In vitro analysis of vessel formation in embryoid bodies.

EBs were generated from control (+/+) and perlecan mutant (−/−) ES cells. (A–F) The data are shown from PECAM-1 immunostainings of 5+12 days EBs cultured in 5% FCS-containing DMEM. A prominent vascular network is formed in wild type EBs (A), while mutants form endothelial networks that are less dense and smaller (B). (C–D) Treatment with 20 ng/ml VEGF165 stimulates the formation of large PECAM-positive vascularized areas both in control and mutant EBs. (E–F) Treatment with 20 ng/ml FGF-2 strongly induces the formation of extensive vascular networks in both control and perlecan-deficient EBs. (G) Binding of FGFR1-AP to control (+/+) and perlecan mutant (−/−) 5+9 days EBs. The EBs were grown until confluency in 96-well tissue culture plates and fixed. The fixed EBs were incubated with increasing concentration of FGF-2 and exposed to FGFR1-AP at a concentration of 300 µg/ml. After washing to remove unbound receptor bound FGFR1-AP was measured with AP chromogenic substrate. The data are means of 8 EBs per data-point. Statistical differences were tested by Students t-test (***, p