JP7173496B2 - METHOD FOR SELECTING PLIPOTENTIAL STEM CELLS THAT HAVE DIFFERENTIATION TROPENCY INTO CARDIFERIC CELLS - Google Patents

Description

The present invention provides specific differentiation-induced cells, particularly pluripotent stem cells having a differentiation tropism to cardiomyocytes, embryoid bodies induced from the pluripotent stem cells, and differentiation-induced cells induced from the pluripotent stem cells. a method for screening the pluripotent stem cells; a method for treating a subject using the medical composition; a method for screening an effective agent using the medical composition; The present invention relates to quality control methods and the like in the production of compositions.

Adult myocardial cells have poor self-renewal ability, and when myocardial tissue is damaged, it is extremely difficult to repair it. In recent years, in order to repair damaged myocardial tissue, attempts have been made to transplant a graft containing myocardial cells produced by a cell engineering technique to an affected area (Patent Document 1, Non-Patent Document 1). Cardiomyocytes derived from pluripotent stem cells such as embryonic stem cells (ES cells) and induced pluripotent stem cells (iPS cells) have recently attracted attention as cardiomyocytes used for the preparation of such grafts. Attempts have been made to prepare sheet-like cell cultures containing cardiomyocytes derived from such pluripotent stem cells and to conduct therapeutic experiments on animals (Non-Patent Documents 2 and 3). However, the development of sheet-like cell cultures containing cardiomyocytes derived from pluripotent stem cells has just begun, and many aspects of their functional characteristics and factors affecting them are still unknown.

When preparing differentiation-inducing cells from pluripotent stem cells, for example, when preparing cardiomyocytes, first, embryoid bodies are formed while giving the direction of differentiation from pluripotent stem cells to mesoderm. The cardiomyocytes are collected by inducing differentiation of the mimoids into cardiomyocytes and dispersing them into single cells (for example, Patent Document 2, etc.).

In recent years, research on pluripotent stem cells such as ES cells and iPS cells has progressed, and while many cell lines have been established, it has been found that there are differences in differentiation characteristics between cell lines. For example, Patent Literature 2 and Non-Patent Literature 4 describe iPS cells having a differentiation tropism into cells of the nervous system.

Japanese Patent Publication No. 2007-528755 WO2013/187416

Shimizu et al., Circ Res. 2002 Feb 22;90(3):e40-e48 Matsuura et al., Biomaterials. 2011 Oct;32(30):7355-62 Kawamura et al., Circulation. 2012 Sep 11;126(11 Suppl 1):S29-37 Kojima et al., Cell Stem Cell 14, 107–120 (2014)

TECHNICAL FIELD The present invention relates to a method for selecting pluripotent stem cells having differentiation tropism toward cardiomyocytes.

The present inventors, while studying the preparation of sheet-like cell cultures using cardiomyocytes derived from pluripotent stem cells, found that even using a similar differentiation induction method, the proportion of cardiomyocytes in the final preparation was I was faced with a very different problem. As a result of intensive research to solve this problem, there are pluripotent stem cell lines that easily differentiate into cardiomyocytes and strains that do not easily differentiate into cardiomyocytes. We found that the percentage of cardiomyocytes in the final preparation was increased. Therefore, as a result of continuing research on these cell lines, we succeeded in identifying genes that are characteristically expressed in cell lines that are likely to differentiate into cardiomyocytes. Arrived.

That is, the present invention relates to:
[1] A method for determining a differentiation tropism marker for evaluating the differentiation tropism of pluripotent stem cells into specific differentiation-inducing cells, comprising:
(1) measuring gene expression levels in multiple pluripotent stem cell lines;
(2) measuring the expression level of miRNA in the plurality of pluripotent stem cell lines;
(3) Extracting a gene with a significantly different expression level between a pluripotent stem cell line with high differentiation tropism to the specific differentiation-inducing cell and a pluripotent stem cell line with low differentiation tropism;
(4) extracting miRNAs with significantly different expression levels between a pluripotent stem cell line with high differentiation tropism to the specific differentiation-inducing cell and a pluripotent stem cell line with low differentiation tropism; and (5) Selecting a gene associated with the miRNA extracted in (4) from the genes extracted in (3);
The above method, comprising

[2] A method for indexing the differentiation propensity of a pluripotent stem cell line, comprising:
(a) measuring the expression level of at least one differentiation-oriented marker gene in the pluripotent stem cells of interest; and (b) comparing the expression level of the gene measured in (a) with a reference. .
[3] the differentiation-oriented marker gene is a gene selected from the group consisting of WNT signaling regulatory factors, mitochondria-related genes, TGFβ signaling regulatory factors, mesoderm-related genes, cardiomyocyte-related genes and undifferentiated cell-related genes; Yes, the method of [2].

[4] WNT signaling regulators are PF4, TMEM64, KDM6A, APC, β-catenin, Axin, CK1, Dsh, GSK-3β, Dkk, WIF, FRP, Cerberus, TCF, Krn, WNT1, WNT2, WNT3, WNT4 , WNT5A, WNT7A, WNT7B, WNT8B, WNT10B, WNT11, WNT2B, WNT9A, WNT9B, LRP5 and LRP6.
[5] mitochondria-related genes selected from the group consisting of CHCHD2, SFXN3, CREB1, PPARGC1A, PPARGC1B, CAMK4, PPP3CA, MYEF2, PPRC1, PKA, NRF1, GABPA, GABPB2, ESRRA, TFB2M, TFB1M, TFAM, POLRMT and MTERF The method of [2] to [4], wherein at least one gene is a

[6] the TGFβ signaling regulator is SKIL, THBS1, CD3, TLR2, SMAD1, SMAD2, SMAD3, SMAD4, SMAD5, SMAD6, SMAD7, SMAD9, TGFBR1, TGFBR2, MAPK1, MAPK3, ROCK1, BMP2, BMP4, BMP5, The method of [2] to [5], wherein at least one gene is selected from the group consisting of BMP6, BMP7, BMP8B, BMPR1A and BMPR1B.
[7] The mesoderm gene is FLK1, BRACHYURY, GOOSECOID, PDGFR-a, IGF2, CD34, CLL1, HHEX, INHBA, LEF1, SRF, T, TWIST1, ADIPOQ, MME, KIT, ITGAL, Tbx1, Gata1, Klf1, The method of [2]-[6], wherein at least one gene is selected from the group consisting of Csf1r, CD45 and Ter119.

[8] cardiomyocyte-related genes consist of TNT2, ML2, GATA4, MYH6, MYH7, Nkx2.5, SCN5A, RYR2, PPARGC1, MYL2, HCN4, CACNa1C, ATP2A2, Actc1, Cx43, TEF-1 and Tbx-5 The method of [2]-[7], wherein at least one gene is selected from the group.
[9] Undifferentiated cell-related genes are Oct-4, Nanog, Lin28, SOX2, c-Myc, Klf4, TRA-1-60, SSEA-4, Oct3/4, Nanog, Cripto, Dax1, ERAs, Fgf4, At least one selected from the group consisting of Esg1, Rex1, Zfp296, UTF1, GDF3, Sall4, Tbx3, Tcf3, DNMT3L, DNMT3B, Tra-1-81, miR-290 cluster miRNAs and miR-302 cluster miRNAs The method of [2]-[8], which is a gene.

[10] The differentiation-directed marker gene is from PF4, CHCHD2, AMMECR1, API5, BCOR, BRWD1, CLEC4G, GLIPR1, HELB, KDM6A, LOC388796, NKTR, POMZP3, ZP3, PRUNE2, RBMX, RC3H1, SKIL, SORBS2 and SRSF11 The method of [2], wherein at least one gene is selected from the group consisting of
[11] The method of [10], wherein if the measured expression level is significantly higher than the reference, the subject pluripotent stem cells are determined to be a cell line with a high differentiation tropism to cardiomyocytes.

[12] The method of [2], wherein the differentiation-directed marker gene is selected from the group consisting of TMEM64, ACTN3, LOC284373, LOC441666, PLCB1, SYNPR, TMEM163, U2AF1L4, VWDE, ZNF229 and ZNF354C.
[13] The method of [12], wherein when the measured expression level is significantly lower than the reference, the subject pluripotent stem cell is determined to be a cell line with high cardiomyocyte differentiation tropism.
[14] A method for culturing pluripotent stem cells, comprising PF4, CHCHD2, AMMECR1, API5, BCOR, BRWD1, CLEC4G, GLIPR1, HELB, KDM6A, LOC388796, NKTR, POMZP3, ZP3, PRUNE2, RBMX, RC3H1, SKIL, The above method, wherein the culture is performed in a medium containing at least one protein selected from the group consisting of SORBS2 and SRSF11.

[15] The method of [14], wherein the at least one protein is PF4.
[16] The method of [14] or [15], wherein the culture is for forming embryoid bodies from pluripotent stem cells.
[17] The method of [16], wherein the embryoid body is a mesodermal embryoid body.
[18] A medical composition comprising differentiation-inducing cells derived from pluripotent stem cells cultured by the method of [14] to [17].
[19] The medical composition of [18], which is a composition for drug screening.
[20] The medical composition of [18], which is a composition for transplantation.
[21] The medical composition of [19] or [20], which is a sheet-like cell culture.

[22] A method for quality control of a medical composition containing cardiomyocytes differentiated from pluripotent stem cells, comprising mesoderm genes, endoderm genes and / Or the above method, comprising measuring the expression level of an ectodermal gene.
[23] The mesoderm gene is FLK1, BRACHYURY, GOOSECOID, PDGFR-a, IGF2, CD34, CLL1, HHEX, INHBA, LEF1, SRF, T, TWIST1, ADIPOQ, MME, KIT, ITGAL, Tbx1, Gata1, Klf1, The method of [22], wherein the method is selected from the group consisting of at least one gene selected from the group consisting of Csf1r, CD45 and Ter119.
[24] The method of [22] or [23], wherein the endoderm gene is selected from the group consisting of AMN, SOX7, SOX17 and HNF3, and the ectoderm gene is selected from the group consisting of SOX1, PAX6 and ZIC1.
[25] The method of [22] to [24], further comprising obtaining morphological characteristics of pluripotent stem cells and embryoid bodies.

