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Bone marrow-derived mesenchymal stem cells expressing the Shh transgene promotes functional recovery after spinal cord injury in rats
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Yijia Jia a , Dou Wu b , Ruiping Zhang c , Weibing Shuang d , Jiping Sun b , Haihu Hao b , Qijun An b , Qiang Liu b,∗ a
Department of Orthopaedics, First Clinical Medical College of Shanxi Medical University, Taiyuan 030001, PR China Department of Orthopaedics, Dayi Hospital of Shanxi Medical University, Taiyuan 030032, PR China c Department of Radiology, First Clinical Medical College of Shanxi Medical University, Taiyuan 030001, PR China d Department of Urology, First Clinical Medical College of Shanxi Medical University, Taiyuan 030001, PR China b
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Shh-BMSCs treatment provided long-term stable expression of Shh. More BMSCs were detected by Shh-BMSCs treatment. Shh-BMSCs treatment significantly improved functional recovery. Shh-BMSCs treatment increased the expression of bFGF and VEGF in BMSCs after SCI. Shh-BMSCs treatment promoted NF200 expression and reduced GFAP expression.
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Article history: Received 26 February 2014 Accepted 6 May 2014 Available online xxx
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Keywords: Bone marrow-derived mesenchymal stem cells Rat Shh Spinal cord injury
Spinal cord injury (SCI) is one of the most disabling diseases. Cell-based gene therapy is becoming a major focus for the treatment of SCI. Bone marrow-derived mesenchymal stem cells (BMSCs) are a promising stem cell type useful for repairing SCI. However, the effects of BMSCs transplants are likely limited because of low transplant survival after SCI. Sonic hedgehog (Shh) is a multifunctional growth factor which can facilitate neuronal and BMSCs survival, promote axonal growth, prevent activation of the astrocyte lineage, and enhance the delivery of neurotrophic factors in BMSCs. However, treatment of SCI with Shh alone also has limited effects on recovery, because the protein is cleared quickly. In this study, we investigated the use of BMSCs overexpressing the Shh transgene (Shh-BMSCs) in the treatment of rats with SCI, which could stably secrete Shh and thereby enhance the effects of BMSCs, in an attempt to combine the advantages of Shh and BMSCs and so to promote functional recovery. After Shh-BMSCs treatment of SCI via the subarachnoid, we detected significantly greater damage recovery compared with that seen in rats treated with phosphate-buffered saline (PBS) and BMSCs. Use of Shh-BMSCs increased the expression and secretion of Shh, basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF), improved the behavioral function, enhanced the BMSCs survival, promoted the expression level of neurofilament 200 (NF200), and reduced the expression of glial fibrillary acidic protein (GFAP). Thus, our results indicated that Shh-BMSCs enhanced recovery of neurological function after SCI in rats and could be a potential valuable therapeutic intervention for SCI in humans. © 2014 Elsevier Ireland Ltd. All rights reserved.
1. Introduction
∗ Corresponding author at: Department of Orthopaedics, Dayi Hospital of Shanxi Medical University, 99 Longcheng Street, Taiyuan 030032, Shanxi, PR China. Tel.: +86 0351 8379001; fax: +86 0351 8379099. E-mail address: [email protected] (Q. Liu).
Spinal cord injury (SCI), markedly leads to neurological deficits in survivors, who then have a lifelong disability. Currently, treatments such as steroids, protein kinase, and metalloproteinase inhibition and regeneration techniques, have been used to improve the symptoms of patients with SCI, but with limited efficacy [13].
http://dx.doi.org/10.1016/j.neulet.2014.05.010 0304-3940/© 2014 Elsevier Ireland Ltd. All rights reserved.
Please cite this article in press as: Y. Jia, et al., Bone marrow-derived mesenchymal stem cells expressing the Shh transgene promotes functional recovery after spinal cord injury in rats, Neurosci. Lett. (2014), http://dx.doi.org/10.1016/j.neulet.2014.05.010
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Cell-based gene therapy has been considered as potential treatment for SCI [16]. Among various types of candidate stem cells, bone marrow-derived mesenchymal stem cells (BMSCs) are a promising stem cell type for repairing SCI [8]. However, the effects of BMSCs transplants are likely limited because of low transplant survival after SCI [18]. Sonic hedgehog (Shh), a protein that is produced by the notochord and floor plate, is a member of the family of hedgehog proteins and is essential for the development and differentiation of the nervous system [11]. Shh can upregulate the expression and secretion of basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) in BMSCs [4], improving the lesion microenvironment after SCI. In addition, Shh can promote the survival of neurons and BMSCs and act as a guidance factor for axon growth in the process of development [2,5]. Shh can inhibit activation of the astrocyte lineage; hence, it reduces scar formation after SCI [1]. Hence, Shh may aid functional recovery from SCI. However, an injection of Shh protein after SCI does not have a beneficial effect on functional behavioral recovery [3]. That was because of the fast clearance of Shh protein from the spinal cord, and long-term release of Shh may be more effective in the repair process [14]. On the basis of previous studies, we speculated that transplantation of BMSCs that have been transfected with a Shh transgene, which can provide long-term stable expression and secretion of high levels of Shh and so enhance the effects of BMSCs therapy, would facilitate repair and recovery after SCI. In this study, we investigated the therapeutic effects of transplanting BMSCs transfected with Shh in rats after SCI.
