Cytotherapy, 2015; 17: 25e37
ORIGINAL PAPERS
Restorative benefits of transplanting human mesenchymal stromal cells overexpressing arginine decarboxylase genes after spinal cord injury
YU MI PARK1,*, SUN HYUP HAN1,*, SU KYUNG SEO1,2,3, KYUNG AH PARK1, WON TAEK LEE1 & JONG EUN LEE1,2,3 1
Department of Anatomy and 2BK21 PLUS Project for Medical Science and 3Brain Research Institute, Yonsei University College of Medicine, Seoul, Republic of Korea Abstract Background aims. Mesenchymal stromal cells (MSCs) promote functional recovery in central nervous system (CNS) injury. Neuroprotective effects of MSCs are being tested in clinical trials for the treatment of CNS injury; however, the underlying mechanisms remain unclear. Arginine decarboxylase (ADC) is a rate-limiting enzyme of agmatine synthesis and is known to exist in the CNS of mammals. The present study investigated whether transplantation of ADC-overexpressing human MSCs (ADC-hMSCs) after spinal cord injury (SCI) could increase the production of neurotrophic factors and promote cell survival, differentiation, axonal regeneration and the restoration of functional recovery. Methods. Retroviral human ADC was constructed with the use of an LXSN vector. After compression injury in thoracic level 9, PKH26-labeled ADC-hMSCs were transplanted into the dorsolateral funiculus 1 mm rostral and caudal to the lesion site. The tissues were sampled at 2, 4 and 10 weeks after SCI. Results. Behavioral analysis revealed that locomotor functions of the ADC-hMSC group were significantly restored. Histological analysis showed that the fibrotic scar volume was smaller in the ADC-hMSCeinjected group than in any other group. Brain-derived neurotrophic factor level was significantly higher in the ADC-hMSCeinjected group than in any other group throughout 10 weeks. Terminal deoxynucleotidyl transferase-mediated nick-end labeling assay showed decreased cell death, and co-localization analysis showed significant increase in the number of neurons and oligodendrocytes originating from transplanted hMSCs when they had been transduced with the ADC gene. Conclusions. The results suggested that ADC-hMSCs are a more suitable candidate than hMSCs for stem cell therapy after SCI. Key Words: arginine decarboxylase, mesenchymal stromal cells, spinal cord injury, transplantation
Introduction Spinal cord injury (SCI) involves the loss of motor and sensory functions in all levels below the site of injury and is typically irreversible and long-lasting. SCI leads to neuronal and glial cell death, inducing cystic cavities and inhibiting axonal regeneration and remyelination [1e3]. Oligodendrocytes provide the myelin sheath that wraps around the axons of neurons to enable them to conduct electrical impulses and produce neurotrophic factors for the maintenance of nerve cells. Oligodendrocytes are lost during SCI, resulting in the loss of myelinated motor nerve fibers and motor function that can cause paralysis in animals [3e5].
A therapeutic strategy to treat patients with SCI through the use of mesenchymal stromal cell (MSC) transplantation has recently received attention for its potential restorative benefits in the treatment of SCI in animal models. MSCs are widely used as transplantable material because they can be easily obtained from adult bone marrow. Additionally, MSC transplantation does not conflict with ethical issues, and there is less chance of immune rejection after transplantation because MSCs are harvested from autologous bone marrow [6]. MSCs can also reduce oxidative stress and inflammation-induced apoptotic cell death because they release neurotrophic factors that stimulate immunomodulation after transplantation [7].
*These authors contributed equally to this work. Correspondence: Jong Eun Lee, PhD, Department of Anatomy, BK21 PLUS Project for Medical Science, and Brain Research Institute, Yonsei University College of Medicine, 50 Yonsei-ro, Seodaemun-gu, Seoul, 120e752, Republic of Korea. E-mail:
[email protected] (Received 17 January 2014; accepted 12 August 2014) http://dx.doi.org/10.1016/j.jcyt.2014.08.006 ISSN 1465-3249 Copyright Ó 2015, International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.
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SCI induces the inflammatory response that renders the surrounding environment of the lesion site unsuitable for survival of MSCs after transplantation. Hydrogen peroxide, one of the major inflammatory response molecules, contributes to the increase in oxidative stress on the transplanted cells [8]. Such a hostile environment exerts detrimental effects on transplanted cells, in which the viability of transplanted MSCs is greatly reduced. Although MSCs exhibit enhanced resilience toward oxidative damage, the unfavorable environment limits the capacity of MSCs to differentiate into neurons and glial cells [9e12]. Cell survival is critical for successful transplantation therapy, and transgenic stem cells have been speculated as a viable solution to achieve a higher cell survival rate [13]. Human MSCs (hMSCs) overexpressing arginine decarboxylase (ADC) have been considered to be one of the most promising candidates for stem cell transplantation therapy. ADC is a ratelimiting enzyme of agmatine synthesis and is known to exist in the central nervous system (CNS) of mammals, regulating agmatine concentration in response to the level of stress [14,15]. An in vitro study of transgenic hMSCs reported that hMSCs overexpressing ADC showed a significantly higher survival rate after injury and that the overexpression of ADC stimulated the release of pro-survival factors that confer resistance against oxidative stress, and, consequently, increase cell survival [16]. ADC overexpression minimizes the loss of neurons after SCI by releasing a series of neurotrophic factors [16]. ADC facilitates the production of brain-derived neurotrophic factors (BDNFs) to halt the progress of degeneration and mobilize neural recovery [16,17]. In a recent study, the capacity of agmatine to regulate the release of neurotrophic factors was examined. The results suggested that agmatine treatment exerts neuroprotective effects by inducing oligodendrogenesis, neuronal protection and suppression of fibrotic scar. Therefore, the present study investigated the effects of transplanting ADC-overexpressing hMSCs transplanted after SCI in animal models. The transplantation of hMSCs overexpressing ADC showed an increased survival rate of transplanted cells in the SCI model, enhanced neuroprotective effects on neural cells and improved recovery of motor function.
