Method for improving induction efficiency of transdifferentiation from astrocytes to neurons

Abstract

The invention belongs to the field of biotechnology medicine, and particularly relates to a method for improving the induction efficiency of transdifferentiation from astrocytes to neurons, which comprises the following steps: preparing primary astrocytes: separating cortical tissues to prepare a cell suspension, and culturing and purifying to obtain the astrocytes. And transdifferentiation induction: transducing the astrocytes by using lentivirus, changing a differential medium and adding BDNF for culture to promote the astrocytes to be transdifferentiated into mature neurons. Animal model and targeted induction: establishing a cerebral infarction model, and injecting AAV-Neurog2-Pax6 virus into a focus area to realize specific transdifferentiation of astrocytes; the method comprises the following steps: performing in-vivo induction and sample treatment: performing perfusion fixation, dehydration, embedding and freezing slicing after taking materials, retaining the integrity of transdifferentiated cells, and performing overexpression on Neurog2 and Pax6 to realize transdifferentiation of the astrocytes to neurons and improve the induction efficiency of transdifferentiation of the astrocytes to the neurons.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology medicine, and particularly relates to a method for improving the efficiency of transdifferentiation of astrocytes into neurons. BACKGROUND

[0002] Cerebral infarction is one of the diseases with the highest disability rate in central nervous injury in the world, and its pathological characteristics are massive loss of neurons and destruction of neural circuits caused by ischemia and hypoxia. Although current thrombolytic therapy and intravascular intervention can partially rescue the ischemic penumbra in the acute phase, the narrow therapeutic time window and the characteristics of irreversible damage to neurons still make most patients with cerebral infarction leave irreversible neurological deficits. Traditional rehabilitation therapy has limited effect on promoting neural remodeling, and innovative strategies are urgently needed to further achieve in situ regeneration and functional repair of neurons. In recent years, direct reprogramming technology provides an innovative idea for neural repair after cerebral infarction: through endogenous glial cell in situ transdifferentiation, directly supplementing the lost neurons and reestablishing synaptic connections.

[0003] Neurog2-mediated astrocyte-neuron reprogramming can partially restore the motor function of cerebral ischemia model animals. In addition, astrocytes can generate neuron subtypes with specific projection characteristics through reprogramming due to the radial glial cell regionalization characteristics retained during development, which provides a unique advantage for precise repair of damaged neural circuits.

[0004] Although studies have shown that Neurog2 single factor can induce astrocytes to transdifferentiate into neurons, the induction efficiency using a single factor Neurog2 is low. SUMMARY

[0005] The purpose of the present application is to overcome the above-mentioned deficiencies, and provide a method for improving the efficiency of transdifferentiation of astrocytes into neurons.

[0006] In order to solve the above technical problems, the present application provides the following technical scheme: a method for improving the efficiency of transdifferentiation of astrocytes into neurons, comprising the following steps: The primary astrocyte preparation step: the cell suspension is prepared by isolating the cortical tissue, and the astrocytes are obtained after culturing and purifying the cells; The transdifferentiation induction step: after the lentivirus is prepared by HEK293T cells, concentrated and detected for titer, the lentivirus is added to the astrocytes for transduction, and then the differentiation medium is replaced, BDNF is added regularly for continuous culture, so as to promote the transdifferentiation of astrocytes into mature neurons; Animal model and targeted induction steps: After establishing a focal cerebral infarction model, AAV-Neurog2-Pax6 virus was injected into the lesion area at a specific time to achieve specific transdifferentiation of astrocytes in the lesion area. Sample processing steps after in vivo induction: After in vivo induction, samples are taken at specific time points, and then subjected to cardiac perfusion fixation, dehydration, OCT embedding, and frozen sectioning to preserve the integrity of transdifferentiated cells.

[0007] Furthermore, the primary astrocyte preparation steps include: Tissue isolation and purification steps: Tissue was isolated from the mouse cerebral cortex, and the subventricular region, striatum and hippocampus were removed. The cortical tissue was placed in frozen HBSS and gently separated using an autoclave to obtain a cell suspension. Centrifugation and resuspension steps: Centrifuge the cell suspension, and resuspend the pellet in culture medium after centrifugation; Culture and purification steps: The cells are cultured in an incubator until they merge. After merging, they are mechanically shaken to selectively remove impurities and purify astrocytes. The remaining purified cells are digested with proteases. Subculture and plating steps: Place cell smears in culture plates in advance and coat them with PDL. When subculturing, discard the old culture medium and wash with PBS. Add an appropriate amount of trypsin to digest the cells and then stop the reaction with culture medium. Centrifuge to resuspend the cells and count them. Seed the cells into culture plates at an appropriate density.

[0008] Furthermore, the culture medium used in the centrifugation and resuspension steps is DMEM / F12 medium containing inactivated fetal bovine serum and penicillin-streptomycin.

[0009] Furthermore, in the culture and purification steps, the enzymatic hydrolysis uses 0.25% trypsin-EDTA.

[0010] Furthermore, in the aforementioned passaging and plating step, 1×10 4 Inoculation density per well.

[0011] Furthermore, the differentiation medium contains DMEM / F-12, adjusted to 50 mL, comprising 0.5% FBS, 2% B27, 1% Glutamax, and 1% penicillin-streptomycin antibiotics.

[0012] Furthermore, the specific time periods refer to the acute and subacute phases of infarction.

[0013] Furthermore, the concentration of the AAV-Neurog2-Pax6 virus is 2.0 × 10⁻⁶. 12 vg / mL.

