Comparative Computational RNA Analysis of Cardiac-Derived Progenitor Cells and Their Extracellular Vesicles
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE196556 | GEO | 2022/11/21
REPOSITORIES: GEO
Similar Datasets
Project description:We aimed to examine differences between c-kit+ progenitor cells (CPCs) and CPC-derived extracellular vesicles (EV) RNA content. We isolated CPCs from cardiac biopsies of patients with congenital heart disease using magnetic bead sorting. We expanded CPCs, reduced serum for 24hrs, and collected secreted EVs from conditioned media with sequential ultracentrifugation. We isolate RNA from CPCs and CPC-EVs and sequence total RNA and miRNA. Sequencing was performed in at two different facilities: "E" and "N".
2022-12-31 | GSE202347 | GEO
Project description:We aimed to examine differences between c-kit+ progenitor cells (CPCs) and CPC-derived extracellular vesicles (EV) RNA content. We isolated CPCs from cardiac biopsies of patients with congenital heart disease using magnetic bead sorting. We expanded CPCs, reduced serum for 24hrs, and collected secreted EVs from conditioned media with sequential ultracentrifugation. We isolate RNA from CPCs and CPC-EVs and sequence total RNA and miRNA. Sequencing was performed in at two different facilities: "E" and "N".
2022-12-31 | GSE202345 | GEO
Project description:Stem- and progenitor cell transplantation therapy holds great promise for regenerating damaged heart tissue. Several lines of evidence suggest that its efficacy is mainly caused by secreted extracellular vesicles (EVs). EVs are small, cell-derived lipid bilayer enclosed particles that play a role in intercellular communication through their enrichment in specific components like nucleic acids and proteins. Indeed, cardiac progenitor cell (CPC)-derived EVs have been shown to protect the myocardium against ischemia/reperfusion injury. However, the underlying mechanisms for CPC-EV-mediated cardioprotection remain elusive. Here, we utilized the proteomic composition of CPC-EVs released during different culture conditions, to unravel protein-mediated effects of CPC-EVs on the endothelium. CPCs were stimulated with calcium ionophore (ca ion-EVs), previously shown to influence EV release, or vehicle (control-EVs) for 24 hours and EVs were isolated using size exclusion chromatography (SEC). EV concentration and size was assessed using NTA and proteomic composition was profiled using mass spectrometry in triplicate. EVs isolated from SKOV-3 cells were included as a non-stem cell control. Proteomic analysis identified multiple proteins uniquely expressed or enriched in control-EVs compared with ca ion-EVs and SKOV-3 EVs. To better understand the effect that these EVs have on cardioprotection, endothelial cells HMEC-1 were stimulated with CPC-EVs, CaIon-EVs or PBS for 30 min. Phosphorylation of ERK1/2 and AKT, and wound closure was increased in HMEC-1 after stimulation with CPC-EVs but not with Ca ion-EVs. To gain further detail in the pathway activation in HMEC-1 cells, we also performed (phospho)proteomic analysis of these cells upon EV-stimulation. The proteomic and phosphoproteomic changes derived from EV-stimulation in HMEC-1 were quantified by mass spectrometry and validated the activation of PI3K-AKT-mTOR and (Insulin/IGF-) MAPK signaling pathways.
2023-07-13 | PXD030779 | Pride
Project description:Extracellular vesicles (EVs) have gained significant attention in the field of extracellular communication, holding promise as intercellular messengers and diagnostic biomarkers. However, the challenges in isolating and purifying EVs from complex biological samples have hindered their widespread utility. This study presents a novel EV isolation strategy leveraging bioinformatics, utilizing the pH-low insertion peptide (pHLIP) to capture and release EVs under controlled conditions. The pHLIP-magnetic bead approach was developed to achieve efficient EV isolation, addressing the persistent issue of EV isolation method comparability. EVs from human plasma were isolated and characterized using various techniques, offering insights into the proposed isolation method's efficacy. A data science-driven statistical metric integrating multiple characterizations was applied to enable direct comparisons between different isolation methods. Next generation sequencing was applied to examine how the observed RNA-population differences across isolated EVs may lead researchers to different conclusions. This research contributes to the advancement of reliable and rigorous EV isolation methods and their potential clinical applications.
2024-01-31 | GSE246925 | GEO
Project description:In the research field of extracellular vesicles (EVs), the use of EV-depleted fetal bovine serum (FBS) for in vitro studies is highly recommended to eliminate the confounding effects of media derived EVs. EV-depleted FBS may either be prepared by ultracentrifugation or bought commercially, nevertheless these depletion methods do not guarantee an RNA-free preparation. In this study we have addressed the RNA contamination issue in FBS, ultracentrifuged EV-depleted FBS, commercially available EV-depleted FBS, and also from our recently developed filtration based EV depleted FBS. Commercially available serum-free, xeno-free defined media were also screened for RNA contamination.