Methods Human MSC culture Human MSCs were obtained from Yonsei Cell Therapy center of Severance Hospital. The hMSCs were analyzed by means of flow cytometry to confirm the phenotypic characteristics pertaining for the
identification of hMSCs. The hMSCs were seeded in T-75 flasks and cultured at 37 C in a humid incubator with 95% of O2 and 5% of CO2. The following day, the non-adherent cells were removed from the flasks and the adherent cells were treated with fresh Dulbecco’s modified Eagle’s mediumelow glucose (Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (Gibco) and 100 unit/mL penicillin and 100 mg/mL streptomycin (Gibco). Cells were then allowed to proliferate for 4 weeks. Medium was changed every 3 days. Cells whose passage number is 5 were used in the experiments [16]. Construction of human ADC and infection of hMSCs with retroviral vector Retroviral vectors carrying human ADC (hADC) complementary DNA (cDNA) (Gene Bank Accession No. AY325129) were constructed as described previously [16]. Briefly, hADC cDNA was amplified by means of polymerase chain reaction. The amplified hADC gene was ligated to retroviral vector pLXSN (Clontech, Mountain View, CA, USA) and introduced into Escherichia coli DH5a. The presence of hADC within vector was confirmed by restriction digestion analysis. The hADC-pLXSN plasmids and empty retrovirus pLXSN vector containing neomycin resistance gene were transfected into PT67 packaging cell with the use of Lipofectamine 2000 (Sigma, St Louis, MO, USA) and were selected in culture media (Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum) by adding G418 (200 mg/mL; Sigma). PT67 packaging cell infected by retroviral vector containing hADC genes and retrovirus containing empty retrovirus pLXSN vector (LXSN) were cultured at 37 C in a humid incubator with 5% of CO2 for 1 week. The virus-containing medium was filtered through a 0.45-mm-pore polysulfonic filter (Millipore, Billerica, MA, USA). The hMSCs were infected with empty retroviral vector (LXSN) or hADC gene with the use of polybrene reagent (6 mg/mL, Sigma) for 24 h [16]. Spinal cord injury Male Imprinting Control Region (ICR) mice (8 weeks of age; weight, 30e35 g; Orient, Gyeong Gigo, South Korea) were used in this study. All animal experiments were performed in accordance with the Korean Food and Drug Administration guidelines. Protocols were reviewed and approved by the Institutional Animal Care and Use Committee of the Yonsei Laboratory Animal Research Center (permit No. 2010e0350). All mice were maintained in the specific pathogen-free facility of the Yonsei Laboratory Animal Research Center.
ADC-hMSCs for spinal cord injury To construct the SCI model, laminectomy was performed and the spinal cord was exposed at thoracic levels 8e10 without any damage or compression to the surrounding dura mater. An injury was induced at the spinal cord at thoracic level 9 by means of a microvascular clamp (Fine Science Tools Inc, British Columbia, Canada) for 1 min. After SCI, the animal was allowed to recover on a 37 C heating pad. This surgery resulted in paralysis of the hind limbs in all animals. Their bladders were manually pressed daily twice for urination. PKH26 labeling and hMSC transplantation into the damaged spinal cord To trace transplanted cells, all cells were pre-labeled with PKH26, a membrane dye, according to the manufacturer’s instructions (Sigma). Cells were centrifuged and washed twice in serum-free medium. Cells were pelleted and suspended in dye solution. The efficiency of cellular labeling (routinely 100%) was examined with the use of a fluorescence microscope to detect and trace the stained cells [18]. At 1 week after SCI (1 WPS), SCI-model mice were divided into 1 experimental group (ADChMSCs) and 3 control groups (EC (experimental control), hMSCs and LXSN-hMSCs) by the type of cells transplanted or solutions injected in replacement. Mice were anesthetized with the use of Zoletil 50 (Virbac, Carroscedex, France) and rompun (Bayer Korea, GyeongGi-do, South Korea), and the spinal cord was re-exposed. For the ADC-hMSC, LXSNhMSC and hMSC groups, 0.5 mL of ADC-hMSCs, LXSN-hMSCs, and hMSCs (approximately 5 104 cells) were each transplanted at rostral and caudal sides, 1 mm apart from the injury site, with the use of a Hamilton syringe (Hamilton Company, Reno, NV, USA). The EC group received 0.5 mL of phosphatebuffered saline (PBS) (WelGENE, Daegu, South Korea) by use of the same method. After the injection, the muscle, subcutaneous and skin layers were closed and the bladder was manually pressed.
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hMSCs infected with control retroviral vector (LXSNhMSC group) and (iv) SCI followed by transplantation of transgenic hMSCs infected with retroviral vector carrying human ADC cDNA (ADC-hMSC group). Behavior test (Basso Mouse Scale score) Behavior tests were performed on all groups to measure their functional recovery of the hind limbs. All experimental mice were evaluated through the use of the Basso Mouse Scale (BMS) score. No ankle movement was scored as 0; slight ankle movement, 1; extensive ankle movement, 2; plantar placing of the paw with or without weight support or occasional weight-bearing during dorsal stepping but not during plantar stepping, 3; occasional plantar stepping, 4; frequent or consistent plantar stepping with or without some coordination, 5; frequent or consistent plantar stepping mostly coordinated, 6; frequent or consistent plantar stepping, mostly coordinated with or without significant trunk instability and paws parallel at initial contact and liftoff, 7; frequent or consistent plantar stepping, mostly coordinated with minor or no trunk instability, paws parallel at initial contact and liftoff and tail up and down movement, 8; frequent or consistent plantar stepping, mostly coordinated, paws parallel at initial contact and liftoff, normal trunk stability and tail always up, 9 [19]. Tissue processing The mice (n ¼ 5 in each group and each experimental period) were anesthetized and transcardially perfused with PBS and 4% paraformaldehyde. The thoracic spinal cord was removed, fixed in 4% paraformaldehyde for 4 h and cryoprotected in 30% sucrose solution at 4 C overnight. Extracted tissues were embedded in OCT compound (Thermo Fisher Scientific Inc, Waltham, MA, USA) and kept at 80 C. Embedded spinal cords were cut sagittally (20 mm) with the use of the cryocut microtome.
