BL-CFC and endothelial potential of Hex-deficient ES cells. (A) BL-CFC potential of Hex^+/+, Hex^+/-, and Hex^-/- EBs at the indicated time points. Numbers represent colonies per 2.5 × 10⁴ cells plated. Data represent means ± SEMs (n = 3). (B) BL-CFC potential of Hex^+/+ (□) and Hex^-/- (▨) Flk-1⁺ cells isolated from day-4 EBs. Data represent means ± SEMs (n = 3). (C) RT-PCR expression analysis of Hex^+/+ day-4 Flk-1⁺ cells cultured in VEGF and bFGF for varying periods of time. The 3′ cDNA was prepared by RT-PCR from the Flk-1⁺ population at the time of isolation (0) or following 3 or 5 days of culture. The cDNA samples were run on a 1.5% agarose gel, blotted, and probed separately with 3′ probes from the indicated genes. L32 was included as an internal control. Control populations include a pool of blast colonies (blast), a pool of primitive erythroid colonies (Ep), and the D4T endothelial cell line. (D) Flk-1⁺ cells (1 × 10⁴ cells) isolated from day 4 Hex^+/+ and Hex^-/- were cultured on matrigel-coated wells with VEGF and bFGF. Cells were harvested and counted at days 3 and 5 of culture. Data represent means ± SEMs (n = 3). (E) Immunohistochemistry showing CD31⁺ endothelial cell clusters (red) and SMA-positive smooth muscle cells (green) (× 50) at 5 days of culture. (F) The size of the CD31⁺ area in 8 separate fields of each culture was evaluated by image analyzer. The mean size of the CD31⁺ area in Hex^+/+ cultures was adjusted to 1. Data represent means ± SEM.