Method for producing brown fat cells

Abstract

The present invention addresses the problem of providing a novel production method whereby somatic cells can be directly converted or induced into brown adipocytes using a low-molecular compound without performing gene transfer.　As an example of the method according to the present invention, citation can be made of a method for producing brown adipocytes by directly inducing differentiation of somatic cells into brown adipocytes, said method being characterized by comprising a step for culturing the somatic cells in a differentiation-inducing serum-free medium in the presence of a selective PPARγ agonist and a cAMP inducing agent.　According to the invention, somatic cells can be directly converted or induced into brown adipocytes at a high efficiency without performing gene transfer. The brown adipocytes obtained by the invention are useful in regenerative medicine and as a human adipocyte model or a human beige cell model, etc.

Description

[Technical field]

[0001] (CROSS REFERENCE TO RELATED APPLICATIONS) This application claims the benefit of Japanese Application No. 2019-211990, filed with the Japan Patent Office on November 25, 2019. The Japanese application is hereby incorporated by reference for all purposes as if the entire application documents (specification, claims, drawings, abstract) were set forth herein. The present invention belongs to the technical field of regenerative medicine. In the technical field, the present invention relates to a method for producing brown adipocytes from somatic cells using a low molecular weight compound, and to low molecular weight compound-induced brown adipocytes (ciBAs) produced by the production method. The present invention further relates to an induction medium composition that can be used for the method for producing brown adipocytes. [Background technology]

[0002] In recent years, a method of directly converting somatic cells such as fibroblasts into other cells using low molecular weight compounds without gene transfer (Direct Reprogramming) has been actively studied. For example, in Patent Document 1, several low molecular weight compounds such as ALK5 inhibitors and ALK6 inhibitors are combined, and somatic cells, particularly human fibroblasts, are cultured in the presence of the compounds, and then directly induced to become brown fat cells, etc. As in the invention of Patent Document 1, it would be very beneficial if common fibroblasts could be directly induced to become cells such as brown fat cells using easily available low molecular weight compounds. For example, since other autologous cells can be easily produced from autologous fibroblasts, the application to regenerative medicine will increase. In addition, it will become possible to easily produce cells as experimental materials for new drug development.

[0003] In Non-Patent Document 1, human dermal fibroblasts are cultured in the presence of a combination of several low molecular weight compounds, as in Patent Document 1, and brown adipocytes are directly induced from the fibroblasts. Specifically, in the invention of Non-Patent Document 1, human dermal fibroblasts are cultured in the presence of low molecular weight compounds such as SB431542 (ALK5 inhibitor), LDN193189 (ALK2 / 3 inhibitor), dorsomorphin (ALK2 / 3 / 6 inhibitor), forskolin (cAMP inducer), and rosiglitazone (PPARγ agonist), and brown adipocytes are directly induced from the fibroblasts. Such a combination of low molecular weight compounds is also disclosed in Patent Document 1.

[0004] The differentiation of somatic cells into brown fat cells and the like is always performed by culturing them under a medium containing various other compounds and nutrients. Examples of the medium include general media such as MEM (minimum essential medium), DMEM (Dulbecco's Modified Eagle Medium), DMEM / F12, and modified media thereof, which are commercially available. Usually, appropriate components (serum, proteins, amino acids, sugars, vitamins, fatty acids, antibiotics, etc.) are appropriately added to these media in consideration of the cells to be induced to differentiate into cells. Among these, serum such as fetal bovine serum (FBS) contains components necessary for cell growth such as hormones, growth factors, and adhesion factors, so that differentiation is often induced by adding serum. In the inventions of Patent Document 1 and Non-Patent Document 1, differentiation is induced by a medium containing FBS. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] International Publication No. 2018 / 062269 [Non-patent literature]

[0006] [Non-Patent Document 1] Takeda Y.,Harada Y.,Yoshikawa T.,and Dai P.,Sci.Rep.,7,4304(2017) Summary of the Invention [Problem to be solved by the invention]

[0007] The main objective of the present invention is to provide a new method for directly converting or inducing somatic cells into brown adipocytes using a low molecular weight compound without gene transfer. Another objective of the present invention is to provide a new composition for producing brown adipocytes by direct conversion by culturing somatic cells in the presence of a low molecular weight compound. [Means for solving the problem]

[0008] As a result of extensive investigations, the present inventors have found that the above-mentioned problems can be solved by using a combination of certain low molecular weight compounds in a serum-free state, and have thus completed the present invention.

[0009] The present invention can include, for example, the following. [1] A method for producing brown fat cells by directly inducing differentiation from somatic cells, characterized in that it includes a step of culturing somatic cells in a serum-free differentiation induction medium in the presence of a selective PPARγ agonist and a cAMP inducer. [2] The method for producing brown adipocytes described in [1] above, wherein the step further comprises culturing somatic cells in the presence of BMP7. [3] The method for producing brown adipocytes described in [1] or [2] above, wherein the step further comprises culturing somatic cells in the absence of an ALK2 / 3 inhibitor and an ALK6 inhibitor. [4] The method for producing brown adipocytes according to any one of [1] to [3] above, wherein the step further comprises culturing somatic cells in the absence of an ALK5 inhibitor. [5] The method for producing brown adipocytes according to any one of the above [1] to [4], wherein the selective PPARγ agonist is rosiglitazone, or the cAMP inducer is forskolin. [6] The method for producing brown adipocytes according to any one of [1] to [5] above, wherein the somatic cells are fibroblasts. [7] Brown adipocytes produced by the production method described in any one of [1] to [6] above.

[0010] [8] A composition for producing brown adipocytes by directly inducing differentiation from somatic cells, the composition comprising a selective PPARγ agonist and a cAMP inducer, and being serum-free. [9] The composition for inducing differentiation described in [8] above, further comprising BMP7.

[10] The composition for inducing differentiation described in [8] or [9] above, wherein the selective PPARγ agonist is rosiglitazone, or the cAMP inducer is forskolin.

