An Ultrasensitive Calcium Reporter System via CRISPR-Cas9-Mediated Genome Editing in Human Pluripotent Stem Cells
Yuqian Jiang,
Yuxiao Zhou,
Xiaoping Bao,
Chuanxin Chen,
Lauren N. Randolph,
Jing Du,
Xiaojun Lance Lian
Affiliations
Yuqian Jiang
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
Yuxiao Zhou
Department of Mechanical and Nuclear Engineering, The Pennsylvania State University, University Park, PA 16802, USA
Xiaoping Bao
Department of Chemical and Biomolecular Engineering, University of California, Berkeley, CA 94720, USA
Chuanxin Chen
Department of Biomedical Engineering, The Pennsylvania State University, University Park, PA 16802, USA; College of Chemical and Biological Engineering, Zhejiang University, Hangzhou, Zhejiang 310027, China
Lauren N. Randolph
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
Jing Du
Department of Mechanical and Nuclear Engineering, The Pennsylvania State University, University Park, PA 16802, USA
Xiaojun Lance Lian
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 Biology, The Pennsylvania State University, University Park, PA 16802, USA; Corresponding author
Summary: 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. : Cell Engineering; Optical Imaging; Specialized Functions of Cells Subject Areas: Cell Engineering, Optical Imaging, Specialized Functions of Cells
