Small extracellular vesicles enhance the survival of Sca-1+ cardiac stem cells against ROS-induced ischemic-reoxygenation injury in vitro

Figures

• 

  Fig. 1. Flow chart of the study design and consecutive steps conducted in the study. BMMSCs: Bone marrow mesenchymal stem cells; CFU: Colony forming unit; CSCs: Cardiac stem cells; DLS: Dynamic light scattering; sEVs: sEVs-treated group; HIF-1α: Hypoxia-inducible factor-1α; MDA: Malondialdehyde; OGD: Oxygen glucose deprivation; OGD/R: Oxygen glucose deprivation/reoxygenation; PI: Propidium iodide; TAC: Total antioxidant capacity; TEM: Transmission electron microscope

• 

  Fig. 2. Characterization of BMMSCs-extracted sEVs. (A) The size of the isolated sEVs by the dynamic light scattering (DLS). The intensity analysis curve shows the average peak size of sEVs to be ~ 49.7 nm. (B) The TEM (120 kV) confirms the presence of sEVs with a heterogeneous size, ranging from 18 to 50 nm (scale bars 200 and 50 nm, respectively). (C) The flow cytometric analysis of fluorescently labeled sEVs shows 97.7% of CD63+, 98.6% of CD81+/CD9+, and negatively mitotracker-stained vesicles. The representative flow cytometry plots are among triplicate runs, with the number of acquired vesicles per single flow reading equal to 5000 events

• 

  Fig. 3. Characterization of CSCs/Sca-1+. (A) Representative phase contrast microscopic images of CSCs (scale bar 100 μm) at passages P0, P3, and P6, with 90% confluence from an initial seeding density of 25 × 104 CSCs in a six-well plate. (B) Confocal microscopy images of CSCs GATA4 cardiac transcriptional factor followed by Hoechst staining (representative of five random confocal microscopic images examined with a scale bar of 23.2 μm). GATA4 fluorescence is green, counterstaining of the nucleus with Hoechst is blue, and overlaid images represent co-localization. (C) Flow cytometry assay displays the percentage expression of CSCs markers (Sca1, CD90, CD117, OCT4, CD105, and CD45). The flow cytometer readings were analyzed in triplicate with the acquisition of 10,000 events

• 

  Fig. 4. HIF-1α expression in the sEVs CSCs/Sca-1+ group in different experimental groups.(A) Fluorescent microscopic images (scale bar 25 μm) show the HIF-1α immunofluorescent staining of CSCs/Sca-1+ under control normoxic conditions and OGD, with significant** (p < 0.01) visible signal in OGD-CSCs as documented in the bar chart of the immunofluorescent analysis. (B) The representative dot plots of flow cytometric analysis of HIF-1α expression reveal its percentage in the gated cells of untreated and treated groups, where sEVs- CSCs/Sca-1+ express the HIF-1α significantly**** higher than the control as displayed in the associated graph bar. (C) The bar chart of the gene expression analysis of Hif-1α mRNA RT-qPCR. Relative mRNA expression of Hif-1α in control, OGD, OGD/R, and sEVs CSCs/Sca-1+ groups. The results are presented as fold change relative to the control group, and data are normalized against Gapdh. The data in all bar charts are the mean ± SD of three independent experiments, each performed in triplicate (from 25 × 104 CSCs/Sca-1+ seeded into three wells of six-well plates). Unpaired t-test was used to analyze fluorescence intensity and one-way ANOVA with Tukey’s multiple…

• 

  Fig. 5. Reoxygenation/sEVs treatment increased the proliferative capacity of CSCs/Sca-1+. (A) Cell cycle analysis of different CSCs/Sca-1+ groups detected by PI/RNase staining with the representative DNA histograms and bar chart displays the distribution and percentages of cell cycle phases. (B and C) Representative dot plots of the flow cytometric expression of proliferative Ki67 and EdU antigens staining incorporation in CSCs/Sca-1+ showing the expression percentage of the gated cells exposed to OGD and reoxygenation conditions with the corresponding bar charts of the percentage of cell populations of the different reoxygenated groups relative to control and OGD groups. Each graph represents the mean ± SD from three independent experiments; each experiment consisted of triplicates per treatment group, group (each replica consists of 25 × 104 CSCs/Sca-1+ seeded into three wells of six-well plates). The significance is marked by *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001, while p > 0.05 denotes non-significance (ns) from two-way ANOVA for cell cycle analysis and one-way ANOVA for Ki67 and EdU analyses followed by Tukey’s multiple comparisons test in all analyses

• 

  Fig. 6. Flow cytometry analysis of apoptosis in CSCs/Sca-1+ under three conditions (OGD, OGD/R, and sEVs) in relation to normal cells. (A) Representative dot plots of Annexin V/PI expression and the corresponding bar charts (B) of different CSCs/Sca-1+ groups reveal that reoxygenation*** and sEVs treatment induce significant apoptosis rather than cell death compared with hypoxic CSCs/Sca-1+. The data of bar plots are the mean ± SD of three independent experiments, each performed in triplicates for each group (each replica consists of 25 × 104 CSCs/Sca-1+ seeded into three wells of six-well plates ), with *p < 0.05, **p < 0.01, and ***p < 0.001, while p > 0.05 of non-significance (ns) from two-way ANOVA followed by Tukey’s multiple comparisons test

• 

  Fig. 7. Oxidative stress index and PTEN /AKT/p-AKT expression in the CSCs/Sca-1+ groups. Oxidative stress is indicated by the overall oxidative stress index (A). The representative western blots and the corresponding densitometric quantification show PTEN 54kDa (B, C), AKT 60 kDa (B, D), p-AKT 60 kDa (B, E), the ratio of p-AKT to AKT (F). and β -Actin 45 kDa (B) in control, OGD, OGD/R, and sEVs CSCs groups. β-actin is the loading control. Data in each bar chart are representative of mean ± SD of three independent experiments performed in triplicates each (each replica consists of 25 × 104 CSCs/Sca-1+ seeded into three wells of six-well plates), where one-way ANOVA followed by Tukey’s multiple comparisons test reveals *p < 0.05, **p < 0.01, and ***p < 0.001, while ns means non-significance of p > 0.05