In Vitro Comparison of Early and Late Phase Endothelial Progenitor Cells Induced from Adipose-derived Stem Cells

Abstract

Adipose-derived stem cells (ADSCs) are multipotent cells that can differentiate into endothelial progenitor cells (EPCs), which are essential for new vessel formation. EPCs can also be incorporated into blood vessels to promote neovascularization. This study aims to investigate the characteristics of the EPCs differentiated from ADSCs and identifying early and late phase EPCs-distinguishing characteristics for our future in vivo neovascularization study. ADSCs were isolated from adipose tissue and cultured to passage 1 (ADSCs P1) in complete DMEM. ADSCs P1 were then induced to differentiate into EPCs by culturing in EGM-2 BulletKit for 9 weeks. A microscopic examination of ADSCs P1 and EPCs collected at the 3-week (EPCs-3w) and 9-week (EPCs-9w) time points revealed differences in morphology between these 3 subsets. The gene expression levels of CD31, vWF, VE-cadherin, and VEGFR-2 were also different. The proliferation rate of EPCs was higher than that of ADSCs P1. Notably, EPCs-3w showed the highest proliferation rate, which then decreased with time of differentiation. Based on these comparisons, EPCs-3w and EPCs-9w were defined as the early phase EPCs and the late phase EPCs, respectively. Fluorescence staining with 1, 1ʹ-Dioctadecyl-3, 3, 3ʹ, 3ʹ-Tetramethylindocarbocyanine (Dil) was also performed. Cells were observed 1-and 3-d after Dil labeling to evaluate their ability to take up Dil. In conclusion, we compared EPCs from successive weeks of differentiation from ADSCs, and identified two phases (early phase and late phase) EPCs with distinct differences.