Abstract Current protocols for in vitro culture of human iPSC-CMs, including 3D tissue-engineering techniques, produce cells and tissues with immature structural and functional properties characteristic of fetal rather than adult myocardium. This lack of maturity significantly limits therapeutic potential of hiPSC-CMs by increasing their arrhythmogenic risks and hinders their use in disease modeling and drug development applications. Despite the large body of work to improve the maturation state of hiPSC-CMs, one important aspect - cell polyploidy - has been largely understudied. In vitro cultured hiPSC-CMs are predominantly mononuclear and diploid, while the adult human myocardium is comprised of nearly 90% polyploid CMs. Polyploidy is a conserved trait in mammalian CMs and is strongly associated with postnatal heart maturation. However, its physiological roles are largely unknown. Specifically, it remains unclear whether polyploidization drives maturation of the heart via specific transcriptomic changes, or if polyploidization is a consequence of maturation. The main hypothesis of my project is that polyploidy drives cardiac maturation, and that 3D engineered cardiac tissues (ECTs) made from primarily polyploid hiPSC-CMs will have increased functionality compared to tissues made from primarily diploid CMs. My promising preliminary results show that hiPSC-CM polyploidy induced genetically via cytokinesis failure or cell fusion yields increased size, mitochondria content, and conduction velocity of hiPSC-CMs and force generation of ECTs. In this project, I will thoroughly characterize process of genetically induced CM polyploidization and determine transcriptomic (RNAseq) and epigenetic (ATACseq) differences between polyploid and diploid hiPSC-CMs. Furthermore, I will examine roles of CM polyploidization in structural, functional, and metabolic maturation of ECTs and determine if polyploidy endows hiPSC-CMs with increased resistance to oxidative stress in vitro and enhanced therapeutic potential in vivo. By combining basic biology and bioengineering approaches, I hope to uncover new mechanistic links between CM polyploidy and maturation and provide innovative strategies to improve safety and efficacy of hiPSC-CM therapies for ischemic heart disease.