CN113388574A

CN113388574A - Serum-free and feed layer-free culture medium and culture method for effectively inhibiting stem cell differentiation

Info

Publication number: CN113388574A
Application number: CN202110516573.XA
Authority: CN
Inventors: 陈东煌; 陈海佳; 张兆清; 姜交华; 李学家
Original assignee: Guangdong Guoke Cell Technology Co ltd; Bioisland Laboratory
Current assignee: Guangdong Guoke Cell Technology Co ltd; Bioisland Laboratory
Priority date: 2021-05-12
Filing date: 2021-05-12
Publication date: 2021-09-14

Abstract

The invention relates to a serum-free and feeder layer-free culture medium and a culture method for effectively inhibiting stem cell differentiation. Stem cells maintained in an undifferentiated state are also contemplated. Previous cultures of human embryonic stem cells required feeder cells and serum-containing media to maintain the stem cells in an undifferentiated state. It was found in the present invention that if TGF-. beta.and Activin are added to a medium for culturing stem cells, the stem cells can be maintained in an undifferentiated state for at least 10 passages indefinitely without the need for feeder cells and serum.

Description

Serum-free and feed layer-free culture medium and culture method for effectively inhibiting stem cell differentiation

Technical Field

The invention relates to the technical field of cell culture, in particular to a serum-free and feeder-layer-free culture medium and a culture method for effectively inhibiting stem cell differentiation.

Background

In adult animals, many tissues, such as skin, blood and small intestine epithelium, have a short cell life and need to be constantly replaced by corresponding new cells. One of the ways in which mature individuals produce new differentiated cells is by simple multiplication of existing differentiated cells to form new differentiated cells, i.e., the differentiated cells divide to form two daughter cells of the same type, such as new endothelial cells in blood vessels, which are produced in this manner. However, during differentiation, cells often lose their ability to re-divide by being highly differentiated and eventually go towards senescent death. To compensate for this deficiency, the body also retains a portion of undifferentiated blasts, i.e., stem cells, during development. Once physiologically desired, these stem cells can undergo division to produce differentiated cells according to developmental pathways.

Stem cells are cells from embryonic, fetal or adult bodies that have the ability to self-renew and proliferate and differentiate without restriction under certain conditions, and can produce daughter cells with the same phenotype and genotype as well as the same cells themselves, and also can produce specialized cells that make up body tissues and organs, and can differentiate into progenitor cells. In brief, a stem cell is a type of cell that has unlimited or immortal self-renewal capacity, and is capable of producing at least one type of highly differentiated progeny cell.

Human embryonic stem cells (hESCs) are pluripotent cells isolated from the blastocyst stage and of embryonic origin that confer the ability of hESCs to proliferate indefinitely in vitro while retaining the ability to differentiate towards ectoderm, mesoderm and endoderm. While hESCs cells have unlimited self-renewal potential, they are readily differentiated spontaneously during culture, severely affecting the quality of the stem cells. Understanding the molecular mechanism and recognizing factors that inhibit differentiation at an early stage are essential conditions for maintaining undifferentiated stem cells.

Research shows that hESCs use different signal pathways to maintain their pluripotency, and hESCs key pluripotency factor, Nanog, is involved in intracellular quality control and maintain proliferation and self-renewal of embryonic stem cells. Nanog limits transcriptional activity of the Smad2/3 cascade, preventing its differentiation towards the extraembryonic endoderm and trophectoderm. Down-regulation of Nanog leads to loss of hESCs pluripotency, expressing ectodermal and endodermal markers after multiple passages. Also, down-regulation of Nanog also resulted in significant down-regulation of Oct4 and loss of hESCs surface antigen, consistent with a loss of pluripotency.

