Safe and efficient non-gene editing neural stem cell and application thereof in treatment of nervous system related diseases

Abstract

The invention belongs to the technical field of biological medicine, and discloses a safe and efficient non-gene editing neural stem cell and application thereof in treatment of nervous system related diseases. The invention provides a non-gene-edited neural stem cell (hCiPSC-NSCs) derived from human induced pluripotent stem cells, the neural stem cell is obtained by reprogramming human umbilical cord mesenchymal stem cells through a small molecule compound (such as a TGF-beta / GSK-3beta inhibitor), exogenous gene introduction is not involved, and the neural stem cell has a normal karyotype, no tumorigenicity and relatively high safety; and the efficient implantation of the non-tumorigenicity hippocampal region is realized in three months. The neural stem cell can improve the autism-like phenotype of a VPA-exposed rat by targeting the intestinal-brain axis at the same time.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to safe and efficient non-gene-edited neural stem cells and their application in the treatment of nervous system-related diseases. Background Technology

[0002] Autism Spectrum Disorder (ASD) is a complex neurodevelopmental disorder with a global prevalence of 1.5-3% in children. Its characteristic social deficits, repetitive behaviors, and cognitive impairments still lack a cure. Current stem cell therapies have significant limitations: while mesenchymal stem cells (MSCs) can modulate peripheral inflammation, they cannot penetrate the blood-brain barrier and have no effect on improving hippocampal microglia activation and synaptic ultrastructural damage. Clinical data show that they only reduce repetitive behaviors but fail to restore social abilities (no statistically significant difference in interaction time between unfamiliar mice in a three-compartment experiment). While gene-edited neural stem cells (NSCs) have potential for neural repair, they face serious safety risks—CRISPR-Cas9-induced NSCs have a tumor incidence rate as high as 22% in animal models, and viral vector-transduced iPSC-NSCs suffer from genomic instability. Single-target therapies have also yielded limited efficacy: neuroinflammation inhibitors (such as anti-IL-6 antibodies) can reduce intracranial inflammation but have no regulatory effect on gut microbiota dysbiosis (the firmicutes / bacteroidota ratio remains unchanged); probiotic interventions (such as lactobacillus) increase bacteroidota abundance but cannot repair synapses across the blood-brain barrier (hippocampal synaptic vesicle density remains unchanged) and have limited improvement on core behavioral symptoms. Therefore, there is an urgent need in this field for a safe, efficient treatment approach that can simultaneously target the dual pathological mechanisms of the brain-gut axis—one that avoids the tumorigenic risks of gene editing while overcoming the limitations of single-pathway drug delivery, ultimately achieving a synergistic effect of neural synaptic repair and gut microbiota remodeling. Summary of the Invention

[0003] This invention aims to address at least one of the technical problems existing in the prior art. It provides a non-gene-edited human chemically induced pluripotent stem cell-derived neural stem cells (hCiPSC-NSCs) that are highly safe and can simultaneously improve neuroinflammation (inhibiting microglia activation and increasing anti-inflammatory factors), synaptic damage (increasing synaptic vesicle density in the hippocampal CA1 region), and gut microbiota dysbiosis (reducing Firmicutes / Bacteroidota and increasing Alloprevotella abundance), and synergistically regulate the brain-gut axis, as well as its application in the preparation of drugs for neurological diseases such as ASD.

[0004] The first aspect of this invention aims to provide a neural stem cell. The second aspect aims to provide the application of the neural stem cells of the first aspect in the preparation of a medicament for treating nervous system-related diseases. The third aspect aims to provide a medicament. The fourth aspect aims to provide a method for treating autism spectrum disorder.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0006] In a first aspect, the present invention provides a neural stem cell (i.e., hCiPSC-NSCs) obtained by induced differentiation of human chemically induced pluripotent stem cells.

[0007] In some embodiments of the present invention, the human chemically induced pluripotent stem cells are obtained by chemical reprogramming mesenchymal stem cells; the chemical reprogramming includes the following steps: mesenchymal stem cells are sequentially induced and cultured using a first-stage culture medium, a second-stage culture medium, a third-stage culture medium, and a fourth-stage culture medium. In some embodiments of the present invention, the human chemically induced pluripotent stem cells are obtained by chemical reprogramming mesenchymal stem cells; the chemical reprogramming includes the following steps: mesenchymal stem cells are sequentially induced and cultured using a first-stage culture medium, a second-stage culture medium, a third-stage culture medium, and a fourth-stage culture medium, cultured for 10-12 days, 10-12 days, 6-9 days, and 6-8 days, respectively. In some embodiments of the present invention, the first-stage culture medium includes KnockOutDMEM, knockout serum substitute, N2 additive, B27 additive, GlutaMax, NEAA, 2-mercaptoethanol, L-ascorbic acid 2-phosphate, LiCl, nicotinamide, AlbuMax-II, GSK3β inhibitor, TGFβR inhibitor, RAR activator, and VEGFR / PDGFR inhibitor. In some embodiments of the present invention, the two-segment culture medium includes KnockOutDMEM, KSR, N2 additive, B27 additive, GlutaMax, NEAA, 2-mercaptoethanol, Vc2p, LiCl, NAM, bFGF, GSK3β inhibitor, TGFβR inhibitor, RAR activator, C-jun N-terminal kinase inhibitor, MAO inhibitor, Smoothened receptor agonist, and DNA methyltransferase inhibitor. In some embodiments of the present invention, the three-segment culture medium includes KnockOutDMEM, N2 additive, B27 additive, GlutaMax, NEAA, 2-mercaptoethanol, Vc2p, AlbuMax-II, recombinant human heregulinβ-1, GSK3β inhibitor, TGFβR inhibitor, ROCK inhibitor, MEK1 / 2 inhibitor, MAO inhibitor, DOT1L histone methyltransferase inhibitor, and HDAC inhibitor. In some embodiments of the present invention, the four-segment culture medium includes KnockOutDMEM, N2 additive, B27 additive, GlutaMax, NEAA, 2-mercaptoethanol, Vc2p, recombinant human heregulinβ-1, GSK3β inhibitor, MEK1 / 2 inhibitor, ROCK inhibitor, and HDAC inhibitor.

[0008] In some embodiments of the present invention, the induced differentiation includes the following step: culturing human chemically induced pluripotent stem cells in N2B27 medium. In some embodiments of the present invention, the N2B27 medium is composed of a 1:1 volume mixture of N2 medium and B27 medium. In some embodiments of the present invention, the N2 medium includes N2 supplement, DMEM / F-12, non-essential amino acids (NEAA), GlutaMAX, insulin, L-glutamine, and 2-mercaptoethanol. In some embodiments of the present invention, the B27 medium comprises Neurobasal medium, B27 supplement, a TGF-β receptor inhibitor (such as SB431542), and dorsomorphin. In some embodiments of the present invention, the induced differentiation further includes the following steps: when neural rosettes appear in the cells during the induced differentiation process, basic fibroblast growth factor (BFGF) (final concentration of BFGF is 1-3 μg / mL) is added to B27 medium, and the cells are cultured for another 2-3 days; the cells are washed, digested into single cells, and neural stem cells can be obtained by continuing to use B27 medium.

[0009] A second aspect of the present invention provides the use of neural stem cells of the first aspect of the present invention in the preparation of medicaments for treating nervous system-related diseases.

[0010] In some embodiments of the present invention, the neurological-related diseases are gut-brain axis-related neurological-related diseases. In some embodiments of the present invention, the neurological-related diseases include at least one of Alzheimer's disease, autism spectrum disorder, vascular dementia, Parkinson's disease, amyotrophic lateral sclerosis, multiple sclerosis, autoimmune encephalitis, meningitis, epilepsy, migraine, fibromyalgia, spinal muscular atrophy, mitochondrial encephalomyopathy, depression, anxiety, schizophrenia, attention deficit hyperactivity disorder, and Cushing's syndrome; preferably, autism spectrum disorder.

[0011] A third aspect of the present invention provides a medicament comprising neural stem cells according to the first aspect of the present invention.

[0012] In some embodiments of the present invention, the drug further includes a combination drug for treating neurological diseases. In some embodiments of the present invention, the combination drug includes exosomes. In some embodiments of the present invention, the exosomes are mesenchymal stem cell exosomes. In some embodiments of the present invention, the exosomes are prepared by the following method: umbilical cord mesenchymal stem cells are cultured in conditioned medium, and the cell culture supernatant is centrifuged using differential ultracentrifugation. In some embodiments of the present invention, the differential ultracentrifugation includes the following steps: centrifugation at 300–350 × g for 8–12 minutes to remove residual cells; followed by centrifugation at 2,000–2,500 × g for 8–12 minutes to remove cell debris; then centrifugation at 10,000–15,000 × g for 20–35 minutes to precipitate microvesicles. The clarified supernatant is filtered through a polyether sulfone (PES) membrane and finally ultracentrifuged at 100,000–150,000 × g for 1–3 hours. In some embodiments of the present invention, the drug comprises a number of 1 × 10⁻⁶ live neural stem cells. 5 ~1×10 8 cells / mL or 1×10 5 ~1×10 8 per g.

[0013] In some embodiments of the present invention, the drug is a pediatric or adult dosage form. In some embodiments of the present invention, the drug is a gastrointestinal or non-gastrointestinal dosage form. In some embodiments of the present invention, the gastrointestinal dosage form includes at least one of powders, tablets, granules, capsules, sustained-release formulations, solutions, dry suspensions, effervescent tablets, emulsions, suspensions, syrups, drops, and chewable tablets. In some embodiments of the present invention, the non-gastrointestinal dosage form includes at least one of injectable, respiratory, transdermal, mucosal, and cavity dosage forms. In some embodiments of the present invention, the composition is an oral or injectable formulation. In some embodiments of the present invention, the composition is any one of powders, granules, tablets, capsules, gels, suspensions, drops, pills, injections, suppositories, aerosols, oral liquids, ointments, emulsions, and irrigants.

[0014] In some embodiments of the present invention, the group of drugs further includes excipients. In some embodiments of the present invention, the excipients further include at least one selected from fillers, diluents, disintegrants, binders, lubricants, flow aids, surfactants, solvents, flavoring agents, and preservatives.

[0015] In some embodiments of the present invention, the drug is used as follows: intravenous injection of the neural stem cells; and, in the later stages of treatment, enhanced injection of the neural stem cells into the ventricles, while simultaneously administering exosomes via nasal cavity. In some preferred embodiments of the present invention, the drug is used as follows: intravenous injection of the neural stem cells, 1×10⁻⁶ per dose. 6 ~9×10 6 One dose per week for 4-5 weeks; during the final treatment, the aforementioned neural stem cells are injected intravenously (using the anterior fontanelle as a reference coordinate system (anterior fontanelle -0.8mm, lateral to the midline ±1.6mm, subdural -3.8mm)) at a dose of 2-8 × 10⁶. 5 Cells / μL, and exosomes (100-200 μg exosomes) were administered intranasally.

[0016] In some embodiments of the present invention, the neurological-related diseases include at least one of Alzheimer's disease, autism spectrum disorder, vascular dementia, Parkinson's disease, amyotrophic lateral sclerosis, multiple sclerosis, autoimmune encephalitis, meningitis, epilepsy, migraine, fibromyalgia, spinal muscular atrophy, mitochondrial encephalomyopathy, depression, anxiety, schizophrenia, attention deficit hyperactivity disorder, Cushing's syndrome, and persistent postural-perceptual dizziness; preferably autism spectrum disorder.

