Base Editing of Human Pluripotent Stem Cells for Modeling Long QT Syndrome

Abstract

Human pluripotent stem cells (hPSCs) have great potential for disease modeling, drug discovery, and regenerative medicine as they can differentiate into many different functional cell types via directed differentiation. However, the application of disease modeling is limited due to a time-consuming and labor-intensive process of introducing known pathogenic mutations into hPSCs. Base editing is a newly developed technology that enables the facile introduction of point mutations into specific loci within the genome of living cells without unwanted genome injured. We describe an optimized stepwise protocol to introduce disease-specific mutations of long QT syndrome (LQTs) into hPSCs. We highlight technical issues, especially those associated with introducing a point mutation to obtain isogenic hPSCs without inserting any resistance cassette and reproducible cardiomyocyte differentiation. Based on the protocol, we succeeded in getting hPSCs carrying LQTs pathogenic mutation with excellent efficiency (31.7% of heterozygous clones, 9.1% of homozygous clones) in less than 20 days. In addition, we also provide protocols to analyze electrophysiological of hPSC-derived cardiomyocytes using multi-electrode arrays. This protocol is also applicable to introduce other disease-specific mutations into hPSCs.

Keywords: Base editing; Disease model; LQT; Point mutation; hPSCs.

Fig. 1

Overview of base editing of hPSCs for modeling LQT syndrome

Fig. 2

Schematic diagram of cell transfection and antibiotic screening

Fig. 3

Schematic diagram of single cell clone screening

Fig. 4

Cardiomyocytes differentiation and MEA testing. a Schematic diagram of the process of cardiomyocytes differentiation and photographs of cells at different days. b Cardiomyocytes plated on MEA plates and diagram of MEA results analysis