Differentiation and proliferation of endothelial progenitor cells from canine peripheral blood mononuclear cells.
BACKGROUND
The isolation, differentiation, and expansion of endothelial progenitor cells (EPCs) from peripheral blood have potential applicability in areas of therapeutic neovascularization, vascular repair, and tissue engineering. The purpose of the current study was to elucidate a simple method of isolation and differentiation of EPCs by defining the endothelial morphology, surface marker expression, and proliferative capacity of EPC outgrowth from canine peripheral blood mononuclear cells (PBMCs).


METHODS
PBMCs were isolated from fresh canine blood and cultured in fibronectin-coated plates in which EPCs were identified from cell morphology and outgrowth characteristics. Cell surface markers were determined with flow cytometry analysis to identify differentiation of cultured and subcultured colonies. A hematologic counter with phase contrast microscopy was used to study cell growth curves of EPCs as compared with mature human coronary artery endothelial cells.


RESULTS
During the first week of canine PBMC culture, cells were morphologically round and varied in size, but in the course of the second and third week of culture, the cells, respectively, became spindle-shaped and displayed an endothelium-like cobblestone morphology with outgrowth. CD34 was significantly decreased at 21 days as compared with 7 days culture (36.04% to 21.37%), whereas vWF (from 77.26% to 96.37%) and eNOS (from 0% to 14.97%) were significantly increased. VEGFR-2 was slightly increased, and P1H12 (CD146) was unchanged. Subcultured canine EPCs displayed a higher proliferation rate as compared to mature human coronary artery endothelial cells in the same culture conditions.


CONCLUSIONS
These data demonstrate that canine EPCs can be isolated and cultured from the canine PBMC fraction. These outgrowth cells displayed characteristics of endothelial morphology with endothelial cell-specific surface markers. Furthermore, it was revealed that canine EPCs have a greater growth potential as compared to mature endothelial cells. This study suggests that PBMCs could be used as a source of EPCs for potential applications in tissue engineering and vascular therapy.
Wu
Huakang
H
Molecular Surgeon Research Center, Division of Vascular Surgery and Endovascular Therapy, Michael E. DeBakey Department of Surgery, Baylor College of Medicine, Houston, Texas, USA.
Riha
Gordon M
GM
Yang
Hui
H
Li
Min
M
Yao
Qizhi
Q
Chen
Changyi
C
D000818:Animals; D015703:Antigens, CD / Q000378:metabolism; D015415:Biomarkers / Q000378:metabolism; D051929:CD146 Antigen; D002452:Cell Count; D002454:Cell Differentiation; D002462:Cell Membrane / Q000378:metabolism; D049109:Cell Proliferation; D002478:Cells, Cultured; D004285:Dogs; D042783:Endothelial Cells / Q000166:cytology* / Q000378:metabolism; D009000:Monocytes / Q000166:cytology* / Q000378:metabolism; D019006:Neural Cell Adhesion Molecules / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000378:metabolism; D052250:Nitric Oxide Synthase Type III; D013234:Stem Cells / Q000166:cytology* / Q000378:metabolism; D013997:Time Factors; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000378:metabolism; D014841:von Willebrand Factor / Q000378:metabolism
D015703:Antigens, CD; D015415:Biomarkers; D051929:CD146 Antigen; D019006:Neural Cell Adhesion Molecules; D014841:von Willebrand Factor; D019001:Nitric Oxide Synthase; D052250:Nitric Oxide Synthase Type III; D040301:Vascular Endothelial Growth Factor Receptor-2