Method for reprogramming cells

Abstract

The present invention relates to a method for producing reprogrammed cells from somatic cells without gene introduction. The method comprises: (a) a step of culturing somatic cells in a culture medium containing a histone deacetylase inhibitor; and (b) a step for producing a reprogrammed cell by culturing the cell cultured in step (a) in a medium containing an OCT3 / 4 transcription stimulating factor.

Description

[0001] Related Applications

[0002] This application is a divisional application of an application with an application date of November 12, 2020, application number 202080093262.0, and invention name “Cell Reprogramming Method”, and claims priority based on Japanese patent application 2019-206270 (filed on November 14, 2019), and its contents are incorporated into this specification as a reference. Technical Field

[0003] The present invention relates to a method for reprogramming cells, and more particularly to a method for reprogramming cells by using chemical substances without introducing genes from the outside. Background Art

[0004] Since embryonic stem cells (ES cells) were established from mouse embryos in 1981 (Non-patent Document 1), ES cells have been widely used as a material for tissue regeneration research. Pluripotent stem cells such as ES cells are characterized by having differentiation pluripotency, and it is believed that various tissues can be regenerated by utilizing this property. Specifically, there is an expectation for the treatment of neurodegenerative diseases such as Parkinson's disease, spinal cord injury, cerebral infarction, diabetes, cirrhosis, cardiomyopathy and other diseases that were previously difficult to cure.

[0005] However, the transplantation of ES cells or tissues regenerated from ES cells is allogeneic transplantation, so there is the problem of rejection after transplantation, just like organ transplantation. In addition, the establishment of ES cells requires fertilized eggs or early embryos at the stage from fertilized eggs to blastocysts. In the case of humans, using human fertilized eggs as materials will destroy the buds of life, which has also been pointed out as an ethical problem.

[0006] On the other hand, in 2006, the production technology of epoch-making artificial pluripotent stem cell (iPS cell) was proposed by people such as Shan Zhong, and the technology is to set up pluripotent stem cell (non-patent literature 2) by introducing 4 kinds of genes (Oct3 / 4 gene, Sox gene, c-Myc gene and Klf gene) in somatic cell. In iPS cell, because of utilizing somatic cell, autologous transplantation can be achieved, and there is no need to destroy the embryo that is regarded as the problem on ethics, so the problem point of ES cell is solved. Therefore, it is a well-known fact that regenerative medicine technology is rapidly advanced by this iPS cell. In 2007, human iPS cell (non-patent literature 3,4) was also successfully established, and about iPS cell, a large number of reports have been available so far.

[0007] Now it can be seen that the method of establishing iPS cells is making great progress compared to when it was first discovered. Since the initial report, the retrovirus used for gene introduction in the early stage was considered to have the problem of the possibility of causing cancer due to insertion mutations in the somatic cell genome, but now the Sendai virus belonging to RNA virus is being developed as a representative, and methods such as methods that do not introduce mutations in the somatic cell genome using free vectors or synthetic mRNA are being developed (non-patent literature 5-8). In addition, various improvements have been made on the types of genes introduced, such as the development of methods that do not require genes other than Oct3 / 4 genes and Sox genes (non-patent literature 9).

[0008] In addition to iPS cells, multipotent / pluripotent stem cells called Muse cells have been proposed (Patent Document 1). Muse cells are pluripotent stem cells that do not have tumors present in biological tissues such as bone marrow or skin, and are characterized by being SSEA-3 (stage specific embryonic antigen-3) positive. Regarding Muse cells, it has been reported that they can differentiate into a variety of cells in all three germ layers, such as hepatocytes, muscles, nerves, glial cells, skin pigment cells (melanocytes), epidermis, and blood vessels. In this way, pluripotent stem cells in regenerative medicine have attracted much attention, and higher technologies for making pluripotent stem cells are sought to be established.

[0009] Prior art literature

[0010] Patent Literature

[0011] Patent Document 1: International Publication No. 2012 / 133948

[0012] Non-patent literature

[0013] Non-patent document 1: Martin GR, Proc Natl Acad Sci US A. 1981 Dec; 78(12): 7634-8.

[0014] Non-patent document 2: Takahashi K et al., Cell. 2006Aug 25; 126(4): 663-76.

[0015] Non-patent document 3: Takahashi K et al., Cell. 2007 Nov 30; 131(5): 861-72.

[0016] Non-patent document 4: Yu J et al., Science. 2007 Dec 21; 318(5858): 1917-20.

[0017] Non-patent document 5: Okita K et al., Nat Methods. 2011 May; 8(5): 409-12.

[0018] Non-patent document 6: Agu CA et al., Stem Cell Reports. 2015 Oct 13; 5(4): 660-71.

[0019] Non-patent document 7: Warren L et al., Cell Stem Cell. 2010 Nov 5; 7(5): 618-30.

[0020] Non-patent document 8: Yakubov E et al., Biochem Biophys Res Commun. 2010 Mar 26; 394(1): 189-93.

[0021] Non-patent literature 9: Shi Y et al., Cell Stem Cell. 2008 Jun 5; 2(6): 525-8. Summary of the invention

[0022] Problems to be solved by the invention

[0023] When making multipotent (multipotent / pluripotent) stem cells, from the viewpoint of clinical application, it is extremely important to use somatic cells as raw materials and not to perform artificial gene manipulation. Although the possibility, cost, production efficiency and production period of genetic modification have been gradually improved so far, further improvement is still a topic required in the future. In addition, in the present invention, as a part of the production technology of pluripotent stem cells, the purpose is to provide a method for reprogramming cells, more specifically, the purpose is to provide a method for reprogramming cells without performing an introduction operation of an artificial gene from outside.

[0024] Technical solutions to problems

[0025] As a result of in-depth research conducted by the inventors of this case to solve the above-mentioned problems, it was found that if somatic cells were cultured in a medium containing a DNA dysfunction substance, i.e., a histone deacetylase inhibitor (e.g., 2-mercaptoethanol), and then cultured in a medium containing a transcriptional stimulator of OCT3 / 4 (e.g., LIF, CCL2, and IL-6), reprogramming would be performed in the somatic cells. The inventors of this case completed the present invention based on the above-mentioned findings.

[0026] The present invention is preferably carried out according to the embodiments described below, but is not limited thereto.

[0027] [1] A method for producing reprogrammed cells from somatic cells without gene introduction, comprising:

[0028] (a) a step of culturing somatic cells in a medium containing a histone deacetylase inhibitor; and

[0029] (b) A step of preparing reprogrammed cells by culturing the cells cultured in step (a) in a medium containing an OCT3 / 4 transcription stimulating factor.

[0030] [2] The method according to [1], characterized in that the OCT3 / 4 transcriptional stimulator is selected from any one or two or more of the group consisting of LIF, CCL2 and IL-6.

[0031] [3] The method according to [1] or [2], characterized in that the histone deacetylase inhibitor is 2-mercaptoethanol.

[0032] [4] A method for producing reprogrammed cells from somatic cells without gene introduction, comprising:

[0033] (a) a step of culturing somatic cells in a medium containing 2-mercaptoethanol; and

[0034] (b) A step of preparing reprogrammed cells by culturing the cells cultured in step (a) in a medium containing LIF.

[0035] Preferably, the culturing time of step (a) is 3 days or more, more preferably 3-7 days, and the culturing time of step (b) is preferably 3 days or more, more preferably 3-9 days. It is desirable to carry out cell culturing until spheroid formation is confirmed.

[0036] [5] The method according to any one of [1] to [4], characterized in that the culture medium of step (a) contains 10 μM-0.2 mM, preferably 10 μM-100 μM, and more preferably 10 μM-50 μM 2-mercaptoethanol.

