JP6876502B2 - Undifferentiated cell remover - Google Patents

Description

The present invention relates to an undifferentiated cell removing agent for removing undifferentiated cells such as stem cells.

When a tissue of the human body is lost, regenerative medicine is performed to restore the function by using stem cells or the like. In regenerative medicine, for example, a method using pluripotent stem cells such as embryonic stem cells (ES cells) and induced pluripotent stem cells (iPS cells) is known. Pluripotent stem cells are cells that have the self-renewal ability to increase the number of the same cells and the pluripotency to differentiate into any cell. For example, differentiated pluripotent stem cells produced from a patient's cells can be used for transplantation to the patient or the like. Among pluripotent stem cells, fertilized eggs are used to produce ES cells, so due to ethical issues, methods using iPS cells are currently attracting worldwide attention.

However, when pluripotent stem cells are induced to differentiate, undifferentiated cells that have not differentiated and remain pluripotent remain in the differentiated cell group, which causes tumorigenesis. In order to utilize pluripotent stem cells for regenerative medicine, it is essential to solve this problem of tumorigenesis.

As a method for removing undifferentiated cells remaining in the cell group in which pluripotent stem cells have been induced to differentiate, for example, a method for obtaining good iPS cells that easily differentiate into the target cells, or a method for obtaining only the target differentiated cells using a cell sorter. There is a way.

In addition, several methods have been reported for selectively removing undifferentiated cells from the cell group in which pluripotent stem cells have been induced to differentiate. For example, Patent Document 1 describes a method for preparing differentiated cells by removing undifferentiated iPS cells, and describes quercetin as an active ingredient for removing undifferentiated iPS cells. Patent Document 2 describes a method for removing stem cells that cause tumorigenesis from a cell source to be transplanted, an undifferentiated cell removing agent for that purpose, and a drug-fused lectin as an active ingredient.

Patent Document 3 describes a method for inducing differentiation of iPS cells while suppressing tumorigenesis, and describes statin as an active ingredient for suppressing tumorigenesis. Patent Document 4 describes a method for removing tumorigenic cells in stem cells, and describes adenovirus as an active ingredient. Patent Document 5 describes a method for separating and obtaining a cell culture consisting of hepatocytes from a cell group containing hepatocytes induced to differentiate from iPS cells and undifferentiated iPS cells, and for culturing the cell group. The medium composition is described.

International Gazette No. 2014/058274 International Gazette No. 2014/126146 International Gazette No. 2013/100080 International Gazette No. 2012/133674 Japanese Unexamined Patent Publication No. 2014-128210

Not limited to the conventional methods described above, there is a demand for the development of undifferentiated cell removing agents that can be used more safely. Therefore, an object of the present invention is to provide a novel undifferentiated cell removing agent that can be used more safely.

As a result of diligent studies to solve the above problems, the present inventors have found that arctigenin does not affect the survival of differentiated human skin fibroblasts, while iPS cells are undifferentiated cells. The present invention has been completed by newly finding that it can be selectively removed.

The present invention provides an undifferentiated cell remover containing arctigenin as an active ingredient.

The present invention also provides the above-mentioned undifferentiated cell removing agent for removing undifferentiated stem cells remaining in the cell group in which stem cells have been induced to differentiate.

The present invention also provides an undifferentiated cell removing agent in which the stem cells are iPS cells.

The present invention also provides the above-mentioned undifferentiated cell removing agent further containing a glucose metabolism antagonist.

The present invention also provides a kit for removing undifferentiated cells, which comprises any of the above-mentioned undifferentiated cell removing agents and a low glucose medium.

The present invention also provides a method for removing undifferentiated cells, which comprises the step of contacting arctigenin with undifferentiated cells.

The present invention also provides a method in which the undifferentiated cells are undifferentiated stem cells remaining in the cell group in which the stem cells have been induced to differentiate in the above method.

The present invention also provides the above method for contacting undifferentiated cells with arctigenin in a low glucose medium in the contacting step.

The present invention also provides the above method of contacting an undifferentiated cell with a glucose metabolism antagonist in the contacting step.

Since the present invention contains arctigenin, which is a natural component contained in plants and the like, as an active ingredient, it is possible to provide an undifferentiated cell removing agent that can be used more safely.

