Method for preparing brown adipocytes

Abstract

The subject of the present invention is to provide a new manufacturing method that can directly transform or induce somatic cells to brown adipocytes using low molecular weight compounds without gene introduction. As the present invention, for example, a method for preparing brown adipocytes can be cited, which is a method for preparing brown adipocytes from somatic cells by direct differentiation induction, the method comprising: 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. According to the present invention, direct transformation or induction from somatic cells to brown adipocytes can be effectively performed without gene introduction. The brown adipocytes obtained using the present invention are useful as regenerative medicine, human brown adipocytes and human beige cell models, etc.

Description

Technical Field

[0001] (Related Applications)

[0002] This application claims the benefit of Japanese Application No. 2019-211990 filed with the Japan Patent Office on November 25, 2019. This Japanese application is incorporated herein by reference in its entirety (specification, claims, drawings, abstract) for all purposes as disclosed in this specification.

[0003] The present invention belongs to the technical field of regenerative medicine. The present invention relates to a method for preparing brown adipocytes from somatic cells using low molecular weight compounds in the technical field, and low molecular weight compound-induced brown adipocytes (ciBAs: chemical compound-induced Brown Adipocytes) prepared by the preparation method. In addition, the present invention also relates to an induction medium composition that can be used in the method for preparing the brown adipocytes. Background Art

[0004] In recent years, methods for directly converting somatic cells such as fibroblasts into other cells using low molecular weight compounds without gene introduction (direct reprogramming: Direct Reprogramming) are being actively studied. For example, in Patent Document 1, several low molecular weight compounds such as ALK5 inhibitors and ALK6 inhibitors are combined, somatic cells, especially human fibroblasts, are cultured in their presence, and directly induced into brown fat cells, etc. As in the invention of Patent Document 1, it would be very beneficial if ordinary fibroblasts could be directly induced into cells such as brown fat cells using easily available low molecular weight compounds. For example, since other autologous cells can be easily prepared from autologous fibroblasts, their application in regenerative medicine is improved. In addition, cells that are used as experimental materials for the development of new drugs can be easily prepared.

[0005] Non-patent document 1 is also similar to patent document 1, in which human skin fibroblasts are cultured in the presence of a combination of several low molecular weight compounds, and the fibroblasts are directly induced into brown adipocytes. Specifically, in the invention of non-patent document 1, human skin 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. Patent document 1 also discloses the combination of the low molecular weight compounds.

[0006] Differentiation induction from such somatic cells as described above to brown adipocytes etc. must be carried out by culturing in a culture medium containing other compounds, nutrients etc. As the culture medium, for example, MEM (minimum essential medium), DMEM (Dulbecco's Modified Eagle Medium, Dulbecco modified Eagle medium), DMEM / F12, or culture mediums obtained by improving them as common culture mediums, which are commercially available. In addition, appropriate components (serum, protein, amino acids, sugars, vitamins, fatty acids, antibiotics, etc.) are usually added to these culture media to induce differentiation of cells in consideration of the cells to be induced. Among them, since serum such as fetal bovine serum (FBS) contains hormones, growth factors, adhesion factors and other components necessary for cell proliferation, serum is often added for differentiation induction. The inventions of patent document 1 and non-patent document 1 also use culture medium added with FBS for differentiation induction.

[0007] Prior art literature

[0008] Patent Literature

[0009] Patent Document 1: International Publication No. 2018 / 062269

[0010] Non-patent literature

[0011] Non-patent document 1: Takeda Y., Harada Y., Yoshikawa T., and Dai P., Sci. Rep., 7, 4304 (2017) Summary of the invention

[0012] Problems to be solved by the invention

[0013] The main object of the present invention is to provide a novel production method capable of directly converting or inducing somatic cells into brown adipocytes using low molecular weight compounds without gene introduction. In addition, the present invention also provides a novel composition for culturing somatic cells in the presence of low molecular weight compounds and preparing brown adipocytes by direct conversion.

[0014] Solution to the problem

[0015] The present inventors have conducted intensive studies and found that the above-mentioned problems can be solved by using a combination of certain low-molecular compounds in a serum-free state, thereby completing the present invention.

[0016] As examples of the present invention, the following inventions can be cited.

[0017] [1] A method for preparing brown adipocytes, which is a method for preparing brown adipocytes from somatic cells by direct differentiation induction, the method comprising:

[0018] 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.

[0019] [2] The method for producing brown adipocytes according to [1] above, wherein the step further comprises culturing the somatic cells in the presence of BMP7.

[0020] [3] The method for preparing brown adipocytes according to [1] or [2] above, wherein the above step is a step of further culturing the somatic cells in the absence of an ALK2 / 3 inhibitor and an ALK6 inhibitor.

[0021] [4] The method for producing brown adipocytes according to any one of [1] to [3] above, wherein the step is a step of further culturing the somatic cells in the absence of an ALK5 inhibitor.

[0022] [5] The method for producing brown adipocytes according to any one of [1] to [4] above, wherein the selective PPARγ agonist is rosiglitazone, or the cAMP inducer is forskolin.

[0023] [6] The method for producing brown adipocytes according to any one of [1] to [5] above, wherein the somatic cells are fibroblasts.

[0024] [7] Brown adipocytes produced by the method described in any one of [1] to [6] above.

[0025] [8] A composition for inducing differentiation, which is a composition for preparing brown adipocytes from somatic cells by direct differentiation induction, wherein the composition for inducing differentiation comprises a selective PPARγ agonist and a cAMP inducer and is serum-free.

[0026] [9] The composition for inducing differentiation according to [8] above, further comprising BMP7.

[0027]

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

[0028]

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

[10] above, wherein the somatic cells are fibroblasts.

[0029]

[12] A method for screening a drug that acts on brown adipocytes, using brown adipocytes prepared by the preparation method described in any one of [1] to [6] above, or the brown adipocytes described in [7] above.

