Rationale Embryonic stem cells (ESCs) hold great promise for cardiac regeneration but are susceptible to various concerns. Recently, salutary effects of stem cells have been connected to exosome secretion. ESCs have the ability to produce exosomes however their effect in the context of the heart is unknown. Objective Determine the effect of ESC-derived exosome for the repair of ischemic myocardium and whether c-kit+ CPCs function can be enhanced with ESC exosomes Methods and Results This study demonstrates that mouse ESC derived exosomes (mES Ex) possess ability to augment function in infarcted hearts. mES Ex enhanced neovascularization, cardiomyocyte survival and reduced fibrosis post infarction consistent with resurgence of cardiac proliferative response. Importantly, mES Ex augmented cardiac progenitor cell (CPC) survival, proliferation and cardiac commitment concurrent with increased c-kit+ CPCs in vivo 8 weeks after in vivo transfer along with formation of bonafide new cardiomyocytes in the ischemic heart. miRNA array revealed significant enrichment of miR290–295 cluster and particularly miR-294 in ESC exosomes. The underlying basis for the beneficial effect of mES Ex was tied to delivery of ESC specific miR-294 to CPCs promoting increased survival, cell cycle progression and proliferation. Conclusions mES Ex provide a novel cell free system that utilizes the immense regenerative power of ES cells while avoiding the risks associated with direct ES or ES derived cell transplantation and risk of teratomas. ESC exosomes possess cardiac regeneration ability and modulate both cardiomyocyte and CPC based repair programs in the heart.

Rational Transplantation of human CD34+ stem cells to ischemic tissues has been associated with reduced angina, improved exercise time and reduced amputation rates in phase 2 clinical trials and has been shown to induce neo-vascularization in pre-clinical models. Previous studies have suggested that paracrine factors secreted by these pro-angiogenic cells are responsible, at least in part, for the angiogenic effects induced by CD34+ cell transplantation. Objective Our objective was to investigate the mechanism of CD34+ stem cell induced pro-angiogenic paracrine effects and to examine if exosomes, a component of paracrine secretion, are involved. Methods and Results Exosomes collected from the conditioned media of mobilized human CD34+ cells had the characteristic size (40–90 nm; determined via dynamic light scattering), cup-shaped morphology (electron microscopy), expressed exosome-marker proteins CD63, phosphatidylserine (flow cytometry) and TSG101 (immunoblotting), besides expressing CD34+ cell lineage marker protein, CD34. In vitro, CD34+ exosomes replicated the angiogenic activity of CD34+ cells by increasing endothelial cell viability, proliferation and tube formation on Matrigel. In vivo, the CD34+ exosomes stimulated angiogenesis in Matrigel plug and corneal assays. Interestingly, exosomes from CD34+ cells, but not from CD34+ cell-depleted mononuclear cells had angiogenic activity. Conclusions Our data demonstrate that human CD34+ cells secrete exosomes that have independent angiogenic activity both in vitro and in vivo. CD34+ exosomes may represent a significant component of the paracrine effect of progenitor-cell transplantation for therapeutic angiogenesis.

Rationale Paracrine secretions appear to mediate therapeutic effects of human CD34+ stem cells locally transplanted in patients with myocardial and critical limb ischemia as well as in animal models. Earlier, we had discovered that paracrine secretion from human CD34+ cells contains pro-angiogenic, membrane-bound nano-vesicles called exosomes (CD34Exo). Objective Here, we investigated the mechanisms of CD34Exo-mediated ischemic tissue repair and therapeutic angiogenesis by studying their miRNA content and uptake. Methods and Results When injected into mouse ischemic hindlimb tissue, CD34Exo, but not the CD34exo-depleted conditioned media, mimicked the beneficial activity of their parent cells by improving ischemic limb perfusion, capillary density, motor function and their amputation. CD34Exo were found to be enriched with pro-angiogenic miRNAs such as miR-126-3p. Knocking down miR-126-3p from CD34exo abolished their angiogenic activity and beneficial function both in vitro and in vivo. Interestingly, injection of CD34Exo increased miR-126-3p levels in mouse ischemic limb, but did not affect the endogenous synthesis of miR-126-3p suggesting a direct transfer of stable and functional exosomal miR-126-3p. miR-126-3p enhanced angiogenesis by suppressing the expression of its known target, SPRED1; simultaneously modulating the expression of genes involved in angiogenic pathways such as VEGF, ANG1, ANG2, MMP9, TSP1 etc. Interestingly, CD34Exo, when treated to ischemic hindlimbs, were most efficiently internalized by endothelial cells relative to smooth muscle cells and fibroblasts demonstrating a direct role of stem cell-derived exosomes on mouse endothelium at the cellular level. Conclusions Collectively, our results have demonstrated a novel mechanism by which cell-free CD34Exo mediates ischemic tissue repair via beneficial angiogenesis. Exosome-shuttled angiomiRs may signify amplification of stem cell function and may explain the angiogenic and therapeutic benefits associated with CD34+ stem cell therapy.

We hypothesized that a small molecule CXCR4 antagonist, AMD3100 (AMD), could augment the mobilization of bone marrow (BM)-derived endothelial progenitor cells (EPCs), thereby enhancing neovascularization and functional recovery after myocardial infarction. Single-dose AMD injection administered after the onset of myocardial infarction increased circulating EPC counts and myocardial vascularity, reduced fibrosis, and improved cardiac function and survival. In mice transplanted with traceable BM cells, AMD increased BM-derived cell incorporation in the ischemic border zone. In contrast, continuous infusion of AMD, although increasing EPCs in the circulation, worsened outcome by blocking EPC incorporation. In addition to its effects as a CXCR4 antagonist, AMD also up-regulated VEGF and matrix metalloproteinase 9 (MMP-9) expression, and the benefits of AMD were not observed in the absence of MMP-9 expression in the BM. These findings suggest that AMD3100 preserves cardiac function after myocardial infarction by enhancing BM-EPC-mediated neovascularization, and that these benefits require MMP-9 expression in the BM, but not in the ischemic region. Our results indicate that AMD3100 could be a potentially useful therapy for the treatment of myocardial infarction.angiogenesis | stem cell | vasculogenesis