Matrigel Mattress: A Method for the Generation of Single Contracting Human-Induced Pluripotent Stem Cell-Derived Cardiomyocytes.
Matrigel Mattress: A Method for the Generation of Single Contracting Human-Induced Pluripotent Stem Cell-Derived Cardiomyocytes.
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DOI：
10.1161/circresaha.115.307580
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发表时间：
2015-12-04
影响因子：
20.1
通讯作者：
Hong CC
中科院分区：
文献类型：
--
作者：
Feaster TK;Cadar AG;Wang L;Williams CH;Chun YW;Hempel JE;Bloodworth N;Merryman WD;Lim CC;Wu JC;Knollmann BC;Hong CC
人诱导多能干细胞衍生的心肌细胞（hiPSC-CM）的可测量的单细胞收缩性的缺乏目前限制了hiPSC-CM用于评估基础研究和药物发现的收缩性能的效用。开发一种培养方法，该方法可快速产生收缩的单个hiPSC-CM，并允许使用用于研究成人心肌细胞（CM）的标准设备定量细胞缩短。将单个hiPSC-CM在0.4 - 0.8mm厚的未稀释基质胶垫（“垫hiPSC-CM”）上培养5 - 7天，并与维持在对照底物（<0.1mm厚的1：60稀释基质胶，“对照hiPSC-CM”）上的hiPSC-CM进行比较。与对照hiPSC-CM相比，床垫式hiPSC-CM具有更多的杆状形态，并且肌节长度显著增加。用基于视频的边缘检测测量的床垫hiPSC-CM的收缩参数与新鲜分离的成年兔心室CM的收缩参数相当。床垫hiPSC-CM的形态和收缩特性在另一个机构独立生成的冻存hiPSC-CM中是一致的。与对照hiPSC-CM不同，床垫式hiPSC-CM对正性肌力剂如肌丝钙敏化剂显示出稳健的收缩反应。床垫hiPSC-CM表现出分子变化，包括成熟标志物心肌肌钙蛋白I的表达增加和由于钠电流（INa）增加2倍而导致的动作电位上搏速度显著增加。Matrigel床垫法能够快速生成强健收缩的hiPSC-CM并增强成熟。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。
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 cardiac myocytes (CMs). Single hiPSC-CMs were cultured for 5 – 7 days on a 0.4 – 0.8 mm thick mattress of undiluted Matrigel (“mattress hiPSC-CM”) and compared to hiPSC-CMs maintained on control substrate (<0.1 mm thick 1:60 diluted matrigel, “control hiPSC-CM”). Compared to control hiPSC-CM, 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 was comparable to that of freshly isolated adult rabbit ventricular CMs. Morphological and contractile properties of mattress hiPSC-CM were consistent across cryopreserved hiPSC-CMs generated independently at another institution. Unlike control hiPSC-CM, 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 due to 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.