Method for producing neuronal cells from dedifferentiated adipocytes derived from mammal

Abstract

The present invention relates to a method for producing neuronal cells from dedifferentiated adipocytes derived from a mammal, the method comprising a step for culturing dedifferentiated adipocytes derived from a mammal using a medium that contains a basic fibroblast growth factor (bFGF), retinoic acid, and a neuronal differentiation inducer, but does not substantially contain an epigenetic inhibitor.

Description

Method for producing nerve cells from dedifferentiated adipocytes derived from mammals

[0001] The present invention relates to a method for producing nerve cells from dedifferentiated adipocytes derived from mammals, and to a culture medium for inducing differentiation of dedifferentiated adipocytes derived from mammals into nerve cells.

[0002] Central nervous system diseases caused by central nervous system disorders, such as cerebral infarction (including chronic cerebral infarction), spinal cord injury, and Parkinson's disease, cause motor and sensory disorders and reduce patients' quality of life. However, central nervous system function involves complex neural circuits, and it is known that these circuits have a low ability to recover.

[0003] In recent years, the development of treatments aimed at restoring neural function by transplanting neural cells generated using pluripotent stem cells such as human iPS cells has been progressing. However, when using human iPS cells to generate neural cells, the necessary genetic manipulation carries risks such as cell tumorigenesis, genetic mutation, and excessive immune response. Therefore, there is a need for a method for easily and efficiently producing neural cells without genetic manipulation outside of a living body.

[0004] The present inventors have previously developed a method for producing neurons from mammalian-derived dedifferentiated adipocytes using a medium containing an epigenetic inhibitor (Patent Document 1). In the method disclosed in Patent Document 1, a medium containing basic fibroblast growth factor (bFGF), retinoic acid, a neuronal differentiation inducer, and an epigenetic inhibitor is used as essential components in order to control the characteristics of dedifferentiated adipocytes. Furthermore, it is known that epigenetic factors such as DNA methylation, histone modification, and chromatin remodeling are involved in adipocyte differentiation (Non-Patent Document 1).

[0005] Japanese Patent Application Laid-Open No. 2019-103423

[0006] Lee, Ji-Eun et al. “Transcriptional and Epigenomic Regulation of Adipogenesis.” Molecular and cellular biology vol. 39,11 e00601-18. 14 May. 2019, doi:10.1128 / MCB.00601-18

[0007] The nerve cells obtained by the method described in Patent Document 1 have room for improvement in the nerve cell-specific functions of the cells. An object of the present invention is to provide a method for easily and efficiently producing nerve cells having nerve cell-specific functions.

[0008] As a result of extensive research, the present inventors discovered that, while epigenetic inhibitors are an essential component of the method described in Patent Document 1, differentiation of mammalian-derived dedifferentiated adipocytes into neurons can be induced by culturing them in a medium substantially free of epigenetic inhibitors, and thus completed the present invention. The present invention is as follows: [1] A method for producing neurons from mammalian-derived dedifferentiated adipocytes, comprising culturing mammalian-derived dedifferentiated adipocytes in a medium containing basic fibroblast growth factor (bFGF), retinoic acid, and a neuronal differentiation inducer, and substantially free of epigenetic inhibitors. [2] The method described in [1], wherein the neuronal differentiation inducer is ISX9. [3] The method described in [1] or [2], wherein the neuronal differentiation inducer is at a concentration of 50 μM or less. [4] The method described in any of [1] to [3], wherein the mammal is a human. [5] A culture medium for inducing differentiation from mammalian-derived dedifferentiated adipocytes into neurons, comprising basic fibroblast growth factor (bFGF), retinoic acid, and a neuronal differentiation inducer, and substantially free of epigenetic inhibitors. [6] The culture medium according to [5], wherein the neuronal differentiation inducer is ISX9. [7] The culture medium according to [5] or [6], wherein the neuronal differentiation inducer has a concentration of 50 μM or less. [8] A kit for inducing differentiation from mammalian-derived dedifferentiated adipocytes into neurons, comprising the culture medium according to any one of [5] to [7].

[0009] According to the present invention, nerve cells having nerve cell-specific functions can be produced simply and efficiently.

