A method for promoting the differentiation of MSCs into oligodendrocyte precursor cells

Abstract

The present invention provides a method for promoting the differentiation of mesenchymal stem cells (MSCs) into oligodendrocyte progenitor cells, which includes primary culturing the isolated MSCs, followed by secondary passage culturing, then treating them with a gene expression vector containing the "key gene for oligodendrocyte progenitor cell differentiation", and further performing differentiation culture using the "oligodendrocyte progenitor cell differentiation medium". Through specific cell differentiation treatment and culture processes, the present invention uses a cell culture matrix treated with recombinant human laminin to culture MSCs and promote their differentiation, so as to obtain oligodendrocyte progenitor cells derived from MSCs in a relatively short time. The present invention further provides a method for efficiently harvesting oligodendrocyte progenitor cells. Specifically, through a rate-limiting shaking method, oligodendrocyte progenitor cells are harvested and allowed to further proliferate, and a relatively large amount of oligodendrocyte progenitor cells with high purity can be harvested in a short time for the production of cell therapy products.

Description

Technical Field

[0001] The present invention relates to the technical field of cell biology, and particularly relates to a method for promoting the differentiation of mesenchymal stem cells (MSCs) into oligodendrocyte precursor cells. Background Art

[0002] Injury or dysplasia of the central nervous system can cause apoptosis of nerve cells, defect of nerve tissue, and defects in abilities such as movement and thinking, ultimately leading to lifelong disability or even shortened lifespan. The self-repair ability of the central nervous system is extremely low. To treat such diseases, cell replacement therapy is required to supplement the apoptotic and defective nerve cells and tissues, thereby restoring the function of the nervous system.

[0003] 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 can directly lead to serious diseases such as leukodystrophy and cerebral palsy in children, and is also related to the deterioration of injuries such as spinal cord injury and stroke, posing a major threat to human health. Previous studies have shown that transplantation of OPs can repair myelin and 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, restricting their clinical application. The isolation of adult OPs depends on histological sites, and it is generally almost impossible to obtain samples from adult humans. The isolation of OPs from the brain tissue of aborted fetuses is restricted by ethics, and the OPs obtained from each batch of brain tissue samples are heterogeneous in quality, affecting the treatment effect. In recent years, great progress has been made in the research on the differentiation mechanism of stem cells, and it has been found that both embryonic stem cells and induced pluripotent stem cells have the potential to differentiate into oligodendrocyte precursor cells. However, the application of embryonic stem cells is also restricted by ethics, and the process of differentiating embryonic stem cells and induced pluripotent stem cells into oligodendrocyte precursor cells is time-consuming, costly, has a high failure rate, and has large quality fluctuations between batches, which is not conducive to drug development and clinical application.

[0004] Mesenchymal stem cells (MSCs) can be collected from various tissues such as fat, umbilical cord, and bone marrow. They are the most abundant adult stem cells in terms of sample resources and are the most promising seed cells for stem cell-based regenerative medicine. Currently, the methods for differentiating mesenchymal stem cells into oligodendrocyte precursor cells are extremely limited and are limited to using a variety of growth factors to induce MSCs to first differentiate into neural stem cells and then into oligodendrocyte precursor cells. The cell growth factor induction method has disadvantages such as low induction efficiency, long induction period, large quality fluctuations between batches, and low yield. Therefore, it is still necessary to develop improved technologies in this field to improve the differentiation efficiency, the quality and yield of oligodendrocyte precursor cells. Summary of the Invention

[0005] Aiming at the defects in the prior art, the present invention proposes a method for promoting the differentiation of MSCs into oligodendrocyte progenitor cells. Specifically, it is a method for culturing MSCs by epigenetic means, which can promote the differentiation of MSCs into oligodendrocyte progenitor cells with high efficiency, shortening the differentiation time while improving the quality and yield of oligodendrocyte progenitor cells.

[0006] The present invention first provides a method for promoting the yield of mesenchymal stem cells differentiating into oligodendrocyte progenitor cells, including the following steps:

[0007] (1) Isolate mesenchymal stem cells and perform primary culture;

[0008] (2) Subculture the mesenchymal stem cells after primary culture and inoculate them on the surface of the cell culture substrate, add cell culture medium, and perform the first subculture. The cell culture substrate has been treated with "promoting cell adhesion";

[0009] (3) Subculture the mesenchymal stem cells after the first subculture again and inoculate them on the surface of the cell culture substrate, add cell culture medium, and perform the second subculture. The cell culture substrate has not been treated with "promoting cell adhesion";

[0010] (4) Treat the mesenchymal stem cells after the second subculture with a gene expression vector containing the "key gene for oligodendrocyte progenitor cell differentiation", and then subculture and inoculate them on the surface of the cell culture substrate again, add the "oligodendrocyte progenitor cell differentiation medium" for differentiation culture to complete the differentiation of oligodendrocyte progenitor cells. The cell culture substrate has been treated with "promoting cell adhesion" and "recombinant human laminin", and the "oligodendrocyte progenitor cell differentiation medium" is a xeno-free formulation.

