从血液高效的iPS细胞生成使用附加体和HDAC抑制剂
Summary
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。
Abstract
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。
Introduction
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。
Protocol
Representative Results
Discussion
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…
Divulgations
The authors have nothing to disclose.
Acknowledgements
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）。
Materials
| Name of Reagent/ Equipment | Company | Catalog Number | Comments/Description |
| Ficoll-Paque PLUS | Fisher Scientific | 45-001-750 | Store at 4°C. Warm to room temperature before use. |
| DPBS | Life Technologies | 14190-250 | Store at 4°C. |
| RPMI 1640 | Life Technologies | 11875-093 | Store at 4°C. |
| fetal bovine serum (FBS) | Life Technologies | 10437028 | Store at -20°C until needed. Thaw, aliquot, store at 4°C. |
| dimethyl sulfoxide (DMSO) | Sigma-Aldrich | 154938 | Store at room temperature. |
| QBSF-60 serum-free medium | Fisher Scientific | 50-983-234 | Store at 4°C. |
| penicillin/streptomycin | Life Technologies | 15140122 | Store at 4°C. |
| Cell Line Nucleofector Kit V | Lonza | VCA-1003 | Store at 4°C. |
| 2% gelatin solution | Sigma-Aldrich | G1393 | Store at 4°C, liquify in 37°C water bath before use. |
| Hank's Balanced Saline Solution (HBSS) | Life Technologies | 14175-103 | Store at 4°C. |
| Dulbecco's Modified Eagle Medium (DMEM) | Life Technologies | 11965-092 | Store at 4°C. |
| Iscove's Modified Dulbecco's Medium (IMDM) | Life Technologies | 12440-061 | Store at 4°C. |
| L-glutamine, 200 mM | Life Technologies | 25030-081 | Aliquot, freeze at -20°C. |
| non-essential amino acids (NEAA), 100X | Life Technologies | 11140-050 | Store at 4°C, away from light. |
| DMEM/Ham's F12, 1:1 | Fisher Scientific | SH30023.02 | Store at 4°C. |
| KnockOut Serum Replacement | Life Technologies | 10828-028 | Aliquot, freeze at -20°C. |
| sodium pyruvate, 100 mM | Life Technologies | 11360-070 | Store at 4°C. |
| sodium bicarbonate, 7.5% | Life Technologies | 25080094 | Store at 4°C. |
| basic fibroblast growth factor (bFGF) | Life Technologies | PHG0263 | Make 10 mg/mL stock in DPBS, aliquot, freeze at -20°C. Once thawed, store at 4°C. |
| sodium butyrate | Sigma-Aldrich | B5887-1G | Make 2000X stock (400 mM) in DPBS, aliquot, freeze at -20°C. Once thawed, store at 4°C. |
| hES Cell Cloning & Recovery Supplement | Stemgent | 01-0014-500 | Store at -20°C until needed. Once thawed, store at 4°C. |
| ESC-qualified BD matrigel | BD Biosciences | 35-4277 | Thaw overnight on ice at 4°C, aliquot into pre-chilled tubes using pre-chilled pipette tips. Store at -20°C until needed. Thaw at 4°C, use immediately. |
| StemSpan SFEM | STEMCELL Technologies | 9650 | Aliquot, freeze at -20°C. |
| ascorbic acid, powdered | Sigma-Aldrich | A4403-100MG | Make 5 mg/mL stock in DPBS, sterile filter, store at 4°C. |
| recombinant human stem cell factor (SCF) | R & D Systems | 255-SC-010 | Make 100 μg/mL stock in SFEM, aliquot, freeze at -20°C. Once thawed, store at 4°C. |
| recombinant human interleukin 3 (IL-3) | R & D Systems | 203-IL-010 | Make 100 μg/mL stock in SFEM, aliquot, freeze at -20°C. Once thawed, store at 4°C. |
| erythropoietin (EPO) | R & D Systems | 287-TC-500 | Make 1000 U/mL stock in SFEM, aliquot, freeze at -20°C. Once thawed, store at 4°C. |
| recombinant human insulin-like growth factor 1 (IGF-1) | R & D Systems | 291-G1-200 | Make 100 μg/mL stock in SFEM, aliquot, freeze at -20°C. Once thawed, store at 4°C. |
| β-mercaptoethanol | Sigma-Aldrich | M7522 | Make 1000X stock (100 mM) in DPBS. |
| dexamethasone | Sigma-Aldrich | D4902-25MG | Make 50X stock (50 μM) in DPBS, sterile filter, store at 4°C. |
| SAHA (vorinostat) | Cayman Chemical | 149647-78-9 | Make 2000X stock (400 mM) in DPBS, aliquot, freeze at -20°C. Once thawed, store at 4°C. |
| 12 well tissue culture plate | Fisher Scientific | 08-772-29 | |
| 15 mL conical tube | Sarstedt | 62553002 | |
| 1.5 mL Eppendorf tube | Fisher Scientific | 05-408-129 | |
| 6 well tissue culture plate | Fisher Scientific | 08-772-1B | |
| 35 mm tisue culture plates | BD Biosciences | 353001 | |
| 10 mL disposable serological pipettes | Fisher Scientific | 13-675-20 | |
| 5 mL disposable serological pipettes | Fisher Scientific | 13-675-22 | |
| 2 mL disposable serological pipettes | Fisher Scientific | 13-675-17 | |
| 20 μL pipette tips, barrier tips | Genessee | 24-404 | |
| glass Pasteur pipettes | Fisher Scientific | 13-678-20D | |
| pipette aid | Fisher Scientific | 13-681-15 | |
| pCXLE-hOCT3/4-shp53-F | Addgene | 27077 | |
| pCXLE-hSK | Addgene | 27078 | |
| pCXLE-hUL | Addgene | 27080 | |
| pCXLE-EGFP | Addgene | 27082 | |
| pCXWB-EBNA1 | Addgene | 37624 |
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