Gui-Long Zhang, Zhi-Han Zhu, Ye-Zhong Wang

Gui-Long Zhang, Ye-Zhong Wang, Department of Neurosurgery, The Second Affiliated Hospital of Guangzhou Medical University, Guangzhou 510260, Guangdong Province, China

Zhi-Han Zhu, Department of Neurosurgery, Zhongda Hospital, School of Medicine, Southeast University, Nanjing 210009, Jiangsu Province, China

Abstract

Key words: Ischemic stroke; Neural stem cells; Transplantation; Cytokines; Exosomes

BRAIN ISCHEMIC STROKE

Globally, stroke is one of the top three common causes of death and disability.It is divided into the following two types: Ischemic and hemorrhagic stroke, of which ischemic stroke accounts for more than three-quarters of cases (about 80%-85%)[1].Following stroke onset, patients can suffer from various neurological dysfunctions that renders them unable to take care of themselves.This seriously affects their quality of life, impacts on their patients and their families physically and mentally, as well socially and economically.Recently, numerous preclinical and clinical studies[2,3]have discovered many drugs or molecules that may potentially exert certain beneficial effects in the treatment of ischemic stroke; however, these drugs exhibit limited or no therapeutic efficacy under clinical applications; thus, a novel drug for this purpose still needs to be explored.

Ischemic stroke can induce some harmful pathophysiological changes around the ischemic area of the brain[4-6].This event is mainly characterized as acute ischemia and hypoxia of nerve tissue after blood interruption to local brain tissue in a short period of time.This is followed by a series of cascade reactions, including the production of local oxygen free radicals and reactive oxygen species, increase in local tissue permeability, generation of cerebral edema, local immune inflammatory cell infiltration, development of neuroinflammation, and blood-brain barrier destruction.These lesions significantly expand in a short period of time, leading to local nerve cell damage and necrosis, reduction of functional neurons, and axon network disruption.Subsequently, due to the extremely poor microenvironment around the peri-ischemic cell regions, secondary brain damage occurs, resulting in an irreversible neural tissue injury.The limits self-repair of nerve tissue.Even after recovery of blood flow to the ischemic area, enough nerve cells and an appropriate immune microenvironment to supplement and repair the functional nerve tissue that died are still not available[4-6].This eventually leads to permanent local nerve tissue loss and nerve function defects of the brain (Figure 1).

However, the treatment of brain ischemic stroke is individualized and involves heterogeneous approaches, most of which are closely related to the location of ischemia, the age of patients, and the ability of local nerve self-repair.The main purpose of clinical treatment of stroke is to restore local cerebral blood perfusion and reduce the occurrence and degree of disability or dysfunction after stroke onset as early as possible[2,7].Due to the weaker regeneration ability of endogenous nerve cells and the harmful effects of an ischemic microenvironment, there are currently no effective methods or strategies available for treatment.Recombinant tissue plasminogen activator (r-tPA) is the only effective class 1A recommended drug for the treatment of acute ischemic stroke approved by the American Food and Drug Administration, but it has a very narrow treatment window (generally within 4.5 h,and no more than 6 h) and it has various treatment-related contraindications or complications (such as increased risk of cerebral hemorrhage)[8-10].Therefore, its application has been markedly limited; only a small percentage of patients (no more than 5% of patients) can receive this timely and effective treatment.In addition,intravascular interventional therapy (such as endovascular thrombectomy) can be used as an adjuvant or replacement therapy for early reocclusion of large vessels in patients with contraindications or complications of intravenous thrombolysis[11-13].This adjuvant therapy can extend the time window of potential treatment to 12 h, and it can also improve the functional independence and vascular remodeling rate of the patient compared to tPA treatment alone.However, the treatment time still has an important effect on the efficacy of this intravascular treatment, because just a 30 min delay beyond the timeframe can significantly reduce the functional independence of patients.Moreover, current endovascular interventions are still difficult to generalize,since only regional stroke centers with neurological intervention capabilities can perform this operation, and only less than 10% of stroke patients are eligible to undergo this effective treatment[11,14].

