Culture medium with composition

Abstract

The present application provides a novel medium composition for use in inducing differentiation of somatic cells into brown adipocytes. As the present application, for example, there can be mentioned a medium composition for use in inducing differentiation of somatic cells into brown adipocytes, which is characterized by comprising seven components of a thyroid hormone receptor agonist, a glucocorticoid receptor agonist, a phosphodiesterase inhibitor, insulin, an ascorbic acid derivative, albumin, and an antibiotic. According to the present application, differentiation induction of somatic cells into brown adipocytes can be effectively performed. In addition, according to the present application, brown adipocytes can be effectively maintained.

Description

Technical Field

[0001] (Related Application)

[0002] This application claims the benefit of Japanese Application No. 2019-211991, filed with the Japan Patent Office on November 25, 2019. For all purposes, that Japanese application is incorporated herein by reference in its entirety, including all its application documents (specification, claims, drawings, and abstract).

[0003] This invention belongs to the technical field of regenerative medicine. Specifically, it relates to culture medium compositions that can be used in inducing differentiation from somatic cells into brown adipocytes, and to culture medium compositions for maintaining differentiated brown adipocytes. Background Technology

[0004] In recent years, there has been active research into methods for directly transforming somatic cells, such as fibroblasts, into other cell types using low-molecular-weight compounds during gene introduction into somatic cells (Direct Reprogramming). For example, in Patent Document 1, several low-molecular-weight compounds, such as ALK5 inhibitors and ALK6 inhibitors, are combined and cultured in the presence of these compounds to directly induce brown adipocytes and other cells. As in the invention of Patent Document 1, it would be highly beneficial to be able to directly induce brown adipocytes and other cells from ordinary fibroblasts using readily available low-molecular-weight compounds. For example, since other autologous cells can be easily prepared from autologous fibroblasts, its application in regenerative medicine is improved. Furthermore, cells that can be easily prepared as experimental materials for new drug development can be readily prepared.

[0005] Similar to Patent Document 1, Non-Patent Document 1 also involves culturing human skin fibroblasts in the presence of a combination of several low-molecular-weight compounds, directly inducing the fibroblasts 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), trichodin (cAMP inducer), and rosiglitazone (PPARγ agonist), directly inducing the fibroblasts into brown adipocytes. The combination of these low-molecular-weight compounds is also disclosed in Patent Document 1.

[0006] The induction of differentiation from somatic cells to brown adipocytes, etc., must be carried out by culturing in a culture medium containing other compounds, nutrients, etc. Examples of such culture media include, for instance, MEM (minimum essential medium), DMEM (Dulbecco modified Eagle medium), DMEM / F12, or modified versions thereof, which are commercially available. Furthermore, appropriate components (serum, proteins, amino acids, sugars, vitamins, fatty acids, antibiotics, etc.) are typically added to these media to induce cell differentiation, taking into account the cells to be induced. Since fetal bovine serum (FBS) contains hormones, growth factors, adhesion factors, and other components essential for cell proliferation, serum is frequently added for differentiation induction. Patent Document 1 and Non-Patent Document 1 also utilize culture media containing added FBS for differentiation induction.

[0007] Existing technical documents

[0008] Patent documents

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

[0010] Non-patent literature

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

[0012] The problem that the invention aims to solve

[0013] The main objective of this invention is to provide a novel culture medium composition that can be used to induce differentiation from somatic cells into brown adipocytes. Furthermore, this invention also aims to provide a novel culture medium composition for maintaining brown adipocytes obtained through differentiation induction.

[0014] Methods for solving problems

[0015] The inventors conducted in-depth research and found that the above-mentioned problems can be solved by using a culture medium composition containing certain low-molecular-weight compounds, thus completing the present invention.

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

[0017] [1] A composition for a differentiation induction culture medium, which is a composition for a culture medium used to induce differentiation from somatic cells to brown adipocytes, comprising the following seven components.

[0018] · Thyroid hormone receptor agonists

[0019] Glucocorticoid receptor agonists

[0020] Phosphodiesterase inhibitors

[0021] ·insulin

[0022] Ascorbic acid derivatives

[0023] ·albumin

[0024] ·antibiotic

[0025] [2] The differentiation induction culture medium composition according to [1] above, wherein the differentiation induction is carried out directly without introducing genes into somatic cells.

[0026] [3] The composition for differentiation induction culture medium according to [1] or [2] above, wherein the thyroid hormone receptor agonist is triiodothyronine, the glucocorticoid receptor agonist is dexamethasone, the phosphodiesterase inhibitor is isobutylmethylxanthine, the ascorbic acid derivative is L-ascorbic acid 2-phosphate, the albumin is albumin bound with linoleic acid and oleic acid or fatty acid unbound albumin, or the antibiotic is penicillin and / or streptomycin.

[0027] [4] The composition for differentiation induction medium according to any one of [1] to [3] above further comprises a selective PPARγ agonist, or a selective PPARγ agonist and cAMP inducer and / or BMP7.

[0028] [5] The composition for differentiation induction medium according to [4] above, wherein the selective PPARγ agonist is rosiglitazone or the cAMP inducer is salamiin.

[0029] [6] A differentiation induction medium comprising the differentiation induction medium composition described in any one of [1] to [5] above, and without serum.

[0030] [7] The differentiation induction medium according to [6] above is made by adding the differentiation induction medium composition described in any one of [1] to [5] above to DMEM medium.

[0031] [8] The differentiation induction medium described in [7] above, wherein the DMEM medium is a high glucose DMEM medium.

[0032] [9] The differentiation induction medium according to [7] or [8] above, wherein the DMEM medium contains L-glutamine.

[0033]

[10] The differentiation induction culture medium composition or differentiation induction culture medium according to any one of [1] to [9] above, wherein the somatic cells are fibroblasts.

[0034]

[11] A brown adipocyte prepared using the differentiation-inducing medium composition or differentiation-inducing medium described in any one of [1] to

[10] above.

[0035]

[12] A composition for maintaining culture medium, which is a composition for maintaining brown adipocytes prepared by somatic cell differentiation induction, comprising the following five components.

[0036] Selective PPARγ agonists

[0037] Glucocorticoid receptor agonists

[0038] ·insulin

[0039] ·albumin

[0040] ·antibiotic

[0041]

[13] The composition for maintaining culture medium according to

[12] above, wherein the selective PPARγ agonist is rosiglitazone, the glucocorticoid receptor agonist is dexamethasone, the albumin is albumin bound with linoleic acid and oleic acid or fatty acid unbound albumin, or the antibiotic is penicillin and / or streptomycin.

[0042]

[14] A maintenance culture medium comprising the maintenance culture medium composition described in

[12] or

[13] above, and serum-free.

[0043]

[15] The maintenance medium according to

[14] is made by adding the maintenance medium composition described in

[12] or

[13] to DMEM medium.

