Journal: Advanced Science

Article Title: Endometrial Assembloid Model Reveals Endometrial Gland Development Regulation by Estradiol‐Driven WNT7B Suppression

doi: 10.1002/advs.202509664

Figure Lengend Snippet: Transcriptome analyses for tubular gland development from EOs. (A) Schematic illustration for single‐cell RNA sequencing analysis and bulk transcriptome analysis for identifying WNT7B and other genes regulating tubular gland formation (Figure ). (B) UMAP plot for the 3D EO/HESC co‐culture model on day 1 and day 9 in single‐cell RNA sequencing analysis. Colors indicate different cellular clusters (0–7). (C) Violin plot for EPCAM (epithelial marker) and VIMENTIN (fibroblast marker) expression in cellular clusters 0–7. (D) UMAP plot for EPCAM expression in day 1 and day 9 EO/HESC co‐culture model and zoomed UMAP plot for Cluster 7 on day 1 and day 9. (E) Enriched GO analysis of biological functions for differentially expressed genes (DEGs) identified in EOs (Cluster 7) and HESCs (Cluster 0–6) on day 9 versus day 1. The red boxes highlight the biological functions related to gland development. (F) Clustered heatmap for DEGs in EOs with and without tubular gland development capacity in bulk transcriptome analysis. n = 2. (G) Enriched GO analysis of biological functions for DEGs in EOs with and without tubular gland development capacity. The red boxes highlight the biological functions related to gland development.

Article Snippet: The membrane was blocked with 5% milk for 1 h, then incubated in primary antibody against WNT7B (1:100, AF3460, R&D Systems, RRID: AB_2834898).

Techniques: Single Cell, RNA Sequencing, Co-Culture Assay, Marker, Expressing

Journal: Advanced Science

Article Title: Endometrial Assembloid Model Reveals Endometrial Gland Development Regulation by Estradiol‐Driven WNT7B Suppression

doi: 10.1002/advs.202509664

Figure Lengend Snippet: Reduced WNT7B expression stimulates assembloid‐derived tubular gland development. (A) RT‐qPCR analysis for relative mRNA expression level of WNT7B in EOs with/without tubular gland development capacity. ****: p <0.0001. n = 4. (B) Immunohistochemical staining for WNT7B in human endometrial tissue. Nuclei were counterstained with Hematoxylin. Red box: magnified section of the field. Scale bars, 100 µm. (C) Schematic illustration for CRISPR Cas9 system targeting WNT7B exon 2. (D) Representative images of EOs after WNT7B ‐targeted lentivirus transduction for CRISPR Cas9. Green fluorescence indicates GFP expression. Scale bars, 100 µm. (E) Western blot analysis (upper) and RT‐qPCR analysis (lower) for WNT7B protein and mRNA expression levels in wildtype EOs (WT), non‐targeting lentivirus transduced EOs (lenti‐GFP‐Cas9) and WNT7B knockdown EOs (lenti‐ WNT7B sgRNA‐Cas9). ***: p <0.001. ns: not significant. n = 4. (F) Proportion of WNT7B knockdown EOs with tubular gland formation compared to wildtype EOs (WT) and non‐targeting lentivirus transduced EOs (GFP‐plasmid). ***: p <0.001. ns: not significant. n = 4. (G) Representative images of non‐targeting lentivirus transduced EOs (GFP‐plasmid) and WNT7B knockdown EOs in the 3D co‐culture model on day 1 and day 10. Merged images were generated from bright‐field image plus green fluorescence image. Scale bars, 100 µm. All data was presented as mean ± standard deviation. All the results were analyzed by the Kolmogrov‐Smirnov normality test. Statistical comparison was conducted using the Student t‐test for two groups or one‐way ANOVA with multiple comparison for more than three groups of variables.

Article Snippet: The membrane was blocked with 5% milk for 1 h, then incubated in primary antibody against WNT7B (1:100, AF3460, R&D Systems, RRID: AB_2834898).

