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

Orlando Chirikian,Beth L Pruitt,Wesley Mckeithan,William R Goodyer,Soah Lee,Joseph C Wu,Alexandre J S Ribeiro,Jan W Buikema,Haodi Wu,Guang Li,Sean M Wu,Vahid Serpooshan,Markus Merk,Aimee Beck,Mark Mercola,Elda Dzilic,Francisco Galdos,Sharon Paige

doi:10.1038/s41598-021-81860-x

Abstract

Generating 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 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.

Highlights

Generating cardiomyocytes (CMs) from human induced pluripotent stem cells has represented a significant advance in our ability to model cardiac disease
The donor plasmid was constructed using the P2A self-cleaving peptide-based multi-gene expression system allowing for the co-expression of the target genes and their corresponding fluorophores based on endogenous promoter activity without disruption to their biological function
Using single cell traction force microscopy (TFM), we identified greater contractile force generated in tdTomato+ hiPSCCMs as compare to tdTomato− human induced pluripotent stem cells (hiPSCs)-CMs (Fig. 3G)

Summary

Introduction

Generating cardiomyocytes (CMs) from human induced pluripotent stem cells (hiPSCs) has represented a significant advance in our ability to model cardiac disease. Using CRISPR-Cas[9] technology, we successfully generated three categories of fluorescent reporter lines including ventricular (MYL-2-tandem dimer Tomato (tdTomato) only), atrial (SLN-cyan fluorescent protein (CFP) only), and a double atrial-ventricular (MYL-2-tdTomato; SLN-CFP) reporter lines by targeting fluorescent reporter constructs to these highly-conserved, chamber-specific genes. These tools provide novel insights into the morphologic, functional and biological differences in chamber-specific cardiomyocytes, and provide an effective method for purifying atrial and ventricular populations from the same hiPSC line for further analysis and high-throughput screening

Methods

Results

Conclusion