[0044]

[16] The maintenance medium according to

[15] above, wherein the DMEM medium is a high glucose DMEM medium.

[0045]

[17] The maintenance medium according to

[15] or

[16] above, wherein the DMEM medium contains L-glutamine and pyruvate.

[0046]

[18] The composition for maintaining culture medium or the maintaining culture medium according to any one of

[12] to

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

[0047] The effects of the invention

[0048] According to the present invention, differentiation induction from somatic cells to brown adipocytes can be effectively induced. Furthermore, according to the present invention, brown adipocytes obtained from somatic cell differentiation induction can be effectively maintained. Attached Figure Description

[0049] Figure 1 The expression level of the Ucp1 gene is shown. Symbol 1 shows Comparative Example 6 (control), Symbol 2 shows Example 1, Symbol 3 shows Comparative Example 1, Symbol 4 shows Comparative Example 2, Symbol 5 shows Comparative Example 3, Symbol 6 shows Comparative Example 4, and Symbol 7 shows Comparative Example 5. The vertical axis shows the relative value of this mRNA level to the expression level of the internal control gene (Tbp).

[0050] Figure 2 The expression levels of the Fapp4 gene are shown. The symbols and vertical axis are shown in the diagram. Figure 1 same.

[0051] Figure 3 The expression level of the Ucp1 gene is shown. Symbol 1 shows Example 2, symbol 2 shows Example 3, symbol 3 shows Example 4, symbol 4 shows Comparative Example 7, symbol 5 shows Example 5, symbol 6 shows Comparative Example 8, and symbol 7 shows Example 6. The vertical axis shows the relative value of this mRNA level to the expression level of the internal control gene (Tbp).

[0052] Figure 4 The expression levels of the Fapp4 gene are shown. The symbols and vertical axis are shown in the diagram. Figure 3 same.

[0053] Figure 5 The images show cultured cells directly induced from fibroblasts using a combination of compounds (Example 7), cells incubated in a maintenance medium (Example 8), and images of these cells after immunostaining of mitochondria (red), UCP1 protein (green), and cell nuclei (blue). Due to... Figure 5 It is monochrome, therefore it does not display various colors such as red, green, and blue, but Figure 5 The original photo shows a variety of colors.

[0054] Figure 6 The gene expression levels are shown. In each figure, the vertical axis shows the relative value of the mRNA level to the mRNA level of the internal control gene (Tbp). The horizontal axis values ​​represent time (weeks).

[0055] Figure 7 The gene expression levels are shown. In each figure, the vertical axis shows the relative value of the mRNA level to the mRNA level of the internal control gene (Tbp). The horizontal axis values ​​indicate time (weeks).

[0056] Figure 8 The expression level of the Ucp1 gene is shown. Symbol 1 indicates the control, symbol 2 indicates Example 11, symbol 3 indicates Example 12, symbol 4 indicates Comparative Example 13, and symbol 5 indicates Example 14. The vertical axis shows the relative value of this mRNA level to the expression level of the internal control gene (Tbp).

[0057] Figure 9 The expression levels of the Ckmt1 gene are shown. The symbols and the vertical axis are related to... Figure 8 same. Detailed Implementation

[0058] The present invention will now be described in detail.

[0059] 1. Composition for Differentiation Induction Culture Medium

[0060] The differentiation induction culture medium composition of the present invention (hereinafter referred to as the "induction composition of the present invention") is a culture medium composition used for inducing differentiation from somatic cells to brown adipocytes, and it contains the following 7 components.

[0061] · Thyroid hormone receptor agonists

[0062] Glucocorticoid receptor agonists

[0063] Phosphodiesterase inhibitors

[0064] ·insulin

[0065] Ascorbic acid derivatives

[0066] ·albumin

[0067] ·antibiotic

[0068] Each of the above ingredients can be used alone, or in combination of two or more.

[0069] Differentiation induction from somatic cells to brown adipocytes can be based on so-called direct reprogramming, which involves the direct conversion using low-molecular-weight compounds without artificial gene introduction into somatic cells, or it can be based on methods involving artificial gene introduction into somatic cells. The induction composition of this invention is preferably used for differentiation induction performed directly without gene introduction into somatic cells.

[0070] In addition, as part of the present invention, a differentiation induction medium containing the induction composition of the present invention and free of serum (hereinafter referred to as "the induction medium of the present invention") can be cited.

[0071] Here, "serum-free" means that it does not substantially contain unadjusted or unpurified serum. In this invention, even if purified blood-derived components or animal tissue-derived components are mixed into the differentiation induction medium or maintenance medium, as long as it does not substantially contain unadjusted or unpurified serum, it can still be called a serum-free differentiation induction medium or maintenance medium.

[0072] The "brown adipocytes" in this invention are obtained through artificial differentiation and induction from somatic cells, possessing a large number of mitochondria and lipid droplets, and expressing the UCP1 (uncoupling protein-1) gene. Additionally, these brown adipocytes also include so-called human beige cells or bright cells induced from white adipocytes or their precursor cells. They also include so-called chemical compound-induced brown adipocytes (ciBAs) prepared by direct conversion using low-molecular-weight compounds without artificial gene introduction into somatic cells. Furthermore, in addition to terminally differentiated brown adipocytes, they also include precursor cells destined to differentiate into brown adipocytes.

[0073] The induction composition or induction medium of the present invention may further contain a selective PPARγ agonist, or a selective PPARγ agonist and cAMP inducer and / or BMP7 as effective components for inducing differentiation into brown adipocytes. Preferably, it further contains a selective PPARγ agonist; more preferably, it further contains both a selective PPARγ agonist and a cAMP inducer, or all three components: a selective PPARγ agonist, a cAMP inducer, and BMP7.

[0074] 1.1 About somatic cells

[0075] Biological cells can be classified into somatic cells and germ cells. The preparation method of this invention can use any somatic cell as its starting material. Somatic cells are not particularly limited and can be primary cells collected from an organism or any cell from an established cell line. In the preparation method of this invention, somatic cells at various stages of differentiation can be used, such as terminally differentiated somatic cells (e.g., fibroblasts, umbilical vein endothelial cells (HUVECs), hepatocytes, bile duct cells, pancreatic α cells, pancreatic acinar cells, pancreatic ductal cells, intestinal crypt cells, etc.), somatic cells undergoing 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 acquired pluripotency, as well as somatic cells differentiated from or in the process of differentiating from iPS cells. As somatic cells that can be used in the preparation method of the present invention, any type of somatic cell can be listed, such as cells of the hematopoietic system (various lymphocytes, macrophages, dendritic cells, bone marrow cells, etc.), organ-derived cells (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, mesenchymal cells, adipocytes (white adipocytes, etc.), embryonic stem cells (ES cells), etc. Furthermore, precursor cells and cancer cells of these cells can also be used in the preparation method of the present invention. Fibroblasts are preferably used.

