Acceleration of endothelial cell differentiation from CD14-positive monocytes

Abstract

Introduction. In vitro differentiation of endothelial cells has potential applications in vascular tissue engineering and cell-based therapy for many diseases. The purpose of this study was to develop a new strategy that utilizes cytokines and LPS to accelerate the process of endothelial cell differentiation. Methods. CD14-positive monocytes were purified from human peripheral blood mononuclear cells (PBMC) by MACS separator. The purified cells were cultured in EBM2 endothelial cell-culture medium supplemented with GM-CSF and IL-4 with or without LPS for 4 days. The cells were then continuously cultured in EBM2 medium for additional 10 days (a total 14 days culture). Monocyte, dendritic cell, macrophage, endothelial cell, as well as smooth muscle cell markers were analyzed by real-time PCR, flow cytometry, and immunofluorescence staining. Results. In the LPS-treated group, most of suspended CD14-positive monocytes were attached and distinguishably changed in morphology to endothelial-like cells (ELCs) as fast as 4 days, but no morphology change was observed in the absence of LPS treatment. At day 8, the ELCs expressed high levels of endothelial cell markers CD31 (67.7%), vWF (93.9%), and VEGF receptor 1 (94.9%), but no smooth muscle cell markers were detected. At day 14, the ELCs grew to form cobblestone morphology and formed outgrowth colonies, which are typical endothelial cell growth characteristics. Moreover, there was gradually decreased expression of CD14 during the course of the differentiation. There were nearly no dendritic cell maturation marker CD83 (1.5%) and macrophage marker CD68 (2.1%). Importantly, eNOS expression on ELC was substantially increased from 4 to 86%. Furthermore, ELCs showed endothelial functional characteristics of uptaking acetylated LDL and binding ulex-lectin. Conclusions. These data demonstrate that LPS may trigger the switch of CD14-positive monocytes to endothelial cell differentiation in the endothelial culture medium. After the switch, cells continue to fully differentiate into endothelial cells in a relative short period of 14 days.