A neural stem cell and preparation method thereof

Abstract

The present invention provides a neural stem cell that simultaneously expresses the genes of Pdgfrβ and Prom1. The present invention also provides a method for preparing the above-mentioned neural stem cells, including using transgenic mice Pdgfrβ-Cre transgenic mice and Cag-Frt-Stop-Frt-Lox-Stop-Lox-tdTomato reporter gene mice, hybridizing to produce Pdgfrβ-Cre; Ai65D double heterozygous experimental mice; constructing pAAV-mP2-FLP-WPRE viruses; isolating Pdgfrβ-Cre; Ai65D double heterozygous transgenic mice lateral ventricle lateral wall neural stem cells, after in vitro culture and passage, adding pAAV-mP2-FLP-WPRE viruses and tamoxifen's active metabolite 4-OH-tamoxifen; obtaining neural stem cells that simultaneously express Pdgfrβ and Prom1 genes. The present invention found that Pdgfrβ and Prom1 double-positive neural stem cells have the function of repairing nerve damage.

Description

Technical Field

[0001] The present invention belongs to the field of bioengineering and relates to a neural stem cell, in particular to a neural stem cell and a preparation method thereof. Background Art

[0002] There are many types of neurological diseases, such as Alzheimer's disease (AD), characterized by tau pathology; Parkinson's disease (PD), characterized by Lewy bodies; Amyotrophic lateral sclerosis (ALS), characterized by microglial activation and motor neuron apoptosis; Stroke, characterized by ischemia or hemorrhage; and spinal cord injury and autism, which are caused by multiple complex factors. [2] Neurological diseases have a high incidence rate and the disease cycle covers almost all age groups. There is still no effective treatment. One of the important reasons is that the nervous system is very difficult to regenerate after birth. Neurogenesis refers to the process in which neural stem cells proliferate to become directed progenitor cells, gradually migrate and differentiate into neurons and participate in the establishment of circuits to produce neural functions. [3] Recent studies have found that adult neurogenesis in mammals continues throughout life and is mainly found in two specific areas: the lateral wall of the lateral ventricle and the dentate gyrus of the hippocampus. Adult hippocampal neurogenesis plays a very important role in emotional regulation and cognitive function, while neurogenesis in the lateral wall of the lateral ventricle plays an important role in maintaining olfactory function, establishing olfactory neural circuits, and regulating social, cognitive, and emotional development that is dependent on olfaction. [4] .

[0003] Exogenous neural stem cell therapy has been applied to a variety of neurological diseases. For example, mouse embryonic stem cells (ESCs) can be integrated into the striatum of rat PD models, differentiate into dopamine (DA) neurons, and restore motor function loss. [5] Human induced pluripotent stem cells (iPSCs) can differentiate into DA precursor cells, migrate to the substantia nigra of PD rodent models, and further differentiate into DA neurons to function. [6] Although it is currently believed that there are multiple factors that play a role in exogenous stem cell transplantation, including providing support for endogenous stem cells, new cell integration, immune regulation, nutritional effects, neuroprotection, and stimulating angiogenesis, the specific molecular mechanisms that regulate the effects of exogenous stem cells have not yet been elucidated. Issues such as the survival efficiency, rejection reaction, and tumorigenicity of exogenous stem cells in the body also need to be addressed.

[0004] Activating and regulating endogenous stem cells in patients with neurological diseases is a relatively safe and effective treatment method, which provides broad treatment prospects for many neurological diseases. However, the identification and isolation of neural stem cells are still very difficult.

[0005] Neural stem cells (NSCs) are multipotent stem cells of the nervous system that can differentiate into a variety of nerve cells. NSCs can be isolated from specific regions of the mammalian brain and spinal cord and are capable of reconstructing neural circuits and repairing neural tissue. Therefore, NSCs hold broad research value in areas such as neurodegenerative diseases, hereditary central nervous system disorders, stroke, and spinal cord injury.

