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Comparative Computational RNA Analysis of Cardiac-Derived Progenitor Cells and Their Extracellular Vesicles

ABSTRACT: 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".

ORGANISM(S): Homo sapiens

PROVIDER: GSE196556 | GEO | 2022/11/21

REPOSITORIES: GEO

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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.

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Comparative Computational RNA Analysis of Cardiac-Derived Progenitor Cells and Their Extracellular Vesicles

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