Monolentivirus vector for inducing differentiation of glutamatergic neurons, preparation method and medical use

Abstract

The present disclosure belongs to the field of biotechnology and life science, and discloses a single lentivirus vector for inducing differentiation of glutamatergic neurons, a preparation method and medical use. In view of the technical defects of the existing double lentivirus induction differentiation system, the reverse tetracycline-regulated transcriptional activator rtTA3, the Ngn2 expression element containing the TRE promoter, and the puromycin resistance screening gene Puro are integrated in a single lentivirus vector. The present disclosure develops a single lentivirus vector-based high-efficiency induction method of glutamatergic neurons, which reduces the cost and workload of lentivirus preparation compared with the double virus transfection strategy. Through the "single infection + pre-screening" strategy, the large-scale preparation of glutamatergic neurons with high survival rate, high purity and high uniformity is realized, and a good cell model is provided for the pathogenesis and drug screening of neurodevelopmental diseases.

Description

Technical Field

[0001] This disclosure pertains to the fields of biotechnology and life sciences, and specifically relates to a single lentiviral vector for inducing differentiation of glutamatergic neurons, its preparation method, and its medical applications. Background Technology

[0002] The pathogenesis of neurological diseases is complex, and obtaining living human neurons presents challenges such as difficulty in acquiring samples, scarcity of samples, and ethical restrictions, making it difficult to directly apply them to research on neurodevelopmental mechanisms, pathological mechanism analysis, drug screening, and cell replacement therapy experiments. Human induced pluripotent stem cells (hiPSCs), possessing unlimited self-renewal capacity and multi-directional differentiation potential, can be directed to differentiate into functional neurons in vitro. This avoids the difficulty of obtaining primary human neurons and has become a core tool for studying the pathogenesis of neurodevelopmental diseases, high-throughput drug screening, and the development of neurocellular replacement therapies, demonstrating extremely high scientific research value and promising prospects for clinical translation.

[0003] Currently, the conventional induction method for the directed differentiation of hiPSCs into neurons is mainly the chemical small molecule induction method. This method usually employs a combination of dual SMAD inhibitors to mimic the physiological process of neural development in vivo, inhibiting non-neural lineage differentiation and thus promoting the differentiation of hiPSCs into neural cells. However, this traditional differentiation technique has significant technical shortcomings: the differentiation cycle is lengthy, often requiring several months to cultivate mature functional neurons; the differentiation process has poor controllability, significant batch-to-batch variability, unstable differentiation efficiency, and high cellular heterogeneity, making it difficult to meet the experimental requirements for large-scale, standardized neuron preparation, which seriously restricts the advancement of basic neuroscience research and drug development.

[0004] To overcome the drawbacks of traditional chemical induction methods, such as long cycles and low efficiency, current mainstream technologies employ a neurogenin-2 (Ngn2) forced expression system to achieve rapid and targeted conversion of hiPSCs into functional neurons. Ngn2 is a key transcription factor in neurogenesis, which can efficiently drive the targeted differentiation of stem cells into neuronal lineages. This differentiation technique has the advantages of short differentiation cycle, high conversion efficiency, and simple operation. The differentiated neurons can form normal functional synaptic structures and possess good neurobiological activity. It is a high-quality cell model for studying neuronal morphological development, synaptic function, and modeling nervous system diseases, providing a reliable experimental tool for elucidating the pathogenic mechanisms of neurodevelopment-related diseases.

[0005] The most widely used Ngn2-induced differentiation strategy currently is the dual-lentiviral co-infection strategy, a classic approach published by Yingsha Zhang et al. in *Neuron* in 2013 (DOI: 10.1016 / j.neuron.2013.05.029). This dual-viral system contains two functionally distinct lentiviral vectors: Virus 1 expresses the inverse tetracycline-regulated transcriptional activator protein rtTA; Virus 2 contains the Ngn2 expression element of the TRE promoter and a puromycin resistance selection gene. The mechanism of action is as follows: HiPSCs are typically transfected simultaneously with rtTA lentivirus and TetO / TRE-Ngn2-Puro lentivirus, allowing cells to simultaneously acquire both the rtTA regulatory element and the Dox-inducible Ngn2 expression element. Subsequently, doxycycline (Dox) is added to the culture system. Dox binds to rtTA, enabling rtTA to recognize and bind to the TetO / TRE sequence, thereby activating the transcription of Ngn2 and related puromycin resistance genes. Inducible Ngn2 expression can rapidly initiate neuronal transcription programs, driving hiPSCs to be directed into excitatory-induced neurons; at the same time, cells successfully transduced and expressing Ngn2 can be enriched through puromycin screening.

