The generation of mature ventricular cardiomyocytes (CMs) resembling adult CMs from human pluripotent stem cells (hPSCs) is necessary for disease modeling and drug discovery. To investigate the effect of self-organizing capacity on the generation of mature cardiac organoids (COs), we generated cardiac mesoderm cell-derived COs (CMC-COs) and CM-derived COs (CM-COs) and evaluated COs. CMC-COs exhibited more organized sarcomere structures and mitochondria, well-arranged t-tubule structures, and evenly distributed intercalated discs. Increased expressions of ventricular CM, cardiac metabolic, t-tubule formation, K⁺ ion channel, and junctional markers were confirmed in CMC-COs. Mature ventricular-like function such as faster motion vector speed, decreased beats per min, increased peak-to-peak duration, and prolonged APD₅₀ and APD₉₀ were observed in CMC-COs. Transcriptional profiling revealed that extracellular matrix-integrin, focal adhesion, and LEFTY-PITX2 signaling pathways are upregulated in CMC-COs. LEFTY knockdown affected ECM-integrin-FA signaling pathways in CMC-COs. Here, we found that high self-organizing capacity of CMCs is critical for the generation of mature and ventricular COs. We also demonstrated that LEFTY-PITX2 signaling plays key roles for CM maturation and specification into ventricular-like CM subtype in CMC-COs. CMC-COs are an attractive resource for disease modeling and drug discovery.

从人类多能干细胞 (hPSC) 生成类似于成人 CM 的成熟心室心肌细胞 (CM) 对于疾病建模和药物发现是必要的。为了研究自组织能力对成熟心脏类器官（CO）生成的影响，我们生成了心脏中胚层细胞衍生的 CO（CMC-CO）和 CM 衍生的 CO（CM-CO）并评估了 CO。 CMC-CO表现出更有组织的肌节结构和线粒体、排列良好的T管结构以及均匀分布的闰盘。在 CMC-CO 中证实了心室 CM、心脏代谢、T 管形成、K ⁺离子通道和连接标志物的表达增加。在 CMC-CO 中观察到成熟的心室样功能，例如更快的运动矢量速度、每分钟心跳减少、峰峰值持续时间增加以及 APD ₅₀和 APD ₉₀延长。转录分析显示，细胞外基质整合素、粘着斑和 LEFTY-PITX2 信号通路在 CMC-CO 中上调。 LEFTY 敲低影响了 CMC-CO 中的 ECM-整合素-FA 信号通路。在这里，我们发现 CMC 的高自组织能力对于成熟和心室 CO 的生成至关重要。我们还证明，LEFTY-PITX2 信号对于 CM 成熟和规范为 CMC-CO 中的心室样 CM 亚型起着关键作用。 CMC-CO 是疾病建模和药物发现的有吸引力的资源。