Method for inducing dopaminergic neuron differentiation by overexpression transcription factor

Abstract

The invention discloses a method for inducing dopaminergic neuron differentiation by overexpression of a transcription factor. The method comprises the following steps: introducing the transcription factor into a human pluripotent stem cell for overexpression, and culturing to obtain the dopaminergic neuron. And the transcription factors are EBF4, FOSL1, MEIS2, YAP1, NHP2, CDX2, PAX7, ZNF415 and EBF3. A flow analysis detection experiment proves that the 10 transcription factors are simultaneously overexpressed, so that the differentiation of dopaminergic neurons can be efficiently induced. The method for inducing the dopaminergic neurons to differentiate by overexpressing the transcription factor, provided by the invention, is high in differentiation efficiency. The dopaminergic neurons cultured based on the differentiation method can be used for screening transcription factors by a high-throughput method, constructing pathological models of nervous system diseases, developing drugs for the nervous system diseases or screening the drugs for the nervous system diseases, and a new experimental basis is provided for subsequent research of Parkinson's disease.

Description

Technical Field

[0001] The present application relates to the field of bioengineering technology, and in particular to a method for inducing dopaminergic neuron differentiation by overexpressing transcription factors. Background Art

[0002] Stem cells are self-renewing populations with the ability to differentiate into specific lineages. Human pluripotent stem cells (hPSCs) include human embryonic stem cells (hESCs) and human induced pluripotent stem cells (iPSCs) generated by Shinya Yamanaka in 2006 using a combination of four factors (Myc, Oct4, Sox2 and Klf4), which provide a renewable source of human cells at a very early stage of development and have the potential to form any cell type in the adult body. Therefore, human pluripotent stem cells provide an expandable source of cells from which standardized and quality-controlled cell derivatives can be obtained for the treatment of various diseases.

[0003] Parkinson's disease (PD) is the second most common progressive neurodegenerative disease in the world. The neuropathological characteristics of Parkinson's disease are the progressive death of dopaminergic neurons in the substantia nigra pars compacta and the depletion of dopamine levels. Therefore, replacing dopaminergic neurons in a single local brain of Parkinson's patients is expected to alleviate their defects.

[0004] In the prior art, the production of neurons induced by human pluripotent stem cells mainly includes directed differentiation and induced differentiation. Directed differentiation is mainly achieved step by step by using exogenous factors (such as recombinant proteins or small molecules) to stimulate or antagonize signal pathways known to be important in the development of target cell types. The development process from stem cells to neurons is realized. In contrast, genetic engineering technology is used to "turn on" the expression of neurogenic factors, thereby directly generating post-mitotic neurons from stem cells to achieve induced differentiation. This process bypasses or greatly shortens the development process and shortens the time required to generate neurons. Not only is the induction speed beneficial, but the induced neuronal populations are often more homogeneous than the neuronal populations produced by directed differentiation, both within and between batches. There are currently many schemes for inducing dopaminergic neurons, but the number of dopaminergic neurons obtained by these schemes is small. Therefore, it is urgent to provide an efficient induction scheme for high-throughput drug screening and potential cell sources for cell replacement therapy. Summary of the invention

[0005] The present application provides a method for inducing dopaminergic neuron differentiation by overexpressing transcription factors, dopaminergic neurons and applications thereof, which can solve the problem of low efficiency in inducing differentiation of dopaminergic neurons in the prior art.

