WO2019085994A1 - Nutritional supplement for human nervous system cell culture and for human pluripotent stem cells differentiating into nervous system cells - Google Patents

Abstract

A nutritional supplement for a human nervous system cell culture and for human pluripotent stem cells differentiating into nervous system cells, comprising the following components: 0.1-20 mg/L of human insulin, 10-200 mg/L of vitamin C, 10-100 mg/L of glutathione, 0.05-5 mg/L of linolenic acid, 0.2-20 mg/L of carnitine, 5-500 μM of N-acetylcysteine, 0.01-10 mg/L of ethanolamine, and 0.05-5 mg/L of linoleic acid. Also disclosed is an application of the supplement in culturing the human nervous system cells and inducing the human pluripotent stem cells to differentiate into the nervous system cells.

Description

A nutritional additive for cell culture of human nervous system and differentiation of human pluripotent stem cells into nervous system cells Technical field

The invention relates to the field of cell culture technology. More specifically, it relates to a nutritional additive for cell culture of human nervous system and differentiation of human pluripotent stem cells into cells of the nervous system.

Background technique

The incidence of various neurological and degenerative neurological diseases is increasing year by year. It is difficult to completely cure these diseases by traditional medical methods. Cell replacement therapy is the most effective means to completely cure such diseases.

Human pluripotent stem cells have unlimited proliferative capacity and the potential to differentiate into various types of somatic cells in the human body, and are ideal seed resource cells for cell replacement therapy. Under certain conditions of induction, human pluripotency can differentiate into neural stem cells, various types of neural precursor cells, neurons, astrocytes, and oligodendrocytes, which are the damage and degeneration of the nervous system. Cellular alternative therapies for disease continue to provide cells.

At present, in the process of differentiation of human pluripotent stem cells into nervous system cells, in the process of culturing neural stem cells, neural precursor cells and neurons, researchers basically adopt serum-free medium, ie, basic culture. The base nutrient additive is responsible for providing inorganic salts, vitamins, amino acids, glucose, organic substances and the like necessary for cell growth. The nutritive additive is responsible for providing proteins, hormones, trace elements and the like necessary for cell growth. Currently, the most used nutritional supplements are commercially available N2 and B27. The N2 component is simple and can only satisfy the process of differentiation of human pluripotent stem cells into neural stem cells. Human pluripotent stem cells differentiate into various types of neural precursor cells and neurons, and the cultivation of neural stem cells, neural precursor cells, and neurons requires the use of more nutritious B27. However, the complex B27 component, containing the animal protein bovine serum albumin, not only causes batch instability, but also the risk of introducing potential animal-borne viruses, posing a potential safety hazard for cell replacement therapy.

In order to make serum-free medium additives safer and more stable between batches, scientists have made a lot of efforts in removing animal or human extract proteins, and have achieved some results (such as patents CN107119010A and CN 106282114A). However, in order to achieve the same culture effect while removing serum albumin, CN107119010A added banana peel extract with unclear chemical composition, and CN 106282114 A used ginseng extract ginseng saponin with unclear chemical composition, not only the composition of the additive. It has become relatively complicated, and there are also problems in that stability between batches is difficult to control.

Therefore, there is a need to provide a novel nutritional additive for cell culture of human nervous system and differentiation of human pluripotent stem cells into cells of the nervous system.

Summary of the invention

It is an object of the present invention to provide a novel nutritional additive for human neural system cell culture and differentiation of human pluripotent stem cells into nervous system cells, which is simple in formulation, low in cost, chemically defined, and free of animal sources. Sexual ingredients, free of protein extracts and hydrolysates.

Another object of the present invention is to provide an application of the above nutritional additive for culturing human nervous system cells and inducing differentiation of human pluripotent stem cells into nervous system cells.

