Method for producing mature cardiomyocytes

Abstract

Disclosed is a method for producing mature cardiomyocytes, the method including a step (1) for inducing the promotion of the expression of miR-208b-3p and / or inducing a decrease in the expression of SIX1 in immature cardiomyocytes, and a method for inducing the maturation of cardiomyocytes, the method including (1) a step for inducing the promotion of the expression of miR-208b-3p and / or inducing a decrease in the expression of SIX1 in immature cardiomyocytes.

Description

Method for producing mature cardiomyocytes

[0001] The present disclosure relates to a method for producing mature cardiomyocytes, a method for inducing maturation of cardiomyocytes, and the like.

[0002] Human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes (iPSC-CM) are promising and powerful tools for drug screening, disease modeling, and regenerative therapy. However, currently available differentiation protocols produce iPSC-CM with an immature fetal phenotype or a mixture of ventricular, atrial, and nodal subtypes, significantly limiting their future practical application. To overcome these limitations, several mature or subtype-specific protocols have been reported. However, no method exists yet for selecting iPSC-CM according to maturity and subtype. Methods for obtaining mature iPSC-CM and iPSC-CM of each subtype are still under development. Contamination with non-CM is also a challenge that must be overcome. Immature cardiomyocytes pose a risk of arrhythmias upon transplantation. Furthermore, more mature cardiomyocytes are considered useful for drug discovery and pathological elucidation, as they are more reproducible of phenomena occurring in living cells.

[0003] Several methods for purifying hiPSC-CM have been reported. Among these methods, the present inventors have previously developed a miRNA-responsive synthetic mRNA encoding a fluorescent reporter protein and a sequence complementary to a target miRNA, which they call miRNA switch (miR-switch) (Cell Stem Cell, 2015.16(6):pp.699-711). Transfecting miR-switch into cells carrying the target miRNA suppresses expression of the reporter protein. Therefore, by using miR-switch and mRNA encoding another reporter protein as a control, these cells can be identified and selected using a cell sorter. The present inventors have reported that the miR-208a switch can be used to specifically separate iPSC-CM from iPSC-nonCM with a purity of 95% or higher. The transfected miR-switch is degraded within a short period of time, eliminating the risk of genome modification. The present inventors have already confirmed that purified iPSC-CMs using the miR-208a switch can be efficiently engrafted into the mouse heart and improve its function (Cell Stem Cell, 2015.16(6):pp.699-711, Stem Cell Reports, 2022.17(7):pp.1772-1785).

[0004] However, because miR-208a switch reacts with all subtypes of iPSC-CM, including ventricular, atrial, and nodal, at an early stage, approximately one week after induction, it is not possible to purify mature iPSC-CM or iPSC-CM of each subtype, as with other methods.

[0005] Although there is a method for modifying the liquid medium used to culture human iPS cell-derived cardiomyocytes to promote their maturation (Non-Patent Document 1), immature cardiomyocytes still remain.

[0006] Furthermore, Patent Document 1 reports a method for inducing the maturation of cardiomyocytes, which method includes a step of inducing two or more of the following in immature cardiomyocytes: overexpression of Let7i microRNA (miRNA), overexpression of miR-452, decreased expression of miR-122, and decreased expression of miR-200a; and in the inducing step, a composition is used that includes two or more of a nucleic acid construct encoding Let7i microRNA, a nucleic acid construct encoding miR-452, a nucleic acid construct that is an oligomer that hybridizes to or encodes a portion of a sequence encoding miR-122, and a nucleic acid construct that is an oligomer that hybridizes to or encodes a portion of a sequence encoding miR-200a.

[0007] Japanese Patent No. 7279953

[0008] Nat Commun. 2021;12(1):3155

[0009] To solve the above problems, it is necessary to develop a method for maturing immature cardiomyocytes.

[0010] An object of the present disclosure is to provide a method for producing mature cardiomyocytes, a method for inducing the maturation of cardiomyocytes, and the like.

[0011] As a result of extensive research to achieve the above-mentioned object, the inventors have discovered that the expression of miR-208b-3p increases over time in ventricular muscle cells as they mature from the start of differentiation induction, that promoting the expression of miR-208b-3p in ventricular muscle cells promotes the maturation of ventricular muscle cells, that miR-208b-3p targets SIX1, and that suppressing the expression of SIX1 increases the expression of ventricular muscle maturation-related genes.

[0012] The present disclosure was completed based on these findings and after further investigation, and provides the following method for producing mature cardiomyocytes, mature cardiomyocytes, pharmaceutical compositions, methods for inducing cardiomyocyte maturation, etc.

[0013] [1] A method for producing mature cardiomyocytes, comprising the following steps: (1) promoting expression of miR-208b-3p and / or inducing decreased expression of SIX1 in immature cardiomyocytes. [2] The method according to [1], wherein the step (1) uses an expression construct containing a nucleic acid encoding miR-208b-3p and / or a miRNA mimic corresponding to miR-208b-3p. [3] The method according to [1], wherein the step (1) uses double-stranded RNA having an RNAi effect on the SIX1 gene. [4] The method according to [1], wherein the step (1) involves knocking out the SIX1 gene. [5] The method according to any one of [1] to [4], wherein the immature cardiomyocytes are stem cell-derived cells. [6] The method according to [5], wherein the stem cells are embryonic stem cells, pluripotent stem cells, or induced pluripotent stem cells. [7] The method according to [5], wherein the stem cells are induced pluripotent stem cells. [8] The method according to any one of [1] to [7], wherein the mature cardiomyocytes are ventricular cardiomyocytes. [9] Mature cardiomyocytes obtained by the method according to any one of [1] to [8].

[10] A pharmaceutical composition comprising the mature cardiomyocytes according to [9].

[11] The pharmaceutical composition according to

[10] , which is a composition for cell transplantation.

[12] A method for inducing maturation of cardiomyocytes, comprising the following steps: (1) inducing promotion of expression of miR-208b-3p and / or reduction of expression of SIX1 in immature cardiomyocytes.

[0014] According to the present disclosure, it is possible to induce maturation of cardiomyocytes by promoting the expression of miR-208b-3p and reducing the expression of SIX1 in immature cardiomyocytes. As a result, it is possible to use the mature cardiomyocytes obtained according to the present disclosure in transplantation therapy, drug discovery, and disease models.

