Generation of Endothelial Progenitor Cells from Amniotic Fluid Stem Cells

Recently it has been shown that human amniotic fluid contains stem cells of fetal origin (AFS) that express embryonic and adult stem cell markers. AFS can be expanded extensively in culture and give rise to multiple differentiated cells such those of myogenic, osteogenic, neurogenic and endothelial lineages, all of them with potential therapeutic value. In this study, we investigated the generation of endothelial progenitor cells (EPC) from AFS. For this purpose, remnant amniotic fluid was obtained from mothers undergoing amniocentesis for medical reasons at 18 weeks of pregnancy. Cells were collected by centrifugation and cultured in DMEM, supplemented with Chang medium and fetal bovine serum. Adherent cells were expanded and characterized by flow cytometry at various times using antibodies for CD34, CD45, CD133, CD117, KDR (VEGFR-2) and CD31. Cultures were also carried out in endothelial growth medium and analyzed for EPC markers (CD133, KDR, CD31). Adherent cells showed, at 8 and 17 days of culture, fibroblastoid features. Flow cytometry analysis, at 2 weeks of culture, showed cells expressing CD 133 (37%), CD 34 (1%), CD 117 (3%) and KDR (12%). Cultures carried out in endothelial differentiation medium (EDM), at 33 days of culture, showed a significant increase of cells expressing EPC markers: CD133 (40%), CD34 (3%) KDR (24%), KDR/CD31 (9%). These cells formed blood vessel-like structures on collagen-coated plates. The in vivo angiogenic capacity of these cells was demonstrated by inoculating them into collagen microspheres and implanting them subcutaneously into immunocompromised mice. Histologic examination revealed that these cells formed microvessels on implantation. Our data indicates that EPC can be generated and expanded from AFS, and they might be useful for therapeutic angiogenesis in ischemic tissues.