Figure 1 The pathological processes of brain ischemic stroke.

Thus, treatment of subacute and chronic phases of stroke is important, and it is mainly based on the clinical rehabilitation of patients with symptoms (such as different rehabilitation methods according to different stages and dysfunctions)[2,7,15-17].This includes subject stroke unit nursing, home or hospital rehabilitation training,task-oriented training, mandatory exercise therapy, different high-intensity training,repetitive task training, spatial sensation, and language ability training.Although the abovementioned rehabilitation methods can improve the symptoms of some patients to a certain extent after long-term adherence, the effect of rehabilitation is limited, and it is still not enough to improve the overall prognosis of stroke patients.Therefore, the therapeutic prognosis of patients with brain ischemic stroke is closely related to the permanent loss of local functional neurons and networks, the repair of local glial scars, and the lack of activated endogenous neural stem cells[7,18-20].

NEURAL STEM CELLS

In 1992, Reynoldset al[21]isolated a cell population with self-renewal abilities and a multi-directional differentiation potential from the striatum of adult mice; these were proposed as neural stem cells (NSCs).Subsequently, in 1997, Mckayet al[22]officially defined the concept of NSCs as a self-renewing cell population with pluripotent abilities that can differentiate into neurons, astrocytes, and oligodendrocytes.Recent studies[23,24]have confirmed that NSCs are present in the lateral ventricle (subventricular zone, SVZ) and hippocampal dentate gyrus (subgranular zone, SGZ) of adult animals.NSCs in the SVZ region mainly migrate along the rostral migratory stream to the olfactory bulb, whereas stem cells in the SGZ migrate to the granule cell layer, and finally differentiate into various neural cells and integrate into nerve networks[23-26].In addition, with the exception of SVZ and SGZ, the human brain possibly consists of another stem cell pool in the deep ventral region of the prefrontal cortex, due to the highly developed prefrontal lobe in the human brain.

Furthermore, endogenous NSCs existin vivo, whereas exogenous NSCs are culturedin vitro.Under normal conditions, endogenous NSCs in the body are in a static, undifferentiated dormant state (called quiescent NSCs, qNSCs), and maintain a dynamic balance in the stem cell pools[27-29].Once they are exposed to external stimuli such as brain damage, qNSCs can be activated to proliferate, migrate, and differentiate, thereby participating in the repair process of damaged nerve tissue[29-31].Usually, the number of endogenous cells activated is very limited, and a large proportion normally differentiates into glial cells.Glial scars are involved in tissue repair, so that the loss of functional neurons (neural network) is not enough to supplement[32-34].Moreover, significant neurological dysfunction still persist for a long time after brain injury, indicating that the neuroregenerative ability of endogenous cells is largely insufficient, and is not enough to replace damaged functional nerve tissue[7,35,36].

With the development of stem cell therapy research, adult stem cells have received more and more attention and provide new directions for future clinical treatment of refractory diseases including ischemic stroke[37-40].NSCs have many advantages, such as self-renewal, low immunogenicity, and good histocompatibility, as well as multidirectional differentiation potential; they can differentiate into three types of nerve cells to maintain and repair damaged brain tissue.Thus, NSCs act as a natural active resource and are considered to be a good tool for treating nervous system diseases.Numerous preclinical studies[39,40]have found that transplantation of exogenous NSCs can significantly complement or replace damaged tissues and treat various neurological diseases.Studies[36,41-44]have also shown that after treatment of neurological diseases with exogenous NSC transplantation, the deficiency of endogenous NSCs was not only supplementedin vivo, but the immune microenvironment around the tissue injury area was also improved.Many preclinical studies[45-47]have demonstrated that exogenous NSCs have a certain therapeutic effect in neurological diseases, and certain studies have found that the therapeutic functions of exogenous NSCs can be enhanced by combining or overexpressing them with some cytokines [such as brain-derived neurotrophic factor (BDNF), vascular endothelial growth factor (VEGF), or NGFetc].However, the efficiency of exogenous stem cell transplantation therapy is still controversial, mainly due to the low grafting efficiency of exogenous stem cells in the brain (less than 5%).Furthermore, the inflammatory immune microenvironment is very severe.Nowadays, the mechanisms of treatment with stem cell transplantation are not entirely clear, and further research methods are needed.

