KR20110078234A - Generation composition for induced pluripotent stem cells with purmorphamine treatment and oct4, and method of manufacturing induced pluripotent stem cells using the same - Google Patents

Abstract

PURPOSE: A composition for inducing redifferentiation from somatic cells to embryonic stem cell-like cells is provided to treat various diseases. CONSTITUTION: A composition contains purmorphamine; and a virus which expresses Oct4 protein by introduction of a nucleic acid molecule encoding Oct4 protein. A method for preparing embryonic stem cell-like cells comprises: a step of culturing fibroblast in a medium; a step of performing transfection of a vector to the cultured fibroblasts and infecting packaging cells; and a step of culturing the fibroblasts.

Description

Composition composition for inducing pluripotent stem cells with purmorphamine treatment and Oct4, and method of inducing differentiation of somatic cells from embryonic stem cell-like cells using permophamine and occ-4 manufacturing induced pluripotent stem cells using the same}

The present invention relates to a composition for inducing the differentiation of somatic cells from embryonic stem cell-like cells using permopamine and Oct4, and to a method for producing embryonic stem cell-like cells using the same. More specifically, the present invention uses a novel composition that induces cells, such as embryonic stem cells, through reprogramming from somatic cells using permorphamine (purmorphamine), an analogue of Shh, a higher regulator of Bmi1, and Oct4, a derivatization inducer. And to suggest a method for producing embryonic stem cell-like cells using the same.

Stem cells, unlike normal somatic cells, have the capacity to divide indefinitely and to differentiate into specific cells under suitable environmental conditions. The differentiation capacity is defined as pluripotency, multipotency, or unipotency depending on the characteristics of the stem cells. Therefore, the technique of proliferating stem cells through culture and then differentiating them into specific cells has the potential to treat various diseases in terms of cell therapy.

Stem cells such as hematopoietic stem cells, bone marrow stem cells, and neural stem cells are also present in adults and can be extracted from the patient's own body. There is an advantage to overcome the problem. This advantage can overcome the difficulty of securing long-term providers in the existing organ transplantation.

However, according to the known opinion, adult stem cells are only multipotent and have a limited ability to differentiate. This means that tissue-specific stem cells can only differentiate into cells of the same tissue type, including the central nervous system (Science 255, 1707-1710 1992; Science 287, 1433-1438 2000), bone marrow ( Science 276, 71-74, 1997; Science 287, 1442-1446, 2000; Science 284, 143-147, 1999), retina (Science 287, 2032-2036, 2000) and skeletal muscle (Proc Stem cells isolated from Natl Acad. Sci. USA 96, 14482-14486, 1999; Nature 401, 390-394, 1999) also indicate that the differentiation capacity can only be differentiated into similar tissue lineages. For example, hematopoietic stem cells are blood-related cells, neural stem cells are neurons or glial cells, and bone marrow stem cells are mesoderm. Differentiation into mesodermal cells is possible. Not only are the adult stem cells capable of proliferating in theory in theory, but reports up to now have difficulty growing these adult stem cells in vitro, and it is also difficult to separate large amounts of cells from actual patients. .

Pluripotent stem cells are an excellent resource for overcoming many of the disadvantages of adult stem cells listed above. These cells are capable of differentiating into all kinds of cells that make up the human body and can infinitely multiply in vitro. Known pluripotent stem cells to date include embryonic stem cells, embryonic germ cells, and embryonic carcinoma cells, of which embryonic stem cells are the most studied. The differentiation method and animal disease model in the furnace have been studied and demonstrated the potential for treatment of various diseases in clinical use.

However, embryonic stem cells also have disadvantages that must be overcome for clinical use like adult stem cells. First of all, in order to obtain embryonic stem cells, it is necessary to destroy fertilized embryos, so there is an ethical problem, and when the cells differentiated from embryonic stems are transplanted into a patient, an immune rejection reaction occurs.

Therefore, various approaches have been attempted to overcome these problems of embryonic stem cells, the most prominent of which is the reprogramming of differentiated cells from undifferentiated cells. Reverse differentiation collectively refers to the generation of pluripotent stem cells, such as embryonic stem cells, using differentiated cells, such as 1) nuclear transfer, 2) cell fusion, and 3) cell extracts. Cell extract treatment, and 4) induced pluripotent stem cell (iPS cell) technology (Cell 132, 567-582, 2008).

iPS cell technology has succeeded in producing cells that are closer to embryonic stem cells than any other technology that has been studied so far, and numerous research papers have been pouring out since 2006 when the technology was first published to prove these results and mechanisms. . Basically, four genes (de-differentiation inducer; Oct4, Sox2, Klf4, and C-Myc / Oct4, Sox2, Nanog, Lin28) were introduced into mouse or human somatic cells and cultured in embryonic stem cell culture conditions for a long time. In this case, stem cells with similar characteristics to embryonic stem cells could be established, which are three times in gene expression, epigenetics, and in vitro / in vivo. Three germ layer differentiation, teratoma formation ability, chimeric mouse generation, and germline transmission of chimeric mice have been shown to be quite similar (Cell 126, 663-). 676, 2006; Science 318, 1917-1920, 2007).