[0015] Figure 1 shows the results of miRNA expression analysis in iPSC-CM using subtype-specific protocols. (A) A graph showing the miR-208b-3p positivity rate (%) in two different iPSC lines using the ventricular protocol. Left bar: 201B7, right bar: 1390D4. n = 3 independent experiments for each group. (B) A graph showing the miR-208b-3p positivity rate in the ventricular protocol, atrial protocol, and nodal protocol. n = 3 independent experiments for each group. All bars represent mean ± SEM. *p < 0.05, ***p < 0.001, ***p < 0.0001, one-way ANOVA followed by Dunnett's test. Figure 1 shows the characteristics of miR-208b-3p-positive iPSC-CM. (A) The left image shows miR-208b-3p-positive and -negative iPSC-CM immunostained with α-actinin. The white line indicates the length of one sarcomere. The graph on the right shows the sarcomere length for each iPSC-CM group. n = 20 independent experiments for each group. The center line indicates the mean. (B) Graph showing the roundness index of miR-208b-3p-positive and -negative iPSC-CM. n = 15 independent experiments for each group. The center line indicates the mean. (C) Representative transmission electron microscopy images of miR-208b-3p-positive and -negative iPSC-CM. I: I-band, M: M-band, Mt: mitochondria, Z: Z-line. (D) Graph showing the MYL2 positivity rate for miR-208b-3p-positive and -negative iPSC-CM. For each group, n = 3 independent experiments. All bars represent mean ± SEM. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, unpaired t-test. Figures showing the characteristics of miR-208b-3p-positive iPSC-CM. (E) Representative action potential waveforms of miR-208b-3p-positive and -negative iPSC-CM. (F) Graph showing the action potential duration (APD) at 90% repolarization (APD90) and 50% repolarization (APD50), as well as the APD90 / 50 ratio, of miR-208b-3p-positive and -negative iPSC-CM. For each group, n = 8 independent experiments. All bars represent mean ± SEM. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, unpaired t-test.(G) Hierarchical clustering analysis using genes classified under the Gene Ontology (GO) term "cell cycle." (H) Flow cytometry analysis showing the percentage of S-phase miR-208b-3p-positive and -negative iPSC-CM. n = 5 independent experiments per group. All bars represent mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ***p < 0.0001, unpaired t-test. (A) MiR-208b-3p-positive iPSC-CM in the maturation-inducing protocol. (A) MiR-208b-3p positivity rates in iPSC-CM from standard and maturation protocols in two different cell lines. Left bar: 201B7, right bar: 1390D4. (N = 3 independent experiments for each group.) (B) Graph showing the results of qPCR analysis of ventricular maturation-related genes. (N = 6 independent experiments for each group.) (C) Schematic diagram of the modified maturation protocol. All graph bars represent mean ± SEM. (A) **p<0.01, unpaired t-test. (B) *p<0.05, **p<0.01, ***p<0.001, ***p<0.0001, Dunnett's test after one-way ANOVA. (D) Graph showing the miR-208b-3p positivity rate of iPSC-CM using the modified maturation protocol. (N = 9 independent experiments for each group.) (E) Representative flow cytometry dot plots showing moderately and strongly miR-208b-positive iPSC-CM using the modified maturation protocol. (F) Graph showing the results of qPCR analysis of miR-208b-3p expression levels in iPSC-CMs according to their responsiveness to the miR-208b-3p switch. n=3 independent experiments for each group. All graph bars represent mean ± SEM. (D), (F), *p<0.05, **p<0.01, ***p<0.001, ***p<0.0001, Dunnett's test after one-way ANOVA. Figure showing iPSC-CM maturation by miR-208b-3p overexpression. (A) Gene set enrichment analysis (GSEA) using AAV-based miR-208b-3p-overexpressing iPSC-CMs.(B) Graphs showing the action potential duration (APD) at 90% repolarization (APD90) and 50% repolarization (APD50), as well as the APD90 / 50 ratio, in miR mimic-based miR-208b-3p-overexpressing iPSC-CM and control iPSC-CM. n = 5 independent experiments per group. All bar graphs represent mean ± SEM. *p < 0.05, **p < 0.01, unpaired t-test. Figure 1 shows the maturation of iPSC-CM by miR-208b-3p overexpression. (C) The left image is a representative image of staining for the mitochondrial probe JC-1 (top: miR mimic control, bottom: miR-208b-3p mimic). The right graph shows the red / green ratio of JC-1 as determined by flow cytometry analysis. n = 7 independent experiments per group. (D) Contractile force of the artificial cardiac tissue (μN / mm. ２ (A) Venn diagram showing the overlap between 456 candidate genes from AAV-based RNA-seq data and 212 candidate genes predicted by TargetScan. (B) Graph showing the results of dual-luciferase assays of miR-208b-3p mimics and miR mimic controls. (C) Graph showing the relative expression levels of SIX1, MYL2, MYH7, IRX4, TNNI3, and MYH6 using si-SIX1. (D) Graph showing the relative expression levels of SIX1, MYL2, MYH7, IRX4, TNNI3, and MYH6 using si-SIX1. (E) Graph showing the relative expression levels of SIX1, MYL2, MYH7, IRX4, TNNI3, and MYH6 using si-SIX1. (F) Graph showing the relative expression levels of SIX1, MYL2, MYH7, IRX4, TNNI3, and MYH6 using si-SIX1. (G) Graph showing the relative expression levels of SIX1, MYL2, MYH7, IRX4, TNNI3, and MYH6 using si-SIX1. (H) Graph showing the relative expression levels of SIX1, MYL2, MYH7, IRX4, TNNI3, and MYH6 using si-SIX1. (I) Graph showing the relative expression levels of SIX1, MYL2, MYH6 using si-SIX1. (I) Graph showing the relative expression levels of SIX1, MYL2, MYH7, IRX4, TNNI3, and MYH6 using si-SIX1. (I) Graph showing the relative expression levels of SIX1, MYL2, MYH6 using si-SIX1. (F) Graph showing the relative expression levels of SIX1, MYL2, MYH7, IRX4, TNNI3, and MYH6 using *p<0.05, **p<0.01, ****p<0.0001, unpaired t-test.

[0016] Hereinafter, embodiments of the present disclosure will be described in detail.

[0017] As used herein, "culturing" refers to maintaining and / or growing cells in an in vitro environment. "Culturing" refers to maintaining and / or growing cells outside a tissue or body, for example, in a cell culture dish or flask.

[0018] As used herein, "expression" is defined as the transcription and / or translation of a particular nucleotide sequence driven by a promoter within a cell.

[0019] As used herein, the term "pluripotent stem cells" refers to embryonic stem cells (ES cells) and cells that have the same pluripotency, i.e., the potential to differentiate into various tissues in the body (all of the endoderm, mesoderm, and ectoderm). Cells that have the same pluripotency as ES cells include "induced pluripotent stem cells" (sometimes referred to as "iPS cells" in this specification).

[0020] From the viewpoint of therapeutic application, the various cells used in the present disclosure are preferably cells that comply with GMP (Good Manufacturing Practice) standards.

[0021] Examples of pluripotent stem cells include embryonic stem cells (ES cells), induced pluripotent stem cells (iPS cells), embryonic tumor cells (EC cells), embryonic germ stem cells (EG cells), and Muse cells, and preferably iPS cells (more preferably human iPS cells). When the pluripotent stem cells are ES cells or any cells derived from a human embryo, the cells may be cells produced by destroying an embryo or cells produced without destroying an embryo, and preferably cells produced without destroying an embryo.

[0022] As for "ES cells," in the case of mouse ES cells, various mouse ES cell lines established by the inGenious targeting laboratory, RIKEN (Institute of Physical and Chemical Research), etc. can be used, and in the case of human ES cells, various human ES cell lines established by the University of Wisconsin, NIH, RIKEN, Kyoto University, National Center for Child Health and Development, Cellartis, etc. can be used. For example, human ES cell lines that can be used include CHB-1 to CHB-12 strains, RUES1 strain, RUES2 strain, HUES1 to HUES28 strains, etc. distributed by ESI Bio, H1 strain, H9 strain, etc. distributed by WiCell Research, and KhES-1 strain, KhES-2 strain, KhES-3 strain, KhES-4 strain, KhES-5 strain, SSES1 strain, SSES2 strain, SSES3 strain, etc. distributed by RIKEN.

[0023] "Induced pluripotent stem cells" refer to cells obtained by reprogramming mammalian somatic cells or undifferentiated stem cells by introducing specific factors (nuclear reprogramming factors). Currently, there are various types of "induced pluripotent stem cells," including iPS cells 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), human-derived iPS cells established by introducing the same four factors into human fibroblasts (Takahashi K, Yamanaka S., et al., Cell, (2007) 131: 861-872), and Nanog-iPS cells established by selecting cells using Nanog expression as an indicator after introducing the above four factors (Okita, K., Ichisaka, T., and Yamanaka, S. (2007). Nature 448, 313-317.), iPS cells produced by a method that does not contain c-Myc (Nakagawa M, Yamanaka S., et al. Nature Biotechnology, (2008) 26, 101-106), and iPS cells established by introducing six factors using a virus-free method (Okita K et al. Nat. Methods 2011 May; 8(5): 409-12, Okita K et al. Stem Cells. 31(3): 458-66.) can also be used. In addition, induced pluripotent stem cells established by introducing four factors, OCT3 / 4, SOX2, NANOG, and LIN28, produced by Thomson et al. (Yu J., Thomson JA. et al., Science (2007) 318: 1917-1920.), induced pluripotent stem cells produced by Daley et al. (Park IH, Daley GQ. et al., Nature (2007) 451: 141-146), induced pluripotent stem cells produced by Sakurada et al. (Japanese Patent Laid-Open Publication No. 2008-307007), and the like can also be used.

[0024] In addition, all published papers (e.g., Shi Y., Ding S., et al., Cell Stem Cell, (2008) Vol. 3, Issue 5, 568-574; Kim JB., Scholeer HR., et al., Nature, (2008) 454, 646-650; Huangfu D., Melton DA., et al., Nature Biotechnology, (2008) 26, No. 7,795-797), or patents (e.g., JP 2008-307007 A, JP 2008-283972 A, U.S. Patent Application Publication No. 2008 / 2336610, U.S. Patent Application Publication No. 2009 / 047263, WO 2007 / 069666, WO 2008 / 118220, WO 2008 / 124133, WO 2008 / 151058, WO 2009 / 006930, WO 2009 / 006997, WO 2009 / 007852) Any of the induced pluripotent stem cells known in the art can be used.

[0025] As induced pluripotent stem cell lines, various iPS cell lines established by the NIH, RIKEN, Kyoto University, etc. can be used. Examples of human iPS cell lines include RIKEN's HiPS-RIKEN-1A, HiPS-RIKEN-2A, HiPS-RIKEN-12A, and Nips-B2 lines, and Kyoto University's 253G1, 201B7, 409B2, 454E2, 606A1, 610B1, 648A1, 1231A3, 1390D4, and 1390C1 lines.

[0026] "Cardiomyocytes" are the smallest unit of contractile cells that constitute cardiac muscle, and include atrial cardiomyocytes, ventricular cardiomyocytes, etc. Identification of cells as atrial cardiomyocytes or ventricular cardiomyocytes can be performed by conventionally known methods, such as electrophysiological analysis, marker expression analysis, and drug response analysis.

[0027]

[0028] Although the cardiomyocytes are not particularly limited, they are preferably derived from mammals (e.g., mice, rats, hamsters, rabbits, cats, dogs, cows, sheep, pigs, monkeys, and humans), and more preferably from humans.

[0029] As used herein, immature cardiomyocytes refer to cardiomyocytes in which the expression level of TNNI3 is very low and in which TNNI1 is predominantly expressed. Immature cardiomyocytes are also called fetal-like cardiomyocytes.

[0030] As used herein, mature cardiomyocytes refer to cardiomyocytes in which the expression level of TNNI1 is very low and TNNI3 is predominantly expressed. Mature cardiomyocytes are also called adult-like cardiomyocytes. For example, when the expression levels of TNNI3 and TNNI1 are measured at the gene level or protein level and compared after standardization using the expression levels of constitutive expression markers, cardiomyocytes in which the expression level of TNNI3 is 5 times or more, more preferably 10 times or more, even more preferably 25 times or more, and particularly preferably 100 times or more the expression level of TNNI3 in fetal cardiomyocytes can be considered mature cardiomyocytes.