[0030]

[13] A method for evaluating the degree of enhancement of the browning tendency of cells caused by a test substance, the method comprising:

[0031] A test substance is added to brown adipocytes prepared by the preparation method of any one of claims [1] to [6] or cells in the process of preparation, or brown adipocytes according to claim [7], and the degree of enhancement of the browning tendency of the cells caused by the test substance is evaluated.

[0032] Effects of the Invention

[0033] According to the present invention, direct conversion or direct differentiation induction from somatic cells to brown adipocytes can be efficiently performed in a serum-free state without gene introduction. The brown adipocytes obtained by the present invention are useful as regenerative medicine, human brown adipocyte models, and the like. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 The following are photos of cultured cells and immunostained mitochondria (red), UCP1 protein (green), and nuclei (blue) of the cells. Figure 1 It is monochrome, so although it does not show the various colors of red, green and blue, it Figure 1 The original photo shows various colors. The upper 4 figures show the results of the combination of compounds of Example 1, and the lower 4 figures show the results of the combination of compounds of Example 2.

[0035] Figure 2 The ratio of adipocytes and UCP1-positive cells is shown. The two columns on the left show the results of the combination of compounds of Example 1, and the two columns on the right show the results of the combination of compounds of Example 2. In the results of each example, the left column shows the ratio of adipocytes, and the right column shows the ratio of UCP1-positive cells. The vertical axis shows the ratio (%) of these cells relative to the cells positive for nuclear staining (DAPI).

[0036] Figure 3 Figure A shows the expression level of the Ucp1 gene, Figure B shows the expression level of the Fabp4 gene, Figure C shows the ratio of Ucp1 / Fabp4, and Figure D is an electrophoresis photograph for quantification of the UCP1 protein. Symbol 1 represents Comparative Example 3 (control), symbol 2 represents Example 1, and symbol 3 represents Example 2. The vertical axis represents the relative value of the mRNA amount relative to the mRNA amount of the internal standard gene (Tbp).

[0037] Figure 4 The left figure shows the expression level of Ucp1 gene, and the right figure is an electrophoresis photograph of quantification of UCP1 protein. Symbol 1 represents Comparative Example 3 (control), symbol 2 represents Example 3, and symbol 3 represents Example 2. The vertical axis represents the relative value of the mRNA amount relative to the mRNA amount of the internal standard gene (Tbp).

[0038] Figure 5 The expression level of Ucp1 gene is shown. Symbol 1 represents Comparative Example 3 (control), symbol 2 represents Example 3, symbol 3 represents Comparative Example 2, symbol 4 represents Comparative Example 1, and symbol 5 represents Example 4. The vertical axis represents the relative value of the mRNA amount relative to the mRNA amount of the internal standard gene (Tbp).

[0039] Figure 6 Figure A shows the expression level of the Ucp1 gene, and Figure B shows the expression level of the Fabp4 gene. In the results of skin fibroblasts derived from different human samples, each left column shows the result without the compound (Comparative Example 3), and each right column shows the result of the combination of the compound of Example 3. The vertical axis shows the relative value of the mRNA amount relative to the mRNA amount of the internal standard gene (Tbp).

[0040] Figure 7 The following are photos of cultured cells, photos of immunostained mitochondria (red) and UCP1 protein (green) of the cells, and photos of stained nuclei (blue) combined with these. Figure 7 It is monochrome, so it does not show green, red, and blue, but Figure 7 In the original photo, green, red and blue are shown.

[0041] Figure 8 The ratios of adipoid cells and UCP1-positive cells are shown. In the results of each human fibroblast, each left column shows the ratio of adipoid cells, and each right column shows the ratio of UCP1-positive cells.

[0042] Fig. 9 Activation of Ucp1 gene expression is shown. C represents the expression level of control cells (no compound added, comparative example 3). H2O 6h represents the result after adding control H2O (water) to the obtained cells after induction using the combination of compounds of Example 3 and culturing for 6 hours in serum-free brown fat maintenance medium (SFBAMaM). Iso 6h represents the result after adding isoproterenol (Isoproterenol), a β-adrenergic receptor agonist, instead of the previous H2O and culturing for 6 hours. The vertical axis represents the relative value of the mRNA amount relative to the mRNA amount of the internal standard gene (Tbp).

[0043] Fig.10 Figure A shows the changes in oxygen consumption rate. The gray circles represent the results of control cells (no compound added, Comparative Example 3), and the black diamonds represent the results of fibroblast-derived brown adipocytes caused by the combination of compounds in Example 3. In the figure, the dotted line indicates the addition of oligomycin, FCCP or antimycin A / rotenone. The horizontal axis represents time (minutes). Figure B shows the oxygen consumption rate corresponding to each parameter on the horizontal axis. In the results of each parameter, each left column represents the result without compound (Comparative Example 3), and each right column represents the result of the combination of compounds in Example 3. The vertical axis represents the oxygen consumption rate OCR (pmol / min / 10 4 cell).

[0044] Fig.11 The expression level of the gene is shown. In each figure, the vertical axis represents the relative value of the mRNA amount to the mRNA amount of the internal standard gene (Tbp). The horizontal axis value represents time (week). DETAILED DESCRIPTION

[0045] Hereinafter, the present invention will be described in detail.

[0046] 1. Preparation of brown adipocytes

[0047] The method for preparing brown adipocytes of the present invention (hereinafter referred to as "the preparation method of the present invention") is a method for preparing brown adipocytes from somatic cells by direct differentiation induction, and the method comprises the step of culturing somatic cells in a serum-free differentiation induction medium in the presence of a selective PPARγ agonist and a cAMP inducer.

[0048] Here, "serum-free" means that it does not substantially contain unadjusted or unpurified serum. In the present invention, even if purified blood-derived components or animal tissue-derived components are mixed into the differentiation induction medium, as long as it does not substantially contain unadjusted or unpurified serum, it can be called a serum-free differentiation induction medium.

