Cell reprogramming method

Abstract

To provide a method for producing reprogrammed cells from somatic cells without gene transfer.SOLUTION: (a) culturing somatic cells of a subject in a first medium free of LIF, a CCL2, and IL-6 and containing 2-mercaptoethanol to obtain precursors of pluripotent stem cells, and (b) culturing the precursors of pluripotent stem cells obtained in step (a) in a second medium containing any one or more selected from the group consisting of LIF, a CCL2, and IL-6 to produce pluripotent stem cells which are reprogrammed cells.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] Related Applications: This application claims priority based on Japanese Patent Application No. 2019-206270 (filed November 14, 2019), the contents of which are incorporated herein by reference. Technical fields: The present invention relates to a method for reprogramming cells, and more particularly to a method for reprogramming cells by using chemicals without exogenous gene introduction. [Background technology]

[0002] 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 to this day. Pluripotent stem cells such as ES cells are characterized by their pluripotency, and it is believed that this property can be utilized to regenerate various tissues. Specifically, they are expected to be useful in treating diseases that have been difficult to cure until now, such as neurodegenerative diseases such as Parkinson's disease, spinal cord injury, cerebral infarction, diabetes, liver cirrhosis, and cardiomyopathy.

[0003] However, transplantation of ES cells or tissues regenerated from ES cells involves the problem of causing post-transplant rejection, similar to organ transplantation, because it is an allotransplant. Furthermore, the establishment of ES cells requires fertilized eggs or early embryos at the blastocyst stage, which is a stage of development beyond the fertilized egg. In particular, in the case of humans, the use of human fertilized eggs as a source of tissue destroys the seeds of life, which has been pointed out as an ethical problem.

[0004] Meanwhile, in 2006, Yamanaka et al. reported a groundbreaking technology for producing induced pluripotent stem cells (iPS cells), in which pluripotent stem cells are established by introducing four genes (Oct3 / 4, Sox, c-Myc, and Klf) into somatic cells (Non-Patent Document 2). Because iPS cells use somatic cells, autotransplantation is possible, and the ethically questionable embryo destruction is not required, thereby eliminating the problems associated with ES cells. It is therefore widely known that iPS cells have dramatically advanced regenerative medicine technology. Human iPS cells were also successfully established in 2007 (Non-Patent Documents 3 and 4), and numerous reports have been published on iPS cells to date.

[0005] Currently, methods for establishing iPS cells appear to have made dramatic advances since their discovery. Retroviruses, which were used for gene transfer in the early stages after their first reports, were criticized for their potential for oncogenicity due to insertional mutations in the somatic cell genome. However, methods that do not introduce mutations into the somatic cell genome have now been developed, including methods using the RNA virus Sendai virus, episomal vectors, and synthetic mRNA (Non-Patent Documents 5-8). Furthermore, various improvements have been made in the types of genes to be transferred, including the development of a method that does not require any genes other than the Oct3 / 4 and Sox genes (Non-Patent Document 9).

[0006] In addition to iPS cells, multipotent (pluripotent) stem cells known as Muse cells have been reported (Patent Document 1). Muse cells are non-tumorigenic pluripotent stem cells present in biological tissues such as bone marrow and skin, and are characterized by being SSEA-3 (stage specific embryonic antigen-3) positive. It has been reported that Muse cells can differentiate into a variety of cells across all three germ layers, including hepatocytes, muscle, nerve, glial cells, skin pigment cells (melanocytes), epidermis, and blood vessels. As such, pluripotent stem cells are of great interest in regenerative medicine, and there is a need to further establish technologies for generating pluripotent stem cells. [Prior art documents] [Chartered documents]

[0007]

Patent Document 1

Non-licensed literature

[0008] [Non-licensed document 1] Martin GR, Proc Natl Acad Sci US A. 1981 Dec;78(12):7634-8. [Non-licensed document 2] Takahashi K et al., Cell. 2006 Aug 25;126(4):663-76. [Non-licensed document 3] Takahashi K et al., Cell. 2007 Nov 30;131(5):861-72.

Non-licensed Document 4

Non-licensed Document 5

Non-licensed Document 6

Non-licensed Document 7

Non-licensed literature 9

[0009] In order to generate pluripotent (multipotent / pluripotent) stem cells, it is extremely important from the perspective of clinical application to use somatic cells as the starting material and to avoid artificial genetic manipulation. Although improvements have been made to date in terms of the feasibility of genetic modification, cost, production efficiency, and production time, further improvements remain a challenge. Based on this, the present invention aims to provide a method for cell reprogramming as part of the technology for generating pluripotent stem cells. More specifically, it aims to provide a method for cell reprogramming without the need for external artificial gene introduction. [Means for solving the problem]

[0010] As a result of intensive research aimed at solving the above-mentioned problems, the present inventors have found that reprogramming of somatic cells occurs when the cells are cultured in a medium containing a histone deacetylase inhibitor (e.g., 2-mercaptoethanol), which is a DNA dysfunctioning substance, and then cultured in a medium containing OCT3 / 4 transcription stimulatory factors (e.g., LIF, CCL2, and IL-6). Based on this finding, the present inventors have completed the present invention.

[0011] The present invention is preferably carried out in the manner described below, but is not limited thereto. [1] A method for producing reprogrammed cells from somatic cells without gene transfer, comprising: (a) culturing somatic cells in a medium containing a histone deacetylase inhibitor; and (b) producing reprogrammed cells by culturing the cells cultured in step (a) in a medium containing an OCT3 / 4 transcription stimulatory factor. [2] The method according to [1], wherein the OCT3 / 4 transcription stimulatory factor is any one or more selected from the group consisting of LIF, CCL2, and IL-6. [3] The method according to [1] or [2], wherein the histone deacetylase inhibitor is 2-mercaptoethanol. [4] A method for producing reprogrammed cells from somatic cells without gene transfer, comprising: (a) culturing somatic cells in a medium containing 2-mercaptoethanol; and (b) producing reprogrammed cells by culturing the cells cultured in step (a) in a medium containing LIF. Preferably, the culture period in step (a) is 3 days or more, more preferably 3 to 7 days, and the culture period in step (b) is preferably 3 days or more, more preferably 3 to 9 days. It is desirable to culture the cells until spheroid formation is confirmed. [5] The method according to any one of [1] to [4], wherein the medium in the step (a) contains 10 μM to 0.2 mM, preferably 10 μM to 100 μM, more preferably 10 μM to 50 μM, of 2-mercaptoethanol. [6] The method according to [4] or [5], wherein the medium in step (b) further contains LIF. Preferably, the medium in step (b) contains LIF at a concentration of 1 to 100 ng / mL (titer: 100 to 5000 units / mL). [7] The method according to any one of [1] to [6], wherein the medium in step (b) further contains one or more selected from ACTH, bFGF, and a GSK3β inhibitor. [8] The method according to any one of [1] to [7], wherein the medium in step (a) does not contain an OCT3 / 4 transcription stimulating factor. Preferably, the medium in step (a) does not contain cytokines or hormones. [9] The method according to any one of [1] to [8], wherein the reprogrammed cells are multipotent stem cells that can differentiate into three germ layers. The pluripotent stem cells may be pluripotent.

