选择和隔离殖民地的人类诱导多能干细胞重新编程成年成纤维细胞
Summary
我们提出了一个高效的人体细胞重编程为人类诱导多能干细胞（hiPSC）用OCT3 / 4，SOX2，KLF4和c-myc（OSKM）的逆转录病毒载体编码和识别正确地重新编程，现场染色hiPSC协议茶1-81抗体。
Abstract
在此，我们提出了一个人类成年成纤维细胞重编程为人类诱导多能干细胞使用编码在场的丁酸钠1-3 OCT3 / 4，SOX2，KLF4和c-myc（OSKM）的逆转录病毒载体（hiPSC）协议。我们用这个方法来改编年底通过（P10）成人成纤维细胞（GM03665，Coriell库），弗里德的共济失调患者。重编程的方法包括高效转协议，使用重复离心成纤维细胞中含有病毒，媒体的存在。重新编程hiPSC殖民地确定了使用现场染色TRA-1-81，多能干细胞的表面标志，从非重编程成纤维细胞分离和手动4,5代。然后转移到这些hiPSC被基底膜板，直接重新编程板，生长在馈线的条件。从第一代开始，hiPSC殖民地展示特点胚胎-LIKE形态。使用该协议可以超过70％选择殖民地成功地扩大到细胞系的建立。建立hiPSC线显示特点的多能性标志物，包括表面标志TRA-1-60和SSEA-4，以及核标记OCT3 / 4，Sox2和Nanog的。这里介绍的协议已经成立，并进行弗里德的共济失调患者和控制个人6，人类新生儿成纤维细胞，以及人类角质细胞获得成年成纤维细胞。
Protocol
1。病毒生产和传导板的凤凰Ampho细胞〜7 8×10的6％DMEM培养基（DMEM高糖，10％胎牛血清的热灭活，L-谷氨酰胺2毫米，无抗生素）在10毫升10厘米的板密度。在孵化器和文化过夜的地方，在37℃，5％的CO 2。 第二天染凤凰细胞使用12微克的矢量编码要么OCT3 / 4，SOX2，KLF4和c-myc，或绿色荧光蛋白基因（质粒Addgene 17217，17218，17219，17220）和35μLFugene 6。准备在500μLDMEM培…
Discussion
研究人类，特别是神经系统和神经退行性疾病，已特别具有挑战性由于交通不便足够的人体细胞模型。容易取得的体细胞重新编程为诱导多能干细胞和潜在能力，分化成多种细胞类型，开了一家可能造成遗传性疾病的细胞模型。此外，iPS细胞保持在再生医学的未来大有希望。因此，它是必不可少的重编程体细胞全能性的可靠，高效，安全的方法来开发和优化。
从治疗应用的?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作得到了弗里德的共济失调研究联盟和阿诺德家庭基金会和MD安德森癌症中心的干细胞和发育生物学中心的试点补助金。
Materials
| Reagent | Company | Catalog number |
| DMEM | Invitrogen | 11965 |
| DMEM/F12 | Invitrogen | 11330 |
| KSR | Invitrogen | 10828 |
| Non-essential aminoacids | Invitrogen | 11140 |
| Sodium butyrate | Sigma | B5887 |
| Y27632 | Stemgent | 04-0012 |
| bFGF | Stemgent | 03-0002 |
| Tra-1-81 antibody | Stemgent | 09-0069 |
| Oct3/4 antibody | Santa Cruz | sc-8628 |
| Nanog antibody | Cell Signaling Technology | 4903S |
| Tra-1-60 antibody | Millipore | MAB4360 |
| Sox2 antibody | Cell Signaling Technology | 3579S |
| SSEA4 | Millipore | MAB4304 |
| CF1 MEFs | Globalstem | GSC-6201G |
| Objective marker | Nikon | MBW10010 |
| Matrigel | BD Biosciences | 354277 |
| mTeSR1 | StemCell Technologies | 05850 |
| β-mercaptoethanol | Sigma | M7522 |
| Fugene 6 | Roche | 11814443001 |
| polybrene | Sigma | H9268 |
| Object marker | Nikon | MBW10010 |
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Tags
SelectingIsolatingColoniesHuman Induced Pluripotent Stem CellsReprogrammingAdult FibroblastsRetroviral VectorsOct3/4Sox2Klf4C-mycSodium ButyrateEfficient Transduction ProtocolVirus-containing MediaLive ImmunostainingTra-1-81Pluripotent CellsManually PassagedMatrigel PlatesFeeder-free ConditionsHES-like MorphologyExpansionCell LinesPluripotency MarkersTRA-1-60SSEA-4Oct3/4Sox2Nanog
Cite This Article
Polak, U., Hirsch, C., Ku, S., Gottesfeld, J., Dent, S. Y., Napierala, M. Selecting and Isolating Colonies of Human Induced Pluripotent Stem Cells Reprogrammed from Adult Fibroblasts. J. Vis. Exp. (60), e3416, doi:10.3791/3416 (2012).