Method for producing selected cell population and selection culturing medium

Abstract

Provided are a method for producing a selected cell population and a selection culturing medium, with which it is possible to easily select for atrial myocytes and pacemaker cells. This method for producing a selected cell population comprises a selection step for culturing a first cell population including ventricular myocytes, atrial myocytes, and pacemaker cells in a selection culturing medium to obtain a second cell population in which the ratio of the total number of the atrial myocytes and the pacemaker cells with respect to the total number of the ventricular myocytes, the atrial myocytes, and the pacemaker cells is larger than that in the first cell population. The selection culturing medium does not substantially contain glutamine and glucose, and contains α-ketoglutaric acid in a concentration at which survival of the atrial myocytes and the pacemaker cells is maintained and death of the ventricular myocytes is induced during the culturing in the selection step.

Description

Method for producing selected cell population and selection medium

[0001] The present invention relates to a method for producing a selected cell population and a selection medium.

[0002] Patent Document 1 describes a method for inducing cell death in undifferentiated stem cells by culturing them in a cell culture medium to which lactic acid, pyruvic acid, or fatty acids are added, which does not contain sugars, and which does not contain glutamine in its amino acid composition, and a method for selectively selecting cardiomyocytes from a mixture of cardiomyocytes and non-cardiomyocytes.

[0003] Patent Document 2 describes a method for removing undifferentiated stem cells, which comprises culturing a cell mixture containing undifferentiated stem cells and differentiated cells in the presence of an agent for removing undifferentiated stem cells, which contains at least one agent selected from the group consisting of a fatty acid synthesis inhibitor, a fatty acid utilization inhibitor, and a cholesterol synthesis inhibitor.

[0004] Patent Document 3 describes a method for culturing pluripotent stem cells, which comprises culturing pluripotent stem cells in a pluripotent stem cell culture medium containing L-tryptophan or an L-tryptophan derivative at a concentration of 176 μM or more.

[0005] Patent Document 4 discloses a method for producing a cardiomyocyte cell population, the method comprising: i. 1. incubating cardiovascular mesoderm cells in a cardiac induction medium comprising a BMP component, optionally greater than a selected amount of BMP4, and retinoic acid (RA), and optionally one or more FGF inhibitors, WNT inhibitors, optionally IWP2, VEGF, and activin / nodal inhibitors, optionally SB-431542, for a period of time to generate cardiovascular progenitor cells expressing TBX18; and 2. incubating the cardiovascular progenitor cells in a basal medium comprising VEGF for a period of time to generate a cardiomyocyte cell population enriched in sinoatrial node-like pacemaker cardiomyocytes (SANLCM); or ii. 1. Incubating for a period of time in a cardiac induction medium containing one or more WNT inhibitors, optionally IWP2, and VEGF; and optionally an FGF component and / or an activin / nodal inhibitor, optionally SB-431542, to generate cardiovascular progenitor cells expressing NKX2-5; and 2. Incubating the cardiovascular progenitor cells for a period of time in a basal medium containing VEGF to generate cardiovascular progenitor cells expressing NKX2-5.ｐｏｓ cTnT ｐｏｓ Methods are described that include generating a cardiomyocyte population that is enriched for these cells and substantially devoid of SANLCM; and optionally isolating the cardiomyocyte population using one or more cardiomyocyte-specific surface markers, optionally wherein the markers are signal-regulatory protein alpha (SIRPA) and thymocyte differentiation antigen 1 (THY-1 / CD90).

[0006] Patent Document 5 discloses a method for producing a cardiomyocyte cell population enriched for sinoatrial node-like pacemaker cardiomyocytes (SANLPCs), comprising: (a) providing a starting population of human cardiomyocytes; (b) contacting the human cardiomyocytes with an agent that specifically binds to CD34; and (c) isolating cells bound by the agent from cells not bound by the agent, thereby producing a CD34 cell population enriched for SANLPCs. ＋ obtaining a population of cardiomyocytes.

[0007] International Publication No. 2016 / 010165 International Publication No. 2018 / 074457 International Publication No. 2018 / 181342 International Publication No. 2016 / 131137 International Publication No. 2021 / 220248

[0008] However, it has been difficult to simply select atrial myocytes and pacemaker cells from a cell population containing these cells.

[0009] The present invention has been made in view of the above problems, and one of its objects is to provide a method for producing a selected cell population that allows for easy selection of atrial myocytes and pacemaker cells, and a selection medium.

[0010] [1] A method for producing a selected cell population according to one embodiment of the present invention for solving the above-mentioned problems includes a selection step of culturing a first cell population containing ventricular muscle cells, atrial muscle cells, and pacemaker cells in a selection medium to obtain a second cell population in which the ratio of the total number of atrial muscle cells and pacemaker cells to the total number of ventricular muscle cells, atrial muscle cells, and pacemaker cells is greater than that of the first cell population, wherein the selection medium is substantially free of glutamine and glucose and contains α-ketoglutaric acid at a concentration that induces death of the ventricular muscle cells and maintains survival of the atrial muscle cells and pacemaker cells during the culture in the selection step. According to the present invention, a method for producing a selected cell population that allows for easy selection of atrial muscle cells and pacemaker cells is provided.

[0011] [2] In the method of [1], the first cell population may be a cell population obtained by in vitro differentiation induction of stem cells capable of differentiating into cardiomyocytes. [3] In the method of [1] or [2], the selection medium may contain the α-ketoglutaric acid at a concentration equal to or greater than a lower limit specified within a range of more than 8 mM. [4] In any of the methods of [1] to [3], the selected cell population for transplantation may be produced. [5] In the method of [4], the selected cell population for transplantation may be produced for the treatment of bradyarrhythmia.

[0012] [6] One embodiment of the present invention for solving the above-mentioned problems provides a selection medium that is used to culture a first cell population containing ventricular muscle cells, atrial muscle cells, and pacemaker cells to obtain a second cell population in which the ratio of the total number of atrial muscle cells and pacemaker cells to the total number of ventricular muscle cells, atrial muscle cells, and pacemaker cells is greater than that of the first cell population, the selection medium being substantially free of glutamine and glucose and containing α-ketoglutaric acid at a concentration that induces death of the ventricular muscle cells and maintains survival of the atrial muscle cells and pacemaker cells during the culture. According to the present invention, a selection medium is provided that allows for easy selection of atrial muscle cells and pacemaker cells.

[0013] [7] In the selection medium of [6], the first cell population may be a cell population obtained by in vitro differentiation induction of stem cells capable of differentiating into cardiomyocytes. [8] The selection medium of [6] or [7] may contain the α-ketoglutaric acid at a concentration equal to or greater than a lower limit specified within a range of more than 8 mM.

[0014] According to the present invention, a method for producing a selected cell population and a selection medium that allow for easy selection of atrial myocytes and pacemaker cells are provided.

[0015] 1 is an explanatory diagram showing the results of immunostaining of a cardiomyocyte population obtained by inducing differentiation of pluripotent stem cells. 2 is an explanatory diagram showing the results of flow cytometry analysis of a cardiomyocyte population obtained by inducing differentiation of pluripotent stem cells. 3 is an explanatory diagram showing the results of immunostaining of pacemaker cells contained in a cardiomyocyte population obtained by inducing differentiation of pluripotent stem cells. 4 is an explanatory diagram showing the results of fluorescent imaging observation of a cardiomyocyte population cultured in an α-ketoglutaric acid-added medium. 5 is an explanatory diagram showing the results of quantitative evaluation by fluorescent imaging observation of the culture area of ​​a cardiomyocyte population cultured in an α-ketoglutaric acid-added medium. 6 is an explanatory diagram showing the results of immunostaining of a cardiomyocyte population cultured in an α-ketoglutaric acid-added medium.

