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M2 microglia-derived small extracellular vesicles modulate NSC fate after ischemic stroke via miR-25-3p/miR-93-5p-TGFBR/PTEN/FOXO3 axis

Journal of Nanobiotechnology volume 23, Article number: 311 (2025) Cite this article

Abstract

Background

Endogenous neurogenesis could promote stroke recovery. Furthermore, anti-inflammatory phenotypical microglia (M2-microglia) could facilitate Neural Stem Cell (NSC)-mediated neurogenesis following Ischemic Stroke (IS). Nonetheless, the mechanisms through which M2 microglia influence NSC-mediated neurogenesis post-IS remain unclear. On the other hand, M2 microglia-derived small Extracellular Vesicles (M2-sEVs) could exert phenomenal biological effects and play significant roles in cell-to-cell interactions, highlighting their potential involvement in NSC-mediated neurogenesis post-IS, forming the basis of this study.

Methods

M2-sEVs were first isolated from IL-4-stimulated microglia. For in vivo tests, M2-sEVs were intravenously injected into mice every day for 14 days after transient Middle Cerebral Artery Occlusion (tMCAO). Following that, the infarct volume and neurological function, as well as NSC proliferation in the Subventricular Zone and dentate gyrus, migration, and differentiation in the infarct area, were examined. For in vitro tests, M2-sEVs were administered to NSC subjected to Oxygen-Glucose Deprivation (OGD) and then reoxygenation, after which NSC proliferation and differentiation were assessed. Finally, M2-sEVs were subjected to microRNA sequencing to explore the regulatory mechanisms.

Results

Our findings revealed that M2-sEVs reduced the infarct volume and increased the neurological score in mice post-tMCAO. Furthermore, M2-sEV treatment promoted NSC proliferation and neuronal differentiation both in vivo and in vitro. Additionally, microRNA sequencing revealed miR-93-5p and miR-25-3p enrichment in M2-sEVs. Inhibitors of these miRNAs prevented TGFBR, PTEN, and FOXO3 downregulation in NSC, reversing M2-sEVs’ beneficial effects on neurogenesis and sensorimotor recovery.

Conclusions

M2-sEVs increased NSC proliferation and neuronal differentiation, and protected against IS, at least partially, via delivering miR-25-3p and miR-93-5p to downregulate TGFBR, PTEN, and FOXO3 expression in NSC.

Graphical abstract

Introduction

Ischemic stroke (IS), a severe medical condition resulting from cerebral artery blockage, significantly impacts the Central Nervous System (CNS), leading to permanent neurofunctional impairments [1]. Notably, the global IS-related death rate rose from 2.04 million in 1990 to 3.29 million in 2019 and is expected to reach ~5 million by 2030 [2], imposing a substantial economic burden on both families and society [3]. Despite significant advancements in preventive, diagnostic, and treatment strategies, ~50% of IS survivors still experience neurological deficits [4, 5], making the development of more effective treatment strategies imperative.

Sudden ischemia and hypoxia following IS have been established to primarily impact neurons, before causing secondary damages such as microglial activation, Blood-Brain Barrier (BBB) breakdown, and an inflammation cascade, which, in turn, could exacerbate neural tissue injury [6]. In the meantime, self-repair mechanisms including astrogliosis, microgliosis, and neurogenesis are often activated. Neurogenesis involves the creation of neurons via Neural Stem Cell (NSC) proliferation, migration, and differentiation [7]. Neurogenesis could accelerate neuronal regeneration and brain functional restoration in various CNS diseases, potentially alleviating neurological damage [8]. Nonetheless, continuous inflammatory responses post-IS could inhibit NSC survival and differentiation, greatly impeding the recovery processes [9].

Resident microglia are the primary immune cells and initial responders to IS-induced pathophysiological changes in the brain. In this regard, microglia activation could be crucial for NSC-mediated neurogenesis [10]. For instance, microglia-secreted Insulin-like Growth Factor (IGF-1), an atrophic factor, regulated neuronal survival sustenance [11]. Additionally, activated microglia with ramified or intermediate morphologies in the ipsilateral Sub-Ventricular Zone (SVZ) facilitated NSC proliferation and migration post-stroke [12]. Moreover, IL-4- or IL-13-stimulated microglia can communicate with NSC, promoting neurogenesis and facilitating brain tissue repair [13, 14]. It is also noteworthy that IL-4-stimulated microglia could enhance NSC proliferation and differentiation following IS via TGF-α delivery [15]. However, the mechanisms underlying microglia-induced modulation of NSC fate post-IS remain largely unknown.

Microglia-secreted paracrine substances are an important mechanism underlying their effects. Furthermore, small Extracellular Vesicles (sEVs), also known as exosomes, have recently been established to play vital roles in mediating the crosstalk between cells [16, 17]. Diverse cell types could secrete sEVs—membrane-enclosed vesicles 50–150 nm in size, encapsulating and delivering functional molecules including microRNAs (miRNAs), mRNAs, and proteins to recipient cells for cell-to-cell communication [18]. Microglia-derived sEVs also play significant biological roles. For instance, sEVs from IL-4 or IL-13-stimulated microglia, also referred to as M2 microglia-derived sEVs (M2-sEVs), demonstrated neuroprotective functions by modulating various cell fates across multiple CNS diseases [19, 20]. Furthermore, M2-sEVs could inhibit astrocyte proliferation and promote astrocyte transformation into neuronal progenitor cells, thus reducing glial scar formation post-stroke [21]. Additionally, M2-sEVs can repair injured pericytes and cerebrovascular endothelial cells by restoring BBB integrity and reducing tight junction breakage, thereby attenuating brain edema and injury [22]. Moreover, M2-sEVs could enhance Oligodendrocyte Precursor Cell (OPC) proliferation, survival, and differentiation, thus aiding in myelin repair post-IS [23]. Therefore, considering the significance of M2 microglia in neurogenesis regulation following CNS injury and the fact that microglia-derived sEVs might exert multifaceted biological effects, we speculated that M2-sEVs could promote neurogenesis post-IS.

Herein, we aimed to comprehensively establish whether and how M2-sEVs affect NSC fate post-IS. Our findings revealed that the exogenous supplementation of M2-sEVs enhanced NSC-mediated neurogenesis both in vivo and in vitro post-IS, thus accelerating neurofunctional recovery in mice following tMCAO. Mechanistically, our findings also suggested that M2-sEVs might regulate NSC fate by delivering miR-25-3p and miR-93-5p, which partially inhibit TGFBR, PTEN, and FOXO3 expression. Overall, this study revealed that sEVs crucially modulate the crosstalk between microglia and NSC post-IS, highlighting a potential therapeutic target for treating IS among other CNS diseases.