The graph which shows the cell viability (%) of the normal human skin fibroblast by the treatment with arctigenin or quercetin. The figure which shows the microscopic image of the culture medium 48 hours after treatment with arctigenin or quercetin. The graph which shows the cell viability (%) of the iPS cell by the treatment with arctigenin or quercetin. The figure which shows the photograph which stained the undifferentiated cell treated with arctigenin or quercetin. The graph which shows the cell viability (%) of each differentiated cell by the arctigenin treatment. In the figure, arctigenin is described as "AG". The graph which shows the cell viability (%) of each undifferentiated cell by the arctigenin treatment. In the figure, arctigenin is described as "AG". The graph which shows the cell viability (%) of the differentiated cell when treated with arctigenin and 2-deoxy-D-glucose. In the figure, arctigenin is described as "AG" and 2-deoxy-D-glucose is described as "2-DG". The graph which shows the cell viability (%) of the undifferentiated cell when treated with arctigenin and 2-deoxy-D-glucose. In the figure, arctigenin is described as "AG" and 2-deoxy-D-glucose is described as "2-DG". The graph which shows the cell viability (%) of the undifferentiated cell when treated with arctigenin in a low glucose medium. In the figure, arctigenin is described as "AG".

The present invention provides an undifferentiated cell removing agent capable of selectively removing undifferentiated cells. The undifferentiated cell remover of the present invention contains arctigenin as an active ingredient.

As used herein, the term "undifferentiated cell" refers to a cell having pluripotency (pluripotency) capable of differentiating into any cell and not differentiating, and is, for example, a stem cell. Stem cells include pluripotent stem cells such as embryonic stem cells (ES cells) and induced pluripotent stem cells (iPS cells). Further, in the present specification, the term "differentiated cell" means a differentiated cell having no pluripotency, for example, an undifferentiated cell having pluripotency differentiated into an arbitrary somatic cell or the like to completely or partially differentiate the pluripotency. A cell that has been lost.

The undifferentiated cell removing agent of the present invention can, for example, remove only undifferentiated cells from a cell group in which differentiated cells and undifferentiated cells coexist. The undifferentiated cell removing agent of the present invention can be used, for example, to remove undifferentiated stem cells remaining in a cell group in which stem cells have been induced to differentiate into arbitrary cells. Stem cells may be iPS cells.

As used herein, "removal of undifferentiated cells" means not only complete removal of undifferentiated cells, but also reduction of the number or proportion of undifferentiated cells. Also, "removal of undifferentiated cells" includes removal or reduction of undifferentiated cells in vitro or in vivo.

Arctigenin is one of the diphenylpropanoids (lignans) contained in plants such as burdock. As the arctigenin, chemically synthesized arctigenin may be used, or arctigenin isolated from a plant may be used. Moreover, you may use the extract extracted from the plant containing arctigenin as arctigenin. Plants containing arctigenin include, for example, thistle (sprout, leaf, rhizome, goboushi), thistle (flower, leaf, fruit, rhizome), thistle (flower, leaf, fruit, rhizome), and thistle (flower, leaf, fruit).・ Rhizome), Sinarenkyo (flowers, leaves, fruits, rhizomes), Benibana, Yagurumagiku, American thistle, Santorisou (Gibana thistle), Cardon, Gorotsuki thistle, Aniuro koazami, Sesame, Momiji hirugao, Shinchikuhimehagi, Chosenteikazura Includes, Munin Teika Kazura, Hime Teika Kazura, Tokyo Chikuto, Keteika Kazura, Ryo Kaou, Oketade, Yamazakura, White Inunazuna, Thistle, Walnut, Embaku, Spellata Comgi, Soft Wheat, Mexican Itosugi and Kaya. Of these, burdock (particularly burdock and burdock sprout) and forsythia (particularly leaves) are preferable because of their high content of arctigenin. When the plant itself is used, it can be raw or dried and chopped, or dried and powdered.

When an extract extracted from a plant is used as arctigenin, the extract may be prepared from the plant by, for example, the following method. The extract used in the present invention may be extracted from a plant containing, for example, arctigenin by two steps of an enzyme conversion step and an extraction step with an organic solvent.

