In the present study, male Sprague-Dawley (SD) rats (weight, 80-120 g; age, 2-4 weeks) were used for MSC isolation and culture. One rat was required for each T25 cell culture flask of primary MSCs collected, and 33 male SD rats were used for MSC isolation. The MCAO model was established using 45 male SD rats (weight, 260-300 g; age, 6-8 weeks). All rats were provided by Zhejiang Chinese Medical University Animal Center (Laboratory Animal Certificate: SCXK 2018-0006; Hangzhou, China). The rats were maintained at a temperature of 20±2°C, a humidity of 55±5%, under a 12-h light/dark cycle, and were given free access to food and water. All animal experiments were approved by the Animal Ethical and Welfare Committee of Zhejiang Chinese Medical University (approval no. 20210524-05, review no. IACUC-202108-05). All SD rats in all experiments were euthanized by intravenous injection of an overdose of pentobarbital (100 mg/kg), which was confirmed by observing their vital signs 10 min after administration.

Wnt3a recombinant cytokine (cat. no. 96-315-20-10) was purchased from PeproTech; Thermo Fisher Scientific, Inc. and the small molecule inhibitor PTC209 (cat. n. S7372) was purchased from Selleck Chemicals. Before use, Wnt3a powder was dissolved in sterile water, and PTC209 powder was dissolved in dimethyl sulfoxide (DMSO). A quantity of 10 mg PTC209 powder was dissolved in 1.14 ml DMSO to create a 2 mmol/l master mix.

MSCs were obtained from rat bone marrow by flushing the contents of the tibia and femur with phosphate-buffered saline (PBS) in a sterile environment, and were then transferred to a centrifuge tube to be centrifuged at 10,000 × g for 5 min at room temperature. After centrifugation, the supernatant was discarded, and the cells were resuspended in MSC medium prepared with DMEM/F-12 (cat. no. TBD10565; Tianjin Haoyang Biological Products Technology Co., Ltd.) containing 10% fetal bovine serum (FBS; cat. no. 10091-148; Gibco; Thermo Fisher Scientific, Inc.) and 1% penicillin-streptomycin solution, and were inoculated in T25 cell culture flasks at a temperature of 37°C in a 5% CO₂ incubator. Cell morphology was observed under an inverted fluorescence microscope (ECLIPSE TE2000-U; Nikon Corporation).

MSCs were cultured in 96-well plates with varying concentrations of recombinant cytokine Wnt3a (0, 10, 50 and 100 ng/ml) and a small molecule inhibitor PTC209 (0, 1, 2.5, 5 and 10 μmol/l). Each group was subjected to a 24-h stimulation period within a 37°C incubator, comprising five replicate wells at varying concentrations. Subsequently, the cells were exposed to 5 mg/ml MTT for 4 h, the blue-violet formazan crystals were dissolved in DMSO and the absorbance was measured at 450 nm using an enzyme-linked instrument. The MTT kit was purchased from Abcam (cat. no. ab211091).

The 3rd-5th generation MSCs in logarithmic growth phase were collected and inoculated in two 6-well plates at a cell density of 1×10⁵ cells/ml. The cells were randomly divided into four groups (n=3 wells each): Normal control (NC) group, β-mercaptoethanol (BME) group, Wnt3a group and PTC209 group. The NC group comprised normal cultured cells without induction of neural differentiation; the BME group was pre-induced with neural differentiation pre-induction medium for 24 h, followed by induction with neural differentiation medium for 3-6 h. The Wnt3a group and the PTC209 group were treated with neural differentiation medium containing 10 ng/ml Wnt3a and 1 μmol/ml PTC209, respectively, and the incubation steps were the same as that of the BME group. The neural differentiation pre-induction medium consisted of DMEM/F-12 supplemented with 20% FBS, 1 mmol/l BME and 1% penicillin-streptomycin solution. The neural differentiation induction medium was prepared by adding 5 mmol/l BME and 1% penicillin-streptomycin solution to DMEM/F-12.

