NSC-derived exosomes enhance therapeutic effects of NSC 1 transplantation on cerebral ischemia in mice 2

: Transplantation of neural stem cells (NSCs) has been proved to promote 16 functional rehabilitation of brain lesions including ischemic stroke. However, the 17 therapeutic effects of NSC transplantation is limited by the low survival and 18 differentiation rates of NSCs due to the harsh environment in the brain after ischemic 19 stroke. Here, we employed NSCs derived from human induced pluripotent stem cells 20 (iPSCs) together with exosomes extracted from NSCs to treat cerebral ischemia 21 induced by middle cerebral artery occlusion/reperfusion (MCAO/R) in mice. The 22


INTRODUCTION
Stroke is the second leading cause of death worldwide, which usually causes motor and cognitive impairments that require long-term rehabilitation (2021).The commonly used treatments of stroke in clinic include tissue plasminogen activator (t-PA) thrombolytic therapy and thrombus clearance surgery, but both are limited by inability to repair damaged neural circuits, and only 10% of stroke patients meet the treatment standards (Fugate andRabinstein 2015, Nagaraja et al., 2020).Stem-cell based therapy is a progressing and promising method to treat ischemic stroke.Many stem cell types including neural stem cells (NSCs) (Sakata et al., 2012), embryonic stem cells (ESCs) (Meamar et al., 2013), mesenchymal stem cells (MSCs) (Toyoshima et al., 2017), bone marrow mononuclear cells (BMMCs) (Yang et al., 2012), and iPSCs (induced pluripotent stem cells) (Duan et al., 2021) have been tested in preclinical and clinical research, which showed encouraging therapeutic effects.Both endogenous and exogenous NSCs have remarkable capacity to maintain self-renewal while differentiating into various cell types including neurons and glial cells in nervous system (Dong et al., 2012).iPSCs can be an ideal resource to acquire NSCs, which voids both ethical problems and immune rejection, and has a potential to provide genetically identical "patient-specific" cells for stroke patients (Baker et al., 2019).On the other hand, the low survival rate of transplanted NSCs, largely due to chronic inflammation and oxidative stress of the microenvironment after stroke (Koutsaliaris et al., 2022), and the poor differentiation of NSCs (Zhang et al., 2019) limited its application.
NSC-derived exosomes are enriched in specific miRNAs that mediate multiple functions in physiological and pathological conditions (Luo et al., 2022).NSC-derived exosomes, and have been proven useful for treating multiple neurological diseases due to their anti-inflammatory, neurogenic and neurotrophic effects as well as the interaction with the microenvironment of the brain tissue (Vogel et al., 2018).Previous studies suggested that application of NSC-derived exosomes could promote the differentiation of NSCs through miRNAs in vitro (Yuan et al., 2021).However, the effect of exosomes on grafted NSCs in vivo remains elusive.We propose that the combined treatment of exosomes and NSCs can effectively ameliorate harsh lesion conditions to help the NSCs survival and differentiation, achieving optimal treatment effects.
In this study, we established ischemic stroke in mice with MCAO/R, and tested different treatment strategies using transplantation of iPSC-induced NSCs and NSCderived exosomes.Our results indicated that NSC-derived exosomes could promote NSCs differentiation, reduce the oxidative stress and inflammation, and alleviate the formation of glial scars after ischemia and reperfusion, and as a result, could enhance the therapeutic effects of NSC transplantation.We further explored the molecular mechanisms through profiling the miRNAs of the NSC-derived exosomes.RESULTS