According to the present invention, it is possible to easily select and obtain a pluripotent stem cell line having differentiation tropism toward specific differentiation-inducing cells, for example, cardiomyocytes. It becomes possible to efficiently prepare cardiomyocytes by induction. In addition, it is possible to determine whether or not a final preparation with a high proportion of cardiomyocytes can be obtained at the early stage of differentiation induction such as pluripotent stem cells and embryoid bodies, so it is possible to efficiently provide a high-quality medical composition. can be done. Furthermore, by providing a novel culture method for differentiating into desired differentiation-induced cells, such as cardiomyocytes, desired differentiation-induced cells can be obtained more efficiently.

FIG. 1 is a photograph of iPS cell culture and embryoid bodies used in Example 1. FIG. A is a photograph of cells in culture, and B is a photograph of an embryoid body. Figure 2 shows the positive rate of troponin T when the iPS cells used in Example 1 were induced to differentiate into cardiomyocytes. This is the positive rate when FIG. 3 shows the beating rate of the culture when the iPS cells used in Example 1 were induced to differentiate into cardiomyocytes. Red and blue bars show results on day 8 and day 17 of culture, respectively.

FIG. 4 shows the survival rate of undifferentiated cells when each iPS cell was induced to differentiate into cardiomyocytes. Blue and red bars are the results when activin was added at 6 ng/mL and 12 ng/mL, respectively. FIG. 5 shows the expression levels of genes related to each germ layer in embryoid bodies formed from each iPS cell line. FIG. 6 is a heat map of cardiomyocyte-related gene expression in cell cultures induced to differentiate from each iPS cell.

FIG. 7 shows expression levels of cardiomyocyte-related genes in cell cultures induced to differentiate from iPS cells. FIG. 8 shows the results of miRNA expression analysis in each iPS cell line. A is a graph plotting changes in miRNA expression levels between an iPS cell line with a high differentiation tropism and an iPS cell line with a low differentiation tropism. B is a graph showing the difference in the expression levels of 5 miRNAs identified as miRNAs with reduced expression levels between the high differentiation tropic strain and the low differentiation tropic strain. C is a distribution map in which the expression levels of all the analyzed miRNAs in a highly differentiation-tropic strain and a low differentiation-tropic strain are plotted on the vertical axis and the horizontal axis.

FIG. 9 is a diagram showing the results of pathway analysis of identified miRNAs and test results of the expression of two identified genes (PF4 and TMEM64). A represents the expression levels of 10 mRNAs with significantly high expression levels and 10 mRNAs with significantly low expression levels between iPS cell lines with high differentiation tropism and iPS cell lines with low differentiation tropism. graph. B is a graph listing signaling pathways involving miRNAs and mRNAs identified by miRNA and gene expression analyses. C is a graph showing the difference in cTnT expression levels in the cell populations obtained when a high differentiation tropic strain and a low differentiation tropism strain were each induced to differentiate into cardiomyocytes. D is a graph showing the expression levels of PF4 and TMEM64 in a high differentiation tropic strain and a low differentiation tropism strain, respectively. E is a graph showing the correlation between the expression levels of PF4 and TMEM64 in each iPS cell line and the cTnT expression level when the same iPS cell line was induced to differentiate into cardiomyocytes. FIG. 10 is a graph showing A: the expression level of cTnT and B: the expression levels of PF4 and TMEM64 when differentiation was induced by adding various agents to the medium.

The present invention will be described in detail below.
Unless defined otherwise herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. All patents, applications and other publications and information referenced herein are hereby incorporated by reference in their entirety. In addition, if there is any discrepancy between the publications referred to in this specification and the description in this specification, the description in this specification shall take precedence.
In the present disclosure, a nucleotide sequence (including mRNA, miRNA, etc.) represented by a specific gene name means a sequence registered in a database known in the art such as GenBank. A person skilled in the art can immediately know what sequence is represented from such a gene name.

In the present disclosure, the term "pluripotent stem cell" is a term well known in the art and is capable of differentiating into cells of all lineages belonging to the three germ layers: endoderm, mesoderm and ectoderm. means a cell having Non-limiting examples of pluripotent stem cells include embryonic stem cells (ES cells), nuclear transfer embryonic stem cells (ntES cells), induced pluripotent stem cells (iPS cells), and the like. Normally, when inducing differentiation of pluripotent stem cells into specific cells, the pluripotent stem cells are first cultured in suspension to form aggregates of cells of any of the above three germ layers, and then cells that form aggregates. to differentiate into specific cells of interest. Alternatively, pluripotent stem cells are adherently cultured at high density and induced to differentiate. In the present disclosure, "embryoid bodies" means aggregates of such cells. In the present disclosure, in particular, an embryoid body having a differentiation tropism to cells of the endodermal lineage is referred to as an "endodermal embryoid body", and an embryoid body having a differentiation tropism to a mesodermal cell is referred to as a "mesoderm embryo." Embryoid bodies having a differentiation tropism to cells of the ectodermal lineage are sometimes referred to as "ectodermal embryoid bodies".

In the present disclosure, a "differentiation-induced cell" means any cell that has been treated to induce differentiation from a pluripotent stem cell into a specific type of cell. Differentiation-induced cells include tissue-constituting adhesive cells such as cardiomyocytes and skeletal myoblasts, and non-adhesive cells such as blood cells. Non-limiting examples of differentiation-induced cells include muscle cells such as cardiomyocytes and skeletal myoblasts, nervous cells such as neuronal cells, oligodendrocytes, and dopamine-producing cells, retinal cells such as retinal pigment epithelial cells, and blood cells. cells, hematopoietic cells such as bone marrow cells, immune-related cells such as T cells, NK cells, NKT cells, dendritic cells, and B cells, organ-composing cells such as hepatocytes, pancreatic β cells, and renal cells, In addition to chondrocytes and germ cells, it includes progenitor cells and somatic stem cells that differentiate into these cells. Typical examples of such progenitor cells and somatic stem cells include mesenchymal stem cells in cardiomyocytes, multipotent cardiac progenitor cells, unipotent cardiac progenitor cells, neural stem cells in nervous system cells, hematopoietic cells and immune cells. Related cells include hematopoietic stem cells and lymphoid stem cells. Differentiation induction of pluripotent stem cells can be performed using any known technique. For example, induction of differentiation from pluripotent stem cells to cardiomyocytes is described in Miki et al., Cell Stem Cell 16, 699-711, June 4, 2015, WO2014/185358, Shugo Tohyama et al., Stem Cell Report, 9, 1-9, Nov 14, 2017.

In the present disclosure, "differentiation tropism" means the property of pluripotent stem cells to easily differentiate into specific differentiation-inducing cells, and the higher the differentiation tropism to a specific differentiation-inducing cell, the more likely it becomes the differentiation-inducing cell. means easy. Therefore, a pluripotent stem cell line with a high differentiation tropism into a specific cell is compared to a pluripotent stem cell line with a low differentiation tropism when differentiation is induced by a method of inducing differentiation into the specific cell. , it is expected that a larger number of differentiation-induced cells can be obtained even with the same differentiation-inducing method.

In the present disclosure, "cardiomyocyte" means a cell having cardiomyocyte characteristics. Features of cardiomyocytes include, but are not limited to, expression of cardiomyocyte markers, presence of autonomous beating, and the like. Non-limiting examples of cardiomyocyte markers include, for example, c-TNT (cardiac troponin T), CD172a (aka SIRPA or SHPS-1), KDR (aka CD309, FLK1 or VEGFR2), PDGFRA, EMILIN2, VCAM, and the like. . In one aspect, the pluripotent stem cell-derived cardiomyocytes are c-TNT positive and/or CD172a positive.

One aspect of the present disclosure relates to a method of determining a differentiation tropism marker for evaluating differentiation tropism into a specific differentiation-induced cell. In the present disclosure, "differentiation-directed marker" or "differentiation-directed marker gene" is a marker (gene) expressed in pluripotent stem cells, and the amount of expression determines the differentiation tropism of the pluripotent stem cells. means that it is possible to evaluate For example, in the case of PF4, which has been confirmed by the present inventors to be a differentiation-oriented marker, when the expression level of PF4 is higher in a certain pluripotent stem cell than in a standard pluripotent stem cell, the pluripotent stem cell is It is evaluated to have a high differentiation tropism to cardiomyocytes. In the present disclosure, the “standard expression level” includes, but is not limited to, the average expression level of genes in multiple pluripotent stem cell lines. The average expression level may be, for example, the average expression level of the gene to be measured in a predetermined number (eg, 5, 10, 15, etc.) of pluripotent stem cell lines that are randomly sampled. .

The method of determining a differentiation-tropic marker of the present disclosure includes the following steps:
(1) measuring gene expression levels in multiple pluripotent stem cell lines;
(2) measuring the expression level of miRNA in the plurality of pluripotent stem cell lines;
(3) Extracting genes with significantly different expression levels between a pluripotent stem cell line with high differentiation tropism to a specific differentiation-inducing cell and a pluripotent stem cell line with low differentiation tropism;
(4) extracting miRNAs with significantly different expression levels between a pluripotent stem cell line with high differentiation tropism to the specific differentiation-inducing cell and a pluripotent stem cell line with low differentiation tropism; and (5) Selecting a gene associated with the miRNA extracted in (4) from the genes extracted in (3).

