WO2017097007A1

WO2017097007A1 - Differentiation medium and use thereof in preparation of neural stem cells

Info

Publication number: WO2017097007A1
Application number: PCT/CN2016/099630
Authority: WO
Inventors: 裴端卿; 蔡景蕾; 刘朋飞
Original assignee: 中国科学院广州生物医药与健康研究院
Priority date: 2015-12-07
Filing date: 2016-09-21
Publication date: 2017-06-15
Also published as: CN105420193B; CN105420193A

Abstract

Provided is a differentiation medium capable of inducing ES cells and/or iPS cells into neural stem cells. The differential medium is a conventional medium comprising 1% of N2 additive, the conventional medium being at least one of RPMI1640 medium, DMEM/F12 medium, high glucose DMEM medium and á-MEM medium. Also provided are a use of the differential medium in the preparation of neural stem cells and a method for preparing neural stem cells. ES cells and/or iPS cells can be rapidly and stably induced into neural stem cells by using the differential medium.

Description

Differentiation medium and use thereof in preparing neural stem cells

Priority information

Priority is claimed on Japanese Patent Application No. 201510897362.X filed on Dec. 7, 2015, the entire disclosure of which is hereby incorporated by reference.

Technical field

The present invention relates to the field of biology. Specifically, the present invention relates to a medium and an application thereof. More specifically, the present invention relates to a use of a differentiation medium, a differentiation medium for preparing a neural stem cell, and a method for preparing a neural stem cell.

Background technique

Stem cells are the initial source of the human body and its various tissue cells. Its most prominent biological characteristics are its ability to self-renew and proliferate, as well as the potential for multi-directional differentiation. Stem cells are classified into somatic stem cells and embryonic stem cells (ES cells) according to different sources. Adult stem cells include bone marrow mesenchymal stem cells, pancreatic stem cells, neural stem cells, and the like, which are present in adult tissues.

In 1981, the isolation and culture of ES cells was first successful in mice, and it is the most widely studied and mature stem cell system to date. Human embryonic stem cells began in 1998, and the University of Wisconsin scientist James A. Thomson led the research team to extract embryonic stem cells (Embryonic Stem Cell, ES Cell) from human embryonic tissue for the first time. Strain, and confirmed that this strain cell has pluripotent stem cell characteristics [Thomson, JA, et al. Embryonic stem cell lines derived from human blastocysts. Science, 282 (1998): 1145-1147.]. This paper marks the arrival of an era, and Thomson is also known as the "father of stem cell research."

The application prospect of human embryonic stem cell (hES) cell research is mainly in the field of regenerative medicine. In the field of tissue engineering, hES cells are used as seed cells, which can provide a large amount of materials for clinical transplantation of cells, tissues or organs. Specific tissue cell types can be obtained by controlling the differentiation environment of hES cells and transfecting key molecular genes that can promote the differentiation of ES cells. Such cells are used for transplantation therapy and will bring new hopes for the treatment of diseases such as diabetes, Parkinson's disease, spinal cord injury, leukemia, myocardial injury, renal failure, and cirrhosis.

However, hES cell research has been faced with many problems and controversies, including the following aspects: (1) The source of donor oocytes is difficult, and the efficiency of hES cell establishment is low. In addition, the immature SCNT technology will inevitably require further consumption of human oocytes, so its source is difficult to guarantee; (2) immune rejection, unless the SCNT technology is used, the patient's differentiation from hES cells Cells and tissues still have immune rejection; (3) hES cells are tumorigenic and may develop into tumors after transplantation into recipients, even with SCNT technology. Responses such as suicide genes in cells may not be able to solve this problem well; (4) Maintain hES risk in vitro. Similarly, lentiviral transfection techniques may have similar risks.

To circumvent the ethical debate on hES cells and therapeutic cloning research, an alternative approach is needed to convert human somatic cells directly into pluripotent stem cells, providing patients with "personalized" autologous stem cells. In 2003, the Gurdon team found that after the cells of fully differentiated mouse thymocytes or adult peripheral blood lymphocytes were injected into Xenopus oocytes, the differentiation markers of mammalian nuclei were lost, while the most characteristic of mammalian stem cells. The high expression of Oct4, a marker of sex, suggests that the mammalian nucleus can be directly remodeled by the nucleus of the amphibian oocyte to express Oct4 [Byrne JA, et al.Nuclei of adult mammalian somatic cells are directly reprogrammed to oct-4stem cell Gene expression by amphibian oocytes. Curr Biol 2003; 13: 1206-1213.].

