Methods for producing myocardium

Abstract

The present invention provides a method for producing atrial muscle cells from early cardiac muscle cells, comprising (a) a step of culturing a cell population containing early cardiac muscle cells in the presence of a Notch signal inhibitor. The invention also provides a cell population containing atrial muscle cells obtained by the method of the invention. The present invention provides the use of Notch signal inhibitors in the production of atrial muscle cells from early cardiac muscle cells.

Description

[Technical Field]

[0001] The present invention relates to a method for efficiently generating atrial cardiomyocytes from early cardiomyocytes, and a cell population containing atrial cardiomyocytes obtained by this method. [Background Art]

[0002] Techniques for inducing the differentiation of stem cells (such as induced pluripotent stem cells (iPSCs) and embryonic stem cells (ESCs)) into cardiomyocytes are expected to be applied to the regenerative medicine of heart diseases and drug discovery using experimental systems of heart disease models. Cardiomyocytes include various subtypes of cells, such as ventricular cardiomyocytes, atrial cardiomyocytes, and pacemaker cells, each having different functions. Currently, a cell population containing cardiomyocytes induced from stem cells contains a mixture of various subtypes of cardiomyocytes and may also contain non-cardiomyocytes such as undifferentiated cells. Therefore, technological development is underway with the aim of selectively obtaining specific subtypes of cells according to the application purpose.

[0003] Patent Document 1 discloses a method for obtaining a cell population rich in ventricular cardiomyocytes or atrial cardiomyocytes by using the expression level of CD151 as an index. However, such conventional techniques do not disclose a technique for selectively obtaining atrial cardiomyocytes from early cardiomyocytes.

[0004] [Citation List]

[0005] [Patent Document]

[0006] [Patent Document 1]

[0007] WO WO2021 / 033699 [Summary of the Invention]

[0008] [Technical Problem]

[0009] An object of the present invention is to provide a method for efficiently generating atrial cardiomyocytes from early cardiomyocytes. The present invention also aims to provide a cell population mainly containing atrial cardiomyocytes produced by the above method.

[0010] [Solution to the Problem]

[0011] The present inventors conducted research to achieve the above object and found that a cell population mainly containing atrial cardiomyocytes is obtained by culturing a cell population containing early cardiomyocytes in the presence of a Notch signal inhibitor, thereby completing the present invention.

[0012] To solve the above problems, the present invention provides the following [1] to

[10] .

[0013] [1]A method for generating atrial myocytes from early cardiomyocytes, which comprises the step of (a) culturing a cell population containing early cardiomyocytes in the presence of a Notch signaling inhibitor.

[0014] [2]The method according to [1], wherein the early cardiomyocytes are derived from pluripotent stem cells.

[0015] [2a]The method according to [2], wherein the pluripotent stem cells are induced pluripotent stem cells.

[0016] [3]The method according to any one of [1] to [2a], wherein the Notch signaling inhibitor is a γ-secretase inhibitor.

[0017] [4]The method according to [3], wherein the Notch signaling inhibitor is at least one compound selected from the group consisting of: LY411575, deshydroxy LY-411575, DAPT, dibenzoazepine , L-685,458, RO4929097, Compound E, Tarenflurbil, Avagacestat, Nirogacestat, Crenigacestat, MK0752, MRK003, BPN-15606, CB-103, RBPJ Inhibitor 1, SAHM1, IMR-1A and IMR-1 and their derivatives, and their salts, solvates and isomers having Notch signal transduction pathway inhibitory properties.

[0018] [5]The method according to any one of [1] to [4], which comprises the step of (b) enriching the atrial myocytes from the cell population by using the expression level of CD151 as an index.

[0019] [6]A cell population containing atrial myocytes, which is obtained by the method according to any one of [1] to [5].

[0020] [7]A method for generating atrial myocytes from stem cells, which comprises

[0021] (A) the step of inducing a cell population containing early cardiomyocytes from the above stem cells,

[0022] (B) the step of culturing the cell population containing early cardiomyocytes in the presence of a Notch signaling inhibitor, and

[0023] (C) the step of enriching the atrial myocytes from the cell population by using the expression level of CD151 as an index.

[0024] [8]A reagent for generating atrial myocytes from early cardiomyocytes, comprising a Notch signaling inhibitor.

[0025] [9]The reagent according to [8], comprising a probe for detecting CD151.

[0026]

[10] Use of a Notch signaling inhibitor in generating atrial myocytes from early cardiomyocytes.

[0027] [Advantageous effects of the invention]

[0028] According to the present invention, atrial myocytes can be efficiently generated from early cardiomyocytes by including the step of culturing a cell population containing early cardiomyocytes in the presence of a Notch signaling inhibitor. This makes it possible to obtain a cell population mainly containing atrial myocytes. [Brief description of the drawings]

[0029] Figure 1 Two-dimensional flow cytometer plot of the analysis results of CD151 expression in EmGFP-positive cells, wherein the EmGFP-positive cells are obtained by staining a cell population containing atrial myocytes with an anti-CD151 antibody and a corresponding Alexa (registered trademark) 647-labeled secondary antibody. The cell population containing atrial myocytes is obtained by culturing a human iPS cell line knocked-in with EmGFP at the TNNI1 locus in cardiomyocyte induction medium 3 containing a Notch signaling inhibitor (LY411575) or DMSO from day 8 to day 20 of differentiation. On the right is the cell population cultured in cardiomyocyte induction medium 3 containing a Notch signaling inhibitor (LY411575), and in the middle is the cell population cultured in cardiomyocyte induction medium 3 containing DMSO as a control. On the left is a two-dimensional flow cytometer plot of the analysis results of CD151 expression in EmGFP-positive cells as a negative control, wherein the EmGFP-positive cells are obtained by staining a cell population cultured in cardiomyocyte induction medium 3 containing DMSO with an IgG1 isotype antibody instead of an anti-CD151 antibody and an Alexa (registered trademark) 647-labeled secondary antibody against the antibody.

[0030] Figure 2-1 Bar graph showing the results of the expression analysis of the target gene (HEY2) of Notch signaling transduction using total RNA extracted from each cell subset of "DMSO / CD151-high", "DMSO / CD151-low", "Notch inhibitor / CD151-high", and "Notch inhibitor / CD151-low" obtained from Experimental Example 2. Relative values (mean ± standard error) are shown, with the expression level of HEY2 in Notch inhibitor / CD151-high being 1.​​

[0031] Figure 2-2 A bar graph showing the results of expression analysis of atrial myocyte marker genes (NR2F1, NR2F2, NPPA, KCNA5, KCNJ3, TBX5, and MYL7) using total RNA extracted from each cell subset of "DMSO / CD151-high", "DMSO / CD151-low", "Notch inhibitor / CD151-high", and "Notch inhibitor / CD151-low" obtained from Experimental Example 2. The relative values (mean ± standard error) are shown, with the expression level of each gene in cells treated with DMSO set to 1.

