Induction culture medium for promoting stem cells to differentiate into neural stem cells and culture method

Abstract

The invention provides an induction culture medium for promoting stem cells to differentiate into neural stem cells and a culture method, the induction culture medium comprises lycium barbarum polysaccharide, and the addition concentration of the lycium barbarum polysaccharide in the induction culture medium is 0.25 mg / mL-1mg / ml. When the lycium barbarum polysaccharide in the concentration range is added into an induction culture medium of the stem cells, the differentiation efficiency of the stem cells into the neural stem cells can be remarkably improved, meanwhile, the lycium barbarum polysaccharide has the advantages of being wide in source and low in price, and compared with a traditional induction method only depending on expensive cell factors, the method has the advantages that the induction effect is guaranteed, and meanwhile the differentiation efficiency of the stem cells into the neural stem cells is greatly improved. By improving the culture efficiency, the cost of the culture medium is reduced, and possibility is provided for obtaining a large number of neural stem cells.

Description

Technical Field

[0001] The present invention relates to the field of cell-related technologies, and in particular to an induction culture medium and a culture method for promoting stem cells to differentiate into neural stem cells. Background Art

[0002] Stem cells are a type of cell with the potential for self-renewal and multidirectional differentiation. They play an extremely important role and have broad application prospects in the biomedical field. Stem cells can differentiate into various types of functional cells, providing new ideas and methods for tissue repair, organ regeneration and the treatment of various diseases. In particular, neural stem cells, because they can differentiate into neural cells such as neurons, astrocytes and oligodendrocytes, have great potential value for the treatment of nervous system diseases such as Parkinson's disease, Alzheimer's disease, spinal cord injury, stroke, etc.

[0003] Neural stem cells play a key role in the development, repair and regeneration of the nervous system. Therefore, inducing stem cells to differentiate into neural stem cells has broad application prospects in neuroscience research, disease treatment and regenerative medicine.

[0004] The preparation of culture medium is one of the key links in inducing stem cell differentiation. The existing induction culture medium has the problems of low efficiency, long time consumption and low differentiation purity, which makes it difficult to obtain a large number of neural stem cells, limiting the rapid development and wide application of stem cell technology in the field of neurological disease treatment. Summary of the invention

[0005] The present invention aims to provide an induction culture medium and a culture method for promoting stem cells to differentiate into neural stem cells, so as to solve the problems of low culture efficiency and low differentiation purity of stem cells induced to differentiate into neural stem cells in the prior art.

[0006] As a first aspect of the present invention, the present invention provides an induction medium for promoting differentiation of stem cells into neural stem cells, wherein the induction medium comprises Lycium barbarum polysaccharide, and the added concentration of Lycium barbarum polysaccharide in the induction medium is 0.25 mg / mL-1 mg / ml.

[0007] Lycium barbarum polysaccharide is a natural active substance extracted from wolfberry. It is composed of six monosaccharide components, including arabinose, glucose, galactose, mannose, xylose, and rhamnose. Studies have shown that it has multiple biological activities and pharmacological effects. It has the effects of enhancing immunity, anti-oxidation and anti-aging, regulating blood sugar, and improving vision.

[0008] The inventors of the present invention have found that adding it to the induction medium can be used to promote the induction differentiation of stem cells into neural stem cells, wherein the concentration of Lycium barbarum polysaccharide in the induction medium is 0.25mg / mL-1mg / ml, that is, the content of Lycium barbarum polysaccharide in each milliliter of the induction medium is between 0.25 mg and 1 mg. For example, in the induction medium, the concentration of Lycium barbarum polysaccharide can be 0.25mg / mL, 0.5mg / mL, 0.75mg / mL, 1mg / mL.

[0009] The inventors of the present invention have found that Lycium barbarum polysaccharides within this concentration range, when added to the induction culture medium of stem cells, can significantly improve the differentiation efficiency of stem cells into neural stem cells. At the same time, Lycium barbarum polysaccharides have the advantages of wide sources and low prices. Compared with the traditional induction method that only relies on expensive cytokines, the present invention reduces the cost of the culture medium by improving its culture efficiency while ensuring the induction effect, thereby providing the possibility for the large-scale acquisition of neural stem cells and providing support for the subsequent clinical research and large-scale use of neural stem cells.

[0010] Specifically, it may be that Lycium barbarum polysaccharides interact with receptors on the cell surface, activating the growth factor signaling pathway in the cell. At the same time, Lycium barbarum polysaccharides can provide energy for cell differentiation, and its antioxidant and anti-apoptotic effects can provide a relatively stable redox environment, reducing the interference of oxidative damage on cell differentiation.