[0011] Further, the mesenchymal stem cells described in step (1) are derived from humans. Mesenchymal stem cells derived from humans are used to obtain human oligodendrocyte progenitor cells and can be applied to the treatment of human myelin sheath injury diseases.

[0012] Further, the sources of the mesenchymal stem cells include bone marrow, cord blood, umbilical cord, placenta, and adipose tissue. These tissues are all tissues from which it is easy to obtain mesenchymal stem cells in the human body.

[0013] Further, the culture medium used in the primary culture described in step (1) and the cell culture media described in steps (2) and (3) are both MSC basal media, and the components are MEM-alpha + 5% human platelet extract.

[0014] Further, 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 flask.

[0015] Further, the starting density of the mesenchymal stem cells in step (4) is 0.1×10 4 / cm 2 ~1.0×10 4 / cm 2 .

[0016] Further, the starting density of the mesenchymal stem cells described in step (4) is 1.0×10 4 / cm 2 . The yield of oligodendrocyte progenitor cells obtained at this starting density is higher.

[0017] Further, the gene expression vector containing the "key gene for oligodendrocyte progenitor cell differentiation" in step (4) can be a viral vector such as a lentivirus, a plasmid vector, a free vector or an mRNA vector.

[0018] Further, the genes contained in the gene expression vector of the "key gene for oligodendrocyte progenitor cell differentiation" described in step (4) include Olig2 and Sox10.

[0019] Further, the specific steps of treating with the gene expression vector containing the "key gene for oligodendrocyte progenitor cell differentiation" in step (4) are: transfecting the plasmid vector into the mesenchymal stem cells in the form of a cell suspension after the second passage of subculture, and electroporating 5-10 μg of Olig2 and 5-10 μg of Sox10 plasmids for every 5×10 6 cells. More preferably, 10 μg of Olig2 and 10 μg of Sox10 plasmids are electroporated for every 5×10 6 cells, and the purity and yield of oligodendrocyte progenitor cells obtained under this condition are higher.

[0020] Further, the differentiation culture time in step (4) is 15-25 days.

[0021] Further, the differentiation culture time in step (4) is 20 days.

[0022] The present invention also provides a method for proliferating the oligodendrocyte progenitor cells obtained by the foregoing method, comprising the following steps:

[0023] S1. After the differentiation of oligodendrocyte progenitor cells is completed, place the cell culture substrate on a shaking device. After ensuring sealing, perform horizontal shaking at 37 °C at 120 - 240 times per minute with an amplitude of 1.5 - 4.5 cm for 15 - 20 hours to promote the separation of oligodendrocyte progenitor cells from the surface of the cell culture substrate, while the remaining cells remain adherent.

[0024] S2. After the shaking is completed, collect the culture medium containing oligodendrocyte progenitor cells and inject it onto the surface of a new cell culture substrate (sub - culture of oligodendrocyte progenitor cells), and add the "oligodendrocyte progenitor cell proliferation medium" for proliferation culture to complete the proliferation of oligodendrocyte progenitor cells. The cell culture substrate has been treated with "promote cell adhesion" and "recombinant human laminin".

[0025] Further, the shaking conditions in step S1 are 120 - 240 times per minute, with an amplitude of 1.5 - 3.0 cm and a time of 15 - 20 hours.

[0026] Further, the shaking conditions in step S1 are 180 times per minute, with an amplitude of 3.0 cm and a time of 20 hours. At these parameters, the separation quantity and purity of oligodendrocyte progenitor cells are relatively high.

[0027] Further, the cell culture substrate in step S1 is a sealable cell culture flask; the cell culture substrates in step S2 are cell culture dishes, cell culture plates or cell factories.

[0028] Further, the seeding density of the oligodendrocyte progenitor cells in the proliferation culture in step S2 is 0.5×10 4 / cm 2 ~1.5×10 4 / cm 2 .

