Division of Regenerative Medicine, Center for Molecular Medicine, Jichi Medical University.
Division of Regenerative Medicine, Center for Molecular Medicine, Jichi Medical University; Department of Pediatrics, Jichi Medical University.
J Vis Exp. 2021 Mar 3(169). doi: 10.3791/62129.
Pluripotent stem cell-derived cardiomyocytes (PSC-CMs) can be produced from both embryonic and induced pluripotent stem (ES/iPS) cells. These cells provide promising sources for cardiac disease modeling. For cardiomyopathies, sarcomere shortening is one of the standard physiological assessments that are used with adult cardiomyocytes to examine their disease phenotypes. However, the available methods are not appropriate to assess the contractility of PSC-CMs, as these cells have underdeveloped sarcomeres that are invisible under phase-contrast microscopy. To address this issue and to perform sarcomere shortening with PSC-CMs, fluorescent-tagged sarcomere proteins and fluorescent live-imaging were used. Thin Z-lines and an M-line reside at both ends and the center of a sarcomere, respectively. Z-line proteins - α-Actinin (ACTN2), Telethonin (TCAP), and actin-associated LIM protein (PDLIM3) - and one M-line protein - Myomesin-2 (Myom2) - were tagged with fluorescent proteins. These tagged proteins can be expressed from endogenous alleles as knock-ins or from adeno-associated viruses (AAVs). Here, we introduce the methods to differentiate mouse and human pluripotent stem cells to cardiomyocytes, to produce AAVs, and to perform and analyze live-imaging. We also describe the methods for producing polydimethylsiloxane (PDMS) stamps for a patterned culture of PSC-CMs, which facilitates the analysis of sarcomere shortening with fluorescent-tagged proteins. To assess sarcomere shortening, time-lapse images of the beating cells were recorded at a high framerate (50-100 frames per second) under electrical stimulation (0.5-1 Hz). To analyze sarcomere length over the course of cell contraction, the recorded time-lapse images were subjected to SarcOptiM, a plug-in for ImageJ/Fiji. Our strategy provides a simple platform for investigating cardiac disease phenotypes in PSC-CMs.
多能干细胞衍生的心肌细胞 (PSC-CMs) 可由胚胎和诱导多能干细胞 (ES/iPS) 产生。这些细胞为心脏疾病建模提供了有前途的来源。对于心肌病，肌节缩短是用于评估成人心肌细胞疾病表型的标准生理评估之一。然而，现有的方法并不适合评估 PSC-CMs 的收缩性，因为这些细胞的肌节发育不良，在相差显微镜下不可见。为了解决这个问题并对 PSC-CMs 进行肌节缩短测量，我们使用了荧光标记的肌节蛋白和荧光活细胞成像。Z 线和 M 线分别位于肌节的两端和中心。Z 线蛋白 - α-辅肌动蛋白 (ACTN2)、Telethonin (TCAP) 和肌节相关 LIM 蛋白 (PDLIM3) - 和一个 M 线蛋白 - Myomesin-2 (Myom2) - 被荧光蛋白标记。这些标记的蛋白可以从内源性等位基因中作为敲入或从腺相关病毒 (AAV) 中表达。在这里，我们介绍了将小鼠和人多能干细胞分化为心肌细胞、产生 AAV 以及进行和分析活细胞成像的方法。我们还描述了用于制作聚二甲基硅氧烷 (PDMS) 印章的方法，用于 PSC-CMs 的图案化培养，这有助于使用荧光标记蛋白分析肌节缩短。为了评估肌节缩短，在电刺激（0.5-1Hz）下以高帧率（每秒 50-100 帧）记录跳动细胞的延时图像。为了分析细胞收缩过程中的肌节长度，将记录的延时图像提交给 SarcOptiM，这是一个用于 ImageJ/Fiji 的插件。我们的策略为研究 PSC-CMs 中的心脏疾病表型提供了一个简单的平台。
