人胚胎干细胞H9诱导心脏类器官的方法研究

The Method Study of Human Embryonic Stem Cells H9 Inducing and Differentiating into Cardiac Organoids

目的探索人胚胎干细胞H9诱导心脏类器官方法的研究。方法首先,我们从碱性磷酸酶以及干细胞干性标志物鉴定人胚胎干细胞(human embryonic stem cell,hESC)的全能性。然后,采用U型孔诱导H9向中胚层(mesoderm,MES)分化,进而继续诱导向心脏类器官分化。在分化过程中,我们观察不同时间点的心脏类器官形态变化,并选取相应时间点,采用real-time PCR、western blotting、细胞免疫荧光化学技术鉴定诱导分化方法的合理性。结果光镜下,H9细胞呈克隆状生长,ALP显示呈蓝紫色且干细胞干性标志物OCT4在细胞核内表达呈强阳性(绿色)。Real-time PCR结果显示,随着时间的推移,心肌细胞标志基因ACTN2、cTnT和MYL2以及非心肌细胞标志物在一定的时间节点后呈爆发式上升;western blotting显示第5天后开始递增表达;细胞免疫荧光显示,分化18天的心脏类器官ACTN2阳性表达(绿色),肌节排列不规则且肌丝分布不均匀;而诱导40天时的心脏类器官中心肌细胞呈杆状,肌节排列整齐,肌丝分布于整体心肌细胞,CX43高表达,趋向于成熟。结论此方法成功诱导H9向心脏类器官分化。

Objective To explore the method of inducing cardiac organoids from human embryonic stem cells H9.Methods We first identified the totipotency of human embryonic stem cells(hESCs,H9)from alkaline phosphatase and stem cell markers.In addition,U-shaped wells were used to induce H9 differentiation into mesoderm(MES),and then to induce cardiac organoids differentiation.During differentiation process,morphological changes of cardiac organoids at different time points were observed,and the rationality of induction differentiation method was identified by real-time PCR,western blotting as well as cellular immunofluorescence.Results Under light microscope,H9 cells presented clones,and ALP was blue and purple,as well as the expression of stem cell marker OCT4 was strongly positive in the nucleus(green).Real-time PCR result showed that cardiomyocyte marker genes ACTN2,cTnT and MYL2 with non-cardiac markers presented explosive increases after a certain time node.Western blotting showed that the expression of cardiac markers began to increase after 5 days.Cellular immunofluorescence showed positive expression of ACTN2(green)in the cardiac organoids after 18 days of differentiation,with irregular sarcomere arrangement and uneven distribution of myofilaments.On the other hand,after 40 days of induction,the cardiomyocytes in the cardiac organoids were rod-shaped,and the sarcomeres were neatly arranged,and the myofilaments were distributed in the whole cardiomyocytes,with CX43 upregulating,which tended to mature.Conclusion The differentiation of H9 into cardiac organoids can be successfully induced by this method.