Culture medium composition

Abstract

The present invention addresses the problem of providing a novel composition which is for a medium and can be used to induce differentiation from somatic cells into brown adipocytes.　For example, the present invention may be a composition which is for a differentiation-inducing medium and is used to induce differentiation from somatic cells into brown adipocytes, the composition being characterized by containing seven components: 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 invention, the differentiation of somatic cells into brown adipocytes can be effectively induced. Moreover, according to the present invention, the brown adipocytes can be effectively maintained.

Description

[Technical field]

[0001] (CROSS REFERENCE TO RELATED APPLICATIONS) This application claims the benefit of Japanese Application No. 2019-211991, filed with the Japan Patent Office on November 25, 2019. The Japanese application is hereby incorporated by reference for all purposes as if the entire application documents (specification, claims, drawings, abstract) were set forth herein. The present invention belongs to the technical field of regenerative medicine. In the technical field, the present invention relates to a medium composition that can be used when inducing differentiation of somatic cells into brown adipocytes, and a medium composition for maintaining the differentiated brown adipocytes. [Background technology]

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

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

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

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

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

[0007] The main object of the present invention is to provide a novel medium composition that can be used when inducing differentiation of somatic cells into brown adipocytes. Another object of the present invention is to provide a novel medium composition for maintaining the differentiated brown adipocytes. [Means for solving the problem]

[0008] As a result of extensive investigations, the present inventors have found that the above-mentioned problems can be solved by a medium composition containing a certain low molecular weight compound, and have thus completed the present invention.

[0009] The present invention can include, for example, the following. [1] A medium composition used for inducing differentiation of somatic cells into brown adipocytes, comprising the following seven components: Thyroid hormone receptor agonists Glucocorticoid receptor agonist Phosphodiesterase inhibitors Insulin Ascorbic acid derivatives ·albumin ·Antibiotics [2] The differentiation-inducing medium composition according to the above-mentioned [1], wherein the differentiation induction is performed directly without introducing a gene into a somatic cell. [3] A differentiation-inducing medium composition 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 having linoleic acid and oleic acid bound thereto or fatty acid-unbound albumin, or the antibiotic is penicillin and / or streptomycin. [4] The differentiation-inducing medium composition according to any one of [1] to [3] above, further comprising a selective PPARγ agonist, or a selective PPARγ agonist and a cAMP inducer and / or BMP7. [5] The differentiation-inducing medium composition according to the above [4], wherein the selective PPARγ agonist is rosiglitazone, or the cAMP inducer is forskolin.

[0010] [6] A differentiation-inducing medium comprising the composition for a differentiation-inducing medium according to any one of the above [1] to [5], and which is serum-free. [7] The differentiation-inducing medium according to [6] above, in which the composition for a differentiation-inducing medium according to any one of [1] to [5] above is added to a DMEM medium. [8] The differentiation-inducing medium according to [7] above, wherein the DMEM medium is a high-glucose DMEM medium. [9] The differentiation-inducing medium according to [7] or [8] above, wherein the DMEM medium contains L-glutamine.

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

[11] A brown adipocyte produced by using the composition for a differentiation-inducing medium or the differentiation-inducing medium according to any one of [1] to

[10] above.

[0011]

[12] A medium composition for maintaining brown adipocytes produced by inducing differentiation from somatic cells, comprising the following five components: Selective PPARγ agonist Glucocorticoid receptor agonist Insulin ·albumin ·Antibiotics

[13] The maintenance medium composition according to the above

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

[14] A maintenance medium comprising the composition for a maintenance medium according to

[12] or

[13] above, and which is serum-free.

[15] The maintenance medium according to

[14] above, in which the maintenance medium composition according to

[12] or

[13] above is added to a DMEM medium.

[16] The maintenance medium according to

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

[17] The maintenance medium according to

[15] or

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

[18] The composition for a maintenance medium or the maintenance medium according to any one of

[12] to

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

[0012] According to the present invention, it is possible to effectively induce differentiation of somatic cells into brown adipocytes. Also, according to the present invention, it is possible to effectively maintain brown adipocytes induced to differentiate from somatic cells. [Brief description of the drawings]

[0013] [Figure 1] The figures show the expression level of the Ucp1 gene. Symbol 1 indicates Comparative Example 6 (control), symbol 2 indicates Example 1, symbol 3 indicates Comparative Example 1, symbol 4 indicates Comparative Example 2, symbol 5 indicates Comparative Example 3, symbol 6 indicates Comparative Example 4, and symbol 7 indicates Comparative Example 5. The vertical axis indicates the relative value of the mRNA amount to the expression level of the internal standard gene (Tbp). [Diagram 2]The expression level of the Fabp4 gene is shown. The symbols and vertical axis are the same as in FIG. [Diagram 3] The figures show the expression level of the Ucp1 gene. Reference numeral 1 indicates Example 2, reference numeral 2 indicates Example 3, reference numeral 3 indicates Example 4, reference numeral 4 indicates Comparative Example 7, reference numeral 5 indicates Example 5, reference numeral 6 indicates Comparative Example 8, and reference numeral 7 indicates Example 6. The vertical axis indicates the relative value of the amount of the mRNA to the expression level of the internal control gene (Tbp). [Figure 4] The expression level of the Fabp4 gene is shown. The symbols and vertical axis are the same as in FIG. [Diagram 5] Photographs of cultured cells directly induced from fibroblasts with a combination of compounds (Example 7) and cells incubated in a maintenance medium (Example 8), as well as photographs of immunostaining of mitochondria (red), UCP1 protein (green), and cell nuclei (blue) of these cells. Figure 5 is monochrome, so the colors red, green, and blue are not displayed, but the original photograph of Figure 5 shows each color. [Figure 6] The expression level of a gene is shown. In each figure, the vertical axis indicates the relative value of the amount of mRNA of the gene to the amount of mRNA of the internal standard gene (Tbp). The horizontal axis indicates time (weeks). [Figure 7] The expression level of a gene is shown. In each figure, the vertical axis indicates the relative value of the amount of mRNA of the gene to the amount of mRNA of the internal standard gene (Tbp). The horizontal axis indicates time (weeks). [Figure 8] The expression level of the Ucp1 gene is shown. Reference numeral 1 indicates the control, reference numeral 2 indicates Example 11, reference numeral 3 indicates Example 12, reference numeral 4 indicates Comparative Example 13, and reference numeral 5 indicates Example 14. The vertical axis indicates the relative value of the amount of the mRNA to the expression level of the internal control gene (Tbp). [Figure 9] The expression level of the Ckmt1 gene is shown. The symbols and vertical axis are the same as in Figure 8. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

