Method for producing myocardial model, myocardial disease model, and method for screening therapeutic agents against myocardial diseases

Abstract

Provided is a method for producing a myocardial model, the method comprising a step for culturing mature heart tissue in the presence of inflammatory cytokines for two or more days to obtain a myocardial model, wherein the mature heart tissue is obtained by maturing immature heart tissue obtained by co-culturing immature myocardial cells and epicardial cells. Further provided are a myocardial disease model and a method for screening therapeutic agents against myocardial diseases.

Description

Method for producing myocardial model, myocardial disease model, and method for screening therapeutic agents for myocardial disease

[0001] The present invention relates to a method for producing a myocardial model, a myocardial disease model, and a method for screening for a therapeutic agent for myocardial disease. This application claims priority to Japanese Patent Application No. 2024-148666, filed on August 30, 2024, the contents of which are incorporated herein by reference.

[0002] More than 90% of new therapeutics that undergo human clinical trials fail due to insufficient therapeutic efficacy or toxicity issues. This is partly due to the low clinical applicability of animal models. Furthermore, the use of animal models is also subject to ethical issues. Therefore, there is a need for in vitro human tissues that offer a high degree of clinical applicability as an alternative to animal models.

[0003] For example, Non-Patent Document 1 describes the formation of a population of fibroblasts that reproduce the behavior of cardiac fibroblasts in vivo by organoid culture of epicardial cells and ventricular cardiomyocytes derived from human pluripotent stem cells. Also, Non-Patent Document 2 describes the generation of mature compact ventricular cardiomyocytes from human pluripotent stem cells.

[0004] Muscular dystrophy is a disease in which a gene necessary for the formation and maintenance of muscle cells is mutated, resulting in the gradual weakening of muscle cells. For example, patients with Duchenne muscular dystrophy (DMD), a type of muscular dystrophy, have a mutation in the dystrophin gene, which prevents the normal production of dystrophin, a protein that maintains the structure of muscle cells, resulting in a gradual decline in muscle strength. Cardiomyopathy is known to be a major cause of death in DMD patients.

[0005] Fernandes I., Funakoshi S. et al., Modeling cardiac fibroblast heterogeneity from human pluripotent stem cell-derived epicardial cells, Nat Commun., 14, 8183, 2023.Funakoshi S., et al., Generation of mature compact ventricular cardiomyocytes from human pluripotent stem cells, Nat Commun., 12, 3155, 2021.

[0006] However, it is difficult to generate in vitro human tissue that reproduces the phenotype of DMD cardiomyopathy. An object of the present invention is to provide a technique for generating a myocardial model that can reproduce the in vivo phenotype.

[0007] The present invention includes the following aspects: [1] A method for producing a myocardial model, comprising the step of culturing mature cardiac tissue in the presence of an inflammatory cytokine for two or more days to obtain a myocardial model, wherein the mature cardiac tissue is obtained by maturing immature cardiac tissue obtained by co-culturing immature cardiomyocytes and epicardial cells. [2] The production method according to [1], wherein the inflammatory cytokine includes interleukin (IL)-6, tumor necrosis factor (TNF)-α, or interferon (IFN)-γ. [3] The production method according to [1] or [2], wherein the step further comprises applying a periodic electrical stimulus to the mature cardiac tissue. [4] The production method according to any one of [1] to [3], wherein the immature cardiomyocytes and the epicardial cells are cells induced to differentiate from pluripotent stem cells. [5] The production method according to any one of [1] to [4], wherein the immature cardiomyocytes or the epicardial cells have a mutation in a myocardial disease-related gene. [6] The manufacturing method according to [5], wherein the myocardial disease-associated gene is the dystrophin (DMD) gene. [7] The manufacturing method according to any one of [1] to [6], wherein the myocardial model is a cardiac organoid or an engineered heart tissue (EHT). [8] A myocardial disease model comprising mature cardiomyocytes having a mutation in a myocardial disease-associated gene and exhibiting a myocardial disease phenotype. [9] The myocardial disease model according to [8], wherein the method comprises the step of culturing mature cardiac tissue in the presence of an inflammatory cytokine for two days or more to obtain a myocardial disease model, wherein the mature cardiac tissue is obtained by maturing immature cardiac tissue obtained by co-culturing immature cardiomyocytes and epicardial cells, and wherein the immature cardiomyocytes and the epicardial cells have a mutation in a myocardial disease-associated gene.

