Quantitative shotgun proteomics (TMT) of EVs in human MSCs
Ontology highlight
ABSTRACT: Quantitative shotgun proteomic analysis (TMT) of the effect of inhibition of MIR21 in the EV protein cargo of human, Umbilical cord-derived Mesenchymal Stem Cells.
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.
Project description:Mesenchymal stem cells (MSC) have emerged as potent therapeutic tool for a number of pathologies, including immune ones. However, unwelcome effects of MSC on the blood coagulation were revealed in some cases, which require more in-depth analysis. In this study, we explored the trombotic properties of human MSC from umbilical cord. We revealed strong procoagulant effects of umbilical cord MSC toward human and rat whole blood and platelets-free plasma using rotational thromboelastometry and thrombodynamics tests. The similar potentiation of clotting was demonstrated for MSC-derived extracellular vesicles (EV). In order to suggest approaches to avoid unwanted effects we studied the impact of heparin supplement on MSC/EV procoagulation properties. We found that therapeutic doses of unfractionated heparin injected in the patient's blood (administered in vivo) did not abrogate the procoagulant properties of MSC. Mass-spectrometry analysis of proteins of MSC and EV involved in coagulation-associated pathways was used to evaluate mechanisms of protrombotic effects.
Project description:Human umbilical cord mesenchymal stem cells maintained multipotency and immunosuppressive ability when being cultured in chemical defined serum free medium, but gained different gene expression profile. We used microarrays to identify the transcriptional difference between human umbilical cord mesenchymal stem cells cultured in serum containing medium and chemical defined serum free medium. human umbilical cord mesenchymal stem cells were cultured in conventional serum containing medium and chemical defined serum free medium separately. Total RNA was extracted and hybridized on Affymetrix microarrays.
Project description:screening of signature deterimes the individual variations in the therapeutic efficacy of human umbilical cord blood-derived mesenchymal stem cells There is paucity of information whether human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs) from separate donors might have different effects on improving myocardial repair after myocardial infarction (MI).
Project description:We report that mesenchymal stem cells isolated from umbilical cord tissue differentiate more robustly into the myogenic lineage compared to mesenchymal stem cells from cord blood isolated from the same individual
Project description:Human umbilical cord mesenchymal stem cells maintained multipotency and immunosuppressive ability when being cultured in chemical defined serum free medium, but gained different gene expression profile. We used microarrays to identify the transcriptional difference between human umbilical cord mesenchymal stem cells cultured in serum containing medium and chemical defined serum free medium.