Table1_Small extracellular vesicles derived from four dimensional-culture of mesenchymal stem cells induce alternatively activated macrophages by upregulating IGFBP2/EGFR to attenuate inflammation in the spinal cord injury of rats.xlsx

Effectively reducing the inflammatory response after spinal cord injury (SCI) is a challenging clinical problem and the subject of active investigation. This study employed a porous scaffold-based three dimensional long-term culture technique to obtain human umbilical cord mesenchymal stem cell (hUC-MSC)-derived Small Extracellular Vesicles (sEVs) (three dimensional culture over time, the “4D-sEVs”). Moreover, the vesicle size, number, and inner protein concentrations of the MSC 4D-sEVs contained altered protein profiles compared with those derived from 2D culture conditions. A proteomics analysis suggested broad changes, especially significant upregulation of Epidermal Growth Factors Receptor (EGFR) and Insulin-like Growth Factor Binding Protein 2 (IGFBP2) in 4D-sEVs compared with 2D-sEVs. The endocytosis of 4D-sEVs allowed for the binding of EGFR and IGFBP2, leading to downstream STAT3 phosphorylation and IL-10 secretion and effective induction of macrophages/microglia polarization from the pro-inflammatory M1 to anti-inflammatory M2 phenotype, both in vitro and in the injured areas of rats with compressive/contusive SCI. The reduction in neuroinflammation after 4D-sEVs delivery to the injury site epicenter led to significant neuroprotection, as evidenced by the number of surviving spinal neurons. Therefore, applying this novel 4D culture-derived Small Extracellular Vesicles could effectively curb the inflammatory response and increase tissue repair after SCI.

有效减轻脊髓损伤（spinal cord injury, SCI）后的炎症反应是一项极具挑战性的临床难题，亦是当前活跃的研究课题。本研究采用基于多孔支架的三维长期培养技术，制备得到人脐带间充质干细胞（human umbilical cord mesenchymal stem cell, hUC-MSC）来源的小细胞外囊泡（Small Extracellular Vesicles, sEVs）（该长期三维培养方式制备的囊泡被称为“4D-sEVs”）。与二维（2D）培养条件下获得的sEVs相比，4D-sEVs的囊泡尺寸、数量及内部蛋白浓度均存在显著差异，且蛋白谱发生广泛改变。蛋白质组学分析结果显示，相较于2D-sEVs，4D-sEVs中表皮生长因子受体（Epidermal Growth Factors Receptor, EGFR）与胰岛素样生长因子结合蛋白2（Insulin-like Growth Factor Binding Protein 2, IGFBP2）的表达上调尤为显著。4D-sEVs经内吞作用进入细胞后，可与EGFR及IGFBP2结合，激活下游STAT3磷酸化通路并促进IL-10分泌，进而在体外实验及压迫/挫伤性脊髓损伤大鼠的损伤区域中，有效诱导巨噬细胞/小胶质细胞从促炎的M1表型极化为抗炎的M2表型。将4D-sEVs递送至损伤中心后，可减轻脊髓损伤后的神经炎症，最终通过存活脊髓神经元的数量改善体现出显著的神经保护作用。因此，应用这种新型4D培养来源的小细胞外囊泡，可有效抑制脊髓损伤后的炎症反应并促进组织修复。