WO2012165740A1

WO2012165740A1 - Composition for improving dedifferentiation of cells and method for producing inducted pluripotent stem cells using the same

Info

Publication number: WO2012165740A1
Application number: PCT/KR2011/010319
Authority: WO
Inventors: Eun Ju Lee
Original assignee: Seoul National University Hospital
Priority date: 2011-06-02
Filing date: 2011-12-29
Publication date: 2012-12-06
Also published as: KR20120134360A

Abstract

The present invention relates to a composition for improving the dedifferentiation of cells and a method for producing induced pluripotent stem cells using the same, and more particularly to a composition for improving the dedifferentiation of cells, which comprises endothelin or an endothelin as an active ingredient, and a method for producing induced pluripotent stem cells using the same.

Description

COMPOSITION FOR IMPROVING DEDIFFERENTIATION OF CELLS AND METHOD FOR PRODUCING INDUCTED PLURIPOTENT STEM CELLS USING

THE SAME

BACKGROUND OF THE INVENTION FIELD OF THE INVENTION

The present invention relates to a composition for improving the dedifferentiation of cells and a method for producing induced pluripotent stem cells using the same, and more particularly to a composition for improving the dedifferentiation of cells, which comprises endothelin or an endothelin analog as an active ingredient, and a method for producing induced pluripotent stem cells using the same.

BACKGROUND OF TECHNIQUE

Stem cells are defined as undifferentiated cells that can self-renew indefinitely and differentiate into any type of cell in the body. Studies on stem cells have received a great deal of attention in regenerative medicine, the development of cell therapeutic drugs such as new drugs, the pathology and treatment of human diseases, and the process of development of the human body.

Totipotent stem cells are defined as cells which developed to the 8-cell stage after fertilization of oocytes and sperm, and when these cells are isolated and implanted into the uterus, these can develop into a complete individual. Pluripotent stem cells are derived the inner cell mass located inside inside of blastocysts, generated 4-5 days after fertilization. These cells can differentiate into various other tissue cells but cannot form new living organisms. Multipotent stem cells are stem cells capable of differentiating into only cells specific to their tissue and organ. Embryonic stem cells are made from the inner cell mass of pre-implantation embryos, can differentiate into more than 200 types of cells in a suitable environment and can also make the whole organ (Nagy et al., Development, 110:815-821, 1990). However, the use of embryonic stem cells as cell therapeutic agents has ethical problems in that the preparation thereof requires the use of oocytes and the destruction of embryos. Also, embryonic stem cells have various problems, including immune rejection which makes it difficult to use these cells in clinical applications.

In attempts to overcome these problems, induced pluripotent stem cells have recently been reported. As used herein, the term "induced pluripotent stem cells" refers to cells having pluripotency, which result from dedifferentiation of differentiated cells. The induced pluripotent stem cells have the capability to self- renew and differentiate into any type of cell in the body, similar to embryonic stem cells. To date, it has been reported that the induced pluripotent stem cells exhibit substantially the same characteristics as embryonic pluripotent stem cells with respect to the ability to express a gene and differentiate (Takahashi and Yamanaka, Cell, 126:663-676, 2006).

Methylation of the gene promoter is the main mechanism regulating the expression of the gene, and it is known that, when the promoter region of a certain gene is methylated, the expression of the gene is suppressed. Embryologically, "primordial germ cells" which are classified as the cells generated prior to the pluripotent stem cell stage are present in a stage in which they have no genomic DNA methylation. When the gene promoter is methylated during the development and differentiation of primordial germ cells following the fertilization procedure in order to regulate the specific expression of genes, differentiated somatic cells are formed and an individual is completed. Thus, demethylating the promoter methylated during the development and differentiation procedures is necessary for the dedifferentiation of the cells. Meanwhile, induced pluripotent stem cells can be produced by various methods, including a method of inducing dedifferentiation by treatment with a pluripotent cell extract, or a method inducing dedifferentiation factors.

However, the method of inducing dedifferentiating somatic cells by introducing the pluripotent cell extract into the somatic cells cannot exclude the possibility of pathogenic infection, because the extract is not an autologous protein, but is a homologous or heterologous protein. Also, this method has a problem in that the experimental results greatly depend on the experimental conditions, because the experiment is carried out in a state in which the key pluripotency protein of the extract was not identified. In addition, like dedifferentiation-inducing methods known to date, the above method has low efficiency, which limits the clinical application of the method.

