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• W2912092982 abstract "Abstract Rationale-Adult human cardiomyocytes (CMs) do not complete cytokinesis despite passing through the S-phase of the cell cycle. As a result polyploidization and multinucleation occur. In order to get a deeper understanding of the mechanisms surrounding division of CMs there is a crucial need for a technique to isolate CMs that complete cell division/cytokinesis. Objective-Markers of cell cycle progression based on DNA content cannot distinguish between mitotic CMs that fail to complete cytokinesis from those cells that undergo true cell division. With the use of molecular beacons (MB) targeting specific mRNAs we aimed to identify truly proliferative CMs derived from hiPSCs. Methods and Results-Fluorescence activated cell-sorting combined with molecular beacons was performed to sort CM populations enriched for mitotic cells. Expressions of cell-cycle specific genes were confirmed by means of RT-qPCR, single-cell RNA sequencing (scRNA-seq). We further characterized the sorted groups by proliferation assays and time-lapse microscopy which confirmed the proliferative advantage of MB-positive cell populations relative to MB-negative and G2/M populations. Gene expression analysis revealed that the MB-positive CM subpopulation exhibited patterns consistent with the biological processes of nuclear division, chromosome segregation, and transition from M to G1 phase. The use of dual-MBs targeting CDC20 and SPG20 mRNAs ( CDC20 + SPG20 + ) enabled the enrichment of cytokinetic events. Interestingly, cells that did not complete cytokinesis and remained binucleated were found to be CDC20 − SPG20 + while polyploid CMs that replicated DNA but failed to complete karyokinesis were found to be CDC20 − SPG20 − . Conclusions-This study demonstrates a novel alternative to existing DNA content-based approaches for sorting CMs with true mitotic potential that can be used to study in detail the unique dynamics of CM nuclei during mitosis. Together with high-throughput scRNA-seq, our technique for sorting live CMs undergoing cytokinesis would provide a basis for future studies to uncover mechanisms underlying the development and regeneration of heart tissue." @default.
• W2912092982 created "2019-02-21" @default.
• W2912092982 creator A5012319289 @default.
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• W2912092982 date "2019-02-13" @default.
• W2912092982 modified "2023-09-26" @default.
• W2912092982 title "Isolation of cardiomyocytes undergoing mitosis with complete cytokinesis" @default.
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• W2912092982 doi "https://doi.org/10.1101/549212" @default.
• W2912092982 hasPublicationYear "2019" @default.
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