Method for promoting MSC (mesenchymal stem cells) to be differentiated into oligodendrocyte precursor cells

Abstract

The invention provides a method for promoting mesenchymal stem cells (MSC) to be differentiated into oligodendrocyte precursor cells, which comprises the following steps of: performing primary culture and secondary subculture on separated MSC, and performing first-stage culture by using an oligodendrocyte precursor cell culture medium # 1 containing a differentiation-promoting proliferation small molecule compound; and carrying out second-stage culture by adopting an oligodendrocyte precursor cell culture medium # 2, so as to obtain the oligodendrocyte precursor cells with relatively high purity. According to the invention, through a specific cell differentiation treatment and culture process, the MSC is cultured by using a cell culture medium treated by recombinant human laminin, so that the MSC is promoted to differentiate and proliferate, and the MSC-derived oligodendrocyte precursor cells are obtained in a relatively short time. Compared with the traditional IPSC which requires 100-200 days of induction culture, the method disclosed by the invention has the advantages that the oligodendrocyte precursor cells with relatively high purity can be harvested in a short time of 8-10 days and are used for producing cell treatment products.

Description

Technical Field

[0001] The invention relates to the technical field of cell biology, and in particular to a method for promoting mesenchymal stem cells (MSC) to differentiate into oligodendrocyte precursor cells. Background Art

[0002] Oligodendrocyte precursor cells (OPs) are responsible for the production of myelin during the development of the central nervous system and play a key role in the normal development and operation of the central nervous system. OP or myelin damage caused by various reasons is an important cause of abnormal development or function of the central nervous system, which will directly lead to serious diseases such as leukodystrophy and cerebral palsy in children. It is also related to the deterioration of injuries such as spinal cord injury and stroke, and poses a major threat to human health. Previous studies have shown that transplanting OPs can repair myelin, fundamentally treat diseases such as leukodystrophy and cerebral palsy in children, and can also significantly promote the recovery of injuries such as spinal cord injury and stroke. However, the source of OPs is extremely limited, which limits its clinical application. The isolation of adult OPs depends on the histological site, and it is almost impossible to obtain materials from adults. The isolation of OPs from aborted fetal brain tissue is subject to ethical restrictions. The quality of OPs isolated from each batch of brain tissue samples is uneven, which affects the treatment effect. In recent years, great progress has been made in the research on the mechanism of stem cell differentiation. It has been found that embryonic stem cells and induced pluripotent stem cells have the potential to differentiate into oligodendrocyte precursor cells. However, the applicability of embryonic stem cells is also subject to ethical restrictions. In addition, the process of differentiating oligodendrocyte precursor cells from embryonic stem cells and induced pluripotent stem cells takes 100-200 days, is costly, has a high failure rate, and has large quality fluctuations between batches, which is not conducive to drug development and clinical application (for relevant literature and intellectual property rights, see Nature-Biotechnology 2022; New York Stem Cell Foundation, Inc.-V. Fossati P. Daovalas 2017).

[0003] Mesenchymal stem cells (MSC) can be collected from a variety of tissues such as fat, umbilical cord, and bone marrow. They are the adult stem cells with the richest sample resources and the most promising seed cells based on stem cell regenerative medicine. The current methods used to differentiate mesenchymal stem cells into oligodendrocyte precursor cells are extremely limited, and are limited to the use of a variety of growth factors to induce MSCs to differentiate into neural stem cells first, and then differentiate into oligodendrocyte precursor cells. The cell growth factor induction method has the disadvantages of low induction efficiency, long induction cycle, large quality fluctuations between batches, and low yield. Therefore, it is necessary to develop improved technologies in this field to improve differentiation efficiency, improve the quality and yield of oligodendrocyte precursor cells.

