Neurotrophin-3 gene modified mesenchymal stem cells promote remyelination and functional recovery in the demyelinated spinal cord of rats

Journal of the Neurological Sciences 313 (2012) 64–74

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Neurotrophin-3 gene modified mesenchymal stem cells promote remyelination and functional recovery in the demyelinated spinal cord of rats Yu-Jiao Zhang a, 1, Wei Zhang a, 1, Cheng-Guang Lin e, Ying Ding a, Si-Fan Huang a, Jin-Lang Wu b, Yan Li a, Hongxin Dong f, Yuan-Shan Zeng a, c, d,⁎ a

Department of Histology and Embryology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, China Department of Electron Microscope, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, China Key Laboratory for Stem Cells and Tissue Engineering, Ministry of Education, Sun Yat-sen University, Guangzhou, China d Institute of Spinal Cord Injury, Sun Yat-sen University, Guangzhou, China e Department of Radiation Oncology, Cancer Center, Sun Yat-sen University, Guangzhou, China f Department of Psychiatry and Behavioral Sciences, Northwestern University, Feinberg School of Medicine, Chicago, USA b c

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Article history: Received 27 December 2010 Received in revised form 14 September 2011 Accepted 20 September 2011 Available online 13 October 2011 Keywords: Demyelination Remyelination Bone marrow mesenchymal stem cells Neurotrophin-3 Gene transfection

a b s t r a c t Multiple sclerosis (MS) is a debilitating neurodegenerative disease characterized by axonal/neuronal damage that may be caused by defective remyelination. Current therapies aim to slow the rate of degeneration, however there are no treatment options that can stop or reverse the myelin sheath damage. Bone marrow mesenchymal stem cells (MSCs) are a potential candidate for the cell implantation-targeted therapeutic strategies, but the proremyelination effects of MSCs when directly injected into a demyelinated cord lesion have been questioned. Neurotrophin-3 (NT-3) has been shown to serve a crucial role in the proliferation, differentiation and maturation of oligodendrocyte lineages. Here, we showed that implantation of NT-3 gene-modified MSCs via a recombinant adenoviral vector (Adv) into a region of ethidium bromide (EB)-induced demyelination in the spinal cord resulted in significant improvement of locomotor function and restoration of electrophysiological properties in rats. The morphological basis of this recovery was evidenced by robust myelin basic protein (MBP) expression and the extensive remyelination. AdvNT-3-MSC implants promote the endogenous remyelinating cells to participate directly in myelination, which was confirmed under light and electron microscopy. Our study suggested that genetically modified MSCs could be a potential therapeutic avenue for improving the efficacy of stem cell treatment for neurodegenerative diseases such as MS. © 2011 Elsevier B.V. All rights reserved.

1. Introduction Multiple sclerosis (MS) is a devastating neurodegenerative disease that affects up to 150 per 100,000 humans [1]. The major pathological change of MS is demyelination, resulting in axonal damage in the central nervous system, which eventually causes permanent disability. The traditional therapies for MS cannot effectively prevent the chronic and irreversible progression of this disease as they fail to prevent demyelination. Therefore, developing remyelination is the key target of experimental studies of MS, especially since recent reports demonstrated that remyelination also plays a role in axonal protection [2–4]. ⁎ Corresponding author at: Department of Histology and Embryology, Zhongshan School of Medicine, Sun Yat-sen University, 74# Zhongshan 2nd Road, Guangzhou 510080, China. Tel./fax: + 86 20 87332698. E-mail addresses: [email protected] (Y.-J. Zhang), [email protected] (W. Zhang), [email protected] (C.-G. Lin), [email protected] (Y. Ding), [email protected] (S.-F. Huang), [email protected] (J.-L. Wu), [email protected] (Y. Li), [email protected] (H. Dong), [email protected] (Y.-S. Zeng). 1 These authors contributed equally to this manuscript. 0022-510X/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.jns.2011.09.027

The advantage of therapeutic approaches based on cell replacement for demyelinating diseases has been confirmed. Experimental studies indicated that using myelinogenic cell populations, such as stem cells, oligodendrocyte precursor cells (OPCs), Schwann cells, and olfactory ensheathing cells, could provide a seemingly unlimited cell supply for transplantation and can be directed to myelinogenic phenotypes under controlled conditions [5,6]. Bone marrow mesenchymal stem cells (MSCs) are considered to be one of the most promising candidates for adult stem cell-based therapy for nervous system diseases due to their ease of collection, rapid proliferation, readily genetic manipulation, and their potential to be utilized as autografts [7]. In addition, there are a number of features that make MSCs beneficial for cell implantation therapies for multiple sclerosis, including immunomodulation, neuroprotection, and cell replacement [8]. Many studies have shown that MSC implantation exerts a therapeutic effect in experimental autoimmune encephalomyelitis (EAE) or toxin-induced demyelinated models, which is supported by evidence of functional recovery, immunomodulation and neuroprotection [9,10]. However, a recent study indicated that direct intralesional injection of multipotent MSCs did not lead to remyelination, but instead the transplanted

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MSCs may migrate into areas of normal tissue, deposit collagen, and be associated with axonal damage [11]. Thus, studies to further evaluate and to improve the efficacy of MSCs in treatment of demyelinated lesions are necessary. Neurotrophin-3 (NT-3) is known for its role in neuronal survival, differentiation and axonal regeneration. Recently, compelling evidence indicates NT-3 is involved in normal oligodendrocyte development [12,13]. NT-3 induces OPC proliferation and survival in both in vitro and in vivo studies [12,14,15]. In vitro studies demonstrate that NT-3 promotes OPC maturation and myelination [16,17]. In experimental demyelination models, NT-3 was shown to promote remyelination [18] and functional recovery [19] by enhancing OPC proliferation and differentiation. In the present study, we aim to investigate whether the implantation of NT-3 gene-transfected MSCs into an EB-induced demyelinated spinal cord in adult rats could advance remyelination and functional recovery. In addition, identifying and uncovering the possible mechanisms inducing this change may provide a therapeutic strategy for future clinical practice. 2. Materials and methods