[11] The composition for inducing differentiation according to any one of the above [8] to

[10] , wherein the somatic cells are fibroblasts.

[0011]

[12] A method for screening drugs that act on brown fat cells, characterized by using brown fat cells produced by the production method described in any one of [1] to [6] above, or brown fat cells described in [7] above.

[13] A method for evaluating the degree to which a test substance enhances the browning tendency in brown fat cells produced by the production method described in any one of [1] to [6] above or cells in the process of being produced, or brown fat cells described in [7] above, is added to the brown fat cells. Effect of the Invention

[0012] According to the present invention, it is possible to effectively convert somatic cells into brown adipocytes or directly induce their differentiation in a serum-free state without gene transfer. The brown adipocytes obtained according to the present invention are useful in regenerative medicine and as a human brown adipocyte model. [Brief description of the drawings]

[0013] [Figure 1] The photographs show cultured cells and immunostained mitochondria (red), UCP1 protein (green), and cell nuclei (blue). Because FIG. 1 is monochrome, the colors red, green, and blue are not displayed, but the original photograph of FIG. 1 shows the colors. The top four figures show the results of the combination of compounds in Example 1, and the bottom four figures show the results of the combination of compounds in Example 2. [Diagram 2] The figures show the proportions of fat-like cells and UCP1-positive cells. The two left columns show the results of the combination of compounds in Example 1, and the two right columns show the results of the combination of compounds in Example 2. In the results of each Example, the left column shows the proportion of fat-like cells, and the right column shows the proportion of UCP1-positive cells. The vertical axis shows the proportion (%) of these cells relative to cells positive for nuclear staining (DAPI). [Diagram 3] Panel A shows the expression level of Ucp1 gene, panel B shows the expression level of Fabp4 gene, panel C shows the Ucp1 / Fabp4 ratio, and panel D shows an electrophoretic photograph of quantification of UCP1 protein. Reference numeral 1 indicates Comparative Example 3 (control), reference numeral 2 indicates Example 1, and reference numeral 3 indicates Example 2. The vertical axis indicates the relative value of the amount of the mRNA to the amount of mRNA of the internal standard gene (Tbp). [Figure 4] The left panel shows the expression level of the Ucp1 gene, and the right panel shows an electrophoretic photograph of the quantification of UCP1 protein. Reference numeral 1 indicates Comparative Example 3 (control), reference numeral 2 indicates Example 3, and reference numeral 3 indicates Example 2. The vertical axis indicates the relative value of the amount of the mRNA to the amount of the internal standard gene (Tbp) mRNA. [Diagram 5] The figures show the expression level of the Ucp1 gene. Reference symbol 1 indicates Comparative Example 3 (control), reference symbol 2 indicates Example 3, reference symbol 3 indicates Comparative Example 2, reference symbol 4 indicates Comparative Example 1, and reference symbol 5 indicates Example 4. The vertical axis indicates the relative value of the mRNA amount to the mRNA amount of the internal standard gene (Tbp). [Figure 6]Figure A shows the expression level of Ucp1 gene, and Figure B shows the expression level of Fabp4 gene. In the results for skin fibroblasts derived from different human specimens, each left column shows the results without compound (Comparative Example 3), and each right column shows the results with the combination of compounds of Example 3. The vertical axis shows the relative value of the amount of the mRNA to the amount of the internal standard gene (Tbp) mRNA. [Figure 7] The photographs are of cultured cells, and of the cells immunostained for mitochondria (red) and UCP1 protein (green), and of stained cell nuclei (blue), overlaid with each other. Figure 7 is monochrome, so green, red, and blue are not visible, but in the original photograph of Figure 7, green, red, and blue are visible. [Figure 8] Shows the percentage of adipocytes and UCP1-positive cells. For each result for human fibroblasts, the left column shows the percentage of adipocytes, and the right column shows the percentage of UCP1-positive cells. [Figure 9] It shows the activation of Ucp1 gene expression. C shows the expression level in control cells (no compound added, Comparative Example 3). H2O6h shows the result after 6 hours of culture with the addition of control H2O (water) to cells cultured in serum-free brown fat maintenance medium (SFBAMaM) after induction with the combination of compounds in Example 3. Iso6h shows the result after 6 hours of culture with the addition of isoproterenol, a β-adrenergic receptor agonist, instead of H2O. The vertical axis shows the relative value of the amount of mRNA in question to the amount of mRNA of the internal standard gene (Tbp). [Figure 10]Figure A shows the change in oxygen consumption rate. Grey circles show the results for control cells (no compound added, Comparative Example 3), and black diamonds show the results for fibroblast-derived brown adipocytes with the combination of compounds in Example 3. In the figure, the dotted lines indicate the addition of oligomycin, FCCP, or antimycin A / rotenone. The horizontal axis shows time (minutes). Figure B shows the oxygen consumption rate corresponding to each parameter on the horizontal axis. In the results for each parameter, the left columns show the results without compound (Comparative Example 3), and the right columns show the results with the combination of compounds in Example 3. The vertical axis shows the oxygen consumption rate OCR (picomoles / min / 104 cells). [Figure 11] The expression level of a gene is shown. In each figure, the vertical axis indicates the relative value of the amount of mRNA of the gene to the amount of mRNA of the internal standard gene (Tbp). The horizontal axis indicates time (weeks). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] The present invention will be described in detail below.

[0015] 1. How to produce brown fat cells The method for producing brown adipocytes according to the present invention (hereinafter referred to as the "method of the present invention") is a method for producing brown adipocytes by directly inducing differentiation from somatic cells, and is characterized by including a step of culturing somatic cells in a serum-free differentiation induction medium in the presence of a selective PPARγ agonist and a cAMP inducer.