The culture medium in the cell plates was aspirated and discarded, and the cells were washed with PBS and fixed with 4% paraformaldehyde at room temperature for 10 min. The cells were washed again with PBS and cell membranes were ruptured with a permeabilizing solution of 0.5% TritonX-100 (cat. no. V900502; MilliporeSigma) at room temperature for 10 min. After further washing with PBS, the cells were blocked in PBS containing 2% BSA (cat. no. 9048-46-8; Shanghai Macklin Biochemical Co., Ltd.) and 0.1% Tween-20 (cat. no. 9005-64-5; Shanghai Macklin Biochemical Co., Ltd.) for 1 h at room temperature. The appropriate primary antibody dilutions were then added and incubated overnight at 4°C. Subsequently, the primary antibody was removed, the cells were washed three times with PBS (8 min each) and the corresponding secondary antibody dilution was added and incubated in the dark for 1 h at room temperature. Both primary and secondary antibodies were prepared at a ratio of 1:200. Finally, DAPI staining solution was added in the dark, and the cells were observed and images were captured under an inverted phase contrast fluorescence microscope to detect neuronal cell markers. The intermediate filament protein nestin is a common specific marker for NSCs, which is mainly expressed in the cytoplasm; NeuN is a neuron-specific nuclear protein; Olig2 is an oligodendrocyte-specific marker; glial fibrillary acidic protein (GFAP) is primarily found in astrocytes within the central nervous system and is a commonly used cellular marker. The primary antibodies anti-GFAP (cat. no. ab7260), anti-NeuN (cat. no. ab177487), anti-nestin (cat. no. ab254048) and anti-Olig2 (cat. no. ab254043), and the Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) secondary antibody (cat. no. ab150077) were obtained from Abcam. The antibodies were diluted with 5% BSA solution according to the instructions.

The rats were deprived of food for 12 h prior to surgery and administered an intraperitoneal injection of 3% sodium pentobarbital (45 mg/kg, provided by the Animal Experiment Center of Zhejiang University of Traditional Chinese Medicine) for anesthesia. The rat MCAO model was created using the modified Zea Longa wire bolus method. The rats were immobilized in the supine position, and an incision was made in the midline of the neck. The right common carotid artery (CCA), external carotid artery (ECA) and internal carotid artery (ICA) were sequentially isolated, and threads (4-0 sutures) were placed distal and proximal to the CCA and at the ECA. The ICA was temporarily clamped with a microarterial clip, and then the CCA and ECA were ligated proximally. Subsequently, a small opening was cut with scissors 4 mm from the bifurcation of the CCA, the prepared suture was inserted into the ICA, and the suture was tied securely with a thin wire wrapped around the distal end of the CCA when the marked point on the suture reached the bifurcation. The wound was covered with a saline-soaked cotton ball to keep it moist, and after 1.5 h of embolization, the suture was removed, the vessels were ligated, and the wound was sutured and disinfected. Rats were scored according to the Longa 5-point scale (26) on day 3 after modeling, and modeling was considered successful in rats with ≥1 point.

The 3rd-5th generation MSCs in logarithmic growth phase were collected, and after discarding the original medium, they were rinsed twice with PBS. Staining solution was prepared by adding the labeling solution to the medium in a ratio of 1 ml MSC medium: 1 μl CM-Dil labeling solution (cat. no. C7000; Invitrogen; Thermo Fisher Scientific, Inc.). The cells were incubated in a CO₂ incubator at 37°C for 10-20 min after the addition of the staining solution. At the end of the incubation, the cells were rinsed twice with PBS and then incubated again at 4°C for 15 min to avoid staining of the nuclei. The MSCs that had been labeled with CM-Dil were cultured for a further 24 h, and were then observed and images were captured under an inverted fluorescence microscope, which was used to detect the survival of MSCs in brain tissues after subsequent cell transplantation.

On day 3 after modeling, the rats were anesthetized by inhalation of 4% isoflurane through a mask, fixed into a brain stereotaxic apparatus, sterilized and a 10 μl suspension of MSCs (10⁷ cells/ml) was injected into the injection sites of the hippocampus at a rate of 1 μl/min. The rats were observed for infection and mortality.

