WO2019078342A1 - Method for selecting pluripotent stem cell having directivity of differentiation to cardiomyocyte - Google Patents

Abstract

The present disclosure addresses the problem of providing a method for selecting a pluripotent stem cell having directivity of differentiation to a cardiomyocyte. The problem can be solved by a method including (a) measuring the expression amount of at least one gene selected from the group consisting of a mitochondrion-related gene, a WNT signal transduction regulation factor, a TGFβ signal transduction regulation factor, a mesoblast-related gene, a cardiomyocyte-related gene and an undifferentiated cell-related gene in a pluripotent stem cell of interest and (b) comparing the expression amount of the gene which is measured in step (a) with a reference value, wherein it is determined that the pluripotent stem cell of interest is a cell of a cell strain having high directivity of differentiation to a cardiomyocyte when the measured expression amount is significantly higher than the reference value.

Description

Method for Selecting Pluripotent Stem Cells Directed to Differentiation into Cardiomyocytes

The present invention relates to pluripotent stem cells having a directivity toward differentiation into specific differentiation-inducing cells, particularly cardiomyocytes, embryoid bodies derived from the pluripotent stem cells, differentiation-inducing cells derived from the pluripotent stem cells , A method of screening the pluripotent stem cells, a method of treating a subject using the medical composition, a method of screening an effective drug using the medical composition, the medical use The present invention relates to a quality control method and the like in the production of a composition.

Adult cardiomyocytes are poorly capable of self-renewal, and their repair is extremely difficult if myocardial tissue is damaged. In recent years, in order to repair damaged myocardial tissue, an attempt has been made to transplant a graft containing cardiomyocytes prepared by a cell engineering method 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) are recently attracting attention as cardiomyocytes used for the preparation of such grafts. Such preparation of a sheet-like cell culture containing cardiomyocytes derived from such pluripotent stem cells and treatment experiments with animals have been attempted (Non-patent documents 2 to 3). However, development of sheet-like cell cultures containing cardiomyocytes derived from pluripotent stem cells has only just begun, and there are still many unclear parts about their functional characteristics and factors affecting them.

When preparing differentiation-inducing cells from pluripotent stem cells, for example, when preparing cardiomyocytes, first, an embryoid body is formed while giving directionality of differentiation of pluripotent stem cells to mesoderm, and such embryos The cardiomyocytes are recovered by inducing differentiation of the body into cardiomyocytes and dispersing them into single cells (for example, Patent Document 2 and the like).

In recent years, research on pluripotent stem cells such as ES cells and iPS cells has been advanced, and it has been found that there are differences in differentiation characteristics among cell lines while many cell lines are being established. For example, Patent Document 2 and Non-Patent Document 4 describe iPS cells having a differentiation directivity to cells of the nervous system.

Japanese Patent Application Publication No. 2007-528755 International Publication No. 2013/187416

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)

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

While the present inventors are studying preparation of a sheet-like cell culture using pluripotent stem cell-derived cardiomyocytes, the proportion of cardiomyocytes in the final preparation is Faced with the task of being very different. As a result of intensive research aimed at solving this problem, pluripotent stem cell lines have strains that are easy to differentiate into cardiomyocytes and strains that are difficult to differentiate exist, and using pluripotent stem cell lines that are easy to differentiate into cardiomyocytes, It was found that the proportion of cardiomyocytes in the final preparation was high. Therefore, when we continued research on these cell lines, we succeeded in identifying genes characteristically expressed in cell lines that are easily differentiated into cardiomyocytes, and as a result of further research, we completed the present invention. It reached.

That is, the present invention relates to the following:
[1] A method of determining a differentiation tropism marker for evaluating differentiation tropism of pluripotent stem cells to a specific differentiation induction cell,
(1) measuring gene expression levels in a plurality of pluripotent stem cell lines;
(2) measuring the expression level of miRNA in the plurality of pluripotent stem cell lines;
(3) extracting a gene having a significant difference in expression amount between the highly directed pluripotent stem cell line and the low pluripotent stem cell line directed to the specific differentiation-inducing cell;
(4) extracting a miRNA having a significantly different expression level between the highly directed pluripotent stem cell line and the low pluripotent stem cell line directed to the specific differentiation-inducing cell; and (5) Selecting a gene involved with the miRNA extracted in (4) from the genes extracted in (3);
Said method.

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

[4] The WNT signaling modulators are PF4, TMEM64, KDM6A, APC, β-catenin, Axin, CK1, Dsh, GSK-3β, Dkk, WIF, FRP, Cerberus, TCF, Krn, WNT1, WNT2, WNT3, WNT4 The method of [2] or [3], which is at least one gene selected from the group consisting of WNT5A, WNT7A, WNT7B, WNT8B, WNT10B, WNT11, WNT2B, WNT9A, WNT9B, LRP5 and LRP6.
[5] The mitochondrial related gene is 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], which is at least one gene selected.

[6] TGFβ signaling modulators include 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], which is at least one gene selected from the group consisting of BMP6, BMP7, BMP8B, BMPR1A and BMPR1B.
[7] The mesodermal genes are 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] to [6], which is at least one gene selected from the group consisting of Csf1 r, CD45 and Ter119.

[8] A cardiomyocyte related gene consists of TNT2, ML2, GATA4, MYH6, MYH7, Nkx2.5, SCN5A, RYR2, PPARGC1, MYL2, HCN4, CACNalC, ATP2A2, Actc1, Cx43, TEF-1 and Tbx-5 The method of [2] to [7], which is at least one gene selected from the group.
[9] The 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 member selected from the group consisting of miRNAs of Esg1, Rexl, Zfp296, UTF1, GDF3, Sall4, Tbx3, Tcf3, DNMT3L, DNMT3B, Tra-1-81, miR-290 cluster and miR-302 cluster The method of [2] to [8] which is a gene.

[10] Differentiation-oriented marker genes 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], which is at least one gene selected from the group consisting of
[11] The method according to [10], wherein the pluripotent stem cell of interest is judged to be a cell line highly directed to cardiomyocytes when the measured expression level is significantly higher than the standard.

[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 the pluripotent stem cell of interest is judged to be a cell line highly directed to cardiomyocytes when the measured expression level is significantly lower than the standard.
[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, Culturing 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 a culture 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 pluripotent stem cell-derived differentiation-inducing cells cultured by the method of [14]-[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 comprising cardiomyocytes induced to differentiate from pluripotent stem cells, which is a mesodermal gene, endodermal gene and embryoid body obtained by culturing pluripotent stem cells, and And / or measuring the expression level of the ectoderm gene.
[23] The mesodermal genes are 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], which is selected from the group consisting of at least one gene selected from the group consisting of Csf1 r, CD45 and Ter119.
[24] The method of [22] or [23], wherein the endodermal 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]-[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 with respect to specific differentiation-inducing cells such as cardiomyocytes, so that differentiation can be performed using such a pluripotent stem cell line. It will be possible to efficiently prepare cardiomyocytes by induction. In addition, since it can be determined whether or not a final preparation with a high proportion of cardiomyocytes can be obtained at an early stage of differentiation induction such as pluripotent stem cells and embryoid bodies, efficiently providing a high quality medical composition. Can. Furthermore, by providing a novel culture method for differentiating into desired differentiation-inducing cells, such as cardiomyocytes, it is possible to obtain desired differentiation-inducing cells more efficiently.

FIG. 1 is a photograph of a culture of iPS cells used in Example 1 and a embryoid body. A is a photograph of cells in culture, and B is a photograph of embryoid bodies. FIG. 2 shows the positive rate of troponin T when iPS cells used in Example 1 were induced to differentiate into cardiomyocytes, and blue bars and red bars each added 12 ng / mL when activin was added at 6 ng / mL. Positive rate when FIG. 3 shows the beating rate of the culture when the iPS cells used in Example 1 are induced to differentiate into cardiomyocytes. The red and blue bars show the results on day 8 and day 17 of culture, respectively.