[0017] In some embodiments of the present invention, the drug prevents and / or treats nervous system diseases through at least one of the following pathways: inhibiting neuroinflammation, inhibiting microglia activation, repairing synaptic ultrastructure, and regulating gut microbiota.

[0018] In some embodiments of the present invention, the inhibition of neuroinflammation includes downregulating the expression of pro-inflammatory cytokines such as IL-1β, IL-6, and TNF-α in the hippocampus and prefrontal cortex, and upregulating the expression of the anti-inflammatory cytokine IL-10 in the hippocampus and prefrontal cortex; increasing the level of oxidative stress in the prefrontal cortex, such as increasing the activity of CAT, GSH-Px, GSH, and SOD, and decreasing the content of MDA, T-NOS, and NO, thereby enhancing the body's antioxidant defense capacity and reducing oxidative damage. The inhibition of microglia activation includes reducing the level of Iba1 protein in the CA1 region and prefrontal cortex, as well as reducing the level of Iba1 in the hippocampus. + Microglial cell density. The repair of synaptic ultrastructure includes increasing the density of synaptic vesicles in the CA1 region of the hippocampus. The regulation of gut microbiota includes reducing the Firmicutes / Bacteroidota ratio, increasing the relative abundance of Alloprevotella, and decreasing the relative abundance of Desulfovibrionales.

[0019] A fourth aspect of the present invention provides a method for treating autism spectrum disorder, comprising administering to a subject neural stem cells of the first aspect of the present invention or a drug of the third aspect of the present invention for treatment.

[0020] In some embodiments of the present invention, the neural stem cells and / or drugs are administered at least once a week, each time at the same or different doses. In some embodiments of the present invention, the route of administration includes at least one of oral administration, sublingual administration, nasal administration, rectal administration, inhalation administration, transdermal administration, intraperitoneal injection, subcutaneous injection, intradermal injection, intramuscular injection, intravenous injection, and intraventricular injection. In some embodiments of the present invention, the route of administration is: intravenous injection: 1×10 6 ~9×10 6 Cells / time, once a week for 4 weeks; Intraventricular injection: 1×10 5 ~8×10 5 Cells / side, administered 4 weeks after intravenous administration.

[0021] The beneficial effects of this invention are:

[0022] This invention provides non-gene-edited human induced pluripotent stem cell neural stem cells (hCiPSC-NSCs) obtained by reprogramming human umbilical cord mesenchymal stem cells with small molecule compounds (such as TGF-β / GSK-3β inhibitors). This process does not involve the introduction of exogenous genes, exhibits a normal karyotype, is non-tumorigenic, and demonstrates high safety. Highly efficient, non-tumorigenic hippocampal implantation was achieved at 3 months. These neural stem cells can simultaneously improve autism-like phenotypes in VPA-exposed rats by targeting the gut-brain axis. Behavioralally, treatment significantly improves social interaction, reduces repetitive behaviors, and enhances spatial memory. Mechanistically, these improvements are associated with reduced neuroinflammation, synaptic ultrastructural repair, and gut microbiota balance. Crucially, microbiota restoration drives oxidative stress relief, indicating that the transplanted neural stem cells and the gut ecosystem communicate paracrine signaling through immunomodulatory factors. Attached Figure Description

[0023] Figure 1Characterization of the pluripotency and neuronal differentiation potential of hCiPSCs (human induced pluripotent stem cells). A shows immunofluorescence detection of hCiPSC pluripotency genes (including NANOG, OCT4, and SOX2). B shows RT-qPCR detection of hCiPSC pluripotency genes. C shows the morphology of hCiPSCs under a microscope. D shows HE staining analysis of hCiPSCs as teratomas. E shows chromosome karyotype and G-banding analysis, indicating that hCiPSCs have a normal karyotype. F shows immunofluorescence detection of the hCiPSC-derived neural stem cell markers PAX6 and NESTIN. G shows flow cytometry detection of the hCiPSC-derived neural stem cell markers PAX6 and NESTIN. H shows a typical neural rosette structure. I shows the expression levels of the neural stem cell markers PAX6 and NESTIN (day 16) detected by RT-qPCR (n=3). Compared to the hCiPSCs stage, the expression of neural stem cell markers is increased in the NSCs stage. J shows immunofluorescence staining of neurons differentiated from hCiPSCs-NSCs, displaying markers of mature cortical neurons (TUJ1, MAP2). Cell nuclei are stained with DAPI. K shows patch-clamp detection of neuronal electrophysiological function. L shows histological section analysis of myocardium, liver, spleen, lung, kidney, brain, stomach, small intestine, pancreas, and muscle. Scale bar = 50 μm.

[0024] Figure 2 This study analyzed the open field test and repetitive stereotyped behaviors in SD rats. A represents the number of times the rats crossed the central region. B represents the distance traveled through the central region. C represents the time spent in the central region. D represents the time spent grooming. E represents the number of glass beads buried. F represents the number of times the rats stood upright. G represents the representative open field trajectory of the control group. H represents the representative open field trajectory of the ASD model group. I represents the representative open field trajectory of the hCiPSC-NSCs treatment group. Data are presented as mean ± standard deviation (SD). (n=12) indicates significance. Statistical significance: Compared with the VPA group, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0025] Figure 3This study analyzed the social behavior of Sprague-Dawley rats in three compartments. A represents the social competence stage indicators: time spent in compartment 1 with a stranger, time spent in an empty compartment, time spent interacting with a stranger rat, and time spent interacting with objects. B represents the social novelty preference stage indicators: time spent in compartment 1 with a stranger, time spent in compartment 2 with a stranger, time spent interacting with a stranger rat, and time spent interacting with a stranger rat. C represents a representative heatmap of the social competence stage (0–10 minutes) in the ASD model group. D represents a representative movement trajectory diagram of the social competence stage (0–10 minutes) in the control group. E represents a representative movement trajectory diagram of the social competence stage (0–10 minutes) in the hCiPSC-NSCs treatment group. F represents a representative movement trajectory diagram of the social novelty stage (10–20 minutes) in the ASD model group. G represents a representative movement trajectory diagram of the social novelty stage (10–20 minutes) in the control group. H represents a representative movement trajectory diagram of the social novelty stage (10–20 minutes) in the hCiPSC-NSCs treatment group. Data are expressed as mean ± standard deviation (n = 12). Compared with the VPA group, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0026] Figure 4 This study presents a behavioral analysis of the Morris water maze. A represents the time spent in the target quadrant during the exploration test. B represents the total swimming distance (motor function control). C represents the escape latency during the acquisition phase. D represents the number of times the platform was traversed during the exploration test. E represents the distribution of time spent in each quadrant during the exploration test. F represents the representative acquisition phase movement path: VPA group. G represents the representative acquisition phase movement path: control group. H represents the representative acquisition phase movement path: hCiPSC-NSCs treatment group. I represents the representative exploration test movement trajectory: VPA group. J represents the representative exploration test movement trajectory: control group. K represents the representative exploration test movement trajectory: hCiPSC-NSCs treatment group. Data are expressed as mean ± standard deviation (n = 12). Compared with the VPA group, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

[0027] Figure 5This study analyzed the levels of neuroinflammation and oxidative stress in the prefrontal cortex. A represents IL-1β level. B represents IL-6 level. C represents TNF-α level. D represents IL-10 level. E represents GSH-Px (glutathione peroxidase) activity. F represents GSH (glutathione) content. G represents SOD (superoxide dismutase) activity. H represents MDA (malondialdehyde) content. I represents T-NOS (total nitric oxide synthase) activity. J represents NO (nitric oxide) content. K represents CAT (catalase) activity. Data are expressed as mean ± standard deviation (n = 12). Compared with the VPA group, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

[0028] Figure 6 This study analyzed the activation status of microglia in the prefrontal cortex and CA1 region of the hippocampus in rats from the control, VPA, and treatment groups. A–C represent the CA1 region of the hippocampus in the control group, showing resting microglia with a branched morphology and low Iba1 expression. D–F represent the CA1 region of the hippocampus in the VPA group, showing activated microglia characterized by enlarged cell bodies, thickened processes, and strong Iba1 immunoreactivity. G represents quantitative Iba1 fluorescence intensity (any unit, AU), confirming that the VPA group showed significantly stronger microglia activation than the control group, and that the activation status returned to normal after treatment (P<0.0001 compared to the VPA group). H–J represent the CA1 region of the hippocampus in the hCiPSC-NSCs treatment group, showing decreased microglia activation. K–M represent the prefrontal cortex of the control group, showing resting microglia with weak Iba1 signal. N represents the quantitative fluorescence of Iba1 in the cortex, showing significantly enhanced activation in the VPA group (P<0.0001 compared to the control group), and weakened activation after intervention (P<0.0001 compared to the VPA group). O–Q represent the prefrontal cortex of the VPA group, showing strong Iba1 expression, indicating pro-inflammatory activation. R–T represent the prefrontal cortex of the treatment group, showing decreased Iba1 immunoreactivity. Data are expressed as mean ± standard deviation (n = 6 biological replicates); intensity analysis was performed using ImageJ software. Immunofluorescence: Iba1 (green), DAPI nuclear counterstaining (blue), scale bar: 50 μm (consistent across all panels).

[0029] Figure 7The ultrastructural pathological changes and repair of neurons in the PFC and CA1 region of the hippocampus are shown in the following figures: A: Mitochondrial ultrastructure in the control group: regular cristae structure and intact membrane. B: Mitochondrial pathological changes in the VPA group: disordered cristae structure and membrane rupture. C: Mitochondrial repair in the hCiPSC-NSCs treatment group: ultrastructure returned to normal. D: Synaptic ultrastructure in the control group: narrow synaptic cleft, dense postsynaptic dense area (PSD), and synaptic vesicle aggregation. E: Synaptic pathological changes in the VPA group: widened synaptic cleft, blurred membrane structure, and reduced vesicles. F: Synaptic repair in the hCiPSC-NSCs treatment group: cleft structure reorganization and vesicle re-aggregation. G: Synaptic vesicle density in the CA1 region of the hippocampus at PND60: significantly decreased after VPA exposure (compared to the control group); partially recovered after hCiPSC-NSCs treatment (compared to the VPA group). H represents the density of PND60 cerebral cortical synaptic vesicles: VPA exposure led to decreased density (compared to the control group); hCiPSC-NSCs treatment mediated density recovery (compared to the VPA group). Representative images are from n=3 independent experiments, with quantitative analysis of synaptic vesicles from 30 nerve endings per animal; data are expressed as mean ± standard error (mean ± SEM); statistical comparisons: *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0030] Figure 8 This study analyzed the composition and diversity of gut microbiota in different experimental groups. Figures A through E show stacked bar charts illustrating the species composition distribution at the class (A), family (B), genus (C), order (D), and phylum (E) levels in the VPA-induced ASD rat model group, normal control group, and hCiPSCs-NSCs treatment group. Taxonomic units with a relative abundance of less than 1% were categorized as "Others." Figure F shows a Venn diagram illustrating shared and unique operational taxonomic units (OTUs) among the groups. Data analysis was performed using the WekemoBioincloud platform.

