Rationale: Adult human cardiomyocytes do not complete cytokinesis despite passing through the S-phase of the cell cycle. As a result, polyploidization and multinucleation occur. To get a deeper understanding of the mechanisms surrounding division of cardiomyocytes, there is a crucial need for a technique to isolate cardiomyocytes that complete cell division/cytokinesis. Objective: Markers of cell cycle progression based on DNA content cannot distinguish between mitotic cardiomyocytes that fail to complete cytokinesis from those cells that undergo true cell division. With the use of molecular beacons (MBs) targeting specific mRNAs, we aimed to identify truly proliferative cardiomyocytes derived from human induced pluripotent stem cells. Methods and Results: Fluorescence-activated cell sorting combined with MBs was performed to sort cardiomyocyte populations enriched for mitotic cells. Expressions of cell cycle specific genes were confirmed by means of reverse transcription-quantitative polymerase chain reaction and single-cell RNA sequencing (scRNA-seq) combined with gene signatures of cell cycle progression. We 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 cardiomyocyte subpopulation exhibited patterns consistent with the processes of nuclear division, chromosome segregation, and transition from M to G1 phase. The use of dual-MBs targeting CDC20 and SPG20 mRNAs enabled the enrichment of cytokinetic events ( CDC20 high SPG20 high ). Interestingly, cells that did not complete cytokinesis and remained binucleated were found to be CDC20 low SPG20 high while polyploid cardiomyocytes that replicated DNA but failed to complete karyokinesis were found to be CDC20 low SPG20 low . Conclusions: This study demonstrates a novel alternative to existing DNA content-based approaches for sorting cardiomyocytes with true mitotic potential that can be used to study the unique dynamics of cardiomyocyte nuclei during mitosis. Our technique for sorting live cardiomyocytes undergoing cytokinesis would provide a basis for future studies to uncover mechanisms underlying the development and regeneration of heart tissue.
As both sentinels and effectors of disease response, peripheral blood mononuclear cells are an accessible and attractive target for clinical application of high throughput, fluidics based single cell RNASeq (scRNASeq). However, new analytic tools required by unique characteristics of scRNASeq data lack validation in acutely ill patients. We report scRNASeq analysis of ~1,000 20 cells from each of 38 patients requiring veno-arterial extracorporeal life support (VA-ECLS)-a diverse group of critically ill patients experiencing circulatory collapse as a common endpoint to wide ranging diseases. We established an analysis pipeline capturing major biological signals from theses samples, confirmed by flow cytometry. Further, we found that these patients appeared immunologically poised at the outset of treatment as either reactive or permissive, and 25 this balance predicted their survival. Annotated code detailing the analysis is available at https://github.com/vanandelinstitute/va_ecls.
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