JP5572814B2 - Marker for detecting myocardial differentiation activity of undifferentiated cells, nucleic acid molecule, primer pair, kit, myocardial differentiation inhibitor, method for detecting myocardial differentiation activity of undifferentiated cells, method for isolating cells having myocardial differentiation activity, myocardium of undifferentiated cells Method for monitoring differentiation activity and method for inhibiting myocardial differentiation of undifferentiated cells - Google Patents

Description

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  This patent application is accompanied by a priority claim based on Japanese Patent Application No. 2006-109858 (application date: April 12, 2006) which is a previously filed Japanese patent application. The entire disclosure of this earlier patent application is hereby incorporated by reference.

  The present invention relates to a marker useful for accurate and simple detection of myocardial differentiation activity of cells. The present invention also relates to a nucleic acid molecule, primer pair, kit, myocardial differentiation inhibitor, method for detecting myocardial differentiation activity of undifferentiated cells, method for isolating cells having myocardial differentiation activity, and method for monitoring myocardial differentiation activity of undifferentiated cells. And a method for inhibiting myocardial differentiation of undifferentiated cells.

  Cardiomyocytes actively divide and proliferate before birth but with autonomous pulsation. However, cardiomyocytes usually lose their ability to divide immediately upon birth and, unlike hepatocytes, etc., they usually never acquire the ability to divide again.

  In recent years, it has been clarified that mature myocardial tissue also contains cardiomyocyte progenitor cells. However, the number of cardiomyocyte progenitor cells is very small, and it is not sufficient to completely restore cardiac function by supplementing the cardiomyocytes when they are damaged. Therefore, when a load or necrosis occurs in the myocardium due to ischemic heart disease or cardiomyopathy, the cardiomyocytes are adapted mainly in the form of cell hypertrophy rather than cell division. Cardiomyocyte hypertrophy is considered to be a physiological adaptation phenomenon to the load in the initial stage, but it increases the coexistence of coexisting cardiac fibroblasts and fibrosis of the interstitium, leading to a decrease in the expansion function of the heart itself and a decrease in the contractile function. It becomes connected and begins to exhibit heart failure.

  Treatment of heart failure due to ischemic heart disease, cardiomyopathy, etc. is centered on symptomatic treatment such as enhancement of cardiac contractility, reduction of pressure load / volume load with vasodilators, reduction of fluid volume with diuretics, etc. . In addition, the fundamental treatment for severe heart failure includes heart transplantation. However, heart transplantation has many problems such as lack of organ donors, difficulty in determining brain death, rejection, and medical expenses. Therefore, development of a method for reacquiring lost myocardium is required for appropriate treatment of heart disease.

  One method for reacquiring the myocardium is a method for reacquiring the ability to divide and proliferate cardiomyocytes. In order to carry out this method, it is required to investigate the mechanism by which cardiomyocytes lose their division ability after birth. Then, it is expected that such a mechanism will be clarified, and that cardiomyocytes are reacquired for division ability by gene therapy or the like (Non-patent Document 1).

  Another method for reacquiring the myocardium is a differentiation induction method in which cells other than cardiomyocytes are converted into cardiomyocytes. Murry et al. Reported that non-cardiomyocytes (fibroblasts) can be converted to skeletal muscle cells by introducing MyoD, a master key gene for skeletal muscle differentiation, into the heart in vitro ( Non-patent document 2). In cardiomyocytes, various cardiomyocyte-specific transcription factors such as Nkx2.5, GATA4, and MEF2C have been cloned. However, there is no report that cardiomyocytes have been produced by introducing these genes into other cells alone or in plural. Therefore, it can be said that there is still a need for a gene that can be used to control myocardial differentiation of cells in elucidation of the details of myocardial differentiation mechanism and in the treatment of heart disease.

  Another method for treating heart disease is cardiomyocyte transplantation. In recent years, a method of transplanting stem cells or myocardial progenitor cells into myocardial injury sites and differentiating them into cardiomyocytes, or a method of transplanting stem cells or myocardial progenitor cells into myocardial cells in vitro and then transplanting them into myocardial injury sites, It is attracting attention from the viewpoint of regenerative medicine. Since this method transplants cells derived from the patient himself, there is no fear of rejection.

  However, in cardiomyocyte transplantation, there is a risk of causing arrhythmia or heart failure if the transplanted cells do not acquire a phenotype suitable for the transplant site and differentiate into cells of an unintended type. Therefore, in order to improve the safety of cardiomyocyte transplantation therapy, it is indispensable to appropriately predict the ease of differentiation of cells into cardiomyocytes, that is, “cardiac myocyte differentiation activity”.

  Searches for markers that serve as indicators of myocardial differentiation activity of cells have been actively conducted in Japan and overseas (Non-patent Document 3). A marker serving as an index of myocardial differentiation activity is expected to be applied to a technique for separating undifferentiated cells having a relatively high myocardial differentiation ability, and a specific and efficient cardiomyocyte differentiation induction technique. However, there is still no marker that can be used in the undifferentiated stage and serves as an index of myocardial differentiation activity of cells. In previous studies, after a myocardial differentiation-inducing stimulus has been applied to undifferentiated cells, a method of searching for a gene whose expression fluctuates early in the differentiation process has been taken. For this reason, although the obtained marker is a marker of “an early stage of cardiomyocyte differentiation that has already begun”, it is not a marker for “differentiation activity of cardiomyocytes possessed by undifferentiated cells” before differentiation induction. Absent. In order to appropriately treat heart diseases, it is necessary to evaluate the quality of undifferentiated cells and to separate undifferentiated cells having high myocardial differentiation ability. Therefore, there is still a need for a marker that can be used to detect myocardial differentiation activity of cells in the undifferentiated stage.

Tamamori et al., Critical role of cyclin D1 nuclear import in cardiomyocyte proliferation, Circ Res. 2003, Jan 10, 92 (1), e12-9 Murryet al., Muscle differentiation during repair of myocardial necrosis in rats via gene transfer with MyoD, J Clin Invest., 1996, Nov 15, 98 (10), 2209-17 Peng et al., Microarray analysis of global changes in gene expression during cardiac myocyte differentiation, Physiol Genomics., 2002, 9 (3), 145-55, Kyoko Hidaka et al., `` Search for myocardial differentiation-related genes expressed in ES cells・ Construction of analysis system '', `` Regenerative medicine '', 2005, 4 (Suppl), 88, Yuji Akimaru, etc., `` Analysis of signals that control myocardial differentiation of ES cells '', `` Regenerative medicine '', 2005, 4 (Suppl) , 89

  The present inventors have now found that the expression level of a plurality of specific genes (hereinafter referred to as “CMP (cardiomyogenesis predictor) gene”) in a cell shows a significant correlation with the myocardial differentiation activity of the cell at an undifferentiated stage. I got the knowledge. Furthermore, the present inventors have obtained the knowledge that the myocardial differentiation activity of cells can be accurately detected by using the CMP gene and its gene product as a marker. The present invention is based on such knowledge.

  Accordingly, an object of the present invention is to provide a marker for detecting myocardial differentiation activity of undifferentiated cells.

And the gene marker for detecting the myocardial differentiation activity of the undifferentiated cell before differentiation induction by this invention consists of a polynucleotide which consists of a nucleotide sequence represented by sequence number 3.

  According to the present invention, it is possible to accurately detect the myocardial differentiation activity of cells in an undifferentiated stage by using the above marker. Therefore, the marker according to the present invention can be advantageously used in the quality evaluation of undifferentiated cells and the isolation of undifferentiated cells having high myocardial differentiation ability. Further, by controlling the expression of the polynucleotide constituting the gene marker according to the present invention, it becomes possible to control the myocardial differentiation of cells. Therefore, the polynucleotide in the present invention can be advantageously used for elucidating the details of the myocardial differentiation mechanism and producing cardiomyocytes. Therefore, it can be said that the provision of the present invention is significant in the treatment of heart diseases.

