Abstract
Current organoid models are limited by their inability to mimic mature organ architecture and associated tissue microenvironments1,2. Here we create multilayer bladder ‘assembloids’ by reconstituting tissue stem cells with stromal components to represent an organized architecture with an epithelium surrounding stroma and an outer muscle layer. These assembloids exhibit characteristics of mature adult bladders in cell composition and gene expression at the single-cell transcriptome level, and recapitulate in vivo tissue dynamics of regenerative responses to injury. We also develop malignant counterpart tumour assembloids to recapitulate the in vivo pathophysiological features of urothelial carcinoma. Using the genetically manipulated tumour-assembloid platform, we identify tumoural FOXA1, induced by stromal bone morphogenetic protein (BMP), as a master pioneer factor that drives enhancer reprogramming for the determination of tumour phenotype, suggesting the importance of the FOXA1–BMP–hedgehog signalling feedback axis between tumour and stroma in the control of tumour plasticity.
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Data availability
All relevant data are included in this Article and its Supplementary Information files. RNA-seq, ChIP–seq, ATAC-seq and scRNA-seq data have been uploaded to the Gene Expression Omnibus with accession codes GSE141356 and GSE141348, respectively. These data (RNA-seq, ChIP–seq, ATAC-seq and scRNA-seq) are also available on the Korean Nucleotide Archive with accession ID PRKJA200011 upon request to the corresponding authors. WES data have been uploaded to the Sequence Read Archive with accession code PRJNA647991. The MSigDB v.7.0 is available at https://www.gsea-msigdb.org/gsea/msigdb. Source data are provided with this paper.
Code availability
The code used for data analysis is available at https://github.com/sysgenlab/BladderAssembloid.
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Acknowledgements
We thank I. Weissman at Stanford University for their generous provision of the Rainbow mouse. This research was supported by grants from the National Research Foundation of Korea (NRF-2020R1A2B5B01001490, NRF-2017M3C7A104787521, NRF-2017R1A5A101536614 and NRF-2014M3C9A3064548), Gyeongbuk Science and Technology Promotion Center of Korea (SF317001A), POSCO (2018Y060), and the BK21 Plus and BK21 FOUR Research Fellowship.
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Authors and Affiliations
Contributions
E.K. and K.S. conceived ideas and experimental design. E.K., S.C., Y.K. and S.E.K. performed the overall experiments. B.K., H.-M.K., E.K. and T.-Y.R. performed RNA-seq, ATAC-seq, ChIP–seq and scRNA-seq analysis and wrote the relevant Methods. J.K., E.K. and S.K. analysed data from WES and scRNA-seq. W.H.Y., H.-R.L. and S.J. performed the 3D printing experiments. S.J. provided advice on 3D bioprinting. C.Y. and Y.J.L. helped with FACS analysis. T.-Y.R. and S.K. provided advice on bioinformatic analyses. H.S.L., M.K. and J.H.K. provided patient specimens and helped analyse data. E.K. and K.S. wrote the manuscript.
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Extended data figures and tables
Extended Data Fig. 1 Single-cell-derived bladder organoids were maintained for over a year by short-term serial passaging or long-term culture.
a, Schematic diagram illustrating the experimental strategies for the short- and long-term culture of mouse normal bladder organoids. b, Time-course images showing that single urothelial cells successfully generated a bladder organoid. Scale bar, 100 μm. c, Quantification of organoid size when cultured for 9 days at each passage. d, Quantification of the organoid-forming efficiency at each passage. e, Bright-field images of serially passaged short-term (day 9) bladder organoids from passage 1 to 20. Scale bars, 100 μm. f, Representative images of serially passaged short-term (day 18) bladder organoids analysed by bright-field images and immunostaining. Scale bars, 100 μm. g, Average size of long-term cultured bladder organoids at indicated day. h, Representative images of bladder organoids at indicated times during long-term culture, analysed by bright-field images and immunostaining. Scale bars, 100 μm. i, Short-term (day 14) and long-term (day 81) organoids compared with embryonic (E16) bladders and adult (p8week) bladders. Dotted lines demarcate the border between epithelium and stroma (white arrowheads, umbrella cells). Scale bars, 20 μm. j, The relative expression of Upk2, Upk1a, Foxa1, and Gata3 in short-term (day 14) organoids, as compared to long-term (day 81) organoids. Data are mean ± s.e.m. For details on statistics, sample sizes (n), and numbers of replications, see ‘Statistics and reproducibility’ (Methods).
