MiRNA profiling of murine mesenchymal stem cell (mMSC)-derived extracellular vesicles (EVs) before and after freeze-drying
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PROVIDER: PRJNA589877 | ENA |
REPOSITORIES: ENA
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Project description:Purpose: The goal of this study was to determine the microRNA (miRNA) content of extracellular vesicles (EVs) derived from murine mesenchymal stem cells (mMSC), and evaluate reproducibility among distinct EV productions. We also aimed at assessing the effect of freeze-drying on EV miRNA content, by performing sequencing on freeze-dried EVs and calculating statistical difference between unmodified and freeze-dried EVs. Methods: mMSC-derived EVs were obtained from mMSC in culture in reduced serum medium Opti-MEM by differential centrifugation, with a final step at 100,000 g for 110 min at 4°C. EV pellets (freeze-dried (n=3) or not (n=2)) were resuspended in Qiazol lysis buffer and RNA was extracted following RNeasy Micro kit. cDNA libraries for sequencing were prepared using the TruSeq Small RNA Sample Preparation Kit. Amplified cDNA constructs were purified on 6 % PAGE gel and DNA molecules corresponding to 15–50 nucleotide transcripts were excised, eluted from gel, and concentrated. Image analyses and base calling were performed using the HiSeq Control Software and Real-Time Analysis component (Illumina). Before statistical analysis, genes with less than 15 reads (cumulating all the analysed samples) were filtered out. Differentially expressed miRNA were identified using three Bioconductor packages: edgeR, DESeq and DESeq2. Results: Considering miRNAs detected with at least 5 counts (in terms of normalised counts), 339 miRNAs were identified and miRNA content was highly conserved among the two batches tested, with 237 miRNAs out of the 339 present in both batches (70%). Statistical analysis did not evidence statistical difference between unmodified EVs (n=2) and freeze-dried EVs (n=3) (DESeq2, p<0.05). No statistical difference was found using other Bioconductor packages DESeq and edgeR. These results indicated conservation of miRNA content following freeze-drying. Conclusion: mMSC-EV miRNA content was comparable between the two EV productions analysed, indicating reproducibility. Some of the miRNAs identified were consistent with previously published results on MSC-derived EVs. Freeze-drying conserved miRNA content.
2019-12-09 | GSE140479 | GEO
Project description:Under physiological conditions, extracellular vesicles (EVs) are present simultaneously in the extracellular compartment together with cytokines. Thus, we hypothesized that EVs in combination with cytokines induce different responses of monocyte cells compared to EVs or cytokines alone. Human monocyte U937 cells were incubated with EV-containing or EV-free CCRF human T-cell supernatant, with or without the addition of TNF. U937 cells cultured in EV-free supernatant, supernatant containing CCRF t-cell derived EVs, TNF or both. Each treatment option was measured in 3 replicates.
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Project description:Purpose: Lactococcus lactis is the primary constituent of many industrial starter cultures used in food fermentations. However, this bacterium is exposed to several stressful conditions during the industrial process. L. lactis TOMSC161 has been shown to resist better to freeze-drying and storage when cells are harvested at the late stationary compared to the early stationary phase. Methods: In this work, the physiological changes that occur during growth (early and late stationary phases) were studied using transcriptomic, proteomic and cytometric approaches in order to elucidate the cell mechanisms involved in the stress tolerance of L. lactis TOMSC161 to freeze-drying and storage processes. Results: The majority of altered abundances for genes determined by transcriptomic analysis indicated a slowdown at the late stationary phase in general metabolism and in the incomplete reduction of O2 leading to reactive oxygen species (ROS), in comparison to the early stationary phase. Furthermore, the expression of genes involved in general and oxidative stress responses was over-expressed at the late stationary phase when compared to the early stationary phase, thus explaining the better resistance to freeze-drying and the storage of cells harvested after six hours of stationary phase. Superoxide anion detection was performed by flow cytometry and related to the ROS production of starters. Superoxide anion concentration was lower for cells harvested at the late stationary phase when compared to cells harvested at the early stationary phase. Conclusions: L. lactis TOMSC161 was thus able to develop an oxidative stress “pre-adaptation” during the stationary phase making it possible to better resist freeze-drying and storage stresses and especially oxidative stress.
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Project description:The vascular wall from diverse human organs is a source of mesenchymal progenitor cells that are able to induce skeletal repair, primarily by paracrine mechanisms. To investigate these paracrine mechanisms, FACS purified human perivascular stem cells (PSC) were observed to induce mitogenic, pro-migratory, and pro-osteogenic effects on osteoprogenitor cells while in non-contact co-culture, and did so via elaboration of extracellular vesicles (EVs). PSC-derived EVs shared mitogenic, pro-migratory, and pro-osteogenic properties of their parent cell. EVs represent a mixture of protein, lipid, and RNA that may affect cellular processes of the recipient cell. To begin to examine this, the RNA content of PSC-EVs was examined in comparison to the parent cell using total RNA sequencing.
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Project description:Exosomes and microvesicles (i.e., extracellular vesicles; EVs) have been identified within ovarian follicular fluid, and recent evidence suggests that EVs are able to elicit profound effects on ovarian cell function. While existence of miRNA within EVs has been reported, it remains unknown if EV size and concentration as well as their cargos (i.e., proteins and RNA) change during antral follicle growth. Extracellular vesicles isolated from follicular fluid of small, medium and large bovine follicles were similar in size, while concentration of EVs decreased progressively as follicle size increased. Electron microscopy indicated a highly purified population of the lipid bilayer enclosed vesicles that were enriched in exosome biomarkers including CD81 and Alix. Small RNA sequencing identified a large number of known and novel miRNAs that changed in the EVs of different size follicles. Ingenuity Pathway Analysis (IPA) indicated that miRNA abundant in small follicle EV preparations were associated with cell proliferation pathways, while those miRNA abundant in large follicle preparations were related to inflammatory response pathways. These studies are the first to demonstrate that EVs change in their levels and makeup during antral follicle development and point to the potential for a unique vesicle-mediated cell-to-cell communication network within the ovarian follicle. Examination of small RNA population in bovine follicular fluid extracellular vesicles isolated from antral follicles
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