人类体细胞重编程为诱导多能干细胞（iPS细胞）用绿色荧光蛋白逆转录病毒载体
Summary
的方法来产生人类诱导多能干细胞（iPS细胞）通过逆转录病毒介导的异位表达Oct4的，SOX2的，KLF4和MYC的描述。基于GFP的表达，以确定人类IPSC的殖民地一个切实可行的办法进行了讨论。
Abstract
人类胚胎干细胞（胚胎干细胞），多能干细胞在体外疾病建模和再生医学的宝贵来源。它已被先前的研究显示，人体细胞可以被重新编程为多能性异位表达四个转录因子（OCT4，SOX2，KLF4和 Myc），成为诱导多能干细胞（iPS细胞）2-4。胚胎干细胞一样，人类iPS细胞是多能干细胞和自体细胞的潜在来源。在这里，我们描述的协议进行重新编程人类成纤维细胞克隆到含有GFP的逆转录病毒的骨干4四个重编程因素 。使用下面的协议，我们生成人类胚胎培养条件下的人类iPS细胞在3-4周。人类IPSC的殖民地酷似胚胎干细胞形态和逆转录病毒转基因沉默的结果显示，GFP荧光的损失。 IPSC的殖民地下的荧光microsco机械分离体育作为人类胚胎干细胞类似的方式表现。在这些细胞中，我们检测的多的多能性基因的表达和表面标志。
Protocol
1。逆转录病毒重新编程，重编程因子的表达人成纤维细胞培养成纤维细胞培养基（10％与PEN /链球菌培养液胎牛血清）。 感染前的一天，1×10板5到6孔板人成纤维细胞。 抽吸介质，去除死皮细胞和新鲜的成纤维细胞培养基中添加2毫升。在终浓度为5微克/毫升，加入硫酸鱼精蛋白。 仔细地添加适量的每个表达GFP病毒的感染复数（MOI）5。 感染后的?…
Discussion
四个转录因子重编程iPSCs的人类成纤维细胞中的表达。作了许多尝试使用非集成或者非遗传方法来生成临床安全iPSCs的人类iPS细胞生成。到目前为止，这些方法效率极低，需要进一步优化，提高了重复性11-14。复古或慢病毒的方法，很容易获得和运用人类iPS细胞， 在体外的疾病模型，这是减少病毒整合所造成的安全问题上的依赖。重新编程的方法，这里描述的是人类iPS细胞的效率推?…
Divulgaciones
The authors have nothing to disclose.
Acknowledgements
这项工作是由耶鲁大学的查尔斯·胡德基金会的医学和儿童健康研究奖的学校。
Materials
| Name | Concentration | Company | Catalogue Number |
| hESC medium | |||
| DMEM/F12 | 80% | Invitrogen | 11330057 |
| Knockout Serum Replacer | 20% | Invitrogen | 10828-028 |
| L-Glutamine (200 mM) | 2 mM | Invitrogen | 25030081 |
| Nonessential Amino Acids (10 mM) | 0.1 mM | Invitrogen | 11140050 |
| β-Mercaptoethanol (14.3 M) or MTG | 0.1 mM | Invitrogen | M-6250 |
| bFGF-2 10 μg/ml | 4 ng/ml | GIBCO/BRL | GF003AF |
| Penicillin/Streptomycin | 1% | Millipore | 15140-122 |
| Fiboblasts Medium | |||
| DMEM | 90% | Invitrogen | 11965118 |
| FBS | 10% | Invitrogen | 10407028 |
| Penicillin/Streptomycin | 1% | Millipore | 15140-122 |
Table 1. Culture Medium
| Name | Concentration | Company | Catalogue Number |
| Antibodies | |||
| OCT4 | 1:500 | Abcam | Ab19857 |
| SSEA3 | 1:100 | Milipore | MAB4303 |
| SSEA4 | 1:100 | BD Biosciences | BD560218 |
| Tra-1-81 | 1:100 | BD Biosciences | BD560173 |
| Tra-1-60 | 1:100 | BD Biosciences | BD560174 |
| NANOG | 1:500 | Abcam | Ab21624 |
| Alexa-Flur 488 | 1:1000 | Invitrogen | A11008 |
| Alexa-Flur 555 | 1:1000 | Invitrogen | A21422 |
| DAPI | 1:5000 | Invitrogen | D1306 |
| Plasmids | |||
| pMIG-OCT4 | Addgene | 17225 | |
| pMIG-SOX2 | Addgene | 17226 | |
| pMIG-KLF4 | Addgene | 17227 | |
| pMIG-MYC | Addgene | 18119 | |
| Other Materials | |||
| Collagenase type IV | 1mg/ml | Invitrogen | 17104019 |
| Gelatin, Porcine | 0.1% | Sigma | G 1890 |
| Triton | 0.2% | Sigma | X100-500ML |
| Paraformaldehyde | 4% | Sigma | 47608 |
| BSA | 3% | American Bioanalytical | AB01800 |
| MEF feeder cells | Millipore | PMEF-N | |
| Cell Lifter | Corning | 3008 | |
| Equipment | |||
| Fluorescent microscopy: inverted microscope with GFP filter | |||
Table 2. Reagents and equipment.