[0037] [6] The method according to [4] or [5], characterized in that the culture medium in step (b) further contains LIF.

[0038] Preferably, the culture medium of step (b) contains 1-100 ng / mL LIF (with a titer of 100-5000 units / ml).

[0039] [7] The method according to any one of [1] to [6], characterized in that the culture medium of step (b) further contains one or more selected from ACTH, bFGF and GSK3β inhibitors.

[0040] [8] The method according to any one of [1] to [7], wherein the culture medium in step (a) does not contain an OCT3 / 4 transcription stimulator. Preferably, the culture medium in step (a) does not contain cytokines and hormones.

[0041] [9] The method according to any one of [1] to [8], wherein the reprogrammed cells are pluripotent stem cells that can differentiate into three germ layers. The pluripotent stem cells may also be pluripotent.

[0042]

[10] The method according to any one of [1] to [8], wherein the somatic cells are fibroblasts, preferably skin fibroblasts. In step (a), the cells are preferably cultured until morphological changes of the fibroblasts are confirmed.

[0043]

[11] The method according to any one of [1] to [8], wherein the somatic cells are human fibroblasts, preferably human skin fibroblasts. The method according to [9] or

[10] is performed.

[0044]

[12] A kit for reprogramming somatic cells, comprising the following A and a reagent B:

[0045] Reagent A) a reagent for constituting a culture medium not containing 2-mercaptoethanol;

[0046] Reagent B) Reagents for constituting a culture medium containing LIF, CCL2 and IL-6.

[0047] Reagent A and reagent B may be prepared when used by packaging the culture medium and components separately, or may be in a state where each component is already contained in the culture medium. In addition, the culture medium composed of reagent A does not contain OCT3 / 4 transcription stimulators (LIF, CCL2, IL-6).

[0048]

[13] The kit according to

[12] , characterized in that reagent B further comprises one or more components selected from ACTH, bFGF and GSK3β inhibitors.

[0049]

[14] A method for producing target cells from somatic cells, comprising:

[0050] (i) a step of producing reprogrammed cells from somatic cells by the method according to any one of [1] to

[11] ; and

[0051] (ii) A step of inducing differentiation of the reprogrammed cells into target cells.

[0052]

[15] A method for producing a cell preparation, comprising:

[0053] (i) a step of producing reprogrammed cells from somatic cells by the method according to any one of [1] to

[11] ;

[0054] (ii) a step of inducing differentiation of the reprogrammed cells into target cells; and

[0055] (iii) a step of preparing a cell preparation comprising the differentiation-induced cells and a pharmacologically acceptable carrier.

[0056]

[16] A cell induced from a somatic cell by the method according to

[14] .

[0057]

[17] A cell preparation prepared by the method according to

[15] , comprising cells and a pharmacologically acceptable carrier.

[0058] Effects of the Invention

[0059] According to the present invention, a method for reprogramming cells without introducing an artificial gene from the outside can be provided. The method provided by the present invention can be used to make pluripotent (multipotent / pluripotent) stem cells. In the method of the present invention, since there is no need to introduce an artificial gene from the outside, the risk of immunogenicity or carcinogenicity risks that may affect somatic cells can be suppressed to a very low level. In addition, according to the method of the present invention, since only HDAC inhibitors are used when culturing cells, cell reprogramming can be performed simply and stably, and further, regarding the reprogramming of cells and the production of pluripotent stem cells, the implementation cost can be suppressed. From these viewpoints, by utilizing the method of the present invention, the research on pluripotent stem cells becomes easier, and a great contribution can be made to the research and development of tissue regeneration technology in the future. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] Figure 1 The figure shows the cell morphology of human skin fibroblasts after culture. Figure 1 A shows the morphology of human skin fibroblasts in a normal state after culture (low cell density). Figure 1 B is a diagram showing a state in which the cell density of human skin fibroblasts is high after culture.

[0061] Figure 2 The figure shows spheroids formed by culture of human skin fibroblasts. Figure 2 A is a low-magnification photo of the spheroids formed by cultured human skin fibroblasts. Figure 2 B is a further enlarged photograph of the spheroid.

[0062] Figure 3 It is a graph showing the alkaline phosphatase reaction of cultured cells.

[0063] Figure 4 It is a figure showing the results of PCR experiments to examine the expression of undifferentiation markers of Nanog, Klf4, Oct4 and Sox2. The predetermined sizes of Nanog, Klf4, Oct4 and Sox2 are 406pb, 396bp, 143bp and 150bp, respectively. Electrophoresis lane 1 shows a human skin fibroblast sample not treated with 2-mercaptoethanol, electrophoresis lane 2 shows a human skin fibroblast sample cultured in an incubator that has not been treated with 2-mercaptoethanol and has been subjected to step (a), electrophoresis lane 3 shows a sample obtained by treating human skin fibroblasts with 2-mercaptoethanol to reprogram them, and electrophoresis lane 4 shows a negative control sample in which water is added to replace RNA.

[0064] Figure 5 It is a figure which shows the result of differentiation into adipocytes. Figure 5 A is a magnified photo of the cells after differentiation into adipocytes. Figure 5 B is a photograph of differentiated adipocytes after fluorescent staining.

[0065] Figure 6 This is a diagram showing the results of differentiation into neural cells. Figure 6 AC is a photo of cells after differentiation into neurons. Figure 6 D is a photograph of differentiated neural cells immunostained with an antibody (anti-neurofilament antibody).

[0066] Figure 7 The figure shows the result of differentiation into hepatocytes. Figure 7 A shows the PAS staining of differentiated hepatocytes. Figure 7 B shows that differentiated hepatocytes produce albumin.

[0067] Figure 8 This is a diagram showing the results for differentiated hepatocytes. Figure 8 A shows the uptake of ICG (indocyanine green) in differentiated hepatocytes. Figure 8 B shows that the differentiated hepatocytes present bile duct-like structures and ICG accumulates in the bile duct-like structures. Figure 8 C is a photo taken 1 hour after ICG ingestion. Figure 8 D shows that ICG almost disappeared after 20 hours.

[0068] Fig. 9 The figure shows the result of differentiation into chondrocytes. Fig. 9 A is a photograph of differentiated chondrocytes. Fig. 9 B is a HE-stained photograph of differentiated chondrocytes. Fig. 9 C is Fig. 9 Enlarged photograph of B. Fig. 9 D is a photograph of differentiated chondrocytes stained with Alcian blue.

[0069] Fig.10 This is a diagram showing the results regarding differentiated chondrocytes. Fig.10 A is a magnified photo of differentiated chondrocytes. Fig.10 B is a photograph showing the fibrocartilage of the human articular labrum. Fig.10 A and Fig.10 B shows that the morphology of differentiated chondrocytes and fibrocartilage of human articular lip is pathologically similar to each other. Fig.10 C shows partial ossification in differentiated chondrocytes. Fig.10 D is a photograph of the bone tissue of a human bone. Fig.10 C and Fig.10 D shows the ossified part of differentiated chondrocytes and the morphology of human bone tissue in pathology.

[0070] Fig.11 The figure shows the results of staining differentiated chondrocytes and chondrocytes in human clinical specimens with various antibodies or chemical solutions. Differentiated chondrocytes in human clinical specimens closely resemble fibrocartilage in terms of their biological characteristics.

[0071] Fig.12 The figure shows the results of alkaline phosphatase staining of differentiated osteoblasts. Fig.12 A shows the control group (skin fibroblasts), Fig.12 B shows cells that have been induced to differentiate into osteoblasts after reprogramming. The differentiated cells are alkaline phosphatase positive.

