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.
This is a preview of subscription content, access via your institution
Access Nature and 54 other Nature Portfolio journals
Get Nature+, our best-value online-access subscription
$29.99 / 30 days
cancel any time
Subscribe to this journal
Receive 12 print issues and online access
$259.00 per year
only $21.58 per issue
Rent or buy this article
Prices vary by article type
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
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.
Takebe, T. & Wells, J. M. Organoids by design. Science 364, 956–959 (2019).
Lancaster, M. A. et al. Cerebral organoids model human brain development and microcephaly. Nature 501, 373–379 (2013).
Sato, T. et al. Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche. Nature 459, 262–265 (2009).
McCracken, K. W. et al. Modelling human development and disease in pluripotent stem-cell-derived gastric organoids. Nature 516, 400–404 (2014).
Ouchi, R. et al. Modeling steatohepatitis in humans with pluripotent stem cell-derived organoids. Cell Metab. 30, 374–384.e6 (2019).
Takebe, T. et al. Vascularized and functional human liver from an iPSC-derived organ bud transplant. Nature 499, 481–484 (2013).
Sato, T. & Clevers, H. Growing self-organizing mini-guts from a single intestinal stem cell: mechanism and applications. Science 340, 1190–1194 (2013).
Takebe, T. et al. Vascularized and complex organ buds from diverse tissues via mesenchymal cell-driven condensation. Cell Stem Cell 16, 556–565 (2015).
Xiang, Y. et al. hESC-derived thalamic organoids form reciprocal projections when fused with cortical organoids. Cell Stem Cell 24, 487–497.e7 (2019).
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).
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).
Koike, H. et al. Modelling human hepato-biliary-pancreatic organogenesis from the foregut-midgut boundary. Nature 574, 112–116 (2019).
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).
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).
Wells, J. M. & Watt, F. M. Diverse mechanisms for endogenous regeneration and repair in mammalian organs. Nature 557, 322–328 (2018).
Zorn, A. M. & Wells, J. M. Vertebrate endoderm development and organ formation. Annu. Rev. Cell Dev. Biol. 25, 221–251 (2009).
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).
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).
Nissim, S. et al. Iterative use of nuclear receptor Nr5a2 regulates multiple stages of liver and pancreas development. Dev. Biol. 418, 108–123 (2016).
Spence, J. R. et al. Sox17 regulates organ lineage segregation of ventral foregut progenitor cells. Dev. Cell 17, 62–74 (2009).
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).
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).
Sumazaki, R. et al. Conversion of biliary system to pancreatic tissue in Hes1-deficient mice. Nat. Genet. 36, 83–87 (2004).
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).
Birey, F. et al. Assembly of functionally integrated human forebrain spheroids. Nature 545, 54–59 (2017).
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).
Rivron, N. C. et al. Blastocyst-like structures generated solely from stem cells. Nature 557, 106–111 (2018).
Spence, J. R. et al. Directed differentiation of human pluripotent stem cells into intestinal tissue in vitro. Nature 470, 105–109 (2011).
Wu, F. et al. Generation of hepatobiliary organoids from human induced pluripotent stem cells. J. Hepatol. 70, 1145–1158 (2019).
Vyas, D. et al. Self-assembled liver organoids recapitulate hepatobiliary organogenesis in vitro. Hepatology 67, 750–761 (2018).
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).
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).
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).
Okita, K. et al. A more efficient method to generate integration-free human iPS cells. Nat. Methods 8, 409–412 (2011).
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).
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.
The authors declare no competing interests.
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.
Key reference using this protocol
Koike, H. et al. Nature 574, 112–116 (2019): https://doi.org/10.1038/s41586-019-1598-0
About this article
Cite this article
Koike, H., Iwasawa, K., Ouchi, R. et al. Engineering human hepato-biliary-pancreatic organoids from pluripotent stem cells. Nat Protoc 16, 919–936 (2021). https://doi.org/10.1038/s41596-020-00441-w
This article is cited by
Cell & Bioscience (2023)
Nature Reviews Gastroenterology & Hepatology (2023)