JP2019103423A - Method for producing neurons from mammalian-derived dedifferentiated adipocytes, and kit for inducing differentiation of mammalian-derived dedifferentiated adipocytes into neurons - Google Patents

Abstract

To provide methods for producing neurons from mammalian-derived dedifferentiated adipocytes, which is capable of simply and efficiently producing a large number of neurons having a function specific to neurons.SOLUTION: Provided is a method for producing neurons from mammalian-derived dedifferentiated adipocytes which comprises culturing mammalian-derived dedifferentiated adipocytes using a medium containing basic a fibroblast growth factor (bFGF), retinoic acid, a neural differentiation inducer, and an epigenetic inhibitor.SELECTED DRAWING: None

Description

  The present invention relates to a method for producing neural cells from mammalian-derived dedifferentiated adipocytes, and a kit for inducing differentiation of mammalian-derived dedifferentiated adipocytes into neural cells.

  The central nervous system generally has a low ability to recover, and once damaged it does not function autonomously. Therefore, many patients suffer from severe central nervous system diseases such as spinal cord injury and Parkinson's disease.

  For these diseases, neuroregenerative medicine has been attempted in which pluripotent stem cells such as human iPS cells and nerve cells prepared by direct reprogramming technology (for example, see Patent Document 1 etc.) are transplanted. In order to perform neuroregenerative medicine, a basic technology to induce differentiation of underlying cells into neurons in vitro is required. However, human iPS cells and direct reprogramming technology need to carry out gene transfer for generating neural cells. Therefore, it can not exclude the risk of tumorigenesis of cells, unexpected gene mutation, excessive immune response and the like. Therefore, in order to realize neuroregenerative medicine, a method for inducing differentiation into neurons in vitro without gene transfer and a cell source suitable for the method are required.

  On the other hand, the present inventors have so far developed a method for producing dedifferentiated adipocytes in which mature adipocytes derived from animal adipose tissue are dedifferentiated (see, for example, Patent Documents 2 and 3). In addition, Patent Document 3 discloses a method using Dulbecco's modified Eagle's medium (DMEM) containing 1-10 mM β-mercaptoethanol and serum to differentiate this dedifferentiated adipocytes into neurons. There is.

WO 2010/140698 Patent No. 5991687 gazette Patent No. 5055613 gazette

  Although neural cells obtained by the method described in Patent Document 3 have been confirmed to be neural cells by their appearance traits, they are specific to nerve cells such as action potential and sensitivity to neurotransmitters. The function has not been considered. Therefore, there has been a demand for a method that can produce nerve cells having a function specific to nerve cells efficiently and efficiently.

  The present invention has been made in view of the above circumstances, and it is possible to easily and efficiently produce a large number of nerve cells having a function specific to nerve cells from mammalian-derived dedifferentiated adipocytes. Provided are a method for producing neural cells and a kit for inducing differentiation of mammalian-derived dedifferentiated adipocytes into neural cells.

The present invention includes the following aspects.
A method for producing a nerve cell from a mammal-derived dedifferentiated adipocyte according to the first aspect of the present invention comprises a basic fibroblast growth factor (bFGF), a retinoic acid, and a nerve differentiation inducer And a step of culturing mammalian-derived dedifferentiated adipocytes using a medium containing an epigenetics inhibitor.

  In the manufacturing method according to the first aspect, the nerve differentiation inducer may be ISX9.

  In the manufacturing method according to the first aspect, the epigenetics inhibitor may be IBET-151.

  In the manufacturing method according to the first aspect, the concentration of the neural differentiation inducing agent may be less than 50 μM.

  In the method according to the first aspect, the mammal may be a human.

  A kit for inducing differentiation of a mammal-derived dedifferentiated adipocytes into neurons according to the second aspect of the present invention comprises a culture medium for culturing neurons, bFGF, retinoic acid, and an agent for inducing nerve differentiation. And an epigenetics inhibitor.

  In the kit of the second aspect, the nerve differentiation inducer may be ISX9.

  In the kit of the second aspect, the epigenetics inhibitor may be IBET-151.

  In the kit of the second aspect, the mammal may be a human.

