Enhanced iPSC-Cardiomyocyte Maturation via Combined 3D-Culture and Metabolic Cues

Rebs, S.; Eberl, H.; Wagensohner, N.; Dybkova, N.; Unsoeld, J. K.; Dudek, J.; Costa, P. M.; Fernandez, M. C.; Rog-Zielinska, E. A.; Schneider-Warme, F.; Maack, C.; Sossalla, S.; Streckfuss-Boemeke, K.

doi:10.1101/2025.10.29.684362

bioRxiv · 10.1101/2025.10.29.684362

Enhanced iPSC-Cardiomyocyte Maturation via Combined 3D-Culture and Metabolic Cues

Rebs, S.·Eberl, H.·Wagensohner, N.·Dybkova, N.·Unsoeld, J. K.·Dudek, J.·Costa, P. M.·Fernandez, M. C.·Rog-Zielinska, E. A.·Schneider-Warme, F.·Maack, C.·Sossalla, S.·Streckfuss-Boemeke, K.

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Abstract

Human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CM) have become an invaluable tool for disease modelling and drug testing. However, while many etiologies of heart failure involve defects in excitation-contraction coupling, mitochondrial energetics or both, iPSC-CM are limited by the developmental immaturity of these processes. Here, we report a combinatorial strategy to enhance the maturation of human iPSC-CM by integrating three-dimensional (3D) spheroid culture conditions with a defined hormone- and fatty acid-enriched maturation medium (MM). A comprehensive analysis of structural, electrophysiological and Ca2+ handling parameters was performed to evaluate cellular and functional maturation. The iPSC-CM generated under these conditions (3D_MM) exhibit many phenotypic characteristics that resemble those of isolated adult human CM, including (i) a rod-shaped morphology, (ii) cardiac ultrastructural features such as aligned myofilaments, unidirectional organized sarcomeres, and the presence of transverse (t)-tubules, (iii) refined action potential (AP) parameters and Ca2+ handling, and (iv) {beta}-adrenergic responsiveness and a positive force-frequency relationship. Compared with long-term (LT) monolayer cultures of 90 days or the individual cues (3D or MM alone), the 3D_MM protocol achieves mostly superior or at the least non-inferior maturation effects. This systematic investigation further demonstrates that while 3D culturing or MM alone improved specific aspects of maturation, only their synergistic combination produced a comprehensive enhancement of key CM processes, such as excitation-contraction coupling and mitochondrial energetics.

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Rebs, S., Eberl, H., Wagensohner, N., Dybkova, N., Unsoeld, J. K., Dudek, J., Costa, P. M., Fernandez, M. C., Rog-Zielinska, E. A., Schneider-Warme, F., Maack, C., Sossalla, S., Streckfuss-Boemeke, K.. 2025-10-30. Enhanced iPSC-Cardiomyocyte Maturation via Combined 3D-Culture and Metabolic Cues. https://doi.org/10.1101/2025.10.29.684362

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