Bridging sciatic nerve gap using tissue-engineered nerves constructed with neural tissue-committed stem cells derived from bone marrow

BACKGROUND： Schwann cells are the most commonly used cells for tissue-engineered nerves. However, autologous Schwann cells are of limited use in a clinical context, and allogeneic Schwann cells induce immunological rejections. Cells that do not induce immunological rejections and that are relatively easy to acquire are urgently needed for transplantation. OBJECTIVE： To bridge sciatic nerve defects using tissue engineered nerves constructed with neural tissue-committed stem cells （NTCSCs） derived from bone marrow; to observe morphology and function of rat nerves following bridging; to determine the effect of autologous nerve transplantation, which serves as the gold standard for evaluating efficacy of tissue-engineered nerves. DESIGN, TIME AND SETTING： This randomized, controlled, animal experiment was performed in the Anatomical Laboratory and Biomedical Institute of the Second Military Medical University of Chinese PLA between September 2004 and April 2006. MATERIALS： Five Sprague Dawley rats, aged 1 month and weighing 100-150 g, were used for cell culture. Sixty Sprague Dawley rats aged 3 months and weighing 220-250 g, were used to establish neurological defect models. Nestin, neuron-specific enolase （NSE）, glial fibrillary acidic protein （GFAP）, and S-100 antibodies were provided by Santa Cruz Biotechnology, Inc., USA. Acellular nerve grafts were derived from dogs. METHODS： All rats, each with 1-cm gap created in the right sciatic nerve, were randomly assigned to three groups. Each group comprised 20 rats. Autograft nerve transplantation group： the severed 1-cm length nerve segment was reverted, but with the two ends exchanged; the proximal segment was sutured to the distal sciatic nerve stump and the distal segment to the proximal stump. Blank nerve scaffold transplantation group： a 1-cm length acellular nerve graft was used to bridge the sciatic nerve gap. NTCSC engineered nerve transplantation group： a 1-cm length acellular nerve graft, in which NTCSCs were inoculated, was used to bridge the sciatic nerve gap. MAIN OUTCOME MEASURES： Following surgery, sciatic nerve functional index and electrophysiology functions were evaluated for nerve conduction function, including conduction latency, conduction velocity, and action potential peak. Horseradish peroxidase （HRP, 20%） was injected into the gastrocnemius muscle to retrogradely label the 1-4 and L5 nerve ganglions, as well as neurons in the anterior horn of the spinal cord, in the three groups. Positive expression of nestin, NSE, GFAP, and S-100 were determined using an immunofluorescence double-labeling method. RESULTS： NTCSCs differentiated into neuronal-like cells and glial-like cells within 12 weeks after NTCSC engineered nerve transplantation. HRP retrograde tracing displayed a large amount of HRP-labeled neurons in I-45 nerve ganglions, as well as the anterior horn of the spinal cord, in both the autograft nerve transplantation and the NTCSC engineered nerve transplantation groups. However, few HRP-labeled neurons were detected in the blank nerve scaffold transplantation group. Nerve bridges in the autograft nerve transplantation and NTCSC engineered nerve transplantation groups exhibited similar morphology to normal nerves. Neither fractures or broken nerve bridges nor neuromas were found after bridging the sciatic nerve gap with NTCSCs-inoculated acellular nerve graft, indicating repair. Conduction latency, action potential, and conduction velocity in the NTCSC engineered nerve transplantation group were identical to the autograft nerve transplantation group （P 〉 0.05）, but significantly different from the blank nerve scaffold transplantation group （P 〈 0.05）. CONCLUSION＂ NTCSC tissue-engineered nerves were able to repair injured nerves and facilitated restoration of nerve conduction function, similar to autograft nerve transplantation. ＂