[0029] Further, the seeding density of the oligodendrocyte progenitor cells in the proliferation culture in step S2 is 1.0×10 4 / cm 2 . At these parameters, the proliferation efficiency of oligodendrocyte progenitor cells is relatively high.

[0030] Further, the time of the proliferation culture in step S2 is 6 - 12 days.

[0031] Further, the time of the proliferation culture in step S2 is 9 days. At these parameters, the proliferation efficiency of oligodendrocyte progenitor cells is relatively high.

[0032] In summary, compared with the prior art, the present invention has achieved the following technical effects:

[0033] 1. The method of the present invention is highly efficient and time-consuming, and oligodendrocyte precursor cells can be obtained in 15-25 days.

[0034] 2. The method for differentiating oligodendrocyte precursor cells of the present invention reduces the use of chemical inducers or cytokines, making the product purer and free from the incorporation of toxic substances, with higher safety.

[0035] 3. The method for differentiating oligodendrocyte precursor cells of the present invention does not use materials derived from animals or human blood, making the product purer and safer.

[0036] 4. The method of the present invention separates high-purity oligodendrocyte precursor cells from the differentiation culture vessel by a shaking method. While improving the purity and survival rate of oligodendrocyte precursor cells, it avoids the use of animal-derived digestive enzymes commonly used in traditional methods.

[0037] 5. The method of the present invention only requires an additional 15-20 hours of shaking time and 6-12 days of proliferation culture to proliferate a large number of oligodendrocyte precursor cells.

[0038] 6. The method of the present invention does not use materials derived from animals or human blood during the proliferation of oligodendrocyte precursor cells, making the product purer and safer.

[0039] 7. The oligodendrocyte precursor cells obtained by the method of the present invention have a stable phenotype and the ability to generate myelin sheaths.

[0040] 8. The oligodendrocyte precursor cells obtained by the preparation method of the present invention can be used to treat various diseases related to myelin sheath damage. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following will briefly introduce the drawings required for the embodiments. It should be understood that the following drawings only show some embodiments of the present invention, and therefore should not be regarded as limiting the scope. For those of ordinary skill in the art, other related drawings can be obtained based on these drawings without creative efforts.

[0042] Figure 1 Morphological diagram of MSC-derived oligodendrocyte precursor cells prepared by the present invention (after shaking purification);

[0043] Figure 2 Fluorescence imaging detection diagram of oligodendrocyte precursor cell markers A2B5 and PDGFRα (after shaking purification);

[0044] Figure 3 Fluorescence imaging detection diagram of in vitro myelin sheath formation of MSC-derived oligodendrocyte precursor cells prepared by the present invention, where MBP is a myelin sheath formation marker and NF200 is a nerve axon marker;

[0045] Figure 4 This is the physical diagram of the shaking device adopted in Embodiment 2 of the present invention. Detailed implementation manners

[0046] In order to enable those skilled in the art to better understand the solution of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative efforts shall fall within the protection scope of the present invention.

[0047] Existing methods for producing oligodendrocyte precursor cells mainly differentiate embryonic stem cells or induced pluripotent stem cells in stages by using a plurality of culture media with different formulations, and finally obtain oligodendrocyte precursor cells. Such methods generally take a long time and are costly. At the same time, due to the complex process, not only the probability of production failure is relatively high, but also the obtained oligodendrocyte precursor cells are not ideal in terms of quantity and purity, and the cost performance is relatively low.

[0048] The present invention discloses a method for culturing MSCs by epigenetic means, which can promote the differentiation of MSCs into oligodendrocyte precursor cells with high efficiency, shorten the differentiation time, and improve the quality and yield of oligodendrocyte precursor cells. During the process of stem cells differentiating into oligodendrocyte precursor cells, the expression of "differentiation key genes" (hereinafter referred to as key genes) is a necessary condition for promoting differentiation. The present invention uses epigenetic means to transiently and highly express key genes in MSCs through vector transfection to promote the efficient differentiation of MSCs into oligodendrocyte precursor cells, and provides a method for separating and proliferating oligodendrocyte precursor cells, which greatly simplifies the overall process, saves time and money costs while improving the differentiation efficiency, and further enhances the batch-to-batch stability of the product, facilitating industrialized and standardized production, and accelerating the clinical transformation of oligodendrocyte precursor cells.

[0049] In the specific implementation manner of the present invention, MSCs are of human origin, specifically from healthy human bone marrow, umbilical cord blood, umbilical cord, placenta and adipose tissue, and oligodendrocyte precursor cells can be obtained by the method of the present invention from all of them.

