The percentage of -gal-positive cells from the total number of cells was calculated (tube formation on Matrigel has been used as a strategy to characterize the angiogenic potential of ECs. P-Selectin etc. These cells achieved unlimited proliferation potential subsequent to inactivation of the cyclin-dependent kinase inhibitor p21, but failed to form colonies on soft agar. We also show their enhanced growth and survival on vascular biomaterials compared to parental cultures in late population doubling. These immortalized EPCs can be used as a cellular model system for studying the biology of these cells, gene manipulation experiments, cellCbiomaterial interactions, as well as a variety of tissue-engineering applications. Introduction Endothelial progenitor cells (EPCs), the circulating pool of progenitor cells with potential to differentiate into functional endothelial cells (ECs), have been proposed as a vital cell source for therapeutic neovascularization, vascular repair, studies on tumor angiogenesis, tissue-engineering efforts, and as a biomarker for a variety of disease says.1C5 Adult peripheral blood as well as umbilical cord blood (UCB) have been used as a good source for various kinds of stem cells, including hematopoietic stem cells, mesenchymal stem cells, and EPCs. The identification and characterization of adult circulating EPCs by Asahara led to the expansion in the field of circulating EC biology.6 Later studies substantiate the existence of Manidipine 2HCl these cells, their bone marrow origin, and their contribution in the regeneration of new blood vessels in adults.7,8 They are characterized by their unique surface feature of CD34+, AC133+, and VEGFR2+expression and the loss of AC133 upon endothelial maturation in culture.9,10 The physiological role of EPCs is not yet clearly established. These cells play a key role in promoting neovascularization of ischemic tissues in nude mice and in developing into neovessels when seeded in decellularized grafts.11 They also express a variety of potent vasoactive regulators of functional ECs such as nitric oxide (NO), VE Cadherin, von Willebrand factor (vWF), and are involved in the protection against tissue degeneration due to vascular damage or dysfunction. 12 The number of EPCs is usually inversely correlated with the risk for coronary Manidipine 2HCl artery diseases, indicating their potential use as a prognostic tool for cardiovascular diseases.13 Several studies substantiated the importance of circulating EPCs in tumor vascularization.14C18 All these properties make this unique cell population an ideal choice for various tissue-engineering applications as well as to study variety of vascular diseases in conditions and in tumor vascularization models.19C21 Currently, there are two subsets of EPCs that have been identified from peripheral blood and UCB. The early EPCs are more like mature ECs with enhanced angiogenic ability and less multiplication potential and assumed to be of myeloid origin than the late EPCs with enhanced proliferation potential.22 Even though late EPCs possess enhanced proliferation potential in defined culture conditions than the early progenitors, it is difficult to maintain these cells in culture beyond 100 days, necessitating repeated isolation of EPCs.23 A clear definition of their identity, functional characterization, and other tissue-engineering Rabbit Polyclonal to SPINK5 applications involving this unique cell type requires a defined homogenous functional cell line with unlimited proliferation potential. Overexpression of human telomerase and viral oncoproteins such as E6, E7, and SV40 has been evaluated for immortalization of a variety of diploid cells, including ECs.24C26 Even though the introduction of telomerase is considered as Manidipine 2HCl a promising method to immortalize normal cells compared to other approaches, several studies indicate that telomerase can only extend lifespan of cells.27C29 In this work, we have generated two EC lines by stably introducing SV40T large antigen using retroviral infection. Both the cell lines retained the functional house of mature ECs and proliferated continually in culture without any manifestation of senescence-like phenotype for more than 150 population doubling (PD) compared to the vector control cells that failed to grow beyond 40C50PD.