[0032] Figure 3 Bar graphs of the proportions of atrial myocytes (ACM) and other cells in the "Notch inhibitor / CD151-high" and "Notch inhibitor / CD151-low" cardiomyocyte subsets obtained in Experimental Example 2, and bar graphs of the proportions of atrial myocytes (ACM) and other cells in the "untreated / CD151-high" and "untreated / CD151-low" cardiomyocyte subsets obtained under conditions without a Notch inhibitor. The graph of "CD151 高 ACM" shows the results of "Notch inhibitor / CD151-high", where "NT" means the condition without a Notch inhibitor, and "LY411575" means the condition containing the Notch inhibitor LY411575. The graph of "CD151 低 ACM" shows the results of "Notch inhibitor / CD151-low", where "NT" means the condition without a Notch inhibitor, and "LY411575" means the condition containing the Notch inhibitor LY411575. In each graph, the proportion of atrial myocytes (ACM) is shown in gray. [Detailed Description]

[0033] The present invention provides a method for generating atrial myocytes from early cardiomyocytes.

[0034] ​​In this specification, a "stem cell" refers to a cell that has both the potential for differentiation and proliferation (particularly the potential for self-renewal) while maintaining the potential for differentiation. According to the differentiation ability, stem cells include subpopulations such as pluripotent stem cells, multipotent stem cells, and unipotent stem cells. A pluripotent stem cell refers to a stem cell that has the ability (pluripotency) to differentiate into all the cells that make up an organism (tissues derived from the three germ layers (ectoderm, mesoderm, and endoderm)). A multipotent stem cell refers to a stem cell that has the ability to differentiate into multiple (although not all) types of tissues and cells. A unipotent stem cell refers to a stem cell that has the ability to differentiate into a specific tissue and cell. The "pluripotent stem cells" that can be used in the present invention are not particularly limited and include, for example, embryonic stem cells (ESCs), embryonic stem cells derived from cloned embryos obtained by nuclear transfer, spermatogonial stem cells, embryonic germ cells, and induced pluripotent stem cells (sometimes referred to as "iPSCs" in this specification). In addition, the "pluripotent stem cells" that can be used in the present invention refer to stem cells that have the ability to differentiate into a limited number of cell lineages. Preferred pluripotent stem cells are ESCs and iPSCs.

[0035] Regarding "embryonic stem cells (ESCs)", various mouse ESC lines established by inGenious targeting laboratory, RIKEN, etc. can be used for mouse ESCs, and various human ESC lines established by the University of Wisconsin, NIH, RIKEN, Kyoto University, National Center for Child Health and Development, Cellartis, etc. can be used for human ESCs. For example, as human ESC lines, CHB-1 to CHB-12, RUES1, RUES2, and HUES1 to HUES28 lines distributed by ESI Bio, H1 and H9 lines distributed by WiCell Research, and KhES-1, KhES-2, KhES-3, KhES-4, KhES-5, SSES1, SSES2, and SSES3 lines distributed by RIKEN can be utilized.

[0036] "Induced pluripotent stem cells" refer to cells obtained by reprogramming mammalian somatic cells or undifferentiated stem cells by introducing specified factors (nuclear reprogramming factors). Currently, there are various types of "induced pluripotent stem cells", and it is also possible to use iPSCs established by Yamanaka et al. by introducing four factors, Oct3 / 4, Sox2, Klf4, and c-Myc, into mouse fibroblasts (Takahashi K, Yamanaka S., Cell, (2006) 126:663-676), and human cell-derived iPSCs established by introducing the same four factors into human fibroblasts (Takahashi K, Yamanaka S. et al. Cell, (2007) 131:861-872.), Nanog-iPSCs established by using Nanog expression as an indicator for selection after introducing the above four factors (Okita, K., Ichisaka, T., and Yamanaka, S. (2007). Nature 448, 313-317.), iPSCs generated by a method that does not include c-Myc (Nakagawa M, Yamanaka S. et al. Nature Biotechnology, (2008) 26, 101-106), iPSCs established by introducing six factors using a virus-free method (Okita K et al. Nat. Methods May 2011; 8(5):409-12, Okita et al. Stem Cells. 31(3):458-66.), etc. In addition, it is also possible to use induced pluripotent stem cells established by introducing four factors, OCT3 / 4, SOX2, NANOG, and LIN28, created by Thomson et al. (Yu J., Thomson JA. et al., Science (2007) 318:1917-1920.), induced pluripotent stem cells generated by Daley et al. (Park IH, Daley GQ. et al., Nature (2007) 451:141-146), and induced pluripotent stem cells generated by Sakurada et al. (JP-A-2008-307007).

[0037] In addition, any artificial pluripotent stem cells known in the art and described in all published papers (e.g., Shi Y., Ding S. et al., Cell Stem Cell, (2008) Vol. 3, No. 5, pp. 568-574; Kim JB., Scholer HR. et al., Nature, (2008) 454, 646-650; Huangfu D., Melton, DA. et al., Nature Biotechnology, (2008) 26, No. 7, pp. 795-797) or patents (e.g., JP-A-2008-307007, JP-A-2008-283972, US2008-2336610, US2009-047263, WO2007-069666, WO2008-118220, WO2008-124133, WO2008-151058, WO2009-006930, WO2009-006997, WO2009-007852) can be used. As artificial pluripotent stem cell lines, various iPSC cell lines established by NIH, RIKEN, Kyoto University, etc. can be used. For example, human iPSC cell lines include the HiPS-RIKEN-1A cell line, HiPS-RIKEN-2A cell line, HiPS-RIKEN-12A cell line, Nips-B2 cell line, etc. of RIKEN, and the 253G1 cell line, 201B7 cell line, 409B2 cell line, 454E2 cell line, 606A1 cell line, 610B1 cell line, 648A1 cell line, 1231A3 cell line, 1390D4 cell line, 1390C1 cell line, etc. of Kyoto University, and the 1390D4 cell line and 1390C1 cell line are more preferred. Alternatively, clinical-grade cell lines provided by Kyoto University, CellularDynamics International, etc. can also be used, as well as research and clinical cell lines prepared using these cell lines.

[0038] "Cardiomyocyte" refers to the cells that constitute the myocardium and are the smallest units with contractile ability, and includes atrial cardiomyocytes, ventricular cardiomyocytes, etc.

[0039] Identifying cells as atrial myocytes or ventricular myocytes can be carried out by conventionally known techniques such as electrophysiological analysis, marker expression analysis, and drug response analysis described below.

[0040] For example, any one of the following (1) to (7) performed using the patch clamp method can be used for identification by electrophysiological analysis.

[0041] (1) Cells with an action potential waveform where the ratio of the action potential duration at 30% repolarization (APD30) to the action potential duration at 90% repolarization (APD90) (APD30 / 90) is 0.3 or greater and the maximum rate of rise of the waveform (dv / dtmax) is 10 or greater are defined as ventricular myocytes. On the other hand, cells with an action potential waveform showing APD30 / 90 less than 0.3 and dv / dtmax of 10 or greater are defined as atrial myocytes. In this case, cells with an action potential waveform showing dv / dtmax less than 10 and a spontaneous action potential duration of 1 s or longer are defined as immature cardiomyocytes that are not classified as ventricular myocytes or atrial myocytes (Cell Stem Cell, 2017, 21, 179 - 194).

