Organogenesis is a complex and interconnected process that is orchestrated by multiple boundary tissue interactions1,2,3,4,5,6,7. However, it remains unclear how individual, neighbouring components coordinate to establish an integral multi-organ structure. Here we report the continuous patterning and dynamic morphogenesis of hepatic, biliary and pancreatic structures, invaginating from a three-dimensional culture of human pluripotent stem cells. The boundary interactions between anterior and posterior gut spheroids differentiated from human pluripotent stem cells enables retinoic acid-dependent emergence of hepato-biliary-pancreatic organ domains specified at the foregut–midgut boundary organoids in the absence of extrinsic factors. Whereas transplant-derived tissues are dominated by midgut derivatives, long-term-cultured microdissected hepato-biliary-pancreatic organoids develop into segregated multi-organ anlages, which then recapitulate early morphogenetic events including the invagination and branching of three different and interconnected organ structures, reminiscent of tissues derived from mouse explanted foregut–midgut culture. Mis-segregation of multi-organ domains caused by a genetic mutation in HES1 abolishes the biliary specification potential in culture, as seen in vivo8,9. In sum, we demonstrate that the experimental multi-organ integrated model can be established by the juxtapositioning of foregut and midgut tissues, and potentially serves as a tractable, manipulatable and easily accessible model for the study of complex human endoderm organogenesis.
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Sequence data used in this study have been deposited in the Gene Expression Omnibus with accession number GSE121830. The data that support the findings of this study are available from the corresponding author upon reasonable request.
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The authors thank A. Kodaka for graphics, the Hydroid team at Amana and H. Nakazawa for video, H. Nakauchi, V. Hwa and A. Asai and Takebe, Wells, Zorn and Helmrath laboratory members for their support and technical assistance, D. Louis for administrative and technical assistance, the confocal imaging core for microscopy and the Transgenic Animal and Genome Editing Core at CCHMC for guide RNA design, construction and validation. This work was supported by Cincinnati Children’s Research Foundation grant, Ono Pharmaceutical, Ltd Grant and PRESTO grant from Japan Science and Technology Agency (JST) to T.T. This work was also supported by an Institutional Clinical and Translational Science Award, NIH/NCRR Grant Number UL1TR001425, 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, and Takeda Science Foundation grants. T.T. is a New York Stem Cell Foundation–Robertson Investigator.
The authors declare no competing interests related to this manuscript.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Peer review information Nature thanks Dominic Grün, Meritxell Huch and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
Extended data figures and tables
Extended Data Fig. 1 Anterior- and posterior-gut cell specification and boundary organoid formation.
a, Flow cytometry of EpCAM in day 7 anterior and posterior gut cells using TkDA3 human iPSCs and 72_3 human iPSCs. The gating strategy was forward scatter (FSC) area (A)/side scatter (SSC) A, FSC height (H)/FSC width (W), SSC H/SSC W, propidium iodide (PI)/FSC A and EpCAM–BV421/SSC A. Representative image of three independent experiments showing similar results. b, Whole-mount immunostaining, flow cytometry showing percentage of the population in each quadrant, qPCR for SOX2 and CDX2, and organoid images at each time point. Data are mean ± s.d.; n = 3 independent experiments. Unpaired, two-tailed t-test. Scale bars, 50 µm. c, Image of day-11 boundary organoids. Anterior and posterior gut spheroids were differentiated from H1 ESCs or 1383D6 iPSCs, mixed and transferred into Matrigel. Independent experiments were repeated twice for each line with similar results. Scale bar, 200 μm. d, Whole-mount Immunofluorescent staining of PDX1, CDX2, FOXF1 and HHEX in boundary organoids derived from 72_3 iPSCs at day 12. Representative image of n = 30 independent organoids showing similar results. The arrowhead indicates the boundary of the organoid. Scale bar, 100 μm. e, Whole-mount immunofluorescent staining of CDX2, E-cadherin and HHEX in the boundary region of organoids derived from H1 ESCs at day 12. Representative image of n = 6 independent organoids showing similar results. Scale bar, 50 μm.
