一种通过CRISPR-Cas9介导的人类多能干细胞基因组编辑实现的超灵敏钙报告系统。

An Ultrasensitive Calcium Reporter System via CRISPR-Cas9-Mediated Genome Editing in Human Pluripotent Stem Cells.

作者信息

Jiang Yuqian, Zhou Yuxiao, Bao Xiaoping, Chen Chuanxin, Randolph Lauren N, Du Jing, Lian Xiaojun Lance

机构信息

Department of Biomedical Engineering, The Pennsylvania State University, University Park, PA 16802, USA; The Huck Institutes of the Life Sciences, The Pennsylvania State University, University Park, PA 16802, USA.

Department of Mechanical and Nuclear Engineering, The Pennsylvania State University, University Park, PA 16802, USA.

出版信息

iScience. 2018 Nov 30;9:27-35. doi: 10.1016/j.isci.2018.10.007. Epub 2018 Oct 12.

Genetically encoded calcium indicator (GCaMP) proteins have been reported for imaging cardiac cell activity based on intracellular calcium transients. To bring human pluripotent stem cell (hPSC)-derived cardiomyocytes (CMs) to the clinic, it is critical to evaluate the functionality of CMs. Here, we show that GCaMP6s-expressing hPSCs can be generated and used for CM characterization. By leveraging CRISPR-Cas9 genome editing tools, we generated a knockin cell line that constitutively expresses GCaMP6s, an ultrasensitive calcium sensor protein. We further showed that this clone maintained pluripotency and cardiac differentiation potential. These knockin hPSC-derived CMs exhibited sensitive fluorescence fluctuation with spontaneous contraction. We then compared the fluorescence signal with mechanical contraction signal. The knockin hPSC-derived CMs also showed sensitive response to isoprenaline treatment in a concentration-dependent manner. Therefore, the GCaMP6s knockin hPSC line provides a non-invasive, sensitive, and economic approach to characterize the functionality of hPSC-derived CMs.

据报道，基于细胞内钙瞬变，基因编码钙指示剂（GCaMP）蛋白可用于成像心脏细胞活性。为了将人多能干细胞（hPSC）衍生的心肌细胞（CM）应用于临床，评估CM的功能至关重要。在此，我们表明可以生成表达GCaMP6s的hPSC，并将其用于CM表征。通过利用CRISPR-Cas9基因组编辑工具，我们生成了一个组成型表达超灵敏钙传感器蛋白GCaMP6s的敲入细胞系。我们进一步表明，该克隆保持了多能性和心脏分化潜能。这些敲入hPSC衍生的CM表现出随着自发收缩而产生的敏感荧光波动。然后，我们将荧光信号与机械收缩信号进行了比较。敲入hPSC衍生的CM对异丙肾上腺素治疗也表现出浓度依赖性的敏感反应。因此，GCaMP6s敲入hPSC系为表征hPSC衍生CM的功能提供了一种非侵入性、灵敏且经济的方法。