Nestin expression during differentiation of fetal endothelial progenitor cells and hypoxic culture of human umbilical vein endothelial cells

Abstract

OBJECTIVE: The aim of this study was to assess whether nestin is expressed during differentiation of endothelial progenitor cells (EPCs) obtained from human umbilical cord blood (HUCB), and nestin expression is changed in hypoxia-conditioned culture of human umbilical vein endothelial cells (HUVECs).

METHODS: Among deliveries at our institute, 20 normal pregnant women who delivered by cesarean section at 37-40 weeks' gestation were selected. HUCB mononuclear cells (MNCs) from HUCB were isolated and cultivated. After characterization of CXCR4/KDR/CD34 positive cells (EPCs) by flow cytometry and fluorescent chemical staining, EPC culture was continued through day 10 for differentiation to outgrowth endothelial cell (OEC). For identification of EPC and OEC, RT-PCR was performed for each specific cell markers, such as AC133, CD45, CXCR4, CDH5, vWF, eNOs, CD34, and Flt 1. HUVECs were isolated from human umbilical cord veins by collagenase treatment. Culture of HUVEC in hypoxic and normoxic conditions was performed for 24 h. Nestin expression in EPCs, OECs and HUVECs was detected by RT-PCR and Western blotting.

RESULTS: Western blot analysis and RT-PCR revealed that nestin was not expressed in EPC, but well expressed in OECs and HUVECs. During 24 h of HUVEC culture, time course gene expression of VEGF was significantly increased, but nestin was not changed.

CONCLUSIONS: Our results showed that nestin could be used as a new differentiation marker of EPCs, and hypoxic stimuli did not directly affect nestin gene expression