Human ESC-derived mesenchymal stem cell (MSC)-conditioned medium (CM) was previously shown to mediate cardioprotection during myocardial ischemia/reperfusion injury through large complexes of 50-100 nm. Here we show that these MSCs secreted 50- to 100-nm particles. These particles could be visualized by electron microscopy and were shown to be phospholipid vesicles consisting of cholesterol, sphingomyelin, and phosphatidylcholine. They contained coimmunoprecipitating exosome-associated proteins, e.g., CD81, CD9, and Alix. These particles were purified as a homogeneous population of particles with a hydrodynamic radius of 55-65 nm by size-exclusion fractionation on a HPLC. Together these observations indicated that these particles are exosomes. These purified exosomes reduced infarct size in a mouse model of myocardial ischemia/reperfusion injury. Therefore, MSC mediated its cardioprotective paracrine effect by secreting exosomes. This novel role of exosomes highlights a new perspective into intercellular mediation of tissue injury and repair, and engenders novel approaches to the development of biologics for tissue repair.

We have previously identified exosomes as the paracrine factor secreted by mesenchymal stem cells. Recently, we found that the key features of reperfusion injury, namely loss of ATP/NADH, increased oxidative stress and cell death were underpinned by proteomic deficiencies in ischemic/reperfused myocardium, and could be ameliorated by proteins in exosomes. To test this hypothesis in vivo, mice (C57Bl6/J) underwent 30 min ischemia, followed by reperfusion (I/R injury). Purified exosomes or saline was administered 5 min before reperfusion. Exosomes reduced infarct size by 45% compared to saline treatment. Langendorff experiments revealed that intact but not lysed exosomes enhanced viability of the ischemic/reperfused myocardium. Exosome treated animals exhibited significant preservation of left ventricular geometry and contractile performance during 28 days follow-up. Within an hour after reperfusion, exosome treatment increased levels of ATP and NADH, decreased oxidative stress, increased phosphorylated-Akt and phosphorylated-GSK-3β, and reduced phosphorylated-c-JNK in ischemic/reperfused hearts. Subsequently, both local and systemic inflammation were significantly reduced 24h after reperfusion. In conclusion, our study shows that intact exosomes restore bioenergetics, reduce oxidative stress and activate pro-survival signaling, thereby enhancing cardiac function and geometry after myocardial I/R injury. Hence, mesenchymal stem cell-derived exosomes are a potential adjuvant to reperfusion therapy for myocardial infarction.

Although paracrine effects of mesenchymal stem cells (MSCs) have been suggested previously, cardioprotection by human MSC secretions has never been demonstrated. Human MSC-conditioned medium (CM) was collected by following a clinically compliant protocol. In a porcine model of ischemia and reperfusion injury, intravenous and intracoronary MSC-CM treatment significantly reduced myocardial nuclear oxidative stress as determined by immunostaining for 8-hydroxy-2'-deoxyguanosine. In addition, expression levels of phospho-SMAD2 and active caspase 3 were diminished following CM treatment, suggesting that TGF-beta signaling and apoptosis were reduced. This was associated with a 60% reduction in infarct size and marked improvement of systolic and diastolic cardiac performance as assessed with echocardiography and pressure volume loops. Fractionation studies revealed that only the fraction of the CM containing products >1000 kDa (100-220 nm) provided cardioprotection in a mouse model of ischemia and reperfusion injury. This indicates that the responsible paracrine factor of human MSCs is likely a large complex rather than a single small molecule. These data identify human MSC-CM as a promising therapeutic option to reduce myocardial infarct size in patients with acute MI and suggest that the use of stem cell secretions could extend the applicability of stem cells for therapeutic purposes.

These data identify exenatide as a potentially effective compound to reduce infarct size in adjunction to reperfusion therapy in patients with acute MI.