Abstract
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.
Immunohistochemical Study Of Fibronectin, Tenascin and Type I Collagen In Tongue And Lip carcinomas