[10] The myocardial disease model according to [8] or [9], wherein the myocardial disease-associated gene is the DMD gene.

[11] The myocardial disease model according to any one of [8] to

[10] , wherein the myocardial disease phenotype comprises a decrease in contractile force over time or DNA damage.

[12] The myocardial disease model according to any one of [8] to

[11] , wherein the myocardial disease model is a cardiac organoid or EHT.

[13] A screening method for a therapeutic agent for myocardial disease, comprising the steps of culturing the myocardial disease model according to any one of [8] to

[12] in the presence of a test substance, and evaluating the myocardial disease phenotype of the myocardial disease model, wherein the myocardial disease phenotype approaching a wild-type phenotype in the presence of the test substance compared to the absence of the test substance indicates that the test substance is a therapeutic agent for myocardial disease.

[0008] According to the present invention, a technique for producing a myocardial model capable of reproducing an in vivo phenotype can be provided.

[0009] FIG. 1 is a schematic diagram of the experimental process of Test Example 1. The left side of FIG. 2 is a cross-sectional view illustrating the structure of a device for artificial cardiac tissue. The right side of FIG. 2 is a schematic diagram of an artificial cardiac tissue. FIG. 3 is a graph showing the measurement results of the contractile force of the artificial cardiac tissue in Test Example 1. FIG. 4 is a schematic diagram of the experimental process of Test Example 2. FIG. 5 is a graph showing the measurement results of the contractile force of the artificial cardiac tissue in Test Example 2. FIG. 6 is a graph showing the measurement results of the contractile force of the artificial cardiac tissue in Test Example 2. FIG. 7 is a schematic diagram of the experimental process of Test Example 3. FIG. 8 is a graph showing the measurement results of the contractile force of the artificial cardiac tissue in Test Example 3. FIG. 9 is a graph showing the measurement results of the contractile force of the artificial cardiac tissue in Test Example 3.

[0010] [Method for producing a myocardial model] In one embodiment, the present invention provides a method for producing a myocardial model, comprising a step of culturing mature cardiac tissue in the presence of inflammatory cytokines for two or more days to obtain a myocardial model, wherein the mature cardiac tissue is obtained by maturing immature cardiac tissue obtained by co-culturing immature cardiomyocytes and epicardial cells.

[0011] In the production method of this embodiment, "culturing mature cardiac tissue in the presence of inflammatory cytokines" means culturing mature cardiac tissue in the culture medium intentionally containing inflammatory cytokines in an amount sufficient to obtain the effects of the present invention, rather than accidentally contaminating the culture medium with inflammatory cytokines. For example, this excludes unintentional contamination with trace amounts of inflammatory cytokines that do not obtain the effects of the present invention. In one aspect, "culturing mature cardiac tissue in the presence of inflammatory cytokines" may mean culturing mature cardiac tissue in a medium containing inflammatory cytokines at a concentration of 10 pg / mL or higher.

[0012] Furthermore, in the production method of this embodiment, "co-culturing immature cardiomyocytes and epicardial cells" does not mean culturing immature cardiomyocytes and epicardial cells in a state where they accidentally coexist, but means intentionally mixing and co-culturing immature cardiomyocytes and epicardial cells in a number of cells sufficient to form immature cardiac tissue. For example, this does not mean a state where immature cardiomyocytes and epicardial cells accidentally coexist in a number of cells sufficient to prevent the formation of immature cardiac tissue. In one aspect, "co-culturing immature cardiomyocytes and epicardial cells" means, for example, culturing immature cardiomyocytes and epicardial cells in a number of cells sufficient to form immature cardiac tissue. ３ This means that at least 10 immature cardiomyocytes and epicardial cells are mixed and cultured in a cell number ratio of 5:1 to 1:5.

[0013] As used herein, the term "cardiomyocyte" refers to a cell expressing at least one marker gene selected from the group consisting of cardiac troponin (cTNT), αMHC (α myosin heavy chain, MYH6), and βMHC (MYH7).

[0014] Examples of cTNT include NCBI accession numbers NM_000364 for humans and NM_001130176 for mice. Examples of αMHC include NCBI accession numbers NM_002471 for humans and NM_001164171 for mice. Examples of βMHC include NCBI accession numbers NM_000257 for humans and NM_080728 for mice.

[0015] It is known that fetal immature cardiomyocytes undergo isoform switching, in which the expression of troponin I1 (TNNI1) decreases and the expression of troponin I3 (TNNI3) increases as they mature into postnatal mature cardiac tissue. As used herein, "immature cardiomyocytes" refer to cardiomyocytes with low expression of TNNI3.

