Markers of dopaminergic neuronal precursor cells and uses thereof

Abstract

The present application relates to the field of medical biotechnology, and provides a dopaminergic neuron precursor cell surface marker F3 and / or SSTR2, and high-purity dopaminergic neuron precursor cells are obtained by screening the dopaminergic neuron precursor cells using the marker, and the dopaminergic neuron precursor cells have a higher efficiency of differentiating into dopaminergic neurons.

Description

Technical Field

[0001] This invention relates to the field of medical biotechnology, specifically to biomarkers of dopaminergic neuron precursor cells and their applications. Background Technology

[0002] Parkinson's disease (PD) is a common neurodegenerative disorder primarily caused by the degeneration of dopaminergic neurons in the substantia nigra and striatum. Currently, there is no effective treatment for PD, and treatment strategies focus on symptom management through the restoration of dopaminergic activity, such as levodopa therapy and deep brain stimulation. These treatments have extremely limited efficacy and cannot halt disease progression. Cell transplantation and replacement are considered the most promising new therapies for PD, potentially offering a cure. In 1987, Lund University in Sweden first used fetal dopaminergic neurons for cell transplantation. However, in addition to ethical issues surrounding fetal cell transplantation, the availability of donor cells and immune rejection problems hinder the development of this treatment. In recent years, with the development of biotechnology, represented by pluripotent stem cells, human pluripotent stem cells (hPSCs), including human embryonic stem cells (hESCs) and human induced pluripotent stem cells (iPSCs), can be induced to differentiate in vitro into functional dopaminergic neurons. The induction and differentiation process involves floor plate induction differentiation to generate proliferating progenitor cells (including floor plate progenitors), which then undergo neurogenic transformation to differentiate into postmitotic dopaminergic neuron progenitors, and subsequently differentiate into mature dopaminergic neurons (Kriks et al. (2011) Nature 480, 547-551; Kikuchi et al. (2017) Nature 548, 592-596). In PD cell therapy, cell populations mainly composed of proliferating dopaminergic neuron progenitors and / or postmitotic dopaminergic neuron progenitors are typically transplanted. These cells can differentiate into grafts containing dopaminergic neurons in vivo. However, the donor cells differentiated from hPSCs exhibit heterogeneity; they are not a single type of neural progenitor cell but a mixture of various types and fates of neural progenitor (progenitor) cells and glial progenitor (progenitor) cells. This could affect in vivo cell evaluation and even increase the risk of side effects, hindering its widespread clinical application. Therefore, establishing highly purified and stable midbrain dopaminergic neuron precursor cells and / or precursor cell donor cells is crucial for the clinical application of PD cell replacement therapy.

[0003] Through the analysis of the in vitro induced differentiation of pluripotent stem cells and the development of dopaminergic neurons, several genes selectively expressed in progenitor cells of dopaminergic neurons have been identified and reported, such as Lrp4 / CORIN (WO2004 / 065599, WO 2006 / 009241), NATO3 (WO 2007 / 021003), and MSX1 / 2 (WO 2007 / 021004). Considering the risk of tumorigenesis in progenitor cells, and the fact that progenitor cells of dopaminergic neurons possess multiple fates and differentiation potentials, the terminal cells of differentiation include not only dopaminergic neurons but also other types of neurons, glial cells, and ependymal cells (Jerber et al. (2021) Nat Genet. 53, 304–312). Postmitotic dopaminergic neuronal precursor cells are better able to differentiate into dopaminergic neurons, therefore, postmitotic dopaminergic neuronal precursor cells are superior to proliferating precursor cells in cell therapy. Several markers for dopaminergic neuronal precursor cells have been reported, such as LMX1A (WO 2005 / 052190) and various markers including CLSTN2 and PTPRO (WO2022 / 222974). However, these genes lack specificity, being expressed in both proliferating and postmitotic dopaminergic neuronal precursor cells, making it difficult to effectively enrich postmitotic dopaminergic neuronal precursor cells using these markers. Therefore, identifying and defining specific surface markers for dopaminergic neuronal precursor cells, optimizing techniques for isolating and purifying dopaminergic neuronal precursor cells, and developing products for PD cell therapy are of great significance. Summary of the Invention

[0004] In view of this, the technical problem to be solved by the present invention is to provide surface markers of dopaminergic neuronal precursor cells and their applications.

[0005] A first aspect of the invention provides specific surface markers for dopaminergic neuronal precursor cells, said surface markers including at least one of F3 or SSTR2.

[0006] In this invention, F3 (coagulation factor III), also known as tissue factor or CD142, is a single-channel type I membrane protein belonging to the tissue factor family. Human F3 consists of 295 amino acids, is approximately 47 kDa in size, and contains a transmembrane region (residues 252-274).

[0007] In this invention, SSTR2 is a member of the somatostatin receptor (SSTR) family, belonging to the G protein-coupled receptor family, and its natural ligand is somatostatin (SST). Human SSTR2 consists of 369 amino acids and contains 7 transmembrane domains.

[0008] The surface markers described in this invention are obtained by single-cell sequencing analysis of the differentiation products at multiple time points during the differentiation process after inducing differentiation from two conventional human pluripotent stem cells into dopaminergic neurons using two conventional methods. These markers exhibit significant cell specificity. The first differentiation method is a method developed by Lorenz Studer's team in 2011 that utilizes a substrate to induce in vitro directed differentiation of pluripotent stem cells into dopaminergic neurons (Kriks et al. (2011) Nature 480, 547-551). Currently, other induction differentiation methods are adjustments based on this method. The second differentiation method is an optimization of the first method reported by Lorenz Studer's team in 2021, which uses "CHIR-boost" to perform biphasic activation of the WNT signaling pathway during differentiation (Kim et al. (2021) Cell Stem Cell 28, 343-355). The inventors of this application discovered that the F3 gene is specifically expressed in post-mitotic dopaminergic neuron precursor cells obtained by the first differentiation method. This population of post-mitotic dopaminergic neuron precursor cells also specifically expresses genes such as ASCL1, TH, and TPH1, and can be directed to differentiate into dopaminergic neurons. The SSTR2 gene is specifically expressed in post-mitotic dopaminergic neuron precursor cells obtained by the second differentiation method. This population of post-mitotic dopaminergic neuron precursor cells also specifically expresses neuroblast characteristic genes NEUROD1 / 4, NHLH1 / 2, and NEUROG1 / 2 (La Manno et al. (2016) Cell 167, 566–580; Jerber et al. (2021) Nat Genet. 53, 304–312), and can also be directed to differentiate into dopaminergic neurons. Therefore, this invention names F3 and / or SSTR2-positive dopaminergic neuron precursor cells as directed dopaminergic neuron precursor cells. F3 and SSTR2 surface markers can be used to isolate, purify, or detect and identify dopaminergic neuronal precursor cells with directed differentiation capabilities.