TRANSPLANTED NEURAL STEM CELLS THERAPY FOR BRAIN ISCHEMIC STROKE

Due to the ethical, therapeutic efficacy, and safety issues, exogenous NSC transplantation therapies have a long way to go to reach the stage of clinical application.At present, only a few clinical studies have been conducted; however, a number of preclinical animal experiments have been conducted[38,40,48-50].A large number of these studies[37-40]have evaluated the therapeutic efficacy and safety of transplanted exogenous NSCs in preclinical animals with brain ischemic stroke.Their results demonstrated that exogenous NSCs could significantly improve the prognosis of cerebral ischemic animals, and not only the functional outcome was improved, but also the histological infarct volume was significantly reduced, with no obvious security issues.The following two main mechanisms have been approved for the activity of exogenous NSCs in the treatment of brain ischemic stroke[36,41-44,51,52]according to the first mechanism, transplantation of exogenous stem cells compensate for the deficiency of endogenous stem cells and activate more endogenous cells to repair the nerve damage; the second mechanism reveals that transplantation of exogenous stem cells improves the inflammatory immune microenvironment around the ischemic regions, which then mediates neural network reconstruction based on the bystander effect (Figure 2).

Animal experiments

Currently, the results of NSC transplantation for brain ischemic stroke in animal models are satisfactory.Moreover, the efficacy and safety of stem cell transplantation has also been confirmed.Leeset al[53]and Vuet al[54]used meta-analyses to evaluate the therapeutic efficacy of stem cell transplantation (including NSCs) in 117 and 46 preclinical animal models with cerebral ischemic stroke, respectively.After treatment,the neurological function of cerebral ischemic animals is improved significantly, and the volume of cerebral infarction reduced.Furthermore, the degree of prognosis improvement was correlated with the source of stem cells, injection route, injection timing, and dose of injection[53,54].Chenet al[40]collated and analyzed animal studies of NSC therapy for the treatment of brain ischemic stroke.A total of 37 studies and 54 independent intervention groups were analyzed and meta-analyzed.The results showed that transplantation of NSCs significantly improved neurological function and histological structure outcomes of cerebral ischemic animals.Of the studies analyzed, 36 reported neurological improvement, 22 reported improved histology,and 21 reported beneficial outcomes in both neurological function and histological structure.They also found that the degree of improvement in prognosis function of ischemic animals had a certain correlation with the injection time of NSCs, the sourceof stem cells, and whether immunosuppressive agents had been used[40].No significant safety problems were found.Although some differences in research quality and different degrees of publication bias between the different animal experiments exist[55-59], the overall results suggest that NSCs can effectively improve neurological function of cerebral ischemic stroke animals.They can reduce the area of ischemic infarction, proliferate, migrate, and differentiate into neuronsin vivo.In addition,some neurons may be integrated into the neural network of the brain.

After exogenous NSCs were transplanted into the ischemic brain, they can induce the endogenous NSCs activation of SVZ region.Then both exogenous and endogenous NSCs migrated to the cortical site of ischemic injury, differentiated to glia cells and neurons which contributed to glial scar formation and nerve tissue repair.Furthermore, it had two main mechanisms for reconstructing ischemic neural tissue in vivo: exogenous and activated endogenous stem cells directly compensated the lost nerve tissue by differentiating into nerve cells, transplanted NSCs improved the harmful inflammatory microenvironment around the ischemic regions via the bystander effect.NSCs: Neural stem cells; SVZ:Subventricular zone.