FIG. 1A is a diagram showing the time course of the number of contractile nodules in cell lines (P19, CL6, G26, G36, G45 and G52). FIG. 1B is a diagram showing the time course of contraction nodule size in cell lines (P19, CL6, G26, G36, G45 and G52). FIG. 2A is a diagram showing the time course of MLC2a gene expression in cell lines (P19, CL6, G26, G36, G45 and G52). FIG. 2B is a diagram showing the time course of MLC2v gene expression in cell lines (P19, CL6, G26, G36, G45 and G52). FIG. 2C is a diagram showing the time course of αMHC gene expression in cell lines (P19, CL6, G26, G36, G45 and G52). FIG. 2D shows the time course of βMHC gene expression in cell lines (P19, CL6, G26, G36, G45 and G52). FIG. 2E shows the time course of Nkx2.5 gene expression in cell lines (P19, CL6, G26, G36, G45 and G52). FIG. 2F is a diagram showing the time course of GATA4 gene expression in cell lines (P19, CL6, G26, G36, G45 and G52). FIG. 2G is a diagram showing the time course of MEF2C gene expression in cell lines (P19, CL6, G26, G36, G45 and G52). FIG. 3A is a diagram showing a scree plot when principal component analysis is performed on mRNA expression data of a cardiomyocyte marker gene in cell lines (P19, CL6, G26, G36, G45 and G52) in which cardiomyocyte differentiation has been induced. FIG. 3B is a diagram showing a variable plot when principal component analysis was performed on the mRNA expression data of the cardiomyocyte marker gene in cell lines (P19, CL6, G26, G36, G45 and G52) in which cardiomyocyte differentiation was induced. . FIG. 4A shows the time course of the first principal component score when principal component analysis was performed on the mRNA expression level data of the cardiomyocyte marker gene in cell lines (P19, CL6, G26, G36, G45 and G52) in which myocardial differentiation was induced. FIG. FIG. 4B shows the time of the second principal component score when principal component analysis was performed on the mRNA expression level data of the cardiomyocyte marker gene in cell lines (P19, CL6, G26, G36, G45 and G52) in which myocardial differentiation was induced. It is a figure which shows progress. FIG. 5A is a diagram showing changes in the expression level of the CMP2 gene in G52 cells due to RNAi treatment. FIG. 5B shows changes in the expression level of the CMP13 gene in G52 cells due to RNAi treatment. FIG. 6 is a diagram showing the time course of myocardial differentiation activity of G52 cells when the expression of CMP2 or CMP13 gene is suppressed by RNAi. 7A and 7B are diagrams showing changes in the expression level of CMP1-24 gene in G52 cells by RNAi treatment. 8A to 8C are diagrams showing the time course of myocardial differentiation activity of G52 cells when the expression of CMP1 to 3 genes was suppressed by RNAi. 8D to 8F are diagrams showing the time course of myocardial differentiation activity of G52 cells when the expression of CMP4 to 6 genes was suppressed by RNAi. FIGS. 8G to I are diagrams showing the time course of myocardial differentiation activity of G52 cells when the expression of CMP7-9 gene is suppressed by RNAi. 8J-L are diagrams showing the time course of myocardial differentiation activity of G52 cells when the expression of CMP10-13 genes was suppressed by RNAi. 8M to O are diagrams showing the time course of myocardial differentiation activity of G52 cells when the expression of the CMP14-16 gene was suppressed by RNAi. FIGS. 8P to 8R are diagrams showing the time course of myocardial differentiation activity of G52 cells when the expression of CMP17-20 gene was suppressed by RNAi. 8S to U are diagrams showing the time course of myocardial differentiation activity of G52 cells when the expression of CMP21-24 gene was suppressed by RNAi.

Genetic marker The genetic marker according to the present invention comprises the CMP genes described below as CMP1-24. By measuring the expression level of such a gene marker in cells, it becomes possible to easily detect the myocardial differentiation activity of the cells.

  Details of the sequence information of the CMP gene can be obtained in a known database, for example, CMP1 corresponds to the accession number NM_027547 in the RefSeq database of the National Center for Biotechnology Information (NCBI), CMP2 corresponds to the same accession number NM_178737, CMP3 corresponds to the same accession number NM_144888, CMP4 corresponds to the same accession number NM_182991, CMP5 corresponds to the same accession number NM_029537, and CMP6 corresponds to the same accession number. It corresponds to NM_009982, CMP7 corresponds to the same accession number NM_010169, CMP8 corresponds to the same accession number NM_011348, CMP9 corresponds to the same accession number NM_030700, CMP10 corresponds to the same accession number NM_025582, and C MP11 corresponds to the same accession number NM_133362, CMP12 corresponds to the same accession number NM_025422, CMP13 corresponds to the same accession number NM_021457, CMP14 corresponds to the same accession number NM_130895, and CMP15 corresponds to the same accession number. It corresponds to NM_010331, CMP16 corresponds to the same accession number NM_018763, CMP17 corresponds to the same accession number AK036481, CMP18 corresponds to the same accession number XM_619720, CMP20 corresponds to the same accession number NM_010363, CMP21 Corresponds to the same accession number AK003902, CMP22 corresponds to the same accession number NM_031998, CMP23 corresponds to the same accession number NM_010447, CMP24 corresponds to the same accession number NM_009627, and CMP19 corresponds to the international nucleotide sequence database. the International Nucleotide Sequence Databases: corresponding to the accession number BB119527 in INSD).

  Specific examples of the nucleotide sequence of the CMP gene include the following sequences. That is, CMP1 is represented by SEQ ID NO: 1, CMP2 is represented by SEQ ID NO: 3, CMP3 is represented by SEQ ID NO: 5, and CMP4 is represented by SEQ ID NO: 7. CMP5 is represented by SEQ ID NO: 9, CMP6 is represented by SEQ ID NO: 11, CMP7 is represented by SEQ ID NO: 13, and CMP8 is represented by SEQ ID NO: 15. CMP9 is represented by SEQ ID NO: 17, CMP10 is represented by SEQ ID NO: 19, CMP11 is represented by SEQ ID NO: 21, and CMP12 is represented by SEQ ID NO: 23. CMP13 is represented by SEQ ID NO: 25, CMP14 is represented by SEQ ID NO: 27, and CMP15 is represented by SEQ ID NO: 29. CMP16 is represented by SEQ ID NO: 31, CMP17 is represented by SEQ ID NO: 33, CMP18 is represented by SEQ ID NO: 34, and CMP19 is represented by SEQ ID NO: 36. CMP20 is represented by SEQ ID NO: 37, CMP21 is represented by SEQ ID NO: 39, CMP22 is represented by SEQ ID NO: 41, and CMP23 is represented by SEQ ID NO: 43. CMP24 is represented by SEQ ID NO: 45. Therefore, according to one embodiment of the present invention, the gene marker is SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 37, SEQ ID NO: 39, A polynucleotide comprising the nucleotide sequence represented by SEQ ID NO: 41, SEQ ID NO: 43, or SEQ ID NO: 45.

  Among the CMP genes, CMP1-6, CMP8-10, CMP13, CMP18, CMP20 and CMP24 are characterized in that the expression level in the cells increases when the myocardial differentiation activity of the cells increases. Therefore, according to a preferred embodiment of the present invention, the gene marker is SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19 , Comprising a nucleotide sequence represented by SEQ ID NO: 25, SEQ ID NO: 34, SEQ ID NO: 37 or SEQ ID NO: 45, wherein the expression level in the cell increases when the myocardial differentiation activity of the cell increases Is.

  Among the CMP genes, CMP7, CMP12, CMP16, CMP17, CMP21 and CMP23 are characterized in that the expression level in the cells decreases when the myocardial differentiation activity of the cells increases. Therefore, according to another preferred embodiment of the present invention, the gene marker consists of a nucleotide sequence represented by SEQ ID NO: 13, SEQ ID NO: 23, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 39 or SEQ ID NO: 43. In addition, when the myocardial differentiation activity of a cell increases, the expression level in the cell decreases.

  The gene marker according to the present invention also includes polynucleotides functionally equivalent to CMP1-24. Here, “functionally equivalent” preferably means that it correlates with the myocardial differentiation activity of cells, as in the case of the CMP gene. Furthermore, the polynucleotide according to the present invention can be used for myocardial differentiation control. Therefore, a polynucleotide functionally equivalent to such a polynucleotide is more preferably one having cell myocardial differentiation control activity equivalent to that of the CMP gene. The functional equivalence between the polynucleotide and the CMP gene is determined by, for example, measuring the expression level of the polynucleotide using a nucleic acid molecule or primer according to the present invention, which will be described later, and the number of cells that cause pulsation. The correlation with the size can be easily determined by determining by a known statistical method and comparing it with that of the CMP gene.

  Moreover, the gene marker according to the present invention may have sequence homology with the CMP gene as long as it serves as an index of myocardial differentiation activity. And according to a preferred embodiment of the present invention, the gene marker consists of a nucleotide sequence having at least 70% homology with the nucleotide sequence encoding the CMP gene, and consisting of a polynucleotide functionally equivalent to the CMP gene. Further, such sequence homology is more preferably 75% or more, still more preferably 80% or more, and further preferably 85% or more.

  In the present invention, the homology between the nucleotide sequence and the amino acid sequence is determined by a known method. Examples of such a method include algorithm BLAST [Proc. By Karlin and Altschul]. Natl. Acad. Sci. USA, 90, 5873-5877 (1993)]. Based on this algorithm, programs called BLASTN and BLASTX have been developed [Altschul et al. J. et al. Mol. Biol. , 215, 403-410 (1990)], and can also be used in the present invention. Furthermore, another preferred method is a method using genetic information processing software GENETYX (manufactured by Genetics). When GENETYX is used, in addition to BLAST analysis, homology analysis by Lipman-Pearson method can be performed, which can be advantageously used in determining homology in the present invention.

