Small extracellular vesicles from hypoxia-preconditioned bone marrow mesenchymal stem cells attenuate spinal cord injury via miR-146a-5p-mediated regulation of macrophage polarization

Spinal cord injury is a disabling condition with limited treatment options.Multiple studies have provided evidence suggesting that small extracellular vesicles(SEVs)secreted by bone marrow mesenchymal stem cells(MSCs)help mediate the beneficial effects conferred by MSC transplantation following spinal cord injury.Strikingly,hypoxia-preconditioned bone marrow mesenchymal stem cell-derived SEVs(HSEVs)exhibit increased therapeutic potency.We thus explored the role of HSEVs in macrophage immune regulation after spinal cord injury in rats and their significance in spinal cord repair.SEVs or HSEVs were isolated from bone marrow MSC supernatants by density gradient ultracentrifugation.HSEV administration to rats via tail vein injection after spinal cord injury reduced the lesion area and attenuated spinal cord inflammation.HSEVs regulate macrophage polarization towards the M2 phenotype in vivo and in vitro.Micro RNA sequencing and bioinformatics analyses of SEVs and HSEVs revealed that mi R-146a-5p is a potent mediator of macrophage polarization that targets interleukin-1 receptor-associated kinase 1.Reducing mi R-146a-5p expression in HSEVs partially attenuated macrophage polarization.Our data suggest that HSEVs attenuate spinal cord inflammation and injury in rats by transporting mi R-146a-5p,which alters macrophage polarization.This study provides new insights into the application of HSEVs as a therapeutic tool for spinal cord injury.

Bone marrow mesenchymal stem cells and exercise restore motor function following spinal cord injury by activating PI3K/AKT/mTOR pathway

Although many therapeutic interventions have shown promise in treating spinal cord injury, focusing on a single aspect of repair cannot achieve successful and functional regeneration in patients following spinal cord injury. In this study, we applied a combinatorial approach for treating spinal cord injury involving neuroprotection and rehabilitation, exploiting cell transplantation and functional sensorimotor training to promote nerve regeneration and functional recovery. Here, we used a mouse model of thoracic contusive spinal cord injury to investigate whether the combination of bone marrow mesenchymal stem cell transplantation and exercise training has a synergistic effect on functional restoration. Locomotor function was evaluated by the Basso Mouse Scale, horizontal ladder test, and footprint analysis. Magnetic resonance imaging, histological examination, transmission electron microscopy observation, immunofluorescence staining, and western blotting were performed 8 weeks after spinal cord injury to further explore the potential mechanism behind the synergistic repair effect. In vivo, the combination of bone marrow mesenchymal stem cell transplantation and exercise showed a better therapeutic effect on motor function than the single treatments. Further investigations revealed that the combination of bone marrow mesenchymal stem cell transplantation and exercise markedly reduced fibrotic scar tissue, protected neurons, and promoted axon and myelin protection. Additionally, the synergistic effects of bone marrow mesenchymal stem cell transplantation and exercise on spinal cord injury recovery occurred via the PI3 K/AKT/mTOR pathway. In vitro, experimental evidence from the PC12 cell line and primary cortical neuron culture also demonstrated that blocking of the PI3 K/AKT/mTOR pathway would aggravate neuronal damage. Thus, bone marrow mesenchymal stem cell transplantation combined with exercise training can effectively restore motor function after spinal cord injury by activating the PI3 K/AKT/mTOR pathway.

Cell transplantation therapies for spinal cord injury focusing on bone marrow mesenchymal stem cells:Advances and challenges

Spinal cord injury(SCI)is a devastating condition with complex pathological mechanisms that lead to sensory,motor,and autonomic dysfunction below the site of injury.To date,no effective therapy is available for the treatment of SCI.Recently,bone marrow-derived mesenchymal stem cells(BMMSCs)have been considered to be the most promising source for cellular therapies following SCI.The objective of the present review is to summarize the most recent insights into the cellular and molecular mechanism using BMMSC therapy to treat SCI.In this work,we review the specific mechanism of BMMSCs in SCI repair mainly from the following aspects:Neuroprotection,axon sprouting and/or regeneration,myelin regeneration,inhibitory microenvironments,glial scar formation,immunomodulation,and angiogenesis.Additionally,we summarize the latest evidence on the application of BMMSCs in clinical trials and further discuss the challenges and future directions for stem cell therapy in SCI models.