[0009] The dopaminergic neuron precursor cell surface marker F3 described in this invention is a specific surface receptor for dopaminergic neuron precursor cells, used for purifying dopaminergic neuron precursor cells. In some specific embodiments of this invention, by using this specific surface receptor to sort dopaminergic neuron precursor cells, dopaminergic neuron precursor cells can be effectively enriched; and these dopaminergic neuron precursor cells can be efficiently differentiated into dopaminergic neurons, with a differentiation efficiency of over 30%, preferably over 40%, more preferably over 50%, more preferably over 60%, more preferably over 70%, and more preferably over 80%; in some specific embodiments of this invention, the differentiation efficiency is as high as 69%.

[0010] The SSTR2 marker for dopaminergic neuron precursor cells described in this invention is a specific surface receptor for dopaminergic neuron precursor cells, used for purifying dopaminergic neuron precursor cells. In some specific embodiments of this invention, by using this specific surface receptor to sort dopaminergic neuron precursor cells, dopaminergic neuron precursor cells can be effectively enriched; and these dopaminergic neuron precursor cells can be efficiently differentiated into dopaminergic neurons, with a differentiation efficiency of over 30%, preferably over 40%, more preferably over 50%, more preferably over 60%, more preferably over 70%, and more preferably over 80%; in some specific embodiments of this invention, the differentiation efficiency is as high as 66%.

[0011] Furthermore, in other embodiments of the present invention, the dopaminergic neuron precursor cell markers F3 and SSTR2, compared with other dopaminergic neuron precursor cell markers, can more accurately label dopaminergic neuron precursor cells, and the dopaminergic neuron precursor cells obtained by screening have a high differentiation efficiency when cultured and differentiated into dopaminergic neurons; and compared with other markers, the dopaminergic neuron precursor cell markers F3 and SSTR2 have higher cell selectivity.

[0012] A second aspect of the invention provides the use of the aforementioned surface markers in the identification, isolation, and / or enrichment of dopaminergic neuronal precursor cells. These dopaminergic neuronal precursor cells are capable of differentiating into dopaminergic neurons both in vivo and in vitro.

[0013] Furthermore, the identification includes:

[0014] To determine whether candidate cells possess the following markers on their surface: F3 and / or SSTR2,

[0015] Candidate cells possessing the aforementioned markers are identified as dopaminergic neuron precursor cells.

[0016] Preferably, the candidate cells are neuronal precursor cells.

[0017] Preferably, the neuronal precursor cells are obtained by isolating fetal or human brain tissue, inducing differentiation of human pluripotent stem cells, and transdifferentiating human tissue cells;

[0018] Preferably, the human pluripotent stem cells include human embryonic stem cells and / or induced pluripotent stem cells.

[0019] Furthermore, the induced differentiation of human pluripotent stem cells includes the following steps:

[0020] Human pluripotent stem cells were contacted with the first inducing agent to induce differentiation into midbrain lamina precursor cells.

[0021] The obtained midbrain basal plate precursor cells were contacted with a second inducing agent to induce differentiation into dopaminergic neuron precursor cells.

[0022] In this invention, the first inducing agent includes BMP signaling pathway inhibitors, TGF-β signaling pathway inhibitors, SHH signaling pathway agonists, pomegranate, GSK-3 signaling pathway inhibitors, and fibroblast growth factor.

[0023] Preferably, the first inducing agent comprises 100–250 nM of a BMP signaling pathway inhibitor, 1–10 μM of a TGF-β signaling pathway inhibitor, 100–500 ng / mL of a SHH signaling pathway agonist, 0.7–7.5 μM of a GSK-3 signaling pathway inhibitor, 100 ng / mL of fibroblast growth factor, and / or 1–2 μM of puromorphamine;

[0024] More preferably, the first inducing agent comprises: 100-250 nM of the BMP signaling pathway inhibitor LDN193189, 1-10 μM of the TGF-β signaling pathway inhibitor SB431542, 100-500 ng / mL of the SHH signaling pathway agonist SHHC25II, 0.7-7.5 μM of the GSK-3 signaling pathway inhibitor CHIR99021, 100 ng / mL of fibroblast growth factor FGF8a and / or 1-2 μM of purinemorphine;

[0025] The second inducing agent includes BDNF, GDNF, dibutyryl cyclic adenosine monophosphate, TGF-β3, DAPT, vitamin C and / or GSK-3 signaling pathway inhibitors;

[0026] Preferably, the second inducing agent comprises 20 ng / mL BDNF, 20 ng / mL GDNF, 200 μM–500 μM dibutyryl cyclic adenosine monophosphate, 1 ng / mL TGF-β3, 10 μM DAPT, 200 μM vitamin C and / or 3 μM GSK-3 signaling pathway inhibitor;

[0027] More preferably, the second inducing agent comprises 20 ng / mL BDNF, 20 ng / mL GDNF, 200 μM–500 μM dibutyryl cyclic adenosine monophosphate, 1 ng / mL TGF-β3, 10 μM DAPT, 200 μM vitamin C and / or 3 μM GSK-3 signaling pathway inhibitor CHIR99021.

[0028] In the application described in this invention, the determination includes contacting the candidate cells with a reagent targeting F3 and / or SSTR2, and then detecting the expression and / or activity levels of F3 and / or SSTR2 in the candidate cells.

[0029] Furthermore, the reagents targeting F3 and / or SSTR2 include reagents targeting nucleic acid molecules encoding F3 and / or SSTR2, and / or reagents targeting F3 and / or SSTR22 proteins.

[0030] Furthermore,

[0031] The reagents targeting nucleic acid molecules encoding F3 and / or SSTR2 include primers capable of specifically amplifying nucleic acid molecules encoding F3 and / or SSTR2 and / or probes capable of specifically recognizing nucleic acid molecules encoding F3 and / or SSTR2.

[0032] The reagents targeting F3 and / or SSTR22 proteins include reagents capable of specifically binding to F3 and / or SSTR2 proteins and / or reagents capable of measuring the activity of F3 and / or SSTR2 proteins.

[0033] Furthermore,

[0034] The reagents that specifically bind to F3 and / or SSTR2 proteins include antibodies against F3 and / or SSTR2, or anti-antibodies against F3 and / or SSTR2.

[0035] Preferably, the amount of antibody used in F3 is 0.15–0.2 μg per 1 million candidate cells;

[0036] Preferably, the amount of SSTR2 antibody used is 10 μL per 1 million candidate cells;

[0037] Preferably, the reagent is labeled with fluorescein.

[0038] A third aspect of the invention provides reagents for identifying, isolating, or enriching dopaminergic neuronal precursor cells, which target the surface markers described in the invention.

[0039] Furthermore, the reagents include: reagents targeting nucleic acid molecules encoding F3 and / or SSTR2, and / or reagents targeting F3 and / or SSTR22 proteins.

[0040] Furthermore,

[0041] The reagents targeting nucleic acid molecules encoding F3 and / or SSTR2 include primers capable of specifically amplifying nucleic acid molecules encoding F3 and / or SSTR2 and / or probes capable of specifically recognizing nucleic acid molecules encoding F3 and / or SSTR2.

