MicroRNA changes of bone marrow-derived mesenchymal stem cells differentiated into neuronal-like cells by Schwann cell-conditioned medium

Bone marrow-derived mesenchymal stem cells differentiate into neurons under the induction of Schwann cells. However, key microRNAs and related pathways for differentiation remain unclear. This study screened and identified differentially expressed microRNAs in bone marrow- derived mesenchymal stem cells induced by Schwann cell-conditioned medium, and explored targets and related pathways involved in their differentiation into neuronal-like cells. Primary bone marrow-derived mesenchymal stem cells were isolated from femoral and tibial bones, while primary Schwann cells were isolated from bilateral saphenous nerves. Bone marrow-derived mesenchymal stem cells were cultured in unconditioned (control group) and Schwann cell-conditioned medium (bone marrow-derived mesenchymal stem cell + Schwann cell group). Neuronal differentiation of bone marrow-derived mesenchymal stem cells induced by Schwann cell-conditioned medium was observed by time-lapse imaging. Upon induction, the morphology of bone marrow-derived mesencaymal stem cells changed into a neural shape with neurites. Results of quantitative reverse transcription-polymerase chain reaction revealed that nestin mRNA expression was upregulated from 1 to 3 days and downregulated from 3 to 7 days in the bone marrow-derived mesenchymal stem cell + Schwann cell group. Compared with the control group, microtubule-associated protein 2 mRNA expression gradually increased from 1 to 7 days in the bone marrow-derived mesenchymal stem cell + Schwann cell group. After 7 days of induction, microRNA analysis iden:ified 83 significantly differentially expressed microRNAs between the two groups. Gene Ontology analysis indicated enrichment of microRNA target genes for neuronal projection development, regulation of axonogenesis, and positive regulation of cell proliferation. Kyoto Encyclopedia of Genes and Genomes pathway analysis demonstrated that Hippo, Wnt, transforming growth factor-beta, and Hedgehog signaling pathv/ays were potentially associated with neural differentiation of bone marrow-derived mesenchymal stem cells. This study, which carried out successful microRNA analysis of neuronal-like cells differentiated from bone marrow-derived mesenchymal stem cells by Schwann cell induction, revealed key microRNAs and pathways involved in neural differentiation of bone marrow-derived mesenchymal stem cells. All protocols were approved by the Animal Ethics Committee of Institute of Radiation Medicine, Chinese Academy of Medical Sciences on March 12, 2017 (approval number: DWLI-20170311).

TrkA regulates the regenerative capacity of bone marrow stromal stem cells in nerve grafts

We previously demonstrated that overexpression of tropomyosin receptor kinase A(TrkA)promotes the survival and Schwann celllike differentiation of bone marrow stromal stem cells in nerve grafts,thereby enhancing the regeneration and functional recovery of the peripheral nerve.In the present study,we investigated the molecular mechanisms underlying the neuroprotective effects of TrkA in bone marrow stromal stem cells seeded into nerve grafts.Bone marrow stromal stem cells from Sprague-Dawley rats were infected with recombinant lentivirus vector expressing rat TrkA,TrkA-shRNA or the respective control.The cells were then seeded into allogeneic rat acellular nerve allografts for bridging a 1-cm right sciatic nerve defect.Then,8 weeks after surgery,hematoxylin and eosin staining showed that compared with the control groups,the cells and fibers in the TrkA overexpressing group were more densely and uniformly arranged,whereas they were relatively sparse and arranged in a disordered manner in the TrkA-shRNA group.Western blot assay showed that compared with the control groups,the TrkA overexpressing group had higher expression of the myelin marker,myelin basic protein and the axonal marker neurofilament 200.The TrkA overexpressing group also had higher levels of various signaling molecules,including TrkA,pTrkA(Tyr490),extracellular signal-regulated kinases 1/2(Erkl/2),pErk1/2(Thr202/Tyr204),and the anti-apoptotic proteins Bcl-2 and Bcl-xL.In contrast,these proteins were downregulated,while the pro-apoptotic factors Bax and Bad were upregulated,in the TrkA-shRNA group.The levels of the TrkA effectors Akt and pAkt(Ser473)were not different among the groups.These results suggest that TrkA enhances the survival and regenerative capacity of bone marrow stromal stem cells through upregulation of the Erk/Bcl-2 pathway.All procedures were approved by the Animal Ethical and Welfare Committee of Shenzhen University,China in December 2014(approval No.AEWC-2014-001219).