Animals and experimental groups During the 10-week period after SCI, MSC transplantation was conducted at 1 WPS, and all tissue sample preparations and analytical experiments were conducted at 2, 4 and 10 WPS. These time frames were selected according to the behavioral test results in which animals exhibited the most extensive motor function recovery after SCI. Subject animals were divided into 4 experimental groups to be examined and compared in the study: (i) SCI followed by injection of culture media (EC group), (ii) SCI followed by hMSC transplantation (hMSC group), (iii) SCI followed by transplantation of
Immunohistochemical analysis To evaluate the cellular characteristics of transplanted cells in vivo, immunohistochemical analysis was performed. To identify the characterization of transplanted cells and confirm trophic factors, tissue sections were blocked with 10% normal serum at room temperature for 1 h. Sections were processed for immunolabeling with monoclonal NeuN (1:500; Cell Signaling TECHNOLOGY, Danvers, MA), monoclonal GFAP (glial fibrillary acidic protein) (1:500; Thermo Fisher Scientific), polyclonal Olig-2 antibody (1:500; Abcam, Cambridge, MA) and
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monoclonal 5-HT (5-hydroxytrypamine) (1:500; Abcam) at 4 C overnight. Tissues were washed thoroughly and were then incubated with appropriate secondary antibodies tagged with Alexa Fluor 488 or Alexa Fluor 594 (1:250, Millipore) for 2 h at room temperature. Cell nuclei were stained with 40 ,6-diamidino-2-phenylindole (DAPI; Vector, Burlingame, CA). To evaluate the percentage of antibody-positive cells, 5e20 random fields per experiment were examined. The number of positive cells was expressed as a percentage of the total number. Tissue slides were observed through the use of confocal microscope (LSM710; Carl Zeiss, Gottingen, Germany). Western blot analysis For quantification of BDNF, Western blot analysis was performed on the subject mice from all groups (n ¼ 5, each group and each experimental period). Approximately 1 cm of each spinal cord segment including caudal and rostral parts 1 mm from the injury site was collected. The samples were homogenized in 300 mL of RIPA (Radioimmunoprecipitation assay) buffer on ice for 2 h and then were centrifuged at 13,200 rpm for 60 min at 4 C. The supernatants were collected and quantified with the use of Bradford Protein assay. To analyze protein levels, equal amounts of proteins (50 mg) were subjected to electrophoresis on 10% sodium dodecyl sulfateepolyacrylamide gels. Separated proteins were then electro-transferred onto Immunobilion-NC membrane (Millipore). The membranes were incubated with 5% bovine serum albumin in tris-buffered saline and 0.1% Tween-20 to block nonspecific binding. The membranes were incubated overnight with primary antibodies at 4 C. After washing 3 times with Tween-20 for 5 min, the membranes were incubated for 1 h at room temperature with peroxidase-conjugated anti-mouse immunoglobulin (Ig)G or anti-rabbit IgG. Secondary antibodies were applied in the same condition for 1 h at room temperature. Finally, proteins were visualized by use of an enhanced chemiluminescent (Thermo Fisher Scientific) protein detection kit according to the manufacturer’s instructions. Hematoxylin and eosin staining and fibrotic scar area assessment To quantify the fibrotic scar area in the groups (n ¼ 4), 20-mm-thick spinal cord sections were stained with hematoxylin and eosin. The fibrotic scar areas within 2 mm rostral or caudal to the injury site were measured. We used converted grayscale type to emphasize the histologic difference between normal tissue and fibrotic scar. Also, major fibrotic scar areas are marked with stars. The fibrotic scar area
around the lesion site was measured by use of a computer-associated scanning image analysis system (Optimas version 6.1; Optimas, Bothwell, WA, USA). Deoxynucleotidyl transferase-mediated nick-end labeling assay The deoxynucleotidyl transferase-mediated nick-end labeling (TUNEL) technique was used to analyze existing DNA fragmentation in the tissue samples. All samples were analyzed with the use of TUNEL assay according to the manufacturer’s instructions (R&D Systems, Mineapolis, MN). Frozen sections were covered in freshly prepared 1% paraformaldehyde solution in PBS, pH 7.4. The sections were washed and stored at 4 C until ready to proceed with staining. Slides were covered with permeabilization solution (post-fix in cooled ethanol:acetic acid ¼ 2:1) at 20 C and were washed with PBS. Immediately, the Working Strength TdT enzyme was added and the samples were incubated in humidified chamber at 37 C for 1 h. After incubation, the sections were agitated for 15 seconds in Working Strength Stop/ Wash Buffer and then incubated for another 10 min. An aliquot of anti-digoxigenin conjugate, warmed to room temperature in the dark, was added and further incubated in the dark, humidified light-proof box at room temperature for 30 min. The samples were mounted with the use of DAPI, sealed with coverslips and stored at 20 C. Seven random fields (20 fields per section) at magnification 100 from each experimental and control specimen were analyzed. Images were analyzed with the use of the Image J 1.47 Program. 5-HT quantification Tissue sections directly below the injured area (T10e11) were extracted and rinsed with PBS containing Tween-20 and blocked with 10% normal serum. The sections were then incubated with a rabbit anti-5HT antibody, rinsed thoroughly and incubated with a goat anti-rabbit IgG conjugated with fluorescein isothiocyanate. A stacked image of the caudal area was captured (LSM710, Carl Zeiss confocal microscope), and the integrated density of 5-HT immunoreactivity was quantified with the use of Image J software. Results were averaged over 5e7 tissue sections per subject samples for all experimental groups. Cell counting All samples were observed under a confocal microscope (Carl Zeiss LSM710), with the use of Zen
