Differential Roles of Notch Receptors in Regulating Activation of Intact Adult Mouse Spinal Cord-derived NSCs

Differential Roles of Notch Receptors in Regulating Activation of Intact Adult Mouse Spinal Cord-derived NSCs | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Differential Roles of Notch Receptors in Regulating Activation of Intact Adult Mouse Spinal Cord-derived NSCs Juan Li, Fen Ju, Zhao-Yang Huang, Liang Yu, Jie Qin, Yu-Bing Yang, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7540357/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Neural stem cells (NSCs) play a crucial role in neural regeneration following spinal cord injury (SCI) owing to their self-proliferative and multidirectional differentiation capabilities. This study examined the biological properties of adult spinal cord-derived NSCs (sp-NSCs) and the role of Notch receptors in regulating their activation. NSCs were isolated from the spinal cords of 8-week-old C57/BL6 mice, and their biological properties, including the gene expression profile of Notch receptors, were subsequently analyzed using single-cell RNA sequencing (scRNA-Seq) and bioinformatics. The NSCs were subsequently infected with lentiviral vectors encoding Notch1 shRNA, Notch2 shRNA, and a combination of Notch1 and Notch2 shRNA sequences, and evaluated using Sphere assays and EdU staining to determine their activation effects. The expression levels of downstream genes, NICD and Rbpj, in the Notch signaling pathway, as well as the related target genes, Hes1 and Hey1, were subsequently quantified using Western blot analysis. Intact adult mouse sp-NSCs predominantly existed in a quiescent state, with their population increasing significantly with age. Notch receptors served as critical regulators of adult sp-NSC activation. Notably, Notch1 expression was significantly elevated compared to Notch2 and Notch3, demonstrating its predominant role in sustaining NSC activation. In contrast, Notch2 and Notch3 were primarily responsible for maintaining NSCs in a quiescent state. Overall, both Notch1 and Notch2 signals are involved in different regulatory roles that facilitate the activation and fate determination of NSCs via NICD-Rbpj. spinal cord injury neural stem cells Notch receptors Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1. Introduction Spinal cord injury (SCI) is a severe central nervous system (CNS) disorder characterized by disruption of spinal cord continuity, neuronal death, and myelin disintegration [ 1 ] . Despite its severity, SCI lacks effective clinical treatment. Neural stem cells (NSCs) possess self-renewal and multipotent differentiation capabilities [ 2 ] . They play a crucial role in nerve regeneration following SCI, but their application in SCI repair is hindered by the limited quantity, restricted activation, and differentiation capacity of spinal cord-derived NSCs (sp-NSCs) in adult mammals [ 3 ] . Elucidating the biological properties and activation of regulatory mechanisms of sp-NSCs is thus a critical step that should be addressed before their application in the treatment of various neurological diseases. Sp-NSCs are governed by complex regulatory mechanisms during development and maturation [ 4 ] . These NSCs display significant heterogeneity, with quiescent and activated states coexisting under various physiological conditions [ 5 ] . Nonetheless, the interactions between these diverse states and the regulatory factors governing their transformation are not fully understood, limiting their activation potential and impairing neural repair. Studies postulate that NSC activation post-SCI is regulated by multiple signaling pathways [ 6 ] . Of note, the Notch signaling pathway plays a crucial role in early activation and differentiation. It primarily involves Notch receptors, ligands, CSL proteins, and downstream target genes [ 7 ] . Studies postulate that Notch receptors have distinct roles in activating and differentiating NSCs within the adult mammalian nervous system. Notch1 sustains the activated state of NSCs and promotes their differentiation into neurons [ 8 ] . In contrast, Notch2 and Notch3 preserve the quiescent state of NSCs. The specific expression patterns of Notch receptors can potentially influence the activation states of NSCs, especially within the CNS, exerting asymmetric effects on NSC activation. This study aimed to characterize the biological properties of sp-NSCs in intact adult mice and examine their activation by Notch receptors to provide new insights for enhancing neurorestorative strategies following SCI. 2. Method and Materials 2.1 Isolation, culture, and differentiation of intact adult mouse sp-NSCs This study was approved by the Medical Ethics Committee of Xijing Hospital, Air Force Medical University (No. XJYYLL-20240320), and all efforts were made to minimize animal suffering and reduce animals used. It was conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals, revised in 1996 (NIH Publications No. 80 − 23). The NSC culture system was modified, building upon the existing in vitro culture methods to achieve a stable and abundant population of intact adult mouse sp-NSCs [ 9 ] . Briefly, 8-week-old and 16-day-pregnant C57/BL6 mice were anaesthetized using 5% isoflurane under aseptic conditions. Subsequently, the mice were euthanized by cervical dislocation. Under aseptic conditions, E16.5 embryos were immediately dissected, and spinal cords were exposed via ventral laminectomy using fine spring scissors. The T1–T12 spinal cord segments were excised, transferred to a culture dish containing pre-cooled phosphate-buffered saline (PBS), and cut into approximately 1 mm³ pieces. Accutase was subsequently added, and the tissues incubated for 10 minutes in a water bath set at 37°C. NSCs growth medium was then added to terminate enzymatic activity after digestion. The mixture was centrifuged to obtain the cells, which were then resuspended in NSCs medium and dissociated into single cells by gentle pipetting approximately 30 times with a Pasteur glass pipette. The cells were then filtered through a 70 µm cell strainer and their viability assessed through Trypan blue staining. Briefly, the cells were seeded at a density of 1 × 10⁶ cells/mL in NSCs growth medium and cultured in an incubator set at 37°C and 5% CO₂. NSCs growth medium comprised DMEM/F12, 2 mM GlutaMAX-I, 100 IU/mL Penicillin-Streptomycin, 20 ng/mL EGF, 20 ng/mL bFGF, B27, and N2. Growth factors were added every 3 days, and the medium was refreshed every 6 days. Cell passaging was performed mechanically using a pipette on day 12. All experiments were conducted between the third and seventh cell passage. The cells were then maintained for at least a day in a medium containing 10 µg/ml laminin and 0.01% poly-L-lysine. Specific differentiation media were used in place of the NSCs growth medium to induce differentiation. The differentiation media included the neuronal, astrocyte, and oligodendrocyte differentiation media. The neuronal differentiation medium contained Neurobasal media (21103049, Gibco), GlutaMAX-I Supplement (2 mM), and B-27 Supplement (17504044, Gibco); the astrocyte differentiation medium contained DMEM (10569010, Gibco), N-2 Supplement, GlutaMAX-I Supplement (2 mM), and FBS (10091148, 1%, Gibco); while the oligodendrocyte differentiation medium contained Neurobasal Medium, GlutaMAX-I Supplement (2 mM), B-27 Supplement, T3 (30 ng/ml, T2877, Sigma), Ascorbic Acid (200 µM, PHR1008, Sigma), and NT-3 (5 ng/ml, 45003, PeproTech). The cells were incubated in the differentiation media for seven days and then fixed with 4% paraformaldehyde (PFA) for immunofluorescence labeling. 2.2 Immunofluorescence Staining The fixed cells were blocked for 1 hour at room temperature using PBS supplemented with 10% normal goat serum and 0.3% Triton X-100. The cells were then incubated with primary antibodies at 4°C overnight, washed with PBS, and incubated with a secondary antibody (1:5000, Abcam) for 2 hours at room temperature in the dark. The cells were then stained with Hoechst for 5 minutes at room temperature in the dark, washed with PBS, mounted onto microscope slides and examined using fluorescence microscopy. The following primary antibodies were used: anti-Nestin (NSC marker, 1:200, Chemicon), anti-Sox2 (NSC marker, 1:200, Abcam), anti-Tuj1 (neuronal marker, 1:200, Millipore), anti-GFAP (astrocyte marker, 1:400, Dako), anti-O4 (oligodendrocyte marker, 1:50, Chemicon), anti-Notch1 (1:200, Abcam), anti-Notch2 (1:200, Abcam), and anti-Notch3 (1:200, Proteintech). The percentage of positive cells was calculated based on three biological replicates. 2.3 Sphere assay [ 10 ] Suspension-cultured NSCs were seeded in 6-cm Petri dishes, and micrographs were randomly captured at 10× magnification following previously published protocols. Three biological replicates were prepared for each treatment group. The diameters of individual neurospheres in each biological replicate were randomly selected and measured using ImageJ software. 2.4 Western blotting NSCs were collected, washed with pre-cooled PBS, and lysed using RIPA buffer supplemented with protease inhibitors. RIPA buffer contained 150 mM NaCl, 50 mM Tris-HCl, 1% Triton X-100, 2% SDS, and 1% sodium deoxycholate. The cell homogenates were centrifuged at 12,000 rpm for 5 minutes at 4°C to collect the supernatant for protein concentration determination using a NanoDrop spectrophotometer (ND-1000, Thermo Fisher). The protein samples (20 µg) were separated on a 10% SDS-PAGE, and the resultant bands transferred onto polyvinylidene fluoride (PVDF) membranes. The membranes were then blocked with 5% skimmed milk at room temperature and incubated with primary antibodies at 4°C overnight. The primary antibodies included rabbit anti-Notch1 (1:1000, Abcam), rabbit anti-Notch2 (1:1000, Proteintech), rabbit anti-NICD (1:1000, CST), rabbit anti-Rbpj (1:1000, Cell Signaling Technology), rabbit anti-Hes1 (1:500, Abcam), rabbit anti-Hey1 (1:500, Abcam), and anti-β-actin (1:2000, Cell Signaling Technology). The membranes were then washed with TBST to remove the excess primary antibodies and subsequently incubated with horseradish peroxidase-conjugated secondary antibody (1:2000, ab205718, Abcam, Cambridge, UK) for 2 hours at room temperature. The chemiluminescent signals were subsequently scanned and analyzed quantitatively using ImageJ software (version 1.45, NIH, USA). 2.5 EdU Staining NSCs were seeded into 96-well cell culture plates. Pre-warmed 10 µM EdU (at 37°C) was then added to the plates and incubated in a cell culture incubator for 2 hours to label the S-phase cells. EdU was pipetted from the plates, and the cells were fixed for 15 minutes at room temperature by adding 100 µL of 4% paraformaldehyde to each well. The cells were washed with 3% BSA and then incubated with 0.3% Triton X-100 permeabilization solution for 15 minutes at room temperature. The permeabilization solution was pipetted and the cells washed with 3% BSA. Click reaction solution (100 µL) containing Click Reaction Buffer, Click Additive Solution, CuSO₄, and Azide 555 in a ratio of 430:50:20:1 was then added to each well, and the cells incubated for 30 minutes at room temperature in the dark. The reaction solution was pipetted out, and the cells were washed thrice with 3% BSA. Hoechst 33342 diluted 1,000-fold with 1× PBS was added, and the cells incubated for 10 minutes at room temperature in the dark. The Hoechst 33342 solution was pipetted out, and the cells were washed thrice with 3% BSA. The bottom of the 96-well plates was then gently wiped using a microscope paper to prepare for fluorescence imaging and analysis. 2.6 Single-cell RNA sequencing (scRNA-seq) Neurospheres were enzymatically digested to dissociate into a single-cell suspension. The single-cell suspension preparations were observed under an electron microscope, followed by cell counting and viability assessments. The prepared single-cell suspensions were transported to Shanghai Jingzhou Genomics Inc., Ltd, at room temperature, where the single cells were isolated and individually trapped from the suspension using droplet microfluidics technology (BD Rhapsody). The cells were subsequently lysed, and the nucleic acids extracted and amplified. After library construction, high-throughput sequencing was performed using the paired-end mode of the Illumina sequencing platform. 2.7 Bioinformatics analysis NSCs were clustered using t-distributed Stochastic Neighbour Embedding (t-SNE) [ 11 ] . RNA velocity analysis was subsequently performed using scVelo software. The level of statistical significance was set at P < 0.05. 