Meanwhile, the method of preparing induced pluripotent stem cells using the . dedifferentiation-inducing factor necessarily includes a step of transferring the dedifferentiation-inducing factor into somatic cells. Specifically, induced pluripotent stem cells can be prepared either by introducing genes encoding the dedifferentiation-inducing factors into retroviruses and transferring the introduced genes into somatic cells (Takahashi et al., Cell, 131:861-872, 2007) or by introducing genes encoding the dedifferentiation-inducing factors into lentiviruses and transferring the introduced genes into somatic cells (Yu et al., Induced Pluripotent Stem Cell Lines Derived from Human Somatic Cell, Science New York, NY, 2007).

However, because each gene is introduced into somatic cells using retroviruses or lentiviruses, the viruses can be irregularly inserted into the genome to cause a mutation, and can activate an oncogene or inhibit the expression of an anticancer gene to cause a tumor. In addition, there is a problem in that the efficiency of preparation of induced pluripotent stem cells is low, because viruses expressing different genes are less likely to be introduced into one somatic cell. For example, about 10 induced pluripotent stem cells are obtained from 5x105 human fibroblasts (Takahashi et al., Cell, 131:861-872, 2007).

Accordingly, the present inventors have found that, when cells are treated with endothelin, the expression level of genes, including pluripotent marker genes, in the cells, will generally be increased due to the demethylation of the genomic DNA, and as a result, have recognized that the efficiency of existing methods for producing induced pluriopotent stem cells can be increased by treating differentiated cells with endothelin, thereby completing the present invention.

Throughout this application, various publications and patents are referred and citations are provided in parentheses. The disclosures of these publications and patents in their entities are hereby incorporated by references into this application in order to fully describe this invention and the state of the art to which this invention pertains.

SUMMARY OF THE INVENTION

The present inventors have made intensive studies to develop a system for improving dedifferentiation of cells to obtain induced pluripotent stem cells efficiently. As results, the present inventors have discovered that providing a cell with endothelin or an endothelin analog leads to greatly enhanced dedifferentiating abilities.

Accordingly, it is an object of the present invention to provide a composition for improving cell dedifferentiation, which can make cells having an increased ability to dedifferentiate.

It is another object of this invention to provide a method for producing induced pluripotent stem cells with increased efficiency, and a composition for producing induced pluripotent stem cells.

Other objects and advantages of the present invention will become apparent from the following detailed description together with the appended claims and drawings.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 shows the change in the expression levels of genes in mesenchymal stem cells after treatment with endothelin-1.

FIG. 2 shows the demethylation of the gene promoter in mesenchymal stem cells after treatment with endothelin-1.

FIG. 3 shows the expression levels of pluripotency genes in mesenchymal stem cells after treatment with endothelin-1.

FIG. 4 shows the expression level of Oct-4 in mesenchymal stem cells after treatment with endothelin-1.

DETAILED DESCRIPTION OF THE INVENTION

Unless otherwise defined, all the technical and scientific terms used here have the same meaning as that usually understood by an ordinary specialist in the field to which this invention belongs. Generally, the nomenclature used herein and the laboratory procedures are well known and commonly employed in the art. Hereinafter, the present invention will be described in detail.

"Endohelin" which is used in the present invention is a peptide of 21 amino acid residues, produced in vascular endothelial cells (Yanagisawa M et al.,Nature, 332:411-415, 1988), and is known as a vasoconstrictor peptide. Endothelin has two S-S bonds in one molecule thereof and is produced by modification of an endothelin precursor.

Most mammals have isopeptides of endothelin-1, endothelin-2 and endothelin-3. The three kinds of endothelin isopeptides have similar functions and effects with respect to transient vasodilation and continuous vasoconstriction. The term "endothelin analog" as used herein refers to a peptide which comprises an active region consisting of amino acid residues 16-21 (SEQ ID NO: 1) in endothelin consisting of 21 amino acid residues and contains a modification or deletion of at least one amino acid of amino acid residues 1-15. The endothelin analog is also called "endothelin derivative". The endothelin analog has a function and effect similar to endothelin binding to its receptor (European Journal of Pharmacology, 174:23-31, 1989; Bioorgmic & Medicinal Chemistry Letters, 4:561- 512, 1994).

SEQ ID NO: 1: His-Leu-Asp-Ile-Ile-Trp

As used herein, the term "modification" means that one or more of amino acid residues 1-15 of endothelin are substituted with other amino acids, and the term "deletion" means that one or more amino acids of amino acid resides 1-15 of endothelin are deleted.

