WO2019078342A1 - Procédé de sélection d'une cellule souche pluripotente présentant une directivité de différenciation en cardiomyocyte - Google Patents

Procédé de sélection d'une cellule souche pluripotente présentant une directivité de différenciation en cardiomyocyte Download PDF

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WO2019078342A1
WO2019078342A1 PCT/JP2018/038951 JP2018038951W WO2019078342A1 WO 2019078342 A1 WO2019078342 A1 WO 2019078342A1 JP 2018038951 W JP2018038951 W JP 2018038951W WO 2019078342 A1 WO2019078342 A1 WO 2019078342A1
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gene
differentiation
pluripotent stem
cells
stem cell
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Japanese (ja)
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繁 宮川
芳樹 澤
文哉 大橋
鮫島　正
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国立大学法人大阪大学
テルモ株式会社
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/34—Muscles; Smooth muscle cells; Heart; Cardiac stem cells; Myoblasts; Myocytes; Cardiomyocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions

Definitions

  • the present invention relates to pluripotent stem cells having a directivity toward differentiation into specific differentiation-inducing cells, particularly cardiomyocytes, embryoid bodies derived from the pluripotent stem cells, differentiation-inducing cells derived from the pluripotent stem cells , A method of screening the pluripotent stem cells, a method of treating a subject using the medical composition, a method of screening an effective drug using the medical composition, the medical use
  • the present invention relates to a quality control method and the like in the production of a composition.
  • Non-patent Document 1 Non-patent Document 1
  • ES cells embryonic stem cells
  • iPS cells induced pluripotent stem cells
  • preparing differentiation-inducing cells from pluripotent stem cells for example, when preparing cardiomyocytes, first, an embryoid body is formed while giving directionality of differentiation of pluripotent stem cells to mesoderm, and such embryos The cardiomyocytes are recovered by inducing differentiation of the body into cardiomyocytes and dispersing them into single cells (for example, Patent Document 2 and the like).
  • Patent Document 2 and Non-Patent Document 4 describe iPS cells having a differentiation directivity to cells of the nervous system.
  • the present invention relates to a method for selecting pluripotent stem cells having differentiation tropism for cardiomyocytes.
  • the present invention relates to the following: [1] A method of determining a differentiation tropism marker for evaluating differentiation tropism of pluripotent stem cells to a specific differentiation induction cell, (1) measuring gene expression levels in a plurality of pluripotent stem cell lines; (2) measuring the expression level of miRNA in the plurality of pluripotent stem cell lines; (3) extracting a gene having a significant difference in expression amount between the highly directed pluripotent stem cell line and the low pluripotent stem cell line directed to the specific differentiation-inducing cell; (4) extracting a miRNA having a significantly different expression level between the highly directed pluripotent stem cell line and the low pluripotent stem cell line directed to the specific differentiation-inducing cell; and (5) Selecting a gene involved with the miRNA extracted in (4) from the genes extracted in (3); Said method.
  • a method for indexing the differentiation tropism of pluripotent stem cell lines comprising: (A) measuring the expression level of at least one differentiation-oriented marker gene in the pluripotent stem cells of interest (b) comparing the expression level of the gene measured in (a) with a reference .
  • a differentiation-oriented marker gene is a gene selected from the group consisting of WNT signaling regulatory factor, mitochondrial related gene, TGF ⁇ signaling regulatory factor, mesoderm related gene, cardiomyocyte related gene and undifferentiated cell related gene There is a way [2].
  • the WNT signaling modulators are PF4, TMEM64, KDM6A, APC, ⁇ -catenin, Axin, CK1, Dsh, GSK-3 ⁇ , Dkk, WIF, FRP, Cerberus, TCF, Krn, WNT1, WNT2, WNT3, WNT4
  • the mitochondrial related gene is selected from the group consisting of CHCHD2, SFXN3, CREB1, PPARGC1A, PPARGC1B, CAMK4, PPP3CA, MYEF2, PPRC1, PKA, NRF1, GABPA, GABPB2, ESRRA, TFB2M, TFB1M, TFAM, POLRMT and MTERF
  • TGF ⁇ signaling modulators include SKIL, THBS1, CD3, TLR2, SMAD1, SMAD2, SMAD3, SMAD4, SMAD5, SMAD6, SMAD7, SMAD9, TGFBR1, TGFBR2, MAPK1, MAPK3, ROCK1, BMP2, BMP4, BMP5, The method of [2] to [5], which is at least one gene selected from the group consisting of BMP6, BMP7, BMP8B, BMPR1A and BMPR1B.
  • the mesodermal genes are FLK1, BRACHYURY, GOOSECOID, PDGFR-a, IGF2, CD34, CLL1, HHEX, INHBA, LEF1, SRF, T, TWIST1, ADIPOQ, MME, KIT, ITGAL, Tbx1, Gata1, Klf1,
  • a cardiomyocyte related gene consists of TNT2, ML2, GATA4, MYH6, MYH7, Nkx2.5, SCN5A, RYR2, PPARGC1, MYL2, HCN4, CACNalC, ATP2A2, Actc1, Cx43, TEF-1 and Tbx-5
  • the undifferentiated cell-related genes are Oct-4, Nanog, Lin28, SOX2, c-Myc, Klf4, TRA-1-60, SSEA-4, Oct3 / 4, Nanog, Cripto, Dax1, ERas, Fgf4, At least one member selected from the group consisting of miRNAs of Esg1, Rexl, Zfp296, UTF1, GDF3, Sall4, Tbx3, Tcf3, DNMT3L, DNMT3B, Tra-1-81, miR-290 cluster and miR-302 cluster.
  • the method of [2] to [8] which is a gene.
  • a method for culturing pluripotent stem cells comprising PF4, CHCHD2, AMMECR1, API5, BCOR, BRWD1, CLEC4G, GLIPR1, HELB, KDM6A, LOC388796, NKTR, POMZP3, ZP3, PRUNE2, RBMX, RC3H1, SKIL, Culturing in a medium containing at least one protein selected from the group consisting of SORBS2 and SRSF11.
  • a medical composition comprising pluripotent stem cell-derived differentiation-inducing cells cultured by the method of [14]-[17].
  • the medical composition of [18] which is a composition for drug screening.
  • a method for quality control of a medical composition comprising cardiomyocytes induced to differentiate from pluripotent stem cells, which is a mesodermal gene, endodermal gene and embryoid body obtained by culturing pluripotent stem cells, and And / or measuring the expression level of the ectoderm gene.
  • the mesodermal genes are FLK1, BRACHYURY, GOOSECOID, PDGFR-a, IGF2, CD34, CLL1, HHEX, INHBA, LEF1, SRF, T, TWIST1, ADIPOQ, MME, KIT, ITGAL, Tbx1, Gata1, Klf1,
  • the method of [22] which is selected from the group consisting of at least one gene selected from the group consisting of Csf1 r, CD45 and Ter119.
  • the present invention it is possible to easily select and obtain a pluripotent stem cell line having differentiation tropism with respect to specific differentiation-inducing cells such as cardiomyocytes, so that differentiation can be performed using such a pluripotent stem cell line. It will be possible to efficiently prepare cardiomyocytes by induction. In addition, since it can be determined whether or not a final preparation with a high proportion of cardiomyocytes can be obtained at an early stage of differentiation induction such as pluripotent stem cells and embryoid bodies, efficiently providing a high quality medical composition. Can. Furthermore, by providing a novel culture method for differentiating into desired differentiation-inducing cells, such as cardiomyocytes, it is possible to obtain desired differentiation-inducing cells more efficiently.
  • FIG. 1 is a photograph of a culture of iPS cells used in Example 1 and a embryoid body.
  • A is a photograph of cells in culture
  • B is a photograph of embryoid bodies.
  • FIG. 2 shows the positive rate of troponin T when iPS cells used in Example 1 were induced to differentiate into cardiomyocytes, and blue bars and red bars each added 12 ng / mL when activin was added at 6 ng / mL.
