WO2014167943A1 - Protein-containing component for inducing cell re-programming, method for manufacturing pluripotent cells using said component, and medium comprising said component - Google Patents

Protein-containing component for inducing cell re-programming, method for manufacturing pluripotent cells using said component, and medium comprising said component Download PDF

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WO2014167943A1
WO2014167943A1 PCT/JP2014/056948 JP2014056948W WO2014167943A1 WO 2014167943 A1 WO2014167943 A1 WO 2014167943A1 JP 2014056948 W JP2014056948 W JP 2014056948W WO 2014167943 A1 WO2014167943 A1 WO 2014167943A1
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cells
protein
cell
containing component
lactic acid
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Japanese (ja)
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訓正 太田
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国立大学法人熊本大学
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    • C12P1/04—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes by using bacteria
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    • C12N2510/00—Genetically modified cells

Definitions

  • the present invention relates to protein-containing components that induce cell reprogramming.
  • the present invention also relates to a method for producing pluripotent cells using such protein-containing components.
  • the invention further relates to a medium comprising such protein-containing components.
  • ES cells called embryonic stem cells, were discovered from mouse embryos in 1981 and from rabbit embryos in 1998.
  • ES cells have the ability to change into various types of cells other than the cells that make up the placenta (pluripotency), and studies have been mainly conducted to construct tissues and organs using these cells.
  • ES cells have a large ethical problem because they use fertilized eggs that become life if they grow smoothly.
  • Another major problem is the problem of rejection. Even if differentiated cells or organs created based on ES cells are transplanted into patients, the immune system may recognize them as non-self and attack them.
  • iPS cells Since the cells used at this time are derived from somatic cells such as the patient's own differentiated skin, even if cells differentiated from iPS cells are transplanted into the patient, the immune system recognizes the organ as self and the transplant is rejected. There is nothing. The discovery of iPS cells also cleared the issue of “bioethics” that ES cells had.
  • iPS cells are attracting worldwide attention as a trump card for regenerative medicine, but there remains a technical problem that cells become cancerous.
  • One of the causes of canceration is due to the c-Myc gene introduced into the cell, but recently, iPS cells were also produced by three factors other than the c-Myc gene.
  • the introduction of genes into cells has been made one step closer to the production of iPS cells that are safer and more practical by using iPS cells using adenoviruses and plasmids, rather than using mouth-opening viruses.
  • some artificial genes are forcedly expressed in cells that have undergone cell differentiation, the possibility that these cells will become cancerous in the future cannot be denied.
  • Patent Document 1 proposes a method for producing reprogrammed embryonic stem cells (ES) -like cells using Mycobacterium leprae or its components.
  • Patent Document 1 describes a method of producing reprogrammed ES-like cells by contacting and infecting adult differentiated cells with Mycobacterium leprae itself, and cells produced by this method. Yes. It is described that the contacted / infected bacteria are present in the produced ES-like cells.
  • Mycobacterium leprae is a leprosy, and there is a safety concern for its application to regenerative medicine.
  • Patent Document 2 a method for producing pluripotent cells from somatic cells by infecting somatic cells with lactic acid bacteria or natto bacteria, which are fermentative bacteria, has been reported by the present inventor (Patent Document 2).
  • An object of the present invention is to provide a novel substance that induces cell reprogramming and a composition containing the substance. Another object of the present invention is to provide a pluripotent cell that is highly safe in application to regenerative medicine, and a method for producing the same. It is to provide a pluripotent cell with less safety concerns and a method for producing the same. Another object of the present invention is to provide a medium that can be used in the production method.
  • the present inventor has paid attention to bacteria having fermentation ability such as lactic acid bacteria and natto bacteria for a long time, and as a result of investigating the relationship between the bacteria having fermentation ability and cells, It was found that when each bacterium was infected, a cell mass was formed like ES cells and iPS cells and stained by alkaline phosphatase staining.
  • the present inventor reprograms chick skin cells that have undergone cell differentiation to form cell masses like ES cells and iPS cells, even if they are protein-containing components derived from lactic acid bacteria rather than living lactic acid bacteria themselves As a result, the present invention has been completed. Moreover, differentiation was confirmed when these cell masses were induced to differentiate into various cells.
  • a pluripotent cell derived from an isolated mammalian (eg, human or mouse) somatic cell comprising the following steps: (A) a protein-containing component that induces reprogramming of a cell that can be obtained from a homogenate of a bacterium having fermentation ability into the somatic cell, and having the following properties: (I) binds to m-aminophenylboronic acid; Culturing or contacting with a protein-containing component having at least one, preferably at least two, and most preferably all of (ii) not binding to concanavalin A and (iii) not binding to wheat germ lectin Maintaining, and (b) recovering the formed cell mass, A pluripotent cell produced by a production process comprising.
  • a protein-containing component that induces reprogramming of a cell that can be obtained from a homogenate of a bacterium having fermentation ability into the somatic cell, and having the following properties: (I) binds to m-aminophenyl
  • the protein-containing component comprises (a) a step of precipitating a homogenate obtained by crushing a bacterium having fermentation ability at an ammonium sulfate concentration of 20 to 60% to obtain an ammonium sulfate precipitate, and (b) the ammonium sulfate precipitate.
  • (8) A cell induced to differentiate by culturing the pluripotent cell according to any one of (1) to (7) above in a differentiation-inducing medium. (9) The cell according to (8), wherein the cell is an adipocyte, bone cell, chondrocyte, nerve cell, cardiomyocyte, hepatocyte, pancreatic cell, or blood cell.
  • a method for producing pluripotent cells from somatic cells of an mammal eg, human or mouse (isolated or in vivo), A protein-containing component that induces reprogramming of cells that can be obtained from a bacterial homogenate having fermentation ability into the somatic cells, and having the following properties: (I) binds to m-aminophenylboronic acid; Contacting a protein-containing component that has at least one, preferably at least two, and most preferably all that does not bind to (ii) concanavalin A and (iii) does not bind to wheat germ lectin.
  • a method for producing pluripotent cells from somatic cells are described in situs.
  • the protein-containing component is a component that is obtained by concentrating bacterial homogenate using an ammonium sulfate precipitation method and does not pass through a 100 KDa ultrafiltration filter.
  • the protein-containing component comprises a protein having a molecular weight of about 55 to about 65 KDa (more preferably about 60 KDa) as a main band in SDS-PAGE.
  • the bacterium having fermentation ability is lactic acid bacteria or natto bacteria.
  • the protein-containing component comprises (a) a step of precipitating a homogenate obtained by crushing bacteria having fermentation ability at an ammonium sulfate concentration of 20 to 60% to obtain an ammonium sulfate precipitate, and (b) the ammonium sulfate precipitate.
  • a protein-containing component that induces reprogramming of cells that can be obtained from a bacterial homogenate having fermentation ability, and having the following properties: (I) binds to m-aminophenylboronic acid; Cell reprogramming containing a protein-containing component that does not bind to concanavalin A and (iii) does not bind to wheat germ lectin, preferably at least two, most preferably all Inducing composition. (18) The composition according to (17), wherein the protein-containing component is a component that is obtained by concentrating bacterial homogenate using an ammonium sulfate precipitation method and does not pass through a 100 KDa ultrafiltration filter. object.
  • composition according to (17) or (18), wherein the protein-containing component comprises a protein having a molecular weight of about 55 to about 65 KDa (more preferably about 60 KDa) as a main band in SDS-PAGE.
  • the bacterium having fermentation ability is lactic acid bacteria or natto bacteria.
  • the lactic acid bacterium is a lactic acid bacterium of the genus Lactococcus, Streptococcus, or Lactobacillus.
  • the protein-containing component comprises (a) precipitating a homogenate obtained by crushing bacteria having fermentation ability at an ammonium sulfate concentration of 20 to 60% to obtain an ammonium sulfate precipitate; and (b) the ammonium sulfate precipitate.
  • a solution for example, buffer
  • a 100 KDa ultrafiltration filter preferably (c) the non-permeate fraction using anion exchange chromatography
  • the composition according to any one of the above (17) to (21) which is a protein-containing component obtained by a process comprising the step of (23)
  • An anticancer agent comprising the composition according to any one of (17) to (23).
  • a reprogramming medium for mammalian (eg, human or mouse) somatic cells comprising a homogenate or crude product from lactic acid bacteria or Bacillus natto.
  • the homogenate or crude product has the following properties: (I) binds to m-aminophenylboronic acid; Characterized in that it comprises a component having at least one, preferably at least two, and most preferably all of (ii) not binding to concanavalin A and (iii) not binding to wheat germ lectin.
  • DMEM Dulbecco's modified Eagle medium
  • EME Eagle's minimal essential
  • IMDM Iskov modified Dulbecco medium
  • ⁇ -MEM alpha-minimal essential medium
  • RPMI 1640 Ham-F-12
  • DMEM Dulbecco's modified Eagle medium
  • EME Eagle's minimal essential
  • IMDM Iskov modified Dulbecco medium
  • ⁇ -MEM alpha-minimal essential medium
  • RPMI 1640 Ham-F-12
  • Ham-F-12 The medium according to any one of (25) to (28), selected from the group consisting of MCDB and modified medium thereof.
  • Induced protein-containing component with the following properties: (I) binds to m-aminophenylboronic acid; Use of a protein-containing component that has at least one, preferably at least two, and most preferably all of (ii) does not bind to concanavalin A and (iii) does not bind to wheat germ lectin.
  • the protein-containing component is a component having a size that does not pass through a 100 KDa ultrafiltration filter obtained by concentrating a bacterial homogenate using an ammonium sulfate precipitation method. .
  • the protein-containing component comprises a protein showing a molecular weight of about 55 to about 65 KDa (more preferably about 60 KDa) as a main band in SDS-PAGE.
  • the bacterium having fermentation ability is lactic acid bacteria or natto bacteria.
  • the fraction includes (a) a step of precipitating a homogenate solution obtained by crushing bacteria having fermentation ability at an ammonium sulfate concentration of 20 to 60% to obtain an ammonium sulfate precipitate, and (b) dissolving the ammonium sulfate precipitate.
  • the obtained solution for example, buffer
  • a non-permeate fraction preferably (c) the non-permeate fraction is separated using anion exchange chromatography.
  • pluripotent stem cells can be produced from somatic cells without using gene introduction and forced expression into somatic cells, or introduction of bacteria themselves.
  • pluripotent stem cells can be produced using a composition containing a component (protein-containing component) obtained from a bacterium having fermentation ability such as lactic acid bacteria that exists in the human body and coexists with cells.
  • the method for producing pluripotent cells according to the present invention includes the medical field (drug discovery research and drug safety, efficacy and side effect testing), disease research (elucidation of the cause of intractable diseases, development of treatments and prevention methods), Useful in regenerative medicine (function repair of nerves, blood vessels, organs) and food.
  • HDF cells cultured with lactic acid bacteria components after 3 days in culture. After culturing HDF cells treated with lactic acid bacteria components in a medium that induces differentiation into adipocytes, bone cells, and chondrocytes, each cell is subjected to Oil Red O staining (fat), Alizarin Red S staining (bone), and The result of Alcian Blue staining (cartilage) is shown. A549 cells cultured with lactic acid bacteria components (after 14 days in culture) are shown. The results of culturing A549 cells treated with lactic acid bacteria components in a medium that induces differentiation into adipocytes and bone cells, followed by Oil Red O staining (fat) and Alizarin Red S staining (bone) are shown.
  • HepG2 cells cultured with lactic acid bacteria components (after 14 days in culture) are shown. Shows the result of culturing HepG2 cells treated with lactic acid bacteria components in a medium that induces differentiation into adipocytes and bone cells, and then staining each cell with Oil Red O staining (fat) and Alizarin Red S staining (bone) .
  • MCF7 cells cultured with lactic acid bacteria components (after 14 days in culture) are shown. MCF7 cells treated with lactic acid bacteria components were cultured in a medium that induces differentiation into adipocytes and bone cells, and then each cell was stained with OilORed O (fat) and Alizarin Red S (bone). .
  • a protein-containing component capable of inducing cell reprogramming or “a cell reprogramming-inducing protein-containing component” refers to an isolated mammalian somatic cell (normal somatic cell, derived from somatic cell). For example, by contacting with epithelial cells, somatic cells can be transformed into pluripotent cells having the ability to differentiate into various cells, similar to ES cells and iPS cells.
  • a protein-containing component that can transform a character is not particularly limited as long as the derived protein-containing component can be contacted with the somatic cell, but preferably, the component is present in the environment in which the somatic cell survives (for example, a medium).
  • the derived protein-containing component here has the following properties: (i) binds to m-aminophenylboronic acid, (ii) does not bind to concanavalin A, and (iii) binds to wheat germ lectin A protein-containing component having at least one, preferably at least two, and most preferably all.
  • the protein-containing component of the present invention is preferably a component that does not permeate a 100 KDa ultrafiltration filter. More preferably, the protein-containing component of the present invention contains, as a main band, a protein showing a molecular weight of about 55 to about 65 KDa (more preferably about 60 KDa) on SDS-PAGE.
  • the protein may exist in a single state, or may be in a state in which another substance is covalently or non-covalently bound, for example, in a complex with another substance.
  • the derived protein-containing component may be purified to some extent or may be roughly purified. For example, as shown in the present invention, since the derived protein-containing component is present in lactic acid bacteria, it may be contacted with somatic cells as a homogenate or crude product of lactic acid bacteria.
  • composition or “cell reprogramming induction composition” is not particularly limited as long as it is a form containing an inducible protein-containing component as a part thereof, and unless otherwise specifically used in the specification.
  • the method for producing pluripotent cells from somatic cells comprises contacting a somatic cell with a protein-containing component or a composition comprising the same that can induce reprogramming of a cell obtainable from a bacterium capable of fermentation.
  • a step of causing Such a protein-containing component can be obtained, for example, by concentrating a bacterial cell extract having fermentation ability by an ammonium sulfate precipitation method. For example, it can be obtained as an ammonium sulfate precipitate fraction precipitated at an ammonium sulfate concentration of 10 to 80% saturation, preferably 20 to 70% saturation, more preferably 20 to 60% saturation.
  • the ammonium sulfate precipitation fraction thus obtained can be obtained from a fraction that does not permeate a 100 KDa ultrafiltration filter.
  • protein-containing components can be made more pure by, for example, further purifying the fraction using anion chromatography, hydrophobic interaction chromatography, gel filtration chromatography, or a combination thereof.
  • An example of a protein contained in the protein-containing component used in the present invention is preferably a glycoprotein, more preferably a glycoprotein exhibiting a molecular weight of about 55 to 65 KDa in SDS-PAGE, more preferably about a protein in SDS-PAGE. It is a glycoprotein showing a molecular weight of 60 KDa.
  • glycoproteins contained in protein-containing components are preferably (1) bound to m-aminophenylboronic acid, (2) not bound to concanavalin A, or (3) A glycoprotein having one or more properties that do not bind to wheat germ lectin, and most preferably a glycoprotein having all these properties.
  • somatic cell used for initialization in the present invention is not particularly limited, and any somatic cell can be used. That is, the somatic cells referred to in the present invention include all cells other than germ cells among the cells constituting the living body, and may be differentiated somatic cells or undifferentiated stem cells.
  • epithelial cells For example, but not limited to, epithelial cells, endothelial cells, fibroblasts (skin cells, etc.), intestinal cells, hepatocytes, spleen cells, pancreatic cells, kidney cells, hair cells, muscle cells, brain cells, lung cells, Examples include adipocytes, gastric mucosa cells, and differentiated cells such as lymphocytes, somatic stem cells such as neural stem cells, hematopoietic stem cells, mesenchymal stem cells, and dental pulp stem cells, and tissue precursor cells.
  • epithelial cells for example, but not limited to, epithelial cells, endothelial cells, fibroblasts (skin cells, etc.), intestinal cells, hepatocytes, spleen cells, pancreatic cells, kidney cells, hair cells, muscle cells, brain cells, lung cells, Examples include adipocytes, gastric mucosa cells, and differentiated cells such as lymphocytes, somatic stem cells such as neural stem cells, hematopo
  • the origin of the somatic cell is not particularly limited as long as it is a mammal, but it is preferably a rodent such as a mouse, or a primate such as a baboon, and particularly preferably a rabbit or a mouse.
  • a rodent such as a mouse
  • a primate such as a baboon
  • any fetal, neonatal or adult somatic cells may be used.
  • somatic cells isolated from the patient suffering from the disease it is preferable to use somatic cells isolated from the patient suffering from the disease.
  • cancer cells can be used as somatic cells.
