Neural stem cells for treating alzheimer's disease and preparation process

Abstract

The present application relates to the technical field of biological medicine, in particular to neural stem cells for treating Alzheimer's disease and a preparation process, the preparation process is prepared through separation process optimization, expansion system strengthening and directional differentiation means innovation, the prepared neural stem cells have the characteristics of high purity, high stemness and high cholinergic differentiation efficiency, and the neural stem cells prepared by the process are expected to provide reliable seed cell sources for cell therapy of AD.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to neural stem cells for the treatment of Alzheimer's disease and their preparation process. Background Technology

[0002] Alzheimer's disease (AD) is a progressive neurodegenerative disease first reported by the German neurologist Alzheimer in 1906. The clinical features of this disease are progressive deficits in cognition and memory. The pathological mechanism of AD is complex, with core characteristics including abnormal aggregation of β-amyloid protein (Aβ) forming senile plaques, hyperphosphorylation of tau protein leading to neurofibrillary tangles (NFTs), accompanied by neuroinflammation, synaptic damage, and neuronal degeneration and death, ultimately resulting in memory loss, cognitive impairment, and loss of daily living abilities.

[0003] Currently, clinical treatment for Alzheimer's disease (AD) primarily relies on symptomatic medications, including cholinesterase inhibitors (such as donepezil and rivastigmine) and N-methyl-D-aspartate (NMDA) receptor antagonists (such as memantine). These drugs, by regulating neurotransmitters (such as acetylcholine) or inhibiting glutamate excitotoxicity, can only provide short-term relief for some symptoms (such as memory and behavioral abnormalities), but they cannot block Aβ deposition, the pathological progression of tau protein, or repair damaged neurons, thus failing to fundamentally reverse the disease progression. Furthermore, while monoclonal antibodies targeting Aβ (such as lencanezumab) can reduce plaque deposition, their efficacy is limited only in the early stages of the disease, and they pose safety concerns such as cerebral microbleeds. Therefore, developing novel therapies that can repair neurological damage and reverse pathological progression is an urgent need in the field of AD treatment.

[0004] Neural stem cells (NSCs) are a type of undifferentiated cell with self-renewal capacity and multi-lineage differentiation potential. They can differentiate into neurons, astrocytes, and oligodendrocytes, and regulate the microenvironment, inhibit inflammatory responses, and promote synaptic regeneration by secreting neurotrophic factors (such as brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF), showing promise for the treatment of Alzheimer's disease. The principle is that although the brain can undergo limited neurogenesis and glial cell development through endogenous NSPCs for repair, these processes decline with age, becoming insufficient to regenerate brain cells and counteract the neurodegenerative changes in AD. Therefore, using exogenous NSPCs to support endogenous NSPCs is an effective strategy for repairing central nervous system damage caused by AD. Studies have found that NSPC transplantation can play an important role by secreting various NFs, including nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and glial cell-derived neurotrophic factor (GDNF).

[0005] NSPC treatment for Alzheimer's disease still faces many problems and challenges. For example, existing NSC preparation processes suffer from problems such as loss of stemness during in vitro expansion and low efficiency of directed differentiation, which limit their clinical application. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention aims to provide a highly active, highly functional neural stem cell that targets AD pathology and its preparation process. By optimizing the isolation, expansion, and differentiation conditions, it solves the problems of low NSC preparation efficiency and insufficient function in the prior art, providing a reliable source of seed cells for AD cell therapy.

[0007] To achieve the above-mentioned technical effects, the present invention adopts the following technical solution: In a first aspect, the present invention provides a process for preparing neural stem cells for treating Alzheimer's disease, comprising the following steps: S1: Primary separation and purification: Human embryonic hippocampal tissue or neural progenitor cells differentiated from induced pluripotent stem cells were obtained, enzymatically digested using a complex enzyme digestion system, and then purified by density gradient centrifugation to obtain primary neural stem cells, i.e., purified neural stem cells. S2: In vitro expansion and stemness maintenance: Purified neural stem cells were seeded into low-adhesion culture plates and expanded using basal medium containing functional enhancement additives to obtain expanded neural stem cells.