[0006] Although the above-mentioned Ngn2 differentiation system mediated by dual lentivirus co-infection has significant advantages over traditional small molecule induction techniques, there are still some technical defects that cannot be ignored in actual experimental applications, as follows: (1) High cost of experimental consumables: This scheme requires the separate preparation and purification of two different types of lentiviruses. The virus packaging, amplification and identification process is cumbersome, which greatly increases the experimental time and economic costs; (2) High cytotoxicity: The experiment requires high virus titers to complete the dual infection operation. The virus infection process will cause mechanical damage and biotoxicity to hiPSCs, which can easily induce apoptosis and cause a large number of cells to die. (3) Complex transfection operation and unstable induction efficiency: The dual-virus co-transfection process is cumbersome, and the infection efficiency of the two viruses is difficult to keep consistent. The infection ratio of the two viruses needs to be repeatedly adjusted and optimized during the experiment. The human operation error is large, resulting in significant differences in differentiation induction effect between batches and poor reproducibility; (4) Difficulty in cell line construction and poor experimental reusability: The puromycin drug screening of the existing dual-virus system can only be carried out after Dox-induced differentiation is initiated. It is impossible to complete the screening of positive transfected cells in the hiPSC stem cell stage and obtain a stable hiPSC cell line integrating dual viruses. If the experiment requires a large number of neurons or repeated induction experiments, the hiPSC dual-virus transfection operation needs to be repeated. The experimental process is redundant and cumbersome, which greatly reduces the overall experimental efficiency.

[0007] In summary, there is an urgent need to develop a highly efficient method for inducing glutamatergic neurons based on a single lentiviral vector, which would simplify the viral construction and infection process, reduce cytotoxicity, enable positive cell screening at the hiPSC stem cell stage, stably improve Ngn2-mediated neuronal differentiation efficiency, and overcome many shortcomings of existing dual-virus induction techniques. Summary of the Invention

[0008] The purpose of this disclosure is to provide a single lentiviral vector for inducing the differentiation of glutamatergic neurons, its preparation method and medical applications, simplifying the virus construction and infection process, reducing cytotoxicity, enabling positive cell screening at the hiPSC stem cell stage, stably improving the efficiency of Ngn2-mediated neuronal differentiation, and overcoming many defects of existing dual-virus induction technologies.

[0009] The objective of this disclosure is achieved through the following technical solution: In a first aspect of this disclosure, a lentiviral vector for inducing differentiation of glutamatergic neurons is provided, characterized in that it comprises, sequentially along the 5' to 3' direction: a TRE promoter, a Neurog2 coding sequence, a 3×FLAG tag coding sequence, a P2A self-cleaving peptide coding sequence, an EGFP coding sequence, an EF1 promoter, an rtTA3 coding sequence, an IRES element, a puromycin resistance gene, and a WPRE element; wherein, the TRE promoter drives the tandem Neurog2 coding sequence, the 3×FLAG tag coding sequence, the P2A self-cleaving peptide coding sequence, and the EGFP coding sequence to induce transcription and translation, respectively generating a Neurog2-3×FLAG fusion protein and an EGFP protein, wherein the Neurog2-3×FLAG is a fusion protein in which a 3×FLAG tag is fused to the C-terminus of the Neurog2 protein, and the two types of proteins are separated by cleavage by the P2A self-cleaving peptide; the EF1 promoter drives the transcription of the rtTA3 coding sequence, the IRES element, and the Puro gene to form a single mRNA transcript, and mediates the co-expression of the rtTA3 protein and the puromycin resistance protein through the IRES.

[0010] In some specific embodiments of this disclosure, the EF1 promoter is a constitutive promoter whose transcriptional activity is not regulated by expression elements containing TRE promoters.

[0011] In a second aspect of this disclosure, a method for inducing glutamatergic neurons using the aforementioned single lentiviral vector is provided, comprising the following steps: (1) The single lentiviral vector was transfected into hiPSC cells to obtain transfected mixed cells; (2) Under conditions without the addition of Doxycycline inducer, the mixed cells were screened by puromycin pressure to remove negative cells that did not integrate the foreign gene and obtain a hiPSC cell line with stable gene integration. (3) Doxycycline was added to stable hiPSC cell lines for induction culture, and glutamatergic neurons were obtained by directed differentiation.