As used herein, the term "prepro ET (prepro Endothelin)" means a 212-amno- acid peptide produced first produced by transcription of a gene encoding endothelin. The prepro ET produces "big ET (big Endothelin)" consisting of 38 amino acids by the action of an enzyme. Endothelin is produced by removal of the C-terminal amino acids of the big ET through the action of endothelin-converting enzyme (ECM) (Yanagisawa M et al., Nature, 332:411-415, 1988).

As used herein, the term "cells" refers to somatic cells or stem cells from diverse genetic backgrounds and/or origins. For example, somatic cells, mesenchymal stem cells, ectodermal stem cells and endodermal stem cells may be used in the present invention. Preferably, umbilical cord blood-derived stem cells and adipose-derived stem cells may be used in the present invention.

As used herein, the term "somatic cells" refers to differentiated cells, which are not pluripotent or can differentiate into limited cell types. As used herein, the term "differentiation" refers to a phenomenon in which the structure or function of cells is specialized during the division, proliferation and growth thereof, that is, the feature or function of cell or tissue of an organism changes in order to perform work given to the cell or tissue. Somatic cells which are used in the present invention may be naturally occurring somatic cells or genetically modified somatic cells.

As used herein, the term "stem cells" refers to undifferentiated cells having the capability to differentiate into various tissue types. Stem cells can be divided into embryonic stem cells and adult stem cells. The term "embryonic stem cells" refers to undifferentiated cells having pluripotency, which can differentiate into various cell types under suitable conditions. In a broad sense, embryonic stem cells also include embryonic stem cell-derived embryoid bodies. The term "adult stem cells" refers to cells having limited ability to differentiate, which are unable to differentiate into all tissue types, but are able to differentiate into their target tissues. In addition, the term "ability to differentiate" means that parts of an embryo corresponding to the early development of an organism are able to differentiate into various organs or tissues under specific developmental conditions.

In the present invention, mesenchymal stem cells were treated with endothelin or an endothelin analog. As a result, it was found that the dedifferentiation of the mesenchymal stem cells was stimulated.

In one aspect, the present invention is directed to a composition for improving dedifferentiation of cells, which comprises endothelin or an endothelin analog as an active ingredient, and a composition for facilitating the demethylation of promoters of various genes in the cells.

As used herein, the term "dedifferentiation" refers to an epigenetic reprogramming process in which partially or terminally differentiated cells revert to an undifferentiated state, such as a pluripotent or multipotent state, so that they can differentiate into new tissue. This dedifferentiation phenomenon is possible because epigenetic changes in the cell genome are not fixed, but can be reversibly erased and recovered. Dedifferentiation which is also called "reprogramming" is a process in which the genetic and phenotypic profiles of partially or terminally differentiated cells are changed to be similar to those of embryonic stem cells. For example, the changes include a change in methylation pattern, a change in the expression level of pluripotency genes, and the like.

In one Examples of the present invention, mesenchymal stem cells were treated with endothelin-1, and 24 hours after the treatment, a cDNA array was performed in order to examine the difference in gene expression between the endothelin-l-treated group and a vehicle-treated group. As a result, it was found that 97% of marker genes having high reliability values were turned on (FIG. 1).

As used herein, the term "methylation" means that a methyl group is attached to a base, and the term "demethylation" means that the methyl group is removed from the base. This methylation can result in a change in the expression pattern of the gene. Methylation of a gene interferes with gene expression by preventing the activity of expression proteins or recruiting enzymes that interfere with expression. Thus, methylation of a gene silences the gene by preventing transcription. Promoters of pluripotency- associated genes, including Oct4, Rexl, and Nanog, may be demethylated in induced pluripotent stem cells, indicating their promoter activity and the expression of pluripotency-associated genes in the induced pluripotent stem cells.

As described above, the results of the cDNA array showed the mechanism in which most of the cells were turned on. The present inventors considered that, in this mechanism, general demethylation of the genes is induced by endothelin-1. To verify this consideration, a CpG array was carried out (FIG. 2).

The region in which the two bases cytosine and guanine are consecutively present in the base sequence of the genome is defined as "CpG". The CpG is highly methylated at the cytosine, and 3-5% of all the cytosines in the human genome are methylated. The number of such CpGs has gradually decreased during the evolutionary process, and the methylation degree and pattern of CpG in the genome vary depending on the species of mammal and the type of tissue. The base sequences of mammals including humans contain a cluster of CpGs, which has a length of about 0.5-4 kb. The CpG islands have a close connection with the genes of the mammalian genome, because they are located near the promoter region which regulates the transcription of the genes. Most CpG islands are not methylated, and if the CpG island in the promoter region that initiates the expression of a specific gene, it means that the expression of the specific gene is inhibited. In other words, methylation of the CpG island can be considered as a mechanism developed into a specific molecular network regulating gene expression for specific purposes.

