Developing receptive endometrial assembloids in vitro

(A) Human endometrial assembloids constructed from adult stem cells were treated with expansion medium (ExM) (CTRL) or subjected to hormonal stimulation. Timeline of endometrial assembloid cultured by ExM (CTRL), ovarian steroid hormones simulating secretory phase (SEC), ovarian steroid hormones combining PRL and placental hormones to mimic the window of implantation (WOI).

(B) Endometrial assembloids in the CTRL, SEC and WOI groups displayed similar growth patterns during the culture period. Scale bar = 200 μm.

(C) The dynamic changes of the counts of assembloids over time in each hormone regimen.

(D) The dynamic changes of the area of assembloids over time in each hormone regimen.

(E) Heatmap showing receptivity related gene expression profile of assembloids in each hormone regimen. The color represents log-transformed fold change of gene expression.

(F) Validation of receptivity markers (IGFBP1, MAOA and DPP4) with immunofluorescence (IF) in the CTRL, SEC and WOI endometrial assembloids in vitro. Nuclei were counterstained with DAPI. Scale bar = 30 μm. The bar chart displaying the quantitative comparison of receptivity markers among three groups. *P ≤ 0.05, **P ≤ 0.005, ***P ≤ 0.0005, ****P ≤ 0.0001.

Receptive endometrial assembloids mimicked the implantation - window endometrium

(A) T-SNE plot of scRNA-seq data from three individual endometrial assembloids of the CTRL, SEC and WOI groups (left). T-SNE plot of combined scRNA-seq data from the three kinds of assembloids and mid-secretory endometrium (right).

(B) Exhibition of stromal cell marked by vimentin of CTRL assembloid through whole-mount clearing, immunostaining and light sheet microscopy imaging. Nuclei were counterstained with DAPI. The arrowhead indicates stromal cells. Scale bar = 40 μm (left), Scale bar = 30 μm (right).

(C) Whole-mount immunofluorescence showed that Vimentin+ F-actin+ cells (stromal cells) were arranged around the glandular spheres that were only F-actin+. Scale bar = 50 μm.

(D) Exhibition of immune cell marked by CD45 and CD44, and endometrial gland marked by FOXA2 of CTRL assembloid through whole-mount clearing, immunostaining and light sheet microscopy imaging. Nuclei were counterstained with DAPI. The arrowhead indicates immune cells. Scale bar = 50 μm (left), Scale bar = 10 μm (right).

(E) Flow cytometric analysis of T cells and macrophages in the CTRL endometrial assembloid. Gating strategy used for determining white blood cells (WBC) (CD45+ cells), T cells (CD45+CD3+ cells) and macrophages (CD45+CD68+CD11b+ cells).

(F) Electron micrograph of the CTRL (top), SEC (middle) and WOI (bottom) endometrial assembloid showing pinopodes (P), glycogen granule (asterisk), microvilli (white arrows) and cilia (orange arrows). Scale bar = 1 μm. Quantitative comparison of pinopodes, glycogen, microvilli, and cilia in the CTRL, SEC and WOI assembloids. *P ≤ 0.05, **P ≤ 0.005, ***P ≤ 0.0005, ****P ≤ 0.0001.

(G) Heatmap and bubble diagram illustrating highly expressed genes as well as GO functions enriched in both assembloids during the WOI and mid secretory endometrial tissue in terms of SOX9+ proliferative epithelium, stem-derived epithelium, secretory epithelium, proliferative epithelium, unciliated epithelium, stromal cells and EMT-derived cells. The color of heatmap represents log-transformed fold change of gene expression.

(H) Heatmaps showing differentially expressed TFs of endometrial assembloids and endometrium in the secretory epithelium (left) and EMT-derived cells (right). The color represents log-transformed fold change of gene expression.

Receptive endometrial assembloids recapitulate WOI-associated hormone response

(A) Principal component analysis (PCA) plot computed with differentially expressed genes in the bulk transcriptome of endometrial assembloids belonging to the CTRL, SEC and WOI groups.

(B) Heatmap showing that enrichment of differentially expressed genes for the terms of hormone response, monocarboxylic acid metabolism, lipid metabolism, and negative regulation of cell differentiation. The color represents log-transformed fold change of gene expression.