In order to achieve the above object, the present invention adopts the following technical solutions:

The present invention provides a nutritional additive for human neural system cell culture and human pluripotent stem cell differentiation into nervous system cells, the nutritional additive comprising the following components: human insulin, vitamin C, glutathione, linolenic acid Carnitine, N-acetylcysteine, ethanolamine and linoleic acid;

Among them, the final concentration of each component is: human insulin 0.1-20mg / L, vitamin C 10-200mg / L, glutathione 10-100mg / L, linolenic acid 0.05-5mg / L, carnitine 0.2 -20 mg/L, N-acetylcysteine 5-500 μM, ethanolamine 0.01-10 mg/L, and linoleic acid 0.05-5 mg/L.

Preferably, the final use concentration of each component of the nutritional additive is: human insulin 0.5-15 mg/L, vitamin C 20-100 mg/L, glutathione 20-90 mg/L, linolenic acid 0.1-4 mg/ L, carnitine 0.5-15 mg / L, N-acetylcysteine 10-400 μM, ethanolamine 0.05-8 mg / L, linoleic acid 0.1-4 mg / L.

Most preferably, the final concentration of each component of the nutritional additive is: human insulin 1-10 mg / L, vitamin C 30-80 mg / L, glutathione 30-80 mg / L, linolenic acid 1-3 mg /L, carnitine 1-10 mg / L, N-acetylcysteine 50-100 μM, ethanolamine 0.1-5 mg / L, linoleic acid 1-3 mg / L.

The human nervous system cells may be neural stem cells, neurons, neural precursor cells, glial cells, and the like.

The preparation of the nutritional additive of the present invention is carried out according to the conventional method of the present invention; in a specific embodiment of the present invention, a concentrated stock solution is prepared by concentrating 100 times of the final use concentration of each component in the nutritional additive in a 100-stage aseptic environment. At the time of use, a concentrated stock solution of 100× nutritional supplement was added to the basal medium in proportion.

The present invention further provides the use of the above nutritional additive for culturing human nervous system cells and inducing differentiation of human pluripotent stem cells into neural cells.

Further, the application is for use with the nutritional supplement in combination with a basal medium.

Further, the basal medium is one or both of DMEM/F12 medium and Neurobasal medium; preferably, DMEM/F12 medium and Neurobasal medium having a volume ratio of 1:1.

Further, the nutritional additive may also be used in combination with growth factors and/or small molecules that promote growth of human nervous system cells and growth factors and/or small molecules that promote differentiation of human pluripotent stem cells into nervous system cells.

Wherein the growth factor for promoting cell growth of the human nervous system is basic fibroblast growth factor (bFGF), epithelial cell growth factor (EGF), glial-derived neurotrophic factor (GDNF), brain-derived neurotrophic factor ( a combination of one or more of BDNF) and a sound factor (SHH);

The small molecule that promotes cell growth in the human nervous system activates the associated small molecule and the retinoic acid signaling pathway to activate small molecules involved in the SHH signaling pathway.

The growth factor for promoting differentiation of human pluripotent stem cells into nervous system cells is a combination of one or both of Wnt signaling pathway activation-related growth factors and SHH signaling pathway activation-related growth factors;

The small molecule that promotes differentiation of human pluripotent stem cells into nervous system cells is a small molecule involved in TGFβ signaling pathway inhibition, Wnt signaling pathway activates related small molecules, SHH signaling pathway activates related small molecules, and retinoic acid signaling pathway activates related small molecules. One or a combination of several.

specific,

The nutritional additive may be used in combination with basic fibroblast growth factor and epithelial cell growth factor for culturing human neural stem cells; the human neural stem cell source may be obtained by in vitro isolation and culture, and induced by human pluripotent stem cell differentiation. , obtained by somatic cell transdifferentiation.

The nutritional additive may be used in combination with a glial-derived neurotrophic factor and a brain-derived neurotrophic factor for culturing human neurons; the source of the human neuron may be obtained by in vitro isolation and culture, and induce human pluripotent stem cell differentiation. Obtained, obtained by somatic cell transdifferentiation, and the like.