[0014] Furthermore, in the in vivo induction and sample processing steps, fixation is performed by placing the sample in paraformaldehyde, followed by rinsing with PBS solution, and dehydration is carried out using a gradient dehydration method with sucrose solution.

[0015] Compared with existing technologies, the beneficial effects achieved by this invention are as follows: the stable state of astrocytes depends on the expression of glial cell-specific genes such as Olig2. Pax6 can directly bind to the promoter region of Olig2, inhibiting its transcription and thus blocking the further differentiation of astrocytes into the glial cell lineage. Meanwhile, Neurog2 can indirectly inhibit signaling pathways related to glial cell maintenance by regulating downstream target genes. The combined effect of these two technologies weakens the cellular identity characteristics of astrocytes.

[0016] Neurog2, as a neuropromoting gene, can initiate the expression of neuronal precursor cell marker genes such as Tbr2, promoting the transition of cells to the neuronal precursor stage. It can also induce the expression of glutamatergic neuron-specific genes, determining the specific subtype attributes of neurons. Pax6 can activate a series of genes related to neuronal fate determination during neural development, and interact with signaling pathways such as Wnt, working synergistically with Neurog2 to construct the gene expression network required for neuronal development.

[0017] Neurog2 and Pax6 can synergistically regulate key signaling pathways such as Notch and Wnt. For example, inhibiting the Notch1 signaling pathway, which normally hinders astrocyte transdifferentiation, can further upregulate the expression of neuron-related transcription factors such as Neurod1. At the same time, activating the Wnt / β-catenin signaling pathway can promote cell differentiation into neurons and inhibit their reversion into glial cells, ultimately driving astrocytes to form neuronal process structures morphologically and possess the electrophysiological characteristics of neurons functionally.

[0018] In summary, overexpression of Neurog2 and Pax6 can induce transdifferentiation of astrocytes into neurons, thereby improving the efficiency of astrocyte transdifferentiation into neurons. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1 This is a purity identification diagram of primary astrocytes cultured in vitro according to an embodiment of the present invention; (AC) Immunofluorescence was used to identify the expression of glial fibrillary acidic protein (GFAP) and neuronal markers in primary astrocytes. GFAP is a green signal, while microtubule-associated protein 2 (Map2), neuronal nuclear antigen (NeuN), and ion-calcium-binding adaptor molecule 1 (Iba1) are red signals. Scale bar = 100 μm. (D) Cultured cells expressed astrocyte markers but not neuronal or microglia markers. The figure shows the mean percentage of cells expressing the indicator markers (mean ± standard error (SEM), n = 3 independent experiments).

[0021] Figure 2 These are morphological changes in cells of the control group and overexpression group in this embodiment of the invention. (A) Control group: Cell morphology of the control group observed under a phase-contrast microscope 4 days after viral infection. Scale bar = 100 μm. (B) Overexpression group: Cell morphology of the overexpression group observed under a phase-contrast microscope 4 days after viral infection.

[0022] Figure 3 is an immunofluorescence analysis of the neuronal marker dicortin (DCX) in the process of Neurog2-Pax6-induced astrocytes transforming into neurons according to an embodiment of the present invention. The results were obtained at 4 days (4-DPI) and 7 days (7-DPI) post-infection (the size of the figure has been adjusted to ensure that the content is clear and legible). (A) Immunofluorescence analysis of DCX expression 4 days post-infection (4 dpi); (B) Immunofluorescence analysis of DCX expression 7 days post-infection (7 dpi). Green signal represents green fluorescent protein (GFP), red signal represents red fluorescent protein (mCherry), and blue signal represents DCX. Scale bar = 50 μm.

[0023] Figure 4 shows the immunofluorescence analysis of β3-tubulin (TUBB3) and neuronal nuclear antigen (NeuN) during the conversion of astrocytes into neurons induced by Neurog2-Pax6 in this embodiment of the invention. (A) Immunofluorescence analysis of TUBB3 expression 7 days post-infection (7 DPI); (B) Immunofluorescence analysis of NeuN expression 7 days post-infection (7 DPI); (C) Immunofluorescence analysis of NeuN expression 14 days post-infection (14 DPI). Green signal represents green fluorescent protein (GFP), red signal represents red fluorescent protein (mCherry), and blue signal represents TUBB3 or NeuN. Scale bar = 50 μm.

[0024] Figure 5 This is an immunofluorescence analysis of NeuN and MAP2 28 days after Neurog2-Pax6 induced astrocytes to transform into neurons according to an embodiment of the present invention. (A) Immunofluorescence analysis of NeuN expression at 28 days post-infection (28 DPI); (B) Immunofluorescence analysis of MAP2 expression at 28 days post-infection (28 DPI). Green signals represent green fluorescent protein (GFP), red signals represent red fluorescent protein (mCherry), and blue signals represent NeuN and MAP2. Scale bar = 20 μm.

[0025] Figure 6 shows the mRNA expression levels of neuronal markers in the control group and the overexpression group at different time points after viral infection in an embodiment of the present invention. (A) mRNA expression levels of glial fibrillary acidic protein (Gfap), dicortin (Dcx), and β3-tubulin (Tubb3) in the control group and overexpression group 4 days post-infection (4 DPI); (B) mRNA expression levels of Gfap, Dcx, Tubb3, and neuronal nuclear antigen-associated gene (Rbfox3) in the control group and overexpression group 7 days post-infection (7 DPI).