Hypoxia-preconditioned bone marrow-derived mesenchymal stem cells protect neurons from cardiac arrest-induced pyroptosis

Cardiac arrest can lead to severe neurological impairment as a result of inflammation,mitochondrial dysfunction,and post-cardiopulmonary resuscitation neurological damage.Hypoxic preconditioning has been shown to improve migration and survival of bone marrow–derived mesenchymal stem cells and reduce pyroptosis after cardiac arrest,but the specific mechanisms by which hypoxia-preconditioned bone marrow–derived mesenchymal stem cells protect against brain injury after cardiac arrest are unknown.To this end,we established an in vitro co-culture model of bone marrow–derived mesenchymal stem cells and oxygen–glucose deprived primary neurons and found that hypoxic preconditioning enhanced the protective effect of bone marrow stromal stem cells against neuronal pyroptosis,possibly through inhibition of the MAPK and nuclear factor κB pathways.Subsequently,we transplanted hypoxia-preconditioned bone marrow–derived mesenchymal stem cells into the lateral ventricle after the return of spontaneous circulation in an 8-minute cardiac arrest rat model induced by asphyxia.The results showed that hypoxia-preconditioned bone marrow–derived mesenchymal stem cells significantly reduced cardiac arrest–induced neuronal pyroptosis,oxidative stress,and mitochondrial damage,whereas knockdown of the liver isoform of phosphofructokinase in bone marrow–derived mesenchymal stem cells inhibited these effects.To conclude,hypoxia-preconditioned bone marrow–derived mesenchymal stem cells offer a promising therapeutic approach for neuronal injury following cardiac arrest,and their beneficial effects are potentially associated with increased expression of the liver isoform of phosphofructokinase following hypoxic preconditioning.

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.

Mesenchymal Stromal Cells Derived from Human Embryonic Stem Cells, Fetal Limb and Bone Marrow Share a Common Phenotype but Are Transcriptionally and Biologically Different

Mesenchymal stromal cells (MSCs) can be obtained from several sources and the significant differences in their properties make it crucial to investigate the differentiation potential of MSCs from different sources to determine the optimal source of MSCs. We investigated if this biological heterogeneity in MSCs from different sources results in different mechanisms for their differentiation. In this study, we compared the gene expression patterns of phenotypically defined MSCs derived from three ontogenically different sources: Embryonic stem cells (hES-MSCs), Fetal limb (Flb-MSCs) and Bone Marrow (BM-MSCs). Differentially expressed genes between differentiated cells and undifferentiated controls were compared across the three MSC sources. We found minimal overlap (5% - 16%) in differentially expressed gene sets among the three sources. Flb-MSCs were similar to BM-MSCs based on differential gene expression patterns. Pathway analysis of the differentially expressed genes using Ingenuity Pathway Analysis (IPA) revealed a large variation in the canonical pathways leading to MSC differentiation. The similar canonical pathways among the three sources were lineage specific. The Flb-MSCs showed maximum overlap of canonical pathways with the BM-MSCs, indicating that the Flb-MSCs are an intermediate source between the less specialised hES-MSC source and the more specialised BM-MSC source. The source specific pathways prove that MSCs from the three ontogenically different sources use different biological pathways to obtain similar differentiation outcomes. Thus our study advocates the understanding of biological pathways to obtain optimal sources of MSCs for various clinical applications.

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.

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.

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.

Exosomal miR-23b from bone marrow mesenchymal stem cells alleviates oxidative stress and pyroptosis after intracerebral hemorrhage

Our previous studies showed that miR-23b was downregulated in patients with intracerebral hemorrhage(ICH). This indicates that miR-23b may be closely related to the patho-physiological mechanism of ICH, but this hypothesis lacks direct evidence. In this study, we established rat models of ICH by injecting collagenase Ⅶ into the right basal ganglia and treating them with an injection of bone marrow mesenchymal stem cell(BMSC)-derived exosomal miR-23b via the tail vein. We found that edema in the rat brain was markedly reduced and rat behaviors were improved after BMSC exosomal miR-23b injection compared with those in the ICH groups. Additionally, exosomal miR-23b was transported to the microglia/macrophages, thereby reducing oxidative stress and pyroptosis after ICH. We also used hemin to mimic ICH conditions in vitro. We found that phosphatase and tensin homolog deleted on chromosome 10(PTEN) was the downstream target gene of miR-23b, and exosomal miR-23b exhibited antioxidant effects by regulating the PTEN/Nrf2 pathway. Moreover, miR-23b reduced PTEN binding to NOD-like receptor family pyrin domain containing 3(NLRP3) and NLRP3 inflammasome activation, thereby decreasing the NLRP3-dependent pyroptosis level. These findings suggest that BMSC-derived exosomal miR-23b exhibits antioxidant effects through inhibiting PTEN and alleviating NLRP3 inflammasome-mediated pyroptosis, thereby promoting neurologic function recovery in rats with ICH.