[0004] In the inventor's previous research, oligodendrocyte precursor cells were obtained by treating with a gene expression vector containing "oligodendrocyte precursor cell differentiation key genes" and then performing differentiation culture for 15-25 days. However, the differentiation time is still relatively long, and how to further shorten the differentiation culture time while taking into account the yield and quality of oligodendrocyte precursor cells still needs further research and solution by the inventor. Summary of the invention

[0005] In view of the deficiencies in the prior art, the present invention proposes a method for promoting MSC differentiation into oligodendrocyte precursor cells. Specifically, the method mainly uses specific small molecule compounds to induce MSC differentiation, which can promote MSC to differentiate into oligodendrocyte precursor cells with high efficiency, greatly shorten the differentiation time, and improve the quality of oligodendrocyte precursor cells.

[0006] The present invention first provides a method for promoting mesenchymal stem cells to differentiate into oligodendrocyte precursor cells, comprising the following steps:

[0007] (1) Isolation of mesenchymal stem cells and primary culture;

[0008] (2) seeding the primary cultured mesenchymal stem cells on the surface of a cell culture substrate, adding a cell culture medium, and performing a first subculture, wherein the cell culture substrate has been treated to "promote cell adhesion";

[0009] (3) inoculating the mesenchymal stem cells after the first subculture onto the surface of a cell culture substrate again, adding cell culture medium, and performing a second subculture, wherein the cell culture substrate has not been treated with "promoting cell adhesion";

[0010] (4) The mesenchymal stem cells after the second subculture are subcultured again and inoculated on the surface of the cell culture substrate. First, the first stage of culture is carried out with "oligodendrocyte precursor cell culture medium #1" containing "small molecule compounds that promote differentiation and proliferation", and then replaced with "oligodendrocyte precursor cell culture medium #2" for the second stage of culture to obtain oligodendrocyte precursor cells. The cell culture substrate is treated with "promoting cell adhesion" and "recombinant human laminin", and the "oligodendrocyte precursor cell differentiation medium" is an animal-free formula.

[0011] Furthermore, the mesenchymal stem cells described in step (1) are derived from humans. The mesenchymal stem cells derived from humans are used to obtain human oligodendrocyte precursor cells, which can be applied to the treatment of human myelin damage diseases.

[0012] Furthermore, the sources of the mesenchymal stem cells include bone marrow, umbilical cord blood, umbilical cord, placenta and adipose tissue. The tissues are all tissues from which the human body can easily obtain mesenchymal stem cells.

[0013] Furthermore, the culture medium used for the primary culture in step (1) and the cell culture medium in steps (2) and (3) are all MSC basal culture medium, the components of which are MEM-alpha + 5% human platelet extract.

[0014] Furthermore, the cell culture substrate in steps (2) and (3) is a cell culture dish, a cell culture plate or a cell factory; and the cell culture substrate in step (4) is a cell culture dish or a cell culture flask.

[0015] Furthermore, the initial density of mesenchymal stem cells in step (4) is 0.1×10 4 / cm 2 ~1.5×10 4 / cm 2 .

[0016] Furthermore, the initial density of the mesenchymal stem cells in step (4) is 1.0×10 4 / cm 2 .

[0017] Furthermore, the first stage of culturing in the "oligodendrocyte precursor cell culture medium #1" containing the "small molecule compound promoting differentiation and proliferation" in step (4) is performed for a period of less than or equal to 4 days, for example, 2 to 4 days (48 to 96 hours).

[0018] Furthermore, in the "oligodendrocyte precursor cell culture medium #1" containing the "small molecule compound that promotes differentiation and proliferation" in step (4), the "small molecule compound that promotes differentiation and proliferation" includes LDN193189 and SB431542.

[0019] Further, the concentration of LDN193189 is 25-250nM, and the concentration of SB431542 is 10-100nM. In our previous experimental studies, it was found that when the concentration of LDN193189 and / or SB431542 exceeded the above range, the effect would be deteriorated or even cell death would occur. More preferably, the concentration of LDN193189 is 100nM, and the concentration of SB431542 is 10nM.

