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Cited by 71Pluripotent Stem Cells ResearchLiver physiology and pathologyTissue Engineering and Regenerative Medicine

Multi-zonal liver organoids from human pluripotent stem cells

Open access
Reza, Hasan Al, Santangelo, Connie, Iwasawa, Kentaro, Reza, Abid Al, Sekiya, Sachiko, Glaser, Kathryn, Bondoc, Alexander, Merola, Jonathan +1 more·2025·Nature
DOI 10.1038/s41586-025-08850-1PMC PMC12488051
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Abstract

Distinct hepatocyte subpopulations are spatially segregated along the portal-central axis and are critical to understanding metabolic homeostasis and injury in the liver 1. Although several bioactive molecules, including ascorbate and bilirubin, have been described as having a role in directing zonal fates, zonal liver architecture has not yet been replicated in vitro 2,3. Here, to evaluate hepatic zonal polarity, we developed a self-assembling zone-specific liver organoid 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 the urea cycle, glutathione synthesis and glutamate synthesis. Single-nucleus RNA-sequencing analysis of these zonally patterned organoids identifies a hepatoblast differentiation trajectory that dictates periportal, interzonal and pericentral human hepatocytes. Epigenetic and transcriptomic analysis showed that zonal identity is orchestrated by ascorbate- or bilirubin-dependent binding of EP300 to TET1 or HIF1α. Transplantation of the self-assembled zonally patterned human organoids improved survival of immunodeficient rats who underwent bile duct ligation by ameliorating the hyperammonaemia and hyperbilirubinaemia. 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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Registry

Crossref · 10.1038/s41586-025-08850-1

Cites

66 of 66 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

11 of 16 cited sources checked · 2 supported · 0 flagged · accuracy not yet measured

  • Premisecitation

    The epigenetic landscape controls the differentiation of hepatocytes into liver zones.

    “The epigenetic landscape determines differentiation into zonal hepatocytes in the liver56.”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [56] Brosch, M. et al. Epigenomic map of human liver reveals principles of zonated morphogenic and metabolic control. Nat. Commun. 9, 4150 (2018). source

      In 'Signatures of transcriptional and epigenetic zonation' (page not provided), the paper links zonal DNA methylation to gene-expression gradients and identifies potential epigenetically marked drivers of zonation. The Discussion (page not provided) proposes an epigenetic layer of zonation control. It does not establish that the epigenetic landscape determines hepatocyte differentiation into liver zones; that causal claim is stronger than the evidence presented. (LLM, conf=0.84)

      Experimental — accuracy not yet measured
  • Premisecitation

    Zonal liver architecture has not yet been replicated in vitro.

    “zonal liver architecture has not yet been replicated in vitro2,3.”
    Quote in the paperSubstantial claim2 of 2 sources checked
    • cites [2] Kang, J. et al. Simultaneous deletion of the methylcytosine oxidases Tet1 and Tet3 increases transcriptome variability in early embryogenesis. Proc. Natl Acad. Sci. USA 112, E4236–E4245 (2015). source

      The early-development passages (pp. 3–4) concern Tet1/3-deficient embryos and transcriptome variability, not liver architecture. Searches for zonal liver architecture and in vitro liver zonation found no passage addressing whether it has been replicated. (LLM, conf=0.77)

      Experimental — accuracy not yet measured
    • cites [3] Kim, S. G. et al. Bilirubin activates transcription of HIF-1α in human proximal tubular cells cultured in the physiologic oxygen content. J. Kor. Med. Sci. 29, S146–S154 (2014). source

      The Cell culture section describes human kidney proximal tubular cells grown at different oxygen levels, not liver architecture. Searches for the claim’s wording and for liver zonation or its replication in vitro found no passage addressing the claim. (LLM, conf=0.76)

      Experimental — accuracy not yet measured
  • Findingcitation

    When mZ-HLOs are treated with inhibitors targeting HIF1A and TET1, zonal identities are lost.

    “Following such developmental patterns, mZ-HLOs exposed to inhibitors specific to HIF1A and TET1 lead to the loss of zonal identities2,14.”
    Quote in the paperSubstantial claim2 of 2 sources checked
    • cites [2] Kang, J. et al. Simultaneous deletion of the methylcytosine oxidases Tet1 and Tet3 increases transcriptome variability in early embryogenesis. Proc. Natl Acad. Sci. USA 112, E4236–E4245 (2015). source

      The Significance section (p. 2) reports increased transcriptome variability in mouse embryos lacking Tet1 and Tet3, not inhibitor-treated mZ-HLOs. The Discussion (pp. 8–9) addresses TET loss of function but does not report HIF1A inhibition or loss of zonal identities. Searches for mZ-HLOs, HIF1A inhibitors, liver organoids, and zonation found no passage addressing the claimed result. (LLM, conf=0.76)