However, the mechanism of this dedifferentiation process is so small that it is difficult to elucidate the detailed mechanism because the number of genes used to induce dedifferentiation is too small. In addition, despite the establishment of dedifferentiation induction technology, a technically simple and efficient method must be continuously developed for actual therapeutic application, and all of the developed technologies must be evaluated in terms of efficiency and safety.

Recently published studies have reported that inactivation of the cancer suppressor gene p53 increases the production efficiency of iPS cells (Nature 460, 1132-1135, 2009). The p53 gene is regulated by the Ink4a / Arf locus, which is regulated by selective splicing. p19 ^Arf produced by ^Arf is the p53 gene and p16 ^Ink4a produced by ^Ink4a is the Rb gene. It is known to induce the expression of. Inhibition of the expression of these two genes, p16 ^Ink4a and p19 ^Arf , has been reported to increase iPS production efficiency compared to the inhibition of p19 ^Arf alone (Nature, 460, 1140-1144, 2009).

Polycomb group (PcG) protein is an epigenetic gene silencer. It is ^reported that Bmi1, one of the PcG proteins, inhibits the expression of p53 and Rb by inhibiting the expression of p16 ^Ink4a and p19 ^Arf (Genes Dev). , 2678-2690, 1999). In addition, Bmi1 is known to inhibit the expression of target genes by altering the structure of chromatin, and this property is responsible for the self-renewal of neural stem cells and hematopoietic stem cells. It is known to play an important role. Accordingly, the present inventors reported that Bmi1 was overexpressed in the astrocytes of the mouse and succeeded in the differentiation into neural stem cells. The characteristics of the de-differentiated neural stem cells in the mouse It was similar to the isolated neural stem cells. Among them, the expression of Sox2, one of the de-differentiation inducing factors and an important role in the self-renewal of neural stem cells, was increased (Biochem Biophys Res Commun. 371, 267-272, 2008).

Normal somatic cells require 4 genes (Oct4, Sox2, Klf4, C-Myc) or 3 genes (Oct4, Sox2, Klf4) in order to dedifferentiate, but to endogenously expressing cells It is known that no additional introduction of genes is necessary. Representative among them is the result of the research that established only differentiated stem cells by introducing only one Oct4 gene into mouse / human neural stem cells, because neural stem cells intrinsically express Sox2, Klf4, and C-Myc (Nature, 461, 649). -653, 2009). However, there is no known method for producing embryonic stem cell-like cells having pluripotency using only Oct4 inducer from somatic cells.

The present inventors hypothesized that de-differentiated induced pluripotent stem cells could be established by inducing expression of Sox2 gene and overexpression of Bmi1, which can suppress the expression of p16 ^Ink4a and p19 ^Arf with Oct4 overexpression in mouse somatic cells. Built up. Based on this hypothesis, the introduction of Oct4 and Bmi1 genes and induction of differentiation led to the induction into cells having the same characteristics as embryonic stem cells. Based on these results, if Bmi1 is induced by higher regulators that can substitute for Bmi1, we could provide a technique that can be used to establish a technique for inducing into differentiated stem cells without transduction. Based on this hypothesis, we used Shh hedgehog signaling to overexpress Bmi1. Shh is an important cytokine in neural stem cell self-renewal and directly regulates Gli1 protein and is important for neural stem cells including Bmi1 and Sox2. It has been reported to increase the expression of factors (Curr Mol Med. 9, 873-886, 2009; Crit Rev Oncol Hematol. 65, 43-53, 2008). In order to induce the Shh signal, permophamine capable of inducing Shh was used (Stem cells 27, 703-713, 2009). When Permopamine (Purmorphamine) was treated to mouse fibroblasts in the culture condition of neural stem cells, it was confirmed that the expression of Bmi1 gene was increased, and the expression of Sox2 was increased at this time. In addition, the expression of p16 ^Ink4a and p19 ^Arf genes, the target genes of the ^Bmi1 gene, was reduced, indicating that Bmi1 and Sox2 were regulated by the treatment of permorphamine, and the expression of target genes of Bmi1 was regulated. I could confirm it. In addition, the cells induced by the treatment of permorphamine (purmorphamine) showed the shape of neural stem cells and the expression of nestin and sox2, the representative markers of neural stem cells was confirmed. Since this is the same result as the system overexpressing Bmi1, the method by treating Shh analog (purmorphamine), which is an Shh analogue, is considered appropriate, and this approach can reduce the number of genes. After introducing Oct4 gene under the condition of adding permophamine and culturing under the culture condition of embryonic stem cells, it was possible to establish a cell population similar in shape to embryonic stem cells. Epigenetics, three germ layer differentiation in vitro / in vivo, teratoma-forming ability, chimeric mouse generation The present invention was completed by confirming that the embryonic stem cells are very similar.

