A method to promote the dedifferentiation of satellite glial cells into neural stem cell-like cells

Abstract

This invention relates to the field of cell dedifferentiation, and more particularly to a method for promoting the dedifferentiation of satellite glial cells into neural stem cell-like cells. First, a three-dimensional composite hydrogel of nervonic acid-modified chitosan is constructed. Then, satellite glial cells are seeded and cultured in this hydrogel, inducing dedifferentiation to obtain a cell population with a neural stem cell-like phenotype. The hydrogel constructs a basic network through covalently linked amide bonds between nervonic acid and chitosan, and utilizes the hydrophobic association domains formed by the long-chain alkyl groups of nervonic acid to self-assemble into a covalently cross-linked + hydrophobically associated dual network structure. This simulates the softness, deformability, and local mechanical gradient properties of the neural tissue microenvironment, providing ideal dedifferentiation conditions for satellite glial cells to differentiate into neural stem cell-like cells. During the dedifferentiation process, markers such as Nestin, SOX2, and PAX6 are expressed, showing promising applications in neural regeneration, disease model construction, and cell therapy.

Description

Technical Field

[0001] This invention relates to the field of cell dedifferentiation, and more particularly to a method for promoting the dedifferentiation of satellite glial cells into neural stem cell-like cells. Background Technology

[0002] The peripheral nervous system (PNS) exhibits significantly greater regenerative capacity compared to the central nervous system (CNS), a phenomenon closely related to its unique cellular microenvironment. Satellite glial cells (SGCs) in the dorsal root ganglion (DRG) have been identified as key participants in the PNS regeneration process. Studies have shown that SGCs not only support neuronal survival after nerve injury by clearing debris and providing neurotrophic factors, but also demonstrate the potential to be induced to differentiate into neurons. This plasticity suggests that SGCs may have the potential to "dedifferentiate" into neural stem cell-like cells, thus providing an ideal source of seed cells for neural regeneration, disease modeling, and cell therapy.

[0003] Currently, the mainstream strategies for inducing dedifferentiation in terminally differentiated cells (such as SGCs) mainly rely on the following methods:

[0004] Transcription factor reprogramming: This method involves introducing exogenous transcription factors such as OCT4, SOX2, KLF4, and c-Myc via viral vectors to forcibly alter cell fate. However, this method suffers from drawbacks including the risk of integration mutations, potential tumorigenicity, immune rejection, and low transfection efficiency, resulting in poor safety profiles for clinical application.

[0005] Small molecule compound induction: Using chemical cocktails (such as those containing small molecules like CHIR99021, 616452, and Y27632) to regulate cell signaling pathways. Although this avoids gene manipulation, most small molecules are cytotoxic, easily leading to abnormal cell cycle arrest (such as G1 phase arrest), functional abnormalities, and impure induction lineages, raising concerns about long-term safety.

[0006] Growth factor / cytokine regulation: Adding growth factors such as bFGF, EGF, and BDNF. This method is relatively safe, but for terminally differentiated SGCs, single or combined growth factors often have difficulty overcoming their strong epigenetic barrier, resulting in low induction efficiency, high cost, and easy degradation of the factors.

[0007] Epigenetic regulation: using DNA methyltransferase inhibitors or histone deacetylase inhibitors. These methods have complex mechanisms of action, are highly nonspecific, may globally activate harmful genes, and have poor controllability.

[0008] In recent years, the "material-induced" strategy, which utilizes three-dimensional biomaterials to simulate the in vivo microenvironment and regulate cell fate, has attracted much attention. In particular, constructing three-dimensional culture systems using hydrogels can simulate the natural tissue environment from multiple dimensions, including physical constraints, mechanical signals, and biochemical signals, providing new ideas for cell reprogramming. Among these approaches, functionalizing biomaterials (such as chitosan) with natural active molecules (such as nervonic acid) is an effective way to enhance the bioactivity of materials.

[0009] Nervonic acid, a monounsaturated long-chain fatty acid abundant in the mammalian nervous system, has been proven to play an important role in neural development, myelination, and plasticity; however, its applications are mostly limited to nutritional supplementation or neuroprotective agents. Chitosan, as a natural polysaccharide, is often used as a cell culture scaffold or drug carrier due to its good biocompatibility and modifiability. Current research typically involves chemically cross-linking chitosan with hydrophilic polysaccharides such as hyaluronic acid (HA) to prepare hydrogels. However, these materials mainly form a single covalent network structure, and their microenvironment differs significantly from the lipid bilayer-rich and dynamically interacting microenvironment of real neural tissue, making it difficult to provide the complex and dynamic physicochemical signals required to induce neurogenic cell fate transitions.

[0010] Therefore, the core technical challenge in this field is: how to construct a three-dimensional culture system that can highly simulate the natural microenvironment of neural tissue while avoiding the safety risks brought about by the introduction of exogenous genetic / chemical substances, so as to achieve efficient, safe and controllable dedifferentiation of SGCs and thus obtain functional neural stem cell-like cells.

[0011] To date, there have been no reports of methods that use nervonic acid as a structural signaling molecule to covalently bind with chitosan and utilize its hydrophobic self-assembly properties to construct a "covalent cross-linking + hydrophobic association" dual-network biomimetic hydrogel, which is then specifically used to induce dedifferentiation of peripheral nerve-derived SGCs. Summary of the Invention

[0012] This invention provides a method for promoting the dedifferentiation of satellite glial cells (SGCs) into neural stem cell-like cells. The method involves preparing a hydrogel by covalently linking nervonic acid and chitosan via amide bonds, and then utilizing the self-assembly of hydrophobic chains of nervonic acid to form a dual network structure of "covalent cross-linking + hydrophobic association," mimicking the microenvironment of neural tissue. The hydrogel provided by this invention exhibits excellent biocompatibility, antibacterial properties, and genetic safety, and can efficiently and safely induce SGCs to dedifferentiate into neural stem cell-like cells, expressing stem cell markers such as Nestin, SOX2, and PAX6, and activating the WNT / NOTCH signaling pathway. It shows promising application prospects in neural regeneration, disease model construction, and cell therapy.

[0013] To solve the above-mentioned technical problems, the technical solution of the present invention is as follows: A method for promoting the dedifferentiation of satellite glial cells into neural stem cell-like cells, comprising the following steps: Step (1) Construct a three-dimensional composite hydrogel containing chitosan modified with nervonic acid; the hydrogel constructs a basic network by covalently linking nervonic acid and chitosan through amide bonds, and uses the long-chain alkyl group of nervonic acid to form hydrophobic association domains to exert hydrophobic effects and self-assemble into a "covalent crosslinking + hydrophobic association" dual network structure, which simulates the soft, deformable and local mechanical gradient characteristics of the microenvironment of nerve tissue, and provides ideal dedifferentiation conditions for SGCs; Step (2) Inoculate satellite glial cells in a three-dimensional composite hydrogel; without introducing exogenous transcription factors or dedifferentiation-promoting small molecule compounds, induce the satellite glial cells to dedifferentiate to obtain a cell population with a neural stem cell-like phenotype.