      Experimental — accuracy not yet measured
    • cites [14] Kolbe, E. et al. Mutual zonated interactions of Wnt and Hh signaling are orchestrating the metabolism of the adult liver in mice and human. Cell Rep. 29, 4553–4567.e4557 (2019). source

      The Discussion (p. 12) addresses metabolic liver zonation through Wnt and Hedgehog signaling, and the in vitro experiments (p. 12) involve GLI overexpression in mouse hepatocytes. Searches for mZ-HLOs, HIF1A, TET1, and their inhibitors found no passage reporting loss of zonal identities after the claimed treatment. (LLM, conf=0.77)

      Experimental — accuracy not yet measured
  • Findingown data

    Preconditioned hepatocyte-like cells exhibited zone-specific functions associated with urea cycle, glutathione synthesis and glutamate synthesis.

    “We found that preconditioned hepatocyte-like cells exhibited zone-specific functions associated with the urea cycle, glutathione synthesis and glutamate synthesis.”
    Quote in the paperSubstantial claim
  • Findingown data

    A self-assembling zone-specific liver organoid was developed by co-culturing ascorbate- and bilirubin-enriched hepatic progenitors derived from hiPSCs.

    “we developed a self-assembling zone-specific liver organoid by co-culturing ascorbate- and bilirubin-enriched hepatic progenitors derived from human induced pluripotent stem cells.”
    Quote in the paperSubstantial claim
  • May combine two claims. 11
  • Classified as probably not a substantial claim. 11

  • May combine two claims.

    Premisecitation

    Loss of Tet1 impairs periportal identity and function and represses pericentral characteristics regulated through Hedgehog signalling.

    “Developmentally, Tet1 deletion impairs periportal identity and function, while repressing the pericentral characteristics regulated through Hedgehog signalling2,14.”
    Quote in the paperSubstantial claim2 of 2 sources checked
    • cites [2] Kang, J. et al. Simultaneous deletion of the methylcytosine oxidases Tet1 and Tet3 increases transcriptome variability in early embryogenesis. Proc. Natl Acad. Sci. USA 112, E4236–E4245 (2015). source

      The Discussion (p. 9) reports down-regulation of Hedgehog-signaling genes in Tet1/Tet3 double-knockout blastocysts, and the results (p. 8) report reduced Hedgehog-pathway components in later embryos. The paper does not establish that Tet1 deletion alone impairs periportal identity or function, or that it represses pericentral characteristics. (LLM, conf=0.86)

      Experimental — accuracy not yet measured
    • cites [14] Kolbe, E. et al. Mutual zonated interactions of Wnt and Hh signaling are orchestrating the metabolism of the adult liver in mice and human. Cell Rep. 29, 4553–4567.e4557 (2019). source

      The Summary (p. 2) and Figure 7 discussion (p. 14) support a role for Hedgehog signalling in liver metabolic zonation. They do not report Tet1 deletion or its developmental effects. They also associate pericentral functions primarily with Wnt signalling and periportal functions with Hedgehog signalling, rather than describing pericentral characteristics as Hedgehog-regulated. (LLM, conf=0.82)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Premisecitation

    Bilirubin can signal through HIF and Wnt cascades, which are important for pericentral hepatocytes.

    “Conversely, recent studies have demonstrated that bilirubin possesses signalling properties that can activate HIF and Wnt signalling cascades3,57, which are important for pericentral hepatocytes58.”
    Quote in the paperNot a substantial claim3 of 3 sources checked
    • cites [3] Kim, S. G. et al. Bilirubin activates transcription of HIF-1α in human proximal tubular cells cultured in the physiologic oxygen content. J. Kor. Med. Sci. 29, S146–S154 (2014). source
    • cites [57] Xu, W. et al. Hypoxia activates Wnt/β-catenin signaling by regulating the expression of BCL9 in human hepatocellular carcinoma. Sci. Rep. 7, 40446 (2017). source

      The Discussion reports that hypoxia promotes Wnt/β-catenin signaling through HIF1α-dependent BCL9 expression in hepatocellular carcinoma. Our copy does not address whether bilirubin activates either pathway; searches for bilirubin and pericentral hepatocytes found no relevant passage. The pericentral-hepatocyte clause is attached to a different citation. (LLM, conf=0.76)

      Experimental — accuracy not yet measured
    • cites [58] Kietzmann, T. Metabolic zonation of the liver: the oxygen gradient revisited. Redox Biol. 11, 622–630 (2017). source

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    EP300 is a histone acetyltransferase that acetylates enhancer regions and activates transcription, which promotes hepatoblast differentiation.

