Mesenchymal stromal cells (MSCs) are potent regulators of immune responses largely through paracrine signaling. MSC secreted extracellular vesicles (MSC-EVs) are increasingly recognized as the key paracrine factors responsible for the biological and therapeutic function of MSCs. We report the first comprehensive study demonstrating the immunomodulatory effect of MSC-EVs on dendritic cell (DC) maturation and function. MSC-EVs were isolated from MSC conditioned media using differential ultracentrifugation. Human monocyte-derived DCs were generated in the absence or presence of MSC-EVs (20 ug/ml) then subjected to phenotypic and functional analysis in vitro. MSC-EV treatment impaired antigen uptake by immature DCs and halted DC maturation resulting in reduced expression of the maturation and activation markers CD83, CD38, and CD80, decreased secretion of pro-inflammatory cytokines IL-6 and IL-12p70 and increased production of anti-inflammatory cytokine TGF-β. MSC-EV treated DCs also demonstrated a diminished CCR 7 expression after LPS stimulation, coupled with a significantly reduced ability to migrate toward the CCR7-ligand CCL21, although they were still able to stimulate allogeneic T cell proliferation in vitro. Through microRNA profiling we have identified 49 microRNAs, which were significantly enriched in MSC-EVs compared to their parent MSCs. MicroRNAs with known effect on DC maturation and functions, including miR-21-5p, miR-142-3p, miR-223-3p, and miR-126-3p, were detected within the top 10 most enriched miRNAs in MSC-EVs, with MiR-21-5p as the third highest expressed miRNA in MSC-EVs. In silico analysis revealed that miR-21-5p targets the CCR7 gene for degradation. To verify these observations, DCs were transfected with miR-21-5p mimics and analyzed for their ability to migrate toward the CCR7-ligand CCL21 in vitro. MiR-21-5p mimic transfected DCs showed a clear trend of reduced CCR7 expression and a significantly decreased migratory ability toward the CCL21. Our findings suggest that MSC-EVs are able to recapitulate MSC mediated DC modulation and MSC-EV enclosed microRNAs may represent a novel mechanism through which MSCs modulate DC functions. As MSCs are currently used in clinical trials to treat numerous diseases associated with immune dysregulation, such as graft-versus-host disease and inflammatory bowel disease, our data provide novel evidence to inform potential future application of MSC-EVs as a cell-free therapeutic agent.

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Summary:wide may reduce the present delay between the initiation of a search for an unrelated donor and actual transplantation that occurs using existing unrelated marrow donor panels. 3 Cord blood transplantation has been proven to be a suitable form of treatment for a variety of diseases in Separation and processing of cord blood samples in large numbers for storage in cord blood banks ideally needs to childhood and more recently in an increasing number of adult patients. Banks of cord blood cryopreserved after be partially automated to allow large numbers of samples to be processed efficiently. A closed system reduces the risk HLA testing are required in order to provide various HLA types for unrelated transplantation. To optimize of bacterial contamination after collection. The processing method must allow adequate recovery of nucleated cells storage space cord blood needs to be stored as a separated product. Several early methods of cord blood sepand progenitors to enable engraftment. Early attempts at separating cord blood by density gradient techniques led to aration resulted in a significant loss of progenitor cells. We used a separation procedure where the donation loss of mononuclear cells and to the suggestion that cord blood should be stored unseparated. 4,5 These donations, was separated by centrifugation into a buffy coat fraction, a red cell fraction, and a plasma fraction. Twentycryopreserved as whole blood, have been transplanted successfully. 1,2,5 However, for cord blood banks to be econfive samples, (mean initial volume 81 ml) were assessed. Nucleated cells were recovered in the buffy coat fracomical and efficient, volumes smaller than that of whole packs need to be stored. Furthermore, clinical problems tion. Recoveries of nucleated cell count, total progenitors and CD34-positive cells in the buffy coat were 90%, could arise with red cell lysis and some form of red cell depletion is necessary to reduce the risk of transfusion reac-88% and 100%, respectively. The buffy fraction was tested for sterility by aerobic and anaerobic culture.tions in cases of ABO incompatibility between donor cord blood and recipient. A study using the density gradient Using this closed bag system, volume reduction was achieved while maintaining sterility and retaining progenimethod to separate cord blood 6 did not find a major loss of progenitor cells, and a later study 7 suggested a modifitor cells in a final mean buffy coat volume of 44 ml. Red cell and plasma fractions were available for ABO cation of the common density gradient separation methods as suitable for processing cord blood before cryopreservgrouping, virology testing and cryopreservation. The results show that cord blood can be effectively volumeation. Falkenburg et al 6 compared cell separation of cord blood and bone marrow by red cell lysis, methylcellulose reduced using simple and readily available blood banking techniques.sedimentation and density gradients, and showed similar recovery of nucleated cells from both cell sources provided

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