A total of 130 C57BL/6J mice (half male and half female), aged 6 to 8 weeks, were euthanized by cervical dislocation following the inhalation of 5% isoflurane. Subsequently, the muscles were removed to expose and collect the femur and tibia. The isolation of mouse BMSCs was performed as previously described (25). In brief, bone marrow was obtained by flushing the marrow cavity using DMEM (Wuhan Servicebio Technology Co., Ltd.) containing 15% fetal bovine serum (FBS). The collected cell suspension was filtered using a 70-mm filter to remove bone spurs, muscle and cell masses. Red cell lysis buffer (cat. no. R1010, Beijing Solarbio Science & Technology Co., Ltd.) was added to the cells and centrifuged at 500 × g for 5 min to collect the precipitated cells. The cells were cultured in DMEM supplemented with 15% FBS and 1% penicillin-streptomycin for 24 h, after which the medium was replaced with fresh medium. The cells at passage three were collected for use in subsequent experiments.

BMSCs at passage three were used for characterization. As previously reported (26), BMSCs were cultured for 3 weeks with conditional media from osteoblasts [complete medium + 100 nM dexamethasone (Shanghai Aladdin Biochemical Technology Co., Ltd.) + 50 μg/ml L-ascorbic acid (Macklin) + 10 mM β-sodium glycerophosphate (Macklin)], lipoblasts (complete medium + 100 nM dexamethasone + 50 μg/ml indomethacin (Macklin) + 50 μg/ml L-ascorbic acid) and chondroblasts [(complete medium +1% insulin-transferrin-selenium additive (ITS) (iCell) +10⁻⁷ M dexamethasone +100 μM L-ascorbic acid + 10 ng/ml TGF-β1 (Sino Biological)], respectively, and stained with Alizarin red, Oil Red O and toluidine blue, respectively, and then observed for osteogenic, lipogenic and chondrogenic differentiation. The cell suspensions were incubated with CD44 (cat. no. E-AB-F1100D), CD90 (cat. no. E-AB-F1283D), CD31 (cat. no. E-AB-F1180D) and CD45 (cat. no. E-AB-F1136D) antibodies (Elabscience Biotechnology, Inc.) conjugated to the fluorescent protein Phycoerythrin (PE), and the expression of cell surface markers was analyzed by flow cytometry. The established MSC markers CD44 and CD90 exhibited strong positivity, whereas the endothelial cell marker, CD31, and the hematopoietic cell marker, CD45, exhibited minimal positivity, indicating that the isolation of BMSCs was successful (27).

The present study employed the 2nd generation lentiviral systems. The coding sequence (CDS) of Six3 (NM_011381: 407-1408 bp) was cloned into the pCDH-CMV-MCS-EF1-GFP-T2A-puro plasmid (cat. no. BR318; Hunan Fenghui Biotechnology Co., Ltd.) (restriction enzyme cutting site, XbaI/NheI) for the construction of a Six3 overexpression plasmid. The CDS of Btg2 (NM_007570: 103-579 bp) was cloned into the pLVX-IRES-puro plasmid (cat. no. BR025; Hunan Fenghui Biotechnology Co., Ltd.) (restriction enzyme cutting site, EcoRI/BamHI), and the short hairpin RNA sequence targeting Btg2 (5'-ATGGCCGTCTCCAGCTAGATA-3') was cloned into the pLKO.1-EGFP-puro plasmid (cat. no. BR524, Hunan Fenghui Biotechnology Co., Ltd.) (restriction enzyme cutting site, AgeI/EcoRI). The empty plasmids without any foreign fragments were used as a negative control. The 14 μg recombinant shuttle-plasmid, together with the 10.5 μg pSPAX2 and 3.5 μg pMD2.G plasmids (cat. no. BR036 and BR037, Hunan Fenghui Biotechnology Co., Ltd.), were transfected into 293T cells (iCell) using Lipofectamine 3000 (cat. no. L3000015; Thermo Fisher Scientific, Inc.) to package the infectious lentiviral particles. The 293T cells were cultured in DMEM (Wuhan Servicebio Technology Co., Ltd.) supplemented with 10% FBS at 37°C and 5%CO₂, and the cell supernatant was collected and filtered through a 0.45-μm filter at 48 and 72 h following transfection. BMSCs were cultured in DMEM supplemented with 15% FBS and 1% penicillin-streptomycin at 37°C and 5% CO₂. When the confluency of BMSCs reached 60%, they were infected with lentivirus (MOI=20) for 48 h and then observed under a fluorescence microscope. Cell activity was measured using MTT assay.