[0072] Fig.13 The figure shows the cell morphology of human skin fibroblasts cultured in a D-MEM medium containing 2-mercaptoethanol and then in a medium containing LIF / CCL2. Fig.13 A shows the usual fibroblasts. Fig.13 B shows fibroblasts when only LIF was used. Fig.13 C shows fibroblasts when LIF+CCL2 (10 ng / mL) was used. Fig.13 D shows fibroblasts when LIF+CCL2 (100 ng / mL) was used.

[0073] Fig.14 Demonstrates induction of differentiation into adipocytes. Fig.14A shows the cells after culturing in D-MEM medium containing 2-mercaptoethanol for 7 days. Fig.14 B shows the cells after culturing in medium containing LIF + CCL2 (10 ng / mL) for 7 days. Fig.14 C of Fig.14 D shows the cells on the 25th day after induction of differentiation into adipocytes (4 times and 20 times, respectively).

[0074] Fig.15 Shows the induction of differentiation into adipocytes. Fig.15 A shows the cells after culturing in D-MEM medium containing 2-mercaptoethanol for 7 days. Fig.15 B shows the cells after culturing in medium containing LIF + CCL2 (100 ng / mL) for 7 days. Fig.15 C of Fig.15 D shows the cells on the 25th day after induction of differentiation into adipocytes (4 times and 20 times, respectively). Detailed implementation mode

[0075] One embodiment of the present invention is a method for reprogramming cells, which includes: the step of culturing cells in a medium containing a histone deacetylase inhibitor (for example, 2-mercaptoethanol); and the step of culturing in a medium containing an OCT3 / 4 transcriptional stimulator (for example, LIF, CCL2, and IL-6).

[0076] In this specification, reprogramming refers to reverting differentiated cells back to multipotent / pluripotent stem cells again. The reprogramming of cells is also called the initialization of cells. Biologically, reprogramming means eliminating or reconstituting epigenetic marks such as DNA methylation. The iPS cell production technology that introduces initialization factors into somatic cells is a representative example of cell reprogramming. In the present invention, instead of gene introduction, reprogramming is carried out using a DNA-disrupting substance, that is, an HDAC inhibitor. In addition, in this specification, the step of culturing cells in a medium containing a DNA-disrupting substance (HDAC inhibitor) is hereinafter referred to as step (a).

[0077] <DNA-disrupting substance>

[0078] A DNA-disrupting substance means a substance that can bind to DNA (deoxyribonucleic acid) or act directly or indirectly on DNA to hinder any one of the functions of DNA, namely DNA synthesis, DNA transcription (RNA synthesis), and DNA replication.

[0079] <Histone deacetylase inhibitor>

[0080] In the present invention, histone deacetylase inhibitors are used as DNA dysfunction substances. Histone deacetylase (HDAC: Histone Deacetylase) is an enzyme that deacetylates histones, which are the main components of the chromatin structure. HDAC inhibitors control gene transcription by inhibiting this HDAC. More specifically, in cells in a differentiated state, histones exist in an aggregated state, but HDAC inhibitors relax the aggregated chromatin structure to allow the transcription of the stopped initialization-related genes to start transcription.

[0081] Examples of the HDAC inhibitor used in the present invention include 2-mercaptoethanol, ethylene oxide, butyric acid, Apicidin histone deacetylase inhibitor, valproic acid, trichostatin A, and vorinostat.

[0082] 2-Mercaptoethanol is a compound represented by the structural formula HS-CH2-CH2-OH, and is also called β-mercaptoethanol or thioethylene glycol. The CAS number of 2-mercaptoethanol is 60-24-2. 2-Mercaptoethanol can be synthesized by reacting hydrogen sulfide and ethylene oxide, and preferably, an industrially produced commercial product can be used. For example, reagents or products of Fujifilm Wako Pure Chemical Industries, Ltd. can be used.

[0083] Ethylene oxide is a compound represented by the molecular formula C2H4O, and is also called epoxyethane, oxirane, propylene oxide, ethylene oxide, and EO for short. In addition, the CAS number of ethylene oxide is 75-21-8. Ethylene oxide can be synthesized by reacting ethylene and oxygen, and preferably, commercial products produced industrially can be used. For example, products of Mitsubishi Chemical, SHOWADENKO GAS PRODUCTS CO.LTD., Taiyo Nippon Sanso, etc. can be used. When using ethylene oxide, it can be contained in the culture medium of the cell, but it is preferred to blow ethylene oxide gas to the culture vessel (culture container, etc.) of the cell.

[0084] In the present invention, not only the above-mentioned 2-mercaptoethanol and ethylene oxide but also other HDAC inhibitors can be used in the same manner as these compounds, but 2-mercaptoethanol is preferably used.

[0085] <Cell>

[0086] The cells cultured in the method of the present invention are preferably somatic cells. The somatic cells in the present invention are not particularly limited as long as they are differentiated cells, and any cell or cell group other than germ cells can be used. As its type, for example, fibroblasts, adipocytes, nerve cells, muscle cells (cardiac muscle cells, smooth muscle cells, skeletal muscle cells, etc.), skin cells, epithelial cells, endothelial cells, blood cells (neutrophils, eosinophils, basophils, monocytes, lymphocytes, etc.), liver cells, kidney cells, lung cells, pancreatic cells, hair matrix cells (skin hair matrix cells, body hair matrix cells, etc.), oral cells (oral mucosal cells, etc.) and other differentiated cells and hematopoietic stem cells, mesenchymal stem cells, neural stem cells, adult stem cells such as stem cells from fat, or various precursor cells, etc., but are not limited to these. In the present invention, from the viewpoint of the simplicity of obtaining cells, preferably fibroblasts (more preferably skin fibroblasts), oral cells and hair matrix cells, more preferably fibroblasts, particularly preferably skin fibroblasts.

[0087] Somatic cells can be collected from mammals, birds and other animals. As mammals (mammals), primates such as humans, monkeys, chimpanzees, gorillas, orangutans, rodents such as mice and rats, rabbits, dogs, cats, cows, pigs, goats, sheep, horses, etc., as birds, for example, chickens, ducks, etc., but are not limited to these. Somatic cells can be somatic cells in the fetal period, or they can be mature somatic cells. In addition, somatic cells can be primary culture cells or passage cells. When the obtained reprogrammed cells or cells or tissues differentiated therefrom are transplanted, it is preferred to use somatic cells collected from the animal as the transplant object (that is, the somatic cells of the animal itself) or somatic cells collected from animals of the same species as the animal. In addition, when the transplant involves the treatment of a disease, it is preferably somatic cells of tissues involved in the disease.

[0088] <Cell Culture>

[0089] The present invention includes a step of culturing cells in a medium containing an HDAC inhibitor, and by at least performing this step, the cells can be reprogrammed. The cells can be cultured in a state of contact with a medium containing an HDAC inhibitor, preferably in a medium containing an HDAC inhibitor.

[0090] The content of the HDAC inhibitor in the culture medium is not particularly limited. For example, it can be expressed as an amount such that the ratio of the number of cells after 1 week of initial culture to the number of cells when cultured for 1 week without the HDAC inhibitor (cultured for 1 week under the same conditions except without the HDAC inhibitor) is 70% or more (preferably 80% or more, more preferably 90% or more, further preferably 95% or more). Also, it can be expressed as an amount such that a part of the cells after 1 week of initial culture exhibits a cell aggregate-like morphology (spheroid). This content can be appropriately set according to the type of HDAC inhibitor, culture conditions, etc., but for example, it is 0.01 μM - 50 mM, preferably 0.1 μM - 10 mM, more preferably 0.5 μM - 7 mM, further preferably 1 μM - 5 mM.