[10] The method according to any one of [1] to [8], wherein the somatic cells are fibroblasts, preferably dermal fibroblasts. In step (a), it is preferable to culture the cells until a morphological change in the fibroblasts is confirmed.

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

[12] A kit for reprogramming somatic cells, comprising the following Reagent A and Reagent B: Reagent A) Reagent for constituting a medium without 2-mercaptoethanol; Reagent B) Reagent for constituting a medium containing LIF, CCL2, and IL-6. Reagent A and Reagent B may be prepared immediately after use by packaging the medium and the components separately, or each component may be contained in the medium. The medium composed of Reagent A does not contain OCT3 / 4 transcription stimulatory factors (LIF, CCL2, IL-6).

[13] The kit according to

[12] , wherein Reagent B further comprises one or more components selected from ACTH, bFGF, and a GSK3β inhibitor.

[14] A method for producing a target cell from a somatic cell, comprising: (i) preparing a reprogrammed cell from a somatic cell by the method according to any one of [1] to

[11] ; and (ii) inducing differentiation of the reprogrammed cells into cells of interest.

[15] A method for producing a cell preparation, comprising: (i) preparing reprogrammed cells from somatic cells by the method according to any one of [1] to

[11] ; (ii) inducing differentiation of the reprogrammed cells into cells of interest; and (iii) preparing a cell preparation comprising the differentiation-induced cells and a pharmacologically acceptable carrier.

[16] Cells derived from somatic cells by the method described in

[14] .

[17] A cell preparation comprising cells prepared by the method described in

[15] and a pharmacologically acceptable carrier. [Effects of the Invention]

[0012] The present invention provides a method for reprogramming cells without the need for external artificial gene introduction. The method provided by the present invention can be used to generate multipotent (pluripotent) stem cells. Because the method of the present invention does not require external artificial gene introduction, it is possible to significantly reduce the risk of affecting somatic cells, such as immunogenicity and carcinogenicity. Furthermore, the method of the present invention requires only the use of an HDAC inhibitor during cell culture, allowing for simple and stable cell reprogramming. Furthermore, the implementation costs of cell reprogramming and generation of pluripotent stem cells can be kept low. From these perspectives, the use of the method of the present invention will facilitate research on pluripotent stem cells and will make a significant contribution to future research and development of tissue regeneration technologies. [Brief explanation of the drawings]

[0013] [Figure 1] Figure 1 shows the morphology of human dermal fibroblasts after culture. Figure 1A shows the morphology of human dermal fibroblasts in a normal state (low cell density) after culture. Figure 1B shows the morphology of human dermal fibroblasts in a high cell density state after culture. [Figure 2] Figure 2 shows spheroids formed after culturing human dermal fibroblasts. Figure 2A is a slightly magnified photograph of the spheroids formed after culturing human dermal fibroblasts, and Figure 2B is a larger magnified photograph of the spheroids. [Figure 3] FIG. 3 shows the alkaline phosphatase reaction of cells after culturing. [Figure 4]Figure 4 shows the results of a PCR experiment examining the expression of undifferentiation markers, Nanog, Klf4, Oct4, and Sox2. The estimated sizes of Nanog, Klf4, Oct4, and Sox2 are 406 bp, 396 bp, 143 bp, and 150 bp, respectively. Lane 1 shows a human dermal fibroblast sample not treated with 2-mercaptoethanol. Lane 2 shows a human dermal fibroblast sample cultured in an incubator following step (a) without treatment with 2-mercaptoethanol. Lane 3 shows a human dermal fibroblast sample reprogrammed by treatment with 2-mercaptoethanol. Lane 4 shows a negative control sample in which water was added instead of RNA. [Figure 5] Figure 5 shows the results of differentiation into adipocytes: Figure 5A is an enlarged photograph after differentiation into adipocytes, and Figure 5B is a photograph of the differentiated adipocytes stained with fluorescent dye. [Figure 6] Figure 6 shows the results of differentiation into neurons. Figures 6A to 6C are photographs after differentiation into neurons, and Figure 6D is a photograph of differentiated neurons immunostained with an antibody (anti-neurofilament antibody). [Figure 7] Figure 7 shows the results of differentiation into hepatocytes. Figure 7A shows that the differentiated hepatocytes were stained with PAS, and Figure 7B shows that the differentiated hepatocytes produced albumin. [Figure 8] Figure 8 shows the results for differentiated hepatocytes. Figure 8A shows that ICG (indocyanine green) is taken up by differentiated hepatocytes. Figure 8B shows that differentiated hepatocytes exhibit bile duct-like structures and that ICG is accumulated within the bile duct-like structures. Figure 8C is a photograph taken 1 hour after ICG uptake, and Figure 8D shows that ICG has almost disappeared 20 hours later. [Figure 9]Figure 9 shows the results of differentiation into chondrocytes. Figure 9A is a photograph of the differentiated chondrocytes. Figure 9B is a photograph of the differentiated chondrocytes stained with HE. Figure 9C is an enlarged photograph of Figure 9B. Figure 9D is a photograph of the differentiated chondrocytes stained with Alcian blue. [Figure 10] Figure 10 shows the results for differentiated chondrocytes. Figure 10A is a magnified photograph of differentiated chondrocytes, and Figure 10B is a photograph showing human labral fibrocartilage. Figures 10A and 10B show that the differentiated chondrocytes and the human labral fibrocartilage have pathologically similar morphologies. Figure 10C shows that some differentiated chondrocytes have ossified. Figure 10D is a photograph of bone tissue from a human femoral head. Figures 10C and 10D show that the ossified portion of the differentiated chondrocytes has a morphology pathologically similar to that of human bone tissue. [Figure 11] Figure 11 shows the results of staining differentiated chondrocytes and chondrocytes in human clinical specimens with various antibodies and chemical solutions. Based on their biological characteristics, differentiated chondrocytes closely resemble fibrocartilage in human clinical specimens. [Figure 12] Figure 12 shows the results of alkaline phosphatase staining of differentiated osteoblasts. Figure 12A shows a control (skin fibroblasts), and Figure 12B shows cells induced to differentiate into osteoblasts after reprogramming. The differentiated cells are alkaline phosphatase positive. [Figure 13] Figure 13 shows the morphology of human dermal fibroblasts cultured in D-MEM medium containing 2-mercaptoethanol followed by culture in medium containing LIF / CCL2. Figure 12A shows normal fibroblasts, Figure 12B shows fibroblasts treated with LIF alone, Figure 12C shows fibroblasts treated with LIF + CCL2 (10 ng / mL), and Figure 12D shows fibroblasts treated with LIF + CCL2 (100 ng / mL). [Figure 14]Figure 14 shows the induction of differentiation into adipocytes. Figure 14A shows the cells after culturing for 7 days in D-MEM medium containing 2-mercaptoethanol, Figure 14B shows the cells after culturing for 7 days in a medium containing LIF + CCL2 (10 ng / mL), and Figures 14C and D show the cells on the 25th day after the induction of differentiation into adipocytes (4-fold and 20-fold magnification, respectively). [Figure 15] Figure 15 shows the induction of differentiation into adipocytes. Figure 15A shows the cells after culturing for 7 days in D-MEM medium containing 2-mercaptoethanol, Figure 15B shows the cells after culturing for 7 days in a medium containing LIF + CCL2 (100 ng / mL), and Figures 15C and D show the cells on the 25th day after the induction of differentiation into adipocytes (4-fold and 20-fold magnification, respectively).