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

[0015] 1. Differentiation-inducing medium composition The differentiation-inducing medium composition of the present invention (hereinafter referred to as the "inducing composition of the present invention") is a medium composition used when inducing differentiation from somatic cells to brown adipocytes, and is characterized by containing the following seven components: Thyroid hormone receptor agonists Glucocorticoid receptor agonist Phosphodiesterase inhibitors Insulin Ascorbic acid derivatives ·albumin ·Antibiotics

[0016] The above-mentioned components may be used alone or in combination of two or more. The differentiation of somatic cells into brown fat cells may be induced by so-called direct reprogramming, which involves direct conversion using a low molecular weight compound without artificially introducing genes into somatic cells, or by a method involving artificially introducing genes into somatic cells. Among these, the inducing composition of the present invention is preferably used for direct differentiation induction without introducing genes into somatic cells. The present invention also includes a differentiation-inducing medium that contains the inducing composition of the present invention and is serum-free (hereinafter referred to as the "inducing medium of the present invention").

[0017] Here, "serum-free" means that it is substantially free of unconditioned or unpurified serum. In the present invention, even if a differentiation-inducing medium or a maintenance medium contains purified blood-derived components or animal tissue-derived components, it is called a serum-free differentiation-inducing medium or maintenance medium as long as it does not substantially contain unconditioned or unpurified serum. The "brown adipocytes" in the present invention are artificially induced to differentiate from somatic cells, have many mitochondria and many lipid droplets, and express the UCP1 (uncoupling protein-1) gene. The brown adipocytes also include so-called human beige cells and bright cells induced from white adipocytes or their precursor cells. They also include so-called low molecular weight compound-induced brown adipocytes (ciBAs), which are produced by a method of direct conversion using low molecular weight compounds without artificially introducing genes into somatic cells. Furthermore, in addition to terminally differentiated brown adipocytes, they also include precursor cells destined to differentiate into brown adipocytes.

[0018] The inducing composition or the inducing medium of the present invention may further contain a selective PPARγ agonist, or a selective PPARγ agonist and a cAMP inducer and / or BMP7 as an active ingredient for inducing differentiation into brown adipocytes. Among these, it is preferable to further contain a selective PPARγ agonist, and it is more preferable to further contain two ingredients, a selective PPARγ agonist and a cAMP inducer, or three ingredients, a selective PPARγ agonist, a cAMP inducer and BMP7.

[0019] 1.1 About somatic cells The cells of an organism can be classified into somatic cells and germ cells. In the method of the present invention, any somatic cells can be used as the starting material. There is no particular limitation on the somatic cells, and they may be either primary cells collected from a living organism or established cells. In the method of the present invention, somatic cells at various stages of differentiation, for example, terminally differentiated somatic cells (e.g., fibroblasts, umbilical vein endothelial cells (HUVECs), hepatocytes, biliary cells, pancreatic α cells, pancreatic acinar cells, pancreatic ductal gland cells, intestinal crypt cells, etc.), somatic cells in the process of terminal differentiation (e.g., mesenchymal stem cells, neural stem cells, endodermal progenitor cells, etc.), or somatic cells such as iPS cells that have been reprogrammed to acquire pluripotency, and somatic cells that have differentiated from iPS cells or are in the process of differentiation, can be used. Somatic cells that can be used in the method of the present invention include any somatic cells, such as hematopoietic cells (various lymphocytes, macrophages, dendritic cells, bone marrow cells, etc.), organ-derived cells (hepatocytes, spleen cells, pancreatic cells, kidney cells, lung cells, etc.), muscle tissue cells (skeletal muscle cells, smooth muscle cells, myoblasts, cardiac muscle cells, etc.), fibroblasts, nerve cells, osteoblasts, chondrocytes, endothelial cells, interstitial cells, adipocytes (white adipocytes, etc.), embryonic stem cells (ES cells), etc. The method of the present invention can also be applied to precursor cells of these cells and cancer cells. Fibroblasts can be preferably used.

[0020] Fibroblasts that can be used in the present invention are not particularly limited, and examples include dermal fibroblasts, adventitial fibroblasts, cardiac fibroblasts, pulmonary fibroblasts, uterine fibroblasts, and villous mesenchymal fibroblasts, which are major cellular components that make up connective tissue in various tissues and organs and produce collagen fibers.

[0021] Examples of sources of the above somatic cells include, but are not limited to, humans, non-human mammals, and non-mammalian animals (birds, reptiles, amphibians, fish, etc.). Preferred sources of somatic cells are humans and non-human mammals, and humans are particularly preferred. When inducing differentiation of brown adipocytes for administration to humans, somatic cells collected from a donor having the same or similar histocompatibility antigen type as the recipient can be preferably used. Somatic cells collected from the recipient themselves may also be provided.

[0022] 1.2 About each ingredient 1.2.1 Thyroid hormone receptor agonists The thyroid hormone receptor agonist is not particularly limited as long as it binds to the thyroid hormone receptor and has a hormone-like action, and examples thereof include the following compounds. Preferred examples include triiodothyronine and thyroxine.

