Driven maturation of human pluripotent stem cell derived cardiomyocyte in engineered cardiac tissue model

Abstract

Human pluripotent stem cell derived cardiomyocytes (hPSC-CMs) represent ideal cell-based platforms of drug screening, cardiotoxicity testing, disease modelling and regenerative medicine. However, these hPSC-CMs are immature, as indicated by their lack of structural alignment, immature electrophysiology, poor calcium handling properties, weak contractility and insensitive drug response. Various pro-maturational factors, including microgroove (M)-induced alignment, hormonal treatment using triiodothyronine (T3), electromechanical conditioning (EC) and prolonged culture, have been used to induce hPSC-CM maturation. For instance, T3 was reported to improve the action potential and calcium handling properties, contractility and mitoKATP activity in single cell and human ventricular microtissue (hvCMT) models. Although the actions of these factors have previously reported in separate studies, examination of their combinatorial effects remains limited. Furthermore, 3D cultivating environment may also promote the maturation of hPSC-CM by providing the physiological niche. In this dissertation, it was sought to integrate these pro-maturational factors in (i) 2D micropatterned human ventricular cardiac anisotropic sheet (hvCAS) and (ii) 3D human ventricular cardiac organoid chamber (hvCOC), to drive hPSC-CM maturation and to demonstrate new approaches in understanding hPSC-CM biology. In 2D-hvCAS, the additive microgroove-triiodothyronine-electromechanical conditioning combined treatment (M-T3-EC) significantly promoted anisotropic ratio (AR) from 1.02 to 1.49-1.78. Also, M-T3-EC increased the incident rate of quiescent hvCAS by 5-fold (over 70%). The phenotypical changes were associated with the increase in fast inward sodium current and decrease in funny current and confirmed by changes in SCN1B and HCN4 expressions. Furthermore, the expression of calcium handling genes was upregulated, which lead to reduction in calcium rise time (27.5-29.1%) and 50% decay time (17.7-18.1%). Using next generation RNA sequencing and pathway mapping, the downregulation of genes in TGF-β signalling pathway was identified as a cause of M-T3-EC driven maturation. To demonstrate the mechanism of M-T3-EC, upregulated or downregulated TGF-β signalling activities by TGFβ1 or SB431542, the agonist or antagonist of TGF-β receptor, could partially revert M-T3-EC induced quiescence or reduce spontaneous contraction frequency, respectively. The pro-maturational effects of prolonged culture and T3 were tested in 3D-hvCOC. In this model, 30-day hvCOC showed better morphology comparing to that of 10-day one. On the other hand, T3 treatment reduced the spontaneous contraction frequency by 23.1% and increased the spontaneous developed pressure by 25.6%. Also, both the T3-treated hvCOC and hvCOC with 30-day culture showed improved cardiac output at 2Hz when compared to 10-day untreated controls. More importantly, the T3-hvCOC displayed higher sensitivity to the positive chronotropic and inotropic effects induced by dobutamine, a β-adrenergic agonist. Taken collectively, it is concluded that 1) combinatorial T3-EC treatment via downregulation of TGF- β signalling pathway can drive hPSC-CM maturation in hvCAS, as shown by improved AR, reduced incidence of spontaneous contraction and matured calcium handling properties, and 2) prolonged culture or T3 can promote the maturation of hvCOC by improving the contractility-frequency response and increasing β-adrenergic sensitivity. The orchestration of these pro-maturational factors advances the formation of artificial cardiac tissue for in vitro drug screening and disease modelling and better understanding in hPSC-CM biology.