[0026] Figure 7 This is a diagram showing the cell type diversity induced by Neurog2-Pax6 (NP) in the process of astrocyte-to-neuron (AtN) conversion in an embodiment of the present invention; (A) Grouping information for each cell on UMAP at 4 DPI. Neurog in each group. and Pax Cells are shown on the right. (B) Grouping information for each cell on UMAP at 7 DPI. Neurog in each group. and Pax The cells are shown on the right.

[0027] Figure 8 This is a graph showing the proportion of cell types and the expression of biomarkers at different time points after infection according to an embodiment of the present invention; (A) Proportional distribution of all cell types in each sample at 4 DPI (Days Post Infection); (B) Proportional distribution of all cell types in each sample at 7 DPI (Days Post Infection).

[0028] Figure 9 This is a pseudo-time analysis and gene expression pattern diagram of 4 days and 7 days during the reprogramming of astrocytes into neurons according to an embodiment of the present invention; (A) Pseudo-temporal scores of cells on the uniform manifold approximation and projection (UMAP). Two distinct branches were identified at 4 days; (B) Pseudo-temporal scores of cells on the UMAP. Two distinct branches were identified at 7 days.

[0029] Figure 10 This is a diagram illustrating the unique intercellular interactions between different astrocytes and neuronal clusters four days after infection, according to an embodiment of the present invention. Figure 11 This is a diagram illustrating the unique intercellular interactions between different astrocytes and neuronal clusters 7 days after infection, according to an embodiment of the present invention. (A) A circular diagram showing the differences in the number and intensity of interactions between different cell types; (B) A network diagram of the bone morphogenetic protein (BMP) signaling pathway; (C) Major participants in the BMP signaling pathway; (D) A network diagram of the platelet-reactive protein (THBS) signaling pathway.

[0030] Figure 12 This is an electrophysiological recording during the reprogramming of astrocytes into neurons according to an embodiment of the present invention; (A) Differential interference contrast (DIC) images of whole-cell recordings of induced neurons 30 days after infection; (B) Current clamp recordings of induced neurons.

[0031] Figure 13 This is a diagram illustrating a focal cerebral infarction model constructed according to an embodiment of the present invention; (A) Terminal deoxynucleotidyl transferase-mediated dUTP nick-end marker (TUNEL) cell expression; green signal represents TUNEL, and blue signal represents 4',6-diamidindo-2-phenylindole (DAPI). Scale bar = 100 μm; (B) Comparison of apoptosis rates between the control and cerebral infarction groups; (C) Balance beam experiment results between the control and cerebral infarction groups; (D) Cylinder experiment results between the control and cerebral infarction groups. Data are expressed as mean ± standard error (n = 4). Significance level: P < 0.05; P < 0.0001.

[0032] Figure 14 This is a diagram illustrating the detection of virus infection types in Embodiment 5 of the present invention; (A) Day 14 post-injection, glial fibrillary acidic protein (GFAP) was expressed in the control group. The green signal represents enhanced green fluorescent protein (EGFP), the red signal represents red fluorescent protein (mCherry), and the blue signal represents GFAP. Scale bar = 100 μm; (B) Day 28 post-injection, neuronal nuclear antigen (NeuN) was expressed in the control group. The green signal represents EGFP, the red signal represents mCherry, and the blue signal represents NeuN. Scale bar = 100 μm; (C) Day 4 post-injection, GFAP was expressed in the overexpression group. The green signal represents EGFP, the red signal represents mCherry, and the blue signal represents GFAP. Scale bar = 100 μm.

[0033] Figure 15 This is a graph showing the expression levels of neurogenin 2 (Neurog2) and pairing cassette gene 6 (Pax6) mRNA in the control group and the overexpression group, according to an embodiment of the present invention. (A) Neurog2 mRNA expression level on day 4 after viral injection; (B) Neurog2 mRNA expression level on day 7 after viral injection; (C) Neurog2 mRNA expression level on day 14 after viral injection; (D) Neurog2 mRNA expression level on day 28 after viral injection; (E) Pax6 mRNA expression level on day 4 after viral injection; (F) Pax6 mRNA expression level on day 7 after viral injection; (G) Pax6 mRNA expression level on day 14 after viral injection; (H) Pax6 mRNA expression level on day 28 after viral injection.

[0034] Figure 16 This is an expression diagram of neuronal markers in the overexpression group 14 days after viral injection, as detected by immunofluorescence in an embodiment of the present invention. (A) Expression of dicortin (DCX), a marker of immature neurons, in the overexpression group on day 14 post-viral injection. Green signal represents enhanced green fluorescent protein (EGFP), red signal represents red fluorescent protein (mCherry), and blue signal represents DCX. Scale bar = 100 μm; (B) Expression of neuronal nuclear antigen (NeuN), a marker of mature neurons, in the overexpression group on day 14 post-viral injection. Green signal represents EGFP, red signal represents mCherry, and blue signal represents NeuN. Scale bar = 100 μm.

[0035] Figure 17 This is an expression diagram of neuronal markers overexpressing the virus at 14 and 28 days after viral injection, as detected by immunofluorescence in an embodiment of the present invention. (A) Expression of the neuronal marker Cleavage Box 1 (Cux1) in the overexpression group on day 14 post-viral injection. Green signal represents enhanced green fluorescent protein (EGFP), red signal represents red fluorescent protein (mCherry), and blue signal represents Cux1. Scale bar = 100 μm; (B) Expression of the mature neuronal marker Neuronal Nuclear Antigen (NeuN) in the overexpression group on day 28 post-viral injection. Green signal represents EGFP, red signal represents mCherry, and blue signal represents NeuN. Scale bar = 100 μm.