[0014] The "no introduction of exogenous transcription factors or dedifferentiation-promoting small molecule compounds" mentioned in this invention means that no transcription factors (such as Oct4, Sox2, Klf4, c-Myc, etc.) or known dedifferentiation-promoting small molecule compounds (such as Y27632, CHIR99021, VPA, 616452, etc.) for direct reprogramming of cell fate or known dedifferentiation-promoting small molecule compounds (such as Y27632, CHIR99021, VPA, 616452, etc.) are added to the culture medium during the entire culture process.

[0015] As a further preferred embodiment of the above scheme, the preparation method of the three-dimensional composite hydrogel is as follows: S1 dissolves chitosan in acetic acid solution to obtain chitosan solution; S2 dissolves nervonic acid in an organic solvent, adds a carbodiimide crosslinking agent and N-hydroxysuccinimide for light-protected activation, and obtains an activated nervonic acid solution. S3 involves adding the activated nervonic acid solution dropwise to the chitosan solution and mixing, adjusting the pH of the reaction system to 5.5-6.0, and reacting at 20-30℃ for 6-12 hours to allow the carboxyl groups of nervonic acid to form amide bonds with the amino groups of chitosan. S4 purifies, dialyzes, and freeze-dries the reaction product to obtain nervonic acid-modified chitosan; preferably, the obtained reaction solution is placed in a dialysis bag with a molecular weight cutoff of 14 kDa and dialyzed in deionized water for 2-4 days, during which the dialysis solution is replaced regularly to remove unreacted small molecules and residual reagents.

[0016] S5 dissolves nervonic acid-modified chitosan in DMEM / F12 medium to obtain a three-dimensional composite hydrogel with a concentration of 1%-10% (w / w).

[0017] The storage modulus (G′) of the three-dimensional composite hydrogel under conditions of 37°C, 1Hz frequency, and 1% strain is 100 Pa-5000 Pa, preferably 500 Pa-2000 Pa. To obtain the preferred range of storage modulus, the storage modulus is first increased by increasing the hydrogel concentration and decreased by decreasing the polymer concentration. This application demonstrates that the storage modulus of 2.5% (w / w) NA-CS hydrogel is ≈10 Pa. 2 -10 3 Pa is within the preferred range.

[0018] Secondly, there are the cross-linking mechanisms and components. HA-CS forms amide bonds through EDC / NHS, which is a purely chemical cross-linking; NA-CS forms hydrophobic association domains in water through long-chain hydrophobic groups, which is a physical self-assembly. Physical self-assembly is superior to chemical cross-linking in terms of regulating energy storage modulus, as the latter can be rapidly adjusted by temperature, concentration, salt ions, pH, etc.; while the former has high strength and is not easily changed by time, temperature, or stress.

[0019] Finally, NA-CS can controllably adjust the energy storage modulus. This application demonstrates that: firstly, the energy storage modulus can be kept within a controllable range by adjusting the amount of NA grafting: it is clear that the amount of NA affects the hydrophobic association density; secondly, it is found that the ideal grafting rate can be obtained under the conditions of pH 5.8, 25℃ and 6h reaction, which indirectly affects the energy storage modulus; and thirdly, NA-CS forms a self-supporting three-dimensional porous network with a clear structure, which indirectly reflects that NA-CS has good porosity and a high degree of self-assembly.

[0020] In summary, the purpose of covalent cross-linking and physical self-assembly is to provide both basic network stiffness to ensure structural stability and tunability and responsiveness, so that NA-CS hydrogels are both stable and have adjustable energy storage modulus. This is also the design concept and principle of NA-CS hydrogels.

[0021] The three-dimensional composite hydrogel exhibits an interconnected porous structure under a scanning electron microscope, with an average pore size of 10 μm to 100 μm. These structural parameters are key to achieving the functions of "simulating the microenvironment of neural tissue" and "suitable for cell growth and dedifferentiation," and are also objective characteristics that distinguish the hydrogel of this invention from ordinary hydrogels.

[0022] The composite hydrogel is formed by the synergistic effect of covalent cross-linking and hydrophobic association of nervonic acid-modified chitosan polymers under aqueous conditions. Nervonic acid is grafted onto the chitosan backbone through an amidation reaction between its carboxyl groups and the amino groups on the chitosan molecular chain. The composite hydrogel internally forms hydrophobic microdomains generated by the self-assembly of long hydrophobic chains of nervonic acid, thus constructing a lipid-like biomimetic microenvironment.

[0023] As a further preferred embodiment of the above scheme, the carbodiimide crosslinking agent is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and the mass ratio of the carbodiimide crosslinking agent to N-hydroxysuccinimide is (0.3-1.0):(0.2-0.6); the organic solvent is dimethyl sulfoxide, and the mass-volume ratio of nervonic acid to dimethyl sulfoxide is 1 g:(5-15) mL.

[0024] As a further preferred embodiment of the above scheme, the mass-to-volume ratio of chitosan to acetic acid is (1.0-2.0) g: 100 mL; the mass ratio of chitosan to nervonic acid is (2-5): 1.

[0025] As a further preferred embodiment of the above scheme, the concentration of the three-dimensional composite hydrogel is 2-3% (w / w).

[0026] As a further preferred embodiment of the above scheme, the satellite glial cells are prepared at a concentration of 1 × 10⁻⁶ cells per milliliter of hydrogel. 5 -5×10 6 The satellite glial cells were seeded at a density of 1,000 cells in a three-dimensional composite hydrogel and cultured for 48-120 hours to induce dedifferentiation and obtain cell spheres with a neural stem cell-like phenotype.

[0027] As a further preferred embodiment of the above scheme, the satellite glial cells are derived from primary satellite glial cells of the dorsal root ganglion of the mammalian peripheral nervous system.

[0028] The present invention also provides the application of the three-dimensional composite hydrogel in inducing satellite glial cell dedifferentiation, constructing neural stem cell-like cells or neural regeneration-related cell models.

[0029] The present invention also provides neural stem cell-like cells prepared by the method, wherein the cells express neural stem cell-related markers, including Nestin, SOX2, PAX6 and / or CD133; the dedifferentiation process is accompanied by activation of the WNT signaling pathway and / or the NOTCH signaling pathway.

[0030] The features of this invention are as follows: In the prior art, nervonic acid is mostly used for nutritional supplementation and neuroprotection; chitosan is mostly used for scaffolds or carriers; this invention is the first to use nervonic acid as a functional signaling molecule, directly participating in the regulation of SGC cell fate through material means rather than chemical induction, and the first to propose nervonic acid-chitosan for inducing SGC dedifferentiation. The hydrogel provided by this invention simultaneously possesses a three-dimensional microenvironment of covalent cross-linking + hydrophobic association dual network. In the covalent cross-linking + hydrophobic association dual network structure, the covalently cross-linked amide bonds provide long-term structural stability and mechanical strength of the hydrogel skeleton, while the dynamic hydrophobic association domains formed by the self-assembly of nervonic acid hydrophobic chains not only simulate the lipid microenvironment of nerve cell membranes, but also provide a dynamic and reconfigurable interface for cell-material interactions. This static-dynamic combined network structure synergistically simulates the viscoelasticity and biophysical signals of the natural extracellular matrix of nerve tissue, which is key to inducing SGC fate reprogramming; it is significantly different from traditional single chemically cross-linked hydrogels such as HA-CS.

