单层分步法诱导人胚胎干细胞定向肝样细胞分化

DIFFERENTIATION OF HUMAN EMBRYONIC STEM CELLS INTO HEPATOCYTE-LIKE CELLS IN AN ADHERENT CULTURE SYSTEM WITH SINGLF-STEP INDUCTION

目的采用单层分步法诱导人胚胎干细胞(human embryonic stem cells,hESCs)分化为肝样细胞,建立hESCs分化为肝细胞的诱导体系。方法将hESC株H1细胞接种到Matrigel包被的培养板上,早期(细胞贴壁4d后)加入含有细胞因子活化素A(Activin A)的分化液诱导5d;中期将诱导液换为含FGF-1和BMP-4的肝分化液诱导6d;最后添加肝细胞生长因子(hepatocyte growth factor,HGF)和制癌蛋白M(Oncostatin M,OSM)继续诱导分化5～7d。采用免疫细胞化学染色、RT-PCR、过碘酸-雪夫(Periodicacid-Schiff,PAS)反应鉴定分化细胞的性质。结果经Activin A、FGF-1、BMP-4、HGF和OSM连续诱导的hESCs呈多角形、卵圆形或圆形,排列紧密;部分细胞出现二核或三核,呈现肝细胞所特有的形态。分化细胞在诱导早期表达SOX17、叉头框(Forkhead box,FOX)A2、GATA-4,中期表达甲胎蛋白(αfetoprotein,AFP)、白蛋白、角蛋白18,晚期表达成熟肝细胞特异性基因乙醇脱氢酶1C、细胞色素P4501B1。免疫细胞化学染色示,Activin A诱导后,细胞呈SOX17和FOXA2阳性;经FGF-1和BMP-4诱导后,呈AFP和α1抗胰蛋白酶阳性。HGF和OSM诱导后,PAS糖原染色检测阳性。结论采用单层分步法逐步添加细胞因子,可诱导hESCs分化为肝样细胞。该分化体系可为肝细胞移植、肝组织工程等临床治疗提供一个有效的细胞来源。

Objective To establish a new induction method from human embryonic stem cells（hESCs） differentiating into hepatocyte-like cells using an adherent culture system with single-step induction.Methods Undifferentiated hESCs were cultured on Matrigel-coated culture plates for 4 days,hepatic differentiation was initiated at 60%-70% confluence by adding Activin A for 5 days.Then the induction medium was replaced by hepatocyte induction medium（HIM） supplemented with fibroblast growth factor 1（FGF-1） and bone morphogenetic protein 4（BMP-4） for another 6 days.Finally,the cells were treated with HIM adding hepatocyte growth factor（HGF） and Oncostatin M（OSM） for 5-7 days.The characteristics of differentiated cells were determined by morphology,immuno fluorescence staining,RT-PCR,and PeriodicacidSchiff（PAS） test.Results Differentiated cells treated with Activin A,FGF-1,BMP-4,HGF,and OSM sequentially were morphologically larger and became spherical,oval or polygon.Some cells had 2 or 3 nuclei,suggesting that the cells have a hepatocyte-like morphology.Differentiated cells at first induction stage could be stained positive by SOX17 and Forkhead（FOX）A2 after induction by Activin A.Then they turned to be α fetoprotein（AFP） and α1 antitripsin positive cells at second induction stage after induction by FGF-1 and BMP-4.Finally,the differentiated cells treated with HGF and OSM showed PAS possitive for glycogen detection.The differentiated cells at various stages also expressed at early（SOX17,FOXA2,and GATA-4）,middle（AFP,albumin,and cytokeratin 18）,and mature（alcohol dehydrogenase 1C and Cytochrome P4501B1） stage hepatic genes,respectively.Conclusion Using a simple-step induction method and by supplied with cytokines consequently,hESCs can be induced to differentiate into hepatocyte-like cells.The differentiation method can provide seed cells for hepatic tissue engineering or cell-therapy.