[0012] In some specific embodiments of this disclosure, the puro resistance selection gene is configured with an independent promoter regulatory element EF1, which is induced to express independently of the Ngn2 expression element containing the TRE promoter.

[0013] In some specific embodiments of this disclosure, the puromycin screening treatment on hiPSC cells lasts for 3-7 days, and the working concentration of puromycin is 0.75-1 ug / ml.

[0014] In some specific embodiments of this disclosure, the doxycycline induction concentration is 2 µg / mL, and the doxycycline neuron induction culture is continuously induced.

[0015] In some specific embodiments of this disclosure, the induction culture adopts a graded culture medium system, which includes at least stem cell maintenance medium, neural induction medium, and neuron maturation medium. The replacement time points of each culture medium are as follows: Day 0 uses stem cell maintenance medium, Day 1-4 uses neural induction medium, and Day 5 onwards uses neuron maturation medium.

[0016] In some specific embodiments of this disclosure, the method uses CMV2 forward sequencing primers with nucleotide sequences as shown in SEQ ID NO:4 and EGFP reverse sequencing primers as shown in SEQ ID NO:5 to complete vector molecule identification.

[0017] In a third aspect of this disclosure, a glutamatergic neuron prepared according to the foregoing method is provided, wherein the glutamatergic neuron expresses at least one of the mature neuron markers vGLUT1, TUBB3, MAP2, and NeuN.

[0018] In a fourth aspect, this disclosure provides the use of the aforementioned lentiviral vector in the preparation of a kit for inducing differentiation of glutamatergic neurons.

[0019] In a fifth aspect of this disclosure, the present disclosure provides the application of glutamatergic neurons as described above in the preparation of cell models for modeling neurological diseases, evaluating the efficacy of neurotoxic drugs, and detecting the safety of neurotoxicity.

[0020] The technical solution provided in this disclosure has the following technical contributions: This disclosure addresses the technical shortcomings of existing dual-lentiviral differentiation-inducing systems by integrating the inverse tetracycline-regulated transcriptional activator protein rtTA3, the Ngn2 expression element containing the TRE promoter, and the puromycin resistance selection gene into a single lentiviral vector. Compared to traditional dual-virus dispersive element delivery schemes, this invention integrates all functional elements into a single vector, avoiding the problems of uneven infection ratios, varying integration efficiency, poor batch reproducibility, and high cytotoxicity caused by multi-virus co-infection. It simplifies virus preparation and cell transduction procedures, significantly improving system stability and experimental reproducibility. The inverse tetracycline-regulated transcriptional activator protein rtTA3 and the Ngn2 expression element containing the TRE promoter together constitute a controllable inducible expression system, achieving precise temporal regulation dependent on doxycycline (Dox). This enables the directed differentiation of hiPSCs into glutamatergic neurons under artificial control, with a simple regulatory method and strong controllability of induction.

[0021] Meanwhile, this invention configures an independently regulated puromycin resistance selection gene. This selection marker does not rely on the Ngn2 expression element containing the TRE promoter for induction of expression. It can complete the positive cell selection in advance at the hiPSC undifferentiated stem cell stage, eliminate negative cells that have not successfully integrated the exogenous gene, and obtain a stem cell line with stable gene integration.

[0022] This pre-screening mode avoids problems such as low purity and large background interference caused by mixed negative cells after differentiation, and effectively improves the purity and uniformity of the differentiated glutamatergic neurons.

[0023] In summary, this invention utilizes an integrated vector arrangement of rtTA3, the Ngn2 expression element of the TRE promoter, and the puromycin resistance selection gene to construct a hiPSC directed differentiation system that is easy to operate, highly safe, allows for pre-screening, achieves high differentiation purity, and is highly controllable, providing a stable and reliable technical platform for neurodevelopment research, neurological disease modeling, and drug screening. Attached Figure Description

[0024] Figure 1 It is a single lentiviral recombinant shuttle vector pLenti-TRE-Neurog2-3xFLAG-P2A-EGFP-EF1-rtTA3- IRES-Puro-WPRE.

[0025] Figure 2 It involves the induction and identification of neuronal cells. Figure 2 A is the process of induced differentiation. Figure 2 B represents changes in cell morphology at different time points. Figure 2C is an immunofluorescence map for neuronal identification (transfected with lentiviral fluorescent gene EGFP). Immature neurons in D7 and D14 are positive for MAP2 staining, while mature neurons in D21 express the presynaptic marker Synapsin 1. Detailed Implementation

[0026] The present invention will be further described in detail below with reference to specific embodiments to enable those skilled in the art to understand it. It should be noted that the following embodiments are for illustrative purposes only and are not intended to limit the scope of protection of the present invention.