Referencias
- Murry, C. E., Keller, G. Differentiation of embryonic stem cells to clinically relevant populations: lessons from embryonic development. Cell. 132, 661-680 (2008).
- Takahashi, K., Tanabe, K., Ohnuki, M., Narita, M., Ichisaka, T., Tomoda, K., Yamanaka, S. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell. 131, 861-872 (2007).
- Yu, J., Vodyanik, M. A., Smuga-Otto, K., Antosiewicz-Bourget, J., Frane, J. L., Tian, S., Nie, J., Jonsdottir, G. A., Ruotti, V., Stewart, R. Induced pluripotent stem cell lines derived from human somatic cells. Science. 318, 1917-1920 (2007).
- Park, I. H., Zhao, R., West, J. A., Yabuuchi, A., Huo, H., Ince, T. A., Lerou, P. H., Lensch, M. W., Daley, G. Q. Reprogramming of human somatic cells to pluripotency with defined factors. Nature. 451, 141-146 (2008).
- Park, I. H., Lerou, P. H., Zhao, R., Huo, H., Daley, G. Q. Generation of human-induced pluripotent stem cells. Nature Protocols. 3, 1180-1186 (2008).
- Park, I. H., Zhao, R., West, J. A., Yabuuchi, A., Huo, H., Ince, T. A., Lerou, P. H., Lensch, M. W., Daley, G. Q. Reprogramming of human somatic cells to pluripotency with defined factors. Nature. 451, 141-146 (2008).
- Hotta, A., Ellis, J. Retroviral vector silencing during iPS cell induction: an epigenetic beacon that signals distinct pluripotent states. Journal of Cellular Biochemistry. 105, 940-948 (2008).
- Matsui, T., Leung, D., Miyashita, H., Maksakova, I. A., Miyachi, H., Kimura, H., Tachibana, M., Lorincz, M. C., Shinkai, Y. Proviral silencing in embryonic stem cells requires the histone methyltransferase ESET. Nature. 464, 927-931 (2010).
- Wolf, D., Goff, S. P. Embryonic stem cells use ZFP809 to silence retroviral DNAs. Nature. 458, 1201-1204 (2009).
- Chan, E. M., Ratanasirintrawoot, S., Park, I. H., Manos, P. D., Loh, Y. H., Huo, H., Miller, J. D., Hartung, O., Rho, J., Ince, T. A. Live cell imaging distinguishes bona fide human iPS cells from partially reprogrammed cells. Nat. Biotechnol. 27, 1033-1037 (2009).
- Yu, J., Hu, K., Smuga-Otto, K., Tian, S., Stewart, R., Slukvin, ., Thomson, J. A. Human induced pluripotent stem cells free of vector and transgene sequences. Science. 324, 797-801 (2009).
- Kim, D., Kim, C. H., Moon, J. I., Chung, Y. G., Chang, M. Y., Han, B. S., Ko, S., Yang, E., Cha, K. Y., Lanza, R. Generation of human induced pluripotent stem cells by direct delivery of reprogramming proteins. Cell Stem Cell. 4, 472-476 (2009).
- Warren, L., Manos, P. D., Ahfeldt, T., Loh, Y. H., Li, H., Lau, F., Ebina, W., Mandal, P. K., Smith, Z. D., Meissner, A. Highly efficient reprogramming to pluripotency and directed differentiation of human cells with synthetic modified mRNA. Cell Stem Cell. 7, 618-630 (2010).
- Ban, H., Nishishita, N., Fusaki, N., Tabata, T., Saeki, K., Shikamura, M., Takada, N., Inoue, M., Hasegawa, M., Kawamata, S. Efficient generation of transgene-free human induced pluripotent stem cells (iPSCs) by temperature-sensitive Sendai virus vectors. Proceedings of the National Academy of Sciences of the United States of America. 108, 14234-14239 (2011).
- Wolf, D., Goff, S. P. TRIM28 mediates primer binding site-targeted silencing of murine leukemia virus in embryonic cells. Cell. 131, 46-57 (2007).
- Park, I. H., Arora, N., Huo, H., Maherali, N., Ahfeldt, T., Shimamura, A., Lensch, M. W., Cowan, C., Hochedlinger, K., Daley, G. Q. Disease-specific induced pluripotent stem cells. Cell. 134, 877-886 (2008).
- Kim, K. Y., Hysolli, E., Park, I. H. Neuronal maturation defect in induced pluripotent stem cells from patients with Rett syndrome. Proceedings of the National Academy of Sciences of the United States of America. 108, 14169-14174 (2011).
Tags
ReprogrammingHuman Somatic CellsInduced Pluripotent Stem CellsRetroviral VectorGFPHuman Embryonic Stem CellsPluripotentIn Vitro Disease ModelingRegenerative MedicineTranscription FactorsOct4Sox2Klf4MycAutologous CellsFibroblast CellsGFP-containing Retroviral BackboneESC Culture ConditionMorphologyRetroviral Transgene SilencingFluorescence MicroscopePluripotency GenesSurface Markers
Citar este artículo
Kim, K., Hysolli, E., Park, I. Reprogramming Human Somatic Cells into Induced Pluripotent Stem Cells (iPSCs) Using Retroviral Vector with GFP. J. Vis. Exp. (62), e3804, doi:10.3791/3804 (2012).