Superparamagnetic Iron Oxide Labeling of Spinal Cord Neural Stem Cells Genetically Modified by Nerve Growth Factor-β

This study established superparamagnetic iron oxide （SPIO）-labeled nerve growth fac-tor-β （NGF-β） gene-modified spinal cord-derived neural stem cells （NSCs）. The El4 rat embryonic spinal cord-derived NSCs were isolated and cultured. The cells of the third passage were transfected with plasmid pcDNA3-hNGFβ by using FuGENE HD transfection reagent. The expression of NGFβ was measured by immunocytochemistry and Western blotting. The positive clones were selected, allowed to proliferate and then labeled with SPIO, which was mediated by FuGENE HD transfection reagent. Prussian blue staining and transmission electron microscopy （TEM） were used to identify the SPIO particles in the cells. The distinctive markers for stem cells （nestin）, neuron （β-Ⅲ-tubulin）, oligodendrocyte （CNPase） and astrocyte （GFAP） were employed to evaluate the differentiation ability of the labeled cells. The immunocytochemistry and western blotting showed that NGF-β was expressed in spinal cord-derived NSCs. Prussian blue staining indicated that numerous blue-stained particles appeared in the cytoplasma of the labeled cells. TEM showed that SPIO particles were found in vacuolar structures of different sizes and the cytoplasma. The immunocytochemistry demonstrated that the labeled cells were nestin-positive. After differentiation, the cells expressed β-Ⅲ-tubulin, CNPase and GFAP. It was concluded that the SPIO-labeled NGF-β gene-modified spinal cord-derived NSC were successfully established, which are multipotent and capable of self-renewal.

Survival of transplanted neurotrophin-3 expressing human neural stem cells and motor function in a rat model of spinal cord injury

BACKGROUND： Many methods have been attempted to repair nerves following spinal cord injury, including peripheral nerve transplantation, Schwann cell transplantation, olfactory ensheathing cell transplantation, and embryonic neural tissue transplantation. However, there is a need for improved outcomes. OBJECTIVE： To investigate the repair feasibility for rat spinal cord injury using human neural stem cells （hNSCs） genetically modified by lentivirus to express neurotrophin-3. DESIGN, TIME AND SETTING： In vitro cell biological experiment and in vivo randomized, controlled genetic engineering experiment were performed at the Third Military Medical University of Chinese PLA and First People＇s Hospital of Yibin, China from March 2006 to December 2007. MATERIALS： A total of 64 adult, female, Wistar rats were used for the in vivo study. Of them, 48 rats were used to establish models of spinal cord hemisection, and were subsequently equally and randomly assigned to model, genetically modified hNSC, and normal hNSC groups. The remaining 16 rats served as normal controls. METHODS： hNSCs were in vitro genetically modified by lentivirus to secrete both green fluorescence protein and neurotrophin-3. Neurotrophin-3 expression was measured by Western blot. Genetically modified hNSC or normal hNSC suspension （5 × 10^5） was injected into the rat spinal cord following T10 spinal cord hemisection. A total of 5μL Dulbecco＇s-modified Eagle＇s medium was infused into the rat spinal cord in the model grop. Transgene expression and survival of transplanted hNSCs were determined by immunohistochemistry. Motor function was evaluated using the Basso, Beattie, and Bresnahan （BBB） scale. MAIN OUTCOME MEASURES： The following parameters were measured： expression of neurotrophin-3 produced by genetically modified hNSCs, transgene expression and survival of hNSCs in rats, motor function in rats. RESULTS： hNSCs were successfully genetically modified by lentivirus to stably express neurotrophin-3. The transplanted hNSCs primarily gathered at, or around, the injection site two weeks following transplantation, and gradually migrated towards the surrounding tissue. Transplanted hNSCs were observed 7.0-8.0 mm away from the injection site. In addition, hNSCs were observed 10 weeks after transplantation. At week 4, BBB locomotor scores were significantly greater in the genetically modified hNSC and normal hNSC groups, compared with the model group （P 〈 0.05）, and scores were significantly greater in the genetically modified hNSC group compared with the normal hNSC group （P 〈 0.05）. CONCLUSION： hNSCs were genetically modified with lentivirus to stably secrete neurotrophin-3. hNSCs improved motor function recovery in rats following spinal cord injury.

