WO2021172542A1

WO2021172542A1 - Mature-cardiomyocyte production method

Info

Publication number: WO2021172542A1
Application number: PCT/JP2021/007466
Authority: WO
Inventors: 善紀吉田; カカセアントニオルセナ
Original assignee: 国立大学法人京都大学
Priority date: 2020-02-28
Filing date: 2021-02-26
Publication date: 2021-09-02
Also published as: JPWO2021172542A1

Abstract

Provided is a mature-cardiomyocyte production method including a step for increasing the expression level of cyclin-dependent kinase inhibitor 1 in immature cardiomyocytes.

Description

Manufacturing method of mature cardiomyocytes

The present invention relates to a method for producing mature cardiomyocytes.

In recent years, attempts have been made to induce differentiation of pluripotent stem cells such as induced pluripotent stem (iPS) cells and embryonic stem (ES) cells into various somatic cells and use them in regenerative medicine and the like. Although some systems that induce differentiation from pluripotent stem cells to cardiomyocytes have been reported, most of the obtained cardiomyocytes are immature cardiomyocytes similar to fetal cardiomyocytes and mature for practical use. The operation of conversion is necessary.

The following methods have been known as methods for maturing cardiomyocytes.
1) Long-term culture (for example, Non-Patent Documents 1 and 2)
2) Culture on an undiluted Matrigel layer (Matrigel mattress) (for example, Non-Patent Document 3)
3) Combination of three-dimensional culture (formation of biowire) and electrical stimulation (for example, Non-Patent Documents 4 to 6)
4) Addition of cardiomyocyte maturation promoter (for example, Patent Document 1)
5) Transient expression of Sall1 gene and Mesp1 gene (for example, Patent Document 2)

International Publication 2019/189554 Pamphlet JP-A-2017-60422

As mentioned above, some techniques for maturing cardiomyocytes have been reported, but there is room for improvement in terms of cost and technology.
Therefore, an object of the present invention is to provide a method for efficiently obtaining mature cardiomyocytes that can be used for drug discovery screening and cell transplantation in a short period of time.

The present inventors have conducted diligent studies to solve the above problems, and first, in the process of cardiomyocyte maturation using a cardiomyocyte maturation promoter (Patent Document 1), CDKN1A (cyclin-dependent kinase inhibitor 1, also known as p21). ) Was found to increase. Then, they found that increasing the expression level of CDKN1A in immature cardiomyocytes efficiently obtained mature cardiomyocytes in a short period of time, and completed the present invention.

The gist of the present invention is as follows.
[1] A method for producing mature cardiomyocytes, which comprises a step of increasing the expression level of cyclin-dependent kinase inhibitor 1 in immature cardiomyocytes.
[2] The method for producing mature cardiomyocytes according to [1], wherein the increase in the expression level is maintained for 3 days or more.
[3] The method for producing a mature cardiomyocyte according to [1] or [2], wherein the cardiomyocyte is a human cardiomyocyte.
[4] The method for producing a mature cardiomyocyte according to any one of [1] to [3], wherein the immature cardiomyocyte is an immature cardiomyocyte in which differentiation is induced from a pluripotent stem cell.
[5] The method for producing mature cardiomyocytes according to [4], wherein the pluripotent stem cells are induced pluripotent stem cells.
[6] Mature cardiomyocytes proliferated by the method according to any one of [1] to [5].

According to the present invention, cardiomyocytes can be efficiently matured in a short period of time without using a special culture medium or the like. In particular, when combined with the induction of differentiation from pluripotent stem cells, mature cardiomyocytes can be easily and mass-produced.