[0011] As an optional embodiment of the first aspect of the present invention, the induction medium also includes a cytokine induction medium.

[0012] As an optional embodiment of the first aspect of the present invention, the cytokine induction medium comprises: 10-30 ng / ml EGF, 10-30 ng / ml bFGF, 0.5%-2% B27, 0.1%-1% N2 and DMEM / F12.

[0013] Specifically, the cytokine induction medium provides nutrition and necessary conditions for the proliferation culture process, and assists in the reaction with Lycium barbarum polysaccharides to induce stem cells to differentiate into neural stem cells. In an embodiment of the present invention, the cytokine induction medium is composed of 10-30 ng / ml EGF, 10-30 ng / ml bFGF, 0.5%-2% B27, 0.1%-1% N2 and DMEM / F12.

[0014] Among them, EGF (epidermal growth factor) is an important growth factor that can promote the proliferation of stem cells. It promotes the cycle entry and DNA synthesis of stem cells by activating related signaling pathways (such as the MAPK pathway and the PI3K / Akt pathway), thereby increasing the number of cells. Adding an appropriate amount of EGF to the culture medium helps to maintain the proliferation ability of stem cells and provide sufficient cellular basis for the induced differentiation of neural stem cells. In the induction medium, the EGF is added at a concentration of 10~30ng / ml, that is, the content of EGF in each milliliter of induction medium is between 10ng and 30ng. Specifically, it can be 10ng / ml, 15ng / ml, 20ng / ml, 25ng / ml, 30ng / ml.

[0015] bFGF (basic fibroblast growth factor) is a strong mitogenic factor and morphogenetic differentiation induction factor. It can not only promote cell proliferation, but also induce stem cells to differentiate in a specific direction. In the induction process of neural stem cells, bFGF can work synergistically with other growth factors to promote the differentiation of stem cells into neural stem cells. In addition, bFGF also has the effects of promoting angiogenesis, wound healing and repair, and tissue regeneration, all of which contribute to the growth and development of neural stem cells. In the induction medium, the bFGF is added at a concentration of 10-30 ng / ml, that is, the content of bFGF per milliliter of induction medium is between 10 ng and 30 ng. Specifically, it can be 10 ng / ml, 15 ng / ml, 20 ng / ml, 25 ng / ml, 30 ng / ml.

[0016] B27 and N2 are neurotrophic factor supplements that provide a variety of growth factors, hormones and other nutrients required for the growth and differentiation of nerve cells. These supplements can support the growth and differentiation of neural stem cells, promote the formation of neurons and the establishment of synaptic connections. Adding an appropriate amount of B27 and N2 to the stem cell culture medium helps to improve the induction efficiency and quality of neural stem cells. In the induction medium, the concentration percentage of B27 is 0.5%~2%, specifically 0.5%, 1%, 1.5%, 2%; the concentration percentage of N2 is 0.1%~1%, specifically 0.1%, 0.3%, 0.5%, 0.7%, 1%.

[0017] DMEM / F12 medium is a mixture of DMEM and Ham's F-12 medium in a 1:1 ratio. It combines the advantages of the two culture media and provides various nutrients required for cell growth, including sugars, amino acids, vitamins, inorganic salts, and growth factors. This culture medium has the characteristics of comprehensive nutrition, wide application, strong buffering capacity, and low toxicity, and can provide a stable and suitable growth environment for stem cells. In the induction process of neural stem cells, DMEM / F12 medium can support the proliferation and differentiation of stem cells and promote the generation and development of neural stem cells.

[0018] In other embodiments, the cytokine induction medium may be of different combinations, such as the corresponding basal medium may be Neurobasal medium, and antioxidants may be added to the corresponding components.

[0019] As a second aspect of the present invention, the present invention seeks to protect the use of Lycium barbarum polysaccharides in an induction medium for differentiating stem cells into neural stem cells.

[0020] Adding Lycium barbarum polysaccharides to the induction culture medium of stem cells is beneficial to their induction and differentiation into neural stem cells.

[0021] As the third aspect of the present invention, the present invention seeks to protect the use of any induction medium as described in the first aspect of the present invention in promoting the induction and differentiation of stem cells into neural stem cells.

[0022] The corresponding induction culture medium is beneficial to promoting the differentiation of stem cells into neural stem cells.