ADC-hMSCs for spinal cord injury 2009 software (Carl Zeiss Micro Imaging GmbH). The photomicrographs were taken with the use of 100 objective lenses. Five animals from each group were examined at 2, 4 and 10 WPS. The sections investigated included 2-mm margins both rostral and caudal to the injury site of the spinal cord. For individual quantification, at least 4 selected areas were examined from each slide. The TUNEL assayepositive cells were quantified at 2 and 4 WPS. The PKH26/NeuN, PHK26/GFAP and PKH26/Olig2 proliferative cells were quantified at 4 and 10 WPS. A sampling grid that comprised 140 140-mm2 squares was laid over each section. Cells were counted at magnifications lower than 100 in a 60 60mm2-square counting frame within each square of sampling grid. Statistical analysis All statistical analyses were performed with the use of SPSS 18.0. The data were presented as mean SE. One-way analysis of variance with post hoc Bonferroni’s test was used to compare the number of positive cells, fibrotic scar area, intensity of 5-HT, BDNF expression and BMS score at each time point among the groups. Differences were considered statistically significant at a value of P < 0.05.
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Results Migration of transplanted hMSCs, LXSN-hMSCs and ADC-hMSCs to the injury site in the spinal cord Photomicrographs of the immunofluorescence staining results were taken, and quantitative analyses were conducted 2 weeks after SCI (WPS) (Figure 1). The immune fluorescence staining study showed PKH26-labeled transplanted cells (red) among the DAPI-stained cells (blue) in the hMSC, LXSNhMSC and ADC-hMSC groups (Figure 1A). The ADC-hMSC group showed the strongest PKH26 fluorescence compared with the other groups in the injury site. The results suggested that grafted cells migrated from the injection site toward the damage site in the spinal cord. Accordingly, in the quantitative analysis, the ADC-hMSC group showed that 54% of original transplanted cells survived after transplantation, a value that was the highest among all the experimental groups (Figure 1B). Percentages of surviving cells in the hMSC and the LXSN-hMSC groups were 15% and 20%, respectively. The survival rate of transplanted cells in the ADC-hMSC group was significantly higher than that in the hMSC group. Similarly, the survival rate of the ADC-hMSC group was significantly higher than that of the LXSN-hMSC group.
Figure 1. Engraftment of transplanted hMSCs, LXSN-hMSCs and ADC-hMSCs labeled with PKH26 after SCI. (A) For the double immunofluorescence (IF) study, transplanted stem cells were stained with PKH26 (red) and the tissue was stained with DAPI (blue) at 2 weeks WPS. Engrafted hMSCs, LXSN-hMSCs and ADC-hMSCs labeled with PKH26 migrated from the injection site to the lesion site. (B) Quantitative analysis showed that in the ADC-hMSCs group, 54% of total transplanted (TP) cells survived after transplantation. In the hMSCs group and the LXSN-hMSCs group, 15% and 20% survived, respectively. Values are expressed as mean SE. *, P < 0.05 for the hMSCs group versus the ADC-hMSCs group; #, P < 0.05 for the LXSN-hMSCs group versus the ADC-hMSCs group. Scale bar is 100 mm.
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Figure 2. Quantitative analysis of TUNEL assay after SCI. Transplantation of ADC-hMSCs led to significant decrease in the number of apoptotic cells at (A) 2 WPS and (C) 4 WPS. Transplanted cells were labeled with PKH26 (red) and co-localization with TUNEL (green) was tested in the hMSCs, the LXSN-hMSCs and the ADC-hMSCs groups at 2 and 4 WPS. In the quantitative analysis, the percentages of TUNELpositive cells among PKH26 cells were acquired at (B) 2 WPS and (D) 4 WPS. Values are expressed as mean SE. *, P < 0.05 for the hMSCs group versus the ADC-hMSCs group; #, P < 0.05 for the LXSN-hMSCs group versus the ADC-hMSCs group. Scale bar is 400 mm.
Apoptotic death of transplanted cells after SCI The terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay was performed to detect DNA fragmentation that resulted from cellular apoptosis after SCI (Figure 2). The ADChMSC group showed a significantly smaller number of TUNEL-positive PKH26 cells than did the hMSC and LXSN-hMSC groups at 2 WPS (Figure 2A,B) and 4 WPS (Figure 2C,D). Quantitative analysis was performed to evaluate the percentage of successfully transplanted PKH26 cells that tested positive in the TUNEL co-localization test. At 2 WPS, the quantitative analysis showed that the ADC-hMSC group had 27% of PKH26 cells that underwent apoptotic cell death, whereas the hMSC and the LXSN-hMSC groups had 73% and 62% of apoptotic PKH26 cells,
respectively (Figure 2A,B). At 4 WPS, the percentage of TUNEL-positive PKH26 cells in the ADC-hMSC group was 8%, the percentage in the LXSN-hMSC group was 42% and the percentage in the hMSC group was 64%. The ADC-hMSC group showed a significantly reduced percentage of apoptotic cells compared with the LXSN-hMSC and hMSC groups (Figure 2C,D). The TUNEL assay identifies apoptotic events by detecting the chromatin condensation and is therefore only significant during the early stages of apoptosis. Whereas TUNEL assay data were significant at 2 and 4 WPS, the assay did not show any significant differences among experimental groups at 10 WPS because most of apoptotic events had already taken place or were at the final stages of apoptosis.