[0031] Existing SGC dedifferentiation methods often rely on transcription factors (with poor safety) or combinations of small molecules (toxicity, G1 blockade). This invention, however, relies entirely on the self-activation of the three-dimensional material microenvironment and endogenous signaling pathways, without transcription factors or small molecule-induced dedifferentiation pathways. This solves the long-standing safety and transformational barriers in the field of cell dedifferentiation. This invention is validated through a transcriptome + PPI + CO-IP system; it clearly proposes that the interaction between Axin2 and Lef1 is a crucial molecular basis for maintaining stemness after dedifferentiation; and it provides a theoretical basis for subsequent drug target and material optimization.

[0032] Compared with the prior art, the present invention has the following beneficial effects: 1. This invention innovatively integrates nervonic acid as a functional signaling molecule into a hydrogel system, directly regulating the fate of satellite glial cells through material-based rather than chemically induced methods, avoiding the toxicity, immune rejection, and genetic risks associated with exogenous transcription factors or small molecule compounds, and significantly improving safety.

[0033] 2. This invention constructs a three-dimensional microenvironment with covalent cross-linking and hydrophobic association dual networks, which not only ensures the structural stability of the hydrogel, but also forms a lipid-like biomimetic microenvironment through the self-assembly of nervonic acid hydrophobic chains, which is closer to the natural structure of nerve tissue and provides ideal physical and biochemical support for the dedifferentiation of SGCs.

[0034] 3. The induction process of this invention relies entirely on the self-activation of the three-dimensional material microenvironment and endogenous signaling pathways, without the need for exogenous transcription factors or small molecule compounds. This effectively solves the problems of poor safety and abnormal cell function in traditional dedifferentiation methods, and improves cell functional maturity and the transformation potential for subsequent applications.

[0035] 4. This invention systematically verified the molecular mechanism in the dedifferentiation process and clarified the core role of the interaction between AXIN2 and LEF1 in maintaining cell stemness, providing a theoretical basis for subsequent drug target development and material optimization.

[0036] 5. The hydrogel system provided by this invention has good biocompatibility, antibacterial properties and genetic safety. It is superior to traditional hydrogel systems in terms of cell survival, proliferation, dedifferentiation efficiency and inflammation regulation. It is suitable for constructing neural regeneration-related cell models and seed cells for regenerative medicine, and has broad clinical application prospects. Attached Figure Description

[0037] Figure 1 Characterization of NA-CS hydrogel; A is the two-dimensional FTIR thermogram of NA-CS hydrogel captured at ~1735 cm⁻¹. -1 ×1735 cm -1 A significant high-intensity correlation signal was observed in the region; B represents the NA-CS hydrogel. 1 H NMR characteristic signals and structure assignment; C is the molecular weight distribution of NA-CS hydrogel; D is the rheological analysis of NA-CS hydrogel (different colors represent different concentrations); E is the inverted experiment of 2.5% (w / w) NA-CS hydrogel; F is the scanning electron microscope image of NA-CS hydrogel (scale bar = 10µm). Figure 2 A schematic comparison of the synthesis of NA-CS hydrogel and HA-CS hydrogel; A shows that NA-CS hydrogel is a dual network of chemical cross-linking and hydrophobic association, while B shows that HA-CS hydrogel is a single network of chemical cross-linking. Figure 3 For the cell compatibility assay of NA-CS hydrogel: A is cell viability assay using CCK8 (**p<0.01), B is cell viability assay, with PI red staining indicating dead cells (scale bar = 100µm), C is antibacterial assay (*p<0.05), and D is ELISA assay for the expression of pro-inflammatory and anti-inflammatory factors (*p<0.05). Figure 4 To explore the concentration for primary and three-dimensional culture of SGCs; A shows the immunofluorescence identification of primary SGCs (scale bar = 100µm); B shows the optimal culture concentration for NA-CS hydrogel screening using CCK8. ** p<0.01); Figure 5 This section compares the effects of NA-CS hydrogel and common culture environments on SGC proliferation. A represents the effect of CCK8 assay on the viability of various culture environments for SGCs. ** p<0.01), B represents the effect of different culture environments on the cell cycle of SGCs observed by flow cytometry. ** p<0.01); Figure 6 To compare the effects of NA-CS hydrogel and common culture environments on SGC dedifferentiation, A shows the observation of spheroidal cells formed by SGC dedifferentiation under white light microscopy (scale bar = 100µm), B shows the identification of spheroidal cells by immunofluorescence (scale bar = 100µm), and C shows the identification of surface markers of spheroidal cells by flow cytometry. * p<0.05); Figure 7 To detect the expression levels of related factor mRNAs in NA-CS hydrogel after SGC dedifferentiation by qRT-PCR, A represents the expression level of stemness factor mRNA. ** p<0.01), B represents the mRNA expression levels of growth and proliferation support-related factors (p<0.01). * p<0.05, ** p<0.01); Figure 8 To explore the mechanism by which NA-CS hydrogel promotes the dedifferentiation of SGCs in the preliminary mRMA sequencing of the transcriptome, A is a volcano diagram of differentially expressed genes (orange represents upregulated differentially expressed genes, and blue represents downregulated differentially expressed genes), B is the KEGG analysis of differentially expressed genes, C is the VENN diagram showing common differentially expressed genes in the key stemness pathways NOTCH signaling pathway, WNT signaling pathway, and axon guidance based on KEGG analysis, D is the PPI interaction diagram of common differentially expressed genes, and E is the CO-IP experiment to verify the strongly interacting proteins inferred from PPI. Detailed Implementation

[0038] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments, but the present invention is not limited to the following technical solution. Unless otherwise specified, the reagents and raw materials used in the following embodiments were all purchased from the market. Among them, nervonic acid is cis-15-tetracosenoic acid with a purity ≥95%, purchased from Shanghai Yuanye Biotechnology Co., Ltd. (item number: B28317).

[0039] Example 1: Synthesis and Characterization of Nervate-Modified Chitosan Composite Hydrogel To determine the optimal coupling conditions, the reaction conditions between nervonic acid and chitosan were screened using the grafting rate as an evaluation index. The change in free amino group content before and after the chitosan reaction was determined using the TNBS method, and the grafting rate was calculated based on the reduction in amino groups. The calculation formula is as follows: Grafting rate (%) = (Amino content before reaction - Amino content after reaction) / Amino content before reaction × 100%The effects of nervonic acid dosage, EDC / NHS ratio, reaction pH, and reaction time on the coupling reaction were investigated using grafting rate as the evaluation index. The results showed that when the nervonic acid dosage increased from 0.2 g to 0.5 g, the grafting rate increased from 11.8% to 27.9%; further increases to 0.7 g and above resulted in little or no increase in grafting rate. When the EDC and NHS dosages were 0.5 g and 0.3 g, respectively, the grafting rate was moderate (approximately 28%), and the reaction system pH was 5.8 with a reaction time of 6 h, resulting in relatively stable grafting efficiency (Table 1).