[0031] Figure 9 Heatmap analysis of gut microbiota composition in different experimental groups. A through D are hierarchical clustering heatmaps showing the relative abundance patterns of the VPA-induced ASD rat model group (VPA), the normal control group (Control), and the hCiPSCs-NSCs treatment group (hCiPSCs) at the phylum (D), order (C), family (A), and genus (B) levels. Darker colors represent higher abundance.

[0032] Figure 10This study presents a hierarchical analysis of gut microbiota changes in autism model rats and stem cell-treated rats. A represents a family-level phylogenetic clade: red / green branches indicate enriched / depleted taxa in the VPA group (autism model group) relative to the control group. B represents the family-level LDA score: bar length represents the effect size (log10 transformed value) of significantly different bacterial families. C represents the genus-level phylogenetic clade: phylogenetic distribution of enriched (red) and depleted (green) genera. D represents the genus-level LDA score: key genera driving inter-group differences. E represents the phylum-level phylogenetic clade: extensive taxonomic variations between groups. F represents the phylum-level LDA score: major phylum-level changes quantified by effect size.

[0033] Figure 11 Comparative analysis of gut microbiota dysbiosis using volcano plots (DESeq2). In this analysis, A represents genus-level differences (hCiPSCs-NSCs treatment group vs. control group); B represents phylum-level differences (hCiPSCs-NSCs treatment group vs. control group); C represents genus-level differences (hCiPSCs-NSCs treatment group vs. VPA group); D represents phylum-level differences (hCiPSCs-NSCs treatment group vs. VPA model group); E represents genus-level differences (control group vs. VPA group); and F represents phylum-level differences (control group vs. VPA group).

[0034] Figure 12 The figures represent the α-diversity indices of the gut microbiota in the experimental groups. Box plots A through E show the Chao1 index (Species Richness) (A), FaithPD index (Phylogenetic Diversity) (B), Observed Features (Richness) (C), Shannon index (Evenness) (D), and Simpson index (Dominance) (E) for the control group, hCiPSCs-NSCs treatment group, and VPA-induced ASD group. Higher Chao1, FaithPD, Observed Features, and Shannon values ​​indicate higher diversity; lower Simpson values ​​indicate decreased dominance. F shows the dilution curve (based on the Shannon index) illustrating the change in species richness with sequencing depth. The control group reached a plateau at higher values, indicating sustained high species richness.

[0035] Figure 13This study analyzed the β-diversity of gut microbiota among the experimental groups. A represents the Bray-Curtis dissimilarity analysis between the control group and the hCiPSCs-NSCs treatment group. B represents the Bray-Curtis dissimilarity comparison between the VPA group and the hCiPSCs-NSCs treatment group. C–D represent unweighted UniFrac analyses, showing differences based on species presence / absence. E–F represent weighted UniFrac analyses, including species abundance information. Box plots show the interquartile range (boxes) and median (inner box lines) of differences between groups; statistical evaluation was performed using ANOSIM (analysis of similarity).

[0036] Figure 14 Analysis of gut microbiota composition in different experimental groups. A is a phylogenetic tree heatmap with species identifiers (phylogenetic_tree_heatmap_ID). B is a phylogenetic tree heatmap (phylogenetic_tree_heatmap).

[0037] Figure 15 This section presents the functional and taxonomic analysis of the gut microbiota in the experimental group. A represents the bar chart for the primary classification (L1) of the KEGG pathway. B represents the bar chart for the secondary classification (L2) of the KEGG pathway. C represents the bar chart for the tertiary classification (L3) of the KEGG pathway. D represents the bar chart for the MetaCyc pathway. Detailed Implementation

[0038] The following will describe the concept and technical effects of the present invention clearly and completely with reference to the embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all of them. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall be followed. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0039] KnockOutDMEM was purchased from Gibco, catalog number 10829018; knockout serum substitute (KSR) was purchased from Gibco, catalog number 10828028; N2 additive was purchased from Gibco, catalog number 17502-048; B27 additive was purchased from Gibco, catalog number 17504-044; L-ascorbic acid 2-phosphate (Vc2P) was purchased from Sigma-Aldrich, catalog number A8960; LiCl was purchased from Sigma-Aldrich. The following were purchased: ich (product number L4408); nicotinamide (NAM) from Sigma-Aldrich (product number 72340); AlbuMax-II from Gibco (product number 11021045); bFGF from Origene (product number TP750002); recombinant human heregulinβ-1 (HRG) from PeproTech (product number 100-03); and Neurobasal medium from Gibco (product number 21103049). All experimental protocols involved in this invention were approved by the Animal Ethics Committee (IACUC) of the Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences (Approval No.: IACUC-2022-0063; CAS Ethics No.: IACUC:2023081). Experiments were conducted in an AAALAC-certified SPF-level facility, strictly following the ARRIVE guidelines (Perciedu Sert et al., 2020) and the Guide for the Care and Use of Laboratory Animals (National Academies Press, 8th edition, 2011). Euthanasia and anesthesia protocols complied with the 2020 American Veterinary Medical Association (AVMA) Guidelines for Euthanasia of Animals. Prohibited methods (such as chloral hydrate, ether, or chloroform) were not used. Every effort was made to minimize animal suffering, including the use of appropriate analgesia protocols and humane endpoints. Data from this invention were analyzed using GraphPad Prism software, and results are expressed as mean ± standard deviation (SD) or standard error (SEM). One-way ANOVA was used for comparisons between groups, followed by Tukey's post-hoc test. Statistical significance was defined as *P < 0.05. The features and performance of the present invention are further described in detail below with reference to embodiments.

[0040] Example 1: Preparation of human chemically induced pluripotent stem cells

[0041] This embodiment provides a preparation method for directly inducing human umbilical cord mesenchymal stem cells (hUC-MSCs) into human chemically induced pluripotent stem cells (hCiPSCs) using small molecule compounds, including the following steps:

[0042] (1) Induce hUC-MSCs in the first stage (for 12 days)

[0043] Take out the hUC-MSCs target cells (WHELAB (primary agent), product number: FC1181) from the incubator, remove the original culture medium, and add a culture medium (KnockOut DMEM, 15% KnockOut Serum Replacement, 2% N2 supplement, 3% B27 supplement, 15% FBS, 2% GlutaMax, 2% NEAA, 0.1 mM 2-mercaptoethanol, 50 μg / mL L-ascorbic acid 2-phosphate, 5 mM LiCl, 1 mM nicotinamide, 2 mg / mL AlbuMax-II, 30 μM TGFβR inhibitor (SB-431542, STEMCELL TECHNOLOGIES (distributor), product number: HY-10431), 35 μM GSK3β inhibitor (CHIR99021, brand: HARVEYBIO / China Haiwei (primary agent), product number: CHIR99021), 6 μM RAR activator (CD2314, brand: MedChemExpress (MCE), product number: HY-108533-10 mg), 3 μM VEGFR / PDGFR inhibitor (SU6668, brand: Macklin / Maclean, product number: SU6668)). Add 1 mL of the culture medium to each well of a 12-well plate, mix well, and place it in a 37°C carbon dioxide incubator; change the medium every 4 days, and continuously observe the cell deformation during the process. Usually, after about 4 days of induction, monolayer epithelial-like cells begin to appear. Continue to induce until the confluence of monolayer epithelial cells approaches 100%, and end the first stage of induction.

[0044] (2) Induce in the second stage (for 12 days)

[0045] Remove cells from the incubator after the first stage of induction, discard the original culture medium, and add the second stage culture medium (KnockOut DMEM, 15% KSR, 2% N2 additive, 3% B27 additive, 2% GlutaMax, 2% NEAA, 0.1mM 2-mercaptoethanol, 50μg / mL VLC2p, 5mM LiCl, 2mM NAM, 50ng / mL bFGF, 40μM MSK3β inhibitor (CHIR99021), 35μM MGFβR inhibitor (SB-431542), 6μM MRAR activator (CD2314), 3μM MC-jun N-terminal kinase inhibitor (SP600125, brand: Sellec). k, catalog number: SP600125), 35 μM MAO inhibitor (MAO-A, brand: SAB (first-level agent), catalog number: MAO-A), 1.5 μM M smoothed receptor agonist (SAG, brand: HARVEYBIO / Huazhong Haiwei, catalog number: SAG), 30 μM DNA methyltransferase inhibitor (VPA, brand: Selleck (distributor), catalog number: Valproicacid)). Add 1 mL of culture medium to each well of a 12-well plate, mix well, and place in a 37°C CO2 incubator. Change the medium every 4 days. Usually, after about 4 days of induction, multilayer cell clones will appear. Continue culturing until a large number of multilayer clones are produced, and end the second stage of induction.

[0046] (3) Third stage induction (lasting 8 days)

[0047] Cells that had completed the second stage of induction were removed from the incubator, the original culture medium was removed, and three-stage culture medium was added (KnockOutDMEM, 2% N2 additive, 3% B27 additive, 3% GlutaMax, 2% NEAA, 0.1mM 2-mercaptoethanol, 50μg / mL VLC2p, 5mg / mL AlbuMax-II, 20ng / mL recombinant human heregulin β-1 (HRG), 3μM MSK3β inhibitor (CHIR99021), 30μM MGFβR inhibitor (SB-431542), and 35μM ROCK inhibitor (Y27632, CAS:129830-38-2). Add 1 mL of culture medium to each well of a 12-well plate (3 μM MEK1 / 2 inhibitor (CI-1040, brand: MedChemExpress (MCE), catalog number: HY-50295-10mg), 30 μM MAO inhibitor (MAO-A), 0.6 μM MOT1L histone methyltransferase inhibitor (SGC0946, brand: TargetMol / Taoshu, catalog number: T3082-50mgSGC0946), and 1500 μM HDAC inhibitor (BRD3308, brand: Cayman, catalog number: 34875). Mix well and place in a 37°C CO2 incubator. Change the medium every 4 days.

[0048] (4) Fourth stage induction (lasting 6-8 days)

[0049] Remove the cells from the incubator after the second stage of induction, remove the original culture medium, and add four-stage culture medium (KnockOutDMEM, 2% N2 additive, 3% B27 additive, 3% GlutaMax, 2% NEAA, 0.1mM 2-mercaptoethanol, 50μg / mL VLC2p, 20ng / mL recombinant human heregulin β-1 (HRG) 3μM MSK3β inhibitor (CHIR99021), 3μM MEK1 / 2 inhibitor (CI-1040), 1500μM MDAC inhibitor (BRD3308, brand: Cayman, catalog number: 34875), 30μM ROCK inhibitor (Y27632)). Add 1mL of culture medium to each well of a 12-well plate, mix well, and place in a 37℃ CO2 incubator. Change the medium every 4 days (generally, smooth, raised pluripotent stem cell clones can be observed in about 6-8 days).

[0050] (5) Single-cell passage establishment

[0051] 1) Prepare the substrate-coated culture plates: This process needs to be completed one day in advance. Prepare the Laminin-521-coated plates according to the instructions (Laminin-521 extracellular matrix purchased from Stemcell Technologies, catalog number 77004); thaw one 1mL vial of Laminin-521 (100μg) at 4°C; dilute Laminin-521 with calcium and magnesium at a ratio of 1:40, i.e., add 1mL of Laminin-521 to 39mL PBS (Dulbecco's Phosphate-Buffered Saline). After mixing, add 700μL to each well of a 12-well plate (add the same volume to other wells, resulting in a final mass of 1.75μg per well). Incubate overnight at 37°C in a CO2 incubator. After coating, use directly or seal and store at 4°C (store at 4°C for no more than one week).