[0008] Preferably, the complex enzyme digestion system in step S1 consists of 0.05-0.15% collagenase IV (w / v, g / 100mL), 0.03-0.05% (w / v, g / 100mL) neutral protease, and 0.01-0.03% (w / v, g / 100mL) DNase I. Regarding the dosage, for example, a complex enzyme digestion system containing 0.05-0.15% collagenase IV, w / v, g / 100mL, indicates that each 100mL of the complex digestive enzyme system contains 0.05-0.12% g of collagenase IV.

[0009] Preferably, the enzymatic hydrolysis conditions in step S1 are: digestion at 37°C and 5% CO2 for 25-35 minutes.

[0010] Preferably, the method for obtaining primary neural stem cells in step S1 is as follows: After digestion, the cell suspension was purified by centrifugation with Percoll gradient solution of density 1.05-1.07 g / mL at 800g for 20 minutes. The intermediate layer cells were collected as primary neural stem cells.

[0011] Preferably, the basal culture medium in step S2 is DMEM / F12 culture medium, containing 2% B27 additive, 20 ng / mL EGF and 10 ng / mL bFGF; More preferably, the functional enhancement additive is 2.5 μg / mL CHIR99021 and 10 μg / mL human umbilical cord mesenchymal stem cell exosomes hUC-MSC-Exo.

[0012] Preferably, the amplification culture conditions in S2 are: controlling the inoculation density to be 3 × 10⁻⁶. 4 cells / cm², change half the amount of medium every 3 days.

[0013] Preferably, the diameter of the neurosphere is controlled at 150-200 μm during passaging.

[0014] Secondly, the present invention also provides a cholinergic neuron, which is obtained by three-stage induced differentiation of the neural stem cells for treating Alzheimer's disease provided in the first aspect above. The three-stage induced differentiation specifically includes: (a) Induction phase 1, days 0-7: Neural stem cells were seeded into polylysine-coated culture plates and differentiated using a differentiation medium containing 1-3 μM retinoic acid and 10-20 ng / mL FGF-8, which was DMEM / F12 with 2% N2 additive. (b) Induction phase 2, days 8-14: Add 20-40 ng / mL BDNF and 10-15 ng / mL GDNF to the culture medium; (c) Induction phase 3, days 15-21: Add 5-10 μM taurine to the culture medium.

[0015] Most preferably, the three-stage induced differentiation in S3 specifically comprises: (a) Induction phase 1, days 0-7: Neural stem cells were seeded in polylysine-coated culture plates and differentiated using a differentiation medium containing 2 μM retinoic acid and 15 ng / mL FGF-8, which was DMEM / F12 with 2% N2 additive. (b) Induction phase 1, days 8-14: 30 ng / mL BDNF and 12 ng / mL GDNF were added to the culture medium; (c) Induction phase 1, days 15-21: 6 μM taurine was added to the culture medium.

[0016] Thirdly, the present invention also provides the application of the neural stem cells for treating Alzheimer's disease described in the first aspect or the cholinergic neurons provided in the second aspect in the treatment of Alzheimer's disease.

[0017] Fourthly, the present invention also provides a cell preparation for treating Alzheimer's disease, comprising the neural stem cells for treating Alzheimer's disease described in the first aspect or the cholinergic neurons provided in the second aspect, and a pharmaceutically acceptable carrier.

[0018] Preferably, the concentration of live cells in the cell preparation is ≥1×10⁻⁶. 7 cells / mL.

[0019] Compared with the prior art, the beneficial effects of the present invention are as follows: First, traditional NSC isolation often uses single-enzyme digestion, which easily damages cell surface receptors, leading to reduced viability. Furthermore, the lack of a purification step makes it easy for fibroblasts to contaminate the cells, affecting the purity of subsequent expansion. This invention employs a complex enzyme system of collagenase IV, neutral protease, and DNase I, which can gently dissociate tissues and reduce cell damage. Utilizing the density difference between NSCs and impurity cells, combined with Percoll density gradient centrifugation, the purity and viability of primary cells can be significantly improved, providing high-quality seed cells for subsequent expansion.

