JP2024024550A - Cell death inducer for undifferentiated stem cells and method for purifying differentiated cells - Google Patents

Abstract

To provide methods for the induction of apoptosis in undifferentiated stem cells, using less quantities of material, applicable even to cells in a spheroidal shape, and agents therefor.SOLUTION: The present invention provides an agent for the induction of apoptosis in pluripotent stem cells or cells with differentiation potential induced from the pluripotent stem cells, comprising as an active ingredient at least one compound selected from the group consisting of TVB-3166 or analogs thereof, TVB-3664 or analogs thereof, TVB-2640 or analogs thereof, FAS-IN-1 tosylate or analogs thereof, and FT113 or analogs thereof.SELECTED DRAWING: None

Description

The present invention provides for the use of a compound selected from the group consisting of TVB-3166 or an analog thereof, TVB-3664 or an analog thereof, TVB-2640 or an analog thereof, FAS-IN-1 tosylate or an analog thereof, and FT113 or an analog thereof. The present invention relates to a cell death inducer for pluripotent stem cells and undifferentiated stem cells containing at least one or more compounds containing as an active ingredient, and a method for purifying differentiated cells using the cell death inducer. The present invention also relates to a method for producing highly purified cardiomyocytes and cardiomyocyte spheres derived from pluripotent stem cells, and a pharmaceutical composition for treating heart diseases.

In recent years, research on pluripotent stem cells such as embryonic stem cells (ES cells) and induced pluripotent stem cells (iPS cells) has been progressing. Since these cells have pluripotency, it is becoming possible to differentiate and induce cells to produce cells differentiated into a desired lineage and use them in transplantation medicine.

Differentiation and induction of pluripotent stem cells such as embryonic stem cells and induced pluripotent stem cells are usually performed in vitro.
However, in vitro, it is difficult to induce differentiation of the desired lineage in all pluripotent stem cells, and some undifferentiated stem cells may remain even after differentiation and induction operations. Such undifferentiated stem cells have proliferative activity and can differentiate into many types of cells, so when transplanted into a living body, there is a risk of forming a teratoma (for example, see Non-Patent Document 1) . For these reasons, it is difficult to directly transplant a cell population produced by differentiating and inducing pluripotent stem cells such as embryonic stem cells or induced pluripotent stem cells into a living body and use it for treatment.

Therefore, in order to safely transplant cells differentiated and induced from pluripotent stem cells such as embryonic stem cells and induced pluripotent stem cells into a living body and to obtain the ideal therapeutic effect, undifferentiated stem cells must be removed. It is necessary. Furthermore, if the undifferentiated stem cells are transplanted while remaining, it is necessary to suppress the proliferation of the undifferentiated stem cells in the living body.

So far, methods have been reported that are said to be able to selectively remove undifferentiated stem cells under culture conditions (see, for example, Non-Patent Documents 2 to 4). The research group of the present inventors has also developed a method for selecting cardiomyocytes from non-cardiomyocytes and undifferentiated stem cells (see Patent Documents 1 to 7 and Non-Patent Document 5).

International Publication No. 2006/022377 International Publication No. 2007/088874 International Publication No. 2009/017254 International Publication No. 2010/114136 International Publication No. 2011/052801 International Publication No. 2016/010165 International Publication No. 2018/074457

Miura et al., (2009) Nature Biotech., 8, 743-745. Ben-David,et al., (2013) Cell Stem Cell, 12, 167-179. Wang,et al., (2009) Science, 325, 435-439. Shiraki, et al., (2014) Cell Metabolism, 19, 780-794. Tohyama, et al., (2016) Cell Metabolism, 23, 663-674.

The methods that have been reported so far that are said to be able to selectively remove undifferentiated stem cells are based on the use of a high optimal concentration of the drug used, or when cells become spheroids in three-dimensional culture, etc. There were problems such as the inability to induce cell death. Therefore, an object of the present invention is to provide a method for inducing cell death of undifferentiated stem cells in a smaller amount and even when the cells are in the form of a spheroid, and a drug for the same.

The present inventors screened various drugs in order to solve the above problems. As a result, they discovered that a certain compound can selectively induce cell death in undifferentiated stem cells. We have diligently investigated conditions under which these compounds can selectively induce cell death in undifferentiated stem cells without adversely affecting differentiated cells, and as a result, we have established suitable conditions and completed the present invention. Ta.
That is, the present invention is as follows.
[1] Selected from the group consisting of TVB-3166 or its analogs, TVB-3664 or its analogs, TVB-2640 or its analogs, FAS-IN-1 tosylate or its analogs, and FT113 or its analogs A cell death inducer for pluripotent stem cells and undifferentiated stem cells derived from the pluripotent stem cells, which contains at least one compound as an active ingredient.
[1A] The inducing agent according to [1] above, wherein the pluripotent stem cells are induced pluripotent stem cells.
[2] By culturing a cell mixture containing pluripotent stem cells and/or undifferentiated stem cells derived from the pluripotent stem cells, and differentiated cells in the presence of the cell death inducing agent described in [1] above. , a method to purify differentiated cells in a cell mixture.
[3] The purification method according to [2] above, wherein the cell mixture containing pluripotent stem cells and/or undifferentiated stem cells derived from the pluripotent stem cells and differentiated cells is in the form of a cell mass.
[4] The purification method according to [2] or [3] above, wherein the differentiated cells are cardiomyocytes.
[5] The purification method according to any one of [2] to [4] above, wherein the pluripotent stem cells are induced pluripotent stem cells.
[6] A method for producing highly purified pluripotent stem cell-derived cardiomyocytes, comprising:
A cell mixture containing pluripotent stem cells and/or undifferentiated stem cells derived from the pluripotent stem cells, and cardiomyocytes,
Subjected to the step of implementing the purification method described in any one of [2] to [5] above,
The proportion of pluripotent stem cells and/or undifferentiated stem cells derived from the pluripotent stem cells in the cell mixture is 10% or less;
Method.
[7] A cell mixture containing pluripotent stem cells and/or undifferentiated stem cells derived from the pluripotent stem cells, and cardiomyocytes is grown in a culture medium with a glucose and/or glutamine content of 10% or less of a normal medium and a lactic acid content. The method for producing cardiomyocytes according to item [6] above, which comprises culturing in an added medium.
[8] A method for producing pluripotent stem cell-derived cardiomyocyte spheres, further comprising a step of aggregating pluripotent stem cell-derived cardiomyocytes produced by the method described in [6] or [7] above.
[9] A pharmaceutical composition for treating heart disease, comprising cardiomyocyte spheres derived from pluripotent stem cells, produced by the method described in [8] above.
[10] A method for producing differentiated cells derived from highly purified pluripotent stem cells, comprising:
A cell mixture containing pluripotent stem cells and/or undifferentiated stem cells derived from the pluripotent stem cells, and differentiated cells,
Subjected to the step of implementing the purification method described in any one of [2] to [5] above,
The proportion of pluripotent stem cells and/or undifferentiated stem cells derived from the pluripotent stem cells in the cell mixture is 10% or less;
Method.

It is possible to induce cell death in undifferentiated stem cells using a smaller amount than existing undifferentiated stem cell death inducers and even when the target cells are in a spheroid state. Therefore, high efficacy and safety can be expected in the entire process of inducing differentiation from pluripotent stem cells to differentiated cells, subsequent purification of differentiated cells, and even transplantation.