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

FIG. 7 shows the expression level of each cardiomyocyte associated gene in cell cultures induced to differentiate from each iPS cell. FIG. 8 shows the results of miRNA expression analysis in each iPS cell line. A is a graph plotting changes in the expression level of miRNA between a differentiation-oriented iPS cell line and a low differentiation-oriented iPS cell line. B is a graph showing the difference in the expression level of the 5 types of miRNAs identified as the miRNA whose expression level has been reduced, between the highly differentiation-oriented strain and the low differentiation-oriented strain. C is a distribution map in which the expression levels in the high differentiation-oriented strain and the low differentiation-oriented strain are plotted as the vertical axis and the horizontal axis for all the analyzed miRNAs.

FIG. 9 shows the results of pathway analysis of the identified miRNAs and the test results for the expression of the identified two genes (PF4 and TMEM64). A represents the expression levels of 10 mRNAs whose expression levels are significantly higher between the differentiation-oriented iPS cell line and the low differentiation-oriented iPS cell line and 10 mRNAs whose expression levels are significantly lower It is a graph. B is a graph listing signal transduction pathways involving miRNAs and mRNAs identified by miRNA analysis and gene expression analysis. C is a graph showing the difference in cTnT expression amount in the obtained cell population when differentiation-promoting and low-derivative targeting strains are induced to differentiate into cardiomyocytes, respectively. D is a graph showing the expression level of PF4 and TMEM64 in the high differentiation-oriented strain and the low differentiation-oriented 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 iPS cell line is differentiated into cardiomyocytes. FIG. 10 is a graph showing the expression level of A: cTnT and the expression levels of B: PF4 and TMEM 64 when differentiation was induced by adding various agents to the medium.

Hereinafter, the present invention will be described in detail.
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, in the case of conflict between the publication referred to in the present specification and the description in the present specification, the description in the present specification shall prevail.
In the present disclosure, a base sequence (including mRNA and miRNA) represented by a specific gene name means a sequence registered in a database known in the art such as GenBank. Those skilled in the art can immediately know what sequence is represented from such a gene name.

In the present disclosure, the term "pluripotent stem cells" is a term well known in the art and capable of differentiating into cells of all lineages belonging to three germs, ie endoderm, mesoderm and ectoderm. Means cells with Non-limiting examples of pluripotent stem cells include, for example, embryonic stem cells (ES cells), nuclear transplanted embryonic stem cells (ntES cells), induced pluripotent stem cells (iPS cells) and the like. Usually, when inducing differentiation of pluripotent stem cells into specific cells, first, pluripotent stem cells are suspended and cultured to form aggregates of any of the above three germ layers and then to form aggregates. To induce differentiation into specific cells of interest. Alternatively, pluripotent stem cells are adherently cultured at high density to induce differentiation. In the present disclosure, "embryoid body" means an aggregate of such cells. In the present disclosure, in particular, an embryoid body having differentiation tropism for endodermal cells is referred to as "endodermal embryoid body", and an embryoid body having differentiation tropism for mesodermal cells is referred to as mesodermal embryo An embryoid body that has differentiation tropism to ectoderm cells may be referred to as "ectodermal embryoid body".

In the present disclosure, “differentiation-inducing cells” mean any cells that have been subjected to differentiation-inducing treatment to differentiate from pluripotent stem cells into specific types of cells. Differentiation-inducing cells include adherent cells constituting tissues such as cardiomyocytes and skeletal myoblasts, and non-adherent cells such as blood cells. Non-limiting examples of differentiation-inducing cells include muscle cells such as cardiac muscle cells and skeletal myoblasts, neural cells such as neuronal cells, oligodendrocytes and dopamine producing cells, retinal cells such as retinal pigment epithelial cells, and blood cells Cells, cells of hematopoietic lineage such as bone marrow cells, T cells, NK cells, NKT cells, dendritic cells, immune related cells such as B cells, cells constituting organs such as liver cells, pancreatic β cells, kidney cells, etc. Besides chondrocytes, germ cells and the like, precursor cells and somatic stem cells that differentiate into these cells are included. As typical examples of such precursor cells and somatic stem cells, for example, mesenchymal stem cells in cardiomyocytes, multipotent cardiac progenitor cells, unipotent cardiac progenitor cells, neural stem cells in cells of nervous system, cells of hematopoietic system and immunity Hematopoietic stem cells and lymphoid stem cells in related cells can be mentioned. Differentiation induction of pluripotent stem cells can be performed using any known method. For example, differentiation induction from pluripotent stem cells to cardiomyocytes can be performed by Miki et al., Cell Stem Cell 16, 699-711, June 4, 2015 or WO 2014/185358, Shugo Tohyama et al., Stem Cell Report, 9, It can be performed based on the method described in 1-9, Nov 14, 2017.

In the present disclosure, “differentiation-directed” means the property that pluripotent stem cells are more likely to differentiate into specific differentiation-inducing cells, and the higher the differentiation directivity into specific differentiation-inducing cells, the more differentiation-inducing cells become It means easy. Therefore, a pluripotent stem cell line highly directed to differentiation into a specific cell is induced to differentiate by the differentiation induction method to the specific cell, as compared to a pluripotent stem cell line not highly directed to differentiation. It is expected that more differentiation-inducing cells can be obtained even by the same differentiation-inducing method.

In the present disclosure, "cardiomyocytes" mean cells having characteristics of cardiomyocytes. Characteristics of cardiomyocytes include, but are not limited to, for example, the expression of cardiomyocyte markers, the presence of an autonomous beat, and the like. Non-limiting examples of cardiomyocyte markers include, for example, c-TNT (cardiac troponin T), CD172a (also known as SIRPA or SHPS-1), KDR (also known as CD309, FLK1 or VEGFR2), PDGFRA, EMILIN2, VCAM, etc. . In one embodiment, 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 to a specific differentiation-inducing cell. In the present disclosure, the “differentiation-directed marker” or the “differentiation-directed marker gene” is a marker (gene) expressed in pluripotent stem cells, and the differentiation directivity of the pluripotent stem cells according to the difference in expression amount. It means something that can be evaluated. For example, in the case of PF4 confirmed to be a differentiation-directed marker by the present inventors, when the expression level of PF4 in a given pluripotent stem cell is larger than that of a standard pluripotent stem cell, the pluripotent stem cell is It is evaluated that differentiation to cardiomyocytes is highly directed. In the present disclosure, the “standard expression amount” is not limited to this, but includes, for example, an average value of gene expression amounts in a plurality of 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 randomly selected. .

The method of determining a differentiation directed marker of the present disclosure includes the following steps:
(1) measuring gene expression levels in a plurality of pluripotent stem cell lines;
(2) measuring the expression level of miRNA in the plurality of pluripotent stem cell lines;
(3) extracting a gene having a significant difference in expression amount between a highly directed pluripotent stem cell line and a low pluripotent stem cell line directed to a specific differentiation-inducing cell;
(4) extracting a miRNA having a significantly different expression level between the highly directed pluripotent stem cell line and the low pluripotent stem cell line directed to the specific differentiation-inducing cell; and (5) Select genes involved with miRNA extracted in (4) from genes extracted in (3).

In the step (1), the expression levels of genes in a plurality of pluripotent stem cell lines are each quantitatively measured. The measurement of the gene expression level may be performed exhaustively. A plurality of pluripotent stem cell lines have low directivity to at least one pluripotent stem cell line known to be highly directed to specific differentiation-inducing cells and to the specific differentiation-induced cells. And at least one pluripotent stem cell line known to be. The directionality of differentiation into specific differentiation-inducing cells can be identified using a method known in the art, a method described in the present disclosure, or the like. The expression level of the gene 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 miRNA in a plurality of pluripotent stem cell lines are each quantitatively measured. The plurality of pluripotent stem cell lines are pluripotent stem cell lines identical to the plurality of pluripotent stem cell lines whose gene expression levels were measured in step (1). The measurement of the expression level of miRNA can be performed using methods known in the art, such as real-time PCR method, microarray method, high-throughput sequencing method and the like.

