在组织工程和再生医学领域，体外构建精确模拟人体肝脏结构和功能的肝脏类器官一直是研究的热点和挑战。肝脏能够高效执行五百多种代谢功能，这与其独特的解剖结构密切相关。肝脏结构的最小单元是呈六边形的肝小叶，六边形顶角的门静脉、胆管与肝动脉的血流汇合，经过肝血窦后在六边形中心的中央静脉汇合流出肝脏。肝细胞和肝血窦内皮细胞等沿着门-中央小叶轴线坐标的变化，展现出基因表型和代谢功能在空间分布上的区域特异性现象，被称为肝脏分区（liver zonation）。一般肝小叶可以分为中央静脉区（peri-central zone，PC）、门脉区（peri-portal zone，PP）和它们中间的中叶区。部分肝脏疾病的发生与区域特异性代谢有关，比如非酒精性脂肪肝（non-alcoholic fatty liver disease，NAFLD）和药物过量造成的肝细胞损伤通常起始于PC区。肝脏分区对于发育和疾病研究都非常重要，但现有的类器官模型中缺乏区域特异性特征。本论文首次分化出人胚胎干细胞来源的具有PC区或PP区特征的肝窦内皮细胞，分别与胚胎干细胞来源的肝细胞共培养，构建出肝脏区域特异性类器官，即PC类器官和PP类器官。类器官的培养系统是一种新研制的低吸附微阵列孔板，可以实现肝窦内皮细胞和肝细胞可控均匀稳定自组装。肝血窦内皮细胞通过ID1/WNT2/β-catenin信号通路调控肝细胞出现区域特异性的表型，PC类器官在谷氨酰胺合成、脂肪合成、CYP450酶活性以及异物质代谢等方面活性高于PP类器官，而PP类器官在蛋白质分泌和尿素生成方面更强。胰高血糖素样肽-1受体（glucagon-like peptide-1 receptor, GLP-1R）激动剂在NAFLD临床试验中显示出显著的治疗效果，但其具体治疗机制尚不明确。基于不同区域特异性类器官在脂质代谢方面表现出的差异，本研究诱导出NAFLD类器官，并进一步探讨GLP-1R激动剂的作用机制。研究结果表明，GLP-1R激动剂主要通过靶向肝窦内皮细胞上表达的GLP-1R，而非肝细胞，从而有效缓解NAFLD的病理症状。综上所述，本研究首次实现了区域特异性肝脏类器官的构建。类器官的构建模式和GLP-1R激动剂在NAFLD类器官中的作用机制双重证明肝血窦内皮细胞在调控肝脏微环境中的关键作用，为非实质细胞特别是内皮细胞在体外复现器官微环境异质性和药物发现等方面的重要性提供了证据。
In the fields of tissue engineering and regenerative medicine, constructing liver organoids that precisely mimic the structure and function of the human liver remains a focal point and challenge. The unique architecture of liver, comprised of tens of thousands of basic units is called hepatic lobules, enables it to perform numerous vital physiological functions which are essential for maintaining body homeostasis. The lobule can be divided into periportal and pericentral zones, with mid-lobule zone lies in between, exhibiting spatial diversity known as zonation, characterized by variations in transcriptome profiles and functions among hepatocytes and non-parenchymal cells. Such spatially heterogeneic distribution of biological activities guarantees the general liver function and enables flexible response to changes. Many liver diseases also showed zonal pathologies. For example, liver parenchymal damage resulted from drug overdose and non-alcoholic fatty liver disease （NAFLD） usually initiate from pericentral regions. Liver zonation, which is essential for diverse physiological functions, is lacking in existing organoid models, hindering their ability to recapitulate liver development and pathogenesis. To address this gap, we explored the feasibility of achieving zonated organoid by co-culturing human embryonic stem cells （hESCs） derived hepatocytes with hESCs derived liver sinusoidal endothelial cells （LSECs） exhibiting characteristics of either the pericentral （PC） or periportal （PP） regions of liver lobules. A novel three-dimensional organoid culture system that facilitates the uniform aggregation of LSECs and hepatocytes was constructed by utilizing a propylene glycol methyl ether acetate （PEGMEA） grafted polydimethylsiloxane （PDMS） microwell chip. Zonated LSECs subtly regulate hepatocyte zonation through the ID1/WNT2/β-catenin signaling pathway, resulting in noticeable metabolic function changes. PC organoids demonstrated higher activities in glutamine synthesis, lipid synthesis, CYP450 enzyme activity, and xenobiotic metabolism compared to PP organoids, which excelled in protein secretion and urea production, closely mirroring the in vivo characteristics of hepatocyte zonation. Considering the variations of lipid metabolism in PC and PP organoids, we constructed biomimetic zonated NAFLD organoids and revealed that glucagon-like peptide-1 receptor （GLP-1R） agonist target LSECs, but not hepatocytes, indicating potential therapeutic mechanisms of GLP-1R agonist in NAFLD alleviation. In summary, this organoid is the first reported model to achieve liver zonation in vitro and this study highlights the crucial role of non-parenchymal cells in organoids for recapitulating niche heterogeneity, offering insights for drug discovery and in vitro modeling of organ heterogeneity.