[0042] (2) Cells with an action potential waveform showing a significantly longer value of the action potential duration at 50% repolarization (APD50) or APD90 are defined as ventricular myocytes, and cells with an action potential waveform showing a significantly shorter value are defined as atrial myocytes (JCI Insight, 2018; 3(12):e99941, Eur. Heart J. 2017, 38, 292 - 301).

[0043] (3) Cells with an action potential waveform showing a significantly longer value of the action potential duration at 20% repolarization (APD20), APD50, or APD90 and a significantly larger amplitude of the action potential plateau (APAPlat) are defined as ventricular myocytes, and cells with an action potential waveform showing a significantly shorter value of APD20, APD50, or APD90 and a significantly smaller APAPlat are defined as atrial myocytes (Stem Cell Reports. December 12, 2017; 9(6):1765 - 1779).

[0044] (4) Cells with an action potential waveform showing a significantly large ratio of APD20 to the action potential duration at 80% repolarization (APD80) (APD20 / 80) are defined as ventricular myocytes, and cells with an action potential waveform showing a significantly small ratio are defined as atrial myocytes (JCI Insight, 2018; 3(12):e99941).

[0045] (5) Cells with an action potential waveform showing a significantly small ratio of APD90 to APD50 (APD90 / 50) are defined as ventricular myocytes, and cells with an action potential waveform showing a significantly large ratio are defined as atrial myocytes (Eur. Heart J., 2017, 38, 292 - 301, Eur. Heart J., 2011, 32, 952 - 962).

[0046] (6) Cells showing an action potential waveform with APD90 / 50 less than 1.4 were defined as ventricular myocytes, and cells showing an action potential waveform with APD90 / 50 greater than 1.7 were defined as atrial myocytes. In this case, cells showing an action potential waveform with APD90 / 50 of 1.4 or greater and 1.7 or less were defined as pacemaker cells (Eur. Heart J., 2011, 32, 952 - 962).

[0047] (7) Cells showing an action potential waveform with a plateau phase of 50 ms or longer, a membrane potential change of 20 mV or less, dv / dtmax greater than 50, an action potential amplitude (APA) greater than 85 mV, and APD90 / 50 less than 2.3 were defined as ventricular myocytes. Cells showing an action potential waveform that differed from the above - mentioned action potential waveform only in that it lacked a plateau phase were defined as atrial myocytes, and cells showing an action potential waveform that differed in that it lacked a plateau phase and had an APD90 / 50 greater than 2.3 were defined as pacemaker cells (PNAS, 2017, E8372 - E8381).

[0048] Identification by marker expression analysis involves measuring the expression of a marker protein or a marker gene, and when a cell strongly expresses an atrial cardiomyocyte marker and preferably does not express or weakly expresses a ventricular cardiomyocyte marker, it can be determined that the cell is an atrial cardiomyocyte. When the expression of the marker gene can be quantitatively measured, a relative expression value corrected by the expression level of a housekeeping gene can be calculated, and it can be determined that a cell with a high relative expression level of the atrial cardiomyocyte marker and a low relative expression level of the ventricular cardiomyocyte marker is an atrial cardiomyocyte. Conversely, when a cell expresses a ventricular cardiomyocyte marker and preferably does not express an atrial cardiomyocyte marker, it can be determined that the cell is a ventricular cardiomyocyte. Alternatively, when the expression of the marker gene can be quantitatively measured, a relative expression value corrected by the expression level of a housekeeping gene can be calculated, and it can be determined that a cell with a high relative expression level of the ventricular cardiomyocyte marker and a low relative expression level of the atrial cardiomyocyte marker is a ventricular cardiomyocyte. Known marker genes for atrial cardiomyocytes include KCNA5 (potassium voltage-gated channel subfamily A member 5), KCNJ3 (potassium voltage-gated channel subfamily J member 3), NPPA (natriuretic peptide A), NR2F1 (nuclear receptor subfamily 2 group F member 1), NR2F2 (nuclear receptor subfamily 2 group F member 2), TBX5 (T-Box 5), and MYL7 (myosin light chain 7), and known marker genes for ventricular cardiomyocytes include HEY2 (Hes-related family BHLH transcription factor with YRPW motif 2), MYL2 (myosin light chain 2), and IRX4 (Iroquois family homeobox 4). As housekeeping genes, GAPDH, β-actin, etc. are known. To ensure higher accuracy, a housekeeping gene that does not fluctuate much during the differentiation of early cardiomyocytes into atrial cardiomyocytes and / or ventricular cardiomyocytes is selected, and the expression levels of the marker genes of ventricular cardiomyocytes and atrial cardiomyocytes are corrected with the expression level of the housekeeping gene. For example, software such as geNorm described in Joey St-Pierre et al. (Scientific Reports 7:16923 (2017)) can be used to select the housekeeping gene for correction.

[0049] In addition, cardiomyocytes including atrial cardiomyocytes and ventricular cardiomyocytes refer to cells that express at least one cardiomyocyte marker (at least one marker selected from the group consisting of cardiac troponin (cTNT), αMHC (α-myosin heavy chain, MYH6), βMHC (MYH7), and NKX2.5 (NK-2 transcription factor-related locus 5)). The cTNT gene is exemplified by NCBI accession number NM_000364 for humans and NM_001130176 for mice. The αMHC gene is exemplified by NCBI accession number NM_002471 for humans and NM_001164171 for mice. The βMHC gene is exemplified by NCBI accession number NM_000257 for humans and NM_080728 for mice.

[0050] The labeled protein can be detected by immunoassays (such as ELISA, immunostaining, flow cytometry, etc.) using antibodies specific to the labeled protein. The labeled gene can be detected by nucleic acid amplification methods and / or nucleic acid detection methods known in the art (such as RT-PCR, microarray, biochip, etc.).

[0051] Identification by drug response analysis includes detecting the response of cells to drugs that activate or inhibit channels specifically expressed in atrial or ventricular myocytes. For example, carbachol is a drug that activates the muscarinic K channel (IK,Ach) specifically expressed in atrial myocytes. When carbachol is added to the cells, it only shortens the action potential duration (APD) of atrial myocytes, but does not affect the APD of ventricular myocytes. In addition, 4-aminopyridine is a drug that inhibits the ultrarapidly activating delayed rectifier K channel (IKur). When added to the cells, it only prolongs APD20 in atrial myocytes, but does not affect the APD of ventricular myocytes (Stem Cell Reports, 2018; 11(6): 1378-1390). Atrial or ventricular myocytes can also be identified by detecting the reactivity of the cells to such drugs.

[0052] The method for generating atrial myocytes from early cardiomyocytes of the present invention includes (a) a step of culturing a cell population containing early cardiomyocytes in the presence of a Notch signal inhibitor.