Mode for Carrying Out the Invention

[0014] One aspect 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 (e.g., 2-mercaptoethanol) and the step of culturing in a medium containing an OCT3 / 4 transcriptional stimulator (e.g., LIF, CCL2, and IL-6).

[0015] In the present specification, reprogramming refers to returning differentiated cells back to multipotent / pluripotent stem cells again, and the reprogramming of cells is also referred to as the initialization of cells. Biologically, reprogramming means the elimination or reconstitution of epigenetic marks such as DNA methylation. Technologies for creating iPS cells by introducing reprogramming factors into somatic cells are representative examples of cell reprogramming. In the present invention, reprogramming is performed using an HDAC inhibitor, which is a DNA-damaging substance, without performing gene transfer. In the present specification, the step of culturing cells in a medium containing a DNA dysfunction substance (HDAC inhibitor) is hereinafter referred to as step (a).

[0016] <DNA dysfunction substance> A substance that impairs DNA function means a substance that can impair any of the DNA functions of DNA synthesis, DNA transcription (RNA synthesis), or DNA replication by binding to DNA (deoxyribonucleic acid) or acting directly or indirectly on DNA.

[0017] <Histone deacetylase inhibitor> In the present invention, a histone deacetylase inhibitor is used as a DNA dysfunction substance. Histone deacetylase (HDAC) is an enzyme that deacetylates histones, which are major components of chromatin structure. HDAC inhibitors control gene transcription by inhibiting HDAC. More specifically, in differentiated cells, histones exist in a condensed state, and HDAC inhibitors relax the condensed chromatin structure, thereby enabling the transcription of paused reprogramming-related genes to begin transcription.

[0018] Examples of HDAC inhibitors used in the present invention include 2-mercaptoethanol, ethylene oxide, butyric acid, apicidin, valproic acid, trichostatin A, and vorinostat.

[0019] 2-mercaptoethanol is a compound represented by the structural formula HS-CH-CH-OH and is also known as β-mercaptoethanol or thioglycol. The CAS registry number for 2-mercaptoethanol is 60-24-2. 2-mercaptoethanol can be synthesized by reacting hydrogen sulfide with ethylene oxide, and preferably, commercially available industrially produced products can be used. For example, reagents and products from Fujifilm Wako Pure Chemical Industries can be used.

[0020] Ethylene oxide is a compound represented by the molecular formula C2H4O and is also known as epoxyethane, oxirane, oxacyclopropane, ethylene oxide, or ethylene oxide, and is abbreviated as EO. The CAS registration number for ethylene oxide is 75-21-8. Ethylene oxide can be synthesized by reacting ethylene with oxygen, and preferably, an industrially produced, commercially available product can be used. For example, products from Mitsubishi Chemical, Showa Denko Gas Products, Taiyo Nippon Sanso, etc. can be used. When using ethylene oxide, it may be added to the cell culture medium, but it is preferable to spray ethylene oxide gas onto the cell culture equipment (culture vessel, etc.).

[0021] 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 it is preferable to use 2-mercaptoethanol.

[0022] <cell> The cells cultured in the method of the present invention are preferably somatic cells. The somatic cells used in the present invention are not particularly limited as long as they are differentiated cells, and any cell or cell population thereof other than germ cells can be used. Examples of somatic cells include, but are not limited to, already differentiated cells such as fibroblasts, adipocytes, nerve cells, muscle cells (e.g., cardiac muscle cells, smooth muscle cells, skeletal muscle cells), skin cells, epithelial cells, endothelial cells, blood cells (e.g., neutrophils, eosinophils, basophils, monocytes, lymphocytes), hepatocytes, kidney cells, lung cells, pancreatic cells, hair matrix cells (e.g., skin hair matrix cells, body hair matrix cells), and oral cells (e.g., oral mucosal cells); somatic stem cells such as hematopoietic stem cells, mesenchymal stem cells, neural stem cells, and adipose-derived stem cells; and various progenitor cells. In the present invention, from the viewpoint of ease of obtaining cells, fibroblasts (more preferably skin fibroblasts), oral cells, and hair matrix cells are preferred, with fibroblasts being more preferred, and skin fibroblasts being particularly preferred.

[0023] Somatic cells can be collected from animals such as mammals and birds. Mammals (mammals) include, for example, primates such as humans, monkeys, chimpanzees, gorillas, and orangutans; rodents such as mice and rats; rabbits, dogs, cats, cows, pigs, goats, sheep, and horses; and birds include, but are not limited to, chickens and ducks. Somatic cells may be fetal somatic cells or mature somatic cells. Furthermore, somatic cells may be primary cultured cells or passaged cells. When the resulting reprogrammed cells or differentiated cells or tissues are transplanted, it is preferable to use somatic cells collected from the target animal (i.e., autologous somatic cells) or somatic cells collected from an animal of the same species. Furthermore, when the transplantation is involved in the treatment of a disease, it is preferable to use somatic cells from a tissue involved in the disease.

[0024] <Cell culture> The present invention comprises a step of culturing cells in a medium containing an HDAC inhibitor, and by carrying out at least this step, cells can be reprogrammed. The cells may be cultured in contact with a medium containing an HDAC inhibitor, and preferably in a medium containing an HDAC inhibitor.

[0025] The content of HDAC inhibitor in the medium is not particularly limited, but can be expressed, for example, as the amount at which the proportion of cells one week after the start of culture is 70% or more (preferably 80% or more, more preferably 90% or more, and even more preferably 95% or more) of the number of cells cultured for one week without the HDAC inhibitor (one week of culture under the same conditions except for the absence of the HDAC inhibitor). It can also be expressed as the amount of cells that exhibit a cell aggregate-like morphology (spheroids) one week after the start of culture. The content can be appropriately set depending on the type of HDAC inhibitor, culture conditions, etc., and is, for example, 0.01 μM to 50 mM, preferably 0.1 μM to 10 mM, more preferably 0.5 μM to 7 mM, and even more preferably 1 μM to 5 mM.