  A part of the gene marker according to the present invention consists of the nucleotide sequence of the protein coding region of the CMP gene. That is, among CMP genes, CMP1-16, 18, 20-24 encode polypeptides. Specifically, in the CMP gene, SEQ ID NO: 1 (CMP1: especially a nucleotide sequence represented by nucleotides 64 to 1863) encodes the amino acid sequence represented by SEQ ID NO: 2, and SEQ ID NO: 3 (CMP2: In particular, the nucleotide sequence represented by nucleotides 230 to 2644) encodes the amino acid sequence represented by SEQ ID NO: 4, and SEQ ID NO: 5 (CMP3: In particular, represented by nucleotides 277 to 1788) Nucleotide number sequence) encodes the amino acid sequence represented by SEQ ID NO: 6, and SEQ ID NO: 7 (CMP4: in particular, nucleotide sequence represented by nucleotides 42 to 1055) is represented by SEQ ID NO: 8. SEQ ID NO: 9 (CMP5: in particular, the 99th to 779th nucleotides) The nucleotide sequence represented by SEQ ID NO: 10 encodes the amino acid sequence represented by SEQ ID NO: 10, and SEQ ID NO: 11 (CMP6: in particular, the nucleotide sequence represented by nucleotides 70 to 1458) is represented by SEQ ID NO: 12 SEQ ID NO: 13 (CMP7: in particular, the nucleotide sequence represented by nucleotides 60 to 1352) encodes the amino acid sequence represented by SEQ ID NO: 14. SEQ ID NO: 15 (CMP8: in particular, the nucleotide sequence represented by nucleotides 610 to 2943) encodes the amino acid sequence represented by SEQ ID NO: 16, and SEQ ID NO: 17 (CMP9: in particular, 77th to Nucleotide sequence represented by 1927 nucleotides) is the amino acid sequence represented by SEQ ID NO: 18. SEQ ID NO: 19 (CMP10: in particular, nucleotide sequence represented by nucleotides 40 to 645) encodes the amino acid sequence represented by SEQ ID NO: 20, and SEQ ID NO: 21 (CMP11: In particular, the nucleotide sequence represented by nucleotides 234 to 767) encodes the amino acid sequence represented by SEQ ID NO: 22, and SEQ ID NO: 23 (CMP12: in particular, the nucleotide represented by nucleotides 301 to 960). Sequence) encodes the amino acid sequence represented by SEQ ID NO: 24, and SEQ ID NO: 25 (CMP13: in particular, the nucleotide sequence represented by nucleotides 392 to 2320) is the amino acid represented by SEQ ID NO: 26. SEQ ID NO: 27 (CMP14: in particular, 396-250 Nucleotide sequence represented by 1 nucleotide) encodes the amino acid sequence represented by SEQ ID NO: 28, and SEQ ID NO: 29 (CMP15: in particular, nucleotide sequence represented by nucleotides 97 to 1962) SEQ ID NO: 31 (CMP16: in particular, nucleotide sequence represented by nucleotides 470 to 1921) encodes the amino acid sequence represented by SEQ ID NO: 32 SEQ ID NO: 34 (CMP18: in particular, nucleotide sequence represented by nucleotides 92 to 1774) encodes the amino acid sequence represented by SEQ ID NO: 35, and SEQ ID NO: 37 (CMP20: Nucleotide sequence represented by 113-763 nucleotides) is represented by SEQ ID NO: 38 SEQ ID NO: 39 (CMP21: in particular, nucleotide sequence represented by nucleotides 221 to 457) encodes the amino acid sequence represented by SEQ ID NO: 40, and SEQ ID NO: 39 41 (CMP22: in particular, the nucleotide sequence represented by the 14th to 1135th nucleotides) encodes the amino acid sequence represented by SEQ ID NO: 42, and SEQ ID NO: 43 (CMP23: in particular, the 25th to 987th nucleotides). The nucleotide sequence represented) encodes the amino acid sequence represented by SEQ ID NO: 44, and SEQ ID NO: 45 (CMP24: in particular, the nucleotide sequence represented by nucleotides 176 to 730) is represented by SEQ ID NO: 46. It encodes the amino acid sequence represented.

  Therefore, according to another preferred embodiment of the present invention, the gene marker is SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 35, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44 Or it consists of the nucleotide sequence which codes the amino acid sequence represented by sequence number 46.

  As described above, among the CMP genes having a protein coding region, CMP1-6, CMP8-10, CMP13, CMP18, CMP20, and CMP24 have an increased expression level in cells when the myocardial differentiation activity of the cells increases. . Therefore, according to a preferred embodiment of the present invention, the gene markers are SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20 , SEQ ID NO: 26, SEQ ID NO: 35, SEQ ID NO: 38, or SEQ ID NO: 46, which comprises a nucleotide sequence encoding the amino acid sequence, and when the myocardial differentiation activity of the cell increases, the expression level in the cell increases. It is characterized by this.

  Among CMP genes having a protein coding region, CMP7, CMP12, CMP16, CMP21 and CMP23 have a decreased expression level in cells when the myocardial differentiation activity of the cells is increased. Therefore, according to another preferred embodiment of the present invention, the gene marker consists of a nucleotide sequence encoding the amino acid sequence represented by SEQ ID NO: 14, SEQ ID NO: 24, SEQ ID NO: 32, SEQ ID NO: 40 or SEQ ID NO: 44 And when the myocardial differentiation activity of the cells increases, the expression level in the cells decreases.

  The gene marker according to the present invention is an amino acid sequence in which one or several amino acids are deleted, substituted, inserted and / or added in the amino acid sequence, and is functionally equivalent to the amino acid sequence. A polynucleotide comprising a nucleotide sequence encoding an amino acid sequence. Here, the “one or several” is preferably 1 to 5, more preferably 1 to 2.

  Furthermore, according to another preferred embodiment of the present invention, the genetic marker encodes an amino acid sequence having at least 60% homology with the amino acid sequence and functionally equivalent to the amino acid sequence. A polynucleotide comprising a nucleotide sequence. Furthermore, the homology in the gene marker is more preferably 70% or more, and still more preferably 80% or more.

  The gene marker may be a CMP gene or a fragment of a polynucleotide functionally equivalent to the gene as long as it can be used as an index of myocardial differentiation activity. Therefore, according to another aspect of the present invention, the gene marker consists of a CMP gene or a partial sequence of a polynucleotide functionally equivalent to the gene. The number of nucleotide residues of such a polynucleotide is not limited as long as it can be used as an index of myocardial differentiation activity, but is, for example, 100 nucleotide residues or more.

  The gene marker described above may be DNA or RNA. More specifically, the gene marker may be DNA existing in the genome or cDNA generated for mRNA that is a transcription product thereof. In addition, the above mRNA may be directly used as a gene marker, and the present invention includes such an embodiment. Therefore, according to another aspect of the present invention, a gene marker is obtained from a ribonucleotide sequence obtained by substituting a uracil residue for a thymine residue in the above nucleotide sequence encoding a CMP gene or a functionally equivalent gene thereof. A polynucleotide comprising:

  Further, the polynucleotide constituting the gene marker may be a single strand or a double strand in which a complementary strand is hybridized.

Polypeptide Marker According to the present invention, the expression product of CMP gene can be used as a marker for detecting myocardial differentiation activity of cells. And as this marker, the marker which consists of polypeptide encoded by said CMP gene 1-16, 18, 20-24 is mentioned. Therefore, according to another aspect of the present invention, polypeptide markers for detecting myocardial differentiation activity of cells are SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12. , SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 35, SEQ ID NO: 38, SEQ ID NO: It comprises a polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 40, SEQ ID NO: 42, SEQ ID NO: 44 or SEQ ID NO: 46.

  In addition, among the CMP genes having protein coding regions, polypeptides encoded by CMP1-6, CMP8-10, CMP13, CMP18, CMP20 and CMP24 increase the expression level in cells when the myocardial differentiation activity of the cells increases. sell. Therefore, according to a preferred embodiment of the present invention, the polypeptide marker comprises SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 16, SEQ ID NO: 18, sequence No. 20, SEQ ID NO: 26, SEQ ID NO: 35, SEQ ID NO: 38 or a nucleotide sequence encoding the amino acid sequence represented by SEQ ID NO: 46, and when the myocardial differentiation activity of the cell is increased, the expression level in the cell is It is characterized by increasing.

