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Long-term, hormone-responsive organoid cultures of human endometrium in a chemically defined medium

Abstract

In humans, the endometrium, the uterine mucosal lining, undergoes dynamic changes throughout the menstrual cycle and pregnancy. Despite the importance of the endometrium as the site of implantation and nutritional support for the conceptus, there are no long-term culture systems that recapitulate endometrial function in vitro. We adapted conditions used to establish human adult stem-cell-derived organoid cultures to generate three-dimensional cultures of normal and decidualized human endometrium. These organoids expand long-term, are genetically stable and differentiate following treatment with reproductive hormones. Single cells from both endometrium and decidua can generate a fully functional organoid. Transcript analysis confirmed great similarity between organoids and the primary tissue of origin. On exposure to pregnancy signals, endometrial organoids develop characteristics of early pregnancy. We also derived organoids from malignant endometrium, and so provide a foundation to study common diseases, such as endometriosis and endometrial cancer, as well as the physiology of early gestation.

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Figure 1: Long-term 3D organoid cultures can be established from human non-pregnant endometrium and decidua.
Figure 2: Established human endometrial organoids recapitulate the molecular signature of glands in vivo.
Figure 3: Human endometrial organoids respond to sex hormones.
Figure 4: Signals from decidualized stroma and the placenta can further stimulate differentiation of human endometrial organoids.
Figure 5: Human endometrial organoids have clonogenic ability and are bipotent.
Figure 6: Derivation of organoids from endometrial carcinomas.

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Acknowledgements

The authors are grateful to patients for donating tissue for research. We thank D. Moore, R. Remadevi, M. Baumgarten, M. Jimenez-Linan, D. S. Charnock-Jones, Department of Obstetrics and Gynaecology and NHS Tissue Bank staff at Addenbrooke’s Hospital, Cambridge; H. Skelton for her invaluable histological services and technical advice; I. Pshenichnaya, K. Bird and A. Starling at the Stem Cell Institute for their histological services; J. Bauer and Cambridge Genomic Services for microarray analysis; I. Simonic at Medical Genetics Laboratory, Cambridge University Hospital for CGH analysis; J. N. Skepper for electron microscopic analysis; N. Miller for flow cytometry sorting; H. W. Yung and A. Sharkey for technical help and advice; J. Cross and Y. W. Loke provided much helpful discussion and all members of the Moffett laboratory were supportive throughout. This work was supported by the Medical Research Council (MR/L020041/1), the Centre for Trophoblast Research, University of Cambridge and the Wellcome Trust (RG60992). M.Y.T. has received funding from the E.U. 7th Framework Programme for research, technological development and demonstration under grant agreement no PIEF-GA-2013-629785. J.H. was supported by a Wellcome Trust vacation scholarship. B.-K.K. is supported by a Sir Henry Dale Fellowship from the Wellcome Trust and the Royal Society (101241/Z/13/Z) and receives a core support grant from the Wellcome Trust and MRC to the WT-MRC Cambridge Stem Cell Institute.

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M.Y.T. and L.G. designed and carried out all experiments and data analyses; J.H. and T.C.-D. assisted with experiments and data analyses; M.J.G. performed microarray analysis; M.Hollinshead performed EM analysis and assisted with confocal analysis; J.J.B. and H.O.C. provided endometrial specimens and input for the manuscript; L.F. and S.G.E.M. assisted with experiments; A.M. and B.-K.K. assisted with experimental design, analyses of results and preparation of manuscript; B.D.S. and M.Hemberger assisted with analyses of results and preparation of manuscript; M.Y.T., A.M. and G.J.B. wrote the manuscript.

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Correspondence to Margherita Y. Turco or Graham J. Burton.

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Integrated supplementary information

Supplementary Figure 1 Growth factor requirements of established endometrial organoids.