[0020] Furthermore, in the step (4), the time for the second stage culture using the "oligodendrocyte precursor cell culture medium #2" is less than or equal to 6 days, for example, 4 to 6 days (96 to 144 hours).

[0021] Furthermore, the total culture time in step (4) is 6 to 10 days.

[0022] Furthermore, the total culture time in step (4) is 8 days.

[0023] In summary, compared with the prior art, the present invention achieves the following technical effects:

[0024] 1. The method of the present invention is highly efficient and time-consuming, and oligodendrocyte precursor cells can be obtained in 10 days or even less than 10 days.

[0025] 2. The oligodendrocyte precursor cell differentiation method of the present invention avoids the gene editing commonly used in the prior art, avoids the risk of gene damage including off-target, and does not use various vectors including viruses, making the product purer and safer.

[0026] 3. The oligodendrocyte precursor cell differentiation method of the present invention does not use animal-derived or human blood-derived materials, making the product purer and safer.

[0027] 4. The oligodendrocyte precursor cells obtained by the method of the present invention have a stable phenotype and the ability to generate myelin.

[0028] 5. The oligodendrocyte precursor cells obtained by the preparation method of the present invention can be used to treat various myelin damage-related diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0030] Figure 1 This is a morphological diagram of MSC-derived oligodendrocyte precursor cells prepared in the present invention;

[0031] Figure 2 This is a flow cytometry statistical diagram of MSC-derived oligodendrocyte precursor cells prepared in the present invention;

[0032] Figure 3 This is a fluorescence imaging detection diagram of myelin sheath formation after the MSC-derived oligodendrocyte precursor cells prepared by the present invention were transplanted into the brain of demyelinating mice, wherein MBP is a myelin sheath formation marker and NF200 is a nerve axon marker;

[0033] Figure 4 This is an electron microscopic image of the myelin sheath formed after animal transplantation. The black myelin sheath is formed and wraps around the axon. DETAILED DESCRIPTION

[0034] In order to enable those skilled in the art to better understand the scheme of the present invention, the technical scheme in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only embodiments of a part of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work should fall within the scope of protection of the present invention.

[0035] The existing methods for producing oligodendrocyte precursor cells mainly use multiple culture media with different formulations to differentiate embryonic stem cells or induced pluripotent stem cells in stages to finally obtain oligodendrocyte precursor cells. Such methods are generally time-consuming and costly. At the same time, due to the complex process, not only is the probability of production failure high, but the quantity and purity of the oligodendrocyte precursor cells obtained are not as ideal, and the cost-effectiveness is low.

[0036] The present invention discloses a method for culturing MSCs by epigenetic means, which can promote MSCs to differentiate into oligodendrocyte precursor cells with high efficiency, shorten the differentiation time, and improve the quality and yield of oligodendrocyte precursor cells. In the process of stem cells differentiating into oligodendrocyte precursor cells, the use of "differentiation small molecule compounds" (hereinafter referred to as compounds) is a necessary condition for promoting differentiation. The present invention uses specific small molecule compounds to promote MSCs to efficiently differentiate into oligodendrocyte precursor cells, greatly simplifying the overall process, while improving differentiation efficiency, saving time and money costs, and enhancing the batch-to-batch stability of the product, which is conducive to industrialized and standardized production, and can accelerate the clinical transformation of oligodendrocyte precursor cells.

[0037] In a specific embodiment of the present invention, MSCs are derived from humans, specifically from healthy human bone marrow, umbilical cord blood, umbilical cord, placenta and adipose tissue. Since human tissue MSCs contain a certain number (no more than 20%) of THY1-expressing hi EGFR + PDGFRA + Glial precursor cells can be artificially cultured by the method of the present invention to promote their proliferation and lock into oligodendrocyte precursor cells in a short period of time (while avoiding the proliferation of other cells that do not have the potential of glial precursor cells, forming a system that only promotes the production of oligodendrocyte precursor cells, thereby greatly improving the purity of the product).

