PMA体外诱导小鼠多能干细胞向心肌细胞的分化

Phorbol myristate acetate promotes differentiation of mouse induced pluripotent stem cells into cardiomyocytes in vitro

目的研究丙二醇甲醚醋酸酯(PMA)对小鼠诱导多能干细胞体外分化为心肌细胞的影响,以建立一种高效安全的体外诱导i PSC分化为心肌细胞的实验方法。方法用悬滴法形成拟胚体(EBs),PMA诱导其向心肌细胞定向分化。免疫细胞学标记检测心肌肌钙蛋白T(c Tn T)和α横纹肌辅肌动蛋白(α-actinin)的表达;RT-PCR和q-PCR检测Brachyury,c Tn T,MLC2a,NKX2.5和GATA4等mRNA的表达。以添加相应DMSO作为对照组,观察各组出现搏动拟胚体的数量,计算分化比率。结果 PMA诱导小鼠诱导多能干细胞分化为心肌细胞的最佳浓度为100 nmol/L,此时拟胚体搏动率可达53%,显著高于对照组(12%),且PMA诱导产生的心肌细胞表达多种心肌蛋白及基因,具有心肌细胞的结构特征。结论 PMA能够促进mi PSC在体外定向分化为心肌样细胞。

Objective To investigate the effect of PMA on the differentiation of mouse induced pluripotent stem cells into cardiomyocytes in vitro, and to establish an efficient and safe protocol for cardiac differentiation in vitro. Methods Embryoid bodies （EBs） were prepared by classical hanging drop method, and were differentiated to car- diomyocyte with PMA treatment; Furthermore, expression of cardiac troponin T and α-Sarcomeric actinin was ob- served by immunofluorescent staining, mRNA expression levels of the related genes Brachyury, cTnT, MLC2a, NKX2. 5, GATA4 were analyzed by qPCR and RT-PCR. Control group was treated by DMSO, number of beating embryoid bodies was calculated. Results The best concentration of PMA to induce the cardiac differentiation of miPSC was 100 nmol/L and the beating area was found in 53% of embryoid bodies, compared with the control group （ beating area was found in 12% of embryoid bodies） , Differentiation efficiency was higher in PMA treatment group. IPS-CM expressed serve cardic related proteins and genes, and had the Structure characteristics of myocar- dium cell. Conclusions PMA can promote miPSC to differentiate into cardiomyocyte in vitro.