(a) Bright field (BF) images of spheroid formation assay for endometrial organoids at d 7. Single factors were omitted from ExM (control) as indicated. Noggin (NG), Rspondin-1 (RSPO1) and Nicotinamide (NIC). Scale bar, 500 μm. Representative of experiments performed with 3 decidual organoids derived from different donors. (b) BF images of endometrial organoids after 4 passages in ENR, ENR + A83-01, ENR + Nicotinamide, ENR + A83-01 + Nicotinamide and ExM. Scale bar, 500 μm. Representative of experiments performed with 3 endometrial organoids derived from different donors. (c) BF images of organoids derived from atrophic post-menopausal endometrium 10 d after plating and cultured under ENR, ENR + A83-01 + Nic and ExM conditions. Scale bar, 500 μm. Only 1 tissue sample obtained for this experiment. (d) BF images of endometrial organoids at early passage 2, (p2) and late passage 8 (p8). Scale bar, 100 μm. (e) Analysis of genetic stability of cultures with CGH array. A representative whole-genome array CGH plot generated using Agilent Cytogenomics software. Genomic DNA from early passage (p2) endometrial organoids (red) is compared to genomic DNA from original sample (blue). Each spot is a single probe. Log ratios of the average signal intensity of each probe on the Y-axis along its position on the chromosomes (1-22, X and Y) on the x-axis. A log signal ratio of 0 represents equivalent copy number. High signal in the Y chromosome region is not significant and is due to absence of the Y chromosome in the samples. Repeated with 4 different decidual organoids and 2 endometrial organoids. (f) Genomic DNA from late passage (p8) endometrial organoids (red) compared to genomic DNA from early passage (p2) organoids (blue). The plot represents data described in the same way as in e. Repeated with 4 different decidual organoids and 2 endometrial organoids.

Supplementary Figure 2 Cell isolation procedure from endometrium enriches for glands and stromal cells.

(a) BF image of gland fragments generated from endometrium. Scale bar, 500 μm. (b) IF staining on cytospin smears of gland isolates shows enrichment for MUC1-positive fragments. Scale bar, 100 μm. Experiment repeated twice with endometrial digests from different donors. (c) Phase-contrast image of endometrial stromal cultures isolated from endometrium showing typical fibroblast morphology (passage 2). Scale bar, 200 μm. (d) IF staining of endometrial stromal cultures shows cells are uniformly positive for mesenchymal marker VIMENTIN (far left, scale bar, 50 μm). Image at higher magnification (centre, scale bar 50 μm) and zoom-in image (far right) show filamentous staining pattern typical of VIMENTIN (white arrowheads). Experiment repeated twice with stromal cells isolated from endometrial samples from different patients. (e) Clustered heatmap of 376 genes upregulated in stromal cells compared to gland digests and organoids derived from endometrium (n = 7 independent donors). Genes descriptive of fibroblast function (magenta), genes that encode for signalling pathways (blue) and genes that encode for fibroblast markers (cyan). (f) Gene ontology (GO) analysis of upregulated genes from (e) using HumanMine v2.2 database for GO Terms Molecular Function with Benjamini Hochberg test correction with maximum P-value of 0.05. The top eight significantly enriched GO terms for each category are shown with the –log of their P-values on the x-axis. (g) GO analysis of upregulated genes from (e) using HumanMine v2.2 database for GO Terms Cellular Components with Benjamini Hochberg test correction with maximum P-value of 0.05. The top eight significantly enriched GO terms are shown with the –log of their P-values on the x-axis.

Supplementary Figure 3 Expression of stem cell markers LRIG1, AXIN2 and SSEA-1 in human endometrium.

(a) ISH for LRIG1 on functional and basal layers of proliferative endometrium. LRIG1 transcripts are detected in both luminal (LE) and glandular (GE) compartments (positive signal, in brown, DAB and counterstain in purple with Carazzi’s hematoxylin). Stroma (St) and myometrium (M) are negative. Positive control probe is for PPIB. Negative control probe is for the bacterial gene dapB. Scale bars, 50 μm. Representative of 3 different endometrial samples. (b) ISH to localize AXIN2 expression in human endometrium (positive signal, in brown, DAB). AXIN2 transcripts are found within GE and not in St. Scale bars, 50 μm (main image) and 10 μm (inset). Representative of 3 different endometrial samples. (c) IHC for SSEA-1 on endometrial organoids showing a small population of positive cells. Representative of 3 different endometrial samples. (d) FACS sorting gates for SSEA-1 on endometrial organoids. Organoids were processed to obtain single cells and stained for SSEA-1-PE and 7-AAD. Gating strategy (from left to right); debris was gated out on forward/side scatter (cells, R1), singlets were gated on (R2) and live cells based on negativity for 7-AAD (R3). To ensure sorted SSEA-1 +/- cells are pure, gating between them was distanced by a log difference. Negative control using mouse IgG-PE was used for gating positive populations. Specificity of SSEA-1 staining was tested on peripheral blood where it is expressed on granulocytes and monocytes. Percentages of SSEA-1 + cells from organoid cultures derived from 4 different donors are shown in table below.

