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 7

The JAG1 mutation has a dominant-negative effect.

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The JAG1 mutation has a dominant-negative effect. (A) The strategy for p...
(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.