[0049] The "brown adipocytes" (ciBAs) prepared by the method of the present invention are equivalent to so-called brown adipocytes that have a large number of mitochondria and a large number of fat droplets and express the UCP1 (uncoupling protein-1) gene. Therefore, they can be simply called brown adipocytes or brown fat-like cells. In addition, they can also be called a model of so-called human beige cells and bright cells.

[0050] The above step in the production method of the present invention is more preferably a step of culturing somatic cells in the presence of BMP7.

[0051] The selective PPARγ agonist and cAMP inducer may be used alone or in combination of two or more.

[0052] 1.1 About somatic cells

[0053] Biological cells can be classified into somatic cells and germ cells. The preparation method of the present invention can use any somatic cell as its starting material. Somatic cells are not particularly limited and can be any cells in primary cells collected from organisms or established cell lines. In the preparation method of the present invention, somatic cells in various stages of differentiation can be used, for example, terminally differentiated somatic cells (e.g., fibroblasts, umbilical vein endothelial cells (HUVEC), hepatocytes (Hepatocytes), bile duct cells (Biliary cells), pancreatic α cells (Pancreaticαcells), pancreatic acinar cells (Acinar cells), pancreatic ductal cells (Ductal cells), intestinal crypt cells (Intestinal crypt cells) etc.), somatic cells in the process of terminal differentiation (e.g., mesenchymal stem cells, neural stem cells, endoderm progenitor cells, etc.), or somatic cells such as iPS cells that have been initialized and obtained pluripotency and somatic cells differentiated from iPS cells or in the process of differentiation. As the somatic cell that can be used for the preparation method of the present invention, any somatic cell can be listed, for example, cells of the hematopoietic system (various lymphocytes, macrophages, dendritic cells, bone marrow cells, etc.), cells of organ origin (hepatocytes, spleen cells, pancreatic cells, kidney cells, lung cells, etc.), cells of the muscle tissue system (skeletal muscle cells, smooth muscle cells, myoblasts, cardiomyocytes, etc.), fibroblasts, nerve cells, osteoblasts, chondrocytes, endothelial cells, interstitial cells, adipocytes (white adipocytes, etc.), embryonic stem cells (ES cells), etc. In addition, the precursor cells and cancer cells of these cells can also be used for the preparation method of the present invention. Preferably, fibroblasts can be used.

[0054] The fibroblasts that can be used in the present invention are not particularly limited, and examples thereof include dermal fibroblasts, adventitial fibroblasts, cardiac fibroblasts, pulmonary fibroblasts, uterine fibroblasts, chorionic mesenchymal fibroblasts, etc., which are the main cell components that constitute connective tissue in various tissues and organs and are cells that produce collagen fibers.

[0055] As the supply source of the above-mentioned somatic cells, humans, mammals other than humans, and animals other than mammals (birds, reptiles, amphibians, fish, etc.) can be exemplified, but are not limited thereto. As the supply source of somatic cells, humans and mammals other than humans are preferred, and humans are particularly preferred. In the case of preparing brown adipocytes by the preparation method of the present invention with the goal of administering to humans, somatic cells collected from donors having the same or similar types of tissue compatibility antigens as the recipient can be preferably used. Somatic cells collected from the recipient itself can be used for the preparation of brown adipocytes.

[0056] 1.2 Selective PPARγ agonists

[0057] PPARγ (Peroxisome proliferator-activated Receptorγ) is a protein belonging to the nuclear receptor superfamily. It is a transcription factor that controls gene expression by forming a heterodimer with the retinoid X receptor (RXR) and binding to the promoter region of the target gene. There are three subtypes of PPAR, namely α, δ (β), and γ. In the present invention, an agonist that selectively acts on γ is used.

[0058] The term "in the presence of a selective PPARγ agonist" means culture under conditions that allow the culture to selectively exert an agonist effect on PPARγ. The form of the culture is not particularly limited, and generally, a compound that selectively exerts an agonist effect on PPARγ can be used.

[0059] In the present invention, the selective PPARγ agonist includes, but is not particularly limited to, the following compounds or salts thereof: Preferred are thiazolidine-based selective PPARγ agonists, specifically rosiglitazone, pioglitazone, and troglitazone.

[0060] Rosiglitazone (CAS No.: 122320-73-4)

[0061] [Chemical formula 1]

[0062]

[0063] Rosiglitazone maleate (CAS No.: 155141-29-0)

[0064] Ciglitazone (CAS No.: 74772-77-3)

[0065] Iglidezone (CAS No.: 213411-83-7)

[0066] GW1929 hydrochloride (CAS No.: 1217466-21-1)

[0067] nTZDpa (CAS No.: 118414-59-8)

[0068] Pioglitazone hydrochloride (CAS No.: 112529-15-4)

[0069] S26948 (CAS No.: 353280-43-0)

[0070] Troglitazone (CAS No.: 97322-87-7)

[0071] Baglitazone (CAS No.: 199113-98-9)

[0072] Rivolitazone (CAS No.: 185428-18-6)

[0073] The concentration of the selective PPARγ agonist varies depending on the type of the agonist, the somatic cells used, etc., and is not particularly limited and can be appropriately determined. 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.

[0074] 1.3 About cAMP inducers

[0075] cAMP (cyclic adenosine monophosphate) is a substance that participates in various intracellular signal transductions as a second messenger. cAMP is generated in cells by cyclizing adenosine triphosphate (ATP) using adenylate cyclase.

[0076] "In the presence of a cAMP inducer" means culture conditions that can induce cAMP, and the method is not particularly limited. For example, any method that can increase the intracellular cAMP concentration can be used. In addition to substances that can directly act on adenylate cyclase, an enzyme related to the production of cAMP, and substances that can promote the expression of adenylate cyclase, substances that inhibit phosphodiesterase, an enzyme that decomposes cAMP, etc. can also be used as a method for increasing the intracellular cAMP concentration. Dibutyryl cAMP (dibutyryl cAMP) and 8-bromo-cAMP (8-bromo-cAMP), which are structural analogs of cAMP and have the same effect as cAMP in cells, can also be used.