NSC-derived exosomes facilitated post-stroke recovery after NSC transplantation in MCAO/R mice
We first characterized the NSCs derived from iPSCs by examining the expression of NSC marker genes including SOX2 and PAX6 by immunocytochemistry staining.
The results showed that the cells used for subsequent transplantation expressed high level of NSC marker genes (Figure 1 -supplement 1A), indicating that NSCs were efficiently induced from iPSCs.We isolated exosomes from the same NSCs and examined the expression of exosomal markers including TSG101, CD63, and CD9 (Figure 1 -supplement 1B).Furthermore, the results of transmission electron microscopy (TEM) showed that the particle size of exosomes mixture was less than 200 nm, and Nanoparticle Tracking Analysis (NTA) confirmed the typical distribution of particle diameter of exosomes (Figure 1 -supplement 1C and D).
We next examined the effects of different treatment strategies on the brain lesion after cerebral ischemia and reperfusion in MCAO/R mice.To examine the presence and persistence of cerebral edema, TTC staining was performed 1 and 7 days after MCAO/R (Figure 1 -supplement 1E).Two doses of NSC transplantation, 2×10 5 and 5×10 5 , were first tested and the results of survival curve (Figure 1 -supplement 1F) and rotarod test (Figure 1 -supplement 1G) showed a dose-dependent effects of transplanted NSCs.Therefore, we determined to use the dose of 5×10 5 NSCs for the subsequent treatments to achieve a robust therapeutic effect.Mice were randomly divided into five groups (Sham, PBS, Exo, NSC and NSC+Exo).Except Sham group, mice in all the other four groups received standard MCAO/R surgery.Lateral ventricle injections of 5 µL PBS (PBS group), 10 µg exosomes in 5 µL PBS (Exo group), 5 × 10 5 NSCs in 5 µL PBS (NSC group), and 5 × 10 5 NSCs + 10 µg exosomes in 5 µL PBS (NSC+Exo group) were performed at 7 days post-MCAO/R (Figure 1A).The levels of reactive oxygen species (ROS) and inflammation were measured in focal brain tissues at 3 days post treatment; behavioral assessments were performed at 0 to 8 weeks post treatment; histological examinations were analyzed at 8 weeks post treatment (Figure 1A).To ensure the successful establishment of cerebral ischemia, the cerebral blood flow was examined before, during and after MCAO/R (Figure 1B).Neurological functions were evaluated by balance beam, ladder lung, rotarod test and Modified Neurological Severity Score (mNSS) up to 8 weeks after treatment (Figure 1C and  combined with exosomes began to take effect starting at 4 weeks after treatment, as evidenced by the results of balance beam, ladder lung and mNSS test, and significantly worked better than that solely with NSCs at 8 weeks post treatment (Figure 1C).The infarct area in the ipsilateral hemisphere was determined by MRI (Figure 1E) at 8 weeks post treatment.Compared to the severe damages of brain tissues in PBS group, mice treated by NSCs combine with exosomes showed significantly reduced infarct areas (Figure 1F).Meanwhile, the combination of NSCs and exosomes showed better protective effects on the brain tissue than either alone (Figure 1D), which was further confirmed by the results of brain weight analysis (Figure 1G).Therefore, our results indicated that NSC-derived exosomes could significantly enhance the therapeutic effects of NSCs on motor dysfunction and brain infarction in MCAO/R mice.
Furthermore, the NSCs-mediated therapeutic effects were greatly accelerated by addition of exosomes.

NSC-derived exosomes enhanced the therapeutic effects of NSCs on neuronal damage
We next examined the recovery of ischemia-induced neuronal damage of cerebral cortex in different treatment groups.The results of NeuN staining revealed that, compared to the mice treated solely with NSCs, the combination NSCs and exosomes significantly reduced the tissue loss from 14.32±3.52% to 7.57±2.59%(Figure 2A, B), while administration of exosomes alone showed no significant difference compared to PBS treatment (Figure 2B).The reduction of infarct area after combined treatment was also accompanied with improved dendritic density and length (Figure 2C, D), alleviated spines loss (Figure 2E), and increased complexity of neuronal projections (Figure 2F) in the cerebral cortex.Interestingly, although exosome treatment did not show robust therapeutic effects on behavior impairment and infarct area, the number of dendritic spines was significantly increased by exosome treatment in Exo group compared to that of PBS group (Figure 2E), suggesting that exosomes might play an important role in the recovery of neuronal complexity.MCAO/R mice had damaged pyramidal and granular cells with fuzzy cell contours as shown by Nissl staining (Figure 2G and Figure 2 -supplement 1).The addition of exosomes could further reduce the neuronal loss in the ipsilesional hemisphere on top of the effects of NSC transplantation.

