直接贴壁法诱导人胚胎干细胞分化为心肌细胞

摘要：目的利用直接贴壁法体外诱导人胚胎干细胞分化为心肌细胞，并检测其分化效率。方法人胚胎干细胞以1×10’个／cm2的细胞密度传代到铺备有基质胶的培养皿中培养，用带8ng／ml碱性成纤维细胞生长因子（bFGF）的条件培养基培养6d后，更换为RPMI1640．B27培养基，同时加入100ng／ml的人重组activinA处理24h，接着再加入10ng／ml的人重组骨形态发生蛋白4（BMP4）处理4d，之后更换为不带诱导因子的RPMI1640．B27培养基，每隔2～3d换1次培养基，持续2～3周。在光学显微镜下观察记录出现跳动心肌细胞的时间和跳动频率，并计算跳动克隆百分比，24孔板一组，共记录4组96孔；用免疫荧光染色法检测心肌细胞特异标志物心肌肌钙蛋白T（cTnT）；膜片钳实验检测心肌细胞自发性动作电位；跳动心肌细胞经过24h缺氧刺激后，用凋亡试剂盒检测心肌细胞凋亡比例。结果大量的自发跳动心肌细胞在诱导分化13d左右开始出现。分化出现自发跳动心肌细胞的时间为（13．0±1．1）d，百分比为66．7％，跳动频率为（63．0±7．0）次／分；跳动心肌细胞cTnT染色阳性；跳动心肌细胞检测到自发性动作电位；跳动心肌细胞缺氧24h后检测到凋亡比率为8．0％±0．5％。结论国内首次利用直接贴壁法在体外诱导人胚胎干细胞分化为心肌细胞，分化效率达到66．7％，分化时间13d左右。

Inducing Human Embryonic Stem Cells to Become Cardiomyocytes by Direct Adherence Method

Objective To use direct adherent method to induce human embryonic stem cells （hESCs） to become cardiomyocytes in vitro and examine their differentiation rate. Methods Undifferentiated hESCs were seeded onto Matrigel-coated plates at a density of 1 ~ 105 cells/cm2 and cultured in MEF-conditioned medium （MEF-CM） with 8 ng/ml basic fibroblast growth factor （bFGF） for 6 days. Then MEF-CM was replaced with RPMI 1640/1327 medium supplemented with 100 ng/ml human recombinant activin A for 24 hours in hESCs culture, followed by supplementation of 10 ng/ml hu- man recombinant bone morphogenetic protein 4 （BMP4） for 4 days in hESCs culture. The medium was then replaced with RPMI 1640/B27 medium without supplementary cytokines, and hESCs were refed every 2-3 days for 2-3 additional weeks. Self-beating cardiomyocytes and the beating frequency were observed under the microscope, and the percentage of colonies showing beating cardiomyocytes was calculated. Cardiac troponin T （cTnT）, a specific marker of cardiomyocytes, was examined by immunofluorescence. Spontaneous action potentials of cardiomyocytes were measured with patch clamp technique. Apoptotic rate of cardiomyocytes was detected with apoptosis-hoechst staining kit after beating cardiomyocytes were cultured under hypoxia for 24 hours. Results A large number of spontaneous beating cardiomyocytes were observed 13 days after induction. The average time to show beating cardiomyocytes was 13.0_＋ 1.1 days after induction, the percentage of colonies showing beating cardiomyocytes was 66.7%, and the beating frequency of cardiomyocytes was 63.0_＋ 7.0 times/ minutes. Beating cardiomyocytes were cTnT-positive. Spontaneous action potentials were detected in beating cardiomyocytes. Apoptotic rate of cardiomyocytes was 8.0%_＋0.5% after beating cardiomyocytes were cultured under hypoxia for 24 hours. Conclusion It＇s the first time to use direct adherent method to induce hESCs to become cardiomyocytes in vitro in China with the differentiation rate of 66.7% and differentiation time of 13 days.