[0077] In the present invention, for example, forskolin (forskolin: CAS No.: 66575-29-9), forskolin derivatives (for example, Japanese Patent Application Laid-Open No. 2002-348243), and the following compounds can be cited as cAMP inducers (adenylate cyclase activators), but are not particularly limited. Forskolin can be preferably used.

[0078] Forskolin(CAS No.:66428-89-5)

[0079] [Chemical formula 2]

[0080]

[0081] Isoproterenol (CAS No.: 7683-59-2)

[0082] NKH477 (CAS No.: 138605-00-2)

[0083] PACAP1-27 (CAS No.: 127317-03-7)

[0084] PACAP1-38(CAS No.: 137061-48-4)

[0085] The concentration of the cAMP inducer varies depending on the type of the inducer, the somatic cells used, etc., and is not particularly limited and can be appropriately determined. 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.

[0086] 1.4 About BMP7

[0087] BMP (Bone Morphogenetic Protein: Bone Morphogenetic Protein) 7 is one of the BMP family. Human BMP7 is composed of 117 amino acids, binds to receptors on the cell surface in the form of a homodimer, and activates the BMP signaling pathway. The six species from BMP2 to BMP7 are classified as the TGFb superfamily, each encoded by a different gene. In the present invention, it can be BMP7 of human origin, BMP7 of animal origin, or their recombinants. Among them, it is preferred to use BMP7 of human origin.

[0088] The concentration of BMP7 varies depending on the type of BMP7, the somatic cells used, etc., and is not particularly limited and can be appropriately determined. 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.

[0089] 1.5 Optimal culture conditions

[0090] In the present invention, although not particularly limited, in the above-mentioned steps in the preparation method of the present invention, it is more preferred to culture the somatic cells in the absence of an ALK2 / 3 inhibitor (an agent that inhibits both ALK2 and 3) and an ALK6 inhibitor, culture the somatic cells in the absence of an ALK5 inhibitor, or culture the somatic cells in the absence of an ALK2 / 3 inhibitor, an ALK6 inhibitor and an ALK5 inhibitor.

[0091] The condition in which an ALK2 / 3 inhibitor, an ALK6 inhibitor or an ALK5 inhibitor is absent means that these inhibitors are substantially absent, and includes not only the case where these inhibitors are completely absent but also the case where they are present in an amount that does not exert their effect.

[0092] ALK2 is a receptor serine / threonine kinase of the ALK family member, located upstream of the signal transduction pathway associated with SMAD proteins, especially SMAD1 / 5 / 8. Through the activation of the ALK2-Smad1 pathway, the motility of prostate cancer cells is reduced by endoglin. The ALK2 gene is the main gene associated with fibrodysplasia ossificans progressiva (FOP), a rare autosomal dominant congenital disease characterized by progressive heterotopic ossification in muscle tissue.

[0093] ALK3 is a member of the transmembrane serine-threonine kinase family. The ALK3 gene functions as a secondary susceptibility gene in PTEN (phosphatase and tensin homologue deleted on chromosome 10) mutation-negative Cowden disease (multiple hamartoma syndrome). ALK3 trafficking plays an important role in lesion formation in FOP and is also associated with human T-cell differentiation.

[0094] Specific ALK2 / 3 inhibitors include, for example, 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).

[0095] ALK6, also known as BMPR1B, is a transmembrane serine / threonine kinase member of the bone morphogenetic protein (BMP) receptor family, very similar to ACVR1 and ACVR2, which are activin receptors. ALK6 is primarily involved in endochondral bone formation and embryogenesis.

[0096] Specific examples of ALK6 inhibitors include Dorsomorphin (CAS No.: 866405-64-3) and K02288 (CAS No.: 1431985-92-0).

[0097] 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 the type II TGFβ receptor upon binding to TGFβ, transducing the TGFβ signal from the cell surface to the cytoplasm.

[0098] Specific ALK5 inhibitors include, for example, 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).

[0099] 1.6 Culture of somatic cells

[0100] The somatic cells in the production method of the present invention are cultured by selecting a medium, temperature and other conditions according to the type of somatic cells to be used, and are cultured by a conventional method using a serum-free differentiation induction medium in the presence of the above-mentioned selective PPARγ agonist and cAMP inducer.

[0101] In the production method of the present invention, by culturing somatic cells in a serum-free differentiation induction medium containing the aforementioned selective PPARγ agonist and cAMP inducer, brown adipocytes can be produced from somatic cells in a one-step culture.

[0102] In addition, by selecting easily cultured cells as somatic cells to be used, somatic cells whose cell number has been increased in advance to a substantially confluent state can be converted into brown adipocytes. Therefore, it is also easy to prepare brown adipocytes on a large scale.

[0103] The differentiation induction medium or the medium for subculture of somatic cells used as the basis for implementing the present invention can be selected from known mediums or commercially available mediums. For example, 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 (Iscov's Modified Dulbecco's Medium), or modified mediums thereof can be used by adding appropriate components (serum, protein, amino acid, sugar, vitamins, fatty acids, antibiotics, etc.).

[0104] As culture conditions, the conditions of common cell culture can be appropriately selected. Examples include conditions of 37°C and 5% CO2. It is preferred to replace the culture medium at appropriate intervals (preferably once every 1 to 5 days, more preferably once every 2 to 3 days) during the culture. When the preparation method of the present invention is implemented using fibroblasts as materials, brown fat cells begin to appear at about 3 to 7 days under the conditions of 37°C and 5% CO2, and the number of brown fat cells continues to increase to about 21 to 28 days.

[0105] The culture of somatic cells can use cell culture containers such as culture plates, culture dishes, cell culture flasks, and cell culture bags. It should be noted that, as cell culture bags, preferably have air permeability. When a large number of cells are needed, a large culture tank can be used. Cultivation can be implemented in any one of an open system or a closed system. When the brown fat cells obtained are administered to humans, etc. as a goal, it is preferably cultivated in a closed system.