[0053] In this specification, an "early cardiomyocyte" is a cell in a stage of being directed towards cardiomyocytes during the process of differentiating from non-cardiomyocytes into cardiomyocytes, and refers to, for example, a cell in a stage where any one or more of cardiomyocyte-specific genes (cTNT, NKX2.5, MYH6, MYH7, MYL7, etc.), the above-mentioned atrial muscle marker genes (except KCNJ3), and the above-mentioned ventricular muscle marker genes (except MYL2) start to be expressed. Early cardiomyocytes can be derived from stem cells, such as embryonic stem cells, adult stem cells, and iPS cells, or can be early cardiomyocytes directly isolated from an organism. Early cardiomyocytes are preferably derived from pluripotent stem cells, and more preferably from iPS cells. For example, when generating atrial cardiomyocytes from iPS cells under atrial cardiomyocyte differentiation conditions known in the art (see, for example, EMBO Mol Med (2015) 7:394 - 410 and Patent Document 1), embryoid bodies generated from iPSCs are cultured in a basal medium containing BMP4, activin A, and bFGF for two days, and then cultured in a basal medium containing VEGF, a Wnt inhibitor, a TGF-β inhibitor, and retinoic acid for three days. In addition, the differentiation of atrial cardiomyocytes is directed by culturing in a basal medium containing VEGF. In this method, specifically, the expression of Notch 4 increases in early cardiomyocytes at about day 8, and the start of iPS cell culture is day 0. In the method for generating atrial cardiomyocytes from early cardiomyocytes of the present invention, a cell population containing early cardiomyocytes is cultured in the presence of a Notch signal inhibitor.

[0054] In this specification, a "cell population" means a collection of two or more cells. A "cell subset" means a collection of cells that constitute a cell population and have at least one common characteristic.

[0055] "Culturing" means maintaining cells, causing cells to proliferate (grow), and / or differentiate in an in vitro environment. "Culturing" means maintaining cells, causing cells to proliferate (grow), and / or differentiate outside of tissues or in vitro, for example, in a cell culture dish, plate, flask, or culture tank.

[0056] "Sustain" means culturing a desired cell population while maintaining its quantity. The maintenance of the cell quantity can be achieved by the survival of cells without proliferation, or by balancing the increase in quantity due to proliferation and the decrease in quantity due to cell death. The maintenance of the cell quantity does not need to be achieved by maintaining exactly the same cell quantity, but for the purposes of the present invention, it is sufficient that the cell quantity is substantially the same.

[0057] For culturing a cell population, the basal medium is not particularly limited, but for example, StemPro-34 SFM (Thermo Fisher Scientific), STEMdiff APEL2 medium (STEMCELL Technologies, ST-05275), TeSR1 medium, and chemically defined medium (CDM) are preferably used. In addition, BME medium, BGJb medium, CMRL 1066 medium, Glasgow MEM medium, modified MEM (IMEM) medium, modified IMDM (IMDM) medium, Medium 199 medium, Eagle's MEM medium, αMEM medium, DMEM medium (high glucose, low glucose), DMEM / F12 medium, Ham's medium, RPMI 1640 medium, Fischer's medium, and their mixed media can also be used. The CDM medium is not particularly limited, but for example, a medium prepared from Iscove's modified Dulbecco's medium (GE Healthcare) can be used. The basal medium can be supplemented with substances commonly used for cell culture, such as Ham's F-12 nutrient mixture, albumin such as human serum albumin, polyvinyl alcohol (PVA), deionized BSA, linoleic acid, linolenic acid, cholesterol, insulin, transferrin apoferritin, selenium, ethanolamine, monothioglycerol, protein-free hybridoma mix II (PFHMII), ascorbic acid, L-alanyl-L-glutamine, and / or antibiotics.

[0058] In the present specification, a Notch signal inhibitor refers to a drug that inhibits the signal transduction pathway activated by the direct interaction between a Notch protein (which is a receptor expressed on the cell membrane) and a Notch ligand (such as Delta or Jagged) expressed on the membrane of an adjacent cell.

[0059] The Notch signal inhibitor is not particularly limited as long as it can inhibit Notch-mediated signal transduction. It can be any one of nucleic acids, proteins, and low molecular weight organic compounds. Examples of such substances include a functionally defective Notch receptor and ligand, a substance that inhibits Notch processing (S1 cleavage), a substance that inhibits the glycosylation of Notch and Notch ligand, a substance that inhibits cell membrane migration, a substance that inhibits the processing (S2 cleavage, S3 cleavage) of the intracellular domain (NICD) of Notch (γ-secretase inhibitor), a substance that degrades NICD, and a substance that inhibits NICD-dependent transcription.

[0060] Among Notch signal inhibitors, many γ-secretase inhibitors are known. This is because γ-secretase, which is involved in the processing (S2 cleavage, S3 cleavage) of the intracellular domain of Notch (NICD), is also involved in the production of amyloid-β protein, which is involved in Alzheimer's disease. Examples of γ-secretase inhibitors that can be used in the present invention include LY411575, dehydroxy LY-411575, DAPT, dibenzoazepine , L-685,458, RO4929097, Compound E, telipofib, agastat, nilogastat, krenigastat, MK0752, CB-103, MRK003, BPN-15606, etc., but are not limited to these, as long as the activity of Notch signal inhibitors is present.

[0061] Among them, LY411575 (CAS No. 209984-57-6) is one of the most effective γ-secretase inhibitors and the most effective Notch signal inhibitors. Dehydroxy LY-411575 (CAS No. 209984-56-5) is a derivative of LY411575 and is also known by other names, such as γ-secretase inhibitor (GSI)-XX, etc. DAPT (CAS No. 208255-80-5) is the abbreviation of N-[2S-(3,5-difluorophenyl)acetyl]-L-alanyl-2-phenyl-1,1-dimethylethyl-glycine and is also known by other names, such as γ-secretase inhibitor (GSI)-IX, LY-374973, etc. Dibenzoazepine (CAS No. 209984-56-5) is also known by other names, such as YO-01027, etc. The chemical name of L-685,458 (CAS No. 292632-98-5) is (5S)-(tert-butoxycarbonylamino)-6-phenyl-(4R)hydroxy-(2R)benzylhexanoyl)-L-leu-L-phe-amide. RO4929097 (CAS No. 847925-91-1) is also known by other names, such as KK8645V7LE, RG-4733, etc. Compound E (CAS No. 209986-17-4) is also known by other names, such as γ-secretase inhibitor (GSI)-XXI, etc. Teliflurbine (CAS No. 51543-40-9) is also known by other names, such as (R)-flurbiprofen, etc. Agastat (CAS No. 1146699-66-2) is also known by other names, such as BMS-708163, etc. Nilotastat (CAS No. 1290543-63-3) is also known by other names, such as PF-3084014 hydrobromide, etc. Krenitastat (CAS No. 1421438-81-4) is also known by other names, such as LY3039478, etc. MK0752 (CAS No. 471905-41-6) is also known by other names, such as UNII-9JD9B4S53T, etc. The chemical name of MRK003 (CAS No. 623165-93-5) is (4R,10'S)-2-(2,2,2-trifluoroethyl)-5'-[(E)-3-[4-(trifluoromethyl)piperidin-1-yl]prop-1-enyl]spiro[1,2,5-thiadiazolidine-4,13'-tricyclo[8.2.1.03,8]tridec-3(8),4,6-triene]1,1-dioxide. BPN-15606 (CAS No. 1914989-49-3) and is also known by other names, such as HY-117482, etc.