The culture medium of hES cells is generally DMEM/F12, and Serum Replacement (SR), 2-mercaptoethanol, basic fibroblast growth factor (bFGF), glutamine, and non-essential amino acids are added, and the culture medium can be divided into two systems including a feeder layer and a feeder layer-free system according to the presence or absence of the feeder layer. Since Thomson et al successfully established lines using mitomycin C or gamma-inactivated Mouse Embryonic Fibroblasts (MEFs) as feeder layers, feeder layer-containing culture systems have been accepted by most researchers, and to date have been the most common system for hES cell culture. The state of hES cells cultured in the system is relatively stable, but MEF has limited in-vitro culture generations (generally about 8 generations, and about 4 th generation is commonly used for a feeder layer), the consumption of manpower and material resources is high, and the murine substances bring potential safety hazards to the clinical application of hES cells. To avoid xeno-contamination, korean scholars use human amniotic fluid cells (HAF) as a feeder layer and culture hES cells using SR instead of serum, which has made hES cells free of animal-derived contamination. Besides, researchers also use amniotic fluid mesenchymal stem cells, bone marrow mesenchymal cells, hair follicle mesenchymal cells and fibroblasts as a feeding layer, and a foundation is laid for clinical application. Although the feeder layer culture system is mature, the system still has many insurmountable defects, such as inconvenient feeder layer material taking, complex preparation, large batch-to-batch difference, difficult sharing and purification, heterologous pollution, easy initiation of immune reaction and the like, and the quality of feeder layer cells and the consistency of culture conditions play a critical role in generating high-quality and stable-performance hES cells, thereby greatly limiting the clinical application of the hES cells.

Therefore, researchers hope to establish a feeder-free hES cell culture method that can be applied clinically, gradually departing from the conventional feeder-dependent cell culture method. The feeder-free cell culture system is a system for culturing hES cells on an extracellular matrix by using a conditioned medium or a conditioned medium, and factors affecting the establishment of the feeder-free cell culture system include extracellular matrix, serum, cytokines, inhibitors, and the like. Conditioned medium is a medium recovered when the cells are in the logarithmic growth phase and reused after filter sterilization, but this medium cultures cells that are easily differentiated. Another method for preventing heterologous contamination is to use well-defined media, i.e.serum-free culture systems.

In general, the mechanism of self-renewal and differentiation in hES cell culture systems is not clear, the chemical composition required is not clear, and some animal materials, such as serum, need to be used. Such culture systems are not conducive to quality control, are more likely to be contaminated by some pathogens, and are very dangerous for clinical use. Therefore, the use of a culture system with a completely defined chemical composition is of interest to many researchers, trying to grow feeder-free cells and develop serum-free media.

Chinese patent application No. 200580029936.6 discloses a cell culture medium containing sufficient amounts of albumin, salts, minerals, vitamins, amino acids, glucose, transferrin, insulin, fibroblast growth factor, transforming growth factor beta, gamma aminobutyric acid, 2-pipecolic acid, and lithium, whereby human embryonic stem cells cultured in the medium are maintained in an undifferentiated state through multiple culture passages. The medium is feeder cells free and never exposed to feeder cells. Wherein the lithium is LiCl. However, the pluripotent stem cell was not effectively maintained by using only LiCl, and among these factors, bFGF, LiCl, gamma aminobutyric acid, 2-pipecolic acid and TGF- β were finally contained in TeSR1, and removing any of these 5 factors reduced the effect of culture, and although these additives supported the growth of human ES cells in culture for at least several passages, many failed to maintain ES cells in an undifferentiated state for subsequent passages.

Chinese patent application No. 200580014907.2 discloses a method for maintaining undifferentiated stem cells by providing a medium rich in TGF- β protein family members, FGF family member proteins, and/or nicotinamide, without the use of fibroblast feeder layers, conditioned medium, or leukemia inhibitory factor, comprising exposing the stem cells to a sufficient amount of TGF- β protein family members, Fibroblast Growth Factor (FGF) protein family members, or Nicotinamide (NIC) to maintain the cells in an undifferentiated state for a sufficient period of time to achieve the desired result. bFGF and TGF beta 1 are commonly used in the culture of embryonic stem cells, but bFGF has instability (half-life is only about 10 h), the stability of a culture medium cannot be ensured by simply using the bFGF and the TGF beta 1, and nicotinamide serving as a vitamin has an unobvious effect on maintaining the pluripotency of cells and is easy to cause insufficient nutrition of the cells. In addition, the redox reaction in the culture medium of this patent may also cause the deterioration of the cell state.

Chinese patent application No. 200610004075.2 discloses the use of Activin A for feeder-free cell culture of human embryonic stem cells. ActivinA is necessary and sufficient for maintaining self-renewal and pluripotency of human embryonic stem cells, can induce the expression of transcription factors Oct4 and Nanog, and the stem cells still have the differentiation potential of forming teratoma in a mouse body after 10 generations of culture by using an invigilated layer cell culture medium containing 5ng/ml of ActivinA, and still maintain normal karyotype after more than 150 days of culture and more than 20 generations of culture through chromosome grouping analysis. Although the use of activinA in this patent maintains the pluripotency of embryonic stem cells, the use of serum replacement in this patent is complicated in composition and not promising in application.