Clinical trials

At present, there are very few clinical trials with NSCs registered in the Clinical Trial Database (ClinicalTrials.gov) for brain ischemic stroke.The first study is a Phase II clinical trial (NCT02117635) conducted at the Queen Elizabeth University Hospital of the University of Glasgow, UK.The current status of the trial shows that it has been completed, but no publications have been made for the Phase II clinical trial to date.They published the Phase I clinical trial (NCT01151124) of human NSC therapy for chronic cerebral ischemic stroke in the Lancet issue from August 2016[38].This clinical study was an unblinded, single-center transplantation study that involved different doses of CTX0E03 cells (an immortalized NSC line, ReNeuron).Allogeneic transplantation of immortalized NSCs CTX0E03 was performed by stereotactic injection.A total of 11 patients were enrolled.Four groups were injected with different doses of exogenous NSCs.The patients were followed up for 2 years and evaluated by the NIHSS (National Institute of Health stroke scale) and Barthel Index methods.All of the evaluated methods and MRI images confirmed that the exogenous NSCs significantly improved the neurological function of patients after NSC transplantation, with no related adverse reactions[38].This study was the first exogenous NSC transplantation performed in clinical patients with brain ischemic stroke[38].None of the patients used immunosuppressants due to human leukocyte antigen (HLA) mismatch, and the results showed that some patients had significant neurological improvement.However, because the number of patients included in a Phase I trial are small, and no control or placebo groups are enrolled (which may relate to ethical issues), more detailed data in the Phase II clinical trial are expected.The second study is a Phase I clinical trial registered with Beijing Bayi Brain Hospital(NCT03296618).In this study, Exogenous NSCs were used from Neuralstem’s NSI-566 cell line derived from primary human fetal spinal cord tissue.The results of this Phase I clinical trial have not yet been published.

OPTIMIZING THE THERAPEUTIC EFFICACY OF NEURAL STEM CELL TRANSPLANTATION

Although preclinical studies have confirmed the efficacy and safety of NSC transplantation for the treatment of ischemic stroke, there are still some controversies.Since the grafting efficiency or survival rate of stem cells is less than 5%in vivo, there are still many problems that should to be addressed before this treatment can be used for clinical applications[60-63].The most important issue is the grafting efficiency and differentiation ratio of NSCsin vivoafter transplantation.In addition, both endogenous and exogenous NSCs differentiate into glial cellsin vivoin a significantly higher ratio than that of neurons[64-66].Thus, many studies have attempted to modify the gene expressions or protein levels of NSCs using different strategies such as virus transfection to express specific genes, pretreatment of cells with inflammatory immune factors, and combination with cytokines to increase the therapeutic effects of transplanted cells.

Gene overexpression

BDNF can promote the differentiation of transplanted NSCs into neurons and increase their survival[67,68].Therefore, studies have attempted to overexpress the BDNF gene in NSCs for improving the therapeutic potential of stem cellsin vivo[67,68].After transplantation of human NSCs that overexpress the BDNF gene in the ischemic striatal region of MCAO rats, the contralaterally transplanted NSCs were found to migrate to the infarcted areaviaMRI images.Neurobehavioral functions of ischemic rats were also significantly improved, and the transplanted cells co-localized with Nestin, DCX, and MAP2 positive cells, indicating that the transplanted NSCs participated in nerve regeneration and functional recoveryin vivo.Lastly, protein expression of BDNF was also high in the ischemic regions[46].

Neurotrophin-3 (NT-3) belongs to a family of neurotrophic factors and has been found to be involved in mediating stem cell survival and inducing neural differentiation[69,70].The lentiviral vector (LV) encoding human NT-3 was constructed and transfected into NSCs, and then transplanted into the ipsilateral striatum region of MCAO rats.After 2 to 4 wk of transplantation, secretion of NT-3 protein was significantly higher than that in the control group.Concurrently, the neurobehavioral function in cerebral ischemia rats was significantly improved, when compared to the control group[71].

VEGF is an important angiogenic factor, and it is involved in mediating angiogenesis and nutrient supplementation[72,73].After transplantation of NSCs transfected with the VEGF121 gene into the ischemic surroundings of stroke rats,studies found that the cells survived and migrated in the ischemic area for up to 12 wk after transplantation.At the same time, the neurological function of the cerebral ischemia rats was significantly improved, compared to the untransfected NSC group,indicating that VEGF121 transfection additionally increased the therapeutic efficacy of transplanted NSCs[74].