[0052] 2) Single-cell replate culture: Remove cells (the cells obtained in step (4)) from the incubator, observe them under a microscope, select wells with good condition and relatively many clones, add 0.5 mL / well of basal medium DF12, and wash once; add 0.5 mL of preheated 37℃ Accutase digestive enzyme to each well of a 12-well plate, and place in a 37℃ CO2 incubator for 5-8 minutes; add an equal volume of basal medium DF12, and repeatedly pipette until there are no obvious clumps; collect the cell suspension, centrifuge at 400g for 5 minutes; carefully aspirate the supernatant, resuspend the cells in four-segment culture medium, and seed them onto coated Laminin521 plates; depending on the number of cells and clones, usually according to 1*10 5 Inoculate at a suitable density; shake well, and incubate in a 37°C CO2 incubator, changing the medium every three days. Obvious clones can usually be observed after 3-6 days; typically, after 6 days, when the clones have grown slightly, the medium can be replaced with mTESR. TM Continue culturing in Plus medium for 6 days, changing the medium every 2 days. When the clone grows to a relatively large size, usually greater than 80 μm in diameter, the next step can be carried out.

[0053] 3) Selecting single clones to establish cell lines: Remove cells from the incubator and observe the clones under a microscope. Mark the single clones to be selected with a marker pen. The selection criteria are: well-formed, dense, clearly defined, undifferentiated homogeneous clones, with a size of 8–150 μm. Choose clones that are as far apart as possible for easier handling. Place the stereomicroscope in a biosafety cabinet beforehand and sterilize it with ultraviolet light for 30 minutes. Prepare the inoculation culture medium: mTESR TMAdd Y27632 (CAS: 129830-38-2) to a final concentration of 10 μm in the Plus medium; aspirate the original medium, add 0.5 mL of DF12 basal medium, wash once to remove residual dead cells, etc.; add 0.3 mL / well (12-well plate) of digestion buffer ReleSR (STEMCELL Technologies 05872 ReLeSR). TM Cell digestion solution (catalog number 05872), shake well, digest at room temperature for 2 minutes; aspirate the digestion solution, digest at 37°C for 2-3 minutes; under a stereomicroscope, carefully scrape off the clones with a small pipette tip or glass needle, aspirate with 20 μL of inoculation culture medium, and disperse into small clones; aspirate the coating medium from the substrate-coated culture plate, add inoculation mTeSRPlus (catalog number 100-0276), 0.5 mL per 1 / 24 well; inoculate each small clone piece into the substrate-coated culture plate, usually one clone per 1 / 24 well; usually pick 10-20 clones, but it is possible that the final total number of clones is less than 10; after picking, resuspend all remaining clones in inoculation mTeSRPlus, take 1 mL (1*10) 5 Inoculate onto a coated plate, mix and culture for future clone picking; shake well and place in an incubator; the next day, observe clone adhesion; usually, almost all clones adhere well; change the medium, aspirate the liquid, and add fresh mTESR. TM Plus culture medium; change the medium every 1-2 days according to the growth rate and condition of the cells until the clones grow large enough to be passaged to obtain hCiPSCs.

[0054] The pluripotency and neuronal differentiation potential of hCiPSCs were characterized using RT-qPCR, flow cytometry, karyotype analysis, and teratoma formation assays, as follows: RT-qPCR: Total RNA was extracted using Trizol reagent and purified by chloroform extraction and isopropanol precipitation. Reverse transcription was performed to generate cDNA. Quantitative PCR amplification was performed on a real-time quantitative PCR system using gene-specific primers and SYBR Green-based PCR premix, with fluorescence monitored during thermal cycling. GAPDH was used as an internal control gene, and the pluripotency and neuronal differentiation potential of hCiPSCs were characterized by RT-qPCR, flow cytometry, karyotype analysis, and teratoma formation assays. -ΔΔCTThe relative gene expression levels were calculated using the following primers: hGapdh: forward primer (F): 5'-ACCCACTCCTCCACCTTTG-3' (SEQ ID NO:1), reverse primer (R): 5'-CTGTTGCTGTAGCCAAATTC-3' (SEQ ID NO:2); hNanog: F: 5'-TGAACCTCAGCTACAAACAG-3' (SEQ ID NO:3), R: 5'-TGGTGGTAGGAAGAGTAAAG-3' (SEQ ID NO:4); hOct4: F: 5'-CCTCACTTCACTGCACTGTA-3' (SEQ ID NO:5), R: 5'-CAGGTTTTCTTTCCCTAGCT-3' (SEQ ID NO:6); hSox2: F: 5'-CCCAGCAGACTTCACATGT-3' (SEQ ID NO:7), R: 5'-CCTCCCATTTCCCTCGTTTT-3' (SEQ ID NO:6). NO: 8); hPax6: F: 5'-TTGCTTGGGAAAATCCGAG-3' (SEQ ID NO: 9), R: 5'-TGCCCGTTCAACATCCTT-3' (SEQ ID NO: 10); hNestin: F: 5'-CCACCCTGCAAAGGGAATCT-3' (SEQ ID NO: 11), R: 5'-GGTGAGCTTGGGCACAAAAG-3' (SEQ ID NO:12).

[0055] Flow cytometry analysis: Cells were washed twice with cold PBS and resuspended in staining buffer (PBS containing 1% FBS). The cell suspension was incubated with antibodies conjugated to fluorescent dyes (CD34, CD45) at 4°C in the dark for 30 minutes. After washing, cells were fixed with 4% paraformaldehyde (at room temperature for 15 minutes). Samples were analyzed using a BDFACSCalibur flow cytometer, with a single-stain compensation control. Data were processed using FlowJo software.

[0056] Karyotype analysis: Cells at 80%–90% confluence were treated with 20 μg / mL colchicine for 2 hours to arrest metaphase. After trypsin digestion and centrifugation, the cell pellet was resuspended in hypotonic solution (37°C, 30 min), fixed with methanol:glacial acetic acid (3:1), and dropped onto pre-chilled slides. The air-dried slides were stained with 1% Giemsa stain for 10 min. Karyotype analysis was performed under a microscope, and images were recorded.

[0057] Teratoma formation experiment: 1×10 6One hCiPSC (dissolved in 100 μL PBS: Matrigel mixture, 1:1 ratio) was subcutaneously injected into the dorsal inguinal region of 6-week-old SCID mice. Tumors were excised 7–8 weeks later, fixed with 4% paraformaldehyde, and embedded in paraffin. Tissues from ectoderm, mesoderm, and endoderm lineages were identified by hematoxylin-eosin (H&E) staining. All procedures were performed according to IACUC-approved protocols.

[0058] Immunofluorescence staining: Cell slide preparation: In a biosafety cabinet, sterilize round glass coverslips by flame and place them in a 24-well plate. Spread 300 μL of L Atrigel on each coverslip, then seed cells to achieve a confluence density of 60-80%. Cell fixation: Gently wash cells twice with 500 μL DPBS per well, then fix with 250 μL of 4% paraformaldehyde at room temperature (RT) for 20 minutes. Washing: Discard the fixative and wash cells three times with 500 μL DPBS per well (5 minutes each time). Permeabilization and blocking: Add 250 μL of permeabilization / blocking buffer (DPBS containing 0.2% Triton X-100, 5% goat serum, and 1% bovine serum albumin (BSA)) to each well and incubate at room temperature for 45 minutes. Primary antibody incubation: After removing the buffer, add the primary antibody (dilution ratios see Table 1) and incubate overnight at 4°C. Use a humidified chamber to reduce evaporation. Secondary antibody washing: Discard the primary antibody and wash the cells three times with PBS (8 minutes each time). Secondary antibody incubation: Add species-matched secondary antibody (diluted according to manufacturer's instructions) and incubate at room temperature in the dark for 1 hour. Discard unbound antibody and wash three times with PBS (8 minutes each time). Nuclear staining: Add 40 μL DAI solution (2 μg / mL) to each well, incubate at room temperature for 2 minutes, then wash twice with PBS (5 minutes each time). Mounting: Place a coverslip (cell side down) on a slide, add a drop of 90% glycerol, and immediately image and observe under a fluorescence microscope.

[0059] Table 1 Antibody Information and Dilution Factors

[0060] Antibody name Brand + Item Number Dilution ratio Rabbitanti-PAX6 Sigma-Aldrich+SAB5700837 1:400 Mouseanti-Nestin Sigma-Aldrich+ZMS1022 1:400 Mouseanti-MAP2 Sigma-Aldrich+ZMS1013 1:400 Anti-BetaIII Tubulin-488 Millipore+ab15708A4 1:400 Rabbitanti-Sox2 Sigma-Aldrich+SAB3500363 1:400 Rabbitanti-OCT4 Sigma-Aldrich+SAB5700848 1:400 Rabbitanti-Nanog Beyotime+AF1912 1:400 Mouseanti-IBA1 Sigma-Aldrich+SAB2702364 1:400 Anti-Mouse-488 HUABIO+HA1125 1:500 Anti-Rabbit-594 HUABIO+HA1122 1:500 Anti-Mouse-594 HUABIO+HA1126 1:500 Anti-Rabbit-488 HUABIO+HA1121 1:500

[0061] To obtain a safe, reliable, and clinically transplantable neural stem cell that is not genetically edited, the inventors first induced human umbilical cord mesenchymal stem cells using small molecule compounds, successfully preparing safe, reliable, and unmodified hCiPSCs (human chemically induced pluripotent stem cells). First, the morphology of hCiPSCs was observed using an inverted microscope, revealing a high degree of similarity to human embryonic stem cells. Figure 1(C). Given the genetic similarity between hCiPSCs and embryonic stem cells, the inventors used immunofluorescence (IF) and RT-qPCR techniques to detect the expression of pluripotency genes in hCiPSCs. Immunofluorescence results showed that hCiPSCs highly expressed NANOG, OCT4, and SOX2 (…). Figure 1 (A); RT-qPCR results further confirmed that the expression levels of pluripotency genes NANOG, SOX2 and OCT4 in hCiPSCs were significantly increased (P<0.0001); Figure 1 These results collectively demonstrate that hCiPSCs possess pluripotency characteristics similar to embryonic stem cells. To verify the similarity in differentiation potential between hCiPSCs and embryonic stem cells, the inventors evaluated the in vivo differentiation capacity of hCiPSCs through a teratoma formation experiment. Approximately 8 weeks after injecting hCiPSCs into the subcutaneous tissue of the mouse axilla or hind leg muscle tissue, tumor masses were observed to form. Hematoxylin-eosin (H&E) staining analysis revealed that these masses contained representative structures from three germ layers: endoderm-like epithelial structures, mesodermal chondrocytes, and ectoderm neural rosette-like structures. Figure 1 (D). This result confirms that hCiPSCs possess differentiation potential comparable to embryonic stem cells. Furthermore, the inventors performed karyotype analysis on hCiPSCs, and the results showed that the reprogrammed hCiPSCs maintained a normal karyotype (D). Figure 1 (E).

