WO2013111515A1 - Method for inducing/activating cardiac stem/progenitor cell using specific factor - Google Patents

Abstract

The present invention addresses the problem of discovering a regulation factor which can regulate all of cardiac progenitor cells and cardiac stem cells and can direct the differentiation/induction of a cardiac progenitor cell from an embryonic stem cell. The present invention also addresses the problem of developing a method which can provide a cardiac progenitor cell or a cardiac stem cell with high efficiency using the regulation factor. The present inventors have made the studies on screening of candidate factors which are expressed characteristically in a cardiac cell. From the results of the search obtained on the basis of the co-expression with a marker characteristic of a cardiac muscle cell or the like, 15 candidate factors are picked up finally and it is found that factors belonging to specific 3 families among the 15 candidate factors are factors common to the differentiation/induction into a cardiac progenitor cell or a cardiac stem cell.

Description

Induction / activation method of cardiac stem / progenitor cells by specific factors

The present invention relates to providing a control factor that can commonly control cardiac progenitor cells and cardiac stem cells isolated and reported so far, and that can be destined from embryonic stem cells to cardiac progenitor cells. The present invention also relates to providing a method for inducing myocardial progenitor cells / cardiac stem cells with high efficiency using such a regulator.

Congenital heart disease in newborns and heart disease, such as heart failure in adults, are serious diseases that occur at a relatively high frequency, resulting in loss of functional parts due to cell damage or necrosis, or morphological It is known to have serious symptoms of loss. In particular, heart failure is the second largest disease in adult mortality, and heart / myocardial regeneration research aiming at elucidating the cause and restoring function is regarded as an important issue.

The treatment for such a heart disease is to make up the damaged area and the lost part with living cells, but since the heart is a tissue with low regeneration ability, it is difficult to recover by its own power. Therefore, many drugs such as β-blockers, vasodilator ACE inhibitors, Ca antagonists, and cardiotonic agents administered during congestive heart failure and atrioventricular block have been developed and used. However, in the prognosis of a patient suffering from a ventricular cell injury / necrosis region due to congenital heart disease or myocardial infarction, there is currently no way to cope with it by performing a surgical operation or refraining from burdensome activities.

In order to treat such patients, the method of transplanting differentiated cells and stem cells is considered to be one of the effective tools, and until now, skeletal myoblast transplantation, bone marrow cell transplantation, embryonic stem cells Techniques such as transplantation have been tried. However, since there are problems such as low adhesion, low differentiation efficiency, risk of other cell differentiation, etc., no significant effect has been reported. It is also known that embryonic stem cells transplanted into the heart form teratomas, and it is necessary to clear such a risk of canceration for practical use. Further, iPS cells, which are expected to have a future, are considered to have the same risk as embryonic stem cells. Therefore, it is a big wall for clinical application that the risk which such a stem cell must be eliminated must be eliminated.

¡The myocardial sheet method has been devised as one of the methods to eliminate such risks. Although this method is also a promising method, the risk of arrhythmia with the patient's original cell group, functional problems with the sheet layer structure, and problems due to the single myocardium remain. . In other words, transplantation of cardiomyocytes alone is an effective means that transient improvement of symptoms is observed in the acute phase, but for long-term improvement, the heart constituent cells at the disease site are efficiently induced and ordered. You must think about how to place it. In 2011, a method for directly inducing cardiomyocytes from fibroblasts was devised, but the efficiency of transdifferentiation is low, it is difficult to differentiate into mature myocardium, and the time required for transdifferentiation is very long. The issue has not been cleared.

In recent years, the presence of cardiac progenitor cells (Islet1 ⁺ , Wt1 · Tbx18 ⁺ ) in mouse embryos and humans, and tissue stem cells (Sca-1 ⁺ , c− that can differentiate into cardiomyocytes and vascular cells in the adult heart. The presence of cardiac progenitor cells and cardiac stem cells that work for cardiac regeneration, such as the presence of Kit ⁺ ), was revealed. However, such cardiac progenitor cells / heart stem cells have differentiation limitations when differentiated from embryonic stem cells or artificial stem cells, and cannot efficiently induce cardiomyocytes. On the other hand, if cardiac progenitor cells and cardiac stem cells can be efficiently proliferated and transplanted, many patients with myocardial infarction and patients with congenital heart disease can be saved.

Furthermore, understanding the differences between stem cells contained in embryonic stem cells, bone marrow cells and umbilical cord blood and cardiac progenitor cells and cardiac stem cells is considered to be very useful for clinical application of somatic stem cells. For that purpose, the cardiac progenitor cells and cardiac stem cells that have been isolated and reported so far can be controlled in common, and control factors that fate the differentiation and induction of embryonic stem cells into cardiac progenitor cells and cardiac stem cells It is important to find out.

However, so far, no absolute cell surface antigen, inducer / maintenance factor as in the case of hematopoietic stem cells has been established in cardiac stem cell research.

The present invention can control a group of cardiac progenitor cells and cardiac stem cells in common, and is destined to differentiate and induce undifferentiated cells such as embryonic stem cells and induced pluripotent stem cells into cardiac progenitor cells and cardiac stem cells. The challenge is to find a control factor. Another object of the present invention is to develop a method capable of providing cardiac progenitor cells and cardiac stem cells with high efficiency from undifferentiated cells using such control factors.

The inventors of the present invention have conducted screening studies of candidate factors that are characteristically expressed in heart cells. Finally, 15 candidate factors are picked up from the results of searches such as co-expression with markers characteristic of cardiomyocytes, and 2 factors belonging to family X in particular are cardiac progenitor cells and cardiac stem cells. We found that it is a common factor for fateing differentiation and induction into

Specifically, protein C (SEQ ID NO: 2 amino acid sequence, SEQ ID NO: 4 amino acid sequence, or similar amino acid sequence) or protein D (SEQ ID NO: 6 amino acid sequence, SEQ ID When a protein belonging to family X selected from proteins having the amino acid sequence of NO: 8 or an amino acid sequence similar thereto is expressed in an undifferentiated cell, the undifferentiated cell may become a cardiac progenitor cell in the future.・ Directed differentiation and induction into cardiac stem cells, and found that it will differentiate into cardiac cells with high probability in the future.

Based on such findings, in one aspect, the present invention is a protein C (SEQ ID NO :) having a function of directing differentiation / induction into cardiac progenitor cells / heart stem cells when expressed in undifferentiated cells. 2 amino acid sequence, SEQ ID NO: 4 amino acid sequence, or similar amino acid sequence), or protein D (SEQ ID NO: 6 amino acid sequence, SEQ ID NO: 8 amino acid sequence, or similar to these) It was shown that an agent for differentiation / induction into cardiac progenitor cells and cardiac stem cells, which is composed of a protein belonging to family X selected from proteins having an amino acid sequence) can be provided.