Extended Data Fig. 2 Bladder assembloids recapitulate tissue architecture of in vivo bladders.
a, Experimental scheme for reconstituting mouse and human bladder assembloids. b, Representative photos of 3D printed, 12-well spinning bioreactor. c, Representative images of the urothelial organoid compared with the reconstituted bladder organoid with stroma. Dotted lines demarcate the border between epithelium and stroma. Scale bars, 100 μm. d, e, Bladder organoids (d) or reconstituted bladder organoids with stroma (e) were analysed by immunostaining. Dotted lines demarcate the border between epithelium and stroma (L, lumen). Scale bars, 100 μm. f, g, Bladder assembloids (f) or wild-type bladders (g) were analysed by immunostaining. Dotted lines demarcate the borders between epithelium and stroma in the left panels, and stroma and muscle in the right panels (L, lumen; S, stroma; M, muscle). Scale bars, 100 μm. h, Representative images of human bladder organoids and human reconstituted bladder organoid with stroma. Dotted lines demarcate the border between epithelium and stroma. Scale bars, 100 μm. i, Representative images of normal fibroblasts derived from the P-9 benign urothelial samples (left) and iPSCs generated from the P-9 normal fibroblasts (right). Scale bars, 100 μm. j, Experimental strategy for stepwise differentiation of hiPSCs derived from human fibroblasts into contractile human smooth muscle cells (hSMCs). Representative images at indicated times during stepwise differentiation process are shown. Scale bar, 100 μm. k, hSMCs differentiated from hiPSCs at day 14 were analysed by immunostaining. Scale bar, 100 μm. For details on statistics, sample sizes (n), and numbers of replications, see ‘Statistics and reproducibility’ (Methods).
Extended Data Fig. 3 scRNA-seq and functional analysis of bladder assembloids.
a, t-SNE plots of human cells (HULECs) in 1-day and 7-day bladder assembloids. b, Heat map representation of gene expression for the top 15 markers expressed in wild-type endothelial cell cluster A3 and A5 compared with human endothelial cell (HULECs) cluster of 1-day and 7-day bladder assembloids. Gene expression of HULECs in bladder assembloids are represented by orthologous genes (the list of orthologous genes is from ENSEMBL BioMart database). c, Split dot plot showing gene expression for the top 15 markers expressed in endothelial cell cluster A3 or A5 of wild-type bladder compared with endothelial clusters of 1-day or 7-day assembloid (circle size, proportion of cells; degree of shading, expression level). d, Comparative analysis between wild-type bladder and bladder assembloids for the proportions of individual cell types. e, Hierarchical clustering of distinct cell types shared by wild-type bladder, 1-day-old, and 7-day-old bladder assembloids using cell type-specific top 50 genes. f, Heat map representation of gene expression between clusters in four individual cell populations for top 15 markers expressed in wild-type bladder. g, Re-clustering of epithelial cell populations in 1-day and 7-day bladder assembloids from Fig. 2a identified five and four epithelial sub-clusters, respectively. h, The proportions of each cluster of 1-day and 7-day bladder assembloids shown in panel g. i, Violin plots for selected eight basal and luminal markers in each cell cluster shown in panel g. j, Bladder assembloids treated with acetylcholine (Ach) or noradrenaline (NA) were analysed using bright-field images and immunostaining. Scale bars, 100 μm. k, Transmission electron microcopy (TEM) and immunostaining analysis for tight junctions and urothelial plaques in bladder assembloids compared with wild-type bladder (black and white arrowheads, tight junctions; L, lumen; UP; urothelial plaques). Scale bars, 1 μm (TEM) or 10 μm (immunostaining). l, Immunostaining of ZO-1 for tight junctions in human bladder assembloids (white arrowheads, tight junctions; L, lumen). Scale bars, 20 μm. For details on statistics, sample sizes (n), and numbers of replications, see ‘Statistics and reproducibility’ (Methods).