The combined application of stem cells and three-dimensional bioprinting scaffolds for the repair of spinal cord injury

Spinal cord injury is considered one of the most difficult injuries to repair and has one of the worst prognoses for injuries to the nervous system.Following surgery,the poor regenerative capacity of nerve cells and the generation of new scars can make it very difficult for the impaired nervous system to restore its neural functionality.Traditional treatments can only alleviate secondary injuries but cannot fundamentally repair the spinal cord.Consequently,there is a critical need to develop new treatments to promote functional repair after spinal cord injury.Over recent years,there have been seve ral developments in the use of stem cell therapy for the treatment of spinal cord injury.Alongside significant developments in the field of tissue engineering,three-dimensional bioprinting technology has become a hot research topic due to its ability to accurately print complex structures.This led to the loading of three-dimensional bioprinting scaffolds which provided precise cell localization.These three-dimensional bioprinting scaffolds co uld repair damaged neural circuits and had the potential to repair the damaged spinal cord.In this review,we discuss the mechanisms underlying simple stem cell therapy,the application of different types of stem cells for the treatment of spinal cord injury,and the different manufa cturing methods for three-dimensional bioprinting scaffolds.In particular,we focus on the development of three-dimensional bioprinting scaffolds for the treatment of spinal cord injury.

Mesenchymal stem cells,extracellular vesicles,and transcranial magnetic stimulation for ferroptosis after spinal cord injury

Spinal cord injury is characte rized by diffe rent aetiologies,complex pathogenesis,and diverse pathological changes.Current treatments are not ideal,and prognosis is generally poor.After spinal cord injury,neurons die due to various forms of cell death.Among them,fe rroptosis causes dysfunction after spinal cord injury,and no existing traditional treatments have been indicated to block its occurrence.Meanwhile,emerging therapies using mesenchymal stem cells,extracellular vesicles,and transcranial magnetic stimulation therapy are promising for reve rsing spinal co rd neuronal ferroptosis after spinal cord injury.However,no definitive studies have demonstrated the effectiveness of these approaches.This review summarizes the existing research on the mechanisms of ferroptosis;fe rroptosis after spinal cord injury;treatment of spinal cord injury with mesenchymal stem cells,extracellular vesicles,and transc ranial magnetic stimulation;and treatment of ferroptosis using mesenchymal stem cells,extracellular vesicles,and transc ranial magnetic stimulation.Inhibiting ferroptosis can promote the reversal of neurological dysfunction after spinal cord injury.In addition,mesenchymal stem cells,extracellular vesicles,and transc ranial magnetic stimulation can reve rse adverse outcomes of spinal cord injury and regulate ferroptosis-related fa ctors.Thus,it can be inferred that mesenchymal stem cells,extracellular vesicles,and transcranial magnetic stimulation have the potential to inhibit fe rroptosis after spinal cord injury.This review serves as a reference for future research to confirm these conclusions.

Therapeutic Potential of Bone Marrow Derived Mesenchymal Stem Cells in Modulating Astroglyosis of Surgical Induced Experimental Spinal Cord Injury

Background: Spinal cord injury (SCI) unsuccessful regeneration was due to glial scar development. It was a major obstacle to axonal restoration. Safe therapeutic intervention by the use of bone marrow derived stem cells (BMMSCs) transplantation applied in the present study could reduce spinal disability. Material and methods: Forty male albino rats were divided into four groups: GI: negative control (n = 10 rats);GII: positive control after SCI (n = 10 rats);GIII: SCI + BM - MSCs intravenous injected and GIV: SCI + BM - MSCs intra lesion injected (n = 10 rats in each group). The samples were taken from spinal cord tissues around the region of injury and were subjected to histological, immunohistochemical assessment. RNA extraction and real time PCR for detection of nerve regeneration and astrocyte response to the injury were also performed. Results: Clinical improvement occurred by the enhancement in the Basso, Beattie and Bresnahan (BBB) score after SCI. Histological examinations showed positive regenerative responses in GIV compared to GIII. Conclusion: BM-MSCs transplantation has a promising role in enhancing the microenvironment for nerve regeneration through stumbling the glial scaring formation and inflammatory response after chronic spinal cord injury especially by using intra-lesion route injection.

