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

Abstract

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.

Hydrogel-encapsulated extracellular vesicles for the regeneration of spinal cord injury

“…In addition, four-dimensional cultured hUCMSC-derived sEVs combined with GelMA hydrogel attenuated neuroinflammation through polarization of the macrophage/microglia from M1 to M2 phenotype, resulting in enhanced survival of spinal neurons after injury. These EVs promoted polarization of the M2 phenotype via upregulation of IGFBP2/EGFR protein, leading to phosphorylation of STAT3 ( Wang et al, 2023 ). Another study used a combination of Berberine-loaded hUC-MSCs-sEVs with GelMA hydrogel, which resulted in attenuated local inflammation and created a favorable microenvironment for nerve regeneration and axonal regrowth ( Wang et al, 2023 ).…”

Mesenchymal Stem Cells-based Cell-free Therapy Targeting Neuroinflammation

“…The reciprocal communication between MSCs and the immune system is facilitated by extracellular vesicles, particularly across extended distances [ 65 - 67 , 70 ]. Of particular interest are small extracellular vesicles (sEVs) derived from human umbilical cord mesenchymal stem cells (hUC-MSCs).…”

“…These sEVs have demonstrated effectiveness in modulating the inflammatory response and promoting neuro-regenerative processes in SCI. Furthermore, these vesicles have been observed to induce polarization from the proinflammatory M1 macrophage phenotype to the anti-inflammatory M2 subtype, both in vitro and within lesion sites in rat models of compressive and contusive SCI [ 67 , 71 ].…”

“…Exosomes derived from MSCs, when primed using physical and chemical methodologies such as hypoxia, traditional Chinese medicine, gene editing, and three-dimensional culture, exhibit enhanced functional characteristics [ 65 - 67 , 70 - 72 ].…”