2.8 Statistical analysis Data was analyzed using SPSS 20.0 software (SPSS, Chicago, IL, USA) and expressed as means ± standard deviation. The student’s t-test was employed to compare the two groups; analysis of variance (ANOVA) was used for comparison in more than two groups, followed by Bonferroni or Tukey’s Honestly Significant Difference (HSD) to conduct a post hoc test. The level of significance for all comparisons was set at P < 0.05. 3. Results 3.1 Culture and biological characterization of intact adult mouse sp-NSCs The isolated NSCs from the spinal cords of 8-week-old adult mice appeared as small, round, suspended cells under an inverted microscope during the initial culture. These cells remained in suspension or adhered to the culture substrate 48–72 hours post-isolation, exhibiting typical morphology with prominent refractive halos in medium containing EGF and bFGF. The suspended cells spontaneously aggregated into three-dimensional spherical structures during culture. The adherent cells appeared pike-shaped or ovoid, with protrusions extending into the periphery. Immunofluorescence staining was subsequently performed using Nestin and Sox2 antibodies to identify adherent cultured NSCs. The cultured cells exhibited high expression of NSC-specific markers (92.43 ± 0.58% Nestin-positive and 98.35 ± 0.69% Sox2-positive, Fig. 1 A), confirming their identity as NSCs. Differentiation was induced using specific differentiation medium to assess the differentiation potential of the NSCs, followed by identifying the cell types using TUJ-1, GFAP, and O4 antibodies (Fig. 1 C). Notably, the cultured NSCs could be induced to differentiate into neurons (TUJ-1), astrocytes (GFAP), and oligodendrocytes (O4), demonstrating their multidirectional differentiation capacity. These findings demonstrate that adult sp-NSCs obtained using the modified culture system possess self-renewal and multidirectional differentiation potential. The system provides a stable and abundant source of NSCs, making it a valuable resource for subsequent experiments. 3.2 The heterogeneity of intact adult mouse sp-NSCs ScRNA-Seq was performed on cultured NSCs from the intact adult mouse spinal cord in vitro to investigate the heterogeneity of adult sp-NSCs (Fig. 2 A). t-SNE clustering analysis revealed six subpopulations within the cultured adult sp-NSCs system: dormant qNSCs (dNSCs), early primed qNSCs (pNSCs early), pNSCs late, active NSCs (aNSCs), transit-amplifying progenitors (TAPs), and neuroblasts (NBs). Adult sp-NSCs were predominantly distributed among the dNSCs (50.45%) and pNSCs (22.51%), while a smaller proportion identified as aNSCs (27.04%) (Fig. 2 A). Fetal sp-NSCs were primarily localized within pNSCs (43.62%). However, there were lower proportions of dNSCs (30.77%) and aNSCs (25.60%) (Fig. 2 A). The specific marker Clu was subsequently selected for quiescent state detection to further explore the biological properties of adult sp-NSCs. Notably, the majority of Nestin-positive cells expressed the qNSCs marker Clu (59.79 ± 0.85% Clu-positive, Fig. 1 B), indicating that most adult sp-NSCs existed in a quiescent state. 3.3 Intact adult mouse sp-NSCs are predominantly in a quiescent state Previous experimental results suggest that most adult sp-NSCs are in a quiescent state. Of note, the activation capacity of NSCs decreases significantly with age [ 12 ] . The activation abilities of adult sp-NSCs and fetal sp-NSCs were compared using the Sphere assay and EdU staining to elucidate the age-related decline in the activation capacity of sp-NSCs. Noteworthy, there were no statistically significant differences in neurosphere diameters between the Fet-Spi-NSCs and Adu-Spi-NSCs groups during the early stages of culture ( P > 0.05, Fig. 2 A). However, the neurosphere diameter in the Adu-Spi-NSCs group was significantly smaller than that in the Fet-Spi-NSCs group by day 5 (Adu-Spi-NSCs (Day5) 36.78 ± 3.59 µm vs Fet-Spi-NSCs (Day5) 69.11 ± 5.58 µm, P < 0.05, Fig. 2 A). EdU staining results were consistent with these findings, revealing a significantly lower percentage of EdU-positive cells in adult sp-NSCs compared to fetal sp-NSCs (Adu-Spi-NSCs 8.13 ± 0.79% vs Fet-Spi-NSCs 22.09 ± 1.25%, P < 0.05, Fig. 2 B). ScRNA-Seq and bioinformatics analyses further revealed significantly lower expression levels of Mki67 and Ccna2 genes in adult sp-NSCs compared to fetal sp-NSCs (Adu-Spi-NSCs-Mki67 13.20% vs Fet-Spi-NSCs-Mki67 55.40%, Adu-Spi-NSCs-Ccna2 15.53% vs Fet-Spi-NSCs-Ccna2 58.97%, P < 0.05, Fig. 2 C). Further assays confirmed that most adult sp-NSCs remained quiescent, a cell state carrying important implications for understanding their properties and activation potential. 3.4 Notch signaling is a vital mechanism for activating intact adult mouse sp-NSCs The Notch signaling pathway is a critical mechanism regulating NSC quiescence and activation, contributing to NSC heterogeneity. Nestin was co-stained with Notch1, Notch2, and Notch3 to investigate Notch receptor expression in adult sp-NSCs. Notch1 was highly expressed in adult sp-NSCs relative to Notch2 and Notch3 (79.01 ± 4.83%, P < 0.05, Fig. 3 A). Analysis of the gene expression profiles of Notch receptors in different subpopulations using scRNA-Seq and bioinformatics (Fig. 3 B) revealed that Notch1 was expressed in six subpopulations: dNSCs, pNSCs early, pNSCs late, aNSCs, TAPs, and NBs. Notably, it had relatively high expression levels, suggesting a close association between Notch1 and the regulation of activation and self-renewal in NSCs (Fig. 3 B). Notch2 and Notch3 were highly expressed in the dNSCs (Fig. 3 B). Further assays suggested that differential expressions of Notch receptors potentially play a significant role in regulating NSC activation in the intact adult mouse spinal cord. 3.5 Differential expression of the Notch receptors affects the activation of intact adult mouse sp-NSCs Lentiviral vectors containing Notch1 shRNA, Notch2 shRNA, and Notch1 + Notch2 shRNA were constructed, and their interference efficacy was characterized to investigate the role of Notch receptors in regulating adult sp-NSCs activation. The expression levels of Notch1 and Notch2 were significantly reduced compared to the controls (CTL 1.00 vs Notch1 shRNA (Notch1) 0.29 ± 0.03 vs Notch2 shRNA (Notch2) 0.55 ± 0.01, P < 0.05, Fig. 4 A) 5 days post-transfection of NSCs with Notch1 and Notch2 shRNA viruses. Similarly, the expression levels of both Notch1 and Notch2 were significantly decreased relative to controls (CTL 1.00 vs Notch1 shRNA + Notch2 shRNA (Notch1) 0.51 ± 0.03 vs Notch1 shRNA + Notch2 shRNA (Notch2) 0.56 ± 0.02, P < 0.05, Fig. 4 A) 5 days post co-transfection of NSCs with Notch1 and Notch2 shRNA viruses. These results indicated that Notch1 shRNA and Notch2 shRNA did not affect each other's RNA silencing efficacy. The activation capacity of these cells was evaluated using the Sphere assay and EdU staining to investigate the roles of Notch1 and Notch2 in regulating adult sp-NSCs activation. Sphere assay results revealed a significant reduction in the neurosphere-forming capacity of NSCs following viral interference with Notch1 shRNA alone (CTL 134.02 ± 1.88 µm vs Notch1 shRNA 88.11 ± 3.71 µm, P < 0.05, Fig. 4 B). Conversely, interference with Notch2 shRNA significantly increased the neurosphere-forming capacity (CTL 134.02 ± 1.88 µm vs Notch2 shRNA 152.70 ± 7.95 µm, P < 0.05, Fig. 4 B). These findings suggested that Notch1 can facilitate NSC activation, while Notch2 can sustain their quiescent state in vitro . Noteworthy, the neurosphere-forming capacity was significantly reduced following combined interference with Notch1 and Notch2 shRNAs (CTL 134.02 ± 1.88 µm vs Notch1 shRNA + Notch2 shRNA 106.65 ± 5.99 µm, P 0.05, Fig. 4 B). EdU staining results were consistent with these findings, revealing a significant decrease in the percentage of Nestin + /EdU + cells following Notch1 shRNA interference (CTL 11.42 ± 1.05% vs Notch1 shRNA 5.43 ± 0.30%, P < 0.05, Fig. 4 C). However, interference with Notch2 shRNA significantly increased the percentage of positive cells (CTL 11.42 ± 1.05% vs Notch2 shRNA 12.33 ± 1.70%, P < 0.05, Fig. 4 C). The proportion of EdU-positive cells was significantly decreased following combined interference with Notch1 and Notch2 shRNAs (CTL 11.42 ± 1.05% vs Notch1 shRNA + Notch2 shRNA 8.33 ± 0.67%, P < 0.05, Fig. 4 C). The expression levels of NICD and Rbpj genes downstream of the Notch signaling pathway and their related target genes, Hes1 and Hey1, were further examined. Western blot results revealed a significant decrease in NICD, Rbpj, Hes1, and Hey1 expression levels in adult sp-NSCs following transfection with Notch1 shRNA virus compared to the control group. The expression of Hes1 decreased to 0.38 ± 0.02, Hey1 to 0.16 ± 0.01, Rbpj to 0.19 ± 0.02, and NICD to 0.09 ± 0.01 compared to the control levels set at 1.00 (all P < 0.05, Fig. 4 D), indicating effective inhibition of the Notch signaling pathway in adult sp-NSCs post-transfection. Similarly, the expression levels of Rbpj, Hes1, and Hey1 in adult sp-NSCs transfected with Notch2 shRNA were significantly reduced compared to the controls (CTL 1.00 vs Notch2 shRNA (Rbpj) 0.73 ± 0.02, CTL 1.00 vs Notch2 shRNA (Hes1) 0.51 ± 0.05, CTL 1.00 vs Notch2 shRNA (Hey1) 0.41 ± 0.03, P 0.05, Fig. 4 D). The expression levels of NICD, Rbpj, Hes1, and Hey1 were significantly decreased compared to controls when both Notch1 and Notch2 shRNAs were used ( P < 0.05, Fig. 4 D). However, the expression of Hes1 and Hey1 when both Notch1 and Notch2 shRNAs were used did not differ significantly from those observed with Notch1 shRNA was used alone (Notch1 shRNA (Hes1) 0.38 ± 0.02 vs Notch1 shRNA + Notch2 shRNA (Hes1) 0.33 ± 0.03, Notch1 shRNA (Hey1) 0.16 ± 0.01 vs Notch1 shRNA + Notch2 shRNA (Hey1) 0.17 ± 0.01, P > 0.05, Fig. 4 D). These findings suggested that Notch receptors potentially promote the activation of adult sp-NSCs in vitro via the Notch signaling pathway. 4. Discussion In this study, optimization of the culture system of intact adult mouse sp-NSCs resulted in an abundant supply of NSCs that served as a stable cell source for subsequent experiments. ScRNA-seq revealed unique heterogeneity of sp-NSCs. The majority of adult mouse sp-NSCs remained in a quiescent state, which is significantly less activated compared to fetal mouse sp-NSCs. Moreover, characterization of the expression patterns of Notch receptors in sp-NSCs revealed asymmetric interaction between Notch1 and Notch2 receptors, which induced different activation states in adult mouse sp-NSCs. This study provides a basis for further elucidation of the regulatory mechanisms governing the activation of adult mouse sp-NSCs. NSCs are predominantly found in the CNS of adult mammals. In vitro culture and differentiation characteristics of NSCs derived from the cerebral cortex, hippocampus, and striatum have been extensively studied. However, systematic research on adult sp-NSCs remains limited. The first in vitro isolation and culture of adult mouse sp-NSCs was performed by Weiss et al [ 13 ] . However, the lengthy digestion time potentially impacted cell viability. Notably, the method described in Methods in Molecular Biology for isolating and culturing adult mouse sp-NSCs is cumbersome [ 14 ] . It involves harsh culture conditions, with an experimental duration typically exceeding four hours, which limits its widespread application. An optimized culture system for intact adult mouse sp-NSCs was developed based on the in vitro culture method for fetal mouse brain-derived NSCs detailed in the Gibco Neurobiology Handbook. The optimized system reduced the number of isolation steps, decreased enzyme digestion time, and increased the initial cell seeding concentration. Assessment of the differentiation capacity of adult mouse sp-NSCs confirmed their ability to be induced to differentiate into neurons, astrocytes, and oligodendrocytes. Stable and abundant populations of adult mouse sp-NSCs with multipotent differentiation capacity were obtained following the modification of the culture system. The optimized method strongly demonstrates effectiveness for isolating and culturing adult mouse sp-NSCs. Sp-NSCs exhibit considerable heterogeneity, with marker expression varying among subpopulations. Llorens-Bobadilla et al. reported that NSCs in the subventricular zone (SVZ) of the lateral ventricles in adult mice significantly express GLAST (also known as Slc1a3) and Prom1 (also known as CD133) [ 15 ] . Of note, GLAST is closely associated with protein synthesis, and its expression in qNSC1 is significantly higher than in qNSC2 [ 15 ] . EGFR is not expressed in either quiescent or activated NSCs [ 15 ] . Dulken et al. postulated that Prom1 and GLAST are continually expressed throughout the lifespan of brain-derived NSCs [ 16 ] . As such, they co-expressed EGFR and Prom1 to distinguish different NSC states [ 16 ] . Beckervordersandforth et al. distinguished SVZ-NSCs from astrocytes in adult hGFAP-GFP transgenic mice using Prom1 [ 17 ] . However, they could not differentiate between quiescent and activated NSCs [ 17 ] . Herein, six subpopulations of intact adult mouse sp-NSCs cultured in vitro were identified through scRNA-Seq and bioinformatics analyses. The subpopulations included dNSCs, pNSCs early, pNSCs late, aNSCs, TAPs, and NBs. Analysis of GLAST and Prom1 gene expression profiles revealed that GLAST was highly expressed across all subpopulations of adult mouse sp-NSCs. However, Prom1 exhibited low expression in sp-NSCs, contrary to the findings in brain-derived NSCs [ 15 – 17 ] . Consistent with our findings, Zywitza et al. and Sachewsky et al. also reported that Prom1 expression is limited in the SVZ of the adult mouse brain-derived NSCs [ 18 , 19 ] . The validity of Prom1 as a specific NSC marker remains controversial. Co-expression of Nestin and Sox2 was thus employed for NSC identification in this study. Specific markers, such as GFAP and S100b, have been employed to investigate the activation potential of adult sp-NSCs to determine whether NSCs are quiescent [ 19 – 22 ] . Zhang et al. proposed that GFAP positivity can be a marker for qNSCs because it exhibits radial glial morphology [ 22 ] . Gengatharan et al. postulated that GFAP-positive cells comprise both NSCs and astrocytes [ 20 ] . GFAP + /S100b + cells are identified as astrocytes, while GFAP + /S100b − cells are identified as qNSCs [ 20 ] . Shah et al. reported that S100b is also expressed in qNSCs [ 21 ] . In addition, Zywitza et al. reported that NSCs and most astrocytes are negative, while mature oligodendrocytes (MOLs) and ependymal cells are positive for S100b [ 19 ] . A significance analysis of differential gene expression conducted to characterize qNSCs revealed predominant expression of GFAP and S100b in the dNSCs but low detection in the pNSCs. Using S100b and GFAP as sole markers for qNSCs may thus result in the omission of certain “shallow dormant” NSCs, indicating that these markers alone are insufficient for the specific identification of qNSCs. Herein, Clu was regarded as a reliable marker. Co-staining with Nestin revealed that most adult sp-NSCs were quiescent. ScRNA-Seq analysis revealed that approximately 72.96% of the NSCs in the adult mouse spinal cord belong to the qNSCs. Among them, dNSCs comprised 69.15% of the qNSCs, suggesting limited activation capacity of adult sp-NSCs. The “deep dormancy” exhibited by dNSCs potentially impeded their transition to aNSCs. Sp-NSCs can be derived from specific subtypes of astrocytes and ependymal cells [ 11 , 23 , 24 ] . Jonas et al. reported that a subpopulation of Troy-positive ependymal cells within the central canal region of the spinal cord exhibits NSCs properties [ 24 ] . Frederico et al. reported that a subpopulation of fetal DNGR-1-positive ependymal cells retains NSC characteristics into adulthood [ 23 ] . Analysis of the Foxj1 gene revealed uniform expression of Foxj1 in the cultured intact adult mouse sp-NSCs system, suggesting that ependymal cells constitute a significant component of NSCs. Noteworthy, a subset of ependymal cells and astrocytes can be converted into NSCs and exhibit various activation states 3 days post-SCI [ 11 ] . The activation capacity of NSCs is highly dependent on age-related changes [ 12 ] . Thymidine labeling assays demonstrate that SVZ-NSCs of aged mice exhibit a lower division rate than those of young mice [ 25 ] . Similarly, Kalamakis et al. observed a significant decline in brain-derived NSCs in aged mice [ 26 ] . Notably, NSCs from young and aged mice demonstrate similar activation and differentiation capacities upon activation [ 26 ] . ScRNA-Seq analysis conducted by Shi et al. revealed a significant reduction in the proliferative capacity across all NSCs and neural progenitor cells (NPCs) in aged SVZ [ 27 ] . This phenomenon was attributed to a highly inflammatory microenvironment in the aged SVZ [ 27 ] . Sphere assay and EdU staining conducted to investigate whether the activation capacity of sp-NSCs decreases with age revealed reduced neurosphere-forming ability of adult sp-NSCs. Moreover, the number of EdU-positive cells was significantly lower than that of fetal sp-NSCs. These findings underscored the diminishing activation potential of adult sp-NSCs. Gene expression analysis of proliferation markers, Mki67 and Ccna2, via scRNA-Seq and bioinformatics revealed significantly lower expression levels in adult sp-NSCs compared to fetal sp-NSCs, indicating reduced activation capacity in adult sp-NSCs. Of note, adult sp-dNSCs constituted 50.45%, while fetal sp-dNSCs constituted 30.77% of NSCs. This finding indicates that the quantity of sp-NSCs in the “deep dormant” state increases considerably with age, and their conversion into aNSCs remains relatively challenging. Adult sp-pNSCs comprised 30.85% of qNSCs, consistent with findings from studies on brain-derived NSCs. In contrast, fetal sp-pNSCs comprised 58.63% of qNSCs, suggesting that adult sp-NSCs possess fewer reserves for converting qNSCs to aNSCs, and exhibit diminished activation potential compared to fetal sp-NSCs. Notably, adult sp-aNSCs accounted for 27.04%, while fetal sp-aNSCs accounted for 25.60% of NSCs upon activation of qNSCs. However, this difference was not statistically significant. Herein, sp-NSCs across different ages demonstrated similar activated capacities consistent with the findings of Kalamakis et al [ 26 ] . Previous studies postulate that dNSCs transiently proliferate after activation and primarily differentiate into astrocytes following SCI [ 24 ] . Xu et al. reported that, unlike dNSCs, certain Oct4 + pNSCs exhibit multidirectional differentiation potential following activation and can differentiate into neurons, oligodendrocytes, and astrocytes [ 28 ] . Herein, the activation potential of sp-NSCs varied across different age groups. However, future studies should investigate the differentiation capacity of qNSCs following activation. The Notch signaling pathway is a critical mechanism for regulating the quiescence and activation of NSCs and is a crucial factor contributing to NSC heterogeneity [ 6 ] . The pathway primarily comprises Notch receptors, ligands, CSL proteins, and downstream target genes [ 7 ] . Notch receptors play different roles in regulating NSC activation in the adult mammalian CNS [ 29 – 31 ] . Basak et al. investigated the role of Notch1 in brain-derived NSCs using a conditional Notch1 knockout mouse model and concluded that Notch1 primarily functions in aNSCs by maintaining the activation state and promoting differentiation into neurons [ 29 ] . Moreover, the study concluded that Notch1 exhibits distinct functions in NSCs depending on their proliferative state [ 29 ] . Engler et al. reported that Notch2 preserves the quiescent state of NSCs by repressing cell cycle-related genes [ 30 ] . In contrast, Notch1 primarily sustains the activated state [ 30 ] . Kawai et al. reported that Notch3 is highly expressed in SEZ-qNSCs of the adult mouse selectively, while Notch1 is predominantly expressed in aNSCs and TAPs [ 31 ] . The study further concluded that Notch3 maintains NSC quiescence via knockdown experiments [ 31 ] . In this study, co-expression staining of Nestin and Notch receptors revealed heterogeneous expression of Notch1, Notch2, and Notch3 in adult sp-NSCs. Notably, Notch1 demonstrated significantly higher co-expression with Nestin than Notch2 and Notch3. However, Notch1 did not co-stain with Nestin in some cases. Previous studies postulate that Notch 1 is potentially expressed in radial glial cells, which account for the absence of positive staining. ScRNA-seq and bioinformatics analyses performed to characterize the gene expression profiles of Notch receptors in adult sp-NSCs revealed that Notch1 is expressed across multiple subpopulations, including dNSCs, pNSCs, and aNSCs, with notably high expression in the aNSCs. Moreover, Notch2 and Notch3 were highly expressed in the dNSCs. These findings suggest that Notch1 is potentially involved in regulating NSC activation and self-renewal, while Notch2 and Notch3 potentially contribute to maintaining NSC quiescence. Notably, Notch1 was highly expressed in both qNSCs and aNSCs. However, the mechanisms underlying its functional heterogeneity warrant further investigation [ 29 ] . Previous studies have extensively explored the role of Notch receptors in regulating NSC activation using lineage tracing experiments and conditional knockout models [ 22 , 29 , 30 ] . For instance, Basak et al. reported that conditional knockout of Notch1 (Notch1 cKO) causes a selective loss of aNSCs without affecting the activation of qNSCs [ 29 ] . Engler et al. reported that GFP + /GFAP + qNSCs are activated and enter the cell cycle in Notch2 cKO, Notch1/Notch2 double cKO, and Rbpj cKO mice [ 30 ] . Zhang et al. observed that conditional knockout of Notch2 increases proliferation of DG-NSCs and enhances the generation of newborn neurons [ 22 ] . Herein, lentiviral vectors carrying Notch1 shRNA and Notch2 shRNA were constructed and used to assess the activation capacity of Notch1 and Notch2 in adult sp-NSC using Sphere assays and EdU staining. Of note, the neurosphere-forming capacity was reduced, and the number of EdU-positive cells was significantly lower following transfection with Notch1 shRNA viruses compared to controls. Conversely, transfection with Notch2 shRNA viruses yielded opposite effects. These findings suggested that interference with Notch1 impairs NSC activation, whereas disruption of Notch2 may promote their transition into an activated state. Notably, co-transfection with Notch1 and Notch2 shRNA viruses reduced the neurosphere-forming capacity and EdU-positive cell counts compared to the controls. However, the co-transfection effect was not significantly different to Notch1 shRNA transfection alone. These findings imply that the dominant role of Notch1 in NSC activation is attenuated when both receptors are disrupted simultaneously because of the higher expression level of Notch1 compared to Notch2 in adult sp-NSCs. The signaling pathways involved in NSC activation by Notch3 were not explored because of its overall low expression. However, future studies should aim to elucidate its effects. Western blotting was employed to validate the expression levels of downstream components of the Notch signaling pathway, including NICD, Rbpj, and the target genes, Hes1 and Hey1. Notably, Hes1 and Hey1 proteins were significantly decreased in adult sp-NSCs transfected with Notch1 shRNA virus compared to the control group, indicating effective inhibition of the Notch signaling pathway in these cells. The expression levels of Hes1, Hey1, Rbpj, and NICD in adult sp-NSCs transfected with Notch2 shRNA virus were also downregulated relative to the control group. However, these expression levels remained significantly higher than those in NSCs transfected with Notch1 shRNA virus. These findings were consistent with those of Sahoo et al., who reported that specific inhibition of the Notch2 gene resulted in the activation of the transcription of Notch1 and typical gene targets, Hes1, Hey1, and Hey2 [ 32 ] . These reports suggest that the enhancement of Notch1 signaling potentially facilitates the activation of adult sp-NSCs following Notch2 interference. Nevertheless, the precise mechanisms underlying the synergistic regulation of adult neurogenesis by Notch1 and Notch2 require further investigation. 