FIG. 1 is a diagram showing the results of investigating the effects of TVB-3664, TVB-3166, TVB-2640, and orlistat (low concentration) on iPS cells in a cell cluster state. FIG. 2 is a diagram showing the results of investigating the effects of TVB-3664, TVB-3166, TVB-2640, and orlistat (high concentration) on iPS cells in a cell cluster state. FIG. 3 is a diagram showing the results of flow cytometry analysis of the effects of TVB-3664, TVB-3166, TVB-2640, and orlistat (high concentration) on a mixed mass of iPS cells and cardiomyocytes. FIG. 4 is a diagram showing the results of flow cytometry analysis of the effects of TVB-3664, TVB-3166, TVB-2640, and orlistat (low concentration) on a mixed mass of iPS cells and cardiomyocytes.

1. Definition etc. In this specification, "undifferentiated stem cells" may be used as a concept that includes pluripotent stem cells having pluripotency, including embryonic stem cells (ES cells), induced pluripotent stem cells, etc. It may include induced pluripotent stem cells (iPS cells) and pluripotent cells derived from these stem cells. That is, in this specification, "undifferentiated stem cells" are synonymous with "pluripotent stem cells or cells with pluripotency derived from the pluripotent stem cells." Undifferentiated stem cells are not particularly limited as long as they have pluripotency, and include unknown cells having properties equivalent to the above-mentioned ES cells and iPS cells, and cells derived therefrom.

In the present invention, pluripotent stem cells among undifferentiated stem cells can be determined from the presence or absence of properties specific to pluripotent stem cells, the expression of various markers specific to pluripotent stem cells, and the like. For example, properties specific to pluripotent stem cells include the ability to self-renew and to differentiate into different types of cells with properties different from those of pluripotent stem cells. Furthermore, the ability to form teratoma and the ability to form chimeric mice are also listed as properties specific to pluripotent stem cells.

Various markers specific to pluripotent stem cells (hereinafter referred to as "undifferentiated stem cell markers") are factors that are specifically expressed in pluripotent stem cells, such as Oct3/4, Nanog, Sox2, SSEA. -1, SSEA-3, SSEA-4, TRA1-60, TRA1-81, Lin28, Fbx15, etc. When expression of at least one of these pluripotent stem cell markers is observed, the cell can be determined to be a pluripotent stem cell.
In this specification, "undifferentiated stem cells derived from pluripotent stem cells" refers to cells that have started to differentiate from the pluripotent stem cells, but have not yet reached the level of differentiated cells described below, and whose proliferation or survival There are no particular limitations as long as the cells are essential for fatty acid metabolism. In one embodiment, cells expressing Oct3/4 may be determined to be undifferentiated stem cells. Note that the expression of undifferentiated stem cell markers in cells can be confirmed using known methods such as RT-PCR and microarrays.

Pluripotent stem cells and undifferentiated stem cells may be mammalian pluripotent stem cells and undifferentiated stem cells, may be of rodent origin, may be of primate origin, or may be of human origin. It may be. An example of pluripotent stem cells is human-derived pluripotent stem cells, and more specific examples include human iPS cells and human ES cells.

As used herein, "differentiated cells" refer to cells that do not have the properties of the above-mentioned "pluripotent stem cells" and "undifferentiated stem cells." Differentiated cells may be cells induced and differentiated from pluripotent stem cells, but do not have pluripotency or differentiation ability. Furthermore, in the present invention, it refers to cells that do not require fatty acid metabolism for their proliferation or survival. The differentiated cells may be, for example, cells differentiated from ES cells or cells differentiated from iPS cells. Examples of differentiated cells include cardiomyocytes, muscle cells, fibroblasts, nerve cells, immune cells such as lymphocytes, vascular cells, eye cells such as retinal pigment epithelial cells, blood cells such as megakaryocytes and red blood cells, and other cells. Examples include tissue cells and their precursor cells.
"Cardiomyocytes", which is one type of differentiated cells, are characterized by the fact that expression of fatty acid synthase is extremely low compared to pluripotent stem cells and undifferentiated stem cells, and fatty acid metabolism is not essential for the proliferation and survival of the cells. have Furthermore, it has the characteristic that the energy necessary for its growth and survival can be generated from lactic acid rather than from the thawing system.

2. Cell Death Inducer In one embodiment, the present invention provides a cell death inducer for pluripotent stem cells and undifferentiated stem cells (hereinafter also referred to as "cell death inducer of the present invention").
The cell death inducing agent of the present invention contains a compound capable of inducing cell death of pluripotent stem cells and undifferentiated stem cells as an active ingredient. The compounds include TVB-3166 or its analogs, TVB-3664 or its analogs, TVB-2640 or its analogs, FAS-IN-1 tosylate or its analogs, and FT113 or its analogs.
The present invention will be described below based on embodiments.

TVB-3166 and its analogues

The compound represented by is called TVB-3166 and is known as a therapeutic agent for diseases in which the fatty acid synthesis pathway is dysregulated, specifically as a therapeutic agent for viral infections and a therapeutic agent for cancer. The pharmacological action of TVB-3166 is explained in detail in Ventura R., EBioMedicine. 2015 Jul 2;2(8):808-24. Examples of analogs of the compound include the following compound (I) and its pharmaceutically acceptable salts disclosed in Patent No. 6522007 (International Publication No. 2015/105860).

In the formula, the definition of each symbol is as described in International Publication No. 2015/105860.

TVB-3664 and its analogues

The compound represented by is called TVB-3664 and is known as a fatty acid synthase (FASN) inhibitor. Examples of analogs of the compound include the following compound (II) disclosed in Patent No. 6109934 (International Publication No. 2014/008197) and pharmaceutically acceptable salts thereof.

In the formula, the definition of each symbol is as described in International Publication No. 2014/008197.

TVB-2640 and its analogues

The compound represented by is also known as TVB-2640, Denifanstat, or FASN-IN-2, and is known as a fatty acid synthase (FASN) inhibitor. Examples of analogs of the compound include the following compound (III) disclosed in Patent No. 5973473 (International Publication No. 2012/122391) and pharmaceutically acceptable salts thereof.

In the formula, the definition of each symbol is as described in International Publication No. 2012/122391.

FAS-IN-1 tosylate and its analogues

The compound represented by is also called FAS-IN-1tosylate and is known as a potent fatty acid synthase (FASN) inhibitor. Examples of the compound and its analogs include the following compound (I) disclosed in International Publication No. 2012/064642) and pharmaceutically acceptable salts thereof.

In the formula, the definition of each symbol is as described in International Publication No. 2012/064642. FAS-IN-1 corresponds to the compound of Example 29 in the publication. The pharmacological action of FAS-IN-1 is explained in detail in Chu J., Nat Metab. 2021 Nov;3(11):1466-1475.

FT113 and its analogs

The compound represented by is also called FT113 and is known as a potent fatty acid synthase (FASN) inhibitor. Examples of the compound and its analogs include the following compound (ID) disclosed in International Publication No. 2014/164749) and pharmaceutically acceptable salts thereof.

In the formula, the definition of each symbol is as described in International Publication No. 2014/164749. The pharmacological action of FT113 is explained in detail in Martin MW, et al. Bioorg Med Chem Lett. 2019 Apr 15;29(8):1001-1006.

The cell death inducing agent of the present invention includes TVB-3166 or its analog, TVB-3664 or its analog, TVB-2640 or its analog, FAS-IN-1 tosylate or its analog, and FT113 or its analog ( (Hereinafter, also collectively referred to as "compounds of the present invention.") One type or a combination of two or more types of compounds can be contained.