Bone marrow mesenchymal stem cells repair spinal cord ischemia/reperfusion injury by promoting axonal growth and anti-autophagy

Bone marrow mesenchymal stem cells can differentiate into neurons and astrocytes after trans- plantation in the spinal cord of rats with ischemia/reperfusion injury. Although bone marrow mesenchymal stem cells are known to protect against spinal cord ischemia/reperfusion injury through anti-apoptotic effects, the precise mechanisms remain unclear. In the present study, bone marrow mesenchymal stem cells were cultured and proliferated, then transplanted into rats with ischemia/reperfusion injury via retro-orbital injection. Immunohistochemistry and immunofluorescence with subsequent quantification revealed that the expression of the axonal regeneration marker, growth associated protein-43, and the neuronal marker, microtubule-as- sociated protein 2, significantly increased in rats with bone marrow mesenchymal stem cell transplantation compared with those in rats with spinal cord ischemia/reperfusion injury. Fur- thermore, the expression of the autophagy marker, microtubule-associated protein light chain 3B, and Beclin 1, was significantly reduced in rats with the bone marrow mesenchymal stem cell transplantation compared with those in rats with spinal cord ischemia/reperfusion injury. Western blot analysis showed that the expression of growth associated protein-43 and neuro- filament-H increased but light chain 3B and Beclin 1 decreased in rats with the bone marrow mesenchymal stem cell transplantation. Our results therefore suggest that bone marrow mes- enchymal stem cell transplantation promotes neurite growth and regeneration and prevents autophagy. These responses may likely be mechanisms underlying the protective effect of bone marrow mesenchymal stem cells against spinal cord ischemia/reperfusion injury.

Transplantation of neurotrophin-3-transfected bone marrow mesenchymal stem cells for the repair of spinal cord injury

Bone marrow mesenchymal stem cell transplantation has been shown to be therapeutic in the repair of spinal cord injury. However, the low survival rate of transplanted bone marrow mesen- chymal stem cells in vivo remains a problem. Neurotrophin-3 promotes motor neuron survival and it is hypothesized that its transfection can enhance the therapeutic effect. We show that in vitro transfection of neurotrophin-3 gene increases the number of bone marrow mesenchymal stem cells in the region of spinal cord injury. These results indicate that neurotrophin-3 can promote the survival of bone marrow mesenchymal stem cells transplanted into the region of spinal cord injury and potentially enhance the therapeutic effect in the repair of spinal cord injury.

Chondroitinase ABC plus bone marrow mesenchymal stem cells for repair of spinal cord injury

As chondroitinase ABC can improve the hostile microenvironment and cell transplantation is proven to be effective after spinal cord injury, we hypothesized that their combination would be a more effective treatment option. At 5 days after T8 spinal cord crush injury, rats were injected with bone marrow mesenchymal stem cell suspension or chondroitinase ABC 1 mm from the edge of spinal cord damage zone. Chondroitinase ABC was first injected, and bone marrow mesenchymal stem cell suspension was injected on the next day in the combination group. At 14 days, the mean Basso, Beattie and Bresnahan score of the rats in the combination group was higher than other groups. Hematoxylin-eosin staining showed that the necrotic area was significantly reduced in the combination group compared with other groups. Glial fibrillary acidic protein-chondroitin sulfate proteoglycan double staining showed that the damage zone of astrocytic scars was significantly reduced without the cavity in the combination group. Glial fibrillary acidic protein/growth associated protein-43 double immunostaining revealed that positive fibers traversed the damage zone in the combination group. These results suggest that the combination of chondroitinase ABC and bone marrow mesenchymal stem cell transplantation contributes to the repair of spinal cord injury.