[0091] When the HDAC inhibitor is 2-mercaptoethanol, its content in the culture medium is, for example, 0.1 μM - 10 mM, preferably 0.1 μM - 2 mM, more preferably 0.1 μM - 5 mM, and more preferably 10 μM - 3 mM. Considering the influence on cells, within the range where reprogramming can be carried out, the amount of 2-mercaptoethanol is preferably less. From this point of view, 2-mercaptoethanol can be 10 μM - 0.2 mM, or 10 μM - 100 μM, 10 μM - 50 μM. In addition, since the concentration of 2-mercaptoethanol usually used for culturing stem cells is 100 μM, it can be said that the reprogramming of the present invention can be carried out with 2-mercaptoethanol at a concentration much lower than this.

[0092] As described below, in the present invention, the order of making an HDAC inhibitor such as 2-mercaptoethanol act and then making an OCT3 / 4 transcriptional stimulatory factor (LIF, CCL2, IL-6, etc.) act is important for cell reprogramming. Therefore, the culture medium in step (a) does not contain an OCT3 / 4 transcriptional stimulatory factor. Preferably, the culture medium in step (a) does not contain any of cytokines or hormones such as ACTH, bFGF, and GSK3β inhibitor.

[0093] <OCT3 / 4 transcriptional stimulatory factor>

[0094] The so-called OCT3 / 4 transcriptional stimulatory factor refers to a factor that stimulates the transcription of OCT3 / 4 and activates it, and examples thereof include LIF (leukemia inhibitory factor), CCL2 (chemokine (CC motif) ligand 2), and IL-6 (interleukin 6). Among them, it is preferable to use LIF, and more preferably to use LIF and CCL2.

[0095] In the present invention, the chromatin structure is relaxed by an HDAC inhibitor, and then the transcription of OCT3 / 4 is activated by the action of an OCT3 / 4 transcription stimulating factor, and the cells are shifted to an undifferentiated state by the action of proteins centered around OCT3 / 4.

[0096] In the method of the present invention (step (a)), the conditions for culturing cells are not particularly limited. For example, the culture temperature can be set to 30-40° C., preferably 35-39° C., and more preferably 36-38° C. In addition, the CO2 concentration is, for example, 1-10%, preferably 1.5-8%, and more preferably 2-5%.

[0097] The cell culture time in step (a) is not particularly limited, and can be appropriately set according to the type of cells used or the culture conditions. The cell culture time in the present invention is, for example, more than 1 day, and as a specific culture time, 1-10 days can be cited. The preferred culture time is also not particularly limited, but is, for example, 2-9 days, preferably more than 3 days, and more preferably 3-7 days.

[0098] The composition of the culture medium used for cell culture can also be appropriately set according to the type of cells used or culture conditions, etc., and is not particularly limited. For example, as its basic culture medium, it is not particularly limited, but Eagle's culture medium (BM, MEM, DMEM, etc.), McCoy's culture medium (McCoy5A, McCoy7A, etc.), Ham's culture medium (F10, F12, etc.), 199 culture medium, RPMI1640 culture medium, NCTC culture medium (NCTC109, NCTC135, etc.) can be used. In addition, various basic culture media can also be mixed and used as needed.

[0099] The above-mentioned basic culture medium may also be added with serum (fetal bovine serum, human serum, etc.), serum substitutes (KSR, B27 supplement, etc.), amino acids (alanine, arginine, cystine, histidine, etc.), vitamins (vitamin B7, vitamin B12, etc.), antibiotics (amphotericin B, kanamycin, etc.), adhesion factors (collagen type I, gelatin, fibronectin, etc.), fatty acids (oleic acid, arachidonic acid, linolenic acid, etc.), adenine, guanosine, hypoxanthine, thymidine, cholesterol, and other additives that are usually required for maintaining cells.

[0100] In the present invention, as a result of the culture in step (a) above, changes in cell morphology may be observed. Here, the term "change in cell morphology" in this specification means that the appearance of the cell group after culture changes from the state at the beginning of culture. For example, Figure 1 As shown in the photograph B of FIG. 1 , it can be observed that the cultured cells begin to aggregate from the surroundings. After confirming the morphological changes of the cells in the above state, the above-mentioned culture step can be stopped.

[0101] <Cultivation in the presence of OCT3 / 4 transcriptional stimulators>

[0102] The method of the present invention further comprises, after the above-mentioned culturing step, a step of culturing cells in a medium containing an OCT3 / 4 transcription stimulating factor, such as one or more selected from the group consisting of LIF, CCL2 and IL-6 (hereinafter referred to as step (b) in this specification).

[0103] The culture medium used in step (b) may contain, in addition to OCT3 / 4 transcription stimulators such as LIF, any one or more of ACTH (adrenocorticotropic hormone), bFGF (basic fibroblast growth factor), and a GSK3β inhibitor. In the present invention, it is preferred to use LIF or LIF and CCL2 in combination with ACTH and Bfgf, and it is most preferred to use LIF or LIF and CCL2 in combination with ACTH, bFGF, and a GSK3β inhibitor.

[0104] In the present invention, the so-called GSK3β inhibitor refers to a substance having an inhibitory activity against GSK3β (glycogen synthase kinase 3β). Examples of GSK3β inhibitors include AR-A014418 (N-(4-methoxybenzyl)-N'-(5-nitro-1,3-thiazol-2-yl)urea), CHIR99021 (6-[[2-[[4-(2,4-dichlorophenyl)-5-(5-methyl-1H-imidazol-2-yl)-2-pyrimidinyl]amino]ethyl]amino]-3-cyanopyridine), CHIR98014 (N-6-[2-[[4-(2,4-dichlorophenyl)-5-(1H -imidazol-1-yl)-2-pyrimidinyl]amino]ethyl]-3-nitro-2,6-pyridinediamine), SB415286 (3-[(3-chloro-4-hydroxyphenyl)-amino]-4-(2-nitrophenyl)-1H-pyrrole-2,5-dione), SB216763 (3-(2,4-dichlorophenyl)-4-(1-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione), BIO (6-bromoindirubin-3-oxime) and valproic acid, etc., but are not limited to these.

[0105] The source animals of OCT3 / 4 transcription stimulators (LIF, CCL2, IL-6, etc.), ACTH, bFGF, and GSK3β inhibitors are not particularly limited, and OCT3 / 4 transcription stimulators, ACTH, bFGF, and GSK3β inhibitors from any animal such as humans, mice, rats, rabbits, sheep, pigs, and cattle can also be used. In addition, OCT3 / 4 transcription stimulators, ACTH, bFGF, and GSK3β inhibitors can all be recombinants. These can all use commercially available reagents. The OCT3 / 4 transcription stimulators, ACTH, bFGF, and GSK3β inhibitors used in the present invention are preferably materials from humans, and are preferably recombinants.

[0106] The contents of OCT3 / 4 transcription stimulators (LIF, CCL2, IL-6, etc.), ACTH, bFGF, and GSK3β inhibitor in the culture medium in step (b) are not particularly limited and can be appropriately set depending on the type of cells used, culture conditions, and the like.

[0107] The content of LIF in the culture medium is not particularly limited, but is, for example, 0.01-5000 ng / mL, preferably 0.1-1000 ng / mL, and more preferably 1-100 ng / mL. Also, although not particularly limited, in a cell growth promotion assay using a mouse ES cell line (D3 line), when 1 / 20 of the amount of the growth range that provides 50% of the maximum growth range is set as 1 unit, the content of LIF in the culture medium is, for example, 10-10000 units / ml, preferably 50-8000 units / ml, and more preferably 100-5000 units / ml. In addition, when using LIF from Fujifilm Wako Pure Chemical Industries, Ltd. (Product No. 129-05601), it can be set to an amount diluted 500-2000 times relative to the amount of the culture medium.