[0040] As shown in Table 1, the optimal mass ratio of chitosan, nervonic acid, EDC, and NHS is 1:(0.13~0.67):(0.20~0.67):(0.13~0.40). The preferred mass ratio is 1:(0.33~0.47):(0.33~0.67):(0.20~0.40); this optimal ratio ensures a grafting rate of over 27%. The grafting rate is sensitive to pH, with an optimal pH range of 5.5~6.0 and an optimal value of 5.8. Too low a pH (≤5.0) or too high a pH (≥6.5) will significantly reduce the grafting rate. The reaction temperature is 20-30℃, preferably 25℃.

[0041] Table 1 Comparison of grafting rates of different components

[0042] Chitosan (CS, 1.5 g) was dissolved in 100 mL of 1% (v / v) acetic acid solution and magnetically stirred until completely dissolved at room temperature. From the determined final components, nervonic acid (NA, 0.5 g) was dissolved in DMSO, followed by the addition of EDC (0.5 g) and NHS (0.3 g), and activated at room temperature in the dark for 30-60 min. The activated NA solution was slowly added dropwise to the chitosan solution under continuous stirring, and the pH of the reaction system was adjusted to 5.8 using NaOH solution. The reaction was continued at 25 °C for 6 h to promote the formation of amide bonds between the carboxyl groups of nervonic acid and the amino groups of chitosan. After the reaction, the resulting reaction solution was placed in a dialysis bag with a molecular weight cutoff of 14 kDa and dialyzed in deionized water for 3 days, with the dialysate being changed periodically to remove unreacted small molecules and residual reagents. Finally, the purified sample was freeze-dried to obtain nervonic acid-modified chitosan hydrogel (NA-CS).

[0043] Synthesis of nervonic acid-modified chitosan hydrogel

[0044] The chemical structure of NA-CS was analyzed using nuclear magnetic resonance (¹H NMR, Av400, Bruker) and Fourier transform infrared spectroscopy (FTIR, AVATAR370, Nicolet). The molecular weight of NA-CS was determined by gel permeation chromatography (GPC, P230) using a gel-OH hybrid column at 40 °C and a flow rate of 1.0 mL / min. The solid NA-CS was dissolved in DMEM / F12 medium. To improve powder solubility, 0.1 M dilute hydrochloric acid was slowly added dropwise with gentle stirring to slightly lower the pH to 6.0-6.5. Solutions with final concentrations of 1%-10% (w / w) were prepared at 50 °C. When the temperature was lowered to <37 °C, a three-dimensional composite hydrogel formed. Tensile tests were performed on the obtained NA-CS hydrogel. The rheological properties of NA-CS hydrogels were determined by oscillation shear tests performed on a rheometer (HAAKE MARS III). The oscillation frequency was 1 Hz, the strain was 1%, and the test temperature was 25℃ or 37℃. The cross-sectional morphology of the freeze-dried hydrogels was observed using a scanning electron microscope (SEM, JEOL JSM 5600LV, Japan).

[0045] 1 ¹H NMR spectroscopy results showed that unmodified chitosan (CS) exhibited a distinct multiplet signal of sugar ring protons (H₂-H₆) in the 3.0-4.0 ppm region, and an angioproton characteristic peak was observed at 4.6-4.8 ppm, consistent with the typical spectroscopic characteristics of the chitosan backbone structure. After modification with nervonic acid (NA), the aforementioned sugar ring and angioproton signals remained clearly present in the NA-CS spectrum, indicating that the chitosan backbone backbone remained intact during grafting. Compared with CS, NA-CS showed new characteristic signals in the low-field and high-field regions: a terminal -CH₃ peak at 0.85 ppm, a broad peak of long-chain -(CH₂)n- in the 1.20-1.35 ppm region, a carboxyl ortho-CO-CH₂- signal at 2.25-2.35 ppm, and a weak olefin proton (C=CH) signal at 5.30-5.40 ppm. These newly added peaks are typical features of the long-chain unsaturated fatty acid structure of NA, indicating that NA has been successfully grafted onto the CS molecular chain via covalent bonding. Figure 1 (A). Fourier transform infrared (FTIR) spectroscopy analysis showed that CS retained the typical vibrational features of the polysaccharide structure. Compared with CS, NA-CS showed vibrational characteristics at approximately 1735 cm⁻¹. -1 The presence of a significantly enhanced C=O stretching vibration peak in the ester group indicates that NA has been successfully grafted onto the CS molecular backbone via esterification. Simultaneously, the amide I region (approximately 1655 cm⁻¹) shows a similar peak. -1The vibrational signals in the amide II region were enhanced; however, no obvious ester characteristic peaks were observed in the HA-CS spectrum. Figure 1 (B). In summary, the successful introduction of long-chain alkyl structures (-CH3 and -(CH2)n-) into the NA molecule has endowed the chitosan molecule, which was originally dominated by a hydrophilic polysaccharide structure, with obvious hydrophobic side chain characteristics.

[0046] The molecular weight distribution of NA-CS was evaluated using gel permeation chromatography. For example... Figure 1 As shown in Figure C, unmodified chitosan exhibits a unimodal molecular weight distribution with a polydispersity index (PDI) of 1.68. After nervonic acid modification, the molecular weight distribution of NA-CS shifted towards the higher molecular weight range, while the distribution also slightly broadened (PDI = 1.90). In summary, the moderate increase in molecular weight and polydispersity index indicates that nervonic acid has been successfully covalently grafted onto the chitosan backbone, and this modification process did not cause significant polymer degradation or disordered crosslinking.

[0047] Figure 1 Rheological testing results from the D-phase showed that all NA-CS hydrogels exhibited a clearly identifiable linear viscoelastic region, within which the storage modulus (G′) remained essentially constant. As the concentration of the three-dimensional composite hydrogel increased from 1% (w / w) to 10% (w / w), G′ increased significantly, indicating a marked increase in gel network stiffness due to increased crosslinking density. Simultaneously, the critical strain (γc, defined as the strain at which G′ begins to decay) gradually decreased with increasing concentration, suggesting that while high-concentration hydrogels possess higher mechanical strength, they are more prone to network structure failure under large deformation conditions. The 2.5% (w / w) NA-CS hydrogel exhibited ideal injectability (G′ ≈ 10). 2 -10 3 Pa, γc ≈ 5-10%, characterized by moderate storage modulus and sufficient critical strain tolerance. Under the same polymer concentration (2.5% (w / w)), the two hydrogels exhibited significantly different gelation behaviors at room temperature. NA-CS rapidly formed a self-supporting gel, while HA-CS hydrogel remained in a near-liquid state, a phenomenon verified by inverted test tube experiments. Figure 1 (E). Scanning electron microscopy (SEM) analysis revealed that NA-CS possesses a well-structured three-dimensional porous network with interconnected pores. Figure 1 (F).

