Development of lymphatic endothelial cells from ES cells. The Tie2⁺Flk1⁺ fraction of ES cell–derived cells formed erythroid colonies (A) and granulocyte-macrophage colonies (B) in methylcellulose. Vascular and lymphatic endothelial cells were stained with PECAM-1 mAb (C) and LYVE-1 mAb (D), respectively. PECAM-1⁺ cells (green, panel E) and LYVE-1⁺ cells (green, panel F) took up acetyl-LDL (red, panels E and F). Scale bars, 10 μm. (I) Expression of LYVE-1 and Tie2 in differentiated cells derived from ES cells on OP9 cells on day 6 of culture was analyzed by flow cytometry. The percentages of cells in each quadrant are indicated. (J) Lymphatic-specific genes, Pdpn, VEGFR-3, and Prox-1 in LYVE-1⁺ and LYVE-1^- cells were analyzed by RT-PCR. (K) Immunostaining at high magnification (× 200) showed that LYVE-1⁺ cells (green) coexpressed Prox-1 (red). TOTO3 (blue) was used to stain nuclei. (L) In the presence of VEGF-C and VEGF-D (100 ng/mL each), the number of LYVE-1⁺ colonies increased to 2 times that of mock-treated cells. Colonies were also larger in the presence of VEGF-C and VEGF-D (panels G and H, respectively). Results are expressed as the mean ± SD. (M) OP9 and ES cell–derived LYVE-1^- cells expressed Ang1. LYVE-1⁺ cells expressed low levels of Ang1 and Ang2. Ang1, Ang2, and Ang3 were not detectable in ES cells and lymphatic precursors (Flk1⁺ cells derived from ES cells).