The cells or tissues were incubated in TRIpure lysis buffer (5~10×10⁶ cells/ml; cat. no. RP1001; BioTeke Corporation), and the mixture was extracted using chloroform. The water phase was collected, and isopropyl alcohol and ethanol were added to it to precipitate the total RNA. The RNA concentration was determined using an ultraviolet spectrophotometer. cDNA was obtained by reverse transcription using All-in-One First-Strand SuperMix according to the manufacturer's instructions. RT-qPCR analysis was performed on a fluorescence quantifier (Bioneer Corporation) in a system containing cDNA, primers and SYBR-Green Master Mix (Beijing Solarbio Science & Technology Co., Ltd.). The thermal cycling conditions are as follows: 95°C, for 5 min (pre-degeneration); 95°C, for 10 sec; 60°C, for 10 sec; 72°C, for 15 sec. A total of 40 cycles were conducted. Actin beta (Actb) was regarded as a regulatory gene. The primer sequences were as follows: Btg2 forward, 5'-GCACTGACCGATCATTACAAA-3' and reverse, 5'-GCAGGTGAGGAGCCCATA-3'; Six3 forward, 5'-GCTGTCGTCGCCTTCCA-3' and reverse, 5'-CGCACGCGGTACTTGTC-3'; Actb forward, 5'-CATCCGTAAAGACCTCTATGCC-3' and reverse, 5'-ATGGAGCCACCGATCCACA3'. Relative gene expression was calculated using the 2^−ΔΔCq method (28).

The cells or tissues were lysed in a lysate buffer (cat. no. P0013; Beyotime Institute of Biotechnology) supplemented with PMSF, and the mixture was centrifuged (10,000 × g) at 4°C for 5 min to separate the supernatant. The protein content in the supernatant was quantified using a BCA protein concentration assay kit and diluted to a uniform concentration using a PBS buffer. The protein solution was mixed with a 5X Loading Buffer and boiled to denature the protein. The prepared protein samples were loaded onto polyacrylamide gel (~30 μg protein per lane) and separated by SDS-PAGE (5% spacer gel and 12% separation gel). The protein bands were transferred to PVDF membranes and incubated in the blocking solution (cat. no. P0023; Beyotime Institute of Biotechnology) for 1 h at room temperature (~26°C). PVDF membranes were then incubated with primary antibodies for 12 h at 4°C and secondary antibodies for 1 h at room temperature and then visualized using ECL chemiluminescence reagents (cat. no. P0018; Beyotime Institute of Biotechnology). The optical density values of the blots were analyzed using Gel-Pro Analyzer 4.0 software (Media Cybernetics, Inc.). Antibody information is provided in Table SI.

The cells were cultured until fused, treated with mitomycin C (MilliporeSigma) for 1 h under serum-free conditions, and then scratched using a pipette tip. The cell surface was cleaned using serum-free medium and then images were captured under a light microscope. Cell migration was observed following 24 h of culture.

Tissues or cell proliferating on glass coverslips were fixed in 4% paraformaldehyde for 24 h or 15 min at room temperature. The tissues were embedded in OCT embedding agent and pre-cooled, then cut into 10-μm-thick slices. The prepared samples were then incubated with primary and fluorescently labeled secondary antibodies. DAPI (1 mg/ml) was used for nuclear staining. The staining results were observed under a fluorescence microscope. Antibody information is provided in Table SI.

The levels of brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) in the cell supernatant were determined using corresponding ELISA kits (cat. nos. EM0020 and EM0148; FineBio; http://www.finebio.cn/) according to the manufacturer's instructions.

