利用诱导多能干细胞构建流产毒性药物的体外筛查模型

妊娠期用药不可避免,如何帮助孕妇合理用药以减少自发流产、早产和低体重儿的发生,对于医务工作者至关重要。本研究建立了药物流产毒性的检测模型,可筛除疑似流产的药物,避免用药不当导致的孕妇流产。通过三维悬浮培养诱导多能干细胞(induced pluripotent stem cells,iPSCs)的方式,诱导其形成拟胚体(embryoid bodies,EBs)和类皮质器官,在体外模拟早期胚胎从内细胞团向三胚层体系的分化过程。根据流产药物米非司酮(RU486)暴露浓度的不同,随机将EBs分为3组:空白对照组(control)、低浓度组(L-RU486,10μg·mL^-1)和高浓度组(HRU486,20μg·mL^-1)。借助末端脱氧核苷酸转移酶介导的dUTP缺口末端标记测定法terminal dexynucleotidyl transferase(TdT)-mediated dUTP nick end labeling,TUNEL]和免疫荧光染色等技术观察第5、8和11天的不同组别EBs的大小、细胞凋亡和胚层分化的情况。研究结果表明:iPSCs体外可聚集成三维结构的EBs,在特异性神经诱导因子作用下,EBs外周分化出神经玫瑰花状结构,形成大脑皮质类器官;米非司酮暴露导致EBs发育迟缓,EBs直径减少(P<0.01),促进EBs内部细胞凋亡(P<0.01),并影响EBs胚层发育,抑制胚胎干细胞增殖和外胚层细胞分化(P<0.01),促进中胚层细胞发育(P<0.05)。本研究提示,iPSCs可作为药物流产毒性的筛选模型,而EBs的大小、细胞凋亡率和胚层细胞分化率可作为药物筛选的评价指标。

Construction of a drug screening model for miscarriage from induced pluripotent stem cells in vitro

Drug use during pregnancy is unavoidable.Therefore,it is vitally important for medical workers to help pregnant women take drugs correctly to reduce the incidence of spontaneous abortion,premature birth,and low birth weight.In our study,drug screening model with induced pluripotent stem cells(iPSCs)was used to find some improper drugs which will result in woman’s abortion.With 3 D culture in vitro,iPSCs can form embryoid bodies(EBs)and cerebral organoids,which simulated in vitro development of early embryos,from inner cell mass to germ-layer differentiation.In the experiment,EBs were exposed to mifepristone(RU486),and three experimental groups were divided randomly.They were control group(without RU486),low-dose group(L-RU486,10μg·mL^-1),and high-dose group(H-RU486,20μg·mL^-1).After mifepristone exposure,EBs were observed at days 5,8,and 11,including size of EB,cell apoptosis,and differentiation of germ layers,by using inverted optical microscope,TUNEL assay,and immunofluorescent staining.The results showed that through 3 D culture,iPSCs could develop into embryoid bodies,neural rosettes,and finally cerebral organoids.After mifepristone exposure,EBs’sizes were decreased(P<0.01);the levels of cell apoptosis in EBs were increased after mifepristone exposure(P<0.01);the development of EBs’germ layer was affected.Mifepristone exposure could inhibit the proliferation of embryonic stem cells,reduce the differentiation of ectoderm(P<0.01)and promote the development of mesoderm(P<0.05).In conclusion,iPSCs can be used as a screening model for abortion drug,and EBs’diameter,cell apoptosis,and differentiation changes of the germ layers can serve as criteria of abortion drug screening.