In step (1), the expression levels of genes in a plurality of pluripotent stem cell lines are quantitatively measured. Such gene expression level measurement may be performed comprehensively. The plurality of pluripotent stem cell lines includes at least one pluripotent stem cell line known to have a high differentiation tropism to specific differentiation-inducing cells and a low differentiation tropism to the specific differentiation-inducing cells. and at least one pluripotent stem cell line known to A differentiation propensity toward a particular differentiation-induced cell can be identified using methods known in the art, methods described in this disclosure, and the like. Gene expression levels can be measured using methods known in the art, such as real-time PCR, microarray, high-throughput sequencing, and the like.

In step (2), the expression levels of miRNAs in multiple pluripotent stem cell lines are quantitatively measured. Such multiple pluripotent stem cell lines are the same pluripotent stem cell lines as the multiple pluripotent stem cell lines whose gene expression levels were measured in step (1). Measurement of miRNA expression levels can be performed using methods known in the art, such as real-time PCR, microarray, high-throughput sequencing, and the like.

In step (3), a gene having a significantly different expression level between a pluripotent stem cell line with high differentiation tropism into a specific differentiation-inducing cell and a pluripotent stem cell line with low differentiation tropism is extracted. For example, comparing a pluripotent stem cell line with high differentiation tropism and a pluripotent stem cell line with low differentiation tropism, 1.5 times or more, 2 times or more, 2.5 times or more, 3 times or more, 5 times or more A gene with a difference in expression level of 10-fold or more, or 1-fold or more, may be extracted. Such an expression level may be high in either pluripotent stem cell line, for example, it may be highly expressed in a pluripotent stem cell line with high differentiation tropism, or may be highly expressed in a pluripotent stem cell line with low differentiation tropism It may be highly expressed. In a preferred embodiment, genes that are significantly highly expressed in a pluripotent stem cell line with high differentiation tropism are extracted. In another preferred embodiment, genes that are significantly highly expressed in pluripotent stem cell lines with low differentiation tropism are extracted.

In step (4), miRNAs with significantly different expression levels are extracted between a pluripotent stem cell line with high differentiation tropism into specific differentiation-induced cells and a pluripotent stem cell line with low differentiation tropism. For example, comparing a pluripotent stem cell line with high differentiation tropism and a pluripotent stem cell line with low differentiation tropism, 1.5 times or more, 2 times or more, 2.5 times or more, 3 times or more, 5 times or more A miRNA having a difference in expression level of 10-fold or more, or 1-fold or more, may be extracted. Such an expression level may be high in either pluripotent stem cell line, for example, it may be highly expressed in a pluripotent stem cell line with high differentiation tropism, or may be highly expressed in a pluripotent stem cell line with low differentiation tropism It may be highly expressed. In a preferred embodiment, miRNAs that are significantly highly expressed in pluripotent stem cell lines with high differentiation tropism are extracted. In another preferred embodiment, miRNAs that are significantly highly expressed in pluripotent stem cell lines with low differentiation tropism are extracted.

In step (5), genes associated with the miRNA extracted in step (4) are selected from among the genes extracted in step (3). As a method for identifying a gene associated with a miRNA, a method known in the art may be used, and such methods include, for example, a pathway analysis method, miRNA target gene database (TargetScan), and the like. By this step, genes with significant differences in expression levels due to significant differences in miRNA expression are selected.

Another aspect of the present disclosure relates to a method of indexing differentiation propensity to specific differentiation-inducing cells. The indexing method of the present disclosure includes the following steps:
(a) at least one differentiation-directed marker gene in the pluripotent stem cells of interest, such as mitochondria-associated genes, WNT signaling regulators, TGFβ signaling regulators, genes associated with each germ layer and genes associated with undifferentiated cells; (b) comparing the expression level of the differentiation-directed marker gene measured in (a) with a reference;

"Genes related to each germ layer" means genes related to the germ layer from which differentiation-inducing cells are derived. For example, if the differentiation-inducing cells are cardiomyocytes, mesoderm-related genes; In the case of cells of the gastrointestinal tract, it means endoderm-related genes.
In the step of measuring the gene expression level, the expression level of a gene related to specific differentiation-inducing cells may be measured. For example, if the differentiation-induced cells are cardiomyocytes, cardiomyocyte-related genes may be further measured.

The present invention will be described in detail below, taking as an example the case where the differentiation-inducing cells are myocardial cells.
<1> Indexing Method of the Present Disclosure One aspect of the present disclosure relates to a method of indexing differentiation tropism into specific differentiation-inducing cells, particularly cardiomyocytes. The indexing method of the present disclosure includes the following steps (a) and (b):
(a) at least one selected from the group consisting of mitochondria-related genes, WNT signaling regulators, TGFβ signaling regulators, mesoderm-related genes, cardiomyocyte-related genes and undifferentiated cell-related genes in the pluripotent stem cells of interest (b) comparing the expression level of the gene measured in (a) with a standard;

In step (a), the expression level of a differentiation-oriented marker gene in pluripotent stem cells is measured. Gene expression levels can be measured using conventional methods known in the art. Examples of such methods include, but are not limited to, real-time PCR methods, microarray methods, high-throughput sequencing methods, and the like.

Differentiation-oriented marker genes whose expression levels are measured are selected from the group consisting of mitochondria-related genes, WNT signaling regulatory factors, TGFβ signaling regulatory factors, mesoderm-related genes, cardiomyocyte-related genes and undifferentiated cell-related genes. at least one gene that
Examples of mitochondria-related genes include, but are not limited to, CHCHD2, SFXN3, CREB1, PPARGC1A, PPARGC1B, CAMK4, PPP3CA, MYEF2, PPRC1, PKA, NRF1, GABPA, GABPB2, ESRRA, TFB2M, TFB1M, TFAM, POLRMT or MTERF and the like. Thus, in one aspect, in step (a), The expression level of at least one mitochondria-related gene selected from the group is measured. In a preferred embodiment, the expression levels of CHCHD2 and/or SFXN3 are measured as mitochondria-related genes.

特に好ましいＷＮＴシグナル伝達調節因子としては、ＰＦ４、ＴＭＥＭ６４、ＫＤＭ６Ａ、ＡＰＣ、βカテニン、Ａｘｉｎ、ＣＫ１、Ｄｓｈ、ＧＳＫ－３β、Ｄｋｋ、ＷＩＦ、ＦＲＰ、Ｃｅｒｂｅｒｕｓ、ＴＣＦ、Ｋｒｎ、ＷＮＴ１、ＷＮＴ２、ＷＮＴ３、ＷＮＴ４、ＷＮＴ５Ａ、ＷＮＴ７Ａ、ＷＮＴ７Ｂ、ＷＮＴ８Ｂ、ＷＮＴ１０Ｂ、ＷＮＴ１１、ＷＮＴ２Ｂ、ＷＮＴ９Ａ、ＷＮＴ９Ｂ、ＬＲＰ５またはＬＲＰ６などが挙げられる。したがって一態様において、工程（ａ）において、ＰＦ４、ＴＭＥＭ６４、ＫＤＭ６Ａ、ＡＰＣ、βカテニン、Ａｘｉｎ、ＣＫ１、Ｄｓｈ、ＧＳＫ－３β、Ｄｋｋ、ＷＩＦ、ＦＲＰ、Ｃｅｒｂｅｒｕｓ、ＴＣＦ、Ｋｒｎ、ＷＮＴ１、ＷＮＴ２、ＷＮＴ３、ＷＮＴ４、ＷＮＴ５Ａ、ＷＮＴ７Ａ、ＷＮＴ７Ｂ、ＷＮＴ８Ｂ、ＷＮＴ１０Ｂ、ＷＮＴ１１、ＷＮＴ２Ｂ、ＷＮＴ９Ａ、ＷＮＴ９Ｂ、ＬＲＰ５およびＬＲＰ６からなる群から選択される少なくとも１種のＷＮＴシグナル伝達調節因子の発現量が計測される。好ましい一態様において、ＷＮＴシグナル伝達調節因子としてＰＦ４またはＴＭＥＭ６４の発現量が、より好ましい一態様においてＰＦ４の発現量が計測される。WNT signaling modulators include, but are not limited to, those listed in the table below.

Particularly preferred WNT signaling modulators include PF4, TMEM64, KDM6A, APC, β-catenin, Axin, CK1, Dsh, GSK-3β, Dkk, WIF, FRP, Cerberus, TCF, Krn, WNT1, WNT2, WNT3, WNT4 , WNT5A, WNT7A, WNT7B, WNT8B, WNT10B, WNT11, WNT2B, WNT9A, WNT9B, LRP5 or LRP6. Thus, in one aspect, in step (a), PF4, TMEM64, KDM6A, APC, β-catenin, Axin, CK1, Dsh, GSK-3β, Dkk, WIF, FRP, Cerberus, TCF, Krn, WNT1, WNT2, WNT3, The expression level of at least one WNT signaling regulator selected from the group consisting of WNT4, WNT5A, WNT7A, WNT7B, WNT8B, WNT10B, WNT11, WNT2B, WNT9A, WNT9B, LRP5 and LRP6 is measured. In a preferred embodiment, the expression level of PF4 or TMEM64 as a WNT signaling regulator is measured, and in a more preferred embodiment, the expression level of PF4 is measured.