[0023] Triiodothyronine (CAS No.: 6893-02-3)

[0024] [ka]

[0025] 3,5-Diiodothyronine dihydrate (CAS No.: 18835-59-1) Chiratricol (CAS No.:51-24-1) Liothyronine Sodium (CAS No.: 55-06-1) L-Thyroxine (CAS No.: 51-48-9)

[0026] The concentration of the thyroid hormone receptor agonist varies depending on the type of agonist and the somatic cells used, but is not particularly limited and may be determined appropriately. Preferably, the concentration is in the range of 1 nmol / L to 10 μmol / L, and more preferably 0.01 μmol / L to 1 μmol / L.

[0027] 1.2.2 Glucocorticoid receptor agonists The glucocorticoid receptor agonist is not particularly limited as long as it binds to the glucocorticoid receptor and has a glucocorticoid-like action, and examples thereof include the following compounds. Preferred examples include dexamethasone, hydrocortisone, corticosterone, and cortisone.

[0028] Dexamethasone (CAS No.:50-02-2)

[0029] [ka]

[0030] Dexamethasone sodium phosphate (CAS No.: 55203-24-2) Dexamethasone acetate (CAS No.: 1177-87-3) Meprednisone (CAS No.: 1247-42-3) Fluocinolone acetonide (CAS No.:67-73-2) Budesonide (CAS No.: 51333-22-3) Desoxymetasone (CAS No.: 382-67-2) Protopanaxatriol (CAS No.:34080-08-5) Triamcinolone (CAS No.: 124-94-7) Beclomethasone dipropionate (CAS No.: 5534-09-8) Prednisolone (CAS No.: 50-24-8) Hydrocortisone (CAS No.: 50-23-7) Deflazacort (CAS No.: 14484-47-0) Triamcinolone acetonide (CAS No.: 76-25-5) Fluocinonide (CAS No.: 356-12-7) Methylprednisolone sodium succinate (CAS No.: 2921-57-5) Ciclesonide (CAS No.: 126544-47-6) Flumethasone (CAS No.: 2135-17-3) Fluorometholone (CAS No.: 426-13-1) Betamethasone dipropionate (CAS No.: 5593-20-4) Betamethasone valerate (CAS No.: 2152-44-5) Mometasone furoate (CAS No.:83919-23-7) Fluticasone propionate (CAS No.: 80474-14-2) Cortisone acetate (CAS No.: 50-04-4) Cortodoxone (CAS No.: 152-58-9) Loteprednol (CAS No.: 82034-46-6) Betamethasone (CAS No.: 378-44-9) Methylprednisolone (CAS No.: 83-43-2) Corticosterone (CAS No.: 50-22-6) Cortisone (CAS No.: 53-06-5)

[0031] The concentration of the glucocorticoid receptor agonist varies depending on the type of agonist and the somatic cells used, but is not particularly limited and may be determined appropriately. It is preferably used in the range of 0.01 μmol / L to 30 μmol / L, preferably 0.1 μmol / L to 10 μmol / L.

[0032] 1.2.3 Phosphodiesterase inhibitors The phosphodiesterase inhibitor is not particularly limited as long as it is capable of increasing the intracellular cAMP concentration by inhibiting phosphodiesterase (PDE), particularly PDE3, PDE4, and PDE5, and examples thereof include the following compounds. Preferred examples include isobutylmethylxanthine (IBMX) and rolipram.

[0033] Isobutylmethylxanthine (CAS No.: 28822-58-4)

[0034] [ka]

[0035] Roflumilast (CAS No.:162401-32-3) Sildenafil (CAS No.: 139755-83-2) Tadalafil (CAS No.:171596-29-5) Vardenafil hydrochloride hydrate (CAS No.: 330808-88-3) Pimobendan (CAS No.: 74150-27-9) GSK256066(CAS No.:801312-28-7) Rolipram (CAS No.: 61413-54-5) Apremilast (CAS No.:608141-41-9) Cilostazol (CAS No.: 73963-72-1) Milrinone (CAS No.: 78415-72-2) Avanafil (CAS No.:330784-47-9) Rolipram (CAS No.:85416-73-5) Fenspiride (CAS No.: 5053-08-7) Udenafil (CAS No.:268203-93-6) Cilostamide (CAS No.: 68550-75-4) Ibudilast (CAS No.: 50847-11-5) Luteolin (CAS No.: 491-70-3)

[0036] The concentration of the phosphodiesterase inhibitor varies depending on the type of inhibitor and the somatic cells used, but is not particularly limited and may be determined appropriately. It is preferably used in the range of 1 μmol / L to 1 mmol / L, preferably 10 μmol / L to 0.5 mmol / L.

[0037] 1.2.4 Ascorbic acid derivatives The ascorbic acid derivative is not particularly limited as long as it is vitamin C (L-ascorbic acid) or a derivative thereof with improved instability. Specific examples include the following compounds, such as vitamin C (L-ascorbic acid) and L-ascorbic acid 2-phosphate (2PAA).

[0038] Sodium L-ascorbyl 2-phosphate (CAS No.: 66170-10-3)

[0039] [ka]

[0040] Ascorbyl palmitate (CAS No.: 137-66-6) Sodium Ascorbate (CAS No.: 134-03-2)

[0041] The concentration of the ascorbic acid derivative varies depending on the type of derivative and the somatic cells used, but is not particularly limited and may be determined appropriately. It is preferably used in the range of 1 μg / mL to 1 mg / mL, preferably 10 μg / mL to 250 μg / mL.

[0042] 1.2.5 Insulin, fatty acid-bound albumin, and antibiotics Insulin is a peptide hormone secreted from β cells of the pancreas, and in the present invention, either animal-derived insulin or human-derived insulin can be used, as well as chemically synthesized insulin and recombinant insulin produced by gene recombination technology. In the present invention, it is preferred to use human insulin.

[0043] The concentration of insulin varies depending on the type of insulin, the somatic cells used, etc., but is not particularly limited and may be appropriately determined, and can be preferably used within the range of 100 ng / mL to 20 μg / mL.

[0044] The albumin may be fatty acid-bound albumin to which fatty acids are bound, or fatty acid-free (fatty acid-free) albumin from which fatty acids have been removed. Examples of the albumin include albumin purified from animal (e.g., bovine) serum, albumin obtained by binding known fatty acids to the purified albumin, and recombinant albumin expressed using animal or plant cells. In particular, recombinant albumin that has been confirmed to be free of animal-derived components and purified albumin to which known fatty acids are bound are preferred. Human-derived albumin is also preferred.

