Stiffness-Tunable Hydrogel-Sandwich Culture Modulates the YAP-Mediated Mechanoresponse in Induced-Pluripotent Stem Cell Embryoid Bodies and Augments Cardiomyocyte Differentiation.

Abstract

Microenvironmental factors, including substrate stiffness, regulate stem cell behavior and differentiation. However, the effects of substrate stiffness on the behavior of induced pluripotent stem cell- (iPSC) derived embryoid bodies (EB) remain unclear. To investigate the effects of mechanical cues on iPSC-EB differentiation, we developed a three-dimensional hydrogel-sandwich culture (HGSC) system that controls the microenvironment surrounding iPSC-EBs using a stiffness-tunable polyacrylamide hydrogel assembly. Mouse iPSC-EBs were seeded between upper and lower polyacrylamide hydrogels of differing stiffness (Young's modulus [E'] = 54.3 ± 7.1kPa [hard], 28.1 ± 2.3kPa [moderate], and 5.1 ± 0.1kPa [soft]) and cultured for 2 days. HGSC induced stiffness-dependent activation of the yes-associated protein (YAP) mechanotransducer and actin cytoskeleton rearrangement in the iPSC-EBs. Moreover, moderate-stiffness HGSC specifically upregulated the mRNA and protein expression of ectoderm and mesoderm lineage differentiation markers in iPSC-EBs via YAP-mediated mechanotransduction. Pretreatment of mouse iPSC-EBs with moderate-stiffness HGSC promoted cardiomyocyte differentiation and structural maturation of myofibrils. Our proposed HGSC system provides a viable platform for investigating the role of mechanical cues on the pluripotency and differentiation of iPSCs that could be beneficial for research into tissue regeneration and engineering. This article is protected by copyright. All rights reserved.