Fig. 2: ARPKD organoids develop characteristic features of ARPKD liver disease. | Nature Communications

Fig. 2: ARPKD organoids develop characteristic features of ARPKD liver disease.

From: A human multi-lineage hepatic organoid model for liver fibrosis

Fig. 2

a Left: A diagram shows how a piggyBac transposon and CRISPR/Cas9 were used to achieve seamless genome editing of iPSC lines to introduce a homozygous ARPKD mutation (PKHD136Met). Right: iPSCs from 3 normal donors were modified to produce isogenic ARPKD iPSC lines, which enable comparisons to be made between isogenic control and mutant lines as well as between the lines generated from different donors. b, d HOs prepared from control (C1–3) or corresponding ARPKD (A1–3) iPSCs were stained with H&E (b) or Trichrome (d). The trichrome stain shows the marked increase in collagen-rich connective tissue (blue regions, indicated by arrows) that appeared throughout all ARPKD HOs. Control organoids had a thin layer of connective tissue (indicated by arrowheads) located at the organoid periphery. The dotted circles surround bile ducts. Scale bars are 100 μm. c, e The total area fraction within control and ARPKD HOs occupied by bile ducts (H&E stain, n = 20 per group, C1 vs A1 p = 1.2 × 10−13, C2 vs A2 p = 4.5 × 10−13, C3 vs A3 p = 1.5 × 10−14) or collagen (trichrome, n = 20 per group, C1 vs A1 p = 2.2 × 10−13, C2 vs A2 p = 3.3 × 10−12, C3 vs A3 p = 7.2 × 10−14). f, g HOs were immunostained with COL1A, CK8, CK19, HNF4A, and Albumin (ALB) antibodies. A marked increase in collagen is seen in ARPKD organoids. CK8 counterstaining indicates the marked increase in the size and number of ductal structures within the ARPKD organoids. In (g), the ducts in control organoids have a simple columnar CK19+ epithelium (indicated by arrows), while the ductal epithelium in ARPKD organoids is thickened (arrowheads) and abnormal. Scale bars are 50 μm. h HOs were immunostained with antibodies to ZO-1 and HNF4A, EZRIN and CK19. While control organoids have a localized pattern of ZO-1 and EZRIN expression that surrounds the apical side of the duct lumen; expression in ARPKD organoids is diffuse and is not oriented around the ducts. i HOs were immunostained with antibodies to VANGL1 and CK19. VANGL1 is highly expressed in CK19+ cells in control HOs, but its expression is decreased, and it is expressed more diffusely in the cytoplasm of cells in ARPKD organoids. j The level of expression of multiple mRNAs associated with PCP (FZD6, CELSR1, VANGL2, PRICKLE2, DVL2) or primary cilium (ARL13B, PKD2, PKD1, PKDH1) are decreased in ARPKD organoids relative to their isogenic control organoids, while the percentages of cells expressing each of these mRNAs are not altered. k Top: Transverse views of ARL13B:GFP fusion protein expression were constructed from 10 stacked images obtained from each control and ARPKD organoid. Bottom: Representative images show the cilia structure within individual cells in control and ARPKD HOs. Scale bar is 5 μm. l A graph showing the percentage of cells with primary cilium in control (C1, C2, C3) and ARPKD organoids (A1, A2, A3). These box plots show measurements made on >100 stacked images (n = 3 per group, C1 vs A1 p = 0.0039, C2 vs A2 p = 0.016, C3 vs A3 p = 0.001). Box plots in c, e, and l (center line, median; box limits, upper and lower quartiles; whiskers, 1.5 × interquartile range). Statistical differences between the groups were determined by unpaired two-tailed t-test. *p < 0.05; **p < 0.01; and ***p < 0.001.

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