Effect of chronic lymphocytic leukemia-derived exosomes on human bone marrow mesenchymal stem cells
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ABSTRACT: Transcription profiling of human bone marrow mesenchymal stem cells treated with exosomes isolated from chronic lymphocytic leukemia cell line MEC-1 supernatant. Cells were left untreated or treated with CLL-exosomes for 6h at 37C.
Project description:Exosomes derived from mesenchymal stem cells (MSCs) have shown to have effective application prospects in the medical field, but exosome yield is very low. The production of exosome mimetic vesicles (EMVs) by continuous cell extrusion leads to more EMVs than exosomes, but whether the protein compositions of MSC-derived EMVs (MSC-EMVs) and exosomes (MSC-exosomes) are substantially different remains unknown. The purpose of this study was to conduct a comprehensive proteomic analysis of MSC-EMVs and MSC-exosomes and to simply explore the effects of exosomes and EMVs on wound healing ability. This study provides a theoretical basis for the application of EMVs and exosomes.In this study, EMVs from human umbilical cord MSCs (hUC MSCs) were isolated by continuous extrusion, and exosomes were identified after hUC MSC ultracentrifugation. A proteomic analysis was performed, and 2,315 proteins were identified. The effects of EMVs and exosomes on the proliferation, migration and angiogenesis of human umbilical vein endothelial cells (HUVECs) were evaluated by cell counting kit-8, scratch wound, Transwell and tubule formation assays. A mouse mode was used to evaluate the effects of EMVs and exosomes on wound healing . Bioinformatics analyses revealed that 1,669 proteins in both hUC MSC-EMVs and hUC MSC-exosomes play roles in retrograde vesicle-mediated transport and vesicle budding from the membrane. The 382 proteins unique to exosomes participate in extracellular matrix organization and extracellular structural organization, and the 264 proteins unique to EMVs target the cell membrane. EMVs and exosomes can promote wound healing and angiogenesis in mice and promote the proliferation, migration and angiogenesis of HUVECs.
Project description:Bone marrow (BM) niches provide an optimal substrate for multiple myeloma (MM) cell lodgement and growth. Nevertheless, little is known about the putative mechanisms by which the BM microenvironment can lead to initiation or progression of oncogenesis in this disease. We have demonstrated that BM mesenchymal stromal cell-derived exosomes transfer their miRNA and protein content to clonal plasma cells, thus acting as synaptic vesicles responsible for molding the microenvironment surrounding multiple myeloma (MM) cells, leading to MM growth, dissemination and, therefore, disease progression. We used microarray to detail the changes in microRNA expression in MM-BM mesenchymal stromal cell (MSC)-derived exosomes, compared to normal- and monoclonal gammopathy of undetermined significance- BM-MSC-derived exosomes. Exosomes have been isolated from cell culture supernatant of BM-MSCs (MM=7; MGUS=2; Normal=4), and subsequently evaluated at ultrastructural level by using electron microscopy and immunogolf labeling. RNA was extracted; and miRNA profiling has been assessed by using TaqMan human miRNA profiling. Mean miRNA expression value has been used for miRNA RT-qPCR data normalization, as described (Mestdagh et al., 2009).
Project description:We hypothesized that miRNAs in the bone maroow mesenchymal stem cells (BM-MSC)-derived exosomes contributed to the phenotype change of breast cancer cells through exosome transfer. We analyzed the miRNA expression signature in BM-MSC-derived exosomes. We compared the miRNA expression levels in exosomes between BM-MSCs and adult fibroblasts (as a control). In this study, miRNA expression including in bone-marrow mesenchymal cell (BM-MSC)-derived exosomes was examined, and compared with that of exosomes derived from adult fibroblast cells or the BM-MSC cells. In addition, miRNA expression of BM-MSC exosomes was also compared with that of breast cancer cells with or without cancer stem cell marker.
Project description:microRNA profiles of Exosomes from Pooled NPC Patients serum comparing Control Exosomes from Healthy donors serum Two-condition experiment, Exosomes from Pooled Healthy donors serum vs. Exosomes from Pooled NPC Patients serum. Biological replicates: 1 Exosomes from Pooled Healthy donors serum, 1 Exosomes from Pooled NPC Patients serum,
Project description:Clinical study of critically ill patients with sepsis and sepsis-related ARDS with whole blood RNA collected within the first 24 hours of admission Goal of the study was to determine whether biologically relevant genes were identified to be differentially expressed genes in patients with sepsis alone and sepsis with ARDS Prospective observational study, case cohort design
Project description:MicroRNA microarray profilling analysis was performed on exosomes derived from 4T1 mammary epithelial cancer cells in two culture conditions, hypoxia and normoxia.