[0076] The fibroblasts that can be used in this invention are not particularly limited, and examples include: dermal fibroblasts, adventitial fibroblasts, cardiac fibroblasts, pulmonary fibroblasts, uterine fibroblasts, villous mesenchymal fibroblasts, etc., which are the main cellular components of connective tissue in various tissues and organs and are cells that produce collagen fibers.

[0077] Examples of sources of somatic cells for supply include humans, mammals other than humans, and animals other than mammals (birds, reptiles, amphibians, fish, etc.), but these are not limited to these. Humans and mammals other than humans are preferred sources of somatic cells, with humans being particularly preferred. In the case of differentiating and inducing brown adipocytes for administration to humans, somatic cells collected from donors with the same or similar types of histocompatibility antigens as the recipient are preferably used. Somatic cells collected directly from the recipient may also be provided.

[0078] 1.2 Regarding each component

[0079] 1.2.1 Thyroid hormone receptor agonists

[0080] As a thyroid hormone receptor agonist, any compound that binds to the thyroid hormone receptor and has hormone-like effects is acceptable; there are no particular limitations. Examples include the following compounds. Preferred examples include triiodothyronine and thyroxine.

[0081] Triiodothyronine (CAS No.: 6893-02-3)

[0082] [Chemical Formula 1]

[0083]

[0084] 3,5-Diiodothyronine dihydrate (CAS No.: 18835-59-1)

[0085] Tilatrol (CAS No.: 51-24-1)

[0086] Sodium iodideronine (CAS No.: 55-06-1)

[0087] L-thyroxine (CAS No.: 51-48-9)

[0088] The concentration of thyroid hormone receptor agonists varies depending on the type of agonist and the somatic cells used, and is not particularly limited. It can be appropriately determined, and is preferably used in the range of 1 nmol / L to 10 μmol / L, and more preferably in the range of 0.01 μmol / L to 1 μmol / L.

[0089] 1.2.2 Glucocorticoid receptor agonists

[0090] As a glucocorticoid receptor agonist, any compound that binds to glucocorticoid receptors and has glucocorticoid-like effects is acceptable; there are no particular limitations. Examples include the following compounds. Preferred examples include: dexamethasone, hydrocortisone, corticosteroids, and cortisone.

[0091] Dexamethasone (CAS No.: 50-02-2)

[0092] [Chemical Formula 2]

[0093]

[0094] Dexamethasone sodium phosphate (CAS No.: 55203-24-2)

[0095] Dexamethasone acetate (CAS No.: 1177-87-3)

[0096] Methylprednisolone (CAS No.: 1247-42-3)

[0097] Fluocinolone acetonide (CAS No.: 67-73-2)

[0098] Budina (CAS No.: 51333-22-3)

[0099] Deshydroxymethasone (CAS No.: 382-67-2)

[0100] Protopanaxadiol (CAS No.: 34080-08-5)

[0101] Triamcinolone (CAS No.: 124-94-7)

[0102] Beclomethasone dipropionate (CAS No.: 5534-09-8)

[0103] Prednisolone (CAS No.: 50-24-8)

[0104] Hydrocortisone (CAS No.: 50-23-7)

[0105] Diftokite (CAS No.: 14484-47-0)

[0106] Triamcinolone (CAS No.: 76-25-5)

[0107] Fluocinolone (CAS No.: 356-12-7)

[0108] Methylprednisolone sodium succinate (CAS No.: 2921-57-5)

[0109] Loop Sonnet (CAS No.: 126544-47-6)

[0110] Flumethasone (CAS No.: 2135-17-3)

[0111] Fluoromethol (CAS No.: 426-13-1)

[0112] Betamethasone dipropionate (CAS No.: 5593-20-4)

[0113] Betamethasone valerate (CAS No.: 2152-44-5)

[0114] Mometasone furoate (CAS No.: 83919-23-7)

[0115] Fluticasone propionate (CAS No.: 80474-14-2)

[0116] Cortisone acetate (CAS No.: 50-04-4)

[0117] Tortosone (CAS No.: 152-58-9)

[0118] Clotiprenor (CAS No.: 82034-46-6)

[0119] Betamethasone (CAS No.: 378-44-9)

[0120] Methylprednisolone (CAS No.: 83-43-2)

[0121] Corticosterone (CAS No.: 50-22-6)

[0122] Cortisone (CAS No.: 53-06-5)

[0123] The concentration of glucocorticoid receptor agonists varies depending on the type of agonist and the somatic cells used, and is not particularly limited. It can be appropriately determined, preferably in the range of 0.01 μmol / L to 30 μmol / L, and more preferably in the range of 0.1 μmol / L to 10 μmol / L.

[0124] 1.2.3 Phosphodiesterase inhibitors

[0125] As a phosphodiesterase inhibitor, any compound that can increase intracellular cAMP concentration by inhibiting phosphodiesterases (PDEs), particularly PDE3, PDE4, and PDE5, is acceptable; there are no particular limitations. Examples of such compounds include isobutylmethylxanthine (IBMX) and leuprolide.

[0126] Isobutylmethylxanthine (CAS No.: 28822-58-4)

[0127] [Chemical Formula 3]

[0128]

[0129] Roflumilast (CAS No.: 162401-32-3)

[0130] Sildenafil (CAS No.: 139755-83-2)

[0131] Tadalafil (CAS No.: 171596-29-5)

[0132] Vardenafil hydrochloride hydrate (CAS No.: 330808-88-3)

[0133] Pimobendan (CAS No.: 74150-27-9)

[0134] GSK256066(CAS No.: 801312-28-7)

[0135] Glypranine (CAS No.: 61413-54-5)

[0136] Apsom (CAS No.: 608141-41-9)

[0137] Cilostazol (CAS No.: 73963-72-1)

[0138] Milrinone (CAS No.: 78415-72-2)

[0139] Avanavir (CAS No.: 330784-47-9)

[0140] Glypran (CAS No.: 85416-73-5)

[0141] Fensipril (CAS No.: 5053-08-7)

[0142] Uldenafil (CAS No.: 268203-93-6)

[0143] Cilolactone (CAS No.: 68550-75-4)

[0144] Isobutylsterol (CAS No.: 50847-11-5)

[0145] Luteolin (CAS No.: 491-70-3)

[0146] The concentration of phosphodiesterase inhibitors varies depending on the type of inhibitor and the somatic cells used, and is not particularly limited. It can be appropriately determined, preferably in the range of 1 μmol / L to 1 mmol / L, and more preferably in the range of 10 μmol / L to 0.5 mmol / L.