[0042] The reagents targeting F3 and / or SSTR22 proteins include reagents capable of specifically binding to F3 and / or SSTR2 proteins and / or reagents capable of measuring the activity of F3 and / or SSTR2 proteins.

[0043] Furthermore, the reagents that specifically bind to F3 and / or SSTR2 proteins include antibodies against F3 and / or SSTR2, or anti-antibodies against F3 and / or SSTR2.

[0044] Preferably, the amount of antibody used in F3 is 0.15–0.2 μg per 1 million candidate cells;

[0045] Preferably, the amount of SSTR2 antibody used is 10 μL per 1 million candidate cells;

[0046] Preferably, the reagent is labeled with fluorescein.

[0047] A fourth aspect of the present invention provides a kit for identifying, isolating, or enriching dopaminergic neuronal precursor cells, comprising excipients and the reagents described herein.

[0048] Furthermore, the excipients include any one or more of culture media, inhibitors, activators, growth factors, buffer solutions, and / or culture instruments.

[0049] The fifth aspect of the present invention provides a method for isolating and / or enriching dopaminergic neuronal precursor cells, which uses the reagents and / or kits described in the present invention to isolate and / or enrich dopaminergic neuronal precursor cells.

[0050] Furthermore, the separation and enrichment methods include any one of the following: fluorescently labeled cell sorting, immunomagnetic bead separation, or immunoadsorption column separation.

[0051] In a specific embodiment of the present invention, the method for isolating and / or enriching dopaminergic neuron precursor cells specifically includes the following steps:

[0052] Step 1: Provide a population of neuronal precursor cells;

[0053] Step 2: Isolate and / or enrich dopaminergic neuronal precursor cells possessing the following markers from the neuronal precursor cell population: F3 or SSTR2.

[0054] Furthermore, the methods for isolating and / or enriching dopaminergic neuron precursor cells in step 2 include fluorescently labeled cell sorting, immunomagnetic bead separation, or immunoadsorption column separation; preferably, these methods use antibodies that specifically bind to F3 and / or SSTR2, or antibodies that are linked to fluorescently labeled, magnetic materials, or adsorption columns to F3 and / or SSTR2, or antibodies that specifically bind to F3 and / or SSTR2; more preferably, antibodies that specifically bind to F3 and / or SSTR2 are used.

[0055] This invention provides dopaminergic neuron precursor cells, which are obtained by the method described in this invention.

[0056] This invention provides dopaminergic neuron cells obtained by culturing and differentiating the aforementioned dopaminergic neuron precursor cells.

[0057] This invention provides the application of at least one of the following (i) to (ii) in the study of the pathogenesis pathways of neurodegenerative diseases and in the preparation of drugs for the prevention and treatment of neurodegenerative diseases:

[0058] i) The dopaminergic neuron precursor cells described in this invention;

[0059] ii) The dopaminergic neurons described in this invention.

[0060] Furthermore, the neurodegenerative disease includes at least one of cerebral ischemia, brain injury, Parkinson's disease, brain atrophy, Alzheimer's disease, or amyotrophic lateral sclerosis.

[0061] This invention provides a method for preventing, treating, or alleviating neurological diseases or conditions, the method comprising the following steps:

[0062] Determine whether candidate cells possess the following markers on their surface: F3 or SSTR2;

[0063] Select cells that possess the aforementioned markers; and

[0064] Administer an effective dose of cells containing the biomarker to the subjects who require it.

[0065] The beneficial effects of this invention are:

[0066] This invention discovers that F3 and SSTR2 are specific surface markers for dopaminergic neuron precursor cells. F3-positive dopaminergic neuron precursor cells possess the bidirectional differentiation ability into both dopaminergic and serotonergic neurons, while SSTR2-positive dopaminergic neuron precursor cells can be directed to differentiate into dopaminergic neurons. Therefore, compared to other dopaminergic neuron precursor cell markers, using F3 and / or SSTR2 as surface markers can provide high-purity, more homogeneous dopaminergic neuron precursor cells and the dopaminergic neurons differentiated from these precursor cells.

[0067] Dopaminergic neuron precursor cells can be rapidly isolated and purified by using F3 and / or SSTR2 as surface markers in combination with flow cytometry or magnetic bead sorting.

[0068] The F3 and / or SSTR2-positive dopaminergic neuronal precursor cells and their differentiated dopaminergic neurons provided by this invention have a wide range of applications. They provide an ideal cell model for seed cells in cell therapy for neurodegenerative diseases, disease mechanism research, drug screening, and developmental studies of dopaminergic neurons.

[0069] The F3 and / or SSTR2 positive dopaminergic neuron precursor cells provided by this invention are post-mitotic cells, which reduce the risk of tumor development and are more in line with the role and requirements of cell therapy donor cells. Attached Figure Description

[0070] Figure 1 The cell differentiation of the first differentiation method (protocol 1) is shown. Among them, a) is the single-cell sequencing UMAP map of pluripotent stem cells differentiated into dopaminergic neurons at multiple time points during the differentiation process of pluripotent stem cells using the first differentiation method into dopaminergic neurons; b) is the expression of dopaminergic neuron precursor cells and the characteristic genes NEUROD1, ASCL1, NR4A2, and TH of dopaminergic neurons.

[0071] Figure 2 The cell differentiation of the second differentiation method (protocol 2) is shown. Among them, a) is the single-cell sequencing UMAP map of pluripotent stem cells differentiated into dopaminergic neurons at multiple time points during the differentiation process of pluripotent stem cells using the second differentiation method into dopaminergic neurons; b) is the expression of dopaminergic neuron precursor cells and the characteristic genes NEUROD1, ASCL1, NR4A2, and TH of dopaminergic neurons.

[0072] Figure 3 The expression of F3 and SSTR2 genes in cells obtained by the first and second differentiation methods is shown.

[0073] Figure 4Flow cytometry sorting of F3 and SSTR2 positive cells increased the differentiation rate of dopaminergic neurons, while F3 and SSTR2 negative cells could not differentiate into dopaminergic neurons. In the diagram, A shows the gene expression of F3 in the first differentiation method; B shows the flow cytometry sorting of F3 positive cells during the differentiation of D22 cells into dopaminergic neuron precursor cells using the first differentiation method, with a positive rate of 40.7%; C shows the results of the sorted F3 negative cells after differentiation, with the left side showing bright field staining and the right side showing immunofluorescence staining results. DAPI is the nuclear dye, indicating the total number of cells, and TUJ1 is the β-tubulin marker. Figure 1 shows the proportion of nerve cells. Tyrosine hydroxylase (TH), a marker of dopaminergic neurons, is shown, indicating the proportion of dopaminergic neurons and highlighting the extremely low neural differentiation efficiency of F3-negative cells. Figure 2 shows the differentiation of sorted F3-positive cells into dopaminergic neurons, revealing a clear mature neural network and high specific expression of the dopaminergic neuron-specific gene TH. Figure 3 shows the statistical analysis of Figures 4 and 5, demonstrating that F3-positive cells can differentiate into nerve cells, especially dopaminergic neurons, with 69.39% of F3-positive cells differentiating into TH-positive cells. The percentage of SSTR2-positive cells differentiating into TH cells was only 7.29%; F shows the SSTR2 gene expression in the second differentiation method; G shows the flow cytometry sorting of SSTR2-positive cells when differentiating D16 cells into dopaminergic neuronal precursor cells using the second differentiation method, with a positive rate of 11.1%; H shows the results of the sorted SSTR2-negative cells after differentiation, with the left side showing bright field and the right side showing immunofluorescence staining results, indicating that SSTR2-negative cells cannot differentiate into neural cells, exhibiting extremely low differentiation efficiency. The immunofluorescence staining results show that only DAPI indicates the distribution of the cell nucleus, while TU... The absence of J1 and TH expression indicates that SSTR2-negative cells cannot differentiate into nerve cells, let alone dopaminergic neurons. Figure I shows the results of sorted SSTR2-positive cells differentiating into dopaminergic neurons, revealing that complex cells have formed a complex neural network. Figure J is a statistical comparison of Figures H and I, showing that SSRT2-positive cells can differentiate into nerve cells, especially dopaminergic neurons, with 66.25% of SSRT2-positive cells differentiating into TH-positive cells, while only 1.32% of F3-negative cells differentiated into TH-positive cells.