The nutritional additive can be used in combination with SHH signaling pathway activators (such as small molecule SAG, purmorphamine or growth factor sonic factor) and retinoic acid signaling pathway activators (such as retinoic acid) for various types of human neural precursors. The culture of the cells; the source of the human neural precursor cells may be obtained by in vitro isolation and culture, induced by differentiation of human pluripotent stem cells, obtained by somatic cell transdifferentiation, and the like.

The nutritional additive can cooperate with TGFβ signaling pathway to inhibit related small molecules such as SB431542, LDN193189, etc., Wnt signaling pathway activates related small molecules such as CHIR99021, 6-BIO, etc., SHH signaling pathway activates related small molecules such as SAG, purmorphamine, etc., retinoic acid Signaling pathway activation related small molecules such as retinoic acid, Wnt signaling pathway activation-related growth factors such as WNT3a and SHH signaling pathway activation-related growth factors such as SHH are used to induce human pluripotent stem cells to differentiate into human nervous system cells.

The invention proves through experiments that the nutritional additive of the invention has better effect than the currently reported chemical composition of the human neutrophils and the differentiation of the human pluripotent stem cells into the nervous system cells, and contains the human transfer iron. A nutritional supplement for protein extracts such as proteins and human serum albumin.

In addition, the reagents and components used in the present invention are commercially available, unless otherwise specified, and any range recited in the present invention includes any value between the end value and the end value, and Any subrange of arbitrary values between end values or end values.

The beneficial effects of the present invention are as follows:

The nutrient additive for human neural cell culture and human pluripotent stem cell differentiation into nervous system cells is obtained by large-scale screening and optimization, and has simple formula, low cost, clear chemical composition and no animal origin. Ingredients, free of protein extracts and hydrolysates, are stable and safer between batches. In addition, the nutritional additive has no risk of potentially introducing animal-derived and human-derived pathogenic microorganisms, is safer, and can be applied to the cultivation of cells required for clinical research and clinical experiments.

DRAWINGS

The specific embodiments of the present invention will be further described in detail below with reference to the accompanying drawings.

Figure 1 shows a neural stem cell morphology map (experimental group, control group).

Figure 2 shows a flow chart of the neural stem cell marker gene Nestin (experimental group, control group).

Figure 3 shows the immunofluorescence map of the neural stem cell marker gene Nestin (experimental group).

Fig. 4 is a view showing the morphological map (experimental group, control group) on the eighth day of differentiation of human pluripotent stem cells into spinal motor neural precursor cells.

Fig. 5 is a flow chart (experimental group, control group) of the spinal cord motor neural precursor cell marker gene Olig2 on the eighth day of differentiation of human pluripotent stem cells into spinal motor nerve precursor cells.

Figure 6 shows an immunofluorescence pattern of the spinal cord motor neural precursor marker gene Olig2 (experimental group) on the eighth day of differentiation of human pluripotent stem cells into spinal motor nerve precursor cells.

Detailed ways

In order to explain the present invention more clearly, the present invention will be further described in conjunction with the preferred embodiments and the accompanying drawings. Similar components in the drawings are denoted by the same reference numerals. It should be understood by those skilled in the art that the following detailed description is intended to be illustrative and not restrictive.

The basal medium DMEM/F12 medium and Neurobasal medium, B27 used in the examples in the present invention were purchased from Life Technologies; growth factors and small molecules were purchased from Sigma, Stemgent, Enzo, Tocris, and other materials were commonly used in the art. material.

Example 1 Nutritional additive for human neural system cell culture and differentiation of human pluripotent stem cells into nervous system cells

A nutritional additive for human neural system cell culture and human pluripotent stem cells to differentiate into nervous system cells, the composition and final concentration of the nutritional additive are: human insulin 0.1 mg / L, vitamin C 10 mg / L Glutathione 10 mg/L, linolenic acid 0.05 mg/L, carnitine 0.2 mg/L, N-acetylcysteine 5 μM, ethanolamine 0.01 mg/L, linoleic acid 0.05 mg/L.

