Abstract: Breakdown of blood-brain barrier, formed mainly by brain microvascular endothelial cells (BMECs), rep- resents the major cause of mortality during early phases of ischemic strokes. Hence, discovery of novel agents that can efectively replace dead or dying endothelial cells to restore blood-brain barrier integrity is of paramount importance in stroke medicine. Although endothelial progenitor cells (EPCs) represent one such agents, their rarity in peripheral blood severely limits their adequate isolation and therapeutic use for acute ischemic stroke which necessitate their ex vivo expansion and generate early EPCs and outgrowth endothelial cells (OECs) as a result. Functional analyses of these cells, in the present study, demonstrated that only OECs endocytosed DiI-labelled acetylated low-density lipoprotein and formed tubules on matri- gel, prominent endothelial cell and angiogenesis markers, respectively. Further analyses by fow cytometry demonstrated that OECs expressed specifc markers for stemness (CD34), immaturity (CD133) and endo- thelial cells (CD31) but not for hematopoietic cells (CD45). Like BMECs, OECs established an equally tight in vitro model of human BBB with astrocytes and pericytes, suggesting their capacity to form tight junc- tions. Ischemic injury mimicked by concurrent deprivation of oxygen and glucose (4 hours) or deprivation of oxygen and glucose followed by reperfusion (20 hours) afected both barrier integrity and function in a similar fashion as evidenced by decreases in transendothelial electrical resistance and increases in para- cellular fux, respectively. Wound scratch assays comparing the vasculoreparative capacity of cells revealed that, compared to BMECs, OECs possessed a greater proliferative and directional migratory capacity. In a triple culture model of BBB established with astrocytes, pericytes and BMEC, exogenous addition of OECs effectively repaired the damage induced on endothelial layer in serum-free conditions. Taken together, these data demonstrate that OECs may efectively home to the site of vascular injury and repair the damage to maintain (neuro)vascular homeostasis during or afer a cerebral ischemic injury

Key words: cell-based therapy, endothelial progenitor cells, endothelium, ischemic stroke, neurodegeneration, novel therapeutics, outgrowth endothelial cells, regenerative medicine, stem cells, translational medicine