In the step (3), a gene having a significant difference in expression amount between a highly pluripotent stem cell line and a low pluripotent stem cell line that are highly directed to specific differentiation-inducing cells is extracted. For example, comparing a pluripotent stem cell line highly differentiation-directed with a pluripotent stem cell line low differentiation-directed, 1.5 times or more, 2 times or more, 2.5 times or more, 3 times or more, 5 Genes having a difference in expression amount such as twice or more and 10 times or more may be extracted. Such expression level may be high in either pluripotent stem cell line, for example, may be high expression in a pluripotent stem cell line highly directed for differentiation, or in a pluripotent stem cell line low for directed differentiation. It may be highly expressed. In a preferred embodiment, genes that are significantly expressed at high differentiation-oriented pluripotent stem cell lines are extracted. In another preferred embodiment, genes that are significantly highly expressed in low differentiation-oriented pluripotent stem cell lines are extracted.

In step (4), miRNA having a significant difference in expression amount between pluripotent stem cell lines highly directed to specific differentiation-inducing cells and low pluripotent stem cell lines is extracted. For example, comparing a pluripotent stem cell line highly differentiation-directed with a pluripotent stem cell line low differentiation-directed, 1.5 times or more, 2 times or more, 2.5 times or more, 3 times or more, 5 It is possible to extract miRNA having a difference in expression amount such as twice or more and 10 times or more. Such expression level may be high in either pluripotent stem cell line, for example, may be high expression in a pluripotent stem cell line highly directed for differentiation, or in a pluripotent stem cell line low for directed differentiation. It may be highly expressed. In a preferred embodiment, miRNAs that are significantly highly expressed in highly differentiation-directed pluripotent stem cell lines are extracted. In another preferred embodiment, miRNAs that are highly highly expressed in low differentiation-oriented pluripotent stem cell lines are extracted.

In the step (5), among the genes extracted in the step (3), a gene involved with the miRNA extracted in the step (4) is selected. As a method for identifying a gene involved with a certain miRNA, a method known in the art may be used, and for example, a pathway analysis method, a database of target genes of miRNA (TargetScan) and the like can be mentioned. By this step, genes having a significant difference in expression amount due to a significant difference in miRNA expression will be selected.

Another aspect of the present disclosure relates to a method of indexing differentiation toward a specific differentiation inducing cell. The indexing method of the present disclosure includes the following steps:
(A) At least one differentiation-oriented marker gene in the pluripotent stem cells of interest, such as mitochondrial related gene, WNT signal transduction regulator, TGFβ signal transduction regulator, associated gene of each germ and undifferentiated cell associated gene, etc. Measuring the expression level of (b) (a) comparing the expression level of the differentiation-oriented marker gene measured in (a) with the standard.

The “related gene of each germ” means a related gene of germ that the differentiation inducing cell is derived from, for example, a mesoderm related gene if the differentiation inducing cell is a cardiomyocyte, an ectoderm related gene if it is a cell of nervous system, The cells of digestive tract mean endoderm related genes.
In the step of measuring the expression level of the gene, the expression level of the gene associated with a specific differentiation-inducing cell may be measured. For example, if the differentiation-inducing cells are cardiomyocytes, cardiomyocyte related genes may be further measured.

The present invention will be described in detail below, taking the case where the differentiation-inducing cells are cardiomyocytes as an example.
<1> Indexing Method of the Present Disclosure One aspect of the present disclosure relates to a method of indexing a differentiation directivity to a specific differentiation-inducing cell, particularly a cardiomyocyte. The indexing method of the present disclosure includes the following steps (a) and (b):
(A) At least one selected from the group consisting of mitochondrial related genes, WNT signal transduction regulators, TGFβ signal transduction regulators, mesoderm associated genes, cardiomyocyte associated genes and undifferentiated cell associated genes in pluripotent stem cells of interest Measuring the expression level of the differentiation-directed marker gene of the species (b) comparing the expression level of the gene measured in (a) with the reference.

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

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

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。 Examples of WNT signaling modulators include, but are not limited to, those described in the following table.

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 and the like. Thus, in one embodiment, in step (a), PF4, TMEM64, KDM6A, APC, β-catenin, Axin, CK1, Dsh, GSK-3β, Dkk, WIF, FRP, Cerberus, TCF, Krn, WNT1, WNT2, WNT3, Wnt3, The amount of expression of at least one WNT signaling modulator 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。 The TGFβ signaling regulator includes, but is not limited to, for example, those described in the following table.

Particularly preferred TGFβ signaling modulators include SKIL, THBS1, CD3, TLR2, SMAD1, SMAD2, SMAD3, SMAD4, SMAD5, SMAD7, SMAD9, TGFD1, TGFBR2, MAPK1, MAPK3, ROCK1, BMP2, BMP4, BMP5, Examples include BMP6, BMP7, BMP8B, BMPR1A or BMPR1B. Thus, in one embodiment, in step (a), SKIL, THBS1, CD3, TLR2, SMAD1, SMAD2, SMAD3, SMAD4, SMAD5, SMAD6, SMAD7, SMAD9, TGFBR1, TGFBR2, MAPK1, MAPK3, ROCK1, BMP2, BMP4, BMP5 The expression level of at least one TGFβ signaling regulator selected from the group consisting of BMP6, BMP7, BMP8B, BMPR1A and BMPR1B is measured. In a preferred embodiment, the expression level of SKIL is measured as a TGFβ signaling regulator.

Examples of mesodermal 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 and the like. Thus, in one embodiment, in step (a), FLK1, BRACHYURY, GOOSECOID, PDGFR-a, IGF2, CD34, CLL1, HHEX, INHBA, LEF1, SRF, T, TWIST1, ADIPOQ, MME, KIT, ITGAL, Tbx1, Gata1 The amount of expression of at least one mesodermal gene selected from the group consisting of Klf1, Csf1 r, CD45 and Ter119 is measured. In a preferred embodiment, the expression level of FLK1, BRACHYURY, GOOSECOID and / or PDGFR-a is measured as a mesodermal gene.

As a cardiomyocyte related gene, although it does not limit to this, for example, TNT2, MYL2, GATA4, MYH6, MYH7, Nkx2.5, SCN5A, RYR2, PPARGC1, MYL2, HCN4, CACNalC, ATP2A2, Actc1, Cx43, TEF-1 or Tbx-5 may, for example, be mentioned. Thus, in one embodiment, 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 In a preferred embodiment, the expression levels of TNT2, MYL2, GATA4, MYH6, MYH7, NKx2.5, SCN5A, RYR2, PPARGC1, MYL2, HCN4, CACNalC and / or ATP2A2 as cardiomyocyte related genes are measured.

Examples of 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 miRNA of miR-290 cluster, miRNA of miR-302 cluster, etc. It can be mentioned. Thus, in one embodiment, 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 at least one selected from the group consisting of miR-302 cluster miRNAs The expression level of undifferentiated cell-related genes of In a preferred embodiment, the expression level of Oct-4, Nanog and / or Lin28 is measured as an undifferentiated cell-related gene.

In a more preferred embodiment, the expression levels of at least two genes selected from the aforementioned gene group are measured. That is, CHCHD2, SFXN3, KDM6A, SKIL, FLK1, BRACHYURY, GOOSECOID, PDGFR-a, TNT2, ML2, GATA4, MYH6, MYH7, NHx2.5, SCN5A, RYR2, PPARGC1, MYL2, HCN4, CACNa1Ct, ATP2 The expression levels of at least two genes selected from 4, Nanog and Lin28 are measured.

In another preferred embodiment, the differentiation-oriented 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 a differentiation-oriented marker gene The quantity is measured.

