Circulating re-entrant waves promote maturation of hiPSC-derived cardiomyocytes in self-organized tissue ring

Human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes are a promising resource for tissue engineering and drug testing, but their immature phenotype limits translation. This study introduces a simple ring-template culture platform that rapidly (≤2 days) self-assembles hiPSC-derived cardiomyocytes into three-dimensional self-organized tissue rings (SOTRs) that spontaneously develop circulating re-entrant waves (ReWs) of electrical activity. Calcium imaging demonstrated robust, long-lived ReWs that pace tissues at high frequencies (~2–4 Hz) without external electrical stimulation, avoiding common drawbacks of chronic electrode pacing. The authors show that ReW number and beating frequency depend on ring diameter and interpret ReW origination and stability using a mathematical excitable-media model that reproduces wave behaviors and scaling trends. After 2 weeks, ReW-trained SOTRs exhibited multi-modal cardiomyocyte maturation: improved myofibrillar alignment along the ring axis, increased sarcomere length, upregulated maturation-associated cardiac genes (e.g., MYH7/β-MHC, ACTN2, MYL2/3, PLN, ADRB1), enhanced Ca2+ handling (caffeine responses), increased mitochondrial respiration capacity, and higher contractile force with a Frank–Starling-like dependence on stretch. Overall, the platform provides an economical, scalable alternative to external pacing for producing more mature hiPSC-cardiomyocyte tissues for drug screening and regenerative engineering.