Background: Stem cells/progenitors are important for the development of cell therapies for vascular ischemic diseases. The crucial step in rescuing tissues from ischemia is improvement of vascularization that can be achieved by promoting neovascularization or growth of new blood vessels. Endothelial Progenitor Cells (EPCs) are the best candidates for developing such an approach due to their ability to self-renew, circulate and differentiate into mature endothelial cells (ECs). However, the successful clinical application of such therapy is limited by low quantities of EPCs that can be generated from patient and by the lack of adequate non-invasive imaging approach for in vivo monitoring bio-distribution of transplanted cells. Hypothesis: Ability of CB AC133+ EPCs to differentiate, in vitro and in vivo, towards mature ECs will not be affected by long term in vitro expansion and cryopreservation and when magnetically labeled, these cells will create sufficient T2 and T2* shortening (MRI parameters) to be detected by MRI. Methods and Results: AC133+ cells were collected from CB using MidiMACS system. Cells were maintained in culture for 5 to 30 days at the cell concentration of 1x106/ml. At days 10-15 and 25-30 of the primary in vitro culture, angiogenic properties of these cells were assessed in vitro and in vivo. For the in vitro experiments, cells were plated on fibronectin coated surface, induced to differentiate for two weeks, and analyzed by flow-cytometry and immunohistochemistry for the expression of mature EC markers and ability to incorporate DiI-acetylated-low density lipoprotein (DiI-Ac-LDL). In vivo differentiation was assessed in mouse matrigel angiogenic and rat glioma models. At days 10-15 and 25-30 of the primary culture, cells were magnetically (FePro) and fluorescently (DiI) labeled and IV injected into the mice or cryopreserved. Cryopreserved cells were then thawed, cultured for 30 min-2h and IV administered to glioma bearing rats. Seven days after injection, animals were analyzed by MRI that showed the migration and neovascular localization of administered EPCs. The presence of labeled EPCs within the neovasculature was confirmed by tissue immunohistochemistry. Conclusion: This study demonstrated that long term in vitro expanded cells preserved their angiogenic properties that were exhibited under in vitro and in vivo conditions and these properties were not abolished by cryopreservation.