In addition to the two protein factors belonging to the family X described above, the inventors of the present invention can further select two protein factors belonging to the family Y and / or two members belonging to the family Z from the 15 candidate factors described above. It was also found that protein factors are common factors for fateing differentiation / induction from undifferentiated cells into cardiac progenitor cells and cardiac stem cells.

In particular,
Protein H (SEQ ID NO: 10 amino acid sequence, SEQ ID NO: 12 amino acid sequence or amino acid sequence similar thereto) or Protein I (SEQ ID NO: 14 amino acid sequence, SEQ ID NO: 16 amino acids) A protein belonging to family Y selected from proteins having the sequence, or an amino acid sequence similar thereto, and / or protein J (SEQ ID NO: 18 amino acid sequence, SEQ ID NO: 20 amino acid sequence, or these A protein belonging to the family Z selected from the proteins having the amino acid sequence similar to the above) or protein K (SEQ ID NO: 22 amino acid sequence, SEQ ID NO: 24 amino acid sequence, or an amino acid sequence similar thereto),
Is expressed in undifferentiated cells, the undifferentiated cells will be directed to differentiation and induction into cardiac progenitor cells and cardiac stem cells in the future, and will be differentiated into cardiac cells with high probability in the future. I found it.

In another embodiment of the present invention, a protein belonging to family X in an undifferentiated cell (a protein having an amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, or SEQ ID NO: 8) Protein belonging to family Y (SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, or protein having SEQ ID NO: 16 amino acid sequence), or protein belonging to family Z (SEQ ID NO: 18, a progenitor cell / stem stem cell characterized by selecting a cell that expresses SEQ ID NO: 20, SEQ ID NO: 22, or a protein having an amino acid sequence of SEQ ID NO: 24) Can provide a way And it is shown also.

Cardiac progenitor cells and cardiac stem cells are in a phase where differentiation and induction have advanced compared to pluripotent stem cells such as embryonic stem cells and induced pluripotent stem cells. It has the feature of differentiating into cells. In adults, it is also possible to differentiate into heart cells.

FIG. 1 is a diagram comparing the expression of genes A to E with the expression of Islet1 gene and Mlc2a gene among the 15 genes found. FIG. 2 is a diagram showing that gene C and gene D generate a unimodal peak later than the expression peak of the mesoderm marker factor and earlier than the expression peak of the myocardial marker. FIG. 3 is a diagram showing the results of phenotypic observation in embryos of double homo knockout mice of gene C and gene D. FIG. 4 is a diagram showing that Islet1-positive cells were also lost in the gene C / gene D double knockout mice. FIG. 5 is a diagram showing a comparison of expression patterns between gene C and Islet1 and Nkx2-5, which are representative of embryonic cardiac progenitor cell markers. FIG. 6 is a diagram showing the effect on the expression of Islet1 when siRNA for gene C, siRNA for gene H, siRNA for gene I, siRNA for gene J, and siRNA for gene K are used alone or in combination. is there. FIG. 7 shows the expression of genes that should be expressed in various differentiation phases in cells that differentiate from the mesoderm to the heart using three genes, gene C / gene I / gene K, using siRNA. It is the figure which showed how it changes when it inhibits. FIG. 8 shows the result of developing GFP ⁺ cells with cKit and PDGFRa, which are cell surface antigens specific to embryonic cardiac progenitor cells. FIG. 9 is a diagram showing the results of examining whether or not cardiomyocytes can be induced using an in vitro embryonic stem cell differentiation system. FIG. 10 is a diagram showing potential changes collected from pulsatile cells obtained after expressing gene C. FIG. FIG. 11 is a diagram showing that cells functioning as pacemaker cells also exist among pulsating cells. FIG. 12 is a diagram showing that cells once expressing gene C / gene D have differentiated throughout the ventricle. FIG. 13 is a diagram showing that cells once expressing gene C / gene D have differentiated throughout the ventricle. FIG. 14 is a diagram suggesting that the expression site of gene C and the expression site of Islet1 overlap, and that expression of Islet1 occurs after expression of gene C. FIG. 15 is a diagram showing that typical cardiac muscle markers such as PDGFRa and cKit are also expressed in cells in which gene C is expressed. FIG. 16 is a diagram showing that differentiation into myocardium proceeds in inverse proportion to the relative expression level of gene C expressed in cells. FIG. 17 shows that the area of cardiac progenitor cells in the developing heart extends over a wider area than previously known. FIG. 18 shows that gene C is expressed in cardiac endothelial cells. FIG. 19 shows gene C, gene I and gene K expressed exogenously in the embryo. The ectopic expression sites of these genes and the ectopic nature of endogenous myocardial markers Islet1 and Baf6oc. It is a figure which shows that the expression site | part of this corresponded completely.

A number of surface markers have been investigated as markers for cardiomyocytes. As such cardiomyocyte markers, Islet1, Nkx2-5, α-heart actin, Brachyury T (BryT), Flk1, Sca-1, c-kit, PDGFRa, Mesp1, Gsc and the like are known.

Among them, after selecting Flk1 + cells based on the fact that the heart cells are mesoderm-derived cells, cells having properties of Islet1 +, Nkx2-5 weak, troponin (cTnt)-, and Tbx5- Furthermore, we aimed to isolate myocardial progenitor cells and stem cells by selection.

In the cells thus obtained, the aim is to find a regulatory factor having an important function for differentiation / induction from undifferentiated cells into cardiomyocytes among marker molecules other than known cardiomyocyte markers. As a result of microarray analysis of strongly expressed genes, we succeeded in isolating 15 interesting genes.

Confirming the expression pattern of these 15 genes in more detail, at the same time or slightly later than the expression peak of known mesodermal markers (eg, BryT, Mesp1, Gsc, etc.) in early embryos, and thereafter Under the condition that the expression peak is reached before the expression peak such as a cardiac muscle cell marker (for example, Islet1, Tbx5, Nkx2-5) that reaches the peak, a gene whose gene expression is remarkably increased is selected. As molecules satisfying the conditions, two types of genes, gene C and gene D, have been identified.

When these genes C and D were analyzed, the gene C is a protein having an amino acid sequence of SEQ ID NO: 2 or an amino acid sequence of SEQ ID NO: 4, and this protein is a nucleotide sequence of SEQ ID NO: 1. Or it became clear that it was encoded by the nucleotide sequence of SEQ ID NO: 3. Further, gene D is a protein having an amino acid sequence of SEQ ID NO: 6 or an amino acid sequence of SEQ ID NO: 8, and this protein has a nucleotide sequence of SEQ ID NO: 5 or a nucleotide sequence of SEQ ID NO: 7. It became clear that it was coded. Gene C and gene D have very similar sequences to each other, and it has been revealed that they form family X.

