KR20120134360A

KR20120134360A - Composition for improving dedifferentiation of cells and method for producing induced pluripotent stem cells

Info

Publication number: KR20120134360A
Application number: KR1020110053213A
Authority: KR
Inventors: 이은주
Original assignee: 서울대학교병원
Priority date: 2011-06-02
Filing date: 2011-06-02
Publication date: 2012-12-12
Also published as: WO2012165740A1

Abstract

PURPOSE: A method for preparing induced pluripotent stem cells using a composition for enhancing cell dedifferentiation is provided to promote promoter demethylation of various genes in the cells and to enhance limited dedifferentiation. CONSTITUTION: A composition for enhancing cell dedifferentiation contains endothelin, endothelin modifier, Big endothelin, or prepro endothelin as an active ingredient. The cells are somatic cells or stem cells. An endothelin analog is prepared by modifying or deleting one or more amino acids among 1-15th amino acids. A composition for promoting demethylation of genes in cells contains the endothelin, endothelin analog, Big-endothelin, or prepro endothelin as an active ingredient. The genes are E-cadherin, ERAS, STAT3, oct-4, sox2, Klf4, c-Myc, Nanog, or Lin28. A method for preparing the induced pluripotent stem cells comprises a step of treating the cells with endothelin, endothelin analog, Big-endothelin, or prepro endothelin.

Description

Composition for Improving Cell Differentiation and Method for Producing Induced Pluripotent Stem Cells Using the Same {Composition for Improving Dedifferentiation of Cells and Method for Producing Induced Pluripotent Stem Cells}

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

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 refer to cells from the egg and sperm to the 8-cell stage after fertilization. When these cells are isolated 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 that contain the cells.

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, as a cell therapy, embryonic stem cells have to be made using an egg, have an ethical problem that can be obtained by destroying the embryo, and also have various problems such as difficulty in clinical use due to an immune rejection reaction.

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 can differentiate into all types of cells of the body with the ability to self-renewal similarly to embryonic stem cells. Has characteristics. To date, induced pluripotent stem cells have been reported to exhibit almost the same characteristics as embryonic stem cells, pluripotent stem cells, in gene expression and differentiation ability (Takahashi and Yamanaka, Cell, 126: 663-676, 2006).

Methylation of gene promoters is a major mechanism that regulates expression of genes, and it is known that the expression of genes is lost when the promoter portion of a gene is methylated. Embryologically, 'raw germ cells', which are classified as all stages of pluripotent stem cells, exist with all genomic DNA methylation blank and regulate the specific expression of genes during development and differentiation during fertilization. As a result of promoter methylation, differentiated somatic cells are formed and the individual is completed. Therefore, eliminating promoter methylation through development and differentiation is an essential gateway for cell differentiation.

On the other hand, induced pluripotent stem cells can be prepared by a variety of methods, including a method of inducing reverse differentiation by a pluripotent cell extract treatment or a method using a reverse differentiation factor.

However, since the method of introducing pluripotent cells into differentiated somatic cells to de-differentiate somatic cells is derived from allogeneic or heterologous rather than autologous proteins, the possibility of pathogenic infection cannot be excluded and the core protein is not identified. There is a problem that the variation of the conditions according to the experiment is severe. In addition, it has a low efficiency, as is known to the differentiation induction methods known to date, there is a limitation in clinical applications.

In addition, the method for producing induced pluripotent stem cells using the reverse differentiation inducer essentially includes the step of delivering the reverse differentiation inducer to the differentiated cells. Specifically, induced pluripotent stem cells transfer genes encoding dedifferentiation inducers into retroviruses to transfer to somatic cells (Takahashi et al., Cell, 131: 861-872, 2007), or genes encoding dedifferentiation inducers. Each can be prepared by the method of delivery to somatic cells (Yu et al., Induced Pluripotent Stem Cell Lines Derived from Human Somatic Cell, Science New York, NY, 2007).