In FIG. 2, the negative values indicate demethylated regions, and the positive values indicate methylated regions. As can be seen in FIG. 2, the negative values are predominant in all the 24 chromosomes. Thus, it can be seen that an increase in gene expression due to an increase in demethylation in mesenchymal stem cells is attributable to endothelin.

In another Example of the present invention, real-time PCR was performed. As a result, it could be seen that, when mesenchymal stem cells were treated with endothelin-1, the expression levels of pluripotency genes, including E-cadherin, ERAS and STATS, in the treated mesenchymal stem cells, were increased compared to those in naive mesenchymal stem cells not treated with endothelin-1 (see FIG. 3).

As used herein, the term "pluripotent stem cells" refers to cells capable of differentiating into all cells related to three germ layers, including mesoderm, endoderm and ectoderm, which are required for the formation of an organism. Also, the pluripotency genes refer to either markers exhibiting this capability or genes capable of inducing pluripotency, and thus are also classified as dedifferentiation-inducing factors.

E-cadherin is known to be involved in the pluripotency and self-renewal- related signaling pathways in embryonic stem cells and induced pluripotent stem cells, and ERAS is understood to stimulate the production and proliferation of a teratoma. The teratoma is a kind of tumor that consists of various types of cells and tissues. This teratoma may be used to verify the ability of embryonic stem cells to differentiate. Because of the ability of embryonic stem cells to differentiate into all types of cells and tissues, when embryonic stem cells are injected into experimental animals such as rats, the embryonic stem cells will differentiate into various types of cells and tissues to form teratomas. Therefore, formation of teratomas in the last step of the preparation of embryonic stem cells demonstrates that embryonic stem cells were prepared. Also, STAT3 is known to maintain embryonic stem cells in an undifferentiated state.

In addition, it could be seen that, when mesenchymal stem cells were treated with endothelin-1, the expression of the dedifferentiation-inducing factor Oct-4 in the treated cells was increased compared to that in the naive group (see FIG. 4).

The Oct-4 transcription factor, an octamer transcription factor, is expressed only in undifferentiated embryonic stem cells and embryonic tumor cells, and plays an important role in maintaining the pluripotency of stem cells (Okamato et al., Cell, 60:461-472, 1990). If the expression level of Oct-4 is normal, the self-renewal and pluripotency of embryonic stem cells will be maintained, but if the expression level of Oct-4 increases abnormally, embryonic stem cells will differentiate into primitive endoderm and primitive mesoderm, and if the expression level of Oct-4 decreases abnormally, embryonic stem cells will develop into trophectodermal cells (Niwa et al., Nat Genet, 24:372-376, 2000). Thus, it can be seen that Oct-4 is an important factor that determines the pluripotency of embryonic stem cells and regulates the differentiation of embryonic stem cells. It is known that Oct-4 interacts with the Sox2 transcription factor to increase the expression of the downstream genes of Oct- 4, including Fgf4 (Yuan et al., Genes Dev, 9:2635-2645, 1995), Utf-1 (Nishimoto et al., Mol Cell Biol, 19:5453-5465, 2000) and Sox2 (Tamioka et al., Nucleic Acids Res, 30:3202-3213, 2002). Other genes belonging to the Oct family, including Oct-1 and Oct-6 having structures similar to Oct-4, have no capability to cause differentiated cells to dedifferentiate, and Oct-4 is involved in the dedifferentiation process (Lowry et al., PNAS, 105:2883-2888, 2008).

As described above, in the present invention, it is illustrated that, when mesenchymal stem cells are treated with endothelin or an endothelin analog, demethylation will be facilitated to stimulate the dedifferentiation of the cells. However, it will also be obvious to those skilled in the art that, when any conventional method of producing induced pluripotent stem cells from somatic cells or stem cells additionally comprises treating differentiated cells with endothelin, the efficiency of dedifferentiation of the cells will increase. Thus, treating differentiated cells with endothelin to increase the efficiency of dedifferentiation of the cells falls within the scope of the invention.

