PFIC2 Disease Model
Progressive familial intrahepatic cholestasis (PFIC) is a heterogeneous group of autosomal recessive liver diseases, characterized by mutations in genes involved in hepatocellular bile acid secretion. Amongst these, PFIC2, caused by mutations in the ABCB11 gene, represents half of the total PFIC cases, and patients with PFIC2 present a range of symptoms, including liver failure, cirrhosis, and hepatocellular carcinoma. Despite the importance of the disease, there are currently no licensed treatments, mainly due to the lack of appropriate pre-clinical models in drug discovery. DefiniGEN have developed a novel iPSC-derived hepatocyte system that recapitulates the human PFIC2 phenotype in-a-dish, offering, for the first time, an effective pre-clinical PFIC2 disease model for hit-lead drug screening studies.
Advantages
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Functional bile acid transport pathways with high expression levels of ABCB11 and MRP2
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Disease circuit verified carrying the D482G mutation in the ABCB11 gene
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Optimized bioassays measuring bile acid transport as end-point assay in Opti-HEP
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Suitable in vitro platforms for screening of compound and gene therapy systems
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Standardized cell products containing iPSC-derived human hepatocytes producing reproducible and biologically relevant data
DefiniGEN CRISPR-derived PFIC2 iPSCs carry the D482G mutation without any effect in pluripotency status
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Figure 1: A) Sanger sequencing showing wild-type (top sequence) and mutated iPSCs (bottom sequence) carrying the D482G mutation (GAT>GTT) in the ABCB11 gene. The codon change is highlighted with yellow. B) mRNA expression levels of the key pluripotency markers NANOG, SOX2, and OCT4 in wild-type (WT) and CRISPR-derived ABCB11 iPSCs (PFIC2). mRNA data were normalized to GAPDH and are presented as mean±SEM of n=3 biological replicates.
DefiniGEN CRISPR-derived PFIC2 iPSCs successfully differentiate to Opti-HEP
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Figure 2: A) Representative images demonstrating the characteristic hepatocyte cobblestone morphology in wild-type (WT) and PFIC2 Opti-HEP. B) mRNA expression levels of the hepatocyte maturity markers albumin (ALB), alpha-1-antitrypsin (A1AT), and hepatocyte nuclear factor 4A (HNF4A) in wild-type (WT) and PFIC2 Opti-HEP. mRNA data were normalized to PPIA and are presented as mean±SEM of n=2 biological replicates.
DefiniGEN CRISPR-derived PFIC2 Opti-HEP demonstrate reduced ABCB11 mRNA and protein expression
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Figure 3: A) mRNA expression levels of ABCB11 in wild-type (WT) and PFIC2 Opti-HEP cultured in transwell culture system. B) Protein expression levels of ABCB11 in transwell-cultured WT and PFIC2 Opti-HEP in comparison to primary human hepatocytes (PHH). mRNA data were normalized to 18S rRNA, and protein data to β-actin. All data are presented as mean±SEM of n=3-4 biological replicates.
DefiniGEN CRISPR-derived PFIC2 Opti-HEP exhibit disrupted ABCB11 localization
Figure 4: Immunocytochemistry analysis of MRP2 (red) and ABCB11 (green) in wild-type (WT) and PFIC2 Opti-HEP cultured in transwell culture system. Scale bar: 50 µm.
DefiniGEN CRISPR-derived PFIC2 Opti-HEP demonstrate reduced bile acid transport
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Figure 5: (A) Schematic depiction of Opti-HEP polarization cultured in transwell culture system. (B) Taurocholic acid (TCA) quantification in the upper and lower transwell compartments of WT and PFIC2 Opti-HEP following 48 hours of TCA addition (10 μM) to the lower compartment, indicating dysfunctional ABCB11 activity in the PFIC2 cells. Data are presented as mean±SEM of n=4 biological replicates.