120 Chapter 5 higher incidence of cytokinesis from mononucleated cells in our system also appears to be in line with the concept that diploid cells are the most powerful for endogenous heart regeneration.10 The observation that hiPSC-CMs disassemble their sarcomeres followed by temporary quiescence is potentially explanatory for the low number of cell division in the adult human heart, which cannot allow itself “to skip a beat”. Besides the heart, the process of multinucleation as a result of a cytokinetic cell division also occurs in skeletal muscle and forms of cancer. Previous studies have demonstrated that this multinucleation process is reflected in hiPSC-derived muscle progenitor and cancer cell line models.33-35 Additionally, our non-viral vector gene transfection studies show that efficiency is highly related to the proliferative status of hiPSC-CMs. Although multiple methods of genetic modifications exist (i.e., lipofectamine-mediated transfection, and viral-based transduction), their efficiency, cytotoxicity, safety, and cost remain unsatisfactory and often below 10– 20%.26 We used our live hiPSC-CM imaging setup to study the transfection in proliferating (canonical Wnt activation) versus non-proliferating hiPSC-CMs. We found that hiPSC-CMs after chromosomal segregation (mitotic figure) were the first cells expressing a non-viral vector-based fluorescent protein. A smaller fraction of cells incorporates non-viral vector DNA in a cell cycle-independent manner. This improved approach for gene transfection is directly usable for research involving molecular studies and cardiac tissue engineering applications. Several limitations should be considered when interpreting the results of our study. Although we used expanding hiPSC-CMs reaching purity of up to 98%, for our time-lapse videos we were not able to live trace sarcomere, and therefore, there is potentially a risk of other cell types being recorded and analyzed. Secondly, CHIR99021, a potent GSK3β inhibitor present in our expansion culture system, could potentially also have non-cell cycle-related effects leading to increased transfection rates. Our data in Figure 5, however, indicates that activation of the cell cycle and M phase is needed for early incorporation of non-viral vectors. Lastly, the lack of environmental cues in our in vitro system put limitations on the interpretation of the described event of self-duplication as a potential route for heart regeneration. This study demonstrates that there is a complex landscape in hiPSC-CM proliferation, multinucleation and self-duplication. Moreover, enhanced incorporation of non-viral vectors is related to Wnt activation and cell cycle activity of hiPSC-CMs, which forms a strong tool for molecular gene studies in hiPSC-CMs and cardiac tissue engineering. ACKNOWLEDGEMENTS We thank Joseph C. Wu from Stanford University for providing lines SCVI-111 and SCVI-273. We thank Amsterdam UMC (location VUmc; O2 building) Microscopy and Cytometry Core Facility (MCCF) for the technological support.
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