[0027] Terminology Explanation Unless otherwise defined, all technical and scientific terms used in this disclosure have the same meaning as commonly understood by one of ordinary skill in the art. While similar or equivalent methods and materials to those described herein may be used in the practice or testing of this invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated herein by reference in their entirety. In case of conflict, this specification (including definitions) shall prevail. Furthermore, materials, methods, and examples are illustrative only and not intended to be limiting.

[0028] In this disclosure, the terms “comprising” or “including” are open-ended expressions used to refer to the phrase “including but not limited to” and are used interchangeably with it, meaning that they include the contents specified in this disclosure but do not exclude other contents.

[0029] The present disclosure is further illustrated below with specific embodiments. However, it should be understood that these embodiments are merely for the purpose of more detailed illustration and should not be construed as limiting the present disclosure in any way.

[0030] Declaration of Experimental Materials: The hiPSC cell line (hereinafter referred to as hiPSC) used in this disclosure was constructed and / or preserved by our laboratory to verify the expression regulation and function of the target gene in hiPSC and its differentiation system.

[0031] It should be clearly stated that the aforementioned cell lines are not improvements of this invention, nor does this invention rely on their specific properties. Those skilled in the art can easily purchase parental hiPSC lines commercially and obtain equivalent tools with the same function using methods known in the art (such as lentiviral packaging, cell fusion, etc.) without any inventive effort.

[0032] The technical solution and its effects of this invention have been verified in multiple independent experiments, and the results are consistent. Therefore, the screening method and the biological function of the obtained genes described in this application do not depend on individual differences in a specific cell line and have universal applicability.

[0033] Unless otherwise stated, all reagents, reagent consumables, and instruments used in this disclosure are commercially available. The main reagents are shown in Table 1.

[0034] Table 1. Main Reagents Unless otherwise stated, the various sequences used in this disclosure are provided by Heyuan Biotechnology Co., Ltd., and the various sequences are shown in Table 2.

[0035] Table 2. Various sequences 1. Construction of single-virus vectors and virus preparation Mouse neurogenin-2 (Ngn2, sequence shown in SEQ ID NO:1) was inserted via EcoRI into the empty lentiviral vector pLenti-TRE-EGFP-EF1-rtTA3-IRES-Puro- WPRE (vector derived from Heyuan Biotechnology, such as...) Figure 1 As shown), the recombinant shuttle vector pLenti-TRE-Neurog2-3xFLAG was constructed. -P2A-EGFP-EF1-rtTA3-IRES-Puro-WPRE. The single lentiviral recombinant shuttle vector, along the 5' to 3' direction, contains, in sequence: a TRE promoter, a Neurog2 coding sequence, a 3×FLAG tag coding sequence, a P2A autocleavage peptide coding sequence, an EGFP coding sequence, an EF1 promoter, an rtTA3 coding sequence, an IRES element, a puromycin resistance gene, and a WPRE element. The TRE promoter drives the tandem Neurog2 coding sequence, 3×FLAG tag coding sequence, P2A autocleavage peptide coding sequence, and EGFP coding sequence to induce transcription and translation, generating the Neurog2-3×FLAG fusion protein and the EGFP protein, respectively. The Neurog2-3×FLAG fusion protein is a Neurog2 protein with a 3×FLAG tag fused to its C-terminus. The two proteins are separated by cleavage with the P2A autocleavage peptide. The EF1 promoter drives the transcription of the rtTA3 coding sequence, the IRES element, and the Puro gene to form a single mRNA transcript, and mediates the co-expression of the rtTA3 protein and the puromycin resistance protein via the IRES.

[0036] The recombinant lentiviral vector was molecularly identified using CMV2 forward sequencing primers and EGFP reverse sequencing primer pairs (sequences shown in SEQ ID NO:4-5).

[0037] The vector virus was prepared using a lentiviral three-plasmid packaging system, which included a shuttle vector, a packaging helper plasmid, and a VSVG envelope plasmid. HEK293T cells were co-transfected with these three plasmids. After viral assembly and release, the cell culture supernatant was collected 48 hours after transfection. The supernatant was concentrated and purified by ultracentrifugation to enrich the viral particles. The physical titer of the purified viral stock solution was determined using qPCR. The target viral titer was 2.90 E+08 TU / ml.

