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.

Background Bone marrow cell-based treatment for critical limb ischemia (CLI) in diabetic patients yielded a modest therapeutic effect due to cell dysfunction. Therefore, approaches that improve diabetic stem/progenitor cell functions may provide therapeutic benefits. Here, we tested the hypotheses that restoration of hydrogen sulfide (H2S) production in diabetic bone marrow cells (BMCs) improves their reparative capacities. Methods Mouse BMCs were isolated by density-gradient centrifugation. Unilateral hind limb ischemia (HLI) was conducted in 12- to 14-week old db/+ and db/db mice by ligation of left femoral artery. H2S level was measured by either gas chromatography or staining with florescent dye sulfidefluor 7AM. Results Both H2S production and cystathionine γ-lyase (CSE), an H2S enzyme, levels were significantly decreased in BMCs from diabetic db/db mice. Administration of H2S donor diallyl trisulfide (DATS) or overexpression of CSE restored H2S production and enhanced cell survival and migratory capacity in high glucose (HG)-treated BMCs. Immediately after HLI surgery, the db/+ and db/db mice were administrated with DATS orally and/or local intramuscular injection of GFP-labeled BMCs or RFP-CSE-overexpressing BMCs (CSE-BMCs). Mice with HLI were divided into six groups: 1) db/+; 2) db/db; 3) db/db+BMCs; 4) db/db+DATS; 5) db/db+DATS+BMCs; 6) db/db+CSE-BMCs. DATS and CSE overexpression greatly enhanced diabetic BMCs retention in ischemic hind limbs (IHL) followed by improved blood perfusion, capillary/arteriole density, skeletal muscle architecture and cell survival, and decreased perivascular CD68+ cell infiltration in IHL of diabetic mice. Interestingly, DATS or CSE overexpression rescued HG-impaired migration, tube formation and survival of BMCs or mature human cardiac microvascular endothelial cells (HCMVECs). Mechanistically, DATS restored nitric oxide production and decreased eNOS-pT495 levels in HCMVECs, and improved BMC angiogenic activity under HG condition. Finally, silencing CSE by siRNA significantly increased eNOS-pT495 levels in HCMVECs. Conclusions Decreased CSE-mediated H2S bioavailability is an underlying source of BMC dysfunction in diabetes. Our data indicate that H2S and overexpression of CSE in diabetic BMCs may rescue their dysfunction and open novel avenues for cell-based therapeutics of CLI in diabetic patients.

Poor survival and function of transplanted cells in ischemic and inflamed myocardium likely compromises the functional benefit of stem cell based therapies. We have earlier reported that co-administration of IL-10 and BMPAC enhances cell survival and improves LV (LV) functions after AMI in mice. We hypothesized that IL-10 regulates miR-375 signaling in BMPACs to enhance their survival and function in ischemic myocardium after MI and attenuates left ventricular dysfunction after MI. MicroRNA-375 expression is significantly up regulated in BMPACs upon exposure to inflammatory/hypoxic stimulus and also after MI. IL-10 KO mice display significantly elevated miR-375 levels. We report that ex vivo miR-375 knock down in BMPAC before transplantation in the ischemic myocardium after MI significantly improve the survival and retention of transplanted BMPACs and also BMPAC-mediated post-infarct repair, neovascularization and LV functions. Our in vitro studies revealed that knockdown of miR-375 enhanced BMPAC proliferation and tube formation and inhibited apoptosis; over expression of miR-375 in BMPAC had opposite effects. Mechanistically, miR-375 negatively regulated 3-phosphoinositide-dependent protein kinase 1 (PDK-1) expression and PDK-1-mediated activation of PI3Kinase/AKT signaling. Interestingly, BMPAC isolated from IL-10-deficient mice showed elevated basal levels of miR-375 and exhibited functional deficiencies, which were partly rescued by miR-375 knockdown, enhancing BMPAC function in vitro and in vivo. Taken together, our studies suggest that miR-375 is negatively associated with BMPAC function and survival and IL-10-mediated repression of miR-375 enhances BMPAC survival and function.