Exosomes promoted the differentiation of transplanted NSCs
To investigate the regulatory effects of NSC-derived exosomes on transplanted NSCs, the differentiation rates of NSCs in the cerebral cortex in vivo were evaluated at 8 weeks after transplantation.Compared to the NSC group, the number of tdTomato positive NSCs was significantly increased in the NSC+Exo combined treatment group (Figure 3A, C).Among the tdTomato positive cells, Nestin + /tdTomato + cells were less in NSC+Exo group than the other groups (Figure 3A, D), while the number of Tuj1 + /tdTomato + cells was significantly higher in NSC+Exo group, which implied that exosomes could promote the differentiation of NSCs into neurons (Figure 3B, E).The TUNEL staining indicated that the excessive apoptosis cells after MCAO/R were also reduced by exosome transplantation (Figure 3 -supplement 1).Therefore, our data indicated that co-transplantation of exosomes could effectively facilitate the differentiation of transplanted NSCs in MCAO/R mice.

Exosomes promoted the microenvironment remodeling
Oxidative stress and global brain inflammation are closely involved in the progressing pathology after stroke (Hurn et al., 2007, Shi et al., 2019), which challenges the survival and colonization of transplanted NSCs (Li et al., 2017).We employed oxygen and glucose deprivation (OGD)/re-oxygenation (OGD/R) procedure on cultured NSCs to simulate the main pathogenesis of stroke, ischemia-reperfusion (Zhang et al., 2017, Yu et al., 2018).The results showed that OGD/R treatment could induce high level of oxidative stress in NSCs, whereas exosomes could reduce the production of ROS after OGD/R (Figure 4A and 4B).We further examined the expression of oxidative stress-related genes.The mRNA expression level of CHOP (endoplasmic reticulum stress marker) was reduced by exosome treatment after OGD/R (Figure 4C).Meanwhile, exosome treatment increased the expression of antioxidant genes NRF2, NQO1 and SOD2 (Figure 4C).Besides the in vitro OGD/R experiments, the level of oxidative stress in vivo was also determined at 3 days post stroke, and the results showed that the MDA content was significantly decreased in exosome-treated mice (Figure 4D).Therefore, our data suggested that NSCs-derived exosomes could ameliorate oxidative stress, which could potentially facilitate the survival, colonization and differentiation of transplanted NSCs.
Heterologous stem cells transplantation could induce robust inflammatory response.
It has been reported that the proliferation of immune cells reaches the peak during the acute phase post-transplantation (Graf andStern 2012, Boncoraglio et al., 2019).Interestingly, our results showed that exosomes could reduce the expression of inflammatory cytokines including TNF-α and IL-1β, while increase the expression of anti-inflammatory cytokine IL-10 in NSCs after OGD/R (Figure 4F) suggesting that exosomes could alleviate the elevated immune response after NSC transplantation.
After brain tissue damages caused by conditions such as cerebral ischemia and reperfusion, glial scars are usually formed, which can reestablish the physical and chemical integrity of the brain tissue by generating a barrier across the injured area, but inhibit the neuronal recovery as well (Michinaga and Koyama 2021).Astrocytes are the main cellular component of the glial scar, and our results indicated that astrocytes were prone to form glial scars during the chronic phase after stroke (Figure 4E and Figure 4 -supplement 1A).We subsequently investigated the effects of different treatments on the formation of glial scars in MCAO/R mice.The results suggested that the combined treatment of NSCs and exosomes significantly decreased the glia scars in the subacute phase (Figure 4 -supplement 1B) and the chronic phase (Figure 4E and 4G).