[0045] Examples of fatty acids bound to albumin include representative saturated and unsaturated fatty acids such as linoleic acid, oleic acid, linolenic acid, palmitic acid, arachidonic acid, eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), etc. A mixed fatty acid of linoleic acid and oleic acid is preferred. Specific examples of fatty acid-bound albumin include albumin purified from animal serum and linoleic acid- and oleic acid-bound albumin. In the present invention, commercially available fatty acid-bound bovine serum albumin (e.g., L9655: manufactured by Sigma-Aldrich) can be used. In addition, in the experimental results described below, the expression level of the Ucp1 gene was much higher in brown adipocytes induced using albumin that did not bind to fatty acids than in those induced using albumin that bound to fatty acids (see Figures 8 and 9). In addition, when inducing brown adipocytes from fibroblasts, it was preferable not to add fatty acids. These are not seen in adipocytes derived from mesenchymal stem cells, which have high fat biosynthesis activity.

[0046] The concentration of albumin varies depending on the type of albumin and the somatic cells used, but is not particularly limited and may be determined appropriately. For example, the concentration can be in the range of 0.1 mg / mL to 10 mg / mL, preferably 0.5 mg / mL to 4 mg / mL.

[0047] The antibiotics are not particularly limited as long as they can be used in animal cell culture, and examples thereof include β-lactam antibiotics such as penicillins, cephalosporins, and monobactams, aminoglycosides, macrolides, tetracyclines, peptides, nucleic acids, and polyenes. Specific examples thereof include actinomycin D, amphotericin B, ampicillin, antimycin A, bafilomycin A1, bleomycin, carbenicillin, chloramphenicol, concanamycin B, erythromycin, G418, gentamicin, hygromycin, kanamycin, mitomycin C, neomycin, oligomycin, penicillin, puromycin, rapamycin, streptomycin, tetracycline, tobramycin, and valinomycin. Among these, the combined use of penicillin and streptomycin is suitable in the present invention.

[0048] The antibiotic content varies depending on the type of antibiotic, somatic cells used, etc., but is not particularly limited and may be appropriately determined, and may be used in the range of, for example, 10 units / mL to 1000 units / mL, preferably 50 units / mL to 200 units / mL. Depending on the type of antibiotic, it may be used in the range of 10 μg / mL to 1000 μg / mL, preferably 50 μg / mL to 200 μg / mL.

[0049] 1.2.6 Selective PPARγ agonists PPARγ (Peroxisome proliferator-activated receptor γ) is a protein belonging to the nuclear receptor superfamily, and is a transcription factor that controls gene expression by forming a heterodimer with retinoid X receptor (RXR) and binding to the promoter region of the target gene. There are three subtypes of PPAR: α, δ (β), and γ. In the present invention, an agonist that selectively acts on γ is used.

[0050] Although not particularly limited in the present invention, the selective PPARγ agonist may be, for example, the following compounds or salts thereof. Preferably, the selective PPARγ agonist may be a thiazolidine-type agent, specifically, rosiglitazone, pioglitazone, or troglitazone.

[0051] Rosiglitazone (CAS No.: 122320-73-4)

[0052] [ka]

[0053] Rosiglitazone maleate (CAS No.: 155141-29-0) Ciglitazone (CAS No.: 74772-77-3) Edaglitazone (CAS No.: 213411-83-7) GW1929 hydrochloride (CAS No.: 1217466-21-1) nTZDpa (CAS No.: 118414-59-8) Pioglitazone hydrochloride (CAS No.:112529-15-4) S26948(CAS No.:353280-43-0) Troglitazone (CAS No.: 97322-87-7) Balaglitazone (CAS No.: 199113-98-9) Rivoglitazone (CAS No.: 185428-18-6)

[0054] The concentration of the selective PPARγ agonist varies depending on the type of agonist and the somatic cells used, but is not particularly limited and may be determined appropriately. For example, it can be used in the range of 0.1 μmol / L to 20 μmol / L, preferably 0.5 μmol / L to 5 μmol / L.

[0055] 1.2.7 cAMP inducers cAMP (cyclic adenosine monophosphate) is a substance that is involved in various intracellular signal transduction as a second messenger. cAMP is generated in cells by the cyclization of adenosine triphosphate (ATP) by adenylate cyclase.

[0056] Although not particularly limited in the present invention, examples of cAMP inducers (adenylate cyclase activators) include forskolin (CAS No.: 66575-29-9), forskolin derivatives (e.g., JP-A-2002-348243), and the following compounds. Forskolin can be preferably used.

[0057] Forskolin (CAS No.: 66428-89-5)

[0058] [ka]

[0059] Isoproterenol (CAS No.: 7683-59-2) NKH477(CAS No.:138605-00-2) PACAP1-27(CAS No.:127317-03-7) PACAP1-38(CAS No.:137061-48-4)

[0060] The concentration of the cAMP inducer varies depending on the type of derivative and the somatic cells used, but is not particularly limited and may be determined appropriately. For example, it can be used in the range of 0.1 μmol / L to 100 μmol / L, preferably 0.5 μmol / L to 30 μmol / L.

[0061] 1.2.8 About BMP7 BMP (Bone Morphogenetic Protein) 7 is a member of the BMP family. Human BMP7 is composed of 117 amino acid residues and binds to a receptor on the cell surface as a homodimer, activating the BMP signal pathway. Six types, from BMP2 to BMP7, are classified as the TGFb superfamily and are each encoded by a different gene. In the present invention, human-derived BMP7 or animal-derived BMP7 may be used, or a recombinant form thereof may be used. Of these, it is preferable to use human-derived BMP7.

[0062] The concentration of BMP7 varies depending on the type of BMP7 and the somatic cells used, but is not particularly limited and may be determined appropriately. For example, it can be used in the range of 0.1 ng / mL to 200 ng / mL, preferably 1 ng / mL to 50 ng / mL.

