Sub-Confluent Culture of Mouse Embryonic Stem Cell-derived Ventricular Cardiomyocytes On & In Gels - Enhancement of Maturation Phenotype Relative to Tissue Culture Polystyrene via Enabling of Auxotonic Contraction

Cardiac myocytes (CMs) obtained by differentiating embryonic stem cells (ES-CMs) have an immature phenotype and promoting the maturation of such PSC-derived cardiomyocytes remains a major limitation in the development of stem cell models of human cardiovascular disease. We cultured murine ES-CMs in a collagen gel (3D) at a low density, or on collagen-coated polystyrene (2D) and found that 3D culture results in dramatic improvement of the maturation rate and end-state gene expression of ES-CMs. There are two main differences between CMs cultured in 3D versus 2D; in 3D the mechanical stiffness of the environment is lower, enabling auxotonic instead of isometric contraction; and, in 3D the amount of cell-cell interaction is higher. To isolate the contributions, we first cultured ES-CMs on gels (2D substrates) that are softer than tissue culture plastic, enabling auxotonic contraction, while controlling for dimensionality and cell interaction. This indeed promoted a mature gene expression profile, while also enabling the maintenance of sarcomeres. Next, we determined that increased cell-cell interaction inhibits the mature gene expression of ES-CMs. Thus, auxotonic contraction is the likely mechanism for improved gene expression in sub-confluent 3D culture. However, 2D auxotonic contraction may offer a suitable compromise between obtaining enhanced gene expression and morphology. After 6 weeks of culture on gels, via Di-8-ANEPPs and WGA staining we also detected CMs forming a t-tubule network. Collectively these results demonstrate that 3D and 2D cultures that enable auxotonic contraction enhance aspects of the maturation of ES-CMs.