Effect of stem cell niche elasticity/ECM protein on the self-beating cardiomyocyte differentiation of induced pluripotent stem (iPS) cells at different stages.

Stem cell-based myocardial regeneration therapies have emerged as alternative strategies to heart transplantation for serious heart diseases, but autologous beating mature cardiomyocytes are not available. Here we investigated the effect of culture substrates on the cardiomyocyte differentiation of induced pluripotent stem cells (iPSs) in vitro by separately evaluating the following continuous three steps: (1) cardiac marker gene expression, (2) contractile gene expression and self-beating, and (3) beating duration. To this end, we used iPS cells to study the cardiac differentiation, and neonatal rat cardiomyocytes (NCMs) to study beating behavior. These cells were cultured on substrates with different natures, i.e., an elastic substrate (Es) with the modulus of 9, 20, or 180 kPa, and hard tissue culture polystyrene dishes (TCPS) coated with collagen type I (Col), gelatin (Gel), or fibronectin (FN). The results revealed that the effective niches in each step were very different. The cardiac marker gene (GATA4, Tbx5, MEF2C) expression of iPSs at the 1st step was very high on the TCPS coated with FN or Gel, whereas on the FN-coated Es (especially with the 9 kPa modulus), the undifferentiated marker gene (Nanog) expression of iPSs was maintained. The expression of the contractile genes α-MHC, TnC1, and TnT2 and the self-beating (the 2nd step) of the NCMs were high on FN-coated TCPS and Col-coated Es. The 3rd step (beating duration) of the NCMs was effective on the Es, and at 21 days both the iPSs and NCMs stopped beating on the TCPS but were still beating on the Es. Overall, cardiac differentiation 'preferred' ECM-rigid culture substrates, and beating-behavior 'preferred' Col-soft culture substrates. These results are important for understanding and designing cardiac differentiation niches for regenerative medicine, and they suggest that a single culture substrate is not suitable for preparing self-beating cardiomyocytes.

STATEMENT OF SIGNIFICANCE: The transplantation of beating cardiomyocytes (BCMs) is expected to be made more effective for serious heart diseases. The identification of the appropriate engineering processes and suitable culture substrates for inducing stem cell differentiation into BCMs is thus indispensable. The differentiation can be divided into three major processes, the cardiac differentiation step, the beating-induction step and the beating-duration step. A protocol with the higher efficiency in all of the steps must be useful. In this study, we separately evaluated the effect of culture substrates at each three step. We clarified that the biological and the physical properties of the culture substrates required at these steps were different. We found useful criteria for effective cardiac cell niche systems design.