Total RNA was extracted using the TRNzol universal reagent (cat. no. DP424, Tiangen Biotech Co., Ltd.), and mRNA was enriched from total RNA using Oligo (dT) magnetic beads (cat. no. N403-01; Vazyme Biotech Co., Ltd.), and then fragmented in fragmentation buffers. cDNA was synthesized from mRNA template and purified using AMPure XP beads. Following terminal repair, PolyA tail addition, and sequencing aptamer connection, the purified DNA fragments were screened using AMPure XP beads. PCR was performed to construct the DNA library, and the qPCR program was used to examine the concentration of DNA libraries. The original concentration of the library was ~4 μM. The integrity of the genomic DNA was analyzed using an Agilent 5400 fragment analyzer (Agilent Technologies, Inc.). The qualified DNA libraries were sequenced (nucleotide length: 150 bp; paired-end) using NovaSeq 6000 high-throughput sequencing platform (Illumina Inc.). The loading volume was 5 μl and the concentration was ~20 pM. Data filtering was performed using the fastp software (version 0.23.1; HaploX), and alignment with the reference genome was conducted using the HISAT2 software (version 2.2.1, Johns Hopkins University) (29). The HTSeq software (version 2.0.2, European Molecular Biology Laboratory) (30) was used for analyzing gene expression, and the R package DESeq2 (version 1.24.0, European Molecular Biology Laboratory) (31) was used for analyzing differentially expressed genes.

The nucleotide sequence containing the Btg2 promoter region was cloned into the pGL3-Basic plasmid vector (restriction enzyme cutting site, NheI/HindIII) and co-transfected into 293T cells with the Six3 overexpression plasmid using Lipofectamine 3000 (cat. no. L3000015; Thermo Fisher Scientific, Inc.). Following 48 h, the luciferase activity was detected using a dual luciferase assay kit (cat. no. KGE3302; Nanjing KeyGen Biotech Co., Ltd.). The ratio of Firefly luciferase activity to sea kidney luciferase activity was calculated.

The cells were incubated in formaldehyde at 37°C for 10 min to cross-link proteins and DNA. After washing, the cells were re-suspended in an SDS Lysis Buffer and incubated on ice for 10 min to be fully lysed. The lysate was ultrasound treated to cut the DNA. The lysates were incubated with Six3 antibody at 4°C overnight and then with Protein A+G Agarose/Salmon Sperm DNA at 4°C for 1 h. The DNA-protein complex was eluted from Protein A+G Agarose/Salmon Sperm DNA and was used for PCR. The primer sequences designed for the potential binding sites of Six3 in the Btg2 promoter region were as follows: Forward, 5'-GGTACTCCGAGTTAAAGA-3' and reverse, 5'-CTGACCAGCAAGACATT-3'. PCR products were examined using agarose gel electrophoresis.

DNA pulldown kit (cat. no. Bes5004; Bersinbio) was used for the DNA pull-down assay according to the method provided by the manufacturer. The biotinylated DNA probes were added to streptavidin magnetic beads and incubated in the DNA beads buffer at 25°C for 30 min to prepare the DNA probe-bead complex. Agarose beads (40 μl) were added to the protein samples for pre-cleaning and then incubated with DNA probe-bead complex in 500 μl binding buffer, 5 μl poly (dI-dC), 5 μl protease inhibitor, 5 μl DTT, 9 μl EDTA and 4.5 μl EGTA at 4°C for 1 h. Magnetic beads were then collected using a magnetic separation device and washed in 0.8 ml binding buffer and 4 μl DTT. The DNA-protein complex was eluted using 60 μl Protein Elution buffer and 0.6 μl DTT at 37°C for 2 h. After removing the magnetic beads, the supernatant was obtained, which was used for western blot analysis.

Male C57BL/6J mice (22-25 g), aged 10 to 12 weeks, were housed in a 12-h light/dark cycle environment with a temperature of 22±1°C and a humidity of 45-55%. They were also allowed free access to food and water. The mice were anesthetized with 5% isoflurane for induction and 1.0% isoflurane for maintenance. The mice were subjected to MCAO according to the previously reported methods (32), maintained for 90 min and followed by reperfusion for 24 h. At the same time as the initiation of reperfusion, 0.3 ml PBS containing 1×10⁵ BMSCs was injected into the tail vein once (33). The mice in the sham-operated group underwent the same surgical procedure, but without the insertion of sutures. During the operation, throughout the recovery period, and within 24 h after reperfusion, a constant-temperature heating pad was used to maintain the animal's body temperature. Moist feed was placed at the bottom of the cage to facilitate feeding. After mice underwent MCAO, continuous observation was conducted throughout the recovery period until they regained spontaneous breathing and basic activities. Subsequently, the animals' spontaneous breathing, righting reflex and bleeding were checked every 30 min. Following reperfusion, continuous monitoring continued until the mice regained consciousness, with assessments every 30 min during the first 0-2 h, every hour from 2 to 6 h, and every 4 h from 6 to 24 h. The ischemic nerve defect score was assessed following reperfusion (33). Two weeks after the MCAO procedure, the mice underwent the cylinder test (34). For mice subjected to long-term behavioral assessments, monitoring was performed every 12 h starting 24 h after reperfusion, focusing primarily on body weight, food and water intake, activity, and reactivity. Subsequently, the animals were euthanized by cervical dislocation following the inhalation of 5% isoflurane, and brain tissues were collected for use in further experiments. The humane termination criteria were determined earlier, including persistent or recurrent convulsions that do not subside, coma unresponsive to stimulation, respiratory distress, and weight loss exceeding 20%. Once any of these criteria are met, euthanasia should be performed immediately. Death was confirmed by the absence of a heartbeat (assessed via chest palpation), cessation of spontaneous respiratory movements for more than 30 sec, and the absence of both the corneal reflex and response to a vigorous toe pinch. The animal experiments were approved by the Animal Ethics Committee of the First Affiliated Hospital of Zhengzhou University (approval no. 2025-KY-1279; Zhengzhou, China).

