109 Sarcomere Disassembly and Transfection E iciency in Proliferating Human iPSC-Derived Cardiomyocyt 5 INTRODUCTION The contractile tissue of the heart is composed of a mixture of different cells, including approximately 35% cardiomyocytes (CMs).1 Proper contraction of CMs is dictated by the architecture of the sarcomeres and appears together with polyploidy inversely correlated with self-renewal. Higher sarcomere architectural organization is related to maturity of the heart2, while immature organization of sarcomeres correlates to the capacity of CMs to proliferate.3 Previous studies in rodent hearts have shown that during embryonic and early postnatal hyperplastic growth phases the CMs gradually disassemble their sarcomeres during mitosis to ensure cytokinesis.4-6 Unlike in fish7, mammals display hyperplastic growth shortly after birth, which is subsequentially silenced leading to heart growth predominantly originating from cellular hypertrophy.8 The hypertrophic growth dynamic is accompanied by increased ploidy and multinucleation of the adult heart.9-10 The exact dynamics of human CM multinucleation and self-duplication in relation to sarcomere architecture remain largely unknown. Moreover, reliable determination of CM cytokinesis, multinucleation, self-duplication and nuclear ploidy is challenging.11-13 The advances in human-induced pluripotent stem cells (hiPSCs) biology allow for efficient directed differentiation into CMs (hiPSC-CMs), thereby relying on biphasic Wnt signal modulation and resulting in up to 95% pure populations of hiPSC-CMs.14-16 Currently, hiPSCCMs are widely used in mechanistic studies, drug testing and tissue engineering.17-18. We and others demonstrated that a third phase of Wnt signal modulation in immature CMs results in a maturation block while prolongating the window for CM proliferation.3,9,20-22 Using this strategy, it now becomes feasible to maintain 30–50% of Ki67 positive cells and up to 10% of mitotic CMs after day 12 of hiPSC-CM differentiation.3,21 Here we utilized live imaging in a hiPSC-CM culture system to follow the sequence of sarcomere breakdown during the mitotic phases of CM cell division. We observed that cytokinesis in hiPSC-CMs originates from mononuclear and binuclear CMs and for both routes of duplication the sarcomere breakdown was present during the mitotic phases. Moreover, we observed that a mitotic cell figure, suggestive for the sequence of sarcomere breakdown, was also present during the process of binucleation. Molecular genetic modification in hiPSC-CMs represents an essential tool for the mechanistic validations and functions of genes and proteins, but is limited by technical challenges to transfect low-proliferative hiPSC-CMs.23 Here, we show that Wnt activation in hiPSC-CMs results in sarcomere breakdown during mitosis and that increased cell cycle activity facilitates increased efficiency of non-viral vector incorporation. These findings give an insight in the regulation of sarcomere homeostasis during mitotic cell phases and provide a tool for further molecular and engineering studies.
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