In 2006, the Yamanaka research team at Kyoto University in Japan used in vitro gene transfection technology to screen four transcription factors including Oct4, Sox2, c-Myc, and Klf4 from 24 factors, and introduced the above four transcription factors into the embryo by retrovirus. Mouse fibroblasts or adult mouse tail skin fibroblasts, obtained a Fbx15+ pluripotent stem cell line under the condition of mouse ES cells, which is in cell morphology, growth characteristics, surface markers, and teratogenesis Tumors and the like are very similar to mouse ES cells, but are different from mouse ES cells in gene expression profiling, DNA methylation and formation of chimeric animals, so they are named as induced pluripotent stem cells (iPS cells). ).

The Yamanaka team used the same technique to introduce the same four transcription factors into human skin fibroblasts, and successfully obtained iPS cells. Primary human fibroblast-like synoviocytes and cell lines derived from neonatal fibroblasts can also be reconstituted into iPS cells. Such iPS cells are similar to hES cells in terms of cell morphology, proliferative capacity, surface antigen markers, gene expression profiles, epigenetic status of pluripotent stem cell-specific genes, and telomerase activity, and are cultured in vitro and in vitro. Different types of cells in three germ layers can be differentiated in teratoma formation in mice [Takahashi K et al, Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 2007; 131:861-872.]. At the same time, the University of Wisconsin Thomson research team also reported successful induction of fetal fibroblasts into human iPS cells with the basic characteristics of hES cells, except that they used lentivirus as a vector and selected among 14 candidate genes. Four genes, such as Oct4, Sox2, Nanog, and Lin28, were transduced [Yu J et al, Induced pluripotent stem cell lines derived from human somatic cells. Science 2007, 318: 1917-1920]. This major breakthrough, known as the “milestone” of the biological sciences, is expected to help scientists bypass the ethical and moral disputes of cloning technology and open the door to medical applications.

Following this, iPS has made important breakthroughs in the methods of induction of reprogramming, induction efficiency, induction of cell source and biosafety. In the re-differentiation of iPS cells, some development has also been made. At present, a stable differentiation system has been established in epithelial cell differentiation and hepatocyte differentiation of iPS cells [Ahmad Sm, et al. Differentiation of human embryonic stem cells into corneal epithelial-like cells by in vitro] Replication of the corneal epithelial stem cell niche. Stem Cells, 2007, 25(5): 1145-55.; Metallo CM, et al. Directed differentiation of human embryonic stem cells to epidermal progenitors. Methods Mol Biol, 2010, 585: 83 -92.; Song ZH, et al. Efficient generation of hepatocyte-like cells from human induced pluripotent stem cells. Cell Res, 2009, 19(11), 1233-1242.]. In the differentiation of neural stem cell cells, iPS cells and ES cells have also achieved certain research results [Kim DS, et al. Robust enhancement of neural differentiation from human ES and iPS cells regardless of their innate difference in differentiation propensity. Stem Cell Rev, 2010, 6(2): 270-281.].

Neural stem cells are a kind of mother cells with the ability to divide and self-renew, which can produce various types of cells of nerve tissue through unequal division. Neural stem cells have the ability to differentiate into neuronal neurons, astrocytes, and oligodendrocytes, which can self-renew, providing a large number of functional cells for nerve tissue, and have important applications in a variety of neurological diseases. Value and research significance. However, the source of neural stem cells is very limited: primary separation from neural tissue is largely limited by tissue sources; neural precursor cells differentiated from other types of adult stem cells have functional limitations. The limitation, which is mainly due to the limited differentiation ability of adult stem cells themselves, makes it difficult to completely regenerate functional neural stem cells. Therefore, the development of neural stem cells has always been a problem that neuroscience needs to solve.

The establishment of ES and iPS technology has developed a new research direction for the regeneration of neural stem cells. These two cells have strong self-renewal ability and high differentiation ability. At present, many research groups have established ES and iPS cells. Technical methods for differentiation into neural stem cells, for example, EB induction, PA6 cell co-culture, etc. [Bain G, et al. Embryonic stem cells express neuronal properties in vitro. Dev Biol. 1995, 168(2): 342-357. Okabe S, et al. Kang HC, et al. Behavioral improvement after transplantation of neural precursors derived from embryonic stem cells into the globally ischemic brain of adolescent rats. Brain & development. 2010, 32(8): 658-668. However, which method is more suitable for the clinical development of this technology is still inconclusive.