[0010] Figure 1A is a graph showing the expression level of mRNA for the neuronal marker NF-L in cells (number of cells = 3) cultured using a differentiation-inducing medium. ct, ABI, and ABIB represent control, NA medium + retinoic acid + bFGF + ISX9, and ABI + IBET151, respectively. The numbers 0, 7, 14, and 21 following ct, ABI, and ABIB represent the number of days elapsed since the start of culture (the same applies to Figure 1B). Figure 1B is a graph showing the expression level of mRNA for the neuronal marker NF-H in cells (number of cells = 3) cultured using a differentiation-inducing medium. Figure 2A shows the intracellular Ca2+ response to depolarization stimulation in cells cultured using a differentiation-inducing medium (on the 21st day of culture). ２＋ Figure 2B shows the change in intracellular Ca concentration upon stimulation with acetylcholine in cells cultured using a differentiation-inducing medium (21 days after culture). ２＋ 2C is a graph showing the change in intracellular Ca concentration upon stimulation with dopamine in cells cultured using a differentiation-inducing medium (on the 21st day of culture). ２＋ Figure 3A shows the change in intracellular Ca concentration upon stimulation with acetylcholine in atropine (a muscarinic receptor antagonist)-treated (gray) and untreated (black) neurons. ２＋ Figure 3B shows the time course of changes in intracellular Ca concentration in neurons treated with a D1 receptor antagonist (gray) and untreated (black). ２＋ 1 is a graph comparing changes in concentration over time.

[0011] [Method for Producing Neurons from Dedifferentiated Adipocytes Derived from Mammal] The method for producing neurons from dedifferentiated adipocytes derived from mammals according to this embodiment is a production method comprising a step of culturing dedifferentiated adipocytes derived from mammals (hereinafter, sometimes referred to as a "culture step") using a medium (hereinafter, sometimes referred to as a "differentiation-inducing medium") containing the following components (a) to (c) and substantially free of epigenetic inhibitors: (a) basic fibroblast growth factor (bFGF); (b) retinoic acid; and (c) a neuronal differentiation inducer.

[0012] According to the production method of this embodiment, by using the differentiation-inducing medium described above, nerve cells having functions specific to nerve cells can be produced simply and efficiently.

[0013] As used herein, "neuron-specific function" refers to the expression of mRNA for a neuron marker (e.g., neurofilament L (NF-L), neurofilament H (NF-H), etc.) and sensitivity to neurotransmitters (acetylcholine, dopamine, etc.). The degree of neuron-specific function can be determined by the expression level of the neuron marker mRNA. For example, the higher the expression level of the neuron marker mRNA, the more neuron-specific function can be determined to be. In addition to the above, "neuron-specific function" may also be confirmed by the expression of a marker previously known as a neuron marker (e.g., microtubule-associated protein 2 (MAP2)), or sensitivity to neurotransmitters.

[0014] As used herein, the phrase "substantially free" of an epigenetic inhibitor in a differentiation-inducing medium means that the differentiation-inducing medium is not prevented from exhibiting epigenetic activity by the addition of an epigenetic inhibitor during the culture process. Therefore, it does not preclude the addition of an epigenetic inhibitor at a concentration that does not exert its inhibitory effect. That is, when a differentiation-inducing medium is "substantially free" of an epigenetic inhibitor, it means that the epigenetic inhibitor may be contained in a range or concentration that does not exert epigenetic inhibitory activity during the culture process. For example, the concentration of the epigenetic inhibitor in the differentiation-inducing medium may be less than 0.1 μM, 0.05 μM or less, or 0.01 μM or less. Furthermore, the phrase "substantially free" of an epigenetic inhibitor in a differentiation-inducing medium may mean that the differentiation-inducing medium is completely free of an epigenetic inhibitor. The phrase "substantially free" of an epigenetic inhibitor in a differentiation-inducing medium may mean that the differentiation-inducing medium contains an epigenetic inhibitor at a concentration of less than 0.1 μM, 0.05 μM or less, or 0.01 μM or less. In the production method of this embodiment, a differentiation-inducing medium substantially free of an epigenetic inhibitor is used. However, the inventors of the present invention continued to closely observe the reprogramming performed using a differentiation-inducing medium containing an epigenetic inhibitor in a conventional method, and observed that cell survival was somewhat poor. The inventors suspected that this result was due to the influence of the epigenetic inhibitor. Since epigenetic modifications regulate various gene expression in addition to cell reprogramming, they performed reprogramming using a differentiation-inducing medium that did not contain an epigenetic inhibitor, even though it was added in the conventional method, and found that neurons with neuron-specific functions could be produced simply and efficiently.