However, retroviruses or lentiviruses were used to introduce individual genes into the cell, so that the viruses could be intermittently inserted into the genome, causing mutations, activating carcinogenic genes or inhibiting the expression of anticancer genes. May cause tumors. In addition, since the viruses expressing different genes are less likely to enter the same cell at the same time, there is a problem that the efficiency of producing induced pluripotent stem cells is low. For example, about 10 induced pluripotent stem cells are obtained from 5 × 10 ⁵ human cell fibroblasts (Takahashi et al., Cell, 131: 861-872, 2007).

Accordingly, the present inventors confirmed that when endothelin was treated in cells, the expression of genes as well as pluripotency marker genes present in the cells was increased in general, which was due to the dimethylation of genomic DNA, resulting in endothelin treatment. By recognizing that it can enhance the efficiency of the conventional induced pluripotent stem cell production method has been completed the present invention.

It is an object of the present invention to provide a composition for promoting reverse differentiation that can make cells having enhanced reverse differentiation ability.

Another object of the present invention is to provide a method for producing induced pluripotent stem cells having enhanced formation efficiency and a composition for inducing induced pluripotent stem cells.

In order to achieve the above object, the present invention provides a composition for enhancing the differentiation of cells comprising an endothelin or an endothelin analog as an active ingredient and a method for producing induced pluripotent stem cells using the same.

The present invention also provides a composition for inducing pluripotent stem cells comprising an endothelin or an endothelin analog as an active ingredient.

According to the present invention, when the cell is treated with an endothelin or an endothelin analog, promoter dimethylation of various genes in the cell is promoted to obtain a cell having enhanced de-differentiation ability, thereby inducing a conventional induced pluripotent stem cell. It is useful for enhancing the limited dedifferentiation capacity, which has been pointed out as a limitation.

1 shows a change in gene expression when endothelin-1 is treated to mesoderm stem cells.
Figure 2 shows the dimethylation (demethylation) of the gene promoter when treated with endothelin-1 to mesoderm stem cells.
3 shows the expression rate of pluripotency genes when endothelin-1 is treated to mesoderm stem cells.
Figure 4 shows the expression rate of Oct-4 when treated with endothelin-1 to mesoderm stem cells.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein is well known and commonly used in the art.

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

Most mammals have isopeptide endothelins of endothelin-1, endothelin-2 and endothelin-3. The three isopeptide endothelins have similar functions and effects in transient vasodilation and continuous vasoconstriction.

As used in the present invention, "Endothelin Modifier" (Endothelin Modifier) is an endothelin consisting of 21 amino acids, including the active part 16-21 amino acids (SEQ ID NO: 1), 1 to 15 of the amino acids It means a form in which one or more is modified or deleted, and means a modified or deleted form except for the amino acids 16-21, the main part responsible for the endothelin activity, also called Endothelin Derivative. Endothelin analogs have similar functions and effects as endothelin binds to receptors (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

In the present invention, "modified" means that one or more amino acids of amino acids 1-15 of the endothelin is substituted with another amino acid, "deleted" means one or more of the amino acids 1-15 of constituting the endothelin Amino acids have been deleted.

In the present invention, "prepro ET (prepro Endothelin)" means 212 amino acids that are first generated by the gene encoding the endothelin, prepro ET is composed of 38 amino acids by the action of the enzyme "big ET (big) Endothelin) ". Endothelin is produced when the ET converting enzyme (Endothelin Converting Enzyme: ECM) acts on the big ET and the amino acid of the C-terminal is released (Yanagisawa M et al., Nature, 332: 411-415, 1988).

As used herein, "cell" refers to somatic or stem cells of various genetic backgrounds and / or origins. For example, it is possible to use somatic cells, mesoderm stem cells, ectoderm stem cells and endoderm stem cells, preferably cord blood derived stem cells, adipose derived stem cells.

"Somatic cells" refers to cells that have completed differentiation and are not pluripotent or limited, and "differentiation" refers to a phenomenon in which structures or functions are specialized during the division and proliferation of cells. Changes in form or function to accomplish a task given to a person. Somatic cells used in the present invention may be naturally occurring somatic cells or genetically modified somatic cells.

"Stem cells" are cells that have the ability to differentiate into different types of body tissues, i.e., 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 differentiate into various organs or tissues according to a given developmental condition in the early development of the organism.

