TW200808967A - Method for de-differentiate a differentiated cell and method for re-programming differentiated cells - Google Patents

Abstract

The subject invention relates to a method for de-differentiate a differentiated cell comprising treating the differentiated cell with an undifferentiated cell extract. The invention also relates to a method for re-programming differentiated cells, which comprises de-differentiatinwdifferentiated cells with the afore-said method. The invention further relates to a composition for de-differentiating a differentiated cell.

Description

200808967 IX. Description of the invention: [Technical field to which the invention pertains] ' The present invention relates to a technique for cell reprogramming. In particular, the present invention relates to a method and composition for de-differentiation of differentiated cells. ^ [Prior Art] Mammalian fertilized eggs and early embryos have a differentiation of totipotency. During embryonic development, the differentiated cells of their derived cells have the ability to generate genomic genes during various tissues and organs. The phenomenon of epigenetic modification is gradually restricted, and finally specialized into somatic cells with special shapes and specific physiological functions. This gradual restriction of cell differentiation due to genomic specialization has now been shown to be reversed rather than an irreversible process. Somatic cell reprogramming or de-differentiation (16-differentiation) refers to a genetic modification that alters the nuclei of a differentiated somatic cell, such that it exhibits cellular or molecular characteristics of different cell lineages, or Restoration of pluripotency and even pluripotency of differentiation. 'The differentiation potential of embryonic stem cells, for the development of cell, tissue or organ transplantation. The regeneration medicine model has the potential to be established. Combining embryonic stem cell technology with somatic cells Therapeutic cloning developed by replication technology may produce tailor-made stem cells for medical use in autotransplantation. Currently using nuclear transfer or cell The method of cell fusion has the purpose of changing the performance of the genome. Nowadays, in the study of the body fine 105006.doc Ding 33134 1〇5〇〇6 005389664 200808967 Cell nuclear translocation and animal tanning, 75% of the research animals For the replication of species, 10% of the mice were studied, and the remaining 15% were targeted at about 20 species. Back (Oback and Wells, Trends Biotechnol. 2〇03; 21(10): 428-432). However, since the publication of the Taoli sheep, the success rate of animal reproduction has been around 3 to 5 ° / ' 'efficiency and Not high, and there are many developmental abnormalities in replicating animals (Chavatte-Palmer et al., Cloning Stem Cells· 2004; 6(2)··94_1 00); and the success rate of producing embryonic stem cell lines from blastocysts is also 10% or less (Chen et al., Cell Res·2〇〇3; 13(4):251-263) 'Shows that the use of nuclear translocation operations to re-emphasize the overall cellular genome, and even the development of medical replication is still greatly improved Furthermore, the replication of blastocysts by replication technology requires a large number of eggs, and the production of embryonic stem cells from replicating blastocysts or the isolation of germ cells from the primitive glands of early fetuses must destroy and sacrifice embryoid bodies in human applications. There is a moral controversy. Although adult stem cells isolated from autologous tissue have no moral controversy, 'however', due to the small number of adult stem cells in the body, the difficulty of separation and the difficulty of cultivation, there are still many practical applications. Difficulties must be overcome. On the other hand, from Among the results of nuclear fusion cells, it has been shown that fusion of differentiated pluripotent cells with differentiated cells can cause differentiated cells to regain pluripotency and allow heterogeneous nuclear fusion cells to behave like stem cells. The heterogeneous nuclear fusion cell itself is tetraploid (tetrapl〇id^s cell' and its genomic instability is not suitable for in vivo treatment of cell transplantation. SUMMARY OF THE INVENTION Summary of the Invention 105006.doc T33134 1〇5〇〇6 005389664 200808967 In addition to a method for successfully providing a large number of cells for obtaining cells, the present invention provides a method for producing undifferentiated cells, which does not have other undifferentiated cells. Inconvenient and moral controversy. Ben: Month: - The purpose is to provide an extract of undifferentiated cells from the differentiated cells to treat the differentiated cells. The purpose of this month is to provide a method of reforming differentiated cells, which is to dedifferentiate a differentiated cell according to the above method.