[0062] Example 2: Induction of hCiPSCs cell line into neural stem cells

[0063] This embodiment provides a method for inducing hCiPSCs cell lines to differentiate into neural stem cells, specifically including the following steps:

[0064] (1) Take hCiPSCs with a growth density of 90% from a 6-well plate (Example 1), digest with EDTA for 5-8 minutes, passage 1:1 into a 12-well plate pre-coated with Matrigel (5 μL / mL, Corning, New York, USA), add 1 μL LY-27632, and after 12 hours, replace with normal mTeSR1 medium (Stemcell Technologies, Vancouver, Canada). (2) Once the cell density reaches 100% on the second day, replace the medium with N2B27+2i (composed of N2 medium and B27 medium mixed in a 1:1 volume ratio, where N2 medium contains 1×N2 additive, DMEM / F-12, 1×non-essential amino acids (NEAA), 1×GlutaMAX, 5 μg / mL insulin, 1 mL M glutamine, and 100 μM 2-mercaptoethanol; B27 medium contains Neurobasal medium, 1×B27 additive, 5 μM MSB431542 (CAS: 301836-41-9), and 5 μM dorsomorphin (CAS: 866405-64-3)). Incubate at 37°C in a CO2 incubator for induced differentiation, changing the medium daily. Add more medium initially. Be gentle when changing the medium to avoid poking cells with the pipette tip and causing them to clump together. (3) After about 7 days, when the cell morphology has changed slightly, wash the cells once, add 1 mL of LDMMEM / F12, scrape the cells off with a 1 mL pipette tip, trying to keep large cell clumps, centrifuge, and transfer to a new pre-coated 6-well plate (1 mL seed volume, containing 1*10^6 live cells). 5 The next day, replace the medium with N2B27 (composed of N2 medium and B27 medium mixed in a 1:1 volume ratio, where B27 medium contains only Neurobasal medium and 1×B27 additive, and does not contain SB431542 and dorsomorphin) to continue inducing differentiation. Change the medium regularly every day. (4) After about 14 days, the cell clumps will be noticeably raised. After washing the cells once, add 1 mL of LDMMEM / F12, scrape off the raised cell clumps with a 1 mL pipette tip, centrifuge, and transfer to a new pre-coated 6-well plate. Continue to use N2B27 medium (1 mL inoculum, containing 1*10^9 viable cells). 5Continue to induce differentiation. Change the medium regularly every day. After about 3 days, the appearance of neural rosettes can be clearly observed. At this time, BFGF (2μg / mL) can be added and removed after two days to avoid premature differentiation of neural rosettes. (5) After culturing for 2-3 days, discard the supernatant, wash the cells once, add Accutase to digest for 2-5 minutes to digest the cells into single cells. After centrifugation, transfer to a new uncoated 6-well plate for suspension culture and continue to use N2B27 medium (inoculum 1mL, containing 1*10 live cells). 5 (6) On the second day, cells can be observed to grow into spheres. About 3 days later, the cells can be digested, passaged, resuspended, or cryopreserved, or digested into single-cell adherent neurons, thus obtaining neural stem cells derived from human chemically induced pluripotent stem cells, denoted as hCiPSC-NSCs.

[0065] hCiPSC-NSCs are digested into single-cell adherent neurons, which can be cultured and electrical signals can be observed in about 40 days, at which point patch-clamp experiments can be performed on the neurons.

[0066] hCiPSCs-derived neural stem cells (hCiPSCs-NSCs) were characterized by immunofluorescence, flow cytometry, and RT-qPCR, and their tumorigenicity was assessed. Further electrophysiological assays were performed after differentiating hCiPSCs-NSCs into neurons. The immunofluorescence, flow cytometry, and RT-qPCR experiments were the same as in Example 1.

[0067] Tumorigenicity assessment: To comprehensively evaluate the safety of cell therapy, 1×10⁻⁶ cells were used. 7 One hCiPSC-NSC was injected into the left quadriceps femoris muscle region and subcutaneous abdominal tissue of 8-week-old NOD-SCID immunodeficient mice. Three months after cell transplantation, the mice were euthanized, and samples were systematically collected from muscle, skin, liver, kidney, myocardium, small intestine, brain, spleen, and lungs. After paraffin embedding and sectioning, histopathological examination was performed using hematoxylin and eosin (H&E) staining. All animal experimental procedures strictly adhered to the guidelines established by the institution's animal ethics committee.

[0068] Electrophysiological detection: Induced neurons were subjected to electrophysiological experiments using whole-cell voltage clamp and current clamp at room temperature, as follows: (1) Whole-cell patch clamp solution (i.e. electrode solution) was prepared with ultrapure water. After the reagent was completely dissolved, the pH was adjusted to 7.2-7.3 with KOH. It was filtered with a 0.22 μM filter membrane, aliquoted into 500 μL tubes, and stored at -20℃. (2) Preparation of amphotericin B: 3 mg of amphotericin B was dissolved in 50 μL of DMSO and sonicated for 10 minutes using an ultrasonic cleaner to assist in its dissolution. 1 mL of the aliquoted electrode solution was added to 30 μL of amphotericin B and sonicated to dissolve. After the dissolution was completed, it was stored away from light and used within 3 hours if possible. (3) The slides containing neurons were placed in a bath containing extracellular fluid. The glass electrodes contained intracellular fluid with amphotericin B. After fixation, the slides were slowly immersed in water. Signals were recorded using an Axopatch 200B amplifier (Axon Instruments, USA) and acquired / analyzed using Clampfit 10.2 software (Molecular Devices, USA). The resistance of the glass microelectrodes used in whole-cell recording was approximately 6–10 MΩ. A water resistance of 10–20 MΩ was preferred. After the electrode contacts the cell surface and seals, the Rm value rises rapidly, reaching a maximum of over 1 GΩ. (4) In voltage-clamp I / V-clamp mode, Na+ can be recorded. + K + Ca 2+ Voltage-gated ion channel currents, as well as presynaptic and postsynaptic currents. Upon administration of depolarizing stimuli, Na+ ion channel currents can be induced. + and Ca 2+ The inward current of the ion channel, and K + Outward current of ion channels. (5) Spontaneous action potentials can be recorded in the current clamp Vm / I-clamp Normal mode. Action potentials can be induced after depolarization stimulation. Note: (1) Preparation of electrode solution (electrode resistance is 4-8 MΩ after filling): contains 140 mM potassium methanesulfonate, 10 mM HEPES, 5 mM NaCl, 1 mM CaCl2, 0.2 mM MEGTA, 3 mM ATP-Na2 and 0.4 mM MTP-Na2 (adjust pH to 7.2-7.3 with KOH). (2) Preparation of cell bath solution: contains 127 mM NaCl, 3 mM KCl, 1 mM MgSO4, 26 mM NaHCO3, 1.25 mM NaH2PO4, 10 mM MD-glucose and 2 mM CaCl2 (adjust pH to 7.4 with NaOH).

[0069] The inventors characterized hCiPSC-derived neural stem cells (hCiPSCs-NSCs) using immunofluorescence, flow cytometry, and RT-qPCR. Immunofluorescence results showed that hCiPSCs-NSCs highly expressed the neural stem cell markers PAX6 and NESTIN. Figure 1 (F); Flow cytometry analysis further confirmed that the PAX6 positivity rate reached 80.5% ( Figure 1 In the study of G (G), the positive rate for Nestin reached 90.1%. Figure 1 (G). Typical neural rosette structures can be observed during hCiPSCs-NSCs culture. Figure 1 RT-qPCR data showed that, compared with undifferentiated hCiPSCs, the expression levels of PAX6 and NESTIN in hCiPSCs-NSCs were significantly increased (P<0.0001). Figure 1 (I). To evaluate the tumorigenicity of hCiPSCs-NSCs, the inventors used 1×10 7 Cells were transplanted into the left quadriceps femoris muscle of 6-10 week old NOD-SCID mice. Histological analysis 3 months after transplantation showed no tumor formation in the myocardium, liver, spleen, lungs, kidneys, brain, stomach, small intestine, pancreas, and muscle tissue. Figure 1 (L). Subsequently, the inventors further differentiated neural stem cells into neurons and detected classic neuronal markers. Immunofluorescence results showed that the differentiated cells simultaneously highly expressed the early neuronal marker MAP2 and the mature neuronal marker β-III-tubulin (L). Figure 1 (J). At approximately 40 days of neuronal culture, the electrophysiological function of hCiPSC-derived neurons was assessed using patch-clamp techniques. Figure 1 (K). The results showed that these neurons could spontaneously generate sodium, potassium, and calcium ion currents, and postsynaptic currents and spontaneous action potentials were recorded. The above experiments confirmed that neurons differentiated from hCiPSCs not only express typical neuronal markers, but also possess characteristic electrophysiological characteristics (K). Figure 1 (J and K). These results demonstrate that hCiPSCs can successfully differentiate into functional neurons, laying the foundation for subsequent research on the use of hCiPSC-derived neural stem cells to treat VPA-induced autism model mice and the underlying mechanisms.

[0070] Example 3: hCiPSCs-NSCs transplantation experiment for the treatment of ASD (autism syndrome).

[0071] The animal experimental methods involved in this embodiment are as follows:

[0072] Animal Experiment Design and Manipulation: All procedures were performed in accordance with the guidelines of the Institutional Animal Care Committee of the Guangdong Academy of Sciences Institute of Biological and Medical Engineering, and under specific pathogen-free (SPF) conditions certified by the International Committee for Assessment and Accreditation of Laboratory Animals (AAALAC). Animal Husbandry and Reproduction: Adult Sprague-Dawley rats (24 females and 12 males; initial weight 200–250 g) were housed in a controlled environment (22±1℃, relative humidity 55±5%) with a 12-hour light / dark cycle (lighting started at 07:00). Animals had free access to sterilized water and standard rodent feed. For mating, male and female rats were housed together at 17:00, and vaginal plugs were examined at 09:00 the following morning. Female rats with vaginal plugs were defined as gestational day 0.5 (GD0.5).

[0073] Establishment of a prenatal valproic acid (VPA) exposure model of autism spectrum disorder (ASD): Twenty-four confirmed pregnant female rats were randomly assigned to two groups. During the period from GD11.5 to GD13.5 (corresponding to the peak period of neocortical neurogenesis in rats), the VPA exposure group (n=16 female rats) received daily intraperitoneal injections of sodium valproate dissolved in endotoxin-free saline (dose: GD11.5: 400 mg / kg, GD12.5: 450 mg / kg, GD13.5: 400 mg / kg). The control group (n=8 female rats) received an equal volume of 0.9% NaCl injection.

[0074] Postnatal grouping: Male offspring were cross-fostered on day 21 (P21) to eliminate litter effect and were randomly assigned to three groups: Control group (n=12): Offspring from control mothers who received saline treatment. VPA group (n=12): Offspring exposed to VPA. hCiPSC-NSCs treatment group (n=12): Offspring exposed to VPA and treated with hCiPSC-NSCs. Specifically, the hCiPSC group received combination therapy via a dual administration route: (1) Systemic administration: Systemic delivery was given weekly via tail vein injection, with each injection being 5 × 10 6 (2) Intraventricular booster injection: Intraventricular (ICV) booster injection was performed in the fourth week after systemic treatment. This procedure involved stereotactic surgery under 3% isoflurane anesthesia (RWDLifeScience). During the surgery, hCiPSC-NSCs (2×10⁻⁶ hCiPSC-NSCs) were injected into the intraventricular (ICV) area using coordinates with the anterior fontanelle as a reference (0.8 mm posterior to the anterior fontanelle, ±1.6 mm lateral to the midline, and -3.8 mm subdurally). 5(3) Intranasal delivery: Immediately after needle withdrawal, intranasal administration is performed: using a calibrated micropipette tip inserted 2 mm deep into the nasal cavity, 100 μg of exosomes suspended in 20 μL PBS.

