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, a 3D hydrogel-sandwich culture (HGSC) system is developed that controls the microenvironment surrounding iPSC-EBs using a stiffness-tunable polyacrylamide hydrogel assembly. Mouse iPSC-EBs are seeded between upper and lower polyacrylamide hydrogels of differing stiffness (Young's modulus [E'] = 54.3 ± 7.1 kPa [hard], 28.1 ± 2.3 kPa [moderate], and 5.1 ± 0.1 kPa [soft]) and cultured for 2 days. HGSC induces stiffness-dependent activation of the yes-associated protein (YAP) mechanotransducer and actin cytoskeleton rearrangement in the iPSC-EBs. Moreover, moderate-stiffness HGSC specifically upregulates 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 promotes cardiomyocyte (CM) differentiation and structural maturation of myofibrils. The proposed HGSC system provides a viable platform for investigating the role of mechanical cues on the pluripotency and differentiation of iPSCs that can be beneficial for research into tissue regeneration and engineering.
Keywords: cardiomyocyte; differentiation; embryoid body; hydrogel; induced pluripotent stem cells; stiffness.
© 2023 The Authors. Macromolecular Bioscience published by Wiley-VCH GmbH.