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, 20 (18)

The Effect of Wharton Jelly-Derived Mesenchymal Stromal Cells and Their Conditioned Media in the Treatment of a Rat Spinal Cord Injury

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The Effect of Wharton Jelly-Derived Mesenchymal Stromal Cells and Their Conditioned Media in the Treatment of a Rat Spinal Cord Injury

Milada Chudickova et al. Int J Mol Sci.

Abstract

The transplantation of Wharton's jelly derived mesenchymal stromal cells (WJ-MSCs) possesses therapeutic potential for the treatment of a spinal cord injury (SCI). Generally, the main effect of MSCs is mediated by their paracrine potential. Therefore, application of WJ-MSC derived conditioned media (CM) is an acknowledged approach for how to bypass the limited survival of transplanted cells. In this study, we compared the effect of human WJ-MSCs and their CM in the treatment of SCI in rats. WJ-MSCs and their CM were intrathecally transplanted in the three consecutive weeks following the induction of a balloon compression lesion. Behavioral analyses were carried out up to 9 weeks after the SCI and revealed significant improvement after the treatment with WJ-MSCs and CM, compared to the saline control. Both WJ-MSCs and CM treatment resulted in a higher amount of spared gray and white matter and enhanced expression of genes related to axonal growth. However, only the CM treatment further improved axonal sprouting and reduced the number of reactive astrocytes in the lesion area. On the other hand, WJ-MSCs enhanced the expression of inflammatory and chemotactic markers in plasma, which indicates a systemic immunological response to xenogeneic cell transplantation. Our results confirmed that WJ-MSC derived CM offer an alternative to direct stem cell transplantation for the treatment of SCI.

Keywords: Wharton’s jelly; cell secretome; cell therapy; conditioned medium; mesenchymal stem cells; spinal cord injury.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure A1
Figure A1
The effect of WJ-MSC-derived CM on neurite growth of rat dorsal root ganglia (DRG) neurons. (A) The area (%) of beta III tubulin positive staining in the presence of conditioned (CM) and non-conditioned (cM) medium (n = 3 in duplicates, * p < 0.05). (B) DRG neurons cultured in the presence of control and conditioned or medium (beta III tubulin/DAPI staining).
Figure 1
Figure 1
Time schedule of experiments. Following a spinal cord injury (SCI), the rats were treated by saline (ctrl), non-conditioned (control) medium (cM), conditioned medium (CM) and Wharton’s jelly derived mesenchymal stromal cells (WJ-MSCs) in the 1st, 2nd and 3rd week. The rats were behaviorally tested weekly up to the 9th week and analysed using qPCR (4th and 9th week), proteomic (4th week) and histological (9th week) analyses.
Figure 2
Figure 2
Proteomic analysis of the CM derived from WJ-MSCs.
Figure 3
Figure 3
Results of behavioral testing. The locomotor and sensory function of animals were tested by a beam walk time measurement (A), beam walk score (B), Basso, Beattie and Bresnahan test (BBB) test (C) and plantar test (D). The number of animals in the groups is as follows: Saline treated control group n = 16, cM group n = 11, CM group n = 10 and WJ-MSCs group n = 12. Asterisks (*) and (#) above ctrl (saline) group columns show statistical significance of ctrl (saline) group vs. all particular treatments, which was * p < (0.05–0.01) and # p < 0.001. The significance between the treated groups: * p < 0.05, ** p< 0.01 and *** p < 0.001.
Figure 4
Figure 4
White matter (A) and gray matter (B) sparing expressed as spared area (mm2) per slice through the lesion area. Asterisks show the statistical significance of a particular treatment when compared to the saline treated control group. The number of animals in the groups is as follows: Saline treated control group n = 7, cM group n = 9, CM group n = 9 and WJ-MSCs group n = 9. (C) Representative images of spinal cord cross sections 5 mm cranially from the centre of lesion were stained using cresyl-violet luxol fast blue.
Figure 5
Figure 5
Glial scar formation analyses. (A) The size of glial scar expressed as the % to spared tissue. (B) Number of reactive astrocytes (RA) counted from the glial fibrillary acidic protein (GFAP) staining as shown in Figure 6. Number of animals: Saline treated control group n = 4, cM group n = 5, CM group n = 5 and WJ-MSCs group n = 5. Asterisks (*) show statistical significance of particular treatment, when compared to saline treated control group, crosses marks (+) show statistically significant difference between WJ-MSCs group and CM group (orange cross) and between WJ-MSCs group and cM group (gray cross), respectively. * p < 0.05, ** p < 0.01 and + p < 0.05.
Figure 6
Figure 6
(AD) Representative fluorescent images of GFAP staining of cross sections 9 weeks after the SCI for evaluation of the glial scar in the cranial part of the lesion, 5 mm from the centre. (EH) highlight the details of the marked area in (AD), with reactive astrocytes depicted by white arrows.
Figure 7
Figure 7
(A) The average number of GAP43+ fibers in the treated groups presented as relative when compared to the control (saline), which was set as 100% (depicted with a black interrupted line). Number of animals (five sections per animal): Saline treated control group n = 11, cM group n = 5, CM group n = 9 and WJ-MSCs group n = 7, ** p < 0.01 and *** p < 0.001. (B) Representative fluorescent images of GAP43 staining of cross sections 9 weeks after the SCI in the cranial part of the lesion, 5 mm from the centre.
Figure 8
Figure 8
Relative expression of selected genes in the lesion 4 (A,B) and 9 weeks (C) after SCI. The graphs show the log2-fold changes of the ΔΔCt values of the indicated genes in comparison to the animals treated with the saline. n = 4 per group, asterisks show statistical significance * p < 0.05, ** p < 0.01 and # p < 0.001 of a particular treatment, when compared to the saline treated control group. The significance between treated groups is * p < 0.05, ** p < 0.01 and *** p < 0.001.
Figure 9
Figure 9
Protein levels in cerebrospinal fluid (CSF) and blood serum taken 4 weeks after SCI. The number of animals: n = 3 for healthy control, n = 4 for control (saline), n = 4 for cM, n = 5 for CM and n = 5 for WJ-MSCs. * p < 0.05, ** p < 0.01 and *** p < 0.001.

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