WO2009157610A1 - Selenium dedifferentiated cell, preparation method and usage thereof - Google Patents

Selenium dedifferentiated cell, preparation method and usage thereof Download PDF

Info

Publication number
WO2009157610A1
WO2009157610A1 PCT/KR2008/004453 KR2008004453W WO2009157610A1 WO 2009157610 A1 WO2009157610 A1 WO 2009157610A1 KR 2008004453 W KR2008004453 W KR 2008004453W WO 2009157610 A1 WO2009157610 A1 WO 2009157610A1
Authority
WO
WIPO (PCT)
Prior art keywords
cells
selenium
dedifferentiated
differentiated
cell
Prior art date
Application number
PCT/KR2008/004453
Other languages
French (fr)
Inventor
Soo Kyung Kang
Jeong Hwan Kim
Original Assignee
Pusan National University Industry-University Cooperation Foundation
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from KR1020080074630A external-priority patent/KR101041914B1/en
Application filed by Pusan National University Industry-University Cooperation Foundation filed Critical Pusan National University Industry-University Cooperation Foundation
Priority to US12/294,228 priority Critical patent/US20100233131A1/en
Publication of WO2009157610A1 publication Critical patent/WO2009157610A1/en

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0662—Stem cells
    • C12N5/0667—Adipose-derived stem cells [ADSC]; Adipose stromal stem cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/05—Inorganic components