The above studies suggest that exogenous genes can be introduced into NSCs by LVs.The modified NSCs carry the therapeutic-related genes to the damaged areas and can express them effectively, and finally increasing the repairing effects of transplanted NSCs.

Preconditioning of NSCs with cytokines

Although overexpression of genes in NSCs leads to better transplantation outcomes by promoting trophic or survival signaling to cells, more beneficial, simpler, and safer methods need to be developed for future clinical applications.Consequently, studies have introduced immunological correlations where NSCs are pretreated with cytokines or inflammatory factors for treatment[75,76].

Interleukin-6 (IL-6) is a pro-inflammatory cytokine involved in the pathogenesis of various neurological diseases including stroke[77,78].Serum IL-6 levels in patients with clinical ischemic stroke are associated with poor infarct volume and long-term prognosis[79].When NSCs were pretreated with IL-6, results showed that stem cells were reprogrammed, and the signal transduction and transcription activator 3-mediated manganese superoxide dismutase (SOD2) was significantly up-regulated in the cells[60].Expression of SOD2 promoted cell survival in the ischemic area; pretreated stem cells also induced the secretion of VEGF, promoted microvascularization, and significantly reduced the infarct size of cerebral ischemia, as well as improved neurological functions.These results demonstrated that pretreatment with IL-6 can properly reprogram NSCs to withstand the oxidative stress environment after an ischemia-reperfusion injury, induce angiogenesis, and ultimately improve the effectiveness of transplanted stem cells for the treatment of ischemic stroke[60].

In addition, expression of inflammatory cytokines such as IL-1, IL-15, and Interferon-gamma (IFN-γ) normally increase after central nervous system injury.When NSCs were pretreated with anti-inflammatory factors such as IL-4 or IL-10, the results were consistent with those using a pretreatment of IL-6.These cytokines mainly played a neuroprotective role and promoted the migration of cells to the injury site.The pretreatment method dominantly provided a more favorable microenvironment for the proliferation of NSCs after transplantation[80,81].Additionally, BDNF-pretreated with NSCs produced similar results, where increased cell survival, migration, and improved neurological function were seen in cerebral ischemic animals after transplantation[82].

Thus, a better understanding of the relationship between neuro-inflammation and neurogenesis, and an understanding of the potential mechanisms of inflammatory stimulation in cerebral ischemia is essential.In vitropretreated stem cells with cytokines or inflammatory factors may further induce the migration of NSCs to inflammatory regions, increase the neuroprotection of NSCs, and more effectively increase the therapeutic effects of stem cells.

Co-transplantation with factors

Cytokines can regulate the self-renewal, proliferation, and differentiation of stem cells, but to maximize the therapeutic potential of stem cells and ameliorate the damage, regulation of the microenvironment may be crucial.Currently, the main direction of NSC-based research is to explore new tools for nerve regeneration.Viral vectors and gene therapy may have certain deficiencies, such as potential tumor formation and lack of efficiency.Studies[81,83,84]have attempted to deliver therapeutic drugs through implanted pumps for sustained release, but deficiencies still persist with these strategies.

Neurotrophic factors can increase the survival of NSCs and promote their proliferation or differentiation.VEGF plays various roles in the CNS, including proangiogenesis, neurogenesis, and neurotrophic and neuroprotective effects[73,85,86].Study have attempted to investigate the feasibility of co-administration of VEGF with human NSCs[87].The results showed that VEGF and transplanted NSCs had a certain synergistic effect in cerebral ischemia.The combination-treatment group expressed a better behavioral recovery than single-treatment groups, and the degree of brain atrophy in the cerebral cortex and striatum was significantly decreased.However, the distribution of VEGF was not co-localized with NSCs, suggesting that VEGF promoted the therapeutic efficacy of NSC transplantation through pro-angiogenic effects[87].IFN-γ is a mediator of the pro-inflammatory pathway and plays an important role in the ischemic brain.However, IFN-γ does not hamper the ability of NSCsin vitro.Study[88]have shown that co-delivery of IFN-γ (50 ng) enhanced the effects of transplanted NSCs in ischemic rats.The study found that in the combination-treatment group, neurogenesis was significantly increased when compared to other groupsin vivo.Moreover, co-treatment with IFN-γ and NSCs provided additional beneficial neurological outcomes.Thus, low concentrations of IFN-γ can mediate NSC functions and facilitate their ability for neurological repair[88].