  • Protein-containing components that can induce reprogramming of cells that can be obtained from bacteria having fermentation ability into cancer cells for example, components containing proteins having the properties described above (from cell extracts of bacteria having fermentation ability)
  • a non-cancer cell can be produced from a cancer cell by contacting with a non-cancer cell.
  • the step of bringing a somatic cell (including a cancer cell) into contact with a protein-containing component that can induce reprogramming of a cell that can be obtained from a bacterium having fermentation ability can be performed in vitro. Can also be done in the body.
  • the pluripotent cell referred to in the present invention has a self-replicating ability over a long period of time under a predetermined culture condition, and various cells (ectodermal cells, mesodermal cells, Or a cell having pluripotency into an endoderm cell or the like (such a cell is also referred to as a stem cell).
  • various cells ectodermal cells, mesodermal cells, Or a cell having pluripotency into an endoderm cell or the like (such a cell is also referred to as a stem cell).
  • the kind of bacteria having fermentative ability that can obtain the reprogramming-inducing protein-containing component used in the present invention is not particularly limited, and may be an aerobic bacterium such as lactic acid bacterium or natto bacterium, or an anaerobic bacterium such as bifidobacteria.
  • lactic acid bacteria are particularly preferred.
  • the kind of lactic acid bacteria used in the present invention is not particularly limited as long as it has fermentation ability.
  • Lactic acid bacteria is a general term for bacteria having the ability to produce lactic acid from sugars by fermentation.
  • Representative lactic acid bacteria include Lactobacillus, Bifidobacterium, Enterococcus, Lactococcus, Pediococcus, and Pediococcus.
  • lactic acid bacteria belonging to the genus Lactococcus, Streptococcus, or Lactobacillus can be used.
  • Lactococcus Lactis subsp. Lactis, Streptococcus salivarius subsp. Thermophilus, Lactobacillus sp., Or Lactobacillus acidophilus can be used particularly preferably.
  • the present invention by using a normal medium for cell culture, culturing somatic cells in the presence of a protein-containing component that can induce reprogramming of cells that can be obtained from bacteria having fermentation ability.
  • the pluripotent cells or non-cancer cells of the present invention (cells that are non-cancerous by reprogramming cancer cells) can be isolated and cultured.
  • the medium for producing and culturing the pluripotent cells of the present invention is not particularly limited, and any medium that can be used for culturing pluripotent cells can be used, for example, but not limited thereto, Dulbecco's modified Eagle's medium (DMEM), Eagle's minimal essential (EME) medium, Iskov's modified Dulbecco medium (IMDM), alpha-minimal essential medium ( ⁇ -MEM), RPMI 1640, Ham-F-12, MCDB, and modifications thereof Medium can be raised.
  • DMEM Dulbecco's modified Eagle's medium
  • EME Eagle's minimal essential
  • IMDM Iskov's modified Dulbecco medium
  • ⁇ -MEM alpha-minimal essential medium
  • RPMI 1640 Ham-F-12
  • MCDB and modifications thereof Medium
  • the medium is preferably a serum-free medium from the viewpoint of subsequent use of the produced pluripotent cells and induction efficiency, and further, if necessary, various growth factors, cyto force-in, hormones and the like (for example, FGF- 2, TGF ⁇ -1, Activin A, Nanoggin, BDNF, NGF, NT-1, NT-2, NT-3 and other components involved in the growth and maintenance of chick ES cells may be added) .
  • growth factors, cyto force-in, hormones and the like for example, FGF- 2, TGF ⁇ -1, Activin A, Nanoggin, BDNF, NGF, NT-1, NT-2, NT-3 and other components involved in the growth and maintenance of chick ES cells may be added.
  • Such a medium is also part of the present invention.
  • the differentiation ability and proliferation ability of the separated pluripotent cells can be confirmed by using confirmation means known for ES cells.
  • the uses of the pluripotent cells and non-cancer cells produced by the method of the present invention are not particularly limited, and can be used for various tests / researches and disease treatments.
  • a growth factor such as retinoic acid, EGF, or glucocorticoid
  • desired differentiated cells for example, nerve cells, cardiomyocytes, hepatocytes, pancreas
  • stem cell therapy by autologous cell transplantation can be achieved by returning the differentiated cells thus obtained to the patient.
  • Examples of central nervous system diseases that can be treated using the pluripotent cells of the present invention include Parkinson's disease, Alzheimer's disease, multiple sclerosis, cerebral infarction, spinal cord injury and the like.
  • pluripotent cells can be differentiated into dopaminergic new mouth and transplanted into the striatum of Parkinson's disease patients. Differentiation into dopaminergic new mouth can be promoted by co-culturing mouse stromal cell line PA6 cells and the pluripotent cells of the present invention under serum-free conditions.
  • the pluripotent cells of the present invention can be induced to differentiate into neural stem cells and then transplanted to the site of injury.
  • the pluripotent cells of the present invention can be used for the treatment of liver diseases such as hepatitis, cirrhosis and liver failure.
  • the pluripotent cells of the present invention can be differentiated into hepatocytes or hepatic stem cells and transplanted.
  • Hepatocytes or hepatic stem cells can be obtained by culturing the pluripotent cells of the present invention in the presence of activin A for 5 days and then culturing with hepatocyte growth factor (HGF) for about 1 week.
  • HGF hepatocyte growth factor
  • the pluripotent cells of the present invention can be used for the treatment of pancreatic diseases such as type I diabetes.
  • pancreatic diseases such as type I diabetes
  • the pluripotent cells of the present invention can be differentiated into pancreatic ⁇ cells and transplanted into the pancreas.
  • the method of differentiating pluripotent cells of the present invention into pancreatic ⁇ cells can be performed according to the method of differentiating ES cells into pancreatic ⁇ cells.
  • the pluripotent cells of the present invention can be used for the treatment of heart failure associated with ischemic heart disease.
  • the pluripotent cells of the present invention are preferably differentiated into cardiomyocytes and then transplanted to the site of injury.
  • the pluripotent cells of the present invention can obtain cardiomyocytes in about 2 weeks after the formation of embryoid bodies by adding noggin 3 days before the formation of embryoid bodies and adding it to the medium.
  • non-cancer cells can be transformed from cancer cells by contacting the cancer cells with a protein-containing component that can induce reprogramming of cells that can be obtained from bacteria having fermentation ability.
  • a protein-containing component that can induce reprogramming of cells that can be obtained from bacteria having fermentation ability.
  • the cell reprogramming-inducing protein-containing component used in the present invention or a composition containing the same is useful as an anticancer agent.
  • the protein-containing component capable of inducing reprogramming of cells provided by the present invention can initialize cells that have undergone abnormal differentiation such as differentiated cells and cancer cells, so that it can be used as an additive to pharmaceuticals and cosmetics. Can be used.
  • the reprogramming-inducing protein-containing component of the present invention is safe because it is a substance derived from lactic acid bacteria or the like.
  • Example 1 Preparation of lactic acid bacteria component Lactic acid bacteria were purchased from the Microbial Materials Development Office, RIKEN BioResource Center. After sterilizing the MRS medium with high-pressure steam, lactic acid bacteria (Lactobacillus acidophilus; JCM1021) were inoculated into 20 ml of the MRS medium and cultured with shaking at 37 ° C. for 2 to 3 days. Next, the cells were inoculated into a 3 L shake flask containing 1 L of the sterilized medium and cultured at 37 ° C. for 3 to 4 days. Bacteria were collected from 1 L of the obtained culture broth by centrifugation at 10,000 rpm for 10 min.
  • lactic acid bacteria Lactic acid bacteria
  • the dialyzed sample was then collected and filtered through a 100 KDa ultrafiltration filter (Millipore) to collect a concentrated sample that did not permeate the membrane.
  • the protein fraction (or protein complex) was quantified using Protein Assay Kit (Bio Rad) and used as the “lactic acid bacteria component” in the following experiments.
  • the protein concentration was 5 mg / ml.
  • Example 2 Culture of HDF cells in the presence of lactic acid bacteria components
  • HDF cells Human Dermal Fibroblasts, CELL APPLICATIONS, INC. Cat No. 106-05a
  • CMF Fibroblast Growth Medium
  • the cells were washed with 10 ml of CMF (Ca 2+ Mg 2+ free buffer), and 1 ml of 0.25% trypsin solution (containing 1 mM EDTA) was added to spread the whole.
  • the cells were placed in a CO 2 incubator (37 ° C.) for 5 minutes, suspended by adding 3 ml of a trypsin inhibitor solution (CELL APLICATION INC.), And the number of cells was counted.
  • Lactobacillus acidophilus (JCM1021) -derived lactic acid bacterium component (50 ⁇ l) obtained in Example 1 per well was placed in advance in a 6-well plate, and 5 ⁇ 10 5 HDF cells were added. Lactic acid bacteria were purchased from the Microbial Materials Development Department, RIKEN BioResource Center. The cells were cultured as they were in a 34 ° C., 5% CO 2 incubator. As a result, cell clusters could be observed after several days. The photograph in FIG. 1 shows the state of cells 3 days after culturing.
  • Example 3 Induction of differentiation of cells (lactic acid bacteria component-treated HDF cells)
  • the HDF cell mass (A) prepared in Example 2 was cultured in the presence of lactic acid bacteria components for 2 weeks to obtain fat cells (B) and bone cells (C).
  • the medium was replaced with a culture medium (GIBCO; A10070-01, A10072-01, A10071-01) that induces differentiation induction in the chondrocytes (D), and further cultured for 2 to 3 weeks.
  • GIBCO culture medium that induces differentiation induction in the chondrocytes (D), and further cultured for 2 to 3 weeks.
  • the results are shown in FIG. As shown in FIG.
  • the HDF cell mass to which the lactic acid bacteria component was allowed to act was cultured in a differentiation-inducing medium, and (B) Oil Red O staining (fat), (C) Alizarin Red S staining (bone), (D ) Stained by Alcian Blue staining (cartilage), and differentiation of cells into each cell could be confirmed.
  • Example 4 Culture of lung cancer cell line (A549) in the presence of lactic acid bacteria components
  • a lung cancer cell line (A549; RBRC-RCB0098) was obtained from BioResource Center.
  • 50 ⁇ l of lactic acid bacteria per well was placed in a 6-well plate in advance, and 5 ⁇ 10 5 lung cancer cells (A549) were added.
  • the cells were cultured as they were at 34 ° C. in a 5% CO 2 incubator. As a result, cell clusters could be observed after several days.
  • the photograph in FIG. 3 shows the state of cells 14 days after culturing.
  • Example 5 Differentiation induction of cells (lactic acid bacteria component-treated A549 cells)
  • a lung cancer cell line (A549; RBRC-RCB0098) was obtained from RIKEN BioResource Center.
  • 50 ⁇ l of lactic acid bacteria per well was placed in a 6-well plate in advance, and 5 ⁇ 10 5 lung cancer cells (A549) were added.
  • the cells were cultured as they were at 34 ° C. in a 5% CO 2 incubator.
  • Photo (A) is from 14 days after culturing.
  • a cell mass derived from a lung cancer cell line (A549) in which a lactic acid bacterium component is allowed to act is cultured in a differentiation-inducing medium, and then stained with (B) Oil Red O staining (fat), (C) Alizarin Red S staining (bone), Cell differentiation was confirmed.
  • Photo (D) is an enlargement of the square part of photo (C).
  • Example 6 Culture of a hepatoma cell line (HepG2) in the presence of lactic acid bacteria components
  • a hepatoma cell line (HepG2; RBRC-RCB1648) was obtained from BioResource Center.
  • 50 ⁇ l of lactic acid bacteria per well was placed in a 6-well plate in advance, and 5 ⁇ 10 5 liver cancer cells (HepG2) were added.
  • the cells were cultured as they were at 34 ° C. in a 5% CO 2 incubator. As a result, cell clusters could be observed after several days.
  • the photograph in FIG. 5 shows the state of the cells 14 days after culturing.
  • Example 7 Differentiation induction of cells (lactic acid bacteria component-treated HepG2 cells)
  • 50 ⁇ l of lactic acid bacteria components per well were previously placed in a 6-well plate, and 5 ⁇ 10 5 hepatoma cells (HepG2 ) was added.
  • the cells were cultured as they were at 34 ° C. in a 5% CO 2 incubator.
  • the formed cell mass is cultured in the presence of lactic acid bacteria components for 2 weeks, and replaced with a culture solution (GIBCO; A10070-01, A10072-01) that induces differentiation induction in fat cells (B) and bone cells (C),
  • the cells were further cultured for 2 to 3 weeks.
  • the results are shown in FIG. Photo (A) is from 14 days after culturing.
  • HepG2 hepatoma cell line treated with lactic acid bacteria components is cultured in differentiation-inducing medium, and then stained with (B) Oil Red O staining (fat), (C) Alizarin Red S staining (bone) And cell differentiation was confirmed.
  • Example 8 Culture of Breast Cancer Cell Line (MCF7) in the Presence of Lactic Acid Bacteria Components
  • MCF7 breast cancer cell line
  • RBRC-RCB1904 RBRC-RCB1904
  • 50 ⁇ l of lactic acid bacteria component per well was placed in a 6 well plate in advance, and 5 ⁇ 10 5 breast cancer cells (MCF7) were added.
  • the cells were cultured as they were at 34 ° C. in a 5% CO 2 incubator. As a result, cell clusters could be observed after several days.
  • the photograph in FIG. 7 shows the state of the cells 14 days after culturing.
  • Example 9 Induction of differentiation of cells (lactic acid bacteria component-treated MCF7 cells)
  • 50 ⁇ l of lactic acid bacteria components per well were previously placed in a 6-well plate, and 5 ⁇ 10 5 breast cancer cells (MCF7) were added. added.
  • the cells were cultured as they were at 34 ° C. in a 5% CO 2 incubator.
  • the formed cell mass is cultured in the presence of lactic acid bacteria components for 2 weeks, and replaced with a culture solution (GIBCO; A10070-01, A10072-01) that induces differentiation induction in fat cells (B) and bone cells (C),
  • the cells were further cultured for 2 to 3 weeks.
  • the results are shown in FIG. Photo (A) is from 14 days after culturing.
  • a cell mass derived from a breast cancer cell line (MCF7) treated with a lactic acid bacteria component is cultured in a differentiation induction medium, and then stained with (B) Oil Red O staining (fat), (C) Alizarin Red S staining (bone), Cell differentiation was confirmed.
  • Example 10 Differentiation induction of cells (lactic acid bacteria component-treated HepG2 cells)
  • 50 ⁇ l of lactic acid bacteria components per well were previously placed in a 6-well plate, and 5 ⁇ 10 5 hepatoma cells (HepG2 ).
  • the cells were cultured as they were at 34 ° C. in a 5% CO 2 incubator.
  • the cell mass is cultured in the presence of lactic acid bacteria components for 2 weeks, then transferred to a cover glass coated with poly-L-lysine and laminin, cultured for 7 days, anti- ⁇ -fetoprotein antibody (endoderm marker), anti-NeuroFilament antibody (ectodermal) , Nerve cell marker).
  • FIG. 9 the results are shown in FIG. As shown in FIG. 9, the cells after differentiation induction were recognized by each antibody.
  • Example 11 Pluripotency marker of lung cancer cell line (A549) cultured in the presence of lactic acid bacteria component
  • the same experiment as in Example 4 was performed using the lung cancer cell line (A549) in the presence or absence of lactic acid bacteria component.
  • the experiment was conducted. Collect cells on day 14 of culture and use various pluripotency markers (Oct3 / 4, Sox2, Nanog, GDF-3, Rex1, ECAT, FGF4) and early aging markers (p15, p16, ARF) RT-PCR was performed.
  • the results are shown in FIG. Interestingly, expression of Nanog, which is said to be a pluripotent master gene, was strongly induced when A549 cells and lactic acid bacteria components were allowed to act (arrows).
  • Example 12 Identification of reprogramming factors
  • the concentrated lactic acid bacteria components 50 ⁇ l, protein amount 0.25 mg obtained in Example 1 were further separated by anion exchange chromatography ( QEF column).
  • QEF column anion exchange chromatography
  • the active fraction (0.1 M to 0.4 M) was collected.
  • HDF cells the effect of the active fraction was confirmed in the same manner as in Example 2. The results are shown in FIG. It was revealed that cell fraction forming activity exists in fractions 12 to 18.
