Engineering human hepato-biliary-pancreatic organoids from pluripotent stem cells


Human organoids are emerging as a valuable resource to investigate human organ development and disease. The applicability of human organoids has been limited, partly due to the oversimplified architecture of the current technology, which generates single-tissue organoids that lack inter-organ structural connections. Thus, engineering organoid systems that incorporate connectivity between neighboring organs is a critical unmet challenge in an evolving organoid field. Here, we describe a protocol for the continuous patterning of hepatic, biliary and pancreatic (HBP) structures from a 3D culture of human pluripotent stem cells (PSCs). After differentiating PSCs into anterior and posterior gut spheroids, the two spheroids are fused together in one well. Subsequently, self-patterning of multi-organ (i.e., HBP) domains occurs within the boundary region of the two spheroids, even in the absence of any extrinsic factors. Long-term culture of HBP structures induces differentiation of the domains into segregated organs complete with developmentally relevant invagination and epithelial branching. This in-a-dish model of human hepato-biliary-pancreatic organogenesis provides a unique platform for studying human development, congenital disorders, drug development and therapeutic transplantation. More broadly, our approach could potentially be used to establish inter-organ connectivity models for other organ systems derived from stem cell cultures.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Fig. 1: Overview of the protocol.
Fig. 2: Representative images of PSC differentiation into definitive endoderm and anterior/posterior gut cells.
Fig. 3: Representative images of anterior or posterior gut cells and boundary organoids.
Fig. 4: Long-term culture–induced HBPO maturation.

Data availability

Data to show validation of this protocol are included in the main article, the Supplementary Information files and the supporting primary research article12. The underlying raw datasets will be provided from the corresponding author upon reasonable request.


  1. 1.

    Takebe, T. & Wells, J. M. Organoids by design. Science 364, 956–959 (2019).

    CAS  Article  Google Scholar 

  2. 2.

    Lancaster, M. A. et al. Cerebral organoids model human brain development and microcephaly. Nature 501, 373–379 (2013).

    CAS  Article  Google Scholar 

  3. 3.

    Sato, T. et al. Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche. Nature 459, 262–265 (2009).

    CAS  Article  Google Scholar 

  4. 4.

    McCracken, K. W. et al. Modelling human development and disease in pluripotent stem-cell-derived gastric organoids. Nature 516, 400–404 (2014).

    CAS  Article  Google Scholar 

  5. 5.

    Ouchi, R. et al. Modeling steatohepatitis in humans with pluripotent stem cell-derived organoids. Cell Metab. 30, 374–384.e6 (2019).

    CAS  Article  Google Scholar 

  6. 6.

    Takebe, T. et al. Vascularized and functional human liver from an iPSC-derived organ bud transplant. Nature 499, 481–484 (2013).

    CAS  Article  Google Scholar 

  7. 7.

    Sato, T. & Clevers, H. Growing self-organizing mini-guts from a single intestinal stem cell: mechanism and applications. Science 340, 1190–1194 (2013).

    CAS  Article  Google Scholar 

  8. 8.

    Takebe, T. et al. Vascularized and complex organ buds from diverse tissues via mesenchymal cell-driven condensation. Cell Stem Cell 16, 556–565 (2015).

    CAS  Article  Google Scholar 

  9. 9.

    Xiang, Y. et al. hESC-derived thalamic organoids form reciprocal projections when fused with cortical organoids. Cell Stem Cell 24, 487–497.e7 (2019).

    CAS  Article  Google Scholar 

  10. 10.

    Xiang, Y. et al. Fusion of regionally specified hPSC-derived organoids models human brain development and interneuron migration. Cell Stem Cell 21, 383–398.e7 (2017).

    CAS  Article  Google Scholar 

  11. 11.

    Song, L. et al. Assembly of human stem cell-derived cortical spheroids and vascular spheroids to model 3-D brain-like tissues. Sci. Rep. 9, 5977 (2019).

    Article  Google Scholar 

  12. 12.

    Koike, H. et al. Modelling human hepato-biliary-pancreatic organogenesis from the foregut-midgut boundary. Nature 574, 112–116 (2019).

    CAS  Article  Google Scholar 

  13. 13.

    Franklin, V. et al. Regionalisation of the endoderm progenitors and morphogenesis of the gut portals of the mouse embryo. Mech. Dev. 125, 587–600 (2008).

    CAS  Article  Google Scholar 

  14. 14.

    Molkentin, J. D., Lin, Q., Duncan, S. A. & Olson, E. N. Requirement of the transcription factor GATA4 for heart tube formation and ventral morphogenesis. Genes Dev. 11, 1061–1072 (1997).

    CAS  Article  Google Scholar 

  15. 15.

    Wells, J. M. & Watt, F. M. Diverse mechanisms for endogenous regeneration and repair in mammalian organs. Nature 557, 322–328 (2018).

    CAS  Article  Google Scholar 

  16. 16.

    Zorn, A. M. & Wells, J. M. Vertebrate endoderm development and organ formation. Annu. Rev. Cell Dev. Biol. 25, 221–251 (2009).

    CAS  Article  Google Scholar 

  17. 17.

    Udager, A., Prakash, A. & Gumucio, D. L. Dividing the tubular gut: generation of organ boundaries at the pylorus. Prog. Mol. Biol. Transl. Sci. 96, 35–62 (2010).

    CAS  Article  Google Scholar 

  18. 18.

    McLin, V. A., Rankin, S. A. & Zorn, A. M. Repression of Wnt/beta-catenin signaling in the anterior endoderm is essential for liver and pancreas development. Development 134, 2207–2217 (2007).

    CAS  Article  Google Scholar 

  19. 19.

