Application of neural stem cells derived from mesenchymal umbilical cord stem cells in treatment of nervous system injury

Abstract

The invention discloses a method for preparing neuronal cells and astrocytes by inducing neural stem cells from umbilical cord mesenchymal stem cells and differentiating the neural stem cells. According to the invention, mesenchymal stem cells are used as initial cells, and neuronal cells and astrocytes are prepared through operations such as pre-culture, induction culture and differentiation culture by using NSE and GFAP immunofluorescence identification. The invention provides an induction culture medium. The invention further finds that a differential culture medium containing cerebrospinal fluid can improve the differentiation level of neural stem cells. In-vitro experiments show that the prepared neural stem cells are beneficial to recovery of rats with spinal cord injury and have a certain treatment effect on nervous system diseases.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to an application of neural stem cells derived from mesenchymal umbilical cord stem cells in treating nervous system damage. Background Art

[0002] Stem cells are a type of cells that have the ability to self-replicate and have the potential for multidirectional differentiation. Mesenchymal stem cells (MSCs) are adult stem cells derived from the mesoderm, which have the ability to self-renew and have the potential for multidirectional differentiation. They also have unique immunoregulatory abilities and important paracrine effects. Due to these characteristics, mesenchymal stem cells are widely used in the research of cell therapy and regenerative medicine.

[0003] For a long time, people believed that the nerve cells in the human brain live for life, and when the nerve cells die, they can only be filled by glial cells, because mature nerve cells do not divide and regenerate. It was not until the last decade of the 20th century that this traditional concept was finally broken. In 1989, Panov first proposed the concept of neural stem cells (NSCs): it is a type of multipotent stem cell that can self-renew for a long time and has the multipotential characteristics of differentiating into neurons, astrocytes and oligodendrocytes. Subsequently, through research, scholars gradually summarized the characteristics of this stem cell. In 2000, Gage described the characteristics of NSCs as: ① can form neural tissue or develop from the nervous system; ② have the ability to self-renew; ③ can produce cells different from themselves through asymmetric division.

[0004] Neural stem cells are neural precursor cells that exist in the central nervous system of developing embryos and adult mammals and have the potential to differentiate into neurons, astrocytes, and oligodendrocytes. NSCs have attracted widespread attention for their strong proliferation ability, cryopreservation, low immunogenicity, homology with spinal cord tissue, and easy integration with host neural tissue after differentiation. NSCs are widely available, and NSCs exist in the hippocampus, dentate gyrus, subventricular zone, striatum, and central canal region of the spinal cord in the adult mammalian brain. Human neural stem cells (hNSCs) are mainly derived from the brain tissue of aborted embryos or fetuses. They have the ability to self-renew and differentiate into almost all neural cells, and have great potential in the treatment of brain damage caused by various causes such as trauma, neuronal degeneration, and stroke. At present, people have been able to isolate and culture multipotential NSCs from umbilical cord blood, the subventricular zone, striatum, ependymal zone, and hippocampus of embryonic or adult animals. Cells in different parts have their own genetic characteristics due to different differentiation directions and executive functions.

[0005] However, there are two problems that limit the clinical application of hNSCs. First, hNSCs obtained from embryonic or fetal brain tissue are immunogenic, which can cause immune rejection when transplanted into patients, and transplanted patients need to take immunosuppressants for life. Second, the source of hNSCs is limited and ethically controversial. If there is a cell with a wide source that does not cause transplant rejection and can produce hNSCs through appropriate methods, then the above problems will be solved.

[0006] NSCs are affected by a variety of signal molecules inside and outside the cells during differentiation. Although NSCs can be isolated and cultured in vivo and in vitro, the biological characteristics and differentiation regulation of NSCs are still in the research stage. The multidirectionality and uncertainty of their differentiation also bring certain difficulties to their clinical application. Exploring and establishing a stable experimental condition that promotes their directional differentiation into neurons is the research direction of scholars. Serum can significantly promote the differentiation of NSCs into astrocytes and oligodendrocytes. Although serum-free culture medium can increase the proportion of NSCs differentiating into neurons, it does not conform to the physiological environment in vivo.