  According to the manufacturing method and kit of the above aspect, a large number of nerve cells having a function specific to nerve cells can be easily and efficiently manufactured.

It is a graph which shows a time-dependent change of the relative ratio of the expression level of the mRNA of the various markers (MAP2, NF-L, and Ascl1) of the nerve cell in the cell cultured using the nerve cell differentiation induction medium in Example 1. It is a graph which shows the action potential of the cell (culture 21st day) cultured using the neural cell differentiation induction medium in Example 1. It is an image which shows the change of the intracellular calcium ion concentration before and behind depolarization stimulation in the cell (culture 21st day) cultured using the nerve cell differentiation induction medium in Example 1. FIG. It is an image which shows the change of the intracellular calcium ion concentration before and behind acetylcholine stimulation in the cell (culture 21st day) cultured using the nerve cell differentiation induction medium in Example 1. FIG. It is an image which shows the change of the intracellular calcium ion concentration before and behind dopamine stimulation in the cell (culture 21st day) cultured using the nerve cell differentiation induction medium in Example 1. FIG. It is the graph which compared the relative ratio of the expression level of the mRNA of the nerve cell marker (NF-L) in the cell (culture 14th day) cultured using the culture medium containing the various low molecular weight compounds in Example 2. FIG. 16 is a graph comparing cell viability of cells cultured on the neural differentiation induction medium containing different concentrations of ISX9 in Example 3 (culture day 21). The relative ratio of the expression levels of mRNA of various markers (NF-L and MAP2) of nerve cells in cells cultured on the 14th day of culture using culture medium for inducing differentiation of nerve cells containing different concentrations of ISX9 in Example 3 It is a graph compared. It is the graph which compared the time-dependent change of the intracellular calcium ion concentration by the acetylcholine stimulation in the nerve cell obtained in Example 1 of atropine (muscarinic receptor antagonist) process and untreated in Experiment 1. FIG. It is the graph which compared the time-dependent change of the intracellular calcium ion concentration by the dopamine stimulation in the neuron obtained in Example 1 of D1 receptor antagonist treatment and untreated in Experiment 1. FIG.

<< Method for producing nerve cells from mammalian-derived dedifferentiated adipocytes >>
The method for producing neural cells from mammalian-derived dedifferentiated adipocytes according to this embodiment is a medium containing the following components (a) to (d) (hereinafter referred to as “media for inducing differentiation into neural cells”) It is a manufacturing method provided with the process (Hereinafter, a "culture process" may be called.) Which culture | cultivates a mammal-derived dedifferentiated adipocyte using (it may).
(A) basic fibroblast growth factor (bFGF);
(B) retinoic acid;
(C) nerve differentiation inducer;
(D) Epigenetics inhibitor

  According to the manufacturing method of the present embodiment, a large amount of nerve cells having a function specific to nerve cells can be simply and efficiently manufactured by using the culture medium having the above configuration.

  In the present specification, “a function specific to a nerve cell” means that mRNA of a nerve cell marker (eg, MAP2, NF-L, Ascl1 etc.) is expressed, having an action potential, and , Being sensitive to neurotransmitters (acetylcholine, dopamine etc.).

  As shown in Examples described later, it has been confirmed that nerve cells obtained by the manufacturing method of the present embodiment have a function specific to the above-described nerve cells. In addition, since it does not have nicotine sensitivity, it is thought that the nerve cell obtained by the manufacturing method of this embodiment is a nerve cell of a central system. Therefore, the obtained neural cells can be used for screening of drugs effective for central nervous system cells. In addition, a large amount of differentiated adipocytes can be produced from an individual's adipocytes, and further, a large amount of nerve cells can be produced. Therefore, it can also be used as a cellular source of customized regenerative medicine for diseases of central nervous tissues (eg, spinal cord injury, Parkinson's disease, etc.).

<Culturing process>
The culture step is a step of culturing the dedifferentiated adipocytes using a differentiation induction medium to nerve cells.

  The culture temperature can be, for example, 25 ° C. or more and 40 ° C. or less, and can be, for example, 30 ° C. or more and 37 ° C. or less.

