Method for induced directional differentiation of iPS pluripotent stem cells into neural stem cells and application

Abstract

The invention provides a method for induced directional differentiation of iPS pluripotent stem cells into neural stem cells and application, and belongs to the technical field of stem cells. Comprising the following steps: S1, preparation of a culture medium: adding silk fibroin peptide and mitomycin C into a DMEM culture medium to prepare the culture medium; s2, subculture: inoculating the human iPS cells into a culture medium, culturing, digesting, blowing down colonies, adding into a new culture medium again, carrying out differential attachment, and collecting suspension cells; s3, induction: re-dispersing the suspension cells in the culture medium, adding an inducer, and performing induction culture to obtain differentiated neural stem cells. The method is simple, wide in raw material source and mild in condition, can induce directional differentiation of ips pluripotent stem cells into neural stem cells, can realize induction culture of neural stem cells, and has a wide application prospect.

Description

Technical Field

[0001] The present invention relates to the technical field of stem cells, and in particular to a method and application of inducing directed differentiation of iPS pluripotent stem cells into neural stem cells. Background Art

[0002] Neural stem cells are multipotent cells in the central nervous system that have the ability to self-renew and differentiate into mature brain cells. In 1992, Rynolds et al. first isolated neural stem cells from the striatum of adult rats. They can continue to proliferate in vitro and have the potential to differentiate into neurons, astrocytes and oligodendrocytes. Subsequently, multipotent stem cells were also isolated from the spinal cord, cerebellum, olfactory bulb, lateral ventricle, hippocampus and striatum. In 1998, Erikson et al. confirmed that neural stem cells also exist in the adult brain.

[0003] The establishment of in vitro stem cell isolation, expansion and differentiation technology and the realization of lateral differentiation of stem cells between different germ layers provide new strategies for the treatment of central nervous system damage, neurodegenerative diseases or inherited metabolic diseases, neurodegenerative diseases, brain tumors and cerebrovascular diseases. At present, stem cell therapy, as a new biological treatment method, is mainly developed and researched in the following aspects: first, direct cell transplantation for the dual effects of cell replacement therapy and gene therapy; second, through the study of growth factors and cytokines, endogenous stem cells are induced to proliferate and differentiate, and perform nerve self-repair.

[0004] The clinical application of stem cells is still immature. There is an urgent need to deepen the understanding of the biological characteristics of stem cells and solve problems such as maintaining the strong proliferation ability of stem cells cultured in vitro and inducing the directed differentiation of stem cells, as well as understanding the factors that control and influence the diversified transformation pathways of neural stem cells into neuronal cells. Summary of the invention

[0005] The purpose of the present invention is to propose a method for inducing directed differentiation of iPS pluripotent stem cells into neural stem cells and its application. The method is simple, has a wide source of raw materials, and has mild conditions. It can induce directed differentiation of iPS pluripotent stem cells into neural stem cells and realize the induced culture of neural stem cells, and has broad application prospects.

[0006] The technical solution of the present invention is achieved in this way:

[0007] The present invention provides a method for inducing directed differentiation of iPS pluripotent stem cells into neural stem cells, comprising the following steps:

[0008] S1. Preparation of culture medium: adding silk fibroin peptide and mitomycin C to DMEM culture medium to prepare culture medium;

[0009] S2. Subculture: Inoculate human iPS cells in culture medium, culture, digest, blow off colonies, add new culture medium, adhere to the wall at differential speed, and collect suspended cells;

[0010] S3. Induction: Re-disperse the suspended cells in the culture medium, add the inducer, induce the culture, and obtain differentiated neural stem cells.

[0011] As a further improvement of the present invention, in step S1, the concentration of the silk fibroin peptide is 30-50 mg / L, and the concentration of the mitomycin C is 10-12 mg / L.

[0012] As a further improvement of the present invention, the culture conditions in step S2 are 36-38° C., 100-120 r / min, 5 v / v% CO2 concentration, and culture for 24-36 hours.

[0013] As a further improvement of the present invention, the digestion in step S2 is performed using type IV collagenase and trypsin for 10-15 minutes.

[0014] As a further improvement of the present invention, the added amount of the type IV collagenase is 0.5-1wt%, and the added amount of the trypsin is 0.1-0.3wt%.

[0015] As a further improvement of the present invention, the time of differential wall adhesion in step S2 is 20-40 minutes.