In the present invention, as a result of treatment of endothelin or endothelin analogs to mesoderm stem cells, it was confirmed that dedifferentiation of mesoderm stem cells is enhanced.

The present invention relates to a composition for promoting reverse differentiation of a cell comprising endothelin or an endothelin analog as an active ingredient and a composition for promoting dimethylation of intracellular genes.

"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 methylation patterns, changes in expression rates of stem cell genes, and the like.

In an embodiment of the present invention, 24 hours after endothelin-1 treatment to mesoderm stem cells, 97 of the gene markers having high reliability were obtained as a result of performing a cDNA array to determine the difference between the vehicle treatment group and gene expression. It was confirmed that the gene of% turned on (Fig. 1).

In the present invention, "methylation" means attaching a methyl group to the base, and "demethylation" means falling off the methyl group from the base. Such methylation may alter gene expression patterns. Methylation of genes interferes with the expression of the gene by disrupting the activity of the expressed protein or by aggregating enzymes that interfere with expression. In other words, methylation of genes prevents transcription and silences the genes. In induced pluripotent stem cells, promoters of genes related to stem cellularity such as Oct 3, Oct 4, and Nanog have been dimethylated, which demonstrates the activity of the promoter and expression of stem cell related genes in induced pluripotent stem cells.

In another embodiment of the present invention, a CpG array was performed to prove that the dimethylation induced by endothelin-1 was induced by the mechanism of turning on most genes as a result of cDNA array (FIG. 2). .

Two consecutive bases of cytosine and guanine in the nucleotide sequence of the genome are called CpG. Cytosine is highly methylated in this sequence, and in human genomes, 3-5% of all cytosine is methylated. have. The CpG has been gradually reduced in evolution, and the degree and pattern of methylation of CpG present in the genome is very specific depending on the species of mammal and the tissue. Mammalian sequences, including humans, contain dense CpG sites that range from 0.5 to 4 kb. CpG islands are closely related to genes in the mammalian genome because they are located near the promoter, a region that regulates gene transcription. Most of the CpG islands are not methylated, and if the CpG islands of the promoter region that initiate expression of a particular gene are methylated, it means that the expression of that particular gene is inhibited. In other words, the methylation of CpG islands is a mechanism developed by special molecular networks that regulate gene expression for specific purposes.

In FIG. 2, a negative value labels a dimethylated region and a positive value labels a methylated region. As a result, it can be seen that the label is biased by a negative value in all 24 chromosomes, resulting in an increase in dimethylation in mesoderm stem cells. The increase in gene expression was confirmed to be due to endothelin.

In another embodiment of the present invention, when real-time PCR is performed to process endothelin-1 to mesoderm stem cells, pluripotent stem cell control compared to the mesoderm stem cell control (naive) not treated with endothelin-1. It was confirmed that the expression of E-cadherin, ERAS and STAT3, which are known as (pluripotency) genes, were increased (FIG. 3).

As used herein, "pluripotent stem cells" refers to the ability to differentiate into all the germ-derived cells, including mesoderm, endoderm and ectoderm, which are necessary for individual formation, and represent the ultimate ability of stem cells. In addition, pluripotent stem cell genes are classified as inducers of dedifferentiation because they mean markers indicating this ability or genes capable of inducing pluripotency.

E-cadherin is known to be involved in signaling pathways related to pluripotency and self-renewal in embryonic stem cells and induced pluripotent stem cells. ERAS is understood to promote the production and proliferation of teratoma in stem cells. Terratoma is a type of tumor composed of many kinds of cells and tissues. These teratomas are also used to verify the differentiation capacity of embryonic stem cells. Due to the characteristics of embryonic stem cells that differentiate into cells of all tissues, when embryonic stem cells are injected into experimental animals such as mice, embryonic stem cells differentiate into various types of cells and tissues to form teratomas. Therefore, if teratomas were made in the final stages of embryonic stem cell proliferation, it proves that stem cells were made. In addition, STAT3 is known to play a role in maintaining embryonic stem cells undifferentiated ability.

In addition, when endothelin-1 was treated to mesoderm stem cells, it was confirmed that the expression of Oct-4 gene, which is a reverse differentiation inducer, was increased as compared to the control (naive) (FIG. 4).