[0006] In a first aspect, the present application provides a method for inducing dopaminergic neuron differentiation by overexpressing a transcription factor, comprising the steps of introducing a transcription factor into human pluripotent stem cells for overexpression, and obtaining dopaminergic neurons by culturing, specifically comprising the following steps:

[0007] (1) Selecting transcription factors that induce human pluripotent stem cells to differentiate into dopaminergic neurons;

[0008] (2) constructing a viral vector carrying transcription factors to mediate transcription factor transfer;

[0009] (3) infecting human pluripotent stem cells with the viral vector of step (2);

[0010] (4) Identification of dopaminergic neurons;

[0011] The above-mentioned transcription factors include EBF4, FOSL1, MEIS2, YAP1, NHP2, CDX2, PAX7, ZNF415 and EBF3. Among them, the cDNA sequence of EBF4 is shown in SEQ ID NO.1; the cDNA sequence of FOSL1 is shown in SEQ ID NO.2; the cDNA sequence of MEIS2 is shown in SEQ ID NO.3; the cDNA sequence of YAP1 is shown in SEQ ID NO.4; the cDNA sequence of NHP2 is shown in SEQ ID NO.5; the cDNA sequence of CDX2 is shown in SEQ ID NO.6; the cDNA sequence of PAX7 is shown in SEQ ID NO.7; the cDNA sequence of ZNF415 is shown in SEQ ID NO.8; and the cDNA sequence of EBF3 is shown in SEQ ID NO.9.

[0012] Preferably, the transcription factor-carrying viral vector in the above step (2) is a lentiviral vector carrying genes encoding EBF4, FOSL1, MEIS2, YAP1, NHP2, CDX2, PAX7, ZNF415 and EBF3.

[0013] Preferably, the virus vector infects human pluripotent stem cells in step (3) above by the following method: after infecting human pluripotent stem cells in vitro with the constructed virus carrying the transcription factor, the cells are inoculated into a cell culture medium for culture.

[0014] Preferably, the identification of dopaminergic neurons in the above step (4) is performed by the following method: identifying enzymes related to dopamine production by immunofluorescence staining.

[0015] Preferably, the above-mentioned enzyme related to dopamine production includes tyrosine hydroxylase.

[0016] In a second aspect, the present application provides a dopaminergic neuron induced by the above method.

[0017] In a third aspect, the present application provides the use of the above-mentioned dopaminergic neurons in the development of drugs for nervous system diseases.

[0018] Preferably, the above-mentioned applications include at least one of high-throughput screening of transcription factors, construction of pathological models of nervous system diseases, and screening of drugs for nervous system diseases.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] The method for inducing dopaminergic neuron differentiation by overexpressing transcription factors based on the present application is to introduce specific transcription factors into human pluripotent stem cells and overexpress them, and use flow cytometry to detect differentiation to confirm that overexpressing 10 transcription factors simultaneously can efficiently induce the differentiation of dopaminergic neurons. A transcription factor induction scheme with high differentiation efficiency and positive rate of dopaminergic neurons is provided.

[0021] The dopaminergic neurons cultured by the method of overexpressing transcription factors to induce dopaminergic neuron differentiation according to the present application can be used for high-throughput screening of transcription factors, construction of pathological models of nervous system diseases, development of drugs for nervous system diseases or screening of drugs for nervous system diseases, providing a new experimental basis for subsequent research on Parkinson's disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the experimental examples of the present application or the technical solutions in the prior art, the drawings required for use in the experimental examples or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some experimental examples of the present application. For those skilled in the art, other drawings can be obtained based on these drawings without paying any creative work.

[0023] Figure 1 Schematic diagram for the establishment of tetracycline-inducible ASCL1 overexpression iPSCs cell line;

[0024] Figure 2 Flow chart for the establishment of tetracycline-induced ASCL1 overexpression iPSCs cell line (top) and PCR gel validation of gene knock-in in the cell line (bottom);

[0025] Figure 3 To identify the expression of ASCL1 protein induced by tetracycline by immunofluorescence staining;

[0026] Figure 4 To identify the maintenance of stemness of cell lines by immunofluorescence staining;

[0027] Figure 5Workflow established for the TH-GFP reporter cell line (top) and PCR gel validation of gene knock-in (bottom);

[0028] Figure 6 The best monoclonal TH-GFP cell line was selected after gene knock-in by strong activation of CrisprA and flow cytometry analysis (top); the removal of the selection marker after Cre in the TH-GFP reporter cell line was verified by PCR gel running (middle); the co-localization and quantitative analysis results of TH and GFP protein expression in the TH-GFP reporter cell line were verified by immunofluorescence staining (bottom);