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2. Materials and methods
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2.1. Spinal cord contusion injury model
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All animal experiments were approved and overseen by the Animal Center of Shanxi Medical University. In this study, 48 adult female Sprague–Dawley rats weighing 200–220 g were used. Animals were anesthetized with 10% chloral hydrate (0.3 ml/100 g, intraperitoneally) [10]; the SCI model was then established using Allen’s method [13], with some modifications. In the present study, after spinal cords were exposed through performing laminectomy at T10, a moderate SCI was induced by dropping a 10-g rod (diameter, 2.5 mm) from a height of 25 mm onto an impounder positioned on the spinal cord.
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2.2. Cell culture and generation of BMSCs expressing Shh
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BMSCs were isolated and expanded as described previously [23], and then identified by morphological features, osteogenetic and adipogenic differentiation, and flow cytometric analysis. The cells were cultured in low glucose Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum at 37 ◦ C under a 5% CO2 atmosphere. At passage 3, BMSCs were prepared for the subsequent experiments. To trace grafted cells in vivo, BMSCs were transduced with a red fluorescent protein (DsRed) reporter gene. The rat Shh sequence (NM 017221) was synthesized and verified by sequencing. A lentiviral vector containing the DsRed or the Shh-DsRed gene was packaged into DsRed and Shh-DsRed lentiviruses by cotransfection with three helper plasmids, in 293T cells, using Lipofectamine 2000. The titers of the Shh-DsRed lentivirus and DsRed lentivirus ranged from 4 × 108 TU/ml to 8 × 108 TU/ml, as assessed using hole-by-hole dilution method. BMSCs were infected with lentiviruses following the standard procedures of infection. BMSCs containing the DsRed gene and those containing the ShhDsRed gene (Shh-BMSCs) were harvested. The effects of Shh-DsRed
and DsRed transduction were detected using a fluorescence microscope. The Shh protein expression in cells was analyzed by western blot analysis. 2.3. Cell treatment and experimental groups Forty-eight female adult Sprague–Dawley rats were randomly assigned to three groups (n = 16): (1) phosphate-buffered saline (PBS) group, (2) BMSCs group, (3) Shh-BMSCs group. Seven days after SCI, a second laminectomy was performed at lumbar level 4. We therefore microinjected 50 L PBS solution containing 1 × 106 BMSCs or 1 × 106 Shh-BMSCs into the model rats intrathecally using a microsyringe at 1 week after SCI. In the control group, 50 L PBS alone was injected. 2.4. Behavioral testing Rats were examined by three trained examiners for six weeks after SCI in a blinded manner. The Basso–Beattie–Bresnahan (BBB) locomotor rating score was widely used to evaluate hind-limb motor function in an open field environment [20]. Briefly, rats were allowed to go around freely in a circular field for 4 min in order to observe the movements of the hind limbs. An inclined-plane test was detected to evaluate the animal’s ability to maintain postural stability as described previously [7]. Briefly, animals were positioned on an inclined plane, and the maximum inclination at which the rat could remain in a constant position for 5 s was recorded as the final angle. 2.5. Preparations for histology and immunohistochemistry On day 28 after treatment, the animals were anesthetized and rapidly perfused with 4% paraformaldehyde in PBS. Spinal cords were dissected out, post-fixed in 4% paraformaldehyde overnight, dehydrated in 30% sucrose, and then continuously sectioned at 20-m thickness using a cryostat. Tissue was sliced parasagitally for the segments (8–10 mm in length) encompassing the injury site. For immunofluorescence staining, sections were incubated with primary antibodies overnight at 4 ◦ C. The following primary antibodies were used: mouse anti-neurofilament 200 antibody (NF200, 1:50, Santa Cruz Biotechnology) and mouse anti-glial fibrillary acidic protein antibody (GFAP, 1:50, Santa Cruz Biotechnology). Followed by washing with PBS, sections were then incubated with goat anti-mouse IgG antibody for 1 h at room temperature, in a dark place. These sections were observed under a fluorescence microscope and all images were analyzed using image J software. The region of interest was an area of 2 mm centered at the contusion epicenter for quantitation. These values of three regions of interest in a longitudinal section were averaged to obtain the average GFAP and NF200 staining intensity for a section; and these were averaged for the five sections to obtain the average GFAP and NF200 staining intensity for each animal. The number of BMSCs in the five sections was averaged to obtain the number of BMSCs per section in each animal. Observers were blinded during measurements. For immunohistochemical staining, the sections were washed with PBS, blocked with goat serum (10%) in PBS for 30 min, and then incubated overnight at 4 ◦ C with primary antibodies. The following primary antibodies were used: rabbit anti-Shh (1:150, Cell Signaling Technology), rabbit anti-bFGF (1:100, Santa Cruz Biotechnology) and rabbit anti-VEGF (1:100, Santa Cruz Biotechnology). Immunoreactivity was viewed by staining with diaminobenzidine (DAB), and the sections were observed under an invert microscope.
Please cite this article in press as: Y. Jia, et al., Bone marrow-derived mesenchymal stem cells expressing the Shh transgene promotes functional recovery after spinal cord injury in rats, Neurosci. Lett. (2014), http://dx.doi.org/10.1016/j.neulet.2014.05.010
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Fig. 1. Identification of the BMSCs, transduction of Shh into BMSCs with lentivirus, and the behavioral results. (A) The morphology of BMSCs at passage 3 (scale bar = 100 m). (B) The cells were induced to differentiate into osteocytes after 28 d in osteogenic media, and were assessed by alizarin red staining (scale bar = 100 m). (C) Adipogenic differentiation after 23 d in specific adipogenic-induction media. The cultured cells were stained by oil red-O solution (scale bar = 100 m). (D) Cell surface marker CD29 (+), CD34 (−), CD44 (+), CD45 (−) were detected using secondary antibodies labeled with FITC or PE. (E) DsRed expression in BMSCs 5 days after Shh-DsRed lentivirus transduction (scale bar = 100 m). (F) Western blot detection of Shh protein expression in vitro (Group 1, BMSCs; Group 2, Shh-BMSCs). (G) and (H) The behavioral results. From week 2 to week 6 after SCI, treatment with Shh-BMSCs resulted in a BBB score and inclined plane score that were significantly higher compared with those in the other two groups (n = 6, *P < 0.05); and the BBB and inclined plane scores of the PBS group were significantly lower than those of the BMSCs group (n = 6, *P < 0.05).
