Differentiation induction of mouse embryonic stem cells into sinus node-like cells by suramin.
Differentiation induction of mouse embryonic stem cells into sinus node-like cells by suramin.
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DOI：
10.1016/j.ijcard.2009.08.021
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发表时间：
2011-02-17
影响因子：
3.5
通讯作者：
Wobus AM
中科院分区：
文献类型：
--
作者：
Wiese C;Nikolova T;Zahanich I;Sulzbacher S;Fuchs J;Yamanaka S;Graf E;Ravens U;Boheler KR;Wobus AM
胚胎干细胞（ES）分化为心脏表型，代表早期起搏器，心房，心室，和窦房结样细胞，然而，ES衍生的规范到窦房结细胞还不清楚。通过使用尿素的萘胺衍生物苏拉明，我们能够跟踪心脏特化为窦房结样细胞的过程。在拟胚体形成的第5 - 7天，用苏拉明（500 μM）处理分化的小鼠ES细胞，并通过形态学分析、流式细胞术、RT-PCR、免疫组织化学和膜片钳分析来分析细胞的分化潜力。应用苏拉明导致心肌细胞数量增加，但抑制神经元、骨骼肌和定形内胚层分化。苏拉明处理后，中内胚层分化立即减少，Brachyury，FGF 10，Wnt 8和Wnt 3a转录水平显著下调，随后中胚层和心脏祖细胞特异性标志物BMP 2，GATA 4/5，Wnt 11，Isl 1，Nkx 2.5和Tbx 5在去除该物质后立即减少。随着继续分化，观察到Brachyury、FGF 10和GATA 5转录物水平的显著上调，而Nkx2.5、Isll、Tbx 5、BMP 2和Wnt 11水平被标准化为对照水平。在晚期分化阶段，窦房结特异性HCN 4，Tbx 2和Tbx 3转录水平显著上调。免疫荧光和膜片钳分析证实窦结样细胞的数量增加，和电生理分析显示，心房和心室样心肌细胞的数量较低的苏拉明治疗后。我们得出结论，苏拉明干扰心脏分化过程修改中胚层和心脏特异性基因表达，从而增强窦结样细胞的形成。
Embryonic stem (ES) cells differentiate into cardiac phenotypes representing early pacemaker-, atrial-, ventricular-, and sinus node-like cells, however, ES-derived specification into sinus nodal cells is not yet known. By using the naphthylamine derivative of urea, suramin, we were able to follow the process of cardiac specialization into sinus node-like cells. Differentiating mouse ES cells were treated with suramin (500 μM) from day 5 to 7 of embryoid body formation, and cells were analysed for their differentiation potential via morphological analysis, flow cytometry, RT-PCR, immunohistochemistry and patch clamp analysis. Application of suramin resulted in an increased number of cardiac cells, but inhibition of neuronal, skeletal muscle and definitive endoderm differentiation. Immediately after suramin treatment, a decreased mesendoderm differentiation was found. Brachyury, FGF10, Wnt8 and Wnt3a transcript levels were significantly down-regulated, followed by a decrease in mesoderm- and cardiac progenitor-specific markers BMP2, GATA4/5, Wnt11, Isl1, Nkx2.5 and Tbx5 immediately after removal of the substance. With continued differentiation, a significant up-regulation of Brachyury, FGF10 and GATA5 transcript levels was observed, whereas Nkx2.5, Isl1, Tbx5, BMP2 and Wnt11 levels were normalized to control levels. At advanced differentiation stages, sinus node-specific HCN4, Tbx2 and Tbx3 transcript levels were significantly up-regulated. Immunofluorescence and patch-clamp analysis confirmed the increased number of sinus node-like cells, and electrophysiological analysis revealed a lower number of atrial- and ventricular-like cardiomyocytes following suramin treatment. We conclude that the interference of suramin with the cardiac differentiation process modified mesoderm- and cardiac-specific gene expression resulting in enhanced formation of sinus node-like cells.