Protective effect of thodioloside and bone marrow mesenchymal stem cells infected with HIF-1-expressing adenovirus on acute spinal cord injury

Rhodioloside has been shown to protect cells from hypoxia injury,and bone marrow mesenchymal stem cells have a good effect on tissue repair.To study the effects of rhodioloside and bone marrow mesenchymal stem cells on spinal cord injury,a rat model of spinal cord injury was established using the Infinite Horizons method.After establishing the model,the rats were randomly divided into five groups.Rats in the control group were intragastrically injected with phosphate buffered saline(PBS)(5μL).PBS was injected at 6 equidistant points around 5 mm from the injury site and at a depth of 5 mm.Rats in the rhodioloside group were intragastrically injected with rhodioloside(5 g/kg)and intramuscularly injected with PBS.Rats in the mesenchymal stem cell(MSC)group were intramuscularly injected with PBS and intramuscularly with MSCs(8×10^6/mL in a 50-μL cell suspension).Rats in the Ad-HIF-MSC group were intragastrically injected with PBS and intramuscularly injected with HIF-1 adenovirus-infected MSCs.Rats in the rhodioloside+Ad-HIF-MSC group were intramuscularly injected with MSCs infected with the HIF-1 adenovirus and intragastrically injected with rhodioloside.One week after treatment,exercise recovery was evaluated with a modified combined behavioral score scale.Hematoxylin-eosin staining and Pischingert’s methylene blue staining were used to detect any histological or pathological changes in spinal cord tissue.Levels of adenovirus IX and Sry mRNA were detected by real-time quantitative polymerase chain reaction and used to determine the number of adenovirus and mesenchymal stem cells that were transfected into the spinal cord.Immunohistochemical staining was applied to detect HIF-1 protein levels in the spinal cord.The results showed that:(1)compared with the other groups,the rhodioloside+Ad-HIF-MSC group exhibited the highest combined behavioral score(P<0.05),the most recovered tissue,and the greatest number of neurons,as indicated by Pischingert’s methylene blue staining.(2)Compared with the PBS group,HIF-1 protein expression was greater in the rhodioloside group(P<0.05).(3)Compared with the Ad-HIF-MSC group,Sry mRNA levels were higher in the rhodioloside+Ad-HIF-MSC group(P<0.05).These results confirm that rhodioloside combined with bone marrow mesenchymal stem cells can promote the recovery of spinal cord injury and activate the HIF-1 pathway to promote the survival of bone marrow mesenchymal stem cells and repair damaged neurons within spinal cord tissue.This experiment was approved by the Animal Ethics Committee of Gansu University of Traditional Chinese Medicine,China(approval No.2015KYLL029)in June 2015.

Injection of bone marrow mesenchymal stem cells by intravenous or intraperitoneal routes is a viable alternative to spinal cord injury treatment in mice

In spite of advances in surgical care and rehabilitation, the consequences of spinal cord injury （SCI） are still challenging. Several experimental therapeutic strategies have been studied in the SCI field, and recent advances have led to the development of therapies that may act on the inhibitory microenvironment. Assorted lineages of stem cells are considered a good treatment for SCI. This study investigated the effect of systemic transplantation of mesenchymal stem cells （MSCs） in a compressive SCI model. Here we present results of the intraperitoneal route, which has not been used previously for MSC administration after compressive SCI. We used adult female C57BL/6 mice that underwent laminectomy at the T9 level, followed by spinal cord compression for 1 minute with a 30-g vascular clip. The animals were divided into five groups： sham （anesthesia and laminectomy but without compression injury induction）, MSC i.p. （intraperitoneal injection of 8×10^5 MSCs in 500 μL of DMEM at 7 days after SCI）, MSC i.v. （intravenous injection of 8 × 10^5 MSCs in 500μL of DMEM at 7 days after SCI）, DMEM i.p. （intraperitoneal injection of 500μL of DMEM at 7 days after SCI）, DMEM i.v. （intravenous injection of 500 μL of DMEM at 7 days after SCI）. The effects of MSCs transplantation in white matter sparing were analyzed by luxol fast blue staining. The number of preserved fibers was counted in semithin sections stained with toluidine blue and the presence of trophic factors was analyzed by immunohistochemistry. In addition, we analyzed the locomotor performance with Basso Mouse Scale and Global Mobility Test. Our results showed white matter preservation and a larger number of preserved fibers in the MSC groups than in the DMEM groups. Furthermore, the MSC groups had higher levels of trophic factors （brain-derived neurotrophic factor, nerve growth factor, neurotrophin-3 and neurotrophin-4） in the spinal cord and improved locomotor performance. Our results indicate that injection of MSCs by either intraperitoneal or intravenous routes results in beneficial outcomes and can be elected as a choice for SCI treatment.