In addition, vascular progenitor cells (VPC) was co-transplanted with NSCs.It was found that co-transplantation of NSCs and VPC enhanced the differentiation ratio of neurons and microvessel formationin vivo; furthermore, it significantly improved motor function and reduced the infarct volume in rats with cerebral ischemia[44].Thus,co-repair of nerve and blood vessel may be more effective[44].Moreover, the combination of BDNF and NSC transplantation resulted in enhanced therapeutic effects when compared to transplantation of NSCs alone[89].However, the combination of these cytokines with cells may have some unavoidable problems such as dosage and injection methods; thus, a safe dosage must be first established to avoid harmful side effects, and secondly, a proper balance between cytokines and cells should be determined.

POTENTIAL PROBLEMS WITH NSC TRANSPLANTATION

Although both preclinical and clinical studies have confirmed that transplantation of exogenous NSCs can treat various refractory nervous system diseases such as ischemic stroke and neurodegenerative diseases, there are still some limitations and potential side effects including a large-scale production bottleneck of stem cells,potential allogeneic rejection of cells, risk of cell tumorigenicity, grafting or survival efficiency of cell transplantation, difficulties with the administration route of cells, and targeting problems.All of those limit the rapid clinical transformation and application of NSCs.

The first limitation pertains to the source of NSCs.Given that they only exist in a specific stem cell pool of the brain[22,25], it is difficult to obtain a large number of homogenous cellsin vitro.Additionally, other limitations in procuring NSC also exist such as the gradual aging of culture cells, repeated extraction and infusion of cells,cost, safety, and ethical issues.Secondly, the obtained exogenous stem cells should be allogeneic, a potential obstacle in the application of SCs is immune-rejection after cell transplantation[90,91].Similar to any tissue or organ transplantation, allogeneic stem cells can be rejected by the host immune system.The main reason for this is that the transplanted tissue does not match the HLA in the host.Typically, host T lymphocytes recognize MHC class 1-protein antigens on other cells, if they do not match, the immune system is activated and begins to attack the transplanted cells.Although the brain has historically been considered an immune privileged area,current studies have found that immune cells can also produce immune rejection in the brain[92,93].

Furthermore, the most prominent problem of cell therapy is the potential tumorigenicity of transplanted exogenous stem cells.Stem cells, especially embryonic stem (ES) cells or induced pluripotent stem (iPS) cells, are pluripotent cells that may form teratomas or malignant tumors when implanted into a living host[94,95].Studies have found that iPS cells derived from B6 mouse embryonic fibroblasts undergo immunological rejection after transplantation into B6 mice and also produce teratomas.ES cells derived from B6 mice also produce teratogenesis in mice, even though no significant immune rejection occurs[95,96].Simultaneously, the tumorigenicity of iPS cells is also related to the mutagenesis of the c-Myc gene insertion site and the persistent expression of reprogrammed exogenous genes[96-98].A small number of contaminated exogenous cells may also induce tumorigenesis in allogeneic transplantation, and even very small amounts of contaminating undifferentiated ES cells have been found to produce tumors in nude mice[99].Therefore, the safety regarding the use of pluripotent cells cannot be ignored.Although NSCs have not yet been found to be tumorigenic, their abnormal proliferation afterin vivotransplantation may lead to tumor formation.

