Development of a one-step embryonic stem cell-based assay for the screening of sprouting angiogenesis

BACKGROUND: 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. RESULTS: 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. CONCLUSION: 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

Development of a one-step embryonic stem cell-based assay for the screening of sprouting angiogenesis-1

<p><b>Copyright information:</b></p><p>Taken from "Development of a one-step embryonic stem cell-based assay for the screening of sprouting angiogenesis"</p><p>http://www.biomedcentral.com/1472-6750/7/20</p><p>BMC Biotechnology 2007;7():20-20.</p><p>Published online 16 Apr 2007</p><p>PMCID:PMC1858686.</p><p></p>ded at day 0. EBs angiogenic sprouts were analyzed at day 11 of differentiation. vWF immunoreactivity (red fluorescence) located in Weibel-Palade bodies can be observed in several sprouting CD31-positive cells (green fluorescence) (upper panels). Elongated NG2 proteoglycan-positive cells (red fluorescence) can be seen close to CD31-positive cells constituting endothelial sprouts (lower panels). Scale bar = 50 μm

Development of a one-step embryonic stem cell-based assay for the screening of sprouting angiogenesis-3

<p><b>Copyright information:</b></p><p>Taken from "Development of a one-step embryonic stem cell-based assay for the screening of sprouting angiogenesis"</p><p>http://www.biomedcentral.com/1472-6750/7/20</p><p>BMC Biotechnology 2007;7():20-20.</p><p>Published online 16 Apr 2007</p><p>PMCID:PMC1858686.</p><p></p>FGF2 (100 ng/ml). For the second growth factor addition at day 6, VEGF (50 ng/ml) and FGF2 (100 ng/ml) were added alone or in combination. The mean total sprout length of EBs exhibiting endothelial sprouts (angiogenic EBs) was measured at day 11, after CD31 immunostaining, and image analysis with MetaMorph Offline software. Data represent the mean values ± SD resulting from at least 25 angiogenic EBs obtained in one differentiation experiment

Development of a one-step embryonic stem cell-based assay for the screening of sprouting angiogenesis-4

<p><b>Copyright information:</b></p><p>Taken from "Development of a one-step embryonic stem cell-based assay for the screening of sprouting angiogenesis"</p><p>http://www.biomedcentral.com/1472-6750/7/20</p><p>BMC Biotechnology 2007;7():20-20.</p><p>Published online 16 Apr 2007</p><p>PMCID:PMC1858686.</p><p></p> day 0 and at day 6 as indicated in the Methods. At day 6, EBs were also treated by the indicated angiogenesis inhibitors or by their respective vehicle (VHL). Analysis of the mean total sprout length of angiogenic EBs was performed at day 11 on 39, 25, 26, 18, 55 and 76 EBs treated with VHL TGFβ, 10 ng/ml TGFβ, VHL PF4, 2.5 μg/ml PF4, VHL angiostatin and 2.5 μg/ml angiostatin, respectively. In each condition, data represent the mean values ± SE from one out of two differentiation experiments performed in collagen gel with CJ7 ES cells. * p < 0.05, *** p < 0.001; significantly different from respective control values by unpaired Student's test