Method for induced differentiation of hair follicle stem cells into neural stem cells

Abstract

A method for induced differentiation of hair follicle stem cells into neural stem cells, comprising: inoculating hair follicle stem cells into a complete culture solution, culturing for 1-2 days, then using a differentiation culture solution A for induced culture for 2-5 days, then using a differentiation culture solution B for secondary induced culture for 3-5 days, and using a differentiation culture solution C for ternary induced culture for 4-6 days to obtain target neural stem cells, wherein before the ternary induced culture, a magnetic field treatment is carried out for 10-30 s with a magnetic field intensity of 2-8 mT. By providing a mode of stage-based induced differentiation culture, the usage amounts of differentiation culture medium components are reduced; in addition, by applying a low-intensity magnetic field, the hair follicle stem cells can be induced and differentiated into neural stem cells. The induction time is relatively short, the induced differentiation is uniform, the obtained hair follicle-derived neural stem cells have good activity, the proliferative activity of fibroblasts can be effectively promoted, and a certain foundation can be provided for tissue repair and remodeling.

Description

A method for inducing hair follicle stem cells to differentiate into neural stem cells

[0001] Technical Field

[0002] The present invention relates to the technical field of cell culture and differentiation, and in particular to a method for inducing hair follicle stem cells to differentiate into neural stem cells.

[0003] Background Art

[0004] Nerve injury refers to the destruction of the integrity of nerve tissue and the impairment of its function. There are many causes of nerve injury, such as trauma, disease, tumors, genetics, infection, etc. Treatment methods mainly include surgery, physical therapy, medication, etc. However, as people's understanding of nerve injury deepens, exploring methods of nerve regeneration and repair has become a hot topic in the medical community. Neuroregeneration refers to the process of re-growth and restoration of function of damaged nerve tissue after the nervous system is damaged, through its own repair ability or external intervention; nerve repair refers to the use of technical means such as stem cells, biomaterials, and gene therapy to promote the repair and regeneration of damaged nerves.

[0005] For example, regarding the treatment of multiple sclerosis: Multiple sclerosis (MS) is an autoimmune disease characterized by inflammatory demyelinating lesions in the white matter of the central nervous system. The most common sites of involvement are the periventricular white matter, optic nerves, spinal cord, brainstem, and cerebellum. The main clinical features are scattered, multifocal lesions in the white matter of the central nervous system, with remissions and relapses over the course of the disease, and spatial and temporal variations in symptoms and signs. Violaine K. et al. reported a method for treating MS using neural stem cells derived from mesenchymal stem cells. Angela Genchi et al. reported a method for treating MS using embryonic neural stem cells. Regarding the treatment of stroke: Cerebral stroke, also known as "stroke" or "cerebral vascular accident" (CVA), is an acute cerebrovascular disease caused by sudden rupture of a cerebral blood vessel or blockage of a vascular blockage, resulting in brain damage. Keith W. Muir et al. reported a method for using embryonic neural stem cells to treat brain damage caused by stroke. Regarding Parkinson's disease treatment: Parkinson's disease (PD) is a common neurodegenerative disorder that is prevalent in the elderly. Its clinical manifestations primarily include resting tremor, bradykinesia, muscle rigidity, and postural and gait disorders. Patients may also experience non-motor symptoms such as depression, constipation, and sleep disturbances. I. Madrazo et al. reported a method for using embryonic neural stem cells to treat PD. Regarding Alzheimer's disease treatment: Alzheimer's disease (AD) is a progressive neurodegenerative disease with an insidious onset. Clinically, it is characterized by global dementia symptoms such as memory impairment, aphasia, apraxia, agnosia, impaired visual-spatial skills, executive dysfunction, and personality and behavioral changes. Sofia Essayan-Perez et al. reported a review of research using neural cells derived from induced pluripotent stem cells to treat AD.