[0036] Figure 18 This is an expression diagram of related neuronal genes in the empty vector group and the overexpression group at different time points after viral injection in an embodiment of the present invention; (A) mRNA expression levels of dicortin (Dcx) and β3-tubulin (Tubb3) in the empty vector group and overexpression group on day 4 after viral injection; (B) mRNA expression levels of Dcx, Tubb3 and neuronal nuclear antigen-associated gene (Rbfox3) in the empty vector group and overexpression group on day 7 after viral injection; (C) mRNA expression levels of Dcx, microtubule-associated protein 2 (Map2) and Rbfox3 in the empty vector group and overexpression group on day 14 after viral injection; (D) mRNA expression levels of Tubb3, Map2 and Rbfox3 in the empty vector group and overexpression group on day 28 after viral injection.

[0037] Figure 19 This invention includes single-cell metabolic analysis and reactive oxygen species analysis at different time points after virus injection in an embodiment of the invention. (A) Single-cell metabolic analysis of the overexpression group on day 4 after viral injection; (B) Single-cell metabolic analysis of the overexpression group on day 7 after viral injection; (C) Reactive oxygen species (ROS) level on day 4 after viral injection.

[0038] Figure 20 This is a graph showing the recovery of motor function in mice at different time points after viral injection in an embodiment of the present invention.

[0039] (AB) Balance beam test: to assess the balance ability and motor coordination of mice; (CD) Cylinder test: to assess the frequency of use and symmetry of the forelimbs of mice, used to analyze the recovery of motor function. Detailed Implementation

[0040] The embodiments of the present invention will be described in detail below with reference to the accompanying drawings. These embodiments are implemented based on the technical solution of the present invention and provide detailed implementation methods and specific operation processes. However, the scope of protection of the present invention is not limited to the following embodiments.

[0041] This embodiment discloses a method for improving the efficiency of transdifferentiation induction of astrocytes into neurons, comprising the following steps: I. Core Steps of In Vitro Transdifferentiation Induction Preparation of primary astrocytes (ensuring cell purity and viability): Tissue isolation and purification: Tissue was isolated from the cerebral cortex of C57BL / 6 mice 5-7 days after birth. The subventricular region, striatum and hippocampus were strictly removed (to avoid contamination with neural progenitor cells or other glial cells and to ensure the purity of astrocytes). The cortical tissue was placed in frozen HBSS (Hankes balanced salt solution, used to maintain cell low-temperature activity) and gently separated using autoclaving instruments (to reduce mechanical damage).

[0042] Centrifugation and resuspension: The cell suspension was centrifuged at 1000×g and 4°C for 5 minutes (low-speed centrifugation avoids cell rupture and low temperature inhibits metabolism). The pellet was resuspended in DMEM / F12 (Durbeco modified Eagle medium / Ham F-12 nutrient mixture) medium containing 10% inactivated fetal bovine serum + 1% penicillin-streptomycin (serum provides growth factors, penicillin-streptomycin dual antibiotics prevent contamination and lay the foundation for cell proliferation).

[0043] Culture and purification: Seeds were placed in T-25 culture flasks coated with 10 μg / mL PDL (poly-D-lysine) (PDL promotes cell adhesion, and astrocytes are highly adhesion-dependent), and cultured at 37°C in a humidified incubator with 5% CO2 for 7-10 days until confluence; after confluence, the cells were mechanically agitated (180-200 rpm for 1-2 hours) to selectively remove microglia and oligodendrocyte precursor cells with weak adhesion (purifying astrocytes, with a purity of over 90%); the remaining cells were enzymatically digested with 0.25% trypsin-EDTA (37°C for 1-2 minutes, avoiding over-digestion) to obtain single, dispersed astrocytes.

[0044] Phenotypic identification of cultured astrocytes was performed using immunofluorescence staining. Immunofluorescence results showed that the cultured astrocytes highly expressed glial fibrillary acidic protein (GFAP), a specific marker of astrocytes, and no neuronal markers (microtubule-associated protein 2, Map2; neuronal nuclear antigen, NeuN) or microglia marker (ion-calcium-binding adapter molecule 1, Iba1) were detected. The results revealed that the cultured cells were predominantly astrocytes, without contamination by other types of neurons. Figure 1 AC). Cell purity was assessed through quantitative analysis, and the proportion of GFAP-positive cells was calculated. It was found that over 90% of the cultured cells expressed GFAP (AC). Figure 1 D).

[0045] Under a bright-field microscope, the cells exhibited typical spindle-shaped or polygonal morphology, consistent with the morphological characteristics of astrocytes. Figure 1 E). This morphological feature further supports the results of immunofluorescence staining, indicating that high-purity astrocytes were successfully isolated and cultured.

[0046] Subculturing and Plating: The night before, place cell smears into 24-well plates and coat them with PDL (poly-D-lysine) (to ensure uniform cell adhesion). Discard the old culture medium during subculturing, wash three times with PBS (to remove residual serum and avoid affecting trypsin activity), add 1 mL of 0.25% trypsin, gently agitate to cover the bottom of the flask, digest at 37°C for 1 minute, and immediately add 2 mL of culture medium to stop the reaction (precisely control the digestion time to maintain cell viability). Centrifuge at 1000 rpm for 5 minutes, resuspend, and count cells using a cell counting chamber at 1×10⁻⁶. 4 Seeding density per well (moderate density to avoid overcrowding or sparseness of cells, which is beneficial for subsequent transduction).