[0006] Currently, identifying true neural stem cells from neural tissue remains a significant challenge. Some studies suggest that neural stem cells can be identified based on morphological characteristics, specific antigen expression, and differentiation potential. However, the in vitro culture environment for stem cells differs significantly from the in vivo stem cell microenvironment, making it difficult for cells to maintain their stem cell properties over the long term. Furthermore, most neural stem cells identified in vitro cannot be tracked long-term in vivo, making it difficult to determine their true role in vivo.

[0007] The Prom1 gene encodes CD133, the first discovered five-transmembrane glycoprotein. [7] Initially used as a marker for hematopoietic progenitor cells and tumor stem cells, CD133 can mediate PI3K / Akt, Akt, Src-FAK and other signaling pathways, affecting the function of stem cells. [8] CD133 is not only a marker for human glioma stem cells, but also a marker for human neural stem cells. In 2008, a study revealed that adult Prom1+ neural stem cells can proliferate, migrate, and differentiate in vivo[1]

[0008] The Pdgfrβ gene is the platelet-derived growth factor receptor β (Pdgfrβ), also known as CD140b, and its structural homologous protein Pdgfrα is a member of the receptor tyrosine kinase (RTK) class III subfamily. Pdgf is an important mitogen that can link the Pdgfr-α chain and the Pdgfr-β chain through disulfide bonds to form homo- or heterodimeric proteins. Pdgfrβ binds to Pdgf-BB with high affinity. Pdgfrβ is highly expressed in the perivascular cells of the nervous system, participates in the formation of the neurovascular unit, and maintains the blood-brain barrier (BBB). Studies have found that Pdgfrβ is highly expressed in adult resting neural stem cells, and as the stem cells gradually become activated, the expression of Pdgfrβ gradually decreases. [9] .

[0009] The present invention focuses on the classification and identification of neural stem cells, invents a new method for identifying and isolating neural stem cells, and through long-term lineage tracing in vivo, identifies that this group of neural stem cells plays an important role in the body. Summary of the Invention

[0010] In response to the above technical problems in the prior art, the present invention provides a neural stem cell and a preparation method thereof, which aims to solve the technical problem in the prior art that neural stem cells are not effective in treating nerve damage.

[0011] The present invention provides a neural stem cell, which simultaneously expresses Pdgfrβ and Prom1 genes.

[0012] The present invention also provides a method for preparing neural stem cells, comprising the following steps:

[0013] 1) Two strains of transgenic mice, Pdgfrβ-Cre transgenic mice and Cag-Frt-Stop-Frt-Lox-Stop-Lox-tdTomato (this strain of mice is abbreviated as Ai65D) reporter gene mice, are used. All cells expressing the Pdgfrβ gene in the Pdgfrβ-Cre transgenic mice express Cre recombinase; all cells expressing the Cag gene in the brain of the Cag-Frt-Stop-Frt-Lox-Stop-Lox-tdTomato (Ai65D) reporter gene mice express a Frt-Stop-Frt-Lox-Stop-Lox-tdTomato sequence. The two transgenic mice are hybridized to produce Pdgfrβ-Cre;Ai65D double heterozygous experimental mice;

[0014] 2) A step of constructing a "pAAV-mP2-FLP-WPRE" virus, wherein mP2 is a promoter specific to the Prom1 gene, and a vector is constructed, wherein the vector backbone is "Ef1α-FLP-WPRE-hGH-polyA", and the restriction sites at both ends of Ef1α are MluI and BamhI, respectively. A large mP2 promoter fragment containing these two restriction sites is synthesized, and the "EF1α" promoter in the vector backbone is replaced with the "mP2 promoter" through enzyme digestion and enzyme ligation reactions, ultimately constructing the "mP2-FLP-WPRE-hGH-polyA" plasmid. Subsequently, an AAV viral vector with serotype 9 is selected for viral coating to obtain the "pAAV-mP2-FLP-WPRE" tool virus;

[0015] 3) Neural stem cells from the lateral ventricle wall of Pdgfrβ-Cre;Ai65D double heterozygous transgenic mice were isolated. After the neural stem cells were cultured and passaged in vitro, the pAAV-mP2-FLP-WPRE virus and tamoxifen's active metabolite 4-OH-tamoxifen (4-OH-TAM) were added to obtain neural stem cells that simultaneously expressed the Pdgfrβ and Prom1 genes.