Oct-4 transcription factor is one of the Octamer Transcription Factor, which is expressed only in undifferentiated embryonic stem cells and embryonic tumor cells and plays an important role in maintaining pluripotency of stem cells (Okamato et al., Cell , 60: 461-472, 1990). Normal expression of Oct-4 maintains autogenous reproduction and pluripotency of embryonic stem cells, but when it is raised above normal level, it differentiates into primitive endoderm and primitive mesoderm, and when it is lowered to normal level, it becomes trophic ectoderm cell. Develop (Niwa et al., Nat Genet, 24: 372-376, 2000). Therefore, Oct-4 is an important factor in determining the pluripotency of embryonic stem cells and regulating differentiation. Oct-4 interacts with Sox2 transcription factors, leading to 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 in the Oct family, including Oct-1 and Oct-6, which have a structure similar to Oct-4, do not have the ability to dedifferentiate differentiated somatic cells, and only Oct-4 is involved in this process ( Lowry et al., PNAS, 105: 2883-2888, 2008).

As described above, in the present invention, when endothelin or endothelin analogs are treated in mesoderm stem cells, dimethylation is promoted, but the reverse differentiation of cells is enhanced, but the conventional method for preparing induced pluripotent stem cells from somatic cells and stem cells In the method, it will be apparent to those skilled in the art that when the cells are treated with endothelin, the reverse differentiation efficiency will be within the scope of the present invention.

In another aspect, the present invention relates to a method for producing induced pluripotent stem cells comprising the step of treating cells with endothelin and a composition for inducing induced pluripotent stem cells comprising endothelin as an active ingredient.

In the present invention, "induced pluripotent stem cells" refers to cells having pluripotency, which can be obtained by reprogramming differentiated cells.

Conventional methods for preparing induced pluripotent stem cells are as follows.

Extracts of pluripotent cells may be introduced into differentiated somatic cells to reverse differentiate the somatic cells. After the cells are permeabilized with streptolysin O, the pluripotent cell extracts are treated and cultured to observe the nuclear differentiation. Direct exposure of human somatic cells to extracts of Xenopus eggs, embryonic germ cells, embryonic carcinoma cells, or embryonic stem cells reexpresses undifferentiated marker genes (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). The pluripotent cell extract generally refers to an embryonic stem cell extract, and refers to a material obtained by pulverizing embryonic stem cells cultured in vitro through physical and chemical methods and then separating them by centrifugation or the like.

In addition, induced pluripotent stem cells can be prepared by using a differentiation factor. Takahashi and Yamanaka succeeded in inducing differentiation with a screening of 24 candidate groups, which are likely to induce somatic pluripotency. They isolate only four factors, Oct-4, Sox2, Klf4 and c-Myc, and express them in mouse fibroblasts through retroviral system, resulting in epigenetic dedifferentiation and pluripotency, leading to similar characteristics as embryonic stem cells. Revealed that. Mice fibroblasts in which neomycin antibiotic resistance genes were expressed by the promoter of Fbx15 gene, which is expressed only in pluripotent cells, were used for inducing differentiation. Thus, dedifferentiated cells can be easily selected using neomycin antibiotics. In addition, expression of Oct-4, Sox2, Klf4 and c-Myc genes introduced externally for reverse differentiation is inhibited by epigenetic methods after reverse differentiation (Takahashi and Yamanaka, Cell, 126: 663-676). , 2006).

The Yamanaka group succeeded in redifferentiating human somatic cells using the same four differentiation factors of Oct-4, Sox2, Klf4 and c-Myc as in mice. Human cells were fibroblasts that had not been genetically manipulated, and the resulting dedifferentiated stem cells were able to differentiate into neurons or heart cells under appropriate differentiation conditions (Takahashi et al., Cell, 131: 861-872). , 2007).

In addition, the Thomson group, which produced human embryonic stem cells for the first time, focused on the reverse differentiation of human embryonic stem cells and bone marrow precursor cells. I separated it. Based on the information obtained here, four factors, Oct-4, Sox2, Nanog and Lin28, were introduced into the lentiviral system to produce dedifferentiated stem cells (Yu et al., Induced Pluripotent Stem Cell Lines Derived from Human). Somatic Cell, Science New York, NY, 2007).