Autophagy in neural stem cells and glia for brain health and diseases

Autophagy is a multifaceted cellular process that not only maintains the homeostatic and adaptive responses of the brain but is also dynamically involved in the regulation of neural cell generation,maturation,and survival.Autophagy facilities the utilization of energy and the microenvironment for developing neural stem cells.Autophagy arbitrates structural and functional remodeling during the cell differentiation process.Autophagy also plays an indispensable role in the maintenance of stemness and homeostasis in neural stem cells during essential brain physiology and also in the instigation and progression of diseases.Only recently,studies have begun to shed light on autophagy regulation in glia(microglia,astrocyte,and oligodendrocyte)in the brain.Glial cells have attained relatively less consideration despite their unquestioned influence on various aspects of neural development,synaptic function,brain metabolism,cellular debris clearing,and restoration of damaged or injured tissues.Thus,this review composes pertinent information regarding the involvement of autophagy in neural stem cells and glial regulation and the role of this connexion in normal brain functions,neurodevelopmental disorders,and neurodegenerative diseases.This review will provide insight into establishing a concrete strategic approach for investigating pathological mechanisms and developing therapies for brain diseases.

Neural stem cells promote neuroplasticity: a promising therapeutic strategy for the treatment of Alzheimer’s disease

Recent studies have demonstrated that neuroplasticity,such as synaptic plasticity and neurogenesis,exists throughout the normal lifespan but declines with age and is significantly impaired in individuals with Alzheimer’s disease.Hence,promoting neuroplasticity may represent an effective strategy with which Alzheimer’s disease can be alleviated.Due to their significant ability to self-renew,differentiate,and migrate,neural stem cells play an essential role in reversing synaptic and neuronal damage,reducing the pathology of Alzheimer’s disease,including amyloid-β,tau protein,and neuroinflammation,and secreting neurotrophic factors and growth factors that are related to plasticity.These events can promote synaptic plasticity and neurogenesis to repair the microenvironment of the mammalian brain.Consequently,neural stem cells are considered to represent a potential regenerative therapy with which to improve Alzheimer’s disease and other neurodegenerative diseases.In this review,we discuss how neural stem cells regulate neuroplasticity and optimize their effects to enhance their potential for treating Alzheimer’s disease in the clinic.

Long non-coding RNA H19 regulates neurogenesis of induced neural stem cells in a mouse model of closed head injury

Stem cell-based therapies have been proposed as a potential treatment for neural regeneration following closed head injury.We previously reported that induced neural stem cells exert beneficial effects on neural regeneration via cell replacement.However,the neural regeneration efficiency of induced neural stem cells remains limited.In this study,we explored differentially expressed genes and long non-coding RNAs to clarify the mechanism underlying the neurogenesis of induced neural stem cells.We found that H19 was the most downregulated neurogenesis-associated lnc RNA in induced neural stem cells compared with induced pluripotent stem cells.Additionally,we demonstrated that H19 levels in induced neural stem cells were markedly lower than those in induced pluripotent stem cells and were substantially higher than those in induced neural stem cell-derived neurons.We predicted the target genes of H19 and discovered that H19 directly interacts with mi R-325-3p,which directly interacts with Ctbp2 in induced pluripotent stem cells and induced neural stem cells.Silencing H19 or Ctbp2 impaired induced neural stem cell proliferation,and mi R-325-3p suppression restored the effect of H19 inhibition but not the effect of Ctbp2 inhibition.Furthermore,H19 silencing substantially promoted the neural differentiation of induced neural stem cells and did not induce apoptosis of induced neural stem cells.Notably,silencing H19 in induced neural stem cell grafts markedly accelerated the neurological recovery of closed head injury mice.Our results reveal that H19 regulates the neurogenesis of induced neural stem cells.H19 inhibition may promote the neural differentiation of induced neural stem cells,which is closely associated with neurological recovery following closed head injury.