Definitions

  • the present invention relates to cells dedifferentiated by selenium, and more particularly to a composition for cell dedifferentiation containing selenium, a method of dedifferentiating cells by treating the cells with selenium, dedifferentiated cells obtained using the method, a composition for cell therapy containing the dedifferentiated cells, cells re-differentiated from the dedifferentiated cells, and a composition for cell therapy containing the re- differentiated cells.
  • Human embryonic stem cells have the capability to differentiate into all types of human cells, and thus show the capability to treat various existing diseases.
  • dedifferentiation technology receives remarkable attention in the stem cell field worldwide, because it can accurately substitute for the use of cloned embryonic stem cells and is not related to ethical concerns.
  • iPS induced pluripotent stem
  • the term "dedifferentiated stem cells” refers to cells were dedifferentiated into the undifferentiated state by inserting a specific gene using somatic cells and have the capability to grow into all types of human cells, just like embryonic stem cells.
  • iPS induced pluripotent stem
  • the present inventors have treated adipose tissue stromal cells with selenium and, as a result, have found that the cells express sternness genes, the growth characteristics of the cells are extended, and the cells have multipotency to re-differentiate into other types of cells, thereby completing the present invention.
  • the present invention is to provide a composition for cell dedifferentiation containing selenium.
  • the present invention is also to provide a method of dedifferentiating cells by treating the cells with selenium.
  • the present invention is also to provide dedifferentiated cells obtained using said method and a composition for cell therapy containing the differentiated cells.
  • the present invention is also to provide cells re- differentiated from the dedifferentiated cells and a composition for cell therapy containing the re-differentiated cells .
  • dedifferentiation is a term familiar in the art and is disclosed in, for example,
  • differentiated cell into a stem cell state, that is, a state which can be transferred or programmed into plural types of cells.
  • the term “dedifferentiation” also means that multipotency increases. Namely, it means that the types of cells which can re-differentiate increase.
  • the dedifferentiation of cells is performed by treating the cells with selenium.
  • Selenium which is an essential trace element in organisms is an important constituent of antioxidant enzymes which protect cells from free radicals generated during normal oxygen metabolism. This indicates that selenium is safe for the human body.
  • organic selenium e.g., selenomethionine and selenocysteine
  • inorganic selenium e.g., sodium selenite
  • cells to be dedifferentiated in the present invention cells isolated from mammals, preferably humans, may be used. Also, the cells are preferably isolated from a subject to be treated with either dedifferentiated cells or cells re-differentiated therefrom. Thus, desired treatment can be achieved without immune rejection. Specifically, cells which are used in the present invention may be adult cells originated from a variety of different tissues, including the cumulus, skin, oral mucosa, blood bone marrow, liver, lungs, kidneys, muscles, reproductive tract, fat, etc.
  • Examples of cells which can be used in the present invention include, but are not limited to, cumulus cells, epithelial cells, fibroblasts, neural cells, keratinocytes, hematopoietic cells, melanocytes, chondrocytes, erythrocytes, macropharges, monocytes, muscle cells, B lymphocytes, T lymphocytes, adipose tissue stromal cells, etc.
  • adipose tissue stromal cells are particularly preferably used.
  • the adipose tissue stromal cells can be derived from adipose tissue. Adipose tissue can be harvested using any conventional method known in the art.
  • adipose tissue can be harvested from the abdomen by liposuction.
  • This process for obtaining adipose tissue has advantages in that it is carried out in a manner much easier than the prior art process for obtaining embryonic stem cells and is free from ethical issues.
  • Cells including adipose tissue stromal cells can generally be collected from the tissue harvested from an individual.
  • the collected tissue is finely cut to remove unnecessary portions excluding the desired cells, and the cells are separated into single cells.
  • the tissue can be finely cut by physical means using a homogenizer, a mortar and pestle, a blender, a scalpel, forceps or an ultrasonic device.
  • an enzyme may also be used. Examples of the enzyme that can be used in the present invention include, but are not limited to, serine protease, elastase and collagenase.
  • physical means and enzymatic treatment may be used in combination.
  • the cells obtained as described above may be immediately treated with selenium.
  • the cells may also be cultured during a predetermined period, and then treated with selenium.
  • the latter is preferable.
  • Cells can be cultured in suitable media under suitable conditions depending on species from which the cells have been derived. When cells are of mammalian origin, the cells can be cultured in mammalian culture medium. Suitable media which can be used in the present invention are commercially available and may be prepared according to published compositions (for example, the catalog of the American Type Culture Collection) .
  • Examples of medium which can be used in the present invention include, but are not limited to, Ham medium, IMDM Iscove's medium, Leibovitz L15 medium, May Coy 5A medium, M199 medium, Melnick's medium, MEM medium, NCTN medium, Puck's medium, RPMI medium, Swim S77 medium, Trowell T8 medium, Waymouth medium, Williams medium, DMEM medium, F12 medium, etc.
  • ⁇ -MEM medium is preferably used.
  • the above-described medium may also be supplemented with serum (e.g., FBS), antibiotics (e.g., kanamycin, streptomycin, penicillin, etc.), growth factors (e.g., EGF, PDGF, V ⁇ GF, FGF, TGF, LTF, etc.), cytokines (e.g., insulin, estradiol, interleukin, corticosterone, etc.), if necessary.
  • serum e.g., FBS
  • antibiotics e.g., kanamycin, streptomycin, penicillin, etc.
  • growth factors e.g., EGF, PDGF, V ⁇ GF, FGF, TGF, LTF, etc.
  • cytokines e.g., insulin, estradiol, interleukin, corticosterone, etc.
  • the cells are preferably starved.
  • the starvation is performed in order to eliminate the effects of various components in serum during dedifferentiation.
  • a step of culturing the cells in a medium containing serum at a concentration of 1-3%, and preferably 2%, is additionally carried out.
  • the present invention relates to a method of dedifferentiating cells by treating the cells with selenium.
  • the phrase "treating cells with selenium” has the same meaning as bringing cells into contact with selenium.
  • the present invention may use all methods capable of bringing selenium and cells into contact with each other, including a method of treating cells in suitable buffer solution directly with selenium, a method of treating a culture of cells with selenium, or a method of adding selenium to a culture medium of cells and culturing the cells.
  • selenium is contained at a concentration of 0.1-20 ng/ml, preferably 1-15 ng/ml, and more preferably 5 ng/ml. If the selenium concentration is higher than 20 ng/ml, it will cause cytotoxicity, and if it is lower than 0.1 ng/ml, sufficient cell dedifferentiation will not occur.
  • the culture of cells is performed for 12 hours to 10 days, preferably 1 to 5 days, and more preferably 3 days.
  • the selenium concentration and the culture time are not limited to the above-described values, because it is possible to induce dedifferentiation by extending the treatment period, if the selenium concentration is relatively low, and shortening the treatment period, if the selenium concentration is relatively high.
  • the dedifferentiated cells After treating cells with selenium, whether the cells have been dedifferentiated by selenium is confirmed, and the dedifferentiated cells can be separated from the buffer solution, medium or culture according to a conventional method such as filtration or centrifugation. Cells differentiated by selenium and cells untreated with selenium
  • the dedifferentiated cells show a difference in the expression level of a specific gene.
  • the difference in expression level between the dedifferentiated cells and the differentiated cells is at least 5%, preferably at least 10%, more preferably at least 20%, and even more preferably at least 30%.
  • the dedifferentiated cells of the present invention have the following characteristics.
  • the dedifferentiated cells of the present invention have an increased expression of a sternness gene as compared to differentiated cells.
  • sternness gene refers to a gene which is remarkably expressed in stem cells.
  • the sternness gene is at least one selected from the group consisting of REXl, Nanog, 0ct4, Sox2, Runx3, CDKl, CDK2, Nestin, VEGF and FGFRl.
  • the dedifferentiated cells of the present invention have an increased expression of growth-related factor as compared to differentiated cells. Examples of the growth-related factor include, but are not limited to, c-Myc.
  • the dedifferentiated cells of the present invention have increased telomerase activity, and preferably about 2- fold increased telomerase activity, as compared to differentiated cells. Also, in the dedifferentiated cells of the present invention, the expression level of a gene which is expressed specifically in differentiated cells is decreased compared to that in differentiated cells, and the expression level of a cell proliferation suppressor gene (or tumor growth suppressor gene) is increased compared to that in differentiated cells.
  • the gene which is expressed specifically in the differentiated cells is at least one of GFAP and Tuj , and the cell growth inhibitory gene is at least one of p53 and p31.
  • the expression level of PI3K is increased compared to that in differentiated cells, and the activities of Rac, c-Raf, MEK, ERK, Stat3 and Akt that are mediators of PI3K are increased, while the expression of p-SAPK/JNK, an apoptosis-associated gene, which is induced by reactive oxygen species (ROS) , is inhibited.
  • ROS reactive oxygen species
  • the level of methylation in the promoter region of a sternness gene in the dedifferentiated cells of the present invention is lower than that in differentiated cells.
  • the sternness gene is at least one selected from the group consisting of REXl, Nanog, 0ct4 and Sox2.
  • the expression level of a cell migration-related gene is increased compared to that in differentiated cells. For this reason, the migration of the cells is activated.
  • the cell migration-related gene is at least one selected from the group consisting of MMPl, MMP3, SDFl, VEGF and CXCR4.
  • Specific methods for analyzing the expression profiles of such genes include, but are not limited to, RT-PCR, competitive RT-PCR, real-time RT-PCR, RNase protection assay, Northern blotting, DNA chip assay, etc.