In step (b), the amount of expression of the gene measured in (a) is compared with a reference. The criteria to be compared are, but not limited to, for example, the expression amount of the same gene in a pluripotent stem cell line known to have low directivity for cardiomyocyte differentiation, directivity for cardiomyocyte differentiation The average expression level of the same gene in multiple pluripotent stem cell lines known to be low, the expression level of the same gene in pluripotent stem cell lines known to be highly directional to cardiomyocytes, cardiomyocytes To the average of the expression level of the same gene in multiple pluripotent stem cell lines known to be highly directed to differentiation, to the average expression level of the same gene in multiple pluripotent stem cell lines, to mesodermal lineage tissue The expression level of the same gene in multiple pluripotent stem cell lines known to have low directivity for differentiation, and the same gene in multiple pluripotent stem cell lines known to be highly directed to differentiation into mesodermal lineage tissue Expression level, inside Expression levels of the same gene in multiple pluripotent stem cell lines that are known to have low directionality of differentiation into leaf lineage tissue, and multiple pluripotent stem cell lines that are known to have high directionality of differentiation into endoderm lineage tissue It is known that the expression level of the same gene in the case, the expression level of the same gene in multiple pluripotent stem cell lines known to be highly directed to differentiation to ectodermal lineage tissue, and the directivity to differentiation to ectodermal lineage tissue is low The expression amount of the same gene in multiple pluripotent stem cell lines of

These criteria may use numerical values known in the art or values previously measured and recorded, or may be measured when performing the indexing method of the present disclosure in conjunction with the step (a). It is also good. Differentiation directional marker of the target pluripotent stem cell line is indexed by comparing with these criteria, and whether the target pluripotent stem cell line is a line with high directivity to cardiomyocytes based on such index Can be determined.
When comparing the expression level measured in (a) with a reference, both values are preferably values measured by the same method, but it is not limited thereto. If the values are measured by different methods, the values may be converted to allow direct comparison.

In one aspect of the present disclosure, a plurality of pluripotency known to exhibit low expression levels of the same gene in a pluripotent stem cell line known to have low directivity toward cardiomyocytes or low directivity to cardiomyocytes. The comparison is made on the basis of the average value of the expression level of the same gene in stem cell lines. In this case, when the expression level measured in (a) is significantly higher than the reference value, it can be determined that the pluripotent stem cell line of interest has high directivity to cardiomyocytes.

In another aspect of the present disclosure, a plurality of multiple genes known to have high expression levels of the same gene in cardiogenic cells or pluripotent stem cell lines that are known to have a high directivity for cardiomyocyte differentiation. The average value of the expression level of the same gene in potential stem cell lines is compared as a standard. 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 pluripotent stem cell line of interest is highly directional to cardiomyocytes . In the present disclosure, "significantly" means that the difference is statistically significant. For example, it is considered to be "significant" when a certain measured value indicates a numerical value that deviates extremely from a certain statistic.

In still another aspect of the present disclosure, comparison is made on the basis of the average value of the expression level of the same gene in multiple pluripotent stem cell lines. In this case, when the expression level measured in (a) is significantly higher than the reference value, it can be determined that the pluripotent stem cell line of interest has high directivity to cardiomyocytes.

In one embodiment, the differentiation-oriented marker gene includes PF4, CHCHD2, AMMECR1, API5, BCOR, BRWD1, CLEC4G, GLIPR1, HELB, KDM6A, LOC388796, NKTR, POMZP3, ZP3, PRUNE2, RBMX, RC3H1, SKIL, SORBS2 and SRSF11 The amount of expression of at least one gene selected from the group consisting of In this case, when the expression level measured in (a) is significantly higher than the reference value, it can be determined that the pluripotent stem cell line of interest has high directivity to cardiomyocytes. In another embodiment, 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 is used. measure. In this case, when the expression level measured in (a) is significantly lower than the reference value, it can be determined that the pluripotent stem cell line of interest has a high directivity to cardiomyocytes.

<2> Pluripotent Stem Cells of the Present Disclosure According to the present inventors, there exist cell lines with high directivity to cardiomyocytes in pluripotent stem cells, particularly induced pluripotent stem cells (iPS cells), and The genetic features of pluripotent stem cell lines were found for the first time. Therefore, one aspect of the present disclosure relates to at least one member selected from the group consisting of a mitochondrial related gene, a WNT signaling regulator, a TGFβ signaling regulator, a mesoderm related gene, a cardiomyocyte related gene and an undifferentiated cell related gene. It includes pluripotent stem cells highly directed to cardiomyocyte differentiation characterized by high gene expression levels.

As described above, in the pluripotent stem cell line highly directed to cardiomyocytes, the present inventors have found that mitochondrial related genes, WNT signal transduction regulatory factor, TGF beta signal transduction regulatory factor, mesoderm related gene, cardiomyocyte related gene And found that the undifferentiated cell-related gene is highly expressed.

In the present disclosure, “high expression level” or “high expression” means that the expression level of a certain gene is higher than a predetermined value. As the "predetermined value", typically, an average value of the expression amount of the gene, etc. may be mentioned. The average value of the expression level may be, for example, the average value of the expression level of the gene to be measured in a predetermined number (eg, 5, 10, 15 etc.) of pluripotent stem cell lines randomly selected.

The genes highly expressed in the pluripotent stem cell line of the present disclosure include mitochondria related genes, WNT signal transduction regulatory factor, TGFβ signal transduction regulatory factor, mesoderm related genes, cardiomyocyte related genes and undifferentiated cell related genes It can be mentioned. Specific examples of these genes include those described in the above <1>.

In a preferred embodiment of the present disclosure, the pluripotent stem cells are iPS cells, more preferably human iPS cells. It has been pointed out that iPS cells may differ in their cell line characteristics depending on the somatic cell origin, type of reprogramming factor and introduction method, etc. Therefore, the differentiation tropism is also cell line by cell line It is expected to be different. In addition, as a merit when iPS cells are used for regenerative medicine, it is possible to establish a cell line using autologous cells to be treated. Therefore, according to the present invention, it is also possible to screen iPS cells produced from autologous cells of interest from those having a high directivity toward cardiomyocytes and establish them as new cell lines.

When preparing differentiation-inducing cells from pluripotent stem cells, it is necessary that the cells be highly undifferentiated. This is also confirmed by the high expression of undifferentiated cell-related genes in the pluripotent stem cells of the present disclosure. That is, high undifferentiating ability in pluripotent stem cells is considered to facilitate differentiation into desired differentiation-inducing cells. According to the tests of the present inventors, it has been obtained that it is considered that the undifferentiated nature of pluripotent stem cells is not directly related to the cardiopatic differentiation per se.

<3> Embryoid body of the present disclosure The embryoid body obtained by culturing pluripotent stem cells highly directional to cardiomyocytes by the present inventors is also highly directional to cardiomyocytes highly It was found. Therefore, one aspect of the present disclosure relates to differentiation into cardiomyocytes characterized by high expression of at least one mesodermal gene and low expression of at least one endodermal gene and / or ectodermal gene. Includes highly oriented embryoid bodies.

The embryoid body of the present disclosure can be obtained by culturing pluripotent stem cells, preferably pluripotent stem cells described in <2> above, by methods known in the art. Specifically, for example, human iPS cells are cultured for 1 day in StemFit AK03 medium (Ajinomoto) containing Y27632 (Wako Pure Chemical Industries, Ltd.), then for 2 days in StemFit AK03 medium not containing Y27632, and then BMP4 is It can be obtained by culturing in a medium containing it.

The embryoid bodies of the present disclosure highly express at least one mesodermal gene. Specific examples of mesodermal genes include those described in the above <1>. The embryoid bodies of the present disclosure also have low expression levels of at least one endodermal gene and / or ectoderm gene. Here, "low expression level" means that the expression level of a certain gene is lower than a predetermined value, contrary to the above "high expression level". As the "predetermined value", typically, an average value of the expression amount of the gene, etc. may be mentioned. The average value of the expression levels may be, for example, the average value of the expression levels of the measurement target genes in a predetermined number (for example, 5, 10, 15 etc.) of embryoid bodies randomly extracted.

