Marker of dopaminergic neuron precursor cells and application thereof

Abstract

The invention relates to the technical field of medical biology, and provides a dopaminergic neuron precursor cell surface marker F3 and / or SSTR2, and the dopaminergic neuron precursor cell surface marker F3 and / or SSTR2 can be used for screening dopaminergic neuron precursor cells to obtain high-purity dopaminergic neuron precursor cells. The dopaminergic neuron precursor cell has relatively high efficiency of differentiating to the dopaminergic neuron.

Description

Technical Field

[0001] The present invention relates to the field of medical biotechnology, and in particular to markers of dopaminergic neuron precursor cells and applications thereof. Background Art

[0002] Parkinson's disease (PD) is a common neurodegenerative disease, mainly caused by degenerative changes in dopaminergic neurons in the substantia nigra and striatum. At present, no effective treatment for Parkinson's disease has been found, and the treatment strategy still focuses on symptom management through the restoration of dopaminergic activity, such as: drug treatment with levodopa and deep brain stimulation. The therapeutic effects of these treatments are extremely limited and cannot prevent the progression of the disease. Cell transplantation and replacement are considered to be the most promising and potential new treatments for PD, which may cure Parkinson's disease. In 1987, Lund University in Sweden used fetal dopaminergic neurons for cell transplantation for the first time. However, in addition to the ethical issues in fetal cell transplantation, the source of donor cells and immune rejection issues have hindered 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 to obtain functional dopaminergic neurons. The induced differentiation process includes floor plate induced differentiation to generate proliferative progenitor cells (including floor plate progenitors), which are then differentiated into postmitotic dopaminergic neuron progenitors through neurogenic transformation, and then differentiated 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 containing proliferative progenitor cells of dopaminergic neurons and / or postmitotic dopaminergic neuron progenitor cells are usually transplanted. After transplantation, these cells can differentiate into grafts containing dopaminergic neurons in vivo. However, the donor cells differentiated from hPSCs are heterogeneous and are not a single type of neural progenitor cells, but a mixture of neural progenitor (progenitor) cells and glial progenitor (progenitor) cells of various types and fates. This may affect the in vivo evaluation of cells and even increase the risk of side effects, hindering their widespread clinical application. Therefore, establishing a method to generate 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 differentiation of pluripotent stem cells and the development of dopaminergic neurons, it has been found that some genes selectively expressed in dopaminergic neuron proliferative precursor cells have been 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 proliferative precursor cells, dopaminergic neuron proliferative precursor cells also have multiple fate differentiation potentials. In addition to dopaminergic neurons, the differentiated terminal cells also include other types of neurons and various cell types such as glial cells and ependymal cells (Jerber et al. (2021) Nat Genet. 53, 304-312). Post-mitotic dopaminergic neuron precursor cells can better differentiate into dopaminergic neurons, so the use of post-mitotic dopaminergic neuron precursor cells in cell therapy is better than the use of proliferative precursor cells. Some markers of dopaminergic neuron precursor cells, such as LMX1A (WO 2005 / 052190) and multiple markers including CLSTN2 and PTPRO (WO2022 / 222974), have also been reported. However, these genes lack specificity and are expressed in both dopaminergic neuron proliferative precursor cells and post-mitotic dopaminergic neuron precursor cells. These marker molecules cannot effectively enrich post-mitotic dopaminergic neuron precursor cells. Therefore, it is of great significance to find and identify specific dopaminergic neuron precursor cell surface markers, optimize the technology of separating and purifying dopaminergic neuron precursor cells, and provide products for PD cell therapy. 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 neuron precursor cells and applications thereof.

[0005] The first aspect of the present invention provides specific surface markers of dopaminergic neuron precursor cells, wherein the surface markers include at least one of F3 or SSTR2.

[0006] In the present invention, F3 (coagulation factor III) is also called tissue factor, CD142, and belongs to the single-channel type I membrane protein of the tissue factor family. Human F3 consists of 295 amino acids, has a size of about 47 kDa, and includes a transmembrane region (residues 252 to 274).