[0016] According to the known information provided by Jackson Laboratory, neural stem cells isolated from Pdgfrβ-Cre; Ai65D double heterozygous transgenic mice have both Pdgfrβ-Cre and Cag-Frt-Stop-Frt in their genomes.

[0017] -Lox-Stop-Lox-tdTomato two sequences are expressed. Therefore, the FLP sequence contained in the cell virus infected with pAAV-mP2-FLP-WPRE is shown in Appendix 1, and the mP2 sequence is provided in the literature. [1,10] .

[0018] Table 1 FLP sequence: 5'→3'

[0019]

[0020]

[0021] Table 2mP2 sequence: 5'→3'

[0022]

[0023] If the Prom1 gene is expressed, the Prom1-specific promoter "mP2" is expressed, inducing the subsequent expression of the FLP sequence and the synthesis of FLP recombinase, which excises the pre-existing Frt-Stop sequence in the mouse cell genome. Cre recombinase is expressed in the cytoplasm of all cells expressing the Pdgfrβ gene. TAM injection allows Cre recombinase to enter the nucleus and excise the Lox-Stop sequence.

[0024] Only when the Pdgfrβ and Prom1 genes are expressed in the cells at the same time can the two Stop sequences in the genome be removed at the same time, and the tdTomato fluorescent protein can be transcribed and translated. From then on, the neural stem cells expressing the Pdgfrβ and Prom1 genes and the daughter cells of this neural stem cell also express the tdTomato fluorescent protein.

[0025] For neural stem cells that only express the Pdgfrβ gene, after TAM injection, the Cre recombinase can only remove the Lox-Stop sequence, and the Frt-Stop sequence still prevents the expression of tdTomato fluorescent protein, and the cells cannot be labeled with red fluorescence.

[0026] Neural stem cells that only express the Prom1 gene are infected with pAAV-mP2-FLP-WPRE. FLP recombinase is expressed and the Frt-Stop sequence is removed. However, the Lox-Stop sequence still prevents the expression of tdTomato fluorescent protein, and the cells cannot be labeled with red fluorescence.

[0027] Compared with existing technologies, the present invention offers significant and positive technical benefits. The present invention discovered that Pdgfrβ and Prom1 double-positive neural stem cells can proliferate and differentiate both in vivo and in vitro. Following traumatic brain injury, Pdgfrβ and Prom1 double-positive cells migrate to the injury site, proliferate, and differentiate. These Pdgfrβ and Prom1 double-positive neural stem cells possess the ability to repair neural damage, providing new insights into the treatment of neurological diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 : The large fragment of the mP2 promoter contains 265 base pairs. The Ef1α promoter on the vector was removed using two DNA enzymes, BamH1 and Mlu1. The large fragment of the enzyme-digested vector was recovered and then subjected to enzyme ligation reaction with the mP2 promoter to finally construct the "mP2-FLP-WPRE-hGH-polyA" plasmid for viral packaging.

[0029] Figure 2 :Neural stem cells that simultaneously express Prom1 and Pdgfrβ grow into spheres and differentiate in vitro; image results show that tdTomato cells grow into spheres and can be passaged, differentiate after growing on the wall, and eventually differentiate into Gfap+ astrocytes and Tuj1+ neurons.

[0030] Figure 3 : Schematic diagram of the labeling method for neural stem cells expressing both Prom1 and Pdgfrβ in transgenic mice;

[0031] Double heterozygous experimental mice were bred from Pdgfrβ-Cre mice and Ai65D mice, and TAM was injected intraperitoneally and intracerebroventricularly.

[0032] pAAV-mP2-FLP-WPRE, which enables cells expressing both Prom1 and Pdgfrβ genes to express tdTomato fluorescent protein, and their offspring will also be labeled with tdTomato; TAM (tamoxifen), mP2-Promoter (promoter of the Prom1 gene).