5. Conclusion This investigation demonstrated the majority of adult mouse sp-NSCs remained in a quiescent state, which is significantly less activated compared to fetal mouse sp-NSCs. Moreover, this study further reveals the characterization of the expression patterns of Notch receptors in adult mouse sp-NSCs. Our results also indicate that both Notch1 and Notch2 signals are involved in different regulatory roles that facilitate the activation and fate determination of sp-NSCs via NICD-Rbpj. Abbreviations Adu-Spi-NSCs, adult spinal cord-derived NSCs; aNSCs, active neural stem cells; ANOVA, analysis of variance; CNS, central nervous system; dNSCs, dormant NSCs; Fet-Spi-NSCs, fetal spinal cord-derived NSCs; MOLs, mature oligodendrocytes; NBs, neuroblasts; Notch1 cKO, conditional knockout of Notch1; NPCs, neural progenitor cells; NSCs, neural stem cells; PBS, phosphate-buffered saline; PFA, paraformaldehyde; pNSCs, primed qNSCs; PVDE, polyvinylidene fluoride; qNSCs, quiescent NSCs; SCI, spinal cord injury; scRNA-Seq, single-cell RNA sequencing; Sp-NSCs, Spinal cord-derived NSCs; SVZ, subventricular zone; TAPs, transit amplifying progenitors; t-SNE, t-distributed Stochastic Neighbor Embedding; Tukey’s HSD, Tukey’s Honestly Significant Difference. Declarations Funding This work was supported by Shaanxi Science and Technology Foundation (grant nos. 2024JC-YBMS-620). Authors contributions Juan Li: conceptualization, validation, methodology, investigation, formal analysis, data curation, writing – original draft, writing – review and editing. Fen Ju: conceptualization, validation, methodology, investigation, formal analysis, data curation, writing – original draft, writing – review and editing. Zhao-Yang Huang: software, investigation, formal analysis, data curation, writing – review and editing. Liang Yu: validation, supervision, software, resources, methodology, formal analysis, data curation, conceptualization, writing – review and editing. Jie Qin: validation, supervision, resources, methodology, conceptualization, writing – review and editing. Yu-Bing Yang: resources, investigation, writing – review and editing. Wei Sun: writing – review and editing, visualization, validation, investigation. Yu-Qiang Ji: visualization, resources, project administration, conceptualization, writing – review and editing. Chen-Guang Zhao: validation, supervision, project administration, methodology, funding acquisition, formal analysis, conceptualization, writing – review and editing, writing – original draft. Hua Yuan: supervision, project administration, methodology, funding acquisition, conceptualization, writing – review and editing. Acknowledgements We also thank Fei Fang for technical support. This work was supported by Shaanxi Science and Technology Foundation (grant nos. 2024JC-YBMS-620). Conflicts of Interest The authors declare no conflicts of interest. Data Availability Statement The data that support the findings of this study are available from the corresponding author upon reasonable request. References Gilbert Ea, B, Lakshman N, Lau KSK, Morshead CM (2022) Regulating Endogenous Neural Stem Cell Activation to Promote Spinal Cord Injury Repair [J]. Cells, 11(5) Barnabé-Heider F, Frisén J (2008) Stem cells for spinal cord repair [J]. 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Am J Physiol Heart Circ Physiol 321(3):H542–h557 Additional Declarations No competing interests reported. Supplementary Files highlights.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7540357","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":512908843,"identity":"07fa4045-2be5-4f2e-b867-20f73300daea","order_by":0,"name":"Juan Li","email":"","orcid":"","institution":"Northwest University","correspondingAuthor":false,"prefix":"","firstName":"Juan","middleName":"","lastName":"Li","suffix":""},{"id":512908844,"identity":"146e9703-7936-418a-973b-e0c27e92b3e2","order_by":1,"name":"Fen Ju","email":"","orcid":"","institution":"Xijing Hospital, Air Force Military Medical University","correspondingAuthor":false,"prefix":"","firstName":"Fen","middleName":"","lastName":"Ju","suffix":""},{"id":512908845,"identity":"b4bf6348-5ec6-49e6-858a-a21645ee5c42","order_by":2,"name":"Zhao-Yang Huang","email":"","orcid":"","institution":"Xidian University","correspondingAuthor":false,"prefix":"","firstName":"Zhao-Yang","middleName":"","lastName":"Huang","suffix":""},{"id":512908846,"identity":"9e0e3ef7-10ee-4dc0-aa8e-0c699ff18f13","order_by":3,"name":"Liang Yu","email":"","orcid":"","institution":"Xidian University","correspondingAuthor":false,"prefix":"","firstName":"Liang","middleName":"","lastName":"Yu","suffix":""},{"id":512908847,"identity":"bd95c483-b4c1-4bd6-820b-94bbc2d725f1","order_by":4,"name":"Jie Qin","email":"","orcid":"","institution":"The Second Affiliated Hospital of Xi’an Jiaotong University","correspondingAuthor":false,"prefix":"","firstName":"Jie","middleName":"","lastName":"Qin","suffix":""},{"id":512908848,"identity":"7ce47231-4049-41af-bd6a-887b2700cf11","order_by":5,"name":"Yu-Bing Yang","email":"","orcid":"","institution":"The Second Affiliated Hospital of Xi’an Jiaotong University","correspondingAuthor":false,"prefix":"","firstName":"Yu-Bing","middleName":"","lastName":"Yang","suffix":""},{"id":512908849,"identity":"46d18e09-a583-4b22-b491-5743cbaec451","order_by":6,"name":"Wei Sun","email":"","orcid":"","institution":"Xijing Hospital, Air Force Military Medical University","correspondingAuthor":false,"prefix":"","firstName":"Wei","middleName":"","lastName":"Sun","suffix":""},{"id":512908850,"identity":"62a054bd-f267-4019-891e-fb9b7bd3fe0e","order_by":7,"name":"Yu-Qiang Ji","email":"","orcid":"","institution":"The First Hospital of Xi’an","correspondingAuthor":false,"prefix":"","firstName":"Yu-Qiang","middleName":"","lastName":"Ji","suffix":""},{"id":512908851,"identity":"6d92c595-da23-459f-97a8-5b859ce43bfc","order_by":8,"name":"Chen-Guang Zhao","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABC0lEQVRIiWNgGAWjYDACCTB5AMa1AWLmBiQBwlrSGBjYGEnTcpiwFvnZzQ8fMNTckTPnX2P4ueDXeTmD+42NDz6cYZDnF8Ouj3HOMWMDhmPPjC1nvDGWntl329jgGGOz4YwbDIYzZydg1cIskWAmwdhwOHHDjTMG0rw9txM3HGNsk+b5wJBgcBu7FjaJ9G8gLfVALca/eXvOEdbCI5EDtiXB4HyPmTTPjwNQLTdwa5GQyCkG+uWw4YYbbGXWvA3JxpLHEoF+OSOB0y/yM9I3AkPssLzB+cObb/P8sZPjO3z44IMPx2zk+aWxawEHwR+wfUAFjG0I63EqRwD+A0DiDxEKR8EoGAWjYMQBAN/UZaIdf2/9AAAAAElFTkSuQmCC","orcid":"","institution":"Xijing Hospital, Air Force Military Medical University","correspondingAuthor":true,"prefix":"","firstName":"Chen-Guang","middleName":"","lastName":"Zhao","suffix":""},{"id":512908853,"identity":"36878964-126d-44a9-aa29-89b7bbc9006f","order_by":9,"name":"Hua Yuan","email":"","orcid":"","institution":"Xijing Hospital, Air Force Military Medical University","correspondingAuthor":false,"prefix":"","firstName":"Hua","middleName":"","lastName":"Yuan","suffix":""}],"badges":[],"createdAt":"2025-09-05 03:38:04","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7540357/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7540357/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":91111852,"identity":"c0fa4e98-5e46-4e64-bb0f-a766f5e447df","added_by":"auto","created_at":"2025-09-11 16:35:30","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":742518,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCulture and biological characterization of intact adult mouse sp-NSCs. \u003c/strong\u003e(A): In vitro cultured sp-NSCs expressing NSC-specific markers, including Nestin (green) and Sox2 (red). (B): Clu immunofluorescence staining (red) showing co-localization of the NSC marker Nestin (green). The arrows indicate Clu-positive and Nestin-positive cells. (C): NSCs express markers for neurons (TUJ-1, red), astrocytes (GFAP, red), and oligodendrocytes (O4, red) following induced differentiation. (D): Clustering of intact adult mouse sp-NSCs performed using t-SNE. Distinct colors represent different cell populations. The gene expression profiles of selected marker genes in intact adult mouse sp-NSCs are depicted. (Bar=100 μm). All of the analysis was calculated based on three biological replicates.\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7540357/v1/7b7b719b691ff0f56b86a241.jpg"},{"id":91112982,"identity":"d15f0f97-679c-4484-9ce5-7363e6f66d9a","added_by":"auto","created_at":"2025-09-11 16:51:30","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1932108,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe heterogeneity of intact adult mouse sp-NSCs. \u003c/strong\u003e(A): Clustering of intact adult mouse sp-NSCs performed using t-SNE. Distinct colors represent different cell populations. The gene expression profiles of selected marker genes in intact adult mouse sp-NSCs are depicted. (B): A heatmap of genes up-regulated or enriched in each cluster of intact adult mouse sp-NSCs was shown via scRNA-Seq and bioinformatics. All of the analysis was calculated based on three biological replicates.\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7540357/v1/56225f32d3cc27eb80d15fb1.jpg"},{"id":91111862,"identity":"5d0ef61b-99a2-4756-b4d5-9b02eb20c223","added_by":"auto","created_at":"2025-09-11 16:35:30","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1387718,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIntact adult mouse sp-NSCs are predominantly in a quiescent state. \u003c/strong\u003e(A): The in vitro culture status of sp-NSCs over a continuous period of 10 days. (B): EdU-positive NSCs in the Fet-Spi-NSCs and Adu-Spi-NSCs groups. (C): Expression levels of cell proliferation-related genes Mki67 and Ccna2 in NSCs derived from different sources. (Bar=100 μm). n = 3 animals per group, repeated measures ANOVA with Bonferroni post hoc correction for neurosphere-forming capacity, unpaired two-tailed Student’s t-test for EdU staining and gene expression profiles. *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05. Results are expressed as the mean ± SD.\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7540357/v1/c679eb6f1ae5a4db0184c0a3.jpg"},{"id":91113919,"identity":"e2f7a4ba-3e92-41ff-bce6-3f322f9953f6","added_by":"auto","created_at":"2025-09-11 16:59:30","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1631934,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNotch signaling is a key mechanism for activating intact adult mouse sp-NSCs. \u003c/strong\u003e(A): Immunofluorescence staining of Notch1, Notch2, and Notch3 (red), demonstrating co-expression with the NSC marker Nestin (green). (B): Analysis of the expression levels of Notch receptors across different cell populations. (Bar=100 μm). *\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05. Results are expressed as the mean ± SD. All of the analysis was calculated based on three biological replicates.\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7540357/v1/7b562eb8f9c94e187982f001.jpg"},{"id":91111858,"identity":"c7e49e83-3f8c-45b2-896a-473d813440a8","added_by":"auto","created_at":"2025-09-11 16:35:30","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1715777,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDifferential expression of the Notch receptors affects the activation of intact adult mouse sp-NSCs.\u003c/strong\u003e (A): Notch1 shRNA significantly reduces the expression level of Notch1 in NSCs without affecting the expression of Notch2. (B): Sphere assay results demonstrating a significant reduction in neurosphere-forming capacity following Notch1 interference. (C): EdU staining results showing a significant downregulation in the proportion of EdU-positive cells upon Notch1 interference. (D): Downstream effects of the Notch receptors in adult sp-NSCs: The expression levels of the Notch signaling pathway downstream genes NICD and Rbpj, as well as those of related target genes, Hes1 and Hey1, were detected using Western blotting after transfection with Notch1 shRNA and Notch2 shRNA. (Bar=100 μm). n = 3 animals per group, ANOVA with Tukey’s HSD post hoc correction for neurosphere-forming capacity, EdU staining, and Western blotting. *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05. Results are expressed as the mean ± SD.\u003c/p\u003e","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7540357/v1/5d9cae75a5d59f71f20c3afe.jpg"},{"id":92416584,"identity":"4a607378-1d00-402e-9132-9daf76bfc6f8","added_by":"auto","created_at":"2025-09-29 13:32:11","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":8360200,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7540357/v1/9dc74fbc-8952-443f-873b-f751847a3eca.pdf"},{"id":91111856,"identity":"fa34b39e-4c9b-4338-9ab4-41df9864fecf","added_by":"auto","created_at":"2025-09-11 16:35:30","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":19204,"visible":true,"origin":"","legend":"","description":"","filename":"highlights.docx","url":"https://assets-eu.researchsquare.com/files/rs-7540357/v1/3d2b18cc471c7b4233770996.