[0038] Table 3. Single Viral Vector Construction Information 2. hiPSC induces differentiation into neurons (1) Preliminary preparation ①Matrigel coating plate: Prepare a working solution by diluting Matrigel with pre-cooled DMEM / F12 at a ratio of 1:100, coat the plate with the working solution, and incubate at 37°C for at least 1 hour.

[0039] ② Polyethyleneimine (PEI) / Laminin coated plate: Coat the culture plate with 0.07% PEI, incubate at room temperature for more than 1 hour, rinse 3 times with water, and air dry overnight in a clean bench (do not turn on UV during this period); coat the cell culture plate with 500ul Laminin (10µg / ml) and incubate at 37℃ for more than 2 hours (note that the cell culture plate should not dry out).

[0040] ③ Culture medium preparation: A. mTeSR medium containing Y-27632: Prepare mTeSR according to the instructions, and add Y-27632 to a final concentration of 10µM before use. B. KSR medium: 41.5mL KnockOut™ DMEM + 7.5mL Knockout™ SR+ 500uL MEM Non-EssentialAmino Acids Solution + 500uL GlutaMAX TM + 50uL β-mercaptoethanol C. N2B medium: 50mL DMEM / F12 + 750uL 20% Dextrose + 500uL GlutaMAX TM + 500uL N2 supplement B D. NBM medium: 48.5mL Neurobasal Medium (NBM) + 750uL 20% Dextrose + 500uL GlutaMAX TM + 250uL MEM Non-Essential Amino Acids Solution (2) Single-vector lentivirus transfection of hiPSC The hiPSC cell line used in this experiment was constructed in our laboratory. This cell line passed the pluripotency marker and karyotype identification and was used solely as the experimental material for in vitro differentiation in this invention. The starting material for this hiPSC can be obtained through commercial or ethical channels. Those skilled in the art can construct equivalent cell lines with the same function using methods known in the art.

[0041] ①hiPSC counting plate: When the hiPSC clone confluence reaches 70-80%, incubate with Accutase digestion solution at 37°C for 3-5 minutes; add an equal volume of DMEM / F12 to terminate digestion, gently pipette to form a single-cell suspension, centrifuge to collect the cell pellet; resuspend the cells in mTeSR medium containing Y-27632, count the cells, and then centrifuge at 3.8 × 10⁻⁶ cells / mL. 5 Cells were seeded at a density of 12-well plates in Matrigel-coated culture plates and incubated overnight at 37°C in a 5% CO2 incubator.

[0042] ② Single-vector lentivirus transfection: The next day, after observing good cell growth, the medium was replaced with mTeSR medium (MOI=7) containing a single-vector lentivirus and placed back into the incubator for continued culture.

[0043] ③Puro screening Remove the old culture medium containing the virus, rinse once with DPBS, add fresh mTeSR medium containing 1ug / ml Puromycin for screening, and screen continuously for 3-7 days.

[0044] ④ After screening, hiPSCs that have been transfected with lentivirus were amplified, cultured, and cryopreserved.

[0045] (3) Induction of differentiation of hiPSC neurons transfected with lentivirus ①Day 0: Plate the hiPSCs that have been transfected with lentivirus (Matrigel plate) and incubate them.

[0046] ②Day 1: Replace the culture medium with KSR medium containing Doxycycline (Dox, 2 µg / mL) and return it to the incubator to continue culturing.

[0047] ③Day 2: Replace the entire culture medium with a KSR medium:N2B medium (volume ratio of 1:1) mixed medium, and add Puromycin (1 µg / mL) and Dox (2 µg / mL) to this medium.

[0048] Day 3: Replace the culture medium with N2B medium and add B27 (1:100), Puromycin (1 μg / mL) and Doxycycline (2 μg / mL).

[0049] ④Day 4: Cell counting and passage. After counting, the cells were seeded into PEI / Laminin-coated culture plates at different cell densities according to experimental requirements. The culture medium was changed to NBM medium + B27 (1:50) + Y-27632 (10µM) + BDNF / GDNF / CNTF (10µg / ml) + Puromycin (1 µg / mL) + Dox (2ug / mL). Cells can also be cryopreserved at this stage.

[0050] ⑤Day 5: Complete medium replacement with NBM medium + B27 (1:50) + BDNF / GDNF / CNTF (10µg / ml) + Puromycin (1 µg / mL) + Dox (2ug / mL).