[0038] In a specific embodiment of the present invention, the components of the "oligodendrocyte precursor cell culture medium #1" containing "differentiation and proliferation promoting small molecule compounds (LDN193189 and SB431542)" are shown in Table 1 below, and the "oligodendrocyte precursor cell culture medium #1" prepared within the concentration range of its various components can be used for the differentiation culture of the present invention.

[0039] Table 1 Oligodendrocyte Precursor Cell Culture Medium #1 Composition

[0040] Serial number Element Concentration range 1 DMEM / F12 / 2 Human serum albumin 500–5000 μg / mL 3 Catalase 0.5–10 μg / mL 4 insulin 1–20 μg / mL 5 holotransferrin 2.5–100 μg / mL 6 Triiodothyronine 0.001~0.01μg / mL 7 L-Carnitine 0.5–10 μg / mL 8 Ethanolamine 0.25–5 μg / mL 9 D-Galactose 2.5–100 μg / mL 10 Putrescine 10–300 μg / mL 11 Sodium selenate 0.01~0.1μg / mL 12 Superoxide dismutase 0.5–10 μg / mL 13 Glutathione 0.25–5 μg / mL 14 Corticosteroids 5–50 ng / mL 15 Linoleic acid 0.2–20 μg / mL 16 α-linolenic acid 0.2–20 μg / mL 17 Lipoic Acid 2–20 ng / mL 18 Progesterone 2–20 ng / mL 19 Retinol 0.02–4 μg / mL 20 Vitamin A 0.02–4 μg / mL 21 Vitamin E 0.2–20 μg / mL 22 dl-α-Tocopheryl acetate 0.2–20 μg / mL 23 LDN193189 25~250nM 24 SB431542 10~100nM

[0041] LDN193189 used in the present invention, whose Chinese name is 4-[6-(4-(piperazine-1-yl)phenyl]pyrazolo[1,5-A]pyrimidin-3-yl)quinoline, is a very strong small molecule inhibitor of bone morphogenetic protein (BMP) type I receptor ALK2 and ALK3. SB431542 is a member of the benzamide class, has the function of EC2.7.10.1 (receptor protein-tyrosine kinase) inhibitor, and can selectively inhibit transforming growth factor β (TGFβ) type I receptor activin receptor-like kinase ALK5, ALK4 and ALK7. LDN193189 and SB431542 are currently widely used to promote the differentiation of PSC into neural stem cells. There is no report on the use of these two small molecule compounds in promoting the differentiation of MSC into oligodendrocyte precursor cells. However, after preliminary screening, the present invention finally determined to use these two small molecule compounds as "small molecule compounds that promote differentiation and proliferation" and added them to "oligodendrocyte precursor cell culture medium #1" to culture MSC cells, and achieved very good results. The mechanism of the present invention is: by i) promoting THY1 contained in MSC hi EGFR + PDGFRA + ii) restricting THY1 hi EGFR + PDGFRA + glial precursor cell differentiation pathway, locking and inducing their differentiation into oligodendrocyte precursor cells, iii) avoiding the proliferation of other cells that do not have the potential of glial precursor cells, thereby greatly improving the purity of the product.

[0042] In a specific embodiment of the present invention, the components and concentration ranges of the used "oligodendrocyte precursor cell culture medium #2" are shown in Table 2. "Oligodendrocyte precursor cell culture medium #2" prepared within the concentration range of its components can be used for the proliferation culture of the present invention.

[0043] Table 2 Oligodendrocyte Precursor Cell Culture Medium #2 Composition

[0044]

[0045]

[0046] In a specific embodiment of the present invention, the cell culture substrate used for primary MSC culture, first subculture and second subculture can be a cell culture dish, a cell culture plate or a cell factory; the cell culture substrate used for MSC differentiation culture can be a sealable cell culture bottle.

