Fig 1.
Isolation and characterization of amniotic fluid exosomes from rat fetal alcohol exposure (FAE) model.
(A) Rat FAE model schematic. Timed pregnant rats were treated with water or 2.5 g/kg of 20% EtOH by oral gavage at E5, 8, 10, 12 and 15. Animals were euthanized at E16 or E19 for embryo collection (n = 3 each). (B) Comparison of amniotic exosome and exosomal RNA isolation by ultracentrifugation and two commercial kits (Total Exosome Isolation Reagent from Thermo Fisher and ExoQuick-TC from SBI). Cell pellet (300 x g) fraction and RNA isolation by direct lysis of AF are included for comparison. Graphs shown at the bottom are Bioanalyzer profiles of extracted exosomal RNA using a Nano kit. (C) Electron microscopic examination of amniotic exosomes purified by ultracentrifugation and Total Exosome Isolation Reagent (Thermo Fisher) (bar = 100 nm). (D) Immunochemical assessment of exosome purification by staining exosomal proteins with anti-Calnexin (endoplasmic marker), anti-Actin and exosomal markers (anti-CD9, CD24 and CD63). The level of the markers in the cell pellet (300 x g) fraction and ultracentrifugation fraction (UC) was compared.
Fig 2.
Bioinformatics analysis of miRNA-Seq data on amniotic exosomal miRNAs.
(A) Processed RNA-seq data was mapped and found piRNA enriched in rat amniotic fluids compared to miRNA. Error bars represent 95% confidence interval. (B) Dendrogram was built to examine correlation among miRNAs in samples depending on the experiment (Exp1 and 2), collection time points (E16 and E19) and treatment (control vs. EtOH). Correlation among AF exosomal miRNAs for (C) E16 samples from Exp1 and 2 and (D) E19 samples from Exp1 and 2. (E) DESeq2 differential expression analysis for E16 samples from Exp1 and 2. (F) DESeq2 differential expression analysis for E19 samples from Exp1 and 2. (G) Selected six miRNAs with p < 0.05 differentially expressed in E16 samples from Exp1 and 2 are shown with circle. (H) Selected three miRNAs with p < 0.05 differentially expressed in E19 samples from Exp1 and 2 are shown with circle.
Fig 3.
Verification of amniotic exosomal miRNA targets affected by FAE.
Differential expression of amniotic exosomal miRNAs identified in FAE by RNA-Seq analysis were verified by qRT-PCR analysis for (A) downregulated in FAE and (B and C) upregulated in FAE. Summary of comparison between RNA-Seq and qRT-PCR analysis is shown in (D). The p value was determined by one-way ANOVA and the error bars represent the standard error in triplicate samples.
Fig 4.
Effects of FAE on miRNAs and osteomarker expression in fetal embryo tissues.
Whole rat fetal embryos at E16 from FAE model were used for total RNA isolation and (A) the levels of selected miRNAs and (B) osteogenic markers were determined by qRT-PCR analysis. The results of ΔCt values were compared between control group (n = 5) and EtOH group (n = 5). The p value was determined by one-way ANOVA and the error bar represents the standard error.
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.
Fig 6.
Effects of miRNAs upregulated in rat AF exosome by FAE on osteogenic differentiation of rat bone marrow stem cells.
Molecular effects of miR-199a-3p, miR-214-3p and let-7g on osteogenic differentiation of rBMSCs were determined by transfection study to undifferentiated rBMSCs. Effects of (A) miR-199a-3p, (B) miR-214 and (C) let-7g on osteogenic markers in the presence of osteo-induction (for 24 hrs) were examined. The p value (<0.05) was determined by one-way ANOVA and the error bar represents the standard error in triplicate samples.