Poster Presentation Australasian Cytometry Society 43rd Annual Conference and Workshop

Modelling Human Cardiomyocyte Cell Cycle Using Multiparametric Flow Cytometry (#103)

Osvaldo Contreras 1 2 , Chris Thekkedam 2 , Richard P Harvey 2 3
  1. St. Vincent’s Clinical School, Faculty of Medicine, UNSW Sydney, Sydney, Kensington, Australia
  2. Stem cell and Developmental Biology Division, Victor Chang Cardiac Research Institute, Sydney, NSW, Australia
  3. School of Biotechnology and Biomolecular Science, UNSW Sydney, Sydney, Kensington, Australia

Congenital heart disease (CHD) is a leading cause of death among infants. Several CHDs are characterized by altered cardiac tissue architecture and the loss of cardiomyocytes, with some of these associating with increased DNA damage and abnormal myocyte proliferation. However, the study of the human cardiomyocyte cell cycle is challenging, and therefore, its behavior and progression remain elusive. Human-induced pluripotent stem cells offer an unlimited resource to study the cardiomyocyte cell cycle. Here, we first compared the efficiency of different broadly-use cardiac dissociation strategies to obtain single and viable cardiomyocytes via flow cytometry analysis, applying them to 2D or 3D hiPSC-derived cardiogenic cultures. Our results show that papain excels in generating single viable cardiomyocytes. We also explored the cardiomyocyte cell cycle progression at two different hiPSC-derived cardiogenic differentiation time points. We used DNA ploidy, Ki67, EdU, and PH3 staining, coupled to CellTrace, to understand cardiomyocyte cell cycle and division using multiparametric flow cytometry. A large majority of cardiomyocytes are in GO/G1 phases, even though a significant fraction of them is positive for Ki67. EdU effectively labels replicating cardiomyocytes, whereas PH3 labels cells only in G2/M phases. Thus, our data highlight the accuracy and high-throughput potential of flow cytometry to better understand the human cardiomyocyte cell cycle and provide an entry point for deeper analysis of cardiac cell cycle behavior in homeostasis and disease.