miRNA profiling and functional enrichment analysis of NSC-derived exosomes
To explore the underlying molecular mechanisms of exosomes regulating the transplanted NSCs and the microenvironment, we proposed that the exosomes might regulate target genes through the release of miRNAs, components of the key functional molecules carried by exosomes.Therefore, we profiled the miRNA expression of NSCderived exosomes using miRNA microarray.A total of 850 known miRNAs were detected, and the top 10 miRNAs with the highest read counts were displayed and verified by qPCR (Figure 5A and Figure 5 -supplement 1A).Targetscan, miRcode and miRDB databases were used to predict the downstream targets of the top 10 abundant miRNAs, and 17 potential relative target genes were selected, which have been proved to play important roles in neural modulation.The interactive network of the exosomal miRNAs and the selected target genes were analyzed and visualized using Cytoscape (Figure 5B).Target genes were predicted to be regulated by multiple miRNAs, among which hsa-miR-30a-5p and hsa-miR-7-5p were involved in multiple regulation.We next examined the effects of exosome treatment on the expression of candidate target genes in NSCs after OGD/R by RT-qPCR using STAT3, PTPN1 and CHUK as examples (Figure 5 -supplement 1B) (Park et al., 2012, Wang et al., 2018, Culley et al., 2019).The results showed that exosomes reduced the expression of downstream genes in NSCs, which was consistent with our hypothesis that exosomes regulate the recipient cells through carrying miRNAs that downregulate the target genes.
To further understand the regulatory effects of exosomal miRNAs on NSCs and the microenvironment, we performed GO enrichment analysis and KEGG pathway analysis on all the potential target genes.GO enrichment analysis, in terms of biological process (BP, Figure 5C), molecular function (MF, Figure 5D) and cellular component (CC, Figure 5E), disclosed that the potential target genes were enriched in functions .that were correlated with cellular and microenvironmental homeostasis of the central nervous system such as regulation of neuron death and neurogenesis, stress−activated protein kinase signaling cascade, cytokine receptor binding, and neuron spine.KEGG pathway analysis suggested that the target genes were mainly involved in inflammation and apoptosis-related signaling pathways (Figure 5F).Therefore, the predicted target genes of exosomal miRNAs were concentrated in the functions and pathways that could regulate the cellular behavior of transplanted NSCs as well as the microenvironment remodeling.
Taken together, our findings suggested that NSC-derived exosomes might regulate the transplanted NSCs and the surrounding microenvironment through carrying the miRNAs which could further modulate the downstream genes and pathways in both the NSCs and the surrounding cells (Figure 6).

DISCUSSION
Stem cell-based therapy is an emerging and promising method to treat stroke, due to its effectiveness in cell replacement, neuroprotection, angiogenesis, and modulation on inflammation and immune response (Hao et al., 2014a), but poor survival and differentiation of grafted cells have limited its efficacy and application (Jiang et al., 2019).In the present study, we co-transplanted NSCs-derived exosomes with NSCs in MCAO/R -induced cerebral ischemia in mice.Consistent with previous reports (Wei et al., 2017, Zhang et al., 2019), our results confirmed that NSCs could effectively promote the recovery of motor function post stroke in mice.Importantly, we demonstrated that exosomes could promote the repairment of the damaged brain tissue The pathological process of ischemia-reperfusion includes the generation of ROS, brain edema, and the increased levels of inflammation, which leads to the tough microenvironment for the transplanted stem cells to survive and differentiate (Zhang et al., 2019).In addition, transplanted allogeneic stem cells also exacerbate oxidative stress levels.Yahata, T et al found that transplantation of human hematopoietic stem cells triggers replication stress and induces increasing ROS levels in mice (Yahata et al., 2011).Bone marrow mesenchymal stem cells (BMSCs) transplantation has also been reported to increase oxidative stress levels in mouse muscle cells (Liu et al., 2019).
Besides the oxidative stress, the chemokines released by macrophages and endothelial cells after stroke, such as chemokine group CXC ligand 1 (CXCL1), recruit peripheral immune cells to flood into the damaged brain, which causes immune-inflammatory damage (Ormstad et al., 2011).Meanwhile, transplantation of exogenous stem cells could aggravate the inflammatory response around infract area due to the immune-rejection (Hao et al., 2014b).Xia et al reported that ESC-derived exosomes decrease the inflammatory response, alleviate neuronal death, and improve long-term recovery after MCAO/R through increasing regulatory T cells (Xia et al., 2021).Due to the properties of regulating signaling pathways in target cells, and remodeling the microenvironment (Vogel et al., 2018), NSCs-derived exosomes have been demonstrated to improve a variety of neurological diseases, such as Alzheimer's disease (Liu et al., 2020), spinal cord injury (Ma et al., 2019), and ischemic stroke (Sun et al., 2019).Recent evidence demonstrated that exosomes promote the maturation of both neuron and glial cells in vitro (Yuan et al., 2021).Furthermore, excessive initiation of apoptosis has also been implicated in stroke (Hwang et al., 2013).Here we showed that NSC-derived exosomes could reduce the oxidative stress and the inflammatory response, and promote the differentiation of transplanted NSCs and reduce excessive apoptosis in MCAO/R mice.Therefore, our results indicated that exosomes could promote the therapeutic effects of transplanted NSCs at multiple levels.
Previous studies have shown that stem cell-derived exosomes had neural protective effects and could promote recovery after ischemic stroke (Webb et al., 2018, Sun et al., 2019, Xia et al., 2021).However, we did not observe significant therapeutic effects with solely exosome treatment, which could be due to the dose of exosomes, the treatment timing and frequency.Considering the fact that cell transplantation requires a relatively stable microenvironment (Nih et al., 2017, Lee et al., 2018), we transplanted NSCs and exosomes at 7 days after stroke without subsequent delivery of exosomes in .CC-BY 4.0 International license perpetuity.It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in this study.Although the delivery of exosomes alone used in this study did not show significant neural protective effects, it indeed ameliorated oxidative and inflammatory lesion conditions, promoted neuronal repairment, and potentiated the therapeutic power of transplanted NSCs, suggesting that the application of exosomes could be an effective adjuvant for NSC-based therapy.Besides, as exosomes are ideal carriers for drug delivery (Chen et al., 2021), modifications of exosomes by adding drugs or other functional molecules could potentially further enhance the beneficial effects of exosome treatment.
As miRNAs were reported to be one of the major exosomal components, we profiled the miRNAs from NSCs-derived exosomes to explore the molecular basis for the effects of exosomes as we observed in this study.Bioinformatic enrichment analysis in this study suggested that the predicted garget genes of exosomal miRNAs were concentrated in the functions and pathways that could regulate the NSCs' behavior and the microenvironment.Interestingly, inflammation and oxidative stress-related genes and signaling pathways were also highly enriched in the target genes, consistent with the antioxidant role of exosomes as disclosed by our study.We, therefore, provided clues and a useful resource of exosomal miRNAs and predicted target genes for understanding the mechanisms underlying the function of exosomes in the NSC-based therapy for ischemic stroke.The roles and working model of the exosomal miRNAs as well as predicted target genes demand further exploration.