The cell death inducing agent of the present invention may contain the compound of the present invention in a concentration of 10 nM to 5 μM, preferably 10 nM to 500 nM. In addition, in this specification, "M" as a unit means mol/L. Furthermore, in this specification, "in the subject" means in the culture medium or in the blood.

When the compound of the present invention is TVB-3166 or an analogue thereof, particularly TVB-3166, the concentration of TVB-3166 in the application target is preferably 10 nM to 1 μM, more preferably 40 nM to 1 μM, and even more preferably 50 nM to 1 μM. preferable. Furthermore, when the undifferentiated stem cells are in the form of a cell mass as described below, the concentration of TVB-3166 in the application target is preferably 50 nM to 1 μM, more preferably 100 nM to 1 μM, and even more preferably 150 nM to 1 μM.

When the compound of the invention is TVB-3664 or an analogue thereof, particularly TVB-3664, the concentration of TVB-3664 in the application is preferably between 10 nM and 1 μM, more preferably between 15 nM and 1 μM. Furthermore, when the undifferentiated stem cells are in the form of a cell mass as described below, the concentration of TVB-3664 in the application target is preferably 50 nM to 1 μM, more preferably 100 nM to 1 μM, and even more preferably 150 nM to 1 μM.

When the compound of the present invention is TVB-2640 or an analogue thereof, particularly TVB-2640, the concentration of TVB-2640 in the application target is preferably 10 nM to 1 μM, more preferably 40 nM to 1 μM, and even more preferably 50 nM to 1 μM. preferable. Furthermore, when the undifferentiated stem cells are in the form of a cell mass as described below, the concentration of TVB-2640 in the application target is preferably 50 nM to 1 μM, more preferably 100 nM to 1 μM, and even more preferably 150 nM to 1 μM.

When the compound of the present invention is FAS-IN-1 tosylate or an analog thereof, particularly FAS-IN-1 tosylate, the concentration of FAS-IN-1 tosylate in the application target is preferably 200 nM to 3 μM, more preferably 300 nM to 3 μM. , 400 nM to 3 μM is more preferred. Furthermore, when the undifferentiated stem cells are in the form of a cell mass as described below, the concentration of FAS-IN-1 tosylate in the application target is preferably 500 nM to 5 μM, more preferably 1 μM to 5 μM, and even more preferably 2 μM to 5 μM.

When the compound of the present invention is FT113 or an analog thereof, particularly FT113, the concentration of FT113 in the application target is preferably 200 nM to 3 μM, more preferably 300 nM to 3 μM, even more preferably 400 nM to 3 μM. Furthermore, when the undifferentiated stem cells are in the form of a cell mass described below, the concentration of FT113 in the application target is preferably 500 nM to 5 μM, more preferably 1 μM to 5 μM, and even more preferably 2 μM to 5 μM.

The cell death inducing agent of the present invention is derived from TVB-3166 or its analog, TVB-3664 or its analog, TVB-2640 or its analog, FAS-IN-1 tosylate or its analog, and FT113 or its analog. It may consist of at least one compound selected from the group consisting of at least one compound, and may contain other optional components as long as it has the ability to induce cell death of pluripotent stem cells and undifferentiated stem cells. .

The cell death inducing agent of the present invention can be used in a culture medium, a method for purifying differentiated cells, a method for producing cardiomyocytes, and a cardiomyocyte sphere, which will be described later. It can also be used as a drug to induce cell death in undifferentiated stem cells transplanted into a patient's body.

The cell death inducing agent of the present invention can also be used to induce cell death in pluripotent stem cells and undifferentiated stem cells transplanted into a patient's body by administering it to a patient who has undergone cell transplantation. In such cases, administration may be performed parenterally or orally by methods known to those skilled in the art. Parenteral administration methods include intraarterial injection, intravenous injection, subcutaneous injection, etc., as well as intranasal, transbronchial, intramuscular, or percutaneous administration. Although the dosage varies depending on the patient's weight, age, administration method, etc., those skilled in the art can appropriately select an appropriate dosage.

By administering the cell death inducing agent of the present invention, transplanted cells or pluripotent stem cells and undifferentiated stem cells remaining in tissues can be killed and removed in vivo. Thereby, diseases caused by proliferation of pluripotent stem cells and undifferentiated stem cells, such as canceration of transplanted cells or tissues, can be prevented or treated.

The pharmaceuticals to be administered to the above-mentioned patients may include other ingredients other than the compound of the present invention, including, but not limited to, pharmaceutically acceptable carriers, transfection promoters, buffers, excipients, and stabilizers. , antioxidants, osmotic pressure regulators, pH regulators, chelating agents, and the like.

The dosage form of the cell death inducing agent of the present invention is not particularly limited, and may be in various forms such as liquid, powder, granule, gel, solid, and the like. Further, it may be in the form of an emulsion in which one or some combination of the compounds of the present invention is encapsulated in micelles, or in the form of liposomes in which it is encapsulated in liposomes.

3. Method for Purifying Differentiated Cells In one embodiment, the present invention provides a method for purifying pluripotent stem cells and/or a cell mixture containing undifferentiated stem cells and differentiated cells derived from pluripotent stem cells in the presence of the cell death inducing agent of the present invention. Provided is a method for purifying differentiated cells in a cell mixture by culturing in a cell mixture (hereinafter also referred to as "purification method of the present invention").
As used herein, a "cell mixture" is a cell population containing two or more types of cells present in a culture system in which pluripotent stem cells are induced to differentiate into specific differentiated cells.
A "cell mixture" may include two or more types of cells as well as components of a culture medium and the like. The form of the "cell mixture" is not particularly limited, and may be aggregated, dispersed, adhered to a culture vessel, adhered to adhesion factors such as extracellular matrix, sheet, lump, colony, embryoid body, or cells. It can be a mass, tissue, organ, etc.

When pluripotent stem cells are subjected to differentiation/induction treatment, differentiated cells are induced from the pluripotent stem cells. However, in vitro, it is difficult to induce all pluripotent stem cells into differentiated cells, and it is usually difficult to induce all pluripotent stem cells into differentiated cells. The stem cells themselves remain and a cell mixture of these cells and differentiated cells is formed. In the method of this embodiment, pluripotent stem cells and undifferentiated stem cells derived from pluripotent stem cells are selectively removed from such a cell mixture, and only differentiated cells are taken out, that is, the differentiated cells are purified. I can do it. In addition, in the method of this embodiment, the cell mixture containing pluripotent stem cells and/or undifferentiated stem cells and differentiated cells is composed of pluripotent stem cells and/or undifferentiated stem cells and differentiated cells derived from pluripotent stem cells. It may be a mixture of the following.

In the purification method of the present invention, the pluripotent stem cells are not particularly limited, but are preferably iPS cells or ES cells, more preferably iPS cells.

In the purification method of the present invention, the differentiated cells are not particularly limited, but are preferably differentiated and induced from pluripotent stem cells of the same type as the undifferentiated stem cells present. Several methods for differentiating and inducing differentiated cells have been reported so far, and differentiated cells can be induced using these known methods.

