Expansion of canine bone marrow-derived endothelial progenitor cells and dynamic observation.
We cultured bone marrow-derived endothelial progenitor cells by a simple ex vivo expansion method and observed the expansion efficacy. Bone marrow mononuclear cells from five mongrel adult dogs were cultured with EGM-2MV medium in culture flasks coated with fibronectin. Morphology was observed with phase contrast microscopy, and a growth curve was constructed to evaluate the efficacy of expansion. Incorporation of Dil-ac-LDL was tested to evaluate the function. At different time points, immunocytochemical staining for flk-1, CD133, and factor VIII-related antigens was done and compared to staining of endothelial cells and mesenchymal stem cells and percentages of CD133, vascular endothelial growth factor receptor 2 (VEGFR-2), and the double-positive cells measured with flow cytometry to identify the quality and efficacy of expansion. Cluster-like attached cells grew to confluence at an average time of 10 days, and the mean number of cells harvested from 1 mL of bone marrow was (1.3 +/- 0.3) x 10(6). The cells presented a cobblestone-like appearance and took up Dil-ac-LDL. Immunocytochemistry showed that flk-1, CD133, and factor VIII-related antigens were positive. Flow cytometry showed that VEGFR-2 and CD133 double-positive cells augmented 242-fold at the tenth day. Ex vivo expansion can effectively proliferate endothelial progenitor cells from bone marrow; the expansion efficacy could meet the requirements for tissue engineering of blood vessels.
Wu
YingFeng
Y
Department of Vascular Surgery, XuanWu Hospital and the Institute of Vascular Surgery, Capital University of Medical Sciences, Beijing, People's Republic of China. wyingfeng@yahoo.com
Zhang
Jian
J
Gu
YongQuan
Y
Li
JianXin
J
Chen
Bing
B
Guo
LianRui
L
Luo
Tao
T
Wang
ZhongGao
Z
D000071916:AC133 Antigen; D000818:Animals; D015703:Antigens, CD / Q000032:analysis; D001854:Bone Marrow Cells / Q000166:cytology* / Q000276:immunology / Q000378:metabolism; D002232:Carbocyanines / Q000378:metabolism; D002454:Cell Differentiation; D049109:Cell Proliferation; D048430:Cell Shape; D002478:Cells, Cultured; D004285:Dogs; D042783:Endothelial Cells / Q000166:cytology* / Q000276:immunology / Q000378:metabolism; D006023:Glycoproteins / Q000032:analysis; D059630:Mesenchymal Stem Cells / Q000166:cytology* / Q000276:immunology / Q000378:metabolism; D010455:Peptides / Q000032:analysis; D013997:Time Factors; D023822:Tissue Engineering / Q000379:methods; D014841:von Willebrand Factor / Q000032:analysis
D000071916:AC133 Antigen; D015703:Antigens, CD; D002232:Carbocyanines; D006023:Glycoproteins; D010455:Peptides; D014841:von Willebrand Factor; C024286:3,3'-dioctadecylindocarbocyanine