Seung-Woo Cho PhD, Sung-Hwan Moon MS, Soo-Hong Lee PhD, Sun-Woong Kang MS, Jumi Kim MS, Jae Min Lim MS, Hyo-Soo Kim MD, PhD, Byung-Soo Kim PhD^*, and Hyung-Min Chung PhD^*

From the Department of Bioengineering (S.C., B.K.), and Department of Chemical Engineering (S.K., J.M.L.), Hanyang University, Seoul, Korea; Department of Chemical Engineering (S.C.), Massachusetts Institute of Technology, Cambridge, Mass; Stem Cell Research Laboratory (S.M., S.L., J.K., H.C.), CHA Stem Cell Institute & CHA Biotech, Pochon CHA University, Seoul, Korea; and Department of Internal Medicine (H.K.), Seoul National University College of Medicine, Seoul, Korea.

^* To whom correspondence should be addressed.

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<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<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.

Key words: angiogenesis, stem cell transplantation, endothelial cells, ischemia, embryonic stem cells