The higher the efficiency with which the pluripotent stem cells and undifferentiated stem cells in the cell mixture can be selectively removed, the better. When a cell mixture containing pluripotent stem cells and/or undifferentiated stem cells and differentiated cells is cultured in the presence of the cell death inducing agent of the present invention described above to obtain a cell mixture, (number of pluripotent stem cells + undifferentiated stem cells + undifferentiated stem cells) The survival rate of pluripotent stem cells, etc. expressed as (differentiated stem cell number)/total cell number x 100 is preferably less than 10%, more preferably less than 1%, and less than 0.1%. More preferably, it is less than 0.01%. Note that dead cells are not included in the above cell number.
Pluripotent stem cells and undifferentiated stem cells remaining in the cell mixture can be detected and determined based on the expression of various markers specific to the pluripotent stem cells and undifferentiated stem cells. Alternatively, the number of differentiated cells can be specified using various markers specific to differentiated cells in the cell mixture, and other cells can be determined as pluripotent stem cells and undifferentiated stem cells.

Differentiated cells include cardiomyocytes, muscle cells, fibroblasts, nerve cells, immune cells (e.g., lymphocytes, etc.), vascular cells, eye cells (e.g., retinal pigment epithelial cells, etc.), and blood cells (e.g., megakaryocytes). , red blood cells, etc.), other tissue cells, and their precursor cells. Among these, cardiomyocytes and nerve cells are preferred, and cardiomyocytes are more preferred.

When cardiomyocytes are derived from pluripotent stem cells, it is thought that as differentiation into cardiomyocytes progresses, they differentiate into cardiomyocytes through undifferentiated mesoderm, cardiac mesoderm (or prospective cardiomyocytes). Here, undifferentiated mesoderm refers to a stage at which expression of Brachyury protein specific to undifferentiated mesoderm is observed. On the other hand, cardiac mesoderm (or presumptive cardiomyocytes) refers to the expression of undifferentiated mesoderm-specific proteins such as Brachyury, and the expression of cardiomyocyte-specific proteins such as Nkx2.5 and actinin in the same cells. This refers to cells that do not require the addition of new substances to the culture medium and have the ability to differentiate exclusively into cardiomyocytes. Cardiomyocytes, which are an embodiment of differentiated cells in the present invention, do not rely on fatty acid metabolism for the energy necessary for their own proliferation and survival, and more preferably do not require sugars such as glucose, and do not require lactic acid or pyruvate. It has the characteristics that it can be used as an energy source. Troponin, Nkx2.5, GATA4, actinin, etc. are used as markers for cardiac muscle cells. As used herein, the term "cardiomyocyte" is used to include cardiomyocytes and cardiac mesoderm (or prospective cardiomyocytes).

Differentiation and induction of pluripotent stem cells into cardiomyocytes are described, for example, in International Publication No. 2007/088874, International Publication No. 2008/150030, Tohyama et al., Cell Stem Cell, 12, 127 (2013), Tohyama et al. , Cell Metabolism, 23, 663 (2016), Tohyama et al., Stem Cell Reports, 9, 1406 (2017), etc. Specifically, for example, differentiation and induction into cardiomyocytes may be performed by adding a substance that induces differentiation into cardiomyocytes to a medium in which pluripotent stem cells are cultured.
Such substances include, for example, mesoderm-inducing factors activin A, BMP4, bFGF, VEGF, SCF, GSK3 inhibitors such as CHIR99021, and Wnt signal inhibitors Dkk1, IWR-1, IWP-2, and IWP. -4, XAV, etc., BMP signal inhibitors such as NOGGIN, TGFβ/activin/NODAL signal inhibitors such as SB431542, retinoic acid signal inhibitors, and cardiac differentiation factors such as VEGF, bFGF, and DLL15.

The purification method of the present invention involves converting a cell mixture containing pluripotent stem cells and/or undifferentiated stem cells and differentiated cells existing in the process of differentiating and inducing differentiated cells such as cardiomyocytes from the pluripotent stem cells into the cells of the present invention. The method includes a step of culturing in the presence of a death-inducing agent. This step can be carried out by adding the cell death inducer of the present invention to a cell culture medium as described above and culturing the cell mixture. Alternatively, a cell death inducer for undifferentiated stem cells may be added to a medium in which a cell mixture containing pluripotent stem cells and/or undifferentiated stem cells and differentiated cells is cultured.
Specifically, after performing the step of differentiating and inducing differentiated cells such as cardiomyocytes from pluripotent stem cells, the medium is replaced with a medium containing a cell death inducer for undifferentiated stem cells of the present invention, and further culture is performed. This can be done by Thereafter, the cardiomyocytes can be further purified by changing the culture medium to a medium containing lactic acid and having a glucose and/or glutamine content of 10% or less of the normal medium. (International Publication No. 2007/088874, International Publication No. 2016/10165).

The amount of the cell death inducer of the present invention added to the medium is not particularly limited, but when the compound of the present invention is TVB-3166 or an analog thereof, particularly TVB-3166, the concentration of TVB-3166 in the medium is , preferably 10 nM to 1 μM, more preferably 40 nM to 1 μM, even more preferably 50 nM to 1 μM. Furthermore, when the undifferentiated stem cells are in the form of a cell mass described below, the concentration of TVB-3166 in the medium is preferably 50 nM to 1 μM, more preferably 100 nM to 1 μM, and even more preferably 150 nM to 1 μM.

When the compound of the invention is TVB-3664 or an analog thereof, particularly TVB-3664, the concentration of TVB-3664 in the medium is preferably 10 nM to 1 μM, more preferably 15 nM to 1 μM. Furthermore, when the undifferentiated stem cells are in the form of a cell mass as described below, the concentration of TVB-3664 in the medium is preferably 50 nM to 1 μM, more preferably 100 nM to 1 μM, and even more preferably 150 nM to 1 μM.

When the compound of the present invention is TVB-2640 or an analogue thereof, particularly TVB-2640, the concentration of TVB-2640 in the medium is preferably 10 nM to 1 μM, more preferably 40 nM to 1 μM, even more preferably 50 nM to 1 μM. . Furthermore, when the undifferentiated stem cells are in the form of a cell mass as described below, the concentration of TVB-2640 in the medium is preferably 50 nM to 1 μM, more preferably 100 nM to 1 μM, and even more preferably 150 nM to 1 μM.

When the compound of the present invention is FAS-IN-1 tosylate or an analog thereof, particularly FAS-IN-1 tosylate, the concentration of FAS-IN-1 tosylate in the medium is preferably 200 nM to 3 μM, more preferably 300 nM to 3 μM, More preferably 400 nM to 3 μM. Furthermore, when the undifferentiated stem cells are in the form of a cell mass described below, the concentration of FAS-IN-1 tosylate in the medium is preferably 500 nM to 5 μM, more preferably 1 μM to 5 μM, and even more preferably 2 μM to 5 μM.

When the compound of the present invention is FT113 or an analog thereof, particularly FT113, the concentration of FT113 in the medium is preferably 200 nM to 3 μM, more preferably 300 nM to 3 μM, even more preferably 400 nM to 3 μM. Further, when the undifferentiated stem cells are in the form of a cell mass described below, the concentration of FT113 in the medium is preferably 500 nM to 5 μM, more preferably 1 μM to 5 μM, and even more preferably 2 μM to 5 μM.

The cell mixture may be cultured in the presence of the cell death inducing agent of the present invention at a temperature commonly used for cell culture. For example, the culture temperature can be 20 to 40°C, preferably 25 to 38°C, and more preferably 30 to 37°C.

The culture time of the cell mixture in the presence of the cell death inducing agent of the present invention is not particularly limited, but is preferably 24 hours or more, specifically 1 to 7 days, preferably 3 to 5 days. If necessary, the cells may be subcultured. The composition of the medium before and after passage may be the same or different as long as it contains a cell death inducing agent.

