Live cell imaging distinguishes bona fide human iPS cells from partially reprogrammed cells
Live cell imaging distinguishes bona fide human iPS cells from partially reprogrammed cells
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DOI：
10.1038/nbt.1580
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发表时间：
2009-11-01
影响因子：
46.9
通讯作者：
Schlaeger, Thorsten M.
中科院分区：
文献类型：
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作者：
Chan, Elayne M.;Ratanasirintrawoot, Sutheera;Schlaeger, Thorsten M.
体细胞可通过转录因子的强制表达而被重新编程为诱导性多能干细胞。利用对重新编程的人成纤维细胞进行的一系列实时成像，我们鉴定了不同的集落类型，这些集落类型在形态上类似于胚胎干细胞，但在分子表型和分化潜力上不同。通过分析多能性标记的表达、OCT4和NANOG启动子的甲基化以及畸胎瘤的分化，我们确定只有一种集落类型代表真正的iPS细胞，而其他克隆类型代表重编程中间产物。前病毒沉默和TRA-1-60、DNMT3b和REx1的表达可以区分完全重编程状态，而碱性磷酸酶、SSEA-4、GDF3、hTERT和NANOG的表达不足以作为标志。我们还表明，使用化学定义的介质进行重新编程有利于形成完全重新编程的菌落，而不是部分重新编程的菌落。我们的数据定义了完全重新编程状态的分子标记，并强调了对假定的iPS细胞进行严格表征和标准化的必要性。
Somatic cells can be reprogrammed into induced pluripotent stem (iPS) cells by enforced expression of transcription factors. Using serial live imaging of human fibroblasts undergoing reprogramming, we identified distinct colony types that morphologically resemble embryonic stem (ES) cells yet differ in molecular phenotype and differentiation potential. By analyzing expression of pluripotency markers, methylation at the OCT4 and NANOG promoters and differentiation into teratomas, we determined that only one colony type represents true iPS cells, whereas the others represent reprogramming intermediates. Proviral silencing and expression of TRA-1-60, DNMT3B and REX1 can be used to distinguish the fully reprogrammed state, whereas alkaline phosphatase, SSEA-4, GDF3, hTERT and NANOG are insufficient as markers. We also show that reprogramming using chemically defined medium favors formation of fully reprogrammed over partially reprogrammed colonies. Our data define molecular markers of the fully reprogrammed state and highlight the need for rigorous characterization and standardization of putative iPS cells.