  • the analysis methods include, but are not limited to, Western blotting, enzyme linked immunosorbent assay (ELISA) , radioimmunoassay (RIA) , ouchterlony immunodiffusion, rocket Immunoelectrophoresis, immunohistostaining, imrnunoprecipitation assay, complement fixation assay, FACS, and protein chip assay.
  • the present invention relates to dedifferentiated cells obtained by the above-described method of dedifferentiating cells by treating the cells with selenium and to a composition for cell therapy containing the dedifferentiated cells.
  • the inventive dedifferentiated cells themselves can be used to treat disease.
  • the differentiated cells can be re- differentiated into a specific type of cells by direct contact with a cell population of this type in vivo.
  • the dedifferentiated cells themselves can be applied to a desired type of tissue in order to treat disease, and the kind of disease which can be treated by the dedifferentiated cells is not limited.
  • tissue engineering Methods for producing tissue using cells which can be re-differentiated (“tissue engineering") are known in the art. Wang, X. et al. have shown that even certain adult cells of the pancreas in mice could be converted into hepatocytes in FAH (fumaroy-laceto-acetate hydrolase) -deficient mice (Wang X. et al. "Liver repopulation and correction of metabolic liver disease by transplanted adult mouse pancreatic cells” Am. J. Pathol. 158 (2) :571-579) . Lagasse et al.
  • hematopoietic stem cells from bone marrow could be converted into hepatocytes after in vivo transfer into FAH-deficient mice (Lagasse et al. "Purified hematopoietic stem cells can differentiate into hepatocytes in vivo" Nature Medicine, 6(11) ; 1229-1234) .
  • Preferable forms of application for the in vivo re- differentiation of the dedifferentiated cells according to the present invention are injection, infusion or implantation of the cells into a specific type of cell population in the body.
  • the dedifferentiated cells can be re- differentiated into a specific type of cells by direct contact with a cell group of this type.
  • the dedifferentiated cells of the present invention is preferably in the form of a cell composition containing one or more diluents which protect and maintain the cells and facilitate the use of the cells in injection, infusion or implantation into target tissue.
  • the diluents may include buffer solutions, such as physiological saline, PBS (phosphate buffered saline) or HBSS (Hank's balanced salt solution), and plasma or blood components.
  • the dedifferentiated cells of the present invention may be immediately re-differentiated into a desired type of cells or be stored in medium for several days.
  • cytokine or LIF leukemia inhibitory factor
  • the cells may be freeze-dried and stored to maintain the ability to re- differentiate.
  • the present invention relates to cells re- differentiated from the dedifferentiated cells and a composition for cell therapy containing the re-differentiated cells as an active ingredient.
  • the dedifferentiated cells of the present invention can be re-differentiated into a variety of different types of cells.
  • the dedifferentiated cells of the present invention can be re-differentiated into a specific type of cells according to any method known in the art. Reference is made to, for example, Weissman I. L., Science 287:1442-1446 (2000); Insight Review Articles Nature 414: 92-131(2000); Handbook "Methods of Tissue Engineering ", Eds. Atala, A., Lanza, R. P., Academic Press, ISBN 0-12-436636-8; and Library of Congress Catalog Card No. 200188747.
  • the dedifferentiated cells of the present invention can be re-differentiated into a specific type of cells by bringing the dedifferentiated cells into contact with this type of cell population as described above, the dedifferentiated cells can be re-differentiated into a desired type of cells by bringing the dedifferentiated cells into contact with this type of cell group in vitro.
  • the dedifferentiated adipose tissue stromal cells of the present invention can be differentiated into mesenchymal-derived cells, including bone cells, chondrocytes and muscle cells, neurons, adiocytes, and insulin-producing cells (pancreatic Langerhans islet B cells) .
  • the dedifferentiated cells of the present invention can be used to treat, but not limited to, various diseases such as cancer, osteoporosis, arthritis, neurodegenerative disease, and diabetes.
  • the re-differentiation of dedifferentiated adipose tissue stromal cells can be confirmed, for example, in the following manner. Differentiation into bone cells, chondrocytes and muscle cells can be confirmed through a method employing dyes which are stained specifically on the respective cells. Differentiation into bone cells and adipocytes can be confirmed by measuring the formation of bone nodules and lipids. Also, differentiation into bone cells can be confirmed by analyzing the expression levels of osteonectin, RXR and osteopontin. Differentiation into adipocytes can be confirmed by analyzing the expression levels of AP and PPAR- ⁇ , and differentiation into neurons can be confirmed by analyzing the expression levels of Tuj , GFAP, MAP2ab and NF160. In addition, differentiation into insulin- producing cells can be confirmed by analyzing the expression level of insulin.
  • the re-differentiated cells of the present invention may be applied to target tissue in the same manner as the case of the above-described dedifferentiated cells to treat disease.
  • Preferable forms of application may be injection, infusion, and implantation.
  • a cell composition containing the re-differentiated cells may comprise one or more diluents, which protect and maintain the cells and facilitate the use of the cells in injection, infusion or implantation into target tissue, in the same manner as the cell composition containing the dedifferentiated cells.
  • dedifferentiated somatic cells which can be differentiated into a variety of different types of cells can be obtained by treating cells with selenium.
  • somatic cells including adipose tissue stromal cells are easier to collect than embryonic stem cells and can be free from ethical issues. Also, when cells are collected from a subject to be treated by the cells of the present invention, the desired therapeutic effect can be achieved without immune rejection.
  • the dedifferentiated cells of the present invention can be differentiated into a variety of different types of cells, and thus can be used to treat various diseases.
  • FIG. 1 shows the proliferation efficiency of dedifferentiated adipose tissue stromal cells according to the present invention.
  • FIG. 2 shows the telomerase activity of dedifferentiated adipose tissue stromal cells according to the present invention.
  • FIG. 3 shows an increase in the proliferation potential of dedifferentiated adipose tissue stromal cells according to the present invention.
  • FIG. 4 shows the expression levels of sternness genes in dedifferentiated adipose tissue stromal cells according to the present invention.
  • FIG. 5 shows the expression levels of cell proliferation stimulating or suppressing genes in dedifferentiated adipose tissue stromal cells according to the present invention.
  • FIG. 6 shows the degree of activation of signals associated with the growth of dedifferentiated adipose tissue stromal cells according to the present invention.
  • FIG. 7 shows the effect of a p38 inhibitor on the growth of dedifferentiated adipose tissue stromal cells according to the present invention.
  • FIG. 8 shows results indicating the effect of an MEK inhibitor on the growth of dedifferentiated adipose tissue stromal cells according to the present invention.
  • FIG. 9 shows results indicating that reactive oxygen species in the cytoplasm are decreased by treatment with selenium.
  • FIG. 10 shows results indicating the proliferation activity of adipose tissue stromal cells transformed with Rexl siRNA.
  • FIG. 11 shows results indicating that the sternness factor Rexl plays an important role in the dedifferentiation of adipose stem cells.
  • FIG. 12 is a schematic diagram simply showing the pathway of dedifferentiation of adipose tissue stromal cells by selenium.
  • FIG. 13 shows methylation in the promoters of sternness genes in dedifferentiated adipose tissue stromal cells according to the present invention.
  • FIG. 14 shows the results of a cell migration assay conducted using a transwell plate in vitro for dedifferentiated adipose tissue stromal cells according to the present invention.
  • FIG. 15 shows the results of a wound model assay for dedifferentiated adipose tissue stromal cells according to the present invention.
  • FIG. 16 shows the expression levels of cell migration- related genes in dedifferentiated adipose tissue stromal cells according to the present invention.
  • FIG. 17 shows the results obtained by examining whether dedifferentiated adipose tissue stromal cells according to the present invention re-differentiate into mesenchymal- derived cells.
  • FIG. 18 shows the results obtained by examining whether dedifferentiated adipose tissue stromal cells according to the present invention differentiate into mesenchymal-derived cells in vivo.
  • FIG. 19 shows the results obtained by examining whether dedifferentiated adipose tissue stromal cells according to the present invention re-differentiate into neurons (Sel2: 2 ng/ml of selenium, and Sel5: 5 ng/ml of selenium).
  • Example 1 Isolation and culture of adipose tissue stromal cells and treatment of the cells with selenium
  • adipose tissue stromal cells fat was extracted from the human abdomen in a hospital, and the extracted fat was washed with phosphate buffer saline (PBS) and incubated at 37 ° C for 30 minutes with 0.075% collagenase (Sigma, St. Louis, MO, USA). After neutralization, the stromal cell pellets were collected via centrifugation, and incubated overnight at 37 ° C in a CO 2 incubator in 10% FBS- containing ⁇ -MEM medium. The medium was replaced first at 48 hours and then replaced at a 4-day interval. When the primary cultured cells reached a confluency of 70-80% after 48-72 hours of culture, the cells were subcultured with 0.0025% trypsin solution.
  • PBS phosphate buffer saline
  • the cultured adipose tissue stromal cells were seeded into 10 cm dishes at a density of 5 x 10 5 cells and cultured in 2% FBS-containing ⁇ - MEM medium for 8 hours at 37 ° C in a CO 2 incubator. The cells were then treated with sodium selenite (Na 2 SeO 3 ; Sigma) at various concentrations for 3 days. The optimum concentration of selenium was selected on the basis of the results obtained from cytotoxicity studies using a broad concentration range for this reagent. Cell viability was evaluated via visual cell counts in conjunction with trypan blue exclusion. In all viability assays, triplicate wells were used for each condition, and each experiment was repeated at least three times .
  • Example 2 Verification of whether dedifferentiation of adipose tissue stromal cells was induced by treatment with selenium
  • ATSCs dedifferentiated adipose tissue stromal cells
  • Adipose tissue-derived stromal cells were treated with different concentrations of selenium (0, 5, 10, 15, and 20 ng/ml) for 3 days, and the proliferation of the ATSCs was evaluated via trypan blue exclusion. When the ATSCs were treated with 5 ng/ml of selenium for 3 days, the proliferation activity of the ATSCs was significantly increased.