The ectoderm gene includes, but is not limited to, for example, SOX1, PAX6 or ZIC1. The endodermal genes include, but are not limited to, for example, AMN, SOX7, SOX17, HNF3 or ZIC1.

As described above, the embryoid body of the present disclosure is characterized in that the expression level of at least one mesodermal gene is high and the expression level of at least one endodermal gene and / or ectoderm gene is low, Thus, it has a high differentiation directivity to somatic cells of mesodermal origin, in particular cardiomyocytes. Such features are particularly prominent in embryoid bodies produced from the pluripotent stem cells of the present disclosure having high differentiation tropism for cardiomyocytes. Therefore, in a preferred embodiment, the embryoid body of the present disclosure is produced from the pluripotent stem cells of the present disclosure described in the above <2>.

<4> Differentiation Inducing Method of the Present Disclosure The present inventors have found genetic characteristics of pluripotent stem cells highly directed to cardiomyocyte differentiation, and differentiated cardiomyocytes using pluripotent stem cells having such characteristics. It was found that cardiomyocytes can be obtained with high efficiency by induction. Thus, the present disclosure, in one aspect, encompasses methods of inducing cardiomyocytes to differentiate from pluripotent stem cells with high efficiency.

The differentiation induction method of the present disclosure uses, in a preferred embodiment, the pluripotent stem cells or embryoid bodies of the present disclosure. In such an embodiment, the differentiation induction technique itself may use any technique known in the art. There are various known methods for inducing cardiomyocytes differentiation from pluripotent stem cells (for example, Burridge et al., Cell Stem Cell. 2012 Jan 6; 10 (1): 16-28). In any method, a mesodermal inducer (eg, activin A, BMP4, bFGF, VEGF, SCF, etc.), a cardiac specification factor (eg, VEGF, DKK1, a Wnt signal inhibitor (eg, IWR-1) , IWP-2, IWP-3, IWP-4 etc.), BMP signal inhibitors (eg NOGGIN etc.), TGFβ / activin / NODAL signal inhibitors (eg SB431542 etc.), retinoic acid signal inhibitors etc.) and cardiac differentiation factors (eg For example, enhancing induction efficiency by sequentially acting VEGF, bFGF, DKK1 etc.) It can be. In one embodiment, cardiomyocyte induction treatment from pluripotent stem cells is carried out by causing BMP4 to act on embryoid bodies formed by (1) combining BMP4 and bFGF and activin A, (2) VEGF and IWP-3, And (3) sequentially acting on the combination of VEGF and bFGF.

As a known method for obtaining cardiomyocytes from human iPS cells, for example, the following steps:
(1) maintaining and culturing human iPS cells in a culture solution containing no feeder cells (feeder free method),
(2) forming an embryoid body from the obtained iPS cells,
(3) culturing the obtained embryoid body in a culture solution containing activin A, bone morphogenetic protein (BMP) 4 and basic fibroblast growth factor (bFGF),
(4) culturing the obtained embryoid body in a culture solution containing a Wnt inhibitor, a BMP4 inhibitor and a TGFβ inhibitor, and (5) the obtained embryoid body in a culture solution containing VEGF and bFGF A method comprising the step of culturing in

In the step of (1), for example, as described in WO 2017038562, StemFit AK03 (Ajinomoto) can be used as a culture medium, and iPS cells can be cultured and adapted on iMatrix 511 (Nippi) to perform maintenance culture. In addition, as described in, for example, Nakagawa M., et al. A novel efficient feeder-free culture system for the derivation of human induced pluripotent stem cells. Sci Rep. 2014; 4: 3594, every 7 to 8 days, as described in iPS cells. Alternatively, passage may be performed as a single cell using TrypLE® Select (Thermo Fisher Scientific). After the steps (1) to (5), optionally, the step of purifying the obtained cardiomyocytes may be selectively performed. Purification of cardiomyocytes includes a method of reducing non-cardiomyocytes using a glucose free medium, a method of reducing undifferentiated cells using heat treatment as described in WO 2017/038562, and the like.

One aspect of the present disclosure may include the step of performing the indexing method of the present disclosure described in the above <1> before and / or after the step of the above (1). In addition, after the step (2), a step of measuring the gene expression amount of the obtained embryoid body may be included. The method further includes the steps of comparing the measured gene expression level with a reference value, and excluding embryoid bodies other than the embryoid bodies judged to be the embryoid bodies described in the above <3> as a result of the comparison. May be. As such a reference value, for example, those described as the "predetermined value" in the above <2> can be mentioned.

As described above, according to the present inventors, in pluripotent stem cells having cardiotropic differentiation tropism, a mitochondrial associated gene, a WNT signal transduction regulator, a TGF beta signal transduction regulator, a mesoderm associated gene, a cardiomyocyte associated The expression level of at least one gene selected from the group consisting of the gene and the undifferentiated cell-related gene was found to be significantly different as compared with pluripotent stem cells having no directivity toward cardiomyocytes. It was issued. Therefore, in each step of the above (1) to (5), cardiomyocyte differentiation can be induced by applying treatment to enhance or reduce the expression of these genes. Non-limiting examples of such treatment include, for example, addition of WNT signal inhibitor, TGFβ signal inhibitor, modulation of TGFβ signal or WNT signal, modulation of mitochondrial activity by addition of MitoBlock, etc.

Therefore, in one aspect of the present disclosure, pluripotent stem cells comprising enhancing or reducing the expression level of a differentiation-directed marker, or enhancing or reducing the action of a protein that is an expression product of a differentiation-directed marker gene. It also relates to a method for inducing differentiation.

Examples of methods for enhancing the expression level of the differentiation directed marker include inhibition of repressor of the differentiation directed marker gene, addition of an enhancer of the differentiation directed marker gene, and the like. Examples of methods for reducing the expression level of the differentiation directed marker include addition of repressor, introduction of antisense nucleic acid such as siRNA, and the like.

Examples of methods for enhancing the action of a protein that is an expression product of a differentiation-oriented marker gene include addition of the expression product protein to a medium, and the like. As a method of reducing the action of a protein which is an expression product of a differentiation-oriented marker gene, for example, addition to a medium such as an inhibitor for the protein or an inhibitory antibody can be mentioned.

For example, in the case of induction of differentiation into cardiomyocytes, expression products of differentiation-oriented marker expression that are significantly increased, such as PF4, CHCHD2, AMMECR1, API5, BCOR, BRWD1, CLEC4G, GLIPR1, HELB, KDM6A, LOC388796, NKTR, It is expected that the induction of differentiation into cardiomyocytes is promoted by culturing in a medium containing proteins such as POMZP3, ZP3, PRUNE2, RBMX, RC3H1, SKIL, SORBS2 and SRSF11. Therefore, the differentiation-inducing method of the present disclosure includes the expression of differentiation-oriented marker expression products significantly increased as described above, such as PF4, CHCHD2, AMMECR1, API5, BCOR, BRWD1, CLEC4G, GLIPR1, HELB, KDM6A, LOC388796. Also included is a method for culturing pluripotent stem cells, which comprises using a medium containing a protein such as NKTR, POMZP3, ZP3, PRUNE2, RBMX, RC3H1, SKIL, SORBS2 and SRSF11. Such a culture method is preferably used in the culture 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 differentiation induction method of the present disclosure is more than about 50%, more than about 60%, more than about 70%, more than about 75%, more than about 80%, More than about 85%, more than about 86%, more than about 87%, more than about 88%, more than about 89%, more than about 90%, more than about 91%, more than about 92%, more than about 93%, more than about 94%, It may be more than about 95%, more than about 96%, more than about 97%, more than about 98%, more than about 99%, and so on. In one aspect, the pluripotent stem cell-derived cardiomyocytes in the present disclosure is a cardiomyocyte population having a cardiomyocyte purity of greater than 90%. In another embodiment, the cardiomyocyte content is, for example, 50 to 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 survival 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 may be, for example, 0.01% to 1%.

