WO2015057015A1 - Method of redifferentiating adult cells into induced pluripotent stem cells using electromagnetic field - Google Patents

Abstract

The present invention relates to a method of redifferentiating adult cells into induced pluripotent stem cells using an electromagnetic field. According to the present invention, the method of redifferentiation into induced pluripotent stem cells using an electromagnetic field has excellent efficacy for redifferentiating adult cells into induced pluripotent stem cells, and induced pluripotent stem cells can be readily obtained. Also, the induced pluripotent stem cells prepared by the method have a large amount of redifferentiation factor expression, and when the stem cells are grafted to a mouse, pluripotency is exhibited. Thus, the redifferentiated induced pluripotent stem cells can be usefully applied to the development of a cell therapeutic agent and studies in the regenerative medicine field.

Description

How to reverse differentiate adult cells into induced pluripotent stem cells

The present invention relates to a method for dedifferentiating adult cells using electromagnetic fields into induced pluripotent stem cells.

Stem cells are undifferentiated cells that can infinitely regenerate and differentiate into cells of all tissues of the body. Stem cell research is an important research area for research on the development of cellular medicines such as regenerative medicine, new drug development, the causes and treatment of human diseases, and the development of human bodies. Pluripotent stem cells are cells up to 8 cells after fertilization of eggs and sperm. When these cells are separated and transplanted into the uterus, they can develop into one complete individual. Pluripotent stem cells originate from the inner cell mass located inside the blastocytes, which appear after 4-5 days of fertilization, and can occur with a variety of cells and tissues, but do not form new organisms. can not do it. Multipotent stem cells are stem cells that can only differentiate into cells specific to the tissues and organs in which they are contained. Embryonic stem cells of stem cells are made from the inner cell mass of embryos before implantation, can differentiate into more than 200 cells under appropriate circumstances, and can make whole organs (Nagy et al., Development, 110). : 815-821, 1990). However, embryonic stem cells as cell therapy have ethical problems that must be made by using eggs, and can be obtained only by destroying the embryos.

Recently, induced pluripotent stem cells have been reported as a supplement. Induced pluripotent stem cells refer to cells having pluripotency by dedifferentiating differentiated cells, and are capable of differentiating into all types of cells of the body with the ability to self-renewal similarly to embryonic stem cells. Has To date, induced pluripotent stem cells have been reported to have very similar characteristics in embryonic stem cells, pluripotent stem cells, in terms of genetic and epigenetic properties and differentiation in gene expression and differentiation capacity (Takahashi and Yamanaka, Cell, 126: 663-676). , 2006). To date, intracellular introduction of dedifferentiation factors is most effective to date by means of intracellular delivery using viral vectors. However, the use of viral genomes in the production of induced pluripotent stem cells for therapeutic purposes poses a potential risk. In addition, since it is inserted into the genome in the cell at random and very stable, various problems such as mutation of genes are always inherent. In addition, the production efficiency of dedifferentiated stem cells is very low, less than 1%, and there is a risk of tumor formation. Many research teams have reported studies to reduce the reprogramming factors by replacing carcinogens or by using cell types that are more favorable for reprogramming. However, this also has the disadvantage of injecting various factors or isolating specific cell types.

Although research on dedifferentiated stem cells is currently a global topic, there have been few reports in Korea presenting new opinions on the reprogramming method or confirming its mechanism. In particular, Japan is a global leader in the field of dedifferentiated stem cells. Of course, there have been many reports of significant research cases in China in recent years, but the research is insufficient in Korea and is falling behind in the world.

On the other hand, research is being conducted on techniques for incorporating wave energy into biology. A technique that combines known waves into biology is a device for applying low-frequency energy of about 10 Hz or less to brain tissue. Device (public patent US20060205993). Zheng developed a technique (JP 2008-543388) to improve brain function by combining high frequency or multiple frequency components to give magnetic stimulation to the central nervous system. Riken manufactures nerve cells by electropulsing embryonic stem cells. Technology (US200740065941) has been developed. Gliner et al. Developed a technique for producing neurons by treating the cells with electric pulses (US20050075679). However, there is no research on the technique of de-differentiating adult cells using wave energy.

Accordingly, the present inventors continue to study a technique for efficiently reprogramming cells to dedifferentiate them into induced pluripotent stem cells, and when culturing adult cells in an electromagnetic field having a specific frequency, the dedifferentiation efficiency is significantly high. By confirming that the present invention was completed.