Accordingly, an object of the present invention is to provide a composition for inducing the differentiation of somatic cells into embryonic stem cell-like cells by using the morphomorphic factor Shh analogue of Bmi1, purmorphamine, and Oct4 gene, which is a reverse differentiation inducer. will be.

It is another object of the present invention to provide a method for producing pluripotent dedifferentiated stem cells having characteristics similar to embryonic stem cells from somatic cells using permopamine and Oct4.

Still another object of the present invention is to provide an embryonic stem cell-like cell produced by the above method.

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

The present inventors treated with permopamine (purmorphamine) in somatic cells to introduce into cells such as neural stem cells while introducing the Oct4 gene and cultured in the culture conditions of embryonic stem cells, somatic cells that are already differentiated cell type has a pluripotent capacity Eggplants were found to be de-differentiated into embryonic stem cell-like cells and completed the present invention.

In one aspect, the present invention provides a composition for inducing reverse differentiation from somatic cells to embryonic stem cell-like cells, including:

a) purmorphamine; And

b) Oct4 protein or nucleic acid molecule encoding Oct4 protein.

As used herein, the term "embryonic stem cell (ESC) -like cell" refers to a cell having pluripotency, and is derived from all three embryonic layers without proliferation, endless growth, self-reproduction and transformation without transformation. Refers to the characteristics of embryonic stem cells, including but not limited to the ability to develop into any cell. Embryonic stem cell-like cells in the present invention may also be described as embryonic stem cells, induced pluripotent stem cells, or induced pluripotent stem cells.

In inducing embryonic stem cell-like cells of the present invention, the type of starting somatic cell is not particularly limited, and any somatic cell may be used. For example, mature somatic cells may be used in addition to somatic cells of the embryonic period. When embryonic stem cell-like cells are used for the treatment of a disease, it is preferable to use somatic cells isolated from the patient. For example, somatic cells involved in the disease or somatic cells involved in the disease treatment can be used. Preferably, the somatic cells are fibroblasts, and in the present invention, the fibroblasts include all fibroblasts derived from animals such as humans, mice, horses, sheep, pigs, goats, camels, antelopes, and dogs.

Purmorphamine of the present invention is a purine compound and is known to be involved in the Shh signaling system.

The permopamine is not particularly limited as long as it can induce the Shh signal, and various derivatives may be used. For example, 2- (1-Naphthoxy) -6- (4-morpholinoanilino) -9-cyclohexylpurin) can be purchased commercially.

The permophamine may be treated with a medium commonly used to induce differentiation into neural stem cell-like cells. By treating Shh analog permophamine, it is possible to easily induce Bmi1, thereby minimizing the number of genes to be introduced to induce differentiation.

The permophamine is included in the medium at an effective concentration. Effective concentrations of permopamine may be affected by factors well known in the art, such as media type and culture method. In a specific example of the present invention, the treatment was carried out in an amount of 0.5 μM to 1 μM.

Oct4 of the present invention is provided in the form of a protein or a nucleic acid encoding the protein thereof. Oct4 used in the composition of the present invention includes all Oct4s derived from humans and animals such as horses, sheep, pigs, goats, camels, antelopes, dogs, and preferably human Oct4. In addition, the Oct4 protein of the present invention used for reverse differentiation into embryonic stem cell-like cells includes variants of the Oct4 protein as well as proteins having their wild type amino acid sequences.

Variants of Oct4 protein refer to proteins in which the native amino acid sequence of Oct4 and one or more amino acid residues have different sequences by deletion, insertion, non-conservative or conservative substitution, or a combination thereof. The variant may be a functional equivalent exhibiting the same biological activity as the native protein or a variant in which the physicochemical properties of the protein are modified as necessary. It is a variant with increased structural stability to physicochemical environment or enhanced physiological activity.

It is also provided in the form of a nucleic acid having a nucleotide sequence encoding Oct4 protein.

The nucleotide sequence encoding the Oct4 protein is a nucleotide sequence encoding Oct4 protein in wild-type or variant form as described above, wherein one or more bases can be mutated by substitution, deletion, insertion, or a combination thereof, It may be prepared using chemical synthesis.

The nucleic acid having the nucleotide sequence encoding the Oct4 protein described above may be single or double stranded, and may be a DNA molecule (genome, cDNA) or an RNA molecule.

In one preferred embodiment, the composition for inducing differentiation of somatic cells into embryonic stem cell-like cells in the present invention comprises a vector expressing an Oct4 protein comprising a nucleic acid encoding an Oct4 protein.

As used herein, the term "vector" refers to a gene construct, which is an expression vector capable of expressing a protein of interest in a suitable host cell, and which contains essential regulatory elements operably linked to express the gene insert.

As used herein, the term "operably linked" refers to a functional linkage of a nucleic acid expression control sequence and a nucleic acid sequence encoding a protein of interest to perform a general function. Operational linkage with recombinant vectors can be prepared using genetic recombination techniques well known in the art, and site-specific DNA cleavage and ligation employs enzymes commonly known in the art.