[0047] 2. Key Operations for Transdifferentiation Induction (Precision Delivery)

[0048] Lentiviral preparation and concentration (ensuring viral activity and titer): HEK293T cells were seeded into 10 cm culture dishes. When the confluence reached 70%-80%, the packaging plasmid was transfected (using high-efficiency transfection reagents such as Lipofectamine 3000, with a transfection efficiency >80%). Viral supernatant was collected twice, at 48 and 72 hours post-transfection (double collection increases viral yield). The supernatant was filtered through a 0.45 μm filter (to remove cell debris) and ultracentrifuged at 25,000 rpm (100,000 × g) for 2 hours at 4°C (high-speed centrifugation concentrates viral particles, avoiding repeated freeze-thaw cycles that could affect activity). Fluorescence-activated cell sorting (FACS) was used to detect the fluorescence intensity of transduced HEK293T cells, confirming a viral titer of 1 × 10⁻⁶. 8 -1×10 9Infectious particles / mL (too low a titer will result in insufficient transduction efficiency, while too high a titer may cause cytotoxicity).

[0049] Viral transduction (enhancing transdifferentiation factor delivery efficiency): Astrocytes at 4 × 10 4 Cells were seeded at a density of cells / well in 24-well PDL-coated plates (cell density matched with virus dosage to ensure each cell could come into contact with virus particles). 24 hours after seeding (when cells are in the logarithmic growth phase and transduction efficiency is highest), lentivirus was added and the multiplicity of infection (MOI) was set to 20 (preliminary experiments verified that the transduction efficiency at this MOI was >70% and there was no obvious cytotoxicity).

[0050] Differentiation medium and neurotrophic factor intervention (to promote transdifferentiation): 24 hours after transduction, the medium was completely replaced with differentiation medium: DMEM / F (Dürbeco modified Eagle medium / Ham F-12 nutrient mixture)-12 + 0.5% FBS (fetal bovine serum) + 2% B27 (B27 cell culture additive) + 1% GlutaMax (L-glutamine substitute (GlutaMax)) + 1% penicillin / streptomycin. The B27 (B27 cell culture additive) additive contains multiple neurotrophic components (such as vitamin A, insulin, transferrin), which inhibit glial cell proliferation and promote neuronal differentiation. 50% of the medium was replaced every 4 days (to avoid depletion of medium components and maintain a stable microenvironment), and 20 ng / mL neurotrophic factor (BDNF) was added at the same time (BDNF can promote neuronal survival, neurite growth and synapse formation, and significantly improve the maturity of transdifferentiated neurons). The medium was cultured for about 30 days (the peak expression of mature neuronal markers usually occurs around 30 days).

[0051] II. Core Steps of In Vivo Transdifferentiation Induction

[0052] 1. Animal models and targeted induction (precise localization of lesion areas)

[0053] Establishment of a focal cerebral infarction model (simulating a pathological environment): ALDH1L1-Cre mice (astrocytocyte-specific Cre tool mice, ensuring that transdifferentiation factors are expressed only in astrocytes) were anesthetized by intraperitoneal injection of 1.25% Avertin (0.2 mL / 10 g) and fixed in a stereotactic apparatus; the projection areas of the cerebral cortex were located (AP: +0.2 mm; ML: -1.35 mm; DV: -0.5 mm, corresponding to the right limb motor area, infarction of this area can simulate the motor function impairment model after stroke), and 0.5 μL of ET-1 (endothelin-1, which constricts blood vessels to cause local ischemia and form a stable infarct) was injected at a rate of 0.05 μL / min using a 33G microsyringe. The needle was left in place for 5 minutes after injection and then slowly withdrawn (to avoid backflow of the drug and ensure that the size of the infarct is uniform).

[0054] TUNEL staining results showed that the apoptosis rate in the cerebral infarction group was significantly higher than that in the control group, and the difference between the two groups was statistically significant (P<0.05). Figure 14 AB). The number of neuronal apoptosis was significantly increased in the cerebral infarction model group, suggesting that cerebral infarction induces significant cell damage. Behavioral experiments showed that, compared with the control group, the stroke group performed significantly worse in the balance beam and cylinder tests. Figure 14 (C) further confirmed that the motor function of the mice in the cerebral infarction model group was impaired. The results of TUNEL staining and behavioral experiments together indicate that a stable model of cerebral infarction has been successfully constructed.

[0055] Targeted viral injection (to achieve specific transdifferentiation of astrocytes in the lesion area): 4 and 14 days after the establishment of the infarction model (corresponding to the acute and subacute phases of infarction, respectively, during which astrocytes are activated and have a high transdifferentiation efficiency), AAV-Neurog2-Pax6 virus (Neurog2 and Pax6 are key transcription factors for neurogenesis, and their synergistic effect can significantly improve the transdifferentiation efficiency of astrocytes into neurons) was injected at the same stereotactic coordinates. The viral concentration was 2.0 × 10¹² vg / mL (higher viral concentrations are required in vivo to improve the transduction efficiency of astrocytes in the lesion area), the injection rate was 0.05 μL / min, the total volume was 0.5 μL, and the needle was left in place for 5 minutes (to ensure that the virus fully diffuses into the astrocytes in the lesion area).

