Human hepatic organoids for the analysis of human genetic diseases

Abstract

We developed an in vitro model system where induced pluripotent stem cells (iPSCs) differentiate into 3-dimensional human hepatic organoids (HOs) through stages that resemble human liver during its embryonic development. The HOs consist of hepatocytes, and cholangiocytes, which are organized into epithelia that surround the lumina of bile duct-like structures. The organoids provide a potentially new model for liver regenerative processes, and were used to characterize the effect of different JAG1 mutations that cause: (a) Alagille syndrome (ALGS), a genetic disorder where NOTCH signaling pathway mutations impair bile duct formation, which has substantial variability in its associated clinical features; and (b) Tetralogy of Fallot (TOF), which is the most common form of a complex congenital heart disease, and is associated with several different heritable disorders. Our results demonstrate how an iPSC-based organoid system can be used with genome editing technologies to characterize the pathogenetic effect of human genetic disease-causing mutations.

Keywords: Genetic diseases; Genetics; Hepatology.

Figure 1. Generation of HOs from iPSCs.

(A) A schematic representation of the in vitro culture system and the growth factors used to direct the differentiation of iPSCs into HO1s. Then, the cells in a HO1 are dissociated, and (B and C) bright-field and immunostaining images taken on day 0 (iPSCs), day 3 (definitive endoderm, EN), day 6 (foregut, FG), day 9 (hepatoblast, HB), day 20 (HO1), and day 62 (HO2) show the morphological and cell marker changes that occur during the development of HOs. The arrows indicate where duct-like structures are located. (D) H&E staining shows the appearance of 3 different types of HO1. (E and F) Immunostained sections prepared from day 50 parenchymal, ductal, or mixed organoids show the pattern of expression of hepatocyte (ALB, CK8, A1AT), cholangiocyte (CK19), or proliferation (Ki67) markers. Scale bars: 50 μm.

Figure 2. HOs have morphological and functional similarities to human liver.

(A) Immunostained sections prepared from day 20 and 50 HO1s show that they express ZO-1. The insert (bottom left) shows a low-power view of the field that is enlarged in the right image. (B) Immunostaining with an antibody against acetylated α-tubulin indicates that primary cilia are present in a CK19⁺ cholangiocyte adjacent to a ductal structure in the HO1. (C) Day 50 HO1s were stained with Alcian blue and periodic acid–Schiff stain for mucus and glycogen (upper), and by Hall’s method, which stains a bile component (bilirubin) green after it is oxidized to biliverdin (lower). (D) The abundance of the following bile acids were measured in the supernatants of a day 50 HO1: glycochenodeoxycholic acid (GCDCA), glycodeoxycholic acid (GDCA), glycocholate (GCA), taurochenodeoxycholate (TCDCA), taurocholate (TCA), and taurodeoxycholate (TDCA). The red dashed line indicates the amounts of the corresponding bile acids measured in the supernatants of 3 different control iPSC lines (all were below the level of quantification). Each measurement is the mean ± SD of 3 supernatants, and was normalized relative to that of iPSC supernatants. (E) The human albumin levels in supernatants obtained from day 50 HO1s and PHHs. The supernatants were collected after 24 hours, and the measurements were normalized per 10⁶ cells. (F) The amounts of 2 clemizole metabolites (M1, M6) produced by day 50 control (C1, C2) organoid cultures were measured 24 hours after incubation with clemizole (3.6 μg/ml). The amount of metabolites produced by recently plated PHH and iPSC lines was also measured. (G) Confocal images show the cells that surround luminal structures in day 50 organoids. The cells were stained with antibodies against CK7 (red) and SOX9 (green) (upper), or with antibodies against CK7 (red) and Ki67 (green) (lower), and were counterstained with DAPI (blue). (H and I) H&E staining and bright-field images show HO2s on day 6 in growth medium (GM) regenerated from dissociated HO1s (lower and higher magnification) (H), or HO2s on day 12 in differentiation medium (DM) (lower and higher magnification) (I). The lower panel is enlarged to show a single HO2. (J) Fluorescence and bright-field merged images show that there is abundant bile acid accumulation in the lumen of HO2s after differentiation. (K and L) Confocal images examining the expression of liver lineage markers by HO2s in GM or DM. Scale bars: 50 μm.

Figure 3. JAG1 expression and NOTCH signaling is altered in ALGS HOs.