[0016] Here, "serum-free" means that it is substantially free of unconditioned or unpurified serum. In the present invention, even if the differentiation-inducing medium contains purified blood-derived components or animal tissue-derived components, it is called a serum-free differentiation-inducing medium as long as it does not substantially contain unconditioned or unpurified serum. The "brown fat cells" (ciBAs) produced by the method of the present invention have many mitochondria and many lipid droplets, and are equivalent to so-called brown fat cells that express the UCP1 (uncoupling protein-1) gene. Therefore, they can be simply called brown fat cells or brown fat-like cells. They can also be a model for so-called human beige cells and bright cells.

[0017] The above step in the method of the present invention is preferably a step of further culturing somatic cells in the presence of BMP7. The selective PPARγ agonists and cAMP inducers may each be used alone or in combination of two or more.

[0018] 1.1 About somatic cells The cells of an organism can be classified into somatic cells and germ cells. In the method of the present invention, any somatic cells can be used as the starting material. There is no particular limitation on the somatic cells, and they may be either primary cells collected from a living organism or established cells. In the method of the present invention, somatic cells at various stages of differentiation, for example, terminally differentiated somatic cells (e.g., fibroblasts, umbilical vein endothelial cells (HUVECs), hepatocytes, biliary cells, pancreatic α cells, pancreatic acinar cells, pancreatic ductal gland cells, intestinal crypt cells, etc.), somatic cells in the process of terminal differentiation (e.g., mesenchymal stem cells, neural stem cells, endodermal progenitor cells, etc.), or somatic cells such as iPS cells that have been reprogrammed to acquire pluripotency, and somatic cells that have differentiated from iPS cells or are in the process of differentiation, can be used. Somatic cells that can be used in the method of the present invention include any somatic cells, such as hematopoietic cells (various lymphocytes, macrophages, dendritic cells, bone marrow cells, etc.), organ-derived cells (hepatocytes, spleen cells, pancreatic cells, kidney cells, lung cells, etc.), muscle tissue cells (skeletal muscle cells, smooth muscle cells, myoblasts, cardiac muscle cells, etc.), fibroblasts, nerve cells, osteoblasts, chondrocytes, endothelial cells, interstitial cells, adipocytes (white adipocytes, etc.), embryonic stem cells (ES cells), etc. The method of the present invention can also be applied to precursor cells of these cells and cancer cells. Fibroblasts can be preferably used.

[0019] Fibroblasts that can be used in the present invention are not particularly limited, and examples include dermal fibroblasts, adventitial fibroblasts, cardiac fibroblasts, pulmonary fibroblasts, uterine fibroblasts, and villous mesenchymal fibroblasts, which are major cellular components that make up connective tissue in various tissues and organs and produce collagen fibers.

[0020] Examples of sources of the above somatic cells include, but are not limited to, humans, non-human mammals, and non-mammalian animals (birds, reptiles, amphibians, fish, etc.). Preferred sources of somatic cells are humans and non-human mammals, and humans are particularly preferred. When producing brown adipocytes by the method of the present invention for administration to humans, somatic cells collected from a donor whose histocompatibility antigen type matches or is similar to that of the recipient can be preferably used. Somatic cells collected from the recipient themselves may be used for producing brown adipocytes.

[0021] 1.2 Selective PPARγ agonists PPARγ (Peroxisome proliferator-activated receptor γ) is a protein belonging to the nuclear receptor superfamily, and is a transcription factor that controls gene expression by forming a heterodimer with retinoid X receptor (RXR) and binding to the promoter region of the target gene. There are three subtypes of PPAR: α, δ (β), and γ. In the present invention, an agonist that selectively acts on γ is used.

[0022] "In the presence of a selective PPARγ agonist" refers to culture conditions under which an agonist effect can be selectively exerted on PPARγ. The means for this are not particularly limited, but typically, a compound that selectively exerts an agonist effect on PPARγ can be used.

[0023] Although not particularly limited in the present invention, examples of selective PPARγ agonists include the following compounds or salts thereof. Preferably, selective PPARγ agonists of the thiazolidine derivatives can be included, specifically, rosiglitazone, pioglitazone, and troglitazone can be included.

[0024] Rosiglitazone (CAS No.: 122320-73-4)

[0025] [ka]

[0026] Rosiglitazone maleate (CAS No.: 155141-29-0) Ciglitazone (CAS No.: 74772-77-3) Edaglitazone (CAS No.: 213411-83-7) GW1929 hydrochloride (CAS No.: 1217466-21-1) nTZDpa (CAS No.: 118414-59-8) Pioglitazone hydrochloride (CAS No.:112529-15-4) S26948(CAS No.:353280-43-0) Troglitazone (CAS No.: 97322-87-7) Balaglitazone (CAS No.: 199113-98-9) Rivoglitazone (CAS No.: 185428-18-6)

[0027] The concentration of the selective PPARγ agonist varies depending on the type of agonist and the somatic cells used, but is not particularly limited and may be determined appropriately. For example, it can be used in the range of 0.1 μmol / L to 20 μmol / L, preferably 0.5 μmol / L to 5 μmol / L.

[0028] 1.3 cAMP inducers cAMP (cyclic adenosine monophosphate) is a substance that is involved in various intracellular signal transduction as a second messenger. cAMP is generated in cells by the cyclization of adenosine triphosphate (ATP) by adenylate cyclase.

[0029] "In the presence of a cAMP inducer" refers to culture conditions under which cAMP can be induced, and the means for induction are not particularly limited. For example, any means capable of increasing the intracellular cAMP concentration can be used. Substances capable of directly acting on and inducing adenylate cyclase, an enzyme involved in the production of cAMP, substances capable of promoting the expression of adenylate cyclase, as well as substances inhibiting phosphodiesterase, an enzyme that breaks down cAMP, can be used as a means for increasing the intracellular cAMP concentration. Dibutyryl cAMP and 8-bromo-cAMP, which are structural analogues of cAMP and have the same effect as cAMP in cells, can also be used.

[0030] Although not particularly limited in the present invention, examples of cAMP inducers (adenylate cyclase activators) include forskolin (CAS No.: 66575-29-9), forskolin derivatives (e.g., JP-A-2002-348243), and the following compounds. Forskolin can be preferably used.

