Dopaminergic neuron progenitor cell as well as preparation method and application thereof

Abstract

The invention relates to the technical field of genetic engineering stem cell transformation and induced differentiation, in particular to a dopaminergic neuron progenitor cell as well as a preparation method and application thereof. The preparation method comprises the following steps: construction of lentiviral vector plasmids, in-vitro culture of human induced pluripotent stem cells, lentiviral infection, acquisition of primitive nerve epithelial cells and acquisition of dopaminergic neuron progenitor cells. The dopaminergic neuron progenitor cells are obtained by induced differentiation of human induced pluripotent stem cells carrying DA synthesis key enzyme TH, DDC and GCH1 genes. The dopaminergic neuron progenitor cell obtained by the method provided by the invention can survive for a long time in a Parkinson rat brain microenvironment, has an obvious repair effect on rat dyskinesia, and lays a foundation for treating Parkinson's disease by stem cell transplantation.

Description

Technical Field

[0001] The present invention relates to the technical field of genetic engineering transformation of stem cells and induced differentiation, and in particular to a dopaminergic neuron progenitor cell and a preparation method and application thereof. Background Art

[0002] Parkinson's disease (PD) is a neurodegenerative disease caused by the massive death of dopaminergic (DA) neurons in the substantia nigra and the formation of Lewy bodies containing α-synuclein. The main manifestations of Parkinson's disease are movement disorders and non-motor symptoms. The main treatment method at present is drug therapy. Common drugs include compound levodopa, MAO-B inhibitors and amantadine, but drugs can only relieve symptoms and cannot cure the disease, and complications may occur. Deep brain stimulation surgery is a common treatment method, but it requires the implantation of electrodes in the patient's skull, which may bring certain burdens and risks. Therefore, although it can improve the patient's condition, it will also cause some burdens on life. At the same time, there are also problems such as patient tolerance to stimulation, electrode displacement, and high surgical costs.

[0003] The synthesis of DA mainly depends on the action of several key enzymes. First, tyrosine is converted into L-dopa by tyrosine hydroxylase (TH), and then further catalyzed into DA by dopamine decarboxylase (DDC). Among them, the activity of TH requires the presence of the auxiliary factor tetrahydrobiopterin (BH4), and the rate-limiting enzyme in the synthesis of BH4 is GTP cyclohydrolase 1 (GCH1).

[0004] Human induced pluripotent stem cells (HiPSCs) are stem cells with multipotentiality, strong self-renewal and differentiation capabilities, including differentiation into dopaminergic neurons. The advantage of HiPSCs is that they can be mass-produced and cryopreserved, which is convenient for use as needed and has no ethical issues. However, the efficiency of HiPSCs in inducing differentiation into dopaminergic neurons is low, resulting in a low amount of DA synthesis and secretion, which limits its application in the treatment of Parkinson's disease.

[0005] Therefore, there is an urgent need to find a method to synthesize and secrete more DA dopaminergic neuron progenitor cells. Summary of the invention

[0006] To solve the above technical problems, the present invention provides a dopaminergic neuron progenitor cell and a preparation method and application thereof, which can be induced to differentiate into dopaminergic neuron progenitor cells that can synthesize and release a large amount of dopamine neurotransmitters. The cells can survive for a long time in the brain microenvironment of Parkinson's rats and have a significant repair effect on the motor dysfunction of rats, providing a factual basis and theoretical foundation for the treatment of related neurodegenerative diseases.

[0007] The present invention provides a method for preparing dopaminergic neuron progenitor cells, comprising the following steps:

[0008] Construction of lentiviral vector plasmids: construct lentiviral vector plasmid LV-TH carrying human TH gene and lentiviral vector plasmid LV-DDC-GCH1 carrying human DDC and GCH1 genes respectively;

[0009] In vitro culture of human induced pluripotent stem cells: culturing human induced pluripotent stem cells in vitro to obtain cultured human induced pluripotent stem cells;

[0010] Lentiviral infection: Under the action of the transfer agent polybrene, the cultured human induced pluripotent stem cells were infected by half-dosage infection using the constructed lentiviral vector plasmids LV-TH and LV-DDC-GCH1, and the human induced pluripotent stem cell line LV-TDG-HiPSCs was obtained by antibiotic screening;

[0011] Derivation of primitive neuroepithelial cells: Human induced pluripotent stem cell line LV-TDG-HiPSCs were cultured in mTeSR medium supplemented with DMH1, SB431542, SHH, FGF8, Purmorphamine, and CHIR99021. TM Induced to differentiate into primitive neuroepithelial cells LV-TDG-iNESCs under the action of Basal Medium;

[0012] Acquisition of dopaminergic neuron progenitor cells: Collagenase was added to the primitive neuroepithelial cells LV-TDG-iNESCs for digestion, followed by centrifugation to remove the collagenase, and then neural differentiation medium was added to induce differentiation to obtain dopaminergic neuron progenitor cells LV-TDG-DAPs.

[0013] Furthermore, the amount of the transfer aid polybrene is 4-6 μg / mL, preferably 5 μg / mL.

[0014] Furthermore, the multiplicity of infection of the lentiviral vector plasmids LV-TH and LV-DDC-GCH1 is 10.0.

[0015] Furthermore, the antibiotics are puromycin and blasticidin; the concentration of puromycin is 1.5-3 μg / mL, preferably 2 μg / mL; the concentration of blasticidin is 1-2 μg / mL, preferably 1.5 μg / mL.

[0016] Furthermore, the specific process of obtaining the primitive neuroepithelial cells is as follows: when the human induced pluripotent stem cell line proliferates to 30%, the mTeSR TM After culturing on BasalMedium medium for 3 days, CHIR99021 was added and cultured until the 5th day, then the medium was changed to DF / 12 medium, SB431542 and FGF8 were removed and cultured until the 7th day, SHH and Purmorphamine were removed and cultured until the 11th day, and finally the medium was changed to Neurobasal TM The medium was used to remove DMH1 and cultured until the 14th day to obtain primitive neuroepithelial cells.

[0017] Furthermore, the amounts of DMH1, SB431542, sound-healing factor (SHH), fibroblast growth factor 8 (FGF8), purmorphamine and CHIR99021 are 10 mM, 10 mM, 200 ng / mL, 100 ng / mL, 2 μM and 3 μM, respectively.

