Cardiomyocytes (CMs) from human induced pluripotent stem cells (hiPSCs) are functionally immature, but this is improved by incorporation into engineered tissues or forced contraction. Here, we showed that tri-cellular combinations of hiPSC-derived CMs, cardiac fibroblasts (CFs), and cardiac endothelial cells also enhance maturation in easily constructed, scaffold-free, three-dimensional microtissues (MTs). hiPSC-CMs in MTs with CFs showed improved sarcomeric structures with T-tubules, enhanced contractility, and mitochondrial respiration and were electrophysiologically more mature than MTs without CFs. Interactions mediating maturation included coupling between hiPSC-CMs and CFs through connexin 43 (CX43) gap junctions and increased intracellular cyclic AMP (cAMP). Scaled production of thousands of hiPSC-MTs was highly reproducible across lines and differentiated cell batches. MTs containing healthy-control hiPSC-CMs but hiPSC-CFs from patients with arrhythmogenic cardiomyopathy strikingly recapitulated features of the disease. Our MT model is thus a simple and versatile platform for modeling multicellular cardiac diseases that will facilitate industry and academic engagement in high-throughput molecular screening.

来自人诱导的多能干细胞（hiPSC）的心肌细胞（CM）在功能上不成熟，但这可以通过整合入工程组织或强制收缩而得到改善。在这里，我们表明，hiPSC衍生的CM，心脏成纤维细胞（CF）和心脏内皮细胞的三细胞组合也增强了易于构建，无支架的三维显微组织（MT）的成熟度。具有CFs的MTs中的hiPSC-CMs显示具有T管的肌节结构改善，收缩力增强和线粒体呼吸，并且在电生理上比不含CFs的MTs更成熟。介导成熟的相互作用包括hiPSC-CM和CF之间通过连接蛋白43（CX43）间隙连接和细胞内环状AMP（cAMP）的偶联。跨系和分化的细胞批次可高度重现数千个hiPSC-MT的大规模生产。含有健康对照hiPSC-CM但来自心律失常性心肌病患者的hiPSC-CF的MT明显概括了该疾病的特征。因此，我们的MT模型是用于建模多细胞心脏病的简单而通用的平台，这将有助于行业和学术界参与高通量分子筛查。