S Hospital, IRCCS, Rome, Italy; 3Department of Cell Biology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Krakow, Poland; CCR5 Proteins Synonyms Malopolska Centre of Biotechnology, Krakow, Poland; 4Electron Microscopy Platform, Mossakowski Healthcare Investigation Centre, PAS, Warsaw, Poland; 5Laboratory of Advanced Microscopy Strategies, Mossakowski Health-related Study Centre, PAS, Warsaw, Poland; 6Department of Women’s and Children’s Health, University of Padua, Padua, Italy; 7NeuroRepair Department, Mossakowski Healthcare Study Centre, PAS, Warsaw, Poland; Russel H. Morgan Department of Radiology and Radiological Science, Division of MR Research, The Johns Hopkins University College of Medicine, Baltimore, USAIntroduction: Mesenchymal stem cells (MSCs) have shown each antiinflammatory and pro-regenerative activity within a selection of issues. Current research support the notion that the signals responsible for these therapeutic effects are at the very least partially conveyed by extracellular vesicles (EVs). In spite of increasing interest in EVs as therapeutic tools, little info is accessible around the fate of these nanoparticles following in vivo administration since of methodological hurdles. The aim with the study was to Ubiquitin-Specific Protease 1 Proteins custom synthesis optimize the method of EVs visualization for in vitro and in vivo biodistribution studies. Solutions: The experiments had been performed employing human bone marrow mesenchymal stem cells (hBM-MSCs) (Lonza). hBM-MSCs had been labelled with PKH26 (Sigma) and iron nanoparticles conjugated with rhodamine (Molday, BioPAL) and co-stained with anti-CD9, -CD63 and -CD81 (tetraspanins) and MSCs antibodies. EVs had been isolated in the culture media of previously labelled hBM-MSCs. The size, number, morphology and biomarker expression of hBM-MSC-EVs were identified by Nanosight analysis, high-resolution flow cytometry, transmission electron microscopy, superresolution illumination microscopy and MRI. The in vivo studies were performed in adult male Wistar rats with focal brain injury of 1l/50nmol ouabain injection into the appropriate hemisphere. Two days soon after the brain insult1.3x109hBM-MSC-EVs labelled with Molday or stained with PKH26 have been infused in to the ideal internal carotid artery and analysed in rat brain immunohistochemically utilizing confocal microscopy. Results: In vitro research revealed the presence of intracellular vesicles positively stained with Molday ION or PKH26 visible inside hBM-MSCs co-expressed CD44, CD73, CD90, CD9, CD63 and CD81 markers. The isolated EVs represented heterogeneous population of different size (50300 nm) and kept their markers soon after isolation. hBM-MSC-EVs transplanted intraarterially in focal brain injured rats migrated in to the proper hemisphere close to the ischemic injury. Summary/Conclusion: PKH26 and Molday ION allow to visualize hBM-MSC-EVs in vitro and in vivo after their intra-arterial transplantation. Molday ION tagging could enable further imaging of EVs delivery utilizing MRI.Introduction: It can be now firmly established that mature 22nt miRNAs are detected in populations of extracellular vesicles (EVs) and exosomes. Exosomal miRNAs have physiological effects in recipient cells but the question remains no matter whether they are able to non-cell autonomously modulate gene expression. Presumably, loading of a single guide strand of miRNAs into RISC (a prerequisite for active repression of mRNA translation) is inefficient in comparison with loading of miRNA duplexes which are normally not located in exosomes. Possibly a chaperone method exists that could c.