  • Comparative Example 1 Comparison with E. coli component The same experiment as in Comparative Example 12 was performed using E. coli (XLI-blue: Stratagene), and fractions (0.1 M to 0.4 M NaCl fraction) were collected. This fraction was allowed to act on HDF cells, and the same experiment as in Example 2 was performed to confirm the cell mass forming activity. The results are shown in FIG. As a result, it was clarified that the cell mass-forming activity does not exist in the E. coli-derived fraction.
  • Example 13 Purification of lactic acid bacteria components
  • the lactic acid bacteria components obtained in Example 1 were further purified using the following steps (1) to (3).
  • Ion exchange chromatography HiPrep Q FF 16/10, GE Healthcare
  • Hydrophobic interaction chromatography HiTrap Phenyl FF (low sub) 5 ml, GE Healthcare
  • (3) Gel filtration chromatography HiLoad Superdex 200 prep grade, GE Healthcare
  • the sample after gel filtration chromatography having reprogramin activity was then subjected to SDS-PAGE. As a result, three bands stained with CBB were detected.
  • the molecular weight of the main band was about 60 KDa.
  • Example 14 Examination of physicochemical properties of cell reprogramming-inducing protein Using the purified product obtained in Example 13 with m-aminophenylboronic acid-agarose (Glycoprotein Enrichment Resin, Takara) that binds to glycoprotein And purified. As a result, binding with m-aminophenylboronic acid was confirmed. Further, using the purified product obtained in Example 13, the binding property was confirmed using concanavalin A and wheat germ lectin, which are lectins generally known to bind to glycoproteins. As a result, it did not bind to these lectins. Furthermore, when the refined product obtained in Example 13 was treated with Proteinase K, the reprogramming induction activity was lost.
  • m-aminophenylboronic acid-agarose Glycoprotein Enrichment Resin, Takara
  • Example 15 Cultivation of intestinal cells in the presence of lactic acid bacteria components
  • experiments were performed using intestinal cells.
  • cell clusters were observed after several days.
  • the present invention is useful as a method for producing pluripotent cells from somatic cells.

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Abstract

The purpose of the present invention is to provide a pluripotent cell that is very safe in regenerative medical treatment applications, and a manufacturing method therefor. The purpose of the present invention is also to provide a pluripotent cell with fewer safety concerns, particularly problems such as the malignant transformation of the cells or the presence of microbes in the cells, and a manufacturing method therefor. The present invention provides a method for manufacturing pluripotent cells from somatic cells. Said method comprises a process for bringing somatic cells into contact with a glycoprotein-containing component that can be obtained from fermentative microbes and is capable of inducing cell re-programming.

Description

細胞の再プログラミングを誘導するタンパク質含有成分、並びに該成分を用いた多能性細胞の製造方法および該成分を含む培地Protein-containing component that induces cell reprogramming, pluripotent cell production method using the component, and medium containing the component
本発明は、細胞の再プログラミングを誘導するタンパク質含有成分に関する。本発明はまた、そのようなタンパク質含有成分を用いて多能性細胞を製造する方法に関する。本発明はさらには、そのようなタンパク質含有成分を含む培地に関する。 The present invention relates to protein-containing components that induce cell reprogramming. The present invention also relates to a method for producing pluripotent cells using such protein-containing components. The invention further relates to a medium comprising such protein-containing components.
　ES細胞は、胚性幹細胞と呼ばれ、1981年にはマウスの胚から、1998年にはヒ卜の胚から発見された。ES細胞は胎盤を構成する細胞以外のさまざまな種類の細胞に変化する能力(多能性)を持つ細胞として、それを用いた組織や器官を構築する研究が主に行われてきた。しかしながら、ES細胞は順調に成長すれば生命になる受精卵を利用しているため、倫理的に大きな問題を抱えている。もうひとつの大きな問題として、拒絶の問題がある。ES細胞を元に作製した分化細胞や臓器を患者に移植しても、免疫系はこれらを非自己と認識し攻撃する可能性がある。 ES cells, called embryonic stem cells, were discovered from mouse embryos in 1981 and from rabbit embryos in 1998. ES cells have the ability to change into various types of cells other than the cells that make up the placenta (pluripotency), and studies have been mainly conducted to construct tissues and organs using these cells. However, ES cells have a large ethical problem because they use fertilized eggs that become life if they grow smoothly. Another major problem is the problem of rejection. Even if differentiated cells or organs created based on ES cells are transplanted into patients, the immune system may recognize them as non-self and attack them.
　これらES細胞の問題を解決するために、京都大学の山中伸弥教授のグループは、通常は他の機能を持つ細胞に分化しない皮膚細胞からさまざまな種類の細胞に変化する能力を持つ細胞を開発し、iPS細胞と名付けた。山中ファクターと呼ばれる4つの因子(Qct 3/4， Sox2， KLf4，c-Myc) をマウスやヒ卜の皮膚細胞にレ卜口ウイルスベクターを使って導入すると、細胞の初期化がおこり、ES細胞と同じく多能性を持つ細胞が作り出せることを示した［非特許文献１ ;及び非特許文献２］。この時に用いる細胞は、患者自身の分化した皮膚などの体細胞に由来するため、iPS細胞から分化させた細胞を患者に移植しても免疫系はその臓器を自己と認識し移植が拒絶されることはない。iPS細胞の発見によりES細胞が抱えていた「生命倫理」という問題もクリアされた。 To solve these ES cell problems, Professor Shinya Yamanaka's group at Kyoto University has developed cells with the ability to change from various types of skin cells that do not normally differentiate into cells with other functions. Named iPS cells. When four factors called Yamanaka Factor (Qct 3/4, Sox2, KLf4, c-Myc) are introduced into the skin cells of mice and chicks using a mouth-mouth virus vector, cell reprogramming occurs and ES cells It was shown that a cell having pluripotency can be produced as well [Non-patent Document 1; and Non-patent Document 2]. Since the cells used at this time are derived from somatic cells such as the patient's own differentiated skin, even if cells differentiated from iPS cells are transplanted into the patient, the immune system recognizes the organ as self and the transplant is rejected. There is nothing. The discovery of iPS cells also cleared the issue of “bioethics” that ES cells had.
　上記の通り、iPS細胞は再生医療の切り札として世界的に注目されているが、細胞が癌化してしまうという技術的な問題が残されている。癌化の原因のひとつは、細胞に導入したc-Myc遺伝子によるものだが、最近では、c-Myc遺伝子を除く3つの因子でもiPS細胞が作製された。また、遺伝子の細胞への導入もレ卜口ウイルスを使うのではなく、アデノウイルスやプラスミドを用いてiPS細胞を作製することで、より安全で実用化に近いiPS細胞の作製に一歩近づいた。しかし、人工的にいくつかの遺伝子を、細胞分化を終えた細胞に強制発現させる手法をとることから、将来、これらの細胞が癌化する可能性は否定できない。 As described above, iPS cells are attracting worldwide attention as a trump card for regenerative medicine, but there remains a technical problem that cells become cancerous. One of the causes of canceration is due to the c-Myc gene introduced into the cell, but recently, iPS cells were also produced by three factors other than the c-Myc gene. In addition, the introduction of genes into cells has been made one step closer to the production of iPS cells that are safer and more practical by using iPS cells using adenoviruses and plasmids, rather than using mouth-opening viruses. However, since some artificial genes are forcedly expressed in cells that have undergone cell differentiation, the possibility that these cells will become cancerous in the future cannot be denied.
　また、特許文献１において、マィコバクテリウム・レプラエ菌またはその成分を用いて、再ブログラミングされた胚幹細胞(ES) 様細胞を産生する方法が提案されている。特許文献１には、マイコバクテリウム・レプラエ菌そのものを、成人の分化細胞に接触・感染させることによる、再プログラミングされたES様細胞を産生する方法、この方法によって産生された細胞が記載されている。接触・感染させた菌は、産生されたＥＳ様細胞中に存在することが記載されている。しかしながら、マイコバクテリウム・レプラエ菌はらい菌であり、再生医療への応用には安全性の懸念がある。 Further, Patent Document 1 proposes a method for producing reprogrammed embryonic stem cells (ES) -like cells using Mycobacterium leprae or its components. Patent Document 1 describes a method of producing reprogrammed ES-like cells by contacting and infecting adult differentiated cells with Mycobacterium leprae itself, and cells produced by this method. Yes. It is described that the contacted / infected bacteria are present in the produced ES-like cells. However, Mycobacterium leprae is a leprosy, and there is a safety concern for its application to regenerative medicine.
　また、本発明者により、発酵能を有する細菌である乳酸菌や納豆菌を、体細胞に感染させて、体細胞から多能性細胞を製造する方法が報告されている（特許文献２）。 In addition, a method for producing pluripotent cells from somatic cells by infecting somatic cells with lactic acid bacteria or natto bacteria, which are fermentative bacteria, has been reported by the present inventor (Patent Document 2).
米国特許出願公開ＵＳ２００６／０２２２６３６Ａ１号明細書US Patent Application Publication US2006 / 0222636A1 国際公開公報ＷＯ２０１３／００８８０３号公報International Publication WO2013 / 008803
　本発明の目的は、細胞の再プログラミングを誘導する新規な物質および該物質を含む組成物を提供することである。本発明の目的はまた、再生医療への応用において安全性が高い多能性細胞、およびその製造方法を提供することであり、特には細胞の癌化の問題や、細胞内での菌の存在という、安全性に対する懸念がより少ない多能性細胞、およびその製造方法を提供することである。本発明の目的はまた、その製造方法に用いることができる培地を提供することである。 An object of the present invention is to provide a novel substance that induces cell reprogramming and a composition containing the substance. Another object of the present invention is to provide a pluripotent cell that is highly safe in application to regenerative medicine, and a method for producing the same. It is to provide a pluripotent cell with less safety concerns and a method for producing the same. Another object of the present invention is to provide a medium that can be used in the production method.
　本発明者は、以前より乳酸菌や納豆菌などの発酵能を有する細菌に着目し、発酵能を有する細菌と細胞との関係を調べた結果、細胞分化を終えたヒ卜皮膚細胞に乳酸菌または納豆菌をそれぞれ感染させると、ES細胞やiPS細胞のように細胞塊を形成し、アルカリホスファターゼ染色法で染色されることを見出した。
　本発明者は、生きた乳酸菌そのものではなく乳酸菌から由来するタンパク質含有成分であっても、細胞分化を終えたヒ卜皮膚細胞を再プログラミングし、ES細胞やiPS細胞のように細胞塊を形成させることを見出し、本発明を完成した。また、これらの細胞塊を種々の細胞へと分化誘導させたところ、分化が確認された。 The present inventor has paid attention to bacteria having fermentation ability such as lactic acid bacteria and natto bacteria for a long time, and as a result of investigating the relationship between the bacteria having fermentation ability and cells, It was found that when each bacterium was infected, a cell mass was formed like ES cells and iPS cells and stained by alkaline phosphatase staining.
The present inventor reprograms chick skin cells that have undergone cell differentiation to form cell masses like ES cells and iPS cells, even if they are protein-containing components derived from lactic acid bacteria rather than living lactic acid bacteria themselves As a result, the present invention has been completed. Moreover, differentiation was confirmed when these cell masses were induced to differentiate into various cells.
　即ち、以下の発明が提供される。
（１）単離された哺乳類動物（例えば、ヒトまたはマウス）の体細胞から誘導された多能性細胞であって、以下の工程、
（ａ）前記体細胞に、発酵能を有する細菌のホモジネートから得ることができる細胞を再プログラミング誘導するタンパク質含有成分であって、以下の性質：
（i）ｍ－アミノフェニルボロン酸に結合する、
（ii）コンカナバリンＡに結合しない、および
（iii）小麦胚芽レクチン（Wheat germ lectin）に結合しない
の少なくとも一つ、好ましくは少なくとも２つ、最も好ましくは全てを有するタンパク質含有成分を接触させて培養または維持する工程、および
（ｂ）形成された細胞塊を回収する工程、
含む製造工程により生産される多能性細胞。
（２）前記タンパク質含有成分が、細菌のホモジネートを硫安沈殿法を用い濃縮することにより得られた、１００ＫＤａの限外ろ過フィルターを透過しない大きさの成分である、前記（１）に記載の多能性細胞。
（３）前記タンパク質含有成分が、SDS-PAGEにおいて約５５～約６５ＫＤａ（より好ましくは約６０ＫＤａ）の分子量を示すタンパク質をメインバンドとして含む、前記（１）または（２）に記載の多能性細胞。
（４）前記発酵能を有する細菌が乳酸菌または納豆菌である前記（１）～（３）のいずれか一つに記載の多能性細胞。
（５）前記乳酸菌が、Lactococcus属、Streptococcus属、またはLactobacillus属の乳酸菌である前記（４）に記載の多能性細胞。
（６）前記タンパク質含有成分が、（a）発酵能を有する細菌を破砕することにより得られるホモジネートを硫安濃度２０～６０％飽和で沈殿させて硫安沈殿として得る工程、および（b）該硫安沈殿を溶解した溶液（例えば、緩衝液）を１００ＫＤａの限外ろ過フィルターを透過させ、透過しない画分を得る工程、（好ましくはさらに、（c）該透過しない画分を、陰イオン交換クロマトグラフィーを用いて分画した溶出画分から得る工程、）を含む工程により得られるタンパク質含有成分である、前記（１）～（５）のいずれか一つに記載の多能性細胞。
（７）前記体細胞が、ヒト由来の、正常体細胞またはがん細胞である前記（１）～（６）のいずれか一つに記載の多能性細胞。
（８）前記（１）～（７）のいずれか一つに記載の多能性細胞を分化誘導培地で培養することにより、分化誘導された細胞。
（９）前記細胞が、脂肪細胞、骨細胞、軟骨細胞、神経細胞、心筋細胞、肝細胞、膵臓細胞、または血球細胞である、前記（８）に記載の細胞。 That is, the following invention is provided.
(1) A pluripotent cell derived from an isolated mammalian (eg, human or mouse) somatic cell, comprising the following steps:
(A) a protein-containing component that induces reprogramming of a cell that can be obtained from a homogenate of a bacterium having fermentation ability into the somatic cell, and having the following properties:
(I) binds to m-aminophenylboronic acid;
Culturing or contacting with a protein-containing component having at least one, preferably at least two, and most preferably all of (ii) not binding to concanavalin A and (iii) not binding to wheat germ lectin Maintaining, and (b) recovering the formed cell mass,
A pluripotent cell produced by a production process comprising.
(2) The protein according to (1), wherein the protein-containing component is a component that is obtained by concentrating a bacterial homogenate using an ammonium sulfate precipitation method and does not permeate a 100 KDa ultrafiltration filter. Capable cells.
(3) The pluripotency according to (1) or (2) above, wherein the protein-containing component contains a protein showing a molecular weight of about 55 to about 65 KDa (more preferably about 60 KDa) as a main band in SDS-PAGE cell.
(4) The pluripotent cell according to any one of (1) to (3), wherein the bacterium having fermentation ability is lactic acid bacteria or natto bacteria.
(5) The pluripotent cell according to (4), wherein the lactic acid bacterium is a lactic acid bacterium of the genus Lactococcus, Streptococcus, or Lactobacillus.
(6) The protein-containing component comprises (a) a step of precipitating a homogenate obtained by crushing a bacterium having fermentation ability at an ammonium sulfate concentration of 20 to 60% to obtain an ammonium sulfate precipitate, and (b) the ammonium sulfate precipitate. A step of allowing a solution (for example, buffer) dissolved in the solution to pass through a 100 KDa ultrafiltration filter to obtain a non-permeate fraction (preferably (c) further subjecting the non-permeate fraction to anion exchange chromatography. The pluripotent cell according to any one of the above (1) to (5), which is a protein-containing component obtained by a process comprising the step of obtaining from the eluted fraction fractionated using
(7) The pluripotent cell according to any one of (1) to (6), wherein the somatic cell is a human-derived normal somatic cell or cancer cell.
(8) A cell induced to differentiate by culturing the pluripotent cell according to any one of (1) to (7) above in a differentiation-inducing medium.
(9) The cell according to (8), wherein the cell is an adipocyte, bone cell, chondrocyte, nerve cell, cardiomyocyte, hepatocyte, pancreatic cell, or blood cell.
