肝脏类器官的自律性形成和具有人源化免疫系统的人肝嵌合大鼠的构建

Induced pluripotential stem cells (iPSCs) have been proved to be a great therapeutic promise in regenerative and translational medicine. However, due to the limited approaches of in vitro differentiation and the insufficient knowledge in differentiating and developing of embryonic stem cell, tissue and organ, the physiological function of either induced or reprogrammed hepatic cells is so weak and could not fully replace human adult hepatocytes to meet current needs. Moreover, the limited resources of hepatocytes also hind the transplantation of clinical therapies. Recently, mimic the physiological conditions ex vivo to generate functional organoids has been provided a new strategy to handle the problem. In this study, we propose to isolate human amniotic stem cells, human vascular endothelial progenitor cells, mesenchymal stem cells and hematopoietic stem cells from human placenta tissue, combined with endoderm cells derived from iPSCs; to apply a 3D micro-pattern cultural system to simulate a microenvironment, early cell lineages, multiple cellular community and their interaction and so on during liver developing of embryogenesis; and finally, to generate and optimize the vascularized liver organoids. The constructed organoids were transplanted into triplicate-gene knockout, sever immune-deficient FRG rat through portal vein, then the humanized liver is established and confirmed followed by the pre-clinical lethal rescue experiment. Transplant of human umbilical cord blood hematopoietic stem cells would totally reconstruct and replace the original hematopoietic system of rat after destroyed recipient hematogenesis function after radiation treatment, to establish a humanized immune system in rats. It should promote and remodel the organ development, disease process, drug screening and cell transplantation, and be a useful tool to study the interactive effects among liver, blood as well as immune system.

iPS细胞研究在再生医学与干细胞移植医疗中颇具潜力和发展前景。但受制于体外分化诱导技术和对胚胎干细胞分化及组织器官发生发育的认识局限，诱导分化之肝细胞其功能有限，直接重编程之肝细胞亦无法替代成熟肝细胞，且适用于移植治疗用肝细胞匮乏。肝脏类器官的构建则提供了新思维。本研究将纯化的胎盘组织由来羊膜干细胞，间充质干细胞，内皮祖细胞，和造血干细胞等体性干细胞，结合iPSC由来的肝性内胚层细胞，利用三维微孔培养系统，模拟胚胎发生期的多细胞构成与序列协同作用，构建并优化具血管网的肝性类器官。生成的类器官经肝门静脉移植至基因剔除的严重免疫缺陷型致死性肝损伤FRG大鼠体内，进行临床前挽救实验并生成新型人肝嵌合动物。人脐带造血干细胞的移植，则可置换骨髓放射性损伤的大鼠血液，生成具有人源免疫系统的嵌合大鼠，为再生移植医疗，药物研发和进行人源性肝脏免疫，血液与肝脏协同作用等研究提供亟需的重要模型。

当今创新医药研发中利用具人源化脏器的动物模型已是大势所趋。相对于小鼠10倍体液的大鼠，展现出重要魅力。由于人体与动物体内药物作用机理和代谢产物差异较大，因而为依托动物模型评估药物安全性和有效性带来了障碍。构建具人源化肝脏的动物模型必将成为药物研发和忠实模拟人类肝病的必然选择。.从前期研究开始经6年多的不懈努力，取得了本项目的决定性成果。.利用CRISPR/Cas9基因编辑技术构建了重症免疫缺陷兼肝损伤的FRG大鼠模型；开发了可控程序化肝损伤与肝再生模式，确保长期存活率下的慢性肝损伤维持，实现供体人肝细胞移植后在大鼠肝脏内可持续性增殖；建立了完整人肝细胞经肝门静脉的原位高效移植体系和稳定可操作的实施方案。所构建的超30%肝脏人源化率的大鼠，除表达人类特异性代谢功能相关基因外，大鼠血液中的人源白蛋白分泌水平达1.5mg/ml。同时，抗病毒药物齐多夫定（zidovudine）在人源化大鼠体内具有明确的人类药物代谢特征。.本项目还利用人诱导多能性干细胞（hiPSC）获得肝细胞，与羊膜干细胞、脐静脉内皮祖细胞和间充质干细胞经三维系统自律构建人肝性类器官。该类器官中肝细胞特征基因和蛋白表达水平显著高于传统二维体系中的肝样细胞；与此同时，细胞色素酶CYP3A4活性在肝性类器官中也显著加强和提高。本项目研究结果表明，肝性类器官的体外功能已极其趋近人原代肝细胞，将该hiPSC肝类器官植入肝损模型可显著改善肝功能，可缓解急性肝衰竭。.本研究着眼于构建兼具人源化肝脏大鼠模型，使药物分析变得更加准确，模拟人类疾病也会更加接近真实人体状态。本模型的使用也使实时解析基因、转录因子、细胞因子和蛋白等疾病特征成为可能。有利于创新药物研发过程中发现新的药物作用靶点，推动其他多领域边际学科研究之快速发展。随着模型移植置换率的提高，人源化周期的缩短，其在药物研发，产业化应用和临床研究中的价值无疑将会带来新变革新浪潮。