[0051] ⑥ Day 6-21, change half of the medium every 3-4 days. The culture medium is NBM medium + B27 (1:50) + BDNF / GDNF (10µg / ml) + Puromycin (1 µg / mL) + Dox (2ug / mL).

[0052] 3. Experimental Results: Using a single lentiviral vector (pLenti-TRE-Neurog2-3xFLAG-P2A-EGFP-EF1-rtTA3-IRES-Puro-) WPRE successfully induced hiPSCs into neurons. Figure 2 Demonstrates the process of induced differentiation ( Figure 2 A) and changes in cell morphology at different time points ( Figure 2 B). Figure 2 C showed positive MAP2 staining in immature neurons from D7 and D14, and D21. + Neuronal cells express the presynaptic marker Synapsin 1.

[0053] The above specific embodiments are merely illustrative of the content of this disclosure and do not represent a limitation thereof. For those skilled in the art, various modifications and improvements can be made without departing from the spirit and substance of this disclosure, and these modifications and improvements are also considered to be within the scope of protection of this disclosure.

Claims

1. A lentiviral vector for inducing the differentiation of glutamatergic neurons, characterized in that: The structure, arranged sequentially from 5' to 3', includes: a TRE promoter, a Neurog2 coding sequence, a 3×FLAG tag coding sequence, a P2A self-cleaving peptide coding sequence, an EGFP coding sequence, an EF1 promoter, an rtTA3 coding sequence, an IRES element, a puromycin resistance gene, and a WPRE element. The TRE promoter drives the tandem Neurog2 coding sequence, 3×FLAG tag coding sequence, P2A self-cleaving peptide coding sequence, and EGFP coding sequence to induce transcription and translation, generating the Neurog2-3×FLAG fusion protein and the EGFP protein, respectively. The Neurog2-3×FLAG fusion protein is a Neurog2 protein with a 3×FLAG tag fused to its C-terminus. The two proteins are separated by cleavage with the P2A self-cleaving peptide. The EF1 promoter drives the transcription of the rtTA3 coding sequence, the IRES element, and the Puro gene to form a single mRNA transcript, and mediates the co-expression of the rtTA3 protein and the puromycin resistance protein via the IRES.

2. The lentiviral vector according to claim 1, characterized in that: The EF1 promoter is a constitutive promoter, and its transcriptional activity is not regulated by expression elements containing TRE promoters.

3. A method for inducing glutamatergic neurons using a single lentiviral vector as described in claim 1 or 2, characterized in that, Includes the following steps: (1) The single lentiviral vector was transfected into hiPSC cells to obtain transfected mixed cells; (2) Under conditions without the addition of Doxycycline inducer, the mixed cells were screened by puromycin pressure to remove negative cells that did not integrate the foreign gene and obtain a hiPSC cell line with stable gene integration. (3) Doxycycline was added to stable hiPSC cell lines for induction culture, and glutamatergic neurons were obtained by directed differentiation.

4. The method according to claim 3, characterized in that: The puromycin screening treatment lasted for 3-7 days, and the working concentration of puromycin was 0.75-1 ug / ml.

5. The method according to claim 3, characterized in that: The doxycycline induction concentration was 2 µg / mL, and the doxycycline neuron induction was continuously induced during the culture period.

6. The method according to claim 3, characterized in that: The induction culture adopts a graded culture medium system, which includes at least stem cell maintenance medium, neural induction medium and neuron maturation medium. The replacement time points of each culture medium are as follows: Day 0 uses stem cell maintenance medium, Day 1-4 uses neural induction medium, and Day 5 onwards uses neuron maturation medium.

7. The method according to claim 3, characterized in that: The method uses CMV2 forward sequencing primers with nucleotide sequences as shown in SEQ ID NO:4 and EGFP reverse sequencing primers as shown in SEQ ID NO:5 to complete the identification of vector molecules.

8. The glutamatergic neurons prepared by the method according to any one of claims 3 to 7, characterized in that: The glutamatergic neurons express at least one neuronal marker among vGLUT1, TUBB3, MAP2, NeuN, and Synapsin 1.

9. The use of a lentiviral vector as described in claim 1 or 2 in the preparation of a kit for inducing differentiation of glutamatergic neurons.

10. The application of the glutamatergic neuron as described in claim 8 in the preparation of cell models for modeling neurological diseases, evaluating the efficacy of neurotoxic drugs, and detecting the safety of neurotoxicity.

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