患者iPSC来源肝组织构成细胞的高效诱导扩增及其功能性肝脏类器官的构建与移植评估

肝脏类器官是具有类似肝组织微环境的肝芽，有望用于晚期肝病的移植治疗。患者iPSC来源的肝实质细胞能够克服原代细胞来源短缺与异体移植免疫排斥的缺点。然而，传统诱导技术获得的肝实质细胞无法维持长期体外扩增与相应功能。在前期研究中，我们建立了由人iPSC来源的肝实质细胞、间充质干细胞和血管内皮祖细胞自组装形成的肝脏类器官。本研究拟在此基础上，利用化学小分子调节细胞增殖和衰老相关信号通路，建立具有可塑性及增殖性的高效肝实质细胞诱导技术；利用多细胞因子组合协同刺激同步诱导肝非实质性细胞；利用肝实质细胞和多种肝非实质性支持细胞的三维悬浮共培养技术，构建患者iPSC来源的高度模拟人体肝脏组织构成的肝脏类器官；最终通过建立“别吃我”免疫缺陷型肝损大鼠模型，评估肝脏类器官移植后的肝功能改善及生存挽救效果。本研究以移植治疗为目标，建立新型肝脏类器官构建技术，探索肝脏修复与重建机理，为临床转化应用提供重要参考。

Transplantation of liver organoids with liver-like micro-environment promises to be one of the major strategies for late-stage liver disease treatment in future. Hepatocytes derived from patient induced pluripotent stem cells (iPSC) may overcome the donor shortage and immune rejection of the primary human hepatocytes, with great prospective in clinical applications. However, the failure of enduring the multiple passages and long-term expansions in vitro of human iPSC (hiPSC)-derived hepatocytes which are obtained by using conventional technique as well as their significant functional deficiencies could not meet the requirement of clinical uses. Our team has succeeded in establishing a self-organized liver tissue-like organoid system with hiPSC-derived hepatocytes, human umbilical cord derived mesenchymal stem cells and vein endothelial progenitor cells. Based on the previous experiences, we aim to generate a new liver organoid model consisting of liver parenchymal and non-parenchymal cells included induced hepatocytes, hepatic stellate cells, liver sinusoidal endothelia cells and Kupffer cells which are all derived from iPSCs of a single donor patient. We seek to establish a strategy for the efficient expansion of hepatocytes by modulating the signalling pathways regarding cell proliferation and senescence using small molecules, to induce the differentiation of non-parenchymal cells synchronously by synergic stimulation of multiple cytokines and growth factors and to establish a novel liver organoid highly resembling the human liver by co-culturing with the parenchymal and non-parenchymal cells in a suspended 3D system. Lastly, we aim to establish a novel severe immunodeficient rat model with hSIRPa knock-in to establish “don’t eat me” environment for the evaluation of therapeutic effects of liver organoids which will be transplanted through portal vein to generate humanized liver and to analyse the repopulation rate, liver functions and survival rescue in the liver damage model. With a goal of clinical applications, this project aims to establish a stable and effective strategy for generating liver organoids, to explore their roles involved in injure repair and liver regeneration, and to provide significant evidences for feasibility and safety studies.