特に好ましいＴＧＦβシグナル伝達調節因子としては、ＳＫＩＬ、ＴＨＢＳ１、ＣＤ３、ＴＬＲ２、ＳＭＡＤ１、ＳＭＡＤ２、ＳＭＡＤ３、ＳＭＡＤ４、ＳＭＡＤ５、ＳＭＡＤ６、ＳＭＡＤ７、ＳＭＡＤ９、ＴＧＦＢＲ１、ＴＧＦＢＲ２、ＭＡＰＫ１、ＭＡＰＫ３、ＲＯＣＫ１、ＢＭＰ２、ＢＭＰ４、ＢＭＰ５、ＢＭＰ６、ＢＭＰ７、ＢＭＰ８Ｂ、ＢＭＰＲ１ＡまたはＢＭＰＲ１Ｂなどが挙げられる。したがって一態様において、工程（ａ）において、ＳＫＩＬ、ＴＨＢＳ１、ＣＤ３、ＴＬＲ２、ＳＭＡＤ１、ＳＭＡＤ２、ＳＭＡＤ３、ＳＭＡＤ４、ＳＭＡＤ５、ＳＭＡＤ６、ＳＭＡＤ７、ＳＭＡＤ９、ＴＧＦＢＲ１、ＴＧＦＢＲ２、ＭＡＰＫ１、ＭＡＰＫ３、ＲＯＣＫ１、ＢＭＰ２、ＢＭＰ４、ＢＭＰ５、ＢＭＰ６、ＢＭＰ７、ＢＭＰ８Ｂ、ＢＭＰＲ１ＡおよびＢＭＰＲ１Ｂからなる群から選択される少なくとも１種のＴＧＦβシグナル伝達調節因子の発現量が計測される。好ましい一態様において、ＴＧＦβシグナル伝達調節因子として、ＳＫＩＬの発現量が計測される。Examples of TGFβ signaling modulators include, but are not limited to, those listed in the table below.

Particularly preferred TGFβ signaling modulators include SKIL, THBS1, CD3, TLR2, SMAD1, SMAD2, SMAD3, SMAD4, SMAD5, SMAD6, SMAD7, SMAD9, TGFBR1, TGFBR2, MAPK1, MAPK3, ROCK1, BMP2, BMP4, BMP5, BMP6, BMP7, BMP8B, BMPR1A or BMPR1B and the like. Thus, in one aspect, in step (a), SKIL, THBS1, CD3, TLR2, SMAD1, SMAD2, SMAD3, SMAD4, SMAD5, SMAD6, SMAD7, SMAD9, TGFBR1, TGFBR2, MAPK1, MAPK3, ROCK1, BMP2, BMP4, BMP5 , BMP6, BMP7, BMP8B, BMPR1A and BMPR1B. In a preferred embodiment, the expression level of SKIL is measured as a TGFβ signaling regulator.

Examples of mesoderm genes include, but are not limited to, FLK1, BRACHYURY, GOOSECOID, PDGFR-a, IGF2, CD34, CLL1, HHEX, INHBA, LEF1, SRF, T, TWIST1, ADIPOQ, MME, KIT , ITGAL, Tbx1, Gata1, Klf1, Csf1r, CD45 or Ter119. Thus, in one aspect, in step (a), FLK1, BRACHYURY, GOOSECOID, PDGFR-a, IGF2, CD34, CLL1, HHEX, INHBA, LEF1, SRF, T, TWIST1, ADIPOQ, MME, KIT, ITGAL, Tbx1, Gata1 , Klf1, Csf1r, CD45 and Ter119. In a preferred embodiment, the expression levels of FLK1, BRACHYURY, GOOSECOID and/or PDGFR-a are measured as mesoderm genes.

Examples of cardiomyocyte-related genes include, but are not limited to, TNT2, MYL2, GATA4, MYH6, MYH7, Nkx2.5, SCN5A, RYR2, PPARGC1, MYL2, HCN4, CACNa1C, ATP2A2, Actc1, Cx43, Examples include TEF-1 or Tbx-5. Thus, in one aspect, in step (a), TNT2, MYL2, GATA4, MYH6, MYH7, Nkx2.5, SCN5A, RYR2, PPARGC1, MYL2, HCN4, CACNalC, ATP2A2, Actc1, Cx43, TEF-1 and Tbx-5 The expression level of at least one cardiomyocyte-related gene selected from the group consisting of is measured. In a preferred embodiment, expression levels of cardiomyocyte-related genes TNT2, MYL2, GATA4, MYH6, MYH7, Nkx2.5, SCN5A, RYR2, PPARGC1, MYL2, HCN4, CACNa1C and/or ATP2A2 are measured.

Undifferentiated cell-related genes include, but are not limited to, Oct-4, Nanog, Lin28, SOX2, c-Myc, Klf4, TRA-1-60, SSEA-4, Oct3/4, Nanog , Cripto, Dax1, ERas, Fgf4, Esg1, Rex1, Zfp296, UTF1, GDF3, Sall4, Tbx3, Tcf3, DNMT3L, DNMT3B, Tra-1-81 or miR-290 cluster miRNA, miR-302 cluster miRNA, etc. mentioned. Thus, in one aspect, in step (a), Oct-4, Nanog, Lin28, SOX2, c-Myc, Klf4, TRA-1-60, SSEA-4, Oct3/4, Nanog, Cripto, Dax1, ERAs, Fgf4 , Esg1, Rex1, Zfp296, UTF1, GDF3, Sall4, Tbx3, Tcf3, DNMT3L, DNMT3B, Tra-1-81, miR-290 cluster miRNA and miR-302 cluster miRNA at least one selected from the group consisting of The expression level of the undifferentiated cell-related gene is measured. In a preferred embodiment, the expression levels of Oct-4, Nanog and/or Lin28 are measured as undifferentiated cell-related genes.

In a more preferred embodiment, the expression levels of at least two genes selected from the gene group described above are measured. That is, CHCHD2, SFXN3, KDM6A, SKIL, FLK1, BRACHYURY, GOOSECOID, PDGFR-a, TNT2, ML2, GATA4, MYH6, MYH7, Nkx2.5, SCN5A, RYR2, PPARGC1, MYL2, HCN4, CACNa1C, ATP2A2, Oct- 4, the expression levels of at least two genes selected from Nanog and Lin28 are measured.

In another preferred embodiment, the differentiation-directed marker gene includes PF4, CHCHD2, AMMECR1, API5, BCOR, BRWD1, CLEC4G, GLIPR1, HELB, KDM6A, LOC388796, NKTR, POMZP3, ZP3, PRUNE2, RBMX, RC3H1, SKIL, The expression level of at least one gene selected from the group consisting of SORBS2 and SRSF11 is measured.
In yet another preferred embodiment, expression of at least one gene selected from the group consisting of TMEM64, ACTN3, LOC284373, LOC441666, PLCB1, SYNPR, TMEM163, U2AF1L4, VWDE, ZNF229 and ZNF354C as differentiation-directed marker genes quantity is measured.

In step (b), the gene expression level measured in (a) is compared with a reference. Standards for comparison include, but are not limited to, expression levels of the same genes in pluripotent stem cell lines known to have low cardiomyocyte differentiation tropism, cardiomyocyte differentiation tropism Mean expression level of the same gene in multiple pluripotent stem cell lines known to have low cardiomyocyte expression levels, expression level of the same gene in pluripotent stem cell lines known to have a high differentiation Mean expression level of the same gene in multiple pluripotent stem cell lines known to have a high differentiation tropism, Mean expression level of the same gene in multiple pluripotent stem cell lines, Mesodermal lineage tissue The expression level of the same gene in multiple pluripotent stem cell lines known to have low differentiation tropism, and the expression level of the same gene in multiple pluripotent stem cell lines known to have high differentiation tropism toward mesodermal lineage The expression level, the expression level of the same gene in multiple pluripotent stem cell lines known to have low differentiation tropism toward the endodermal lineage, and the expression level of the same gene in multiple pluripotent stem cell lines known to have high The expression level of the same gene in a potent stem cell line, the expression level of the same gene in multiple pluripotent stem cell lines known to have a high differentiation tropism to the ectodermal lineage, Examples include the expression level of the same gene in multiple pluripotent stem cell lines known to be low.

For these criteria, numerical values known in the art or previously measured and recorded values may be used, or when performing the indexing method of the present disclosure in conjunction with step (a) above, good too. By comparing with these criteria, the target pluripotent stem cell line differentiation tropism marker is indexed, and whether the target pluripotent stem cell line has a high cardiomyocyte differentiation tropism based on the index can be determined.
When comparing the expression level measured in (a) with a reference, both values are preferably measured by the same method, but are not limited to this. If the values are measured in different ways, the values may be transformed to allow direct comparison.

In one aspect of the present disclosure, the expression level of the same gene in a pluripotent stem cell line known to have a low differentiation tropism to cardiomyocytes or a plurality of pluripotent cells known to have a low differentiation tropism to cardiomyocytes The average value of the expression level of the same gene in stem cell lines is used as a standard for comparison. In this case, if the expression level measured in (a) is significantly higher than the reference value, it can be determined that the subject pluripotent stem cell line has a high cardiomyocyte differentiation tropism.

In another aspect of the present disclosure, the expression level of the same gene in a pluripotent stem cell line known to have a high cardiomyocyte differentiation tropism or a plurality of multipotent cells known to have a high cardiomyocyte differentiation tropism The average value of the expression level of the same gene in the potential stem cell line is used as a standard for comparison. In this case, if the expression level measured in (a) is equal to or significantly higher than the reference value, it can be determined that the subject pluripotent stem cell line has a high cardiomyocyte differentiation tropism. . In the present disclosure, "significantly" means a statistically significant difference. For example, it is assumed that a measured value is “significant” when it shows a numerical value that is extremely deviated from a certain statistic.

In yet another aspect of the present disclosure, comparison is made using the average expression level of the same gene in a plurality of pluripotent stem cell lines as a reference. In this case, if the expression level measured in (a) is significantly higher than the reference value, it can be determined that the subject pluripotent stem cell line has a high cardiomyocyte differentiation tropism.