The enzyme conversion step is a step of enzymatically converting arctiine, which is a precursor of arctigenin contained in the plant, into arctigenin by β-glucosidase, which is an enzyme inherent in the plant. Specifically, by keeping a dried and cut plant at an appropriate temperature, the endogenous β-glucosidase acts to promote the reaction from arctiine to alktigenin. For example, the plant can be kept at an arbitrary temperature by adding an arbitrary solution such as water to the cut plant and stirring the mixture at a temperature of around 30 ° C. (20 to 50 ° C.).

The extraction step with an organic solvent is a step of extracting arctigenin from a plant using any suitable organic solvent. That is, it is a step of extracting an extract from a plant by adding an appropriate solvent in a state where the content of arctigenin is high by the above-mentioned enzyme conversion step. For example, the appropriate solvent is added to the plant and the extract is extracted by heating and stirring for an appropriate period of time. In addition to heating and stirring, the extract can be extracted using any extraction method known to those skilled in the art, such as heating reflux, drip extraction, immersion extraction, or pressure extraction.

Since arctigenin is poorly soluble in water, the yield of arctigenin can be improved by adding an organic solvent. Any organic solvent can be used as the organic solvent. For example, alcohols such as methanol, ethanol and propanol, as well as acetone can be used. In consideration of safety, it is preferable to use 30% ethanol as the organic solvent in the method for producing the extract used in the present invention. Distilling off the solvent from the extract gives a paste-like concentrate, and further drying the concentrate gives a dried product.

The shape of the undifferentiated cell remover of the present invention may be, for example, solid, liquid, granular, granular, powdery or paste.

The undifferentiated cell remover of the present invention may further contain a glucose metabolism antagonist. The glucose metabolism antagonist may be a substance capable of inhibiting glucose metabolism. By further containing a glucose metabolism antagonist, the effect of removing undifferentiated cells of arctigenin can be enhanced and the treatment rate can be accelerated. Therefore, it is possible to remove undifferentiated cells faster, more, and preferably completely.

Glucose metabolism antagonists include, for example, 2-deoxy-D-glucose, 5-thio-D-glucose, 3-O-methylglucose, 1,5-anhydro-D-glucitol, 2,5-anhydro-D-glucitol, 2,5-Anhydro-D-mannitol, mannoheptulose and the like can be used. The undifferentiated cell remover of the present invention may contain only one type of glucose metabolism antagonist, or may contain two or more types.

The undifferentiated cell remover of the present invention may further contain any component other than arctigenin and a glucose metabolism antagonist. For example, the undifferentiated cell remover of the present invention may be a pharmaceutically acceptable base, carrier, excipient, binder, disintegrant, lubricant, colorant, pH adjuster, depending on its use. It may further contain buffers, stabilizers, solubilizers and the like.

In addition, the undifferentiated cell removing agent of the present invention may contain any component having an undifferentiated cell removing action other than arctigenin. In addition, the undifferentiated cell removing agent of the present invention may be used in combination with any other drug having an undifferentiated cell removing action or a method for removing undifferentiated cells. This allows for faster, more, preferably complete removal of undifferentiated cells. For example, quercetin, YM155, drug-fused lectin, 2-deoxyglucose, metformin, perbalanol A, flavopyridiol, ALA, N-oleoylserinol, paclitaxel, benzethonium chloride, for example , Metformin chloride and PluriSIns # 1. In addition, examples of the method for removing undifferentiated cells include a method of reducing the glucose concentration in the medium and adding lactic acid or glutamic acid, and a method of raising the temperature during culturing to 42 ° C.

In the undifferentiated cell remover of the present invention, arctigenin may be bound to an antibody. For example, arctigenin may be bound to an antibody that specifically recognizes iPS cells. Binding of arctigenin to an antibody that specifically recognizes iPS cells allows undifferentiated cells to be eliminated faster, more, and preferably completely by lower concentrations of arctigenin.

Since the undifferentiated cell removing agent of the present invention can remove the undifferentiated cells remaining when the stem cells are differentiated into arbitrary cells, it is possible to suppress tumorigenesis by the undifferentiated cells. Therefore, by using the undifferentiated cell removing agent of the present invention, it becomes possible to obtain a transplant material in which tumorigenesis is suppressed in regenerative medicine.