[0007] In the present invention, SSTR2 is a member of the somatostatin receptor (SSTR) family, belongs to the G protein-coupled receptor, 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 the present invention are obtained by single-cell sequencing analysis of the differentiation products at multiple time points in the differentiation process after inducing differentiation using two conventional methods for directed differentiation of human pluripotent stem cells into dopaminergic neurons, and have significant cell specificity. The first differentiation method is a method developed by the Lorenz Studer team in 2011 for directed differentiation of pluripotent stem cells into dopaminergic neurons in vitro using floor plate induction (Kriks et al. (2011) Nature 480, 547-551). Currently, other induced differentiation methods are adjustments based on this method. The second differentiation method is an optimization of the first method reported by the Lorenz Studer team in 2021. "CHIR-boost" is used to perform biphasic activation of the WNT signaling pathway during differentiation (Kim et al. (2021) Cell Stem Cell 28, 343-355). The inventors of the present application have found that the F3 gene is specifically expressed in the post-mitotic dopaminergic neuron precursor cells obtained by the first differentiation method, and the dopaminergic neuron precursor cell group also specifically expresses the ASCL1, TH, and TPH1 genes, and can be directed to differentiate into dopaminergic neurons. The SSTR2 gene is specifically expressed in the post-mitotic dopaminergic neuron precursor cells obtained by the second differentiation method, and the dopaminergic neuron precursor cell group also specifically expresses the characteristic genes NEUROD1 / 4, NHLH1 / 2, and NEUROG1 / 2 of neuroblasts (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, the present invention names the F3 and / or SSTR2-positive dopaminergic neuron precursor cells as directed dopaminergic neuron precursor cells. The F3 and SSTR2 surface markers can be used to separate, purify, detect, or identify dopaminergic neuron precursor cells with directed differentiation ability.

[0009] The dopaminergic neuron precursor cell surface marker F3 of the present invention is a dopaminergic neuron precursor cell specific surface receptor, which is used to purify dopaminergic neuron precursor cells. In some specific embodiments of the present invention, dopaminergic neuron precursor cells can be effectively enriched by using the specific surface receptor to sort dopaminergic neuron precursor cells; and the dopaminergic neuron precursor cells can be efficiently differentiated into dopaminergic neurons using the dopaminergic neuron precursor cells, and the differentiation efficiency can reach more than 30%, and the preferred differentiation rate can reach more than 40%, more preferably more than 50%, more preferably more than 60%, more preferably more than 70%, and more preferably more than 80%; in some specific embodiments of the present invention, the differentiation efficiency is as high as 69%.

[0010] The dopaminergic neuron precursor cell marker SSTR2 of the present invention is a dopaminergic neuron precursor cell-specific surface receptor, which is used to purify dopaminergic neuron precursor cells. In some specific embodiments of the present invention, dopaminergic neuron precursor cells can be effectively enriched by using the specific surface receptor to sort dopaminergic neuron precursor cells; and the dopaminergic neuron precursor cells can be efficiently differentiated into dopaminergic neurons using the dopaminergic neuron precursor cells, and the differentiation efficiency can reach more than 30%, and as a preferred differentiation rate, it can reach more than 40%, more preferably more than 50%, more preferably more than 60%, more preferably more than 70%, and more preferably more than 80%; in some specific embodiments of the present invention, the differentiation efficiency is as high as 66%.

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

[0012] The second aspect of the present invention provides the use of the surface marker in identifying, isolating and / or enriching dopaminergic neuron precursor cells. The dopaminergic neuron precursor cells can differentiate into dopaminergic neurons in vivo and in vitro.

[0013] Further, the identification includes:

[0014] Determine whether the candidate cells have the following markers on their surface: F3 and / or SSTR2,

[0015] Candidate cells possessing the 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 inducing human pluripotent stem cell differentiation comprises the following steps:

[0020] contacting the human pluripotent stem cells with a first induction agent to perform a first induction differentiation to differentiate into midbrain floor plate progenitor cells;

[0021] The obtained midbrain floor plate precursor cells are contacted with a second induction agent to perform a second induction differentiation, and differentiate into dopaminergic neuron precursor cells.

[0022] In the present invention, the first induction agent includes a BMP signaling pathway inhibitor, a TGF-β signaling pathway inhibitor, a SHH signaling pathway agonist, purmorphamine, a GSK-3 signaling pathway inhibitor, and a fibroblast growth factor;

[0023] Preferably, the first induction 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 a fibroblast growth factor, and / or 1-2 μM of purmorphamine;

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

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

[0026] Preferably, the second induction agent comprises 20 ng / mL BDNF, 20 ng / mL GDNF, 200 μM to 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 induction agent includes 20 ng / mL BDNF, 20 ng / mL GDNF, 200 μM to 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 of the present invention, the determination comprises contacting the candidate cells with a reagent targeting F3 and / or SSTR2, and then detecting the expression and / or activity level of F3 and / or SSTR2 in the candidate cells.

