Table1_Comparative analysis of the cardiomyocyte differentiation potential of induced pluripotent stem cells reprogrammed from human atrial or ventricular fibroblasts.pdf

Background: We had shown that cardiomyocytes (CMs) were more efficiently differentiated from human induced pluripotent stem cells (hiPSCs) when the hiPSCs were reprogrammed from cardiac fibroblasts rather than dermal fibroblasts or blood mononuclear cells. Here, we continued to investigate the relationship between somatic-cell lineage and hiPSC-CM production by comparing the yield and functional properties of CMs differentiated from iPSCs reprogrammed from human atrial or ventricular cardiac fibroblasts (AiPSC or ViPSC, respectively).
Methods: Atrial and ventricular heart tissues were obtained from the same patient, reprogrammed into AiPSCs or ViPSCs, and then differentiated into CMs (AiPSC-CMs or ViPSC-CMs, respectively) via established protocols.
Results: The time-course of expression for pluripotency genes (OCT4, NANOG, and SOX2), the early mesodermal marker Brachyury, the cardiac mesodermal markers MESP1 and Gata4, and the cardiovascular progenitor-cell transcription factor NKX2.5 were broadly similar in AiPSC-CMs and ViPSC-CMs during the differentiation protocol. Flow-cytometry analyses of cardiac troponin T expression also indicated that purity of the two differentiated hiPSC-CM populations (AiPSC-CMs: 88.23% ± 4.69%, ViPSC-CMs: 90.25% ± 4.99%) was equivalent. While the field-potential durations were significantly longer in ViPSC-CMs than in AiPSC-CMs, measurements of action potential duration, beat period, spike amplitude, conduction velocity, and peak calcium-transient amplitude did not differ significantly between the two hiPSC-CM populations. Yet, our cardiac-origin iPSC-CM showed higher ADP and conduction velocity than previously reported iPSC-CM derived from non-cardiac tissues. Transcriptomic data comparing iPSC and iPSC-CMs showed similar gene expression profiles between AiPSC-CMs and ViPSC-CMs with significant differences when compared to iPSC-CM derived from other tissues. This analysis also pointed to several genes involved in electrophysiology processes responsible for the physiological differences observed between cardiac and non-cardiac-derived cardiomyocytes.
Conclusion:AiPSC and ViPSC were differentiated into CMs with equal efficiency. Detected differences in electrophysiological properties, calcium handling activity, and transcription profiles between cardiac and non-cardiac derived cardiomyocytes demonstrated that 1) tissue of origin matters to generate a better-featured iPSC-CMs, 2) the sublocation within the cardiac tissue has marginal effects on the differentiation process.

研究背景：我们此前已证实，当人类诱导多能干细胞（human induced pluripotent stem cells, hiPSCs）由心脏成纤维细胞重编程获得时，其向心肌细胞（cardiomyocytes, CMs）的分化效率要高于由皮肤成纤维细胞或外周血单个核细胞重编程的hiPSCs。本研究进一步探究体细胞谱系与hiPSC-CM生成的关联，通过比较由人类心房或心室心脏成纤维细胞重编程获得的诱导多能干细胞（分别记为AiPSC与ViPSC）分化得到的CMs的产出率与功能特性。

研究方法：从同一患者体内获取心房与心室心脏组织，将其分别重编程为AiPSCs与ViPSCs，随后通过已确立的实验方案将这两种细胞分化为CMs（分别为AiPSC-CMs与ViPSC-CMs）。

研究结果：在分化过程中，AiPSC-CMs与ViPSC-CMs的多能性基因（OCT4、NANOG及SOX2）、早期中胚层标志物Brachyury、心脏中胚层标志物MESP1与Gata4以及心血管祖细胞转录因子NKX2.5的表达时序整体相似。流式细胞术检测心肌肌钙蛋白T的表达结果显示，两种hiPSC-CM群体的纯度相当（AiPSC-CMs：88.23% ± 4.69%，ViPSC-CMs：90.25% ± 4.99%）。尽管ViPSC-CMs的场电位时程显著长于AiPSC-CMs，但两种hiPSC-CM群体在动作电位时程、搏动周期、峰电位幅度、传导速度及钙瞬变峰值幅度上均无显著差异。不过，本研究中源自心脏的iPSC-CMs的ADP与传导速度均高于此前文献报道的非心脏组织来源的iPSC-CMs。对比iPSC与iPSC-CMs的转录组数据显示，AiPSC-CMs与ViPSC-CMs的基因表达谱相似，且二者与其他组织来源的iPSC-CMs均存在显著差异。该分析还鉴定出多个参与电生理过程的基因，这些基因与心脏来源与非心脏来源心肌细胞间观察到的生理差异密切相关。

研究结论：AiPSCs与ViPSCs向CMs的分化效率相当。在心脏来源与非心脏来源心肌细胞间检测到的电生理特性、钙处理活性及转录谱差异表明：1）细胞起源组织对获得功能特性更优异的iPSC-CMs具有重要影响；2）心脏组织内的亚定位对分化过程仅存在微弱影响。