In one embodiment, PF4, CHCHD2, AMMECR1, API5, BCOR, BRWD1, CLEC4G, GLIPR1, HELB, KDM6A, LOC388796, NKTR, POMZP3, ZP3, PRUNE2, RBMX, RC3H1, SKIL, SORBS2 and SRSF11 as differentiation-directed marker genes The expression level of at least one gene selected from the group consisting of is measured. In this case, if the expression level measured in (a) is significantly higher than the reference value, it can be determined that the subject pluripotent stem cell line has a high cardiomyocyte differentiation tropism. In another aspect, the expression level of at least one gene selected from the group consisting of TMEM64, ACTN3, LOC284373, LOC441666, PLCB1, SYNPR, TMEM163, U2AF1L4, VWDE, ZNF229 and ZNF354C as a differentiation-oriented marker gene measure. In this case, if the expression level measured in (a) is significantly lower than the reference value, it can be determined that the subject pluripotent stem cell line has a high cardiomyocyte differentiation tropism.

<2> Pluripotent Stem Cells of the Present Disclosure The present inventors have found that pluripotent stem cells, particularly induced pluripotent stem cells (iPS cells), exist in cell lines that are highly oriented toward cardiomyocyte differentiation, and that such cell lines exist. The genetic signature of a pluripotent stem cell line has been found for the first time. Accordingly, one aspect of the present disclosure provides at least one selected from the group consisting of mitochondria-related genes, WNT signaling regulators, TGFβ signaling regulators, mesoderm-related genes, cardiomyocyte-related genes and undifferentiated cell-related genes. It includes pluripotent stem cells with high differentiation tropism to cardiomyocytes characterized by high gene expression levels.

As described above, the present inventors have found that in a pluripotent stem cell line highly oriented toward cardiomyocyte differentiation, mitochondria-related genes, WNT signaling regulatory factors, TGFβ signaling regulatory factors, mesoderm-related genes, cardiomyocyte-related genes and undifferentiated cell-related genes were found to be highly expressed.

In the present disclosure, "highly expressed" or "highly expressed" means that the expression level of a gene is higher than a predetermined value. Such a "predetermined value" typically includes the average expression level of the gene. The average expression level may be, for example, the average expression level of the gene to be measured in a predetermined number (for example, 5, 10, 15, etc.) of randomly-extracted pluripotent stem cell lines.

Genes that are highly expressed in the pluripotent stem cell lines of the present disclosure include mitochondria-related genes, WNT signaling regulators, TGFβ signaling regulators, mesoderm-related genes, cardiomyocyte-related genes, and undifferentiated cell-related genes. mentioned. Specific examples of these genes include those described in <1> above.

In one preferred aspect of the present disclosure, the pluripotent stem cells are iPS cells, more preferably human iPS cells. It has been pointed out that the characteristics of each iPS cell may differ depending on the origin of the somatic cell used as the base, the type of reprogramming factor, and the method of introduction. expected to be different. Another advantage of using iPS cells in regenerative medicine is that it is possible to establish cell lines using autologous cells to be treated. Therefore, according to the present invention, iPS cells prepared from autologous cells of a subject can be screened for those with a high differentiation tropism to cardiomyocytes, and can be established as new cell lines.

When preparing differentiation-induced cells from pluripotent stem cells, they must be highly undifferentiated. This is also supported by the high expression of undifferentiated cell-related genes in the pluripotent stem cells of the present disclosure. That is, it is thought that the high undifferentiated nature of pluripotent stem cells facilitates differentiation into desired differentiation-inducing cells. The present inventors' tests have shown that the cardiomyocyte differentiation propensity itself is not directly related to the undifferentiated nature of pluripotent stem cells.

<3> Embryoid bodies of the present disclosure The present inventors have found that embryoid bodies obtained by culturing pluripotent stem cells with a high differentiation tropism to cardiomyocytes also have a high differentiation tropism to cardiomyocytes. Found. Therefore, one aspect of the present disclosure is differentiation into cardiomyocytes, characterized by high expression of at least one mesoderm gene and low expression of at least one endoderm gene and/or ectoderm gene. It contains highly oriented embryoid bodies.

Embryoid bodies of the present disclosure are obtained by culturing pluripotent stem cells, preferably the pluripotent stem cells described in <2> above, by a method known in the art. Specifically, for example, human iPS cells are cultured in StemFit AK03 medium (Ajinomoto) containing Y27632 (Wako Pure Chemical Industries) for 1 day, then cultured in StemFit AK03 medium containing no Y27632 for 2 days, and then BMP4 is added. Obtained by culturing in the containing culture medium.

The embryoid bodies of the present disclosure highly express at least one mesoderm gene. Specific examples of mesoderm genes include those described in <1> above. Embryoid bodies of the present disclosure also have reduced expression of at least one endoderm gene and/or ectoderm gene. Here, "low expression level" means that the expression level of a certain gene is lower than a predetermined value, which is the opposite of "high expression level". Such a "predetermined value" typically includes the average expression level of the gene. The average expression level may be, for example, the average expression level of the gene to be measured in a predetermined number (for example, 5, 10, 15, etc.) of randomly sampled embryoid bodies.

Examples of ectodermal genes include, but are not limited to, SOX1, PAX6, or ZIC1. Endoderm genes include, but are not limited to, for example, AMN, SOX7, SOX17, HNF3 or ZIC1.

As described above, the embryoid bodies of the present disclosure are characterized by high expression levels of at least one mesoderm gene and low expression levels of at least one endoderm gene and/or ectoderm gene. Therefore, it has a high differentiation tropism to somatic cells derived from mesoderm, especially cardiomyocytes. Such characteristics are particularly conspicuous in embryoid bodies produced from the pluripotent stem cells of the present disclosure, which have a high differentiation tropism for cardiomyocytes. Therefore, in a preferred embodiment, the embryoid body of the present disclosure is produced from the pluripotent stem cell of the present disclosure described in <2> above.

<4> Differentiation-inducing method of the present disclosure The present inventors discovered genetic characteristics of pluripotent stem cells with a high differentiation tropism to cardiomyocytes, and differentiated cardiomyocytes using pluripotent stem cells having such characteristics. It was found that cardiomyocytes can be obtained with high efficiency by induction. Accordingly, in one aspect, the present disclosure includes a method for highly efficient differentiation induction of cardiomyocytes from pluripotent stem cells.

In a preferred embodiment, the differentiation-inducing method of the present disclosure uses the pluripotent stem cells or embryoid bodies of the present disclosure. In such an embodiment, any method known in the art may be used for the method of inducing differentiation. Various methods are known for inducing the differentiation of cardiomyocytes from pluripotent stem cells (for example, Burridge et al., Cell Stem Cell. 2012 Jan 6;10(1):16-28). In either method, mesoderm-inducing factors (e.g., activin A, BMP4, bFGF, VEGF, SCF, etc.), cardiac specification factors (e.g., VEGF, DKK1, Wnt signaling inhibitors (e.g., IWR-1 , IWP-2, IWP-3, IWP-4, etc.), BMP signal inhibitors (e.g., NOGGIN, etc.), TGFβ/activin/NODAL signal inhibitors (e.g., SB431542, etc.), retinoic acid signal inhibitors, etc.) and cardiac differentiation factors ( For example, VEGF, bFGF, DKK1, etc.) can be caused to act sequentially to increase induction efficiency. In one embodiment, cardiomyocyte induction treatment from pluripotent stem cells is performed on embryoid bodies formed by the action of BMP4 (1) a combination of BMP4, bFGF and activin A, (2) VEGF and IWP-3, and (3) sequentially acting a combination of VEGF and bFGF.

Known methods for obtaining cardiomyocytes from human iPS cells include, for example, the following steps:
(1) a step of maintaining and culturing human iPS cells in a culture medium that does not contain feeder cells (feeder-free method);
(2) forming embryoid bodies from the obtained iPS cells;
(3) culturing the obtained embryoid bodies in a medium containing activin A, bone morphogenetic protein (BMP) 4 and basic fibroblast growth factor (bFGF);
(4) culturing the obtained embryoid bodies in a medium containing a Wnt inhibitor, a BMP4 inhibitor and a TGFβ inhibitor; and (5) culturing the obtained embryoid bodies in a medium containing VEGF and bFGF. and culturing at.

In step (1), for example, as described in WO2017038562, StemFit AK03 (Ajinomoto) is used as a medium and iPS cells are cultured on iMatrix511 (Nippi) for adaptation and maintenance culture. In addition, for example, as described in Nakagawa M., et al. A novel efficient feeder-free culture system for the derivation of human induced pluripotent stem cells. In addition, passages can be performed as single cells using TrypLE® Select (Thermo Fisher Scientific). After steps (1) to (5) above, a step (6) of purifying the obtained cardiomyocytes may optionally be performed. Examples of cardiomyocyte purification include a method of reducing cells other than cardiomyocytes using a glucose-free medium, a method of reducing undifferentiated cells using heat treatment as described in WO2017/038562, and the like.

One aspect of the present disclosure may include a step of implementing the indexing method of the present disclosure described in <1> above before and/or after step (1) above. Moreover, after the step (2), a step of measuring the gene expression level of the obtained embryoid bodies may be included. Furthermore, the step of comparing the measured gene expression level with a reference value, and the step of excluding embryoid bodies other than the embryoid bodies determined to be the embryoid bodies described in <3> above as a result of the comparison. It's okay. Examples of such a reference value include those described as "predetermined value" in <2> above.

As described above, the present inventors found that, in pluripotent stem cells with a differentiation tropism to cardiomyocytes, mitochondria-related genes, WNT signaling regulatory factors, TGFβ signaling regulatory factors, mesoderm-related genes, cardiomyocyte-related It was found that the expression level of at least one gene selected from the group consisting of genes and undifferentiated cell-related genes is significantly different compared to pluripotent stem cells that do not have cardiomyocyte differentiation tropism. served. Therefore, in each of steps (1) to (5) above, differentiation into cardiomyocytes can be induced by performing treatment that enhances or reduces the expression of these genes. Non-limiting examples of such treatments include, for example, addition of WNT signal inhibitors, TGFβ signal inhibitors, modulation of TGFβ and WNT signals, modulation of mitochondrial activity, such as addition of MitoBlock.