[0033] Figure 4 : Migration of neural stem cells expressing both Prom1 and Pdgfrβ in mice; fluorescence imaging results showed that tdTomato cells migrated along the ventricular wall to the corpus callosum and along the RMS to the olfactory bulb.

[0034] Figure 5 : Differentiation of neural stem cells expressing both Prom1 and Pdgfrβ in mice; fluorescence imaging results showed that tdTomato cells differentiated into Gfap+ astrocytes, NeuN+ neurons, and CNPase+ oligodendrocytes. DETAILED DESCRIPTION

[0035] Example 1

[0036] like Figure 1 As shown in FIG, the steps for constructing the "pAAV-mP2-FLP-WPRE" virus are as follows:

[0037] mP2 is a specific promoter of the Prom1 gene, and the mP2 promoter fragment contains 265 base pairs.

[0038] The vector model PT-0144 purchased from Wuhan Shumi Brain Science Technology Co., Ltd. was used. The backbone of the vector model PT-0144 was "rAAV-EF1α-FLP-WPRE-hGH The polyA promoter is Ef1α. The restriction sites at either end of Ef1α are MluI and BamhI, respectively. A large mP2 promoter fragment containing these two restriction sites was synthesized. The Ef1α promoter on the PT-0144 vector was excised by restriction digestion. DNA gel electrophoresis was used to confirm the successful digestion reaction and recover the large vector fragment. T4 ligase was used to enzymatically ligate the large vector fragment with the mP2 promoter, replacing the "EF1α" promoter in the vector backbone with the "mP2" promoter. DNA gel electrophoresis was used to confirm successful ligation, ultimately constructing the "mP2-FLP-WPRE-hGH-polyA" plasmid. Subsequently, an AAV serotype 9 viral vector was selected for viral coating to obtain the "pAAV-mP2-FLP-WPRE" virus.

[0039] Example 2

[0040] The present invention also provides a method for preparing neural stem cells, comprising the following steps:

[0041] Transgenic mice purchased from The Jackson Laboratory, Pdgfrβ-Cre (Jax#030201) and Cag-FRF-LSL-tdTomato (Jax#021875, Ai65D), were bred to generate Pdgfrβ-Cre; Ai65D double heterozygous transgenic experimental mice.

[0042] Specifically, all cells expressing the Pdgfrβ gene in the Pdgfrβ-Cre transgenic mice express Cre recombinase (see https: / / www.jax.org / strain / 030201 for details); all cells expressing the Cag gene in the brain of the Cag-Frt-Stop-Frt—Lox-Stop-Lox-tdTomato (Ai65D) reporter gene mice express a Frt-Stop-Frt—Lox-Stop-Lox-tdTomato sequence (see https: / / www.jax.org / strain / 021875 for details). The two transgenic mice were hybridized to produce Pdgfrβ-Cre;Ai65D double heterozygous experimental mice.

[0043] 1) To isolate primary neural stem cells from wild-type (WT) fetal rats, intact brain tissue was dissected in pre-chilled phosphate-buffered saline (PBS). Under a stereomicroscope, the meninges and blood vessels on the cortical surface were removed. The olfactory bulb and partial cortical tissue from the telencephalon were collected. The tissue was minced with spring scissors and Accutase (stem cell digestion enzyme) was added. The cells were incubated at 37°C for 15 minutes, and culture medium was added to disperse the tissue into single-cell cultures.

[0044] 2) Neural stem cell culture medium consisted of DMEM:F12 (Teromo Fisher, Catalog No. 11320033) supplemented with 0.1 mg / ml penicillin-streptomycin solution (Biyuntian Biotechnology, Catalog No. C0222) and 2% serum replacement B27 (Teromo Fisher, Catalog No. 17504044). Basic fibroblast growth factor (bFgf) (Teromo Fisher, Catalog No. 13256-029) and epidermal growth factor (Egf) (Teromo Fisher, Catalog No. 315-09) were supplemented daily at 20 ng / ml each. During co-culture, the two cells shared the same culture medium but were not in contact with each other.