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Differential Roles of Notch Receptors in Regulating Activation of Intact Adult Mouse Spinal Cord-derived NSCs","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eSpinal cord injury (SCI) is a severe central nervous system (CNS) disorder characterized by disruption of spinal cord continuity, neuronal death, and myelin disintegration\u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]\u003c/sup\u003e. Despite its severity, SCI lacks effective clinical treatment. Neural stem cells (NSCs) possess self-renewal and multipotent differentiation capabilities\u003csup\u003e[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e. They play a crucial role in nerve regeneration following SCI, but their application in SCI repair is hindered by the limited quantity, restricted activation, and differentiation capacity of spinal cord-derived NSCs (sp-NSCs) in adult mammals\u003csup\u003e[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e. Elucidating the biological properties and activation of regulatory mechanisms of sp-NSCs is thus a critical step that should be addressed before their application in the treatment of various neurological diseases.\u003c/p\u003e\u003cp\u003eSp-NSCs are governed by complex regulatory mechanisms during development and maturation\u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/sup\u003e. These NSCs display significant heterogeneity, with quiescent and activated states coexisting under various physiological conditions\u003csup\u003e[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/sup\u003e. Nonetheless, the interactions between these diverse states and the regulatory factors governing their transformation are not fully understood, limiting their activation potential and impairing neural repair. Studies postulate that NSC activation post-SCI is regulated by multiple signaling pathways\u003csup\u003e[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/sup\u003e. Of note, the Notch signaling pathway plays a crucial role in early activation and differentiation. It primarily involves Notch receptors, ligands, CSL proteins, and downstream target genes\u003csup\u003e[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]\u003c/sup\u003e. Studies postulate that Notch receptors have distinct roles in activating and differentiating NSCs within the adult mammalian nervous system. Notch1 sustains the activated state of NSCs and promotes their differentiation into neurons\u003csup\u003e[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/sup\u003e. In contrast, Notch2 and Notch3 preserve the quiescent state of NSCs. The specific expression patterns of Notch receptors can potentially influence the activation states of NSCs, especially within the CNS, exerting asymmetric effects on NSC activation. This study aimed to characterize the biological properties of sp-NSCs in intact adult mice and examine their activation by Notch receptors to provide new insights for enhancing neurorestorative strategies following SCI.\u003c/p\u003e"},{"header":"2. Method and Materials","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1 Isolation, culture, and differentiation of intact adult mouse sp-NSCs\u003c/h2\u003e\u003cp\u003eThis study was approved by the Medical Ethics Committee of Xijing Hospital, Air Force Medical University (No. XJYYLL-20240320), and all efforts were made to minimize animal suffering and reduce animals used. It was conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals, revised in 1996 (NIH Publications No. 80\u0026thinsp;\u0026minus;\u0026thinsp;23). The NSC culture system was modified, building upon the existing \u003cem\u003ein vitro\u003c/em\u003e culture methods to achieve a stable and abundant population of intact adult mouse sp-NSCs\u003csup\u003e[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]\u003c/sup\u003e. Briefly, 8-week-old and 16-day-pregnant C57/BL6 mice were anaesthetized using 5% isoflurane under aseptic conditions. Subsequently, the mice were euthanized by cervical dislocation. Under aseptic conditions, E16.5 embryos were immediately dissected, and spinal cords were exposed via ventral laminectomy using fine spring scissors. The T1\u0026ndash;T12 spinal cord segments were excised, transferred to a culture dish containing pre-cooled phosphate-buffered saline (PBS), and cut into approximately 1 mm\u0026sup3; pieces. Accutase was subsequently added, and the tissues incubated for 10 minutes in a water bath set at 37\u0026deg;C. NSCs growth medium was then added to terminate enzymatic activity after digestion. The mixture was centrifuged to obtain the cells, which were then resuspended in NSCs medium and dissociated into single cells by gentle pipetting approximately 30 times with a Pasteur glass pipette. The cells were then filtered through a 70 \u0026micro;m cell strainer and their viability assessed through Trypan blue staining. Briefly, the cells were seeded at a density of 1 \u0026times; 10⁶ cells/mL in NSCs growth medium and cultured in an incubator set at 37\u0026deg;C and 5% CO₂. NSCs growth medium comprised DMEM/F12, 2 mM GlutaMAX-I, 100 IU/mL Penicillin-Streptomycin, 20 ng/mL EGF, 20 ng/mL bFGF, B27, and N2. Growth factors were added every 3 days, and the medium was refreshed every 6 days. Cell passaging was performed mechanically using a pipette on day 12. All experiments were conducted between the third and seventh cell passage.\u003c/p\u003e\u003cp\u003eThe cells were then maintained for at least a day in a medium containing 10 \u0026micro;g/ml laminin and 0.01% poly-L-lysine. Specific differentiation media were used in place of the NSCs growth medium to induce differentiation. The differentiation media included the neuronal, astrocyte, and oligodendrocyte differentiation media. The neuronal differentiation medium contained Neurobasal media (21103049, Gibco), GlutaMAX-I Supplement (2 mM), and B-27 Supplement (17504044, Gibco); the astrocyte differentiation medium contained DMEM (10569010, Gibco), N-2 Supplement, GlutaMAX-I Supplement (2 mM), and FBS (10091148, 1%, Gibco); while the oligodendrocyte differentiation medium contained Neurobasal Medium, GlutaMAX-I Supplement (2 mM), B-27 Supplement, T3 (30 ng/ml, T2877, Sigma), Ascorbic Acid (200 \u0026micro;M, PHR1008, Sigma), and NT-3 (5 ng/ml, 45003, PeproTech). The cells were incubated in the differentiation media for seven days and then fixed with 4% paraformaldehyde (PFA) for immunofluorescence labeling.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2 Immunofluorescence Staining\u003c/h2\u003e\u003cp\u003eThe fixed cells were blocked for 1 hour at room temperature using PBS supplemented with 10% normal goat serum and 0.3% Triton X-100. The cells were then incubated with primary antibodies at 4\u0026deg;C overnight, washed with PBS, and incubated with a secondary antibody (1:5000, Abcam) for 2 hours at room temperature in the dark. The cells were then stained with Hoechst for 5 minutes at room temperature in the dark, washed with PBS, mounted onto microscope slides and examined using fluorescence microscopy. The following primary antibodies were used: anti-Nestin (NSC marker, 1:200, Chemicon), anti-Sox2 (NSC marker, 1:200, Abcam), anti-Tuj1 (neuronal marker, 1:200, Millipore), anti-GFAP (astrocyte marker, 1:400, Dako), anti-O4 (oligodendrocyte marker, 1:50, Chemicon), anti-Notch1 (1:200, Abcam), anti-Notch2 (1:200, Abcam), and anti-Notch3 (1:200, Proteintech). The percentage of positive cells was calculated based on three biological replicates.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3 Sphere assay\u003csup\u003e[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/sup\u003e\u003c/h2\u003e\u003cp\u003eSuspension-cultured NSCs were seeded in 6-cm Petri dishes, and micrographs were randomly captured at 10\u0026times; magnification following previously published protocols. Three biological replicates were prepared for each treatment group. The diameters of individual neurospheres in each biological replicate were randomly selected and measured using ImageJ software.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4 Western blotting\u003c/h2\u003e\u003cp\u003eNSCs were collected, washed with pre-cooled PBS, and lysed using RIPA buffer supplemented with protease inhibitors. RIPA buffer contained 150 mM NaCl, 50 mM Tris-HCl, 1% Triton X-100, 2% SDS, and 1% sodium deoxycholate. The cell homogenates were centrifuged at 12,000 rpm for 5 minutes at 4\u0026deg;C to collect the supernatant for protein concentration determination using a NanoDrop spectrophotometer (ND-1000, Thermo Fisher). The protein samples (20 \u0026micro;g) were separated on a 10% SDS-PAGE, and the resultant bands transferred onto polyvinylidene fluoride (PVDF) membranes. The membranes were then blocked with 5% skimmed milk at room temperature and incubated with primary antibodies at 4\u0026deg;C overnight. The primary antibodies included rabbit anti-Notch1 (1:1000, Abcam), rabbit anti-Notch2 (1:1000, Proteintech), rabbit anti-NICD (1:1000, CST), rabbit anti-Rbpj (1:1000, Cell Signaling Technology), rabbit anti-Hes1 (1:500, Abcam), rabbit anti-Hey1 (1:500, Abcam), and anti-β-actin (1:2000, Cell Signaling Technology). The membranes were then washed with TBST to remove the excess primary antibodies and subsequently incubated with horseradish peroxidase-conjugated secondary antibody (1:2000, ab205718, Abcam, Cambridge, UK) for 2 hours at room temperature. The chemiluminescent signals were subsequently scanned and analyzed quantitatively using ImageJ software (version 1.45, NIH, USA).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.5 EdU Staining\u003c/h2\u003e\u003cp\u003eNSCs were seeded into 96-well cell culture plates. Pre-warmed 10 \u0026micro;M EdU (at 37\u0026deg;C) was then added to the plates and incubated in a cell culture incubator for 2 hours to label the S-phase cells. EdU was pipetted from the plates, and the cells were fixed for 15 minutes at room temperature by adding 100 \u0026micro;L of 4% paraformaldehyde to each well. The cells were washed with 3% BSA and then incubated with 0.3% Triton X-100 permeabilization solution for 15 minutes at room temperature. The permeabilization solution was pipetted and the cells washed with 3% BSA. Click reaction solution (100 \u0026micro;L) containing Click Reaction Buffer, Click Additive Solution, CuSO₄, and Azide 555 in a ratio of 430:50:20:1 was then added to each well, and the cells incubated for 30 minutes at room temperature in the dark. The reaction solution was pipetted out, and the cells were washed thrice with 3% BSA. Hoechst 33342 diluted 1,000-fold with 1\u0026times; PBS was added, and the cells incubated for 10 minutes at room temperature in the dark. The Hoechst 33342 solution was pipetted out, and the cells were washed thrice with 3% BSA. The bottom of the 96-well plates was then gently wiped using a microscope paper to prepare for fluorescence imaging and analysis.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e2.6 Single-cell RNA sequencing (scRNA-seq)\u003c/h2\u003e\u003cp\u003eNeurospheres were enzymatically digested to dissociate into a single-cell suspension. The single-cell suspension preparations were observed under an electron microscope, followed by cell counting and viability assessments. The prepared single-cell suspensions were transported to Shanghai Jingzhou Genomics Inc., Ltd, at room temperature, where the single cells were isolated and individually trapped from the suspension using droplet microfluidics technology (BD Rhapsody). The cells were subsequently lysed, and the nucleic acids extracted and amplified. After library construction, high-throughput sequencing was performed using the paired-end mode of the Illumina sequencing platform.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e2.7 Bioinformatics analysis\u003c/h2\u003e\u003cp\u003eNSCs were clustered using t-distributed Stochastic Neighbour Embedding (t-SNE)\u003csup\u003e[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/sup\u003e. RNA velocity analysis was subsequently performed using scVelo software. The level of statistical significance was set at \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e2.8 Statistical analysis\u003c/h2\u003e\u003cp\u003eData was analyzed using SPSS 20.0 software (SPSS, Chicago, IL, USA) and expressed as means\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation. The student\u0026rsquo;s t-test was employed to compare the two groups; analysis of variance (ANOVA) was used for comparison in more than two groups, followed by Bonferroni or Tukey\u0026rsquo;s Honestly Significant Difference (HSD) to conduct a post hoc test. The level of significance for all comparisons was set at \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e3.1 Culture and biological characterization of intact adult mouse sp-NSCs\u003c/h2\u003e\u003cp\u003eThe isolated NSCs from the spinal cords of 8-week-old adult mice appeared as small, round, suspended cells under an inverted microscope during the initial culture. These cells remained in suspension or adhered to the culture substrate 48\u0026ndash;72 hours post-isolation, exhibiting typical morphology with prominent refractive halos in medium containing EGF and bFGF. The suspended cells spontaneously aggregated into three-dimensional spherical structures during culture. The adherent cells appeared pike-shaped or ovoid, with protrusions extending into the periphery.