[0048] This invention also compared the properties of two hydrogels: NA-CS (nervonic acid-modified chitosan) and HA-CS (hyaluronic acid-modified chitosan). The synthesis materials and cross-linking mechanisms can be seen from (Table 2 and...). Figure 2NA-CS hydrogels do not introduce additional ionic or highly toxic chemical crosslinking agents. Figure 2 The stability of the network structure of hydrogel A mainly comes from two aspects: first, the covalent amide bond formed between the nervonic acid carboxyl group and the chitosan amino group through EDC / NHS mediation; second, the self-assembly of the long-chain hydrophobic groups of nervonic acid grafted onto the chitosan backbone in an aqueous environment to form hydrophobic association domains, thereby further enhancing the structural stability of the hydrogel and promoting the formation of the gel network. In contrast, the HA-CS hydrogel system mainly relies on the covalent cross-linking between hyaluronic acid and chitosan to form a network structure ( Figure 2 (B), but hyaluronic acid itself does not provide significant hydrophobic interactions or additional non-covalent physical association mechanisms.

[0049] Table 2 Comparison of the synthesis of NA-CS hydrogel and HA-CS hydrogel

[0050] Overall, the NA-CS hydrogel achieves good structural stability and gelation properties through a physical-chemical dual network structure of covalent crosslinking and hydrophobic association, under mild crosslinking conditions. Therefore, this system offers mild crosslinking conditions and low residual toxicity risk, making it more suitable for constructing primary neural cell-related biomedical materials that are highly sensitive to material biosafety and microenvironment stability.

[0051] Example 2: Evaluation of the biocompatibility and safety of hydrogels

[0052] CCK8 assay to detect the activity of SGCs in hydrogels Hyaluronic acid-modified chitosan hydrogels (control group) and nervonic acid-modified chitosan hydrogels, used for three-dimensional culture of SGCs, were cut into uniformly sized pieces and placed in 96-well plates, with one cell-containing hydrogel in each well. Then, 100-200 μL of CCK-8 working solution (complete culture medium containing 10% CCK-8 reagent) was added to each well, and the plates were incubated at 37°C with 5% CO2 for 4-6 hours. After incubation, the absorbance was measured at 450 nm using a microplate reader. Cell viability was calculated using the following formula: Cell viability (%) = [(Experimental group absorbance - Blank hydrogel absorbance) / (Control group absorbance - Blank hydrogel absorbance)] × 100%.

[0053] Live and dead staining to assess cell viability Cell viability was assessed using a live / dead staining kit. Staining solutions were prepared by diluting calcein AM (2 μM) and propidium iodide (PI, 4.5 μM) in 1× Assay Buffer. SGCs were co-cultured in both hydrogels with 0.5 mL of this solution at 37°C for 20 minutes in the dark. The stained samples were washed three times with PBS and observed using a fluorescence microscope (Nikon DS-Vi1 and AZ100). Green fluorescence indicated live cells (Calcein AM), and red fluorescence indicated apoptotic or dead cells (PI).

[0054] Antibacterial test (using Staphylococcus aureus as a model, comparing the antibacterial rates of NA-CS and HA-CS) Staphylococcus aureus was inoculated into liquid culture medium and cultured with shaking at 37°C until the logarithmic growth phase. After centrifugation, the culture was resuspended in sterile PBS and the bacterial concentration was adjusted to approximately 1 × 10⁻⁶. 6 CFU / mL. Incubate 1 mL of bacterial suspension with the pre-sterilized hydrogel sample (37℃, shaking for 6 h). After incubation, vortex thoroughly to mix the system, and perform a 10-fold serial dilution of the supernatant or the entire system. Then prepare solid culture medium, mold it into plates and allow it to solidify. Spread 100 µL of the diluted solution evenly on the plate, using a sterile spatula to spread it horizontally until fully covered, and allow the surface to dry slightly. Pre-sterilize the prepared NA-CS hydrogel dried material sample under UV light, and dilute it to 1% (w / w), 2.5% (w / w), and 5% (w / w), then drop 10 µL onto the plate to form a localized sample area. The control group also used 1% (w / w), 2.5% (w / w), and 5% (w / w) HA-CS hydrogel. Invert the plates and incubate at 37℃ for 18-24 h. After incubation, the number of countable colonies on the plates was counted, and the number of viable bacteria per unit volume (CFU / mL) was calculated. Antibacterial effect is expressed as the colony reduction rate. The formula for calculating the inhibition rate is: Inhibition rate (%) = [1 - (CFU / mL)] 样本 ] / [ CFU 对照 )]× 100%.

[0055] Genotoxicity testing (Ames test) evaluates the mutagenicity of materials. To evaluate the potential genotoxicity of NA-CS and HA-CS, a bacterial reverse mutation assay (Ames assay) was used, designed according to the OECD TG471 guidelines. Salmonella typhimurium strains TA98 and TA100 were selected to detect frameshift and base substitution mutations, respectively. After sterilization, hydrogel samples were extracted in sterile PBS at the surface area / volume ratio (0.2 g / mL) recommended by ISO 10993-12 for 72 hours (37±1℃, horizontal shaker, 50 rpm). The extracted solution was then sterilized using a 0.22 μm filter. Three concentration gradients were set for each sample: stock solution, 1 / 2 dilution, and 1 / 4 dilution, all with a ±S9 metabolic activation system (S9 being liver microsomes).

[0056] The experiment employed the plate incorporation method. 100 μL of bacterial suspension (logarithmic growth phase), 100 μL of NA-CS (or HA-CS) extract, and 500 μL of S9 mixture (+S9 group) or PBS (-S9 group) were mixed with 2 mL of soft agar (containing histidine-biotin mixture, incubated at 45°C) and quickly poured onto preheated minimum glucose agar plates. Each group was replicated in triplicate. Positive controls included 2-aminofluorene (TA98, +S9) and NaN3 (TA100, -S9), while the negative control was PBS. Plates were incubated at 37°C for 48 hours, after which colony counts were performed.

[0057] The number of revertant colonies on each plate was recorded, and the mean and standard deviation for each group were calculated. The criteria for a positive result were: the colony count in the sample group was more than twice that of the negative control, and a dose-response relationship was observed; a slight increase in colony count without statistical significance was considered suspicious; and no significant increase was considered negative.