[0031] Forskolin (CAS No.: 66428-89-5)

[0032] [ka]

[0033] Isoproterenol (CAS No.: 7683-59-2) NKH477(CAS No.:138605-00-2) PACAP1-27(CAS No.:127317-03-7) PACAP1-38(CAS No.:137061-48-4)

[0034] The concentration of the cAMP inducer varies depending on the type of inducer and the somatic cells used, but is not particularly limited and may be determined appropriately. For example, it can be used in the range of 0.1 μmol / L to 100 μmol / L, preferably 0.5 μmol / L to 30 μmol / L.

[0035] 1.4 About BMP7 BMP (Bone Morphogenetic Protein) 7 is a member of the BMP family. Human BMP7 is composed of 117 amino acid residues and binds to a receptor on the cell surface as a homodimer, activating the BMP signal pathway. Six types, from BMP2 to BMP7, are classified as the TGFb superfamily and are each encoded by a different gene. In the present invention, human-derived BMP7 or animal-derived BMP7 may be used, or a recombinant form thereof may be used. Of these, it is preferable to use human-derived BMP7.

[0036] The concentration of BMP7 varies depending on the type of BMP7 and the somatic cells used, but is not particularly limited and may be determined appropriately. For example, it can be used in the range of 0.1 ng / mL to 200 ng / mL, preferably 1 ng / mL to 50 ng / mL.

[0037] 1.5 Preferred Culture Conditions Although not particularly limited in the present invention, it is more preferable to culture somatic cells in the above steps of the method of the present invention in the absence of an ALK2 / 3 inhibitor (an agent that inhibits both ALK2 and 3) and an ALK6 inhibitor, to culture somatic cells in the absence of an ALK5 inhibitor, or to culture somatic cells in the absence of an ALK2 / 3 inhibitor, an ALK6 inhibitor, and an ALK5 inhibitor. The absence of an ALK2 / 3 inhibitor, an ALK6 inhibitor, or an ALK5 inhibitor means that they are substantially absent, and includes not only the case where they are completely absent, but also the case where they are present in amounts that do not produce the effect.

[0038] ALK2 is a receptor serine / threonine kinase that is a member of the ALK family and is upstream of signaling pathways involving SMAD proteins, particularly SMAD1 / 5 / 8. Endoglin reduces prostate cancer cell motility through activation of the ALK2-Smad1 pathway. The ALK2 gene is the major gene involved in fibrodysplasia ossificans progressiva (FOP), a rare autosomal dominant congenital disorder characterized by progressive ectopic bone formation in muscle tissue.

[0039] ALK3 is a member of the transmembrane serine-threonine kinase family. The ALK3 gene acts as a minor susceptibility gene in PTEN (phosphatase and tensin homologue deleted on chromosome 10) mutation-negative Cowden disease. ALK3 trafficking plays an important role in the pathogenesis of FOP and is also involved in human T cell differentiation.

[0040] Specific examples of ALK2 / 3 inhibitors include LDN193189 (CAS No.: 1062368-24-4), dorsomorphin (CAS No.: 866405-64-3), K02288 (CAS No.: 1431985-92-0), LDN212854 (CAS No.: 1432597-26-6), LDN193189 hydrochloride (CAS No.: 1062368-62-0), ML347 (CAS No.: 1062368-49-3), and LDN214117 (CAS No.: 1627503-67-6).

[0041] ALK6, also known as BMPR1B, is a transmembrane serine / threonine kinase member of the bone morphogenetic protein (BMP) receptor family and is highly similar to the activin receptors ACVR1 and ACVR2. ALK6 is primarily involved in endochondral bone formation and embryogenesis. Specific examples of ALK6 inhibitors include dorsomorphin (CAS No.: 866405-64-3) and K02288 (CAS No.: 1431985-92-0).

[0042] ALK5 is a member of the TGFβ receptor subfamily, also known as TGFβR1 (transforming growth factor β receptor 1). ALK5 is a serine / threonine kinase that forms a heterodimeric complex with type II TGFβ receptor upon binding to TGFβ, and transmits the TGFβ signal from the cell surface to the cytoplasm.

[0043] Specific examples of ALK5 inhibitors include SB431542 (CAS No.: 301836-41-9), A83-01 (CAS No.: 909910-43-6), Repsox (CAS No.: 446859-33-2), D4476 (CAS No.: 301836-43-1), LY364947 (CAS No.: 396129-53-6), SB525334 (CAS No.: 356559-20-1), and SD208 (CAS No.: 627536-09-8).

[0044] 1.6 Somatic cell culture In the method of the present invention, somatic cells can be cultured in a conventional manner using a serum-free differentiation induction medium in the presence of the selective PPARγ agonist and cAMP inducer described above, by selecting the medium, temperature, and other conditions according to the type of somatic cells used. In the method of the present invention, brown adipocytes can be produced from somatic cells in a single-stage culture by culturing somatic cells in a serum-free differentiation-inducing medium containing the selective PPARγ agonist and cAMP inducer described above. In addition, by selecting somatic cells that are easy to culture, it is possible to increase the number of cells in advance and convert somatic cells that have reached a nearly confluent state into brown fat cells, making it easy to produce scaled-up brown fat cells.

[0045] The differentiation-inducing medium or the medium for subculturing somatic cells, which is the basis for carrying out the present invention, can be selected from known or commercially available media. For example, general basal media such as MEM (Eagle's minimum essential medium), GMEM (Glasgow's minimum essential medium), DMEM (Dulbecco's modified Eagle's medium), Ham's F-12, DMEM / F12, RPMI1640, IMDM (Iscove's modified Dulbecco's medium), or modified media thereof can be used by adding appropriate components (serum, proteins, amino acids, sugars, vitamins, fatty acids, antibiotics, etc.).