  The culture time can be, for example, 10 days to 30 days, and for example, 14 days to 28 days.

The CO ₂ concentration in the culture step can be, for example, 5% CO ₂ .

[Dedifferentiated adipocytes]
The dedifferentiated adipocytes used in the culture step can be obtained by dedifferentiating mature adipocytes using known methods developed by the present inventors (see, for example, Patent Documents 2 and 3). Specifically, first, mature adipocytes are isolated from adipose tissue by collagenase treatment or the like. Then, dedifferentiated adipocytes are obtained by culturing the isolated mature adipocytes by the ceiling culture method.

  Moreover, the dedifferentiated adipocytes used in the production method of the present embodiment may be those derived from a mammal. Examples of mammals include humans, monkeys, dogs, cats, chickens, rabbits, pigs, cows, goats, sheep, mice, rats, guinea pigs, hamsters and the like. Among them, humans are preferable as mammals.

  In addition, the dedifferentiated adipocytes used in the culture step may be cultured in advance using a low nutrient medium to be starved. Specific examples of the nutrient-poor medium include Neurobasal (registered trademark) -A Medium (manufactured by Thermo Fisher) containing 2% of B-27 Serum-Free Supplement (manufactured by Thermo Fisher). Thereby, differentiation into neural cells can be induced with high efficiency.

[Differentiation induction medium to nerve cells]
The medium for inducing differentiation into neural cells used in the culture step is a medium containing the components (a) to (d) above. Each component will be described in detail below.

((A) Basic fibroblast growth factor (bFGF))
In general, "basic fibroblast growth factor (bFGF)", also called FGF2, has two isoforms, low molecular weight (LWL) and high molecular weight (HWL). The low molecular weight FGF2 is mainly present in the cytoplasm and acts in an autocrine manner (autocrine). On the other hand, high molecular weight FGF2 is in the nucleus and exhibits activity by an intracrine mechanism that acts in cells. The bFGF contained in the medium may be a low molecular weight type or a high molecular weight type.

  bFGF may be derived from an animal of the same species as the mammal from which the dedifferentiated adipocytes are derived, or may be derived from an animal of a different species. Preferably, it is derived from an animal of the same species as the mammal. For example, when using human-derived dedifferentiated adipocytes, it is preferable to use bFGF also from human.

  The concentration of bFGF contained in the medium is preferably 10 μg / mL or more and 500 μg / mL or less, more preferably 30 μg / mL or more and 300 μg / mL or less, and preferably 50 μg / mL or more and 150 μg / mL or less More preferable.

((B) Retinoic acid)
In general, "retinoic acid" is a metabolite of vitamin A (retinol), and is known to mediate the function of vitamin A necessary for growth and development.

  Retinoic acid may be cis or trans. Among them, retinoic acid is preferably an all-trans isomer (ie, tretinoin) in which all double bonds are in trans form.

  The concentration of retinoic acid contained in the medium is preferably 1 μM or more and 100 μM or less, more preferably 3 μM or more and 50 μM or less, and still more preferably 5 μM or more and 15 μM or less.

((C) Neural differentiation inducer)
The nerve differentiation inducer is not particularly limited, but is preferably a low molecular weight compound having an isoxazole ring capable of inducing nerve differentiation.

  The "neural differentiation induction ability" referred to herein means the property of inducing differentiation of dedifferentiated adipocytes into nerve cells having the above-mentioned function.

  Specific examples of such low molecular weight compounds include ISX9 and its derivatives.

  Among them, ISX9 is preferable as a nerve differentiation inducer.

  The concentration of the neural differentiation inducer contained in the medium is preferably less than 50 μM, more preferably 5 μM or more and less than 50 μM, still more preferably 10 μM or more and 20 μM or less, and particularly preferably 20 μM .

  In particular, when the concentration of the neural differentiation inducer contained in the culture medium is less than 50 μM, dedifferentiated adipocytes can be efficiently induced to neural cells while keeping the cell survival rate higher.

((D) Epigenetics inhibitor)
The epigenetics inhibitor is not particularly limited, but is preferably a low molecular weight compound having an isoxazole ring capable of inhibiting epigenetics.

