Controlled Functional Zonation of Hepatocytes In Vitro by Engineering of Wnt Signaling.,ACS Synthetic Biology

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Controlled Functional Zonation of Hepatocytes In Vitro by Engineering of Wnt Signaling.
ACS Synthetic Biology ( IF 3.7 ) Pub Date : 2020-06-18 , DOI: 10.1021/acssynbio.9b00435
Tom Wahlicht ₁ , Gabrielle Vièyres ₂ , Svenja A Bruns _{3,

4} , Nadja Meumann ₅ , Hildegard Büning _{5,

6} , Hansjörg Hauser ₇ , Ingo Schmitz _{3,

4} , Thomas Pietschmann ₂ , Dagmar Wirth _{1,

8}

Affiliation

Key liver functions, including protein synthesis, carbohydrate metabolism, and detoxification, are performed by specific populations of hepatocytes that are defined by their relative positions within the liver lobules. On a molecular level, the functional heterogeneity with periportal and pericentral phenotypes, so-called metabolic liver zonation, is mainly established by a gradient of canonical Wnt signaling activity. Since the relevant physiological cues are missing in in vitro liver models, they fail to reflect the functional heterogeneity and thus lack many liver functions. We synthetically re-engineered Wnt signaling in murine and human hepatocytes using a doxycycline-dependent cassette for externally controlled digital expression of stabilized β-catenin. Thereby, we achieved adjustable mosaic-like activation of Wnt signaling in in vitro-cultured hepatocytes that was resistant to negative-feedback loops. This allowed the establishment of long-term-stable periportal-like and pericentral-like phenotypes that mimic the heterogeneity observed in vivo. The in vitro-zonated hepatocytes show differential expression of drug-metabolizing enzymes and associated differential toxicity and higher levels of autophagy. Furthermore, recombinant adeno-associated virus and hepatitis C virus preferentially transduce the pericentral-like zonation phenotype, suggesting a bias of these viruses that has been unappreciated to date. These tightly controlled in vivo-like systems will be important for studies evaluating aspects of liver zonation and for the assessment of drug toxicity for mouse and man.

中文翻译：

Wnt信号工程技术可控制的肝细胞体外功能区划。

关键的肝功能，包括蛋白质合成，碳水化合物的代谢和排毒，是由特定的肝细胞种群来完成的，这些特定的肝细胞群是由它们在肝小叶内的相对位置决定的。在分子水平上，具有门静脉周围和周围中心表型的功能异质性，即所谓的代谢性肝区带化，主要是通过规范的Wnt信号传导活性梯度来建立的。由于体外缺少相关的生理学线索肝模型，它们无法反映功能异质性，因此缺乏许多肝功能。我们使用强力霉素依赖性盒对稳定的β-catenin进行外部控制的数字表达，在鼠和人肝细胞中合成了Wnt信号，并对其进行了重新设计。因此，我们在体外培养的肝细胞中实现了对负反馈环具有抗性的Wnt信号的可调镶嵌样激活。这允许建立长期稳定的门静脉样和中心周样表型，以模仿体内观察到的异质性。在体外分区肝细胞显示药物代谢酶的差异表达以及相关的差异毒性和更高的自噬水平。此外，重组腺相关病毒和丙型肝炎病毒优先转导中央周围样带状表型，表明这些病毒迄今尚未发现偏见。这些严格控制的类体内系统对于评估肝区带的研究以及对小鼠和人类的药物毒性评估都将是重要的。

更新日期：2020-07-17

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