Transplantation of fibrin-thrombin encapsulated human induced neural stem cells promotes functional recovery of spinal cord injury rats through modulation of the microenvironment

Recent studies have mostly focused on engraftment of cells at the lesioned spinal cord,with the expectation that differentiated neurons facilitate recovery.Only a few studies have attempted to use transplanted cells and/or biomaterials as major modulators of the spinal cord injury microenvironment.Here,we aimed to investigate the role of microenvironment modulation by cell graft on functional recovery after spinal cord injury.Induced neural stem cells reprogrammed from human peripheral blood mononuclear cells,and/or thrombin plus fibrinogen,were transplanted into the lesion site of an immunosuppressed rat spinal cord injury model.Basso,Beattie and Bresnahan score,electrophysiological function,and immunofluorescence/histological analyses showed that transplantation facilitates motor and electrophysiological function,reduces lesion volume,and promotes axonal neurofilament expression at the lesion core.Examination of the graft and niche components revealed that although the graft only survived for a relatively short period(up to 15 days),it still had a crucial impact on the microenvironment.Altogether,induced neural stem cells and human fibrin reduced the number of infiltrated immune cells,biased microglia towards a regenerative M2 phenotype,and changed the cytokine expression profile at the lesion site.Graft-induced changes of the microenvironment during the acute and subacute stages might have disrupted the inflammatory cascade chain reactions,which may have exerted a long-term impact on the functional recovery of spinal cord injury rats.

Metabolic and proteostatic differences in quiescent and active neural stem cells

Adult neural stem cells are neurogenesis progenitor cells that play an important role in neurogenesis.Therefore,neural regeneration may be a promising target for treatment of many neurological illnesses.The regenerative capacity of adult neural stem cells can be chara cterized by two states:quiescent and active.Quiescent adult neural stem cells are more stable and guarantee the quantity and quality of the adult neural stem cell pool.Active adult neural stem cells are chara cterized by rapid proliferation and differentiation into neurons which allow for integration into neural circuits.This review focuses on diffe rences between quiescent and active adult neural stem cells in nutrition metabolism and protein homeostasis.Furthermore,we discuss the physiological significance and underlying advantages of these diffe rences.Due to the limited number of adult neural stem cells studies,we refe rred to studies of embryonic adult neural stem cells or non-mammalian adult neural stem cells to evaluate specific mechanisms.

Ethanol changes Nestin-promoter induced neural stem cells to disturb newborn dendritic spine remodeling in the hippocampus of mice

Adolescent binge drinking leads to long-lasting disorders of the adult central nervous system,particularly aberrant hippocampal neurogenesis.In this study,we applied in vivo fluorescent tracing using NestinCreERT2::Rosa26-tdTomato mice and analyzed the endogenous neurogenesis lineage progression of neural stem cells(NSCs)and dendritic spine formation of newborn neurons in the subgranular zone of the dentate gyrus.We found abnormal orientation of tamoxifen-induced tdTomato+(tdTom^(+))NSCs in adult mice 2 months after treatment with EtOH(5.0 g/kg,i.p.)for 7 consecutive days.EtOH markedly inhibited tdTom^(+)NSCs activation and hippocampal neurogenesis in mouse dentate gyrus from adolescence to adulthood.EtOH(100 mM)also significantly inhibited the proliferation to 39.2%and differentiation of primary NSCs in vitro.Adult mice exposed to EtOH also exhibited marked inhibitions in dendritic spine growth and newborn neuron maturation in the dentate gyrus,which was partially reversed by voluntary running or inhibition of the mammalian target of rapamycinenhancer of zeste homolog 2 pathway.In vivo tracing revealed that EtOH induced abnormal orientation of tdTom+NSCs and spatial misposition defects of newborn neurons,thus causing the disturbance of hippocampal neurogenesis and dendritic spine remodeling in mice.

Neural stem cell-derived exosomes regulate cell proliferation,migration,and cell death of brain microvascular endothelial cells via the miR-9/Hes1 axis under hypoxia