[0018] Furthermore, the neural differentiation medium is Neurobasal TM The medium culture medium was supplemented with 1% N 2 , 2% B27 by volume, 10 ng / mL brain-derived neurotrophic factor (BDNF), 10 ng / mL glial cell-derived neurotrophic factor (GDNF), 200 μM ascorbic acid (AA), 1 ng / mL transforming growth factor-β (TGF-β), 1 μM cyclic adenosine monophosphate (cAMP), and 1% L-glutamine (GlutaMax) by volume.

[0019] The present invention also provides a dopaminergic neuron progenitor cell, and the dopaminergic neuron progenitor cell is prepared by the preparation method.

[0020] Furthermore, the dopaminergic neuron progenitor cells are obtained by inducing differentiation of human induced pluripotent stem cells carrying genes of TH, DDC and GCH1, key enzymes in DA synthesis.

[0021] The present invention also provides an application of the dopaminergic neuron progenitor cells as a cell model for screening Parkinson's disease drugs.

[0022] The present invention also provides an application of the dopaminergic neuron progenitor cells in preparing a drug for treating Parkinson's disease.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] (1) The method provided by the present invention successfully constructed a dopaminergic neuron progenitor cell that can express the key enzyme gene for DA synthesis. The cell can stably express genetic expression and secrete more DA without causing the target gene level to decrease with the increase in the number of passages.

[0025] (2) The dopaminergic neuron progenitor cells provided by the present invention can synthesize and secrete a larger amount of DA transmitter. The DA content in the cell supernatant is detected by Elisa method, indicating that LV-TDG-DAPs can secrete more DA than DAPs without the target gene. The obtained dopaminergic neuron progenitor cells can be used to treat Parkinson's disease to compensate for the insufficient DA secretion caused by the death of DA neurons.

[0026] (3) The present invention detects tyrosine hydroxylase expression by brain slice immunohistochemistry, effectively verifying the survival, migration and differentiation of transplanted dopaminergic neuron progenitor cells (LV-TDG-DAPs) in the host brain; using apomorphine (Apomorphine, APO) induced rotation test, open field test and fatigue rotarod test, it is effectively shown that transplanted dopaminergic neuron progenitor cells (LV-TDG-DAPs) have an effective and significant effect on Parkinson's disease rats. HPLC detection shows that transplanted dopaminergic neuron progenitor cells (LV-TDG-DAPs) significantly upregulate DA levels in the brain transplantation area and serum. The dopaminergic neuron progenitor cells obtained by the method provided by the present invention can survive for a long time in the brain microenvironment of Parkinson's rats, and have a significant repair effect on rat motor dysfunction, laying a foundation for the treatment of Parkinson's disease with stem cell transplantation.

[0027] (4) The present invention aims to develop an enhanced two-dimensional-based induction system to generate midbrain dopamine neuron progenitor cells by dual SMAD inhibition (DMH1 and SB431542) and optimizing the concentrations of SHH, purmorphamine, FGF8 and CHIR. LDN193189 was replaced by the BMP inhibitor DMH1, which significantly reduced the expression of pluripotency markers OCT4 and Nanog. Compared with the traditional method, the improved two-dimensional monolayer culture method produced healthier dopamine neural precursor cells. In the initial stage of induction, CHIR was identified as a key factor inducing FOXA2 / LMX1A co-expression. The co-expression of FOXA2 / LMX1A requires the use of purmorphamine alone or in combination with recombinant SHH to activate the SHH signaling pathway. Dual SMAD inhibition (DMH1 and SB431542) leads to the generation of FOXA2 / LMX1A expressing cells. The second stage of the induction process involves lineage-specific induction in neural differentiation medium. At day 28, induced DAPs were identified as TH+ / TUJ1+ cells that co-expressed LMX1A and FOXA2, as well as the nuclear receptor Nurr1. Given that dopaminergic neurons in the midbrain are derived from neurons in the floor plate, DAPs were defined as cells that co-expressed FOXA2 (a floor plate marker) and TUJ1. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0029] Figure 1 This is the map of the lentiviral vector plasmid LV-TH of the present invention.

[0030] Figure 2 This is a map of the lentiviral vector plasmid LV-DDC-GCH1 of the present invention.

[0031] Figure 3 The HiPSCs cell line is a lentivirus-infected HiPSCs cell line after antibiotic screening according to the present invention;

[0032] In the figure, A is a fluorescent protein image of a cell line infected with a lentivirus;

[0033] B is the expression of TH, DDC and GCH1 proteins in infected cells detected by Western blotting;

[0034] C is the expression of TH, DDC and GCH1 genes in infected cells detected by RT-PCR.

[0035] Figure 4 The whole process of inducing the differentiation of induced pluripotent stem cells into dopaminergic neuron progenitor cells by the inventors;

[0036] In the figure, A shows the morphological changes of primitive neuroepithelial cells obtained from human induced pluripotent stem cells during differentiation from day 0 to day 13;

[0037] B shows the changes in cell morphology during the differentiation of the induced primitive neuroepithelial cells into dopaminergic neuron progenitor cells from 16 to 28 days.

[0038] Figure 5 The immunofluorescence images (200×) of primitive neuroepithelial cells obtained by inducing differentiation of four groups of human induced pluripotent stem cells of the present invention to day 14;

[0039] In the figure, A is the control group that is not infected with the virus and can express neuroepithelial cell markers SOX2, Nestin, and Pax6;

[0040] B shows that primitive neuroepithelial cells infected with LV-TDG can express SOX2, Nestin, Pax6, mCherry and ZsGreen;

[0041] C shows that primitive neuroepithelial cells infected with LV-DDC-GCH1 can express SOX2, Nestin, Pax6 and ZsGreen;

[0042] D shows that primitive neuroepithelial cells infected with LV-TH can express SOX2, Nestin, Pax6, and mCherry;

[0043] E is a statistical graph of the proportion of SOX2-positive cells, with the ordinate: Percentage of SOX2+cells indicating the percentage of SOX2-positive cells;

[0044] F is a statistical graph of the proportion of Nestin-positive cells, and the ordinate: Percentage of Nestin+cells indicates the percentage of Nestin-positive cells;

[0045] G is a statistical graph of the proportion of Pax6 positive cells, and the vertical axis: Percentage of Pax6+cells represents the percentage of Pax6 positive cells.

