Journal: Cell Communication and Signaling : CCS

Article Title: Sex hormone-responsive human vaginal epithelial organoids: a novel in vitro platform for studying Chlamydia trachomatis infection

doi: 10.1186/s12964-026-02685-7

Figure Lengend Snippet: Long-term genetically stable 3D human vaginal epithelial organoid (VEOs) cultures. A Schematic diagram illustrating the workflow for deriving vaginal epithelial organoids. B Representative bright-field images comparing the morphology of VEOs under different culture medium conditions: COM-W, ExM, M-VOM, EOM. Scale bar: 200 µm. C Representative images of vaginal epithelial organoids at passage 0, after three passages, and after nine passages in COM-W medium. Scale bar: 200 µm. D Bright-field images of VEOs cultured in different medium components. Scale bar: 200 µm. E Genomic stability assessment by CGH. The plot displays the log2 ratio of sequencing coverage for each chromosome across different passages (Passage2, Passage9) of VEOs. Differently colored dots represent individual data points for each passage. A uniform distribution of data points around the baseline (log2 ratio = 0) indicates a diploid state without major chromosomal aberrations. F Transmission electron microscopy (TEM) images showing microvilli (white arrows), secretory vesicles (red arrows), and the keratinized layer (above dashed lines) in both VEOs and native vaginal epithelium (VE). Scale bar: 5 µm

Article Snippet: After polymerization, the VEOs were treated with COM-W medium supplemented with clarithromycin (HY-17508, MedChemExpress), doxycycline (HY-N0565, MedChemExpress), or azithromycin (HY-17506, MedChemExpress).

Techniques: Cell Culture, Sequencing, Transmission Assay, Electron Microscopy

Journal: Cell Communication and Signaling : CCS

Article Title: Sex hormone-responsive human vaginal epithelial organoids: a novel in vitro platform for studying Chlamydia trachomatis infection

doi: 10.1186/s12964-026-02685-7

Figure Lengend Snippet: Human VEOs respond to sex hormonal stimulation. A Experimental design for hormonal stimulation. Three groups were included: (1) CM (untreated), (2) E (VEOs grown in COM-W, primed with E2 for 96 h starting on day 4 (D4)), and (3) EP (VEOs grown in COM-W, primed with E2 for 48 h on D4, followed by stimulation with P4 and cyclic AMP (cAMP) for 48 h). B Quantitative RT-PCR analysis for ESR1. C Quantitative RT-PCR analysis for PGR. D Hierarchical clustered heatmap of selected genes from organoids grown in COM-W (CM), COM-W with E2 ( E ), or COM-W with E2, P4, and cAMP (EP). Data are from n =3 donors. E Venn diagram showing overlap of 85 genes significantly upregulated in EP group, showing a fold change ≥2 ( P <0.05) relative to E and CM groups. F Venn diagram showing overlap of 47 genes significantly downregulated in EP group, showing a fold change ≥2 ( P <0.05) relative to E and CM groups. G GO analysis of the 85 genes from ( E ) for Biological processes. The four significantly enriched GO terms are shown with –log10( P -value). H GO analysis of the 47 genes from ( F ) for Biological processes. I GSEA revealed enrichment of VEOs (CM group) in “antimicrobial peptides” and “keratinization”. Genes in “antigen processing cross presentation” and “keratinization” pathways were enriched in E group. EP-VEOs showed enhanced expression of genes related to oxidative stress-induced senescence. J - K Western blot (WB) analysis of indicated proteins (e.g., CK5, IVL, NRF2, iNOS) in VEOs from CM, E, and EP groups. Data are representative of three independent experiments. L Expression levels of immune-related (PGLYRP3, CEACAM6, PI3) and keratinization-related (S100A9, IVL, SPRR1B) genes as determined by RNA sequencing ( n =3 per group). M Quantitative RT-PCR analysis for differentiation markers from ( J ). Statistical significance was determined by one-way ANOVA; data are presented as mean ± SEM values, * P < 0.05, ** P < 0.01, *** P < 0.001, ns: no significant difference

Article Snippet: After polymerization, the VEOs were treated with COM-W medium supplemented with clarithromycin (HY-17508, MedChemExpress), doxycycline (HY-N0565, MedChemExpress), or azithromycin (HY-17506, MedChemExpress).

Techniques: Quantitative RT-PCR, Expressing, Western Blot, RNA Sequencing

Journal: Cell Communication and Signaling : CCS

Article Title: Sex hormone-responsive human vaginal epithelial organoids: a novel in vitro platform for studying Chlamydia trachomatis infection

doi: 10.1186/s12964-026-02685-7

Figure Lengend Snippet: Chlamydia trachomatis (CT) infection in VEOs under different sex hormonal conditions. A Bright-field images of control (Ctrl) and CT-infection (CTN) group. Scale bar: 100 µm. B Transmission electron microscopy (TEM) images showing Chlamydia trachomatis inclusions (red arrows). Scale bar: 2 µm. C Experimental design for CT infection. Five groups were included: (1) Ctrl (Untreated control, COM-W from D0-D7), (2) CTN (CT-infected, hormone-free, COM-W from D0-D7 + CT infection from D5-D7), (3) noCT (E2 treatment without CT infection, COM-W from D0 + E2 from D1-D7), (4) CTE (CT-infected under E2 treatment, COM-W from D0 + E2 from D1-D7 + CT infection from D5-D7), and (5) CTP (CT-infected under sequential E2/P4 treatment, COM-W from D0 + E2 from D1-D7 + P4 and cAMP from D3-D7 + CT infection from D5-D7). D Principal component analysis (PCA) of RNA-sequencing resulted from Ctrl, CTN, noCT, CTE, and CTP samples ( n =3 per group) (fold change ≥ 2 and P < 0.05). E Venn diagram showing overlap of 1851 differentially expressed genes identified between the Ctrl group and the CTN group on Day 1 and Day 2. F Venn diagram showing overlap of 658 differentially expressed genes identified between the CTN group and the CTE group on Day 1 and Day 2. G GO analysis of the 658 genes from ( F ) for Biological processes. H Volcano plot showing differentially expressed genes (DEGs) between noCT and CTE groups at Day 1 (D1) and Day 2 (D2). Red and blue points indicate upregulated and downregulated DEGs, respectively (fold change ≥ 2, P < 0.05). I GSEA revealed enrichment of specific signal pathways in CTE group compared to noCT group. J Gene expression analysis showing upregulation of genes related to “mitochondrial gene expression” and “translation” in CTP group compared to CTE group at D2

Article Snippet: After polymerization, the VEOs were treated with COM-W medium supplemented with clarithromycin (HY-17508, MedChemExpress), doxycycline (HY-N0565, MedChemExpress), or azithromycin (HY-17506, MedChemExpress).

Techniques: Infection, Control, Transmission Assay, Electron Microscopy, RNA Sequencing, Gene Expression