[0016] An embodiment of the present invention will be described below, although the present invention is not limited to this embodiment.

[0017] The method for producing a selected cell population according to this embodiment (hereinafter referred to as "this method") includes a selection step of culturing a first cell population containing ventricular muscle cells, atrial muscle cells, and pacemaker cells in a selection medium to obtain a second cell population in which the ratio of the total number of atrial muscle cells and pacemaker cells to the total number of ventricular muscle cells, atrial muscle cells, and pacemaker cells is greater than that of the first cell population, and the selection medium is substantially free of glutamine and glucose, and contains α-ketoglutaric acid at a concentration that induces death of the ventricular muscle cells and maintains survival of the atrial muscle cells and pacemaker cells during the culture in the selection step.

[0018] Furthermore, the selection medium according to this embodiment is a selection medium that is used to culture a first cell population containing ventricular muscle cells, atrial muscle cells, and pacemaker cells, to obtain a second cell population in which the ratio of the total number of atrial muscle cells and pacemaker cells to the total number of ventricular muscle cells, atrial muscle cells, and pacemaker cells is greater than that of the first cell population, and is substantially free of glutamine and glucose, and contains α-ketoglutaric acid at a concentration that induces the death of the ventricular muscle cells and maintains the survival of the atrial muscle cells and pacemaker cells during the culture.

[0019] That is, the inventors of the present invention have conducted extensive research into technical means for selecting some subtypes of cardiomyocytes from a cell population containing multiple subtypes of cardiomyocytes, and unexpectedly and independently discovered that atrial myocytes and pacemaker cells can be easily selected from the cell population by culturing the cell population in a medium having a specific composition, thereby completing the present invention.

[0020] In the selection step of this method, a first cell population is cultured, which includes ventricular myocytes (hereinafter referred to as "VM cells"), atrial myocytes (hereinafter referred to as "AM cells"), and pacemaker cells (hereinafter referred to as "PM cells"), all of which are subtypes of cardiomyocytes.

[0021] The VM cells contained in the first cell population are cardiomyocytes (ventricular myocytes) that constitute the ventricular wall of the heart of a living organism, and / or differentiation-induced cells corresponding to the ventricular myocytes. That is, the VM cells are ventricular myocytes collected from a living organism, or cardiomyocytes that are induced to differentiate in vitro from stem cells capable of differentiating into cardiomyocytes and express specific markers. Specifically, VM cells are identified as cardiomyocytes that express one or more markers selected from the group consisting of MYL2, IRX4, and MLC2v, which are not expressed by AM cells and PM cells.

[0022] The AM cells contained in the first cell population are cardiomyocytes (atrial myocytes) that constitute the atrial wall of the heart of a living body, and / or differentiation-induced cells corresponding to the atrial myocytes. That is, AM cells are cardiomyocytes that express specific markers and are differentiated in vitro from atrial myocytes collected from a living body and / or stem cells capable of differentiating into cardiomyocytes. Specifically, AM cells may be identified as cardiomyocytes that express one or more markers selected from the group consisting of NPPA, NR2F2, and MLC2a, which are not expressed by VM cells, or as cardiomyocytes that express one or more markers selected from the group consisting of NPPA and NR2F2, which are not expressed by VM cells and PM cells. It should be noted that VM cells with a low degree of differentiation may express MLC2a.

[0023] The PM cells contained in the first cell population are cardiomyocytes (pacemaker cells) (sometimes called sinoatrial node cells) that are present in the sinoatrial node of a living heart and generate automatic action potentials, and / or differentiation-induced cells corresponding to the pacemaker cells. That is, PM cells are cardiomyocytes that are induced to differentiate in vitro from pacemaker cells collected from a living body and / or stem cells capable of differentiating into cardiomyocytes and express specific markers. Specifically, PM cells are identified as cardiomyocytes that express one or more markers selected from the group consisting of SHOX2, HCN4, and MLC2a, which are not expressed by VM cells, and are identified as cardiomyocytes that express one or more markers selected from the group consisting of SHOX2 and HCN4, which are not expressed by VM cells and AM cells.

[0024] Another known subtype of cardiomyocytes is the cardiomyocytes present in the atrioventricular node of the heart (atrioventricular nodal cells). Atrioventricular nodal cells are identified as cardiomyocytes that express one or more markers selected from the group consisting of MSX2 and TBX2, which are not expressed by VM cells, AM cells, and PM cells.

[0025] Cardiomyocytes are identified as cells that express one or more markers (cardiomyocyte markers) commonly expressed by VM cells, AM cells, and PM cells, such as α-actinin, troponin I (TnI), and TNNT2 (cardiac troponin T).

[0026] The first cell population is not particularly limited as long as it is a cell population containing VM cells, AM cells, and PM cells, and may be a cell population collected from a living body, but is preferably a cell population induced to differentiate in vitro from stem cells that have the ability to differentiate into cardiomyocytes.

[0027] Here, the stem cells used for differentiation induction to obtain the first cell population are not particularly limited as long as they are stem cells capable of differentiating into a cardiomyocyte population including VM cells, AM cells, and PM cells. For example, it is preferable that the stem cells are pluripotent stem cells (e.g., one or more selected from the group consisting of iPS (induced pluripotent stem) cells, ES (embryonic stem) cells, ntES (nuclear transfer embryonic stem) cells, EG (embryonic germ) cells, or Muse (multilineage-differentiating stress-ending) cells), or stem cells other than the pluripotent stem cells (e.g., stem cells induced from pluripotent stem cells, stem cells obtained by reprogramming a differentiated cell, or somatic stem cells).

[0028] The method for inducing differentiation of a first cell population in vitro from stem cells capable of differentiating into cardiomyocytes is not particularly limited as long as it is a method that can obtain the first cell population. For example, known methods for inducing differentiation of stem cells in vitro into cardiomyocytes (for example, the methods described in Patent Document 1 (WO 2016 / 010165), Patent Document 2 (WO 2018 / 074457), Patent Document 4 (WO 2016 / 131137), or Patent Document 5 (WO 2021 / 220248)) or modified methods thereof are preferably used.

[0029] That is, the first cell population can be obtained, for example, by culturing stem cells capable of differentiating into cardiomyocytes in a medium containing a component that induces differentiation of the stem cells into cardiomyocytes. Specifically, when inducing differentiation of iPS cells into cardiomyocytes, for example, a nerve cell culture supplement (e.g., B-27) that does not contain insulin is first added to a basal medium (e.g., RPMI medium). ＴＭ supplement, minus insulin (A18956-02, Gibco TM The iPS cells are cultured (differentiation-inducing culture) in a medium prepared by adding a GSK-3β inhibitor (e.g., CHIR-99021) and BMP-4 to a supplement-added medium (serum-free medium) prepared by adding a Wnt signal inhibitor (e.g., IWR-1) to the supplement-added medium (serum-free medium), and then, on the first day from the start of the differentiation-inducing culture (preferably 24 hours from the start of the culture), the medium is replaced with a supplement-added medium (medium not containing a GSK-3β inhibitor or BMP-4). On the third day from the start of the differentiation-inducing culture, the medium is replaced with a supplement-added medium containing a Wnt signal inhibitor (e.g., IWR-1). and retinoic acid, and on day 6 from the start of the differentiation-inducing culture, the culture medium is replaced with a medium prepared by adding retinoic acid to a supplement-added medium (a medium not containing a Wnt signal inhibitor), on day 7 from the start of the differentiation-inducing culture, the culture medium is replaced with a medium prepared by adding retinoic acid to a serum-added medium (for example, a medium prepared by adding fetal bovine serum (FBS) to MEMα medium), and on day 11 from the start of the differentiation-inducing culture, the culture medium is replaced with the serum-added medium (a medium not containing retinoic acid), and the culture is preferably carried out.

