Postnatal vasculogenesis has been implicated as an important mechanism for neovascularization via bone marrow-derived endothelial progenitor cells (EPCs) circulating in peripheral blood. In preparation of the utilization of EPCs in clinical protocols, we have generated blood-derived EPCs according to two established protocols by culturing either nonadherent mononuclear cells on fibronectin or adherent mononuclear cells on collagen. To explore the feasibility of these EPCs for their potential clinical use as target cells for genetic transduction to enhance their thromboresistance, newly designed retroviral and lentiviral gene ontology expression vectors were tested. Whereas cell clusters derived from the nonadherent cells demonstrated an only limited proliferative potential, cell colonies derived from collagen-adherent cells expanded more than a million-fold. Characterization of the exponentially growing cells by surface antigen and gene expression profiling revealed a consistently strong expression of characteristic endothelial markers, whereas expression of leukocyte markers was gradually lost. Using a single-step transduction protocol, we were able to achieve gene transfer efficiency of up to 99%. Our results suggest that the generated blood-derived EPC population might be attractive target cells for tissue engineering and gene therapy protocols due to their well defined phenotype, extensive proliferative potential, and efficient genetic transducibility, three important qualities that need to be defined prior to any clinical use.

Blood endothelial outgrowth cells (BEOC), bone marrow derived cells circulating in adult peripheral blood, may play an important role in postnatal vasculogenesis. Overexpression of antithrombotic genes in BOEC might enhance thromboresistance and open new avenues for the treatment of prosthetic and vascular thromboembolic disease. To genetically modify endothelial progenitor cells (EPC), we have used two different culture conditions for the generation of EPC from peripheral blood. Mononuclear cells harvested from peripheral blood of healthy volunteers by Ficoll-density gradient centrifugation were cultured (1) in fibronectin (FN)-coated plates with Endocult medium or (2) in collagen-coated plates with EBM2-MV medium. Colonies were counted at day 5 or between days 14 – 25, respectively. Cells were phenotypically analyzed on days 7, 14, 21, 28 or between days 30 – 60, respectively, for the following antigens: leukocyte markers CD45 and CD14, and endothelial markers CD31, CD34, CD105, CD141, CD144, CD146, and von-Willebrand (vW) antigen. Stem cell microarray hybridization was used to analyze the gene expression pattern. Proliferative potential was evaluated by determination of replating efficiency, growth kinetics, and CFSE dilution analysis. Using nonadherent cells cultured on FN-coated plates, colonies appeared on days 4–5. They consisted of central round cells with elongated cells sprouting at the periphery. These cells slowly disappeared after days 8–10. FACS analysis of these cells showed strong expression of CD45 and CD14, weak expression of CD31, but no expression of CD105, CD34, and vW-antigen. Using cells adherent to collagen, cells appeared at a median of 20 ± 5 days (range 9 – 29 days). These cells rapidly formed colonies with a cobblestone-like appearance and subsequently a confluent monolayer. After a lag phase, cell expanded exponentially (21 population doublings in 50–60 days). These cells showed strong expression of CD31, CD105, and CD146, intermediate expression of CD141, weak expression of CD34, and no expression of CD45 or CD14. Cells stained positive for VE-cadherin and vW-antigen. Microarray analysis displayed upregulated expression of endothelial genes such as PAI-1, uPA, eNOS, vWF, CD105, CD141, CD144, CD146, EphrinB4, TIE-1, VEGF-R2, endothelin and other genes such as CTGF, MMP2, ID1, ID3, TBX1, GATA2, FKHL16, ROBO4, but downregulation of CD11c, CD18, and L-selectin. An eGFP-encoding retroviral SFFV vector (cell free supernatant from PG13; titer 1.5x106 cfu) and a novel lentiviral LeGO vector expressing the red chomophore tdTomato (cell-free supernatant from PhoenixGP or 293T, packaging plasmids pMDLg/pRRE, pRSV-Rev; GALV env; 16x106 cfu) were used for centrifugation-enhanced transduction. BEOC were transduced with high efficiency, 62% – 80% and 91% – 99%, using the murine retroviral SF-eGFP and the human LeGo-T lentiviral vectors, respectively. Our results suggest that nonadherent cells cultured on FN-coated plates have a low proliferative potential and display an angiogenic macrophage-like phenotype. In contrast, adherent cells cultured on collagen-coated plates have a high proliferative potential, display an endothelial phenotype without coexpression of leukocyte antigens, and can be very efficiently transduced using retroviral vectors. Blood endothelial outgrowth cells may be an excellent autologous biomaterial source for vascular graft and device coatings, as well as for antithrombotic gene therapy.

Endothelial progenitor cells (EPC), bone marrow derived cells circulating in adult peripheral blood, may play an important role in postnatal vasculogenesis. We have used two different culture conditions for the isolation and generation of EPC from peripheral blood. Mononuclear cells (MNC) were harvested from peripheral blood (PB) of healthy volunteers by Ficoll-density gradient centrifugation. In the first method according to Hill et al., NEJM2003, 348: 593, cells were plated at a density of 5x106 cells/well in fibronectin-coated 6-well plates and cultured in Endocult (CellSystems). After a preplating step of 48 hours, non-adherent cells were cultured in fibronectin-coated 24-well plates at a density of 1x106/well. Emerging colonies (cell mass composed of a central cord of round cells with elongated spindle-shaped cells sprouting at the periphery) were counted at day 5. In the second method according to Lin et al., J Clin Invest2000, 105: 71, cells were plated at a density of 10x106 cells/well in collagen type I-coated 6-well plates and cultured in EBM2-MV (Cambrex) medium containing 5% FCS, VEGF, bFGF, R3-IGF-1, hEGF, hydrocortisone, and ascorbic acid. Emerging colonies were counted at day 14–25. Colony cells were phenotypically analyzed on day 7, 14, 21, 28 (method 1), and between day 40 – 55 (method 2) for the following antigens: leukocyte markers CD45 and CD14, and endothelial markers CD31 (PE-CAM), CD105 (endoglin), von-Willebrand factor, and CD34. Furthermore, the proliferative potential was evaluated by counting and determining the replating efficiency. Using method 1, colonies appeared on day 4–5. They consisted of centrally located round cells with elongated spindle-shaped cells sprouting at the periphery. The colonies disappeared after day 8–10; after 4 to 5 weeks the spindle-shaped cells degenerated to foamy cells. FACS analysis of the cells on day 5, 12, and 21 showed strong expression of CD45 and CD14, weak expression of CD31, but no expression of CD 105, CD34, and von-Willebrand factor. Using method 2, we observed cells with a different morphology and growth-pattern in 50% of the wells between 14–25 days. These cells rapidly replicated to form colonies with a cobblestone-like appearance which later formed a confluent monolayer. The cells continued growing for more 60 days. By FACS analysis, these cells showed strong expression of CD31 and CD105, weak expession of CD34, and no expression of CD45 or CD14. The cells stained positive for von-Willebrand factor. Our results suggest that cells which were cultured according to method 1 (fibronectin-coated plates, preplating step, use of non-adherent cells) display an angiogenic macrophage-like phenotype. These cells have a low proliferative potential. In contrast, cells which were cultured according to method 2 (collagen-type I-coated plates, no preplating, adherent cells) have a high proliferative potential and display an endothelial phenotype with no coexpression of leukocyte antigens. Since transduction with retroviral vectors depends on replication of target cells, isolated late endothelial outgrowth cells (EOC) generated according to the second method will be used for genetic modification to enhance the vasculogenetic properties of endothelial progenitor cells.