  As used herein, "epigenetics inhibitory ability" refers to the inhibition of DNA methylation, histone acetylation and chemical modification of histones such as methylation, or proteins that recognize such chemical modifications. It means the nature. Specifically, for example, inhibiting DNA methyltransferase (DNMT), inhibiting methylated DNA binding protein, inhibiting histone acetyltransferase (HAT), promoting histone deacetylase To inhibit bromo domain protein (BRD), to inhibit histone lysine methyltransferase (KMT), to promote histone lysine demethylase, to inhibit methylated lysine binding protein (MKBP) And the like.

  Specific examples of such low molecular weight compounds include DNMT inhibitors, HAT inhibitors, BRD inhibitors, KMT inhibitors, MKBP inhibitors and the like.

  DNMT inhibitors include, for example, azacitidine, decitabine and their derivatives.

  Examples of HAT inhibitors include C646, acetyl-CoA analogues 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 the MKBP inhibitor include UNC1215 and derivatives thereof.

  Among them, as an epigenetics inhibitor, it is preferable to be IBET-151.

  The concentration of the epigenetics inhibitor contained in the culture medium is preferably 0.1 μM or more and 10 μM or less, more preferably 0.3 μM or more and 5 μM or less, still more preferably 0.5 μM or more and 3 μM or less And 2 μM are particularly preferred.

((E) Other components)
The medium may further contain (e) other components in addition to the components (a) to (d).

  The other components are not particularly limited, and include components (inorganic salts, carbohydrates, hormones, essential amino acids, non-essential amino acids, vitamins) and the like necessary for survival growth of cells.

  Inorganic salts contained in the medium are intended to help maintain the cell's osmotic balance and to regulate the membrane potential.

  The inorganic salt is not particularly limited and includes, for example, salts of calcium, copper, iron, magnesium, potassium, sodium, zinc and the like. The salts are usually used in the form of chloride, phosphate, sulfate, nitrate and bicarbonate.

  Generally, the osmolality of the medium can be, for example, 200 mOsm / kg or more and 400 mOsm / kg or less, for example, 290 mOsm / kg or more and 350 mOsm / kg or less, such as 280 mOsm / kg or more and 310 mOsm / kg or less. For example, it can be 280 mOsm / kg or more and less than 300 mOsm / kg (specifically, 280 mOsm / kg).

  The carbohydrate is not particularly limited, and examples thereof include glucose, galactose, maltose, fructose and the like.

  Generally, the concentration of carbohydrate (preferably, D-glucose) in the culture medium is preferably 0.5 g / L or more and 2 g / L.

  The amino acids are 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 Etc.

  In general, the concentration of glutamine contained in the medium is 0.05 g / L or more and 1 g / L or less (usually 0.1 g / L or more and 0.75 g / L or less). Each amino acid other than glutamine contained in the medium is 0.001 g / L or more and 1 g / L (usually 0.01 g / L or more and 0.15 g / L or less). The amino acids may be of synthetic origin.

  The vitamins are not particularly limited. For example, thiamine (vitamin B1), riboflavin (vitamin B2), niacinamide (vitamin B3), D-pantothenic acid hemi-calcium, (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, myo-inositol and the like can be mentioned.

  The medium may further contain an antibiotic, serum, hormone or the like.

  Antibiotics include, for example, gentamicin, amphotericin, ampicillin, minomycin, kanamycin, penicillin, streptomycin, gentacin, tylosin, aureomycin etc., which are used for culture of normal animal cells. These antibiotics may be contained singly or in combination of two or more.

  Generally, the concentration of the antibiotic contained in the medium is not particularly limited, and can be, for example, 0.1 μg / mL or more and 100 μg / mL or less.

  Examples of the serum include, but not limited to, FBS / FCS (Fetal Bovine / Calf Serum), NCS (Newborn Calf serum), CS (Calf Serum), HS (Horse Serum), and the like.

  In general, the concentration of serum contained in the medium can be, for example, 2% by mass or more and 10% by mass or less.

  Examples of hormones include insulin, glucagon, triiodothyronine, adrenocortical hormone (hydrocortisone etc.) and the like. These hormones may be contained singly or in combination of two or more.