[0046] Figure 6 The immunofluorescence images (200×) of the in vitro differentiation potential of dopaminergic neuron progenitor cells obtained by inducing differentiation of four groups of human induced pluripotent stem cells of the present invention to day 28 are shown;

[0047] In the figure, A shows that LV-TH-DAPs can express TH, FOXA2, Nurr1, Tuj1, and Cherry, specific markers of midbrain dopaminergic neurons;

[0048] B: LV-DDC-GCH1-DAPs can express TH, FOXA2, Nurr1, Tuj1 and ZsGreen;

[0049] C: DAPs can express TH, FOXA2, Nurr1, and Tuj1;

[0050] D is LV-TDG-DAPs that can express TH, FOXA2, Nurr1, Tuj1, ZsGreen, and Cherry;

[0051] E is the detection of DA concentration in the culture supernatant of DAPs in each group by ELISA method, and the vertical axis: DA level insupernatant by Elisa (pg / mL) indicates the DA level in the supernatant determined by Elisa method (pg / mL);

[0052] Figure 7 It is a statistical graph of the in vitro differentiation potential identification of dopaminergic neuron progenitor cells obtained by inducing differentiation of four groups of human induced pluripotent stem cells to day 28 of the present invention;

[0053] In the figure, A is a statistical graph of the proportion of TH-positive cells, and the vertical axis: Percentage of TH+cells represents the percentage of TH-positive cells;

[0054] B is a statistical graph of the proportion of FOXA2-positive cells, with the ordinate: Percentage of FOXA2+cells indicating the percentage of FOXA2-positive cells;

[0055] C is a statistical graph of the proportion of Tuj1-positive cells, with the ordinate: Percentage of Tuj1+cells indicating the percentage of Tuj1-positive cells;

[0056] D is a statistical graph of the proportion of Nurr1-positive cells, and the vertical axis: Percentage of Nurr1+cells represents the percentage of Nurr1-positive cells.

[0057] Figure 8 Behavioral testing of Parkinson's disease model rats after transplantation of dopaminergic neuron progenitor cells of the present invention;

[0058] In the figure, A is the two points on the marked coordinates where cells were stereo-transplanted into the right forebrain medial bundle of each model rat; a is the sagittal plane site of the two injection sites; b is the coronal plane site of the first injection site: ①AP-4.4mm, ML-1.2mm, DV-7.8mm; c is the coronal plane site of the second injection site: ②AP-4.0mm, ML-0.8mm, DV-8.0mm;

[0059] B: rat fixed in a stereotaxic apparatus;

[0060] C is the damage of the right medial forebrain bundle induced by 6-OHDA;

[0061] D is the fatigue rotating rod test;

[0062] E is the APO-induced rotation experiment;

[0063] F is an open field experiment.

[0064] Fig. 9 These are the behavioral statistics of rats 4 weeks (4W) and 8 weeks (8W) after the cells of the present invention were transplanted;

[0065] In the figure, A is a statistical graph of APO-induced rotation experiment, and the vertical axis: Rotations / 30minutes represents rotation time / 30 minutes;

[0066] B is the statistical graph of the open field test, the ordinate: Total distance of movement (em) represents the total movement distance (em);

[0067] C is a statistical diagram of the fatigue rotating rod test, and the vertical axis: Retention time (s) represents the retention time (s).

[0068] Fig.10 This is a fluorescence image of the transplanted cells of the present invention surviving, migrating and establishing connections with neurons in the brain 8 weeks (8W);

[0069] In the figure, A shows that transplanted cells can survive and migrate in the rat brain for a long time; a is the fluorescence image at 200 μm, and b is the fluorescence image at 100 μm;

[0070] B is TH-positive cells in Healthy control and PD Model rats;

[0071] C: Transplanted LV-TDG-DAPs can survive in the brain and express neuronal markers Tuj1, SYN, and PSD95.

[0072] Fig.11 The cells transplanted by the present invention can survive in the brain for a long time up to 16 weeks (16W) and migrate to the surrounding areas (fluorescence image).

[0073] Fig.12 The immunofluorescence image is of the differentiation potential of the transplanted cells of the present invention in the brain;

[0074] In the figure, A shows that LV-TDG-DAPs can express the DA neuron marker FOXA2;

[0075] B: LV-TDG-DAPs can express the DA neuron marker LMX1A;

[0076] C: LV-TDG-DAPs can express DA neuron marker TH;

[0077] D: LV-TDG-DAPs can express the DA neuron marker Nurr1;

[0078] E: LV-TDG-DAPs can express GABA, a GABAergic neuron marker;

[0079] F: LV-TDG-DAPs can express the astrocyte marker GFAP.

[0080] Fig.13 DA levels in the brain transplant area and plasma 8 weeks after transplantation of the present invention;

[0081] In the figure, A is the HPLC chromatogram of DA, and the abscissa Time:min represents time: minutes;

[0082] B is a statistical graph of DA levels in the brain transplant area detected by HPLC, with the ordinate: DA in Midbrain (ng / mg) representing DA in the brain transplant area (ng / mg);

[0083] C is a statistical graph of DA levels in plasma detected by HPLC, and the vertical axis: DA in Serum represents DA in plasma (ng / mg). DETAILED DESCRIPTION

[0084] The present invention is described in detail below in conjunction with the accompanying drawings and specific examples, but should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources.

[0085] Lentiviral vector information involved in the embodiments of the present invention:

[0086] pHBLV-CMV-MCS-3flag-EF1-mCherry-T2A-Puro, product number: LV006, was purchased from Hanheng Biotechnology (Shanghai) Co., Ltd.

[0087] pHBLV-CMV-EF1-ZsGreen-P2A-BSD, product number: LV-060, was purchased from Hanheng Biotechnology (Shanghai) Co., Ltd.

[0088] Example 1: Construction of lentiviral vector plasmids LV-TH and LV-DDC-GCH1.

[0089] 1. Construction of lentiviral vector plasmid LV-TH

[0090] The full-length RNA sequence of human tyrosine hydroxylase (TH) was obtained from the NCBI gene library (GeneID: NM_199292). The full-length TH gene sequence is 1587 bp (SEQ ID NO. 1);

[0091] Then the TH gene was inserted into the CMV promoter of the lentiviral vector pHBLV-CMV-MCS-3flag-EF1-mCherry-T2A-puro to construct the lentiviral vector plasmid pHBLV-CMV-TH-3flag-EF1-mCherry-T2A-puro (LV-TH), the map of which is shown in Figure 1 .