[0147] 1.2.4 Ascorbic acid derivatives

[0148] As an ascorbic acid derivative, any derivative of vitamin C (L-ascorbic acid) obtained by modifying its instability is acceptable, without particular limitation. Specifically, examples include compounds such as vitamin C (L-ascorbic acid) and L-ascorbic acid 2-phosphate (2PAA).

[0149] L-Ascorbic acid-2-phosphate sodium (CAS No.: 66170-10-3)

[0150] [Chemical Formula 4]

[0151]

[0152] Ascorbate palmitate (CAS No.: 137-66-6)

[0153] Sodium ascorbate (CAS No.: 134-03-2)

[0154] The concentration of ascorbic acid derivatives varies depending on the type of derivative, the somatic cells used, etc., and is not particularly limited. It can be appropriately determined, preferably in the range of 1 μg / mL to 1 mg / mL, and more preferably in the range of 10 μg / mL to 250 μg / mL.

[0155] 1.2.5 Insulin, fatty acid-bound albumin, and antibiotics

[0156] Insulin is a peptide hormone secreted by the β cells of the pancreas. In this invention, either animal-derived insulin or human-derived insulin can be used. Alternatively, it can be chemically synthesized insulin or recombinant insulin manufactured using genetic recombination technology.

[0157] In this invention, human insulin is preferably used.

[0158] The concentration of insulin varies depending on the type of insulin and the somatic cells used, and there is no particular limitation. It can be appropriately determined, but it is preferred to use it in the range of 100 ng / mL to 20 μg / mL.

[0159] Albumin can be fatty acid-bound albumin, which contains fatty acids, or fatty acid-unbound (fatty acid-free) albumin, which is albumin from which fatty acids have been removed. Examples of such albumin include, for instance, albumin purified from animal (e.g., bovine) serum, albumin formed by binding the purified albumin to known fatty acids, and recombinant albumin expressed using animal or plant cells. Particularly preferred are recombinant albumin that has been confirmed to be free of animal-derived components, and albumin formed by binding purified albumin to known fatty acids. Furthermore, albumin of human origin is preferred.

[0160] Fatty acids that bind to albumin can include, for example, linoleic acid, oleic acid, linolenic acid, palmitic acid, arachidonic acid, eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA), among other representative saturated and unsaturated fatty acids. A mixture of linoleic acid and oleic acid is preferred.

[0161] Specific examples of fatty acid-bound albumin include albumin purified from animal serum, linoleic acid-bound albumin, and oleic acid-bound albumin. In this invention, commercially available fatty acid-bound bovine serum albumin (e.g., L9655: manufactured by Sigma-Aldrich) can be used.

[0162] It should be noted that, in the experimental results described later, the expression level of the Ucp1 gene in brown adipocytes induced with fatty acid-unbound albumin was significantly higher than that induced with fatty acid-bound albumin (see reference). Figure 8 , Figure 9 Furthermore, in the induction of brown adipocytes from fibroblasts, it is preferable not to add fatty acids. These were not observed in adipocytes derived from mesenchymal stem cells, which have high adipogenic biosynthesis activity.

[0163] The concentration of albumin can vary depending on the type of albumin and the somatic cells used, and is not particularly limited. It can be appropriately determined, for example, it can be used in the range of 0.1 mg / mL to 10 mg / mL, preferably in the range of 0.5 mg / mL to 4 mg / mL.

[0164] As an antibiotic, any antibiotic suitable for animal cell culture is acceptable, without particular limitation. Examples include: penicillins, cephalosporins, β-lactam antibiotics such as monocyclic lactams, aminoglycosides, macrolides, tetracyclines, peptides, nucleic acids, and polyenes. Specifically, examples include: actinomycin D, amphotericin B, ampicillin, antimycin A, bafloxacin A1, bleomycin, carbenicillin, chloramphenicol, concanavalin B, erythromycin, G418, gentamicin, hygromycin, kanamycin, mitomycin C, neomycin, oligomycin, penicillin, puromycin, rapamycin, streptomycin, tetracycline, tobramycin, and valproic acid. In this invention, the combination of penicillin and streptomycin is appropriate.

[0165] The concentration of antibiotics varies depending on the type of antibiotic and the somatic cells used, and is not particularly limited; it can be appropriately determined. For example, it can be used in the range of 10 units / mL to 1000 units / mL, preferably 50 units / mL to 200 units / mL. Furthermore, depending on the type of antibiotic, it can be used in the range of 10 μg / mL to 1000 μg / mL, preferably 50 μg / mL to 200 μg / mL.

[0166] 1.2.6 Regarding selective PPARγ agonists

[0167] PPARγ (Peroxisome proliferator-activated receptor γ) is a protein belonging to the nuclear receptor superfamily. It is a transcription factor that controls gene expression by forming a heterodimer with the retinol X receptor (RXR) and binding to the promoter region of target genes. PPARs exist in three isoforms: α, δ (β), and γ. In this invention, an agonist that selectively interacts with the γ isoform is used.

[0168] In this invention, as selective PPARγ agonists, compounds such as those listed below or their salts may be used, but there are no particular limitations. Preferably, thiazolidinediones are selective PPARγ agonists, specifically including rosiglitazone, pioglitazone, and troglitazone.

[0169] Rosiglitazone (CAS No.: 122320-73-4)

[0170] [Chemical Formula 5]

[0171]

[0172] Rosiglitazone maleate (CAS No.: 155141-29-0)

[0173] Cycloglitazone (CAS No.: 74772-77-3)

[0174] Iglesias (CAS No.: 213411-83-7)

[0175] GW1929 hydrochloride (CAS No.: 1217466-21-1)

[0176] nTZDpa (CAS No.: 118414-59-8)

[0177] Pioglitazone hydrochloride (CAS No.: 112529-15-4)

[0178] S26948 (CAS No.: 353280-43-0)

[0179] Troglitazone (CAS No.: 97322-87-7)

[0180] Bagliflozin (CAS No.: 199113-98-9)

[0181] Linaglitazone (CAS No.: 185428-18-6)

[0182] The concentration of selective PPARγ agonists varies depending on the type of agonist, the somatic cells used, etc., and is not particularly limited. It can be appropriately determined, for example, it can be used in the range of 0.1 μmol / L to 20 μmol / L, preferably in the range of 0.5 μmol / L to 5 μmol / L.

[0183] 1.2.7 Regarding cAMP inducers

[0184] cAMP (cyclic adenosine triphosphate) is a substance that participates in various intracellular signal transductions as a second messenger. cAMP is produced in cells by cyclizing adenosine triphosphate (ATP) using adenylate cyclase.

[0185] In this invention, for example, trichodin (trichodin: CAS No.: 66575-29-9), trichodin derivatives (e.g., Japanese Patent Application Publication No. 2002-348243), and the following compounds can be used as cAMP inducers (adenylate cyclase activators), but there are no particular limitations. Trichodin is preferred.