Techniques: Expressing, Derivative Assay, Quantitative RT-PCR, Immunohistochemical staining, Staining, CRISPR, Transduction, Fluorescence, Western Blot, Knockdown, Plasmid Preparation, Co-Culture Assay, Generated, Standard Deviation, Comparison

Journal: Advanced Science

Article Title: Endometrial Assembloid Model Reveals Endometrial Gland Development Regulation by Estradiol‐Driven WNT7B Suppression

doi: 10.1002/advs.202509664

Figure Lengend Snippet: TGFβ1‐VDR interaction between HESCs and EOs regulates WNT7B expression and assembloid‐derived tubular gland development. (A) Dot plot for predicted ligand‐receptor pairs between HESCs (Cluster 0–6) and EOs (Cluster 7) according to single‐cell RNA sequencing analysis. The red box highlights TGFβ1‐VDR interaction. (B) Enriched GO analysis of biological functions in ligand‐receptor pairs between EOs and HESCs by CellPhone DB. The red boxes highlight the terms related to gland development, glandular structure morphogenesis and epithelium branching. (C) UMAP plot for TGFβ1 and VDR expression in single‐cell RNA sequencing analysis of day 1 and day 9 3D model. The red box highlights Cluster 7 (EOs/tubular EOs); blue box highlights Cluster 0‐6 (stromal cells). (D) Recombinant VDR or lysate of EO were extracted and were incubated with recombinant TGFβ1. Anti‐VDR and Protein G beads were used to co‐immunoprecipitated the VDR‐interacting protein complexes. The captured complex was analyzed by Western blotting using anti‐TGFβ1 and ‐VDR antibodies. n = 3. Figure shows a representative image from three independent experiments. (E) H&E staining of 3D EO/HESC co‐culture model on day 1 after co‐culture. Scale bars, 100 µm (top) and 50 µm (bottom). (F) Immunofluorescence staining for VDR (green) and VIMENTIN (red, fibroblast marker) in 3D EO/HESC co‐culture model on day 1 after co‐culture. Nuclei were counterstained with DAPI (blue). Scale bars, 20 µm. (G) Immunohistochemical staining for TGFβ1 in 3D EO/HESC co‐culture model on day 1 after co‐culture. Nuclei were counterstained with Hematoxylin. The bar chart indicates the quantified staining intensity of TGFβ1 in comparison to isotype control. Scale bars, 50 µm. (H) RT‐qPCR analysis for relative mRNA expression level of WNT7B in EOs after TGFβ1 treatment at 10 ng mL −1 for 48 h. **: p <0.01. n = 4. (I) Average tubular length of assembloid‐derived tubular structure in the co‐culture model from EOs pretreated with 10 ng mL −1 of TGFβ1 for 48 h. **: p <0.01. n = 5. All data was presented as mean ± standard deviation. All the results were analyzed by the Kolmogrov‐Smirnov normality test. Statistical comparison was conducted using the Student t‐test for two groups or one‐way ANOVA with multiple comparison for more than three groups of variables.

Article Snippet: The membrane was blocked with 5% milk for 1 h, then incubated in primary antibody against WNT7B (1:100, AF3460, R&D Systems, RRID: AB_2834898).

Techniques: Expressing, Derivative Assay, Single Cell, RNA Sequencing, Recombinant, Incubation, Immunoprecipitation, Western Blot, Staining, Co-Culture Assay, Immunofluorescence, Marker, Immunohistochemical staining, Comparison, Control, Quantitative RT-PCR, Standard Deviation

Journal: Advanced Science

Article Title: Endometrial Assembloid Model Reveals Endometrial Gland Development Regulation by Estradiol‐Driven WNT7B Suppression

doi: 10.1002/advs.202509664

Figure Lengend Snippet: Ablation of WNT7B expression in mouse endometrium results in increased endometrial gland number at proestrus phase. (A) Schematic illustration for establishment of endometrial‐specific WNT7B knockout mouse by Cre‐loxP system. Genotyping for Wnt7b f/f ; Pgr Cre/+ mouse via PCR and gel electrophoresis. WT: wildtype control ( Wnt7b f/f ; Pgr +/+ ); KO: conditional knockout ( Wnt7b f/f ; Pgr Cre/+ ). (B) Expression of WNT7B (green) in mouse endometrium in wildtype and conditional knockout mice. Nuclei were counterstained with DAPI (blue). Scale bars, 20 µm. (C) Vaginal smear staining for mouse at proestrus phase. Nuclei were counterstained with Hematoxylin. Scale bars, 25 µm. (D) Representative images for uteri of wildtype mouse (WT) and WNT7B conditional knockout mouse (KO) at 6 weeks at proestrus phase. (E) Uterine weight for wildtype (WT) and WNT7B conditional knockout (KO) mice at 6 weeks at proestrus phase. The uterine weight was divided by the body weight. ns: not significant. n = 4. (F) Average endometrial gland number in wildtype (WT) and WNT7B conditional knockout (KO) mice at 6 weeks at proestrus phase. **: p <0.01. n = 4. (G) H&E staining and immunohistochemical staining for FOXA2 in endometrial tissue of wildtype (WT) and WNT7B conditional knockout (KO) mice at 6 weeks at proestrus phase. Nuclei were counterstained with Hematoxylin. Scale bars, 100 µm. All data was presented as mean ± standard deviation. All the results were analyzed by the Kolmogrov‐Smirnov normality test. Statistical comparison was conducted using the Student t‐test for two groups or one‐way ANOVA with multiple comparison for more than three groups of variables.