It is an object of the present invention to provide a method for dedifferentiating adult cells, including culturing adult cells under electromagnetic fields, into induced pluripotent stem cells.

The present invention also provides an induced pluripotent stem cell dedifferentiated by the above method.

The present invention provides a method for dedifferentiating adult cells into induced pluripotent stem cells, including culturing adult cells under electromagnetic fields.

The present invention also provides induced pluripotent stem cells dedifferentiated by the above method.

Hereinafter, the present invention will be described in detail.

In the present invention, "electromagnetic field" refers to a phenomenon in which electromagnetic fields whose intensity changes periodically are propagated into space, and have the same meaning as electromagnetic waves, and the electromagnetic fields used in the present invention may include both pulse wave forms and continuous wave (sine wave) forms. have.

In the present invention, the frequency of the electromagnetic field may be 1 to 200 Hz, preferably 50 Hz, and the strength of the electromagnetic field may be 0.1 to 15 mT, preferably 0.5 to 2 mT.

In the present invention, "stem cells" are cells that have the ability to differentiate into various types of body tissues, that is, undifferentiated cells and can be classified into embryonic stem cells and adult stem cells. "Embryonic stem cells" are undifferentiated cells that have differentiation ability, but have not yet differentiated, and mean cells with pluripotency capable of differentiating into various tissue cells if appropriate conditions are set in such an undifferentiated state. In its broadest sense, it also includes embryoid bodies derived from embryonic stem cells. "Adult stem cell" means a cell having a limited differentiation capacity that cannot differentiate into all tissues but can differentiate into each target organ. In addition, "differentiation capacity" refers to the ability of a part of the embryo to be differentiated into various organs or tissues according to a given developmental condition in the initial development of the organism. In the present invention, "induced pluripotent stem cells" refers to cells having pluripotency by dedifferentiating differentiated cells, and have all the types of cells of the body with the ability to self-renewal similarly to embryonic stem cells. It is characterized by being capable of differentiation and is also referred to as "dedifferentiated stem cells". Induced pluripotent stem cells have almost the same characteristics as embryonic stem cells, specifically, have similar cell morphology, similar gene and protein expression patterns, pluripotency in vitro and in vivo, and teratoma. When formed and inserted into the blastocyst of the mouse, chimera mice are formed and germline transmission of the gene is possible.

In addition, in the present invention, "adult cell" refers to a cell derived from an adult born and alive, as opposed to embryonic cells. The genetic background of the adult cells used in the present invention is not limited, but canine, feline, boar, bovine, deer and animal, giraffe and animal, pelican, camel, hippopotamus, horse and animal, May be derived from one or more selected from the group consisting of veins, rhinos, animals, weasels, rabbits, rodents and primates. The adult cells may be autologous, allogeneic or xenogeneic, and limit their types such as fat cells, fibroblasts, fibroblasts, muscle cells, heart cells, blood cells, bone marrow cells, germ cells, etc. I never do that.

"Dedifferentiation" in the present invention refers to an epigenetic retrograde process that allows partial or final differentiated cells to return to an undifferentiated state, such as pluripotency or pluripotency, to allow formation of new differentiated tissue. This reverse differentiation is possible because epigenetic changes in the cell genome are not fixed but are reversible processes that can be erased and re-formed. Reverse differentiation, also called "reprogramming," relates to the process of changing the genetic and expressive profile of partially or finally differentiated cells to be similar to that of embryonic stem cells. For example, such changes include changes in the methylation pattern, changes in the expression rate of stem cell genes, and the like.

Artificial dedifferentiation process is performed by the use of virus-mediated or non-viral vectors with retroviruses and lentiviruses, introduction of non-virus-mediated dedifferentiation factors using protein and cell extracts, or by stem cell extracts, compounds, etc. Although it includes a reverse differentiation process, it is preferable to use a lentiviral in the present invention.

Reverse differentiation factors in the present invention are Oct4, Sox2, Klf4, c-Myc, Nanog and Lin28, preferably Oct4, Sox2, Klf4 and C-Myc, but is not limited thereto.