Materials and methods

M2 microglia culture and identification

The BV2 microglia cells used in this study were acquired from Shanghai Zhong Qiao Xin Zhou Biotechnology Company (#ZQ-0397, ZQXZ-bio, China). The microglia were stimulated with 1 ug/ml Lipopolysaccharides (LPS) (#L2880, Sigma) for 48 h and set aside as the control group. Another batch was activated with 20 ng/ml IL-4 (#CK74, Novoprotein) for 48 h and transformed into M2 microglia. The proportion of M2-microglia was then assessed using flow cytometry, Immunofluorescence (IF) analysis, and quantitative Real-time Polymerase Chain Reaction (RT-qPCR). Finally, the microglia were cultured in DMEM supplemented with 10% Fetal Bovine Serum (FBS) without sEVs (#10099141, Gibco). To remove the EVs, the FBS was centrifuged at 100,000×g for 18 h and then inactivated via 30-minute incubation at 56℃.The cell culture supernatants were collected for EVs isolation [23]. Before isolating EVs, all conditioned media were kept at − 80 °C.

M2-sEV isolation

EV Isolation: EVs were isolated and purified via differential ultracentrifugation, conducted as previously described [24]. Briefly, the supernatants from M2-microglia cultures were first centrifuged at 300×g for 10 min, and then at 2000×g for another 10 min to clear out the cells. Following that, the collected supernatants were centrifuged at 10,000×g for 30 min to remove cell debris. Subsequently, they were filtered with a 0.22 μm filter and centrifuged at 100,000×g for 90 min. The resulting pellets were transferred to a new tube and then centrifuged again at 100,000×g for 90 min. The pellets were ultimately resuspended in Phosphate-Buffered Saline (PBS) or stored at − 80 ℃, awaiting subsequent tests, which were conducted within two weeks. All centrifugations were performed at 4 ℃ using a tabletop ultracentrifuge (Optima MAX-XP, Beckman Coulter, USA).

Animal and MCAO procedures

All animal experiments adhered to the guidelines of the Institutional Animal Care and Use Committees of the Central South University, Hunan, China (No. 202110093). Animal experiments were performed at Xiangya Hospital using 70 male C57/BL6 mice (age = 2–3 months; weight = 25–30 g), which were housed at the hospital’s animal facility. The animals were randomly assigned to three groups (Sham, MCAO, and MCAO + M2-sEVs-treatment) using a lottery box. Another assistant blinded to the groupings performed the MCAO procedure. The animals were then subjected to a transient MCAO (tMCAO) procedure to replicate IS as described in previous research [25]. Briefly, anesthesia was first administered to the mice using 5% isoflurane. After achieving surgical anesthesia (confirmed by the absence of toe-pinch reflex), the isoflurane concentration was reduced to 1.5% during the MCAO procedure. To stop blood perfusion, a silicon suture was placed into the middle cerebral artery for 60 min, followed by reperfusion. After surgery, the mice were returned to their cages and accorded the necessary care. Regional Cerebral Blood Flow (rCBF) was recorded during MCAO surgery. The death rate was ~10%, and mice that died following MCAO or had an rCBF reduction < 50% post-suture insertion were omitted. Following reperfusion, 100 µL PBS or a serial M2–sEV concentration (1.0 × 10^7, 5.0 × 10^7, 1.0 × 10^8, 1.0 × 10^9, and 1.0 × 10^10 in 100 µL of PBS) was administered intravenously on days 0, 2, 4, 6, 8, 10, 12, and 14. The animals were euthanized at designated time points for tissue collection as follows: Ten/group on day 14, eight/group on day 24 for immunostaining studies, and four/group on day 28 for protein extraction.

Oxygen-glucose deprivation (OGD), reoxygenation, and sEV treatment

To mimic ischemic-like conditions in vitro, NSC were cultured in glucose-free DMEM supplemented as in the proliferation medium. They were then exposed to anaerobic conditions (5% CO2, 95% N2) for 8 h to induce OGD. Subsequently, OGD was terminated by changing the medium with glucose and restoring normoxic conditions. The NSC were then cultured for an additional 24 h, either without M2-sEVs (vehicle) or with them at a concentration of 1 × 10^9 particles/mL. A separate group of control NSC which received no treatment was maintained under normal conditions. The cells were finally harvested for further analysis.

M2-sEV MicroRNA expression profiling and bioinformatics analysis

First, miRNAs from M2-sEVs were extracted using an miRNeasy Mini Kit (Qiagen, Hilden, Germany) as outlined in our previous study [17]. Microarray analysis was then conducted on an Illumina NextSeq 500 platform (Illumina, San Diego, CA, USA) after confirming the RNA samples’ concentration and purity. Subsequently, the results were examined using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). Gene Ontology (GO) enrichment analysis was performed using FunRich software (Version 3.1.3). Neuronal-related miRNAs were identified based on annotations from miRTarBase, and neurogenesis-related miRNAs were obtained as outlined in previous research [17].

Delivery of MiRNA inhibitors to M2-sEVs

Inhibitors targeting miR-25-3p and miR-93-5p, and a negative control were acquired from GenePharma (Shanghai, China). Each nucleotide in the inhibitors had 2’-O-Me modifications at every base and included an amino linker with 5’-Cy3. Table 1 details the sequences of the miRNA inhibitors, which were transferred into M2-sEVs using an Exo-Fect™ siRNA/miRNA Transfection Kit (System Biosciences, Mountain View, CA, USA), per the manufacturer’s instructions.

Table 1 Sequences of MiRNA inhibitor
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Other methods

Supporting information covers other details, including Primary NSC isolate and culture, flow cytometry analysis, M2-sEV identification, EdU administration, rCBF measurement, immunostaining, infarct volume evaluation, behavioral test neurological score measurement, uptake of DiI-labelled M2-sEVs by NSC, and uptake of DiR-labelled M2-sEVs in mice, detection of NSC proliferation both in vitro and in vivo, Western Blot (WB) analysis, and RT-qPCR.

