Department of Cell and Molecular Biology, Karolinska Institutet, Stockholm, Sweden.
Liver Int. 2024 Feb;44(2):541-558. doi: 10.1111/liv.15789. Epub 2023 Nov 28.
BACKGROUND & AIMS: Alagille syndrome (ALGS) manifests with peripheral intrahepatic bile duct (IHBD) paucity, which can spontaneously resolve. In a model for ALGS, Jag1 mice, this occurs with distinct architectural mechanisms in hilar and peripheral IHBDs. Here, we investigated region-specific IHBD characteristics and addressed whether IGF1, a cholangiocyte mitogen that is downregulated in ALGS and in Jag1 mice, can improve biliary outcomes.
Intrahepatic cholangiocyte organoids (ICOs) were derived from hilar and peripheral adult Jag1 and Jag1 livers (hICOs and pICOs, respectively). ICOs were grown in Matrigel or microwell arrays, and characterized using bulk RNA sequencing, immunofluorescence, and high throughput analyses of nuclear sizes. ICOs were treated with IGF1, followed by analyses of growth, proliferation, and death. CellProfiler and Python scripts were custom written for image analyses. Key results were validated in vivo by immunostaining.
Cell growth assays and transcriptomics demonstrated that Jag1 ICOs were less proliferative than Jag1 ICOs. IGF1 specifically rescued survival and growth of Jag1 pICOs. Jag1 hICOs were the least proliferative, with lower Notch signalling and an enrichment of hepatocyte signatures and IGF uptake/transport pathways. In vitro (Jag1 hICOs) and in vivo (Jag1 hilar portal tracts) analyses revealed ectopic HNF4a hepatocytes.
Hilar and peripheral Jag1 ICOs exhibit differences in Notch signalling status, proliferation, and cholangiocyte commitment which may result in cholangiocyte-to-hepatocyte transdifferentiation. While Jag1 pICOs can be rescued by IGF1, hICOs are unresponsive, perhaps due to their hepatocyte-like state and/or expression of IGF transport components. IGF1 represents a potential therapeutic for peripheral bile ducts.
Alagille 综合征（ALGS）表现为肝内外周胆管（IHBD）稀少，可自发缓解。在 ALGS 的 Jag1 小鼠模型中，肝门部和外周部 IHBD 存在明显的结构机制。在这里，我们研究了区域特异性 IHBD 特征，并探讨了胰岛素样生长因子 1（IGF1）是否可以改善胆管结局，IGF1 是 ALGS 和 Jag1 小鼠中下调的胆管细胞有丝分裂原。
从 Jag1 和 Jag1 肝脏的肝门部和外周部（hICOs 和 pICOs）中分离出肝内胆管细胞类器官（ICOs）。ICOs 在 Matrigel 或微井阵列中生长，并通过批量 RNA 测序、免疫荧光和核大小高通量分析进行表征。IGF1 处理后，分析生长、增殖和死亡。使用 CellProfiler 和 Python 脚本编写了自定义图像分析脚本。关键结果通过免疫染色在体内进行了验证。
细胞生长测定和转录组学表明，Jag1 ICOs 的增殖能力低于 Jag1 ICOs。IGF1 特异性挽救了 Jag1 pICOs 的存活和生长。Jag1 hICOs 的增殖能力最低，Notch 信号通路较低，富含肝细胞特征和 IGF 摄取/转运途径。体外（Jag1 hICOs）和体内（Jag1 肝门部门脉区）分析显示异位 HNF4a 肝细胞。
Jag1 hICOs 和 pICOs 在 Notch 信号状态、增殖和胆管细胞分化方面存在差异，这可能导致胆管细胞向肝细胞转化。虽然 Jag1 pICOs 可以被 IGF1 挽救，但 hICOs 无反应，这可能是由于其肝细胞样状态和/或 IGF 转运成分的表达。IGF1 可能是外周胆管的潜在治疗药物。