In order to investigate how genes encoding proteins belonging to family X are involved in heart development during embryogenesis, knockout animals lacking gene C or gene D were created. When embryogenesis of these animals was performed, it was revealed that the development of the heart was significantly suppressed compared to the wild type. Such characteristic suppression of cardiac development showed a very similar form to that of cardiac development that occurred in the knockout animal of Islet1, a cardiomyocyte marker.

From these results, it was shown that gene C and gene D encoding proteins belonging to family X transiently increased in the early stage of embryogenesis and played an important function in the development of the heart. .

From these findings, in one aspect, the present invention is an amino acid of protein C (SEQ ID NO: 2) having a function of directing differentiation / induction into cardiac progenitor cells / heart stem cells when expressed in undifferentiated cells. Sequence, SEQ ID NO: 4 amino acid sequence, or a protein having an amino acid sequence similar to these), or protein D (SEQ ID NO: 6 amino acid sequence, SEQ ID NO: 8 amino acid sequence, or these It was shown that a differentiation / inducing agent for cardiac progenitor cells and cardiac stem cells composed of a protein having a similar amino acid sequence can be provided.

Among these, it is particularly preferable to use protein C (SEQ ID NO: 4) which is an amino acid sequence derived from human and gene C (SEQ ID NO: 3) encoding the same.

The inventor of the present invention investigated whether a substance capable of further promoting differentiation / induction from undifferentiated cells into cardiac progenitor cells / heart stem cells by combining with proteins belonging to family X was examined. Specifically, from the 15 genes described above, four types of genes, gene H and gene I encoding proteins belonging to family Y, and gene J and gene K encoding proteins belonging to family Z are selected. Applying knockdown using short double stranded interfering RNA (siRNA) in various combinations, it is exerted on markers of myocardial induction such as expression of Islet1 when expression is inhibited during embryogenesis The effect was investigated.

As a result, even when each of gene H and gene I encoding proteins belonging to family Y and gene J and gene K encoding proteins belonging to family Z are independently inhibited, Islet1 which is a cardiomyocyte marker Although it was shown to inhibit the expression, it was shown that when the protein belonging to family X, the protein belonging to family Y, and the protein belonging to family Z were combined, the inhibitory action on the expression of Islet1 was greatly increased. . Therefore, not only the protein belonging to family X described above, but also the protein belonging to family Y and the protein belonging to family Z are also expressed in differentiated cells, so that differentiation / induction into cardiac progenitor cells / heart stem cells is achieved. The possibility of having the function of directing was shown.

Here, proteins belonging to Family Y include protein H (SEQ ID NO: 10 amino acid sequence, SEQ ID NO: 12 amino acid sequence, or similar amino acid sequence) or protein I (SEQ ID NO: 14 amino acid sequence). Amino acid sequence, SEQ ID NO: 16 amino acid sequence, or amino acid sequence similar to these), among them, protein I (SEQ ID NO: 16), which is an amino acid sequence derived from a human, and the code thereof It is preferable to use Gene I (SEQ ID NO: 15).

Proteins belonging to Family Z include protein J (SEQ ID NO: 18 amino acid sequence, SEQ ID NO: 20 amino acid sequence, or similar amino acid sequence) or protein K (SEQ ID NO: 22 amino acid sequence). Sequence, SEQ ID NO: 24 amino acid sequence, or an amino acid sequence similar thereto), among them, protein K (SEQ ID NO: 24), which is an amino acid sequence derived from a human, and the like. It is preferable to use gene K (SEQ ID NO: 23).

When specifying the protein of the present invention, mouse protein and human protein are disclosed for all proteins. For example, with regard to protein C, the amino acid sequence of SEQ ID NO: 2 is a mouse-derived sequence. The amino acid sequence of SEQ ID NO: 4 indicates a human-derived sequence. When the term “amino acid sequence similar to these” is used, when using an amino acid sequence analysis tool generally used by those skilled in the art (for example, NCBI BLAST), the amino acid sequence of SEQ ID NO: 2 and SEQ ID NO : Means a protein derived from all mammals other than mice and humans, which is judged to be homologous to both amino acid sequences of 4 and specifically, SEQ ID NO: 2 amino acid sequence or SEQ Compared with the amino acid sequence of ID NO: 4, a protein having an amino acid sequence in which one or several amino acids are deleted, substituted or added, or a protein having 80% amino acid sequence identity, and undifferentiated When expressed in cells, it has the effect of promoting differentiation and induction into cardiac progenitor cells and cardiac stem cells It means that the protein.

When specifying the genes of the present invention, mouse genes and human genes are disclosed for all genes. For example, with regard to gene C, the nucleotide sequence of SEQ ID NO: 1 is a mouse-derived sequence. The nucleotide sequence of SEQ ID NO: 3 is a human-derived sequence. When the term “nucleotide sequence similar to these” is used, the nucleotide sequence of SEQ ID NO: 1 and SEQ when using nucleotide sequence analysis tools generally used by those skilled in the art (for example, NCBI BLAST and GENETX). This means a nucleotide sequence derived from all mammals other than mice and humans, which is judged to be homologous to both the nucleotide sequence of ID NO: 3. Specifically, the nucleotide of SEQ ID NO: 1 When hybridizing under stringent conditions with a polynucleotide consisting of a nucleotide sequence complementary to a polynucleotide consisting of the sequence or SEQ ID NO: 3 nucleotide sequence and expressed in undifferentiated cells, Differentiation and induction into cardiac stem cells A polynucleotide that encodes a protein having a promoting action; or at least 80% identity with a nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 3 and expressed in an undifferentiated cell And a polynucleotide encoding a protein having an action of promoting differentiation / induction into cardiac progenitor cells and cardiac stem cells.

The stringent conditions used in the present specification can be easily determined by those skilled in the art having general techniques based on, for example, the length of DNA. The basic conditions are Sambrook et al. , Molecular Cloning: A Laboratory Manual, 3rd edition, Cold Spring Harbor Laboratory Press, (2001). For example, moderately stringent conditions for nitrocellulose filters are 5 × SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0) pre-wash solution, about 40 ° C. to 60 ° C., preferably about Other similar hybridization solutions, such as Stark's solution, in 1 × SSC to 6 × SSC, preferably 2 × SSC (or about 50% formamide at about 42 ° C. at 50 ° C. ) And about 60 ° C., 0.5 × SSC, 0.1% SDS wash conditions. For example, when the hybridization solution contains about 50% formamide, the hybridization temperature is about 15 ° C. to 20 ° C. lower. Highly stringent conditions can also be readily determined by one skilled in the art based on, for example, the length of the DNA. In general, highly stringent conditions include hybridization at higher temperatures and / or lower salt concentrations and / or washing conditions than the above moderately stringent conditions, such as about 60 ° C. to Hybridization conditions of 0.1 × SSC to 0.2 × SSC at 65 ° C. and / or wash conditions of about 65 ° C. to 68 ° C., 0.2 × SSC, 0.1% SDS. One skilled in the art will recognize that the temperature and wash solution salt concentration may be adjusted as necessary according to factors such as the length of the probe.