[0063] 1.3 Somatic cell culture The inducing composition of the present invention can be used as a composition for producing brown adipocytes by inducing differentiation from somatic cells. The inducing composition of the present invention or the inducing medium of the present invention can also be used as a medium for producing brown adipocytes by inducing differentiation from somatic cells.

[0064] Somatic cells can be cultured in a conventional manner, for example, by adding somatic cells to a basal medium prepared by mixing components necessary for cell culture and a medium containing the induction composition of the present invention having components effective for inducing differentiation into brown adipocytes, or to an induction medium (serum-free) of the present invention having components effective for inducing differentiation into brown adipocytes, and appropriately selecting the temperature and other conditions according to the type of somatic cells used. The components effective for inducing differentiation into brown adipocytes may be contained at a concentration effective for producing brown adipocytes, and the concentration can be appropriately determined by those skilled in the art. The above-mentioned basal medium can be selected from known media or commercially available media. For example, the common basal medium MEM (Eagle's minimum essential medium), GMEM (Glasgow's minimum essential medium), DMEM (Dulbecco's modified Eagle's medium), Ham's F-12, DMEM / F12, RPMI1640, IMDM (Iscove's modified Dulbecco's medium) or modified media thereof can be used as the basal medium. One embodiment of the induction medium of the present invention is a differentiation induction medium obtained by adding the induction composition of the present invention to a DMEM medium. "DMEM medium" is a basal medium widely used in the culture of mammalian cells and well known to those skilled in the art, and basically contains amino acids, inorganic salts, D-glucose, and vitamins. In the present invention, it is preferable to use a high-glucose DMEM medium or a DMEM medium containing L-glutamine and pyruvate.

[0065] In some cases, using the induction composition or induction medium of the present invention, it is possible to produce brown fat cells by inducing differentiation from somatic cells through a single-step culture, or by selecting somatic cells that are easy to culture, it is also possible to increase the cell number in advance and induce differentiation of somatic cells that have reached a nearly confluent state to produce brown fat cells.

[0066] Culture conditions can be selected from those generally used for cell culture, such as conditions at 37°C and 5% CO2. It is preferable to change the medium at appropriate intervals during culture (preferably once every 1 to 5 days, more preferably once every 2 to 3 days).

[0067] For the culture of somatic cells, cell culture containers such as plates, dishes, cell culture flasks, cell culture bags, etc. can be used. In addition, gas-permeable cell culture bags are preferable. When a large amount of cells is required, a large culture tank may be used. The culture can be performed in either an open or closed system, but when the obtained brown adipocytes are intended to be administered to humans, it is preferable to perform the culture in a closed system.

[0068] 1.4 Detection and identification of brown fat cells Brown adipocytes produced using the induction composition or induction medium of the present invention can be detected, confirmed and isolated, for example, by utilizing morphological changes in cells, characteristic properties and specific markers of brown adipocytes. Adipocytes accumulate fat within the cells, and therefore can be detected by staining the intracellular fat with Oil Red O. Specific markers for brown adipocytes include, but are not limited to, UCP1, EVOL3 (Elongation of very long chain fatty acid protein 3), PGC1A (PPAR gamma coactivator 1-alpha), PRDM16 (PRD1-BF1-RIZ1 homologous domain containing 16), CITED1 (CBP / p300 interacting transactivator with Glu / Asp rich carboxy-terminal domain 1), etc. UCP1 is a type of uncoupling protein.

[0069] Specific markers can be detected by diagnostic methods (antibody detection), but protein molecules can also be detected by quantifying the amount of mRNA. Antibodies that recognize specific markers for brown adipocytes are also useful for isolating and purifying brown adipocytes obtained using the induction composition or induction medium of the present invention.

[0070] The brown adipocytes produced using the induction composition of the present invention or the induction medium of the present invention can be used for, for example, tissue repair after surgical treatment. The brown adipocytes produced using the induction composition of the present invention or the induction medium of the present invention can be used to produce a pharmaceutical composition for tissue repair. In addition, transplantation or administration of brown adipocytes to a living body is expected to have effects on improving metabolism of the living body, preventing obesity, etc.

[0071] When brown fat cells are used as a pharmaceutical composition, the brown fat cells may be mixed with a medicamentically acceptable carrier in a conventional manner to prepare a formulation suitable for administration to an individual. Examples of the carrier include isotonic distilled water for injection made by adding physiological saline, glucose, or other auxiliary drugs (e.g., D-sorbitol, D-mannitol, sodium chloride, etc.). In addition, buffers (e.g., phosphate buffer, sodium acetate buffer), soothing agents (e.g., benzalkonium chloride, procaine hydrochloride, etc.), stabilizers (e.g., human serum albumin, polyethylene glycol, etc.), preservatives, antioxidants, etc. may be added. Brown adipocytes produced using the induction composition or induction medium of the present invention can also be made into a composition in which they are combined with other cells or components that are effective in improving the function or engraftment of brown adipocytes.

[0072] Furthermore, the brown adipocytes produced using the induction composition of the present invention or the induction medium of the present invention can be used for screening of drug candidate compounds acting on brown adipocytes and for safety evaluation of the drug candidate compounds. Therefore, the brown adipocytes produced using the induction composition of the present invention or the induction medium of the present invention can be used to screen for drugs that enhance the sugar and lipid metabolism and heat production function of brown adipocytes. The drug candidate compounds for the screening include, for example, existing drugs such as so-called lifestyle-related disease treatment drugs, metabolic disease treatment drugs, and other library drugs. Food ingredients include, for example, vitamins, sugars, amino acids, minerals, and other food nutrients, functional food ingredients, Chinese medicines, herbs, and other low molecular weight compounds, proteins, lipids, and the like in foods.