The undifferentiated cell remover of the present invention can be provided, for example, as a reagent for addition to a medium. The undifferentiated cell removing agent of the present invention remains undifferentiated by being added to, for example, a medium for culturing differentiated cells obtained by inducing differentiation of stem cells or a medium for inducing differentiation of stem cells. Cells can be removed.

Further, the undifferentiated cell removing agent of the present invention may be added to, for example, a transplant material for regenerative medicine and used to suppress tumorigenesis by undifferentiated cells after transplantation. Arctigenin, which is an active ingredient in the undifferentiated cell remover of the present invention, is a natural compound contained in plants and the like, and has extremely low toxicity. Arctigenin shows little toxicity to differentiated cells, as shown in the examples described below. Therefore, the undifferentiated cell remover of the present invention can be safely used as a transplant material for regenerative medicine.

The present invention also provides a kit for removing undifferentiated cells, which comprises the above-mentioned undifferentiated cell removing agent and a low glucose medium. The low glucose medium may be a medium having a glucose concentration lower than usual, for example, a glucose concentration of less than 1 mg / ml, preferably 0.5 mg / ml or less, more preferably 0.3 mg / ml or less, still more preferably 0.1 mg / ml. The following medium may be used. Further, the low glucose medium may have a glucose concentration of 0 mg / ml, that is, may not contain glucose. The kit of the present invention can remove undifferentiated cells by treating undifferentiated cells with an undifferentiated cell removing agent in a low glucose medium and culturing the cells, for example. The kit of the present invention can remove undifferentiated cells faster, more, and preferably completely by using a low glucose medium.

The present invention also provides a method for removing undifferentiated cells, which comprises contacting the undifferentiated cells with arctigenin. The undifferentiated cells may be undifferentiated stem cells remaining in the cell group in which the stem cells have been induced to differentiate. Moreover, the undifferentiated cell may be an iPS cell.

In the method of the present invention, the step of contacting arctigenin with undifferentiated cells comprises contacting arctigenin with undifferentiated cells in vitro or in vivo. For example, arctigenin can be brought into contact with undifferentiated stem cells remaining in the cell group by adding the stem cells to the cell group obtained by inducing differentiation. Arctigenin may be added directly to the differentiated cells, or may be added to a medium for culturing the differentiated cells or a medium for inducing differentiation of stem cells. In addition, arctigenin can be brought into contact with undifferentiated cells contained in the transplant material, for example, by adding it to the transplant material for regenerative medicine.

The concentration of arctigenin when the arctigenin is brought into contact with the undifferentiated cells may be any concentration as long as it can remove the undifferentiated cells, and can be appropriately optimized and used depending on the cell type and the medium composition. The concentration of arctigenin is preferably 0.01 μM or more, more preferably 0.1 μM or more, still more preferably 1 μM or more, and most preferably 3 μM or more. When the concentration of arctigenin is 0.01 μM or more, it is preferable from the viewpoint that undifferentiated cells can be efficiently removed. The upper limit of the concentration of arctigenin is not particularly limited, but can be, for example, 100 μM.

In the step of contacting arctigenin with undifferentiated cells, arctigenin may be contacted with undifferentiated cells in a low glucose medium. The low glucose medium may be a medium having a glucose concentration lower than usual, for example, a glucose concentration of less than 1 mg / ml, preferably 0.5 mg / ml or less, more preferably 0.3 mg / ml or less, still more preferably 0.1 mg / ml. The following medium may be used. In this step, for example, arctigenin may be added to undifferentiated cells cultured in a low glucose medium and further cultured. In addition, undifferentiated cells may be inoculated into a low glucose medium, and arctigenin may be added and cultured. Further, undifferentiated cells may be inoculated into a low glucose medium containing arctigenin and cultured. By contacting the undifferentiated cells with arctigenin in a low glucose medium, the undifferentiated cells can be removed faster, more, and preferably completely.