Extended Data Fig. 2 Cell–cell contact of anterior–posterior gut spheroids induced HBP marker expression.
a, Anterior and posterior spheroids were mixed on day 8, fused on day 9, cultured, and collected on day 12 for quantitative RT–qPCR. The spheroids that did not fuse were also collected on day 12 for comparison. Independent experiments were repeated twice with similar results. b, Gene expression of PDX1 and HHEX in fused, non-fused, posterior spheroids (day 8) and iPSCs. Data are mean ± s.d. from two independent experiments. c, Comparison of different combinations of anterior and posterior gut spheroids. Immunofluorescent staining of CDX2, HHEX, and PDX1 in AP, AA and PP spheroids at day 12. Images are representative of n = 4 (AA and PP) and n = 6 (AP) independent organoids showing similar results. Scale bar, 200 μm.
a, Starting on day (d)0, unlabelled iPSCs were differentiated into anterior spheroids and GFP-labelled (GFP sequence inserted into AAVS1 (Adeno-associated virus integration site 1) locus) iPSCs were differentiated into posterior spheroids. Top, bright-field and GFP fluorescence image during boundary organoid formation. Middle and bottom, whole-mount immunostaining for HHEX and PDX1 at day 13. HHEX expression overlapped with GFP expression. Images are representative of n = 3 independent organoids showing similar results. Scale bar, 200 μm. b, H2B–GFP labelled and unlabelled PROX1::tdTomato reporter iPSCs were differentiated into anterior and posterior spheroids, respectively. tdTomato expression was detected only in unlabelled original posterior gut spheroids. Independent experiments were repeated twice with similar results. Scale bar, 200 μm. c, Anterior and posterior gut spheroids were differentiated from unlabelled iPSCs and PROX1::tdTomato reporter iPSCs. Reporter cell-derived anterior and unlabelled cell posterior spheroid (left), or unlabelled cell-derived anterior and reporter cell-derived posterior spheroid (right) were examined for tdTomato expression. Top, bright-field image; bottom, tdTomato fluorescence. Images are representative of n = 3 independent organoids showing similar results. Scale bar, 200 μm.
a, Schematic of PROX1-tdTomato reporter generation using the CRISPR-Cas9 system. b, PROX1 reporter activity in boundary organoids. All images are boundary organoids derived from the PROX1::tdTomato reporter line at day 12. Images are oriented with anterior at the top and posterior at the bottom. Arrowheads indicate PROX1::tdTomato expression at the boundary of each spheroid. Independent experiments were repeated three times with similar results. Scale bar, 100 μm. c, Transcriptomic characterization of dissected anterior, boundary and posterior domains by RNA-seq. Heat map shows downstream gene expression related to FGF, BMP, hedgehog, Notch and retinoic acid signalling pathways selected by gene ontology (GO) term and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway category. The heat map was separated into eight groups (C1–C8) by hierarchical clustering. d, Default developmental potential of transplanted boundary organoids. Middle, H&E staining and immunohistochemistry; right, immunofluorescence. The experiment was repeated with three independent samples with similar results. Scale bar, 100 µm.
Extended Data Fig. 5 Depletion of HHEX, PDX1 and PROX1 by exposure of human boundary organoids and mouse embryos to retinoic acid receptor.
a, Anterior or posterior spheroids pretreated with the retinoic-acid-receptor antagonist BMS493 were fused to induce HBP anlage formation and were whole-mount-stained for HHEX, PDX1 and CDX2. Expression of HHEX and PDX1 in the BMS493-pretreated posterior spheroid was inhibited at the boundary, suggesting that retinoic-acid-receptor function in the posterior side is important to establish the HBP boundary organoid. Images are representative of n = 4 independent organoids showing similar results. Scale bar, 200 μm. b, c, PROX1 inhibition by BMS493 exposure with embryonic day (E) 9.0 Prox1::eGFP reporter mouse embryo explant culture. The whole embryo was cultured in the rotator-type bottle culture system for 24 h. The group treated with retinoic acid receptor antagonist BMS493 was compared with the control (DMSO only) group. b, Bright-field image and GFP fluorescence of the embryo after culture. Images are representative of n = 3 independent embryos showing similar results. c, The area of GFP-expressing regions was quantified from images represented in b. Data were mean ± s.d. (n = 3). P = 0.0035 by unpaired, two-tailed Student’s t-test. Scale bar, 1 mm.