[0020] Secondly, NSCs are prone to losing stemness during long-term amplification due to weakened Wnt / β-catenin signaling, generally manifested as decreased nestin expression and increased differentiation tendency. In the technical solution of this invention, the addition of CHIR99021 and hUC-MSC-Ex can maintain a nestin positivity rate of ≥85% in NSCs up to the 10th generation, which is significantly higher than that of traditional culture methods, and there are no obvious karyotype abnormalities, thus significantly improving amplification stability.

[0021] Finally, the significant loss of cholinergic neurons in the hippocampus and basal forebrain in AD patients is a key cause of memory impairment. This invention mimics the temporal regulation of embryonic neural development: in the early stage, RA+FGF-8 activates telencephalon-specific transcription factors, defining the cholinergic lineage; in the middle stage, BDNF+GDNF promotes neuronal axonal growth and synapse formation; in the later stage, taurine enhances the expression of acetylcholine synthase ChAT through receptor activation, which can promote the directed differentiation of NSCs, thus significantly improving the targeted therapeutic potential of NSCs for AD.

[0022] In summary, the neural stem cells for treating Alzheimer's disease provided by this invention and their preparation process, through optimization of the separation process, enhancement of the expansion system, and innovation of directed differentiation methods, produce neural stem cells with high purity, high stemness, and high cholinergic differentiation efficiency, which are expected to provide a reliable source of seed cells for cell therapy of AD. Detailed Implementation

[0023] The following embodiments are only used to more clearly illustrate the technical solutions of the present invention, and are therefore merely examples and should not be used to limit the scope of protection of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Specific conditions not specified in the embodiments are performed according to conventional conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially. Example 1

[0024] The purpose of this embodiment is to provide a neural stem cell for treating Alzheimer's disease and its preparation process. The specific preparation process is as follows: S1: Obtain human embryonic hippocampal tissue (after ethical review), cut it into 1 mm³ pieces, add a compound enzyme digestion solution to completely cover the embryonic hippocampal tissue, and then place it in a 37°C, 5% CO2 incubator for 30 minutes for digestion. After digestion is terminated, centrifuge at 800g for 5 minutes to collect the cell suspension. Add the suspension to a Percoll gradient solution with a density of 1.06 g / mL, centrifuge at 800g for 20 minutes, and collect the intermediate layer cells as primary neural stem cells.

[0025] The complex enzyme digestion solution contains, by mass-volume percentage (g / 100mL), 0.1% collagenase IV, 0.04% neutral protease, and 0.02% DNase I, with collagenase IV activity of 160 U / mg, neutral protease activity of 50 U / mg, and DNase I activity of 2000 Kunitz units / mg.

[0026] S2: The purified neural stem cells were processed at 3×10 4 The cells / cm² were seeded in low-adhesion culture plates and cultured in a basal medium consisting of DMEM / F12 + 2% B27 additive + 20 ng / mL EGF + 10 ng / mL bFGF. The medium was further expanded by adding 2.5 μg / mL CHIR99021 (CAS No.: 252917-06-9) and 10 μg / mL human umbilical cord mesenchymal stem cell exosomes hUC-MSC-Exo. The medium was changed at half volume every 3 days. During passage, the diameter of the neurospheres was controlled at 150-200 μm. The expanded neural stem cells were then collected.

[0027] Furthermore, this embodiment also provides a method for preparing cholinergic neurons, the preparation process of which is as follows: The expanded neural stem cells were seeded into poly-L-lysine-coated culture plates and induced to differentiate in three stages, specifically including: Induction phase 1 (days 0-7) was performed using DMEM / F12+2% N2 medium containing 2 μM retinoic acid and 15 ng / mL FGF-8 (i.e., 2 mL N2 supplement was added to every 100 mL DMEM / F12 basal medium). During induction phase 2 (days 8-14), 30 ng / mL BDNF and 12 ng / mL GDNF were added. During induction phase 3 (days 15-21), 6 μM bovine alkaloids were added.

[0028] Cholinergic neurons were collected after three stages of induced differentiation. Example 2

[0029] The purpose of this embodiment is to provide a neural stem cell for treating Alzheimer's disease and its preparation process. The specific preparation process is as follows: S1: Human embryonic hippocampal tissue (ethically reviewed) was collected, minced to 1 mm³, and then digested with a compound enzyme digestion solution at 37°C and 5% CO2 for 25 minutes. After digestion was terminated, the cell suspension was collected by centrifugation at 800g for 5 minutes, added to a Percoll gradient solution with a density of 1.05 g / mL, and centrifuged at 800g for 20 minutes to collect the intermediate layer cells.