[0186] Trichosanthesin (CAS No.: 66428-89-5)

[0187] [Chemical Formula 6]

[0188]

[0189] Isoproterenol (CAS No.: 7683-59-2)

[0190] NKH477 (CAS No.: 138605-00-2)

[0191] PACAP1-27 (CAS No.: 127317-03-7)

[0192] PACAP1-38(CAS No.: 137061-48-4)

[0193] The concentration of the cAMP inducer varies depending on the type of derivative, the somatic cells used, etc., and is not particularly limited. It can be appropriately determined, for example, it can be used in the range of 0.1 μmol / L to 100 μmol / L, preferably in the range of 0.5 μmol / L to 30 μmol / L.

[0194] 1.2.8 About BMP7

[0195] BMP (Bone Morphogenetic Protein) 7 is one of the BMP family. Human BMP7 consists of 117 amino acid residues and binds to receptors on the cell surface as a homodimer, activating the BMP signaling pathway. The six BMPs, from BMP2 to BMP7, are classified as the TGFb superfamily, each encoded by a different gene. In this invention, BMP7 can be of human origin, animal origin, or a recombinant of both. Human-derived BMP7 is preferred.

[0196] The concentration of BMP7 varies depending on the type of BMP7 and the somatic cells used, and is not particularly limited. It 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.

[0197] 1.3 Somatic cell culture

[0198] The inducing composition of the present invention can be used as a composition for preparing brown adipocytes by inducing differentiation from somatic cells. Additionally, the inducing composition or the inducing medium of the present invention can also be used as a culture medium for preparing brown adipocytes by inducing differentiation from somatic cells.

[0199] For somatic cell culture, for example, somatic cells can be added to a basal culture medium prepared with the components required for the culture of mixed cells, a culture medium containing the induction composition of the present invention which has components effective for inducing differentiation into brown adipocytes, or an induction culture medium of the present invention (serum-free) which has components effective for inducing differentiation into brown adipocytes, and the temperature and other conditions are appropriately selected according to the type of somatic cells used, and the culture is carried out by conventional methods.

[0200] The components effective in inducing differentiation into brown adipocytes need only be included at concentrations effective for the preparation of brown adipocytes, and those skilled in the art can appropriately determine their concentrations. The aforementioned basal culture medium can be selected from known or commercially available media. For example, MEM (Eagle Minimum Essential Medium), GMEM (Glasgow's Minimum Essential Medium), DMEM (Dulbecco Modified Eagle Medium), Ham's F-12, DMEM / F12, RPMI 1640, IMDM (Iscov's Modified Dulbecco Medium), or modified versions thereof, can be used as the basal medium.

[0201] As one example of the induction medium of the present invention, a differentiation induction medium prepared by adding the induction composition of the present invention to DMEM medium can be cited. "DMEM medium" is a basal medium widely used in the culture of mammalian cells and is well-known to those skilled in the art, basically containing amino acids, inorganic salts, D-glucose, and vitamins. In the present invention, high-glucose DMEM medium or DMEM medium containing L-glutamine and pyruvate is preferred.

[0202] Brown adipocytes can sometimes be prepared by inducing differentiation from somatic cells in a one-step culture using the induction composition or induction medium of the present invention. Alternatively, brown adipocytes can be prepared by inducing differentiation from somatic cells that have been pre-increased in number to a state of near confluence by selecting easily cultured cells as the somatic cells to be used.

[0203] As for culture conditions, typical cell culture conditions can be selected. Examples include 37°C and 5% CO2. During culture, it is preferable to change the culture medium at appropriate intervals (preferably once every 1 to 5 days, more preferably once every 2 to 3 days).

[0204] Somatic cell culture can be performed using cell culture containers such as culture plates, culture dishes, cell culture flasks, and cell culture bags. It should be noted that air-permeable cell culture bags are preferred. Large culture tanks can be used when a large number of cells are required. Culture can be carried out in either an open or closed system; however, when the goal is to administer the obtained brown adipocytes to humans, a closed system is preferred.

[0205] 1.4 Detection / confirmation of brown adipocytes, etc.

[0206] Brown adipocytes prepared using the induction composition or induction culture medium of the present invention can be detected, identified, and isolated by utilizing changes in cell morphology, characteristic properties of brown adipocytes, and specific markers.

[0207] Adipocytes store fat within their cells. Therefore, adipocytes can be detected by staining intracellular fat with Oil Red O.

[0208] Specific markers for brown adipocytes include: 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., but are not limited to these. UCP1 is a type of uncoupling protein.

[0209] Detection of specific markers can be performed using quarantine methods (antibody-based detection), and for protein molecules, detection can also be performed by quantifying their mRNA levels. Antibodies that recognize specific markers of brown adipocytes are useful in the isolation and purification of brown adipocytes obtained using the induction composition or induction medium of the present invention.

[0210] Brown adipocytes prepared using the induction composition or induction medium of the present invention can be used, for example, for tissue repair after surgical treatment. Using brown adipocytes prepared using the induction composition or induction medium of the present invention, pharmaceutical compositions for tissue repair, etc., can be manufactured. Furthermore, it is anticipated that transplantation and application of brown adipocytes to organisms will improve metabolism and prevent obesity.

[0211] When brown adipocytes are formulated into pharmaceutical compositions, a formulation suitable for individual administration can be prepared by mixing the brown adipocytes with a pharmaceutically acceptable carrier using conventional methods. Examples of carriers include isotonic distilled water for injection containing physiological saline, glucose, and other adjuvants (e.g., D-sorbitol, D-mannitol, sodium chloride, etc.). Further formulations may include buffers (e.g., phosphate buffer, sodium acetate buffer), analgesics (e.g., benzalkonium chloride, procaine hydrochloride, etc.), stabilizers (e.g., human serum albumin, polyethylene glycol, etc.), preservatives, antioxidants, etc.

[0212] Brown adipocytes prepared using the induction composition or induction culture medium of the present invention can also be formulated into compositions that are further combined with other cells or components that are effective in enhancing the function of brown adipocytes and improving transplantability.

[0213] Furthermore, brown adipocytes prepared using the inducing composition or inducing medium of the present invention can also be used for screening pharmaceutical candidate compounds that act on brown adipocytes and for evaluating the safety of pharmaceutical candidate compounds. Therefore, using brown adipocytes prepared using the inducing composition or inducing medium of the present invention, agents that enhance the glucose / lipid metabolism and thermogenesis of brown adipocytes can be screened. Pharmaceutical candidate compounds used for this screening include, for example, existing drugs such as so-called lifestyle disease treatments, metabolic disease treatments, and other pharmacopoeia drugs. Food components include, for example, a wide range of food nutrients such as vitamins, sugars, amino acids, and minerals, components of functional foods, traditional Chinese medicine, herbs, low-molecular-weight compounds in other foods, and proteins and lipids.

