Lady Davis Institute for Medical Research, Jewish General Hospital, McGill University, Montreal, Quebec, Canada.
Cell Transplant. 2011;20(9):1431-43. doi: 10.3727/096368910X557164. Epub 2011 Feb 3.
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.
内皮祖细胞 (EPCs) 由两个不同的亚群组成，分别称为早期 (eEPCs) 和晚期 EPCs (lEPCs)，它们分别来源于 CD14(+) 和 CD14(-) 循环细胞。这些细胞通常在纤维连接蛋白包被的表面上，在含有胰岛素样生长因子 (IGF-1)、血管内皮生长因子 (VEGF)、表皮生长因子 (EGF) 和成纤维细胞生长因子 (FGF) 的内皮基础培养基 (EBM)-2 中培养。我们开发了一种新的、简化的方法来培养来自外周血的人 EPCs，并测试了它们在梗塞后维持心脏功能的能力。我们首先证明，源自人外周血单核细胞 (PBMCs) 的 eEPCs 在纤维连接蛋白包被的表面上培养，在 EBM-2 培养基中补充自体血清 (10%)，在 IGF-1 (50ng/ml) 存在下培养，其分泌组与在常规条件下用 IGF-1、VEGF、EGF 和 FGF 培养的 eEPCs 相似。我们的数据还表明，IGF-1 以剂量依赖的方式调节 PBMC 的分泌组。在另一系列实验中，我们表明在纤维连接蛋白和 IGF-1 补充的基础培养基中悬浮培养在袋中的 PBMC (S-PBMCs) 分泌大量干细胞因子 (SCF，31.3 ± 3.1 pg/ml)、肝细胞生长因子 (HGF，438.6 ± 41.4 pg/ml)、可溶性肿瘤坏死因子受体 1 (sTNFR1，127.1 ± 9.9 pg/ml)、VEGF(139.3 ± 9.6 pg/ml) 和 IGF-1(147.2 ± 46.1 pg/ml)，但 TNF-α 的水平非常低 (13.4 ± 2.5 pg/ml)。静脉注射到 NOD SCID 小鼠体内的 S-PBMCs迁移到受损的心肌，减少心肌纤维化，增强血管生成，并在心肌梗塞 (MI) 后以类似于在标准条件下培养的 eEPCs 的方式保存心脏功能。总之，我们在这项研究中展示了一种改良和优化的培养 eEPCs 的方法。我们的数据表明，S-PBMC 由包括 eEPCs 在内的几个细胞群体组成，并且它们分泌大量的抗细胞凋亡、抗炎和促血管生成因子，能够在 MI 后保存心脏功能。