Induction of Functional Recovery by Co-transplantation of Neural Stem Cells and Schwann Cells in a Rat Spinal Cord Contusion Injury Model

Objective To study the transplantation efficacy of neural stem cells （NSCs） and Schwann cells （SC） in a rat model of spinal cord contusion injury. Methods Multipotent neural stem cells （NSCs） and Schwann cells were harvested from the spinal cords of embryonic rats at 16 days post coitus and sciatic nerves of newborn rats, respectively. The differential characteristics of NSCs in vitro induced by either serum-based culture or co-culture with SC were analyzed by immunofluorescence. NSCs and SCs were co-transplanted into adult rats having undergone spinal cord contusion at T9 level. The animals were weekly monitored using the Basso-Beattie-Bresnahan locomotor rating system to evaluate functional recovery from contusion-induced spinal cord injury. Migration and differentiation of transplanted NSCs were studied in tissue sections using immunohistochemical staining. Results Embryonic spinal cord-derived NSCs differentiated into a large number of oligodendrocytes in serum-based culture upon the withdrawal of mitogens. In cocultures with SCs, NSCs differentiated into neuron more readily. Rats with spinal cord contusion injury which had undergone transplantation of NSCs and SCs into the intraspinal cavity demonstrated a moderate improvement in motor functions. Conclusions SC may contribute to neuronal differentiation of NSCs in vitro and in vivo. Transplantation of NSCs and SCs into the affected area may be a feasible approach to promoting motor recovery in patients after spinal cord injury.

Bridging the injured spinal cord with neural stem cells

Spinal cord injury （SCI） damages not only the gray matter neurons, but also the white matter axonal tracts that carry signals to and from the brain, re- suiting in permanent loss of function below injury. Neural stem cells （NSCs） have high therapeutic potential for reconstruction of the injured spinal cord since they can potentially fnrm neuronal relays to bridge functional con-nectivity between separated spinal cord segments. This requires host axonal regeneration into and connectivity with donor neurons, and axonal growth and connectivity of donor neurons to host central nervous system （CNS） circuitry. In this mini-review, we will discuss key studies that explore novel neuronal relay formation by grafting NSCs in models of SCI, with emphasis on long-distance axonal growth and connectivity of NSCs grafted into in-jured spinal cord.

Transplantation of erythropoietin gene-modified neural stem cells improves the repair of injured spinal cord

The protective effects of erythropoietin on spinal Here, the eukaryotic expression plasmid pcDNA3.1 cord injury have not been well described. human erythropoietin was transfected into rat neural stem cells cultured in vitro. A rat model of spinal cord injury was established using a free falling object. In the human erythropoietin-neural stem cells group, transfected neural stem cells were injected into the rat subarachnoid cavity, while the neural stem cells group was inject- ed with non-transfected neural stem cells. Dulbecco＇s modified Eagle＇s medium/F12 medium was injected into the rats in the spinal cord injury group as a control. At 1-4 weeks post injury, the motor function in the rat lower limbs was best in the human erythropoietin-neural stem ceils group, followed by the neural stem cells group, and lastly the spinal cord injury group. At 72 hours, compared with the spinal cord injury group, the apoptotic index and Caspase-3 gene and protein expressions were apparently decreased, and the bd-2 gene and protein expressions were noticeably increased, in the tissues surrounding the injured region in the human erythro- poietin-neural stem cells group. At 4 weeks, the somatosensory evoked potential latencies were cavities were clearly smaller and the motor and remarkably shorter in the human erythropoi- etin-neural stem cells group and neural stem cells group than those in the spinal cord injury group. These differences were particularly obvious in the human erythropoietin-neural stem cells group. More CM-Dil-positive cells and horseradish peroxidase-positive nerve fibers and larger amplitude motor and somatosensory evoked potentials were found in the human erythro- poietin-neural stem cells group and neural stem cells group than in the spinal cord injury group. Again, these differences were particularly obvious in the human erythropoietin-neural stem cells group. These data indicate that transplantation of erythropoietin gene-modified neural stem cells into the subarachnoid cavity to help repair spinal cord injury and promote the recovery of spinal cord function better than neural stem cell transplantation alone. These findings may lead to significant improvements in the clinical treatment of spinal cord injuries.