[0047] Example 1

[0048] This embodiment provides a method for differentiating MSCs into oligodendrocyte precursor cells, and the specific steps are as follows:

[0049] (1) Primary MSC culture

[0050] 1mL of healthy adult bone marrow stock solution was diluted with 9mL of MSC basal medium and cultured on a 100mm diameter round culture dish on the surface of TCT (abbreviation for "cell adhesion treatment") for 3 days. All the culture medium was removed and replaced with 10mL of new MSC basal medium. MSC at this stage is defined as P0. P0 MSCs were cultured with MSC basal medium for another 10 days, with the medium replaced every 2 days, 10mL each time. The MSC basal medium consists of MEM-alpha + 5% human platelet extract.

[0051] (2) First subculture

[0052] After 10 days of culture, P0 MSCs were digested and collected with TrypLE Express and then passaged to a 100 mm diameter round dish on the surface of TCT at a ratio of 1:3. These MSCs were defined as P1, and P1 MSCs were cultured with 10 mL of MSC basal medium for 2 days.

[0053] (3) Second subculture

[0054] After culturing for 2 days, P1 MSCs were digested and collected with TrypLE Express and then cultured at 2×10 4 cells / cm 2 The cells were seeded at a density of 100 mm and subcultured onto a 100 mm diameter circular culture dish on the surface of TCT. These MSCs were defined as P2, and P2 MSCs were cultured for 2 days in 10 mL of MSC basal medium.

[0055] (4) MSC differentiation into oligodendrocyte precursor cells

[0056] After culturing for 2 days, P2 MSCs were digested and collected with TrypLE Express and then cultured at 1.0×10 4 cells / cm 2The cells were seeded at a density of 100 μg / cm2 and passaged to a 6-well culture dish on a TCT surface treated with "recombinant human laminin". The cells were cultured for 3 days (72 hours) in "oligodendrocyte precursor cell culture medium #1" containing "small molecule compounds that promote differentiation and proliferation (LDN193189 and SB431542)". The culture medium was then replaced with "oligodendrocyte precursor cell culture medium #2" for 5 days (120 hours, with a change of medium at the 72nd hour) to obtain oligodendrocyte precursor cells. After 3 days of culture at the concentrations of each combination of the "small molecule compounds that promote differentiation and proliferation" and the use of each combination of the "small molecule compounds that promote differentiation and proliferation" in "oligodendrocyte precursor cell culture medium #1", Olig2 + The results of cell % determination are shown in Table 5.

[0057] The specific step of treating with "recombinant human laminin" is: soak the surface of the culture plate with a 5-30 μg / mL recombinant human laminin solution for 30 minutes at a temperature of 37 degrees Celsius. In this embodiment, a recombinant human laminin solution with a concentration of 20 μg / mL is used. As can be seen from the results in Table 5, the combination of two small molecules (combination 5 and combination 6) of LDN193189 at 10-100 nM and SB431542 at 10 nM can obtain the highest proportion of Olig2 + Oligodendrocyte precursor cells, Olig2 + The percentage of oligodendrocyte precursor cells can reach more than 47%. When a single small molecule (combination 2 to combination 4) or SB431542 with too high a concentration (combination 7 and combination 8) is used, the obtained Olig2 + The percentage of oligodendrocyte precursor cells was less than 30%.

[0058] After 8 days of differentiation culture using a combination of 6 concentrations of small molecule compounds, analysis showed that the harvested cells had oligodendrocyte morphology ( Figure 1 ), and the percentage of positive oligodendrocyte precursor cell marker O4 was 97.20±0.10% ( Figure 2 The components of the “oligodendrocyte precursor cell culture medium #1” and “oligodendrocyte precursor cell culture medium #2” containing “differentiation and proliferation promoting small molecule compounds (LDN193189 and SB431542)” used in this example are detailed in Table 3, Table 4 and Table 5.