In another aspect, the present invention is directed to a method for producing induced pluripotent stem cells, which comprises a step of treating cells^with endothelin, and to a composition for producing induced pluripotent stem cells, which comprises endothelin as an active ingredient.

As used herein, the term "induced pluripotent stem cells" refers to cells having pluripotency, which can be generated by reprogramming differentiated cells.

A conventional method for producing induced pluripotent stem cells will now be described.

An extract from pluripotent cells can be introduced into differentiated somatic cells to cause the somatic cells to dedifferentiate. The dedifferentiation of nuclei can be observed by permeabilizing cells with streptolysin O, treating the permeabilized cells with an extract from pluripotent cells and culturing the treated cells. When human somatic cells are exposed directly to an extract from Xenopus oocytes, embryonic germ cells, embryonic carcinoma cell or embryonic stem cells, the undifferentiated marker genes in the cells will be expressed (Freberg et al., Mol Biol Cell, 18:1543-1553, 2007; Hansis et al., Curr Biol, 14:1475-1480, 2004; Taranger et al., Mol Biol Cell, 16:5719-5735). An extract from pluripotent stem cells is generally an extract from embryonic stem cells and refers to a substance obtained by disrupting cells, cultured in vitro, by a physical or chemical method, and separating the disrupted cells by centrifugation or the like.

In addition, induced pluripotent stem cells may be produced using a dedifferentiation-inducing factor. Takahashi and Yamanaka succeeded in inducing dedifferentiation with 24 selected candidates which have the capability to induce the pluripotency of somatic cells. They discovered the fact that, when four separated factors (Oct-4, Sox2, Klf4 and c-Myc) are introduced into mouse fibroblasts by a retrovirus system and expressed in the fibroblasts, the epigenetic dedifferentiation of the cells will occur, and the cells will acquire pluripotency and have properties similar to those of embryonic stem cells. To induce dedifferentiation, mouse fibroblasts expressing a neomycin antibiotic-resistance gene by the Fbxl5 promoter which is expressed only in pluripotent cells were used. Thus, the dedifferentiated cells can be easily selected using the neomycin antibiotic. In addition, the expression of the foreign genes Oct-4, Sox2, Klf4 and c-Myc introduced for dedifferentiation is inhibited by an epigenetic method after dedifferentiation has occurred (Takahashi and Yamanaka, Cell, 126:663-676, 2006).

Yamanaka's group succeeded in dedifferentiating human somatic cells by using four dedifferentiation factors (Oct-4, Sox2, Klf4 and c-Myc) which were the same as those used in the above-described experiment carried out using the mice. The human cells used were non-genetically engineered fibroblasts, and the produced dedifferentiated stem cells could differentiate into nerve cells or cardiac cells under suitable differentiation conditions (Takahashi et al., Cell, 131:861-872, 2007).

Also, Thomson's group that first produced human embryonic stem cells paid attention to the attention that cell dedifferentiation occurs when human embryonic stem cells are fused with bone marrow precursor cells. Based on this fact, Thomson's group isolated factors which are overexpressed in human embryonic stem cells compared to bone marrow precursor cells. Based on this information, differentiated stem cells were successfully constructed by introducing four factors (Oct-4, Sox2, Nanog and Lin28) into lentivirus systems (Yu et al., Induced Pluripotent Stem Cell Lines Derived from Human Somatic Cell, Science New York, NY, 2007).

Thus, in order to make induced pluripotent stem cells by reprogramming, a step of transferring dedifferentiation-inducing factors into somatic cells is necessarily carried out. Specifically, genes encoding the differentiation-inducing factors Oct-4, Sox2, Klf4 and c-Myc should be transferred into somatic cells using retroviruses as transporters (Takahashi. K. et al, Cell, 131:861-872, 2007). Alternatively, genes encoding the differentiation-inducing factors Oct-4, Sox2, Klf4 and c-Myc should be transferred into somatic cells using lentiviruses as transporters.