<5> Medical composition of the present disclosure Another aspect of the present disclosure includes a medical composition containing differentiation-inducing cells, for example, cardiac muscle cells, which are induced by the method described in the above <4>.
In the present disclosure, “medical composition” means a composition used for medical purposes, and is not limited thereto, and for example, a pharmaceutical composition, a therapeutic composition, a composition for transplantation, etc. Besides compositions used for direct treatment of a subject, compositions used in drug development and the like, such as, for example, compositions for drug screening, are also included.

The composition containing cardiomyocytes induced by the differentiation induction method of the present disclosure is a composition having a high cardiomyocyte content and can be said to be very useful in medical treatment. For example, when a composition for transplantation is prepared using a composition containing cardiomyocytes induced by the differentiation induction method of the present disclosure, the content of cardiomyocytes is high, so the amount of cardiomyocytes contained is large, and An implant composition suitable for implant of the present invention can be prepared. Thus, in one aspect, the medical composition of the present disclosure is a composition for implantation. In such embodiments, the cardiomyocyte content is greater than about 50%, about 60%, about 70%, about 75%, about 80%, about 85%, about 86%, about 87%, or more. More than about 88%, more than about 89%, more than about 90%, more than about 91%, more than about 92%, more than about 93%, more than about 94%, more than about 95%, more than about 96%, more than about 97% Such as greater than about 98%, greater than about 99%, and the like. In one aspect, the pluripotent stem cell-derived cardiomyocytes in the present disclosure is a cardiomyocyte population having a cardiomyocyte purity of greater than 90%. In another embodiment, the cardiomyocyte content is, for example, 50 to 99%, preferably 50 to 70%, or 75 to 99%.

In recent years, cell transplantation has been considered useful for recovery of cardiac function in patients with severe heart failure, and clinical application / research with autologous skeletal myoblasts and iPS cell-derived cardiomyocytes has already been started. As an example, by using a temperature-responsive culture dish to which tissue engineering is applied, a three-dimensional cell culture applicable to the heart including cells derived from parts other than adult myocardium has been developed. . The medical composition of the present disclosure, in particular the composition for transplantation, can also be such a cell culture. Thus, in a preferred embodiment, the medical composition of the present disclosure is a sheet-like cell culture.

In the present disclosure, “sheet-like cell culture” refers to cells in which cells are linked to each other into a sheet. The cells may be linked to each other directly (including via cell components such as adhesion molecules) and / or via an intermediary substance. The mediator is not particularly limited as long as it is a substance capable of at least physically (mechanically) connecting cells to each other, and examples include extracellular matrix and the like. The mediator is preferably of cell origin, in particular of the cells constituting the cell culture. The cells are at least physically (mechanically) linked, but may be further functionally linked, for example, chemically or electrically. The sheet-like cell culture is composed of one cell layer (monolayer), but is composed of two or more cell layers (layered (multilayer) body, for example, two or three layers, It may be four layers, five layers, six layers, etc.).

The sheet-like cell culture preferably does not contain a scaffold (support). Scaffolds may be used in the art to attach cells on and / or within their surface and maintain the physical integrity of sheet cell cultures, such as polyvinylidene difluoride (eg, Membranes and the like made of PVDF) are known, but the sheet-like cell culture in the present disclosure may be capable of maintaining its physical integrity even without such scaffolds. In addition, the sheet-like cell culture preferably comprises only the substance derived from cells constituting the cell culture, and does not contain any other substance.

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

The cells forming the sheet-like cell culture may be xenogeneic cells or allogeneic cells. Here, "heterologous cell" means a cell derived from an organism of a species different from that of the recipient when a sheet-like cell culture is used for transplantation. For example, when the recipient is a human, cells derived from monkeys or pigs correspond to xenogeneic cells. Also, "allogeneic derived cells" mean cells derived from an organism of the same species as the recipient. For example, when the recipient is human, human cells correspond to allogeneic cells. Allogeneic cells include autologous cells (also referred to as autologous cells or autologous cells), ie cells derived from a recipient and allogeneic non-autologous cells (also referred to as allogeneic cells). Autologous cells are preferred in the present disclosure as transplantation does not result in rejection. However, it is also possible to use heterologous cells or allogeneic non-autologous cells. When xenogeneic cells or allogeneic non-autologous cells are used, immunosuppressive treatment may be required to suppress rejection. In the present specification, cells other than autologous cells, ie, xenogeneic cells and allogeneic nonautologous cells may be collectively referred to as nonautologous cells. In one aspect of the present disclosure, the cells are autologous cells or allogeneic cells. In one aspect of the present disclosure, the cells are autologous cells. In another aspect of the present disclosure, the cells are allogeneic cells.

The autologous or allogeneic pluripotent stem cells are not limited, for example, collected autologous or allogeneic cells (eg, skin cells (fibroblasts, keratinocytes, etc.) and blood cells (peripheral blood mononuclear cells, etc.), etc.) It can be obtained by introducing a gene such as OCT3 / 4, SOX2, KLF4 or C-MYC into autologous or allogeneic iPS cells. 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, etc.).

The medical composition and the sheet-like cell culture of the present disclosure contain cardiomyocytes in a high degree as described above, and therefore, use of the composition and the sheet-like cell culture act on cardiomyocytes. The effects of drugs can be effectively tested. Thus, in another aspect of the present disclosure, the medical composition of the present disclosure is a composition for drug screening. In particular, since the composition of the present disclosure can prepare cardiomyocytes derived from a specific subject, it becomes possible to screen an agent that effectively acts on the 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 an improvement in the quality of the medical composition. However, it is usually difficult to predict how much cardiomyocytes are contained unless differentiation is induced from pluripotent stem cells to finally 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 mesodermal genes, endodermal genes and / or ectoderm genes in embryoid bodies at the stage of culture from pluripotent stem cells to form embryoid bodies, such embryoid bodies are myocardium. It is possible to test whether the cells have differentiation tropism. Therefore, one aspect of the present disclosure relates to pluripotent stem cells comprising measuring expression levels of mesodermal genes, endodermal genes and / or ectoderm genes in embryoid bodies obtained by culturing pluripotent stem cells. The present invention also includes a method of controlling the quality of a medical composition containing differentiation-induced cardiomyocytes, and a method of producing a medical composition including such a method of quality control.

Specific examples of the mesodermal gene, endodermal gene and ectodermal gene in the present method include those described in the above <3>. Any method known in the art can be used to measure the expression level of these genes.
In one embodiment, the method of the present aspect further comprises comparing the measured gene expression amount with a reference value, and excluding the embryoid body judged to be the embryoid body as described in the above <3> as a result of the comparison. It may include excluding the embryoid bodies of The reference value typically includes an average value of the expression level of the gene, and the like. The average value of the expression levels may be, for example, the average value of the expression levels of the measurement target genes in a predetermined number (for example, 5, 10, 15 etc.) of embryoid bodies randomly extracted.

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

The method of producing the medical composition of the present disclosure includes the following steps:
(A) Differentiating and culturing pluripotent stem cells to form an embryoid body,
(B) Directing cardiomyocyte differentiation, including measuring the expression levels of mesodermal genes, endodermal genes and / or ectodermal genes in the obtained embryoid body, and comparing such measured values with reference values Selecting an embryo-like body of high sex,
(C) a step of inducing differentiation and culturing the selected embryoid body to obtain a cell population containing cardiomyocytes.

In step (A), embryoid bodies are formed from pluripotent stem cells. For embryoid body formation, methods known in the art can be used, and specifically, for example, the method described in the above <3> can be used. The pluripotent stem cells that can be used are preferably, but not limited to, allogeneic cells to be treated (eg, human) with the medical composition. When the medical composition is used for transplantation etc., pluripotent stem cells prepared from autologous cells such as autologous iPS cells are preferable. In addition, before the step (A), the pluripotent stem cells to be used may further be screened by the method of the present disclosure.

