Treatment with Neuronal-Induced Human Mesenchymal Stem Cells Improves Functional Recovery of Acute Spinal Cord Injury through Attenuating Astrogliosis and Neurotoxic Astrocyte Activation
Article information
Abstract
Objective
Most of the preclinical studies have been focusing on inhibition of astrogliosis which is known as a major mechanism that inhibits recovery from spinal cord injury (SCI). Although mesenchymal stem cells (MSCs) have been widely studied as therapeutic candidates for SCI treatment, the role of MSCs on astrogliosis remains unclear. Furthermore, recent studies revealed that astrogliosis also has a protective role in SCI. The purpose of this study was to determine whether neural-induced human adipose tissue-derived MSCs (NI-hADSCs) promote SCI repair through astrogliosis modulation.
Methods
NI-hADSCs were transplanted into the perilesional spinal cord in an acute severe SCI rat model. Functional recovery was evaluated serially on postoperative day 1 and weekly thereafter for 6 weeks using the Basso-Beattie-Bresnahan (BBB) locomotor rating scale. Western blot analysis was performed to assess protein levels of gliosis markers and neuroinflammatory pathways at 6 weeks post-injury. Histopathological examination was conducted at 6 weeks post-injury to evaluate astrogliosis and astrocyte phenotypic changes.
Results
NI-hADSC transplantation significantly improved functional recovery compared with the SCI group, as demonstrated by a greater cumulative BBB locomotor score over 6 weeks (area under the curve : 23.65 vs. 13.58; p=0.026). At 6 weeks post-injury, the levels of glial fibrillary acidic protein (0.52±0.11 vs. 1.00±0.12; p=0.012), vimentin (0.54±0.01 vs. 1.00±0.08; p=0.006), and complement component 3 (C3; 0.38±0.05 vs. 1.00±0.07; p<0.001) in the NI-hADSC-treated group were markedly decreased, whereas the levels of signal transducer and activator of transcription 3 (STAT3)/phosphorylated STAT3 proteins were markedly increased (1.51±0.18 vs. 1.00±0.14; p<0.05). The levels of S100 calcium-binding protein A10 and nuclear factor kappa B (NFκB)/phosphorylated NFκB expression showed no significant differences between groups. Immunohistochemistry confirmed reduced astrogliosis and fewer C3-expressing astrocytes in the NI-hADSC-treated group, predominantly in the dorsal perilesional spinal cord. Substantially increased B-cell lymphoma-2 (Bcl-2) expression (14.30±2.13 vs. 5.86±0.66; p<0.01) and decreased Bcl-2-associated X protein expression (58.64%±2.82% vs. 69.46%±1.81%; p<0.01) indicated that NI-hADSC treatment reduced neuronal apoptosis.
Conclusion
NI-hADSC transplantation improved functional recovery in SCI by modulating astrogliosis. The modulation of astrogliosis was likely to be achieved through selective suppression of detrimental A1 astrocytes, potentially via STAT3 signaling. However, the protective effect of NI-hADSCs on astrogliosis diminished during the subacute SCI period and was spatially restricted to the perilesional area.
INTRODUCTION
Spinal cord injury (SCI) is a devastating neurological condition that imposes lifelong disabilities, and resulting significant physical, social, and economic burdens on patients [1]. The incidence of SCI is estimated at approximately 23.8 per million people globally [26], with a similar rate of about 26.4 per million reported in South Korea [9]. Given its high socioeconomic burden, there has been considerable interest, research, and efforts in developing treatments for SCI. However, to date, all clinical studies have failed to improve neurological outcome due to the complex pathophysiology of SCI.
After the primary injury to the spinal cord by direct trauma, a secondary injury persists for weeks to months [1]. Extensive cell death and degeneration in the early stages of SCI lead to the ex vacuo formation of cystic cavities at the lesion site [1]. Reactive astrocytes tightly interweave their processes, creating an inhibitory mesh-like barrier at the perilesional zone. Also, activated microglia, astrocytes and macrophages secrete extracellular matrix proteins, such as chondroitin sulfate proteoglycans, tenascin, and neural/glial antigen 2 proteoglycan in the early phase of SCI that are inhibitory to axonal growth, are condensed with astrocytes in the glial scar [1]. A glial scar forms at the perilesional zone months after SCI and acts as a formidable barrier preventing axonal regeneration, which has been considered a major obstacle to overcome for SCI recovery [27,44]. Because astrocytes play a major role in glial scar formation, most preclinical and clinical studies focused on inhibiting astrogliosis [25].
