PKHD1-deficient human induced pluripotent stem (iPS) cell-derived cholangiocyte-like cells (PKHD1-KO CCs) vs heterozygously mutated PKHD1 human iPS cell-derived cholangiocyte-like cells (PKHD1-Hetero CCs)

Transcriptional profiling of PKHD1-KO and PKHD1-Hetero CCs. PKHD1-deficient (PKHD1-KO) and heterozygously mutated PKHD1 iPS clones were established by RNA-guided genome editing using CRISPR/Cas9 system. The iPS clones were differentiated into cholangiocyte-like cells in cysts (cholangiocytic cysts, CCs) in a 3D-culture system. Two-condition experiment, PKHD1-KO and PKHD1-Hetero CCs. PKHD1-deficient (PKHD1-KO) and heterozygously mutated PKHD1 (PKHD1-Hetero) iPS clones were established by RNA-guided genome editing using CRISPR/Cas9 system. PKHD1-Hetero iPS clones obtained using same gRNA as PKHD1-KO iPS clones were used as control cell lines in this study to exclude the possibility that an off-target mutation by the genome editing method affected the phenotype of such clones. Targeted homologous recombination of the gene cassette into TkDA3-4 iPS cells (established from dermal fibroblasts of a normal healthy human) was performed by electroporation. The genotype of the clones was determined by PCR. Clones carrying the mutant fragment were selected and the DNA sequences of the wild type fragment were determined by direct sequencing. Clones with a premature stop codon in PKHD1 exon 2 were selected as the PKHD1-KO cell line and the loss of PKHD1 protein in the KO lines was confirmed by immunostaining. The iPS clones were differentiated into cholangiocyte-like cells in cysts (cholangiocytic cysts, CCs) in a 3D-culture system for 10 days, and the cells were harvested.