Regenerative Medicine Institute, School of Medicine, College of Medicine, Nursing and Health Science, University of Galway, Galway, Ireland.
Department of Pharmacology, Northwestern University Feinberg School of Medicine, Chicago, IL, USA.
Stem Cell Rev Rep. 2023 Nov;19(8):2774-2789. doi: 10.1007/s12015-023-10602-5. Epub 2023 Aug 31.
Human induced pluripotent stem cells (hiPSCs) have been widely used in cardiac disease modelling, drug discovery, and regenerative medicine as they can be differentiated into patient-specific cardiomyocytes. Long QT syndrome type 3 (LQT3) is one of the more malignant congenital long QT syndrome (LQTS) variants with an SCN5A gain-of-function effect on the gated sodium channel. Moreover, the predominant pathogenic variants in LQTS genes are single nucleotide substitutions (missense) and small insertion/deletions (INDEL). CRISPR/Cas9 genome editing has been utilised to create isogenic hiPSCs to control for an identical genetic background and to isolate the pathogenicity of a single nucleotide change. In this study, we described an optimized and rapid protocol to introduce a heterozygous LQT3-specific variant into healthy control hiPSCs using ribonucleoprotein (RNP) and single-stranded oligonucleotide (ssODN). Based on this protocol, we successfully screened hiPSCs carrying a heterozygous LQT3 pathogenic variant (SCN5A) with high efficiency (6 out of 69) and confirmed no off-target effect, normal karyotype, high alkaline phosphatase activity, unaffected pluripotency, and in vitro embryonic body formation capacity within 2 weeks. In addition, we also provide protocols to robustly differentiate hiPSCs into cardiomyocytes and evaluate the electrophysiological characteristics using Multi-electrode Array. This protocol is also applicable to introduce and/or correct other disease-specific variants into hiPSCs for future pharmacological screening and gene therapeutic development.
人诱导多能干细胞（hiPSCs）已广泛应用于心脏病建模、药物发现和再生医学，因为它们可以分化为患者特异性心肌细胞。长 QT 综合征 3 型（LQT3）是恶性先天性长 QT 综合征（LQTS）变异型之一，其钠通道门控具有 SCN5A 功能获得效应。此外，LQTS 基因中的主要致病性变异是单核苷酸取代（错义）和小插入/缺失（INDEL）。CRISPR/Cas9 基因组编辑已用于创建同基因 hiPSCs，以控制相同的遗传背景，并分离单个核苷酸变化的致病性。在这项研究中，我们描述了一种使用核糖核蛋白（RNP）和单链寡核苷酸（ssODN）将杂合 LQT3 特异性变异引入健康对照 hiPSCs 的优化和快速方案。基于该方案，我们成功筛选出了携带有杂合 LQT3 致病性变异（SCN5A）的 hiPSCs，效率高（6 个中有 69 个），且未发现脱靶效应、正常核型、高碱性磷酸酶活性、未受影响的多能性以及 2 周内体外胚胎体形成能力。此外，我们还提供了将 hiPSCs 稳健分化为心肌细胞的方案，并使用多电极阵列评估其电生理特征。该方案还适用于将其他疾病特异性变异引入 hiPSCs，用于未来的药物筛选和基因治疗开发。
