Background

Human sinoatrial cardiomyocytes are essential building blocks for cell therapies of conduction system disorders. However, current differentiation protocols for deriving nodal cardiomyocytes from human pluripotent stem cells (hPSCs) are very inefficient.

Methods

By employing the hPSCs to cardiomyocyte (CM) in vitro differentiation system and generating E2A-knockout hESCs using CRISPR/Cas9 gene editing technology, we analyze the functions of E2A in CM differentiation.

Findings

We found that knockout of the transcription factor E2A substantially increased the proportion of nodal-like cells in hESC-derived CMs. The E2A ablated CMs displayed smaller cell size, increased beating rates, weaker contractile force, and other functional characteristics similar to sinoatrial node (SAN) cells. Transcriptomic analyses indicated that ion channel-encoding genes were up-regulated in E2A ablated CMs. E2A directly bounded to the promoters of genes key to SAN development via conserved E-box motif, and promoted their expression. Unexpect enhanced activity of NOTCH pathway after E2A ablation could also facilate to induct ventricle workingtype CMs reprogramming into SAN-like cells.

Interpretation

Our study revealed a new role for E2A during directed cardiac differentiation of hESCs and may provide new clues for enhancing induction efficiency of SAN-like cardiomyocytes from hPSCs in the future.

Funding

This work was supported by the NSFC (No.82070391, N.S.; No.81870175 and 81922006, P.L.), the National Key R&D Program of China (2018YFC2000202, N.S.; 2017YFA0103700, P.L.), the Haiju program of National Children's Medical Center EK1125180102, and Innovative research team of high-level local universities in Shanghai and a key laboratory program of the Education Commission of Shanghai Municipality (ZDSYS14005).

中文翻译：

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E2A消融提高人胚胎干细胞心脏特异性分化中结节样心肌细胞的比例

背景

人类窦房心肌细胞是传导系统疾病细胞疗法的重要组成部分。然而，目前从人类多能干细胞 (hPSCs) 中提取淋巴结心肌细胞的分化方案效率非常低。

方法

通过将 hPSCs 用于心肌细胞 (CM) 体外分化系统并使用 CRISPR/Cas9 基因编辑技术生成 E2A 敲除 hESCs，我们分析了 E2A 在 CM 分化中的功能。

发现

我们发现转录因子 E2A 的敲除显着增加了 hESC 衍生的 CM 中节点样细胞的比例。E2A 消融的 CM 显示出更小的细胞尺寸、更高的搏动率、更弱的收缩力以及与窦房结 (SAN) 细胞相似的其他功能特征。转录组学分析表明离子通道编码基因在 E2A 消融的 CM 中上调。E2A通过保守的E-box基序直接与SAN发育关键基因的启动子结合，促进它们的表达。E2A 消融后 NOTCH 通路的意外增强活性也可能有助于诱导心室工作型 CM 重编程为 SAN 样细胞。

解释

我们的研究揭示了 E2A 在 hESCs 定向心脏分化过程中的新作用，并可能为未来提高 hPSCs 中 SAN 样心肌细胞的诱导效率提供新线索。

资金

这项工作得到了国家自然科学基金 (No.82070391, NS; No.81870175 和 81922006, PL)、国家重点研发计划 (2018YFC2000202, NS; 2017YFA0103700, PL)、国家儿童医学中心海聚计划 EK1125180102、上海市地方高水平高校创新研究团队和上海市教委重点实验室项目（ZDSYS14005）。