[0007] Spinal cord injury (SCI) is often accompanied by softening and necrosis of nerve tissue, autolysis and loss of nerve cells, and cavitation. Spinal cord injury is a common clinical severe central nervous system injury, which can lead to partial or complete motor, sensory and autonomic dysfunction below the injury plane, causing great physical and mental pain and heavy economic burden to patients. The consequences of spinal cord injury are very serious. Many patients with high cervical spinal cord injury die from central respiratory and cardiac failure or suffer from lifelong quadriplegia. Even patients with thoracic spinal cord injury often suffer from severe disabilities such as paralysis of both lower limbs, incontinence, and loss of sexual function, which brings serious burdens to family members and society. In addition, craniocerebral trauma, tumors, and strokes often cause brain tissue defects, and the number of such patients is also very large.

[0008] Even today, with the rapid progress of medical technology, complex treatments and intensive functional training may lead to mild recovery, but the treatment process is slow, the effect is limited, and depends on the severity of the spinal cord injury. The main reason is that the neurons of adult mammals lack the ability to regenerate. There is still a lack of effective treatments that can achieve complete functional recovery.

[0009] Cell therapy may be a more promising treatment for spinal cord injury. Currently, cell therapy for spinal cord injury mainly focuses on stem cell therapy. Since bone marrow mesenchymal stem cells can regulate immune response in the acute phase of spinal cord injury, it is suggested that neural stem cells can promote the repair of neurons and the regrowth of axons. Since the 1980s, foreign scholars have discovered that neural stem cells exist in the central nervous system, which can differentiate into mature neural tissue cells such as neurons, astrocytes, and oligodendrocytes under in vitro culture conditions, which brings hope for the treatment of central nervous system injuries.

[0010] The isolation, culture, differentiation, induction and transplantation of NSCs will provide important experimental basis for the treatment of central nervous system diseases such as spinal cord injury and Parkinson's disease. However, the existing in vitro induction and culture methods of neural stem cells, especially the methods for preparing neural stem cells with normal differentiation function, are still insufficient. Summary of the invention

[0011] The purpose of the present invention is to provide a method for inducing neural stem cells in view of the problem that the methods for separating and preparing neural stem cells in the prior art are limited.

[0012] Specifically, the present invention discloses a method for inducing neural stem cells from mesenchymal umbilical cord stem cells.

[0013] In order to solve the above technical problems, the present invention adopts the following technical solutions: The present invention discloses a method for inducing neural stem cells from umbilical cord mesenchymal stem cells, comprising the following steps: 1) Umbilical cord mesenchymal stem cells were obtained, digested with trypsin, and then washed with sterile PBS; 2) Collect the cells in step 1) and inoculate them in 10% FBS-DMEM / F12 medium for pre-culture for 24 hours; 3) Replace with induction medium and continue culturing, replenish the induction medium every 3-4 days, and culture for 7 days. The induction medium is prepared as follows: 2-8g ITS, 5-20μg bFGF, 5-20μg EGF and 5-20mL B27 are dissolved in 480-495mL DMEM / F12 medium.

[0014] Preferably, the trypsin digestion treatment is performed using 0.2% trypsin in PBS at room temperature for 15 min-60 min.

[0015] Preferably, the induction medium is prepared by dissolving 5 g ITS, 10 μg bFGF, 15 μg EGF and 10 mL B27 in 490 mL DMEM / F12 medium.

[0016] The present invention discloses a method for preparing neuronal cells and astrocytes, comprising the following steps: 1) Umbilical cord mesenchymal stem cells were obtained, digested with trypsin, and then washed with sterile PBS; 2) Collect the cells from step 1) and inoculate them in 10% FBS-DMEM / F12 medium for pre-culture for 24 hours; 3) Replace with induction medium and continue culturing, replenish induction medium every 3-4 days, and culture for 7 days. The induction medium is prepared as follows: 2-8g ITS, 5-20μg bFGF, 5-20μg EGF and 5-20mL B27 are dissolved in 480-495mL DMEM / F12 medium; 4) Collecting the neural stem cells in step 3) and performing differentiation culture using a differentiation medium, wherein the differentiation medium is prepared by adding 5%-15% cerebrospinal fluid to the induction medium described in step 3).

[0017] Preferably, the amount of cerebrospinal fluid added is 10%.

[0018] Preferably, the induction medium is prepared by dissolving 5 g ITS, 10 μg bFGF, 15 μg EGF and 10 mL B27 in 490 mL DMEM / F12 medium.