[0026] When the HDAC inhibitor is 2-mercaptoethanol, its content in the medium is, for example, 0.1 μM to 10 mM, preferably 0.1 μM to 2 mM, more preferably 0.1 μM to 5 mM, and even more preferably 10 μM to 3 mM. Considering the influence on cells, within the limit where reprogramming is possible, it is preferable that the amount of 2-mercaptoethanol is less. From such a perspective, 2-mercaptoethanol may be 10 μM to 0.2 mM, 10 μM to 100 μM, or 10 μM to 50 μM. 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 a much lower concentration of 2-mercaptoethanol than that.

[0027] As described below, in the present invention, the order of allowing an HDAC inhibitor such as 2-mercaptoethanol to act and then allowing an OCT3 / 4 transcriptional stimulator (such as LIF, CCL2, IL-6) to act is important for cell reprogramming. Therefore, the medium in step (a) does not contain an OCT3 / 4 transcriptional stimulator. Preferably, the medium in step (a) does not contain any cytokines or hormones such as ACTH, bFGF, and a GSK3β inhibitor.

[0028] <OCT3 / 4 transcriptional stimulator> The OCT3 / 4 transcriptional stimulator is a factor that stimulates and activates the transcription of OCT3 / 4, 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 it is more preferable to use LIF and CCL2.

[0029] In the present invention, the chromatin structure is relaxed by an HDAC inhibitor, and then, by allowing an OCT3 / 4 transcriptional stimulator to act, the transcription of OCT3 / 4 is activated, and the function of proteins centered on OCT3 / 4 is used to shift the cells to an undifferentiated state.

[0030] In the method of the present invention (step (a)), the conditions for culturing the cells are not particularly limited. For example, the culture temperature can be 30 to 40°C, preferably 35 to 39°C, and more preferably 36 to 38°C. The CO2 concentration is, for example, 1 to 10%, preferably 1.5 to 8%, and more preferably 2 to 5%.

[0031] The cell culture period in step (a) is not particularly limited and can be appropriately set depending on the type of cells used, culture conditions, etc. The cell culture period in the present invention is, for example, one day or more, and a specific culture period can be 1 to 10 days. The preferred culture period is also not particularly limited, but is, for example, 2 to 9 days, preferably 3 days or more, and more preferably 3 to 7 days.

[0032] The composition of the medium used for cell culture is not particularly limited and can be appropriately determined depending on the type of cells used, the culture conditions, etc. For example, the basal medium may be, but is not particularly limited to, Eagle's medium (BM, MEM, DMEM, etc.), McCoy's medium (McCoy5A, McCoy7A, etc.), Ham's medium (F10, F12, etc.), 199 medium, RPMI1640 medium, NCTC medium (NCTC109, NCTC135, etc.), etc. Furthermore, various basal media may be mixed and used as needed.

[0033] The above-mentioned basal medium may also contain additives normally required for cell maintenance, such as 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 (type I collagen, gelatin, fibronectin, etc.), fatty acids (oleic acid, arachidonic acid, linolenic acid, etc.), adenine, guanosine, hypoxanthine, thymidine, cholesterol, etc.

[0034] In the present invention, by performing the culture in the above step (a), as a result, morphological changes of cells may be observed. Here, in this specification, the morphological change of cells means that the appearance of the cell population after culture changes from the state at the start of culture. For example, as shown in the photograph of FIG. 1B, a state may be observed in which the cells after culture begin to aggregate from the periphery. After confirming the morphological change of cells in such a state, the above culture step can be stopped.

[0035] <Culture in the presence of OCT3 / 4 transcriptional stimulator> The method of the present invention further includes, following the above culture step, a step of culturing cells in a medium containing one or more selected from the group consisting of OCT3 / 4 transcriptional stimulators such as LIF, CCL2, and IL-6 (hereinafter referred to as step (b) in this specification).

[0036] In addition to OCT3 / 4 transcriptional stimulators such as LIF, the medium used in step (b) may contain any one or more of ACTH (adrenocorticotropic hormone), bFGF (basic fibroblast growth factor), and GSK3β inhibitor. In the present invention, it is preferable to use ACTH and bFGF in combination with LIF or LIF and CCL2, and most preferably to use ACTH, bFGF, and GSK3β inhibitor in combination with LIF or LIF and CCL2.

[0037] In the present invention, the term "GSK3β inhibitor" refers to a substance having 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-pyridinecarbonitrile), CHIR98014 (N-6-[2-[[4-(2,4-dichlorophenyl)-5-(1H-imidazol-1-yl)-2-pyrimidinyl]amino]ethyl]amino]-3-pyridinecarbonitrile), [3-(2,4-dichlorophenyl)-4-(1-methyl-1H-indol-3-yl)-1H-pyrrole-2,5-dione), 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.

[0038] The animals from which the OCT3 / 4 transcription stimulators (such as LIF, CCL2, and IL-6), ACTH, bFGF, and GSK3β inhibitors are derived are not particularly limited, and OCT3 / 4 transcription stimulators, ACTH, bFGF, and GSK3β inhibitors derived from any animal, such as humans, mice, rats, rabbits, sheep, pigs, or cows, can be used. Furthermore, the OCT3 / 4 transcription stimulators, ACTH, bFGF, and GSK3β inhibitors may all be recombinant. Commercially available reagents can be used for all of these. The OCT3 / 4 transcription stimulators, ACTH, bFGF, and GSK3β inhibitors used in the present invention are preferably derived from human-derived materials, and are preferably recombinant.

[0039] The contents of OCT3 / 4 transcription stimulatory factors (such as LIF, CCL2, and IL-6), ACTH, bFGF, and GSK3β inhibitor in the medium in step (b) are not particularly limited and can be set appropriately depending on the type of cells used, culture conditions, etc.

[0040] The amount of LIF contained in the medium is not particularly limited, but is, for example, 0.01 to 5,000 ng / mL, preferably 0.1 to 1,000 ng / mL, and more preferably 1 to 100 ng / mL. Furthermore, although not particularly limited, the amount of LIF contained in the medium is, for example, 10 to 10,000 units / mL, preferably 50 to 8,000 units / mL, and more preferably 100 to 5,000 units / mL, where 1 unit is 1 / 20 of the amount that gives 50% of the maximum proliferation rate in a cell proliferation promotion assay using a mouse ES cell line (D3 strain). When using LIF from Fujifilm Wako Pure Chemical Industries (product number 129-05601), the amount can be diluted 500 to 2,000 times with respect to the volume of the medium.

[0041] The content of CCL2 in the medium is not particularly limited, but is, for example, 0.1 to 5000 ng / mL, preferably 1 to 1000 ng / mL, more preferably 10 to 500 ng / mL, 10 to 400 ng / mL, 10 to 300 ng / mL, 50 to 500 ng / mL, 50 to 400 ng / mL, or 50 to 300 ng / mL.

