IL-3 induces arterial specification of CD45⁺ cells acting as vasculogenic cells. (A) Q-RT-PCR was performed on CD45⁺ cells cultured with or without IL-3. The indicated arterial and venous markers were evaluated. Expression levels are presented as fold increase (logarithmic scale) in comparison with baseline levels and were normalized by using GAPDH as housekeeping gene. The mRNA isolated from arterial or venous samples were used as positive control (+; *P < .05, day 2 of culture vs days 4, 6, 8, and 12). (B) IL-3–expanded CD45⁺ cells were stained with CD31 (red), with EphrinB1 (green; top panel) or with EphB4 (green; bottom panel). Colocalization of CD31 and EphrinB1 or CD31 and EphB4 is reported in merge. DAPI staining was used as nuclear marker. Magnification, 20×. (C) Sections of Matrigel plugs containing TECs, recovered from SCID mice injected (panel ii) with prelabeled IL-3–cultured CD45⁺ cells, were analyzed by immunohistochemistry (IH). Panel i shows representative functional vessel formed by TECs. Black arrows indicate the incorporated CD45⁺ cells. The number of incorporated cells per vessel was reported on the right (4 nonsequential sections for each experiment were analyzed). Magnification, 40×. (D) IH of Matrigel plugs containing IL-3 and labeled CD45⁺ cells implanted subcutaneously into SCID mice. Panels i,iii, Matrigel recovered after 5 days; panels ii and iv, Matrigel recovered after 15 days. Prelabeled cells were detected by Fluorescein/Oregon Green Antibody (panels i,ii). Ephrin B1 staining was also performed (panels iii,iv). Black arrows indicate positive CD45⁺ cells. Magnification, 20× (panels i and iii) and 40× (panels ii and iv). (E) Matrigel plugs recovered after 15 days were stained with anti–human HLA I (red) or with anti–mouse MHC II (green) antibodies. DAPI staining was used as nuclear marker. Magnification, 20×. The number of human or host-derived vessels is reported in the histogram. Three different experiments were performed with comparable results.