\u003c/p\u003e\u003cp\u003eImmunofluorescence staining was subsequently performed using Nestin and Sox2 antibodies to identify adherent cultured NSCs. The cultured cells exhibited high expression of NSC-specific markers (92.43\u0026thinsp;\u0026plusmn;\u0026thinsp;0.58% Nestin-positive and 98.35\u0026thinsp;\u0026plusmn;\u0026thinsp;0.69% Sox2-positive, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA), confirming their identity as NSCs. Differentiation was induced using specific differentiation medium to assess the differentiation potential of the NSCs, followed by identifying the cell types using TUJ-1, GFAP, and O4 antibodies (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). Notably, the cultured NSCs could be induced to differentiate into neurons (TUJ-1), astrocytes (GFAP), and oligodendrocytes (O4), demonstrating their multidirectional differentiation capacity. These findings demonstrate that adult sp-NSCs obtained using the modified culture system possess self-renewal and multidirectional differentiation potential. The system provides a stable and abundant source of NSCs, making it a valuable resource for subsequent experiments.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003e3.2 The heterogeneity of intact adult mouse sp-NSCs\u003c/h2\u003e\u003cp\u003eScRNA-Seq was performed on cultured NSCs from the intact adult mouse spinal cord \u003cem\u003ein vitro\u003c/em\u003e to investigate the heterogeneity of adult sp-NSCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). t-SNE clustering analysis revealed six subpopulations within the cultured adult sp-NSCs system: dormant qNSCs (dNSCs), early primed qNSCs (pNSCs early), pNSCs late, active NSCs (aNSCs), transit-amplifying progenitors (TAPs), and neuroblasts (NBs). Adult sp-NSCs were predominantly distributed among the dNSCs (50.45%) and pNSCs (22.51%), while a smaller proportion identified as aNSCs (27.04%) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Fetal sp-NSCs were primarily localized within pNSCs (43.62%). However, there were lower proportions of dNSCs (30.77%) and aNSCs (25.60%) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). The specific marker Clu was subsequently selected for quiescent state detection to further explore the biological properties of adult sp-NSCs. Notably, the majority of Nestin-positive cells expressed the qNSCs marker Clu (59.79\u0026thinsp;\u0026plusmn;\u0026thinsp;0.85% Clu-positive, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB), indicating that most adult sp-NSCs existed in a quiescent state.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003e3.3 Intact adult mouse sp-NSCs are predominantly in a quiescent state\u003c/h2\u003e\u003cp\u003ePrevious experimental results suggest that most adult sp-NSCs are in a quiescent state. Of note, the activation capacity of NSCs decreases significantly with age\u003csup\u003e[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]\u003c/sup\u003e. The activation abilities of adult sp-NSCs and fetal sp-NSCs were compared using the Sphere assay and EdU staining to elucidate the age-related decline in the activation capacity of sp-NSCs. Noteworthy, there were no statistically significant differences in neurosphere diameters between the Fet-Spi-NSCs and Adu-Spi-NSCs groups during the early stages of culture (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). However, the neurosphere diameter in the Adu-Spi-NSCs group was significantly smaller than that in the Fet-Spi-NSCs group by day 5 (Adu-Spi-NSCs (Day5) 36.78\u0026thinsp;\u0026plusmn;\u0026thinsp;3.59 \u0026micro;m vs Fet-Spi-NSCs (Day5) 69.11\u0026thinsp;\u0026plusmn;\u0026thinsp;5.58 \u0026micro;m, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). EdU staining results were consistent with these findings, revealing a significantly lower percentage of EdU-positive cells in adult sp-NSCs compared to fetal sp-NSCs (Adu-Spi-NSCs 8.13\u0026thinsp;\u0026plusmn;\u0026thinsp;0.79% vs Fet-Spi-NSCs 22.09\u0026thinsp;\u0026plusmn;\u0026thinsp;1.25%, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). ScRNA-Seq and bioinformatics analyses further revealed significantly lower expression levels of Mki67 and Ccna2 genes in adult sp-NSCs compared to fetal sp-NSCs (Adu-Spi-NSCs-Mki67 13.20% vs Fet-Spi-NSCs-Mki67 55.40%, Adu-Spi-NSCs-Ccna2 15.53% vs Fet-Spi-NSCs-Ccna2 58.97%, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). Further assays confirmed that most adult sp-NSCs remained quiescent, a cell state carrying important implications for understanding their properties and activation potential.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003e3.4 Notch signaling is a vital mechanism for activating intact adult mouse sp-NSCs\u003c/h2\u003e\u003cp\u003eThe Notch signaling pathway is a critical mechanism regulating NSC quiescence and activation, contributing to NSC heterogeneity. Nestin was co-stained with Notch1, Notch2, and Notch3 to investigate Notch receptor expression in adult sp-NSCs. Notch1 was highly expressed in adult sp-NSCs relative to Notch2 and Notch3 (79.01\u0026thinsp;\u0026plusmn;\u0026thinsp;4.83%, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Analysis of the gene expression profiles of Notch receptors in different subpopulations using scRNA-Seq and bioinformatics (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB) revealed that Notch1 was expressed in six subpopulations: dNSCs, pNSCs early, pNSCs late, aNSCs, TAPs, and NBs. Notably, it had relatively high expression levels, suggesting a close association between Notch1 and the regulation of activation and self-renewal in NSCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Notch2 and Notch3 were highly expressed in the dNSCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Further assays suggested that differential expressions of Notch receptors potentially play a significant role in regulating NSC activation in the intact adult mouse spinal cord.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003e3.5 Differential expression of the Notch receptors affects the activation of intact adult mouse sp-NSCs\u003c/h2\u003e\u003cp\u003eLentiviral vectors containing Notch1 shRNA, Notch2 shRNA, and Notch1\u0026thinsp;+\u0026thinsp;Notch2 shRNA were constructed, and their interference efficacy was characterized to investigate the role of Notch receptors in regulating adult sp-NSCs activation. The expression levels of Notch1 and Notch2 were significantly reduced compared to the controls (CTL 1.00 vs Notch1 shRNA (Notch1) 0.29\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03 vs Notch2 shRNA (Notch2) 0.55\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA) 5 days post-transfection of NSCs with Notch1 and Notch2 shRNA viruses. Similarly, the expression levels of both Notch1 and Notch2 were significantly decreased relative to controls (CTL 1.00 vs Notch1 shRNA\u0026thinsp;+\u0026thinsp;Notch2 shRNA (Notch1) 0.51\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03 vs Notch1 shRNA\u0026thinsp;+\u0026thinsp;Notch2 shRNA (Notch2) 0.56\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA) 5 days post co-transfection of NSCs with Notch1 and Notch2 shRNA viruses. These results indicated that Notch1 shRNA and Notch2 shRNA did not affect each other's RNA silencing efficacy.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe activation capacity of these cells was evaluated using the Sphere assay and EdU staining to investigate the roles of Notch1 and Notch2 in regulating adult sp-NSCs activation. Sphere assay results revealed a significant reduction in the neurosphere-forming capacity of NSCs following viral interference with Notch1 shRNA alone (CTL 134.02\u0026thinsp;\u0026plusmn;\u0026thinsp;1.88 \u0026micro;m vs Notch1 shRNA 88.11\u0026thinsp;\u0026plusmn;\u0026thinsp;3.71 \u0026micro;m, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Conversely, interference with Notch2 shRNA significantly increased the neurosphere-forming capacity (CTL 134.02\u0026thinsp;\u0026plusmn;\u0026thinsp;1.88 \u0026micro;m vs Notch2 shRNA 152.70\u0026thinsp;\u0026plusmn;\u0026thinsp;7.95 \u0026micro;m, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). These findings suggested that Notch1 can facilitate NSC activation, while Notch2 can sustain their quiescent state \u003cem\u003ein vitro\u003c/em\u003e. Noteworthy, the neurosphere-forming capacity was significantly reduced following combined interference with Notch1 and Notch2 shRNAs (CTL 134.02\u0026thinsp;\u0026plusmn;\u0026thinsp;1.88 \u0026micro;m vs Notch1 shRNA\u0026thinsp;+\u0026thinsp;Notch2 shRNA 106.65\u0026thinsp;\u0026plusmn;\u0026thinsp;5.99 \u0026micro;m, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). However, this decrease was not significantly different compared to that induced by Notch1 shRNA interference alone (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). EdU staining results were consistent with these findings, revealing a significant decrease in the percentage of Nestin\u003csup\u003e+\u003c/sup\u003e/EdU\u003csup\u003e+\u003c/sup\u003e cells following Notch1 shRNA interference (CTL 11.42\u0026thinsp;\u0026plusmn;\u0026thinsp;1.05% vs Notch1 shRNA 5.43\u0026thinsp;\u0026plusmn;\u0026thinsp;0.30%, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). However, interference with Notch2 shRNA significantly increased the percentage of positive cells (CTL 11.42\u0026thinsp;\u0026plusmn;\u0026thinsp;1.05% vs Notch2 shRNA 12.33\u0026thinsp;\u0026plusmn;\u0026thinsp;1.70%, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). The proportion of EdU-positive cells was significantly decreased following combined interference with Notch1 and Notch2 shRNAs (CTL 11.42\u0026thinsp;\u0026plusmn;\u0026thinsp;1.05% vs Notch1 shRNA\u0026thinsp;+\u0026thinsp;Notch2 shRNA 8.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.67%, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC).\u003c/p\u003e\u003cp\u003eThe expression levels of NICD and Rbpj genes downstream of the Notch signaling pathway and their related target genes, Hes1 and Hey1, were further examined. Western blot results revealed a significant decrease in NICD, Rbpj, Hes1, and Hey1 expression levels in adult sp-NSCs following transfection with Notch1 shRNA virus compared to the control group. The expression of Hes1 decreased to 0.38\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02, Hey1 to 0.16\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01, Rbpj to 0.19\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02, and NICD to 0.09\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01 compared to the control levels set at 1.00 (all \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD), indicating effective inhibition of the Notch signaling pathway in adult sp-NSCs post-transfection. Similarly, the expression levels of Rbpj, Hes1, and Hey1 in adult sp-NSCs transfected with Notch2 shRNA were significantly reduced compared to the controls (CTL 1.00 vs Notch2 shRNA (Rbpj) 0.73\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02, CTL 1.00 vs Notch2 shRNA (Hes1) 0.51\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05, CTL 1.00 vs Notch2 shRNA (Hey1) 0.41\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). In contrast, the expression of NICD did not differ significantly from that of the control group (CTL 1.00 vs Notch2 shRNA (NICD) 0.97\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). The expression levels of NICD, Rbpj, Hes1, and Hey1 were significantly decreased compared to controls when both Notch1 and Notch2 shRNAs were used (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). However, the expression of Hes1 and Hey1 when both Notch1 and Notch2 shRNAs were used did not differ significantly from those observed with Notch1 shRNA was used alone (Notch1 shRNA (Hes1) 0.38\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02 vs Notch1 shRNA\u0026thinsp;+\u0026thinsp;Notch2 shRNA (Hes1) 0.