Endothelial progenitor cells (EPCs) play a reparative role during vascular regeneration in ischemic pathologies such as myocardial infarction, stroke, peripheral vascular disease, and ischemic retinopathies. For autologous therapy, EPCs need to be expanded in vitro in order to obtain sufficient numbers for therapy. Many EPC sub-classes have been described and this creates confusion surrounding EPC nomenclature. For this study, we have concentrated on the subpopulation known as Outgrowth endothelial cells (OECs) because these EPCs have unequivocal endothelial phenotype and significant proliferative potential. Here, we examined implications that this long term in vitro expansion step has on human OEC biology. OECs were isolated from human peripheral and umbilical cord blood and expanded in culture until they reached their Hayflick limit. Cellular senescence was characterised by growth curves, cell cycle analysis, senescence-associated beta-galactosidase evaluation and DNA damage quantification. Furthermore, changes in gene/protein expression associated with OEC senescence were assessed by transcriptome, proteome and secretome analysis. OEC function was evaluated in vitro by migration and tubulogenesis assays. The therapeutic potential of senescent OECs was evaluated in vivo using a mouse model of ischaemic retinopathy. Proliferation capacity in late passage-OEC diminished significantly, with more than 70% of cells arrested in G0/G1 phase of the cell cycle. These late passage-OECs displayed a significant increase in cytoplasmic volume, increased β-galactosidase activity and accumulated γ-H2AX foci (p<0.001). There was also a significant decrease in telomerase activity coupled with telomere shortening. Senescent OECs demonstrated impaired migratory and tubulogenic capacity compared to early-passage OECs (p<0.001). Inflammatory signalling pathways were identified as major components of the OEC senescence programme by transcriptome analysis. IL8 was found significantly upregulated on the secretome of senescent OECs. shRNA mediated knock-down of IL8 in OECs significantly extended ex-vivo lifespan, delayed senescence and enhanced cellular function. Using the in-vivo animal model of ischaemic retinopathy, both early and late-passage OECs significantly reduced retinal avascular area compared to vehicle treated eyes (p<0.001). However, senescent OECs showed reduced ability to integrate into the retinal vasculature compared to early-passage OECs. Furthermore, only early-passage OECs significantly reduced the area of pathological pre-retinal neovascularisation (p<0.001). Ex-vivo expansion of human OECs ultimately leads to replicative senescence linked to cellular dysfunction in vitro and in vivo. Modulation of OEC senescence is an important strategy to improve reparative potential and usage as an autologous cell therapy.