The cell density of the cell mixture in the presence of the cell death inducing agent of the present invention is such that cells other than differentiated cells in the cell mixture tend to proliferate more easily than differentiated cells, so that cells other than differentiated cells are unnecessary. It is adjusted to the extent that it does not proliferate. Specifically, for example, at the time of adding the cell death inducer of the present invention, it is preferable to seed 1 to 6×10 ⁷ cells as a cell mixture in a culture dish with a bottom area of 150 cm ² , and 2 to 6× 10 ⁷ pieces are more preferable, and 3 to 5×10 ⁷ pieces are even more preferable.

Furthermore, in the purification method of the present invention, the cell mixture may be cultured two-dimensionally using a culture dish plate or the like, or may be cultured three-dimensionally. Examples of three-dimensional culture methods include the methods described in Kempf et al., Nature Protocol, vol.10, 1345 (2015) and Halloin et al., Stem Cell Reports, vol.13,1 (2019). etc.
When cultured in three dimensions, cells are known to aggregate and form cell clusters. However, the cell death inducing agent of the present invention was difficult to induce cell death in pluripotent stem cells, etc. in such clustered cells. can induce cell death in pluripotent stem cells etc. even when the cell mixture is in an aggregated state.

By the purification method of the present invention, the cell mixture from which pluripotent stem cells and the like have been removed has a reduced proportion of pluripotent stem cells and the like and is composed exclusively of differentiated cells. That is, the present invention provides a method for purifying differentiated cells. According to the differentiated cell purification method of the present embodiment, it is possible to obtain a cell mixture that does not substantially contain pluripotent stem cells or the like or has a reduced proportion of pluripotent stem cells and the like. Therefore, the cell mixture obtained by the method of this embodiment can be suitably used as cells for transplantation into a living body.

In other aspects, the present invention also provides TVB-3166 or an analog thereof, TVB-3664 or an analog thereof, TVB-2640 or an analog thereof, FAS-IN- 1tosylate or an analog thereof, and FT113 or an analog thereof.

4. Method for producing cells for transplantation In one embodiment, the present invention provides a method for producing differentiated cells derived from highly purified pluripotent stem cells, preferably cardiomyocytes, comprising the following steps (i) to (iii). I will provide a. The highly purified differentiated cells, preferably cardiomyocytes, are used as cells for transplantation.
(i) inducing desired differentiated cells from pluripotent stem cells;
(ii) obtaining a cell mixture containing pluripotent stem cells and/or cells with differentiation potential derived from the pluripotent stem cells (undifferentiated stem cells) and differentiated cells by the step (i); and ( iii) A step of culturing the cell mixture obtained in step (ii) in the presence of a cell death inducer for pluripotent stem cells and undifferentiated stem cells.

The cell death inducing agent used in the method for producing cells for transplantation of the present embodiment is the same as that described in "2. Cell death inducing agent" above.

Step (i) in the method of this embodiment is a step of inducing desired differentiated cells from pluripotent stem cells. The pluripotent stem cells in step (i) are not particularly limited, but are preferably iPS cells or ES cells, more preferably iPS cells.

In step (i), the type of differentiated cells induced from pluripotent stem cells is not particularly limited, and may be induced into desired differentiated cells. As a method for inducing differentiated cells from pluripotent stem cells, known methods can be appropriately selected and used depending on the desired differentiated cells. Examples of differentiated cells derived from pluripotent stem cells include cardiomyocytes, myocytes, fibroblasts, nerve cells, immune cells (e.g., lymphocytes, etc.), vascular cells, eye cells (e.g., retinal pigment epithelium, etc.). Examples include, but are not limited to, blood cells (eg, megakaryocytes, red blood cells, etc.), other tissue cells, and their precursor cells. Examples of suitable differentiated cells include cardiomyocytes. Pluripotent stem cells can be induced into cardiomyocytes by the method exemplified in "3. Method for purifying differentiated cells" above. Further, step (ii) can also be performed by the method exemplified in "3. Method for purifying differentiated cells" above.

Step (iii) in the production method of this embodiment is a step of culturing the cell mixture obtained in step (ii) in the presence of the cell death inducing agent of the present invention. The culture in step (iii) can be performed by the method exemplified in "3. Method for purifying differentiated cells" above.

The manufacturing method of this embodiment may include other steps in addition to the steps (i) to (iii) above. Other steps include, for example, a step of purifying differentiated cells, a step of collecting differentiated cells, and the like. When these steps are added, these steps are performed between steps (i) and (ii) above, between steps (ii) and (iii) above, or after step (iii) above.
When the differentiated cells are cardiomyocytes, the method may further include a step of aggregating the obtained cardiomyocytes after step (iii). By adding this step, cardiomyocyte spheres can be manufactured.

For the purification process and recovery process of differentiated cells, an appropriate method can be selected depending on the type of differentiated cells. For example, when the differentiated cells are cardiomyocytes, the methods described in International Publication No. 2006/022377, International Publication No. 2007/088874, and International Publication No. 2016/010165 may be applied to the purification process. good. Further, as the recovery step, a centrifugation method or the like may be applied. The process of aggregating cardiomyocytes is described in International Publication No. 2009/017254, Hattori et al., Nat Methods, 7(1), 61-6(2010), Morita et al. Methods Mol Biol.2320:11-21( 2021), Tabei et al. J Heart Lung Transplant. 38(2):203-214 (2019) may be applied.
The cardiomyocyte sphere of the present invention has a diameter of 50 to 500 μm, preferably 100 to 300 μm, and more preferably 100 to 200 μm.

In the cells for transplantation obtained by the production method of the present embodiment, the purity of the differentiated cells expressed as number of differentiated cells/total number of cells x 100 may be, for example, 70% or more, or 80% or more. , 90% or more, 95% or more, 99% or more. Note that dead cells are not included in the above cell number.

The cells for transplantation produced by the production method of this embodiment have pluripotent stem cells etc. selectively removed, so the proportion of pluripotent stem cells etc. is reduced and is composed exclusively of differentiated cells. . Therefore, according to the manufacturing method of the present embodiment, it is possible to obtain cells for transplantation that substantially do not contain pluripotent stem cells or the like or have a reduced proportion of pluripotent stem cells and the like. Therefore, even when the cells for transplantation are transplanted into a living body, the risk of teratoma formation, etc. is reduced.

The present invention provides TVB-3166 or an analog thereof, TVB-3664 or an analog thereof, TVB-2640 or an analog thereof, FAS-IN-1 tosylate or an analog thereof, and FT113 or an analog thereof, for producing cells for transplantation. Use of at least one selected from the group consisting of analogs thereof.

5. Pharmaceutical Composition In one embodiment, the present invention provides the above-mentioned 3. Or 4. The present invention provides a pharmaceutical composition containing differentiated cells derived from highly purified pluripotent stem cells obtained by the method described in , preferably cardiomyocytes, or cell spheres obtained by aggregating these differentiated cells. When the differentiated cells are cardiomyocytes, the pharmaceutical composition preferably contains the cardiomyocytes in the form of cardiomyocyte spheres.

