Method for evaluating differentiation state of cell, method for determining differentiation state of cell, method for producing cardiomyocyte, and reagent kit

Abstract

To provide means for evaluating a differentiation state of a cell with higher accuracy.SOLUTION: Provided is a method of inducing the differentiation of a pluripotent stem cell into a cardiomyocyte in a liquid culture medium by first induction treatment of inducing the differentiation of a pluripotent stem cell into a mesodermal cell and second induction treatment of inducing the differentiation of a mesodermal cell into a cardiomyocyte, collecting supernatant of a liquid culture medium including the cell whose differentiation is induced by the second induction treatment, measuring miRNA-3p of miR-1 / 133a cluster in the supernatant, and determining a differentiation state of the cell on the basis of the miRNA-3p measured value.SELECTED DRAWING: Figure 10

Description

[Technical field]

[0001] The present invention relates to a method for evaluating the differentiation state of a cell.The present invention relates to a method for determining the differentiation state of a cell.The present invention relates to a method for producing cardiomyocytes.The present invention provides a reagent kit for use in these methods. [Background technology]

[0002] In recent years, regenerative medicine using cardiomyocytes obtained by inducing differentiation of pluripotent stem cells has been attracting attention as an alternative treatment to heart transplantation. However, it is difficult to obtain a large amount of cardiomyocytes with high efficiency by differentiation induction. For example, the efficiency of differentiation into cardiomyocytes varies due to the influence of differences in the quality of the pluripotent stem cells used. In order to apply cardiomyocytes obtained by differentiation induction to patients, it is necessary to evaluate the differentiation state of the cells. Conventionally, cardiac troponin T (cTnT), a marker of cardiomyocytes, has been used to evaluate the differentiation state from pluripotent stem cells to cardiomyocytes. In this evaluation method, first, a portion of cells after differentiation induction is collected and immunostained with a fluorescently labeled anti-cTnT antibody. Then, cells expressing cTnT are detected by flow cytometry, and the proportion of cells expressing cTnT (cTnT positive rate) among the cells after differentiation induction is calculated. Conventionally, the differentiation state into cardiomyocytes was evaluated based on this cTnT positive rate. However, since fluorescently labeled cells cannot be used for transplantation into living bodies, this evaluation method can be called destructive testing. Since pluripotent stem cells and cells prepared from them are valuable, it is not preferable to consume a portion of them for quality evaluation.On the other hand, as an example of non-destructive testing, Non-Patent Document 1 discloses that the differentiation state of human embryonic stem cells was evaluated by measuring the amount of miRNA in the culture supernatant during the process of inducing differentiation of the cells into cardiomyocytes. [Prior art documents] [Patent documents]

[0003] [Non-Patent Document 1] Zhang Y. et al., A non-invasive method to determine the pluripotent status of stem cells by culture medium microRNA expression detection (2016) Scientific Reports, vol.6:22380 Summary of the Invention [Problem to be solved by the invention]

[0004] In Non-Patent Document 1, in order to evaluate the differentiation state of human embryonic stem cells into cardiomyocytes, miRNAs known to be expressed only in mice and miRNAs known to be expressed in small amounts are also measured. Therefore, there are concerns about the accuracy of the evaluation of the differentiation state. The present invention aims to provide a means that enables the evaluation of the differentiation state of cells with higher accuracy. [Means for solving the problem]

[0005] The present inventors have found that the differentiation state of cells can be evaluated by measuring miRNA-3p of the miR-1 / 133a cluster in the supernatant of a liquid medium containing differentiation-induced cells, and have completed the present invention. Thus, the following inventions [1] to

[24] are provided.

[0006] [1] A method for evaluating the differentiation state of a cell, comprising the steps of inducing the differentiation of pluripotent stem cells into cardiomyocytes in a liquid medium by a first induction treatment for inducing the differentiation of pluripotent stem cells into mesodermal cells and a second induction treatment for inducing the differentiation of the mesodermal cells into cardiomyocytes, collecting the supernatant of the liquid medium containing the cells induced to differentiate by the second induction treatment, and measuring miRNA-3p of the miR-1 / 133a cluster in the supernatant, wherein the miRNA-3p is at least one selected from the group consisting of miR-1-3p and miR-133a-3p, and the measured value of the miRNA-3p is an indicator of differentiation into cardiomyocytes.

[0007] [2] The method according to [1], wherein the miRNA-3p is the miR-1-3p, and when the measured value of the miR-1-3p is less than a first threshold, it is suggested that the differentiated cells are not suitable for transplantation.

[0008] [3] The method according to [1], wherein the miRNA-3p is the miR-133a-3p, and when the measured value of the miR-133a-3p is less than a second threshold, it is suggested that the differentiated cells are not suitable for transplantation.

[0009] [4] The method according to [1], wherein when the miRNA-3p is the miR-1-3p and the miR-133a-3p, and the measured value of the miR-1-3p is less than a first threshold value and the measured value of the miR-133a-3p is less than a second threshold value, it is suggested that the differentiated cells are not suitable for transplantation.

[0010] [5] The method according to [1], wherein the miRNA-3p is the miR-1-3p and the miR-133a-3p, and when the measured value of the miR-1-3p is less than a first threshold value or the measured value of the miR-133a-3p is less than a second threshold value, it is suggested that the differentiated cells are not suitable for transplantation.

[0011] [6] The method according to any one of [1] to [5], wherein the first induction treatment comprises using a liquid medium containing at least one of a substance that promotes a BMP signaling pathway and a substance that promotes a Wnt signaling pathway.

[0012] [7] The method according to any one of [1] to [6], wherein the second induction treatment comprises using a liquid medium containing a substance that inhibits the Wnt signaling pathway.

[0013] [8] The method according to any one of [1] to [7], further comprising the steps of collecting a supernatant of a liquid culture medium containing cells that have been induced to differentiate by the first induction treatment and have not yet been subjected to the second induction treatment, and measuring miR-489-3p in the supernatant, wherein the measured value of miR-489-3p is an index of differentiation into mesodermal cells.

[0014] [9] The method according to [8], wherein when the measured value of the miR-489-3p is equal to or greater than a third threshold, it is suggested that the pluripotent stem cells have been differentiated into mesodermal cells.

[0015]

[10] A method for determining the differentiation state of a cell, comprising: a differentiation induction step including a first induction treatment for inducing the differentiation of pluripotent stem cells into mesodermal cells in a liquid medium and a second induction treatment for inducing the differentiation of the mesodermal cells into cardiomyocytes; a step of collecting a supernatant of a liquid medium containing cells induced to differentiate by the second induction treatment; a step of measuring miRNA-3p of the miR-1 / 133a cluster in the supernatant; and a step of determining that the differentiated cells are unsuitable for transplantation when the measured value of the miRNA-3p is below the threshold value, wherein the miRNA-3p is at least one selected from miR-1-3p and miR-133a-3p.

[0016]

[11] The method according to

[10] , wherein the miRNA-3p is the miR-1-3p, the threshold is a first threshold, and in the determination step, when the measured value of the miR-1-3p is less than the first threshold, the differentiated cells are determined to be unsuitable for transplantation.

[0017]

[12] The method according to

[10] , wherein the miRNA-3p is the miR-133a-3p, the threshold is a second threshold, and in the determination step, when the measured value of the miR-133a-3p is less than the second threshold, the differentiated cells are determined to be unsuitable for transplantation.

[0018]

[13] The method according to

[10] , wherein the miRNA-3p is the miR-1-3p and the miR-133a-3p, the thresholds are a first threshold and a second threshold, and when, in the determination step, the measured value of the miR-1-3p is less than the first threshold and the measured value of the miR-133a-3p is less than the second threshold, the differentiated cells are determined to be unsuitable for transplantation.

[0019]

[14] The method according to

[10] , wherein the miRNA-3p is the miR-1-3p and the miR-133a-3p, the thresholds are a first threshold and a second threshold, and when, in the determination step, the measured value of the miR-1-3p is less than the first threshold or the measured value of the miR-133a-3p is less than the second threshold, the differentiated cells are determined to be unsuitable for transplantation.

[0020]

[15] The method according to any one of

[10] to

[14] , wherein the first induction treatment includes an induction treatment using a liquid medium containing at least one of a substance that promotes a BMP signaling pathway and a substance that promotes a Wnt signaling pathway.

[0021]

[16] The method according to any one of

[10] to

[15] , wherein the second induction treatment comprises using a liquid medium containing a substance that inhibits the Wnt signaling pathway.

[0022]

[17] The method according to any one of

[10] to

[16] , further comprising the steps of: collecting a supernatant of a liquid culture medium containing cells that have been induced to differentiate by the first induction treatment and before being subjected to the second induction treatment, and measuring miR-489-3p in the supernatant; and determining that the pluripotent stem cells have differentiated into mesodermal cells when the measured value of miR-489-3p is equal to or greater than the third threshold.

[0023]

[18] The method according to any one of

[10] to

[17] , further comprising the steps of: collecting a supernatant of a liquid culture medium containing cells that have been induced to differentiate by the first induction treatment but before being subjected to the second induction treatment, and measuring miR-489-3p in the supernatant; and determining that differentiation of the pluripotent stem cells into mesodermal cells is insufficient when the measured value of miR-489-3p is less than a third threshold value.

[0024]

[19] A method for producing cardiomyocytes, comprising: (1) a step of inducing differentiation of mesodermal cells into cardiomyocytes in a liquid medium by an induction treatment for inducing differentiation of mesodermal cells into cardiomyocytes; (2) a step of collecting a supernatant of the liquid medium containing the cells induced in the step (1); (3) a step of measuring miRNA-3p of the miR-1 / 133a cluster in the supernatant collected in the step (2); and (4) a step of culturing the cells induced to differentiate in the step (1) to obtain cardiomyocytes when the measured value of miRNA-3p is equal to or higher than the threshold value, wherein the miRNA-3p is at least one selected from miR-1-3p and miR-133a-3p.

[0025]

[20] The method according to

[19] , wherein in the step (4), when the measured value of the miRNA-3p is below the threshold, culturing of the cells induced to differentiate in the step (1) is discontinued.

[0026]

[21] The production method according to

[19] or

[20] , comprising the steps of: (i) inducing the differentiation of pluripotent stem cells into mesodermal cells in a liquid medium by an induction treatment for inducing the differentiation of the pluripotent stem cells into mesodermal cells; (ii) collecting a supernatant of the liquid medium containing the cells induced in the step (i); and (iii) measuring miR-489-3p in the supernatant collected in the step (ii), wherein when the measured value of miR-489-3p is equal to or greater than a third threshold, the cells of the step (ii) are used as the mesodermal cells of the step (1).

[0027]

[22] The method according to

[21] , wherein the culturing of the cells in step (ii) is discontinued when the measured value of the miR-489-3p is below the threshold value.