[0092] 2. Construction of lentiviral vector plasmid LV-DDC-GCH1

[0093] The full-length RNA sequences of human dopamine decarboxylase (DDC) and cyclohydrolase 1 (GCH1) were obtained from the NCBI gene library (Gene ID: NM_001082971 and NM_001024024);

[0094] Then the tail end of the nucleotide sequence encoding DDC was connected to the nucleotide sequence encoding GCH1 through T2A to form a DDC-T2A-GCH1 fusion gene, and the full sequence length of the fusion gene was 2319 bp (SEQ ID NO. 2);

[0095] Then the fusion gene was inserted into the CMV promoter of pHBLV-CMV-EF1-ZsGreen-P2A-BSD to construct the lentiviral vector plasmid pHBLV-CMV-DDC-T2A-GCH1-EF1-ZsGreen-P2A-BSD (LV-DDC-GCH1), the map of which is shown in Figure 2 .

[0096] Example 2: Construction of human induced pluripotent stem cell lines stably genetically expressing TH, DDC, and GCH1 (LV-TH-HiPSCs, LV-DDC-GCH1-HiPSCs, and LV-TDG-HiPSCs).

[0097] 1. In vitro culture of HiPSCs

[0098] Human induced pluripotent stem cells (HiPSCs) derived from human neonatal foreskin cells were purchased from the Stem Cell Bank of the Chinese Academy of Sciences (Catalog No. SCSP-1301). When culturing HiPSCs in vitro, 1 mL of matrix gel was first added to the T25 culture flask in advance to ensure that the bottom of the flask was evenly covered, and then placed in 5% CO 2 Incubate in the incubator for 1 hour and set aside. Next, take out the HiPSCs with a confluency of about 80%, remove the old culture medium with a pipette, and then wash twice with PBS. Then, add 1 mL of preheated Tryple TM Express enzyme (Gibco, 12604039), digested in a 37°C incubator for 1 min, and then TM After the Express enzyme is aspirated, add 1 mL of mTeSR TM Basal Medium culture medium, use a cell scraper to collect the cells into a 15mL centrifuge tube, gently resuspend the cells (be careful not to blow into single cells), and obtain a cell suspension. Finally, inoculate the cell suspension in the centrifuge tube into a T25 culture flask and add 3mL of mTeSR TM Basal Medium was supplemented with Y27632 to a final concentration of 10 μM and incubated at 37°C with 5% CO 2 Incubator, discard the old culture medium on the second day and replace it with mTeSR TM Basal Medium culture medium was used, and the culture medium was replaced every two days to obtain cultured HiPSCs.

[0099] 2. Lentiviral infection of HiPSCs

[0100] 1. Double infection of LV-TH and LV-DDC-GCH1 lentiviruses: Select HiPSCs with good growth status by half-infection, add LV-TH (infection multiplicity MOI: 10.0) and LV-DDC-GCH1 (infection multiplicity MOI: 10.0) lentiviral vector plasmids for double lentiviral infection experiment. TM Medium culture medium was used for lentiviral infection. The multiplicity of infection remained the same as that of single lentiviral infection. The cells to be infected were washed twice with PBS and half volume of Neurobasal TM The cells were infected with medium and cultured in a 37°C cell culture incubator for 4 h, then supplemented with half the amount of Neurobasal TMAfter 24 h of infection, the HiPSCs were cultured in the culture medium.

[0101] 2. Single infection with LV-TH lentivirus: Select HiPSCs with good growth status after culture by half-infection, add LV-TH (infection multiplicity MOI: 10.0) lentiviral vector plasmid for experiment. Subsequent operations are the same as above.

[0102] 3. Single infection with LV-DDC-GCH1 lentivirus: Select HiPSCs with good growth status after culture by half-infection, and add LV-DDC-GCH1 (infection multiplicity MOI: 10.0) lentiviral vector plasmid for experiment. Subsequent operations are the same as above.

[0103] 3. Antibiotic Selection of Lentivirus-Infected HiPSCs

[0104] (1) Lentiviral dual-infected cell line (LV-TDG-HiPSCs): using mTeSR TM HiPSCs were screened for 2 days after dual infection of LV-TH and LV-DDC-GCH1 (LV-TH+LV-DDC-GCH1) lentiviruses in Basal Medium (containing 2 μg / mL puromycin (PURO)), and then cultured for 2 days in medium containing 1 μg / mL PURO, followed by stable culture for 3 days. TM The cells were screened and cultured in Basal Medium for 2 days, followed by replacement with medium containing 0.75 μg / mL BSD for another 2 days. TM The cells were stably cultured in Basal Medium for 3 days to obtain the purified lentivirus-infected HiPSCs cell line (LV-TH+DDC-GCH1-HiPSCs, referred to as LV-TDG-HiPSCs).

[0105] (2) Lentiviral single infection cell line (LV-TH-HiPSCs): using mTeSR TM HiPSCs infected with LV-TH lentivirus were screened for 2 days using Basal Medium (containing 2 μg / mL puromycin (PURO)). Subsequent operations were the same as above to obtain purified lentivirus-infected cell lines (LV-TH-HiPSCs).

[0106] (3) Lentiviral single infection cell line (LV-DDC-GCH1-HiPSCs): using mTeSR TMHiPSCs infected with LV-DDC-GCH1 lentivirus were screened for 2 days using Basal Medium (containing 2 μg / mL puromycin (PURO)). Subsequent operations were the same as above to obtain purified lentivirus-infected cell lines (LV-DDC-GCH1-HiPSCs).

[0107] HiPSCs were infected with lentivirus, and fluorescence began to appear after 24 hours, reaching the peak of fluorescence expression at 72 hours, and the infection efficiency reached 60%. After antibiotic screening, almost all cells strongly expressed the fluorescent protein carried by the lentivirus ( Figure 3 A). HiPSCs not infected with lentivirus were used as blank control group (Control), and Western blotting and RT-PCR were used to detect the expression of genes and proteins in cells. Figure 3 The results of B showed that compared with the control group HiPSCs (Control), the expression of DDC and GCH1 proteins was significantly upregulated in HiPSCs infected with LV-DDC-GCH1 lentivirus alone; the expression of TH protein was also significantly upregulated in HiPSCs infected with LV-TH lentivirus alone, and the increase in TH protein expression led to a significant increase in GCH1 protein expression; the expression of TH, DDC and GCH1 proteins was also significantly increased in HiPSCs infected with LV-TH + LV-DDC-GCH1 lentiviruses. RT-PCR analysis showed consistent results at the mRNA level with those at the protein level ( Figure 3 C).