Further refinement of the invention - an object of the invention is to provide a composition for dedifferentiating differentiated cells, which comprises an extract of undifferentiated cells. DETAILED DESCRIPTION OF THE INVENTION The present invention is first directed to a method of dedifferentiating differentiated cells comprising treating the differentiated cells with an extract of undifferentiated cells.乙 The term “de-differentiation” in this article refers to the change of the gene expression of the differentiated somatic cell nuclei, so that it can restore the pluripotency and even the omnipotent differentiation ability. Means a cell that has been specialized into a particular shape and/or specific physiological function. According to the method of the present invention, preferably, the differentiated cell line is selected from the group consisting of a precursor cell of an incompletely differentiated adult cell and a fully differentiated somatic cell. As used herein, the term "undifferentiated cells" refers to cells that are not specialized in any particular shape or specific physiological function, especially cells that have pluripotency or even pluripotency, or according to the method of the present invention. Differentiation and return to the undifferentiated state. Suitable for the undifferentiated cells of the present invention are preferably (IV) a group of free oocytes, imprints, embryonic stem cells, adult stem cells and incompletely differentiated cells; more preferably, cells. According to the present invention, the cultured cells of the undifferentiated cell are cultured and differentiated. The extract of the undifferentiated cell can be taken from any spun yarn t cell. Preferably, it is prepared from rapidly differentiated cells in cell culture to achieve optimal results. According to the method of the present invention, the detachment cover 彳b έ^ ° is the condition of the chemical cell, and the cytoplasm and the replicator are exemplified, for example, the commercial cell culture solution can be regarded as Need to add the required medicine. Comparative: The undifferentiated cell line is a toxic oxidative metabolic alcohol produced by the original culture medium in the culture medium containing the reducing agent to help the growth of the reduced cells. In a specific embodiment of the present invention, the reducing agent is a chloroethyl sulphate (10) anol), and the k-based thiol can be used as a reducing agent to provide a basal group necessary for the formation of glutamic acid (gi gihi). It can also protect the cells of the enzymes and proteins from oxidation, thereby promoting the attachment of undifferentiated cells and the formation of cell clusters. Preferably, the undifferentiated cell line is scented in a culture medium comprising a acidity regulator. The acid test regulator can adjust the cell culture process in the cell culture process: the acidity change generated, so that the culture solution is not caused by cell metabolism, in the specific embodiment, the acid test regulator system It is sodium bicarbonate' to slow down the peracid phenomenon in the medium produced during cell culture. Preferably, the undifferentiated cell line is cultured in a culture medium containing a differentiation inhibitor. The differentiation inhibitor can inhibit the differentiation of undifferentiated cells in culture. 105006.doc Τ33134 1〇5〇〇6 005389664 200808967 1 发明= invention-specifically, the differentiation inhibitor is leukemia inhibitor (Leukemia inhibit) faetGr, UF), which suppresses

