Journal: Bioactive Materials

Article Title: Microfluidic chip-integrated vascularized endometrial complexes: Mitochondrial function and paracrine crosstalk enhance regenerative potential

doi: 10.1016/j.bioactmat.2025.08.035

Figure Lengend Snippet: Single-cell RNA sequencing (scRNA-seq) analysis of the dynamically cultured EO and HEO complexes. a. UMAP visualization of 12 samples across four culture conditions: HEOe (HEO + estrogen), HEOp (HEO + estrogen + progesterone + cAMP), EOe (EO + estrogen), EOp (EO + estrogen + progesterone + cAMP). Endometrial epithelial cell clusters were annotated as main Epi (mEpi), ribosome-related Epi (rEpi), proliferative Epi (pEpi), and ciliated Epi (cEpi). Endometrial stromal cell clusters were annotated as main Str (mStr), ribosome-related Str (rStr), proliferative Str (pStr), and a second proliferative Str cluster (pStr2); the endothelial cell clusters included main endothelial cells (mEndo) and ribosome-related endothelial cells (rEndo). b–c. Relative abundance of epithelial (b) and stromal (c) clusters within their respective compartments across groups.]d. Comparison of the percentage of proliferative epithelial cells (pEpi, left panel) and proliferative stromal cells (pStr, right panel) between the dynamically cultured EO and HEO complexes under estrogen culture.e. Validation of enhanced proliferation in HEO complexes by flow cytometry. Significantly higher proportions of EdU + cells in epithelial cells of HEO vs. EO complexes under estrogen culture.f. Ligand-receptor pairs exhibiting significant differences in communication probability between HEOe and EOe complexes. Pathways of biological interest include WNT7A, WNT5A , BMP6 , and LGALS9. Dot size represents the maximum communication probability across compared groups; dot color indicates statistical significance ( P < 0.05). Cluster abbreviations: Epi (endometrial epithelial cells), Str (endometrial stromal cells) and Endo (endothelial cells).g. Circle plot showing the BMP6, LGALS9, WNT7A, WNT5A signaling networks among different cell types in EOe and HEOe complexes.

Article Snippet: The spheroids were stimulated with 100 μL of ECM with different treatments, including WNT5A (200 ng/mL, CSB-EP026138HU, CUSABIO, Wuhan, China), WNT7A (100 ng/mL, P06680 , Solarbio), DKK1 (200 ng/mL, C12B, Novoprotein), BOX5 (HY-123071A, MCE), anti-WNT5A neutralizing antibody (2 μg/mL, MAB645, R&D), anti-WNT7A neutralizing antibody (10 μg/mL, sc-365665, Santa Cruz), or VEGFA (10 ng/mL, C744, Novoprotein) as a positive control, by adding dropwise onto the collagen matrix.

Techniques: RNA Sequencing, Cell Culture, Comparison, Biomarker Discovery, Flow Cytometry

Journal: Bioactive Materials

Article Title: Microfluidic chip-integrated vascularized endometrial complexes: Mitochondrial function and paracrine crosstalk enhance regenerative potential

doi: 10.1016/j.bioactmat.2025.08.035

Figure Lengend Snippet: Endometrial epithelial and stromal cells enhance the formation of vascular networks in HUVECs within the HEO complex through WNT7A and WNT5A signaling pathways. a. Representative images of sprouting assays using HUVEC spheroids treated as indicated. HUVEC spheroids cultured with basic ECM medium are shown as the blank group (Control). VEGFA added to the basic ECM medium served as the positive control. Scale bar: 400 μm. b. Quantification of sprouting number was determined by Image J software. ∗ P < 0.05 versus Control; # P < 0.05 versus WNT7A; $ P < 0.05 versus DKK1; ^ P < 0.05 versus WNT5A; & P < 0.05 versus BOX5; one-way ANOVA was conducted for statistical analysis. c. Representative images of the tube formation of HUVECs on Matrigel surface treated as indicated. HUVECs cultured with basic ECM medium are shown as the blank (Control). VEGFA added to the basic ECM medium served as the positive control. Scale bar: 400 μm. d. Quantification of branch number using Image J software. ∗ P < 0.05 versus Control; $ P < 0.05 versus DKK1; &P < 0.05 versus BOX5; one-way ANOVA was conducted for statistical analysis.e. Vascular networks of complex with HUVECs and ESCs (HE), complex with HUVECs and EEOs (HO), and HEO treated with WNT7A (or its inhibitor DKK1) and WNT5A (or its inhibitor BOX5) as indicated. Vascular networks are depicted in green. Scale bar: 100 μm.f. Quantification of the vessel-positive area within the formed vascular network across different groups. ∗P < 0.05, ∗∗P < 0.01, Student's t-test.g. The electrical resistance of HEO and HE with DKK1 and WNT7A treatments, respectively. ∗∗∗P < 0.001, two-way ANOVA.h. The electrical resistance of HEO and HO with BOX5 and WNT5A treatments, respectively. ∗∗∗P < 0.001, two-way ANOVA.