[0058] ELISA detection of the expression of TNF-α, IL-1β, IL-13 and other factors in culture supernatant NA-CS containing SGCs were placed in multi-well plates containing complete culture medium, allowing the hydrogel to fully impregnate the cells. The supernatant was collected at 24 h, 48 h, and 72 h. The collected culture was centrifuged at 12,000 rpm for 10 min at 4 °C to remove impurities and cell debris, and the supernatant was stored at -80 °C. The concentration of inflammatory factors in the culture medium was detected using an ELISA kit. The standard dilution, sample addition, incubation, washing, color development, and reaction termination procedures were performed according to the manufacturer's instructions. OD values ​​were read at 450 nm. A standard curve was plotted, and the concentration of inflammatory factors (pg / mL) in the samples was calculated. All experiments were performed with ≥3 biological replicates and 3 technical replicates. Statistical analysis was performed using GraphPad Prism 9.

[0059] CCK8 results showed that after 72 hours of continuous observation, the activity of SGCs cultured in NA-CS hydrogel was superior to that in HA-CS hydrogel (**p<0.01). Figure 3 (A). Live / dead staining results confirmed the activity of SGCs in the NA-CS hydrogel, with lower red fluorescence expression levels than those in SGCs cultured in the HA-CS hydrogel. Figure 3 (B) Antibacterial tests showed that NA-CS hydrogels at concentrations of 2.5% (w / w) and 5% (w / w) had higher antibacterial rates than HA-CS hydrogels (*p<0.05). Figure 3 (C). ELISA was used to detect the expression of pro-inflammatory and anti-inflammatory factors in SGCs cultured in two hydrogels. The results showed that the expression levels of pro-inflammatory factors TNF-α and IL-1β were increased in the HA-CS hydrogel group (*p<0.05), while the expression level of anti-inflammatory factor IL-13 was increased in the NA-CS hydrogel group (*p<0.05). Figure 3 (D).

[0060] The Ames test results (Table 3) showed that the number of revertant mutant colonies in the NA-CS group was close to that of the negative control group in all tested strains (TA98 and TA100), at each dose group (undiluted, 1 / 2, and 1 / 4 dilution), and under conditions with and without metabolic activation (±S9), indicating no significant mutation-inducing effect. Furthermore, no obvious dose-response trend was observed among the concentration groups, further suggesting that the NA-CS material does not possess genotoxicity or mutagenic potential. In contrast, under the same experimental conditions, the HA-CS group showed some signs of genotoxicity. Particularly in the TA98 and TA100 strains, the number of revertant mutant colonies in the undiluted and 1 / 2 dilution groups was significantly higher than that in the negative control (p<0.05), showing a preliminary dose-response trend, suggesting that the material may have a mild mutagenic effect. This may be due to residual cross-linking agents used in the HA-CS synthesis process, high surface reactivity of the material, or intermediate products generated by HA itself during metabolic activation; these factors may all cause DNA damage or gene mutations. Therefore, NA-CS showed good genetic safety in the Ames experiment, while HA-CS had a certain risk of mutation and its synthesis process needs to be further optimized to reduce biosafety risks.

[0061] Table 3 Ames Trials (n=3, X - ±S)

[0062] * indicates a significantly higher colony count than the negative control. * (P < 0.05)

[0063] The above results indicate that NA-CS hydrogels exhibit good biocompatibility and safety. At the cellular level, NA-CS significantly improves the survival and viability of SGCs, reduces the expression of pro-inflammatory factors, and enhances the level of anti-inflammatory factors, demonstrating a good ability to regulate the anti-inflammatory microenvironment. At the material level, NA-CS exhibits excellent antibacterial properties, and no mutagenicity was observed in the Ames mutagenicity test, suggesting that it has low toxicity and genetic safety. In contrast, although HA-CS has certain biological activity, it may pose a mild mutagenic risk due to residual cross-linking agents, and its synthesis process needs further optimization to ensure material safety.

[0064] In summary, NA-CS can be used as a low-toxicity hydrogel rich in neurotrophic components for culturing primary neural cells. It not only has good biocompatibility and cell support capabilities, but also has certain anti-inflammatory and antibacterial effects, and has good application prospects in tissue engineering and nerve regeneration.

[0065] Example 3: Dedifferentiation induction and phenotypic verification of satellite glial cells (SGCs)

[0066] Concentration Exploration in Primary and Three-Dimensional Cultures of SGCs C57BL / 6 mice were anesthetized by intraperitoneal injection of 1% sodium pentobarbital (30 mg / kg), then decapitated and their spines were quickly removed. The dorsal root ganglia (DRG) were isolated under a dissecting microscope, the epineurium and fibrous tissue were dissected, and the tissue was minced and placed in poly-L-lysine-coated culture plates containing DRG-SGCs-specific culture medium (the specific formula of DRG-SGCs can be found in CN106635990A or CN110982788A), and cultured at 37°C and 5% CO2.

[0067] After 3 days of culture, the expression of glial fibrillary acidic protein GFAP and glutamine synthase GS was detected by immunofluorescence staining. The results showed that the vast majority of cells were double-positive for both GFAP and GS. Figure 4 (A) indicates that the primary SGCs were successfully cultured.

[0068] Establishment and concentration optimization of three-dimensional culture system The NA-CS powder prepared in Example 1 was dissolved in DMEM / F12 medium. To improve powder solubility, 0.1 M dilute hydrochloric acid was slowly added dropwise with gentle stirring to slightly lower the pH of the system to 6.0–6.5, with the pH value monitored in real time throughout the process. During dissolution, the solution could be heated briefly at 50°C until completely dissolved. After preparing the dissolved hydrogel solution to the desired concentration (1%, 2%, 2.5%, 5%, 10% w / w), it was appropriately mixed with the original PBS (pH 7.4) to restore it to near-neutral pH (approximately 7.2–7.4) and allowed to cool naturally to <37°C to form a gel. Subsequently, primary SGCs were cultured at 5 × 10⁻⁶... 5 Cells were seeded at a density of cells / mL in hydrogels of different concentrations and cultured for 72 hours. During the culture period, the temperature was kept ≤37℃ to ensure cell survival and uniform distribution.

[0069] CCK-8 assays showed that GFAP and GS are markers of satellite glial cell activation, which can characterize DRG-SGCs, indicating successful primary DRG-SGC culture. CCK-8 results showed that SGCs cultured in 2.5% (w / w) NA-CS hydrogel exhibited the best proliferation ability after 72 hours of continuous observation. Figure 4 Since the concentration of B was p < 0.01, this concentration was used in all subsequent experiments.

[0070] Effects of different culture systems on the viability and proliferation of SGCs Experimental Groups ① NA-CS hydrogel group (2.5%): Take 0.025g of dried nervonic acid modified chitosan (NA-CS) and add 1mL of DMEM / F12 medium; ② HA-CS hydrogel group (2.5%): Take 0.025g of dried HA-CS and add 1mL of DMEM / F12 medium; ③ Ordinary two-dimensional culture dish group (DRG-SGCs medium); ④ Two-dimensional culture dish + nervonic acid group (2 µM); ⑤ Two-dimensional culture dish + small molecule compound induction group (Y27632, 616452, CHIR99021, 3.5 µM each).