  Generally, the concentration of hormone contained in the medium is not particularly limited, and can be, for example, 1 ng / mL or more and 10 μg / mL or less.

  In addition, Bovine Pituitary Extract (BPE) may be used as a medium additive containing a hormone.

  The medium for inducing differentiation into neural cells used in the culture step may be prepared by mixing the above-mentioned various components, and usually, the medium for culturing neural cells comprises the components (a) to (d) above; And you may prepare by adding the component etc. of said (e) as needed.

  Examples of the culture medium for culturing nerve cells include Neurobasal (registered trademark) -A Medium (manufactured by Thermo Fisher) to which B-27 Serum-Free Supplement (manufactured by Thermo Fisher) is added.

«A kit for inducing differentiation from mammalian-derived dedifferentiated adipocytes to neurons»
The kit for inducing differentiation of a mammal-derived dedifferentiated adipocytes into neurons according to this embodiment comprises a culture medium for culturing neurons, bFGF, retinoic acid, an agent for inducing differentiation of nerves, and epigenetics inhibition. And an agent.

  According to the kit of the present embodiment, a large number of nerve cells having a function specific to nerve cells can be easily and efficiently produced.

  The details of each component of the kit of the present embodiment will be described below.

<Media for Cultivating Neurons>
Examples of the culture medium for culturing neural cells included in the kit of the present embodiment include the same media as those exemplified in the above-mentioned "Method for producing neural cells from dedifferentiated adipocytes derived from mammals".

<BFGF>
Examples of bFGF contained in the kit of the present embodiment include the same ones as exemplified in the above-mentioned “Method for producing nerve cells from mammalian-derived dedifferentiated adipocytes”.

<Retinoic acid>
Examples of retinoic acid contained in the kit of the present embodiment include the same ones as exemplified in the above-mentioned “Method for producing neural cells from dedifferentiated adipocytes derived from mammals”. Among them, retinoic acid is preferably an all-trans isomer (ie, tretinoin) in which all double bonds are in trans form.

<Neural differentiation inducer>
As the neural differentiation inducer contained in the kit of the present embodiment, the same ones as exemplified in the above-mentioned “Method for producing neural cells from mammalian-derived dedifferentiated adipocytes” can be mentioned. Among them, ISX9 is preferable as a nerve differentiation inducer.

<Epigenetics inhibitor>
Examples of the epigenetics inhibitor contained in the kit of the present embodiment include the same as those exemplified in the above-mentioned “Method for producing nerve cells from dedifferentiated adipocytes derived from mammals”. Among them, as an epigenetics inhibitor, it is preferable to be IBET-151.

<Other configuration>
The kit of the present embodiment may further have other configurations in addition to the above configuration.

  The other components included in the kit of the present embodiment include the same ones as those exemplified as the “other components” in the above-mentioned “method for producing nerve cells from dedifferentiated adipocytes derived from mammals”. .

  By mixing the above-described components contained in the kit of the present embodiment to the concentrations described in “Method for producing nerve cells from dedifferentiated adipocytes derived from mammals” in the above-described “components”, A medium for inducing differentiation into neural cells can be prepared. By using this differentiation induction medium, a large number of nerve cells having a function specific to nerve cells can be easily and efficiently produced from dedifferentiated adipocytes.

  Examples of the dedifferentiated adipocytes cultured using the kit of the present embodiment include the same ones as exemplified in the above-mentioned “Method for producing nerve cells from dedifferentiated adipocytes derived from mammals”. Among them, the dedifferentiated adipocytes are preferably of human origin.

  In addition, it has been confirmed that nerve cells obtained using the kit of the present embodiment have a function specific to the above-described nerve cells. In addition, since it does not have nicotine sensitivity, it is thought that the nerve cell obtained using the kit of this embodiment is a nerve cell of a central system. Therefore, the obtained neural cells can be used for screening of drugs effective for central nervous system cells. In addition, a large amount of differentiated adipocytes can be produced from an individual's adipocytes, and further, a large amount of nerve cells can be produced. Therefore, it can also be used as a cellular source of customized regenerative medicine for diseases of central nervous tissues (eg, spinal cord injury, Parkinson's disease, etc.).

