The importance of matrix in cardiomyogenesis: Defined substrates for maturation and chamber specificity

Human embryonic stem cell-derived cardiomyocytes (hESC-CM) are a promising source of cardiac cells for disease modelling and regenerative medicine. However, current protocols invariably lead to mixed population of cardiac cell types and often generate cells that resemble embryonic phenotypes. Here we developed a combinatorial approach to assess the importance of extracellular matrix proteins (ECMP) in directing the differentiation of cardiomyocytes from human embryonic stem cells (hESC). We did this by focusing on combinations of ECMP commonly found in the developing heart with a broad goal of identifying combinations that promote maturation and influence chamber specific differentiation. We formulated 63 unique ECMP combinations fabricated from collagen 1, collagen 3, collagen 4, fibronectin, laminin, and vitronectin, presented alone and in combinations, leading to the identification of specific ECMP combinations that promote hESC proliferation, pluripotency, and germ layer specification. When hESC were subjected to a differentiation protocol on the ECMP combinations, it revealed precise protein combinations that enhance differentiation as determined by the expression of cardiac progenitor markers kinase insert domain receptor (KDR) and mesoderm posterior transcription factor 1 (MESP1). High expression of cardiac troponin (cTnT) and the relative expression of myosin light chain isoforms (MLC2a and MLC2v) led to the identification of three surfaces that promote a mature cardiomyocyte phenotype. Action potential morphology was used to assess chamber specificity, which led to the identification of matrices that promote chamber-specific cardiomyocytes. This study provides a matrix-based approach to improve control over cardiomyocyte phenotypes during differentiation, with the scope for translation to cardiac laboratory models and for the generation of functional chamber specific cardiomyocytes for regenerative therapies.