ADC-hMSCs for spinal cord injury Differentiation of transplanted cells in the injured spinal cord To evaluate the neural cell differentiation potential of transplanted hMSCs, LXSN-hMSCs and ADChMSCs in the spinal cord environment, hMSCs were labeled with PKH26 (red) before transplantation, and PKH26-positive cells in the tissue samples were stained with NeuN (a marker for neurons), Olig-2 (a marker for oligodendrocytes), and GFAP (a marker for astrocytes) antibodies (green) and counterstained with DAPI (nucleus) at 4 and 10 WPS (Figure 3). Cells that tested positive in co-localization tests are highlighted in boxes in Figure 3A,C. The number of PKH26-positive cells that were stained for NeuN (Figure 3A-7) and Olig-2 (Figure 3A-9) was significantly higher in the ADC-hMSC group than in the hMSC (Figure 3A-1,A-3) and the LXSN-hMSC (Figure 3A-4,A-6) groups at 4 WPS. The statistical analysis showed that the ADC-hMSC group showed
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significantly higher percentages of NeuN-positive and Olig-2epositive cells at 4 WPS (Figure 3B) and 10 WPS (Figure 3D) compared with the hMSC and the LXSN-hMSC groups. The immunohistochemical staining showed a small number of GFAP-positive cells in the hMSC and the LXSN-hMSC groups at 4 WPS (Figure 3A-2,A-5) and 10 WPS (Figure 3C-2,C5), but none was observed in the field of view of the ADC-hMSC group (Figure 3A-8,C-8). The statistical analysis of the entire injury area showed that the number of GFAP-expressing transplanted cells in the ADC-hMSC group was significantly lower compared with the hMSC and the LXSN-hMSC groups at both 4 WPS (Figure 3B) and 10 WPS (Figure 3D). At 2 WPS, 1 week after the transplantation of stem cells, the stem cells exhibited immature differentiation in which the cellular characteristics did not fully manifest according to their respective lineage. The immunofluorescence study of NeuN, Olig-2
Figure 3. Representative immunofluorescence images of NeuN-, Olig-2e and GFAP-positive cells within the injured spinal cord. Immunofluorescence analysis was performed by measuring the expression levels of target proteins with the use of their respective antibodies. Red fluorescence represents PKH26-labeled cells; green, cells expressing neural markers (NeuN, Olig-2 or GFAP) at 4 WPS (A, B) and 10 WPS (C, D). The ADC-hMSCs group exhibited significantly increased expression of NeuN and Olig-2 compared with other groups at 4 and 10 WPS (B, D). The ADC-hMSCs group showed statistically significant reduction in the expression of GFAP at 4 and 10 WPS compared with other groups (B, D). Values are expressed as mean SE. *, P < 0.05 for the hMSCs group versus the ADC-hMSCs group; #, P < 0.05 for the LXSN-hMSCs group versus the ADC-hMSCs group; y, P < 0.05 for the hMSCs group versus the LXSN-hMSCs group. Scale bar is 400 mm.
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Figure 4. Quantification of fibrotic scar area after SCI. Tissue sections were stained with hematoxylin and eosin and the average fibrotic scar area (mm2) in each experimental group was measured at 2 WPS. Visual confirmations identified the following histological features to define glial scar area in the tissue samples: hemorrhagic foci, dissolved neuronal structures without nucleus, necrotic and cyst formations, and large number of vacuoles. We converted the images into grayscale to emphasize the histologic difference between normal tissue and fibrotic scar. Major fibrotic scar areas (white) are marked with stars (+). At 2 WPS, compared with the EC and the hMSCs groups, the ADC-hMSCs group showed significantly reduced fibrotic scar area. The ADC-hMSCs group also showed the best tissue preservation in the hematoxylin and eosin staining sections. Values are expressed as mean SE. *, P < 0.05 for the EC group versus the ADC-hMSCs group; #, P < 0.05 for the hMSCs group versus the ADC-hMSCs group. Scale bar is 1 mm.
and GFAP showed no significant differences among the experimental groups at 2 WPS, and the 2-WPS data were omitted because of lack of significance (Figure 3AeD). Quantification of fibrotic scar area in the injured spinal cord At 2 WPS, visual confirmation of the hematoxylin and eosinestained tissue sections showed that the ADC-hMSC group formed the smallest fibrotic scar area (Figure 4). The EC group showed the largest fibrotic scar formation at the lesion site. Additionally, the ADC-hMSC group showed almost intact tissue in which the remaining tissue area was the largest compared with those of the other groups. The EC, hMSC and LXSN-hMSC groups showed extensive loss of tissue in the lesion site at 2 WPS. The quantitative analysis of fibrotic scar formation measured with the use of a computer-associated scanning image analysis system showed that the average fibrotic scar area was approximately 1.4 mm2 in the EC group, 1.1 mm2 in the hMSC group, 0.70 mm2 in the LXSNhMSC group and 0.50 mm2 in the ADC-hMSC group. The ADC-hMSC group showed a statistically significant reduction in fibrotic scar formation compared with the hMSC and the LXSN-hMSC groups.