It is a schematic diagram which shows one aspect of the differentiation induction method of this invention. “Day” indicates the number of days from the start of differentiation induction. It is a schematic diagram of the change detected in the process of differentiation and maturation of the reporter iPS cells (1390D4 strain) used in the examples into cardiomyocytes. Since EmGFP is inserted under the promoter control of the TNNI1 gene and mCherry is inserted under the promoter control of the TNNI3 gene, when differentiation is induced into cardiomyocytes, immature cardiomyocytes that are EmGFP-positive and mCherry-negative are first produced. After that, as the maturation progresses, the expression level of EmGFP decreases and the expression of mCherry is induced, so that the mature cardiomyocytes eventually become EmGFP-negative mCherry-positive mature cardiomyocytes. About mature cardiomyocytes (maturation-promoting agent-treated cells) obtained from reporter iPS cells using a cardiomyocyte maturation promoter, and immature cardiomyocytes (promoter-non-treated cells) obtained without treatment with the promoter. The gene ontology (GO) enrichment analysis results (A) of accelerator-treated cells for accelerator-treated cells are shown. FIG. 3B is a list of genes included in “Kinase inhibitor activity” in FIG. 3A. Time course of expression (mRNA) of CDKN1A and TNNI3 genes in mouse heart development using the National Center for Biotechnology Information (NCBI) gene expression information database (GEO, Acc. No. GSE51483) (A) This is the result of analyzing the correlation (B) between the expression levels of both genes. The analysis samples in each stage of A are as follows; E8.5 heart tubes, left and right ventures at E9.5, E12.5, E14.5, E18.5, 3 days after birth (Postnatal 3d), adult heart (mCM) The results of analyzing the mRNA amounts of the CDKN1A and HOPX genes of human ES cells and mature cardiomyocytes obtained from the cells using the cardiomyocyte maturation promoter are shown (A, C). B is a graph obtained by analyzing the correlation between the expression levels of the CDKN1A gene and the HOPX gene in human cardiomyocytes using GEO: GSE46224. D shows the result of analyzing the mRNA amount of the CDKN1A gene in human adult and human fetal cardiomyocytes using the above GEO: GSE50704. HOPX is a gene that has been suggested to contribute to heart development by regulating serum response factor (SRF) -dependent heart-specific gene expression. It is a graph which analyzed the correlation of the expression level of the CDKN1A gene and the TNNI3 gene (A), and the expression level of the CDKN1A gene and the HIC1 gene (B) in human heart development using the above GEO: GSE46224, respectively. For mature cardiomyocytes (accelerator-treated cells) and immature cardiomyocytes (accelerator-untreated cells) obtained from reporter iPS cells, cardiomyocyte-specific genes (Myh6: Myosin) of accelerator-treated cells relative to accelerator-untreated cells heavy chain, α isoform (MHC-α), Myl7: myosin light chain 7, Ttn: titin, cTnT (= TNNT): troponin T2, cardiac type) and fibroblast-specific genes (Tgfbi: transforming growth factor, beta- Induced, 68 kDa, Col11a1: collagen type XI alpha 1 chain, Fbln1: fibulin 1, Thy: thymidylate synthase) expression level (A) and main component analysis 7 days, 14 days, and 21 days after induction of differentiation The result (B) of (PCA, n = 3) is shown. In the process of differentiating from reporter iPS cells to mature myocardial cells with a myocardial cell differentiation inducer, analysis results of genes whose expression levels change between Day7 vs. Day14 and Day7 vs. Day21 (heat map: A), expressed on Day7 vs. Day21 It shows the analysis result of the number of genes whose expression was increased or decreased (Ben diagram: B), and the time course of the mRNA amount of the TNNI3 or CDKN1A gene (C). It is a graph which analyzed the mRNA amount of CDKN1A and TNNI3 at the time point (Day40, Day60) which a long time passed from the start of differentiation induction about the mature cardiomyocyte obtained from the reporter iPS cell by the maturation accelerator treatment. The results of analysis of cardiomyocyte maturation-related genes and CDKN1A in the mouse fetal heart using GEO (Gene Expression Omnibus) GSE1479 data are shown. A is a graph showing the time course of CD36 gene expression (n = 6), B is a graph showing the correlation analysis (Pearson correlation) between CD36 and CDKN1A, and C is a graph showing the correlation analysis (Pearson correlation) between HOPX and CDKN1A. It is a graph which shows the result of having analyzed the expression variation gene (differentially expressed genes; DEGs) in the pathway associated with CDKN1A in the development of fetal myocardium using the data of GEO GSE51483. * Indicates p-value <0.05, ** indicates p-value <0.01, and *** indicates p-value <0.001 (pearson r). It is a heat map which shows the result of having divided into the atrium and the ventricle of the heart of a mouse fetal. A shows the time course of maturation-related gene expression in the atrial muscle, and B shows the time-course change of maturation-related gene expression in the ventricles. In the step of producing mature cardiomyocytes from the reporter iPS cells by the method shown in FIG. 1 (method of introducing and expressing the exogenous CDKN1A gene into immature cardiomyocytes to mature them), the exogenous CDKN1A gene is introduced (expression started). It is the analysis result of the cell 4 or 5 days after (Day 20 or Day 21). A is a graph showing the analysis results of the fluorescence intensities of mCherry and GFP. The image of B is a fluorescence micrograph in which fluorescence of mCherry and GFP is detected, and the graph of B is a graph obtained by analyzing the amount of mRNA of a group of genes involved in cardiomyocyte maturation. “CDKN1A” in A and B represents a cell infected with a lentivirus (virus particle) encoding the CDKN1A gene. In addition, "pLenti6.3" in A and "Control" in B both represent cells infected with a lentivirus (virus particle) that does not encode the CDKN1A gene (negative control). This is the result of analysis of mature cardiomyocytes obtained from reporter iPS cells by the method shown in FIG. A represents a heat map that analyzes the amount of mRNA for negative control cells for the gene set involved in cardiomyocyte maturation. B represents a phase-contrast microscopic image, TNN3 and / or TNN1 fluorescence observation image of mature cardiomyocytes on Day 60 (all co-field images). The results of evaluation of the maturation-inducing effect of CDKN1A on more immature early cardiomyocytes are shown. The workflow of the CDKN1A expression experiment in early immature cardiomyocytes is shown in A. “Day” indicates the number of days from the start of differentiation induction. CDKN1A was expressed in cardiomyocytes on Day 14. B is a graph in which the mRNA expressions of CDKN1A and TNNI3 on Day 30 were analyzed by quantitative PCR.

The method for producing mature cardiomyocytes of the present invention includes a step of increasing the expression level of cyclin-dependent kinase inhibitor 1: CDKN1A in immature cardiomyocytes.

In the present invention, cardiomyocytes are cells expressing at least one marker gene selected from the group consisting of myocardial troponin (cTNT), αMHC (α myosin heavy chain, MYH6) and βMHC (MYH7). means. For cTNT, NCBI accession number NM_000364 is exemplified in the case of humans, and NM_001130176 is exemplified in the case of mice. For αMHC, NCBI accession number NM_002471 is exemplified in the case of humans, and NM_001164171 is exemplified in the case of mice. For βMHC, NCBI accession number NM_000257 is exemplified in the case of humans, and NM_080728 is exemplified in the case of mice.
The cardiomyocytes are not particularly limited, but are preferably derived from mammals (eg, mice, rats, hamsters, rabbits, cats, dogs, cows, sheep, pigs, monkeys, humans), and more preferably derived from humans. Is.

It is known that as cardiomyocytes mature, the expression of troponin I1 (TNNI1) decreases, and an isoform switch occurs in which the expression of troponin I3 (TNNI3) increases (Fikru B. Bedada, (2014) 3 (Fikru B. Bedada, (2014) 3 ( 4): 594-605.).

In the present specification, the immature cardiomyocyte means a cardiomyocyte in which the expression level of TNNI3 is very low and TNNI1 is predominantly expressed. Immature cardiomyocytes are sometimes called fetal-like cardiomyocytes.

In the present specification, the mature cardiomyocyte means a cardiomyocyte in which the expression level of TNNI1 is very low and TNNI3 is predominantly expressed. Mature cardiomyocytes are sometimes called adult-like cardiomyocytes. For example, when the expression levels of TNNI3 and TNNI1 were measured at the gene level or protein level, and standardized and compared with the expression levels of constitutive expression markers, respectively, the expression level of TNNI3 was the expression of TNNI3 in fetal myocardial cells. A mature myocardial cell can be a myocardial cell having an amount of 5 times or more, more preferably 10 times or more, still more preferably 25 times or more, and particularly preferably 100 times or more.