[0030] The VM cells, AM cells, and PM cells contained in the first cell population are preferably mammalian cells, and may be cells of a mammal other than a human (e.g., a primate (e.g., a monkey), a rodent (e.g., a mouse, a rat, a hamster, a guinea pig, or a rabbit), a carnivore (e.g., a dog, a cat), or an ungulate (e.g., a pig, a cow, a horse, a goat, or a sheep)), but are particularly preferably human cells.

[0031] Specifically, for example, when producing a selected cell population for transplantation into humans as described below, the first cell population cultured in a selection medium in this method is a human cell population containing human VM cells, AM cells, and PM cells. In this case, the second cell population obtained in the selection step of this method and the selected cell population finally obtained by this method are also human cell populations. Furthermore, when the first cell population is a cell population obtained by in vitro differentiation induction of iPS cells, the iPS cells are preferably iPS cells derived from an HLA-homologous donor.

[0032] The proportion of the number of VM cells to the total number of VM cells, AM cells, and PM cells in the first cell population (a proportion calculated by dividing the number of VM cells contained in the cell population by the sum of the number of VM cells, the number of AM cells, and the number of PM cells contained in the cell population and multiplying the obtained value by 100) (hereinafter referred to as "VM cell proportion") may be, for example, 1% or more, 3% or more, 5% or more, 7% or more, 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, or 95% or more.

[0033] Furthermore, the proportion of VM cells in the first cell population may be, for example, 97% or less, 95% or less, 93% or less, 90% or less, 85% or less, 80% or less, 75% or less, 70% or less, 65% or less, 60% or less, 55% or less, 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, or 5% or less. The proportion of VM cells in the first cell population may be specified by any combination of any of the above-mentioned lower limit values ​​and any of the above-mentioned upper limit values.

[0034] The ratio of the total number of AM cells and PM cells to the total number of VM cells, AM cells, and PM cells in the first cell population (the ratio calculated by dividing the total number of AM cells and PM cells contained in the cell population by the total number of VM cells, AM cells, and PM cells contained in the cell population, and multiplying the obtained value by 100) (hereinafter referred to as "AM+PM cell ratio") may be, for example, 1% or more, 3% or more, 5% or more, 7% or more, or 10% or more. It may be 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, or 95% or more.

[0035] The proportion of AM+PM cells in the first cell population may be, for example, 97% or less, 95% or less, 93% or less, 90% or less, 85% or less, 80% or less, 75% or less, 70% or less, 65% or less, 60% or less, 55% or less, 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, or 5% or less. The proportion of AM+PM cells in the first cell population may be specified by any combination of any of the above-mentioned lower limit values ​​and any of the above-mentioned upper limit values.

[0036] The ratio of the number of PM cells to the total number of VM cells, AM cells, and PM cells in the first cell population (a ratio calculated by dividing the number of PM cells contained in the cell population by the sum of the number of VM cells, the number of AM cells, and the number of PM cells contained in the cell population and multiplying the obtained value by 100) (hereinafter referred to as "PM cell ratio") may be, for example, 1% or more, 3% or more, 5% or more, 7% or more, 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, or 95% or more.

[0037] The proportion of PM cells in the first cell population may be, for example, 97% or less, 95% or less, 93% or less, 90% or less, 85% or less, 80% or less, 75% or less, 70% or less, 65% or less, 60% or less, 55% or less, 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, or 5% or less. The proportion of PM cells in the first cell population may be specified by any combination of any of the above-mentioned lower limit values ​​and any of the above-mentioned upper limit values.

[0038] The first cell population preferably does not contain undifferentiated cells, but may contain undifferentiated cells. The ratio of the number of undifferentiated cells to the total number of cells in the first cell population (the ratio calculated by dividing the number of undifferentiated cells contained in the cell population by the total number of cells contained in the cell population and multiplying the result by 100) (hereinafter referred to as the "undifferentiated cell ratio") may be, for example, 90% or less, 85% or less, 80% or less, 75% or less, 70% or less, 65% or less, 60% or less, 55% or less, 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, 5% or less, 3% or less, or 1% or less. The undifferentiated cells are identified as cells that express one or more markers selected from the group consisting of Oct3 / 4, Nanog, Sox2, SSEA-1, SSEA-3, SSEA-4, TRA1-60, TRA1-81, Lin28, and Fbx15.

[0039] The first cell population preferably does not contain cells other than cardiomyocytes (undifferentiated cells and / or differentiated cells other than cardiomyocytes) (hereinafter referred to as "non-cardiomyocytes"), but may contain non-cardiomyocytes. The ratio of the number of non-cardiomyocytes to the total number of cells in the first cell population (the ratio calculated by dividing the number of non-cardiomyocytes contained in the cell population by the total number of cells contained in the cell population and multiplying the result by 100) (hereinafter referred to as "non-cardiomyocyte ratio") may be, for example, 90% or less, 85% or less, 80% or less, 75% or less, 70% or less, 65% or less, 60% or less, 55% or less, 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, 5% or less, 3% or less, or 1% or less. Non-cardiomyocytes are identified as cells that do not express the above-mentioned cardiac muscle cell markers, for example.

[0040] The selection medium used in the culture (hereinafter referred to as "selection culture") to obtain a second cell population from a first cell population in the selection step of this method has a composition necessary to induce the death of VM cells and maintain the survival of AM cells and PM cells in the selection culture.

[0041] That is, the selection medium contains α-ketoglutaric acid (hereinafter referred to as "aKG") at a concentration that induces the death of VM cells and maintains the survival of AM and PM cells during selection culture in the selection medium. Here, in the present invention, aKG is not limited to α-ketoglutaric acid (also known as 2-oxoglutaric acid) having CAS Registry Number 328-50-7, but may be an α-ketoglutaric acid derivative that acts on cells substantially as α-ketoglutaric acid, such as a derivative with improved cell permeability, such as dimethyl-α-ketoglutaric acid (also known as dimethyl-2-oxoglutaric acid) having CAS Registry Number 13192-04-6.

[0042] That is, the aKG contained in the selection medium is not particularly limited as long as the effects of the present invention are obtained, but cell-permeable aKG is preferred. The cell-permeable aKG is not particularly limited as long as it is taken up into cells in the selection medium, but for example, α-ketoglutaric acid ester is preferred. The α-ketoglutaric acid ester may be α-ketoglutaric acid monoester or α-ketoglutaric acid diester. The α-ketoglutaric acid ester is preferably an α-ketoglutaric acid alkyl ester. The α-ketoglutaric acid alkyl ester is preferably an alkyl ester having an alkyl group containing 1 to 15 carbon atoms, more preferably an alkyl ester having an alkyl group containing 1 to 12 carbon atoms, and particularly preferably an alkyl ester having an alkyl group containing 1 to 10 carbon atoms. Specific examples of commercially available cell-permeable aKG include dimethyl-α-ketoglutaric acid, diethyl-α-ketoglutaric acid, and 5-octyl-α-ketoglutaric acid.

[0043] The aKG concentration in the selection medium is not particularly limited as long as it is within a range in which the effects of the present invention can be obtained, and it is sufficient that the concentration is such that the death of VM cells is induced and the survival of AM cells and PM cells is maintained during selection culture using the selection medium.

[0044] The aKG concentration of the selection medium may be statistically determined, for example, by conducting a preliminary selection culture test using multiple first cell populations with different preparation conditions (for example, in the case of planning to carry out selection culture of first cell populations induced to differentiate from pluripotent stem cells, multiple first cell populations with different conditions for the differentiation induction (for example, the origin or strain of the pluripotent stem cells used for the differentiation induction)), and determining the aKG concentration at which the death of VM cells is induced and the survival of AM cells and PM cells is maintained in the selection culture test.

