人源诱导多能干细胞体外定向分化为功能性肝细胞的方法研究

Study on the Method of Directional Differentiation of Human Induced Pluripotent Stem Cells into Functional Hepatocytes in Vitro

目的:研究开发一种简易、快速在体外使多能诱导干细胞(induced pluripotent stem cells,i PSCs)定向分化为功能性肝样细胞的培养方法。方法:根据正常肝细胞在体内的发育规律,设计简化诱导方法使i PS细胞定向分化为内胚层细胞,应用qPCR和流式细胞术鉴定其纯度后进一步诱导分化为肝样细胞,并通过qPCR、ELISA、免疫荧光等技术鉴定肝细胞的性状和功能。结果:i PS细胞诱导7天后,OCT4和NANOG的表达水平显著下降,内胚层细胞相关基因CXCR4、FOXA2和HNF4A表达水平明显升高。内胚层细胞继续诱导培养15天后,肝细胞特异性标志基因ALB、TDO2、RBP4、G6PC和肝药酶基因CYPs等显著上调,同时产生高水平的白蛋白和尿素;PAS糖原染色为阳性,能主动摄取和释放吲哚菁绿,证实诱导成的肝样细胞具备正常肝细胞的部分功能。结论:该诱导方案能够在体外使i PS细胞遵循正常肝脏发育通路简易、高效地分化为功能性肝细胞。本研究为大量获得iPS来源的肝细胞及其在细胞疗法和药筛模型中的运用提供了可能性。

Objective:This study is to research and develop a simple and rapid method to directly differentiate the induced pluripotent stem cells(i PS)into functional hepatocytes in vitro.Methods:According to the development of normal hepatocytes in vitro,a simplified method was designed for differentiation of i PS cells into endodermal cells.The phenotypes of endodermal cells was identified by q PCR and flow cytometry,and then these cells were further differentiated into hepatocyte-like cells.The characteristics and functions of hepatocytes were identified by qPCR,ELISA and immunofluorescence.Results:The results showed that the m RNA/protein expression of OCT4 and NANOG were decreased significantly,and the m RNA/protein expression of endodermal cell-related genes,CXCR4,FOXA2and HNF4A,were increased significantly at day 7 after i PS cell induction.Hepatocyte specific marker genes ALB,TDO2,RBP4,G6PC and hepatocyte drug enzyme genes CYPs were significantly upregulated at day 15,and high levels of albumin and urea were produced simultaneously.Positive PAS glycogen staining and active uptake and release of indocyanine green were observed in the induced hepatocytes.It was confirmed that the hepatocyte-like cells had the partial functions of primary hepatocytes.Conclusion:This easy and efficient induction method can be used in differentiation of i PS cells into functional hepatocytes by mimicking the process of liver organogenesis in vitro.This study provides the possibility for massive production of i PS-derived hepatocytes and their applications in cellular therapy and drug screening models.