[0108] Example 3: Method for Differentiating HiPSCs into Dopaminergic Neuron Progenitor Cells

[0109] The method for differentiating HiPSCs into dopaminergic neuron progenitor cells provided by the present invention is divided into two stages. The first stage is to induce HiPSCs for 14 days with SB431542, DMH1, sound-preserving factor (SHH), fibroblast growth factor 8 (FGF8), purmorphamine and CHIR99021 to produce intermediate cells similar to primitive neuroepithelial cells ( Figure 4 A), the second stage is to digest the original neuroepithelial cells and re-seed them in PLO-Laminin coated culture plates to continue inducing for 14 days to produce dopaminergic neuron progenitor cells ( Figure 4 B).

[0110] (1) Obtaining primitive neuroepithelial cells (iNESCs)

[0111] Four groups of cells, HiPSCs, LV-TH-HiPSCs, LV-DDC-GCH1-HiPSCs and LV-TDG-HiPSCs, were induced simultaneously. After the proliferation of HiPSCs reached 30%, they were cultured in mTeSR TM DMH1 (10 mM), SB431542 (10 mM), SHH (200 ng / mL), fibroblast growth factor 8 (FGF8, 100 ng / mL) and purmorphamine (2 μmol / L) were added to the Basal Medium culture medium to induce the cells for 3 days. On the fourth day, CHIR99021 was added to the culture medium and cultured until the fifth day. The culture medium was then replaced with neural induction medium NIM (replace mTeSR TM BasalMedium medium (DMEM / F12 basal medium, N2, DMH1, SHH, Purmorphamine, CHIR99021) was used to culture cells until day 7. In the next 4 days, SHH and Purmorphamine were removed, and then DMH1 was removed and cultured until day 14, and 4 groups of primitive neuroepithelial cells (iNESCs, LV-TH-iNESCs, LV-DDC-GCH1-iNESCs, LV-TDG-iNESCs) were obtained.

[0112] (2) Obtaining dopaminergic progenitor cells (DAPs)

[0113] During the second stage of induction, 1 mL of pre-warmed type IV collagenase was used in a 5% CO 2 , digested in an incubator at 37°C for 1 min. Afterwards, the four groups of primitive neuroepithelial cells obtained were collected into centrifuge tubes by cell scraping, and then the primitive neuroepithelial cells were inoculated into PLO-Laminin-coated culture plates. At this stage, the neural differentiation medium used was Neurobasal medium, and 1% N2, 2% B27, 10ng / mL brain-derived neurotrophic factor (BDNF), 10ng / mL glial cell-derived neurotrophic factor (GDNF), 200μM ascorbic acid (AA), 1ng / mL transforming growth factor-β (TGF-β), 1μM cyclic adenosine monophosphate (cAMP), and 1% L-glutamine were added. The cells were cultured until the 28th day, and the medium was changed every 2 days to obtain four groups of dopaminergic neuron progenitor cells (DAPs, LV-TH-DAPs, LV-DDC-GCH1-DAPs and LV-TDG-DAPs).

[0114] Immunofluorescence was used to detect the expression of specific surface markers in four groups of primitive neuroepithelial cells in the first stage of induction and dopaminergic neuron progenitor cells in the second stage. The results showed that the control group without virus infection could highly express neuroepithelial cell markers SOX2, Nestin and PAX6 ( Figure 5 A); Primitive neuroepithelial cells infected with LV-TH can express markers mCherry, SOX2, Nestin and PAX6 ( Figure 5 D), the expression of mCherry gene in HiPSCs did not decrease during the differentiation of HiPSCs, indicating that TH gene was integrated in HiPSCs and could be stably inherited; primitive neuroepithelial cells infected with LV-DDC-GCH1 could express ZsGreen, SOX2, Nestin and PAX6 ( Figure 5 C); Primitive neuroepithelial cells infected with LV-TDG can express mCherry, ZsGreen, SOX2, Nestin and PAX6 ( Figure 5 B), all four groups of cells were successfully induced into primitive neuroepithelial cells. Among them, the proportion of cells expressing SOX2 in the control group without virus infection, the LV-TH group, the LV-DDC-GCH1 group and the LV-TDG group was 93.1±1.2%, 94.5±1.3%, 93.4±1.5% and 93.5±1.0% ( Figure 5 The percentages of cells expressing Nestin were 94.2±1.5%, 93.8±1.8%, 94.6±1.8% and 94.6±1.5% ( Figure 5 F); the proportion of cells expressing PAX6 was 93.9±1.6%, 94.3±1.6%, 92.9±1.0% and 93.0±0.8% ( Figure 5 G).

[0115] Immunofluorescence analysis of dopaminergic neuron progenitor cells in the second stage of induction showed that the control group without virus infection expressed dopaminergic neuron progenitor cell markers TH, FOXA2, Tuj1 and Nurr1 ( Figure 6 C). Dopaminergic neuron progenitor cells infected with LV-TH-DAPs can express TH, FOXA2, Tuj1, Nurr1 and Cherry ( Figure 6 A). Dopaminergic neuron progenitor cells infected with LV-DDC-GCH1-DAPs can express TH, FOXA2, Tuj1, Nurr1 and ZsGreen ( Figure 6 B). Dopaminergic neuron progenitor cells infected with LV-TDG-DAPs can express TH, FOXA2, Tuj1, Nurr1, Cherry and ZsGreen ( Figure 6D), all four groups of cells were successfully induced into dopaminergic neuron progenitor cells. The gene carried by the plasmid was well expressed in the differentiated DAPs, and the expression level did not decrease with the continuous differentiation process. Among them, the proportion of cells expressing TH in the control group without virus infection, the LV-TH group, the LV-DDC-GCH1 group and the LV-TDG group was 94.2±1.5%, 94.1±1.2%, 93.8±1.6% and 93.3±1.5% ( Figure 7 A); the proportions of cells expressing FOXA2 were 93.2±1.0%, 92.0±1.5%, 92.5±1.2% and 92.5±1.4% ( Figure 7 B); the proportion of cells expressing Tuj1 was 93.6±1.2%, 94.4±1.4%, 93.3±1.1% and 92.4±1.2% ( Figure 7 C); the proportion of cells expressing Nurr1 was 93.8±1.4%, 93.0±1.2%, 92.8±1.2% and 92.0±1.4% ( Figure 7 D).