[0006] Currently, neural stem cells used in research are mostly derived from the patient's own induced pluripotent stem cells (iPS). These have drawbacks such as lengthy preparation cycles, low safety, low differentiation efficiency, and high production costs. Some studies have also used neural stem cells derived from umbilical cord-derived mesenchymal stem cells, but as these cells are allogeneic, they lack long-term survival in the patient's body. Furthermore, embryonic-derived neural stem cells carry a high risk of tumorigenesis and raise ethical concerns.

[0007] Summary of the Invention

[0008] In view of this, the purpose of the present invention is to propose a method for inducing the differentiation of hair follicle stem cells into neural stem cells. To address the above problems, the present invention provides a method for using a patient's own hair follicle stem cells to differentiate into neural stem cells. The method has the advantages of easy material acquisition, minimal damage to the donor, short preparation cycle, high safety, no rejection reaction, low preparation cost, and no ethical controversy.

[0009] Hair follicle stem cells are adult stem cells found in the outer root sheath of the human hair follicle. While quiescent in vivo, they exhibit remarkable proliferation in vitro. Studies have shown that hair follicle stem cells possess multipotential differentiation potential, capable of developing into epidermal, hair follicle, sebaceous gland, and neural cells. They are widely used in research areas such as hair growth, wound repair, and damaged nerve repair. Neural stem cells derived from hair follicle stem cells are autologous, avoiding rejection and ethical concerns. They will become important research materials in the future for neural repair and regeneration.

[0010] The technical solution of the present invention is achieved as follows:

[0011] A method for inducing hair follicle stem cells to differentiate into neural stem cells, comprising the following steps:

[0012] S1 raw material: Hair follicles were harvested, digested, and then complete culture medium was added to terminate the digestion. The cells were centrifuged, and then complete culture medium was added again to resuspend the cells. The cells were subcultured to obtain hair follicle stem cells.

[0013] S2 induction: Hair follicle stem cells are prepared into a hair follicle stem cell suspension and inoculated into complete culture medium. After 1-2 days of culture, differentiation culture medium A is used for induction culture. After 2-5 days of induction culture, differentiation culture medium B is used for secondary induction culture. After 3-5 days of secondary induction culture, differentiation culture medium C is used for tertiary induction culture. Before the tertiary induction culture, magnetic field treatment is performed at a magnetic field strength of 2-8 mT for 10-30 seconds. After tertiary induction culture for 4-6 days, the target neural stem cells are obtained.

[0014] Furthermore, the components of the differentiation culture medium A include epidermal growth factor;

[0015] The components of the differentiation culture medium B include epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), B27 additive and glutamine;

[0016] The components of the differentiation culture medium C include epidermal growth factor, glutamine and crocin.

[0017] Furthermore, the components of the differentiation culture medium A include 0.1-30 ng / mL of epidermal growth factor;

[0018] The components of the differentiation culture medium B include 0.1-30 ng / mL of epidermal growth factor, 0.1-50 ng / mL of basic fibroblast growth factor, 0.5-5 nM of B27 additive, and 0.5-5 mM of glutamine;

[0019] The components of the differentiation culture medium C include 0.1-30 ng / mL of epidermal growth factor, 0.5-5 mM of glutamine, and 0.1-5 μg / mL of crocin.

[0020] Furthermore, the components of the differentiation culture medium A include 10-15 ng / mL of epidermal growth factor;

[0021] The components of the differentiation culture medium B include 5-10 ng / mL of epidermal growth factor, 5-10 ng / mL of basic fibroblast growth factor, 1-1.5 nM of B27 additive, and 0.5-1 mM of glutamine;

[0022] The components of the differentiation culture medium C include 5-8 ng / mL of epidermal growth factor, 0.5-1 mM of glutamine, and 1-3 μg / mL of crocin.

[0023] Furthermore, the components of the complete culture medium include 20-50 ng / mL of basic fibroblast growth factor and 1 wt%-4 wt% of serum substitute (Ultroser G).

[0024] Furthermore, in step S2, the hair follicle stem cells are seeded at a density of 500 to 6000 cells / cm 2 .

[0025] Furthermore, in step S1, the hair follicle stem cells are hair follicle stem cells that have been subcultured to passages 2 to 4.