In the step of contacting arctigenin with undifferentiated cells, a glucose metabolism antagonist may be brought into contact with undifferentiated cells. As the glucose metabolism antagonist, the above-mentioned substances can be used in the description of the undifferentiated cell removing agent. Glucose metabolism antagonists may be added to undifferentiated cells at the same time as arctigenin, or may be added either before or after contact with arctigenin. Further contact with a glucose metabolism antagonist can enhance the effect of removing undifferentiated cells of arctigenin and accelerate the treatment rate. Therefore, it is possible to remove undifferentiated cells faster, more, and preferably completely.

The concentration of the glucose metabolism antagonist when the glucose metabolism antagonist is brought into contact with the undifferentiated cells may be a concentration that inhibits the metabolism of glucose and does not inhibit the growth of the differentiated cells. The concentration of the glucose metabolism antagonist can be, for example, 1 mM or more, preferably 3 mM or more. The upper limit of the concentration of the glucose metabolism antagonist is not particularly limited and can be, for example, 1 M or less.

In the method of the present invention, the above-mentioned undifferentiated cell removing agent can be used as arctigenin.

According to the present invention, it is possible to obtain differentiation-inducing cells with no or little contamination of undifferentiated cells. Therefore, according to the present invention, a transplant material for regenerative medicine in which tumorigenesis is suppressed can be safely and easily obtained.

[Test 1: Effect of arctigenin on differentiated cells]
In order to investigate the effect of arctigenin on differentiated cells (human skin fibroblasts), the cell viability when human skin fibroblasts were treated with arctigenin or quercetin was examined. Quercetin is reported in Patent Document 1 described above to have an effect of removing undifferentiated cells.

Under aseptic conditions (in a clean bench), normal human skin fibroblasts (KF-4009, Kurabo) were ^{inoculated into a 6-well plate at a density of 0.7 × 10 4} cells / ml (number of living cells) at 37 ° C. for 24 hours. It was cultured. As the medium, Dulbecco's Modified Eagle's Medium (Sigma, D6046) supplemented with 10% Fatal Bovine Serum (biowest, S1820) and 1% Antibiotic-Antimycotic (Thermo Fisher Scientific Inc., 15240-062) was used.

Arctigenin or quercetin was then treated with 0, 1, 5, 25 μM and cultured for 48 hours. Arctigenin and quercetin were each dissolved in dimethyl sulfoxide and treated to a dimethyl sulfoxide concentration of 0.1% in all samples. As a control sample, a sample treated with only a solvent was used.

The cytotoxicity of arctigenin to human skin fibroblasts was evaluated. The number of cells was measured with Cell Counting Kit-8 (DOJINDO, 343-07623), and the relative value when the result of the control sample was 100 was calculated as the cell viability (%) (n = 3). FIG. 1 shows the cell viability (%) of human skin fibroblasts treated with arctigenin or quercetin. Quercetin treatment reduced cell viability when treated with 5 μM and 25 μM, whereas arctigenin treatment reduced cell viability very little.

In addition, a microscopic image of the medium 48 hours after treatment with arctigenin or quercetin is shown in FIG. The microscopic images shown in FIG. 2 also showed that arctigenin had little effect on the survival of human skin fibroblasts.

From the above results, it was found that arctigenin has almost no effect on the survival of differentiated cells (skin fibroblasts).

[Test 2: Effect of arctigenin on undifferentiated cells]
Next, in order to investigate the effect of arctigenin on undifferentiated cells, the cell viability when iPS cells were treated with arctigenin or quercetin was examined.

IPS cells were cultured in wells pre-coated with a cell culture matrix (Geltrex ™ LDEV-Free, hESC-Qualified, Reduced Growth Factor Basement Membrane Matrix (Thermo Fisher Scientific Inc., A1413302)).

The iPS cells were seeded on a 6-well plate under aseptic conditions (in a clean bench), and after 24 hours, the medium was changed. iPS cells were cultured to 50% confluence. As the medium, Essential 8 ™ medium (Thermo Fisher Scientific Inc., A1517001) was used.

Arctigenin or quercetin (0, 1, 5 μM) was treated and cultured for 72 hours. Since the nutrients in the medium were depleted in 24 hours, the medium was changed and treated with arctigenin or quercetin every 24 hours. Arctigenin and quercetin were each dissolved in dimethyl sulfoxide and treated to a dimethyl sulfoxide concentration of 0.1% in all samples. As a control sample, a sample treated with only a solvent was used.

