生物材料介导工程化人肝类器官构建及其调控机制的研究

In order to solve the histological structural randomness and improve the functions of liver organoid, in this study, the polymer microbeads delivering cell adhesion factors and cell growth factors are designed for constructing engineered liver organoid by mediating hUC-MSCs aggregates and inducing the homogeneous hepatic differentiation of the hUC-MSCs. Based on the endothelial cells could promote the hepatoblasts formation and differentiation into hepatocytes and cholangiocytes at the early stage of liver development, the hVE-cad/VEGF delivering microbeads are prepared by Fc domain of the fusion protein binding on the surfaces of PLGA/Chitosan/Heparin complex microbeads, which rapidly induce the hUC-MSCs differentiation into endothelial cell. Meanwhile, hE-cad/HGF delivering microbeads are prepared by heparin-specific binding of HGF in the PLGA/Chitosan/Heparin complex microbeads and hE-cad-Fc binding on the surfaces of the complex microbeads, which induce hUC-MSCs differentiation into hepatoblasts-like cells, and cooperating with the endothelial cells regulate the differentiation of hepatoblasts-like cells into hepatocyte and cholangiocytes within the hUC-MSCs aggregates. The spatio-temporal controlled delivery of hVE-cad/VEGF and hE-cad/HGF by the polymer microbeads further regulate the secretion functions of hUC-MSCs and the bionic construction of dynamic extracellular microenvironment through the form of “inside-out” within the hUC-MSCs aggregates, which drive the homogeneous formation of hepatic histological structures and construct the engineered liver organoids. We will study the spatial and temporal effect of the functionalized polymeric microspheres system on the regulation of endogenous information molecules secreted by the hUC-MSC aggregates and exogenous information molecules derived from the biomaterials, further illuminate the molecular mechanism of the bionic construction extracellular microenvironment by biomaterials and innovate the biotechnology of engineered liver organoid with hUC-MSCs. This research has extremely important scientific significance and application value on developing drug screening platform, bioartifitial liver system and cell therapy.

为了解决肝类器官多细胞聚集体中组织结构随机性和提高肝功能表达，仿生肝脏发育早期内皮细胞促进肝芽形成以及调控肝祖细胞向肝细胞与胆管上皮细胞定向分化的作用，本项目拟设计并制备上皮、内皮细胞钙粘素与细胞生长因子联合可控递送聚合物微球系统，构建由聚合物微球系统介导的hUC-MSCs聚集体，研发聚集体内基于内皮钙粘素和VEGF联合快速诱导MSC向内皮细胞分化，进一步与上皮钙粘素和HGF协同调控类肝祖细胞增殖及其向肝细胞和胆管细胞的定向分化、驱动细胞迁移、并时序性驱动类肝芽结构的可控组装及其发育成熟并表达肝功能，阐明功能化聚合物微球系统对MSC聚集体中细胞分泌的内源性信息分子和材料导入的外源信息分子的时空协同调控作用及其仿生构建细胞外微环境的分子机制，创新MSC源工程化人肝类器官的可控构建理论与技术。该研究对肝病的新药以及生物人工肝和细胞疗法的研发具有极其重要的科学意义和应用价值。

近年基于干细胞的肝类器官研究已成为多学科交叉领域的前沿性研究热点，其中肝类器官的血管化不足及其结构随机性一直是肝类器官应用转化的技术瓶颈。本项目系统研究了人上皮-钙黏素胞外域与人IgG-Fc结构域融合蛋白（hE-Cad-Fc）和人内皮-钙黏素胞外域与人IgG-Fc结构域融合蛋白（hVE-Cad-Fc）基质材料对人脐带间充质干细胞（hMSCs）分泌及分化特性的影响。结果显示，hE-Cad-Fc/hVE-Cad-Fc基质可以分别激活EGFR/VEGFR及其磷酸化，显著促进YAP蛋白在细胞质中的滞留，抑制Wnt/β-catenin信号通路，促进hMSCs向肝实质、胆管上皮及内皮细胞的分化效率。进一步研制了钙黏素融合蛋白修饰的缓释生长因子的PLGA/Chitosan-heparin复合材料微球系统（Cad/GF-MPs），优化了其批量制备的微流控系统参数；成功构建了钙黏素功能化生物活性材料微球介导的人脐带间充质干细胞聚集体。微球本体缓释HGF和FGF-2、表面联合固定VEGF-Fc和hE/VE-Cad-Fc的Cad/GF-MP微球，在hMSCs聚集体中实现了细胞黏附因子钙黏素与细胞生长因子的时空仿生递送，在体外仿生肝脏发生发育过程，成功调控hMSCs的黏附、分泌及分化特性，在聚集体内形成了有着均匀血管和胆小管网络的肝类器官；其不仅具有分泌和贮存功能，而且高表达与物质代谢密切相关的极性膜蛋白和代谢酶，显著提高了hMSCs源材料/细胞聚集体型肝类器官对经典肝毒性模型药物（异烟肼、乙酰氨基酚）的剂量敏感性。同时，将构建的类器官植入肝衰Fah-/-Rag2-/-IL-2Rγc-/-SCID（FRGS）小鼠体内显示，其显著提高了hMSCs人源白蛋白的表达并促进肝脏再生。该研究初步揭示了钙黏素融合蛋白基质仿生细胞与细胞间相互作用，综合调控细胞与细胞外基质及细胞与细胞生长因子间的相互作用，仿生构建细胞外微环境，提高hMSCs的增殖与分化能力，促进组织工程血管化及肝类器官的构建，不仅为药物肝毒性评价提供了一种可用的体外模型，对再生医学相关生物材料结构设计与功能调控研究提供理论与实践指导。