[0026] Furthermore, in step S1, the digestion includes: adding collagenase to the hair follicles and digesting at 35-38° C. for 10-120 min.

[0027] Furthermore, the amount of collagenase added is 50-500 μg / mL.

[0028] The present invention provides an application of inducing hair follicle stem cells to differentiate into neural stem cells. The application of the neural stem cells in promoting the proliferation activity of fibroblasts can provide a certain basis for tissue repair and remodeling.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] The present invention utilizes a staged induction differentiation culture model, combined with a simple and convenient external application of a low-intensity magnetic field, to induce the differentiation of hair follicle stem cells into neural stem cells. This results in a short induction time, uniform differentiation, and high activity and induction rates of the resulting hair follicle-derived neural stem cells. Furthermore, the present invention reduces the amount of differentiation medium components used during the induction differentiation of hair follicle stem cells into neural stem cells, thereby obtaining hair follicle-derived neural stem cells while maintaining their activity.

[0031] In addition, the hair follicle-derived stem cells provided by the present invention can effectively promote the proliferation activity of fibroblasts and provide a certain basis for tissue repair and remodeling.

[0032] BRIEF DESCRIPTION OF THE DRAWINGS

[0033] FIG1 is a diagram of primary inoculated hair follicle stem cells according to Example 1 of the present invention;

[0034] FIG2 is a diagram of hair follicle stem cells before passage according to Example 1 of the present invention;

[0035] FIG3 is a diagram of neural stem cells induced from hair follicle stem cells according to Example 1 of the present invention;

[0036] Figure 4 is an immunofluorescence image of Nestin protein detection, where the left is a negative control and the right is hair follicle-derived neural stem cells.

[0037] DETAILED DESCRIPTION

[0038] In order to better understand the technical content of the present invention, specific examples are provided below to further illustrate the present invention.

[0039] Unless otherwise specified, the experimental methods used in the examples of the present invention are all conventional methods.

[0040] Unless otherwise specified, the materials, reagents, etc. used in the examples of the present invention can be obtained from commercial sources.

[0041] Example 1

[0042] (1) Isolation and culture of hair follicle stem cells: Hair follicles need to be extracted in the operating room under local anesthesia using a hair follicle extractor. The extracted hair follicles need to be stored in normal saline containing penicillin, streptomycin, and amphotericin B and sent to the laboratory for preparation within 1 hour;

[0043] (2) Prepare complete culture medium:

[0044]

[0045] (3) After washing the hair follicles twice with saline containing antibiotics, they were digested with 250 μg / mL collagenase at 37°C for 60 min. The cells were then centrifuged at 300 g for 5 min. After centrifugation, the cells were resuspended in complete culture medium and inoculated into T25 flasks. The cells were passaged when the confluence reached approximately 85%.

[0046] (4) Cell passaging: Cells are passaged when their confluence reaches approximately 85%;

[0047] Cell passaging quality standards:

[0048]

[0049] (5) Cell cryopreservation: When the cell confluence reaches about 85%, the cells are frozen and sent for inspection for sterility, mycoplasma, endotoxin, and cell phenotype. Cells that meet the quality requirements can be used for subsequent differentiation experiments;

[0050] Hair follicle stem cell cryopreservation quality standards:

[0051]

[0052] Flow cytometry results of P2 hair follicle stem cells:

[0053]

[0054] (6) Preparation of differentiation culture medium:

[0055] Differentiation medium 1:

[0056]

[0057] Differentiation medium 2:

[0058]

[0059] Differentiation medium 3:

[0060]

[0061] (7) Recovery of frozen hair follicle stem cells: Remove the cells from the liquid nitrogen tank and quickly thaw them in a 37°C water bath. Wash the cell suspension once with 10 times the volume of normal saline, count the cells, centrifuge, discard the supernatant, take the precipitate, and add normal saline to prepare the hair follicle stem cell suspension.