[0031] In addition to the expression levels of TNNI3 and TNNI1, the maturity of cardiomyocytes may be assessed using indicators such as increased expression of CD36, MYL2, etc., decreased expression of MYH6, morphology and structure (e.g., sarcomeres, mitochondria), properties (e.g., pulsation state, electrophysiological maturity), energy metabolism, and cell cycle. For example, the depth of the resting membrane potential measured by patch clamp or the like can be used as an indicator of electrophysiological maturity. Indicators of the sarcomere ultrastructure and mitochondria can be observed using an electron microscope, analyzed using a microscope or flow cytometer with fluorescent labeling, or functionally analyzed using an extracellular flux analyzer or the like. These indicators can also be compared with control mature cardiomyocytes such as adult cardiomyocytes and control immature cardiomyocytes such as fetal cardiomyocytes to determine whether the cardiomyocytes being evaluated are mature or immature.

[0032] The term "nucleic acid" refers to any molecule formed by polymerizing nucleotides and molecules having functions equivalent to those nucleotides, such as RNA, which is a polymer of ribonucleotides; DNA, which is a polymer of deoxyribonucleotides; a mixed polymer of ribonucleotides and deoxyribonucleotides; and a nucleotide polymer containing a nucleotide analogue. Nucleic acids may also be single-stranded or double-stranded nucleic acids. Double-stranded nucleic acids also include double-stranded nucleic acids in which one strand hybridizes to the other strand under stringent conditions.

[0033] The nucleotide analogue may be any molecule obtained by modifying ribonucleotides, deoxyribonucleotides, RNA, or DNA to improve or stabilize nuclease resistance, increase affinity with a complementary nucleic acid strand, increase cell permeability, or enable visualization, compared to RNA or DNA. The nucleotide analogue may be a naturally occurring molecule or a non-natural molecule, and examples thereof include sugar-modified nucleotide analogues (e.g., nucleotide analogues substituted with 2'-O-methylribose, nucleotide analogues substituted with 2'-O-propylribose, nucleotide analogues substituted with 2'-methoxyethoxyribose, nucleotide analogues substituted with 2'-O-methoxyethylribose, nucleotide analogues substituted with 2'-O-[2-(guanidium)ethyl]ribose, nucleotide analogues substituted with 2'-fluororibose, bridged artificial nucleic acid (BNA), locked artificial nucleic acid (LNA), ethylene bridged artificial nucleic acid (ENA), and the like. acid), peptide nucleic acid (PNA), oxypeptide nucleic acid (OPNA), peptide ribonucleic acid (PRNA)), nucleotide analogs modified with a phosphodiester bond (e.g., nucleotide analogs substituted with a phosphorothioate bond, nucleotide analogs substituted with an N3'-P5' phosphoamidate bond), etc.

[0034] The nucleic acid derivative may be any molecule in which another chemical substance is added to the nucleic acid in order to improve nuclease resistance, stabilization, affinity with a complementary nucleic acid strand, cell permeability, or visualization, compared to nucleic acids. Specific examples include 5'-polyamine-added derivatives, cholesterol-added derivatives, steroid-added derivatives, bile acid-added derivatives, vitamin-added derivatives, Cy5-added derivatives, Cy3-added derivatives, 6-FAM-added derivatives, and biotin-added derivatives.

[0035] In the present disclosure, "miRNA" refers to a 15-25 base non-coding RNA that is transcribed as a hairpin-like RNA precursor, cleaved by a dsRNA cleaving enzyme with RNase III cleavage activity, incorporated into a protein complex called RISC, and involved in the translational repression of mRNA. "miRNA" encompasses not only "miRNA" represented by a specific base sequence, but also precursors of the "miRNA" (pre-miRNA, pri-miRNA), and miRNAs with biological functions equivalent to the "miRNA." The base sequences of such miRNAs can be identified using publicly known databases (e.g., miRBase: http: / / www.mirbase.org / ).

[0036] The method for producing mature cardiomyocytes and the method for inducing cardiomyocyte maturation of the present disclosure (hereinafter, these may be simply referred to as the "method of the present disclosure") are characterized by comprising the following steps: (1) a step of inducing promotion of miR-208b-3p expression and / or reduction of SIX1 expression in immature cardiomyocytes.

[0037] Preferably, miR-208b-3p is human miRNA (hsa-miR-208b-3p). The "hsa-miR-208b-3p" used in the present disclosure has the nucleotide sequence set forth in SEQ ID NO: 7 (miRBaseAccession No. MIMAT0004960), and may be a variant of the miRNA having the nucleotide sequence set forth in SEQ ID NO: 7, as long as it has an equivalent biological function (for example, as long as it can exert the function of maturing immature cardiomyocytes). Furthermore, "hsa-miR-208b-3p" is known to have a precursor, "hsa-mir-208b" (miRBaseAccession No. MI0005570, SEQ ID NO: 8), which has a hairpin-like structure. SEQ ID NO: 7: AUAAGACGAACAAAAGGUUUGU SEQ ID NO: 8: CCUCUCAGGGAAGCUUUUUGCUCGAAUUAUGUUUCUGAUCCGAAUAUAAGACGAACAAAAGGUUUGUCUGAGGGCAG

[0038]

[0039] As used herein, the term "mutant" refers to, for example, a nucleic acid consisting of a base sequence that has 70% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identity to a base sequence registered in the above database, or an amino acid sequence registered in the above database, or a protein consisting of an amino acid sequence.

[0040] The identity (%) of the base sequences can be calculated using an analytical tool that is commercially available or available via an electric communication line (Internet). For example, it can be determined using a program commonly used in the field, such as BLAST or FASTA, with the default settings.

[0041] As used herein, "inducing the promotion of miR-208b-3p expression" refers to increasing the expression level of miR-208b-3p RNA, specifically, increasing the expression level by 5 times or more, preferably 10 times or more, more preferably 15 times or more, and even more preferably 20 times or more. Furthermore, the increase may be based on, for example, the expression level of miR-208b-3p in immature cardiomyocytes. The miR-208b-3p RNA may be derived from either an endogenous or exogenous miR-208b (miR-208b-3p) gene.

[0042] When increasing the expression level of miR-208b-3p RNA, the expression level of the miR-208b gene present endogenously in immature cardiomyocytes may be increased, or the miR-208b-3p gene may be exogenously introduced into immature cardiomyocytes to be expressed, or a miRNA mimic corresponding to miR-208b-3p may be introduced into immature cardiomyocytes. To exogenously introduce and express the miR-208b-3p gene into immature cardiomyocytes, an expression construct containing a nucleic acid encoding miR-208b-3p can be used. To exogenously express the miR-208b-3p gene, the miR-208b gene, which is a precursor of miR-208b-3p, may be introduced and expressed with the aim of ultimately expressing the miR-208b-3p gene. The expression construct is not particularly limited as long as it can express miR-208b-3p, and preferably includes a promoter, a terminator, etc. in addition to the nucleic acid encoding miR-208b-3p, and more preferably functions in the cells into which these are introduced. The expression construct can also be an expression vector, as described below. Furthermore, miRNA mimics corresponding to miR-208b-3p can also be prepared using various known techniques.

[0043] As used herein, "inducing a decrease in SIX1 expression" refers to reducing the expression level of SIX1 RNA and / or protein, specifically to 1 / 2 or less, preferably 1 / 3 or less, more preferably 1 / 5 or less, and even more preferably 1 / 10 or less. The reduction may be based on, for example, the expression level of SIX1 in immature cardiomyocytes.

[0044] When reducing the expression level of SIX1 RNA and / or protein, double-stranded RNA (e.g., siRNA, shRNA, dsRNA, and expression vectors expressing these) that have an RNAi effect on the SIX1 gene may be used, as well as miRNA, antisense nucleic acids, and expression vectors expressing these. Another method for reducing the expression level of SIX1 RNA and / or protein is to knock out the SIX1 gene. Methods known in the art, such as genome editing using ZFN, TALEN, or the CRISPR / Cas system, can be used to knock out such genes.

[0045] RNAi (RNA interference) refers to a phenomenon in which double-stranded RNA consisting of sense RNA with a sequence identical to the mRNA sequence of a target gene and antisense RNA with a sequence complementary to that of the sense RNA is introduced into a cell, thereby inducing destruction of the mRNA of the target gene and inhibiting translation into protein, thereby inhibiting expression of the target gene. Although the details of the RNAi mechanism are still unclear, an enzyme called DICER (a member of the RNase III nuclease family) comes into contact with the double-stranded RNA and degrades the double-stranded RNA into small fragments called small interfering RNA (siRNA). siRNA is incorporated into argonaute (Ago), and the sense strand (passenger strand) is removed, leaving only the antisense strand (guide strand) incorporated into Ago to form mature RNA-induced silencing complexes (RISCs). RISCs are thought to bind to and cleave target mRNAs with a sequence completely complementary to the guide strand.