[0071] The reason why the hydrogel group did not use the DRG-SGCs special medium in the above experiments was to eliminate the interference of the medium and lock the induction effect of the hydrogel. The special medium itself has the effect of promoting growth and differentiation.

[0072] The original SGCs were divided into 5×10 5 Cells were seeded at densities of cells / mL into different experimental groups. After culturing for 72 hours, cell viability was assessed using CCK-8 assay. Cells were also collected, digested with collagenase I / hyaluronidase, and single-cell suspensions were prepared. Cell cycle distribution was analyzed using PI staining combined with flow cytometry.

[0073] Flow cytometry detection of cell surface antigens Add 1 × 10 to each flow cytometry tube 5 Cells (approximately 50 μL in volume) were collected. 50 μL of diluted primary antibody GFAP and CD133 were added to the experimental group, while 50 μL of Staining Buffer was added to the blank control group. After gentle mixing, the cells were incubated for 20 min in the dark on ice. After incubation, the cells were centrifuged at 1000 rpm for 5 min, the supernatant was discarded, and the cells were resuspended in Staining Buffer and washed three times to remove unbound antibodies. Finally, the cells were resuspended in 500 μL of Staining Buffer, filtered through a 40 μm filter, and analyzed by flow cytometry.

[0074] like Figure 5 As shown in Figure A, flow cytometry results indicated that the NA-CS hydrogel group exhibited the best cell viability, with a significant increase in S phase compared to the normal culture dish group (**p<0.01). In contrast, the small molecule compound-induced group showed significant G1 phase arrest, indicating the strong cytotoxicity of the small molecule compound. Figure 5 (B) (**p<0.01).

[0075] Identification of dedifferentiated cell morphology and phenotype Morphological observation After 72 hours of culture, observation under an optical microscope revealed spherical cell aggregates in both the NA-CS group and the small molecule-induced group, but the spherical aggregates in the NA-CS group were more regular in shape and had clearer boundaries. Figure 6 (A)

[0076] Immunofluorescence staining Spherical cells were collected and fixed by soaking in 4% paraformaldehyde solution for 20 min; washed three times with 0.01 mol / L PBS for 10 min each time. Blocked with 5% sheep serum in PBS at room temperature for 1 h; washed three times with 0.01 mol / L PBS for 10 min each time; primary antibodies GFAP, GS, Nestin, and p75NTR (1:500) were added and incubated overnight in a humidified chamber at 4°C. The chamber was removed and allowed to stand for 30 min; washed three times with PBST for 10 min each time; fluorescent secondary antibody (1:500) was added and incubated at room temperature for 2 h (protected from light); washed three times with PBST for 10 min each time; and mounted with DAPI. Images were acquired using an inverted fluorescence microscope.

[0077] The results showed that globular cells highly expressed neural stem / precursor cell markers Nestin and p75NTR ( Figure 6 (B)

[0078] Flow cytometry for surface marker detection Globular cells were digested into single-cell suspensions and stained by flow cytometry using anti-CD133 antibody. Results showed that the cell spheres formed in the NA-CS hydrogel group had more regular morphology, and the CD133 positivity rate (approximately 51%) was higher than that in the HA-CS group (approximately 36%) and the small molecule compound-induced group (approximately 42%). Figure 6 (C)

[0079] Molecular expression profiling analysis qRT-PCR detection Total RNA was extracted from cells in each group, reverse transcribed, and then subjected to real-time quantitative PCR. Primer sequences are shown in SEQ ID NO: 1-SEQ ID NO: 20, as presented in Table 4.

[0080] After discarding the culture medium, add 2 mL of sterile 1× PBS to each cell sample and gently wash once to remove residual culture medium and serum components, then discard the PBS. Next, add 2 mL of 0.25% trypsin-EDTA solution, gently agitate the culture dish to ensure the trypsin fully covers the hydrogel surface, and digest at 37°C for 3-5 min until cells loosen and detach from the hydrogel matrix. Add 4 mL of fetal bovine serum (FBS) to terminate the digestion reaction, and gently pipette repeatedly to fully disperse the cells. Transfer the cell suspension to nuclease-free EP tubes, centrifuge at 1100 rpm for 5 min, discard the supernatant, and collect the cell pellet. Add 500 μL of RNA lysis buffer to the pellet, mix thoroughly with a pipette, and transfer to a 1.5 mL nuclease-free EP tube. Then add 500 μL of RNA diluent and incubate at room temperature for 3-5 min to fully lyse the cells and release RNA. Centrifuge the sample at 12000 rpm for 5 min, aspirate the supernatant and add 0.5 volume of anhydrous ethanol, then pipette 20-25 times to thoroughly mix. Transfer the mixture to an RNA centrifuge column, centrifuge at 12000 rpm for 1 min, and discard the filtrate. Add 50 μL of DNase I incubation solution to the column membrane and incubate at room temperature for 15 min to remove genomic DNA contamination. Then add 600 μL of RNA wash buffer, centrifuge at 12000 rpm for 1 min, and discard the filtrate; repeat the washing once. Transfer the centrifuge column to a new elution tube, add 200 μL of nuclease-free water, incubate at room temperature for 2 min, centrifuge at 12000 rpm for 1 min, and collect the total RNA obtained from the elution.

[0081] The extracted RNA was used for subsequent reverse transcription. Using RNA as a template, a cDNA synthesis reaction system was prepared on ice, gently mixed, and then placed in a PCR amplification instrument for reverse transcription. Subsequently, a qRT-PCR reaction system was prepared, and real-time quantitative PCR amplification was performed to obtain melting curves and Ct values. The gene expression level was analyzed using the 2-ΔΔCt method.

[0082] Table 4 Primer Sequences

[0083] qRT-PCR results showed that the expression levels of stem cell factors Nestin, SOX2, and PAX6 mRNA in spherical cells of the NA-CS hydrogel group were significantly increased by approximately 1.2 times compared to the small molecule compound-induced group. Figure 7(**p<0.01); The expression levels of growth and proliferation support-related factors bFGF, EGF, BDNF, Wnt3a, Shh, and c-Myc mRNA were significantly increased by approximately 1.05-1.25 times compared to the small molecule compound-induced group. Figure 7 (B) (**p<0.01).

[0084] Preliminary Exploration of Transcriptome Sequencing and Mechanisms Total RNA was extracted from the samples using the methods described above. RNA integrity and contamination were assessed by agarose gel electrophoresis, and RNA purity (OD260 / 280 and OD260 / 230) was determined using a NanoPhotometer. RNA library construction was performed using the NEBNext® Ultra™ RNA Library Prep Kit for Illumina®. Fragmented mRNA was used as a template, and double-stranded cDNA was synthesized via reverse transcription. End repair, A-tailing, and adapter ligation were then performed. Fragments of approximately 250-300 bp were screened using AMPure XP beads, amplified and purified by PCR to obtain sequencing libraries, which were then subjected to high-throughput sequencing on the Illumina platform. Quality control was performed on the raw Fastq data, removing adapter sequences, N-containing reads, and low-quality reads to obtain clean reads. These clean reads were aligned to a reference genome, and their distribution in exons, introns, and intergenic regions was analyzed. Gene expression quantification was performed on reads with unique alignments and an alignment quality value >10, and normalization was performed using the FPKM method. The reliability of biological replicates was assessed using sample correlation analysis and principal component analysis (PCA). Differentially expressed genes were visualized using volcano plots and hierarchical clustering. Further KEGG pathway enrichment analysis and protein-protein interaction (PPI) network analysis based on the STRING database were conducted to screen key core genes.