The blood-brain barrier remains porous to blood and serum components for up to 14 days after brain or spinal cord injury, and the areas of greatest glial scarring correlate well with areas of most extensive blood-brain barrier breakdown as well as the largest numbers of activated macrophages [20]. Accordingly, the glial scar formation was probably completed in the injured spinal cord by 2 WPS, at which we measured the glial scar area (Figure 4). The reference in support of such finding is further explained in the primary author’s previous study published in Plos One [3]. Regeneration of descending axon in the injured spinal cord after SCI Regeneration of the descending serotonergic raphespinal axons is the hallmark of motor function recovery in SCI. The serotonergic raphespinal axons were analyzed with the use of the 5-HT antibody (Figure 5). Immunofluorescence analysis showed that the ADC-hMSC group exhibited the highest number of 5-HTepositive nerve fibers compared with the other groups at 10 WPS. The integrated density of the 5-HTepositive cells was used to represent the number of raphespinal axons present in the tissue sections. The quantitative analysis showed that the ADC-hMSC group showed significantly higher density of 5-HT positivity compared with the
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Figure 5. Regeneration of descending axon by transplanted cells after SCI. Immunofluorescent images of 5-HTestained spinal cord tissue represent reconnected descending axons at the caudal side of injury site at 10 WPS. The results showed that the ADC-hMSCs group showed the larger number of 5-HTepositive fibers (indicated by white arrows) compared with the hMSCs and the LXSN-hMSCs groups. Values are expressed as mean SE. *, P < 0.05 for the EC group versus the ADC-hMSCs group; #, P < 0.05 for the hMSCs group versus the ADChMSCs group; y, P < 0.05 for the LXSN-hMSCs group versus the ADC-hMSCs group. Scale bar is 400 mm.
EC, the hMSC and the LXSN-hMSC groups. Transplantation of ADC-hMSCs induced extensive in-growth of serotonin-positive raphespinal axons into the damage site.
Changes in BDNF protein content after SCI Western blot results and quantitative analysis showed that the ADC-hMSC group exhibited a significant
Figure 6. BDNF expression in the spinal cord and locomotor function after SCI. (A) The level of BDNF expression was examined in the EC, hMSCs, LXSN-hMSCs and ADC-hMSCs groups by means of Western blot analysis at 2, 4 and 10 WPS. Histograms show the mean optical density for each band relative to the corresponding b-actin level for each groups. The ADC-hMSCs group showed increased BDNF protein level at 2 and 4 WPS. (B) The functional recovery of locomotor function was examined by means of BMS score. Values are expressed as mean SE. *, P < 0.05 for the EC group versus the ADC-hMSCs group; #, P < 0.05 for the hMSCs group versus the ADC-hMSCs group; y, P < 0.05 for the LXSN-hMSCs group versus the ADC-hMSCs group; z, P < 0.05 for the hMSCs group versus LXSN-hMSCs group.
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increase in BDNF expression compared with other groups at 2, 4 and 10 WPS (Figure 6A). Western blot analysis showed more prominent bands for the ADChMSC group, and quantitative analysis confirmed that the level of BDNF expression was increased in the ADC-hMSC group at a statistically significant level. At 10 WPS, the BDNF expression level in the ADC-hMSC group was greatly diminished to the same level as the other groups. Western blot analysis showed less prominent bands for all the experimental groups at 10 WPS. Quantitative analysis also showed no statistically significant difference in the BDNF expression level among the hMSC, LXSN-hMSCs and ADC-hMSC groups at 10 WPS.
Effects of ADC-hMSC transplantation on behavioral recovery after SCI The open-field test was performed with the use of the BMS system. The control hMSC, LXSN-hMSC, ADC-hMSC and EC groups were tested for motor function recovery once per week for 10 WPS (Figure 6B). The EC group showed a slight increase in locomotion throughout 10 weeks. By 10 WPS, the average score of the EC group increased from 0 to 1.9 in the BMS score. The control hMSC group also showed a similar pattern of locomotor improvement, increasing from 0 to 1.9 in the BMS score during 10 WPS. However, at 5 WPS, a sharp increase up to 2.5 in the scale was observed, which subsequently subsided to 1.75 in the following week. The LXSNhMSC group exhibited a similar pattern of gradual increase from 0 to 2.1 in the BMS score. At 10 WPS, mice observed in the EC, hMSC and LXSN-hMSC groups showed extensive ankle movement in which their hind limbs moved more than half of the ankle joint excursion (BMS score 2). The ADC-hMSC group showed significantly greater increase in the BMS score, with an average of 5.3 recorded by 10 WPS. The ADC-hMSC group showed a significantly higher BMS score at 2, 4, 6, 7, 8, 9 and 10 WPS than the other experimental groups. The rate of recovery of the ADC-hMSC group, measured by means of the slope, was notably high at 2 and 4 WPS. At 2 WPS, the rate was 1.4 BMS points per week and, at 4 WPS, 1.3 points per week. At 10 WPS, the mice in the ADC-hMSC group showed frequent plantar stepping with some coordination between both paws (BMS score 5). Both paws in the hind limbs were externally rotated during stepping and lift off (BMS score 5).