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2.6. Western blotting Animals were anesthetized with an overdose of 10% chloral hydrate at day 28 after treatment. 1-cm spinal cord tissue segments were collected from the lesion center. Proteins from the cells and spinal cord samples were used for Western blot analysis. The membrane with proteins was incubated with primary antibodies for Shh (1:1000, Cell Signaling Technology), bFGF (1:500, Santa Cruz Biotechnology) and VEGF (1:500, Santa Cruz Biotechnology) overnight at 4 ◦ C and followed by incubation with secondary antibodies for 2 h at room temperature. Protein signals were detected by electrochemiluminescence. The density of bands on the blots was analyzed using Image J software.
2.7. Statistical analysis All data were expressed as mean ± SD. One-way analysis of variance (ANOVA) was used to compare mean values, followed by Bonferroni post hoc analysis. Repeated measures ANOVA with the Tukey–Kramer test was used to analyze weekly BBB and inclined plane scores in different groups. Statistical analyses were
performed using SPSS 16.0 software. Statistical evaluations were considered significant at P < 0.05.
3. Results 3.1. Identification of BMSCs and Shh expression after transfection in vitro The morphological features of the cells were observed (Fig. 1A). We evaluated osteogenic differentiation in osteogenic media by alizarin red staining, at day 28 (Fig. 1B). The adipogenic differentiation in specific adipogenic-induction media was assessed at day 23 by staining with oil red-O solution (Fig. 1C). As shown in Fig. 1D, the cells were found to express cell markers CD29 and CD44, but CD34 and CD45 were not detected by flow cytometry. Cell surface marker CD29 (+), CD34 (−), CD44 (+) and CD45 (−) indicate that the cells may be BMSCs. Based on the above factors, we deduced that the cells were BMSCs. We identified red fluorescence in BMSCs at day 5 after ShhDsRed lentivirus transfection (Fig. 1E). To examine the protein levels expressed from the transferred Shh in vitro, we used
Please cite this article in press as: Y. Jia, et al., Bone marrow-derived mesenchymal stem cells expressing the Shh transgene promotes functional recovery after spinal cord injury in rats, Neurosci. Lett. (2014), http://dx.doi.org/10.1016/j.neulet.2014.05.010
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Fig. 2. Western blotting and immunohistochemical staining results. The expression of Shh, bFGF and VEGF was detected by Western blotting (A) and immunochemical staining (B) on day 28 after treatment. In Shh-BMSCs group, strong immunohistochemical staining for Shh, bFGF and VEGF were observed in the cytoplasm of cells around the injury site; and less immunohistochemical staining were detected in the other two groups (B, scale bar = 20 m). By Western blotting analysis, the protein levels of Shh, bFGF, and VEGF were significantly higher in Shh-BMSCs group compared to those in BMSCs and PBS groups (C, n = 6, *P < 0.05); and the protein levels in BMSCs group were significantly higher than those in PBS group (C, n = 6, *P < 0.05).
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western blot analysis (Fig. 1F). The level of Shh expression in ShhBMSCs was significantly increased compared with the BMSCs.
P < 0.05), and the Shh, VEGF, and bFGF protein expression in the BMSCs group increased significantly in comparison with the levels in the PBS group (n = 6, P < 0.05).
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3.2. Behavioral tests As shown in Fig. 1G and H, from week 2 to week 6 after injury, BBB and inclined plane tests showed highly significant motor improvement in rats that had been treated with Shh-BMSCs compared with those treated with PBS or BMSCs (n = 6, P < 0.05), and the BBB and inclined plane scores of the BMSCs group were significantly greater than those of the PBS group (n = 6, P < 0.05). 3.3. Shh, VEGF, and bFGF protein levels The Shh, VEGF, and bFGF protein levels were determined at day 28 after treatment, using western blot analysis and immunohistochemical staining (Fig. 2A and B). As shown in Fig. 2C, the protein levels of Shh, VEGF, and bFGF in the Shh-BMSCs group were markedly higher than those in the PBS and BMSCs groups (n = 6,
3.4. NF 200 and GFAP expression and BMSCs survival On day 28 after transplantation, the expression of NF200 in the Shh-BMSCs group was significantly higher than that in the other two groups, and the staining in the PBS group was significantly lower than that in the BMSCs group (Fig. 3A–C, and I, n = 6, P < 0.05). The expression of GFAP on day 28 after transplantation was markedly weaker in the Shh-BMSCs group versus the other two groups (Fig. 3D, E, F and J, n = 6, P < 0.05), and the staining between the PBS and BMSCs groups did not differ significantly (Fig. 3D, E and J, n = 6, P > 0.05). Moreover, the number of BMSCs present at this time point in the Shh-BMSCs group was significantly more than those in the BMSCs group on day 28 after transplantation (Fig. 3 G, H and K, n = 6, P < 0.05).
