利用人诱导多能干细胞的衍生细胞在体外构建可用于移植的血管化及胆管化的功能性肝组织

Liver transplantation has been established as the most effective treatment modality for end-stage liver diseases, while its application is severely limited due to the critical shortage of donor organs. Efforts have made to construct artificial hepatic tissues in order to replace donor livers for the applications of transplantation and in vitro liver modeling. However, it remains challenging to generate a functional liver tissue that possesses the physiologically relevant cell combination and architecture, which is largely due to the limited availability of the appropriate hepatic cell types and the lack of advanced tissue engineering tools. The fact that the induced pluripotent stem cells (iPSCs) can generate various functional hepatic cells in large quantities makes them the most promising candidate for developing patient-specific human liver tissues and organs in vitro, and the advanced 3D bioprinting technologies have paved the way for assembling these cells into liver tissues that can recapitulate the structural and functional integrity of the liver. Here we propose to construct functional and transplantable artificial liver tissues with vascular and biliary networks by mimicking the native liver structure, and by integrating the advanced directed differentiation systems of iPSCs and the cutting-edge 3D bioprinting technologies. In particular, for the tissue engineering, an iPSC-derived endoderm stem cell (EP cell) line, which is more efficient than iPSCs to make the functional hepatocytes and cholangiocytes, will be used to generate the parenchymal liver cells in large quantities, and the non-parenchymal endothelial and mesenchymal cells will be derived from the same iPSC line. The advanced 3D multi-material combinatorial bioprinting system will be harnessed for the cell seeding and tissue scaffold assembling. The resultant personalized artificial liver tissues are expected to be functional and to be amenable for transplantation, and can therefore be used in drug screening, liver pathophysiology studies and ultimately clinical applications.

重症肝病是危害我国人民健康的重要杀手，造成了巨大的社会经济损失。肝移植是治疗终末期肝病的最有效手段，但供体肝脏来源匮乏严重制约其临床应用。利用组织工程技术在体外构建人工肝脏器官有望满足肝病临床治疗、体外模拟与药物毒性预测的重大需求。本项目拟利用人诱导多能干细胞（human iPSCs）及其衍生的内胚层干细胞（EP cells）制备肝脏主要细胞类型，结合包括3D生物打印在内的多种组织工程学先进技术，通过模拟肝脏生理结构，着重解决人工肝组织的血管化和胆管化难点，在体外构建可用于移植、体外疾病模拟及药物筛选的新型个体化人工肝脏，并利用动物模型评估其体内功能。

重症肝病是危害人民健康的重要杀手，目前除肝脏移植外尚缺乏真正有效的治疗手段。体外构建人工肝脏器官可有效缓解供肝匮乏的现状，满足肝病临床治疗、肝病体外模拟与药物毒性预测的重大需求。而体外构建人工肝的关键因素是实现体外人工肝的血管化与胆管化。血管化保证人工肝在体外的氧气和养分的供应。而胆管则负责将肝细胞产生的胆汁酸排出，保证肝脏存活以及正常代谢功能。我们首先创立了 “结构诱导分化”（architecture-based in vitro differentiation）的思路，通过模拟体内发育过程，实现基于血管化的胆管化肝组织组织构建。由于受限于3D打印技术在精度和生物打印材料局限等的瓶颈问题，我们改用微流控芯片及层堆叠等先进组织工程技术，技术尝试人工肝组织构建。项目实施期间，我们完成了如下工作：1）通过设计制造模具构建了体外灌流系统，并采用胶原材料制造仿生血管，得到具有高强韧性同时适宜细胞贴附、生长、迁移的胶原血管支架，为解决中小尺度人造器官移植的体内连接问题做好铺垫；2）利用组织芯片技术，将包含HUVEC和辅助细胞诱导为可灌流的微血管网络；3）利用细胞堆叠技术模拟体内致密组织3D环境，筛选同时支持血管化及肝细胞存活及功能维持的培养基；4）利用体外扩增肝细胞ProliHH、HUVEC等细胞在器官芯片上构建了微尺度血管化肝组织，并分析细胞共定位、结构及可灌流性；5）利用内胚层干细胞及肝向干细胞来源的肝母细胞或胆管前体细胞进行基于芯片的胆管化尝试，并获得了胆管网络。上述工作，利用芯片技术着重解决肝组织体外构建的血管化及胆管化两个关键问题，初步构建了具备血管网络或胆管网络的微尺度肝组织，为最终实现功能性肝组织的体外构建奠定了扎实的基础。