干细胞定向诱导肝向分化亟需完善，但受限于缺乏肝细胞特异的分子鉴定标记，由肝母细胞到成熟肝细胞过程中关键因子迄今未明。 申请人在前期工作中，稳定定向分化人胚胎干细胞为肝细胞 (hESC-Heps)，成功建立肝炎性病毒感染研究模型，真实的呈现了人原代肝细胞的特性。在此基础上，本项目将基于肝炎病毒的嗜肝性寻找hESC-Heps分化过程中关键因子： 1）稳定定向分化人胚胎干细胞获得肝细胞hESC-Hep，通过HBVpp/HCVpp系统和肝炎病毒的产生来筛选未成熟的和成熟的hESC-Heps；2）对筛选获得的hESC-Heps进行基因表达谱的比对分析获得候选因子；3）运用CRISP/CAS9基因敲除和高表达候选基因等技术确定hESC-Heps分化过程中关键因子。项目预期通过已建立的干细胞定向分化平台，揭示hESC-Heps分化过程中关键因子，为干细胞来源的肝细胞的发展完善提供重要参考依据。

It is necessary to improve the directed differentiation of human stem Cells into hepatocytes. But the key factors during differentiation from hepatoblast to mature hepatocytes are still unknown due to the restriction of deficiency of hepatocyte-specific markers. In our previous work, we have.successfully differentiated human embryonic stem cell into hepatocytes (hESC-Heps) and modeled hepatitis viral infection, which reflected the characteristics of primary human hepatocytes authentically (Zhou XL, et al. Stem Cell Reports, 2014). This project aims to find out.the key factors during differentiation of hESC-heps based on the hepatotropism of hepatitis virus (HBV/HCV). It includes three parts 1) stably differentiate hESC into hESC-Heps and sort out the mature and immature hESC-Heps by HBVpp/HCVpp systems and production of hepatitis virus using FACS; 2) analyze gene expression profile between these hESC-Heps and select out the candidates 3) apply the technologies of CRISP/CAS9 and gene over expression etc to identify the key factors during the process. The project is aimed at disclosing the key factors using the current platform of stem-cell-directed differentiation, and therefore providing the important data for development of stem cell-derived hepatocytes.