Please cite this article in press as: Y. Jia, et al., Bone marrow-derived mesenchymal stem cells expressing the Shh transgene promotes functional recovery after spinal cord injury in rats, Neurosci. Lett. (2014), http://dx.doi.org/10.1016/j.neulet.2014.05.010
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Fig. 3. Immunofluorescence for NF200 and GFAP and the number of BMSCs survival on longitudinal sections. NF200 expression in Shh-BMSCs group (C) was significantly greater than those in BMSCs (B) and PBS (A) groups, and NF200 expression in BMSCs group (B) was significantly higher than that in PBS group (A) on day 28 after treatment (I, n = 6, *P < 0.05, scale bar = 50 m). Little GFAP expression was observed in Shh-BMSCs group (F) and there were no differences between PBS (D) and BMSCs (E) groups in the expression of GFAP on day 28 after treatment (J, n = 6, *P < 0.05, scale bar = 50 m). More BMSCs were observed in Shh-BMSCs group (H) than in BMSCs group (G) on day 28 after treatment (K, n = 6, *P < 0.05, scale bar = 200 m).
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4. Discussion Although there are many controversies over BMSCs and their use, many studies have reported that BMSCs transplantation after SCI showed consistent benefit in preclinical models [8]. However, the survival of BMSCs after SCI remains problematic [18]. BMSCs transplants may be lost due to various events including ischemia, hypoxia, inflammation, immunological rejection and so on after SCI [18,21]. Therefore, the efficiency of treatment by BMSCs alone is limited. During spinal cord regeneration, Shh is a candidate pleiotropic beneficial environmental factor. However, the Shh signaling pathway is well conserved [15]; only a little Shh expression is observed after SCI. In the present study, we demonstrated that BMSCs expressing the Shh transgene, which can combine the advantages
of Shh and BMSCs, were delivered into the subarachnoid space to accelerate functional recovery in a rat SCI model. In our study, BMSCs were transfected with a Shh transgene using a lentiviral vector, resulting in high Shh protein levels by western blot analysis in vitro. After migrating to the lesion, BMSCs can secrete the Shh protein after ischemic injury [24], but the expression of Shh is low. By day 28 after treatment, Shh expression in the spinal cord tissues in the Shh-BMSCs group was significantly higher than those in the BMSCs and PBS groups by western blot analysis. We found that Shh-BMSCs had a high transduction efficiency and contained a high level of Shh protein in vivo. Thus, release of Shh into the injured region was successful. Previous study has shown that BMSCs expressing the Shh transgene maximize their survival after transplantation [2]. At day 28 after treatment, more BMSCs were present in the injured
Please cite this article in press as: Y. Jia, et al., Bone marrow-derived mesenchymal stem cells expressing the Shh transgene promotes functional recovery after spinal cord injury in rats, Neurosci. Lett. (2014), http://dx.doi.org/10.1016/j.neulet.2014.05.010
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tissue in the Shh-BMSCs group than in the BMSCs group. This indicates that cell transplantation can be more successful when BMSCs expressing the Shh transgene. More survival of BMSCs in the Shh-BMSCs group could secrete more Shh protein and play a better role in promoting repair damage of the spinal cord. Although cell-based gene therapy may lead the carcinogenicity of exogenous gene, Shh-BMSCs did not result in the formation of tumors during the five weeks after the transplantation. The repair mechanisms of BMSCs transplantation have not been clarified clearly [19]. After SCI, neural differentiation of BMSCs is still quite controversial [19]. Trophic factors may play an important role in promoting a microenvironment conductive to enhancing functional recovery [19]. bFGF is well-studied trophic factor, which can promote the survival of neurons and axon growth [12]. VEGF is particularly effective in blood vessel development, neural populations, and decreased apoptosis in SCI, and plays a key role in neuroprotection [22]. After migrating to the lesion, BMSCs can secrete these trophic factors to improve the microenvironment [6,13], but the expression of these factors is low. Shh can increase the expression and secretion of bFGF and VEGF in BMSCs [4]. In our study, we found that the Shh secretion in the Shh-BMSCs group improved bFGF and VEGF expression within the SCI region. By promoting the expression of bFGF and VEGF, Shh-BMSCs could improve the microenvironment in the lesions and enhance functional recovery. GFAP is considered a marker protein of astrocytes. The role of astrocytes at a later stage after SCI may be suppressed because of mechanical and chemical impedence [17]. Our study indicated that GFAP expression at the injury site in the Shh-BMSCs group was decreased compared with that in the other two groups by day 28 after treatment, during the astrocyte suppression period, because of the effect of Shh. Reduction of the scar formation is beneficial for axon regeneration both physically and chemically. NF200 is considered to be a marker of the axon. Shh can promote axon growth and potentiate neural cell survival. In addition, the reduction of the scar and secretion of trophic factors will be beneficial for axon regeneration. In our study, we found that Shh-BMSCs demonstrated a higher expression of NF200 than did the other two groups by day 28 after treatment. This finding suggests that ShhBMSCs could enhance these biological effects in the recovery from SCI. The BBB and inclined plane scores in the Shh-BMSCs group showed a greater statistically significant improvement compared with the recovery in the other groups. We demonstrated a progressive functional recovery over time. Previous studies have shown that treatment of BMSCs significantly improves functional recovery after SCI [9]. In our study, there were greater persistence of BMSCs, more trophic factors, more axon regeneration and less scar formation in the Shh-BMSCs group; therefore, Shh-BMSCs can enhance the functional recovery of SCI in rats. In conclusion, our results suggest that transplanting BMSCs transfected with a Shh transgene can promote functional recovery through complex processes after the occurrence of SCI and could be a potential valuable therapeutic intervention for SCI in humans.
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This work was supported by the National Natural Science Foundation of China (Grant number: 81371628).