Extended Data Fig. 4 Bladder assembloids mimic the pathophysiology and represent tissue dynamics during urinary tract infection.
a, Schematic diagram for establishment of in vivo and in vitro models of UTI. b, UTI89 labelled with the fluorescent protein, AmCyan, was microinjected into the lumen bladder assembloids. Scale bars, 100 μm. c, d, Wild-type bladders (c) and bladder assembloids (d) were analysed by immunostaining. Scale bars, 50 μm. e, Wild-type bladders and bladder assembloids were analysed by immunostaining (white arrowheads, intracellular bacterial aggregates within superficial cells). Scale bars, 50 μm. f, Wild-type bladders and bladder assembloids were immunostained for KRT5, KRT18 and vimentin to highlight basal epithelial cells, luminal epithelial cells, and stromal cells, respectively. Scale bars, 100 μm. g, h, Bladder assembloids reconstituted with different cell types from Col1a2CreER;Smofl/fl mice (g, wild-type Smo; h, Smo ablated) analysed by immunostaining. The quantification of epithelial and stromal cell proliferations is shown in the right panels. Scale bars, 100 μm. i, Schematic representation of the Rainbow allele. After Cre-mediated excision of three lox sites (lox2272, loxN, and loxP) induced by tamoxifen (TM), each cell expresses either EGFP (before recombination) or mCerulean, mOrange, or mCherry (after recombination). j, Single-cell-labelled normal bladder or bladder organoid from Rainbow mouse after TM treatment. Scale bars, 100 μm. k, Experimental scheme for in vivo and in vitro lineage tracing of KRT5-expressing basal epithelial cells. l, Bladders from TM-injected Krt5CreERT2;Rosa26Rainbow/WT mice and bladder assembloids derived from Krt5CreERT2;Rosa26Rainbow/WT mice treated with 4-OHT were analysed before (UPEC day 0) and after (UPEC day 7) bacterial injection using four-colour fluorescence. Scale bars, 100 μm. m, Model for clonal relationship during UTI-induced urothelial regeneration. For details on statistics, sample sizes (n), and numbers of replications, see ‘Statistics and reproducibility’ (Methods).
Extended Data Fig. 5 Generation of bladder tumour assembloids recapitulating the histopathology, genomic alterations and tumour subtypes of human urothelial carcinomas.
a, Eight bladder tumour organoid lines were established from patient-derived invasive urothelial carcinoma samples. Resulting tumour organoids were analysed by H&E staining and immunostaining to mark basal (KRT5) and luminal (KRT18) cells. Scale bars, 100 μm. b–i, The relative expressions of luminal (UPK1A, UPK2, ERBB2, FOXA1, and GATA3) and basal (KRT5, KRT14, CDH3, and KRT6A) markers in eight patient-derived bladder tumour organoids. j, Summary of molecular subtypes of eight bladder tumour organoids based on gene expression analysis. k, Experimental scheme for reconstituting patient-derived bladder tumour assembloids. l, m, Histopathology of tumour organoids, tumour assembloids, xenografts, and parental tumours derived from P-3 and P-6 were analysed by H&E staining and immunostaining. Scale bars, 100 μm. n, Bladder tumour assembloids derived from P-1 were analysed by immunostaining to mark tumour cells, CAFs, and endothelial cells (arrowheads, interconnected regions). Scale bars, 100 μm. o, Tumour growth was quantified by measuring tumour areas using the Image J program. p, Bladder tumour assembloids reconstituted without or with endothelial cells were analysed by immunostaining. Scale bars, 100 μm. q, Comparative analysis for mutations detected by WES of parental tumours, tumour organoids, and tumour assembloids. r, Parental tumours, tumour organoids at late passages (late organoid), and tumour assembloids at late passages (late assembloid) were analysed by immunostaining. Scale bars, 50 μm. s, Relative expression of luminal (UPK1A, UPK2, ERBB2, FOXA1, and GATA3) and basal (KRT5, KRT14, CDH3, and KRT6A) markers in parental tumours, late organoids, and late assembloids. Data are mean ± s.e.m. For details on statistics, sample sizes (n), and numbers of replications, see ‘Statistics and reproducibility’ (Methods).