[0042] The content of ACTH in the medium is not particularly limited, but is, for example, 0.1 to 200 μmol / L, preferably 1 to 100 μmol / L, and more preferably 5 to 50 μmol / L.

[0043] The content of bFGF in the medium is not particularly limited, but is, for example, 0.01 to 5 nmol / L, preferably 0.05 to 3 nmol / L, and more preferably 0.1 to 1 nmol / L.

[0044] The content of the GSK3β inhibitor in the medium is not particularly limited, but is, for example, 0.1 to 50 μM, preferably 0.5 to 30 μM, and more preferably 1 to 10 μM.

[0045] The medium composition used for cell culture in step (b) can also be appropriately determined depending on the type of cells used, culture conditions, etc. Basically, the medium is not particularly limited as long as it is a medium that can maintain reprogrammed cells. For example, the basal medium can be, but is not particularly limited to, Eagle's medium (BM, MEM, DMEM, etc.), McCoy's medium (McCoy5A, McCoy7A, etc.), Ham's medium (F10, F12, etc.), 199 medium, RPMI1640 medium, NCTC medium (NCTC109, NCTC135, etc.), stem cell medium, etc. Furthermore, various basal media can be mixed and used as needed.

[0046] In addition to OCT3 / 4 transcription stimulatory factors (such as LIF, CCL2, and IL-6), ACTH, bFGF, and a GSK3β inhibitor, the above-mentioned basal medium may also contain additives favorable for maintaining stem cells, such as serum (fetal bovine serum, human serum, etc.), serum substitutes (such as KSR), amino acids (such as alanine, arginine, cystine, and histidine), vitamins (such as vitamin B7 and vitamin B12), antibiotics (such as amphotericin B and kanamycin), adhesion factors (such as type I collagen, gelatin, and fibronectin), growth factors (such as EGF, PDGF, and TGF-α), cytokines (such as IL-2, IL-3, IL-4, IL-5, and IL-6), hormones (such as insulin, glucagon, and progesterone), fatty acids (such as oleic acid, arachidonic acid, and linolenic acid), adenine, guanosine, hypoxanthine, thymidine, and cholesterol.

[0047] In step (b), a medium already prepared as a stem cell medium can also be used. Examples include, but are not limited to, Wako Pure Chemical Industries' StemSure® hPSC Medium Δ, Kohjin Bio's ADSC-4 Medium, ReproCell's StemFit, and StemCell Technologies' TeSR series. Commercially available stem cell media may contain OCT3 / 4 transcription stimulator, ACTH, bFGF, or a GSK-3β inhibitor. It is not necessary to change the medium from step (a) in step (b). The medium used in step (a) can be used as is, and step (b) can be performed by adding one or more selected from the group consisting of LIF, ACTH, bFGF, and a GSK-3β inhibitor to the medium.

[0048] In step (b), the conditions for culturing the cells are not particularly limited. For example, the culture temperature can be 30 to 40°C, preferably 35 to 39°C, and more preferably 36 to 38°C. The CO2 concentration is, for example, 1 to 10%, preferably 1.5 to 8%, and more preferably 2 to 5%.

[0049] The cell culture period in step (b) is not particularly limited and can be appropriately set depending on the type of somatic cells used, the culture conditions, etc. The cell culture period in step (b) is, for example, one day or more, and a specific culture period can be 1 to 10 days. The preferred culture period is also not particularly limited, but is, for example, 2 to 10 days, preferably 3 days or more, and more preferably 3 to 9 days.

[0050] In the present invention, further morphological changes in the cells may be observed as a result of the culture in step (b). For example, as shown in Figure 2, the cells after culture may aggregate to form a cell aggregate. Such a cell aggregate may also be called a cell mass (spheroid). After confirming such a morphological change in the cells, the culture in step (b) may be stopped.

[0051] In the present invention, as described above, a preferred embodiment is to perform step (b) following step (a). Without being bound by any particular theory, if a drug that disrupts DNA itself or a DNA function (e.g., DNA replication or RNA synthesis, including indirect functions of RNA (specifically, the series of functions for synthesizing proteins from RNA)) is defined as a "triggering drug" (a drug that provides input for reprogramming to cells), and a drug used to maintain reprogrammed cells (e.g., LIF or ACTH) is defined as a "drug necessary for the progression of reprogramming," it is believed that the order of administering the "triggering drug" and then the "drug necessary for the progression of reprogramming" to cells is important, rather than administering the "triggering drug" and the "drug necessary for the progression of reprogramming" to cells simultaneously. The concentration of the "triggering drug" used is a concentration that does not affect cell viability.

[0052] <Cell passage> In the present invention, cells can be passaged in the culture of steps (a) and (b). The cell passage procedure is not particularly limited and can be performed appropriately depending on the type of cells used, the culture conditions, etc. For example, when it is confirmed that the cells are in a confluent state by microscopic observation, the medium can be removed from the culture vessel, a buffer solution such as PBS(-) can be added to the culture vessel to wash the cell surface, and a protease such as trypsin can then be added to the vessel to recover the cells. The recovered cells can be added to new medium, and the cells can be passaged through this series of operations.

[0053] The number of times the cells are passaged can be 1 to 10, but is not particularly limited thereto, and can be appropriately set depending on the type of somatic cells used, culture conditions, etc. Although not particularly limited, in the present invention, the number of times the cells are passaged is preferably 1 to 5, more preferably 1 to 3, even more preferably 1 or 2, and most preferably 1.

[0054] The present invention provides a method for producing reprogrammed cells from somatic cells without gene introduction, comprising: (a) culturing somatic cells in a medium containing a histone deacetylase inhibitor; and (b) culturing the cells cultured in step (a) in a medium containing an OCT3 / 4 transcription stimulator, thereby producing reprogrammed cells.

[0055] The reprogrammed cells are multipotent stem cells that can differentiate into three germ layers. The pluripotent stem cells can be pluripotent.

[0056] <Multipotent / pluripotent stem cells> As used herein, "pluripotent stem cells" refer to cells that have the ability to self-renew by dividing and proliferating while maintaining an undifferentiated state, and the ability to differentiate into cells of multiple lineages. Preferably, the pluripotent stem cells of the present invention have the pluripotency to differentiate into cell lineages belonging to the three germ layers (endoderm, mesoderm, and ectoderm).

[0057] As used herein, pluripotent stem cells refer to cells that have the ability to self-renew, that is, to proliferate by dividing while maintaining an undifferentiated state, and the pluripotency to differentiate into all cell lineages belonging to the three germ layers (endoderm, mesoderm, and ectoderm). Pluripotent stem cells have the ability to form teratomas and chimeras.

[0058] The terms "multipotent" and "pluripotent" are not necessarily clearly distinguished. In this specification, the reprogrammed cells of the present invention are described as "multipotent" because they are unlikely to have teratoma-forming ability (tumor-forming ability). However, this does not exclude the cells from being "pluripotent" in the commonly used sense.