(A) Flow cytometric analyses showing the proportion of JAG1⁺ cells present in day 20 control HO1s. (B) Immunofluorescence images of E17 liver show that JAG1 expression was colocalized with a cholangiocyte marker (Pan-CK). (C) Quantitative RT-PCR analyses showing the dynamic changes in the expression level for 4 mRNAs in day 0, 3, 9, 20 (HO1), and 62 (HO2) organoid cultures. For comparison, the expression of these 4 mRNAs was also measured in primary human hepatocytes (PHHs) and in human liver tissue. The mRNAs analyzed are involved in NOTCH signaling (JAG1, NOTCH2, HEY1, and HES1). (D) Immunofluorescence images indicate that day 9 control HO1s simultaneously express the cholangiocyte marker CK7 and the NOTCH signaling ligand JAG1. There is also evidence of Notch pathway activation, since the intracellular domain of NOTCH1 is detected in these cells (right middle panel). Scale bars: 25 μm. (E) Immunofluorescence images show the change in the pattern of JAG1 expression from day 9 to day 50 in control HO1s. JAG1⁺ cells were initially localized within rosette-like clusters of cells on day 9, and then appeared in regions surrounding the developing bile ductal structures. (F) Immunofluorescence images show the change in the pattern of JAG1 and NICD expression in growth media and differentiation media of HO2s. (G) Flow cytometric analyses showing the proportion of CK19⁺ and JAG1⁺ cells in control HOs1, HO2s, and PHHs. Scale bars: 50 μm.

Figure 4. HOs recapitulate the impaired bile duct formation that is characteristic of ALGS liver pathology.

(A) H&E–stained sections of normal (N) and ALGS liver tissue. The boxed area within the ALGS liver section is enlarged in the right panel to show an area with cholestasis and fibrosis. Scale bars: 100 μm. (B) Quantitative RT-PCR analyses for mRNAs encoding cholangiocyte differentiation (SOX9, CK7, CK19, and CFTR), hepatocyte (ALB, A1At, GGT, and HNF1b), and NOTCH signaling markers (HEY1, HES1, JAG1, and NOTCH2) in normal and ALGS liver tissues. * P < 0.05 for comparison of the gene expression level (2^^–Ct) in normal versus ALGS liver tissue. (C) A schematic representation of the C829X mutation site (ALGS1) within an EGF-like domain of the JAG1 protein. (D) Bright-field images of ALGS and control HO1 cultures on day 30. While control iPSCs form intact HO1s, ALGS iPSCs formed large (>2 mm) fluid-filled vesicles. Scale bars: 1 mm. (E) The number of vesicles and organoids formed in day 30 HO1 cultures generated from 3 control or 2 ALGS iPSC lines are shown. Each bar is the average of 3 independent determinations, and each culture had more than 100 evaluable structures. (F) Immunofluorescence images showing CK7 and CK18 expression in control and ALGS HO1s. (G) Flow cytometric analyses showing the proportion of cells expressing hepatocyte (ALB) or cholangiocyte (CD26, CK7, CK19, and CFTR) markers in day 20 HO1s prepared from control and ALGS iPSCs. (H) Quantitative RT-PCR analyses for CK19, CK7, GGT, CFTR, and NOTCH2 mRNA expression in day 20 control and ALGS organoids. (I) Quantitative RT-PCR analyses for JAG1 mRNA expression in iPSCs, endodermal spheres, hepatoblasts, and HO1s formed from 3 control and 2 ALGS individuals. The actual P values for the difference in expression between control and ALGS cells are indicated at each comparison. FC, fold change. (J) Immunofluorescence images indicate that JAG1 is expressed in the cells surrounding the lumen of control HO1s, while JAG1 expression is reduced in ALGS1 HO1s. Representative images from n = 6 technical replicates are shown. (K) Bright-field images of HO2s formed 12 days after control or ALGS organoids were dissociated. The number of intact HO2s formed within these cultures is shown. The results are normalized relative to 1,000 cells plated. Scale bars: 50 μm (J and K).

Figure 5. Characterization of iPSC lines with a CRISPR-engineered ALGS1 mutation (C829X).

(A and B) Bright-field images and H&E–stained cryosections of day 25 HO1 cultures. The cultures were generated from 2 control (C1, C2) iPSC lines prepared and from the following CRISPR-engineered iPSC: C1^mu and C2^mu, which have the heterozygous C829X mutation; ALGS1^rev, where the ALGS1 mutation was reverted to wild type; and ALGS1^revcon, which has the ALGS1 mutation but is a control for the genome engineering process. (C) Flow cytometric analyses showing the proportion of cells expressing hepatocyte (ALB) or cholangiocyte (CD26, CK7, CK19, and CFTR) markers in day 20 HO1s prepared from C1, C1^mu, C2, C2^mu, ALGS1^rev, and ALGS^revcon iPSC lines. (D) The JAG1 allele determines the efficiency of formation of intact HO1s. The number of fluid-filled vesicles and intact organoids in HO1 cultures generated from the indicated iPSC lines was measured on day 30. The proportion of fluid-filled vesicles formed by iPSCs with a mutated JAG1 (C1^mu, C2^mu, ALGS^revcon) was much greater than by those with an un-mutated JAG1 (C1, C2, ALGS1^rev). Each bar is the average of 3 independent determinations, and each culture had more than 100 evaluable structures. (E and F) Immunofluorescence staining of the HO1 cultures with anti–human albumin, anti–human CK8, and anti–human CK7 antibodies. The sections were also stained with DAPI to indicate the nuclei. Irrespective of whether they had a normal or mutated JAG1 allele, all HO1s expressed albumin and CK8. However, CK7⁺ cholangiocytes are only abundant in HO1s prepared from lines (C1, C2 or ALGS1^rev) with a wild-type JAG1. Each analysis was reproduced in 3 independent experiments. (G) Quantitative RT-PCR analysis of JAG1 mRNA expression in day 9 HO1s prepared from different iPSCs. Each bar is the average of 3 independent cultures, and the results were normalized relative to control iPSCs. JAG1 mRNA levels were 10-fold lower in C1^mu, C2^mu, and ALGS1^revcon HO1s relative to wild-type HO1s (P < 0.01), but unaltered in ALGS1^revcon iPSCs (P > 0.05). FC, fold change. (H) The fluorescence intensity within the lumina of the indicated HO1 was normalized relative to the area. The red line is the average of 3 independent measurements, and the box indicates the first and third quartiles. All HO1s with a JAG1 mutation were significantly impaired in their ability to transport rhodamine 123 into the ductal lumen. Scale bars: 50 μm (all panels).