Vectors of the present invention include signal or leader sequences for membrane targeting or secretion in addition to expression control elements such as promoters, operators, initiation codons, termination codons, polyadenylation signals, enhancers, and can be prepared in various ways depending on the purpose. The promoter of the vector may be constitutive or inducible. In addition, the expression vector includes a selectable marker for selecting a host cell containing the vector and, in the case of a replicable expression vector, a replication origin. Vectors can self replicate or integrate into host DNA.

Vectors include plasmid vectors, cosmid vectors, viral vectors, and the like. Preferably, it is a viral vector. Viral vectors are retroviruses such as Human immunodeficiency virus HIV (Murineleukemia virus) Avian sarcoma / leukosis (ASLV), Spleen necrosis virus (SNV), Rous sarcoma virus (RSV), and Mouse mammary tumor viruses, such as, but are not limited to, vectors derived from Adenovirus, Adeno-associated virus, Herpes simplex virus, and the like. In a specific embodiment of the present invention, for the Oct4 gene, a pBabe neo Oct4 vector containing a selection marker for neomycin was used as the MLV-based-virus vector.

Nucleic acids encoding Oct4 proteins can be delivered intracellularly by methods known in the art, such as naked DNA in the form of vectors (Wolff et al. Science, 1990: Wolffet al. J Cell Sci. 103: 1249-59, 1992). ), Liposomes, cationic polymers (Cationic polymer) can be delivered to the cell. Liposomes are phospholipid membranes prepared by mixing cationic phospholipids such as DOTMA or DOTAP for gene transfer. Nucleic acid-liposomal complexes are formed when cationic liposomes and anionic nucleic acids are mixed at a predetermined ratio.

In another preferred embodiment, the composition inducing reverse differentiation from somatic cells to embryonic stem cell-like cells in the present invention comprises a virus expressing Oct4 protein comprising a nucleic acid encoding Oct4 protein.

As used herein, the term "virus" refers to a virus expressing Oct4 produced by transforming and infecting a viral vector containing a nucleic acid encoding Oct4 protein with packaging cells.

Viruses that can be used to prepare viruses expressing Oct4 proteins of the invention include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes simplex viruses, and the like. Preferably, it is a retrovirus. In a specific embodiment of the present invention, a vector (pBabe neo Oct4) prepared by inserting a nucleotide sequence (SEQ ID NO: 1) encoding a human Oct4 protein into a pBabe neo vector generates a high titer virus capable of infecting a wide range of mammalian host cells. PT67 cells, which are packaging cells, were transformed to prepare viruses expressing Oct4 protein to infect fibroblasts.

In another aspect, the present invention provides a method for producing embryonic stem cell-like cells from somatic cells comprising the step of treating the somatic cells with permorphamine (purmorphamine) and introducing the Oct4 gene into the somatic cells.

More specifically, (i) culturing the fibroblast cells in the medium, (ii) infecting the cultured fibroblasts with packaging cells transfected with the vector treated with permophamine and inserted Oct4 gene, ( iii) culturing the infected fibroblasts under embryonic stem cell culture conditions.

The medium for culturing fibroblasts in step (i) of the method includes all media commonly used for culturing fibroblasts in the art. The medium used for the culture generally contains a carbon source, a nitrogen source and a trace element component. In a specific embodiment of the present invention, DMEM (high glucose, w / o sodium pyruvate) + 10% FBS (Fetal Bovine Serum) + 0.1 mM non-essential amino acid + 1% penicilin / streptomycin + 0.1 mM β-mercaptoethanol) Incubated.

In step (ii) of the method, the permopamine is treated under neural stem cell culture conditions, and it is preferable to introduce Oct4 gene into fibroblasts while inducing them into cells such as neural stem cells. It is preferable to continue permophamine treatment after permophamine treatment for 1 day, followed by three times of infection with the Oct4 virus at 16 hour intervals. In this case, it is preferable to maintain the neural stem cell culture condition while continuing the permophamine treatment for a total of 72 hours, and then change the embryonic stem cell culture condition to increase the efficiency of dedifferentiation into embryonic stem cell-like cells.

PT67 cell, which is a packaging cell that generates a high titer virus capable of infecting a wide range of mammalian host cells by incorporating a nucleic acid molecule encoding human Oct4 protein into pBabe neo vector in step (ii) of the method Is transformed into a virus expressing Oct4 protein to infect fibroblasts. The nucleic acid which is the Oct4 protein or the nucleotide sequence encoding the Oct4 protein may be any Oct4 derived from humans and animals such as horses, sheep, pigs, goats, camels, antelopes and dogs, and includes wild-type and variants. In a specific embodiment of the present invention, human Oct4 (NCBI accession No. NM_002701; SEQ ID NO: 1) was used.

Embryonic stem cell culture conditions of step (iii) of the method includes all of the medium commonly used in the culture of embryonic stem cells in the art. In a specific embodiment of the present invention, 15% FBS (Fetal bovine serum) + 0.1 mM nonessential amino acid + 1% penicillin / streptomycin + 0.1 mM β-mercaptoethanol + 1000 unit / ml mouse in high glucose DMEM in the presence of supporting cells Subcultures were performed every 2-3 days while incubated in LIF (leukemia inhibitory factor) media.

