x stock (400 mM) in DPBS, aliquot, freeze at -20 &amp;deg;C. Once thawed, store at 4 &amp;deg;C.</td></tr><tr><td>12 well tissue culture plate</td><td>Fisher Scientific</td><td>08-772-29</td><td></td></tr><tr><td>15 ml conical tube</td><td>Sarstedt</td><td>62553002</td><td></td></tr><tr><td>1.5 ml Eppendorf tube</td><td>Fisher Scientific</td><td>05-408-129</td><td></td></tr><tr><td>6 well tissue culture plate</td><td>Fisher Scientific</td><td>08-772-1B</td><td></td></tr><tr><td>35 mm tisue culture plates</td><td>BD Biosciences</td><td>353001</td><td></td></tr><tr><td>10 ml disposable serological pipettes</td><td>Fisher Scientific</td><td>13-675-20</td><td></td></tr><tr><td>5 ml disposable serological pipettes</td><td>Fisher Scientific</td><td>13-675-22</td><td></td></tr><tr><td>2 ml disposable serological pipettes</td><td>Fisher Scientific</td><td>13-675-17</td><td></td></tr><tr><td>20 &amp;mu;l pipette tips, barrier tips</td><td>Genessee</td><td>24-404</td><td></td></tr><tr><td>glass Pasteur pipettes</td><td>Fisher Scientific</td><td>13-678-20D</td><td></td></tr><tr><td>pipette aid</td><td>Fisher Scientific</td><td>13-681-15</td><td></td></tr><tr><td>pCXLE-hOCT3/4-shp53-F</td><td>Addgene</td><td>27077</td><td></td></tr><tr><td>pCXLE-hSK</td><td>Addgene</td><td>27078</td><td></td></tr><tr><td>pCXLE-hUL</td><td>Addgene</td><td>27080</td><td></td></tr><tr><td>pCXLE-EGFP</td><td>Addgene</td><td>27082</td><td></td></tr><tr><td>pCXWB-EBNA1</td><td>Addgene</td><td>37624</td><td></td></tr></tbody></table>

efficient ips cell generation from blood using episomes and hdac inhibitors

此稿件说明了一个协议，用于建立高效集成的无人类诱导多能干细胞从外周血中使用游离质粒和组蛋白去乙酰化酶（HDAC）抑制剂细胞​​（iPS细胞）。这种方法的优点包括:（1）使用的外周血作为源材料的最小量; （2）nonintegrating重编程载体; （3）具有成本效益的方法，用于产生向量自由iPSCs的; （4）单次转染;及（5）使用的小分子，以促进表观重编程。简言之，将外周血单核细胞（PBMC）从常规采血样品中分离，然后培养中所定义的生长因子，以产生高度增殖红细胞祖细胞群是非常适合进行再编程。 Nonintegrating，表达OCT4，SOX2，KLF4，MYCL，LIN28A和p53的短发夹（SH）RNA非传播游离质粒introdu通过单核转染土木工程署到来自红细胞。表达增强的绿色荧光蛋白的游离基因（绿色荧光蛋白）的共转染允许容易识别转染细胞。表达爱泼斯坦-巴尔核抗原1（EBNA1）一个单独的复制缺陷型的质粒也将被添加到该反应混合物中游离的蛋白的表达增加。然后转染的细胞接种在层照射的小鼠胚胎成纤维细胞（的iMEFs），用于持续重新编程。只要iPSC的样集落核转染后出现在大约十二天后，HDAC抑制剂被加入到培养基中，以方便后生重塑。我们已经发现，包含HDAC抑制剂的常规由2倍增加的完全重新编程的iPSC集落的生成。一次的iPSC集落呈现出典型的人类胚胎干细胞（胚胎干细胞）的形态，它们被轻轻转移到个人IMEF涂覆组织培养板中进行的持续增长和扩展。

核转染后三天和电镀nucleofected细胞上的iMEFs之前，成功的核转染的效率，应通过对eGFP的荧光显微术进行估计。 图1显示了一个典型的核转染实验用表达绿色荧光蛋白的总细胞群的约5-10％。 重新编程的iPSC克隆将开始核转染后出现大约两个星期。该菌落是具有明确定义的边界大致为圆形的，并且可以由人类胚胎干细胞特性的形态包括:（1）小的，紧凑的细胞具有较高...

Watch this Scientific Journal Video about Efficient iPS Cell Generation from Blood Using Episomes and HDAC Inhibitors at JoVE.com

Efficient iPS Cell Generation from Blood Using Episomes and HDAC Inhibitors

在这里，我们描述了一种协议，用于使用基于附加体重编程的策略和组蛋白脱乙酰酶抑制剂的外周血产生的诱导的人多能干细胞。

从血液高效的iPS细胞生成使用附加体和HDAC抑制剂

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

efficient-ips-cell-generation-from-blood-using-episomes-hdac

Research

JoVE Journal

Biology

12.7K 次观看.  Fred Hutchinson Cancer Research Center. 在这里，我们描述了一种协议，用于使用基于附加体重编程的策略和组蛋白脱乙酰酶抑制剂的外周血产生的诱导的人多能干细胞。 

视频: Efficient iPS Cell Generation from Blood Using Episomes and HDAC Inhibitors

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, ...