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03, Notch1 shRNA (Hey1) 0.16\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01 vs Notch1 shRNA\u0026thinsp;+\u0026thinsp;Notch2 shRNA (Hey1) 0.17\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD). These findings suggested that Notch receptors potentially promote the activation of adult sp-NSCs \u003cem\u003ein vitro\u003c/em\u003e via the Notch signaling pathway.\u003c/p\u003e\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eIn this study, optimization of the culture system of intact adult mouse sp-NSCs resulted in an abundant supply of NSCs that served as a stable cell source for subsequent experiments. ScRNA-seq revealed unique heterogeneity of sp-NSCs. The majority of adult mouse sp-NSCs remained in a quiescent state, which is significantly less activated compared to fetal mouse sp-NSCs. Moreover, characterization of the expression patterns of Notch receptors in sp-NSCs revealed asymmetric interaction between Notch1 and Notch2 receptors, which induced different activation states in adult mouse sp-NSCs. This study provides a basis for further elucidation of the regulatory mechanisms governing the activation of adult mouse sp-NSCs.\u003c/p\u003e\u003cp\u003eNSCs are predominantly found in the CNS of adult mammals. \u003cem\u003eIn vitro\u003c/em\u003e culture and differentiation characteristics of NSCs derived from the cerebral cortex, hippocampus, and striatum have been extensively studied. However, systematic research on adult sp-NSCs remains limited. The first \u003cem\u003ein vitro\u003c/em\u003e isolation and culture of adult mouse sp-NSCs was performed by Weiss et al\u003csup\u003e[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/sup\u003e. However, the lengthy digestion time potentially impacted cell viability. Notably, the method described in Methods in Molecular Biology for isolating and culturing adult mouse sp-NSCs is cumbersome\u003csup\u003e[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/sup\u003e. It involves harsh culture conditions, with an experimental duration typically exceeding four hours, which limits its widespread application. An optimized culture system for intact adult mouse sp-NSCs was developed based on the \u003cem\u003ein vitro\u003c/em\u003e culture method for fetal mouse brain-derived NSCs detailed in the Gibco Neurobiology Handbook. The optimized system reduced the number of isolation steps, decreased enzyme digestion time, and increased the initial cell seeding concentration. Assessment of the differentiation capacity of adult mouse sp-NSCs confirmed their ability to be induced to differentiate into neurons, astrocytes, and oligodendrocytes. Stable and abundant populations of adult mouse sp-NSCs with multipotent differentiation capacity were obtained following the modification of the culture system. The optimized method strongly demonstrates effectiveness for isolating and culturing adult mouse sp-NSCs.\u003c/p\u003e\u003cp\u003eSp-NSCs exhibit considerable heterogeneity, with marker expression varying among subpopulations. Llorens-Bobadilla et al. reported that NSCs in the subventricular zone (SVZ) of the lateral ventricles in adult mice significantly express GLAST (also known as Slc1a3) and Prom1 (also known as CD133)\u003csup\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e. Of note, GLAST is closely associated with protein synthesis, and its expression in qNSC1 is significantly higher than in qNSC2\u003csup\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e. EGFR is not expressed in either quiescent or activated NSCs\u003csup\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e. Dulken et al. postulated that Prom1 and GLAST are continually expressed throughout the lifespan of brain-derived NSCs\u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e. As such, they co-expressed EGFR and Prom1 to distinguish different NSC states\u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e. Beckervordersandforth et al. distinguished SVZ-NSCs from astrocytes in adult hGFAP-GFP transgenic mice using Prom1\u003csup\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e. However, they could not differentiate between quiescent and activated NSCs\u003csup\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e. Herein, six subpopulations of intact adult mouse sp-NSCs cultured \u003cem\u003ein vitro\u003c/em\u003e were identified through scRNA-Seq and bioinformatics analyses. The subpopulations included dNSCs, pNSCs early, pNSCs late, aNSCs, TAPs, and NBs. Analysis of GLAST and Prom1 gene expression profiles revealed that GLAST was highly expressed across all subpopulations of adult mouse sp-NSCs. However, Prom1 exhibited low expression in sp-NSCs, contrary to the findings in brain-derived NSCs\u003csup\u003e[\u003cspan additionalcitationids=\"CR16\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/sup\u003e. Consistent with our findings, Zywitza et al. and Sachewsky et al. also reported that Prom1 expression is limited in the SVZ of the adult mouse brain-derived NSCs\u003csup\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/sup\u003e. The validity of Prom1 as a specific NSC marker remains controversial. Co-expression of Nestin and Sox2 was thus employed for NSC identification in this study.\u003c/p\u003e\u003cp\u003eSpecific markers, such as GFAP and S100b, have been employed to investigate the activation potential of adult sp-NSCs to determine whether NSCs are quiescent\u003csup\u003e[\u003cspan additionalcitationids=\"CR20 CR21\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/sup\u003e. Zhang et al. proposed that GFAP positivity can be a marker for qNSCs because it exhibits radial glial morphology\u003csup\u003e[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/sup\u003e. Gengatharan et al. postulated that GFAP-positive cells comprise both NSCs and astrocytes\u003csup\u003e[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/sup\u003e. GFAP\u003csup\u003e+\u003c/sup\u003e/S100b\u003csup\u003e+\u003c/sup\u003e cells are identified as astrocytes, while GFAP\u003csup\u003e+\u003c/sup\u003e/S100b\u003csup\u003e\u0026minus;\u003c/sup\u003e cells are identified as qNSCs\u003csup\u003e[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/sup\u003e. Shah et al. reported that S100b is also expressed in qNSCs\u003csup\u003e[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]\u003c/sup\u003e. In addition, Zywitza et al. reported that NSCs and most astrocytes are negative, while mature oligodendrocytes (MOLs) and ependymal cells are positive for S100b\u003csup\u003e[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/sup\u003e. A significance analysis of differential gene expression conducted to characterize qNSCs revealed predominant expression of GFAP and S100b in the dNSCs but low detection in the pNSCs. Using S100b and GFAP as sole markers for qNSCs may thus result in the omission of certain \u0026ldquo;shallow dormant\u0026rdquo; NSCs, indicating that these markers alone are insufficient for the specific identification of qNSCs. Herein, Clu was regarded as a reliable marker. Co-staining with Nestin revealed that most adult sp-NSCs were quiescent. ScRNA-Seq analysis revealed that approximately 72.96% of the NSCs in the adult mouse spinal cord belong to the qNSCs. Among them, dNSCs comprised 69.15% of the qNSCs, suggesting limited activation capacity of adult sp-NSCs. The \u0026ldquo;deep dormancy\u0026rdquo; exhibited by dNSCs potentially impeded their transition to aNSCs.\u003c/p\u003e\u003cp\u003eSp-NSCs can be derived from specific subtypes of astrocytes and ependymal cells\u003csup\u003e[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/sup\u003e. Jonas et al. reported that a subpopulation of Troy-positive ependymal cells within the central canal region of the spinal cord exhibits NSCs properties\u003csup\u003e[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/sup\u003e. Frederico et al. reported that a subpopulation of fetal DNGR-1-positive ependymal cells retains NSC characteristics into adulthood\u003csup\u003e[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/sup\u003e. Analysis of the Foxj1 gene revealed uniform expression of Foxj1 in the cultured intact adult mouse sp-NSCs system, suggesting that ependymal cells constitute a significant component of NSCs. Noteworthy, a subset of ependymal cells and astrocytes can be converted into NSCs and exhibit various activation states 3 days post-SCI\u003csup\u003e[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eThe activation capacity of NSCs is highly dependent on age-related changes\u003csup\u003e[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]\u003c/sup\u003e. Thymidine labeling assays demonstrate that SVZ-NSCs of aged mice exhibit a lower division rate than those of young mice\u003csup\u003e[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]\u003c/sup\u003e. Similarly, Kalamakis et al. observed a significant decline in brain-derived NSCs in aged mice\u003csup\u003e[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]\u003c/sup\u003e. Notably, NSCs from young and aged mice demonstrate similar activation and differentiation capacities upon activation\u003csup\u003e[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]\u003c/sup\u003e. ScRNA-Seq analysis conducted by Shi et al. revealed a significant reduction in the proliferative capacity across all NSCs and neural progenitor cells (NPCs) in aged SVZ\u003csup\u003e[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]\u003c/sup\u003e. This phenomenon was attributed to a highly inflammatory microenvironment in the aged SVZ\u003csup\u003e[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]\u003c/sup\u003e. Sphere assay and EdU staining conducted to investigate whether the activation capacity of sp-NSCs decreases with age revealed reduced neurosphere-forming ability of adult sp-NSCs. Moreover, the number of EdU-positive cells was significantly lower than that of fetal sp-NSCs. These findings underscored the diminishing activation potential of adult sp-NSCs. Gene expression analysis of proliferation markers, Mki67 and Ccna2, via scRNA-Seq and bioinformatics revealed significantly lower expression levels in adult sp-NSCs compared to fetal sp-NSCs, indicating reduced activation capacity in adult sp-NSCs.\u003c/p\u003e\u003cp\u003eOf note, adult sp-dNSCs constituted 50.45%, while fetal sp-dNSCs constituted 30.77% of NSCs. This finding indicates that the quantity of sp-NSCs in the \u0026ldquo;deep dormant\u0026rdquo; state increases considerably with age, and their conversion into aNSCs remains relatively challenging. Adult sp-pNSCs comprised 30.85% of qNSCs, consistent with findings from studies on brain-derived NSCs. In contrast, fetal sp-pNSCs comprised 58.63% of qNSCs, suggesting that adult sp-NSCs possess fewer reserves for converting qNSCs to aNSCs, and exhibit diminished activation potential compared to fetal sp-NSCs. Notably, adult sp-aNSCs accounted for 27.04%, while fetal sp-aNSCs accounted for 25.60% of NSCs upon activation of qNSCs. However, this difference was not statistically significant. Herein, sp-NSCs across different ages demonstrated similar activated capacities consistent with the findings of Kalamakis et al\u003csup\u003e[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]\u003c/sup\u003e. Previous studies postulate that dNSCs transiently proliferate after activation and primarily differentiate into astrocytes following SCI\u003csup\u003e[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/sup\u003e. Xu et al. reported that, unlike dNSCs, certain Oct4\u003csup\u003e+\u003c/sup\u003epNSCs exhibit multidirectional differentiation potential following activation and can differentiate into neurons, oligodendrocytes, and astrocytes\u003csup\u003e[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]\u003c/sup\u003e. Herein, the activation potential of sp-NSCs varied across different age groups. However, future studies should investigate the differentiation capacity of qNSCs following activation.