  • Positive rate when FIG. 3 shows the beating rate of the culture when the iPS cells used in Example 1 are induced to differentiate into cardiomyocytes. The red and blue bars show the results on day 8 and day 17 of culture, respectively.
  • FIG. 4 shows the survival rate of undifferentiated cells when differentiation induction of each iPS cell into cardiomyocytes is performed. Blue bars and red bars are the results when 6 ng / mL of activin was added and 12 ng / mL, respectively.
  • FIG. 5 shows the expression levels of related genes of each germ in embryoid bodies formed from each iPS cell line.
  • FIG. 6 is a heat map of cardiomyocyte associated gene expression in cell cultures induced to differentiate from each iPS cell.
  • FIG. 7 shows the expression level of each cardiomyocyte associated gene in cell cultures induced to differentiate from each iPS cell.
  • FIG. 8 shows the results of miRNA expression analysis in each iPS cell line.
  • A is a graph plotting changes in the expression level of miRNA between a differentiation-oriented iPS cell line and a low differentiation-oriented iPS cell line.
  • B is a graph showing the difference in the expression level of the 5 types of miRNAs identified as the miRNA whose expression level has been reduced, between the highly differentiation-oriented strain and the low differentiation-oriented strain.
  • C is a distribution map in which the expression levels in the high differentiation-oriented strain and the low differentiation-oriented strain are plotted as the vertical axis and the horizontal axis for all the analyzed miRNAs.
  • FIG. 9 shows the results of pathway analysis of the identified miRNAs and the test results for the expression of the identified two genes (PF4 and TMEM64).
  • A represents the expression levels of 10 mRNAs whose expression levels are significantly higher between the differentiation-oriented iPS cell line and the low differentiation-oriented iPS cell line and 10 mRNAs whose expression levels are significantly lower It is a graph.
  • B is a graph listing signal transduction pathways involving miRNAs and mRNAs identified by miRNA analysis and gene expression analysis.
  • C is a graph showing the difference in cTnT expression amount in the obtained cell population when differentiation-promoting and low-derivative targeting strains are induced to differentiate into cardiomyocytes, respectively.
  • D is a graph showing the expression level of PF4 and TMEM64 in the high differentiation-oriented strain and the low differentiation-oriented strain, respectively.
  • E is a graph showing the correlation between the expression levels of PF4 and TMEM64 in each iPS cell line and the cTnT expression level when the iPS cell line is differentiated into cardiomyocytes.
  • FIG. 10 is a graph showing the expression level of A: cTnT and the expression levels of B: PF4 and TMEM 64 when differentiation was induced by adding various agents to the medium.
  • a base sequence (including mRNA and miRNA) represented by a specific gene name means a sequence registered in a database known in the art such as GenBank. Those skilled in the art can immediately know what sequence is represented from such a gene name.
  • pluripotent stem cells is a term well known in the art and capable of differentiating into cells of all lineages belonging to three germs, ie endoderm, mesoderm and ectoderm.
  • Means cells with Non-limiting examples of pluripotent stem cells include, for example, embryonic stem cells (ES cells), nuclear transplanted embryonic stem cells (ntES cells), induced pluripotent stem cells (iPS cells) and the like.
  • ES cells embryonic stem cells
  • ntES cells nuclear transplanted embryonic stem cells
  • iPS cells induced pluripotent stem cells
  • pluripotent stem cells are suspended and cultured to form aggregates of any of the above three germ layers and then to form aggregates. To induce differentiation into specific cells of interest.
  • pluripotent stem cells are adherently cultured at high density to induce differentiation.
  • embryoid body means an aggregate of such cells.
  • endodermal embryoid body an embryoid body having differentiation tropism for endodermal cells
  • mesodermal embryoid body an embryoid body having differentiation tropism for mesodermal cells
  • mesodermal embryo An embryoid body that has differentiation tropism to ectoderm cells may be referred to as "ectodermal embryoid body”.
  • differentiation-inducing cells mean any cells that have been subjected to differentiation-inducing treatment to differentiate from pluripotent stem cells into specific types of cells.
  • Differentiation-inducing cells include adherent cells constituting tissues such as cardiomyocytes and skeletal myoblasts, and non-adherent cells such as blood cells.
  • Non-limiting examples of differentiation-inducing cells include muscle cells such as cardiac muscle cells and skeletal myoblasts, neural cells such as neuronal cells, oligodendrocytes and dopamine producing cells, retinal cells such as retinal pigment epithelial cells, and blood cells Cells, cells of hematopoietic lineage such as bone marrow cells, T cells, NK cells, NKT cells, dendritic cells, immune related cells such as B cells, cells constituting organs such as liver cells, pancreatic ⁇ cells, kidney cells, etc. Besides chondrocytes, germ cells and the like, precursor cells and somatic stem cells that differentiate into these cells are included.
  • somatic stem cells for example, mesenchymal stem cells in cardiomyocytes, multipotent cardiac progenitor cells, unipotent cardiac progenitor cells, neural stem cells in cells of nervous system, cells of hematopoietic system and immunity Hematopoietic stem cells and lymphoid stem cells in related cells can be mentioned.
  • Differentiation induction of pluripotent stem cells can be performed using any known method.
  • differentiation induction from pluripotent stem cells to cardiomyocytes can be performed by Miki et al., Cell Stem Cell 16, 699-711, June 4, 2015 or WO 2014/185358, Shugo Tohyama et al., Stem Cell Report, 9, It can be performed based on the method described in 1-9, Nov 14, 2017.
  • differentiation-directed means the property that pluripotent stem cells are more likely to differentiate into specific differentiation-inducing cells, and the higher the differentiation directivity into specific differentiation-inducing cells, the more differentiation-inducing cells become It means easy. Therefore, a pluripotent stem cell line highly directed to differentiation into a specific cell is induced to differentiate by the differentiation induction method to the specific cell, as compared to a pluripotent stem cell line not highly directed to differentiation. It is expected that more differentiation-inducing cells can be obtained even by the same differentiation-inducing method.
  • cardiomyocytes mean cells having characteristics of cardiomyocytes. Characteristics of cardiomyocytes include, but are not limited to, for example, the expression of cardiomyocyte markers, the presence of an autonomous beat, and the like. Non-limiting examples of cardiomyocyte markers include, for example, c-TNT (cardiac troponin T), CD172a (also known as SIRPA or SHPS-1), KDR (also known as CD309, FLK1 or VEGFR2), PDGFRA, EMILIN2, VCAM, etc. . In one embodiment, pluripotent stem cell-derived cardiomyocytes are c-TNT positive and / or CD172a positive.
  • the “differentiation-directed marker” or the “differentiation-directed marker gene” is a marker (gene) expressed in pluripotent stem cells, and the differentiation directivity of the pluripotent stem cells according to the difference in expression amount. It means something that can be evaluated.
  • PF4 confirmed to be a differentiation-directed marker by the present inventors, when the expression level of PF4 in a given pluripotent stem cell is larger than that of a standard pluripotent stem cell, the pluripotent stem cell is It is evaluated that differentiation to cardiomyocytes is highly directed.
  • the “standard expression amount” is not limited to this, but includes, for example, an average value of gene expression amounts in a plurality of pluripotent stem cell lines.
  • the average expression level may be, for example, the average expression level of the gene to be measured in a predetermined number (eg, 5, 10, 15 etc.) of pluripotent stem cell lines randomly selected. .
  • the method of determining a differentiation directed marker of the present disclosure includes the following steps: (1) measuring gene expression levels in a plurality of pluripotent stem cell lines; (2) measuring the expression level of miRNA in the plurality of pluripotent stem cell lines; (3) extracting a gene having a significant difference in expression amount between a highly directed pluripotent stem cell line and a low pluripotent stem cell line directed to a specific differentiation-inducing cell; (4) extracting a miRNA having a significantly different expression level between the highly directed pluripotent stem cell line and the low pluripotent stem cell line directed to the specific differentiation-inducing cell; and (5) Select genes involved with miRNA extracted in (4) from genes extracted in (3).