Sox family genes are known to play an important role in maintaining pluripotency, similar to Oct-4. However, the Oct-4 gene is involved only in pluripotent stem cells, whereas the Sox family genes are also involved in multipotent stem cells or unipotent stem cells. The Sox-2 (SRY-type high mobility group box 2) transcription factor plays an important role in maintaining the pluripotency of embryonic stem cells, unlike other proteins of the Sox-2 family proteins (Avilion et al., Genes Dev, 17:126-140, 2003). Like the case of Oct-4, when the expression of Sox-2 in mouse embryonic stem cells is inhibited, the differentiation of the cells will be induced (Ivanova et al., Nature, 442:533-538, 2006). Also, Sox-2-binding sites found in the promoter regions of downstream genes of Sox-2 are frequently present adjacent to the Oct-4 and Nanog binding sites (Boyer et al., Cell, 122:947-956, 2005). Thus, it is expected that the interaction between the Sox-2 transcription factor and the Oct-4 transcription factor will provide a framework for maintaining induced pluripotent stem cells in an undifferentiated state and for causing induced pluripotent stem cells to exhibit the characteristics of embryonic stem cells (Lewitzky and Yamanaka, Current Opinion in Biotechnology, 18:467-473, 2007). c-Myc is an oncogene that performs various intracellular functions, cell growth, differentiation, proliferation, programmed cell death, transformation into cancer cells. In addition, it was found to be the downward gene of the LIF (Leukemia Inhibitory Factor)/STAT3 and Wnt signaling mechanism that is important in maintaining pluripotency (Sears et al., Genes Dev, 14:2501-2514, 2000). The c- Myc transcription factor is expected to prevent inhibition of the proliferation of induced pluripotent stem cells (Seoane et al., Nature, 419:729-734, 2002). Also, c- Myc binds to the Myc recognition site in the genome and changes the chromatin structure so that Oct-4 and Sox-2 can easily bind to their target genes (Lewitzky and Yamanaka, Current Opinion in Biotechnology, 18:467-473, 2007).

Klf4 is involved in the inhibition of growth and serves to regulate the cell cycle. According to recent studies, it was found that Klf4 acts as the downward gene of STAT3 in embryonic stem cells, similar to c-Myc, and inhibits the differentiation of mouse embryonic stem cells by maintaining the expression Oct-4 when it is overexpressed (Li et al., Blood, 105:635-637, 2005).

Nanog is an embryo-specific gene and is required to maintain the pluripotency of embryonic stem cells, similar to Oct-4 or Sox-2. Also, LIN28 is an mRNA-binding protein and is known to be expressed in embryonic stem cells and embryonic tumor cells and to be involved in the differentiation and proliferation of the cells (Yu et al., Induced Pluripotent Stem Cell Lines Derived from Human Somatic Cell, Science New York, NY, 2007).

The Oct-4, Sox2, Klf4, c-Myc, Nanog and Lin28 genes as described above are called "dedifferentiation-inducing factors". The term "dedifferentiation-inducing factors" means genes capable of reprogramming completely differentiated cells. Particularly, Oct-4, Sox2, Klf4 and c-Myc are called "Yamanaka factors".

Hereinafter, the present invention will be described in further detail with reference to examples. It will be obvious to those skilled in the art that these examples are illustrative purposes only and are not to be construed to limit the scope of the present invention.

Particularly, in EExamples of the present invention, it was illustrated that, when stem cells were treated with endithelin-1, promoter demethylation was facilitated to improve the dedifferentiation of the cells. However, it will also be obvious to those skilled in the art that, when any conventional method of producing induced pluripotent stem cells from somatic cells or stem cells additionally comprises treating dedifferentiated cells with endothelin, the efficiency of dedifferentiation of the cells will increase. Thus, treating differentiated cells with endothelin to increase the efficiency of dedifferentiation of the cells falls within the scope of the invention.

In addition, in Examples of the present invention, it was illustrated that, when stem cells were treated with endithelin, demethylation was facilitated to improve the dedifferentiation of the cells. However, it will also be obvious to those skilled in the art that, when any conventional method of producing induced pluripotent stem cells from somatic cells or stem cells additionally comprises treating«dedifferentiated cells with an endothelin analog, the efficiency of dedifferentiation of the cells will increase. Thus, treating differentiated cells with an endothelin analog to increase the efficiency of dedifferentiation of the cells falls within the scope of the invention.

EXAMPLES

Example 1ί Preparation of mesenchymal stem cells

In the present invention, human mesenchymal stem cells purchased from Lonza were used. The cells were selected through MSC identification and showed the following marker expressions: more than 95% for positive markers (CD29, CD44, CD73, CD105, CD166, and H LA-ABC); and less than 5% for (CD34, CD45, and HLA- DR). Also, the cells was confirmed to have the multipotency of mesenchymal stem cells and identified as human umbilical blood-derived stem cells. Example 2: Analysis of difference in gene expression by cDNA array

The mesenchymal stem cells were adhesion-cultured in a-MEM (Minimum Essential Medium, Invitrogen) and 10% FBS (Fetal Bovine Serum) medium (5xl05/60@ dish). After 24-48 hours of culture, 0.025 ^g/ml of endothelin-1 was added to the medium, after which the cells were cultured for 24 hours. The culture was carried out in a C02 incubator at 37 °C , and the cells were cultured for 7-9 passages. Then, the cells were sampled, and RNA was collected from the cell samples and subjected to a cDNA array (Aagillent, USA).