A total of 45 SD rats were randomly assigned to five groups: Sham, MCAO, MSCs, Wnt3a-MSCs and PTC209-MSCs (n=9 rats/group). The Sham and MCAO groups were transplanted with 10 μl PBS on the postoperative day 3, the MSCs group was transplanted with MSCs on postoperative day 3, the Wnt3a-MSCs group was transplanted with 10 ng/ml Wnt3a-treated MSCs on day 3 after modeling, and the PTC209-MSCs group was transplanted with 1 μmol/l PTC209-treated MSCs on postoperative day 3. MSCs in each group were pretreated for 48 h.

The neurological injury severity in rats was assessed using the Neurological Severity Score (NSS) on days 1, 3, 5 and 7 after MSC transplantation. The rat tail was lifted 30 cm off the ground and the forelimbs were observed. Rats with both forelimbs symmetrically extended to the ground, with the left shoulder internally rotated and the left forelimb internally retracted were considered normal and rated 4 points, otherwise they were rated 0 points. In addition, the rat was placed on a smooth floor and resistance to movement was examined by pushing the left (or right) shoulder to the opposite side, respectively. Rats with resistance that was clearly symmetrical on both sides were considered normal (0 points); when the resistance was decreased, this was rated 1-3 points according to the degree of decrease. Furthermore, the two forelimbs of the rats were placed on a metal net and the tension of the two forelimbs was observed. When the tension of the two forelimbs was obviously symmetrical, this was considered normal (0 points); when the muscle tension of the left forelimb decreased, this was rated 1-3 points according to the degree of decrease. Based on the aforementioned scores out of 10, the higher the score, the more severe the behavioral disorder.

Grip assays were also performed on days 1, 3, 5 and 7 after transplantation of MSCs. In order to measure grip force, the grip force measuring instrument was placed on a horizontal table and levelled so that the grip force plate was in a horizontal direction. The rat was then gently placed on the grip plate and the tail was pulled back horizontally to record the maximum grip force of the rat.

On day 7 after MSC transplantation, intact brain tissues were obtained from three randomly selected rats in each group after euthanasia.; the animals were euthanized as aforementioned. These tissues were placed in a pre-cooled tank (4°C), rapidly frozen at −20°C for 20 min, and 2-mm coronal sections were cut and placed in 2% TTC dye at 37°C away from light for 15 min. After staining, images were captured, and were processed using the Image-Pro Plus 6.0 image analysis system software (Media Cybernetics, Inc.) to calculate the infarction rate of the rat brain tissue.

On day 14 after the transplantation of MSCs, three rats in each group were randomly selected for the removal of intact brain tissues, which were subjected to H&E staining to observe the pathological changes in the CA1 area of the hippocampus in each group. The animals were euthanized as aforementioned. Brain tissues were fixed with 4% paraformaldehyde at 4°C for 24 h, embedded in paraffin and were cut into 5-10 μm slices on a sectioning machine at 4°C. Tissue sections were deparaffinized with xylene two times (10 min each), hydrated with a descending series of ethanol concentrations, washed with water and distilled water, and stained with hematoxylin for 5 min. Subsequently, the sections were washed with water, incubated with 1% hydrochloric acid and 70% ethanol for 3 sec, washed with water for 10 min and stained with 1% eosin for 1 min. Finally, the sections were washed with water, dehydrated in an ascending series of ethanol concentrations, permeabilized twice with xylene (1 min each) and sealed with neutral gum. Finally, the sections were observed and images were captured under a light microscope.

On day 14 after transplantation of MSCs, three brain tissue sections were randomly collected from each group. Rat brain tissues were fixed in 4% paraformaldehyde for 8 h at room temperature., dehydrated in 30% sucrose solution, and embedded in OCT embedding agent, before being frozen at −80°C. Subsequently, the tissues were sectioned into 5-10 μm slices. Subsequently, 3% H₂O₂ was added dropwise to the sections and incubated at 37°C for 10 min to block endogenous peroxidase, after which, the sections were rinsed with distilled water three times (5 min each). The sections were then placed in boiling sodium citrate buffer solution and reacted at 92-98°C for 20 min for antigen retrieval. After cooling, the slices were washed three times with PBS solution (2 min each). Immunofluorescence was then performed as aforementioned for MSCs, using the same antibodies.