(C) Responsiveness to progesterone and estrogen was evaluated by IF to PRA/B and OLFM4 with IF respectively. Scale bar = 40 μm, **P ≤ 0.005.

(D) Exhibition of implantation marker (FOXO1) and endometrial gland marker (FOXA2) through combination of assembloid clearing, IF and light sheet microscopy. Nuclei were counterstained with DAPI. **P ≤ 0.005.

(E) Bar graph exhibiting various percentages of each cell type in the three groups.

(F) GSEA between the SEC and WOI groups for secretory epithelium.

(G) Pseudotime trajectory showing the transformation between proliferative and secretory epithelium in the CTRL, SEC and WOI groups. Arrows indicate the direction of the pseudotime trajectory. The black dot indicates the key differentiation node.

Receptive endometrial assembloids possess enhanced energy metabolism

(A) The Mfuzz trend analysis displayed the transcriptional variation trends of five clusters from CTRL, SEC, and WOI groups (with a focus on the differences between SEC and WOI assembloids). The heatmap illustrated corresponding gene expression profile (where color represents Z-score). The bubble plot showed the associated GO functions (with bubble size representing the number of genes and bubble color indicating the P value).

(B) The circular heatmap illustrated the functional differences of SEC and WOI assembloids at the proteomic level. The color represents protein expression levels, and the innermost circle color represents GO functions.

(C) Transmission electron microscopy images displayed the mitochondrial morphology of CTRL, SEC, and WOI assembloids, along with a quantitative comparison of mitochondrial area. Scale bar = 1 μm.

(D) RT-qPCR assessed the expression levels of mitochondrial function-related genes in the three assembloid groups.

(E) Quantitative comparison of the concentration of ATP (left) and IL8 (right) released by CTRL, SEC and WOI assembloids. *P<0.05<**P<0.005<***P<0.0005<****P<0.0001.

Receptive endometrial assembloids increased the ciliary assembly and motility

(A) The heatmap illustrated the expression of cilia-related genes across the CTRL, SEC, and WOI assembloids. The color represents Z-score, while the leftmost block indicates various characteristic functions related to cilia.

(B) RT-qPCR assessed the expression levels of cilia-related genes in the three assembloids.

(C) The histogram showed the expression of cilia-related proteins in the three groups of assembloids. The color of the longitudinal protein names corresponds to the color of cilia-related functional blocks in Fig 5A.

(D) IF analysis of cilia assembly marked by acetyl-α-tubulin. Nuclei were counterstained with DAPI. The arrowhead indicates cilia. Scale bar = 50 μm.

(E) GSEA between the SEC and WOI groups for ciliated epithelium.

(F) Pseudotime trajectory showing the transformation between ciliated and unciliated epithelium in the CTRL, SEC and WOI groups. Arrows indicate the direction of the pseudotime trajectory.

(G) Dot plots demonstrating the Cellphone DB analysis of relevant receptors and ligands of ciliated epithelium with other cell types. The size of the dot represents the level of significance. The color of the dot indicates the mean of the average expression level of interacting molecule 1 in ciliated epithelium and molecule 2 in other cell types.

(H) Proximity ligation assay (PLA) validating the interactions of ROR2-Wnt5A and CD74-COPA in the CTRL, SEC and WOI assembloids. Red signals the interaction of two proteins. Nuclei were counterstained with DAPI. Scale bar = 20 μm. *P<0.05<**P<0.005<***P<0.0005<****P<0.0001.

The receptive endometrial assembloids possess the potential for embryo implantation.

(A) Diagram illustrated the co-culture model of endometrial assembloids with blastoids (the blastoid stage corresponds to a 6-day post-fertilization human embryo, referred to as Day 6 here).

(B) Bright-field images of the co-culture of CTRL, SEC, and WOI assembloids with blastoids (Day 9) (yellow arrows indicate the blastoids). Scale bar = 100μm.

(C) Whole-mount fluorescence staining of Day 9 co-cultured embryoid and assembloid. OCT4 indicates the epiblast, GATA6 indicates the hypoblast, and KRT18 indicates the trophoblast. Scale bar = 40μm and 20μm (the rightmost image).

(D) Comparison of the survival rates of Day 9 embryoid in CTRL, SEC, and WOI assembloids.

