Derivation of functional endothelial progenitor cells from human umbilical cord blood mononuclear cells isolated by a novel cell filtration device.
Endothelial progenitor cells (EPCs) can differentiate from mononuclear cells (MNCs) of adult human peripheral blood, bone marrow, and cord blood during culture. Although MNCs are usually isolated by a Ficoll gradient centrifuge method, this method is time-consuming, and blood is easily contaminated. We developed a novel cell filtration device (StemQuickE, Asahi Kasei Medical, Oita, Tokyo, Japan) to isolate MNCs from human cord blood and examined whether functional EPCs could differentiate from MNCs isolated by this device. Recovery rates of MNCs, CD34(+) and CD133(+) progenitor cells, were significantly greater in the StemQuickE method than in the Ficoll method. During MNC culture, spindle-shaped attaching cells developed, and most of these cells incorporated DiI-acetylated low-density lipoprotein and showed positive binding to fluorescein isothiocyanate-lectin. Reverse transcription-polymerase chain reaction analysis revealed that attaching cells expressed various progenitor and endothelial lineage markers such as KDR, CD31, endothelial cell nitric oxide synthase, CD133, and LOX-1. Culture-expanded EPCs were isolated and labeled with a green fluorescent dye, PKH2-GL, and cocultured with human umbilical vein endothelial cells (HUVECs). EPCs formed angiogenesis-like networks together with HUVECs in 3D matrix gel. Finally, EPCs (3 x 10(5)) were implanted into ischemic hindlimb of nude rats (n = 3), and laser Doppler blood flowmetry (LDBF) revealed that the ratio of ischemic to normal limb LDBF was significantly greater in EPC-transplanted animals compared with controls receiving saline. In conclusion, the novel cell filtration device, StemQuickE, is a useful tool to isolate MNCs from human cord blood. Moreover, MNCs obtained by this filter system can give rise to functional EPCs.
Aoki
Mika
M
Department of Cardiology, Nagoya University Graduate School of Medicine, 65 Tsurumai, Showa-ku, Nagoya 466-8550, Japan.
Yasutake
Mikitomo
M
Murohara
Toyoaki
T
D000071916:AC133 Antigen; D000818:Animals; D015703:Antigens, CD; D018952:Antigens, CD34 / Q000096:biosynthesis; D001854:Bone Marrow Cells / Q000166:cytology; D018929:Cell Culture Techniques / Q000379:methods*; D019070:Cell Lineage; D002469:Cell Separation; D002478:Cells, Cultured; D018920:Coculture Techniques; D004730:Endothelium, Vascular / Q000166:cytology*; D005312:Fetal Blood / Q000166:cytology*; D005434:Flow Cytometry; D016650:Fluorescein-5-isothiocyanate / Q000494:pharmacology; D006023:Glycoproteins / Q000096:biosynthesis; D006801:Humans; D017078:Laser-Doppler Flowmetry; D017493:Leukocyte Common Antigens / Q000096:biosynthesis; D007963:Leukocytes, Mononuclear / Q000166:cytology*; D008856:Microscopy, Fluorescence; D009389:Neovascularization, Pathologic; D019001:Nitric Oxide Synthase / Q000096:biosynthesis; D052250:Nitric Oxide Synthase Type III; D010455:Peptides; D019408:Platelet Endothelial Cell Adhesion Molecule-1 / Q000096:biosynthesis; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D011923:Rats, Nude; D011973:Receptors, LDL / Q000096:biosynthesis; D051131:Receptors, Oxidized LDL; D020133:Reverse Transcriptase Polymerase Chain Reaction; D051127:Scavenger Receptors, Class E; D013234:Stem Cells / Q000166:cytology*; D013997:Time Factors; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000096:biosynthesis
D000071916:AC133 Antigen; D015703:Antigens, CD; D018952:Antigens, CD34; D006023:Glycoproteins; C495137:OLR1 protein, human; C495136:OLR1 protein, rat; C000606023:PROM1 protein, human; D010455:Peptides; D019408:Platelet Endothelial Cell Adhesion Molecule-1; C000606025:Prom1 protein, rat; D012333:RNA, Messenger; D011973:Receptors, LDL; D051131:Receptors, Oxidized LDL; D051127:Scavenger Receptors, Class E; C496314:NOS3 protein, human; D019001:Nitric Oxide Synthase; D052250:Nitric Oxide Synthase Type III; C496316:Nos3 protein, rat; D040301:Vascular Endothelial Growth Factor Receptor-2; D017493:Leukocyte Common Antigens; D016650:Fluorescein-5-isothiocyanate