The culture-expanded AFS cells at 3 104/mL were induced in NH OsteoDiff Medium (Miltenyi Biotec) for 3 weeks. 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 AZD4017 levels (12 dyne/cm2) of shear pressure. Culture in hypoxic conditions (5% O2) 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 pressure, 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. == Introduction == Numerous stem and progenitorcell types demonstrate the potential to differentiate into cardiovascular cell-like phenotypes, particularly those derived from the bone marrow, cord and peripheral blood, and adipose tissue [17]. Presently, human adult bone marrow is the most common source of mesenchymal stem cells for use in regenerative medicine purposes. Unfortunately, the frequency and potential of cells derived from human adult bone marrow decreases with donor age and morbidity [810], potentially limiting their use in certain patient populations. Recently, several groups have isolated pluripotent stem cells from human amniotic fluid [1115]. These stem cells possess several potential advantages over both embryonic and adult stem cells. Considered to be at an intermediate stage between these two basic stem cell types, amniotic fluidstem (AFS) cells express both types of stem cell markers, proliferate rapidly, and differentiate into cells of all three embryonic germ layers [1620]. Kaviani et al. exhibited that these cells proliferate more rapidly than comparable fetal and adult cells in culture and attach strongly to polyglycolic acid polymer [17]. This study suggested that small amounts of amniotic fluid yield enough stem cells to create a tissue-engineered construct ready for implantation immediately after the birth of the child. Due to their higher proliferative capacity and ability to maintain pluripotency at higher passage numbers, these cells may prove to be a readily available source for large numbers of cells for other types of cardiovascular regenerative medicine applications. Both endothelial growth factors and shear pressure mediate endothelial differentiation and vascular development. Recently, the effect of shear on embryonic stem, endothelial progenitor, and mesenchymal stem cells has been investigated [2124]. Shear pressure increases proliferation, differentiation, capillary tube formation, vascular endothelial growth factor (VEGF) receptor expression, and production of tissue plasminogen activator and nitric oxide in these cells [2528]. Currently, the effect of shear on amniotic fluid-derived stem cell differentiation into vascular cells is usually unknown. Understanding this effect may provide us with an additional stimulus to use in developing the clinical potential for this unusual stem cell. The purpose of this study is usually to further evaluate the potential use of amniotic fluid stem cells for use in cardiovascular regeneration strategies. Specifically, we evaluate the isolation of these cells from minute amounts of amniotic fluid and their subsequent acquisition of endothelial cell characteristics after stimulation with both endothelial growth factors and shear pressure. Additionally, since hypoxia modulates proangiogenic cytokine production, we also evaluate the effect of hypoxia around the function of AFS cells in this AZD4017 setting. == Materials AZD4017 and Methods == == Human AFS cell isolation and culture == Under an IRB-approved protocol, human amniotic fluid samples (14 cc/specimen) were obtained from patients undergoing amniocentesis for clinical indications at 1518 weeks of gestation (n= 10). The procedure was described by Kim et al. [11] with some modifications. After centrifugation the AFS cells were plated in a 24-well culture dishes and produced in AmnioMAX II Complete (Gibco, Grand Island, NY) medium at 37C with 5% CO2atmosphere. Non-adherent cells were removed after 5 days and the medium was subsequently replaced twice weekly. When the cells reached confluence, they were subcultured in T-25 cm2flasks made up of expansion medium (M199 media with HEPES buffer, heparin (7.5 U/mL), and 10% of PlGF-2 FBS). For experiments involving hypoxic culture conditions (5% O2), a hypoxia workstation (Invivo2, Ruskinn Technology, Cleveland, OH) was utilized, allowing for accurate and uninterrupted hypoxic conditions. All cells used for experimentation were between passages 4 and 8. == Confirmation of multipotency == While the purpose of this study is usually to further define the endothelial.