[0016] As a further improvement of the present invention, the inducer in step S3 is ginsenoside Rg3 and icariin, with a mass ratio of 10:2-3.

[0017] As a further improvement of the present invention, the addition amount of the inducer in step S3 is 1-2 wt %.

[0018] As a further improvement of the present invention, the induction culture conditions in step S3 are 36-38° C., 100-120 r / min, 5 v / v% CO2 concentration, and culture for 10-12 h.

[0019] The present invention further protects the use of neural stem cells obtained by the above method in preparing medicines for treating nervous system diseases.

[0020] The present invention has the following beneficial effects:

[0021] Ginsenoside Rg3 is a diol ginsenoside, a rare saponin, which has neuroprotective and repairing effects on many nervous system diseases such as Parkinson's disease, Alzheimer's disease, and stroke. Icariin is the main active ingredient of Epimedium, which can also have a good repair and protection effect on nervous system diseases through antioxidant effects. The inducer prepared by the synergistic effect of the two can well induce the directional differentiation of iPS pluripotent stem cells into neural stem cells, which further helps to differentiate into specific nerve cells, playing a good role in protecting and repairing the nervous system.

[0022] The method of the invention is simple, has a wide source of raw materials, and is mild under mild conditions. It can induce ips pluripotent stem cells to differentiate into neural stem cells, can achieve the induced culture of neural stem cells, and has broad application prospects. DETAILED DESCRIPTION

[0023] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. 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.

[0024] Collagenase type IV, brand: Sigma.

[0025] Trypsin, 100,000 U / g, was purchased from Xiasheng (Beijing) Biotechnology Development Co., Ltd.

[0026] Example 1

[0027] A method for inducing directed differentiation of iPS pluripotent stem cells into neural stem cells, comprising the following steps:

[0028] S1. Preparation of culture medium: adding silk fibroin peptide and mitomycin C to DMEM culture medium to prepare culture medium; the concentration of the silk fibroin peptide is 30 mg / L, and the concentration of mitomycin C is 10 mg / L;

[0029] S2. Subculture: human iPS cells were inoculated into culture medium, cultured at 36°C, 100 rpm, 5 v / v% CO2 concentration for 24 h, digested with type IV collagenase and trypsin, the amount of type IV collagenase added was 0.5 wt%, the amount of trypsin added was 0.1 wt%, the time was 10 min, the colonies were blown off, re-added into new culture medium, differentially adhered for 20 min, and the suspended cells were collected;

[0030] S3. Induction: Re-disperse the suspended cells in the culture medium, add the inducing agent, and culture for 10 hours at 36°C, 100 rpm, and 5 v / v% CO2 concentration to obtain differentiated neural stem cells;

[0031] The inducer is ginsenoside Rg3 and icariin, the mass ratio is 10:2, and the addition amount of the inducer is 1wt%.

[0032] Example 2

[0033] A method for inducing directed differentiation of iPS pluripotent stem cells into neural stem cells, comprising the following steps:

[0034] S1. Preparation of culture medium: adding silk fibroin peptide and mitomycin C to DMEM culture medium to prepare culture medium; the concentration of the silk fibroin peptide is 50 mg / L, and the concentration of mitomycin C is 12 mg / L;

[0035] S2. Subculture: human iPS cells were inoculated into culture medium, cultured at 38°C, 120r / min, 5v / v% CO2 concentration for 36h, digested with type IV collagenase and trypsin, the amount of type IV collagenase added was 1wt%, the amount of trypsin added was 0.3wt%, the time was 15min, the colonies were blown off, re-added into new culture medium, differentially adhered for 40min, and suspended cells were collected;

[0036] S3. Induction: The suspended cells were redispersed in the culture medium, and the induction agent was added. The culture was carried out at 38°C, 120 r / min, and 5 v / v% CO2 concentration for 12 h to obtain differentiated neural stem cells;

[0037] The inducer is ginsenoside Rg3 and icariin, the mass ratio is 10:3, and the addition amount of the inducer is 2wt%.