[0062] Among Notch signal inhibitors, CB-103 (CAS No. 218457-67-1) is not a γ-secretase inhibitor, but a substance that inhibits NICD-dependent transcription by preventing the migration of the Notch intracellular domain (NICD) into the nucleus to form a transcriptional activation complex with γ-secretase.

[0063] RBPJ inhibitor 1 (CAS No. 2682114-39-0) inhibits Notch-mediated signal transduction by blocking the functional interaction between RBPJ (the main effector of Notch signal transduction) and the scaffold protein SHARP.

[0064] SAHM1 (CAS No. 2050906-89-1), IMR-1A (CAS No. 331862-41-0), and IMR-1 (CAS No. 310456-65-6) are all dominant-negative mutant peptide mimics of mastermind-like (MAML) proteins and inhibit Notch-mediated signal transduction.

[0065] In the present invention, the concentration of the Notch signal inhibitor added to the medium of the cell population containing early cardiomyocytes is generally 0.1 μM or higher, preferably 1 μM or higher. There is no particular upper limit for the concentration of the Notch signal inhibitor as long as it does not adversely affect the differentiation into atrial myocytes. From the aspect of culture cost, it is generally 1000 μM or lower, preferably 100 μM or lower. In one embodiment, the concentration of the Notch signal inhibitor in the medium is generally from 0.1 μM to 1000 μM, preferably from 1 μM to 100 μM (for example, 10 μM).

[0066] In the present invention, under the conventionally known atrial myocyte differentiation conditions (see, for example, EMBO Mol Med (2015) 7:394-410), the time to start adding the Notch signal inhibitor to the medium of the cell population containing early cardiomyocytes is when the expression of Notch 4 in the early cardiomyocytes increases, for example, around day 8, with the start of iPS cell culture being day 0, but different times may be appropriate depending on the conditions.

[0067] The period for adding the Notch signal inhibitor to the medium of the cell population containing early cardiomyocytes is not particularly limited. The period is, for example, from 7 days to 40 days, and can be, for example, 120 days, 90 days, 60 days, 30 days, 28 days, 21 days, 14 days, or 7 days depending on the purpose.

[0068] In the present invention, when inducing a cell population containing early cardiomyocytes from stem cells, the method is described, for example, in Patent Document 1. Here, the atrial myocyte differentiation conditions include, for example, culturing embryoid bodies generated from iPSCs in a basal medium containing BMP4, activin A, and bFGF for two days, and then culturing in a basal medium containing VEGF, a Wnt inhibitor, a TGF-β inhibitor, and retinoic acid for three days. By further culturing in a basal medium containing VEGF, a cell population containing atrial myocytes can be obtained.

[0069] A Wnt inhibitor (Wnt signaling inhibitor) is a substance that inhibits the signal transduction pathway mediated by Wnt, and for example, IWP-2, IWP-3, IWP-4, 2-(4-trifluoromethylphenyl)-7,8-dihydro-5H-thiopyrano[4,3-d]pyrimidin-4(3H)-one, IWR1, G-CSF, IGFBP4, Dkk1, Cerberus, anti-Wnt antibody, Wnt agonist (Wnt receptor inhibitor), soluble Wnt receptor protein (such as Frzb-1), dominant negative form, etc. can be used. Two or more of these can be used in combination.

[0070] Examples of TGF-β inhibitors include SB431542 (4-[4-(1,3-benzodioxol-5-yl)-5-(2-pyridyl)-1H-imidazol-2-yl]-benzamide), A83-01 (4-[4-(1,3-benzodioxol-5-yl)-5-(2-pyridyl)-1H-imidazol-2-yl]-benzamide), LDN193189 (4-[6-[4-(1-piperazinyl)phenyl]pyrazolo[1,5-a]pyrimidin-3-yl]-quinoline), GW788388 (4-[4-[3-(2-pyridyl)-1H-pyrazol-4-yl]-2-pyridyl]-N-(tetrahydro-2H-pyran-4-yl)-benzamide), SM16 (4-[4-(1,3-benzodioxol-5-yl)-5-(6-methyl-2-pyridyl)-1H-imidazol-2-yl]-bicyclo[2.2.2]octane-1-carboxamide), IN-1130 (3-[[5-(6-methyl-2-pyridyl)-4-(6-quinoxalinyl)-1H-imidazol-2-yl]methyl]-benzamide), GW6604 (2-phenyl-4-[3-(pyridin-2-yl)-1H-pyrazol-4-yl]pyridine), SB505124 (2-[4-(1,3-benzodioxol-5-yl)-2-(1,1-dimethylethyl)-1H-imidazol-5-yl]-6-methyl-pyridine), etc. Two or more of these can be used in combination.

[0071] In addition to or after the above steps (a) and (b), the method for generating atrial myocytes from early cardiomyocytes of the present invention may include a step of enriching atrial myocytes from the above cell population using the expression level of CD151 as an index.

[0072] "Enrich" and "enrichment" refer to increasing the amount of a specific constituent component in a composition such as a cell composition. "Enriched", when used to describe a composition of cells, such as a cell population, means that the amount of a specific constituent component is increased compared to the proportion of such component in the cell population before enrichment. For example, a composition such as a cell population can be enriched for a target cell type such that the proportion of the target cell type is increased compared to the proportion of target cells present in the cell population before enrichment. A cell population can also be enriched for a target cell type by cell selection and sorting methods known in the art. A cell population can also be enriched by certain selection or sorting methods described in this specification. In a specific embodiment of the present invention, relative to the cell population before enrichment, the enriched cell population is enriched by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 97%, 98% or 99% relative to the target cell population. In a specific embodiment of the present invention, the enriched cell population contains at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% of the target cell population. In this specification, a "cell population mainly containing atrial myocytes" can refer to a cell population containing at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% atrial myocytes.

[0073] In a cell population derived from stem cells and comprising cardiomyocytes, the cell subset identified by high CD151 expression contains a higher proportion of ventricular myocytes than the cell subset identified by low CD151 expression. Thus, atrial myocytes can be enriched in the cell population by removing the cell subset identified by high CD151 expression from a cell population derived from stem cells and comprising cardiomyocytes, or by recovering the cell subset identified by low CD151 expression from a cell population comprising cardiomyocytes.

[0074] "CD151" is a tetraspanin belonging to the tetraspanin family. CD151 plays an important role in signal transduction related to cell differentiation, proliferation and motility. It is also known that CD151 forms a complex with integrins and is involved in functions such as cell adhesion and fusion. In the present invention, CD151 is used as a marker for enriching atrial myocytes or ventricular myocytes in a cell population derived from stem cells and comprising cardiomyocytes.

[0075] "Marker" is "marker protein" or "marker gene" and refers to a protein or its gene that is specifically expressed on the cell surface of a given cell type, in the cytoplasm and / or nucleus of a given cell type. The marker can be a positive selection marker or a negative selection marker. Preferably, the marker is a cell surface marker, and the cell surface selection marker can enrich, isolate and / or detect live cells.