[0019] The invention discloses a culture medium for preparing neuronal cells and astrocytes. The culture medium comprises a culture medium 1 and a culture medium 2. The culture medium 1 is an induction culture medium, and the induction culture medium is prepared by the following method: 2-8g ITS, 5-20μg bFGF, 5-20μg EGF and 5-20mL B27 are dissolved in 495-480mL DMEM / F12 culture medium; the culture medium 2 is a differentiation culture medium.

[0020] Preferably, the differentiation medium is obtained by adding cerebrospinal fluid to the induction medium.

[0021] Preferably, the volume of cerebrospinal fluid added is 5%-15%.

[0022] The invention discloses the use of the culture medium in preparing neuronal cells and astrocytes.

[0023] The present invention uses an induction medium and a differentiation medium to prepare umbilical cord mesenchymal stem cells into neuronal cells and astrocytes through neural stem cell differentiation. The present invention further finds that a differentiation medium containing cerebrospinal fluid can improve the differentiation level of neural stem cells. By establishing a rat spinal cord injury model, it is verified that the prepared neural stem cells contribute to the recovery of rats with spinal cord injury and have a certain therapeutic effect on nervous system diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Umbilical cord mesenchymal stem cell marker detection results.

[0025] Figure 2 Identification of neural stem cells by nestin immunostaining.

[0026] Figure 3 Differentiation of neural stem cells in culture medium 3 produces neuronal cells.

[0027] Figure 4 NSE immunofluorescence staining of neural stem cell differentiation Figure 4 A is the cell nucleus stained with HOECHST33258, Figure 4 B is NSE positive cells. Figure 4 C is Figure 4 A and Figure 4 B Fusion diagram.

[0028] Figure 5 GFAP immunofluorescence staining of neural stem cell differentiation Figure 5 A is the cell nucleus stained with HOECHST33258, Figure 5 B is GFAP positive cells, Figure 5 C is Figure 5 A and Figure 5 B Fusion diagram.

[0029] Figure 6 Evaluation of BBB motility in rats of different treatment groups.

[0030] Figure 7 HE staining of paraffin sections of rat spinal cord specimens. DETAILED DESCRIPTION

[0031] The specific implementation of the present invention is described in detail below in conjunction with the accompanying drawings. It should be understood that the specific implementation described here is only used to illustrate and explain the present invention, and is not used to limit the present invention.

[0032] Example 1 Isolation and identification of human umbilical cord mesenchymal stem cells With the informed consent of the pregnant woman, the umbilical cord of a healthy full-term infant was collected under sterile conditions. The cord was repeatedly rinsed with PBS (containing penicillin and streptomycin) until clean, the arteries and veins and the outer membrane of the umbilical cord were removed, the Wharton's jelly was peeled off, and the cord was cut into uniform pieces with a size of about 3.5 mm, and spread flat in the culture bottle.

[0033] Add 10% FBS-DMEM / F12 culture medium at a volume of 0.5 mL / cm umbilical cord and culture in an incubator at 37°C and 5% CO2. Replace half of the medium after 48 hours and then replace the full medium every 3 days. Observe cell fusion under an inverted microscope and perform subculture when cell fusion reaches about 90%. Discard the culture medium in the culture flask, wash the cells twice with 5mL PBS, add 3mL 0.25% trypsin solution, and gently shake until the trypsin completely covers the cell surface. After the cells are completely digested (under the microscope, the cells completely fall off from the bottom of the bottle and appear round), add 15mL of 10% FBS-DMEM / F12 culture medium to terminate the digestion, pipette and blow evenly, then transfer to a 50mL centrifuge tube, centrifuge at 300g / 10min, discard the supernatant, add an appropriate amount of 10% FBS-DMEM / F12 culture medium, blow evenly, transfer to a new 175T culture flask for continued culture, and record it as P1 generation. When the cells fill the culture flask, continue to repeat the above steps to pass to the third generation, which is marked as P3 generation, and observe the cell morphology under the microscope.

[0034] Flow cytometry (FCM) is a technology that performs multi-parameter, rapid quantitative analysis and sorting of cells or other biological particles arranged in a single file in a liquid flow at the cellular molecular level.