[0050] In the specific implementation manner of the present invention, the composition of the "oligodendrocyte precursor cell differentiation medium" is shown in Table 1 below, and the "oligodendrocyte precursor cell differentiation medium" prepared within the concentration range of each component can be used for the differentiation culture of the present invention.

[0051] Table 1 Composition of oligodendrocyte precursor cell differentiation medium

[0052] Serial number Component Concentration range 1 DMEM / F12 / 2 Non-essential amino acids (100x) 0.1 - 5x, i.e., 0.1 - 5% (volume ratio) 3 Insulin 0.1 - 10 μg / mL 4 Transferrin holoferric 2 - 100 μg / mL 5 Putrescine 5 - 200 μg / mL 6 Human serum albumin 250 - 4000 μg / mL 7 Superoxide dismutase 1 - 10 μg / mL 8 Glutathione 0.1 - 10 μg / mL 9 Progesterone 1 - 10 ng / mL 10 Retinol 0.01 - 2 μg / mL 11 Vitamin A 0.01 - 2 μg / mL 12 dl-α-Tocopheryl acetate 0.1 - 10 μg / mL 13 Vitamin E 0.1 - 10 μg / mL 14 Linoleic acid 0.1 - 10 μg / mL 15 α-Linolenic acid 0.1 - 10 μg / mL 16 Lipoic acid 1 - 10 ng / mL 17 bFGF 0.5 - 10 ng / mL 18 PDGF-AA 0.5 - 10 ng / mL

[0053] In a specific embodiment of the present invention, the composition of the "oligodendrocyte progenitor cell proliferation medium" used is substantially the same as the composition and concentration range in Table 1 above, except that the concentration range of bFGF used in the "oligodendrocyte progenitor cell proliferation medium" is 1-15 ng / mL, and the concentration range of PDGF-AA is 1-15 ng / mL. The "oligodendrocyte progenitor cell proliferation medium" prepared within the concentration range of each of its components can be used for the proliferation culture of the present invention.

[0054] In a specific embodiment of the present invention, the cell culture substrates used for primary MSC culture, first passage culture, and second passage culture can be cell culture dishes, cell culture plates, or cell factories; the cell culture substrate used for the differentiation culture of MSC can be a sealable cell culture flask.

[0055] In a specific embodiment of the present invention, the cell culture substrates used for the proliferation culture of differentiated oligodendrocyte progenitor cells can be cell culture dishes, cell culture plates, or cell factories.

[0056] Example 1

[0057] This example provides a method for differentiating MSC into oligodendrocyte progenitor cells, and the specific steps are as follows:

[0058] (1) Primary MSC culture

[0059] After diluting 1 mL of healthy adult bone marrow stock solution with 9 mL of MSC basal medium, it was cultured in a 100 mm diameter round culture dish on the TCT (abbreviation for "treated to promote cell adhesion") surface for 3 days, then all the medium was removed and replaced with 10 mL of fresh MSC basal medium. MSC at this stage is defined as P0. P0 MSC was cultured in MSC basal medium for another 10 days, with the medium changed every 2 days, 10 mL each time. The composition of the MSC basal medium is MEM-alpha + 5% human platelet extract.

[0060] (2) First passage culture

[0061] After culturing P0 MSC for 10 days, it was digested and collected with TrypLE Express, and then passaged at a ratio of 1:3 to a 100 mm diameter round culture dish on the TCT surface. These MSC are defined as P1, and P1 MSC was cultured in 10 mL of MSC basal medium for 2 days.

[0062] (3) Second passage culture

[0063] After culturing P1 MSC for 2 days, it was digested and collected with TrypLE Express, and then 2×104 cells / cm 2 At an inoculation density of 4 , the cells were passaged into a non-treated cell adhesion (Non Tc-Treated, abbreviated as nTCT) polystyrene 6-well cell culture plate, with 2 mL of medium in each well. These MSCs were defined as P2, and the P2 MSCs were cultured in MSC basal medium for 2 days.

[0064] (4) Differentiation of MSCs into oligodendrocyte progenitor cells - vector treatment

[0065] After culturing the P2 MSCs for 2 days, they were digested and collected using TrypLE Express. First, the MSCs were treated with an "oligodendrocyte progenitor cell differentiation vector" containing an epigenetic gene expression vector. As described above, the vector can be a viral vector such as a lentivirus, a plasmid vector, a free vector, or an mRNA vector. In this example, a plasmid vector was used, and its architecture can refer to the following publicly available vectors:

[0066] Olig2: https: / / www.addgene.org / 32933 /

[0067] Sox10: https: / / www.addgene.org / 167791 /

[0068] The MSCs were transfected with the plasmid vector in the form of a cell suspension. The transfection process used a nucleofector (e.g., a Lonza product: https: / / bioscience.lonza.com / lonza_bs / CH / en / Catalogue / Products / Transfection / c / 6). For every 5×10 6 cells, 5 - 10 μg of Olig2 and 5 - 10 μg of Sox10 plasmids were transfected.