Mild hypothermia combined with a scaffold of Ng Rsilenced neural stem cells/Schwann cells to treat spinal cord injury

Because the inhibition of Nogo proteins can promote neurite growth and nerve cell differentiation, a cell-scaffold complex seeded with Nogo receptor （NgR）-silenced neural stem cells and Schwann cells may be able to improve the microenvironment for spinal cord injury repair. Previous studies have found that mild hypothermia helps to attenuate secondary damage in the spinal cord and exerts a neuroprotective effect. Here, we constructed a cell-scaffold complex consisting of a poly（D,L-lactide-co-glycolic acid） （PLGA） scaffold seeded with NgR-silenced neural stem cells and Schwann cells, and determined the effects of mild hypothermia combined with the cell-scaffold complexes on the spinal cord hemi-transection injury in the T9 segment in rats. Compared with the PLGA group and the NgR-silencing cells ＋ PLGA group, hindlimb motor function and nerve electrophysiological function were dearly improved, pathological changes in the injured spinal cord were attenuated, and the number of surviving cells and nerve fibers were increased in the group treated with the NgR-silenced cell scaffold ＋ mild hypothermia at 34℃ for 6 hours. Furthermore, fewer pathological changes to the injured spinal cord and more surviving cells and nerve fibers were found after mild hypothermia therapy than in injuries not treated with mild hypothermia. These experimental results indicate that mild hypothermia combined with NgR gene-silenced cells in a PLGA scaffold may be an effective therapy for treating spinal cord injury.

Electroacupuncture in the repair of spinal cord injury:inhibiting the Notch signaling pathway and promoting neural stem cell proliferation

Electroacupuncture for the treatment of spinal cord iniury has a good dinical curative effect, but the underlying mechanism is unclear. In our experiments, the spinal cord of adult Sprague-Daw- ley rats was clamped for 60 seconds. Dazhui （GV14） and Mingmen （GV4） acupoints of rats were subjected to electroacupuncture. Enzyme-linked immunosorbent assay revealed that the expres- sion of serum inflammatory factors was apparently downregulated in rat models of spinal cord injury after electroacupuncture. Hematoxylin-eosin staining and immunohistochemistry results demonstrated that electroacupuncture contributed to the proliferation of neural stem cells in rat injured spinal cord, and suppressed their differentiation into astrocytes. Real-time quantitative PCR and western blot assays showed that electroacupuncture inhibited activation of the Notch signaling pathway induced by spinal cord injury. These findings indicate that electroacupuncture repaired the injured spinal cord by suppressing the Notch signaling pathway and promoting the proliferation of endogenous neural stem ceils.

Repair of spinal cord injury by neural stem cells modified with BDNF gene in rats

Objective To explore repair of spinal cord injury by neural stem cells （NSCs） modified with brain derived neurotrophic factor （BDNF） gene （BDNF-NSCs） in rats. Methods Neural stem cells modified with BDNF gene were transplanted into the complete transection site of spinal cord at the lumbar 4 （L4） level in rats. Motor function of rats＇ hind limbs was observed and HE and X-gal immunoeytochemical staining, in situ hybridization, and retrograde HRP tracing were also performed. Results BDNF-NSCs survived and integrated well with host spinal cord. In the transplant group, some X-gal positive, NF-200 positive, GFAP positive, BDNF positive, and BDNF mRNA positive cells, and many NF-200 positive nerve fibers were observed in the injury site. Retrograde HRP tracing through sciatic nerve showed some HRP positive cells and nerve fibers near the rostral side of the injury one month after transplant and with time, they increased in number. Examinations on rats＇ motor function and behavior demonstrated that motor function of rats＇ hind limbs improved better in the transplant group than the injury group. Conclusion BDNF-NSCs can survive, differentiate, and partially integrate with host spinal cord, and they significantly ameliorate rats＇ motor function of hind limbs, indicating their promising role in repairing spinal cord injury.