[0023] In the application of the third aspect of the present invention, the stem cells include one or more of hematopoietic stem cells, embryonic stem cells, mesenchymal stem cells, and induced pluripotent stem cells.

[0024] Specifically, in the present invention, the corresponding stem cells include one or more of hematopoietic stem cells, embryonic stem cells, mesenchymal stem cells, and induced pluripotent stem cells that can differentiate into neural stem cells. For example, when the hematopoietic stem cells are used, they can be simultaneously added to neural stem cells for co-culture, and the corresponding mesenchymal stem cells can be bone marrow mesenchymal stem cells, adipose mesenchymal stem cells, and umbilical cord mesenchymal stem cells.

[0025] As a fourth aspect of the present invention, the present invention further claims a method for inducing mesenchymal stem cells to differentiate into neural stem cells, the method comprising adding mesenchymal stem cells to any induction medium as described in the first aspect of the present invention for culturing.

[0026] Specifically, in the fourth aspect of the present invention, the corresponding induction method is claimed, which is to add the corresponding mesenchymal stem cells to the induction medium as described above for culturing to obtain the corresponding neural stem cells. The corresponding mesenchymal stem cells are preferably umbilical cord mesenchymal stem cells, which are more likely to differentiate into neural stem cells than adipose mesenchymal stem cells, and are easier to obtain than bone marrow mesenchymal stem cells.

[0027] An optimized scheme of the induction method of the fourth aspect of the present invention includes the steps of: culturing P2 mesenchymal stem cells, and collecting the cells by digesting with enzymes until the cell fusion degree reaches 70% to 80%, resuspending the collected mesenchymal stem cell precipitates with the induction medium, and inoculating them in a six-well plate for culturing.

[0028] When inducing the mesenchymal stem cells to differentiate into neural stem cells, the P3 mesenchymal stem cells are cultured with the above-mentioned induction medium to obtain neural stem cells.

[0029] Specifically, P2 generation mesenchymal stem cells are subcultured, wherein the specific steps of culturing P2 generation stem cells to obtain P3 generation stem cells are as follows: when the cell fusion degree reaches 70% to 80%, the cells are digested with Tryple, the cell precipitates are collected, resuspended with the induction medium, and then inoculated in a six-well plate for culture.

[0030] In an optimized solution of the induction method of the fourth aspect of the present invention, the inoculation density is 3*10 5 / well, and the medium was fully or half replaced every 2 to 3 days during the culture process.

[0031] Specifically, the P3 mesenchymal stem cells were seeded into a six-well plate at a seeding density of 3*10 5 / well, during the culture process, full or half of the medium was replaced every 2 to 3 days to ensure that the cell culture had an adequate nutritional environment.

[0032] In an optimized solution of the induction method of the fourth aspect of the present invention, the mesenchymal stem cells are umbilical cord mesenchymal stem cells, and the step before the step further includes: isolating the umbilical cord mesenchymal stem cells and culturing them to P2 generation cells.

[0033] Specifically, the present invention uses umbilical cord mesenchymal stem cells, and before culturing, it also includes the separation and subculture of the umbilical cord mesenchymal cells.

[0034] Among them, umbilical cord mesenchymal stem cells can be separated to obtain primary cells and then cultured to the third generation for use or commercially resuscitated for use, and the corresponding separation and culture methods are consistent with existing methods.

[0035] For example, in a specific embodiment, human umbilical cord mesenchymal stem cells are cut into pieces, separated and cultured. During the process of subculturing the mesenchymal stem cells to the P3 generation cells, the culture medium used is a common culture medium.

[0036] Beneficial Effects The induction culture medium proposed by the present invention, with the addition of Lycium barbarum polysaccharide, can effectively improve the efficiency of stem cells differentiating into neural stem cells, thus providing the possibility for obtaining a large number of neural stem cells.

[0037] The induction culture medium is used to culture stem cells, and the induction differentiation efficiency is greatly improved. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0039] Figure 1 This is an example of SOX2 flow cytometry detection on day 3; Figure 2 This is an example of Nestin flow detection on day 3; Figure 3 This is an example of SOX2 flow cytometry detection on day 8; Figure 4 This is an example of Nestin flow detection on day 8; Figure 5 This is an example of SOX2 flow cytometry detection on day 14; Figure 6 This is an example of Nestin flow cytometry detection on day 14; Figure 7 This is a general example diagram of nestin flow cytometry detection; Figure 8 This is a general example diagram of SOX2 flow cytometry detection; Fig. 9 Examples of microscopic images taken for each group of induced differentiation. DETAILED DESCRIPTION

[0040] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be clearly and completely described below in conjunction with the drawings of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0041] The invention provides an induction culture medium containing wolfberry polysaccharide, thereby promoting stem cells to differentiate into neural stem cells.