Article Snippet: The membrane was blocked with 5% milk for 1 h, then incubated in primary antibody against WNT7B (1:100, AF3460, R&D Systems, RRID: AB_2834898).

Techniques: Expressing, Knock-Out, Nucleic Acid Electrophoresis, Control, Staining, Immunohistochemical staining, Standard Deviation, Comparison

Journal: Advanced Science

Article Title: Endometrial Assembloid Model Reveals Endometrial Gland Development Regulation by Estradiol‐Driven WNT7B Suppression

doi: 10.1002/advs.202509664

Figure Lengend Snippet: Estradiol stimulates endometrial gland development with WNT7B downregulation in assembloid‐derived tubular gland, estradiol stimulated mouse and human IVF clinical models. (A) Proportion of EOs with tubular gland formation in estradiol (E2) pretreatment and E2 treatment in HESC/assembloid co‐culture model. Scale bars, 100 µm. ***: p <0.001; ****: p <0.0001. n = 3. (B) Western blot analysis ( n = 5, left) and RT‐qPCR analysis ( n = 8, right) and for relative protein and mRNA expression level of WNT7B in wildtype EOs (control) and EOs treated with 10 nM/100 nM of estradiol (E2) for 7 days. *: p <0.05; **: p <0.01; ***: p <0.001****: p <0.0001. n = 8. (C) Schematic illustration for estradiol‐stimulated mouse model establishment. H&E staining for endometrial sections of mice with i.p. PBS injection (control) or 100 µg kg −1 of estradiol injection (E2) for 14 days. i.p.: Intraperitoneal. PBS: PBS injection for control group; E2: estradiol injection. Scale bars, 100 µm. Dosing timeline: Estrous‐cycle monitoring for approximately 2 weeks prior to day 0 is not depicted. Day 0: proestrus confirmed by late‐afternoon vaginal smear; day 1–14: daily i.p. PBS or 100 µg kg −1 E2; day 15: tissue collection. (D) Immunohistochemical staining for WNT7B in endometrial sections of mice with i.p. PBS injection (control) or 100 µg kg −1 of estradiol injection (E2) with quantified expression intensity. Nuclei were counterstained with Hematoxylin. **: p <0.01. Scale bars, 100 µm. n = 3. (E) Schematic illustration for controlled ovarian stimulation protocol in female participants undergoing IVF treatment. GnRH: gonadotrophin releasing hormone; hMG: human menopausal gonadotrophin. (F) Age and serum estradiol concentrations in female participants with natural menstrual cycle and controlled ovarian stimulation for IVF treatment. Data represents mean ± standard deviation. ***: p <0.001. n = 15. (G) Immunohistochemical staining for WNT7B in endometrial tissue of female participants with natural menstrual cycle and controlled ovarian stimulation with quantified expression intensity. Nuclei were counterstained with Hematoxylin. **: p <0.01. Scale bars, 100 µm. n = 3. All data was presented as mean ± standard deviation. All the results were analyzed by the Kolmogrov‐Smirnov normality test. Statistical comparison was conducted using the Student t‐test for two groups or one‐way ANOVA with multiple comparison for more than three groups of variables.

Article Snippet: The membrane was blocked with 5% milk for 1 h, then incubated in primary antibody against WNT7B (1:100, AF3460, R&D Systems, RRID: AB_2834898).

Techniques: Derivative Assay, Co-Culture Assay, Western Blot, Quantitative RT-PCR, Expressing, Control, Staining, Injection, Immunohistochemical staining, Standard Deviation, Comparison