  • the expression levels of genes in a plurality of pluripotent stem cell lines are each quantitatively measured.
  • the measurement of the gene expression level may be performed exhaustively.
  • a plurality of pluripotent stem cell lines have low directivity to at least one pluripotent stem cell line known to be highly directed to specific differentiation-inducing cells and to the specific differentiation-induced cells. And at least one pluripotent stem cell line known to be.
  • the directionality of differentiation into specific differentiation-inducing cells can be identified using a method known in the art, a method described in the present disclosure, or the like.
  • the expression level of the gene can be measured using methods known in the art, such as real-time PCR, microarray, high-throughput sequencing and the like.
  • step (2) the expression levels of miRNA in a plurality of pluripotent stem cell lines are each quantitatively measured.
  • the plurality of pluripotent stem cell lines are pluripotent stem cell lines identical to the plurality of pluripotent stem cell lines whose gene expression levels were measured in step (1).
  • the measurement of the expression level of miRNA can be performed using methods known in the art, such as real-time PCR method, microarray method, high-throughput sequencing method and the like.
  • a gene having a significant difference in expression amount between a highly pluripotent stem cell line and a low pluripotent stem cell line that are highly directed to specific differentiation-inducing cells is extracted. For example, comparing a pluripotent stem cell line highly differentiation-directed with a pluripotent stem cell line low differentiation-directed, 1.5 times or more, 2 times or more, 2.5 times or more, 3 times or more, 5 Genes having a difference in expression amount such as twice or more and 10 times or more may be extracted.
  • Such expression level may be high in either pluripotent stem cell line, for example, may be high expression in a pluripotent stem cell line highly directed for differentiation, or in a pluripotent stem cell line low for directed differentiation. It may be highly expressed.
  • genes that are significantly expressed at high differentiation-oriented pluripotent stem cell lines are extracted.
  • genes that are significantly highly expressed in low differentiation-oriented pluripotent stem cell lines are extracted.
  • step (4) miRNA having a significant difference in expression amount between pluripotent stem cell lines highly directed to specific differentiation-inducing cells and low pluripotent stem cell lines is extracted. For example, comparing a pluripotent stem cell line highly differentiation-directed with a pluripotent stem cell line low differentiation-directed, 1.5 times or more, 2 times or more, 2.5 times or more, 3 times or more, 5 It is possible to extract miRNA having a difference in expression amount such as twice or more and 10 times or more. Such expression level may be high in either pluripotent stem cell line, for example, may be high expression in a pluripotent stem cell line highly directed for differentiation, or in a pluripotent stem cell line low for directed differentiation. It may be highly expressed. In a preferred embodiment, miRNAs that are significantly highly expressed in highly differentiation-directed pluripotent stem cell lines are extracted. In another preferred embodiment, miRNAs that are highly highly expressed in low differentiation-oriented pluripotent stem cell lines are extracted.
  • a gene involved with the miRNA extracted in the step (4) is selected.
  • a method for identifying a gene involved with a certain miRNA a method known in the art may be used, and for example, a pathway analysis method, a database of target genes of miRNA (TargetScan) and the like can be mentioned.
  • a pathway analysis method a database of target genes of miRNA (TargetScan) and the like can be mentioned.
  • the indexing method of the present disclosure includes the following steps: (A) At least one differentiation-oriented marker gene in the pluripotent stem cells of interest, such as mitochondrial related gene, WNT signal transduction regulator, TGF ⁇ signal transduction regulator, associated gene of each germ and undifferentiated cell associated gene, etc. Measuring the expression level of (b) (a) comparing the expression level of the differentiation-oriented marker gene measured in (a) with the standard.
  • A At least one differentiation-oriented marker gene in the pluripotent stem cells of interest, such as mitochondrial related gene, WNT signal transduction regulator, TGF ⁇ signal transduction regulator, associated gene of each germ and undifferentiated cell associated gene, etc.
  • the “related gene of each germ” means a related gene of germ that the differentiation inducing cell is derived from, for example, a mesoderm related gene if the differentiation inducing cell is a cardiomyocyte, an ectoderm related gene if it is a cell of nervous system,
  • the cells of digestive tract mean endoderm related genes.
  • the expression level of the gene associated with a specific differentiation-inducing cell may be measured. For example, if the differentiation-inducing cells are cardiomyocytes, cardiomyocyte related genes may be further measured.
  • One aspect of the present disclosure relates to a method of indexing a differentiation directivity to a specific differentiation-inducing cell, particularly a cardiomyocyte.
  • the indexing method of the present disclosure includes the following steps (a) and (b): (A) At least one selected from the group consisting of mitochondrial related genes, WNT signal transduction regulators, TGF ⁇ signal transduction regulators, mesoderm associated genes, cardiomyocyte associated genes and undifferentiated cell associated genes in pluripotent stem cells of interest Measuring the expression level of the differentiation-directed marker gene of the species (b) comparing the expression level of the gene measured in (a) with the reference.
  • step (a) the expression level of a differentiation-oriented marker gene in pluripotent stem cells is measured.
  • the measurement of the expression level of the gene can be performed using a conventional method known in the art. Such methods include, but are not limited to, for example, real time PCR method, microarray method, high throughput sequencing method and the like.
  • Differentiation-oriented marker genes whose expression levels are measured are selected from the group consisting of mitochondrial related genes, WNT signaling regulatory factor, TGF ⁇ signaling regulatory factor, mesoderm related genes, cardiomyocyte related genes and undifferentiated cell related genes At least one gene.
  • Mitochondria-related genes include, but are not limited to, CHCHD2, SFXN3, CREB1, PPARGC1A, PPARGC1B, PPARGC1B, CAMK4, PPP3CA, MYEF2, PPRC1, PKA, NRF1, GABPA, GABPB2, ESRRA, TFB2M, TFB1M, TFAM, for example.
  • POLRMT or MTERF are examples of a cell related genes.
  • step (a) CHCHD2, SFXN3, CREB1, PPARGC1A, PPARGC1B, CAMK4, PPP3CA, MYEF2, PPRC1, PKA, NRF1, GABPA, GABPB2, ESRRA, TFB2M, TFB1M, TFAM, POLRMT and MTERF
  • the expression level of at least one mitochondrial related gene selected from the group is measured.
  • the expression level of CHCHD2 and / or SFXN3 is measured as a mitochondrial associated gene.
  • WNT signaling modulators include, but are not limited to, those described in the following table. Particularly preferred WNT signaling modulators include PF4, TMEM64, KDM6A, APC, ⁇ -catenin, Axin, CK1, Dsh, GSK-3 ⁇ , Dkk, WIF, FRP, Cerberus, TCF, Krn, WNT1, WNT2, WNT3, WNT4 , WNT5A, WNT7A, WNT7B, WNT8B, WNT10B, WNT11, WNT2B, WNT9A, WNT9B, LRP5 or LRP6 and the like.
  • step (a) PF4, TMEM64, KDM6A, APC, ⁇ -catenin, Axin, CK1, Dsh, GSK-3 ⁇ , Dkk, WIF, FRP, Cerberus, TCF, Krn, WNT1, WNT2, WNT3, Wnt3,
  • the amount of expression of at least one WNT signaling modulator selected from the group consisting of WNT4, WNT5A, WNT7A, WNT7B, WNT8B, WNT10B, WNT11, WNT2B, WNT9A, WNT9B, LRP5 and LRP6 is measured.
  • the expression level of PF4 or TMEM64 as a WNT signaling regulator is measured, and in a more preferred embodiment, the expression level of PF4 is measured.
  • the TGF ⁇ signaling regulator includes, but is not limited to, for example, those described in the following table.
  • Particularly preferred TGF ⁇ signaling modulators include SKIL, THBS1, CD3, TLR2, SMAD1, SMAD2, SMAD3, SMAD4, SMAD5, SMAD7, SMAD9, TGFD1, TGFBR2, MAPK1, MAPK3, ROCK1, BMP2, BMP4, BMP5, Examples include BMP6, BMP7, BMP8B, BMPR1A or BMPR1B.
