Amniotic fluid holds great promise as a stem cell source, especially in neonatal applications where autologous
cells can be isolated and used. This study examined chemical-mediated differentiation of amniotic fluid-derived
stem cells (AFSC) into endothelial cells and verified the function of AFSC-derived endothelial cells (AFSC-EC).
AFSC were isolated from amniotic fluid obtained from second trimester amnioreduction as part of therapeutic
intervention from pregnancies affected with twin-twin transfusion syndrome. Undifferentiated AFSC were of
normal karyotype with a subpopulation of cells positive for the embryonic stem cell marker SSEA4, hematopoietic
stem cell marker c-kit, and mesenchymal stem cell markers CD29, CD44, CD73, CD90, and CD105.
Additionally, these cells were negative for the endothelial marker CD31 and hematopoietic differentiation
marker CD45. AFSC were cultured in endothelial growth media with concentrations of vascular endothelial
growth factor (VEGF) ranging from 1 to 100 ng/mL. After 2 weeks, AFSC-EC expressed von Willebrand factor,
endothelial nitric oxide synthase, CD31, VE-cadherin, and VEGF receptor 2. Additionally, the percentage of cells
expressing CD31 was positively correlated with VEGF concentration up to 50 ng/mL, with no increase at higher
concentrations. AFSC-EC showed a decrease in stem cells markers c-kit and SSEA4 and were morphologically
similar to human umbilical vein endothelial cells (HUVEC). In functional assays, AFSC-EC formed networks and
metabolized acetylated low-density lipoprotein, also characteristic of HUVEC. Nitrate levels for AFSC-EC, an
indirect measure of nitric oxide synthesis, were significantly higher than undifferentiated controls and significantly
lower than HUVEC. These results indicate that AFSC can differentiate into functional endothelial-like cells
and may have the potential to provide vascularization for constructs used in regenerative medicine strategies