[0035] 10 mL of P3 cells with good growth status were selected, the culture medium was discarded, and after centrifugation, they were washed 3 times with 1×PBS buffer, and digested with 5 mL of preheated trypsin cell digestion solution (0.25% trypsin solution). After centrifugation, the supernatant was removed, and 1×PBS buffer was used to wash twice. 1.5 mL of PBS solution was resuspended to make a uniform single cell suspension. Under light-proof conditions, fluoresceine isothiocyanate (FITC)-labeled anti-human CD34, CD45 and HLA-DR monoclonal antibodies and phycoerythrin (PE)-labeled anti-human CD73, CD105, CD90 monoclonal antibodies were added, and negative controls were set up. After incubation at 37°C in the dark for 30 min, flow cytometry was used for detection. The test results are shown in the table. Figure 1 .

[0036] Example 2 Differentiation and identification of human umbilical cord mesenchymal stem cells into neural stem cells P3 human umbilical cord mesenchymal stem cells were obtained, treated with 0.2% trypsin in PBS at room temperature for 30 min for digestion, washed twice with sterile PBS, inoculated into culture bottles, and cultured in 10% FBS-DMEM / F12 medium for 24 hours before replacement with NSCs induction medium (500 mL DMEM / F12 containing 5 g ITS (insulin-transferrin-selenium), 10 μg bFGF, 15 μg EGF and 10 mL B27). Culture was continued, with fluid replenishment every 3 days for about 7 days in an incubator at 37°C and 5% CO2.

[0037] The differentiated NSCs spheres were adhered to coverslips treated with poly-lysine, and after fixation, the expression of the NSCs marker nestin was detected by immunocytochemical staining. After the cells were fixed with 4% paraformaldehyde for 15 minutes, they were washed three times with 0.1M PBS for 4 minutes each time. 0.5% Triton X-100 was used for 10 minutes, and the cells were blocked with 1% BSA for 30 minutes at 37°C. Rabbit anti-human nestin (1:300) was added, incubated at 37°C for 1.5 hours, and washed three times with 0.1M PBS for 5 minutes each time. Labeled secondary antibody goat anti-rabbit (1:500) was added, incubated at 37°C for 40 minutes, and washed three times with 0.1M PBS for 5 minutes each time. The slides were sealed with glycerol buffer, observed under a fluorescence microscope, and photographed. Positive nestin staining indicates that the differentiated proliferating spheres are neural stem cells. For test results, see Figure 2 .

[0038] Example 3 Cultivation and differentiation of neural stem cells Cerebrospinal fluid was obtained from patients with brain trauma in the Department of Neurosurgery, and the family members gave informed consent. The fluid was collected in sterile centrifuge tubes and stored in a -20°C refrigerator. No abnormalities were found in biochemical, routine, and bacterial tests.

[0039] The neural stem cells cultured for 7 days were cultured for differentiation. They were cultured in 4 different culture media in an incubator at 37°C and 5% CO2. Culture media 1 was the induction culture media, culture media 2 was the induction culture media + 5% cerebrospinal fluid, culture media 3 was the induction culture media + 10% cerebrospinal fluid, and culture media 4 was the induction culture media + 15% cerebrospinal fluid. The cultured differentiated cells were observed under a microscope. The results are shown in Figure 3 .

[0040] Immunofluorescence identification of NSCs differentiation by different culture media. NSCs were induced to differentiate on the 7th day, and the fluorescent dye Hoechst33258 was added to the culture medium to a concentration of 2μg / mL. After continued culture at 37°C and 5% CO2 for 30 min, the slides were removed and rabbit anti-NSE (neuron-specific enolase) and rabbit anti-GFAP (glial fibrillary acidic protein) (1:120) were used as primary antibodies to mark neurons and astrocytes, respectively, on different cell slides. In the negative control group, PBS was used instead of the primary antibody, and the secondary antibody was RBITC-labeled goat anti-rabbit IgG. Immunofluorescence detection of NSCs differentiation ability was performed. Five fields of view were randomly selected in both the experimental group and the normal group to count the percentages of differentiated neurons and astrocytes, and then the average values ​​were calculated to compare the differences in the differentiation ability of NSCs in the differentiation groups induced by different culture medium conditions. For the results of fluorescence detection, see Figure 4 and Figure 5 The detection and counting results of each culture medium are shown in Tables 1 and 2.