The message-transfer pathway of cell differentiation is maintained to maintain its undifferentiated properties. Knife Since the specific substance f contained in the undifferentiated cells is present in the cytoplasm, in a preferred embodiment, the extract of the undifferentiated cells takes the cytoplasm of the cells. The method for preparing the extract is a method for differentiating the cytoplasm of the cell, and preferably, the undifferentiated cell is obtained by disrupting the undifferentiated cell. In a preferred embodiment of the present invention, the undifferentiated cells are disrupted by cell disruption, ultrasonic shock I or reverse freezing to obtain the extract. 7 according to the method of the present invention, the conditions for culturing the differentiated cells are such that the cells are normal physiology and replication, and do not interfere with the dedifferentiation process, and the commercial cell culture solution can be used as needed. Add the required medicine. Preferably, the differentiated cell line is cultured in a culture medium comprising a reducing agent, an acid-base regulator or a sputum inhibitor, wherein the reducing agent, the acid-base regulator, and the differentiation inhibitor are as described above. According to the method of the present invention, the differentiated cells can be subjected to dedifferentiation treatment in any cell culture period; preferably, in the rapid growth phase of cell culture, the differentiated cells are treated with an extract of undifferentiated cells to achieve The best effect. In order to allow the undifferentiated cell extract to sufficiently treat the differentiated cells, preferably, the differentiated cells are treated with a cell membrane permeabilizing agent to make the cell membrane permeability before being treated with the undifferentiated cell extract. The increase is made so that the 105006.doc 200808967 undifferentiated cell extract can have an effect in the differentiated cells. The cell membrane permeabilizing agent according to the present invention is an agent for increasing the permeability of a cell membrane. In one embodiment of the present invention, the cell membrane permeabilizing agent is streptolysin-0 or digitalis. Digitonin or Leading peptide-fused protein. Streptococcal hemolysin 0 can temporarily perforate the cell membrane to temporarily cause pores in the cell membrane, so that the extract of the undifferentiated cells can enter the differentiated cells. On the other hand, after adding the cell membrane permeabilizing agent, it may be further treated with a cell membrane repairing agent to seal the pores produced by the cell membrane permeabilizing agent. In one embodiment of the present invention, the cell membrane repairing agent is calcium ion. . It has been reported that the process of dedifferentiation is a highly energy-consuming process (Novak et al., Biol Reprod. 2004; 71:1279-1289). In order for the dedifferentiation process to proceed smoothly, the method according to the invention preferably further comprises energy regeneration. The systemic promoter treats the differentiated cells. Wherein the energy regeneration system promoter is selected from the group consisting of ATP, GTP, CTP, GIT, creatine phosphate and creatine kinase, wherein ATP, GTP, CTP, GTP are intracellular High energy molecules that provide energy during dedifferentiation; phosphocreatine and creatine kinase act as batteries, helping ATP, GTP, CTP, and GTP high energy molecules reconnect to phosphate and return to high energy. use. The methylation of DNA molecules also affects the effect of dedifferentiation. In general, the more glycosylated DNA molecules, the worse the dedifferentiation effect. Therefore, the method according to the present invention preferably further comprises DNA demethylation. Treatment of the differentiated cells, a number of DNA demethylating agents have been developed (Selkwe, Proc. 105006.doc -11 - T33134 105006 005389664 200808967