（１０）（単離されたまたは生体内の）哺乳類動物（例えば、ヒトまたはマウス）の体細胞から多能性細胞を製造するための方法であって、
　前記体細胞に、発酵能を有する細菌のホモジネートから得ることができる細胞を再プログラミング誘導するタンパク質含有成分であって、以下の性質：
（i）ｍ－アミノフェニルボロン酸に結合する、
（ii）コンカナバリンＡに結合しない、および
（iii）小麦胚芽レクチン（Wheat germ lectin）に結合しない
の少なくとも一つ、好ましくは少なくとも２つ、最も好ましくは全てを有するタンパク質含有成分を接触させることを含む体細胞から多能性細胞を製造する方法。
（１１）前記タンパク質含有成分が、細菌のホモジネートを硫安沈殿法を用い濃縮することにより得られた、１００ＫＤａの限外ろ過フィルターを透過しない大きさの成分である、前記（１０）に記載の方法。
（１２）前記タンパク質含有成分が、SDS-PAGEにおいて約５５～約６５ＫＤａ（より好ましくは約６０ＫＤａ）の分子量を示すタンパク質をメインバンドとして含む、前記（１０）または（１１）に記載の方法。
（１３）前記発酵能を有する細菌が乳酸菌または納豆菌である前記（１０）～（１２）のいずれか一つに記載の方法。
（１４）前記乳酸菌が、Lactococcus属、Streptococcus属、またはLactobacillus属の乳酸菌である前記（１３）に記載の方法。
（１５）前記タンパク質含有成分が、（a）発酵能を有する細菌を破砕することにより得られるホモジネートを硫安濃度２０～６０％飽和で沈殿させて硫安沈殿として得る工程、および（b）該硫安沈殿を溶解した溶液（例えば、緩衝液）を１００ＫＤａの限外ろ過フィルターを透過させ、透過しない画分を得る工程、（好ましくはさらに、（c）該透過しない画分を、陰イオン交換クロマトグラフィーを用いて分画した溶出画分から得る工程、）を含む工程により得られるタンパク質含有成分である、前記（１０）～（１４）のいずれか一つに記載の方法。
（１６）前記体細胞が、ヒト由来の、正常体細胞またはがん細胞である前記（１０）～（１５）のいずれか一つに記載の方法。 (10) A method for producing pluripotent cells from somatic cells of an mammal (eg, human or mouse) (isolated or in vivo),
A protein-containing component that induces reprogramming of cells that can be obtained from a bacterial homogenate having fermentation ability into the somatic cells, and having the following properties:
(I) binds to m-aminophenylboronic acid;
Contacting a protein-containing component that has at least one, preferably at least two, and most preferably all that does not bind to (ii) concanavalin A and (iii) does not bind to wheat germ lectin. A method for producing pluripotent cells from somatic cells.
(11) The method according to (10), wherein the protein-containing component is a component that is obtained by concentrating bacterial homogenate using an ammonium sulfate precipitation method and does not pass through a 100 KDa ultrafiltration filter. .
(12) The method according to (10) or (11) above, wherein the protein-containing component comprises a protein having a molecular weight of about 55 to about 65 KDa (more preferably about 60 KDa) as a main band in SDS-PAGE.
(13) The method according to any one of (10) to (12), wherein the bacterium having fermentation ability is lactic acid bacteria or natto bacteria.
(14) The method according to (13), wherein the lactic acid bacterium is a lactic acid bacterium of the genus Lactococcus, Streptococcus, or Lactobacillus.
(15) The protein-containing component comprises (a) a step of precipitating a homogenate obtained by crushing bacteria having fermentation ability at an ammonium sulfate concentration of 20 to 60% to obtain an ammonium sulfate precipitate, and (b) the ammonium sulfate precipitate. A step of allowing a solution (for example, buffer) dissolved in the solution to pass through a 100 KDa ultrafiltration filter to obtain a non-permeate fraction (preferably (c) further subjecting the non-permeate fraction to anion exchange chromatography. The method according to any one of the above (10) to (14), which is a protein-containing component obtained by a step comprising the step of obtaining from an eluted fraction fractionated using.
(16) The method according to any one of (10) to (15), wherein the somatic cells are human-derived normal somatic cells or cancer cells.
（１７）発酵能を有する細菌のホモジネートから得ることができる細胞を再プログラミング誘導するタンパク質含有成分であって、以下の性質：
（i）ｍ－アミノフェニルボロン酸に結合する、
（ii）コンカナバリンＡに結合しない、および
（iii）小麦胚芽レクチン（Wheat germ lectin）に結合しない
の少なくとも一つ、好ましくは少なくとも２つ、最も好ましくは全てを有するタンパク質含有成分を含有する細胞再プログラミング誘導組成物。
（１８）前記タンパク質含有成分が、細菌のホモジネートを硫安沈殿法を用い濃縮することにより得られた、１００ＫＤａの限外ろ過フィルターを透過しない大きさの成分である、前記（１７）に記載の組成物。
（１９）前記タンパク質含有成分が、SDS-PAGEにおいて約５５～約６５ＫＤａ（より好ましくは約６０ＫＤａ）の分子量を示すタンパク質をメインバンドとして含む、前記（１７）または（１８）に記載の組成物。
（２０）前記発酵能を有する細菌が乳酸菌または納豆菌である前記（１７）～（１９）のいずれか一つに記載の組成物。
（２１）前記乳酸菌が、Lactococcus属、Streptococcus属、またはLactobacillus属の乳酸菌である前記（２０）に記載の組成物。
（２２）前記タンパク質含有成分が、（a）発酵能を有する細菌を破砕することにより得られるホモジネートを硫安濃度２０～６０％飽和で沈殿させて硫安沈殿として得る工程、および（b）該硫安沈殿を溶解した溶液（例えば、緩衝液）を１００ＫＤａの限外ろ過フィルターを透過させ、透過しない画分を得る工程、（好ましくはさらに（c）該透過しない画分を、陰イオン交換クロマトグラフィーを用いて分画した溶出画分から得る工程、）を含む工程により得られるタンパク質含有成分である、前記（１７）～（２１）のいずれか一つに記載の組成物。
（２３）前記体細胞が、ヒト由来の、正常体細胞またはがん細胞であるである前記（１７）～（２２）のいずれか一つに記載の組成物。
（２４）前記（１７）～（２３）のいずれか一つに記載の組成物を含む抗がん剤。 (17) A protein-containing component that induces reprogramming of cells that can be obtained from a bacterial homogenate having fermentation ability, and having the following properties:
(I) binds to m-aminophenylboronic acid;
Cell reprogramming containing a protein-containing component that does not bind to concanavalin A and (iii) does not bind to wheat germ lectin, preferably at least two, most preferably all Inducing composition.
(18) The composition according to (17), wherein the protein-containing component is a component that is obtained by concentrating bacterial homogenate using an ammonium sulfate precipitation method and does not pass through a 100 KDa ultrafiltration filter. object.
(19) The composition according to (17) or (18), wherein the protein-containing component comprises a protein having a molecular weight of about 55 to about 65 KDa (more preferably about 60 KDa) as a main band in SDS-PAGE.
(20) The composition according to any one of (17) to (19), wherein the bacterium having fermentation ability is lactic acid bacteria or natto bacteria.
(21) The composition according to (20), wherein the lactic acid bacterium is a lactic acid bacterium of the genus Lactococcus, Streptococcus, or Lactobacillus.
(22) a step in which the protein-containing component comprises (a) precipitating a homogenate obtained by crushing bacteria having fermentation ability at an ammonium sulfate concentration of 20 to 60% to obtain an ammonium sulfate precipitate; and (b) the ammonium sulfate precipitate. A step of obtaining a non-permeate fraction by allowing a solution (for example, buffer) dissolved therein to pass through a 100 KDa ultrafiltration filter (preferably (c) the non-permeate fraction using anion exchange chromatography) The composition according to any one of the above (17) to (21), which is a protein-containing component obtained by a process comprising the step of
(23) The composition according to any one of (17) to (22), wherein the somatic cell is a human-derived normal somatic cell or cancer cell.
(24) An anticancer agent comprising the composition according to any one of (17) to (23).
（２５）乳酸菌または納豆菌からのホモジネートまたは粗精製物を含む、哺乳類動物（例えば、ヒトまたはマウス）体細胞の再プログラミング培地。
（２６）前記ホモジネートまたは粗精製物が、以下の性質：
（i）ｍ－アミノフェニルボロン酸に結合する、
（ii）コンカナバリンＡに結合しない、および
（iii）小麦胚芽レクチン（Wheat germ lectin）に結合しない
の少なくとも一つ、好ましくは少なくとも２つ、最も好ましくは全てを有する成分を含むことを特徴とする、前記（２５）に記載の培地。
（２７）前記成分が、SDS-PAGEにおいて約５５～約６５ＫＤａ（より好ましくは約６０ＫＤａ）の分子量を示すタンパク質をメインバンドとして含む、前記（２６）に記載の培地。
（２８）前記乳酸菌が、Lactococcus属、Streptococcus属、またはLactobacillus属の乳酸菌である前記（２５）～（２７）のいずれか一つに記載の培地。
（２９）前記培地が、ダルベッコ改変イーグル培地（ＤＭＥＭ）、イーグル最少必須（ＥＭＥ）培地、イスコフ改変ダルベッコ培地（ＩＭＤＭ）、アルファ－最少必須培地（α－ＭＥＭ）、ＲＰＭＩ　１６４０、Ｈａｍ－Ｆ－１２、ＭＣＤＢおよびそれらの改変培地からなる群より選ばれる、前記（２５）～（２８）のいずれか一つに記載の培地。 (25) A reprogramming medium for mammalian (eg, human or mouse) somatic cells comprising a homogenate or crude product from lactic acid bacteria or Bacillus natto.
(26) The homogenate or crude product has the following properties:
(I) binds to m-aminophenylboronic acid;
Characterized in that it comprises a component having at least one, preferably at least two, and most preferably all of (ii) not binding to concanavalin A and (iii) not binding to wheat germ lectin. The medium according to (25) above.
(27) The medium according to (26), wherein the component contains a protein having a molecular weight of about 55 to about 65 KDa (more preferably about 60 KDa) as a main band in SDS-PAGE.
(28) The medium according to any one of (25) to (27), wherein the lactic acid bacterium is a lactic acid bacterium of the genus Lactococcus, Streptococcus, or Lactobacillus.
(29) Dulbecco's modified Eagle medium (DMEM), Eagle's minimal essential (EME) medium, Iskov modified Dulbecco medium (IMDM), alpha-minimal essential medium (α-MEM), RPMI 1640, Ham-F-12 The medium according to any one of (25) to (28), selected from the group consisting of MCDB and modified medium thereof.
（３０）（単離されたまたは生体内の）哺乳類動物（例えば、ヒトまたはマウス）の体細胞を多能性細胞に誘導するための、発酵能を有する細菌から得ることができる細胞を再プログラミング誘導するタンパク質含有成分であって、以下の性質：
（i）ｍ－アミノフェニルボロン酸に結合する、
（ii）コンカナバリンＡに結合しない、および
（iii）小麦胚芽レクチン（Wheat germ lectin）に結合しない
の少なくとも一つ、好ましくは少なくとも２つ、最も好ましくは全てを有するタンパク質含有成分の使用。
（３１）前記タンパク質含有成分が、細菌のホモジネートを硫安沈殿法を用い濃縮することにより得られた、１００ＫＤａの限外ろ過フィルターを透過しない大きさの成分である、前記（３０）に記載の使用。
（３２）前記タンパク質含有成分が、SDS-PAGEにおいて約５５～約６５ＫＤａ（より好ましくは約６０ＫＤａ）の分子量を示すタンパク質をメインバンドとして含む、前記（３０）または（３１）に記載の使用。
（３３）前記発酵能を有する細菌が乳酸菌または納豆菌である前記（３０）～（３２）のいずれか一つに記載の使用。
（３４）（単離されたまたは生体内の）哺乳類動物の体細胞を多能性細胞に誘導するための、発酵能を有する細菌の抽出液からの精製画分の使用、ここで、該精製画分は、（a）発酵能を有する細菌を破砕することにより得られるホモジネート溶液を硫安濃度２０～６０％飽和で沈殿させて硫安沈殿硫安沈殿として得る工程、および（b）該硫安沈殿を溶解した溶液（例えば、緩衝液）を１００ＫＤａの限外ろ過フィルターを透過させ、透過しない画分を得る工程、（好ましくはさらに（c）該透過しない画分を、陰イオン交換クロマトグラフィーを用いて分画した溶出画分から得る工程、）を含む工程により得られる画分である。
（３５）前記発酵能を有する細菌が乳酸菌または納豆菌である前記（３４）の使用。 (30) Reprogramming cells obtainable from fermentable bacteria to induce somatic cells of an isolated (in vivo or in vivo) mammal (eg, human or mouse) into pluripotent cells Induced protein-containing component with the following properties:
(I) binds to m-aminophenylboronic acid;
Use of a protein-containing component that has at least one, preferably at least two, and most preferably all of (ii) does not bind to concanavalin A and (iii) does not bind to wheat germ lectin.
(31) The use according to (30), wherein the protein-containing component is a component having a size that does not pass through a 100 KDa ultrafiltration filter obtained by concentrating a bacterial homogenate using an ammonium sulfate precipitation method. .
(32) The use according to (30) or (31) above, wherein the protein-containing component comprises a protein showing a molecular weight of about 55 to about 65 KDa (more preferably about 60 KDa) as a main band in SDS-PAGE.
(33) The use according to any one of (30) to (32), wherein the bacterium having fermentation ability is lactic acid bacteria or natto bacteria.
(34) Use of a purified fraction from a fermentable bacterial extract to induce mammalian somatic cells (isolated or in vivo) into pluripotent cells, wherein the purification The fraction includes (a) a step of precipitating a homogenate solution obtained by crushing bacteria having fermentation ability at an ammonium sulfate concentration of 20 to 60% to obtain an ammonium sulfate precipitate, and (b) dissolving the ammonium sulfate precipitate. The obtained solution (for example, buffer) through a 100 KDa ultrafiltration filter to obtain a non-permeate fraction (preferably (c) the non-permeate fraction is separated using anion exchange chromatography. A fraction obtained by a step including a step obtained from a fractionated elution fraction).
(35) Use of (34) above, wherein the bacterium having fermentation ability is lactic acid bacteria or natto bacteria.
　本発明においては、体細胞への遺伝子の導入および強制発現、または細菌そのものの導入を用いることなく、体細胞から多能性幹細胞を製造することができる。本発明では、人体に存在して細胞と共存している乳酸菌などの発酵能を有する細菌から得られる成分（タンパク質含有成分）を含む組成物を用いて多能性幹細胞を製造することができる。本発明による多能性細胞の製造方法は、医療分野(創薬研究、並びに医薬品の安全性、有効性及び副作用の試験)、疾患研究(難病の原因解明、治療法や予防法の開発)、再生医療(神経、血管、臓器の機能修復)、並びに食品分野において有用である。 In the present invention, pluripotent stem cells can be produced from somatic cells without using gene introduction and forced expression into somatic cells, or introduction of bacteria themselves. In the present invention, pluripotent stem cells can be produced using a composition containing a component (protein-containing component) obtained from a bacterium having fermentation ability such as lactic acid bacteria that exists in the human body and coexists with cells. The method for producing pluripotent cells according to the present invention includes the medical field (drug discovery research and drug safety, efficacy and side effect testing), disease research (elucidation of the cause of intractable diseases, development of treatments and prevention methods), Useful in regenerative medicine (function repair of nerves, blood vessels, organs) and food.
