Functional characterization of porcine adipose-derived stem cells into endothelial cells under hypoxic condition

Abstract

Background: Endothelial cells (EC) that make up the inner wall of blood vessels play an important role in angiogenesis and vascular recovery. Cardiovascular disease caused by dysfunction of ECs has been reported as a major cause of death worldwide. Despite significant research so far, the underlying mechanism of dysfunction of ECs in cardiovascular disease progression is not yet fully understood. Although therapeutic transplantation of autologous ECs is limited due to lack of cell availability, adiposederived stem cells (ADSCs), known for their ease of procurement and high potential for differentiation, will provide promising solutions to generate autologous ECs. Methods: This study investigated the optimal differentiation of ADSCs into ECs under EBM-2 culture conditions supplemented with VEGF and BMP-4 in hypoxia (2% O2). Results: During 14 days of in vitro differentiation, cells cultured in EBM-2 supplemented with VEGF showed the characteristics of early vascular ECs and some cells adopted polygonal forms. Conversely, cells cultured in EBM-2 and hypoxia supplemented with both VEGF and BMP-4 differentiated into the typical cobblestone morphology that appears in vascular ECs. As a result of immunostaining against the vascular ECs marker CD-31, CD-31 expression was increased under EBM-2 culture conditions with VEGF and VEGF + BMP-4 in hypoxia, but expression was insufficient in normal oxygenation (21% O2). In the flow cytometry analysis, high expression of CD-31 expression was observed under conditions including both VEGF and BMP-4 of hypoxia. Interestingly, in gene expression, the pluripotency marker OCT-3/4 was significantly reduced under hypoxic conditions, but SOX2 and NANOG expression were higher than under normal oxygen conditions. However, CD-31 expression was significantly higher under differentiation conditions in which VEGF and BMP-4 were added under hypoxia conditions. In a functional analysis, CD-31-positive ADSC-derived ECs differentiated under hypoxia had excellent tube formation and Dil-Ac-LDL uptake, which are important for vascular repair and function. Conclusions: These findings confirmed the therapeutic usefulness of ECs derived from ADSC for the treatment of cardiovascular disease due to the synergy effect of hypoxia and BMP-4.