Discussion In the present study, the effects of transplanting transgenic hMSCs overexpressing ADC gene were
investigated by evaluating the survival rate, neuroprotective effects and recovery of motor function after transplantation of ADC-hMSCs. The ADC-hMSC group showed the highest number of PKH26-positive cells exhibiting colocalization with DAPI staining. The results suggested that the ADC-hMSC group had the largest percentage of the original stem cells successfully engrafted. Accordingly, the quantitative analysis showed the highest engraftment success rate at 53% in the ADC-hMSC group compared with 15% and 20% in the hMSC and LXSN-hMSC groups, respectively. To account for the discrepancy in the transplantation success rate, cell survival after transplantation was investigated. The quantitative analysis of the TUNEL assay suggested that the number of TUNEL-positive cells in the ADC-hMSC group was significantly reduced compared with those in the hMSC and the LXSN-hMSC groups. The transplantation of ADC-hMSCs showed significantly improved survival rates. Our previous published in vitro study presented high-performance liquid chromatography results that showed the differential expression levels of agmatine after oxidative injury among experimental groups, the hMSC, the LXSN-hMSC and the ADC-hMSC groups [16]. Compared with the reference expression level of agmatine when there was no injury done to the cells, only in the ADC-hMSC group was there a statistically significant increase in agmatine expression on oxidative injury. The increase in the agmatine level after injury was not significant in other groups. The increased production of agmatine led to an inhibition of caspase 3, which triggers cellular apoptosis. The study confirmed that the increased production of endogenous agmatine by transgenic hMSCs overexpressing ADC confers cellular resistance against oxidative stress by inhibiting apoptotic signal [16]. In this study, the same transgenic stem cells investigated in the in vitro study were used for engraftment. A similar oxidative environment observed in the in vitro study was created after SCI, triggering a series of inflammatory cascade. The inflammation might serve as the oxidative insult to stimulate the grafted ADC-hMSCs to produce an increased level of agmatine. In the present study, no experiment to quantify the level of agmatine in vivo was conducted. However, increased agmatine secretion in response to an inflammatory environment served as the most plausible explanation for the increased survival rate of grafts in the ADC-hMSC group. On the basis of the findings in the present study, the increased production of agmatine in the ADC-hMSCs showed more extensive resistance against oxidative stress following transplantation into the injured spinal cord.
ADC-hMSCs for spinal cord injury A study showed that agmatine prevents neuronal cell death and facilitates neuronal regeneration and oligodendrogenesis by stimulating the release of neurotrophic factors [3]. To investigate the neural cell differentiation potential of the transplanted ADC-hMSCs, we measured the expression levels of marker proteins for neurons (NeuN), oligodendrocytes (Olig-2) and astrocytes (GFAP). At both 4 and 10 WPS, the ADC-hMSC group showed the highest expression of NeuN and Olig-2, which suggests an increased number of surviving neurons and oligodendrocytes. Among the salvaged neural cells were raphespinal nerves. A previous study confirmed that the transplantation of stem cells in animal SCI models exhibited an effective neural preservation in the injury site, showing an increased number of 5HT-positive nerve fibers stretching across the lesion site from rostral to caudal [21]. Consistently, the transplantation of ADC-hMSCs showed improved neural preservation in which the ADC-hMSC group showed a significantly larger number of 5HT-positive fibers than did the hMSC group at 10 WPS. In the ADC-hMSC group, the raphespinal axon regeneration mostly occurred in the first 4 WPS (data not shown), whereas the regeneration in other groups occurred gradually over the period of 10 weeks. Because we wanted to compare results when the recovery process had fully taken place, only the 10 WPS data were considered to reflect the extent of motor function recovery. The transplantation of ADC-hMSCs also suppressed the fibrotic scar formation. A study showed that the release of neurotrophic factors by agmatine inhibits accumulation of astrocyte, allowing neuronal regeneration to occur [3]. In this study, the hematoxylin and eosin staining results showed that the ADC-hMSC group showed the least fibrosis among other experimental groups. The ADC-hMSC group demonstrated extensive neuroprotective effects on neurons and oligodendrocytes and suppression of astrocyte activation. The ultimate purpose of this study was to investigate the efficacy of ADC-hMSCs in treating SCI and enhancing motor function recovery. The therapeutic benefits of transplanting ADC-hMSCs were evaluated by locomotive behavior tests. The functional motor recovery was quantified by means of the BMS score in the open-field test. Our results showed that the ADC-hMSC group exhibited the fastest and most extensive motor function recovery compared with the other groups. At 2 and 4 WPS, the recovery accelerated significantly, and, after 5 WPS, the ADC-hMSC group maintained a gradual recovery. In comparison, the motor function recovery in the EC, hMSC and LXSN-hMSC groups reached a plateau after 5 WPS. In summary, the present study suggests that the increased agmatine synthesis in ADC-hMSCs
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promoted anti-apoptotic effects, neuronal regeneration, oligodendrogenesis, fibrotic scar suppression and ultimately, improved motor function recovery. Studies have linked agmatine with neurotrophic factors, namely BDNF [16,17,22], in that agmatine stimulates its release to adjacent cells. It has been confirmed that the hMSCs also increase the production of BDNF within the host spinal cord and exert a paracrine effect [23]. Increased BDNF level has been associated with neuronal survival and replacement of damaged neurons in SCI [23e26]. Therefore, the BDNF expression levels of all experimental groups were also investigated in this present study. Our Western blotting results suggested that in the ADChMSC group, the expression of BDNF was significantly increased compared with those in the hMSC and the LXSN-hMSC groups. On the basis of our results, we speculated that increased synthesis of agmatine in ADC-hMSCs might have amplified the secretion of BDNF, inducing an augmented paracrine effect. These findings may also elucidate the paracrine effects of BDNF as a key role in facilitating neuronal regeneration, oligodendrogenesis, inhibition of fibrotic scar formation and ultimately, improved motor function recovery after SCI. Our histological, molecular