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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.

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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.

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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.


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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.

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Authors and Affiliations



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.

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The authors declare no competing interests.

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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.

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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

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Koike, H., Iwasawa, K., Ouchi, R. et al. Engineering human hepato-biliary-pancreatic organoids from pluripotent stem cells. Nat Protoc 16, 919–936 (2021).

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