    Nissim, S. et al. Iterative use of nuclear receptor Nr5a2 regulates multiple stages of liver and pancreas development. Dev. Biol. 418, 108–123 (2016).

    CAS  Article  Google Scholar 

  20. 20.

    Spence, J. R. et al. Sox17 regulates organ lineage segregation of ventral foregut progenitor cells. Dev. Cell 17, 62–74 (2009).

    CAS  Article  Google Scholar 

  21. 21.

    Bort, R., Martinez-Barbera, J. P., Beddington, R. S. & Zaret, K. S. Hex homeobox gene-dependent tissue positioning is required for organogenesis of the ventral pancreas. Development 131, 797–806 (2004).

    CAS  Article  Google Scholar 

  22. 22.

    Bredenoord, A. L., Clevers, H. & Knoblich, J. A. Human tissues in a dish: the research and ethical implications of organoid technology. Science 355, eaaf9414 (2017).

    Article  Google Scholar 

  23. 23.

    Sumazaki, R. et al. Conversion of biliary system to pancreatic tissue in Hes1-deficient mice. Nat. Genet. 36, 83–87 (2004).

    CAS  Article  Google Scholar 

  24. 24.

    Bagley, J. A., Reumann, D., Bian, S., Levi-Strauss, J. & Knoblich, J. A. Fused cerebral organoids model interactions between brain regions. Nat. Methods 14, 743–751 (2017).

    CAS  Article  Google Scholar 

  25. 25.

    Birey, F. et al. Assembly of functionally integrated human forebrain spheroids. Nature 545, 54–59 (2017).

    CAS  Article  Google Scholar 

  26. 26.

    Harrison, S. E., Sozen, B., Christodoulou, N., Kyprianou, C. & Zernicka-Goetz, M. Assembly of embryonic and extraembryonic stem cells to mimic embryogenesis in vitro. Science 356, eaal1810 (2017).

    Article  Google Scholar 

  27. 27.

    Rivron, N. C. et al. Blastocyst-like structures generated solely from stem cells. Nature 557, 106–111 (2018).

    CAS  Article  Google Scholar 

  28. 28.

    Spence, J. R. et al. Directed differentiation of human pluripotent stem cells into intestinal tissue in vitro. Nature 470, 105–109 (2011).

    Article  Google Scholar 

  29. 29.

    Wu, F. et al. Generation of hepatobiliary organoids from human induced pluripotent stem cells. J. Hepatol. 70, 1145–1158 (2019).

    Article  Google Scholar 

  30. 30.

    Vyas, D. et al. Self-assembled liver organoids recapitulate hepatobiliary organogenesis in vitro. Hepatology 67, 750–761 (2018).

    CAS  Article  Google Scholar 

  31. 31.

    Takeishi, K. et al. Assembly and function of a bioengineered human liver for transplantation generated solely from induced pluripotent stem cells. Cell Rep. 31, 107711 (2020).

    CAS  Article  Google Scholar 

  32. 32.

    Ma, X. et al. Deterministically patterned biomimetic human iPSC-derived hepatic model via rapid 3D bioprinting. Proc. Natl Acad. Sci. USA 113, 2206–2211 (2016).

    CAS  Article  Google Scholar 

  33. 33.

    Bauer, S. et al. Functional coupling of human pancreatic islets and liver spheroids on-a-chip: towards a novel human ex vivo type 2 diabetes model. Sci. Rep. 7, 14620 (2017).

    Article  Google Scholar 

  34. 34.

    Okita, K. et al. A more efficient method to generate integration-free human iPS cells. Nat. Methods 8, 409–412 (2011).

    CAS  Article  Google Scholar 

  35. 35.

    McCracken, K. W., Howell, J. C., Wells, J. M. & Spence, J. R. Generating human intestinal tissue from pluripotent stem cells in vitro. Nat. Protoc. 6, 1920–1928 (2011).

    CAS  Article  Google Scholar 

Download references


We express sincere gratitude to Hiro Nakazawa for helping with the audio in the video abstract, and the other Takebe laboratory members and the Wells-Zorn laboratory members for their support and excellent technical assistance. This work was supported by a Cincinnati Children’s Research Foundation grant, an NIH Director’s New Innovator Award (DP2 DK128799-01) and a PRESTO grant from Japan Science and Technology Agency (JST) to T.T. This work was also supported by NIH grant UG3 DK119982, a Cincinnati Center for Autoimmune Liver Disease Fellowship Award, PHS Grant P30 DK078392 (Integrative Morphology Core and Pluripotent Stem Cell and Organoid Core) of the Digestive Disease Research Core Center in Cincinnati, a Takeda Science Foundation Award, a Mitsubishi Foundation award, AMED JP19fk0210037, JP19bm0704025, JP19fk0210060, 20ta0127003h0001 and JP19bm0404045, and JSPS JP18H02800 and 19K22416. T.T. is a New York Stem Cell Foundation Robertson Investigator.

Author information




H.K. and T.T. conceived the study and experimental design. H.K., K.I., W.L.T. and T.T. wrote the manuscript. H.K., K.I., R.O., M.M. and M.K. analyzed the data and performed experiments. A.K. and S.N. illustrated and animated the graphics for the figures and video.

Corresponding author

Correspondence to Takanori Takebe.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Peer review information Nature Protocols thanks Hans Clevers, Kenneth S. Zaret and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Related links

Key reference using this protocol

Koike, H. et al. Nature 574, 112–116 (2019):

Supplementary information

Reporting Summary

Supplementary Video 1

Animated overview of the HBPO protocol

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Koike, H., Iwasawa, K., Ouchi, R. et al. Engineering human hepato-biliary-pancreatic organoids from pluripotent stem cells. Nat Protoc (2021).

Download citation


By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.


Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing