悬浮培养诱导人胚胎干细胞分化为心肌细胞的研究

Research of human embryonic stem cells into cardiomyocytes by suspension method

目的:利用不加任何诱导剂的悬浮培养法,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:人胚胎干细胞克隆用200U/mL胶原酶Ⅳ,370C处理10min后,挑起克隆碎片转移到低黏附性培养皿中悬浮培养以形成EB(拟胚体),4d后将EB转移到基质胶处理过的6孔板中贴壁培养(1-3EBs/cm2),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;免疫荧光染色检测心肌细胞特异标志物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:在悬浮培养14d左右开始出现大量的自发跳动心肌细胞,分化出现自发跳动心肌细胞的平均时间(13.9±0.9)d,百分比为20.8%,平均跳动频率为(63.8±5.6)次/min;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的凋亡比例为(8.1±0.4)%。结论:不加诱导剂的悬浮培养可以诱导人胚胎干细胞分化为心肌细胞,分化效率达到20.8%,分化时间14d左右。为进一步的干细胞移植治疗动物心肌梗死模型提供种子细胞。

Objective：Using the suspension-culture method which dosent using inducers to induce the differentiation of human embryonic stem cells into cardiomyocytes in vitro and detecting its differentiation efficiency.Methods：Undifferentiated hES cells were dissociated into clumps using 200U/mL collagenase Ⅳ at 37℃ for 10 min and cultured in suspension using low attachment plates to form EBs（Embryoid body）.After 4 d in suspension,EBs were plated on 0.2 matrigel coated plates to score the contracting cardiac clusters （1-3EBs/cm2）.Observing and counting the time of appearing beating cardiomyocytes,the percentage of beating colonies and the beating frequency of cardiomyocytes under the microscope; Staining the specific marker cTnT of cardiomyocytes by immolunofluorescence; Detecting the electrophysiological function of cardiomyocytes by patch clamp experiment.Using apoptosis-hoechst staining kit to detect the apoptosis ratio of beating cardiomyocytes which had been treated by hypoxia for 24 hours.Results：Widespread spontaneous beating cardiomyocytes was typically observed by day 14 after differentiation,and the average time of appearing beating cardiomyocytes was （13.9 ± 0.9） days,the percentage of beating colonies was 20.8 ％,the beating frequency of cardiomyocytes （63.8 ± 5.6）times/min; Beating cardiomyocytes were positive to cTnT staining; Spontaneous action potential of beating cardiomyocytes was detected ; The apoptosis ratio of beating cardiomyocytes which had been treated by hypoxia for 24h was（8.1 ± 0.4） ％.Conclusion：The suspension-culture method wbich dosent using inducers can successfully induce the differentiation of human embryonic stem cells into cardiomyocytes,and the differentiation efficiency reached 20.8％,and the differentiation time was about 14 days.Providing seed cells for further stem cell transplantation to animal model of myocardial infarction.