Collagen-chitosan scaffold impregnated with bone marrow mesenchymal stem cells for treatment of traumatic brain injury

Combinations of biomaterials and cells can effectively target delivery of cells or other therapeutic factors to the brain to rebuild damaged nerve pathways after brain injury.Porous collagen-chitosan scaffolds were prepared by a freeze-drying method based on brain tissue engineering.The scaffolds were impregnated with rat bone marrow mesenchymal stem cells.A traumatic brain injury rat model was established using the 300 g weight free fall impact method.Bone marrow mesenchymal stem cells/collagen-chitosan scaffolds were implanted into the injured brain.Modified neurological severity scores were used to assess the recovery of neurological function.The Morris water maze was employed to determine spatial learning and memory abilities.Hematoxylin-eosin staining was performed to measure pathological changes in brain tissue.Immunohistochemistry was performed for vascular endothelial growth factor and for 5-bromo-2-deoxyuridine(BrdU)/neuron specific enolase and BrdU/glial fibrillary acidic protein.Our results demonstrated that the transplantation of bone marrow mesenchymal stem cells and collagen-chitosan scaffolds to traumatic brain injury rats remarkably reduced modified neurological severity scores,shortened the average latency of the Morris water maze,increased the number of platform crossings,diminished the degeneration of damaged brain tissue,and increased the positive reaction of vascular endothelial growth factor in the transplantation and surrounding areas.At 14 days after transplantation,increased BrdU/glial fibrillary acidic protein expression and decreased BrdU/neuron specific enolase expression were observed in bone marrow mesenchymal stem cells in the injured area.The therapeutic effect of bone marrow mesenchymal stem cells and collagen-chitosan scaffolds was superior to stereotactic injection of bone marrow mesenchymal stem cells alone.To test the biocompatibility and immunogenicity of bone marrow mesenchymal stem cells and collagen-chitosan scaffolds,immunosuppressive cyclosporine was intravenously injected 12 hours before transplantation and 1-5 days after transplantation.The above indicators were similar to those of rats treated with bone marrow mesenchymal stem cells and collagen-chitosan scaffolds only.These findings indicate that transplantation of bone marrow mesenchymal stem cells in a collagen-chitosan scaffold can promote the recovery of neuropathological injury in rats with traumatic brain injury.This approach has the potential to be developed as a treatment for traumatic brain injury in humans.All experimental procedures were approved by the Institutional Animal Investigation Committee of Capital Medical University,China(approval No.AEEI-2015-035)in December 2015.

Hydrogel loaded with bone marrow stromal cell-derived exosomes promotes bone regeneration by inhibiting inflammatory responses and angiogenesis

BACKGROUND Bone healing is a complex process involving early inflammatory immune regu-lation,angiogenesis,osteogenic differentiation,and biomineralization.Fracture repair poses challenges for orthopedic surgeons,necessitating the search for efficient healing methods.AIM To investigate the underlying mechanism by which hydrogel-loaded exosomes derived from bone marrow mesenchymal stem cells(BMSCs)facilitate the process of fracture healing.METHODS Hydrogels and loaded BMSC-derived exosome(BMSC-exo)gels were charac-terized to validate their properties.In vitro evaluations were conducted to assess the impact of hydrogels on various stages of the healing process.Hydrogels could recruit macrophages and inhibit inflammatory responses,enhance of human umbilical vein endothelial cell angiogenesis,and promote the osteogenic differen-tiation of primary cranial osteoblasts.Furthermore,the effect of hydrogel on fracture healing was confirmed using a mouse fracture model.RESULTS The hydrogel effectively attenuated the inflammatory response during the initial repair stage and subsequently facilitated vascular migration,promoted the formation of large vessels,and enabled functional vascularization during bone repair.These effects were further validated in fracture models.CONCLUSION We successfully fabricated a hydrogel loaded with BMSC-exo that modulates macrophage polarization and angiogenesis to influence bone regeneration.