[0029] Furthermore, the agent targeting F3 and / or SSTR2 includes an agent targeting a nucleic acid molecule encoding F3 and / or SSTR2, and / or an agent targeting F3 and / or SSTR22 protein.

[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 that can specifically bind to F3 and / or SSTR2 proteins and / or reagents that can measure the activity of F3 and / or SSTR2 proteins.

[0033] Furthermore,

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

[0035] Preferably, the dosage of the F3 antibody is 0.15-0.2 μg per 1 million candidate cells;

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

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

[0038] The third aspect of the present invention provides a reagent for identifying, isolating or enriching dopaminergic neuron precursor cells, which targets the surface markers of the present 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 that can specifically bind to F3 and / or SSTR2 proteins and / or reagents that can measure the activity of F3 and / or SSTR2 proteins.

[0043] Furthermore, the reagent that specifically binds to F3 and / or SSTR2 protein includes an antibody to F3 and / or SSTR2, or an anti-antibody to F3 and / or SSTR2;

[0044] Preferably, the dosage of the F3 antibody is 0.15-0.2 μg per 1 million candidate cells;

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

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

[0047] The fourth aspect of the present invention provides a kit for identifying, isolating or enriching dopaminergic neuron precursor cells, which comprises auxiliary materials and the reagent of the present invention.

[0048] Furthermore, the auxiliary materials include any one or more of culture medium, inhibitors, activators, growth factors, buffers and / or culture equipment.

[0049] A fifth aspect of the present invention provides a method for isolating and / or enriching dopaminergic neuron precursor cells, which uses the reagent and / or the kit described in the present invention to isolate and / or enrich dopaminergic neuron precursor cells.

[0050] Furthermore, the separation and enrichment method includes: any one of a fluorescent labeling cell sorting method, an immunomagnetic bead separation method or an immunoadsorption column separation method.

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

[0052] Step 1, providing a neuronal precursor cell population;

[0053] Step 2: Isolating and / or enriching dopaminergic neuron precursor cells having the following markers from the neuron precursor cell population: F3 or SSTR2.

[0054] Furthermore, the method for separating and / or enriching dopaminergic neuron precursor cells in step 2 includes fluorescent labeling cell sorting, immunomagnetic bead separation or immunoadsorption column separation; preferably, these methods use antibodies that specifically bind to F3 and / or SSTR2, or antibodies to F3 and / or SSTR2 linked to fluorescent labels, magnetic substances or adsorption columns, or antibodies that specifically bind to F3 and / or SSTR2 antibodies, and more preferably, antibodies that specifically bind to F3 and / or SSTR2 are used.

[0055] The present invention provides dopaminergic neuron precursor cells, which are obtained by the method of the present invention.

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

[0057] The present invention provides the use of at least one of the following i) to ii) in the study of the pathogenesis pathway of neurodegenerative diseases and the preparation of drugs for preventing and treating neurodegenerative diseases:

[0058] i) the dopaminergic neuron precursor cells of the present invention;

[0059] ii) The dopaminergic neuron cell of the present 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] The present invention provides a method for preventing, treating or alleviating a nervous system disease or disorder, the method comprising the following steps:

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

[0063] selecting cells having the marker; and

[0064] An effective dose of cells having the marker is administered to a subject in need thereof.

[0065] Beneficial effects of the present invention:

[0066] The present invention finds that F3 and SSTR2 are specific surface markers of dopaminergic neuron precursor cells, and that F3-positive dopaminergic neuron precursor cells have the bidirectional differentiation ability to differentiate into dopaminergic neurons and serotonergic neurons, while SSTR2-positive dopaminergic neuron precursor cells can be directionally differentiated into dopaminergic neurons. Thus, compared with other dopaminergic neuron precursor cell markers, using F3 and / or SSTR2 as surface markers can provide high-purity, more uniform dopaminergic neuron precursor cells and dopaminergic neurons differentiated from the precursor cells.

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

[0068] The F3 and / or SSTR2 positive dopaminergic neuron precursor cells and the dopaminergic neurons differentiated therefrom provided by the present invention have a wide range of uses and can be used as seed cells for cell therapy of neurodegenerative diseases, and provide an ideal cell model for disease mechanism research, drug screening, and development research of dopaminergic neurons.

