Huber Irit, Itzhaki Ilanit, Caspi Oren, Arbel Gil, Tzukerman Maty, Gepstein Amira, Habib Manhal, Yankelson Lior, Kehat Izhak, Gepstein Lior
Sohnis Family Research Laboratory for the Regeneration of Functional Myocardium and the Rappaport Family Institute for Research in the Medical Sciences, The Bruce Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, Haifa, Israel.
FASEB J. 2007 Aug;21(10):2551-63. doi: 10.1096/fj.05-5711com. Epub 2007 Apr 13.
Human embryonic stem cells (hESC) are pluripotent lines that can differentiate in vitro into cell derivatives of all three germ layers, including cardiomyocytes. Successful application of these unique cells in the areas of cardiovascular research and regenerative medicine has been hampered by difficulties in identifying and selecting specific cardiac progenitor cells from the mixed population of differentiating cells. We report the generation of stable transgenic hESC lines, using lentiviral vectors, and single-cell clones that express a reporter gene (eGFP) under the transcriptional control of a cardiac-specific promoter (the human myosin light chain-2V promoter). Our results demonstrate the appearance of eGFP-expressing cells during the differentiation of the hESC as embryoid bodies (EBs) that can be identified and sorted using FACS (purity>95%, viability>85%). The eGFP-expressing cells were stained positively for cardiac-specific proteins (>93%), expressed cardiac-specific genes, displayed cardiac-specific action-potentials, and could form stable myocardial cell grafts following in vivo cell transplantation. The generation of these transgenic hESC lines may be used to identify and study early cardiac precursors for developmental studies, to robustly quantify the extent of cardiomyocyte differentiation, to label the cells for in vivo grafting, and to allow derivation of purified cell populations of cardiomyocytes for future myocardial cell therapy strategies.
人胚胎干细胞（hESC）是多能细胞系，能够在体外分化为包括心肌细胞在内的所有三个胚层的细胞衍生物。这些独特的细胞在心血管研究和再生医学领域的成功应用受到了阻碍，因为难以从分化细胞的混合群体中识别和选择特定的心脏祖细胞。我们报告了使用慢病毒载体生成稳定的转基因hESC系，以及在心脏特异性启动子（人类肌球蛋白轻链-2V启动子）的转录控制下表达报告基因（eGFP）的单细胞克隆。我们的结果表明，在hESC分化为胚状体（EB）的过程中出现了表达eGFP的细胞，这些细胞可以使用荧光激活细胞分选术（FACS）进行识别和分选（纯度>95%，活力>85%）。表达eGFP的细胞对心脏特异性蛋白呈阳性染色（>93%），表达心脏特异性基因，显示心脏特异性动作电位，并且在体内细胞移植后能够形成稳定的心肌细胞移植体。这些转基因hESC系的产生可用于识别和研究发育研究中的早期心脏前体细胞，以可靠地量化心肌细胞分化的程度，为体内移植标记细胞，并允许获得纯化的心肌细胞群体，用于未来的心肌细胞治疗策略。