[0214] The above screening can be carried out, for example, by adding the test substance to brown adipocytes prepared using the inducing composition or the inducing medium of the present invention, or cells during the preparation process thereof, and evaluating the degree of enhancement of the browning tendency of the cells induced by the test substance. In this evaluation, for example, the expression levels of the Ucp1 gene and the Fap4 gene can be quantified using real-time PCR, and their ratio can be used as an indicator of browning tendency. The pharmaceutical candidate compounds and food ingredients that enhance browning tendency analyzed in this way can become powerful candidate compounds capable of increasing brown adipocytes (beige cells) in the human body.

[0215] 2. Regarding the composition for maintaining the culture medium

[0216] The culture medium composition of the present invention (hereinafter referred to as the "culture medium composition of the present invention") is a culture medium composition for maintaining brown adipocytes produced by differentiation induction from somatic cells, and comprises the following five components.

[0217] Selective PPARγ agonists

[0218] Glucocorticoid receptor agonists

[0219] ·insulin

[0220] ·albumin

[0221] ·antibiotic.

[0222] Each of the above ingredients can be used alone, or in combination of two or more.

[0223] In addition, as part of the present invention, a serum-free maintenance culture medium containing the maintenance composition of the present invention (hereinafter referred to as "the maintenance culture medium of the present invention") can be cited.

[0224] Here, terms related to "somatic cells," "differentiation induction," "serum-free," "brown adipocytes," "selective PPARγ agonists," "glucocorticoid receptor agonists," "insulin," "albumin," "antibiotics," and other components have the same meaning as described above.

[0225] The retention composition of the present invention can be used as a composition for maintaining brown adipocytes prepared by differentiation induction from somatic cells. Additionally, the retention composition or the retention medium of the present invention can also be used as a culture medium for maintaining brown adipocytes prepared by differentiation induction from somatic cells.

[0226] For the preservation of these brown adipocytes, for example, they can be added to a basal culture medium containing the preservation composition of the present invention, or to the preservation culture medium of the present invention (serum-free), and cultured using conventional methods, with appropriate selection of temperature and other conditions depending on the type of brown adipocytes being preserved.

[0227] The aforementioned basal media can be selected from known media or commercially available media. For example, MEM (Eagle Minimum Essential Medium), GMEM (Glasgow's Minimum Essential Medium), DMEM (Dulbecco Modified Eagle Medium), Ham's F-12, DMEM / F12, RPMI 1640, IMDM (Iscov's Modified Dulbecco Medium), or media modified from them, which are commonly used as basal media, can be used as basal media.

[0228] As one method of maintaining the culture medium according to the present invention, a maintenance medium prepared by adding the maintenance composition of the present invention to DMEM medium can be cited. In the present invention, high-glucose DMEM medium or DMEM medium containing L-glutamine and pyruvate is preferred.

[0229] As for the culture conditions used for this maintenance, typical cell culture conditions can be selected. Examples include 37°C and 5% CO2. During culture, it is preferable to change the culture medium at appropriate intervals (preferably once every 1 to 5 days, more preferably once every 2 to 3 days).

[0230] The brown adipocytes can be cultured using cell culture containers such as culture plates, culture dishes, cell culture flasks, and cell culture bags. It should be noted that air-permeable cell culture bags are preferred. Large culture tanks can be used when a large number of cells are required. Culture can be carried out in either an open or closed system; however, when the goal is to administer the obtained brown adipocytes to humans, a closed system is preferred.

[0231] Example

[0232] The present invention will be specifically described below through examples, comparative examples and test examples, but the present invention is not limited to the scope of the examples, etc.

[0233] [Experimental Example A] Discussion on the composition or differentiation-inducing medium used in differentiation-inducing media

[0234] (1) Cell Culture

[0235] The human fibroblasts used as the material were purchased from DS PHARMA BIOMEDICAL. They were fibroblasts derived from the skin of a 38-year-old person.

[0236] The aforementioned human fibroblasts were seeded into 35 mm culture dishes coated with gelatin (Cat#: 190-15805, manufactured by Wako Pure Chemical Industries, Ltd.), with 5 × 10⁶ cells seeded per dish. 4 One fibroblast was 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 it reached 80-90% confluence.

[0237] (2) Differentiation induction from human fibroblasts to brown adipocytes

[0238] The culture medium used for inducing differentiation into brown adipocytes was serum-free medium. To achieve the final concentration, the components of Table 1 were added to DMEM medium (Cat#: 11965092; manufactured by Gibco, hereinafter referred to as "DMEM") in the combination shown in Table 2, thereby preparing (Example 1, Comparative Examples 1-6).

[0239] [Table 1]

[0240]

[0241] [Table 2]

[0242]

[0243] In Table 2, "+" indicates that the component was added to the culture medium, and "-" indicates that the component was not added to the culture medium.

[0244] In addition, the compounds shown in Table 3 were added to the culture medium above in a manner that achieved the final concentration.

[0245] [Table 3]

[0246]

[0247] Then, after the human fibroblasts reached 80-90% confluence, the culture medium in the culture dish was replaced with the serum-free medium described above, and the human fibroblasts were cultured in this medium for 3 weeks. The culture medium was changed every 3 days.

[0248] (3) RNA isolation and QRT-PCR

[0249] To quantify gene expression, total RNA was extracted from fibroblasts treated with various compounds using the FastGene RNA basic kit (manufactured by Nippon Genetics). Real-time PCR analysis was then performed using Power SYBR Green PCR Master Mix (Thermo Fisher Scientific) after reverse transcription using ReverTraAce PCR RT Master Mix with gDNA Remover (manufactured by Toyobo Co., Ltd.). The reaction was performed in triplicate for each level under the following conditions: 40 cycles of 95°C for 10 min, 95°C for 15 sec, and 60°C for 60 sec. All results were normalized to Tbp mRNA levels. Primers used in QRT-PCR are shown in Table 4 below.

[0250] [Table 4]

[0251]

[0252] (4) Results

[0253] The experimental results are shown in Figure 1 and Figure 2 It should be noted that in all the figures, the data represent the mean ± SD (n = 3), and "***", "**" and "*" indicate that the differences are statistically significant at p < 0.001, p < 0.01 and p < 0.05, respectively, according to Student's t-test.

[0254] The culture medium composition added in Example 1 was a combination of triiodothyronine, dexamethasone, IBMX, insulin, 2PAA, linoleic acid-oleic acid-albumin, and penicillin / streptomycin. Comparative Examples 1-5 were combinations from which any of the following components were excluded: triiodothyronine, dexamethasone, IBMX, insulin, and 2PAA. Furthermore, Comparative Example 6 did not include any of these components (all components shown in Table 1) in the culture medium composition.