[0059] Table 3 Oligodendrocyte precursor cell culture medium #1 composition (this embodiment)

[0060] Serial number Element Concentration range 1 DMEM / F12 / 2 Human serum albumin 2500 μg / mL 3 Catalase 5 μg / mL 4 insulin 10 μg / mL 5 holotransferrin 15 μg / mL 6 Triiodothyronine 0.002 μg / mL 7 L-Carnitine 2 μg / mL 8 Ethanolamine 1 μg / mL 9 D-Galactose 15 μg / mL 10 Putrescine 30 μg / mL 11 Sodium selenate 0.02 μg / mL 12 Superoxide dismutase 2.5 μg / mL 13 Glutathione 1 μg / mL 14 Corticosteroids 2ng / mL 15 Linoleic acid 1 μg / mL 16 α-linolenic acid 1 μg / mL 17 Lipoic Acid 5ng / mL 18 Progesterone 6ng / mL 19 Retinol 0.1 μg / mL 20 Vitamin A 0.1 μg / mL 21 Vitamin E 1 μg / mL 22 dl-α-Tocopheryl acetate 1 μg / mL 23 LDN193189 0~100nM 24 SB431542 0~50nM

[0061] Table 4 Oligodendrocyte precursor cell culture medium #2 composition (this embodiment)

[0062]

[0063]

[0064] Table 5 Concentration of each combination of "small molecule compounds promoting differentiation and proliferation" and the Olig2 + Cell% Test Data

[0065]

[0066] The results of the examples show that the technology disclosed in this patent can produce oligodendrocyte precursor cells with high purity in a relatively short period of time (8 days) using a simple cell culture method. Secondly, the technology disclosed in this patent does not use gene modification, gene overexpression and other means at all, avoiding the risk of off-target gene damage. Thirdly, the technology disclosed in this patent does not use serum or animal-derived materials at all, and the chemical composition of the culture medium is clear, which better ensures product safety and batch-to-batch stability.

[0067] The above culture method has been verified and can be extended to eight passages of mesenchymal stem cells (MSC) and successfully provides verified oligodendrocyte precursor cells.

[0068] To further confirm the function of the harvested oligodendrocyte precursor cells, the oligodendrocyte precursor cells obtained after differentiation and culture with a combination of 6 concentrations of small molecule compounds were transplanted into the brain of the demyelinating disease model "shiverer" mouse to observe the development of oligodendrocyte precursor cells into oligodendrocytes and the generation of myelin. The results are as follows Figure 3 As shown, oligodendrocytes expressing myelin markers (MBP, green fluorescent signal) and axons (NF200, red fluorescent signal) highly overlap; Figure 4 This is an electron microscope image of myelin sheaths formed after animal transplantation. The black myelin sheaths are formed and wrap around the axons. This proves that oligodendrocyte precursor cells are functional and can develop into oligodendrocytes and generate myelin sheaths, which has the potential to repair demyelinating damage in the nervous system and treat demyelinating diseases.

[0069] The above results show that by treating MSCs with "oligodendrocyte precursor cell differentiation medium" and then culturing them with "oligodendrocyte precursor cell proliferation medium", oligodendrocyte precursor cells with a purity of ≥85% can be obtained within 8 days. The present invention not only greatly shortens the production time of oligodendrocyte precursor cells, but also successfully discovers a new method for differentiating mesenchymal stem cells into oligodendrocyte precursor cells, while solving the two major problems of ethics and production time. The present invention uses animal-free materials, and the cells produced are suitable for future clinical applications.

[0070] Application Example 1 Oligodendrocyte precursor cells prepared by the method of the present invention can be used to treat neurological diseases

[0071] 1. White matter dystrophy

[0072] Using leukodystrophy mice as a disease model, oligodendrocyte precursor cells were minimally injected into the corpus callosum of the left and right brains, with 3×10 cells transplanted on each side. 5 Cells can regenerate white matter, restore brain function, and extend the lifespan of animals.

[0073] 2. Stroke

[0074] The stroke model was established in SD rats by middle cerebral artery occlusion. Oligodendrocyte precursor cells were minimally invasively injected near the stroke area. The number of transplanted cells was 3×10 6 cells, can reduce inflammation, repair nerves, and restore brain function.