and behavioral analyses indicated that the LXSN-hMSC group showed marginal, although not to a statistically significant level, neuroprotective effects. The BDNF level of the LXSN-hMSCs was higher than that of the hMSCs at 2 WPS. The engraftment success rate was also higher in the LXSN-hMSC group by 5 percentage points. Axonal regeneration and oligodendrogenesis were also slightly more prominent in the LXSN-hMSC group. TUNEL assays showed that the LXSNhMSC group showed less apoptotic cell death compared with the hMSC group. Despite the lack of statistical significance, these marginal improvements of the LXSN-hMSCs in cell survival and neural regeneration must be clarified. The presence of promoter and enhancer sequences in the viral vector might transiently be integrated into the genes promoting cell proliferation and anti-apoptotic signals [27,28]. Although such phenomena occur randomly and transiently, the viral induction clearly has merits in enhancing neuroprotective effects of stem cells. Before conducting the present study, complications associated with implanting human stem cells into immune-competent animal models had to be considered. Significant immune reactions to xenogeneic transplantation could render the experiment inconclusive because most of the stem cell grafts would be eliminated within the host nervous system. The suitability of xenogeneic transplantation had to be considered carefully. In this study, we used bone marrowederived hMSCs. MSCs are well known for
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their immune modulatory capabilities. Their immunosuppressive property allows allogeneic or even xenogeneic transplantations into immune-competent recipients without the use of immune suppressants [29]. The bone marrowederived stem cell (BMSC) xenotransplantation study was published in 2003, in which intracerebral transplantation of human BMSCs improved neurological functions in a cerebral ischemic rat model [30]. Interestingly, despite being a xenogeneic transplantation, the investigators observed no evidence of inflammation or rejection. They thus offered several explanations, including the brain being partially immune privileged and BMSCs lacking major histocompatibility complex II expression. According to the findings elaborated in the references, we concluded that xenogeneic transplantation was a viable option for this study. Had immune rejections taken place in the injured spinal cord, the stem cell grafts would have been eliminated and functional improvements not observed. Although symptoms associated with immune rejection were not observed during the experiment, no pathological study was done to investigate immune reactivity at cellular and molecular levels. We could not rule out that the immunomodulatory effect of MSCs simply overshadowed the relatively weak immune reactions. Nevertheless, the ADC-hMSC transplantation resulted in a significantly improved motor function recovery compared with the EC group, which confirmed that the engraftment was a success in this study. On a similar note, significant neuroprotective effects were exerted by the hMSC and the LXSN-hMSC groups compared with those of the EC group. In the hMSC and the LXSN-hMSC groups, marginal increases in the integrated density of serotonergic nerves (Figure 5) and BDNF expression (Figure 6A) and decrease in fibrotic scar area (Figure 4) were observed compared with those in the EC group. However, the behavioral study (Figure 6B) showed no significance in the degree of recovery among the hMSC, the LXSN-hMSC and the EC groups. The focus of this study was centered on the effects of transplanting genetically modified stem cells on motor function recovery after SCI. Although it was interesting to see the marginal benefits exerted by the hMSC and the LXSN-hMSC groups compared with those by the EC group, the investigators considered an extensive report on the marginal benefits of hMSCs and LXSN-hMSCs without significant benefit on motor function recovery beyond the scope and purpose of this study. In conclusion, the present study confirmed that transplantation of hMSCs overexpressing ADC after SCI has therapeutic benefits in the recovery of motor function. The transplantation of hMSCs overexpressing ADC increases the survival rate of stem cells upon transplantation. Additionally, neurotrophic
factors are stimulated, and neural cells are salvaged by preventing apoptotic cell death. Furthermore, ADCoverexpressing hMSC transplantation suppresses fibrotic scar formation and allows for neuronal regeneration and ogligodendrogenesis. All these effects combined ultimately improve the recovery of motor function after SCI. The results of the present study indicate that transplantation of hMSCs overexpressing ADC may be beneficial in the treatment of SCI. Acknowledgements This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korean government (MEST) (NRF-2011e0017276). The authors thank Professor Hosung Jung (Yonsei University College of Medicine) and Dr Jae Hwan Kim (Yonsei University College of Medicine) for their critical reading of the manuscript and assistance in revision. Disclosure of interests: The authors have no commercial, proprietary, or financial interest in the products or companies described in this article. References [1] Rasouli A, Bhatia N, Dinh P, Cahill K, Suryadevara S, Gupta R. Resection of glial scar following spinal cord injury. J Orthop Res 2009;27(7):931e6. [2] McDonald JW, Belegu V. Demyelination and remyelination after spinal cord injury. J Neurotrauma 2006;23(3-4):345e59. [3] Park YM, Lee WT, Bokara KK, Seo SK, Park SH, Kim JH, et al. The multifaceted effects of agmatine on functional recovery after spinal cord injury through Modulations of BMP-2/4/7 expressions in neurons and glial cells. PloS One 2013;8(1):e53911. [4] Kotter MR, Stadelmann C, Hartung HP. Enhancing remyelination in diseaseecan we wrap it up? Brain 2011; 134(Pt 7):1882e900. [5] Kulbatski I, Mothe AJ, Keating A, Hakamata Y, Kobayashi E, Tator CH. Oligodendrocytes and radial glia derived from adult rat spinal cord progenitors: morphological and immunocytochemical characterization. J Histochem Cytochem 2007;55(3):209e22. [6] Rice CM, Scolding NJ. Autologous bone marrow stem cellsproperties and advantages. J Neurol Sci 2008;265(1-2): 59e62. [7] Forostyak S, Jendelova P, Sykova E. The role of mesenchymal stromal cells in spinal cord injury, regenerative medicine and possible clinical applications. Biochimie 2013;95(12): 2257e70. [8] Chen X, Li Y, Wang L, Katakowski M, Zhang L, Chen J, et al. Ischemic rat brain extracts induce human marrow stromal cell growth factor production. Neuropathology 2002; 22(4):275e9. [9] Parr AM, Tator CH, Keating A. Bone marrow-derived mesenchymal stromal cells for the repair of central nervous system injury. Bone Marrow Transplant 2007;40(7):609e19.
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