[0030] The complex enzyme digestion solution contains, by mass-volume percentage (g / 100mL), 0.08% collagenase IV, 0.04% neutral protease, and 0.02% DNase I, with collagenase IV activity of 160 U / mg, neutral protease activity of 50 U / mg, and DNase I activity of 2000 Kunitz units / mg.

[0031] S2: Neural stem cells were transported at a rate of 3 × 10 4 The cells / cm² were seeded in low-adhesion culture plates, and the expansion medium was the same as in Example 1. Half of the medium was changed every 3 days. During passage, the diameter of the neurosphere was controlled at 150-200 μm, and the expanded neural stem cells were collected.

[0032] Furthermore, this embodiment also provides a method for preparing cholinergic neurons, the preparation process of which is as follows: Referring to Example 1, the expanded neural stem cells were subjected to a three-stage induction differentiation process, specifically as follows: Induction phase 1 (days 0-7): DMEM / F12+2% N2 medium containing 1 μM RA and 10 ng / mL FGF-8 was used; Induction phase 2 (days 8-14): Add 20 ng / mL BDNF and 10 ng / mL GDNF; Induction phase 3 (days 15-21): Add 5 μM bovine alkaloid.

[0033] Cholinergic neurons were collected after three stages of induced differentiation. Example 3

[0034] The purpose of this embodiment is to provide a neural stem cell for treating Alzheimer's disease and its preparation process. The specific preparation process is as follows: S1: Human embryonic hippocampal tissue (ethically reviewed) was collected, minced to 1 mm³, and then digested with a compound enzyme digestion solution at 37°C and 5% CO2 for 35 minutes. After digestion was terminated, the cell suspension was collected by centrifugation at 800g for 5 minutes, added to a Percoll gradient solution with a density of 1.07 g / mL, and centrifuged at 800g for 20 minutes. The intermediate layer cells were collected as primary cells.

[0035] The complex enzyme digestion solution contains, by mass-volume percentage (g / 100mL), 0.12% collagenase IV, 0.03% neutral protease, and 0.03% DNase I, with collagenase IV activity of 160 U / mg, neutral protease activity of 50 U / mg, and DNase I activity of 2000 Kunitz units / mg.

[0036] S2: The expansion culture conditions are the same as in Example 1, and the expanded neural stem cells are collected.

[0037] Furthermore, this embodiment also provides a method for preparing cholinergic neurons, the preparation process of which is as follows: Referring to Example 1, the expanded neural stem cells were subjected to a three-stage induction differentiation process, specifically as follows: Induction phase 1 (days 0-7): DMEM / F12+2% N2 medium containing 3 μM RA and 20 ng / mL FGF-8 was used; Induction phase 2 (days 8-14): Add 40 ng / mL BDNF and 15 ng / mL GDNF; Induction phase 3 (days 15-21): Add 10 μM bovine alkaloid.

[0038] Cholinergic neurons were collected after three stages of induced differentiation. Comparative Example 1

[0039] The purpose of this comparative study was to verify the synergistic effect of CHIR99021 and hUC-MSC-Exo on the stemness maintenance capacity and proliferation efficiency of neural stem cells (NSCs). The experimental design and results are as follows: 1.1 Experimental Methods: Primary neural stem cells from the same batch as in Example 1 (unless otherwise specified, all subsequent comparative examples use the same source batch as in Example 1) were selected and divided into 3 groups for expansion culture (passaged to the 10th generation) to form controls with Examples 1-3. Other conditions (seeding density 3×10⁶) were also used. 4 The cell / cm² ratio and basal culture medium (DMEM / F12+2% B27+20 ng / mL LEGGF+10 ng / mL bFGF) were consistent with Example 1, with only the functional enhancement additives adjusted, including the following groups: Control group 1: without CHIR99021 and hUC-MSC-Exo (basal culture medium only); Control group 2: Based on the basal culture medium, only the raw material CHIR99021 was added, and the amount added was 12.5 μg / mL (without hUC-MSC-Exo). Control group 3: Based on the basal culture medium, only the raw material hUC-MSC-Exo was added, and the amount added was 12.5 μg / mL (without CHIR99021 added). 1.2 Detection Indicators and Significance: The detection indicators include nestin positivity rate, cell proliferation rate, and apoptosis rate.