Results

Isolation and identification of M2-sEVs

First, we determined the characteristics of M2-microglia cells that were induced with IL-4. LPS induction to obtain M1-microglia cells. Microglia polarization across different groups was assessed using flow cytometry, IF staining, and RT-qPCR. According to flow cytometry analysis, CD86 (an M1 marker) was expressed in 76.6% of cells in the LPS-treated group and in only 0.69% of cells in the IL-4-treated group. Conversely, CD206 (an M2 marker) was only expressed in 23.4% of cells in the LPS-treated group, compared to 83.1% of cells in the IL-4-treated group (Fig. 1A). Meanwhile, CD206 and iNOS expression was assessed via IF staining. Results showed CD206 was profoundly expressed while iNOS was almost undetectable in IL-4-treated microglia cells. Conversely, in LPS-treated microglia cells, iNOS was markedly upregulated while CD206 was almost undetectable (Fig. 1B). Furthermore, the transcriptional expression levels of polarization-related markers were quantified using RT-qPCR. According to the results, LPS-treated microglia cells exhibited a significant upregulation of M1-related genes (iNOS, TNF-α, IL-1β, and IL-6), from 10-fold to over 100-fold. Conversely, IL-4-treated cells exhibited no change in the expression of these genes. On the other hand, IL-4-treated microglia cells exhibited a significant upregulation of M2-related genes (Arginase-1 and CD206), from 15-fold to over 100-fold, whereas LPS-treated cells exhibited a much lower expression of these genes. These findings collectively suggest that IL-4 successfully induced microglia cell polarization towards the M2 phenotype (Fig. 1C-D). Subsequently, we isolated and identified M2-sEVs as outlined in Fig. 1E. Nano flow analysis revealed that a large portion of M2-sEVs exhibited diameters ranging between 30 and 120 nm (Fig. 1F). Additionally, WB analysis was employed to examine the expression of sEV-specific markers (CD63, CD9, and TSG101) in cellular and M2-sEV lysates, revealing significant CD9, CD63, and TSG101 expression in both fractions. Conversely, GM130, a Golgi membrane marker, was prominently detected in cellular lysates but was absent in the M2-sEV fraction (Fig. 1G). Transmission Electron Microscopy (TEM) further confirmed that the sEVs exhibited a characteristic cup-shaped membrane vesicle morphology.

Fig. 1
figure 1

Isolation and identification of M2-sEVs. (A-D) IL-4 was used to induce M2 phenotype differentiation of BV2 cells in vitro. PBS was added in the culture medium as control group and LPS was applied to induce M1 phenotype differentiation. (A) Representative images showing flow cytometry analysis of CD86+ or CD206+ cells in control, LPS and IL-4 groups. (B) Representative images of F4/80+/CD206+ M2 microglia and F4/80+/iNOS+ M1 microglia in control, LPS and IL-4 groups, respectively. Scale bar, 50 μm. (C-D) Quantification of mRNA expression of M1-related genes including iNOS, IL-6, IL-1β, and TNF-α and M2-related genes including Arg-1 and CD206 among control, LPS, and IL-4 groups. (E) illustration of isolation and identification of M2-sEVs. (F) Nano flow analysis of the size distribution of M2-sEVs. (G) WB analysis of GM130, TSG101, CD63, and CD9 in BV2 cells and M2-sEVs. (H) Representative image of transmission electron microscope (TEM) showing the morphology of M2-sEVs. Scale bar at the lower left, 200 nm; Scale bar at the upper right, 100 nm

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M2-sEVs reduced infarct volume and improved sensorimotor function recovery for up to 14 days after stroke

We subsequently assessed the neuroprotective role of M2-sEVs against IS. To establish whether M2-sEV treatment impacted brain atrophy in sham-operated mice, PBS or M2-sEVs were administered via tail veil injection from 6 h after sham operation and every day until day 14. According to the Nissl staining results, the Sham + PBS and Sham + M2-sEVs groups showed no significant difference in brain atrophy (Figure S1). Given that sEVs can home to injured tissues [18], we further sought to establish whether M2-sEVs localized to injured brain tissues by injecting the mice with DiR or DiO-labelled M2-sEVs. After 24 h, we examined multiple key organs and found that the liver, lungs, spleen, and intestines contained most of the fluorescent signals (Figure S2). A partial fluorescence signal was detected ipsilateral to the injury site, suggesting that intravenously delivered M2-sEVs can traverse the BBB, selectively localize to damaged brain regions, and be internalized by NSCs (Figures S2 and S3). To determine the optimal dose, MCAO mice received several doses of M2-sEVs via tail vein injection (Fig. 2 A). According to MRI findings, the animals exhibited no notable differences in cerebral infarction three days post-MCAO. Furthermore, based on the Nissl staining results, compared to mice injected with PBS or a lower dose of sEVs (< 1 × 10^9 particles), mice treated with 1 × 10^9 and 1 × 10^10 particles of M2-sEVs exhibited a significant reduction in cerebral infarction volume 14 days post-MCAO. Additionally, treatment group mice that received 1 × 10^9 and 1 × 10^10 particles of M2-EVs showed no significant difference in cerebral infarction volume reduction. Therefore, 1 × 10^9 particles of sEVs were adopted as the optimal dose for subsequent experiments (Fig. 2B-D). To confirm M2-sEVs’ beneficial role in sensorimotor function recovery, behavioral experiments including cylinder and adhesive tests were performed on days 3, 5, 7, 10, and 14 post-stroke, while MAP-2 staining was conducted at 14 days post-stroke (Fig. 2E). First, rCBF was assessed before, during, and at 5 min after the MCAO procedure, revealing no significant variation in rCBF at the infarct core in the Ipsilateral Side (IL) between the PBS and M2-sEV groups (Fig. 2F, I). This finding suggested a comparable change in blood flow in the two groups before, during, and after IS. On the other hand, the MAP-2 staining results revealed that compared to the MCAO + PBS group, the MCAO + M2-sEVs group showed significantly lower levels of brain atrophy at 14 days post-MCAO (Fig. 2G-H). We further evaluated neurological function, revealing no significant difference in the Neurological Score (NS) from days 1 to 3 between the MCAO + PBS and MCAO + M2-sEVs groups after MCAO (Fig. 2J). In the cylinder test, compared to the PBS group, the M2-sEV group exhibited a significantly lower asymmetric rate for up to 14 days post-MCAO (Fig. 2K). Additionally, in the adhesive test, compared to the MCAO + PBS group, the MCAO + M2-sEVs group spent significantly less time in contacting and removing the sticker for up to 14 days post-stroke (Figs. 2L-M). Overall, these findings suggest that M2-sEVs provided substantial protection against brain atrophy and enhanced neurofunctional recovery in mice with stroke.