[0074] Figure 5 This indicates that some CORIN genes are expressed in proliferating precursor cells of dopaminergic neurons;

[0075] Figure 6 The results showed that PTPRO gene expression was universal in both differentiation methods and did not exhibit cell selectivity. Detailed Implementation

[0076] This invention provides surface markers for dopaminergic neuronal precursor cells and their applications. A detailed description is provided below with reference to the accompanying drawings and specific embodiments. It should be understood that these embodiments are for illustrative purposes only and not for limiting the scope of the invention. After reading this invention, any similar substitutions and modifications made by those skilled in the art are considered to be included within the scope of this invention. All test materials used in this invention are common commercially available products.

[0077] The present invention will be further illustrated below with reference to the embodiments:

[0078] Example 1: Obtaining surface markers of dopaminergic neuron precursor cells

[0079] This invention provides a method for preparing high-quality dopaminergic neuron precursor cells using single-cell sequencing technology. During the process of inducing pluripotent stem cells to differentiate into dopaminergic neurons, different differentiation methods produce dopaminergic neuron precursor cells with different characteristics. F3 is a marker for dopaminergic neuron precursor cells produced by one commonly used differentiation method, and SSTR2 is a marker for dopaminergic neuron precursor cells produced by another commonly used differentiation method. High-purity dopaminergic neuron precursor cells can be obtained using these two markers.

[0080] I. Acquisition of surface marker F3 (first differentiation method)

[0081] The first aspect of the present invention provides an analytical method for identifying tissue factor F3 as a marker on the surface of dopaminergic neuronal precursor cells obtained as a method of directed differentiation, comprising the following steps:

[0082] A: Human pluripotent stem cells that have grown to a cell density of 70%–80% are digested into single cells, at a rate of 300,000 cells / cm³. 2 The density was transferred into Matrigel (Corning, 354277) coated 24-well plates and cultured in mTeSR medium (STEMCELL, 85850) with 10 μM MRock inhibitor Y-27632 (ALX-270-333-M005) added.

[0083] B: On day 0, the culture medium was replaced with SRM medium (Table 1) and supplemented with 100 nM of BMP inhibitor LDN193189 (Stemgent, 04-0074) and 10 μM of TGF-β inhibitor SB431542 (R&D, 1614).

[0084] Table 1. Basal culture medium for dopaminergic neuron differentiation

[0085]

[0086] C: On days 1-2, the culture medium was replaced with SRM medium and 100 nM LDN193189, 10 μM SB431542 and 100 ng / mL Sonic Hedgehog signaling pathway agonist SHH C25II (R&D, 464-SH), 100 ng / mL FGF8a (R&D, 4745-F8) and 2 μM purinemorphine (STEMCELL, 72202) were added.

[0087] D: On days 3-4, replace the culture medium with SRM medium and add 100 nM LDN193189, 10 μM SB431542, 100 ng / mL SHH C25II, 100 ng / mL FGF8a, 2 μM purinemorphine and 3 μM GSK-3 inhibitor CHIR99021;

[0088] E: On days 5-6, the culture medium was replaced with 75% SRM / 25% N2B27 medium (Table 2), and 100 nM DN193189, 100 ng / mL SHH C25II, 100 ng / mL FGF8a, 2 μM purinemorphine and 3 μM CHIR99021 (R&D, 4432) were added.

[0089] Table 2. Basal culture medium for dopaminergic neuron differentiation

[0090]

[0091]

[0092] F: On days 7-8, replace the culture medium with 50% SRM / 50% N2B27 medium and add 100 nM LDN193189 and 3 μM CHIR99021;

[0093] G: On days 9-10, replace the culture medium with 25% SRM / 75% N2B27 medium and add 100 nM LDN193189 and 3 μM CHIR99021;

[0094] H: On days 11-12, the culture medium was replaced with N2B27 medium and 3 μM CHIR99021, 20 ng / mL GDNF (Peptrotech, 450-10), 20 ng / mL BDNF (R&D, 248-BD), 500 μM dibutyryl cyclic adenosine monophosphate (Sigma, 4043), 1 ng / mL TGF-β3 (R&D, 243-B3), 10 μM DAPT (R&D, 2634) and 200 μM vitamin C (Sigma, 4034) were added.

[0095] I: On day 13, cells were digested into single cells using Accutase (Innovative Cell Technologies, AT104-500), centrifuged, and resuspended in N2B27 medium. 10 μM Y27632, 20 ng / mL GDNF, 20 ng / mL BDNF, 500 μM dibutyryl cyclic adenosine monophosphate, 1 ng / mL TGF-β3, 10 μM DAPT, and 200 μM vitamin C were added to the medium. Cells were then seeded in 12-well plates pretreated with poly-L-orthinine (Sigma, P3655) and laminin (R&D, 3400-010-1).

[0096] J: On day 14, change the culture medium to N2B27 medium and add 20 ng / mL GDNF, 20 ng / mL BDNF, 500 μM dibutyryl cyclic adenosine monophosphate, 1 ng / mL TGF-β3, 10 μM DAPT and 200 μM vitamin C to the medium. Continue culturing, changing the medium daily. Subculture once on day 22 and continue culturing until day 51, changing the medium every other day.

[0097] K: During the differentiation process, samples were collected at multiple time points after differentiation (D0, D1, D3, D5, D7, D9, D11, D13, D15, D16, D18, D20, D22, D25, D30, D40, D51) for single-cell RNA-seq analysis to obtain the cell populations that appeared during the differentiation of dopaminergic neurons. Figure 1 The dynamic changes in the transcriptome were analyzed, and key dopaminergic neuron precursor cells were identified through trajectory analysis. Differential gene analysis of the cell populations produced during differentiation identified the surface molecular marker F3 (F3) specifically expressed in dopaminergic neuron precursor cells. Figure 3 ).

