Hermant Bastien, Desroches-Castan Agnès, Dubessay Marie-Laure, Prandini Marie-Hélène, Huber Philippe, Vittet Daniel
Inserm, U882, Grenoble, F-38054 France.
BMC Biotechnol. 2007 Apr 16;7:20. doi: 10.1186/1472-6750-7-20.
Angiogenesis assays are important tools for the identification of regulatory molecules and the potential development of therapeutic strategies to modulate neovascularization. Although numerous in vitro angiogenesis models have been developed in the past, they exhibit limitations since they do not recapitulate the entire angiogenic process or correspond to multi-step procedures that are not easy to use. Convenient, reliable, easily quantifiable and physiologically relevant assays are still needed for pharmacological screenings of angiogenesis.
Here, we have optimized an angiogenesis model based on ES cell differentiation for screening experiments. We have established conditions leading to angiogenic sprouting of embryoid bodies during ES cell differentiation in type I three-dimensional collagen gels. Immunostaining experiments carried out during these cultures showed the formation of numerous buds comprising CD31 positive cells, after 11 days of culture of ES cells. Moreover, this one-step model has been validated in response to activators and inhibitors of angiogenesis. Sprouting was specifically stimulated in the presence of VEGF and FGF2. Alternatively, endothelial sprouting induced by angiogenic activators was inhibited by angiogenesis inhibitors such as angiostatin, TGFbeta and PF4. Sprouting angiogenesis can be easily quantified by image analysis after immunostaining of endothelial cells with CD31 pan-endothelial marker.
Taken together, these data clearly validate that this one-step ES differentiation model constitutes a simple and versatile angiogenesis system that should facilitate, in future investigations, the screening of both activators and inhibitors of angiogenesis.
血管生成检测是鉴定调控分子以及开发调节新生血管形成治疗策略的重要工具。尽管过去已经开发出众多体外血管生成模型，但它们存在局限性，因为它们无法重现整个血管生成过程，或者对应于不易使用的多步骤程序。对于血管生成的药理学筛选，仍需要方便、可靠、易于量化且与生理相关的检测方法。
在此，我们基于胚胎干细胞（ES细胞）分化优化了一种用于筛选实验的血管生成模型。我们建立了在I型三维胶原凝胶中ES细胞分化过程中诱导胚状体血管生成芽的条件。在这些培养过程中进行的免疫染色实验显示，ES细胞培养11天后形成了许多包含CD31阳性细胞的芽。此外，该一步模型已针对血管生成激活剂和抑制剂进行了验证。在VEGF和FGF2存在的情况下，芽生受到特异性刺激。相反，血管生成抑制剂如血管抑素、TGFβ和PF4可抑制血管生成激活剂诱导的内皮芽生。在用CD31全内皮标记物对内皮细胞进行免疫染色后，通过图像分析可轻松量化芽生血管生成。
综上所述，这些数据清楚地验证了这种一步ES分化模型构成了一个简单且通用的血管生成系统，在未来的研究中应有助于血管生成激活剂和抑制剂的筛选。