  Hereinafter, the present invention will be described by way of examples, but the present invention is not limited to the following examples.

Example 1
1. Preparation of Dedifferentiated Adipocytes First, human dedifferentiated adipocytes were prepared from human mature adipocytes using a known method (see, for example, Patent Document 2). Specifically, first, collagenase (Type II) (manufactured by SIGMA) was added to a fat tissue of 5 g collected from the buccal fat body located under human buccal mucosa so as to have a final concentration of 0.02 w / v%. It was placed in NaHCO ₃ -containing Dulbecco's modified Eagle's medium (DMEM) (manufactured by SIGMA), and collagenase treatment was performed. After collagenase treatment, it was filtered through a nylon mesh to obtain a cell suspension. The obtained cell suspension is centrifuged at 700 G for 1 minute, the monocellular fat fraction separated in the upper layer is added to fresh DMEM supplemented with 10% FBS, and centrifugation at 700 G is repeated 3 times for 1 minute. Then, mature adipocytes were obtained as mononuclear adipocytes.

Then, the obtained mononuclear adipocytes are transferred to a tissue culture flask (Falcon, 3107), and the inside of the flask is completely filled with DMEM supplemented with 20% FBS, 1% penicillin and streptomycin, 37 ° C., 5% CO ₂ , The flask was allowed to stand for 7 days in a carbon dioxide gas culture apparatus adjusted to a gas phase of 95% air with the bottom of the flask facing up.

  After 4 days of culture, most of the cells firmly adhered to the top surface of the flask, and transformed into multivesicular fat cells having fat droplets of various sizes around large fat droplets. After 6 days of culture, the lipid droplets became further smaller, and a large number of cells were observed to be transformed into fibroblast-like morphology without any lipid droplets.

  After 7 days of culture, the medium in the flask was replaced with DMEM supplemented with 20% FBS, and culture was continued for 10 days in a carbon dioxide gas culture apparatus with the cell-adhesion surface at the bottom. Medium change was performed every 4 days. The cells without lipid droplets proliferated actively, and after 10 days of culture, the cells in the flask became fibroblast-like cells only, and reached confluence.

Since this fibroblast-like cell has an active proliferation ability, and also has the differentiation ability to redifferentiate into fat cells having fat droplets by differentiation inducers such as DEX, INS, IBMX, etc. It was created as a cell-derived dedifferentiated adipocyte.

Then, using 1% penicillin and streptomycin and 20% FBS-added DMEM (high concentration of glucose), the dedifferentiated adipocytes produced are brought to a concentration of 1 × 10 ⁶ cells / 5 mL / 75-cm ² flask. It was added and cultured. Medium change was performed once every two to three days.

The cells were passaged once a week and subcultured four times. Then, after the fourth subculture, the cells were washed with 5 mL of PBS. Then, it was treated at 37 ° C. for 2 minutes with 2 mL of trypsin-EDTA. The treated cells were then harvested and centrifuged at 300 g for 1 minute at room temperature. Then, cells were seeded at a concentration of 1 × 10 ⁶ cells / 5 mL / 75-cm ² flask and cultured.

2. Preparation of Differentiation-Inducing Medium Subsequently, each component was mixed so as to have the composition shown in Table 1 below, and a medium for inducing differentiation into neural cells was prepared.

3. Induction of Differentiation of Dedifferentiated Adipocytes into Neurons Next, the dedifferentiated adipocytes prepared in “1.” were washed with 5 mL of PBS. Then, it was treated at 37 ° C. for 2 minutes with 2 mL of trypsin-EDTA. The treated cells were then harvested and centrifuged at 300 g for 1 minute at room temperature. Then, the cells were seeded in a 6-well plate so as to be 3.0 × 10 ⁵ cells / well and cultured for 24 hours.

  It was then washed with 1 mL PBS. Then, the cells were cultured under starvation for 24 hours using 1 mL of 2% B27-containing Neurobasal®-A medium (hereinafter sometimes abbreviated as “NA medium”).