[0069] The F3 and / or SSTR2-positive dopaminergic neuron precursor cells provided by the present invention are post-mitotic cells, which reduce the risk of tumor occurrence and are more in line with the role and requirements of donor cells for cell therapy. BRIEF DESCRIPTION OF THE DRAWINGS

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

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

[0072] Figure 3 Shows the expression of F3 and SSTR2 genes in cells obtained by the first and second differentiation methods, respectively;

[0073] Figure 4Figure 3 shows that flow sorting of F3 and SSTR2 positive cells can increase the differentiation ratio of dopaminergic neurons, while F3 and SSTR2 negative cells cannot differentiate into dopaminergic neurons; A is the gene expression of F3 in the first differentiation method; B is the flow sorting of F3 positive cells when they differentiate into dopaminergic neuron precursor cells in the first differentiation method at D22, and the positive rate is 40.7%; C is the result of the sorted F3 negative cells after differentiation, the left side is the bright field, and the right side is the immunofluorescence staining result, where DAPI is a nuclear dye, showing the number of all cells, and TUJ1 is a β-tubulin marker Figure 1 shows the proportion of nerve cells. Tyrosine hydroxylase (TH) is a dopaminergic neuron marker, showing the proportion of dopaminergic neurons, indicating that the F3-negative cells have a very low neural differentiation efficiency; D shows that the sorted F3-positive cells differentiate into dopaminergic neuron cells, and it can be seen that the mature neural network and the specific high expression of the dopaminergic neuron-specific gene TH can be seen. E is the statistics of Figure C and Figure D, indicating that F3-positive cells can efficiently differentiate into nerve cells, especially dopaminergic neurons, among which the proportion of F3-positive cells differentiated into TH-positive cells is 69.39%, while F The percentage of SSTR2-negative cells differentiated into TH-positive cells was only 7.29%; F is the gene expression of SSTR2 in the second differentiation method; G is the flow cytometry sorting of SSTR2-positive cells when they differentiated into dopaminergic neuron precursor cells at D16 in the second differentiation method, and the positive rate was 11.1%; H is the result of the sorted SSTR2-negative cells after differentiation, the left side is the bright field, and the right side is the immunofluorescence staining result, indicating that SSTR2-negative cells cannot differentiate into neurons and show extremely low differentiation efficiency. The cell immunofluorescence staining results show that only DAPI indicates the distribution of cell nuclei, and TU J1 and TH were not expressed, indicating that SSTR2-negative cells could not differentiate into nerve cells, let alone dopaminergic neurons; I is the result of SSTR2-positive cells being differentiated into dopaminergic neurons, and it can be seen that complex cells have formed a complex neural network; J is a statistical analysis of H and I, indicating that SSRT2-positive cells can efficiently differentiate into nerve cells, especially dopaminergic neurons, among which the proportion of SSRT2-positive cells differentiated into TH-positive cells was 66.25%, while the proportion of F3-negative cells differentiated into TH-positive cells was only 1.32%;

[0074] Figure 5 It showed that some CORIN genes were expressed in proliferating precursor cells of dopaminergic neurons;

[0075] Figure 6 These results indicate that the ubiquitous expression of PTPRO gene in the two differentiation methods is not cell-selective. DETAILED DESCRIPTION

[0076] The present invention provides surface markers of dopaminergic neuron precursor cells and their applications, which are now described in detail in conjunction with the accompanying drawings and specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and are not used to limit the scope of the present invention. After reading the present invention, various similar replacements and modifications of the present invention by those skilled in the art are deemed to be included in the present invention. The test materials used in the present invention are all common commercial products and can be purchased on the market.

[0077] The present invention will be further described below in conjunction with embodiments:

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

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

[0080] 1. 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 surface marker of dopaminergic neuron precursor cells obtained by a directed differentiation method, which comprises the following steps:

[0082] A: Human pluripotent stem cells grown to a cell density of 70% to 80% were digested into single cells at a rate of 300,000 cells / cm 2 The cells were transferred into a 24-well plate coated with Matrigel (Corning, 354277) at a density of 1.5 μM and cultured in mTeSR medium (STEMCELL, 85850) supplemented with 10 μM Rock inhibitor Y-27632 (ALX-270-333-M005);

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

[0084] Table 1. Dopaminergic neuron differentiation basal medium

[0085]

[0086] C: On day 1-2, the culture medium was replaced with SRM medium, and 100 nM LDN193189, 10 μM SB431542, 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 purmorphamine (STEMCELL, 72202) were added;

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

[0088] E: On days 5-6, the culture medium was changed to 75% SRM / 25% N2B27 medium (Table 2), and 100 nM LDN193189, 100 ng / mL SHH C25II, 100 ng / mL FGF8a, 2 μM purmorphamine, and 3 μM CHIR99021 (R&D, 4432) were added;

[0089] Table 2. Dopaminergic neuron differentiation basal medium

[0090]

[0091]

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

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

[0094] H: On days 11-12, the culture medium was changed to N2B27 medium supplemented with 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);

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

[0096] J: On the 14th day, the culture medium was replaced with N2B27 medium and 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. The culture was continued, the medium was changed every day, and the cells were subcultured once on the 22nd day. The culture was continued until the 51st day, and the medium was changed every other day.