In another aspect, the present invention provides embryonic stem cell-like cells prepared by the above method.

Embryonic stem cell-like cells prepared by the reverse differentiation method of the present invention show positive responses to antibodies against alkaline phosphatase and SSEA1, Oct4, and Sox2, which are embryonic stem cell-specific markers. Expression of genes (Oct4, Sox2, Nanog, c-myc, Klf4), which are important for the maintenance of self-renewal ability, is expressed in a similar pattern to embryonic stem cells. In addition, it was confirmed to have the same pluripotency as the general embryonic stem cells. Furthermore, embryonic stem cell-like cells prepared by the reverse differentiation method of the present invention have the characteristics of self-renewal.

Embryonic stem cell-like cells of the present invention are good sources for providing various types of cells. For example, the embryonic stem cell-like cells may be induced to differentiate into hematopoietic cells, neurons, beta cells, hepatocytes, chondrocytes, epithelial cells, urinary tract cells and similar cells by culturing in a medium for cell differentiation. .

Media conditions and methods for differentiation of embryonic stem cell-like cells are described in Palacios, et al., PNAS. USA, 92: 7530-7537 (1995), Pedersen, J. Reprod. Fertil. Dev., 6; 543-552 (1994), and Bain et al., Dev. Biol, 168: 342-357 (1995). The embryonic stem cell-like cells can be applied to numerous treatments through transplantation. The embryonic stem cell-like cells are diabetes, Parkin's disease, Alzheimer's disease, cancer, spinal cord injury, multiple sclerosis, amyotrophic lateral sclerosis (Lou Gehrig's disease), muscular dystrophy, liver disease, hypercholesterolemia, heart disease, cartilage replacement, wound, It can be applied to the treatment of numerous diseases or disorders such as foot ulcers, gastrointestinal diseases, vascular diseases, kidney diseases, urinary tract diseases, aging-related diseases and conditions. In addition, the embryonic stem cell-like cells of the present invention can be applied to evaluation during drug development.

As described above, according to the present invention, somatic cells can be induced into dedifferentiated stem cells having pluripotency by introducing only Oct4 gene while treating perhamine, an analog of Shh. Embryonic stem cell-like cells differentiated by the present invention are differentiated into cells such as cardiomyocytes, insulin-producing cells, or neurons, for example, stems for various diseases such as heart failure, insulin-dependent diabetes mellitus, Parkinson's disease or spinal cord injury. It can be used for cell transplantation and is extremely useful because it can avoid ethical problems using human embryos and rejection after transplantation. In addition, various cells (for example, cardiomyocytes, hepatocytes, etc.) generated by differentiating induced pluripotent stem cells can be usefully used as a system for evaluating the efficacy and toxicity of compounds, drugs, and poisons.

In addition, the present invention can be used to produce a differentiation embryonic stem cell-like cells using only Oct4 gene by using a chemical called permophamine, a Shh analog that is a higher regulator of the Bmi1 gene, to replace the reverse differentiation inducer. Furthermore, it can be said to be a very useful invention in the field of stem cells that provides a technology based on establishing a cell line without gene introduction.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example 1 Culture of Mouse Fibroblasts

To prepare embryonic stem cell-like cells, mouse fibroblasts of mouse somatic cells were used. Mouse fibroblasts were prepared for embryos at the 13.5th day of CF1 strain mouse pregnancy and DMEM (high glucose, w / o sodium pyruvate) + 3rd passage after incubation in a tissue culture flask with 10% FBS (Fetal Bovine Serum) + 0.1 mM non-essential amino acid + 1% penicilin / streptomycin + 0.1 mM β-mercaptoethanol 2 x 10 ⁵ cells were seeded in 6 well plates.

Example 2 Bmi1 Induction and Oct4 Gene Introduction by Permophamine (Shh analog) Treatment

Fibroblasts prepared in Example 1 were treated with permopamine, an analog of Shh (Sonic hedgehog), to confirm whether Bmi1 is induced. Figure 1a shows the results confirmed by the RT-PCR method that the lower genes are regulated when treated with permophamine (purmorphamine), an analog of Shh (Sonic hedgehog) known as the upper regulator of the Bmi1 gene. Permopamine (purmorphamine, Calbiochem cat.no.540220) at 0.5μM and 1μM culture condition of neural stem cells (DMEM / F12 + B27 + N2 + 1% penicillin / streptomycin + 20 ng / ml bFGF + 20 ng / ml EGF) Gli1 and Bmi1 were induced when treated at, and the expression of p16 ^Ink4a and p19 ^Arf , the target genes of ^Bmi1 , was reduced compared to mouse fibroblasts. In addition, as the shape of the cells (aggregation) it can be seen that the neuron-like morphology (Fig. 1b). Thus induced cells such as neural stem cells can be confirmed that the expression of Nestin, Sox2 by immunochemical staining method, showing positive results in SSEA1 and AP staining (Fig. 1c). Through these results, it was confirmed that the Bmi1-introduced cells exhibited similar characteristics to those of Bmi1-introduced cells by analogue treatment of Shh, an upper regulator capable of controlling Bmi1.