[0046] The means for introducing a gene into the cells is not particularly limited, and various known or common means can be used. Typically, a gene (nucleic acid) is introduced into a cell using an expression vector and expressed. The expression vector may be linear or circular, and may be a non-viral vector such as a plasmid, a viral vector, or a transposon-based vector.

[0047] The technique for introducing an expression vector into cells can be an appropriate one depending on the embodiment. For example, the expression vector can be introduced into cells by known methods such as viral infection, calcium phosphate precipitation, lipofection, microinjection, and electroporation. The expression vector can be prepared in a form suitable for use in each technique by known means or using a commercially available kit (according to its instructions).

[0048] The expression vector can be introduced into cells by viral infection. Examples of viral vectors include retroviral vectors, lentiviral vectors, adenoviral vectors, and adeno-associated viral vectors. When using these viral vectors, a vector containing a gene (nucleic acid) for expression and a packaging vector (plasmid) for each virus can be transfected into host cells using a corresponding commercially available kit to produce a recombinant virus, and then the resulting recombinant virus can be used to infect cells.

[0049] The period for "promoting miR-208b-3p expression and / or inducing decreased SIX1 expression" in immature cardiomyocytes is not particularly limited as long as it is a period sufficient for the immature cardiomyocytes to change into mature cardiomyocytes, and is, for example, 3 days or more, 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 the state of increased miR-208b-3p expression and / or decreased SIX1 expression may be maintained even after the immature cardiomyocytes have changed into mature cardiomyocytes, or the state of increased miR-208b-3p expression and / or decreased SIX1 expression may not be maintained continuously after the immature cardiomyocytes have changed into mature cardiomyocytes.

[0050] In this way, by culturing immature cardiomyocytes in a state in which the expression level of miR-208b-3p is increased and / or the expression level of SIX1 is decreased, it is possible to induce the immature cardiomyocytes into mature cardiomyocytes. The mature cardiomyocytes obtained by the method of the present disclosure are, in particular, ventricular cardiomyocytes.

[0051] As a medium used for culturing or maintaining immature cardiomyocytes in a state in which "the expression level of miR-208b-3p is increased and / or the expression level of SIX1 is decreased," any known medium can be used without any particular limitation. The basal medium is not particularly limited as long as it can be used for culturing animal cells, and examples thereof include StemFit (e.g., StemFit AK03N, StemFit AK02N) (Ajinomoto Co., Inc.), StemPro-34 (Thermo Fisher Scientific), PECM (Primate ES Cell Medium), Essential 6 medium (Thermo Fisher Scientific), AIM V, X-VIVO-15, NeuroBasal, EGM2, TeSR, BME, BGJb, and CMRL. Examples of such media include 1066, Glasgow MEM, improved MEM zinc option, IMDM, 199 medium, Eagle's MEM, αMEM, DMEM, Ham, RPMI-1640, and Fisher's medium. Any one of these media may be used alone, or two or more may be used in combination.

[0052] The medium may contain serum or may be serum-free. The medium may also contain a serum substitute (e.g., albumin, transferrin, Knockout Serum Replacement (KSR), fatty acids, insulin, collagen precursors, trace elements, 2-mercaptoethanol, 3'-thiolglycerol, ITS supplement, B27™ supplement, N2 supplement, etc.). One or more types of serum substitutes may be used.

[0053] Furthermore, the medium may also contain one or more substances such as lipids, amino acids (e.g., non-essential amino acids), L-glutamine, vitamins, growth factors, cytokines, antibiotics, antioxidants, pyruvic acid, buffers, inorganic salts, etc. It is desirable to use a chemically defined medium that does not contain unknown components such as serum, as this reduces differences between medium lots and allows the preparation of cells of stable quality.

[0054] Furthermore, compounds such as cytokines such as activin A, BMP4, bFGF, and VEGF, GSK-3β inhibitors, and Wnt inhibitors may be added as appropriate.

[0055] As shown in the Examples, enhanced expression of miR-208b-3p and / or decreased expression of SIX1 can also be induced by using a maturation induction protocol in which compounds such as palmitic acid, dexamethasone, T3 hormone, or a PPARα agonist (e.g., GW7647) are appropriately added (preferably to a medium containing low glucose). In this case, by reducing the concentration of T3 hormone (preferably to 50% or less, more preferably to 25% or less) about one week after the start of culture, enhanced expression of miR-208b-3p and / or decreased expression of SIX1 can be further induced.

[0056] The pH of the medium is usually 7.0 to 7.8, preferably 7.2 to 7.6. Before use, the medium is preferably sterilized by filtration, ultraviolet irradiation, heat sterilization, radiation irradiation, or other methods to prevent contamination.

[0057] The culture may be carried out in the presence or absence of feeder cells. The method of the present disclosure is preferably carried out in the absence of feeder cells, since it allows stable production of mature cardiomyocytes with uniform properties without contamination by unknown components.

[0058] The culture conditions are not particularly limited. The culture temperature is, for example, about 30 to 40°C, preferably about 36 to 38°C, and CO ２ The concentration is, for example, 2 to 5%, and the oxygen concentration is, for example, 5 to 20%.

[0059] The culture period is also not particularly limited and can be determined appropriately, for example, 3 days or more, 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, similar to the period for "promoting miR-208b-3p expression and / or inducing a decrease in SIX1 expression." The upper limit of the culture period is not particularly limited, and the culture can be continued as long as the properties of mature cardiomyocytes are maintained even after the immature cardiomyocytes have been transformed into mature cardiomyocytes. In the culture, passages may be performed as many times as necessary to obtain a desired amount of mature cardiomyocytes, and the culture medium may be added and replaced. The culture can be carried out using known CO ２ The culture vessel is not particularly limited, and can be appropriately selected from plates, dishes, petri dishes, flasks, bags, bottles, tanks (culture vessels), bioreactors, etc.

[0060] The immature cardiomyocytes may be cardiomyocytes isolated from a living body or immature cardiomyocytes induced to differentiate from stem cells, and are preferably immature cardiomyocytes induced to differentiate from stem cells. Examples of stem cells include embryonic stem cells, pluripotent stem cells, and induced pluripotent stem cells, and induced pluripotent stem cells are preferred. The induced pluripotent stem cells may also be universalized iPS cells (i.e., iPS cells with a specific HLA type that does not cause immune rejection in many patients, or iPS cells with HLA knockout) that are not induced to differentiate from stem cells.

[0061] When immature cardiomyocytes induced to differentiate from stem cells are used as immature cardiomyocytes, the method of the present disclosure may further include step (0) of inducing differentiation of (immature) cardiomyocytes from stem cells prior to step (1).

[0062] Immature cardiomyocytes can be produced from stem cells by known methods. Examples of methods for inducing differentiation from pluripotent stem cells into immature cardiomyocytes include the methods disclosed in the following documents: 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

[0063] Other examples include, but are not limited to, a method for producing cardiomyocytes by forming cell masses (embryoid bodies) from induced pluripotent stem cells in suspension culture (WO 2016 / 104614), a method for producing cardiomyocytes in the presence of a substance that suppresses Bone Morphogenic Protein (BMP) signaling (WO 2005 / 033298), a method for producing cardiomyocytes by sequentially adding Activin A and BMP (WO 2007 / 002136), a method for producing cardiomyocytes in the presence of a substance that promotes activation of the canonical Wnt signal pathway (WO 2007 / 126077), and a method for isolating FLk / KDR-positive cells from induced pluripotent stem cells and producing cardiomyocytes in the presence of cyclosporin A (WO 2009 / 118928).

[0064] In addition, there are methods for inducing cardiomyocyte differentiation using cytokines in embryoid body formation (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), and methods for inducing cardiomyocyte differentiation 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 U S A., 2012 July 3; 109(27):E1848-57), and a method of inducing cardiomyocyte differentiation without using cytokines by combining adherent culture and suspension culture (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 November 1999). 29;2(5):1448-60) have also been proposed.

[0065] As a medium used for inducing differentiation of (immature) cardiomyocytes from pluripotent stem cells, known media can be used without particular limitation. Examples of basal media include StemFit (e.g., StemFit AK03N, StemFit AK02N) (Ajinomoto Co., Inc.), StemPro-34 (Thermo Fisher Scientific), PECM (Primate ES Cell Medium), Essential 6 medium (Thermo Fisher Scientific), AIM V, X-VIVO-15, NeuroBasal, EGM2, TeSR, BME, BGJb, and CMRL. Examples of such media include 1066, Glasgow MEM, improved MEM zinc option, IMDM, 199 medium, Eagle's MEM, αMEM, DMEM, Ham, RPMI-1640, and Fisher's medium. Any one of these media may be used alone, or two or more may be used in combination.

[0066] The medium may contain serum or may be serum-free. The medium may also contain a serum substitute (e.g., albumin, transferrin, Knockout Serum Replacement (KSR), fatty acids, insulin, collagen precursors, trace elements, 2-mercaptoethanol, 3'-thiolglycerol, ITS supplement, B27™ supplement, N2 supplement, etc.). One or more types of serum substitutes may be used.

[0067] Furthermore, the medium may also contain one or more substances such as lipids, amino acids (e.g., non-essential amino acids), L-glutamine, vitamins, growth factors, cytokines, ROCK inhibitors, antibiotics, antioxidants, pyruvic acid, buffers, inorganic salts, etc. It is desirable to use a chemically defined medium that does not contain unknown components such as serum, as this reduces differences between medium lots and allows the preparation of cells of stable quality.