[0046] Culture conditions can be appropriately selected from general cell culture conditions. Examples include conditions of 37°C and 5% CO2. It is preferable to change the medium at appropriate intervals during culture (preferably once every 1 to 5 days, more preferably once every 2 to 3 days). When the method of the present invention is carried out using fibroblast cells as the material, brown adipocytes begin to appear in about 3 to 7 days under conditions of 37°C and 5% CO2, and the number of brown adipocytes continues to increase for about 21 to 28 days.

[0047] For the culture of somatic cells, cell culture containers such as plates, dishes, cell culture flasks, cell culture bags, etc. can be used. In addition, gas-permeable cell culture bags are preferable. When a large amount of cells is required, a large culture tank may be used. The culture can be performed in either an open or closed system, but when the obtained brown adipocytes are intended to be administered to humans, it is preferable to perform the culture in a closed system.

[0048] In addition, dexamethasone (glucocorticoid receptor agonist), insulin, 3-isobutyl-1-methylxanthine (phosphodiesterase inhibitor), triiodothyronine (thyroid hormone receptor agonist, also called T3), L-ascorbic acid 2-phosphate (ascorbic acid derivative, L-ascorbic acid-2-phophate), etc. are known as substances effective in inducing differentiation into adipocytes. As a substance effective in inducing differentiation into brown adipocytes, for example, a commercially available differentiation inducer can also be used. In the production method of the present invention, it is preferable to culture somatic cells in the presence of one or more, preferably three or more, more preferably four or more, and even more preferably five substances selected from the above-mentioned substances. Preferably, dexamethasone, insulin, 3-isobutyl-1-methylxanthine, T3, and L-ascorbic acid 2-phosphate can be used.

[0049] When dexamethasone is used, the concentration of dexamethasone is not particularly limited, but it can be used preferably in the range of 0.01 μmol / L to 30 μmol / L, and more preferably 0.1 μmol / L to 10 μmol / L.

[0050] When insulin is used, the concentration of insulin is not particularly limited, but it can be used preferably in the range of 1 ng / ml to 100 μg / ml, more preferably 100 ng / ml to 20 μg / ml. When 3-isobutyl-1-methylxanthine is used, the concentration of 3-isobutyl-1-methylxanthine is not particularly limited, but it can be preferably used in the range of 1 μmol / L to 1 mmol / L, and more preferably 10 μmol / L to 0.5 mmol / L. When T3 is used, the concentration of T3 is not particularly limited, but it can be used preferably in the range of 1 nmol / L to 10 umol / L, and more preferably 0.01 μmol / L to 1 μmol / L. When L-ascorbic acid 2-phosphate is used, the concentration of L-ascorbic acid 2-phosphate is not particularly limited, but can be preferably used in the range of 1 μg / mL to 1 mg / mL, and more preferably 10 μg / mL to 250 μg / mL.

[0051] 2 Brown fat cells The above-mentioned method of the present invention can obtain a cell population containing brown adipocytes. The brown adipocytes produced by the method of the present invention are also within the scope of the present invention. The brown adipocytes produced by the method of the present invention may be terminally differentiated cells or precursor cells destined to differentiate into brown adipocytes. The brown fat cells produced by the method of the present invention are so-called low molecular weight compound-induced brown fat cells (ciBAs) that are directly induced to differentiate from somatic cells by low molecular weight compounds, and are distinct from brown fat cells induced to differentiate by gene transfer. The brown fat cells produced by the method of the present invention may be beige cell-like or bright cell-like.

[0052] Brown adipocytes produced by the method of the present invention can be detected, identified and separated, for example, by utilizing morphological changes in cells, characteristic properties of brown adipocytes and specific markers. Adipocytes accumulate fat within the cells, and therefore can be detected by staining the intracellular fat with Oil Red O.

[0053] Specific markers for brown adipocytes include, but are not limited to, UCP1, EVOL3 (Elongation of very long chain fatty acid protein 3), PGC1A (PPAR gamma coactivator 1-alpha), PRDM16 (PRD1-BF1-RIZ1 homologous domain containing 16), CITED1 (CBP / p300 interacting transactivator with Glu / Asp rich carboxy-terminal domain 1), etc. UCP1 is a type of uncoupling protein.

[0054] Specific markers can be detected by diagnostic methods (antibody detection), but protein molecules can also be detected by quantifying the amount of mRNA. Antibodies that recognize specific markers for brown adipocytes are also useful for isolating and purifying brown adipocytes obtained by the method of the present invention.

[0055] The brown fat cells produced by the method of the present invention can be used for, for example, tissue repair after surgical treatment. The brown fat cells produced by the method of the present invention can be used to produce a pharmaceutical composition for tissue repair. In addition, transplantation or administration of brown fat cells to a living body is expected to have effects on improving metabolism and preventing obesity in the living body.

[0056] When brown fat cells are used as a pharmaceutical composition, the brown fat cells may be mixed with a medicamentically acceptable carrier in a conventional manner to prepare a formulation suitable for administration to an individual. Examples of the carrier include isotonic distilled water for injection made by adding physiological saline, glucose, or other auxiliary drugs (e.g., D-sorbitol, D-mannitol, sodium chloride, etc.). In addition, buffers (e.g., phosphate buffer, sodium acetate buffer), soothing agents (e.g., benzalkonium chloride, procaine hydrochloride, etc.), stabilizers (e.g., human serum albumin, polyethylene glycol, etc.), preservatives, antioxidants, etc. may be added. Brown adipocytes can also be made into a composition in which they are combined with other cells or components that are effective in improving the function and engraftment of brown adipocytes.

[0057] Furthermore, the brown fat cells produced by the method of the present invention can be used for screening of candidate pharmaceutical compounds or food ingredients acting on brown fat cells, and for safety evaluation of candidate pharmaceutical compounds. Therefore, using the brown fat cells produced by the method of the present invention, it is possible to screen for drugs that enhance the sugar and lipid metabolism and heat production function of brown fat cells. The candidate pharmaceutical compounds for the screening include, for example, existing drugs such as so-called lifestyle-related disease drugs, metabolic disease drugs, and other library drugs. The food ingredients include, for example, vitamins, sugars, amino acids, minerals, and other food nutrients, functional food ingredients, Chinese medicines, herbs, and other low molecular weight compounds, proteins, lipids, and the like in foods.