[0040] Among them, the nestin positivity rate was detected by flow cytometry (APC-labeled nestin antibody). Nestin is a specific marker for neural stem cells, and the higher the positivity rate, the more complete the stem cell preservation. The cell proliferation fold was calculated by the CCK-8 assay to determine the total proliferation fold from generation 1 to generation 10, reflecting the in vitro expansion efficiency of neural stem cells. The higher the proliferation fold, the stronger the process economy. The apoptosis rate was detected by Annexin V / PI double staining flow cytometry, reflecting the cell viability. The lower the apoptosis rate, the less cell loss during the expansion process.

[0041] 1.3 The experimental results are shown in Table 1: Table 1. Differences in stem cell stemness and proliferation efficiency among the groups (n=3) Control group 1 (no additives) 58.12±1.72 4.18±0.21 17.93±0.79 Control group 2 (CHIR only) 71.92±1.75 6.05±0.13 12.11±0.63 Control group 3 (Exo only) 74.87±1.54 6.46±0.14 9.89±0.42 Example 1 88.93±0.41 8.29±0.21 4.98±0.15 Example 2 86.23±0.37 8.11±0.16 5.26±0.23 Example 3 85.51±0.39 7.89±0.22 5.74±0.57 1.3 Results Analysis: The above experimental results show that when only one component (CHIR99021 or hUC-MSC-Exo) is added, the nestin positivity rate and proliferation rate are significantly improved compared with the group without addition, but still inferior to the technical effects of Examples 1-3; while the combined addition group (Examples 1-3) has the highest nestin positivity rate and proliferation rate and the lowest apoptosis rate, indicating that CHIR99021 and hUC-MSC-Exo have a synergistic effect.

[0042] Furthermore, this comparative study further discussed the effect of the ratio balance of CHIR99021 to hUC-MSC-Exo on the stemness maintenance capacity and proliferation efficiency of neural stem cells (NSCs). The experimental design and results are briefly described below: Primary neural stem cells from the same batch as in Example 1 were selected and divided into two groups for expansion culture (passaged to the 10th generation) to form a control group with Example 1. All other conditions (seeding density 3×10⁴ cells / cm², basal culture medium DMEM / F12+2%B27+20ng / mLEGF+10ng / mLbFGF) were consistent with Example 1, only the ratio of CHIR99021 to hUC-MSC-Exo was adjusted. The specific groups are shown in Table 2. Table 2 Group Settings Experimental Group A 1.25 11.25 1:9 Experimental Group B 5 7.5 2:3 Experimental group C 3.75 8.75 3:7 Experimental group D 6.25 6.25 1:1 Similar to section 1.2 above, the detection indicators included nestin positivity rate, cell proliferation rate, and apoptosis rate. The experimental results are shown in Table 3: Table 3. Nestin positivity rate, cell proliferation rate, and apoptosis rate (n=3) Example 1 88.93±0.41 8.29±0.21 4.98±0.15 Experimental Group A 79.8 ± 1.0* 7.12 ± 0.19* 7.85 ± 0.42* Experimental Group B 74.6 ± 1.2* 6.51 ± 0.18* 10.10 ± 0.55* Experimental group C 76.5 ± 1.1* 6.83 ± 0.17* 9.42 ± 0.48* Experimental group D 70.3 ± 1.5* 5.97 ± 0.16* 12.30 ± 0.60* *Note: Compared with Example 1 group, *p < 0.05 (one-way ANOVA + Tukey test) The above experimental results show that, under the premise of a constant total dose (the total amount of CHIR99021 and hUC-MSC-Exo added is 12.5 μg / mL), deviations from the CHIR99021:hUC-MSC-Exo ratio of 1:4 to some extent lead to a significant decrease in the maintenance of neural stem cell stemness, proliferation efficiency, and survival rate. On the one hand, when the proportion of CHIR99021 is too low (experimental group A, 1:9), the Wnt / β-catenin signaling is insufficient, the nestin positivity rate drops to 79.8%, the proliferation fold decreases to 7.12-fold, and the apoptosis rate increases to 7.85%. On the other hand, when the proportion of CHIR99021 is too high (experimental group B, 2:3 or experimental group D, 1:1), overactivation occurs, leading to a tendency for premature differentiation. The nestin positivity rate is as low as 70.3%, the proliferation fold decreases to 5.97-fold, and the apoptosis rate further increases to 12.30%. Even with only minor adjustments to the ratio (experimental group C, 3:7), stemness maintenance (76.5%) and proliferation (6.83-fold) failed to recover to the levels of the positive control. In summary, the 1:4 mass ratio of CHIR99021 to hUC-MSC-Exo is within a narrow "optimal window". Comparative Example 2