Fig. 2
figure 2

M2-sEVs reduced infarct volume and improved sensorimotor function recovery up to 14 days after stroke. (A-D) Different concentrations of M2-sEVs and PBS were administered via tail vein injection starting 6 h after MCAO and every day for 14 days, MRI scanning was performed at day 3, and Nissil staining was conducted at day 14 after MCAO. (A) The design of the experiment. (B) Representative images of the coronal T2 MRI scanning of mouse brain 3 days after MCAO and Nissil staining 14 days after MCAO. Scale bar, 1 mm. (C-D) Quantification of the infarct volume among different groups 3 days after MCAO by T2 MRI scanning (C) and 14 days after MCAO by Nissil staining (D). (E-M) A different batch of mice were used, PBS or M2-sEVs (1 × 109 particles per injection) was injected via the tail veil as described above. Behavior tests including cylinder test and adhesive test were performed at day 3, 5, 7, 10, and 14 post-stroke, MAP-2 staining was conducted 14 days after stroke. (E) Illustration of experimental design. Representative images (F) and the corresponding quantification (I) of rCBF at the infarct core in ipsilateral side (IL) and the corresponding area at the contralateral side (CL) 5 min before suture insertion (pre), during suture blocking (during), and 5 min after suture removal (after). White and black dotted lines indicate ROI in the left and right brain, respectively. Scale bar, 1 mm. n = 7–8 per group. (G) Representative images of MAP-2 staining of serial coronal brain sections relative to the bregma 14 days after MCAO. The yellow dotted line indicates infarct border. Scale bar, 1 mm. (H) Quantification of brain atrophy 14 days after stroke. n = 10 per group. (J) Quantification of the neurological score on day 1, 2, and 3 after MCAO. n = 10 per group. (K-M) Quantification of asymmetric rate in the cylinder test (K), touch time (L) and removal time (M) in the adhesive test 3, 5, 7, 10, and 14 days following stroke. n = 10 per group

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M2-sEVs increased NSC proliferation and the number of new neurons at the SVZ at 14 days post-stroke

The number of neurons correlates positively with an enhanced neurological function following brain injury. Furthermore, NSC at the SVZ and Subgranular Zone (SGZ) can proliferate and differentiate into mature neurons [26]. Therefore, we evaluated NSC proliferation and maturation at the SVZ and SGZ. First, we examined the cell proliferation rate post-MCAO in different groups (Sham + PBS, MCAO + PBS, and MCAO + M2-sEVs). The EDU staining results revealed that M2-sEVs significantly increased cell proliferation around the SVZ (Figure S4 A-C). Furthermore, the number of EDU+ cells correlated positively with sensorimotor function recovery post-stroke (Figure S4 D-F). Subsequently, we explored the types of proliferating cells using Immunofluorescence (IF) staining to detect proliferating NSC (SOX2+/EdU+) and newborn neurons (DCX+/EdU+). According to the results, compared to the Sham + PBS group, M2-sEV treatment resulted in a significantly higher number of proliferating NSC and newborn neurons in the SVZ (Fig. 3A-F) and SGZ post-stroke (Figure S5). We also observed that the number of newborn neurons (DCX+/EdU+) and proliferating NSC (SOX2+/EdU+) correlated positively with sensorimotor function recovery post-stroke (Fig. 3G-L). These findings collectively suggest that M2-sEVs can promote NSC proliferation and maturation, thus ameliorating MCAO-induced neurological deficits.

Fig. 3
figure 3

M2-sEVs increased proliferated NSC and new-born neurons at subventricular zone (SVZ) 14 days following stroke. (A-B) Representative immunofluorescence staining images of Sox2/EdU (A) and DCX/EdU (B) in SVZ in the ipsilateral side of the brain 14 days after MCAO or Sham operation. ROI indicates region of interest. Scale bar, 50 μm. n = 8–10 per group (C) Illustration of EdU+ cells in the ROI. CC indicates corpus callosum. White dotted line delineate CC. Red dotted line represent infarct border. scale bar, 500 μm. (D-E) Quantification SOX2+/EdU + proliferated NSC (D) and DCX+/EdU + new-born neurons (E) in ROI 1, ROI 2, and ROI 3 respectively 14 days after MCAO or Sham operation. (F-G) Quantification of total SOX2+/EdU + NSC (F) and total DCX+/EdU + new-born neurons in SVZ. (G-I) Correlation analysis between the number of DCX+/EdU + cells and adhesive touch time (G), adhesive removal time (H), and asymmetric rate in the cylinder test (I) at day 14 after stroke. (J-L) Correlation analysis between the number of Sox2+/EdU + cells and adhesive touch time (J), adhesive removal time (K), and asymmetric rate in the cylinder test (L) at day 14 after stroke respectively

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M2-sEVs promoted the migration of new-born neurons from the SVZ to the infarct area and increased the number of mature neurons post-stroke

NSC’ neurological repair function correlates with their differentiation into newborn neurons, migration to the target destination, and neuronal maturation [27]. In this regard, we sought to establish whether M2-sEVs could enhance the migration of newborn neurons from the SVZ to the infarct area at 14 days post-stroke and promote neuronal maturation by day 28 post-stroke. First, we quantified newborn neurons (DCX+) migrating from the SVZ to the infarct area using IF. According to the results, compared to the Sham + PBS group, the MCAO procedure resulted in a higher number of newborn neurons in the SVZ, which was even significantly higher after M2-sEV treatment (Figure S6). Additionally, compared to the Sham + PBS group, the MCAO procedure enhanced the migration of new neurons from the SVZ to the edge of the infarct region, with the MCAO + M2-sEVs group exhibiting a significant increase. Furthermore, M2-sEV treatment increased the number of newborn neurons within the infarct core (Fig. 4A-C). Additionally, the number of newborn neurons (DCX+) in the SVZ and SGZ correlated positively with sensorimotor function recovery post-stroke (Fig. 4D-F). Subsequently, we quantified mature neurons (NeuN+) migrating from the SVZ to the infarct area using IF stain. Compared to the Sham + PBS group, the MCAO procedure resulted in a significantly lower number of mature neurons at the edge of the infarct lesion, which increased significantly following M2-sEV treatment. We also conducted EdU and NeuN co-staining to determine the origin of mature neurons. Results showed that there were higher levels of proliferating mature neurons (NeuN+/EdU+) within the border of the infarct lesion in the MCAO + M2-sEVs group. However, such proliferation was barely detected in the Sham + PBS or MCAO + PBS groups (Fig. 4G-J, Figure S7). These findings collectively suggest that M2-sEVs could regulate the migration of new neurons from the SVZ to the injured area and enhance their maturation within that area, thus facilitating neurological repair.