  • step (a) SKIL, THBS1, CD3, TLR2, SMAD1, SMAD2, SMAD3, SMAD4, SMAD5, SMAD6, SMAD7, SMAD9, TGFBR1, TGFBR2, MAPK1, MAPK3, ROCK1, BMP2, BMP4, BMP5
  • the expression level of at least one TGF ⁇ signaling regulator selected from the group consisting of BMP6, BMP7, BMP8B, BMPR1A and BMPR1B is measured.
  • the expression level of SKIL is measured as a TGF ⁇ signaling regulator.
  • mesodermal genes include, but are not limited to, FLK1, BRACHYURY, GOOSECOID, PDGFR-a, IGF2, CD34, CLL1, HHEX, INHBA, LEF1, SRF, T, TWIST1, ADIPOQ, MME, KIT , ITGAL, Tbx1, Gata1, Klf1, Csf1r, CD45 or Ter119 and the like.
  • step (a) FLK1, BRACHYURY, GOOSECOID, PDGFR-a, IGF2, CD34, CLL1, HHEX, INHBA, LEF1, SRF, T, TWIST1, ADIPOQ, MME, KIT, ITGAL, Tbx1, Gata1
  • the amount of expression of at least one mesodermal gene selected from the group consisting of Klf1, Csf1 r, CD45 and Ter119 is measured.
  • the expression level of FLK1, BRACHYURY, GOOSECOID and / or PDGFR-a is measured as a mesodermal gene.
  • TNT2, MYL2, GATA4, MYH6, MYH7, Nkx2.5, SCN5A, RYR2, PPARGC1, MYL2, HCN4, CACNalC, ATP2A2, Actc1, Cx43, TEF-1 or Tbx-5 may, for example, be mentioned.
  • step (a) TNT2, MYL2, GATA4, MYH6, MYH7, Nkx2.5, SCN5A, RYR2, PPARGC1, MYL2, HCN4, CACNalC, ATP2A2, Actc1, Cx43, TEF-1 and Tbx-5
  • the expression level of at least one cardiomyocyte related gene selected from the group consisting of
  • the expression levels of TNT2, MYL2, GATA4, MYH6, MYH7, NKx2.5, SCN5A, RYR2, PPARGC1, MYL2, HCN4, CACNalC and / or ATP2A2 as cardiomyocyte related genes are measured.
  • undifferentiated cell-related genes include, but are not limited to, Oct-4, Nanog, Lin28, SOX2, c-Myc, Klf4, TRA-1-60, SSEA-4, Oct3 / 4, Nanog , Cripto, Dax1, ERas, Fgf4, Esg1, Rex1, Zfp296, UTF1, GDF3, Sall4, Tbx3, Tcf3, DNMT3L, DNMT3B, Tra-1-81 or miRNA of miR-290 cluster, miRNA of miR-302 cluster, etc. It can be mentioned.
  • step (a) Oct-4, Nanog, Lin28, SOX2, c-Myc, Klf4, TRA-1-60, SSEA-4, Oct3 / 4, Nanog, Cripto, Dax1, ERas, Fgf4 , Esg1, Rex1, Zfp296, UTF1, GDF3, Sall4, Tbx3, Tcf3, DNMT3L, DNMT3B, Tra-1-81, miR-290 cluster miRNA and at least one selected from the group consisting of miR-302 cluster miRNAs
  • the expression levels of at least two genes selected from the aforementioned gene group are measured. That is, CHCHD2, SFXN3, KDM6A, SKIL, FLK1, BRACHYURY, GOOSECOID, PDGFR-a, TNT2, ML2, GATA4, MYH6, MYH7, NHx2.5, SCN5A, RYR2, PPARGC1, MYL2, HCN4, CACNa1Ct, ATP2
  • the expression levels of at least two genes selected from 4, Nanog and Lin28 are measured.
  • the differentiation-oriented marker gene includes PF4, CHCHD2, AMMECR1, API5, BCOR, BRWD1, CLEC4G, GLIPR1, HELB, KDM6A, LOC388796, NKTR, POMZP3, ZP3, PRUNE2, RBMX, RC3H1, SKIL,
  • the expression level of at least one gene selected from the group consisting of SORBS2 and SRSF11 is measured.
  • expression of at least one gene selected from the group consisting of TMEM64, ACTN3, LOC284373, LOC441666, PLCB1, SYNPR, TMEM163, U2AF1L4, VWDE, ZNF229 and ZNF354C as a differentiation-oriented marker gene The quantity is measured.
  • step (b) the amount of expression of the gene measured in (a) is compared with a reference.
  • the criteria to be compared are, but not limited to, for example, the expression amount of the same gene in a pluripotent stem cell line known to have low directivity for cardiomyocyte differentiation, directivity for cardiomyocyte differentiation.
  • the average expression level of the same gene in multiple pluripotent stem cell lines known to be low the expression level of the same gene in pluripotent stem cell lines known to be highly directional to cardiomyocytes, cardiomyocytes
  • Differentiation directional marker of the target pluripotent stem cell line is indexed by comparing with these criteria, and whether the target pluripotent stem cell line is a line with high directivity to cardiomyocytes based on such index Can be determined.
  • both values are preferably values measured by the same method, but it is not limited thereto. If the values are measured by different methods, the values may be converted to allow direct comparison.
  • a plurality of pluripotency known to exhibit low expression levels of the same gene in a pluripotent stem cell line known to have low directivity toward cardiomyocytes or low directivity to cardiomyocytes is made on the basis of the average value of the expression level of the same gene in stem cell lines. In this case, when the expression level measured in (a) is significantly higher than the reference value, it can be determined that the pluripotent stem cell line of interest has high directivity to cardiomyocytes.
  • a plurality of multiple genes known to have high expression levels of the same gene in cardiogenic cells or pluripotent stem cell lines that are known to have a high directivity for cardiomyocyte differentiation is compared as a standard. In this case, if the expression level measured in (a) is equal to or significantly higher than the reference value, it can be determined that the pluripotent stem cell line of interest is highly directional to cardiomyocytes .
  • "significantly” means that the difference is statistically significant. For example, it is considered to be "significant" when a certain measured value indicates a numerical value that deviates extremely from a certain statistic.
  • comparison is made on the basis of the average value of the expression level of the same gene in multiple pluripotent stem cell lines.
  • the expression level measured in (a) is significantly higher than the reference value, it can be determined that the pluripotent stem cell line of interest has high directivity to cardiomyocytes.
  • the differentiation-oriented marker gene includes PF4, CHCHD2, AMMECR1, API5, BCOR, BRWD1, CLEC4G, GLIPR1, HELB, KDM6A, LOC388796, NKTR, POMZP3, ZP3, PRUNE2, RBMX, RC3H1, SKIL, SORBS2 and SRSF11
  • the amount of expression of at least one gene selected from the group consisting of when the expression level measured in (a) is significantly higher than the reference value, it can be determined that the pluripotent stem cell line of interest has high directivity to cardiomyocytes.
  • the expression level of at least one gene selected from the group consisting of TMEM64, ACTN3, LOC284373, LOC441666, PLCB1, SYNPR, TMEM163, U2AF1L4, VWDE, ZNF229 and ZNF354C as a differentiation-oriented marker gene is used. measure.
  • the expression level measured in (a) is significantly lower than the reference value, it can be determined that the pluripotent stem cell line of interest has a high directivity to cardiomyocytes.
  • Pluripotent Stem Cells of the Present Disclosure there exist cell lines with high directivity to cardiomyocytes in pluripotent stem cells, particularly induced pluripotent stem cells (iPS cells), and The genetic features of pluripotent stem cell lines were found for the first time. Therefore, one aspect of the present disclosure relates to at least one member selected from the group consisting of a mitochondrial related gene, a WNT signaling regulator, a TGF ⁇ signaling regulator, a mesoderm related gene, a cardiomyocyte related gene and an undifferentiated cell related gene. It includes pluripotent stem cells highly directed to cardiomyocyte differentiation characterized by high gene expression levels.