(E) Comparison of the interaction ratios between Day 9 embryoid and endometrial assembloids in the CTRL, SEC, and WOI groups. *P < 0.05, **P < 0.005.

Schematic diagram displaying the establishment and validation of receptive endometrial assembloids, and summarizing the characteristic biological events of implantation-window endometrium.

Developing receptive endometrial assembloids in vitro

(A) Brightfield of endometrial assembloids on day2, day4 and day11. Scale bar = 200 μm.

(B) Brightfield of endometrial assembloids in the primary generation (P0) and first generation (P1). Scale bar = 200 μm.

(C) Screenshot of video S1 showing endometrial glands gradually developing into a vesicular shape, and the surrounding stromal cells arranging in a fibrous pattern in the CTRL endometrial assembloids (100x, up-left) (The yellow arrows indicate stromal cells and the white arrows indicate endometrial glands). Screenshot of video S2 displaying the stromal cells growing in fibrous pattern and forming an extensive network in the CTRL endometrial assembloids (200x, up-right) (The white arrows indicate stromal cells). The epithelial cells arrange like paving stones (middle and down, left). Stromal cells formed an extensive network (middle and down, right) (The arrowhead indicates stromal cells). Scale bar = 100 μm (middle), Scale bar = 50 μm (down).

(D) Validation of epithelial, stromal cell and endometrial gland markers (E-cadherin, vimentin and FOXA2, respectively) with immunofluorescence (IF) in the endometrium in vivo and CTRL endometrial assembloids in vitro. Nuclei were counterstained with DAPI. Scale bar = 40 μm. Periodic acid-schiff staining (PAS) of endometrium in vivo and endometrial assembloid in vitro. Scale bar = 20 μm.

(E) IF analysis of proliferation and apoptosis indicated by Ki67 and cleaved caspase-3 in the CTRL assembloids, respectively. Nuclei were counterstained with DAPI. Scale bar = 40 μm.

(F) Verification of hormone responsiveness by the expression levels of ERα and PRA/B (E-cadherin indicated the marker of epithelial cell). Scale bar = 40 μm. ***P ≤ 0.0005.

(G) Relative expression of PGR, PAEP, EGR1, and OLFM4 in the CTRL and hormone-treated assembloids by RT-qPCR. *P ≤ 0.05, **P ≤ 0.005, ***P ≤ 0.0005.

(H) The dynamic changes of the average intensity of assembloids over time in each hormone regimen.

(I) PAS of CTRL, SEC and WOI assembloids. Scale bar = 20 μm. Quantitative comparison of glycogen staining area in the CTRL, SEC and WOI assembloids. *P ≤ 0.05, ****P ≤ 0.0001.

(J) Endometrial receptivity evaluation of endometrium and their derived WOI assembloids through ERT. Asterisks indicate individual samples.

Various functions performed by all kinds of cells identified with scRNA-seq.

(A) Box plot of the gene numbers detected, UMI numbers, and ratios of mitochondrial gene expression in single cells of the CTRL, SEC and WOI groups.

(B) Bubble diagram showing the distribution of marker gene expression in each cluster.

(C∼D) Comparison of cell composition between CTRL, SEC, WOI endometrial assembloids and mid-secretory endometrium demonstrating samples (C) and cell types (D).

(E) Bubble diagram and heatmap showing the corresponding upregulated genes and GO function of each cluster of endometrial cells.

(F) Bubble diagram and heatmap showing corresponding upregulated genes and KEGG functions of SOX9+ proliferative epithelium, stem-derived epithelium, secretory epithelium, proliferative epithelium, stromal cells, ciliated epithelium and unciliated epithelium. Color is proportional to log-transformed fold change of gene expression.

(G) GSEA between the SEC and WOI groups for proliferative epithelium.

(H) The single-cell pseudotime trajectory of SOX9+ proliferative epithelium, secretory epithelium and proliferative epithelium. Cells start at proliferative epithelium and progress to SOX9+ proliferative epithelium and secretory epithelium (left). There are seven major states over pseudotime (right). The black spot indicates the differentiation node between state 5 and state 6, indicating the direction from proliferative epithelium to SOX9+ proliferative epithelium and secretory epithelium, respectively.