Therefore, in order to form induced pluripotent stem cells for reprogramming, it is necessary to pass the induction of dedifferentiation inducers into the somatic cells. Genes encoding genes were transferred to somatic cells using retroviruses as transporters (Takahashi. K. et al, Cell, 131: 861-872, 2007), or induction of reverse differentiation of Oct-4, Sox2, Nanog and Lin28. Each gene encoding the factor should be delivered to somatic cells using lentiviruses as transporters.

Sox family genes are known to play an important role in maintaining pluripotency, similar to Oct-4. But while the Oct-4 gene is only involved in pluripotent stem cells, the Sox family of genes is also associated with pluripotent stem cells or pluripotent stem cells. Sox-2 (SRY-type high mobility group box 2) 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 ). As with Oct-4, inhibition of Sox-2 expression in mouse embryonic stem cells leads to differentiation (Ivanova et al., Nature, 442: 533-538, 2006). In addition, Sox-2 binding sites found in the promoter regions of several Sox-2 subgenes often exist adjacent to Oct-4 and NAnog binding sites (Boyer et al., Cell, 122: 947-956, 2005). Therefore, the interaction between Sox-2 transcription factor and Oct-4 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 shown to be a subgene of the major mechanisms that maintain pluripotency, LIF (Leukemia Inhibitory Factor) / STAT3 and Wnt signaling mechanisms (Sears et al., Genes Dev, 14: 2501-2514, 2000). c-Myc transcription factors are expected to play a role in inhibiting cell proliferation in induced pluripotent stem cells (Seoane et al., Nature, 419: 729-734, 2002). In addition, c-Myc not only binds to Myc recognition sites in the genome, but also changes the chromatin structure to help Oct-4 and Sox-2 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 subgene of STAT3 in embryonic stem cells and inhibits differentiation of mouse embryonic stem cells by maintaining Oct-4 expression when overexpressed (Li et al., Blood, 105: 635-637, 2005).

Nanog is an embryo-specific gene that, like Oct-4 and Sox-2, 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, Oct-4, Sox2, Klf4, c-Myc, Nanog and Lin28 genes are referred to as "reprogramming-inducing genes", which can reprogram differentiated cells. Genes present. In particular Oct-4, Sox2, Klf4 and c-Myc are called Yamanaka factors.

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.

Particularly, in the present embodiment, when endothelin-1 is treated to stem cells, dimethylation is promoted to reverse cell differentiation. However, in the conventional method for preparing induced pluripotent stem cells from somatic cells and stem cells, When the endothelin is treated in cells, it will also be apparent to those skilled in the art that the efficiency of reverse differentiation will fall within the scope of the present invention.

In addition, in the present embodiment, the endothelial dimethylation is promoted when the endothelin is treated in the stem cells, but the reverse differentiation of the cells is enhanced, but in the conventional method for preparing induced pluripotent stem cells from somatic cells and stem cells, the cells It will also be apparent to those skilled in the art that the treatment of endothelin analogs will increase their dedifferentiation efficiency, which will be within the scope of the present invention.

Preparation of Mesoderm-Derived Stem Cells

In the present invention, human mesoderm stem cells purchased from Lonza were used. The cells were selected by performing human mesodermal stem cell identification experiments, and expressed more than 95% of positive cell markers (CD29, CD44, CD73, CD105, CD166, HLA-ABC) and negative cell markers (CD34, CD45). , HLA-DR) was identified and classified as human umbilical cord blood-derived stem cells after confirming uniform expression of less than 5%, and confirming the multiple differentiation capacity of mesoderm stem cells.

Gene expression difference analysis using cDNA array

The hMSCs were cultured attached using α-MEM (Minimum Essential Medium, Invitrogen) + 10% FBS (Fetal Bovine Serum) medium ^{(5 × 10 5/60 @} dish). After 24-48 hours of incubation, 0.025 μg / ml of endothelin-1 was added to the medium and incubated for 24 hours. Cultivation was performed in a 37 ° C. CO ₂ incubator and cells were used with passages between 7-9. Cells were then sampled and RNA was collected, followed by cDNA array (Aagillent, USA).