In the step (B), embryoid bodies highly directional to cardiomyocytes are selected. Examples of embryoid bodies highly directed to cardiomyocyte differentiation are as described in <3> above. In this step, an embryoid body having a particularly high expression level of mesodermal genes and a low expression level of ectodermal genes and / or endodermal genes is selected. Specific examples and reference values of ectoderm gene, endoderm gene, mesoderm gene may be those described in <3>.

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

After the step (C), it may further include the step of optionally modifying the medical composition. As such a step, for example, a sheeting step for forming a sheet-like cell culture, a step of cryopreserving a medical composition and the like can be mentioned.

The present disclosure will be described in more detail with reference to the following examples, which illustrate specific embodiments of the disclosure and the disclosure is not limited thereto.

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. Differentiation to cardiomyocytes Differentiation of highly directed iPS cell lines to cardiomyocyte differentiation In order to investigate the genetic characteristics of highly directed iPS cells, first try to identify highly directed iPS cell lines to cardiomyocytes The
(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 line immediately before induction of differentiation was extracted according to procoll using miRNeasy Mini Kit (QIAGEN). Using SuperScript ^TM VILO (Invitrogen) for RT-PCR, we were synthesized cDNA. Using PCR primers for SYBR Green and SYBR Green PCR master mixes (Applied Biosystems) or Taqman probe and Taqman Gene Expression Master Mix (Applied Biosystems) described in Table 4, PCR with ViiA 7 Real-Time PCR System (Applied Biosystems) Carried out. GAPDH was used as a housekeeping gene for analysis of gene expression. Gene expression analysis was performed using ViiA 7 Sysytem. In TaqMan Gene Expression Assays, temperature cycling conditions were as follows: hold at 95 ° C. for 20 seconds, cycle: 95 ° C. for 1 second, 60 ° C. for 20 seconds. SYBR Green temperature cycling conditions hold: 95 ° C for 20 seconds, cycle: 1 second at 95 ° C, 40 cycles of 20 seconds at 60 ° C, and 15 seconds at 95 ° C, 1 minute at 60 ° C, 15 seconds at 95 ° C I went there.

These iPS cell lines are referred to methods described in Matsuura, et al., Biochemical and Biophysical Research Communications 425 (2012) 321-327, Miki K. Cell Stem Cell (2015), WO 2014/185358 A1 and WO 2017/038562, etc. In addition, cardiomyocytes were induced to differentiate. Specifically, undifferentiated human iPS cells are treated on a feeder cell mitomycin C-treated MEF (ReproCell), which is a feeder cell, using 5 ng / mL bFGF added to Primate ES medium (ReproCell) as the undifferentiated maintenance medium. Cultures were performed and passaged once every 3-4 days. Differentiation induction dissociates human iPS cells with Dissociation solution (ReproCell) and Accumax (Innovation Cell Technologies), StemPro 34 (Life Technologies) supplemented with 0.5 ng / mL BMP-4 and 10 μM Y27632 (Rock inhibitor) The suspension was suspended with and cultured for 1 day with EZSPHERE (IWAKI) to form a mass. The obtained embryoid body is cultured in a culture solution containing activin A, bone morphogenetic protein (BMP) 4 and basic fibroblast growth factor (bFGF), and further in a culture solution 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 induction directivity The troponin positive rate and beating rate in cardiomyocyte-containing embryoid bodies induced to differentiate from each iPS cell line, cardiomyocyte related gene expression amount is measured, and the myocardium of each iPS cell line The differentiation orientation to cells was ranked.
The troponin positive rate was determined by dispersing the embryoid bodies using TrypLE Select, and then dispersing the dispersed cells using BD Cytofix / Cytoperm® Fixation / Permeabilization Solution Kit (BD Bioscience) and permeabilizing the cells, then anti-human. After a troponin antibody (Thermo Fisher Scientific) and a labeled secondary antibody (Thermo Fisher Scientific) were sequentially reacted, the measurement was performed using a flow cytometer.

The beating rate is obtained by moving picture imaging of a germinal body after induction of differentiation from each iPS cell line to a cardiomyocyte by cell motion imaging (Sony) and pulsing among those observed. It counted and calculated.

The results are shown in FIG. 2 and FIG. Particularly high troponin positive rates and beat rates were calculated in the three cell lines 201B7, 253G1 and 409B2. Therefore, these three cell lines were identified as cell lines highly directed to cardiomyocytes.

(3) Residual rate of undifferentiated cells The residual rate of undifferentiated cells in cardiomyocyte cultures prepared from the three cell lines identified as cell lines highly directed to cardiomyocyte differentiation in (2) above. As a percentage of the number of cells expressing Lin28, which is an undifferentiated cell marker, it was measured by quantitative PCR.
The results are shown in FIG. In the three cell lines identified as highly directed to cardiomyocyte differentiation, the residual rate of undifferentiated cells tended to be significantly lower than in other cell lines.

Example 2. Gene expression in embryoid bodies Next, SOX2, PAX6, ZIC1, BRACHYURY, FLK1, PDGFR-a, GOOSECOID, HNF3, SOX17, SOX7, AMN in each embryoid body at the 4th day of culture in Example 1 above The expression of various genes was measured.

The results are shown in FIG. The three cell lines 201B7, 253G1 and 409B2, which are cell lines highly directed to cardiomyocytes, express a large amount of mesodermal genes, and the expression levels of endodermal genes and ectodermal genes were low. Pearson's correlation coefficient between the expression level and the troponin T positive rate after differentiation induction of each cell line was calculated for each gene, and the result is as shown in the table below.

Example 3. Principal component analysis in each cell line Total RNA was extracted from each iPS cell line using RNeasy Kit (QIAGEN). Quality assessment of each RNA was confirmed by calculating the 28S to 18S rRNA ratio 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 according to the product protocol using GeneChip 3 'IVT Express kit (Affymetrix).

First, double-stranded cDNA synthesis including T7 promoter sequence was performed from total RNA, and biotin labeled aRNA using cDNA as a template was synthesized by in vitro reverse transcription reaction. Next, calcium random degradation using a hammerhead reaction was performed to generate an ̃100-120 nt aRNA fragment. Biotin-labeled aRNA prepared in Genechip Array Human Genome U133 Plus 2.0 Array (Affymetrix) was hybridized using GeneChip Hybridization Oven (Affymetrix). After hybridization, washing and phycoerythrin staining were performed using GeneChip Wash and Stain Kit (Affymetrix) and GeneChip Fluidics Station 450 (Affymetrix). Thereafter, a 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 MAS5 algorithm and MSK file (Affymetrix).

Differentiation to cardiomyocytes Using 201B7, 253G1 and 409B2 as highly directional cell lines, and using RIKEN-1A, RIKEN-2A, and RIKEN-12A as low directional cell lines to cardiomyocytes We analyzed genes that are characteristically expressed in highly directed cell lines.

(1) Cardiac muscle related gene expression comparison Each cell line was differentiated to cardiac muscle cells in the same manner as in Example 1, and the expression levels of cardiac muscle related genes were compared. The results are shown in FIG. 6 and FIG.
Differentiation of cardiomyocytes into 3 cell lines highly induced expression of high cardiac muscle related genes was confirmed in all cell cultures induced to differentiate. In addition, the troponin positive rate was also significantly higher in the three cell lines highly directed to cardiomyocytes.

(2) Microarray analysis Next, differences in gene expression between 3 cell lines with high directivity to cardiomyocytes and low 3 cell lines were analyzed using microarray analysis using Affymetrix GeneChip (R) Arrays. The
Among the 3300 genes that can be analyzed with Affymetrix GeneChip (R) Arrays, the expression level was more than twice as high in the highly directed group as compared with the highly directed group and the low directed group. Eighty four genes were identified and analyzed to determine which signal transduction pathway the gene cluster is. The results are shown in the table below.

