Despite decades of effort, there is a pressing need for better in vitro cardiac and cardiotoxicity models. Cardiovascular diseases (CVD) remain the leading global cause of death and cardiotoxicity is responsible for a third of all pre-clinical pharmaceutical regulatory failures.  While traditional models (ex vivo and in vivo animal models, primary human tissue and immortalised cell lines) have provided valuable insights, there remains a translational gap between the “bench and bedside” due to issues with human-relevance, alongside well-characterized challenges with availability, throughput and cost.

Researchers have therefore been turning to human iPSC-derived cardiomyocytes for their potential to provide a limitless source of consistent, human-relevant cardiac cells. By taking donor cells, reprogramming them to an iPSC state and then differentiating them into cardiomyocytes, researchers can mimic human physiology in a scalable format, using these cells to power advanced in vitro cardiac disease and cardiotoxicity models.

However, standard differentiation protocols can lead to less mature cardiomyocyte phenotypes, impairing functional performance and therefore the utility of human iPSC-based models. Key measures of immature phenotype include the following six parameters; their sarcomere alignment, cardiac maturity marker expression, lower levels of spontaneous beating, longer Field Potential Duration (FPD), slower Conduction Velocity (CV) and a switch from glycolysis to fatty acid metabolism.

Axol Bioscience, in collaboration with partner organizations, have developed a new metabolic maturation media to enhance the maturity of human iPSC-derived cardiomyocytes, enabling better models for cardiac research, disease and cardiotoxicity. 

This new metabolic maturation media has been validated on two healthy control-derived ventricular cardiomyocyte lines: a newborn male line (ax2508) and a middle-aged female line (ax5858).  This media drives more mature cardiac morphology as measured by ICC, with improved sarcomere alignment and increased expression of the key cardiac maturity markers Troponin T and Connexin-43 by DIV8.  Transcriptomics analysis demonstrates improvement in the expression of cardiac maturity markers including PLN, SCN1B, KCNJ2, KCNJ3 and KCNQ1, with a Log2fold change from 2.6 to 3.8 for KCNJ2 and corresponding decreases in the expression of immaturity markers including HCN2.   Functionally, the maturation media drives more mature electrical activity measured via cardiac electrophysiology, with a reduction in beat rate from 44 ± 1 bpm to 23 ± 2 bpm, increases in CV from 0.40 ± 0.04 mm/ms to 0.70 ± 0.04 mm/ms and a shortening of FPD from 491 ± 55 ms to 312 ± 14 ms (all n=7-8, p<0.05-0.0001, unpaired t-test with Welch’s correction) by DIV14.  The maturation media also incorporates a factor which selects against cells utilising glycolysis in favour of cells utilising fatty acid metabolism causing the die-back of any remaining immature cardiomyocytes as detected by microscopy and confluence measurements. These improvements represent a more mature cardiomyocyte phenotype across the six parameters described previously.

            With these improvements, human iPSC-derived cardiomyocytes can be produced with enhanced structural and functional maturity to power advanced in vitro models of cardiac disease and cardiotoxicity. This addresses a key challenge to the wider adoption of iPSC-derived cardiomyocytes for cardiac research, drug testing and cardiotoxicity.