However, recent studies have revealed the dual role of astrocytes in SCI, demonstrating not only their detrimental effects but also their protective role in reducing scar formation and promoting axonal regeneration [2,10,33]. Anderson et al. [2] reported that newly generated immature scar-forming astrocytes produced after SCI aid axon regeneration rather than being hostile. Several in vivo studies showed that the loss of reactive astrocytes during the early phase of SCI results in poor functional outcomes and more extensive tissue destruction [2,10,12]. Furthermore, accumulating evidence demonstrates that astrocytes do not serve fixed roles but can modulate their functions based on different time points, triggering conditions, and response pathways, thereby generating diverse biological outcomes [21,23,45]. Therefore, selective astrocyte modulation at optimal timing and through proper methods after injury may offer therapeutic potential in SCI treatment by reducing neural injury and improving neuroprotection.
Mesenchymal stem cells (MSCs) have been widely studied as therapeutic candidates for SCI treatment due to their characteristics such as self-renewal capacity, pluripotency, and plasticity, as well as their immunomodulatory properties [14,19,31]. Most studies with stem cells have been conducted during the subacute and chronic phase of SCI because of poor cell survival during the acute period [31,37]. Therefore, the mechanisms by which MSCs modulate astrogliosis are poorly understood in acute SCI, which is a critical period for patient prognosis. We previously reported that MSCs improved the functional outcomes of acute SCI by modulating astrogliosis [17]. The present study aimed to elucidate the mechanisms by which MSCs affect reactive astrocytes using neural-induced human adipose tissue-derived MSCs (NI-hADSCs). Given that selective astrocyte modulation through stem cell transplantation may offer therapeutic potential in SCI by reducing neural injury and improving neuroprotection, we hypothesized that NI-hADSCs influence reactive astrocyte phenotypes during the acute phase of SCI and sought to determine their potential in modulating astrogliosis.
MATERIALS AND METHODS
Preparation of neural induced NI-hADSCs
Adipose tissues from healthy human donors of both sexes aged 4 to 20 years were collected individually in accordance with the regulations of the Ethics Committee at the Chonnam National University Medical School (IRB : I-2009-03-016). hADSCs were cultured in an incubator (5% CO2/95% air, 37℃) with Dulbecco’s modified Eagle’s medium (Cytiva, Marlborough, MA, USA) containing 10% fetal bovine serum (Cat. No. SV3020702; Cytiva), and 1% penicillin-streptomycin (Cat. No. LS202-02; WELGENE, Gyeongsan, Korea), and 0.2% amphotericin B (Cat. No. 15290-018; Thermo Fisher Scientific, Waltham, MA, USA). The hADSCs were induced into neuronal cells (abbreviated as NI-hADSCs) in accordance with our previous studies [15,16].
Animals
All animal experiments were approved and conducted in accordance with the regulations of the Institutional Animal Care and Use Committee (CNU IACUC-H-2022-36) of Chonnam National University Medical School, Hwasun-gun, Jeollanamdo, Republic of Korea. Forty female Sprague-Dawley (SD) rats (weight, 200 g; age, 12 weeks) were obtained from Damool Science (Daejeon, Korea) and maintained in an animal facility under constant environmental conditions (20℃±2℃, 50%±10% humidity, 12 h light/dark cycle) with ad libitum access to food and water. No more than three rats were kept in each cage. Conventional practices for handling animals were performed before functional outcome measurements to minimize the stress experienced by the animal during human interaction [28,42]. Forty rats were randomly assigned to two groups of 20 each using a random allocation sequence prior to surgery : a SCI group and a SCI with NI-hADSC transplanted group (SCI+NI group). In addition to these groups, three rats were assigned for sham procedure. During surgery, three rats in the SCI+NI group and one rat in the SCI group died. Therefore, functional outcome measurements were performed for 19 rats from the SCI group and 17 rats from the SCI+NI group. Every effort was made to alleviate the pain in these animals. Six rats per group were maintained for 6 weeks post-surgery, and their spinal cord tissues were subsequently processed for analysis : three specimens for western blot analysis and three for immunohistochemical evaluation.
SCI model and NI-hADSC transplantation
The surgical procedure was performed using the clipping compression technique, as previously described [17,38,40]. Briefly, SD rats were anesthetized with 50 mg/kg pentothal sodium (intraperitoneal injection; JW Pharmaceutical, Seoul, Korea) and maintained under isoflurane USP (Troikaa Pharmaceuticals Ltd., Ahmedabad, India) during surgery. After serial dissection of the fascia and paraspinal muscles, the T10 and T11 laminae were completely removed. Severe SCI was induced by compressing the full thickness of the spinal cord with aneurysmal clips (occlusion pressure : 75 g, Asculap, Oakville, Canada) for 1 minute before removal. The sham-operated animals were handled the same way, exposing the spinal cord without clamping.
