Synthetic hydrogels support robust and reproducible cardiomyocyte differentiation†

Cardiomyocyte manufacturing from human pluripotent stem cells is limited by the variability of differentiation efficiencies, partly attributed to the widespread use of the tumor-derived substrate Matrigel. Here, we describe a screening approach to identify fully-defined synthetic PEG hydrogels that support iPSC-derived cardiac progenitor cell (iPSC-CPC) adhesion, survival, and differentiation into iPSC-derived cardiomyocytes (iPSC-CMs). Our PEG hydrogels supported superior iPSC-CM differentiation efficiency, with a 24% increase in cTnT expression, and greater reproducibility when compared to cells cultured on Matrigel. By combining our 5-level, 3-variable full factorial screening approach with multi-variate analysis, we showed that all substrate variables manipulated here (adhesion ligand type/concentration, stiffness) had a significant influence on iPSC-CPC confluency and that iPSC-CM differentiation was significantly influenced by adhesion ligands. These results highlight the benefit of synthetic, tunable cell culture substrates and multi-variate screening studies to identify substrate formulations for a targeted cell behavior.