[0015] As shown in the examples below, neurons obtained by the manufacturing method of this embodiment have been confirmed to have neuron-specific functions. In particular, neurons obtained by the manufacturing method of this embodiment are responsive to dopamine and acetylcholine, and therefore may be dopaminergic neurons or acetylcholinergic neurons. That is, neurons obtained by the manufacturing method of this embodiment may be dopaminergic neurons, acetylcholinergic neurons, or dopaminergic and acetylcholinergic neurons. Neurons obtained by the manufacturing method of this embodiment may be central system neurons. This embodiment also provides a method for screening drugs corresponding to neuronal activity using the obtained neurons. Furthermore, neurons can be mass-produced by producing a large number of differentiated adipocytes derived from individual adipocytes and using the manufacturing method of this embodiment. Furthermore, since dopamine and acetylcholine are neurotransmitters, neurons obtained by the manufacturing method of this embodiment may be used as customized regenerative medicine drugs for diseases related to the dopamine and acetylcholine transmission pathways, taking advantage of their agonism toward these substances. They may also be used as a cell source for custom-made regenerative medicine for diseases of the central nervous tissue (for example, cerebral infarction (including chronic cerebral infarction), spinal cord injury, Parkinson's disease, etc.).

[0016] <Culturing Step> The culturing step is a step of culturing dedifferentiated adipocytes using a differentiation-inducing medium.

[0017] The culture temperature can be, for example, 25°C or higher and 40°C or lower, for example, 30°C or higher and 37°C or lower.

[0018] The culture time can be, for example, 10 days or more and 30 days or less, for example, 14 days or more and 28 days or less.

[0019] CO in the culture process ２ The concentration is, for example, 5% CO ２ It can be said that:

[0020] <Dedifferentiated Adipocytes> The dedifferentiated adipocytes used in the culture process are obtained by dedifferentiating mature adipocytes using a known method developed by the present inventors (see, for example, Japanese Patent Nos. 5,991,687 and 5,055,613). Specifically, mature adipocytes are first isolated from adipose tissue by collagenase treatment or the like. The isolated mature adipocytes are then cultured by the ceiling culture method to obtain dedifferentiated adipocytes.

[0021] In this embodiment, the dedifferentiated adipocytes are derived from mammals, such as humans, monkeys, dogs, cats, chickens, rabbits, pigs, cows, goats, sheep, mice, rats, guinea pigs, hamsters, etc. Among these, humans are preferred as mammals.

[0022] Dedifferentiated adipocytes may be cultured in advance in a low-nutrient medium to starve them. Specific examples of low-nutrient medium include Neurobasal®-A Medium (manufactured by Thermo Fisher) (sometimes abbreviated as "NA medium" herein) containing 2% B-27 Serum-Free Supplement (manufactured by Thermo Fisher). This allows for highly efficient induction of differentiation into neurons.

[0023] [Differentiation-inducing medium] The differentiation-inducing medium is a medium containing the following components (a) to (c) and substantially free of epigenetic inhibitors:

[0024] (a) Basic fibroblast growth factor (bFGF) Generally, "basic fibroblast growth factor (bFGF)" is also called FGF2 and has two isoforms: low molecular weight (LWL) and high molecular weight (HWL). Low molecular weight FGF2 is mainly present in the cytoplasm and acts in an autocrine manner. On the other hand, high molecular weight FGF2 is present in the nucleus and exhibits activity via an intracellular intracrine mechanism. The bFGF contained in the differentiation-inducing medium may be either the low molecular weight type or the high molecular weight type.

[0025] The bFGF may be derived from an animal of the same species as the mammal from which the dedifferentiated adipocytes are derived, or from an animal of a different species, but is preferably derived from an animal of the same species as the mammal from which the dedifferentiated adipocytes are derived. For example, when dedifferentiated adipocytes derived from humans are used, it is preferable to use bFGF derived from humans.

[0026] The concentration of bFGF contained in the differentiation-inducing medium is preferably 10 ng / mL or more and 500 ng / mL or less, more preferably 30 ng / mL or more and 300 ng / mL or less, and even more preferably 50 ng / mL or more and 150 ng / mL or less.

[0027] (b) Retinoic Acid Generally, "retinoic acid" is a metabolite of vitamin A (retinol) and is known to mediate the functions of vitamin A, which is necessary for growth and development.