In order to form induced pluripotent stem cells for reprogramming in the present invention, the step of delivering dedifferentiation inducers into the somatic cells is essential. Specifically, Oct4, Sox2, Klf4 and retroviruses are used as transporters. Genes encoding c-Myc's dedifferentiation inducers are transferred to somatic cells (Takahashi. K. et al, Cell, 131: 861-872, 2007), or using lentiviruses as transporters, Oct4, Sox2, Klf4 and Genes encoding c-Myc dedifferentiation inducers can be transferred to somatic cells.

Sox family genes are known to play an important role in maintaining pluripotency, similar to Oct4. But while the Oct4 gene is only involved in pluripotent stem cells, the Sox family of genes is also associated with pluripotent stem cells or pluripotent stem cells. The SRY-type high mobility group box 2 (Sox2) transcription factor is the only Sox family protein that plays an important role in maintaining pluripotency of embryonic stem cells (Avilion et al., Genes Dev, 17: 126-140, 2003). Inhibition of Sox-2 expression in mouse embryonic stem cells, as in Oct4, induces differentiation (Ivanova et al., Nature, 442: 533-538, 2006). In addition, Sox-2 binding sites found in the promoter regions of several Sox2 subgenes often exist adjacent to Oct4 and Nanog binding sites (Boyer et al., Cell, 122: 947-956, 2005). Therefore, the interaction between Sox2 transcription factor and Oct4 transcription factor is expected to provide a basic framework for induced pluripotent stem cells to maintain their undifferentiated state and to characterize embryonic stem cells (Lewitzky and Yamanaka, Current Opinion in Biotechnology, 18). : 467-473, 2007).

c-Myc is a carcinogenic gene that performs a variety of intracellular functions such as cell growth, differentiation, proliferation, cell death and transformation into cancer cells. It has also been identified as a subgene of LIF (Leukemia Inhibitory Factor) / STAT3 and Wnt signaling mechanisms, which are key mechanisms for maintaining pluripotency (Sears et al., Genes Dev, 14: 2501-2514, 2000). The c-Myc transcription factor is expected to play a role in inhibiting the proliferation of cells in induced pluripotent stem cells. In addition, c-Myc not only binds to Myc recognition sites in the genome, but also changes the chromatin structure to help Oct4 and Sox2 bind well to target genes (Lewitzky and Yamanaka, Current Opinion in Biotechnology, 18: 467-473, 2007).

Klf4 is involved in growth inhibition and regulates the cell cycle. Recent studies have shown that, similar to c-Myc, it acts as a sub-gene of STAT3 in embryonic stem cells and inhibits the differentiation of mouse embryonic stem cells by maintaining Oct4 expression upon overexpression (Li et al., Blood, 105: 635-637, 2005).

Nanog is an embryo-specific gene that, like Oct4 and Sox2, is required for maintaining pluripotency of embryonic stem cells. LIN28 is also an mRNA binding protein expressed in embryonic stem cells and embryonic tumor cells and is known to be involved in differentiation and proliferation (Yu et al., Induced Pluripotent Stem Cell Lines Derived from Human Somatic Cell, Science New York, NY, 2007 ).

As described above, Oct4, Sox2, Klf4, c-Myc, Nanog, and Lin28 genes are referred to as "reprogramming-inducing genes," which are genes that can reprogram differentiated cells. I mean. In particular Oct4, Sox2, Klf4 and c-Myc are called Yamanaka factors.

In the present invention, the adult cells can be cultured in FBS (Fetal Bovine serum) and penicillin / streptomycin-added medium.

The medium may be DMEM (Dulbecco's Modified Eagle Medium), but is not limited thereto. 5-15% (v / v) Fetal Bovine serum (FBS), preferably 10% FBS, 0.1-5% (v / v) penicillin / streptomycin, preferably 1% penicillin / streptomycin Adult cells can be cultured in the medium to which the medium is added, and the culture can be cultured for 1 to 10 days, preferably 5 days.

When the adult cells are adult cells derived from a mouse, in a medium containing FBS (Fetal Bovine serum), non-essential amino acids, penicillin / streptomycin, glutamine, -mercaptoethanol and leukemia inhibitory factor (LIF) Adult cells can be further cultured.