For injection of NI-hADSC, rats were anesthetized with pentobarbital sodium and positioned on a stereotaxic injector (Model KDS-310; Muromachi Kikai Co., Tokyo, Japan). The T9 and T12 spinous processes were then fixed horizontally using forceps from a fixation device. NI-hADSCs (20 μL) at a concentration of 10000 cells/μL were injected at 5 mm above the SCI site within 1 hour from injury through a Hamilton syringe (Cat. No. 788130; KD scientific, Seoul, Korea) at an infusion rate of 5 μL/min over 4 minutes. The needle was left in place for 5 minutes after injection. After the needle was removed, the muscle, fascia and skin were closed layer by layer. Animals in the SCI group were handled in the same manner, except for the injection procedure.
After surgery, the rats were allowed to recover for 1 week before they were subjected to behavioral testing, and were observed every week until the end of the study. The rats were then sacrificed, and their spinal cords were collected. The spinal cord tissues were fixed in 4% paraformaldehyde (PFA; Cat. No. SMP01-100; GeneAll, Seoul, Korea) for histological analysis or phosphate-buffered saline (PBS; Biosesang, Yongin, Korea) for western blotting analysis.
Functional outcome measurements
Functional outcomes were analyzed serially using the Basso-Beattie-Bresnahan (BBB) scale at postoperative day 1 and weekly for 6 weeks [4,49]. It is a widely used method for evaluating hindlimb locomotor performance such as the recovery of the magnitude and speed of hindlimb movement, forelimbhindlimb coordination, and trunk stability in rats. In this study, two well-trained investigators who were blind to the study were invited to observe the behavior of rats for 4 minutes.
Tissue preparations
At the end of the experiments, tissue samples were prepared. For protein analysis, rats were anesthetized and perfused with PBS (Biosesang), and tissues were collected and stored at -80℃. For immunohistochemistry (IHC), rats were anesthetized and perfused with PBS, followed by 4% PFA. After the spinal cord was harvested, it was fixed in 4% PFA and maintained in 30% sucrose solution. The tissues were embedded in optimal cutting temperature compound (Cat. No. 3801480; Leica Biosystems, Nussloch, Germany). Serial sections were prepared on a slide glass by using a cryostat (DE/CM1860 cryostat; Leica Biosystems) with 15 μm sections for staining.
Western blot analysis
For western blot analysis, a 20-mm longitudinal segment of the spinal cord centered on the injury site was harvested from each animal (n=3 per group) to evaluate total protein expression. The samples were then homogenized in radioimmunoprecipitation assay lysis buffer supplemented with a protease inhibitor cocktail on ice. Protein samples (20 μg each) were then separated by 6–10% sodium-dodecyl-sulfate polyacrylamide gels. The separated proteins were electroblotted onto nitrocellulose transfer membranes, and membranes were blocked with 5% skim milk in Tris-buffered saline supplemented with 0.1% Tween 20 buffer for 1 hour. The membranes were incubated with primary and secondary antibodies in order. All antibodies used are listed in Table 1. β-actin was used as the primary endogenous loading control for western blotting. Immobilon Crescendo Western HRP substrate (Cat. No. WBLUR0500; Merck Millipore, Burlington, MA, USA) was used to visualize the immunoblotted bands, and quantitative analysis was conducted using Image J software (National Institutes of Health, Bethesda, MD, USA).
List of the antibodies
IHC analysis
For immunohistochemical staining (n=3 per group), slides were fixed and blocked in 10% normal goat serum (Vector Laboratory, Newark, CA, USA) for 30 minutes at room temperature. The slides were then incubated with primary and secondary antibodies (Table 1). Signals were visualized with 0.03% 3,3′-diaminobenzidine (Cat. No. ENZ-KIT159-0150; Enzo Life Science, New York, NY, USA), and the nuclei were counterstained with hematoxylin (Cat. No. S3309; Agilent, Santa Clara, CA, USA). For IHC quantification, the ventral horn of the gray matter and dorsal column of the spinal cord within the perilesional area was designated as the primary region of interest (ROI) to assess cellular phenotypic changes. All images were taken under a microscope (ZEISS Axio Vert.A1, Oberkochen, Germany), and the immunoreactive area was quantified using Image J software (National Institutes of Health).
Statistical analysis
Functional assessments, western blot densitometric quantification, and immunohistochemical image analyses were performed by investigators blinded to group allocation. Blinded analysis was maintained throughout data acquisition and quantification to minimize experimental bias. GraphPad Prism 10 (GraphPad Software, La Jolla, CA, USA) was used to perform statistical analyses and generate graphical illustrations. To assess the statistical significance of the differences between the SCI and SCI+NI groups, a two-tailed unpaired Student’s t-test was used. When the sample size was small or assumptions for parametric tests were violated, the Mann-Whitney U test was employed.
