Wakimizu Takayuki, Morikawa Kumi, Fukumura Kenta, Yuki Tetsuo, Adachi Takashi, Kurata Yasutaka, Miake Junichiro, Hisatome Ichiro, Tsuneto Motokazu, Shirayoshi Yasuaki
Division of Regenerative Medicine and Therapeutics, Department of Genetic Medicine and Regenerative Therapeutics, Tottori University Graduate School of Medical Science, 86 Nishi-cho, Yonago, Tottori 683-8503, Japan.
Center for Promoting Next-Generation Highly Advanced Medicine, Tottori University Hospital, 36-1 Nishi-cho, Yonago, Tottori 683-8504, Japan.
Regen Ther. 2022 Aug 23;21:239-249. doi: 10.1016/j.reth.2022.07.012. eCollection 2022 Dec.
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 () and hyperpolarization-activated cyclic nucleotide-gated cation channel 4 () 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 and as SAN markers.
We developed an HCN4-EGFP/SHOX2-mCherry dual reporter cell line derived from human induced pluripotent stem cells (hiPSCs), and and 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.
窦房结（SAN）细胞功能障碍会导致心律失常，许多患者需要植入人工心脏起搏器。然而，SAN自律性受损的机制尚不清楚，体外生成人类SAN细胞可能为理解SAN功能障碍的发病机制提供一个平台。矮小同源框2（SHOX2）基因和超极化激活的环核苷酸门控阳离子通道4（HCN4）基因在SAN细胞中特异性表达，对SAN的发育和自律性很重要。在本研究中，我们旨在以SHOX2和HCN4作为SAN标志物，在体外纯化并鉴定人类SAN样细胞。
我们开发了一种源自人类诱导多能干细胞（hiPSC）的HCN4-EGFP/SHOX2-mCherry双报告基因细胞系，分别使用荧光蛋白EGFP和mCherry来可视化HCN4和SHOX2基因的表达。该双报告基因细胞系是使用基于HCN4-EGFP细菌人工染色体的半敲入系统和带有SHOX2-mCherry靶向载体的CRISPR-Cas9依赖性敲入系统建立的。进行了流式细胞术、逆转录-聚合酶链反应（RT-PCR）和全细胞膜片钳分析以鉴定SAN样细胞。
流式细胞术分析和细胞分选分离出了HCN4-EGFP单阳性（HCN4/SHOX2）和HCN4-EGFP/SHOX2-mCherry双阳性（HCN4/SHOX2）细胞。RT-PCR分析表明，SAN相关基因在HCN4/SHOX2细胞中富集。此外，电生理分析表明，约70%的HCN4/SHOX2细胞表现出SAN样电生理特征，这是由最大上升速度和动作电位持续时间的动作电位参数定义的。
本研究中开发的HCN4-EGFP/SHOX2-mCherry双报告基因hiPSC系统能够在分化的心脏细胞混合的HCN4/SHOX2群体中富集SAN样细胞。这种新型细胞系有助于进一步富集人类SAN样细胞。它可能有助于再生医学，例如生物起搏器，以及测试新型候选药物的心脏毒性和变时作用。