[0068] Examples of initial additives for inducing differentiation into cardiomyocytes using the above-mentioned medium include a combination of activin A, BMP4, and bFGF, or a Wnt signal activator (e.g., Wnt protein, GSK3β inhibitor (e.g., BIO, CHIR99021)). After adding such additives, vascular endothelial growth factor (VEGF) and a Wnt inhibitor (further, an AMPK inhibitor (e.g., dorsomorphin) and an ALK5 inhibitor (e.g., SB431542)) can be added to the medium to induce differentiation into cardiomyocytes.

[0069] Examples of Wnt inhibitors include DKK1 protein (for example, in humans, RefSeq accession No.: NP_036374 (SEQ ID NO: 13)), sclerostin (for example, in humans, RefSeq accession No.: NP_079513 (SEQ ID NO: 14)), IWR-1 (Merck Millipore), IWP-2 (Sigma-Aldrich), IWP-3 (Sigma-Aldrich), IWP-4 (Sigma-Aldrich), PNU-74654 (Sigma-Aldrich), XAV939 (Sigma-Aldrich), and derivatives thereof, of which IWP-3, IWP-4, and IWR-1 are preferably used. SEQ ID NO: 13: MMALGAAGATRVFVAMVAAALGGHPLLGVSATLNSVLNSNAIKNLPPPLGGAAGHPGSAVSAAPGILYPGGNKYQTIDNYQPYPCAEDEECGTDEYCASPTRGGDAGVQICLACRKRRKRCMRHAMCCPGNYCKNGICVSSDQNHFRGEIEETITESFGNDHSTLDGYSRRTTLSSKMYHTKGQEGSVCLRSSDCASGLCCARHFWSKICKPVLKEGQVCTKHRRKGSHGLEIFQRCYCGEGLSCRIQKDHHQASNSSRLHTCQRH SEQ ID NO: 14: MQLPLALCLVCLLVHTAFRVVEGQGWQAFKNDATEIIPELGEYPEPPPELENNKTMNRAENGGRPPHHPFETKDVSEYSCRELHFTRYVTDGPCRSAKPVTELVCSGQCGPARLLPNAIGRGKWWRPSGPDFRCIPDRYRAQRVQLLCPGGEAPRARKVRLVASCKCKRLTRFHNQSELKDFGTEAARPQKGRKPRPRARSAKANQAELENAY

[0070] The culture conditions are not particularly limited. The culture temperature is, for example, about 30 to 40°C, preferably about 36 to 38°C, and CO ２ The concentration is preferably 2 to 5%. The cells may be cultured under hypoxic conditions, for example, hypoxic conditions having an oxygen concentration of 1 to 15%, such as 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, and 1%.

[0071] Mature cardiomyocytes obtained by the method of the present disclosure are preferably used as a pharmaceutical composition (e.g., a cell transplantation composition) (hereinafter, this may be simply referred to as the "pharmaceutical composition of the present disclosure") to be transplanted into patients in need of cardiomyocyte transplantation. Patients in need of cardiomyocyte transplantation include, for example, patients with diseases caused by cardiomyocyte deficiency, such as myocarditis, myocardial infarction, and myocardial injury. The amount of cells to be transplanted is selected appropriately depending on the type and severity of the disease, and the number of transplants may be one or multiple. The transplantation method is also not limited, and may be injection into the diseased area, or a cardiomyocyte cell sheet may be prepared and applied to the diseased area.

[0072] In one embodiment, mature cardiomyocytes obtained by the method of the present disclosure can be used in cardiac regenerative medicine. For example, a composition containing a cell mass of cardiomyocytes produced by the method of the present disclosure can be administered to the heart of a patient suffering from cardiac disease. Specifically, the cardiomyocytes obtained by the method of the present disclosure can be transplanted into the heart of a patient with cardiac disease either as a cell suspension or in the form of a myocardial sheet (single or multi-layered). Depending on the intended use and form, the pharmaceutical composition of the present disclosure may contain other components such as pharmaceutically acceptable carriers and additives according to known methods (e.g., methods described in the Japanese Pharmacopoeia). Examples of carriers and additives include isotonicity agents, thickeners, sugars, sugar alcohols, preservatives, bactericides or antibacterial agents, pH adjusters, stabilizers, chelating agents, oily bases, gel bases, surfactants, suspending agents, fluidizing agents, dispersing agents, buffers, antioxidants, etc.

[0073] When carrying out such cell therapy, in order to avoid rejection reactions, it is preferable that the subject from whom the cells to be used in the production of mature cardiomyocytes are isolated has an HLA type that matches that of the subject to which the mature cardiomyocytes will be administered, and it is even more preferable that the subject is the same as the subject to which the mature cardiomyocytes will be administered.

[0074] The pharmaceutical compositions of the present disclosure are intended for use in mammals, including humans.

[0075] In another embodiment, the mature cardiomyocytes obtained by the method of the present disclosure are uniformly mature and can be used for drug discovery and pathological models for the treatment of cardiac diseases. For example, the efficacy and toxicity of a test drug can be evaluated by administering a test drug to the mature cardiomyocytes obtained by the method of the present disclosure and examining the response of the cardiomyocytes.

[0076] "Comprise(s)" or "comprising" means the inclusion of, but is not limited to, the elements that follow the phrase. Thus, it implies the inclusion of the elements that follow the phrase, but not the exclusion of any other elements. "Consist(s) of" or "consisting of" means inclusive of and limited to any elements that follow the phrase. Thus, the phrase "consisting of" indicates that the listed elements are required or essential, with other elements being substantially absent. "Consist(s) essentially of" or "consisting essentially of" means inclusive of any elements that follow the phrase, and is limited to other elements that do not affect the activity or function of the element identified in this disclosure. Thus, the phrase "consisting essentially of" indicates that the recited elements are required or essential, but that other elements are optional and may or may not be present depending on whether they affect the activity or function of the recited elements.

[0077] Furthermore, the various characteristics (properties, structures, functions, etc.) described in each embodiment of the present disclosure above may be combined in any way to identify the subject matter encompassed by the present disclosure, i.e., the present disclosure encompasses all subject matter consisting of any combination of the combinable characteristics described herein.

[0078] Examples will be given below to further explain the present disclosure, but the present disclosure is not limited to these examples.

[0079] <Methods> Human iPSC lines, cell culture, and cardiac differentiation. The human iPSC lines used in this study (201B7 and 1390D4) were established at CiRA. 201B7 was maintained on SNL feeder cells in primate ES cell medium (ReproCELL, RCHEMD001) supplemented with 4 ng / mL rh bFGF (Fujifilm Wako Pure Chemical Industries, Ltd., 060-04543). 1390D4 was maintained on iMatrix-511 (Nippi Corporation, 892021)-coated dishes in StemFit AK02N medium (Ajinomoto Co., Inc., AK02N). Cardiac differentiation was performed using the embryoid body (EB) method. Briefly, on day 0, iPS cells were dissociated using Acuumax (Nacalai Tesque, Inc., 17087-54) for 201B7 and 0.5x TrypLE select (Thremo Fisher Scientific, 12563029) (diluted with 0.5 mM EDTA) for 1390D4, and then diluted with 2 mM L-glutamine (Life Technologies), 4x10 －４ 2 × 10 cells were cultured in 1.5 mL / well StemPro-34 (Invitrogen, 10640019) supplemented with M monothioglycerol (MTG; Sigma-Aldrich), 50 μg / mL ascorbic acid (AA; Sigma-Aldrich), 150 μg / mL transferrin (Roche), 10 mM Y-27632 (Fujifilm Wako Pure Chemical Industries, Ltd., 253-00513), and 2 ng / mL rh BMP4 (R&D Systems, 314-BP). ６ The cells were suspended in a 6-well low-adhesion plate at 100 cells / well to form EBs. After 24 hours, the cells were incubated with 2 mM L-glutamine, 4 x 10 －４StemPro-34 supplemented with M MTG, 50 μg / mL AA, 150 μg / mL transferrin, 10 ng / mL bFGF (R&D Systems, 233-FB), 18 ng / mL rh BMP4, and 12 ng / mL rh activin A (R&D Systems, 338-AC) was added at 1.5 mL / well. In the atrial and nodal protocols, rh BMP4 was at 4 ng / mL and rh activin A at 2 ng / mL. On day 3, EBs were washed with Iscove's modified Dulbecco's medium (IMDM; Thermo Fisher Scientific, 12440061) and resuspended in 2 mM L-glutamine, 4 × 10 －４ The cells were suspended in 1.5 mL / well StemPro-34 containing MTG, 50 μg / mL AA, 150 μg / mL transferrin, 10 ng / mL rhVEGF (R&D Systems, 293-VE), and 1 mM IWP-3 (Stemgent, 04-0035). For 1390D4, 0.6 μM dorsomorphin (Sigma-Aldrich, P5499-5MG) and 5.4 μM SB431542 (Sigma-Aldrich, S4317-5MG) were also added. For the atrial protocol, 2 mM all-trans retinoic acid (RA; Fujifilm Wako Pure Chemical Industries, Ltd., 182-01116) was added. For the nodulation protocol, 2 mM RA, 0.25 ng / mL rh BMP4, 5.4 μM SB431542, and 200 nM FGFR inhibitor PD 173074 (Chemscene, CS-0182) were added. On day 6, the medium was supplemented with 2 mM L-glutamine, 4 × 10 －４ The medium was changed to 2.0 mL / well of StemPro-34 supplemented with MTG, 50 μg / mL AA, 150 μg / mL transferrin, and 5 ng / mL rh VEGF. Thereafter, the cultures were maintained in the same medium every 2–3 days. For the first 10 days, the cells were maintained in a hypoxic environment (5% O ２) and then transferred to a normoxic environment. For the maturation protocol, on day 15, the medium was changed to 2.0 mL / well of low-glucose (2 g / L) DMEM supplemented with 200 μM palmitic acid (Sigma-Aldrich), 100 ng / mL dexamethasone (Bioshop), 4 nM T3 hormone (Sigma-Aldrich), and 1 μM PPARA agonist GW7647 (Sigma-Aldrich). Cultures were maintained in the same medium every 2–3 days.