  In addition, among the CMP genes having a protein coding region, the polypeptide encoded by CMP7, CMP12, CMP16, CMP21 and CMP23 can decrease the expression level in cells when the myocardial differentiation activity of the cells increases. Therefore, according to another preferred aspect of the present invention, the polypeptide marker is a nucleotide sequence encoding the amino acid sequence represented by SEQ ID NO: 14, SEQ ID NO: 24, SEQ ID NO: 32, SEQ ID NO: 40 or SEQ ID NO: 44. When the myocardial differentiation activity of a cell increases, the expression level in the cell decreases.

  Furthermore, a polypeptide encoded by a gene functionally equivalent to the CMP gene can also be used as an indicator of cell myocardial differentiation activity. Therefore, according to another preferred embodiment of the present invention, the polypeptide marker consists of an amino acid sequence in which one or several amino acids are deleted, substituted, inserted and / or added in the amino acid sequence, and It comprises a polypeptide that is functionally equivalent to the polypeptide encoded by the CMP gene.

  According to another preferred embodiment of the present invention, the polypeptide marker comprises an amino acid sequence having at least 60% homology with the amino acid sequence and is functionally equivalent to a polypeptide encoded by the CMP gene. Comprising a polypeptide.

  In addition, the polypeptide marker according to the present invention does not need to have the entire sequence of the polypeptide encoded by the CMP gene or a functionally equivalent polynucleotide as long as it can be used as an index of myocardial differentiation activity. There may be. Thus, a polypeptide marker according to the present invention encompasses fragments of the aforementioned polypeptides. And the amino acid residue which such a fragment has is made into 13 or more, for example.

Cells Both the above gene marker and polypeptide marker can be used as an index of myocardial differentiation activity of cells in the undifferentiated stage. Therefore, the cell in the present invention is an undifferentiated cell. The cell in the present invention is preferably a mammal-derived cell, more preferably a human, mouse or monkey-derived cell.

Nucleic acid molecule / probe Further , in order to detect the myocardial differentiation activity of a cell using the gene marker according to the present invention as an indicator, the polynucleotide constituting the gene marker according to the present invention or a polynucleotide complementary thereto is used as a target, And nucleic acid molecules that can hybridize under stringent conditions. Here, complementary means that two nucleotides can be paired under hybridization conditions, for example, the relationship between adenine (A) and thymine (T) or uracil (U), It refers to the relationship between cytosine (C) and guanine (G).

  In the present invention, “hybridize” means that the nucleic acid molecule according to the present invention hybridizes to a gene marker under normal hybridization conditions, preferably under stringent hybridization conditions, to nucleotide molecules other than gene markers. Means not hybridized. Stringent conditions can be determined depending on the melting temperature Tm (° C.) of the duplex between the nucleic acid molecule of the present invention and its complementary strand, the salt concentration of the hybridization solution, etc., for example, J. Sambrook , EF Frisch, T. Maniatis; Molecular Cloning 2nd edition, Cold Spring Harbor Laboratory (1989), and the like. For example, when hybridization is performed at a temperature slightly lower than the melting temperature of the nucleic acid molecule to be used, the nucleic acid molecule can be specifically hybridized to the gene marker. In addition, the nucleic acid molecule that hybridizes to the gene marker according to the present invention does not need to be completely complementary to the gene marker, as long as specific hybridization is possible. It shall comprise the sequence of all or part of a complementary nucleotide molecule.

  In the present invention, “nucleic acid molecule” is used to mean DNA, RNA, and PNA (peptide nucleic acid). According to a preferred embodiment of the invention, the nucleic acid molecule is DNA or RNA.

  The nucleotide sequence of the nucleic acid molecule according to the present invention can be appropriately designed by those skilled in the art. As described above, the nucleic acid molecule according to the present invention targets a gene marker, but when the detection target is a genome, the nucleic acid molecule according to the present invention hybridizes not only to a portion that hybridizes to an exon, but also to an intron. A portion to be included.

  The nucleic acid molecule according to the present invention can be used as a nucleic acid probe in the detection of the gene marker. For this purpose, the nucleic acid probe may be prepared as a synthetic oligonucleotide using a commercially available oligonucleotide synthesizer or the like, or as a double-stranded DNA fragment obtained by restriction enzyme treatment or the like. May be.

  When the nucleic acid molecule according to the present invention is used as a nucleic acid probe, the chain length of the nucleic acid molecule is preferably 18 nucleotides or more, and preferably 30 nucleotides or less, more preferably 24 nucleotides or less.

  Furthermore, when the nucleic acid molecule according to the present invention is used as a nucleic acid probe, it is preferable to appropriately label the nucleic acid molecule. As a labeling method, a method of labeling by phosphorylating the 5 ′ end of the oligonucleotide with 32P using T4 polynucleotide kinase, and a DNA polymerase such as Klenow enzyme, a random hexamer oligonucleotide or the like as a primer And a method of incorporating a substrate base labeled with an isotope such as 32P, a fluorescent dye, biotin or the like (random prime method or the like).

  Moreover, the nucleic acid molecule according to the present invention can be used as a primer for nucleic acid amplification in the detection of the gene marker according to the present invention. When the nucleic acid molecule according to the present invention is used as a primer for nucleic acid amplification, the chain length of the nucleic acid molecule is preferably 18-30 nucleotides, more preferably 18-24 nucleotides.

  The nucleic acid amplification method is usually performed using a pair of primers. Therefore, according to the present invention, there is provided a primer pair for detecting the gene marker, which can amplify all or part of the gene marker according to the present invention. As the two primers constituting such a primer pair, the nucleic acid molecule according to the present invention can be used. Such a primer can be appropriately designed by those skilled in the art based on the nucleotide sequence of the region to be amplified. For example, one primer of the primer pair has a 5 ′ terminal sequence in the nucleotide sequence of the amplification target region, and the other primer has a 5 ′ terminal sequence in the nucleotide sequence of the complementary strand of the amplification target region. It can have an array.

  According to a preferred embodiment of the present invention, a method known as TaqMan PCR method is used as a method for detecting the gene marker. In the TaqMan PCR method (Livak KJ. Genet. Anal. Vol.14, pp143-149, (1999)), the probe specifically hybridizes to the gene marker according to the present invention, and the fluorescent labeling substance has a 5 ′ end. A quencher (quenching substance) for the fluorescent label is attached to the 3 ′ end, and a probe (TaqMan probe) in which the 3 ′ end is phosphorylated is used, and these are added to the PCR reaction solution. Then, a PCR reaction is carried out using a cell-derived nucleic acid sample as a template. As the TaqMan probe and the PCR primer, the nucleic acid molecule according to the present invention can be used, and the specific nucleotide sequence thereof can be appropriately determined by those skilled in the art. The fluorescent labeling substance, the quencher, and combinations thereof can be those known to those skilled in the art. For example, examples of the fluorescent labeling substance include FAM and VIC, and examples of the quencher include MGB, TAMRA etc. In such a PCR reaction, the TaqMan probe is first hybridized to the gene marker according to the present invention, and when the extension reaction from the primer reaches the hybridization region, the fluorescent labeling substance is released by the action of Taq DNA polymerase. Since the released fluorescent labeling substance is not affected by the quencher, it emits fluorescence. Therefore, according to this method, each fluorescence having an intensity corresponding to the expression level of the gene marker can be observed, whereby the expression level of the gene marker can be easily determined.

  In addition, in order to detect the myocardial differentiation activity of cells, the nucleic acid molecules and / or primer pairs as described above can be collected together with necessary reagents to form a kit. Therefore, according to another aspect of the present invention, there is provided a kit comprising at least the nucleic acid molecule and / or primer pair.

The myocardial differentiation regulator and the CMP gene of the present invention or a polynucleotide functionally equivalent thereto can be used to suppress or induce myocardial differentiation of cells by controlling its expression in cells. In particular, when targeting mRNA expressed by a CMP gene or a polynucleotide functionally equivalent thereto, RNAi (RNA interference) can suppress or induce myocardial differentiation of cells.

  Among the CMP genes, CMP1-6, CMP8-10, CMP13, CMP18, CMP20 and CMP24 expression levels positively correlated with increased myocardial differentiation activity of cells, and when the expression of these genes was suppressed by RNAi, It can suppress myocardial differentiation of cells. Therefore, according to one embodiment of the present invention, RNAi can suppress the expression of a polynucleotide comprising CMP1-6, CMP8-10, CMP13, CMP18, CMP20 or CMP24 or a polynucleotide functionally equivalent thereto. A cell myocardial differentiation inhibitor comprising a single-stranded RNA molecule as an active ingredient, wherein the antisense strand of the double-stranded RNA molecule comprises a nucleotide sequence that specifically hybridizes with the polynucleotide in the cell. The myocardial differentiation inhibitor is provided.