[0069] (5) Differentiation of MSCs into oligodendrocyte progenitor cells - differentiation culture

[0070] After the vector treatment was completed, the cells were inoculated at an initial density of 0.1×10 4 / cm 2 -1.0×10 4 / cm 2 into a polystyrene 6-well cell culture plate that had been treated with "promoting cell adhesion" and "recombinant human laminin", with 2 mL of medium in each well. Then, "oligodendrocyte progenitor cell differentiation medium" was used for 20 days of differentiation culture.

[0071] The step of performing the "recombinant human laminin" treatment is specifically as follows: Soak and plate the surface of the culture plate with a recombinant human laminin solution at a concentration of 5 - 30 μg / mL for 30 minutes at a temperature of 37 °C. In this example, a recombinant human laminin solution with a concentration of 20 μg / mL is specifically used.

[0072] Table 2 shows the differentiation results of MSCs into oligodendrocytes under different initial MSC densities and different vector concentrations. After 20 days of differentiation culture and confirmation that the plasmid vector is not present inside the cells, analyze the percentage of positive Olig2, a marker for oligodendrocyte progenitor cells, and whether the cells have the typical morphology of oligodendrocyte progenitor cells. Take the criteria for successful differentiation as being positive for Olig2 and having the typical morphology of oligodendrocyte progenitor cells, then calculate the cell yield to determine the optimal differentiation conditions. The "oligodendrocyte progenitor cell differentiation vector" combinations are listed in Table 3, and the components of the "oligodendrocyte progenitor cell differentiation medium" are listed in Table 4.

[0073] Table 2 Detection of the positive rate of markers for MSC oligodendrocyte progenitor cells

[0074]

[0075]

[0076] Table 3 Differentiation vector combinations

[0077] Group Concentration of key gene Olig2 vector Concentration of key gene Sox10 vector 01 <![CDATA[5 μg / 1×10 6 cells]]> <![CDATA[5 μg / 1×10 6 cells]]> 02 <![CDATA[10 μg / 1×10 6 cells]]> <![CDATA[5 μg / 1×10 6 cells]]> 03 <![CDATA[5 μg / 1×10 6 cells]]> <![CDATA[10 μg / 1×10 6 cells]]> 04 <![CDATA[10 μg / 1×10 6 cells]]> <![CDATA[10 μg / 1×10 6 cells]]>

[0078] Table 4 Components of the oligodendrocyte progenitor cell differentiation medium

[0079] Serial number Component Concentration 1 DMEM / F12 / 2 Non-essential amino acids (100x) 1x, i.e., 1% (volume ratio) 3 Insulin 5 μg / mL 4 Transferrin holoferric 5 μg / mL 5 Putrescine 20 μg / mL 6 Human serum albumin 2500 μg / mL 7 Superoxide dismutase 2 μg / mL 8 Glutathione 1 μg / mL 9 Progesterone 5 ng / mL 10 Retinol 0.2 μg / mL 11 Vitamin A 0.2 μg / mL 12 dl-α-Tocopheryl acetate 1 μg / mL 13 Vitamin E 1 μg / mL 14 Linoleic acid 1 μg / mL 15 α-Linolenic acid 1 μg / mL 16 Lipoic acid 10 ng / mL 17 bFGF 5 ng / mL 18 PDGF-AA 5 ng / mL

[0080] From the results in Table 2, it was found that at different culture time points with different initial densities, oligodendrocyte progenitor cells with a purity (i.e., the percentage positive for Olig2 and having a typical morphology) ≥ 8% can be obtained using different "oligodendrocyte progenitor cell differentiation vectors". The highest yield of Olig2 - positive cells per cm 2 can reach 10466.42 ± 1209.59 / cm 2 , among which the yield of oligodendrocyte progenitor cells using the "oligodendrocyte progenitor cell differentiation vector" combination 04 is the highest at different seeding densities. Finally, the combination of "oligodendrocyte progenitor cell differentiation vector" combination 04 and a seeding density of 1.0×10 4 / cm 2 was preferably selected.