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1 mL medium at 37 °C for 2 h. Medium was then added to the flask and cultured continuously until 48 h had elapsed since Adv infection. At this point, the medium was removed, and the cells were re-cultured with fresh medium for another 24 h. The expression of LacZ in Advtransfected MSCs was evaluated by X-gal staining, while that of NT-3 was determined by immunocytochemisty. 2.3. Experimental groups and cell transplantation Prior to transplantation, cultured MSCs were treated as follows (n = 5, each group): (1) without treatment, “MSC group”; (2) transfection with the LacZ gene, “AdvLacZ-MSC group”, where MSCs were transfected with AdvLacZ for 72 h; (3) transfection with the NT-3 gene, “AdvNT-3-MSC group”, treated similarly to the AdvLacZMSC group except that AdvNT-3 transfection was performed. Before transplantation, 10 μg/mL Hoechst 33342 (Sigma) was used to label MSCs in the different treatment groups for 2 h. Three days after EB injection, rats were anesthetized again, 1 μL suspension of MSCs (1 × 10 5/μL) was directly injected into the EB lesion site. However, both in the saline group and EB group, 1 μL DMEM was injected instead of cell suspension.

2.1. Induction of demyelinating lesion 2.4. Locomotion Adult female Sprague–Dawley rats (n = 25, 220–280 g, 9– 10 weeks old) were used and all procedures were performed in accordance with the Public Health Service Guide for the Care and Use of Laboratory Animals, Sun Yat-sen University. Prior to experimental commencement, animals were allowed to acclimate to the housing facilities and were handled daily for at least one week. Animals had free access to food and water throughout the study. Focal demyelinating lesions were induced by injection of EB into the dorsal funiculus (DF) of the rat spinal cord, according to previous reports [21–23]. Briefly, animals were anesthetized with 1% sodium pentobarbital (40 mg/kg, i.p.). A T9 laminectomy was performed and the spinal cord was exposed. Using a Hamilton syringe, the dorsal dura was sheared open and 1 μL EB (0.1 mg/mL in saline) was injected into the dorsal funiculus of the T10 thoracic cord at a depth of 1.8 mm by microsyringe using a stereotaxic apparatus. In the saline control (saline group), 1 μL of saline was injected into the same region of the T10 thoracic cord according to the same procedure. Three days later, rats were anesthetized as described above, and the T10 thoracic cord was exposed again for cell transplantation. 2.2. Isolation of bone marrow mesenchymal stem cells and adenovirus vector transfection

A total of 25 SD female rats were used for this test, and a 2 m long and 15 mm diameter beam was used to test the locomotor activity. The training and scoring methods were in accordance with the criteria previously used in the EB-induced demyelinated rats [24,25]. Briefly, a portion of the beam was measured and marked; a video camera was positioned in the same horizontal plane for recording during the tests. Prior to EB injection, the rats were trained to cross the beam without stalling, and they were offered a reward (sugar) upon completion of the task. After cell transplantation, the locomotor behavior was tested twice a week for 3 weeks. In each test, the rats traversed the beam twice in each direction. The video recordings of the movement were scored in slow motion by trained observers. Error scores were given according to the following scheme: 0 =‘normal’: foot positioned on top of the beam, no slippage; 1 = ‘minor error’: foot slip so that part of the foot was visible below the lower surface of beam, or the foot dragged along beam surface 2 = ‘major error’: whole foot slipped below lower surface of the beam [24]. The total score for each time at each test was calculated and used for analysis. The observer was blind to the identification of each experimental group throughout the experiments. 2.5. Spinal cord evoked potentials (SCEP)

MSCs were prepared from bone marrow according to our previous reports [20]. Briefly, the cells were obtained from the femurs of 5–10 day old male Sprague–Dawley rats and seeded at 1 × 107/mL in a 50 mL tissue culture flask with low glucose Dulbecco's modified Eagle's medium (DMEM, Gibco) supplemented with 10% fetal bovine serum (FBS, TBD, Zhanchen) and 100 units/mL penicillin and 100 units/mL streptomycin. Then the cells were incubated at 37 °C in a humidified atmosphere containing 5% CO2. The medium was changed at 48 h and floating cells were removed. When the adherent primary MSCs grew to confluence (passage 0), the cells were passaged using 0.25% trypsin/0.02% EDTA at the ratio of 1:3, and MSCs from passages 3–6 were used for all experiments. The replication-deficient adenoviral (Adv) vector carrying human NT-3 (AdvNT-3) under the control of the CMV promoter was constructed by Wang et al. as previously described [20]. Another Adv vector (AdvLacZ) serving as the reporter gene was generously provided by Dr. Wen-lin Huang's laboratory (Cancer Center, Sun Yat-sen University, Guangzhou, China). Adv-mediated gene transfection was performed as previously described [20]. Briefly, 80% confluent MSCs in a 50 mL culture flask were exposed to AdvNT-3 or AdvLacZ (MOI= 300) in