The medium for further culture may be DMEM (Dulbecco`s Modified Eagle Medium), 5-20% (v / v) of FBS (Fetal Bovine serum), preferably 15% of FBS, 0.1-5% (v / v) non-essential amino acids, preferably 1% non-essential amino acids, 0.1-5% (v / v) penicillin / streptomycin, preferably 1% penicillin / streptomycin, 0.1-5% (v / v), preferably 1% (v / v) glutamine (100 mM to 500 mM, preferably 200 mM), 0.1 to 5% (v / v), preferably 1% (v / v) medium containing -mercaptoethanol of v) and Leukemia Inhibitory factor (LIF) of 10 ^-6 to 10 ^-5 % (v / v), preferably 4 10 ^-6 % (v / v) Adult cells can be further cultured in, and the culture can be cultured for 10 to 20 days, preferably 15 days.

When the adult cells are adult cells derived from humans, DF12 culture medium may be used as a medium for further culture, and 10-30% (v / v), preferably 20% (v / v) serum substitute ( serum replacer), 0.1% to 5% (v / v), preferably 1% (v / v) non-essential amino acids, 0.1% to 5% (v / v), preferably 1% penicillin / strepto Mycin, a mixture of 0.1 to 5% (v / v), preferably 1% (v / v) of glutamine and -mercaptoethanol and 10 ^-5 to 10 ^-4 % (v / v), preferably 1.4 Adult cells may be additionally cultured in a medium containing 10 ⁻⁵ (v / v)% bFGF (bovine fibroblast growth factor), and the culturing may be performed for 20 to 40 days, preferably 30 days.

Induced pluripotent stem cells differentiated according to the method of the present invention have a high expression level of dedifferentiation factor, and the promoter region of dedifferentiation factor is demethylated. In addition, when the dedifferentiated induced pluripotent stem cells are transplanted into mice, pluripotency is shown. Therefore, the dedifferentiated induced pluripotent stem cells can be usefully used for cell therapy and research in regenerative medicine.

In the present invention, a cell therapy product refers to a cell autologous, allogenic, or xenogenic cell in vitro to proliferate or otherwise restore the tissue and function of the cell. It is a medicine used for the purpose of treatment, diagnosis and prevention through a series of actions such as changing the biological characteristics of the cell by the method. The field of cellular therapeutics in which the induced pluripotent stem cells induced by the method of the present invention can be used is not limited. For example, tumors, neurodegenerative diseases, immune diseases, nervous system diseases, physical organ damage, nerve damage, diabetes , Cerebrovascular disease, spinal cord injury, osteoblastic metastasis, hematopoietic dysfunction, osteoarthritis, leukocytosis, and the like.

The method for reverse differentiation into induced pluripotent stem cells using the electromagnetic field according to the present invention has an excellent efficiency of dedifferentiating adult cells into induced pluripotent stem cells, and can easily obtain induced pluripotent stem cells. In addition, the induced pluripotent stem cells prepared by the above method have a high expression level of dedifferentiation factor, and when transplanted into mice, pluripotency is shown. Therefore, the dedifferentiated induced pluripotent stem cells can be usefully used for cell therapy and research in regenerative medicine.

Figure 1 shows the GFP-positive induced pluripotent stem cell colonies when 50 Hz electromagnetic wave treatment after introducing the differentiation factors (Oct4, Sox2, c-Myc, Klf4) into the fibroblasts of Oct4-GFP Knock-in mouse It is also.

Figure 2 shows the GFP-positive cell population using FACS, when 50 Hz electromagnetic wave is treated after introducing the differentiation factors (Oct4, Sox2, c-Myc, Klf4) into fibroblasts of Oct4-GFP Knock-in mice This is the figure analyzed.

Figure 3 shows the introduction of dedifferentiation factors (Oct4, Sox2, c-Myc, Klf4) into fibroblasts of Oct4-GFP Knock-in mice, followed by time-dependent pluripotency marker gene (Oct4, Sox2 and Nanog) is a diagram showing the change in mRNA expression over time.

Figure 4 shows the differentiation factor (Oct4, Sox2, c-Myc, Klf4) introduced into fibroblasts of Oct4-GFP Knock-in mouse, and then treated with 50 Hz electromagnetic wave, the pluripotency marker gene (by immunostaining method) Oct4, Sox2, Nanog) is a diagram confirming the expression at the protein level.

FIG. 5 shows the propensity for methylation of Oct4 and Nanog promoters when 50 Hz electromagnetic wave was introduced after the introduction of reverse differentiation factors (Oct4, Sox2, c-Myc, Klf4) into fibroblasts of Oct4-GFP Knock-in mice. It is also.