[0080] Immunostaining and flow cytometry. Immunostaining was performed using the following primary antibodies: mouse anti-cardiac troponin T (Thermo Fisher Scientific, MS-295-P; 1:400), rabbit anti-MLC2V (abcam, ab79935; 1:400), and mouse anti-α-actinin (Sigma-Aldrich, A7811; 1:400). Secondary antibodies used in this study were donkey anti-rabbit IgG-Alexa Fluor 488 (Life Technologies, cat. no. A-21206; 1:400) and donkey anti-mouse IgG-Alexa Fluor 546 (Life Technologies, cat. no. A-10036; 1:400). Hoechst 33342 (Life Technologies, H3570; 1:10,000) was used for nuclear counterstaining. Stained cells were observed under a confocal microscope (Olympus FV3000). Flow cytometer samples were prepared using the following antibodies: rabbit anti-MLC2V (abcam, ab79935; 1:800) and goat anti-rabbit IgG-BV421 (BD Horizon, 565014). Stained cells were analyzed and sorted using a FACSAria II (BD Biosciences). Data were analyzed using FlowJo v10.6.1 analysis software (BD Biosciences).

[0081] Measurement of cell roundness index and sarcomere length 5 × 10 ４iPSC-CMs were plated on 35 mm glass-bottom dishes. Cardiac troponin T and α-actinin were stained, and the roundness index and sarcomere length were examined. Images were acquired using an FV3000. Roundness index and sarcomere length were measured using ImageJ software (National Institutes of Health).

[0082] Transmission Electron Microscopy. iPSC-CMs were fixed at room temperature using 2.0% glutaraldehyde and 4.0% paraformaldehyde in 0.1 M sodium cacodylate buffer (CB) and placed on ice. After 48 hours, post-fixation was performed with 1% osmium tetroxide in 0.15 M cold CB for 30 minutes. Cells were washed five times with 0.15 M cold CB for 2 minutes each. Samples were then dehydrated in a cold gradient ethanol series (50%, 70%, 90%, 100%, 100%; 3 minutes each) and rinsed with absolute ethanol at room temperature for 3 minutes. Infiltration into Durcupan ACM (Sigma-Aldrich, 44611) resin was performed using a 1:1 mixture of absolute ethanol and resin for 30 minutes, followed by 100% resin for 1 hour each, three times. Then, the cells were polymerized with fresh resin in a vacuum oven at 60°C for 48 hours. Images were acquired using an H-7650 (Hitachi, Ltd.).

[0083] Action potential recording and Ca imaging. ５ iPSC-CMs were plated in 35 mm glass-bottom dishes. The medium was replaced with Gey's Balanced Salt Solution (Sigma-Aldrich, G9779) and 0.1% FluoVolt (Thermo Fisher Scientific, F10488). After 20 minutes of incubation at 37°C, the medium was replaced with Gey's Balanced Salt Solution without FluoVolt and incubated at 37°C for 1 hour. Action potentials were then recorded using an FV3000. For Ca imaging, the same protocol was used, except that 1 μM Fluo-4 (Thermo Fisher Scientific, F14201) was used instead of FluoVolt.

[0084] Quantitative PCR: After cell lysis with QIAzol Lysis Reagent (QIAGEN, 79306), total RNA was extracted using the RNeasy Mini Kit (QIAGEN, 74104). The extracted RNA was reverse transcribed to cDNA using ReverTra Ace qPCR RT Master Mix with gDNA Remover (Toyobo Co., Ltd., FSQ-301). qPCR analysis was performed using TaqMan probes (Thermo Fisher Scientific). Samples were analyzed using QuantStudio 3 (Applied Biosystems). Fold changes in gene expression were calculated using the ddCt method.

[0085] Cell cycle analysis. EdU assays were performed using the Click-iT EdU Pacific Blue Flow Cytometry Assay Kit (Thermo Fisher Scientific, C10418) according to the manufacturer's protocol to detect cells in the S phase of the cell cycle. Twenty-four hours after transfection of miR-208a and miR-208b-3p switch, 1 × 10 ６ Cells were incubated with 10 μM EdU at 37°C for 2 hours, then fixed with 10 μL of Click-iT fixative for 15 minutes at room temperature. Then, cells were permeabilized with 10 μL of Click-iT saponin-based permeabilization solution for 15 minutes. Then, cells were resuspended in 43.8 μL of PBS, 1 μL of CuSO , and 1 μL of PBS. ４ 50 μL of Click-iT reaction cocktail containing 0.25 μL of fluorescent dye azide and 5 μL of 1× reaction buffer additive was added. After incubation at room temperature for 30 minutes, flow cytometry analysis (BD Biosciences, FACSAria II) was performed.

[0086] Overexpression of miR-208b-3p using adeno-associated virus (AAV). AAV serotype 6.2 expressing either the miR-208b-3p sequence or a scrambled control under the control of the human U6 promoter was commercially produced (VectorBuilder). It also contained a cassette containing the EF1A promoter followed by a fluorescent protein (mCherry). Dissociated iPSC-CMs on day 14 were cultured at 1 × 10 ４ GC / cell were transduced with either AAV. After 14 days, maturation analysis was performed.

[0087] Mitochondrial membrane potential analysis: The MitoProbe JC-1 assay (Thermo Fisher Scientific, M34152) was used according to the manufacturer's protocol. Briefly, 1 × 10 ６ The cells were incubated with 2 μM JC-1 at 37°C for 30 minutes. After washing twice with warm phosphate-buffered saline (PBS), they were observed under a confocal microscope (Olympus Corporation, FV3000) and analyzed by flow cytometry (BD Biosciences, FACSAria II). The ratio of mean red to green fluorescence intensity was quantified using FlowJo v10.6.1 analysis software (BD Biosciences).

[0088] Fabrication of engineered cardiac tissue (EHT) and measurement of contractile force were performed using a modified version of a previously reported protocol ( Nat Protoc, 2017.12(6):pp.1177-1197 ). Briefly, the following normal cardiomyocyte medium (NCM), EHT medium, and 2x DMEM were prepared. NCM contained DMEM (Biochrom, F0415), 10% heat-inactivated FBS (Sigma-Aldrich, 9048-46-8), 1% glutamine, and 1% penicillin / streptomycin. EHT medium contained DMEM, 10% heat-inactivated horse serum (Life Technologies, 26050088), 0.1% aprotinin (Sigma-Aldrich, A1153), 0.1% insulin (Sigma-Aldrich, I9278), and 1% penicillin / streptomycin. 2x DMEM contained 20% 10x DMEM (Gibco, 52100-021), 20% heat-inactivated horse serum, 58% sterile water, and 2% penicillin / streptomycin. The master mix contained 81.9 μL of cold NCM, 2.5 μL of fibrinogen stock (Sigma-Aldrich, F8630), 10 μL of Matrigel basement membrane matrix (BD bioscience, 354234), 5.5 μL of 2x DMEM, and 0.1 μL of Y-27632 (Fujifilm Wako Pure Chemical Industries, Ltd., 036-24023), for a total of 100 μL for 1 EHT. After pipetting until the fibrinogen was completely dissolved, 100 μL of the master mix and 3 μL of the thrombin aliquot were mixed to obtain a 1x10 ６The cells were quickly transferred to a container (Sumitomo Bakelite Co., Ltd., BS-X9606) containing iPSC-CMs. A silicone rack (DNABIOS, C0001) was placed on top of the cell master mix. The mixture solidified after 2 hours of incubation at 37°C. Then, the cells were transferred to a 24-well plate containing 1.5 mL / well of preheated EHT medium. EHT cultures were maintained every 2–3 days with the same medium. After several days, EHTs began to contract. The contractile function of EHTs was monitored on day 7. The moving tip of each flexible post was captured using a BZ-X 700 (Keyence Corporation) under 1 Hz electrical stimulation with a C-Pace EM (ION OPTIX). The contraction distance (δ) and EHT tissue width (D) were measured using ImageJ software (National Institutes of Health). The contractile force was calculated from the following formula: (3πER ４ δ / 4L ３ ) / A, where the pole Young's modulus (E) is 1.7 MPa, the diameter is 1.0 mm, the length is 12 mm, and the tissue area (A) is πD ２ / 4.