In view of the above technical problems, it is highly desirable to provide a serum-free feeder-free medium that effectively maintains the undifferentiated state of stem cells, and that realizes rapid proliferation of ESCs while maintaining the undifferentiated state of cells.

Disclosure of Invention

Stem cells can be classified into embryonic stem cells and adult stem cells. Embryonic stem cells are cells selected from the inner cell mass of an embryo or primordial germ cells by in vitro culture with inhibition. In addition, embryonic stem cells can also be obtained using somatic cell nuclear transfer techniques. Embryonic stem cells have development totipotency, and theoretically can be induced and differentiated into all kinds of cells in an organism; embryonic stem cells can be expanded, screened, cryopreserved and revived in vitro in large quantities without losing their original characteristics. Adult stem cells refer to undifferentiated cells present in an already differentiated tissue, which are capable of self-renewal and of specialization to form the cells that make up that type of tissue. Adult stem cells are present in various tissues and organs of the body. The adult stem cells found so far are mainly: hematopoietic stem cells, bone marrow mesenchymal stem cells, neural stem cells, hepatic stem cells, muscle satellite cells, skin epidermal stem cells, intestinal epithelial stem cells, retinal stem cells, pancreatic stem cells, and the like.

In the present invention, the stem cell is an embryonic stem cell.

The invention provides a human embryonic stem cell culture medium without serum or a feeding layer, aiming at solving the problems that the embryonic stem cells are easy to have spontaneous differentiation and the like during in vitro culture, improving the fault tolerance rate during the culture process and more stably maintaining the good state of the cells. The culture medium has definite components and no animal source, can realize the rapid proliferation of ESCs, and simultaneously maintains the undifferentiated state of cells.

The invention aims to provide a composition, which comprises TGF-beta and Activin, and is used for the application of serum-free and feeder-layer-free cell culture of human embryonic stem cells.

The composition is added into a serum-free culture medium through the mutual matching of TGF-beta and Activin, so that feeder cells are not needed, and the undifferentiated state of stem cells in the serum-free culture medium can be maintained; the composition contains no serum, and has the advantages of clear components, high safety, and low cost. Wherein TGF-beta is transforming growth factor-beta and Activin is Activin.

Preferably, the TGF-beta is TGF-beta 1, the working concentration of the TGF-beta 1 is 1-20 mu g/L, such as 1 mu g/L, 5 mu g/L, 10 mu g/L, 15 mu g/L or 20 mu g/L, and the like, and other specific values in the range can be selected, which are not described in detail herein, and preferably 1-10 mu g/mL.

Preferably, the Activin is activinA, the working concentration of the activinA is 1-30 μ g/L, for example, 1 μ g/L, 5 μ g/L, 10 μ g/L, 20 μ g/L or 30 μ g/L, and the like, and other specific point values in the range can be selected, which are not described in detail herein, and preferably 10 μ g/L-20 μ g/L.

The composition further comprises a basic fibroblast growth factor.

Preferably, the basic fibroblast growth factor is a recombinant human basic fibroblast growth factor bFGF; the working concentration of the recombinant human basic fibroblast growth factor bFGF is 5-150 mu g/L; preferably, the working concentration of the recombinant human basic fibroblast growth factor bFGF is 40 mu g/L.

The composition comprises amino acids selected from one or more of reduced glutathione, L-glutamine, NEAA; the working concentration of the reduced glutathione is 1-10 mg/L; the working concentration of the L-glutamine is 100-1000 mu M; preferably, the working concentration of the NEAA is 10-500 μ M; the working concentration of the L-glutamine is 400 mu M; preferably, the working concentration of the NEAA is 100 mu M, and the working concentration of the reduced glutathione is 2 mg/L.

The composition comprises an antioxidant;

preferably, the antioxidant is sodium ascorbate; the working concentration of the sodium ascorbate is 1-100 mg/L; preferably, the working concentration of sodium ascorbate is 64 mg/L.

The composition further comprises NaHCO₃Sodium selenite, iron ethylenediaminetetraacetate and zinc sulfate.

Said composition comprising NaHCO₃The working concentration is 100-1000mg/L, the working concentration of sodium selenite is 1-50 mu g/L, the working concentration of iron ethylenediaminetetraacetate is 100-400 mu M, and the working concentration of zinc sulfate is 1-50 mu g/L; preferably, NaHCO₃The working concentration is 500mg/L, the working concentration of sodium selenite is 30 mug/L, the working concentration of iron ethylenediaminetetraacetate is 300 mug/L, and the working concentration of zinc sulfate is 10 mug/L.

