Microvesicles (MVs) are membrane-enclosed cytoplasmic fragments released by normal and activated cells that have been described as important mediators of cell-to-cell communication.Although the ability of human induced pluripotent stem cells (hiPSCs) to participate in tissue repair is being increasingly recognized, the use of hiPSC-derived MVs (hiPSC-MVs) in this regard remains unknown. Accordingly, we investigated the ability of hiPSC-MVs to transfer bioactive molecules including mRNA, microRNA (miRNA), and proteins to mature target cells such as cardiac mesenchymal stromal cells (cMSCs), and we next analyzed effects of hiPSC-MVs on fate and behavior of such target cells. The results show that hiPSC-MVs derived from integration-free hiPSCs cultured under serum-free and feeder-free conditions are rich in mRNA, miRNA, and proteins originated from parent cells; however, the levels of expression vary between donor cells and MVs. Importantly, we found that transfer of hiPSC components by hiPSC-MVs impacted on transcriptome and proteomic profiles of target cells as well as exerted proliferative and protective effects on cMSCs, and enhanced their cardiac and endothelial differentiation potential. hiPSC-MVs also transferred exogenous transcripts from genetically modified hiPSCs that opens new perspectives for future strategies to enhance MV content. We conclude that hiPSC-MVs are effective vehicles for transferring iPSC attributes to adult somatic cells, and hiPSC-MV-mediated horizontal transfer of RNAs and proteins to injured tissues may be used for therapeutic tissue repair. In this study, for the first time, we propose a new concept of use of hiPSCs as a source of safe acellular bioactive derivatives for tissue regeneration. STEM CELLS 2015;33:2748-2761 SIGNIFICANCE STATEMENTOur results show, for the first time, that human induced pluripotent stem cells (hiPS cells) may serve as a source of bioactive microvesicles (MVs) that could be potentially utilized for future safe applications in tissue regeneration. For the first time, we extensively characterized bioactive content of MVs released by hiPS cells (hiPS-MVs) on both transcriptomic and proteomic levels and we established their impact on functions and differentiation potential of mature target cells from human heart. These results have obvious translational relevance for developing potential new iPS cell-based strategies in tissue regeneration by employing thier safe acellular bioactive derivatives.

Abstract. Mechanisms leading to inhibitory effects of an anti-GD2 ganglioside (GD2) 14G2a mouse monoclonal antibody (mAb) and PI3K/Akt/mTOR pathway inhibitors on human neuroblastoma cell survival were studied in vitro. We have recently shown on IMR-32, CHP-134, and LA-N-1 neuroblastoma cells that targeting GD2 with the mAb decreases cell viability of the cell lines. In this study we used cytotoxicity assays, proteomic arrays and immunoblotting to evaluate the response of the three cell lines to the anti-GD2 14G2a mAb and specific PI3K/Akt/mTOR pathway inhibitors. We show here that the mAb modulates intracellular signal transduction through changes in several kinases and their substrates phosphorylation. More detailed analysis of the PI3K/Akt/ mTOR pathway showed significant decrease in activity of Akt, mTOR, p70 S6 and 4E-BP1 proteins and transient increase in PTEN (a suppressor of the pathway), leading to inhibition of the signaling network responsible for stimulation of translation and proliferation. Additionally, combining the GD2-specific 14G2a mAb with an Akt inhibitor (perifosine), dual mTOR/PI3K inhibitors (BEZ-235 and SAR245409), and a pan-PI3K inhibitor (LY294002) was shown to enhance cytotoxic effects against IMR-32, CHP-134 and LA-N-1 cells. Our study extends knowledge on mechanisms of action of the 14G2a mAb on the neuroblastoma cells. Also, it stresses the need for further delineation of molecular signal orchestration aimed at more reasonable selection of drugs to target key cellular pathways in quest for better cure for neuroblastoma patients.

The current evidence suggests that beneficial effects of mesenchymal stem cells (MSCs) toward myocardial repair are largely due to paracrine actions of several factors. Although Monocyte chemoattractant protein-induced protein 1 (MCPIP1) is involved in the regulation of inflammatory response, apoptosis and angiogenesis, whether MCPIP1 plays any role in stem cell-induced cardiac repair has never been examined. By employing retroviral (RV)-transduced overexpression of MCPIP1, we investigated the impact of MCPIP1 on viability, apoptosis, proliferation, metabolic activity, proteome, secretome and differentiation capacity of murine bone marrow (BM) - derived MSCs. MCPIP1 overexpression enhanced angiogenic and cardiac differentiation of MSCs compared with controls as indicated by elevated expression of genes accompanying angiogenesis and cardiomyogenesis in vitro. The proangiogenic activity of MCPIP1-overexpressing MSCs (MCPIP1-MSCs) was also confirmed by increased capillary-like structure formation under several culture conditions. This increase in differentiation capacity was associated with decreased proliferation of MCPIP1-MSCs when compared with controls. MCPIP1-MSCs also expressed increased levels of proteins involved in angiogenesis, autophagy, and induction of differentiation, but not adverse inflammatory agents. We conclude that MCPIP1 enhances endothelial and cardiac differentiation of MSCs. Thus, modulating MCPIP1 expression may be a novel approach useful for enhancing the immune-regulatory, anti-apoptotic, anti-inflammatory and regenerative capacity of BM-derived MSCs for myocardial repair and regeneration of ischemic tissues.

Very small embryonic-like stem cells (VSELs) represent a unique rare population of adult stem cells (SCs) sharing several structural, genetic, biochemical, and functional properties with embryonic SCs and have been identified in several adult murine and human tissues. However, rat bone marrow- (BM-) derived SCs closely resembling murine or human VSELs have not been described. Thus, we employed multi-instrumental flow cytometric approach including classical and imaging cytometry and we established that newly identified population of nonhematopoietic cells expressing CD106 (VCAM-I) antigen contains SCs with very small size, expressing markers of pluripotency (Oct-4A and Nanog) on both mRNA and protein levels that indicate VSEL population. Based on our experience in both murine and human VSEL isolation procedures by fluorescence-activated cell sorting (FACS), we also optimized sorting protocol for separation of CD45−/Lin−/CD106+ rat BM-derived VSELs from wild type and eGFP-expressing rats, which are often used as donor animals for cell transplantations in regenerative studies in vivo. Thus, this is a first study identifying multiantigenic phenotype and providing sorting protocols for isolation VSELs from rat BM tissue for further examining of their functional properties in vitro as well as regenerative capacity in distinct in vivo rat models of tissue injury.