Abstract
Background: Human induced pluripotent stem cells (hiPSCs) are widely used in studies of developmental and regenerative biomedicine involving various cell types, including cardiomyocytes (CMs). Understanding the cellular and molecular processes during hiPSC-CM differentiation and maturation will be crucial to develop heart regeneration therapies. In addition, while bioengineered cellular cues have been shown to modulate cellular morphology and phenotypes, little is known regarding their effects on molecular and functional maturity of hiPSC-CMs. Aims: To determine the expression of cardiac and cell cycle genes and the electrophysiological properties of hiPSC-CMs during their differentiation and maturation on unpatterned (Flat) or isotropic/nanopatterned (Nano) cell culture surface. Methods and Results: Using small molecules, WTC-11 hiPSC were differentiated into CMs efficiently (92.5% TNNT2 + ; 95% NKX2-5 + ). The resultant hiPSC-CMs were re-plated in Flat or Nano surfaces and harvested at different time points. Cardiac genes Myh7, Tnnt2, Serca2a, Ryr2, Cacna1c , and Kcnj2 gradually and significantly increased during differentiation; this was accompanied by reduced expression of cell cycle genes. While myofilament genes expressions were similar between Nano and Flat cultured hiPSC-CMs, ion channel genes Scn5a, Cacna1c , and Kcnj2 were significantly higher in Nano group, suggesting that Nano cultured CMs were more matured. In addition, fewer hiPSC-CMs were proliferative (EdU + ) in 2-week Nano group compared to Flat group. This was associated with decreased expression of active cell cycle genes Ccne1, Cdk4, Cdk14, Ki67 and Plk1 in Nano 2-week CMs. Micro-electrode array (MEA) analysis demonstrated that Beat Period, Spike Amplitude, and Field Potential Duration were increased in the Nano group. Fluo-4 Ca 2+ imaging assay revealed improved Ca 2+ transition activities in isotropically cultured hiPSC-CMs. Conclusion: These results demonstrate a significant upregulation of cardiac genes along with a down-regulation of cell cycle genes during the differentiation and maturation of hiPSC-CM on Nano surfaces. Bioengineered nanotopographically patterned substrates promoted the maturation and electrophysiological functions of hiPSC-CMs.
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