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Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Bone marrow-derived mesenchymal stem cells expressing the Shh transgene promotes functional recovery after spinal cord injury in rats
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Yijia Jia a , Dou Wu b , Ruiping Zhang c , Weibing Shuang d , Jiping Sun b , Haihu Hao b , Qijun An b , Qiang Liu b,∗ a
Department of Orthopaedics, First Clinical Medical College of Shanxi Medical University, Taiyuan 030001, PR China Department of Orthopaedics, Dayi Hospital of Shanxi Medical University, Taiyuan 030032, PR China c Department of Radiology, First Clinical Medical College of Shanxi Medical University, Taiyuan 030001, PR China d Department of Urology, First Clinical Medical College of Shanxi Medical University, Taiyuan 030001, PR China b
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Shh-BMSCs treatment provided long-term stable expression of Shh. More BMSCs were detected by Shh-BMSCs treatment. Shh-BMSCs treatment significantly improved functional recovery. Shh-BMSCs treatment increased the expression of bFGF and VEGF in BMSCs after SCI. Shh-BMSCs treatment promoted NF200 expression and reduced GFAP expression.
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Article history: Received 26 February 2014 Accepted 6 May 2014 Available online xxx
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Keywords: Bone marrow-derived mesenchymal stem cells Rat Shh Spinal cord injury
Spinal cord injury (SCI) is one of the most disabling diseases. Cell-based gene therapy is becoming a major focus for the treatment of SCI. Bone marrow-derived mesenchymal stem cells (BMSCs) are a promising stem cell type useful for repairing SCI. However, the effects of BMSCs transplants are likely limited because of low transplant survival after SCI. Sonic hedgehog (Shh) is a multifunctional growth factor which can facilitate neuronal and BMSCs survival, promote axonal growth, prevent activation of the astrocyte lineage, and enhance the delivery of neurotrophic factors in BMSCs. However, treatment of SCI with Shh alone also has limited effects on recovery, because the protein is cleared quickly. In this study, we investigated the use of BMSCs overexpressing the Shh transgene (Shh-BMSCs) in the treatment of rats with SCI, which could stably secrete Shh and thereby enhance the effects of BMSCs, in an attempt to combine the advantages of Shh and BMSCs and so to promote functional recovery. After Shh-BMSCs treatment of SCI via the subarachnoid, we detected significantly greater damage recovery compared with that seen in rats treated with phosphate-buffered saline (PBS) and BMSCs. Use of Shh-BMSCs increased the expression and secretion of Shh, basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF), improved the behavioral function, enhanced the BMSCs survival, promoted the expression level of neurofilament 200 (NF200), and reduced the expression of glial fibrillary acidic protein (GFAP). Thus, our results indicated that Shh-BMSCs enhanced recovery of neurological function after SCI in rats and could be a potential valuable therapeutic intervention for SCI in humans. © 2014 Elsevier Ireland Ltd. All rights reserved.
1. Introduction
∗ Corresponding author at: Department of Orthopaedics, Dayi Hospital of Shanxi Medical University, 99 Longcheng Street, Taiyuan 030032, Shanxi, PR China. Tel.: +86 0351 8379001; fax: +86 0351 8379099. E-mail address: [email protected] (Q. Liu).
Spinal cord injury (SCI), markedly leads to neurological deficits in survivors, who then have a lifelong disability. Currently, treatments such as steroids, protein kinase, and metalloproteinase inhibition and regeneration techniques, have been used to improve the symptoms of patients with SCI, but with limited efficacy [13].
http://dx.doi.org/10.1016/j.neulet.2014.05.010 0304-3940/© 2014 Elsevier Ireland Ltd. All rights reserved.
Please cite this article in press as: Y. Jia, et al., Bone marrow-derived mesenchymal stem cells expressing the Shh transgene promotes functional recovery after spinal cord injury in rats, Neurosci. Lett. (2014), http://dx.doi.org/10.1016/j.neulet.2014.05.010
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Cell-based gene therapy has been considered as potential treatment for SCI [16]. Among various types of candidate stem cells, bone marrow-derived mesenchymal stem cells (BMSCs) are a promising stem cell type for repairing SCI [8]. However, the effects of BMSCs transplants are likely limited because of low transplant survival after SCI [18]. Sonic hedgehog (Shh), a protein that is produced by the notochord and floor plate, is a member of the family of hedgehog proteins and is essential for the development and differentiation of the nervous system [11]. Shh can upregulate the expression and secretion of basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) in BMSCs [4], improving the lesion microenvironment after SCI. In addition, Shh can promote the survival of neurons and BMSCs and act as a guidance factor for axon growth in the process of development [2,5]. Shh can inhibit activation of the astrocyte lineage; hence, it reduces scar formation after SCI [1]. Hence, Shh may aid functional recovery from SCI. However, an injection of Shh protein after SCI does not have a beneficial effect on functional behavioral recovery [3]. That was because of the fast clearance of Shh protein from the spinal cord, and long-term release of Shh may be more effective in the repair process [14]. On the basis of previous studies, we speculated that transplantation of BMSCs that have been transfected with a Shh transgene, which can provide long-term stable expression and secretion of high levels of Shh and so enhance the effects of BMSCs therapy, would facilitate repair and recovery after SCI. In this study, we investigated the therapeutic effects of transplanting BMSCs transfected with Shh in rats after SCI.