Key resources table
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Identifiers Additional information
Cell line (Homo sapiens) iPSCs (Cai et al., 2021) Commercial Male C57BL/6 mice (age: 7 -8 w, weight: 22 -24 g) were selected due to estrogen and progesterone have recognized neuroprotective effects (Newton et al., 2022).All animal procedures were performed in compliance with guidelines for the care and use of animals and were approved by the University Huazhong Agriculture Institutional Animal Care and Use Committee (Approval number: HZAUMO-2021-0111).Mice were assigned to MCAO/R or sham operation, and accepted NSCs or exosome treatment.

NSCs induction and culture
NSCs induction was performed by iRegene Therapeutics, Wuhan, China as previously reported (Cai et al., 2021).Briefly, Human iPSCs were cultured with STEMdiff Neural Induction Medium (Stemcell Technologies).The medium was replaced daily until day 9.After the first passage, Y-27632 was added to the medium .on day 1 to ensure the cell attachment and then removed from the medium on day 2. NSCs were cultured in STEMdiff Neural Progenitor Medium (Stemcell Technologies) to maintain cell growth after passage.NSCs were identified by immunofluorescence staining and qPCR.

Exosomes isolation and detection
Cellular debris were removed from cell culture supernatant at 2000 g for 10 min.
The supernatants were centrifuged at 2,0000 g for 30 min.Then, exosomes were collected by ultracentrifugation (Beckman, America) at 100,000g for 120 min.Finally, exosomes were washed in 12 ml PBS and collected again for 90 min.Exosomes were resuspended in PBS and protein concentration was measured by BCA assay (Biosharp, China).For observation by transmission electron microscopy, exosomes were fixed in 2.5% glutaraldehyde at 4 °C overnight and then mounted on a copper grid, stained with 2% uranyl acetate, and examined with a transmission electron microscope (Japan) with 100 kV.For nanoparticle tracking analysis, exosomes were examined by Malvern Nano ZS90 as previously described (Shi et al., 2018).Exosomes were diluted in PBS and 1.0 mL suspension was loaded into a cuvette to measure and analyze.