[0108] The content of CCL2 in the culture medium is not particularly limited, but is, for example, 0.1-5000 ng / mL, preferably 1-1000 ng / mL, more preferably 10-500 ng / mL, 10-400 ng / mL, 10-300 ng / mL, 50-500 ng / mL, 50-400 ng / mL, 50-300 ng / mL.

[0109] The content of ACTH in the culture medium is not particularly limited, but is, for example, 0.1-200 μmol / L, preferably 1-100 μmol / L, and more preferably 5-50 μmol / L.

[0110] The content of bFGF in the culture medium is not particularly limited, but is, for example, 0.01-5 nmol / L, preferably 0.05-3 nmol / L, and more preferably 0.1-1 nmol / L.

[0111] The content of the GSK3β inhibitor in the culture medium is not particularly limited, but is, for example, 0.1-50 μM, preferably 0.5-30 μM, and more preferably 1-10 μM.

[0112] The composition of the culture medium used for cell culture in step (b) can also be appropriately set according to the type of cells used or culture conditions, etc. Basically, as long as the culture medium can maintain the culture medium of the reprogrammed cells, it is not particularly limited. For example, as the basic culture medium, although not particularly limited, Eagle's culture medium (BM, MEM, DMEM, etc.), McCoy's culture medium (McCoy5A, McCoy7A, etc.), Ham's culture medium (F10, F12, etc.), 199 culture medium, RPMI1640 culture medium, NCTC culture medium (NCTC109, NCTC135, etc.), stem cell culture medium, etc. can be used. In addition, various basic culture media can also be mixed and used as needed.

[0113] In addition to OCT3 / 4 transcription stimulators (LIF, CCL2, IL-6, etc.), ACTH, bFGF and GSK3β inhibitors, the above-mentioned basic culture medium may also contain serum (fetal bovine serum, human serum, etc.), serum substitutes (KSR, etc.), amino acids (alanine, arginine, cystine, histidine, etc.), vitamins (vitamin B7, vitamin B12, etc.), antibiotics (amphotericin B, kanamycin, etc.), adhesion factors (collagen type I, gelatin, fibronectin, etc.), growth factors (EGF, PDGF, TGF-α, etc.), cytokines (IL-2, IL-3, IL-4, IL-5, IL-6, etc.), hormones (insulin, glucagon, progesterone, etc.), fatty acids (oleic acid, arachidonic acid, linolenic acid, etc.), adenine, guanosine, hypoxanthine, thymidine, cholesterol, and other preferred additives for maintaining stem cells.

[0114] Furthermore, in step (b), a culture medium that has been prepared as a culture medium for stem cells can also be used. For example, the StemSure (registered trademark) hPSC culture medium Δ of Wako Pure Chemical Industries, the ADSC-4 culture medium of Kohjin Biotechnology Co., Ltd., the StemFit of REPROCELL, the TeSR series of STEMCELL Technologies, etc. can be used, but are not particularly limited to these. Commercially available culture medium for stem cells may also contain OCT3 / 4 transcription stimulators, ACTH, bFGF or GSK3β inhibitors. In addition, in step (b), it is not necessary to change the culture medium from step (a), and the culture medium used in step (a) can be used directly, and the culture medium contains one or more selected from the group consisting of LIF, ACTH, bFGF and GSK3β inhibitors to perform step (b).

[0115] In step (b), the conditions for culturing cells are not particularly limited. For example, the culture temperature can be set to 30-40° C., preferably 35-39° C., and more preferably 36-38° C. In addition, the CO2 concentration is, for example, 1-10%, preferably 1.5-8%, and more preferably 2-5%.

[0116] The cell culture time in step (b) is not particularly limited, and can be appropriately set according to the type of somatic cells used or the culture conditions. The cell culture time in step (b) is, for example, more than 1 day, and as a specific culture time, 1-10 days can be cited. The preferred culture time is also not particularly limited, but is, for example, 2-10 days, preferably more than 3 days, and more preferably 3-9 days.

[0117] In the present invention, further morphological changes of cells may be observed as a result of culturing in step (b). Figure 2 As shown, it can be observed that the cells after culture aggregate to form a cell aggregate. The cell aggregate is also called a cell mass (spheroid). After confirming the morphological changes of the cells in this state, the culture of step (b) can be stopped.

[0118] In the present invention, as described above, a preferred embodiment is to perform step (b) after step (a). Although not bound by a specific theory, when an agent that interferes with the function of DNA itself or DNA (e.g., DNA replication or RNA synthesis, etc. In addition, it also includes an indirect effect, that is, a function of RNA (specifically, a series of functions of synthesizing proteins from RNA)) is used as a "trigger agent" (agent that provides reprogramming input to cells), and an agent used to maintain reprogrammed cells (e.g., LIF or ACTH, etc.) is used as a "drug required for initialization", it is believed that the order of providing the "trigger agent" and the "drug required for initialization" to the cells after the "trigger agent" is important compared to providing the cells with the "trigger agent" and the "drug required for initialization" at the same time. In addition, the concentration of the "trigger agent" is a concentration that does not affect the survival of the cells.

[0119] <Cell Passaging>

[0120] In the present invention, cells can be passaged in the culture of step (a) and step (b). The passage operation of cells is not particularly limited and can be appropriately performed according to the type of cells or culture conditions used. For example, as long as it can be confirmed by microscopic observation that the cells are in a fused state, the culture medium can be taken out from the culture container, a buffer solution such as PBS (-) is added to the culture container to clean the cell surface, and then a protease such as trypsin is added to the container to recover the cells. The recovered cells can be added to a new culture medium, and the passage of cells is performed by a series of operations.

[0121] The number of times of cell passage can be set to 1-10 times, but is not particularly limited thereto, and can be appropriately set according to the type of somatic cells used or the culture conditions, etc. Although not particularly limited, in the present invention, the number of times of cell passage is preferably 1-5 times, more preferably 1-3 times, further preferably 1 or 2 times, and most preferably 1 time.

[0122] The present invention is a method for producing reprogrammed cells from somatic cells without gene introduction, and comprises: (a) a step of culturing somatic cells in a culture medium containing a histone deacetylase inhibitor; and (b) a step of producing reprogrammed cells by culturing the cells cultured in step (a) in a culture medium containing an OCT3 / 4 transcription stimulator.

[0123] The reprogrammed cells are pluripotent stem cells that can differentiate into the three germ layers. The pluripotent stem cells can be pluripotent.

[0124] <Multipotent / pluripotent stem cells>

[0125] In this specification, the so-called pluripotent stem cells refer to cells that have the ability to self-replicate and grow by dividing while maintaining an undifferentiated state and the ability to differentiate into cells of various systems. Preferably, the pluripotent stem cells of the present invention have differentiation pluripotency to differentiate into a cell series belonging to the three germ layers (endoderm, mesoderm, ectoderm).

[0126] In this specification, the so-called pluripotent stem cells refer to cells that have the ability to self-replicate and grow by dividing while maintaining an undifferentiated state and the ability to differentiate into a full range of cells belonging to the three germ layers (endoderm, mesoderm, ectoderm). Pluripotent stem cells have the ability to form teratomas and chimeras.

[0127] There is not necessarily a clear distinction between "multipotent" and "pluripotent." In this specification, the reprogrammed cells of the present invention are described as "multipotent" because the possibility of having teratoma-forming ability (tumor-forming ability) is low, but in the general sense, the cells are not excluded from being "pluripotent."