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

[0098] 2. Acquisition of surface marker SSTR2 (second differentiation method)

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

[0100] A: Human pluripotent stem cells grown to a cell density of 70% to 80% were digested into single cells at a rate of 300,000 cells / cm 2 The cells were transferred into a 24-well plate coated with Matrigel (Corning, 354277) at a density of 1.5 μM and cultured overnight in mTeSR medium supplemented with 10 μM Rock inhibitor Y-27632.

[0101] B: Differentiation started the next day. On days 0 to 3, the culture medium was replaced with N2B27 medium supplemented with 250 nM LDN193189, 10 μM SB431542, 500 ng / mL SHH C25II, and a low concentration of 0.7 μM CHIR99021.

[0102] C: On days 4 to 6, the culture medium was changed to N2B27 medium supplemented with 250 nM LDN193189, 10 μM SB431542, 500 ng / mL SHH C25II, and a high concentration of 7.5 μM CHIR99021;

[0103] D: On days 7 to 9, the culture medium was replaced with N2B27 medium and supplemented with a high concentration of 7.5 μM CHIR99021;

[0104] E: On day 10, the culture medium was changed to N2B27 medium supplemented with 3 μM CHIR99021 and 20 ng / mL GDNF, 20 ng / mL BDNF, 200 μM dibutyryl-cyclic AMP, 1 ng / mL TGF-β3, and 200 μM vitamin C at medium concentrations;

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

[0106] G: On day 12, the culture medium was changed to N2B27 medium supplemented with 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;

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

[0108] J: On the 14th day, 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 to the medium. The culture was continued, and the medium was changed every day. The cells were subcultured once on the 16th to 17th day. The culture was continued until the 51st day, and the medium was changed every other day.

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

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

[0111] In the second differentiation method, the proportion of SSTR2-positive cells was 11.1% on the 16th day after flow cytometry sorting. The cells after flow cytometry sorting were further cultured and differentiated into dopaminergic neurons. The efficiency of SSTR2-positive cells differentiating into dopaminergic neurons was as high as 66%, and SSTR2-negative cells had no ability to differentiate into neural cells ( Figure 4 F~J).

[0112] 3. Comparison with other markers

[0113] The reported marker CORIN (WO 2004 / 065599, WO 2006 / 009241) is a marker for dopaminergic neuron proliferative precursor cells (including floor plate precursor cells). Single-cell sequencing of the in vivo and in vitro dopaminergic neuron differentiation process found that during the differentiation process, proliferative precursor cells can undergo neurogenic transformation and differentiate into post-mitotic dopaminergic neuron precursor cells, including neuroblasts. The marker SSTR2 in the present invention is a neuroblast marker, and neuroblasts can be directed to differentiate into dopaminergic neurons. F3 is another dopaminergic neuron precursor cell marker generated by proliferative precursor cells, and the precursor cells can also be directed to differentiate into dopaminergic neurons. We also found that during the in vitro differentiation process, some CORIN-positive cells cannot differentiate into dopaminergic neurons, such as the glioma cells produced by the first differentiation method and a floor plate precursor cell population produced by the second differentiation method (such as Figure 5 ), therefore, the marker of the present invention more accurately marks the directed differentiated dopaminergic neuron precursor cells.

[0114] In addition, some 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 the present invention, CLSTN2 is not expressed, and LMX1A and PTPRO are universally expressed without cell selectivity (such as Figure 6 ).

[0115] Example 2 Using F3 as a marker to separate and purify dopaminergic neuron precursor cells

[0116] The purification of dopaminergic neuron precursor cells obtained by inducing differentiation of hPSCs includes hPSC culture, dopaminergic neuron precursor cell induction, flow cytometry sorting, etc. The specific steps are as follows:

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

[0118] (2) When the cell density reached 70% to 80%, the cells were digested into single cells using Accutase, counted using a cell counter, and resuspended in mTeSR medium containing 10 μM Y-27632 at 300,000 cells / cm 2 The cells were seeded in a 24-well plate at a density of 100 μg / mL and cultured in a 37°C incubator with 5% CO2. The cells were observed under a microscope the next day. The cells should have covered the entire plate. The cells will be used for dopaminergic neuron differentiation.