  Among the CMP genes, the expression levels of CMP7, CMP12, CMP16, CMP17, CMP21 and CMP23 showed a negative correlation with the increase in myocardial differentiation activity of cells, and when the expression of these genes was suppressed by RNAi, Myocardial differentiation can be induced. Therefore, according to one embodiment of the present invention, a double-stranded RNA molecule capable of suppressing the expression of a polynucleotide consisting of CMP7, CMP12, CMP16, CMP17, CMP21 and CMP23 or a polynucleotide functionally equivalent thereto by RNAi. A myocardial differentiation inducing agent for cells comprising an active ingredient as an active ingredient, wherein the antisense strand of a double-stranded RNA molecule specifically comprises a nucleotide sequence that hybridizes specifically with the polynucleotide in the cell. An inducer is provided.

  RNAi refers to a phenomenon in which double-stranded RNA molecules suppress their expression by degrading target RNA containing the same sequence. Here, the above-mentioned double-stranded RNA molecule refers to an RNA molecule obtained by hybridizing two single-stranded RNA molecules in whole or in part. Furthermore, in the double-stranded RNA molecule, a nucleotide chain having a sequence homologous to the target sequence is referred to as a sense strand, and a nucleotide chain having a sequence complementary to the target sequence is referred to as an antisense strand. In addition, “specifically hybridize” means that the above-described antisense strand suppresses the expression of a CMP gene or a functionally equivalent polynucleotide in a cell in which the CMP gene or a functionally equivalent polynucleotide is expressed. It means not hybridizing to nucleic acid molecules other than nucleotides and their transcripts. Such an antisense strand is said to contain a sequence corresponding to the target sequence in the CMP gene or a functionally equivalent polynucleotide, but need not have a sequence that is completely complementary to the target sequence, It may contain non-complementary nucleotides so long as it specifically hybridizes to the sequence. However, the antisense strand in the myocardial differentiation inhibitor is preferably composed of a sequence complementary to the target sequence.

  The double-stranded RNA molecule may be composed of different strands and may be provided by a single RNA stem-loop structure. In addition, the chain length of the single-stranded RNA molecule in the double-stranded RNA molecule has at least 19 residues, preferably 19 to 27 residues, in view of obtaining an appropriate RNAi effect. In addition, the sense strand and the antisense strand may have the same chain length, but by providing an overhang of about 2 bases on the 3 ′ side of each strand, the gene expression suppression effect can be enhanced. it can.

  The double-stranded RNA molecule is synthesized, for example, by a known method using an oligonucleotide synthesizer or the like with a sense strand homologous to a CMP gene or a target sequence of a polynucleotide functionally equivalent thereto and an antisense strand thereof. , Can be created by annealing each. In addition, regarding the target sequence and the length and structure of the double-stranded RNA molecule as described above, those skilled in the art can try various modifications using known techniques, and have the strongest gene expression suppressing effect. It is possible to optimize double stranded RNA molecules.

  Another method for controlling myocardial differentiation of cells is a method of suppressing the expression of a CMP gene or a polynucleotide functionally equivalent thereto using antisense, ribozyme, etc. in the present invention. Such an embodiment is also included.

  In addition, as described above, the expression levels of CMP1-6, CNP8-10, CMP13, CMP18, CMP20, and CMP24 have a positive correlation with an increase in myocardial differentiation activity of cells. Therefore, according to another aspect of the present invention, myocardial differentiation induction comprising CMP1-6, CNP8-10, CMP13, CMP18, CMP20 and CMP24 or a functionally equivalent polynucleotide thereof in a functional form. An agent is provided. In addition, the expression levels of CMP7, CMP12, CMP16, CMP17, CMP21 and CMP23 have a negative correlation with an increase in myocardial differentiation activity of cells. Therefore, according to another preferred aspect of the present invention, there is provided a myocardial differentiation inhibitor comprising functionally the CMP7, CMP12, CMP16, CMP17, CMP21 and CMP23 or a functionally equivalent polynucleotide thereof. Is done.

  Examples of the myocardial differentiation controlling agent, that is, a myocardial differentiation inducing agent or inhibitor, include, for example, a vector in which the polynucleotide is inserted in a functional manner. As used herein, “comprising in a functional manner” means in a manner that allows expression of the polynucleotide (DNA) under the control of appropriate regulatory elements (eg, promoters, enhancers, transcription terminators, etc.) It means that the polynucleotide is inserted into the vector.

  Such vectors can be constructed by standard methods well known in the art, eg, Sambrook, J. et al., “Molecular Cloning: a laboratory manual”, Cold Spring Harbor Laboratory Press, New York (1989). ).

Method for Detecting Cell Myocardial Differentiation Activity As described above, the gene marker according to the present invention is used as an indicator of cell myocardial differentiation activity. Therefore, according to another aspect of the present invention, there is provided a method for detecting myocardial differentiation activity of a cell, comprising measuring the expression level of the polypeptide marker or gene marker in the cell, and the expression in the cell. A method is provided wherein the amount correlates with the level of myocardial differentiation activity of the cell.

  In addition, as described above, the expression level of CMP1-6, CMP8-10, CMP13, CMP18, CMP20 and CMP24 positively correlated with increased myocardial differentiation activity of cells, CMP7, CMP12, CMP16, CMP17, CMP21 and CMP23 Is negatively correlated with an increase in myocardial differentiation activity of cells. These properties are advantageous in accurately detecting the myocardial differentiation activity of cells. Therefore, according to another preferred embodiment of the present invention, in cells of a marker consisting of CMP1-6, CMP8-10, CMP13, CMP18, CMP20 or CMP24, a polynucleotide functionally equivalent thereto, or a gene product thereof. Measuring the expression level, and when the expression level is higher than a preset threshold value with reference to the expression level of the marker in a control cell having no myocardial differentiation activity, A method of having activity is provided. According to another preferred embodiment of the present invention, the expression level of a marker consisting of CMP7, CMP12, CMP16, CMP17, CMP21 or CMP23, a functionally equivalent polynucleotide, or a marker comprising these gene products is measured. A cell having a myocardial differentiation activity when the expression level is lower than a preset threshold value with reference to the expression level of the marker in a control cell having no myocardial differentiation activity A method is provided.

  Measurement of the expression level of the marker is performed by, for example, collecting a cell-derived biological sample, extracting a nucleic acid sample such as genomic DNA or mRNA from the obtained sample, and, if necessary, cDNA from the mRNA by reverse transcriptase. The nucleic acid amplification method is carried out using the obtained nucleic acid sample as a template and the nucleic acid molecule or primer pair according to the present invention, and the expression level of the obtained amplification product is analyzed and quantified. . Any method known in the art may be used as the nucleic acid amplification method and the marker detection method using the nucleic acid amplification method. For example, as a nucleic acid amplification method, a normal PCR method or the like can be used when genomic DNA or cDNA is used as a template, and an RT-PCR method or NASBA method or the like can be used when mRNA is used as a template. Can do.

  Moreover, the threshold value of the expression level of the marker in the control cell can be set by a known statistical method with reference to the quantitative value of the marker measured in advance by the above method. Specific examples of the method for setting the expression level include (average value of marker expression level in control cells ± standard deviation) or ROC (Receiver Operating Characteristic) analysis.

  Moreover, the said detection method can be utilized for the efficient and simple isolation of the cell which has myocardial differentiation activity. Therefore, according to another preferred embodiment of the present invention, there is provided a method for isolating cells having myocardial differentiation activity, wherein cells having myocardial differentiation activity are detected and isolated by the above detection method. A method comprising is provided. Cells isolated by such a method can be advantageously used as transplant cells for the treatment of refractory heart diseases such as cardiomyopathy and ischemic heart disease.

  In the above method, the isolation step is appropriately performed under culture conditions known to those skilled in the art. In addition, as a method for isolating the cells, for example, a method of isolating cells by flow cytometry using an antibody against the marker (FACS), or using a magnetic bead carrying an antibody, specific cells can be isolated. Examples include a method of collecting and isolating with a magnet (MACS).

  Further, by measuring the expression level of the marker according to the present invention in the cells over time, the differentiation state of the cells into the myocardium can be monitored. Therefore, according to another preferred embodiment of the present invention, there is provided a method for monitoring myocardial differentiation activity of a cell, comprising CMP1-6, CMP8-10, CMP13, CMP18, CMP20 or CMP24, a polynucleotide functionally equivalent thereto, Alternatively, there is provided a method comprising measuring the expression level of a marker comprising these gene products in a cell, and when the expression level increases with time, the myocardial differentiation activity of the cell is increased. According to another preferred embodiment of the present invention, there is provided a method for monitoring myocardial differentiation activity of a cell, comprising CMP7, CMP12, CMP16, CMP17, CMP21 or CMP23, a polynucleotide functionally equivalent thereto, or these Measuring the expression level of the marker comprising the gene product in a cell, and when the expression level decreases with time, a method is provided in which the myocardial differentiation activity of the cell is increased.

