Preparation of VEGFR-3⁺ ECs from ES cells.

(A) The experimental procedure. ES cells maintained in the presence of LIF were transferred onto OP9. Five days later VE-cadherin⁺PECAM-1⁺ ECs were sorted and recultured on OP9 for 3 days in the presence or absence of exogenous reagents. (B) Expression of VEGFR-3 in the EC. VE-cadherin⁺ PECAM-1⁺ ECs were analyzed by flow cytometry at the time of sorting (left panel) and after 3 days' culture (right panel). OP9 cells were easily distinguishable from ES cell–derived cells in forward and side scatter so that they could be excluded from the cells analyzed here. The numbers indicate the percentage of cells that appeared in each quadrant. (C) Expression of the angiogenetic factors was analyzed by RT-PCR with RNA extracted from entire EC cultures (ECs and OP9), OP9 stromal cells without cocultured ECs, and sorted VE-cadherin⁺ PECAM-1⁺ ECs cultured on OP9. PCR reactions were carried out using cDNAs obtained from 100 and 20 cells. PCR products were electrophoresed on 1% agarose gels and detected by staining with ethidium bromide.