[0106] It should be noted that dexamethasone (glucocorticoid receptor agonist), insulin, 3-isobutyl-1-methylxanthine (phosphodiesterase inhibitor), triiodothyronine (thyroid hormone receptor agonist, also known as T3), L-ascorbic acid 2-phosphate (ascorbic acid derivative, L-Ascorbic acid-2-phophate) and the like are known to be effective substances for inducing differentiation into adipocytes. As substances effective for inducing differentiation into brown adipocytes, for example, commercially available products can also be used as differentiation inducers. In the preparation method of the present invention, somatic cells are preferably cultured in the presence of one or more, preferably three or more, more preferably four or more, and further 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.

[0107] 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, more preferably 0.1 μmol / L to 10 μmol / L.

[0108] 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.

[0109] 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.

[0110] When T3 is used, the concentration of T3 is not particularly limited, but it can be preferably used in the range of 1 nmol / L to 10 μmol / L, and more preferably 0.01 μmol / L to 1 μmol / L.

[0111] When L-ascorbic acid 2-phosphate is used, the concentration of L-ascorbic acid 2-phosphate is not particularly limited, but it 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.

[0112] 2 Brown adipocytes

[0113] By the above-mentioned preparation method of the present invention, a cell group containing brown adipocytes can be obtained. The brown adipocytes prepared by the preparation method of the present invention are also within the scope of the present invention. The brown adipocytes prepared by the preparation method of the present invention can be precursor cells destined to differentiate into brown adipocytes in addition to terminally differentiated cells.

[0114] The brown adipocytes prepared by the preparation method of the present invention are so-called low molecular weight compound-induced brown adipocytes (ciBAs) that are directly differentiated and induced from somatic cells using low molecular weight compounds, and are distinguished from brown adipocytes differentiated and induced by gene introduction. In addition, the brown adipocytes prepared by the preparation method of the present invention can be beige cell-like or bright cell-like.

[0115] The brown adipocytes prepared by the preparation method of the present invention can be detected, confirmed and separated by using changes in cell morphology, characteristic properties of brown adipocytes and specific markers.

[0116] Adipocytes store fat inside the cells. Therefore, adipocytes can be detected by staining intracellular fat using Oil Red O.

[0117] 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 coactivator1-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.

[0118] Specific markers can be detected by quarantine methods (antibody-based detection), and protein molecules can also be detected by quantifying their mRNA levels. Antibodies that recognize specific markers of brown adipocytes are useful for isolating and purifying brown adipocytes obtained by the preparation method of the present invention.

[0119] The brown adipocytes prepared by the preparation method of the present invention can be used, for example, for tissue repair after surgical treatment. Using the brown adipocytes prepared by the preparation method of the present invention, a pharmaceutical composition for tissue repair can be prepared. In addition, the transplantation and administration of brown adipocytes to a living body is expected to have effects such as improving the metabolism of the living body and preventing obesity.

[0120] When brown adipocytes are prepared into a pharmaceutical composition, the brown adipocytes can be mixed with a pharmaceutically acceptable carrier by a conventional method to prepare a preparation in a form suitable for administration to an individual. As a carrier, for example, isotonic distilled water for injection can be added with physiological saline, glucose, and other adjuvants (e.g., D-sorbitol, D-mannitol, sodium chloride, etc.). It can also be further combined with a buffer (e.g., phosphate buffer, sodium acetate buffer), an analgesic (e.g., benzalkonium chloride, procaine hydrochloride, etc.), a stabilizer (e.g., human serum albumin, polyethylene glycol, etc.), a preservative, an antioxidant, etc.

[0121] Brown adipocytes may be prepared as a composition in which brown adipocytes are further combined with other cells or components that are effective in exerting the functions of brown adipocytes and improving transplantability.

[0122] In addition, the brown adipocytes prepared by the preparation method of the present invention can also be used for the screening of candidate medical compounds or food ingredients acting on brown adipocytes, and the safety evaluation of candidate medical compounds. Therefore, using the brown adipocytes prepared by the preparation method of the present invention, it is possible to screen for agents that enhance the sugar / lipid metabolism and heat production function of brown adipocytes. The candidate medical compounds used for the screening include, for example, existing drugs such as so-called lifestyle disease treatment drugs, metabolic disease treatment drugs, and other drug library drugs. Food ingredients include, for example, nutrients of a wide range of foods such as vitamins, sugars, amino acids, and minerals, ingredients of functional foods, Chinese medicines, herbs, low molecular weight compounds and proteins, lipids, etc. in other foods.

[0123] The above screening can be implemented, for example, in the following manner: adding a test substance to the brown adipocytes prepared by the preparation method of the present invention or the cells in the preparation process thereof, and evaluating the degree of enhancement of the browning tendency of the cells caused by the test substance. In the evaluation, for example, the expression of the Ucp1 gene as a marker specific to brown adipocytes and the Fabp4 gene expressed in all adipocytes can be quantified by, for example, real-time PCR, and the ratio thereof is used as an index of the browning tendency. The pharmaceutical candidate compounds and food ingredients that enhance the browning tendency thus analyzed can become powerful candidate compounds that can increase brown adipocytes (beige cells) in the human body.

[0124] According to the preparation method of the present invention, since a large number of brown adipocytes can be obtained through a one-time operation, reproducible research results can be obtained without being affected by batch differences of cells.

[0125] 2 Composition

[0126] The induction composition of the present invention (hereinafter referred to as "the composition of the present invention") is a composition for preparing brown adipocytes from somatic cells by direct differentiation induction, and is characterized by comprising a selective PPARγ agonist and a cAMP inducer and being serum-free.

[0127] In the present invention, the composition of the present invention further comprising BMP7 is preferred.

[0128] The selective PPARγ agonist and cAMP inducer may be used alone or in combination of two or more.

[0129] The aforementioned selective PPARγ agonist, cAMP inducer and serum-free agent have the same meanings, specific examples, preferred examples and the like as those described above.