The expression levels of TNNI3 and TNNI1 are measured, for example, by measuring the mRNA levels of these genes using PCR or the like; analyzing the protein expression levels by Western blotting or the like; analyzing by the expression levels of reporter molecules; or fluorescent labeling or It can be analyzed by a method such as analysis by a microscope or a flow cytometer based on the fluorescence intensity of the fluorescent reporter.

In addition to the above-mentioned expression levels of TNNI3 and TNNI1, the maturity of cardiomyocytes is indexed by morphology, structure (eg, sarcomere, mitochondria), properties (eg, pulsatile state, potential physiological maturity), etc. You may. For example, the depth of the resting membrane potential by a patch clamp or the like can be used as an index of the potential physiological maturity. The microstructure of sarcomere and mitochondrial indicators can be observed with an electron microscope; analyzed with a microscope or flow cytometer with a fluorescent label; or functionally analyzed with an extracellular flux analyzer or the like. These indicators are compared with control mature cardiomyocytes such as adult cardiomyocytes and control immature cardiomyocytes such as fetal cardiomyocytes to determine whether the cardiomyocytes to be evaluated are mature or immature. It can also be determined.

In the present specification, CDKN1A means cyclin-dependent kinase inhibitor 1 (also known as p21), and its sequence and origin as long as it can exert the function of maturing immature myocardial cells. There are no particular restrictions on the species to be used, but for example, as the human CDKN1A gene, a gene registered as Gene ID 1026 of NCBI can be exemplified (https://www.ncbi.nlm.nih.gov). / gene / 1026).
The nucleotide sequence of this CDKN1A gene is shown in SEQ ID NO: 1, and the encoding amino acid sequence is shown in SEQ ID NO: 2. The CDKN1A gene has stringent conditions (eg, 65 ° C., 0.1 × SSC, 0 after hybridization) with the polynucleotide having the complementary nucleotide sequence of SEQ ID NO: 1 as long as it can exert the function of maturing immature cardiomyocytes. It may be a gene that hybridizes under the condition of washing with 1% SDS). In addition, the CDKN1A protein is a protein having an amino acid sequence having 90% or more, 95% or more, or 98% or more identity of the amino acid sequence of SEQ ID NO: 2 as long as it can exert the function of maturing immature cardiomyocytes. It may be there.

As used herein, "increasing the expression level of CDKN1A" means increasing the expression level of CDKN1A mRNA and / or CDKN1A protein, specifically, 5 times or more, preferably 10 times or more, and more. It means that the increase is preferably 20 times or more, more preferably 50 times or more, and most preferably 100 times or more. Further, the increase may be based on, for example, the expression level of CDKN1A in immature cardiomyocytes having a TNNI1 / TNNI3 expression level ratio of 10 or more or fetal cardiomyocytes.
In addition, the CDKN1A mRNA or CDKN1A protein may be derived from either an endogenous or exogenous CDKN1A gene.

When increasing the expression level of CDKN1A mRNA and / or CDKN1A protein, the CDKN1A gene endogenously present in immature myocardial cells may be activated, or the CDKN1A gene may be exogenously introduced into immature myocardial cells for expression. You may let me. Activation of the CDKN1A gene endogenously present in immature cardiomyocytes can be performed, for example, by modifying the expression regulation mechanism of the endogenously present CDKN1A gene.

The method for introducing the CDKN1A gene into immature cardiomyocytes exogenously is not particularly limited, but for example, the following method can be used.

When introducing in the form of a gene (DNA), for example, a vector such as a virus, a plasmid, or an artificial chromosome can be introduced into immature myocardial cells by a method such as lipofection, liposome, or microinjection. Examples of the viral vector include a retrovirus vector, a lentiviral vector, an adenovirus vector, an adeno-associated virus vector, and a Sendai virus vector. Further, the artificial chromosome vector includes, for example, a human artificial chromosome (HAC), a yeast artificial chromosome (YAC), a bacterial artificial chromosome (BAC, PAC) and the like. As the plasmid, a plasmid for mammalian cells can be used. The vector can contain regulatory sequences such as promoters, enhancers, ribosome binding sequences, terminators, polyadenylation sites, etc. so that the gene of interest can be expressed, and if desired, drug resistance genes (eg, canamycin). It can include selection marker sequences such as resistance gene, ampicillin resistance gene, puromycin resistance gene, etc.), thymidin kinase gene, diphtheriatoxin gene, fluorescent protein, β-glucuronidase (GUS), reporter gene sequence such as FLAG, and the like. As promoters, SV40 promoter, LTR promoter, CMV (cytomegalovirus) promoter, RSV (Rous sarcomavirus) promoter, MoMuLV (Moloney mouse leukemia virus) LTR, HSV-TK (herpes simpleplex virus thymidine kinase) promoter, EF-α promoter, CAG An example is a promoter and a TRE promoter (a CMV minimum promoter having a Tet response sequence in which the tetO sequence is consecutive 7 times). When using the TRE promoter, it is desirable to simultaneously express the fusion protein with tetR and VP16AD or the fusion protein with reverse tetR (rtetR) and VP16AD in the same cell. Here, a vector having a TRE promoter and capable of expressing a fusion protein with reverse tetR (rtetR) and VP16AD is an example of a drug-responsive induction vector. In addition, the above vector has a transposon sequence before and after the expression cassette in order to incorporate the expression cassette consisting of the promoter and the CDKN1A gene that binds to the chromosome of the pluripotent cell into the chromosome, and to excise it if necessary. But it may be. The transposon sequence is not particularly limited, but piggyBac is exemplified. In another embodiment, LoxP sequences may be present before and after the expression cassette for the purpose of removing the expression cassette.