25 days after EB induction, 25 rats (n = 5 in each group) were anesthetized with ketamine (40 mg/kg) and 1% sodium pentobarbital (30 mg/kg). Each rat was stereotaxically fixed and its T1–L1 vertebrae were completely exposed. According to previous studies [26,27], the stimulation electrode was inserted into the T1–T2 interspinous ligaments, and a pair of recording electrodes was inserted into the interspinous ligaments of Ti3 to L1. These electrodes were then connected to the BL-410E Data Acquisition Analysis System for Life Science (Taimeng, China). The Parameter Settings of the SCEP signal are as follows: gain parameter 2000, time constant 0.01 s, and filtering 300 Hz. Each pulse stimulation is 50 ms in duration at a frequency of 5.1 Hz and with a 1 mV voltage density. 100 SCEP responses were collected and averaged for each rat in order to obtain high-quality waveforms. 2.6. Processing of tissue Following the recordings of evoked potentials, the rats in all 5 groups were transcardially perfused with 4% paraformaldehyde in

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0.1 M phosphate buffer (pH 7.4) under deep anesthesia by sodium pentobarbital (60 mg/kg i.p.). The length of spinal cord containing the lesion was removed and separated into rostral and caudal halves. One-half of the tissue from the center of the lesion was separated for cryosectioning to look for morphological and immunohistocytochemistry analysis, and the other half was prepared for semi-thin sectioning for analysis under the electron microscope.

(Additional Fig. B). Normal myelin, preserved demyelinated axons, and remyelinated axons (thin myelin) were counted within a unit area of 50 μm × 50 μm in each layer at a 200× magnification. All counts were performed blindly by one investigator. The total number of three axon types for 12 unit areas per rat was calculated and averaged. The percentage of remyelinated axons of the total axons was determined and compared.

2.7. Morphological analysis and immunohistochemistry

2.9. Quantification of differentiated phenotype from transplanted cells

X-gal histochemical staining was performed to identify the expression of the LacZ gene in AdvLacZ-MSC-implanted spinal cord. Cryosections were incubated in 20 mM K ferrocyanide, 2 mM MgCl2, and 1 mg/mL X-gal (Sangon, China) prepared with 0.01 M PBS, pH 7.4 overnight at 37 °C in darkness. Luxol Fast Blue staining was used to show the distribution of myelin. Following washing and dehydration in 95% ethanol, cryostat sections of the cords were placed into a solution containing 1% Luxol fast blue, 95% ethanol, and 10% acetic acid overnight at 56 °C. After serial rinsing in 95% and 70% ethanol and rehydration in water, sections were differentiated first in 0.05% lithium-carbonate and then in 70% ethanol until the contrast between gray and white matter was maximal, and finally, these sections were evaluated under a light microscope. For immunohistochemistry, the rostral halves were post-fixed in fresh paraformaldehyde and cryoprotected in 30% PB-sucrose at 4 °C. Transverse cryostat sections of the cords were cut at 20 μm and mounted on slides coated with gelatin. Primary antibodies were used as follows: mouse monoclonal anti-glial fibrillary acidic protein (GFAP, 1:200, Millipore), rabbit polyclonal anti-NG2 (a marker for oligodendrocyte progenitor cells, 1:200, Chemicon), mouse monoclonal anti-adenomatous polyposis coli (APC, labels cytoplasm of mature oligodendrocytes, 1:200, Chemicon), rabbit polyclonal anti-myelin basic protein (MBP, a structural protein of myelin, 1:200, Santa Cruz), mouse monoclonal anti-neurofilament (NF, 1:300, Boster) and rabbit polyclonal anti-NT-3 (1:300, Santa Cruz). Cy3-conjugated goat anti-mouse or anti-rabbit IgG (1:800, Jackson ImmunoResearch Labs, Inc.) and FITC-conjugated goat anti-mouse or anti-rabbit IgG (1:200, Jackson ImmunoResearch) were used as secondary antibodies. The IHC technique was previously described [30]. Briefly, cryosections were washed three times with 0.01 M PBS (pH 7.4) and incubated with 10% normal goat serum with 0.3% Triton X-100 in PBS for 30 min at 37 °C. The sections were incubated overnight in the appropriate primary antibodies at 4 °C. They were then washed repeatedly with PBS, incubated in their respective secondary antibodies for 1 h at 37 °C, washed with PBS, coverslipped, and finally examined with a fluorescent microscope (Leica, Germany).

Six transverse sections from each cell-implantation group were randomly selected to undergo NG2 and APC IHC staining to evaluate the differentiation of implanted-MSCs into oligodendrocyte lineage. The dorsal funiculus (DF) of the cord in each section was imaged at a 100× magnification and captured by a Leica DC300 camera mounted on a Leica microscope. A united area of 180 μm × 160 μm in the epicenter of the demyelinated lesion was set to count the total number of NG2- and APC-positive cells, the number of cells with NG2- or APCpositive and Hoechst33342 labeling, and the total number of cells labeled with Hoechst 33342. Only NG2- and APC-positive cells with clearly defined cell bodies were counted. Data were expressed as cells/mm 2, and the ratio of double-labeled cells (NG2 or APC and Hoechst33342) to the total Hoechst33342-labeled cells was calculated and compared between different MSC-implantation groups.