Effects of interleukin-10 treated macrophages on bone marrow mesenchymal stem cells via signal transducer and activator of transcription 3 pathway

BACKGROUND Alveolar bone defects caused by inflammation are an urgent issue in oral implant surgery that must be solved.Regulating the various phenotypes of macrophages to enhance the inflammatory environment can significantly affect the progression of diseases and tissue engineering repair process.AIM To assess the influence of interleukin-10(IL-10)on the osteogenic differentiation of bone marrow mesenchymal stem cells(BMSCs)following their interaction with macrophages in an inflammatory environment.METHODS IL-10 modulates the differentiation of peritoneal macrophages in Wistar rats in an inflammatory environment.In this study,we investigated its impact on the proliferation,migration,and osteogenesis of BMSCs.The expression levels of signal transducer and activator of transcription 3(STAT3)and its activated form,phos-phorylated-STAT3,were examined in IL-10-stimulated macrophages.Subsequently,a specific STAT3 signaling inhibitor was used to impede STAT3 signal activation to further investigate the role of STAT3 signaling.RESULTS IL-10-stimulated macrophages underwent polarization to the M2 type through substitution,and these M2 macrophages actively facilitated the osteogenic differentiation of BMSCs.Mechanistically,STAT3 signaling plays a crucial role in the process by which IL-10 influences macrophages.Specifically,IL-10 stimulated the activation of the STAT3 signaling pathway and reduced the macrophage inflammatory response,as evidenced by its diminished impact on the osteogenic differentiation of BMSCs.CONCLUSION Stimulating macrophages with IL-10 proved effective in improving the inflammatory environment and promoting the osteogenic differentiation of BMSCs.The IL-10/STAT3 signaling pathway has emerged as a key regulator in the macrophage-mediated control of BMSCs’osteogenic differentiation.

Neuronal-like cell differentiation of non-adherent bone marrow cell-derived mesenchymal stem cells

Non-adherent bone marrow cell-derived mesenchymal stem cells from C57BL/6J mice were sepa- rated and cultured using the ＂pour-off＂ method. Non-adherent bone marrow cell-derived mesen- chymal stem ceils developed colony-forming unit-fibroblasts, and could be expanded by supple- mentation with epidermal growth factor. Immunocytochemistry showed that the non-adherent bone marrow cell-derived mesenchymal stem cells exposed to basic fibroblast growth factor/epidermal growth factor/nerve growth factor expressed the neuron specific markers, neurofilament-200 and NeuN, in vitro. Non-adherent bone marrow cell-derived mesenchymal stem cells from 13-galactosidase transgenic mice were also transplanted into focal ischemic brain （right corpus striatum） of C57BL/6J mice. At 8 weeks, cells positive for LacZ and 13-galactosidase staining were observed in the ischemic tissues, and cells co-labeled with both 13-galactosidase and NeuN were seen by double immunohistochemical staining. These findings suggest that the non-adherent bone marrow cell-derived mesenchymal stem cells could differentiate into neuronal-like cells in vitro and in vivo.

Advances and perspectives on cellular therapy in acquired bone marrow failure diseases

Acquired bone marrow failure diseases(ABMFD) are a class of hematopoietic stem cell diseases with a commonality of non-inherited disruption of hematopoiesis that results in pancytopenia. ABMFDs also are a group of heterogeneous diseases with different etiologies and treatment options. The three most common ABMFDs are aplastic anemia, myelodysplastic syndrome, and paroxysmal nocturnal hemoglobinuria. Stem cell transplantation is the only treatment that can cure these diseases. However, due to high therapy-related mortality, stem cell transplantation has rarely been used as a first line treatment in treating ABMFD. With the advance of personalized medicine and precision medicine, various novel cellular therapy strategies are in trial to increase the efficiency and efficacy of ABMFD treatment. This article aims to review current available stem cell transplantation protocols and promising cellular therapy research in treating ABMFD.