Finally, a series of cascades following cerebral ischemia, neuro-inflammation, and immune responses can severely affect the survival of cells after transplantation,reduce their ability of replacing damaged neurons, and eliminate the therapeutic effect of cell transplants[80,90,100].In addition, inflammatory factor could promote glial differentiation of NSCs, resulting in the generation of GFAP-positive cells.And the immune response produced after cell transplantation may also facilitate NSC differentiation into glial cells.This suggests that inflammation may inhibit neuronal generation.However, stem cell-based therapies can modulate the host inflammatory response to recreate a favorable cellular microenvironment and prevent further endogenous cell death.Through regulation of immune inflammation, transplanted cells can increase the chances of endogenous cell survival, but the mechanisms are not clear and need to be further explored.

PROSPECT-STEM CELL-BASED CELL-FREE THERAPY STRATEGY

Currently, stem cell-based therapy is the most promising method for the treatment of refractory diseases.A large number of studies[38,40,48-50]have confirmed the effectiveness and feasibility of stem cell therapies for refractory neurological diseases including ischemic stroke.Although transplanted exogenous NSCs can provide neuroprotective effects after acute stroke and supplement lost nerve tissue for chronic stroke (through direct cell replacement and enhanced endogenous repair), the ultimate goal of complete recovery is not reached, and there are still some problems that need to be resolved.

Several studies[101,102]have confirmed that the paracrine effect plays a major role in stem cell transplantation, and the main molecules responsible for this paracrine effect are extracellular vesicles (EVs).Exosomes are small molecules of exocytic vesicles derived from cells that play a major role in EVs.Exosomes are about 30-150 nm in diameter and can be secreted by all cells including proteins, lipids, and RNAs;furthermore, they play a very important role in cell-to-cell communication[103-105].Since exosomes, in part, possess active ingredients and functional properties of the cells from which they are derived, they can be used to develop a new type of cell-free treatment.Relevant preclinical and clinical studies[106-108]have confirmed that exosomes derived from stem cells are safe and effective; not only can they cross biological barriers (such as the blood-brain barrier, BBB), but they also exhibit immune tolerance and are relatively stable (no immune rejection)in vivo.At the same time, the tumorigenicity of exogenous cells can be avoided (due to the cell-free treatment).In addition, they can be modified by nanomedicine technologies to enhance their therapeutic effects, and facilitate the route of administration or the targeting of treatment[106-108].

Exosomes have been developed as early diagnostic markers for some diseases (such as tumors) and are developing into novel molecules with potential targeted therapeutic effects.Although exosomes were discovered 30 years ago, their clinical relevance has significantly increased in recent years[107-109].Furthermore, there is growing evidence demonstrating that exosomes are critical for the benefits of cell therapy[106,110-113].Since exosomes can overcome many of the risks and difficulties associated with cell therapy, they can be developed as a new strategy to replace stem cell therapy (stem cell-based cell-free treatment method)[101,110,111,114-117].However, the therapeutic potential and mechanism of exosomes in the nervous system, especially NSC-derived exosomes, has not been studied extensively.For example, the dynamic migration and kinetics of transplanted exosomes derived from NSCsin vivo, the type of exosome acting on cells, and the action mode with the local immune microenvironment need to be further studied.Moreover, the specific components of exosomes (such as proteins or miRNAs or lncRNAs) that have a significant potential of action are also unknown and need to be further explored.

In conclusion, currently the most promising treatment approach for refractory neurological diseases including ischemic stroke is based on stem cell transplantation(Figure 3).As a special type of stem cells that are present only in the nervous system,NSCs play a very important role in repairing neurological diseases.NSCs are a type of natural seed resource that not only can supplement necrotic nerve cells or tissues, but also participate in endogenous repair mechanisms.Furthermore, exosomes derived from NSCs have similar functional properties, and may serve as a new research topic in the field of stem cell research.Thus, using stem cell-based cell-free treatment,exosomes can be developed as a new therapeutic strategy, and they may play a more important role in the future.

Figure 3 Exosomes derived from neural stem cells are critical for the benefits of stem cell-based therapy.