2.8. Ultrastructural analysis and evaluation of remyelination in vivo As for the semi-thin sections and ultrastructural analysis, the caudal halves of the spinal cord were cut into 3-mm blocks (Additional Fig. A) and post-fixed in a fixative solution containing 2.5% glutaraldehyde. After being washed several times in PBS, the tissues were then post-fixed with 2% osmium tetraoxide for 1 h at room temperature in darkness, dehydrated in ascending ethanol solutions and absolute acetone, immersed in 50% Epon812 in acetone and embedded in Epon812. Semi-thin sections (1 μm) were cut and stained with toluidine blue. For ultrastructural analysis, ultrathin sections were cut, stained with lead citrate and uranyl acetate, and then observed under a transmission electron microscope (Olympus, Japan). Two blocks near the demyelinated lesion were processed for semithin sectioning and toluidine blue staining. Two sections in which the lesion occupied the whole dorsal funiculus were selected from each block to perform remyelination analysis. A total of ten sections were selected from each group (n = 5 per group). The depth of the dorsal funiculus in the selected sections was divided into three layers

2.10. Statistical analysis All data are expressed as mean ± SD. One-way ANOVA or repeatedmeasure ANOVA was used to test for differences among groups. If equal variances were assumed, the least-significant difference (LSD) test was applied. Otherwise, the Kruskal–Wallis test was used. Significance levels were set at P b 0.05. 3. Results 3.1. Focal demyelination in dorsal funiculus of spinal cord EB, a nucleic acid chelator, causes the death of affected cells. When EB is injected into the central region of the dorsal funiculus (DF) of the adult spinal cord, it kills the glial cells (including the centrally myelin-forming oligodendrocytes) in the DF area due to its diffusion from the injection point. 3 weeks after EB injection, the DF region of the EB-injected cord was easily distinguished from the other white matter regions of the cord under phase contrast microscopy (Fig. 1A), where a focal demyelinated lesion area was also detected as confirmed by Luxol Fast Blue (LFB) staining (Fig. 1B). Hematoxylin–eosin (HE)-stained sections showed an area of pale staining (Fig.1C) in this region due to the deficiency of glias (Fig. 1D and E) and demyelinated axons (Fig. 1C and F). The lesions nearly covered the entire dorso-ventral extent of the DF and extended between 4 and 8 mm longitudinally. The lesions were mainly occupied by densely-packed demyelinated axons and islands of cellular debris and macrophages (Fig. 2A and B). Many degenerated axons were also found in the lesion area (Fig. 2B and Fig. 1F). A few remyelinated axons were observed in the lesion area, especially at the peripheral boundaries of the lesion zone (Fig. 2A and B), characterized by a very thin myelin sheath. However, the saline-injected dorsal funiculus was replete with normal myelinated axons (Fig. 2C and D). 3.2. Identification of AdvNT-3-MSCs in vitro and in vivo MSCs isolated from the bone marrow of adult rats displayed a typical morphology of MSCs (Fig. 3A), which was consistent with previous reports [20,28]. The MSCs expressed the cell surface marker CD29, but not CD34 or CD45, based on the fluorescence-activated cell sorter (FACS) analysis (data not shown). They also exhibited

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Fig. 1. A focal demyelinated area elicited by a direct EB injection into the dorsal funiculus (DF) of the spinal cord at 3 weeks after EB injection. (A) The image of the EB-injected DF region of the spinal cord (surrounded by black dash line) shown under phase contrast microscopy. Both Luxol fast blue (LFB) staining (B) and HE (C) indicated a focal pale-staining area when compared with the other white matter regions in the spinal cord. (D–E) This area also displayed a glia-free environment especially at the central region of the DF. The white dashed line in D and E labels the border of GFAP- and APC-positive expression, respectively. (F) Ultrastructural analysis showed some demyelinated axons in the DF region of the spinal cord (arrows). Scale bars, A–C = 320 μm; D and E = 160 μm; and F = 1 μm.

mesodermic and neuroectodermal differentiation [20] when placed in a proper environment in vitro. At MOI 300, cultured MSCs were successfully transfected with the foreign report gene (LacZ) and neurotrophin-3 (NT-3) gene via recombinant Adv (Fig. 3B and C). Three days after EB was injected into the spinal cord, bone marrow MSCs labeled with Hoechst33342 were implanted into the DF lesion zone of the cord (boxed area in the light microscopic image of Fig. 3D1). At 3 weeks post-injury, fluorescence-labeled MSCs

were detected and migrated circumferentially from the injected site at a distance not more than 0.5 mm (Fig. 3D). In the rats receiving Adv-transfected MSC (both AdvNT-3 and AdvLacZ) implants, X-gal staining and NT-3 immunofluorescence detected X-gal-positive (Fig. 3E) and NT-3-positive (double-labeled with Hoechst33342) MSCs (arrows indicated, Fig. 3F) in the lesion zone, which confirmed Adv-mediated foreign genes were still expressed in vivo at this time.

Fig. 2. Morphology of the dorsal funiculus (DF) after EB (A–B) or saline (C–D) injection. (B, D) Magnified views are shown in the area (asterisk) in A and C, respectively. Demyelination and many debris-laden macrophages are found predominantly in the central part of the DF region of the EB-injected cord. Some remyelinated axons were mainly at the lateral edge of the lesion (A–B). However, nearly normal DF of the spinal cord is shown in the cord injected by saline (D). Abbreviation: DF – dorsal funiculus; DH – dorsal horn. Scale bars, A and C = 80 μm; B and D = 40 μm.