[0028] Retinoic acid may be in the cis form or the trans form, and is preferably an all-trans isomer in which all double bonds are in the trans form (i.e., tretinoin).

[0029] The concentration of retinoic acid contained in the differentiation-inducing medium is preferably 1 μM or more and 100 μM or less, more preferably 3 μM or more and 50 μM or less, and even more preferably 5 μM or more and 15 μM or less.

[0030] (c) Neuronal Differentiation Inducer Although there are no particular limitations on the neuronal differentiation inducer, it is preferably a low-molecular-weight compound having an isoxazole ring and having the ability to induce neuronal differentiation. Here, "neuronal differentiation inducer" refers to the property of inducing differentiation of dedifferentiated adipocytes into neurons having the above-mentioned functions.

[0031] Specific examples of such low molecular weight compounds include ISX9 and its derivatives. Among them, ISX9 is preferred as a neuronal differentiation inducer. ISX9 is a compound also known as isoxazole 9, CAS No. 832115-62-5.

[0032] The concentration of the neuronal differentiation inducer contained in the differentiation-inducing medium is preferably 50 μM or less, more preferably 5 μM to 50 μM, and even more preferably 10 μM to 30 μM. The concentration of the neuronal differentiation inducer may be 15 μM to 25 μM.

[0033] In particular, by keeping the concentration of the neuronal differentiation inducer contained in the differentiation-inducing medium at 50 μM or less, it is possible to efficiently induce dedifferentiated adipocytes into neurons while maintaining a high cell viability.

[0034] (d) Other Components In addition to the components (a) to (c) above, the differentiation-inducing medium may further contain (d) other components, as long as it does not substantially contain epigenetics inhibitors.

[0035] The other components are not particularly limited, and examples thereof include components necessary for cell survival and proliferation (inorganic salts, carbohydrates, hormones, essential amino acids, non-essential amino acids, vitamins, etc.).

[0036] The inorganic salts contained in the differentiation-inducing medium are not particularly limited, and may be used, for example, to help maintain the osmotic equilibrium of the cells and to help regulate the membrane potential.

[0037] The inorganic salt is not particularly limited, and examples thereof include salts of calcium, copper, iron, magnesium, potassium, sodium, zinc, etc. The salt is not particularly limited, and is used in the form of, for example, chloride, phosphate, sulfate, nitrate, bicarbonate, etc.

[0038] The osmolality of the differentiation-inducing medium is not particularly limited, and may be, for example, 200 mOsm / kg or more and 400 mOsm / kg or less, 290 mOsm / kg or more and 350 mOsm / kg or less, 280 mOsm / kg or more and 310 mOsm / kg or less, or 280 mOsm / kg or more and less than 300 mOsm / kg (specifically, 280 mOsm / kg).

[0039] The carbohydrate is not particularly limited, and examples thereof include glucose, galactose, maltose, and fructose.

[0040] The concentration of carbohydrate (preferably D-glucose) in the differentiation-inducing medium is not particularly limited, and is preferably, for example, 0.5 g / L or more and 2 g / L or less.

[0041] The amino acid is not particularly limited, and examples thereof include L-alanine, L-arginine, L-asparagine, L-aspartic acid, L-cysteine, L-cystine, L-glutamic acid, L-glutamine, L-glycine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-proline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine, and combinations thereof.

[0042] The concentration of glutamine contained in the differentiation-inducing medium is not particularly limited and may be, for example, 0.05 g / L to 1 g / L (usually 0.1 g / L to 0.75 g / L). The concentrations of each amino acid other than glutamine contained in the differentiation-inducing medium are not particularly limited and may be, for example, 0.001 g / L to 1 g / L (usually 0.01 g / L to 0.15 g / L). The amino acids may be synthetically derived.

[0043] The vitamins are not particularly limited, and examples thereof include thiamine (vitamin B1), riboflavin (vitamin B2), niacinamide (vitamin B3), D-hemicalcium pantothenate (vitamin B5), pyridoxal / pyridoxamine / pyridoxine (vitamin B6), folic acid (vitamin B9), cyanocobalamin (vitamin B12), ascorbic acid (vitamin C), calciferol (vitamin D2), DL-α-tocopherol (vitamin E), biotin (vitamin H), menadione (vitamin K), choline chloride, and myo-inositol.

[0044] The differentiation-inducing medium may further contain antibiotics, serum, hormones, or the like.