Project description:The goal of this study is to report that breast cancer associated exosomes contain microRNAs (miRNAs) associated with the RISC Loading Complex (RLC) and display cell-independent capacity to process precursor microRNAs (pre-miRNAs) into mature miRNAs. Pre-miRNAs, along with Dicer, AGO2, and TRBP, are present in exosomes of cancer cells. CD43 mediates the accumulation of Dicer specifically in cancer exosomes. Cancer exosomes mediate an efficient and rapid silencing of mRNAs to reprogram the target cell transcriptome. Exosomes derived from cells and sera of patients with breast cancer instigate non-tumorigenic epithelial cells to form tumors in a Dicer-dependent manner. These findings offer opportunities for the development of exosomes based biomarkers and therapies. Exosomes from cancer cells and non-tumorigenic cells were isolated using established ultracentrifugation methods. The global miRNA content of cancer exosomes and normosomes was investigated. Profiling of cells themselves was also used as a control. Exosomes with Dicer down regulation (MCF10AshDicer and MDA-MB-231shDicer exosomes), as well as MDA-MB-231 exosomes that contain a Dicer antibody inside were used to study the function Dicer protein in the microRNA biogenesis in exosomes.
Project description:Exosomes are molecular entities derived from membrane vesicles of endocytic origin secreted by most cell types. These vesicles are implicated in cell-to-cell communication, deliver proteins and mRNA molecules between cells. Recent studies have shown that exosomes are found in body fluids such as saliva, blood, urine, amniotic fluid, malignant ascites, bronchoalveolar lavage fluid, synovial fluids and breast milk. Exosomes secreted through human saliva contain mRNA may potentially be useful for diagnostic purposes. Although the exact protective mechanism of saliva RNA is a topic of debate, the consensus is that the enrichment of mRNAs in these nano-vesicles in one of the features of the biomarker discoveries. Our aim was to determine if exosomes are present in human saliva and to nano-characterize their transcriptomic content. Exosomes were purified by differential ultracentrifugation, identified by immunoelectron microscopy, flow cytometry and western blot using a CD-63 antibody. Atomic force microscopy studies revealed ultra structural analysis of both size and density of exosomes. Microarray analysis revealed the presence of 590 mRNA core transcripts are relatively stable inside the exosomes, which can be of saliva mRNA biomarkers. Exosomal mRNA stability was determined by detergent lyses with treatment of RNase. Under in vitro conditions fluorescent dye labeled saliva exosomes were able to communicate between human oral keratinocytes studied by using fluorescence microscopy. The RNA from saliva exosomes can transfer their genetic information to human oral keratinocytes and alters gene expression in the new location. Together, these results suggest that saliva is involved in mRNA trafficking via exosomes, and provides a mechanism for cargoing passenger mRNAs. Our findings are consistent with proposal that exosomes can shuttle RNAs between cells and mRNA is protected inside these vesicles may be a possible resource for biomarker discovery. Experiment Overall Design: Human saliva exosomes were purified through differential centrifugation followed by RNA extraction and hybridization on Affymetrix microarrays. We were able to obtain normal human subjects saliva which are pooled and subjected to ultracentrifugation. The protocol was approved by UCLA Institutional review board. 1 ml of saliva exosomes were used to extract RNA followed by two rounds of amplification by Actorus Amp kit. The amplified RNA was biotin labled and hybridized with Affymetrix protocol.
Project description:The goal of this study is to report that breast cancer associated exosomes contain microRNAs (miRNAs) associated with the RISC Loading Complex (RLC) and display cell-independent capacity to process precursor microRNAs (pre-miRNAs) into mature miRNAs. Pre-miRNAs, along with Dicer, AGO2, and TRBP, are present in exosomes of cancer cells. CD43 mediates the accumulation of Dicer specifically in cancer exosomes. Cancer exosomes mediate an efficient and rapid silencing of mRNAs to reprogram the target cell transcriptome. Exosomes derived from cells and sera of patients with breast cancer instigate non-tumorigenic epithelial cells to form tumors in a Dicer-dependent manner. These findings offer opportunities for the development of exosomes based biomarkers and therapies. Exosomes from cancer cells were isolated using established ultracentrifugation methods. The global miRNA content of non-tumorigenic cells was investigated before and after exosomes treatment to study the role of microRNA biogenesis in exosomes for cancer progression and the transformation process of normal cells.