[0098] II. Obtaining the surface marker SSTR2 (Second differentiation method)

[0099] A second aspect of the present invention provides an analytical method for identifying somatostatin receptor SSTR2 as a dopaminergic neuron precursor cell surface marker obtained by another directed differentiation method, comprising the following steps:

[0100] A: Human pluripotent stem cells that have grown to a cell density of 70%–80% are digested into single cells, at a rate of 300,000 cells / cm³. 2 The density was transferred into Matrigel (Corning, 354277) coated 24-well plates and cultured overnight in mTeSR medium with 10 μM MRock inhibitor Y-27632 added;

[0101] B: Differentiation begins the next day. On days 0-3, the culture medium is replaced with N2B27 medium and 250 ng / mL DN193189, 10 μM SB431542, 500 ng / mL SHH C25II and a low concentration of 0.7 μM CHIR99021 are added.

[0102] C: On days 4 to 6, the culture medium was replaced with N2B27 medium and 250 nM LDN193189, 10 μM MSB431542, 500 ng / mL SHH C25II and a high concentration of 7.5 μM CHIR99021 were added.

[0103] D: On days 7-9, replace the culture medium with N2B27 medium and add a high concentration of 7.5μM CHIR99021;

[0104] E: On day 10, the culture medium was changed to N2B27 medium and a moderate concentration of 3 μM CHIR99021 and 20 ng / mL GDNF, 20 ng / mL BDNF, 200 μM dibutyryl cyclic adenosine monophosphate, 1 ng / mL TGF-β3 and 200 μM vitamin C were added.

[0105] F: On day 11, the culture medium was changed to N2B27 medium and 20 ng / mL GDNF, 20 ng / mL BDNF, 200 μM dibutyryl cyclic adenosine monophosphate, 1 ng / mL TGF-β3, and 200 μM vitamin C were added.

[0106] G: On day 12, the culture medium was changed to N2B27 medium and 20 ng / mL GDNF, 20 ng / mL BDNF, 200 μM dibutyryl cyclic adenosine monophosphate, 1 ng / mL TGF-β3, 200 μM vitamin C, and 10 μM DAPT were added.

[0107] H: On day 13, cells were digested into single cells, centrifuged, and resuspended in N2B27 medium. 10 μM Y-27632, 20 ng / mL GDNF, 20 ng / mL BDNF, 200 μM dibutyryl cyclic adenosine monophosphate, 1 ng / mL TGF-β3, 200 μM vitamin C, and 10 μM DAPT were added to the medium. Cells were then seeded in 12-well plates pretreated with poly-L-orthinine (Sigma, P3655) and laminin (R&D, 3400-010-1).

[0108] J: On day 14, change the culture medium to N2B27 medium and add 20 ng / mL GDNF, 20 ng / mL BDNF, 200 μM dibutyryl cyclic adenosine monophosphate, 1 ng / mL TGF-β3, 200 μM vitamin C, and 10 μM DAPT to the medium. Continue culturing, changing the medium daily. Subculture once on days 16-17 and continue culturing until day 51, changing the medium every other day.

[0109] K: Samples were collected at specific time points (D0, D1, D3, D5, D7, D9, D11, D13, D15, D16, D18, D20, D22, D25, D30, D40, D51) during the differentiation process, and single-cell RNA-seq analysis was performed to obtain the cellular states that occurred during the differentiation of dopaminergic neurons. Figure 2 The dynamic changes in the transcriptome were analyzed, and key dopaminergic neuron precursor cells were identified through trajectory analysis. Differential gene analysis of the cell populations generated during differentiation identified the surface molecular marker SSTR2 (SSTR2) specifically expressed in dopaminergic neuron precursor cells. Figure 3 ).

[0110] F3 and SSTR2, as surface markers of dopaminergic neuron precursor cells, can be used to identify, isolate, and purify dopaminergic neuron precursor cells. In the first differentiation method, F3-positive cells accounted for 40.7% by flow cytometry on day 22. These sorted cells were further cultured and differentiated into dopaminergic neurons. The differentiation efficiency of F3-positive cells into dopaminergic neurons was as high as 69%, while F3-negative cells did not have the ability to differentiate into neural cells. Figure 4 (A to E in the original text).

[0111] In the second differentiation method, on day 16, SSTR2-positive cells accounted for 11.1% of the cells sorted by flow cytometry. These cells were further cultured and differentiated into dopaminergic neurons. The differentiation efficiency of SSTR2-positive cells into dopaminergic neurons was as high as 66%, while SSTR2-negative cells did not have the ability to differentiate into neural cells. Figure 4 (F~J in the middle).

[0112] III. Comparison with other markers

[0113] The previously reported biomarkers CORIN (WO 2004 / 065599, WO 2006 / 009241) are markers for proliferating precursor cells (including basal plate precursor cells) of dopaminergic neurons. Single-cell sequencing of the differentiation process of dopaminergic neurons in vivo and in vitro revealed that proliferating precursor cells can differentiate into post-mitotic dopaminergic neuronal precursor cells via neurogenic transformation, including neuroblasts. The biomarker SSTR2 in this invention is a neuroblast marker, and neuroblasts can be directed to differentiate into dopaminergic neurons. F3 is another marker for dopaminergic neuronal precursor cells generated from proliferating precursor cells, which can also be directed to differentiate into dopaminergic neurons. We also found that some CORIN-positive cells could not differentiate into dopaminergic neurons during in vitro differentiation, such as glioblasts generated by the first differentiation method and a basal plate precursor cell population generated by the second differentiation method (e.g., Figure 5 Therefore, the markers of this invention more accurately label the dopaminergic neuron precursor cells of directed differentiation.

[0114] In addition, several dopaminergic neuron precursor cell markers have been reported, such as LMX1A (WO 2005 / 052190), CLSTN2, and PTPRO (WO 2022 / 222974). In the two differentiation methods of this invention, CLSTN2 is not expressed, and LMX1A and PTPRO are universally expressed without cell selectivity (e.g., ...). Figure 6 ).

[0115] Example 2 uses F3 as a marker to isolate and purify dopaminergic neuronal precursor cells.

[0116] Purification of dopaminergic neuron precursor cells obtained from hPSC-induced differentiation includes hPSC culture, dopaminergic neuron precursor cell induction, and flow cytometry sorting. The specific steps are as follows:

[0117] (1) hPSCs were cultured in 6-well plates with mTeSR medium in a 5% CO2 incubator at 37°C. The medium was changed daily, and the cells were digested with 0.5 mM EDTA or Accutase every 3 to 4 days. The cells were passaged at a ratio of 1:3 to 1:5.

[0118] (2) When the cell density reaches 70%–80%, the cells are digested into single cells using Accutase, counted using a cell counter, and resuspended in mTeSR medium containing 10 μM Y-27632 at a density of 300,000 cells / cm³. 2 Cells were seeded at a density suitable for dopaminergic neuron differentiation in 24-well plates and cultured in a 37°C incubator with 5% CO2. The cells were observed under a microscope the following day; ideally, the cells should completely cover the entire plate. These cells will be used for dopaminergic neuron differentiation.

