Human Epidural AD-MSC Exosomes Improve Function Recovery after Spinal Cord Injury in Rats

Abstract

Spinal cord injury (SCI) interferes with the normal function of the autonomic nervous system by blocking circuits between the sensory and motor nerves. Although many studies focus on functional recovery after neurological injury, effective neuroregeneration is still being explored. Recently, extracellular vesicles such as exosomes have emerged as cell-free therapeutic agents owing to their ability of cell-to-cell communication. In particular, exosomes released from mesenchymal stem cells (MSCs) have the potential for tissue regeneration and exhibit therapeutic effectiveness in neurological disorders. In this study, we isolated exosomes from human epidural adipose tissue-derived MSCs (hEpi AD-MSCs) using the tangential flow filtration method. The isolated exosomes were analyzed for size, concentration, shape, and major surface markers using nanoparticle tracking analysis, transmission electron microscopy, and flow cytometry. To evaluate their effect on SCI recovery, hEpi AD-MSC exosomes were injected intravenously in SCI-induced rats. hEpi AD-MSC exosomes improved the locomotor function of SCI-induced rats. The results of histopathological and cytokine assays showed that hEpi AD-MSC exosomes regulated inflammatory response. Genetic profiling of the rat spinal cord tissues revealed changes in the expression of inflammation-related genes after exosome administration. Collectively, hEpi AD-MSC exosomes are effective in restoring spinal functions by reducing the inflammatory response.

Keywords: exosomes; extracellular vesicles; mesenchymal stem cells; spinal cord injury.

Figure 1

Characterization of human epidural adipose tissue-derived mesenchymal stem cells (hEpi AD–MSCs): (a) representative image of passage 5 of human epidural AD–MSCs; (b) expression of positive markers (CD105, CD90, and CD73) of human AD–MSCs analyzed by flow cytometry (upper). Expression of negative markers (CD45, CD34, and CD14) of human AD–MSCs analyzed by flow cytometry (below). The horizontal axis represents the fluorescence intensity (FL1 = FITC, FL2 = PE), and the vertical axis indicates the cell count.

Figure 2

Isolated exosomes from hEpi AD–MSCs: (a) representative TEM images of hEpi AD–MSC exosomes; (b) the isolated exosomes were analyzed for particle number and size by nanoparticle tracking analysis (NTA); (c) the surface positive markers CD63 and CD81 (tetraspanins) expression in exosomes was analyzed by flow cytometry.

Figure 3

In vivo evaluation of hEpi AD–MSC exosomes for SCI treatment: (a) schematic representation of SCI rat model with exosome injection; (b) hindlimb locomotor function was measured using the BBB scoring method for 28 d. Score ranged from 0 to 21 depending on the function of hindlimb (*** p ≤ 0.001, **** p ≤ 0.0001 between the Vehicle and High-Exo group); (c) mean body weight in non-treated and SCI-induced rat model (n = 6, per group).

Figure 4

Histopathology and immunohistochemistry analysis of Iba-1 and glial fibrillary acidic protein (GFAP) in the rat spinal cord: (a) representative images of spinal cord gross findings after sacrifice. The spinal cord tissues were damaged by compression stimulation; (b) the spinal cord tissues were sectioned longitudinally and stained with hematoxylin and eosin. (Scale bar = 100 μm); (c) immunodetection of Iba-1 in the non-treated and SCI-induced rats (scale bar = 200 μm); (d) quantification of Iba-1 expression by DAB staining; (e) representative images of Iba-1 microglia states (scale bar = 20 μm, black arrow: ramified, white arrow: ameboid); (f) quantification of Iba-1 morphology ratio (ameboid and ramified microglia); (g) immunodetection of GFAP in the non-treated and spinal cord injured rats. (Scale bar = 200 μm); (h) quantification of GFAP expressing intensity by DAB staining. (* p ≤ 0.05, ** p ≤ 0.001, ns = not significant).

Figure 5

Gene expression levels in rats of the control and SCI groups: (a,b) expression patterns of BDNF and VEGF measured by qPCR (* p ≤ 0.05, *** p ≤ 0.001, **** p ≤ 0.0001, ns = not significant).

Figure 6

Cytokine levels in rats of the control and SCI groups: (a–f) levels of cytokines IL-1β, IL-2, IL-10, IL-13, TNF-α, and IFN-γ were analyzed in the serum of the control and SCI-induced rats (* p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001, ns = not significant).

Figure 7

Comparison of gene expression level by mRNA sequencing: (a) gene category chart of the control and vehicle groups. The ratio of significant genes according to each gene category; (b) scatter plot of normalized data (log2N) for each gene. X-axis represents control (log2N), and y-axis represents vehicle value (log2N), upregulated genes (red) and downregulated genes (green); (c) clustering heatmap for genes with significantly altered expression; (d) top 10 Gene Ontology (GO) biological process (BP) terms of genes with significantly altered expression in the exosome-treated groups, compared with that in the vehicle group (** p < 0.01, *** p < 0.001).