CRISPR/Cas9-based targeting of fluorescent reporters to human iPSCs to isolate atrial and ventricular-specific cardiomyocytes

Orlando Chirikian, William R. Goodyer, Elda Dzilic, Vahid Serpooshan, Jan W. Buikema, Wesley McKeithan, HaoDi Wu, Guang Li, Soah Lee, Markus Merk, Francisco Galdos, Aimee Beck (+6 others)
2021 Scientific Reports  
AbstractGenerating cardiomyocytes (CMs) from human induced pluripotent stem cells (hiPSCs) has represented a significant advance in our ability to model cardiac disease. Current differentiation protocols, however, have limited use due to their production of heterogenous cell populations, primarily consisting of ventricular-like CMs. Here we describe the creation of two chamber-specific reporter hiPSC lines by site-directed genomic integration using CRISPR-Cas9 technology. In the MYL2-tdTomato
more » ... the MYL2-tdTomato reporter, the red fluorescent tdTomato was inserted upstream of the 3′ untranslated region of the Myosin Light Chain 2 (MYL2) gene in order faithfully label hiPSC-derived ventricular-like CMs while avoiding disruption of endogenous gene expression. Similarly, in the SLN-CFP reporter, Cyan Fluorescent Protein (CFP) was integrated downstream of the coding region of the atrial-specific gene, Sarcolipin (SLN). Purification of tdTomato+ and CFP+ CMs using flow cytometry coupled with transcriptional and functional characterization validated these genetic tools for their use in the isolation of bona fide ventricular-like and atrial-like CMs, respectively. Finally, we successfully generated a double reporter system allowing for the isolation of both ventricular and atrial CM subtypes within a single hiPSC line. These tools provide a platform for chamber-specific hiPSC-derived CM purification and analysis in the context of atrial- or ventricular-specific disease and therapeutic opportunities.
doi:10.1038/s41598-021-81860-x pmid:33542270 pmcid:PMC7862643 fatcat:buytddgi3bdc5dwwoy3ix7szqm