[0058] The above screening can be carried out, for example, by adding a test substance to brown fat cells produced by the method of the present invention or cells in the process of producing the cells, and evaluating the degree of enhancement of the browning tendency in the cells by the test substance. In such evaluation, for example, the expression levels of the Ucp1 gene, which is a marker specific to brown fat cells, and the Fabp4 gene, which is expressed in fat cells in general, can be quantified by real-time PCR, and the ratio can be used as an index of the browning tendency. The drug candidate compounds and food ingredients that enhance the browning tendency analyzed in this way can be potential candidate compounds that can increase brown fat cells (beige cells) in the human body. According to the method of the present invention, a large number of brown adipocytes can be obtained in a single operation, making it possible to obtain reproducible research results without being affected by differences in cell lots.

[0059] 2 Composition The induction composition of the present invention (hereinafter referred to as the "composition of the present invention") is a composition for producing brown adipocytes by directly inducing differentiation from somatic cells, and is characterized by containing a selective PPARγ agonist and a cAMP inducer and being serum-free.

[0060] In the present invention, the composition of the present invention further comprises BMP7. The selective PPARγ agonists and cAMP inducers may each be used alone or in combination of two or more.

[0061] The meanings, specific examples, preferred examples, etc. of the selective PPARγ agonist, cAMP inducer, and serum-free agent described above are the same as those described above.

[0062] The composition of the present invention can be used as a composition for producing brown adipocytes from somatic cells. The composition of the present invention can also be used as a medium for producing brown adipocytes from somatic cells.

[0063] Examples of media used for producing brown adipocytes from somatic cells include a basal medium (serum-free) produced by mixing components necessary for cell culture, and a selective PPARγ agonist and a cAMP inducer are contained as active ingredients. The above-mentioned active ingredients may be contained at a concentration effective for producing brown adipocytes, and the concentration can be appropriately determined by those skilled in the art. The basal medium can be selected from known media or commercially available media. For example, common media such as MEM (Eagle's minimum essential medium), GMEM (Glasgow's minimum essential medium), DMEM (Dulbecco's modified Eagle's medium), Ham's F-12, DMEM / F12, RPMI1640, and IMDM (Iscove's modified Dulbecco's medium) can be used as the basal medium.

[0064] The medium may further contain known medium components as described above in this specification, such as serum, proteins (albumin, transferrin, growth factors, etc.), amino acids, sugars, vitamins, fatty acids, antibiotics, etc.

[0065] The medium may further contain substances effective for inducing differentiation into adipocytes, such as dexamethasone, insulin, and 3-isobutyl-1-methylxanthine, T3, L-ascorbic acid 2-phosphate, as described herein above. EXAMPLES

[0066] The present invention will be specifically described below with reference to examples, comparative examples, and test examples, but the present invention is not limited to the scope of the examples, etc.

[0067] [Example] Production of brown adipocytes (1)Cell culture The human fibroblasts used as the material were purchased from DS Pharma Biomedical Co., Ltd. The fibroblasts were derived from the skin of a 38-year-old human. The human fibroblasts were plated at 5 × 10 in a 35 mm dish coated with gelatin (Cat#: 190-15805, Fujifilm Wako Pure Chemical Industries, Ltd.). 4The cells were seeded one at a time and cultured in DMEM medium (Cat#: 11965092; Gibco) supplemented with 10% fetal bovine serum (FBS), 100 U / mL penicillin, and 100 μg / mL streptomycin at 37°C and 5% CO2 until they became 80-90% confluent.

[0068] (2) Direct conversion of human fibroblasts to brown adipocytes (direct induction of differentiation) The serum-free medium used for direct conversion to brown fat cells (serum-free brown fat induction medium) was prepared by adding each component in Table 1 to DMEM medium (Cat#: 11965092; manufactured by Gibco), and further adding each compound shown in Table 2 in the combination of compounds shown in Table 3 so as to achieve the corresponding final concentrations. Hereinafter, this will be referred to as serum-free brown fat induction medium "SFBAM."

[0069] [Table 1]

[0070] [Table 2]

[0071] [Table 3]

[0072] In Table 3, "+" indicates the presence of the compound in question in the medium, and "-" indicates the absence of the compound in question in the medium.

[0073] After the human fibroblasts reached 80-90% confluence, the medium in the dish was replaced with the above-mentioned SFBAM, and the human fibroblasts were cultured in the SFBAM for 3 weeks. The medium was replaced every 3 days. For example, in a specific experiment related to FIG. 9, the chemically induced brown adipocyte-like cells (ciBAs) were matured by further culturing for 1 week in serum-free brown fat maintenance medium (hereinafter referred to as "SFBAMaM") prepared by adding each component shown in Table 4 to DMEM medium (Cat#: 11965092; manufactured by Gibco).

[0074] [Table 4]

[0075] (3) Immunostaining Immunocytochemistry was performed as previously reported (Dai P, et al. J Clin Biochem Nutr. 2015;56(3):166-70). First, cells were fixed with 4% paraformaldehyde (Nacalai Tesque). After washing three times with phosphate-buffered saline (PBS), cells were incubated with PBS containing 0.1% Triton X-100 for 10 min. Then, cells were blocked with PBS containing 3% skim milk for 1 h at room temperature. UCP-1 antibody (ab10983, Abcam, Cambridge, UK) was diluted 1 / 500 in blocking solution. Cells were incubated with the antibody overnight at 4°C. After washing three times with PBS, cells were incubated with Alexa Fluor 488 Donkey anti-Rabbit IgG (A-21206, Thermo Fisher Scientific) for 1 h at room temperature. Cell nuclei were stained with DAPI solution (Dojindo Laboratories). All images were acquired using a fluorescence microscope (Axio Vert.A1, Carl Zeiss, Oberkochen, Germany). Mitochondria were stained with MitoTracker® Red CMXRos (Thermo Fisher Scientific).