[0116] The dopaminergic neuron progenitor cells of each group were centrifuged at 3000r / min and 4℃ for 20 minutes to obtain the supernatant, which was collected in a sterile tube. The dopamine enzyme linked immunosorbent assay (ELISA) was used to determine the concentration of substantia nigra dopaminergic (DA) in the supernatant of each group. The results showed that the DA concentrations of the DAPs group, LV-TH-DAPs group, LV-DDC-GCH1-DAPs group, and LV-TDG-DAPs group were (1502.20±75.80) pg / mL, (2474.00±121.40) pg / mL, (1859.50±57.40) pg / mL, and (2832.00±70.30) pg / mL, respectively. Compared with the DAPs control group that was not infected with the virus, the DA content in the LV-TH-DAPs infected group, LV-DDC-GCH1-DAPs group, and LV-TDG-DAPs group was higher than that in the control group (P<0.05) ( Figure 6 E) ELISA results showed that when DAPs differentiated from HiPSCs carried the three genes TH, DDC and GCH1, they could secrete more DA in vitro.

[0117] Example 4: In vivo functional detection of DAPs, LV-TH-DAPs, LV-DDC-GCH1-DAPs and LV-TDG-DAPs

[0118] 1. Experimental Methods

[0119] (1) Construction of PD model

[0120] Prepare male SD rats weighing about 200 g (provided by Liaoning Changsheng Biotechnology Co., Ltd.), calculate the corresponding amount of pentobarbital based on 3% of the rat's body weight, and fix its head horizontally on a brain stereotaxic instrument after the rat is fully anesthetized ( Figure 8 B). Use iodine to disinfect the rat's head, shave the hair on the top of the head, cut the scalp longitudinally, peel off the periosteum to expose the rat skull, adjust the height of the ear bar and nose clip to balance the anterior and posterior fontanelles and left and right sides of the skull. Use a small cranial drill to drill the skull, and select the medial forebrain bundle (MFB) of the right brain as the injection target. Referring to Paxson's rat brain stereotaxic atlas, take the rat's anterior fontanelle as the base point, and select two points in the medial forebrain bundle of the right brain as the injection sites: ①AP-4.4mm, ML-1.2mm, DV-7.8mm; ②AP-4.0mm, ML-0.8mm, DV-8.0mm ( Figure 8 A). The skull of the SD rat was drilled with a skull drill, and 4 μL of hexahydroxydopa (6-OHDA) was injected into the two sites, respectively. Figure 8 C), inject once at each point, the injection time is 8 minutes, stop the injection for 8 minutes after the end, suture the incision, and inject penicillin 20wU per mouse intramuscularly for three consecutive days.

[0121] (2) Behavioral testing

[0122] 1. Ipsilateral ratations: APO induces rotational behavior on the contralateral side of the injured side to evaluate motor dysfunction. Two weeks after modeling, 0.5 mg / kg of APO was intraperitoneally injected into the modeling rats, and the number of contralateral rotations of the rats was recorded in 30 minutes. If the number of rotations reached > 210 in 30 minutes, the modeling was considered successful. Figure 8 E).

[0123] 2. Rotarod test: Before the experiment, the test set the rotarod speed to 10r / min for 30min. Three rats were tested simultaneously on three channels of the rotarod device. Each animal was tested three times. The rotarod device automatically records the number of revolutions and the time spent on the rotarod. Figure 8 D).

[0124] 3. Open field assay: The open field box is 100 cm long, 100 cm wide, and 40 cm high, and the inner wall of the box is completely black. Before the experiment, each rat to be tested was subjected to two pre-experiments to adapt to the open field environment. The rat was placed in the center of the open field box, and the Ethovision XT 10.1S system of the open field instrument (Noldus, Leesburg, Netherlands) automatically captured and tracked the hot spots of the rat, and recorded the total movement distance of the rat within 5 minutes. After each experiment, the experimental traces left by the rat were cleaned up, and the next set of experiments was carried out after waiting for about 5 minutes. Figure 8 F).

[0125] (3) Stereotaxic transplantation of DAPs, LV-TH-DAPs, LV-DDC-GCH1-DAPs, and LV-TDG-DAPs into Parkinson's disease model rats

[0126] DAPs transplantation group, LV-TH-DAPs transplantation group, LV-DDC-GCH1-DAPs transplantation group and LV-TDG-DAPs transplantation group: DAPs, LV-TH-DAPs, LV-DDC-GCH1-DAPs and LV-TDG-DAPs in good growth state were collected and digested with preheated type IV collagenase solution into centrifuge tubes. Then, the type IV collagenase solution was removed by centrifugation, and DMEM-F / 12 (Gibco, 21331020) solution was added to blow single cells and counted to ensure that the cell concentration of the cell suspension was 1.25x10 4 / μL.

[0127] Healthy control group: SD rats of the same age as the cell-transplanted PD rats were selected and no treatment was performed.

[0128] Model control group (Vehicle, PD Model): Take the successfully established PD rat model and add only the same volume of DF / 12 solution as that of the cell transplantation group.

[0129] Stereotaxic transplantation of Parkinson's model rats: The successfully established PD rat model was selected, and DAPs, LV-TH-DAPs, LV-DDC-GCH1-DAPs and LV-TDG-DAPs were transplanted into the right medial forebrain bundle of PD rats (DAPs transplantation group, LV-TH-DAPs transplantation group, LV-DDC-GCH1-DAPs transplantation group and LV-TDG-DAPs transplantation group) using the same method as the modeling.

[0130] (4) Tissue sampling

[0131] Male SD rats were randomly selected and killed directly to obtain the whole brain. 3% sodium pentobarbital was injected intraperitoneally for general anesthesia. After the rats were anesthetized, the abdominal skin was clamped with forceps, the abdominal cavity was quickly cut open with scissors, and the thoracic cavity was broken from the diaphragm. After exposing the heart, the perfusion needle was inserted from the left atrium of the rat, passed through the heart and entered the rat aorta, and then the right atrial appendage was cut. Normal saline was used for rapid perfusion to replace the whole body blood until clear liquid flowed out of the right atrial appendage, about 300 mL per rat. Then 200 mL of pre-cooled 4% polyformaldehyde fixative was slowly perfused to fix the brain tissue. After fixation, the rat brain tissue was carefully removed to ensure the integrity of the brain tissue. After removing the brain tissue, the brain tissue was fixed in 4% PFA for 4 hours. After fixation, the brain tissue was transferred to 20% sucrose solution for preliminary dehydration, and then the brain tissue was transferred to 25% sucrose solution for further dehydration until the brain tissue settled. After removing the brain tissue, it was placed at -80℃ for storage.