Extended Data Fig. 6 Transplantation of dissected PROX1-expressing domain from human organoid and mouse embryo.
a, Dissected PROX1-positive boundary domain at day 13 was transplanted into an immunodeficient mouse, and formed a duct-like structure in the tissue, expressing PROX1 and duct marker SOX9 after one month. Images are representative of n = 2 independent transplants showing similar results. b, E9.0 PROX1–GFP mouse embryonic HBP domain was transplanted and formed limited tissue expressing PROX1 (GFP) or PDX1 seven days after transplantation. Images are representative of n = 2 independent transplants showing similar results.
a, Illustration of the dissection strategy of the PROX1-positive region from organoids with representative images. The imaging experiments were repeated with 12 independent samples with similar results. Scale bar, 100 µm. b, Optimization of the organoid culture system, comparing: (1) floating, (2) embedding in Matrigel, (3) embedding in Matrigel and culture in Transwells from day 13 and (4) dissection followed by embedding in Matrigel, and culture in Transwell from day 13. Left, the typical morphology of invaginating or branching organoid. The imaging experiments were repeated with 12 independent samples with similar results. Scale bar, 100 µm. c, Illustration of optimization of in vitro culture system. We compared various culture formats to enhance morphological change, such as invagination and branching morphogenesis, of PROX1-positive HBP domains. At day 7, anterior and posterior gut spheroids were mixed after 24 h of culture. Connected spheroids were transferred into a Matrigel drop or a low-binding culture plate to compare between non-floating and floating conditions during emergence of the HBP domain. Organoids in the Matrigel-embedded group started to express tdTomato at day 11. The tdTomato-positive region was manually dissected under the microscope according to the fluorescent signal and transferred into a Matrigel again or a Transwell to compare the effect of various agonists and antagonists in the medium. d, Morphogenesis of boundary organoids during 2 days from day 13. Imaging experiments were repeated independently three times with similar results. Scale bar, 100 µm.
Extended Data Fig. 8 Comparison of organoid size, PROX1-positive area, branching and invagination in various conditions.
a, Comparison of PROX1-tdTomato expression in AP, AA and PP spheroids at day 50 of culture. Images are representative image of n = 6 independent organoids showing similar results. Scale bar, 500 μm. b, c, Quantification of entire spheroid surface area (b) and of PROX1-positive region (c). n = 11 (AP), 6 (AA) and 7 (PP). Box plots show the mean, box edges show the 25th and 75th percentiles and whiskers show the range of values. In b, P = 0.0278 (AP versus AA), 0.0052 (AP versus PP) and 0.8566 (AA versus PP); in c, P = 0.0011 (AP versus AA), 0.0022 (AP versus PP) and 0.9063 (AA versus PP). One-way ANOVA, followed by Tukey’s test. d, Percentage of branching and invaginating PROX1-positive organoids, defined as in Extended Data Fig. 7b. The AP combination produced spheroids with branching and invagination, whereas other two combinations did not. e, Failure to branch and invaginate from the posterior region of HBPOs. Dissected posterior region from day 11 organoid cultured until day 30. Whereas HBPO formed a PROX1-expressing branching structure, the posterior dissected region of HBPO that shows PDX1 expression did not form this structure. Images are representative of two independent experiments with similar results. Scale bar, 200 μm.
a, Immunofluorescent staining for AFP, albumin and HHEX at day 30. AFP and albumin were expressed in the same region but HHEX was not expressed. Expression of HHEX, a hepatocyte progenitor marker, was lost at this late stage. The experiment was repeated twice independently with similar results. b, Immunofluorescent staining for NKX6.1, NKX6.3 and PDX1. NKX6.3 was expressed in the vicinity of pancreatic markers PDX1 and NKX6.1. The experiment was repeated three times independently with similar results. c. Immunofluorescent staining for EpCAM, PROX1, SOX9 and CLF. Representative image of n = 3 independent organoids showing similar results. Scale bar, 100 μm.
Extended Data Fig. 10 Upregulation of pancreatic marker genes and depletion of bile duct markers in HES−/− organoids.
a, Gene-targeting strategy for HES1-knockout (KO) line using the CRISPR–Cas9 system. b, Confirmation of modified gene sequence of control and HES1 knockout (Del #11). c, Representative image of n = 3 HES1−/− iPSC culture showing similar results. Scale bar, 500 µm, d, HES1 expression in an organoid at day 20. Data are mean ± s.d.; n = 6 independent organoids. Unpaired two-tailed t-test. e, Heat map shows gene-expression profile of pancreas-associated markers at day 22 of HES1+/+ and HES1−/− HBPOs. These data correspond to Fig. 4c. f, Connected structure is disrupted in long-term cultured HES1-knockout organoids. Whole-mount staining of DBA and SOX9 in HES1−/− and HES1+/+ organoids. DBA and SOX9 signal was lost in HES1−/− organoids. The experiment was repeated three times independently with similar results. Scale bar, 200 μm.