Transplantation of placenta-derived mesenchymal stem cell-induced neural stem cells to treat spinal cord injury

Because of their strong proliferative capacity and multi-potency, placenta-derived mesenchymal stem cells have gained interest as a cell source in the field of nerve damage repair. In the present study, human placenta-derived mesenchymal stem ceils were induced to differentiate into neural stem cells, which were then transplanted into the spinal cord after local spinal cord injury in rats. The motor functional recovery and pathological changes in the injured spinal cord were observed for 3 successive weeks. The results showed that human placenta-derived mesenchymal stem cells can differentiate into neuron-like cells and that induced neural stem cells contribute to the restoration of injured spinal cord without causing transplant rejection. Thus, these cells promote the recovery of motor and sensory functions in a rat model of spinal cord injury. Therefore, human placenta-derived mesenchymal stem cells may be useful as seed cells during the repair of spinal cord injury.

Neural stem cell transplantation in a double-layer collagen membrane with unequal pore sizes for spinal cord injury repair

A novel double-layer collagen membrane with unequal pore sizes in each layer was designed and tested in this study. The inner, loose layer has about 100-μm-diameter pores, while the outer, compact layer has about 10-μm-diameter pores. In a rat model of incomplete spinal cord injury, a large number of neural stem cells were seeded into the loose layer, which was then adhered to the injured side, and the compact layer was placed against the lateral side. The results showed that the transplantation of neural stem cells in a double-layer collagen membrane with unequal pore sizes promoted the differentiation of neural stem cells, attenuated the pathological lesion, and signiifcantly improved the motor function of the rats with incomplete spinal cord injuries. These experimental ifndings suggest that the transplantation of neural stem cells in a double-lay-er collagen membrane with unequal pore sizes is an effective therapeutic strategy to repair an injured spinal cord.

miR-124 and miR-128 differential expression in bone marrow stromal cells and spinal cord-derived neural stem cells

BACKGROUND： MicroRNA （miRNA） expression in stem cells provides important clues for the molecular mechanisms of stem cell proliferation and differentiation. Bone marrow stromal cells and spinal cord-derived neural stem cells exhibit potential for neural regeneration. However, miRNA expression in these cells has been rarely reported. OBJECTIVE： To explore differential expression of two nervous system-specific miRNAs, miR-124 and miR-128, in bone marrow stromal cells and spinal cord-derived neural stem cells. DESIGN, TIME AND SETTING： An In vitro, cell biology experiment was performed at the Department of Biotechnology, Shanxi Medical University from June 2008 to June 2009. MATERIALS： TaqMan miRNA assays were purchased from Applied Biosystems. METHODS： Rat bone marrow stromal cells were isolated and cultured using the whole-bone marrow method, and rat spinal cord-derived neural stem cells were obtained through neurosphere formation. TaqMan miRNA assays were used to measure miR-124 and miR-128 expression in bone marrow stromal cells and spinal cord-derived neural stem cells. MAIN OUTCOME MEASURES： Morphology of bone marrow stromal cells and spinal cord-derived neural stem cells were observed by inverted microscopy. Expression of the neural stem cell-specific marker, nestin, the bone marrow stromal cell surface marker, CD71, and expression of miR-124 and miR-128, were detected by real-time polymerase chain reaction. RESULTS： Cultured bone marrow stromal cells displayed a short fusiform shape. Flow cytometry revealed a large number of CD71-positive cells （〉 95%）. Cultured spinal cord-derived neural stem cells formed nestin-positive neurospheres, and quantitative detection of miRNA demonstrated that less miR-124 and miR-128 was expressed in bone marrow stromal cells compared to spinal cord-derived neural stem cells （P 〈 0.05）. CONCLUSION： Bone marrow stromal cells and spinal cord-derived neural stem cells exhibited differential expression of miR-124 and miR-128, which suggested different characteristics in miRNA expression.