[0042] Specifically, in a specific embodiment of the present invention, umbilical cord mesenchymal stem cells are cultured for verification.

[0043] In the embodiment of the present invention, the following experimental group and control group are included: Blank group 1: blank control culture medium group Model group 2: cytokine model culture medium group Experimental group 3: Umbilical cord mesenchymal stem cell group containing low concentration of Lycium barbarum polysaccharide + cytokine mixed culture medium Experimental group 4: Umbilical cord mesenchymal stem cell group containing medium concentration of Lycium barbarum polysaccharide + cytokine mixed culture medium Experimental group 5: Umbilical cord mesenchymal stem cell group containing high concentration of Lycium barbarum polysaccharide + cytokine mixed culture medium Specifically, the blank control group was an induction medium containing DMEM / F12 and FBS; the model group 2 was a cytokine induction medium without Lycium barbarum polysaccharide, and the specific cytokine induction medium contained 20ng / ml EGF, 20ng / ml bFGF, 1.5% B27, 0.5% N2, and DMEM / F12. Experimental groups 3-5 were induction mediums with Lycium barbarum polysaccharide added to model group 2, wherein the concentrations of Lycium barbarum polysaccharide in experimental groups 3, 4, and 5 were 0.25mg / mL, 0.5mg / ml, and 1mg / ml, respectively, and the concentrations of the other components in experimental groups 3-5 were the same as those in model group 2.

[0044] The culture method is as follows: culture P2 umbilical cord mesenchymal stem cells, wait until the cell confluence reaches 70%-80%, digest and collect the cells with Tryple digestive enzyme, resuspend the collected umbilical cord mesenchymal stem cell pellet with the respective induction culture medium group, and inoculate in a six-well plate at a density of 3*10 5 / well, and perform full or half medium change every 2 to 3 days.

[0045] During the culture process of the embodiment of the present invention, the cell status was recorded, and the status diagram was taken on the 3rd, 8th and 14th days of culture. The cells were collected, and anti-Nestin and anti-Sox2 fluorescent labeled antibodies were added for flow cytometry detection.

[0046] Among them, the flow chart is as follows Figure 1-6 The streaming results are shown in Table 1-6, and the state diagram is shown in Figure 4 In the figure, group 1 is a blank control culture medium group; group 2 is a cytokine model culture medium group; group 3 is a low-lycium barbarum polysaccharide + cytokine mixed culture medium group; group 4 is a medium-lycium barbarum polysaccharide + cytokine mixed culture medium group; group 5 is a high-lycium barbarum polysaccharide + cytokine mixed culture medium group.

[0047] Table 1. Day 3 Sox2 positive rate / %

[0048] Note: a means that compared with the blank control group, the cytokine induction medium group and the Lycium barbarum polysaccharide groups at various concentrations have extremely significant differences, P < 0.001; compared with the cytokine induction control group, there are no significant differences in the Lycium barbarum polysaccharide groups at various concentrations, P > 0.5.

[0049] Table 2. Day 3 Nestin positive rate / %

[0050] Note: a means that compared with the blank control group, the cytokine induction medium group and the Lycium barbarum polysaccharide groups at various concentrations have extremely significant differences, P < 0.001; b means that compared with the cytokine induction control group, the Lycium barbarum polysaccharide groups at various concentrations have extremely significant differences, P < 0.001.

[0051] Table 3. Day 8 Sox2 positive rate / %

[0052] Note: a means that compared with the blank control group, the cytokine induction medium group and the Lycium barbarum polysaccharide groups at various concentrations have extremely significant differences, P < 0.001; b means that compared with the cytokine induction control group, the Lycium barbarum polysaccharide groups at various concentrations have extremely significant differences, P < 0.001.

[0053] Table 4. Day 8 Nestin positive rate / %

[0054] Note: a means that compared with the blank control group, the cytokine induction medium group and the Lycium barbarum polysaccharide groups at various concentrations have extremely significant differences, P < 0.001; b means that compared with the cytokine induction control group, the Lycium barbarum polysaccharide groups at various concentrations have extremely significant differences, P < 0.001.