[0062] (8) The hair follicle stem cell suspension was inoculated into complete culture medium at a density of 2000 cells / cm 2The next day, the medium was fully replaced. After 3 days of culture in Differentiation Medium 1, the medium was fully replaced with Differentiation Medium 2. After 4 days of culture, the medium was fully replaced and Differentiation Medium 3 was added. The cells were then subjected to a low-intensity magnetic field at 5 mT for 20 seconds and cultured for another 5 days. In Differentiation Medium 2, the cells differentiated into neural stem cells and proliferated rapidly. The cells were harvested when the cell clusters expanded to a point where light could not penetrate the central region. The harvested cells were plated in a 6-well plate and immunofluorescence was used to detect the neural stem cell marker Nestin protein, identifying hair follicle-derived neural stem cells.

[0063] Nestin protein detection standard:

[0064]

[0065] Example 2

[0066] (1) Isolation and culture of hair follicle stem cells: Hair follicles need to be extracted in the operating room under local anesthesia using a hair follicle extractor. The extracted hair follicles need to be stored in normal saline containing penicillin, streptomycin, and amphotericin B and sent to the laboratory for preparation within 1 hour;

[0067] (2) Prepare complete culture medium:

[0068]

[0069] (3) After washing the hair follicles twice with saline containing antibiotics, they were digested with 250 μg / mL collagenase at 37°C for 60 min. The cells were then centrifuged at 300 g for 5 min. After centrifugation, the cells were resuspended in complete culture medium and inoculated into T25 flasks. The cells were passaged when the confluence reached approximately 85%.

[0070] (4) Cell passaging: According to the cell passaging quality standards of Example 1, cells were passaged when the confluence reached about 85%;

[0071] (5) Cell cryopreservation: According to the hair follicle stem cell cryopreservation quality standards of Example 1, cells are cryopreserved when the cell confluence reaches about 85%, and when the cell confluence reaches 80% to 90%. The cells are then tested for sterility, mycoplasma, endotoxin, and cell phenotype. Cells that meet the quality requirements can be used for subsequent differentiation experiments;

[0072] (6) Preparation of differentiation culture medium:

[0073] Differentiation medium 1:

[0074]

[0075] Differentiation medium 2:

[0076]

[0077] Differentiation medium 3:

[0078]

[0079] (7) Recovery of frozen hair follicle stem cells: Remove the cells from the liquid nitrogen tank and quickly thaw them in a 37°C water bath. Wash the cell suspension once with 10 times the volume of normal saline, count the cells, centrifuge, discard the supernatant, take the precipitate, and add normal saline to prepare the hair follicle stem cell suspension.

[0080] (8) The hair follicle stem cell suspension was inoculated into complete culture medium at a density of 2000 cells / cm 2 The next day, the medium was fully replaced. After 3 days of culture in Differentiation Medium 1, the medium was fully replaced with Differentiation Medium 2. After 4 days of culture, the medium was fully replaced and Differentiation Medium 2 was added, along with crocin (final concentration 3 μg / mL). The cells were subjected to a low-intensity magnetic field at 5 mT for 20 s and cultured for another 5 days. In Differentiation Medium 2, the cells differentiated into neural stem cells and proliferated rapidly. The cells were harvested when the cell clusters expanded to a point where light could not penetrate the central region. The harvested cells were plated in 6-well plates and immunofluorescence was used to detect the neural stem cell marker Nestin protein, identifying hair follicle-derived neural stem cells.

[0081] Example 3

[0082] The difference between this embodiment and embodiment 1 is that, in step (6), crocin is replaced with kaempferol in the components of differentiation culture medium 3. The specific steps are as follows:

[0083] (6) Preparation of differentiation culture medium:

[0084] Differentiation medium 1:

[0085]

[0086] Differentiation medium 2:

[0087]

[0088] Differentiation medium 3:

[0089]

[0090] (7) Recovery of frozen hair follicle stem cells: Remove the cells from the liquid nitrogen tank and quickly thaw them in a 37°C water bath. Wash the cell suspension once with 10 times the volume of normal saline, count the cells, centrifuge, discard the supernatant, take the precipitate, and add normal saline to prepare the hair follicle stem cell suspension.