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

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

[0121] (5) Days 3-4: Same as Day 2, except that 3 μM CHIR99021 was added.

[0122] (6) Day 5-6: Discard the supernatant and slowly add 2 mL of culture medium in the ratio of 75% SRM / 25% N2B27 culture medium (Table 1) to prevent cells from falling off due to excessive force. Supplement with 100 nM LDN193189, 100 ng / mL SHH C25II, 100 ng / mL FGF8a, 2 μM purmorphamine, and 3 μM CHIR99021, and culture in a 37°C incubator with 5% CO2.

[0123] (7) Day 7-8: Discard the supernatant, 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 culture in a 37°C incubator with 5% CO2.

[0124] (8) Days 9-10: Discard the supernatant, 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 culture in a 37°C incubator with 5% CO2.

[0125] (9) Day 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 TGF-β3, and 10 μM DAPT, and culture in a 37°C incubator with 5% CO2.

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

[0127] (11) Days 14 to 21: Change the medium every day with 2 mL of fresh N2B27 medium per well, 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, and culture in a 37°C incubator with 5% CO2.

[0128] (12) On the 22nd day, 0.5 mL of Accutase was added to each well and digested at 37°C for 10-15 min. 0.3 mL of 3% BSA (mass percentage) was added to terminate the digestion. The cells were gently pipetted to resuspend the cells. The cell suspension was transferred to a 1.5 mL EP tube and centrifuged at 300 g for 5 min. The supernatant was aspirated and the cells were resuspended with 0.5 mL of 3% BSA (mass percentage) and blocked at room temperature for 15 min. F3-PE antibody (CD142 Monoclonal Antibody (HTF-1), PE, ThermoFisher, 12-1429-42) was added (10 6 Cells were incubated in a rotating mixer at room temperature in the dark for 90 min. After centrifugation at 300 g for 5 min, the cells were washed three times with 0.5% BSA (mass percentage) after discarding the antibody, and resuspended with 1.0 mL 0.5% BSA (mass percentage) and transferred to a flow tube.

[0129] (13) Use a flow cytometer (model: BECKMAN COULTER, MoFlo Astrios or BD Biosciences FACSAria) to sort and obtain PE-positive cells. The percentage of F3-positive dopaminergic neuron precursor cells is approximately 40.7% ( Figure 4 B)

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

[0131] (1) Inoculate the flow-sorted F3-positive cells into 24-well plates pretreated with poly-L-orthinine and laminin (300,000 to 500,000 cells per well), continue to culture in N2B27 medium, and supplement 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, and culture in a 37°C incubator with 5% CO2;

[0132] (2) The next day, the culture medium was replaced with N2B27 medium 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, the efficiency of dopaminergic neuron differentiation was detected by immunofluorescence staining. 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 using a blocking solution containing 3% BSA (mass percentage) and 0.2% Triton-X100 (volume percentage) (Sigma, X100) in PBS. Tyrosine hydroxylase (TH) antibody (Abcam, ab75875) was diluted 1:500 and β-tubulin III (TUJ1) antibody (Sigma, T8660) was diluted 1:1000 with 3% BSA (mass percentage), and the cells were incubated at 4°C overnight. The cells were then washed three times with PBS on a shaker for 5 minutes each time. Then, the fluorescently labeled secondary antibodies Goat anti-Mouse IgG, Alexa Fluor488 (ThermoFisher Scientific, A-11001) and Goat anti-Rabbit IgG, Alexa Fluor568 (ThermoFisher Scientific, A-11011) were diluted with 3% BSA (mass percentage) at a multiple of 1:1000 and incubated at room temperature in the dark for 1 to 2 hours. After that, the secondary antibodies were discarded, and the cells were washed 3 times with PBS on a shaker for 5 minutes each time. DAPI was diluted with blocking solution at 1:5000 and incubated with the cells for 5 minutes. After the incubation, the cells were washed with PBS on a shaker for 5 minutes, and then observed and photographed with a fluorescence microscope.

[0134] (4) Statistics on differentiation efficiency showed that F3 positive cells had a high efficiency of differentiation into DA neurons, with TH expression as high as 70% and TUJ1 expression as high as 90% ( Figure 4 D, E), while F3 negative cells had very low ability to differentiate into neural cells, with TH expression at 7.29% and TUJ expression at 13.6% ( Figure 4 C, E).

[0135] Example 4 Using SSTR2 as a marker to separate and purify dopaminergic neuron precursor cells

[0136] The specific steps are as follows:

[0137] (1) Human pluripotent stem cells grown to a cell density of 70% to 80% were digested into single cells and cultured at 300,000 cells / cm 2 The cells were transferred into Matrigel-coated 24-well plates at a density of 1.5 μM and cultured overnight in mTeSR medium supplemented with 10 μM Rock inhibitor Y-27632.