For functional outcome analysis, longitudinal functional recovery which evaluated with BBB scores was analyzed using a linear mixed model with restricted maximum likelihood estimation to handle repeated measures and missing data. Fixed effects included group, time, and the group×time interaction. Additionally, the area under the curve (AUC) was calculated to assess the cumulative therapeutic effect over the 6-week period [8,13,41,47]. Differences in AUC between the SCI and SCI+NI groups were analyzed using the Mann-Whitney U test. For this analysis, Python (version 3.11.5; Python Software Foundation, Beaverton, OR, USA), and statsmodels (version 0.14.0; statsmodels developers, Open-source project) and SciPy (version 1.11.3; statsmodels developers, Open-source project) libraries were used.
Data are shown as means±standard error (SE). Statistical significance was set at p<0.05. The levels of significance were as follows : ***p<0.001, **p<0.01, and *p<0.05.
RESULTS
NI-hADSC treatment enhances functional outcomes in rats with acute SCI
To demonstrate the effects of NI-hADSCs, a functional assessment using BBB locomotor score was performed weekly for 6 weeks postoperatively. All rats presented with noticeable hind limb paralysis compared with that in the sham group. BBB score differences between the SCI and SCI+NI groups emerged 3 weeks post-SCI, with significant differences observed at both 3 and 6 weeks. Although statistical significance was lost at weeks 4 and 5, the SCI+NI group consistently showed a trend toward higher mean BBB scores than those of the SCI group throughout the observation period (Fig. 1). The loss of statistical significance in BBB score differences at weeks 4 and 5 remains unclear.
Locomotor function results. A : Longitudinal evaluation of locomotor recovery was assessed using the Basso-Beattie-Bresnahan (BBB) score weekly for 6 weeks post-injury. Number of rats per group at each time point (SCI vs. SCI+NI) : week 1 (n=19 vs. 17), week 2 (n=17 vs. 11), week 3 (n=14 vs. 11), week 4 (n=12 vs. 11), week 5 (n=6 vs. 5), week 6 (n=6 vs. 5). Week 3 (p=0.025; Student t-test, two-tailed) and week 6 (p=0.049; Mann-Whitney U test, two-tailed) showed statistical significance. B : Cumulative functional recovery represented as the area under the curve (AUC) of BBB scores over 6 weeks. The SCI+NI group showed significantly greater AUC compared to the SCI group (SCI group, n=6; SCI+NI group, n=5; p=0.026; Mann-Whitney U test). Data are presented as mean±standard error of the mean. SCI : spinal cord injury, SCI+NI : SCI with neural-induced human adipose tissue-derived mesenchymal stem cells transplanted. *p<0.05 compared with the SCI group.
To examine the longitudinal recovery trajectory, we performed a linear mixed model analysis. Both groups showed significant improvement over time (p<0.001). Although the SCI+NI group displayed a trend toward higher BBB scores compared to the SCI group, the difference in the rate of recovery (group×time interaction) did not reach statistical significance (p=0.252).
However, given the consistently higher scores observed in the SCI+NI group across the observation period, we further evaluated the overall therapeutic efficacy by calculating the AUC, which represents the cumulative functional recovery of each animal. The AUC analysis revealed that the SCI+NI group achieved a significantly greater magnitude of functional recovery compared to the control group over the 6-week period (mean AUC, 23.65 vs. 13.58; p=0.026; Mann-Whitney U test; Fig. 1B). This indicates that while the weekly rate of improvement was similar between the groups, NI-hADSC transplantation conferred a significantly sustained functional benefit throughout the acute and subacute phases.
NI-hADSC treatment decreases astrogliosis and increases the level of phosphorylation of signal transducer and activator of transcription 3 (STAT3) after SCI
To determine whether NI-hADSC treatment after SCI attenuates astrogliosis, astrogliosis levels were analyzed via western lot analysis of spinal cord tissues. As shown in Fig. 2, the expression of gliosis markers, such as glial fibrillary acidic protein (GFAP) and vimentin, was substantially lower in the SCI+NI group than in the SCI group. In addition, the level of STAT3 phosphorylation, which is a protective pathway in astrogliosis, was higher in the spinal cord of NI-hADSC-treated rats than in SCI rats. However, the level of nuclear factor kappa B (NFκB) phosphorylation, which is a detrimental pathway, was similar between the two groups. At 6 weeks post-SCI, active inflammation had subsided, which may explain why no difference was observed in the inflammatory transcriptional factor NFκB. Therefore, the protein levels of complement component 3 (C3) and S100 calcium-binding protein A10 (S100A10), which are markers of neurotoxic A1- and neuroprotective A2-type reactive astrocytes, respectively, were examined. C3 levels were significantly lower in the SCI+NI group than in the SCI group, whereas S100A10 levels were higher in the SCI+NI group than in the SCI group, although this difference was not statistically significant. While western blot analysis of bulk spinal cord tissue (perilesional 20 mm segment) showed a non-significant increase in S100A10 levels, this may reflect the diluted signal from the heterogeneous cellular environment of the entire cord segment. All uncropped gel images are shown in the Supplementary Fig. 1. Therefore, these data suggest that NI-hADSCs prevented damage and induced recovery by inhibiting astrogliosis through STAT3 pathway in SCI.