[0089] Dual-luciferase reporter assay. The human 3'UTR of SIX1 (1858 bp; reference gene accession number, NM_001425142.1) was inserted into pmirGLO Dual-Luciferase miRNA Target Expression Vector (Promega, E133A) to construct a dual-luciferase reporter vector. 0.8 μg of vector and 25 nM miR-208b-3p mimic or mimic control were transfected at 5 × 10 ng / ml using Lipofectamine MessengerMAX Transfection Reagent. ５ The co-transfected 293T cells were cultured for 24 hours after transfection, and then lysed. Luciferase activity was measured using the Dual-Luciferase Reporter Assay system (Promega, E910) on an EnVision 2104 (PerkinElmer). Firefly luciferase activity was normalized to Renilla luciferase activity according to the manufacturer's protocol.

[0090] Preparation of DNA templates for in vitro transcription (IVT) of mRNA and miR switches. DNA templates for IVT were generated by PCR using a KOD-Plus-Neo (Toyobo Co., Ltd., KOD-401). The forward primer contained a T7 promoter sequence, and the reverse primer contained a 120-base poly-T tract transcribed into a poly-A tail. For miR switches, template oligo DNA containing each miRNA sequence was added to the PCR materials. The PCR products were treated with DpnI restriction enzyme (Toyobo Co., Ltd., DPN-101) at 37°C for 30 minutes and then purified using the MinElute PCR Purification Kit (QIAGEN, 28006) according to the manufacturer's protocol.

[0091] Preparation of mRNA and miR switches IVT of mRNA and miR-switches was performed using MEGAScript T7 Transcription Kit (Ambion, AMB13345). Template DNA, T7 enzyme, ATP, GTP, CTP, N1-methylpseudouridine-5'-triphosphate (m1pU), 5-methyl-CTP (Tri-Link Bio Technologies, N-1081-10), and Anti Reverse Cap Analog (Tri-Link Bio Technologies, N-7003-10) were reacted at 37°C for 16 hours. The transcribed RNA was then treated with TURBO DNase (Thermo Fisher Scientific, AM1907) and purified using the RNeasy MinElute Cleanup Kit (QIAGEN, 74204). The purified RNA was treated with Antarctic Phosphatase (New England Biolabs, M0289S) and purified again using the RNeasy MinElute Cleanup Kit.

[0092] Transfection of mRNA, miR switch, miR mimic, and siRNA. mRNA, miR switch, and miR mimic were transfected into dissociated iPSC-CMs using Lipofectamine MessengerMAX Transfection Reagent (Thermo Fisher Scientific, LMRNA008) according to the manufacturer's protocol with modifications. The amount of mRNA or miR-switch was 150 ng / 10 ６ For the iPSCs, the volume of Lipofectamine MessengerMAX Transfection Reagent was 7.5 μL. For mRNA and miR-switch, flow cytometry analysis was performed 24 hours after transfection. For miR-208b-3p overexpression, 25 nM of mirVana miRNA mimic (Thermo Fisher Scientific; hsa-miR-208b-3p, MC12444; mimic-control, 4464058) was transfected into iPSC-CMs dissociated on day 14 using the same amount of Lipofectamine MessengerMAX transfection reagent as miR-switch. One week after transfection, maturation analysis was performed.

[0093] Lipofectamine RNAiMAX Transfection Reagent (Thermo Fisher Scientific, 13778030) was used for siRNA. The amount of siRNA was 6 pmol / 1.5 × 10 ５ The cells (Thermo Fisher Scientific; SIX1 siRNA, s529249; negative control, 4390843) were transfected with Lipofectamine RNAiMAX Transfection Reagent in a volume of 1 μL. qPCR analysis was performed one week after transfection.

[0094] RNA sequencing and analysis: 201B7 iPSC-CM were used to identify subtype-specific miRNAs. Thirty days after induction, 201B7 iPSC-CM were selected for the miR-208a switch using each subtype-specific protocol. The cells were lysed using QIAzol Lysis Reagent (QIAGEN, 79306), and RNA was extracted using the mirVana miRNA Isolation Kit (Thermo Fisher Scientific, AM1560).

[0095] 1390D4 iPSC-CMs were used to evaluate miR-208b-3p positive / negative iPSC-CMs and miR-208b-3p overexpression. Cells were lysed with QIAzol Lysis Reagent, and total RNA was extracted using the RNeasy Mini Kit (QIAGEN, 74104). Library construction and sequencing were performed using the TruSeq Stranded Total RNA Library Prep Kit with Ribo-Zero Gold Set A and B (Illumina, RS-122-2301 and RS-122-2302) and the NextSeq 500 / 550 High Output Kit v2 (75 cycles) (Illumina, TG-160-2005). The quality of the raw single-end reads was assessed using RSeQC (ver. 2.6.4). Prior to mapping, raw reads were trimmed using cutadapt (ver. 1.14) (https: / / doi.org / 10.14806 / ej.17.1.200), and the trimmed reads were mapped to human rRNA and tRNA using bowtie2 (ver. 2.2.5). The trimmed reads were mapped to the human genome (GRCh38) using STAR (ver. 2.5.3a). Alignment was performed using HTSeq (ver. 0.9.1) and GENCODE annotation (ver. 27). In R (version 4.3.1), counts were normalized using the DESeq2 package, GO and KEGG enrichment analysis and GSEA were performed using the clusterProfiler package, and clustered heatmaps were generated using the pheatmap package. Batch effects between datasets were removed using the ComBat algorithm in the sva package. Statistical tests and multiple comparison procedures were performed using these packages with default settings. All RNA-seq results were obtained from three biologically independent experiments.

[0096] Results: Identification of miR-208b-3p using subtype-specific protocols. Although subtype-specific protocols have been reported to efficiently differentiate ventricular, atrial, and nodal iPSC-CM-enriched populations, respectively, these protocols fail to induce completely pure subtype-specific populations. Furthermore, we previously reported that the miR-208a switch responds to all three subtypes of iPSC-CM (Stem Cell Reports, 2022.17(7):pp.1772-1785).

[0097] Among iPSC-CMs, miR-208b-3p-positive iPSC-CMs appeared only in the ventricular protocol, and their proportion increased with increasing days after induction, reaching approximately 50% positivity on day 40 in two different iPSC lines (201B7 and 1390D4) (Figure 1A). This increase in miR-208b-3p positivity was observed only in the ventricular protocol, but not in the atrial or nodal protocols, suggesting that miR-208b-3p may be a marker of mature ventricular iPSC-CMs (Figure 1B).

[0098] Characteristics of miR-208b-3p-Positive iPSC-CM Morphological Analysis of miR-208b-3p-Positive iPSC-CM First, we performed a morphological evaluation of miR-208b-positive iPSC-CM. Immunostaining revealed that miR-208b-3p-positive iPSC-CM had significantly longer sarcomere lengths and significantly lower roundness indices than miR-208b-3p-negative iPSC-CM (Figures 2A and 2B). Furthermore, transmission electron microscopy data revealed that miR-208b-3p-positive iPSC-CM had clearly aligned sarcomeres with M-bands, I-bands, and Z-lines. In contrast, no M-bands were detected in miR-208b-3p-negative iPSC-CM (Figure 2C).

[0099] Gene Expression and Metabolic Analysis of miR-208b-3p-Positive iPSC-CM Next, we compared gene expression in miR-208b-3p-positive and -negative iPSC-CM. Quantitative PCR (qPCR) analysis revealed significantly increased expression of myocardial sarcomere- and metabolism-related genes, such as TNNI3 and CD36, and ventricular maturation-related genes, such as MYL2, in miR-208b-3p-positive iPSC-CM compared to miR-208b-3p-negative iPSC-CM. MYH6, a gene known to be associated with ventricular immaturity, was significantly less expressed in miR-208b-3p-positive iPSC-CM compared to miR-208b-3p-negative iPSC-CM. Regarding MYL2, a ventricle-specific sarcomere component encoded by MLC2V, flow cytometry analysis and immunostaining revealed that miR-208b-3p-positive iPSC-CM were broadly positive for MYL2, whereas miR-208b-3p-negative iPSC-CM were partially negative for MYL2 (Fig. 2D).

[0100] Furthermore, we performed RNA sequencing (RNA-seq) analysis comparing miR-208b-3p-positive and -negative iPSC-CMs. Gene set enrichment analysis (GSEA) between the two populations revealed a high enrichment of gene ontology (GO) terms related to mitochondrial energy metabolism. Furthermore, the most enriched KEGG pathway in GSEA was oxidative phosphorylation. These findings indicated that miR-208b-3p-positive iPSC-CMs are highly mature in terms of energy metabolism.

[0101] Electrophysiological properties of miR-208b-3p-positive iPSC-CM. The electrophysiological properties of miR-208b-3p-positive iPSC-CM were analyzed. ２＋Transient analysis revealed that miR-208b-3p-positive iPSC-CMs had superior Ca handling capabilities, including peak amplitude, upstroke velocity, and decay rate. Additionally, action potential analysis using FluoVolt dye demonstrated that both miR-208b-3p-positive and -negative iPSC-CMs had ventricular characteristics (long plateau phase and typical action potential duration (APD) ratio; APD90 / APD50 < 1.4) (Nat Methods, 2014.11(8):pp.855-60). However, action potential durations at 50% and 90% repolarization (APD50 and APD90) were significantly longer in miR-208b-3p-positive iPSC-CMs (Figures 2E and F).

