We used a screening strategy to test for reprogramming factors for the conversion of human cardiac progenitor cells (CPCs) into Pacemaker-like cells. Human transcription factors SHOX2, TBX3, TBX5, TBX18, and the channel protein HCN2, were transiently induced as single factors and in trio combinations into CPCs, first transduced with the connexin 30.2 (CX30.2) mCherry reporter. Following screens for reporter CX30.2 mCherry gene activation and FACS enrichment, we observed the definitive expression of many pacemaker specific genes; including, CX30.2, KCNN4, HCN4, HCN3, HCN1, and SCN3b. These findings suggest that the SHOX2, HCN2, and TBX5 (SHT5) combination of transcription factors is a much better candidate in driving the CPCs into Pacemaker-like cells than other combinations and single transcription factors. Additionally, single-cell RNA sequencing of SHT5 mCherry+ cells revealed cellular enrichment of pacemaker specific genes including TBX3, KCNN4, CX30.2, and BMP2, as well as pacemaker specific potassium and calcium channels (KCND2, KCNK2, and CACNB1). In addition, similar to human and mouse sinoatrial node (SAN) studies, we also observed the down-regulation of NKX2.5. Patch-clamp recordings of the converted Pacemaker-like cells exhibited HCN currents demonstrated the functional characteristic of pacemaker cells. These studies will facilitate the development of an optimal Pacemaker-like cell-based therapy within failing hearts through the recovery of SAN dysfunction.

我们使用了一种筛选策略来测试将人类心脏祖细胞（CPC）转换为起搏器样细胞的重编程因子。人类转录因子SHOX2，TBX3，TBX5，TBX18和通道蛋白HCN2作为单一因子被瞬时诱导，并以三重组合的形式被导入CPC，首先由连接蛋白30.2（CX30.2）mCherry报告员转导。筛选记者CX30.2 mCherry基因激活和FACS富集后，我们观察到了许多起搏器特异性基因的确定表达。其中，CX30.2，KCNN4，HCN4，HCN3，HCN1和SCN3b。这些发现表明，与其他组合和单个转录因子相比，转录因子的SHOX2，HCN2和TBX5（SHT5）组合是将CPC驱入Pacemaker样细胞的更好候选者。此外，SHT5 mCherry +细胞的单细胞RNA测序揭示了起搏器特异性基因（包括TBX3，KCNN4，CX30.2和BMP2）以及起搏器特异性钾和钙通道（KCND2，KCNK2和CACNB1）的细胞富集性。此外，类似于人和小鼠窦房结（SAN）的研究，我们还观察到NKX2.5的下调。转换后的起搏器样细胞的膜片钳记录显示HCN电流证明了起搏器细胞的功能特征。这些研究将通过SAN功能障碍的恢复，促进在衰竭心脏内开发最佳的基于Pacemaker样细胞的疗法。