[0076] (4) RNA isolation and QRT-PCR To quantify gene expression, total RNA was extracted from fibroblasts treated with each compound using FastGene® RNA basic kit (Nihon Genetics). As a control, fibroblasts were cultured in parallel using SFBAM, and total RNA was extracted from compound-induced brown adipocytes and control cells. After reverse transcription using ReverTraAce® PCR RT Master Mix with gDNA Remover (Toyobo), real-time PCR analysis was performed using Power SYBR Green PCR Master Mix (Thermo Fisher Scientific). Reactions were performed in triplicate for each level under the following conditions: 95°C for 10 minutes, followed by 40 cycles of 95°C for 15 seconds and 60°C for 60 seconds. All results were normalized to the amount of Tbp mRNA. The primers used in QRT-PCR are shown in Table 5 below.

[0077] [Table 5]

[0078] (5) Results The experimental results are shown in the figures. In the figures, the data are shown as mean ± SD (n=3), "***", "**" and "*" indicate significant differences at p<0.001, p<0.01 and p<0.05, respectively, according to the Student's t-test, and "NS" indicates that there is no significant difference.

[0079] As shown in Figure 1, in serum-free medium SFBAM, sufficient direct differentiation induction (direct conversion) from human fibroblasts to brown adipocytes was observed by a combination of compounds including rosiglitazone (selective PPARγ agonist) and forskolin (cAMP inducer). Also, as shown in Figures 1 and 2, in serum-free medium, the combination of compounds in Example 2, which excludes LDN193189 (ALK2 / 3 inhibitor) and dorsomorphin (ALK2 / 3 / 6 inhibitor) from the combination of compounds in Example 1 and adds BMP7, improved the conversion efficiency to UCP1-positive cells, i.e., brown adipocytes, compared with the combination of compounds in Example 1.

[0080] As shown in FIG. 3, in serum-free medium, the expression of Fabp4, an adipocyte-specific gene, was increased compared to the control (Comparative Example 3, no compound), but no significant difference was observed between Example 1 and Example 2 (FIG. 3B). However, the expression level and ratio of Ucp1, a brown adipocyte-specific gene, was significantly increased in the case of the combination of compounds in Example 2 compared to the case of the combination of compounds in Example 1 (FIG. 3A). In addition, the ratio of the expression level of Ucp1 to the expression level of Fabp4 was significantly increased in the case of Example 2 compared to Example 1 (FIG. 3C). In addition, in response to the increase in the expression level of Ucp1 mRNA, an increase in UCP1 protein was observed in the case of Example 2 compared to Example 1 (FIG. 3D).

[0081] 4, in serum-free medium, the expression of Ucp1, a brown adipocyte-specific gene, was further increased by the combination of the compounds of Example 3 (forskolin, rosiglitazone, and BMP7), which was the combination of the compounds of Example 2 except for SB431542 (ALK5 inhibitor). Correspondingly, the amount of UCP1 protein was also increased in the case of Example 3.

[0082] The results of omitting one of the compounds from the combination of compounds in Example 3 (forskolin, rosiglitazone, and BMP7) are shown in Figure 5. As shown in Figure 5, in serum-free medium, when rosiglitazone (selective PPARγ agonist) was omitted from the combination of compounds in Example 3 (symbol 3, Comparative Example 2), almost no direct conversion to brown adipocytes was observed. On the other hand, when forskolin (cAMP inducer) was omitted from the combination of compounds in Example 3 (symbol 4, Comparative Example 1) and when BMP7 was omitted (symbol 5, Example 4), a small amount of direct conversion to brown adipocytes was observed, but direct conversion was more difficult when forskolin was omitted.

[0083] Therefore, in the direct conversion of human fibroblasts to brown adipocytes in a serum-free medium, it is considered that rosiglitazone (selective PPARγ agonist) has the highest conversion efficiency, followed by forskolin (cAMP inducer).

[0084] The direct conversion efficiency into adipocyte-like cells in serum-free medium was evaluated for various combinations of compounds based on the results shown in Figures 1 to 5. The results are shown in Table 6 below. Note that Figure 1 shows the results of immunostaining observed under a phase contrast microscope, but the efficiency was determined comprehensively taking into account the state of the cells. The more + marks there are, the higher the conversion efficiency. For example, ++++ indicates that most of the cells converted into adipocytes were converted into UCP1-positive brown adipocytes. - marks indicate that the cells were not converted into brown adipocytes.

[0085] [Table 6]

[0086] [Test Example 1] Direct conversion of different human fibroblasts to brown adipocytes As in the above Examples (1) and (2), three different human skin fibroblasts were cultured for three weeks in serum-free induction medium SFBAM containing the compounds of Example 3 (rosiglitazone, forskolin and BMP7). Then, multiple marker genes specific to adipocytes and brown adipose tissue were quantified by QRT-PCR.

[0087] The human dermal fibroblasts used in the experiment were purchased from DS Pharma Biomedical Co., Ltd. The cell information is shown in Table 7 below.

[0088] [Table 7]

[0089] The results are shown in Figure 6. Ucp1 mRNA was significantly induced in all fibroblasts in serum-free medium compared to the control without the addition of the compound (Comparative Example 3) (Figure 6A). In serum-free medium, the expression of Fabp4, an adipogenic differentiation marker, was increased in all fibroblasts in brown adipocytes induced with the compound (Figure 6B).

[0090] We also performed immunocytochemical analysis to analyze the increase in mitochondria and UCP1 protein expression, and the efficiency of brown adipocyte conversion (Figures 7 and 8). The results showed that the majority of adipocytes were UCP1 positive, indicating that they had characteristics of brown adipocytes. Furthermore, UCP1 staining was fully overlapped with the increase in mitochondrial signal.

