Proximity proteomics for serum extracellular vesicles from EML4-ALK transgenic mouse
Ontology highlight
ABSTRACT:
ORGANISM(S): Mus Musculus (mouse)
SUBMITTER: Norihiro Kotani
PROVIDER: PXD023672 | JPOST Repository | Mon Apr 06 00:00:00 BST 2026
REPOSITORIES: jPOST
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Project description:Gene expression profile of lung tumor tissues from EML4-ALK transgenic mouse (generous gift from Prof. H. Mano, Univ. of Tokyo) compared with lung normal tissues
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Project description:The ALK^F1174L mutation is associated with intrinsic and acquired resistance to crizotinib and cosegregates with MYCN in neuroblastoma. In this study, we generated a mouse model overexpressing ALK^F1174L in the neural crest. Comapred to mice expressing ALK^F1174L or MYCN alone, combined expression of the two aberrations led to development of neuroblastoma with a shorter latency and higher penetrance. Here, we evaluated the transcriptional profiles of MYCN-driven neuroblastomas with or without the expression of ALK^F1174L to determine the pathogenic consequences of the ALK^F1174L/MYCN interaction in neuroblastoma. 10 mice were analysed in this study. Five ALK^F1174L/MYCN tumors were compared with five MYCN tumors. Total RNA was extracted, samples were labeled and processed using the Agilent Low Input Quick Amp two color Cy3(sample) and Cy5 (mouse reference) labeling kit and hybridized to Agilent SurePrint G3 Mouse Gene Expression arrays.
2012-07-04 | E-GEOD-35560 | biostudies-arrayexpress
Project description:Osteolineage cells represent one of the critical bone marrow niche components that support maintenance of hematopoietic stem and progenitor cells (HSPCs). Recent studies demonstrate that extracellular vesicles (EVs) regulate stem cell development via horizontal transfer of bioactive cargo, including microRNAs (miRNAs). Here, we characterize the miRNA profile of EVs secreted by human osteoblasts and study their biological effect of on human umbilical cord blood-derived CD34+ HSPCs by sequencing, gene expression and biochemical analyses. Using next-generation sequencing we show that osteoblast-derived EVs contain highly abundant miRNAs specifically enriched in EVs, including critical regulators of hematopoietic proliferation (e.g., miR-29a). EV treatment of CD34+ HSPCs alters the expression of candidate miRNA targets, such as HBP1, BCL2 and PTEN. Furthermore, EVs enhance proliferation of CD34+ cells and their immature subsets in growth factor-driven ex vivo expansion cultures. Importantly, EV-expanded cells retain their differentiation capacity in vitro and show successful engraftment in NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice in vivo. These discoveries reveal a novel osteoblast-derived EV-mediated mechanism for regulation of HSPC proliferation and warrant consideration of EV-miRNAs for the development of expansion strategies to treat hematological disorders.
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Project description:The present studies tested the hypothesis that the elongating ovine conceptus and uterus produces EVs with the potential to mediate conceptus-maternal communication during early pregnancy. In Study One, EVs were purified from uterine luminal fluid (ULF) of day 14 cyclic sheep. The EVs were fluorescently labeled with PKH67 dye and infused into the uterine lumen of pregnant sheep for 6 days using an osmotic pump. On day 14, labeled EVs were observed in the conceptus trophectoderm and uterine epithelia, but not in the uterine stroma or myometrium. In Study Two, day 14 conceptuses were cultured ex vivo for 24 hours and found to release EVs into the culture medium. Isolated EVs from conceptuses were fluorescently labeled with PKH67 and infused into the uterine lumen of cyclic sheep for 6 days using an osmotic pump. On day 14, labeled EVs were observed in the uterine epithelia, but not in the uterine stroma or myometrium. No evidence of EV escape from the uterine lumen was observed by analysis of the ovary and other maternal tissues. Proteomics analysis of the day 14 conceptus-derived EVs identified 231 proteins that were enriched for extracellular space and several protein classes including proteases, protease inhibitors, chaperones and chaperonins. RNA-sequencing of day 14 conceptus-derived EVs detected expression of 512 mRNAs. The top expressed genes were overrepresented in ribosomal functions and components. These studies support the ideas that EVs emanate from both the conceptus trophectoderm and uterine epithelia and are involved in intercellular communication during the establishment of pregnancy. Transcriptional profiles from day 14 conceptus extracellular vesicles isolated from 24 hour conceptus-conditioned culture media (n=3) were generated by sequencing on the Illumina HiSeq 2500 platform.
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Project description:Liquid biopsies contain multiple analytes that can be mined to improve the detection and management of cancer. Beyond cell-free DNA (cfDNA), mutations have been detected in DNA associated with extracellular vesicles (EV-DNA). The genome-wide composition and structure of EV-DNA remains poorly characterized, and whether circulating EVs are enriched in tumor signal compared to unfractionated cfDNA is still unclear.
Here, using whole genome sequencing from selected lung cancer patients with a relatively high cfDNA tumor content >5%, we determined that the tumor fraction and heterogeneity are comparable between DNA associated with small (<200 nm) EVs and matched plasma cfDNA. DNA in EV fractions, obtained with standardized size-exclusion chromatography, are comprised of short ~150-180 bp fragments and long >1000 bp fragments that are poor in tumor signal. Other fractions only exhibit short fragments with similar tumor DNA content. The composition in bases at the end of EV-DNA fragments, as well as their fragmentation patterns are similar to total plasma cfDNA. Mitochondrial DNA however is relatively enriched in EV fractions.
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| EGAS00001006848 | EGA
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.
2024-10-02 | E-MTAB-13966 | biostudies-arrayexpress
Project description:Phenotypic changes induced by extracellular vesicles (EVs) have been implicated in the recovery of acute kidney injury (AKI) induced by mesenchymal stromal cells (MSCs). miRNAs are potential candidates for cell reprogramming towards a pro-regenerative phenotype. The aim of the present study was to evaluate whether miRNA de-regulation inhibits the regenerative potential of MSCs and derived-EVs in a model of glycerol-induced AKI in SCID mice. For this purpose, we generated MSCs depleted of Drosha, a critical enzyme of miRNA maturation, to alter miRNA expression within MSCs and EVs. Drosha knock-down MSCs (MSC-Dsh) maintained the phenotype and differentiation capacity. They produced EVs that did not differ from those of wild type cells in quantity, surface molecule expression and internalization within renal tubular epithelial cells. However, EVs derived from MSC-Dsh (EV-Dsh) showed global down-regulation of miRNAs. Whereas, wild type MSCs and derived EVs were able to induce morphological and functional recovery in AKI, MSC-Dsh and EV-Dsh were ineffective. RNA sequencing analysis showed that genes deregulated in the kidney of AKI mice were restored by treatment with MSCs and EVs but not by MSC-Dsh and EV-Dsh. Gene Ontology analysis showed that down-regulated genes in AKI were associated with fatty acid metabolism. The up-regulated genes in AKI were involved in inflammation, ECM-receptor interaction and cell adhesion molecules. These alterations were reverted by treatment with wild type MSCs and EVs, but not by the Drosha counterparts. In conclusion, miRNA depletion in MSCs and EVs significantly reduced their intrinsic regenerative potential in AKI, suggesting a critical role of miRNAs. RNA-seq
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2014-10-28 | E-GEOD-56172 | biostudies-arrayexpress