(I)(L) The horizontal axis is the pseudotime point, and the vertical axis is the gene expression level. The solid line represents states 1, 2, 4, and 5 corresponding to Fig. S2H (I) or Fig. S2K (L). Different colors represent samples in the CTRL, SEC and WOI groups.

(J)(M) Heatmap of genes at the branch node regulating differentiation into SOX9+ proliferative epithelium or secretory epithelium (J), and ciliated epithelium or unciliated epithelium (M). The horizontal axis is the pseudo-time point (the pseudo-time point gradually increases from the middle to both sides). The vertical axis is the gene expression level, representing two differential directions on the left and right sides. Clusters represent the gene sets with a similar branch gene expression trend. Different colors represent the level of gene expression.

(K) Pseudotime trajectory of ciliated and unciliated epithelium. Cells start at ciliated epithelium and progress to unciliated epithelium (left). There are seven major states over pseudotime (right). The black spot indicates the differentiation node between state 5 and state 6, indicating the direction of ciliated and unciliated epithelium, respectively. Arrows indicate the direction of the pseudotime trajectory.

Comparisons between CTRL, SEC and WOI assembloids at the level of transcriptome and proteome.

(A) Venn diagram showing differential genes in pairs among the three groups. Log2 FC (Fold Change)>1.2 or<-1.2, q value<0.05.

(B) Histogram showing pairwise comparison of differential gene expression (DEG) from bulk RNA-seq among the CTRL, SEC and WOI groups. qvalue<0.05, |logFC|>1.

(C) PCA plot computed with differentially expressed proteins in the micro proteomics of endometrial assembloids belonging to the CTRL, SEC and WOI groups.

(D) Venn diagram showing differential proteins in pairs among the three groups. Log2 FC>1.2 or <-1.2, q value<0.05.

(E) Histogram showing pairwise comparison of differential proteins in micro proteomics among the CTRL, SEC and WOI groups.

(F) Scatterplot depicting the correlation between the transcriptome and proteome as for the WOI group and CTRL group. For transcriptome, FC>2 and p value<0.05 were defined as significantly upregulated. For proteome, FC>1.2 and p value<0.05 were defined as significantly upregulated.

(G) Circle diagram showing the functions of genes upregulated in both the transcriptome and proteome as for the WOI group compared to CTRL group.

(H) Verification of hypoxia response by the expression levels of HIF1α. Scale bar = 50 μm. *P ≤ 0.05, **P ≤ 0.005.

(I) Verification of lipid metabolism by the expression levels of SLC25A1. Scale bar = 50 μm. ****P ≤ 0.0001.

Receptive endometrial assembloids experienced epithelial-mesenchymal transition (EMT)

(A) GSEA between the SEC and WOI groups for proliferative epithelium.

(B) The single-cell pseudotime trajectory of proliferative epithelium, stromal cell, EMT derived cell and stem derived epithelium. Cells start at proliferative epithelium and progress to EMT derived cell. There are seven major states over pseudotime. The black spot indicates the differentiation node between state 4, 5 and state 6, indicating the direction from proliferative epithelium to EMT derived cell. Arrows indicate the direction of the pseudotime trajectory.

(C) The horizontal axis is the pseudotime point, and the vertical axis is the gene expression level. The solid line represents states 1, 2, 4, and 5 corresponding to Fig. S4B. Different colors represent samples in the CTRL, SEC and WOI groups.

(D) Heatmap of genes at the branch node regulating differentiation into EMT derived cell and stem-derived epithelium. The horizontal axis is the pseudo-time point (the pseudo-time point gradually increases from the middle to both sides). The vertical axis is the gene expression level, representing two differential directions on the left and right sides. Clusters represent the gene sets with a similar branch gene expression trend. Different colors represent the level of gene expression.

(E-F) Dot plots demonstrating the Cellphone DB analysis of relevant receptors and ligands of EMT derived cell (E) or stromal cell (F) with other cell types. The size of the dot represents the level of significance. The color of the dot indicates the mean of the average expression level of interacting molecule 1 in EMT derived cells (E) or stromal cells (F) and molecule 2 in other cell types.

(H) Proximity ligation assay (PLA) validating the interactions of SEMA3A-NRP1 and CD46-JAG1 in the CTRL, SEC and WOI assembloids. Red signals the interaction of two proteins. Nuclei were counterstained with DAPI. Scale bar = 20 μm.