    “EP300, a histone acetyltransferase, is one such epigenetic modifier that acetylates enhancer regions and activates transcription leading to hepatoblast differentiation41,42,.”
    Quote in the paperNot a substantial claim1 of 2 sources checked
    • cites [41] Thakur, A. et al. Hepatocyte nuclear factor 4-alpha is essential for the active epigenetic state at enhancers in mouse liver. Hepatology 70, 1360–1376 (2019). source

      The Introduction identifies CBP/p300 as a histone acetylase, and the Discussion says its interaction with HNF4A may account for H3K27 acetylation at enhancers. The Results link increased enhancer acetylation with gene expression during hepatoblast-to-hepatocyte differentiation, but the paper attributes that regulation to HNF4A; it does not establish that EP300 itself drives the differentiation. (LLM, conf=0.86)

      Experimental — accuracy not yet measured
    • cites [42] Raisner, R. et al. Enhancer activity requires CBP/P300 bromodomain-dependent histone H3K27 acetylation. Cell Rep. 24, 1722–1729 (2018). source

  • May combine two claims.

    Findingnone given

    Zone 1- or zone 3-primed organoid transplantation improved either hyperammonaemia or hyperbilirubinaemia but resulted in reduced survival benefit.

    “Notably, zone 1- or zone 3-primed organoid transplant offer improvements in either hyperammonaemia or hyperbilirubinaemia, resulting in reduced survival benefit.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingcitation

    A rat small interfering RNA model showed that EP300 is integral to zonal development.

    “Moreover, a rat small interfering RNA model revealed that EP300 is integral to zonal development.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    mZ-HLOs integrated into the rat liver parenchyma with tubular morphology and maintained zonal characteristics.

    “Furthermore, the mZ-HLOs integrated into the parenchyma of the rat liver in a tubular morphology, while retaining their zonal characteristics.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Integrated RNA-seq and ChIP-seq identified HIF1A as the top target in Dox-treated Z1-HLOs and TET1 as the top target in bilirubin-treated Z3-HLOs.

    “The integrated RNA-seq and ChIP–seq showed that the top targets were HIF1A and TET1 in the Dox-treated Z1-HLOs and bilirubin-treated Z3-HLOs, respectively.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Z1-HLOs preferentially integrated near the portal vein, whereas Z3-HLOs showed higher affinity for the central vein region.

    “The Z1-HLOs showed a preferential integration near the portal vein, whereas the Z3-HLOs exhibited higher affinity for the central vein region.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    mZ-HLOs engrafted into the resident liver and maintained zonal-specific functionality.

    “Finally, mZ-HLOs engrafted into the resident liver, maintained zonal-specific functionality and augmented the survival rate in rats following bile duct ligation.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    The authors' data suggest that EP300 binds enhancers lying upstream of zonal genes in a context-dependent way, differentially activating gene expression, as seen in prior primary human hepatocyte studies.

    “Similarly, our data suggest that EP300 binds to enhancers upstream of zonal genes in a context-dependent manner to differentially activate gene expression similar to previously published PHH studies.”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim.

    Premisecitation

    Hepatocyte transplantation is a treatment for liver diseases.

    “Hepatocyte transplantation has been used to treat liver diseases, but the difficulty in obtaining compatible primary human hepatocytes makes this an impractical approach59,60.”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [59] Iansante, V., Mitry, R. R., Filippi, C., Fitzpatrick, E. & Dhawan, A. Human hepatocyte transplantation for liver disease: current status and future perspectives. Pediatr. Res. 83, 232–240 (2018). source
    • cites [60] Reza, H. A., Okabe, R. & Takebe, T. Organoid transplant approaches for the liver. Transpl. Int. 34, 2031–2045 (2021). source

  • May combine two claims.

    Interpretationown data

    Self-assembled organoids contain diverse cell types that express zonal hepatocyte markers.

    “Thus, our self-assembled organoids contain diverse cell types that express zonal hepatocyte markers.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductown data

    A post hoc power analysis was conducted for each experiment to determine whether the design had sufficient power.

    “For data from each experiment, we also conducted a post hoc power analysis to determine whether our design had sufficient power.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Study design and conductnone given

    Human samples, including foreskin fibroblasts and liver samples, were obtained with patient informed consent and IRB approval from Cincinnati Children's Hospital Medical Center.

    “All human samples including human foreskin fibroblasts and human liver samples were collected with informed consent from the corresponding patients and the approval of the Institutional Review Board (IRB) of the Cincinnati Children’s Hospital Medical Center.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductown data

    With G*Power, the minimum sample size for each experiment was based on preliminary effect sizes, alpha 0.05, power 0.8.

    “Using G*Power software, for each experiment, we determined the minimum sample size to collect the data for using the preliminary effect sizes, α = 0.05 and power = 0.8.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    The image analyses were non-blinded.