Effect of spinal cord extracts after spinal cord injury on proliferation of rat embryonic neural stem cells and Notch signal pathway in vitro

Objective:To investigate the effect of the spinal cord extracts(SCE)after spinal cord injuries(SCIs)on the proliferation of rat embryonic neural stem cells(NSCs)and the expressions of mRNA of Notch1 as well as of Hes1 in this process in vitro.Methods:The experiment was conducted in 4 different mediums:NSCs+PBS(Group A-blank control group),NSCs+SCE with healthy SD rats(Croup B-normal control group),NSCs+SCE with SD rats receiving sham-operation treatment(Croup C-sham-operation group)and NSCs+SCE with SCIs rats(Group D-paraplegic group).Proliferative abilities of 4 different groups were analyzed by MTT chromatometry after co-culture for 1,2,3,4 and 5 d,respectively.The expressions of Notch 1 and Hes1 mRNA were also detected with RT-PCR after co-culture for 24 and 48 h,respectively.Results:After co-culture for 1,2,3,4 and 5 d respectively,the MTT values of group D were significantly higher than those of group A,group B and group C(P<0.05).However,there were no significantly differences regarding MTT values between group A,group B and group C after co-culture for 1,2,3,4 and 5 d,respectively(P>0.05).Both the expressions of Notch1 and Hes1 mRNA of group D were significantly higher than those of other 3 groups after co-culture for 24 h and 48 h as well(P<0.05).But there was no difference oin expressions of Notch1 and Hes1 mRNA among group A,group B and group C after co-culture for 24 h and 48 h(P>0.05).There was no difference in expressions of Notch1and Hes1 mRNA between 24 h and 48 h treatment in group D.Conclusions:SCE could promote the proliferation of NSCs.It is demonstrated that the microenvironment of SCI may promote the proliferation of NSCs.Besides,SCE could increase the expression of Notch1 and Hes1 mRNA of NSC.It can be concluded that the Notch signaling pathway activation is one of the mechanisms that locally injured microenvironment contributes to the proliferation of ENSC after SCIs.This process may be performed by up-regulating the expressions of Notch1 and Hes1 gene.

Effects of microtubule-associated protein tau expression on neural stem cell migration after spinal cord injury

Our preliminary proteomics analysis suggested that expression of microtubule-associated protein tau is elevated in the spinal cord after injury. Therefore, the first aim of the present study was to examine tau expression in the injured spinal cord. The second aim was to determine whether tau can regulate neural stem cell migration, a critical factor in the successful treatment of spinal cord injury. We established rat models of spinal cord injury and injected them with mouse hippocampal neural stem cells through the tail vein. We used immunohistochemistry to show that the expression of tau protein and the number of migrated neural stem cells were markedly increased in the injured spinal cord. Furthermore, using a Transwell assay, we showed that neural stem cell migration was not affected by an elevated tau concentration in the outer chamber, but it was decreased by changes in intracellular tau phosphorylation state. These results demonstrate that neural stem cells have targeted migration capability at the site of injury, and that although tau is not a chemokine for targeted migration of neural stem cells, intracellular tau phosphorylation/dephosphorylation can inhibit cell migration.

Effect of valproic acid on endogenous neural stem cell proliferation in a rat model of spinal cord injury