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2. Materials and methods
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2.1. Spinal cord contusion injury model
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All animal experiments were approved and overseen by the Animal Center of Shanxi Medical University. In this study, 48 adult female Sprague–Dawley rats weighing 200–220 g were used. Animals were anesthetized with 10% chloral hydrate (0.3 ml/100 g, intraperitoneally) [10]; the SCI model was then established using Allen’s method [13], with some modifications. In the present study, after spinal cords were exposed through performing laminectomy at T10, a moderate SCI was induced by dropping a 10-g rod (diameter, 2.5 mm) from a height of 25 mm onto an impounder positioned on the spinal cord.
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2.2. Cell culture and generation of BMSCs expressing Shh
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BMSCs were isolated and expanded as described previously [23], and then identified by morphological features, osteogenetic and adipogenic differentiation, and flow cytometric analysis. The cells were cultured in low glucose Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum at 37 ◦ C under a 5% CO2 atmosphere. At passage 3, BMSCs were prepared for the subsequent experiments. To trace grafted cells in vivo, BMSCs were transduced with a red fluorescent protein (DsRed) reporter gene. The rat Shh sequence (NM 017221) was synthesized and verified by sequencing. A lentiviral vector containing the DsRed or the Shh-DsRed gene was packaged into DsRed and Shh-DsRed lentiviruses by cotransfection with three helper plasmids, in 293T cells, using Lipofectamine 2000. The titers of the Shh-DsRed lentivirus and DsRed lentivirus ranged from 4 × 108 TU/ml to 8 × 108 TU/ml, as assessed using hole-by-hole dilution method. BMSCs were infected with lentiviruses following the standard procedures of infection. BMSCs containing the DsRed gene and those containing the ShhDsRed gene (Shh-BMSCs) were harvested. The effects of Shh-DsRed
and DsRed transduction were detected using a fluorescence microscope. The Shh protein expression in cells was analyzed by western blot analysis. 2.3. Cell treatment and experimental groups Forty-eight female adult Sprague–Dawley rats were randomly assigned to three groups (n = 16): (1) phosphate-buffered saline (PBS) group, (2) BMSCs group, (3) Shh-BMSCs group. Seven days after SCI, a second laminectomy was performed at lumbar level 4. We therefore microinjected 50 L PBS solution containing 1 × 106 BMSCs or 1 × 106 Shh-BMSCs into the model rats intrathecally using a microsyringe at 1 week after SCI. In the control group, 50 L PBS alone was injected. 2.4. Behavioral testing Rats were examined by three trained examiners for six weeks after SCI in a blinded manner. The Basso–Beattie–Bresnahan (BBB) locomotor rating score was widely used to evaluate hind-limb motor function in an open field environment [20]. Briefly, rats were allowed to go around freely in a circular field for 4 min in order to observe the movements of the hind limbs. An inclined-plane test was detected to evaluate the animal’s ability to maintain postural stability as described previously [7]. Briefly, animals were positioned on an inclined plane, and the maximum inclination at which the rat could remain in a constant position for 5 s was recorded as the final angle. 2.5. Preparations for histology and immunohistochemistry On day 28 after treatment, the animals were anesthetized and rapidly perfused with 4% paraformaldehyde in PBS. Spinal cords were dissected out, post-fixed in 4% paraformaldehyde overnight, dehydrated in 30% sucrose, and then continuously sectioned at 20-m thickness using a cryostat. Tissue was sliced parasagitally for the segments (8–10 mm in length) encompassing the injury site. For immunofluorescence staining, sections were incubated with primary antibodies overnight at 4 ◦ C. The following primary antibodies were used: mouse anti-neurofilament 200 antibody (NF200, 1:50, Santa Cruz Biotechnology) and mouse anti-glial fibrillary acidic protein antibody (GFAP, 1:50, Santa Cruz Biotechnology). Followed by washing with PBS, sections were then incubated with goat anti-mouse IgG antibody for 1 h at room temperature, in a dark place. These sections were observed under a fluorescence microscope and all images were analyzed using image J software. The region of interest was an area of 2 mm centered at the contusion epicenter for quantitation. These values of three regions of interest in a longitudinal section were averaged to obtain the average GFAP and NF200 staining intensity for a section; and these were averaged for the five sections to obtain the average GFAP and NF200 staining intensity for each animal. The number of BMSCs in the five sections was averaged to obtain the number of BMSCs per section in each animal. Observers were blinded during measurements. For immunohistochemical staining, the sections were washed with PBS, blocked with goat serum (10%) in PBS for 30 min, and then incubated overnight at 4 ◦ C with primary antibodies. The following primary antibodies were used: rabbit anti-Shh (1:150, Cell Signaling Technology), rabbit anti-bFGF (1:100, Santa Cruz Biotechnology) and rabbit anti-VEGF (1:100, Santa Cruz Biotechnology). Immunoreactivity was viewed by staining with diaminobenzidine (DAB), and the sections were observed under an invert microscope.
Please cite this article in press as: Y. Jia, et al., Bone marrow-derived mesenchymal stem cells expressing the Shh transgene promotes functional recovery after spinal cord injury in rats, Neurosci. Lett. (2014), http://dx.doi.org/10.1016/j.neulet.2014.05.010
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Fig. 1. Identification of the BMSCs, transduction of Shh into BMSCs with lentivirus, and the behavioral results. (A) The morphology of BMSCs at passage 3 (scale bar = 100 m). (B) The cells were induced to differentiate into osteocytes after 28 d in osteogenic media, and were assessed by alizarin red staining (scale bar = 100 m). (C) Adipogenic differentiation after 23 d in specific adipogenic-induction media. The cultured cells were stained by oil red-O solution (scale bar = 100 m). (D) Cell surface marker CD29 (+), CD34 (−), CD44 (+), CD45 (−) were detected using secondary antibodies labeled with FITC or PE. (E) DsRed expression in BMSCs 5 days after Shh-DsRed lentivirus transduction (scale bar = 100 m). (F) Western blot detection of Shh protein expression in vitro (Group 1, BMSCs; Group 2, Shh-BMSCs). (G) and (H) The behavioral results. From week 2 to week 6 after SCI, treatment with Shh-BMSCs resulted in a BBB score and inclined plane score that were significantly higher compared with those in the other two groups (n = 6, *P < 0.05); and the BBB and inclined plane scores of the PBS group were significantly lower than those of the BMSCs group (n = 6, *P < 0.05).