Background:Our previous study found that mouse embryonic neural stem cell(NSC)-derived exosomes(EXOs)regulated NSC differentiation via the miR-9/Hes1 axis.However,the effects of EXOs on brain microvascular endothelial cell(BMEC)dysfunction via the miR-9/Hes1 axis remain unknown.Therefore,the current study aimed to determine the effects of EXOs on BMEC proliferation,migration,and death via the miR-9/Hes1 axis.Methods:Immunofluorescence,quantitative real-time polymerase chain reaction,cell counting kit-8 assay,wound healing assay,calcein-acetoxymethyl/propidium iodide staining,and hematoxylin and eosin staining were used to determine the role and mechanism of EXOs on BMECs.Results:EXOs promoted BMEC proliferation and migration and reduced cell death under hypoxic conditions.The overexpression of miR-9 promoted BMEC prolifera-tion and migration and reduced cell death under hypoxic conditions.Moreover,miR-9 downregulation inhibited BMEC proliferation and migration and also promoted cell death.Hes1 silencing ameliorated the effect of amtagomiR-9 on BMEC proliferation and migration and cell death.Hyperemic structures were observed in the regions of the hippocampus and cortex in hypoxia-induced mice.Meanwhile,EXO treatment improved cerebrovascular alterations.Conclusion:NSC-derived EXOs can promote BMEC proliferation and migra-tion and reduce cell death via the miR-9/Hes1 axis under hypoxic conditions.Therefore,EXO therapeutic strategies could be considered for hypoxia-induced vascular injury.

Neurotrophins and neural stem cells in posttraumatic brain injury repair

Traumatic brain injury(TBI)is the main cause of disability,mental health disorder,and even death,with its incidence and social costs rising steadily.Although different treatment strategies have been developed and tested to mitigate neurological decline,a definitive cure for these conditions remains elusive.Studies have revealed that vari-ous neurotrophins represented by the brain-derived neurotrophic factor are the key regulators of neuroinflammation,apoptosis,blood-brain barrier permeability,neurite regeneration,and memory function.These factors are instrumental in alleviating neu-roinflammation and promoting neuroregeneration.In addition,neural stem cells(NSC)contribute to nerve repair through inherent neuroprotective and immunomodulatory properties,the release of neurotrophins,the activation of endogenous NSCs,and in-tercellular signaling.Notably,innovative research proposals are emerging to combine BDNF and NSCs,enabling them to synergistically complement and promote each other in facilitating injury repair and improving neuron differentiation after TBI.In this review,we summarize the mechanism of neurotrophins in promoting neurogen-esis and restoring neural function after TBI,comprehensively explore the potential therapeutic effects of various neurotrophins in basic research on TBI,and investigate their interaction with NSCs.This endeavor aims to provide a valuable insight into the clinical treatment and transformation of neurotrophins in TBI,thereby promoting the progress of TBI therapeutics.

Induced neural stem cells regulate microglial activation through Akt-mediated upregulation of CXCR4 and Crry in a mouse model of closed head injury

Microglial activation that occurs rapidly after closed head injury may play important and complex roles in neuroinflammation-associated neuronal damage and repair.We previously reported that induced neural stem cells can modulate the behavior of activated microglia via CXCL12/CXCR4 signaling,influencing their activation such that they can promote neurological recovery.However,the mechanism of CXCR4 upregulation in induced neural stem cells remains unclear.In this study,we found that nuclear factor-κB activation induced by closed head injury mouse serum in microglia promoted CXCL12 and tumor necrosis factor-αexpression but suppressed insulin-like growth factor-1 expression.However,recombinant complement receptor 2-conjugated Crry(CR2-Crry)reduced the effects of closed head injury mouse serum-induced nuclear factor-κB activation in microglia and the levels of activated microglia,CXCL12,and tumor necrosis factor-α.Additionally,we observed that,in response to stimulation(including stimulation by CXCL12 secreted by activated microglia),CXCR4 and Crry levels can be upregulated in induced neural stem cells via the interplay among CXCL12/CXCR4,Crry,and Akt signaling to modulate microglial activation.In agreement with these in vitro experimental results,we found that Akt activation enhanced the immunoregulatory effects of induced neural stem cell grafts on microglial activation,leading to the promotion of neurological recovery via insulin-like growth factor-1 secretion and the neuroprotective effects of induced neural stem cell grafts through CXCR4 and Crry upregulation in the injured cortices of closed head injury mice.Notably,these beneficial effects of Akt activation in induced neural stem cells were positively correlated with the therapeutic effects of induced neural stem cells on neuronal injury,cerebral edema,and neurological disorders post–closed head injury.In conclusion,our findings reveal that Akt activation may enhance the immunoregulatory effects of induced neural stem cells on microglial activation via upregulation of CXCR4 and Crry,thereby promoting induced neural stem cell–mediated improvement of neuronal injury,cerebral edema,and neurological disorders following closed head injury.