Expression of the signal transducer and activator of transcription 3 (STAT3), nuclear factor kappa B (NFκB) pathways, and astrogliosis markers. The levels of astrogliosis, STAT3 and NFκB markers were visualized via western blotting analysis (A) and quantified (B). β-actin was used as a control. Number of rats per each group was 3. Con : control, MSC : mesenchymal stem cell, C3 : complement component 3, pSTAT3 : phosphorylated STAT3, pNFκB : phosphorylated NFκB, S100A10 : S100 calcium-binding protein A10, GFAP : glial fibrillary acidic protein. *p<0.05, **p<0.01, and ***p<0.001 compared with the spinal cord injury (SCI) group.
NI-hADSC treatment reduces apoptotic cell death in peri-injury spinal cord
To confirm the protective effect of NI-hADSCs, the levels of B-cell lymphoma-2 (Bcl-2) and Bcl-2-associated X (Bax), which are representative markers of apoptosis, were measured in the spinal cord tissue of each group using IHC. The SCI site showed severe tissue destruction and accumulation of inflammatory cells, rendering objective comparison difficult. Therefore, the peri-lesion area near the injury site, specifically the ventral horn of gray matter of the spinal cord, was designated as the ROI for comparison. As shown in Fig. 3A, Bax-positive cells decreased and Bcl-2-positive cells increased after treatment of NI-hADSCs compared with those in the SCI group. The number of Bax-positive cells in each group was 69.46±1.81 and 58.64±2.82 (mean±SE, %) in the SCI and SCI+NI groups, respectively (**p<0.01; Fig. 3B). Furthermore, the level of Bcl-2 expression in the SCI group was decreased (5.86±0.66), whereas NI-hADSC treatment increased the Bcl-2 expression (14.30±2.13; **p<0.01 compared with the SCI group, Fig. 3C). The number of apoptotic cells increased after SCI and decreased after injection of NI-hADSCs in injured animals. These results indicate that NI-hADSC treatment reduced apoptosis after SCI.
Expression levels of apoptosis markers. Spinal cord tissues were incubated overnight with primary antibodies, B-cell lymphoma-2 (Bcl-2, 1 : 500) and Bcl-2-associated X (Bax, 1 : 3000), and secondary one. The tissues were visualized under 3,3'-diaminobenzidine (DAB) staining as a brown. A : Immunohistochemical images showed the Bcl-2- or Bax-positive cells in the ventral gray horn of the spinal cord. Compared with SCI controls, the NI-hADSC group showed fewer Bax-positive cells - particularly neurons - accompanied by markedly stronger Bcl-2 immunoreactivity with a diffuse cytoplasmic pattern. The number of Bax-positive cells (B) and Bcl-2-positive cells (C) were calculated. Scale bar represents 50 µm. Number of rats per each group was 3. MSC : mesenchymal stem cell, SCI : spinal cord injury. **p<0.01 compared with the SCI group.
NI-hADSC treatment in the acute phase of SCI reduces astrogliosis locally, with effects limited to areas close to the treated region
To determine the effects of NI-hADSC, the levels of astrogliosis markers, such as GFAP, vimentin, and C3, in the perilesional spinal cord were assessed 6 weeks after SCI. Following IHC, the immunoreactivity of GFAP and vimentin was markedly reduced in the SCI+NI group (11.05%±1.03% and 3.01%±0.20%, respectively; ***p<0.001) compared with those in the SCI group (44.08%±2.11% and 8.42%±0.96%, respectively; ***p<0.001, Fig. 4A-D). Similarly, immunoreactivity for C3 was significantly decreased in the SCI+NI group (4.60%±0.71%) compared with those in the SCI group (16.67%±1.47%; ***p<0.001; Fig. 4E and F). These findings suggest that NI-hADSC treatment suppresses astrogliosis after SCI, thereby conferring neuroprotective effects. Notably, the anti-astrogliotic effects were spatially restricted to the dorsal spinal cord rather than distributed throughout the entire cord (Fig. 4). In contrast to the western blot findings, IHC analysis focused specifically on the ventral horn of the perilesional gray matter as the ROI. Interestingly, in this localized region, S100A10-positive cells (a marker for A2 reactive astrocytes) were significantly decreased in the SCI+NI group (6.26%±0.89%) compared to the SCI group (8.93%±0.46%; *p<0.05; Fig. 4G and H). These results demonstrate that NI-hADSC treatment effectively suppresses detrimental A1 reactive astrocytes in SCI. However, the therapeutic benefits are spatially restricted and its modulatory effects on A2 reactive astrocytes remain inconclusive.