乳酸菌成分と一緒に培養したHDF細胞（培養３日後）を示す。1 shows HDF cells cultured with lactic acid bacteria components (after 3 days in culture). 乳酸菌成分で処理したHDF細胞を、脂肪細胞、骨細胞、および軟骨細胞へ分化誘導する培地で培養した後、それぞれの細胞を、Oil Red O染色（脂肪）、Alizarin Red S染色（骨）、およびAlcian Blue染色（軟骨）した結果を示す。After culturing HDF cells treated with lactic acid bacteria components in a medium that induces differentiation into adipocytes, bone cells, and chondrocytes, each cell is subjected to Oil Red O staining (fat), Alizarin Red S staining (bone), and The result of Alcian Blue staining (cartilage) is shown. 乳酸菌成分と一緒に培養したA549細胞（培養１４日後）を示す。A549 cells cultured with lactic acid bacteria components (after 14 days in culture) are shown. 乳酸菌成分で処理したA549細胞を、脂肪細胞、および骨細胞へ分化誘導する培地で培養した後、それぞれの細胞を、Oil Red O染色（脂肪）、およびAlizarin Red S染色（骨）した結果を示す。The results of culturing A549 cells treated with lactic acid bacteria components in a medium that induces differentiation into adipocytes and bone cells, followed by Oil Red O staining (fat) and Alizarin Red S staining (bone) are shown. . 乳酸菌成分と一緒に培養したHepG2細胞（培養１４日後）を示す。HepG2 cells cultured with lactic acid bacteria components (after 14 days in culture) are shown. 乳酸菌成分で処理したHepG2細胞を、脂肪細胞、および骨細胞へ分化誘導する培地で培養した後、それぞれの細胞を、Oil Red O染色（脂肪）、およびAlizarin Red S染色（骨）した結果を示す。Shows the result of culturing HepG2 cells treated with lactic acid bacteria components in a medium that induces differentiation into adipocytes and bone cells, and then staining each cell with Oil Red O staining (fat) and Alizarin Red S staining (bone) . 乳酸菌成分と一緒に培養したMCF7細胞（培養１４日後）を示す。MCF7 cells cultured with lactic acid bacteria components (after 14 days in culture) are shown. 乳酸菌成分で処理したMCF7細胞を、脂肪細胞、および骨細胞へ分化誘導する培地で培養した後、それぞれの細胞を、Oil Red O染色（脂肪）、およびAlizarin Red S染色（骨）した結果を示す。MCF7 cells treated with lactic acid bacteria components were cultured in a medium that induces differentiation into adipocytes and bone cells, and then each cell was stained with OilORed O (fat) and Alizarin Red S (bone). . 乳酸菌成分で処理したHepG2細胞を、分化誘導培地で培養した後、それぞれ細胞を、抗α-fetoprotein抗体（内胚葉マーカー）、および抗NeuroFilament抗体（外胚葉、神経細胞マーカー）で染色した結果を示す。Shown are the results of culturing HepG2 cells treated with lactic acid bacteria components in a differentiation-inducing medium and then staining the cells with anti-α-fetoprotein antibody (endoderm marker) and anti-NeuroFilament antibody (ectodermal, neuronal marker), respectively. . 乳酸菌成分で処理したA549細胞における、多能性マーカーの発現をRT-PCR法を用いて確認した結果である。矢印は、Nanogの発現を示す。It is the result of having confirmed the expression of the pluripotency marker in the A549 cell processed with the lactic-acid-bacteria component using RT-PCR method. Arrows indicate Nanog expression. 実施例１で得た乳酸菌成分を、陰イオン交換クロマトグラフィーで分離した溶出パターンを示す。The elution pattern which isolate | separated the lactic-acid-bacteria component obtained in Example 1 by the anion exchange chromatography is shown. 大腸菌成分と一緒に培養したHDF細胞（培養３日後）を示す。HDF cells cultured with E. coli components (after 3 days in culture) are shown.
　以下、本発明について更に詳細に説明する。
　本発明において、「細胞を再プログラミング誘導することができるタンパク質含有成分」または「細胞再プログラミング誘導タンパク質含有成分」とは、単離された哺乳類動物の体細胞（正常な体細胞、体細胞由来のがん細胞のいずれも含む意味である）、例えば、上皮細胞と接触させることにより、体細胞を、ＥＳ細胞やｉＰＳ細胞と同様の、種々の細胞に分化する能力を有する多能性細胞へと形質を変換させることができるタンパク質含有成分をいう。ここで接触とは、体細胞に誘導タンパク質含有成分が接触できる限り特に制限されないが、好ましくは、体細胞が生存する環境（例えば、培地）中に該成分を存在させることにより、該成分が体細胞に作用できる状態におくことを言う。
　ここでいう誘導タンパク質含有成分とは、以下の性質：（i）ｍ－アミノフェニルボロン酸に結合する、（ii）コンカナバリンＡに結合しない、および（iii）小麦胚芽レクチン（Wheat germ lectin）に結合しない、の少なくとも一つ、好ましくは少なくとも２つ、最も好ましくは全て、を有するタンパク質含有成分である。本発明のタンパク質含有成分は、好ましくは、１００ＫＤａの限外ろ過フィルターを透過しない大きさの成分である。本発明のタンパク質含有成分は、さらに好ましくは、SDS-PAGEにおいて約５５～約６５ＫＤａ（より好ましくは約６０ＫＤａ）の分子量を示すタンパク質をメインバンドとして含む。該成分中においてタンパク質は、単独の状態で存在しても、また他の物質が共有結合または非共有結合で結合した状態、例えば、他の物質と複合体を形成した状態でもよい。また、誘導タンパク質含有成分は、ある程度精製された状態であっても粗精製の状態であってもよい。例えば、本発明に示されるように、誘導タンパク質含有成分が乳酸菌中に存在するので、乳酸菌のホモジネートまたは粗精製物として体細胞と接触させても良い。粗精製物としては、これに限定されないが、例えば、硫安沈殿、限外ろ過、ゲルろ過、イオン交換クロマトグラフィー、疎水性相互作用クロマトグラフィー、ゲルろ過クロマトグラフィーまたはこれらを組み合わせて得られる画分から得られるものをあげることができる。本明細書において「組成物」または「細胞再プログラミング誘導組成物」とは、誘導タンパク質含有成分をその一部に含む形態であれば特に制限されず、特に明細書中で区別して用いられない限り、あるいは文脈の意味から限定されない限り、ある程度精製されたまたは粗精製された誘導タンパク質含有成分を任意の成分と混合したもの、ある程度の精製または粗精製誘導タンパク質含有成分を添加した培地、さらには、前記の粗精製画分そのものを含む意味で用いられる。 Hereinafter, the present invention will be described in more detail.
In the present invention, “a protein-containing component capable of inducing cell reprogramming” or “a cell reprogramming-inducing protein-containing component” refers to an isolated mammalian somatic cell (normal somatic cell, derived from somatic cell). For example, by contacting with epithelial cells, somatic cells can be transformed into pluripotent cells having the ability to differentiate into various cells, similar to ES cells and iPS cells. A protein-containing component that can transform a character. Here, the contact is not particularly limited as long as the derived protein-containing component can be contacted with the somatic cell, but preferably, the component is present in the environment in which the somatic cell survives (for example, a medium). Saying to be able to act on cells.
The derived protein-containing component here has the following properties: (i) binds to m-aminophenylboronic acid, (ii) does not bind to concanavalin A, and (iii) binds to wheat germ lectin A protein-containing component having at least one, preferably at least two, and most preferably all. The protein-containing component of the present invention is preferably a component that does not permeate a 100 KDa ultrafiltration filter. More preferably, the protein-containing component of the present invention contains, as a main band, a protein showing a molecular weight of about 55 to about 65 KDa (more preferably about 60 KDa) on SDS-PAGE. In the component, the protein may exist in a single state, or may be in a state in which another substance is covalently or non-covalently bound, for example, in a complex with another substance. In addition, the derived protein-containing component may be purified to some extent or may be roughly purified. For example, as shown in the present invention, since the derived protein-containing component is present in lactic acid bacteria, it may be contacted with somatic cells as a homogenate or crude product of lactic acid bacteria. Although it is not limited to this as a crudely purified product, for example, it is obtained from fraction obtained by ammonium sulfate precipitation, ultrafiltration, gel filtration, ion exchange chromatography, hydrophobic interaction chromatography, gel filtration chromatography or a combination thereof. I can give you. In the present specification, the “composition” or “cell reprogramming induction composition” is not particularly limited as long as it is a form containing an inducible protein-containing component as a part thereof, and unless otherwise specifically used in the specification. Or, unless otherwise limited by context, a mixture of some purified or crude purified derived protein-containing component with any component, a medium supplemented with some purified or crude derived protein-containing component, It is used in the meaning including the aforementioned crudely purified fraction itself.
　本発明による体細胞から多能性細胞を製造する方法は、体細胞に、発酵能を有する細菌から得ることができる細胞を再プログラミング誘導することができるタンパク質含有成分またはそれを含む組成物を接触させる工程を含むことを特徴とする。このようなタンパク質含有成分は、例えば、発酵能を有する細菌の細胞抽出液を、硫安沈殿法により濃縮することにより得ることができる。例えば、硫安濃度、１０～８０％飽和、好ましくは２０～７０％飽和、より好ましくは２０～６０％飽和で沈殿させた硫安沈殿画分として得ることができる。さらに好ましくは、このようにして得た硫安沈殿画分であって、１００ＫＤａの限外ろ過フィルターを透過しない画分から得ることができる。さらには、このようなタンパク質含有成分は、例えば、前記画分を、陰イオンクロマトグラフィー、疎水性相互作用クロマトグラフィー、ゲルろ過クロマトグラフィー、またはこれらの組み合わせを用いてさらに精製することにより、より純度が高いタンパク質含有成分として得ることができる。
　本発明で用いるタンパク質含有成分に含まれるタンパク質の例は、好ましくは、糖タンパク質であり、さらに好ましくは、ＳＤＳ－ＰＡＧＥにおいて約５５～６５ＫＤａの分子量を示す糖タンパク質、より好ましくはＳＤＳ－ＰＡＧＥにおいて約６０ＫＤａの分子量を示す糖タンパク質である。また、本発明で用いることができるタンパク質含有成分に含まれる糖タンパク質の例は、好ましくは、（１）ｍ－アミノフェニルボロン酸に結合する、（２）コンカナバリンＡに結合しない、または（３）小麦胚芽レクチン（Wheat germ lectin）に結合しない、の一つまたは複数の性質を有する糖タンパク質であり、最も好ましくは、これら全ての性質を有する糖タンパク質である。 The method for producing pluripotent cells from somatic cells according to the present invention comprises contacting a somatic cell with a protein-containing component or a composition comprising the same that can induce reprogramming of a cell obtainable from a bacterium capable of fermentation. A step of causing Such a protein-containing component can be obtained, for example, by concentrating a bacterial cell extract having fermentation ability by an ammonium sulfate precipitation method. For example, it can be obtained as an ammonium sulfate precipitate fraction precipitated at an ammonium sulfate concentration of 10 to 80% saturation, preferably 20 to 70% saturation, more preferably 20 to 60% saturation. More preferably, the ammonium sulfate precipitation fraction thus obtained can be obtained from a fraction that does not permeate a 100 KDa ultrafiltration filter. Further, such protein-containing components can be made more pure by, for example, further purifying the fraction using anion chromatography, hydrophobic interaction chromatography, gel filtration chromatography, or a combination thereof. Can be obtained as a high protein-containing component.
An example of a protein contained in the protein-containing component used in the present invention is preferably a glycoprotein, more preferably a glycoprotein exhibiting a molecular weight of about 55 to 65 KDa in SDS-PAGE, more preferably about a protein in SDS-PAGE. It is a glycoprotein showing a molecular weight of 60 KDa. In addition, examples of glycoproteins contained in protein-containing components that can be used in the present invention are preferably (1) bound to m-aminophenylboronic acid, (2) not bound to concanavalin A, or (3) A glycoprotein having one or more properties that do not bind to wheat germ lectin, and most preferably a glycoprotein having all these properties.
　本発明で初期化のために用いる体細胞の種類は特に限定されず、任意の体細胞を用いることができる。即ち、本発明で言う体細胞とは、生体を構成する細胞のうち生殖細胞以外の全ての細胞を包含し、分化した体細胞でもよいし、未分化の幹細胞でもよい。例えば、これに制限されないが、上皮細胞、内皮細胞、線維芽細胞（皮膚細胞等）、腸細胞、肝細胞、脾細胞、膵細胞、腎細胞、毛細胞、筋肉細胞、脳細胞、肺細胞、脂肪細胞、胃粘膜細胞、およびリンパ球等の分化した細胞、神経幹細胞、造血幹細胞、間葉系幹細胞、歯髄幹細胞等の体性幹細胞、組織前駆細胞をあげることができる。体細胞の由来は、哺乳動物であれば特に限定されないが、好ましくはマウスなどのげっ歯類、またはヒ卜などの霊長類であり、特に好ましくはヒ卜またはマウスである。また、ヒ卜の体細胞を用いる場合、胎児、新生児または成人の何れの体細胞を用いてもよい。本発明の方法で製造される多能性細胞を再生医療など疾患の治療に用いる場合には、該疾患を患う患者自身から分離した体細胞を用いることが好ましい。また、本発明では体細胞としてがん細胞を用いることができる。がん細胞に、発酵能を有する細菌から得ることができる細胞を再プログラミング誘導することができるタンパク質含有成分、例えば、前記した性質を有するタンパク質を含む成分（発酵能を有する細菌の細胞抽出液からの粗精製画分を含む）を接触させることによって、がん細胞から非がん細胞を製造することができる。本発明において、体細胞(がん細胞である場合を含む)に、発酵能を有する細菌から得ることができる細胞を再プログラミング誘導することができるタンパク質含有成分を接触させる工程は、インビトロでもまた生体内でも行うことができる。 The type of somatic cell used for initialization in the present invention is not particularly limited, and any somatic cell can be used. That is, the somatic cells referred to in the present invention include all cells other than germ cells among the cells constituting the living body, and may be differentiated somatic cells or undifferentiated stem cells. For example, but not limited to, epithelial cells, endothelial cells, fibroblasts (skin cells, etc.), intestinal cells, hepatocytes, spleen cells, pancreatic cells, kidney cells, hair cells, muscle cells, brain cells, lung cells, Examples include adipocytes, gastric mucosa cells, and differentiated cells such as lymphocytes, somatic stem cells such as neural stem cells, hematopoietic stem cells, mesenchymal stem cells, and dental pulp stem cells, and tissue precursor cells. The origin of the somatic cell is not particularly limited as long as it is a mammal, but it is preferably a rodent such as a mouse, or a primate such as a baboon, and particularly preferably a rabbit or a mouse. In addition, when using chick somatic cells, any fetal, neonatal or adult somatic cells may be used. When the pluripotent cells produced by the method of the present invention are used for treatment of diseases such as regenerative medicine, it is preferable to use somatic cells isolated from the patient suffering from the disease. In the present invention, cancer cells can be used as somatic cells. Protein-containing components that can induce reprogramming of cells that can be obtained from bacteria having fermentation ability into cancer cells, for example, components containing proteins having the properties described above (from cell extracts of bacteria having fermentation ability) A non-cancer cell can be produced from a cancer cell by contacting with a non-cancer cell. In the present invention, the step of bringing a somatic cell (including a cancer cell) into contact with a protein-containing component that can induce reprogramming of a cell that can be obtained from a bacterium having fermentation ability can be performed in vitro. Can also be done in the body.
　本発明で言う多能性細胞とは、所定の培養条件下において長期にわたって自己複製能を有し、また所定の分化誘導条件下において多種の細胞(外胚葉系の細胞、中胚葉系の細胞、または内胚葉系の細胞など)への多分化能を有する細胞(このような細胞のことは、幹細胞とも称する)のことを言う。 The pluripotent cell referred to in the present invention has a self-replicating ability over a long period of time under a predetermined culture condition, and various cells (ectodermal cells, mesodermal cells, Or a cell having pluripotency into an endoderm cell or the like (such a cell is also referred to as a stem cell).
　本発明で用いる再プログラミング誘導タンパク質含有成分を得ることができる発酵能を有する細菌の種類は特に限定されず、乳酸菌、納豆菌などの好気性細菌でもよいし、ビフィズス菌などの嫌気性細菌でもよいが、乳酸菌が特に好ましい。また、本発明で用いる乳酸菌の種類は、発酵能を有する限り特に限定されない。乳酸菌とは、発酵によって糖類から乳酸を産生する能力を有する菌の総称である。代表的な乳酸菌としては、ラク卜バシラス属(Lactobacillus)、ビフィドバクテリウム属(Bifidobacterium)、エンテ口コッ力ス属(Enterococcus)、ラク卜コッ力ス属(Lactococcus) 、ペディオコッ力ス属(Pediococcus)、リューコノストック属(Leuconostoc) 、ス卜レプトコッ力ス属(Streptococcus) などに属する乳酸菌が挙げられ、本発明で用いる誘導タンパク質含有成分を得るために、これらの乳酸菌を使用することができる。好ましくは、Lactococcus属、Streptococcus属、またはLactobacillus属の乳酸菌を使用することができる。乳酸菌としては、特に好ましくは、Lactococcus Lactis subsp. Lactis、Streptococcus salivarius subsp. thermophilus、Lactobacillus sp.、またはLactobacillus acidophilusを使用することができる。 The kind of bacteria having fermentative ability that can obtain the reprogramming-inducing protein-containing component used in the present invention is not particularly limited, and may be an aerobic bacterium such as lactic acid bacterium or natto bacterium, or an anaerobic bacterium such as bifidobacteria. However, lactic acid bacteria are particularly preferred. Moreover, the kind of lactic acid bacteria used in the present invention is not particularly limited as long as it has fermentation ability. Lactic acid bacteria is a general term for bacteria having the ability to produce lactic acid from sugars by fermentation. Representative lactic acid bacteria include Lactobacillus, Bifidobacterium, Enterococcus, Lactococcus, Pediococcus, and Pediococcus. ), Leuconostoc spp., Streptococcus spp. And other lactic acid bacteria, and these lactic acid bacteria can be used to obtain the derived protein-containing component used in the present invention. Preferably, lactic acid bacteria belonging to the genus Lactococcus, Streptococcus, or Lactobacillus can be used. As the lactic acid bacteria, Lactococcus Lactis subsp. Lactis, Streptococcus salivarius subsp. Thermophilus, Lactobacillus sp., Or Lactobacillus acidophilus can be used particularly preferably.