When a drug-responsive induction vector is used, the expression time of the introduced CDKN1A gene can be controlled. That is, the CDKN1A gene can be expressed by introducing the CDKN1A gene into cells in advance and adding a drug at a required time. For example, when immature cardiomyocytes are obtained from pluripotent stem cells as described later, the CDKN1A gene is introduced at the stage of pluripotent stem cells, and when differentiation is induced into immature cardiomyocytes, a drug is used. It may be added to express the CDKN1A gene. As the drug, an appropriate drug can be used in relation to the drug-responsive promoter, and for example, doxycycline and the like are used.
When a vector having a LoxP sequence is used, it is also possible to stop the expression by introducing Cre into the cell after the lapse of a desired period.

When introduced in the form of RNA, it may be introduced into pluripotent stem cells by a method such as electroporation, lipofection, or microinjection.

When introduced in the form of a protein, it may be introduced into pluripotent stem cells by a method such as lipofection, fusion with a cell membrane penetrating peptide (for example, HIV-derived TAT and polyarginine), or microinjection.

The period of "increasing the expression level of CDKN1A" in immature cardiomyocytes may be a period sufficient for the immature cardiomyocytes to change into mature cardiomyocytes, and is not particularly limited. More than a day, more than 5 days, more than 6 days, more than 7 days, more than 8 days, more than 9 days, or more than 10 days. There is no particular upper limit, and the state in which the expression level of CDKN1A is increased may be maintained even after the immature cardiomyocytes are transformed into mature cardiomyocytes, but the CDKN1A gene is not always required after the immature cardiomyocytes are transformed into mature cardiomyocytes. It is not necessary to continue the state in which the expression level of is increased.

The operation of "increasing the expression level of CDKN1A" such as the introduction of the CDKN1A gene into the cell can be performed once or multiple times. For example, when CDKN1A is introduced in the form of a gene product, if the half-life of the gene product is short, the introduction may be performed multiple times. The number of introductions can be appropriately set, but for example, twice. Three times, four times, five times or more are exemplified.

The operation of "increasing the expression level of CDKN1A" in immature cardiomyocytes can be started at an appropriate timing. For example, it may be started immediately after differentiation into immature cardiomyocytes occurs. At this time, the differentiation into immature cardiomyocytes is at least one cardiomyocyte marker gene selected from the group consisting of, for example, TNNI1, myocardial troponin (cTNT), αMHC (α myosin heavy chain, MYH6) and βMHC (MYH7). It can be confirmed by the expression of, but is not limited to this, and can be confirmed by an appropriate means. The operation of "increasing the expression level of CDKN1A" may be performed, for example, on cells expressing at least one cardiomyocyte marker gene.

By culturing immature cardiomyocytes in a state where the expression level of CDKN1A is increased, immature cardiomyocytes can be converted (induced) into mature cardiomyocytes.
The culture conditions may be those used for normal cardiomyocyte culture, and are, for example, 30 to 40 ° C, preferably 36 to 38 ° C, and more preferably about 37 ° C. Further, the culture is preferably carried out in an atmosphere containing oxygen and carbon dioxide, and the oxygen concentration is preferably about 5 to 20%, and the carbon dioxide concentration is preferably about 2 to 5%.

The culture period may be a period sufficient for the immature cardiomyocytes to be transformed into mature cardiomyocytes, and is not particularly limited. 4 days or more, 5 days or more, 6 days or more, 7 days or more, 8 days or more, 9 days or more, or 10 days or more. There is no particular upper limit, and even after the immature cardiomyocytes are transformed into mature cardiomyocytes, the culture may be continued as long as the properties of the mature cardiomyocytes can be maintained.
Although it depends on the expression level of CDKN1A, as a guide, mature cardiomyocytes are obtained about 12 to 13 days after "increasing the expression level of CDKN1A" (about Day 30 by the method described in FIG. 1). be able to. After that, the proportion of mature cardiomyocytes continued to increase, and after about 22 to 23 days (by the method shown in FIG. 1, about Day 40), 70% or more, 80% or more, and 90% or more of all cells matured. Become a cardiomyocyte.

As the medium used for culturing and maintaining immature cardiomyocytes in the state of "increased expression level of CDKN1A", a medium known per se can be used without particular limitation. For example, IMDM medium, Medium 199 medium, Eagle's Minimum Essential Medium (EMEM) medium, αMEM medium, Dulbecco's modified Eagle's Medium (DMEM) medium, Ham's F12 medium, RPMI 1640 medium, Fischer's medium, Neurobasal Medium (Life Technologies), StemPro34 ( Invitrogen), StemFit AK02 medium (AJINOMOTO), Essential 6 medium (Thermo Fischer Scientific) and a mixed medium thereof are used.
Additives known per se can be added to these media for each cell and culture condition. For example, the medium may contain serum or may be serum-free. Further, if desired, media include, for example, albumin, transferase, Knockout Serum Replacement (KSR), N2 supplement (Invitrogen), B27 supplement (Invitrogen), fatty acids, insulin, It may contain one or more serum substitutes such as collagen precursors, trace elements, 2-mercaptoethanol, 1-thiolglycerol, lipids, amino acids, L-glutamine, Glutamax (Invitrogen), non-essential amino acids, vitamins, It may contain one or more substances such as growth factors, low molecular weight compounds, antibiotics, antioxidants, serum, buffers, inorganic salts and the like.
In addition, cytokines such as activin A, BMP4, bFGF, VEGF, and VEGF, and compounds such as GSK-3β inhibitors and Wnt inhibitors may be appropriately added.

The immature cardiomyocyte may be a cardiomyocyte isolated from a living body, but is preferably an immature cardiomyocyte whose differentiation is induced from a pluripotent stem cell.

Pluripotent stem cells are stem cells that have pluripotency that can differentiate into many cells existing in the living body and also have proliferative ability, and include arbitrary cells induced in the primitive endoderm. Will be done. The pluripotent stem cells are not particularly limited, and are, for example, embryonic stem (ES) cells, induced pluripotent stem (iPS) cells, sperm stem cells (“GS cells”), and embryonic germ cells (“EG cells””. ), Cultured fibroblasts, pluripotent cells derived from bone marrow stem cells (Muse cells), and the like. Preferred pluripotent stem cells are iPS cells and ES cells. The pluripotent stem cells are preferably derived from mammals, more preferably from primates, and even more preferably from humans.

