Neural progenitor cell-derived exosomes in ischemia/reperfusion injury in cardiomyoblasts

Figures

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  Fig. 1. Complete Workflow. Supernatant was collected at multiple time points from 1 neurospheres, 2 adherent neural stem cell cultures, and 3 from cells grown in CL 1000 bioreactor, for serial ultracentrifugation steps. 20k and 110k harvests were resuspended and frozen for further analysis. H9c2 cardiomyoblasts were cultured in FX seahorse plates, subjected to 4-hour and 6-hour oxygen glucose deprivation, followed by treatment with exosomes. Supernatant was collected for LDH assay, and mitochondrial respiration was measured using FX seahorse

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  Fig. 2. Neural stem cell phenotype quality control. At day 3 in a chamber slide culture, there were groups of cells forming neurosphere like gatherings, expressing GFAP (green), DXC (red) and Nestin (green), and DAPI (blue), indicating the progenitor cells expressed characteristics of neural stem cells, neurons and astrocytes early in the process. Scale bar is 100 μm

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  Fig. 3. Culture growth conditions. The cells were cultured as neurospheres and adherent cultures, and in one liter CL1000 bioreactor. Neurosphere cultures showed typical floating spheres, which later upon touching the flask bottom attached to the bottom and continued growing. There were no spheres in adherent culture flask. We could not investigate the conditions inside CELLline CL 1000 bioreactor. Scale bar is 200 μm

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  Fig. 4. Nanoparticle tracking analysis. A representative image from Nanoparticle Tracking Analysis from the same sample showing (A) 20k yield and (B) 110k yield, from neurosphere culture labeled Exo1. The particle density was higher in 110k samples, with more small EVs. The left side represents technical NTA replicates for each measurement from a single sample. The rights side represents averaged finite track length adjustment (FTLA) Concentration / Size graphs for NTA analysis. Error bars indicate ± 1 standard error of the mean

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  Fig. 5. Electron microscopy. A representative image from electron microscopy findings. A and B show typical yeild of small extracellular vesicles (red circle) from Exo1 sample, neurosphere culture 110k. C. EM showed some atypical structures on the background in all samples (dark string like structures)

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  Fig. 6. Western blot. A1 and A2 are gels from the samples 1–8, B1 and B2 are gels from samples 9–16, and sample17 is technical control showing positive for calnexin at 90 kDa (negative antibody control, B1, line 17) and TSG101 at 25 kDa (positive control, B2, line 17). L stands for ladder. 1 = Bioreactor 20k, 2 = Bioreactor 110k, 3 = Neurosphere 20k, 4 = Neurosphere 110k, 5 = Adherent cells 20k, 6 = Adherent cells 110k, 7 = Bioreactor 20k, 8 = Bioreactor 110k, 9 = Adherent cells 20k, 10 = Adherent cells 110k, 11 = Neurospheres 20k, 12 = Neurospheres 110k, 13 = Bioreactor 20k, 14 = Bioreactor 110k, 15 = Neurosphere 20k, 16 = Neurosphere 110k, 17 = control. Only three samples of our exosomes returned positive findings in Western blot analysis. These were labeled as our study groups as follows: 12 = Exo 1 Neurospheres 110k, 14 = Exo 2 Bioreactor 110k and 16 = Exo 3 Neurospheres 110k

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  Fig. 7. LDH assay. LDH release at 20 h post treatment with exosomes. The results are expressed as means. Error bars show 95% confidence interval. Statistical analysis was performed as One-Way ANOVA multivariate with Dunnett’s test. P-value 0.05 or lower (p ≤ 0.05) was set as a threshold for statistically significant difference level

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  Fig. 8. Mitochondrial respiration. Data presented as mean +- Standard Error of Mean (SEM). Statistical analysis with One-Way Anova with Dunnetts test, signifigance level p < 0.05, no statistical differences observed in treatment groups when compared to the control group