Project Leader Tokyo Metropolitan Institute of Medical Science Tokyo, Japan
Abstract: Human induced pluripotent stem cell (iPSC)-derived cardiomyocytes (iPS-CMs) have great promise for disease modeling, drug development, and regenerative medicine. However, the immaturity of iPS-CMs resembling the human fetal state remains a significant challenge for broader applications. We focused on the polyploidy of human mature cardiomyocytes to address this issue. While over 80% of human cardiomyocytes become tetraploid during maturation, only 10% of iPS-CMs become 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. We successfully established 4N-iPSCs by Sendai virus-mediated fusion of diploid iPSCs and then differentiated them into cardiomyocytes using a general protocol. Almost all cardiomyocytes derived from 4N-iPSCs (4N-iPS-CMs) were tetraploid. As we expected, 4N-iPS-CMs exhibited more matured phenotypes, including post-mitotic gene expression signatures, higher mitochondrial content, higher contractile force, and faster upstroke velocity compared to the conventional iPS-CMs derived from diploid iPSCs. These experiments were initially done by fusion of the same diploid iPSC lines. However, if we could generate 4N-iPS-CMs from two different diploid iPSC lines derived from different individuals, we would be able to analyze interactions of two different genetic backgrounds within iPS-CMs. In this study, we established 4N-iPSC lines by fusion of diploid iPSCs derived from a Japanese male (WTC11) and a Caucasian female (HPS0076). We were able to differentiate these hybrid 4N-iPSCs into cardiomyocytes. These hybrid 4N-iPS-CMs also exhibited organized sarcomere and regular beating signatures. We also conducted RNAseq analysis and found that the gene expression profile of hybrid 4N-iPS-CMs showed intermediate features between those of WTC11 iPS-CMs and HPS0076 iPS-CMs. Therefore, our strategy to generate 4N-iPSCs and 4N-iPS-CMs serves as a versatile platform to study genetic interactions between two different genetic backgrounds in cardiomyocytes.
Funding Source: JSPS, Grant-in-Aid for Scientific Research (B) (24K02028). JCRF, Bayer Scholarship for Cardiovascular Research. SENSHIN Medical Research Foundation Grant. TERUMO Life Science Foundation Research Grant.