First, among a total of 440,000 marker genes, 1086 genes showing a variation in gene expression of 1.5 times or more and a high reliability value (< P value 0.05) relative to a vehicle after treatment of the cells with endothelin-1 were selected. As shown in FIG. 1. 1,053 genes were turned on when the cells were treated with endothelin-1.

Example 3: Analysis of demethylation by CpG array (Aagillent, USA)

Genomic DNA was extracted from each of a group treated with endothelin-1 and a group not treated with endothelin-1 and immunoprecipitated using 5-methyl cytosine antibody (Diagenode), after which each extract was purified and subjected to a CpG array (Aagillent, USA) with a probe in order to synthesize CpG genomic sequences. Then, a comparison of intensity between the synthesized sequences was performed.

As a result, as shown in FIG. 2, most of the genes were demethylated upon treatment with endothelin. In FIG. 2, the negative values indicate demethylated regions, and the positive values indicate methylated regions. As can be seen in FIG. 2, the negative values were predominant in all the 24 chromosomes. Thus, it could be seen that the increase in gene expression resulting from the increase in demethylation in the mesenchymal stem cells was attributable to endothelin. Example 4: Analysis of increase in expression levels of pluripotency genes by real-time PCR

The effect of endothelin-1 on the expression of pluripotency genes (E- cadherin, ERAS, and STAT3) in improving the dedifferentiation of stem cells was examined. In order to examine the change in gene expression at the RNA level, RT-PCR (real-time PCR) was performed. Specifically, primers specific for each of E- cadherin (SEQ ID NOS: 2 and 3), ERAS (SEQ ID NOS: 4 and 5) and STAT3 (SEQ ID NOS: 6 and 7) genes were constructed, after which the stem cells prepared in Example 1 were treated with endothelin-1. After 24 hours, RNA was extracted from the cells, and then complementary cDNA was synthesized from the extracted RNA. RT-PCR was performed using the synthesized cDNA as a template with the constructed primers.

SEQ ID NO: 2: F-GTTCACCATTAACGAGAACA

SEQ ID NO: 3: R-AATGCCATCGTTGTTCACTG

SEQ ID NO: 4: F-AACAAAGCCTGGCACCTT

SEQ ID NO: 5: R-TTCAGAATGCAGTCCCCACT

SEQ ID NO: 6: F-CCTTTGGAACGAAGGGTACA

SEQ ID NO: 7: R-G I I G I I CAGCTGCTGCTTT

The results of the RT-PCR are shown in FIG. 3. As can be seen therein, the expression levels of the pluripotency genes (E-cadherin, ERAS, and STAT3) in the group treated with endothelin-1 increased compared to those in the naive group.

In the same manner as above, RT-PCR was performed using primers of SEQ ID NOS: 8 and 9 in order to examine the effect of endothelin-1 on the expression of the Oct-4 gene. As a result, as can be seen in FIG. 4, the expression of Oct-4 in the group treated with endothelin-1 increased compared to that in the naive group.

SEQ ID NOS: 8: F-GAGGCAACCTGGAGAATTTG

SEQ ID NOS: 9: R-TAGCCTGGGGTACCAAAATG Although the present invention has been described in detail with reference to the specific features, it will be apparent to those skilled in the art that this description is only for a preferred embodiment and does not limit the scope of the present invention. Thus, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

Industrial Applicability

As described above, when cells are treated with endothelin or an endothelin analog according to the present invention, the demethylation of promoters of various genes in the cells can be facilitate, thus obtaining cells having an increased ability to dedifferentiate. Thus, the present invention is useful for improving the limited ability of existing induced pluripotent stem cells to dedifferentiate.

Claims

What is Claimed is:

1. A composition for improving dedifferentiation of cells, which comprises a polypeptide having the amino acid sequence of SEQ ID NO: l or a nucleotide sequence encoding the polypeptide having the amino acid sequence of SEQ ID NO:l as an active ingredient.

2. The composition according to claim 1, wherein the polypeptide having the amino acid sequence of SEQ ID NO:l is selected from the group consisting of Endothelin, Endothelin Analog, Big Endothelin and prepro Endothelin.