First, 1086 genes were selected from among 440,000 gene markers, which showed more than 1.5-fold change in gene expression and high reliability (<P value 0.05) after endothelin-1 treatment. As shown in FIG. 1, 1053 genes were turned on upon endothelin-1 treatment.

CpG array (Aagillent, USA ) Trend of demethylation

Genomic DNA from endothelin-1 and non-treated groups was extracted, immunoprecipitated with 5-methyl cytosine antibody (Diagenode), purified and synthesized in CpG array (Aagillent, USA) with probe. Derived and intensity were compared.

As a result, as shown in Figure 2, it was confirmed that most genes are dimethylated during the endothelin treatment. Negative values mark the dimethylated region and positive values mark the methylated region. As a result, the label is shifted to the negative value in all 24 chromosomes. Therefore, the gene expression increases due to the increase of dimethylation in mesoderm stem cells. Was confirmed to be due to endothelin.

Analysis of Increased Expression of Pluripotency Genes Using Real-time PCR

In promoting stem cell reverse differentiation, the effects of endothelin-1 on the expression of Pluripotency genes (E-cadherin, ERAS, STAT3) were examined. To confirm changes in RNA levels, RT-PCR (Real-time PCR) was performed. Specifically, E-cadherin (SEQ ID NOs: 2 and 3); ERAS (SEQ ID NOs: 4 and 5); And primers specific for each of the STAT3 (SEQ ID NOs: 6 and 7) genes, and then treated with endothelin-1 in the stem cells prepared in Example 1 and extracted RNA after 24 hours to synthesize complementary cDNA, RT-PCR was performed as a template.

SEQ ID NO: F-GTTCACCATTAACGAGAACA

SEQ ID NO: R-AATGCCATCGTTGTTCACTG

SEQ ID NO 4: F-AACAAAGCCTGGCACCTT

SEQ ID NO: 5: R-TTCAGAATGCAGTCCCCACT

SEQ ID NO: F-CCTTTGGAACGAAGGGTACA

SEQ ID NO: R-GTTGTTCAGCTGCTGCTTTG

As a result, as shown in Figure 3, when the endothelin-1 treatment, it was found that the expression of pluripotent stem cell genes (E-cadherin, ERAS, STAT3) increased compared to the control (naive).

In the same way, RT-PCR was performed using primers SEQ ID NOs: 8 and 9 to examine the effect of endothelin-1 on the expression of Oct-4 gene. As a result, as shown in Figure 4, it was found that the expression of Oct-4 increased compared to the control (naive).

SEQ ID NO: F-GAGGCAACCTGGAGAATTTG

SEQ ID NO: R-TAGCCTGGGGTACCAAAATG

While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will readily appreciate that many modifications are possible in the exemplary embodiments without materially departing from the novel teachings and advantages of this invention. something to do. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

<110> SEOUL NATIONAL UNIVERSITY HOSPITAL <120> Composition for Improving Dedifferentiation of Cells and Method for Producing Induced Pluripotent Stem Cells <130> P11-B045 <160> 9 <170> Kopatentin 2.0 <210> 1 <211> 6 <212> PRT <213> Mus musculus <400> 1 His Leu Asp Ile Ile Trp 1 5 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> E-cadherin forward primer <400> 2 gttcaccatt aacgagaaca 20 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> E-cadherin reverse primer <400> 3 aatgccatcg ttgttcactg 20 <210> 4 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> ERAS forward primer <400> 4 aacaaagcct ggcacctt 18 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> ERAS reverse primer <400> 5 ttcagaatgc agtccccact 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> STAT3 forward primer <400> 6 cctttggaac gaagggtaca 20 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> 223 STAT3 reverse primer <400> 7 gttgttcagc tgctgctttg 20 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Oct-4 foward primer <400> 8 gaggcaacct ggagaatttg 20 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Oct-4 reverse primer <400> 9 tagcctgggg taccaaaatg 20

Claims

A composition for enhancing dedifferentiation of cells comprising at least one component selected from the group consisting of endothelin, endothelin modifier, big end (big endothelin) and prepro end tooth (prepro endothelin) as an active ingredient.

