Induction medium and culture method for promoting differentiation of stem cells into neural stem cells

Abstract

The present invention provides an induction medium and a culture method for promoting the differentiation of mesenchymal stem cells into neural stem cells. The induction medium comprises 0.25 mg / mL to 1 mg / mL Lycium barbarum polysaccharide.

Description

Induction culture medium and culture method for promoting stem cell differentiation into neural stem cells Technical Field

[0001] This invention relates to the field of cell-related technologies, and in particular to an induction culture medium and culture method for promoting the differentiation of stem cells into neural stem cells. Background Technology

[0002] Stem cells are a type of cell with self-renewal and multi-lineage differentiation potential, holding an extremely important position and 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. Neural stem cells, in particular, because they can differentiate into neurons, astrocytes, and oligodendrocytes, have enormous potential value in the treatment of neurological diseases such as Parkinson's disease, Alzheimer's disease, spinal cord injury, and stroke.

[0003] Neural stem cells play a crucial 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 steps in inducing stem cell differentiation. Existing induction media suffer from low efficiency, long time consumption, and low differentiation purity, making it difficult to obtain large quantities of neural stem cells and limiting the rapid development and widespread application of stem cell technology in the treatment of neurological diseases. Summary of the Invention

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

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

[0007] Goji berry polysaccharides are natural active substances extracted from goji berries. They are composed of six monosaccharide components, including arabinose, glucose, galactose, mannose, xylose, and rhamnose. Studies have shown that they have a variety of biological activities and pharmacological effects, such as enhancing immunity, anti-oxidation and anti-aging, regulating blood sugar, and improving vision.

[0008] The inventors of this invention discovered that adding it to an induction culture medium can promote the induced differentiation of stem cells into neural stem cells. In this induction culture medium, the concentration of Lycium barbarum polysaccharide is 0.25 mg / mL to 1 mg / mL, that is, the content of Lycium barbarum polysaccharide in each milliliter of induction culture medium is between 0.25 mg and 1 mg. For example, the concentration of Lycium barbarum polysaccharide in the induction culture medium can be 0.25 mg / mL, 0.5 mg / mL, 0.75 mg / mL, or 1 mg / mL.

[0009] The inventors of this invention have discovered that adding Lycium barbarum polysaccharides within this concentration range to the stem cell induction culture medium can significantly improve the differentiation efficiency of stem cells into neural stem cells. At the same time, Lycium barbarum polysaccharides have the advantages of being widely available and inexpensive. Compared with traditional induction methods that rely solely on expensive cytokines, this invention reduces the cost of the culture medium by improving its culture efficiency while ensuring the induction effect, thus making it possible to obtain neural stem cells in large quantities and providing support for subsequent clinical research and large-scale use of neural stem cells.

[0010] Specifically, it is possible that the interaction between Lycium barbarum polysaccharides and receptors on the cell surface activates intracellular growth factor signaling pathways. At the same time, Lycium barbarum polysaccharides can provide energy for cell differentiation, and their antioxidant and anti-apoptotic effects can provide a relatively stable redox environment, reducing the interference of oxidative damage on cell differentiation.

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

[0012] In an optional embodiment of the first aspect of the present invention, the cytokine induction culture 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-inducing culture medium provides nutrients and necessary conditions for the proliferation culture process, and assists in the interaction with Lycium barbarum polysaccharides to induce the differentiation of stem cells into neural stem cells. In this embodiment of the invention, the cytokine-inducing culture medium consists of 10-30 ng / ml EGF, 10-30 ng / ml bFGF, 0.5%-2% B27, 0.1%-1% N2, and DMEM / F12.

[0014] Epidermal growth factor (EGF) is an important growth factor that promotes stem cell proliferation. It increases cell number by activating related signaling pathways (such as the MAPK and PI3K / Akt pathways) to promote stem cell cell cycle entry and DNA synthesis. Adding an appropriate amount of EGF to the culture medium helps maintain the proliferative capacity of stem cells, providing a sufficient cellular basis for the induced differentiation of neural stem cells. In the induction medium, the concentration of EGF added is 10-30 ng / ml, meaning that the content of EGF in each 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, or 30 ng / ml.

[0015] bFGF (basic fibroblast growth factor) is a potent mitotic factor and morphogenetic differentiation inducer. It not only promotes cell proliferation but also induces stem cells to differentiate in specific directions. During the induction of neural stem cells, bFGF can synergistically act with other growth factors to promote the differentiation of stem cells into neural stem cells. Furthermore, bFGF also promotes 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 concentration of bFGF added is 10-30 ng / ml, meaning that the content of bFGF in each 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, or 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 neural cells. These supplements support the growth and differentiation of neural stem cells, promoting neuronal formation and the establishment of synaptic connections. Adding appropriate amounts of B27 and N2 to the stem cell culture medium helps improve the induction efficiency and quality of neural stem cells. In the induction culture medium, the concentration percentage of B27 is 0.5%–2%, specifically 0.5%, 1%, 1.5%, or 2%; the concentration percentage of N2 is 0.1%–1%, specifically 0.1%, 0.3%, 0.5%, 0.7%, or 1%.