[0043] The purpose of this comparative study was to verify the key role of adding 30 ng / mL BDNF + 12 ng / mL GDNF in cholinergic neuron differentiation efficiency and glial cell inhibition during phase 2 (days 8-14). The experimental design and results are as follows: 2.1 Experimental Methods: Neural stem cells expanded from the same batch as in Example 1 (nestin positivity rate of 87.03% after 10 passages) were selected and divided into 3 groups for three-stage differentiation culture (stage 1 and stage 3 conditions were fixed), with only the BDNF and GDNF addition levels adjusted in stage 2 (days 8-14). The group settings are as follows: Control group 1: Phase 2 without the addition of BDNF and GDNF; Control group 2: Phase 2 supplemented with low doses (15 ng / mL BDNF + 6 ng / mL GDNF). Control group 3: Phase 2 was supplemented with a low dose (30 ng / mL BDNF + 24 ng / mL GDNF). 2.2 Detection indicators and their significance: The detection indicators include ChAT positivity rate, GFAP positivity rate, and synaptic density.

[0044] The ChAT positivity rate, detected by immunofluorescence (FITC-labeled ChAT antibody), reflects the differentiation efficiency of cholinergic neurons; the GFAP positivity rate, detected by immunofluorescence (Cy3-labeled GFAP antibody), reflects the differentiation ratio of glial cells. ChAT is acetylcholine synthase, a specific marker for cholinergic neurons; a higher positivity rate indicates more complete differentiation. GFAP is a specific marker for glial cells; a lower GFAP ratio indicates higher cell purity.

[0045] 2.3 The experimental results are shown in Table 4: Table 4. Comparison of differentiation efficiency of neural stem cells into cholinergic neurons and inhibition of glial cells among different groups (n=3) Control group 1 45.08±1.45 27.89±1.22 Control group 2 61.92±1.44 17.64±0.78 Control group 3 63.15±1.02 19.16±1.01 Example 1 77.64±1.61 7.98±0.29 Example 2 75.12±2.03 8.23±0.32 Example 3 78.35±2.19 8.41±0.30 2.4 Results Analysis BDNF and GDNF, as neurotrophic factors, can promote neuronal survival and inhibit glial cell differentiation. In control group 1, which received no BDNF, neural stem cells tended towards glial cell differentiation, and cholinergic neurons showed low maturity. In control group 2, although low doses were added, nutritional support was insufficient. In contrast, the experimental groups (Examples 1-3) showed significantly higher ChAT positivity rates and significantly lower GFAP positivity rates than the control groups. We hypothesize that excessively high concentrations of BDNF and GDNF may lead to endocytosis or downregulation of their receptors (such as TrkB), triggering signal desensitization and thus weakening their role in promoting neuronal differentiation and survival. Comparative Example 3

[0046] The purpose of this comparative study was to verify the regulatory effect of adding 2 μM RA during the induction phase 1 (days 0-7) on the directional differentiation and forebrain specificity of cholinergic lineages during the preparation of cholinergic neurons. The experimental design and results are as follows: 3.1 Experimental Methods: Neural stem cells from the same batch (87.03% nestin positivity rate after 10 passages) were selected and divided into 3 groups for three-stage differentiation culture (stages 2 and 3 were kept under fixed conditions). Only the concentration of RA added in stage 1 (days 0-7) was adjusted. The specific groupings are as follows: Control group 1: No RA in phase 1 (0 μM); Control group 2: Phase 1 low-dose RA (1 μM); Control group 3: Phase 1 high-dose RA (3 μM).