Oct4 gene was introduced into the permopamine-treated fibroblasts using retrovirus. Virus particles were prepared using PT67 packaging cell lines. More specifically, pBabe neo Oct4 vector prepared by inserting human Oct4 (NCBI accession No. NM_002701) gene into pBabe neo vector was transfected into PT67 packaging cell line (Clontech) using turbofect (Turbofect, Fermentas). After transfection, the mice were selected with niomycin (1000 μg / ml) (BD biosciences). The PT67 packaging cell line is a cell that produces high titer viruses capable of infecting a wide range of mammalian host cells.

After confirming the expression of Oct4 gene by RT-PCR method, the cell debris was obtained by filtering supernatant and filtering with 0.45㎛ (Millipore) filter when 80% of the cell lines were made. After removal, polybrene (polygrene, 6 µg / ml) (sigma) was added to the mouse fibroblasts isolated at 16 hour intervals, and the cells were repeatedly infected three times.

At this time, the permophamine treatment was performed for 1 day, followed by introducing the Oct4 gene three times at 16 hour intervals for 48 hours using a retroviral system to continue the permophamine treatment. Permophamine was continued for a total of 72 hours, and neural stem cell culture conditions were maintained during treatment with permophamine. Subsequently, reverse differentiation was induced while changing to culture conditions of mouse embryonic stem cells (FIG. 1D). As a result, a modified protocol was established based on the conditions that induced reverse differentiation only with the introduction of existing genes and the conditions that induced reverse differentiation by chemical treatment of recently reported Oct4 and Sox2. Induction of reverse differentiation under these conditions led to the appearance of colony-like morphologies after 10-14 days.

Example 3 While Treating Shh Analogue Permophamine Establishment of Differentiated Stem Cell Line by Oct4 Gene Introduction

According to the modified protocol shown in Example 2 (FIG. 1D), Retrograded stem cell lines could be established by introducing Oct4 using retroviruses while treating Shh analog permorphamine (purmorphamine) and transferring them to culture conditions of mouse embryonic stem cells. Culture conditions of mouse embryonic stem cells were 15% FBS (Fetal bovine serum) + 0.1 mM nonessential amino acid + 1% penicillin / streptomycin + 0.1 mM β-mercaptoethanol + 1000 unit / ml in high glucose DMEM under supporting cells. Subcultures were performed every 2-3 days while incubated in a culture of mouse LIF (leukemia inhibitory factor). After 10-14 days of culture, cells of similar morphology (morphology) began to appear, and after culturing, colonies similar to embryonic stem cells were obtained. The dedifferentiated stem cell line established through this method was named PO-iPS.

PO-iPS was compared with embryonic stem cells and characterized. Comparative expression analysis of representative genes known as important genes in embryonic stem cells showed similar expression patterns as embryonic stem cells (FIG. 2A). At this time, the expression of the exogenous inserted Oct4 gene was shown to be silent. In addition, it was confirmed through the genomic DNA PCR (genomic DNA PCR) method that the Oct4 gene is integrated on the genomic DNA (genomic DNA) (FIG. 2B). Such cells showed similar expression levels to embryonic stem cells when the expression of genes important for maintaining self-renewal capacity was quantified by real-time PCR (FIG. 2C). The FACS analysis method showed that SSEA1 and Oct4 were expressed in% of embryonic stem cells at a similar level (FIG. 2D). The shape of the cells was similar to that of embryonic stem cells, and AP staining was positive. In addition, when the expression of representative markers was confirmed by immunochemical staining, it was confirmed that the expression of Oct4, SSEA1, Sox2, and Nanog was similar to that of embryonic stem cells (FIG. 2E).

Example 4. Epigenetic Characterization of PO-iPS

The bisulfite sequencing method was used to analyze the methylation of the promoter region of Oct4 and Nanog, which is known to be important for maintaining the self-renewal capacity of embryonic stem cells compared to embryonic stem cells. It was. It was confirmed that the promoter region of Oct4 and Nanog was demethylated (FIG. 3A). In addition, it was confirmed that histone H3 (acetylation) was acetylated by chromatin immunoprecipitation (FIG. 3B). In contrast, mouse fibroblasts were found to be demethylated in lysine 9 (dimethylation). This is the result of comparative analysis by real-time PCR using a primer of the promoter region of the corresponding Oct4, Sox2, and Nanog gene after the ChIP assay (ChIP assay).

Example 5 Comparative Analysis of Global Gene Expression with Embryonic Stem Cells

PO-iPS was compared to embryonic stem cells by DNA microarray analysis. Scatter plots indicated that most genes were contained within 2-fold, especially Oct4, Sox2, Nanog, and c-myc, which are known to be important factors in inducing differentiation and important genes in embryonic stem cells. , Klf4 was expressed to about the same level as the embryonic stem cells. It was confirmed that the same result was obtained in cells induced by treating the concentration of permophamine differently. In addition, as a result of quantifying the similarity through the Pearson correlation (pearson correlation) method, it was confirmed that the similarity is shown as high as 0.97 and 0.95 (Fig. 4).

