Generation of blood vessel organoids from human embryonic stem cells

To establish and characterize vascular organoids based on the capacity of definitive differentiation and multiple cell self-organization of human embryonic stem cells (hESCs).

The hESCs were subjected to a stepwise induction protocol to sequentially differentiate into mesoderm progenitors, lateral plate mesoderm progenitors, and endothelial cells over a 5-day period. The resulting endothelial cell population was harvested and embedded in Matrigel to undergo three-dimensional culture of 6-8 days, allowing for self-organization into vascular organoids with branched luminal networks. The organoids were characterized via morphological analysis, flow cytometry, immunofluorescence staining, and transmission electron microscopy.

The generated organoids formed complex networks of branched lumens, containing (41.10±4.51)% CD31⁺ endothelial cells and (6.88%±0.40)% CD140b⁺ pericytes. Among the endothelial cells, (43.00±0.21)% were CD31⁺CD184⁺ arterial endothelial cells and (3.66±0.25)% were CD31⁺CD184^-CD73⁺ venous endothelial cells. Immunofluorescence analysis found that the vascular organoids were composed of VE-cadherin⁺Sox17⁺ arterial endothelial cells, VE-cadherin⁺NR2F2⁺ venous endothelial cells, PDGFR-β⁺ pericytes, α-SMA⁺ smooth muscle cells, and collagen I. Tight junctions were observed between endothelial cells, and characteristic Weibel-Palade body ultrastructures were identified within the cytoplasm that resembled the structures of human microvascular units.

We have established a robust method for generating hESCs-based vascular organoids that mimic the architecture of functional microvasculature. This three-dimensional vascular organoid can serve as a promising platform for research on vascular development and disease modeling.