The cytotoxicity of arctigenin to iPS cells was evaluated. After culturing for 72 hours, the number of cells was measured with Cell Counting Kit-8 (DOJINDO, 343-07623), and the relative value when the result of the control sample was 100 was calculated as the cell viability (%) (n =). 3). FIG. 3 shows the cell viability (%) of iPS cells treated with arctigenin or quercetin. Quercetin treatment showed little reduction in cell viability when treated with 1 μM and 5 μM, whereas arctigenin treatment reduced cell viability in a concentration-dependent manner.

In addition, in order to distinguish between differentiated cells and undifferentiated cells in the well, the undifferentiated cells were stained using the Blue Alkaline phosphatase substrate kit (Vector, SK-5300). As a result, it was shown that the number of undifferentiated cells decreased by the treatment with arctigenin (Fig. 4).

[Test 3: Effect of arctigenin on differentiated cells]
In order to investigate the effect of arctigenin on differentiated cells, the cell viability when the cells were treated with arctigenin was examined. As differentiated cells, human skin fibroblasts (KF-4009, Kuraboou), human cardiomyocytes (C-12810, Takara Bio) and human iPS-derived cardiomyocytes (Y50015, Takara Bio) were used. Under aseptic conditions (in a clean bench), cells were ^{inoculated into 96 well plates at a density of 2 × 10 4} cells / well (live cell count) and cultured at 37 ° C. for 24 hours. Human iPS-derived cardiomyocytes were cultured in pre-coated wells in a cell culture matrix (Fibronectin from human plasma, F0895, SIGMA).

As the medium, a medium suitable for each cell was used. Human skin fibroblasts are Dulbecco's Modified Eagle's Medium (Sigma, D6046) supplemented with 10% Fatal Bovine Serum (biowest, S1820) and 1% Antibiotic-Antimycotic (Thermo Fisher Scientific Inc., 15240-062). Using. For human cardiomyocytes, a muscle cell proliferation medium kit (C-22170, Takara Bio) was used. For human iPS-derived cardiomyocytes, MiraCell ™ CM Culture Medium (Y50013, Takara Bio) was used.

Arctigenin was then treated with 0, 0.1, 0.5, 1, 3, 5, 8, 10, 25, 50, 100 μM and cultured for 72 hours. Treatment was performed every 24 hours to prevent depletion of nutrients in the medium. Arctigenin was dissolved in dimethyl sulfoxide and treated to a dimethyl sulfoxide concentration of 0.1% in all samples. As a control sample, a sample treated with only a solvent was used.

The cytotoxicity of arctigenin to each differentiated cell was evaluated. The number of cells was measured with Cell Counting Kit-8 (DOJINDO, 343-07623), and the relative value when the result of the control sample was 100 was calculated as the cell viability (%) (n = 6). FIG. 5 shows the cell viability (%) of each differentiated cell treated with arctigenin. Arctigenin treatment did not significantly reduce the cell viability of each differentiated cell. From the above results, it was found that arctigenin has almost no effect on the survival of differentiated cells.

[Test 4: Effect of arctigenin on undifferentiated cells]
Next, in order to investigate the effect of arctigenin on undifferentiated cells, the cell viability when undifferentiated cells (iPS cells) were treated with arctigenin was examined. For undifferentiated cells, three types of iPS cells prepared by different methods, namely RPChiPS771-2 (reprocell), 201B7 (CiRA) and 253G1 (CiRA), were used. Under aseptic conditions (in a clean bench), each cell ^{was inoculated into a 96-well plate at a density of 2 × 10 4} cells / well (number of viable cells) and cultured at 37 ° C. for 24 hours. iPS cells were cultured in wells pre-coated with a cell culture matrix (eye matrix-551 solution, 892011, Nippi). StemFit AK02N (RCAK02N, reprocell) was used as the medium.

Arctigenin was then treated with 0, 0.1, 0.5, 1, 3, 5, 8, 10, 25, 50, 100 μM and cultured for 72 hours. Treatment was performed every 24 hours to prevent depletion of nutrients in the medium. Arctigenin was dissolved in dimethyl sulfoxide and treated to a dimethyl sulfoxide concentration of 0.1% in all samples. As a control sample, a sample treated with only a solvent was used.