  • the present inventors have found that mitochondrial related genes, WNT signal transduction regulatory factor, TGF beta signal transduction regulatory factor, mesoderm related gene, cardiomyocyte related gene And found that the undifferentiated cell-related gene is highly expressed.
  • “high expression level” or “high expression” means that the expression level of a certain gene is higher than a predetermined value.
  • a predetermined value typically, an average value of the expression amount of the gene, etc. may be mentioned.
  • the average value of the expression level may be, for example, the average value of the expression level of the gene to be measured in a predetermined number (eg, 5, 10, 15 etc.) of pluripotent stem cell lines randomly selected.
  • the genes highly expressed in the pluripotent stem cell line of the present disclosure include mitochondria related genes, WNT signal transduction regulatory factor, TGF ⁇ signal transduction regulatory factor, mesoderm related genes, cardiomyocyte related genes and undifferentiated cell related genes It can be mentioned. Specific examples of these genes include those described in the above ⁇ 1>.
  • the pluripotent stem cells are iPS cells, more preferably human iPS cells. It has been pointed out that iPS cells may differ in their cell line characteristics depending on the somatic cell origin, type of reprogramming factor and introduction method, etc. Therefore, the differentiation tropism is also cell line by cell line It is expected to be different. In addition, as a merit when iPS cells are used for regenerative medicine, it is possible to establish a cell line using autologous cells to be treated. Therefore, according to the present invention, it is also possible to screen iPS cells produced from autologous cells of interest from those having a high directivity toward cardiomyocytes and establish them as new cell lines.
  • pluripotent stem cells When preparing differentiation-inducing cells from pluripotent stem cells, it is necessary that the cells be highly undifferentiated. This is also confirmed by the high expression of undifferentiated cell-related genes in the pluripotent stem cells of the present disclosure. That is, high undifferentiating ability in pluripotent stem cells is considered to facilitate differentiation into desired differentiation-inducing cells. According to the tests of the present inventors, it has been obtained that it is considered that the undifferentiated nature of pluripotent stem cells is not directly related to the cardiopatic differentiation per se.
  • Embryoid body of the present disclosure The embryoid body obtained by culturing pluripotent stem cells highly directional to cardiomyocytes by the present inventors is also highly directional to cardiomyocytes highly It was found. Therefore, one aspect of the present disclosure relates to differentiation into cardiomyocytes characterized by high expression of at least one mesodermal gene and low expression of at least one endodermal gene and / or ectodermal gene. Includes highly oriented embryoid bodies.
  • the embryoid body of the present disclosure can be obtained by culturing pluripotent stem cells, preferably pluripotent stem cells described in ⁇ 2> above, by methods known in the art. Specifically, for example, human iPS cells are cultured for 1 day in StemFit AK03 medium (Ajinomoto) containing Y27632 (Wako Pure Chemical Industries, Ltd.), then for 2 days in StemFit AK03 medium not containing Y27632, and then BMP4 is It can be obtained by culturing in a medium containing it.
  • the embryoid bodies of the present disclosure highly express at least one mesodermal gene.
  • mesodermal genes include those described in the above ⁇ 1>.
  • the embryoid bodies of the present disclosure also have low expression levels of at least one endodermal gene and / or ectoderm gene.
  • low expression level means that the expression level of a certain gene is lower than a predetermined value, contrary to the above "high expression level”.
  • predetermined value typically, an average value of the expression amount of the gene, etc. may be mentioned.
  • the average value of the expression levels may be, for example, the average value of the expression levels of the measurement target genes in a predetermined number (for example, 5, 10, 15 etc.) of embryoid bodies randomly extracted.
  • the ectoderm gene includes, but is not limited to, for example, SOX1, PAX6 or ZIC1.
  • the endodermal genes include, but are not limited to, for example, AMN, SOX7, SOX17, HNF3 or ZIC1.
  • the embryoid body of the present disclosure is characterized in that the expression level of at least one mesodermal gene is high and the expression level of at least one endodermal gene and / or ectoderm gene is low, Thus, it has a high differentiation directivity to somatic cells of mesodermal origin, in particular cardiomyocytes.
  • Such features are particularly prominent in embryoid bodies produced from the pluripotent stem cells of the present disclosure having high differentiation tropism for cardiomyocytes. Therefore, in a preferred embodiment, the embryoid body of the present disclosure is produced from the pluripotent stem cells of the present disclosure described in the above ⁇ 2>.
  • the present inventors have found genetic characteristics of pluripotent stem cells highly directed to cardiomyocyte differentiation, and differentiated cardiomyocytes using pluripotent stem cells having such characteristics. It was found that cardiomyocytes can be obtained with high efficiency by induction. Thus, the present disclosure, in one aspect, encompasses methods of inducing cardiomyocytes to differentiate from pluripotent stem cells with high efficiency.
  • the differentiation induction method of the present disclosure uses, in a preferred embodiment, the pluripotent stem cells or embryoid bodies of the present disclosure.
  • the differentiation induction technique itself may use any technique known in the art. There are various known methods for inducing cardiomyocytes differentiation from pluripotent stem cells (for example, Burridge et al., Cell Stem Cell. 2012 Jan 6; 10 (1): 16-28).
  • a mesodermal inducer eg, activin A, BMP4, bFGF, VEGF, SCF, etc.
  • a cardiac specification factor eg, VEGF, DKK1, a Wnt signal inhibitor (eg, IWR-1) , IWP-2, IWP-3, IWP-4 etc.), BMP signal inhibitors (eg NOGGIN etc.), TGF ⁇ / activin / NODAL signal inhibitors (eg SB431542 etc.), retinoic acid signal inhibitors etc.
  • cardiac differentiation factors eg For example, enhancing induction efficiency by sequentially acting VEGF, bFGF, DKK1 etc. It can be.
  • cardiomyocyte induction treatment from pluripotent stem cells is carried out by causing BMP4 to act on embryoid bodies formed by (1) combining BMP4 and bFGF and activin A, (2) VEGF and IWP-3, And (3) sequentially acting on the combination of VEGF and bFGF.
  • a known method for obtaining cardiomyocytes from human iPS cells for example, the following steps: (1) maintaining and culturing human iPS cells in a culture solution containing no feeder cells (feeder free method), (2) forming an embryoid body from the obtained iPS cells, (3) culturing the obtained embryoid body in a culture solution containing activin A, bone morphogenetic protein (BMP) 4 and basic fibroblast growth factor (bFGF), (4) culturing the obtained embryoid body in a culture solution containing a Wnt inhibitor, a BMP4 inhibitor and a TGF ⁇ inhibitor, and (5) the obtained embryoid body in a culture solution containing VEGF and bFGF
  • a method comprising the step of culturing in
  • StemFit AK03 (Ajinomoto) can be used as a culture medium, and iPS cells can be cultured and adapted on iMatrix 511 (Nippi) to perform maintenance culture.
  • iPS cells can be cultured and adapted on iMatrix 511 (Nippi) to perform maintenance culture.
  • iMatrix 511 Nippi
  • passage may be performed as a single cell using TrypLE® Select (Thermo Fisher Scientific).
  • the step of purifying the obtained cardiomyocytes may be selectively performed.
  • Purification of cardiomyocytes includes a method of reducing non-cardiomyocytes using a glucose free medium, a method of reducing undifferentiated cells using heat treatment as described in WO 2017/038562, and the like.
  • One aspect of the present disclosure may include the step of performing the indexing method of the present disclosure described in the above ⁇ 1> before and / or after the step of the above (1).
  • a step of measuring the gene expression amount of the obtained embryoid body may be included.
  • the method further includes the steps of comparing the measured gene expression level with a reference value, and excluding embryoid bodies other than the embryoid bodies judged to be the embryoid bodies described in the above ⁇ 3> as a result of the comparison. May be.
  • a reference value for example, those described as the "predetermined value" in the above ⁇ 2> can be mentioned.