Fig. 4
figure 4

M2-sEVs promote migration of new-born neurons from SVZ to the infarct area and increased mature neurons after stroke. (A) Illustration of DCX+ cells in SVZ and infarct core. CC, CTX, STR, and LV indicate corpus collosum, cortex, striatum, and left ventricle respectively. Red dotted line represents infarct border. Scale bar, 150 μm. (B) Representative images of DCX+ cells in the ROIs 14 days following MCAO. Scale bar, 50 μm. n = 8–10 per group. (C) Quantification of DCX+ cells in the ROIs 14 days following MCAO. (D-F) Correlation analysis between the count of DCX+ cells in the ROIs and adhesive touch time (D), adhesive removal time (E), and asymmetric rate in the cylinder test (F). (G) Illustration of the experimental design. M2-sEVs or PBS were administered every day starting 6 h after MCAO until day 14, EdU was injected intraperitoneally on day 3, 5, 10, and 14, after which the mice were sacrificed 28 days following MCAO. (H) Representative images of NeuN+/EdU+ cells around the lesion border 28 days following stroke. Scale bar, 50 μm. (I-J) Quantification of NeuN+ cells and NeuN+/EdU+ cells. n = 8 per group. Arrow head indicates NeuN+/EdU+ cells

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M2-sEVs increased NSC proliferation and neuronal differentiation after OGD

We further explored the impact of M2-sEVs on NSC under OGD conditions, which mimic IS in vitro [24]. First, we isolated and identified mouse primary NSC which exhibited a spherical morphology and had high Nestin and Sox2 expression (Figure S8). We then monitored NSC’ uptake of M2-sEVs (Dil-labelled). Notably, M2-sEVs were detectable in the vicinity of NSC nuclei (Fig. 5A-B), indicating their effective uptake by NSC. We further subjected the Control + PBS, OGD + PBS, and OGD + M2-sEVs groups to WB and IF analyses to examine the beneficial role of M2-sEVs on NSC proliferation and maturation post-OGD. Following OGD treatment, NSC exhibited a decrease in the expression of the mature neuron marker (MAP-2) and an increase in the expression of the astrocyte marker (GFAP). Nonetheless, incubation with M2-sEVs reversed this effect. Moreover, IF results demonstrated that the OGD-induced suppression of NSC’ proliferative ability restored the M2-sEVs (Fig. 5C-H), implying that the proliferation of NSC could be hampered and that their differentiation might shift away from mature neurons towards astrocytes following OGD. We also found that M2-sEV treatment could rescue NSC proliferation and redirect NSC differentiation towards mature neurons. These findings collectively suggest that M2-sEVs could significantly boost NSC proliferation and neuronal differentiation post-IS.

Fig. 5
figure 5

M2-sEVs increased NSC proliferation and neuronal differentiation after OGD. (A) A diagram showing the experimental design for investigating the uptake of M2-sEVs by NSC in vitro. M2-sEVs or PBS were stained with DiI dye, and then ultracentrifugated to harvest M2-sEVs, and then M2-sEVs-DiI was cocultured with NSC, finally the cells was imaged under microscope. (B) Representative immunofluorescence images of DAPI and DiI in NSC. Scale bar, 50 μm. (C) A diagram showing the experimental design for exploring the proliferation and differentiation of NSC after OGD. (D) Representative WB bands and analysis of MAP-2 and GFAP proteins in NSC among different groups. n = 5 per group. (E) Representative immunofluorescence images of EdU and Nestin in NSC in the indicated groups. Scale bar, 50 μm. (F) Representative immunofluorescence images of GFAP and MAP-2 in NSC in the indicated groups. Scale bar, 50 μm. (G) Quantification of EdU+ proliferated cells and percentage of EdU+Nestin+ proliferated NSC in DAPI + cells. n = 5 per group. (H) Determining the percentage of MAP-2+ cells or GFAP+ cells in DAPI+ cells. n = 5 per group

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MiRNA sequencing analysis of M2-sEVs revealed some neurogenesis-related MiRNAs

Notably, sEVs often contain miRNAs that when secreted could end up modifying the behavior of recipient cells. To establish whether the enhancement of NSC proliferation and neuronal differentiation could be attributed to miRNAs within M2-derived sEVs, we subjected M2-sEVs to miRNA sequencing analysis. Gene Ontology (GO) analysis revealed that numerous miRNAs were enriched in neuronal-related Biological Processes (BPs), including neurogenesis, negative neuronal apoptosis regulation, neuronal migration, and neuronal differentiation(Fig. 6A-B). Furthermore, we intersected the miRNAs in M2-sEVs, encompassing neuronal- and neurogenesis-related miRNAs, and detected 24 shared miRNAs (Fig. 6C-D). We then validated the CT values of the top ten shared miRNAs in M2-sEVs using RT-qPCR. According to the results, miR-25-3p and miR-93-5p exhibited relatively higher expression levels (Fig. 6E). Subsequently, we assessed miR-25-3p and miR-93-5p levels in NSC after incubation with M2-sEVs (Fig. 6F). Compared to the PBS group, M2-sEV treatment resulted in significantly higher levels of miR-25-3p and miR-93-5p in NSC (Fig. 6F), implying that M2-sEVs could deliver miR-25-3p and miR-93-5p to NSC.

Fig. 6
figure 6

miRNA sequence of M2-sEVs revealed some neurogenesis-related miRNAs. (A) GO analysis of various miRNA in M2-sEVs. (B) Neuronal related GO analysis of miRNA in M2-sEVs. Bar represents number of miRNA and dot indicates significance (-log10 [adjusted p value]). (C) Venn diagram showing the neuronal and neurogenesis related miRNA in M2-sEVs. (D) Measurement of co-expressed 24 miRNA in M2-sEVs. (E) Quantification of the CT value of top 10 enriched miRNA in M2-sEVs by qPCR. n = 4 per group. (F) A diagram showing the experimental design and quantification of miR-93-5p and miR25-3p in NSC. Briefly, PBS or M2-sEVs was cocultured with NSC and miRNAs including miR-93-5p and miR25-3p in NSC were evaluated via qPCR. n = 4 per group

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Inhibition of miR93-5p and miR25-3p blocked the M2-sEV-afforded promotion of neurogenesis and infarct volume reduction