Bone marrow mesenchymal stem cells with Nogo-66 receptor gene silencing for repair of spinal cord injury

We hypothesized that RNA interference to silence Nogo-66 receptor gene expression in bone marrow mesenchymal stem cells before transplantation might further improve neurological function in rats with spinal cord transection injury. After 2 weeks, the number of neurons and BrdU-positive cells in the Nogo-66 receptor gene silencing group was higher than in the bone marrow mesenchymal stem cell group, and significantly greater compared with the model group. After 4 weeks, behavioral performance was signiifcantly enhanced in the model group. Af-ter 8 weeks, the number of horseradish peroxidase-labeled nerve ifbers was higher in the Nogo-66 receptor gene silencing group than in the bone marrow mesenchymal stem cell group, and signiifcantly higher than in the model group. The newly formed nerve ifbers and myelinated ner ve ifbers were detectable in the central transverse plane section in the bone marrow mesenchymal stem cell group and in the Nogo-66 receptor gene silencing group.

Effect of intravenous transplantation of bone marrow mesenchymal stem cells on neurotransmitters and synapsins in rats with spinal cord injury

Bone marrow mesenchymal stem cells were isolated, purified and cultured in vitro by Percoll density gradient centrifugation combined with the cell adherence method. Passages 3 5 bone marrow mesenchymal stem cells were transplanted into rats with traumatic spinal cord injury via the caudal vein. Basso-Beattie-Bresnahan scores indicate that neurological function of experimental rats was significantly improved over transplantation time （1-5 weeks）. Expressions of choline acetyltransferase, glutamic acid decarboxytase and synapsins in the damaged spinal cord of rats was significantly increased after transplantation, determined by immunofluorescence staining and laser confocal scanning microscopy. Bone marrow mesenchymal stem cells that had migrated into the damaged area of rats in the experimental group began to express choline acetyltransferase, glutamic acid decarboxylase and synapsins, 3 weeks after transplantation. The Basso-Beattie- Bresnahan scores positively correlated with expression of choline acetyltransferase and synapsins. Experimental findings indicate that intravenously transplanted bone marrow mesenchymal stem cells traverse into the damaged spinal cord of rats, promote expression of choline acetyltransferase, glutamic acid decarboxylase and synapsins, and improve nerve function in rats with spinal cord injury.

Upregulation of UBAP2L in Bone Marrow Mesenchymal Stem Cells Promotes Functional Recovery in Rats with Spinal Cord Injury

Post-translational modifications of cellular proteins with ubiquitin or ubiquitin-like proteins regulate many cellular processes,such as cell proliferation,differentiation,apoptosis, signal transduction,intercellular immune recognition,inflammatory response,stress response,and DNA repair.Nice4/UBAP2L is an important member in the family of ubiquitin-like proteins,and its biological function remains unknown.This study aimed to investigate the effect of UBAP2L on spinal cord injury (SCI).At first,rat bone marrow mesenchymal stem cells (BMSCs)were infected with adeno-associated virus to induce over-expression of Nice4.Subsequently,the infected BMSCs were transplanted into rats suffering from semi-sectioned SCI.The results showed that the over-expression of Nice4 significantly promoted the proliferation and differentiation of BMSCs. In addition,the transplantation of infected BMSCs into the injured area of SCI rats improved the function repair of SCI.Importantly,the immunohistochemical and hematoxylin-eosin staining and RT-PCR results showed that the number of neuronal cells,oligodendrocytes,and astrocytes was significantly increased in the injured area,along with significantly upregulated expression ofcyclin D1 and p38 mitogen-activated protein kinase (MAPK).Meanwhile,the expression of caspase 3 protein was significantly down-regulated.In conclusion,the over-expression of Nice4 gene can promote the functional recovery in SCI rats by promoting cell proliferation and inhibiting apoptosis. The results of this study indicate an alternative option for the clinical treatment of SCI.