[0075] The exosomes described above were human umbilical cord mesenchymal stem cell (hUC-MSC) exosomes, prepared as follows: Exosomes were isolated from hUC-MSC conditioned medium (DMEMF12 + 10% FBS, cultured to 80% saturation) using differential ultracentrifugation, according to the MISEV 2018 guidelines. The cell culture supernatant was centrifuged sequentially: first, centrifuged at 300×g for 10 minutes (4℃) to remove residual cells; then centrifuged at 2,000×g for 10 minutes (4℃) to remove cell debris; followed by centrifugation at 10,000×g for 30 minutes (4℃) to precipitate microvesicles. The clarified supernatant was filtered through a 0.22 μm polyethersulfone (PES) membrane (Millipore) and finally ultracentrifuged at 100,000×g for 2 hours (4℃; OptimaXPN-100, Beckman Coulter). The purified exosome precipitate was resuspended in sterile phosphate-buffered saline (PBS) for subsequent use. During the peri-transplantation period, animals were treated with 0.3% gentamicin for 3 days to mitigate potential immune responses. Behavioral testing began one week after the last treatment.

[0076] All behavioral assessments were performed by experimenters unaware of their group assignments, followed by histological analysis. Data collection and processing were conducted entirely using a group-blinding method.

[0077] (I) Behavioral Testing

[0078] Sprague-Dawley rats were acclimatized in the laboratory for 1 hour before testing.

[0079] 1. Open Field Test: A square open field (100×100×40cm) was used, with a defined central region (60×60cm). After a 10-minute adaptation period (free exploration), a 10-minute test was conducted. A top-mounted camera system recorded the movement trajectory, and NoldusEthoVisionXT software was used to quantify the number of times the limbs entered the central region and the duration of stay in the central region. Recorded parameters included the number of central / peripheral crossings (all limbs crossing the region boundaries) and upright movements (forelimbs raised for ≥2 seconds).

[0080] Results: The open field test assessed autonomous exploratory behavior and anxiety-like responses in novel environments. Valproic acid (VPA) exposure significantly inhibited exploratory activity and increased anxiety-like behaviors in SD rats. Figure 2 hCiPSC-NSCs treatment improved the deficiencies in these VPA-exposed animals, specifically: Exploration behavior: The hCiPSC-NSCs treatment group showed an increased crossing frequency compared to the VPA group (P<0.05). Figure 2 (A). Compared with the control group, the VPA group showed a reduced range of motion in the central region (P<0.0001). Animals treated with hCiPSC-NSCs showed partial recovery, with a significantly greater range of motion than the VPA group (P<0.0001). Figure 2 (B) Anxiety-like behavior: The hCiPSC-NSCs treatment group had a longer central zone dwell time compared to the VPA group (P<0.0001), indicating reduced anxiety ( Figure 2 (C); The vertical activity score of the VPA group was lower than that of the control group (P<0.0001), and the hCiPSC-NSCs treated animals showed improved standing behavior compared with VPA-exposed rats (P<0.001). Figure 2 Trajectory analysis: VPA-exposed rats exhibited restricted movement patterns with minimal exploration of the central region (F). Figure 2 In the H), hCiPSC-NSCs treated animals showed trajectories similar to those of the control group. Figure 2 (G), characterized by increased exploration in the central area and decreased tactile activity (G). Figure 2 Middle I).

[0081] 2. Analysis of repetitive and stereotyped behaviors

[0082] 1) Marble-Burying Test: SD rats were placed individually in polycarbonate test cages containing 5 cm deep fresh corncob bedding. After acclimatization for 3 minutes, the animals were temporarily transferred to a temporary holding cage. Sixteen black glass beads (1.6 mm in diameter) were arranged in a standardized 4×4 grid (3 cm spacing). The rats were returned to their cages for a 10-minute test (50 lux illumination), and the test was recorded on video from orthogonal angles. Three blinded observers independently scored the test based on a glass bead surface coverage area ≥75% as the burial criterion. A consensus was reached with ≥2 / 3 of the observers agreeing; any ambiguities were resolved through frame-by-frame analysis.

[0083] Results: The results of the glass bead burial experiment are as follows: Figure 2 Compared with the VPA exposure group, the repetitive behavior was significantly reduced in the E, hCiPSC-NSCs treatment group (P<0.0001), indicating that the exploratory behavior pattern was partially restored.

[0084] 2) Self-Grooming Test: Rats were placed individually in clean polycarbonate cages (30×20×15cm) under 50 lux illumination. After a 10-minute acclimatization period, spontaneous grooming behavior (facial cleaning, body licking, and genital / tail grooming) was recorded for 10 minutes using a Logitech C920HDPro camera. Three blinded observers analyzed the number of grooming events (discrete grooming events) and the cumulative duration (total time spent performing the stereotyped movement).

[0085] Results: Mice exposed to VPA exhibited excessive self-grooming, a hallmark of repetitive and stereotyped behavior. Figure 2 (D). hCiPSC-NSCs treatment significantly reduced this behavior; compared with the VPA group, animals in the hCiPSC-NSCs treatment group showed a reduced frequency of grooming (P<0.05). Figure 2 (D). Compared with the control group, VPA-exposed SD rats exhibited a significant increase in repetitive stereotyped behaviors (glass bead burying and grooming), suggesting neurodevelopmental abnormalities that may be associated with dysfunction of the basal ganglia-cortical circuit. hCiPSC-NSCs treatment effectively alleviated these behaviors, supporting their therapeutic potential for modulating circuit-specific abnormalities in ASD-like phenotypes.

[0086] 3. Three-Chamber Social Interaction Test

[0087] The three-compartment social interaction experiment assesses social motivation (0–10 minutes) and social novelty preference (10–20 minutes) using experimental animals. This experiment quantifies social ability and cognitive flexibility by measuring the time animals spend exploring a stranger of the same species (stranger 1 / 2) and an empty cage / object. The specific procedures are as follows: Adaptation program: Days 1–7: Animals undergo 2 hours of environmental acclimatization daily (23°C, 50 lux illumination). Days 8–10: Progressive exposure training, including 3 minutes of exploration in the central compartment daily, followed by 3 minutes of free movement throughout the entire area daily. The experimental procedure includes: Baseline period (5 minutes): An empty wire cage is placed in each of the two compartments. Sociality test (10 minutes): An age / sex-matched stranger rat (Stranger 1) is placed in one compartment, and an empty control cage (control group) is placed in the opposite compartment. Social novelty preference test (10 minutes): A new stranger rat (Stranger 2) replaces the control cage, while the original social stimulus (Stranger 1) remains unchanged. Key controls: Stranger rats were housed alone for 48 hours prior to testing to standardize olfactory cues. Interaction time (nose tip <2cm from cage) was quantified using EthoVisionXT 15.0 software (NoldusIT, Wageningen). Indicator definitions: Stranger 1 cage: time spent in the cage containing Stranger 1; Empty cage: time spent in the empty cage; Stranger 1: interaction time with Stranger 1; Object: interaction time with inanimate objects; Stranger 2 cage: time spent in the cage containing Stranger 2; Stranger 2: interaction time with Stranger 2.

[0088] Results: Social motivation phase (0–10 minutes): VPA exposure significantly impaired social motivation in SD rats. Figure 3 (A); trajectory analysis ( Figure 3 (C-E): VPA group avoided unfamiliar mouse 1 and remained fixated on the empty cage ( Figure 3 In the hCiPSC-NSCs treatment group, an exploratory pattern similar to a control group was observed. Figure 3 In the middle D), the increase in unfamiliar mouse approach and the decrease in non-social attention were observed. Figure 3 (E). Conclusion: Rats exposed to VPA exhibited avoidance of social interaction (stranger rat 1) and preference for non-social environments (empty cage), indicating impaired social abilities (P<0.0001). hCiPSC-NSCs treatment reversed these deficits, restoring the balance of exploratory behavior to social stimuli. Social novelty preference phase (10–20 minutes): Spatial exploration such as Figure 3 In China B, social interaction is like Figure 3 Trajectory analysis of B in the middle is as follows: Figure 3 F to H. Conclusion: The VPA group had a novelty preference deficit (fixation in strange rat 1 / avoidance in strange rat 2). hCiPSC-NSCs treatment could restore cognitive flexibility and promote novelty-seeking behavior (P<0.05).

[0089] 4. Assessment of learning and memory abilities

[0090] The Morris Water Maze Assessment (MWM) evaluates spatial learning and memory abilities through a hidden platform training test (acquisition test) and a memory retention test (exploration test). The procedure is as follows: The apparatus consists of a black circular pool (150 cm in diameter) filled with opaque water (23.0 ± 0.5 °C; non-toxic white egg tempera paint) and a hidden platform (14 cm in diameter) located 1.5 cm below the water surface in the target quadrant. Spatial cues include four high-contrast geometric patterns with uniform illumination (50 ± 5 lux). Acquisition phase (5 days): Four tests are conducted daily (maximum 90 seconds, 25-minute intervals), with randomized starting points (North-South-East-West); failed tests without platform location include a 10-second guided platform orientation. Exploration test: Conducted 24 hours after training, the rats swim freely for 60 seconds (without a platform). All testing procedures used EthoVisionXTv15.0 software (Noldus Information Technology) for digital data acquisition, and the positional error was verified to be less than 2% through checkerboard calibration.

[0091] Results: Spatial acquisition experiment: Escape latency as Figure 4 The C-value indicates that the escape latency in the hCiPSC-NSCs treatment group was significantly shorter than that in the VPA group (P<0.01), suggesting improved learning efficiency. Search strategies such as... Figure 4 From F to H, it was indicated that the hCiPSC-NSCs treatment group had significantly improved search efficiency, and its movement trajectory was similar to that of the control group (e.g., reduced circling behavior and more precise approach to the target platform). Spatial exploration test: Memory retention index: Duration in the target quadrant, such as... Figure 4 In China A, the number of times the platform is traversed is as follows: Figure 4 In the middle D quadrant, the preference is as follows Figure 4 E. Exploratory behavior: swimming trajectory as shown Figure 4 Swimming distance data for I-K is as follows: Figure 4 Conclusion: VPA rats exhibited significant spatial memory deficits (prolonged latency, impaired random search strategy, and quadrant preference) independent of motor dysfunction. hCiPSC-NSCs treatment improved these deficits and enhanced spatial learning efficiency (P<0.01) and memory retention (P<0.05).

[0092] (II) Analysis of the level of neuroinflammation and oxidative stress in the prefrontal cortex

[0093] 1. Tissue Collection and Processing: Animals were euthanized after behavioral testing. Anesthesia was induced by intraperitoneal injection of ketamine (100 mg / kg) and toluene-thiazide (10 mg / kg). Sprague-Dawley rats underwent cardiac perfusion: first with 0.9% NaCl, followed by perfusion with 4% paraformaldehyde (PFA; Merck, Germany). The extracted brain was post-fixed in 4% PFA for 24 hours, after which the hippocampus and prefrontal cortex were dissected. The tissues were dehydrated using a gradient of ethanol, embedded in paraffin (Merck, Germany), and sectioned into 5 μm thick sections using a rotary microtome (MicromHM335E, Walldorf, Germany).