Extended Data Fig. 6 3D bioprinting-based, reconstituted bladder tumour assembloids recapitulate the pathophysiology of patient-derived urothelial carcinoma.
a, Schematic diagram for 3D bioprinting-based reconstitution of patient-derived bladder tumour assembloids. b, c, 3D bioprinted bladder tumour organoids and assembloids derived from the P-1 (b) or P-3 (c) lines were analysed by H&E staining and immunostaining. Scale bars, 100 μm. d, e, 3D bioprinted bladder tumour assembloids treated with SAG, FK506, or vehicle control were analysed by immunostaining for KRT18 (luminal subtype; d) or KRT5 (basal subtype; e), and vimentin, which was compared with bladder tumour organoids. Quantification of tumour cell number is shown. Scale bars, 100 μm. f, g, 3D bioprinted bladder tumour assembloids treated with cisplatin were analysed by caspase 3, which was compared with bladder tumour organoids. Quantification of tumour cell apoptosis is shown. Scale bars, 100 μm. h, Experimental strategy for stepwise differentiation of human ES cells into contractile hSMCs. Representative images at indicated times during stepwise differentiation process are shown. Scale bar, 100 μm. i, hSMCs differentiated from human ES cells at day 14 were analysed by immunostaining. Scale bar, 100 μm. j, Experimental scheme to create bladder tumour assembloids containing an outer muscle layer. k, Bladder tumour assembloids containing outer muscle layer derived from P-7 and P-3 were analysed by immunostaining. Dotted lines demarcate the border between the stromal and muscle layer. Scale bar, 100 μm. Data are mean ± s.e.m. For details on statistics, sample sizes (n), and numbers of replications, see ‘Statistics and reproducibility’ (Methods).
Extended Data Fig. 7 Bladder tumour assembloids mimic the pathophysiology of bladder tumours and show the CD8 T cell-mediated tumour cytotoxicity.
a, Experimental scheme for reconstituting mouse bladder tumour assembloids. b, Tumour organoids derived from BBN-induced mouse bladder tumours, reconstituted bladder tumour assembloids, and endogenous BBN-induced mouse bladder tumours were analysed by H&E staining and immunostaining. Scale bars, 100 μm. c, Mouse bladder tumour organoids and tumour assembloids treated with SAG, FK506, or vehicle control were analysed by immunostaining. Tumour growth was quantified by measuring tumour areas using the Image J program. Scale bars, 100 μm. d, Experimental strategy for generating tumour-reactive T cells. e, Gating strategy to analyse CD8 T cells (B220-CD11b-TCRβ+CD4-CD8+) shown in panel f. f, Flow cytometry plots gated on tumour-reactive CD8 T cells expressing CD69 and IFNγ. g, Experimental strategy for generating bladder tumour assembloids containing tumour-reactive T cells. h, Bladder tumour assembloids containing tumour-reactive T cells were analysed by bright-field imaging (dotted lines, tumour areas) and immunostaining. Scale bars, 100 μm. i, Schematic representation for the strategy to systematically discover tumour neoantigens and test tumour reactivity within the tumour-assembloid platform. Tumour-specific mutations in BBN-induced mouse tumours were detected by WES and identified through application of mutation calling algorithms (Mutect). Candidate neoepitopes were predicted using well-validated algorithms (NetMHCpan) and selected based on binding affinity to H-2-Db and H-2-Kb molecules. Tumour reactivity of selected neoantigen-specific T cells was validated by increased Ifng expression. These candidate neoantigen-specific T cells were further reconstituted to test their ability to stimulate tumour-specific T cell responses within the assembloid platform. j, The list of putative neoantigens considered to bind strongly to H-2-Db (left) or H-2-Kb (right). Top 10 neoantigen candidates are displayed in order of binding affinity of mutant peptide relative to wt peptide. Only one mutant, LGFSNYPEL (Olfr512 mutant), is predicted to bind strongly to both H-2-Db and H-2-Kb. k, Relative expression of Ifng in T cells stimulated with wt-Olfr512 peptide compared with the mutant-Olfr512 peptide. l, Bladder tumour assembloids containing neoantigen-reactive T cells were analysed by bright-field images (dotted lines, tumour areas) and immunostaining. Quantifications of tumour size and tumour cell apoptosis are shown. Scale bars, 100 μm. Data are mean ± s.e.m. For details on statistics, sample sizes (n), and numbers of replications, see ‘Statistics and reproducibility’ (Methods).