　本発明においては、細胞培養用の通常の培地を用いて、発酵能を有する細菌から得ることができる細胞を再プログラミング誘導することができるタンパク質含有成分の存在下において体細胞の培養を行うことにより、本発明の多能性細胞または非がん細胞（がん細胞を再プログラミングして非がん化した細胞）を分離及び培養することができる。本発明の多能性細胞を製造および培養するための培地は、特に制限されず、多能性細胞の培養に用いることができる任意の培地を用いることができ、例えば、これに制限されないが、ダルベッコ改変イーグル培地（ＤＭＥＭ）、イーグル最少必須（ＥＭＥ）培地、イスコフ改変ダルベッコ培地（ＩＭＤＭ）、アルファ－最少必須培地（α－ＭＥＭ）、ＲＰＭＩ　１６４０、Ｈａｍ－Ｆ－１２、ＭＣＤＢ、およびそれらの改変培地をあげることができる。培地は、製造した多能性細胞のその後の使用や誘導効率の観点より、無血清培地が好ましく、さらには、必要に応じて、各種の成長因子、サイト力イン、ホルモンなど(例えば、FGF-2、TGFβ-1、アクチビンA、ノギン(Nanoggin) 、BDNF、NGF、NT-1、NT-2、NT-3等のヒ卜ES細胞の増殖・維持に関与する成分)を添加してもよい。このような培地も本発明の一部である。また、分離された多能性細胞の分化能及び増殖能は、ES細胞について知られている確認手段を利用することにより確認することができる。 In the present invention, by using a normal medium for cell culture, culturing somatic cells in the presence of a protein-containing component that can induce reprogramming of cells that can be obtained from bacteria having fermentation ability. The pluripotent cells or non-cancer cells of the present invention (cells that are non-cancerous by reprogramming cancer cells) can be isolated and cultured. The medium for producing and culturing the pluripotent cells of the present invention is not particularly limited, and any medium that can be used for culturing pluripotent cells can be used, for example, but not limited thereto, Dulbecco's modified Eagle's medium (DMEM), Eagle's minimal essential (EME) medium, Iskov's modified Dulbecco medium (IMDM), alpha-minimal essential medium (α-MEM), RPMI 1640, Ham-F-12, MCDB, and modifications thereof Medium can be raised. The medium is preferably a serum-free medium from the viewpoint of subsequent use of the produced pluripotent cells and induction efficiency, and further, if necessary, various growth factors, cyto force-in, hormones and the like (for example, FGF- 2, TGFβ-1, Activin A, Nanoggin, BDNF, NGF, NT-1, NT-2, NT-3 and other components involved in the growth and maintenance of chick ES cells may be added) . Such a medium is also part of the present invention. In addition, the differentiation ability and proliferation ability of the separated pluripotent cells can be confirmed by using confirmation means known for ES cells.
　本発明の方法で製造される多能性細胞及び非がん細胞の用途は特に限定されず、各種の試験・研究や疾病の治療などに使用することができる。例えば、本発明の方法により得られた多能性細胞をレチノイン酸、EGFなどの増殖因子、またはグルココルチコイドなどで処理することにより、所望の分化細胞(例えば神経細胞、心筋細胞、肝細胞、膵臓細胞、血球細胞など)を誘導することができ、そのようにして得られた分化細胞を患者に戻すことにより自家細胞移植による幹細胞療法を達成することができる。 The uses of the pluripotent cells and non-cancer cells produced by the method of the present invention are not particularly limited, and can be used for various tests / researches and disease treatments. For example, by treating pluripotent cells obtained by the method of the present invention with a growth factor such as retinoic acid, EGF, or glucocorticoid, desired differentiated cells (for example, nerve cells, cardiomyocytes, hepatocytes, pancreas) Cells, blood cells, etc.) can be induced, and stem cell therapy by autologous cell transplantation can be achieved by returning the differentiated cells thus obtained to the patient.
　本発明の多能性細胞を用いて治療を行うことができる中枢神経系の疾患としてはパーキンソン病、アルツハイマー病、多発性硬化症、脳梗塞、脊髄損傷などが挙げられる。パーキンソン病の治療のためには、多能性細胞をドーパミン作動性ニュー口ンへと分化しパーキンソン病患者の線条体に移植することができる。ドーパミン作動性ニュー口ンへの分化はマウスのストローマ細胞株であるPA6細胞と本発明の多能性細胞を無血清条件で共培養することで進めることができる。アルツイハイマー病、脳梗塞、脊髄損傷の治療においては本発明の多能性細胞を神経幹細胞に分化誘導した後に、傷害部位に移植することができる。 Examples of central nervous system diseases that can be treated using the pluripotent cells of the present invention include Parkinson's disease, Alzheimer's disease, multiple sclerosis, cerebral infarction, spinal cord injury and the like. For the treatment of Parkinson's disease, pluripotent cells can be differentiated into dopaminergic new mouth and transplanted into the striatum of Parkinson's disease patients. Differentiation into dopaminergic new mouth can be promoted by co-culturing mouse stromal cell line PA6 cells and the pluripotent cells of the present invention under serum-free conditions. In the treatment of Alzheimer's disease, cerebral infarction, and spinal cord injury, the pluripotent cells of the present invention can be induced to differentiate into neural stem cells and then transplanted to the site of injury.
　また、本発明の多能性細胞は肝炎、肝硬変、肝不全などの肝疾患の治療に用いることができる。これら疾患を治療するには、本発明の多能性細胞を肝細胞あるいは肝幹細胞に分化し移植することができる。本発明の多能性細胞をアクチビンA存在下で5日間培養し、その後肝細胞増殖因子(HGF) で1週間程度培養することで肝細胞あるいは肝幹細胞を取得することができる。 In addition, the pluripotent cells of the present invention can be used for the treatment of liver diseases such as hepatitis, cirrhosis and liver failure. In order to treat these diseases, the pluripotent cells of the present invention can be differentiated into hepatocytes or hepatic stem cells and transplanted. Hepatocytes or hepatic stem cells can be obtained by culturing the pluripotent cells of the present invention in the presence of activin A for 5 days and then culturing with hepatocyte growth factor (HGF) for about 1 week.
　さらに本発明の多能性細胞はＩ型糖尿病などの膵臓疾患の治療に用いることができる。I型糖尿病の場合には、本発明の多能性細胞を膵臓β細胞に分化させ、膵臓に移植することができる。本発明の多能性細胞を膵臓β細胞に分化させる方法は、ES細胞を膵臓β細胞に分化させる方法に準じて行うことができる。 Furthermore, the pluripotent cells of the present invention can be used for the treatment of pancreatic diseases such as type I diabetes. In the case of type I diabetes, the pluripotent cells of the present invention can be differentiated into pancreatic β cells and transplanted into the pancreas. The method of differentiating pluripotent cells of the present invention into pancreatic β cells can be performed according to the method of differentiating ES cells into pancreatic β cells.
　さらに本発明の多能性細胞は虚血性心疾患に伴う心不全の治療に用いることができる。心不全の治療には、本発明の多能性細胞を心筋細胞に分化させた後に傷害部位に移植することが好ましい。本発明の多能性細胞は胚様体を形成させる3日前よりノギンを添加し培地中に添加することで、胚様体形成後2週間程度で、心筋細胞を得ることができる。 Furthermore, the pluripotent cells of the present invention can be used for the treatment of heart failure associated with ischemic heart disease. For the treatment of heart failure, the pluripotent cells of the present invention are preferably differentiated into cardiomyocytes and then transplanted to the site of injury. The pluripotent cells of the present invention can obtain cardiomyocytes in about 2 weeks after the formation of embryoid bodies by adding noggin 3 days before the formation of embryoid bodies and adding it to the medium.
　また、本発明によれば、がん細胞に、発酵能を有する細菌から得ることができる細胞を再プログラミング誘導することができるタンパク質含有成分を接触させることによって、がん細胞から非がん細胞を製造することができる。従って、本発明で用いられる細胞再プログラミング誘導タンパク質含有成分またはそれを含む組成物は、抗がん剤として有用である。
　さらに本発明により提供される細胞を再プログラミング誘導することができるタンパク質含有成分は、分化した細胞やがん細胞などの異常分化を起こした細胞を初期化できるので、医薬品や化粧品への添加物として用いることができる。また、本発明の再プログラミング誘導タンパク質含有成分は、乳酸菌等に由来する物質であるので安全である。 In addition, according to the present invention, non-cancer cells can be transformed from cancer cells by contacting the cancer cells with a protein-containing component that can induce reprogramming of cells that can be obtained from bacteria having fermentation ability. Can be manufactured. Therefore, the cell reprogramming-inducing protein-containing component used in the present invention or a composition containing the same is useful as an anticancer agent.
Furthermore, the protein-containing component capable of inducing reprogramming of cells provided by the present invention can initialize cells that have undergone abnormal differentiation such as differentiated cells and cancer cells, so that it can be used as an additive to pharmaceuticals and cosmetics. Can be used. The reprogramming-inducing protein-containing component of the present invention is safe because it is a substance derived from lactic acid bacteria or the like.
　以下の実施例により本発明を具体的に説明するが、本発明は以下の実施例によって限定されるものではない。 The present invention will be specifically described with reference to the following examples, but the present invention is not limited to the following examples.
実施例１：乳酸菌成分の調製
　乳酸菌は、理化学研究所バイオリソースセンター　微生物材料開発室から購入した。MRS 培地を高圧蒸気殺菌後、乳酸菌（Lactobacillus acidophilus; JCM1021）をMRS培地20mlに植菌し、37℃で2～3日間振盪培養した。次に、滅菌済みの前記培地1Lを含んだ3L振盪フラスコに植菌し、37℃で3～4日間、振盪培養した。得られた培養液1Lから遠心分離10,000 rpm、10 minにより菌体を集めた。PBSに懸濁、遠心分離を3回繰り返して菌体を洗浄後、PBS (pH7.0) 30 mlに懸濁した。菌体懸濁液をBranson soniccator 250D 超音波破砕装置（株式会社Branson）でOUTPUT 3 Duty 50% 氷上で30分間破砕した。得られた細胞抽出液（ホモジネート）に3.42gの硫酸アンモニウム（硫安）を添加し（硫安濃度20%）、4℃で2時間冷却後、遠心分離8,000rpm、10min、4℃により菌体残渣を除いた。上清を回収後、硫酸アンモニウム8.28g追加し、一晩冷蔵庫で冷却した。サンプルを遠心分離10,000rpm、20min、4℃により硫安濃度20～60%飽和で沈殿するタンパク質画分（またはタンパク質複合物）を沈殿させた。沈殿を0.02 M Triethanolamine (TEA) 緩衝液（pH 7.5） 5 mlに溶解し（全量で7.5 ml前後になる）、透析膜（Thermo slide A dialyzer pore size 20,000 MW）に入れ、一晩4℃で0.02 M Triethanolamine (TEA) 緩衝液(pH 7.5)に対して透析を行い、低分子の物質を取り除いた。次いで、透析したサンプルを回収し、100 KDaの限外ろ過フィルター(Millipore)でろ過し、膜を透過しなかった濃縮サンプルを回収した。タンパク質画分（またはタンパク質複合物）を、Protein Assay Kit（Bio Rad社）を用いて定量し、「乳酸菌成分」として以下の実験に用いた。タンパク濃度は、５mg/mlであった。 Example 1: Preparation of lactic acid bacteria component Lactic acid bacteria were purchased from the Microbial Materials Development Office, RIKEN BioResource Center. After sterilizing the MRS medium with high-pressure steam, lactic acid bacteria (Lactobacillus acidophilus; JCM1021) were inoculated into 20 ml of the MRS medium and cultured with shaking at 37 ° C. for 2 to 3 days. Next, the cells were inoculated into a 3 L shake flask containing 1 L of the sterilized medium and cultured at 37 ° C. for 3 to 4 days. Bacteria were collected from 1 L of the obtained culture broth by centrifugation at 10,000 rpm for 10 min. After suspending in PBS and centrifuging three times to wash the cells, the suspension was suspended in 30 ml of PBS (pH 7.0). The cell suspension was crushed for 30 minutes on OUTPUT 3 Duty 50% ice with a Branson soniccator 250D ultrasonic crusher (Branson Co., Ltd.). To the obtained cell extract (homogenate), 3.42 g of ammonium sulfate (ammonium sulfate concentration) was added (ammonium sulfate concentration 20%), cooled at 4 ° C for 2 hours, and centrifuged at 8,000 rpm, 10 min, 4 ° C to remove cell residue. It was. After recovering the supernatant, 8.28 g of ammonium sulfate was added and cooled in the refrigerator overnight. The protein fraction (or protein complex) was precipitated by centrifuging the sample at 10,000 rpm, 20 min, 4 ° C. with an ammonium sulfate concentration of 20-60% saturation. Dissolve the precipitate in 5 ml of 0.02 M Triethanolamine (TEA) buffer (pH 7.5) (about 7.5 ml in total), put it in a dialysis membrane (Thermo slide A dialyzer pore size 20,000 MW), and leave at 0.02 overnight at 4 ° C. Dialysis was performed against M Triethanolamine (TEA) buffer (pH 7.5) to remove low-molecular substances. The dialyzed sample was then collected and filtered through a 100 KDa ultrafiltration filter (Millipore) to collect a concentrated sample that did not permeate the membrane. The protein fraction (or protein complex) was quantified using Protein Assay Kit (Bio Rad) and used as the “lactic acid bacteria component” in the following experiments. The protein concentration was 5 mg / ml.
実施例２：乳酸菌成分存在下でのHDF細胞の培養
　10cmシャーレでHDF細胞（Human Dermal Fibroblasts, CELL APPLICATIONS, INC. Cat No.106-05a）をFibroblast Growth Medium（CELL  APLICATION INC.）で培養した。10mlのCMF（Ca2+ Mg2+フリーバッファー）で細胞を洗浄し、0.25%トリプシン溶液（1mM EDTA含）を1ml加えて全体にいきわたらせた。細胞を、CO2インキュベーター（37℃）に5分間入れた後、トリプシン阻害溶液（CELL  APLICATION INC.）3mlを加え懸濁し、細胞数をカウントした。あらかじめ6 well plate に1 wellあたり実施例１で得られたLactobacillus acidophilus (JCM1021)由来の乳酸菌成分（50μl）を入れておき、5 x 105のHDF細胞を加えた。乳酸菌は理化学研究所バイオリソースセンター　微生物材料開発室から購入した。細胞をそのまま34℃、5% CO2インキュベータで培養した。
　その結果、数日後には細胞塊が観察できた。図１の写真は、培養してから3日後の細胞の状態を示している。 Example 2: Culture of HDF cells in the presence of lactic acid bacteria components HDF cells (Human Dermal Fibroblasts, CELL APPLICATIONS, INC. Cat No. 106-05a) were cultured in Fibroblast Growth Medium (CELL APLICATION INC.) In a 10 cm petri dish. The cells were washed with 10 ml of CMF (Ca 2+ Mg 2+ free buffer), and 1 ml of 0.25% trypsin solution (containing 1 mM EDTA) was added to spread the whole. The cells were placed in a CO 2 incubator (37 ° C.) for 5 minutes, suspended by adding 3 ml of a trypsin inhibitor solution (CELL APLICATION INC.), And the number of cells was counted. A Lactobacillus acidophilus (JCM1021) -derived lactic acid bacterium component (50 μl) obtained in Example 1 per well was placed in advance in a 6-well plate, and 5 × 10 5 HDF cells were added. Lactic acid bacteria were purchased from the Microbial Materials Development Department, RIKEN BioResource Center. The cells were cultured as they were in a 34 ° C., 5% CO 2 incubator.
As a result, cell clusters could be observed after several days. The photograph in FIG. 1 shows the state of cells 3 days after culturing.
