Cardiac‐Derived ECM Microspheres for Enhanced hiPSC‐CMs Maturation

ABSTRACT The phenotypic immaturity of human induced pluripotent stem cell‐derived cardiomyocytes (hiPSC‐CMs) remains a critical barrier to their effective use in disease models, drug screening, and cardiac regeneration. Current culture platforms still find it hard to provide a physiologically relevant, stable, and scalable microenvironment to support cardiomyocyte maturation. We report a reproducible heart tissue‐derived cardiac extracellular matrix (ECM) microsphere that integrates native biochemical cues with a porous three‐dimensional (3D) architecture to support cardiac cell culture and maturation. These microspheres supported the attachment and proliferation of C2C12, HL‐1, H9c2, and hiPSC‐CMs culture, suggesting the broad applicability of multiple cell types. Compared with the conventional two‐dimensional (2D) culture system, decellularized cardiac ECM microspheres provided a spherical 3D culture interface and significantly enhanced hiPSC‐CM maturation, as indicated by upregulation of cardiac genes ( ACTA2, TNNT2, GJA1 ), rapid calcium cycling, and synchronized calcium transients. Long‐term culture (up to 8 months) on these ECM microspheres supported hiPSC‐CM maturation, as evidenced by enhanced sarcomere alignment, robust α‐actinin expression, contractile phenotype, elevated gap junction (connexin 43, CX‐43) protein expression, and increased binucleation. This ECM microsphere system represents a scalable and bioactive platform for long‐term cardiomyocyte culture and maturation and broader applications in cardiac tissue engineering.