Effects of Substrate Mechanics On Yield and Contractility of Cardiomyocytes Generated From Pluripotent Stem Cells

The mechanical microenvironment plays a role in proper development and function of heart cells, but implementing mechanical cues in an appropriate context during differentiation of human pluripotent stem cells (hPSCs) to cardiomyocytes remains challenging.  We used polyacrylamide hydrogels as a model system to investigate the effects of substrate mechanics on differentiation and functionality of cardiomyocytes generated from hPSCs.  By changing the concentration of crosslinker in our polyacrylamide hydrogels, we obtained substrates with a physiologically relevant range of stiffnesses.  We plated hPSCs undergoing embryoid body-based differentiation onto the hydrogels and observed that cardiogenesis peaks on substrates of intermediate stiffness.  To investigate the effects of substrate stiffness on contractility, we added fluorescent beads to the surface of the hydrogels, which act as optical trackers for the movement of contracting cells.  We used traction force microscopy to quantify the contraction stress of singularized cardiomyocytes cultured on a range of stiffnesses.  In primary rat cardiomyocytes, we observed that contraction stress increases with substrate stiffness.  We observed the same trend in cardiomyocytes derived from hPSCs, providing an indication of their similarity to native, fully differentiated cardiomyocytes.  These results demonstrate that hPSCs and their cardiomyocyte derivatives can respond to substrate mechanics.  Our approaches for investigating and quantifying this response may aid in the development of novel culture systems for effective differentiation of functional cardiomyocytes from hPSCs.