Natl. Acad. Sci. USA 1998; 95: 9430-9435), in one embodiment of the invention, the DNA demethylating agent is selected from the group consisting of an acetylated protease inhibitor (trich 〇 Statin A, TSA) And the group consisting of %Aza. According to the method of the present invention, wherein the condition of the knifed, and the field cells is treated with the extract of the undifferentiated cells, the condition of the undifferentiated cells and the differentiated cells is succumbed and the 'differentiated cells are treated about 10 From minute to about 2 hours, preferably, the differentiated cells are treated with an extract of undifferentiated cells for about a day. On the other hand, the ratio of the undifferentiated cells contained in the extract of the undifferentiated cells to the differentiated cells is from 丨〇〇〇··1 to 1:1. As shown in the following examples, in accordance with an embodiment of the present invention, mouse and human adult fibroblasts are treated with different concentrations of cell membrane permeabilizing agents to increase their cell membrane permeability, treated cells. More than 9% of the permeability is increased, and 80% of the treated cells are still alive; then the treated mice and human adult fibroblasts are cultured in cell extracts of mouse embryonic stem cells. The results showed that the treated mice and human adult fibroblasts could observe that the cells gradually became larger and rounder from a small spindle-shaped phenotype after 48 hours of culture, and the proportion of the nucleus in the whole cell gradually increased. Performance cultures have a tendency to form cell communities (c〇l〇ny); while control untreated or cells treated only with Hanks' buffered saline solution (HBSS) still maintain a small spindle-like, cell-like list The phenotype of the monolayer indicates that these treatments can perform nuclear remodeling of mouse and human adult fibroblasts. In addition, the stem cell-specific cell markers were analyzed by tissue immunochemistry. It was also found that the treated human and mouse somatic cells can express the expression of SSEA-3/4 embryonic stem cell-specific markers; and then immediately 105006.doc -12- T33134 1 〇5〇〇6 005389664 200808967 Polymerase chain reaction analysis can also detect Oci-4, iV(10)og and i gene expression in treated human and mouse somatic cells. It was also positive by alkaline phosphatase test. It is shown that according to the method of the present invention, the differentiated, cell-derived cells can be dedifferentiated to exhibit an undifferentiated phenotype and genetic characteristics. According to the method of the present invention, the cell extract obtained from the undifferentiated cells is introduced into the dedifferentiation treatment by using the cell extract obtained from the undifferentiated cells by increasing the differentiation and the membrane permeability in vitro. Regaining differentiation, pluripotency and increasing differentiation plasticity, and in addition to providing a large amount of undifferentiated cells, the present invention also avoids the inconvenience and moral controversy of other currently undifferentiated cells. The undifferentiated cells prepared according to the method of the present invention have been successfully dedifferentiated by type, tissue immunochemistry and polymerase chain reaction detection. The present invention also provides a method of reforming differentiated cells by dedifferentiating a differentiated cell according to the above method. The invention further provides a composition for dedifferentiating differentiated cells, wherein φ comprises an extract of undifferentiated cells. The present invention is described in detail by the following examples, which are not intended to be construed as limited only. [Examples] Example 1: Cell membrane permeation test cells of differentiated cells · Mouse embryonic stem cells (ES_D3) used in the present examples and the following examples, mouse adult fibroblasts (mouse) Skin fibroblast cells, Durnii cells, Clone III8C) and human adult fibroblasts (human f〇reskin 105006.doc -13- T33134 105006 005389664 200808967 cells, HS68 cells) were purchased from the Bioresource Conservation and Research Center (Hsinchu, Taiwan) ). The method used in this embodiment is reference to Walev et al. (Proc Natl).

Acad Sci U S 2001· 2001 Mar 13;98(6)··3185-90) and H^kelien et al. (Nat Biotechnol· 2002 May; 20(5): 460-6) revealing mice

And human adult fibroblasts were treated with different concentrations (2〇, 3〇, 40, 5〇, 60 ui/mL) of streptolysin for different time 〇〇, 2〇, 3〇, 40 ' 50 ' 60 minutes) 'to find the best processing conditions (as shown in Figure 1). Preliminary results showed that the conditions of treatment with 30 iu/mL of streptolysin for 1 minute were most suitable, and more than 90% of the treated cells were increased in permeability to propidium iodine (PI, green). Fluorescent light was introduced into the nucleus, and the cells were cultured for 24 hours after re-sealing with CaCb, and then stained with SYBR (red fluorescence), and 80% of the treated cells were still alive (as shown in Fig. 2). W々'U _ Fertilizer I cloud differentiation treatment Culture of non-knife cells: The culture of mouse stem cells is shown in the following table! In the culture solution, the ST 〇 cells are used as a feeding layer (the culture solution is as described in Table 2 below) to provide a substrate for stem cell attachment, and the sputum is used to maintain the undifferentiated stem cells. And in the sound of stem cell culture, the day, treated with 0·1 mL of mitomystamine (nntomycm) for three hours to produce ^~^ to make (the nourishing cells stop dividing, avoiding the growth space of stem cells and consuming too much nutrients. 10$006 .doc T33134 105006 005389664 -14- 200808967 Table i

Ingredient model concentration range DMEM Gibco®11965-092 Fetal bovine serum Gibco®16000-044 10%-20% L-prolinamide (L- Gibco® G7513 plus or minus 0· ImM glutamine) β-ethylthiol Sigma® M6250 0. lmM-0.2mM sodium bicarbonate Sigma®S5761 1.3g-1.7g/L glucose Sigma®G7528 2g-5g leukemia inhibitor Chemicon® ESGRO-1107 500-1000 iu/mL MEM non-essential amino acid M7145 0.1 -0.2 mM sodium pyruvate Sigma® P2256 0.5-lmM (Sodium pyruvate) Penicillin/Glycoside Gibco® 15140-122 P: 50-150 unit/mL S: 0.05-0.15 mg/mL Table 2:

Ingredient Model Concentration Range DMEM Gibco® 11965-092 Fetal Bovine Serum Gibco® 16000-044 5%-15°/〇Pencillin/Streptomycin Gibco® 15140-122 P: 50-150 unit/mL S: 0.05-0.15 mg/ mL mitomycin M0503 0.1 -0.2/mL

When the mouse stem cells were grown to 5 〇 to 15 0 μηη, treated with 0.01% type IV collagenase-EDTA (collagenase IV-EDTA) for two hours, tapping the edge of the culture dish to make the stem cell mass and the underlying support layer STO The cells are separated. I05006.doc -15- T33134 1〇5〇〇6 005389664 200808967 Undifferentiated cell extract: The stem cells obtained by the above culture were disrupted by ultrasonic waves to obtain a mouse embryo stem cell extract. Culture of differentiated cells: The mouse adult adult fibroblast cell line was cultured in the culture solution described in Table 3 below, and human foreskin adult fibroblasts were cultured in the culture solution of Table 4 below. table 3 ··

Maccoy* 5A fetal bovine serum penicillin/keyin

Concentration range

Sigma®8712154 M8403

Gibco® 16000-044 ----------

Gibc〇 (D15! 40-122 5%-15%

P: 50-150 unit/mL S. 0.05-0.15 mg/mL

Table 4: Ingredient DMEM Model Gibco® 11965-092 Concentration range Fetal bovine serum Gibco® 16000-044 5%-15% Penicillin/Gibco 15140-122 P* 50-150 unit/mL S: 0.05-0.15 mg/ mL or human foreskin adult fibroblasts are treated with 3 〇iu/mL of streptococci hemolysis. After ο 分钟 minutes, ❶ dedifferentiation treatment: the tail of the mouse is treated with cell membrane permeabilizing agent The cells or human foreskin adult fibroblasts were cultured in the culture medium described in Table 1, and the mouse embryo stem cell extract prepared as described above and the additives described in Table 5 below were added to culture at 2 to 20 mM. CaCl2 sealed culture 0 105006.doc T33134 1〇5〇〇6 005389664 200808967 Table 5 ··

Ingredient Model Concentration Range ATP Sigma® A33 77 l-10mM GTP Sigma®G8877 0.1-lmM Mixed NTP In vitrogene® 18109-017 1 -1 OmM Phosphocreatine Fluka®27920 10-100mM Phosphoinositide Sigma® C3755 25mM -50 mM Example 3 · Evaluation of dedifferentiation effect: After 48 hours of incubation in Example 2, it was observed that the cells gradually became larger and smaller from the small spindle-shaped phenotype (Fig. 3), and the proportion of the nucleus in the whole cell gradually increased. Increased, and continued to culture until the fourth day, there is a tendency to form cell communities (Figure 4). The control untreated or blank treated cells only with HBSS still maintained a small spindle-like phenotype of cell monolayer. Gene expression: Further, after the cell reformation, the mouse tail adult fibroblasts and adult foreskin adult fibroblasts were divided into mRNA (using PolyATract System 1000, Promega®,

Madison, WI) for the analysis of 〇Cr- by real-time polymerase chain reaction (LightCycler FastStart DNA Master SYBR Green I, Roche®, Indianapolis, IN, and using ABI Prism® 7900HT Light Cycler, ABI®, Lincoln Centre DriveFoster, CA) 4. ΛΓα(10)g and three undifferentiated cell marker genes of ECdM-1. The results are shown in Figures 5 to 7. It can be seen that the dedifferentiated cells restored the ability to express 0 (: 7 ^ 4, 7 \ ^ / 7 〇 与 户 户 ( ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^. Histochemical analysis: The cells after dedifferentiation were fixed and reacted with antibodies, and then reacted with a secondary antibody linked to FITC, followed by fluorescence 105006.doc -17· T33134 1〇5〇〇6 005389664 200808967 Microscopic observation showed that the dedifferentiated cells could express three germ layer-specific markers such as 68 (ectoderm), BMp_4 (mesoderm) and cytokeratin (Cyt〇keratin, endoderm). Enzyme: the cells after dedifferentiation are fixed and (5)