[0130] The composition of the present invention can be used as a composition for preparing brown adipocytes from somatic cells. In addition, the composition of the present invention can also be used as a culture medium for preparing brown adipocytes from somatic cells.

[0131] As a culture medium for preparing brown adipocytes from somatic cells, a culture medium containing a selective PPARγ agonist and a cAMP inducer as an effective ingredient in a basal culture medium (serum-free) prepared by mixing the components required for the culture of the cell can be exemplified. The above-mentioned effective ingredient can be contained at a concentration effective for the preparation of brown adipocytes, and the concentration can be appropriately determined by a person skilled in the art. The basal culture medium can be selected from a known culture medium or a commercially available culture medium. For example, 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 (Iscov's modified Dulbecco's medium) as a common basic culture medium can be used as a basal culture medium.

[0132] The culture medium may further contain known culture medium components described in the present specification, such as serum, proteins (albumin, transferrin, growth factors, etc.), amino acids, sugars, vitamins, fatty acids, antibiotics, and the like.

[0133] The medium may further contain dexamethasone, insulin, 3-isobutyl-1-methylxanthine, T3, L-ascorbic acid 2-phosphate, and other substances effective for inducing differentiation into adipocytes as described in the present specification.

[0134] Example

[0135] Hereinafter, the present invention will be specifically described by way of Examples, Comparative Examples, and Test Examples, but the present invention is not limited to the scope of Examples and the like.

[0136] [Example] Preparation of brown adipocytes

[0137] (1) Cell culture

[0138] Human fibroblasts used as materials were purchased from DS PHARMA BIOMEDICAL Co., Ltd. The fibroblasts were derived from the skin of a 38-year-old human.

[0139] The human fibroblasts were seeded in 35 mm culture dishes coated with gelatin (Cat#: 190-15805, manufactured by FUJIFILM Wako Pure Chemical Co., Ltd.), and 5×10 4The cells were cultured in DMEM medium (Cat#: 11965092; manufactured by 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 reached 80-90% confluence.

[0140] (2) Direct transformation of human fibroblasts into brown adipocytes (direct differentiation induction)

[0141] The serum-free medium (serum-free brown fat induction medium) used for direct conversion to brown adipocytes was prepared by adding the components of Table 1 and the compounds shown in Table 2 to DMEM medium (Cat#: 11965092; manufactured by Gibco) in a combination of compounds shown in Table 3 to achieve the final concentration. Hereinafter, this medium is referred to as serum-free brown fat induction medium "SFBAM".

[0142] [Table 1]

[0143]

[0144] [Table 2]

[0145]

[0146] [Table 3]

[0147]

[0148] In Table 3, "+" indicates that the compound exists in the culture medium, and "-" indicates that the compound does not exist in the culture medium.

[0149] Then, after the human fibroblasts reached 80-90% confluence, the culture medium of the culture dish was replaced with the above-mentioned SFBAM, and the human fibroblasts were cultured in SFBAM for 3 weeks. The culture medium was replaced every 3 days. Fig. 9 In a specific experiment, the components shown in Table 4 were added to DMEM medium (Cat#: 11965092; manufactured by Gibco) to prepare a serum-free brown fat maintenance medium (hereinafter referred to as "SFBAMaM") and the cells were further cultured for one week to allow chemically induced brown adipocyte-like cells (ciBAs) to mature.

[0150] [Table 4]

[0151]

[0152] (3) Immunostaining

[0153] Immunization of cells was performed as 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), the cells were incubated with PBS containing 0.1% Triton X-100 for 10 minutes. Next, the cells were blocked at room temperature for 1 hour with PBS containing 3% skim milk. UCP-1 antibody (ab10983, Abcam, Cambridge, UK) was diluted to 1 / 500 with blocking solution. The cells and antibodies were incubated at 4°C overnight. After washing three times with PBS, the cells were incubated with Alexa Fluor 488 Donkey anti-Rabbit IgG (A-21206, Thermo Fisher Scientific) at room temperature for 1 hour. The cell nuclei were stained with DAPI solution (Dojin Chemical Research Institute). All images were obtained using a fluorescence microscope (Axio Vert.A1, Carl Zeiss, Oberkochen, Germany). Mitochondria were stained using MitoTracker (registered trademark) Red CMXRos (Thermo Fisher Scientific).

[0154] (4) RNA isolation and QRT-PCR

[0155] In order to quantify gene expression, total RNA was extracted from fibroblasts treated with each compound using FastGene (registered trademark) RNA basic kit (manufactured by Nippon Genetics). As a control, fibroblasts were cultured in parallel using SFBAM, and total RNA was extracted from brown adipocytes induced by the compound and control cells. After reverse transcription using ReverTraAce (registered trademark) PCR RT Master Mix with gDNA Remover (manufactured by Toyobo Co., Ltd.), real-time PCR analysis was performed using Power SYBR Green PCR Master Mix (Thermo Fisher Scientific). The reaction was performed 3 times for each level under the following conditions. After 10 minutes at 95°C, 40 cycles were performed at 95°C for 15 seconds and 60 seconds at 60°C. All the results were standardized with the amount of Tbp mRNA. The primers used in QRT-PCR are shown in Table 5 below.

[0156] [Table 5]

[0157]

[0158] (5) Results

[0159] The experimental results are shown in the accompanying drawings. It should be noted that in all the accompanying drawings, the data represent mean ± SD (n = 3), "***", "**" and "*" indicate significant differences at p < 0.001, p < 0.01 and p < 0.05, respectively, by Student's t test, and "NS" indicates no significant difference.

[0160] like Figure 1 As shown in FIG. 1 , it was observed that in the serum-free medium SFBAM, a combination of a compound containing rosiglitazone (a selective PPARγ agonist) and forskolin (a cAMP inducer) sufficiently induced direct differentiation (direct conversion) from human fibroblasts to brown adipocytes. Figure 1 and Figure 2 As shown, in serum-free culture medium, in the combination of compounds of Example 2 in which LDN193189 (ALK2 / 3 inhibitor) and Dorsomorphin (ALK2 / 3 / 6 inhibitor) were excluded from the combination of compounds of Example 1 and BMP7 was added, the conversion efficiency to UCP1-positive cells, i.e., brown adipocytes, was improved compared with the combination of compounds of Example 1.