The MSCs of each group, as well as the ischemic penumbra tissues and hippocampal tissues on the damaged side of the rat brain of each group, were lysed with protein lysis solution (cat. no. 89900; Thermo Fisher Scientific, Inc.) on ice. The supernatant was collected after centrifugation at 10,000 × g for 15 min at 4°C after lysis. Following the extraction of total proteins from the cells and tissues, the protein concentration was detected according to the instructions in the BCA kit (cat. no.AR0146; Boster Biological Technology). Depending on the molecular weight of the protein, a 10 or 12% separation gel was prepared, and 20 μg proteins underwent SDS-PAGE. Proteins were then transferred to polyvinylidene fluoride membranes, which were blocked with a ready-to-use 5% skimmed milk powder sealer. The membranes were then incubated at 4°C overnight with the following primary antibodies: Anti-Bmi1 (cat. no. ab38295), anti-RhoA (cat. no. ab187027), anti-Wnt3a (cat. no. ab219412) (all from Abcam) and anti-β-actin (cat. no. BK7018; Bioker), anti-GFAP (cat. no. ab7260), anti-NeuN (cat. no. ab177487) and anti-nestin (cat. no. ab254048) (all from Abcam). Primary antibodies were diluted and prepared in 5% BSA solution at a ratio of 1:1,000 according to the antibody instructions. Subsequently, the membranes were incubated for 2 h at room temperature with a Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) secondary antibody (cat. no. ab150077; Abcam), which was diluted and prepared according to the antibody instructions. Images of membranes were captured using an ultrasensitive chemiluminescent substrate kit (cat. no. AR1111; Boster Biological Technology) utilizing a chemiluminescent imaging system (cat. no. 6000; Clinx Science Instruments Co., Ltd.) was used to capture images of the membranes, which were subsequently analyzed via ImageJ v1.8.0 analysis software (National Institutes of Health) for semi-quantification.

All experiments were repeated more than three times. The experimental data were statistically analyzed using SPSS 25.0 (IBM Corp.). Experimental data are presented as the mean ± standard deviation and bar graphs were constructed using GraphPad Prism 9 (Dotmatics). A χ² test for variance was performed on the experimental data to assess distribution of data. One-way analysis of variance was used to compare the differences between groups and the data were analyzed by Tukey's post-hoc test for multiple comparisons between groups. The analysis of NSS scores was performed using the Kruskal-Wallis test followed by Dunn's post hoc test. P<0.05 was considered to indicate a statistically significant difference.

Primary MSCs were cultured in T25 cell culture flasks and allowed to adhere to the wall and aggregate for 3 days. By day 7, the cells had formed colonies and were vigorously proliferating and dividing. Under a microscope, the cells were found to be either polygonal or irregularly shuttle-shaped (Fig. 1A-C). When the primary cultured MSCs reached 80-90% fusion, passaging culture was performed. Subsequently, MSCs experienced an increase in proliferative activity, as well as a continuous improvement in purity. Furthermore, their cell morphology gradually changed from an irregular polygonal shape to a shuttle or spindle shape (Fig. 1D). Different concentrations of Wnt3a (0, 10, 50 and 100 ng/ml) and Bmi1 small molecule inhibitor PTC209 (0, 1, 2.5, 5 and 10 μmol/l) were utilized to treat MSCs for 24 h, and the impact of both on cell viability was measured through MTT analysis. The results indicated that the viability of MSCs was normal in all groups of Wnt3a-treated cells, and there was no significant difference in the proliferative activity of MSCs between the different concentration groups, thus indicating that Wnt3a treatment did not affect the activity of MSCs (Fig. 1E). By contrast, in the cell groups treated with PTC209, that 1 μmol/l PTC209 had no significant effect on cell viability, whereas 2.5, 5 and 10 μmol/l PTC209 had a toxic effect on cells (Fig. 1F). Combining the findings of previous studies (27,28) and the present experimental results, 10 ng/ml Wnt3a and 1 μmol/l PTC209 were selected for subsequent experiments in the current study.