[0056] 2. Sample processing after in vivo induction (preserving the integrity of transdifferentiated cells)

[0057] After induction, tissue samples were collected at 7, 14, and 28 days. Mice were anesthetized and perfused with 4% paraformaldehyde (prepared in PBS) via the heart. Brain tissue was immediately fixed in 4% paraformaldehyde pre-cooled at 4°C for 2-6 hours (rapid fixation maintains cell morphology and antigenicity, and avoids apoptosis of transdifferentiated cells). After fixation, the tissue was rinsed three times with PBS (10 minutes each time) and then transferred to a 30% sucrose solution (4°C) for gradient dehydration (the sucrose solution was changed every 24 hours until the tissue was completely settled, to prevent ice crystals from damaging cells during cryosectioning). The tissue was embedded in OCT (optimal cutting temperature) embedding medium and prepared into 15 μm thick serial sections using a -20°C cryostat (the section thickness is moderate, ensuring cell integrity and facilitating subsequent immunofluorescence staining to observe the morphology of transdifferentiated cells).

[0058] Quantitative Polymerase Chain Reaction

[0059] Samples were collected from astrocytes or brain tissue, immediately flash-frozen in liquid nitrogen, and stored at -80°C. RNA extraction was performed using the TransZol Up Plus RNA Kit (Cat. No. ER501), following the kit's instructions. The concentration and purity of the extracted RNA were measured using a NanoDrop spectrophotometer to ensure the RNA quality met the requirements for subsequent experiments. The extracted RNA was reverse transcribed into cDNA using TransScript Uni All-in-One First-Strand cDNA Synthesis SuperMix for qPCR (Cat. No. AU341-02) as a template for qPCR. cDNA synthesis was performed in a 20 μL system containing 4 μL 5×TransScript Uni All-in-One Mix, 1 μL dNTP Mix, 1 μL PrimerScript RT Enzyme Mix, and 4 μL RNA template. The reaction conditions were: 37℃ for 15 minutes, 85℃ for 5 seconds to terminate the reaction, and stored at 4℃ for later use.

[0060] The total volume of the qPCR reaction system was 20 μL, containing 10 μL of 2×qPCR Mix, 0.4 μL of forward and reverse primers (10 μM), 2 μL of cDNA template, and 7.2 μL of ddH2O. The qPCR reaction conditions were: 95℃ pre-denaturation for 3 minutes, followed by 40 cycles of 95℃ denaturation for 15 seconds and 60℃ annealing / extension for 30 seconds. After the reaction, melting curve analysis was performed (fluorescence signal was collected every 0.5℃ from 60℃ to 95℃) to verify the specificity of the amplified products and ensure the absence of non-specific amplification and primer dimer interference.

[0061] Statistical methods

[0062] For immunofluorescence analysis, images were acquired using a BX53 fluorescence microscope (Olympus Corporation), and quantitative analysis was performed using ImageJ software. All imaging parameters and subsequent analysis settings were standardized for each antigen. Under each experimental condition, 15 fields of view were randomly selected from each coverslip for analysis, involving a total of 3 coverslips, and 3 batches of independent samples were evaluated (biological replicates).

[0063] In qPCR analysis, gene expression levels were quantified using the 2^(-ΔΔCt) method and standardized using Gapdh as an internal reference. Results are presented as mean ± standard error (SEM), and data were obtained from three biological replicates. Statistical significance was determined using an unpaired two-tailed t-test (GraphPad Prism 8), with p < 0.05 as the significance threshold.

[0064] Co-overexpression of Neurog2-eGFP and Pax6-mCherry was achieved by infecting astrocytes with lentivirus. The lentiviral vector used a ubiquitin (Ubi) promoter to drive the co-overexpression of Neurog2 and Pax6, and integrated an SV40 enhancer element to enhance transcriptional activity. The control group used a lentiviral vector that co-overexpressed Ubi-MCS-SV40-eGFP and Ubi-MCS-SV40-mCherry to exclude possible interference from the lentiviral transfection process.

[0065] During the experiment, no significant morphological changes were observed in the control group cells under phase-contrast microscopy. Figure 2 A). After overexpression of transcription factors Neurog2 and Pax6 in astrocytes, cell morphology changed: on day 4, the cell bodies became smaller and more compact, exhibiting neurite-like structures. Figure 2 B).

[0066] Immunofluorescence results showed that at 4 days (4 DPI) and 7 days (7 DPI) post-lentiviral infection, no expression of the early neuronal marker dicortin (DCX) was detected in control astrocytes. Conversely, DCX expression was detectable in astrocytes reprogrammed with Neurog2-Pax6 at 4 DPI, and also at 7 DPI. Figure 3 AB). At 7 DPI, cells induced to highly express neuron-specific β-3 tubulin (TUBB3) and simultaneously begin expressing neuronal nuclear antigen (NeuN), a marker of mature neurons. Figure 4 AB). NeuN was induced to persist in cells 14 days post-infection ( ). Figure 4 C), further confirmation of NeuN and microtubule-associated protein 2 (MAP2) expression at 28 DPI (C) Figure 5 AB).

[0067] To elucidate the molecular mechanisms of transdifferentiation, time-series analysis of key gene expression was performed using real-time quantitative PCR (RT-qPCR). At 4 DPI, a significant downregulation of the astrocyte marker glial fibrillary acidic protein (Gfap) was observed, while significant upregulation of early neuronal markers Dcx and Tubb3 was observed, suggesting the initiation of the neuronal differentiation process. Figure 6 A). By the 7 DPI stage, Gfap expression continued to decrease, while Dcx and Tubb3 maintained high expression levels, and the expression of Rbfox3, a marker of mature neurons, significantly increased. Figure 6 B) indicates the continued advancement of neuronal differentiation.

