Vascular potential of FLK-1⁺ cells from Scl^+/+ and Scl^-/- EBs. (A) FLK-1⁺ cells from day 3.0 Scl^+/+ or day 3.5 Scl^-/- EBs were cultured on gelatin-coated wells in the presence of VEGF and bFGF for 3 days. Following culture the cells were harvested and stained for CD31, FLK-1, VECAD, and SMA. Numbers indicate the percentage of the respective populations. (B) Immunohistochemistry of day 3 Scl^+/+ FLK-1⁺ cells (Scl^+/+) and day 3.5 Scl^-/- FLK-1⁺ cells (Scl^-/-) cultured on fibronectin-coated glass coverslips for 3 days. CD31⁺ cells are shown in red, whereas the SMA⁺ cells are indicated in green. Original magnification, × 200. Objective × 20, numerical aperture 0.70. OpenLab software was used to analyze the images. (C) FLK-1⁺ cells from day 3 Scl^+/+ or day 3.5 Scl^-/- EBs were allowed to aggregate by culturing at high density in low-adherence plates. The aggregates were cultured for 3 days in VEGF and bFGF and then processed for CD31, FLK-1, VECAD, and SMA as described in panel A. (D) RT-PCR analyses of day 3.0 Scl^+/+ FLK-1⁺ sorted cells (+/+, S), Scl^+/+ adherent cells (+/+, Ad), Scl^+/+ aggregated cells (+/+, Agg), Scl^-/- day 3.5 FLK-1⁺ sorted cells (-/-, S), Scl^-/- adherent cells (-/-, Ad) and Scl^-/- aggregated cells (-/-, Agg). Hybridization was carried out with probes designed against the 3′ UTR of the indicated genes. (E) RT-PCR analysis of day 3.0 Scl^+/+ FLK-1⁺ sorted cells (+/+, S), Scl^+/+ adherent cells (+/+, Ad), and Scl^+/+ aggregated cells (+/+, Agg) for the expression of the macrophage-specific genes c-fms, f4/80, CD11b, for the erythroid-specific gene, β-major, and for the endothelial-specific gene, VWF. β-Actin was used to normalize the content of cDNA in these populations.