  It was then washed with 1 mL PBS. Subsequently, it culture | cultivated for 21 days using 1 mL of differentiation induction culture medium produced by "2.". Medium change was performed once a week.

4. Confirmation of mRNA Expression of Neuronal Markers Next, 1 mL of Trizol was used to recover total RNA of cells cultured on a differentiation induction medium at "3." (culture 0, 7, 14 and 21 days) . Subsequently, the expression of mRNA of various markers (MAP2, NF-L and Ascl1) of neural cells was confirmed by real time RT-PCR using the primers shown in Table 2 below. The results are shown in FIG. As a control, the expression level of GAPDH mRNA was measured. In FIG. 1, the expression levels of mRNA of various markers (MAP2, NF-L and Ascl1) of neural cells are expressed as relative ratios with respect to the expression level of mRNA of GAPDH.

  From FIG. 1, on the 14th day of culture, the expression of mRNA of various markers of neural cells was significantly increased.

5. Confirmation of action potential Next, the action potential was measured by the patch clamp method about the cell (culture 21st day) cultured using the differentiation induction culture medium by "3." The results are shown in FIG.

  From FIG. 2, action potentials were observed in cells on day 21 of culture.

6. Confirmation of intracellular Ca ²⁺ concentration change in response to external stimulation Then, using calcium imaging method, depolarization stimulation, acetylcholine stimulation and dopamine are carried out using calcium imaging method for cells cultured using a differentiation induction medium in “3.” Changes in intracellular Ca ²⁺ concentration before and after stimulation were observed. The results are shown in FIG. 3A (depolarization stimulation), FIG. 3B (acetylcholine stimulation) and FIG. 3C (dopamine stimulation).

From FIG. 3A to FIG. 3C, in the depolarization stimulation, acetylcholine stimulation and dopamine stimulation, in the cells after stimulation, an increase in intracellular Ca ²⁺ concentration was observed.

  From the above, the cells cultured using the above-mentioned differentiation induction medium express various markers of nerve cells on the 14th day of culture, and on the 21st day of culture, they have a function specific to nerve cells. It was confirmed to have.

Example 2
Subsequently, using a low molecular weight compound other than ISX9 as a nerve differentiation inducer, it was confirmed whether differentiation into nerve cells could be induced.

1. Preparation of Dedifferentiated Adipocytes Dedifferentiated adipocytes were prepared using the same method as in “1.” of Example 1.

2. Preparation of Differentiation-Inducing Medium A medium was prepared using the low molecular weight compounds shown below instead of ISX9, and the other components were the same as in “2.” of Example 1. In addition, as a control, a medium containing no neural differentiation inducer and a medium containing 20 μM ISX9 were prepared.
SP600125 (manufactured by Sigma-Aldrich)
-SU 5402 (manufactured by Sigma-Aldrich)
-FIPI (manufactured by Sigma-Aldrich)
-SU 6656 (manufactured by Sigma-Aldrich)
-YM 254 890 (manufactured by Sigma-Aldrich)
MK2206 (manufactured by Sigma-Aldrich)
・ GSK 525762 (manufactured by Sigma-Aldrich)
-JW 67 (manufactured by Sigma-Aldrich)
-SKF 86002 (manufactured by Sigma-Aldrich)
-SB 239063 (manufactured by Sigma-Aldrich)
-FR180204 (manufactured by Sigma-Aldrich)
U0126 (manufactured by Sigma-Aldrich)

3. Induction of Differentiation of Dedifferentiated Adipocytes into Neurons Differentiated adipocytes are cultured using the same method as in “3.” of Example 1 except that a medium containing various low molecular weight compounds is used. We tried to induce differentiation into cells.

4. Confirmation of mRNA Expression of Neuronal Marker (NF-L) Subsequently, using the same method as in “4.” of Example 1, the cells cultured in the medium containing various low molecular weight compounds (culture day 14) The expression of the neuronal marker NF-L mRNA was confirmed. The results are shown in FIG.

  From FIG. 4, none of the cells cultured using a medium containing a low molecular weight compound other than ISX9 was able to confirm the expression of NF-L mRNA.

[Example 3]
Then, the optimal concentration of ISX9 for inducing differentiation into neural cells was examined.