To establish whether the M2-sEVs-afforded promotion of neurogenesis was attributable to the delivery of miR-25-3p and miR-93-5p to NSC, we co-cultured M2-sEVs with miRNA inhibitors [17]. We aimed to suppress the expression of these miRNAs and verify whether they could block the effects of M2-sEVs on neurogenesis both in vitro post-OGD and in vivo post-MCAO. Consistent with the aforementioned results, M2-sEVs with the negative control inhibitor (M2-sEVs + NC) still had a significantly higher number of Nestin+/EdU+ proliferating NSC than the PBS group (Fig. 7A and E). They also promoted the differentiation of NSC into MAP+ neurons (Fig. 7B-C) instead of GFAP+ astrocytes (Fig. 7B and D) post-OGD. Meanwhile, compared to the OGD + M2-sEVs + NC group, when M2-sEVs-IN were introduced to NSC post-OGD, the NSC exhibited a slightly lower MAP2 expression, higher GFAP expression, and fewer proliferating NSC (Nestin+/Edu+). We then injected M2-sEVs-IN into MCAO mice and conducted IF staining. According to the results, compared to the MCAO + M2-sEVs + NC group, M2-sEVs-IN treatment lowered the ratio of proliferating NSC (SOX+/EdU+), proliferating newborn neurons (DCX+/EdU+), and proliferating cells in the SVZ to similar levels as in the MCAO + PBS group at 14 days post-MCAO (Fig. 7F-H, Figure S9, and Figure S10). Additionally, M2-sEVs-IN decreased the ratio of mature neurons (NeuN+/EdU+) at the border of the infarct lesion after 28 days in MCAO mice. Brain atrophy analysis with MAP-2 staining further revealed that compared to the MCAO + M2-sEVs + NC group, treatment with M2-sEVs-IN resulted in a higher volume of the infarct lesion after 14 days in MCAO mice. These findings collectively suggest that M2-sEVs could influence NSC proliferation, migration, and maturation, in a process partially mediated by miR-25-3p and miR-93-5p. Specifically, miR-25-3p and miR-93-5p inhibition partially blocked the promotional effect of M2-sEVs on NSC neurogenesis.

Fig. 7
figure 7

Inhibition of miR-93-5p and miR-25-3p blocked M2-sEVs-afforded promotion of neurogenesis and reduction of infarct volume. The effects of miR-93-5p and miR-25-3p in M2-sEVs (M2-sEVs-IN) were blocked by their inhibitor, with PBS or M2-sEVs without inhibitor serving as controls. (A) The immunofluorescence images of GFAP/MAP2 and EdU/Nestin in NSC after OGD. Scale bar, 50 μm. (B-D) Representative WB bands (B) and quantification of MAP2 (C) and GFAP (D) in NSC after OGD. (E) Quantification of the percentage of EdU+/Nestin+ proliferated NSC in total DAPI+ cells. (F) Representative immunofluorescence images of Sox2+/EdU+ proliferated NSC, and DCX+/EdU+ new-born neurons in SVZ 14 days following MCAO. Scale bar, 50 μm. (G-H) Quantification of Sox2+/EdU+ and DCX+/EdU+ cells in ROI1. n = 6–8 per group. (I) Immunofluorescence images of NeuN+/EdU+ cells at the lesion border 28 days following MCAO. Scale bar, 50 μm. (J-K) Quantification of NeuN+ (J) and NeuN+/EdU+ (K) cells. n = 8 per group. (L) Representative images of the MAP-2 staining of serial coronal brain Sect. 14 days following MCAO. Scale bar, 1 mm. (M) Quantification of brain atrophy. n = 9 per group

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M2-sEVs transferred miR93-5p and miR25-3p to downregulate TGFBR, PTEN, and FOXO3 in NSC

To explore the mechanisms underlying the miR-25-3p and miR-93-5p-mediated promotion of neurogenesis post-IS, we searched the miRBase database and found that miR-93-5p targets PTEN, TGFBR1, and TGFBR2, while miR-25-3p targets PTEN and TGFBR3. According to research, PTEN could activate FOXO activity and inhibit the insulin/IGF pathway, suppressing neurogenesis [28,29,30,31]. Additionally, TGFβ signaling could negatively regulate NSC proliferation and neurogenesis via TGFβ binding to its receptors (TGFBR1, TGFBR2, and TGFBR3), a process that could lead to TGFβ signal transduction from the cell surface to the cytoplasm [32, 33]. Consequently, we hypothesized that M2-sEVs transferred miR-25-3p and miR-93-5p to NSC, downregulating TGFBR, PTEN, and FOXO3, thus promoting NSC proliferation and differentiation. We further employed WB to assess TGFBR, PTEN, and FOXO3 expression both in vitro and in vivo following M2-sEV treatment. According to the results, there was a striking decrease in TGFBR, PTEN, and FOXO3 expression in the SVZ at 14 days following MCAO and M2-sEV treatment (Fig. 8A-C). Furthermore, TGFBR, PTEN, and FOXO3 expression decreased significantly when M2-sEVs were co-cultured with NSC post-OGD (Fig. 8D-F). However, treatment with M2-sEVs inhibitors (M2-sEVs-IN) partially reversed this downregulation (Fig. 8G-I). These findings suggest that M2-sEVs might enhance NSC proliferation and differentiation via the miR-25-3p- and miR-93-5p-mediated regulation of TGFBR, PTEN, and FOXO3 expression.

Fig. 8
figure 8

M2-sEVs transfer miR93-5p and miR25-3p to decrease the expression of TGFBR, PTEN, and FOXO3 in NSC. (A) A scheme of the experimental design. M2-sEVs or PBS were administered via tail veil injection following MCAO. The expressin of proteins in SVZ was determined by WB. (B-C) Representative WB bands (B) and quantification of protein level (C) of TGFBR, PTEN, and FOXO3 in NSC 14 days following MCAO. n = 4 per group. (D) A demonstration of the experimental design. M2-sEVs or PBS was added into the culture medium of NSC after OGD, followed by measurement of the proteins levels in NSC by WB. (E-F) Representative WB bands (E) and quantification of protein level (F) of TGFBR, PTEN, FOXO3 in NSC after OGD. n = 4 per group. (G) The design of the experiment. Briefly, After OGD, NSC cocultured with PBS, M2-sEV with or without miRNA inhibitor (M2-sEVs + IN or M2-sEVs + NC). (H-I) Representative WB bands (H) and the corresponding quantification of protein levels (I) of TGFBR, PTEN, and FOXO3 in NSC (n = 4 per group)

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Discussion

Microglia, essential immune cells specific to the nervous system, affect brain development and the neural environment and respond to both injury and repair [34]. During IS onset, microglia could react to injury signals and adapt to various polarized phenotypes including the classically activated M1 pro-inflammatory phenotype and the alternatively activated M2 phenotype, which harbor numerous anti-inflammatory factors, including IL-4, IL-13, TGF-β, and Brain-Derived Neurotrophic Factor (BDNF) [35]. According to recent research, M2 microglia-derived sEVs could influence cell fate in the CNS, thereby promoting IS recovery [13, 21,22,23]. Moreover, endogenous NSC-mediated neurogenesis could promote neurofunctional recovery post-IS. Herein, we demonstrated that M2-sEVs increased NSC proliferation and neuronal differentiation in vitro post-OGD and in vivo after MCAO, thereby improving neurofunctional recovery post-IS. Mechanistically, the M2-sEVs-afforded promotion of neurogenesis and neurological rehabilitation was, at least partially, attributable to the delivery of miR-25-3p and miR-93-5p, which, in turn, inhibited TGFBR, PTEN, and FOXO3 expression in NSC. Overall, M2-sEVs could be an effective therapeutic agent for IS patients.

