Human hepatic organoids for the analysis of human genetic diseases
Yuan Guan, Dan Xu, Phillip M. Garfin, Ursula Ehmer, Melissa Hurwitz, Greg Enns, Sara Michie, Manhong Wu, Ming Zheng, Toshihiko Nishimura, Julien Sage, Gary Peltz
Yuan Guan, Dan Xu, Phillip M. Garfin, Ursula Ehmer, Melissa Hurwitz, Greg Enns, Sara Michie, Manhong Wu, Ming Zheng, Toshihiko Nishimura, Julien Sage, Gary Peltz
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Resource and Technical Advance Genetics Hepatology

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.

Authors

Yuan Guan, Dan Xu, Phillip M. Garfin, Ursula Ehmer, Melissa Hurwitz, Greg Enns, Sara Michie, Manhong Wu, Ming Zheng, Toshihiko Nishimura, Julien Sage, Gary Peltz

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Figure 4

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

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HOs recapitulate the impaired bile duct formation that is characteristic...
(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).