[0045] Furthermore, the aKG concentration of the selection medium may be determined, for example, by extracting a portion of a cell population from the first cell population that is to be actually cultured in the selection process, conducting a preliminary selection culture test using the portion of the cell population, and determining the aKG concentration at which the death of VM cells is induced and the survival of AM cells and PM cells is maintained in the selection culture test.

[0046] Specifically, the aKG concentration in the selection medium may be, for example, a concentration equal to or greater than the lower limit defined within the range of more than 8 mM, preferably a concentration equal to or greater than the lower limit defined within the range of more than 8 mM and not more than 15 mM, and particularly preferably a concentration equal to or greater than the lower limit defined within the range of more than 8 mM and not more than 12 mM.

[0047] In other words, the lower limit of the aKG concentration in the selection medium may be determined, for example, by conducting a preliminary selection culture test using multiple candidate media containing αKG at different concentrations within any of the above ranges, and determining the lower limit concentration at which VM cell death is induced and AM and PM cell survival is maintained in the selection culture test.

[0048] More specifically, the aKG concentration of the selection medium may be, for example, more than 8 mM, 8.5 mM or more, 9 mM or more, 9.5 mM or more, 10 mM or more, 10.5 mM or more, 11 mM or more, 11.5 mM or more, or 12 mM or more.

[0049] The aKG concentration of the selection medium may be, for example, 100 mM or less, 70 mM or less, 50 mM or less, 40 mM or less, or 30 mM or less. The aKG concentration of the selection medium may be specified by any combination of any of the above-mentioned lower limit values ​​and any of the above-mentioned upper limit values.

[0050] The selection medium is substantially free of glutamine. In this regard, the selection medium preferably does not contain glutamine, but may contain a trace amount of glutamine as long as the effects of the present invention are achieved.

[0051] That is, the glutamine concentration in the selection medium may be low enough to induce the death of VM cells and maintain the survival of AM and PM cells during selection culture in the selection medium.

[0052] The glutamine concentration of the selection medium is determined to be low enough to induce the death of VM cells and maintain the survival of AM and PM cells in a preliminary selection culture test, similar to the determination of the aKG concentration described above.

[0053] Specifically, the glutamine concentration of the selection medium may be, for example, 0.2 mM or less, preferably 0.1 mM or less, more preferably 0.05 mM or less, even more preferably 0.01 mM or less, even more preferably 5 μM or less, even more preferably 1 μM or less, even more preferably 0.5 μM or less, and particularly preferably 0.1 μM or less.

[0054] The glutamine concentration referred to here refers to the concentration of glutamine, including L-glutamine, in a dipeptide of L-glutamine and another amino acid (e.g., L-alanyl-L-glutamine) used as a substitute for L-glutamine. That is, for example, when the glutamine concentration in the selection medium is 0.1 mM or less, the sum of the glutamine concentration and the concentration of the dipeptide containing L-glutamine in the selection medium is 0.1 mM or less.

[0055] The selection medium is substantially free of glucose. In this regard, the selection medium preferably does not contain glucose, but may contain a trace amount of glucose as long as the effects of the present invention are achieved.

[0056] That is, the glucose concentration in the selection medium may be low enough to induce the death of VM cells and maintain the survival of AM and PM cells during selection culture in the selection medium.

[0057] The glucose concentration of the selection medium is determined to be low enough to induce the death of VM cells and maintain the survival of AM and PM cells in a preliminary selection culture test, similar to the determination of the aKG concentration described above.

[0058] Specifically, the glucose concentration of the selection medium may be, for example, 1 mM or less, preferably 0.5 mM or less, more preferably 0.1 mM or less, even more preferably 0.05 mM or less, even more preferably 0.01 mM or less, even more preferably 5 μM or less, even more preferably 1 μM or less, even more preferably 0.5 μM or less, and particularly preferably 0.1 μM or less.

[0059] When the selection medium is substantially free of glucose, the selection medium may be substantially free of sugars that can be utilized by cells. In this case, the selection medium preferably does not contain sugars that can be utilized by cells, but may contain trace amounts of such sugars as long as the effects of the present invention are achieved.

[0060] In other words, the concentration of sugars available to cells in the selection medium should be low enough to induce the death of VM cells and maintain the survival of AM and PM cells during selection culture in the selection medium.

[0061] The concentration of sugars available to cells in the selection medium is determined, for example, in a preliminary selection culture test, as a concentration low enough to induce the death of VM cells while maintaining the survival of AM and PM cells, similar to the determination of the aKG concentration described above.

[0062] Specifically, the concentration of sugars available to cells in the selection medium may be, for example, 1 mM or less, preferably 0.5 mM or less, more preferably 0.1 mM or less, even more preferably 0.05 mM or less, even more preferably 0.01 mM or less, even more preferably 5 μM or less, even more preferably 1 μM or less, even more preferably 0.5 μM or less, and particularly preferably 0.1 μM or less.

[0063] The selection medium contains other components necessary for inducing the death of VM cells and maintaining the survival of AM and PM cells during selection culture. That is, the selection medium contains, for example, one or more components selected from the group consisting of amino acids, vitamins, inorganic salts, hormones, and growth factors necessary for inducing the death of VM cells and maintaining the survival of AM and PM cells during selection culture. The selection medium may also contain a basal medium substantially free of glutamine and glucose (e.g., one or more components selected from the group consisting of DMEM medium, MEM medium, RPMI medium, and Ham's F12 medium substantially free of glutamine and glucose).

[0064] The selection medium is a medium for culturing cardiomyocytes, not a medium for culturing undifferentiated cells. Therefore, the selection medium may not contain components necessary for maintaining the undifferentiated potential of stem cells (e.g., pluripotent stem cells) or components that suppress stem cell differentiation. Specifically, the selection medium may not contain, for example, bFGF, TGF-β, albumin, or methionine.

[0065] In the selection step of this method, a first cell population is cultured (selection culture) in the above-described selection medium. In the selection culture, the death of VM cells is induced in the selection medium while the survival of AM and PM cells is maintained. That is, in the selection culture, the proportion of AM + PM cells in the cell population cultured in the selection medium is increased by selectively killing VM cells among VM, AM, and PM cells. Thus, in the selection step, a second cell population is obtained in which the proportion of AM + PM cells is greater than that of the first cell population.

[0066] In the selective culture, it is not necessary to kill all VM cells contained in the cell population, and VM cells may remain in the second cell population. However, the more VM cells are killed in the selective culture, the higher the proportion of AM+PM cells in the second cell population (i.e., the higher the purity of AM and PM cells) will be.

[0067] Specifically, the ratio of the AM + PM cell proportion of the second cell population to the AM + PM cell proportion of the first cell population (the value obtained by dividing the AM + PM cell proportion of the second cell population by the AM + PM cell proportion of the first cell population) (hereinafter referred to as the "AM + PM cell proportion increase ratio") may be, for example, 1.02 or more, preferably 1.04 or more, more preferably 1.06 or more, even more preferably 1.10 or more, even more preferably 1.15 or more, even more preferably 1.20 or more, even more preferably 1.25 or more, even more preferably 1.30 or more, even more preferably 1.40 or more, and particularly preferably 1.50 or more. In the selection step, the selection culture may be performed until a second cell population is obtained whose AM + PM cell proportion increase ratio is equal to or greater than any of the above lower limits.

[0068] Furthermore, the proportion of AM+PM cells in the second cell population may be, for example, 50% or more, preferably 55% or more, more preferably 60% or more, even more preferably 65% ​​or more, even more preferably 70% or more, even more preferably 75% or more, even more preferably 80% or more, even more preferably 85% or more, even more preferably 90% or more, even more preferably 95% or more, even more preferably 96% or more, even more preferably 97% or more, even more preferably 98% or more, and particularly preferably 99% or more. In the selection step, the selection culture may be performed until a second cell population having a proportion of AM+PM cells equal to or greater than any of the above lower limits is obtained.