The subject to whom the pharmaceutical composition containing differentiated cells of the present invention, such as cardiomyocytes, particularly cardiomyocyte spheres, is transplanted is not particularly limited, but may be a human or a non-human animal in need of transplantation of the differentiated cells. . For example, the patient may be a patient whose differentiated cells are not functioning normally, or a patient who has a defect, disorder, etc. in the tissue containing the differentiated cells. Animals other than humans include, but are not limited to, mammals. Examples of mammals include primates such as monkeys; rodents such as mice and rats; pet animals such as dogs and cats; livestock such as cows, horses, sheep, and pigs. The subject to which differentiated cells derived from undifferentiated stem cells are transplanted is preferably an organism of the same species as the organism from which the undifferentiated stem cells are derived, and more preferably the same individual as the individual from which the undifferentiated stem cells are derived. . The subject to whom differentiated cells derived from undifferentiated stem cells are transplanted may be a "subject requiring transplantation" described in "6. Transplant method" below.

6. Transplantation method In one embodiment, the present invention also provides a method for transplantation of differentiated cells, comprising the following steps (i) to (iv):
(i) inducing desired differentiated cells from pluripotent stem cells;
(ii) obtaining a cell mixture containing pluripotent stem cells and/or undifferentiated stem cells derived from the pluripotent stem cells, and differentiated cells by the step (i);
(iii) culturing the cell mixture obtained in step (ii) in the presence of a cell death inducer for pluripotent stem cells and undifferentiated stem cells to obtain cells for transplantation, and (iv) obtaining cells for transplantation in the step (iv) A step of transplanting the cells for transplantation obtained in iii) into a subject in need of transplantation.

Steps (i) to (iii) in the transplantation method of this embodiment can be performed in the same manner as steps (i) to (iii) described in "4. Method for producing cells for transplantation" above. Furthermore, the cell death inducing agent of the present invention used in step (iii) of the transplantation method of this embodiment is the same as that described in "2. Cell death inducing agent" above.

Step (iv) in the transplantation method of this embodiment is a step of transplanting the cells for transplantation obtained in step (iii) to a subject requiring transplantation. "Subject requiring transplantation" means that in the subject's body, cells of the same type as the cells for transplantation obtained in step (iii) have defects, damage, etc., and the cells for transplantation are to be transplanted. This is a target that can be expected to improve symptoms caused by cell defects, damage, etc. Transplantation can be performed using common cell transplantation techniques. Specifically, examples include a method using an injection device described in International Publication No. 2020/013125, a method using a catheter, and the like.

The transplantation method of this embodiment may include other steps in addition to the steps (i) to (iv) above. Other steps include, for example, a step of purifying differentiated cells, a step of collecting differentiated cells, and the like. Moreover, when the differentiated cells are cardiomyocytes, the method may further include, and preferably includes, a step of aggregating the cardiomyocytes obtained in step (iii). When these steps are added, these steps are performed between the above steps (iii) and (iv). Such a purification step, a recovery step, or a cardiomyocyte aggregation step can be performed as described in "4. Method for producing cells for transplantation" above.

7. Medium In one embodiment, the present invention provides a medium (hereinafter also referred to as "the medium of the present invention"). The culture medium of this embodiment contains the above-mentioned cell death inducing agent of the present invention. The medium of the present invention can be used for culturing cells. As used herein, "culturing" means rearing or growing cells outside a living body (individual), and includes handling cells in so-called in vitro. "Medium" refers to 3. above. It refers to a liquid or solid substance that provides cells with a culture environment as described in et al.

The medium of the present invention includes, for example, any medium component, TVB-3166 or an analog thereof, TVB-3664 or an analog thereof, TVB-2640 or an analog thereof, FAS-IN-1 tosylate or an analog thereof, and FT113. and at least one compound selected from the group consisting of compounds selected from the group consisting of analogs thereof (i.e., the compound of the present invention). The medium of the present invention is a composition comprising a medium and a compound of the present invention. Examples of the medium include water and buffer solutions. The medium may be one that dissolves or disperses the compound of the present invention. The medium component may contain components effective for cell growth, and examples of such components include various components such as amino acids, vitamins, inorganic salts, saccharides, and growth factors. It may also contain other components such as antibiotics, buffers, chelating agents, and phenol red indicators.

The remaining components of the medium of the present invention, except for the compound of the present invention, can be obtained from a common cell culture medium used as a conventional medium [e.g., Dulbecco's modified Eagle's medium (DMEM), MEM culture medium (e.g., α- MEM, MEM [Hank's BSS]), RPMI culture solution (for example, RPMI 1640, etc.), F12 culture solution, StemPro34, mTeSR1, etc.] may have the same composition. The components have the same composition as general cell culture media used as undifferentiated stem cell maintenance media [e.g., StemFit medium, mTeSR (registered trademark) Essential 8 (registered trademark) medium, StemSure (registered trademark) medium, etc.] You can also use it as Furthermore, since the cell death inducing agent of the present invention induces cell death in pluripotent stem cells and the like by inhibiting fatty acid synthase activity, those that do not contain fatty acids are preferably used.

The concentration of the compound of the present invention in the medium of the present invention may be the same as the concentration of the compound of the present invention in the subject to which the cell death inducing agent of the present invention described above is applied. When the medium of the present invention contains the compound of the present invention at the above concentration, it becomes possible to effectively induce cell death of undifferentiated pluripotent stem cells and the like. In addition, when undifferentiated pluripotent stem cells, etc. and differentiated cells coexist, it is possible to induce cell death and remove the undifferentiated pluripotent stem cells, etc. while keeping the differentiated cells in a good growth state. .

The medium of the present invention preferably does not contain fatty acids.
The medium of the present invention may contain at least one compound selected from the group consisting of glucose, glutamine, and methionine.

In another aspect, the present invention provides TVB-3166 or an analog thereof, TVB-3664 or an analog thereof, TVB-2640 or an analog thereof, FAS- Provided is the use of at least one compound selected from the group consisting of IN-1 tosylate or an analog thereof, and a compound selected from the group consisting of FT113 or an analog thereof.

8. Kit In one embodiment, the present invention provides a kit comprising the above-described cell death inducing agent of the present invention (hereinafter also referred to as "kit of the present invention").

In addition to the cell death inducing agent of the present invention described above, the kit of the present invention further includes reagents, media, cell culture equipment, instructions for use, etc. for inducing differentiated cells from pluripotent stem cells. It's okay. Reagents for inducing differentiated cells can be appropriately selected depending on the differentiated cells to be induced. Reagents for inducing cardiomyocytes include, for example, chromosomal DNA demethylating agents such as demethylase, 5-azacytidine, and DMSO; PDGF, fibroblast growth factor 8 (FGF-8), endothelin 1 (ET1), Cytokines such as Midkine and bone morphogenetic protein 4 (BMP-4), G-CSF; Adhesion molecules such as gelatin, laminin, collagen, fibronectin; Vitamins such as retinoic acid; Nkx2.5/Csx, GATA4, MEF- Transcription factors such as 2A, MEF-2B, MEF-2C, MEF-2D, dHAND, eHAND, TEF-1, TEF-3, TEF-5 and MesP1; extracellular matrix derived from cardiomyocytes; Noggin, cordin, fetuin, Examples include, but are not limited to, BMP antagonists such as follistatin, sclerostin, dan, cerberus, gremlin, dante, and the like. Examples of media include Dulbecco's modified Eagle's culture medium (DMEM), MEM culture medium (α-MEM, MEM [Hank's BSS], etc.), RPMI culture medium (RPMI 1640, etc.), F12 culture medium, StemPro34 , mTeSRI, StemFit medium, mTeSR Essential 8 medium, StemSure medium, etc., but are not limited to these. In addition, examples of cell culture instruments include, but are not limited to, cell culture plates.