[0085] Total RNA was extracted from spherical cells of the NA-CS group and the small molecule-induced group, and mRNA sequencing was performed. Differential gene analysis identified a total of 3456 differentially expressed genes (1874 upregulated and 1582 downregulated). Figure 8 (A)

[0086] KEGG pathway enrichment analysis showed that, compared with spheroid cells induced by small molecule compounds, differentially expressed genes enriched in spheroid cells induced by NA-CS hydrogel culture in classical pathways related to stem cell stemness, such as the NOTCH signaling pathway and WNT signaling pathway, as well as pathways involved in chemokine signaling, cell cycle, and axonal guidance biology. Figure 8 (B)

[0087] The VENN diagram shows 10 common differentially expressed genes in the NOTCH signaling pathway, WNT signaling pathway, and axonal guidance. Figure 8 The network components (C) are AXIN2, LEF1, JAG1, NKD2, SOST, LZTS2, FZD3, DAAM1, CCND3, and PRKCG. PPI network interaction analysis suggests a potentially strong interaction between AXIN2 and LEF1. Figure 8 (D). CO-IP experiments demonstrate the interaction between AXIN2 and LEF1. Figure 8 In the WNT signaling pathway, AXIN2 and LEF1 are key regulators. Their expression is closely related to and involved in the regulation of stemness homeostasis. The negative feedback regulation of AXIN2 can stabilize β-catenin levels, while LEF1, as a downstream transcription activator, can jointly regulate the expression of stemness genes Nestin, SOX2, and PAX6. The AXIN2 and LEF1 signaling axis may play a core regulatory role in the stemness process of spherical cells after SGC dedifferentiation, and at the same time provide a potential molecular basis for NA-CS hydrogel-induced SGC dedifferentiation and self-renewal.

[0088] In summary, NA-CS hydrogel can efficiently and safely induce SGCs to dedifferentiate into neural stem cell-like cells, forming regular spherical structures and highly expressing stemness markers, without the presence of exogenous transcription factors or small molecule compounds. The induction mechanism is closely related to the activation of the WNT / NOTCH signaling pathway, with Axin2-Lef1 protein interaction likely playing a key regulatory role. Compared to traditional small molecule induction, the NA-CS system exhibits significant advantages in cell viability, morphological regularity, molecular expression, and biosafety. It is an ideal three-dimensional culture system for inducing stemness in SGCs and constructing seed cells for regenerative medicine, showing promising prospects for translation and application.

[0089] The above are merely preferred embodiments of the present invention. The scope of protection of the present invention is not limited to the above embodiments. All technical solutions falling within the scope of the present invention's concept are within the scope of protection of the present invention. It should be noted that for those skilled in the art, any modifications, equivalent substitutions, or improvements made without departing from the principles of the present invention should be considered within the scope of protection of the present invention.

Claims

1. A method of promoting the de-differentiation of satellite glial cells into neural stem cell-like cells, characterized in that, Includes the following steps: Step (1) Construct a three-dimensional composite hydrogel containing nervonic acid-modified chitosan; Step (2) Inoculate satellite glial cells in a three-dimensional composite hydrogel; without introducing exogenous transcription factors or dedifferentiation-promoting small molecule compounds, induce the satellite glial cells to dedifferentiate to obtain a cell population with a neural stem cell-like phenotype.

2. The method for promoting the dedifferentiation of satellite glial cells into neural stem cell-like cells according to claim 1, characterized in that, The preparation method of the three-dimensional composite hydrogel is as follows: S1 dissolves chitosan in acetic acid solution to obtain chitosan solution; S2 dissolves nervonic acid in an organic solvent, adds a carbodiimide crosslinking agent and N-hydroxysuccinimide for light-protected activation, and obtains an activated nervonic acid solution. S3 involves adding the activated nervonic acid solution dropwise to the chitosan solution and mixing, adjusting the pH of the reaction system to 5.5-6.0, and reacting at 20-30℃ for 6-12 hours to allow the carboxyl groups of nervonic acid to form amide bonds with the amino groups of chitosan. S4 purified, dialyzed and freeze-dried the reaction product to obtain nervonic acid-modified chitosan; S5 dissolves nervonic acid-modified chitosan in DMEM / F12 medium to obtain a three-dimensional composite hydrogel with a concentration of 1%-10%.

3. The method for promoting the dedifferentiation of satellite glial cells into neural stem cell-like cells according to claim 2, characterized in that, The carbodiimide crosslinking agent is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and the mass ratio of the carbodiimide crosslinking agent to N-hydroxysuccinimide is 0.3-1.0:0.2-0.6; the organic solvent is dimethyl sulfoxide, and the mass-volume ratio of nervonic acid to dimethyl sulfoxide is 1 g:(5-15) mL.

4. The method for promoting the dedifferentiation of satellite glial cells into neural stem cell-like cells according to claim 2, characterized in that, The mass-to-volume ratio of chitosan to acetic acid is (1.0–2.0) g: 100 mL; the mass-to-volume ratio of chitosan to nervonic acid is 2–5:

1.

5. The method for promoting the dedifferentiation of satellite glial cells into neural stem cell-like cells according to claim 2, characterized in that, The concentration of the three-dimensional composite hydrogel is 2-3%.

6. The method for promoting the dedifferentiation of satellite glial cells into neural stem cell-like cells according to claim 1, characterized in that, The satellite glial cells are inoculated into the three-dimensional composite hydrogel at a density of 1 x 10 5 -5 x 10 6 cells per milliliter of hydrogel, and cultured for 48-120 hours to induce dedifferentiation of the satellite glial cells to obtain cell spheres with a neural stem cell-like phenotype.

7. The method for promoting the dedifferentiation of satellite glial cells into neural stem cell-like cells according to claim 1, characterized in that, The satellite glial cells are derived from primary satellite glial cells of the dorsal root ganglion of the mammalian peripheral nervous system.

8. The application of the three-dimensional composite hydrogel according to claim 2 in inducing satellite glial cell dedifferentiation, constructing neural stem cell-like cells or neural regeneration-related cell models.

9. The neural stem cell-like cells prepared by the method according to any one of claims 1-7 are characterized in that, The cells express neural stem cell-related markers, including Nestin, SOX2, PAX6, and / or CD133; the dedifferentiation process is accompanied by activation of the WNT signaling pathway and / or the NOTCH signaling pathway.

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