Reduced graphene oxide-embedded nerve conduits loaded with bone marrow mesenchymal stem cell-derived extracellular vesicles promote peripheral nerve regeneration

We previously combined reduced graphene oxide(rGO)with gelatin-methacryloyl(GelMA)and polycaprolactone(PCL)to create an rGO-GelMA-PCL nerve conduit and found that the conductivity and biocompatibility were improved.However,the rGO-GelMA-PCL nerve conduits differed greatly from autologous nerve transplants in their ability to promote the regeneration of injured peripheral nerves and axonal sprouting.Extracellular vesicles derived from bone marrow mesenchymal stem cells(BMSCs)can be loaded into rGO-GelMA-PCL nerve conduits for repair of rat sciatic nerve injury because they can promote angiogenesis at the injured site.In this study,12 weeks after surgery,sciatic nerve function was measured by electrophysiology and sciatic nerve function index,and myelin sheath and axon regeneration were observed by electron microscopy,immunohistochemistry,and immunofluorescence.The regeneration of microvessel was observed by immunofluorescence.Our results showed that rGO-GelMA-PCL nerve conduits loaded with BMSC-derived extracellular vesicles were superior to rGO-GelMA-PCL conduits alone in their ability to increase the number of newly formed vessels and axonal sprouts at the injury site as well as the recovery of neurological function.These findings indicate that rGO-GelMA-PCL nerve conduits loaded with BMSC-derived extracellular vesicles can promote peripheral nerve regeneration and neurological function recovery,and provide a new direction for the curation of peripheral nerve defect in the clinic.

Migration of bone marrow progenitor cells in the adult brain of rats and rabbits

Neurogenesis takes place in the adult mammalian brain in three areas:Subgranular zone of the dentate gyrus(DG);subventricular zone of the lateral ventricle;olfactory bulb.Different molecular markers can be used to characterizethe cells involved in adult neurogenesis.It has been recently suggested that a population of bone marrow(BM)progenitor cells may migrate to the brain and differentiate into neuronal lineage.To explore this hypothesis,we injected recombinant SV40-derived vectors into the BM and followed the potential migration of the transduced cells.Long-term BM-directed gene transfer using recombinant SV40-derived vectors leads to expression of the genes delivered to the BM firstly in circulating cells,then after several months in mature neurons and microglial cells,and thus without central nervous system(CNS)lesion.Most of transgene-expressing cells expressed NeuN,a marker of mature neurons.Thus,BM-derived cells may function as progenitors of CNS cells in adult animals.The mechanism by which the cells from the BM come to be neurons remains to be determined.Although the observed gradual increase in transgene-expressing neurons over 16mo suggests that the pathway involved differentiation of BM-resident cells into neurons,cell fusion as the principal route cannot be totally ruled out.Additional studies using similar viral vectors showed that BM-derived progenitor cells migrating in the CNS express markers of neuronal precursors or immature neurons.Transgene-positive cells were found in the subgranular zone of the DG of the hippocampus 16 mo after intramarrow injection of the vector.In addition to cells expressing markers of mature neurons,transgene-positive cells were also positive for nestin and doublecortin,molecules expressed by developing neuronal cells.These cells were actively proliferating,as shown by short term BrdU incorporation studies.Inducing seizures by using kainic acid increased the number of BM progenitor cells transduced by SV40vectors migrating to the hippocampus,and these cells were seen at earlier time points in the DG.We show that the cell membrane chemokine receptor,CCR5,and its ligands,enhance CNS inflammation and seizure activity in a model of neuronal excitotoxicity.SV40-based gene delivery of RNAi targeting CCR5 to the BM results in downregulating CCR5 in circulating cells,suggesting that CCR5 plays an important role in regulating traffic of BM-derived cells into the CNS,both in the basal state and in response to injury.Furthermore,reduction in CCR5 expression incirculating cells provides profound neuroprotection from excitotoxic neuronal injury,reduces neuroinflammation,and increases neuronal regeneration following this type of insult.These results suggest that BM-derived,transgeneexpressing,cells can migrate to the brain and that they become neurons,at least in part,by differentiating into neuron precursors and subsequently developing into mature neurons.

