人胚胎干细胞分化为心肌细胞的体外实验(英文)

Differentiation of human embryonic stem cells into cardiomyocytes in vitro

背景:体外诱导胚胎干细胞分化为心肌细胞国内外已做了大量研究。然而,在国内对胚胎干细胞分化为心肌细胞的研究只限于小鼠胚胎干细胞,还未见有将人胚胎干细胞诱导为心肌细胞的报道。目的:将人胚胎干细胞系H14诱导分化为心肌细胞。设计:体外人胚胎干细胞培养细胞,采用悬浮法让其形成拟胚体,在不同的分化时期观察出现有节律性收缩的拟胚体的比率以及心肌特异性基因的表达。单位:香港中文大学公共卫生学院何鸿燊防治传染病研究中心分子生物学实验室。材料:实验所用人胚胎干细胞株H14来源于美国威斯康星大学WiCell研究所并授权使用。方法:于2006-08/12在香港中文大学公共卫生学院何鸿燊防治传染病研究中心分子生物学实验室完成。采用悬浮法培养人胚胎干细胞株H14,让其形成拟胚体。4d后,将拟胚体培养在包被有明胶的6孔培养板内(5～10个胚胎体/孔),让其自发分化为跳动的拟胚体,显微镜下观察有节律性收缩的拟胚体;然后采用反转录聚合酶链反应方法检测心肌特异性基因的表达。主要观察指标:不同的分化时期有节律性收缩的拟胚体的比率以及心肌特异性基因的表达。结果:拟胚体形成8d后,大约有2%的拟胎体出现有节律性的收缩,随着时间的延长,产生收缩的拟胚体越多,到第16天,大约有10%的拟胚体出现节律性跳动,跳动频率为70～100次/min。反转录聚合酶链反应结果显示,有节律性收缩的拟胚体表达心肌特异性的转录因子GATA-4和Nkx2.5,心肌特异性基因Isl-1和α-MHC。结论:国内首次成功使人胚胎干细胞诱导分化为心肌细胞。

BACKGROUND： Differentiation of embryonic stem （ES） cells into cardiomyocytes in vitro has been studied in great detail in the world. However, much of what is currently known about cardiomyocyte differentiation from ES cells has been learned from studies on mouse in China. few studies are on human ES cells. OBJECTIVE： TO investigate the differentiation efficacy of human ES cells into functional cardiomycytes with the human H14 ES cell line. DESIGN： Suspending method was used to form pseudo-embryo proper of human ES cells so as to observe ratio of pseudo-embryo proper with rhythmic contraction and expression of specific gene of myocardium in various differentiated phases. SETTING： Molecular Biology Laboratory of Stanley Ho Center for Emerging Infectious Diseases, School of Public Health, the Chinese University of Hong Kong. MATERIALS： Human ES cell line H14 was obtained from WiCell Research Institute （Wisconsin, USA） with a license agreement. METHODS： The experiments were carried out in the Molecular Biology Laboratory of Stanley Ho Center for Emerging Infectious Diseases, School of Public Health, the Chinese University of Hong Kong from August to December of 2006. The H14 ES cell colony was used to form embryoid bodies （EBs） by using suspending method. Four days later, pseudo-embryo proper was cultured in gelatin-coating 6-well culture plate （5-10 embryo proper/well） and spontaneously differentiated into moving pseudo-embryo proper. Rhythmic contraction was observed under microscope and RT-PCR was used to detect expression of special genes of myocardium. MAIN OUTCOME MEASURES ： Ratio of pseudo-embryo proper with rhythmic contraction and expression of specific gene of myocardium in various differentiated phases RESULTS： Spontaneously contracting cells appeared as cluster and were identified in approximately 2% of EBs at differentiation day 8 and increased to as many as 10% of the EBs by day 16. The beating rate of contracting cells arranged at 70-100 beats per minute, RT-PCR analyses demonstrated that cells isolated from spontaneous beating areas within the EB expressed the cardiac transcription factors GATA-4 and Nkx2.5, cardiac progenitor gene Isl-1 and cardiomyocyte marker gene α-MHC. CONCLUSION ： This is the first time to report human ES cells can effectively differentiate into functional cardiomyocytes in China.