[0081] In the preliminary screening experiments of the present inventors, it was also found that too high an initial density of MSCs would affect the differentiation efficiency. Exceeding 1.0×10 4 / cm2 The output no longer increases, and the yield further decreases. Regarding the concentration of the "oligodendrocyte progenitor cell differentiation vector", it can be predicted that further increasing the vector concentration has the opportunity to increase the yield and output, but there is an upper limit to the vector concentration, and excessive concentration will cause toxic reactions.

[0082] Example 2

[0083] To ensure that there are enough oligodendrocyte progenitor cells for patient treatment and the purity is high enough, this example provides a method for shaking and purifying the obtained oligodendrocyte progenitor cells and amplifying them under animal-free conditions. The specific steps are as follows:

[0084] (1) After completing the differentiation culture of oligodendrocyte progenitor cells using the method of Example 1 (with the seeding density in step (5) being 1.0×10 4 / cm 2 and the "oligodendrocyte progenitor cell differentiation vector" being the combination 04 in Table 2), place the cell culture flask in a shaking device (as shown in Figure 4 , with the equipment model being S41i Incubator Shaker, Eppendorf). After ensuring sealing, perform horizontal shaking at 37°C at 120 - 240 times per minute with an amplitude of 1.5 - 4.5 cm for 15 - 20 hours to promote the separation of oligodendrocyte progenitor cells from the surface of the culture medium matrix, while the remaining cells remain adherent;

[0085] (2) After completing the shaking, collect the culture medium containing oligodendrocyte progenitor cells and inject it onto the surface of a new cell culture medium plate that has been treated with "promoting cell adhesion" and "recombinant human laminin" at an MSC seeding density of 0.25 - 1.5×10 4 / cm 2 (subculture of oligodendrocyte progenitor cells), and add the "oligodendrocyte progenitor cell proliferation medium" for proliferation culture for 6 - 12 days to complete the proliferation of oligodendrocyte progenitor cells.

[0086] Table 5 shows the purity and quantity of oligodendrocytes obtained using different shaking parameters in step (2), where the purity is judged by the positive percentage of the oligodendrocyte progenitor cell marker Olig2. Group 17 in Table 5 is the result obtained without shaking, but only standing in the shaking device for 20 h. Table 6 shows the output and purity of oligodendrocyte progenitor cells after inoculating oligodendrocyte progenitor cells at different seeding densities and completing 6 - 12 days of amplification culture, where the purity is judged by the Olig2 positive percentage. The components of the "oligodendrocyte progenitor cell proliferation medium" are listed in Table 7.

[0087] Table 5 Shaking Parameters and Results for Purifying Oligodendrocyte Progenitor Cells

[0088]

[0089] Table 6 Results of oligodendrocyte progenitor cell expansion

[0090]

[0091]

[0092] Table 7 Components of the oligodendrocyte progenitor cell expansion medium

[0093] Serial number Component Concentration 1 DMEM / F12 / 2 Non-essential amino acids (100x) 1x, i.e., 1% (volume ratio) 3 Insulin 5 μg / mL 4 Transferrin holoferric 5 μg / mL 5 Putrescine 20 μg / mL 6 Human serum albumin 2500 μg / mL 7 Superoxide dismutase 2 μg / mL 8 Glutathione 1 μg / mL 9 Progesterone 5 ng / mL 10 Retinol 0.2 μg / mL 11 Vitamin A 0.2 μg / mL 12 dl-α-Tocopheryl acetate 1 μg / mL 13 Vitamin E 1 μg / mL 14 Linoleic acid 1 μg / mL 15 α-Linolenic acid 1 μg / mL 16 Lipoic acid 10 ng / mL 17 bFGF 10 ng / mL 18 PDGF-AA 10 ng / mL

[0094] From the results in Table 5, it was found that oligodendrocyte progenitor cells with a purity ≥ 70% could be obtained with different combinations of shaking parameters. When no shaking was performed, only oligodendrocyte progenitor cells with a purity of approximately 47% and a yield of only 16.33 ± 4.40 were obtained, indicating that the purity and yield of the obtained oligodendrocyte progenitor cells could be significantly improved by shaking treatment. The highest purity of Olig2-positive cells was obtained under the condition of "120 shakes per minute, shaking amplitude of 1.5 or 3.0 cm, and shaking time of 15 or 20 hours", but this condition was not adopted considering the yield. The highest yield of Olig2-positive cells was obtained under the condition of "240 shakes per minute, shaking amplitude of 1.5 or 3.0 cm, and shaking time of 15 or 20 hours", but the purity was already lower than 80%. Therefore, considering both the purity and yield dual indicators, the optimal shaking condition was "180 shakes per minute, shaking amplitude of 3.0 cm, and shaking time of 20 hours", and the purity of the obtained Olig2-positive cells reached 82.82 ± 1.62% and the yield reached 4388.33 ± 259.93 / cm 2 。