[0045] 3) Pdgfrβ-Cre; Ai65D double heterozygous transgenic experimental mice were treated by cervical dislocation, and the intact brain tissue was immediately dissected and transferred to pre-chilled artificial cerebrospinal fluid (ACSF) (Solebro, Catalog No. H7701). The fresh brain tissue was incised along the sagittal suture under a stereomicroscope, and the olfactory bulb and 2-3 mm of prefrontal cortex tissue were removed. The hippocampus was bluntly dissected with dissecting forceps. The neatly arranged "grapefruit-like" lateral ventricle wall tissue was clearly distinguishable under the stereomicroscope. While ensuring the integrity of the lateral wall, the thinnest 100-200 μm ependymal surface tissue was excised.

[0046] 4) The intact tissue block of the peeled lateral ventricle wall was transferred to 37°C papain (Worthington, catalog number LS003126) and incubated for half an hour; the lateral ventricle wall tissue was gently pipetted in PBS solution to dissociate into single cells.

[0047] 5) The single cell suspension was transferred to a 20% Percoll (Sigma, Catalog No. P1644) solution and subjected to density gradient centrifugation at 400 g for 9 min, with both the acceleration rate of the ascending and descending speeds set to 3.

[0048] 6) Resuspend the cells in 0.1% BSA (Biyuntian, Catalog No. ST023) solution, collect single cells at the bottom of the tube, filter the cell resuspension using a 40 μm cell sieve to remove cell clumps, and transfer to stem cell culture medium for culture.

[0049] 7) Cells were grown as spheres in low-adhesion plates. During the logarithmic growth phase of the second-generation cells (36 hours after subculture), 1 μM 4-OH-TAM (Sigma, H6278) was added for co-incubation. This allowed Cre recombinase to enter the nucleus of cells expressing the Pdgfrβ gene and excise the Lox-Stop sequence in the genome. After 16 hours, the cells were replaced with normal culture medium and cultured overnight.

[0050] 8) When the cell density reached 80%-90%, pAAV-mP2-FLP-WPRE was added to the cells at a concentration of 5E+10 vg / well (96-well plate) for infection. After 72 hours of co-incubation, the cells were replaced with normal culture medium and co-cultured with primary neural stem cells in a co-culture chamber. Fetal rat primary neural stem cells were added to the upper layer of the co-culture chamber, sharing the culture medium but not in contact with each other.

[0051] After 3-5 days of co-culture of the two cell suspensions, it was observed that the neural stem cells in the upper and lower layers of the chamber were able to grow into spheres. After the cell spheres were attached to the wall for observation, tdTomato cell spheres were found, indicating that these cells can continue to proliferate and divide under in vitro culture conditions. After 5-7 days of culturing the tdTomato cells on the wall, it was found that the attached cells could extend abundant protrusions. Immunofluorescence staining was used to identify the expression of the neuronal marker β3-tubulin Tuj1 and the astrocyte marker glial fibrillary acidic protein Gfap in tdTomato cells, indicating that the labeled cells can differentiate into neurons and astrocytes ( Figure 2 ).

[0052] Example 3 Labeling of neural stem cells in mice

[0053] Five days after birth, Pdgfrβ-Cre; Ai65D double heterozygous transgenic experimental mice were injected with pAAV-mP2-FLP-WPRE virus into the lateral ventricle, followed by subcutaneous injection of tamoxifen TAM (Sigma, catalog number T5648) for three consecutive days. Cells expressing both Prom1 and Pdgfrβ genes were labeled, and the labeled cells expressed tdTomato red fluorescent protein.

[0054] For AAV intracerebroventricular injection, mice were anesthetized on ice for 90 seconds. The bregma and sagittal suture of the mouse skull were visible under a cold light source. The skull skin was cleaned with disinfectant alcohol. The injection was performed between the anterior and posterior bregma, 0.8-1 mm on either side of the sagittal suture. The injection depth was approximately 3 mm, and the injection rate was 2 μl / min. The injection volume was 1 μl per lateral ventricle. After the injection, the injection was allowed to rest for 1 minute. The syringe was then slowly withdrawn and the mouse was immediately transferred to a warming pad. After the mouse fully recovered, it was returned to its cage.

