Abstract
Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) hold tremendous promise for in vitro modeling to assess native myocardial function and disease mechanisms, as well as testing drug safety and efficacy. However, current hiPSC-CMs are functionally immature, resembling in vivo CMs of fetal or neonatal developmental states. The use of targeted culture media and organoid formats have been identified as potential high-yield contributors to improve CM maturation. This study presents an hiPSC-CM maturation medium formulation, designed using a differential evolutionary approach targeting metabolic functionality for iterative optimization. Relative to existing high-performing reference formulations, our medium significantly matured morphology, Ca2+ handling, electrophysiology, and metabolism, which was further validated by multi-omic screening, for cells in either pure or co-cultured microtissue formats. Together, these findings not only provide a reliable workflow for highly functional hiPSC-CMs for downstream use, but also demonstrate the power of high-dimensional optimization processes in evoking advanced biological function in vitro.
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Data availability
The data generated in this study are provided in the Supplementary Information/Source Data file. Raw and processed RNAseq data have been deposited in the NCBI Gene Expression Omnibus under the GEO Series accession number GSE214617. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE92 partner repository with the dataset identifier PXD036639. Source data are provided with this paper.
Code availability
The HD-DE code is available at https://doi.org/10.5281/zenodo.18664141.
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Acknowledgments
The authors acknowledge Dr. Michael Laflamme (University Health Network) for constructive conversations in the ideation and realization of this project. The authors also acknowledge The Metabolomics Innovation Center (TMIC; McGill University Node, Montreal QC), the Nanoscale Biological Imaging Facility (NBIF) at the Hospital for Sick Children (Toronto ON), and the Princess Margaret Genomics Center (PMGC, University Health Network, Toronto ON) for their assistance with metabolomics, transmission electron microscopy, and RNA sequencing, respectively. This study was funded by a Canadian Institutes of Health Research (CIHR) Project grant (PJT-175231) to CAS; a Collaborative Health Research Program grant from CIHR (CPG-151946) and the Natural Sciences and Engineering Research Council of Canada (NSERC) (CHRPJ 508366-17) to CAS and FB; a Ted Rogers Center for Heart Research Strategic Innovation Grant to JE, SM, FB, MR, and CAS; a Canada Research Chair in Stem Cell Models of Childhood Disease to JE; and a Heart and Stroke Foundation of Canada / Robert M. Freedom Chair in Cardiovascular Science to SM. NIC and RGI were funded by Vanier Canada Graduate Scholarships from NSERC and CIHR, respectively. LJD was funded by the Translational Biology and Engineering Program, Ted Rogers Center for Heart Research.
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N.I.C. and C.A.S. conceived the study. N.I.C., M.M.K., and J.A. contributed to the iterative optimization workflow. EYW and KW assisted with microtissue method development. N.I.C., L.J.D., W.C., U.K., M.Z.M., Y.D., Z.M., C.R., R.A.G., and R.G.I. collected and analyzed data. J.P.S., A.O.G., F.B., M.R., S.M., J.E., P.H.B., and C.A.S supervised the study. N.I.C., L.J.D., W.C., and R.A.G. prepared display items and drafted the manuscript. All authors edited the manuscript and approved the final version.
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NIC and CAS have assigned their interest in the intellectual property associated with the C16 formulation and its applications to the University of Toronto, which has filed a patent application (US 63/280,388; Maturation Medium for Pluripotent Stem Cell-derived Cardiomyocytes). This IP has been licensed to Censo Biotechnologies Ltd. T/A Axol Bioscience (Cambridge, UK). All other authors declare no competing interests.
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Callaghan, N.I., Durland, L.J., Chen, W. et al. Advanced physiological maturation of human iPSC-derived cardiomyocytes using an algorithm-directed optimization of defined media components. Nat Commun (2026). https://doi.org/10.1038/s41467-026-70550-9
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DOI: https://doi.org/10.1038/s41467-026-70550-9