[0076] Marker proteins can be detected by immunoassays using antibodies specific for the marker protein (such as ELISA, immunostaining, and flow cytometry). As an antibody specific for the marker protein, an antibody that binds to a specific amino acid sequence in the marker protein or to a specific sugar chain that binds to the marker protein can be used. In addition, in the case of a marker protein that is expressed intracellularly and not present on the cell surface (such as a transcription factor or its subunit), a reporter protein is co-expressed with the marker protein, and the reporter protein is detected to detect the marker protein of interest. This method can be preferably used when no suitable cell surface marker is found. Marker genes can be detected using nucleic acid amplification methods and / or nucleic acid detection methods known in the art (such as RT-PCR, microarray, biochip, and RNA-seq).

[0077] "Expression" is defined as the transcription of a specific nucleotide sequence and / or the translation of the transcript driven by a promoter.

[0078] "Positive" or "expressed" means that the protein or mRNA is expressed in an amount detectable by techniques known in the art. Proteins can be detected by immunoassays using antibodies (such as ELISA, immunostaining, and flow cytometry). In addition, in the case of a protein that is expressed intracellularly and not present on the cell surface (such as a transcription factor or its subunit), a reporter protein is co-expressed with the protein, and the reporter protein is detected to detect the protein of interest. mRNA can be detected using nucleic acid amplification methods and / or nucleic acid detection methods (such as RT-PCR, microarray, biochip, and RNA-seq).

[0079] "Negative" or "not expressed" means that the expression level of the protein or gene is below the detection limit of all or any of the above-known techniques. The detection limit for protein or gene expression may vary for each technique.

[0080] The expression level of CD151 in cells can be determined by setting a certain reference value based on the distribution of the expression levels in the cell population, and determining that cells showing an expression level equal to or greater than the reference value are highly expressed, while cells showing an expression level lower than the reference value are lowly expressed. In this case, the reference value can be, for example, the maximum value, average value, median value, or mode value of the expression levels per cell, preferably the maximum value. The reference value can be appropriately set according to the desired cell enrichment rate, and can be set to, for example, a value 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more greater or smaller than the maximum value, average value, median value, or mode value (preferably the maximum value) of the expression levels per cell.

[0081] Alternatively, the expression level of CD151 in cells can also be determined by setting the expression level in stem cells as the reference value, and determining that cells showing an expression level equal to or greater than the reference value are highly expressed, while cells showing an expression level lower than the reference value are lowly expressed. In this case, the reference value can be, for example, the maximum value, average value, median value, or mode value of the expression levels in stem cells, preferably the maximum value. The reference value can be appropriately set according to the desired cell enrichment rate, and can be set to, for example, a value 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more greater or smaller than the maximum value, average value, median value, or mode value (preferably the maximum value) of the expression levels in stem cells.

[0082] Alternatively, the expression level of CD151 in cells can also be determined by setting the signal intensity detected in cells not in contact with the probe (negative control) as the reference value in the same manner as in the cells in contact with the probe, and determining that cells showing a signal intensity equal to or greater than the reference value are highly expressed, while cells showing a signal intensity less than the reference value are lowly expressed. The reference value can be appropriately set according to the desired cell enrichment rate, and can be set to, for example, a value 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more greater or smaller than the maximum value, average value, median value, or mode value (preferably the maximum value) of the expression level of the negative control.

[0083] Recovery of the target cell subset or removal of the non-target cell subset can be carried out under conventionally known conditions, and for example, cell sorting using a flow cytometer can be appropriately applied. For example, first, the cells are reacted with an anti-CD151 primary antibody. The cells are washed to remove the anti-CD151 primary antibody that has not bound to the cells, and then the cells are reacted with a fluorescently labeled secondary antibody. After further washing the cells to remove the fluorescently labeled secondary antibody that has not bound to the primary antibody, the fluorescence intensity of the cells is measured using a flow cytometer, and the cells showing an expression level equal to or higher than the above reference value are sorted into the CD151-high expression cell subset (CD151-high), and the cells showing an expression level lower than the reference value are sorted into the CD151-low expression cell subset (CD151-low).

[0084] The present invention provides a cell population containing atrial myocytes obtained by the method of the present invention. As used herein, "atrial myocytes" and "cell population" are as described above.

[0085] The present invention provides a method for generating atrial myocytes from stem cells, which comprises

[0086] (A) a step of inducing a cell population containing early cardiomyocytes from the above-mentioned stem cells,

[0087] (B) a step of culturing the above-mentioned cell population containing early cardiomyocytes in the presence of a Notch signal inhibitor, and

[0088] (C) a step of enriching the atrial myocytes from the above-mentioned cell population using the expression level of CD151 as an index. Each step of the method of the present invention is as described above.

[0089] The present invention provides a reagent for generating atrial myocytes from stem cells. The reagent of the present invention comprises a Notch signal inhibitor. "Notch signal inhibitor" is as described above.

[0090] The reagent of the present invention may comprise a probe for detecting CD151.

[0091] The expression level of CD151 in cells can be determined using a CD151 detection probe (hereinafter also simply referred to as "probe") having a binding property to CD151. The probe can be, for example, an antibody or a nucleic acid (such as an aptamer), and is preferably an antibody. The probe is preferably labeled so that it can be detected optically, electrically, or magnetically. In this case, the cells are contacted with the probe, and the labeled signal from the probe bound to CD151 is detected optically, electrically, or magnetically, and the expression level of CD151 in the cells can be determined based on the signal intensity. In addition, for example, when the probe is an antibody, the expression level of CD151 in cells can also be determined using a labeled secondary antibody that binds to the antibody.

[0092] In addition, for example, when the probe is an antibody, the expression level of CD151 in cells can also be determined using a labeled secondary antibody that binds to the antibody.

[0093] The present invention provides the use of a Notch signal inhibitor for generating atrial myocytes from early cardiomyocytes. The Notch signal inhibitor herein can be provided in a state of being added to a culture solution of a cell population containing early cardiomyocytes. From the perspective of storage stability, it can be provided in a dry form or in the form of a high-concentration stock solution, and can be prepared at the time of use.

[0094] Atrial myocytes and a cell population containing atrial myocytes produced by the present invention can be applied to a cell drug containing atrial myocytes for treating heart diseases, and a method for treating heart diseases by administering the cell drug. The present invention also provides the use of the above cell population and atrial myocytes for producing the above cell drug, and the use of the above cell population and atrial myocytes for treating heart diseases.

[0095] A cell drug containing atrial myocytes and a cell population containing atrial myocytes produced by the present invention can be used in regenerative medicine for atrial myocyte-specific diseases such as atrial fibrillation and heart diseases such as myocardial infarction, heart failure, ischemic heart disease, cardiomyopathy, myocarditis, hypertrophic cardiomyopathy, dilated hypertrophic cardiomyopathy, and dilated cardiomyopathy.