[0073] The above screening can be carried out, for example, by adding a test substance to brown adipocytes produced using the inducing composition of the present invention or the inducing medium of the present invention or cells in the process of producing the brown adipocytes, and evaluating the degree of enhancement of the browning tendency in the cells by the test substance. In such an evaluation, for example, the expression levels of the Ucp1 gene and the Fabp4 gene can be quantified, for example, by real-time PCR, and the ratio can be used as an index of the browning tendency. The drug candidate compounds or food ingredients that enhance the browning tendency analyzed in this way can be potential candidate compounds that can increase brown adipocytes (beige cells) in the human body.

[0074] 2. Maintenance medium composition The maintenance medium composition of the present invention (hereinafter referred to as "the maintenance composition of the present invention") is a medium composition for maintaining brown adipocytes produced by inducing differentiation from somatic cells, and is characterized by comprising the following five components: Selective PPARγ agonist Glucocorticoid receptor agonist Insulin ·albumin ·Antibiotics.

[0075] The above-mentioned components may be used alone or in combination of two or more. The present invention also includes a maintenance medium that contains the maintenance composition of the present invention and is serum-free (hereinafter referred to as the "maintenance medium of the present invention"). Here, the terms "somatic cells," "differentiation induction," "serum-free," "brown adipose cells," "selective PPARγ agonist," "glucocorticoid receptor agonist," "insulin," "albumin," "antibiotics," and other components are synonymous with those defined above.

[0076] The maintenance composition of the present invention can be used as a composition for maintaining brown adipocytes produced by inducing differentiation from somatic cells. The maintenance composition of the present invention or the maintenance medium of the present invention can also be used as a medium for maintaining brown adipocytes produced by inducing differentiation from somatic cells.

[0077] The brown adipocytes can be maintained, for example, by adding the brown adipocytes to a medium containing the maintenance composition of the present invention in a basal medium prepared by mixing components necessary for cell culture, or to the maintenance medium of the present invention (serum-free), and culturing in a conventional manner by appropriately selecting the temperature and other conditions according to the type of brown adipocytes to be maintained. The above-mentioned basal medium can be selected from known or commercially available media. For example, common basal media such as MEM (Eagle's minimum essential medium), GMEM (Glasgow's minimum essential medium), DMEM (Dulbecco's modified Eagle's medium), Ham's F-12, DMEM / F12, RPMI1640, IMDM (Iscove's modified Dulbecco's medium), or modified media thereof can be used as the basal medium. An embodiment of the maintenance medium of the present invention is a maintenance medium obtained by adding the maintenance composition of the present invention to a DMEM medium. In the present invention, it is preferable to use a high glucose DMEM medium or a DMEM medium containing L-glutamine and pyruvate.

[0078] The culture conditions for maintaining the cells can be selected from general cell culture conditions, such as 37°C and 5% CO2. It is preferable to change the medium at appropriate intervals during the culture (preferably once every 1 to 5 days, more preferably once every 2 to 3 days).

[0079] For the maintenance culture of the brown adipocytes, cell culture containers such as plates, dishes, cell culture flasks, cell culture bags, etc. can be used. In addition, gas-permeable cell culture bags are preferable. When a large amount of cells is required, a large culture tank may be used. The culture can be performed in either an open system or a closed system, but when the obtained brown adipocytes are intended to be administered to humans, it is preferable to culture in a closed system. EXAMPLES

[0080] The present invention will be specifically described below with reference to examples, comparative examples, and test examples, but the present invention is not limited to the scope of the examples, etc.

[0081] [Test Example A] Study of composition for differentiation-inducing medium or differentiation-inducing medium (1)Cell culture The human fibroblasts used as the material were purchased from DS Pharma Biomedical Co., Ltd. The fibroblasts were derived from the skin of a 38-year-old human. The human fibroblasts were cultured at 5 × 10 in a 35 mm dish coated with gelatin (Cat#: 190-15805, Wako Pure Chemical Industries, Ltd.). 4 The cells were seeded one at a time and cultured in DMEM medium (Cat#: 11965092; Gibco) supplemented with 10% fetal bovine serum (FBS), 100 U / mL penicillin, and 100 μg / mL streptomycin at 37°C and 5% CO2 until they became 80-90% confluent.

[0082] (2) Induction of differentiation of human fibroblasts into brown adipocytes The medium used for inducing differentiation into brown adipocytes was a serum-free medium, which was prepared by adding each component in Table 1 to DMEM medium (Cat#: 11965092; manufactured by Gibco, hereinafter referred to as "DMEM") in the combinations shown in Table 2 so as to achieve the corresponding final concentrations (Example 1, Comparative Examples 1 to 6).

[0083] [Table 1]

[0084] [Table 2]

[0085] In Table 2, "+" indicates that the component was added to the medium, and "-" indicates that the component was not added to the medium. Furthermore, each compound shown in Table 3 was added to the above medium to give the corresponding final concentration.

[0086] [Table 3]

[0087] After the human fibroblasts reached 80-90% confluence, the medium in the dish was replaced with each of the serum-free media described above, and the human fibroblasts were cultured in the medium for 3 weeks. The medium was replaced every 3 days.

[0088] (3) RNA isolation and QRT-PCR To quantify gene expression, total RNA was extracted from fibroblasts treated with each compound using FastGene® RNA basic kit (Nihon Genetics). After reverse transcription using ReverTraAce® PCR RT Master Mix with gDNA Remover (Toyobo), real-time PCR analysis was performed using Power SYBR Green PCR Master Mix (Thermo Fisher Scientific). Reactions were performed under the following conditions, with three reactions per level: 95°C for 10 minutes, followed by 40 cycles of 95°C for 15 seconds and 60°C for 60 seconds. All results were normalized to the amount of Tbp mRNA. The primers used in QRT-PCR are shown in Table 4 below.

[0089] [Table 4]

[0090] (4) Results The experimental results are shown in Figures 1 and 2. In addition, throughout the figures, the data are shown as mean ± SD (n = 3), and "***", "**", and "*" indicate significant differences at p<0.001, p<0.01, and p<0.05, respectively, according to the Student's t-test.