Combination of bone marrow mesenchymal stem cells and brain-derived neurotrophic factor for treating spinal cord injury

BACKGROUND： Because bone marrow mesenchymal stem cells （BMSCs） do not secrete sufficient brain-derived neurotrophic factor （BDNF）, the use of exogenous BDNF could improve microenvironments in injured regions for BMSCs differentiation. OBJECTIVE： To analyze recovery of the injured spinal cord following BMSCs venous transplantation in combination with consecutive injections of BDNF. DESIGN, TIME AND SETTING： A randomized, controlled animal experiment was performed at the Central Laboratory of First Hospital and Anatomical Laboratory, Fujian Medical University from October 2004 to May 2006. MATERIALS： Human BDNF was purchased from Sigma, USA. METHODS： A total of 44 New Zealand rabbits were randomly assigned to model （n = 8）, BDNF （n = 12）, BMSC （n= 12）, and BMSC＋BDNF （n= 12） groups. Spinal cord （I-2）injury was established with the dropping method. The model group rabbits were injected with 1 mL normal saline via the ear margin vein; the BDNF group was subdurally injected with 100 μg/d human BDNF for 1 week; the BMSC group was injected with 1 mL BMSCs suspension （2 × 10^6/mL） via the ear margin vein; and the BMSC＋BDNF group rabbits were subdurally injected with 100 μg/d BDNF for 1 week, in addition to BMSCs suspension via the ear margin vein. MAIN OUTCOME MEASURES： BMSCs surface markers were detected by flow cytometry. BMSCs differentiation in the injured spinal cord was detected by immunofluorescence histochemistry. Functional and structural recovery, as well as morphological changes, in the injured spinal cord were respectively detected by Tarlov score, horseradish peroxidase retrograde tracing, and hematoxylin ＆ eosin staining methods at 1, 3, and 5 weeks following transplantation. RESULTS： Transplanted BMSCs differentiated into neuronal-like cells in the injured spinal cord at 3 and 5 weeks following transplantation. Neurological function and pathological damage improved following BMSC ＋ BDNF treatment compared with BDNF or BMSC alone （P 〈 0.01 or P 〈 0.05）. CONCLUSION： BMSCs venous transplantation in combination with BDNF subdural injection benefits neuronal-like cell differentiation and significantly improves structural and function of injured spinal cord compared with BMSCs or BDNF alone.

Does vitamin C have the ability to augment the therapeutic effect of bone marrow-derived mesenchymal stem cells on spinal cord injury？

Methylprednisolone（MP） is currently the only drug confirmed to exhibit a neuroprotective effect on acute spinal cord injury（SCI）. Vitamin C（VC） is a natural water-soluble antioxidant that exerts neuroprotective effects through eliminating free radical damage to nerve cells. Bone marrow mesenchymal stem cells（BMMSCs）, as multipotent stem cells, are promising candidates in SCI repair. To evaluate the therapeutic effects of MP, VC and BMMSCs on traumatic SCI, 80 adult male rats were randomly divided into seven groups： control, SCI（SCI induction by weight-drop method）, MP（SCI induction, followed by administration of 30 mg/kg MP via the tail vein, once every other 6 hours, for five times）, VC（SCI induction, followed by intraperitoneal administration of 100 mg/kg VC once a day, for 28 days）, MP ＋ VC（SCI induction, followed by administration of MP and VC as the former）, BMMSCs（SCI induction, followed by injection of 3 × 10~6 BMMSCs at the injury site）, and BMMSCs ＋ VC（SCI induction, followed by BMMSCs injection and VC administration as the former）. Locomotor recovery was assessed using the Basso Mouse Scale. Injured spinal cord tissue was evaluated using hematoxylin-eosin staining and immunohistochemical staining. Expression of transforming growth factor-beta, tumor necrosis factor-alpha, and matrix metalloproteinase-2 genes was determined using real-time quantitative PCR. BMMSCs intervention better promoted recovery of nerve function of rats with SCI, mitigated nerve cell damage, and decreased expression of transforming growth factor-beta, tumor necrosis factor-alpha, and matrix metalloproteinase-2 genes than MP and/or VC. More importantly, BMMSCs in combination with VC induced more obvious improvements. These results suggest that VC can enhance the neuroprotective effects of BMMSCs against SCI.