  • CFUs colony forming units
  • ATSCs were seeded in 10 cm dishes at a density of 5 x 10 5 cells and cultured in 2% FBS-containing ⁇ -MEM media for 8 hours at 37 °C in a CO2 incubator. The cells were then treated with selenium (5 ng/ml) for 3 days.
  • control ATSCs i.e., ATSCs not treated with selenium
  • selenium-treated ATSCs were seeded in 10 cm dishes at a density of 2 x 10 2 and cultured in 10% FBS-containing ⁇ -MEM media at 37 °C in a C02 incubator. After 15 days, the cells were fixed with 4% paraformaldehyde (PFA) for 30 minutes at room temperature and stained with 0.1% toluidine blue in 1% PFA. The proliferation efficiency of the CFU was assessed via visual colony counts.
  • PFA paraformaldehyde
  • telomere activity is the characteristic of stem cells, and decreased telomerase activity indicates that stem cells are differentiated.
  • Rexl expression in particular, was markedly elevated as the result of selenium treatment.
  • treatment with selenium induced the expression of Nestin and c-Myc and reduced the expression of GFAP, TuJ, p53 and p21.
  • treatment with selenium reduced the acetylation of Histone 3 and Histone 4.
  • treatment with selenium induced the dedifferentiation of ATSCs.
  • Example 3 Examination of whether growth-related signals in dedifferentiated adipose tissue stromal cells were induced by selenium
  • FIG. 6 shows Western blot results for the selenium- treated ATSCs for different lengths of time (0, 3, 6, and 12 hours).
  • selenium induced significant activation of PI3K and its downstream mediators (p-Rac, p-c- Raf, p-MEK, p-ERK, p-Stat3, and p-Akt) in a time-dependent manner.
  • p-Rac, p-c- Raf, p-MEK, p-ERK, p-Stat3, and p-Akt selenium treatment was also shown to reduce the concentration of the apoptosis-relative protein, p- SAPK/JNK, in a time-dependent manner.
  • the selenium-treated ATSCs were treated with SB203580
  • the present inventors examined whether selenium inhibited the generation of ROS in the ATSCs.
  • ROS production in the ATSCs increased the oxidation of 2', 7'- dichlorodihydrofluorescein (DCF) in a concentration-dependent manner, and the increased DCF fluorescence intensity was abolished by selenium treatment (FIG. 9) . Therefore, these results showed that selenium induces the proliferation of ATSCs via the activation of MEK and PI3K signaling pathways and the inhibition of ROS generation.
  • DCF dichlorodihydrofluorescein
  • Rexl sternness transcription factors
  • ATSCs were transfected with Rexl silencing siRNA prior to selenium treatment.
  • the Rexl siRNA- transfected cells were harvested and examined via measurements of cell proliferation activity (FIG. 10) and changes in the expression of Rexl, CDKl, and CDK2 mRNA (FIG. 11) .
  • FIGS. 10, 11 and 3b the Rexl siRNA- transfected cells profoundly inhibited cell growth and Rexl gene expression as compared with the untreated controls.
  • Example 4 Examination of whether epigenetic reprogramming in selenium-treated ATSCs was induced by selenium via DNA demethylation 4-1: Analysis of change in gene expression pattern
  • the genomic DNA of ATSCs was purified via phenol/chloroform/isoamylalcohol extraction, followed by one chloroform extraction, after which the DNA was ethanol- precipitated.
  • the DNA was dissolved in distilled water.
  • Bisulfite conversion was conducted using the EZ DNA Methylation-Gold Kit (Zymo Research, USA) according to the manufacturer's instruction. Specifically, unmethylated cytosines in the DNA were converted into uracil via the heat- denaturation of the DNA and with a specifically designed CT conversion reagent. The DNA was then desulphonated and subsequently cleaned and eluted. The bisulfite-modified DNA was then immediately utilized for PCR or stored at or below - 20 ° C .
  • the converted DNA was amplified via a polymerase chain reaction (PCR) using primers designed with MethPrimer (http: //www. urogene. org/methprimer) .
  • the PCR reactions were conducted in a MyGenie 96 Gradient Thermal Block (Bioneer, Daejeon, South Korea) in accordance with the following protocol: 95 " C for 15 min, 40 cycles of 95 ° C for 20 sec, 43- 58 ° C for 40 sec, 72 "C for 30 sec, followed by extension at 72 ° C for 10 min, and soaking at 4 "C.
  • PCR products were cloned into bacteria (DH5 ⁇ ) by a pGEM T-Easy Vector System I (Promega, Madison, WI, USA) .
  • DNA extracted from the bacterial clones was analyzed via sequencing with the M13 reverse primer, using an ABI 3730XL capillary DNA sequencer (Applied Biosystems, Foster City, CA, USA) and expressed as rows of circles, with each circle symbolizing the methylation state of one CpG.
  • Rexl five amplicons were assessed, collectively converting the potentially methylated CpG dinucleotides within nucleotides - 868 to +7889 relative to the TSS (Fig. 3C) .
  • the Rexl region assessed was methylated in the ATSC control cells and was significantly demethylated in the third region from 72.2% (ATSCs) to 42.2% (selenium-treated ATSCs).
  • Three regions were evaluated in the Nanog promoter and were effectively demethylated in the third region (-86 to +66) relative to the TSS.
  • Three regions were also evaluated in the 0ct4 promoter, encompassing the CpGs within nucleotides -57 to +66 relative to the TSS. This 0ct4 methylation pattern was downregulated in the selenium-treated ATSCs (third region; 30.0%) as compared to the control ATSC cells (third region; 65.7%).
  • Example 5 Examination of whether improvement in migration of dedifferentiated adipose tissue stromal cells was induced by selenium
  • 5-1 Cell migration assay by transwell plate in vitro
  • the cells were seeded in 10 cm dishes at a density of 5 x 10 5 cells and cultured in 2% FBS containing ⁇ -MEM media for 8 hours at 37 ° C in a CO 2 incubator.
  • the cells were then treated with selenium (5 ng/ml) for 3 days.
  • the cultured cells were transferred into Costar transwell membranes (8 ⁇ m pore size) and placed in 6-well plates. Below the membrane, ⁇ MEM medium containing selenium and 2% FBS was added to each well.
  • the cells were incubated for 2 hours at 37 ° C in 2% FBS containing ⁇ -MEM medium, and the plate was incubated overnight at 37 ° C in a CO 2 incubator.
  • the cells on the lower surface were air dried and counterstained with Harris hematoxylin for 20 minutes, followed by washing.
  • the stained inserts were placed on object slides and the number of cells on the lower surfaces was assessed at 200X in an inverted bright field microscope. 10 images were repeatedly measured under a microscope at 20Ox magnification, and the measurements were averaged.
  • Cell migration activity was expressed as the count of cells per field of the spontaneous migration toward the cell bottom.
  • control dish was scraped and incubated in the same fashion as described above without the addition of selenium. Cells that migrated across the marked reference line were photographed under phase contrast microscopy.
  • Example 6 Determination of differentiation potential of adipose tissue stromal cells dedifferentiated by selenium 6-1: Determination of potential of selenium-treated ATSCs to differentiate into mesodermal lineage cells in vitro In order to determine the multipotency of the selenium- treated ATSCs, the osteogenic and adipogenic differentiation potential was assessed.
  • the selenium-treated ATSCs were determined to accumulate significant quantities of calcium and lipid droplets after only 1 week of in vitro osteogenic and adipogenic induction (FIG. 17) .
  • the present inventors quantified the differences in the efficiency of bone nodule and lipid droplet formation between the control ATSCs and the selenium-treated ATSCs by determining the number of stained bone nodules and lipid droplets in 25 random fields. As shown in FIG. 17, von Kossa-positive staining for calcium deposits and Nile red staining for lipid droplets were clearly observed in the selenium-treated ATSCs.
  • the selenium-treated ATSCs showed prominent calcium deposits and fat formation.
  • the selenium-treated ATSCs induced a significant increase in the levels of AP and PPAR- ⁇ mRNA in adipogenesis (FIG. 17) .
  • 6-2 Determination of potential of selenium-treated ATSCs to differentiate into mesodermal lineage cells in vitro
  • the in vivo osteogenesis and chondrogenesis effects of the selenium-treated ATSCs were examined.
  • the control ATSCs and the selenium-treated ATSCs were immobilized in Matrigel (BD Bioscience, San Jose, CA, USA) .
  • mice Approximately 2 x 10 6 cells were mixed with Matrigel and subcutaneously implanted in 6- week-old immunodeficient beige mice (NIH III/bg/nu/xid; Charles River Laboratories, Wilmington, MA, USA) . The procedures were conducted in accordance with the specifications of an approved protocol. The transplants were recovered 6 weeks after transplantation, fixed with 4% formalin, and decalcified with 10% EDTA (pH 8.0) for paraffin embedding. The paraffin-embedded sections were deparaffinized and stained with Alizarin Red (bone) , Masson (muscle and chondrocytes) , and Van Gieson (chondrocytes) stains.
  • neuroprogenitors neurospheres
  • bFGF vascular endothelial growth factor
  • EGF vascular endothelial growth factor
  • BDNF vascular endothelial growth factor
  • FIG. 19 The immunocytochemical data indicated that neuroprogenitors (neurospheres) can be expanded with bFGF, EGF, and BDNF, and more extensive differentiation is caused by the removal of cytokines and growth on PDL-laminin-coated surfaces.
  • GFAP astrocytes
  • MAP2ab neurons after induction of differentiation in vitro.
  • a higher percentage (approximately 9.0% of total cells) of neural differentiation (MAP2ab/total cells) was detected in the selenium-treated ATSCs as compared to the control ATSCs (about 2.4% of total cells; FIG. 19).
  • beta-like cell differentiation cells were cultured in ⁇ X N2 media+NA" containing DMEM/F12 (Gibco- Invitrogen) supplemented with 10 mM nicotinamide, ITS (1:50), B27 (1:50; Invitrogen), and 15% FBS. After 24 hours of culture, the medium was replaced with high glucose (3500 mg/L) differentiation medium for 2 weeks. After the induction of differentiation, double immunocytochemistry using insulin (1:800; Sigma) and c-peptide (1:100; Millipore) antibodies was conducted. As a result, the selenium-treated ATSCs were effectively differentiated into insulin-secreting cells. The selenium-treated differentiated ATSCs secreted a significant amount of insulin with C-peptide in contrast to the differentiated control ATSCs.
  • DMEM/F12 Gibco- Invitrogen
  • cells including adipose tissue stromal cells can be dedifferentiated using selenium safe for the human body.
  • the cells dedifferentiated by selenium and cells re- differentiated from the dedifferentiated cells can be used to treat various diseases.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Zoology (AREA)
  • Developmental Biology & Embryology (AREA)
  • Organic Chemistry (AREA)
  • Biotechnology (AREA)
  • Chemical & Material Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Genetics & Genomics (AREA)
  • Wood Science & Technology (AREA)
  • Microbiology (AREA)
  • Cell Biology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Rheumatology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)