[0095] For expansion, from the results in Table 6, it was found that under the condition of "proliferating for 9 days with an initial density of 1.0×10 4 / cm 2 ", the yield of Olig2-positive cells per cm 2 was relatively high, reaching 8396.28 / cm 2 / day (obtained by dividing the yield by the number of proliferation days), which was the most cost-effective result economically. Under all expansion conditions, the final percentage of Olig2-positive cells reached over 90%.

[0096] To further confirm the identity of the obtained oligodendrocyte progenitor cells, optical microscopy observation and immunofluorescence detection were performed on the oligodendrocyte progenitor cells differentiated from MSCs. The results are as Figure 1 shown, Figure 1 and are the typical morphological features of oligodendrocyte progenitor cells under bright-field optical microscopy observation (after shaking purification, the cell morphology can be seen to be highly consistent, indicating an increase in purity).Figure 2 It is a fluorescence imaging detection diagram of oligodendrocyte progenitor cell markers PDGFRα and A2B5, further proving the identity of the prepared oligodendrocyte progenitor cells.

[0097] To further confirm the purity of the obtained oligodendrocyte progenitor cells, the percentage of Olig2 positivity of the cells after shaking purification and amplification was detected by flow cytometry. The results showed that the percentage of Qlig2 positivity before purification was ≥8%, while the percentage of Qlig2 positivity after purification was ≥70%, and preferably the conditions could reach ≥80%. After amplification of the purified OP, the purity could reach ≥90%.

[0098] To further confirm the function of the obtained oligodendrocyte progenitor cells, the oligodendrocyte progenitor cells differentiated from MSCs were co-cultured with neurons, and it was observed that the oligodendrocyte progenitor cells developed into oligodendrocytes and generated myelin sheaths. The results were as Figure 3 shown. Oligodendrocytes expressing myelin sheath markers (MBP, green fluorescence signal) highly overlapped with axons (NF200, red fluorescence signal), proving that oligodendrocyte progenitor cells are functional, can develop into oligodendrocytes and generate myelin sheaths, and have the potential to repair demyelinating injuries of the nervous system and treat demyelinating diseases.

[0099] The above results indicate that treating MSCs with the "oligodendrocyte progenitor cell differentiation vector" and then culturing them with the "oligodendrocyte progenitor cell differentiation medium" can obtain oligodendrocyte progenitor cells with a purity of ≥8% within 20 days. Coupled with shaking purification and proliferation, only an additional 20 hours of shaking time and 9 days of proliferation time are required to obtain a large number of high-purity (percentage of Olig2 positivity ≥90%) oligodendrocyte progenitor cells. The present invention not only greatly shortens the production time of oligodendrocyte progenitor cells, but also successfully discovers a new method for differentiating mesenchymal stem cells into oligodendrocyte progenitor cells, while solving the two major problems of ethics and production time. The present invention uses animal-free materials, and the produced cells are suitable for future clinical applications.

[0100] Application Example 1 The oligodendrocyte progenitor cells prepared by the method of the present invention can be applied to the treatment of neurological diseases

[0101] I. For leukodystrophy

[0102] Using leukodystrophy mice as a disease model, oligodendrocyte progenitor cells were minimally invasively injected into the corpus callosum of the left and right brains respectively. The number of transplanted cells was 3×10 5 cells per side, which can regenerate white matter, restore brain function, and extend the lifespan of animals.

[0103] II. For stroke

[0104] A stroke disease model was established in SD rats by middle cerebral artery occlusion method. Minimally invasive injection of oligodendrocyte progenitor cells was performed near the stroke area, and the number of transplanted cells was 3×10 6 cells, which can reduce inflammation, repair nerves, and restore brain function.

[0105] Myelin sheath damage occurs in many neurological diseases and is closely related to the loss of nerve function. Oligodendrocyte progenitor cells are a kind of versatile cells that can exert therapeutic effects on various diseases. Therefore, the oligodendrocyte progenitor 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.