The kit of the present invention can be suitably used in the above method for purifying differentiated cells, and can further include instructions for explaining the method for purifying differentiated cells. By preparing a kit containing the above-mentioned cell death inducing agent of the present invention as well as reagents, instructions, etc. used in the method for purifying differentiated cells, it becomes possible to purify differentiated cells more easily and in a shorter time.

All prior art documents cited herein are incorporated by reference.

Next, the present invention will be further explained with reference to Examples, but the present invention is not limited thereto.

(1) Confirmation of the effect on human induced pluripotent stem cells (human iPS cells) Human induced pluripotent stem cells (253 G4 strain) (hereinafter sometimes referred to as "iPS cells") were purchased from Kyoto University, a national university corporation. Obtained from Professor Shinya Yamanaka of the iPS Cell Research Institute. The above iPS cells were cultured to maintain undifferentiation using Matrigel (BD Bioscience cat 354230). StemFit AK02N medium (Ajinomoto Co., Ltd.) was used as the undifferentiated maintenance culture medium. Technologies Inc. cat11875-119) that are commonly used as feeder-free media can be used.In addition, as a matrix, other media that are commonly used as feeder-free matrices such as iMatrix511 (matrixsome) can be used. It can be used as long as it is used in
For subplanting, iPS cell colonies were isolated using Accutase (Innovative Cell Technologies cat. AT-104) at 37°C for 3-5 minutes. Furthermore, 10 μM of CultureSure Y27632 (WAKO cat.034-24024) is added to the medium at the time of seeding, and is not added when changing the medium thereafter.
iPS cells maintained in undifferentiated culture as described above were cultured in a Matrigel-coated 24-well culture plate (Corning) using StemFit AK02N medium (Ajinomoto) + Y27632 (Wako cat.034-24024) at 3.0 to 3.5 × 10 ⁴ The cells were seeded at a density of 2 cells/well and cultured for 3-4 days at 37° C. in a 5% CO ₂ incubator. At 70-80% confluence, the medium was replaced with StemFitAK02N medium containing each compound listed in Table 1 at the final concentration listed in Table 2, and after culturing for an additional 3 days, the medium was fixed with 4% paraformaldehyde. The alkaline phosphatase (ALP) activity of the cells was developed using a StemTAG (registered trademark) alkaline phosphatase staining kit (Sigma 86-R), and cells stained red were confirmed as viable cells. As a control, wells cultured under the same conditions in StemFit AK02N medium (Ajinomoto) to which nothing was added were also prepared. As shown in Table 2, TVB3166 and TVB2640 can induce undifferentiated iPS cell death when added at a concentration of 80 nM, TVB3664 can induce death of undifferentiated iPS cells when added at a concentration of 16 nM, and FAS-IN-1 Tosylate and FT113 can induce death of undifferentiated iPS cells when added at a concentration of 400 nM. Cell death of differentiated iPS cells could be induced. On the other hand, orlistat (Sigma, O4139), which is known as an undifferentiated stem cell inducer, induced cell death when added at a concentration of 6 μM under the same conditions. Furthermore, the IC ₅₀ of the above compound as a fatty acid synthase inhibitor is as shown in Table 1, and the IC 50 of the fatty acid synthase inhibitory activity of the compound is determined by the concentration of each compound that can induce cell death in iPS cells. There was no correlation with ₅₀ .

(2) Confirmation of influence on cardiomyocytes The method for inducing differentiation and purification of iPS cells into cardiomyocytes used in this test is as follows.
For differentiation and induction into cardiomyocytes, when the iPS cells maintained in undifferentiated culture as in (1) become 70-90% confluent, B27 (without insulin, Invitrogen) and CHIR99021 were added to RPMI medium (Wako). The medium was replaced with one containing 6 μM (Selleckchem or Wako) and 1 μg/ml of BMP-4 (R&D Systems cat. 314-BP) (Day 0).
On Day 1, the RPMI medium (Wako) was replaced with a medium containing B27 (without insulin, Invitrogen) (hereinafter sometimes referred to as "RPMI/B27 insulin (-) medium"), and on Day 3, RPMI/B27 (Invitrogen) was added. Culture was performed by replacing the B27 insulin (-) medium with a medium supplemented with 5 μM of IWP2 or 5 μM of IWR-1 (Sigma I0161). On Day 6, the medium was changed to RPMI/B27 insulin (-), and from Day 7 onwards, MEMα (Invitrogen)/5% FBS/2% pyruvate (SIGMA) (hereinafter referred to as "prepared MEMα medium") was used. (Lian, X., et al., Nat Protocol, 2013, 8, 162-175). After detaching the cells using trypsin on Day 10, they were reseeded in a 15 cm collagen I coated dish (IWAKI) and cultured until Day 10-12 in a prepared MEMα medium supplemented with orlistat at 6 μM. From Day 13 onwards, culture in glucose (-) glutamine (-) lactic acid supplemented medium for about 3-4 days according to the method described in the literature (Tohyama, et al., (2016) Cell Metabolism, 23, 663-674.) Cells other than myocardial cells were killed to obtain purified myocardial cells. The purified cardiomyocytes were suspended in Cryostor CS10 (STEMCELL Technologies) and used as a frozen stock. Note that pulsating myocardial cells could be confirmed at the stage of Day 8 to Day 11.
Thaw the frozen stock of cardiomyocytes prepared by the above procedure into a 24-well culture plate (Corning) coated with iM221 (matrixsome) and seed them at 4 x ¹⁰ cells/well using prepared MEMα medium. After culturing in a 5% _CO2 incubator at 37°C for 3 days, the cells were replaced with prepared MEMα medium supplemented with each compound listed in Table 3 at each final concentration, and cultured under the above conditions for an additional 4 days. , LiveDead Viability/Cytotoxicity Kit for mammalian cells (Thermo L3224)) was used to stain live cells and dead cells. As a control, a well cultured under the same conditions in a conditioned MEMα medium to which nothing was added was also prepared. As shown in Table 3, TVB3166, TVB2640, TVB3664, FAS-IN-1 Tosylate, and FT113 had no effect on cardiomyocytes even when added at concentrations of 2 μM, 5 μM, and 10 μM. Moreover, the above-mentioned orlistat (manufactured by Sigma, O4139) had no effect when added at 2 μM, but when added at a concentration of 10 μM, cardiomyocytes died.

(3) Confirmation of influence on neural stem cells iPS cells (253 G4 strain) cultured in the same manner as in (1) above were seeded in a ⁶ -well culture plate (Corning) at a density of 2 x 10 cells/well, and then subjected to STEM diff. Differentiation into neural stem cells was performed using the SMADi Neural Induction kit (STEMCELL Technologies) according to the protocol attached to the kit. Differentiation into neural stem cells was confirmed by immunostaining using PAX6 antibody and Nestin antibody.
Differentiated neural stem cells were seeded onto a Matrigel-coated 24-well culture plate using StemFit AK02N medium (Ajinomoto) at a density of 4 x 10 ⁵ cells/well, and after 2 days, the medium was treated with each compound listed in Table 4 at each final concentration. The culture medium was replaced with StemFit AK02N medium (Ajinomoto Co., Ltd.) added to give the following conditions, and after further culturing for 3 days, live cells and dead cells were stained using LiveDead Viability/Cytotoxicity Kit for mammian cells (Thermo L3224). The results are shown in Table 4.

