DataSheet3_Influence of inflammation on the expression of microRNA-140 in extracellular vesicles from 2D and 3D culture models of synovial-membrane-derived stem cells.PDF

BackgroundIn osteoarthritis (OA), articular homeostasis is regulated by microRNA-140 that inhibits ADAMTS-5, an enzyme that cleaves aggrecan and stimulates the synthesis of other inflammatory mediators. This study aims to evaluate the expression of microRNA-140 in extracellular vesicles (EVs) derived from equine synovial-membrane-derived mesenchymal stem cells (eqSMMSCs) cultured in monolayer (2D) and three-dimensional (3D) culture models under an in vitro inflammatory environment.
MethodsFour experimental groups of eqSMMSC cultures were defined for isolation of the EVs. The 2D and 3D control groups were cultured in a conventional cell culture medium, while the 2D-OA and 3D-OA treatment groups were exposed to an OA-like medium containing IL-1β and TNFα. The culture media samples were collected at 24 h, 72 h, and 120 h time points for EV isolation and characterization using nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM). Reverse transcription quantitative polymerase chain reaction was employed to assess the expressions of microRNA-140 in both the cells and EVs. All statistical analyses were conducted at the 5% significance level.
ResultsEncapsulation of the eqSMMSCs protected the cells from the inflammatory media compared to the monolayer cultures. EVs were found in higher concentrations in the 3D-OA cultures. Additionally, higher expressions of microRNA-140 were observed in the cells of the 3D-OA group at 24 and 72 h, whereas microRNA-140 expressions in the EVs were higher in the 3D group at 72 h and in the 2D-OA group at 120 h (p < 0.001). However, the 3D-OA culture showed higher expression of the mRNA Adamts5 in the EVs at 120 h.
ConclusionThe responses of the eqSMMSCs to inflammatory stimuli involve intracellular expression of microRNA-140 and its subsequent transportation via the EVs, with quicker responses observed in the 3D than 2D cultures. This study sheds light on the behaviors of stem cells in restoring homeostasis in osteoarthritic joints.

背景：在骨关节炎（OA）中，关节稳态由微小RNA-140（microRNA-140）调控，该分子可抑制ADAMTS-5——一种可切割蛋白聚糖并促进其他炎症介质合成的酶。本研究旨在评估体外炎症环境下，单层（2D）与三维（3D）培养的马滑膜来源间充质干细胞（eqSMMSCs）所分泌的细胞外囊泡（EVs）中微小RNA-140的表达水平。
方法：设置四组马滑膜来源间充质干细胞培养体系以分离细胞外囊泡。2D对照组与3D对照组采用常规细胞培养基培养，2D-OA与3D-OA处理组则使用含有IL-1β和TNFα的骨关节炎样培养基培养。分别于24 h、72 h、120 h三个时间点收集培养基样本，通过纳米颗粒跟踪分析（NTA）与透射电子显微镜（TEM）对分离得到的细胞外囊泡进行鉴定与表征。采用逆转录定量聚合酶链反应检测细胞及细胞外囊泡中微小RNA-140的表达水平，所有统计分析均采用5%的显著性水平。
结果：与单层培养相比，三维培养的马滑膜来源间充质干细胞可使细胞免受炎症培养基的损伤。3D-OA组的细胞外囊泡浓度更高。此外，3D-OA组细胞内的微小RNA-140在24 h与72 h时表达水平更高，而细胞外囊泡中的微小RNA-140表达在72 h的3D组与120 h的2D-OA组中更高（p < 0.001）。但在120 h时，3D-OA组细胞外囊泡中的Adamts5 mRNA表达水平更高。
结论：马滑膜来源间充质干细胞对炎症刺激的响应包含细胞内微小RNA-140的表达及其后续通过细胞外囊泡的转运，且三维培养的响应速度快于单层培养。本研究阐明了干细胞在骨关节炎关节稳态修复中的行为机制。