A total of 256 male C57BL/6 mice were used in the stroke study, of which 244 underwent MCAO surgery, and 12 mice underwent sham surgery. During the establishment of the MCAO model, 39 mice succumbed within 24 h after reperfusion (found dead), and the similar mortality rates have been reported before (35). The primary causes of death were intraoperative bleeding or severe brain oedema following reperfusion. The remaining 205 mice that underwent MCAO survived until 24 h after reperfusion. Based on the neurological deficit scores, 166 models were confirmed as successful (scores ≥1) and used for subsequent experiments, while 39 models (scores <1) were classified as unsuccessful and euthanized. Among them, 156 MCAO mice and 12 Sham mice were used to generate valid research data. Before the formal experiment, 10 spare mice were used to explore the antibody incubation conditions and to optimize the cylinder test setup.

Fresh brain tissue was frozen at −20°C and cut into six consecutive slices at a thickness of 1-mm, and then incubated in TTC solution for 15 min. Following staining, the ischemic area appears grayish white, and the normal tissue appears dark red.

The ischemic penumbral cortex tissue was isolated, placed in OCT embedding agent, pre-cooled, and then cut into 10-μm-thick slices. The slices were covered with cresol purple solution (Sinopharm Chemical Regent Co., Ltd.) and incubated for 10 min, then placed in ethanol solution containing 0.25% acetic acid to remove the excess dye. The results of neuron staining were observed under a light microscope.

The data were analyzed using GraphPad Prism software (version 9.3; Dotmatics). The data is presented as the mean ± standard deviation. A one-way ANOVA followed by Tukey's multiple comparisons test was employed to assess differences between groups. Data that do not conform to the rules of ANOVA were analyzed using unpaired t-tests. P<0.05 was considered to indicate a statistically significant difference.

BMSCs were isolated from mouse bone marrow and cultured to the third passage for use in the following experiments (Fig. 1A). Flow cytometry was used to detect the expression of cell surface marker proteins, including CD44, CD90, CD31 and CD45. The results show revealed that the expression levels of CD44 and CD90 on the cell surface were increased, while the expression levels of CD31 and CD45 were decreased (Fig. 1B). Following the induced differentiation culture (Fig. 1C), Alizarin red, Oil Red O and toluidine blue staining yielded strongly positive results, indicating that the cells had the potential to differentiate into osteogenesis, adipogenesis and chondrogenesis (Fig. 1D). These findings indicated that BMSCs were successfully isolated.

As shown in Figs. S1 and S2, the Gene Ontology (GO) enrichment analyses of the differentially expressed genes (DEGs) provided by the GSE8590 and GSE150710 datasets were analyzed. The results revealed that they were generally involved in biological processes such as neural development, stem cell differentiation and neuronal regeneration. Of note, four potential target genes were screened by analyzing the GSE8590 and GSE150710 data sets (Fig. 2). GSE8590 indicated that the expression of Six3 was elevated in neural progenitor cells derived from hESCs. Notably, Six3 exhibited the largest upregulation in expression among the four genes, suggesting that Six3 may be involved in the neural differentiation of MSCs. GSE150710 also revealed that the expression of Six3 was enhanced in the host receiving human neural stem cells (hNSCs), and it was also increased in the transplanted hNSCs, which further emphasized the role of Six3 in stem cell transplantation for the treatment of stroke.