Example 2 Nutritional additive for human neural system cell culture and differentiation of human pluripotent stem cells into nervous system cells

The invention relates to a nutritional additive for human neural system cell culture and differentiation of human pluripotent stem cells into nervous system cells, wherein the composition and final concentration of the nutritional additive are: human insulin 20 mg/L, vitamin C 200 mg/L, Glutathione 100 mg / L, linolenic acid 5 mg / L, carnitine 20 mg / L, N-acetylcysteine 500 μM, ethanolamine 10 mg / L, linoleic acid 5 mg / L.

Example 3 Nutritional additive for human neural system cell culture and differentiation of human pluripotent stem cells into nervous system cells

A nutritional additive for human neural system cell culture and human pluripotent stem cells to differentiate into nervous system cells, the composition and final concentration of the nutritional additive are: human insulin 0.5 mg / L, vitamin C 20 mg / L Glutathione 20 mg/L, linolenic acid 0.1 mg/L, carnitine 0.5 mg/L, N-acetylcysteine 10 μM, ethanolamine 0.05 mg/L, linoleic acid 0.1 mg/L.

Example 4 Nutritional additive for human neural system cell culture and differentiation of human pluripotent stem cells into nervous system cells

A nutritional additive for human neural system cell culture and human pluripotent stem cells to differentiate into nervous system cells, the composition and final concentration of the nutritional additive are: human insulin 15 mg / L, vitamin C 100 mg / L, Glutathione 90 mg/L, linolenic acid 4 mg/L, carnitine 15 mg/L, N-acetylcysteine 400 μM, ethanolamine 8 mg/L, and linoleic acid 4 mg/L.

Example 5 Nutritional additive for human neural system cell culture and differentiation of human pluripotent stem cells into nervous system cells

A nutritional additive for human neural system cell culture and human pluripotent stem cells to differentiate into nervous system cells, the composition and final concentration of the nutritional additive are: human insulin 1 mg / L, vitamin C 30 mg / L, Glutathione 30 mg/L, linolenic acid 1 mg/L, carnitine 1 mg/L, N-acetylcysteine 50 μM, ethanolamine 0.1 mg/L, and linoleic acid 1 mg/L.

Example 6 Nutritional additive for human neural system cell culture and differentiation of human pluripotent stem cells into nervous system cells

The invention relates to a nutritional additive for human neural system cell culture and differentiation of human pluripotent stem cells into nervous system cells, wherein the composition and final concentration of the nutritional additive are: human insulin 10 mg/L, vitamin C 80 mg/L, Glutathione 80 mg/L, linolenic acid 3 mg/L, carnitine 10 mg/L, N-acetylcysteine 100 μM, ethanolamine 5 mg/L, and linoleic acid 3 mg/L.

Example 7 Comparative experiment of neural stem cell culture

1. The formulation of the culture solution for culturing human neural stem cells is as follows:

Base medium (volume ratio): 50% DMEM/F12 medium and 50% Neurobasal medium

Nutritional additive

bFGF (final use concentration): 20ng/mL

EGF (final use concentration): 20ng/mL

among them,

The composition and final use concentration of the nutritional additive of the experimental group are the same as in the embodiment 6;

The nutritional supplement of the control group is a nutritional additive commonly used in human neural stem cell culture, namely B27.

2. Culture of human neural stem cells

The culture of human neural stem cells includes two methods: adherent culture and suspension culture:

2.1 Adherent culture method of neural stem cells

Inoculate at a density of 10 ⁵ cells/mL into a culture dish containing a pre-coated substrate, culture at 37 ° C, 5% CO ₂ saturated humidity incubator, change the solution once every two days, and subculture when the cell confluence reaches 90% or more. .

The coating method of the culture substrate was as follows: Poly-L-ornithine (PLO) was diluted to 15 μg/mL with PBS, and added to the culture dish. The amount of addition was shown in Table 1, and incubated at 37 ° C for 2 h. Or overnight at 4 ° C, be careful not to let the bottom of the dish dry during the incubation. Discard the PLO, rinse it twice with PBS, and rinse it with DMEM/F12. Laminin was diluted to 5 μg/mL with DMEM/F12 and added to the PLO-coated petri dish. The amount of addition is shown in Table 1. Incubate at 37 °C for 2 h or 4 °C overnight, taking care not during incubation. Let the bottom of the dish be dried.

