Feaster Tromondae K, Cadar Adrian G, Wang Lili, Williams Charles H, Chun Young Wook, Hempel Jonathan E, Bloodworth Nathaniel, Merryman W David, Lim Chee Chew, Wu Joseph C, Knollmann Björn C, Hong Charles C
From the Departments of Pharmacology (T.K.F.), Cardiovascular Medicine (Y.W.C., J.E.H., C.C.L., C.C.H.), and Department of Medicine, Divisions of Cardiovascular Medicine and Clinical Pharmacology, Oates Institute for Experimental Therapeutics (L.W., B.C.K.), Department of Molecular Physiology and Biophysics (A.G.C.), Vanderbilt University School of Medicine, Nashville, TN; Departments of Cell and Developmental Biology (C.H.W.) and Biomedical Engineering (N.B., W.D.M.), Vanderbilt University, Nashville, TN; Research Medicine, Veterans Affairs TVHS, Nashville, TN (C.C.H.); and Stanford Cardiovascular Institute, Department of Medicine, Division of Cardiology and Department of Radiology, Stanford University School of Medicine, CA (J.C.W.).
Circ Res. 2015 Dec 4;117(12):995-1000. doi: 10.1161/CIRCRESAHA.115.307580. Epub 2015 Oct 1.
The lack of measurable single-cell contractility of human-induced pluripotent stem cell-derived cardiac myocytes (hiPSC-CMs) currently limits the utility of hiPSC-CMs for evaluating contractile performance for both basic research and drug discovery.
To develop a culture method that rapidly generates contracting single hiPSC-CMs and allows quantification of cell shortening with standard equipment used for studying adult CMs.
Single hiPSC-CMs were cultured for 5 to 7 days on a 0.4- to 0.8-mm thick mattress of undiluted Matrigel (mattress hiPSC-CMs) and compared with hiPSC-CMs maintained on a control substrate (<0.1-mm thick 1:60 diluted Matrigel, control hiPSC-CMs). Compared with control hiPSC-CMs, mattress hiPSC-CMs had more rod-shape morphology and significantly increased sarcomere length. Contractile parameters of mattress hiPSC-CMs measured with video-based edge detection were comparable with those of freshly isolated adult rabbit ventricular CMs. Morphological and contractile properties of mattress hiPSC-CMs were consistent across cryopreserved hiPSC-CMs generated independently at another institution. Unlike control hiPSC-CMs, mattress hiPSC-CMs display robust contractile responses to positive inotropic agents, such as myofilament calcium sensitizers. Mattress hiPSC-CMs exhibit molecular changes that include increased expression of the maturation marker cardiac troponin I and significantly increased action potential upstroke velocity because of a 2-fold increase in sodium current (INa).
The Matrigel mattress method enables the rapid generation of robustly contracting hiPSC-CMs and enhances maturation. This new method allows quantification of contractile performance at the single-cell level, which should be valuable to disease modeling, drug discovery, and preclinical cardiotoxicity testing.
目前，人诱导多能干细胞衍生的心肌细胞（hiPSC-CMs）缺乏可测量的单细胞收缩性，这限制了hiPSC-CMs在基础研究和药物研发中评估收缩性能的应用。
开发一种培养方法，能快速产生收缩的单个hiPSC-CMs，并使用研究成年心肌细胞的标准设备对细胞缩短进行量化。
将单个hiPSC-CMs在0.4至0.8毫米厚的未稀释基质胶床垫上培养5至7天（床垫hiPSC-CMs），并与在对照基质（<0.1毫米厚的1:60稀释基质胶，对照hiPSC-CMs）上培养的hiPSC-CMs进行比较。与对照hiPSC-CMs相比，床垫hiPSC-CMs具有更多的杆状形态，肌节长度显著增加。用基于视频的边缘检测测量的床垫hiPSC-CMs的收缩参数与新鲜分离的成年兔心室心肌细胞相当。在另一个机构独立产生的冷冻保存的hiPSC-CMs中，床垫hiPSC-CMs的形态和收缩特性是一致的。与对照hiPSC-CMs不同，床垫hiPSC-CMs对正性肌力药物（如肌丝钙敏化剂）表现出强烈的收缩反应。床垫hiPSC-CMs表现出分子变化，包括成熟标志物心肌肌钙蛋白I的表达增加，以及由于钠电流（INa）增加两倍导致动作电位上升速度显著增加。
基质胶床垫方法能够快速产生强烈收缩的hiPSC-CMs并促进其成熟。这种新方法允许在单细胞水平上量化收缩性能，这对于疾病建模、药物发现和临床前心脏毒性测试应该是有价值的。