Compared with untreated MSCs, after 3 h of neural differentiation, the cytoplasm of MSCs contracted, longer synapses were formed, tiny branches appeared to converge into a meshwork, the cells grew well and a neuronal cell-like appearance was presented (Fig. 2A and B). MSCs induced by neural differentiation for 3 h could therefore be used for subsequent experiments. The BME, Wnt3a and PTC209 groups demonstrated specific green fluorescence of Nestin, indicating Nestin positive expression (Fig. 2C). In addition, the immunofluorescence results indicated the absence of marked green fluorescence in both the nucleus and cytoplasm following Olig2 staining; double-color staining of the nucleus and cytoplasm was not observed under a microscope, thus establishing that the expression of Olig2 was negative in all cell groups (Fig. 2D). Furthermore, after inducing neural differentiation of MSCs in vitro, the cytoplasm and nucleus emitted green fluorescence following GFAP staining; GFAP-positive cells could be clearly seen under the microscope in the BME and Wnt3a groups, whereas GFAP-positive cells were almost unobservable in the PTC209 group (Fig. 2E).

As determined by western blotting, the protein expression levels of Nestin were enhanced in the BME, Wnt3a and PTC209 groups compared with those in the NC group; however, no significant difference emerged between the three groups (Fig. 3). In addition, the protein levels of NeuN were significantly increased in the BME group compared with those in the NC group, and were significantly higher in the Wnt3a group compared with in the BME group. By contrast, NeuN protein expression levels were significantly lower in the PTC209 group compared with those in the BME group.

These findings indicated that BME stimulated MSCs to differentiate into NSCs in vitro, but not towards downstream oligodendrocyte lineage cells. Furthermore, Wnt3a treatment enhanced the differentiation of MSCs towards neurons, whereas PTC209 treatment impeded the differentiation of MSCs into astrocytes and neurons.

The protein expression levels of Bmi1, Wnt3a and RhoA were significantly increased in the BME group compared with those in the NC group (Fig. 4). In comparison with the BME group, the protein expression levels of Wnt3a and RhoA were increased in the Wnt3a group; however, there was no significant difference in Bmi1 protein expression. By contrast, the protein expression levels of Bmi1, Wnt3a and RhoA were significantly decreased in the PTC209 group compared with those in the BME group. These findings indicated that Bmi1 may be implicated in the neural differentiation process of MSCs by controlling the expression of Wnt3a and RhoA.

In the present study, SD rats were subjected to the experiment shown in Fig. 5A. When cultured in vitro to the third generation, MSCs displayed spindle-shaped morphology with high homogeneity (Fig. 5B), and CM-Dil-labeled MSCs exhibited strong red fluorescence with an uncolored nucleus and high labeling efficiency (Fig. 5C). A total of 7 days after MSCs transplantation the rats were sacrificed and their brain tissues were analyzed using fluorescence detection. The transplanted cells were visibly labeled with red fluorescence after CM-Dil labeling (Fig. 5D), and blue cell fluorescence was observed following DAPI staining (Fig. 5E). The cells that displayed co-localization of red and blue fluorescence were considered MSCs that had been implanted in the brain tissue (Fig. 5F and G). These findings indicated that the MSCs were successfully transplanted into the brain tissues of rats that had IBI and were viable.