Blue RR Salt and Naphthol AS-MX ph〇sphate (Sigma®) were tested for ft h酉夂 enzyme activity. The results are shown in Fig. 8. It is known that the positive nitrite is positive.

In summary, whether from the cell phenotype, gene expression and test (4) acid enzyme activity f, see also the mouse tail adult fibroblasts and adult foreskin adult fibroblasts and other somatic cells, can be made The co-culture of mouse embryonic stem cell extracts is dedifferentiated to restore stem cell characteristics. The above-described embodiments are merely illustrative of the nature of the invention, and are not intended to limit the invention. Modifications and variations of the embodiments described above will be apparent to those skilled in the art without departing from the spirit of the invention. The scope of the invention should be as set forth in the scope of the patent application described hereinafter. BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 is a graph showing the ratio of time to permeability of streptococcal hemolysin treatment in the cell membrane permeability test of Example 1, wherein the triangular symbol line represents the survival rate; the square line represents the permeability. Fig. 2 is a view showing the fluorescence permeation microscope of the cell membrane permeability experiment of Example 1. Figure 3 shows the adult fibroblasts in the tail of the mouse after two days of cell dedifferentiation treatment; b: adult foreskin adult fibroblasts; and the tail of the mice that have not been subjected to cell reformation The cell type of fibroblasts. Figure 4 is a day after cell dedifferentiation treatment: a mouse tail adult fiber formation 105006.doc T33134 1〇5〇〇6 005389664 -18· 200808967 dimensional cells; b: adult foreskin adult fibroblasts, A community pattern that resembles embryonic stem cells is formed after treatment. Figure 5 shows the expression of OCT-4 in mouse tail adult fibroblasts (rDunni) and adult foreskin adult fibroblasts (rHS-68) 4 days after cell dedifferentiation treatment, wherein D3 represents mouse embryonic stem cells. . Fig. 6 shows the expression of mouse tail adult fibroblasts (rDunni) and adult foreskin adult fibroblasts (rHS-68) 4 days after cell dedifferentiation treatment, wherein D3 represents mouse embryonic stem cells. Figure 7 shows the expression of cadaveric five CMM in mouse tail adult adult fibroblasts (rDunni) and adult foreskin adult fibroblasts (rHS-68) 4 days after cell dedifferentiation treatment, wherein D3 represents mouse embryo stem cell. Fig. 8 is a graph showing the results of an activity test of alkaline luciferase after dedifferentiation of mouse tail adult fibroblasts (rDunni). 105006.doc .19- T33134 1〇5〇〇6 005389664

Claims (1)

