Cardiac differentiation of induced pluripotent stem cells on elastin-like protein-based hydrogels presenting a single-cell adhesion sequence

Substrate-dependent cardiac differentiation of induced pluripotent stem cells (iPSCs) has been studied on various extracellular matrix (ECM)-derived substrates, such as collagen type I (Col-I). However, ECM-derived substrates have multiple cell-adhesive amino acid sequences and stimulate various signaling pathways in cells, making it difficult to clarify the mechanism of substrate-dependent stem cell differentiation. A substrate presenting one of these sequences is a powerful tool for elucidating the mechanism. We designed elastin-like proteins (ELPs) composed of repetitive VPGIG sequences with or without the RGD cell adhesion motif (ELP-RGD/ELP-Ctrl) and used a chemical crosslinker to generate hydrogels. By adjusting the ELP and crosslinker concentrations, we obtained ELP-Ctrl and ELP-RGD hydrogels with a Young’s modulus of 0.3？kPa. The ELP-Ctrl and ELP-RGD gels were used as a substrate for the cardiac differentiation of cultured murine iPSCs. Cells on the ELP-RGD gel showed four times higher gene expression of the contractile protein troponin T type 2 than those on a Col-I gel, which is an effective substrate for iPSC cardiac differentiation. The ELP-RGD gel might stimulate integrin-derived signaling pathways in the cells to promote cardiac differentiation. This study showed the potential of ELP hydrogels for studying substrate-dependent iPSC cardiac differentiation by enabling the control of cell-adhesive sequence presentation