[0029] Figure 7 The best single clone of TH-GFP cell line after Cre was selected by strong activation of CrisprA and flow cytometry analysis (top); the maintenance of stemness of TH-GFP reporter cell line was identified by immunofluorescence staining (bottom left); karyotype analysis of TH-GFP reporter iPSCs with ASCL1 overexpression induced by tetracycline (bottom right);

[0030] Figure 8 Flow chart for MORF library screening;

[0031] Fig. 9 The differentiation process and culture medium of MORF screening using Dox-induced ASCL1 overexpression and TH-GFP reporter gene iPSCs cell line (top), and the cell morphology changes at different days (bottom);

[0032] Fig.10 GFP autofluorescence image (left) and flow cytometry analysis results (right) for MORF screening;

[0033] Fig.11 Results of PCR library construction for MORF screening (top and bottom left); NGS sequencing results for MORF screening (bottom right);

[0034] Fig.12 GFP fluorescence images of the FULL group and the minus group on Day 26 of differentiation after overexpression of 10 transcription factors;

[0035] Fig.13 for Fig.12 Corresponding flow analysis results;

[0036] Fig.14 Quantitative statistics of flow cytometry results (upper left); HPLC detection results of dopamine (upper right); qPCR results of dopaminergic neuron maturation markers in the FULL group on day 30 of differentiation (lower);

[0037] Fig.15Current pulses induce action potentials in differentiated neurons (upper left); Differentiated neurons express voltage-gated Na+ (influx) and K+ (efflux) channels (red is an enlarged view of the boxed area in black traces (lower left); Depolarizing current injection (upper black trace) into differentiated neurons blocks action potential generation, which then returns to baseline frequency after termination of current injection stimulation (blue trace is an enlarged view of the boxed area in red traces (upper right); Supercharged current injections cause differentiated neurons to respond to hyperpolarization with a characteristic supercharged sag and a brief rebound delay mediated by current (Ih) (lower right);

[0038] Fig.16 The heat map of RNA-Seq correlation check (left) shows that the experiment is repeatable and has little variation; the FPKM box plot of gene expression levels between the experimental group and the control group (right), the horizontal axis is the sample name, the vertical axis is log10 (FPKM), and the box plots in each area are for five statistics (maximum value, upper quartile, median, lower quartile and minimum value from top to bottom);

[0039] Fig.17 The volcano map of differentially expressed genes (left), red dots of significantly differentially expressed genes indicate up-regulation, blue dots indicate down-regulation, the horizontal axis represents the fold change of gene expression in different samples, and the vertical axis represents the statistical significance of the difference in gene expression, among which the number of up-regulated genes is 2695, and the number of down-regulated genes is 1774; the cluster map of differentially expressed genes (right), clustered by log10 (FPKM+1) value, red indicates highly expressed genes, blue indicates lowly expressed genes, and the color from blue to red indicates higher gene expression;

[0040] Fig.18 GO enrichment histogram (left), the ordinate is the enriched GO term, the abscissa is the number of differentially expressed genes in the term, and different colors are used to distinguish biological processes, cellular components, and molecular functions; GO enrichment P value histogram (right), the ordinate is the enriched GO term, and the abscissa is the term-log10 (p-value);

[0041] Fig.19 The bar chart of significantly enriched KEGG annotation classification (left), the vertical axis represents the pathway name, and the horizontal axis represents the number of genes; the scatter plot of differential gene KEGG enrichment (right), the vertical axis represents the pathway name, the horizontal axis represents the Richfactor, the size of the dot represents the number of differentially expressed genes in this pathway, and the color of the dot corresponds to different Qvalue ranges; DETAILED DESCRIPTION

[0042] In order to make the purpose, technical solution and advantages of the present application more clearly understood, the present application is further described in detail below in conjunction with the accompanying drawings and experimental examples. It should be understood that the specific experimental examples described herein are only used to explain the present application and are not used to limit the present application.