The NSS scores demonstrated that rats in the Sham group obtained a score of 1 on days 1, 3, 5 and 7, and displayed no observable behavioral abnormalities (Fig. 6A). Conversely, rats in the MCAO group exhibited substantial behavioral deficits, including left shoulder internal rotation, decreased unilateral resistance to countermovement, and grip strength reduction on the left side due to IBI. There were no significant differences between the three groups receiving MSC transplantation on days 1, 3 and 5 when compared with the MCAO group; however, on day 7, rats in the MSC and Wnt3a-MSC groups had lower scores compared with in the MCAO group, whereas the PTC209-MSC group showed no significant change compared with the MCAO group. Furthermore, the results suggested that rats in the MCAO group had significantly weaker grasping strength than those in the Sham group on days 1, 3, 5 and 7 (Fig. 6B). On day 7, the MSCs group showed an improvement in grasping strength compared with the MCAO group. Furthermore, the Wnt3a-MSCs group exhibited an improvement in grasp strength on days 5 and 7, whereas the PTC209-MSCs group did not show any significant improvement at all time points. The TTC staining results indicated uniformly symmetric brain tissue coloring without infarction in the Sham group rats, whereas different degrees of infarction conditions were observed in the brain tissues of the MCAO group and the three MSC transplantation groups (Fig. 6C). The infarction rate in brain tissue was significantly decreased in all three groups receiving MSC transplantation compared with that in the MCAO group. Moreover, the Wnt3a-MSCs group had a lower infarction rate than the MSCs group, whereas the PTC209-MSCs group had an increased infarction rate compared with that in the MSCs group. After performing H&E staining on brain tissue sections, a normal and clear cell structure was observed in the Sham group; by contrast, the MCAO group exhibited more deeply stained, degenerated and atrophied neuronal cells, as well as a markedly enlarged tissue gap (Fig. 6D). The three groups that underwent MSC transplantation exhibited fewer cellular wrinkles and necrotic areas compared with the MCAO group, with varying degrees of improvement in tissue damage. However, among these three groups, there were still more wrinkled and deeply stained cells in the PTC209-MSCs group. This suggests that Wnt3a may enhance the neural repair function of MSCs and PTC209 could attenuate the neural repair function of MSCs.

All of the aforementioned findings indicated that the MCAO model rats developed notable behavioral disturbances following ischemic brain damage, whereas neurological function and localized brain infarction symptoms improved following transplantation of MSCs into MCAO model rats. Treatment of MSCs with Wnt3a further improved infarction symptoms in damaged brain tissues, whereas downregulation of Bmi1 using PTC209 hindered the reduction of the cerebral infarction rate, thus suggesting that Bmi1 serves a role in repairing IBI through MSC transplantation.

Sections of the rat hippocampal CA1 area were stained with red fluorescence for NeuN-positive cells and green fluorescence for GFAP-positive cells. The NeuN immunofluorescence results indicated a significant decrease in the number of NeuN-positive cells in the MCAO group as compared with in the Sham group (Fig. 7A). By contrast, the MSCs and Wnt3a-MSCs groups exhibited a significant increase in NeuN-positive cells compared with those in the MCAO group, whereas the PTC209-MSCs group showed no significant difference. When compared with the MSCs group, the Wnt3a-MSCs group showed a significant increase in NeuN staining while the PTC209-MSCs group showed a significant decrease. By contrast, the GFAP immunofluorescence results revealed a notable increase in GFAP-positive cells in the MCAO group when compared with the Sham group (Fig. 7B). Furthermore, the MSCs and Wnt3a-MSCs groups exhibited a significant reduction in the number of GFAP-positive cells compared with in the MCAO group, whereas there was no statistically significant difference in GFAP staining between the PTC209-MSCs group and the MCAO group. The number of GFAP-positive cells was significantly increased in the PTC209-MSCs group compared with the MSCs group.

The results of western blotting indicated that in the MCAO group, NeuN protein expression was lower and GFAP expression was higher compared with that in the Sham group (Fig. 8). Conversely, in the MSCs and Wnt3a-MSCs groups, NeuN protein expression was higher whereas GFAP protein expression was lower than that in the MCAO group. Notably, neither NeuN or GFAP expression was significantly altered in the Wnt3a-MSCs group compared with in the MSCs group. NeuN expression was decreased and GFAP expression was elevated in the PTC209-MSCs group compared with the MSCs group.

The present findings suggested that Wnt3a may promote an increase in the number of neurons in the CA1 region of the rat hippocampus and enhance the neural repair function of MSCs, whereas downregulation of Bmi1 could inhibit the increased neuronal number and the decrease of astrocyte number in the CA1 region of the hippocampus of MSC-transplanted rats, and may thus suppress neural repair.