[0119] (3) Differentiation begins, marked as day 0: aspirate supernatant, add 2 mL of SRM medium (Table 1) to each well of the 24-well plate, and supplement with 100 nM LDN193189 and 10 μM SB431542. Incubate at 37°C in a 5% CO2 incubator.

[0120] (4) Days 1-2: Discard the supernatant, add 2 mL of SRM medium to each well of the 24-well plate, and supplement with 100 nM LDN193189, 10 μM SB431542, 100 ng / mL SHH C25II, 100 ng / mL FGF8a, and 2 μM purinemorphine. Incubate at 37°C in a 5% CO2 incubator.

[0121] (5) Days 3-4: Same as Day 2, but add 3μM CHIR99021 on the basis of Day 2.

[0122] (6) Days 5-6: Discard the supernatant and slowly add 2 mL of culture medium according to the ratio of 75% SRM / 25% N2B27 medium (Table 1) to avoid cell detachment due to excessive stress. Supplement with 100 nM LDN193189, 100 ng / mL SHH C25II, 100 ng / mL FGF8a, 2 μM purinemorphine, and 3 μM CHIR99021, and incubate at 37°C in a 5% CO2 incubator.

[0123] (7) Days 7-8: Discard the supernatant and slowly add 2 mL of culture medium to each well at a ratio of 50% SRM / 50% N2B27. Supplement with 100 nM LDN193189 and 3 μM CHIR99021 and incubate at 37°C in a 5% CO2 incubator.

[0124] (8) Days 9-10: Discard the supernatant and slowly add 2 mL of culture medium to each well at a ratio of 25% SRM / 75% N2B27. Supplement with 100 nM LDN193189 and 3 μM CHIR99021 and incubate at 37°C in a 5% CO2 incubator.

[0125] (9) Days 11-12: Discard the supernatant and slowly add 2 mL of N2B27 medium to each well. Supplement with 3 μM CHIR99021, 20 ng / mL BDNF, 200 μM Vitamin C, 20 ng / mL GDNF, 500 μM dibutyryl cyclic adenosine monophosphate, 1 ng / mL LTGF-β3, and 10 μM DAPT. Incubate at 37°C in a 5% CO2 incubator.

[0126] (10) Day 13: On the day of passage, aspirate the supernatant, add 0.5 mL of Accutase to each well, and digest at 37°C for 15–20 min. Add 1.5 mL of DMEM / F-12 (CORNING, 10-092-CVRV) to each well to terminate the digestion. Resuspend the digested cells in a 15 mL centrifuge tube and centrifuge at 300 g for 3 min. Resuspend the cells in the same culture medium as on day 12 with 10 μM Y-27632. Seed the cells at a 1:1 ratio in 12-well plates pretreated with poly-L-orthinine (Sigma, P3655) and laminin (R&D, 3400-010-1) and incubate at 37°C in a 5% CO2 incubator.

[0127] (11) Days 14-21: Change the medium daily, replacing each well with 2 mL of fresh N2B27 medium and supplementing with 20 ng / mL BDNF, 200 μM vitamin C, 20 ng / mL GDNF, 500 μM dibutyryl cyclic adenosine monophosphate, 1 ng / mL TGF-β3, and 10 μM DAPT. Incubate at 37°C in a 5% CO2 incubator.

[0128] (12) On day 22, add 0.5 mL of Accutase to each well, digest at 37°C for 10–15 min, add 0.3 mL of 3% BSA (w / w) to stop digestion, gently pipette to resuspend the cells, transfer the cell suspension to a 1.5 mL EP tube, centrifuge at 300 g for 5 min. Aspirate the supernatant, resuspend the cells in 0.5 mL of 3% BSA (w / w) and block at room temperature for 15 min. Add F3-PE antibody (CD142 Monoclonal Antibody (HTF-1), PE, ThermoFisher, 12-1429-42) (per 10 wells). 6 Cells were incubated with 0.2 μg of antibody at room temperature in the dark for 90 min on a rotary mixer. After centrifugation at 300g for 5 min, the antibody was discarded, and the cells were washed three times with 0.5% BSA (mass percentage). The cells were resuspended in 1.0 mL of 0.5% BSA (mass percentage) and transferred to flow cytometry tubes.

[0129] (13) PE-positive cells were obtained by sorting using a flow cytometer (BECKMAN COULTER, MoFlo Astrios, or BD Biosciences FACSAria). Approximately 40.7% of the cells were F3-positive dopaminergic neuronal precursors. Figure 4 (B)

[0130] Example 3: F3-positive dopaminergic neuron precursor cells differentiate into dopaminergic neurons.

[0131] (1) F3 positive cells sorted by flow cytometry were seeded into 24-well plates pretreated with poly-L-orthinine and Laminin (300,000 to 500,000 cells per well), and cultured in N2B27 medium supplemented with 10 μM Y-27632, 20 ng / mL BDNF, 200 μM vitamin C, 20 ng / mL GDNF, 500 μM dibutyryl cyclic adenosine monophosphate, 1 ng / mL TGF-β3, and 10 μM DAPT. The cells were then cultured in a 37°C incubator with 5% CO2.

[0132] (2) The next day, the culture medium was changed to N2B27 medium and supplemented with 20 ng / mL BDNF, 200 μM vitamin C, 20 ng / mL GDNF, 500 μM dibutyryl cyclic adenosine monophosphate, 1 ng / mL TGF-β3 and 10 μM DAPT. The culture was continued until differentiation for 30 days, and the medium was changed every other day.

[0133] (3) On day 30, immunofluorescence staining was used to detect the differentiation efficiency of dopaminergic neurons. After washing the cells with 1 mL PBS, the cells were fixed with 4% paraformaldehyde (mass percentage) for 10 min, and then washed three times with 1 mL PBS. The cells were permeabilized and blocked for 1 h at room temperature with blocking buffer containing 3% BSA (mass percentage) and 0.2% Triton-X100 (volume percentage) (Sigma, X100). Tyrosine hydroxylase (TH) antibody (Abcam, ab75875) at a ratio of 1:500 and β-tubulin III (TUJ1) antibody (Sigma, T8660) at a ratio of 1:1000 were diluted with 3% BSA (mass percentage) and incubated overnight at 4 °C. The cells were then washed three times with PBS on a shaker for 5 min each time. Subsequently, fluorescently labeled secondary antibodies, Goat anti-Mouse IgG, Alexa Fluor 488 (ThermoFisher Scientific, A-11001) and Goat anti-Rabbit IgG, Alexa Fluor 568 (ThermoFisher Scientific, A-11011), were diluted 1:1000 with 3% BSA and incubated at room temperature in the dark for 1–2 hours. Afterward, the secondary antibodies were discarded, and the cells were washed three times with PBS on a shaker for 5 minutes each time. DAPI was diluted 1:5000 with blocking buffer and incubated with the cells for 5 minutes. After incubation, the cells were washed with PBS on a shaker for 5 minutes, and then observed and photographed under a fluorescence microscope.