Table 1 Amount of PLO and Laminin required to coat the dish

Petri dish specification	Growing area (cm 2 )	Amount added (mL)
6-well plate	10cm 2 /well	1mL/well
12-well plate	4cm 2 /well	0.4mL/well
24-well plate	2cm 2 /well	0.2mL/well
35mm culture dish	10cm 2	1mL
60mm culture dish	20cm 2	2mL
100mm culture dish	60cm 2	6mL

2.2 Neural stem cell suspension culture method

Inoculate the low-adsorption culture dish at a density of 10 ⁵ cells/mL, incubate at 37 ° C, 5% CO ₂ saturated humidity incubator, change the solution once every two days, transfer the neurospheres and culture solution to a 50 mL centrifuge tube during the change. Naturally settle, carefully discard the culture solution, add fresh culture solution, resuspend the neurosphere, and transfer to a Petri dish. Subculture was carried out when most of the neurospheres reached a diameter of 250 μm.

The culture results are shown in Figure 1-3. Figure 1 shows the morphology of human neural stem cells after five generations of growth in the experimental medium and control medium. It can be seen that the growth state of neural stem cells in the experimental group is higher than that. The growth state in the control medium was better; Figure 2 shows the expression of the neural stem cells Nestin gene after the growth of human neural stem cells in the experimental medium and the control medium for five generations. It can be seen that the neural stem cells are in the experimental group. The positive proportion of Nestin in the culture medium was significantly higher than that of the control group; FIG. 3 is an immunofluorescence picture of Nestin after human neural stem cells were cultured for five generations in the experimental medium.

From this, it can be seen that in the neural stem cell culture, the nutritional additive of the present invention is more effective than the nutritional additive B27 which is complex and widely used, which contains animal or human extract proteins.

Example 8 Comparative experiment of inducing human cell pluripotent stem cells to differentiate into spinal motor neural precursor cells

1. The differentiation basic culture solution for inducing differentiation of human pluripotent stem cells into spinal motor neural precursor cells is as follows:

Base medium (volume ratio): 50% DMEM/F12 medium and 50% Neurobasal medium

Nutritional additive

among them,

The composition and final use concentration of the nutritional additive of the experimental group are the same as in the embodiment 6;

The nutritional supplement of the control group is a nutritional additive commonly used in the process of inducing differentiation of human pluripotent stem cells into the nervous system cells, namely B27.

2. Method for inducing differentiation of human pluripotent stem cells into spinal motor neural precursor cells

D0 digested human embryonic stem cells with good growth into single cells and inoculated them into a culture dish at a density of 10 ⁵ cells/mL. The culture medium was a differentiation medium containing 40 μM SB431542, 100 nM LDN193189, and 3 μM CHIR99021. D2, the culture medium was changed to a differentiation base medium containing 40 μM SB431542, 100 nM LDN193189, 3 μM CHIR99021, 500 nM SAG, 100 nM retinoic acid. After D4, the culture medium was changed to a differentiated basal medium containing 500 nM SAG and 100 nM retinoic acid, and the solution was changed every two days. At the beginning of D8, Olig2-positive spinal motor nerve precursor cells can be obtained.

The results of differentiation are shown in Fig. 4-6. In Fig. 4, in the differentiation basal culture solution described in the experimental group and the control group, the human pluripotent stem cells were induced to differentiate into spinal cord motor neural precursor cells for differentiation and the eighth day. Morphological map. As can be seen from the figure, the growth state of the cells during the differentiation induction did not differ between the experimental group and the control group. Figure 5 shows the differentiation of human pluripotent stem cells into spinal motor nerve precursor cells in the differentiation basal medium described in the experimental group and the control group, respectively. The eighth day of human spinal motor motility neural precursor cell marker gene Olig2 was shown. The expression of human pluripotent stem cells differentiated into spinal motor nerve precursor cells in the experimental group was significantly higher than the differentiation efficiency in the control group. Figure 6 shows the immunofluorescence pattern of the spinal cord motor neural precursor marker gene Olig2 induced by differentiation of human pluripotent stem cells into spinal motor neural precursor cells in the experimental medium.