[0161] like Figure 3 As shown, in the serum-free medium, although the expression of Fabp4, a fat cell-specific gene, was increased compared to the control (Comparative Example 3, no compound), no significant difference was confirmed between Example 1 and Example 2 ( Figure 3 B). However, regarding the expression level and ratio of Ucp1, a brown adipocyte-specific gene, the combination of the compounds in Example 2 was significantly higher than that in Example 1 ( Figure 3 A). In addition, compared with Example 1, the ratio of the expression level of Ucp1 to the expression level of Fabp4 in Example 2 was significantly increased ( Figure 3 C). In addition, corresponding to the increase in the expression level of Ucp1 mRNA, an increase in UCP1 protein was observed in Example 2 compared with Example 1 ( Figure 3 D).

[0162] like Figure 4As shown, in the 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) excluding SB431542 (ALK5 inhibitor) from the combination of the compounds of Example 2. Correspondingly, the amount of UCP1 protein in Example 3 also increased.

[0163] The results of excluding any one compound from the combination of the compounds of Example 3 (forskolin, rosiglitazone, and BMP7) are shown in FIG. Figure 5 .like Figure 5 As shown, in the serum-free medium, when rosiglitazone (selective PPARγ agonist) was excluded from the combination of compounds in Example 3 (symbol 3, Comparative Example 2), direct conversion to brown adipocytes was hardly observed. On the other hand, when forskolin (cAMP inducer) (symbol 4, Comparative Example 1) or BMP7 (symbol 5, Example 4) was excluded from the combination of compounds in Example 3, direct conversion to brown adipocytes was observed, although it was small, but it was difficult to directly convert when forskolin was excluded.

[0164] Therefore, it is believed that in the direct conversion of human fibroblasts to brown adipocytes in a serum-free medium, rosiglitazone (a selective PPARγ agonist) has a higher conversion efficiency, followed by forskolin (a cAMP inducer).

[0165] For various combinations of compounds, according to Figure 1 to Figure 5 The results shown evaluated the efficiency of direct conversion to adipocyte-like cells in serum-free medium. The results are shown in Table 6 below. It should be noted that Figure 1 The results of immunostaining were observed using a phase contrast microscope, and the efficiency was comprehensively judged in combination with the state of the cells. The more + marks there are, the higher the conversion efficiency is. For example, ++++ means that most of the cells converted into adipocytes are converted into UCP1-positive brown adipocytes. - marks indicate that they are not converted into brown adipocytes.

[0166] [Table 6]

[0167] combination efficiency Example 1 ++ Example 2 +++ Example 3 ++++ Example 4 +++ or ++ Comparative Example 1 + Comparative Example 2 － Comparative Example 3 －

[0168] [Test Example 1] Direct transformation from different human fibroblasts to brown adipocytes

[0169] Similar to the above Examples (1) and (2), human skin fibroblasts from three different individuals were cultured for 3 weeks in a serum-free induction medium SFBAM to which the compounds of Example 3 (rosiglitazone, forskolin, and BMP7) were added. Then, multiple marker genes specific to adipocytes and brown adipose tissue were quantified by QRT-PCR.

[0170] The human skin fibroblasts used in the experiment were purchased from DS PHARMA BIOMEDICAL Co., Ltd. The cell information is shown in Table 7 below.

[0171] [Table 7]

[0172] batch# Generation Number BMI age gender Location HDF22 DFM062509 3 29 22 male Chest HDF38 DFM090214 3 23.1 38 male abdomen HDF54 DDFM052010B 3 21.3 54 female abdomen

[0173] The results are shown in Figure 6 Compared with the control without compound addition (Comparative Example 3), Ucp1 mRNA was significantly induced in all fibroblasts in serum-free medium ( Figure 6 A). In the serum-free medium, in brown adipocytes induced with the compound, the expression of Fabp4, a marker of adipogenic differentiation, was increased in all fibroblasts ( Figure 6 B).

[0174] In addition, immunocytochemical analysis was performed to analyze the increase in mitochondria and UCP1 protein expression, as well as the efficiency of conversion to brown adipocytes ( Figure 7 and Figure 8 ). The results showed that most of the adipocytes were UCP1 positive, which showed characteristics of brown adipocytes. In addition, the UCP1 staining overlapped well with the increase of mitochondrial signals.

[0175] [Test Example 2] Evaluation of the thermogenic capacity of brown adipocytes induced by differentiation in the present invention

[0176] Heat production via UCP1 is regulated by the sympathetic nervous system and β-adrenergic receptor signaling pathway in brown adipocytes. In order to evaluate the heat production capacity of differentiation-induced brown adipocytes in the present invention, human fibroblasts were directly transformed in the serum-free medium SFBAM for 3 weeks using the combination of compounds of Example 3 (rosiglitazone, forskolin and BMP7) in the same manner as in the above-mentioned Examples (1) and (2), and the directly transformed cells were then allowed to mature. Therefore, for cells that were further cultured in the serum-free medium SFBAMaM for 1 week, Ucp1 mRNA was quantified after treatment with isoproterenol (β-adrenergic receptor agonist) for 6 hours and after treatment with its solvent H2O for the same period of time. The results are shown in Fig. 9 .

[0177] like Fig. 9 As shown, Ucp1 mRNA was significantly increased by 3.1 times after treatment with isoproterenol compared with the control group supplemented with H2O.

[0178] Therefore, in a serum-free medium, brown adipocytes differentiated and induced using the combination of compounds of Example 3, that is, brown adipocytes differentiated and induced from fibroblasts according to the present invention, were shown to be able to respond to the β-adrenergic receptor signaling pathway induced by thermogenic genes.