[0043] Experimental Example 1 Establishment of Dox-induced ASCL1 overexpression iPSCs cell line carrying TH-GFP reporter gene

[0044] 1. Experimental methods

[0045] 1.1 Neural induction and transfection of Cas9 RNP and Donor DNA:

[0046] On Day 0, human pluripotent stem cells were seeded into cells. Taking a 24-well plate as an example, the number of cells was 1x10 5 In the case of mRNA transfection, the cell culture medium is prepared as shown in Table 1:

[0047] Table 1

[0048]

[0049] Prepare the RNP mixture according to Table 2:

[0050] Table 2

[0051] Components Dosage OptiMEM 19μL Cas9 protein (1uM) 3μL sgRNA (1uM) 3μL

[0052] Tap the tube gently to mix and incubate at room temperature for 5 min to allow the RNP complex to form.

[0053] Cas9 protein (Nearshore protein #E365-02A), sgRNA (synthesized from GenScript), Opti-MEM (Gibco #31985070). Prepare the transfection mixture according to Table 3:

[0054] Table 3

[0055] Tube 1 component Dosage Donor DNA 500ng RNP complex Table 2 All the results Tube 2 components Dosage OptiMEM 25μL Lipofectamine Stem 1μL

[0056] After gently tapping the tube wall to mix, add the mixture in tube 2 to tube 1, tap the tube wall to mix and incubate at room temperature for 10 minutes. Add the mixture to the seeded 250K iPSCs cells (24-well plate), draw a "cross" shape to mix, and immediately place in the cell culture incubator for further culture.

[0057] 1.2 Cell screening and monoclonal selection:

[0058] On the second day after transfection, the cells were digested into single cells and all cells were plated in a six-well plate so that the cells had space to expand. On the third day after transfection, the cells were preliminarily screened by adding the corresponding antibiotics to the culture medium according to the resistance of the Donor DNA to remove cells that were not successfully knocked in. After the screening, the cells were digested into single cells and diluted in a cell density gradient. 200-300 single cells were planted in 10cm, and ClonR 2 (Stem Cell #100-0691) was added to the culture medium. After 7 days of culture, the single cells will be expanded into monoclonal clones, and the monoclonal clones will be digested one by one using collagenase IV (Biofroxx #2091MG100), and they will be sucked out one by one with a gun tip, and transferred to a twenty-four-well plate in two.

[0059] 1.3 Gene knock-in verification:

[0060] Monoclonal genomic DNA was extracted using the Monarch Genomic DNA Extraction Kit (NEB #T3010S), PCR was performed using the primers in Table 4 below, and then the gel was run to verify the bands.

[0061] Table 4

[0062]

[0063] 1.4 Immunofluorescence staining (taking 24-well plate as an example):

[0064] Wash the cells with 500 μl PBS, place on a horizontal shaker and shake slowly for 5 minutes, repeat 3 times. After discarding the PBS, slowly add 500 μl 4% paraformaldehyde along the wall to fix the cells at room temperature for 20 minutes. Wash with 500 μl PBS plus 1% BSA, place on a horizontal shaker and shake slowly for 5 minutes, repeat 3 times. After discarding the PBS, add 250 μl blocking solution and block at room temperature for 1 hour.

[0065] During the blocking process, prepare the primary antibody and dissolve it in the blocking solution according to the recommended ratio. After blocking, add 250 μL to each well and incubate in a 4°C refrigerator overnight. After the primary antibody blocking is completed, wash with 500 μL PBS (containing 1% BSA), place it on a horizontal shaker and shake it slowly for 5 minutes, and repeat 3 times. Dilute the secondary antibody in PBS (1% BSA) according to the recommended ratio, add 250 μL to each well, and incubate at room temperature in the dark for 1 hour. Wash with 500 μL PBS (containing 1% BSA), place it on a horizontal shaker and shake it slowly for 5 minutes, and repeat 3 times. Dilute DAPI in PBS (1% BSA) at 1:1000 and incubate in the dark for 5 minutes. Wash with 500 μL PBS (containing 1% BSA), place it on a horizontal shaker and shake it slowly for 5 minutes, and repeat 3 times. Cover the cells with PBS, store at 4°C in the dark or directly observe the results using a fluorescence microscope. Among them, the blocking solution: PBS + 1% BSA + 0.3% TritonX-100 + 10% Donkey Serum.

