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 6

The G274D JAG1 mutation does not alter HO1 properties.

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The G274D JAG1 mutation does not alter HO1 properties. (A) A schematic r...
(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).