Genetically selected cardiomyocytes from differentiating embryonic stem cells form stable intracardiac grafts
Genetically selected cardiomyocytes from differentiating embryonic stem cells form stable intracardiac grafts
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DOI：
10.1172/jci118769
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发表时间：
1996-07-01
影响因子：
15.9
通讯作者：
Field, LJ
中科院分区：
文献类型：
--
作者：
Klug, MG;Soonpaa, MH;Field, LJ
本研究介绍了一种从分化的小鼠胚胎干细胞(ES细胞)获得相对纯的心肌细胞的简单方法，将由α-心肌肌球蛋白重链启动子和编码氨基糖苷磷酸转移酶的基因组成的融合基因稳定地转染到多能ES细胞中。将所获得的细胞系进行体外分化，并进行G418筛选。免疫细胞学和超微结构分析表明，选定的心肌细胞培养物(纯度为99%)是高度分化的，G418选定的心肌细胞在成年营养不良小鼠的心脏中形成移植物的能力进行了测试。通过抗dystrophin免疫组织学以及肌球蛋白重链氨基糖苷磷酸转移酶转基因特异性引物的聚合酶链式反应(PCR)分析来监测移植细胞的命运。这两项分析都显示，植入后长达7wk的ES来源的心肌细胞移植物的存在，分析了最近的时间点，这些研究表明，这些研究表明，可以使用简单的基因操作来从分化的ES细胞中选择基本上纯的心肌细胞培养，而且所得到的心肌细胞适合于心内移植物的形成。这种选择方法应该适用于所有ES来源的细胞系。
This study describes a simple approach to generate relatively pure cultures of cardiomyocytes from differentiating murine embryonic stem (ES) cells, A fusion gene consisting of the alpha-cardiac myosin heavy chain promoter and a cDNA encoding aminoglycoside phosphotransferase was stably transfected into pluripotent ES cells. The resulting cell lines were differentiated in vitro and subjected to G418 selection. Immunocytological and ultrastructural analyses demonstrated that the selected cardiomyocyte cultures (> 99% pure) were highly differentiated, G418 selected cardiomyocytes were tested for their ability to form grafts in the hearts of adult dystrophic mice. The fate of the engrafted cells was monitored by antidystrophin immunohistology, as well as by PCR analysis with primers specific for the myosin heavy chain-aminoglycoside phosphotransferase transgene. Both analyses revealed the presence of ES-derived cardiomyocyte grafts for as long as 7 wk after implantation, the latest time point analyzed, These studies indicate that a simple genetic manipulation can be used to select essentially pure cultures of cardiomyocytes from differentiating ES cells, Moreover, the resulting cardiomyocytes are suitable for the formation of intracardiac grafts. This selection approach should be applicable to all ES-derived cell lineages.