Research progress of endogenous neural stem cells in injury repair strategies after ischemic stroke

Ischemic stroke is characterized by high incidence,high mortality,and high disability and is the primary cause of death and disability among adults in China.Endogenous neural stem cells(NSCs)are a group of cells that have the potential to self-renew and differentiate into functional nerve cells.Under normal circumstances,NSCs are in a quiet state.When the body is subjected to specific stimulation or injury,NSCs can be activated,proliferate,migrate to the damaged site,and differentiate into functional nerve cells to repair the injured tissue.NSCs induced by ischemic stimulation have limited regenerative capacity and cannot completely restore damaged tissues.Exogenous NSC transplantation has some effect.However,it is limited by the low survival rate of transplanted NSCs,immune rejection,ethics,and risk of tumor formation.Therefore,it is necessary to study further the strategies and mechanisms of endogenous NSC activation to promote nerve function repair after stroke.This article reviews recent advancements in drug therapy,hypoxic/ischemic conditioning,Chinese medicine,and rehabilitation strategies for NSC treatment.Furthermore,it explores new strategies and mechanisms developed recently,offering innovative plans and ideas to enhance clinical stroke treatment.

Emerging strategies for nerve repair and regeneration in ischemic stroke:neural stem cell therapy

Ischemic stroke is a major cause of mortality and disability worldwide,with limited treatment options available in clinical practice.The emergence of stem cell therapy has provided new hope to the field of stroke treatment via the restoration of brain neuron function.Exogenous neural stem cells are beneficial not only in cell replacement but also through the bystander effect.Neural stem cells regulate multiple physiological responses,including nerve repair,endogenous regeneration,immune function,and blood-brain barrier permeability,through the secretion of bioactive substances,including extracellular vesicles/exosomes.However,due to the complex microenvironment of ischemic cerebrovascular events and the low survival rate of neural stem cells following transplantation,limitations in the treatment effect remain unresolved.In this paper,we provide a detailed summary of the potential mechanisms of neural stem cell therapy for the treatment of ischemic stroke,review current neural stem cell therapeutic strategies and clinical trial results,and summarize the latest advancements in neural stem cell engineering to improve the survival rate of neural stem cells.We hope that this review could help provide insight into the therapeutic potential of neural stem cells and guide future scientific endeavors on neural stem cells.

High-frequency repetitive transcranial magnetic stimulation promotes neural stem cell proliferation after ischemic stroke

Prolife ration of neural stem cells is crucial for promoting neuronal regeneration and repairing cerebral infarction damage.Transcranial magnetic stimulation(TMS)has recently emerged as a tool for inducing endogenous neural stem cell regeneration,but its underlying mechanisms remain unclea r In this study,we found that repetitive TMS effectively promotes the proliferation of oxygen-glucose deprived neural stem cells.Additionally,repetitive TMS reduced the volume of cerebral infa rction in a rat model of ischemic stro ke caused by middle cerebral artery occlusion,im p roved rat cognitive function,and promoted the proliferation of neural stem cells in the ischemic penumbra.RNA-sequencing found that repetitive TMS activated the Wnt signaling pathway in the ischemic penumbra of rats with cerebral ischemia.Furthermore,PCR analysis revealed that repetitive TMS promoted AKT phosphorylation,leading to an increase in mRNA levels of cell cycle-related proteins such as Cdk2 and Cdk4.This effect was also associated with activation of the glycogen synthase kinase 3β/β-catenin signaling pathway,which ultimately promotes the prolife ration of neural stem cells.Subsequently,we validated the effect of repetitive TMS on AKT phosphorylation.We found that repetitive TMS promoted Ca2+influx into neural stem cells by activating the P2 calcium channel/calmodulin pathway,thereby promoting AKT phosphorylation and activating the glycogen synthase kinase 3β/β-catenin pathway.These findings indicate that repetitive TMS can promote the proliferation of endogenous neural stem cells through a Ca2+influx-dependent phosphorylated AKT/glycogen synthase kinase 3β/β-catenin signaling pathway.This study has produced pioneering res ults on the intrinsic mechanism of repetitive TMS to promote neural function recove ry after ischemic stro ke.These results provide a stro ng scientific foundation for the clinical application of repetitive TMS.Moreover,repetitive TMS treatment may not only be an efficient and potential approach to support neurogenesis for further therapeutic applications,but also provide an effective platform for the expansion of neural stem cells.