According to claim 1, wherein the cells are somatic cells or stem cells, characterized in that for improving the differentiation.

The method of claim 1 or 2, wherein the endothelin is at least one selected from the group consisting of endothelin-1, endothelin-2, and endothelin-3.

The method of claim 1, wherein the endothelin analogue comprises the amino acid sequence of SEQ ID NO: 1 from amino acids 16 to 21 of the 21 amino acids constituting the endothelin, one or more amino acids of 1 to 15 modified A composition for promoting reverse differentiation of cells, characterized in that the cell is deleted or deleted.

The composition of claim 1, wherein the prepro ET and big ET comprise the amino acid sequence of SEQ ID NO: 1 in sequence.

Endothelin, endothelin analogs, Big ET and prepro ET composition for promoting dimethylation (demethylation) of intracellular genes comprising at least one component selected from the group consisting of an active ingredient.

The method according to claim 6, wherein the gene is at least one selected from the group consisting of E-cadherin, ERAS, STAT3, Oct-4, Sox2, Klf4, c-Myc, Nanog and Lin28 Promotes the dimethylation of intracellular genes Composition.

The composition for promoting dimethylation of intracellular genes according to claim 6 or 7, wherein the cells are somatic cells or stem cells.

8. The composition for promoting dimethylation of intracellular genes according to claim 6 or 7, wherein the endothelin is at least one selected from the group consisting of endothelin-1, endothelin-2, and endothelin-3.

The method according to claim 6, wherein the endothelin analogue comprises the amino acid sequence of SEQ ID NO: 1 from amino acids 16 to 21 among 21 amino acids constituting the endothelin, wherein at least one of amino acids 1 to 15 is modified. A composition for promoting dimethylation of intracellular genes, characterized in that it is deleted or deleted.

The composition for promoting dimethylation of intracellular genes according to claim 6, wherein the prepro ET and big ET comprise the amino acid sequence of SEQ ID NO.

A method for producing induced pluripotent stem cells, comprising treating a cell with at least one component selected from the group consisting of endothelin, endothelin analogs, Big ET and prepro ET.

The method of claim 12, wherein the cells are somatic cells or stem cells.

The method of claim 12, wherein reprogramming-inducing genes present in the cell are dimethylated by the endothelin treatment.

13. The method of claim 12, wherein the cells are treated with pluripotent cell extracts.

13. The method of claim 12, wherein the cell is introduced with a reverse differentiation inducer.

17. The induced pluripotency of claim 14 or 16, wherein the inducible inducer is at least one selected from the group consisting of Oct-4, Sox2, Klf4, c-Myc, Nanog, Lin28, E-cadherin, ERAS, and STAT3. Method for producing stem cells.

The method according to any one of claims 12 to 16, wherein the endothelin is at least one selected from the group consisting of endothelin-1, endothelin-2, and endothelin-3. .

13. The method of claim 12, wherein the endothelin analogue comprises any of the amino acid sequences of SEQ ID NO: 1 from amino acids 16 to 21 of the 21 amino acids constituting the endothelin, at least one of amino acids 1 to 15 Method for producing induced pluripotent stem cells, characterized in that modified or deleted.

The method of claim 12, wherein the prepro ET and big ET comprise the amino acid sequence of SEQ ID NO: 1 in sequence.

Endothelial, endothelin analogs, Big ET and prepro ET composition for inducing pluripotent stem cells comprising at least one component selected from the group consisting of an active ingredient.

22. The composition of claim 21, wherein the induced pluripotent stem cells are derived from somatic cells or stem cells.

23. The composition of claim 21 or 22, wherein the endothelin is at least one selected from the group consisting of endothelin-1, endothelin-2, and endothelin-3.

22. The method according to claim 21, wherein the endothelin analogue comprises the amino acid sequence of SEQ ID No. 1 as it is, among 21 amino acids constituting the endothelin, wherein at least one of amino acids 1 to 15 is modified or deleted. Induced pluripotent stem cell composition for induction.

The composition for inducing pluripotent stem cells according to claim 21, wherein the prepro ET and big ET comprise the amino acid sequence of SEQ ID NO.