[0045] The antibiotics are not particularly limited and include, for example, those commonly used in culturing animal cells, such as gentamicin, amphotericin, ampicillin, minomycin, kanamycin, penicillin, streptomycin, gentamicin, tylosin, aureomycin, etc. These antibiotics may be contained alone or in combination.

[0046] The concentration of the antibiotic contained in the differentiation-inducing medium is not particularly limited, and may be, for example, 0.1 μg / mL or more and 100 μg / mL or less.

[0047] The serum is not particularly limited, and examples thereof include FBS / FCS (Fetal Bovine / Calf Serum), NCS (Newborn Calf Serum), CS (Calf Serum), and HS (Horse Serum).

[0048] The concentration of serum contained in the differentiation-inducing medium is not particularly limited, and may be, for example, 2% by mass or more and 10% by mass or less.

[0049] The hormone is not particularly limited, and examples thereof include insulin, glucagon, triiodothyronine, adrenocortical hormones (hydrocortisone, etc.), etc. These hormones may be contained alone or in combination.

[0050] The concentration of the hormone contained in the differentiation-inducing medium is not particularly limited, and may be, for example, 1 ng / mL or more and 10 μg / mL or less.

[0051] Furthermore, the hormone-containing medium additive is not particularly limited, and for example, bovine pituitary extract (BPE) may be used.

[0052] The differentiation-inducing medium may be prepared by mixing the above components, or may be prepared by adding the above components (a) to (c) and, if necessary, the above component (d) to a medium for culturing nerve cells.

[0053] The basal medium for culturing neurons is not particularly limited, and examples thereof include Neurobasal (registered trademark)-A Medium (manufactured by Thermo Fisher) supplemented with B-27 Serum-Free Supplement (manufactured by Thermo Fisher). The differentiation-inducing medium used in the culture step may contain the basal medium.

[0054] The differentiation-inducing medium used in the culture step is a medium that is substantially free of epigenetic inhibitors. Epigenetic inhibitors are not particularly limited, and examples include low-molecular-weight compounds with epigenetic inhibitory activity. Examples of epigenetic inhibitors include the compounds described in JP 2019-103423 A. Among these, epigenetic inhibitors include DNMT inhibitors, HAT inhibitors, BRD inhibitors, KMT inhibitors, and MKBP inhibitors. Examples of DNMT inhibitors include azacytidine, decitabine, and derivatives thereof. Examples of HAT inhibitors include C646, acetyl-CoA analogs, and derivatives thereof. Examples of BRD inhibitors include IBET-151, IBET-762, and derivatives thereof. Examples of KMT inhibitors include DOT1L, EZH2, and derivatives thereof. Examples of MKBP inhibitors include UNC1215 and derivatives thereof.

[0055] As used herein, the term "epigenetics inhibitory ability" refers to the property of inhibiting chemical modifications of DNA and histones, such as DNA methylation, histone acetylation and methylation, or inhibiting proteins that recognize these chemical modifications. Specific examples of such abilities include inhibiting DNA methyltransferase (DNMT), inhibiting methylated DNA-binding proteins, inhibiting histone acetyltransferase (HAT), promoting histone deacetylase, inhibiting bromodomain protein (BRD), inhibiting histone lysine methyltransferase (KMT), promoting histone lysine demethylase, and inhibiting methylated lysine-binding protein (MKBP).

[0056] [Kit for inducing differentiation of mammalian-derived dedifferentiated adipocytes into neurons] The kit for inducing differentiation of mammalian-derived dedifferentiated adipocytes into neurons of this embodiment includes the above-mentioned "differentiation-inducing medium." This allows for the simple and efficient production of neurons having neuron-specific functions by using the kit for inducing differentiation of mammalian-derived dedifferentiated adipocytes into neurons of this embodiment.

[0057] The kit of this embodiment may further include other components in addition to the differentiation-inducing medium. The other components included in the kit of this embodiment are not particularly limited and include, for example, dedifferentiated adipocytes, a culture medium for dedifferentiated adipocytes, a petri dish, a flask, a plastic bag, etc. The kit of this embodiment may include a basal medium, basic fibroblast growth factor (bFGF), retinoic acid, and a neuronal differentiation inducer. Furthermore, the kit of this embodiment may include, in addition to the differentiation-inducing medium, a package insert explaining protocols and the like.

[0058] The present invention will be described below with reference to examples, but the present invention is not limited to the following examples.

[0059] [Example 1] 1. Preparation of dedifferentiated adipocytes First, human dedifferentiated adipocytes were prepared from human mature adipocytes using a known method (see, for example, Japanese Patent No. 5991687). Specifically, 5 g of adipose tissue collected from the buccal fat pad located under the human buccal mucosa was first dissolved in NaHCO 3 containing collagenase (Type II) (manufactured by SIGMA) at a final concentration of 0.02 w / v%. ３ The cells were placed in Dulbecco's modified Eagle's medium (DMEM) (Sigma) containing collagenase and treated with collagenase. After collagenase treatment, the cells were filtered through a nylon mesh to obtain a cell suspension. The resulting cell suspension was centrifuged at 700 G for 1 minute, and the unicellular fat fraction separated into the upper layer was added to fresh DMEM supplemented with 10% fetal bovine serum (FBS). This was centrifuged three times at 700 G for 1 minute to obtain mature unicellular fat cells.

[0060] The resulting unicellular adipocytes were then transferred to a tissue culture flask (Falcon, 3107), and the flask was completely filled with DMEM supplemented with 20% FBS, 1% penicillin, and 1% streptomycin. The flask was then incubated at 37°C and 5% CO ２ The flask was placed with the bottom facing up in a carbon dioxide culture apparatus adjusted to a gas phase of 95% air, and cultured for 7 days.

[0061] After 4 days of culture, most of the cells firmly adhered to the flask ceiling and transformed into multilocular adipocytes with lipid droplets of various sizes surrounding large lipid droplets. After 6 days of culture, the lipid droplets became even smaller, and many cells were observed to have completely lost lipid droplets and transformed into fibroblast-like cells.

[0062] After 7 days of culture, the medium in the flask was replaced with DMEM supplemented with 20% FBS, and the cells were cultured for 10 days in a carbon dioxide incubator with the cell adhesion surface facing the bottom. The medium was replaced every 4 days. Cells without lipid droplets proliferated actively, and after 10 days of culture, the cells in the flask consisted of only fibroblast-like cells, reaching confluence.

[0063] These fibroblast-like cells have active proliferation ability and the differentiation ability to redifferentiate into fat droplet-containing fat cells in the presence of differentiation inducers such as dexamethasone (DEX), insulin (INS), and isobutylmethylxanthine (IBMX), and were therefore produced as dedifferentiated fat cells derived from unicellular fat cells.

[0064] The dedifferentiated adipocytes were prepared using DMEM (high glucose) supplemented with 1% penicillin and streptomycin and 20% FBS. ６ cells / 5mL / 75-cm ２ The medium was added to the flask to achieve the same concentration as in the flask and cultured. The medium was replaced every 2 to 3 days.

[0065] The cells were subcultured once a week for four passages. After the fourth passage, the cells were washed with 5 mL of PBS. After washing, the cells were treated with 2 mL of trypsin-EDTA at 37°C for 2 minutes. After treatment, the cells were collected and centrifuged at 300 g for 1 minute at room temperature. 1 x 10 ６ cells / 5mL / 75-cm ２ The cells were seeded to the same concentration as in a flask and cultured.

[0066] 2. Preparation of Differentiation-Inducing Medium A differentiation-inducing medium for neurons was prepared by mixing the components to obtain the composition shown in Table 1 below. ABI is a medium containing Neurobasal®-A Medium, B-27 Serum-Free Supplement, bFGF, retinoic acid, and a neuronal differentiation inducer (ISX9) as shown in Table 1, and ABIB is a medium having the same composition as ABI but further containing IBET-151, an epigenetics inhibitor. That is, a medium (ABI) containing retinoic acid, bFGF, and ISX9 in NA medium was used in the examples as the differentiation-inducing medium of this embodiment.

[0067]

[0068] 3. Induction of differentiation of dedifferentiated adipocytes into nerve cells The dedifferentiated adipocytes prepared in "1." were washed with 5 mL of PBS. After washing, the cells were treated with 2 mL of trypsin-EDTA at 37°C for 2 minutes. The treated cells were collected and centrifuged at 300 g for 1 minute at room temperature. 3.0 x 10 ５ The cells were seeded onto a 6-well plate at 100 cells / well and cultured for 24 hours.

[0069] After the culture, the cells were washed with 1 mL of PBS and then cultured in a starved state using 1 mL of 2% B27-containing Neurobasal®-A medium for 24 hours.

[0070] After culturing, the cells were washed with 1 mL of PBS. After washing, the cells were cultured for 21 days using 1 mL of the differentiation-inducing medium prepared in "2." The medium was changed once a week.

[0071] 4. Confirmation of mRNA Expression of Neuronal Markers Total RNA was collected from the cells induced to differentiate in "3." (culture days 0, 7, 14, and 21) using 1 mL of Trizol. The mRNA expression of various neuronal markers (NF-L and NF-H) was confirmed by real-time RT-PCR using the primers shown in Table 2 below. The results are shown in Figures 1A-B. As a control, the expression level of GAPDH mRNA was measured. Figures 1A-B show the relative expression levels of the mRNA of various neuronal markers (NF-L and NF-H) to the expression level of GAPDH mRNA.

[0072]

[0073] 1A-B, the mRNA expression of various neuronal markers increased on days 7, 14, and 21 of culture. In particular, the mRNA expression of various neuronal markers increased significantly on day 14 of culture.

[0074] 5. Intracellular Ca in response to external stimuli ２＋ Confirmation of concentration changes For the cells (cultured on the 21st day) cultured using the differentiation induction medium in "3.", the intracellular Ca concentration was measured before and after depolarization stimulation, acetylcholine stimulation, and dopamine stimulation using calcium imaging.２＋ The concentration change was observed, and the results are shown in Figure 2A (depolarization stimulation), Figure 2B (acetylcholine stimulation), and Figure 2C (dopamine stimulation).

[0075] 2A to 2C, in all of the depolarization stimulation, acetylcholine stimulation, and dopamine stimulation, intracellular Ca ２＋ An increase in concentration was observed.

[0076] From the above, it was confirmed that the cells cultured using the differentiation-inducing medium expressed various neuronal markers on the 21st day of culture, and had functions specific to neuronal cells.

[0077] Test Example 1 Using nerve cells, the effects of various antagonists on acetylcholine or dopamine stimulation were tested.

[0078] Specifically, atropine (a muscarinic receptor antagonist) or a D1 receptor antagonist was added to the neurons obtained in Example 1, and the neurons were cultured for 10 minutes. As a control, cells cultured without the addition of any antagonist were also prepared. Using calcium imaging, the cells to which the muscarinic receptor antagonist had been added were stimulated with acetylcholine, and the cells to which the D1 receptor antagonist had been added were stimulated with dopamine, and the intracellular Ca concentration over time was measured. ２＋ The change in concentration was measured, and the results are shown in Figure 3A (with the addition of atropine (a muscarinic receptor antagonist)) and Figure 3B (with the addition of a D1 receptor antagonist).

[0079] As shown in Figures 3A and 3B, in cells to which atropine (a muscarinic receptor antagonist) or a D1 receptor antagonist was added, the intracellular Ca ２＋ It was confirmed that the increase in concentration was suppressed.

[0080] From the above, it was suggested that neurons obtained by culturing using a differentiation-inducing medium containing bFGF, retinoic acid, and a neuronal differentiation inducer listed in Table 1, but substantially free of epigenetic inhibitors, could be applied to drug screening.

[0081] According to the production method and kit of this embodiment, nerve cells having nerve cell-specific functions can be produced simply and efficiently.

Claims

1. A method for producing neurons from dedifferentiated adipocytes derived from a mammal, comprising the step of culturing the dedifferentiated adipocytes derived from a mammal in a medium containing basic fibroblast growth factor (bFGF), retinoic acid, and a neuronal differentiation inducer, and substantially free of epigenetic inhibitors.

2. The method of claim 1, wherein the neuronal differentiation inducer is ISX9.

3. The method of claim 1, wherein the concentration of the neuronal differentiation inducer is 50 μM or less.

4. The method according to any one of claims 1 to 3, wherein the mammal is a human.

5. A culture medium for inducing differentiation of mammalian-derived dedifferentiated adipocytes into neurons, the culture medium comprising basic fibroblast growth factor (bFGF), retinoic acid, and a neuronal differentiation inducer, and being substantially free of epigenetic inhibitors.

6. The medium according to claim 5, wherein the neuronal differentiation inducer is ISX9.

7. The medium according to claim 5, wherein the concentration of the neuronal differentiation inducer is 50 μM or less.

8. A kit for inducing differentiation of mammalian-derived dedifferentiated adipocytes into nerve cells, comprising the medium according to any one of claims 5 to 7.

Images