Xenografted human amniotic fluid-derived stem cell as a cell source in therapeutic angiogenesis.
Amniotic fluid-derived stem cells (AFSCs) are pluripotent with high renewal capacity and are not tumorigenic. We tested whether AFSCs can function as a cell source for therapeutic angiogenesis in a mouse hindlimb ischemia model. Using a defined culture medium for endothelial lineage cells (ECs), we differentiated human AFSCs into AFSC-derived ECs (AFSC-ECs) in vitro, as evidenced by expression of EC markers, and capillary-like network formation on Matrigel. We assessed the in vivo therapeutic angiogenesis efficacy of AFSC-ECs in an athymic nude mouse model of hindlimb ischemia. One day after high ligation of the external iliac artery in athymic nude mice, AFSC-ECs were intramuscularly injected into ischemic limbs. The AFSC-ECs demonstrated endothelial cell characteristics in vitro. Four weeks later, AFSC-ECs transplantation significantly increased limb salvage (85%), compared to AFSCs (56%), human umbilical vein endothelial cells (HUVECs; 25%), or medium (0%). Laser Doppler perfusion analysis revealed that the ischemic/normal limb blood perfusion ratio significantly improved in the AFSC-EC group. AFSC-EC transplantation significantly increased capillary and arteriole densities as compared to AFSCs, HUVECs, and medium. Transplanted AFSC-ECs were incorporated into vessels in the ischemic region, as confirmed by immunofluorescent staining for human smooth muscle 22α or von Willebrand factor. Matrix metalloproteinase (MMP)-3 and MMP-9 expressions were significantly higher in AFSC-ECs. MMP-9 might activate angiogenesis by regulation of vascular endothelial growth factor. Our study indicated that AFSC-EC transplantation improved limb salvage and blood perfusion by promoting neovascularization. Therefore, AFSC-ECs possess the potential for therapeutic angiogenesis.