Acceleration of endothelial-like cell differentiation from CD14+ monocytes in vitro.
OBJECTIVE
In vitro differentiation of endothelial cells has potential applications in vascular tissue engineering and cell-based therapy for many diseases. The objective of this study was to develop a new strategy that utilizes cytokines and lipopolysaccharide (LPS) to accelerate endothelial-like cell differentiation from peripheral blood CD14(+) monocytes.


METHODS
Peripheral blood CD14(+) monocytes were purified with immunobeads and cultured with an angiogenic growth factor-rich growth medium (EGM-2) with or without initial treatment of LPS in combination of granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4) for 4 days (the day 4 cultures). The cells were then continuously cultured in EGM-2 medium for an additional 4 or 10 days (the day 8 or day 14 cultures). Cell markers were determined by flow cytometry analysis and immunofluorescence staining. Cytokine/chemokine profile was studied by Bio-Plex immunoassay.


RESULTS
In the group of initial treatment of LPS in combination with GM-CSF, IL-4, and EGM-2, the majority of suspended CD14(+) monocytes were attached and changed their morphology to endothelial-like cells, which expressed high levels of endothelial cell markers CD31, von Willebrand factor, and vascular endothelial growth factor receptor-1 as well as two major endothelial tight junction proteins zonula occludens -1 and occludin in the day 8 cultures. Endothelial nitric oxide synthase expression was substantially increased. Endothelial-like cells were also able to uptake acetylated low-density lipoprotein and bind to Ulex europeus lectin. In addition, endothelial-like cells showed a unique cytokine/chemokine profile with substantial increases of macrophage inflammatory protein-1beta, IL-6, granulocyte colony-stimulating factor, and IL-8.


CONCLUSIONS
Initial treatment of LPS in combination with GM-CSF, IL-4, and EGM-2 is an effective strategy for acceleration of endothelial-like cell differentiation from peripheral blood CD14(+) monocytes in vitro.
Zhang
Rongxin
R
Molecular Surgeon Research Center, Division of Vascular and Endovascular Therapy, Michael E. DeBakey Department of Surgery, Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, TX 77030, USA.
Yang
Hui
H
Li
Min
M
Yao
Qizhi
Q
Chen
Changyi
C
D015415:Biomarkers / Q000032:analysis; D001773:Blood Cells; D018929:Cell Culture Techniques / Q000379:methods; D002454:Cell Differentiation / Q000187:drug effects*; D048430:Cell Shape / Q000187:drug effects; D003470:Culture Media / Q000494:pharmacology; D016207:Cytokines / Q000096:biosynthesis / Q000494:pharmacology; D042783:Endothelial Cells / Q000166:cytology*; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000494:pharmacology; D006801:Humans; D015847:Interleukin-4 / Q000494:pharmacology; D018950:Lipopolysaccharide Receptors; D008070:Lipopolysaccharides / Q000494:pharmacology; D009000:Monocytes / Q000166:cytology* / Q000187:drug effects; D019108:Tight Junctions
D015415:Biomarkers; D003470:Culture Media; D016207:Cytokines; D018950:Lipopolysaccharide Receptors; D008070:Lipopolysaccharides; D015847:Interleukin-4; D016178:Granulocyte-Macrophage Colony-Stimulating Factor