Molecular consequences of fetal alcohol exposure on amniotic exosomal miRNAs with functional implications for stem cell potency and differentiation
Fig 5
Uptake and functional effects of FAE amniotic exosomes on osteogenic potency of rat bone marrow stem cells.
(A) After a 24 hr incubation period, PKH26-labeled exosomes were detected in the cytoplasm of β-Actin- and DAPI-labeled rBMSCs. Scale bar = 10 μm. Exosomes from amniotic fluids (control and FAE group) were purified, labeled with PKH26, and incubated with rBMSCs. After 24 hr incubation, cells were extensively washed and stained with anti-β-Actin antibody for cytoskeleton and DAPI for nucleus. Images were obtained with Olympus IX81 fluorescence microscope at 60× magnification. (B) Cells were induced for osteogenic differentiation in the absence or presence of 20 mM EtOH (alternating treatment by two-day treatment and two-day withdrawal) or exosome treatment every third day. After 4 weeks of osteogenic induction with 20 mM EtOH, exosome from control (+Exo Cont) or exosome from FAE (+Exo EtOH) group, cells were stained for alkaline phosphatase or mineral deposit by Alizarin Red staining. (C) Effects of EtOH (0 or 20 mM) 24-hr treatment on osteogenic potency of rBMSCs as determined by qRT-PCR analysis. (D) The effects of exosomes from control group (Exo control) or FAE group (Exo EtOH) on 24-hr osteogenic induction were determined. Relative fold changes in osteogenic markers from (+)Osteo-induction only, (+) Osteo-induction + Exo control and (+) Osteo-induction + Exo EtOH are shown. The p value was determined by one-way ANOVA and the error bar represents the standard error in triplicate samples.