[0041] Table 1: Determination of NSE positive expression rate in NSES-induced differentiation groups under different culture medium conditions (%) project Horizon 1 Horizon 2 Horizon 3 Horizon 4 Horizon 5 average value Medium 1 26.32 31.55 24.66 35.13 28.56 29.24 Medium 2 37.55 42.36 45.13 40.28 48.54 42.77 Medium 3 62.31 72.14 68.56 63.27 65.28 66.32 Medium 4 51.26 55.47 60.18 57.63 53.62 55.63 It can be seen that adding cerebrospinal fluid to the induction medium can provide differentiation of neural stem cells into neurons.

[0042] Table 2: Determination of GFAP positive expression rate in NSES-induced differentiation group under different culture medium conditions (%) project Horizon 1 Horizon 2 Horizon 3 Horizon 4 Horizon 5 average value Medium 1 10.23 9.56 12.31 11.57 10.66 10.87 Medium 2 16.32 14.27 18.52 17.18 15.33 16.32 Medium 3 26.42 28.55 26.38 30.26 25.34 27.39 Medium 4 20.34 22.53 19.67 18.76 23.21 20.90 It can be seen that adding cerebrospinal fluid to the induction medium can provide neural stem cells with differentiation into astrocytes. From the test results, adding 10% cerebrospinal fluid to the induction medium can induce neural stem cells to differentiate into more neurons and astrocytes.

[0043] Example 4 Construction and grouping of spinal cord injury rat model Fifty SD rats were selected and anesthetized with 8% chloral hydrate intraperitoneal injection. The rats were fixed on the operating table in prone position, with their limbs and lower collar fixed, and the 9th-10th thoracic segments were carefully located. The skin of the surgical area was disinfected with 2.5% iodine tincture, and then deiodinated twice with 75% alcohol.

[0044] A disposable surgical towel was laid, and the midline approach was taken from the waist and back. The skin and subcutaneous tissue were cut layer by layer to the spine under sterile conditions to fully expose the 9th thoracic segment. A laminectomy was performed at the level of the 9th segment, and the dura mater was preserved intact. A rat free fall injury model device was used to drop a 10g weight from a height of 25 cm to hit the exposed side of the spine to obtain the SCI rat model (Allen's impact method). The inclusion criteria were that the rat's hind limbs suddenly contracted violently and the tail rotated a circle.

[0045] Completely stop bleeding, then suture the muscles and skin layer by layer. The sham operation only cuts the vertebral plate, without damaging the spinal cord. After the operation, the animals are fed in a clean cage. Penicillin is injected intramuscularly for three consecutive days to prevent infection. The abdomen is squeezed to urinate and defecate once a day. Turn over regularly every day to prevent bedsores.

[0046] One week after the rat model was successfully prepared, rats in good survival condition were selected and divided into the following groups using a completely random number table method: (induction medium + 10% cerebrospinal fluid) treatment group (Group 1: 10 rats), induction medium treatment group (Group 2: 10 rats), normal saline treatment group (Group 3: 10 rats), and sham operation control group (Group 4: 10 rats).

[0047] Example 5 The therapeutic effect of induced neural stem cells on rats with spinal cord injury Collection of neural stem cells: The non-adherent cells in the culture medium were removed and washed with 5 mL of sterile PBS for 3 times to remove the culture medium components. The cells were digested with a sterile PBS solution containing 0.02% EDTA and 0.25% trypsin at room temperature for about 30 minutes, and the supernatant was removed by centrifugation. The cells were resuspended in the induction medium and 10% cerebrospinal fluid as one group (induction medium + 10% cerebrospinal fluid: Group 1), and the induction medium was used as the second group (induction medium treatment group, Group 2). The concentration of neural stem cells was about 10 7 / mL concentration of cell suspension.

[0048] Intrathecal injection of neural stem cells in vitro: On the 7th day after SCI surgery, intrathecal injection of neural stem cells was performed. The specific operation was to slowly insert the Hamilton microsyringe (25µL) into the spine through the L4-L5 intervertebral space and directly reach the subarachnoid space under isoflurane anesthesia (oxygen content 2%). When the needle punctures the subarachnoid space, the rat's tail can be seen swinging. Inject 20µL of cell suspension for 5 minutes at a speed of 4µL / min. Repeat the intrathecal injection once every 48 hours.