BACKGROUND： Valproic acid has been reported to decrease apoptosis, promote neuronal differentiation of brain-derived neural stem cells, and inhibit glial differentiation of brain-derived neural stem cells. OBJECTIVE： To investigate the effects of valproic acid on proliferation of endogenous neural stem cells in a rat model of spinal cord injury. DESIGN, TIME AND SETTING： A randomized, controlled, neuropathological study was performed at Key Laboratory of Trauma, Buming, and Combined Injury, Research Institute of Surgery, Daping Hospital, the Third Military Medical University of Chinese PLA between November 2005 and February 2007. MATERIALS： A total of 45 adult, Wistar rats were randomly divided into sham surgery （n = 5）, injury （n = 20）, and valproic acid （n = 20） groups. Valproic acid was provided by Sigma, USA. METHODS： Injury was induced to the T10 segment in the injury and valproic acid groups using the metal weight-dropping method. The spinal cord was exposed without contusion in the sham surgery group. Rats in the valproic acid group were intraperitoneally injected with 150 mg/kg valproic acid every 12 hours （twice in total）.MAIN OUTCOME MEASURES： Nestin expression （5 mm from injured center） was detected using immunohistochemistry at 1,3 days, 1, 4, and 8 weeks post-injury. RESULTS： Low expression of nestin was observed in the cytoplasm, but rarely in the white matter of the spinal cord in the sham surgery group. In the injury group, nestin expression was observed in the ependyma and pia mater one day after injury, and expression reached a peak at 1 week （P 〈 0.05）. Expression was primarily observed in the ependymal cells, which expanded towards the white and gray matter of the spinal cord. Nestin expression rapidly decreased by 4 weeks post-injury, and had almost completely disappeared by 8 weeks. At 24 hours after spinal cord injury, there was no significant difference in nestin expression between the valproic acid and injury groups. At 1 week, there was a significant increase in the number of nestin-positive cells surrounding the central canal in valproic acid group compared with the injury group （P 〈 0.05）. Expression reached a peak by 4 weeks, and it was still present at 8 weeks. CONCLUSION： Valproic acid promoted endogenous neural stem cell proliferation following spinal cord injury in rats.

Transplantation of neural stem cells, Schwann cells and olfactory ensheathing cells for spinal cord injury:A Web of Science-based literature analysis

OBJECTIVE： To identify global research trends in transplantation of neural stem cells, Schwann cells and olfactory ensheathing cells for spinal cord injury. DATA RETRIEVAL： We performed a bibliometric analysis of studies on transplantation of neural stem cells, Schwann cells and olfactory ensheathing cells for spinal cord injury published from 2002 to 2011 and retrieved from the Web of Science, using the key words spinal cord injury along with either neural stem cell, Schwann cell or olfactory ensheathing cell. SELECTION CRITERIA： Inclusion criteria： （a） peer-reviewed published articles on neural stem cells, Schwann cells or olfactory ensheathing cells for spinal cord injury indexed in the Web of Science; （b） original research articles, reviews, meeting abstracts, proceedings papers, book chapters, editorial materials and news items; and （c） published between 2002 and 2011. Exclusion criteria： （a） articles that required manual searching or telephone access; （b） documents that were not published in the public domain; and （c） corrected papers. MAIN OUTCOME MEASURES： （1）Annual publication output, distribution by journal, distribution by institution and top-cited articles on neural stem cells; （2） annual publication output, distribution by journal, distribution by institution and top-cited articles on Schwann cells; （3） annual publication output, distribution by journal, distribution by institution and top-cited articles on olfactory ensheathing cells. RESULTS： This analysis, based on articles indexed in the Web of Science, identified several research trends among studies published over the past 10 years in transplantation of neural stem cells, Schwann cells and olfactory ensheathing cells for spinal cord injury. The number of publications increased over the 10-year period examined. Most papers appeared in journals with a focus on neurology, such as Journal of Neurotrauma, Experimental Neurology and Gila. Research institutes publishing on the use of neural stem cells to repair spinal cord injury were mostly in the USA and Canada. Those publishing on the use of Schwann cells were mostly in the USA and Canada as well. Those publishing on the use of olfactory ensheathing cells were mostly in the UK, the USA and Canada. CONCLUSION： On the basis of the large number of studies around the world, cell transplantation has proven to be the most promising therapeutic approach for spinal cord injury.

E-cadherin-transfected Neural Stem Cells Transplantation for Spinal Cord Injury in Rats