[0138] (2) Differentiation began the next day. On days 0 to 3, N2B27 basal medium was used and supplemented with 250 nM LDN193189, 10 μM SB431542, 500 ng / mL SHH C25II, and 0.7 μM CHIR99021. The medium was changed every day.

[0139] (3) On days 4 to 6, use N2B27 basal medium and add 250 nM LDN193189, 10 μM SB431542, 500 ng / mL SHH C25II, and 7.5 μM CHIR99021, and change the medium every day;

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

[0141] (5) On day 10, the culture medium was changed to N2B27 medium supplemented with 3 μM CHIR99021, 20 ng / mL GDNF, 20 ng / mL BDNF, 200 μM dibutyryl cyclic adenosine monophosphate, 1 ng / mL TGF-β3, and 200 μM vitamin C;

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

[0143] (7) On day 12, the culture medium was changed to N2B27 medium supplemented with 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;

[0144] (8) On day 13, the cells were digested into single cells with Accutase, centrifuged and resuspended in N2B27 medium, and 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 seeded in a 12-well plate pretreated with poly-L-orthinine and laminin;

[0145] (9) Day 14-15: Change the medium every day, with each well replaced with 2 mL of fresh N2B27 medium 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.

[0146] (10) On day 16, add 0.5 mL of 0.5 mM EDTA to each well and incubate at 37°C for 5 minutes. Add 0.5 mL of 3% BSA (mass percentage) to terminate 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 and resuspend the cells with 0.5 mL of 3% BSA (mass percentage). Block at room temperature for 15 minutes. Add SSTR2-PE antibody (R&D systems, FAB4224P) (10 6 Cells were incubated in a rotating mixer at room temperature in the dark for 90 min. After centrifugation at 300 g for 5 min, the cells were washed three times with 0.5% BSA after discarding the antibody, and resuspended with 1.0 mL 0.5% BSA (mass percentage) and transferred to a flow tube.

[0147] (11) Use a flow cytometer (model: BECKMAN COULTER, MoFlo Astrios or BD Biosciences FACSAria) to sort and obtain PE-positive cells. F3-positive dopaminergic neuron precursor cells account for approximately 11.1% ( Figure 4 G in.

[0148] (12) The SSTR2-positive cells separated by flow cytometry were cultured in N2B27 medium 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 Differentiation of SSTR2-positive dopaminergic neuron precursor cells into dopaminergic neurons

[0150] (1) Inoculate flow cytometry-sorted SSTR2-positive cells into 24-well plates pretreated with poly-L-orthinine and laminin (300,000 to 500,000 cells per well), continue culturing in N2B27 medium, and supplement 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, and culture in a 37°C incubator with 5% CO2;

[0151] (2) The next day, the culture medium was replaced with N2B27 medium 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, the efficiency of dopaminergic neuron differentiation was detected by immunofluorescence staining. 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 using a blocking solution containing 3% BSA (mass percentage) and 0.2% Triton-X100 (volume percentage) (Sigma, X100) in PBS. Tyrosine hydroxylase (TH) antibody (Abcam, ab75875) was diluted 1:500 and β-tubulin III (TUJ1) antibody (Sigma, T8660) was diluted 1:1000 with 3% BSA (mass percentage), and the cells were incubated at 4°C overnight. The cells were then washed three times with PBS on a shaker for 5 minutes each time. Then, the fluorescently labeled secondary antibodies Goat anti-Mouse IgG, Alexa Fluor488 (ThermoFisher Scientific, A-11001) and Goat anti-Rabbit IgG, Alexa Fluor568 (ThermoFisher Scientific, A-11011) were diluted with 3% BSA (mass percentage) at a multiple of 1:1000 and incubated at room temperature in the dark for 1 to 2 hours. After that, the secondary antibodies were discarded, and the cells were washed 3 times with PBS on a shaker for 5 minutes each time. DAPI was diluted with blocking solution at 1:5000 and incubated with the cells for 5 minutes. After the incubation, the cells were washed with PBS on a shaker for 5 minutes, and then observed and photographed with a fluorescence microscope.

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

[0154] The above are only preferred embodiments of the present invention. It should be pointed out that, for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A surface marker of dopaminergic neuron precursor cells, characterized in that: The surface markers include at least one of F3 or SSTR2.