[0096] A cell drug containing atrial myocytes and a cell population containing atrial myocytes produced by the present invention is produced as any form of parenteral preparation, such as a suspension in which the cells are suspended in a suitable solvent, cell aggregates, and cell sheets forming a monolayer or two or more layers. The solvent can be water, physiological saline, various buffer solutions, or cell preservation solutions. The cell aggregates and cell sheets can be composed of cells alone, or can be composed of a suitable biocompatible material and cells. Examples of biocompatible materials include collagen, polyglycolic acid (PGA), polylactic acid, alginate, polyethylene oxide, poly(lactic acid-glycolic acid) copolymer, proteoglycan, glycosaminoglycan, human dermis, or combinations thereof. The biocompatible material can be a membrane (such as a sheet), a porous body (such as a sponge), or a mesh (such as a woven fabric, cloth, or non-woven fabric).

[0097] Depending on the use and form, the cell drug containing atrial myocytes and a cell population containing atrial myocytes produced by the present invention can contain other components, such as pharmaceutically acceptable carriers and additives, according to known methods (for example, methods described in the Japanese Pharmacopoeia, etc.). Examples of carriers and additives include isotonic agents, thickeners, sugars, sugar alcohols, antibacterial agents (preservatives), antimicrobials or antibacterial agents, pH regulators, stabilizers, chelating agents, oily matrices, gel matrices, surfactants, suspending agents, fluidizing agents, dispersing agents, buffer agents, antioxidants, etc.

[0098] A method for treating the above-mentioned diseases is provided, which includes administering to a patient a therapeutically effective amount of a cell drug containing atrial myocytes and a cell population containing atrial myocytes produced by the present invention.

[0099] The therapeutically effective amount is the amount of cells that can provide a therapeutic effect against the above-mentioned diseases when administered to a patient compared to a control that does not receive the cells. The specific therapeutically effective amount can be appropriately determined according to the administration form (for example, cell suspension, cell aggregate, cell sheet), administration method (for example, injection, transplantation), purpose of use, and the age, weight, symptoms, etc. of the patient. For humans (for example, adults), the effective amount per treatment is, for example, 200,000 to 1,000,000 cells / kg body weight.

[0100] The medicament of the present invention is applied to mammals, including humans.

[0101] The present invention will be described in more detail below with reference to examples, but the present invention is not limited by the examples.

[0102] In this specification, the conjunctions "about" or "approximately" used to modify numerical values respectively represent values that vary by adding or subtracting 30%, 25%, 20%, 15%, 10%, 8%, 6%, 5%, 4%, 3%, 2%, or 1% relative to a reference value. Preferably, the terms "about" or "approximately" respectively represent a range of adding or subtracting 15%, 10%, 5%, or 1% relative to a reference value.

[0103] All documents mentioned in this specification are incorporated herein by reference in their entirety.

[0104] The embodiments of the present invention described below are for illustrative purposes only and are not intended to limit the technical scope of the present invention. The technical scope of the present invention is defined only by the claims. Without departing from the spirit of the present invention, the present invention can be modified, such as adding, deleting, and replacing the components of the present invention.

[0105] [Examples]

[0106] [Experimental Example 1: Induction of Atrial Myocyte Population from iPSC]

[0107] A cell population containing cardiomyocytes was induced from iPSC under atrial myocyte differentiation conditions or ventricular myocyte differentiation conditions. As the iPSC, a double knock-in human iPS cell line (1390D4 strain) was used, in which EmGFP was inserted into the TNNI1 locus and mCherry was inserted into the TNNI3 locus as reporter proteins.

[0108] The maintenance culture of iPSC was carried out according to a conventionally known method ("A novel efficient feeder-free culture system for the derivation of human induced pluripotent stem cells", Masato Nakagawa et al., Scientific Reports, 2014, 4: 3594).

[0109] To differentiate iPSC into cardiomyocytes, the iPSC was seeded into a low-adhesion 6-well plate (2x10 6 cells / 1.5 ml / well) and statically cultured at 37 °C and 5% oxygen to form embryoid bodies (day 0). The medium used was StemPro-34 SFM (Thermo Fisher Scientific) supplemented with 1% L-glutamine, 150 μg / ml transferrin, 50 μg / ml ascorbic acid, 4x10 -4 M monothioglycerol, 10 μM Rock inhibitor (Y-27632), 2 ng / ml BMP4, and 0.5% Matrigel.

[0110] On the second day (day 1), 1.5 ml / well of atrial myocyte induction medium 1 was added to each well seeded with cells, and the cells were cultured for another 2 days at 37 °C and 5% oxygen. The atrial myocyte induction medium 1 used was StemPro-34 SFM supplemented with 1% L-glutamine, 150 μg / ml transferrin, 50 μg / ml ascorbic acid, 4 x 10 -4 M monothioglycerol, 4 ng / ml BMP4, 8 ng / ml activin A, and 10 ng / ml bFGF.

[0111] Then (on the third day), the medium was replaced with atrial myocyte induction medium 2, and the cells were cultured at 37 °C under 5% oxygen conditions for three days. As atrial myocyte induction medium 2, StemPro-34 SFM supplemented with 1% L-glutamine, 150 μg / ml transferrin, 50 μg / ml ascorbic acid, 4 x 10 -4 M monothioglycerol, 10 ng / ml VEGF, 1 μM Wnt inhibitor (IWP-3), 5.4 μM TGF-β inhibitor (SB431542), and 1 μM retinoic acid was used.

[0112] On the 6th day of differentiation, the medium was replaced with cardiomyocyte induction medium 3, and starting from the 8th day of differentiation, a γ-secretase inhibitor (LY411575) that inhibits Notch signaling at 3 μM was added when changing the medium, and the cells were cultured at 37 °C under 5% oxygen conditions until the 10th day. Cells supplemented with 0.1% DMSO were also prepared as a control. Thereafter, the medium was changed on the 10th, 13th, 15th, and 17th days, and after changing the medium on the 10th day, the cells were cultured under normal oxygen conditions until the 20th day. As cardiomyocyte induction medium 3, StemPro-34 SFM supplemented with 1% L-glutamine, 150 μg / ml transferrin, 50 μg / ml ascorbic acid, 4 x 10 -4 M monothioglycerol and 5 ng / ml VEGF was used.

[0113] [Experimental Example 2: Sorting of Atrial Myocyte Populations Based on CD151 Expression Level]

[0114] On the 20th day of culture, the cell population containing atrial myocytes was dispersed into single cells and the cell number was counted. An anti-CD151 antibody (BD) was added to the cell suspension and left standing at 4°C for 30 min. A sample added with an IgG1 isotype antibody was also prepared as a negative control. After washing, an Alexa (registered trademark) 647-labeled secondary antibody was added to the cell suspension and the cells were left standing at 4°C for 30 min. CD151 expression in EmGFP-positive cells was analyzed using a flow cytometer (BD FACS Aria Fusion cell sorter). The fluorescence intensity of 95% or more of the negative control cells was set as the threshold value, and the cell subset showing a fluorescence intensity lower than the threshold value was defined as "CD151-low", and the cell subset showing a fluorescence intensity higher than the threshold value was defined as "CD151-high". Then the cell subsets were sorted into "DMSO / CD151-high", "DMSO / CD151-low", "Notch inhibitor / CD151-high", and "Notch inhibitor / CD151-low". The figure of the flow cytometer is shown in Figure 1 and the proportions of the cell subsets are shown in Table 1. The values are shown as the mean ± standard error of the experimental data.

