[Establishment of an in vitro fibrotic endometrial organoid model and investigation of the early intervention effects of placenta-derived mesenchymal stem cell-derived extracellular vesicles (Pd-MSC-EV) on endometrial fibrosis].

Abstract

Objective: To establish an in vitro fibrotic endometrial organoid model and investigate the early intervention effects of placenta-derived mesenchymal stem cell extracellular vesicles (Pd-MSC-EV) on fibrotic injury in endometrial organoids. Methods: Cell clusters were isolated from the endometrial tissues of healthy female mice and used to establish a three-dimensional endometrial organoid culture system. The structure and cellular composition of the organoids were characterized by histological staining and immunofluorescence staining. To construct an in vitro fibrosis model, the organoids were treated with different concentrations of transforming growth factor-β1 (TGF-β1), and model establishment was evaluated based on organoid morphological changes and the mRNA expression of fibrosis-related genes, including collagen type Ⅰ alpha 1 chain (COL1A1), α-smooth muscle actin (α-SMA), and fibronectin (FN). Pd-MSC-EVs were isolated and characterized by transmission electron microscopy, nanoparticle tracking analysis, and western blotting. The effects of Pd-MSC-EVs on TGF-β1-induced fibrotic changes in endometrial organoids were then assessed by morphological observation, real-time quantitative PCR (RT-qPCR) analysis of COL1A1, α-SMA, and FN mRNA expression, and immunofluorescence detection of COL1A1 and α-SMA protein expression. All experiments were repeated three times. Results: Mouse endometrial organoids with lumen-like structures were successfully established and expressed the epithelial markers E-cadherin and CK8 as well as the stromal marker Vimentin. Compared with the control group, TGF-β1 treatment disrupted organoid architecture and increased the mRNA expression levels of fibrosis-related genes, including COL1A1, α-SMA and FN, in a dose-dependent manner (all P<0.001). The isolated Pd-MSC-EV showed typical cup-shaped or disk-like vesicular morphology, with an average particle size of (163.8±50.0) nm, and expressed the exosomal markers CD63, CD81 and TSG101. Compared with the TGF-β1 group, co-treatment with Pd-MSC-EV alleviated organoid structural damage and reduced the mRNA expression levels of COL1A1, α-SMA and FN (all P<0.001). Immunofluorescence showed that COL1A1 protein expression was lower in the Pd-MSC-EV group than in the TGF-β1 group (P<0.005), while α-SMA protein expression showed a decreasing trend. Conclusion: Pd-MSC-EV attenuate TGF-β1-induced fibrotic changes in endometrial organoids in vitro and reduce organoid structural injury, providing experimental evidence for mechanistic studies and potential intervention strategies for endometrial fibrosis-related diseases such as intrauterine adhesions.