Abstract
SummaryGenetically 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.
Highlights
Human pluripotent stem cell-derived cardiomyocytes (CMs) hold tremendous promise for cellbased regenerative therapies, drug screening, and heart disease modeling due to the advances in stem cell differentiation research
CRISPR-Cas9-Mediated Generation of GCaMP6s Knockin Cell Line We previously utilized gene overexpression or knockdown approaches in Human pluripotent stem cell (hPSC) to study gene function during stem cell differentiation (Lian et al, 2012)
We have used lentiviral or PiggyBac strategies to integrate our designed DNA constructs into the genome of hPSCs to study the important role of the Wnt signaling pathway for stem cell differentiation (Lian et al, 2012; Randolph et al, 2017)
Summary
Human pluripotent stem cell (hPSC)-derived cardiomyocytes (CMs) hold tremendous promise for cellbased regenerative therapies, drug screening, and heart disease modeling due to the advances in stem cell differentiation research. The time and cost required to produce the multi-electrode array as well as the lack of single-cell-level measurement sensitivity limit its applicability to CM research. As pointed out in the most recent studies (Smith et al, 2018), chemical fluorescent Ca2+ indicators such as Fluo-4 acetoxymethyl (AM), Rhod-2 AM, and Fura-2 AM loaded into different cell lines suppressed their Na,K-ATPase activity, which is crucial in many physiological and pathological processes. They altered the metabolic status, induced cell swelling, and caused a dose-dependent loss of cell viability.
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