Human-induced pluripotent stem cell-derived neural stem cell exosomes improve blood-brain barrier function after intracerebral hemorrhage by activating astrocytes via PI3K/AKT/MCP-1 axis

Cerebral edema caused by blood-brain barrier injury after intracerebral hemorrhage is an important factor leading to poor prognosis.Human-induced pluripotent stem cell-derived neural stem cell exosomes(hiPSC-NSC-Exos)have shown potential for brain injury repair in central nervous system diseases.In this study,we explored the impact of hiPSC-NSC-Exos on blood-brain barrier preservation and the underlying mechanism.Our results indicated that intranasal delivery of hiPSC-NSC-Exos mitigated neurological deficits,enhanced blood-brain barrier integrity,and reduced leukocyte infiltration in a mouse model of intracerebral hemorrhage.Additionally,hiPSC-NSC-Exos decreased immune cell infiltration,activated astrocytes,and decreased the secretion of inflammatory cytokines like monocyte chemoattractant protein-1,macrophage inflammatory protein-1α,and tumor necrosis factor-αpost-intracerebral hemorrhage,thereby improving the inflammatory microenvironment.RNA sequencing indicated that hiPSC-NSC-Exo activated the PI3K/AKT signaling pathway in astrocytes and decreased monocyte chemoattractant protein-1 secretion,thereby improving blood-brain barrier integrity.Treatment with the PI3K/AKT inhibitor LY294002 or the monocyte chemoattractant protein-1 neutralizing agent C1142 abolished these effects.In summary,our findings suggest that hiPSC-NSC-Exos maintains blood-brain barrier integrity,in part by downregulating monocyte chemoattractant protein-1 secretion through activation of the PI3K/AKT signaling pathway in astrocytes.

Maintaining moderate levels of hypochlorous acid promotes neural stem cell proliferation and differentiation in the recovery phase of stroke

It has been shown clinically that continuous removal of ischemia/reperfusion-induced reactive oxygen species is not conducive to the recovery of late stroke.Indeed,previous studies have shown that excessive increases in hypochlorous acid after stroke can cause severe damage to brain tissue.Our previous studies have found that a small amount of hypochlorous acid still exists in the later stage of stroke,but its specific role and mechanism are currently unclear.To simulate stroke in vivo,a middle cerebral artery occlusion rat model was established,with an oxygen-glucose deprivation/reoxygenation model established in vitro to mimic stroke.We found that in the early stage(within 24 hours)of ischemic stroke,neutrophils produced a large amount of hypochlorous acid,while in the recovery phase(10 days after stroke),microglia were activated and produced a small amount of hypochlorous acid.Further,in acute stroke in rats,hypochlorous acid production was prevented using a hypochlorous acid scavenger,taurine,or myeloperoxidase inhibitor,4-aminobenzoic acid hydrazide.Our results showed that high levels of hypochlorous acid(200μM)induced neuronal apoptosis after oxygen/glucose deprivation/reoxygenation.However,in the recovery phase of the middle cerebral artery occlusion model,a moderate level of hypochlorous acid promoted the proliferation and differentiation of neural stem cells into neurons and astrocytes.This suggests that hypochlorous acid plays different roles at different phases of cerebral ischemia/reperfusion injury.Lower levels of hypochlorous acid(5 and 100μM)promoted nuclear translocation ofβ-catenin.By transfection of single-site mutation plasmids,we found that hypochlorous acid induced chlorination of theβ-catenin tyrosine 30 residue,which promoted nuclear translocation.Altogether,our study indicates that maintaining low levels of hypochlorous acid plays a key role in the recovery of neurological function.