The effects of E-cadherin-transfected neural stem cells（NSCs） transplantation for spinal cord injury（SCI） in rats were investigated. Sixty SD rats were randomly divided into model control group, NSCs group, empty plasmid group and E-cadherin overexpression group（n=15 each）. The animal SCI model was established by using the modified Allen＇s method. NSCs were cultured. Rats in NSCs group were subjected to NSCs transplantation. E-cadherin gene eucaryotic expression vector and pcDNA3.1-E-cadherin were respectively transfected into cultured NSCs, serving as empty plasmid group and E-cadherin overexpression group respectively. At 7th day after transplantation, neurological function of all rats was assessed by Tarlov score. After rats were sacrificed in each group, the number of BrdU and Nestin positive cells was counted by immunohistochemistry. Immumofluorescence method was used to detect the expression of neurofilament protein（NF） and glial fibrillary acidic protein（GFAP）. As compared with model control group, the Tarlov score and the number of of BrdU and Nestin positive cells, and the expression of NF and GFAP in NSCs group, empty plasmid group, and E-cadherin overexpression group were increased significantly（P〈0.05）, and those in the E-cadherin overexpression group were increased more significantly than the other transplantation groups（P〈0.05）. It was suggested that E-cadherin could be conductive to nerve regeneration and repair probably by promoting the proliferation and differentiation of NSCs.

Three-dimensional bioprinting collagen/silk fibroin scaffold combined with neural stem cells promotes nerve regeneration after spinal cord injury

Many studies have shown that bio-scaffolds have important value for promoting axonal regeneration of injured spinal cord.Indeed,cell transplantation and bio-scaffold implantation are considered to be effective methods for neural regeneration.This study was designed to fabricate a type of three-dimensional collagen/silk fibroin scaffold (3D-CF) with cavities that simulate the anatomy of normal spinal cord.This scaffold allows cell growth in vitro and in vivo.To observe the effects of combined transplantation of neural stem cells (NSCs) and 3D-CF on the repair of spinal cord injury.Forty Sprague-Dawley rats were divided into four groups: sham (only laminectomy was performed),spinal cord injury (transection injury of T10 spinal cord without any transplantation),3D-CF (3D scaffold was transplanted into the local injured cavity),and 3D-CF + NSCs (3D scaffold co-cultured with NSCs was transplanted into the local injured cavity.Neuroelectrophysiology,imaging,hematoxylin-eosin staining,argentaffin staining,immunofluorescence staining,and western blot assay were performed.Apart from the sham group,neurological scores were significantly higher in the 3D-CF + NSCs group compared with other groups.Moreover,latency of the 3D-CF + NSCs group was significantly reduced,while the amplitude was significantly increased in motor evoked potential tests.The results of magnetic resonance imaging and diffusion tensor imaging showed that both spinal cord continuity and the filling of injury cavity were the best in the 3D-CF + NSCs group.Moreover,regenerative axons were abundant and glial scarring was reduced in the 3D-CF + NSCs group compared with other groups.These results confirm that implantation of 3D-CF combined with NSCs can promote the repair of injured spinal cord.This study was approved by the Institutional Animal Care and Use Committee of People’s Armed Police Force Medical Center in 2017 (approval No.2017-0007.2).

Effects of neural stem cell transplantation on the motor function of rats with contusion spinal cord injuries:a meta-analysis

Objective:To judge the efficacies of neural stem cell(NSC)transplantation on functional recovery following contusion spinal cord injuries(SCIs).Data sources:Studies in which NSCs were transplanted into a clinically relevant,standardized rat model of contusion SCI were identified by searching the PubMed,Embase and Cochrane databases,and the extracted data were analyzed by Stata 14.0.Data selection:Inclusion criteria were that NSCs were used in in vivo animal studies to treat contusion SCIs and that behavioral assessment of locomotor functional recovery was performed using the Basso,Beattie,and Bresnahan lo-comotor rating scale.Exclusion criteria included a follow-up of less than 4 weeks and the lack of control groups.Outcome measures:The restoration of motor function was assessed by the Basso,Beattie,and Bresnahan locomotor rating scale.Results:We identified 1756 non-duplicated papers by searching the aforementioned electronic databases,and 30 full-text articles met the inclusion criteria.A total of 37 studies reported in the 30 articles were included in the meta-analysis.The meta-analysis results showed that transplanted NSCs could improve the motor function recovery of rats following contusion SCIs,to a moderate extent(pooled standardized mean difference(SMD)=0.73;95%confidence interval(CI):0.47–1.00;P<0.001).NSCs obtained from different donor species(rat:SMD=0.74;95%CI:0.36–1.13;human:SMD=0.78;95%CI:0.31–1.25),at different donor ages(fetal:SMD=0.67;95%CI:0.43–0.92;adult:SMD=0.86;95%CI:0.50–1.22)and from different origins(brain-derived:SMD=0.59;95%CI:0.27–0.91;spinal cord-derived:SMD=0.51;95%CI:0.22–0.79)had similar efficacies on improved functional recovery;however,adult induced pluripotent stem cell-derived NSCs showed no significant efficacies.Furthermore,the use of higher doses of transplanted NSCs or the administration of immunosuppressive agents did not promote better locomotor function recovery(SMD=0.45;95%CI:0.21–0.70).However,shorter periods between the contusion induction and the NSC transplantation showed slightly higher efficacies(acute:SMD=1.22;95%CI:0.81–1.63;subacute:SMD=0.75;95%CI:0.42–1.09).For chronic injuries,NSC implantation did not significantly improve functional recovery(SMD=0.25;95%CI:–0.16 to 0.65).Conclusion:NSC transplantation alone appears to be a positive yet limited method for the treatment of contusion SCIs.