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2.6. Western blotting Animals were anesthetized with an overdose of 10% chloral hydrate at day 28 after treatment. 1-cm spinal cord tissue segments were collected from the lesion center. Proteins from the cells and spinal cord samples were used for Western blot analysis. The membrane with proteins was incubated with primary antibodies for Shh (1:1000, Cell Signaling Technology), bFGF (1:500, Santa Cruz Biotechnology) and VEGF (1:500, Santa Cruz Biotechnology) overnight at 4 ◦ C and followed by incubation with secondary antibodies for 2 h at room temperature. Protein signals were detected by electrochemiluminescence. The density of bands on the blots was analyzed using Image J software.
2.7. Statistical analysis All data were expressed as mean ± SD. One-way analysis of variance (ANOVA) was used to compare mean values, followed by Bonferroni post hoc analysis. Repeated measures ANOVA with the Tukey–Kramer test was used to analyze weekly BBB and inclined plane scores in different groups. Statistical analyses were
performed using SPSS 16.0 software. Statistical evaluations were considered significant at P < 0.05.
3. Results 3.1. Identification of BMSCs and Shh expression after transfection in vitro The morphological features of the cells were observed (Fig. 1A). We evaluated osteogenic differentiation in osteogenic media by alizarin red staining, at day 28 (Fig. 1B). The adipogenic differentiation in specific adipogenic-induction media was assessed at day 23 by staining with oil red-O solution (Fig. 1C). As shown in Fig. 1D, the cells were found to express cell markers CD29 and CD44, but CD34 and CD45 were not detected by flow cytometry. Cell surface marker CD29 (+), CD34 (−), CD44 (+) and CD45 (−) indicate that the cells may be BMSCs. Based on the above factors, we deduced that the cells were BMSCs. We identified red fluorescence in BMSCs at day 5 after ShhDsRed lentivirus transfection (Fig. 1E). To examine the protein levels expressed from the transferred Shh in vitro, we used
Please cite this article in press as: Y. Jia, et al., Bone marrow-derived mesenchymal stem cells expressing the Shh transgene promotes functional recovery after spinal cord injury in rats, Neurosci. Lett. (2014), http://dx.doi.org/10.1016/j.neulet.2014.05.010
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Fig. 2. Western blotting and immunohistochemical staining results. The expression of Shh, bFGF and VEGF was detected by Western blotting (A) and immunochemical staining (B) on day 28 after treatment. In Shh-BMSCs group, strong immunohistochemical staining for Shh, bFGF and VEGF were observed in the cytoplasm of cells around the injury site; and less immunohistochemical staining were detected in the other two groups (B, scale bar = 20 m). By Western blotting analysis, the protein levels of Shh, bFGF, and VEGF were significantly higher in Shh-BMSCs group compared to those in BMSCs and PBS groups (C, n = 6, *P < 0.05); and the protein levels in BMSCs group were significantly higher than those in PBS group (C, n = 6, *P < 0.05).
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western blot analysis (Fig. 1F). The level of Shh expression in ShhBMSCs was significantly increased compared with the BMSCs.
P < 0.05), and the Shh, VEGF, and bFGF protein expression in the BMSCs group increased significantly in comparison with the levels in the PBS group (n = 6, P < 0.05).
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3.2. Behavioral tests As shown in Fig. 1G and H, from week 2 to week 6 after injury, BBB and inclined plane tests showed highly significant motor improvement in rats that had been treated with Shh-BMSCs compared with those treated with PBS or BMSCs (n = 6, P < 0.05), and the BBB and inclined plane scores of the BMSCs group were significantly greater than those of the PBS group (n = 6, P < 0.05). 3.3. Shh, VEGF, and bFGF protein levels The Shh, VEGF, and bFGF protein levels were determined at day 28 after treatment, using western blot analysis and immunohistochemical staining (Fig. 2A and B). As shown in Fig. 2C, the protein levels of Shh, VEGF, and bFGF in the Shh-BMSCs group were markedly higher than those in the PBS and BMSCs groups (n = 6,
3.4. NF 200 and GFAP expression and BMSCs survival On day 28 after transplantation, the expression of NF200 in the Shh-BMSCs group was significantly higher than that in the other two groups, and the staining in the PBS group was significantly lower than that in the BMSCs group (Fig. 3A–C, and I, n = 6, P < 0.05). The expression of GFAP on day 28 after transplantation was markedly weaker in the Shh-BMSCs group versus the other two groups (Fig. 3D, E, F and J, n = 6, P < 0.05), and the staining between the PBS and BMSCs groups did not differ significantly (Fig. 3D, E and J, n = 6, P > 0.05). Moreover, the number of BMSCs present at this time point in the Shh-BMSCs group was significantly more than those in the BMSCs group on day 28 after transplantation (Fig. 3 G, H and K, n = 6, P < 0.05).