The development of a new source of neural stem cells is still an urgent problem in neuroscience.

Summary of the invention

The present invention is directed to solving at least some of the above technical problems or at least providing a commercial choice.

According to an aspect of the present invention, the present invention provides a differentiation medium capable of inducing ES cells and/or iPS cells into neural stem cells, the differentiation medium being a conventional culture containing 1% of an N2 additive The conventional medium is at least one of the following: RPMI1640 medium, DMEM/F12 medium, high glucose DMEM medium, and α-MEM medium.

The differentiation medium of the present invention is a conventional medium supplemented with only 1% of N2 additive, so that ES cells and/or iPS cells can be induced into neural stem cells quickly, efficiently, and economically. The obtained neural stem cells can be used for nerve damage Repair of injuries and treatment of diseases of the nervous system.

According to another aspect of the present invention, the present invention provides the use of the differentiation medium of one aspect of the present invention described above for the preparation of neural stem cells. By culturing ES cells and/or iPS cells using the differentiation medium, ES cells and/or iPS cells can be efficiently and stably induced into neural stem cells.

According to still another aspect of the present invention, the present invention provides a method of producing a neural stem cell, which comprises obtaining the neural stem cell by culturing ES cells and/or iPS cells using the differentiation medium of one aspect of the present invention described above.

The method for preparing a neural stem cell of the present invention can utilize the differentiation medium of one aspect of the present invention, that is, a conventional medium containing only 1% of an N2 additive, to quickly and efficiently, economically and easily, ES cells and/or iPS cells. Induced as neural stem cells. The neural stem cells obtained by the method can be used for the repair of nerve damage and the treatment of diseases of the nervous system.

DRAWINGS

The above and/or additional aspects and advantages of the present invention will become apparent and readily understood from

Figure 1 shows the morphological structure of various stages of differentiation of iPS cells into neural stem cells in an embodiment of the present invention.

Fig. 2 is a view showing qPCR results of pluripotency genes and changes in expression of neural stem cell marker genes at various stages of differentiation of iPS cells into neural stem cells in one embodiment of the present invention.

Fig. 3 shows the results of immunofluorescence of neural stem cell marker gene expression on day 7 when iPS cells were induced to differentiate into neural stem cells in the preparation of neural stem cells according to an embodiment of the present invention.

Figure 4 shows the results of flow cytometry detection of neural stem cell marker gene expression on day 7 of induction of differentiation of iPS cells into neural stem cells according to one embodiment of the present invention.

Specifically, according to one embodiment of the present invention, a differentiation medium capable of directionally inducing differentiation of ES cells and/or iPS cells into neural stem cells, the differentiation medium being a 1% N2 additive Conventional medium is at least one of the following: RPMI1640 medium, DMEM/F12 medium, high glucose DMEM medium, and α-MEM medium.

The differentiation medium in this embodiment is a conventional medium supplemented with only 1% of the N2 additive, and the ES cells and/or iPS cells can be induced into neural stem cells quickly, efficiently, and economically. The obtained neural stem cells can be used for the repair of nerve damage and the treatment of diseases of the nervous system.

The so-called N2 supplement (N2 Supplement) is made up of transferrin, insulin, flavonoids, putrescine and selenite. Made up of sodium. The N2 additive can be formulated by itself, for example, pre-formulated N2 additive containing transferrin, insulin, flavonoids, putrescine and sodium selenite, and the concentration of each component is 1 mM, as a mother liquor storage. It is also possible to purchase a commercially available product, and according to the introduction of the product specification, the differentiation medium in the embodiment is prepared in proportion to the embodiment of the present invention.

According to a preferred embodiment of the invention, the differentiation medium is RPMI 1640 medium comprising 1% N2 additive. RPMI is the abbreviation of Roswell Park Memorial Institute. RPMI is a type of cell culture medium developed by the Institute. 1640 is a medium code containing 10% fetal bovine serum. RPMI1640 medium can be obtained by purchasing a commercially available product or by self-configuration. The RPMI1640 medium containing only 1% of the N2 additive has been tested by the inventors for several times and is suitable for the induction of neural stem cells.