Article Snippet: The spheroids were stimulated with 100 μL of ECM with different treatments, including WNT5A (200 ng/mL, CSB-EP026138HU, CUSABIO, Wuhan, China), WNT7A (100 ng/mL, P06680 , Solarbio), DKK1 (200 ng/mL, C12B, Novoprotein), BOX5 (HY-123071A, MCE), anti-WNT5A neutralizing antibody (2 μg/mL, MAB645, R&D), anti-WNT7A neutralizing antibody (10 μg/mL, sc-365665, Santa Cruz), or VEGFA (10 ng/mL, C744, Novoprotein) as a positive control, by adding dropwise onto the collagen matrix.

Techniques: Protein-Protein interactions, Cell Culture, Control, Positive Control, Software

Journal: Nature Communications

Article Title: Autocrine ECM molecules establish MSC quiescence during incisor development by disrupting WNT ligand trafficking process

doi: 10.1038/s41467-025-65705-z

Figure Lengend Snippet: a – d Knockout of Smoc1/2 leads to elevated Wnt activity. a Gene Ontology and KEGG analyses of differentially expressed genes in the mesenchyme of control versus Smoc1/2 DKO incisors at E16.5. b , c Quantitation of expression levels of Axin2 and Lef within individual apical pulp cells of Smoc2 + /- littermate control and Smoc1/2 DKO. Two-tailed Mann–Whitney U test; Axin2 : control vs DKO, p < 0.0001; Lef1 : control vs DKO, p < 0.0001. d Mip-seq analysis showing RNA expression of Axin2 and Lef1 in the incisor of control and Smoc1/2 DKO mice. Scale bars: 50 μm. Representative images from three independent experiments with similar results. e – g Knockout of Smoc1/2 did not significantly affect Wnt6 and Wnt10a RNA levels but changed their protein distribution. e UMAP feature plots showed comparable transcription levels of Wnt6 and Wnt10a in control versus Smoc1/2 DKO incisors at E16.5. f MIP-seq data showed similar RNA expression levels and patterns of Wnt6 and Wnt10a in incisors of Smoc2 + /- littermate controls and Smoc1/2 DKO mice embryos at E16.5. g Antibody staining of WNT6 and WNT10a in incisors of Smoc2 + /- littermate control and Smoc1/2 KO embryos at E16.5. Scale bars: 100 μm. White arrows indicate RNA probe signals in incisor epithelium, while yellow arrows show RNA probe signals in incisors mesenchyme. White asterisks indicate positive immunostaining signals, while yellow asterisks show low signals in the apical pulp regions of incisors. Representative images from three independent experiments with similar results. h , i Quantitation of panels ( f ) revealed no significant difference in the percentage of cells expressing Wnt6 or Wnt10a RNA between control and Smoc1/2 DKO mice incisors. j , k Quantitation of g showed significantly more pulp cells positive for WNT6+ and WNT10a+ immunostaining in Smoc1/2 DKO mice incisors compared to controls. Data are presented as mean ± SEM. n = 3 biological replicates; unpaired two-tailed Student’s t-test. Wnt6 + cell: control vs DKO, p = 0.2071; Wn10a + cell: control vs DKO, p = 0.6739, WNT6+ cell: control vs DKO, p = 0.0003; WNT10a+ cell: control vs DKO, p = 0.0026; N.S.: not significant. l – v Organ culture of incisor tooth germs at E16.5. l The schematic drawing illustrates the experimental scheme for the explant culture. Created with MedPeer (medpeer.cn). Organ culture experiments were conducted using wild-type control incisor explants incubated with WNT6 beads ( m ), WNT6 + SMOC2 beads ( n ), WNT10a beads ( o ), or WNT10a + SMOC2 beads ( p ). The samples were subsequently stained with WNT6 or WNT10a antibodies, respectively, and the staining results were quantified in ( u ). q – t Similar beads explant experiments were performed using Smoc1/2 DKO incisor embryos at E16.5, and the results were quantified in ( v ). Data are presented as mean ± SEM. n = 3 biological replicates; unpaired two-tailed Student’s t -test. WNT6+ cell: control-WNT6 vs control-WNT6-SMOC2, p = 0.4189, control-WNT6 vs DKO-WNT6, p < 0.0001, DKO-WNT6 vs DKO-WNT6-SMOC2, p = 0.0001; WNT10a+ cell: control-WNT10a vs control-WNT10a-SMOC2, p = 0.8690, control-WNT10a vs DKO-WNT10a, p = 0.0003, DKO-WNT10a vs DKO-WNT10a-SMOC2, p = 0.00; N.S.: not significant. Source data are provided as a Source Data file.

