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Human pluripotent stem cell-derived cardiomyocytes (CMs) are a promising tool for cardiac cell therapy. Although transplantation of induced pluripotent stem cell (iPSC)-derived CMs have been reported in several animal models, the treatment effect was limited, probably due to poor optimization of the injected cells. To optimize graft cells for cardiac reconstruction, we compared the engraftment efficiency of intramyocardially-injected undifferentiated-iPSCs, day 4 mesodermal cells, and day 8, day 20, and day 30 purified iPSC-CMs after initial differentiation by tracing the engraftment ratio (ER) using in vivo bioluminescence imaging. This analysis revealed the ER of day 20 CMs was significantly higher compared to other cells. Transplantation of day 20 CMs into the infarcted hearts of immunodeficient mice showed good engraftment, and echocardiography showed significant functional improvement by cell therapy. Moreover, the imaging signal and ratio of Ki67-positive CMs at 3 months post injection indicated engrafted CMs proliferated in the host heart. Although this graft growth reached a plateau at 3 months, histological analysis confirmed progressive maturation from 3 to 6 months. These results suggested that day 20 CMs had very high engraftment, proliferation, and therapeutic potential in host mouse hearts. They also demonstrate this model can be used to track the fate of transplanted cells over a long time.

Cardiac replacement therapy using induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) holds great promise as a new treatment strategy to cure drug-resistant heart failure. We have previously reported enlargement of engrafted iPSC-CMs for three months after direct injection of the cells into the myocardium of immunodeficienct mouse infarcted hearts with left anterior descending artery (LAD) ligation. We also confirmed the iPSC-CMs are more proliferative in vivo than in vitro.Here, we analyzed the cell cycle of iPSC-CMs for the purpose of enhancing engraftment efficiency after transplantation. We generated an iPSC line that constitutively expressed Fluorescent Ubiquitination-based Cell Cycle Indicator (FUCCI), which enables visualization of the cell cycle phase: Cells in S/G2/M phase (proliferating) emit green fluorescence, while cells in G0/G1 phase (non-proliferating) emit red fluorescence. Next, we compared the engraftment ability between S/G2/M phase iPSC-CMs and G0/G1 phase iPSC-CMs three months after transplantation. Cell cycle activated S/G2/M phase iPSC-CMs, in which the cell cycle is activated, showed significantly higher engraftment efficiency than G0/G1 phase iPSC-CMs, in which the cell cycle is inactivated. High-throughput screening for compounds that activate the cell cycle in iPSC-CMs was performed to realize effective engraftment. Out of 4032 initially screened compounds, we identified 82 candidates. A second screening suggested CCA-1 had the most potent effect. We therefore optimized its administration, which resulted in us increasing the percentage of S/G2/M phase iPSC-CMs from 2.8% to 39.8% according to flow cytometry analysis.We further investigated the proliferative effect of CCA-1. EdU assays showed CCA-1 enhanced DNA replication in iPSC-CMs, indicating an increased proliferation ability, while additional experiments found CCA-1 also increased the cell number count. Finally, CCA-1-treated iPSC-CMs showed enhanced graft size after transplantation into the ischemic hearts of immunodeficient mice in vivo. Altogether, these data demonstrate that the cell cycle plays a key role in the efficiency of iPSC-CM engraftment, and its modification should be considered in novel therapeutic treatments. [ABSTRACT FROM AUTHOR]

Myocardial infarction is caused by a lack of oxygen due to blockage of a coronary artery and is a common cause of heart failure. Despite therapeutic advances, the prognosis of patients with heart failure is poor. One of the reasons is that present therapeutic approaches do not restore the loss of cardiac tissue. Stem cell-based therapies have the potential to regenerate the myocardium, and numerous studies using stem cells have shown improved cardiac function and reduced infarct size. In this chapter, we describe our methodology for transplanting human induced pluripotent stem cell-derived cardiomyocytes into immunodeficient mouse hearts with myocardial infarction., (© 2021. Springer Science+Business Media, LLC, part of Springer Nature.)

Specific cell lineages differentiated from human induced pluripotent stem cells (iPSCs) are promising sources for cell replacement therapy and are useful biomedical research tools for research on disease mechanisms and drug discovery. Among the different lineages, cardiac lineage has been one of the most efficiently differentiated through several established protocols. In this chapter, we describe our reproducible and highly efficient methodology for differentiating iPSCs into cardiomyocytes using embryoid bodies. We also describe methods to dissociate iPSC-derived cardiomyocytes and to evaluate iPSC-derived cardiomyocytes.