According to an embodiment of the present invention, the differentiation medium is a conventional medium containing 1% of an N2 additive, and the conventional medium is at least one of the following: RPMI1640 medium, DMEM/F12 medium, high glucose DMEM medium. And the α-MEM medium, for example, the so-called conventional medium may be one of them, or a mixture of two, three or all four. The so-called differentiation medium is a conventional medium containing only 1% of the N2 additive.

According to another embodiment of the present invention, there is provided the use of the differentiation medium of any of the above embodiments for preparing a neural stem cell. By culturing ES cells and/or iPS cells using the differentiation medium, ES cells and/or iPS cells can be efficiently and stably induced into neural stem cells.

According to still another embodiment of the present invention, there is provided a method of producing a neural stem cell, which comprises obtaining the neural stem cell by culturing ES cells and/or iPS cells using the differentiation medium in any of the above embodiments.

The method for preparing a neural stem cell in the embodiment, using the differentiation medium in any of the above embodiments, that is, using a conventional medium containing only 1% of the N2 additive, can quickly and efficiently market ES cells and/or iPS cells. It is easily induced into neural stem cells. The neural stem cells obtained by the method can be used for the repair of nerve damage and the treatment of diseases of the nervous system.

According to an embodiment of the invention, the ES cells and/or iPS cells in this embodiment are from humans and can be represented as hES and hiPS, respectively.

Unlike the known neural stem cell induction medium and induction technique, the method of the present embodiment does not incorporate a complex signaling pathway inhibitor, nor does it use a known conditioned medium, and only a N2 Supplement is added to the conventional medium. Human ES cells and/or iPS cells can be induced into cells with neural stem cell characteristics, and the induction process is rapid and the method is stable.

The iPS is derived from reprogrammed somatic cells, and the present embodiment does not limit the type and reprogramming manner of the reprogrammed somatic cells. For example, iPS cells are derived from, but are not limited to, at least one of the following cells that are reprogrammed: dermal fibroblasts, periodontal ligament stem cells, dental pulp stem cells, and urine cells. According to one embodiment of the invention, iPS cells are urine in consideration of safety and applicability Cell-derived non-integrated cells.

According to an embodiment of the present invention, before culturing ES cells and/or iPS cells using the differentiation medium in any of the above embodiments, the method comprises: culturing ES cells and/or iPS cells with at least one of the following media to obtain said ES cells and / or iPS cells: mTeSR ^TM 1 medium, E8 KSR medium and conditioned medium. mTeSR ^TM 1 medium, E8 KSR media and conditioned medium can be obtained by conventional commercially available.

According to an embodiment of the present invention, the ES cells and/or iPS cells are cultured using at least one of the following media to obtain the ES cells and/or iPS cells, comprising: culturing on a Petri dish coated with Matrigel The ES cells and/or iPS cells, when the ES cells and/or iPS cells are grown to cover 60-70% of the bottom of the culture dish, digest the ES cells and/or iPS cells to obtain stem cell single cells. According to a preferred embodiment of the invention, the Matrigel is type I collagen; and/or the ES cells and/or iPS cells are digested with Accutase. It is convenient and quick to obtain ES cells and/or iPS cells and their single cells.

According to an embodiment of the present invention, the ES cells and/or iPS cells are cultured using the differentiation medium of any of the above embodiments to obtain the neural stem cells, comprising: using the differentiation medium containing 10 μM ROCK inhibitor The stem cell single cells are resuspended, and seeded in the culture plate at a density of 0.75-1.0×10 ⁵ cells/mL; the cells in the culture plate are exchanged once every two days with the differentiation medium until The neural stem cells are obtained. This condition was determined by the inventors through repeated adjustment attempts and experimental verification to facilitate efficient induction of ES cells and/or iPS into neural stem cells.

In accordance with a preferred embodiment of the invention, the so-called ROCK inhibitor is selected from commercially available Y-27632 for stem cell survival and initiation into a neural stem cell induction program.

According to one embodiment of the present invention, after resuspending in a differentiation medium containing 10 μM Y-27632, the stem cell single cell suspension is inoculated into a 6-well plate at 1.5-2.0×10 ⁵ cells/well to start and Achieve induction.

Embodiments of the present invention are described in detail below. The examples are intended to be illustrative, and not to limit the invention. In the description of the present invention, "a plurality" means two or more unless otherwise stated.

In the present specification, the singular "a", "the" For example, the term "(a) cell" includes a plurality of cells, including mixtures thereof.

