Modelling the impact of decidual senescence on embryo implantation in human endometrial assembloids
- Division of Biomedical Sciences, Warwick Medical School, University of Warwick, United Kingdom
- Centre for Early Life, Warwick Medical School, University of Warwick, United Kingdom
- Centre for Reproductive Medicine, University Hospitals Coventry and Warwickshire NHS Trust, United Kingdom
- Department of Physiology, Development and Neuroscience, University of Cambridge, United Kingdom
- Tommy’s National Centre for Miscarriage Research, University Hospitals Coventry & Warwickshire NHS Trust, United Kingdom
- Synthetic Mouse and Human Embryology Group, California Institute of Technology (Caltech), Division of Biology and Biological Engineering, United Kingdom
Figures
Figure 1
Establishment of endometrial assembloids.
(A) Schematic for establishing endometrial assembloids. (B) Structural appearance of hematoxylin and eosin stained secretory endometrium, E-cadherin labelled gland-like organoids, and E-cadherin and …
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Figure 1—source data 1
Secretion of PRL and CXCL14 by endometrial assembloids.
Secretion of PRL and CXCL 14 (pg/ml) was measured by ELISA in spent medium from assembloids. Supports Figure 1D.
- https://cdn.elifesciences.org/articles/69603/elife-69603-fig1-data1-v2.xlsx
Figure 2
Characterization of a minimal differentiation medium for endometrial assembloids.
Parallel gland-like organoids (red) and assembloids (blue) were established from three endometrial biopsies and decidualized with 8-bromo-cAMP and MPA for 4 days in either expansion medium (ExM), …
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Figure 2—source data 1
RTqPCR data associated with the minimal differentiation medium (MDM) experiments.
Parallel epithelial gland organoids and assembloids were established from endometrial biopsies and decidualized with 8-bromo-cAMP and MPA for 4 days in either expansion medium (ExM), base medium (BM), or BM with each exogeneous factor added back individually (red).
- https://cdn.elifesciences.org/articles/69603/elife-69603-fig2-data1-v2.xlsx
Figure 3 with 3 supplements
Decidualizing assembloids mimic midluteal endometrium.
(A) Schematic overview of experimental design. ExM: expansion medium; MDM: minimal differentiation medium. (B) Uniform Manifold Approximation and Projection (UMAP) visualizing epithelial and stromal …
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Figure 3—source data 1
Epithelial subpopulation markers.
Population markers were generated in Seurat v3 using FindMarkers on specified comparisons. Supports Figure 3C.
- https://cdn.elifesciences.org/articles/69603/elife-69603-fig3-data1-v2.xlsx
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Figure 3—source data 2
GO analysis of differentiated subpopulations.
- https://cdn.elifesciences.org/articles/69603/elife-69603-fig3-data2-v2.xlsx
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Figure 3—source data 3
Stromal sub-population markers.
Population markers were generated in Seurat v3 using FindMarkers on specified comparisons. Supports Figure 3E.
- https://cdn.elifesciences.org/articles/69603/elife-69603-fig3-data3-v2.xlsx
Figure 3—figure supplement 1
Heatmap showing relative expression (Z-scores) of genes encoding the cyclin-dependent kinase inhibitors p16INK4a and p21CIP1 as well as SASP-related genes in epithelial and transitional subpopulations in decidualizing assembloids.
Supports Figure 3C.
Figure 3—figure supplement 2
Heatmap showing relative expression (Z-scores) of epithelial-mesenchymal transition/mesenchymal-epithelial transition (EMT/MET), epithelial and mesenchymal marker genes in the transitional population (TP), epithelial (EpS4-5) and stromal (SS3-5) subpopulations in decidualizing assembloids.
Supports Figure 3C.
Figure 3—figure supplement 3
Heatmap showing relative expression (Z-scores) of genes encoding the cyclin-dependent kinase inhibitors p16INK4a and p21CIP1 as well as secretory and SASP-related genes in stromal subpopulations (SS3-5) in decidualizing assembloids.
Supports Figure 3D.
Figure 4
Putative receptor-ligand interactions in decidualizing assembloids.
(A) Heatmap showing the total number of cell-cell interactions predicted by CellPhoneDB between different subpopulations in decidualizing assembloids. (B) Dot plots of representative ligand-receptor …
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Figure 4—source data 1
CellPhoneDB prediction of cell-cell interactions.
Ligand-receptor interactions were predicted using CellPhoneDB on counts data from Seurat v3.