In the present invention, an undifferentiated cell includes not only an early embryo cell in a living body but also an undifferentiated stem cell in an in vitro system (for example, a pluripotent stem cell such as an embryonic stem cell or an induced pluripotent stem cell). For example, in the case of an early embryo, the phenomenon during embryogenesis is the time when genetic information is accurately replicated and at the same time the state of the cell is rapidly changed from an undifferentiated state to a state in which differentiation is induced, For example, in the case of mice and rats, it means 2 to 5 days after fertilization. Moreover, the in vitro undifferentiated cells refer to, for example, cells that are in the period of 3 to 5 days after the start of myocardial induction treatment on embryonic stem cells.

Gene C and gene D belonging to family X of the present invention are expressed at almost the same timing as Brachyury T (BryT), Mesp1, and Gsc, which are known as mesodermal cell markers. On the other hand, it is expressed early compared to markers such as Islet1, Nkx2-5, and α-heart actin known as cardiomyocyte markers.

On the other hand, the gene H and gene I belonging to the family Y of the present invention are strongly expressed in the early fetal period and disappear after the myocardial differentiation period.

Moreover, the genes J and K belonging to the family Z of the present invention have an expression pattern very similar to that of the X gene, and the expression disappears with myocardial differentiation.

In another embodiment of the present invention, in undifferentiated cells, a factor belonging to family X (protein C amino acid sequence (SEQ ID NO: 2 or SEQ ID NO: 4) or protein D amino acid sequence (SEQ ID NO: 6 or SEQ ID NO: 8)), factors belonging to family Y (protein H amino acid sequence (SEQ ID NO: 10 or SEQ ID NO: 12)) or protein I amino acid sequence (SEQ ID NO: 14 or SEQ ID NO: 16) )) And any of the factors belonging to family Z (protein J amino acid sequence (SEQ ID NO: 18 or SEQ ID NO: 20) or protein K amino acid sequence (SEQ ID NO: 22 or SEQ ID NO: 24)) Or Others and selecting cells expressing any combination, were also shown to be able to provide a method for obtaining cardiac progenitor cells or cardiac stem cells.

As described above, factors belonging to family X (protein C or protein D), factors belonging to family Y (protein H or protein I), and factors belonging to family Z (protein J or protein K) are responsible for the formation of the myocardium. It became clear that it is a gene that plays a central role. Therefore, even with undifferentiated cells, it is possible to select cells that differentiate into cardiomyocytes via cardiac stem cells and cardiac progenitor cells with a high probability triggered by the expression of these genes. Became clear.

Such cell selection can be performed by a cell sorter using a cardiomyocyte surface marker to be identified. For example, a detectable label can be complexed with an antibody against the target cardiomyocyte surface marker, and cells can be selected using the label. Alternatively, a gene encoding a labeled protein such as GFP is fused to the gene of the target cardiomyocyte surface marker, the cell is transfected, and the cell is selected using the labeled protein expressed in the cell. You can also

In the present invention, a factor belonging to family X (protein C or protein D, or gene C or gene D encoding them), a factor belonging to family Y (protein H or protein I, or gene H or genes encoding them) I), and in addition to detecting factors belonging to family Z (protein J or protein K, or gene J or gene K encoding them), production of marker molecules generally known as cardiomyocyte markers Furthermore, it is possible to select cardiac progenitor cells and cardiac stem cells with a higher probability. In general, it is often known that one molecule plays multiple roles when a molecule plays a role in the development of a living organism. Therefore, factors other than the development of the heart also apply to factors belonging to family X (gene C or gene D), factors belonging to family Y (gene H or gene I), and factors belonging to family Z (gene J or gene K). The possibility of being used in is undeniable. Thus, if another cardiomyocyte marker is used at the same time and examined, it becomes possible to select cardiac progenitor cells and cardiac stem cells more accurately.

Examples of such cardiomyocyte markers include Islet1, Nkx2-5, α-heart actin, Brachyury T (BryT), Flk1, Sca-1, c-kit, PDGFRa, Mesp1, and Gsc.

This specification describes the invention in further detail below.

Example 1: Selection method of factors commonly expressed in cardiac progenitor cells and cardiac stem cells 1-1: Isolation of candidate factors using Flow Cytometry In this example, cardiac progenitor cells are derived from mesodermal properties. The target cells were isolated by combining three cell markers.

Since Flk1 is a various progenitor cell marker and also a cardiac progenitor cell marker, it is necessary to isolate cells that are Flk1 ⁺ as one of the cell markers. Next, trans in which a fluorescent tag (YFP or EGFP) is knocked in on the loci of two other cardiac progenitor cell markers, Islet1 (also a stem cell marker for neuromotor neurons) and Wt1 (also known as a hematopoietic stem cell marker) A group of cells that are YFP ⁺ or GFP ⁺ was selected using a transgenic mouse.

As a result of microarray analysis of strongly expressed genes in such a cell group, 15 interesting genes were successfully isolated. That is, as a result of quantitative RT-PCR, it was confirmed that this cell group was Islet1 ⁺⁺ , Nkx2-5 ^+/− , Tbx5 ⁻ , cardiac troponin ⁻ , and a characteristic transcription factor expressed in the second predetermined cardiac region Strong expression was observed in all 15 genes of the group or epigenetic factor group.

1-2: Histological analysis in the embryogenesis process Histological confirmation of the expression pattern of each of the 15 genes found in 1-1 reveals that there are many cardiac progenitor cells in the embryogenesis process. It was very similar to the Islet1 expression region (Fig. 1). FIG. 1 shows the expression of genes A to E among 15 genes compared to the expression of Islet1 gene and Mlc2a gene.

As a result, all of the genes A to E are strongly expressed in the second cardiac predetermined region (the portion surrounded by the red line in the tissue photograph of the genes A to E in FIG. 1), and this expression site is the Islet1 gene. It was similar to the place of expression. Of these genes, genes C and D were found to be expressed particularly from the early embryogenesis.

