Abstract Body: Background
The emergence of functional endothelial and hematopoietic lineages from embryonic stem cells (ESCs) is fundamental to vascular and blood development. Vascular endothelial growth factor A (VEGF-A) is considered a pivotal regulator of angiogenesis, influencing endothelial progenitor cell proliferation, migration, and survival. Traditional two-dimensional (2D) culture systems have improved our understanding of early lineage commitment, but they do not fully capture the three-dimensional (3D) microenvironment that more closely mimics in vivo conditions. Recent studies have emphasized the importance of 3D culture platforms in promoting more physiologically relevant differentiation and cell–cell interactions, yet systematic comparisons of 2D versus 3D approaches in VEGF-mediated endothelial and hematopoietic differentiation remain limited.
Methods
Mouse ESCs underwent a standardized mesoderm induction protocol beginning at day 0. After four days, Flk1⁺ multipotent progenitors were purified by fluorescence-activated cell sorting (FACS) and then cultured either in 2D adherent plates or within a 3D hydrogel matrix.
Results
Flow cytometric analyses revealed that VEGF-A supplementation increased the percentage of CD144⁺ endothelial cells in both 2D and 3D cultures. However, the effect was markedly more pronounced in the 3D system, with a dose-dependent elevation in endothelial markers at VEGF-A concentrations ranging from 1 to 100 ng/mL. Concomitantly, hematopoietic lineage cells (CD41⁺) declined in the presence of VEGF-A, more substantially in 3D cultures, suggesting a shift in lineage commitment favoring an endothelial phenotype. Immunofluorescence staining confirmed robust CD144 expression and outlined the formation of tubular-like networks in 3D cultures receiving VEGF-A, whereas 2D monolayers displayed flattened endothelial cell morphologies with less pronounced cell–cell connections.
Conclusion
Our findings indicate that 3D culture conditions enhance the pro-endothelial effects of VEGF-A on mouse ESC differentiation relative to conventional 2D systems. The 3D environment appears to mimic aspects of embryonic tissue architecture, allowing for improved cellular interactions and factor gradients that promote endothelial lineage specification. These results advance the understanding of how microenvironmental factors modulate vascular commitment and may provide a foundation for tissue engineering strategies that rely on robust vascularization.