Although the first progressive familial intrahepatic cholestasis (PFIC) secondary to the TJP2 mutation patient was reported in early 2014, an effective in vitro model for this disease is still lacking. During my PhD, I generated a human-induced Pluripotent Stem Cell (iPSC)-derived hepatocyte (iHEPs)-based high-throughput functional assay, which mimicked the disease phenotype in patients, and hence, it could be used for drug screening and mechanistic studies. Both PFIC patients with tight junction protein 2 (TJP2) mutation fibroblast-reprogrammed iPSCs and clustered regularly interspaced short palindromic repeats-CRISPR-associated protein (CRISPR-Cas9)-engineered iPSC isogenic control clones with TJP2 knock out (KO) were successfully generated. Additionally, an ATP Binding Cassette Subfamily B Member 4 (ABCB4) homozygous knock-out iPSC clone was generated by using CRISPR-Cas9, but it needs to be characterised further. Both disease iPSC lines were successfully differentiated to hepatocyte-like cells. In addition, functional bile salt export pump (BSEP)/Multidrug-Resistant protein 2 (MRP2) substrates 5-(and-6)-carboxy-2’,7’ – dichlorofluorescein diacetate (CDFDA) – transportation and chicken-wire-like canaliculi structures were observed in healthy iHEP cultured in a Matrigel sandwich. Interestingly, both disease iHEPs demonstrated abnormal CDFDA transportation and an irregular canaliculi shape, which partially resembled the patients’ liver phenotypes. Furthermore, as genome-wide association study (GWAS) data of drug-induced liver injury (DILI) patients and Encyclopedia of DNA Elements (ENCODE) project phase 3 information became available in 2020, in silico analysis was used to curate a list of DILI-related single nucleotide polymorphisms (SNPs) (excluding human leukocyte antigen (HLA)-related SNPs) located within cis-regulated elements (CREs), from which a CRISPR activator/inhibitor (CRISPRa/i) screening library was designed to investigate how those SNPs in CREs caused DILI. There were 26 novel SNPs identified in CRE which could contribute to DILI, but they were unable to be used in the CRISPR screen due to their relatively low quantity. Overall, this study suggested iHEPs can be used to model PFIC with TJP2 mutation, as it demonstrated the relevant patient liver disease phenotype. Moreover, iHEPs combined with in silico analysis and CRISPRa/i screening can be used to model complex disorders, such as DILI.
MODELLING LIVER DISEASE IN VITRO AND IN SILICO
Li, C. (Author). 1 Dec 2022
Student thesis: Doctoral Thesis › Doctor of Philosophy