[0028]

[23] A reagent kit for use in the method according to any one of [1] to [7],

[10] to

[16] ,

[19] and

[20] , comprising an oligonucleotide primer and / or an oligonucleotide probe for detecting miRNA-3p of the miR-1 / 133a cluster.

[0029]

[24] A reagent kit for use in the method according to any one of claims [8], [9],

[17] ,

[18] ,

[21] and

[22] , comprising an oligonucleotide primer and / or an oligonucleotide probe for detecting miRNA-3p of the miR-1 / 133a cluster, and an oligonucleotide primer and / or an oligonucleotide probe for detecting miR-489-3p. Effect of the Invention

[0030] According to the present invention, it is possible to evaluate the differentiation state of cells with high accuracy. [Brief description of the drawings]

[0031] [Figure 1] FIG. 1 shows an example of a schedule for differentiation induction and the time when the supernatant was obtained. [Figure 2A] FIG. 2 is a diagram showing an example of the appearance of a reagent kit. [Figure 2B] FIG. 2 is a diagram showing an example of the appearance of a reagent kit. [Diagram 3] FIG. 2 is a diagram showing an example of the appearance of a reagent kit. [Figure 4] FIG. 1 shows the schedule of differentiation induction and the time of obtaining the supernatant in Example 1. [Figure 5A] 1 is a graph showing the copy number of miR-489-3p in the supernatant obtained during differentiation induction of iPS cells of the 201B7 clone, where Group H is the cells that underwent the first and second induction treatments, and Group L is the cells that underwent only the second induction treatment. [Figure 5B] 1 is a graph showing the copy number of miR-489-3p in the supernatant obtained during the differentiation induction process of iPS cells of the 253G4 clone. [Figure 6] 1 shows an example of an image of cells immunofluorescently stained with anti-brachyury T antibody and a fluorescently labeled secondary antibody for groups H and L. [Figure 7] Graph showing the ratio of Brachyury T positive cells in groups H and L. [Figure 8] For groups H and L, the graph shows the fluorescent signal intensity (indicated as "cTnT" in the figure) and the count (number of cells) on the two axes. [Figure 9] (A) is a box plot showing the relative expression level of miR-1-3p in the supernatant. (B) is a box plot showing the relative expression level of miR-1-5p in the supernatant. "Acceptable products" are cells from lots in which the cTnT positivity rate was 70% or higher on the 10th day (d10 in FIG. 4). "Rejectable products" are cells from lots in which the cTnT positivity rate was less than 70% on the 10th day. [Figure 10] This is a box plot in which the relative expression levels in FIG. 9(A) are converted into copy numbers. [Figure 11] (A) is a box plot showing the relative expression levels of miR-133a-3p and (B) is a box plot showing the relative expression levels of miR-133a-5p in the supernatant. [Figure 12] This is a box plot in which the relative expression levels in FIG. 11(A) are converted into copy numbers. [Figure 13] (A) is a box plot showing the relative expression levels of miR-23b-3p and (B) is a box plot showing the relative expression levels of miR-23b-5p in the supernatant. [Figure 14] This box plot was recreated from the box plot in Figure 13(A) by changing the maximum value of the vertical axis to 80. [Figure 15] (A) is a box plot showing the relative expression levels of miR-26b-3p and (B) is a box plot showing the relative expression levels of miR-26b-5p in the supernatant. [Figure 16]This box plot was recreated from the box plot in Figure 15 by changing the maximum value on the vertical axis to 80. [Figure 17] (A) is a box plot showing the relative expression levels of miR-125b-3p and (B) is a box plot showing the relative expression levels of miR-1254b-5p in the supernatant. [Figure 18] (A) is a box plot showing the relative expression levels of miR-145-3p and (B) is a box plot showing the relative expression levels of miR-145-5p in the supernatant. [Figure 19] (A) is a box plot showing the relative expression levels of miR-499a-3p and (B) is a box plot showing the relative expression levels of miR-499a-5p in the supernatant. [Figure 20] (A) is a box plot showing the relative expression levels of miR-503-3p and (B) is a box plot showing the relative expression levels of miR-503-5p in the supernatant. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0032] In the method for evaluating the differentiation state of cells of this embodiment (hereinafter also referred to as the "evaluation method of this embodiment"), first, pluripotent stem cells are induced to differentiate into cardiomyocytes in liquid medium by a first induction treatment for inducing the differentiation of pluripotent stem cells into mesodermal cells, and a second induction treatment for inducing the differentiation of mesodermal cells into cardiomyocytes.

[0033] Pluripotent stem cells (PSCs) are stem cells that have the ability to differentiate into cells derived from the ectoderm, endoderm, and mesoderm (pluripotency) and have the ability to proliferate. Examples of PSCs include induced pluripotent stem cells (iPS cells), embryonic stem cells (ES cells), nuclear transfer ES cells (ntES cells), and embryonic germ stem cells (EG cells). iPS cells are stem cells that are produced by introducing a specific reprogramming factor (DNA or protein) such as Oct3 / 4, Klf4, c-Myc, or Sox2 into somatic cells and have pluripotency and proliferation capabilities. ES cells are stem cells derived from the inner cell mass of mammalian blastocysts and have pluripotency and proliferation capabilities. ntES cells are ES cells established from the inner cell mass of blastocysts derived from cloned embryos obtained by replacing the nucleus of an unfertilized egg with the nucleus of a somatic cell. ntES cells have almost the same properties as ES cells. EG cells are cells that are established from primordial germ cells during the fetal stage and have the same pluripotency as ES cells. EG cells can be established by culturing primordial germ cells in the presence of substances such as LIF, basic FGF (bFGF), and stem cell factor. Among these, iPS cells are preferred.

[0034] The origin of PSCs is not particularly limited. For example, PSCs are preferably derived from mammals such as humans, monkeys, dogs, cats, horses, mice, rats, hamsters, guinea pigs, rabbits, sheep, pigs, and cows. From the viewpoint of regenerative medicine research and clinical application, human PSCs (hPSCs) are preferred as PSCs.

[0035] The liquid medium itself is known and can be appropriately selected depending on the type of PSC. Examples of the liquid medium include RPMI1640 medium, StemFit™ series (Ajinomoto Co., Inc.), MEMα medium, IMDM medium, Medium 199 medium, EMEM medium, DMEM medium, Ham's F12 medium, Fischer's medium, MEF-CM, StemPro™ 34 (Invitrogen), Essential 8™ (Thermo Fisher Scientific), ReproNaive™ (ReproCELL Co., Ltd.), hPSC Growth Medium DXF™ (Takara Bio Inc.), and mixed media thereof.

[0036] The liquid medium may contain components necessary for the growth and / or maintenance of PSCs, such as, for example, serum, albumin, transferrin, B-27™ supplement (Invitrogen), Knockout™ Serum Replacement (KSR), N2 supplement (Invitrogen), fatty acids, insulin, collagen precursors, 2-mercaptoethanol, thioglycerol, lipids, amino acids, L-glutamine, Glutamax™ (Invitrogen), non-essential amino acids, vitamins, growth factors, antibiotics, antioxidants, pyruvic acid, buffers, inorganic salts, and the like.

[0037] With reference to FIG. 1, the schedule of cell culture and differentiation induction and the time to obtain the supernatant in the evaluation method of this embodiment will be described. However, the present invention is not limited to the example of FIG. 1. In the example of FIG. 1, the day on which the first induction treatment is performed is defined as "Day 0". In FIG. 1, Day 0 is written as "Day 0". FIG. 1 shows on what days the operations performed before and after the first induction treatment are performed, based on Day 0. In the figure, the period shown in gray (Day 0 to Day 7) is an example of the period during which the evaluation method of this embodiment is performed.

[0038] The PSCs used in the evaluation method of this embodiment are preferably subjected to maintenance culture in advance in a liquid medium before the first induction treatment. Maintenance culture refers to culturing PSCs until they proliferate to a number of cells suitable for differentiation induction while maintaining pluripotency. The period of maintenance culture is not particularly limited, but is, for example, 1 day or more and 7 days or less. In the example of FIG. 1, the period of maintenance culture is 4 days from 4 days before day 0 (Day-4) to day 0.

[0039] The liquid medium used for the maintenance culture preferably contains components necessary for maintaining the survival and / or undifferentiation of PSCs. Examples of such components include ROCK inhibitors (e.g., Y-27632), leukemia inhibitory factor (LIF), fibroblast growth factor (FGF), stem cell factor, MEK inhibitors (e.g., PD184352), and the like. When the PSCs are iPS cells, it is preferable to use a liquid medium containing a ROCK inhibitor on the first day of the maintenance culture. The addition of a ROCK inhibitor can reduce cell death when iPS cells are suspended and seeded in a liquid medium. It is preferable to replace the liquid medium with a liquid medium not containing a ROCK inhibitor at least 1 to 4 days after the start of the maintenance culture of PSCs in a liquid medium containing a ROCK inhibitor.

[0040] The first induction treatment is performed by adding a substance that promotes the induction of differentiation from PSCs to mesodermal cells to PSCs in a liquid medium. Such substances themselves are known. For example, in the first induction treatment, a liquid medium containing at least one of a substance that promotes the bone morphogenetic protein (BMP) signaling system and a substance that promotes the Wnt signaling system is used. Specifically, the liquid medium containing the cells (PSCs in this case) is replaced with a liquid medium containing at least one of these substances. Alternatively, at least one of these substances may be added to the liquid medium containing the cells. These substances bind to the receptors of the PSCs and / or are taken up by the PSCs in the liquid medium, thereby inducing the differentiation of the PSCs into mesodermal cells.

[0041] As used herein, the term "liquid medium containing cells" refers to a liquid medium in a culture vessel in contact with cells or in which the cells are immersed. In the liquid medium containing cells, the cells may be suspended in the liquid medium, or may be precipitated or attached to the bottom of the culture vessel in the liquid medium.

[0042] Substances that promote the BMP signal transduction system are not particularly limited, and examples thereof include BMP4, BMP2, activin, etc. Substances that promote the Wnt signal transduction system are not particularly limited, and examples thereof include Wnt signal agonists, activin A, BMP-4, bFGF, etc. Examples of Wnt signal agonists include glycogen synthase kinase (GSK)-3 inhibitors, etc. GSK-3 inhibitors themselves are known, and examples thereof include CHIR99021, TWS119, SB2167363, SB415286, CHIR-98014, etc.

[0043] The timing of starting the first induction treatment is not particularly limited. If the PSCs have proliferated to a cell number suitable for differentiation induction while maintaining pluripotency, the first induction treatment may be performed. In the example of FIG. 1, the first induction treatment is performed 3 or 4 days after the start of maintenance culture. The period of the first induction treatment is not particularly limited, but is, for example, 12 hours or more and 6 days or less, preferably 1 day or more and 6 days or less. In the example of FIG. 1, the first induction treatment is performed until the first day (Day 1). The first induction treatment can be terminated by replacing the liquid medium containing the cells with a liquid medium that does not contain a substance that promotes the BMP signaling system and a substance that promotes the Wnt signaling system. Note that the first induction treatment itself is terminated by medium replacement, but the PSCs continue to differentiate into mesodermal cells even after the medium replacement. When the period of the first induction treatment is determined in days, the time at which the first induction treatment is terminated is preferably ±1 hour from the time at which the first induction treatment is started.

[0044] The second induction treatment is performed by adding a substance that promotes differentiation induction from mesodermal cells to cardiomyocytes to the cells in the liquid medium after the first induction treatment. Such substances themselves are known. For example, in the second induction treatment, a liquid medium containing a substance that inhibits the Wnt signaling pathway is used. Specifically, the liquid medium containing the cells is replaced with a liquid medium containing this substance. Alternatively, this substance may be added to the liquid medium containing the cells. When the substance that inhibits the Wnt signaling pathway is taken up by the cells in the liquid medium, the cells are induced to differentiate into cardiomyocytes.

[0045] The substance that inhibits the Wnt signaling pathway is not particularly limited, but examples thereof include substances that promote the degradation of β-catenin. Such substances are themselves known, and examples thereof include IWR-1, IWP-2, IWP-3, IWP-4, PNU-74654, XAV939, and KY02111.

[0046] The time to start the second induction treatment can be appropriately determined. If a sufficient period of time has passed for PSCs to differentiate into mesodermal cells, the second induction treatment may be performed. Preferably, the second induction treatment is performed 2 to 7 days after the first induction treatment. In the example of FIG. 1, the second induction treatment is performed on the third day (Day 3), that is, 3 days after the first induction treatment. The period of the second induction treatment is not particularly limited, but is, for example, 12 hours to 5 days. In the example of FIG. 1, the second induction treatment is performed from the third day (Day 3) to the fifth day (Day 5). The second induction treatment can be terminated by replacing the liquid medium with a liquid medium that does not contain a substance that inhibits the Wnt signaling system. Note that the second induction treatment itself is terminated by medium replacement, but the mesodermal cells are induced to differentiate into cardiomyocytes even after the medium replacement. When the period of the second induction treatment is determined in days, the time to end the second induction treatment is preferably ±1 hour from the time when the second induction treatment is started.

[0047] The cell culture method during the maintenance culture and differentiation induction period can be appropriately selected from known culture methods depending on the type of PSC. Cell culture is usually performed by suspension culture, adhesion culture, or a combination of these. Suspension culture refers to culture performed under non-adhesive conditions to the culture vessel. In suspension culture, cells do not necessarily need to be dispersed in a liquid medium, and the cells may be precipitated to the bottom of the culture vessel. The culture vessel used for suspension culture is not particularly limited, and examples thereof include flasks, dishes, plates, chambers, and tubes. It is preferable that the culture vessel used for suspension culture is non-adhesive to cells. It is preferable that the culture vessel is made of a hydrophobic material or is coated with a coating agent (e.g., polyhydroxyethyl methacrylate copolymer) to prevent cell adhesion to the surface.

[0048] Adhesion culture refers to culture in which cells are attached to a support. A culture vessel is used as the support. The culture vessel used for adhesion culture is not particularly limited. For adhesion culture, a culture vessel used for suspension culture can be used, but it is preferable that the surface or inside of the culture vessel is covered with a coating agent (also called a cell culture substrate, adhesion substrate, etc.) that allows cells to adhere. Examples of the coating agent include gelatin, laminin, collagen, poly-D-lysine, polyornithine, fibronectin, and vitronectin. A known scaffold can also be used as the support. By using this, PSCs can grow on the scaffold and three-dimensional culture can be performed. As such a scaffold, a commercially available product can be used, and examples thereof include Matrigel (trademark) (Corning), QGel (trademark) MT 3D Matrix (Qgel SA), 3-D Life Biomimetic (Cellendes), Puramatrix (3D MATRIX), and alvetex (Reinnavate).

[0049] If necessary, PSCs may be cultured together with feeder cells. The type of feeder cells is not particularly limited and can be appropriately selected from known feeder cells. Examples include mouse embryonic fibroblasts (MEF), mouse embryonic fibroblast cell line (STO), SL10 cells, and SNL cells.

[0050] The culture conditions for PSCs can be adjusted appropriately depending on the type of PSC. For example, iPS cells can be cultured in a multi-gas incubator at a temperature of about 30° C. to 40° C., preferably 37° C., with CO2 and O2 concentrations of 1% to 10%, preferably 5%. After differentiation into cardiomyocytes, the cells can be cultured in a CO2 incubator at a temperature of about 30° C. to 40° C., preferably 37° C., with CO2 concentrations of 1% to 10%, preferably 5%.

[0051] In the evaluation method of this embodiment, the supernatant of the liquid medium containing the cells induced to differentiate by the second induction treatment is collected. The supernatant is the whole or part of the solution portion of the liquid medium containing the cells, and does not substantially contain the cells. The method of collecting the supernatant from the liquid medium containing the cells is not particularly limited. In the case of suspension culture, the liquid medium containing the cells is centrifuged to obtain the supernatant after centrifugation. In the case of adhesion culture, since the cells are attached to the bottom of the culture vessel, the supernatant in the culture vessel can be obtained by sucking it with a pipette or the like. The collected supernatant is used as a sample for miRNA measurement described later.

[0052] In the evaluation method of this embodiment, the miRNA in the supernatant secreted from the cells is the measurement target. When the collected supernatant is contaminated with cells, it is preferable to remove the cells from the supernatant to avoid measuring the miRNA in the cells. The removal of the cells from the supernatant can be performed by filter filtration and / or centrifugation.

[0053] As shown in FIG. 1, it is known that it usually takes 15 to 17 days from the date of the first induction treatment until the operation of inducing differentiation into cardiomyocytes is completed. In this specification, the "operation of inducing differentiation into cardiomyocytes" includes performing the first and second induction treatments, and culturing the cells in the liquid medium for a period sufficient for the cells to be terminally differentiated into cardiomyocytes. Conventionally, the cTnT positive rate of cells was confirmed after the operation of inducing differentiation into cardiomyocytes was completed. However, the evaluation method of this embodiment can evaluate the differentiation state of cells after the completion of differentiation induction before the operation of inducing differentiation into cardiomyocytes is completed. For example, in the example of FIG. 1, the differentiation state of cells can be evaluated by the measured value of miRNA in the supernatant collected at Day 7. In this specification, "cells induced to differentiate by the second induction treatment" refers to cells after differentiation induction by the second induction treatment and before the operation of inducing differentiation into cardiomyocytes is completed.

[0054] As described above, the evaluation method of this embodiment can evaluate the differentiation state of cells based on the measured value of miRNA in the supernatant collected before the operation of inducing differentiation into cardiomyocytes is completed. Therefore, the time of collecting the supernatant can be before the operation of inducing differentiation into cardiomyocytes is completed. On the other hand, even if the supernatant is collected not long after the second induction treatment, it is considered that the miRNA-3p of the miR-1 / 133a cluster is not sufficiently secreted into the supernatant. In addition, the second induction treatment is terminated by changing the medium. Even if not long after the medium is changed, it is considered that the above-mentioned miRNA-3p is not sufficiently contained in the supernatant. From these facts, the time of collecting the supernatant can be, for example, a time point that is 3 to 4 days after the second induction treatment and before the second induction treatment is completed. The time point before the second induction treatment is completed can be, for example, a time point 1 day before the second induction treatment is completed, or a time point when the old liquid medium is removed for medium replacement. Alternatively, the supernatant can be collected 2 to 3 days after the end of the second induction treatment.

[0055] The time to collect the supernatant may be determined, for example, according to the length of the period of the second induction treatment. When the period of the second induction treatment is 12 hours or more but less than 24 hours, the time to collect the supernatant may be, for example, 3 days or more but 4 days or less from the date of the end of the second induction treatment. When the period of the second induction treatment is 1 or 2 days, the time to collect the supernatant may be, for example, 2 days or more but 3 days or less from the date of the end of the second induction treatment. When the period of the second induction treatment is 3 days or more, the time to collect the supernatant may be, for example, 3 days or more but 4 days or less from the date of the second induction treatment and before the end of the second induction treatment. In the example of FIG. 1, the period of the second induction treatment is 2 days, and the supernatant is collected on the 7th day (Day 7), that is, 2 days after the date of the end of the second induction treatment.

[0056] As a negative control for the measurement of miRNA-3p of the miR-1 / 133a cluster, the supernatant before the second induction treatment (hereinafter also referred to as the "second control sample") may be collected. The second control sample may be a supernatant collected at any time before the second induction treatment. The supernatant collected at such a time contains almost no miRNA-3p of the miR-1 / 133a cluster. The control sample can also be said to be a sample for obtaining a background value for the measurement of the miRNA-3p. Preferably, the second control sample is a liquid medium that is removed when the medium is replaced to perform the second induction treatment. In the example of FIG. 1, the second control sample is a liquid medium containing cells before the medium replacement on the third day (Day 3). The second control sample can be obtained by recovering a part or the whole of the liquid medium before removing it.

[0057] In the evaluation method of this embodiment, miRNA-3p of the miR-1 / 133a cluster in the supernatant is measured. miRNA-3p is a mature miRNA expressed from the 3' end of pre-miRNA, which is a precursor of miRNA. The mature miRNA expressed from the 5' end of pre-miRNA is called miRNA-5p. Genes encoding human miR-1-3p and miR-133a-3p are present on chromosomes 18 and 20, forming the miR-1 / 133a cluster. miRNA-3p of the miR-1 / 133a cluster refers to miR-1-3p and miR-133a-3p. In this specification, "miRNA-3p of the miR-1 / 133a cluster" refers to at least one selected from miR-1-3p and miR-133a-3p. In other words, measuring miRNA-3p of the miR-1 / 133a cluster means measuring miR-1-3p and / or miR-133a-3p.

[0058] Human miR-1-3p and human miR-133a-3p themselves are publicly known, and the nucleotide sequences of these miRNAs are shown in SEQ ID NOs: 1 and 2, respectively. In addition, the genes encoding these miRNAs are each published in the GenBank database as shown in Table 1.

[0059] [Table 1]

[0060] The method of measuring miRNA is not particularly limited, and known methods can be used. For example, a measurement method using hybridization of an oligonucleotide probe, a measurement method using a nucleic acid amplification method, a measurement method using mass spectrometry, a measurement method using sequencing, etc. can be mentioned. Measurement methods using hybridization of a probe include, for example, a microarray method, a Northern hybridization method, and an RNase protection assay. Measurement methods using nucleic acid amplification include, for example, a quantitative RT-PCR method and a quantitative RT-LAMP method. Examples of quantitative RT-PCR include, for example, a SYBR (trademark) Green method and a TaqMan (trademark) method. Since the miRNA to be amplified is short, a stem-loop primer or a poly(A) addition method is usually used in a method using nucleic acid amplification. A commercially available miRNA quantification reagent kit such as TaqMan (trademark) MicroRNA Assay (Thermo Fisher Scientific) may be used.

[0061] The measured value of miRNA-3p of the miR-1 / 133a cluster is an index of differentiation into cardiomyocytes. The measured value of miRNA-3p of the miR-1 / 133a cluster is the measured value of miR-1-3p and / or the measured value of miR-133a-3p. Hereinafter, the "measured value of miRNA-3p of the miR-1 / 133a cluster" is also referred to as the "measured value of miRNA-3p". In this specification, the "measured value" is a value that reflects the amount of miRNA present in the supernatant. For example, the optical measured value, the reaction cycle number (Ct value) or reaction time when the optical measured value reaches a predetermined reference value, the quantitative value of miRNA calculated using a calibration curve, etc. are included. The "measured value" may be a difference or ratio with respect to a background value. Examples of the optical measured value include fluorescence intensity, turbidity, absorbance, etc. Examples of the quantitative value of miRNA include copy number, mass, concentration, etc. The predetermined reference value can be appropriately set depending on the type of value indicating the amount or concentration of miRNA. For example, when quantitative RT-PCR is used, a given Ct value can be used as a reference value to set the amount of change in fluorescence intensity when the nucleic acid amplification reaction shows logarithmic amplification.

[0062] As shown in Example 2 below, the measured value of miRNA-3p in the supernatant collected before the operation of inducing differentiation into cardiomyocytes was completed was significantly higher in cells with a cTnT positive rate of 90% or more than in cells with a cTnT positive rate of less than 70% after the completion of the operation (d10). Therefore, based on the measured value of miRNA-3p, information on the differentiation state of cells induced to differentiate by the second induction treatment when the operation of inducing differentiation is completed can be obtained. Such information can be, for example, information suggesting whether the cells induced to differentiate by the second induction treatment can be applied for transplantation as cardiomyocytes after the operation of inducing differentiation is completed. Here, "applicable for transplantation" means a state in which at least one of the criteria for determining whether a cell is transplantable is satisfied, and if all other criteria are satisfied, the cell can be transplanted into a living body. Even if the method suggests that the cell is "applicable for transplantation", it may be determined that the cell is not transplantable based on other criteria. One indicator of whether cardiomyocytes produced by inducing differentiation of PSCs are suitable for transplantation is the percentage of cells induced to differentiate by the second induction treatment that differentiate into cardiomyocytes after the differentiation induction operation is completed. Thus, information on the differentiation state upon completion of the differentiation induction operation can be, for example, an indication that at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the cells induced to differentiate by the second induction treatment will differentiate into cardiomyocytes.

[0063] For example, when the measurement value of miR-1-3p is obtained as the measurement value of miRNA-3p, if the measurement value of miR-1-3p is less than the first threshold, it may be suggested that the cells induced to differentiate by the second induction treatment are not suitable for transplantation. Also, when the measurement value of miR-1-3p is equal to or greater than the first threshold, it may be suggested that the cells induced to differentiate by the second induction treatment are applicable for transplantation. Also, when the measurement value of miR-1-3p is equal to or greater than the first threshold, it may be suggested that at least 70% of the cells induced to differentiate by the second induction treatment are differentiated into cardiomyocytes. The first threshold is a threshold corresponding to the measurement value of miR-1-3p. Alternatively, when the measurement value of miR-133a-3p is obtained as the measurement value of miRNA-3p, if the measurement value of miR-133a-3p is less than the second threshold, it may be suggested that the cells induced to differentiate by the second induction treatment are not suitable for transplantation. Moreover, when the measured value of miR-133a-3p is equal to or greater than the second threshold, it may be suggested that the cells induced to differentiate by the second induction treatment are suitable for transplantation. Moreover, when the measured value of miR-133a-3p is equal to or greater than the second threshold, it may be suggested that at least 70% of the cells induced to differentiate by the second induction treatment are differentiated into cardiomyocytes. The second threshold is a threshold corresponding to the measured value of miR-133a-3p. By using the above-mentioned evaluation method using the measured value of miR-1-3p or the measured value of miR-133a-3p, it is possible to identify cells that are unsuitable for transplantation or cells that are suitable for transplantation at an early stage, and to determine whether or not to continue the culture at an early stage.

[0064] Alternatively, when the measured values ​​of miR-1-3p and miR-133a-3p are obtained as the measured values ​​of miRNA-3p, if the measured values ​​of miR-1-3p are less than the first threshold and the measured values ​​of miR-133a-3p are less than the second threshold, it may be suggested that the cells induced to differentiate by the second induction treatment are not suitable for transplantation. If the measured values ​​of miR-1-3p are equal to or greater than the first threshold and the measured values ​​of miR-133a-3p are equal to or greater than the second threshold, it may be suggested that the cells induced to differentiate by the second induction treatment are applicable to transplantation. If the measured values ​​of miR-1-3p are equal to or greater than the first threshold and the measured values ​​of miR-133a-3p are equal to or greater than the second threshold, it may be suggested that at least 70% of the cells induced to differentiate by the second induction treatment are differentiated into cardiomyocytes.

[0065] Alternatively, when the measured values ​​of miR-1-3p and miR-133a-3p are obtained as the measured values ​​of miRNA-3p, if the measured value of miR-1-3p is less than the first threshold or the measured value of miR-133a-3p is less than the second threshold, it may be suggested that the cells induced to differentiate by the second induction treatment are not suitable for transplantation. If the measured value of miR-1-3p is equal to or greater than the first threshold or the measured value of miR-133a-3p is equal to or greater than the second threshold, it may be suggested that the cells induced to differentiate by the second induction treatment are applicable to transplantation. If the measured value of miR-1-3p is equal to or greater than the first threshold or the measured value of miR-133a-3p is equal to or greater than the second threshold, it may be suggested that at least 70% of the cells induced to differentiate by the second induction treatment are differentiated into cardiomyocytes.

[0066] The first threshold and the second threshold are not particularly limited and can be set appropriately. For example, the threshold may be set as follows. The first and second induction processes are performed to induce differentiation of PSCs into cardiomyocytes to obtain a plurality of lots of cells. Before the operation of inducing differentiation into cardiomyocytes is completed, a supernatant is collected from a liquid medium containing each lot of cells. After the induction of differentiation into cardiomyocytes is completed, the cTnT positivity of each lot of cells is confirmed. Then, the collected supernatant is classified into a lot of supernatant with a high cTnT positivity (e.g., 70% or more) and a lot of supernatant with a low cTnT positivity (e.g., less than 70%). For each supernatant, a measurement value of miRNA-3p is obtained. Then, a value that can distinguish a lot with a high cTnT positivity from a lot with a low cTnT positivity is obtained from the obtained measurement value, and the value is set as the threshold. It is preferable to consider sensitivity, specificity, positive predictive value, negative predictive value, and the like when setting the threshold.

[0067] As described above, the measured value of miRNA-3p in the supernatant collected before the operation of inducing differentiation into cardiomyocytes is an index suggesting the differentiation state of cells after the operation is completed. Therefore, the evaluation method of this embodiment can be rephrased as a "method of obtaining an index for predicting the differentiation state of cells."

[0068] The evaluation method of this embodiment makes it possible to determine whether the cells induced to differentiate by the second induction process will differentiate well into cardiomyocytes before the operation of inducing differentiation into cardiomyocytes is completed. If it is suggested that the cells induced to differentiate by the second induction process are suitable for transplantation or that at least 70% will differentiate into cardiomyocytes, the culture of the cells can be continued to obtain a sufficient amount of cardiomyocytes. With reference to FIG. 1, the culture may be continued for, for example, less than 200 days.

[0069] The present invention may include a step of comparing the measured value of miRNA-3p with a threshold value, and a step of judging the differentiation state of cells based on the comparison result. In the judging step, the differentiation state of the cells induced to differentiate by the second induction treatment is judged when the operation of inducing differentiation into cardiomyocytes is completed. The differentiation state when the operation is completed may be, for example, either a differentiation state applicable to transplantation or a differentiation state unsuitable for transplantation. Alternatively, the differentiation state when the operation is completed may be at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the cells induced to differentiate by the second induction treatment differentiated into cardiomyocytes.

[0070] For example, when the measurement value of miR-1-3p is obtained as the measurement value of miRNA-3p, if the measurement value of miR-1-3p is less than the first threshold, it may be determined that the cells induced to differentiate by the second induction treatment are not suitable for transplantation. When the measurement value of miR-1-3p is equal to or greater than the first threshold, it may be determined that the cells induced to differentiate by the second induction treatment are applicable for transplantation. When the measurement value of miR-1-3p is equal to or greater than the first threshold, it may be determined that at least 70% of the cells induced to differentiate by the second induction treatment are differentiated into cardiomyocytes. Alternatively, when the measurement value of miR-133a-3p is obtained as the measurement value of miRNA-3p, if the measurement value of miR-133a-3p is less than the second threshold, it may be determined that the cells induced to differentiate by the second induction treatment are not suitable for transplantation. Moreover, when the measured value of miR-133a-3p is equal to or greater than the second threshold, it can be determined that the cells induced to differentiate by the second induction treatment are suitable for transplantation. Moreover, when the measured value of miR-133a-3p is equal to or greater than the second threshold, it can be determined that at least 70% of the cells induced to differentiate by the second induction treatment are differentiated into cardiomyocytes. By using the above-mentioned evaluation method using the measured value of miR-1-3p or the measured value of miR-133a-3p, it is possible to identify cells that are unsuitable for transplantation or cells that are suitable for transplantation at an early stage, and to determine whether or not to continue the culture at an early stage. The first threshold and the second threshold are as described above.

[0071] Alternatively, when the measured values ​​of miR-1-3p and miR-133a-3p are obtained as the measured values ​​of miRNA-3p, if the measured values ​​of miR-1-3p are less than the first threshold and the measured values ​​of miR-133a-3p are less than the second threshold, it may be determined that the cells induced to differentiate by the second induction treatment are not suitable for transplantation. If the measured values ​​of miR-1-3p are equal to or greater than the first threshold and the measured values ​​of miR-133a-3p are equal to or greater than the second threshold, it may be determined that the cells induced to differentiate by the second induction treatment are suitable for transplantation. If the measured values ​​of miR-1-3p are equal to or greater than the first threshold and the measured values ​​of miR-133a-3p are equal to or greater than the second threshold, it may be determined that at least 70% of the cells induced to differentiate by the second induction treatment are differentiated into cardiomyocytes.

[0072] Alternatively, when the measured values ​​of miR-1-3p and miR-133a-3p are obtained as the measured values ​​of miRNA-3p, if the measured value of miR-1-3p is less than the first threshold or the measured value of miR-133a-3p is less than the second threshold, it may be determined that the cells induced to differentiate by the second induction treatment are not suitable for transplantation. If the measured value of miR-1-3p is equal to or greater than the first threshold or the measured value of miR-133a-3p is equal to or greater than the second threshold, it may be determined that the cells induced to differentiate by the second induction treatment are applicable to transplantation. If the measured value of miR-1-3p is equal to or greater than the first threshold or the measured value of miR-133a-3p is equal to or greater than the second threshold, it may be determined that at least 70% of the cells induced to differentiate by the second induction treatment are differentiated into cardiomyocytes.

[0073] The present invention may further include a step of collecting the supernatant of a liquid medium containing cells that have been induced to differentiate by the first induction treatment and before the second induction treatment, and measuring miR-489-3p in the supernatant. Human miR-489-3p itself is publicly known, and the nucleotide sequence of this miRNA is shown in SEQ ID NO: 3. In addition, the gene encoding human miR-489-3p is published in the GenBank database under the accession number NR_030164. The measured value of miR-489-3p is an indicator of differentiation into mesodermal cells. The time of collecting the supernatant is not particularly limited as long as a sufficient period of time has passed for PSCs to differentiate into mesodermal cells. On the other hand, the first induction treatment is terminated by changing the medium. If not much time has passed since the medium was changed, it is considered that the above-mentioned miR-489-3p is not sufficiently contained in the supernatant. Therefore, the time to collect the supernatant can be, for example, one to three days after the end of the first induction treatment and before the second induction treatment is performed.

[0074] The time to collect the supernatant may be determined, for example, according to the length of the period of the first induction treatment. When the period of the first induction treatment is 12 hours or more but less than 24 hours, the time to collect the supernatant may be, for example, a time point when 2 days or more but not exceeding 3 days have passed since the day the first induction treatment was completed and before the second induction treatment is performed. When the period of the first induction treatment is 1 day or 2 days, the time to collect the supernatant may be, for example, a time point when 1 day or more but not exceeding 2 days have passed since the day the first induction treatment was completed and before the second induction treatment is performed. In the example of FIG. 1, the period of the first induction treatment is 1 day, and it is preferable to collect the supernatant on the third day (Day 3), that is, 2 days after the day the first induction treatment is completed.

[0075] As a negative control for miRNA measurement, the supernatant before the first induction treatment (hereinafter also referred to as the "first control sample") may be collected. The first control sample may be a supernatant collected at any time before the first induction treatment. The supernatant collected at such a time contains almost no miR-489-3p. The first control sample can also be said to be a sample for obtaining a background value for the measurement of miR-489-3p. Preferably, the first control sample is a liquid medium that is removed when the medium is replaced to perform the first induction treatment. In the example of FIG. 1, the control sample is a liquid medium containing cells before the medium replacement on Day 0. The first control sample can be obtained by recovering a part or the whole of the liquid medium before removing it.

[0076] As shown in Example 1 described later, the supernatant was collected from the liquid medium containing the iPS cells subjected to the first induction treatment before and after the operation of inducing differentiation into mesodermal cells was completed. In this specification, the "operation of inducing differentiation into mesodermal cells" includes performing the first induction treatment and culturing the cells in the liquid medium for a period sufficient for the cells to differentiate into mesodermal cells. After the operation was completed, the cells were subjected to immunofluorescence staining for Brachyury T, a mesodermal marker. As a result, the measured value of miR-489-3p was significantly higher in the supernatant collected after the completion of differentiation induction than in the supernatant collected before the completion of differentiation induction. In addition, the positive rate of Brachyury T was high in the cells after the completion of differentiation induction, and most of the iPS cells were differentiated into mesodermal cells. Therefore, information on the differentiation state of PSCs into mesodermal cells can be obtained based on the measured value of miR-489-3p. Such information includes, for example, information on whether PSCs have differentiated into mesodermal cells. For example, when the measured value of miR-489-3p is equal to or greater than the third threshold, it may be suggested that the PSCs have differentiated into mesodermal cells. The third threshold corresponds to the measured value of miR-489-3p.

[0077] The present invention may include a step of comparing the measured value of miR-489-3p with a third threshold value, and a step of judging whether or not the PSC has differentiated into a mesodermal cell based on the result of the comparison. For example, when the measured value of miR-489-3p is equal to or greater than the third threshold value, it may be judged that the PSC has differentiated into a mesodermal cell. "Differentiated into a mesodermal cell" means that a sufficient amount of mesodermal cells has been obtained to obtain a quantity of cardiomyocytes applicable for transplantation. "Differentiated into a mesodermal cell" does not mean that all PSCs have differentiated into mesodermal cells. In addition, when the measured value of miR-489-3p is less than the third threshold value, it may be judged that the differentiation of the PSC into a mesodermal cell is insufficient. Since cardiomyocytes differentiate from PSCs via mesodermal cells, if the differentiation of PSCs into mesodermal cells is insufficient at this stage, it is highly likely that cardiomyocytes applicable for transplantation will not be obtained even if differentiation induction is continued. Therefore, when the measured value of miR-489-3p is less than the third threshold, it may be predicted that cardiomyocytes suitable for transplantation will not be obtained. When it is determined that the differentiation of PSCs into mesodermal cells is insufficient or when it is predicted that cardiomyocytes suitable for transplantation will not be obtained, the induction of differentiation into cardiomyocytes can be discontinued.

[0078] The third threshold is not particularly limited and can be set appropriately. For example, the threshold may be set as follows. The first induction treatment is performed on PSCs to obtain cells induced to differentiate. Also, PSCs are cultured without the first induction treatment. When the operation of inducing differentiation into mesodermal cells is completed, the supernatant is collected from the liquid medium containing each cell, and the measurement value of miR-489-3p is obtained. Also, the Brachyury T positivity rate is confirmed for each cell. From the obtained measurement value, a value that can distinguish cells that have been subjected to the first induction treatment from cells that have not been subjected to the first induction treatment is obtained, and the value is set as the third threshold. It is preferable to consider the sensitivity, specificity, positive predictive value, negative predictive value, etc. in setting the threshold.

[0079] The present invention also includes a method for producing cardiomyocytes (hereinafter also referred to as the "production method of this embodiment"). In the production method of this embodiment, cardiomyocytes are obtained by determining that differentiation-induced cells have differentiated into cardiomyocytes with high differentiation efficiency based on the measured value of miRNA-3p. In the production method of this embodiment, first, mesodermal cells are induced to differentiate into cardiomyocytes in a liquid medium by an induction treatment for inducing differentiation of the mesodermal cells into cardiomyocytes. This induction treatment is the same as the second induction treatment described above. The mesodermal cells are not particularly limited, but are preferably cells obtained by subjecting PSCs to the first induction treatment. The liquid medium and culture conditions are as described above.

[0080] Next, the supernatant of the liquid medium containing the cells induced is collected, and the miRNA-3p of the miR-1 / 133a cluster in the supernatant is measured. The timing and method of collecting the supernatant are the same as those of the supernatant from the liquid medium containing the cells induced to differentiate by the second induction treatment. The method of measuring miRNA is as described above. Then, the measured value of the miRNA-3p of the miR-1 / 133a cluster is compared with the threshold value. When the measured value of the miRNA-3p is equal to or greater than the threshold value, it is determined that the cells induced to differentiate by the induction treatment are differentiated into cardiomyocytes at a high rate as described above. Thus, cardiomyocytes can be obtained by culturing the cells. The liquid medium and culture conditions used for culturing the cells induced to differentiate into cardiomyocytes are known. When the measured value of the miRNA-3p is less than the threshold value, it is determined that the cells induced to differentiate by the induction treatment are not suitable for transplantation as cardiomyocytes as described above. The culture of the cells can be stopped based on this determination.

[0081] For example, when the measurement value of miR-1-3p is obtained as the measurement value of miRNA-3p, when the measurement value of miR-1-3p is equal to or greater than the first threshold, the cells induced to differentiate by the induction process can be cultured to obtain cardiomyocytes. When the measurement value of miR-1-3p is less than the first threshold, the culture can be stopped. Alternatively, when the measurement value of miR-133a-3p is obtained as the measurement value of miRNA-3p, when the measurement value of miR-133a-3p is equal to or greater than the second threshold, the cells induced to differentiate by the induction process can be cultured to obtain cardiomyocytes. When the measurement value of miR-133a-3p is less than the second threshold, the culture can be stopped. The first threshold and the second threshold are as described above.

[0082] Alternatively, when the measured values ​​of miR-1-3p and miR-133a-3p are obtained as the measured values ​​of miRNA-3p, when the measured values ​​of miR-1-3p are equal to or greater than a first threshold and the measured values ​​of miR-133a-3p are equal to or greater than a second threshold, the cells induced to differentiate by the induction treatment can be cultured to obtain cardiomyocytes. When the measured values ​​of miR-1-3p are less than the first threshold and the measured values ​​of miR-133a-3p are less than the second threshold, the culture can be discontinued.

[0083] Alternatively, when the measured values ​​of miR-1-3p and miR-133a-3p are obtained as the measured values ​​of miRNA-3p, when the measured value of miR-1-3p is equal to or greater than the first threshold or the measured value of miR-133a-3p is equal to or greater than the second threshold, the cells induced to differentiate by the induction treatment can be cultured to obtain cardiomyocytes. When the measured value of miR-1-3p is less than the first threshold or the measured value of miR-133a-3p is less than the second threshold, the culture can be discontinued.

[0084] The mesodermal cells used in the production method of this embodiment can be cells that have been induced to differentiate from PSCs and determined to have differentiated into mesodermal cells based on the measured value of miR-489-3p. Specifically, first, PSCs are induced to differentiate into mesodermal cells in a liquid medium by an induction treatment for inducing differentiation of PSCs into mesodermal cells. This induction treatment is the same as the first induction treatment described above. The PSCs, liquid medium, and culture conditions are as described above.

[0085] Next, the supernatant of the liquid medium containing the cells induced is collected, and miR-489-3p in the supernatant is measured. The timing and method of collecting the supernatant are the same as those of collecting the supernatant from the liquid medium containing the cells induced to differentiate by the first induction treatment. The method of measuring miRNA is as described above. Then, the measured value of miR-489-3p is compared with the third threshold value. When the measured value of miR-489-3p is equal to or greater than the third threshold value, the cells induced to differentiate by the induction treatment are determined to have differentiated into mesodermal cells. Therefore, the cells can be used as mesodermal cells in the production method of this embodiment. When the measured value of miR-489-3p is less than the third threshold value, the cells induced to differentiate by the induction treatment are determined to have insufficient differentiation into mesodermal cells, or it is predicted that cardiomyocytes applicable to transplantation cannot be obtained. In this case, the induction of differentiation into cardiomyocytes can be discontinued.

[0086] The present invention also includes a reagent kit (hereinafter, also referred to as "reagent kit") used in the method for evaluating the differentiation state of a cell, the method for determining the differentiation state of a cell, or the method for producing cardiomyocytes. The reagent kit includes an oligonucleotide primer and / or an oligonucleotide probe for detecting miRNA-3p of the miR-1 / 133a cluster. The reagent kit may further include an oligonucleotide primer and / or an oligonucleotide probe for detecting miR-489-3p. The oligonucleotide primer and / or the oligonucleotide probe for detecting each miRNA may be bound to a solid phase such as a plate or particles. The reagent kit may include other reagents such as a buffer solution, dNTPs (dATP, dTTP, dGTP, and dCTP), reverse transcriptase, and polymerase.

[0087] The oligonucleotide primer may be a primer for reverse transcription reaction of each miRNA (hereinafter also referred to as "RT primer"), or may be a forward primer and / or a reverse primer for amplifying cDNA synthesized from each miRNA (hereinafter also referred to as "cDNA amplification primer"). The reagent kit may include both an RT primer and a cDNA amplification primer. When measuring miRNA using an oligonucleotide primer, since miRNA has a short nucleotide length, a stem-loop primer can be used as the RT primer and / or the cDNA amplification primer.

[0088] An example of the reagent kit is shown in Figure 2A. In Figure 2A, 10 indicates the reagent kit, 11 indicates a first container containing oligonucleotide primers and / or oligonucleotide probes for detecting miR-1-3p, 12 indicates a packaging box, and 13 indicates an attached document. The attached document may describe the nucleotide sequence of the primers and / or probes, usage methods such as PCR conditions, storage methods, and the like. The first container may contain oligonucleotide primers and / or oligonucleotide probes for detecting miR-133a-3p.

[0089] Another example of the reagent kit is shown in Fig. 2B. In Fig. 2B, 20 denotes a reagent kit, 21 denotes a first container containing an oligonucleotide primer and / or an oligonucleotide probe for detecting miR-1-3p, 22 denotes a second container containing an oligonucleotide primer and / or an oligonucleotide probe for detecting miR-133a-3p, 23 denotes a packaging box, and 24 denotes an attached document.

[0090] Another example of the reagent kit is shown in Figure 3. In Figure 3, 30 indicates the reagent kit, 31 indicates a first container containing oligonucleotide primers and / or oligonucleotide probes for detecting miR-1-3p, 32 indicates a second container containing oligonucleotide primers and / or oligonucleotide probes for detecting miR-133a-3p, 33 indicates a third container containing oligonucleotide primers and / or oligonucleotide probes for detecting miR-489-3p, 34 indicates a packaging box, and 35 indicates an attached document.

[0091] The present invention includes the use of oligonucleotide primers and / or oligonucleotide probes for detecting miRNA-3p of the miR-1 / 133a cluster to manufacture a reagent kit for use in a method for evaluating or determining the differentiation state of a cell, or a method for producing cardiomyocytes. The present invention also includes the use of oligonucleotide primers and / or oligonucleotide probes for detecting miRNA-3p of the miR-1 / 133a cluster and oligonucleotide primers and / or oligonucleotide probes for detecting miR-489-3p to manufacture a reagent kit for use in a method for evaluating or determining the differentiation state of a cell, or a method for producing cardiomyocytes.

[0092] The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples. EXAMPLES

[0093] Example 1: Evaluation of mesoderm differentiation by miR489-3p In the process of inducing differentiation of pluripotent stem cells (PSCs) to obtain cardiomyocytes, we investigated whether miR489-3p could be an indicator showing that PSCs have differentiated into mesodermal cells by the first induction treatment.

[0094] (1) Cell differentiation induction and supernatant collection As PSCs, human iPS cells (253G4 clone and 201B7 clone) were obtained from the Center for iPS Cell Research and Application (CiRA), Kyoto University. In Example 1, two groups of iPS cells were prepared, and each group was treated with a different efficiency of differentiation induction into cardiomyocytes. One group contained cells that had been subjected to the first and second induction treatments that are generally performed to induce differentiation into cardiomyocytes. Hereinafter, these cells will also be referred to as "group H" (details of the induction treatment will be described later). The other group contained cells that had not been subjected to the first induction treatment, but had only been subjected to the second induction treatment. Hereinafter, these cells will also be referred to as "group L". Group L was a group in which the differentiation efficiency was artificially reduced compared to group H.

[0095] With reference to FIG. 4, the procedure for inducing differentiation from iPS cells to cardiomyocytes in Example 1 will be described. FIG. 4 shows the schedule of cell culture and differentiation induction and the time when the supernatant was obtained. In Example 1, the day on which the first induction treatment was performed on the iPS cells was determined as "day 0". In FIG. 4, day 0 is indicated as "d0". "M1" to "M10" in the figure indicate various liquid media used in each period. The black bars in the figure indicate the period in which the liquid media containing each additive were used. The arrows in the figure indicate the time when the supernatant was obtained from the liquid medium containing the cells. With reference to FIG. 4, the period from 4 days before day 0 (d-4) to day 0 (d0) was the period for maintaining and culturing the iPS cells. The period from day 0 (d0) to day 7 (d7) was the period for inducing differentiation of the iPS cells to cardiomyocytes. The period from day 7 (d7) onwards was the period for maintaining and culturing the cardiomyocytes induced to differentiate. To confirm the proportion of cells that had differentiated into cardiomyocytes, a portion of the cells in culture was taken on day 10 (d10) and day 17 (d17) and cTnT-positive cells were detected by the flow cytometry method described below.

[0096] (1.1) Maintenance culture The iPS cells of each clone were maintained and cultured on a plate coated with Matrigel (registered trademark) (Corning Incorporated). StemFit (registered trademark) AS103C (Ajinomoto Co., Inc.) was used as the liquid medium. Referring to FIG. 4, four days before day 0 (d-4), the iPS cells of each clone (1 × 10 5 The iPS cells from each clone were passaged onto a new plate. StemFit AS103C containing 10 μM Y-27632 (FUJIFILM Wako Pure Chemical Corporation) was used as the liquid medium (M1 in the figure). The medium was replaced 3 days before day 0 (d-3), and the cells were cultured until day 0 (d0). The liquid medium after replacement (M2 in the figure) was StemFit AS103C. The iPS cells from each clone were divided into two groups to be used as groups H and L.

[0097] (1.2) First induction treatment (induction of differentiation of iPS cells into mesodermal cells) Referring to FIG. 4, on day 0 (d0), the supernatant (100 μL) was collected from the liquid medium containing the cells, and the cells were washed with Dulbecco's PBS (D-PBS) (FUJIFILM Wako Pure Chemical Corporation). To the cells of group H, RPMI1640 (FUJIFILM Wako Pure Chemical Corporation) containing 2% B-27™ supplement (insulin-free), 6 μM CHIR99021 (FUJIFILM Wako Pure Chemical Corporation) and 1 ng / mL BMP4 (R&D Systems) was added as the liquid medium (M3 in the figure), and the cells were cultured until day 1 (d1). To the cells of group L, StemFit RPMI1640 containing 2% B-27™ supplement (insulin-free) was added as the liquid medium (M3 in the figure), and the cells were cultured until day 1 (d1). The subsequent operations were performed on all groups. On day 1 (d1), the supernatant (100 μL) was collected from the liquid medium containing the cells, and the cells were washed with D-PBS. RPMI1640 containing 2% B-27 supplement (without insulin) was added as the liquid medium (M4 in the figure), and the cells were cultured until day 3 (d3).

[0098] (1.3) Second induction treatment (induction of differentiation of mesodermal cells into cardiomyocytes) Referring to FIG. 4, on the third day (d3), a supernatant (100 μL) was collected from the liquid medium containing the cells, and the cells were washed with D-PBS. RPMI1640 containing 2% B-27 supplement (insulin-free) and 5 μM IWR-1 (Sigma-Aldrich) was added as the liquid medium (M5 in the figure), and the cells were cultured until the fifth day (d5). On the fifth day (d5), a supernatant (100 μL) was collected from the liquid medium containing the cells, and the cells were washed with D-PBS. RPMI1640 containing 2% B-27 supplement (insulin-free) was added as the liquid medium (M6 in the figure), and the cells were cultured until the seventh day (d7). On the seventh day (d7), a supernatant (100 μL) was collected from the liquid medium containing the cells, and the medium was replaced. The liquid medium after replacement (M7 in the figure) was MEMα (Thermo Fisher Scientific) containing 5% FBS and 2 mM sodium pyruvate. The cells were then cultured until day 9 (d9).

[0099] (1.4) Purification and maintenance of cells after differentiation induction Referring to FIG. 4, on the 9th day (d9), a supernatant (100 μL) was collected from the liquid medium containing the cells, and the cells were washed with D-PBS. The cells were dispersed and collected using a solution containing 0.25% trypsin and 1 mM EDTA. The collected cells were resuspended in liquid medium and added to a plate coated with type I collagen (AGC Technoglass Co., Ltd.). The liquid medium used (M8 in the figure) was MEMα containing 5% FBS. The cells were then cultured until the 13th day (d13). On the 13th day (d13), the medium was replaced. The liquid medium after replacement (M9 in the figure) was StemFit AS501 (Ajinomoto Co., Ltd.). The cells were cultured until the 17th day (d17). On the 17th day (d17), the medium was replaced. The liquid medium after replacement (M10 in the figure) was MEMα containing 5% FBS. Thereafter, the liquid medium was changed every other day and the cells were maintained in culture until day 51 (d51).

[0100] (2) Measurement of miR489-3p in the supernatant miRNA was isolated from the collected supernatant (100 μL) by the Boom method (Boom R. et al., J. Clin. Microbiol. vol. 28, pp. 495-503, 1990) using magnetic beads Sicastar-M (CoreFront). cDNA was synthesized from a portion of the obtained miRNA using MultiScribe reverse transcriptase (Applied Biosystems). The reverse transcription reaction was incubated at 16°C for 30 minutes, 42°C for 30 minutes, and 85°C for 5 minutes, followed by maintenance at 4°C. Real-time PCR was performed using standard TaqMan™ PCR protocol with hot start ExTaq PCR enzyme (Takara Bio Inc.) and LightCycler® 96 System (Roche). The PCR conditions were heating at 95°C for 10 minutes, followed by 40 cycles of 95°C for 15 seconds and 60°C for 1 minute. The Ct (threshold cycle) value was defined as the cycle number at which the fluorescence intensity exceeded a certain threshold. The Ct value was converted to copy number using a standard curve. The standard curve was created from the measurement results of synthetic miRNA. Primers and probes for miR489-3p were purchased as TaqMan™ MicroRNA Assay (Thermo Fisher Scientific).

[0101] (3) Immunofluorescence staining of mesodermal markers Immunofluorescence staining of brachyury T as a mesodermal marker was performed on cells of groups H and L on day 1 (d1). The specific procedures were as follows: Cells were washed with D-PBS and fixed with 4% paraformaldehyde. Fixed cells were washed with D-PBS and permeabilized with 0.1% Triton™X. Cells were washed with PBS containing 0.05% Tween™20 (PBS-T) and blocked with ImmunoBlock (KAC) for 1 hour at room temperature. Cells were incubated overnight at 4°C with ImmunoBlock containing anti-Brachyury T antibody (Abcam) at 1:200. Cells were washed with D-PBS and incubated with ImmunoBlock containing secondary antibody Alexa Fluor™ 488-labeled anti-rabbit IgG antibody (Thermo Fisher Scientific) for 2 hours at room temperature. The cells were washed twice with D-PBS and nuclear stained with 5 μg / mL Hoechst 33342 (Thermo Fisher Scientific). The cells were washed with D-PBS and observed under a fluorescent microscope BZ-X710 (Keyence). Based on the observation results, the ratio of Brachyury T positive cells was calculated.

[0102] (4) Detection of cTnT-positive cells On the 10th day, the cells of groups H and L were bound with PE-labeled anti-cardiac troponin T (cTnT) antibody and analyzed by flow cytometry (FCM) to detect cTnT-positive cells. The specific procedures were as follows: After washing the cells with D-PBS, the cells were dispersed and collected using a solution containing 0.25% trypsin and 1 mM EDTA. The cells were fixed with 4% paraformaldehyde. The fixed cells were washed with D-PBS and permeabilized with 0.1% Triton (trademark) X. The cells were washed with PBS-T and incubated in ImmunoBlock containing PE-labeled anti-cTnT antibody (Miltenyi Biotec) or PE-labeled anti-REA control antibody (Miltenyi Biotec) for 15 to 20 minutes in the dark. The cells were washed with D-PBS and analyzed with a Gallios flow cytometer (Beckman Coulter). The cTnT positivity rate was calculated based on the number of cTnT-positive cells detected.

[0103] (5) Results Figures 5A and B show the copy number of miR-489-3p in 100 μL of the supernatant obtained during the differentiation induction process of iPS cells from each clone (n=3, experiments were performed independently for each clone). In the figures, "*" indicates p<0.05, and "**" indicates p<0.01. Error bars indicate standard deviation (SD). As can be seen from Figure 5A, in Group H of clone 201B7, the expression level of miR-489-3p hardly changed on day 1, but the expression level of miR-489-3p rapidly increased on day 3. Then, the expression level of miR-489-3p decreased from day 5 onwards, and the expression of miR-489-3p almost disappeared on day 9. On the other hand, in Group L, no significant increase in the expression level of miR-489-3p was observed between days 0 and 9. Referring to Figure 5B, the same results were obtained in Groups H and L of clone 253G4.

[0104] An example of an image of immunofluorescently stained cells for groups H and L is shown in FIG. 6. In the figure, the scale bar indicates 100 μm. FIG. 7 shows the ratio of Brachyury T positive cells in groups H and L (n=3, the experiment was performed independently for the 253G4 clone). In FIG. 7, "**" indicates p<0.01. The error bars indicate SD. As can be seen from these figures, most of the cells in group H were Brachyury T positive cells. On the other hand, no Brachyury T positive cells were detected in group L. Since Brachyury T is a mesoderm marker, these results indicate that on day 1, the cells in group H were differentiated into mesoderm cells. On the other hand, it was indicated that the cells in group L were not differentiated into mesoderm cells.

[0105] FIG. 8 shows a graph with the fluorescent signal intensity (indicated as "cTnT" in the figure) and count (cell number) on two axes for groups H and L. Cells with high fluorescent signal intensity were cTnT positive cells. The cTnT positive rate of cells in group H was 93.1%, while the cTnT positive rate of cells in group L was 0.1%. Since cTnT is a cardiomyocyte marker, these results indicate that the cells in group H had differentiated into cardiomyocytes on day 10. On the other hand, it was shown that the cells in group L had not differentiated into cardiomyocytes.

[0106] As mentioned above, in group H, most of the iPS cells were induced to differentiate into mesodermal cells one day after the first induction treatment, and the expression level of miR-489-3p increased three days after the first induction treatment. On the other hand, in group L, which did not undergo the first induction treatment, differentiation into mesodermal cells was not observed, and the expression level of miR-489-3p did not increase. In group H, the expression level of miR-489-3p decreased after the second induction treatment on day 5. Then, on day 10, most of the mesodermal cells in group H had become cardiomyocytes. This suggests that the expression of miR-489-3p decreased as mesodermal cells were induced to differentiate into cardiomyocytes. These results suggest that miR-489-3p can be an indicator of the differentiation of PSCs into mesodermal cells. Although immunofluorescence staining with Brachyury T consumes precious PSCs, we found that measuring miR-489-3p in the supernatant allows for non-destructive evaluation of PSC differentiation into mesoderm.

[0107] Example 2: Evaluation of cardiac differentiation by miR-1-3p and miR-133a-3p In the process of inducing differentiation of PSCs into cardiomyocytes, we investigated whether miR-1-3p and miR-133a-3p could be indicators of cardiomyocyte differentiation. For comparison, we also measured various miRNAs known to be related to cardiomyocyte differentiation.

[0108] (1) Induction of cell differentiation, collection of supernatant, and classification of the obtained cells Under clinical grade mass culture conditions, human iPS cells were subjected to the first and second induction treatments to obtain differentiation-induced cells (11 kinds of lots). The procedure of differentiation induction in Example 2 was based on the method of group H in Example 1 (see FIG. 4). On the 3rd and 7th days, supernatants (100 μL) were collected from the liquid medium containing each lot of cells. On the 10th and 17th days, a portion was collected from each lot of cells, and the cTnT positivity rate was calculated by FCM analysis. On the 10th day, cells from the lot with a cTnT positivity rate of 70% or more were classified as "accepted products," and cells from the lot with a cTnT positivity rate of less than 70% were classified as "rejected products." Table 2 shows the cTnT positivity rates of each lot of cells on the 10th day (d10) and the 17th day (d17).

[0109] [Table 2]

[0110] (2) Measurement of miRNA in the supernatant The miRNAs in the supernatants on the 3rd and 7th days were measured in the same manner as in Example 1. The measured miRNAs were miR-1-3p, miR-1-5p, miR-133a-3p, miR-133a-5p, miR-23b-3p, miR-23b-5p, miR-26b-3p, miR-26b-5p, miR-125b-3p, miR-125b-5p, miR-145-3p, miR-145-5p, miR-499a-3p, miR-499a-5p, miR-503-3p and miR-503-5p. MiRNAs other than miR-1 and miR-133a were known to be associated with cardiomyocyte differentiation. miR-23b was known to be involved in cardiac differentiation. miR-26b was known to be involved in myocardial differentiation and the Wnt signaling system. miR-125b was known to be involved in early myocardial differentiation together with LIN28. miR-145 was known to be involved in myocardial differentiation and myocardial-related genes. miR-499a was known to be involved in myocardial differentiation and to form a cluster with miR-208. miR-503 was known to be involved in myocardial differentiation and myocardial-related genes and to form a cluster with miR-322. The above miRNAs were measured in the same manner as in Example 1. Primers and probes for each miRNA were purchased as TaqMan™ MicroRNA Assay (Thermo Fisher Scientific).

[0111] (3) Results 9 to 20 show the relative expression level of each miRNA in 100 μL of the supernatant (passed products n=6, rejected products n=5, experiments were performed independently for each lot). The relative expression level was calculated by the following formula. (Relative expression level) = 2^{(Ct value when measuring miRNA in the supernatant on the 3rd day) - (Ct value when measuring miRNA in the supernatant on the 7th day)}

[0112] As described above, the lot classified as a passing product had a high percentage of cells differentiated into cardiomyocytes, at 90% or more, on the 10th day. In contrast, the lot classified as a failing product had a low percentage of cells differentiated into cardiomyocytes. When the relative expression level of a miRNA shows a significant difference between a passing product and a failing product, the miRNA can be said to be an indicator that makes it possible to distinguish a lot with a high percentage of cells differentiated into cardiomyocytes from a lot with a low percentage. The measurement results of each miRNA are explained below.

[0113] Referring to FIG. 9(A), the relative expression level of miR-1-3p was high in the passed product and low in the failed product. The difference in the relative expression level between the passed product and the failed product was significant (p=0.00299). On the other hand, referring to FIG. 9(B), the relative expression level of miR-1-5p was low in both the passed product and the failed product, and did not show a significant difference (p=0.35079). These results showed that the relative expression level of miR-1-3p made it possible to distinguish between the passed product and the failed product, but the relative expression level of miR-1-5p did not make such a distinction. FIG. 10 shows a box plot in which the relative expression level of FIG. 9(A) was converted to copy number. As can be seen from FIG. 10, the difference between the passed product and the failed product was more prominently shown by displaying the measured value of miR-1-3p as copy number (p=0.00278). Thus, the measurement of miR-1-3p in the supernatant up to day 7 gave the same results as the FCM analysis performed on day 10. Therefore, it was suggested that miR-1-3p can be an indicator of differentiation into cardiomyocytes.

[0114] The measurement results of miR-133a-3p were similar to those of miR-1-3p. Referring to FIG. 11(A), the relative expression level of miR-133a-3p was high in the passed product and low in the failed product. The difference in the relative expression level between the passed product and the failed product was significant (p=0.00245). On the other hand, referring to FIG. 11(B), the relative expression level of miR-133a-5p was low in both the passed product and the failed product, and did not show a significant difference (p=0.24092). FIG. 12 shows a box plot in which the relative expression level of FIG. 11(A) was converted to copy number. As can be seen from FIG. 12, by displaying the measured value of miR-133a-3p as copy number, the difference between the passed product and the failed product was more prominent (p=0.00206). Therefore, it was possible to distinguish between the passed product and the failed product by the relative expression level of miR-133a-3p. miR-133a-3p was suggested to be an indicator of differentiation into cardiomyocytes.