[0017] DMEM / F12 medium is a 1:1 mixture of DMEM and Ham's F-12 medium. It combines the advantages of both media, providing the various nutrients required for cell growth, including sugars, amino acids, vitamins, inorganic salts, and growth factors. This medium is characterized by its comprehensive nutrition, wide applicability, strong buffering capacity, and low toxicity, providing a stable and suitable growth environment for stem cells. During the induction of neural stem cells, DMEM / F12 medium supports stem cell proliferation and differentiation, promoting the generation and development of neural stem cells.

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

[0019] As a second aspect of the invention, the invention claims protection for the use of Lycium barbarum polysaccharide in an induction culture medium for stem cell differentiation into neural stem cells.

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

[0021] As a third aspect of the invention, the invention claims protection for the use of the induction culture medium as described in any of the first aspects of the invention in promoting the induced differentiation of stem cells into neural stem cells.

[0022] The corresponding induction culture medium is beneficial for 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 this invention, the corresponding stem cells include one or more of the following: hematopoietic stem cells, embryonic stem cells, mesenchymal stem cells, and induced pluripotent stem cells, which can differentiate into neural stem cells. For example, when hematopoietic stem cells are used, they can be co-cultured with neural stem cells simultaneously. The corresponding mesenchymal stem cells can be bone marrow mesenchymal stem cells, adipose-derived mesenchymal stem cells, and umbilical cord mesenchymal stem cells, etc.

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

[0026] Specifically, in a fourth aspect of the invention, protection is claimed for a corresponding induction method, which involves adding corresponding mesenchymal stem cells to an induction culture medium as described above for culturing to obtain corresponding neural stem cells. The corresponding mesenchymal stem cells are preferably umbilical cord mesenchymal stem cells, which have a higher probability of differentiating into neural stem cells than adipose-derived mesenchymal stem cells, and are easier to obtain than bone marrow mesenchymal stem cells.

[0027] An optimized embodiment of the induction method in the fourth aspect of the present invention includes the following steps: culturing P2 generation mesenchymal stem cells until the cell confluence reaches 70%~80%, digesting and collecting the cells with enzymes, resuspending the collected mesenchymal stem cell pellet in the induction medium, and seeding it in a six-well plate for culture.

[0028] When inducing the mesenchymal stem cells to differentiate into neural stem cells, the above-mentioned induction medium is used to culture P3 generation mesenchymal stem cells to obtain neural stem cells.

[0029] Specifically, P2 generation mesenchymal stem cells are passaged and cultured. The specific steps for culturing P2 generation stem cells to obtain P3 generation stem cells are as follows: when the cell confluence reaches 70%~80%, the cells are digested with Tryple, the cell pellet is collected and resuspended in the aforementioned induction medium, and then seeded into a six-well plate for culture.

[0030] In an optimized embodiment of the induction method of the fourth aspect of the present invention, the inoculation density is 3*102 5 / well, and perform a full or partial medium change every 2-3 days during the culture process.

[0031] Specifically, the seeding density of the P3 generation mesenchymal stem cells in the six-well plate is 3*10-1. 5 / well, the medium should be completely or partially changed every 2 to 3 days during the culture process to ensure that the cell culture has a sufficient nutrient environment.

[0032] In an optimized embodiment 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 prior to the above step includes: isolation and culture of umbilical cord mesenchymal stem cells to P2 generation cells.

[0033] Specifically, this invention uses umbilical cord mesenchymal stem cells, and before culturing, it also includes the isolation and passage culture of umbilical cord mesenchymal cells.

[0034] Among them, umbilical cord mesenchymal stem cells can be isolated and cultured to the third generation for use or commercially available resuscitation, and the corresponding isolation and culture methods are consistent with existing methods.

[0035] In one specific method, human umbilical cord mesenchymal stem cells are minced, separated, and cultured. During the passage of these mesenchymal stem cells to the P3 generation, a standard culture medium is used. Beneficial effects

[0036] The induction culture medium proposed in this invention, with the addition of Lycium barbarum polysaccharide, can effectively improve the efficiency of stem cell differentiation into neural stem cells, thus providing the possibility for the large-scale acquisition of neural stem cells.