The composition can be used for stem cell culture, and can maintain stem cell undifferentiated state for at least 10 generations. The composition maintains the stem cell undifferentiated state by regulating Nanog signal path.

Another object of the present invention is to provide a stem cell serum-free medium containing TGF-beta and Activin, which comprises a basal medium and the above composition. The basic culture medium is a serum-free culture medium; preferably, the serum-free medium is DMEM/F12; the basic culture medium involved in the invention includes but is not limited to DMEM/F12 medium.

It is still another object of the present invention to provide a method for culturing human embryonic stem cells to maintain the undifferentiated state, the method comprising the steps of: and (3) inoculating the mesenchymal stem cells into the stem cell culture medium for culture without feeder cells.

In one embodiment, NaHCO₃The concentration is 500mg/L, the concentration of sodium selenite is 30ug/L, the concentration of iron ethylenediaminetetraacetic acid is 300uM, the concentration of recombinant human basic fibroblast growth factor (bFGF) is 40ug/L, the concentration of zinc sulfate is 10ug/L, the concentration of L-glutamine is 400uM, the concentration of NEAA is 100uM, the concentration of sodium ascorbate is 64mg/L, the concentration of reduced glutathione is 2mg/L, the concentration of recombinant human transforming growth factor beta 1 is 2 ug/L, and the concentration of activinA is 12.5 ng/ml.

Compared with the prior art, the invention has the following beneficial effects:

the serum-free and feed layer-free culture medium is a culture system with clear culture components and no foreign sources, and can avoid exogenous pollution. On the basis of the existing serum-free and feeder layer-free culture medium of the embryonic stem cells, recombinant human transforming growth factor beta 1 (TGF-beta 1) and ActivinA are added, and the undifferentiated state of the embryonic stem cells is better maintained while a small molecular inhibitor is not used for regulating a Nanog signal path. The downstream effector Smad2/3 of activin A binds to and directly controls the activity of the Nanog gene in hESCs. In addition, TGF-. beta.1 may be bound to the Nanog proximal promoter. The Nanog promoter activity is enhanced by TGF-beta 1/Activin signals and plays an important role in maintaining the self-renewal of hESCs.

Nanog is an ES cell self-renewal maintenance factor that independently supports ES cell self-renewal when LIF/Stat3 is withdrawn. We obtained the above listed media additions after systematically testing several tens of growth factors, but this is not to say that changes in these components cannot be made. For example, other substances that stimulate the Nanog pathway may be used as alternatives to, or equivalents to, the media supplements listed above.

The serum-free and feeder layer-free culture medium is a culture system with clear culture components and no sources of foreign sources, can maintain the undifferentiated state of stem cells for at least 10 generations, and after 10 generations of culture, the cells still have no differentiated cells, the cells in cloning are arranged compactly, the amplification times are obviously increased, and the cell survival rate can reach 98.79%.

Drawings

FIG. 1 is a morphological diagram of huES (H9) cells corresponding to the 46 th passage cultured in examples 1-9;

FIG. 2 is the expression profiles of Oct4, Sox2 and Nanog genes of the 46 th generation of huES (H9) cells cultured in examples 1-9;

FIG. 3 shows the expression levels of Tra-1-60 and Tra-1-81 in the 46 th generation hue (H9) cells cultured in examples 1-9.

Detailed Description

The technical solution of the present invention is further explained by the following embodiments. It should be understood by those skilled in the art that the examples are only for the understanding of the present invention and should not be construed as the specific limitations of the present invention.

Control TeSR^TM-E8

Use of TeSR in the invention^TM-E8 as control group, TeSR^TME8 is a feeder cells-free, animal-derived component-free medium for human embryonic Stem cells from Stem cell, Cat # 05990.

Example 1

The present example is a serum-free feeder-free embryonic stem cell or pluripotent stem cell culture medium, and the formulation thereof is shown in the following table:

the preparation method comprises the following steps: dissolving the additive components in the culture medium according to respective dissolution characteristics, filtering with a filter membrane for sterilization, adding the components into DMEM/F12 basal medium one by one under the aseptic condition at 20 ℃, blowing and uniformly mixing, adjusting the osmotic pressure to 340mOsm/kg with sodium chloride, and storing the prepared culture medium at 4 ℃.

