体内肝细胞来源的肝干/前体细胞鉴定以及再分化机理研究

肝细胞在体外可诱导大量扩增形成类器官，而损伤条件下体内肝细胞仅通过有限的增殖完成修复，未发现某类肝细胞充当干/前体细胞。我们改进构建长期肝损伤模型，发现长期损伤诱发部分肝细胞呈现克隆形成及双向分化能力，符合肝干/前体细胞表型。且成熟肝细胞发生去分化，肝细胞类群分布也发生变迁。本项目拟通过单细胞RNA测序结合细胞谱系追踪技术鉴定哪种转录组表型的肝细胞充当肝干/前体细胞或哪种肝细胞向肝干/前体细胞转化，解析各种功能的肝细胞类群如何产生与变迁。再利用体外类器官体平台进一步验证转化过程和有效的调控靶点。据此优化建立高效产生鼠源和人源肝干/前体细胞及各类群肝细胞的类器官体培养与转化平台。以期进一步解析肝脏再生过程和机理，为开发促进肝脏再生修复的诊疗手段找到治疗靶点。改进的体外产生肝干/前体细胞及各类群肝细胞的平台，可用于体外药物研发、患者个性化药物筛选及自身细胞移植治疗，有直接的应用价值。

Recent reports demonstrated that modest proliferation of hepatocytes distributed throughout the hepatic lobule maintains the hepatocyte mass during liver injury and that most hepatocytes proliferate to regenerate it. The ability to proliferate is broadly distributed among hepatocytes rather than limited to a rare stem cell-like population. We modified traditional liver regeneration models to simulating pathologic changes of chronic human liver diseases, under such conditions, hepatocytes distributed throughout the lobule underwent modest proliferation during early stage of liver injury, while single labeled hepatocytes show clone forming capacity at later stage of long-term injuries. Interestingly, we found cell clusters derived from single labeled pre-existing hepatocyte containing both hepatocytes and biliary epithelial cells at later stage of long-term liver injury, which is consistent with the characters of hepatic progenitor cells. Long-term injury also triggered dedifferentiation of hepatocytes and contribute to change of proportion and identity of some hepatocyte sub-populations. In order to clarify the process and mechanism of hepatic progenitor cells generation, and to clarify how sub-populations of hepatocyte change during chronic liver injuries, we plan to identify sub-populations of hepatocytes based on transcriptome profile revealed by single-cell RNA sequencing, and to find out specific markers for the hepatocyte sub-populations that serve as progenitor cells or underwent converting into hepatic progenitor cells. We will identify their conversion process, and to screen specific genes and signal pathways relating to the conversion between sub-populations. These genes and pathways might be involved in regulating the lineage conversion and could be therapeutic targets. Organoid platform will be adopted to verify the lineage conversion process, and to find out effective regulatory genes or pathways. Based on these, an organoid platform for producing mouse and human hepatic progenitor cells and hepatocytes would be established. In conclusion, the project will further reveal the process of liver regeneration, and will identify molecular targets for the promoting-liver-regeneration-therapies. The platform for generating hepatic progenitor cells and hepatocytes in vitro can be used for drug research, personalized drug screening and autologous cell transplantation therapy, which has direct application value.