科研

发育生物学

Epigenetic Conversion as a Safe and Simple Method to Obtain Insulin-secreting Cells from Adult Skin Fibroblasts

Regenerative medicine requires new, fully functional cells that are delivered to patients in order to repair degenerated or damaged tissues. When such cells are not readily available, they can be obtained using different approaches that include, among the many, reprogramming and trans-differentiation, with advantages and limitations that are specific of the different techniques. Here a new strategy for the conversion of an adult mature fibroblast into an insulin-secreting cell, arbitrarily designated as epigenetic converted cells (EpiCC), is described. The method has been developed, based on the increasing understanding of the mechanisms controlling epigenetic regulation of cell fate and differentiation. In particular, the first step uses an epigenetic modifier, namely 5-aza-cytidine, to drive adult cells into a "highly permissive" state. It then takes advantage of this brief and reversible window of epigenetic plasticity, to re-address cells toward a different lineage. The approach is designated "epigenetic cell conversion". It is a simple and robust way to obtain an efficient, controlled and stable cellular inter-lineage switch. Since the protocol does not involve the use of any gene transfection, it is free of viral vectors and does not involve a stable pluripotent state, it is highly promising for translational medicine applications.

Here, a new method that allows the conversion of adult skin fibroblasts into insulin-secreting cells is presented. This technique is based on epigenetic conversion, does not involve the use of retroviral vectors nor the acquisition of a stable pluripotent state. It is therefore highly promising for translational medicine applications.

后生转换为安全和简单的方法来获得成人皮肤成纤维细胞胰岛素分泌细胞

Regenerative medicine requires new, fully functional cells that are delivered to patients in order to repair degenerated or damaged tissues. When such ...

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., ...

生物学

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 ...

Direct Lineage Reprogramming of Adult Mouse Fibroblast to Erythroid Progenitors

Erythroid cell commitment and differentiation proceed through activation of a lineage-restricted transcriptional network orchestrated by a group of cell fate determining and maturing factors. We previously set out to define the minimal set of factors necessary for instructing red blood cell development using direct lineage reprogramming of fibroblasts into induced erythroid progenitors/precursors (iEPs). We showed that overexpression of Gata1, Tal1, Lmo2, and c-Myc (GTLM) can rapidly convert murine and human fibroblasts directly to iEPs that resemble bona fide erythroid cells in terms of morphology, phenotype, and gene expression. We intend that iEPs will provide an invaluable tool to study erythropoiesis and cell fate regulation. Here we describe the stepwise process of converting murine tail tip fibroblasts into iEPs via transcription factor-driven direct lineage reprogramming (DLR). In this example, we perform the reprogramming in fibroblasts from erythroid lineage-tracing mice that express the yellow fluorescent protein (YFP) under the control of the erythropoietin receptor gene (EpoR) promoter, enabling visualization of erythroid cell fate induction upon reprogramming. Following this protocol, fibroblasts can be reprogrammed into iEPs within five to eight days. 
While improvements can still be made to the process, we show that GTLM-mediated reprogramming is a rapid and direct process, yielding cells with properties of bona fide erythroid progenitor and precursor cells.

Here we present our protocol for producing induced erythroid progenitors (iEPs) from mouse adult fibroblasts using transcription factor-driven direct lineage reprogramming (DLR).

成人小鼠成纤维细胞对红细胞生成子细胞的直接线重编程

Erythroid cell commitment and differentiation proceed through activation of a lineage-restricted transcriptional network orchestrated by a group of cell ...

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

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

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

由Stemgent人类TF慢病毒重新编程人类成纤维细胞生成诱导多能干细胞

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

Hemogenic Reprogramming of Human Fibroblasts by Enforced Expression of Transcription Factors

The cellular and molecular mechanisms underlying specification of human hematopoietic stem cells (HSCs) remain elusive. Strategies to recapitulate human HSC emergence in vitro are required to overcome limitations in studying this complex developmental process. Here, we describe a protocol to generate hematopoietic stem and progenitor-like cells from human dermal fibroblasts employing a direct cell reprogramming approach. These cells transit through a hemogenic intermediate cell-type, resembling the endothelial-to-hematopoietic transition (EHT) characteristic of HSC specification. Fibroblasts were reprogrammed to hemogenic cells via transduction with GATA2, GFI1B and FOS transcription factors. This combination of three factors induced morphological changes, expression of hemogenic and hematopoietic markers and dynamic EHT transcriptional programs. Reprogrammed cells generate hematopoietic progeny and repopulate immunodeficient mice for three months. This protocol can be adapted towards the mechanistic dissection of the human EHT process as exemplified here by defining GATA2 targets during the early phases of reprogramming. Thus, human hemogenic reprogramming provides a simple and tractable approach to identify novel markers and regulators of human HSC emergence. In the future, faithful induction of hemogenic fate in fibroblasts may lead to the generation of patient-specific HSCs for transplantation.

This protocol demonstrates the induction of a hemogenic program in human dermal fibroblasts by enforced expression of the transcription factors GATA2, GFI1B and FOS to generate hematopoietic stem and progenitor cells.

通过转录因子的强制表达，人类纤维细胞的造血再编程

The cellular and molecular mechanisms underlying specification of human hematopoietic stem cells (HSCs) remain elusive. Strategies to recapitulate human ...

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, ...