In this document, all numerical designations, such as pH, temperature, time, concentration, and molecular weight, including ranges, are approximations unless otherwise indicated, for example, they vary (+) or (-) in increments of 0.1. It is to be understood that although the term "about" is not always explicitly stated before all numerical signs are added. It is also to be understood that although not always explicitly stated, the reagents described herein are merely examples, the equivalents of which are known in the art.

Herein, the "induced pluripotent stem cell (iPS cell)" is a cell which can be formed under ES cell culture conditions and in ES cell morphology, growth characteristics, surface marker expression, and transplantation into the skin. Teratoma containing three germ layer tissue structures is very similar to human ES cells, and in the way of DNA methylation, gene table The spectrum and chromatin status are also very similar to human ES cells.

As used herein, the term "induced reprogramming" refers to the process of dedifferentiating somatic cells into pluripotent stem cells. Preferably, the somatic cells are dedifferentiated into pluripotent stem cells by introducing a pluripotency factor required for maintaining stem cell pluripotency into a somatic cell through a non-integrating plasmid. The method of introducing the pluripotency factor into a somatic cell can be a variety of techniques well known to those skilled in the art, including various methods of transferring DNA into cells, such as viral infection, lipofection, electroporation, particle bombardment, and the like. Preferably, plasmid transfection is performed using electroporation.

In the following examples, the differentiation medium component used and its working concentration are preferably: N2 Supplement: 1% by weight, configured on a RPMI1640-based medium.

The experimental methods in the following examples, unless otherwise specified, are conventional methods, for example, refer to "Molecular Cloning Experimental Guide", 3rd edition (2002), edited by Sambrook, Fritsch and Maniatis; "Modern Molecular Biology Experimental Methods" (FMAusubel et al. (1987)); "Academic Press, Inc."; "PCR2: Practical Methods", edited by MJ MacPherson, BD Hames and GRTaylor, (1995); "Antibody", Harlow Edited by Lane, (1988); Manual of Animal Cell Culture Laboratory, edited by RI Freshney, (1987); Handbook of Stem Cells, Vol. 2, edited by W. French Anderson et al.

The test materials used in the following examples, unless otherwise specified, are from conventional commercially available reagent materials.

Embodiment 1

Conventional culture of human ES cells and iPS cells

Materials: H1 human embryonic stem cells (H1-ES cells), urinary cell-derived induced pluripotent stem cells (UC-iPS cells).

Medium: mTeSR ^TM 1 medium (STEMCELL catalog # 05850).

Method steps:

Routine maintenance culture of pluripotency of H1-ES cells and UC-iPS cells requires coating with Metrigel (BD catalog #354277), inoculation of cells, culture with mTeSR ^TM 1 medium; passage for about 4-6 days Once, digestion can be carried out using 2 mg/ml Dispase enzyme (Gibco catalog #17105-041).

Embodiment 2

Materials: H1 human embryonic stem cells (H1-ES cells), urinary cell-derived induced pluripotent stem cells (UC-iPS cells)

Reagent: RPMI1640 medium (Gibco catalog #21870-076) supplemented with N2 Supplement (Gibco #17502-048), wherein the medium contained 1% by weight of N2Supplement; Y-27632 (Sigma catalog #Y0503); Accutase ( Sigma catalog #A6964); Bovine Type I collagen (BD catalog #354231). Y-27632/ROCK inhibitor.

Method steps:

60-70% of H1-ES cells or UC-iPS cells grown to the bottom of the culture dish were digested with Accutase for 3-5 min, blown into single cells, and seeded in a type I collagen-coated 6-well plate at a seeding density of 1.5. -2.0×10 ⁵ cells/well, cultured in a differentiation medium, and added Y-27632 to the medium (there is no need to add Y-27632 after the change), the final concentration is 10 μM, and then the whole amount of liquid is changed every other day for 7 days. A typical neuron-like structure is seen.

It should be noted that the morphology of embryonic stem cells observed under the microscope and the morphological changes and differentiation results during differentiation into neural stem cells are basically the same as those of human induced pluripotent stem cells. The stem cells are described as an example.

The morphological changes of cells during culture were as shown in Figure 1. Before differentiation, UC-iPS cells grew in a clone-like manner. On the first day of differentiation, the single cells after digestion were adherent and showed small clone-like growth; induced differentiation. On the third day, the clones were no longer adherent and spheroidal in suspension. On the fifth day of induced differentiation, the suspended cells were attached again, and the morphology was clearly changed. After 7 days of differentiation, typical nerve garlands appeared in adherent cells. The structure shows that neural stem cells (NSCs) are formed initially. After the rosette structure is separated and purified by mechanical method, it can grow in a spherical shape (the scale is 200 μm in the figure). It can be seen that the method does not require EB (embryonic body) over-extension, and only needs simple differentiation culture to differentiate human ES or iPS cells into neural stem cells.

