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2009;4:e5057. to sustain the activation of Stat1 signaling by EV-associated IFN-/Ifngr1 complexes. Our study identifies a mechanism of cellular signaling regulated by EV-associated IFN-/Ifngr1 complexes, which grafted stem cells may use to communicate with ODM-203 the host immune system. INTRODUCTION The systemic injection of neural stem/precursor cells (NPCs) in laboratory animals with immune-mediated experimental CNS demyelination, stroke, or injuries of the spinal cord leads to remarkable neuroprotection and functional recovery (Martino et al., 2011; Uccelli et al., 2011). While a comprehensive understanding ODM-203 of the mechanisms by which stem cell grafts work is still lacking, it is becoming increasingly accepted that they exert some of their therapeutic effects by secreting a complex array of homeostatic molecules (stem cell secretome) with immune regulatory and tissue trophic functions that ultimately reduce tissue damage and/or enhance endogenous repair (Drago et al., 2013). Partly as drugs and partly as devices, stem cell medicines work like naturally occurring disease-modifying agents that sense signals, migrate to specific areas of the body, make decisions, and execute complex response behaviorsalways in the context of specific microenvironments (Fischbach et al., 2013). Communication between grafted stem cells and the host is delivered via secreted cytokines and/or growth factors or through cellular (Gap) junctional transfer of electrical, metabolic, and immunological information. Furthermore, early work also suggests that extracellular vesicles (EVs) may play a key role when transferred from grafted stem cells to target host neural and nonneural cells (Pluchino and Cossetti, 2013). EVs are complex membranous structures composed of a lipid bilayer that contain transmembrane proteins and enclose soluble hydrophilic components derived from the cytosol of donor cells. EV is a general term that defines different types of Nfatc1 vesicles, including exosomes, microparticles, gesicles (Mangeot et al., 2011), and human endogenous retroviral particles (Balaj et al., 2011). Cells secrete EVs simultaneously, although there are yet no established criteria to distinguish one type of vesicle from another or physical means to separate them once released (Witwer et al., 2013). EVs capture bioactive molecules responsible for direct stimulation (Al-Nedawi et al., 2008) and increased survival of target cells (Frhbeis et al., 2013; Lopez-Verrilli et al., 2013), transmission of infectious agents (Mattei et al., 2009), and horizontal transfer of membrane and/or cargo molecules, which are enriched in specific proteins (Antonyak et al., 2011) and nucleic acids (Mittelbrunn et al., 2011; Valadi et al., 2007). It is well established that this transfer of information affects the physiology of recipient cells in various ways, from the activation versus suppression of immune responses, to promotion of tissue repair and cancer progression (Breakefield et al., 2011; Thry et al., 2009). Furthermore, experimental therapeutics with either unmodified or functionalized EVs/exosomes collected from mesenchymal stem cells (MSCs) or immune cells are being established as a promising anti-inflammatory (Yu et al., 2013; Zhuang et al., 2011), tissue-protective (Xin et al., 2013), stem cell-free alternative approach for brain repair. Here, we focused on defining whether the form of communication mediated by EVs exists for NPCs, on elucidating its molecular signature and functional relevance to target cells, and on identifying the key elements responsible for this mechanism of cellular signaling. We show that NPC EVs primarily consist of exosomes and observe cytokine-regulated pathways that sort proteins and mRNAs into EVs. Moreover, we describe a highly specific induction of the interferon gamma (IFN-) pathway in parental NPCs exposed to proinflammatory cytokines that is mirrored in EVs. We determined that activation of Stat1-dependent signaling in target NIH 3T3 cells happens as a result of the intercellular transfer of IFN- bound to interferon gamma receptor 1 (Ifngr1) on the surface of EVs. Finally, we demonstrate that endogenous Stat1 and Ifngr1 in target cells are indispensable to sustain the activation of Stat1 signaling by EV-associated IFN-/Ifngr1 complexes. Our study sheds light ODM-203 within the mechanisms of intercellular info exchange and demonstrates that EV-mediated cytokine signaling is an important mechanism by which NPCs may propagate some of their immune modulatory activities (Pluchino and Cossetti, 2013). RESULTS NPCs Secrete EVs NPCs were established from your subventricular zone (SVZ) of adult mice, as explained (Pluchino et al., 2005). Scanning electron ODM-203 microscopy (SEM) of NPC surface exposed polarized membranous ODM-203 constructions of small and medium size, which included very long nanotubes and round membrane vesicles (Numbers.