[0055] 2) Subcutaneous injection of TAM. Because fetal mouse skin is thin and the skin on the back and back of the neck develops early, this is where the subcutaneous injection is performed. The TAM concentration is 10 mg / ml, and the mouse dose is 25 mg / kg. The injection is given subcutaneously on the back at a rate of 5 μl / min. After the injection, the needle is slowly withdrawn and the skin is wiped dry with a dry cotton ball before being returned to the cage. Cells in the lateral ventricle that express both Prom1 and Pdgfrβ will express the tdTomato fluorescent protein, and their progeny will be permanently tracked. Figure 3 ).

[0056] Example 4 Tracking the proliferation of labeled neural stem cells in vivo

[0057] Pdgfrβ-Cre; Ai65D double-heterozygous transgenic experimental mice were injected postnatally with the pAAV-mP2-FLP-WPRE virus and TAM. TdTomato cells were used to label neural stem cells that had previously expressed both Prom1 and Pdgfrβ. Brain tissue samples were collected 4, 6, 8, 10, 12, 15, 25, and 56 days after AAV injection. After frozen sections were made, DAPI (Biyuntian, Cat. No. C1002) was used to label cell nuclei. After AAV and TAM administration in adult mice, brain tissue sections were similarly collected and stained to observe tdTomato-labeled cells.

[0058] 1) Mice were intraperitoneally injected with sodium pentobarbital. After deep anesthesia, the limbs were fixed, the chest cavity was opened, and pre-chilled PBS was used for transcardial perfusion. After the blood flow was completed, bleached liver tissue was visible. The perfusion was continued with pre-chilled 4% PFA (Biyuntian, P0099).

[0059] 2) Cut the skull open along the sagittal suture. After peeling the skull, carefully sever the nerves at the base of the skull using blunt forceps. Remove the intact brain tissue and transfer it to pre-chilled 4% PFA solution for fixation overnight at 4°C. The next day, dehydrate the brain tissue in 0.15 g / ml sucrose solution. After overnight at 4°C, allow the brain tissue to sink to the bottom and then transfer it to 0.3 g / ml sucrose solution at 4°C overnight.

[0060] 3) Embed the dehydrated brain tissue in OCT. For coronal sections, remove the cerebellum and brainstem, and embed with the olfactory bulb facing upward. For sagittal sections, cut along the sagittal suture and embed with the medial surface facing downward. After embedding, transfer the brain tissue to ice and allow it to rest. Remove any bubbles around the tissue and freeze it in a -80°C freezer.

[0061] 4) Transfer the OCT tissue blocks frozen overnight to an ice slicer to equilibrate the temperature. Start slicing after one hour, with a slice thickness of 30 μm. Collect brain slices continuously into a 48-well plate containing PBS, or attach the brain slices to adhesive slides.

[0062] 5) Incubate the brain tissue with DAPI staining solution at room temperature. After 5 minutes, wash the brain tissue with PBS, add anti-fluorescence quenching mounting medium, cover with a coverslip, and use laser confocal microscopy to scan and image the brain tissue.

[0063] Six days after AAV injection, cells labeled with tdTomato appeared in the lateral ventricle. As time went on, the number of labeled cells gradually increased, indicating that these cells continued to proliferate and divide in the body.

[0064] Example 5 Tracking the migration of labeled neural stem cells in vivo

[0065] Pdgfrβ-Cre; Ai65D double-heterozygous transgenic mice were injected postnatally with the pAAV-mP2-FLP-WPRE virus and TAM, using tdTomato cells to label neural stem cells expressing both Prom1 and Pdgfrβ. Brain tissue samples were collected at different time points after labeling, and the location of tdTomato-labeled cells was observed microscopically after nuclear staining.

[0066] Results: tdTomato-labeled cells were observed in the rostral migratory stream (RMS). As the sampling time increased, the number of tdTomato-labeled cells in the corpus callosum (CC) and olfactory bulb increased. Figure 4 ).

