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 3

JAG1 expression and NOTCH signaling is altered in ALGS HOs.

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JAG1 expression and NOTCH signaling is altered in ALGS HOs. (A) Flow cyt...
(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.