1. Preparation of Dedifferentiated Adipocytes Dedifferentiated adipocytes were prepared using the same method as in “1.” of Example 1.

2. Preparation of differentiation induction medium The final concentrations of ISX9 are 0, 1, 3, 5, 5, 10, 20, 50, 100 and 200 μM, and the other components are the same as in “2.” of Example 1. The culture medium was prepared to have a composition.

3. Induction of Differentiation of Dedifferentiated Adipocytes into Neurons Dedifferentiated adipocytes are cultured using the same method as in "3." of Example 1 except that a medium containing different concentrations of ISX9 is used to We tried to induce differentiation into cells.

4. Confirmation of Cell Viability Next, using the same method as in “4.” of Example 1, cells cultured in a medium containing different concentrations (0, 20, 50, 100 and 200 μM) of ISX 9 (culture day 21) Cells were stained using the LIVE / DEAD® Cell Viability Kit and live and dead cells were counted to calculate cell viability. The results are shown in FIG.

  From FIG. 5, it was revealed that cell death was caused and the cell survival rate decreased when the concentration of ISX9 was 50 μM or more.

5. Confirmation of mRNA Expression of Neuronal Markers (NF-L and MAP2) Then, using the same method as in "4." of Example 1, different concentrations (0, 1, 3, 5, 5, 10 and 20 μM) The expression of mRNAs of neural cell markers NF-L and MAP2 was confirmed for cells cultured in a medium containing ISX9 of the above (day 14 of culture). The results are shown in FIG.

  It became clear from FIG. 6 that the expression levels of the nerve cell markers NF-L and MAP2 mRNA increased depending on the concentration of ISX9.

[Test Example 1]
Neurons were used to test the effects of various antagonists on acetylcholine or dopamine stimulation.

  Specifically, atropin (muscarinic receptor antagonist) or D1 receptor antagonist was added to the neurons obtained in Example 1 and cultured for 10 minutes. In addition, as a control, cells cultured without adding various antagonists were also prepared. Then, using calcium imaging, cells to which a muscarinic receptor antagonist has been added are given acetylcholine stimulation, and cells to which a D1 receptor antagonist has been added are given dopamine stimulation to obtain intracellular calcium ion concentration over time. The change was measured. The results are shown in FIG. 7A (with atropine (muscarinic receptor antagonist) added) and FIG. 7B (with D1 receptor antagonist added).

  From FIGS. 7A and 7B, it was confirmed that in cells to which atropine (muscarinic receptor antagonist) or D1 receptor antagonist was added, an increase in intracellular calcium ion concentration due to acetylcholine stimulation or dopamine stimulation was suppressed.

  From the above, it is suggested that neural cells obtained by culturing using the above-mentioned differentiation induction medium may be applicable to drug screening.

  According to the manufacturing method and kit of the present embodiment, a large number of nerve cells having a function specific to nerve cells can be simply and efficiently manufactured.

Claims (9)

A method of producing neural cells from mammalian-derived dedifferentiated adipocytes, which comprises:
Using a medium containing basic fibroblast growth factor (bFGF), retinoic acid, a neural differentiation inducer, and an epigenetics inhibitor, mammalian-derived dedifferentiated adipocytes are obtained A manufacturing method comprising the step of culturing.   The method according to claim 1, wherein the nerve differentiation inducer is ISX9.   The method according to claim 1 or 2, wherein the epigenetics inhibitor is IBET-151.   The method according to any one of claims 1 to 3, wherein the concentration of the nerve differentiation inducer is less than 50 μM.   The method according to any one of claims 1 to 5, wherein the mammal is a human. A kit for inducing differentiation of mammalian-derived dedifferentiated adipocytes into neurons, comprising
A medium for culturing nerve cells,
bFGF,
With retinoic acid,
Nerve differentiation inducer,
An epigenetics inhibitor,
Kit.   The kit according to claim 6, wherein the nerve differentiation inducer is ISX9.   The kit according to claim 6 or 7, wherein the epigenetics inhibitor is IBET-151.   The kit according to any one of claims 6 to 8, wherein the mammal is a human.