Figure 2.

Hematopoiesis and angiogenesis in the para‐aortic splanchnopleural (P‐Sp) culture system. (A) P‐Sp culture system. (a) P‐Sp explants were cultured on OP9 stromal cells for 7–10 days. Cultured cells were fixed and stained with anti‐PECAM‐1/CD31 monoclonal antibody to stain the endothelial cells (EC; dark blue). This culture system supports vasculogenesis and angiogenesis. The vascular bed (vb), a sheet‐like structure, first appears near explants and mimics vasculogenesis because its formation depends on the vascular endothelial growth factor (VEGF)–VEGF receptor (VEGFR) system. Subsequently, vascular network (vn) formation mimics the process of fine capillary structure, angiogenesis, because this process depends on the angiopoietin–tyrosine kinase with Ig and EGF homology domain 2 (TIE2) system. (b) Higher magnification of box in (a). Note that there are many hematopoietic cells (HC) beside the EC forming a network‐like structure. (B) Schematized diagram of the P‐Sp cultures using AML1 mutant embryos. P‐Sp explants from AML1 mutant embryos, which lack hematopoietic stem cell (HSC) development, showed impaired vn formation. However, the disruption of vn formation observed in AML1 mutant culture was rescued by the exogenous addition of HSC from wild type embryos. Angiopoietin‐1 (Ang‐1) also rescued vn formation in AML1 mutant embryos, but addition of HSC from Ang‐1 mutant embryos did not, suggesting that Ang‐1 from HSC and HC is involved in vn formation.