Effect of hematopoietic cells expressing NP-1 on vascular development in the P-Sp culture.

(A) The development of ECs in P-Sp cultures from E9.5 NP-1^−/− embryos. Culture plates were fixed after 14 days of culture and stained with anti–PECAM-1 mAb. PECAM-1⁺ cells are visualized as dark blue products. (i) In mutant embryo, formation of the vascular bed and network is defective. (ii) The addition of CD45⁺ cells from the fetal liver of E12.5 GFP embryo (5 × 10³ cells per well), which are also positive for NP-1, rescued the defective formation of the vascular bed (vb) and network (vn); however, these cells did not differentiate to endothelial cells as confirmed by their morphology (iv). (iii) The addition of CD45⁺ cells from the fetal liver of E12.5 NP-1^−/− embryo did not rescue the defective vascular formation. (iv) Localization of GFP⁺ HCs in panel ii. Formation of vb and vn observed in panel ii is comparable to that observed in the culture using P-Sp explants from wild-type embryo. Scale bar indicates 200 μm. (B) Comparison of the area of the vascular bed. The area of the vascular beds in the images in panel A was determined by NIH image 1.62 software. The vascular area per explant is as follows: (i) 0.3 ± 0.1 mm²; (ii) 20.5 ± 3.2 mm²; (iii) 3.5 ± 0.5 mm². Each result was obtained from 3 independent experiments and is expressed as the mean ± SD.