1-3: Time-series expression change in ES cell differentiation induction system Next, in order to confirm whether these genes are also expressed in the ES cell differentiation system before the heart induction period and decrease after muscle differentiation In the example, the myocardial induction was induced using ES cells, and then the time-series expression pattern of major genes was examined. As a result, the expression peak of a factor known as a mesoderm marker factor ((BryT, Mesp1, Gsc) was observed at a very early stage (after 3 to 4 days) after myocardial induction to ES cells. On the other hand, the expression peak of Islet1 gene known as a cardiomyocyte marker was found to occur around 5 to 6.5 days after myocardial induction (FIG. 2 (A ))

On the other hand, when the 15 genes obtained in 1-1 were examined, genes C and D in particular were almost at the same time as the expression peak of the mesodermal marker factor (BryT, Mesp1, Gsc), and It was found to produce a unimodal peak earlier than the expression peak of Islet1. Time-series changes in the expression of genes C and D are expressed later than the mesoderm marker, while the expression of myocardial markers (Tbx5, α-heart actin, Nkx2-5) is increased, and myocardial differentiation occurs. After the 6th day of induction, a characteristic change was observed in which the expression decreased and then disappeared below the measurement limit (FIG. 2 (B)).

From these expression results, genes C and D are considered to work before cardiac induction (induction day 6), that is, cells that express these genes in the early stage of induction are considered to meet the definition of cardiac stem cells. It was.

1-4: Generation of gene C and gene D knockout mice and consideration from morphological abnormality Next, if cells expressing gene C and gene D are cardiac stem cells, by destroying these genes, Anomalies must occur. Therefore, in this example, gene knockout mice for gene C and gene D were prepared, the morphology was observed during high occurrence, and the change in gene expression over time was examined. Regarding gene C and gene D, an attempt was made to create a gene-disrupted mouse using a Baygenome gene trap line mouse.

Since gene C and gene D are factors belonging to the same family, a double homo knockout mouse of gene C and gene D was prepared, and phenotype observation in the embryo was performed. As shown in the right figure of FIG. 3 (B), the gene C / gene D double knockout (DKO) mice are completely deficient in the outflow tract and the right ventricular area and become lethal by embryonic day 9.5. It was. Such a phenotype was very similar to that of the knockout mouse (Islet1KO mouse) of the cardiac progenitor cell marker Islet1 (FIG. 3 (A)).

In fact, in gene C / gene D double knockout mice, Islet1 ⁺ cells in the outflow tract of the heart (FIG. 4A) that existed in normal animals were replaced by Islet1 knockout knockout mice (FIG. 4B). It disappeared as well as (FIG. 4C). From these facts, it was considered that genes C and D are genes located upstream of Islet 1 from a functional viewpoint.

1-5: Expression pattern of gene C and embryonic cardiac progenitor cell Islet1 / Nkx2-5 From these results, to determine whether gene C and gene D are expressed specifically in cardiac progenitor cells / stem cells Next, two experimental systems were constructed. 1) An experimental system for observing the expression patterns of endogenous gene C and gene D. 2) An experimental system for confirming the necessity of re-culturing after preparing a transgenic mouse in which GFP is knocked in at gene C and gene D loci, and isolating GFP ⁺ cells.

In this example, first, the expression patterns of Islet1 and Nkx2-5, which are representative of gene C and embryonic cardiac progenitor cell markers, were observed. For knock-in mice, knock-in GFP mice were generated for gene C, while lacZ knock-in mice were generated for gene D.

When the endogenous expression pattern was examined with an antibody against gene C, it was very similar to Islet1 (red in FIG. 5 (B)) expressed in the second expected cardiac region (SHF) (gene C was shown in FIG. 5 (A)). green). It was expressed more widely in SHF than Nkx2-5 (FIG. 5 (C) white), suggesting the possibility of a cardiac progenitor cell marker. FIGS. 5D to 5F show superimposed images of the respective tendencies, FIG. 5D shows a superimposed image of gene C and Islet1, and FIG. 5E shows Islet1 and Nkx2- 5 and FIG. 5 (F) show the superimposed images of genes C, Islet1 and Nkx2-5, respectively.

Example 2: Selection of additional factors commonly expressed in cardiac progenitor cells and cardiac stem cells In this example, of the 15 proteins found in Example 1 (1-1), other than protein C or protein D The purpose of this study is to investigate whether there are substances that can promote differentiation / induction from undifferentiated cells into cardiac progenitor cells / cardiac stem cells by combining with other factors of protein C and protein D. It was.

Specifically, the 15 factors described above (in addition to gene C encoding protein C, gene H encoding protein H, gene I encoding protein I, gene J encoding protein J, and protein K encoding) (Including four types of factors of gene K) to determine how the induced expression of Islet1, a cardiomyocyte marker, changes when siRNAs for these genes are used alone or in combination It was.

After embryoid body formation from mouse ES cells, myocardial differentiation was induced using a differentiation induction medium. From the second day of differentiation induction, siRNA was used to continuously inhibit the function of a specific gene once every two days for six days, and the expression change of cardiac progenitor cell gene (Islet1) was detected using the qPCR method. . Among them, when the genes C, H, J, and K were combined, the cardiac progenitor cell gene expression was strongly suppressed and myocardial differentiation was suppressed.

Fig. 6 shows the result of the experiment. As shown in this figure, when siRNA for gene C, siRNA for gene H, siRNA for gene I, siRNA for gene J, and siRNA for gene K are used alone, siRNA for gene J is used. Although the expression of Islet1 was significantly reduced, it was only reduced to about 0.7 to 0.8 when siRNA for the other four genes was used.

Next, the inhibitory effect on the induced expression of Islet1 was examined when the expression was inhibited by siRNA combining factors belonging to family X, factors belonging to family Y, and factors belonging to family Z one by one. In the case of gene H / gene J, in the case of gene C / gene H / gene K, in the case of gene C / gene I / gene J, the induced expression of Islet1 is inhibited to about 0.2, and gene C / In the case of gene I / gene K, it was revealed that the induced expression of Islet1 was inhibited to about 0.1.

Therefore, in further experiments in this example, when 3 genes of gene C / gene I / gene K were inhibited using siRNA, various differentiations were observed in cells that differentiated from mesoderm to heart. We examined how the expression of genes that should be expressed in the phase changes.

The genes examined for expression here are Mesp1 and Gsc as mesoderm genes; Dkk1, Flk1, PDGFRa, and Islet1 as cardiac progenitor genes; Mef2c, Nkx2-5 as myocardial transcription factors; Tbx5; And Baf60f; and WT1, Tbx18, and Tnnt2; as pericardial progenitor cell specific factors.

The results are shown in FIG. In this figure, the expression of each gene in NC1 cells was set to 1, and when 3 genes of gene C / gene I / gene K were inhibited using siRNA, the expression of each gene was calculated as a ratio (qRT-PCR). (Data acquisition by quantitative PCR method). As a result, mesoderm marker, myocardial progenitor cell-specific factor, myocardial transcription factor, pericardial progenitor cell-specific factor by inhibiting the expression of 3 genes of gene C / gene I / gene K in undifferentiated cells It was clarified that the expression of any of the myocardial progenitor cells and myocardial stem cell markers decreased. Therefore, it is essential for the undifferentiated cells to be differentiated and induced into myocardial progenitor cells and myocardial stem cells that the gene C / gene I / gene K are expressed in undifferentiated cells derived from mesoderm. It was shown that this process.