Figure 6. The G274D JAG1 mutation does not alter HO1 properties.

(A) A schematic representation of G274D mutation (TOF1), which is located within the EGF-like domain of the JAG1 protein. (B) Bright-field images of day 15 and day 30 HO1s prepared from iPSCs with a heterozygous TOF1 mutation. Right image: H&E–stained cryosection of a day 25 TOF1 organoid. These images indicate that TOF1 organoids have a morphology that is similar to that of control organoids. Scale bars: 1 mm. (C) The number of fluid-filled vesicles and of intact organoids formed on day 30 by HO1s generated from the control (C1) or TOF1 iPSC lines. (D) Immunofluorescence staining of a day 50 TOF1 HO1 with albumin, CK19, and SOX9 antibodies. (E) Quantitative RT-PCR analyses of JAG1 mRNA expression in iPSCs, endodermal spheres (day 3), hepatoblasts (day 9), and HO1s (day 20) formed from C1 and TOF1 iPSCs. Each measurement is the average of 3 independent determinations. FC, fold change. (F) Bright-field images showing the HO2s formed on day 12 by the cells obtained after dissociation of the control (C1) and TOF1 HO1s. The number of HO2s formed in the day 12 cultures was measured, and each bar is the average of 3 independent determinations. Scale bars: 50 μm (D and F).

Figure 7. The JAG1 mutation has a dominant-negative effect.

(A) The strategy for producing iPSC lines with a heterozygous JAG1 knockout. The 3,079-bp puroΔtk cassette was inserted at the JAG1 C829 site in control (C1) or ALGS1 iPSC lines by CRISPR-mediated genomic engineering. After drug treatment, iPSC lines with a heterozygous JAG1 knockout (C1^+/–829 and ALGS1^+/–) were selected for further study. The cassette was also inserted at the JAG1 G274 site in control (C1) iPSCs to produce the C1^+/–274 iPSC line. (B and C) Bright-field images of day 25 HO1s generated from the indicated iPSC line, and the number of vesicles and organoids formed in the HO1s generated from the indicated iPSC lines are shown. Of note, all iPSC lines with a heterozygous JAG1 knockout (C1^+/–829, C1^+/–274, and ALGS1^+/–) could form liver organoids as efficiently as the control (C1) iPSC line. Each bar is the average of 3 independent determinations, and each culture had more than 100 evaluable structures. (D) Immunofluorescence staining of day 50 organoids produced from 2 different iPSC lines with JAG1 heterozygous knockouts (ALGS1^+/– and C1^+/–829). The albumin⁺ and CK19⁺ cells are clearly seen in the C1^+/–829 organoids, and the arrow indicates the location of CK19⁺ cells in an ALGS1^+/– organoid. (E) RT-PCR analysis of JAG1 mRNA expression in day 9 HO1s prepared from the indicated iPSC lines. Of note, the JAG1 mRNA levels in all of the JAG1 heterozygous organoids was equivalent to that of the control (C1) organoid, and was markedly increased relative to that in the ALGS1 organoid. Each bar is the average of 3 independent determinations. FC, fold change.(F) Bright-field images showing HO2s formed from the cells obtained after dissociation of C1, C1^+/–829, ALGS1, ALGS1^+/–, and C1^+/–274 HO1s. The images shown were prepared on day 12 HO2s. Scale bars: 50 μm (all panels). The number of HO2s formed in the day 12 cultures was quantified, and each bar is the average of 3 independent determinations. While the formation of HO2s was impaired in the presence of the ALGS1 mutation, the cells in all organoids with heterozygous JAG1 mutations (C1^+/–829, ALGS1^+/–, and C1^+/–274) could efficiently form HO2s.