Cell Myocardial Differentiation Control Method Also, the myocardial differentiation of cells can be induced or suppressed by introducing the myocardial regulator according to the present invention into cells. Therefore, according to another aspect of the present invention, there is provided a method for inducing myocardial differentiation of a cell, comprising introducing the myocardial differentiation inducing agent according to the present invention into a cell. Moreover, according to another aspect of the present invention, there is provided a method for inhibiting myocardial differentiation of a cell, comprising introducing the myocardial differentiation inhibitor according to the present invention into a cell.

  As the method for introducing the myocardial differentiation regulator, methods known in the art as transfection or transformation methods can be used. For example, calcium phosphate method, electroporation method, microinjection method, DEAE-dextran. Method, a method using a liposome reagent, a lipofection method using a cationic lipid, and a transfection using a virus vector. In this method for controlling myocardial differentiation, the gene product of the polynucleotide constituting the gene marker according to the present invention may be directly introduced into a cell, and the present invention includes such an embodiment.

  EXAMPLES Hereinafter, although an Example demonstrates this invention more concretely, this invention is not limited to these Examples.

  Unless otherwise specified in the examples, J. Sambrook, EF Fritsch & T. Maniatis (Ed.), Molecular cloning, a laboratory manual (3rd edition), Cold Spring Harbor Press, Cold Spring Harbor, New York (2001); Standard protocols such as FM Ausubel, R. Brent, RE Kingston, DD Moore, JG Seidman, JA Smith, K. Struhl (Ed.), Current Protocols in Molecular Biology, John Wiley & Sons Ltd. Or a modified or modified method thereof is used. In addition, when using commercially available reagent kits and measuring devices, unless otherwise explained, protocols attached to them are used.

Example 1: Evaluation of cell differentiation efficiency
Obtaining Cells Fetal cancer cell P19 cells (ATCC number: CRL-1825) derived from mice were donated by the American Type Culture Collection (ATCC). The subline CL6 cells (RCB number: RCB1539) were provided by the RIKEN BioResource Center (RIKEN Cell Bank). In addition, CLDNA cells were sequentially vectorized with a vector having a neomycin resistance gene, pcDNA3.1 (+) (Invitrogen), mouse αMHC promoter, EGFP gene (manufactured by Clontech) and human growth hormone poly A signal in order from the 5 ′ direction. P Bluescript II SK (+) (Stratagene) contained in the cloning site was introduced with Lipofectamine 2000 (Invitrogen) and screened with Geneticine (G418, Sigma). As a result, the CL6 cell sublines CL6G26 (hereinafter referred to as “G26”), CL6G36 (hereinafter referred to as “G36”), CL6G45 (hereinafter referred to as “G45”), CL6G52 (hereinafter referred to as “G52”) containing the neomycin resistance gene. Got.
The following experiment was performed using the above cells.

For the growth of the above undifferentiated cells, 10% fetal calf serum (FCS, fetal calf serum), 2 mM L, inactivated as a basic medium (inactivated complement components by heat treatment at 56 ° C. for 30 minutes) -5% on a 100 mm diameter cell culture dish (Falcon) using MEM medium (Minimum Essential Medium, SIGMA) containing glutamine (SIGMA), penicillin G (100 units / mL) and streptomycin sulfate (100 units / mL) The cells were cultured at 37 ° C. in the presence of CO ₂ so that the cells did not become confluent.

Differentiation induction treatment Next, the undifferentiated cells were cultured and proliferated to about 60 to 70% of the dish area to induce differentiation into cardiomyocytes. As a differentiation induction treatment, first, the basic medium was removed and washed twice with 5 mL of sterilized phosphate-buffered saline (FBS, SIGMA). Next, after gently wetting the whole cell with 1 mL of trypsin-EDTA (Gibco), immediately aspirate trypsin-EDTA and incubate in the presence of 5% CO _{2 at} 37 ° C for 3 minutes and then at room temperature for a little less than 5 minutes to stop the reaction. Therefore, it was suspended in 10 mL of differentiation medium (MEMα medium containing 1% DMSO). The cell suspension was dispensed into a cell culture vessel (Falcon) to obtain differentiation-treated cells. Furthermore, the obtained cells were seeded in a 6-well cell culture multiwell plate (Costar) at a density of 1 × 10 ⁵ cells / well. This was cultured in a 37 ° C. differentiation medium in the presence of 5% CO ₂ , and the medium was changed every two days.

Comparison of contraction nodules For the above cells, observe the appearance date (contraction start date), size and number of contraction nodules every 2 days with a CKX41 or CKX31 culture microscope (OLYMPUS) to evaluate differentiation efficiency. did. When evaluating, the number of shrinkage nodules is 0.016 / cm ² or more (when there is more than one shrinkage per plate), 0.098 / cm ² or more (more than one shrinkage per well) “Sometime” was defined as “medium” and 157 / cm ² or more (when there was one or more contractions per field of view (x200) of the microscope) as “many”. Further, regarding the number of contraction nodules, a score of 0 was given for those with no contraction, 1 was given for “less”, 2 was given for “medium”, and 3 was given for “more”. The shrinkage nodule size should be 6.36 × 10 ^-5 cm ² or more (shrinking to a point size of about 1/100 or more of the microscope field of view (× 200)). `` Small '', those with a size of 3.18 × 10 ⁻³ cm ² or more (shrinking into a sheet with a size of about 1/2 or more of the microscope field of view (× 200)) Defined. Further, regarding the size of the shrinkage nodule, a score of 0 was given to the “no” shrinkage, 1 to the “small” and 2 to the “large”.

  The result using the number of contraction nodules as an index is as shown in FIG. 1A, and the result using the size of contraction nodules as an index is as shown in FIG. 1B. 1A and B, the horizontal axis represents the number of days after induction of myocardial differentiation. From the results of FIGS. 1A and 1B, it was confirmed that there was a difference in the start date of contraction and the number and size of contraction nodules depending on the cell line. In particular, for P19 cells, after differentiation induction, the cells became confluent on the 6th day, and on the 16th day, one or more small contractions were observed for each well of the plate, and the contraction was at least until the 20th day. Was observed to continue. As a result, P19 cells can be differentiated into cardiomyocytes without the formation of embryoid bodies, as in CL6 cells, by culturing in the presence of DMSO under adhesion conditions for a relatively longer period of time than CL6 cells. Was confirmed.

Example 2: Confirmation of cardiomyocyte marker gene expression and principal component analysis
Comparison of expression of cardiomyocyte marker gene Six different cell lines, P19 cells, CL6 cells and CL6 cell sublines including Neomycin resistance gene G26, G36, G45 and G52 were treated with myocardial differentiation in the same manner as in Example 1. And culture. Each cell line was recovered by SV Total RNA Isolation system (Promega) on the 8th, 12th, 16th and 20th days, and total RNA was extracted. As a positive control, ventricular muscles were isolated from 9-week-old male C3H / He mice, and total RNA was extracted with Sepazol (Nacalai Tesque). After extracting the total RNA, the expression of the existing cardiomyocyte marker gene in the RNA was measured by quantitative RT-PCR. As myocardial cell marker genes, select MLC2a (myosin light chain 2a), MLC2v (myosin light chain 2v), αMHC (α myosin heavy chain) and βMHC (β myosin heavy chain) as myocardial fiber genes and transcription related to myocardial differentiation Nkx2.5, GATA4 and MEF2C were selected as factors. Moreover, in order to normalize the expression level of each gene, the expression level of 18S rRNA was also measured. In the quantitative RT-PCR, the primers and TaqMan probe shown in Table 1 below, TaqMan One-Step RT-PCR Master Mix Reagent (Applied Biosystems) and ABI Prism 7000 Sequence Detection System were used. In the Taqman probe, FAM was used as the fluorescent labeling substance at the 5 ′ end, and TAMRA was used as the quencher at the 3 ′ end.

  The results were as shown in FIGS. 2A to 2G. 2A to 2G, the vertical axis represents relative values when mRNA / 18S rRNA values are compared with adult mouse myocardium. Moreover, the value of mRNA / 18S rRNA is an average value (n = 6).

  As shown in FIG. 2A to FIG. 2G, differences in the expression of cardiomyocyte marker genes were confirmed depending on the cell line, and it was quantitatively confirmed that there was a difference in the state of differentiation into myocardium.

Principal component analysis When interpreting the expression of the above cardiomyocyte marker gene in association with cardiomyocyte differentiation, it is not clear how much weight the expression of each cardiomyocyte marker gene should be interpreted. Therefore, principal component analysis was performed based on the mRNA expression data of the above-mentioned cardiomyocyte marker gene obtained for the cell lines P1, CL6, G26, G36, G45 and G52. The principal component analysis was performed using statistical software SYSTAT (SYSTAT Software). The mRNA expression level of the cardiomyocyte marker gene was standardized based on the average value and standard deviation of the samples.