The present study aimed to modify BMSCs through a lentivirus-mediated Six3 overexpression system and then explore the role of Six3 in BMSCs (Fig. 3A). Following 48 h of infection, the successful infection by the lentivirus was observed under a fluorescence microscope (Fig. 3B). Additionally, it was observed that the overexpression of Six3 enhanced the proliferation of BMSCs. RT-qPCR and western blot analysis revealed that Six3 was successfully overexpressed in BMSCs (Fig. 3C and D). The overexpression of Six3 enhanced BMSC viability (Fig. 3E) and promoted BMSC migration in vitro (Fig. 3F). According to the representative immunofluorescence images of neuronal marker proteins, the overexpression of Six3 increased the expression of Nestin (Fig. 3G), microtubule associated protein 2 (MAP2) (Fig. 3H) and neuronal nuclei (NeuN) (Fig. 3I) in BMSCs. The levels of the neuronal growth factors, BDNF and NGF, in the cell supernatant were increased by the overexpression of Six3 (Fig. 3J). These results demonstrate the potential of Six3 to promote the differentiation of BMSCs into neurons.

As demonstrated in Fig. 4A, the results revealed a significant separation of BMSCs and BMSCS-Six3^GFP samples for transcriptomic analysis, and the favorable homogeneity of samples within the groups. The findings depicted in Fig. 4B also demonstrated that the overexpression of Six3 significantly altered the transcriptomes of BMSCs, including 150 downregulated genes and 323 upregulated genes (|Log₂FC|>1, P<0.05); among these, the expression of Six3 was significantly increased (Log₂FC=4.99, P<0.05), indicating that Six3 was successfully overexpressed (Fig. 4C). Through GO annotation and enrichment analysis of the aforementioned DEGs, it was observed that Six3 overexpression targets the genes related to neuronal death (Fig. 4D), including five downregulated genes and 26 upregulated genes (Fig. 4E). In order to further explore the mechanisms of Six3 overexpression, the upregulated or downregulated genes in the results of RNA-sequencing and DEGs in the GSE8590 dataset were comprehensively analyzed (Fig. 4F), and 13 shared genes were obtained. Among these, three genes were found to be enriched in terms related to neuronal death, including Btg2, Igfbp5 and Lrp2. It was observed that Btg2 was significantly increased following the overexpression of Six3 and had the highest basal expression in BMSCs. Therefore, Btg2 was selected for further analyses.

RT-qPCR and western blot analysis further confirmed that the overexpression of Six3 increased the expression of Btg2 (Fig. 5A and B). Dual luciferase report assay revealed that Six3 enhanced Btg2 promoter activity (Fig. 5C), and the ChIP-PCR results indicated that Six3 bound to the Btg2 promoter region (Fig. 5D). The Btg2 promoter binding protein was obtained through DNA pull-down assay, and the binding of Six3 and Btg2 promoter was further demonstrated by western blot analysis (Fig. 5E).

RT-qPCR and western blot analysis demonstrated that Btg2 expression in BMSCs was upregulated by the overexpression of Six3 and downregulated by the BTG2-targeting lentiviral system (Fig. 5F and G). Consistent with the aforementioned findings, the overexpression of Six3 increased the cell viability of BMSCs (Fig. 5H), the expression of neuronal marker proteins (MAP2 and Nestin) (Fig. 5I), and the secretion of BDNF and NGF (Fig. 5J). However, Btg2 knockdown reversed the effects of Six3; these findings indicated that Btg2 mediated the promoting effects of Six3 on the neural differentiation of BMSCs.

Mice subjected to MCAO exhibited moderate to severe neurological deficits (Fig. 6A), such as circling to the contralateral side. Mice transplanted with BMSCs exhibited mild neurological deficits. Representative TTC staining images revealed that BMSC transplantation alleviated cerebral ischemic injury in mice subjected to MCAO (Fig. 6B). Specifically, BMSC transplantation reduced brain swelling and cerebral infarction caused by MCAO. By observing the distribution of GFP in brain tissue, it was also observed that BMSCs were transplanted into the brain tissue (Fig. 6C). The 200-fold magnification images demonstrated GFP expression in the ischemic penumbra, demonstrating the migration of BMSCs to the injured area. As demonstrated in Fig. 6D, the neural deficits were further improved in the mice subjected to MCAO and transplanted with Six3-overexpressing BMSCs. The infusion of BMSCs reduced the limb asymmetry in stroke-stricken mice. Mice infused with Six3 overexpressed BMSCs exhibited more balanced limb use (Fig. 6E). The TTC staining results revealed that compared with BMSC transplantation, the transplantation of Six3-overexpressing BMSCs had a more prominent reducing effect on brain swelling and cerebral infarction (Fig. 6F).