Multiple factors to assist human-derived induced pluripotent stem cells to efficiently differentiate into midbrain dopaminergic neurons

Midbrain dopaminergic neurons play an important role in the etiology of neurodevelopmental and neurodegenerative diseases.They also represent a potential source of transplanted cells for therapeutic applications.In vitro differentiation of functional midbrain dopaminergic neurons provides an accessible platform to study midbrain neuronal dysfunction and can be used to examine obstacles to dopaminergic neuronal development.Emerging evidence and impressive advances in human induced pluripotent stem cells,with tuned neural induction and differentiation protocols,makes the production of induced pluripotent stem cell-derived dopaminergic neurons feasible.Using SB431542 and dorsomorphin dual inhibitor in an induced pluripotent stem cell-derived neural induction protocol,we obtained multiple subtypes of neurons,including 20%tyrosine hydroxylase-positive dopaminergic neurons.To obtain more dopaminergic neurons,we next added sonic hedgehog(SHH)and fibroblast growth factor 8(FGF8)on day 8 of induction.This increased the proportion of dopaminergic neurons,up to 75%tyrosine hydroxylase-positive neurons,with 15%tyrosine hydroxylase and forkhead box protein A2(FOXA2)co-expressing neurons.We further optimized the induction protocol by applying the small molecule inhibitor,CHIR99021(CHIR).This helped facilitate the generation of midbrain dopaminergic neurons,and we obtained 31-74%midbrain dopaminergic neurons based on tyrosine hydroxylase and FOXA2 staining.Thus,we have established three induction protocols for dopaminergic neurons.Based on tyrosine hydroxylase and FOXA2 immunostaining analysis,the CHIR,SHH,and FGF8 combined protocol produces a much higher proportion of midbrain dopaminergic neurons,which could be an ideal resource for tackling midbrain-related diseases.

TAX1BP1 and FIP200 orchestrate non-canonical autophagy of p62 aggregates for mouse neural stem cell maintenance

Autophagy plays a pivotal role in diverse biological processes,including the maintenance and differentiation of neural stem cells(NSCs).Interestingly,while complete deletion of Fip200 severely impairs NSC maintenance and differentiation,inhibiting canonical autophagy via deletion of core genes,such as Atg5,Atg16l1,and Atg7,or blockade of canonical interactions between FIP200 and ATG13(designated as FIP200-4A mutant or FIP200 KI)does not produce comparable detrimental effects.This highlights the likely critical involvement of the non-canonical functions of FIP200,the mechanisms of which have remained elusive.Here,utilizing genetic mouse models,we demonstrated that FIP200 mediates non-canonical autophagic degradation of p62/sequestome1,primarily via TAX1BP1 in NSCs.Conditional deletion of Tax1bp1 in fip200hGFAP conditional knock-in(cKI)mice led to NSC deficiency,resembling the fip200hGFAP conditional knockout(cKO)mouse phenotype.Notably,reintroducing wild-type TAX1BP1 not only restored the maintenance of NSCs derived from tax1bp1-knockout fip200hGFAP cKI mice but also led to a marked reduction in p62 aggregate accumulation.Conversely,a TAX1BP1 mutant incapable of binding to FIP200 or NBR1/p62 failed to achieve this restoration.Furthermore,conditional deletion of Tax1bp1 in fip200hGFAP cKO mice exacerbated NSC deficiency and p62 aggregate accumulation compared to fip200hGFAP cKO mice.Collectively,these findings illustrate the essential role of the FIP200-TAX1BP1 axis in mediating the non-canonical autophagic degradation of p62 aggregates towards NSC maintenance and function,presenting novel therapeutic targets for neurodegenerative diseases.