Example 3: Culture of cardiac progenitor cells and development of high-efficiency cardiac differentiation direction 3-1: Isolation of GFP-positive cells from knock-in GFP mice and evaluation by culture Gene C ⁺ cells have properties as cardiac progenitor cells Therefore, in this example, GFP knock-in mice were prepared and GFP ⁺ cells were isolated using FACS.

When GFP ⁺ cells were developed with embryonic cardiac progenitor cell-specific cell surface antigens cKit and PDGFRa, they were divided into three patterns: GFP +, GFP +; cKit +, GFP +; PDGFRa + (FIG. 8 (A)). Interestingly, strong expression of Islet1 was observed in GFP ⁺ cells (FIG. 8 (B)). From this, it was found that gene C ⁺ / gene D ⁺ cells are factors common to known cardiac stem cell markers. Furthermore, after selecting and isolating the gene C ⁺ / gene D ⁺ cells with a cell sorter, the beating myocardium: cardiac troponin ⁺ cells have been differentiated with a probability of 90%.

3-2: Establishment of an efficient myocardial guidance method using gene C / gene D positive cell differentiation system In this example, it was examined whether cardiomyocytes could be induced using an in vitro embryonic stem cell differentiation system. . The gene C-EGFP was transgenic to ES cells to produce recombinant cells.

When these cells were cultured according to the schedule shown in FIG. 9A, it was examined whether the gene C ⁺ / gene D ⁺ cells differentiated into myocardium at the 10th day of culture. As a result, it was shown that the cells that expressed the gene C were positive for troponin (cTnt) in almost all cells as in the case of 2-1. It was found that cTnt ⁺ cells were very limited in the cells that did not (FIG. 9 (B) bottom).

Furthermore, when the origin of the cells of the pulsating colony was confirmed, it was also shown that 80% of the pulsating colonies were cells expressing the gene C (FIG. 9C).

In addition, when cells expressing the gene C-GFP were selected using a cell sorter and cultured, myocardial markers and cardiac progenitor cell markers that were not expressed on day 0 of culture were expressed on day 4 of culture. (FIG. 9D).

Therefore, based on these results, cells that initially expressed gene C / gene D expressed cardiomyocyte markers, cardiac progenitor cell markers, and the like thereafter, and had the same functions as the innermost functional myocardium. In the future, it is expected that clinical application will be performed by collecting and transplanting gene C ⁺ / gene D ⁺ cells.

Next, when a potential change was collected from the pulsatile cells thus obtained, it became clear that the pulsation was repeated at a constant rhythm (left side of FIG. 10). Barium chloride (BaCl), an inhibitor of I _K1 (inward rectifier K + current that acts on the formation of a plateau layer that maintains the action potential of the membrane quiescent potential) and has myocardial toxicity against these beating cells ₂ ) 0.2 mM was added, the pulsation almost stopped (middle of FIG. 10). After that, when the barium chloride was washed and removed, it was found that the pulsation was restarted and the pulsation after the restart was repeated at a constant rhythm (right side of FIG. 10). Further examination of the state of pulsating cells revealed that there are cells functioning as pacemaker cells (FIG. 11), and cells expressing gene C can also differentiate into sinus nodes. It became clear that.

3-3: Gene C, gene D heart forming region and differentiated cell mouse, insert creER into gene C / gene D locus, and cross with ROSA-RFP (or lacZ) called reporter mouse. Alternatively, it is possible to generate a lineage after differentiation of a cell expressing gene D. As a result, it is possible to clarify which cell has actually differentiated in vivo.

When the differentiation state of the cells that once expressed the gene C / gene D was traced using this method, it was shown that the cells differentiated throughout the ventricle (FIGS. 12 and 13). That is, gene C ⁺ / gene D ⁺ cells are essential factors common to cardiac progenitor cells and cardiac stem cells, and it was shown that heart constituent cells can be established efficiently by using this as an index.

3-4: Analysis of function of gene C during embryogenesis Insertion of creER into the Islet 1 locus (Islet1-cre mouse) and mating with a ROSA26-RFP mouse called a reporter mouse, thereby expressing cells expressing the Islet 1 gene. A mouse capable of generating a lineage after differentiation was created.

On the other hand, using a transgenic mouse (gene C-GFP knock-in mouse) in which a fluorescent tag (EGFP) was knocked in the gene C locus, a cell group that was GFP ⁺ in the embryo was selected.

In these embryos, only the gene C is expressed in the embryo at the developmental stage on the seventh day after fertilization, and the expression of the cardiac progenitor gene Islet1 is not observed. The expression intensity of gene C in the developmental stage of embryos on the 8th day after fertilization has increased. When the expression site and the expression site of Islet1 were examined, the expression site of gene C and the expression site of Islet1 were very similar. (FIG. 14). Similarly, in the embryo 8.5 days after fertilization, the Islet1 gene was weak at the site where the gene C was strongly expressed, and the expression of the Islet1 gene was weak in the weak positive cells. When the expression of the gene C and Islet1 gene was examined with a cell sorter, there was no cell group that expressed only the Islet1 gene, the group that expressed the gene C but not the Islet1 gene, both the gene C and the Islet1 gene Since there was a group expressing both, it was shown that expression of Islet1 occurred after the expression of gene C (FIG. 14).

Based on this finding, from the group of embryos 8.5 days after fertilization, a strong expression group that expresses gene C was collected from the cell group that expresses gene C, and the following examination was performed. Specifically, in a transgenic mouse (gene C-GFP knock-in mouse) in which a fluorescent tag (EGFP) is knocked in on the gene C locus, it is strongly expressed at an expression level of GFP from an embryo 8.5 days after fertilization. Cell groups (ie, gene C ⁺ cells) are collected (the upper left figure in FIG. 15, enclosed area), and the cells are expanded with embryonic cardiac progenitor cell-specific cell surface antigens c-kit and PDGFRa (The upper right figure of FIG. 15). As a result, 86.1% of the cells strongly positive for gene C at 8.5 days after fertilization were determined to be c-kit ⁺ and PDGFRa ^−, and when this cell group was cultured, gene C ⁺ / c− kit ⁺ of the cells showed cardiomyocyte-like shape (FIG. 15 left figure), also to determine the fluorescence image on tissue sections, from gene ^C + / c-kit ⁺ at culture conditions, gene ^{C +} It was suggested that cells of / c-kit ⁺ / Islet1 ⁺ were formed. Therefore, it was suggested that the gene C ⁺ / c-kit ⁺ cell is a more immature cardiac progenitor cell.