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Fig. 3. (A) Cultured rat bone marrow MSCs from passage 3 at confluence show the fusiform shape and arranged in a helical manner. (B–C) Cultured MSCs were successfully transfected and expressed the foreign gene via Adv. (B) X-gal staining indicated β-galactosidase-positive products in the AdvLacZ-MSC group (30–40%, blue). (C) NT-3 immunofluorescence also confirmed AdvNT-3-transfected MSCs successfully expressed NT-3 gene (red). The cells were counterstained with Hoechst33342 (blue). (D) Prior to implantation, MSCs were labeled with Hoechst33342. At 3 weeks after lesion induction, Hoechst-labeled cells could be detected in the DF region, indicated as the light microscopic image (boxed area in D1) of the damaged cord. (E) X-gal-positive cells (black arrows in E1) were detected in the lesion zone that receiving AdvLacZ-MSC implants. E1 is the magnified view of the boxed area in E. (F, F1) It shows NT-3 immunofluorescence in the cross-section of cord from the rat receiving AdvNT-3 implants. Dashed lines label the boundary between DF and the bilateral DH. F is the magnified view of the area labeled with an asterisk in F1. White arrows indicate NT-3-positive cells (red) co-labeled with Hoechst 33342 (blue) in the DF area of the demyelinated cord. Abbreviation: DF – dorsal funiculus, DH – dorsal horn. Scale bar, E = 320 μm; A, D, D1 and F1 = 160 μm; B, E1 and F = 80 μm; and C = 40 μm.

3.3. Locomotor activity after AdvNT-3 MSC transplantation Within the first week post-injection, EB-injected only rats (the EB group) exhibited a staggering and retardant gait with higher behavioral error scores in the beam-walking test (Fig. 4). Rats in all groups showed an immediately elevated error score, even in the saline group, prior to the day 4 considering the effect of the injection procedure on locomotion. Then, the rats showed a gradual improvement up to the experiment end at 21 days, but this effect was more pronounced in the AdvNT-3-MSC group (Fig. 4). There was no significant difference between the AdvNT-3-MSC and saline groups (P N 0.05, Fig. 4), although the mean score in the AdvNT-3-MSC group was slightly higher than the saline group prior to the 10 day post-injury. However, the scores in both groups gradually returned to pre-injection values within three weeks. Compared with the EBEB, MSC or AdvLacZ-MSC groups did not induce distinct functional improvement until 10 days and 17 days, respectively (P b 0.05). But the mean scores in both MSC-implanted rats were lower than the EB group (Fig. 4). 3.4. Spinal cord evoked potentials (SCEP) SCEP response was used as an assessment of the electrophysiological properties of the descending axonal conduction in the DF of the spinal cord. It is important to evaluate the functional remyelination because anatomic myelin repair may not necessarily lead to functional myelination. Representative sample traces showed that SCEP stimulation evoked a short-latency positive–negative–positive wave in the saline group, which is similar to that in other groups or in normal rats, reported previously [27]. EB injection induces a lower wave with significantly prolonged latency and decreased amplitude, compared with that in the saline group (Fig. 5A). Statistical analysis revealed that the latencies of SCEP were significantly shorter, and their

Fig. 4. Comparison of changes in error scores with time after intraspinal injection of EB among different MSC-implanted groups and the saline group (repeated one-way ANOVA used). The rats in all groups showed an immediately elevated error score prior to the 4 day time point (data not shown). AdvNT-3-MSC implants did not evoke the lowest error scores until the 4 day time point in the EB group (including all different recipients and no cell implants). The scores in the rats receiving AdvNT3-MSCs were not significantly different from those in the saline group during this time window, and they even arrived at the same level at the third week post-lesion induction. MSC or AdvLacZ-MSC implants induced a mild functional recovery at the second or the third week post-injury, and there was no significant difference between those two groups at any time point. *P b 0.05, compared with other cell-implanted groups; #P b 0.05, compared with the EB group.

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compared to the MSC group (40%) or AdvLacZ-MSC group (44%), and was higher than the other groups (Fig. 7F). Although the MSC and AdvLacZ-MSC groups also showed a moderate increase in remyelinated axons (Fig. 7F), considerable degenerated axons, myelin debris and macrophages still presented in the DF (Fig. 7C, D and E). It is notable that 20% of axons in the saline-injected controls were remyelinated, which are predictably higher than those in the EB group (11%) (Fig. 7F). It is well understood that the EB-induced tissue damage may prohibit remyelination, as we found EB induced a gliafree environment. However, the saline control axons, which sustained damage solely from injection, may be more readily remyelinated. Indeed, we found the number of demyelinated axons in our controls was significantly lower than in the EB group (data not shown). On the other hand, our ultrastructural results also confirmed the positive effects of AdvNT-3-MSC implants (Fig. 8), consistent with those results that connected and analyzed from semi-thin sections. 3.6. Identification of myelin related cells

Fig. 5. (A) Spinal cord evoked potential (SCEP) shows a representative positive–negative– positive wave with a similar wave-shape among all five groups. EB injection alone evoked a smaller and lower wave than that in the saline group. Various MSC implants (AdvNT-3-MSCs, MSCs or AdvLacZ-MSCs) caused SCEP recovery to different degrees. (B) The amplitude of SCEP was significantly reduced in the EB group as compared to the saline group. Various MSC implants evidently elevated the amplitude values, and the role of AdvNT-3-MSC implants was the most efficient. (C) The latency of SCEP was significantly prolonged in the EB group as compared to the saline group. AdvNT-3-MSC transplantation restored the latency of SCEP in EB-injected rats. However, there was no distinct difference among the three MSC-implanted groups (# indicates P b 0.05, compared with the saline group; * indicates P b 0.05, compared with the EB group; $ indicates P b 0.05, compared with the MSC or AdvLacZ-MSC groups). Data = means± SD.

