Chemically Defined and Small Molecule-Based Generation of Cardiomyocytes

Existing methodologies for human induced pluripotent stem cell (hiPSC) cardiac differentiation are efficient but require the use of complex undefined media constituents that hinder further elucidation of the molecular mechanisms of cardiomyogenesis. Using hiPSCs derived under chemically defined conditions on synthetic matrices, we systematically developed a highly optimized cardiac differentiation strategy employing a chemically defined medium, consisting of just three components: RPMI 1640, L-ascorbic acid 2-phosphate, and rice-derived recombinant human albumin. Along with small molecule-based differentiation induction, this produced contractile sheets of up to 95% TNNT2+ cardiomyocytes at a yield of up to 100 cardiomyocytes for every input pluripotent cell. This methodology was effective in 11 hiPSC lines tested repeatedly from p20 to p83, representing >600 differentiations. Cardiomyocytes produced this way were demonstrated to be of a predominantly atrial phenotype that progressively matured to a ventricular phenotype. The complete absence of any undefined or animal derived product in the entire hiPSC culture and differentiation platform makes it ideal for production of large numbers (>1E9) of cardiomyocytes to cGMP standards. This is the first fully chemically defined platform for cardiac specification of hiPSCs and permits elucidation of cardiomyocyte macromolecular/metabolic requirements, providing a minimally complex system for the study of maturation and subtype specification.