Improvement of neurological function in rats with spinal cord injury after the transplantation of neural stem cells directly differentiated from bone marrow mesenchymal stem cells

Objective To study the effect and mechanism of neurological function recovery in rats with spinal cord injury ( SCI) rats after transplantation of neural stem cells which are directly differentiated from bone marrow mesenchymal stem cells ( BMSC ) ,and to investigate the suitable engraftment time. Methods BMSC at 3rd passage were differentiated into neural stem cells ( NSC) , and immunofluorescence staining was used to

Functional recovery and microenvironmental alterations in a rat model of spinal cord injury following human umbilical cord blood-derived mesenchymal stem cells transplantation

BACKGROUND： Transplantation of human umbilical cord blood-derived mesenchymal stem cells （MSCs） has been shown to benefit spinal cord injury （SCI） repair. However, mechanisms of microenvironmental regulation during differentiation of transplanted MSCs remain poorly understood. OBJECTIVE： To observe changes in nerve growth factor （NGF）, brain-derived neurotrophic factor （BDNF）, and interleukin-8 （IL-8） expression following transplantation of human umbilical cord-derived MSCs, and to explore the association between microenvironment and neural functional recovery following MSCs transplantation. DESIGN, TIME AND SETTING： A randomized, controlled, animal experiment was performed at the Department of Orthopedics, First Affiliated Hospital of Soochow University from April 2005 to March 2007. MATERIALS： Human cord blood samples were provided by the Department of Gynecology and Obstetrics, First Affiliated Hospital of Soochow University. Written informed consent was obtained. METHODS： A total of 62 Wister rats were randomly assigned to control （n = 18）, model （n = 22, SCI ＋ PBS）, and transplantation （n = 22, SCI ＋ MSCs） groups. The rat SCI model was established using the weight compression method. MSCs were isolated from human umbilical cord blood and cultured in vitro for several passages. 5-bromodeoxyuridine （BrdU）-Iabeled MSCs （24 hours before injection） were intravascularly transplanted. MAIN OUTCOME MEASURES： The rats were evaluated using the Basso, Beattie and Bresnahan （BBB） locomotor score and inclined plane tests. Transplanted cells were analyzed following immunohistochemistry. Enzyme-linked immunosorbant assay was performed to determine NGF, BDNF, and IL-8 levels prior to and after cell transplantation. RESULTS： A large number of BrdU-positive MSCs were observed in the SCI region of the transplantation group, and MSCs were evenly distributed in injured spinal cord tissue 1 week after transplantation. BBB score and inclined plane test results revealed significant functional improvement in the transplantation group compared to the model group （P 〈 0.05）, which was maintained for 2-3 weeks. Compared to the model group, NGF and BDNF levels were significantly increased in the injured region following MSCs transplantation at 3 weeks （P 〈 0.05）, but IL-8 levels remained unchanged （P 〉 0.05）. CONCLUSION： MSCs transplantation increased NGF and BDNF expression in injured spinal cord tissue. MSCs could promote neurological function recovery in SCI rats by upregulating NGF expression and improving regional microenvironments.