Ph.D Student Institute of Science Tokyo Setagaya-ku, Tokyo, Japan
Abstract: Human induced pluripotent stem cell (iPSC)-derived cardiomyocytes (iPS-CMs) have great promise for disease modeling, drug development, and regenerative medicine. iPS-CMs have already been used for drug development. Transplantation therapies using iPS-CM-engineered heart sheets have also advanced to clinical stages. However, the immaturity of iPS-CMs resembling the human fetal state, remains a significant challenge for broader applications. To address this issue, we focused on the polyploidy of human mature cardiomyocytes. While over 80% of human cardiomyocytes become tetraploid during maturation, only 10% of iPS-CMs achieve polyploid using conventional methods. This ploidy difference could limit the maturation of iPS-CMs. To overcome this limitation, we completely changed the differentiation strategy from the conventional scheme by first establishing tetraploid iPSCs (4N-iPSCs) and then differentiating them into cardiomyocytes to replicate the polyploidy of human mature cardiomyocytes. In this study, we successfully established 4N-iPSCs by Sendai virus-mediated fusion of diploid iPSCs. 4N-iPSCs had doubled DNA content within a single nucleus without any chromosomal abnormality. These 4N-iPSCs were then differentiated into cardiomyocytes using a general protocol. Almost all cardiomyocytes derived from 4N-iPSCs (4N-iPS-CMs) were tetraploid, replicating the polyploidy of adult human cardiomyocytes. Gene expression analysis revealed reduced expression of mitotic genes in 4N-iPS-CMs compared to diploid iPS-derived cardiomyocytes (2N-iPS-CMs), indicating that 4N-iPS-CMs were more mature, post-mitotic cardiomyocyte-like cells. Besides, the mitochondrial content was higher in 4N-iPS-CMs, suggesting an increased capacity for ATP production. Functionally, 4N-iPS-CMs exhibited higher contractile force and faster upstroke velocity than 2N-iPS-CMs based on electric impedance measurement. Furthermore, when tested for sensitivity to Terfenadine, a hERG channel blocker, 4N-iPS-CMs demonstrated greater resistance. These findings indicate that 4N-iPS-CMs replicate the polyploidy of adult human cardiomyocytes, resulting in greater maturity compared to conventional iPS-CMs. We propose that 4N-iPS-CMs are a promising platform for drug discovery and regenerative medicine.
Funding Source: JSPS, Grant-in-Aid for Scientific Research (B) (24K02028), and for JSPS Research Fellows (24KJ0995). JCRF, Bayer Scholarship for Cardiovascular Research. SENSHIN Medical Research Foundation. TERUMO Life Science Foundation.