Expression of astrogliosis markers. Spinal cord tissues were incubated overnight with primary antibodies, glial fibrillary acidic protein (GFAP) (A, 1 : 2000), vimentin (C, 1 : 1000), C3 (E, 1 : 1000), and S100A10 (G, 1 : 1000) and secondary one. The tissues were visualized under 3,3'-diaminobenzidine (DAB) staining as a brown. The number of marker-positive cells (B, D, F, H) were calculated. Scale bar represents 50 µm. Number of rats per each group was 3. MSC : mesenchymal stem cell, SCI : spinal cord injury. *p<0.05 and ***p<0.001 compared with the SCI group.
DISCUSSION
This study demonstrated that acute transplantation of NI-hADSC contributed to the motor function recovery in SCI rats with reducing apoptosis and gliosis at 6 weeks post-SCI (Fig. 5). A notable finding in this study is phosphorylation levels of STAT3, associated with protective astrogliosis, were higher in the spinal cord of NI-hADSC-treated rats compared with that of SCI rats. In contrast, the levels of NFκB phosphorylation remained similar between two groups at chronic SCI, compared to our previously report which decreased level of MSC treated group at acute SCI [17]. Although MSCs modulate inflammation through diverse mechanisms, including enhancing anti-inflammatory immune cell activity and reducing pro-inflammatory signaling [35], these therapeutic effects on SCI are presumed to arise from the suppression of A1 astrocytes. Although our findings suggest involvement of the STAT3 pathway in astrogliosis modulation, this study does not provide direct mechanistic evidence to confirm this association in our experimental model.
Diagrammatic representation of the proposed mechanisms of the NI-hADSC transplantation in SCI rat. Transplantation of NI-hADSCs after SCI could promote functional recovery through modulation of astrogliosis. NI-hADSCs decrease levels of glial fibrillary acidic protein, vimentin, and complement component 3, while increasing STAT3/phosphorylated STAT3 proteins. This selective suppression of detrimental A1 astrocytes occurs during the subacute period. The NI-hADSC transplantation also reduces neuronal apoptosis via increased Bcl-2 and decreased Bax expression, demonstrating the neuroprotective effect of NI-hADSC therapy in spinal cord injury. The downward (↓) and upward (↑) arrows denote inhibitions and stimulation, respectively. NI-hADSC : neural-induced human adipose tissue-derived mesenchymal stem cells, GFAP : glial fibrillary acidic protein, C3 : complement component 3, S100A10 : S100 calcium-binding protein A10, Bax : Bcl-2-associated X, Bcl-2 : B-cell lymphoma-2, pSTAT3 : phosphorylated signal transducer and activator of transcription 3, SCI : spinal cord injury, STAT3 : signal transducer and activator of transcription 3.
Astrocytes are the most abundant cell type in the adult CNS and play essential roles in neuronal survival, synaptic function, and homeostasis [30]. Following CNS trauma, astrocytes become reactive astrocytes, and through reactive astrogliosis ultimately form glial scar [2,51]. In contrast to previous dogma of reactive astrocytes inhibiting regeneration, recent reports highlighting the promotion of CNS recovery are gaining attention [2,11,24,34,51]. However, the conditions under which astrocytes are beneficial or harmful remain incompletely understood.
Reactive astrocytes are classified into two phenotypes : A1 and A2. Zamanian et al. [51] identified two distinct types of reactive astrocytes through purification and genetic profiling of reactive astrocytes from mice treated with lipopolysaccharide (LPS) or middle cerebral artery occlusion. The nomenclature of A1 and A2 astrocyte phenotypes was later proposed by Liddelow et al. [24] in 2017, drawing parallels to the M1 and M2 phenotype transitions of microglia, which are the first responders to CNS injury [29]. The A1 phenotype is generally associated with detrimental roles, whereas the A2 phenotype is associated with protective roles [23]. When tissue damage occurs, released LPS induces microglia secrete proinflammatory cytokines, such as interleukin-1α, tumor necrosis factor-α, and complement component 1q. These cytokines activate astrocytes and transform them into the detrimental A1 phenotype. These astrocytes then secrete neurotoxic factors, such as the specific complement component C3 and inflammatory cytokines, thereby inducing neuronal and other glial cell death [24]. In contrast, under hypoxic conditions, astrocytes are induced to the A2 phenotype, expressing the specific protein S100A10. A2 astrocytes secrete anti-inflammatory cytokines and various neurotrophic factors, thereby exerting neuroprotective functions [51]. This discovery suggested a therapeutic interest in suppressing A1 astrocytes while promoting A2 astrocytes [21,23], and multiple studies have confirmed that this strategy improves outcomes in SCI [11,45,50].