[0115] Referring to (A) of FIG. 13, the relative expression level of miR-23b-3p was high in the passed product and low in the failed product. The difference in the relative expression level between the passed product and the failed product was significant (p=0.00142). On the other hand, referring to (B) of FIG. 13, the relative expression level of miR-23b-5p did not show a significant difference between the passed product and the failed product (p=0.20920). These results showed that the relative expression level of miR-23b-3p made it possible to distinguish between the passed product and the failed product, but the relative expression level of miR-23b-5p did not make such a distinction. However, compared with miR-1-3p and miR-133a-3p, the relative expression level of miR-23b-3p in the passed product was small. In this regard, for example, in order to compare FIG. 13(A) with FIG. 11(A), the box plot of FIG. 13(A) was redrawn with the maximum value of the vertical axis changed to 80. This is shown in FIG. 14. As can be seen from FIG. 14, the relative expression level of miR-23b-3p was displayed low in both the passed and failed products. This was because the expression level of miR-23b-3p was not significantly different between the 3rd and 7th days even in the passed products. Therefore, it was suggested that miR-1-3p and miR-133a-3p could more clearly distinguish between passed and failed products than miR-23b-3p.

[0116] Referring to FIG. 15(A), the relative expression level of miR-26b-3p was high in the passed product and low in the failed product. The difference in the relative expression level between the passed product and the failed product was significant (p=0.00198). The measurement result of miR-26b-5p was similar to that of miR-26b-3p. Referring to FIG. 15(B), the relative expression level of miR-26b-5p showed a significant difference between the passed product and the failed product (p=0.00061). These results showed that the relative expression levels of miR-26b-3p and miR-26b-5p made it possible to distinguish between passed and failed products. However, compared with miR-1-3p and miR-133a-3p, the relative expression levels of miR-26b-3p and miR-26b-5p in the passed product were small. The box plots in (A) and (B) of Figure 15 were redrawn with the maximum value of the vertical axis changed to 80. These are shown in (A) and (B) of Figure 16. As can be seen from these figures, the relative expression levels of miR-26b-3p and miR-26b-5p were displayed low in both the passed and failed products. This suggests that miR-1-3p and miR-133a-3p can more clearly distinguish between passed and failed products than miR-26b-3p and miR-26b-5p.

[0117] Referring to FIG. 17(A), the relative expression level of miR-125b-3p was low in both the passed and failed products, and no significant difference was observed (p=0.19892). On the other hand, referring to FIG. 17(B), the relative expression level of miR-125b-5p was high in the passed products and low in the failed products. However, miR-125b-5p did not show a significant difference in the relative expression level that could clearly distinguish between the passed and failed products (p=0.05005). These results showed that the relative expression levels of miR-125b-3p and miR-125b-5p could not be used to distinguish between passed and failed products.

[0118] Referring to (A) of FIG. 18, the relative expression level of miR-145-3p was high in the passed product and low in the failed product. However, miR-145-3p did not show a significant difference in relative expression level to clearly distinguish between the passed product and the failed product (p=0.06853). Referring to (B) of FIG. 18, the relative expression level of miR-145-5p did not show a significant difference between the passed product and the failed product (p=0.18337). These results showed that the relative expression levels of miR-145-3p and miR-145-5p could not distinguish between the passed product and the failed product.

[0119] Referring to Figures 19 (A) and (B), the relative expression levels of miR-499a-3p and miR-499a-5p did not show significant differences between the passed and failed products (p=0.88740 and 0.18337). These results indicate that the relative expression levels of miR-499a-3p and miR-499a-5p cannot distinguish between passed and failed products.

[0120] Referring to Figures 20(A) and (B), the relative expression levels of miR-503-3p and miR-503-5p did not show significant differences between the acceptable and unacceptable products (p=0.77954 and 0.89987). These results indicate that the relative expression levels of miR-503-3p and miR-503-5p cannot distinguish between the acceptable and unacceptable products. [Explanation of symbols]

[0121] 10, 20, 30 Reagent Kits 11, 21, 31 1st container 12, 23, 34 Packing box 13, 24, 35 Attachment 22, 32 2nd container 33 Third container

Claims

1. A step of inducing differentiation of pluripotent stem cells into cardiomyocytes in a liquid medium by a first induction treatment of inducing differentiation of pluripotent stem cells into mesodermal cells and a second induction treatment of inducing differentiation of the mesodermal cells into cardiomyocytes; collecting a supernatant of the liquid medium containing the cells induced to differentiate by the second induction treatment; measuring miRNA-3p of the miR-1 / 133a cluster in the supernatant; Including, The miRNA-3p is at least one selected from the group consisting of miR-1-3p and miR-133a-3p; The measured value of the miRNA-3p is an indicator of differentiation into cardiomyocytes. A method for assessing the differentiation state of a cell.

2. the miRNA-3p is the miR-1-3p, The method according to claim 1, wherein when the measured value of miR-1-3p is below a first threshold, it is suggested that the differentiated cells are not suitable for transplantation.

3. the miRNA-3p is the miR-133a-3p, The method according to claim 1, wherein when the measured value of miR-133a-3p is less than a second threshold, it is suggested that the differentiated cells are not suitable for transplantation.

4. the miRNA-3p is the miR-1-3p and the miR-133a-3p, The method of claim 1, wherein when the measurement value of the miR-1-3p is below a first threshold and the measurement value of the miR-133a-3p is below a second threshold, it is suggested that the differentiated cells are not suitable for transplantation.

5. the miRNA-3p is the miR-1-3p and the miR-133a-3p, The method of claim 1, wherein when the measurement value of the miR-1-3p is below a first threshold or the measurement value of the miR-133a-3p is below a second threshold, it is suggested that the differentiated cells are not suitable for transplantation.

6. The method according to claim 1 , wherein the first induction treatment comprises using a liquid medium containing at least one of a substance that promotes a BMP signaling pathway and a substance that promotes a Wnt signaling pathway.

7. The method according to claim 1 , wherein the second induction treatment comprises using a liquid medium containing a substance that inhibits the Wnt signaling pathway.

8. The method according to claim 1, further comprising the steps of collecting a supernatant of a liquid culture medium containing cells that have been induced to differentiate by the first induction treatment and have not yet been subjected to the second induction treatment, and measuring miR-489-3p in the supernatant, wherein the measured value of miR-489-3p is an indicator of differentiation into mesodermal cells.

9. The method according to claim 8, wherein when the measured value of miR-489-3p is equal to or greater than a third threshold, it is suggested that the pluripotent stem cell has differentiated into a mesodermal cell.

10. a differentiation induction step including a first induction treatment of inducing differentiation of pluripotent stem cells into mesodermal cells in a liquid medium, and a second induction treatment of inducing differentiation of the mesodermal cells into cardiomyocytes; collecting a supernatant of the liquid medium containing the cells induced to differentiate by the second induction treatment; measuring miRNA-3p of the miR-1 / 133a cluster in the supernatant; determining that the differentiation-induced cells are not suitable for transplantation when the measured value of the miRNA-3p is below a threshold value; Including, The miRNA-3p is at least one selected from miR-1-3p and miR-133a-3p; A method for determining the differentiation state of a cell.

11. the miRNA-3p is the miR-1-3p, the threshold is a first threshold, The method according to claim 10, wherein in the determination step, when the measured value of miR-1-3p is less than the first threshold, the differentiation-induced cells are determined to be unsuitable for transplantation.

12. the miRNA-3p is the miR-133a-3p, and the threshold is a second threshold; The method according to claim 10, wherein in the determination step, when the measured value of miR-133a-3p is less than the second threshold, the differentiation-induced cells are determined to be unsuitable for transplantation.

13. the miRNA-3p is the miR-1-3p and the miR-133a-3p, the thresholds are a first threshold and a second threshold, The method according to claim 10, wherein in the determination step, when the measured value of miR-1-3p is less than the first threshold and the measured value of miR-133a-3p is less than the second threshold, the differentiated cells are determined to be unsuitable for transplantation.

14. the miRNA-3p is the miR-1-3p and the miR-133a-3p, the thresholds are a first threshold and a second threshold, The method according to claim 10, wherein in the determination step, when the measured value of miR-1-3p is less than the first threshold or the measured value of miR-133a-3p is less than the second threshold, the differentiated cells are determined to be unsuitable for transplantation.

15. The method according to claim 10 , wherein the first induction treatment comprises an induction treatment using a liquid medium containing at least one of a substance that promotes a BMP signaling pathway and a substance that promotes a Wnt signaling pathway.

16. The method according to claim 10 , wherein the second induction treatment comprises using a liquid medium containing a substance that inhibits the Wnt signaling pathway.

17. A step of collecting a supernatant of a liquid medium containing cells that have been induced to differentiate by the first induction treatment and have not yet been subjected to the second induction treatment, and measuring miR-489-3p in the supernatant; determining that the pluripotent stem cells have differentiated into mesodermal cells when the measured value of the miR-489-3p is equal to or greater than a third threshold; The method of claim 10 further comprising:

18. A step of collecting a supernatant of a liquid medium containing cells that have been induced to differentiate by the first induction treatment and have not yet been subjected to the second induction treatment, and measuring miR-489-3p in the supernatant; determining that the differentiation of the pluripotent stem cells into mesodermal cells is insufficient when the measured value of the miR-489-3p is less than a third threshold value; The method of claim 10 further comprising:

19. (1) inducing differentiation of mesodermal cells into cardiomyocytes in a liquid medium by an inducing treatment for inducing differentiation of mesodermal cells into cardiomyocytes; (2) collecting the supernatant of the liquid medium containing the cells induced in the step (1); (3) measuring miRNA-3p of the miR-1 / 133a cluster in the supernatant collected in the step (2); (4) culturing the cells induced to differentiate in the step (1) to obtain cardiomyocytes when the measured value of the miRNA-3p is equal to or greater than a threshold value; wherein the miRNA-3p is at least one selected from miR-1-3p and miR-133a-3p; A method for producing cardiomyocytes.

20. The method according to claim 19, wherein in step (4), when the measured value of miRNA-3p is below the threshold, the culture of the cells induced to differentiate in step (1) is discontinued.

21. (i) inducing differentiation of pluripotent stem cells into mesodermal cells in a liquid medium by an induction treatment for inducing differentiation of the pluripotent stem cells into mesodermal cells; (ii) collecting the supernatant of the liquid medium containing the cells induced in step (i); (iii) measuring miR-489-3p in the supernatant collected in the step (ii); and when the measured value of miR-489-3p is equal to or greater than a third threshold, the cells of step (ii) are used as the mesodermal cells of step (1).

22. The method of claim 21, wherein the culturing of the cells in step (ii) is discontinued when the measured value of miR-489-3p is below a threshold value.

23. A reagent kit for use in the method according to any one of claims 1 to 7, 10 to 16, 19 and 20, comprising an oligonucleotide primer and / or an oligonucleotide probe for detecting miRNA-3p of the miR-1 / 133a cluster.

24. an oligonucleotide primer and / or an oligonucleotide probe for detecting miRNA-3p of the miR-1 / 133a cluster; Oligonucleotide primers and / or oligonucleotide probes for detecting miR-489-3p; A reagent kit for use in the method according to any one of claims 8, 9, 17, 18, 21 and 22, comprising:

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