[0091] The medium composition added in Example 1 was a combination of triiodothyronine, dexamethasone, IBMX, insulin, 2PAA, L,O-Alb, and penicillin / streptomycin. Comparative Examples 1 to 5 were obtained by excluding any one of triiodothyronine, dexamethasone, IBMX, insulin, and 2PAA from this combination. Comparative Example 6 did not include any of the components (all components shown in Table 1) in the medium composition. 1 and 2, both Ucp1 and Fabp4 are significantly expressed in Example 1, whereas their expression is insufficient in the Comparative Example. In particular, in the combination of the compositions of Comparative Examples 1 and 2, Ucp1 is hardly expressed.

[0092] Therefore, it was demonstrated that the combination of Example 1 is preferable as components of a medium for inducing differentiation of human fibroblasts into brown adipocytes.

[0093] [Test Example B] Study of the composition for maintenance medium or maintenance medium (1)Cell culture The same human fibroblasts as those in Test Example A above were cultured under the same conditions until they became 80 to 90% confluent.

[0094] (2) Induction of differentiation of human fibroblasts into brown adipocytes The serum-free medium used for inducing differentiation into brown fat cells (serum-free brown fat induction medium) was prepared by adding the same components as in Example 1 to DMEM medium (Cat#: 11965092; manufactured by Gibco) (hereinafter referred to as "SFBAM"), and further adding the compounds shown in Table 3.

[0095] After the human fibroblasts reached 80-90% confluence, the medium in the dish was replaced with serum-free induction medium SFBAM (containing all the compounds shown in Table 3), and the human fibroblasts were cultured in the medium for 3 weeks. The medium was replaced every 3 days. Each induction cell was further cultured for 1 week in a serum-free maintenance medium containing all the components shown in Table 5 (Example 2), or in a serum-free maintenance medium containing at least rosiglitazone (Examples 3-6, Comparative Examples 7 and 8) to mature the chemically induced brown adipocyte-like cells (ciBAs). The maintenance medium was prepared by adding each component shown in Table 5 below to DMEM medium (Cat#: 11965092; Gibco) in the combination shown in Table 6 so as to reach the final concentration.

[0096] [Table 5]

[0097] [Table 6]

[0098] In Table 6, "+" indicates that the component was added to the medium, and "-" indicates that the component was not added to the medium.

[0099] (3) RNA isolation and QRT-PCR RNA isolation and PCR analysis were performed as in Test Example A above.

[0100] (4) Results The experimental results are shown in Figures 3 and 4. The medium composition added in Examples 2 and 3 is a combination of triiodothyronine, dexamethasone, IBMX, insulin, 2PAA, L,O-Alb, and penicillin / streptomycin. In Examples 4 to 6 and Comparative Examples 7 and 8, any one of triiodothyronine, dexamethasone, IBMX, insulin, and 2PAA is omitted from this combination. 3 and 4, brown adipocyte-specific Ucp1 is significantly expressed in the serum-free maintenance media of Examples 2 to 6. Dexamethasone and insulin were omitted from the serum-free maintenance media of Comparative Examples 7 and 8, respectively, and although Fabp4 specific to all adipocytes was expressed to some extent in these Comparative Examples, Ucp1 was hardly expressed.

[0101] Therefore, it was shown that a combination of dexamethasone, insulin, L,O-Alb and penicillin / streptomycin in addition to rosiglitazone is preferable as a component of a maintenance medium for brown adipocytes.

[0102] [Test Example C] Evaluation of the composition or medium according to the present invention (1)Cell culture The same human fibroblasts as those in Test Example A above were cultured under the same conditions.

[0103] (2) Induction of differentiation of human fibroblasts into brown adipocytes After the human fibroblasts reached 80-90% confluence, the medium in the dish was replaced with serum-free induction medium SFBAM (containing all the compounds shown in Table 3), and the human fibroblasts were cultured in the SFBAM for 4 weeks. The medium was replaced every 3 days. The brown adipocytes obtained at this stage were first subjected to immunostaining (described below) (Example 7). The induced cells were then cultured for another week in serum-free brown fat 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 mature the chemically induced brown fat cell-like cells (ciBAs). The matured brown fat cells were also subjected to immunostaining (described below) (Example 8).

[0104] [Table 7]

[0105] (3) Immunostaining Immunocytochemistry was performed as previously reported (Dai P, et al. J Clin Biochem Nutr. 2015;56(3):166-70). First, cells were fixed with 4% paraformaldehyde (Nacalai Tesque). After washing three times with phosphate-buffered saline (PBS), cells were incubated with PBS containing 0.1% Triton X-100 for 10 min. Then, cells were blocked with PBS containing 3% skim milk for 1 h at room temperature. UCP-1 antibody (ab10983, Abcam, Cambridge, UK) was diluted 1 / 500 in blocking solution. Cells were incubated with the antibody overnight at 4°C. After washing three times with PBS, cells were incubated with Alexa Fluor 488 Donkey anti-Rabbit IgG (A-21206, Thermo Fisher Scientific) for 1 h at room temperature. Cell nuclei were stained with DAPI solution (Dojindo Laboratories). All images were acquired using a fluorescence microscope (Axio Vert.A1, Carl Zeiss, Oberkochen, Germany). Mitochondria were stained with MitoTracker® Red CMXRos (Thermo Fisher Scientific).

[0106] (4) Results The experimental results are shown in Figure 5. As shown in Figure 5, sufficient direct differentiation induction (direct conversion) of human fibroblasts to brown adipocytes was observed in the serum-free induction medium SFBAM. In addition, brown adipocytes with more lipid droplets were observed by incubating in the serum-free maintenance medium SFBAMaM for one week after four weeks of induction in SFBAM.

