Demarcation of stable subpopulations within the pluripotent hESC compartment.
Demarcation of stable subpopulations within the pluripotent hESC compartment.
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DOI：
10.1371/journal.pone.0057276
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发表时间：
2013
期刊：
影响因子：
3.7
通讯作者：
Draper JS
中科院分区：
文献类型：
--
作者：
Bhatia S;Pilquil C;Roth-Albin I;Draper JS
异质性是干细胞群体的一个特征，源于控制分化能力的先天细胞等级。异质性如何影响人类多能干细胞群体直接关系到它们在再生医学中的有效应用。多能性的控制是由核心转录因子网络断言的，其中Oct4是一个必要的成员。在小鼠胚胎干细胞（ESCs）中，锌指转录因子Rex1 （Zfp42）密切跟踪未分化状态，并能够将Oct4阳性的mESCs分离成表达或缺乏Rex1的亚稳态群体。然而，目前对人类多能性区室中异质群体的范围或功能知之甚少。人类ESCs表达REX1转录本，但表达REX1细胞的分布和性质尚未被描述。为了解决这些问题，我们在人类ESCs中使用基因靶向技术，在内源性REX1转录调控元件的控制下插入荧光蛋白Venus和抗生素选择标记，产生了一个敏感的、可选择的多能性报告基因。REX1在OCT4和TRA-1-60阳性hESCs中共表达，并在分化后迅速丢失。重要的是，REX1表达在看似同质的OCT4和TRA-1-60 hESCs群体中显示出显著的异质性。在分化过程中，REX1的表达在OCT4之前就消失了，但是，与小鼠相比，在多能hESCs中，REX1表达的缺失标志着一个稳定的、OCT4阳性谱系引发的状态，在正常条件下不会恢复到REX1阳性。我们发现REX1表达的缺失与REX1位点DNA甲基化模式的改变相关，这意味着表观遗传机制可能会干扰小鼠多能性中常见的亚稳态表型。
Heterogeneity is a feature of stem cell populations, resulting from innate cellular hierarchies that govern differentiation capability. How heterogeneity impacts human pluripotent stem cell populations is directly relevant to their efficacious use in regenerative medicine applications. The control of pluripotency is asserted by a core transcription factor network, of which Oct4 is a necessary member. In mouse embryonic stem cells (ESCs), the zinc finger transcription factor Rex1 (Zfp42) closely tracks the undifferentiated state and is capable of segregating Oct4 positive mESCs into metastable populations expressing or lacking Rex1 that are inter-convertible. However, little is currently understood about the extent or function of heterogeneous populations in the human pluripotent compartment. Human ESCs express REX1 transcripts but the distribution and properties of REX1 expressing cells have yet to be described. To address these questions, we used gene targeting in human ESCs to insert the fluorescent protein Venus and an antibiotic selection marker under the control of the endogenous REX1 transcription regulatory elements, generating a sensitive, selectable reporter of pluripotency. REX1 is co-expressed in OCT4 and TRA-1-60 positive hESCs and rapidly lost upon differentiation. Importantly, REX1 expression reveals significant heterogeneity within seemingly homogenous populations of OCT4 and TRA-1-60 hESCs. REX1 expression is extinguished before OCT4 during differentiation, but, in contrast to the mouse, loss of REX1 expression demarcates a stable, OCT4 positive lineage-primed state in pluripotent hESCs that does not revert back to REX1 positivity under normal conditions. We show that loss of REX1 expression correlates with altered patterns of DNA methylation at the REX1 locus, implying that epigenetic mechanisms may interfere with the metastable phenotype commonly found in murine pluripotency.
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