As shown in Table 4, TVB3166 and TVB3664 induce cell death in neural stem cells derived from iPS cells at a final concentration of 80 nM, and TVB2640 induces cell death in neural stem cells derived from iPS cells at a final concentration of 400 nM. guided. Further, the above-mentioned orlistat (Sigma, O4139) slightly induced cell death in neural stem cells derived from iPS cells at a final concentration of 10 μM, but had no effect at lower concentrations.

(4) Confirmation of influence on iPS cell clusters iPS cells (253 G4 strain) maintained undifferentiated in the same manner as in (1) above were cultured in a 24-well culture plate with small dimples on the bottom (Corning Elplasia multi-well plate cat. 4441) at 1 x ¹⁰ cells/well, cultured for 1 day in StemFit AK02N medium (Ajinomoto), and confirmed that the iPS cells were in a clump under a microscope. ) was replaced with each compound listed in Table 5 added to each final concentration (concentration listed in the first batch). After culturing for an additional 5 days, the cell mass was collected and placed in a 24-well flat bottom culture plate (Corning ), and the state of the cell mass was observed under a microscope. As shown in FIG. 1, TVB3166, TVB3664, and TVB2640 induced cell death in iPS cells in a cell cluster state at a final concentration of 200 nM. In addition, a second test was conducted with the added concentration increased. The final concentrations are as shown in Table 5 (concentrations listed for the second time). In the second test, as a control, wells cultured in StemFit AK02 medium supplemented with DMSO in the same amount as the amount of each compound were also cultured under the same conditions. As shown in FIG. 2 and Table 5, TVB3166, TVB3664, and TVB2640 induced cell death in iPS cells in a cell cluster state at a final concentration of 200 nM or higher. Furthermore, the above-mentioned orlistat (Sigma, O4139) did not induce cell death in iPS cells in a cell cluster state even at a final concentration of 10 μM.

(5) Confirmation of the effect on a cell mixture containing iPS cell-derived cardiomyocytes and iPS cells 5 × 10 ⁵ purified cardiomyocytes prepared by the procedure shown in (2) and undifferentiated by the procedure shown in (1) A cell mixture containing 5×10 ⁴ maintained cultured iPS cells was prepared and seeded onto a 12-well plate coated with iM221 (matrixsome). One day later, StemFit AK02N medium (Ajinomoto Co., Ltd.) was replaced with StemFit AK02N medium supplemented with each compound listed in Table 6 at each final concentration, and DMSO was added as a control, and after further culturing for 3 days, the undifferentiated cells were Immunostaining was performed for the marker OCT4 and the differentiated cardiomyocyte marker Actinin. The results are shown in Table 6.

When TVB3166, TVB3664, and TVB2640 were added at a final concentration of 100 nM, cardiomyocytes expressing Actinin were confirmed as in the control, but iPS cells expressing OCT4 were not confirmed, indicating that cells in an undifferentiated state were specific. It was shown that it can induce cell death. Even at a final concentration of 1 μM for the above-mentioned orlistat (manufactured by Sigma, O4139), many OCT-positive cells were confirmed.

(6) Confirmation of the effect on the mixed mass of iPS cells and cardiomyocytes 8 x 10 ⁵ iPS cells (253 G4 strain) maintained undifferentiated and cultured as described in (1) above (procedures shown in (2)) 4 × 10 ⁶ purified cardiomyocytes prepared using StemFit AK02N medium (Ajinomoto) were mixed, seeded in a 6-well culture plate with small dimples (Corning Elplasia multiwell plate cat. 4440), and cultured for 1 day. After confirming that the cells were clustered using a microscope, the medium was replaced with StemFit AK02N medium (Ajinomoto Co., Ltd.) containing each compound listed in Table 7 at a final concentration of 10 μM, and after further culturing for 3 days, the cell clusters were collected. The collected cell mass was suspended in a 3:1 mixture of Trypsin-EDTA (Thermo cat. 25200056) and Accumax (Innovative Cell Technologies cat. AM105) and placed in a water bath set at 37°C for 5 minutes. After forming single cells with moderate shaking, they were fixed with 4% paraformaldehyde, membrane permeabilized with 0.1% Triton The results are shown in Figure 3. As is clear from Figure 3, TVB3166, TVB3664, and TVB2640 had a final concentration of 10 μM and the proportion of undifferentiated cells was approximately 9. %, each decreased to about 1%, but in the case of orlistat (manufactured by Sigma, O4139), the decrease was only up to 4.4%.

As a second test, a similar test was conducted with each compound added at a concentration of 100 nM, and the results are shown in FIG. As is clear from Figure 4, at a final concentration of 100 nM, TVB3166, TVB3664, and TVB2640 each reduced the proportion of undifferentiated cells to 3 to 5%, compared to approximately 18.5% in the negative control (DMSO). However, with the above-mentioned orlistat (manufactured by Sigma, O4139), the reduction was only up to 14%.

A smaller amount can induce cell death of undifferentiated stem cells even when the cells are in a spheroid state. Therefore, high efficacy and safety can be expected in the entire process of inducing differentiation from pluripotent stem cells to differentiated cells, subsequent purification of differentiated cells, and even transplantation.

Claims (10)

At least one member selected from the group consisting of TVB-3166 or an analog thereof, TVB-3664 or an analog thereof, TVB-2640 or an analog thereof, FAS-IN-1 tosylate or an analog thereof, and FT113 or an analog thereof An agent for inducing cell death of pluripotent stem cells and undifferentiated stem cells derived from the pluripotent stem cells, which contains a compound of the following as an active ingredient. In a cell mixture by culturing a cell mixture containing pluripotent stem cells and/or undifferentiated stem cells derived from the pluripotent stem cells, and differentiated cells in the presence of the cell death inducing agent according to claim 1. A method to purify differentiated cells. 3. The purification method according to claim 2, wherein the cell mixture containing pluripotent stem cells and/or undifferentiated stem cells derived from the pluripotent stem cells and differentiated cells is in the form of a cell mass. The purification method according to claim 2, wherein the differentiated cells are cardiomyocytes. The purification method according to claim 2, wherein the pluripotent stem cells are induced pluripotent stem cells. A method for producing highly purified pluripotent stem cell-derived cardiomyocytes, the method comprising:
A cell mixture containing pluripotent stem cells and/or undifferentiated stem cells derived from the pluripotent stem cells, and cardiomyocytes,
Subjected to the step of implementing the purification method according to claim 4,
The proportion of pluripotent stem cells and/or undifferentiated stem cells derived from the pluripotent stem cells in the cell mixture is 10% or less;
Method. A cell mixture containing pluripotent stem cells and/or undifferentiated stem cells derived from the pluripotent stem cells, and cardiomyocytes was prepared with a glucose and/or glutamine content of 10% or less of the normal medium and lactic acid was added. 7. The method for producing cardiomyocytes according to claim 6, which comprises culturing in a medium. A method for producing pluripotent stem cell-derived cardiomyocyte spheres, further comprising the step of aggregating pluripotent stem cell-derived cardiomyocytes produced by the method according to claim 6 or 7. A pharmaceutical composition for treating heart disease, comprising cardiomyocyte spheres derived from pluripotent stem cells produced by the method according to claim 8. A method for producing differentiated cells derived from highly purified pluripotent stem cells, the method comprising:
A cell mixture containing pluripotent stem cells and/or undifferentiated stem cells derived from the pluripotent stem cells, and differentiated cells,
Subjected to the step of implementing the purification method according to any one of claims 2 to 5,
The proportion of pluripotent stem cells and/or undifferentiated stem cells derived from the pluripotent stem cells in the cell mixture is 10% or less;
Method.