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Background— We established an efficient preparation method to obtain endothelial-like cells (ECs) from human embryonic stem cells (hESCs) and tested whether these hESC-ECs would show therapeutic potential for treatment of hindlimb ischemia.Methods and Results— ECs differentiated from hESCs were obtained by mechanical isolation and cell sorting for von Willebrand factor. The isolated hESC-ECs maintained endothelial cell–specific characteristics such as endothelial marker expression and capillary formation. One day after surgical induction of hindlimb ischemia in athymic mice, hESC-ECs were injected intramuscularly into ischemic limbs. Four weeks after treatment, hESC-EC treatment significantly increased limb salvage (36%) compared with treatment with medium (0%). In addition, laser Doppler imaging showed that the ratio of blood perfusion (ischemic to normal limb) was increased significantly (P &lt;0.01) by hESC-EC treatment (0.511±0.167) compared with medium injection (0.073±0.061). Capillary and arteriole densities were 658±190/mm2 and 30±11/mm2 in the hESC-EC group, respectively, whereas those in the medium group were 392±118/mm2 and 16±8/mm2 , respectively (P &lt;0.01). Reverse-transcription polymerase chain reaction with human-specific primers revealed mRNA expression of human endothelial markers and human angiogenic factors in ischemic mouse tissues. The transplanted hESC-ECs were localized as capillaries near muscle tissues in ischemic regions or incorporated in the vessels between muscle tissues, as confirmed by human nuclear antigen staining with platelet/endothelial cell adhesion molecule or von Willebrand factor.Conclusions— This study demonstrates that hESC-EC transplantation improves blood perfusion and limb salvage by facilitating postnatal neovascularization in a mouse model of hindlimb ischemia. Thus, hESC-ECs might be useful as an alternative cell source for angiogenic therapy.

circulation

Improvement of Postnatal Neovascularization by Human Embryonic Stem Cell–Derived Endothelial-Like Cell Transplantation in a Mouse Model of Hindlimb Ischemia