amplitudes were increased in the AdvNT-3-MSC group compared to the EB group (P b 0.05), while there was no obvious difference of latency or amplitude between the AdvNT-3-MSC and saline groups (P N 0.05). The amplitude of SCEP is partially restored by MSC or AdvLacZ-MSC implants, but the values in both groups were significantly lower than the AdvNT-3-MSC group. However, there was no significant difference in latencies between the three cellimplanted groups, although the mean value of latency was shortened by AdvNT-3-MSC implants compared to that by MSC or AdvLacZ-MSC implants (Fig. 5B and C). 3.5. Remyelination induced by AdvNT-3-MSC implants Immunohistochemistry revealed AdvNT-3-MSC implants induced robust expression of myelin basic protein (MBP) at the site surrounding or within the EB-induced demyelinated area (Fig. 6A), as compared with that of animals without cell implants (Fig. 6C). Some MBP expression was detected at the periphery of NF-positive axons, which demonstrated the formation of myelin structures (Fig. 6D). MBP-positive cells were also found in the lesion site, some of which were co-labeled with Hoechst33342, suggesting they may be from exogenous MSC implants (Fig. 6B). In order to confirm that the increased MBP expression was due to the remyelinated axons being induced by AdvNT-3-MSC-implants, semi-thin section and ultrastructural analysis were performed. After AdvNT-3-MSC implantation, nearly all the DF region was filled with remyelinated axons, while demyelination, axonal degeneration and debris-laden macrophages were rarely seen (Fig. 7A and B). However, in the MSC and AdvLacZ-MSC groups, the tissue repair in the same area was not as robust (Fig. 7C, D and E). Quantified data confirmed our morphologic results. A significant increase in the percentage of remyelinated axons was found in the AdvNT-3-MSC group (63%), as

In the EB-induced demyelinated spinal cord, there are two types of cell-mediated myelination: central oligodendrocyte-like and peripheral Schwann cell-like myelination [29]. Our electron micrographs reflected this oligodendrocyte-like and Schwann cell-like myelination (Fig. 9G and H–I). Oligodendrocyte myelination is characterized by a central body with minimal cytoplasm and a nucleus surrounded by many remyelinated axons (Fig. 9G). In contrast, Schwann cell myelination is characterized by large cytoplasmic and nuclear domains and a basement membrane and is often associated with one axon (Fig. 9H, I). Interestingly, cells morphologically different from oligodendrocytes and Schwann cells were found associated with myelinated axons in the AdvNT-3-MSC group (Fig. 9A, B and E). These cells usually had irregular cellular profiles and many long protrusions extending from the cell body. Their nuclei showed little dense chromatin and had a well-demarcated nucleolus. The cytoplasm contained abundant organelles, including some filamentary structures (Fig. 9C), as indicated by an asterisk in Fig. 9A, B and E. From their features, these cells seem to be of mesodermic origin. Moreover, these cells were only found in the AdvNT-3-MSC group. The processes of these cells extend and approach the axon to be myelinated, but myelin may not form because a unit membrane separates the cell and the axon (Fig. 9A and D). On the other hand, the cell may directly participate in the formation of myelin because no membrane divides the cytoplasm and myelinated axon (Fig. 9B–C and E–F). However, we did not have the direct evidence to identify the origin of these cells. 3.7. MSCs from AdvNT-3-MSC implants differentiated into the phenotypes of oligodendrocytes NG2 and APC are the markers of oligodendrocyte precursor cells (OPCs) and mature oligodendrocytes, respectively. 22 days following EB injection, a few endogenous NG2- and APC-positive cells were found in the demyelinated lesion area (data not shown). Implantation of both single MSCs and genetically modified MSCs could significantly increase the total number of NG2-positive cells in the center of lesion site, especially in the AdvNT-3-MSC group (Fig. 10A, C and E). As compared with the EB group, the number of APC-positive cells was also increased after cell implantation, but no significant difference existed between the three cell-implanted groups (Fig. 10E). In the MSC groups, some NG2- or APC-positive cells double-labeled with Hoechst33342 were detected in the lesion site, which suggests that implanted MSCs differentiate into the oligodendrocyte lineage. Quantitative analysis indicated that there was a significantly higher number of double-labeling with NG2 or APC and Hoechst33342 in the AdvNT-3-MSC group as compared to the MSC or AdvLacZ-MSC groups (all P b 0.05), but there was no significant difference between the MSC and AdvLacZ-MSC groups (P N 0.05, Fig. 10 F).

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Fig. 6. Immunohistochemistry staining showing myelin repair in the longitudinal section of the spinal cord containing the demyelinating lesion between the AdvNT-3-MSC (A, B and D) and EB (C) groups. (A) The expression of myelin basic protein (MBP) was robust near or within the demyelination lesion (asterisk) in the rats that receiving AdvNT-3-MSC implants. (B) It is the magnified image from the area (asterisk) in A. Some Hoechst-labeled cells (blue) are co-expressed with MBP (red, arrows). (C) MBP expression in the group of EB-injection alone was very weak in the demyelinated lesion site (asterisk). (D) Within the demyelinated area of implanted AdvNT-3-MSCs, MBP (red) expressed surrounding NFpositive axons (green, arrows). Scale bars, A and C = 320 μm; B and D = 40 μm.

