Abstract 29: Isolation of Cardiomyocytes Undergoing Mitosis with Complete Cytokinesis

Abstract

Adult cardiomyocytes (CMs) fail to exit the cell cycle and result in polyploidization and multi-nucleation. This extremely limited renewal potential is a key barrier for cardiac regeneration after heart injury. The use of traditional cell cycle assays such as Ki67 or purine analogue administration does not allow for isolation of viable CMs in late-M phase. We hypothesize that a molecular beacon (MB)-based method could be developed to isolate a highly pure population of hiPSC-derived CMs capable of completing mitosis and cytokinesis. We first differentiated human PSCs into cardiomyocytes using the small molecules CHIR and IWR and then applied MBs targeting CDC20 mRNAs, followed by fluorescence-activated cell sorting (FACS). Validation of cell cycle-specific and mitosis-associated gene expressions of cell fractions sorted from G2 and M-phase were performed by RT-qPCR prior to transcriptional profiling by single-cell RNA sequencing. Gene set enrichment analysis (GSEA) confirmed the observation of the gene ontology (GO) analysis. The ‘GO_cytokinesis’ and the ‘GO_cell separation after cytokinesis’ gene sets were highly enriched and predominantly upregulated in the CDC20 (+) (M-phase) population compared with the CDC20 (-) (G2-phase) population. Furthermore, using these data sets we expect to develop robust surface marker based flow cytometry protocols at a number and purity allowing for statistically significant genome-wide integrative transcriptome and epigenome analysis. In conclusion, the isolation of CMs using CDC20 MB and DNA content dye proved to enrich for cells that were confirmed by GSEA to be CMs in cytokinesis.