[0037] Using this induction medium to culture stem cells greatly improves their differentiation induction efficiency. Attached Figure Description

[0038] To more clearly illustrate the technical solutions in this invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0039] Figure 1 shows an example of flow cytometry detection on day 3 SOX2.

[0040] Figure 2 shows an example of nestin flow cytometry detection on day 3.

[0041] Figure 3 shows an example of flow cytometry detection on day 8 SOX2.

[0042] Figure 4 shows an example of nestin flow cytometry detection on day 8.

[0043] Figure 5 shows an example of SOX2 flow cytometry detection on day 14.

[0044] Figure 6 shows an example of nestin flow cytometry detection on day 14.

[0045] Figure 7 shows a general example of Nestin flow cytometry detection;

[0046] Figure 8 shows a general example of SOX2 flow cytometry detection;

[0047] Figure 9 shows examples of microscopic images taken for each group of induced differentiation. Detailed Implementation

[0048] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0049] This invention provides an induction culture medium containing Lycium barbarum polysaccharides, thereby promoting the differentiation of stem cells into neural stem cells.

[0050] Specifically, in a specific embodiment of the present invention, umbilical cord mesenchymal stem cells are used for verification.

[0051] In this embodiment of the invention, the following experimental group and control group are included:

[0052] Blank group 1: Blank control culture medium group

[0053] Model group 2: Cytokine model culture medium group

[0054] Experimental Group 3: Umbilical cord mesenchymal stem cell group containing low concentration of wolfberry polysaccharide + cytokine mixed culture medium

[0055] Experimental Group 4: Umbilical cord mesenchymal stem cell group containing a mixed culture medium of medium concentration of wolfberry polysaccharide and cytokines

[0056] Experimental Group 5: Umbilical cord mesenchymal stem cell group containing high concentration of wolfberry polysaccharide + cytokine mixed culture medium

[0057] Specifically, the blank control group used an induction medium containing DMEM / F12 and FBS; model group 2 used a cytokine induction medium without Lycium barbarum polysaccharide, specifically containing 20 ng / ml EGF, 20 ng / ml bFGF, 1.5% B27, 0.5% N2, and DMEM / F12. Experimental groups 3-5 used an induction medium containing Lycium barbarum polysaccharide in addition to the induction medium of model group 2. The concentrations of Lycium barbarum polysaccharide in experimental groups 3, 4, and 5 were 0.25 mg / mL, 0.5 mg / mL, and 1 mg / mL, respectively, while the concentrations of the other components in experimental groups 3-5 were the same as those in model group 2.

[0058] The culture method is as follows: P2 generation umbilical cord mesenchymal stem cells are cultured until the cell confluence reaches 70%~80%. Cells are then digested and collected using Tryple digestive enzyme. The collected umbilical cord mesenchymal stem cell pellet is resuspended in its respective induction culture medium and seeded into six-well plates at a density of 3*10⁻⁶. 5 / For each well, perform a full or partial fluid change every 2-3 days.

[0059] During the culture process of this embodiment of the invention, the cell state was recorded, and its state images were taken on days 3, 8, and 14 of culture. The cells were collected, and anti-Nestin and anti-Sox2 fluorescently labeled antibodies were added for flow cytometry detection.

[0060] The flow cytometry plots are shown in Figures 1-6, the flow cytometry results are shown in Tables 1-6, and the state diagram is shown in Figure 4. In the figures, group 1 is the blank control culture medium group; group 2 is the cytokine model culture medium group; group 3 is the mixed culture medium containing low Lycium barbarum polysaccharide and cytokines; group 4 is the mixed culture medium containing medium Lycium barbarum polysaccharide and cytokines; and group 5 is the mixed culture medium containing high Lycium barbarum polysaccharide and cytokines.

[0061] Table 1. Day 3 Sox2 Positive Rate (%)

[0062]

[0063] Note: 'a' indicates that compared with the blank control group, the cytokine-inducing culture medium group and the various concentrations of Lycium barbarum polysaccharide groups showed extremely significant differences (P < 0.001); compared with the cytokine-inducing control group, there were no significant differences in the various concentrations of Lycium barbarum polysaccharide groups (P > 0.5).

[0064] Table 2. Day 3 Nestin Positive Rate (%)

[0065]

[0066] Note: a indicates that compared with the blank control group, the cytokine-inducing culture medium group and each concentration of Lycium barbarum polysaccharide group had extremely significant differences (P < 0.001); b indicates that compared with the cytokine-inducing control group, each concentration of Lycium barbarum polysaccharide group had extremely significant differences (P < 0.001).

[0067] Table 3. Day 8 Sox2 Positive Rate (%)

[0068]

[0069] Note: a indicates that compared with the blank control group, the cytokine-inducing culture medium group and each concentration of Lycium barbarum polysaccharide group had extremely significant differences (P < 0.001); b indicates that compared with the cytokine-inducing control group, each concentration of Lycium barbarum polysaccharide group had extremely significant differences (P < 0.001).