[0069] Furthermore, the proportion of PM cells in the second cell population may be, for example, 1% or more, 3% or more, 5% or more, 7% or more, 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, or 95% or more.

[0070] Furthermore, the proportion of PM cells in the second cell population may be, for example, 97% or less, 95% or less, 93% or less, 90% or less, 85% or less, 80% or less, 75% or less, 70% or less, 65% or less, 60% or less, 55% or less, 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, or 5% or less. The proportion of PM cells in the second cell population may be specified by any combination of any of the above-mentioned lower limit values ​​and any of the above-mentioned upper limit values.

[0071] The second cell population preferably does not contain undifferentiated cells, but may contain undifferentiated cells. The proportion of undifferentiated cells in the second cell population may be, for example, 90% or less, 85% or less, 80% or less, 75% or less, 70% or less, 65% or less, 60% or less, 55% or less, 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, 5% or less, 3% or less, or 1% or less.

[0072] The second cell population preferably does not contain non-cardiomyocytes, but may contain non-cardiomyocytes. The proportion of non-cardiomyocytes in the second cell population may be, for example, 90% or less, 85% or less, 80% or less, 75% or less, 70% or less, 65% or less, 60% or less, 55% or less, 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, 5% or less, 3% or less, or 1% or less.

[0073] The time for culturing the cell population in the selection medium in the selection process (the time from the start of culturing the first cell population in the selection medium to the time when the second cell population is obtained) (hereinafter referred to as the "selection culture time") is not particularly limited as long as the effects of the present invention are obtained, but may be, for example, 12 hours or more, preferably 18 hours or more, more preferably 24 hours or more, even more preferably 30 hours or more, even more preferably 36 hours or more, even more preferably 40 hours or more, even more preferably 44 hours or more, and particularly preferably 48 hours or more.

[0074] The selective culture time may be, for example, 750 hours or less, 600 hours or less, 500 hours or less, 400 hours or less, 350 hours or less, 300 hours or less, 250 hours or less, or 200 hours or less. The selective culture time may be specified by any combination of any of the above-mentioned lower limit values ​​and any of the above-mentioned upper limit values. Note that the medium may be replaced at an appropriate time during the selective culture time.

[0075] Other conditions for the selection culture in the selection step can be general culture conditions for cardiomyocytes. That is, since cardiomyocytes are adhesive cells, VM cells, AM cells, and PM cells are cultured in a state of adhering to the surface of a culture substrate. The culture substrate is not particularly limited as long as it is used for culturing cardiomyocytes, and for example, commercially available multi-well culture plates, dishes, flasks, or roller bottles are preferably used.

[0076] The selective culture may be carried out in an atmosphere set at a temperature of 35° C. or higher, preferably 36° C. or higher, and particularly preferably 36.5° C. or higher. Alternatively, the selective culture may be carried out in an atmosphere set at a temperature of 40° C. or lower, preferably 39° C. or lower, more preferably 38° C. or lower, and particularly preferably 37.5° C. or lower. The temperature of the atmosphere in which the selective culture is carried out may be specified by any combination of any of the above-mentioned lower limit values ​​and any of the above-mentioned upper limit values.

[0077] The selective culture of the present method does not require a procedure for detaching cells from the surface of the culture substrate, as in passaging. Therefore, in the selection step of the present method, the VM cells, AM cells, and PM cells adhered to the surface of the culture substrate may be cultured in a selection medium, and a second cell population containing AM cells and PM cells adhered to the surface of the culture substrate may be obtained without a procedure for detaching the AM cells and PM cells from the surface of the culture substrate.

[0078] This method does not require cell separation procedures using antibodies against cell surface markers (e.g., physical cell sorting procedures using a cell sorter or magnetic beads). Therefore, this method may obtain a second cell population with an increased proportion of AM+PM cells without artificially binding antibodies to any of the VM cells, AM cells, and PM cells. In this case, antibodies against the cell surface markers are not bound to the AM cells and PM cells contained in the second cell population.

[0079] In this method, there is no need to artificially modify the genes of the VM cells, AM cells, or PM cells cultured in the selection medium. Thus, in this method, a second cell population with an increased proportion of AM+PM cells may be obtained without artificially modifying the genes of any of the VM cells, AM cells, and PM cells contained in the first cell population.

[0080] The present method may further include another step before the selection step. That is, the present method may include, for example, a pre-culture step of culturing the first cell population in a medium containing glutamine and glucose (hereinafter referred to as a "pre-culture medium") before the selection step. In the pre-culture step, culturing the first cell population in a pre-culture medium containing glutamine and glucose promotes differentiation induction of cells contained in the first cell population and / or promotes maturation of differentiated cells contained in the first cell population.

[0081] The glutamine concentration of the preculture medium may be, for example, 0.5 mM or more, 0.8 mM or more, 1 mM or more, 1.2 mM or more, 1.5 mM or more, or 2 mM or more.

[0082] The glucose concentration of the pre-culture medium may be, for example, 4 mM or more, 5 mM or more, 7 mM or more, 10 mM or more, or 15 mM or more.

[0083] According to this method, a selected cell population in which the proportion of AM+PM cells is effectively increased can be produced. In this method, the second cell population after the selective culture may be directly obtained as the selected cell population.

[0084] In addition, in this method, the second cell population after the selective culture may be subjected to a predetermined treatment, and the treated cell population may be obtained as a selected cell population. That is, for example, if the second cell population after the selective culture contains non-cardiomyocytes, the second cell population may be subjected to a treatment to remove the non-cardiomyocytes, and the treated cell population (i.e., a cell population in which the proportion of non-cardiomyocytes is reduced compared to that of the second cell population) may be obtained as a selected cell population.

[0085] Here, the treatment for removing non-cardiomyocytes is not particularly limited as long as it is a treatment that reduces the proportion of non-cardiomyocytes in the second cell population. For example, as described in the above Patent Document 1 (WO 2016 / 010165) or Patent Document 2 (WO 2018 / 074457), a method of culturing the second cell population in a medium having a composition that selectively kills non-cardiomyocytes is preferably used.

[0086] The selected cell population produced by this method is useful for various applications. For example, the heart of a living body beats as a whole when an action potential generated by PM cells in the sinoatrial node propagates to the atrial wall composed of AM cells, and the action potential propagates further from the atrial wall to the ventricular wall via the atrioventricular node.

[0087] Therefore, if the function of PM cells in a living heart is impaired, the heart will not beat normally, resulting in a condition called bradyarrhythmia. Although patients with bradyarrhythmia may undergo surgery to implant a mechanical pacemaker, a fundamental treatment for bradyarrhythmia has yet to be established. Therefore, in recent years, a treatment called a biological pacemaker has been proposed, in which cardiomyocytes are transplanted into the hearts of patients with bradyarrhythmia.

[0088] In this regard, a selected cell population in which the proportion of AM+PM cells is effectively increased by this method is suitable for transplantation into a living body as a biological pacemaker. Therefore, this method may be used to produce a selected cell population for transplantation, more specifically, a selected cell population for transplantation for the treatment of bradyarrhythmia. That is, this method may be a method for producing a selected cell population for transplantation, more specifically, a selected cell population for transplantation for the treatment of bradyarrhythmia.

[0089] Furthermore, the selected cell population for transplantation produced by this method is expected to function by engrafting into the heart of the living body to which it is transplanted. For this reason, the selected cell population for transplantation produced by this method is preferably a cell population to be transplanted into the heart of a living body, more specifically, a cell population to be transplanted by injection into the heart wall (ventricular wall or atrial wall) of the living body.