Herein, due to efficiency considerations in sEV production and batch-to-batch consistency, we cultured BV2 cells rather than primary microglia to isolate numerous M2-sEVs. According to research, sEVs could easily cross the BBB and enrich the infarct zone, transfer cargo to different types of brain cells, and effectively interact with cells in distant brain areas [36, 37]. In this regard, it is noteworthy that microglial-sEVs, play two distinct roles in various phases of CNS diseases [38,39,40]. They could inhibit NSC neurogenesis within the hippocampal Dentate Gyrus (DG) in pathological processes of depression [37]. Furthermore, in an Alzheimer’s animal model, microglia secreted abundant p-tau+ sEVs, which accelerated amyloid plaque deposition, thus exacerbating tau propagation and worsening the symptoms of the disease [41]. Microglial sEVs could also suppress disease progression. Xin et, al. found that sEVs derived from hypoxic preconditioned microglia could reduce inflammation, astrogliosis, and AQP4 depolarization in mice post-stroke [42]. Furthermore, Huang et, al. found that microglial sEVs can deliver miR-124-3p to neurons, promoting neurite outgrowth, suppressing neuroinflammation, and enhancing neurological function in mice with Traumatic Brain Injury (TBI) [43]. Herein, M2-sEVs enhanced NSC proliferation and neuronal differentiation in vitro post-OGD. Furthermore, the in vivo study revealed that M2-sEVs increased NSC proliferation and the count of newborn neurons in the SVZ and DG, facilitated the migration of newborn neurons from the SVZ to the infarct area and promoted neuronal maturation post-stroke. In an adult brain, NSC are mainly located in two specific niches, the SVZ and DG, where they could differentiate into neurons, astrocytes, or oligodendrocytes [44]. Neurons, astrocytes, and oligodendrocytes originating from NSC could regulate numerous functions, including remyelination, trophic support, neural repair, and regeneration. Following brain injury, NSC proliferate actively, mobilize to the injury site, and regulate differentiation pathways, ultimately fulfilling critical roles during tissue repair [45]. Herein, we found that M2-sEVs promoted NSC proliferation, migration, and differentiation into mature neurons, facilitating neurological recovery post-IS. However, reactive astrocytes, which proliferate extensively, constitute the primary component of glial scars and could inhibit the migration of newborn neurons to the lesion core, potentially hindering tissue repair [46]. Additionally, oligodendrocytes, which provide support and envelope the axons of neurons by forming myelin, are particularly susceptible to ischemic insults and could crucially impact the restoration of neurobehavioral functions post-IS [47, 48]. Therefore, despite our study found M2-sEV-mediated neurogenesis, additional research will still be required in the future to explore whether M2-sEVs can modulate the functions of resident astrocytes and oligodendrocytes, and influence the differentiation of NSC into astrocytes and oligodendrocytes.

In the CNS, sEVs are released by all types of cells and exist as stable vesibles in the cerebrospinal fluid [49]. Specifically, microglia and NSC serete sEVs which facilitate their long-distance communication [50, 51]. MicroRNA (miRNA) is a major component encapsulated in sEVs and participates in the regulation of various biological functions of sEVs. They are defined as a class of non-coding RNAs, with a size ranging from 18 to 25 nucleotides [52]. They can mediate the post-transcriptional gene silencing and regulate gene expression [53]. sEVs can transport miRNAs to recipient cells, thereby modulating the biological activities of the recipient cells [53, 54]. In this study, we employed a miRNA microarray to elucidate the miRNA profile of M2-sEVs and identified miR-25-3p and miR-93-5p as the two most abundant miRNAs. Analysis of the RT-qPCR test results confirmed upregulation of miR-25-3p and miR-93-5p levels in NSC following exposure to M2-sEVs, compared to those treated with PBS. Inhibition of miR-25-3p and miR-93-5p blocked the M2-sEVs-mediated neurogenesis in NSC and decrease of infarct volume in MCAO mice. Future research is required to investigate direct evidence of how sEVs transferred from M2 microglia to NSC in vivo. Additionally, while our findings underscore direct M2-sEV-NSC interactions, parallel immunomodulatory effects, including microglial reprogramming, macrophage infiltration, peripheral immune cell activition (e.g., Treg cells) may synergize with neurogenesis. Systematic investigation of these indirect pathways will refine our understanding of M2-sEV-mediated repair.

Previous studies reported that miR-93-5p expression was significantly upregulated in sEVs secreted from M2 macrophages and confer protective in the recipient cells. For example, knockdown of miR-93-5p in sEVs secreted by M2 macrophage can promote apoptosis of LPS-stimulated podocytes via the TLR4 signaling pathway [55]. In addition, Zhan et, al. found that Bacillus Calmette-Guérin (BCG) infection significantly increased the amount of miR-25-3p and miR-93-5p in macrophage-derived sEVs, which is positively correlated with macrophage apoptosis [56]. As for miR-25-3p, a prevous study found that M2 macrophage secreted exosomal miR-25-3p, which attenuated podocytes injury-induced by high glucose environment [57]. Wang et, al. reported that colorectal cancer cells could transfer exosomal miR-25-3p to macrophages, and thus activated macrophages M2 polarization and cancer metastasis [58]. The findings from these studies indicate that M2 macrophage can secrete sEVs rich in miR-25-3p and/or miR-93-5p, which promote injury repair in the recipient cell or tissue. However, Du et, al. found that exosomal miR-25-3p derived from bone marrow mesenchymal stem cell induced M1 macrophage polarization and aggregate at the injury site following myocardial ischemia reperfusion [59]. Moreover, Wang et al. reported that elevated miR-93-5p exacerbated microglia-mediated neuroinflammation and delayed the recovery of neurological function following intracerebral hemorrhage [60]. These reports suggesting that miR-25-3p and miR-93-5p regulates cellular processes in a disease and environmental-dependent manner. Whether miR-25-3p and miR-93-5p are enriched in the sEVs secreted by IL4-stimulated microglia, and their functions, have not been studied.