[0128] The fact that the cells obtained by the method of the present invention are highly undifferentiated cells can be appropriately confirmed using methods known to those skilled in the art. As one of the confirmation methods, for example, alkaline phosphatase activity can be examined. Alkaline phosphatase activity can be appropriately examined using a commercially available alkaline phosphatase staining kit, etc.

[0129] Identification of multipotent / pluripotent stem cells can be performed, for example, by confirming the expression of Nanog, Klf4, Oct4, Sox2, c-Myc, Lin28, TRA-1-60, SSEA (SSEA-4, SSEA-1, etc.), which are marker genes of pluripotent stem cells, to identify pluripotent stem cells. The expression of marker genes can be confirmed by methods known to those skilled in the art such as RT-PCR, and if it is confirmed at the protein level, it can be performed using antibodies specific to various markers or FACS, etc., which are known devices or methods per se.

[0130] Differentiation pluripotency (multipotent / pluripotent) can also be confirmed by checking the differentiation to the three germ layers of endoderm, mesoderm and ectoderm. Differentiation to the three germ layers can be confirmed by checking the markers of each germ layer. For example, as endoderm markers, Sox17, CXCR4, HNF-3β, FoxA2, ​​AFP, GATA-4, PDX-1, Nkx2.1, etc. can be listed, as mesoderm markers, MSX1, α-SMA, Obt2, Brachyury, etc. can be listed, as ectoderm markers, Pax6, MAP2, Nestin, Otx2, TP63, SOX2, etc. can be listed. As with the confirmation of pluripotent stem cell markers, the confirmation of various markers can be carried out using methods known to those skilled in the art.

[0131] The teratoma forming ability can be evaluated, for example, by injecting cells into the subcutaneous tissue of mice to form teratomas and analyzing their differentiated tissues. Teratomas contain differentiated tissues from the three germ layers of endoderm, mesoderm, and ectoderm. The chimera forming ability can be confirmed, for example, by injecting cells into blastocysts and checking whether chimeric animals are formed from the blastocysts. Both the teratoma forming ability and the chimera forming ability can be checked using methods known to those skilled in the art.

[0132] The differentiation ability can be examined in vitro using a differentiation induction method known to those skilled in the art. Differentiation into various cells can also be performed using a commercially available differentiation induction kit or the like.

[0133] <Method for producing cells>

[0134] The present invention further provides a method for producing cells using the pluripotent stem cells obtained by the above method as another embodiment. Specifically, the present invention provides a method for producing cells, which comprises the following steps (i) and (ii):

[0135] (i) a step of producing reprogrammed cells from somatic cells by the above method;

[0136] (ii) A step of inducing differentiation of the reprogrammed cells into target cells.

[0137] Step (i) may be performed by performing the above step (a) or by combining the above step (a) and step (b). Furthermore, the cell differentiation induction in step (ii) may be appropriately performed using a method known to those skilled in the art depending on the type of target cells.

[0138] The cells obtained by the production method of the present invention are not particularly limited, but examples thereof include adipocytes, nerve cells, muscle cells (cardiac muscle cells, smooth muscle cells, skeletal muscle cells, etc.), skin cells, epithelial cells, endothelial cells, blood cells (neutrophils, eosinophils, basophils, monocytes, lymphocytes, etc.), liver cells, kidney cells, lung cells, pancreatic cells, breast cells, hair cells, etc.

[0139] <Kit and Composition>

[0140] The present invention provides a kit for reprogramming cells as another embodiment, the kit comprising a culture medium containing an HDAC inhibitor. In addition, the present invention provides a composition for reprogramming cells as another embodiment, the composition comprising a culture medium containing an HDAC inhibitor. The HDAC inhibitor and the culture medium containing the HDAC inhibitor are as described in step (a) of the method of the present invention described above.

[0141] The kit of the present invention is composed of the following two reagents:

[0142] Reagent A) a reagent for constituting a culture medium not containing 2-mercaptoethanol;

[0143] Reagent B) Reagents used to constitute a culture medium containing LIF.

[0144] Reagent A and reagent B may be prepared when used by packaging the culture medium and components separately, or may be in a state where each component is already contained in the culture medium. In addition, the culture medium composed of reagent A may further contain CCL2 and / or IL-6 as OCT3 / 4 transcription stimulators.

[0145] Reagent B may contain one or more selected from the group consisting of ACTH, bFGF and GSK3β inhibitor in addition to LIF, CCL2 and IL-6. The culture medium used for reagent A and reagent B is the same as that described in steps (a) and (b) of the method of the present invention.

[0146] <Pharmaceutical composition, cosmetic composition>

[0147] The present invention can provide a cell or a cell group thereof reprogrammed by the method of the present invention. Although the cell of the present invention is not particularly limited, one of its characteristics can be cited, for example, because HDAC inhibitors can be used, it does not contain exogenous gene introduction such as Oct3 / 4, Sox2, Klf4, c-Myc, etc. In addition, the cell group (cell aggregate) of the cell reprogrammed by the method of the present invention is not particularly limited, but as described in the following examples ( Figure 2As shown in B), one of the morphological characteristics is that the boundaries of the cell group are unclear and the interior is uneven.

[0148] The cells reprogrammed by the method of the present invention, the differentiated cells of the cells (cells differentiated by the cells reprogrammed by the method of the present invention), or the extracted components or secreted components of the cells (components extracted from the cells reprogrammed by the method of the present invention or components secreted by the cells) can be used as active ingredients of pharmaceutical compositions or cosmetic compositions. That is, the present invention provides a pharmaceutical composition or cosmetic composition comprising the cells reprogrammed by the method of the present invention, the differentiated cells of the cells, or the extracted components or secreted components of the cells as another embodiment. In addition, the present invention provides a method for producing a pharmaceutical composition or a cosmetic composition as another embodiment, the production method comprising the following steps: the step of mixing the cells reprogrammed by the method of the present invention, the differentiated cells of the cells, or the extracted components or secreted components of the cells.

[0149] The components extracted from the cells reprogrammed by the method of the present invention or the components secreted by the cells are not particularly limited, but examples thereof include gene-related substances such as miRNA, DNA or RNA, proteins, cytokines, extracellular vesicles (including exosomes), miRNA, DNA, RNA, proteins contained in extracellular vesicles, etc. These components may be one or a combination of two or more.

[0150] The use of the pharmaceutical composition is not particularly limited, but examples thereof include the treatment of olfactory disorders, cerebral infarction, diabetes, neurological disorders, cancer, liver diseases, etc. When using cells reprogrammed by the method of the present invention, the cells themselves or cells differentiated from the cells can be used for cell transplantation.

[0151] The pharmaceutical composition or cosmetic composition of the present invention may contain a pharmacologically acceptable carrier according to its form. Examples of pharmacologically acceptable carriers include, but are not limited to, excipients, bonding agents, emulsifiers, tonicity agents (isotonic agents), buffers, solubility aids, preservatives, stabilizers, antioxidants, colorants, coagulants, or coating agents.

[0152] The present invention may also provide a method for treating any of the above-mentioned diseases and a use thereof as another embodiment in relation to the above-mentioned uses. That is, the present invention provides a method for treating any of the above-mentioned diseases as another embodiment, wherein the method uses the above-mentioned cell reprogrammed by the method of the present invention, a differentiated cell of the cell, or an extract or secreted component of the cell. Furthermore, the present invention provides a use of the above-mentioned cell reprogrammed by the method of the present invention, a differentiated cell of the cell, or an extract or secreted component of the cell as another embodiment, wherein the use is used to treat any of the above-mentioned diseases.

[0153] Example

[0154] The present invention will be further described in detail below with reference to Examples. However, these are for illustration only and are not intended to limit the scope of the present invention in any way.