- https://cdn.elifesciences.org/articles/69603/elife-69603-fig4-data1-v2.xlsx
Figure 5
Tyrosine kinase-dependent stress responses determine the fate of decidual cells.
(A) Schematic overview of experimental design. ExM: expansion medium; MDM: minimal differentiation medium. (B) Uniform Manifold Approximation and Projection (UMAP) visualization (left panel) and …
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Figure 5—source data 1
Differentially expressed genes for day 4 populations treated with and without dasatinib.
Specified pairwise comparisons were generated in Seurat v3 using FindMarkers. Supports Figure 5C.
- https://cdn.elifesciences.org/articles/69603/elife-69603-fig5-data1-v2.xlsx
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Figure 5—source data 2
GO analysis for day 4 populations treated with and without dasatinib.
- https://cdn.elifesciences.org/articles/69603/elife-69603-fig5-data2-v2.xlsx
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Figure 5—source data 3
ELISA data.
Secreted levels of key senescent (CXCL8) and decidual stromal cell markers (CXCL14, IL-15, TIMP3) (pg/ml) were examined by ELISA in spent medium from assembloids treated with or without dasatinib. Supports Figure 5D.
- https://cdn.elifesciences.org/articles/69603/elife-69603-fig5-data3-v2.xlsx
Figure 6 with 2 supplements
Impact of decidual senescence in assembloids on co-cultured human blastocysts.
(A) Diagram showing experimental design. ExM: expansion medium; MDM: minimal differentiation medium; EM: embryo medium. (B) Schematic drawing of co-culture method. (C) Representative time-lapse …
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Figure 6—source data 1
Embryo expansion measurement.
Embryo diameters were measured over 72 hr co-culture with assembloids previously differentiated with or without dasatinib. Images were captured using time-lapse imaging. Supports Figure 6D.
- https://cdn.elifesciences.org/articles/69603/elife-69603-fig6-data1-v2.xlsx
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Figure 6—source data 2
Embryo human chorionic gonadotropin (hCG) secretion.
Secreted levels of hCG (pg/ml) by embryos in co-culture with assembloids previously differentiated with or without dasatinib were examined by ELISA in spent medium. Supports Figure 6F.
- https://cdn.elifesciences.org/articles/69603/elife-69603-fig6-data2-v2.xlsx
Figure 6—figure supplement 1
Stromal migration towards the polar trophectoderm of expanding embryos in differentiated endometrial assembloids.
Images were captured by time-lapse microscopy and annotated to indicate the frontier of the stromal egress (white dotted line).
Figure 6—figure supplement 2
Dasatinib prevents disintegration of decidualizing assembloids.
Time-course images of endometrial assembloids in minimal differentiation medium (MDM) supplemented or not with dasatinib. Scale bar = 50 µm. Supports Figure 6C.
Author response image 1
(A) Expression of FOXA2, a glandular epithelial marker (green font), and putative luminal epithelial marker genes (black font) in assembloid subpopulations.
Dot size indicates the proportion of cells expressing the marker, while colour indicates the level of expression. Note that none of the cell populations express a compelling luminal epithelial …
Author response image 2
Comparison of secreted levels of IL-8, CXCL14 and TIMP3 in two independent primary endometrial stromal cells maintained in standard 2D cultures in response to standard growth medium (10% DCC-DMEM+E2) or assembloid expansion medium (ExM+E2) and standard differentiation medium (2% DCC-DMEM+cAMP+MPA) or assembloid differentiation medium (MDM+E2+cAMP+MPA), as indicated.
Author response image 3
Videos
Video 1
Time-lapse microscopy of a human blastocyst embedded in a decidualizing assembloid.
Representative video of a human blastocyst embedded in an assembloid, as imaged by time-lapse microscopy over 72 hr with images captured every 60 min.
Video 2
Time-lapse microscopy of a human blastocyst embedded in a decidualizing assembloid pre-treated with dasatinib.
Representative video of a human blastocyst embedded in an assembloid which had been pre-treated with dasatinib, as imaged by time-lapse microscopy over 72 hr with images captured every 60 min.
Additional files
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Supplementary file 1
Supplementary tables 1-3.
Table 1. Culture media composition. Table 2. Patient demographics for endometrial samples. Table 3. Antibody details for immunofluorescence labelling.
- https://cdn.elifesciences.org/articles/69603/elife-69603-supp1-v2.xlsx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/69603/elife-69603-transrepform1-v2.docx