Combined transplantation of bone marrow mesenchymal stem cells and pedicled greater omentum promotes locomotor function and regeneration of axons after spinal cord injury in rats

BACKGROUND： According to previous studies, the neuroprotective effect of the pedicled greater omentum may be attributed to the secretion of neurotrophic factors and stimulation of angiogenesis. The neurotrophic factors released from the pedicled greater omentum, such as brain-derived neurotrophic factor and neurotrophin 3/4/5 could exert a neuroprotective effect on the damaged host neural and glial cells, and also could induce the transdifferentiation of transplanted bone marrow mesenchymal stem cells （BMSCs） into neural cells. OBJECTIVE： Based on the functions of the omentum of neuro-protection and vascularization, we hypothesize that the transplantation of BMSCs and pedicled greater omentum into injured rat spinal cord might improve the survival rate and neural differentiation of transplanted BMSCs and consequently gain a better functional outcome. DESIGN, TIME AND SETFING： A randomized, controlled animal experiment. The experiments were carried out at the Department of Anatomy, the Secondary Military Medical University of Chinese PLA between June 2005 and June 2007. MATERIALS： Fifteen male inbred Wistar rats, weighing （200±20） g, provided by the Experimental Animal Center of the Secondary Military Medical University of Chinese PLA were used and met the animal ethical standards. Mouse anti-BrdU and mouse anti-NF200 monoclonal antibody were purchased from Boster, China. METHODS： Cell culture： We used inbred Sprague-Dawley rats to harvest bone marrow for culture of BMSCs and transplantation to avoid possible immune rejection. BMSCs were cultured via total bone marrow adherence. Experimental grouping and intervention： The rats were randomly divided into a control group, cell group and combined group, five rats per group. Rats in the control group underwent spinal cord injury （SCI） only, during which an artery clamp with pressure force of 30 g was employed to compress the spinal cord at the Tl0 level for 30 seconds to produce the SCI model. 5 μ L PBS containing 10^5 BMSCs was injected into the injured site of the spinal cord in 60 seconds via a microsyringe in the cell group after SCI. In the combined group, after SCI and BMSC transplantation, an autograft pedicled greater omentum was transplanted onto the injured site of the spinal cord and fixed with a suture. SCI model and transplantation： Control group, SCI model without treatment; cell group, transplantation of BMSCs after SCI; combined group, combined transplantation of BMSCs and pedicled greater omentum after SCI. MAIN OUTCOME MEASURES： At days 1, 7, 14, 21 and 28 PO （post operation）, the Basso, Beattie and Bresnahan （BBB） scale was used to observe and evaluate the recovery of locomotor function. At day 29 PO, after transcardial perfusion using 4% paraformaldehyde, a spinal cord segment of 1 cm around the injury was harvested. A cryostat section was performed longitudinally in the horizontal plane and sections were chosen by systematic random sampling for staining. Anti-BrdU staining and counting was performed to measure survival rate of transplanted BMSCs; anti-BrdU-nestin and BrdU-glial fibrillary acidic protein （GFAP） double staining and counting measured neural differentiation of BMSCs; and anti-NF 200 staining was used to evaluate axonal regeneration. RESULTS： All 15 rats were included in the outcome analysis, without any loss. Changes in BBB scores： Combined transplantation of BMSCs and the pedicled greater omentum produced significantly higher BBB scores at 7-28 days post-injury than in the control group （P 〈 0.05）. BBB scores in the cell group were higher than in the control group at 28 days post-injury （P 〈 0.05）. Survival rate and neural differentiation of transplanted BMSCs： Immunostaining of BrdU demonstrated that transplanted BMSCs survived in the spinal cord and migrated cranially and caudally as far as 0.5 mm from the injection site in the cell group and combined group. Some of the transplanted BMSCs expressed nestin or GFAP which revealed neural differentiation of BMSCs in the combined group and cell group. Axonal regeneration： The areas of axonal NF200 staining in the cell group and control group were lower than that of the combined group （P 〈 0.01 ）. CONCLUSION： It is effective and feasible to transplant BMSCs with the pedicled greater omentum for regeneration of spinal cord after SCI compared with transplanting BMSCs alone. This method results in better locomotor outcomes and axonal regeneration.

Exosomes from bone marrow mesenchymal stem cells are a potential treatment for ischemic stroke