6 is a diagram confirming the pluripotency of induced pluripotent stem cells through the generation of chimeric mice when injected mouse-induced pluripotent stem cells prepared through the electromagnetic field of the present invention in immunodeficient mice.

7 is a diagram confirming the generation of human induced pluripotent stem cells when the electromagnetic field is treated to human fibroblasts.

8 is a diagram showing mRNA expression changes of pluripotency marker genes with time when electromagnetic fields were treated to human fibroblasts.

9 is a diagram confirming the expression of the pluripotency marker gene protein level through immunostaining, when the electromagnetic field is treated to human fibroblasts.

10 is a diagram confirming the pluripotency by analyzing the teratoma ability when the human induced pluripotent stem cells prepared through the electromagnetic field of the present invention injected into the immunodeficiency mouse.

Hereinafter, the present invention will be described in more detail with reference to the following examples. Since these examples are only for illustrating the present invention, it will be apparent to those skilled in the art that the scope of the present invention should not be construed as being limited by these examples.

Example 1 Mouse Adult Cell Reprogramming Using Electromagnetic Field.

1. Check the retrodifferentiation efficiency if the electromagnetic field is investigated

After cutting the tail of OCT4-GFP Knock-in adult mice and performing primary cell culture, a lentivirus into which four reverse differentiation factors (Oct4 / Sox2 / c-Myc / Klf4) were introduced was introduced. Treated. Thereafter, an electromagnetic wave device generating an electromagnetic field (50 Hz, 0.5 to 2 mT) is mounted in the incubator, and the culture dish is placed in the electromagnetic field, and the ratio of 10% (v / v) of FBS, 1% (v / v) to DMEM is placed. Essential amino acids, 1% (v / v) penicillin / streptomycin, 1% (v / v) 200 mM glutamine,

After 5 days incubation in a culture medium containing DMEM, 1% (v / v) FBS, penicillin / streptomycin (exchange culture medium once every 2 days), 1% (15% (v / v) in DMEM v / v) FBS, 1% (v / v) non-essential amino acid, 1% (v / v) penicillin / streptomycin, 200 mM glutamine, 0.7 l -mercaptoethanol, 5 l leukemia The culture medium containing the inhibitor (LIF; Leukemia Inhibitory factor) was incubated for 15 days (culture medium exchange once every two days). Thereafter, GFP positive induced pluripotent stem cell colonies were observed through fluorescence microscopy, and colonies were quantitatively analyzed using FACS. The results are shown in FIGS. 1 and 2.

As shown in FIG. 1 and FIG. 2, it was confirmed that in the group treated with the electromagnetic field of 50 Hz, GFP positive induced pluripotent stem cell colonies increased about 30 times or more compared with the group not treated with the electric field.

2. Determining dedifferentiation factor expression at the molecular level if the electromagnetic field is investigated

The induced pluripotent stem cells prepared in Example 1-1 were passaged.

Specifically, mouse embryonic fibroblasts (embryonic stage) treated with 0.2% gelatin-coated 6-well dish, mitomycin C (10 g / ml) in a CO ₂ incubator maintained at 37 days prior to passage one day. After culturing 13 days after mouse embryonic fibroblast (feeder)), the next day 5000-1000 induced pluripotent stem cells were treated with trypsinase, and then cultured on the fibroblasts.

Thereafter, mRNA was obtained by a conventional method, and after synthesis of cDNA, pluripotency marker genes (Oct4, Sox2, Nanog) were analyzed by quantitative PCR, and the results are shown in FIG. 3. In addition, the results of confirming the expression at the protein level of pluripotency marker genes (Oct4, Sox2, Nanog) through the immunostaining method is shown in FIG. In addition, the trend of demethylation of the promoter region of Oct4, Nanog of the induced pluripotent stem cells of the present invention is shown in FIG. In addition, the results of confirming the pluripotency of the induced pluripotent stem cells of the present invention is shown in FIG.

As shown in FIG. 3, it was confirmed that the expression of pluripotency marker genes (Oct4, Sox2, Nanog) was significantly increased in the cells differentiated through the electromagnetic field.

In addition, as shown in Figure 4, when the electromagnetic field was investigated, it was confirmed that the pluripotency markers (Oct4, Sox2, Nanog) in the induced pluripotent stem cells of the present invention at the protein level.

In addition, as shown in Figure 5, when the electromagnetic field was investigated, it was confirmed that the promoter region of OCt4, Nanog of induced pluripotent stem cells induced by the differentiation of the present invention was demethylated.

