Embryonic stem cells (ES) are a valuable source of endothelial cells. By co-culturing ES cells with the stromal PA6 cells, the endothelial commitment can be achieved by adding exogenous FGF2 or BMP4. In this work, the molecular pathways that direct the differentiation of ES cells toward endothelium in response to FGF2 are evaluated and compared to those activated by BMP4. To this purpose the genes expression profiles of both ES/PA6 co-cultures and of pure cultures of PA6 cells were obtained by microarray technique at different time points. The bioinformatics processing of the data indicated TGFβ1 as the most represented upstream regulator in FGF2-induced endothelial commitment while WNT pathway as the most represented in BMP4-activated endothelial differentiation. Loss of function experiments were performed to validate the importance of TGFβ1 and WNT6 respectively in FGF2 and BMP4-induced endothelial differentiation. The loss of TGFβ1 expression significantly impaired the accomplishment of the endothelial commitment unless exogenous recombinant TGFβ1 was added to the culture medium. Similarly, silencing WNT6 expression partially affected the endothelial differentiation of the ES cells upon BMP4 stimulation. Such dysfunction was recovered by the addition of recombinant WNT6 to the culture medium. The ES/PA6 co-culture system recreates an in vitro complete microenvironment in which endothelial commitment is accomplished in response to alternative signals through different mechanisms. Given the importance of WNT and TGFβ1 in mediating the crosstalk between tumor and stromal cells this work adds new insights in the mechanism of tumor angiogenesis and of its possible inhibition.

胚胎干细胞 (ES) 是内皮细胞的宝贵来源。通过与基质 PA6 细胞共培养 ES 细胞，可以通过添加外源性 FGF2 或 BMP4 来实现内皮细胞的定型。在这项工作中，评估了引导 ES 细胞向内皮细胞分化以响应 FGF2 的分子途径，并将其与 BMP4 激活的分子途径进行了比较。为此目的，通过微阵列技术在不同时间点获得ES/PA6共培养物和PA6细胞纯培养物的基因表达谱。数据的生物信息学处理表明 TGFβ1 是 FGF2 诱导的内皮定向中最具代表性的上游调节因子，而 WNT 途径是 BMP4 激活的内皮分化中最具代表性的。进行功能丧失实验以验证 TGFβ1 和 WNT6 分别在 FGF2 和 BMP4 诱导的内皮分化中的重要性。除非将外源重组 TGFβ1 添加到培养基中，否则 TGFβ1 表达的丧失会显着损害内皮承诺的完成。类似地，沉默 WNT6 表达在 BMP4 刺激后部分影响 ES 细胞的内皮分化。通过向培养基中添加重组 WNT6 可以恢复这种功能障碍。ES/PA6 共培养系统重建了一个体外完整的微环境，在该微环境中，通过不同的机制响应替代信号，完成内皮承诺。