
==== Front
bioRxiv
BIORXIV
bioRxiv
2692-8205
Cold Spring Harbor Laboratory

10.1101/2024.08.30.610426
preprint
1
Article
Self-Assembled Generation of Multi-zonal Liver Organoids from Human Pluripotent Stem Cells
Al Reza Hasan
Santangelo Connie
Al Reza Abid
Iwasawa Kentaro
Sachiko Sachiko
Glaser Kathryn
Bondoc Alexander http://orcid.org/0000-0002-9802-5948

Merola Jonathan
Takebe Takanori http://orcid.org/0000-0002-6989-3041

30 8 2024
2024.08.30.610426https://creativecommons.org/licenses/by-nc-nd/4.0/ This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which allows reusers to copy and distribute the material in any medium or format in unadapted form only, for noncommercial purposes only, and only so long as attribution is given to the creator.
http://biorxiv.org/lookup/doi/10.1101/2024.08.30.610426
nihpp-2024.08.30.610426.pdf
Distinct hepatocyte subpopulations are spatially segregated along the portal-central axis and critical to understanding metabolic homeostasis and liver injury. While several bioactive molecules have been described to play a role in directing zonal fates, including ascorbate and bilirubin, in vitro replication of zonal liver architecture has not been achieved to date. In order to evaluate hepatic zonal polarity, we developed a self-assembling zone-specific liver organoid culture by co-culturing ascorbate and bilirubin enriched hepatic progenitors derived from human induced pluripotent stem cells. We found that preconditioned hepatocyte-like cells exhibited zone-specific functions associated with urea cycle, glutathione synthesis and glutamate synthesis. Single nucleus RNA sequencing analysis of these zonally patterned organoids identifies hepatoblast differentiation trajectory that mimics periportal-, interzonal-, and pericentral human hepatocytes. Epigenetic and transcriptomic analysis showed that zonal identity is orchestrated by ascorbate or bilirubin dependent binding of histone acetyltransferase p300 (EP300) to methylcytosine dioxygenase TET1 or hypoxia-inducible factor 1-alpha (HIF1α). Transplantation of the self-assembled zonally patterned human organoids improved survival of immunodeficient rats who underwent bile duct ligation by ameliorating the hyperammonemia and hyperbilirubinemia. Overall, this multi-zonal organoid system serves as an in vitro human model to better recapitulate hepatic architecture relevant to liver development and disease.
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