Article Snippet: Recombinant Wnt6 (CSB-EP026140MO, CUSABIO) and Wnt10a (CSB-EP026129MO, CUSABIO) proteins were procured for the preparation of Wnt6-beads and Wnt10a-beads.

Techniques: Knock-Out, Activity Assay, Control, Quantitation Assay, Expressing, Two Tailed Test, MANN-WHITNEY, RNA Expression, Staining, Immunostaining, Organ Culture, Incubation

Journal: Nature Communications

Article Title: Autocrine ECM molecules establish MSC quiescence during incisor development by disrupting WNT ligand trafficking process

doi: 10.1038/s41467-025-65705-z

Figure Lengend Snippet: a Expression of active β-Catenin and its downstream targets c-Myc, Cyclin D1, and LEF1 in DPCs after Smoc1 or Smoc2 overexpression, as assessed by western blot. b Expression level of active β-Catenin and its downstream targets c-Myc, and Cyclin D1 in DPCs after Smoc1/2 overexpression alone or simultaneously. c Immunostaining of β-Catenin in control, Wnt agonist WAY and WAY with Smoc1/2 treated groups. Scale bars: 10 μm. Representative images from three independent experiments with similar results. SMOC1/2 interfere with Wnt10a transportation. d The schematic drawing illustrates the experimental scheme of DPCs supplied with beads. Created with MedPeer (medpeer.cn). e Immunostaining of Wnt10a in cultured DPCs supplied with beads. White arrows indicate WNT Dynabeads. Scale bars: 10 μm. Representative images from three independent experiments with similar results. f Expression of active β-Catenin and its downstream targets c-Myc, Cyclin D1, and LEF1 in DPCs after Gpc6 silencing, as assessed by western blot. g Immunostaining of β-Catenin in siRNA negative control (si NC ), WAY with si NC and WAY with si Gpc6 treated groups. Scale bars: 10 μm. Representative images from three independent experiments with similar results. h Co-IP of GPC6 with SMOC1 or SMOC2 in DPCs. i Co-localization analysis of SMOC1/2 with GPC6, as assessed by immunostaining. White arrows indicate co-localization signals. Scale bars: 10 μm. Representative images from three independent experiments with similar results. j Co-IP of GPC6 with WNT10a in DPCs in control , Smoc2 or Smoc1 overexpression groups. Source data are provided as a Source Data file. k Mechanism diagram. SMOC1/2 inhibit WNT trafficking by competitively binding to GPC6, thereby disrupting WNT ligand distribution within the niche. Created with MedPeer (medpeer.cn).

Article Snippet: Recombinant Wnt6 (CSB-EP026140MO, CUSABIO) and Wnt10a (CSB-EP026129MO, CUSABIO) proteins were procured for the preparation of Wnt6-beads and Wnt10a-beads.

Techniques: Expressing, Over Expression, Western Blot, Immunostaining, Control, Cell Culture, Negative Control, Co-Immunoprecipitation Assay, Binding Assay