Development of a one-step embryonic stem cell-based assay for the screening of sprouting angiogenesis.
Development of a one-step embryonic stem cell-based assay for the screening of sprouting angiogenesis.
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DOI：
10.1186/1472-6750-7-20
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发表时间：
2007-04-16
影响因子：
3.5
通讯作者：
Vittet D
中科院分区：
文献类型：
--
作者：
Hermant B;Desroches-Castan A;Dubessay ML;Prandini MH;Huber P;Vittet D
血管生成试验是鉴定调节分子和潜在发展治疗策略以调节新生血管的重要工具。尽管过去已经开发了许多体外血管生成模型，但它们表现出局限性，因为它们不能概括整个血管生成过程或对应于不易使用的多步骤程序。方便，可靠，易于量化和生理相关的分析仍然需要血管生成的药理学筛选。在这里，我们优化了一个基于胚胎干细胞分化的血管生成模型，用于筛选实验。我们已经建立了在I型三维胶原凝胶中胚胎干细胞分化过程中导致胚状体血管生成发芽的条件。在这些培养过程中进行的免疫染色实验显示，胚胎干细胞培养11天后，形成了许多由CD31阳性细胞组成的芽。此外，这种一步模型已经在血管生成激活剂和抑制剂的反应中得到验证。在VEGF和FGF2的存在下，发芽被特异性地刺激。另外，血管生成抑制剂如血管抑制素、TGFβ和PF4可以抑制血管生成激活剂诱导的内皮细胞发芽。内皮细胞用CD31泛内皮标记物免疫染色后，通过图像分析可以很容易地定量血管新生。综上所述，这些数据清楚地证实，这一步ES分化模型构成了一个简单而通用的血管生成系统，在未来的研究中，它应该有助于筛选血管生成的激活剂和抑制剂。
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, TGFβ 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.