[0134] (4) Statistical analysis of differentiation efficiency showed that F3 positive cells had a high efficiency in differentiating into DA neurons, with TH expression reaching as high as 70% and TUJ1 expression reaching as high as 90%. Figure 4 Among them, D and E), while F3 negative cells have a very low ability to differentiate into neural cells, with TH expression at 7.29% and TUJ expression at 13.6% ( Figure 4 (C, E in the text).

[0135] Example 4: Using SSTR2 as a marker to isolate and purify dopaminergic neuronal precursor cells

[0136] The specific steps are as follows:

[0137] (1) Human pluripotent stem cells that have grown to a cell density of 70%–80% are digested into single cells, at a rate of 300,000 cells / cm³. 2 The density was transferred into Matrigel-coated 24-well plates and cultured overnight in mTeSR medium supplemented with 10 μM Rock inhibitor Y-27632;

[0138] (2) Differentiation begins the next day. On days 0 to 3, use N2B27 basal medium and add 250 nM LDN193189, 10 μM SB431542, 500 ng / mL SHH C25II and 0.7 μM CHIR99021. Change the medium daily.

[0139] (3) On days 4 to 6, use N2B27 basal medium and add 250 nM LDN193189, 10 μM MSB431542, 500 ng / mL SHH C25II and 7.5 μM CHIR99021, and change the medium daily;

[0140] (4) On days 7 to 9, use N2B27 basal medium and add 7.5 μM CHIR99021, and change the medium daily;

[0141] (5) On day 10, the culture medium was replaced with N2B27 medium and 3 μM CHIR99021 and 20 ng / mL LGDNF, 20 ng / mL BDNF, 200 μM dibutyryl cyclic adenosine monophosphate, 1 ng / mL TGF-β3 and 200 μM vitamin C were added.

[0142] (6) On day 11, the culture medium was replaced with N2B27 medium and 20 ng / mL GDNF, 20 ng / mL BDNF, 200 μM dibutyryl cyclic adenosine monophosphate, 1 ng / mL TGF-β3, and 200 μM vitamin C were added.

[0143] (7) On day 12, the culture medium was replaced with N2B27 medium and 20 ng / mL GDNF, 20 ng / mL BDNF, 200 μM dibutyryl cyclic adenosine monophosphate, 1 ng / mL TGF-β3, 200 μM vitamin C, and 10 μM DAPT were added.

[0144] (8) On day 13, the cells were digested into single cells with Accutase, centrifuged and resuspended in N2B27 medium. 10 μM Y-27632, 20 ng / mL GDNF, 20 ng / mL BDNF, 200 μM dibutyryl cyclic adenosine monophosphate, 1 ng / mL TGF-β3, 200 μM vitamin C, and 10 μM DAPT were added to the medium. The cells were then seeded in 12-well plates pretreated with poly-L-orthinine and laminin.

[0145] (9) Days 14-15: Change the medium daily, replacing each well with 2 mL of fresh N2B27 medium, and supplement with 20 ng / mL BDNF, 200 μM vitamin C, 20 ng / mL GDNF, 500 μM dibutyryl cyclic adenosine monophosphate, 1 ng / mL TGF-β3, and 10 μM DAPT.

[0146] (10) On day 16, add 0.5 mL of 0.5 mM EDTA to each well, incubate at 37°C for 5 minutes, add 0.5 mL of 3% BSA (w / w) to stop the incubation, gently pipette to resuspend the cells, transfer the cell suspension to a 1.5 mL EP tube, and centrifuge at 300 g for 5 minutes. Aspirate the supernatant, resuspend the cells in 0.5 mL of 3% BSA (w / w) again, and block at room temperature for 15 minutes. Add SSTR2-PE antibody (R&D systems, FAB4224P) (per 10 wells). 6 Add 10 μL of antibody to cells and incubate at room temperature in the dark for 90 min on a rotary mixer. Centrifuge at 300g for 5 min, discard the antibody, wash the cells three times with 0.5% BSA, resuspend the cells in 1.0 mL of 0.5% BSA (mass percentage), and transfer to a flow cytometry tube.

[0147] (11) PE-positive cells were obtained by sorting using a flow cytometer (BECKMAN COULTER, MoFlo Astrios, or BD Biosciences FACSAria). F3-positive dopaminergic neuronal precursor cells accounted for approximately 11.1% ( Figure 4 (G in the middle).

[0148] (12) SSTR2 positive cells sorted by flow cytometry were cultured in N2B27 medium and supplemented with 20 ng / mL BDNF, 200 μM vitamin C, 20 ng / mL GDNF, 200 μM dbcAMP, 1 ng / mL TGF-β3 and 10 μM DAPT.

[0149] Example 5: SSTR2-positive dopaminergic neuron precursor cells differentiate into dopaminergic neurons.

[0150] (1) SSTR2 positive cells sorted by flow cytometry were seeded into 24-well plates pretreated with poly-L-orthinine and Laminin (300,000 to 500,000 cells per well), and cultured in N2B27 medium supplemented with 10 μM Y-27632, 20 ng / mL BDNF, 200 μM vitamin C, 20 ng / mL GDNF, 200 μM dibutyryl cyclic adenosine monophosphate, 1 ng / mL TGF-β3, and 10 μM DAPT. The cells were then cultured in a 37°C incubator with 5% CO2.

[0151] (2) The next day, the culture medium was changed to N2B27 medium and supplemented with 20 ng / mL BDNF, 200 μM vitamin C, 20 ng / mL GDNF, 200 μM dibutyryl cyclic adenosine monophosphate, 1 ng / mL TGF-β3 and 10 μM DAPT. The culture was continued until differentiation for 30 days, and the medium was changed every other day.

[0152] (3) On day 20, immunofluorescence staining was used to detect the differentiation efficiency of dopaminergic neurons. After washing the cells with 1 mL PBS, the cells were fixed with 4% paraformaldehyde (mass percentage) for 10 min, and then washed three times with 1 mL PBS. The cells were permeabilized and blocked for 1 h at room temperature with blocking buffer containing 3% BSA (mass percentage) and 0.2% Triton-X100 (volume percentage) (Sigma, X100). Tyrosine hydroxylase (TH) antibody (Abcam, ab75875) at a ratio of 1:500 and β-tubulin III (TUJ1) antibody (Sigma, T8660) at a ratio of 1:1000 were diluted with 3% BSA (mass percentage) and incubated overnight at 4 °C. The cells were then washed three times with PBS on a shaker for 5 min each time. Subsequently, fluorescently labeled secondary antibodies, Goat anti-Mouse IgG, Alexa Fluor 488 (ThermoFisher Scientific, A-11001) and Goat anti-Rabbit IgG, Alexa Fluor 568 (ThermoFisher Scientific, A-11011), were diluted 1:1000 with 3% BSA and incubated at room temperature in the dark for 1–2 hours. Afterward, the secondary antibodies were discarded, and the cells were washed three times with PBS on a shaker for 5 minutes each time. DAPI was diluted 1:5000 with blocking buffer and incubated with the cells for 5 minutes. After incubation, the cells were washed with PBS on a shaker for 5 minutes, and then observed and photographed under a fluorescence microscope.