  As a result of the principal component analysis, the scree plot shown in FIG. 3A was obtained, and the first principal component with a contribution rate of 65% and the second principal component with a contribution rate of 15% were adopted. Here, the cumulative contribution ratio of the two main components was 80% or more.

  Moreover, the variable plot regarding the said two main components was as FIG. 3B shows. The first principal component was interpreted as an index of myocardial differentiation because the coefficient values of all variables were positive. For the second main component, the coefficient of the cardiomyocyte marker gene functioning relatively early in the development was negative, and the coefficient of the cardiomyocyte marker gene functioning relatively late in the development was positive. , Interpreted as an indicator of maturity.

  In addition, with respect to the two main components, changes with time in the main component scores of each cell line were as shown in FIGS. 4A and 4B. It was confirmed that there were differences in the differentiation state into cardiomyocytes depending on the type of cell line. In FIG. 4A, the vertical axis indicates the first principal component score. In FIG. 4B, the vertical axis represents the second principal component score.

Example 3: Identification of markers for cardiomyocyte differentiation detection
Acquisition of Sequence Tag
Total RNA was extracted from 6 types of cell lines, P19, CL6, G26, G36, G45, and G52, using RNeasy Midi (QIAGEN), taking care not to become confluent before differentiation induction. Next, the obtained RNA was cleaned up twice using RNeasy Mini (QIAGEN). In order to comprehensively analyze gene expression by GeneChip (Affymetrix), cDNA was synthesized from each RNA sample according to the manual of Affymetrix, and then a biotinylated cRNA fragment was synthesized based on the cDNA. Next, the biotinylated cRNA fragment was hybridized to GeneChip using GeneChip Hybridizatioin Oven, and then the hybridized cRNA was visualized by staining with streptavidin-phycoerythrin using GeneChip Fluidics Station. Scanned. The obtained fluorescence data was analyzed using GCOS software (Affymetrix). Here, MOE430A (Sequence Tag number 22,626) and MOE430B (Sequence Tag number 22,511) were used for GeneChip. 2% from the highest signal and 2% from the lower signal were excluded from the extracted genes, and the average value of the remaining Sequence Tags was corrected to 500 by default. The following filters were applied to extract genes correlated with cardiomyocyte differentiation of each cell line.

Filter 1
The signal of each Sequence Tag analyzed by GCOS is the result of Absolute Analysis (Analysis to determine the presence or absence of expression) "Those with expression: P (Present)", "Things with unknown expression: M (Marginal)" or Judgment is made as “no expression: A (Absent)”. About Sequence Tag determined to be P in more than half (that is, 3 or more) of 5 cases in each group of cell lines, it was determined that a gene containing the base sequence of the Sequence Tag was expressed in the cell line. On the other hand, when 5 or less of the 5 cases in each group were 2 or less, it was determined that there was no expression of the gene containing the base sequence of the Sequence Tag in the cell line. Sequence tags in which expression was observed in at least one cell line were subjected to the following filter, and Sequence tags in which expression was not observed in all cell lines were discarded.

Filter 2
Analysis of variance (ANOVA) compares the average gene expression between cell lines, and the null hypothesis was rejected under the condition of 5% significance level, that is, cell lines with significantly different expression levels among 6 cell lines Sequence tags that resulted in the presence of at least one were subjected to the next filter and sequence tags that did not show significant differences across all cell lines were rejected.

Filter 3
Sequence tags that have an average gene expression difference of 50% or more between cell lines, that is, the difference between the lowest average value and the highest average value of 6 cell lines more than 2.5 times, apply the following filter, Those less than 2.5 times were rejected.

Filter 4
For the six types of P19, CL6, G26, G36, G45 and G52, the expression signal of the above-mentioned Sequence Tag obtained by GeneChip before differentiation induction, the number of days until the appearance of automatic beating ability, and the score of the number of cell nodules that automatically beat Calculate Spearman's rank correlation coefficient between the first principal component score or the second principal component score of myocardial gene expression data obtained by quantitative RT-PCR and significant difference under the condition of 5% significance level And a Sequence Tag with a significant correlation was extracted.

  As a result of the above Sequence Tag extraction, 109 Sequence Tags correlated with the first principal component, 342 Sequence Tags correlated with the second principal component, and Sequence Tag correlated with the number of days until the appearance of automatic pulsation ability is 122. 274 Sequence Tags correlated with the number of contraction nodules were extracted. Among these Sequence Tags, there were a total of 24 Sequence Tags significantly correlated with the three elements of the first principal component, which is an index of myocardial differentiation, the number of days until the appearance of automatic pulsation ability, and the number of contraction nodules. The genes corresponding to these 24 Sequence Tags were named CMP1-24. In addition, among CMP1 to 24, there is a significant correlation with the four components of the first principal component (index of cardiomyocyte differentiation), the second principal component (index of cardiomyocyte maturation), the number of days until the appearance of automatic pulsatile capacity and the number of contractile nodules There were 9 genes with.

Function prediction by SOSUI system In addition, the function prediction analysis was performed on the amino acid sequences encoded by the 24 CMP genes using the SOSUI system (Narumi Miyake Laboratory, Nagoya University). As a result of the analysis, 12 genes were determined to be membrane-bound proteins, and 10 genes were determined to be soluble proteins.

  The results of the statistical analysis and the function analysis by the SOSUI system are as shown in Table 2. In Table 2, rs represents Spearman's rank correlation coefficient, and p represents the p-value of Spearman's rank correlation coefficient. In addition, the genes indicated by * (CMP1-6, 8, 11 and 12) are significantly correlated with the four components of the first principal component, the second principal component, the number of days until the appearance of automatic pulsation and the number of contractile nodules. It is what was done.

Example 4: Control of cardiomyocyte differentiation by RNAi 1
CMP2 and CMP13 were set as RNAi targets for the purpose of investigating whether the myocardial differentiation efficiency could be controlled by controlling the expression of the CMP gene. And StealthRNAi (Invitrogen) which has a nucleotide sequence of Table 3 was introduce | transduced into G52 cell by the lipofection method. StealthRNAi Negative Control Medium GC Duplex (Invitrogen) was used as a control (NC).

After the siRNA was introduced into the cells, the expression levels of CMP2 and CMP13 were measured by quantitative RT-PCR using the PCR primers and Taqman probe described below. At this time, 18S rRNA was used as an internal standard in quantitative RT-PCR. In the Taqman probe, FAM was used as the fluorescent labeling substance at the 5 ′ end, and TAMRA was used as the quencher at the 3 ′ end. The specific method of this experiment was the same as in Example 5 below.
CMP2 gene Forward primer: 5'-CCATCCTGAATCCTAGATACCATCTC-3 '(SEQ ID NO: 72)
Reverse primer: 5'-TAGTGGCAACCATGCTGAGTGT-3 '(SEQ ID NO: 73)
Taqman probe: 5'-CCGATGGACGTGAGGACAGTAATCTGGA-3 '(SEQ ID NO: 74)
CMP13 gene Forward primer: 5'-CGCCTCTCTTCGTTTATCTGTTC-3 '(SEQ ID NO: 75)
Reverse primer: 5'-TCTCTGTCTTGGTGCCGTCAT-3 '(SEQ ID NO: 76)
Taqman probe: 5'-ACTTCTTTCCTGCTGGCCGGTTTCGT-3 '(SEQ ID NO: 77)
The results were as shown in FIGS. 5A and B. StealthRNAi suppressed the expression of CMP2 and CMP13 by about 70%. Furthermore, as shown in FIG. 6, myocardial differentiation of cells was suppressed under conditions where the expression of CMP2 and CMP13 was suppressed. In particular, the myocardial differentiation of the cells was almost completely suppressed under the condition where the expression of CMP2 was suppressed.

Example 5: Control of cardiomyocyte differentiation by RNAi 2
RNAi
Of the CL6 cell subline, G52 cells were seeded at 1 × 10 ⁴ cells / well in a multiwell plate (Falcon) for 24-well cell culture without differentiation induction treatment. This was cultured overnight at 37 ° C. and 5% CO ₂ . Each cell in the well was washed twice with 500 μL of an antibiotic-free basic medium, and 400 μL of the same medium was added to each well.
Next, siRNAs having nucleotide sequences described in Tables 4 to 5 below obtained using Stealth RNAi (Invitrogen) or siRNA (QIAGEN) were added to each cell in the well using Lipofectamine 2000 (Invitrogen). And CMP1-24 were knocked down.