実施例３：細胞（乳酸菌成分処理HDF細胞）の分化誘導
　実施例２で作製したHDF細胞塊(A)を、乳酸菌成分存在下で２週間培養し、脂肪細胞（B）、骨細胞（C）、軟骨細胞（D）に分化誘導をうながす培養液（GIBCO; A10070-01, A10072-01, A10071-01）に交換し、さらに2～3週間培養した。
　結果を図２に示す。図２に示されるように、乳酸菌成分を作用させたHDF細胞塊は、分化誘導培地で培養後、(B)Oil Red O染色（脂肪）、(C)Alizarin Red S染色（骨）、(D)Alcian Blue染色（軟骨）により染色され、それぞれの細胞への細胞の分化が確認できた。 Example 3: Induction of differentiation of cells (lactic acid bacteria component-treated HDF cells) The HDF cell mass (A) prepared in Example 2 was cultured in the presence of lactic acid bacteria components for 2 weeks to obtain fat cells (B) and bone cells (C). The medium was replaced with a culture medium (GIBCO; A10070-01, A10072-01, A10071-01) that induces differentiation induction in the chondrocytes (D), and further cultured for 2 to 3 weeks.
The results are shown in FIG. As shown in FIG. 2, the HDF cell mass to which the lactic acid bacteria component was allowed to act was cultured in a differentiation-inducing medium, and (B) Oil Red O staining (fat), (C) Alizarin Red S staining (bone), (D ) Stained by Alcian Blue staining (cartilage), and differentiation of cells into each cell could be confirmed.
実施例４：乳酸菌成分存在下での肺がん細胞株（A549）の培養
　理化学研究所　バイオリソースセンターから肺がん細胞株（A549; RBRC-RCB0098）を入手した。実施例２と同様に、あらかじめ6 well plate に1 wellあたり50μlの乳酸菌成分を入れておき、5 x 105の肺がん細胞（A549）を加えた。そのまま34℃、5% CO2インキュベータで培養した。
　その結果、数日後には細胞塊が観察できた。図３の写真は、培養してから14日後の細胞の状態を示している。 Example 4: Culture of lung cancer cell line (A549) in the presence of lactic acid bacteria components A lung cancer cell line (A549; RBRC-RCB0098) was obtained from BioResource Center. In the same manner as in Example 2, 50 μl of lactic acid bacteria per well was placed in a 6-well plate in advance, and 5 × 10 5 lung cancer cells (A549) were added. The cells were cultured as they were at 34 ° C. in a 5% CO 2 incubator.
As a result, cell clusters could be observed after several days. The photograph in FIG. 3 shows the state of cells 14 days after culturing.
実施例５：細胞（乳酸菌成分処理A549細胞）の分化誘導
　理化学研究所　バイオリソースセンターから肺がん細胞株（A549; RBRC-RCB0098）を入手した。実施例２と同様に、あらかじめ6 well plate に1 wellあたり50μlの乳酸菌成分を入れておき、5 x 105の肺がん細胞（A549）を加えた。そのまま34℃、5% CO2インキュベータで培養した。形成された細胞塊を、乳酸菌成分存在下で２週間培養し、脂肪細胞（B）、骨細胞（C）に分化誘導をうながす培養液（GIBCO; A10070-01, A10072-01）に交換し、さらに2～3週間培養した。
　結果を図４に示す。写真(A)は培養してから14日後のものである。乳酸菌成分を作用させた肺がん細胞株（A549）由来の細胞塊は、分化誘導培地で培養後、(B) Oil Red O染色（脂肪）、(C) Alizarin Red S染色（骨）により染色され、細胞の分化が確認できた。なお、写真(D)は、写真（C）の四角の部分の拡大である。 Example 5: Differentiation induction of cells (lactic acid bacteria component-treated A549 cells) A lung cancer cell line (A549; RBRC-RCB0098) was obtained from RIKEN BioResource Center. In the same manner as in Example 2, 50 μl of lactic acid bacteria per well was placed in a 6-well plate in advance, and 5 × 10 5 lung cancer cells (A549) were added. The cells were cultured as they were at 34 ° C. in a 5% CO 2 incubator. The formed cell mass is cultured in the presence of lactic acid bacteria components for 2 weeks, and replaced with a culture solution (GIBCO; A10070-01, A10072-01) that induces differentiation induction in fat cells (B) and bone cells (C), The cells were further cultured for 2 to 3 weeks.
The results are shown in FIG. Photo (A) is from 14 days after culturing. A cell mass derived from a lung cancer cell line (A549) in which a lactic acid bacterium component is allowed to act is cultured in a differentiation-inducing medium, and then stained with (B) Oil Red O staining (fat), (C) Alizarin Red S staining (bone), Cell differentiation was confirmed. Photo (D) is an enlargement of the square part of photo (C).
実施例６：乳酸菌成分存在下での肝がん細胞株（HepG2）の培養
　理化学研究所　バイオリソースセンターから肝がん細胞株（HepG2; RBRC-RCB1648）を入手した。実施例２と同様に、あらかじめ6 well plate に1 wellあたり50μlの乳酸菌成分を入れておき、5 x 105の肝がん細胞（HepG2）を加えた。そのまま34℃、5% CO2インキュベータで培養した。
　その結果、数日後には細胞塊が観察できた。図５の写真は、培養してから14日後の細胞の状態を示している。 Example 6: Culture of a hepatoma cell line (HepG2) in the presence of lactic acid bacteria components A hepatoma cell line (HepG2; RBRC-RCB1648) was obtained from BioResource Center. In the same manner as in Example 2, 50 μl of lactic acid bacteria per well was placed in a 6-well plate in advance, and 5 × 10 5 liver cancer cells (HepG2) were added. The cells were cultured as they were at 34 ° C. in a 5% CO 2 incubator.
As a result, cell clusters could be observed after several days. The photograph in FIG. 5 shows the state of the cells 14 days after culturing.
実施例７：細胞（乳酸菌成分処理HepG2細胞）の分化誘導
　実施例２と同様に、あらかじめ6 well plate に1 wellあたり50μlの乳酸菌成分を入れておき、5 x 105の肝がん細胞（HepG2）を加えた。そのまま34℃、5% CO2インキュベータで培養した。形成された細胞塊を、乳酸菌成分存在下で２週間培養し、脂肪細胞（B）、骨細胞（C）に分化誘導をうながす培養液（GIBCO; A10070-01, A10072-01）に交換し、さらに2～3週間培養した。
　結果を図６に示す。写真(A)は培養してから14日後のものである。乳酸菌成分を作用させた肝がん細胞株（HepG2）由来の細胞塊は、分化誘導培地で培養後、(B) Oil Red O染色（脂肪）、(C) Alizarin Red S染色（骨）により染色され、細胞の分化が確認できた。 Example 7: Differentiation induction of cells (lactic acid bacteria component-treated HepG2 cells) In the same manner as in Example 2, 50 μl of lactic acid bacteria components per well were previously placed in a 6-well plate, and 5 × 10 5 hepatoma cells (HepG2 ) Was added. The cells were cultured as they were at 34 ° C. in a 5% CO 2 incubator. The formed cell mass is cultured in the presence of lactic acid bacteria components for 2 weeks, and replaced with a culture solution (GIBCO; A10070-01, A10072-01) that induces differentiation induction in fat cells (B) and bone cells (C), The cells were further cultured for 2 to 3 weeks.
The results are shown in FIG. Photo (A) is from 14 days after culturing. Cell mass derived from hepatoma cell line (HepG2) treated with lactic acid bacteria components is cultured in differentiation-inducing medium, and then stained with (B) Oil Red O staining (fat), (C) Alizarin Red S staining (bone) And cell differentiation was confirmed.
実施例８：乳酸菌成分存在下での乳がん細胞株（MCF７）の培養
　理化学研究所　バイオリソースセンターから乳がん細胞株（MCF7; RBRC-RCB1904）を入手した。実施例２と同様に、あらかじめ6 well plate に1 wellあたり50μlの乳酸菌成分を入れておき、5 x 105の乳がん細胞（MCF7）を加えた。そのまま34℃、5% CO2インキュベータで培養した。
　その結果、数日後には細胞塊が観察できた。図７の写真は、培養してから14日後の細胞の状態を示している。 Example 8: Culture of Breast Cancer Cell Line (MCF7) in the Presence of Lactic Acid Bacteria Components A breast cancer cell line (MCF7; RBRC-RCB1904) was obtained from BioResource Center. In the same manner as in Example 2, 50 μl of lactic acid bacteria component per well was placed in a 6 well plate in advance, and 5 × 10 5 breast cancer cells (MCF7) were added. The cells were cultured as they were at 34 ° C. in a 5% CO 2 incubator.
As a result, cell clusters could be observed after several days. The photograph in FIG. 7 shows the state of the cells 14 days after culturing.
実施例９：細胞（乳酸菌成分処理MCF7細胞）の分化誘導
　実施例２と同様に、あらかじめ6 well plate に1 wellあたり50μlの乳酸菌成分を入れておき、5 x 105の乳がん細胞（MCF7）を加えた。そのまま34℃、5% CO2インキュベータで培養した。形成された細胞塊を、乳酸菌成分存在下で２週間培養し、脂肪細胞（B）、骨細胞（C）に分化誘導をうながす培養液（GIBCO; A10070-01, A10072-01）に交換し、さらに2～3週間培養した。
　結果を図８に示す。写真(A)は培養してから14日後のものである。乳酸菌成分を作用させた乳がん細胞株（MCF7）由来の細胞塊は、分化誘導培地で培養後、(B) Oil Red O染色（脂肪）、(C) Alizarin Red S染色（骨）により染色され、細胞の分化が確認できた。 Example 9: Induction of differentiation of cells (lactic acid bacteria component-treated MCF7 cells) In the same manner as in Example 2, 50 μl of lactic acid bacteria components per well were previously placed in a 6-well plate, and 5 × 10 5 breast cancer cells (MCF7) were added. added. The cells were cultured as they were at 34 ° C. in a 5% CO 2 incubator. The formed cell mass is cultured in the presence of lactic acid bacteria components for 2 weeks, and replaced with a culture solution (GIBCO; A10070-01, A10072-01) that induces differentiation induction in fat cells (B) and bone cells (C), The cells were further cultured for 2 to 3 weeks.
The results are shown in FIG. Photo (A) is from 14 days after culturing. A cell mass derived from a breast cancer cell line (MCF7) treated with a lactic acid bacteria component is cultured in a differentiation induction medium, and then stained with (B) Oil Red O staining (fat), (C) Alizarin Red S staining (bone), Cell differentiation was confirmed.
実施例１０：細胞（乳酸菌成分処理HepG2細胞）の分化誘導
　実施例２と同様に、あらかじめ6 well plate に1 wellあたり50μlの乳酸菌成分を入れておき、5 x 105の肝がん細胞（HepG2）を加える。そのまま34℃、5% CO2インキュベータで培養した。細胞塊を、乳酸菌成分存在下で２週間培養した後、ポリLリジンとラミニンでコートしたカバーグラス上に移し7日間培養後、抗α-fetoprotein抗体（内胚葉マーカー）、抗NeuroFilament抗体（外胚葉、神経細胞マーカー）で染色した。
　結果を図９に示す。図９に示されるように分化誘導させた後の細胞は、各々の抗体で認識された。 Example 10: Differentiation induction of cells (lactic acid bacteria component-treated HepG2 cells) In the same manner as in Example 2, 50 μl of lactic acid bacteria components per well were previously placed in a 6-well plate, and 5 × 10 5 hepatoma cells (HepG2 ). The cells were cultured as they were at 34 ° C. in a 5% CO 2 incubator. The cell mass is cultured in the presence of lactic acid bacteria components for 2 weeks, then transferred to a cover glass coated with poly-L-lysine and laminin, cultured for 7 days, anti-α-fetoprotein antibody (endoderm marker), anti-NeuroFilament antibody (ectodermal) , Nerve cell marker).
The results are shown in FIG. As shown in FIG. 9, the cells after differentiation induction were recognized by each antibody.
実施例１１：乳酸菌成分存在下で培養した肺がん細胞株（A549）の多能性マーカー
　実施例４と同様の実験を、肺がん細胞株（A549）を用いて乳酸菌成分の存在下または非存在下で実験を行った。培養から14日目に細胞を回収し、様々な多能性マーカー(Oct3/4, Sox2, Nanog, GDF-3, Rex1, ECAT, FGF4)や初期の老化マーカー(p15, p16, ARF)を用いてRT-PCR法を行った。
　結果を図１０に示す。興味深いことに、A549細胞と乳酸菌成分を作用させると、多能性のマスター遺伝子と言われているNanogの発現が強く誘導された（矢印）。 Example 11: Pluripotency marker of lung cancer cell line (A549) cultured in the presence of lactic acid bacteria component The same experiment as in Example 4 was performed using the lung cancer cell line (A549) in the presence or absence of lactic acid bacteria component. The experiment was conducted. Collect cells on day 14 of culture and use various pluripotency markers (Oct3 / 4, Sox2, Nanog, GDF-3, Rex1, ECAT, FGF4) and early aging markers (p15, p16, ARF) RT-PCR was performed.
The results are shown in FIG. Interestingly, expression of Nanog, which is said to be a pluripotent master gene, was strongly induced when A549 cells and lactic acid bacteria components were allowed to act (arrows).
実施例１２：再プログラミング因子の同定
　乳酸菌由来の再プログラミング因子を同定するために、実施例1で得られた濃縮乳酸菌成分（50μl、タンパク量0.25 mg）をさらに陰イオン交換クロマトグラフィーで分離した（QEF カラム）。0.02 M TEA緩衝液（pH 7.5）で平衡化したHiPrep 16/10-Q-FF（GE health care社製）カラム（16 ml）にチャージし、0～1Mの塩化ナトリウムの直線濃度勾配法により溶出を行ない、活性画分(0.1M～0.4M)を回収した。HDF細胞を用いて、実施例２と同様にして、活性画分の効果を確認した。
　結果を図１１に示す。フラクション12から18に細胞塊形成活性が存在することが明らかになった。 Example 12: Identification of reprogramming factors In order to identify reprogramming factors derived from lactic acid bacteria, the concentrated lactic acid bacteria components (50 μl, protein amount 0.25 mg) obtained in Example 1 were further separated by anion exchange chromatography ( QEF column). Charge to a HiPrep 16 / 10-Q-FF (GE health care) column (16 ml) equilibrated with 0.02 M TEA buffer (pH 7.5) and elute with a linear concentration gradient of 0 to 1M sodium chloride. The active fraction (0.1 M to 0.4 M) was collected. Using HDF cells, the effect of the active fraction was confirmed in the same manner as in Example 2.
The results are shown in FIG. It was revealed that cell fraction forming activity exists in fractions 12 to 18.
比較例１：大腸菌成分による比較
　実施例１２と同様の実験を大腸菌（XLI-blue：Stratagene社）を用いて行い、画分(0.1M～0.4M NaCl画分)を回収した。この画分をHDF細胞に作用させ、実施例２と同様の実験を行い、細胞塊形成活性を確認した。
　結果を図１２に示す。その結果、大腸菌由来のフラクションには、細胞塊形成活性が存在しないことが明らかになった。 Comparative Example 1: Comparison with E. coli component The same experiment as in Comparative Example 12 was performed using E. coli (XLI-blue: Stratagene), and fractions (0.1 M to 0.4 M NaCl fraction) were collected. This fraction was allowed to act on HDF cells, and the same experiment as in Example 2 was performed to confirm the cell mass forming activity.
The results are shown in FIG. As a result, it was clarified that the cell mass-forming activity does not exist in the E. coli-derived fraction.
実施例１３：乳酸菌成分の精製
　実施例１で得られた乳酸菌成分を、さらに以下の工程（１）～（３）を用いて精製した。
（１）イオン交換クロマトグラフィー（HiPrep Q FF 16/10、GEヘルスケア）
（２）疎水性相互作用クロマトグラフィー（HiTrap Phenyl FF (low sub) 5ml、GEヘルスケア）
（３）ゲルろ過クロマトグラフィー（HiLoad Superdex 200 prep grade、GEヘルスケア）
　次いで、再プログラミン活性を有するゲルろ過クロマトグラフィー後のサンプルを、ＳＤＳ－ＰＡＧＥを行った。その結果、ＣＢＢで染色される３本のバンドが検出された。メインのバンドの分子量は、約６０ＫＤａであった。 Example 13: Purification of lactic acid bacteria components The lactic acid bacteria components obtained in Example 1 were further purified using the following steps (1) to (3).