[0132] The brain tissue was embedded and fixed using OCT embedding medium (SAKURA, 4583), ensuring that the brain tissue was in the coronal plane and fixed on a freezing microtome. The slice thickness was set at 12 μm, and continuous slices were made from the striatum to the substantia nigra. The slices were adsorbed on a slide and stored at -20°C.

[0133] (5) Immunofluorescence

[0134] The slices were rewarmed at room temperature for 10 min, and then immunofluorescence staining (cells with red and green fluorescence) was performed to observe the survival, differentiation and migration ability of the transplanted cells in vivo.

[0135] (6) HPLC detection of DA

[0136] Before the experiment begins, solution preparation and sample processing are required. First, prepare the perchloric acid precipitant, including 0.35M perchloric acid, 1.5mM cysteine ​​and ultrapure water (10mL perchloric acid precipitant: prepared from 0.28mL perchloric acid, 1.8175mg cysteine ​​and 9.73mL ultrapure water). Secondly, prepare the DA mother solution. You need to weigh 0.00619g DA hydrochloride, add methanol to dissolve it, and dilute it to 1mL to make a 5mg / mL DA mother solution. Then dilute the DA mother solution with methanol to a working solution with concentrations of 0, 78.13, 156.25, 312.5, 625, 1250, 2500, 5000, and 10000ng / mL, and store it at -20℃. During the working curve processing, take DA mother solutions of different concentrations and mix them with the perchloric acid precipitant for testing.

[0137] For the collection of plasma samples, the posterior ocular venous plexus blood collection method was used. A 1.5 mm capillary was inserted into the inner canthus of the rat eyeball and inserted in a rotational direction toward the fundus. The insertion depth was 4 to 5 mm, and the blood was dripped into the anticoagulant tube. 50 μL of plasma sample was mixed with 250 μL of perchloric acid precipitant and centrifuged at 18000 rpm for 10 minutes, and then the supernatant was taken for testing. All operations were performed under ice bath conditions.

[0138] Weigh 50 mg of brain tissue (including substantia nigra and striatum) with a 1 / 10,000 balance, place in a clean 2 mL centrifuge tube, add 450 μL of perchloric acid precipitant, and evenly mix the tissue with a high-speed tissue homogenizer; after standing for 10 minutes, centrifuge at 18,000 rpm for 10 minutes. Take 300 microliters of the supernatant into a clean 1.5 mL centrifuge tube, centrifuge again at 18,000 rpm for 10 minutes, and transfer the supernatant to a clean injection bottle for testing. The entire processing process needs to be carried out in an ice bath environment.

[0139] The chromatographic experiment was performed using a C18 column (YMC-Triart, 4.6×100 mm, 5 μm). The mobile phase consisted of 50 mM potassium dihydrogen phosphate (KH 2 PO 4 ) and 0.08mM ethylenediaminetetraacetic acid (EDTA) composed of aqueous phase (phase A) and methanol composed of organic phase (phase B). An isocratic elution program of 15% phase B was used, the flow rate was 1mL / mL, the injection volume was 20μL, and the sample analysis time was 10min. The fluorescence detector was operated at an excitation wavelength of 290nm and an emission wavelength of 330nm, and the temperature of the column oven was 40℃.

[0140] 2. Experimental Results

[0141] Healthy normal rats without cell transplantation were used as blank control group (Control). At 4 weeks (4W) and 8 weeks (8W) after cell transplantation, the rats were subjected to APO-induced rotation test, fatigue rotarod test and open field test. Transplanted LV-TDG-DAPs significantly improved the cognitive and motor behavior disorders of Parkinson's model rats. The results of the APO-induced rotation experiment showed that healthy normal rats did not experience lateral rotation after APO injection. Four weeks after cell transplantation, the number of APO-induced rotations in the DAPs transplantation group, LV-TH-DAPs transplantation group, LV-DDC-GCH1-DAPs transplantation group and LV-TDG-DAPs transplantation group was lower than that in the control group, but the effect of the LV-TH-DAPs transplantation group and LV-TDG-DAPs group was the most obvious (P<0.01) ( Fig. 9The results of the fatigue rotarod test showed that the motor coordination ability of the LV-TDG-DAPs transplantation group was improved most significantly (P<0.01), followed by the LV-TH-DAPs transplantation group, while the recovery effect of the LV-DDC-GCH1-DAPs and HiPSCs-DAPs transplantation groups was relatively weak ( Fig. 9 C). The results of the open field test showed that 4 weeks after cell transplantation, the total movement distance of rats in the LV-TH-DAPs transplantation group and the LV-TDG-DAPs transplantation group increased significantly and the effect was the same. However, 8 weeks after cell transplantation, the LV-TDG-DAPs group showed better behavioral improvement than the LV-TH-DAPs group ( Fig. 9 B).

[0142] After LV-TDG-DAPs were transplanted into the MFB region of the right hemisphere of rats for 8 weeks, a large number of cells were observed to survive and migrate approximately 1.8 mm ( Fig.10 A). Immunofluorescence staining was performed on the PD model group (Vehicle) rats and the healthy control group (Healthy control) rats. It can be observed that the number of TH-positive cells in the substantia nigra of the PD model group rats was significantly reduced compared with the normal group rats ( Fig.10 B). The cells transplanted into the brain can express the neuronal marker Tuj1, the neuronal presynaptic membrane protein SYN, and the postsynaptic density protein PSD95. These results show that the transplanted LV-TDG-DAPs can survive in the rat brain and establish connections with the neurons in the host body ( Fig.10 C).

[0143] In order to detect the survival time of transplanted cells in rat brain, rats were observed 2 weeks (2W), 8 weeks (8W) and 16 weeks (16W) after transplantation. Fig.11 As shown in the figure, 2 weeks after cell transplantation, the transplanted cells can be observed to gather around the injury site. 8 weeks after cell transplantation, the transplanted cells began to spread from the injury site to the surrounding areas and showed obvious cell morphology. 16 weeks after cell transplantation, it can be observed that the damaged area during modeling has healed, and a large number of transplanted cells are still alive, which shows that the transplanted cells can survive in the host for more than 16 weeks, and no tumor cells are found.