[0047] 3.2 Detection indicators and their significance: The detection indicators include the positive rates of ChAT, Nkx2.1, Foxg1, and GFAP.

[0048] Among them, the ChAT positivity rate was detected by immunofluorescence (FITC-labeled ChAT antibody) to reflect the differentiation efficiency of cholinergic neurons; the Nkx2.1 positivity rate was detected by immunofluorescence (Alexa 647-labeled Nkx2.1 antibody) to reflect the induction efficiency of telencephalon cholinergic neuron precursors (Nkx2.1 is a specific marker of telencephalon cholinergic precursors, and the higher the positivity rate, the more abundant the precursors); the Foxg1 positivity rate was detected by immunofluorescence (Cy5-labeled Foxg1 antibody) to reflect the degree of forebrain-specific differentiation (Foxg1 is a forebrain-specific transcription factor, and the higher the positivity rate, the more defined the forebrain differentiation); and the GFAP positivity rate was detected by immunofluorescence (Cy3-labeled GFAP antibody) to reflect the differentiation ratio of glial cells (the lower the positivity rate, the higher the cell purity).

[0049] 3.3 The experimental results are shown in Table 5: Table 5. Effects of neural stem cells on the directed differentiation of cholinergic lineages in each group (n=3) Control group 1 38.21±1.51 11.89±0.97 54.93±2.39 24.87±1.26 Control group 2 64.78±1.72 34.65±1.28 77.82±2.36 14.91±0.61 Control group 3 69.85±1.45 27.93±1.14 61.74±1.65 17.89±0.97 Example 1 77.41±0.71 41.53±1.84 84.72±1.49 7.71±0.34 Example 2 79.05±0.83 42.01±1.29 83.13±1.75 8.06±0.44 Example 3 78.42±0.89 43.39±1.20 85.37±1.71 8.24±0.31 3.4 Results Analysis: RA is a key factor regulating the regional differentiation of neural progenitor cells. In the RA-free group (control group 1), due to the lack of inducing signals, cholinergic differentiation was minimal and showed weak forebrain specificity. While low-dose RA (1 μM) could partially induce telencephalon precursors, the differentiation efficiency did not reach its peak. High-dose RA (3 μM) over-activated RA signals, leading to a reduction in telencephalon precursors and inducing glial differentiation. In contrast, the experimental groups (Examples 1-3) showed significantly higher ChAT, Nkx2.1, and Foxg1 positivity rates than the control group, and significantly lower GFAP positivity rates, indicating that "2 μM RA" is the optimal dose for inducing telencephalon cholinergic precursors and inhibiting glial differentiation. Comparative Example 4

[0050] The purpose of this comparative study is to verify the promoting effect of adding 6 μM taurine during the induction phase 3 (days 15-21) on the functional maturation of cholinergic neurons during the preparation of cholinergic neurons.

[0051] 4.1 Experimental Methods: Cholinergic neurons from the same batch (ChAT positivity rate 77.89% after stage 1-2 culture) were selected and divided into 3 groups for stage 3 culture (days 15-21) (stage 1-2 conditions were fixed), with only the concentration of bovine taurine added being adjusted. The experimental design and results are as follows: Control group 1: Phase 3 without bovine alkaloids (0 μM); Control group 2: Phase 3 low-dose bovine syringin (3 μM); Control group 3: Phase 3 high-dose bovine syringin (9 μM); 4.2 Detection indicators and their significance: The detection indicators include acetylcholine secretion, nAChR expression, and cell viability.

[0052] Among them, the acetylcholine secretion level was detected by ELISA to measure the concentration of acetylcholine in the culture medium, which reflects the functional maturity of cholinergic neurons. The higher the acetylcholine secretion level, the more complete the neuronal function. The nAChR expression level was detected by Western blotting (β-actin as internal control), which reflects the activation degree of nicotinic acetylcholine receptors. Cell viability was detected by trypan blue staining, which reflects the toxic effect of taurine on cells.