The cytotoxicity of arctigenin to undifferentiated cells was evaluated. The number of cells was measured with Cell Counting Kit-8 (DOJINDO, 343-07623), and the relative value when the result of the control sample was 100 was calculated as the cell viability (%) (n = 6). FIG. 6 shows the cell viability (%) of each undifferentiated cell treated with arctigenin. In each iPS cell of RPChiPS771-2, 201B7 and 253G1, a decrease in cell viability was observed in a concentration-dependent manner of arctigenin. From the above results, it was shown that arctigenin has little effect on the survival of differentiated cells, while reducing the survival of undifferentiated cells in a concentration-dependent manner. It was also shown that the effect is independent of the type of undifferentiated cells.

[Test 5: Effect of combined use of arctigenin and 2-DG on differentiated cells]
In order to investigate the effect on differentiated cells when arctigenin and 2-deoxy-D-glucose (hereinafter referred to as "2-DG") are used in combination, when cells are treated with both arctigenin and 2-DG. The cell viability was examined. Human iPS-derived cardiomyocytes (Y50015, Takara Bio) were used as differentiated cells. Under aseptic conditions (in a clean bench), cells were ^{inoculated into 96 well plates at a density of 2 × 10 4} cells / well (live cell count) and cultured at 37 ° C. for 24 hours. Human iPS-derived cardiomyocytes were cultured in pre-coated wells in a cell culture matrix (Fibronectin from human plasma, F0895, SIGMA). As the medium, MiraCell ™ CM Culture Medium (Y50013, Takara Bio) was used.

2-Deoxy-D-glucose (D0051, Tokyo Chemical Industry) was used as 2-DG. Here, 2-DG is a glucose molecule in which the 2-hydroxyl group is replaced with a hydrogen atom, and is not metabolized by glycolysis. By adding 2-DG to the medium, the uptake of glucose by cells can be antagonistically inhibited, and the same effect as that of the glucose-depleted medium can be expected.

Then, arctigenin was treated with 10 μM, 2-DG was treated with 1 mM or 3 mM, and cultured for 24 hours. Arctigenin was dissolved in dimethyl sulfoxide and treated to a dimethyl sulfoxide concentration of 0.1% in all samples. As a control sample, a sample treated with only a solvent was used.

The cytotoxicity of arctigenin and 2-DG to differentiated cells was evaluated. The number of cells was measured with Cell Counting Kit-8 (DOJINDO, 343-07623), and the relative value when the result of the control sample was 100 was calculated as the cell viability (%) (n = 3). FIG. 7 shows the cell viability (%) of differentiated cells when treated with arctigenin and 2-DG. Treatment with arctigenin and 2-DG did not significantly reduce the cell viability of differentiated cells.

[Test 6: Effect of combined use of arctigenin and 2-DG on undifferentiated cells]
In order to investigate the effect on undifferentiated cells when arctigenin and 2-DG were used in combination, the cell viability when undifferentiated cells (iPS cells) were treated with both arctigenin and 2-DG was examined. For undifferentiated cells, 201B7 (CiRA) was used. Under aseptic conditions (in a clean bench), cells were ^{inoculated into 96 well plates at a density of 2 × 10 4} cells / well (live cell count) and cultured at 37 ° C. for 24 hours. 201B7 cells, which are undifferentiated cells (iPS cells), were cultured in wells pre-coated with a cell culture matrix (eye matrix-551 solution, 892011, Nippi). StemFit AK02N (RCAK02N, reprocell) was used as the medium. 2-Deoxy-D-glucose (D0051, Tokyo Chemical Industry) was used as 2-DG.

Then, arctigenin was treated with 10 μM, 2-DG was treated with 1 mM or 3 mM, and cultured for 24 hours. Arctigenin was dissolved in dimethyl sulfoxide and treated to a dimethyl sulfoxide concentration of 0.1% in all samples. As a control sample, a sample treated with only a solvent was used.