It can be seen that in the process of inducing differentiation of human pluripotent stem cells into spinal motor nerve precursor cells, the nutritional additive of the present invention is more effective than the complex and widely used nutrients containing animal or human extract proteins. The factor additive B27 works well.

It is apparent that the above-described embodiments of the present invention are merely illustrative of the present invention and are not intended to limit the embodiments of the present invention, and those skilled in the art can also make the above description. It is to be understood that various changes and modifications may be made without departing from the spirit and scope of the invention.

Claims (10)

1. A nutritional additive for human neural system cell culture and differentiation of human pluripotent stem cells into nervous system cells, characterized in that the nutritional additive comprises the following components: human insulin, vitamin C, glutathione, linolenic acid Carnitine, N-acetylcysteine, ethanolamine and linoleic acid;

   Among them, the final concentration of each component is: human insulin 0.1-20mg / L, vitamin C 10-200mg / L, glutathione 10-100mg / L, linolenic acid 0.05-5mg / L, carnitine 0.2 -20 mg/L, N-acetylcysteine 5-500 μM, ethanolamine 0.01-10 mg/L, and linoleic acid 0.05-5 mg/L.
2. The nutritional supplement according to claim 1, wherein the final concentration of each component of the nutritional additive is: human insulin 0.5-15 mg/L, vitamin C 20-100 mg/L, glutathione 20 -90 mg/L, linolenic acid 0.1-4 mg/L, carnitine 0.5-15 mg/L, N-acetylcysteine 10-400 μM, ethanolamine 0.05-8 mg/L, linoleic acid 0.1-4 mg/L.
3. The nutritional supplement according to claim 2, wherein the final use concentration of each component of the nutritional additive is: human insulin 1-10 mg/L, vitamin C 30-80 mg/L, glutathione 30, respectively. -80 mg/L, linolenic acid 1-3 mg/L, carnitine 1-10 mg/L, N-acetylcysteine 50-100 μM, ethanolamine 0.1-5 mg/L, linoleic acid 1-3 mg/L.
4. Use of a nutritional supplement according to any of claims 1-3 for culturing cells of the human nervous system.
5. Use of a nutritional supplement according to any of claims 1-3 for inducing differentiation of human pluripotent stem cells into cells of the nervous system.
6. The use according to claim 4 or 5, characterized in that the application is for use with the nutritional supplement in combination with a basal medium.
7. The use according to claim 6, wherein the basal medium is one or both of DMEM/F12 medium and Neurobasal medium; preferably, DMEM/F12 medium and Neurobasal in a volume ratio of 1:1 Medium.
8. The use according to claim 6, wherein the nutritional additive is further compatible with growth factors and/or small molecules that promote growth of human nervous system cells and small molecules that promote differentiation of human pluripotent stem cells into nervous system cells. / or growth factor use.
9. The use according to claim 8, wherein the growth factor for promoting cell growth of the human nervous system is basic fibroblast growth factor, epithelial cell growth factor, glial source neurotrophic factor, brain-derived neurotrophic A combination of one or more of a factor, a sonic factor; the small molecule that promotes cell growth in the human nervous system activates a small molecule involved in the activation of the SHH signaling pathway and a retinoic acid signaling pathway that activates the associated small molecule.
10. The use according to claim 8, wherein the growth factor for promoting differentiation of human pluripotent stem cells into nervous system cells is one or two of Wnt signaling pathway activation-related growth factors and SHH signaling pathway activation-related growth factors. The combination of the small molecule that promotes the differentiation of human pluripotent stem cells into the nervous system cells is a small molecule involved in TGFβ signaling pathway inhibition, a small molecule involved in activation of Wnt signaling pathway, a small molecule involved in activation of SHH signaling pathway, and a retinoic acid signaling pathway. Activating one or a combination of the relevant small molecules.

Images