Fig. 4

In vitro and in vivo endothelial differentiation and miR expression of AFS cells. a AFS cells were seeded over Matrigel-coated wells, and phase-contrast images showed network formation after 24 hours (scale bar = 100 μm). Image analysis of those network structures (b) produced values that were compared against standard positive control cells (HUVECs, left white bars), and statistical significance is shown among AFS cells and control with asterisks, also between different concentrations of oxygen for the same trimester with hashes (n = 10). Immunofluorescence stainings of endothelial network (c) showed vWF (green signal) expression (scale bar = 100 μm). d Ability of endothelial cells to intake AcLDL: green vesicles are indicated by white arrows, phase contrast (left) and fluorescence detection (right) are shown for better appreciation (scale bar = 200 μm). *P < 0.05, **P < 0.01, ***P < 0.001 ^# P < 0.05, ^### P < 0.001. e Time course of pro angiomiR (miR126, 132 and 210) and anti angiomiR (miR221 and 222) expression after differentiation. f Upper row: Matrigel plugs (n = 3 for each condition. Experiment # = experiment number) underwent hemoglobin (Hb) test: relative quantification of Hb was higher in plugs loaded with AFS cells cultured at 5 %. Lower row: appearance of the harvested plugs with and without cells before Hb quantification. g Representative expression of vWF (green signal) co-localized with a specific human antigen (human mitochondria, red signal) (scale bar = 50 μm). All nuclei in the figure were counterstained with DAPI. AcLDL acetylated-low density lipoprotein, AFS amniotic fluid stem, A.U. arbitrary units, CB cord blood, DAPI 4′,6-diamidino-2-phenylindole, HUVEC human umbilical vein endothelial cell, PBS phosphate-buffered saline, PhC phase contrast, vWF von Willebrand factor