부산대학교 도서관

Introduction Pathological cardiac hypertrophy has not been fully recapitulated in the in vitro settings. Currently used in vitro models are presumably oversimplified since the vast majority are based on iPSC-derived cardiomyocytes (CMs) exclusively and do not include any cardiac non-myocytes. The aim of this study was to generate engineered heart tissues (EHTs) containing five hiPSC-derived cardiac cell types, i.e., CMs, endothelial cells (ECs), macrophages (Mϕs), fibroblasts (FBs), and smooth muscle cells (SMCs) and treat them with prohypertrophic compounds.Methods: Mesoderm induction preceded all cell-type-specific differentiation steps. CMs were then generated by Wnt signaling inhibition and ECs by VEGF stimulation. Mϕ differentiation required treatment with IL-3+M-CSF. FBs and SMCs were generated by Wnt pathway activation followed by bFGF or TGFβ+bFGF stimulation. The characterization of different iPSC-derivatives and EHTs was based on the MICS (MACSima Imaging Cycling Staining) technology and profiling of hallmark genes and proteins. To study the response to prohypertrophic stimuli, EHTs were treated with 20 μM phenylephrine (PE) and 50 nM endothelin-1 (ET-1) for 9-10 days.Results: The expression of cell-type-specific markers and functional assays were the determinants of successful differentiation. For instance, CMs were identified based on cTnT expression and contractile capabilities, Mϕs by expression of CD68 and phagocytic activity, and ECs by CD31 expression and tube formation abilities. Multi-cell-type EHTs were assembled from 60% CMs, 20% ECs, 10% Mϕs, 5% FBs and 5% SMCs. These EHTs displayed accelerated development, i.e., faster remodeling and earlier onset of beating compared to CM-only EHTs. Preliminary results show that multi-cell-type EHTs exhibited higher maximal forces and increased stiffness compared to CM-only controls. The exposure to PE+ET-1 led to a decrease in force, activation of the hypertrophic gene program, and elevation of NT-pro BNP production.Conclusions: We successfully generated functional multi-cell-type EHTs containing five hiPSC-derived cardiac cell types. Upon hypertrophic stimulation, these EHTs developed a pathological phenotype that resembles the hallmarks of a failing human heart.