Early reprogramming regulators identified by prospective isolation and mass cytometry.
Early reprogramming regulators identified by prospective isolation and mass cytometry.
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DOI：
10.1038/nature14274
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发表时间：
2015-05-21
期刊：
影响因子：
64.8
通讯作者：
Wernig M
中科院分区：
文献类型：
--
作者：
Lujan E;Zunder ER;Ng YH;Goronzy IN;Nolan GP;Wernig M
在大多数诱导多能干细胞（iPS）重编程方法的背景下，非生产性和交错生产性中间体的异质群体在不同的重编程时间点出现。尽管最近的报道声称使用遗传修饰的供体细胞显著提高了重编程效率，但前瞻性地分离不同的重编程中间体仍然是破译重编程机制的重要目标。先前鉴定中间细胞群的表面标志物的尝试是基于这样的假设，即在重编程过程中，细胞逐渐失去供体细胞身份并逐渐获得iPS细胞特性。在这里，我们报告说，iPS细胞和上皮标志物，如SSEA1和EpCAM，分别不能预测在早期阶段的重编程。相反，在系统的功能性表面标记物筛选中，我们发现早期重编程倾向细胞表达一组独特的表面标记物，包括成纤维细胞和iPS细胞中不存在的CD73，CD49d和CD200。单细胞团细胞计数和前瞻性分离显示，这些不同的中间体是短暂的，并在早期，可能是随机重编程阶段期间，桥接供体细胞沉默和多能性标记物获取之间的差距。表达谱分析显示，在这种重编程状态下，转录调节因子Nr0b1和Etv5的早期上调，先于关键的多能性调节因子如Rex1、Dppa2、Nanog和Sox2的激活。这两种因子都是产生早期中间状态和完全重编程的iPS细胞所必需的，因此标志着一些最早已知的iPS细胞诱导调节因子。我们的研究对导致多能性获得的一系列分层事件中的第一步进行了解卷积。
In the context of most induced pluripotent stem (iPS) cell reprogramming methods, heterogeneous populations of nonproductive and staggered productive intermediates arise at different reprogramming time points. Despite recent reports claiming substantially increased reprogramming efficiencies using genetically modified donor cells prospectively isolating distinct reprogramming intermediates remains an important goal to decipher reprogramming mechanisms. Previous attempts to identify surface markers of intermediate cell populations were based on the assumption that during reprogramming cells progressively lose donor cell identity and gradually acquire iPS cell properties. Here, we report that iPS cell and epithelial markers, such as SSEA1 and EpCAM, respectively, are not predictive of reprogramming during early phases. Instead, in a systematic functional surface marker screen we find that early reprogramming-prone cells express a unique set of surface markers, including CD73, CD49d and CD200 that are absent in fibroblasts and iPS cells. Single cell mass cytometry and prospective isolation show that these distinct intermediates are transient and bridge the gap between donor cell silencing and pluripotency marker acquisition during the early, presumably stochastic reprogramming phase. Expression profiling revealed early upregulation of the transcriptional regulators Nr0b1 and Etv5 in this reprogramming state, preceding activation of key pluripotency regulators such as Rex1, Dppa2, Nanog and Sox2. Both factors are required for the generation of the early intermediate state and fully reprogrammed iPS cells, and thus mark some of the earliest known regulators of iPS cell induction. Our study deconvolutes the first steps in a hierarchical series of events that lead to pluripotency acquisition.