Supplementary Figure 4 Organoids derived from gestational endometrium (decidua) are similar to endometrial organoids.

(a) Unsupervised hierarchical clustering analysis of global gene expression profiles by microarray of initial glandular digests, primary isolates of stromal cells and corresponding established organoids from endometrial biopsies. Highlighted in red, endometrial organoid_1 to organoid_7, highlighted in yellow: decidual organoid_8 to organoid_13. Analysis based on 20611 probes with sd/mean > 0.1. Glands and organoids derived from endometrium and decidua cluster together whilst the stroma clustered separately. (b) IHC for FOXA2, SOX17 and PAX8 expression identified from the microarray as characteristic of endometrial glands. All gland cells in the decidua and organoids derived from decidua are positive for these markers. Scale bars, 50 μm (main image) and 10 μm (insets). Representative images of negative controls on decidua and organoids using mouse or rabbit IgGs are shown. Representative of 7 decidual samples and organoids derived from 5 different patients. (c) ISH for LRIG1 on decidua and organoids derived from decidua. Positive control probe is for PPIB. Negative control probe is for the bacterial gene dapB. Scale bars, 50 μm (main image) and 10 μm (insets). Representative of 3 decidual samples and organoids derived from 3 different patients.

Supplementary Figure 5 Analysis of genes that are differentially expressed between isolated glands and organoids in comparison to stromal cells.

Genes that are only expressed in one or other group without restriction to a fold change of ≥1.5 were examined. For glands, these are 421/652, and for organoids 286/484. Gene ontology analysis using R package TopGO (stringent Weight method) of 421 genes expressed only in isolated glands show terms that describe interaction of cells with their microenvironment, such as integrins and extracellular matrices. The 268 genes expressed only in organoids contain GO terms describing proliferation and DNA replication, reflecting the expanding in vitro system.

Supplementary Figure 6 Further characterization of endometrial and decidual organoid differentiation.

(a) IHC for ERα and PR on decidual organoids cultured in ExM and after hormonal stimulation. Expression and upregulation of ERα and PR in ExM and after hormonal stimulation is similar to that of endometrial organoids (Fig. 3c). Scale bars, 50 μm (main image) and 10 μm (insets). Representative of stimulation of decidual organoid cultures derived from 3 different patients. (b) QRT-PCR for differentiation markers PAEP and SPP1 in endometrial organoids stimulated with differentiation protocol described in Fig. 3b, in the presence and absence of cAMP. Addition of cAMP to differentiation medium enhances upregulation of these markers. Results from endometrial organoids derived from 3 different donors are shown individually. (c) Effect of conditioned medium from endometrial stromal cells stimulated with hormones on cilia formation in endometrial organoids. Acetylated-α tubulin are shown in brown (DAB) and cells are counterstained with Carazzi’s Hematoxylin. Ciliated cells are present in both ExM + E2 + P4 + cAMP + PRL and d 10 stromal cell conditioned medium Scale bars, 50 μm (main image) and 10 μm (insets). Representative of treatment of endometrial organoids derived from 3 different patients.

Supplementary Figure 7 Unprocessed scans of western blots for Fig. 3f.

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Turco, M., Gardner, L., Hughes, J. et al. Long-term, hormone-responsive organoid cultures of human endometrium in a chemically defined medium. Nat Cell Biol 19, 568–577 (2017). https://doi.org/10.1038/ncb3516

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