Please cite this article in press as: Y. Jia, et al., Bone marrow-derived mesenchymal stem cells expressing the Shh transgene promotes functional recovery after spinal cord injury in rats, Neurosci. Lett. (2014), http://dx.doi.org/10.1016/j.neulet.2014.05.010
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Fig. 3. Immunofluorescence for NF200 and GFAP and the number of BMSCs survival on longitudinal sections. NF200 expression in Shh-BMSCs group (C) was significantly greater than those in BMSCs (B) and PBS (A) groups, and NF200 expression in BMSCs group (B) was significantly higher than that in PBS group (A) on day 28 after treatment (I, n = 6, *P < 0.05, scale bar = 50 m). Little GFAP expression was observed in Shh-BMSCs group (F) and there were no differences between PBS (D) and BMSCs (E) groups in the expression of GFAP on day 28 after treatment (J, n = 6, *P < 0.05, scale bar = 50 m). More BMSCs were observed in Shh-BMSCs group (H) than in BMSCs group (G) on day 28 after treatment (K, n = 6, *P < 0.05, scale bar = 200 m).
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4. Discussion Although there are many controversies over BMSCs and their use, many studies have reported that BMSCs transplantation after SCI showed consistent benefit in preclinical models [8]. However, the survival of BMSCs after SCI remains problematic [18]. BMSCs transplants may be lost due to various events including ischemia, hypoxia, inflammation, immunological rejection and so on after SCI [18,21]. Therefore, the efficiency of treatment by BMSCs alone is limited. During spinal cord regeneration, Shh is a candidate pleiotropic beneficial environmental factor. However, the Shh signaling pathway is well conserved [15]; only a little Shh expression is observed after SCI. In the present study, we demonstrated that BMSCs expressing the Shh transgene, which can combine the advantages
of Shh and BMSCs, were delivered into the subarachnoid space to accelerate functional recovery in a rat SCI model. In our study, BMSCs were transfected with a Shh transgene using a lentiviral vector, resulting in high Shh protein levels by western blot analysis in vitro. After migrating to the lesion, BMSCs can secrete the Shh protein after ischemic injury [24], but the expression of Shh is low. By day 28 after treatment, Shh expression in the spinal cord tissues in the Shh-BMSCs group was significantly higher than those in the BMSCs and PBS groups by western blot analysis. We found that Shh-BMSCs had a high transduction efficiency and contained a high level of Shh protein in vivo. Thus, release of Shh into the injured region was successful. Previous study has shown that BMSCs expressing the Shh transgene maximize their survival after transplantation [2]. At day 28 after treatment, more BMSCs were present in the injured
Please cite this article in press as: Y. Jia, et al., Bone marrow-derived mesenchymal stem cells expressing the Shh transgene promotes functional recovery after spinal cord injury in rats, Neurosci. Lett. (2014), http://dx.doi.org/10.1016/j.neulet.2014.05.010
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tissue in the Shh-BMSCs group than in the BMSCs group. This indicates that cell transplantation can be more successful when BMSCs expressing the Shh transgene. More survival of BMSCs in the Shh-BMSCs group could secrete more Shh protein and play a better role in promoting repair damage of the spinal cord. Although cell-based gene therapy may lead the carcinogenicity of exogenous gene, Shh-BMSCs did not result in the formation of tumors during the five weeks after the transplantation. The repair mechanisms of BMSCs transplantation have not been clarified clearly [19]. After SCI, neural differentiation of BMSCs is still quite controversial [19]. Trophic factors may play an important role in promoting a microenvironment conductive to enhancing functional recovery [19]. bFGF is well-studied trophic factor, which can promote the survival of neurons and axon growth [12]. VEGF is particularly effective in blood vessel development, neural populations, and decreased apoptosis in SCI, and plays a key role in neuroprotection [22]. After migrating to the lesion, BMSCs can secrete these trophic factors to improve the microenvironment [6,13], but the expression of these factors is low. Shh can increase the expression and secretion of bFGF and VEGF in BMSCs [4]. In our study, we found that the Shh secretion in the Shh-BMSCs group improved bFGF and VEGF expression within the SCI region. By promoting the expression of bFGF and VEGF, Shh-BMSCs could improve the microenvironment in the lesions and enhance functional recovery. GFAP is considered a marker protein of astrocytes. The role of astrocytes at a later stage after SCI may be suppressed because of mechanical and chemical impedence [17]. Our study indicated that GFAP expression at the injury site in the Shh-BMSCs group was decreased compared with that in the other two groups by day 28 after treatment, during the astrocyte suppression period, because of the effect of Shh. Reduction of the scar formation is beneficial for axon regeneration both physically and chemically. NF200 is considered to be a marker of the axon. Shh can promote axon growth and potentiate neural cell survival. In addition, the reduction of the scar and secretion of trophic factors will be beneficial for axon regeneration. In our study, we found that Shh-BMSCs demonstrated a higher expression of NF200 than did the other two groups by day 28 after treatment. This finding suggests that ShhBMSCs could enhance these biological effects in the recovery from SCI. The BBB and inclined plane scores in the Shh-BMSCs group showed a greater statistically significant improvement compared with the recovery in the other groups. We demonstrated a progressive functional recovery over time. Previous studies have shown that treatment of BMSCs significantly improves functional recovery after SCI [9]. In our study, there were greater persistence of BMSCs, more trophic factors, more axon regeneration and less scar formation in the Shh-BMSCs group; therefore, Shh-BMSCs can enhance the functional recovery of SCI in rats. In conclusion, our results suggest that transplanting BMSCs transfected with a Shh transgene can promote functional recovery through complex processes after the occurrence of SCI and could be a potential valuable therapeutic intervention for SCI in humans.
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This work was supported by the National Natural Science Foundation of China (Grant number: 81371628).
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