RAD16-I 3D cultured cardioids promote hiPSC-CMs maturation

Objectives RAD16-I (RADARADARADARADA) self-assembling peptides have demonstrated significant potential in supporting the growth and differentiation of various cell types, including the ability to reduce the automaticity of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) within a beating recipient heart. However, when used alone in scaffold-based three-dimensional (3D) culture, RAD16-I has not consistently yielded contractile hiPSC-CMs in vitro. This study aimed to generate beating cardioids using RAD16-I 3D culture. Methods We investigated the synergistic effects of combining suspension culture with RAD16-I 3D culture. The cell types, structural organization and function of cardioids were analyzed. And the functional maturation of hiPSC-CMs was further evaluated. Results Our results indicate that RAD16-I 3D culture promotes hiPSC-CM differentiation and supports spontaneous beating of the derived cardiomyocytes. Importantly, the integration of suspension embryoid body (EB) formation with subsequent RAD16-I 3D culture (SR culture) enabled the generation of complex cardiac organoids (cardioids) containing distinct cavitations, endothelial cells (ECs), smooth muscle cells (SMCs), and endoderm-like cellular components. Within these cardioids, hiPSC-CMs exhibited significantly enhanced functional maturity compared to those differentiated in two-dimensional (2D) culture. This improvement was evidenced by upregulation of cardiac-specific gene expression, improved calcium handling dynamics, and enhanced mitochondrial function. Conclusions Our findings demonstrate that RAD16-I self-assembling peptides can be effectively utilized to generate physiologically relevant cardiac organoids and promote functional maturation of hiPSC-CMs. The SR culture strategy, using RAD16-I peptides, thus represents a novel and effective methodology for targeted cardiac tissue engineering applications.