Patient-derived liver organoids recapitulate epithelial heterogeneity and enable precision disease modelling of alcohol-associated liver disease

Organoids are emerging as a powerful human-based in vitro tool in the biomedical field. However, patient-derived liver organoids fail to recapitulate the liver epithelial heterogeneity and its generation still requires liver surgical resections, thus limiting personalized chronic liver diseases modeling. Here, we report the derivation of organoids from intact liver needle biopsies(b-Orgs) from alcohol-associated liver disease (ALD) patients for precision disease modeling and drug testing. B-Orgs were generated with an efficiency of 80% from patients with early and advanced stages of ALD. b-Orgs show an enriched hepatocyte phenotype as assessed by immunofluorescence, functional studies, and transcriptomics. Single cell RNA-sequencing revealed a heterogeneous epithelial composition comprising hepatocyte, biliary and progenitor hepatobiliary populations, mirroring the epithelial populations found in advanced ALD patients. Moreover, b-Orgs preserve disease-stage features, as b-Orgs from advanced ALD patients showed increased expression of genes related to epithelial-mesenchymal transition, angiogenesis and inflammation. Stimulation of b-Orgs with ethanol and pro-inflammatory mediators,promoted ALD features such asROS production, lipid accumulation, inflammation and decreased proliferation, which were mitigated in response to prednisolone. Overall, we provide a new methodology to obtain b-Orgs showing epithelial complexity and patient specific features, thus expanding organoid-based liver disease modelling for personalized medicine. Overall design: single-cell RNA sequencing 3' (scRNA-seq)

类器官（Organoids）正逐渐成为生物医学领域中极具应用潜力的人类源性体外实验工具。然而，患者来源的肝脏类器官无法复现肝脏上皮异质性，且其构建仍需依赖肝脏手术切除样本，极大限制了慢性肝病个性化建模的发展。本研究报道了一种从酒精相关性肝病（Alcohol-associated liver disease, ALD）患者的完整肝脏穿刺活检组织中构建类器官（b-Orgs）的方法，可用于精准疾病建模与药物筛选。针对早期与晚期ALD患者，b-Orgs的构建效率可达80%。通过免疫荧光实验、功能学研究与转录组学分析证实，b-Orgs具备富集的肝细胞表型。单细胞RNA测序（single cell RNA-sequencing, scRNA-seq）结果显示，b-Orgs的上皮细胞组成具有异质性，涵盖肝细胞、胆管细胞与肝胆祖细胞亚群，其特征与晚期ALD患者体内的上皮细胞群一致。此外，b-Orgs可保留疾病分期相关特征：晚期ALD患者来源的b-Orgs中，上皮间质转化、血管生成与炎症相关基因的表达水平显著上调。用乙醇与促炎介质刺激b-Orgs，可诱导其出现ALD相关病理特征，包括活性氧（Reactive Oxygen Species, ROS）生成增多、脂质蓄积、炎症反应以及增殖能力降低；而泼尼松龙（prednisolone）干预可缓解上述病理变化。综上，本研究建立了一种全新的b-Orgs构建方法，所获类器官兼具上皮细胞复杂性与患者特异性特征，可为个性化医疗领域的肝病类器官建模研究提供新的拓展途径。实验整体设计：3'端单细胞RNA测序（single-cell RNA sequencing 3', scRNA-seq）