[0094] 2. Biochemical Assays: The levels of glutathione (GSH), total superoxide dismutase (T-SOD), glutathione peroxidase (GSH-PX), malondialdehyde (MDA), catalase (CAT), total nitric oxide synthase (T-NOS), and nitric oxide (NO) were quantitatively detected using commercially available kits (Nanjing Jiancheng Bioengineering Institute). The concentrations of interleukin-6 (IL-6), IL-10, IL-1β, and tumor necrosis factor-α (TNF-α) were determined using a rat-specific ELISA kit (Fankel, Shanghai, China).

[0095] Results: Analysis of prefrontal cortical neuroinflammation levels: Enzyme-linked immunosorbent assay (ELISA) showed that valproic acid (VPA) exposure significantly altered the inflammatory cytokine profile of the prefrontal cortex in SD rats. hCiPSC-NSCs treatment effectively restored the pro-inflammatory / anti-inflammatory balance, specifically with the following downregulation of pro-inflammatory cytokines: IL-1β levels in the hCiPSC-NSCs treatment group were significantly lower than those in the VPA group (P<0.0001). Figure 5 (A). The hCiPSC-NSCs treatment group showed a significantly better inhibitory effect on IL-6 overexpression than the VPA group (P<0.0001). Figure 5 (B) The hCiPSC-NSCs treatment group showed a significantly better inhibitory effect on TNF-α levels than the VPA group (P<0.0001). Figure 5 (C). Upregulation of anti-inflammatory cytokines ( Figure 5(D): hCiPSC-NSCs treatment significantly restored IL-10 production compared to the VPA group (P<0.0001). VPA exposure led to pro-inflammatory changes in the prefrontal cortex (increased levels of IL-1β, IL-6, and TNF-α; decreased IL-10 levels). hCiPSC-NSCs treatment significantly reversed this imbalance, inhibited pro-inflammatory cytokines, and enhanced anti-inflammatory IL-10 levels, suggesting that hCiPSC-NSCs have therapeutic potential in alleviating neuroinflammation in autism spectrum disorder (ASD)-like phenotypes. Analysis of prefrontal cortex oxidative stress levels: Enzyme-linked immunosorbent assay (ELISA) results showed that VPA exposure significantly disrupted oxidative stress homeostasis in the prefrontal cortex of SD rats. hCiPSC-NSCs treatment restored redox balance, specifically as follows: Antioxidant system inhibition: hCiPSC-NSCs treatment significantly increased CAT activity compared to the VPA group (P<0.0001). Figure 5 The presence of K in the middle jiao (a traditional Chinese medicine) significantly restored GSH-Px activity compared to the VPA group (P<0.0001). Figure 5 The middle E group significantly increased GSH levels compared to the VPA group (P<0.001). Figure 5 The addition of F significantly enhanced SOD activity compared to the VPA group (P<0.0001). Figure 5 (G). Elevated oxidative damage markers: MDA levels in the hCiPSC-NSCs treatment group were significantly lower than those in the VPA group (P<0.0001). Figure 5 (H). The T-NOS activity in the hCiPSC-NSCs treatment group was significantly inhibited compared with the VPA group (P<0.0001). Figure 5 (I). The NO level in the hCiPSC-NSCs treatment group was significantly lower than that in the VPA group (P<0.0001). Figure 5 (J). Conclusion: VPA exposure induces systemic oxidative stress, characterized by depletion of antioxidants (such as CAT, GSH-Px, GSH, and SOD) and elevated levels of oxidative damage markers (such as MDA, T-NOS, and NO). hCiPSC-NSCs treatment can reverse these effects, enhance the body's antioxidant defense capacity, and reduce oxidative damage. This suggests therapeutic potential through activation of the Nrf2 / ARE pathway and ROS scavenging mechanisms.

[0096] (III) Microglial activation and neuroinflammation in autism spectrum disorders

[0097] Immunofluorescence staining of the hippocampal CA1 region and prefrontal cortex with ionized calcium-binding aptamer 1 (Iba1) was used to assess neuroinflammatory pathological changes in VPA-exposed Sprague-Dawley (SD) rats. Immunofluorescence staining: Brain sections were dewaxed and antigen-retrieval (using standard protocols) followed by washing three times (5 min each) in phosphate-buffered saline (PBS, pH 7.4). Non-specific binding was blocked for 1 h at room temperature using 4% (w / v) bovine serum albumin (BSA) PBS solution containing 0.3% Triton X-100. Rabbit primary antibody (diluted 1:200 with blocking buffer) was incubated at 4°C for 16 h under humidified conditions. After washing three times (10 min each) in PBS containing 0.1% Tween 20 (PBST), sections were incubated in the dark with Alexa Fluor 488-labeled goat anti-rabbit IgG secondary antibody (1:500 dilution) at room temperature for 1 h. Cell nuclei were counterstained with 4',6-diamidinyl-2-phenylindole (DAPI; 1 μg / mL) for 10 min in the dark. Confocal imaging was performed using a TCSSP8STED3X system (Leica Microsystems) with the following parameters normalized: 488 nm laser (12% power), detector gain 650–750 V, and Z-axis scan interval 0.5 μm. Integrated fluorescence density (IntDen) was quantified within the threshold-optimized region of interest (ROI) using ImageJ software (v1.53k, NIH).

[0098] Results: Hippocampal CA1 region: Iba1 fluorescence intensity ( Figure 6 (A-J): Control group ( Figure 6 Resting microglia in the control group (A-C) showed low Iba1 expression. Activated microglia in the VPA group exhibited hypertrophic morphology, and Iba1 fluorescence intensity was significantly increased (P<0.0001 compared to the control group). Figure 6 (D-G). In the hCiPSC-NSCs treatment group, microglial activation was reduced, and Iba1 fluorescence intensity was significantly lower than in the VPA exposure group (P<0.0001) (6 G-J). Prefrontal cortex: Iba1 fluorescence intensity ( Figure 6 K-T): Control group ( Figure 6 Resting microglia (M-K) showed weak Iba1 signaling. VPA group ( Figure 6 Strong Iba1 staining in O-Q cells indicated activation of pro-inflammatory microglia (P<0.0001 compared to the control group). Figure 6 (N). hCiPSC-NSCs treatment group ( Figure 6 Iba1 expression was weakened in the R-T group, and the fluorescence intensity was significantly lower than that in the VPA group (P<0.0001). Figure 6(N). Conclusion: VPA exposure induces microglial overactivation (elevated Iba1 protein levels in the CA1 region and prefrontal cortex), suggesting a pro-inflammatory phenotypic shift. hCiPSC-NSCs treatment significantly inhibits microglial activation, supporting its therapeutic potential to modulate neuroinflammation in autism spectrum disorder by regulating microglial phenotype.

[0099] (iv) Ultrastructural pathological changes and repair of neurons in the prefrontal cortex and CA1 region of the hippocampus

[0100] Transmission electron microscopy: 1) Tissue preparation (PND60 rats): Terminal anesthesia was administered via intraperitoneal injection of ketamine / toluidine (75:5 mg / kg). Cardiac perfusion was performed via the ascending aorta: Vascular irrigation: 200 mL ice-cold PBS (0.01 M sodium / potassium phosphate, pH 7.4); Primary fixation: 300 mL mixed aldehyde fixative (0.1 M sodium dimethylarsenate buffer containing 2% paraformaldehyde + 2.5% glutaraldehyde, pH 7.4; Sigma-Aldrich #G5882); Microdissection: 1 mm tissue was taken. 3 Prefrontal cortex and dorsal hippocampus samples (n=3 / group, 6 regions / animal). 2) Post-processing: Samples were subjected to the following steps in sequence: secondary fixation (fixation in primary fixative at 4°C for 20 hours → fixation in 1% osmium tetroxide (OsO4) / 0.8% potassium ferricyanide (K4[Fe(CN)6]) for 2 hours); ethanol gradient dehydration (50%–100%); propylene oxide transition, EPON TM 812 epoxy resin embedding (polymerization at 60℃ for 48 hours). 3) Imaging: Semi-thin sections (1μm) were stained with toluidine blue for localization. Ultrathin sections (60nm) were placed on a 200-mesh copper grid and stained with uranyl acetate (15 minutes) and lead Reynolds citrate (5 minutes) in sequence. Images were acquired using a JEOL JEM-1200EX transmission electron microscope (80kV) equipped with a MORADACCD camera (OlympusiTEMv5.2 software; 3280×2464 pixels, 8-bit depth). Ultrastructure of the control group: Transmission electron microscopy (TEM) observation of neurons in the prefrontal cortex (PFC) and CA1 region of the hippocampus of the control group rats showed that their ultrastructure was intact and undamaged. Figure 7 The synaptic cleft is narrow, and the postsynaptic dense granules (PSDs) are well-defined and electron-dense. Numerous synaptic vesicles (SVs) are aggregated at the presynaptic terminals; these vesicles are mainly distributed near areas of high neural activity. Figure 7 (D). The axon terminals maintain their membrane structure intact without swelling, while the mitochondria exhibit an orderly arrangement of cristae and a complete membrane structure. Figure 7 (A). Pathological changes in a VPA-induced autism spectrum disorder model: Compared with the control group, the VPA group showed significant synaptic ultrastructural abnormalities ( Figure 7 The specific manifestations include: obvious loss of synaptic vesicles (SVs) at the presynaptic terminal (complete disappearance in local areas); disordered synaptic membrane structure: blurred and thickened synaptic cleft, indistinct presynaptic / postsynaptic boundaries; abnormally thickened postsynaptic diffuse (PSD) with decreased electron density; mitochondrial lesions: disordered cristae structure and membrane rupture, presenting as swelling or atrophy. Figure 7 (B) ; accompanied by astrocyte swelling, suggesting neuroinflammatory activation. Ultrastructure of the hCiPSC-NSC treatment group: Transmission electron microscopy showed that hCiPSC-NSCs treatment improved synaptic / mitochondrial pathology in the prefrontal cortex and CA1 region, with characteristics close to those of the control group ( Figure 7 In the middle C and F): the synaptic cleft recovers its narrow spacing and has a clear PSD; the presynaptic terminal shows an accumulation of SV near the membrane. Figure 7 (D). Quantitative analysis of synaptic vesicles: SV density analysis ( Figure 7 The results showed that the number of SVs in the CA1 region of the hippocampus of rats in the VPA group was significantly reduced (P<0.001 compared with the control group), and the number of SVs increased after hCiPSC-NSC treatment (P<0.05 compared with the VPA group). Figure 7 In the VPA group, the SV count in the cerebral cortex of rats was reduced (P<0.001 compared with the control group), and hCiPSC-NSC treatment restored SV density (P<0.05 compared with the VPA group). Figure 7 (H). Pathophysiological significance: Ultrastructural disorder indicates: synaptic dysfunction: impaired SV release and interrupted postsynaptic signal transduction, altering the excitatory-inhibitory balance. Compromise of bioenergy: mitochondrial structural disorder exacerbates metabolic insufficiency; Cross-influence of neuroinflammation: astrocyte swelling indicates glial cells' involvement in the pathogenesis of ASD.

