Real-time quantitative PCR analysis of human mesenchymal stromal (MSC) cells extracellular vesicles (EV)
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ABSTRACT: Bone Marrow MSC were isolated from 5 donnors, EV were isolated by unltracentrifugation from the conditionned media after 48h without serum. 3 conditions are described: control, interferon priming (25 ng/ml) or hypoxia priming (O2 = 3%)
Project description:RNA-seq was performed on cultured human induced pluripotent cell derived cardiomyocytes (iPSC-CMs). There are four groups, with three samples per group: H1,H2,H3. Negative control. Healthy, normoxic iPSC-CMs D1,D2,D3. Positive control. iPSCs cultured under hypoxic (0-1% O2) for 48h with 2% v/v PBS as vehicle control EV1,EV2,EV3. Treatment 1. iPSCs cultured under hypoxic (0-1% O2) for 48h, with cardiac stromal cell derived extracellular vesicles, provided at a dose of 67ng/µl (2% v/v) EV21, EV23, EV24. Treatment 2 iPSCs cultured under hypoxic (0-1% O2) for 48h, with bone marrow mesenchymal stromal cell (BM-MSC) derived extracellular vesicles, provided at a dose of 67ng/µl (2% v/v) All EVs were isolated from cultured human cells using sequential centrifugation methods. Cells were cultured using commercial EV-depleted FBS to avoid contamination of bovine EVs. EVs were validated for CD81, CD9, ALIX positivity, and visualised by cryoEM. Particle to protein ratios were not different between cardiac and bone marrow EV isolates. Therefore EV doses were standardised so that the same dose by protein (67ng/µl) and EV number (~2,000 EVs per iPSC-CM) were added.
Project description:Characterization of proteins in extracellular vesicles (EV) from cardiosphere-derived cells (CDCs) of a clinically relevant pig model. Additionally, considering that priming stem cells with inflammatory stimuli may increase the therapeutic potential of released vesicles, here we studied the dynamic changes that take place in the EV from IFNγ-primed CDCs.
Project description:The extracellular vesicles released by endMSCs (EV-endMSCs)were characterized by multiplexedquantitative proteomics approach. We hypothesized that inflammatory priming of adult stem cells may contribute to increase their therapeutic effectiveness, thus, extracellular vesicles derived from IFN-γ-primed endMSCs (IFNγ/EV-endMSCs) were compared to control counterparts (n=3 patients).
Project description:To identify gene expression changes associated with treatment of EVs from MDA-MB-231 [231] and MCF10A [10A] cells) in NIH3T3 cells, we analyzed RNA isolated from PBS- or EV-treated NIH3T3 cells. Gene expression in NIH3T3 cells treated with EV from MDA-MB-231 cells was compared to cells treated with PBS or EV from MCF10A cells, both of which served as controls in this experiment.
Project description:To investigate the impact of a pro-inflammatory stimulus (TNFα and IFNγ, 20 ng/ml) on miRNA in SCAP-EV, we isolated EV from non-activated and activated SCAP and then extracted miRNA
Project description:To identify gene expression changes associated with treatment of EV that carry high levels of miR-105 (from MDA-MB-231 and MCF10A/miR-105 cells) in human breast tumor derived CAF, we analyzed RNA isolated from PBS- or EV-treated CAF. Gene expression in CAF treated with EV from MDA-MB-231 or MCF10A/miR-105 cells was compared to cells treated with PBS or EV from MCF10A cells, both of which served as controls in this experiment.
Project description:Treatment of graft failure after allogeneic stem cell transplantation with mesenchymal stromal cells (MSC) is being evaluated clinically. MSC can exert their functions by the release of extracellular vesicles (EV). Our aim is to analyze changes induced in hematopoietic stem cells (HSC) after the incorporation of MSC-EV. MSC were isolated from healthy donors. MSC-EV were isolated by ultracentrifugation and characterized by flow cytometry (FC), Western-blot, electron microscopy and Nanosight. EV incorporation into HSC was analyzed by FC and confocal microscopy. To study HSC gene expression, RT-PCR and arrays were performed. Apoptosis and cell cycle were evaluated by FC and clonal growth by clonogenic assays in methylcellulose. The engraftment capacity of HSC was analyzed four weeks after transplantation in NOD-SCID mice. Our results showed that MSC-EV presented the characteristic morphology, size and inmunophenotype and were able to be incorporated into HSC. MSC-EV incorporation induced an a down-regulation of pro-apoptotic genes, an overexpression of genes involved in colony formation and cell proliferation together with an activation of the JAK-STAT pathway in HSC. A significant decrease in apoptosis (p=0.001), an increase of the percentage of cells in phase S and an increased CD44 expression (p=0.013) were confirmed by FC in HSC with MSC-EV. In addition, these HSC displayed a higher CFU-GM clonogenic potential (p=0.010). Finally, in the xenotransplantation model, the engraftment ability of human HSC with MSC-EV was significantly higher (p= 0.027). In summary, the incorporation of MSC-EV induces genomic and functional changes in HSC, increasing their clonogenic capacity and their engraftment ability.
Project description:To identify gene expression changes in liver associated with circulating EV that carry high levels of miR-122 (from MDA-MB-231 and MCF10A/miR-122 cells), we analyzed RNA isolated from the liver of PBS- or EV-treated female NOD scid gamma (NSG) mice. Mice had received tail-vein injected EV (or PBS) twice a week for 5 weeks (~10 ug EV per injection). Gene expression in liver from mice treated with EV from MDA-MB-231 or MCF10A/miR-122 cells was compared to cells treated with PBS or EV from MCF10A cells, both of which served as controls in this experiment.