[0068] To dissect the molecular events in the AtN reprogramming process, single-cell RNA sequencing (scRNA-seq) was performed to investigate the transcriptome of single cells collected at 4 and 7 days post-infection. Unsupervised dimensionality reduction and visualization were performed using the uniform manifold approximation and projection (UMAP) method, classifying cells into five cell types at 4 days and six cell types at 7 days. Each time point was analyzed as two independent groups: a control group and an NP overexpression (OE) group. Notably, at 4 days, cells were predominantly identified as Ast_1, Ast_2, Ast_3, Neu_1, and Neu_2. In contrast ( Figure 7 A), at 7 days, the cells were mainly identified as Ast_1, Ast_2, Ast_3, Ast_4, Neu_1 and Neu_2 (A). Figure 7 B). Specifically, in the control group, cells were primarily identified as Ast_1 and Ast_3 at day 4, and as Ast_1, Ast_3, and Ast_4 at day 7. In the NP group, cells were primarily identified as Neu_1 and Neu_2 at both days 4 and 7. Figure 8 AB).

[0069] Pseudo-temporal analysis was performed to elucidate the relationships between various cellular subclusters in cell fate determination. This analysis reconstructs the temporal progression of cellular states and identifies key molecular changes driving differentiation. The analysis identified two distinct developmental trajectories originating from the Ast_1 cell type, the initiating cell of transdifferentiation. One trajectory leads to differentiation into astrocytes, while the other leads to differentiation into induced neurons. Figure 9 A, B).

[0070] Using CellChat, we mapped the interactions between all identified cell types based on single-cell RNA sequencing (scRNA-seq) data. We paid particular attention to the communication between Ast_1 cells, which are the initiating cells for astrocyte-to-neuronal transdifferentiation (AtN), and neuronal clusters at 4 and 7 days post-infection (DPI).

[0071] The frequency and intensity of interactions between astrocyte clusters and neuronal clusters were demonstrated. Figure 10 A).

[0072] At 4 days, intercellular communication was primarily regulated by the BMP signaling pathway and the atypical Wnt (ncWnt) signaling pathway. Figure 10 BD). The interaction between astrocyte subsets and neuronal subsets was characterized by its frequency and intensity. Figure 11 A). By day 7, the primary signaling pathway had shifted to the BMP and THBS pathways. Figure 11 BD).

[0073] To assess the functional maturity of reprogrammed neurons, electrophysiological experiments were performed 30 days after astrocyte infection with a virus. The results showed that the reprogrammed neurons exhibited an immature electrophysiological phenotype. Figure 12 (AB). This finding suggests that although cells have successfully transitioned to neuronal fate, they are not yet fully mature in terms of electrophysiological properties. This immature phenotype may be attributed to ongoing synaptic refinement and the need for further cell integration into neural circuits. The observed electrophysiological immaturity is consistent with pathway enrichment data, indicating that reprogrammed neurons are still establishing functional synaptic connections and maturing their intrinsic membrane properties.

[0074] A focal cerebral infarction model was successfully constructed. Figure 13 The cell tropism of adeno-associated virus (AAV) was systematically evaluated by cortical injection of AAV expressing green fluorescent protein (GFP) and red fluorescent protein (mCherry) into a mouse model of focal cerebral infarction. The results showed that on day 14 post-injection, GFP- and mCherry-labeled cells in the control group mice exhibited a positive immunoreactivity to the astrocyte-specific marker glial fibrillary acidic protein (GFAP). Figure 14 A) indicates that adeno-associated virus (AAV) can effectively infect astrocytes. However, on day 28 post-injection, GFP and mCherry-labeled cells in the control group mice did not co-label with the neuron-specific nuclear marker NeuN (A). Figure 14 B), which further confirms that adeno-associated virus primarily infects astrocytes, rather than neurons, in this model. In the experimental group overexpressing Neurog2 and Pax6, the observations on day 4 post-infection were consistent with the control group, namely, the infected cells were mainly astrocytes (B). Figure 14 C). This result indicates that adeno-associated virus (AAV) exhibits a significant tropism towards astrocytes in both the control and experimental groups.

[0075] Real-time quantitative polymerase chain reaction (qPCR) was used to assess the expression levels of the experimental and control mice on days 4, 7, 14, and 28 after injection of the overexpressing virus. Results showed that at all time points, the mRNA expression levels of Neurog2 and Pax6 in the experimental group were significantly higher than those in the control group. Figure 15 Adeno-associated virus (AH)-mediated overexpression of Neurog2 and Pax6 was effectively achieved in the experimental group and was able to maintain high expression levels for a relatively long period of time.

[0076] Immunofluorescence results showed that on day 14 after overexpression of Neurog2 and Pax6, cells induced to express early neuronal marker Dcx, mature neuronal marker NeuN, and cortical neuronal marker Cux1. Figure 16 , Figure 17 A). By day 28, the induced cells continued to express NeuN, a marker of mature neurons. Figure 17 (B) indicates that astrocytes, after overexpressing Neurog2 and Pax6, gradually differentiate into neurons and eventually acquire the phenotypic characteristics of mature neurons. This confirms at the protein level the crucial roles of Neurog2 and Pax6 in the reprogramming of astrocytes into neurons.

