Endothelial progenitor cell culture and differentiation in vitro: a methodological comparison using human umbilical cord blood.
OBJECTIVE
Endothelial progenitor cells (EPC) can contribute to vascular repair and targeted tumour therapy. Little is known about generating EPC from human umbilical cord blood. We therefore compared methods for purification of EPC from human umbilical cord blood.


METHODS
Mononuclear cells were isolated from human umbilical cord blood by density gradient centrifugation and used either unselected or after CD34 preselection. Unselected mononuclear cells were cultured for 9 days. Culture-dish-adherent (CDAC) and non-adherent (CDNAC) CD34+ cells were cultured separately for 4 weeks. Surface markers were assessed by immunofluorescence staining and FACS analysis.


RESULTS
In unselected mononuclear cells, VEGF-R2 and VE-cadherin expression increased up to day 6. They stained positive with UEA-1 and took up acetylated LDL. Expression of CD45 and CD14 decreased over time, but remained strong. CD133 and CD34 were not expressed. CD34+-CDNAC acquired an endothelial phenotype over time with an increase of VEGFR-2 and von Willebrand factor (vWF). CD45 and CD14 decreased, while CD34 and the progenitor-cell marker CD133 remained strongly expressed. CD34(+)-CDAC showed a strong increase in VEGFR-2, CD133, CD34 and vWF, while CD14 decreased, and CD45 did not change.


CONCLUSIONS
Putative EPC can be obtained from human umbilical cord blood. When selected for CD34, cells can be differentiated in culture to express markers of mature endothelial cells, while keeping progenitor markers. In contrast, short-term culture of unselected mononuclear cells leads to an endothelioid-monocytoid phenotype devoid of progenitor markers. Thus, the outgrowth from CD34-selected cells appears to be superior to short-term culture of unselected mononuclear cells with regard to endothelial cell-lineage specific differentiation of cells with a progenitor marker profile.
Eggermann
Juliane
J
Department of Internal Medicine II (Cardiology), Ulm University Medical Centre, Robert-Koch-Str. 8, D-89081, Ulm, Germany.
Kliche
Stefanie
S
Jarmy
Gergely
G
Hoffmann
Karin
K
Mayr-Beyrle
Ulrike
U
Debatin
Klaus Michael
KM
Waltenberger
Johannes
J
Beltinger
Christian
C
D000071916:AC133 Antigen; D015703:Antigens, CD; D018952:Antigens, CD34 / Q000032:analysis; D015415:Biomarkers / Q000032:analysis; D018929:Cell Culture Techniques / Q000379:methods; D002454:Cell Differentiation; D002469:Cell Separation; D000071063:Endoglin; D004730:Endothelium, Vascular / Q000166:cytology*; D005312:Fetal Blood; D006023:Glycoproteins / Q000032:analysis; D006801:Humans; D017493:Leukocyte Common Antigens / Q000032:analysis; D018950:Lipopolysaccharide Receptors / Q000032:analysis; D010455:Peptides / Q000032:analysis; D011956:Receptors, Cell Surface; D013234:Stem Cells / Q000166:cytology* / Q000276:immunology; D013997:Time Factors; D019010:Vascular Cell Adhesion Molecule-1 / Q000032:analysis; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000032:analysis; D014841:von Willebrand Factor / Q000032:analysis
D000071916:AC133 Antigen; D015703:Antigens, CD; D018952:Antigens, CD34; D015415:Biomarkers; C064206:ENG protein, human; D000071063:Endoglin; D006023:Glycoproteins; D018950:Lipopolysaccharide Receptors; C000606023:PROM1 protein, human; D010455:Peptides; D011956:Receptors, Cell Surface; D019010:Vascular Cell Adhesion Molecule-1; D014841:von Willebrand Factor; D040301:Vascular Endothelial Growth Factor Receptor-2; D017493:Leukocyte Common Antigens