[0049] The normal saline group was injected with an equal amount of normal saline (Group 3), and the sham operation control group (Group 4) was not treated. Motor function was assessed according to BBB movement and evaluated one week after the sheath injection. The average evaluation value of each group of mice was taken as the evaluation result, see Figure 6 .

[0050] Eight weeks after the sheath transplantation, the rats were killed, and spinal cord specimens were obtained by perfusion, made into paraffin sections, and HE staining and cavity observation were performed. Among them, the spinal cord tissue of the damaged segment in the normal saline group (Group 3) was incomplete, with many gaps, and no granulation tissue or other tissue filling. It was completely a cavity left after mechanical injury. The damaged spinal cord tissue of the (induction medium + 10% cerebrospinal fluid) treatment group and the induction medium treatment group recovered, the location of the cavity was filled with inflammatory cells and granulation tissue, and the cavity was significantly reduced. The (induction medium + 10% cerebrospinal fluid) treatment group had fewer cavities than the induction medium treatment group, see Figure 7 .

[0051] The present invention illustrates the process method of the present invention through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned process steps, that is, it does not mean that the present invention must rely on the above-mentioned process steps to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of the raw materials selected by the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

Claims

1. A method for inducing neural stem cells from umbilical cord mesenchymal stem cells, characterized in that: The steps include: 1) Umbilical cord mesenchymal stem cells were obtained, digested with trypsin, and washed with sterile PBS; 2) Collect the cells in step 1) and inoculate them in 10% FBS-DMEM / F12 medium for pre-culture for 24 hours; 3) Replace with induction medium and continue culturing, replenish the induction medium every 3-4 days, and culture for 7 days. The induction medium is prepared as follows: 2-8g ITS, 5-20μg bFGF, 5-20μg EGF and 5-20mL B27 are dissolved in 480-495mL DMEM / F12 medium.

2. The preparation method according to claim 1, characterized in that: The trypsin digestion treatment is performed by using 0.2% trypsin in PBS at room temperature for 15 min-60 min.

3. The preparation method according to claim 1, characterized in that: The preparation method of the induction medium is as follows: 5 g ITS, 10 μg bFGF, 15 μg EGF and 10 mL B27 are dissolved in 490 mL DMEM / F12 medium.

4. A method for preparing neuronal cells and astrocytes, characterized in that: The steps include: 1) Umbilical cord mesenchymal stem cells were obtained, digested with trypsin, and washed with sterile PBS; 2) Collect the cells from step 1) and inoculate them in 10% FBS-DMEM / F12 medium for pre-culture for 24 hours; 3) Replace with induction medium and continue culturing, replenish induction medium every 3-4 days, and culture for 7 days. The induction medium is prepared as follows: 2-8g ITS, 5-20μg bFGF, 5-20μg EGF and 5-20mL B27 are dissolved in 480-495mL DMEM / F12 medium; 4) Collecting the neural stem cells in step 3) and performing differentiation culture using a differentiation medium, wherein the differentiation medium is prepared by adding 5%-15% cerebrospinal fluid to the induction medium described in step 3).

5. The method according to claim 4, characterized in that The amount of cerebrospinal fluid added was 10%.

6. The method according to claim 4, characterized in that The induction medium is prepared by dissolving 5 g ITS, 10 μg bFGF, 15 μg EGF and 10 mL B27 in 490 mL DMEM / F12 medium.

7. A method for preparing a culture medium for neurons and astrocytes, wherein the culture medium comprises a culture medium 1 and a culture medium 2, wherein: The culture medium 1 is an induction culture medium, which is prepared by the following method: 2-8 g ITS, 5-20 μg bFGF, 5-20 μg EGF and 5-20 mL B27 are dissolved in 495-480 mL DMEM / F12 culture medium; the culture medium 2 is a differentiation culture medium.

8. The culture medium according to claim 7, characterized in that The differentiation medium is obtained by adding cerebrospinal fluid to the induction medium.

9. The culture medium according to claim 7, characterized in that The volume of cerebrospinal fluid added is 5%-15%.

10. Use of the culture medium according to any one of claims 7 to 9 in preparing neuronal cells and astrocytes.

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