Overexpression of vascular endothelial growth factor enhances the neuroprotective effects of bone marrow mesenchymal stem cell transplantation in ischemic stroke

Although bone marrow mesenchymal stem cells(BMSCs)might have therapeutic potency in ischemic stroke,the benefits are limited.The current study investigated the effects of BMSCs engineered to overexpress vascular endothelial growth factor(VEGF)on behavioral defects in a rat model of transient cerebral ischemia,which was induced by middle cerebral artery occlusion.VEGF-BMSCs or control grafts were injected into the left striatum of the infarcted hemisphere 24 hours after stroke.We found that compared with the stroke-only group and the vehicle-and BMSCs-control groups,the VEGF-BMSCs treated animals displayed the largest benefits,as evidenced by attenuated behavioral defects and smaller infarct volume 7 days after stroke.Additionally,VEGF-BMSCs greatly inhibited destruction of the blood-brain barrier,increased the regeneration of blood vessels in the region of ischemic penumbra,and reducedneuronal degeneration surrounding the infarct core.Further mechanistic studies showed that among all transplant groups,VEGF-BMSCs transplantation induced the highest level of brain-derived neurotrophic factor.These results suggest that BMSCs transplantation with vascular endothelial growth factor has the potential to treat ischemic stroke with better results than are currently available.

Dorsal root ganglion neurons promote proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells

Preliminary animal experiments have confirmed that sensory nerve fibers promote osteoblast differentiation, but motor nerve fibers have no promotion effect. Whether sensory neurons pro- mote the proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells remains unclear. No results at the cellular level have been reported. In this study, dorsal root ganglion neurons （sensory neurons） from Sprague-Dawley fetal rats were co-cultured with bone marrow mesenchymal stem cells transfected with green fluorescent protein 3 weeks after osteo- genic differentiation in vitro, while osteoblasts derived from bone marrow mesenchymal stem cells served as the control group. The rat dorsal root ganglion neurons promoted the prolifera- tion of bone marrow mesenchymal stem cell-derived osteoblasts at B and 5 days of co-culture, as observed by fluorescence microscopy. The levels of mRNAs for osteogenic differentiation-re- lated factors （including alkaline phosphatase, osteocalcin, osteopontin and bone morphogenetic protein 2） in the co-culture group were higher than those in the control group, as detected by real-time quantitative PCR. Our findings indicate that dorsal root ganglion neurons promote the proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells, which pro- vides a theoretical basis for in vitro experiments aimed at constructing tissue-engineered bone.

Gender difference in the neuroprotective effect of rat bone marrow mesenchymal cells against hypoxiainduced apoptosis of retinal ganglion cells

Bone marrow mesenchymal stem cells can reduce retinal ganglion cell death and effectively prevent vision loss. Previously, we found that during differentiation, female rhesus monkey bone marrow mesenchymal stem cells acquire a higher neurogenic potential compared with male rhesus monkey bone marrow mesenchymal stem cells. This suggests that female bone marrow mesenchymal stem cells have a stronger neuroprotective effect than male bone marrow mesenchymal stem cells. Here, we first isolated and cultured bone marrow mesenchymal stem cells from female and male rats by density gradient centrifugation. Retinal tissue from newborn rats was prepared by enzymatic digestion to obtain primary retinal ganglion cells. Using the transwell system, retinal ganglion cells were co-cultured with bone marrow mesenchymal stem cells under hypoxia. Cell apoptosis was detected by flow cytometry and caspase-3 activity assay. We found a marked increase in apoptotic rate and caspase-3 activity of retinal ganglion cells after 24 hours of hypoxia compared with normoxia. Moreover, apoptotic rate and caspase-3 activity of retinal ganglion cells significantly decreased with both female and male bone marrow mesenchymal stem cell co-culture under hypoxia compared with culture alone, with more significant effects from female bone marrow mesenchymal stem cells. Our results indicate that bone marrow mesenchymal stem cells exert a neuroprotective effect against hypoxia-induced apoptosis of retinal ganglion cells, and also that female cells have greater neuroprotective ability compared with male cells.

O-linkedβ-N-acetylglucosaminylation may be a key regulatory factor in promoting osteogenic differentiation of bone marrow mesenchymal stromal cells

Cumulative evidence suggests that O-linkedβ-N-acetylglucosaminylation(OGlcNAcylation)plays an important regulatory role in pathophysiological processes.Although the regulatory mechanisms of O-GlcNAcylation in tumors have been gradually elucidated,the potential mechanisms of O-GlcNAcylation in bone metabolism,particularly,in the osteogenic differentiation of bone marrow mesenchymal stromal cells(BMSCs)remains unexplored.In this study,the literature related to O-GlcNAcylation and BMSC osteogenic differentiation was reviewed,assuming that it could trigger more scholars to focus on research related to OGlcNAcylation and bone metabolism and provide insights into the development of novel therapeutic targets for bone metabolism disorders such as osteoporosis.