[0091] (8) The hair follicle stem cell suspension was inoculated into complete culture medium at a density of 2000 cells / cm 2 The next day, the medium was fully replaced. After culturing for 3 days with differentiation medium 1, the medium was fully replaced again with differentiation medium 2. After culturing for 4 days, the medium was fully replaced and differentiation medium 3 was added. The cells were treated with a low-intensity magnetic field at 5 mT for 20 s and then cultured for another 5 days. The harvested cells were plated in a 6-well plate and the neural stem cell marker Nestin protein was detected by immunofluorescence to obtain hair follicle-derived neural stem cells. The remaining steps (1) to (5) were the same as in Example 1.

[0092] Example 4

[0093] The difference between this embodiment and embodiment 1 is that the components of differentiation culture medium 2 and 3 in step (6) are different. The specific steps are as follows:

[0094] (6) Preparation of differentiation culture medium:

[0095] Differentiation medium 1:

[0096]

[0097] Differentiation medium 2:

[0098]

[0099] Differentiation medium 3:

[0100]

[0101] (7) Recovery of frozen hair follicle stem cells: Remove the cells from the liquid nitrogen tank and quickly thaw them in a 37°C water bath. Wash the cell suspension once with 10 times the volume of normal saline, count the cells, centrifuge, discard the supernatant, take the precipitate, and add normal saline to prepare the hair follicle stem cell suspension.

[0102] (8) The hair follicle stem cell suspension was inoculated into complete culture medium at a density of 2000 cells / cm 2 The next day, the medium was fully replaced. After culturing for 3 days with differentiation medium 1, the medium was fully replaced again with differentiation medium 2. After culturing for 4 days, the medium was fully replaced and differentiation medium 3 was added. The cells were treated with a low-intensity magnetic field at 5 mT for 20 s and then cultured for another 5 days. The harvested cells were plated in a 6-well plate and the neural stem cell marker Nestin protein was detected by immunofluorescence to obtain hair follicle-derived neural stem cells. The remaining steps (1) to (5) were the same as in Example 1.

[0103] Example 5

[0104] The difference between this embodiment and embodiment 1 is that the composition of the differentiation culture medium in step (6) is different, and the induction method in step (8) is different. The specific steps are as follows:

[0105] (6) Preparation of differentiation culture medium:

[0106] Differentiation medium 1:

[0107]

[0108] Differentiation medium 2:

[0109]

[0110] (7) Recovery of frozen hair follicle stem cells: Remove the cells from the liquid nitrogen tank and quickly thaw them in a 37°C water bath. Wash the cell suspension once with 10 times the volume of normal saline, count the cells, centrifuge, discard the supernatant, take the precipitate, and add normal saline to prepare the hair follicle stem cell suspension.

[0111] (8) The hair follicle stem cell suspension was inoculated into complete culture medium at a density of 2000 cells / cm 2 The next day, the medium was fully replaced. After culturing with differentiation medium 1 for 3 days, the medium was fully replaced again. Differentiation medium 1 was added and cultured for another 4 days. The medium was fully replaced and differentiation medium 2 was added. The cells were treated with a low-intensity magnetic field at 5 mT for 20 s and then cultured for another 5 days. The harvested cells were plated in a 6-well plate and the neural stem cell marker Nestin protein was detected by immunofluorescence to obtain hair follicle-derived neural stem cells. The remaining steps (1) to (5) were the same as in Example 1.

[0112] Comparative Example 1

[0113] The difference between this comparative example and Example 1 is that, during the differentiation and culture of hair follicle stem cells in step (8), the magnetic field treatment method is different. The specific step (8) is as follows:

[0114] (8) The hair follicle stem cell suspension was inoculated into complete culture medium at a density of 2000 cells / cm 2 The next day, the medium was fully replaced. After 3 days of culture using differentiation medium 1, the medium was fully replaced again with differentiation medium 2, and the cells were treated with a low-intensity magnetic field of 5 mT for 20 s. After 4 days of culture, the medium was fully replaced, differentiation medium 3 was added, and the cells were treated with a low-intensity magnetic field of 5 mT for 20 s, and then cultured for another 5 days. The harvested cells were plated in a 6-well plate, and the neural stem cell marker Nestin protein was detected by immunofluorescence to obtain hair follicle-derived neural stem cells. The remaining steps (1) to (7) were the same as in Example 1.