[0107] [Test Example D] Examination of induction period for brown fat cells In the same manner as in Test Example C, after the human fibroblasts reached 80-90% confluence, the medium in the dish was replaced with serum-free induction medium SFBAM (containing all the compounds shown in Table 3), and the human fibroblasts were cultured in the SFBAM. The culture experiment in SFBAM containing all the compounds shown in Table 3 was performed as Example 9. The medium was replaced every 3 days. Then, the expression levels of multiple marker genes specific to adipocytes and brown adipose tissue, shown in Table 8 below, were quantified by QRT-PCR. The results are shown in FIG. 6. In the figure, "C" indicates the case where the cells were cultured for 5 weeks in the same manner without containing the compounds shown in Table 3 (compounds necessary for induction into brown adipocytes), and "Ro" indicates the case where the cells were cultured for 5 weeks in the same manner with only rosiglitazone among the compounds shown in Table 3. The genes shown in FIG. 6A are specific to brown adipocytes, and the genes shown in FIG. 6B are specific to adipocytes in general.

[0108] [Table 8]

[0109] As shown in Figure 6, when all the compounds shown in Table 3 were included, all genes began to be expressed within 1 week of culture, and the expression levels continued to increase until the 3rd or 4th week. In consideration of this result, it is considered that brown adipocytes induced to differentiate from fibroblasts by the present invention began to appear within 1 week, and continued to increase until the 3rd or 4th week.

[0110] The medium was then replaced with SFBAMaM, the maintenance medium of the present invention, and the induced cells were allowed to mature for one week. This maintenance culture experiment in SFBAMaM was designated Example 10. The results are shown in Figure 7. The meanings of "C" and "Ro" in the figure are the same as in Figure 6, but the medium was similarly replaced with SFBAMaM and cultured for one week. Similarly, the genes shown in Figure 7C are specific to brown adipocytes, and the genes shown in Figure 7D are specific to adipocytes in general. As shown in Figure 7, brown adipocytes induced in SFBAM containing all of the compounds shown in Table 3 were matured in SFBAMaM for one week, and without the need to induce new brown adipocytes, the expression levels of brown adipocytes and adipocyte-specific genes were maintained without significant reduction, with a minimal medium composition.

[0111] [Test Example E] Examination of the presence or absence of fatty acids in serum-free medium After the human fibroblasts reached 80-90% confluence, the medium in the dish was replaced with serum-free induction medium SFBAM or SFBAM in which the albumin component or fatty acid component was changed to the combination shown in Table 10 of the compounds shown in Table 9 (all containing all the compounds shown in Table 3), and the human fibroblasts were cultured in each medium for 3 weeks (Examples 11-14). The medium was replaced every 3 days. Thereafter, the expression levels of marker genes Ucp1 and Ckmt1 specific to brown adipose tissue were quantified by QRT-PCR. The results are shown in Figures 8 and 9. Symbol 1 (control) in the figure represents the result in SFBAM that does not contain the compounds shown in Table 3 required for induction into brown adipocytes.

[0112] [Table 9]

[0113] [Table 10]

[0114] In Table 10, "+" indicates that the component was added to the medium, and "-" indicates that the component was not added to the medium. As can be seen from Figures 8 and 9, induction under fatty acid-free conditions demonstrated an increased expression level of marker genes specific to brown adipose tissue. [Industrial Applicability]

[0115] The induction medium of the present invention (eg, SFBAM) is useful as a differentiation-inducing medium for human fibroblasts into brown adipocytes, and the maintenance medium of the present invention (eg, SFBAMaM) is useful as a maintenance medium for brown adipocytes.

Claims

1. A serum-free medium composition used for inducing differentiation of fibroblasts into brown adipocytes, comprising the following seven components: 1: Triiodothyronine in the range of 0.01 μmol / L to 1 μmol / L 2: Dexamethasone in the range of 0.1 μmol / L to 10 μmol / L 3: Isobutylmethylxanthine in the range of 10 μmol / L to 0.5 mmol / L 4: Insulin in the range of 100 ng / mL to 20 μg / mL 5: L-ascorbic acid 2-phosphate in the range of 10 μg / mL to 250 μg / mL 6: Albumin within the range of 0.5 mg / mL to 4 mg / mL (limited to albumin bound to linoleic acid and oleic acid or fatty acid-unbound albumin) 7: Antibiotics within the range of 50 units / mL to 200 units / mL (limited to penicillin and / or streptomycin)

2. 2. The differentiation-inducing medium composition according to claim 1, wherein the differentiation is directly induced into fibroblasts without introducing a gene into the fibroblasts.

3. The differentiation-inducing medium composition according to claim 1 or 2, further comprising a selective PPARγ agonist, or a selective PPARγ agonist and a cAMP inducer and / or BMP7.

4. The differentiation-inducing medium composition according to claim 3, wherein the selective PPARγ agonist is rosiglitazone, or the cAMP inducer is forskolin.

5. A differentiation-inducing medium comprising the composition for a differentiation-inducing medium according to any one of claims 1 to 4, and which is serum-free.

6. The differentiation-inducing medium according to claim 5, wherein the composition for differentiation-inducing medium according to any one of claims 1 to 4 is added to a DMEM medium.

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

8. 8. The differentiation-inducing medium according to claim 6, wherein the DMEM medium contains L-glutamine and pyruvate.

9. A serum-free medium composition for maintaining brown adipocytes produced by inducing differentiation from fibroblasts, the maintenance medium composition comprising the following five components: 1: Rosiglitazone in the range of 0.5 μmol / L to 5 μmol / L 2: Dexamethasone in the range of 0.1 μmol / L to 10 μmol / L 3: Insulin in the range of 100 ng / mL to 20 μg / mL 4: Albumin within the range of 0.5 mg / mL to 4 mg / mL (limited to albumin bound to linoleic acid and oleic acid or fatty acid-unbound albumin). 5: Antibiotics within the range of 50 units / mL to 200 units / mL (limited to penicillin and / or streptomycin)

10. A maintenance medium comprising the composition for a maintenance medium according to claim 9 and being serum-free.

11. The maintenance medium according to claim 10, wherein the composition for a maintenance medium according to claim 9 is added to a DMEM medium.

12. The maintenance medium of claim 11 , wherein the DMEM medium is a high glucose DMEM medium.

13. The maintenance medium according to claim 11 or 12, wherein the DMEM medium contains L-glutamine.

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