[0179] [Test Example 3] Measurement of oxygen consumption rate of brown adipocytes induced by differentiation in the present invention

[0180] In order to measure the oxygen consumption rate (OCR) of mitochondria, human fibroblasts were directly transformed in serum-free medium SFBAM on 96-well plates for 3 weeks using the combination of compounds of Example 3 (rosiglitazone, forskolin and BMP7) as in the above Examples (1) and (2). As a control, human fibroblasts were cultured in parallel in serum-free medium SFBAM. After incubation in a non-CO2 incubator at 37°C for 1 hour, the oxygen consumption rate of each well containing brown adipocytes induced with the compound was analyzed using an XF96 Extracellular Flux Analyzer (Seahorse Bioscience Inc., Billerica, MA) according to the operating instructions. In the analysis, oligomycin (オリゴマシン), FCCP (carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone), and antimycin A / rotenone, which are inhibitors of the mitochondrial electron transport system, were added to each well via an injection device to a final concentration of 2 μM, 0.3 μM, and 0.5 μM, respectively.

[0181] The results are shown in Fig.10 .like Fig.10 As shown in A, OCR values ​​typically change with the addition of mitochondrial inhibitors to the assay, with brown adipocytes derived from human fibroblasts directly converted using the combination of compounds of Example 3 showing higher OCR than controls. Fig.10 As shown in B, the OCR values ​​corresponding to the various mitochondrial activities were also increased compared with the control.

[0182] Therefore, it was found that the oxidative metabolism in the mitochondria of the brown adipocytes differentiated by the combination of the compounds of Example 3 in a serum-free medium, that is, the brown adipocytes differentiated from the fibroblasts according to the present invention, was enhanced.

[0183] [Test Example 4] Study on the induction period to brown adipocytes

[0184] Similar to the above-described Examples (1) and (2), human skin fibroblasts were cultured in a serum-free induction medium SFBAM supplemented with the compounds of Example 3 (rosiglitazone, forskolin, and BMP7). Then, QRT-PCR was used to quantify the expression levels of multiple marker genes shown in Table 8 below, which are specific to adipocytes and brown adipose tissue. The results are shown in Fig.11 . In the figure, "C" represents the case where the cells were cultured for 5 weeks without the compounds of Example 3, and "Ro" represents the case where the cells were cultured for 5 weeks with only rosiglitazone among the compounds of Example 3. It should be noted that Fig.11 the gene shown in A is specific to brown adipocytes, Fig.11 and the gene shown in B is specific to all adipocytes.

[0185] [Table 8]

[0186]

[0187] As Fig.11 shown, the expression of any gene started before the 1st week of culture and continued to increase until the 3rd or 4th week. Additionally, based on these results, it can be considered that the brown adipocytes induced to differentiate from fibroblasts using the present invention appeared before the 1st week and continued to increase until the 3rd or 4th week. SEQUENCE LISTING <110> KATAOKA CORPORATION Kyoto Prefectural Public University Corporation <120> Method for Preparing Brown Adipocytes <130> P19028WO <160> 12 <170> PatentIn version 3.5 <210> 1 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Description of artificial sequence: Synthetic DNA <400> 1 actacggggt tatcacctgt gag 23 <210> 2 <211> twenty two <212> DNA <213> Artificial sequence <220> <223> Artificial sequence description: Synthetic DNA <400> 2 gtgcaggagt aggccacatt ac 22 <210> 3 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Artificial sequence description: Synthetic DNA <400> 3 tctacgacac ggtccaggag 20 <210> 4 <211> twenty two <212> DNA <213> Artificial sequence <220> <223> Artificial sequence description: Synthetic DNA <400> 4 gaatactgcc actcctccag tc 22 <210> 5 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> Artificial sequence description: Synthetic DNA <400> 5 gccaggaatt tgacgaagtc a 21 <210> 6 <211> twenty two <212> DNA <213> Artificial sequence <220> <223> Artificial sequence description: Synthetic DNA <400> 6 cccatttctg cacatgtacc ag 22 <210> 7 <211> 19 <212> DNA <213> Artificial sequence <220> <223> Artificial sequence description: Synthetic DNA <400> 7 agcaggaatg gctcgagac 19 <210> 8 <211> twenty two <212> DNA <213> Artificial sequence <220> <223> Artificial sequence description: Synthetic DNA <400> 8 atcctcctca ttcacccaga tc 22 <210> 9 <211> 19 <212> DNA <213> Artificial sequence <220> <223> Artificial sequence description: Synthetic DNA <400> 9 tggcacctca cctgcgaag 19 <210> 10 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Artificial sequence description: Synthetic DNA <400> 10 gcagaatggc cactgctttg 20 <210> 11 <211> twenty one <212> DNA <213> Artificial sequence <220> <223> Artificial sequence description: Synthetic DNA <400> 11 ctggtgagaa gggtgagaaa g 21 <210> 12 <211> twenty two <212> DNA <213> Artificial sequence <220> <223> Artificial sequence description: Synthetic DNA <400> 12 gtttcaccga tgtctccctt ag 22

Claims

1. A method for preparing brown adipocytes, wherein the method prepares brown adipocytes from fibroblasts by direct differentiation induction without gene introduction. The method comprises: A process of culturing fibroblasts in a serum-free differentiation induction medium in the presence of 0.5 μmol / L to 5 μmol / L rosiglitazone, 0.5 μmol / L to 30 μmol / L forskolin and 1 ng / mL to 50 ng / mL BMP7, and in the absence of ALK2 / 3 inhibitors, ALK5 inhibitors and ALK6 inhibitors.

2. A composition for differentiation induction, which is used for preparing brown adipocytes from fibroblasts by direct differentiation induction without gene introduction, The differentiation inducing composition comprises 0.5 μmol / L to 5 μmol / L rosiglitazone, 0.5 μmol / L to 30 μmol / L forskolin and 1 ng / mL to 50 ng / mL BMP7, and does not contain ALK2 / 3 inhibitor, ALK5 inhibitor and ALK6 inhibitor, and the composition is serum-free.

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