The method for producing iPS cells is known in the art, and can be produced by introducing a reprogramming factor into any somatic cell or the like. Here, the reprogramming factors include, for example, Oct3 / 4, Sox2, Sox1, Sox3, Sox15, Sox17, Klf4, Klf2, c-Myc, N-Myc, L-Myc, Nanog, Lin28, Fbx15, Eras, ECAT15. Genes or gene products such as -2, Tcl1, beta-catenin, Lin28b, Sall1, Sall4, Esrrb, Nr5a2, Tbx3 or Glis1 are exemplified, and these reprogramming factors may be used alone or in combination. Is also good. As a combination of reprogramming factors, WO2007 / 069666, WO2008 / 118820, WO2009 / 007852, WO2009 / 032194, WO2009 / 058413, WO2009 / 057831, WO2009 / 075119, WO2009 / 079007, WO2009 / 091659, WO2009 / 101084, WO2009 / 101407, WO2009 / 102983, WO2009 / 114949, WO2009 / 117439, WO2009 / 126250, WO2009 / 126251, WO2009 / 126655, WO2009 / 157593, WO2010 / 009015, WO2010 / 033906, WO2010 / 033920, WO2010 / 042800, WO2010 WO2010 / 056831, WO2010 / 068955, WO2010 / 098419, WO2010 / 102267, WO2010 / 111409, WO2010 / 111422, WO2010 / 115050, WO2010 / 124290, WO2010 / 147395, WO2010 / 147612, Hungfu D, et. (2008), Nat. Biotechnol. , 26: 795-797, Shi Y, et al. (2008), CellStem Cell, 2: 525-528, Eminli S, et al. (2008), Stem Cells. 26: 2467-2474, Hungfu D, et al. (2008), Nat. Biotechnol. 26: 1269-1275, Shi Y, et al. (2008), CellStem Cell, 3,568-574, Zhao Y, et al. (2008), CellStem Cell, 3: 475-479, Marson A, (2008), CellStem Cell, 3,132-135, Feng B, et al. (2009), Nat. Cell Biol. 11: 197-203, R.M. L. Judson et al. , (2009), Nat. Biotechnol. , 27: 459-461, Lyssiotics CA, et al. (2009), Proc Natl Acad Sci U S A. 106: 8912-8917, Kim JB, et al. (2009), Nature. 461: 649-643, Ichida JK, et al. (2009), CellStem Cell. 5: 491-503, Heng JC, et al. (2010), CellStem Cell. 6: 167-74, Han J, et al. (2010), Nature. 463: 1096-100, Mary P, et al. (2010), Stem Cells. 28: 713-720, Maekawa M, et al. (2011), Nature. 474: 225-9. The combinations described in are exemplified.

The somatic cells used to make iPS cells include both fetal (pup) somatic cells, neonatal (pup) somatic cells, and mature healthy or diseased somatic cells, as well as the primary. All of cultured cells, passaged cells, and established cells are included. Specifically, the somatic cells include, for example, (1) tissue stem cells (somatic stem cells) such as nerve stem cells, hematopoietic stem cells, mesenchymal stem cells, and dental pulp stem cells, (2) tissue precursor cells, and (3) blood cells (peripheral). Blood cells, umbilical cord blood cells, etc.), lymphocytes, epithelial cells, endothelial cells, muscle cells, fibroblasts (skin cells, etc.), hair cells, hepatocytes, gastric mucosal cells, intestinal cells, splenocytes, pancreatic cells (pancreatic exocrine cells, etc.) Etc.), differentiated cells such as brain cells, lung cells, renal cells and fat cells are exemplified.

Immature cardiomyocytes can be produced from pluripotent stem cells by a method known per se. As a method for inducing differentiation of pluripotent stem cells into immature cardiomyocytes, for example, the methods disclosed in the following documents are exemplified.
Laflamme MA & Murry CE, Nature 2011, May 19; 473 (7347): 326-35 Review
Funakoshi, S. et al. Sci Rep 8, 19111 (2016)
Miki, K. et al. Cell Stem Cell. 2015 Jun 4; 16 (6): 699-711
In addition to this, although not particularly specified, for example, a method for producing cardiomyocytes by forming cell clusters (embryonic bodies) by suspension culture of induced pluripotent stem cells (WO2016 / 104614), Bone Morphogenic Protein (BMP) signal. Method of producing cardiomyocytes in the presence of a substance that suppresses transmission (WO2005 / 033298), method of producing cardiomyocytes by adding Activin A and BMP in order (WO2007 / 002136), activation of canonical Wnt signal pathway Method for producing cardiomyocytes in the presence of promoting substances (WO2007 / 126077) and method for isolating FLk / KDR-positive cells from induced pluripotent stem cells and producing cardiomyocytes in the presence of cyclosporin A (WO2009 / 118928) Etc. are exemplified.
In addition, a method of inducing differentiation of myocardial cells using cytokines in the embryonic body formation method (Yang L, et al., Human cardiovascular progenitor cells develop from a KDR + embryonic-stem-cell-derived population., Nature., 2008 May 22; 453 (7194): 524-8), Method of inducing differentiation of myocardial cells without using cytokines in adherent culture (Lian X, et al., Robust cardiomyocyte differentiation from human pluripotent stem cells via temporal modulation of canonical Wnt signaling ., Proc Natl Acad Sci US A., 2012 July 3; 109 (27): E1848-57), A method of inducing differentiation of myocardial cells without using cytokines by using adhesive culture and suspension culture together (Minami I) , et al., A small molecule that promotes cardiac differentiation of human pluripotent stem cells under defined, cytokine- and xeno-free conditions., Cell Rep., 2012 Nov 29; 2 (5): 1448-60) ing.