(3) Selection of Marker Candidate Genes Among the 84 genes described above, genes showing a significant difference in expression amount were identified as biomarker candidate genes. As a result, mitochondrial related genes CHCHD2 and SFXN3, WNT signal regulator KDM6A, TGF-β signal related factor SKIL, etc. were identified as biomarker gene candidates. In addition, on the other hand, miRNA-139 and miRNA-204 were identified as the genes that showed significantly high expression in cell lines with low directivity for cardiomyocytes.

Example 4: miRNA expression analysis (1) miRNA microarray From each iPS cell line, total RNA was extracted 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. Biotin-labeled RNA prepared in a miRNA 3.0 array (Affymetrix) was hybridized using GeneChip Hybridization Oven (Affymetrix). After hybridization, washing and phycoerythrin staining were performed using GeneChip Fluidics Station 450 (Affymetrix). Thereafter, a 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).
Differentiation to cardiomyocytes Using 201B7, 253G1 and 409B2 as highly directional cell lines, and using RIKEN-1A, RIKEN-2A, and RIKEN-12A as low directional cell lines to cardiomyocytes We analyzed genes that are characteristically expressed in highly directed cell lines.
The expression of miRNA in various iPS cell lines used in Example 1 was analyzed using a miRNA microarray.
Of the 534 miRNAs that can be analyzed, five miRNAs (ACA24 that showed half or less of the expression level in the high differentiation-oriented group compared with the high differentiation-oriented group and the low differentiation-oriented group , Hsa-miR-629-star, mmi-miR-204, ACA61 and hsa-miR-139-5p) were identified. The results are shown in FIG.
We analyzed which signal transduction pathway the identified miRNA was, and referred to the results analyzed in Example 3 (3) above, and narrowed down the strongly related genes. The results are shown in FIG. We found PF4 as a gene whose expression level is significantly higher in iPS cell lines with strong differentiation-directed to cardiomyocytes, and TMEM64 as a gene whose expression level is significantly lower.

(2) Confirmation of involvement in differentiation tropism to cardiomyocytes In order to confirm the effectiveness of the above gene as a differentiation tropism marker gene, a medium to which DMSO, IWR-1 and 2, CHIR 99021 or MitoBlock 6 were respectively added was used The 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 obtained cell population were measured.
The results are shown in FIG. In particular, when the medium to which CHIR 99021 was added was used, a significant reduction in the cTnT positive rate and the expression level of PF4 was confirmed. In addition, cTnT was significantly reduced also in the culture medium to which MitoBlock-6 was added. In this case, a decreasing tendency was confirmed in PF4 and an increasing tendency was confirmed in TMEM64. Therefore, it was suggested that PF4 may be positively correlated in induction of cardiomyocyte differentiation, and TMEM64 may be negatively correlated.

Claims (25)

A method of determining a differentiation tropism marker for evaluating differentiation tropism of pluripotent stem cells to a specific differentiation induction cell, comprising:
(1) measuring gene expression levels in a plurality of pluripotent stem cell lines;
(2) measuring the expression level of miRNA in the plurality of pluripotent stem cell lines;
(3) extracting a gene having a significant difference in expression amount between the highly directed pluripotent stem cell line and the low pluripotent stem cell line directed to the specific differentiation-inducing cell;
(4) extracting a miRNA having a significantly different expression level between the highly directed pluripotent stem cell line and the low pluripotent stem cell line directed to the specific differentiation-inducing cell; and (5) Selecting a gene involved with the miRNA extracted in (4) from the genes extracted in (3);
Said method. A method for indexing the differentiation directivity of pluripotent stem cell lines, comprising
(A) measuring the expression level of at least one differentiation-oriented marker gene in the pluripotent stem cells of interest (b) comparing the expression level of the gene measured in (a) with a reference . The differentiation directed marker gene is a gene selected from the group consisting of a WNT signaling regulator, a mitochondrial related gene, a TGFβ signaling regulator, a mesoderm related gene, a cardiomyocyte related gene and an undifferentiated cell related gene. The method according to Item 2. 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, WNT5A, The method according to claim 2 or 3, which is at least one gene selected from the group consisting of WNT7A, WNT7B, WNT8B, WNT10B, WNT11, WNT2B, WNT9A, WNT9B, LRP5 and LRP6. The mitochondrial related gene is at least 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 according to any one of claims 2 to 4, which is a single gene. TGFβ signaling modulators include SKIL, THBS1, CD3, TLR2, SMAD1, SMAD2, SMAD3, SMAD4, SMAD5, SMAD6, SMAD7, SMAD9, TGFBR1, TGFK2, MAPK1, MAPK3, ROCK1, BMP2, BMP4, BMP5, BMP6, BMP7 The method according to any one of claims 2 to 5, which is at least one gene selected from the group consisting of BMP8B, BMPR1A and BMPR1B. The mesodermal genes are FLK1, BRACHYURY, GOOSECOID, PDGFR-a, IGF2, CD34, CLL1, HHEX, INHBA, LEF1, SRF, T, TWIST1, ADIPOQ, MME, KIT, ITGAL, Tbx1, Gat1, Klf1, Csf1r, CD45 The method according to any one of claims 2 to 6, which is at least one gene selected from the group consisting of: and Ter119. Cardiomyocyte related genes selected from the group consisting of TNT2, ML2, GATA4, MYH6, MYH7, Nkx2, SCN5A, RYR2, PPARGC1, MYL2, HCN4, CACNalC, ATP2A2, Actc1, Cx43, TEF-1 and Tbx-5 The method according to any one of claims 2 to 7, which is at least one gene selected. The 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, Esg1, Rex1 , Zfp296, UTF1, GDF3, Sall4, Tbx3, Tcf3, DNMT3L, DNMT3B, Tra-1-81, miRNA of miR-290 cluster, and at least one gene selected from the group consisting of miRNA of miR-302 cluster A method according to any one of claims 2-8. The differentiation-oriented marker gene is composed of PF4, CHCHD2, AMMECR1, API5, BCOR, BRWD1, CLEC4G, GLIPR1, HELB, KDM6A, LOC388796, NKTR, POMZP3, ZP3, PRUNE2, RBMX, RC3H1, SKIL, SORBS2 and SRSF11 The method according to claim 2, which is at least one gene selected. 11. The method according to claim 10, wherein the pluripotent stem cell of interest is judged to be a cell line with high directivity for cardiomyocytes when the measured expression level is significantly higher than the standard. 3. The method according to claim 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 according to claim 12, wherein the pluripotent stem cell of interest is judged to be a cell line highly directed to cardiomyocyte differentiation if the expression level measured is significantly lower than the standard. 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, SORBS2 and SRSF11 Culturing in a medium containing at least one protein selected from the group consisting of 15. The method of claim 14, wherein the at least one protein is PF4. The method according to claim 14 or 15, wherein the culture is a culture for forming embryoid bodies from pluripotent stem cells. 17. The method of claim 16, wherein the embryoid body is a mesodermal embryoid body. A composition for medical use, comprising pluripotent stem cell-derived differentiation-inducing cells cultured by the method according to any one of claims 14 to 17. The medical composition according to claim 18, which is a composition for drug screening. The medical composition according to claim 18, which is a composition for transplantation. 21. The medical composition according to claim 19 or 20, which is a sheet-like cell culture. A method for quality control of a medical composition comprising cardiomyocytes induced to differentiate from pluripotent stem cells, which comprises a mesodermal gene, an endodermal gene and / or an exodermoid gene in an embryoid body obtained by culturing the pluripotent stem cells. The above method comprising measuring the expression level of a germ gene. The mesodermal genes are FLK1, BRACHYURY, GOOSECOID, PDGFR-a, IGF2, CD34, CLL1, HHEX, INHBA, LEF1, SRF, T, TWIST1, ADIPOQ, MME, KIT, ITGAL, Tbx1, Gat1, Klf1, Csf1r, CD45 The method according to claim 22, wherein the method is selected from the group consisting of at least one gene selected from the group consisting of and Ter119. The method according to claim 22 or 23, wherein the endodermal 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. The method according to any one of claims 22 to 24, further comprising obtaining morphological characteristics of pluripotent stem cells and embryoid bodies.

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