[0077] To further validate this process at the gene expression level, the expression dynamics of neuron-related genes were detected using qPCR (quantitative real-time polymerase chain reaction). Compared with the control group, the Neurog2-Pax6 group showed significantly higher expression of neuron-related genes at different time points. On day 4, the expression levels of early neuronal markers Dcx and Tubb3 were significantly increased (…). Figure 18 A); On days 7 and 14, the expression levels of Dcx, Map2, and NeuN significantly increased ( Figure 18 BC); On day 28, the expression levels of Tubb3, Map2, and NeuN were also significantly higher than those in the control group (BC). Figure 18 (D) Overexpression of Neurog2 and Pax6 significantly promoted the differentiation of astrocytes into neurons and sequentially activated the expression of early and mature neuronal markers at different time points, revealing the dynamic changes in gene expression during reprogramming. Immunofluorescence and qPCR results at both protein and gene expression levels indicated that overexpression of Neurog2 and Pax6 effectively induced astrocyte reprogramming into neurons, gradually acquiring the phenotypic and gene expression characteristics of mature neurons during differentiation.

[0078] In the early stages of somatic cell reprogramming, reactive oxygen species (ROS) levels rise rapidly. On days 4 and 7 after overexpression, significantly elevated ROS levels were observed in cells induced by the overexpression group, showing a significant difference compared to the control group. Figure 19 AB).

[0079] To evaluate the effect of Neurog2-Pax6 treatment on the recovery of motor function in a mouse model of cerebral infarction, behavioral tests were performed between 14 and 42 days after viral injection. The foot error rate was significantly reduced in the Neurog2-Pax6 treatment group, indicating partial recovery of motor coordination function after treatment. Figure 20 A). The transdifferentiation of stellate glial cells into neurons may help improve motor dysfunction after cerebral infarction.

[0080] However, within the same time period, the Neurog2-Pax6 treatment group did not show significant recovery in behavioral tests involving contact with the lateral wall using the injured forelimb. Figure 20 B). This result indicates that although Neurog2-Pax6 treatment has a certain effect on improving overall motor coordination, its effect on the recovery of forelimb function is limited, and may require a longer treatment period or combination with other treatment strategies to further promote functional recovery.

[0081] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of protection claimed by the present invention. The scope of protection of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for improving the efficiency of astrocyte transdifferentiation induction into neurons, characterized in that, Includes the following steps: Primary astrocyte preparation steps: Isolate the cortical tissue to obtain a cell suspension, and then culture and purify the cells to obtain astrocytes; Transdifferentiation induction steps: After the lentivirus was prepared and concentrated in HEK293T cells and the titer was detected, the lentivirus was added to astrocytes for transduction. Then, the astrocytes were continuously cultured by changing the differentiation medium and adding BDNF regularly to promote the transdifferentiation of astrocytes into mature neurons. Animal model and targeted induction steps: After establishing a focal cerebral infarction model, AAV-Neurog2-Pax6 virus was injected into the lesion area at a specific time to achieve specific transdifferentiation of astrocytes in the lesion area. Sample processing steps after in vivo induction: After in vivo induction, samples are taken at specific time points, and then subjected to cardiac perfusion fixation, dehydration, OCT embedding, and frozen sectioning to preserve the integrity of transdifferentiated cells.

2. The method for improving the efficiency of astrocyte transdifferentiation induction into neurons according to claim 1, characterized in that, The primary astrocyte preparation steps include: Tissue isolation and purification steps: Tissue was isolated from the mouse cerebral cortex, and the subventricular region, striatum and hippocampus were removed. The cortical tissue was placed in frozen HBSS and gently separated using an autoclave to obtain a cell suspension. Centrifugation and resuspension steps: Centrifuge the cell suspension, and resuspend the pellet in culture medium after centrifugation; Culture and purification steps: The cells are cultured in an incubator until they merge. After merging, they are mechanically shaken to selectively remove impurities and purify astrocytes. The remaining purified cells are digested with proteases. Subculture and plating steps: Place cell smears in culture plates in advance and coat them with PDL. When subculturing, discard the old culture medium and wash with PBS. Add an appropriate amount of trypsin to digest the cells and then stop the reaction with culture medium. Centrifuge to resuspend the cells and count them. Seed the cells into culture plates at an appropriate density.

3. The method for improving the efficiency of astrocyte transdifferentiation induction into neurons according to claim 2, characterized in that, The culture medium used in the centrifugation and resuspension steps is DMEM / F12 medium containing inactivated fetal bovine serum and penicillin-streptomycin.

4. The method for improving the efficiency of astrocyte transdifferentiation induction into neurons according to claim 2, characterized in that, In the culture and purification steps, enzymatic hydrolysis was performed using 0.25% trypsin-EDTA.

5. The method for improving the efficiency of astrocyte transdifferentiation induction into neurons according to claim 2, characterized in that, In the aforementioned passaging and tiling step, 1×10 4 Inoculation density per well.

6. The method for improving the efficiency of astrocyte transdifferentiation induction into neurons according to claim 1, characterized in that, The differentiation medium contained DMEM / F-12, brought to a final volume of 50 mL, and included 0.5% FBS, 2% B27, 1% Glutamax, and 1% penicillin-streptomycin antibiotics.

7. The method for improving the efficiency of astrocyte transdifferentiation induction into neurons according to claim 1, characterized in that, The specific time periods referred to are the acute and subacute phases of infarction.

8. The method for improving the efficiency of astrocyte transdifferentiation induction into neurons according to claim 1, characterized in that, The concentration of the AAV-Neurog2-Pax6 virus was 2.0 × 10⁻⁶. 12 vg / mL.

9. The method for improving the efficiency of astrocyte transdifferentiation induction into neurons according to claim 1, characterized in that, In the in vivo induction and sample processing steps, the sample is fixed in paraformaldehyde, then rinsed with PBS solution, and dehydrated using a gradient dehydration method with sucrose solution.

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