[0066] 1.5 Karyotype analysis:

[0067] Completed by Suzhou Qilin Zhijian Cell Biotechnology Co., Ltd., the general steps are as follows: When the stem cells are in the logarithmic growth phase, colchicine is added to keep the cells in the metaphase. The cells are digested and centrifuged to obtain cell pellets, which are then treated with hypotonicity. After hypotonicity, the cells are fixed three times. After fixation, drop sections and Giemsa staining are performed, followed by observation and analysis.

[0068] 2. Experimental results:

[0069] 2.1.1 After determining the basic differentiation scheme, the Tet-on regulatory system and CRISPR-Cas9 system were used to overexpress the ASCL1 gene more conveniently and controllably. On the Donor vector, the ASCL1 gene is located downstream of the TRE3G promoter, and the expression of the ASCL1 gene can only be induced in the presence of Dox. The Donor vector also carries two screening markers, mApple and BSD, to facilitate the selection of positive cells after gene knock-in. Since the screening marker is located between the two LoxP sites, the Cre-LoxP system can be used to delete the screening marker at the end.

[0070] 2.1.2 When constructing this overexpression cell line, the gene is knocked into the CLYBL site by homologous recombination. This site can be used as a safe harbor site for safe gene knock-in and can ensure the normal and stable expression of the transferred gene.

[0071] 2.1.3 Attachment Figure 2(Top) The process of cell line construction is shown. After transfer, BSD administration is performed for preliminary screening, and then two rounds of single-cell sorting and verification are performed to obtain the final positive clones.

[0072] 2.1.4 Attachment Figure 2 (Bottom) is the verification of gene knock-in. After extracting cell genomic DNA, PCR gel verification was performed using the 5' homology arm and 3' homology arm primers shown in the figure. From the PCR gel bands, it can be seen that the selected Clone 16 and Clone 24 have successfully knocked in the ASCL1 gene.

[0073] 2.1.5 To verify the controllability of ASCL1 gene expression, immunofluorescence staining was performed on cells with and without Dox addition. Figure 3 As shown in the figure, when Dox was added, the ASCL1 gene was expressed normally in all cells, but when Dox was not added, the ASCL1 gene was not expressed.

[0074] 2.1.6 To verify that this cell line still has the characteristics of normal stem cells, namely unlimited proliferation and differentiation capabilities, immunofluorescence staining of stemness marker genes was performed. Figure 3 As shown, OCT4, SOX2, and NANOG genes can be expressed normally, proving that this cell line still has stem cell characteristics.

[0075] 2.2.1 In order to screen the MORF library, the GFP gene was knocked into the TH locus based on the Dox-induced ASCL1 overexpression cell line to establish a Dox-induced ASCL1 overexpression iPSCs cell line carrying the TH-GFP reporter gene.

[0076] 2.2.2 As attached Figure 5 As shown in (above), the GFP gene is inserted downstream of the TH gene using the CRISPR-Cas9 system and homologous recombination to achieve a one-to-one correspondence between TH gene expression and GFP gene expression. In this way, the expression of the TH gene can be indicated by the autofluorescence of GFP.

[0077] 2.2.3 Attachment Figure 5 (Bottom) is the verification of GFP gene insertion. From the PCR gel bands, it can be seen that the four selected clones have successfully inserted the GFP gene.