When the immature cardiomyocytes are immature cardiomyocytes induced to differentiate from pluripotent stem cells, the operation of "increasing the expression level of CDKN1A" can be started at an appropriate timing. For example, in any of the above methods for producing cardiomyocytes or inducing differentiation of cardiomyocytes, it may be started immediately after immature cardiomyocytes are induced to differentiate. At this time, the differentiation-induced immature cardiomyocytes are, for example, at least one cardiomyocyte selected from the group consisting of TNNI1, myocardial troponin (cTNT), αMHC (α myosin heavy chain, MYH6) and βMHC (MYH7). It may be a cell in which expression of a marker gene has been confirmed. That is, the operation of "increasing the expression level of CDKN1A" may be performed on cells expressing at least one cardiomyocyte marker gene, for example.

In the present invention, the mature cardiomyocytes obtained by increasing the expression of CDKN1A have excellent characteristics that the transplant engraftment ability is improved when transplanted into myocardial tissue and can function as myocardium. Therefore, cardiomyocytes. It is suitably used as a cell preparation for transplantation to patients in need of transplantation. Patients who require cardiomyocyte transplantation include, but are not limited to, patients with diseases caused by cardiomyocyte deficiency such as myocarditis, myocardial infarction, and myocardial damage. The amount of cells to be transplanted is appropriately selected depending on the type and degree of the disease, and the number of transplants may be one or more. The method of transplantation is not limited, and injection may be performed at the diseased site, or a cardiomyocyte sheet may be prepared and applied to the diseased site.

In one embodiment, the mature cardiomyocytes obtained by the method of the present invention can be used for cardiac regenerative medicine. For example, a composition containing a cell mass of cardiomyocytes produced by the method of the present invention can be administered to the heart of a patient suffering from heart disease. Specifically, the cardiomyocytes obtained by the method of the present invention may be transplanted into the heart of a heart disease patient as it is as a cell suspension or in the form of a myocardial sheet (single layer or multi-layer). For the manufacturing method of myocardial sheet, refer to, for example, WO2012 / 133945, WO2013 / 137491, WO2014 / 192909, WO2016 / 076368.

In another embodiment, the mature cardiomyocytes obtained by the method of the present invention are uniformly mature and can be used for drug screening for the treatment of heart disease and cardiotoxicity evaluation of the drug. For example, the effect and toxicity of the test drug can be evaluated by administering the test drug to the cardiomyocytes obtained by the method of the present invention and examining the response of the cardiomyocytes.

Hereinafter, the present invention will be described in more detail with reference to examples, but the aspects of the present invention are not limited to the following examples.

<Reporter cell>
The reporter cells described in Examples of Patent Document 1 were used as cells for inducing differentiation into cardiomyocytes so that the degree of maturation of cardiomyocytes could be easily detected. The cell is a double knock-in human iPS cell line (1390D4 strain) in which the EmGFP gene sequence is inserted at the TNNI1 locus and the mCherry gene sequence is inserted at the TNNI3 locus via the 2A sequence. Since TNNI1 and EmGFP and TNNI3 and mCherry are polycistronically expressed in the 1390D4 strain, the expression level of TNNI1 should be evaluated using the expression level of EmGFP as an index, and the expression level of TNNI3 should be evaluated using the expression level of mCherry as an index. Can be done.
Therefore, by using the 1390D4 strain, the change from immature cardiomyocytes to mature cardiomyocytes can be detected by the change in fluorescent color (Fig. 2).

Method 1: Induction of differentiation of iPS cells into immature cardiomyocytes StemFit on a culture dish coated with iMatrix511 (Nippi) as described in Nakagawa et al. (Sci. Rep. 2014; 4: 3594.). 1390D4 cells were cultured in AK02N medium (Ajinomoto). Then, the protocol described in Dubois et al. (Nat Biotechnol. 2011 Oct 23; 29 (11): 1011-1018.) was modified to use a 6-well low-adhesion plate (Corning) to cardiomyocytes. Differentiation was induced.
Specifically, in order to dissociate 1390D4 cells into single cells using 0.5 × TrypLE select (Thermo Fisher Scientific) (1 × TrypLE select diluted with 0.5 mM EDTA), and then to form embryonic bodies, 2 mM L-glutamine (Invitrogen), 4 × 10 ^-4 M monothioglycerol (MTG), 50 μg / ml ascorbic acid (AA), 150 μg / ml transtransferase, 10 μM ROCK inhibitor (Y-27632), 0.5% The cells were suspended in a medium supplemented with Matrigel (Corning) and 2 ng / ml BMP4 (R & D Systems) and ^{seeded to 2 × 10 6} cells in 1.5 ml / well (Day 0: day when differentiation induction was started). Hereinafter, X days after the start date of differentiation induction is referred to as DayX.
On

Day

1, 2 mM L-glutamine, 4 × 10 ^-4 MTG, 50 μg / ml AA, 150 μg / ml transferrin, 10 ng / ml bFGF (final 5 ng / ml), 12 ng / ml activin A (final 6 ng / ml) and 18 ng / 1.5 ml of StemPro-34 medium supplemented with ml BMP4 (final 10 ng / ml) was added to the wells.
On Day 3, the formed EB was washed once with Iscove-modified Dulbecco medium (IMDM; Invitrogen), followed by 2 mM L-glutamine, 4 × 10 ^-4 MTG, 50 μg / ml AA, 150 μg / ml transferrin, 10 ng / ml. Incubate in 3 ml StemPro-34 medium supplemented with Vascular Endothelial Growth Factor (VEGF; R & D Systems), 1 μM IWP3 (Stemgent), 0.6 μm dolsomorphin and 5.4 μm SB431542.
On Day 6, the medium was replaced with 2 ml of StemPro-34 medium supplemented with 2 mM L-glutamine, 4 × 10 ^-4 MTG, 50 μg / ml AA, 150 μg / ml transferrin and 5 ng / ml VEGF.
Then, the medium was changed to the medium having the same composition every 2-3 days.
_{The plate was placed in a hypoxic environment (5% O 2} ) from Day 0 to Day 10 and then moved to a normal oxygen environment.
Immature cardiomyocytes were obtained on Day 14.

