SHOX2 refines the identification of human sinoatrial nodal cell population in the in vitro cardiac differentiation

Dysfunction of the sinoatrial node (SAN) cells causes arrhythmias, and many patients require artificial cardiac pacemaker implantation. However, the mechanism of impaired SAN automaticity remains unknown, and the generation of human SAN cells in vitro may provide a platform for understanding the pathogenesis of SAN dysfunction. The short stature homeobox 2 (SHOX2) and hyperpolarization-activated cyclic nucleotide-gated cation channel 4 (HCN4) genes are specifically expressed in SAN cells and are important for SAN development and automaticity. In this study, we aimed to purify and characterize human SAN-like cells in vitro, using HCN4 and SHOX2 as SAN markers.We developed an HCN4-EGFP/SHOX2-mCherry dual reporter cell line derived from human induced pluripotent stem cells (hiPSCs), and HCN4 and SHOX2 gene expressions were visualized using the fluorescent proteins EGFP and mCherry, respectively. The dual reporter cell line was established using an HCN4-EGFP bacterial artificial chromosome-based semi-knock-in system and a CRISPR-Cas9-dependent knock-in system with a SHOX2-mCherry targeting vector. Flow cytometry, RT-PCR, and whole-cell patch-clamp analyses were performed to identify SAN-like cells.Flow cytometry analysis and cell sorting isolated HCN4-EGFP single-positive (HCN4+/SHOX2-) and HCN4-EGFP/SHOX2-mCherry double-positive (HCN4+/SHOX2+) cells. RT-PCR analyses showed that SAN-related genes were enriched within the HCN4+/SHOX2+ cells. Further, electrophysiological analyses showed that approximately 70% of the HCN4+/SHOX2+ cells exhibited SAN-like electrophysiological characteristics, as defined by the action potential parameters of the maximum upstroke velocity and action potential duration.The HCN4-EGFP/SHOX2-mCherry dual reporter hiPSC system developed in this study enabled the enrichment of SAN-like cells within a mixed HCN4+/SHOX2+ population of differentiating cardiac cells. This novel cell line is useful for the further enrichment of human SAN-like cells. It may contribute to regenerative medicine, for example, biological pacemakers, as well as testing for cardiotoxic and chronotropic actions of novel drug candidates.