与胎儿心肌细胞共同培养诱导人胚胎干细胞发生心肌样分化

Cardiac differentiation of human embryonic stem cells by coculturing with human fetal cardiomyocytes

目的了解与胎儿心肌细胞共同培养对人胚胎干细胞（embryonic stem cells，ESCs）向心肌方向分化的影响。方法通过酶消化法从流产胎儿中分离培养出胎儿心肌细胞及成纤维细胞，将后者用丝裂霉素处理后制备成滋养层；收集辅助生育中心施行试管婴儿后所剩余的健康胚胎，取内细胞团接种在滋养层上分离培养出人ESCs。用荧光原位杂交（fluorescent in situ hybridzation，FISH）方法来检测上述两种细胞的XY染色体，选用不同性别的胎儿心肌细胞与2—5代人ESCs以1：2的比例混合，然后以5×10^4／cm^2的密度接种于培养皿中进行共同培养，诱导其向心肌方向的分化。将未与胎儿心肌细胞混合的人ESCs以相同密度接种于培养皿中作为阴性对照。至第5天收集共同培养的细胞，进行双重染色，即FISH染色及心肌特异性蛋白Desmin或cTnⅠ免疫组化染色。结果在与胎儿心肌细胞共同培养5d后，40％～50％的人ESCs在心肌细胞的诱导下表达心肌特异性蛋白Desmin或cTnⅠ，而没有进行共同培养的人ESCs不表达上述蛋白。结论与胎儿心肌细胞的共同培养能诱导人ESCs发生向心肌方向的分化，人ESCs有望成为心肌干细胞移植的细胞材料。

Objective To explore the cardiac differentiation of human embryonic stem cells （ESCs） in coculturing with human fetal cardiomyocytes. Methods Fetal cardiomyocytes and fibroblasts were obtained from the aborted fetus, and human ESCs were from the inner cell mass （ICM） of the surplus embryos for tube babies, with the treated fibroblast as feeder layer. Human ESCs of passage 2 to 5 were cocultured with fetal cardiomyocytes at the ratio of 1 ： 2, and the XY chromosome types of these two kinds of human cells were different to each other in order to be marked. At the same time, human ESCs without coculture were used as negative control. The cocultured cells were harvested 5 days later and were double - stained with human XY chromosome probe and cardiac specific antibody （desmin or cardiac troponin Ⅰ, cTnⅠ）. Results On coculture day 5,40% -50% human ESCs expressed cardiac specific antigens, and human ESCs without coculture did not express those antigens. Conclusion Human ESCs could be induced into cardiac differentiation by the coculture with fetal cardiomyocytes, and hopefully they will be the candidate for cell transplantation of heart.