Extended Data Fig. 8 Prospective model for the development of next-generation assembloids.
Our study provides a conceptual framework for development of multilayered, functional organoids derived from tissue stem cells or tumour cells that mimic the biology of native tissues. These miniature tissues, assembloids, could serve as model systems for various diseases, including cancers and degenerative diseases. With 3D bioprinting technology, this platform may facilitate the establishment of an in vitro organoid system for high-throughput drug screening or repositioning to develop precise and personalized therapies.
Extended Data Fig. 9 Genetically manipulated tumour assembloids show differential epigenomic landscape of chromatin accessibility and enhancer activity between basal and luminal tumours.
a, Basal tumour organoids and luminal tumour organoids were reconstituted with patient-derived CAFs, followed by shaking incubation in a spinning bioreactor. b, Basal tumour organoids infected with lentivirus to overexpress FOXA1 were reconstituted with CAFs. Luminal tumour organoids infected with lentivirus to knockout FOXA1 were reconstituted with CAF. c, Basal tumour organoids were reconstituted with CAFs manipulated to overexpress BMP4 and BMP5. Luminal tumour organoids were reconstituted with CAFs manipulated to knockout BMP4 and BMP5. d, Basal tumour organoids infected with lentivirus to knockout FOXA1 were reconstituted with CAFs manipulated to overexpress BMP4 and BMP5 and were cultured by shaking incubation. Luminal tumour organoids infected with lentivirus to overexpress FOXA1 were reconstituted with CAFs manipulated to knockout BMP4 and BMP5 and were cultured by shaking incubation. e, f, Relative expression of FOXA1 in FOXA1-overexpressing basal tumour organoids was analysed by RT–qPCR. KO, knockout; OE, overexpression. g, h, Relative expression of FOXA1 in FOXA1-knocked out luminal tumour organoids was analysed by RT–qPCR. i, j, Relative expression of BMP4 or BMP5 in BMP4/BMP5-overexpressing CAFs was analysed by RT–qPCR. k, l, Relative expression of BMP4 or BMP5 in BMP4/BMP5-knocked out CAFs was analysed by RT–qPCR. m, Sequences of guide RNAs and target genes for knockout of BMP4, BMP5, and FOXA1 (PAM sequences are marked in red). n, p, Heat map representation of regions with increased chromatin accessibility/H3K27ac signals in luminal (n) or basal (p) in four assembloids (L1 = P-1; L2 = P-6; B1 = P-2; B2 = P-3). Each row; individual line (n = 4). Each column; single region (centre +/− 5Kb). o, q, Pie chart showing the genomic annotations of regions with increased chromatin accessibility/H3K27ac signals in luminal (o) or basal (q) according to the location of a given peak. TSS, “−1-kb to +100-bp” of transcription start sites. TTS, “−100-bp to +1-Kb” of transcription termination sites. r, Top 5 enriched de novo motifs and transcription factors with best matched binding motifs in regions with increased chromatin accessibility/H3K27ac signals in luminal. The most enriched motif shows high similarity with Forkhead box transcription factor binding motifs. s, Representative ATAC-seq, H3K27ac ChIP–seq, and FOXA1 ChIP–seq profiles of the regions at ISX loci in the indicated samples. t, Pathway analysis for genes associated with increased chromatin accessibility/H3K27ac signals in luminal. Only top 10 GO terms ranked by their significance are shown. Data are mean ± s.e.m. For details on statistics, sample sizes (n), and numbers of replications, see ‘Statistics and reproducibility’ (Methods).