  • cardiomyocyte differentiation can be induced by applying treatment to enhance or reduce the expression of these genes.
  • Non-limiting examples of such treatment include, for example, addition of WNT signal inhibitor, TGF ⁇ signal inhibitor, modulation of TGF ⁇ signal or WNT signal, modulation of mitochondrial activity by addition of MitoBlock, etc.
  • pluripotent stem cells comprising enhancing or reducing the expression level of a differentiation-directed marker, or enhancing or reducing the action of a protein that is an expression product of a differentiation-directed marker gene. It also relates to a method for inducing differentiation.
  • Examples of methods for enhancing the expression level of the differentiation directed marker include inhibition of repressor of the differentiation directed marker gene, addition of an enhancer of the differentiation directed marker gene, and the like. Examples of methods for reducing the expression level of the differentiation directed marker include addition of repressor, introduction of antisense nucleic acid such as siRNA, and the like.
  • Examples of methods for enhancing the action of a protein that is an expression product of a differentiation-oriented marker gene include addition of the expression product protein to a medium, and the like.
  • a method of reducing the action of a protein which is an expression product of a differentiation-oriented marker gene for example, addition to a medium such as an inhibitor for the protein or an inhibitory antibody can be mentioned.
  • differentiation-oriented marker expression such as PF4, CHCHD2, AMMECR1, API5, BCOR, BRWD1, CLEC4G, GLIPR1, HELB, KDM6A, LOC388796, NKTR. It is expected that the induction of differentiation into cardiomyocytes is promoted by culturing in a medium containing proteins such as POMZP3, ZP3, PRUNE2, RBMX, RC3H1, SKIL, SORBS2 and SRSF11.
  • PF4 CHCHD2, AMMECR1, API5, BCOR, BRWD1, CLEC4G, GLIPR1, HELB, KDM6A, LOC388796, NKTR It is expected that the induction of differentiation into cardiomyocytes is promoted by culturing in a medium containing proteins such as POMZP3, ZP3, PRUNE2, RBMX, RC3H1, SKIL, SORBS2 and SRSF11.
  • the differentiation-inducing method of the present disclosure includes the expression of differentiation-oriented marker expression products significantly increased as described above, such as PF4, CHCHD2, AMMECR1, API5, BCOR, BRWD1, CLEC4G, GLIPR1, HELB, KDM6A, LOC388796. Also included is a method for culturing pluripotent stem cells, which comprises using a medium containing a protein such as NKTR, POMZP3, ZP3, PRUNE2, RBMX, RC3H1, SKIL, SORBS2 and SRSF11. Such a culture method is preferably used in the culture at the stage of differentiating pluripotent stem cells into embryoid bodies (especially mesodermal embryoid bodies).
  • cardiomyocytes can be efficiently obtained.
  • the cardiomyocyte content (purity) in the cardiomyocyte-containing composition obtained by the differentiation induction method of the present disclosure is more than about 50%, more than about 60%, more than about 70%, more than about 75%, more than about 80%, More than about 85%, more than about 86%, more than about 87%, more than about 88%, more than about 89%, more than about 90%, more than about 91%, more than about 92%, more than about 93%, more than about 94%, It may be more than about 95%, more than about 96%, more than about 97%, more than about 98%, more than about 99%, and so on.
  • the pluripotent stem cell-derived cardiomyocytes in the present disclosure is a cardiomyocyte population having a cardiomyocyte purity of greater than 90%.
  • the cardiomyocyte content is, for example, 50 to 99%. Preferably, it is 50% to 70%, or 75 to 99%.
  • the cardiomyocyte-containing composition obtained by the induction method of the present disclosure is characterized by a low survival rate of undifferentiated cells.
  • the survival rate of undifferentiated cells is, for example, 0.01% to 5%, 0.01% to 4%, 0.01% to 3%, 0.01% to 2%, 0.01% to 1%, It may be, for example, 0.01% to 1%.
  • Medical composition of the present disclosure includes a medical composition containing differentiation-inducing cells, for example, cardiac muscle cells, which are induced by the method described in the above ⁇ 4>.
  • medical composition means a composition used for medical purposes, and is not limited thereto, and for example, a pharmaceutical composition, a therapeutic composition, a composition for transplantation, etc.
  • compositions used for direct treatment of a subject compositions used in drug development and the like, such as, for example, compositions for drug screening, are also included.
  • the composition containing cardiomyocytes induced by the differentiation induction method of the present disclosure is a composition having a high cardiomyocyte content and can be said to be very useful in medical treatment.
  • a composition for transplantation is prepared using a composition containing cardiomyocytes induced by the differentiation induction method of the present disclosure, the content of cardiomyocytes is high, so the amount of cardiomyocytes contained is large, and An implant composition suitable for implant of the present invention can be prepared.
  • the medical composition of the present disclosure is a composition for implantation.
  • the cardiomyocyte content is greater than about 50%, about 60%, about 70%, about 75%, about 80%, about 85%, about 86%, about 87%, or more.
  • the pluripotent stem cell-derived cardiomyocytes in the present disclosure is a cardiomyocyte population having a cardiomyocyte purity of greater than 90%.
  • the cardiomyocyte content is, for example, 50 to 99%, preferably 50 to 70%, or 75 to 99%.
  • the medical composition of the present disclosure in particular the composition for transplantation, can also be such a cell culture.
  • the medical composition of the present disclosure is a sheet-like cell culture.
  • sheet-like cell culture refers to cells in which cells are linked to each other into a sheet.
  • the cells may be linked to each other directly (including via cell components such as adhesion molecules) and / or via an intermediary substance.
  • the mediator is not particularly limited as long as it is a substance capable of at least physically (mechanically) connecting cells to each other, and examples include extracellular matrix and the like.
  • the mediator is preferably of cell origin, in particular of the cells constituting the cell culture.
  • the cells are at least physically (mechanically) linked, but may be further functionally linked, for example, chemically or electrically.
  • the sheet-like cell culture is composed of one cell layer (monolayer), but is composed of two or more cell layers (layered (multilayer) body, for example, two or three layers, It may be four layers, five layers, six layers, etc.).
  • the sheet-like cell culture preferably does not contain a scaffold (support). Scaffolds may be used in the art to attach cells on and / or within their surface and maintain the physical integrity of sheet cell cultures, such as polyvinylidene difluoride (eg, Membranes and the like made of PVDF) are known, but the sheet-like cell culture in the present disclosure may be capable of maintaining its physical integrity even without such scaffolds.
  • the sheet-like cell culture preferably comprises only the substance derived from cells constituting the cell culture, and does not contain any other substance.
  • the cells that make up the sheet-like cell culture can be derived from any organism that can be treated by the sheet-like cell culture. Such organisms include, but are not limited to, humans, non-human primates, dogs, cats, pigs, horses, goats, sheep, rodents (eg, mice, rats, hamsters, guinea pigs, etc.), rabbits, etc. Is included. In one embodiment, the cells constituting the sheet-like cell culture are human cells.
  • the cells forming the sheet-like cell culture may be xenogeneic cells or allogeneic cells.
  • heterologous cell means a cell derived from an organism of a species different from that of the recipient when a sheet-like cell culture is used for transplantation.
  • cells derived from monkeys or pigs correspond to xenogeneic cells.
  • allogeneic derived cells mean cells derived from an organism of the same species as the recipient. For example, when the recipient is human, human cells correspond to allogeneic cells.
  • Allogeneic cells include autologous cells (also referred to as autologous cells or autologous cells), ie cells derived from a recipient and allogeneic non-autologous cells (also referred to as allogeneic cells). Autologous cells are preferred in the present disclosure as transplantation does not result in rejection. However, it is also possible to use heterologous cells or allogeneic non-autologous cells. When xenogeneic cells or allogeneic non-autologous cells are used, immunosuppressive treatment may be required to suppress rejection.
  • cells other than autologous cells, ie, xenogeneic cells and allogeneic nonautologous cells may be collectively referred to as nonautologous cells.