Method 2: Maturation of immature cardiomyocytes using a cardiomyocyte maturation promoter (positive control experiment) Using the following T112 and T623 as maturation promoters, pluripotency by the method described in Test Example 2 of Patent Document 1. Mature cardiomyocytes were obtained from sex stem cells. Specifically, T112 or T623 was added to the medium for 6 days from Day 10 to Day 16 (final concentration 40 nM). In the examples, mature cardiomyocytes obtained by this method are referred to as “accelerator-treated cells”, and cells that have only been induced to differentiate into cardiomyocytes without the accelerator treatment (cardiomyocytes that remain immature) are used. Sometimes referred to as "accelerator-untreated cells". Then, for the comparative analysis of both cells, cells on the same day from the start of differentiation induction were used.

Method 3: CDKN1A gene transfer using a lentiviral vector (Example, FIG. 1)
The immature cardiomyocytes of Day 14 obtained in Method 1 were dissociated, and ^{plated and adhered at a cell density of 1 × 10 6} cells / well for 3 days (= immature cardiomyocytes of Day 17).
In parallel, HEK293FT cells were transfected with pLenti6.3-CDKN1A (pLenti6.3 / V5DEST from which the human CDKN1A gene was cloned; obtained from Thermofisher Scientific) to package lentivirus particles containing the CDKN1A gene. The human CDKN1A cDNA clone was obtained from the DNA type derived from clone ID H04D013O09. The medium was changed 24 hours after transfection, and the culture supernatant containing the virus particles was collected 24 hours later.
The immature cardiomyocytes of Day 17 were cultured in the HEK293FT-derived culture supernatant for 24 hours to infect the virus particles. After that, the culture was continued, and the expression analysis and the expression of the reporter protein at each time point were analyzed. A lentiviral vector encoding blue fluorescent protein (BFP) was used as a positive control for lentiviral infection.

Method 4: CDKN1A gene transfer using a lentiviral vector into earlier immature cardiomyocytes (Example)
In the same manner as in Method 3, a culture supernatant containing lentivirus particles containing the CDKN1A gene was collected from pLenti6.3-CDKN1A.
The immature cardiomyocytes of Day 14 obtained in Method 1 were cultured in the culture supernatant for 24 hours to infect the virus particles. A lentiviral vector encoding blue fluorescent protein (BFP) was used as a positive control for lentiviral infection. After that, the culture was continued, and the expression of CDKN1A and TNNI3 was analyzed by quantitative RT-PCR at Day 30. In addition, GAPDH was used as an internal control for quantitative RT-PCR.

Results <Identification of new genes that contribute to cardiomyocyte maturation>
A comparative analysis of gene expression profiles was performed on mature cardiomyocytes obtained from 1390D4 cells by Method 1 and immature cardiomyocytes not treated with the accelerator. FIG. 3 (A) shows the results of OG enrichment analysis of the gene cluster expressed and enriched in the accelerator-treated cells with respect to the accelerator-untreated cells. Multiple GO Termes that were significantly (p <0.05) enriched were found, and in particular, it was revealed that the expression of genes whose kinase inhibitory activity was GO was significantly enriched. The gene cluster contained 15 types of genes shown in FIG. 3 (FIG. 3 (B)).

Focusing on CDKN1A from various analysis results, we analyzed changes in the expression of CDKN1A gene and TNNI3 gene during cardiac development in mice using NCBI's gene expression information database (GEO, Acc.No. GSE51483) (Fig. 4). ..
The expression levels of both genes increased as cardiac development progressed, and it was revealed that the expression of CDKN1A reached almost the same level as that of adult mice in the late development stage (Fig. 4A). It was also confirmed that the transcription levels of both genes were significantly correlated (Fig. 4B).

Subsequently, mature cardiomyocytes were also produced from human ES cells using the maturation promoter, and the expression level of CDKN1A was analyzed (FIG. 5). The expression of CDKN1A was hardly detected in human ES cells (Fig. 5A), and the expression of HOPX was also very low (Fig. 5C). Has increased significantly (Figs. 5A and C). Furthermore, the expression levels of both genes in human cardiomyocytes were significantly correlated (Fig. 5B). FIG. 5D shows the expression level of CDKN1A in human adult and human fetal cardiomyocytes. The expression level of CDKN1A was significantly increased in human adult cardiomyocytes as compared with human fetal cells.
Therefore, not only in the step of allowing a maturation promoter to act on cardiomyocytes derived from pluripotent stem cells (including iPS cells and ES cells) to mature them, but also in the step of (naturally) maturing in vivo, human myocardium In cells, it was revealed that the expression of CDKN1A increased remarkably with maturation.

FIG. 6 shows the results of analyzing the correlation between the expression levels of the CDKN1A gene and the TNNI3 gene (A) and the CDKN1A gene and the HIC1 gene (tumor suppressor gene) (B) in human heart development using GEO: GSE46224. It can be seen that the expression of the CDKN1A gene increases with cardiac development, and the increase correlates with the increased expression of TNNI3 and HIC1. From this result, it is considered that CDKN1A regulates the expression levels of HIC1 and TNNI3.

In mature cardiomyocytes obtained using a cardiomyocyte maturation promoter, the expression of cardiomyocyte-specific genes is significantly enhanced and the expression of fibroblast-specific genes is significantly higher than that of untreated immature cardiomyocytes. (Fig. 7A). From the results of principal component analysis (Fig. 7B) 7 days, 14 days, and 21 days after the induction of differentiation, it was clarified that the gene expression pattern changed dramatically between Day 7 and Day 14.