MCAO/R model
Ischemic stroke was established with MCAO/R surgery on male C57BL/6 mice (age: 7-8weeks, weight: 22-24g).Mice were anesthetized with 2% isoflurane (RWD, China).For focal cerebral ischemia, a silicon-coated filament (RWD, China) was inserted into the left middle cerebral artery (MCA) to block blood flow.Sixty minutes later, the filament was extracted for reperfusion.Rectal temperature was maintained at .37°C during the entire procedure.Then anesthesia was discontinued and mice were allowed to recover.The cerebral blood flow (rCBF) was detected using a laser doppler flowmetry (Perimed, Sweden).A 55% decrease in the rCBF of the ipsilateral hemisphere, as compared to contralateral hemisphere, was considered the threshold for successful establishment of cerebral ischemia.Mice of the Sham group were performed the same as the MCAO/R procedure without filament insertion.

Delivery of NSCs and exosomes
Eighty-eight MCAO/R mice were randomly divided into 4 groups at 7 days post operation, and twelve mice with low body weight (less than 15g) were excluded.Mice were anesthetized and placed in a mouse stereoscopic apparatus (RWD, China).The skull was drilled to make a burr hole above the lateral ventricle (AP+0, ML-1, DV-2.25 mm) for NSCs and exosomes injection.NSCs were genetically labeled with tdTomato for cell tracking.The five groups were treated as follows: Model (MCAO/R mice treated with 5 µL PBS), Exo (MCAO/R mice treated with 10 µg exosomes in 5 µL PBS), NSC (MCAO/R mice treated with 5×10 5 NSCs in 5 µL PBS), NSC+Exo (MCAO/R mice treated with 5×10 5 NSCs combine with 10 µg exosomes in 5 µL PBS) and Sham (fifteen mice with sham operation not treated).
Stroke leads to damage in the cerebral cortex, the atrophy was more severe without treatment, therefore we chose to observe the neuron recovery corresponding to the atrophied area in the model group.For staining of mice brain tissues, mice were anesthetized and immediately perfused with PBS followed by 10% formalin for 30 min.
Brains were fixed overnight in fixative at 4 °C.Fixed brains were dehydrated in 30% sucrose in PBS for 2 days at 4 °C.Brains were embedded in the OCT compound (SAKURA, Japan).Brain sections were obtained at a thickness of 25 µm using a microtome cryostat (Leica, Germany).Tissues were permeabilized, blocked and incubated as the above-mentioned protocol for cultured cell staining.Tissues were incubated with primary antibodies, including anti-NeuN (1:300, Abcam, catalog ab177487), anti-GFAP (1:1000, Cell signaling technology, catalog 3670S), anti-ß-IIItubulin (1:300, Cell signaling technology, catalog 5568S) or anti-Nestin (1:250, Santa Cruz Biotechnology, catalog Sc-23927).For TUNEL staining, in situ cell death detection kit (Roche, Germany) was used to detect the cell apoptosis according to the manufacturer's instructions.Briefly, 3% BSA incubated sections were incubated with .TUNEL reaction mixture for 1h at 37 °C in the dark.Then sections were incubated with anti-NeuN primary antibody (1:300, Abcam, catalog ab177487) and corresponding secondary antibody successively.

Motor function assessment
Testing on balance beam, ladder rung, rotarod test and mNSS tasks was conducted preoperatively, and at 1 to 8 weeks postoperatively.Investigators were blinded to treatment groups in test.

Balance beam:
The balance beam apparatus used in this study was a 10 mm square wood in width and 50 cm wood in length (Beijing Zhongshi Science, China).Mice were trained to pass through the balance beam 3 days before the MCAO/R procedure.The mice that successfully passed the beam without foot slips were recruited and grouped.anesthetized and sacrificed, and the brains were removed quickly and immersed in the mixture of Solution A and B for 2 weeks at room temperature in the dark.The brain was then transferred into Solution C for 48 h at 4 °C.Sections were cut with 100 µm thickness using a concussion slicer (Lecia, Germany) and stained with D and E mixture.
Images were captured by an inverted microscope using Z-stack images (Lecia, Germany).Golgi-stained neurons were reconstructed using Fiji-Image J.The total dendritic length, the number of dendritic spines and intersections were calculated and analyzed by Sholl analysis according to the previous study (Yang et al., 2020).