The cytotoxicity of arctigenin and 2-DG to undifferentiated cells was evaluated. The number of cells was measured with Cell Counting Kit-8 (DOJINDO, 343-07623), and the relative value when the result of the control sample was 100 was calculated as the cell viability (%) (n = 3). FIG. 8 shows the cell viability (%) of undifferentiated cells when treated with arctigenin and 2-DG. In this example, the treatment time of arctigenin was shorter than that of test 4 (72 hours in test 4 compared to 24 hours in this example). Therefore, when only arctigenin was treated, undifferentiated cells There was almost no decrease in cell viability. On the other hand, when treated with arctigenin and 2-DG in combination, a decrease in cell viability was observed at the treatment time of 24 hours.

From the above results, it was shown that the combined use of arctigenin and 2-DG can promote a decrease in the survival rate of undifferentiated cells while not affecting the survival rate of differentiated cells. Therefore, it was shown that the combined use of arctigenin and 2-DG can shorten the treatment time and remove undifferentiated cells more efficiently.

[Test 7: Effect of arctigenin and glucose concentration in the medium on undifferentiated cells]
In order to investigate the effect of arktigenin and glucose inhibition on undifferentiated cells, the cell viability when undifferentiated cells (iPS cells) were treated with arktigenin in a medium with a low glucose concentration (low glucose medium) was examined. For undifferentiated cells, 201B7 (CiRA) was used. Under aseptic conditions (in a clean bench), cells were ^{inoculated into 96 well plates at a density of 2 × 10 4} cells / well (live cell count) and cultured at 37 ° C. for 24 hours. 201B7 cells, which are undifferentiated cells (iPS cells), were cultured in wells pre-coated with a cell culture matrix (eye matrix-551 solution, 892011, Nippi). As the medium, DMEM / Ham's F-12 (No Glucose) with L-Glu and Sodium Pyruvate (09893-05, Nakarai) was used. As glucose, (D- (+) Glucose solution 100 ml 45% in H2O (G8769-100ML, SIGMA) was used.

The glucose concentration in the medium was then adjusted to 0.05, 0.1, 1 mg / ml. Arctigenin was treated with 0, 0.1, 0.3, 0.5, 0.8, 1 μM and cultured for 24 hours. Arctigenin was dissolved in dimethyl sulfoxide and treated to a dimethyl sulfoxide concentration of 0.1% in all samples. As a control sample, a medium having a glucose concentration of 1 mg / ml treated with a solvent was used.

The cytotoxicity of undifferentiated cells due to the concentration of arctigenin and glucose in the medium was evaluated. The number of cells was measured with Cell Counting Kit-8 (DOJINDO, 343-07623), and the relative value when the result of the control sample was 100 was calculated as the cell viability (%) (n = 3). FIG. 9 shows the cell viability (%) of undifferentiated cells when treated with arctigenin in a low glucose medium. When the glucose concentration in the medium was 1 mg / ml, no decrease in the cell viability of undifferentiated cells was observed at the treatment time of 24 hours. On the other hand, by adjusting the glucose concentration in the medium to 0.1 mg / ml or less, a decrease in the cell viability of undifferentiated cells was observed in the treatment time of 24 hours.

From the above results, it was shown that treatment with arctigenin in a low glucose medium can promote a decrease in the survival rate of undifferentiated cells. Therefore, it was shown that by treating arctigenin in a low glucose medium, the treatment time can be shortened and undifferentiated cells can be removed more efficiently.

Since the undifferentiated cell removing agent of the present invention can be used more safely, it can be suitably used in regenerative medicine, research and development, and the like.

Claims (8)

An iPS cell remover containing arctigenin as an active ingredient. The iPS cell removing agent according to claim 1, for removing undifferentiated iPS cells remaining in the cell group in which iPS cells have been induced to differentiate. The iPS cell scavenger according to claim 1 or 2 , further comprising a glucose metabolism antagonist. A kit for removing iPS cells , which comprises the iPS cell removing agent according to any one of claims 1 to 3 and a low glucose medium. A method of removing iPS cells , which comprises contacting arctigenin with iPS cells in vitro. The iPS cells are undifferentiated iPS cells remaining iPS cells to differentiation induction cell populations The method of claim 5. The method according to claim 5 or 6 , wherein in the contacting step, the arctigenin is contacted with the iPS cells in a low glucose medium. The method according to any one of claims 5 to 7 , wherein in the contacting step, a glucose metabolism antagonist is brought into contact with the iPS cells.

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