[0059] The cells obtained by the method of the present invention can be confirmed as highly undifferentiated cells by using methods known to those skilled in the art. One method for confirmation is, for example, examining alkaline phosphatase activity. Alkaline phosphatase activity can be appropriately examined using a commercially available alkaline phosphatase staining kit or the like.

[0060] Pluripotent stem cells can be identified by confirming the expression of pluripotent stem cell marker genes such as Nanog, Klf4, Oct4, Sox2, c-Myc, Lin28, TRA-1-60, and SSEA (SSEA-4, SSEA-1, etc.). Expression of marker genes can be confirmed using methods known to those skilled in the art, such as RT-PCR, and confirmation at the protein level can be carried out using devices and methods known per se, such as antibodies specific to various markers or FACS.

[0061] Pluripotency can also be confirmed by examining differentiation into the three germ layers of endoderm, mesoderm, and ectoderm. Differentiation into the three germ layers can be confirmed by examining markers for each germ layer. For example, endoderm markers include Sox17, CXCR4, HNF-3β, FoxA2, ​​AFP, GATA-4, PDX-1, and Nkx2.1; mesoderm markers include MSX1, α-SMA, Obt2, and Brachyury; and ectoderm markers include Pax6, MAP2, Nestin, Otx2, TP63, and SOX2. Various markers can be confirmed using methods known to those skilled in the art, similar to the confirmation of pluripotent stem cell markers.

[0062] Teratoma formation ability can be evaluated, for example, by subcutaneously injecting cells into a mouse to form a teratoma and analyzing the differentiated tissue. Teratomas include differentiated tissues derived from the three germ layers of endoderm, mesoderm, and ectoderm. Chimera formation ability can be confirmed, for example, by injecting cells into a blastocyst and examining whether a chimeric animal is formed from the blastocyst. Both teratoma formation ability and chimera formation ability can be examined using methods known to those skilled in the art.

[0063] The differentiation ability can be examined in vitro using a differentiation induction method known to those skilled in the art. Differentiation into various cells may be carried out using a commercially available differentiation induction kit or the like.

[0064] <Cell manufacturing method> In another aspect, the present invention provides a method for producing cells using pluripotent stem cells obtained by the above-mentioned method. Specifically, the present invention provides a method for producing cells, comprising the following steps (i) and (ii): (i) producing reprogrammed cells from somatic cells by the methods described above; (ii) inducing differentiation of the reprogrammed cells into cells of interest; The present invention provides a method for producing cells, comprising:

[0065] Step (i) can be carried out by the above step (a) or by combining step (a) with step (b). In addition, the differentiation induction of cells in step (ii) can be carried out appropriately using methods known to those skilled in the art depending on the type of target cells.

[0066] The cells obtainable by the production method of the present invention are not particularly limited, but examples thereof include fat cells, nerve cells, muscle cells (cardiomyocytes, 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, and hair cells.

[0067] Kits and Compositions In another aspect, the present invention provides a kit for reprogramming cells, comprising a medium containing an HDAC inhibitor. In yet another aspect, the present invention provides a composition for reprogramming cells, comprising a medium containing an HDAC inhibitor. The HDAC inhibitor and the medium containing it are as described in step (a) of the method of the present invention described above.

[0068] The kit of the present invention comprises, for example, the following two reagents: Reagent A) Reagent for constituting a medium without 2-mercaptoethanol; Reagent B) Reagents for constituting a medium containing LIF. Reagent A and Reagent B may be prepared immediately after use by packaging the medium and the components separately, or each component may be contained in the medium. The medium composed of Reagent A may further contain CCL2 and / or IL-6 as OCT3 / 4 transcription stimulatory factors.

[0069] Reagent B may contain one or more selected from the group consisting of ACTH, bFGF, and a GSK3β inhibitor, in addition to LIF, CCL2, and IL-6. The media used in Reagent A and Reagent B are as described in steps (a) and (b) of the method of the present invention.

[0070] <Pharmaceutical compositions, cosmetic compositions> The present invention provides cells or cell populations thereof reprogrammed by the methods of the present invention. The cells of the present invention are not particularly limited, but one of their characteristics is that they are obtained using an HDAC inhibitor and therefore do not contain exogenous genes for Oct3 / 4, Sox2, Klf4, c-Myc, etc. Furthermore, one of the morphological characteristics of the cell populations (cell aggregates) of cells reprogrammed by the methods of the present invention is, but is not particularly limited, that the boundaries of the cell populations are unclear and the interiors are heterogeneous, as shown in the Examples described below (Figure 2B).

[0071] Cells reprogrammed by the methods of the present invention, differentiated cells of such cells (cells differentiated from cells reprogrammed by the methods of the present invention), or extracts or secreted components of such cells (components extracted from or secreted by cells reprogrammed by the methods of the present invention) can be used as active ingredients in pharmaceutical compositions or cosmetic compositions. That is, in another aspect, the present invention provides pharmaceutical compositions or cosmetic compositions containing cells reprogrammed by the methods of the present invention described above, differentiated cells of such cells, or extracts or secreted components of such cells. In another aspect, the present invention provides methods for producing pharmaceutical or cosmetic compositions, comprising the step of incorporating cells reprogrammed by the methods of the present invention described above, differentiated cells of such cells, or extracts or secreted components of such cells.

[0072] Components extracted from or secreted by cells reprogrammed by the methods of the present invention are not particularly limited, and examples include gene-related substances such as miRNA, DNA, or RNA, proteins, cytokines, extracellular vesicles (including exosomes), miRNA, DNA, RNA, and proteins contained in extracellular vesicles, etc. These may be of one type only, or a combination of two or more types.

[0073] The uses of the pharmaceutical composition are not particularly limited, but include, for example, the treatment of olfactory disorders, cerebral infarction, diabetes, nerve disorders, cancer, liver diseases, etc. When cells reprogrammed by the method of the present invention are used, the cells themselves or cells differentiated from the cells can be used for cell transplantation.

[0074] The pharmaceutical or cosmetic composition of the present invention may contain a pharmacologically acceptable carrier depending on its form. Examples of pharmacologically acceptable carriers include, but are not limited to, excipients, binders, emulsifiers, tonicity agents (isotonicity agents), buffers, solubilizers, preservatives, stabilizers, antioxidants, colorants, coagulants, and coating agents.

