Efficient human iPS cell derivation by a non-integrating plasmid from blood cells with unique epigenetic and gene expression signatures.
Efficient human iPS cell derivation by a non-integrating plasmid from blood cells with unique epigenetic and gene expression signatures.
复制标题
作者：
为了鉴定可获得和允许高效获得诱导多能干细胞(IPSCs)的人类细胞类型，我们研究了多种出生后细胞类型的表观遗传学和基因表达特征，如成纤维细胞和血细胞。我们的分析表明，新生儿脐带血(CB)和成人外周血(PB)单个核细胞(MNCs)比年龄匹配的成纤维细胞更接近IPSCs和人类胚胎干细胞(ESCs)，因此血液MNCs是产生无整合IPSCs的有吸引力的细胞选择。使用表达5个重编程因子的改进的EBNA1/ORIP质粒，我们证明了对短暂培养的血MNC进行高效的重编程。在一次转染法的14天内，每200万个CBMNC可产生多达1000个IPSC样集落。从成人外周血单个核细胞中获得IPSCs的效率大约低50倍，但通过加入第二个EBNA1/ORIP质粒以瞬时表达额外的基因，如SV40T抗原，可以提高效率。从成人外周血单核细胞获得真正的IPSC集落的时间比成人成纤维细胞(28-30天)缩短了一半(∼14天)。来自外周血或外周血的9个以上的人IPSC株被广泛鉴定，包括来自一名患有镰状细胞病的成人患者的外周血单核细胞。扩增10~12代后，缺乏V(D)J DNA重排和载体DNA。这种从血液单个核细胞中产生无整合的人类IPSCs的简便方法将加速它们在研究和未来临床应用中的应用。
To identify accessible and permissive human cell types for efficient derivation of induced pluripotent stem cells (iPSCs), we investigated epigenetic and gene expression signatures of multiple postnatal cell types such as fibroblasts and blood cells. Our analysis suggested that newborn cord blood (CB) and adult peripheral blood (PB) mononuclear cells (MNCs) display unique signatures that are closer to iPSCs and human embryonic stem cells (ESCs) than age-matched fibroblasts to iPSCs/ESCs, thus making blood MNCs an attractive cell choice for the generation of integration-free iPSCs. Using an improved EBNA1/OriP plasmid expressing 5 reprogramming factors, we demonstrated highly efficient reprogramming of briefly cultured blood MNCs. Within 14 days of one-time transfection by one plasmid, up to 1000 iPSC-like colonies per 2 million transfected CB MNCs were generated. The efficiency of deriving iPSCs from adult PB MNCs was approximately 50-fold lower, but could be enhanced by inclusion of a second EBNA1/OriP plasmid for transient expression of additional genes such as SV40 T antigen. The duration of obtaining bona fide iPSC colonies from adult PB MNCs was reduced to half (∼14 days) as compared to adult fibroblastic cells (28–30 days). More than 9 human iPSC lines derived from PB or CB blood cells are extensively characterized, including those from PB MNCs of an adult patient with sickle cell disease. They lack V(D)J DNA rearrangements and vector DNA after expansion for 10–12 passages. This facile method of generating integration-free human iPSCs from blood MNCs will accelerate their use in both research and future clinical applications.
登录
查看更多内容
影响因子：
3.7
作者：
Marchetto MC;Yeo GW;Kainohana O;Marsala M;Gage FH;Muotri AR
通讯作者：
Muotri AR
影响因子：
4.4
作者：
Serwold, Thomas;Hochedlinger, Konrad;Weissman, Irving L.
通讯作者：
Weissman, Irving L.
影响因子：
23.9
作者：
Kim D;Kim CH;Moon JI;Chung YG;Chang MY;Han BS;Ko S;Yang E;Cha KY;Lanza R;Kim KS
通讯作者：
Kim KS
DOI：
10.1126/science.1172482
发表时间：
2009-05-08
期刊：
Science (New York, N.Y.)
影响因子：
--
作者：
Yu J;Hu K;Smuga-Otto K;Tian S;Stewart R;Slukvin II;Thomson JA
通讯作者：
Thomson JA
影响因子：
23.9
作者：
Loh YH;Hartung O;Li H;Guo C;Sahalie JM;Manos PD;Urbach A;Heffner GC;Grskovic M;Vigneault F;Lensch MW;Park IH;Agarwal S;Church GM;Collins JJ;Irion S;Daley GQ
通讯作者：
Daley GQ