(1) Ion exchange chromatography (HiPrep Q FF 16/10, GE Healthcare)
(2) Hydrophobic interaction chromatography (HiTrap Phenyl FF (low sub) 5 ml, GE Healthcare)
(3) Gel filtration chromatography (HiLoad Superdex 200 prep grade, GE Healthcare)
The sample after gel filtration chromatography having reprogramin activity was then subjected to SDS-PAGE. As a result, three bands stained with CBB were detected. The molecular weight of the main band was about 60 KDa.
実施例１４：細胞再プログラミング誘導タンパク質の物理化学的性質の検討
　実施例１３で得られた精製品を、糖タンパク質と結合するｍ－アミノフェニルボロン酸－アガロース（Glycoprotein Enrichment Resin、Takara社）を用いて精製した。その結果、ｍ－アミノフェニルボロン酸との結合が確認できた。また、実施例１３で得られた精製品を用いて、糖タンパク質と結合することが一般に知られているレクチンである、コンカナバリンＡおよび小麦胚芽レクチン（Wheat germ lectin）を用いて、結合性を確認したところ、これらのレクチンには結合しなかった。
　さらに、実施例１３で得られた精製品をProteinase Kで処理したところ、再プログラミング誘導活性が失われた。 Example 14: Examination of physicochemical properties of cell reprogramming-inducing protein Using the purified product obtained in Example 13 with m-aminophenylboronic acid-agarose (Glycoprotein Enrichment Resin, Takara) that binds to glycoprotein And purified. As a result, binding with m-aminophenylboronic acid was confirmed. Further, using the purified product obtained in Example 13, the binding property was confirmed using concanavalin A and wheat germ lectin, which are lectins generally known to bind to glycoproteins. As a result, it did not bind to these lectins.
Furthermore, when the refined product obtained in Example 13 was treated with Proteinase K, the reprogramming induction activity was lost.
実施例１５：乳酸菌成分存在下での腸細胞の培養
　実施例２と同様にして、腸細胞を用いて実験を行った。その結果、HDF細胞と同様に、数日後に細胞塊が観察された。 Example 15: Cultivation of intestinal cells in the presence of lactic acid bacteria components In the same manner as in Example 2, experiments were performed using intestinal cells. As a result, similar to HDF cells, cell clusters were observed after several days.
　上記の記載は、本発明の目的及び対象を単に説明するものであり、添付の特許請求の範囲を限定するものではない。添付の特許請求の範囲から離れることなしに、記載された実施態様に対しての、種々の変更及び置換は、本明細書に記載された教示より当業者にとって明らかである。 The above description merely explains the objects and objects of the present invention, and does not limit the scope of the appended claims. Various changes and substitutions to the described embodiments will be apparent to those skilled in the art from the teachings described herein without departing from the scope of the appended claims.
　本発明は、体細胞から多能性細胞を生産する方法として有用である。 The present invention is useful as a method for producing pluripotent cells from somatic cells.

Claims (29)

  1. 　単離された哺乳類動物の体細胞から誘導された多能性細胞であって、以下の工程、
    （ａ）前記体細胞に、発酵能を有する細菌のホモジネートから得ることができる細胞を再プログラミング誘導するタンパク質含有成分であって、以下の性質：
    （i）ｍ－アミノフェニルボロン酸に結合する、
    （ii）コンカナバリンＡに結合しない、および
    （iii）小麦胚芽レクチン（Wheat germ lectin）に結合しない
    の少なくとも一つを有するタンパク質含有成分を接触させて培養または維持する工程、および
    （ｂ）形成された細胞塊を回収する工程、
    含む製造工程により生産される多能性細胞。     A pluripotent cell derived from an isolated mammalian somatic cell, comprising the following steps:
    (A) a protein-containing component that induces reprogramming of a cell that can be obtained from a homogenate of a bacterium having fermentation ability into the somatic cell, and having the following properties:
    (I) binds to m-aminophenylboronic acid;
    (B) formed by contacting and culturing or maintaining a protein-containing component having at least one of (ii) not binding to concanavalin A and (iii) not binding to wheat germ lectin Recovering the cell mass,
    A pluripotent cell produced by a production process comprising.
  2. 　前記タンパク質含有成分が、細菌のホモジネートを硫安沈殿法を用い濃縮することにより得られた、１００ＫＤａの限外ろ過フィルターを透過しない大きさの成分である、請求項１に記載の多能性細胞。 The pluripotent cell according to claim 1, wherein the protein-containing component is a component that is obtained by concentrating a bacterial homogenate using an ammonium sulfate precipitation method and does not permeate a 100 KDa ultrafiltration filter.
  3. 　前記タンパク質含有成分が、SDS-PAGEにおいて約５５～約６５ＫＤａの分子量を示すタンパク質をメインバンドとして含む、請求項１または２に記載の多能性細胞。 The pluripotent cell according to claim 1 or 2, wherein the protein-containing component contains a protein having a molecular weight of about 55 to about 65 KDa as a main band in SDS-PAGE.
  4. 　前記発酵能を有する細菌が乳酸菌または納豆菌である請求項１～３のいずれか一つに記載の多能性細胞。 The pluripotent cell according to any one of claims 1 to 3, wherein the bacterium having fermentation ability is a lactic acid bacterium or a natto bacterium.
  5. 　前記乳酸菌が、Lactococcus属、Streptococcus属、またはLactobacillus属の乳酸菌である請求項４に記載の多能性細胞。 The pluripotent cell according to claim 4, wherein the lactic acid bacterium is a lactic acid bacterium of the genus Lactococcus, Streptococcus, or Lactobacillus.
  6. 　前記タンパク質含有成分が、（a）発酵能を有する細菌を破砕することにより得られるホモジネートを硫安濃度２０～６０％飽和で沈殿させて硫安沈殿として得る工程、および（b）該硫安沈殿を溶解した溶液を１００ＫＤａの限外ろ過フィルターを透過させ、透過しない画分を得る工程、を含む工程により得られるタンパク質含有成分である、請求項１～５のいずれか一つに記載の多能性細胞。 The protein-containing component is (a) a step of precipitating a homogenate obtained by crushing bacteria having fermentative ability at an ammonium sulfate concentration of 20 to 60% to obtain an ammonium sulfate precipitate, and (b) dissolving the ammonium sulfate precipitate. The pluripotent cell according to any one of claims 1 to 5, which is a protein-containing component obtained by a step comprising a step of allowing a solution to pass through a 100 KDa ultrafiltration filter and obtaining a fraction that does not pass through.
  7. 　前記体細胞が、ヒト由来の、正常体細胞またはがん細胞である請求項１～６のいずれか一つに記載の多能性細胞。 The pluripotent cell according to any one of claims 1 to 6, wherein the somatic cell is a human-derived normal somatic cell or cancer cell.
  8. 　請求項１～７のいずれか一つに記載の多能性細胞を分化誘導培地で培養することにより分化誘導された細胞。 A cell induced to differentiate by culturing the pluripotent cell according to any one of claims 1 to 7 in a differentiation-inducing medium.
  9. 　前記細胞が、脂肪細胞、骨細胞、軟骨細胞、神経細胞、心筋細胞、肝細胞、膵臓細胞、または血球細胞である、請求項８に記載の細胞。 The cell according to claim 8, wherein the cell is an adipocyte, bone cell, chondrocyte, nerve cell, cardiomyocyte, hepatocyte, pancreas cell, or blood cell.
  10. 　単離された哺乳類動物の体細胞から多能性細胞を製造するための方法であって、
    　前記体細胞に、発酵能を有する細菌のホモジネートから得ることができる細胞を再プログラミング誘導するタンパク質含有成分であって、以下の性質：
    （i）ｍ－アミノフェニルボロン酸に結合する、
    （ii）コンカナバリンＡに結合しない、および
    （iii）小麦胚芽レクチン（Wheat germ lectin）に結合しない
    の少なくとも一つを有するタンパク質含有成分を接触させることを含む体細胞から多能性細胞を製造する方法。     A method for producing pluripotent cells from isolated mammalian somatic cells, comprising:
    A protein-containing component that induces reprogramming of cells that can be obtained from a bacterial homogenate having fermentation ability into the somatic cells, and having the following properties:
    (I) binds to m-aminophenylboronic acid;
    A method of producing pluripotent cells from somatic cells comprising contacting a protein-containing component having at least one of (ii) not binding to concanavalin A and (iii) not binding to wheat germ lectin .
  11. 　前記タンパク質含有成分が、細菌のホモジネートを硫安沈殿法を用い濃縮することにより得られた、１００ＫＤａの限外ろ過フィルターを透過しない大きさの成分である、請求項１０に記載の方法。 The method according to claim 10, wherein the protein-containing component is a component that is obtained by concentrating a bacterial homogenate using an ammonium sulfate precipitation method and does not pass through a 100 KDa ultrafiltration filter.
  12. 　前記タンパク質含有成分が、SDS-PAGEにおいて約５５～約６５ＫＤａの分子量を示すタンパク質をメインバンドとして含む、請求項１０または１１に記載の方法。 The method according to claim 10 or 11, wherein the protein-containing component contains a protein having a molecular weight of about 55 to about 65 KDa as a main band in SDS-PAGE.
  13. 　前記発酵能を有する細菌が乳酸菌または納豆菌である請求項１０～１２に記載の方法。 The method according to any one of claims 10 to 12, wherein the bacterium having fermentation ability is lactic acid bacteria or natto bacteria.
  14. 　前記乳酸菌が、Lactococcus属、Streptococcus属、またはLactobacillus属の乳酸菌である請求項１３に記載の方法。 The method according to claim 13, wherein the lactic acid bacterium is a lactic acid bacterium of the genus Lactococcus, Streptococcus, or Lactobacillus.
  15. 　前記タンパク質含有成分が、（a）発酵能を有する細菌を破砕することにより得られるホモジネートを硫安濃度２０～６０％飽和で沈殿させて硫安沈殿として得る工程、および（b）該硫安沈殿を溶解した溶液を１００ＫＤａの限外ろ過フィルターを透過させ、透過しない画分を得る工程を含む工程により得られるタンパク質含有成分である、請求項１０～１４のいずれか一つに記載の方法。 The protein-containing component is (a) a step of precipitating a homogenate obtained by crushing bacteria having fermentative ability at an ammonium sulfate concentration of 20 to 60% to obtain an ammonium sulfate precipitate, and (b) dissolving the ammonium sulfate precipitate. The method according to any one of claims 10 to 14, which is a protein-containing component obtained by a step comprising a step of allowing a solution to pass through a 100 KDa ultrafiltration filter and obtaining a non-permeated fraction.
  16. 　前記体細胞が、ヒト由来の、正常体細胞またはがん細胞である請求項１０～１５のいずれか一つに記載の方法。 The method according to any one of claims 10 to 15, wherein the somatic cells are human-derived normal somatic cells or cancer cells.
  17. 　発酵能を有する細菌のホモジネートから得られる細胞を多能性細胞に再プログラミング誘導するタンパク質含有成分であって、以下の性質：
    （i）ｍ－アミノフェニルボロン酸に結合する、
    （ii）コンカナバリンＡに結合しない、および
    （iii）小麦胚芽レクチン（Wheat germ lectin）に結合しない
    の少なくとも一つを有する糖タンパク質含有成分を含有する細胞再プログラミング誘導組成物。     A protein-containing component that induces reprogramming of cells obtained from a bacterial homogenate having fermentability into pluripotent cells, having the following properties:
    (I) binds to m-aminophenylboronic acid;
    A cell reprogramming induction composition comprising a glycoprotein-containing component having at least one of (ii) not binding to concanavalin A and (iii) not binding to wheat germ lectin.
  18. 　前記タンパク質含有成分が、細菌のホモジネートを硫安沈殿法を用い濃縮することにより得られた、１００ＫＤａの限外ろ過フィルターを透過しない大きさの成分である、請求項１７に記載の組成物。 The composition according to claim 17, wherein the protein-containing component is a component that is obtained by concentrating a bacterial homogenate using an ammonium sulfate precipitation method and does not pass through a 100 KDa ultrafiltration filter.
  19. 　前記タンパク質含有成分が、SDS-PAGEにおいて約５５～約６５ＫＤａの分子量を示すタンパク質をメインバンドとして含む、請求項１７または１８に記載の組成物。 The composition according to claim 17 or 18, wherein the protein-containing component contains a protein having a molecular weight of about 55 to about 65 KDa as a main band in SDS-PAGE.
  20. 　前記発酵能を有する細菌が乳酸菌または納豆菌である請求項１７～１９のいずれか一つに記載の組成物。 The composition according to any one of claims 17 to 19, wherein the bacterium having fermentation ability is a lactic acid bacterium or a natto bacterium.
  21. 　前記乳酸菌が、Lactococcus属、Streptococcus属、またはLactobacillus属の乳酸菌である請求項２０に記載の組成物。 The composition according to claim 20, wherein the lactic acid bacterium is a lactic acid bacterium of the genus Lactococcus, Streptococcus, or Lactobacillus.
  22. 　前記タンパク質含有成分が、（a）発酵能を有する細菌を破砕することにより得られるホモジネートを硫安濃度２０～６０％飽和で沈殿させて硫安沈殿として得る工程、および（b）該硫安沈殿を溶解した溶液を１００ＫＤａの限外ろ過フィルターを透過させ、透過しない画分を得る工程、を含む工程により得られるタンパク質含有成分である、請求項１７～２１のいずれか一つに記載の組成物。 The protein-containing component is (a) a step of precipitating a homogenate obtained by crushing bacteria having fermentative ability at an ammonium sulfate concentration of 20 to 60% to obtain an ammonium sulfate precipitate, and (b) dissolving the ammonium sulfate precipitate. The composition according to any one of claims 17 to 21, which is a protein-containing component obtained by a step comprising a step of allowing a solution to pass through a 100 KDa ultrafiltration filter and obtaining a non-permeate fraction.
  23. 　前記細胞が、ヒト由来の、正常体細胞またはがん細胞である請求項１７～２２のいずれか一つに記載の組成物。 The composition according to any one of claims 17 to 22, wherein the cell is a normal somatic cell or cancer cell derived from human.
  24. 　請求項１７～２３のいずれか一つに記載の組成物を含む抗がん剤。 An anticancer agent comprising the composition according to any one of claims 17 to 23.
  25. 　乳酸菌または納豆菌からのホモジネートまたは粗精製物を含む、単離された哺乳類動物体細胞の再プログラミング培地。 An isolated mammalian somatic cell reprogramming medium containing a homogenate or crude product from lactic acid bacteria or natto.
  26. 　前記ホモジネートまたは粗精製物が、以下の性質：
    （i）ｍ－アミノフェニルボロン酸に結合する、
    （ii）コンカナバリンＡに結合しない、および
    （iii）小麦胚芽レクチン（Wheat germ lectin）に結合しない
    の少なくとも一つを有するタンパク質を含むことを特徴とする、請求項２５に記載の培地。     The homogenate or crude product has the following properties:
    (I) binds to m-aminophenylboronic acid;
    26. The medium of claim 25, comprising a protein having at least one of (ii) not binding to concanavalin A and (iii) not binding to wheat germ lectin.
  27. 　前記成分が、SDS-PAGEにおいて約５５～約６５ＫＤａの分子量を示すタンパク質をメインバンドとして含む、請求項２６に記載の培地。 The medium according to claim 26, wherein the component contains a protein having a molecular weight of about 55 to about 65 KDa as a main band in SDS-PAGE.
  28. 　前記乳酸菌が、Lactococcus属、Streptococcus属、またはLactobacillus属の乳酸菌である請求項２５～２７のいずれか一つに記載の培地。 The medium according to any one of claims 25 to 27, wherein the lactic acid bacterium is a lactic acid bacterium of the genus Lactococcus, Streptococcus, or Lactobacillus.
  29. 　前記培地が、ダルベッコ改変イーグル培地（ＤＭＥＭ）、イーグル最少必須（ＥＭＥ）培地、イスコフ改変ダルベッコ培地（ＩＭＤＭ）、アルファ－最少必須培地（α－ＭＥＭ）、ＲＰＭＩ　１６４０、Ｈａｍ－Ｆ－１２、ＭＣＤＢおよびそれらの改変培地からなる群より選ばれる、請求項２５～２８のいずれか一つに記載の培地。 The medium is Dulbecco's Modified Eagle Medium (DMEM), Eagle's Minimal Essential (EME) Medium, Iskov's Modified Dulbecco Medium (IMDM), Alpha-Minimal Essential Medium (α-MEM), RPMI 1640, Ham-F-12, MCDB and The medium according to any one of claims 25 to 28, which is selected from the group consisting of those modified media.
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