Co-culturing hiPSC-cardiomyocytes and cardiac fibroblasts enhances engineered heart tissue structure and function

BACKGROUND: Engineered heart tissues (EHTs) are widely used for cardiac disease modeling and drug screening, but their lack of multicellularity limits translational relevance. Thus, it is essential to incorporate other cardiac cells to improve the reliability and accuracy of the model. OBJECTIVES: To develop a co-culture EHT model from human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes (CMs) and cardiac fibroblasts (CFs) and assess its structural, contractile, electrophysiological and metabolic properties. METHODS: hiPSCs were differentiated into CMs and CFs and combined at a ratio of 3:1 to generate co-culture EHTs. Structural, functional and metabolic features of CM-only and co-culture EHTs were evaluated and compared using immunofluorescence, force analysis, optical mapping and metabolomics. RESULTS: Co-culture EHTs were more compact, generated higher force when stimulated, and displayed improved sarcomere organization compared to CM-only EHTs. They showed reduced hypoxia under high frequency pacing and a more mature, stress resistant metabolic profile, while maintaining stable electrophysiology and reduced arrhythmogenicity. CONCLUSIONS: Incorporating hiPSC-CFs into EHTs enhanced structural and functional properties, improved stress resistance, and reduced variability, making our co-culture EHTs a more physiological and predictive platform for cardiac disease modeling and drug screening.