[0067] Example 6 Tracking the differentiation of labeled neural stem cells in vivo

[0068] Pdgfrβ-Cre;Ai65D double-heterozygous transgenic mice were injected postnatally with the pAAV-mP2-FLP-WPRE virus and tamoxifen (TAM). tdTomato cells were used to label neural stem cells expressing both Prom1 and Pdgfrβ. Immunofluorescence staining was used to identify the expression of glial fibrillary acidic protein (Gfap), a marker for astrocytes; doublecortin (Dcx), a marker for immature neurons; NeuN, a marker for mature neurons; and 2'2'-cyclic nucleotide 3'-phosphodiesterase (CNPase), a marker for oligodendrocytes, in tdTomato-labeled cells.

[0069] 1) Wash the brain tissue on the slide with PBS three times, 5 minutes each time. Use an immunohistochemistry hydrophobic pen to draw a closed circle around the brain tissue, add blocking solution, and incubate at room temperature for two hours.

[0070] 2) Dilute the antibody in blocking solution at an appropriate ratio, add it dropwise to the brain tissue, and place it in a 4°C wet box overnight. The next day, wash it three times with PBS, each time for 10 minutes.

[0071] 3) Dilute the secondary antibody and DAPI in blocking solution at an appropriate ratio and apply dropwise to the brain tissue. Incubate in a humidified chamber at room temperature for two hours. Wash three times with PBS for 10 minutes each. Apply anti-fluorescence quenching mounting medium to the brain tissue, cover with a coverslip, and image the sections using a laser confocal microscope.

[0072] The results showed that tdTomato-labeled cells can be co-labeled with markers such as Gfap, Dcx, NeuN, and CNPase, indicating that the labeled cells can differentiate into multiple neural cell directions; most of the tdTomato-labeled cells in the corpus callosum expressed Gfap and CNPase, indicating that the labeled cells that migrated there mainly differentiated into astrocytes and oligodendrocytes; while in the RMS and olfactory bulb, the vast majority of tdTomato-labeled cells expressed Dcx and NeuN, indicating that most of the tdTomato-labeled cells in this migration direction differentiated into neurons ( Figure 5 ).

[0073] References

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Claims

1. A method for preparing neural stem cells, characterized in that The steps include: 1) Using transgenic mice Pdgfrβ-Cre transgenic mice and Cag-Frt-Stop-Frt-Lox-Stop-Lox-tdTomato reporter gene mice Ai65D, all cells expressing the Pdgfrβ gene in the Pdgfrβ-Cre transgenic mice express Cre recombinase; the Cag-Frt-Stop-Frt-Lox-Stop-Lox-tdTomato reporter gene mice, i.e. Ai65D mice, all Cag gene-expressing cells in their brains express a Frt-Stop-Frt-Lox-Stop-Lox-tdTomato sequence, and the two transgenic mice are hybridized to produce Pdgfrβ-Cre; Ai65D double heterozygous experimental mice; 2) A step of constructing a "pAAV-mP2-FLP-WPRE" virus, where mP2 is a specific promoter of the Prom1 gene, and a vector is constructed, wherein the vector backbone is "Ef1α-FLP-WPRE-hGH-polyA", and the restriction sites at both ends of Ef1α are MluI and BamhI, respectively. A large mP2 promoter fragment containing these two restriction sites is synthesized, and the "EF1α" promoter in the vector backbone is replaced with the "mP2" promoter by restriction digestion and enzyme ligation reactions, and finally the "mP2-FLP-WPRE-hGH-polyA" plasmid is constructed, and subsequently an AAV virus vector of serotype 9 is selected for virus coating to obtain the "pAAV-mP2-FLP-WPRE" tool virus; 3) Isolate neural stem cells from the lateral wall of the lateral ventricle of Pdgfrβ-Cre; Ai65D double heterozygous transgenic mice, add pAAV-mP2-FLP-WPRE virus and tamoxifen's active metabolite 4-OH-tamoxifen after the neural stem cells are cultured and passaged in vitro to obtain neural stem cells that simultaneously express the genes of Pdgfrβ and Prom1.

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