2. Use of the surface marker according to claim 1 in identifying, separating and / or enriching dopaminergic neuron precursor cells.

3. The use according to claim 2, characterized in that: The identification includes: Determine whether the candidate cells have the following markers on their surface: F3 and / or SSTR2, Candidate cells possessing the markers are identified as dopaminergic neuron precursor cells.

4. The use according to claim 3, characterized in that: The candidate cells are neuronal precursor cells.

5. The use according to claim 4, characterized in that: 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; Preferably, the human pluripotent stem cells include human embryonic stem cells and / or induced pluripotent stem cells.

6. The use according to claim 5, characterized in that: The inducing human pluripotent stem cell differentiation comprises the following steps: contacting the human pluripotent stem cells with a first induction agent to perform a first induction differentiation into midbrain floor plate progenitor cells; The obtained midbrain floor plate precursor cells are contacted with a second induction agent to perform a second induction differentiation, and differentiate into dopaminergic neuron precursor cells.

7. The use according to claim 6, characterized in that: The first induction reagent includes a BMP signaling pathway inhibitor, a TGF-β signaling pathway inhibitor, a SHH signaling pathway agonist, purmorphamine, a GSK-3 signaling pathway inhibitor, and a fibroblast growth factor; Preferably, the first induction agent comprises: 100-250 nM BMP signaling pathway inhibitor, 1-10 μM TGF-β signaling pathway inhibitor, 100-500 ng / mL SHH signaling pathway agonist, 0.7-7.5 μM GSK-3 signaling pathway inhibitor, 100 ng / mL fibroblast growth factor, and / or 1-2 μM purmorphamine; The second induction agent includes BDNF, GDNF, dibutyryl cyclic adenosine monophosphate, TGF-β3, DAPT, vitamin C and / or a GSK-3 signaling pathway inhibitor; Preferably, the second induction agent comprises 20 ng / mL BDNF, 20 ng / mL GDNF, 200 μM to 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.

8. The use according to any one of claims 3 to 7, characterized in that: The determination comprises contacting the candidate cells with a reagent targeting F3 and / or SSTR2, and then detecting the expression and / or activity level of F3 and / or SSTR2 in the candidate cells.

9. The use according to claim 8, characterized in that: The agents targeting F3 and / or SSTR2 include agents targeting nucleic acid molecules encoding F3 and / or SSTR2, and / or agents targeting F3 and / or SSTR22 proteins.

10. The use according to claim 9, characterized in that: 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; The reagents targeting F3 and / or SSTR22 proteins include reagents that can specifically bind to F3 and / or SSTR2 proteins and / or reagents that can measure the activity of F3 and / or SSTR2 proteins.

11. The use according to claim 10, characterized in that: The reagents that specifically bind to F3 and / or SSTR2 proteins include antibodies to F3 and / or SSTR2, or anti-antibodies to F3 and / or SSTR2; Preferably, the dosage of the F3 antibody is 0.15-0.2 μg per 1 million candidate cells; Preferably, the amount of the SSTR2 antibody is 10 μL per 1 million candidate cells; Preferably, the reagent is labeled with fluorescein.

12. A reagent for identifying, isolating or enriching dopaminergic neuron precursor cells, characterized in that: The surface marker according to claim 1 is used as a target.

13. A kit for identifying, isolating or enriching dopaminergic neuron precursor cells, characterized in that: The method comprises auxiliary materials and the reagent according to claim 12.

14. A method for isolating and / or enriching dopaminergic neuron precursor cells, characterized in that: Dopaminergic neuron precursor cells are separated and / or enriched using the reagent according to claim 12 and / or the kit according to claim 13.

15. The method according to claim 14, characterized in that The separation and enrichment method includes: any one of a fluorescent labeling cell sorting method, an immunomagnetic bead separation method or an immunoadsorption column separation method.

16. A dopaminergic neuron precursor cell, characterized in that: It is obtained by the method according to claim 14 or 15.

17. Dopaminergic neuron cells obtained by culturing and differentiating the dopaminergic neuron precursor cells according to claim 16.

18. Use of at least one of the following i) to ii) in the study of the pathogenic mechanism pathway of neurodegenerative diseases and in the preparation of drugs for the prevention and treatment of neurodegenerative diseases: i) the dopaminergic neuron precursor cell according to claim 16; ii) The dopaminergic neuron cell according to claim 17.

19. The use according to claim 18, characterized in that The neurodegenerative disease includes at least one of cerebral ischemia, brain injury, Parkinson's disease, brain atrophy, Alzheimer's disease, or amyotrophic lateral sclerosis.

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