Nissl staining
Nissl staining was conducted using the Nissl Stain Solution (Solarbio, America) according to the manufacturer's instructions.Mice brain sections were stained with methylene blue stain for 10 min at 65°C, then differentiated by nissl differentiation solution for 3 min.The brain sections were subsequently treated in ammonium molybdate solution for 5 min followed by a quick rinse quickly in distilled water to avoid decolorizing.Images were taken using an inverted microscope (Lecia, Germany).

Oxygen-glucose deprivation and reoxygenation (OGD/R)
To perform OGD/R on cultured NSCs, the normal culture medium was replaced with Dulbecco's modified of eagle's medium (Solarbio, China).The culture was then incubated in a hypoxia chamber aerated with 5% CO2, 94% N2 and 1% O2 at 37° C for 2 h.Then the NSCs were transferred back into the normal culture medium and incubated in normal culture conditions for 24 h.

Intracellular ROS detection
. ROS level was detected using the fluorescent probe DCF-DA (Invitrogen, America).
Cultured NSCs were incubated with 10 µM DCF-DA for 30 minutes at 37 °C and then fixed with 4% paraformaldehyde.DCF fluorescence was photographed and quantified via a spinning disk confocal microscope (Andor Technology, UK).

MDA level measurement
The MDA level was measured at 3 days after treatment by the TBA method (Nanjing Jiancheng Bioengineering Institute, China) according to the manufacturer's instructions.The ipsilateral brain was homogenized and incubated in the assay solution at 95°C for 80 min.The optical density was measured at 532 nm by a microplate reader.
The value was calculated based on the standard formula.

Microarray analysis of exosomal miRNAs
Sequencing libraries of miRNAs of NSC-derived exosomes were produced using NEBNext Multiplex Small RNA Library Prep Set for Illumina (NEB, United States) following the previous report (Cai et al., 2021).
The downstream target genes of exosomal miRNAs were predicted using three online databases: Targetscan (http://www.targetscan.org/),miRcode (http://www.mircode.org/)and miRDB (http://mirdb.org/).The enrichment analysis of the predicted target genes was performed using ClusterProfiler R package for GO process and KEGG pathway enrichment.The miRNA-mRNA regulatory network was built by Cytoscape software.miRNA was calculated by qPCR.Libraries were prepared by ligating adaptors to the total RNA, PCR amplification and size selection using 6% polyacrylamide gels.Sequencing was performed on Illumina NovaSeq 6000 (Illumina Inc, USA).

Statistics
GraphPad Prism version 7 was used for statistical analyses.Unpaired t-tests (twotailed) were used for single comparisons, and two-way ANOVA was used for multiple comparisons.Survival analysis was performed via the Kaplan-Maier method.All data are presented as mean ± SEM.

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as well as the functional recovery, enhance the differentiation of the grafted NSCs in the infarct area, reduce the oxidative stress and inflammation, and alleviate the formation of glial scars in MCAO/R mice.As a proof-of-concept study on the codelivery of NSCs together with exosomes in the classical animal model of ischemic stroke, our study provided solid rationale supporting the application of exosomes during stem cell-based therapy.On the other hand, whether exosomes from different sources have similar effects on transplanted NSCs and how exosomes regulate other types of stem cells in vivo deem further exploration.

Figure 1 .
Figure 1.NSC-derived exosomes enhanced the therapeutic effects of NSCs on

Figure 1 -
Figure 1 -source data 1.NSC-derived exosomes enhanced the therapeutic effects of

Figure 2 .
Figure 2. Effects of combined treatment with NSCs and exosomes on neuronal

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Figure 2 -source data 1.Effects of combined treatment with NSCs and exosomes

Figure 3 .
Figure 3. Effects of NSC-derived exosomes on the differentiation of transplanted

Figure 4 .
Figure 4. Effects of exosomes on the microenvironment remodeling.

Figure 1 -
Figure 1 -supplement 2. Effects of different treatment strategies of NSCs and

Figure 1 -
Figure 1 -supplement 2-source data 1.Effects of different treatment strategies of

Figure 4 -
Figure 4 -supplement 1.Combined treatment of NSCs and exosomes reduced the

Figure 5 -
Figure 5 -supplement 1. Verification of top 10 abundant miRNAs and the

Figure
Figure 2 -supplement 1 International license perpetuity.It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in International license perpetuity.It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in International license perpetuity.It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in