Abstract

Disclosed herein are a composition for cell dedifferentiation containing selenium, a method of dedifferentiating cells by treating the cells with selenium, dedifferentiated cells obtained using the method, a composition for cell therapy containing the dedifferentiated cells, cells re-differentiated from the dedifferentiated cells, and a composition for cell therapy containing the re- differentiated cells. The disclosed dedifferentiated cells and the cells re-differentiated therefrom can be used as therapeutic agents for treating various diseases.

Description

[DESCRIPTION]
[invention Title]
Selenium Dedifferentiated Cell, Preparation Method and Usage Thereof
[Technical Field]
The present invention relates to cells dedifferentiated by selenium, and more particularly to a composition for cell dedifferentiation containing selenium, a method of dedifferentiating cells by treating the cells with selenium, dedifferentiated cells obtained using the method, a composition for cell therapy containing the dedifferentiated cells, cells re-differentiated from the dedifferentiated cells, and a composition for cell therapy containing the re- differentiated cells.
[Background Art]
Human embryonic stem cells have the capability to differentiate into all types of human cells, and thus show the capability to treat various existing diseases.
Since human embryonic stem cells were established for the first time in the world by the Thomson' s research group (USA) in 1998, a few hundred human embryonic stem cell lines have been established worldwide, and new human embryonic stem cell lines are now being continuously established. In studies on human embryonic stem cells, studies on feeder cells, the establishment of culture conditions excluding animal-derived factors, techniques for differentiation into specific cells, and the like are currently being conducted. However, an approach to clinical trials is still difficult due to the limitations of embryonic stem cells. Because embryonic stem cells are extracted from blastocysts, embryonic stem cells derived from the patient himself cannot be obtained, and thus when they are used for therapeutic purposes, immune rejection will occur. Also, the provision of an alternative method for eliminating the ethical issues raised by the destruction of embryos is required. Currently, studies on tailor-made stem cells created by nuclear transfer are being conducted, but are not yet successful. In addition, due to the ethical problems associated with oocyte donation, studies on the production of tailor-made stem cells by dedifferentiation are being attempted as alternative methods. Dedifferentiation technology receives remarkable attention in the stem cell field worldwide, because it can accurately substitute for the use of cloned embryonic stem cells and is not related to ethical concerns. Recently, the field of dedifferentiated stem cells, so- called induced pluripotent stem (iPS) cells, created using dedifferentiation technology, has started to receive new attention. As used herein, the term "dedifferentiated stem cells" refers to cells were dedifferentiated into the undifferentiated state by inserting a specific gene using somatic cells and have the capability to grow into all types of human cells, just like embryonic stem cells.
Many researchers have attempted to make embryonic stem cells having the same genotype as that of a patient from somatic cells using nuclear transfer technology or cell fusion technology, but have not yet found a proper solution.
Recently, Dr. Sinya Yamanaka (Japan) produced induced pluripotent stem (iPS) cells from somatic cells using a combination of four genes (oct4, sox2, klf4, and c-myc) among genes which are expressed specifically only in mouse embryonic stem cells, and he confirmed that this production is also applied to human cells. In addition, Dr. James Thomson (USA) reported that human somatic cells can be dedifferentiated using other gene combinations of 0ct4, Sox2, Nanog, Lin28, etc.
[Disclosure]
[Technical Problem]
The present inventors have treated adipose tissue stromal cells with selenium and, as a result, have found that the cells express sternness genes, the growth characteristics of the cells are extended, and the cells have multipotency to re-differentiate into other types of cells, thereby completing the present invention.
The present invention is to provide a composition for cell dedifferentiation containing selenium. The present invention is also to provide a method of dedifferentiating cells by treating the cells with selenium.
The present invention is also to provide dedifferentiated cells obtained using said method and a composition for cell therapy containing the differentiated cells.
The present invention is also to provide cells re- differentiated from the dedifferentiated cells and a composition for cell therapy containing the re-differentiated cells .
[Technical Solution]
As used herein, the term "dedifferentiation" is a term familiar in the art and is disclosed in, for example,
Weissman I. L., Cell 100: 157-168, FIG. 4, (2000). The term "differentiation" refers to the regression of a specialized
(i.e., differentiated) cell into a stem cell state, that is, a state which can be transferred or programmed into plural types of cells. The term "dedifferentiation" also means that multipotency increases. Namely, it means that the types of cells which can re-differentiate increase.
In the present invention, the dedifferentiation of cells is performed by treating the cells with selenium. Selenium which is an essential trace element in organisms is an important constituent of antioxidant enzymes which protect cells from free radicals generated during normal oxygen metabolism. This indicates that selenium is safe for the human body. In the present invention, organic selenium (e.g., selenomethionine and selenocysteine) or inorganic selenium (e.g., sodium selenite) may be used for the dedifferentiation of cells. Inorganic selenium is preferably used.
As cells to be dedifferentiated in the present invention, cells isolated from mammals, preferably humans, may be used. Also, the cells are preferably isolated from a subject to be treated with either dedifferentiated cells or cells re-differentiated therefrom. Thus, desired treatment can be achieved without immune rejection. Specifically, cells which are used in the present invention may be adult cells originated from a variety of different tissues, including the cumulus, skin, oral mucosa, blood bone marrow, liver, lungs, kidneys, muscles, reproductive tract, fat, etc. Examples of cells which can be used in the present invention include, but are not limited to, cumulus cells, epithelial cells, fibroblasts, neural cells, keratinocytes, hematopoietic cells, melanocytes, chondrocytes, erythrocytes, macropharges, monocytes, muscle cells, B lymphocytes, T lymphocytes, adipose tissue stromal cells, etc. As cells to be dedifferentiated in the present invention, adipose tissue stromal cells are particularly preferably used. The adipose tissue stromal cells can be derived from adipose tissue. Adipose tissue can be harvested using any conventional method known in the art. For example, adipose tissue can be harvested from the abdomen by liposuction. This process for obtaining adipose tissue has advantages in that it is carried out in a manner much easier than the prior art process for obtaining embryonic stem cells and is free from ethical issues.
Cells including adipose tissue stromal cells can generally be collected from the tissue harvested from an individual. The collected tissue is finely cut to remove unnecessary portions excluding the desired cells, and the cells are separated into single cells. In one embodiment, the tissue can be finely cut by physical means using a homogenizer, a mortar and pestle, a blender, a scalpel, forceps or an ultrasonic device. In another embodiment, an enzyme may also be used. Examples of the enzyme that can be used in the present invention include, but are not limited to, serine protease, elastase and collagenase. In still another embodiment, physical means and enzymatic treatment may be used in combination.
The cells obtained as described above may be immediately treated with selenium. Alternatively, the cells may also be cultured during a predetermined period, and then treated with selenium. The latter is preferable. Cells can be cultured in suitable media under suitable conditions depending on species from which the cells have been derived. When cells are of mammalian origin, the cells can be cultured in mammalian culture medium. Suitable media which can be used in the present invention are commercially available and may be prepared according to published compositions (for example, the catalog of the American Type Culture Collection) . Examples of medium which can be used in the present invention include, but are not limited to, Ham medium, IMDM Iscove's medium, Leibovitz L15 medium, May Coy 5A medium, M199 medium, Melnick's medium, MEM medium, NCTN medium, Puck's medium, RPMI medium, Swim S77 medium, Trowell T8 medium, Waymouth medium, Williams medium, DMEM medium, F12 medium, etc. When the cells are adipose tissue stromal cells, α-MEM medium is preferably used.
The above-described medium may also be supplemented with serum (e.g., FBS), antibiotics (e.g., kanamycin, streptomycin, penicillin, etc.), growth factors (e.g., EGF, PDGF, VΕGF, FGF, TGF, LTF, etc.), cytokines (e.g., insulin, estradiol, interleukin, corticosterone, etc.), if necessary. When the cells are adipose tissue stromal cells, 5-20% FBS is preferably added to the medium. When adipose tissue stromal cells reach a confluency of 60-90%, and preferably 70-80%, during the culture thereof, the cells are treated with an enzyme and subcultured. In the present invention, adipose tissue stromal cells, subcultured 1-10 times, preferably 2-5 times, and more preferably 3 times, are used.
Meanwhile, before cells are treated with selenium according to the present invention, the cells are preferably starved. The starvation is performed in order to eliminate the effects of various components in serum during dedifferentiation. For this purpose, a step of culturing the cells in a medium containing serum at a concentration of 1-3%, and preferably 2%, is additionally carried out.
The present invention relates to a method of dedifferentiating cells by treating the cells with selenium. As used herein, the phrase "treating cells with selenium" has the same meaning as bringing cells into contact with selenium.
The present invention may use all methods capable of bringing selenium and cells into contact with each other, including a method of treating cells in suitable buffer solution directly with selenium, a method of treating a culture of cells with selenium, or a method of adding selenium to a culture medium of cells and culturing the cells. In the present invention, it is preferable to culture cells in medium containing selenium.
In a buffer solution, culture or medium containing cells to be dedifferentiated, selenium is contained at a concentration of 0.1-20 ng/ml, preferably 1-15 ng/ml, and more preferably 5 ng/ml. If the selenium concentration is higher than 20 ng/ml, it will cause cytotoxicity, and if it is lower than 0.1 ng/ml, sufficient cell dedifferentiation will not occur. The culture of cells is performed for 12 hours to 10 days, preferably 1 to 5 days, and more preferably 3 days. However, the selenium concentration and the culture time are not limited to the above-described values, because it is possible to induce dedifferentiation by extending the treatment period, if the selenium concentration is relatively low, and shortening the treatment period, if the selenium concentration is relatively high.
After treating cells with selenium, whether the cells have been dedifferentiated by selenium is confirmed, and the dedifferentiated cells can be separated from the buffer solution, medium or culture according to a conventional method such as filtration or centrifugation. Cells differentiated by selenium and cells untreated with selenium
(or differentiated cells) show a difference in the expression level of a specific gene. The difference in expression level between the dedifferentiated cells and the differentiated cells is at least 5%, preferably at least 10%, more preferably at least 20%, and even more preferably at least 30%. The dedifferentiated cells of the present invention have the following characteristics.
The dedifferentiated cells of the present invention have an increased expression of a sternness gene as compared to differentiated cells. As used herein, the term "sternness gene" refers to a gene which is remarkably expressed in stem cells. The sternness gene is at least one selected from the group consisting of REXl, Nanog, 0ct4, Sox2, Runx3, CDKl, CDK2, Nestin, VEGF and FGFRl. Also, the dedifferentiated cells of the present invention have an increased expression of growth-related factor as compared to differentiated cells. Examples of the growth-related factor include, but are not limited to, c-Myc.
The dedifferentiated cells of the present invention have increased telomerase activity, and preferably about 2- fold increased telomerase activity, as compared to differentiated cells. Also, in the dedifferentiated cells of the present invention, the expression level of a gene which is expressed specifically in differentiated cells is decreased compared to that in differentiated cells, and the expression level of a cell proliferation suppressor gene (or tumor growth suppressor gene) is increased compared to that in differentiated cells. The gene which is expressed specifically in the differentiated cells is at least one of GFAP and Tuj , and the cell growth inhibitory gene is at least one of p53 and p31.
In the dedifferentiated cells of the present invention, the expression level of PI3K is increased compared to that in differentiated cells, and the activities of Rac, c-Raf, MEK, ERK, Stat3 and Akt that are mediators of PI3K are increased, while the expression of p-SAPK/JNK, an apoptosis-associated gene, which is induced by reactive oxygen species (ROS) , is inhibited.
The level of methylation in the promoter region of a sternness gene in the dedifferentiated cells of the present invention is lower than that in differentiated cells. The sternness gene is at least one selected from the group consisting of REXl, Nanog, 0ct4 and Sox2.
In the dedifferentiated cells of the present invention, the expression level of a cell migration-related gene is increased compared to that in differentiated cells. For this reason, the migration of the cells is activated. The cell migration-related gene is at least one selected from the group consisting of MMPl, MMP3, SDFl, VEGF and CXCR4.
Specific methods for analyzing the expression profiles of such genes include, but are not limited to, RT-PCR, competitive RT-PCR, real-time RT-PCR, RNase protection assay, Northern blotting, DNA chip assay, etc. In addition, the analysis methods include, but are not limited to, Western blotting, enzyme linked immunosorbent assay (ELISA) , radioimmunoassay (RIA) , ouchterlony immunodiffusion, rocket Immunoelectrophoresis, immunohistostaining, imrnunoprecipitation assay, complement fixation assay, FACS, and protein chip assay.
The present invention relates to dedifferentiated cells obtained by the above-described method of dedifferentiating cells by treating the cells with selenium and to a composition for cell therapy containing the dedifferentiated cells.