[0255] like Figure 1 and 2 As shown, in Example 1, both Ucp1 and Fapp4 were significantly expressed, while their expression was insufficient in the comparative examples. In particular, in the combination of the compositions of Comparative Examples 1 and 2, Ucp1 was essentially not expressed.

[0256] Therefore, it is shown that the combination of Example 1 is preferred as a component of the differentiation induction medium from human fibroblasts to brown adipocytes.

[0257] [Experimental Example B] Investigation of compositions for maintaining culture media or the use of culture media for maintenance

[0258] (1) Cell Culture

[0259] Human fibroblasts identical to those in Experiment A above were cultured under the same conditions until they reached 80-90% confluence.

[0260] (2) Differentiation induction from human fibroblasts to brown adipocytes

[0261] The serum-free medium (serum-free brown fat induction medium) used for inducing differentiation into brown adipocytes was prepared by adding the same components as in Example 1 to DMEM medium (Cat#: 11965092; manufactured by Gibco) (hereinafter referred to as "SFBAM"). Furthermore, the compounds shown in Table 3 were added.

[0262] Then, after the human fibroblasts reached 80-90% confluence, the culture medium in the culture dish was replaced with serum-free induction medium SFBAM (containing all compounds shown in Table 3), and the human fibroblasts were cultured in this medium for 3 weeks. The medium was changed every 3 days. Each induced cell was further cultured for 1 week in serum-free maintenance medium containing all the components shown in Table 5 (Example 2), or in serum-free maintenance medium containing at least rosiglitazone (Examples 3-6, Comparative Examples 7 and 8) to allow the chemically induced brown adipocyte-like cells (ciBAs) to mature. This maintenance medium was prepared by adding the components shown in Table 5 to DMEM medium (Cat#: 11965092; manufactured by Gibco) in the combination shown in Table 6 to achieve the final concentration.

[0263] [Table 5]

[0264]

[0265] [Table 6]

[0266]

[0267] In Table 6, "+" indicates that the component was added to the culture medium, and "-" indicates that the component was not added to the culture medium.

[0268] (3) RNA isolation and QRT-PCR

[0269] RNA isolation and PCR analysis were performed in the same manner as in Experiment A above.

[0270] (4) Results

[0271] The experimental results are shown in Figure 3 and Figure 4 .

[0272] The culture medium compositions added in Examples 2 and 3 were combinations of triiodothyronine, dexamethasone, IBMX, insulin, 2PAA, linoleic acid-oleic acid-albumin, and penicillin / streptomycin. Examples 4-6 and Comparative Examples 7 and 8 were compositions in which any one of the following components was excluded: triiodothyronine, dexamethasone, IBMX, insulin, and 2PAA.

[0273] like Figure 3 and 4 As shown, in the serum-free maintenance medium of Examples 2-6, Ucp1, specific to brown adipocytes, was significantly expressed. In the serum-free maintenance medium of Comparative Examples 7 and 8, dexamethasone and insulin were excluded, respectively. In these comparative examples, although Fabp4, specific to all adipocytes, was expressed to some extent, Ucp1 was essentially not expressed.

[0274] Therefore, it is indicated that, in addition to rosiglitazone, the combination of dexamethasone, insulin, linoleic acid-oleic acid-albumin, and penicillin / streptomycin is preferred as a component of the culture medium for maintaining brown adipocytes.

[0275] [Experimental Example C] Evaluation of the composition or culture medium of the present invention

[0276] (1) Cell Culture

[0277] Human fibroblasts, identical to those in Experiment A above, were cultured under the same conditions.

[0278] (2) Differentiation induction from human fibroblasts to brown adipocytes

[0279] After the human fibroblasts reached 80-90% confluence, the culture medium in the culture dish was replaced with serum-free induction medium SFBAM (containing all compounds shown in Table 3), and the human fibroblasts were cultured in this SFBAM for 4 weeks. The culture medium was changed every 3 days. The brown adipocytes obtained at this stage were first subjected to immunostaining (described later) (Example 7).

[0280] Next, the induced cells were cultured for another week in serum-free brown adipocyte maintenance medium (hereinafter referred to as "SFBAMaM") prepared by adding the components shown in Table 7 to DMEM medium (Cat#: 11965092; manufactured by Gibco) to allow the chemically induced brown adipocyte-like cells (ciBAs) to mature. Immunostaining was also performed on the matured brown adipocytes (described later) (Example 8).

[0281] [Table 7]

[0282]

[0283] (3) Immunostaining

[0284] Immune cells were used 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 for 10 minutes with PBS containing 0.1% Triton X-100. Next, cells were blocked for 1 hour at room temperature with PBS containing 3% skim milk. UCP-1 antibody (ab10983, Abcam, Cambridge, UK) was diluted to 1 / 500 with blocking solution. Cells and antibody were incubated 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 hour at room temperature. Cell nuclei were stained with DAPI solution (Dongjin Chemical Research Institute). All images were obtained using fluorescence microscopy (Axio Vert.A1, Carl Zeiss, Oberkochen, Germany). Mitochondria were stained using MitoTracker (trademarked) Red CMXRos (Thermo Fisher Scientific).

[0285] (4) Results

[0286] The experimental results are shown in Figure 5 .like Figure 5 As shown, sufficient direct differentiation induction (direct conversion) from human fibroblasts to brown adipocytes was observed in serum-free induction medium SFBAM. Furthermore, brown adipocytes with more lipid droplets were observed after 4 weeks of induction in SFBAM followed by 1 week of incubation in serum-free maintenance medium SFBAM.

[0287] [Experimental Example D] Investigation during induction into brown adipocytes

[0288] Similar to Example C, after human fibroblasts reached 80-90% confluence, the culture medium in the culture dish was replaced with serum-free induction medium SFBAM (containing all compounds shown in Table 3), and the human fibroblasts were cultured in this SFBAM. This culture experiment in SFBAM containing all compounds shown in Table 3 is Example 9. The culture medium was changed every 3 days. Furthermore, the expression levels of several marker genes specific to adipocytes and brown adipose tissue, as shown in Table 8 below, were quantified using QRT-PCR. The results are shown below. Figure 6 In the figure, "C" indicates the case where the cells were cultured for 5 weeks without the compounds shown in Table 3 (the compounds required for induction of brown adipocytes), and "Ro" indicates the case where only rosiglitazone was included in the compounds shown in Table 3 and the cells were cultured for 5 weeks. It should be noted that... Figure 6 The gene shown in A is specific to brown adipocytes. Figure 6 The gene shown in B is specific to all adipocytes.

[0289] [Table 8]

[0290]

[0291] like Figure 6 As shown, if all the compounds listed in Table 3 are included, any gene begins to be expressed before week 1 of culture, and the expression level continues to increase until week 3 or 4. Furthermore, taking this result into consideration, according to the present invention, brown adipocytes induced by fibroblast differentiation begin to appear before week 1 and continue to increase until week 3 or 4.