[0078] 2.2.4 In order to select the best monoclonal clone, the expression of TH gene was strongly activated by CRISPRa technology. Figure 6 As shown in (above), five sgRNAs were designed to implement CRISPRa. The best clones were selected based on the GFP autofluorescence intensity and ratio. Figure 6The flow cytometry results shown in (above) show that under the same strong activation experiment, Clone 5 has the highest GFP positive rate. Therefore, Clone 5 was selected for subsequent experiments.

[0079] 2.2.5 Then the selection marker of Clone 5 was deleted. As before, the Cre-LoxP system was used to delete the Neo selection marker. Figure 6 (Middle) The primers shown were used for PCR gel running verification. The band sizes showed that the selection markers of Clone 1 and Clone 2 selected after Cre were successfully deleted.

[0080] 2.2.6 To prove the one-to-one correspondence between GFP gene and TH gene expression, we performed immunofluorescence staining on cells. Figure 6 The results shown in (bottom) show that the expression of GFP gene and TH gene is co-localized, and the expression of GFP can be used to accurately indicate the expression of TH gene.

[0081] 2.2.7 Select Clone 1 and Clone 2 in the same way as before. Figure 7 (Top) Flow cytometry analysis results show that Clone 2 is more sensitive in expressing the GFP reporter gene.

[0082] 2.2.8 To verify that this cell line still has the characteristics of normal stem cells, namely unlimited proliferation and differentiation capabilities, immunofluorescence staining of stemness marker genes was performed. Figure 7 As shown in (lower left), OCT4, SOX2, and NANOG genes can all be expressed normally, proving that this cell line still has stem cell characteristics.

[0083] 2.2.9 To verify that this cell line still has a normal stem cell karyotype, chromosome karyotype analysis was performed using G banding. Figure 7 (Lower right) The results show that the number of chromosomes is 46, which is normal. The sex chromosomes are composed of XY, which is a female karyotype. There are no abnormalities in the chromosome results.

[0084] Example 2 MORF library screening

[0085] 1. Experimental methods:

[0086] 1.1 Neural induction:

[0087] Day 0: 1.6x10 6 The cells were plated in a 10 cm dish, the culture medium was PGM1 medium (Cellapy#CA1007500), and the Rock inhibitor was Y-27632 (TargetMol#T1870) + Dox.

[0088] Day 1-4: The induction medium is Neural Induction medium (Stem Cell#05835) + LDN (AbMole#M1873) + SB (TargetMol#301836-41-9) + Dox (TargetMol#10016275).

[0089] Day5-18: The induction medium is Neurobasal medium (Thermo#21103049) + B27 supplement minus vitamin A (Gibco#12587010) + BDNF (Peoprotech#450-02) + GDNF (Peoprotech#96-450-10-2) + cAMP (Sigma#D0627).

[0090] 1.2MORF library screening process is as follows:

[0091] First, the packaged MORF library lentivirus was infected with Dox-induced ASCL1 overexpression and iPSCs cell line carrying TH-GFP reporter gene. After 24 hours of virus integration, the cells were screened using the screening marker Puromycin to eliminate uninfected cells. Then neural induction was performed, and the cells were flow sorted on the 18th day. The genomic DNA of the sorted GFP-positive cells was extracted using a micro-genomic DNA extraction kit (Thermo#KIT0103) and PCR library construction was performed. After the library construction was completed, NGS sequencing was performed. Sequencing and analysis were completed by Suzhou Genewise Biotechnology Co., Ltd.

[0092] 2. Experimental results:

[0093] MORF library screening and NGS sequencing were performed on cell lines to select the 10 transcription factors with the highest expression levels.

[0094] Example 3: Transcription factor combination validation

[0095] 1. Experimental methods:

[0096] 1.1 Minus-one screening of transcription factors:

[0097] The cells were divided into a FULL group with all viruses added and a group with one virus minus 10. The cell lines were infected with MOI=5 for neural induction and cultured until day 26 for flow cytometry analysis to determine the most suitable combination.

[0098] 1.2 RNA extraction:

[0099] The total RNA of the sample cells to be analyzed was extracted using a micro RNA extraction kit (Zymo #R1050).

