Figure 4

An exosome-independent mechanism mediates the observed protection of human cardiomyocytes by mouse cMSC. (a) Schematic of exosome capture and detection using anti-CD63-conjugated latex beads plus FITC-anti-CD9. (b,c) Bead-exosome complexes were analysed by flow cytometry. (b) Representative contour plots are shown. The gate defines the CD63⁺CD9⁺ exosomes. The top row shows controls for staining and the bottom row the proportion of exosomes in unfractionated conditioned media versus the depleted (exo-depleted) and enriched (exo-enriched) fractions. FSC forward scatter. More than 5,000 beads were scored for each condition shown. (c) Bar graph of CD9⁺ depletion and enrichment; n = 3. (d) DRAQ7 uptake in vCor.4U human ventricular myocytes after menadione ± cMSC-conditioned media or the indicated fractions. Above, representative images. Scale bar, 50 μm. Below, bar graph of DRAQ7 uptake; n = 10. (e) Thermostability testing of cMSC-conditioned media. Bar graph of DRAQ7 uptake; n = 9. (f) Size-fractionation of cMSC-conditioned media. Bar graph of DRAQ7 uptake; n = 6. For all panels, data are shown as the mean ± SEM. *p < 0.05; **p < 0.001; ***p < 0.0001. (g) Bar graphs of cytokine levels, using low-density membrane arrays, for the factors enriched in cMSC-conditioned medium vs medium only or TTF-conditioned medium. Results are image analysis of integrated density, normalised to the average of anti-streptavidin and anti-HRP controls for each membrane. Enlarged versions of the arrays can be found in Supplementary Fig. S9. Data are mean ± SEM; n = 2; *p < 0.05; **p < 0.01; ***p < 0.001. Unpaired one-tailed t test between cMSC and medium or cMSC and TTF. Graphics were created using Servier Medical Art website, a free medical image database with a licence under Creative Commons Attribution 3.0 Unported License (https://creativecommons.org/licenses/by/3.0/).