Experimental culture condition for endothelial and their progenitor cells from CD34+ hematopoietic stem cells / 대한수혈학회지

ABSTRACT

BACKGROUND: The concept of "neovascularization", which was first applied to describe the pathogenesis of diseases such as diabetic retinopathy or rheumatoid arthritis, has been extended to other fields of study such as myocardial ischemia and tumorigenesis. Endothelial progenitor cells (EPCs), play a critical role in neovascularization and have been reported to also be capable of colonizing vascular grafts. In this study, EPCs were isolated from cord blood, peripheral blood and bone marrow, and then cultured. Various cytokines, such as vascular endothelial growth factor(VEGF), Insulin growth factor(IGF), endothelial growth factor(EGF) fibroblast growth factor-basic(FGF-b), stem cell factor(SCF), flt3-ligand(FL), and thrombopoietin(TPO) were added to the cultures and observed for their effects on endothelial cells for their potential use in antineoplastic therapy or treatment of regional ischemia. METHODS: The mononuclear cells (MNCs) were isolated from cord blood, peripheral blood buffy coat, and bone marrow. They were collected from healthy donors using Ficoll-Hypaque. CD34+ cells were isolated by MACS system. To evaluate the effect of various cytokines, purified CD34+ cells were cultured under conditions of various cytokine combinations including SCF, Fl, TPO, VEGF, EGF, IGF, and FGF-b. After four weeks of culture, umbilical cord blood and bone-marrow derived adherent cells were analyzed for endothelial markers by immunohistochemical stain. RESULTS: Cultured adherent cells expressed the endothelial specific markers, such as KDR, CD34, CD31, CD62E, and CD cadherin but did not express vWF antigen. Typical morphology of endothelial cells was observed, such as the cord-like structure and cobblestone appearance during the culture period, which suggested that the adherent cells were consistent with endothelial cells. CONCLUSION: We described the experimental conditions in which endothelial progenitors were differentiated from CD34+ cells isolated from three hematopoietic stem cell sources bone marrow, peripheral blood and cord blood.