3. The composition according to claim 1, wherein the cells are somatic cells or stem cells.

4. The composition according to claim 2 or 3, wherein the Endothelin is at least one selected from the group consisting of Endothelin-1, Endothelin-2 and Endothelin-3.

5. The composition according to claim 2 or 3, wherein the Endothelin Analog comprises amino acid residues 16-21 described in SEQ ID.NO: l and amino acid residues in which at least one amino acid(s) is/are deleted or modified from the amino acid residues 1-15 in 21 amino acid residues which consist of Endothelin.

6. A composition for facilitating the demethylation of promoters of genes in the cells, which comprises a polypeptide having the amino acid sequence of SEQ ID NO: l or a nucleotide sequence encoding the polypeptide having the amino acid sequence of SEQ ID NO:l as an active ingredient.

7. The composition according to claim 6, wherein the polypeptide having the amino acid sequence of SEQ ID NO:l is selected from the group consisting of Endothelin, Endothelin Analog, Big Endothelin and prepro Endothelin.

8. The composition according to claim 6, the genes are at least one selected from a group consisting of E-cadherim, ERAS, STATS, Oct-4, Sox-2, Klf4, c-Myc, Nanog and Lin28.

9. The composition according to any one of claims 6 to 8, wherein the cells are somatic cells or stem cells.

10. The composition according to claim 7, wherein the Endothelin is at least one selected from a group consisting of Endothelin-1, Endothelin-2 and Endothelin-3.

11. The composition according to claim 7, wherein the Endothelin Analog comprises amino acid residues 16-21 described in SEQ ID.NO: l and amino acid residues in which at least one amino acid(s) is/are deleted or modified from the amino acid residues 1-15 in 21 amino acid residues which consist of Endothelin.

12. A method for producing induced pluripotent stem cells, which comprises a step of treating cells with a polypeptide having the amino acid sequence of SEQ ID NO:l or a nucleotide sequence encoding the polypeptide having the amino acid sequence of SEQ ID NO: l.

13. The method according to claim 12, wherein the polypeptide having the amino acid sequence of SEQ ID NO:l is selected from the group consisting of Endothelin, Endothelin Analog, Big Endothelin and prepro Endothelin.

14. The method according to claim 12 or 13, wherein the cells are somatic cells or stem cells.

15. The method according to claim 12, wherein dedifferentiating-inducing factors in the cells are demethylated by treating cells with a polypeptide having the amino acid sequence of SEQ ID NO:l or a nucleotide sequence encoding the polypeptide having the amino acid sequence of SEQ ID NO:l.

16. The method according to claim 12, wherein the cells are treated with an extract from pluripotent stem cells.

17. The method according to claim 12, wherein dedifferentiating inducing factors are introduced into genes of the cells.

18. The method according to claim 15 or 17, wherein the dedifferentiating-inducing factors are at least one selected from a group consisting of Oct-4, Sox-2, Klf4, c-Myc, Nanog, Lin28, E-cadherim, ERAS and STAT3.

19. The method according to claim 13, wherein the Endothelin is at least one selected from a group consisting of Endothelin-1, Endothelin-2 and Endothelin-3

20. The method according to claim 13, wherein the Endothelin Analog comprises amino acid residues 16-21 described in SEQ ID.NO: l and amino acid residues in which at least one amino acid(s) is/are deleted or modified from the amino acid residues 1-15 in 21 amino acid residues which consist of Endothelin.

21. A composition for producing induced pluripotent stem cells, which comprises a polypeptide having the amino acid sequence of SEQ ID NO: l or a nucleotide sequence encoding the polypeptide having the amino acid sequence of SEQ ID NO:l as an active ingredient.

22. The composition according to claim 21, wherein the polypeptide having the amino acid sequence of SEQ ID NO: l is selected from the group consisting of Endothelin, Endothelin Analog, Big Endothelin and prepro Endothelin.

23. The composition according to claim 21, wherein the induced pluripotent stem cells are induced from somatic cells or stem cells.

24. The composition according to claim 22, wherein the Endothelin is at least one selected from a group consisting of Endothelin-1, Endothelin-2 and Endothelin-3.

25. The composition according to claim 22, wherein the Endothelin Analog comprises amino acid residues 16-21 described in SEQ ID.NO: l and amino acid residues in which at least one amino acid(s) is/are deleted or modified from the amino acid residues 1-15 in 21 amino acid residues which consist of Endothelin.