The inventive dedifferentiated cells themselves can be used to treat disease. The differentiated cells can be re- differentiated into a specific type of cells by direct contact with a cell population of this type in vivo. Thus, the dedifferentiated cells themselves can be applied to a desired type of tissue in order to treat disease, and the kind of disease which can be treated by the dedifferentiated cells is not limited.
Methods for producing tissue using cells which can be re-differentiated ("tissue engineering") are known in the art. Wang, X. et al. have shown that even certain adult cells of the pancreas in mice could be converted into hepatocytes in FAH (fumaroy-laceto-acetate hydrolase) -deficient mice (Wang X. et al. "Liver repopulation and correction of metabolic liver disease by transplanted adult mouse pancreatic cells" Am. J. Pathol. 158 (2) :571-579) . Lagasse et al. have shown that hematopoietic stem cells from bone marrow could be converted into hepatocytes after in vivo transfer into FAH-deficient mice (Lagasse et al. "Purified hematopoietic stem cells can differentiate into hepatocytes in vivo" Nature Medicine, 6(11) ; 1229-1234) .
Preferable forms of application for the in vivo re- differentiation of the dedifferentiated cells according to the present invention are injection, infusion or implantation of the cells into a specific type of cell population in the body. Thus, the dedifferentiated cells can be re- differentiated into a specific type of cells by direct contact with a cell group of this type.
The dedifferentiated cells of the present invention is preferably in the form of a cell composition containing one or more diluents which protect and maintain the cells and facilitate the use of the cells in injection, infusion or implantation into target tissue. The diluents may include buffer solutions, such as physiological saline, PBS (phosphate buffered saline) or HBSS (Hank's balanced salt solution), and plasma or blood components.
The dedifferentiated cells of the present invention may be immediately re-differentiated into a desired type of cells or be stored in medium for several days. In the latter case, cytokine or LIF (leukemia inhibitory factor) is preferably added to the medium in order to prevent loss of the ability of the cells to re-differentiate. Also, the cells may be freeze-dried and stored to maintain the ability to re- differentiate.
The present invention relates to cells re- differentiated from the dedifferentiated cells and a composition for cell therapy containing the re-differentiated cells as an active ingredient.
The dedifferentiated cells of the present invention can be re-differentiated into a variety of different types of cells. The dedifferentiated cells of the present invention can be re-differentiated into a specific type of cells according to any method known in the art. Reference is made to, for example, Weissman I. L., Science 287:1442-1446 (2000); Insight Review Articles Nature 414: 92-131(2000); Handbook "Methods of Tissue Engineering ", Eds. Atala, A., Lanza, R. P., Academic Press, ISBN 0-12-436636-8; and Library of Congress Catalog Card No. 200188747.
Also, because the dedifferentiated cells of the present invention can be re-differentiated into a specific type of cells by bringing the dedifferentiated cells into contact with this type of cell population as described above, the dedifferentiated cells can be re-differentiated into a desired type of cells by bringing the dedifferentiated cells into contact with this type of cell group in vitro.
Particularly, the dedifferentiated adipose tissue stromal cells of the present invention can be differentiated into mesenchymal-derived cells, including bone cells, chondrocytes and muscle cells, neurons, adiocytes, and insulin-producing cells (pancreatic Langerhans islet B cells) . Thus, the dedifferentiated cells of the present invention can be used to treat, but not limited to, various diseases such as cancer, osteoporosis, arthritis, neurodegenerative disease, and diabetes.
The re-differentiation of dedifferentiated adipose tissue stromal cells can be confirmed, for example, in the following manner. Differentiation into bone cells, chondrocytes and muscle cells can be confirmed through a method employing dyes which are stained specifically on the respective cells. Differentiation into bone cells and adipocytes can be confirmed by measuring the formation of bone nodules and lipids. Also, differentiation into bone cells can be confirmed by analyzing the expression levels of osteonectin, RXR and osteopontin. Differentiation into adipocytes can be confirmed by analyzing the expression levels of AP and PPAR-γ, and differentiation into neurons can be confirmed by analyzing the expression levels of Tuj , GFAP, MAP2ab and NF160. In addition, differentiation into insulin- producing cells can be confirmed by analyzing the expression level of insulin.
The re-differentiated cells of the present invention may be applied to target tissue in the same manner as the case of the above-described dedifferentiated cells to treat disease. Preferable forms of application may be injection, infusion, and implantation. Furthermore, a cell composition containing the re-differentiated cells may comprise one or more diluents, which protect and maintain the cells and facilitate the use of the cells in injection, infusion or implantation into target tissue, in the same manner as the cell composition containing the dedifferentiated cells. [Advantageous Effects]
According to the present invention, dedifferentiated somatic cells which can be differentiated into a variety of different types of cells can be obtained by treating cells with selenium.
Particularly, somatic cells including adipose tissue stromal cells are easier to collect than embryonic stem cells and can be free from ethical issues. Also, when cells are collected from a subject to be treated by the cells of the present invention, the desired therapeutic effect can be achieved without immune rejection.
The dedifferentiated cells of the present invention can be differentiated into a variety of different types of cells, and thus can be used to treat various diseases.
[Description of Drawings]
FIG. 1 shows the proliferation efficiency of dedifferentiated adipose tissue stromal cells according to the present invention. FIG. 2 shows the telomerase activity of dedifferentiated adipose tissue stromal cells according to the present invention.
FIG. 3 shows an increase in the proliferation potential of dedifferentiated adipose tissue stromal cells according to the present invention.
FIG. 4 shows the expression levels of sternness genes in dedifferentiated adipose tissue stromal cells according to the present invention.
FIG. 5 shows the expression levels of cell proliferation stimulating or suppressing genes in dedifferentiated adipose tissue stromal cells according to the present invention. FIG. 6 shows the degree of activation of signals associated with the growth of dedifferentiated adipose tissue stromal cells according to the present invention.
FIG. 7 shows the effect of a p38 inhibitor on the growth of dedifferentiated adipose tissue stromal cells according to the present invention.
FIG. 8 shows results indicating the effect of an MEK inhibitor on the growth of dedifferentiated adipose tissue stromal cells according to the present invention. FIG. 9 shows results indicating that reactive oxygen species in the cytoplasm are decreased by treatment with selenium.
FIG. 10 shows results indicating the proliferation activity of adipose tissue stromal cells transformed with Rexl siRNA.
FIG. 11 shows results indicating that the sternness factor Rexl plays an important role in the dedifferentiation of adipose stem cells.
FIG. 12 is a schematic diagram simply showing the pathway of dedifferentiation of adipose tissue stromal cells by selenium.
FIG. 13 shows methylation in the promoters of sternness genes in dedifferentiated adipose tissue stromal cells according to the present invention. FIG. 14 shows the results of a cell migration assay conducted using a transwell plate in vitro for dedifferentiated adipose tissue stromal cells according to the present invention.
FIG. 15 shows the results of a wound model assay for dedifferentiated adipose tissue stromal cells according to the present invention.
FIG. 16 shows the expression levels of cell migration- related genes in dedifferentiated adipose tissue stromal cells according to the present invention.
FIG. 17 shows the results obtained by examining whether dedifferentiated adipose tissue stromal cells according to the present invention re-differentiate into mesenchymal- derived cells.
FIG. 18 shows the results obtained by examining whether dedifferentiated adipose tissue stromal cells according to the present invention differentiate into mesenchymal-derived cells in vivo.
FIG. 19 shows the results obtained by examining whether dedifferentiated adipose tissue stromal cells according to the present invention re-differentiate into neurons (Sel2: 2 ng/ml of selenium, and Sel5: 5 ng/ml of selenium).
[Best Mode]
Hereinafter, the present invention will be described in further detail with reference to examples, but the scope of the present invention is not limited only to these examples. Example 1: Isolation and culture of adipose tissue stromal cells and treatment of the cells with selenium
To isolate adipose tissue stromal cells, fat was extracted from the human abdomen in a hospital, and the extracted fat was washed with phosphate buffer saline (PBS) and incubated at 37 °C for 30 minutes with 0.075% collagenase (Sigma, St. Louis, MO, USA). After neutralization, the stromal cell pellets were collected via centrifugation, and incubated overnight at 37 °C in a CO2 incubator in 10% FBS- containing α-MEM medium. The medium was replaced first at 48 hours and then replaced at a 4-day interval. When the primary cultured cells reached a confluency of 70-80% after 48-72 hours of culture, the cells were subcultured with 0.0025% trypsin solution.
For treatment with selenium, the cultured adipose tissue stromal cells were seeded into 10 cm dishes at a density of 5 x 105 cells and cultured in 2% FBS-containing α- MEM medium for 8 hours at 37 °C in a CO2 incubator. The cells were then treated with sodium selenite (Na2SeO3; Sigma) at various concentrations for 3 days. The optimum concentration of selenium was selected on the basis of the results obtained from cytotoxicity studies using a broad concentration range for this reagent. Cell viability was evaluated via visual cell counts in conjunction with trypan blue exclusion. In all viability assays, triplicate wells were used for each condition, and each experiment was repeated at least three times . Example 2: Verification of whether dedifferentiation of adipose tissue stromal cells was induced by treatment with selenium
2-1: Determination of whether proliferation activity of dedifferentiated adipose tissue stromal cells (ATSCs) was increased by treatment with selenium
Adipose tissue-derived stromal cells (ATSCs) were treated with different concentrations of selenium (0, 5, 10, 15, and 20 ng/ml) for 3 days, and the proliferation of the ATSCs was evaluated via trypan blue exclusion. When the ATSCs were treated with 5 ng/ml of selenium for 3 days, the proliferation activity of the ATSCs was significantly increased.
Also, the present inventors determined the proliferation efficiency of colony forming units (CFU) in the selenium-treated cells. CFUs are single cell-derived populations, and increases in CFU values indicate that selenium can actively stimulates the proliferation of ATSCs. First, ATSCs were seeded in 10 cm dishes at a density of 5 x 105 cells and cultured in 2% FBS-containing α-MEM media for 8 hours at 37 °C in a CO2 incubator. The cells were then treated with selenium (5 ng/ml) for 3 days. For the CFU assay, control ATSCs (i.e., ATSCs not treated with selenium) and selenium-treated ATSCs were seeded in 10 cm dishes at a density of 2 x 102 and cultured in 10% FBS-containing α-MEM media at 37 °C in a C02 incubator. After 15 days, the cells were fixed with 4% paraformaldehyde (PFA) for 30 minutes at room temperature and stained with 0.1% toluidine blue in 1% PFA. The proliferation efficiency of the CFU was assessed via visual colony counts.
As a result, it was confirmed that the selenium-treated ATSCs had a 1.8-fold increase in colony value, an index of cell proliferation (FIG. 1) .
2-2: Determination of whether dedifferentiation of ATSCs was induced by treatment with selenium
To determine the effect of selenium on the removal of ROS from ATSCs, the cells were treated with 5 ng/ml of selenium and then measured for telomerase activity. Increased telomerase activity is the characteristic of stem cells, and decreased telomerase activity indicates that stem cells are differentiated.
When ATSCs were treated with selenium and then measured for telomerase activity, treatment with selenium increased telomerase activity by about 2 times to induce the dedifferentiation of ATSCs (FIG. 2) .
2-3: Examination of whether growth characteristics of ATSCs were extended due to selenium treatment During prolonged culture periods, the population of control ATSCs underwent a progressive reduction in proliferation potential. The cells finally underwent senescence after 21-23 passages (90-100 days in culture) . At the end of the proliferative lifespan, the cells were flatter and larger in morphology in a monolayer similar to that described for senescent fibroblasts. In experimental selenium exposure, selenium-treated ATSCs grew continuously for more than 3 months (>21 passages; FIG. 3) . In addition, ATSCs exposed to selenium retained their inhibition for cellular proliferation via cell-to-cell contact (FIG. 3) . The results showed that the extended growth of stromal cells, as a consequence of exposure to selenium, did not alter cell growth properties.
2-4: Examination of whether expression of sternness genes in ATSCs was increased due to selenium treatment
The expression of gene markers in both the control ATSCs and the selenium-treated ATSCs was verified via real time RT-PCR and Western blot analysis.
As shown in FIG. 4, treatment with selenium induced the overexpression of various sternness genes and functional genes
(REXl, Nanog, Oct4, Sox2, Runx3, CDKl, CDK2, Nestin, VEGF, and FGFRl) . Rexl expression, in particular, was markedly elevated as the result of selenium treatment.
Also, as shown in FIG. 5, treatment with selenium induced the expression of Nestin and c-Myc and reduced the expression of GFAP, TuJ, p53 and p21. In addition, treatment with selenium reduced the acetylation of Histone 3 and Histone 4. Thus, it was confirmed that treatment with selenium induced the dedifferentiation of ATSCs.
Example 3: Examination of whether growth-related signals in dedifferentiated adipose tissue stromal cells were induced by selenium
3-1: Assessment of relevance of selenium-induced growth-related signaling pathways in selenium-treated ATSCs To identify activated signaling molecules involved in cell proliferation occurring after selenium treatment, the total protein levels and phosphorylation status of several proliferation-related proteins were assessed in ATSCs via Western blot analysis.
FIG. 6 shows Western blot results for the selenium- treated ATSCs for different lengths of time (0, 3, 6, and 12 hours). As shown therein, selenium induced significant activation of PI3K and its downstream mediators (p-Rac, p-c- Raf, p-MEK, p-ERK, p-Stat3, and p-Akt) in a time-dependent manner. However, selenium treatment was also shown to reduce the concentration of the apoptosis-relative protein, p- SAPK/JNK, in a time-dependent manner.