  • the cells are autologous cells or allogeneic cells.
  • the cells are autologous cells.
  • the cells are allogeneic cells.
  • the autologous or allogeneic pluripotent stem cells are not limited, for example, collected autologous or allogeneic cells (eg, skin cells (fibroblasts, keratinocytes, etc.) and blood cells (peripheral blood mononuclear cells, etc.), etc.) It can be obtained by introducing a gene such as OCT3 / 4, SOX2, KLF4 or C-MYC into autologous or allogeneic iPS cells. Methods for inducing iPS cells from somatic cells are well known in the art (see, eg, Bayart and Cohen-Haguenauer, Curr Gene Ther. 2013 Apr; 13 (2): 73-92, etc.).
  • the medical composition and the sheet-like cell culture of the present disclosure contain cardiomyocytes in a high degree as described above, and therefore, use of the composition and the sheet-like cell culture act on cardiomyocytes.
  • the effects of drugs can be effectively tested.
  • the medical composition of the present disclosure is a composition for drug screening.
  • the composition of the present disclosure can prepare cardiomyocytes derived from a specific subject, it becomes possible to screen an agent that effectively acts on the specific subject.
  • one aspect of the present disclosure relates to pluripotent stem cells comprising measuring expression levels of mesodermal genes, endodermal genes and / or ectoderm genes in embryoid bodies obtained by culturing pluripotent stem cells.
  • the present invention also includes a method of controlling the quality of a medical composition containing differentiation-induced cardiomyocytes, and a method of producing a medical composition including such a method of quality control.
  • the method of the present aspect further comprises comparing the measured gene expression amount with a reference value, and excluding the embryoid body judged to be the embryoid body as described in the above ⁇ 3> as a result of the comparison. It may include excluding the embryoid bodies of The reference value typically includes an average value of the expression level of the gene, and the like.
  • the average value of the expression levels may be, for example, the average value of the expression levels of the measurement target genes in a predetermined number (for example, 5, 10, 15 etc.) of embryoid bodies randomly extracted.
  • embryoid bodies with high differentiation efficiency may be selected.
  • embryoid bodies with high differentiation efficiency may be selected based on morphological features of the embryoid bodies, such as the size of the formed embryoid bodies, and the manner of aggregation.
  • the method of producing the medical composition of the present disclosure includes the following steps: (A) Differentiating and culturing pluripotent stem cells to form an embryoid body, (B) Directing cardiomyocyte differentiation, including measuring the expression levels of mesodermal genes, endodermal genes and / or ectodermal genes in the obtained embryoid body, and comparing such measured values with reference values Selecting an embryo-like body of high sex, (C) a step of inducing differentiation and culturing the selected embryoid body to obtain a cell population containing cardiomyocytes.
  • step (A) embryoid bodies are formed from pluripotent stem cells.
  • the pluripotent stem cells that can be used are preferably, but not limited to, allogeneic cells to be treated (eg, human) with the medical composition.
  • pluripotent stem cells prepared from autologous cells such as autologous iPS cells are preferable.
  • the pluripotent stem cells to be used may further be screened by the method of the present disclosure.
  • embryoid bodies highly directional to cardiomyocytes are selected. Examples of embryoid bodies highly directed to cardiomyocyte differentiation are as described in ⁇ 3> above.
  • an embryoid body having a particularly high expression level of mesodermal genes and a low expression level of ectodermal genes and / or endodermal genes is selected. Specific examples and reference values of ectoderm gene, endoderm gene, mesoderm gene may be those described in ⁇ 3>.
  • step (C) the embryoid bodies selected in step (B) are induced to differentiate to obtain a medical composition containing cardiomyocytes.
  • a medical composition containing cardiomyocytes for induction of differentiation from embryoid bodies, methods known in the art can be used, and specifically, for example, methods described in the above ⁇ 4> may be used.
  • step (C) it may further include the step of optionally modifying the medical composition.
  • a sheeting step for forming a sheet-like cell culture, a step of cryopreserving a medical composition and the like can be mentioned.
  • Example 1 Differentiation to cardiomyocytes Differentiation of highly directed iPS cell lines to cardiomyocyte differentiation
  • the (1) Differentiation induction Ten types of cells listed in Table 5 were used as iPS cell lines.
  • 201B7, 253G1, 409B2, HiPS-RIKEN-1A, HiPS-RIKEN-2A and HiPS-RIKEN-12A were obtained from RIKEN BioResource Center.
  • ATCC-DYR0100 and ATCC-HYR0103 were obtained from ATCC.
  • mc-iPS was obtained from System Biosciences. Tic was obtained from National Institute of Biomedical Innovation.
  • RNA of human iPS cell line immediately before induction of differentiation was extracted according to procoll using miRNeasy Mini Kit (QIAGEN).
  • SuperScript TM VILO Invitrogen
  • PCR primers for SYBR Green and SYBR Green PCR master mixes (Applied Biosystems) or Taqman probe and Taqman Gene Expression Master Mix (Applied Biosystems) described in Table 4, PCR with ViiA 7 Real-Time PCR System (Applied Biosystems) Carried out. GAPDH was used as a housekeeping gene for analysis of gene expression. Gene expression analysis was performed using ViiA 7 Sysytem. In TaqMan Gene Expression Assays, temperature cycling conditions were as follows: hold at 95 ° C.
  • iPS cell lines are referred to methods described in Matsuura, et al., Biochemical and Biophysical Research Communications 425 (2012) 321-327, Miki K. Cell Stem Cell (2015), WO 2014/185358 A1 and WO 2017/038562, etc.
  • cardiomyocytes were induced to differentiate.
  • undifferentiated human iPS cells are treated on a feeder cell mitomycin C-treated MEF (ReproCell), which is a feeder cell, using 5 ng / mL bFGF added to Primate ES medium (ReproCell) as the undifferentiated maintenance medium. Cultures were performed and passaged once every 3-4 days. Differentiation induction dissociates human iPS cells with Dissociation solution (ReproCell) and Accumax (Innovation Cell Technologies), StemPro 34 (Life Technologies) supplemented with 0.5 ng / mL BMP-4 and 10 ⁇ M Y27632 (Rock inhibitor) The suspension was suspended with and cultured for 1 day with EZSPHERE (IWAKI) to form a mass.
  • FIG. 1 is a photograph of iPS cells in culture.
  • the troponin positive rate was determined by dispersing the embryoid bodies using TrypLE Select, and then dispersing the dispersed cells using BD Cytofix / Cytoperm® Fixation / Permeabilization Solution Kit (BD Bioscience) and permeabilizing the cells, then anti-human. After a troponin antibody (Thermo Fisher Scientific) and a labeled secondary antibody (Thermo Fisher Scientific) were sequentially reacted, the measurement was performed using a flow cytometer.
  • the beating rate is obtained by moving picture imaging of a germinal body after induction of differentiation from each iPS cell line to a cardiomyocyte by cell motion imaging (Sony) and pulsing among those observed. It counted and calculated.
  • Residual rate of undifferentiated cells The residual rate of undifferentiated cells in cardiomyocyte cultures prepared from the three cell lines identified as cell lines highly directed to cardiomyocyte differentiation in (2) above. As a percentage of the number of cells expressing Lin28, which is an undifferentiated cell marker, it was measured by quantitative PCR. The results are shown in FIG. In the three cell lines identified as highly directed to cardiomyocyte differentiation, the residual rate of undifferentiated cells tended to be significantly lower than in other cell lines.
  • Example 2 Gene expression in embryoid bodies Next, SOX2, PAX6, ZIC1, BRACHYURY, FLK1, PDGFR-a, GOOSECOID, HNF3, SOX17, SOX7, AMN in each embryoid body at the 4th day of culture in Example 1 above The expression of various genes was measured.
  • the results are shown in FIG.