[0102] Cell cycle analysis of miR-208b-3p-positive iPSC-CM. Next, we focused on the cell cycle of miR-208b-3p-positive iPSC-CM. Hierarchical clustering analysis using genes classified as "cell cycle" in the Gene Ontology (GO) terminology clearly separated miR-208b-3p-positive and -negative iPSC-CM, demonstrating distinct cell cycle characteristics between the two types of cells (Figure 2G). EdU assays revealed significantly fewer EdU-positive cells in miR-208b-3p-positive iPSC-CM than in -negative iPSC-CM, indicating that miR-208b-3p-positive iPSC-CM are likely in a quiescent state (Figure 2H).

[0103] Taking these findings together, miR-208b-3p-positive iPSC-CMs were considered to be a mature ventricular subtype.

[0104] MiR-208b-3p-Positive iPSC-CM in the Maturation Induction Protocol. Therefore, we decided to use a maturation protocol to induce iPSC-CM. On day 14 after iPSC induction, the normal medium was replaced with a low-glucose maturation medium containing cytokines such as thyroid hormone and dexamethasone, and culture was continued until day 30. As expected, the miR-208b-3p-positive rate of iPSC-CM increased compared to normal medium in two different iPSC lines (201B7 and 1390D4) (Figure 3A). qPCR results showed that miR-208b-3p-positive iPSC-CM expressed higher maturation marker genes than miR-208b-3p-negative iPSC-CM, even within iPSC-CM cultured in maturation medium (Figure 3B). These findings, consistent with previous reports, demonstrate that the maturation protocol efficiently increases the number of mature ventricular miR-208b-3p-positive iPSC-CM.

[0105] However, because a significant amount of miR-208b-3p-negative iPSC-CM remained during the maturation protocol, we attempted to further increase the miR-208b-3p-positive rate of iPSC-CM. The thyroid hormone T3 concentration in the existing maturation medium mimics the transient T3 surge observed during the neonatal period. However, in humans, a transient T3 surge occurs immediately after birth, followed by a rapid decline in T3 concentration, which reaches a steady state within approximately one week (Stem Cell Res, 2014.13(3 Pt B):pp.582-91). Therefore, we modified the maturation protocol to decrease the T3 concentration one week after the start of maturation medium on day 15 (T3: 4 nM from Day 15; 2 nM or 1 nM from Day 22) (Figure 3C). When this modified maturation medium was used, the miR-208b-3p positivity rate of 1390D4 iPSC-CMs further increased, reaching 80% (Fig. 3D).

[0106] Using this modified maturation protocol, we were able to obtain sufficient miR-208b-3p-positive iPSC-CM. Therefore, we next focused on the strongly miR-208b-3p-positive population. First, we investigated whether the miR-208b-3p-negative, moderately positive, and strongly positive profiles correlated with the expression level of miR-208b-3p. As shown in Figures 3E and 3F, strongly miR-208b-3p-positive iPSC-CM expressed higher levels of miR-208b-3p than moderately positive or negative iPSC-CM. Furthermore, we found that the expression of maturation-related genes was even higher in strongly miR-208b-3p-positive iPSC-CM than in moderately positive iPSC-CM.

[0107] In summary, the modified maturation protocol enabled more efficient production of miR-208b-3p-positive mature ventricular iPSC-CMs, and the degree of miR-208b-3p positivity correlated with the degree of maturation.

[0108] Next, we investigated whether overexpression of miR-208b-3p promotes iPSC-CM maturation. To upregulate miR-208b-3p expression, we used an adeno-associated virus (AAV)-based overexpression method. For the overexpression group, iPSC-CM were transduced with an AAV vector encoding the miR-208b-3p sequence, while the control group was transduced with an AAV vector encoding a scrambled sequence. qPCR confirmed that miR-208b-3p expression was more efficiently elevated in the overexpression group than in the control group. For transcriptome analysis, RNA sequencing was performed on both groups. GSEA revealed that GO terms related to myocardial contraction, energy metabolism, and cardiac action potential were significantly increased in the overexpression group (Figure 4A). Furthermore, GO analysis of genes significantly upregulated in the overexpression group revealed significant increases in cardiac contraction-related biological process (BP) GO terms, sarcomere-related molecular function (MF) GO terms, and action potential-related cellular component (CC) GO terms. Furthermore, the overexpression group also showed elevated expression patterns of maturation-related genes. These transcriptome analysis results suggested that miR-208b-3p overexpression promoted iPSC-CM maturation. To confirm this, we performed the following functional analysis using a miR-mimic-based overexpression method to verify whether other miR-208b-3p overexpression methods could also mature iPSC-CM. MiR-208b-3p mimics were transfected on day 15, and action potential, mitochondrial membrane potential, and contractile force analyses were performed one week later.

[0109] FluoVolt dye analysis of action potentials revealed that the APD50 and APD90 were significantly prolonged in the overexpression group compared with the control group, and the waveform and APD90 / 50 ratio <1.4 (Nat. Methods, 2014.11(8):pp.855-60) indicated a ventricular phenotype in both groups (Figure 4B). The red / green fluorescence ratio of the mitochondrial probe JC-1 was significantly higher in the overexpression group, indicating that the mitochondrial membrane potential was relatively well maintained and mitochondria were more mature (Figure 4C). Furthermore, engineered cardiac tissue (EHT) was generated using iPSC-CM from both groups. EHTs composed of miR-208b-3p-overexpressing iPSC-CM contracted well, with calculated contractile force significantly stronger than that of the control group (Figure 4D). Collectively, these data demonstrate that miR-208b-3p overexpression functionally matures iPSC-CM.

[0110] Potential Targets of miR-208b-3p Finally, to elucidate the mechanism of miR-208b-3p maturation, we explored its target genes. Because miRNAs suppress target gene expression, we focused on 456 genes whose expression was significantly reduced in the overexpression group compared to the control group in the AAV-based RNA-seq data. Six of these genes overlapped with 212 target genes predicted by TargetScan (release 8.0, https: / / www.targetscan.org / ) (Figure 5A). None of these six genes had been reported or confirmed as targets of miR-208b-3p. Among these six genes, SIX1 was the most highly expressed and therefore most likely to be functional. Therefore, we performed further analysis of SIX1. We aimed to verify the interaction between miR-208b-3p and the SIX1 3'-UTR using a dual luciferase assay in 293T cells. A reporter plasmid containing the SIX1 3'-UTR sequence was co-transfected with the miR-208b-3p mimic or mimic control into 293T cells. The miR-208b-3p mimic group showed significantly reduced luciferase activity compared to the mimic control group, demonstrating the inhibitory function of miR-208b-3p on SIX1 (Figure 5B). Next, we knocked down SIX1 using siRNA. si-SIX1 effectively downregulated SIX1 expression in iPSC-CMs, resulting in significant upregulation of maturation-related genes (Figure 5C). Therefore, miR-208b-3p appears to target SIX1 to promote iPSC-CM maturation.

[0111] This application is based on Japanese Patent Application No. 2024-148013 (filing date: August 29, 2024), the contents of which are incorporated in their entirety herein.

Claims

1. A method for producing mature cardiomyocytes, comprising the steps of: (1) promoting the expression of miR-208b-3p and / or inducing a decrease in the expression of SIX1 in immature cardiomyocytes.

2. The method according to claim 1, wherein step (1) uses an expression construct containing a nucleic acid encoding miR-208b-3p and / or a miRNA mimic corresponding to miR-208b-3p.

3. The method according to claim 1, wherein step (1) uses double-stranded RNA that has an RNAi effect on the SIX1 gene.

4. The method according to claim 1, wherein the SIX1 gene is knocked out in step (1).

5. The method of claim 1, wherein the immature cardiomyocytes are stem cell-derived cells.

6. The method of claim 5, wherein the stem cells are embryonic stem cells, pluripotent stem cells, or induced pluripotent stem cells.

7. The method of claim 5, wherein the stem cells are induced pluripotent stem cells.

8. The method of claim 1, wherein the mature cardiomyocytes are ventricular myocytes.

9. The method of claim 2, wherein the immature cardiomyocytes are cells derived from induced pluripotent stem cells and the mature cardiomyocytes are ventricular myocytes.

10. The method of claim 3, wherein the immature cardiomyocytes are cells derived from induced pluripotent stem cells and the mature cardiomyocytes are ventricular myocytes.

11. The method of claim 4, wherein the immature cardiomyocytes are cells derived from induced pluripotent stem cells and the mature cardiomyocytes are ventricular myocytes.

12. Mature cardiomyocytes obtained by the method according to any one of claims 1 to 11.

13. A pharmaceutical composition comprising the mature cardiomyocytes of claim 12.

14. The pharmaceutical composition according to claim 13, which is a composition for cell transplantation.

15. A method for inducing maturation of cardiomyocytes, comprising the steps of: (1) inducing enhanced expression of miR-208b-3p and / or decreased expression of SIX1 in immature cardiomyocytes.

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