Human iPSC-derived hepatocyte system models cholestasis with tight junction protein 2 deficiency.
Human iPSC-derived hepatocyte system models cholestasis with tight junction protein 2 deficiency.
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DOI：
10.1016/j.jhepr.2022.100446
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发表时间：
2022-04
期刊：
影响因子：
--
通讯作者：
Asai A
中科院分区：
文献类型：
--
作者：
Li CZ;Ogawa H;Ng SS;Chen X;Kishimoto E;Sakabe K;Fukami A;Hu YC;Mayhew CN;Hellmann J;Miethke A;Tasnova NL;Blackford SJI;Tang ZM;Syanda AM;Ma L;Xiao F;Sambrotta M;Tavabie O;Soares F;Baker O;Danovi D;Hayashi H;Thompson RJ;Rashid ST;Asai A
紧密连接蛋白2 （TJP2）的截断突变导致进行性胆汁淤积、肝功能衰竭和肝细胞癌变。由于缺乏有效的模型系统，目前还没有针对TJP2缺乏症肝脏病理的靶向药物。我们利用患者特异性诱导多能干细胞（iPSC）和CRISPR基因组编辑技术，旨在建立一个概括TJP2缺乏症患者表型的疾病模型。我们将iPSC分化为肝细胞样细胞（iHep），在Transwell膜上形成极化单层。共聚焦显微镜检测极性标记物的免疫荧光染色。测定两腔间胆小管上皮屏障功能和胆汁酸转运。采用荧光探针和活体共聚焦成像技术，在Matrigel夹层系统中培养iHep，观察其胆管形态。从TJP2突变的iPSC分化出来的iHep表现出细胞内包涵体破坏的根尖膜结构，扭曲的小管网络，根尖和基底外侧标记物/转运体的分布改变。在突变的肝细胞中，胆管的定向胆汁酸运输受到损害，类似于在患者肝脏中观察到的疾病表型。我们的ipsc衍生的体外肝细胞系统揭示了TJP2缺乏症肝细胞的管状膜破坏，并证明了TJP2缺乏症胆汁淤积性疾病模型的能力，为进一步的病理生理学研究和药物发现提供了平台。我们研究了一种遗传性肝病，进行性家族性肝内胆汁淤积症（PFIC），由于缺乏TJP2基因，导致新生儿和婴儿严重的肝脏疾病。利用尖端的干细胞技术和基因组编辑方法，我们在细胞培养实验中建立了一种新的疾病建模系统。我们的实验表明，缺乏TJP2会导致细胞极性异常和胆汁酸运输中断。这些发现将导致后续的调查，以进一步了解疾病机制和开发有效的治疗方法。紧密连接蛋白2 （TJP2）缺乏导致婴儿进行性胆汁淤积，其病理生理机制尚不清楚。从TJP2突变患者中获得诱导多能干细胞（iPSCs）。通过CRISPR基因组编辑生成了具有和不具有TJP2缺陷的人iPSCs的等基因对。TJP2缺乏的iPSCs衍生的肝细胞显示胆汁酸运输受损，重现了患者的表型。缺乏tjp2的肝细胞表现出管状膜破坏，导致细胞极性紊乱。
The truncating mutations in tight junction protein 2 (TJP2) cause progressive cholestasis, liver failure, and hepatocyte carcinogenesis. Due to the lack of effective model systems, there are no targeted medications for the liver pathology with TJP2 deficiency. We leveraged the technologies of patient-specific induced pluripotent stem cells (iPSC) and CRISPR genome-editing, and we aim to establish a disease model which recapitulates phenotypes of patients with TJP2 deficiency. We differentiated iPSC to hepatocyte-like cells (iHep) on the Transwell membrane in a polarized monolayer. Immunofluorescent staining of polarity markers was detected by a confocal microscope. The epithelial barrier function and bile acid transport of bile canaliculi were quantified between the two chambers of Transwell. The morphology of bile canaliculi was measured in iHep cultured in the Matrigel sandwich system using a fluorescent probe and live-confocal imaging. The iHep differentiated from iPSC with TJP2 mutations exhibited intracellular inclusions of disrupted apical membrane structures, distorted canalicular networks, altered distribution of apical and basolateral markers/transporters. The directional bile acid transport of bile canaliculi was compromised in the mutant hepatocytes, resembling the disease phenotypes observed in the liver of patients. Our iPSC-derived in vitro hepatocyte system revealed canalicular membrane disruption in TJP2 deficient hepatocytes and demonstrated the ability to model cholestatic disease with TJP2 deficiency to serve as a platform for further pathophysiologic study and drug discovery. We investigated a genetic liver disease, progressive familial intrahepatic cholestasis (PFIC), which causes severe liver disease in newborns and infants due to a lack of gene called TJP2. By using cutting-edge stem cell technology and genome editing methods, we established a novel disease modeling system in cell culture experiments. Our experiments demonstrated that the lack of TJP2 induced abnormal cell polarity and disrupted bile acid transport. These findings will lead to the subsequent investigation to further understand disease mechanisms and develop an effective treatment. Deficiency of tight junction protein 2 (TJP2) causes progressive cholestasis in infants, and the pathophysiology remains unclear. Induced pluripotent stem cells (iPSCs) from patients with TJP2 mutations were generated. Isogenic pairs of human iPSCs with and without TJP2 deficiency were generated by CRISPR genome editing. Hepatocytes derived from iPSCs with TJP2 deficiency showed impaired bile acid transport, recapitulating phenotypes of patients. TJP2-deficient hepatocytes exhibit canalicular membrane disruption that resulted in derangement of cellular polarity.
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DOI：
10.1124/jpet.105.084830
发表时间：
2005-08-01
影响因子：
3.5
作者：
Hirano, M;Maeda, K;Sugiyama, Y
通讯作者：
Sugiyama, Y
影响因子：
13.5
作者：
Hayashi, Hisamitsu;Inamura, Kaori;Sugiyama, Yuichi
通讯作者：
Sugiyama, Yuichi
影响因子：
5.9
作者：
Hayashi H;Osaka S;Sakabe K;Fukami A;Kishimoto E;Aihara E;Sabu Y;Mizutani A;Kusuhara H;Naritaka N;Zhang W;Huppert SS;Sakabe M;Nakamura T;Hu YC;Mayhew C;Setchell K;Takebe T;Asai A
通讯作者：
Asai A
TJP2的突变引起进行性胆汁淤积性肝病。
影响因子：
30.8
作者：
Sambrotta M;Strautnieks S;Papouli E;Rushton P;Clark BE;Parry DA;Logan CV;Newbury LJ;Kamath BM;Ling S;Grammatikopoulos T;Wagner BE;Magee JC;Sokol RJ;Mieli-Vergani G;University of Washington Center for Mendelian Genomics;Smith JD;Johnson CA;McClean P;Simpson MA;Knisely AS;Bull LN;Thompson RJ
通讯作者：
Thompson RJ
DOI：
10.1126/science.aad5227
发表时间：
2016-01-01
期刊：
Science (New York, N.Y.)
影响因子：
--
作者：
Slaymaker IM;Gao L;Zetsche B;Scott DA;Yan WX;Zhang F
通讯作者：
Zhang F