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Human amniotic fluid-derived stem (AFS) cells possess several advantages over embryonic and adult stem cells, as evidenced by expression of both types of stem cell markers and ability to differentiate into cells of all three germ layers. Herein, we examine endothelial differentiation of AFS cells in response to growth factors, shear force, and hypoxia. We isolated human AFS cells from amniotic fluid samples (1-4 cc/specimen) obtained from patients undergoing amniocentesis at 15-18 weeks of gestation (n = 10). Isolates maintained in nondifferentiating medium expressed the stem cell markers CD13, CD29, CD44, CD90, CD105, OCT-4, and SSEA-4 through passage 8. After 3 weeks of culture in endothelial growth media-2 (EGM-2), the stem cells exhibited an endothelial-like morphology, formed cord-like structures when plated on Matrigel, and uptook acetylated LDL/lectin. Additionally, mRNA and protein levels of CD31 and von Willebrand factor (vWF) significantly increased in response to culture in EGM-2, with further up-regulation when stimulated by physiological levels (12 dyne/cm(2)) of shear force. Culture in hypoxic conditions (5% O(2)) resulted in significant expression of vascular endothelial growth factor (VEGF) and placental growth factor (PGF) mRNA. This study suggests that AFS cells, isolated from minute amounts of amniotic fluid, acquire endothelial cell characteristics when stimulated by growth factors and shear force, and produce angiogenic factors (VEGF, PGF, and hepatocyte growth factor [HGF]) in response to hypoxia. Thus, amniotic fluid represents a rich source of mesenchymal stem cells potentially suitable for use in cardiovascular regenerative medicine.

Endothelial Differentiation of Amniotic Fluid–Derived Stem Cells: Synergism of Biochemical and Shear Force Stimuli