[0090] The bradyarrhythmia that can be treated by transplanting the selected cell population produced by this method may be, for example, bradyarrhythmia caused by dysfunction of the sinoatrial node (e.g., sick sinus syndrome) and / or bradyarrhythmia caused by treatment for the treatment of cardiac diseases other than bradyarrhythmia (e.g., surgical procedures).

[0091] The selected cell populations produced by this method are also useful as in vitro test models, for example, for screening drugs that target AM and / or PM cells.

[0092] Next, a specific example according to this embodiment will be described.

[0093] [Preparing pluripotent stem cells] First, mTeSR ＴＭ Human iPS cells (201B7 strain, iPS Academia Japan, Inc.) (hereinafter referred to as "hPSCs") were suspended in a medium prepared by adding Y-27632 (034-24024, Fujifilm Wako Pure Chemical Industries, Ltd.) to a final concentration of 5 μM to Medium 1 (ST-85850, STEMCELL Technologies, Inc.) to prepare a cell suspension.

[0094] Then, cells were plated at a density of 5 × 10 cells in each well of a 6-well plate (3530466 Falcon™, Corning) pre-coated with Matrigel (354230, Corning). ５ hPSCs were seeded by adding a cell suspension in an amount to give cells / well, and cultured until the hPSCs became subconfluent.

[0095] [Differentiation induction: Day 0] Add 10 mL of B-27 to 500 mL of RPMI medium (189-02025, Fujifilm Wako Pure Chemical Industries, Ltd.). ＴＭ supplement, minus insulin (A18956-02, Gibco TM , Thermo Fisher Scientific) was added to prepare a medium (hereinafter referred to as "RPMI+supplement medium").

[0096] Meanwhile, the medium was removed from each well containing the subconfluent hPSCs cultured as described above, and the wells were washed with D-PBS(-) (049-29795, Fujifilm Wako Pure Chemical Industries, Ltd.).

[0097] Next, D-PBS(-) was removed from each well, and 2 mL of a medium prepared by adding CHIR-99021 (034-23103, Fujifilm Wako Pure Chemical Industries, Ltd.) to a final concentration of 6 μM to 12 μM (e.g., 9.2 μM) and BMP-4 (314-BP, R&D Systems) to a final concentration of 1 ng / mL to RPMI+supple medium was added to each well, and differentiation induction culture was initiated.

[0098] [Differentiation induction: Day 1] 24 hours after the start of differentiation induction culture, the medium was removed from each well and washed with D-PBS(-). Next, the D-PBS(-) was removed from each well, and 2 mL of RPMI+supplement medium was added to each well, and culture was continued.

[0099] [Differentiation induction: Day 3] From day 3 onwards, differentiation induction was carried out according to two protocols. Specifically, in some wells, differentiation induction was carried out according to protocol VM, resulting in a cell population with a relatively high content of VM cells (hereinafter referred to as "induced cells VM"). In other wells, differentiation induction was carried out according to protocol AM+PM, resulting in a cell population with a relatively high content of AM and PM cells (hereinafter referred to as "induced cells AM+PM").

[0100] Specifically, in protocol VM, on the third day after the start of differentiation-inducing culture, the medium was removed from each well, and 2 mL of medium prepared by adding IWR-1 (I0161, Sigma-Aldrich) to a final concentration of 5 μM to RPMI+supple medium was added to each well, and the culture was continued.

[0101] On the other hand, in protocol AM+PM, on the third day after the start of differentiation-inducing culture, the medium was removed from each well, and 2 mL of medium prepared by adding IWR-1 to a final concentration of 1 μM and retinoic acid (R2625, Sigma-Aldrich) to a final concentration of 1 μM to RPMI+supple medium was added to each well, and the culture was continued.

[0102] [Differentiation induction: Day 4] In protocol AM+PM, on day 4 from the start of differentiation induction culture, without removing the medium from each well, an additional 2 mL of medium prepared by adding IWR-1 to a final concentration of 1 μM and retinoic acid to a final concentration of 1 μM to RPMI+supple medium was added to each well, and culture was continued with a total medium volume of 4 mL / well.

[0103] [Differentiation induction: Day 6] In Protocol VM, on day 6 from the start of differentiation induction culture, the medium was removed from each well, and 2 mL of RPMI + supple medium was added to each well to continue the culture. On the other hand, in Protocol AM + PM, on day 6 from the start of differentiation induction culture, the medium was removed from each well, and 2 mL of medium prepared by adding retinoic acid to a final concentration of 1 μM to each well was added to each well to continue the culture.

[0104] [Differentiation induction: Days 7 and 9] In Protocol VM, on days 7 and 9 from the start of differentiation induction culture, the medium was removed from each well and replaced with 1 L of MEMα medium (12571-089, Invitrogen). TM Culture was continued by adding 2 mL of a medium prepared by adding 65 mL of inactivated fetal bovine serum (FBS) and 20 mL of sodium pyruvate solution (S8636, Sigma-Aldrich) to MEM+FBS medium (hereinafter referred to as "MEM+FBS medium").

[0105] On the other hand, in protocol AM+PM, on days 7 and 9 from the start of differentiation-inducing culture, the medium was removed from each well, and 2 mL of medium prepared by adding retinoic acid to MEM+FBS medium at a final concentration of 1 μM was added to each well, and the culture was continued.

[0106] [Differentiation Induction: Day 11] In Protocol VM and Protocol AM+PM, on day 11 from the start of differentiation induction culture, cells obtained by differentiation induction were collected from each well and reseeded into the wells of a new 6-well plate. Specifically, cells were first collected from each well using trypsin solution and suspended in MEM+FBS medium. The cell suspension was then centrifuged and the supernatant was removed. MEM+FBS medium was added to the centrifuged cells and suspended to prepare a cell suspension.

[0107] Then, cells were cultured at a cell density of 3 × 10 in each well of a 6-well plate previously coated with iMatrix-221 (892 062, Matrixome Co., Ltd.). ６ The cells were seeded by adding a cell suspension in an amount to give cells / well, and then cultured for 2 days.

[0108] In this way, induced cells VM were obtained by differentiation induction culture according to protocol VM, and induced cells AM+PM were obtained by differentiation induction culture according to protocol AM+PM.

[0109] [Removal of Non-Cardiomyocytes] Further, additional culture was performed to remove non-cardiomyocytes from the induced VM cells and the induced AM+PM cells obtained as described above. Specifically, the medium was removed from each well containing the induced VM cells or the induced AM+PM cells, and PBS was added and incubated for 5 minutes. Next, the PBS was removed from each well, and 2 mL of AS501 medium (Ajinomoto Co., Inc.), which does not contain glucose or glutamine but contains 4 mM lactic acid, was added to each well to initiate culture. This culture was continued for 4 days. The medium was changed daily.

[0110] In this way, by further culturing the induced cells VM, a cardiomyocyte cell population with a relatively high content of VM cells (hereinafter referred to as "cardiomyocytes VM") was obtained, and by further culturing the induced cells AM+PM, a cardiomyocyte cell population with a relatively high content of AM cells and PM cells (hereinafter referred to as "cardiomyocytes AM+PM") was obtained.

[0111] The pulsations of the VM and AM+PM cardiomyocytes were measured on days 30 and 60 after the start of differentiation-inducing culture. The pulsations per unit time of the AM+PM cardiomyocytes were significantly higher than that of the VM cardiomyocytes at both times. Furthermore, the pulsations per unit time of the VM cardiomyocytes on day 60 were reduced to about half of that on day 30, whereas the pulsations per unit time of the AM+PM cardiomyocytes remained almost unchanged between days 30 and 60.