In this study, miRNA sequence analysis revealed that sEVs derived from IL4-stimulated microglia were enriched with miR-25-3p and miR-93-5p. Previously, knockdown of the cluster miR-106b ~ 25 (miR‐106b, miR‐93, and miR‐25) was found to decrease the percentage of proliferating NSC. Otherwise, ectopic expression of miR-106b ~ 25 cluster enhanced NSC proliferation and neuronal differentiation [61]. Particularly, miR-25-3p target TGFBR2 and PTEN to inhibit TGFβ signaling, activate insulin/IGF signaling, and decrease FOXO activity. TGFβ signaling is a canonical signaling pathway that modulates the CNS development, and its inhibition can promote adult neural stem/progenitor cells proliferation and neurogenesis [32, 62]. Activation of the insulin/IGF pathway increases NSC proliferation and self-renewal [28]. PTEN is also a well-characterized negative regulator of the PI3K/Akt pathway. Downregulation of PTEN leads to increased phosphorylation of Akt and mTOR, which promotes NSCs Proliferation and Differentiation [63, 64]. While the FOXO activity have a negative effect on the proliferation and differentiation of NSC [61]. Prior study report NSC isolated from adult FoxO3-/- mice have decreased self-renewal and an impaired ability to generate different neural lineages. Mechanism, FoxO3 binding on promoters of P27 and Ddit4 and mediated a serious of genes transcription, which involved in quiescence, hypoxia, aging, and glucose metabolism [30]. Future studies should directly assess the downstream effectors of PTEN and FOXO3 (e.g., Akt and mTOR) to confirm this axis. Collectively, these studies indicate that miR-25-3p and miR-93-5p can regulate the expression of TGFBR, PTEN and FOXO in NSC, and the corresponding NSC neurogenesis. In this study, we observed that co-culture of M2-sEVs with NSC after OGD significantly downregulated the expression of TGFBR, PTEN, and FOXO3. Furthermore, this down-regulation was partially reversed following M2-sEVs treatment with miR-25-3p and miR-93-5p inhibitors (Fig. 8G-I). These results indicated that miR-25-3p and miR-93-5p in M2-sEVs enhance NSC proliferation and differentiation by modulting the expression of TGFBR, PTEN, and FOXO3. While functional assays robustly support the regulatory role of these miRNAs in targeting PTEN, TGFBR, and FOXO3, direct mechanistic validation, such as luciferase reporter assays or RNA immunoprecipitation (RIP) remains critical to confirm physical miRNA-mRNA interactions in future studies. Moreover, future studies employing PTEN/FOXO3-overexpressing NSC are needed to confirm their indispensable role in M2-sEV-mediated effects. Further, while miR-25-3p and miR-93-5p show preferential roles in neuronal differentiation and proliferation, respectively, their co-delivery via M2-sEVs likely creates a synergistic neurogenic program. Systematic single/double miRNA perturbation studies will elucidate their individual and cooperative mechanisms.

Conclusion

Our findings revealed that M2-microglia-sEVs reduced the infarct volume post-IS via miR-25-3p and miR-93-5p delivery, which downregulated TGFBR, PTEN, and FOXO3, thus increasing NSC proliferation and neuronal differentiation.

Data availability

Data is provided within the manuscript or supplementary information files.

Abbreviations

NSC:

Neural stem cell

M2-sEVs:

Small extracellular vesicles derived from IL-4 stimulated microglia

MCAO:

Middle cerebral artery occlusion

IS:

Ischemic stroke

CNS:

Central nervous system

BBB:

Blood-brain barrier

SVZ:

Sub-ventricular zone

M2-sEVs-IN:

M2-sEVs inhibitors

GO:

Gene Ontology

SGZ:

Sub granular zone

DG:

Dentate gyrus

NS:

Neurological score

SEVs:

Small extracellular vesicles

MiRNAs:

micrornas

OPCs:

Oligodendrocyte precursor cells

TBI:

Traumatic brain injury

SCI:

Spinal cord injury

LPS:

Lipopolysaccharides

RT-qPCR:

Quantitative Real-time polymerase chain reaction

DMEM:

Dulbecco’s Modified Eagle’s Medium

TEM:

Transmission Electron Microscope

MCA:

Middle cerebral artery

RCBF:

Regional cerebral blood flow

FGF2:

Fibroblast growth factor 2

OGD:

Oxygen-glucose Deprivation

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Acknowledgements

None.

Funding

This study was kindly funded by the by National Natural Science Foundation of China (82201543，XYG; 82371407, LJF), National Postdoctoral Program for Innovative Talent (BX20220356, XYG), China Postdoctoral Science Foundation (2022M723562, XYG), Natural Science Foundation of Hunan Province (2024JJ6657, ZQ; 2024JJ5623, CX), Young Foundation of Xiangya hospital (2021Q01, XYG).

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Author notes

  1. Qian Zhang and Yan Yi contributed equally to this work.

Authors and Affiliations

  1. Department of Neurosurgery, Xiangya Hospital Central South University, Changsha, Hunan, CN, 410008, China

    Qian Zhang, Tiange Chen, Ying Ai, Ziyang Chen, Ganzhi Liu, Xin Chen, Jinfang Liu & Yuguo Xia

  2. Reproductive Medicine Center, Xiangya Hospital Central South University, Changsha, Hunan, CN, 410008, China

    Yan Yi

  3. National Clinical Research Center for Geriatric Disorders, Xiangya Hospital Central South University, Changsha, Hunan, CN, 410008, China

    Qian Zhang, Yan Yi, Tiange Chen, Ying Ai, Ziyang Chen, Ganzhi Liu, Xin Chen, Jinfang Liu & Yuguo Xia

  4. School of Graduate Studies, Biomedical Science – Dental Scholars Track Program, Rutgers Biomedical and Health Sciences, Rutgers University, Newark, NJ, 07103, USA

    Zexuan Tang

  5. Bio-Intelligent Manufacturing and Living Matter Bioprinting Center, Research Institute of Tsinghua University in Shenzhen, Tsinghua University, Shenzhen, China

    Jianwei Chen & Tao Xu

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  1. Qian Zhang
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Contributions

Qian Zhang performed in vitro experiments; Yan Yi, Tiange Chen, Zexuan Tang, and Ying Ai performed animal study together; Ziyang Chen, Ganzhi Liu and Xin Chen helped to organize and analyze the data; Qian Zhang, Yan Yi, and Yuguo Xia drafted this manuscript, and corrected it; Jianwei Chen, Tao Xu, Jinfang Liu, and Yuguo Xia supervised this study and provided funding. All authors read and approved the final manuscript.

Corresponding authors

Correspondence to Xin Chen, Jinfang Liu or Yuguo Xia.

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All animal experiments were conducted according to the standard operating procedure and complied with the rules of the Animal Care and Use Committees of the Laboratory Animal Research Center at Xiangya Medical School of Central South University, Hunan, China (No. 202110093).

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The authors declare no competing interests.

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Zhang, Q., Yi, Y., Chen, T. et al. M2 microglia-derived small extracellular vesicles modulate NSC fate after ischemic stroke via miR-25-3p/miR-93-5p-TGFBR/PTEN/FOXO3 axis. J Nanobiotechnol 23, 311 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12951-025-03390-2

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Journal of Nanobiotechnology

ISSN: 1477-3155

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