The immature phenotype of human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) represents a major limitation for their application in disease modelling, identification of cardiotoxic or pro-arrhythmic compounds, and for guiding personalized drug selection for patients.
To address this challenge and to generate iPSC-CMs with an advanced maturation state, we established an approach that combines lipid-enriched maturation medium, nanopatterning of culture surfaces and electrostimulation.
By systematic, parallel testing of these three stimuli we identified electrostimulation as the pivotal factor to enhance mitochondrial development. In combination with MM and NP, ES strongly increased the expression of markers for mitochondrial development, such as PPARGC1α, PPARα, OPA1 and MFN2. In agreement with these findings, we observed an increased Tom20 staining intensity of in iPSC-CMs matured under the influence of ES, indicating an increased mitochondrial mass. RNA-sequencing and gene set enrichment analyses confirmed that ES, combined to NP and MM, strongly enhances the expression of gene involved in oxidative phosphorylation, TCA cycle, fatty acid oxidation, glucose metabolism and mitochondrial biogenesis. Subsequent analysis against the transcription factor targets database revealed that the genes upregulated through ES map to the enrichment of TFAM- and HMCES-related target gene sets. While TFAM (mitochondrial transcription factor A) is essential for the transcription, replication and packaging of mtDNA, HMCES (5-hydroxymethylcytosine binding, embryonic stem cell-specific) acts as a safeguard to maintain genomic and mtDNA integrity during oxidative stress. Our findings demonstrate that both factors are important for the endogenous mechanisms controlling the development of the mitochondrial network in response to contractile activity. In further studies, we aim to investigate the molecular mechanisms of how mitochondrial development and metabolic activity of iPSC-CMs is linked to their structural and electrophysiological maturation.