\u003c/p\u003e\u003cp\u003eThe Notch signaling pathway is a critical mechanism for regulating the quiescence and activation of NSCs and is a crucial factor contributing to NSC heterogeneity\u003csup\u003e[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/sup\u003e. The pathway primarily comprises Notch receptors, ligands, CSL proteins, and downstream target genes\u003csup\u003e[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]\u003c/sup\u003e. Notch receptors play different roles in regulating NSC activation in the adult mammalian CNS\u003csup\u003e[\u003cspan additionalcitationids=\"CR30\" citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]\u003c/sup\u003e. Basak et al. investigated the role of Notch1 in brain-derived NSCs using a conditional Notch1 knockout mouse model and concluded that Notch1 primarily functions in aNSCs by maintaining the activation state and promoting differentiation into neurons\u003csup\u003e[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]\u003c/sup\u003e. Moreover, the study concluded that Notch1 exhibits distinct functions in NSCs depending on their proliferative state\u003csup\u003e[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]\u003c/sup\u003e. Engler et al. reported that Notch2 preserves the quiescent state of NSCs by repressing cell cycle-related genes\u003csup\u003e[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]\u003c/sup\u003e. In contrast, Notch1 primarily sustains the activated state\u003csup\u003e[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]\u003c/sup\u003e. Kawai et al. reported that Notch3 is highly expressed in SEZ-qNSCs of the adult mouse selectively, while Notch1 is predominantly expressed in aNSCs and TAPs\u003csup\u003e[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]\u003c/sup\u003e. The study further concluded that Notch3 maintains NSC quiescence via knockdown experiments\u003csup\u003e[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]\u003c/sup\u003e. In this study, co-expression staining of Nestin and Notch receptors revealed heterogeneous expression of Notch1, Notch2, and Notch3 in adult sp-NSCs. Notably, Notch1 demonstrated significantly higher co-expression with Nestin than Notch2 and Notch3. However, Notch1 did not co-stain with Nestin in some cases. Previous studies postulate that Notch 1 is potentially expressed in radial glial cells, which account for the absence of positive staining. ScRNA-seq and bioinformatics analyses performed to characterize the gene expression profiles of Notch receptors in adult sp-NSCs revealed that Notch1 is expressed across multiple subpopulations, including dNSCs, pNSCs, and aNSCs, with notably high expression in the aNSCs. Moreover, Notch2 and Notch3 were highly expressed in the dNSCs. These findings suggest that Notch1 is potentially involved in regulating NSC activation and self-renewal, while Notch2 and Notch3 potentially contribute to maintaining NSC quiescence. Notably, Notch1 was highly expressed in both qNSCs and aNSCs. However, the mechanisms underlying its functional heterogeneity warrant further investigation\u003csup\u003e[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003ePrevious studies have extensively explored the role of Notch receptors in regulating NSC activation using lineage tracing experiments and conditional knockout models\u003csup\u003e[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]\u003c/sup\u003e. For instance, Basak et al. reported that conditional knockout of Notch1 (Notch1 cKO) causes a selective loss of aNSCs without affecting the activation of qNSCs\u003csup\u003e[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]\u003c/sup\u003e. Engler et al. reported that GFP\u003csup\u003e+\u003c/sup\u003e/GFAP\u003csup\u003e+\u003c/sup\u003eqNSCs are activated and enter the cell cycle in Notch2 cKO, Notch1/Notch2 double cKO, and Rbpj cKO mice\u003csup\u003e[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]\u003c/sup\u003e. Zhang et al. observed that conditional knockout of Notch2 increases proliferation of DG-NSCs and enhances the generation of newborn neurons\u003csup\u003e[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/sup\u003e. Herein, lentiviral vectors carrying Notch1 shRNA and Notch2 shRNA were constructed and used to assess the activation capacity of Notch1 and Notch2 in adult sp-NSC using Sphere assays and EdU staining. Of note, the neurosphere-forming capacity was reduced, and the number of EdU-positive cells was significantly lower following transfection with Notch1 shRNA viruses compared to controls. Conversely, transfection with Notch2 shRNA viruses yielded opposite effects. These findings suggested that interference with Notch1 impairs NSC activation, whereas disruption of Notch2 may promote their transition into an activated state. Notably, co-transfection with Notch1 and Notch2 shRNA viruses reduced the neurosphere-forming capacity and EdU-positive cell counts compared to the controls. However, the co-transfection effect was not significantly different to Notch1 shRNA transfection alone. These findings imply that the dominant role of Notch1 in NSC activation is attenuated when both receptors are disrupted simultaneously because of the higher expression level of Notch1 compared to Notch2 in adult sp-NSCs. The signaling pathways involved in NSC activation by Notch3 were not explored because of its overall low expression. However, future studies should aim to elucidate its effects.\u003c/p\u003e\u003cp\u003eWestern blotting was employed to validate the expression levels of downstream components of the Notch signaling pathway, including NICD, Rbpj, and the target genes, Hes1 and Hey1. Notably, Hes1 and Hey1 proteins were significantly decreased in adult sp-NSCs transfected with Notch1 shRNA virus compared to the control group, indicating effective inhibition of the Notch signaling pathway in these cells. The expression levels of Hes1, Hey1, Rbpj, and NICD in adult sp-NSCs transfected with Notch2 shRNA virus were also downregulated relative to the control group. However, these expression levels remained significantly higher than those in NSCs transfected with Notch1 shRNA virus. These findings were consistent with those of Sahoo et al., who reported that specific inhibition of the Notch2 gene resulted in the activation of the transcription of Notch1 and typical gene targets, Hes1, Hey1, and Hey2\u003csup\u003e[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]\u003c/sup\u003e. These reports suggest that the enhancement of Notch1 signaling potentially facilitates the activation of adult sp-NSCs following Notch2 interference. Nevertheless, the precise mechanisms underlying the synergistic regulation of adult neurogenesis by Notch1 and Notch2 require further investigation.\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eThis investigation demonstrated the majority of adult mouse sp-NSCs remained in a quiescent state, which is significantly less activated compared to fetal mouse sp-NSCs. Moreover, this study further reveals the characterization of the expression patterns of Notch receptors in adult mouse sp-NSCs. Our results also indicate that both Notch1 and Notch2 signals are involved in different regulatory roles that facilitate the activation and fate determination of sp-NSCs via NICD-Rbpj.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eAdu-Spi-NSCs, adult spinal cord-derived NSCs; aNSCs, active neural stem cells; ANOVA, analysis of variance; CNS, central nervous system; dNSCs, dormant NSCs; Fet-Spi-NSCs, fetal spinal cord-derived NSCs; MOLs, mature oligodendrocytes; NBs, neuroblasts; Notch1 cKO, conditional knockout of Notch1; NPCs, neural progenitor cells; NSCs, neural stem cells; PBS, phosphate-buffered saline; PFA, paraformaldehyde; pNSCs, primed qNSCs; PVDE, polyvinylidene fluoride; qNSCs, quiescent NSCs; SCI, spinal cord injury; scRNA-Seq, single-cell RNA sequencing; Sp-NSCs, Spinal cord-derived NSCs; SVZ, subventricular zone; TAPs, transit amplifying progenitors; t-SNE, t-distributed Stochastic Neighbor Embedding; Tukey\u0026rsquo;s HSD, Tukey\u0026rsquo;s Honestly Significant Difference.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by Shaanxi Science and Technology Foundation (grant nos. 2024JC-YBMS-620).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eJuan Li: conceptualization, validation, methodology, investigation, formal analysis, data curation, writing – original draft, writing – review and editing. Fen Ju: conceptualization, validation, methodology, investigation, formal analysis, data curation, writing – original draft, writing – review and editing. Zhao-Yang Huang: software, investigation, formal analysis, data curation, writing – review and editing. Liang Yu: validation, supervision, software, resources, methodology, formal analysis, data curation, conceptualization, writing – review and editing. Jie Qin: validation, supervision, resources, methodology, conceptualization, writing – review and editing. Yu-Bing Yang: resources, investigation, writing – review and editing. Wei Sun: writing – review and editing, visualization, validation, investigation. Yu-Qiang Ji: visualization, resources, project administration, conceptualization, writing – review and editing. Chen-Guang Zhao: validation, supervision, project administration, methodology, funding acquisition, formal analysis, conceptualization, writing – review and editing, writing – original draft. Hua Yuan: supervision, project administration, methodology, funding acquisition, conceptualization, writing – review and editing.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe also thank Fei Fang for technical support.\u003c/p\u003e\n\u003cp\u003eThis work was supported by Shaanxi Science and Technology Foundation (grant nos. 2024JC-YBMS-620).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data that support the findings of this study are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cbr\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eGilbert Ea, B, Lakshman N, Lau KSK, Morshead CM (2022) Regulating Endogenous Neural Stem Cell Activation to Promote Spinal Cord Injury Repair [J]. 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Am J Physiol Heart Circ Physiol 321(3):H542\u0026ndash;h557\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"spinal cord injury, neural stem cells, Notch receptors","lastPublishedDoi":"10.21203/rs.3.rs-7540357/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7540357/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eNeural stem cells (NSCs) play a crucial role in neural regeneration following spinal cord injury (SCI) owing to their self-proliferative and multidirectional differentiation capabilities. This study examined the biological properties of adult spinal cord-derived NSCs (sp-NSCs) and the role of Notch receptors in regulating their activation. NSCs were isolated from the spinal cords of 8-week-old C57/BL6 mice, and their biological properties, including the gene expression profile of Notch receptors, were subsequently analyzed using single-cell RNA sequencing (scRNA-Seq) and bioinformatics. The NSCs were subsequently infected with lentiviral vectors encoding Notch1 shRNA, Notch2 shRNA, and a combination of Notch1 and Notch2 shRNA sequences, and evaluated using Sphere assays and EdU staining to determine their activation effects. The expression levels of downstream genes, NICD and Rbpj, in the Notch signaling pathway, as well as the related target genes, Hes1 and Hey1, were subsequently quantified using Western blot analysis. Intact adult mouse sp-NSCs predominantly existed in a quiescent state, with their population increasing significantly with age. Notch receptors served as critical regulators of adult sp-NSC activation. Notably, Notch1 expression was significantly elevated compared to Notch2 and Notch3, demonstrating its predominant role in sustaining NSC activation. In contrast, Notch2 and Notch3 were primarily responsible for maintaining NSCs in a quiescent state. Overall, both Notch1 and Notch2 signals are involved in different regulatory roles that facilitate the activation and fate determination of NSCs via NICD-Rbpj.\u003c/p\u003e","manuscriptTitle":"Differential Roles of Notch Receptors in Regulating Activation of Intact Adult Mouse Spinal Cord-derived NSCs","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-11 16:35:25","doi":"10.21203/rs.3.rs-7540357/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"0b1e4a41-7745-446a-b8ea-2925414a0d08","owner":[],"postedDate":"September 11th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-09-29T13:23:56+00:00","versionOfRecord":[],"versionCreatedAt":"2025-09-11 16:35:25","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7540357","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7540357","identity":"rs-7540357","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}