Characterization of mixed Hem-End colonies.

(A) Number of Hem-End colonies (% of plated cells) generated by CD34⁺KDR⁺ versus CD34⁺KDR⁻ cells from CB (▵,○) or BM (●) in 6 independent experiments (mean ± SEM values; cell number/well is indicated). See “Results.” (B) Microscopy analysis of a representative Hem-End colony (original magnifications, left × 50; right × 100). (C) Morphology of granulocytes, a large macrophage, and a putative endothelial cell in Hem-End colony (original magnification, × 400). (D) Immunofluorescence analysis of cells in Hem-End colony by double labeling with anti-CD45 (green) and anti-VWF (red) mAb (original magnification, × 600). (E-H) Characterization of mixed Hem-End colonies (representative results). (E,F) RT-PCR analysis of Hem-End colonies from primary Hem-End culture or secondary hematopoietic (Hem) and endothelial (End) culture—that is, a single Hem-End colony was divided in 2, and each half was grown in secondary Hem or End specific medium. (G) Hem-End colonies at day 40 were split into adherent (left) and suspension (right) fractions and analyzed for LDL uptake (red) and CD45 expression (green; original magnification, × 600). (H) Limiting-dilution analysis of Hem-End colony frequency by Poisson single-hit statistics.8