Efficient nonadhesive ex vivo expansion of early endothelial progenitor cells derived from CD34+ human cord blood fraction for effective therapeutic vascularization.
Endothelial progenitor cells (EPCs) have been shown to have therapeutic potential in ischemic disease. However, the number of EPCs for cell therapy is limited. In this study, instead of the typical adherent culture method, we investigated a more efficient, clinically applicable nonadhesive expansion method for early EPCs using cord blood-derived cells to overcome rapid cellular senescence. After a suspension culture of isolated CD34(+) cells in serum-free medium containing each cytokine combination was maintained for 9 d, the number of expanded functional EPCs was assessed by an adherent culture assay. Compared to mononuclear cells, the CD34(+) fraction was superior in its expansion of functional EPCs that could differentiate into acLDL/UEA-1(+) cells without significant cellular senescence, whereas the CD34(-) fraction showed no EPC expansion. Among the cytokine combinations tested for the CD34(+) fraction, a combination (SFIb) consisting of stem cell factor (SCF), FMS-like tyrosine kinase 3 ligand, interleukin-3, and basic fibroblast growth factor resulted in a reproducible 64- to 1468-fold EPC expansion from various cord blood origins. Interestingly, the SFIb combination displayed markedly increased EPC expansion (2.43-fold), with a higher percentage of CD34(+) cells (2.17-fold), undifferentiated blasts (2.38-fold) and CXCR4(+) cells (1.68-fold) compared to another cytokine combination (SCF, thrombopoietin, and granulocyte colony-stimulating factor), although the two cytokine combinations had a similar level of total mononucleated cell expansion (∼ 10% difference). Accordingly, the cells expanded in the SFIb combination were more effective in recovery of blood flow and neovascularization in hind-limb ischemia in vivo. Taken together, these results suggest that the nonadhesive serum-free culture conditions of the CD34(+) fraction provide an effective EPC expansion method for cell therapy, and an expansion condition leading to high percentages of CD34(+) cells and blasts is likely important in EPC expansion.
O
Eunju
E
Cancer Research Institute, Department of Biomedical Sciences, College of Medicine, The Catholic University of Korea, Banpo-dong 505, Seocho-ku, Seoul 137-701, Korea.
Lee
Byung Hun
BH
Ahn
Hyun-Young
HY
Shin
Jong-Chul
JC
Kim
Hyun-Kyung
HK
Kim
Myungshin
M
Park
In-Yang
IY
Park
Yong-Gyu
YG
Joe
Young Ae
YA
D000818:Animals; D018952:Antigens, CD34 / Q000032:analysis*; D002448:Cell Adhesion; D018929:Cell Culture Techniques; D002460:Cell Line; D049109:Cell Proliferation; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000166:cytology* / Q000378:metabolism / Q000637:transplantation; D005312:Fetal Blood / Q000166:cytology; D006614:Hindlimb / Q000098:blood supply; D006801:Humans; D007511:Ischemia / Q000601:surgery; D007963:Leukocytes, Mononuclear / Q000166:cytology / Q000378:metabolism; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D018919:Neovascularization, Physiologic; D019408:Platelet Endothelial Cell Adhesion Molecule-1 / Q000032:analysis; D012039:Regional Blood Flow; D033581:Stem Cell Transplantation / Q000379:methods; D013234:Stem Cells / Q000166:cytology* / Q000378:metabolism; D013997:Time Factors
D018952:Antigens, CD34; D019408:Platelet Endothelial Cell Adhesion Molecule-1