[0115] [Table 1]

[0116]

[0117] [Experimental Example 3: Analysis of the expression of atrial myocyte markers in the cell subsets "CD151-high" and "CD151-low" obtained under Notch inhibition]

[0118] For the cardiomyocyte subsets "DMSO / CD151-high", "DMSO / CD151-low", "Notch inhibitor / CD151-high", and "Notch inhibitor / CD151-low" obtained in Experimental Example 2, the expression of the marker gene indicating Notch signal activity and atrial myocyte markers was analyzed.

[0119] According to the conventional method, total RNA was extracted from each cell subset, and expression analysis was performed on the target gene of Notch signal (HEY2) and the marker genes of atrial myocytes (NR2F1, NR2F2, NPPA, KCNA5, KCNJ3, TBX5, and MYL7). The results are shown in Figure 2. For HEY2, the relative value was shown with the expression level in Notch inhibitor / CD151-high as 1, and for the atrial myocyte marker gene group, the relative value was shown with the expression level in the cells added with DMSO as 1 (mean ± standard error).

[0120] The expression of the HEY2 gene controlled by the Notch signal was decreased by adding a Notch signal inhibitor, and was independent of the expression of CD151. Therefore, it was obvious that the Notch signal was inhibited in both groups. In addition, compared with the control population supplemented with DMSO, the expression of atrial myocyte markers increased in both of the cell subsets, "CD151-high" and "CD151-low", obtained by adding a Notch signal inhibitor to the atrial myocyte differentiation conditions. Therefore, it was found that the inhibition of the Notch signal promoted the expression of genes important for atrial myocyte differentiation.

[0121] [Experimental Example 4: Determination of the proportion of atrial myocytes in the atrial myocyte population differentiated under Notch signal inhibition]

[0122] For the "Notch inhibitor / CD151-high" and "Notch inhibitor / CD151-low" cardiomyocyte subsets obtained in Experimental Example 2, the proportion of atrial myocytes was determined by electrophysiological methods.

[0123] Each cell subset was cultured on a fibronectin-coated coverslip. It was cultured at 37 °C under normal oxygen conditions using StemPro-34 SFM supplemented with 1% L-glutamine, 150 μg / ml transferrin, 50 μg / ml ascorbic acid, 4x10 -4 M monothioglycerol and 5 ng / ml VEGF, and the culture medium was changed every 3 days. Electrophysiological analysis was performed on the cells on the 15th and 16th days of culture.

[0124] Using an Axopatch 200B amplifier (Molecular Devices) and pCLAMP software, analysis was performed using the whole-cell patch clamp method. The electrodes were prepared from glass capillaries (WPI) using a micropipette puller and filled with the intracellular solution (130 mM KOH, 130 mM L-aspartic acid, 20 mM KCl, 5 mM NaCl, 10 mM HEPES, 5 mM Mg-ATP, 10 mM EGTA, 1 mM MgCl 2, pH 7.2). The experiments were conducted at 35 - 37 °C while perfusing Tyrode's balanced salt solution (Sigma) as the extracellular solution. Spontaneous action potentials of each cell were recorded for 1 min in the current clamp mode. APD30, APD90, and the maximum velocity of waveform rise (dv / dtmax) were calculated from the average waveform of 8 - 10 consecutive waveforms. Cells showing waveforms with APD30 / 90 less than 0.3 and dv / dtmax of 10 or greater were defined as atrial myocytes. Cells showing waveforms with APD30 / 90 of 0.3 or greater and dv / dtmax of 10 or greater or dv / dtmax less than 10 were classified as other cells. The results of the comparison between the proportions of atrial myocytes in the "Notch inhibitor / CD151 - high" and "Notch inhibitor / CD151 - low" cardiomyocyte subsets obtained in Experimental Example 2 and the proportions of atrial myocytes in the "CD151 - high" and "CD151 - low" cardiomyocyte subsets obtained by culturing in cardiomyocyte induction medium 3 without Notch inhibitor and solvent DMSO are shown in Table 2.

[0125] [Table 2]

[0126]

[0127] As shown in Table 2 and Figure 3 as shown in the bar graph presenting the results of Table 2, the cell subset "CD151 - low" obtained under the condition of adding a Notch signal inhibitor during atrial myocyte differentiation contains the highest proportion (80%) of atrial myocytes. In addition, the cell subset "CD151 - high" differentiated without adding a Notch signal inhibitor does not contain atrial myocytes, but 32% of the cells were obtained as atrial myocytes by adding a Notch inhibitor. It is clearly seen from the above results that the differentiation into atrial myocytes is promoted by adding a Notch signal inhibitor during the atrial myocyte differentiation process, and a population of atrial myocytes can be efficiently obtained by selecting atrial myocytes with CD151.

[0128] [Industrial Applicability]

[0129] The present invention enables the efficient production of atrial myocytes from stem cells. In addition, the method of the present invention enables the obtaining of a cell population mainly containing atrial myocytes. Therefore, the present invention can contribute to the development of physiological, pharmacological, and / or toxicological analysis and screening techniques for atrial myocytes, aiming to develop regenerative medicine techniques using atrial myocytes, construct disease pathology models using patient cells, etc., and discover therapeutic drugs for heart diseases such as atrial fibrillation.

[0130] This application is based on Japanese Patent Application No. 2022-125789 (filing date: August 5, 2022), the entire content of which is incorporated herein by reference.

Claims

1. A method for generating atrial myocytes from early cardiomyocytes, comprising the step of culturing a cell population containing early cardiomyocytes in the presence of a Notch signaling inhibitor.

2. The method according to claim 1, wherein the early cardiomyocytes are derived from pluripotent stem cells.

3. The method according to claim 1, wherein the Notch signaling inhibitor is a γ-secretase inhibitor.

4. The method according to claim 3, wherein the Notch signal inhibitor is at least one compound selected from the group consisting of: LY411575, deshydroxy LY-411575, DAPT, dibenzoazepine L-685,458, RO4929097, Compound E, telisotuzumab, agomelatine, nilotinib, crenigacestat, MK0752, MRK003, BPN-15606, CB-103, RBPJ inhibitor 1, SAHM1, IMR-1A and IMR-1, and their derivatives, as well as their salts, solvates and isomers having Notch signal transduction pathway inhibitory properties.

5. The method according to claim 1, further comprising the step of (b) enriching the atrial myocytes from the cell population by using the expression level of CD151 as an indicator.

6. A cell population containing atrial myocytes, obtained by the method according to claim 1.

7. A method for generating atrial myocytes from stem cells, comprising (A) a step of inducing a cell population containing early cardiomyocytes from the stem cells, (B) a step of culturing the cell population containing early cardiomyocytes in the presence of a Notch signaling inhibitor, and (C) a step of enriching the atrial myocytes from the cell population by using the expression level of CD151 as an indicator.

8. A reagent for generating atrial myocytes from early cardiomyocytes, comprising a Notch signaling inhibitor.

9. The reagent according to claim 8, further comprising a probe for detecting CD151.

10. Use of a Notch signaling inhibitor in generating atrial myocytes from early cardiomyocytes.

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