Beauchamp Philippe, Moritz Wolfgang, Kelm Jens M, Ullrich Nina D, Agarkova Irina, Anson Blake D, Suter Thomas M, Zuppinger Christian
1 Department of Clinical Research, Cardiology, Bern University Hospital , Bern, Switzerland .
2 InSphero AG , Schlieren, Switzerland .
Tissue Eng Part C Methods. 2015 Aug;21(8):852-61. doi: 10.1089/ten.TEC.2014.0376. Epub 2015 Mar 16.
Cardiomyocytes (CMs) are terminally differentiated cells in the adult heart, and ischemia and cardiotoxic compounds can lead to cell death and irreversible decline of cardiac function. As testing platforms, isolated organs and primary cells from rodents have been the standard in research and toxicology, but there is a need for better models that more faithfully recapitulate native human biology. Hence, a new in vitro model comprising the advantages of 3D cell culture and the availability of induced pluripotent stem cells (iPSCs) of human origin was developed and characterized. Human CMs derived from iPSCs were studied in standard 2D culture and as cardiac microtissues (MTs) formed in hanging drops. Two-dimensional cultures were examined using immunofluorescence microscopy and western blotting, while the cardiac MTs were subjected to immunofluorescence, contractility, and pharmacological investigations. iPSC-derived CMs in 2D culture showed well-formed myofibrils, cell-cell contacts positive for connexin-43, and other typical cardiac proteins. The cells reacted to prohypertrophic growth factors with a substantial increase in myofibrils and sarcomeric proteins. In hanging drop cultures, iPSC-derived CMs formed spheroidal MTs within 4 days, showing a homogeneous tissue structure with well-developed myofibrils extending throughout the whole spheroid without a necrotic core. MTs showed spontaneous contractions for more than 4 weeks that were recorded by optical motion tracking, sensitive to temperature and responsive to electrical pacing. Contractile pharmacology was tested with several agents known to modulate cardiac rate and viability. Calcium transients underlay the contractile activity and were also responsive to electrical stimulation, caffeine-induced Ca(2+) release, and extracellular calcium levels. A three-dimensional culture using iPSC-derived human CMs provides an organoid human-based cellular platform that is free of necrosis and recapitulates vital cardiac functionality, thereby providing a new and promising relevant model for the evaluation and development of new therapies and detection of cardiotoxicity.
心肌细胞（CMs）是成年心脏中终末分化的细胞，缺血和心脏毒性化合物可导致细胞死亡及心脏功能的不可逆衰退。作为测试平台，来自啮齿动物的离体器官和原代细胞一直是研究和毒理学的标准，但需要更好的模型来更忠实地重现天然人类生物学特性。因此，开发并表征了一种新的体外模型，该模型兼具三维细胞培养的优势以及人源诱导多能干细胞（iPSCs）的可用性。对源自iPSCs的人CMs在标准二维培养中以及在悬滴中形成的心脏微组织（MTs）进行了研究。使用免疫荧光显微镜和蛋白质印迹法检测二维培养物，而对心脏MTs进行免疫荧光、收缩性和药理学研究。二维培养的iPSC衍生的CMs显示出肌原纤维形成良好，连接蛋白43阳性的细胞间接触以及其他典型的心脏蛋白。这些细胞对促肥大生长因子有反应，肌原纤维和肌节蛋白大量增加。在悬滴培养中，iPSC衍生的CMs在4天内形成球形MTs，显示出均匀的组织结构，肌原纤维发育良好，贯穿整个球体延伸且无坏死核心。MTs显示出超过4周的自发收缩，通过光学运动跟踪记录，对温度敏感且对电起搏有反应。用几种已知可调节心率和活力的药物测试了收缩药理学。钙瞬变是收缩活动的基础，并且也对电刺激、咖啡因诱导的Ca(2+)释放和细胞外钙水平有反应。使用iPSC衍生的人CMs进行的三维培养提供了一个基于类器官的人体细胞平台，该平台无坏死且重现了重要的心脏功能，从而为新疗法的评估和开发以及心脏毒性检测提供了一个新的且有前景的相关模型。
