Induced pluripotent stem-cells (iPSC) and their subsequent differentiation into cardiomyocytes (CM) and other cardiac-relevant cell types became an invaluable tool in cardiovascular research. Many etiologies of heart failure involve defects in excitation-contraction coupling, mitochondrial energetics or both, but currently available iPSC-CM are limited by the developmental immaturity in respect to these processes. Maturation strategies typically include long-term culture, fatty-acid enriched medium, or mechanical or electrical stimulation. We aim to boost maturation by a novel combinatorial approach of 3D-spheroid culture with a specially composed maturation medium (MM). This MM is composed of three fatty-acids, hormones, and a PPARα-agonist. To evaluate the maturation progress, we analyzed 2D-cultures and 3D-spheroids via molecular and functional read outs including adult CM isoform-switches on mRNA-level via qPCR, sarcomeric and mitochondrial structures via immunostaining, single-cell patch clamp recordings for action potentials, Ca2+ homeostasis with Indo-1-probe imaging, the mitochondrial membrane-potential (ΔΨM) with the TMRM-probe and the mitochondrial Ca2+ content with imaging genetically-encoded Pericam.
This systematic investigation demonstrates that the spheroid+MM protocol produces iPSC-CM with adult-like features yielding rod-shaped cells with the appropriate sarcomeric length, refined action potential shape and parameters concomitant with higher systolic Ca2+ levels, and a high sensitivity to β-adrenergic stimulation. In contrast, the mitochondrial arrangement is not improved, however the mitochondrial function is enhanced, which is demonstrated by higher ΔΨM and mitochondrial Ca2+ levels. To test the assumption that disease traits can be unmasked by enhanced maturation, we investigated the splice-defect in patient-iPSC-CM with a mutation in the splicing factor RBM20. We demonstrated more pronounced mis-splicing of RBM20 target genes CAMK2D and triadin in RBM20-CM vs isogenic rescue-CM cultured as spheroid+MM compared to their long-term culture cohort. Taken together, we present a maturation approach that significantly enhances structural, metabolic and functional parameters and can unmask disease traits in genetically caused cardiomyopathies.