[0101] (V) Taxonomy of Gut Microbiota and Operational Taxonomic Unit (OTU) Profile

[0102] 16S rRNA sequencing and bioinformatics analysis

[0103] (1) Fecal sample collection: To minimize the impact of circadian rhythm, fresh fecal samples were aseptically collected between 09:00 and 11:00. Rats were briefly immobilized, and defecation was induced by gently raising the tail and applying aseptic abdominal pressure. Freshly expelled fecal particles (3-5 particles ≈ 100mg) were transferred to sterile 2mL centrifuge tubes (Axygen, PCR-02-C, USA) using disposable forceps, and were flash-frozen in liquid nitrogen within 5 minutes and stored at -80℃. (2) DNA extraction and amplification: Genomic DNA was extracted using the CTAB method combined with liquid nitrogen homogenization (0.1mm zirconia beads; FastPrep-24 homogenizer). DNA purity (NanoDrop2000 test: A) was measured.260 / A 280 =1.8~1.9, A 260 / A 230 >2.0) normalized to 1 ng / μL. Using barcoded primers 341F (5'-CCTAYGGGRBGCASCAG-3', SEQ ID NO:13) / 806R (5'-GGACTACNNGGGTATCTAAT-3', SEQ ID NO:14) and High-Fidelity PCR Master Mix (New England Biolabs, USA) was used to amplify the V3-V4 region under the following cycling conditions: 98℃ / 1 min; 30 cycles (98℃ / 10 s, 50℃ / 30 s, 72℃ / 30 s); 72℃ / 5 min. (3) Library construction and sequencing: Amplicon (≈550 bp) was purified after verification by agarose gel electrophoresis (TIANgelMidi kit). The library was constructed using the NEB Next Ultra II kit, and the library size distribution (Agilent 5400 Bioanalyzer detection: 450–650 bp) and concentration (Qubit 4.0 detection: ≥20 nM) were verified. Paired-end sequencing (PE250) (50,000 reads per sample) was performed on the Illumina NovaSeq 6000 platform. (4) Bioinformatics analysis: The original sequences were denoised and chimeric removed using DDA2 in QIIME2 (v2023.2) to generate an amplicon sequence variant (ASV) table, and species classification annotation was performed based on the SILVA138.1 database. Alpha diversity analysis: Chao1 index, Shannon index, Faith's PD index; β diversity analysis: Bray-Curtis distance / UniFrac distance (Principal Coordinate Analysis (PCoA) / Non-metric Multidimensional Scale Analysis (NMDS)); Differential species analysis: LEfSe (Linear Discriminant Analysis (LDA) score > 2.0), ANCOM (W value > 0.7), DESeq2 (False Detection Rate (FDR) < 0.05); Functional prediction: KEGG pathway prediction using PICRUSt2 (v2.4.1) (STAMP software: Welch'st-test, FDR correction); Network analysis: Gephi (v0.10.1; Spearman correlation coefficient |ρ| > 0.6, P < 0.01); Multivariate modeling: Redundancy analysis (RDA) (using the vegan package) and partial least squares discriminant analysis (PLS-DA) (using the mixOmics package).

[0104] Results: (1) Dynamic changes in gut microbiota at the phylum, family, genus, order, and class levels, and the distribution of shared / unique species across groups: through relative abundance analysis at the phylum, class, order, family, and genus levels ( Figure 8 Analysis of A to E in conjunction with Venn diagrams ( Figure 8 (2) Taxonomic hierarchical specific changes in gut microbiota composition among experimental groups: Family-level analysis revealed significant clustering patterns among the groups (F). Figure 9 (A) The hCiPSC-NSCs treatment group showed that the abundance of these bacterial families was at an intermediate level, indicating that the microbial community structure had been partially restored. Genus-level resolution further supports group-specific clustering. Figure 9 (B) Dynamic changes in the target level, such as Figure 9 In C, the change in the horizontal position of the gate is as follows Figure 9 (3) Hierarchical analysis of changes in gut microbiota in rats treated with ASD model and hCiPSC-NSCs: family-level phylogenetic tree as follows Figure 10 In the Chinese A-level competition, the LDA score at the science and technology level is as follows: Figure 10 Middle B; genus-level phylogenetic tree as follows Figure 10 In the C-level, the LDA score analysis at the genus level is as follows: Figure 10 Middle D; phylogenetic tree such as Figure 10 In the middle E, the gate-level LDA score analysis is as follows Figure 10 (4) Comparative analysis of gut microbiota composition in the experimental groups: taxonomic changes at the genus and phylum levels based on DESeq2 analysis, the results are as follows: Figure 11 As shown, hCiPSC-NSCs therapy may combat autism spectrum disorder-related dysbiosis by enriching microbiota that produce short-chain fatty acids. (5) Analysis of gut microbiota α diversity in different experimental groups: The results of α diversity analysis are as follows Figure 12 As shown. (6) Beta diversity analysis of gut microbiota based on distance indicators: Bray-Curtis differences are shown in the figure. Figure 13 As shown. (7) Phylogenetic heatmap analysis of gut microbiota composition: Phylogenetic tree heatmap as shown. Figure 14 (8) Functional analysis of intestinal microbial metabolic pathways in the experimental group: The results of KEGG primary pathway analysis are as follows: Figure 15 The results of the analysis of the A and KEGG secondary pathways are as follows: Figure 15 The results of the three-level pathway analysis of B and KEGG are as follows: Figure 15 The results of the analysis of the MetaCyc pathway in the middle are as follows: Figure 15Conclusion: This invention demonstrates that non-gene-edited human induced pluripotent stem cell neural stem cells (hCiPSC-NSCs) delivered via a dual-pathway strategy (intravenous systemic administration + intraventricular bolus injection) can simultaneously improve autism-like phenotypes in VPA-exposed rats by targeting the gut-brain axis. Behavioralally, treatment significantly improved social interaction, reduced repetitive behaviors, and enhanced spatial memory. Mechanistically, these improvements are associated with reduced neuroinflammation, synaptic ultrastructural repair, and gut microbiota balance. Crucially, microbiota restoration drives oxidative stress relief, indicating that transplanted neural stem cells and the gut ecosystem communicate through paracrine signaling via immunomodulatory factors such as BDNF / VEGF detected in conditioned media.

[0105] Conclusions and Future Prospects: This invention establishes non-gene-edited human induced pluripotent stem cells (hCiPSC-NSCs) as a transformative therapy for autism spectrum disorders, demonstrating that they can simultaneously improve core behavioral deficits (social / repetitive behaviors), restore synaptic ultrastructure (upregulate synaptic vesicles), and rebalance the gut microbiota (downregulate Firmicutes / Bacteroidota ratio and upregulate Alloprevotella) by modulating the gut-brain axis. A dual-pathway delivery strategy—combining intravenous systemic administration with intraventricular targeting—achieved efficient, tumorigenic hippocampal implantation at 3 months, highlighting the clinical potential of chemically reprogrammed cells compared to gene therapy. Although the valerate-induced model effectively reproduces key autism phenotypes, future research needs to validate the applicability of these findings to genetic subtypes (such as Shank3 gene knockout) and elucidate the causal interactions between the microbiota and neuroimmunity through fecal transplantation. Extending the biosafety monitoring period (>6 months) and optimizing intranasal administration protocols to suit pediatric applications will drive the clinical translation of this mechanism framework and accelerate the development of stem cell-based neurodevelopmental disorder correction technologies.

Claims

1. A neural stem cell, characterized in that, The neural stem cells were obtained by inducing differentiation of human chemically induced pluripotent stem cells.

2. The neural stem cells according to claim 1, characterized in that, The human chemically induced pluripotent stem cells are obtained by chemical reprogramming of mesenchymal stem cells; the chemical reprogramming includes the following steps: mesenchymal stem cells are sequentially induced and cultured using a first-stage culture medium, a second-stage culture medium, a third-stage culture medium, and a fourth-stage culture medium; Preferably, the culture medium comprises KnockOutDMEM, knockout serum substitute, N2 additive, B27 additive, GlutaMax, NEAA, 2-mercaptoethanol, L-ascorbic acid 2-phosphate, LiCl, nicotinamide, AlbuMax-II, GSK3β inhibitor, TGFβR inhibitor, RAR activator and VEGFR / PDGFR inhibitor. Preferably, the two-stage culture medium includes KnockOutDMEM, KSR, N2 additive, B27 additive, GlutaMax, NEAA, 2-mercaptoethanol, Vc2p, LiCl, NAM, bFGF, GSK3β inhibitor, TGFβR inhibitor, RAR activator, C-jun N-terminal kinase inhibitor, MAO inhibitor, Smoothened receptor agonist, and DNA methyltransferase inhibitor; Preferably, the three-segment culture medium includes KnockOutDMEM, N2 additive, B27 additive, GlutaMax, NEAA, 2-mercaptoethanol, Vc2p, AlbuMax-II, recombinant human heregulinβ-1, GSK3β inhibitor, TGFβR inhibitor, ROCK inhibitor, MEK1 / 2 inhibitor, MAO inhibitor, DOT1L histone methyltransferase inhibitor, and HDAC inhibitor; Preferably, the four-segment culture medium includes KnockOutDMEM, N2 additive, B27 additive, GlutaMax, NEAA, 2-mercaptoethanol, Vc2p, recombinant human heregulinβ-1, GSK3β inhibitor, MEK1 / 2 inhibitor, ROCK inhibitor, and HDAC inhibitor.

3. The neural stem cells according to claim 1, characterized in that, The induced differentiation includes the following steps: culturing human chemically induced pluripotent stem cells in N2B27 medium.

4. The use of neural stem cells according to any one of claims 1 to 3 in the preparation of medicaments for treating nervous system-related diseases.

5. The application according to claim 4, characterized in that, The neurological disorders mentioned are those related to the gut-brain axis. Preferably, the neurological-related diseases include at least one of Alzheimer's disease, autism spectrum disorder, vascular dementia, Parkinson's disease, amyotrophic lateral sclerosis, multiple sclerosis, autoimmune encephalitis, meningitis, epilepsy, migraine, fibromyalgia, spinal muscular atrophy, mitochondrial encephalomyopathy, depression, anxiety, schizophrenia, attention deficit hyperactivity disorder, and Cushing's syndrome.

6. A drug comprising neural stem cells according to any one of claims 1 to 3.

7. The drug according to claim 6, characterized in that, The medication also includes combination therapies used to treat neurological disorders.

8. The medicament according to claim 7, characterized in that, The combination drugs include exosomes.

9. The medicament according to claim 8, characterized in that, The method of using the drug is as follows: intravenous injection of the neural stem cells; in the later stage of treatment, enhanced injection of the neural stem cells into the ventricle, and simultaneously, administration of exosomes via nasal cavity.

10. The medicament according to any one of claims 7 to 9, characterized in that, The neurological disorders mentioned include at least one of the following: Alzheimer's disease, autism spectrum disorder, vascular dementia, Parkinson's disease, amyotrophic lateral sclerosis, multiple sclerosis, autoimmune encephalitis, meningitis, epilepsy, migraine, fibromyalgia, spinal muscular atrophy, mitochondrial encephalomyopathy, depression, anxiety, schizophrenia, attention deficit hyperactivity disorder, Cushing's syndrome, and persistent postural-perceptual dizziness.

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