[0055] Table 5. Day 14 Sox2 positive rate / %

[0056] Note: a means that compared with the blank control group, the cytokine induction medium group and the Lycium barbarum polysaccharide groups at various concentrations have extremely significant differences, P < 0.001; b means that compared with the cytokine induction control group, the Lycium barbarum polysaccharide groups at various concentrations have extremely significant differences, P < 0.001.

[0057] Table 6. Day 14 Nestin positive rate / %

[0058] Note: a means that compared with the blank control group, the cytokine induction medium group and the Lycium barbarum polysaccharide groups at various concentrations have extremely significant differences, P < 0.001; b means that compared with the cytokine induction control group, the Lycium barbarum polysaccharide groups at various concentrations have extremely significant differences, P < 0.001.

[0059] Combined with Fig. 9 ,from Fig. 9 It can be seen that there is no obvious neuron in Group 1, indicating that Group 1 has no effect of inducing differentiation into neural stem cells; in Group 2, the cells first grow attached to the wall, and then gradually cluster and spherical, and then float again into spherical growth, and only on the 14th day can the clustering pattern be clearly seen; between Groups 3 to 5, the Lycium barbarum polysaccharide + cytokine culture medium group has obvious cell changes on the third day, and the cells are mostly clustered, spherical or "sunflower"-like radial growth, indicating that the umbilical cord mesenchymal stem cells have gradually changed their growth pattern and transformed to a suspended growth similar to that of neural precursor cells, and as the culture time increases, the cell sphere gradually increases in size and the center color becomes darker, indicating that the cells are proliferating rapidly.

[0060] Combination Figure 1-8 As shown in Table 1-6, the Sox2 positive rate and Nestin positive rate on the 8th day increased significantly, and were much greater than those in Group 1 and Group 2. Fig. 9 Combined with the morphology of neural stem cells, it can be seen that in Groups 3 to 5 of the present invention, after adding Lycium barbarum polysaccharides, the umbilical cord mesenchymal stem cells differentiated into neural stem cells in the early stage, and the corresponding neural stem cells had rapidly proliferated on the 8th day.

[0061] In summary, the induction culture medium of the present invention can significantly improve the differentiation efficiency of stem cells into neural stem cells.

[0062] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. An induction medium for promoting differentiation of stem cells into neural stem cells, characterized in that: The induction medium includes wolfberry polysaccharide, and the added concentration of the wolfberry polysaccharide in the induction medium is 0.25 mg / mL-1 mg / ml.

2. The induction medium for promoting differentiation of stem cells into neural stem cells according to claim 1, characterized in that: The induction medium also includes a cytokine induction medium.

3. The induction medium for promoting differentiation of stem cells into neural stem cells according to claim 2, characterized in that: The cytokine induction medium includes: 10-30 ng / ml EGF, 10-30 ng / ml bFGF, 0.5%-2% B27, 0.1%-1% N2 and DMEM / F12.

4. Application of Lycium barbarum polysaccharides in the induction culture medium for differentiation of stem cells into neural stem cells.

5. Use of the induction medium as claimed in any one of claims 1 to 3 in differentiating stem cells into neural stem cells.

6. Use of the induction medium according to claim 5 in promoting the induction and differentiation of stem cells into neural stem cells, characterized in that: The stem cells are one or more of hematopoietic stem cells, embryonic stem cells, mesenchymal stem cells, and induced pluripotent stem cells.

7. A method for inducing mesenchymal stem cells to differentiate into neural stem cells, characterized in that: The mesenchymal stem cells are added to the induction medium according to any one of claims 1 to 3 for culture.

8. The method for inducing mesenchymal stem cells to differentiate into neural stem cells according to claim 7, characterized in that: The method comprises the following steps: culturing P2 mesenchymal stem cells, digesting and collecting the cells with enzymes until the cell fusion degree reaches 70% to 80%, resuspending the collected mesenchymal stem cell precipitates with the induction culture medium, and inoculating the cells in a six-well plate for culturing.

9. The method for inducing stem cells to differentiate into neural stem cells according to claim 8, characterized in that: The inoculation density is 3*10 5 / well, and the medium was fully or half replaced every 2 to 3 days during the culture process.

10. The method for inducing stem cells to differentiate into neural stem cells according to claim 7, characterized in that: The mesenchymal stem cells are umbilical cord mesenchymal stem cells, and the method further comprises the steps of isolating the umbilical cord mesenchymal stem cells and culturing them to P2 generation cells before the above step.

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