[0144] Then we tested whether the transplanted LV-TDG-DAPs could differentiate into different neuronal types in rats. Immunofluorescence results showed that most of the transplanted cells expressed the dopamine neuron marker TH ( Fig.12 C), FOXA2( Fig.12 A), LMX1A( Fig.12 B), Nurr1( Fig.12D), a small number of cells can express the astrocyte marker GFAP ( Fig.12 F) and GABA ( Fig.12 E), the results showed that transplanted LV-TDG-DAPs could differentiate into functional neurons and astrocytes in rat striatum.

[0145] 8 weeks after transplantation, the DA levels in the midbrain and serum of each group of rats were detected by high performance liquid chromatography. DA (Dopamine) began to peak at around 2.5 min ( Fig.13 A). Fig.13 B and Fig.13 The results showed that the DA levels in the midbrain and serum of the model control group were 12.741±0.769 ng / mg and 3.371±0.219 ng / mL, respectively; the DA levels in the midbrain and serum of the LV-TDG-DAPs transplantation group were 22.145±1.25 ng / mg and 7.823±0.437 ng / mL, respectively; the DA levels in the midbrain and serum of the LV-TH-DAPs transplantation group were 19.473±1.04 ng / mg and 5.573±0.3 27ng / mL, the DA levels in the midbrain and serum of the LV-DDC-GCH1-DAPs transplantation group were 15.247±0.523ng / mg and 5.052±0.535ng / mL, respectively, and the DA levels in the midbrain and serum of the DAPs transplantation group were 14.523±0.924ng / mg and 4.083±0.527ng / mL, respectively. Compared with the model group, the DA concentrations in the midbrain and serum of the LV-TDG-DAPs transplantation group were significantly increased (P<0.01). The results showed that after the transplantation of LV-TDG-DAPs, the obvious recovery of DA in PD rats reflected the recovery of the DA biosynthesis pathway and function in the body.

[0146] In summary, the present invention successfully constructed a lentiviral vector that overexpressed the three key enzyme genes of the dopamine synthesis pathway, TH, DDC and GCH1, and then obtained a HiPSCs cell line expressing TH, DDC and GCH1 genes. After inducing the differentiation of this cell line into dopaminergic neuron progenitor cells, it can stably synthesize a large amount of DA transmitter. After transplantation of LV-TDG-DAPs, it can survive in the body for a long time, migrate to the surrounding damaged area and differentiate into functional neurons and astrocytes. In addition, it also significantly upregulates the levels of DA and 5-HT in the body, promoting the repair of neurodegenerative diseases and behavioral improvements.

[0147] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0148] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A method for preparing dopaminergic neuron progenitor cells, characterized in that: The following steps are involved: Construction of lentiviral vector plasmids: lentiviral vector plasmid LV-TH carrying the human TH gene and lentiviral vector plasmid LV-DDC-GCH1 carrying the human DDC and GCH1 genes were constructed respectively; In vitro culture of human induced pluripotent stem cells: culturing human induced pluripotent stem cells in vitro to obtain cultured human induced pluripotent stem cells; Lentiviral infection: Under the action of polybrene, cultured human induced pluripotent stem cells were infected with the constructed lentiviral vector plasmids LV-TH and LV-DDC-GCH1 by half-infection. The human induced pluripotent stem cell line LV-TDG-HiPSCs was obtained through antibiotic selection; Obtaining primitive neuroepithelial cells: Human induced pluripotent stem cell line LV-TDG-HiPSCs were cultured in mTeSR medium supplemented with DMH1, SB431542, SHH, FGF8, Purmorphamine, and CHIR99021. TM Induced to differentiate into primitive neuroepithelial cells LV-TDG-iNESCs under the action of Basal Medium; Obtaining dopaminergic neuron progenitor cells: Collagenase was added to the primitive neuroepithelial cells LV-TDG-iNESCs for digestion, followed by centrifugation to remove the collagenase, and then neural differentiation medium was added to induce differentiation to obtain dopaminergic neuron progenitor cells LV-TDG-DAPs.

2. The method for preparing dopaminergic neuron progenitor cells according to claim 1, wherein: The concentration of polybrene was 4-6 μg / mL.

3. The method for preparing dopaminergic neuron progenitor cells according to claim 1, wherein: The multiplicity of infection of the lentiviral vector plasmids LV-TH and LV-DDC-GCH1 was 10.

0.

4. The method for preparing dopaminergic neuron progenitor cells according to claim 1, wherein: The antibiotics are puromycin and blasticidin; the concentration of puromycin is 1.5-3 μg / mL, and the concentration of blasticidin is 1-2 μg / mL.

5. The method for preparing dopaminergic neuron progenitor cells according to claim 1, wherein: The specific process of obtaining the primitive neuroepithelial cells is as follows: after the human induced pluripotent stem cell line proliferates to 30%, the cells are cultured in mTeSR medium supplemented with DMH1, SB431542, SHH, FGF8, and Purmorphamine. TM After culturing on Basal Medium for 3 days, CHIR99021 was added and cultured until the 5th day. Then the medium was replaced with DMEM / F12 medium, SB431542 and FGF8 were removed and cultured until the 7th day. Then SHH and Purmorphamine were removed and cultured until the 11th day. Finally, the medium was replaced with Neurobasal TM The culture medium was depleted of DMH1 and cultured until day 14 to obtain primitive neuroepithelial cells.

6. The method for preparing dopaminergic neuron progenitor cells according to claim 1, wherein: The amounts of DMH1, SB431542, SHH, FGF8, Purmorphamine and CHIR99021 were 10 mM, 10 mM, 200 ng / mL, 100 ng / mL, 2 μM and 3 μM, respectively.

7. The method for preparing dopaminergic neuron progenitor cells according to claim 1, wherein: The neural differentiation medium is Neurobasal TM The medium was supplemented with 1% N2, 2% B27, 10 ng / mL BDNF, 10 ng / mL GDNF, 200 μM AA, 1 ng / mL TGF-β, 1 μM cAMP, and 1% L-glutamine.

8. A dopaminergic neuron progenitor cell, characterized in that: The dopaminergic neuron progenitor cells are prepared by the preparation method according to any one of claims 1 to 7.

9. Use of the dopaminergic neuron progenitor cells according to claim 8 as a cell model for screening drugs for Parkinson's disease.

10. Use of the dopaminergic neuron progenitor cells according to claim 8 in preparing a drug for treating Parkinson's disease.

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