Fig. 5. CD34⁺ CD45^– cells sorted from embryonic tissues develop into endothelial cells in culture. Endothelial cells isolated from umbilical cord veins and CD34⁺ CD45^– cells sorted from the 32-day yolk sac, 32-day AGM and 44-day embryonic liver were grown in EGM2 medium. Cells were observed directly by phase-contrast microscopy (A-D) or, after immunostaining, by fluorescence microscopy (E-T). Fixed cells were stained indirectly with biotinylated UEA-1 (I-L), with an antibody to CD31 (E-H) or with an antibody to vWF, after permeabilization (M- P). After 7 days of culture, confluent layers of cells with typical endothelial shape were obtained from CD34⁺ CD45^– cells sorted from the yolk sac (A), AGM (B) and embryonic liver (C), when compared with HUVEC (D). Endothelial cells developed from CD34⁺ CD45^– cells sorted from the yolk sac (E,I,M), AGM (F,J,N) or embryonic liver (G,K,O), when compared with HUVEC (H,L,P), all express CD31, UEA-1 ligand and vWF. vWF isotype controls on endothelial cells developed in culture from CD34⁺ CD45- cells sorted from the yolk sac (Q), AGM (R), embryonic liver (S) and from the umbilical vein (T). Scale bars: 50 µm in A-D; 5 µm in E-T.