Abstract

Background: Progressive familial intrahepatic cholestasis (PFIC) are a group of recessive disorders linked by the inability to appropriately excrete bile salts (BS) from hepatocytes. PFIC1 results from mutations in ATP8B1 encoding FIC1, an apical membrane aminophospholipid translocase. FIC1 dysfunction is hypothesized to lead to BS retention via decreased farnesoid X receptor (FXR) activation. Unfortunately, a lack of reliable disease models has impeded scientific discovery and clinical advancement. We aim to explore how human induced pluripotent stem cells (hiPSCs) may be used to generate patient-specific liver tissue to model PFIC.

Methods: Fibroblasts of 3 PFIC1 patients were reprogrammed into hiPSCs via nucleofection. hiPSC stemness was determined by qPCR and staining for pluripotency markers. Human induced hepatocytes (hiHeps) were then generated, carrying ATP8B1 mutations. Hepatic differentiation was validated by comparison to primary human hepatocytes (hAH). Staining and Western Blot assessed FIC1 expression and localization. BA assays measured concentrations and investigated BA export. A luciferase assay enabled assessment of FXR activity, and a microarray was run to identify pathway specific alterations in gene expression.

Results: We successfully generated PFIC1-hiPSCs, their stemness status comparable with commercially available hiPSC (OCT3/4 and Nanog positive nucleus > 95%) and differentiate these cells into hepatocyte-like cells. All generated hiHeps showed the expression of adult isoform of the HNF4a (>90%positive nucleus) and albumin (>70% positive cells) and no expression of the alpha fetoprotein. Additional mRNA levels of hepatocyte specific markers were comparable with hAH. Functionally, patient specific PFIC1 hiHeps exported less BA than healthy hiHeps and hAH hepatocytes (basal FXR activity: hiHeps control: 0.96±0.06, PFIC1 hiHeps #1: 0.09±0.05 and hAH: 1.37±0.15). The luciferase assay demonstrated a reduction in FXR. Microarray showed increases in expression of ER stress related genes in the PFIC1 hiHeps, suggesting that the cells are compensating for the mutant FIC1 protein.

Conclusion: PFIC1-specific hiHeps can recapitulate the dysfunctional activity of FIC1 and support the hypothesis that dysregulated FXR contributes to the PFIC1 phenotype. These findings represent a novel model for further interrogation to unlock a deeper understanding of PFIC1 pathophysiology and possible therapeutic targets.