[0115] Comparative Example 2

[0116] The difference between this comparative example and Example 1 is that, during the differentiation and culture of hair follicle stem cells in step (8), no magnetic field treatment is performed. The specific step (8) is as follows:

[0117] (8) The hair follicle stem cell suspension was inoculated into complete culture medium at a density of 2000 cells / cm 2 The next day, the medium was fully replaced. After 3 days of culture using differentiation medium 1, the medium was fully replaced again with differentiation medium 2. The cells were treated with a low-intensity magnetic field at 5 mT for 20 s. After 4 days of culture, the medium was fully replaced and differentiation medium 3 was added. The cells were then cultured for another 5 days. The harvested cells were plated in a 6-well plate and the neural stem cell marker Nestin protein was detected by immunofluorescence to obtain hair follicle-derived neural stem cells. The remaining steps (1) to (7) were the same as in Example 1.

[0118]

[0119] Test indicators

[0120] The hair follicle-derived neural stem cells obtained by culture and differentiation were inoculated into 6-well plates. The culture morphology of the hair follicle-derived neural stem cells was observed under an optical microscope. Three fields of view were randomly selected for image acquisition. The diameter of the hair follicle-derived neural stem cells was analyzed using Image J software. The results are as follows:

[0121] Table 1

[0122]

[0123] The growth of neural stem cells shows an aggregated growth trend. Most neural stem cells aggregate to form spheres or beads. If the diameter is too large, the exchange efficiency of nutrients and gases between internal cells and the outside world will be reduced, which will affect cell activity and easily cause large-scale cell death as the culture time increases. The results in Table 1 above indicate that the diameters of the hair follicle-derived neural stem cells in Examples 1 and 2 were 135.6 μm and 131.7 μm, respectively, which are smaller than those in Comparative Examples 1 and 2. This indicates that during the differentiation culture process, the hair follicle stem cells in Examples 1 and 2 aggregated well, nutrients were sufficient, neurospheres formed quickly, and the internal and external cell activity of the neurospheres was well maintained. Compared with Comparative Examples 1 and 2, treatment with a certain magnetic field dispersed the cells and promoted the utilization efficiency of differentiation culture medium components to a certain extent, thereby improving the differentiation efficiency and cell activity of the hair follicle stem cells. However, premature application of the magnetic field affected the hair follicle stem cells in the early, rapidly dividing state and altered the culture environment, affecting the differentiation process of the hair follicle stem cells in the later stages. Compared with Examples 3 and 4, crocin reduced the degree of damage to the aggregated hair follicle stem cells, helping to maintain cellular homeostasis. The effect was more pronounced when crocin was added during the period of rapid differentiation of the hair follicle stem cells.

[0124]

[0125] In vitro testing

[0126] The CCK-8 method was used to detect the effect of hair follicle-derived stem cells on the proliferation activity of fibroblasts. Fibroblasts with a confluence of about 85% were activated and cultured, and the cell density was adjusted to 1.5×10 5 pieces / cm 2 , seeded into a 96-well culture plate, 200 μL per well, cultured until the cell confluence reached about 75%, the medium was aspirated, and after washing twice with DPBS, DMEM high-glucose medium without serum was added and starvation culture was performed for 24 h. The medium was aspirated, and after washing again twice with DPBS, 100 μL of DMEM high-glucose medium was added to each well (the hair follicle-derived stem cells of Example 1 were diluted to concentrations of 25%, 50%, and 75%, respectively, and then added to DMEM high-glucose medium and mixed). The cells were incubated for 24 h. CCK-8 reagent was added to each well, and the cells were incubated for another 3 h. The absorbance was measured at a wavelength of 450 nm. The DMEM high-glucose medium without hair follicle-derived stem cells was used as the control group. The results are as follows:

[0127] Table 2

[0128]

[0129] The results in Table 2 above show that, compared with the control group, the in vitro proliferation activity of fibroblasts was improved to varying degrees by adding hair follicle-derived stem cells to the fibroblasts. Among them, the proliferation activity of fibroblasts at concentrations of 50% and 75% hair follicle-derived stem cells was higher than that at a concentration of 25%, indicating that hair follicle-derived stem cells can promote the proliferation of fibroblasts and provide a certain basis for tissue repair and remodeling.