Example 2

This example provides a serum-free feeder-free embryonic stem cell or pluripotent stem cell culture medium having the following formulation:

Example 3

Example 4

Example 5

Example 6

Example 7

Example 8

Example 9

Evaluation test:

(1) morphological Observation of ESCs

The 36 th generation of huES (H9) cells were cultured at 1X 10⁵cell/cm²The density of the culture medium is inoculated in a 6-hole plate coated by matrigel, and the culture medium groups are respectively added for culture; and 2, changing the liquid every day, continuously subculturing by using ReleSR every 4-6 days until the culture reaches 46 th generation, observing the forms of the ESCs of each group at the 46 th generation under an inverted microscope, and collecting images. As shown in FIG. 1, only example 1 and the control group showed good cell status, no differentiated cells, and dense cell arrangement in the clones. The clones of example 2 and example 3 had loose cell arrangement around them, a small nucleoplasmic ratio and a tendency to differentiate. Groups 4-6, example 8, began to show distinct differentiated cells around the clone, which was fibrous and not in the typical morphology of embryonic stem cells. Although no differentiated cells appeared around the clones of examples 7 and 9, the cytoplasmic ratio of the nucleus was decreased in the middle of the clone, the three-dimensional effect of the cell was deteriorated, and the quality of the whole clone was seriously deteriorated. It is better to show that the serum-free culture medium of the inventionMaintain the typical morphology of pluripotent stem cells and maintain their pluripotency.

(2) ESCs proliferation Activity assay

The 46 th generation of huES (H9) cells were cultured at 2X 10⁴cell/cm²The density of (d) was seeded in matrigel-coated 24-well plates with 3 replicates per group. Adding the culture medium into each group for culture, changing the culture medium every day from the 2 nd day, culturing till the 7 th day, collecting the cells every day, and calculating the amplification factor and the cell viability of each group. Results Table 1 shows that the amplification factor of examples 1 to 3 in which TGF-. beta.1 and activinA were added was higher than that of the group in which only one of the components was added, and that the amplification factor of example 1 was the highest and reached 49.64 times. The examples in which a single component is added have not been able to achieve high proliferation ability of pluripotent stem cells due to problems such as deterioration of the state of cells and differentiation. The serum-free culture medium has stronger proliferation effect and can better maintain the survival of cells.

TABLE 1 fold expansion and cell viability of 46 th generation of huES (H9) cells

(3) Detection of pluripotent genes of ESCs

The 46 th generation of huES (H9) cells were cultured at 1X 10⁵cell/cm²The density of the culture medium is inoculated in a matrigel coated 6-well plate, the culture medium groups are respectively added for culture, and the culture medium is changed every day from the 2 nd day to the 5 th day. After being digested by 0.25% pancreatin solution, the total RNA of each group of huES (H9) cells is respectively extracted and is reversely transcribed into cDNA, the cDNA is taken as a template, the expression levels of the hESCs pluripotency genes Oct4, Sox2 and Nanog of each group are detected by adopting fluorescence quantitative PCR, and the qPCR primer sequences are shown in Table 2. As shown in FIG. 2, the expression of the pluripotency genes in the groups of examples 1-3 was significantly increased, wherein the genes of Oct4, Sox2 and Nanog in example 1 were all highest values and significantly better than the control TeSR-E8. The cell morphology and the gene level show that the culture medium of the invention not only can maintain the typical morphology of the pluripotent stem cells, but also better keeps the high expression of the pluripotent genes. Namely the inventionThe serum-free medium can better maintain the pluripotency of hESCs.

TABLE 2 hESCs pluripotency gene qPCR primer sequences

(4) Flow detection of specific markers of ESCs cell membranes

The 46 th generation of huES (H9) cells were cultured at 1X 10⁵cell/cm²The density of the culture medium is inoculated in a matrigel coated 6-well plate, the culture medium groups are respectively added for culture, and the culture medium is changed every day from the 2 nd day to the 5 th day. The cell suspensions of each group were collected after digestion with 0.25% pancreatin solution. Two kinds of pluripotent stem cell membrane specific markers including Tra-1-60 and Tra-1-81 are added to identify antibodies, and Tra-1-60 and Tra-1-81 are pluripotent stem cell specific proteins expressed on the surfaces of undifferentiated human Embryonic Stem Cells (ESCs) and induced pluripotent stem cell (iPS) cell lines, and are reduced or lost during cell differentiation. Incubating at 4 ℃ in a dark place for 20min, and detecting the marker expression level on the surface of each group of cells on a computer. As shown in FIG. 3, the ESCs of example 1 showed significantly higher surface marker expression levels, up to 99.4% in percentage, and more concentrated fluorescence intensity of the positive proportion cells, indicating more uniform quality of the positive cells, compared to other experimental groups. And the proportion of the positive cells in other embodiments is reduced to different degrees, a plurality of peak values are generated or the peak values are overlapped with the negative control peak, namely the fluorescence intensity of the positive cells is weak, which indicates that the quality of the cells in other embodiments is poor, and the differentiation phenomenon is generated to reduce the pluripotent marker. The serum-free culture medium can better maintain the pluripotency of ESCs and has stronger self-renewal capacity.