During the differentiation process, the expression changes of pluripotency genes and neural stem cell marker genes at various stages are shown in Figure 2: during the differentiation process, the expression of pluripotency genes is gradually down-regulated, and the expression level of neural stem cell marker genes is gradually increased. Most of the neural stem cell marker genes reached the highest expression level on the 7th day after differentiation; however, Sox2 was both a pluripotency gene and a neural stem cell marker gene, and the change trend was not significant during the whole differentiation process.

For the neural stem cells on the 7th day of differentiation, we performed the protein level analysis of the neural stem cell marker molecules, and the results of immunofluorescence and flow cytometry were shown in Fig. 3 and Fig. 4, respectively.

From the results of immunofluorescence in Fig. 3, it was found that neural stem cells formed on the 7th day after differentiation of iPS cells clearly expressed neural stem cell marker genes (Nestin, Pax6 and Sox1). The left column of the figure shows the results of fluorescence staining observed under a fluorescence microscope, the middle column is the DUD staining result of the corresponding field of view, and the right column is the composite picture of the former two. The scale in the figure is 50 μm.

From the results of flow cytometry in Figure 4, it can be seen that the expression efficiency of neural stem cells Nestin formed on the 7th day after iPS cell differentiation is above 90%, and the expression efficiency of Pax6 is close to 80%, which indicates that the differentiation efficiency of the method is high.

In the description of the present specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" and the like means a specific feature described in connection with the embodiment or example. A structure, material or feature is included in at least one embodiment or example of the invention. In the present specification, the schematic expression of the above terms does not necessarily mean The same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in a suitable manner in any one or more embodiments or examples.

While the embodiments of the present invention have been shown and described, the embodiments of the invention may The scope of the invention is defined by the claims and their equivalents.

Claims

A differentiation medium characterized in that the differentiation medium is capable of inducing ES cells and/or iPS cells into neural stem cells, and the differentiation medium is a conventional medium containing 1% of an N 2 additive.

The conventional medium is at least one of the following: RPMI1640 medium, DMEM/F12 medium, high glucose DMEM medium, and α-MEM medium.
The differentiation medium of claim 1 wherein said differentiation medium is RPMI 1640 medium comprising 1% N2 additive.
Use of a differentiation medium according to any of claims 1-2 for the preparation of neural stem cells.
A method for producing a neural stem cell, characterized in that ES cells and/or iPS cells are cultured using the differentiation medium of claim 1 or 2 to obtain the neural stem cells.
The method of claim 4 wherein said ES cells and/or said iPS cells are from humans.
The method of claim 4 wherein said iPS cells are derived from at least one of the following cells reprogrammed: dermal fibroblasts, periodontal ligament stem cells, dental pulp stem cells, and urine cells.
The method of claim 4, wherein prior to culturing the ES cells and/or iPS cells using the differentiation medium of claim 1 or 2, the method comprises:

Using at least one of the following culture medium of ES cells and / or iPS cells, in order to obtain the ES cells and / or iPS cells: mTeSR TM 1 medium, E8 KSR media and conditioned media.
The method of claim 7, wherein said culturing ES cells and/or iPS cells with at least one of the following vectors to obtain said ES cells and/or iPS cells comprises:

The ES cells and/or iPS cells are cultured on a Matrigel-coated culture dish, and when the ES cells and/or iPS cells are grown to cover 60-70% of the bottom of the culture dish, the ES cells are digested and / or iPS cells, get stem cell single cells.
The method of claim 8 wherein said Matrigel is type I collagen;

Optionally, the ES cells and/or iPS cells are digested with Accutase enzyme.
The method of claim 9, wherein said culturing ES cells and/or iPS cells with the differentiation medium of claim 1 or 2 to obtain said neural stem cells comprises:

The stem cell single cells were resuspended in the differentiation medium containing 10 μM ROCK inhibitor, and seeded in the culture plate at a density of 0.75-1.0×10 5 cells/mL;

The cells in the culture plate were subjected to a liquid exchange every two days using the differentiation medium until the neural stem cells were obtained.