Cardiac tissue engineering to replace damaged areas of the postmitotic heart is still presented with significant challenges, due to the complex and dynamic interplay of electrical, mechanical and biochemical signals involved in the myocardium. The advancement of regenerative approaches is focussed on understanding the underlying regulatory mechanisms involved throughout cardiac development. However, current knowledge of how biophysical cues in the stem cell niche can modulate cell behaviour is limited.
Firstly, polyacrylamide-co-acrylic acid was used as an in vitro stiffness-tuneable platform to test the effect of substrate mechanics on human induced pluripotent stem cell (hiPSC) differentiation into cardiomyocytes (CM). The results showed that the optimum differentiation efficiency level peaked at the embryonic-like stiffness of 560 Pa, with increased upregulation of cardiac genes. Functionally, hiPSC-CMs showed a biphasic relationship with a faster calcium transient and higher force generation at cardiac physiological stiffness. Next, shape was incorporated into the experimental design via CardioArray, a custom-built platform which mimics both the stiffness and shape of an adult human CM. This system can accommodate individual hiPSC-CMs to adopt the 3D geometry of an adult CM, while at the same time providing the relevant stiffness cues from the underlying substrate. The results highlighted the specific contribution of stiffness and 3D shape to α-sarcomeric structure, cell membrane stiffness, single cell gene expression and intracellular calcium cycling. Finally, the electrical microenvironment was investigated as a third infleuncing factor on hiPSC-CM development. A hybrid conductive polyaniline-Scl2 scaffold was fabricated, showing long term electronic stability and no cell toxicity when interfaced with electrosensitive hiPSC-CMs. This could provide electromechanical stability in model studies. Improvement of conduction velocity was observed in an in vitro myocardial slice model. As a whole, this thesis demonstrates the differential effects of substrate mechanics on hiPSC cardiac differentiation, providing a novel crucial understanding of how biophysical cues modulate the stem cell niche during differentiation and in vitro culture.