Human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) represent a key technology to study cardiac pathomechanisms in a human model system. However, a major limitation in the use of iPSC-CMs in comparison to adult cardiomyocytes is their immature phenotype with respect to the structure, metabolism, contractile- as well as electrophysiological function. Supplementation of the culture medium with fatty acids was shown to induce the metabolic switch of iPSC-CMs from glycolysis towards β-oxidation as the major energy-generating pathway, representing the metabolic phenotype in adult cardiomyocytes. The presence of an adult-like metabolic state as well as culture conditions that are close to physiological conditions provide an important basis for the modeling of metabolic diseases such as diabetic cardiomyopathy.

In this study, we aimed to establish a culture medium to induce maturation of iPSC-CMs by mimicking the physiological levels of cardiac energy substrates. We compared the widely used standard medium, which provides mainly glucose as an energy substrate, with different fatty-acid supplemented maturation media containing varying levels of glucose, lactate and insulin. Our results reveal that fatty acid supplementation represents the major stimulus for the maturation of iPSC-CMs, while the concentrations of glucose, lactate and insulin can be adjusted to physiological levels without impairing structural or functional development of iPSC-CMs. Cultivation in maturation medium led to increased cell size as well as increased expression ratios of TNNI3/TNNI1 and MYL2/MYL7, demonstrating structural maturation of iPSC-CMs. Immunostaining for MLC2A and MLC2V confirmed that cultivation in maturation media enhanced the development of iPSC-CMs towards a
ventricular phenotype, as the proportion of MLCA^-/MLC2V⁺ cells was strongly increased in comparison to standard medium. Documentation of the contractile activity of iPSC-CMs revealed an enhanced functional maturation based on reduced spontaneous beating frequency, shortened contraction time and increased contraction velocity. An improved mitochondrial function of iPSC-CMs cultured in maturation media compared to standard medium was demonstrated by an increased mitochondrial membrane potential, increased expression of genes involved in fatty acid metabolism (CPT1B, CD36) and mitochondrial development (PPARGC1α) as well as enhanced mitochondrial maximal respiratory capacity. Interestingly, cell number was slightly increased after 21-day cultivation in maturation medium compared to standard medium, while the proportion of cTNT⁺ cells was comparable. Investigation of the proliferative activity based on EdU-incorporation revealed an increased population of EdU⁺ cells after 7 days in maturation medium, which was strongly reduced at the end of the maturation period after 21 days.

In summary, our results demonstrate that fatty acid supplementation represents the main stimulus to induce maturation of iPSC-CMs, while concentrations of glucose, lactate and insulin can be adjusted to mimic physiological levels without affecting the structural and functional development. Based on the metabolically matured iPSC-CMs in physiological culture medium, future studies aim to investigate the effect of culture conditions mimicking the situation in diabetic patients to recapitulate hallmarks of diabetic cardiomyopathy.