2019-04-30 | GSE120594 | GEO
Project description:Beyond forming bone, osteoblasts play pivotal roles in various biological processes, including hematopoiesis and bone metastasis. Extracellular vesicles (EVs) have recently been implicated in intercellular communication via transfer of proteins and nucleic acids between cells. Here, we focused on the proteomic characterization of non-mineralizing (NMOBs) and mineralizing (MOBs) human osteoblast (SV-HFOs) EVs and investigated their effect on human prostate cancer (PC3) cells by microscopic, proteomic and gene expression analyses. Proteomic analysis showed that 97% of the proteins were shared among NMOB and MOB EVs, and 30% were novel osteoblast-specific EV proteins. Label-free quantification demonstrated mineralization stage-dependent five-fold enrichment of 59 and 451 EV proteins in NMOBs and MOBs, respectively. Interestingly, bioinformatic analyses of the osteoblast EV proteomes and EV-regulated prostate cancer gene expression profiles showed that they converged on pathways involved in cell survival and growth. This was verified by in vitro proliferation assays where osteoblast EV uptake led to two-fold increase in PC3 cell growth compared to cell-free culture medium-derived vesicle controls. Our findings elucidate the mineralization stage-specific protein content of osteoblast-secreted EVs, show a novel way by which osteoblasts communicate with prostate cancer, and open up innovative avenues for therapeutic intervention. PC3 cells were treated with extracellular vesicles from non-mineralizing and mineralizing SV-HFOs for three different incubation times (4hrs, 24hrs, 48hr)
2014-10-28 | E-GEOD-56172 | biostudies-arrayexpress
Project description:The use of cardiac progenitor cell (CPC)-derived small extracellular vesicles (sEVs) has shown potential to stimulate cardiac repair. sEVs are released by cells and play a role in intercellular communication through transfer of their bioactive content. Increasing evidence indicates that sEVs present a heterogeneous population of vesicles. In the context of cardiac regeneration, studying sEV heterogeneity could provide new insights into mechanisms underlying sEV-mediated cardiac repair properties and help to improve the therapeutic application of CPC-derived sEVs. Here we used size-exclusion chromatography to isolate and fractionate EV subpopulations dervied from CPCs. We then applied a bottom-up proteomic approach to characterize the contents of individual EV subpopulations.
2024-01-26 | PXD032334 | Pride
Project description:Baseline gene expression of mouse Cardiac Progenitor Cells (CPC) We used microarrays to analyze the global gene expression of mouse CPCs. Compare the gloable gene expression of mouse CPCs and Cardiomyocytes. Although the chip is two color, we only used Cy5 as the data channel.
2012-12-01 | E-GEOD-41175 | biostudies-arrayexpress
Project description:Extracellular vesicles (EVs) are involved in a wide range of physiological and pathological processes by shuttling material out of and between cells. Tissue EVs may thus lend insights into disease mechanisms and also betray disease when released into easily accessed biological fluids. Since brain-derived EVs (bdEVs) and their cargo may serve as biomarkers of neurodegenerative diseases, we evaluated modifications to a published, rigorous protocol for separation of EVs from brain tissue and studied effects of processing variables on quantitative and qualitative outcomes. To this end, size exclusion chromatography (SEC) and sucrose density gradient ultracentrifugation were compared as final separation steps in protocols involving stepped ultracentrifugation. bdEVs were separated from brain tissues of human, macaque. Effects of post-mortem interval (PMI) before final bdEV separation were probed. MISEV2018-compliant EV characterization was performed, and both small RNA and protein profiling were done. We conclude that the modified, SEC-employing protocol achieves EV separation efficiency roughly similar to a protocol using gradient density ultracentrifugation, while decreasing operator time and, potentially, variability. The protocol appears to yield bdEVs of higher purity for human tissues compared with macaque, suggesting opportunities for optimization. The interval between death/tissue storage/processing and final bdEV separation can also affect bdEV populations and composition and should thus be recorded for rigorous reporting. Different populations of EVs obtained through the modified method reported herein display characteristic RNA and protein content that hint at biomarker potential. To conclude, this study finds that the automatable and increasingly employed technique of SEC can be applied to tissue EV separation, and also reveals more about the importance of species-specific and technical considerations when working with tissue EVs. These results are expected to enhance the use of bdEVs in revealing and understanding brain disease.
2020-05-14 | GSE150460 | GEO
Project description:The regulation of multipotent cardiac progenitor cell (CPC) expansion and subsequent differentiation into cardiomyocytes, smooth muscle, or endothelial cells is a fundamental aspect of basic cardiovascular biology and cardiac regenerative medicine. However, the mechanisms governing these decisions remain unclear. Here, we show that Wnt/β-Catenin signaling, which promotes expansion of CPCs, is negatively regulated by Notch1-mediated control of phosphorylated β-Catenin accumulation within CPCs, and that Notch1 activity in CPCs is required for their differentiation. Notch1 positively, and β-Catenin negatively, regulated expression of the cardiac transcription factors, Isl1, Myocd and Smyd1. Surprisingly, disruption of Isl1, normally expressed transiently in CPCs prior to their differentiation, resulted in expansion of CPCs in vivo and in an embryonic stem (ES) cell system. Furthermore, Isl1 was required for CPC differentiation into cardiomyocyte and smooth muscle cells, but not endothelial cells. These findings reveal a regulatory network controlling CPC expansion and cell fate that involve unanticipated functions of β-Catenin, Notch1 and Isl1 that may be leveraged for regenerative approaches involving CPCs. YFP+ cells marking precardiac mesoderm (Isl1-cre domain) were FACS-sorted (w/wo stabilized Beta-catenin). Their total RNA was amplified with the WT-Ovation Pico RNA Amplification System, fragmented and labeled with the FL-Ovation cDNA Biotin Module V2 (Nugen). The hybridization, staining and scanning of the Affymetrix GeneChips were performed in the Gladstone Genomics Core Lab. Raw data generated from at least three independent experiments were further analyzed by the group of Dr. Ru-Fang Yeh at the Center for Informatics and Molecular Biostatistics, UCSF.
2009-05-13 | E-GEOD-15232 | biostudies-arrayexpress