Human iPSC-derived hepatocyte system models cholestasis with tight junction protein 2 deficiency

Chao Zheng Li,Oliver Tavabie,Hiromi Ogawa,Christopher N Mayhew,Zu Ming Tang,Melissa Sambrotta,Aiko Fukami,Richard J Thompson,Alexander Miethke,Kokoro Sakabe,S Tamir Rashid,Akihiro Asai,Liang Ma,Soon Seng Ng,Hisamitsu Hayashi,Fang Xiao,Samuel J.I Blackford,Jennifer Hellmann,Eriko Kishimoto,Xindi Chen,Davide Danovi,Filipa Soares,Adam M Syanda,Nahrin L Tasnova,O K Baker ,Yueh-Chiang Hu

doi:10.1016/j.jhepr.2022.100446

Abstract

Background & AimsThe 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.MethodsWe 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.ResultsThe 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.ConclusionsOur 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.Lay summaryWe 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.

Highlights

We and others have identified the protein-truncating mutations in the tight junction protein 2 gene (TJP2) to cause progressive familial intrahepatic cholestasis (PFIC)[1][2]
Modeling the bile canalicular function of human hepatocytes has been difficult because transporters on the canalicular membrane and the bile acid profile in human hepatocytes
Human induced pluripotent stem cells (iPSCs) technology provides an alternative path to build a novel model for cholestatic diseases

Summary

Introduction

We and others have identified the protein-truncating mutations in the tight junction protein 2 gene (TJP2) to cause progressive familial intrahepatic cholestasis (PFIC)[1][2]. A clinically relevant disease model that recapitulates the pathophysiology of PFIC with TJP2 deficiency is crucial for the development of effective therapies. The truncating mutations in tight junction protein 2 (TJP2) cause progressive cholestasis, liver failure, and hepatocyte carcinogenesis. We leveraged the technologies of patient-specific induced pluripotent stem cells (iPSCs) and CRISPR genome-editing, and we aim to establish a disease model which recapitulates phenotypes of patients with TJP2 deficiency. Journal Pre-proof and Matrigel sandwich culture systems, the hepatocyte-like cells differentiated from iPSCs with TJP2 mutations exhibited intracellular inclusions of disrupted apical membrane structures, distorted canalicular networks, altered distribution of apical and basolateral markers/transporters. Conclusions: 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

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