Furthermore, the same examination was performed on embryos 8.5 days after fertilization, and the gene C ⁺ cells were expanded with c-kit and PDGFRa using a cell sorter. Here, among the gene C ⁺ cells, c-kit ⁺ / PDGFRa ⁻ cells are referred to as “cell group 1”, c-kit ⁻ / PDGFRa ⁻ cells are referred to as “cell group 2”, and c-kit ⁻ / PDGFRa ⁺ The cells were designated as “cell group 3” (the cell groups 1 to 3 are simply indicated as 1 to 3 in FIG. 16).

Each of these cell groups was examined for expression of gene C, c-kit gene, PDGFRa gene, Islet1 gene (myocardial progenitor cell), Nkx2-5 gene (myocardial progenitor cell), and Actc1 gene (cardiomyocyte). . As a result, at 8.5 days after fertilization, cells expressing gene C express PDGFRa, which is a typical myocardial marker, over time, and gene C ⁺ cells become c-kit ⁺ / from ^state, c-kit ^- - PDGFRa via the state ^{of, c-kit - - / PDGFRa} / migrating was suggested to PDGFRa ^+. In the middle of c-kit ⁻ / PDGFRa ⁻ , it is shown that a myocardial progenitor cell marker (eg, Islet1 gene, Nkx2-5 gene, etc.) or myocardial marker (eg, Actc1 gene) is expressed. It was.

Furthermore, in the region of cardiac progenitor cells in the developing embryo, how the expression of gene C and the expression of Islet1, which is a cardiomyocyte marker, are related to Islet1-cre mice and the gene C-GFP knock-in. The expression of gene C and Islet1 gene during embryogenesis was confirmed using mice obtained from mating with mice. As a result, it was shown that the area of cardiac progenitor cells in the developing heart supported by the expression of gene C covers a wider area than the area known so far (right figure in FIG. 17). It was also shown that gene C is expressed in cardiac endothelial cells (FIG. 18).

Example 4: Function of three genes C / G / I / K in vivo In Example 2, gene C / G / I / G 3 in undifferentiated cells based on in vitro experiments. Since the expression of the gene was shown to be an essential step for the differentiation / induction of undifferentiated cells into myocardial progenitor cells / cardiac stem cells, these three factors are used in this example. However, whether or not it promotes cardiomyocyte differentiation and induction also in vivo was confirmed.

Three genes, gene C, gene I and gene K, were each incorporated into separate CMV vectors and directly transfected into embryos using a microcapillary in embryonic embryo 7.0 (E7.0) mouse embryos. Thereafter, embryos were cultured in a culture solution while rotating a culture tube in a CO ₂ incubator at 37 ° C., and overexpression of each candidate gene was caused within 24 hours while normal embryogenesis proceeded. After culture for 36 hours, the expression and localization of cardiomyocyte markers (Islet1 and Baf6oc) in this embryo were examined.

As a result of such an experiment, a fluorescence image of GFP when exogenously expressing gene C, gene I and gene K introduced exogenously in the embryo, and endogenous Islet1 gene and Baf6oc gene are expressed. It was shown that the localized site was consistent (FIG. 19). From this, when gene C, gene I and gene K were expressed ectopically, the expression of endogenous Islet1 gene and Baf6oc gene was consequently induced ectopically (FIG. 19). That is, it was shown that the expression of three genes, gene C, gene I, and gene K, in the embryo is a factor for inducing cardiomyocytes.

If the present invention is put to practical use, it becomes possible to supply a cell source or bioparts for saving myocardial injury patients / congenital heart disease patients.

In the past, artificial parts such as artificial hearts, pacemaker devices, and artificial valves have already been used for the treatment of heart disease, but when using these artificial parts, it is necessary to replace them almost every five years. It was pointed out that it had to be installed or permanently installed. On the other hand, in the case of a biological part, since it is constructed from a patient's individual tissue, there is no problem of immune rejection and no problem of deterioration over time. In addition, since cardiac progenitor cells and cardiac stem cells can be preserved and have a good cytokine response, an effect that they can be used as needed can be expected.

In addition, expression of the gene C of the present invention allows such cells to efficiently differentiate into cardiac constituent cells from the myocardium to the pacemaker cells. By using these cells, it is effective for screening factors that promote myocardial differentiation, pacemaker-inducing factors, cardiac smooth muscle, and cardiac endothelium. It can also be used to screen for factors that can be loaded after myocardial induction to cause hypertrophy and rescue the effects.

Furthermore, by using the method of the present invention, there is a great merit that cardiac stem cells can be established from the skin or fibroblasts of the heart disease patient himself.

SEQ ID NO: 1: nucleotide sequence encoding mouse-derived gene C;

SEQ ID NO: 2: amino acid sequence of gene C derived from mouse;

SEQ ID NO: 3: nucleotide sequence encoding human-derived gene C;

SEQ ID NO: 4: Amino acid sequence of gene C derived from human.

SEQ ID NO: 5: nucleotide sequence encoding mouse-derived gene D;

SEQ ID NO: 6: amino acid sequence of mouse-derived gene D;

SEQ ID NO: 7: nucleotide sequence encoding human-derived gene D;

SEQ ID NO: 8: amino acid sequence of gene D derived from human.

SEQ ID NO: 9: nucleotide sequence encoding mouse-derived gene H;

SEQ ID NO: 10: amino acid sequence of mouse-derived gene H;

SEQ ID NO: 11: Nucleotide sequence encoding human-derived gene H;

SEQ ID NO: 12: amino acid sequence of gene H derived from human.

SEQ ID NO: 13: nucleotide sequence encoding mouse-derived gene I;

SEQ ID NO: 14: amino acid sequence of mouse-derived gene I;

SEQ ID NO: 15: nucleotide sequence encoding human-derived gene I;

SEQ ID NO: 16: amino acid sequence of gene I derived from human.

SEQ ID NO: 17: nucleotide sequence encoding mouse-derived gene J;

SEQ ID NO: 18: amino acid sequence of mouse-derived gene J;

SEQ ID NO: 19: nucleotide sequence encoding human-derived gene J;

SEQ ID NO: 20: amino acid sequence of gene J derived from human.

SEQ ID NO: 21: nucleotide sequence encoding mouse-derived gene K;

SEQ ID NO: 22: amino acid sequence of mouse-derived gene K;

SEQ ID NO: 23: nucleotide sequence encoding human-derived gene K;

SEQ ID NO: 24: amino acid sequence of gene K derived from human.

