Generation of a bile salt export pump deficiency model using patient-specific induced pluripotent stem cell-derived hepatocyte-like cells

Bile salt export pump (BSEP) plays an important role in hepatic secretion of bile acids and its deficiency results in severe cholestasis and liver failure. Mutation of the ABCB11 gene encoding BSEP induces BSEP deficiency and progressive familial intrahepatic cholestasis type 2 (PFIC2). Because liver transplantation remains standard treatment for PFIC2, the development of a novel therapeutic option is desired. However, a well reproducible model, which is essential for the new drug development for PFIC2, has not been established. Therefore, we attempted to establish a PFIC2 model by using iPSC technology. Human iPSCs were generated from patients with BSEP-deficiency (BD-iPSC), and were differentiated into hepatocyte-like cells (HLCs). In the BD-iPSC derived HLCs (BD-HLCs), BSEP was not expressed on the cell surface and the biliary excretion capacity was significantly impaired. We also identified a novel mutation in the 5′-untranslated region of the ABCB11 gene that led to aberrant RNA splicing in BD-HLCs. Furthermore, to evaluate the drug efficacy, BD-HLCs were treated with 4-phenylbutyrate (4PBA). The membrane BSEP expression level and the biliary excretion capacity in BD-HLCs were rescued by 4PBA treatment. In summary, we succeeded in establishing a PFIC2 model, which may be useful for its pathophysiological analysis and drug development.


RNA isolation and reverse transcription-polymerase chain reaction (RT-PCR)
Total RNA was extracted from human ESC/iPSCs and their derivatives using ISOGEN (NIPPON GENE). The complemented DNA (cDNA) was synthesized using 500 ng of total RNA with a Superscript VILO cDNA synthesis kit (Thermo fisher scientific). Real-time RT-PCR was performed with SYBR Green PCR Master Mix (Applied Biosystems) using a StepOnePlus real-time PCR system (Applied Biosystems). Relative quantification was performed against a standard curve and the values were normalized against the input determined for the housekeeping gene, glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The PCR primer sequences used in this study are described in Table S1. The ABCB11 cDNA synthesized from the RNA of HLCs was amplified by RT-PCR using Ex Taq DNA polymerase. The primers 1F and 263R are listed in Table S2. The expected size of the PCR products was 263 bp for ABCB11 Exon1-2-3-4. The PCR products were separated by electrophoresis on 2% agarose gels and visualized by staining with ethidium bromide ( Figure 3C).

Immunostaining
The cultured cells were fixed with 4% paraformaldehyde (PFA) in PBS for 20 min.
After the incubation with 0.1% Triton X-100 (Wako) in PBS for 10 min, the cells were blocked with PBS containing 10% FBS and 1% bovine serum albumin for 1 hr, and incubated with a primary antibody at 4°C overnight. Finally, the cells were incubated with a secondary antibody at room temperature for 1 hr. The primary and secondary antibodies used in this study are described in Table S4, respectively. The paraffin section was obtained from the liver and fixed with formalin. The liver section was stained for BSEP and MRP2 by using DAB immunohistochemistry.

Transmission electron microscopy
The samples were fixed with 2% paraformaldehyde and 2% glutaraldehyde (GA) in 100 mM phosphate buffer (PB) pH 7.4 at incubation temperature, and then they were placed in a refrigerator for 30 min in order to lower the temperature to 4°C. Thereafter, they were fixed with 2% GA in 100 mM PB at 4°C overnight. After three washes with 100 mM PB, the samples were postfixed with 2% osmium tetroxide in 100 mM PB at 4°C for 1 hr. They were then dehydrated in graded ethanol solutions (50%, 70%, 90%, 100%). The samples were transferred to a resin (Quetol-812; Nisshin EM Co.), and were polymerized at 60°C for 48 hr. Ultrathin sections were stained with 2% uranyl 3 acetate at room temperature for 15 min, then washed in distilled water and secondarily stained with Lead stain solution (Sigma-Aldrich Co.) at room temperature for 3 min.
The grids were observed by a transmission electron microscope (JEM-1400plus; JEOL, Ltd.) at 80 kV. Images were taken with a CCD camera (VELETA; Olympus Soft Imaging Solutions GmbH).

Enzyme-linked immuno-sorbent assay (ELISA)
The culture supernatants, which were incubated for 24 hr after fresh medium was added, were collected and analyzed for the amount of albumin (ALB) secretion by ELISA. Human Albumin ELISA Quantitation Set was purchased from Bethyl Laboratories. ELISA was performed according to the manufacturer's instructions. The amount of ALB secretion was calculated according to each standard followed by normalization to the protein content per well.

Biliary excretion and bile canaliculi formation assay
On day 25 of the hepatocyte differentiation, the Control-HLCs were treated with 5 µM cholyl-lysyl-fluorescein (CLF) (Corning) for 30 min. The cells were then washed with HBSS buffer, and observed with a fluorescence microscope ( Figure S2).

Embryoid body formation and spontaneous differentiation in vitro
To form the embryoid bodies (EBs), human ESCs/iPSCs were cultured with DMEM (Thermo fisher scientific) supplemented with 10% FBS, 120 µg/mL streptomycin, and 200 µg/mL penicillin on low-attachment 96-wel plates for 4 days. The EBs were then plated onto gelatin-coated dishes, and cultured with the same culture medium for 10 days. The culture medium was changed every other day.

Treatment with 4-phenylbutyrate
The HLCs were treated with 1 mM 4-phenylbutyrate (4PBA; Sigma) for 24 hours.    The score less than 0.05 indicates that the amino acid substitution is predicted as damaging.