[0155] Example 1

[0156] 1. Reprogramming of somatic cells

[0157] Normal human skin fibroblasts (adult) (Takara Bio) were seeded in spheroid plates, and after 24-48 hours, the medium was replaced with a D-MEM (Low Glucose) medium to which 2-mercaptoethanol was diluted 1 / 100000 (142 μM). Then, after 3-4 days, the medium was replaced again with the same D-MEM medium as above.

[0158] As mentioned above, after culturing human skin fibroblasts for 1 week, it was confirmed that the morphology of the cells had changed ( Figure 1 ). After confirming the morphological changes of the cells, the medium was replaced with a medium containing LIF (wako, used at 1:1000), ACTH (wako, 10 μmol / L), bFGF (wako, 0.34 nmol / L), and AR-A014418 (Merck, 3 μM) added to ADSC-4 (Kohjin Biotech). After that, after 3-4 days, the medium was replaced again with the same medium as above.

[0159] As described above, cells were cultured for 1 week in an ADSC-4 medium containing LIF, etc., and as a result, the cells further aggregated and formed spheroids ( Figure 2 ).

[0160] 2. Alkaline phosphatase reaction

[0161] The alkaline phosphatase reaction of the cells finally obtained by the above culture was examined. Alkaline phosphatase staining was performed using Alkaline Phosphatase Live Stain (Invitrogen) in accordance with the instructions for use attached to the kit. Specifically, the staining solution (AP Live Stain) attached to the kit was added to D-MEM in a 500-fold dilution to obtain a culture medium, and the cells were cultured with the culture medium for 30 minutes. After that, the culture medium was replaced and the cells were washed for 5 minutes × 2 times. After washing, the cells were observed under a fluorescent microscope using a GFP filter to determine whether they were stained. The results showed that the cells were stained ( Figure 3 ).

[0162] 3. Various conditions research

[0163] (1) 2-Mercaptoethanol concentration

[0164] The same treatment as in the above items 1 and 2 was performed to examine the concentration of 2-mercaptoethanol. Specifically, the same operation as in the above items 1 and 2 was performed except that the concentration of 2-mercaptoethanol was set to 0.142 μM, 1.42 μM, 14.2 μM, 142 μM, 1.42 mM, 14.2 mM, and 142 mM. As a result, when the concentration of 2-mercaptoethanol was 0.142 μM-1.42 mM, spheroid formation and alkaline phosphatase staining of cells were observed, and when the concentration of 2-mercaptoethanol was 14.2 μM-1.42 mM, particularly obvious alkaline phosphatase staining was found. In addition, when the concentration of 2-mercaptoethanol was 14.2 mM or more, cell death was observed.

[0165] (2) Use of culture medium

[0166] The cells were cultured in a D-MEM medium containing 2-mercaptoethanol (142 μM) in the same manner as in item 1 above, and then LIF (wako, used at 1:1000), ACTH (wako, 10 μmol / L), bFGF (wako, 0.34 nmol / L), and AR-A014418 (Merck, 3 μM) were added to the medium and the cells were cultured for another week. As a result, both the formation of cell spheroids and alkaline phosphatase staining were observed. Furthermore, as in the above-mentioned item 1, when the cells were cultured only in D-MEM medium containing 2-mercaptoethanol (142 μM), the formation of cell spheroids and weak alkaline phosphatase staining were observed, but when the cells were cultured only in a medium in which LIF (wako, used at 1:1000), ACTH (wako, 10 μmol / L), bFGF (wako, 0.34 nmol / L) and AR-A014418 (Merck, 3 μM) were added to ADSC-4 medium, neither the formation of cell spheroids nor alkaline phosphatase staining were observed.

[0167] (3) Factors of LIF and ACTH

[0168] In the above item 1, ADSC-4 culture medium was cultured by adding (i) only LIF, (ii) only ACTH, or (iii) only LIF and ACTH. As a result, spheroid formation and alkaline phosphatase staining were observed in all conditions. In the conditions of (i) and (iii), particularly obvious alkaline phosphatase staining was observed.

[0169] 4. Expression of undifferentiated markers

[0170] As undifferentiation markers, whether or not the genes of Nanog, Klf4, Oct4, and Sox2 were expressed was examined. RNA was recovered from the cells using RNeasy Mini Kit (QIAGEN). PCR was performed for the above-mentioned various undifferentiation markers using the method described in Cell. 2007 Nov 30; 131(5): 861-72.

[0171] As a result, all bands of Nanog, Klf4, Oct4, and Sox2 were confirmed in the cultured cells tested near the target band sizes (Nanog: 406 bp, Klf4: 396 bp, Oct4: 143 bp, Sox2: 150 bp). Figure 4 ). In addition, no band was observed in the negative control.

[0172] 5. Induction of differentiation into adipocytes

[0173] The cells obtained in the above item 1 were subjected to a differentiation induction test into adipocytes using a commercially available cell differentiation kit. The test kit used was Mesenchymal Stem Cell Adipogenic Differentiation Medium 2 (PromoCell, product code C-28016). The cell culture solution was replaced with the culture solution attached to the kit, and then the cells were cultured according to the instructions for use attached to the kit.

[0174] The fat droplets observed in the cells that had been induced to differentiate were stained using LipiDye (Funakoshi). Live cell imaging was performed according to the instructions for use of the kit. Specifically, the staining solution provided with the kit was added to D-MEM to a final concentration of 1 μM, and the cells were cultured in the medium for 2 hours. After that, the medium was replaced and the cells were washed for 5 minutes × 2 times. After washing, the cells were observed using a fluorescent microscope using a GFP filter to determine whether they were stained. The results showed that the cells were stained ( Figure 5 ).

[0175] 6. Induction of differentiation into neural cells

[0176] For the cells obtained in the above item 1, a differentiation induction test into neural cells was performed using a commercially available cell differentiation kit. The kit used for the test was Mesenchymal Stem Cell Neurogenic Differentiation Medium (PromoCell, product code C-28015). The cell culture solution was replaced with the culture solution attached to the kit, and then the cells were cultured according to the instructions for use attached to the kit.

[0177] For cells that have undergone differentiation induction, immunostaining was performed using an antibody against neural cells, namely an anti-neurofilament antibody (abcam, ab7255, Anti-68kDa Neurofilament / NF-L antibody [DA2]). Figure 6 ).

[0178] 7. Induction of differentiation into hepatocytes

[0179] A differentiation induction test into hepatocytes was performed on the cells obtained in the above item 1. The cells were cultured in 0.8 μM hexachlorophene / D-MEM medium according to the method described in Scientific Reports (2015) 5:16169.

[0180] PAS staining, Western blotting, and indocyanine green test were performed on the cells that had been induced to differentiate to confirm the properties of the hepatocytes. PAS staining was performed using a commercially available PAS staining kit (Muto Chemical) in accordance with the instructions for use attached to the kit. In the Western blotting method, CellLytic M (Sigma Aldrich) was used to prepare a cell lysate in accordance with the instructions for use attached to the product. The prepared cell lysate was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) according to the Laemmli method, and then transferred to a PVDF membrane using a semi-dry method and subjected to Western blotting analysis. As the primary detection antibody, an anti-albumin rabbit polyclonal antibody (4929: Cell Signaling Technology) and an anti-β-actin mouse monoclonal antibody (A5441: Sigma Aldrich) were used. As the secondary antibody, an HRP-labeled antibody against IgG from an animal in which the primary detection antibody was produced was used. Signal detection was performed using a luminol imaging device LAS4000 (Fujifilm). The indocyanine green test was performed according to the method described in Cloning Stem Cells (2007) Spring; 9(1): 51-62, and observation was performed 1 hour and 20 hours after the start of staining ( Figure 7 , Figure 8 ).

