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Endothelial cells (ECs) play a key role in revascularization within regenerating tissue. Stem cells are often used as an alternative cell source when ECs are not available. Several cell types have been used to give rise to ECs, such as umbilical cord vessels, or differentiated from somatic stem cells, embryonic, or induced pluripotent stem cells. However, the latter carry the potential risk of chronic immune rejection and oncogenesis. Autologous endothelial precursors are an ideal resource, but currently require an invasive procedure to obtain them from the patient's own blood vessels or bone marrow. Thus, the goal of this study was to determine whether urine‐derived stem cells (USCs) could differentiate into functional ECs in vitro. Urine‐derived cells were then differentiated into cells of the endothelial lineage using endothelial differentiation medium for 14 days. Changes in morphology and ultrastructure, and functional endothelial marker expression were assessed in the induced USCs in vitro. Grafts of the differentiated USCs were then subcutaneously injected into nude mice. Induced USCs expressed significantly higher levels of specific markers of ECs (CD31, vWF, eNOS) in vitro and in vivo, compared to nondifferentiated USCs. In addition, the differentiated USC formed intricate tubular networks and presented similar tight junctions, and migration and invasion ability, as well as ability to produce nitric oxide (NO) compared to controls. Using USCs as autologous EC sources for vessel, tissue engineering strategies can yield a sufficient number of cells via a noninvasive, simple, and low‐cost method suitable for rapid clinical translation.  Stem   Cells   Translational   Medicine   2018  S tem  C ells  T ranslational  M edicine   2018;7:686–698

Human Urine‐Derived Stem Cell Differentiation to Endothelial Cells with Barrier Function and Nitric Oxide Production