[0075] Regarding the above-mentioned uses, the present invention can also provide, as another aspect, a method for treating any of the above-mentioned diseases and a use for treating any of the above-mentioned diseases. That is, as another aspect, the present invention provides a method for treating any of the above-mentioned diseases, using cells reprogrammed by the above-mentioned method of the present invention, cells differentiated from such cells, or extracts or secreted components of such cells. Furthermore, as another aspect, the present invention provides use of cells reprogrammed by the above-mentioned method of the present invention, cells differentiated from such cells, or extracts or secreted components of such cells, for treating any of the above-mentioned diseases. [Example]

[0076] The present invention will be explained in more detail below by way of examples, but these are merely illustrative and do not limit the scope of the present invention in any way. Example 1

[0077] 1. Somatic cell reprogramming Normal human dermal fibroblasts (adult) (Takara Bio) were seeded onto spheroid plates, and after 24-48 hours, the medium was replaced with D-MEM (Low Glucose) medium supplemented with 2-mercaptoethanol diluted 1 / 100,000 (142 μM). After 3-4 days, the medium was replaced again with the same D-MEM medium.

[0078] After culturing human dermal fibroblasts for one week as described above, changes in cell morphology were confirmed (Figure 1). After confirming this change in cell morphology, the medium was replaced with a new 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) for ADSC-4 (Kohjin Bio). After 3–4 days, the medium was replaced with the same medium as above.

[0079] As described above, when cells were cultured for one week in ADSC-4 medium containing LIF, etc., the cells further aggregated and formed spheroids (Figure 2).

[0080] 2. Alkaline Phosphatase Reaction The alkaline phosphatase activity of the cells finally obtained by the above culture was examined. Alkaline phosphatase staining was performed using Alkaline Phosphatase Live Stain (Invitrogen) according to the kit's instructions. Specifically, the cells were cultured for 30 minutes in medium containing the staining solution (AP Live Stain) provided with the kit diluted 500-fold in D-MEM. The medium was then replaced and the cells were washed twice for 5 minutes. After washing, the cells were observed under a fluorescent microscope using a GFP filter to determine whether or not they were stained. Staining was confirmed (Figure 3).

[0081] 3. Examination of various conditions (1) 2-mercaptoethanol concentration The same procedures as in items 1 and 2 above were performed to examine the concentration of 2-mercaptoethanol. Specifically, the same procedures as in items 1 and 2 above were performed except that the 2-mercaptoethanol concentrations were 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, cell spheroid formation and alkaline phosphatase staining were observed at 2-mercaptoethanol concentrations of 0.142 μM to 1.42 mM, and particularly strong alkaline phosphatase staining was observed at 2-mercaptoethanol concentrations of 14.2 μM to 1.42 mM. Note that cell death was observed at 2-mercaptoethanol concentrations of 14.2 mM or higher.

[0082] (2) Culture medium used As in Section 1 above, cells were cultured in D-MEM medium containing 2-mercaptoethanol (142 μM). LIF (Wako, used at a 1:1000 ratio), ACTH (Wako, 10 μmol / L), bFGF (Wako, 0.34 nmol / L), and AR-A014418 (Merck, 3 μM) were then added to the medium and cultured for another week. As a result, both cell spheroid formation and alkaline phosphatase staining were observed. Furthermore, when cells were cultured solely in D-MEM medium containing 2-mercaptoethanol (142 μM) as described in item 1 above, spheroid formation and weak alkaline phosphatase staining were observed. However, when cells were cultured solely in ADSC-4 medium supplemented with LIF (Wako, used at 1:1000), ACTH (Wako, 10 μmol / L), bFGF (Wako, 0.34 nmol / L), and AR-A014418 (Merck, 3 μM), neither spheroid formation nor alkaline phosphatase staining was observed.

[0083] (3) LIF and ACTH factors In the above item 1, when ADSC-4 cells were cultured in the presence of (i) LIF only, (ii) ACTH only, or (iii) LIF and ACTH only, spheroid formation and alkaline phosphatase staining were observed under all conditions. Under conditions (i) and (iii), particularly strong alkaline phosphatase staining was observed.

[0084] 4. Expression of undifferentiated markers Gene expression of the undifferentiation markers Nanog, Klf4, Oct4, and Sox2 was examined. RNA was extracted from the cells using the RNeasy Mini Kit (QIAGEN). PCR was performed for the undifferentiation markers using the method described in Cell. 2007 Nov 30;131(5):861-72.

[0085] As a result, bands for Nanog, Klf4, Oct4, and Sox2 were all detected in the cultured cells tested, near the target band sizes (Nanog: 406 bp, Klf4: 396 bp, Oct4: 143 bp, Sox2: 150 bp) (Figure 4). It was also confirmed that no bands were detected in the negative control.

[0086] 5. Induction of adipocyte differentiation The cells obtained in section 1 above were subjected to a test for differentiation induction into adipocytes using a commercially available cell differentiation kit. The kit used for the test was Mesenchymal Stem Cell Adipogenic Differentiation Medium 2 (PromoCell, product code C-28016). The cell culture medium was replaced with the culture medium provided with the kit, and the cells were then cultured according to the instructions provided with the kit.

[0087] Lipid droplets observed in the differentiation-induced cells were stained using LipiDye (Funakoshi). Live cell imaging was performed according to the instructions provided with the kit. Specifically, the cells were cultured for 2 hours in D-MEM medium containing the staining solution provided with the kit at a final concentration of 1 μM. The medium was then replaced and the cells were washed twice for 5 minutes. After washing, the cells were observed under a fluorescence microscope using a GFP filter to determine whether or not they were stained, and staining was observed (Figure 5).

[0088] 6. Induction of differentiation into neural cells The cells obtained in section 1 above were used to conduct a neuronal differentiation test 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 medium was replaced with the culture medium provided with the kit, and the cells were then cultured according to the instructions provided with the kit.

[0089] The differentiated cells were immunostained using an anti-neurofilament antibody (abcam, ab7255, Anti-68kDa Neurofilament / NF-L antibody [DA2]), an antibody specific to neurons (Figure 6).

[0090] 7. Induction of differentiation into hepatocytes A differentiation induction test into hepatocytes was performed on the cells obtained in section 1. The cells were cultured in 0.8 μM hexachlorophene / D-MEM medium according to the method described in Scientific Reports (2015) 5: 16169.

[0091] The differentiated cells were subjected to PAS staining, Western blotting, and indocyanine green assay to confirm their hepatocyte properties. PAS staining was performed using a commercially available PAS staining kit (Muto Chemical) according to the kit's instructions. For Western blotting, cell lysates were prepared using CellLytic M (Sigma-Aldrich) according to the kit's instructions. The prepared cell lysates were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) according to the Laemmli method, then transferred to a PVDF membrane using the semi-dry method for Western blot analysis. Anti-albumin rabbit polyclonal antibody (4929: Cell Signaling Technology) and anti-β-actin mouse monoclonal antibody (A5441: Sigma-Aldrich) were used as primary antibodies. HRP-conjugated antibodies against IgG from the same animal were used as secondary antibodies. Signal detection was performed using a luminometer imaging system LAS4000 (Fujifilm). The indocyanine green test was performed according to the method described in Cloning Stem Cells (2007) Spring;9(1):51-62, and observations were made 1 hour and 20 hours after the start of staining (Figure 8).