[0100] 1.3 Real-time quantitative PCR (RT-qPCR):

[0101] RNA was reverse transcribed into cDNA using HiScript III RT SuperMix for qPCR (+gDNAwiper) (Vazyme#R323-01), and qPCR experiments were performed using AceQ Universal SYBR qPCR Master Mix (Vazyme#Q511-02). GAPDH gene was used for standardization, and RT-qPCR was used to verify the expression of related marker genes. The primer sequences used are shown in Table 5 below:

[0102] Table 5

[0103]

[0104] 1.4 Functional identification of neurons using electrophysiology: completed by Zhang Jian from Liu Ji's laboratory at the University of Science and Technology of China.

[0105] 1.5 Using RNA-seq to analyze neuronal characteristics: completed by Suzhou Genewise Biotechnology Co., Ltd.

[0106] 2. Experimental results:

[0107] The results of flow cytometry analysis showed that the positive rate of overexpressing 10 transcription factors into the cell line for neural induction was greatly improved compared with the basic scheme of ASCL1 induction. The results of HPLC showed that the level of dopamine released by neurons obtained by this scheme was also significantly increased. The results of RT-qPCR showed that the dopaminergic neurons obtained by this scheme were more mature. The results of electrophysiological identification showed that the differentiated neuron type was dopaminergic neuron with electrophysiological function. The results of RNA-Seq analysis showed that the cells induced by this scheme had significantly increased neural-related pathways and genes.

[0108] In summary, the present application provides a transcription factor induction scheme with high differentiation efficiency and positive rate of dopaminergic neurons. The dopaminergic neurons cultured based on the method of inducing dopaminergic neuron differentiation by overexpressing transcription factors in the present application can be used for screening transcription factors using high-throughput methods, constructing pathological models of nervous system diseases, developing drugs for nervous system diseases or screening drugs for nervous system diseases, providing a new experimental basis for subsequent research on Parkinson's disease.

[0109] The above description is only a preferred experimental example of the present application and is not intended to limit the present application. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present application should be included in the protection scope of the present application.

Claims

1. A method for inducing dopaminergic neuron differentiation by overexpressing transcription factors, characterized in that: The method comprises the steps of introducing transcription factors into human pluripotent stem cells for overexpression and obtaining dopaminergic neurons through culture, and specifically comprises the following steps: (1) Selecting transcription factors that induce human pluripotent stem cells to differentiate into dopaminergic neurons; (2) constructing a viral vector carrying transcription factors to mediate transcription factor transfer; (3) infecting human pluripotent stem cells with the viral vector of step (2); (4) Identification of dopaminergic neurons; The transcription factors are EBF4, FOSL1, MEIS2, YAP1, NHP2, CDX2, PAX7, ZNF415 and EBF3.

2. The method according to claim 1, characterized in that The transcription factor-carrying viral vector of step (2) is a lentiviral vector carrying genes encoding EBF4, FOSL1, MEIS2, YAP1, NHP2, CDX2, PAX7, ZNF415 and EBF3.

3. The method according to claim 1, characterized in that The viral vector in step (3) infects human pluripotent stem cells by the following method: After infecting human pluripotent stem cells in vitro with the constructed virus carrying transcription factors, they are inoculated into cell culture medium for culture.

4. The method according to claim 1, characterized in that: The dopaminergic neuron identification in step (4) comprises the following method: (1) Identify enzymes related to dopamine production by immunofluorescence staining; (2) Neurons were verified by electrophysiological validation, qPCR validation, or RNA-seq validation.

5. The method according to claim 4, characterized in that The enzymes involved in dopamine production include tyrosine hydroxylase.

6. A dopaminergic neuron, characterized in that The method is induced by any one of claims 1 to 5.

7. Use of the dopaminergic neurons according to claim 6 in developing drugs for nervous system diseases.

8. The use according to claim 7, characterized in that: The application includes at least one of high-throughput screening of transcription factors, construction of a pathological model of a nervous system disease, or screening of drugs for a nervous system disease.

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