4. Discussion The present study demonstrates the significant advantages of transplanting NT-3 gene-modified MSCs into an EB-induced demyelinated spinal cord, supported by evidence of electrophysiological and behavioral functional recovery. Further morphological evidence via light and electron microscopy indicates that AdvNT-3-MSC transplantation significantly promotes oligodendrocyte genesis and remyelination in the EB-lesion site. The results of this study provide better evidence indicating that genetically modifying MSCs with the NT-3 gene greatly improves the treatment efficacy of neurodegenerative disorders, such as MS. 4.1. EB induces demyelination Direct injection of a demyelinating toxin such as lysolecithin or EB into areas of white matter is a common experimental demyelination model widely used in the study of central nervous system disorders in rodents. The advantages of this approach include easy manipulation and localized demyelination [30]. EB also induces a glia-free environment at the injected area of the spinal cord white matter, which was confirmed by LFB and immunofluorescence staining (see Fig. 1). This effect is apparent after the first 2 weeks following EB injection [29]. 40 Gy

of X-irradiation could delay the endogenous repair process, followed by EB injection into the spinal cord [11,21,22,31]. However, we didn't use this method as it leads to severe animal lethality, resulting in a lack of animals at the desired experimental time points. In addition, the endogenous repair process isn't completely inhibited using X-irradiation [22]. Our results indicate EB-induced demyelination was significant, especially after 3–4 weeks in the central region of the lesion site, which extended 4 and 8 mm longitudinally. In contrast, endogenous remyelination was very limited (see Fig. 7). 4.2. AdvNT-3-MSCs improve remyelination and functional recovery Remyelination is a spontaneous recovery process in certain traumatic injury models and demyelinating diseases, such as MS, but this process is inadequate for complete recovery or reversing disease progression. Current therapies for MS are unable to prevent the progression of this disease. Boosting remyelination is postulated to prevent axonal damage and promote functional recovery [2,3,30]. Although a number of features of MSCs make them good candidates for cell implantation therapies for MS [21,31], one report indicated that direct intra-lesional injection of multipotent MSCs did not lead to remyelination, but inversely resulted in a number of adverse effects, including collagen deposition, extensive axonal degeneration, and disrupted cellular

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Fig. 7. (A–E) Morphology of remyelinated dorsal funiculus (DF) induced by various MSC implants. (B, D) Magnified views are shown in the area (asterisk labeling) in A and C, respectively. (A–B) After AdvNT-3-MSC implantation, almost all the DF region was occupied by remyelinated axons (thinner myelin). (C–D) MSC implants did not effectively improve EB-induced demyelination. (E) MSC or AdvLacZ-MSC implants may cause additional neural tissue injury. Arrows indicate the degenerated axons. (F) Quantitative analysis showed that the percentage of remyelinated axons within the demyelinated area in the AdvNT-3-MSC group was highest among the MSC, AdvLacZ-MSC, and no cell recipient groups. MSC or AdvLacZ-MSC implants moderately increased the percentage of remyelinated axons in the demyelinated area, compared with the saline or EB groups. One way ANOVA used. *P b 0.05, compared with the EB group; #P b 0.05, compared with the saline group; $P b 0.05, compared with the MSC or AdvLacZ-MSC groups. Abbreviation: DF – dorsal funiculus; DH – dorsal horn. Scale bars, A and C = 80 μm; D and E = 40 μm; B = 20 μm.

Fig. 8. Electron micrographs of the remyelinated axons in the dorsal funiculus (DF) of the spinal cord among different groups. (A) Normal DF axons (“n”) in the saline group. (B) Many axons without myelin (demyelinated axons, “d”) in the spinal cord of the EB group. (C) Many remyelinated axons (having thinner myelin, “r”) in the AdvNT-3-MSC group. (D) Remyelinated axons (“r”) and demyelinated axons (“d”) were also found in the MSC group. Scale bars, A, C and D = 1 μm; B = 0.5 μm.

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Fig. 9. Electron micrographs showing a cell morphologically different from endogenous myelin-forming cells (oligodendrocyte and Schwann cell) closely interacting with the remyelinated axons in the dorsal funiculus of the AdvNT-3-MSC-implanted cord. Images of A–F are from the AdvNT-3-MSC group, while G and H–I are from the EB and MSC groups, respectively. D, C and F are the magnified views of boxed area in A, B and E, respectively. After AdvNT-3-MSC implantation, a different cell (irregular cellular profile, many long protrusions and a cytoplasmic filamentary structure) was found to have a close interaction with remyelinated axons (asterisk in A, B and E). (D) The process of this cell is associated with the remyelinated axons separated by a unit membrane (arrows). However, some of this cell might directly participate in myelination since there was no membrane separating cytoplasmic parts from myelin (arrows in C and F). (G–H) Some axons seemed to be remyelinated by oligodendrocyte-like cells or peripheral myelin-forming cells (Schwann cell). (I) is the magnified view in H, clearly indicating a basement membrane (arrows). Also, some collagen deposition was detected in the MSC group. Abbreviation: Oli – oligodendrocyte; SC – Schwann cell; cg – collagen. Scale bars, B = 5 μm; A and E = 2 μm; G and H = 1 μm; C, D, F and I = 0.5 μm.

architecture in the CNS [11]. In our study, we found MSC or AdvLacZMSC implants only moderately increased the ratio of remyelinated axons in the demyelinated cord, and effects on electrophysiological and behavioral recovery were limited. In addition, degenerated axons and collagen deposition were present in these two groups (Fig. 7I and Fig. 9I). We speculate that the deleterious effects might restrict unaltered MSC implantation as an effective therapy. However, we showed that almost all axons in the DF were remyelinated after implanting NT-3 gene-modified MSCs into the EB-induced demyelinated cord. Remyelination is essential for functional recovery in demyelinating disorders [19,24,25,32]. Studies show that remyelination due to exogenous cell transplantation in demyelinated models or myelin-deficient animals results in improved conduction [33]. The new, thin myelin sheaths (remyelinated) and shorter internodes are able to support fast and secure saltatory conduction [34]. Our present study indicated enhanced behavioral recovery and restoration of electrophysiological properties in the AdvNT-3-MSC group, which was associated with extensive remyelination in the lesion site. 4.3. AdvNT-3 promotes endogenous OPCs conversion to oligodendrocytes The main function of oligodendrocytes is to provide support to axons and to produce the myelin sheath that insulates axons [35]. NT-3 has a crucial role in normal oligodendrocyte development and believed to enhance the proliferation and survival of optic nerve- or brain-derived oligodendrocyte progenitor cells (OPCs) [12,16]. However, the proliferation of OPCs induced by NT-3 cannot be sustained