[0092] 8. Induction of differentiation into chondrocytes The cells obtained in section 1 above were subjected to a chondrocyte differentiation test using a commercially available cell differentiation kit. The kit used for the test was Mesenchymal Stem Cell Chondrogenic Differentiation Medium (PromoCell, product code C-28012). The cell culture medium was replaced with the culture medium provided with the kit, and the cells were then cultured according to the instructions provided with the kit.

[0093] The differentiated cells were subjected to HE staining, Alcian blue staining, and immunostaining (S-100 antibody, GFAP antibody, CD34 antibody, SMA antibody, and Vimentin antibody) to evaluate the proteins expressed in the cells (Figure 11). When compared with fibrocartilage and hyaline cartilage from randomly selected human clinical specimens, protein expression similar to that of fibrocartilage was observed, demonstrating that the chondrocytes obtained by differentiation induction are similar to fibrocartilage.

[0094] 9. Induction of osteoblast differentiation Following the procedure described in Section 1 above, cells were cultured in D-MEM medium containing 2-mercaptoethanol, followed by culture in ADSC-4 medium containing LIF, ACTH, and bFGF. A test to induce differentiation into chondrocytes was then performed using a commercially available osteoblast differentiation medium: Mesenchymal Stem Cell Osteogenic Differentiation Medium (PromoCell, product code C-28013).

[0095] After differentiation induction, the cells were stained for alkaline phosphatase and confirmed to be alkaline phosphatase positive (FIG. 12). Example 2 1. Effects of CCL2 The effect of adding CCL2 to LIF on reprogramming was examined. According to Example 1, normal human dermal fibroblasts (adult) (Takara Bio) were cultured for 7 days in D-MEM medium containing 2-mercaptoethanol (142 μM), with the medium effect being observed for 3–4 days. Next, ADSC-4 cells were cultured for an additional 7 days in medium supplemented with (i) LIF alone, (ii) LIF + CCL2 (10 ng / mL), or (iii) LIF + CCL2 (100 ng / mL). LIF was used at a concentration of 1:1000.

[0096] After culturing (i) to (iii), morphological changes were observed in the cells (a change from a spindle-shaped form to a slightly rounded form with slightly depressed protrusions) (Figure 13). In particular, the addition of CCL2 resulted in significant cell aggregation, clearer cell boundaries, and a breakdown of cell adhesion (Figure 13D).

[0097] 2. Induction of differentiation into adipocytes 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 using a commercially available cell differentiation kit according to item 5 of Example 1.

[0098] The cells on day 25 after differentiation induction were stained with LipiDye (Funakoshi) (Figures 14 and 15). Lipid droplets were observed in both cells, but the number and size of lipid droplets formed were increased in cells (iii) compared to cells (ii).

[0099] Furthermore, we examined the differences in differentiation induction into adipocytes between the period of culturing in the presence of 2-mercaptoethanol (step A) and the period of culturing in the presence of LIF (step B), including the case where a period of culturing in medium alone was set between steps A and B. Step A (3-9 days) → Culture period in medium only (0-14 days) → Step B (3-9 days) → Induction of adipogenesis (14 days)

[0100] First, step A and step B were each performed for 3 days, and the culture period in the medium was set to 0, 3, 7, and 10 days. - Differentiation induction from Step A for 3 days to Step B for 3 days resulted in the formation of lipid droplets. Step A 3 days → Culture medium 3 days → Step B Differentiation induction from day 3 resulted in the formation of lipid droplets. Step A: 3 days → 7 days in culture medium → Step B: Differentiation induction from day 3 resulted in the formation of lipid droplets. Step A: 3 days → 10 days in culture medium → Step B: Differentiation induction from day 3 resulted in the formation of lipid droplets. When the culture period in the medium was 0, 3, or 7 days, small but numerous lipid droplets were formed 14 days after differentiation induction, and the lipid droplets became larger and more numerous after 21 days. When the culture period in the medium was 10 days, there were significantly fewer lipid droplets 14 days after differentiation induction.

[0101] The culture period in the medium was set as day 0, and the culture periods in steps A and B were varied. Lipid droplets were formed after differentiation induction from step A for 5 days and step B for 7 days. Lipid droplets were formed after differentiation induction from Step A for 7 days and Step B for 7 days. Lipid droplets were formed after differentiation induction from step A on day 9 and step B on day 7. At 14 days after differentiation induction, the cells with lower density appeared to have more lipid droplets, but after about 21 days, the lipid droplets became larger and more numerous regardless of density.

[0102] From the above results, it was confirmed that a culture period of 3 days for each of Step A and Step B is sufficient. [Industrial Applicability]

[0103] 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, for example, for producing organs for regenerative medicine. Furthermore, since it is possible to transform cells into other organs, in fields other than human medicine, for example, cow skin cells can be transformed into muscle cells for human food.

[0104] All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety.

Claims

1. If the target somatic cells are mammalian somatic cells other than fibroblasts, A method for producing a reprogrammed multipotent stem cell from a subject's somatic cell without gene transfer, comprising: (a) culturing the subject somatic cells in a first medium that does not contain LIF, CCL2, and IL-6, and that contains 2-mercaptoethanol, thereby obtaining precursors of pluripotent stem cells; and (b) culturing the precursors of the pluripotent stem cells obtained in step (a) in a second medium containing one or more selected from the group consisting of LIF, CCL2, and IL-6, thereby producing pluripotent stem cells that are reprogrammed cells.

2. The second medium comprises LIF; The method of claim 1.

3. The method of claim 1, wherein the second culture medium does not contain 2-mercaptoethanol.

4. The method of claim 1, wherein the target somatic cells are one or more of the following: fat cells, nerve cells, muscle cells, skin cells, epithelial cells, endothelial cells, blood cells, liver cells, kidney cells, lung cells, pancreatic cells, hair matrix cells, oral cells, hematopoietic stem cells, and somatic stem cells.

5. The method of claim 1 , wherein the subject somatic cells are hair matrix cells or oral cells.

6. When a target somatic cell is a mammalian somatic cell other than a fibroblast, a method for producing a target cell from the target somatic cell comprises the steps of: (i) preparing pluripotent stem cells, which are reprogrammed cells, from the subject somatic cells by the method according to any one of claims 1 to 5; and (ii) inducing differentiation of the pluripotent stem cells into cells of interest; A method comprising:

7. 1. A method for producing a cell preparation, comprising: If the target somatic cells are mammalian somatic cells other than fibroblasts, (i) preparing pluripotent stem cells, which are reprogrammed cells, from a subject somatic cell by the method according to any one of claims 1 to 5; (ii) inducing differentiation of the pluripotent stem cells into target cells and obtaining the cells after differentiation induction; and (iii) preparing a cell preparation containing the differentiation-induced cells and a pharmacologically acceptable carrier; A method comprising:

Images