Neuroprotective effects of neural stem cells pretreated with neuregulin1β on PC12 cells exposed to oxygen-glucose deprivation/reoxygenation

Studies on ischemia/reperfusion(I/R)injury suggest that exogenous neural stem cells(NSCs)are ideal candidates for stem cell therapy reperfusion injury.However,NSCs are difficult to obtain owing to ethical limitations.In addition,the survival,differentiation,and proliferation rates of transplanted exogenous NSCs are low,which limit their clinical application.Our previous study showed that neuregulin1β(NRG1β)alleviated cerebral I/R injury in rats.In this study,we aimed to induce human umbilical cord mesenchymal stem cells into NSCs and investigate the improvement effect and mechanism of NSCs pretreated with 10 nM NRG1βon PC12 cells injured by oxygen-glucose deprivation/reoxygenation(OGD/R).Our results found that 5 and 10 nM NRG1βpromoted the generation and proliferation of NSCs.Co-culture of NSCs and PC12 cells under condition of OGD/R showed that pretreatment of NSCs with NRG1βimproved the level of reactive oxygen species,malondialdehyde,glutathione,superoxide dismutase,nicotinamide adenine dinucleotide phosphate,and nuclear factor erythroid 2-related factor 2(Nrf2)and mitochondrial damage in injured PC12 cells;these indexes are related to ferroptosis.Research has reported that p53 and solute carrier family 7 member 11(SLC7A11)play vital roles in ferroptosis caused by cerebral I/R injury.Our data show that the expression of p53 was increased and the level of glutathione peroxidase 4(GPX4)was decreased after RNA interference-mediated knockdown of SLC7A11 in PC12 cells,but this change was alleviated after co-culturing NSCs with damaged PC12 cells.These findings suggest that NSCs pretreated with NRG1βexhibited neuroprotective effects on PC12 cells subjected to OGD/R through influencing the level of ferroptosis regulated by p53/SLC7A11/GPX4 pathway.

New insights into the biological roles of immune cells in neural stem cells in post-traumatic injury of the central nervous system

Traumatic injuries in the central nervous system,such as traumatic brain injury and spinal cord injury,are associated with tissue inflammation and the infiltration of immune cells,which simultaneously affect the self-renewal and differentiation of neural stem cells.Howeve r,the tissue repair process instigated by endogenous neural stem cells is incapable of restoring central nervous system injuries without external intervention.Recently,resident/peripheral immune cells have been demonstrated to exert significant effects on neural stem cells.Thus,the resto ration of traumatic injuries in the central nervous system by the immune intervention in neural stem cells represents a potential therapeutic method.In this review,we discuss the roles and possible mechanisms of immune cells on the selfrenewal and differentiation of neural stem cells along with the prognosis of central nervous system injuries based on immune intervention.Finally,we discuss remaining research challenges that need to be considered in the future.Further elucidation of these challenges will fa cilitate the successful application of neural stem cells in central nervous system injuries.

Low-temperature 3D-printed collagen/chitosan scaffolds loaded with exosomes derived from neural stem cells pretreated with insulin growth factor-1 enhance neural regeneration after traumatic brain injury

There are various clinical treatments for traumatic brain injury,including surgery,drug therapy,and rehabilitation therapy;howeve r,the therapeutic effects are limited.Scaffolds combined with exosomes represent a promising but challenging method for improving the repair of traumatic brain injury.In this study,we determined the ability of a novel 3D-printed collagen/chitosan scaffold loaded with exosomes derived from neural stem cells pretreated with insulin-like growth factor-1(3D-CC-INEXOS) to improve traumatic brain injury repair and functional recove ry after traumatic brain injury in rats.Composite scaffolds comprising collagen,chitosan,and exosomes derived from neural stem cells pretreated with insulin-like growth fa ctor-1(INEXOS) continuously released exosomes for 2weeks.Transplantation of 3D-CC-INExos scaffolds significantly improved motor and cognitive functions in a rat traumatic brain injury model,as assessed by the Morris water maze test and modified neurological seve rity scores.In addition,immunofluorescence staining and transmission electron microscopy showed that3D-CC-INExos implantation significantly improved the recove ry of damaged nerve tissue in the injured area.In conclusion,this study suggests that transplanted3D-CC-INExos scaffolds might provide a potential strategy for the treatment of traumatic brain injury and lay a solid foundation for clinical translation.