Extended Data Fig. 10 FOXA1-mediated enhancer reprogramming confers tumour subtype plasticity of urothelial carcinomas through a FOXA1–BMP–hedgehog signalling feedback axis between the epithelial tumour and stroma.
a, c, Heat maps of ATAC-seq, H3K27ac ChIP–seq, and RNA-seq for GAIN/LOSS regions/genes from indicated samples. Hierarchical clustering was performed on ATAC-seq and H3K27ac ChIP–seq. b, d, GSEA of indicated samples to assess differential enrichment of GAIN genes (left) and signature genes for cell differentiation (right). NES, normalized enrichment score. Nominal p value is shown e, f, Relative expression of FOXA1 in basal tumour organoid B1 (e) and B2 (f), treated with BMP4, BMP5, or BMP4 and BMP5 together. Data are mean ± s.e.m. g, h, Representative ATAC-seq, H3K27ac ChIP–seq, and FOXA1 ChIP–seq profiles of the regions at IRX2 loci in the indicated samples. i, j, Molecular subtypes of indicated samples were analysed using the BASE47 together with MDACC classifiers. Heat maps show the normalized gene expression profile organized by the luminal and basal classifier genes. k, l, Heat map representation of unsupervised hierarchical clustering from ATAC-seq (k) or H3K27ac ChIP–seq (l) data for tumour assembloids in each experiment summarized in Extended Data Fig. 9a–d. Samples were clustered based on the Spearman correlation coefficient with single linkage. m, Principal components analysis from RNA-seq data for tumour assembloids in each experiment summarized in Extended Data Fig. 9a–d. n, Representation of motifs enriched at the regions with increased chromatin accessibility/H3K27ac signals in basal. o, Top 5 enriched de novo motifs and transcription factors with best matched binding motifs in regions with increased chromatin accessibility/H3K27ac signals in basal. The most enriched motif shows high similarity with AP-1 transcription factor binding motifs. For details on statistics, sample sizes (n), and numbers of replications, see ‘Statistics and reproducibility’ (Methods).
Supplementary information
Supplementary Table 1
Patient sources of human invasive urothelial carcinoma samples. Invasive urothelial carcinoma samples were obtained from TURB or radical cystectomy patients with disease and treatment histories as shown.
Supplementary Video 1.
Plate-making process for the spinning bioreactor using a 3D printer.
Supplementary Video 2.
Gear-making process for the spinning bioreactor using a 3D printer.
Supplementary Video 3.
Operation of 3D-printed spinning bioreactor.
Supplementary Video 4.
Incubation of reconstituted assembloids with shaking in a 3D-printed spinning bioreactor.
Supplementary Video 5.
Microinjection of UPEC into the lumen of reconstituted bladder assembloids.
Supplementary Video 6.
3D bioprinting-based reconstitution of patient-derived bladder tumour assembloids.
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Kim, E., Choi, S., Kang, B. et al. Creation of bladder assembloids mimicking tissue regeneration and cancer. Nature 588, 664–669 (2020). https://doi.org/10.1038/s41586-020-3034-x
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DOI: https://doi.org/10.1038/s41586-020-3034-x
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