Highly efficient lentiviral transduction of phenotypically and genotypically characterized endothelial progenitor cells from adult peripheral blood

Abstract

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.

Labeling and Qualification of Endothelial Progenitor Cells for Tracking in Tissue Engineering: An in Vitro Study

“…From our findings, the present method achieved a transduction efficiency greater than 93% TdTomato positive cells. Such a high fluorescence yield is a result consistent with that of Stockschlaeder (19), who achieved gene transfer efficiency of up to 99% with endothelial progenitor cell from adult peripheral blood. Yockell-Lelievre et al (17) reported an average transduction efficiency of 68% in human umbilical vein endothelial cells (HUVECs) as well as in human aortic endothelial cells and human microvascular endothelial cells using a double-pulse electroporation method.…”

A Systematic Approach to the Establishment and Characterization of Endothelial Progenitor Cells for Gene Therapy

“…Increasing MOI to 10 and 60 resulted in 80% and 88% transduction efficiency, respectively (De Meyer et al, 2006;van den Biggelaar et al, 2009). The transduction efficiency could be improved to as high as 96% when using a lentiviral vector LeGo with an SFFV-U3-derived internal promoter (Stockschlaeder et al, 2010), demonstrating that transduction efficiencies may vary significantly depending on the particular viral vector, internal promoter, and particularly the MOI used. In addition, we found that transduction efficiency could also be improved from 47% to 79% when transducing EPC colonies containing fewer cells, suggesting that transduction of isolated EPC colonies as early as possible could be an improved way to obtain a large population of transduced EPCs.…”