Recent studies suggest that the NFκB and Janus kinase (JAK)-STAT3 pathways regulate the polarization of A1 and A2 astrocytes, respectively [7,10,11,23]. NFκB is a key transcription factor in neuroinflammation and is necessary for A1 induction [22,24], whereas STAT3 plays a critical role in modulating astrogliosis [7] and functional recovery [2,34]. Wang et al. [46] demonstrated that MSCs and MSC-exosomes effectively reduce neurotoxic A1 astrocyte formation after SCI by inhibiting NFκB subunit p65 nuclear translocation, which suppresses the production of proinflammatory cytokines including tumor necrosis factor-alpha (TNF-α), interleukin (IL)-1α, and IL-1β. This anti-inflammatory mechanism leads to decreased neuronal apoptosis, reduced lesion size, and improved functional recovery in SCI rats, with MSC-exosomes demonstrating comparable therapeutic efficacy to MSCs when administered intravenously. Yang et al. [50] reported that hypoxic-preconditioned MSC-derived small extracellular vesicles promote SCI recovery by transforming A1 to A2 astrocytes through the microRNA-21 (miR-21)/JAK2/STAT3 pathway. miR sequencing revealed high miR-21 expression in these vesicles, and overexpression/knockdown experiments demonstrated that miR-21 increases JAK2 and STAT3 phosphorylation to drive A1-to-A2 astrocyte conversion, with blocking miR-21 reversing this effect. Although this study does not address the NFκB pathway, the JAK2/STAT3 pathway suppresses A1 astrocytes, promotes functional recovery, and reduces apoptosis, which is similar to our findings.
In the current study, increased STAT3 expression was observed at the 6-week post-injury time point, whereas NFκB phosphorylation levels remained comparable between groups. Since it is difficult to find studies in the literature that compare results up to the 6-week time point, we cannot precisely explain the results observed in our study. However, there is indirect evidence suggesting that NFκB activation precedes STAT3 following SCI, with pro-inflammatory gene and protein expression appearing first, followed by anti-inflammatory patterns. Li et al. [20] reported that C3 protein levels, an A1 astrocyte marker, increased rapidly and reached a peak at 1 and 4 days post-injury, whereas S100A10 protein levels, an A2 astrocyte marker, showed no significant change at 1 day post-injury but increased notably and reached a peak at 4 and 7 days post-injury. Kisucká et al. [18] demonstrated that at 1 week post-SCI, the A1 phenotype predominated over the A2 phenotype at the injury site, explaining that this was due to cytokines such as TNF-α, IL-1α, and C1q secreted by activated microglia in the early injury phase inducing A1 transformation. They also reported that A2 marker genes including Tgm1, CD109, and Ptx3 were activated in adjacent areas at 1 week, with their expression further increasing in the cranial region at 2 weeks. Taken together, these results suggest that immediately after SCI, neurotoxic A1 astrocytes predominate and accelerate secondary injury, but subsequently, the proportion of neuroprotective A2 astrocytes gradually increases, reflecting an attempt at tissue repair. Considering the close relationship between A1 and A2 with NFκB and STAT3, respectively, this can be explained as temporal dynamics of neuroinflammation in which NFκB predominates initially and STAT3 predominates subsequently. At the 6-week time point, it is plausible that the acute post-injury phase characterized by NFκB activation had subsided, while STAT3 remained relatively sustained. However, the above explanation is an inference based on observations obtained through previous studies and does not constitute reliable direct evidence, nor provides we have data in our study model to demonstrate this. Therefore, the specific patterns of activation and inhibition of key pathways over the secondary SCI time window should be confirmed through future studies.
Another finding that requires explanation is the discrepancy in A2 reactive astrocyte markers between the two methods : IHC demonstrated a significant decrease in the SCI+NI group, whereas Western blot analysis showed an increase, though not statistically significant. The discrepancy in S100A10 expression between western blot and IHC can be attributed to the different sampling regions. Western blot represents the overall protein changes within a 20 mm spinal cord segment, whereas IHC allowed for a region-specific assessment of the perilesional dorsal column of the spinal cord. These findings suggest that the therapeutic effect of NI-hADSCs on A2 astrocytes may vary depending on the specific anatomical location within the injured spinal cord, with localized changes potentially being outbalanced by the broader tissue response in bulk analysis.
The effect of NI-hADSCs on astrogliosis was confined to the perilesional dorsal spinal cord at 6 weeks post-SCI. Although the survival of transplanted hADSC has not been directly assessed, this localized effect likely reflects the limited persistence of the transplanted cells [36]. The timing of cell delivery is critical for optimal therapeutic efficacy. The acute phase following SCI is characterized by a surge of proinflammatory cytokines and inflammatory cells, creating a hostile microenvironment with low cell survival probability. Neutrophils infiltrate immediately after injury, reaching peak levels within 1 day, followed by macrophages and microglia from day 3, which progressively increase until day 7 [1,5,32]. Consequently, many researchers recommend avoiding the acute period for cell transplantation, with meta-analysis suggesting that the subacute period is more suitable for cell therapy [39,43,48].
