Figure 5

Analysis of secretion. (a) Western blot analysis. Proteins from cell lysate, conditioned medium (CM), and HPLC purified exosomes of E1MSCs or E1-MYC-MSCs were separated on SDS-PAGE and probed with different antibodies to detect MYC (64 kDa), ACTIN (42 kDa), and CD9 (24 kDa). (b) HPLC fractionation and dynamic light scattering of CM from E1-MYC-MSC. CM was fractionated on a HPLC using BioSep S4000, 7.8 mm × 30 cm column. The components in CM were eluted with 20 mM phosphate buffer with 150 mM of NaCl at pH 7.2. The elution mode was isocratic and the run time was 40 minutes. The eluent was monitored for UV absorbance at 220 ηm. Each eluting peak was then analyzed by light scattering. The fastest eluting peak (arrow) was collected for testing in a mouse model of myocardial ischemia/reperfusion injury. (c) 0.3 μg HPLC-purified exosomes was administered intravenously to a mouse model of acute myocardial/ischemia reperfusion injury five minutes before reperfusion. Infarct size (IS) as a percentage of the area at risk (AAR) upon treatment with saline (n = 10), exosomes from E1-MYC 21.1 (n = 5) and exosomes from E1-MYC 16.3 (n = 4) were measured. The relative infarct size (IS/AAR) in mice treated with E1-MYC 21.1 exosome or E1-MYC 16.3 exosome was 23.4 ± 8.2%, and 22.6 ± 4.5%, respectively and their relative infarct sizes were significantly lower than the relative infarct size of 38.5 ± 5.6% in saline-treated mice (p < 0.001 and p < 0.002, respectively).