[0091] [Test Example 2] Evaluation of thermogenesis in brown adipocytes induced to differentiate by the present invention Thermogenesis via UCP1 is regulated by the sympathetic nervous system and β-adrenergic receptor signaling pathway in brown adipocytes. To evaluate the thermogenic ability of brown adipocytes differentiated in the present invention, human fibroblasts were directly converted in serum-free medium SFBAM for 3 weeks with the combination of compounds in Example 3 (rosiglitazone, forskolin, and BMP7) in the same manner as in Examples (1) and (2) above, and then cultured in serum-free medium SFBAMaM for an additional week to mature the directly converted cells. Ucp1 mRNA was quantified in the cells after treatment with isoproterenol (β-adrenergic receptor agonist) for 6 hours and after treatment with the solvent HO for the same period. The results are shown in Figure 9.

[0092] As shown in FIG. 9, Ucp1 mRNA was significantly increased by 3.1-fold after treatment with isoproterenol compared to the control with added HO. Therefore, brown adipocytes induced to differentiate in serum-free medium with the combination of compounds of Example 3, i.e., brown adipocytes induced to differentiate from fibroblasts by the present invention, are shown to be able to respond to the beta-adrenergic receptor signaling pathway in response to induction of thermogenic genes.

[0093] [Test Example 3] Measurement of oxygen consumption rate of brown adipocytes induced to differentiate by the present invention To measure the mitochondrial oxygen consumption rate (OCR), human fibroblasts were directly transformed with the combination of compounds in Example 3 (rosiglitazone, forskolin, and BMP7) in serum-free medium SFBAM on a 96-well plate for 3 weeks in the same manner as in Examples (1) and (2) above. As a control, human fibroblasts were cultured in serum-free medium SFBAM in parallel. After incubation in a non-CO2 incubator at 37°C for 1 hour, the oxygen consumption rate of each well containing compound-induced brown adipocytes was analyzed using an XF96 Extracellular Flux Analyzer (Seahorse Bioscience Inc., Billerica, MA) according to the manufacturer's instructions. During the analysis, mitochondrial electron transport chain inhibitors oligomycin, FCCP (carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone), and antimycin A / rotenone were added to each well via an injection device to final concentrations of 2 μM, 0.3 μM, and 0.5 μM, respectively.

[0094] The results are shown in Figure 10. As shown in Figure 10A, the OCR value typically changes with the addition of mitochondrial inhibitors during analysis, and the brown adipocytes directly converted from human fibroblasts by the combination of the compounds of Example 3 showed higher OCR than the control. As shown in Figure 10B, the OCR values ​​corresponding to each mitochondrial activity also increased compared to the control. Therefore, it is clear that brown adipocytes induced to differentiate in serum-free medium with the combination of the compounds of Example 3, i.e., brown adipocytes induced to differentiate from fibroblasts by the present invention, have enhanced oxidative metabolism in mitochondria.

[0095] [Test Example 4] Examination of induction period for brown adipocytes In the same manner as in the above Examples (1) and (2), human dermal fibroblasts were cultured in serum-free induction medium SFBAM to which the compounds of Example 3 (rosiglitazone, forskolin and BMP7) were added. The expression levels of a number of marker genes specific to adipocytes and brown adipose tissue, as shown in Table 8 below, were quantified by QRT-PCR. The results are shown in FIG. 11. In the figure, "C" indicates a case in which the compound of Example 3 was not included and the cells were cultured for 5 weeks in the same manner, and "Ro" indicates a case in which only rosiglitazone was included among the compounds of Example 3 and the cells were cultured for 5 weeks in the same manner. The genes shown in FIG. 11A are specific to brown adipocytes, and the genes shown in FIG. 11B are specific to adipocytes in general.

[0096] [Table 8]

[0097] As shown in Figure 11, all genes began to be expressed within 1 week of culture, and the expression levels continued to increase until the 3rd or 4th week. Furthermore, from these results, it is believed that brown adipocytes induced to differentiate from fibroblasts by the present invention began to appear within the 1st week, and continued to increase until the 3rd or 4th week.

Claims

1. A method for producing brown adipocytes by directly inducing differentiation from fibroblasts, comprising the step of culturing fibroblasts in a serum-free differentiation-inducing medium in the presence of rosiglitazone in the range of 0.5 μmol / L to 5 μmol / L and forskolin in the range of 0.5 μmol / L to 30 μmol / L.

2. The method for producing brown adipocytes according to claim 1, wherein the step further comprises culturing fibroblasts in the presence of BMP7 in the range of 1 ng / mL to 50 ng / mL.

3. The method for producing brown adipocytes according to claim 1 or 2, wherein the step further comprises culturing fibroblasts in the absence of an ALK2 / 3 inhibitor and an ALK6 inhibitor.

4. The method for producing brown adipocytes according to any one of claims 1 to 3, wherein the step further comprises culturing fibroblasts in the absence of an ALK5 inhibitor.

5. A serum-free mixture comprising brown adipocytes, rosiglitazone in the range of 0.5 μmol / L to 5 μmol / L, and forskolin in the range of 0.5 μmol / L to 30 μmol / L.

6. A composition for producing brown fat cells by directly inducing differentiation from fibroblasts, the composition comprising rosiglitazone in the range of 0.5 μmol / L to 5 μmol / L and forskolin in the range of 0.5 μmol / L to 30 μmol / L, and being serum-free.

7. The differentiation-inducing composition according to claim 6, further comprising BMP7 in the range of 1 ng / mL to 50 ng / mL.

8. A method for screening for a drug acting on brown fat cells, comprising a step of using the serum-free mixture according to claim 5, or carrying out the production method according to any one of claims 1 to 4, and using brown fat cells produced by said step.

9. A method for evaluating the degree of enhancement of browning tendency in brown fat cells or cells in the process of being produced, comprising the step of adding a test substance to the serum-free mixture described in claim 5, or carrying out the production method described in any one of claims 1 to 4, and characterized in that the test substance is added to brown fat cells produced by the step or cells in the process of being produced.

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