Exosomes derived from human bone marrow mesenchymal stem cells(MSC-Exo)are characterized by easy expansion and storage,low risk of tumor formation,low immunogenicity,and anti-inflammatory effects.The therapeutic effects of MSC-Exo on ischemic stroke have been widely explored.However,the underlying mechanism remains unclear.In this study,we established a mouse model of ischemic brain injury induced by occlusion of the middle cerebral artery using the thread bolt method and injected MSC-Exo into the tail vein.We found that administration of MSC-Exo reduced the volume of cerebral infarction in the ischemic brain injury mouse model,increased the levels of interleukin-33(IL-33)and suppression of tumorigenicity 2 receptor(ST2)in the penumbra of cerebral infarction,and improved neurological function.In vitro results showed that astrocyte-conditioned medium of cells deprived of both oxygen and glucose,to simulate ischemia conditions,combined with MSC-Exo increased the survival rate of primary cortical neurons.However,after transfection by IL-33 siRNA or ST2 siRNA,the survival rate of primary cortical neurons was markedly decreased.These results indicated that MSC-Exo inhibited neuronal death induced by oxygen and glucose deprivation through the IL-33/ST2 signaling pathway in astrocytes.These findings suggest that MSC-Exo may reduce ischemia-induced brain injury through regulating the IL-33/ST2 signaling pathway.Therefore,MSC-Exo may be a potential therapeutic method for ischemic stroke.

Direct remyelinating effect of bone marrow mesenchymal stem cells in the rat model of spinal cord injury

Background:Several studies have tried to evaluate the effects of stem cell therapy for spinal cord injury in animal models and human trials.Bone marrow mesenchymal stem cells are one of the safe and available candidate cells with established beneficial therapeutic effects for bone and cartilage repair.However,there are some doubts regarding their therapeutic efficacy for nervous system disorders.Bone marrow mesenchymal stem cells have been used in some clinical trials for spinal cord injury but their precise mechanism of action and also their safety has not been investigated.Available experimental reports do not provide sufficient and convincing evidence to persuade therapists to perform further clinical trials.In the present study we have used detailed staining methods to clearly show the entire structure of the injured spinal cord and the effects of bone marrow mesenchymal stem cells treatment on this tissue.Methods:Intrathecal injection of neutral red stained bone marrow mesenchymal stem cells were done in a rat model of spinal cord injury and the fate of the transplanted cells,immunologic responses in the host tissue,and tumor generation were assessed by using brightfield and fluorescence microscopy.Sensory-motor functions were also evaluated in the animals.Results:Injected cells could migrate to the site of the injury and differentiate into oligodendrocytes and astrocytes.Immuno-histological assays showed no cells expressing CD2,CD3,CD15 and CD45 in host tissues,and sensory-motor assessments revealed that there were some partial improvements.Conclusion:Present study provides strong evidence that mesenchymal stem cell therapy is safe and could be helpful for the remyelination of injured nerve fibers in the acute phase of injury.

Transplantation of BMP-7 gene-transfected bone marrow mesenchymal stem cells for the treatment of spinal cord injury in rats

Background:Spinal cord injury(SCI)is a serious traumatic disease of the central nervous system,and there is currently no effective treatment for SCI because of its complicated pathophysiology.Bone marrow mesenchymal stem cells(BMSCs)have multidirectional differentiation abilities.Our study aims to explore the effects of bone morphogenetic protein 7(BMP-7)-modified BMSCs transplantation on the repair of SCI in rats.Methods:In this study,a rat spinal cord injury model was established with the modified Allen method.Then,BMSCs transfected with the BMP7 gene were transplanted to treat the spinal cord injury in rats.Forty Sprague-Dawley rats were randomly divided into the sham operation group(sham group),spinal cord injury group(model group),BMSC treatment group(BMSC group)and LV-BMP7-BMSC treatment group(LV-BMP7-BMSC group).The Basso,Beattie,and Bresnahan(BBB)score was used to evaluate the recovery of hindlimb function in the rats.The levels of neurofilament protein NF-200(NF-200)and glial fibrillary acidic protein(GFAP)were detected by immunofluorescence,RT-PCR and Western blotting.Results:At 14 d,21 d,and 28 d after treatment,the BBB score of the rats in the LV-BMP7-BMSC group was higher than that of the rats in the model group and BMSC group.The results showed that NF-200 was expressed at the local spinal cord injury site.Compared with that of the sham group,the NF-200 expression level of the BMSC group and LV-BMP7-BMSC group was increased(P<0.05).The results showed that the mRNA expression levels of NF-200 in the spinal cord tissue of the BMSC group and LV-BMP7-BMSC group were increased compared with those of the sham group(P<0.05).The western blotting results further confirmed the PCR results.Conclusion:BMP-7 gene-modified BMSC transplantation can promote the repair of spinal cord functions after SCI in rats.