In Vitro Differentiation Group, Leibniz Institute of Plant Genetics and Crop Plant Research IPK, Corrensstrasse 3, D-06466 Gatersleben, Germany.
Int J Cardiol. 2011 Feb 17;147(1):95-111. doi: 10.1016/j.ijcard.2009.08.021. Epub 2009 Sep 22.
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.
胚胎干细胞（ES 细胞）分化为具有早期起搏细胞、心房细胞、心室细胞和窦房结样细胞特征的心脏表型，但 ES 细胞向窦房结细胞的分化尚不清楚。我们使用尿素的萘基胺衍生物苏拉明，能够跟踪心脏特化形成窦房结样细胞的过程。
从胚状体形成的第 5 天到第 7 天，用苏拉明（500µM）处理分化中的小鼠 ES 细胞，并通过形态分析、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能。
苏拉明的应用导致心脏细胞数量增加，但抑制神经元、骨骼肌和确定内胚层的分化。苏拉明处理后，中胚层分化减少。Brachyury、FGF10、Wnt8 和 Wnt3a 的转录水平显著下调，随后中胚层和心脏祖细胞特异性标志物 BMP2、GATA4/5、Wnt11、Isl1、Nkx2.5 和 Tbx5 的水平在去除该物质后立即下降。随着分化的继续，Brachyury、FGF10 和 GATA5 的转录水平显著上调，而 Nkx2.5、Isl1、Tbx5、BMP2 和 Wnt11 的水平恢复到对照水平。在分化的后期阶段，窦房结特异性 HCN4、Tbx2 和 Tbx3 的转录水平显著上调。免疫荧光和膜片钳分析证实了窦房结样细胞数量的增加，电生理分析显示苏拉明处理后心房样和心室样心肌细胞的数量减少。
我们得出结论，苏拉明对心脏分化过程的干扰改变了中胚层和心脏特异性基因的表达，从而增强了窦房结样细胞的形成。
