Cell alignment widely exists in various in vivo tissues and also plays an essential role in the construction of in vitro models, such as vascular endothelial and myocardial models. Recently, microscale and nanoscale hierarchical topographical structures have been drawing increasing attention for engineering in vitro cell alignment. In the present study, we fabricated a micro-/nanohierarchical substrate based on soft lithography and electrospinning to assess the synergetic effect of both the aligned nanofibrous topographical guidance and the off-ground culture environment provided by the substrate on the endothelium formation and the maturation of human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). The morphology, proliferation, and barrier formation of human umbilical vein endothelial cells (HUVECs) as well as the alignment, cardiac-specific proteins, and maturity-related gene expression of hiPSC-CMs on the aligned-nanofiber/microridge (AN-MR) substrate were studied. Compared with the glass slide and the single-aligned nanofiber substrate, the AN-MR substrate enhanced the proliferation, alignment, and cell−cell interaction of HUVECs and improved the length of the sarcomere and maturation-related gene expression of hiPSC-CMs. Finally, the response of hiPSC-CMs on different substrates to two typical cardiac drugs (isoproterenol and E-4031) was tested and analyzed, showing that the hiPSC-CMs on AN-MR substrates were more resistant to drugs than those in other groups, which was related to the higher maturity of the cells. Overall, the proposed micro-/nanohierarchical substrate supports the in vitro endothelium formation and enhances the maturation of hiPSC-CMs, which show great potential to be applied in the construction of in vitro models and tissue engineering.
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