Claims

1. A composition comprising TGF- β and Activin.

2. The composition of claim 1, wherein the TGF- β is selected from TGF- β 1, TGF- β 2, TGF- β 3, or TGF- β 1 β 2;

preferably, the TGF-beta is TGF-beta 1;

preferably, the TGF-beta concentration is 1-20 mug/L; more preferably, the TGF-beta concentration is 1-10 μ g/L; more preferably, the TGF-beta concentration is 2 μ g/L;

more preferably, the TGF-beta is TGF-beta 1, at a concentration of 2 μ g/L.

3. The composition according to claim 1 or 2, wherein said Activin is selected from the group consisting of Activin a, Activin B, Activin C, Activin AB, and Activin AC;

preferably, the Activin is Activin a; the working concentration of Activin A is 1-30 mug/L; more preferably, the working concentration of Activin A is 10-20 μ g/L; more preferably, the Activin A working concentration is 12.5 μ g/L.

4. The composition of any one of claims 1-3, wherein the composition further comprises a basic fibroblast growth factor;

preferably, the basic fibroblast growth factor is a recombinant human basic fibroblast growth factor bFGF; the working concentration of the recombinant human basic fibroblast growth factor bFGF is 5-150 mu g/L; more preferably, the working concentration of the recombinant human basic fibroblast growth factor bFGF is 40 mug/L.

5. The composition according to any one of claims 1 to 4, wherein the composition comprises an amino acid selected from one or more of reduced glutathione, L-glutamine, NEAA; the working concentration of the reduced glutathione is 1-10 mg/L; the working concentration of the L-glutamine is 100-1000 mu M; preferably, the working concentration of the NEAA is 10-500 μ M; the working concentration of the L-glutamine is 400 mu M; more preferably, the NEAA working concentration is 100 μ M; the working concentration of the reduced glutathione is 2 mg/L.

6. The composition according to any one of claims 1 to 5, comprising an antioxidant;

preferably, the antioxidant is sodium ascorbate; the working concentration of the sodium ascorbate is 1-100 mg/L; more preferably, the working concentration of sodium ascorbate is 64 mg/L.

7. The composition of any one of claims 1-6, wherein the composition further comprises NaHCO3, sodium selenite, iron ethylenediaminetetraacetic acid, zinc sulfate;

preferably, the working concentration of NaHCO3 is 100-;

more preferably, the working concentration of NaHCO3 is 500mg/L, the working concentration of sodium selenite is 30 μ g/L, the working concentration of iron ethylenediaminetetraacetate is 300 μ M, and the working concentration of zinc sulfate is 10 μ g/L.

8. Use of a composition according to any one of claims 1 to 7 in stem cell culture.

9. The use of claim 8, wherein the composition maintains stem cells in an undifferentiated state for at least 10 passages; preferably, the composition maintains the stem cell in an undifferentiated state by modulating the Nanog signaling pathway.

10. A stem cell serum-free medium comprising a basal medium and the composition of any one of claims 1-7;

preferably, the basal medium is a serum-free medium;

more preferably, the serum-free medium is DMEM/F12.

11. A method for culturing stem cells, comprising the steps of: inoculating the stem cells into the serum-free culture medium of the stem cells according to claim 10 for culture;

preferably, the stem cells are human stem cells;

more preferably, the stem cells are human embryonic stem cells or pluripotent stem cells;

preferably, the culture method does not require feeder cells.

12. A cell obtained by the stem cell culture method according to claim 11 or by culturing the cell in the medium according to claim 10;

preferably, the cells are in an undifferentiated state;

more preferably, the cells are selected from embryonic stem cells or pluripotent stem cells.

13. A pharmaceutical composition comprising the cell of claim 12.