Specifically, first, siRNA with OPTI-MEMI Reduced Serum Medium (Gibco) containing BLOCK-iT (Invitrogen) (final concentration 10 pmol / well) as a fluorescent oligo so that siRNA per well becomes 40 pmol. Was diluted to obtain a siRNA solution. Further, Stealth RNAi Negative Control Duplexes (Invitrogen), which is a negative control (NC), was similarly diluted. Then, it diluted with OPTI-MEMI Reduced Serum Medium so that Lipofectamine 2000 might be 1.5 microliter / well, and it incubated at room temperature for 5 minutes, and obtained Lipofectamine 2000 solution. Next, 50 μL of siRNA solution or negative control solution and 50 μL of Lipofectamine 2000 solution were mixed and incubated at room temperature for 20 minutes to form a complex. 100 μL of this complex was added to each well and mixed while gently shaking, and incubated at 37 ° C. for 48 hours under 5% CO ₂ .

After introducing siRNA into cells, the expression level of CMP1-24 was measured by quantitative RT-PCR. Specifically, first, total RNA was extracted from each cell using RNesay Mini (QIAGEN) or BioRobot M48 Workstation (QIAGEN). This total RNA was diluted to 2 ng / μL and used as a measurement sample. As a sample for the calibration curve, undifferentiated CL6G52 was cultured in a cell culture dish (Falcon) having a diameter of 100 mm so as not to become confluent, and Total RNA extracted from the cells using BioRobot M48 Workstation (QIAGEN) was used. Diluted to 20 ng / μl, 6 ng / μl, 2 ng / μl, 0.6 ng / μl, 0.2 ng / μl, 0.06 ng / μl and used to create a calibration curve.
In order to correct the expression level of each gene, 18S rRNA was measured as an internal standard. For 18S rRNA measurement, each measurement sample is diluted to 0.02 ng / μL, and the calibration curve samples are 0.2 ng / μl, 0.06 ng / μl, 0.02 ng / μl, 0.006 g / μl, 0.002 ng / μl And diluted to 0.0006 ng / μl.

  Using the above sample, quantitative RT-PCR was performed by ABI PRISM 7000 Sequence Detection System (Applied Biosystems). In addition, quantitative RT-PCR about CMP1-24 was performed using TaqMan Master Mix and TaqMan One-Step RT-PCR Master Mix Reagents (Applied Biosystems). For CMP19, the measurement using the RQuantiTect Probe RT-PCR Kit (QIAGEN) was performed again in consideration of the detection sensitivity. The obtained results were corrected by the expression value of 18S rRNA, and the fluctuation value of each gene was analyzed by comparing the correction value of each sample with the correction value of the negative control (NC).

  The PCR primers and Taqman probes used in the above quantitative RT-PCR are as shown in Table 6 and Table 7. In the Taqman probe, FAM was used as the fluorescent labeling substance at the 5 'end, and TAMRA was used as the quencher at the 3' end.

  The results were as shown in FIGS. 7A and B. After applying RNAi to cells, the expression levels of CMP1-10, CMP12-18, CMP20-21 and CMP23-24 were significantly reduced compared to control (NC) (Student't-test, p <0 .05).

Measurement of contraction nodule number After introduction of the siRNA, changes in the contraction nodule number in the culture well were observed, and the results were as shown in FIGS.

  In cells treated with RNAi for CMP1-6, CMP9, CMP10, CMP13, CMP18, and CMP24, the number of contractile nodules was significantly lower compared to the control (NC) (repeated measurement two-way analysis of variance (two- way repeated-measures ANOVA), p <0.05). On the other hand, the number of contraction nodules was significantly higher in CMP7, CMP12, CMP16, CMP17, CMP21, and CMP23 than in the control (NC) (two-way repeated-measures ANOVA), p <0.05).

Measurement of expression level of cardiomyocyte marker gene
After introduction of siRNAi into the above cells, the expression level of existing cardiomyocyte marker genes (myocardial fiber genes αMHC, βMHC, MLC2a and MLC2v) is measured by quantitative RT-PCR. , CMP12-13, CMP16-18, CMP20-21, and CMP23-24 expression levels were compared. Quantitative RT-PCR was performed in the same manner as in Example 2 using the primers listed in Table 1 and TaqMan probe.

  After knocking down the CMP gene with RNAi, changes in the expression level of the existing cardiomyocyte marker genes (αMHC, βMHC, MLC2a, MLC2v) were as shown in Table 8. Further, Table 8 also shows changes in the number of contraction nodules after the CMP gene was knocked down.

: Shows that it was significantly knocked down by RNAi (p <0.05 vs. NC).
↓: Indicates a significant decrease after knockdown (p <0.05 vs. NC).
↑: Significantly increased after knockdown (p <0.05 vs. NC).
NS: No significant difference.

  As shown in Table 8, the expression of any of the cardiomyocyte marker genes (αMHC, βMHC, MLC2a or MLC2v) is achieved by knocking down CMP1-6, CMP8, CMP9, CMP10, CMP13, CMP18, CMP20, CMP24 with RNAi. It was confirmed that the number of contraction nodules was suppressed or cardiomyocyte differentiation was suppressed. In addition, knockdown of CMP7, CMP12, CMP16, CMP17, CMP21, and CMP23 by RNAi promotes the expression of any cardiomyocyte marker gene (αMHC, βMHC, MLC2a or MLC2v) or increases the number of contractile nodules It was confirmed that cardiomyocyte differentiation was induced. In particular, for CMP23, the number of contractile nodules was increased, and the cardiomyocyte marker genes αMHC, βMHC, and MLC2v were significantly increased, while MLC2a was significantly decreased. Since MLC2a that characterizes atrial muscle decreased and MLC2v that characterizes ventricular muscle increased, it was confirmed that CMP23 can be used for specific induction of cells into ventricular muscle.

  This is useful for detecting the myocardial differentiation activity of undifferentiated cells.

Sequence number 47,48,50,51,53,54,56,57,59,60,62,63,65,66,72,73,75,76,123,124,126,127,129,130, 132, 133, 135, 136, 138, 139, 141, 142, 144, 145, 147, 148, 150, 151, 154, 156, 157, 159, 160, 162, 163, 165, 166, 168, 169, 171, 172, 174, 175, 177, 178, 180, 181, 183, 184, 186, 187: Primer SEQ ID NOs: 49, 52, 55, 58, 61, 64, 67, 74, 77, 122, 125, 128 131, 134, 137, 140, 143, 146, 149, 152, 155, 158, 161, 164, 167, 170 , 173, 176, 179, 182, 185: Taqman probe SEQ ID NO: 68, 69, 70, 71, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116 , 117, 118, 119, 120, 121: one strand of siRNA

Claims (12)

A genetic marker for detecting myocardial differentiation activity of undifferentiated cells before differentiation induction, polynucleotide or Ranaru consisting of the nucleotide sequence represented by SEQ ID NO: 3, genetic markers.   The gene marker according to claim 1, wherein when the myocardial differentiation activity of the cell increases, the expression level in the cell increases.   A primer pair for detecting myocardial differentiation activity of undifferentiated cells before differentiation induction, which can amplify all or part of the polynucleotide according to claim 1 by a nucleic acid amplification method. A kit for detecting myocardial differentiation activity of undifferentiated cells before differentiation induction, comprising at least the primer pair according to claim 3 .   An anti-myocardial differentiation inhibitor for undifferentiated cells, comprising as an active ingredient a double-stranded RNA molecule capable of suppressing the expression of the polynucleotide according to claim 2 by RNAi, the antisense of the double-stranded RNA molecule A myocardial differentiation inhibitor, wherein the strand comprises a nucleotide sequence that specifically hybridizes with the polynucleotide in an undifferentiated cell. The myocardial differentiation inhibitor of claim 5 , wherein the antisense strand consists of a nucleotide sequence complementary to the polynucleotide of claim 2. A method for detecting myocardial differentiation activity of undifferentiated cells before differentiation induction,
Measuring the expression level of the gene marker of claim 1 in a cell,
The method wherein the expression level correlates with the level of myocardial differentiation activity of the undifferentiated cells. Measuring the expression level of the gene marker of claim 2 in a cell,
The cell according to claim 7 , wherein the expression level is higher than a preset threshold value with reference to the expression level of the marker in a control cell having no myocardial differentiation activity. The method described. The measurement is performed using a primer pair according to claim 3, method according to claim 7. A method for isolating a cell having a myocardial differentiation activity, comprising detecting a cell having a myocardial differentiation activity and isolating the cell by the method according to claim 8 . A method of monitoring myocardial differentiation activity of undifferentiated cells before differentiation induction,
Measuring the expression level of the gene marker according to claim 2 in undifferentiated cells,
A method wherein the myocardial differentiation activity of the undifferentiated cells is increased when the expression level increases with time. The measurement is performed using a primer pair according to claim 3, method according to claim 11.

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