  • the three cell lines 201B7, 253G1 and 409B2 which are cell lines highly directed to cardiomyocytes, express a large amount of mesodermal genes, and the expression levels of endodermal genes and ectodermal genes were low. Pearson's correlation coefficient between the expression level and the troponin T positive rate after differentiation induction of each cell line was calculated for each gene, and the result is as shown in the table below.
  • double-stranded cDNA synthesis including T7 promoter sequence was performed from total RNA, and biotin labeled aRNA using cDNA as a template was synthesized by in vitro reverse transcription reaction.
  • calcium random degradation using a hammerhead reaction was performed to generate an ⁇ 100-120 nt aRNA fragment.
  • Biotin-labeled aRNA prepared in Genechip Array Human Genome U133 Plus 2.0 Array was hybridized using GeneChip Hybridization Oven (Affymetrix). After hybridization, washing and phycoerythrin staining were performed using GeneChip Wash and Stain Kit (Affymetrix) and GeneChip Fluidics Station 450 (Affymetrix).
  • Cardiac muscle related gene expression comparison Each cell line was differentiated to cardiac muscle cells in the same manner as in Example 1, and the expression levels of cardiac muscle related genes were compared. The results are shown in FIG. 6 and FIG. Differentiation of cardiomyocytes into 3 cell lines highly induced expression of high cardiac muscle related genes was confirmed in all cell cultures induced to differentiate. In addition, the troponin positive rate was also significantly higher in the three cell lines highly directed to cardiomyocytes.
  • biomarker candidate genes genes showing a significant difference in expression amount were identified as biomarker candidate genes.
  • mitochondrial related genes CHCHD2 and SFXN3, WNT signal regulator KDM6A, TGF- ⁇ signal related factor SKIL, etc. were identified as biomarker gene candidates.
  • miRNA-139 and miRNA-204 were identified as the genes that showed significantly high expression in cell lines with low directivity for cardiomyocytes.
  • Example 4 miRNA expression analysis (1) miRNA microarray From each iPS cell line, total RNA was extracted using miRNeasy mini kit (QIAGEN). Biotin-labeled RNA was prepared from total RNA containing low molecular weight RNA using FlashTag Biotin HSR RNA labeling kit (Affymetrix) according to the product protocol. Biotin-labeled RNA prepared in a miRNA 3.0 array (Affymetrix) was hybridized using GeneChip Hybridization Oven (Affymetrix). After hybridization, washing and phycoerythrin staining were performed using GeneChip Fluidics Station 450 (Affymetrix).
  • the expression of miRNA in various iPS cell lines used in Example 1 was analyzed using a miRNA microarray. Of the 534 miRNAs that can be analyzed, five miRNAs (ACA24 that showed half or less of the expression level in the high differentiation-oriented group compared with the high differentiation-oriented group and the low differentiation-oriented group , Hsa-miR-629-star, mmi-miR-204, ACA61 and hsa-miR-139-5p) were identified. The results are shown in FIG. We analyzed which signal transduction pathway the identified miRNA was, and referred to the results analyzed in Example 3 (3) above, and narrowed down the strongly related genes. The results are shown in FIG. We found PF4 as a gene whose expression level is significantly higher in iPS cell lines with strong differentiation-directed to cardiomyocytes, and TMEM64 as a gene whose expression level is significantly lower.
  • ACA24 that showed half or less of the expression level in the high differentiation-oriented group compared with the high differentiation-

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Abstract

La présente invention aborde le problème de la fourniture d'un procédé de sélection d'une cellule souche pluripotente présentant une directivité de différenciation en cardiomyocyte. Le problème peut être résolu par un procédé comprenant les étapes consistant à : (a) mesurer la quantité d'expression d'au moins un gène choisi dans le groupe constitué par un gène associé à une mitochondrie, un facteur de régulation de transduction de signal WNT, un facteur de régulation de transduction de signal TGFβ, un gène associé à un mésoblaste, un gène associé à un cardiomyocyte et un gène associé à une cellule indifférenciée dans une cellule souche pluripotente d'intérêt et (b) comparer la quantité d'expression du gène qui est mesurée à l'étape (a) avec une valeur de référence, étant déterminé que la cellule souche pluripotente d'intérêt est une cellule d'une souche cellulaire présentant une directivité de différenciation élevée vis-à-vis d'un cardiomyocytes lorsque la quantité d'expression mesurée est significativement supérieure à la valeur de référence.
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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020149391A1 (fr) * 2019-01-17 2020-07-23 公立大学法人横浜市立大学 Procédé d'évaluation de la résistance à la différenciation de cellules indifférenciées
CN113017650A (zh) * 2021-03-12 2021-06-25 南昌航空大学 一种基于功率谱密度图像的脑电特征提取方法和系统
WO2021145402A1 (fr) * 2020-01-16 2021-07-22 富士フイルム株式会社 Procédé de production de cellules souches pluripotentes capables de se différencier en cellules spécifiques, et application associée
WO2024085251A1 (fr) * 2022-10-21 2024-04-25 住友ファーマ株式会社 Procédé d'évaluation de la qualité d'un implant rétinien

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2016532435A (ja) * 2013-06-10 2016-10-20 プレジデント・アンド・フェロウズ・オブ・ハーバード・カレッジ 多能性幹細胞の有用性および安全性を特徴付けるための初期発生ゲノムアッセイ
WO2017010544A1 (fr) * 2015-07-15 2017-01-19 テルモ株式会社 Procédé de cryoconservation pour cellules myocardiques dérivées de cellules souches pluripotentes ou de cellules souches mésenchymateuses dérivées de tissus adipeux ou de la moelle osseuse

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2016532435A (ja) * 2013-06-10 2016-10-20 プレジデント・アンド・フェロウズ・オブ・ハーバード・カレッジ 多能性幹細胞の有用性および安全性を特徴付けるための初期発生ゲノムアッセイ
WO2017010544A1 (fr) * 2015-07-15 2017-01-19 テルモ株式会社 Procédé de cryoconservation pour cellules myocardiques dérivées de cellules souches pluripotentes ou de cellules souches mésenchymateuses dérivées de tissus adipeux ou de la moelle osseuse

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
BURRIDGE, PW. ET AL.: "Production of De Novo Cardiomyocytes: Human Pluripotent Stem Cell Differentiation and Direct Reprogramming", CELL STEM CELL, vol. 10, 2012, pages 16 - 28, XP028434719, ISSN: 1934-5909, DOI: doi:10.1016/j.stem.2011.12.013 *
LEWANDOWSKI, J. ET AL.: "Techniques for the induction of human pluripotent stem cell differentiation towards cardiomyocytes", JOURNAL OF TISSUE ENGINEERING AND REGENERATIVE MEDICINE, vol. 11, no. 5, 17 January 2016 (2016-01-17) - May 2017 (2017-05-01), pages 1658 - 1674, XP055594702, ISSN: 1932-6254, DOI: 10.1002/term.2117 *
SATO, YOJI: "Development of cell characterization analysis method using differentiation propensity as an indicator", GRANT-IN-AID FROM THE MINISTRY OF HEALTH, LABOR, AND WELFARE OF JAPAN FOR HUMAN ES CELLS OR IPS CELLS, COMPREHENSIVE REGULATORY SCIENCE RESEARCH SUCH AS PHARMACEUTICALS AND MEDICAL DEVICES, 2014, pages 117 - 143 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020149391A1 (fr) * 2019-01-17 2020-07-23 公立大学法人横浜市立大学 Procédé d'évaluation de la résistance à la différenciation de cellules indifférenciées
WO2021145402A1 (fr) * 2020-01-16 2021-07-22 富士フイルム株式会社 Procédé de production de cellules souches pluripotentes capables de se différencier en cellules spécifiques, et application associée
JPWO2021145402A1 (fr) * 2020-01-16 2021-07-22
CN113017650A (zh) * 2021-03-12 2021-06-25 南昌航空大学 一种基于功率谱密度图像的脑电特征提取方法和系统
WO2024085251A1 (fr) * 2022-10-21 2024-04-25 住友ファーマ株式会社 Procédé d'évaluation de la qualité d'un implant rétinien

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