Example 6 Comparative Analysis of Differentiation Ability into Trioderm Cells in Vitro and In Vivo

In order to confirm whether PO-iPS dedifferentiated stem cells have differentiation ability in vitro, first, an embryonic body (embryonic body, EB) was formed. And on the 7th day of EB formation, it was confirmed by RT-PCR method that the genes corresponding to the three germ layers are expressed, showing that the same pattern as the embryonic stem cells (Fig. 5a). Induction of spontaneous differentiation in tissue culture plates coated with 0.1% gelatin-coated embryonic bodies resulted in induction of differentiation into representative cells of the trioderm. This can be confirmed by staining each corresponding marker. It was confirmed that the expression of Tuj1, SMA through immunochemical staining was induced to differentiate into representative cells corresponding to the three germ layers (Fig. 5b).

To determine whether they have differentiation capacity in vivo, 10 ⁶ cells were centrifuged at 8000 rpm for 5 minutes and pellet cultured in embryonic stem cell culture in a 37 ° C incubator for 24 hours before 6 weeks of age. The kidneys of Balb / c nude mice were injected in the form of capsules. After 8-10 weeks, kidneys were separated and differentiated into trioderm to determine whether paraffin blocks were made and H & E staining. As a result, it was confirmed that the differentiation into cells corresponding to the three germ layers (Fig. 5c). After injection into the blastocyst of C57 / BL6 and transplantation into the surrogate mother, chimera formation was observed in F1. When compared with surrogate mice and mice of C57 / BL6, chimera formation was observed (FIG. 5D). As a result, dedifferentiated stem cells have characteristics similar to embryonic stem cells. Could know.

The present invention described above is not limited to the above-described embodiments and drawings, and various substitutions, modifications, and changes can be made without departing from the technical spirit of the present invention. It will be apparent to those who have

Figure 1 shows the results of Bmi1 induction by the treatment of Smorph (sonic hedgehog) analog, purmorphamine (purmorphamine).

Figure 1a shows the results of confirming the expression of subregulatory genes when treated with mouse fibroblasts Shh (Sonic hedgehog) analog, permorphamine (purmorphamine). When treated with permorphamine (purmorphamine) it can be seen that the expression of Gli1 and Bmi1 increased, and at this time the expression of p16 ^Ink4a and p19 ^Arf genes known as target genes of the Bmi1 gene is reduced by RT-PCR method Could.

Figure 1b shows the results of confirming that the change in cells shaped like neurospheres when treated with permorphamine (purmorphamine).

Figure 1c shows the results confirming that the cells formed in the shape of the neurospheres thus formed have the same characteristics as the neural stem cells. Expression of representative markers of Nestin and Sox2 was confirmed by immunochemical staining, and positive results were shown in SSEA1 and AP.

FIG. 1D shows a schematic diagram of a protocol for introducing Oct4 gene while treating permorphamine, and inducing differentiation in embryonic stem cell culture conditions.

Figure 2 shows the results of comparing the characteristics of embryonic stem cells and induced differentiation stem cells induced by introducing the Oct4 gene while treating permorphamine (purmorphamine).

Figure 2a shows the results of comparative analysis of the expression of representative genes expressed in embryonic stem cells by the method of RT-PCR, the results below confirm that the expression of exogenous Oct4 does not appear Indicates.

Figure 2b shows the result of the Oct4 gene fusion (interation) on genomic DNA.

Figure 2c shows the results confirming that similar to the embryonic stem cells when quantifying and comparing the expression of genes important for maintaining the self-renewal ability of embryonic stem cells through the real-time PCR method.

Figure 2d shows the results confirmed that the expression of SSEA1 and Oct4 appear similar to embryonic stem cells by FACS analysis.

Figure 2e shows the results confirming that the expression of SSEA1 and Oct4 is significantly high through the FACS analysis method.

Figure 3 shows the results of comparative analysis of epigenetic characteristics of embryonic stem cell-like cells according to the present invention.

FIG. 3a shows the results of confirming that the promoter regions of Oct4 and Nanog genes are demethylated by bisulfite sequencing method for dedifferentiated stem cells (PO-iPS) and embryonic stem cells. .

Figure 3b is the histone number 3 of the promoters of Oct4, Sox2, and Nanog genes of embryonic stem cells and dedifferentiated stem cells using Chromatin immunoprecipitation (ChIP) is acetylated (acetylation) gene expression This result shows that the result is shown to be activated. In contrast, mouse fibroblasts are demethylated in lysine 9, indicating that the expression of genes is inactivated. Is showing.

Figure 4 shows the results of comparative analysis on the whole gene of embryonic stem cells and de-differentiated stem cells PO-iPS by the DNA microarray method (DNA microarray). When the analysis results were displayed by the scatter plot method, the expression level of most genes was similar to that of embryonic stem cells, and the similarity of 0.97 and 0.95 was shown by the Pearson correlation method. I could confirm that.