[0112] [Evaluation 1] Immunostaining was performed on VM cardiomyocytes and AM+PM cardiomyocytes using MLC2v and MLC2a as markers. Specifically, the medium was removed from each well containing VM cardiomyocytes or AM+PM cardiomyocytes, and the cells were fixed by adding 4% paraformaldehyde.

[0113] The cells were then immunostained using anti-MLC2v antibody (79935 rabbit, Abcam) or anti-MLC2a antibody (311 011 mouse, Synaptic Systems) as the primary antibody, and Alexa488 anti-rabbit IgG (A-21206, Thermo Fisher Scientific) or Alexa594 anti-mouse IgG (A-21203, Thermo Fisher Scientific) as the secondary antibody. Cell nuclei were stained with Hoechst staining. After staining, the cells in the wells were observed under a fluorescence microscope (Keyence Corporation).

[0114] [Result 1] Figure 1 shows fluorescent images of cells in wells obtained by fluorescence microscopy. Specifically, for VM cardiomyocytes ("VM" in the figure) and AM+PM cardiomyocytes ("AM" in the figure), Figure 1 shows a fluorescent image of cells fluorescently stained green with anti-MLC2v antibody ("MLC2v" in the figure), a fluorescent image of cells fluorescently stained red with anti-MLC2a antibody ("MLC2a" in the figure), and a fluorescent image obtained by superimposing these images ("Merge" in the figure).

[0115] As shown in Figure 1, most VM cardiomyocytes were fluorescently stained with anti-MLC2v antibody, and only a few were stained with anti-MLC2a antibody. On the other hand, most AM+PM cardiomyocytes were fluorescently stained with anti-MLC2a antibody, and only a few were stained with anti-MLC2v antibody. This confirms that the majority of VM cardiomyocytes are VM cells, and the majority of AM+PM cardiomyocytes are AM and PM cells.

[0116] [Evaluation 2] Flow cytometry analysis was performed on VM cardiomyocytes and AM+PM cardiomyocytes using TNNT2 and MLC2a as markers. Specifically, VM cardiomyocytes or AM+PM cardiomyocytes were collected from each well using trypsin solution and suspended in MEMα medium supplemented with FBS. The cell suspension was then centrifuged and the supernatant was removed. After centrifugation, 4% paraformaldehyde was added to the cells to fix them.

[0117] The VM cardiomyocytes and AM+PM cardiomyocytes were then divided into an antibody staining group and an isotype control group. The VM cardiomyocytes for isotype control were mixed with the AM+PM cardiomyocytes for isotype control to prepare a mixed cardiomyocyte suspension for isotype control. The VM cardiomyocyte suspension for staining, the AM+PM cardiomyocyte suspension for staining, and the mixed cardiomyocyte suspension for isotype control were then centrifuged, and the supernatants were removed.

[0118] Then, a staining solution prepared by mixing an FITC-labeled anti-TNNT2 antibody (TNNT2-FITC rabbit, Miltenyi Biotec), an APC-labeled anti-MLC2a antibody (MLC2a-APC mouse, Miltenyi Biotec), and ImmunoBlock (KAC Corporation) was added to each of the VM cardiomyocytes for staining and the AM+PM cardiomyocytes for staining.

[0119] On the other hand, a staining solution prepared by mixing an FITC-labeled control antibody (REA control FITC, Miltenyi Biotec), an APC-labeled control antibody (REA control APC, Miltenyi Biotec), and ImmunoBlock was added to the mixed cardiomyocytes for isotype control. The stained cells were then analyzed using a flow cytometer (Beckman Coulter).

[0120] [Result 2] Figure 2 shows the analysis results by flow cytometer for mixed cardiomyocytes ("Negative control" in the figure), VM cardiomyocytes ("VM" in the figure), and AM+PM cardiomyocytes ("AM" in the figure).

[0121] As shown in Figure 2, 92.80% of VM cardiomyocytes were TNNT2-positive and MLC2a-negative, and 7.14% of AM+PM cardiomyocytes were TNNT2-positive and MLC2a-positive. On the other hand, 97.32% of AM+PM cardiomyocytes were TNNT2-positive and MLC2a-positive, and 2.68% of AM+PM cardiomyocytes were TNNT2-positive and MLC2a-negative. That is, it was confirmed that approximately 93% of VM cardiomyocytes were VM cells, and approximately 97% of AM+PM cardiomyocytes were AM cells and PM cells.

[0122] [Evaluation 3] Immunostaining of AM+PM cardiomyocytes was performed using cardiac troponin T and SHOX2 as markers. Specifically, the medium was removed from each well containing AM+PM cardiomyocytes, and 4% paraformaldehyde was added to fix the cells.

[0123] The cells were then immunostained using anti-Cardiac Troponin T antibody (45932 rabbit, Abcam) or anti-SHOX2 antibody (55740 mouse, Abcam) as the primary antibody, and Alexa488 anti-rabbit IgG (A-21206, Thermo Fisher Scientific) or Alexa594 anti-mouse IgG (A-21203, Thermo Fisher Scientific) as the secondary antibody. Cell nuclei were stained with Hoechst staining. The stained cells in the wells were then observed under a fluorescence microscope (Keyence Corporation).

[0124] [Result 3] Figure 3 shows fluorescent images of cells in wells obtained by fluorescence microscopy. Specifically, Figure 3 shows a fluorescent image of AM+PM cardiomyocytes stained green with anti-Cardiac Troponin T antibody (labeled "TNT" in the figure), a fluorescent image of cells stained red with anti-SHOX2 antibody (labeled "SHOX2" in the figure), and a fluorescent image obtained by superimposing these images (labeled "MERGE" in the figure).

[0125] As shown in Figure 3, all cells were fluorescently stained with anti-Cardiac Troponin T antibody, and some of them were also stained with anti-SHOX2 antibody, confirming that AM+PM cardiomyocytes contain PM cells.

[0126] [Selection Culture 1] Selection culture was performed for VM cardiomyocytes, a cell population with a high proportion of VM cells, and AM+PM cardiomyocytes, a cell population with a high proportion of AM+PM cells. Five aKG-supplemented media with different dm-aKG concentrations were prepared by adding dimethyl-α-ketoglutarate (Merck) (hereinafter referred to as "dm-aKG") to a glucose- and glutamine-free DMEM medium (Glc(-), Gln(-), Sartorius) (hereinafter referred to as "Glc(-)Gln(-) medium") at final concentrations of 4 mM, 8 mM, 12 mM, 16 mM, or 20 mM.

[0127] Meanwhile, the medium was removed from each well containing the cardiomyocytes VM or cardiomyocytes AM+PM, and 2 mL of MEMα medium (Thermo Fisher Scientific) containing 5.6 mM glucose and 2 mM glutamine (hereinafter referred to as "Glc(+)Gln(+) medium") was added and cultured.

[0128] Then, the Glc(+)Gln(+) medium was removed from each well, and 2 mL of one of the five aKG-supplemented media was added to each well to initiate selection culture. For comparison, in some wells, after removing the Glc(+)Gln(+) medium, fresh Glc(+)Gln(+) medium was added without adding the aKG-supplemented medium, and the culture was continued.

[0129] [Evaluation 4] On the second day after the start of selection culture, the staining reagent from the LIVEDEAD Viability assay kit (Thermo Fisher Scientific) was added to each well, and live cells were fluorescently stained green with calcein AM. Then, fluorescent imaging of each well was performed using an imaging analyzer (Cell3iMager duos2, SCREEN Holdings Co., Ltd.), and the area of ​​the green-stained portion in each well was measured.