However, the current study aimed to investigate the protective role of NI-hADSCs and their modulatory effects on astrogliosis during the acute phase. Early intervention during this period is critical for mitigating secondary injury and influencing glial scar formation—principles extensively explored in pharmacological SCI clinical trials. Most stem cell research has focused on subacute or chronic phases, resulting in limited information regarding acutely delivered MSCs, which motivated this investigation. Moreover, supporting evidence exists : Kim et al. [17] reported that BDSC transplantation within one hour of injury promoted functional recovery, while Aras et al. [3] demonstrated that immediate versus delayed ADSC administration yielded comparable functional improvements, suggesting that acute-phase MSC treatment strategies warrant further consideration despite theoretical limitations.
Limitation
This study had some limitations. First, SCI pathology is highly dynamic; however, the study analysis was restricted to a single cross-sectional time point at 6 weeks post-injury, which does not accurately reflect temporal changes in inflammatory and reparative processes. Second, we investigated the astrogliosis with related to astrocytes which are the hallmark of astrogliosis, although other cells such as microglia, macrophages, pericytes and oligodendroglial cells, are also responsible for astrogliosis after 7 days of SCI [6]. In addition, we were unable to provide a plausible explanation or supporting data to account for the localized treatment effect in the dorsal spinal cord. Furthermore, our findings suggest the suppression of A1 astrocytes, the potential induction of A2 astrocytes has not been directly validated. While we quantified representative proteins associated with each phenotype, we did not directly assess astrocyte function and behavior. Consequently, the link between protein expression and actual astrocyte functional phenotypes and mechanisms remains incompletely established. Third, a portion of the mechanistic interpretation relied on previous studies and literature rather than direct experimental confirmation. If the STAT3 pathway is the main therapeutic target mechanism of NI-hADSCs in SCI, performing STAT3 inhibition to observe the abolishment of therapeutic effects would provide more conclusive evidence demonstrating that the therapeutic effects of NI-hADSCs occur through the STAT3 pathway. Additionally, the study has limitations due to insufficient additional experimentation and lack of reasonable explanations for findings that do not align well with the experimental hypothesis, including the loss of statistically significant differences in BBB functional scores between the SCI group and the SCI+NI group at 4 and 5 weeks post-injury, and the absence of differences in NFκB protein levels between the two groups. Despite these limitations, this study provides valuable insights into the therapeutic potential of NI-hADSCs in SCI and highlights directions for future research.
CONCLUSION
The study findings suggest that NI-hADSC transplantation could modulate astrogliosis and support functional recovery in SCI, showing an association between increased pSTAT3 and suppression A1 markers. However, direct stem cell transplantation during the acute phase yields only transient and spatially restricted effects, underscoring the limited efficacy of this approach alone. Future strategies should focus on enhancing transplanted cell survival or other alternative approaches such as developing cell-free therapeutic modalities, which may overcome current limitations and provide more sustainable benefits for SCI treatment.
Notes
Conflicts of interest
Chi-Heon Kim has been editorial board of JKNS since January 2023. He was not involved in the review process of this original article. No potential conflict of interest relevant to this article was reported.
Informed consent
This type of study does not require informed consent.
Author contributions
Conceptualization : SL, CK, SJ; Data curation : SL, JH; Formal analysis : SL, JH, HSJ; Funding acquisition : CHK; Methodology : HSJ, CK, SJ; Project administration : CHK CK, SJ; Visualization : SL, JH, SJ; Writing - original draft : SL, CK, SJ; Writing - review & editing : SL CK, SJ
Data sharing
All data needed to evaluate the conclusions in the manuscript are present in the manuscript and the Supplementary Materials. All methods and protocols used are included in the main manuscript. All reagents, antibodies, and resources are listed in the main manuscript or supplementary files. The data sets used and/or analyzed during the current study are available from the corresponding authors on reasonable request.
Preprint
None
Acknowledgements
This work was supported by the Korean Brain and Spinal Cord Research Foundation (KBSCR-024-001).
Supplementary materials
The online-only data supplement is available with this article at https://doi.org/10.3340/jkns.2025.0240.
Unedited images and their molecular markers for respective western blot analysis used in Fig. 2 of this manuscript. Con : control, MSC : mesenchymal stem cell, pSTAT3 : phosphorylated signal transducer and activator of transcription 3, STAT3 : signal transducer and activator of transcription 3, pNFκB : phosphorylated nuclear factor kappa B, NFκB : nuclear factor kappa B, GFAP : glial fibrillary acidic protein, C3 : complement component 3, S100A10 : S100 calcium-binding protein A10.