Neuronal atrophy and loss were detected in the brain tissue of mice subjected to MCAO, which was reduced by BMSC transplantation (Fig. 7A). The transplantation of Six3-overexpressing BMSCs further protected the morphology of neurons in the brain tissue of mice subjected to MCAO and increased the number of neurons. The expression of Six3 and GFP in brain tissue is illustrated in Fig. 7B. The colocalization of Six3 and GFP expression was observed in the ischemic penumbra of mice transplanted with BMSCs. The high expression of Six3 in BMSCs was observed in the brain tissues of mice in the MCAO + BMSCs-Six3 group. The expression of GFP is illustrated in Fig. 7C. Compared with the MCAO + BMSCs group, the overexpression of Six3 promoted the migration of BMSCs to the infarct area in brain tissue. As demonstrated in Fig. 7D and E, the expression of the neuronal markers, NeuN and MAP2, was increased in the BMSCs within the host. Notably, the overexpression of Six3 in BMSCs enhanced enhances the expression of these neuronal markers in BMSCs within the host brain tissue. These findings suggest that the overexpression of Six3 in BMSCs enhances their homing ability and neural differentiation in the damaged brain tissue of mice subjected to MCAO. Additionally, it was observed that the expression of Btg2 in the brain tissues of mice with stroke was increased following the transplantation of Six3-overexpressing BMSCs (Fig. 7F). As demonstrated in Fig. 7G, Btg2 was widely expressed in the brain tissues of mice subjected to MCAO. The colocalization of Btg2 and GFP was detected in the brain tissue of mice transplanted with BMSCs. The increased expression of Btg2 was observed in the Six3-overexpressing BMSCs within the injured brain tissue. The aforementioned evidence indicates that the promoting effect of the Six3-Btg2 axis on the migration and neural differentiation of BMSCs in vitro is also observed in the brain tissues of mice subjected to MCAO that received BMSC transplantation.

The in vivo rescue experiments demonstrated that Btg2 mediated the enhancing effects of Six3 on the therapeutic efficacy of infused BMSCs for the treatment of stroke. The silencing of Btg2 led to an increase in the neurological deficit score (Fig. 8A), an increase in the asymmetry of limb use (Fig. 8B), an increase in cerebral infarction (Fig. 8C) and an increase in brain swelling (Fig. 8D). The double-stained immunofluorescence images confirmed the reduction of Btg2 expression in BMSCs (Fig. 8E). Btg2 knockdown abolished the promoting effects of Six3 on the neural differentiation of infused BMSCs (Fig. 8F).

Kyoto Encyclopedia of Genes and Genomes enrichment analysis of the upregulated DEGs that were identified through RNA sequencing was performed to investigate the regulatory effects of Six3 on downstream signaling pathways. The results revealed that the overexpression of Six3 primarily affected the PI3K-Akt signaling pathway (Fig. 9A). Elevated levels of Akt phosphorylation were observed in brain tissues of mice transplanted with Six3-overexpressing BMSCs (Fig. 9B). The silencing of Btg2 partially suppressed Akt phosphorylation in BMSCs induced by Six3 overexpression (Fig. 9C). It was demonstrated that the Akt signaling pathway was directly activated by the overexpression of Btg2, rather than being secondary to altered cellular states (Fig. S3A-C). In rescue experiments, BMSCs were treated with the PI3K inhibitor, LY294002, to inhibit the activation of the Akt signaling pathway (Fig. 9D). Treatment with LY294002 reversed the promoting effects of Six3 overexpression on BMSC viability (Fig. 9E) and neural differentiation, as evidenced by the suppression of MAP2 and Nestin expression (Fig. 9F) and the reduced secretion of BDNF and NGF (Fig. 9G). To determine whether the Akt signaling pathway is directly regulated by Btg2, rather than being secondary to changes in the cell state, loss-of-function and rescue experiments were conducted. As demonstrated in Fig. S1A and B, Btg2 silencing reversed the effects of Btg2 overexpression, including reducing the viability of BMSCs and inactivating the Akt signaling pathway.