Identification and isolation of embryonic stem cell-derived target cells by adenoviral conditional targeting
Identification and isolation of embryonic stem cell-derived target cells by adenoviral conditional targeting
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DOI：
10.1016/j.ymthe.2006.05.010
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发表时间：
2006-11-01
影响因子：
12.4
通讯作者：
Kosai, Ken-ichiro
中科院分区：
文献类型：
--
作者：
Takahashi, Tomoyuki;Kawai, Takao;Kosai, Ken-ichiro
分离靶细胞的技术局限性限制了多能胚胎干（ES）细胞的应用。例如，早期心脏（即，收缩前）细胞尚未从ES细胞中分离出来。在这里，我们发现，在细胞特异性启动子下直接表达报告基因目前可用的分离缺乏细胞特异性表面标记的细胞的策略对于分离祖细胞是无效的。这是由于细胞特异性启动子的弱活性，特别是在早期阶段的ES细胞中。我们表明，腺病毒条件靶向有效地隔离可行的ES细胞来源的靶细胞，而没有有害的影响。在该策略中，我们采用α-肌球蛋白重链和Nkx2.5启动子分别有效地可视化和纯化心脏谱系的分化细胞和原始细胞。虽然前者的细胞主要表达肌节蛋白，并保持收缩功能，后者表现出这些功能，但相反，表现出原始细胞和心肌细胞的混合物的特征的表达模式。有趣的是，平滑肌肌动蛋白主要在后者细胞中表达，并且系统地鉴定了功能已知和未知的基因，证明了该系统的益处。因此，我们的方法有利于发育和ES细胞衍生的细胞治疗的分子和细胞研究。
The technical limitations of isolating target cells have restricted the utility of pluripotent embryonic stem (ES) cells. For example, early cardiac (i.e., precontractile) cells have not been isolated from ES cells. Here, we find that direct expression of reporter genes under cell-specific promoters-the currently available strategy for isolating cells lacking cell-specific surface markers-is ineffective for isolating progenitor cells. This was due to the weak activity of cell-specific promoters, particularly in ES cells at early stages. We show that adenoviral conditional targeting efficiently isolates viable ES cell-derived target cells without harmful effects. In this strategy, we employ the (x-myosin heavy chain and Nkx2.5 promoter to visualize and purify efficiently differentiated and primitive cells of the cardiac lineage, respectively. While the former cells predominantly expressed sarcomeric proteins and maintained contractile function, the latter demonstrated neither of these features, but rather exhibited expression patterns characteristic of a mixture of primitive cells and cardiomyocytes. Interestingly, smooth muscle actin was predominantly expressed in the latter cells, and both functionally known and unknown genes were systematically identified, demonstrating the benefits of this system. Thus, our method facilitates molecular and cellular studies of development and ES cell-derived cell therapy.