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

• Published in: JHEP reports Vol. 4; no. 4; p. 100446
• Main Authors: Li, Chao Zheng, Ogawa, Hiromi, Ng, Soon Seng, Chen, Xindi, Kishimoto, Eriko, Sakabe, Kokoro, Fukami, Aiko, Hu, Yueh-Chiang, Mayhew, Christopher N., Hellmann, Jennifer, Miethke, Alexander, Tasnova, Nahrin L., Blackford, Samuel J.I., Tang, Zu Ming, Syanda, Adam M., Ma, Liang, Xiao, Fang, Sambrotta, Melissa, Tavabie, Oliver, Soares, Filipa, Baker, Oliver, Danovi, Davide, Hayashi, Hisamitsu, Thompson, Richard J., Rashid, S. Tamir, Asai, Akihiro
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.04.2022; Elsevier
• Subjects: Bile acid transport; Cellular polarity; PFIC (progressive familial intrahepatic cholestasis); DE; BSA-FAF; FGF2; GCDCA; ASGR2; CDFDA; Cellular polarity; NTCP; iHep; MDCKII; RT-qPCR; DILI; BSEP; MRP2; PFIC (progressive familial intrahepatic cholestasis); GCA; HGF; iPSC; ATP1a1; TCDCA; BMP4; TJP1; HCM; ssODN; TCA; PFIC; sgRNA; HNF4a; ALB; Bile acid transport; PI; TEER; HE; TEM; TJP2; PFIC, progressive familial intrahepatic cholestasis; DE, definitive endoderm; HE, hepatic endodermal; MDCKII, Madin–Darby canine kidney II; HNF4a, hepatic nuclear factor 4a; iHep, iPSC-derived hepatocytes; ssODN, single-stranded oligonucleotide-DNA; TJP1, tight junction protein 1; DILI, drug-induced liver injury; CDFDA, 5-(and-6)-carboxy-2′,7′-dichlorofluorescein; BSEP, bile salt export pump; FGF2, fibroblast growth factor 2; ATP1a1, ATPases subunit alpha-1; GCA, glycocholate; TCDCA, taurochenodeoxycholate; NTCP, Na+-TCA cotransporter; iPSC, induced pluripotent stem cell; ASGR2, asialoglycoprotein receptor 2; TEER, transepithelial electrical resistance; HCM, Hepatocyte Culture Medium; PI, propidium iodide; TJP2, tight junction protein 2; MRP2, multidrug resistance-associated protein 2; TEM, transmission electron microscopy; ALB, albumin; RT-qPCR, quantitative reverse transcription PCR; TCA, taurocholic acid; BSA-FAF, bovine serum albumin fatty acid-free; GCDCA, glycochenodeoxycholate; sgRNA, single-guide RNA; BMP4, bone morphogenetic protein 4; HGF, hepatocyte growth factor
• ISSN: 2589-5559; 2589-5559
• DOI: 10.1016/j.jhepr.2022.100446

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. [Display omitted] •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.