[0106] Figure 3 This is the fluorescence imaging detection map of in vitro myelinogenesis of MSC-derived oligodendrocyte progenitor cells prepared by the present invention. Among them, MBP is a myelinogenesis marker (green), and NF200 is a nerve axon marker (red). From Figure 3 the results, it can be seen that after the MSC-derived oligodendrocyte progenitor cells were inoculated into the nerve axon network, they matured into MBP-positive oligodendrocytes, and the green fluorescence signal of MBP overlapped with the red fluorescence signal of NF200, proving that they generated myelin sheaths around the nerve axons.

[0107] In summary, the method of the present invention uses a new differentiation method to treat MSCs with an "oligodendrocyte progenitor cell differentiation vector" and then culture them with an "oligodendrocyte progenitor cell differentiation medium", which can produce oligodendrocyte progenitor cells in a short time, and these oligodendrocyte progenitor cells have the function of generating myelin sheaths.

[0108] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for promoting the differentiation of mesenchymal stem cells into oligodendrocyte precursor cells, characterized in that, It includes the following steps: (1) Isolate mesenchymal stem cells and perform primary culture; (2) Subculture the mesenchymal stem cells after primary culture and inoculate them on the surface of the cell culture matrix, add cell culture medium, and perform the first subculture. The cell culture matrix has been treated with "promoting cell adhesion"; (3) Subculture the mesenchymal stem cells after the first subculture and inoculate them again on the surface of the cell culture matrix, add cell culture medium, and perform the second subculture. The cell culture matrix has not been treated with "promoting cell adhesion"; (4) Treat the mesenchymal stem cells after the second subculture with a gene expression vector containing the "key genes for the differentiation of oligodendrocyte progenitor cells", and then subculture and inoculate them again on the surface of the cell culture matrix, add the "differentiation medium for oligodendrocyte progenitor cells" for differentiation culture to complete the differentiation of oligodendrocyte progenitor cells. The cell culture matrix has been treated with "promoting cell adhesion" and "recombinant human laminin"; The gene expression vector of the "key genes for the differentiation of oligodendrocyte progenitor cells" described in step (4) contains genes including Olig2 and Sox10; The specific steps of the treatment with the gene expression vector containing the "key gene for oligodendrocyte progenitor cell differentiation" described in step (4) are as follows: The mesenchymal stem cells after the second passage are transfected with the plasmid vector in the form of a cell suspension, and 5-10 μg of Olig2 and 5-10 μg of Sox10 plasmids are electrotransfected into every 1×10 6 cells; The "differentiation medium for oligodendrocyte progenitor cells" includes the following components: non-essential amino acids 0.1 - 5%, insulin 0.1 - 10 μg / mL, holo-transferrin 2 - 100 μg / mL, putrescine 5 - 200 μg / mL, human serum albumin 250 - 4000 μg / mL, superoxide dismutase 1 - 10 μg / mL, glutathione 0.1 - 10 μg / mL, progesterone 1 - 10 ng / mL, retinol 0.01 - 2 μg / mL, vitamin A 0.01 - 2 μg / mL, dl-α-tocopheryl acetate 0.1 - 10 μg / mL, vitamin E 0.1 - 10 μg / mL, linoleic acid 0.1 - 10 μg / mL, α-linolenic acid 0.1 - 10 μg / mL, lipoic acid 1 - 10 ng / mL, bFGF 0.5 - 10 ng / mL, PDGF-AA 0.5 - 10 ng / mL.

2. The method for promoting the differentiation of mesenchymal stem cells into oligodendrocyte precursor cells according to claim 1, characterized in that, The mesenchymal stem cells described in step (1) are of human origin.

3. The method for promoting the differentiation of mesenchymal stem cells into oligodendrocyte precursor cells according to claim 2, wherein The sources of the mesenchymal stem cells include bone marrow, cord blood, umbilical cord, placenta, and adipose tissue.

4. The method for promoting the differentiation of mesenchymal stem cells into oligodendrocyte progenitor cells according to claim 1, wherein The cell culture matrix in steps (2) and (3) is a cell culture dish, a cell culture plate, or a cell factory; the cell culture matrix in step (4) is a cell culture dish or a cell culture flask.

5. The method for promoting the differentiation of mesenchymal stem cells into oligodendrocyte precursor cells according to claim 1, wherein The mesenchymal stem cell seeding density described in step (4) is 0.1×10 4 / cm 2 ~1.0×10 4 / cm 2 .

6. The method according to claim 5, wherein The starting density of the mesenchymal stem cells described in step (4) is 1.0×10 4 / cm 2 .

7. The method according to claim 1, wherein The time for the differentiation culture described in step (4) is 15 - 25 days.

8. The method according to claim 7, wherein The time for the differentiation culture described in step (4) is 20 days.

Images