[0053] 4.3 The experimental results are shown in Table 6: Table 6. Effects of botrytis cinerea on the function of cholinergic neurons in neural stem cells. Control group 1 4.18±0.22 0.29±0.02 94.87±0.95 Control group 2 7.79±0.35 0.58±0.03 93.92±0.97 Control group 3 8.06±0.32 0.49±0.02 78.91±1.68 Example 1 10.37±0.44 0.76±0.02 91.64±0.79 Example 2 10.51±0.30 0.79±0.03 92.03±0.94 Example 3 10.58±0.47 0.81±0.03 92.21±0.82 4.4 Analysis of Experimental Results: Boehmite promotes the functional maturation of cholinergic neurons by activating nicotinic acetylcholine receptors (nAChR). In the boehmite-free group (control group 1), the acetylcholine secretion was only 4.18 pmol / well due to unactivated receptors; although the low dose (3 μM) could partially activate the receptors, the secretion did not reach the peak; the high dose (9 μM) caused feedback downregulation due to overactivation of the receptors, and boehmite itself has neurotoxicity, resulting in a significant decrease in cell viability; while the experimental groups (Examples 1-3) had significantly higher acetylcholine secretion and nAChR expression levels than the control group, and the cell viability was considerable.

[0054] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the present invention, and all such modifications and substitutions should be covered within the scope of the claims of the present invention. Technical aspects, shapes, and structures not described in detail in this invention are all well-known technologies.

Claims

1. A process for preparing neural stem cells for treating Alzheimer's disease, characterized in that, Includes the following steps: S1: Primary separation and purification: The neural progenitor cells were enzymatically digested using a complex enzyme digestion system, and then purified by density gradient centrifugation to obtain primary neural stem cells, i.e., purified neural stem cells. S2: In vitro expansion and stemness maintenance: Purified neural stem cells were seeded into low-adhesion culture plates and expanded in basal medium containing functional enhancement additives to obtain expanded neural stem cells. The functional enhancement additives are 2-3 μg / mL CHIR99021 and 8-12 μg / mL human umbilical cord mesenchymal stem cell exosomes.

2. The preparation process of neural stem cells for treating Alzheimer's disease as described in claim 1, characterized in that: The complex enzyme digestion system described in step S1 consists of 0.05-0.15% collagenase IV, 0.03-0.05% neutral protease and 0.01-0.03% DNase I.

3. The preparation process of neural stem cells for treating Alzheimer's disease as described in claim 1, characterized in that: The conditions for enzymatic hydrolysis in step S1 are: digestion at 37°C and 5% CO2 for 25-35 minutes.

4. The preparation process of neural stem cells for treating Alzheimer's disease as described in claim 1, characterized in that: The method for obtaining primary neural stem cells in S1 is as follows: after digestion, the cell suspension is purified by centrifugation with Percoll gradient solution of density 1.05-1.07 g / mL. The centrifugation parameters are 800g and 20 minutes. The intermediate layer cells are collected as primary neural stem cells.

5. The preparation process of neural stem cells for treating Alzheimer's disease as described in claim 1, characterized in that: The amplification culture conditions in S2 are as follows: the inoculation density is controlled at 3 × 10⁻⁶. 4 cells / cm 2 Change half the medium every 3 days, and control the diameter of the neurospheres to 150-200 μm during passage. Alternatively, the functional enhancement additive may be 2.5 μg / mL CHIR99021 and 10 μg / mL human umbilical cord mesenchymal stem cell exosomes.

6. A neural stem cell for treating Alzheimer's disease, prepared by the preparation process according to any one of claims 1-5.

7. A cholinergic neuron, characterized in that, It is obtained by three-stage induced differentiation of neural stem cells for treating Alzheimer's disease as described in claim 6, wherein the three-stage induced differentiation specifically includes: (a) Induction phase 1, days 0-7: Neural stem cells were seeded in polylysine-coated culture plates and differentiated in a medium containing 1-3 μM retinoic acid and 10-20 ng / mL FGF-8. (b) Induction phase 2, days 8-14: Add 20-40 ng / mL BDNF and 10-15 ng / mL GDNF to the culture medium; (c) Induction phase 3, days 15-21: Add 5-10 μM taurine to the culture medium.

8. The use of the neural stem cells for treating Alzheimer's disease as described in claim 6 or the cholinergic neurons as described in claim 7 in the preparation of an agent for treating Alzheimer's disease.