[0075] Myelin damage occurs in many nervous system diseases and is closely related to the loss of neural function. Oligodendrocyte precursor cells are a kind of universal cells that can play a therapeutic effect on a variety of diseases. Therefore, the oligodendrocyte precursor cells prepared by the method of the present invention can also be used to treat a variety of neurological diseases including leukodystrophy, cerebral palsy, multiple sclerosis, stroke, etc.

[0076] In summary, the method of the present invention uses a novel differentiation method, first using "oligodendrocyte precursor cell culture medium #1" containing "small molecule compounds that promote differentiation and proliferation" for the first stage of culture, and then using "oligodendrocyte precursor cell culture medium #2" for the second stage of culture. Oligodendrocyte precursor cells can be produced in a short time of 6-10 days, and these oligodendrocyte precursor cells have the function of generating myelin.

[0077] 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 principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for promoting mesenchymal stem cells to differentiate into oligodendrocyte precursor cells, characterized in that: The steps include: (1) Isolation of mesenchymal stem cells and primary culture; (2) inoculating the primary cultured mesenchymal stem cells onto the surface of a cell culture substrate, adding a cell culture medium, and performing a first subculture, wherein the cell culture substrate has been treated to "promote cell adhesion"; (3) inoculating the mesenchymal stem cells after the first subculture onto the surface of a cell culture substrate again, adding cell culture medium, and performing a second subculture, wherein the cell culture substrate has not been treated with "promoting cell adhesion"; (4) The mesenchymal stem cells after the second subculture are subcultured again and inoculated on the surface of the cell culture substrate. The first stage of culture is performed with "oligodendrocyte precursor cell culture medium #1" containing "small molecule compounds that promote differentiation and proliferation", and then the culture medium is replaced with "oligodendrocyte precursor cell culture medium #2" for the second stage of culture to obtain oligodendrocyte precursor cells. The cell culture substrate is treated with "promoting cell adhesion" and "laminin".

2. The method for promoting mesenchymal stem cells to differentiate into oligodendrocyte precursor cells according to claim 1, characterized in that: The mesenchymal stem cells described in step (1) are derived from humans.

3. The method for promoting mesenchymal stem cells to differentiate into oligodendrocyte precursor cells according to claim 2, characterized in that: The sources of mesenchymal stem cells include bone marrow, cord blood, umbilical cord, placenta and adipose tissue.

4. The method for promoting mesenchymal stem cells to differentiate into oligodendrocyte precursor cells according to claim 1, characterized in that: The cell culture substrate in steps (2) and (3) is a cell culture dish, a cell culture plate or a cell factory; the cell culture substrate in step (4) is a cell culture dish or a cell culture bottle.

5. The method for promoting mesenchymal stem cells to differentiate into oligodendrocyte precursor cells according to claim 1, characterized in that: The initial density of mesenchymal stem cells in step (4) is 0.1×10 4 / cm 2 ~1.5×10 4 / cm 2 .

6. The method according to claim 5, characterized in that The initial density of mesenchymal stem cells in step (4) is 1.0×10 4 / cm 2 .

7. The method according to claim 1, characterized in that The first stage of culture in step (4) using the "oligodendrocyte precursor cell culture medium #1" containing the "differentiation and proliferation promoting small molecule compound" is carried out for a period of less than or equal to 4 days.

8. The method according to claim 1 or 7, characterized in that: The "small molecule compounds promoting differentiation and proliferation" described in step (4) include LDN193189 and SB431542.

9. The method according to claim 8, characterized in that The concentration of LDN193189 is 25-250 nM, and the concentration of SB431542 is 10-100 nM.

10. The method according to claim 1, characterized in that The time for the second stage culture using the "oligodendrocyte precursor cell culture medium #2" in step (4) is less than or equal to 6 days.

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