[0070] Table 4. Day 8 Nestin Positive Rate (%)

[0071]

[0072] Note: a indicates that compared with the blank control group, the cytokine-inducing culture medium group and each concentration of Lycium barbarum polysaccharide group had extremely significant differences (P < 0.001); b indicates that compared with the cytokine-inducing control group, each concentration of Lycium barbarum polysaccharide group had extremely significant differences (P < 0.001).

[0073] Table 5. Sox2 Positive Rate (%) on Day 14

[0074]

[0075] Note: a indicates that compared with the blank control group, the cytokine-inducing culture medium group and each concentration of Lycium barbarum polysaccharide group had extremely significant differences (P < 0.001); b indicates that compared with the cytokine-inducing control group, each concentration of Lycium barbarum polysaccharide group had extremely significant differences (P < 0.001).

[0076] Table 6. Day 14 Nestin Positive Rate (%)

[0077]

[0078] Note: a indicates that compared with the blank control group, the cytokine-inducing culture medium group and each concentration of Lycium barbarum polysaccharide group had extremely significant differences (P < 0.001); b indicates that compared with the cytokine-inducing control group, each concentration of Lycium barbarum polysaccharide group had extremely significant differences (P < 0.001).

[0079] Referring to Figure 9, it can be seen that no obvious neuronal shape was observed in Group 1-1, indicating that Group 1 did not have the effect of inducing differentiation into neural stem cells. In Group 2, the cells first adhered to the culture wall, then gradually clustered, formed spheres, and then floated again to grow in a spherical shape. The clustering pattern was only clearly observed on the 14th day. Between Groups 3 and 5, the changes in cells were clearly visible on the third day in the Lycium barbarum polysaccharide + cytokine culture medium group. The cells mostly grew in clusters, spheres, or radial patterns similar to "sunflowers," indicating that the umbilical cord mesenchymal stem cells had gradually changed their growth mode, transforming into a suspension growth similar to neural progenitor cells. Furthermore, as the culture time increased, the cell spheres gradually enlarged, and the central color darkened, suggesting that the cells were proliferating rapidly.

[0080] Combining Figures 1-8 and Tables 1-6, the positive rates of Sox2 and Nestin on day 8 were significantly increased, and much greater than those in groups 1 and 2. In addition, combining Figure 9 and the morphology of neural stem cells, it can be seen that in groups 3 to 5 of this invention, after the addition of Lycium barbarum polysaccharide, the umbilical cord mesenchymal stem cells differentiated into neural stem cells in the early stage, and the corresponding neural stem cells had already proliferated rapidly on day 8.

[0081] In summary, the induction culture medium of the present invention can significantly improve the differentiation efficiency of stem cells into neural stem cells.

[0082] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate 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 by, The inducing medium comprises wolfberry polysaccharide, and the wolfberry polysaccharide is added in a concentration of 0.25 mg / mL to 1 mg / mL.

2. The induction medium for facilitating differentiation of stem cells into neural stem cells according to claim 1, wherein The inducing medium further comprises a cytokine inducing medium.

3. The induction medium for facilitating differentiation of stem cells into neural stem cells according to claim 2, wherein The cytokine inducing medium comprises 10-30 ng / mL EGF, 10-30 ng / mL bFGF, 0.5%-2% B27, 0.1%-1% N2 and DMEM / F12.

4. Application of wolfberry polysaccharide in an inducing medium for differentiating stem cells into neural stem cells.

5. Application of the inducing medium according to any one of claims 1-3 in differentiating stem cells into neural stem cells.

6. Use of the induction medium according to claim 5 for promoting the 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. An induction method for inducing differentiation of mesenchymal stem cells into neural stem cells, characterized by, The mesenchymal stem cells are added into the inducing medium according to any one of claims 1-3 for culture.

8. The method for inducing mesenchymal stem cells to differentiate into neural stem cells according to claim 7, wherein, The method comprises the steps of culturing P2 generation mesenchymal stem cells, digesting the cells with an enzyme when the cell confluence reaches 70%-80% and collecting the cells, resuspending the collected mesenchymal stem cell pellets in the inducing medium and inoculating the mesenchymal stem cells into a six-well plate for culture.

9. The method for inducing differentiation of stem cells into neural stem cells according to claim 8, wherein The inoculation density is 3*10 5 / well, and full or half medium replacement is performed every 2-3 days during the culture.

10. The method for inducing differentiation of stem cells into neural stem cells according to claim 7, wherein 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 the umbilical cord mesenchymal stem cells to P2 generation cells before the step.

Images