[0292] Next, the culture medium was replaced with the maintenance medium of this invention, namely SFBAMaM, to induce cell maturation for one week. This maintenance culture experiment in SFBAMaM is referred to as Example 10. The results are shown below. Figure 7 The meanings of "C" and "Ro" in the diagram are... Figure 6 The situation is the same; similarly, the culture medium was changed to SFBAMaM, and the culture was carried out for 1 week. Additionally, similarly... Figure 7 The gene shown in C is specific to brown adipocytes. Figure 7 The gene shown in D is specific to all adipocytes.

[0293] like Figure 7 As shown, brown adipocytes induced in SFBAM containing all the compounds shown in Table 3 were maintained without inducing new brown adipocytes after maturation in SFBAM for 1 week using a minimal culture medium composition, without significantly reducing the expression levels of brown adipocyte- and adipocyte-specific genes.

[0294] [Experimental Example E] Investigation into the presence or absence of fatty acids in serum-free culture medium

[0295] After human fibroblasts reached 80-90% confluence, the culture medium in the culture dishes was replaced with serum-free induction medium SFBAM, or SFBAM obtained by changing the albumin or fatty acid components to combinations shown in Table 10 of the compounds shown in Table 9 (all containing all compounds shown in Table 3). Human fibroblasts were cultured in these media for 3 weeks (Examples 11-14). The culture medium was changed every 3 days. Then, the expression levels of the brown adipose tissue-specific marker genes Ucp1 and Ckmt1 were quantified using QRT-PCR. The results are shown in... Figure 8 and 9 Symbol 1 (control) in the figure represents the results of SFBAM without the compounds shown in Table 3 required for induction of brown adipocytes.

[0296] [Table 9]

[0297]

[0298] [Table 10]

[0299] Ingredient Name Example 11 Example 12 Example 13 Example 14 L,O-Alb + － － － FF-BSA － + + + Oleic acid － － + － Palmitic acid － － － +

[0300] In Table 10, "+" indicates that the component was added to the culture medium, and "-" indicates that the component was not added to the culture medium.

[0301] according to Figure 8 and 9 It can be seen that the expression level of the marker gene specific to brown adipose tissue was further increased by induction under fatty acid-free conditions.

[0302] Industrial applicability

[0303] The induction medium of the present invention (e.g., SFBAM) is useful as a differentiation induction medium from human fibroblasts to brown adipocytes. In addition, the maintenance medium of the present invention (e.g., SFBAMaM) is useful as a maintenance medium for brown adipocytes. Sequence Listing <110> KATAOKA CORPORATION Kyoto Prefectural Public University Corporation <120> Composition for Medium <130> P19030WO <160> 12 <170> PatentIn version 3.5 <210> 1 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Artificial Sequence Description: Synthetic DNA <400> 1 actacggggt tatcacctgt gag 23 <210> 2 <211> 22 <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> 22 <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 differentiation-inducing medium composition for use in differentiation induction of a somatic cell into a brown adipocyte, the composition being a serum-free composition, which is composed of the following components: 1: 0.01 to 1 μmol / L of triiodothyronine; 2: 0.1 to 10 μmol / L of dexamethasone; 3: 10 to 0.5 mmol / L of isobutylmethylxanthine; 4: 100 ng / mL to 20 μg / mL of insulin; 5: 10 to 250 μg / mL of L-ascorbic acid 2-phosphate; 6: 0.5 to 4 mg / mL of albumin to which linoleic acid and oleic acid are bound; 7: 50 to 200 units / mL of penicillin and / or streptomycin; 8: 0.5 to 5 μmol / L of rosiglitazone; 9: 0.5 to 30 μmol / L of forskolin; 10: 1 to 50 ng / mL of BMP7.

2. The composition for a differentiation-inducing culture medium according to claim 1, wherein The differentiation induction is directly performed without introducing a gene into the somatic cell.

3. The composition for a differentiation-inducing culture medium according to claim 1 or 2, wherein The somatic cell is a fibroblast.

4. A differentiation-inducing medium comprising the differentiation-inducing medium composition according to any one of claims 1 to 3, and being serum-free.

5. The differentiation-inducing medium according to claim 4, which is prepared by adding the differentiation-inducing medium composition according to any one of claims 1 to 3 to a DMEM medium.

6. The differentiation-inducing medium according to claim 5, wherein, The DMEM medium is a high-glucose DMEM medium.

7. The differentiation-inducing medium according to claim 5 or 6, wherein, The DMEM medium contains L-glutamine and pyruvate.

8. The differentiation-inducing medium according to claim 4, wherein, The somatic cell is a fibroblast.

9. A maintenance medium composition for use in maintaining a brown adipocyte prepared by differentiation induction from a somatic cell, the composition being a serum-free composition, The medium composition is composed of the following components: 1: 0.5 to 5 μmol / L of rosiglitazone; 2: 0.1 to 10 μmol / L of dexamethasone; 3: 100 ng / mL to 20 μg / mL of insulin; 4: 0.5 to 4 mg / mL of albumin to which linoleic acid and oleic acid are bound; 5: 50 to 200 units / mL of penicillin and / or streptomycin; and 6: one or more selected from the group consisting of 0.01 to 1 μmol / L of triiodothyronine, 10 to 0.5 mmol / L of isobutylmethylxanthine, and 10 to 250 μg / mL of L-ascorbic acid 2-phosphate; or The medium composition is composed of the following components: 1: 0.5 to 5 μmol / L of rosiglitazone; 2: 0.5 to 30 μmol / L of forskolin; 3: 1 to 50 ng / mL of BMP7; 4: 0.01 to 1 μmol / L of triiodothyronine; 5: 0.1 μmol / L to 10 μmol / L of dexamethasone; 6: 10 μmol / L to 0.5 mmol / L of isobutylmethylxanthine; 7: 100 ng / mL to 20 μg / mL of insulin; 8: 10 μg / mL to 250 μg / mL of L-ascorbic acid 2-phosphate ester; 9: 0.5 mg / mL to 4 mg / mL of albumin to which linoleic acid and oleic acid are bound; 10: 50 units / mL to 200 units / mL of penicillin and / or streptomycin.

10. The maintenance medium with composition according to claim 9, wherein, The somatic cell is a fibroblast.

11. A maintenance medium comprising the maintenance medium composition according to claim 9, and being serum-free.

12. The maintenance medium according to claim 11, which is prepared by adding the maintenance medium composition according to claim 9 to a DMEM medium.

13. The maintenance medium of claim 12, wherein, The DMEM medium is a high-glucose DMEM medium.

14. The maintenance medium of claim 12, wherein, The DMEM medium contains L-glutamine.

15. The maintenance medium according to any one of claims 11 to 14, wherein, The somatic cell is a fibroblast.

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