3-2: Assessment of relevance of p38 and MEK signaling pathways in control of cell growth in selenium-treated ATSCs
To assess the relevance of the p38 and MEK signaling pathways in the control of cell growth in selenium-treated
ATSCs, the selenium-treated ATSCs were treated with SB203580
(10 μM; an inhibitor of p38) and PD98059 (10 μM; an inhibitor of MEK) and subjected to Western blot analysis and RT-PCR.
As shown in FIG. 7, the results of Western blot analysis indicated that SB203580 induced the downregulation of p-SAPK/JNK and the p53 and p21 proteins and overexpressed c-Myc protein. Also, the results of real time RT-PCR analysis indicated that SB203580 showed upregulation of proliferation-related transcription factors, including CDKl and CDK2. These data show that selenium can directly attenuate the levels of the apoptosis-relative protein, p- SAPK/JNK. As shown in FIG. 8, PD98059 exhibited downregulation of p-ERK and c-Myc proteins. The results of RT-PCR analysis also showed that PD98059 had downregulation of proliferation-related transcription factors, including Rexl, CDKl, and CDK2. These data clearly show that selenium induced ATSCs proliferation via the activation of MEK and PI3K signaling pathways with the direct inhibition of the apoptosis-related protein, p-SAPK/JNK. 3-3; Examination of whether ROS was generated in selenium-treated ATSCs
The present inventors examined whether selenium inhibited the generation of ROS in the ATSCs. ROS production in the ATSCs increased the oxidation of 2', 7'- dichlorodihydrofluorescein (DCF) in a concentration-dependent manner, and the increased DCF fluorescence intensity was abolished by selenium treatment (FIG. 9) . Therefore, these results showed that selenium induces the proliferation of ATSCs via the activation of MEK and PI3K signaling pathways and the inhibition of ROS generation.
3-4: Assessment of relevance of Rexl in growth of selenium-treated ATSCs
The activation of ERK1/2 and Akt in the selenium- treated ATSCs induced the expression of sternness transcription factors, particularly Rexl. In order to evaluate the roles of Rexl in the proliferation of selenium- treated ATSCs, ATSCs were transfected with Rexl silencing siRNA prior to selenium treatment. The Rexl siRNA- transfected cells were harvested and examined via measurements of cell proliferation activity (FIG. 10) and changes in the expression of Rexl, CDKl, and CDK2 mRNA (FIG. 11) . As shown in FIGS. 10, 11 and 3b, the Rexl siRNA- transfected cells profoundly inhibited cell growth and Rexl gene expression as compared with the untreated controls. These results showed that Rexl is a major gene, the expression of which is closely associated with ATSCs proliferation, and that selenium increases the proliferative efficiency of selenium-treated ATSCs via the enhancement of Rexl expression.
Based on these results, the present inventors suggest a model to explain the mechanisms underlying the adipose tissue stromal cell proliferation and dedifferentiation induced by selenium treatment (FIG. 12) .
Example 4: Examination of whether epigenetic reprogramming in selenium-treated ATSCs was induced by selenium via DNA demethylation 4-1: Analysis of change in gene expression pattern
In order to assess the pattern of gene expression, oligonucleotide microarray analysis was performed. The analysis of gene expression levels indicated that less than 6 % of the total genes showed a difference of 2.2 times in expression level between ATSCs and selenium-treated ATSC cells, as indicated by the r value (=0.89). A comparison of the expression of those genes between ATSCs and selenium- treated ATSCs showed that cell proliferation-associated genes were upregulated in selenium-treated ATSCs (42%). 4-2: Analysis of change in DNA methylation in Rexl and Nanog promoter regions
In an effort to determine whether selenium treatment was capable of eliciting epigenetic modifications on exogeneous chromatin templates, changes in DNA methylation in the Rexl and Nanog promoter regions were analyzed. The present inventors also performed bisulfate sequencing analysis in order to establish the 5' -3' CpG methylation profiles across each test gene proximal promoter, the proximal enhancer, and the early transcription start site (TSS) .
The genomic DNA of ATSCs was purified via phenol/chloroform/isoamylalcohol extraction, followed by one chloroform extraction, after which the DNA was ethanol- precipitated. The DNA was dissolved in distilled water. Bisulfite conversion was conducted using the EZ DNA Methylation-Gold Kit (Zymo Research, USA) according to the manufacturer's instruction. Specifically, unmethylated cytosines in the DNA were converted into uracil via the heat- denaturation of the DNA and with a specifically designed CT conversion reagent. The DNA was then desulphonated and subsequently cleaned and eluted. The bisulfite-modified DNA was then immediately utilized for PCR or stored at or below - 20 °C . The converted DNA was amplified via a polymerase chain reaction (PCR) using primers designed with MethPrimer (http: //www. urogene. org/methprimer) . The PCR reactions were conducted in a MyGenie 96 Gradient Thermal Block (Bioneer, Daejeon, South Korea) in accordance with the following protocol: 95 "C for 15 min, 40 cycles of 95 °C for 20 sec, 43- 58 °C for 40 sec, 72 "C for 30 sec, followed by extension at 72 °C for 10 min, and soaking at 4 "C. Following electrophoresis on a 1.5% agarose gel, the remaining PCR products were cloned into bacteria (DH5α) by a pGEM T-Easy Vector System I (Promega, Madison, WI, USA) . DNA extracted from the bacterial clones was analyzed via sequencing with the M13 reverse primer, using an ABI 3730XL capillary DNA sequencer (Applied Biosystems, Foster City, CA, USA) and expressed as rows of circles, with each circle symbolizing the methylation state of one CpG.
As shown in FIG. 13, in the case of Rexl, five amplicons were assessed, collectively converting the potentially methylated CpG dinucleotides within nucleotides - 868 to +7889 relative to the TSS (Fig. 3C) . The Rexl region assessed was methylated in the ATSC control cells and was significantly demethylated in the third region from 72.2% (ATSCs) to 42.2% (selenium-treated ATSCs). Three regions were evaluated in the Nanog promoter and were effectively demethylated in the third region (-86 to +66) relative to the TSS. Three regions were also evaluated in the 0ct4 promoter, encompassing the CpGs within nucleotides -57 to +66 relative to the TSS. This 0ct4 methylation pattern was downregulated in the selenium-treated ATSCs (third region; 30.0%) as compared to the control ATSC cells (third region; 65.7%).
Example 5: Examination of whether improvement in migration of dedifferentiated adipose tissue stromal cells was induced by selenium
5-1: Cell migration assay by transwell plate in vitro In order to evaluate the migration activity of the selenium-treated ATSC cells, the cells were seeded in 10 cm dishes at a density of 5 x 105 cells and cultured in 2% FBS containing α-MEM media for 8 hours at 37 °C in a CO2 incubator. The cells were then treated with selenium (5 ng/ml) for 3 days. The cultured cells were transferred into Costar transwell membranes (8 μm pore size) and placed in 6-well plates. Below the membrane, αMEM medium containing selenium and 2% FBS was added to each well. To the upper chamber, the cells were incubated for 2 hours at 37 °C in 2% FBS containing α-MEM medium, and the plate was incubated overnight at 37 °C in a CO2 incubator. The cells on the lower surface were air dried and counterstained with Harris hematoxylin for 20 minutes, followed by washing. The stained inserts were placed on object slides and the number of cells on the lower surfaces was assessed at 200X in an inverted bright field microscope. 10 images were repeatedly measured under a microscope at 20Ox magnification, and the measurements were averaged. Cell migration activity was expressed as the count of cells per field of the spontaneous migration toward the cell bottom.
As shown in FIG. 14, the selenium-treated ATSCs showed migration efficiency which was significantly increased in a time-dependent manner as compared to the control ATSCs. 5-2: Wound model assay
In order to obtain clearer evidence regarding the role of selenium in ATSCs migration, a simple cell scraped wound model assay was conducted. The cells were seeded into 60 mm culture dishes, and a straight line was gently carved diametrically across the center, outer, and bottom surface of each dish with a scalpel. Adipose tissue stromal cells were incubated overnight in serum-free medium, and then scraped from one side of the marked line and washed three times with medium to remove all loose or dead cells. The cells were then stimulated with 5 ng/ml of selenium and incubated at
37 °C for 24 hours. The control dish was scraped and incubated in the same fashion as described above without the addition of selenium. Cells that migrated across the marked reference line were photographed under phase contrast microscopy.
As shown in FIG. 15, selenium induced the migration of ATSCs across the reference line, thereby indicating an almost 3-fold increase in migration in selenium-treated ATSCs over the untreated control ATSCs. These results were consistent with the increase in the expression of migration-associated transcription factors, including MMPl, MMP3, SDFl, VEGF, and CXCR4 after selenium treatment (FIG. 16) .
[Mode for Invention]
Example 6: Determination of differentiation potential of adipose tissue stromal cells dedifferentiated by selenium 6-1: Determination of potential of selenium-treated ATSCs to differentiate into mesodermal lineage cells in vitro In order to determine the multipotency of the selenium- treated ATSCs, the osteogenic and adipogenic differentiation potential was assessed.
In this Example, the selenium-treated ATSCs were determined to accumulate significant quantities of calcium and lipid droplets after only 1 week of in vitro osteogenic and adipogenic induction (FIG. 17) . The present inventors quantified the differences in the efficiency of bone nodule and lipid droplet formation between the control ATSCs and the selenium-treated ATSCs by determining the number of stained bone nodules and lipid droplets in 25 random fields. As shown in FIG. 17, von Kossa-positive staining for calcium deposits and Nile red staining for lipid droplets were clearly observed in the selenium-treated ATSCs. The selenium-treated ATSCs showed prominent calcium deposits and fat formation. There were as many as five times more nodules and six times more lipid droplets in the selenium-treated ATSCs than observed in the control ATSCs through an elated, dye quantitative assay using spectrophotometry. These results are consistent with the overexpression of osteogenesis- and adipogenesis-related transcription factors after selenium treatment, including osteonectin, RXR, osteopontin, AP, and PPAR-γ (FIG. 17) . The selenium-treated ATSCs induced significant increases in the levels of osteonectin, RXR, and osteopontin mRNA in osteogenesis. Also, the selenium-treated ATSCs induced a significant increase in the levels of AP and PPAR-γ mRNA in adipogenesis (FIG. 17) . 6-2: Determination of potential of selenium-treated ATSCs to differentiate into mesodermal lineage cells in vitro In this Example, the in vivo osteogenesis and chondrogenesis effects of the selenium-treated ATSCs were examined. For this purpose, the control ATSCs and the selenium-treated ATSCs were immobilized in Matrigel (BD Bioscience, San Jose, CA, USA) . Approximately 2 x 106 cells were mixed with Matrigel and subcutaneously implanted in 6- week-old immunodeficient beige mice (NIH III/bg/nu/xid; Charles River Laboratories, Wilmington, MA, USA) . The procedures were conducted in accordance with the specifications of an approved protocol. The transplants were recovered 6 weeks after transplantation, fixed with 4% formalin, and decalcified with 10% EDTA (pH 8.0) for paraffin embedding. The paraffin-embedded sections were deparaffinized and stained with Alizarin Red (bone) , Masson (muscle and chondrocytes) , and Van Gieson (chondrocytes) stains.
As shown in FIG. 18, the results of hematoxylin-eosin and Alizarin Red staining of the implanted tissue sections revealed highly enhanced bone formation by the selenium- treated ATSCs as compared with the control ATSCs. The control ATSCs-implanted tissue sections did not show effective bone and collagen fiber staining. Intensive Masson staining also showed that the selenium-treated ATSCs implants effectively differentiated into muscle fibers. 6-3: Determination of potential of selenium-treated ATSCs to differentiate into neurons and insulin-producing cells
In an attempt to determine the potential of the selenium-treated ATSCs to differentiate into neurons in vitro, very low levels of Nestin protein expression in the selenium- treated ATSCs were detected after the induction of differentiation (FIG. 19) . After neural differentiation, the differentiated selenium-treated ATSCs cells expressed higher levels of TuJ, GFAP, MAP2ab, and NF160 than was observed in the differentiated control ATSCs (FIG. 19) . The control ATSC cells did not undergo efficient neural differentiation under the conditions adopted in this Example, thereby suggesting distinctions in the differentiation capacities between the control ATSCs and the selenium-treated ATSCs. Also, the results of Western blot analysis showed that the selenium- treated ATSCs exhibited overexpression of acetyl-histones 3 and 4 after neural differentiation (FIG. 19) .
The immunocytochemical data indicated that neuroprogenitors (neurospheres) can be expanded with bFGF, EGF, and BDNF, and more extensive differentiation is caused by the removal of cytokines and growth on PDL-laminin-coated surfaces (FIG. 19) . Populations of selenium-treated differentiated ATSCs had morphologic and phenotypic characteristics consistent with astrocytes (GFAP) and neurons (MAP2ab) after induction of differentiation in vitro. A higher percentage (approximately 9.0% of total cells) of neural differentiation (MAP2ab/total cells) was detected in the selenium-treated ATSCs as compared to the control ATSCs (about 2.4% of total cells; FIG. 19). As a result of a postnatal mouse brain transplantation study, considerable numbers of engrafted selenium-treated ATSCs were stably integrated in the hippocampus, striatum, and cortex and they were effectively differentiated into MAP2ab-positive neurons in the hippocampus (FIG. 19) .
Also, for beta-like cell differentiation, cells were cultured in λXN2 media+NA" containing DMEM/F12 (Gibco- Invitrogen) supplemented with 10 mM nicotinamide, ITS (1:50), B27 (1:50; Invitrogen), and 15% FBS. After 24 hours of culture, the medium was replaced with high glucose (3500 mg/L) differentiation medium for 2 weeks. After the induction of differentiation, double immunocytochemistry using insulin (1:800; Sigma) and c-peptide (1:100; Millipore) antibodies was conducted. As a result, the selenium-treated ATSCs were effectively differentiated into insulin-secreting cells. The selenium-treated differentiated ATSCs secreted a significant amount of insulin with C-peptide in contrast to the differentiated control ATSCs.
[industrial Applicability]
As described above, according to the present invention, cells including adipose tissue stromal cells can be dedifferentiated using selenium safe for the human body. The cells dedifferentiated by selenium and cells re- differentiated from the dedifferentiated cells can be used to treat various diseases.