for an extended period of time due to subsequent rapid differentiation into mature oligodendrocytes [19,20]. One study indicated that NT-3 in OPCs introduced via gene transfection could induce the production of MBP and the formation of mature myelin structures when co-cultured with hippocampal neurons [16]. TrkC, the highaffinity NT-3 receptor, is expressed in both OPCs and mature oligodendrocytes (OLs) [13]. In addition, the role of NT-3 on oligodendrocyte lineage has been shown by inducing the phosphorylation of mitogen-activated protein kinase (MAPK) in OPCs and in OLs [13,36]. Studies using NT-3 and TrkC knockout animals also confirmed an important role of NT-3 in the proliferation, survival and differentiation of OPCs in vivo [15]. Furthermore, direct administration of NT-3 or SC grafts that over-express NT-3 into LPC-induced CNS lesions or models of toxic demyelination improves functional recovery and enhances remyelination [18,19]. Previous results from our lab and others indicate that MSCs rarely produce NT-3 [20,37]. However, after the NT-3 gene was transfected into MSCs, the expression of NT-3 was secreted at its highest level on day 4 and gradually reduced until day 14, at which time secretion was maintained at constant levels [38]. Our previous study showed that NT-3 was expressed in the NT-3 gene-modified MSCs for up to two months following implantation into the transected spinal cord [38]. The present study also detected the expression of the Advmediated NT-3 gene three weeks following the EB-induced demyelination lesion in the spinal cord. Increased NT-3 secretion may stimulate the conversion of endogenous OPCs into oligodendrocytes for myelin repair, as we found more NG2-positive cells (a marker for

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Fig. 10. NG2- (A, C) and APC- (B, D) positive cells were shown in the demyelination site of the cord that received AdvNT-3-MSC (A, B) and MSC implants (C, D) at 3 weeks postinjury. Magnified views (A–D) were selected from the boxed area in the inset images of A1–D1, respectively. Some NG2- or APC-positive cells co-labeled with Hoechst33342 could be found in the lesion site, (white arrows, A–D). (E) Comparison of changes in the total number of NG2- and APC-positive cells per mm2 in the demyelination site of cord. Various MSC implants increased NG2- or APC-positive cells, compared with the EB or saline groups, while AdvNT-3-MSC implantation evoked the highest NG2-positive cells among the three cell-implanted groups. (F) Quantitative analysis for the percentage of NG2- and APC-positive cells double-labeled with Hoechst33342 indicated that AdvNT-3-MSC implantation significantly improved the frequency of MSC differentiation into oligodendrocyte lineage phenotypes, compared with the AdvLacZ-MSC or MSC groups. (E–F) * indicates P b 0.05, compared with the EB or saline groups; # indicates P b 0.05, compared with the MSC or AdvLacZ-MSC groups). Scale bar, A1 and C1 = 320 μm; B1 and D1 = 160 μm; A, B, C and D = 40 μm.

OPCs) in the lesion site following AdvNT-3-MSC implantation three weeks post-injury. This result is consistent with Girard's report showing that NT-3 favored proliferation of NG2-positive OPCs in a LPC-induced demyelinated spinal cord, but had no significant role on GFAPpositive astrocytes [22]. However, at this time point we did not find an increase in APC (a marker of mature oligodendrocytes) positive cells in the AdvNT-3-MSC group compared to the other two MSC implant groups, potentially due to a delay in OPC differentiation or maturation. Interestingly, using electron microscopy we found a new cell type, morphologically different from OLs and only detectable in the MSCimplantation groups, particularly in the AdvNT-3 MSC group, but not in the saline or EB groups without implants. In the AdvNT-3MSC group, we found the new cells were directly targeting demyelinated axons. We hypothesize that these cells are young oligodendrocytes from endogenous OPCs. These cells were characterized by irregular cell bodies with many long protrusions. The dense chromatin in their nuclei was sparse, and the nucleolus was clear. Its cytoplasm contained abundant organelles, and filamentary structures

were also detected. This morphology more closely resembled endogenous OPCs. However, we cannot exclude the possibility that some of these cells may came from NT-3-MSCs because bone marrow-derived MSCs can be converted into neurons or glial cells both in vitro and in vivo and further develop the phenotypes of oligodendrocytes in demyelination models [9,22,39]. In the present study, we also found a few remyelinating cells with Schwann-cell like morphology. The cells are probably generated by Schwann cell progenitors that are attracted by NT-3-MSCs into the lesion from the central–peripheral myelin border. In summary, implantation of NT-3 gene-modified MSCs into an EB-induced demyelinated spinal cord lesion in rats indicates that NT-3 overexpression significantly improves the efficacy by which NT-3 promotes endogenous oligodendrocyte genesis, and endogenous remyelinating cells directly participate in myelination. As a result, significant improvements of locomotor function and restoration of electrophysiological properties occurred. These results suggest that MSCs overexpressing NT-3 enhance the remyelination and functional recovery of a demyelinated spinal cord, and this study may

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