Claims (26)

1. Protein C (SEQ ID NO: 2 amino acid sequence, SEQ ID NO: 4 amino acid sequence, or the function of directing differentiation / induction into cardiac progenitor cells and cardiac stem cells when expressed in undifferentiated cells, or Amino acid sequence similar to these), or protein D (SEQ ID NO: 6 amino acid sequence, SEQ ID NO: 8 amino acid sequence, or similar amino acid sequence), belonging to family X selected from proteins A differentiation / induction agent for cardiac progenitor cells and cardiac stem cells composed of proteins.
2. A protein belonging to family X is protein C encoded by gene C (SEQ ID NO: 1 nucleotide sequence, SEQ ID NO: 3 nucleotide sequence, or a similar nucleotide sequence), or gene D (SEQ ID NO) 2. The agent for differentiation / induction into cardiac progenitor cells / cardiac stem cells according to claim 1, which is protein D encoded by a nucleotide sequence of SEQ ID NO: 7 or a nucleotide sequence similar to these: .
3. The agent for differentiation / induction into cardiac progenitor cells and cardiac stem cells according to claim 1, wherein the protein belonging to family X is protein C.
4. The agent for differentiation / induction into cardiac progenitor cells and cardiac stem cells according to claim 3, wherein protein C has an amino acid sequence of SEQ ID NO: 4.
5. The cardiac progenitor cell of claim 2, wherein protein C is encoded by the nucleotide sequence of SEQ ID NO: 3.
   Differentiation / induction agent for cardiac stem cells.
6. In addition to proteins belonging to family X,
   A protein belonging to family Y (protein H (amino acid sequence of SEQ ID NO: 10, amino acid sequence of SEQ ID NO: 12 or amino acid sequence similar thereto) or protein I (amino acid sequence of SEQ ID NO: 14; SEQ Amino acid sequence of ID NO: 16 or an amino acid sequence similar to these)),
   A protein belonging to family Z (protein J (SEQ ID NO: 18 amino acid sequence, SEQ ID NO: 20 amino acid sequence, or amino acid sequence similar thereto) or protein K (SEQ ID NO: 22 amino acid sequence, SEQ ID NO: 24 amino acid sequence, or an amino acid sequence similar to these))), or a combination of a protein belonging to family Y and a protein belonging to family Z,
   The agent for differentiating / inducing cardiac progenitor cells / cardiac stem cells according to any one of claims 1 to 5, further comprising:
7. A protein belonging to family Y is protein H encoded by gene H (SEQ ID NO: 9 nucleotide sequence, SEQ ID NO: 11 nucleotide sequence, or similar nucleotide sequence), or gene I (SEQ ID NO: SEQ ID NO: 9). The agent for differentiation / induction into cardiac progenitor cells / cardiac stem cells according to claim 6, which is protein I encoded by: nucleotide sequence of 13; nucleotide sequence of SEQ ID NO: 15 or a nucleotide sequence similar thereto; .
8. The agent for differentiation / induction into cardiac progenitor cells and cardiac stem cells according to claim 7, wherein the protein belonging to family Y is protein I.
9. The agent for differentiation / induction into cardiac progenitor cells and cardiac stem cells according to claim 8, wherein the protein I has an amino acid sequence of SEQ ID NO: 16.
10. The agent for differentiation / induction into cardiac progenitor cells and cardiac stem cells according to claim 7, wherein protein I is encoded by a nucleotide sequence of SEQ ID NO: 15.
11. A protein belonging to family Z is protein J encoded by gene J (SEQ ID NO: 17 nucleotide sequence, SEQ ID NO: 19 nucleotide sequence, or similar nucleotide sequence), or gene K (SEQ ID NO) The agent for differentiation / induction into cardiac progenitor cells / cardiac stem cells according to claim 6, which is a protein K encoded by a nucleotide sequence of 21: SEQ ID NO: 23, or a nucleotide sequence similar thereto. .
12. The agent for differentiation / induction into cardiac progenitor cells and cardiac stem cells according to claim 6, wherein the protein belonging to family Z is protein K.
13. The agent for differentiation / induction into cardiac progenitor cells and cardiac stem cells according to claim 12, wherein the protein K has an amino acid sequence of SEQ ID NO: 24.
14. The agent for differentiation / induction into cardiac progenitor cells and cardiac stem cells according to claim 11, wherein protein K is encoded by the nucleotide sequence of SEQ ID NO: 23.
15. The agent for differentiation / induction into cardiac progenitor cells / heart stem cells according to any one of claims 1 to 14, wherein the undifferentiated cells are selected from early embryo cells, embryonic stem cells, and induced pluripotent stem cells.
16. A cardioprogenitor cell / cardiac stem cell characterized by selecting a cell that expresses the differentiation / induction agent for cardiac progenitor cell / cardiac stem cell according to any one of claims 1 to 15 as an undifferentiated cell. How to get.
17. A gene C (SEQ ID NO: 1 nucleotide sequence, SEQ ID NO: 3 nucleotide sequence, or a nucleotide sequence similar thereto) or gene D (undifferentiated cell encodes a protein belonging to family X) The method according to claim 16, which is prepared by expressing a SEQ ID NO: 5 nucleotide sequence, a SEQ ID NO: 7 nucleotide sequence, or a nucleotide sequence similar thereto.
18. The method according to claim 17, wherein the undifferentiated cells are prepared by expressing gene C (SEQ ID NO: 3 nucleotide sequence) as a gene encoding a protein belonging to family X.
19. Gene H (SEQ ID NO: 9 nucleotide sequence, SEQ ID NO: 11 nucleotide sequence, or similar nucleotide sequence), or gene I (undifferentiated cell) encodes a protein belonging to family Y. The method according to claim 16, which is created by expressing a SEQ ID NO: 13 nucleotide sequence, a SEQ ID NO: 15 nucleotide sequence, or a nucleotide sequence similar thereto.
20. The method according to claim 19, wherein the undifferentiated cells are prepared by expressing gene I (SEQ ID NO: 15 nucleotide sequence) as a gene encoding a protein belonging to family Y.
21. Gene J (SEQ ID NO: 17 nucleotide sequence, SEQ ID NO: 19 nucleotide sequence, or a similar nucleotide sequence) or gene K (as a gene encoding a protein belonging to family Z by an undifferentiated cell) The method according to claim 4, which is prepared by expressing a nucleotide sequence of SEQ ID NO: 21, a nucleotide sequence of SEQ ID NO: 23, or a nucleotide sequence similar thereto.
22. The method according to claim 21, wherein the undifferentiated cells are prepared by expressing gene K (SEQ ID NO: 23 nucleotide sequence) as a gene encoding a protein belonging to family Z.
23. The method according to any one of claims 16 to 22, wherein the undifferentiated cells are selected from early embryo cells, embryonic stem cells, and induced pluripotent stem cells.
24. The method according to any one of claims 16 to 23, wherein the cells are selected by a cell sorter.
25. The method according to any one of claims 16 to 24, wherein the production of a cardiomyocyte marker is further detected.
26. The cardiomyocyte marker is selected from the group consisting of Islet, Nkx2-5, α-heart actin, Brachyury T (BryT), Flk1, Sca-1, c-kit, PDGFRa, Mesp1, and Gsc. The method of any one of these.

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