Evidence of hematopoietic differentiation, vasculogenesis and angiogenesis in the formation of human choroidal blood vessels

Human fetal eyes 8–40 weeks gestation (WG) were examined using markers to hematopoietic stem cells (HSC), vascular precursor cells (VPC), monocytes/macrophages and endothelial cells (EC). Electron microscopy and bromo-deoxyuridene labeling were undertaken to confirm the existence of solid vascular cords and to demonstrate vasculogenesis and angiogenesis in developing choroidal tissue. Our results demonstrated that the earliest incipient choroid consisted of vimentin⁺ mesenchymal precursor cells which downregulated vimentin expression with maturation. Our observations lead us to conclude that these vimentin⁻/CD34⁺/CD44⁺/CD133⁺ HSCs then differentiated into three distinct lineages: single isolated CD34⁻/CD39⁺ VPCs that formed solid vascular cords which lumenized and became lined with CD34⁺ vascular ECs; CD34⁻⁻⁺/CD14⁺/CD68⁺ monocytes that differentiated into tissue macrophages; and CD133⁺/CD34⁻⁻⁺/α-smooth muscle actin⁺ mural precursor cells that matured into smooth muscle cells and pericytes. Blood vessel formation occurred throughout the whole choroid simultaneously, indicative of in situ differentiation. Vasculogenesis, as evidenced by lumenization of solid vascular cords, was responsible for the formation of the entire choroidal area with angiogenesis, in all three layers of the choroid, only adding to vascular density. These results suggest that formation of the human choroid involves three processes: HSC differentiation, vasculogenesis and angiogenesis. Since vasculogenesis takes place independently of VEGF₁₆₅, further insights regarding the molecular mechanisms of vasculogenesis are required to better inform future treatments of choroidal neovascularization.