Establishment of human pluripotent stem cell-derived pace-making cells using physiological approach and its application to the regenerative medicine

我们尝试建立携带HCN 4-GFP BAC载体的人ES细胞，通过生理学方法分离人多能干细胞来源的起搏细胞。我们构建了BAC载体，将敲除的GFP基因插入HCN 4基因的启动子区，并将其导入人ES细胞（KhES-1），以建立包括克隆#1在内的几个克隆。这些克隆表达多能基因标记和正常核型染色体。来自克隆#1的用于分化心肌细胞的胚状体包括占总人ES细胞5%的HCN 4-GFP阳性细胞。HCN 4-GFP阳性细胞表达心肌收缩蛋白和HCN 4通道。HCN 4-GFP阳性细胞表达的几种离子通道负责其自律性。HCN 4-GFP阳性细胞聚集产生的Ca瞬变进入HL-1心房细胞片层。综上所述，我们建立了携带HCN 4-GFP BAC载体的人ES细胞，并成功分离出HCN 4-GFP阳性的起搏细胞。

We attempted to establish the human ES cells harboringHCN4-GFP BAC vector to isolate human pluripotent stem cell-derived pace-making cells using physiological approach. We construct BAC vector harboring the knocked GFP gene into the promoter region of HCN4 gene and introduced it into human ES cells (KhES-1) to establish the several clones including clone #1. These clones expressed the pluripotent gene markers and normal karyo type of chromosome. The embryoid body derived from clone#1 to differentiate cardiac cells included the HCN4-GFP positive cells at 5% out of total human ES cells. HCN4-GFP positive cells expressed cardiac contractile proteins and HCN4 channels. HCN4-GFP positive cells expressed the several ion channels being responsible for their automaticity. The Ca transient derived from the aggregation of HCN4-GFP positive cells propagated into the HL-1 atrial cell-sheet. Taken together, we establish the human ES cells harboringHCN4-GFP BAC vector and successfully isolate the HCN4-GFP positive pacemakingcells.