In addition, as shown in Figure 6, by confirming that the induced pluripotent stem cells of the present invention to generate a chimeric mouse, it was confirmed that the induced pluripotent stem cells of the present invention has a pluripotent ability.

Example 2 Human Adult Cell Reprogramming Using Electromagnetic Field

1. Check the retrodifferentiation efficiency if the electromagnetic field is investigated

Human fibroblasts were cultivated to treat lentiviral in which the differentiation factor (Oct4 / Sox2 / c-Myc / Klf4) was introduced, an electromagnetic wave device was placed in the incubator, a culture plate was placed in the electromagnetic field, and 10% of DMEM (v / v) incubated for 5 days in a culture medium containing FBS and 1% (v / v) penicillin / streptomycin (incubation medium every 2 days), then 50 ml (v / v) in 200 ml of DF12 medium. v) serum replacer, 2.5 ml of 1% non-essential amino acids, 1% (v / v) penicillin / streptomycin, 1.25 ml [200 mM glutamine and -mercaptoethanol] and 10 l of bFGF Incubated for 30 days in a culture medium containing (exchanging culture medium once daily). The cell generation rates of the treated and untreated electromagnetic fields are shown in FIG. 7.

As shown in FIG. 7, when the electromagnetic field was treated, it was confirmed that induced pluripotent stem cell colonies were rapidly generated as compared with the cells not treated with the electromagnetic field.

2. Determining dedifferentiation factor expression at the molecular level if the electromagnetic field is investigated

For passage culture of the induced pluripotent stem cells prepared in Example 2-1, after culturing feeder cells in a 6-well plate coated with 0.2% gelatin one day before passage, the induced pluripotent stem cells were fed the next day. Cultured on phase.

Thereafter, mRNA was obtained by a conventional method, followed by quantitative PCR of pluripotency marker genes after cDNA synthesis, and the results thereof are shown in FIG. 8. In addition, the results of confirming the expression of the pluripotency marker at the protein level through the immunostaining method is shown in FIG. In addition, the results of confirming the pluripotency through the analysis of the teratoma ability when the human induced pluripotent stem cells of the present invention injected into the immunodeficiency mouse is shown in FIG.

As shown in Fig. 8 to 10, when the electromagnetic field is irradiated to the cells, it was confirmed that the expression of pluripotency markers increased, and also, when the induced induced pluripotent stem cells were irradiated to the immunodeficient mice, teratoma (Teratoma) is By confirming that the three-germ layer is generated, it was confirmed that the induced pluripotent stem cells of the present invention have pluripotency.

Claims (13)

1. Culturing the adult cells under an electromagnetic field; a method for dedifferentiating adult cells into induced pluripotent stem cells.
2. The method of claim 1, wherein the frequency of the electromagnetic field is 1 to 200 Hz.
3. The method of claim 1, wherein the strength of the electromagnetic field is between 0.1 and 15 mT.
4. The method of claim 1, wherein the adult cells are transduced with a differentiation factor.
5. The method of claim 4, wherein the dedifferentiation factor is at least one selected from the group consisting of Oct4, Sox2, KLF4, C-Myc, Nanog, and Lin28.
6. The method of claim 1, wherein the culturing is characterized in that adult cells are cultured in a medium to which Fetal Bovine serum (FBS) and penicillin / streptomycin are added.
7. The method of claim 6, wherein the culturing is incubated for 1 to 10 days.
8. The method according to claim 6, wherein when the adult cells are adult cells derived from a mouse,

   Method for further culturing adult cells in a medium containing FBS (Fetal Bovine serum), non-essential amino acids, penicillin / streptomycin, glutamine, -mercaptoethanol and Leukemia Inhibitory factor (LIF) .
9. The method of claim 8, wherein the culture is incubated for 10 to 20 days.
10. The method according to claim 6, wherein the adult cells are adult human cells,

    Further culturing the adult cells in a medium comprising a serum replacer, a non-essential amino acid, a mixture of penicillin / streptomycin, glutamine and -mercaptoethanol and a bovine fibroblast growth factor (bFGF).
11. The method of claim 10, wherein the culturing is performed for 20 to 40 days.
12. Induced pluripotent stem cells differentiated by the method of any one of claims 1 to 11.
13. A cell therapeutic agent comprising the induced pluripotent stem cell of claim 12.

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