[0130] [Result 4] Figure 4 shows fluorescence images of the entire wells obtained by fluorescence imaging. Specifically, Figure 4 shows fluorescence images (three wells for each condition) of wells cultured in aKG-supplemented medium containing dm-aKG at concentrations of 4 mM, 8 mM, 12 mM, 16 mM, or 20 mM (labeled "4 mM," "8 mM," "12 mM," "16 mM," and "20 mM" in the figure) and wells cultured in Glc(+)Gln(+) medium without dm-aKG (labeled "Glc(+)Gln(+)" in the figure) for VM cardiomyocytes ("VM" in the figure) and AM+PM cardiomyocytes ("AM+SANLPCs" in the figure).

[0131] FIG. 5 also shows the results of measuring the area of ​​the green-stained portion in each well by fluorescent imaging observation. Specifically, Figure 5 shows the results (n = 3) of measuring the area of ​​the green-stained portion (vertical axis: "Area (sq μm) / Sum") in wells cultured in a Glc(+)Gln(+) medium containing no dm-aKG ("Glc(+)_Gln(+)" in the figure) and in wells cultured in aKG-supplemented medium containing dm-aKG at concentrations of 4 mM, 8 mM, 12 mM, 16 mM, or 20 mM ("dm-aKG_4mM," "dm-aKG_8mM," "dm-aKG_12mM," "dm-aKG_16mM," and "dm-aKG_20mM" in the figure).

[0132] As shown in Figures 4 and 5, the number of viable cells in the cardiomyocytes VM tended to decrease as the dm-aKG concentration in the medium increased, and the decrease was particularly pronounced when the dm-aKG concentration in the medium was greater than 8 mM (specifically, 12 mM or greater). In contrast, the number of viable cells in the cardiomyocytes AM+PM did not change with the dm-aKG concentration in the medium.

[0133] In other words, it was confirmed that cells contained in cardiomyocytes VM die when cultured in a medium containing dm-aKG at a relatively high concentration (more than 8 mM, more specifically, 12 mM or more), whereas cells contained in cardiomyocytes AM+PM remain viable even when cultured in a medium containing dm-aKG at this relatively high concentration.

[0134] [Selection Culture 2] A mixed cell population prepared by mixing VM cardiomyocytes and AM+PM cardiomyocytes was subjected to selection culture. Specifically, VM cardiomyocytes and AM+PM cardiomyocytes were collected from each well using trypsin solution and suspended in Glc(+)Gln(+) medium, respectively. The cell suspension was then centrifuged, and the supernatant was removed. Glc(+)Gln(+) medium was added to the VM cardiomyocytes and AM+PM cardiomyocytes after centrifugation, respectively, to prepare VM cardiomyocyte suspensions and AM+PM cardiomyocyte suspensions.

[0135] Furthermore, the VM cardiomyocyte suspension and the AM+PM cardiomyocyte suspension were mixed so that the ratio of the number of VM cardiomyocytes to the number of AM+PM cardiomyocytes was 1:1, and a mixed cell suspension containing the AM cardiomyocytes and the AM+PM cardiomyocytes was prepared.

[0136] Then, each well of a 6-well plate was cultured at a total cell density of 1.0 × 10 ６ The mixed cell population was seeded by adding the mixed cell suspension to each well in an amount to give 10 cells / well, and culture was initiated. On day 53 after the initiation of culture, the Glc(+)Gln(+) medium was removed from each well, and 2 mL of aKG-supplemented medium prepared by adding dm-aKG to a final concentration of 12 mM to the Glc(-)Gln(-) medium was added to each well, and selection culture of the mixed cell population was initiated.

[0137] For comparison, in some wells, after removing the Glc(+)Gln(+) medium, fresh Glc(+)Gln(+) medium was added without adding aKG-supplemented medium, and the culture was continued.

[0138] [Evaluation 5] The mixed cell population after the selective culture was subjected to immunostaining using MLC2v and MLC2a as markers. Specifically, on day 11 from the start of the selective culture, the medium was removed from each well, and the cells were fixed by adding 4% paraformaldehyde.

[0139] The cells were then immunostained using anti-MLC2v antibody (79935 rabbit, Abcam) or anti-MLC2a antibody (311 011 mouse, Synaptic Systems) as the primary antibody, and Alexa488 anti-rabbit IgG (A-21206, Thermo Fisher Scientific) or Alexa594 anti-mouse IgG (A-21203, Thermo Fisher Scientific) as the secondary antibody. Cell nuclei were stained with Hoechst staining. After staining, the cells in the wells were observed under a fluorescence microscope (Keyence Corporation).

[0140] [Result 5] Figure 6 shows fluorescent images of cells in wells obtained by fluorescence microscopy. Specifically, Figure 6 shows a fluorescent image of cells fluorescently stained green with anti-MLC2v antibody ("MLC2v"), a fluorescent image of cells fluorescently stained red with anti-MLC2a antibody ("MLC2a"), and a fluorescent image obtained by overlaying these images ("Merge") for the mixed cell population cultured in Glc(+)Gln(+) medium without aKG ("Glc(+)_Gln(+)" in the figure) and the mixed cell population cultured in aKG-supplemented medium ("dm-aKG_12mM" in the figure).

[0141] As shown in Figure 6, in the mixed cell population cultured in Glc(+)Gln(+) medium, approximately half of the cells were fluorescently stained with anti-MLC2v antibody, and the remaining approximately half of the cells were stained with anti-MLC2a antibody.

[0142] In contrast, the mixed cell population cultured in aKG-supplemented medium showed a significantly reduced cell number compared to the mixed cell population cultured in Glc(+)Gln(+) medium, and most of the cells were fluorescently stained with anti-MLC2a antibody, with only a few cells stained with anti-MLC2v antibody.

[0143] That is, by culturing a mixed cell population containing VM cells, AM cells, and PM cells in a medium supplemented with aKG, it was possible to selectively kill VM cells while maintaining the survival of AM cells and PM cells, thereby easily obtaining a cell population with a significantly increased proportion of AM cells and PM cells.

Claims

1. A method for producing a selected cell population, comprising a selection step of culturing a first cell population containing ventricular muscle cells, atrial muscle cells, and pacemaker cells in a selection medium to obtain a second cell population in which the ratio of the total number of atrial muscle cells and pacemaker cells to the total number of ventricular muscle cells, atrial muscle cells, and pacemaker cells is greater than that of the first cell population, wherein the selection medium is substantially free of glutamine and glucose, and contains α-ketoglutaric acid at a concentration that induces the death of the ventricular muscle cells and maintains the survival of the atrial muscle cells and pacemaker cells during the culture in the selection step.

2. The method for producing a selected cell population according to claim 1, wherein the first cell population is a cell population obtained by inducing differentiation in vitro from stem cells capable of differentiating into cardiomyocytes.

3. The method for producing a selected cell population according to claim 1 or 2, wherein the selection medium contains the α-ketoglutaric acid at a concentration equal to or greater than the lower limit set within a range of more than 8 mM.

4. The method for producing a selected cell population according to claim 1 or 2, which produces the selected cell population for transplantation.

5. The method for producing a selected cell population according to claim 4, which produces the selected cell population for transplantation for the treatment of bradyarrhythmia.

6. A selection medium for use in culturing a first cell population comprising ventricular muscle cells, atrial muscle cells, and pacemaker cells to obtain a second cell population in which the ratio of the total number of atrial muscle cells and pacemaker cells to the total number of ventricular muscle cells, atrial muscle cells, and pacemaker cells is greater than that of the first cell population, the selection medium being substantially free of glutamine and glucose, and containing α-ketoglutaric acid at a concentration that induces the death of the ventricular muscle cells and maintains the survival of the atrial muscle cells and pacemaker cells during the culture.

7. The selection medium according to claim 6, wherein the first cell population is a cell population obtained by inducing differentiation in vitro from stem cells capable of differentiating into cardiomyocytes.

8. The selection medium according to claim 6 or 7, containing α-ketoglutaric acid at a concentration equal to or greater than the lower limit set within the range of more than 8 mM.

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