    “The image analyses were non-blinded.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    The Animal Research Committee of Institute of Science Tokyo approved animal care and experimental procedures (approval number A2023554).

    “Animal care and experimental procedures were approved by the Animal Research Committee of Institute of Science Tokyo (approval number A2023554).”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    The Ethics Committees of Cincinnati Children's Hospital Medical Center approved experiments using hiPS cells.

    “Experiments using hiPS cells were approved by the Ethics Committees of Cincinnati Children’s Hospital Medical Center.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    All animal experiments received approval from the IRB and IACUC at Cincinnati Children's Hospital Medical Center.

    “All animal experiments were conducted with the approval of the Institutional Review Board (IRB) and Institutional Animal Care and Use Committee (IACUC) of the Cincinnati Children’s Hospital Medical Center.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    n refers to biologically independent replicates.

    “n refers to biologically independent replicates.”
    Quote in the paperNot a substantial claim
20 of 20

  • The paper relies on this without support.

    Most therapeutic proof-of-concepts have targeted a single disease parameter to correct monogenic conditions.

    “Most therapeutic proof-of-concepts have aimed at correcting monogenic conditions by targeting one particular disease parameter.”
1 of 1

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

Each contradiction between two passages listed here was confirmed by an independent check.

  • Abstract gives two different top transcriptional regulators for Z1/Z3

    Minor

    One abstract passage says zonal identity is orchestrated by EP300 binding to TET1 or HIF1α, while another abstract passage says the top targets were HIF1A and TET1 in the Dox-treated Z1-HLOs and bilirubin-treated Z3-HLOs, respectively. These statements reverse the stated pairing of regulators and conditions.

  • Discussion misidentifies the rat gene-silencing reagent

    Minor

    The discussion calls the rat experiment a small interfering RNA model, but the results describe an adenovirus encoding short hairpin RNA targeting Ep300.

  • Discussion reverses the zonal assignments of HIF1A and TET1

    Minor

    The results associate EP300-co-occupied HIF1A motifs with Z3-HLOs and TET1 motifs with Z1-HLOs. The discussion assigns those targets to Z1-HLOs and Z3-HLOs, respectively.

3 of 3

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Authors
Reza, H. A., Santangelo, C., Iwasawa, K., Reza, A. A., Sekiya, S., Glaser, K., Bondoc, A., Merola, J., & Takebe, T.
Affiliations
  • Cincinnati Children's Hospital Medical Center (US)
  • Institute of Science Tokyo (JP)
Corresponding author
Takebe, Takanori
ORCID
Venue
Nature
Publisher
Springer Science and Business Media LLC
Published
2025-04-16
Volume
641
Issue
8065
Pages
1258-1267
ISSN
  • 0028-0836 (ISSN-L)
  • 1476-4687
DOI
10.1038/s41586-025-08850-1
PMC
PMC12488051
OpenAlex
W4409485986
Cited by
71
References
66
Citations by year
2026: 49 · 2025: 22
FWCI
21.19
Type
journal article
Language
en
Update notices
correction (2025-05-22)
Open access
Licence
Locations
  • Nature (publishedVersion)
  • PubMed (publishedVersion)
  • PubMed Central (publishedVersion)
  • Europe PMC (PubMed Central) (acceptedVersion)
  • RePEc: Research Papers in Economics (submittedVersion)
Funders
  • National Cancer Institute (R01CA272903)
  • National Institute of Diabetes and Digestive and Kidney Diseases (DP2DK128799, UH3DK119982, UG3DK119982, R01DK135478, P30DK078392)
  • National Center for Advancing Translational Sciences (UM1TR006070)
  • Eunice Kennedy Shriver National Institute of Child Health and Human Development (P01HD093363)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Pluripotent Stem Cells Research
  • Health Sciences › Medicine › Hepatology › Liver physiology and pathology
  • Health Sciences › Medicine › Surgery › Tissue Engineering and Regenerative Medicine
Keywords
liver organoids, hepatocyte subpopulations, vitamin C, bilirubin, induced pluripotent stem cells, hepatoblast differentiation, EP300, TET1, HIF-1α, urea cycle, glutathione synthesis, hyperammonemia, hyperbilirubinemia, bile duct ligation, liver transplantation, single-nucleus RNA sequencing
MeSH terms
Animals, Ascorbic Acid, Bilirubin, Cell Differentiation, Female, Glutathione, Humans, Liver, Male, Organoids, Coculture Techniques, Hepatocytes, Pluripotent Stem Cells, Epigenesis, Genetic, Rats, Hypoxia-Inducible Factor 1, alpha Subunit, Induced Pluripotent Stem Cells

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