Therefore, in one aspect of the present disclosure, the expression level of a differentiation-directed marker is enhanced or reduced, or the action of a protein that is the expression product of a differentiation-directed marker gene is enhanced or reduced. It also relates to a method of inducing differentiation.

Examples of methods for enhancing expression levels of differentiation-directed markers include inhibition of repressors of differentiation-directed marker genes, addition of enhancers of differentiation-directed marker genes, and the like. Examples of methods for reducing the expression level of differentiation-directed markers include addition of repressors, introduction of antisense nucleic acids such as siRNA, and the like.

Methods for enhancing the action of a protein that is an expression product of a differentiation-directed marker gene include, for example, adding the expression product protein to the medium. Methods for reducing the action of a protein that is the expression product of a differentiation-directed marker gene include, for example, adding an inhibitor or an inhibitory antibody against the protein to the medium.

For example, in the case of induction of differentiation into cardiomyocytes, expression products of differentiation-directed markers whose expression is significantly increased, such as PF4, CHCHD2, AMMECR1, API5, BCOR, BRWD1, CLEC4G, GLIPR1, HELB, KDM6A, LOC388796, NKTR, Culturing in a medium containing proteins such as POMZP3, ZP3, PRUNE2, RBMX, RC3H1, SKIL, SORBS2 and SRSF11 is expected to promote differentiation induction into cardiomyocytes. Thus, the differentiation-inducing methods of the present disclosure include the above-described significantly upregulated differentiation-directed marker expression products, such as PF4, CHCHD2, AMMECR1, API5, BCOR, BRWD1, CLEC4G, GLIPR1, HELB, KDM6A, LOC388796, Also included is a method of culturing pluripotent stem cells, characterized by using a medium containing proteins such as NKTR, POMZP3, ZP3, PRUNE2, RBMX, RC3H1, SKIL, SORBS2 and SRSF11. Such a culture method is preferably used in culture, particularly at the stage of differentiating pluripotent stem cells into embryoid bodies (especially mesodermal embryoid bodies).

According to the differentiation induction method of the present disclosure, cardiomyocytes can be efficiently obtained. The cardiomyocyte content (purity) in the cardiomyocyte-containing composition obtained by the method of inducing differentiation of the present disclosure is greater than about 50%, greater than about 60%, greater than about 70%, greater than about 75%, greater than about 80%, greater than about 85%, greater than about 86%, greater than about 87%, greater than about 88%, greater than about 89%, greater than about 90%, greater than about 91%, greater than about 92%, greater than about 93%, greater than about 94%, It can be greater than about 95%, greater than about 96%, greater than about 97%, greater than about 98%, greater than about 99%, and the like. In one aspect, the pluripotent stem cell-derived cardiomyocytes in the present disclosure are cardiomyocyte populations with greater than 90% cardiomyocyte purity. In yet another aspect, the cardiomyocyte content is, for example, 50-99%. Preferably it is 50% to 70%, or 75 to 99%. In another aspect, the cardiomyocyte-containing composition obtained by the induction method of the present disclosure is characterized by a low survival rate of undifferentiated cells. The residual rate of undifferentiated cells is, for example, 0.01% to 5%, 0.01% to 4%, 0.01% to 3%, 0.01% to 2%, 0.01% to 1%, It can be from 0.01% to 1%, and the like.

<5> Medical composition of the present disclosure Another aspect of the present disclosure includes a medical composition containing differentiation-inducing cells, such as cardiomyocytes, induced by the method described in <4> above.
In the present disclosure, the term "medical composition" means a composition used for medical purposes, and is not limited thereto. Compositions used for direct treatment of subjects as well as compositions used in drug development and the like, eg, compositions for drug screening, are included.

A composition containing cardiomyocytes induced by the method of inducing differentiation of the present disclosure is a composition with a high cardiomyocyte content, and can be said to be very useful medically. For example, when a composition for transplantation is prepared using a composition containing cardiomyocytes induced by the method of inducing differentiation of the present disclosure, since the cardiomyocyte content is high, the amount of cardiomyocytes contained increases, leading to a large amount of cardiomyocytes. An implantable composition can be prepared that is suitable for the implantation of a Accordingly, in one aspect, the medical composition of the present disclosure is an implantable composition. In such embodiments, the cardiomyocyte content is greater than about 50%, greater than about 60%, greater than about 70%, greater than about 75%, greater than about 80%, greater than about 85%, greater than about 86%, greater than about 87%. , greater than about 88%, greater than about 89%, greater than about 90%, greater than about 91%, greater than about 92%, greater than about 93%, greater than about 94%, greater than about 95%, greater than about 96%, greater than about 97% , greater than about 98%, greater than about 99%, and the like. In one aspect, the pluripotent stem cell-derived cardiomyocytes in the present disclosure are cardiomyocyte populations with greater than 90% cardiomyocyte purity. In yet another aspect, the cardiomyocyte content is, for example, 50-99%, preferably 50-70%, or 75-99%.

In recent years, cell transplantation has been shown to be useful for recovering cardiac function in patients with severe heart failure, and clinical applications and studies using autologous skeletal myoblasts and iPS cell-derived cardiomyocytes have already begun. As an example, tissue-engineered temperature-responsive culture dishes have been used to develop three-dimensional cell cultures applicable to the heart that contain cells derived from portions other than adult myocardium. . Medical compositions of the present disclosure, particularly compositions for transplantation, can also be such cell cultures. Therefore, in one preferred aspect, the medical composition of the present disclosure is a sheet-like cell culture.

In the present disclosure, a “sheet-like cell culture” refers to a sheet-like cell formed by connecting cells to each other. Cells may be connected to each other directly (including through cellular elements such as adhesion molecules) and/or through intermediaries. The intervening substance is not particularly limited as long as it is a substance capable of at least physically (mechanically) connecting cells to each other, and examples thereof include an extracellular matrix. The mediator is preferably of cell origin, in particular of cells that make up the cell culture. The cells are at least physically (mechanically) linked, but may also be functionally linked, eg chemically, electrically. The sheet-like cell culture may be composed of one cell layer (single layer) or composed of two or more cell layers (laminated (multilayered) body, e.g., two layers, three layers, 4 layers, 5 layers, 6 layers, etc.).

The sheet-like cell culture preferably does not contain a scaffold (support). Scaffolds may be used in the art to attach cells onto and/or into their surfaces and maintain the physical integrity of sheet-like cell cultures, such as polyvinylidene difluoride ( PVDF) membranes and the like are known, but the sheet cell culture in the present disclosure may be able to maintain its physical integrity without such a scaffold. Moreover, the sheet-like cell culture preferably consists of only substances derived from the cells that constitute the cell culture, and does not contain other substances.

The cells that make up the sheet-like cell culture can be derived from any organism that can be treated with the sheet-like cell culture. Such organisms include, but are not limited to, humans, non-human primates, dogs, cats, pigs, horses, goats, sheep, rodents (such as mice, rats, hamsters, guinea pigs, etc.), rabbits, and the like. is included. In one aspect, the cells that make up the sheet-like cell culture are human cells.

Cells forming a sheet-like cell culture may be heterologous or allogeneic. As used herein, "heterologous cells" refer to cells derived from organisms of a species different from that of the recipient when sheet-like cell cultures are used for transplantation. For example, when the recipient is a human, cells derived from monkeys or pigs are examples of heterologous cells. Also, "allogeneic cells" refer to cells derived from the same species of organism as the recipient. For example, if the recipient is human, human cells are allogeneic cells. Allogeneic cells include autologous cells (also referred to as autologous or autologous cells), ie cells derived from the recipient, and allogeneic non-autologous cells (also referred to as allogeneic cells). Autologous cells are preferred in the present disclosure because they do not result in rejection upon transplantation. However, it is also possible to use xenogenic or allogeneic non-autologous cells. Immunosuppressive treatment may be required to prevent rejection when xenogeneic or allogeneic non-autologous cells are used. In this specification, cells other than autologous cells, that is, heterologous cells and allogeneic non-autologous cells may be collectively referred to as non-autologous cells. In one aspect of the disclosure, the cells are autologous or allogeneic. In one aspect of the disclosure, the cells are autologous cells. In another aspect of the disclosure, the cell is an allogeneic cell.

Autologous or heterologous pluripotent stem cells include, but are not limited to, collected autologous or heterologous somatic cells (e.g., skin cells (fibroblasts, keratinocytes, etc.), blood cells (peripheral blood mononuclear cells, etc.), etc.). can be obtained by inducing autologous or allogeneic iPS cells by, for example, introducing genes such as OCT3/4, SOX2, KLF4 and C-MYC. Methods for inducing iPS cells from somatic cells are well known in the art (see, eg, Bayart and Cohen-Haguenauer, Curr Gene Ther. 2013 Apr;13(2):73-92).

Since the medical composition and sheet-like cell culture of the present disclosure contain cardiomyocytes at a high level as described above, the use of such a composition and sheet-like cell culture acts on cardiomyocytes. The effect of drugs can be effectively tested. Accordingly, in another aspect of the present disclosure, the medical composition of the present disclosure is a drug screening composition. In particular, the composition of the present disclosure makes it possible to prepare cardiomyocytes derived from a specific subject, so it becomes possible to screen drugs that effectively act on such a specific subject.

<6> Quality Control Method of the Present Disclosure In the preparation of the medical composition of the present disclosure, efficiently obtaining a high-purity cardiomyocyte-containing composition leads to improved quality of the medical composition. However, it is usually difficult to predict how many cardiomyocytes are contained unless pluripotent stem cells are finally differentiated into cardiomyocytes.