[0130] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for inducing hair follicle stem cells to differentiate into neural stem cells, characterized in that: The steps include: S1 raw material: Hair follicles were harvested, digested, and then complete culture medium was added to terminate the digestion. The cells were centrifuged, and then complete culture medium was added again to resuspend the cells. The cells were subcultured to obtain hair follicle stem cells. S2 induction: Hair follicle stem cells are prepared into a hair follicle stem cell suspension and inoculated into complete culture medium. After 1-2 days of culture, differentiation culture medium A is used for induction culture. After 2-5 days of induction culture, differentiation culture medium B is used for secondary induction culture. After 3-5 days of secondary induction culture, differentiation culture medium C is used for tertiary induction culture. Before the tertiary induction culture, magnetic field treatment is performed at a magnetic field strength of 2-8 mT for 10-30 seconds. After tertiary induction culture for 4-6 days, the target neural stem cells are obtained.

2. The method of inducing hair follicle stem cells to differentiate into neural stem cells according to claim 1, characterized in that: The components of the differentiation culture medium A include epidermal growth factor; The components of the differentiation culture medium B include epidermal growth factor, basic fibroblast growth factor, B27 additive and glutamine; The components of the differentiation culture medium C include epidermal growth factor, glutamine and crocin.

3. The method of inducing hair follicle stem cells to differentiate into neural stem cells according to claim 2, characterized in that: The components of the differentiation culture medium A include 0.1-30 ng / mL of epidermal growth factor; The components of the differentiation culture medium B include 0.1-30 ng / mL of epidermal growth factor, 0.1-50 ng / mL of basic fibroblast growth factor, 0.5-5 nM of B27 additive, and 0.5-5 mM of glutamine; The components of the differentiation culture medium C include 0.1-30 ng / mL of epidermal growth factor, 0.5-5 mM of glutamine, and 0.1-5 μg / mL of crocin.

4. The method of inducing hair follicle stem cells to differentiate into neural stem cells according to claim 3, characterized in that: The components of the differentiation culture medium A include 10-15 ng / mL of epidermal growth factor; The components of the differentiation culture medium B include 5-10 ng / mL of epidermal growth factor, 5-10 ng / mL of basic fibroblast growth factor, 1-1.5 nM of B27 additive, and 0.5-1 mM of glutamine; The components of the differentiation culture medium C include 5-8 ng / mL of epidermal growth factor, 0.5-1 mM of glutamine, and 1-3 μg / mL of crocin.

5. The method of inducing hair follicle stem cells to differentiate into neural stem cells according to claim 1, characterized in that: The components of the complete culture medium include 20-50 ng / mL of basic fibroblast growth factor and 1 wt%-4 wt% of serum substitute.

6. The method of inducing hair follicle stem cells to differentiate into neural stem cells according to claim 1, characterized in that: In step S2, the hair follicle stem cells are seeded at a density of 500 to 6000 cells / cm 2 .

7. The method of inducing hair follicle stem cells to differentiate into neural stem cells according to claim 1, characterized in that: In step S1, the hair follicle stem cells are hair follicle stem cells that have been subcultured to passages 2 to 4.

8. The method of inducing hair follicle stem cells to differentiate into neural stem cells according to claim 1, characterized in that: In step S1, the digestion includes: adding collagenase to the hair follicles and digesting them at 35-38° C. for 10-120 minutes.

9. The method of inducing hair follicle stem cells to differentiate into neural stem cells according to claim 8, characterized in that: The amount of collagenase added is 50-500 μg / mL.

10. A use of hair follicle stem cells to induce differentiation into neural stem cells, characterized in that: Application of the neural stem cells in promoting the proliferation activity of fibroblasts.

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