Claims

[CLAIMS]
[Claim l]
A composition for cell dedifferentiation containing selenium.
[Claim 2]
The composition of Claim 1, wherein the cells are isolated from mammals.
[Claim 3]
The composition of Claim 2, wherein the cells are adipose tissue stromal cells.
[Claim 4] A method for dedifferentiating cells which comprises a step of treating cells with selenium.
[Claim 5]
The method of Claim 4, which additionally comprises, before the step, a step of culturing the cells in medium containing 1-3% FBS.
[Claim 6]
The method of Claim 4, wherein the cells are treated with 0.1-200 ng/ml of selenium for a period of time ranging from 12 hours to 10 days.
[Claim 7]
The method of Claim 1, wherein the cells are isolated from mammals.
[Claim 8]
The method of Claim 7, wherein the cells are adipose tissue stromal cells.
[Claim 9]
The method of Claim 7, wherein the dedifferentiated cells have an increased expression of a sternness gene selected from the group consisting of REXl, Nanog, 0ct4, Sox2, Runx3, CDKl, CDK2, Nestin, VEGF and FGFRl as compared to differentiated cells.
[Claim 10]
The method of Claim 7, wherein the dedifferentiated cells show an increased expression of c-Myc as compared to differentiated cells.
[Claim 11]
The method of Claim 7, wherein the dedifferentiated cells have increased telomerase activity.
[Claim 12]
The method of Claim 7, wherein the dedifferentiated cells have a decreased expression of GFAP and Tuj genes as compared to differentiated cells.
[Claim 13]
The method of Claim 7, wherein the dedifferentiated cells have a decreased expression of p53 and p31 as compared to differentiated cells.
[Claim 14] The method of Claim 7, wherein the dedifferentiated cells have an increased expression of a PI3K gene and an increased phosphorylation of a PI3K mediator selected from the group consisting of Rac, c-Raf, MEK, ERK, Stat3 and Akt as compared to differentiated cells.
[Claim 15]
The method of Claim 7, wherein the dedifferentiated cells have a decreased expression of a p-SAPK/JNK gene as compared to differentiated cells.
[Claim 16]
The method of Claim 7, wherein the dedifferentiated cells have decreased methylation in the promoter region of a sternness gene selected from the group consisting of REXl, Nanog, Oct4 and Sox2 as compared to differentiated cells.
[Claim 17]
The method of Claim 7, wherein the dedifferentiated cells have a decreased expression of a cell migration-related gene selected from the group consisting of the group consisting of MMPl, MMP3, SDFl, VEGF and CXCR4 as compared to differentiated cells.
[Claim 18]
A dedifferentiated cell obtained according to the method of any one of Claims 4 to 17.
[Claim 19] A composition for cell therapy containing the dedifferentiated cell of Claim 18 as an active ingredient
[Claim 20]
A cell re-differentiated from the dedifferentiated cell of Claim 18.
[Claim 21]
The re-differentiated cell of Claim 20, wherein the re- differentiated cell is selected from the group consisting of mesenchymal-derived cells, neurons, adipocytes, and insulin- producing cells.
[Claim 22]
A composition for cell therapy containing the re- differentiated cell of Claim 20 or 21 as an active ingredient
PCT/KR2008/004453 2008-06-26 2008-07-31 Selenium dedifferentiated cell, preparation method and usage thereof WO2009157610A1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
US12/294,228 US20100233131A1 (en) 2008-06-26 2008-07-31 Selenium dedifferentiated cell, preparation method and usage thereof

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
KR10-2008-0061126 2008-06-26
KR20080061126 2008-06-26
KR1020080074630A KR101041914B1 (en) 2008-06-26 2008-07-30 Selenium-Dedifferentiated Cell, Preparation Method and Usage Thereof
KR10-2008-0074630 2008-07-30

Publications (1)

Publication Number Publication Date
WO2009157610A1 true WO2009157610A1 (en) 2009-12-30

Family

ID=41444666

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/KR2008/004453 WO2009157610A1 (en) 2008-06-26 2008-07-31 Selenium dedifferentiated cell, preparation method and usage thereof

Country Status (1)

Country Link
WO (1) WO2009157610A1 (en)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
RU2668803C2 (en) * 2014-05-23 2018-10-02 Ббхк Method for producing stem cells with induced pluripotency of mesenchimal stem cells by using phlorotannin fraction
US20210171524A1 (en) * 2018-06-22 2021-06-10 The Royal Institution For The Advancement Of Learning/Mcgill University Purine compounds and method for the treatment of cancer

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002038741A1 (en) * 2000-11-09 2002-05-16 Bresagen Limited Cell reprogramming
US6900051B2 (en) * 2000-10-02 2005-05-31 Centre Hospitalier Regional Universitaire De Lille Process for obtaining mammalian insulin secreting cells in vitro and their uses
US20050176707A1 (en) * 2003-11-10 2005-08-11 The Scripps Research Institute Compositions and methods for inducing cell dedifferentiation
US7138275B2 (en) * 2002-03-28 2006-11-21 Blasticon Biotechnologische Forschung Gmbh Dedifferentiated, programmable stem cells of monocytic origin, and their production and use
WO2007069666A1 (en) * 2005-12-13 2007-06-21 Kyoto University Nuclear reprogramming factor

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6900051B2 (en) * 2000-10-02 2005-05-31 Centre Hospitalier Regional Universitaire De Lille Process for obtaining mammalian insulin secreting cells in vitro and their uses
WO2002038741A1 (en) * 2000-11-09 2002-05-16 Bresagen Limited Cell reprogramming
US7138275B2 (en) * 2002-03-28 2006-11-21 Blasticon Biotechnologische Forschung Gmbh Dedifferentiated, programmable stem cells of monocytic origin, and their production and use
US20050176707A1 (en) * 2003-11-10 2005-08-11 The Scripps Research Institute Compositions and methods for inducing cell dedifferentiation
WO2007069666A1 (en) * 2005-12-13 2007-06-21 Kyoto University Nuclear reprogramming factor

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
RU2668803C2 (en) * 2014-05-23 2018-10-02 Ббхк Method for producing stem cells with induced pluripotency of mesenchimal stem cells by using phlorotannin fraction
US20210171524A1 (en) * 2018-06-22 2021-06-10 The Royal Institution For The Advancement Of Learning/Mcgill University Purine compounds and method for the treatment of cancer

Similar Documents

Publication Publication Date Title
KR101041914B1 (en) Selenium-Dedifferentiated Cell, Preparation Method and Usage Thereof
JP2022120163A (en) Methods for reprogramming cells and uses thereof
AU2012346645B2 (en) Culture-medium composition for rejuvenating stem cells
JP6316938B2 (en) Method for preparing induced neural stem cells reprogrammed from non-neuronal cells using HMGA2
EP3134513B1 (en) Method of producing skin-derived precursor cells
Kim et al. IFATS collection: Selenium induces improvement of stem cell behaviors in human adipose-tissue stromal cells via SAPK/JNK and stemness acting signals.
US20120003186A1 (en) Method for dedifferentiating adipose tissue stromal cells
CN108300686B (en) Small molecule compound/combination for preventing, delaying or reversing cell, tissue, organ and organism aging, product and application thereof
He et al. Transplantation of miRNA‐34a overexpressing adipose‐derived stem cell enhances rat nerve regeneration
Edamura et al. Recombinant canine basic fibroblast growth factor-induced differentiation of canine bone marrow mesenchymal stem cells into voltage-and glutamate-responsive neuron-like cells
WO2009157610A1 (en) Selenium dedifferentiated cell, preparation method and usage thereof
KR101211624B1 (en) Generation composition for induced pluripotent stem cells with Shh, FGFR tyrosine kinase inhibitor, MEK inhibitor and GSK inhibitor treatment and method of manufacturing induced pluripotent stem cells using the same
WO2009157562A1 (en) Method for establishment of adult pancreatic stem cell, and method for differentiation of adult pancreatic stem cell
KR20180117070A (en) A novel stem cell therapeutics
KR20200001756A (en) A method for inducing transdifferentiation of cardiomyocytes based on exosome
KR20180044760A (en) Methods for improving proliferation of stem cell using chenodeoxycholic acid
RU2575106C1 (en) Culture medium for rejuvenation of stem cells
WO2015098111A1 (en) METHOD FOR ESTABLISHING iPS CELLS AND METHOD FOR LONG-TERM MAINTENANCE OF STEM CELLS
Kim et al. Retracted: IFATS Collection: Selenium Induces Improvement of Stem Cell Behaviors in Human Adipose‐Tissue Stromal Cells via SAPK/JNK and Stemness Acting Signals
CN115052983A (en) Methods and kits for reprogramming somatic cells
WO2009157559A1 (en) Pancreatic cell regeneration/transplantation kit for pancreatic diseases or diabetes
CN115505573A (en) Genetically engineered mesenchymal stem cell with enhanced migration activity and application thereof in stem cell treatment and stem cell cosmetology
JP2015123079A (en) METHOD FOR ESTABLISHING iPS CELLS, AND METHOD FOR LONG-TERM MAINTENANCE OF STEM CELLS
KR20120096150A (en) Process for the differentiation of adipose-derived mesenchymal stem cells to chondrocytes and method for analyzing chondrogenic differentiation efficacy of mesenchymal stem cells
Henning Identification and characterisation of a novel, multi-potent, skeletal muscle-derived stem cell with broad developmental plasticity

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 08792968

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 08792968

Country of ref document: EP

Kind code of ref document: A1