DNA methyltransferase inhibition induces mouse embryonic stem cell differentiation into endothelial cells.
DNA methyltransferase inhibition induces mouse embryonic stem cell differentiation into endothelial cells.
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DOI：
10.1016/j.yexcr.2009.08.011
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发表时间：
2010-01-15
影响因子：
3.7
通讯作者：
Bacanamwo M
中科院分区：
文献类型：
--
作者：
Banerjee S;Bacanamwo M
了解内皮细胞（EC）分化是组织工程，控制血管生成和内皮功能障碍的一个步骤。我们假设，胚胎干细胞（ESC）分化为EC谱系特异性基因的表观遗传激活是一个重要的调解人。通过在存在或不存在特异性DNA甲基转移酶（DNMT）抑制剂5′-氮杂-2 ′-脱氧胞苷（aza-dC）的情况下从维持培养基中去除白血病抑制因子（LIF）来分化小鼠ESC。通过定量PCR、Western、免疫细胞化学和流式细胞术监测EC特异性和标记基因的表达。通过血管生成试验评估分化的EC的功能。通过亚硫酸氢盐测序确定EC分化介质VEGF-A、BMP 4和EPAS-1以及成熟EC标记物VE-钙粘蛋白的近端启动子CpG中的甲基化状态。ESC分化导致在存在和不存在aza-dC处理的情况下OCT 4表达的抑制。然而，显着增加血管生成和EC分化和EC特异性基因的介质的表达，只观察到aza-dC处理的细胞。DNMT抑制介导的EC特异性和标记基因表达的增加与这些基因的去甲基化无关。这些研究表明，DNMT抑制是ESC分化EC的有效诱导剂。
Understanding endothelial cell (EC) differentiation is a step forward in tissue engineering, controlling angiogenesis, and endothelial dysfunction. We hypothesized that epigenetic activation of EC lineage specification genes is an important mediator of embryonic stem cell (ESC) differentiation into EC. Mouse ESC was differentiated by removing leukemia inhibitory factor (LIF) from the maintenance media in the presence or absence of the specific DNA methyltransferase (DNMT) inhibitor 5′-aza-2′-deoxycytidine (aza-dC). Expression of EC specification and marker genes was monitored by quantitative PCR, western, immunocytochemistry, and flow cytometry. Functionality of differentiated EC was assessed by angiogenesis assay. The methylation status in the proximal promoter CpGs of the mediators of EC differentiation VEGF-A, BMP4, and EPAS-1 as well as of the mature EC marker VE-cadherin was determined by bisulfite sequencing. ESC differentiation resulted in repression of OCT4 expression in both the absence and presence of aza-dC treatment. However, significant increase in angiogenesis and expression of the mediators of EC differentiation and EC-specific genes was only observed in aza-dC-treated cells. The DNMT inhibition-mediated increase in EC specification and marker gene expression was not associated with demethylation of these genes. These studies suggest that DNMT inhibition is an efficient inducer of EC differentiation from ESC.
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