[0153] (4) Statistical analysis of differentiation efficiency showed that SSTR2-positive cells had a high efficiency in differentiating into DA neurons, with TH expression reaching as high as 70% and TUJ1 expression reaching as high as 90%. Figure 4 Among them, D and E), while SSTR2-negative cells have a very low ability to differentiate into neural cells, with TH expression at 1.32% and TUJ expression at 3.34% ( Figure 4 (C, E in the text).

[0154] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for isolating and / or enriching dopaminergic neuronal precursor cells, characterized in that, Includes the following steps: (1): Human pluripotent stem cells were cultured in 6-well plates with mTeSR medium in a 5% CO2 incubator at 37°C. The medium was changed daily, and the cells were digested with 0.5 mM EDTA or Accutase every 3 to 4 days. The cells were passaged at a ratio of 1:3 to 1:

5. (2): When the cell density reaches 70%~80%, the cells are digested into single cells with Accutase, and the cells are resuspended in mTeSR medium containing 10 μM Y-27632 at a density of 300,000 cells / cm³. 2 Cells were seeded at a density of 100% in 24-well plates and cultured in a 37°C incubator with 5% CO2. (3) Differentiation begins, marked as day 0: aspirate supernatant, add 2 mL SRM medium to each well of the 24-well plate, and supplement with 100 nM DN193189 and 10 μM SB431542, and incubate in a 37°C incubator with 5% CO2; (4) Days 1-2: Discard the supernatant, add 2 mL of SRM medium to each well of the 24-well plate, and supplement with 100 nM LDN193189, 10 μM MSB431542, 100 ng / mL SHH C25II, 100 ng / mL FGF8a and 2 μM purinemorphine, and incubate in a 37°C incubator with 5% CO2; (5) Days 3-4: Discard the supernatant, add 2 mL of SRM medium to each well of the 24-well plate, and supplement with 100 nM LDN193189, 10 μM MSB431542, 100 ng / mL SHH C25II, 100 ng / mL FGF8a, 2 μM purinemorphine and 3 μM CHIR99021, and incubate in a 37°C incubator with 5% CO2; (6) Days 5-6: Discard the supernatant, add 2 mL of medium slowly according to the ratio of 75% SRM / 25% N2B27 medium, and supplement with 100 nM LDN193189, 100 ng / mL SHH C25II, 100 ng / mL FGF8a, 2 μM purinemorphine and 3 μM CHIR99021, and incubate in a 37℃ incubator with 5% CO2; (7) Days 7-8: Discard the supernatant, add 2 mL of culture medium slowly to each well according to the ratio of 50% SRM / 50% N2B27, and supplement with 100 nM LDN193189 and 3 μM CHIR99021, and incubate in a 37°C incubator with 5% CO2. (8) Days 9-10: Discard the supernatant, add 2 mL of culture medium slowly to each well according to the ratio of 25% SRM / 75% N2B27, and supplement with 100 nM LDN193189 and 3 μM CHIR99021, and incubate in a 37°C incubator with 5% CO2. (9) Days 11-12: Discard the supernatant, slowly add 2 mL of N2B27 medium to each well, and supplement with 3 μM CHIR99021, 20 ng / mL BDNF, 200 μM vitamin C, 20 ng / mL GDNF, 500 μM dibutyryl cyclic adenosine monophosphate, 1 ng / mL TGF-β3 and 10 μM DAPT, and incubate in a 37°C incubator with 5% CO2; (10) Day 13: On the day of passage, aspirate the supernatant, add 0.5 mL of Accutase to each well, digest at 37°C for 15-20 min, and add 1.5 mL of DMEM / F-12 to each well to stop digestion; resuspend the digested cells and centrifuge at 300 g for 3 min, resuspend the cells in 2 mL of N2B27 medium, and supplement with 3 μM CHIR99021, 20 ng / mL BDNF, 200 μM vitamin C, 20 ng / mL GDNF, 500 μM dibutyryl cyclic adenosine monophosphate, 1 ng / mL TGF-β3, 10 μM DAPT and 10 μM Y-27632; seed the cells in 12-well plates pretreated with poly-L-orthinine and Laminin at a ratio of 1:1, and incubate in a 37°C incubator with 5% CO2; (11) Days 14-21: Change the medium daily, replacing each well with 2 mL of fresh N2B27 medium and supplementing with 20 ng / mL BDNF, 200 μM vitamin C, 20 ng / mL GDNF, 500 μM dibutyryl cyclic adenosine monophosphate, 1 ng / mL TGF-β3 and 10 μM DAPT, and incubate in a 37°C incubator with 5% CO2; (12) Day 22: Add 0.5 mL Accutase to each well, digest at 37°C for 10-15 min, add 0.3 mL 3% BSA (mass percentage) to stop digestion, gently pipette to resuspend the cells, centrifuge the cell suspension at 300 g for 5 min, aspirate the supernatant, resuspend the cells with 0.5 mL 3% BSA (mass percentage) and block at room temperature for 15 min; then use F3-PE antibody to separate and enrich to obtain the dopaminergic neuron precursor cells.

2. The method according to claim 1, characterized in that, The human pluripotent stem cells include human embryonic stem cells and / or induced pluripotent stem cells.

3. The method according to claim 2, characterized in that, The dosage of the F3-PE antibody is 0.15~0.2 μg per 1 million candidate cells.

4. Dopaminergic neuron precursor cells, characterized in that, It is obtained by the method described in any one of claims 1 to 3.

5. The dopaminergic neuron cells obtained by culturing and differentiating the dopaminergic neuron precursor cells according to claim 4, characterized in that, The culture and differentiation process includes the following steps: Step 1: Seed the dopaminergic neuron precursor cells into 24-well plates pretreated with poly-L-orthinine and laminin, with a seeding concentration of 300,000 to 500,000 cells per well. Supplement N2B27 medium with 10 μM Y-27632, 20 ng / mL BDNF, 200 μM vitamin C, 20 ng / mL GDNF, 500 μM dibutyryl cyclic adenosine monophosphate, 1 ng / mL TGF-β3 and 10 μM MDAPT, and incubate at 37°C in a 5% CO2 incubator. Step 2: The next day, the culture medium was changed to N2B27 medium, and 20 ng / mL BDNF, 200 μM vitamin C, 20 ng / mL GDNF, 500 μM dibutyryl cyclic adenosine monophosphate, 1 ng / mL TGF-β3, and 10 μM DAPT were added. The cells were cultured for another 30 days until differentiation, and the medium was changed every other day to obtain the dopaminergic neurons.

6. The application of at least one of the following (i) to (ii) in the study of pathogenic pathways of neurodegenerative diseases: i) The dopaminergic neuron precursor cells as described in claim 4; ii) The dopaminergic neuron cells according to claim 5.

7. The application according to claim 6, characterized in that, The neurodegenerative diseases include at least one of cerebral ischemia, brain injury, Parkinson's disease, brain atrophy, Alzheimer's disease, or amyotrophic lateral sclerosis.

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