[0038] Example 3

[0039] A method for inducing directed differentiation of iPS pluripotent stem cells into neural stem cells, comprising the following steps:

[0040] S1. Preparation of culture medium: adding silk fibroin peptide and mitomycin C to DMEM culture medium to prepare culture medium; the concentration of the silk fibroin peptide is 40 mg / L, and the concentration of mitomycin C is 11 mg / L;

[0041] S2. Subculture: human iPS cells were inoculated into culture medium, cultured at 37°C, 110r / min, 5v / v% CO2 concentration for 30h, digested with type IV collagenase and trypsin, the amount of type IV collagenase added was 0.7wt%, the amount of trypsin added was 0.2wt%, the time was 12min, the colonies were blown off, re-added into new culture medium, differentially adhered for 30min, and suspended cells were collected;

[0042] S3. Induction: Re-disperse the suspended cells in the culture medium, add the inducing agent, and culture for 11 hours at 37°C, 110r / min, and 5v / v% CO2 concentration to obtain differentiated neural stem cells;

[0043] The inducer is ginsenoside Rg3 and icariin, the mass ratio is 10:2.5, and the addition amount of the inducer is 1.5wt%.

[0044] Example 4

[0045] Compared with Example 3, the difference is that the inducer is ginsenoside Rg3.

[0046] Example 5

[0047] Compared with Example 3, the difference is that the inducer is icariin.

[0048] Comparative Example 1

[0049] Compared with Example 3, the difference is that no inducer is added.

[0050] Test Example 1 Flow cytometer detection of cell cycle

[0051] The neural stem cells prepared in Examples 1-5 and Comparative Example 1 were taken respectively, and normal human neural stem cells were selected as the control group. The neural stem cells of the third generation and the fourth day of culture were taken, digested into a single cell suspension, washed twice with PBS, fixed with 70% ethanol by volume, incubated at 4°C overnight, washed twice with PBS, digested with 100 μL RNase, and then 0.5 mL PI dye was added, incubated at 4°C for 30 minutes, and the cell cycle was detected by flow cytometry, and the proliferation index (PI) was calculated according to the formula = [(S+G2 / M) / (G0 / G1+S+G2 / M)]×100%. The results are shown in Table 1.

[0052] Table 1

[0053] Group G0 / G1(%) S(％) G2 / M(%) PI(%) Control group 45.22 27.56 27.22 54.78 Example 1 38.93 40.53 20.54 61.07 Example 2 39.12 39.69 21.19 60.88 Example 3 38.33 40.32 21.35 61.67 Example 4 41.25 32.45 26.3 58.75 Example 5 42.92 33.13 23.95 57.08 Comparative Example 1 43.88 36.09 20.03 56.12

[0054] As can be seen from the above table, the neural stem cells prepared in Examples 1-3 of the present invention have more S-phase cells and higher PI values.

[0055] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for inducing directed differentiation of iPS pluripotent stem cells into neural stem cells, characterized in that: The following steps are involved: S1. Preparation of culture medium: adding silk fibroin peptide and mitomycin C to DMEM culture medium to prepare culture medium; S2. Subculture: Inoculate human iPS cells in culture medium, culture, digest, blow off colonies, add new culture medium, adhere to the wall at differential speed, and collect suspended cells; S3. Induction: Re-disperse the suspended cells in the culture medium, add the inducing agent, induce the culture, and obtain differentiated neural stem cells.

2. The method according to claim 1, characterized in that In step S1, the concentration of the silk fibroin peptide is 30-50 mg / L, and the concentration of the mitomycin C is 10-12 mg / L.

3. The method according to claim 1, characterized in that The culture conditions in step S2 are 36-38° C., 100-120 r / min, 5 v / v% CO 2 concentration, and culture for 24-36 hours.

4. The method according to claim 1, characterized in that: The digestion in step S2 is performed using type IV collagenase and trypsin for 10-15 minutes.

5. The method according to claim 2, characterized in that: The added amount of the type IV collagenase is 0.5-1 wt %, and the added amount of the trypsin is 0.1-0.3 wt %.

6. The method according to claim 2, characterized in that The time of differential adhesion in step S2 is 20-40 minutes.

7. The method according to claim 2, characterized in that: The inducers in step S3 are ginsenoside Rg3 and icariin, with a mass ratio of 10:2-3.

8. The method according to claim 2, characterized in that: The amount of the inducer added in step S3 is 1-2 wt %.

9. The method according to claim 2, characterized in that: The induction culture conditions in step S3 are 36-38° C., 100-120 r / min, 5 v / v% CO 2 concentration, and culture for 10-12 h.

10. Use of the neural stem cells obtained by the method according to claim 9 in preparing drugs for treating nervous system diseases.