200808967 X. Patent Application Range: [A method for dedifferentiating differentiated cells, which comprises treating the differentiated cells with an extract of undifferentiated cells. According to the method of the monthly term, wherein the differentiated cell line is selected from the group consisting of a precursor cell which is fully differentiated by an adult cell and a body cell which has been completely differentiated. 3 According to the request, j member 1 $ f, , soil, and sputum, wherein the undifferentiated cell line is selected from the group consisting of "'17, embryonic stem cells, adult stem cells, and incompletely differentiated precursor cells. 4. According to claim 3 $ ^• , ^ ^ ^ cell., /, the undifferentiated cell line is adult dry fine 5 · According to the request item 夕古, 土, 法,八中, the treatment is to differentiate the undifferentiated cell extract Cell 6. Stroke 6 · According to the request, the extract of the undifferentiated cells of the soil, ',,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,,, The undifferentiated cell line of the item 1 , the car φ , and the zhongzhong is obtained by culturing the unrestricted cell J t broth. 8. According to the method of the request, the 未 ~ ~ ^ , Τ δ 亥 未 undifferentiated cells It is obtained by culturing in a culture solution containing an acid-base modulating agent. The method of claim 苴, 苴 Λ Λ δ δ δ δ δ δ δ δ δ δ δ δ δ δ δ δ δ δ δ δ δ δ δ δ δ δ δ δ δ δ δ δ δ δ 根据 根据 根据 根据 根据 根据 根据 根据 根据 根据 根据The extract of undifferentiated cells is the cytoplasm of each differentiated cell. U 11 · According to the method of claim, the extract of 苴 Μ / / knife-shaped cells will be 105006.doc Τ33234 1〇5〇〇6 005389664 - I 200808967 The undifferentiated cells are broken. 12. According to the request item, the cell is broken, the ultrasonic wave is oscillated or the cold is repeated, and the undifferentiated cells are broken by the East. 13 · According to the request item 1 ', wherein the differentiated cell line is cultured in a culture medium containing a reducing agent. 14 · According to claim 1, 'where the differentiated cell line is cultured in a culture medium containing a test regulator. 1 5 According to the request j 苜 夕 夕 , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , 'Unsupplied The differentiated cells are treated with the extract of the cells. According to the method of spray application, wherein the treated cells are treated with the undifferentiated cells, the differentiated cells are first treated with a cell membrane permeabilizing agent. The method of item 17, wherein the cell membrane permeabilizing agent is Strept〇lysin-0, Digitonin or Leading peptide-fused protein. 19. The method according to claim 5, further comprising treating the differentiated cells with an energy regeneration system enhancer. 20. The method of claim 19, wherein the energy regeneration system promoter is selected from the group consisting of ATP, GTP, CTP, GTP, creatine phosphate, and creatine kinase. 21. The method of claim 1, further comprising treating the differentiated cells with a DNA depurinating agent. The method according to claim 21, wherein the DNA demethylating agent is selected from the group consisting of B.105006.doc T33134 1〇5〇〇6 005389664 200808967 tric 蛋白酶 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 Group. 23. The method according to claim 5, further comprising treating the differentiated cells with a cell membrane repair agent. 24. The method according to claim 23, wherein the cell membrane repairing agent is a calcium ion. 25. The method of claim 1, wherein the differentiated cells are treated with an extract of undifferentiated cells for about 1 minute to about 2 hours. 26. The method of claim 25, wherein the towel is treated with the extract of undifferentiated cells for about one hour. 27. The method according to claim 5, wherein the ratio of the undifferentiated cells of the undifferentiated cells + the differentiated cells to the differentiated cells is from 1000··1 to i: 28·-reformed differentiated cells The method comprises dedifferentiating the differentiated cells according to the method of the claim item. 29. A composition for dedifferentiating differentiated cells comprising an extract of undifferentiated cells. 30. The composition of claim 29, wherein the extract of the undifferentiated cells is prepared from undifferentiated cells of the cell culture lip that are rapidly growing. The composition according to claim 29, wherein the undifferentiated cell line is cultured in a culture solution containing a reducing agent. The medium of the alkali regulator is cultured to obtain a composition according to claim 29, wherein the undifferentiated cell line comprises an acid 33. The composition according to claim 29, wherein the undifferentiated cell line comprises the fraction 105006 .doc 200808967 Cultured in a culture medium of inhibitors. Extract system 34. The composition according to claim 29, wherein the undifferentiated cells comprise cytoplasm of undifferentiated cells. 35. The composition according to claim 29, wherein the extract of the undifferentiated cells of the spleen is damaged by the undifferentiated cells. 〒 initial system 36. According to the request item, county + composition, which is composed of fine 朐谕 篇 冷 cold / East material broke undifferentiated cells. shock 105006.doc t33134 1〇5〇〇6 Γ\Λγ*#\ O