A Novel and Simplified Method of Culture of Human Blood-Derived Early Endothelial Progenitor Cells for the Treatment of Ischemic Vascular Disease

• Published in: Cell transplantation Vol. 20; no. 9; pp. 1431 - 1443
• Main Authors: Bouchentouf, M., Forner, K., Cuerquis, J., Boulassel, M. R., Routy, J. P., Waller, E. K., Quyyumi, A. A., Paradis, P., Schiffrin, E. L., Galipeau, J.
• Format: Journal Article
• Language: English
• Published: Los Angeles, CA SAGE Publications 01.01.2011; SAGE Publishing
• Subjects: Angiogenesis Inducing Agents - metabolism; Animals; Apoptosis - drug effects; Blood Cells - cytology; Blood Cells - drug effects; Blood Cells - secretion; Cell Adhesion - drug effects; Cell Culture Techniques - economics; Cell Culture Techniques - methods; Cell Differentiation - drug effects; Cells, Cultured; Endothelial Cells - cytology; Endothelial Cells - drug effects; Endothelial Cells - transplantation; Fibronectins - pharmacology; Heart Function Tests - drug effects; Humans; Inflammation Mediators - metabolism; Injections, Intravenous; Insulin-Like Growth Factor I - pharmacology; Ischemia - complications; Ischemia - physiopathology; Ischemia - therapy; Mice; Mice, Inbred NOD; Mice, SCID; Myocardial Infarction - complications; Myocardial Infarction - pathology; Myocardial Infarction - physiopathology; Myocardial Infarction - therapy; Stem Cell Transplantation; Vascular Diseases - complications; Vascular Diseases - physiopathology; Vascular Diseases - therapy; Angiogenesis; Endothelial progenitor cells (EPCs); Growth factors; Culture method; Myocardial infarction (MI)
• ISSN: 0963-6897; 1555-3892
• DOI: 10.3727/096368910X557164

Endothelial progenitor cells (EPCs) consist of two different subpopulations named early (eEPCs) and late EPCs (lEPCs) that are derived from CD14+ and CD14- circulating cells, respectively. These cells are regularly cultured over fibronectin-coated surfaces in endothelial basal medium (EBM)-2 supplemented with insulin-like growth factor (IGF-1), vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), and fibroblast growth factor (FGF). We have developed a new and simplified method for culturing human EPCs obtained from peripheral blood and tested their ability to preserve cardiac function following infarction. We first demonstrated that eEPCs derived from human peripheral blood mononuclear cells (PBMCs) and cultured in EBM-2 medium supplemented with autologous serum (10%) over fibronectin-coated surfaces (10 μg/ml) in the presence of IGF-1 (50 ng/ml) only, have a secretome similar to eEPCs cultured under regular conditions with IGF-1, VEGF, EGF, and FGF. Our data also indicate that IGF-1 modulates PBMC secretome in a dose-dependent manner. In another series of experiments, we showed that PBMCs cultured in suspension in bags (S-PBMCs) in basal medium supplemented with fibronectin and IGF-1 secrete significant amounts of stem cell factor (SCF, 31.3 ± 3.1 pg/ml)), hepatocyte growth factor (HGF, 438.6 ± 41.4 pg/ml), soluble tumor necrosis factor receptor 1 (sTNFR1, 127.1 ± 9.9 pg/ml), VEGF (139.3 ± 9.6 pg/ml), and IGF-1 (147.2 ± 46.1 pg/ml) but very low levels of TNF-α (13.4 ± 2.5 pg/ml). S-PBMCs injected intravenously into NOD SCID mice migrated to the injured myocardium, reduced cardiac fibrosis, enhanced angiogenesis, and preserved cardiac function after myocardial infarction (MI) in a manner similar to eEPCs cultured under standard conditions. In conclusion, we show in this study a refined and optimized method for culturing eEPCs. Our data indicate that S-PBMCs are composed of several cell populations including eEPCs and that they secrete high amounts of antiapoptotic, anti-inflammatory, and proangiogenic factors capable of preserving cardiac function following MI.