[0181] 8. Induction of differentiation into chondrocytes

[0182] The cells obtained in the above item 1 were subjected to a differentiation induction test into chondrocytes using a commercially available cell differentiation kit. The test kit used was Mesenchymal Stem Cell Chondrogenic Differentiation Medium (PromoCell, product code C-28012). The cell culture solution was replaced with the culture solution provided with the kit, and then the cells were cultured according to the instructions provided with the kit ( Fig. 9 , Fig.10 ).

[0183] The cells that had been induced to differentiate were stained with HE, Alcian blue, and immunostained (antibodies to S-100, GFAP, CD34, SMA, and vimentin), and the proteins expressed in the cells were evaluated. Fig.11 ). Comparison studies with fibrocartilage and hyaline cartilage in randomly selected human clinical specimens confirmed protein expression similar to that of fibrocartilage, and it was clear that the chondrocytes obtained by differentiation induction were similar to fibrocartilage.

[0184] 9. Induction of osteoblast differentiation

[0185] According to the above item 1, the cells were cultured in D-MEM medium containing 2-mercaptoethanol, and then cultured in ADSC-4 medium containing LIF, ACTH, and bFGF. The cells were subjected to a differentiation induction test into chondrocytes using a commercially available osteoblast differentiation medium. The medium used in the test was Mesenchymal Stem Cell Osteogenic Differentiation Medium (PromoCell, product code C-28013).

[0186] The cells after differentiation induction were stained with alkaline phosphatase and confirmed to be alkaline phosphatase positive ( Fig.12 ).

[0187] Example 2

[0188] 1. Effect of CCL2

[0189] CCL2 was added to LIF to verify the effect on reprogramming. According to Example 1, normal human skin fibroblasts (adult) (Takara Bio) were cultured for 3-4 days with D-MEM medium containing 2-mercaptoethanol (142 μM) to confirm the effect of the medium and cultured for 7 days. Then, the medium was cultured for another 7 days in which (i) only LIF was added, (ii) LIF+CCL2 (10 ng / mL) was added, or (iii) LIF+CCL2 (100 ng / mL) was added to the ADSC-4 medium. In addition, LIF was used at 1:1000.

[0190] Morphological changes were observed in the cells after culture (i) to (iii) (from a spindle-shaped morphology to a slightly rounded morphology with a slightly protruding part depressed). Fig.13 ). In particular, the addition of CCL2 made cell aggregation more obvious, the outlines of cells became clear, and the separation of cell adhesion was found ( Fig.13 D).

[0191] 2. Induction of differentiation into adipocytes

[0192] The cells cultured in the medium containing (ii) LIF+CCL2 (10 ng / mL) and (iii) LIF and CCL2 (10 ng / mL) were subjected to a differentiation induction test into adipocytes according to item 5 of Example 1 using a commercially available cell differentiation kit.

[0193] On day 25 after differentiation induction, cells were stained with LipiDye (Funakoshi) ( Fig.14 as well as Fig.15 ). Although fat droplets were observed in all cells, an increase in the number and size of fat droplets was observed in cells of (iii) compared to those in (ii).

[0194] Furthermore, regarding the time of culturing in the presence of 2-mercaptoethanol (step A) and the time of culturing in the presence of LIF (step B), including the case where the time of culturing only with the medium was set between step A and step B, the difference in induction of differentiation into adipocytes was studied.

[0195] Step A (3-9 days) → Culture time with medium only (0-14 days) → Step B (3-9 days) → Adipogenic differentiation induction (14 days)

[0196] First, step A and step B were each set to 3 days, and the culture time in the medium was set to 0 day, 3 days, 7 days, and 10 days.

[0197] · Differentiation induction from step A for 3 days → step B for 3 days to form fat droplets.

[0198] Form fat droplets by inducing differentiation from step A for 3 days → culture medium for 3 days → step B for 3 days.

[0199] · Differentiation induction from step A for 3 days → culture medium for 7 days → step B for 3 days to form fat droplets.

[0200] · Differentiation induction from step A for 3 days → culture medium for 10 days → step B for 3 days to form fat droplets.

[0201] When the culture time was 0 days, 3 days, and 7 days, the fat droplets were small but a large number of fat droplets were formed on the 14th day after differentiation induction, and the fat droplets became larger and the number increased after the 21st day. When the culture time was 10 days, the fat droplets were quite small on the 14th day after differentiation induction.

[0202] The culture time in the medium was set to 0 day, and the culture time in step A and step B was changed.

[0203] · Formation of fat droplets by differentiation induction for 5 days in step A and 7 days in step B.

[0204] Formation of fat droplets by differentiation induction for 7 days in step A and 7 days in step B.

[0205] · Formation of fat droplets by differentiation induction for 9 days in step A and 7 days in step B.

[0206] · At 14 days after differentiation induction, it was found that the smaller the density, the more fat droplets there were, but after about 21 days, the fat droplets became larger and the number increased regardless of the density.

[0207] From the above results, it was confirmed that 3 days is sufficient for the culture time in step A and step B respectively.

[0208] Industrial Applicability

[0209] The cell reprogramming method provided by the present invention is useful for research and development of tissue regeneration technology and can be used in the field of regenerative medicine, such as the preparation of organs for regenerative medicine. In addition, since cells can be transformed into other organs, for example, in fields other than human medicine, cow skin cells can be transformed into muscle cells and used as human food.

[0210] The contents of all publications, patents, and patent applications cited in this specification are incorporated herein by reference as they are.

Claims

1. A method for producing reprogrammed cells, i.e., pluripotent stem cells, from fibroblasts of non-human mammals without gene introduction, characterized in that: include: (a) culturing non-human mammalian fibroblasts in a medium containing 2-mercaptoethanol but not LIF, CCL2 and IL-6 to obtain precursors of pluripotent stem cells; and (b) a step of preparing pluripotent stem cells as reprogrammed cells by culturing the precursors of the pluripotent stem cells obtained in step (a) in a culture medium containing OCT3 / 4 transcriptional stimulatory factors, wherein the OCT3 / 4 transcriptional stimulatory factors are selected from any one or two or more of the group consisting of LIF, CCL2 and IL-6.

2. The method according to claim 1, characterized in that The OCT3 / 4 transcription stimulating factor in step (b) comprises LIF.

3. The method according to claim 1 or 2, characterized in that: The culture medium of step (a) contains 0.1 μM to 2 mM 2-mercaptoethanol.

4. The method according to claim 2, characterized in that: The culture medium of step (b) further comprises CCL2.

5. The method according to claim 1 or 2, characterized in that: The culture medium of step (b) further comprises one or more selected from ACTH, bFGF and GSK3β inhibitor.

6. The method according to claim 1 or 2, characterized in that: The culture medium of step (b) does not contain 2-mercaptoethanol.

7. A method for producing target cells from somatic cells, characterized in that: include: (i) a step of preparing reprogrammed cells, namely pluripotent stem cells, from fibroblasts of a non-human mammal by the method according to any one of claims 1 to 6; and (ii) a step of inducing differentiation of the pluripotent stem cells into target cells.

8. A method for producing a cell preparation, characterized in that: include: (i) a step of preparing reprogrammed cells, namely pluripotent stem cells, from fibroblasts of a non-human mammal by the method according to any one of claims 1 to 6; (ii) a step of inducing differentiation of the pluripotent stem cells into target cells to obtain differentiated cells; and (iii) a step of preparing a cell preparation, wherein the cell preparation comprises the differentiation-induced cells and a pharmacologically acceptable carrier.

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