FIG. 8 shows a heat map analysis (A) of genes whose expression levels change between Day 7 vs. Day 14 or Day 7 vs. Day 21 in accelerator-treated cells, and a Venn diagram of the number of genes whose expression increased or decreased between Day 7 vs. Day 21 (B). ) Is shown. Although the expression of a large number of genes changes (Fig. 8B), it was shown that many of the genes whose expression increased or decreased on Day 21 had already increased or decreased as of Day 14 (Fig. 8B). It was also clarified that the expression level of TNNI3 remained almost unchanged until Day 7, decreased on Day 14, and then increased, whereas CDKN1A continued to increase from Day 7 to Day 21 (Fig. 8C). Furthermore, it was revealed that the expression levels of CDKN1A and TNNI3 were significantly higher on Day 40 than on Day 20, and there was no significant difference between Day 40 and Day 60 (Fig. 9).

Furthermore, using GEO: GSE1479, the correlation between the maturation-related gene of cardiomyocytes and the expression of CDKN1A in the mouse fetal heart was investigated. As a result, as shown in FIG. 10, a significant correlation was found between CDKN1A and the known maturation marker gene CD36 and the transcription factor HOPX1 associated with maturation during cardiac development in mice. Was done.

In addition, GEO: GSE51483 was used to extract genes (DEG; Differently Expressed Genes) whose expression was observed to fluctuate in the development of fetal myocardium, and pathway analysis of signals related to CDKN1A was performed in Fig. 11. show. R2: genomics analysis and visualization platform (http://r2.amc.nl/) was used for the analysis, and the high expression group and the low expression group were defined as having a difference of 1 SD (standard deviation) or more from the mean value. .. According to this result, a significant correlation was found in pathways including those involved in myocardial maturation, such as “cardiac muscle contraction and maturation”.

FIG. 12 shows the time course of maturation-related gene expression analyzed by dividing the atrium and ventricle of the mouse fetal heart using GEO: GSE51483. As a result, it was found that the expression of CDKN1A increased with time in both the atrial and ventricle, like other maturation-related genes.

From the above results, it was suggested that CDKN1A may contribute to the maturation of immature cardiomyocytes and may be involved in the maturation of both atria and ventricles and become a biomarker for maturation.

<Analysis of cardiomyocyte maturation promoting effect of CDKN1A>
According to Method 3, a viral vector expressing the CDKN1A gene was introduced into immature cardiomyocytes obtained by inducing differentiation of reporter iPS cells, and the effect of CDKN1A on cardiomyocyte maturation was investigated.
Four to five days after the introduction (Days 20 to 21), the mCherry fluorescence intensity was significantly higher in the cardiomyocytes into which the CDKN1A expression vector was introduced than in the cardiomyocytes into which the negative control vector was introduced (Fig. 13A left graph), and GFP. The fluorescence intensity was remarkably low (Fig. 13A right graph). In the cells into which the CDKN1A expression vector was introduced, many cardiomyocytes positive for both TNNI1 and TNNI3 were observed at Day 30, but many cardiomyocytes were TNNI1-negative and TNNI3-positive at Day 40 (Fig. 13B). Image panel). Furthermore, when the expression of genes involved in the maturation of cardiomyocytes was analyzed, the expression of the CDKN1A expression vector was significantly increased in the cardiomyocytes introduced with the CDKN1A expression vector as compared with the cardiomyocytes introduced with the negative control vector (Fig. 13B graph). ).
Therefore, it was shown that increasing the expression level of CDKN1A in immature cardiomyocytes remarkably promotes maturation as cardiomyocytes. As a result, 30 days after the start of differentiation induction (Day 30), the expression level of TNNI1 decreased and the expression level of TNNI3 increased remarkably, and 40 days later (Day 40), the expression level of TNNI1 was very low. , It was clarified that mature cardiomyocytes that predominantly express TNNI3 can be obtained.

Subsequently, the expression of a known gene set involved in cardiomyocyte maturation was analyzed. As a result, the expression of all the genes except HCN4 was increased in the cardiomyocytes into which the CDKN1A expression vector was introduced into the cardiomyocytes into which the negative control vector was introduced (FIG. 14A). Therefore, it was strongly suggested that when the expression level of CDKN1A increased in immature cardiomyocytes, the (original) gene expression involved in maturation was basically induced and the maturation was accelerated. It was also suggested that CDKN1A can directly or indirectly regulate the expression of known genes involved in the maturation of cardiomyocytes.
Furthermore, as a result of microscopic observation of cardiomyocytes into which the CDKN1A expression vector was introduced at Day 60, it was found that elongated and deformed TNNI1-negative TNNI3-positive mature cardiomyocytes were close to each other, similar to mature cardiomyocytes in vivo. It was observed (Fig. 14B).

Furthermore, according to Method 4, the maturation-inducing effect of CDKN1A on more immature early cardiomyocytes was evaluated. CDKN1A gene transfer was performed with lentivirus into immature cardiomyocytes on the 14th day after the start of differentiation induction, and gene expression was evaluated on the 30th day. As a result, a marked increase in TNNI3 expression was confirmed ( FIG. 15). From this result, it was confirmed that CDKN1A also has a mature cardiomyocyte-inducing effect in immature cardiomyocytes 14 days after the start of differentiation induction, which is a cell that has just differentiated into cardiomyocytes at an earlier stage.

From the above results, it was clarified that CDKN1A has the effect of promoting the expression of genes required for maturation and promoting maturation of immature cardiomyocytes.

Claims

A method for producing mature cardiomyocytes, which comprises a step of increasing the expression level of cyclin-dependent kinase inhibitor 1 in immature cardiomyocytes.
The method for producing mature cardiomyocytes according to claim 1, wherein the increase in the expression level is maintained for 3 days or more.
The method for producing mature cardiomyocytes according to claim 1 or 2, wherein the cardiomyocytes are human cardiomyocytes.
The method for producing a mature cardiomyocyte according to any one of claims 1 to 3, wherein the immature cardiomyocyte is an immature cardiomyocyte in which differentiation is induced from a pluripotent stem cell.
The method for producing mature cardiomyocytes according to claim 4, wherein the pluripotent stem cells are induced pluripotent stem cells.
Mature cardiomyocytes proliferated by the method according to any one of claims 1 to 5.