J Korean Neurosurg Soc.  2021 Sep;64(5):705-715. 10.3340/jkns.2021.0003.

Optimal Ratio of Wnt3a Expression in Human Mesenchymal Stem Cells Promotes Axonal Regeneration in Spinal Cord Injured Rat Model

• Yoon HH
• Lee HJ
• Min J
• Kim JH
• Park JH
• Kim JH
• Kim SW
• Lee H
• Jeon SR

• KMID: 2519697
• DOI: http://doi.org/10.3340/jkns.2021.0003

Abstract

Objective
: Through our previous clinical trials, the demonstrated therapeutic effects of MSC in chronic spinal cord injury (SCI) were found to be not sufficient. Therefore, the need to develop stem cell agent with enhanced efficacy is increased. We transplanted enhanced Wnt3asecreting human mesenchymal stem cells (hMSC) into injured spines at 6 weeks after SCI to improve axonal regeneration in a rat model of chronic SCI. We hypothesized that enhanced Wnt3a protein expression could augment neuro-regeneration after SCI.
Methods
: Thirty-six Sprague-Dawley rats were injured using an Infinite Horizon (IH) impactor at the T9–10 vertebrae and separated into five groups : 1) phosphate-buffered saline injection (injury only group, n=7); 2) hMSC transplantation (MSC, n=7); 3) hMSC transfected with pLenti vector (without Wnt3a gene) transplantation (pLenti-MSC, n=7); 4) hMSC transfected with Wnt3a gene transplantation (Wnt3a-MSC, n=7); and 5) hMSC transfected with enhanced Wnt3a gene (1.7 fold Wnt3a mRNA expression) transplantation (1.7 Wnt3a-MSC, n=8). Six weeks after SCI, each 5×10⁵ cells/15 µL at 2 points were injected using stereotactic and microsyringe pump. To evaluate functional recovery from SCI, rats underwent Basso-Beattie-Bresnahan (BBB) locomotor test on the first, second, and third days post-injury and then weekly for 14 weeks. Axonal regeneration was assessed using growth-associated protein 43 (GAP43), microtubule-associated protein 2 (MAP2), and neurofilament (NF) immunostaining.
Results
: Fourteen weeks after injury (8 weeks after transplantation), BBB score of the 1.7 Wnt3a-MSC group (15.0±0.28) was significantly higher than that of the injury only (10.0±0.48), MSC (12.57±0.48), pLenti-MSC (12.42±0.48), and Wnt3a-MSC (13.71±0.61) groups (p<0.05). Immunostaining revealed increased expression of axonal regeneration markers GAP43, MAP2, and NF in the Wnt3a-MSC and 1.7 Wnt3a-MSC groups.
Conclusion
: Our results showed that enhanced gene expression of Wnt3a in hMSC can potentiate axonal regeneration and improve functional recovery in a rat model of chronic SCI.

Keyword

Spinal cord regeneration; Recovery of function; Mesenchymal stem cells; Spinal cord injuries; Wnt3a

Figure

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  Fig. 1. Wnt3a mRNA expression in each transfection condition. 2× Wnt3a-MSc showed 1.7 fold Wnt3a mRNA expression level, and 4× Wnt3a-MSc showed 2.4 fold Wnt3a mRNA expression level as compared to 1× Wnt3a-MSc. *p<0.001. MSc : mesenchymal stem cells.

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  Fig. 2. Western blotting analysis of Wnt3a protein expression in each transfection condition. 2× Wnt3a-MSc showed 3.25 fold Wnt3a protein expression, and 4× Wnt3a-MSc showed 2.38 fold Wnt3a as compared to 1× Wnt3a-MSc. *p<0.001. MSc : mesenchymal stem cells.

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  Fig. 3. Western blotting analysis. β-catenin expression in L929 cells indicates functionality of secreted Wnt3a protein. 2× Wnt3a-MSc showed a 2.2 fold increase in β-catenin expression level as compared to 1× Wnt3a-MSc. *p<0.001. MSc : mesenchymal stem cells.

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  Fig. 4. basso-beattie-bresnahan (bbb) locomotor scale test. We transplanted phosphate-buffered saline (injury only group), hMSc (MSc group), hMSc transfected with pLenti vector (pLenti-MSc group), hMSc transfected with Wnt3a gene (Wnt3a-MSc group), and hMSc transfected with enhanced Wnt3a gene (1.7 Wnt3a-MSc group) in each group of rat at 6 weeks after spinal cord injury (new chronic spinal cord injury model). The 1.7 Wnt3a-MSc group showed significant behavioral recovery as compared to all other groups at 8 weeks after transplantation. Data were presented as mean±standard deviation. *p<0.05. MSc : mesenchymal stem cells, hMSc : human mesenchymal stem cells.

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  Fig. 5. Immunofluorescent staining of axonal regeneration marker growth-associated protein 43 (GAP43). confocal microscopic images revealed that anti-GAP43 antibody staining in the spinal cord was greater in the Wnt3a-MSc and 1.7 Wnt3a-MSc groups than in other groups. A : Tile scan image; scale bar : 1000 μm. b : Scale bar : 50 μm (magnification, ×100). c : Quantification of fluorescence intensity. *p<0.05. MSc : mesenchymal stem cells, DAPI : 4',6-diamidino-2-phenylindole.

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  Fig. 6. Immunofluorescent staining of axonal regeneration marker microtubule-associated protein 2 (MAP2). confocal microscopic images revealed that anti-MAP2 antibody staining in the spinal cord was greater in the Wnt3a-MSc and 1.7 Wnt3a-MSc groups than in other groups. A : Tile scan image; scale bar : 1000 μm. b : Scale bar : 50 μm (magnification, ×100). c : Quantification of fluorescence intensity. *p<0.05. MSc : mesenchymal stem cells, DAPI : 4',6-diamidino-2-phenylindole.

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  Fig. 7. Immunofluorescent staining of axonal regeneration marker neurofilament (NF). confocal microscopic images revealed that anti-NF antibody staining in the spinal cord was greater in the Wnt3a-MSc and 1.7 Wnt3a-MSc groups than in other groups. A : Tile scan image; scale bar : 1000 μm. b : Scale bar : 50 μm (magnification, ×100). c : Quantification of fluorescence intensity. *p<0.05. MSc : mesenchymal stem cells, DAPI : 4',6-diamidino-2-phenylindole.

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