Figure 5 shows the results confirming whether the embryonic stem cell-like cells according to the present invention has the ability to differentiate from in vitro and in vivo to trioderm cells.

FIG. 5A shows the formation of an embryonic body (EB) like embryonic stem cells, and the right panel of FIG. 5A shows the expression of representative genes corresponding to the three germ layers through the method of RT-PCR. The confirmed result is shown.

Figure 5b shows the result of confirming the markers by immunochemical staining after induction of spontaneous differentiation in vitro.

Figure 5c shows the results of the analysis by H & E staining that teratoma (teratoma) formation in vivo.

FIG. 5D shows the chimera formation rate after injecting cells into the blastocyst of C57 / BL6 and transplanting into the surrogate mother, thus confirming the chimera formation in the two mice. .

<110> Korea University Industrial and Academic Collaboration Foundation <120> Generation composition for induced pluripotent stem cells with          purmorphamine treatment and Oct4, and method of manufacturing          induced pluripotent stem cells using the same <130> P11-091218-04 <160> 1 <170> KopatentIn 1.71 <210> 1 <211> 1411 <212> DNA <213> Homo sapiens <400> 1 ccttcgcaag ccctcatttc accaggcccc cggcttgggg cgccttcctt ccccatggcg 60 ggacacctgg cttcggattt cgccttctcg ccccctccag gtggtggagg tgatgggcca 120 ggggggccgg agccgggctg ggttgatcct cggacctggc taagcttcca aggccctcct 180 ggagggccag gaatcgggcc gggggttggg ccaggctctg aggtgtgggg gattccccca 240 tgccccccgc cgtatgagtt ctgtgggggg atggcgtact gtgggcccca ggttggagtg 300 gggctagtgc cccaaggcgg cttggagacc tctcagcctg agggcgaagc aggagtcggg 360 gtggagagca actccgatgg ggcctccccg gagccctgca ccgtcacccc tggtgccgtg 420 aagctggaga aggagaagct ggagcaaaac ccggaggagt cccaggacat caaagctctg 480 cagaaagaac tcgagcaatt tgccaagctc ctgaagcaga agaggatcac cctgggatat 540 acacaggccg atgtggggct caccctgggg gttctatttg ggaaggtatt cagccaaacg 600 accatctgcc gctttgaggc tctgcagctt agcttcaaga acatgtgtaa gctgcggccc 660 ttgctgcaga agtgggtgga ggaagctgac aacaatgaaa atcttcagga gatatgcaaa 720 gcagaaaccc tcgtgcaggc ccgaaagaga aagcgaacca gtatcgagaa ccgagtgaga 780 ggcaacctgg agaatttgtt cctgcagtgc ccgaaaccca cactgcagca gatcagccac 840 atcgcccagc agcttgggct cgagaaggat gtggtccgag tgtggttctg taaccggcgc 900 cagaagggca agcgatcaag cagcgactat gcacaacgag aggattttga ggctgctggg 960 tctcctttct cagggggacc agtgtccttt cctctggccc cagggcccca ttttggtacc 1020 ccaggctatg ggagccctca cttcactgca ctgtactcct cggtcccttt ccctgagggg 1080 gaagcctttc cccctgtctc cgtcaccact ctgggctctc ccatgcattc aaactgaggt 1140 gcctgccctt ctaggaatgg gggacagggg gaggggagga gctagggaaa gaaaacctgg 1200 agtttgtgcc agggtttttg ggattaagtt cttcattcac taaggaagga attgggaaca 1260 caaagggtgg gggcagggga gtttggggca actggttgga gggaaggtga agttcaatga 1320 tgctcttgat tttaatccca catcatgtat cacttttttc ttaaataaag aagcctggga 1380 cacagtagat agacacactt aaaaaaaaaa a 1411  

Claims (6)

Compositions that induce differentiation from somatic cells into embryonic stem cell-like cells, including a) purmorphamine; And b) Oct4 protein or nucleic acid molecule encoding Oct4 protein. The method of claim 1, wherein the composition a) purmorphamine; And b) A composition comprising a virus in which a nucleic acid molecule encoding Oct4 protein is introduced to express Oct4 protein. A method of producing embryonic stem cell-like cells from somatic cells comprising treating somatic cells with permorphamine and introducing Oct4 gene into somatic cells. According to claim 3, (i) culturing the fibroblast cells in the medium, (ii) the cultured fibroblasts were treated with permorphamine (purmorphamine) and the packaging cells transfected with the vector transfected with the Oct4 gene Infecting, (iii) A method for producing embryonic stem cell-like cells from somatic cells comprising the step of culturing the infected fibroblasts in embryonic stem cell culture conditions. The embryo from somatic cells of claim 4, wherein in step (ii), the permophamine is treated under neural stem cell culture conditions to introduce Oct4 gene into fibroblasts while inducing into cells such as neural stem cells. Method for producing stem cell-like cells. Embryonic stem cell-like cells produced by the method of any one of claims 3 to 5.

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