Endothelial progenitor cell differentiation using cryopreserved, umbilical cord blood-derived mononuclear cells.
OBJECTIVE
To investigate the endothelial differentiation potentiality of umbilical cord blood (UCB), we induced the differentiation of endothelial progenitor cells (EPC) from cryopreserved UCB-derived mononuclear cells (MNC).


METHODS
MNC from cryopreserved UCB and peripheral blood (PB) were cultured in M199 medium with endothelial cell growth supplements for 14 d. EPC were characterized by RT-PCR, flow cytometry, and immunocytochemistry analysis. The proliferation of differentiated EPC was studied by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay, and vascular endothelial growth factor (VEGF) concentration was measured using an ELISA kit. Characteristics of UCB-derived EPC were compared with those of PB-derived EPC.


RESULTS
A number of round-shaped cells were loosely attached to the bottom after 24 h culture, and numerous spindleshaped cells began to appear from the round-shaped ones on d 7. Those cells expressed endothelial markers such as, Flt-1/VEGFR-1, ecNOS, VE-cadherin, von Willebrand factor, and secreted VEGF. The patterns of endothelial markers of EPC from PB and UCB did not show striking differences. The results of the proliferation and secretion of VEGF were also similar.


CONCLUSIONS
We successfully cultured UCB cells stored at -196 Celsius degree into cells with the quality of endothelial cells. Those EPC could be used for angiogenic therapeutics by activating adjacent endothelial cells and enhancing angiogenesis.
Jang
Jun-Ho
JH
Department of Hematology-Oncology, Ajou University, School of Medicine, Suwon 442749, Korea.
Kim
Sun-Kyung
SK
Choi
Jeong-Eun
JE
Kim
Young-Jin
YJ
Lee
Hyun-Woo
HW
Kang
Seok-Yun
SY
Park
Joon-Seong
JS
Choi
Jin-Hyuk
JH
Lim
Ho-Yeong
HY
Kim
Hugh C
HC
D015415:Biomarkers; D002454:Cell Differentiation; D002478:Cells, Cultured; D015925:Cryopreservation; D042783:Endothelial Cells / Q000502:physiology* / Q000648:ultrastructure; D005312:Fetal Blood / Q000166:cytology / Q000502:physiology*; D006801:Humans; D066298:In Vitro Techniques; D009000:Monocytes / Q000502:physiology*; D013234:Stem Cells / Q000502:physiology* / Q000648:ultrastructure; D042461:Vascular Endothelial Growth Factor A / Q000096:biosynthesis / Q000235:genetics
D015415:Biomarkers; D042461:Vascular Endothelial Growth Factor A