However, by inducing differentiation using the pluripotent stem cells or embryoid bodies of the present disclosure, a composition with a high cardiomyocyte content can be easily obtained. That is, by measuring the expression levels of mesoderm genes, endoderm genes and/or ectoderm genes in embryoid bodies at the stage of culturing pluripotent stem cells to form embryoid bodies, it is possible to determine whether such embryoid bodies are myocardial. It is possible to test whether the cell has differentiation tropism. Therefore, one aspect of the present disclosure includes measuring the expression levels of mesoderm genes, endoderm genes and/or ectoderm genes in embryoid bodies obtained by culturing pluripotent stem cells, from pluripotent stem cells It also includes a method for quality control of a medical composition containing myocardial cells induced to differentiate, and a method for producing a medical composition comprising such a quality control method as a step.

Specific examples of the mesoderm gene, endoderm gene and ectoderm gene in this method include those described in <3> above. Any method known in the art can be used to measure the expression levels of these genes.
In one embodiment, the method of this aspect further comprises comparing the measured gene expression level with a reference value, and embryoid bodies other than embryoid bodies determined to be embryoid bodies according to <3> above as a result of the comparison. excluding embryoid bodies of Typical reference values include the average expression level of the gene, and the like. The average expression level may be, for example, the average expression level of the gene to be measured in a predetermined number (for example, 5, 10, 15, etc.) of randomly sampled embryoid bodies.

In addition, at the stage of embryoid body formation, embryoid bodies with high subsequent differentiation efficiency may be selected. For example, embryoid bodies with high differentiation efficiency may be selected based on the morphological characteristics of embryoid bodies, such as the size of formed embryoid bodies and the manner of aggregation.

A method of manufacturing a medical composition of the present disclosure includes the following steps:
(A) inducing differentiation and culturing pluripotent stem cells to form embryoid bodies;
(B) Measuring the expression levels of mesoderm genes, endoderm genes and/or ectoderm genes in the obtained embryoid bodies, and comparing the measured values with reference values, directed toward cardiomyocyte differentiation selecting embryoid bodies with high potential,
(C) Differentiation induction and culture of the selected embryoid bodies to obtain a cell population containing cardiomyocytes.

In step (A), embryoid bodies are formed from pluripotent stem cells. Techniques known in the art can be used for the formation of embryoid bodies, and specifically, for example, the technique described in <3> above can be used. Pluripotent stem cells that can be used are not particularly limited, but are preferably allogeneic cells to the subject (eg, human) to be treated with the medical composition. When the medical composition is used for transplantation, pluripotent stem cells prepared from autologous cells, such as autologous iPS cells, are preferred. Moreover, it may further include a step of screening the pluripotent stem cells to be used by the method of the present disclosure before step (A).

In step (B), embryoid bodies with a high cardiomyocyte differentiation tropism are selected. Examples of embryoid bodies highly oriented toward cardiomyocyte differentiation are as described in <3> above. In this step, embryoid bodies with particularly high expression levels of mesoderm genes and low expression levels of ectoderm genes and/or endoderm genes are selected. Specific examples and standard values of ectoderm genes, endoderm genes, and mesoderm genes may be those described in <3>.

In step (C), the embryoid bodies selected in step (B) are differentiated to obtain a medical composition containing cardiomyocytes. Methods known in the art can be used for induction of differentiation from embryoid bodies, and specifically, for example, the method described in <4> above may be used.

After step (C), the step of optionally modifying the medical composition may be further included. Such steps include, for example, a sheet forming step for forming a sheet-like cell culture, a step of cryopreserving a medical composition, and the like.

The disclosure will be described in more detail with reference to the following examples, which are intended to be specific examples of the disclosure and are not intended to limit the disclosure.

これらのｉＰＳ細胞株を、Matsuura, et al., Biochemical and Biophysical Research Communications 425 (2012) 321?327、Miki K. Cell Stem Cell (2015)、ＷＯ２０１４／１８５３５８Ａ１およびＷＯ２０１７／０３８５６２などに記載の方法を参考に、心筋細胞まで分化誘導した。具体的には、Primate ES培地（ReproCell）に５ｎｇ／ｍＬのｂＦＧＦを添加したものを未分化維持培地として用い、フィーダー細胞であるマイトマイシンＣ処理済みのMEF（ReproCell）上で未分化ヒトｉＰＳ細胞を培養して、３－４日に１回継代を行った。分化誘導はヒトｉＰＳ細胞をDissociation solution（ReproCell）およびAccumax（イノベーションセルテクノロジーズ）で解離して、０．５ｎｇ／ｍＬのＢＭＰ－４と１０μＭのY27632（Ｒｏｃｋ阻害剤）を添加したStemPro34（ライフテクノロジーズ）で懸濁し、EZSPHERE（IWAKI）で１日培養して集塊を形成させた。得られた胚様体をアクチビンＡ、骨形成タンパク質（ＢＭＰ）４および塩基性線維芽細胞増殖因子（ｂＦＧＦ）を含有する培養液中で培養し、さらにＷｎｔ阻害剤（ＩＷＲ１）を含む培養液中で培養し、その後ＶＥＧＦおよびｂＦＧＦを含む培養液中で培養を行った。図１は培養中のｉＰＳ細胞の写真図である。 Example 1. Identification of iPS cell lines with high cardiomyocyte differentiation tropism In order to investigate the genetic characteristics of iPS cells with high cardiomyocyte differentiation tropism, we first attempted to identify iPS cell lines with high cardiomyocyte differentiation tropism. rice field.
(1) Differentiation Induction Ten types of cells listed in Table 5 were used as iPS cell lines. 201B7, 253G1, 409B2, HiPS-RIKEN-1A, HiPS-RIKEN-2A and HiPS-RIKEN-12A were obtained from RIKEN BioResource Center. ATCC-DYR0100 and ATCC-HYR0103 were obtained from ATCC. mc-iPS was obtained from System Biosciences. Tic was obtained from National Institute of Biomedical Innovation. Total RNA of human iPS cell lines immediately before induction of differentiation was extracted using miRNeasy Mini Kit (QIAGEN) according to the protocol. SuperScript ^™ VILO (Invitrogen) was used for RT-PCR to synthesize cDNA. PCR was performed with the ViiA 7 Real-Time PCR System (Applied Biosystems) using the PCR primers for SYBR Green listed in Table 4 and the SYBR Green PCR Master Mix (Applied Biosystems) or Taqman probe and Taqman Gene Expression Master Mix (Applied Biosystems). carried out. GAPDH was used as a housekeeping gene for gene expression analysis. Gene expression analysis was performed using the ViiA 7 System. In TaqMan Gene Expression Assays, temperature cycling conditions were hold: 95°C for 20 seconds, cycle: 95°C for 1 second, 60°C for 20 seconds. Temperature cycling conditions for SYBR Green are Hold: 95°C for 20 seconds, Cycle: 95°C for 1 second, 60°C for 20 seconds for 40 cycles and 95°C for 15 seconds, 60°C for 1 minute, 95°C for 15 seconds. I went with

These iPS cell lines are referred to Matsuura, et al., Biochemical and Biophysical Research Communications 425 (2012) 321-327, Miki K. Cell Stem Cell (2015), WO2014/185358A1 and WO2017/038562. Then, the cells were induced to differentiate into cardiomyocytes. Specifically, a Primate ES medium (ReproCell) supplemented with 5 ng/mL bFGF was used as an undifferentiated maintenance medium, and undifferentiated human iPS cells were grown on mitomycin C-treated MEFs (ReproCell) as feeder cells. The cells were cultured and passaged once every 3-4 days. For induction of differentiation, human iPS cells were dissociated with Dissociation solution (ReproCell) and Accumax (Innovation Cell Technologies), and 0.5 ng/mL of BMP-4 and 10 μM of Y27632 (Rock inhibitor) were added to StemPro34 (Life Technologies). and cultured in EZSPHERE (IWAKI) for one day to form clumps. The resulting embryoid bodies were cultured in a medium containing activin A, bone morphogenetic protein (BMP) 4 and basic fibroblast growth factor (bFGF), and further cultured in a medium containing a Wnt inhibitor (IWR1). and then cultured in a medium containing VEGF and bFGF. FIG. 1 is a photograph of iPS cells in culture.

(2) Ranking of differentiation-inducing directionality Troponin-positive rate, beating rate, and cardiomyocyte-related gene expression level in cardiomyocyte-containing embryoid bodies differentiated from each iPS cell line were measured, and the myocardium of each iPS cell line A ranking of differentiation tropism into cells was performed.
The troponin-positive rate was determined by dispersing embryoid bodies using TrypLE Select, fixing and permeabilizing the dispersed cells using the BD Cytofix/Cytoperm (registered trademark) Fixation/Permeabilization Solution Kit (BD Bioscience), and then measuring the anti-human A troponin antibody (Thermo Fisher Scientific) and a labeled secondary antibody (Thermo Fisher Scientific) were allowed to react in sequence, and then measurement was performed with a flow cytometer to calculate.

The beating rate was determined by filming the embryoid bodies after differentiation induction from each iPS cell line into cardiomyocytes by cell motion imaging (Sony). counted and calculated.

The results are shown in FIGS. 2 and 3. FIG. Particularly high troponin positive rate and beating rate were calculated in the three cell lines 201B7, 253G1 and 409B2. Therefore, these three cell lines were identified as cell lines with high cardiomyocyte differentiation tropism.

(3) Survival rate of undifferentiated cells , was measured by quantitative PCR as a percentage of the number of cells expressing Lin28, an undifferentiated cell marker.
The results are shown in FIG. In the three cell lines identified as having a high cardiomyocyte differentiation tropism, the survival rate of undifferentiated cells tended to be significantly lower than in the other cell lines.

Example 2. Gene Expression in Embryoid Bodies SOX2, PAX6, ZIC1, BRACHYURY, FLK1, PDGFR-a, GOOSECOID, HNF3, SOX17, SOX7, and AMN 11 in each embryoid body on day 4 of culture in Example 1 above. The expression of different genes was measured.

The results are shown in FIG. The three cell lines 201B7, 253G1 and 409B2, which are cell lines highly oriented toward cardiomyocyte differentiation, all expressed large amounts of mesoderm genes, and expressed low levels of endoderm genes and ectoderm genes. For each gene, Pearson's correlation coefficient between the expression level and the troponin T positive rate after induction of differentiation of each cell line was calculated, and the results are shown in the table below.

Example 3. Principal Component Analysis of Each Cell Line Total RNA was extracted from each iPS cell line using RNeasy Kit (QIAGEN). The purity of each RNA was confirmed by calculating the rRNA ratio of 28S and 18S using Agilent RNA 6000 Nano Assay (Agilent Technologies). The extracted RNA samples were stored frozen at -80°C. Biotin-labeled cRNA synthesis of RNA samples was performed using the GeneChip 3' IVT Express kit (Affymetrix) according to the manufacturer's protocol.

First, double-stranded cDNA containing a T7 promoter sequence was synthesized from total RNA, and biotin-labeled aRNA was synthesized using the cDNA as a template by an in vitro reverse transcription reaction. ˜100-120 nt aRNA fragments were then generated by calcium random digestion using the hammerhead reaction. A GeneChip Hybridization Oven (Affymetrix) was used to hybridize the biotin-labeled aRNA prepared to a Genechip array Human Genome U133 Plus 2.0 Array (Affymetrix). After hybridization, washing and phycoerythrin staining were performed using the GeneChip Wash and Stain Kit (Affymetrix) and GeneChip Fluidics Station 450 (Affymetrix). After that, the fluorescence image of the Genechip array was scanned using GeneChip Scanner 3000 7G (Affymetrix) to obtain an image. The obtained fluorescence intensity data was analyzed using Expression Console Ver.1.1 (Affymetrix). Signal normalization was performed using the MAS5 algorithm and MSK files (Affymetrix).

201B7, 253G1 and 409B2 were used as cell lines with high cardiomyocyte differentiation tropism, and RIKEN-1A, RIKEN-2A and RIKEN-12A were used as cell lines with low cardiomyocyte differentiation tropism. We analyzed the genes that are characteristically expressed in cell lines with high differentiation tropism.

(1) Comparison of myocardial-related gene expression Each cell line was differentiated into cardiomyocytes in the same manner as in Example 1, and the expression levels of myocardial-related genes were compared. The results are shown in FIGS. 6 and 7. FIG.
High myocardial-related gene expression was confirmed in all of the cell cultures induced to differentiate from the three cell lines with high differentiation tropism into cardiomyocytes. In addition, the troponin-positive rate was significantly higher in the three cell lines with high cardiomyocyte differentiation tropism.

(2) Microarray analysis Next, microarray analysis was performed using Affymetrix GeneChip (R) Arrays for differences in gene expression levels between 3 cell lines with high cardiomyocyte differentiation tropism and 3 cell lines with low cardiomyocyte differentiation tropism. rice field.
Among the 3,300 genes that can be analyzed with Affymetrix GeneChip(R) Arrays, the expression level was more than doubled in the group with high differentiation tropism compared to the group with low differentiation tropism. We identified 84 genes and analyzed which signal transduction pathway the gene group was associated with. The results are shown in the table below.

(3) Narrowing down marker candidate genes Among the 84 genes described above, genes that showed a particularly remarkable difference in expression level were identified as biomarker candidate genes. Mitochondria-associated genes CHCHD2 and SFXN3, WNT signal regulator KDM6A, TGF-β signal-associated factor SKIL, etc., were thereby identified as candidate biomarker genes. Conversely, miRNA-139 and miRNA-204 were identified as genes that showed significantly high expression in cell lines with low cardiomyocyte differentiation tropism.

Example 4: miRNA expression analysis (1) miRNA microarray Total RNA was extracted from each iPS cell line using miRNeasy mini kit (QIAGEN). Biotin-labeled RNA was prepared from Total RNA containing low-molecular-weight RNA using FlashTag Biotin HSR RNA labeling kit (Affymetrix) according to the product protocol. A GeneChip Hybridization Oven (Affymetrix) was used to hybridize the biotin-labeled RNA prepared to the miRNA 3.0 array (Affymetrix). After hybridization, washing and phycoerythrin staining were performed using the GeneChip Fluidics Station 450 (Affymetrix). After that, the fluorescence image of the Genechip array was scanned using GeneChip Scanner 3000 7G (Affymetrix) to obtain an image. The obtained fluorescence intensity data was analyzed using Expression Console Ver.1.1 (Affymetrix). Signal normalization was performed using the miRNA array RMA+DABG analysis and Expression Console software (Affymetrix).
201B7, 253G1 and 409B2 were used as cell lines with high cardiomyocyte differentiation tropism, and RIKEN-1A, RIKEN-2A and RIKEN-12A were used as cell lines with low cardiomyocyte differentiation tropism. We analyzed the genes that are characteristically expressed in cell lines with high differentiation tropism.
The miRNA expression in the various iPS cell lines used in Example 1 above was analyzed using a miRNA microarray.
Of the 534 miRNAs that can be analyzed, 5 miRNAs (ACA24 , hsa-miR-629-star, mmi-miR-204, ACA61 and hsa-miR-139-5p). The results are shown in FIG.
It was analyzed which signal transduction pathway the identified miRNA was associated with, and the results analyzed in Example 3(3) above were referred to narrow down the strongly associated genes. The results are shown in FIG. PF4 was found as a gene with a significantly high expression level, and TMEM64 was found as a gene with a significantly low expression level in an iPS cell line with a strong differentiation tropism to cardiomyocytes.

(2) Confirmation of involvement in cardiomyocyte differentiation tropism In order to confirm the effectiveness of the above genes as differentiation tropism marker genes, a medium supplemented with DMSO, IWR-1 and 2, CHIR99021 or MitoBlock6 was used. iPS cells were induced to differentiate into cardiomyocytes, and the cTnT positive rate, the expression level of PF4, and the expression level of TMEM64 in the resulting cell population were measured.
The results are shown in FIG. In particular, a significant decrease in the cTnT positive rate and the expression level of PF4 was confirmed when using the medium supplemented with CHIR99021. In addition, cTnT was significantly reduced in the medium supplemented with MitoBlock-6, and in this case, a decreasing tendency was confirmed with PF4, and an increasing tendency was confirmed with TMEM64. Therefore, it was suggested that PF4 is positively correlated with cardiomyocyte differentiation induction, and that TMEM64 is negatively correlated.

Claims (9)

A method for determining a differentiation tropism marker for evaluating the differentiation tropism of human induced pluripotent stem cells into cardiomyocytes , comprising:
(1) measuring gene expression levels in multiple human induced pluripotent stem cell lines;
(2) measuring the expression level of miRNA in the plurality of human induced pluripotent stem cell lines;
(3) Extracting a gene with a significantly different expression level between a human induced pluripotent stem cell line with a high cardiomyocyte differentiation tropism and a human induced pluripotent stem cell line with a low differentiation tropism;
(4) ACA24, hsa-miR-629-star, which has a significant difference in the expression level between the human induced pluripotent stem cell line with high cardiomyocyte differentiation tropism and the human induced pluripotent stem cell line with low differentiation tropism , mmi-miR-204, ACA61 and hsa-miR-139-5p ; and (5) ACA24, hsa-miR-629-star extracted in (4) from the genes extracted in (3). PF4 and TMEM64 associated with , mmi-miR-204, ACA61 and hsa-miR-139-5p ;
The above method, comprising In step (1), at least one human induced pluripotent stem cell line known to have a high differentiation tropism to cardiomyocytes, and a plurality of human induced pluripotent stem cell lines to cardiomyocytes. 2. The method of claim 1, comprising at least one human induced pluripotent stem cell line known to have a low differentiation propensity. In step (2), the plurality of human induced pluripotent stem cell lines are the same human induced pluripotent stem cell lines as the plurality of human induced pluripotent stem cell lines whose gene expression levels were measured in step (1). 3. The method of claim 1 or 2, wherein: In step (3), a gene that is significantly highly expressed in a human induced pluripotent stem cell line with high differentiation tropism or a gene that is significantly highly expressed in a human induced pluripotent stem cell line with low differentiation tropism A method according to any one of claims 1 to 3, wherein is extracted. In step (4), miRNAs that are significantly highly expressed in human induced pluripotent stem cell lines with high differentiation tropism, or miRNAs that are significantly highly expressed in human induced pluripotent stem cell lines with low differentiation tropism A method according to any one of claims 1 to 4, wherein is extracted. The method according to any one of claims 1 to 5, wherein in step (5), a gene having a significant difference in expression level due to a significant difference in miRNA expression is selected. A method for indexing the cardiomyocyte differentiation propensity of a human induced pluripotent stem cell line, comprising:
(a) measuring the expression level of PF4 and/or TMEM64 in human induced pluripotent stem cells of interest
(b) comparing the expression level of PF4 and/or TMEM64 measured in (a) with a reference
The above method, comprising 8. The method according to claim 7, wherein when the measured expression level of PF4 is significantly higher than the reference, the subject human induced pluripotent stem cells are determined to be a cell line with a high cardiomyocyte differentiation tropism. 9. The method according to claim 7 or 8, wherein when the measured expression level of TMEM64 is significantly lower than the reference, the subject human induced pluripotent stem cells are determined to be a cell line with high cardiomyocyte differentiation tropism.

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