[0016] As used herein, "epicardial cells" refers to WT1-positive, ALDH1A2-positive cells. WT1 is exemplified by NCBI accession numbers NM_000378 for humans and NM_144783 for mice. ALDH1A2 is exemplified by NCBI accession numbers NM_003888 for humans and NM_009022 for mice.

[0017] The immature cardiomyocytes and epicardial cells are derived from mammals, such as rodents (e.g., mice, rats, hamsters, guinea pigs, etc.), lagomorphs (e.g., rabbits, etc.), ungulates (e.g., pigs, cattle, goats, horses, sheep, etc.), carnivores (e.g., dogs, cats, etc.), and primates (e.g., humans, monkeys, rhesus monkeys, cynomolgus monkeys, marmosets, orangutans, chimpanzees, etc.), with humans being particularly preferred.

[0018] The immature cardiomyocytes and epicardial cells may be cells induced to differentiate from pluripotent stem cells, such as embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). Among these, iPSCs are preferred as pluripotent stem cells.

[0019] The method for inducing differentiation of pluripotent stem cells into immature cardiomyocytes is not particularly limited, and examples include a method in which mesodermal cardiac progenitor cells are produced by adding optimal concentrations of activin and BMP4 (high concentration, activin 6-10 ng / mL, BMP4 10-12 ng / mL) to a culture medium for pluripotent stem cells, and then Wnt signaling is inhibited and VEGF is added and cultured to obtain fetal-type immature cardiomyocytes.

[0020] The method for inducing differentiation of pluripotent stem cells into epicardial cells is not particularly limited, and examples include a method in which activin and BMP4 are added at optimal concentrations (low concentration, activin 2 to 4 ng / mL, BMP4 3 to 4 ng / mL) to a culture medium for pluripotent stem cells to produce mesodermal cardiac progenitor cells, and then BMP4, retinol, a GSK3 inhibitor, and a TGF-β inhibitor are added to differentiate them into epicardial progenitor cells, and then WT1-positive, ALDH1A2-positive epicardial cells are obtained by further culturing them in the presence of a TGF-β inhibitor.

[0021] As described below in the Examples, immature cardiac tissue can be obtained by co-culturing immature cardiomyocytes and epicardial cells for 7 to 10 days, and mature cardiac tissue can be obtained by culturing the immature cardiac tissue in a metabolic maturation medium for 3 to 10 days.

[0022] Indicators of cardiac tissue maturity include the expression level of marker genes, cardiac tissue morphology and structure (sarcomeres, mitochondria), and properties (pulsation state, electrophysiological maturity). For example, "immature cardiac tissue" expresses marker genes such as troponin I1 (TNNI1). "Mature cardiac tissue" expresses marker genes such as troponin I3 (TNNI3).

[0023] A myocardial model can be obtained by culturing mature cardiac tissue for two or more days in the presence of inflammatory cytokines. Examples of inflammatory cytokines include interleukin (IL)-6, tumor necrosis factor (TNF)-α, and interferon (IFN)-γ. These inflammatory cytokines may be used alone or in combination of two or more.

[0024] The concentration of each inflammatory cytokine in the medium is preferably 10 pg / mL to 50 ng / mL. Furthermore, culturing mature cardiac tissue in the presence of inflammatory cytokines for 2 days or longer allows for the production of a myocardial model capable of reproducing an in vivo phenotype. The upper limit of the period for culturing mature cardiac tissue in the presence of inflammatory cytokines is not particularly limited, but may be, for example, 20 days or less.

[0025] As described later in the Examples, the myocardial model may be in the form of a cardiac organoid or an artificial cardiac tissue (Engineered Heart Tissue, EHT). Cardiac organoids are three-dimensional cellular tissues that contain multiple types of cells and reproduce a cellular environment similar to that of an actual organ. Examples of cells contained in cardiac organoids include cardiomyocytes (TNNT2-positive cells), epicardial cells (WT1-positive cells), endocardial cells (NFATC1-positive cells), cardiac fibroblasts (VIM, THY1-positive cells), and the like. The artificial cardiac tissue is formed by placing cardiac tissue at the tips of a pair of elastically deformable support pillars. The right side of Figure 2 is a schematic diagram of the artificial cardiac tissue. The artificial cardiac tissue includes a substrate 210, a pair of elastically deformable support pillars 220 suspended from the substrate 210, and cardiac tissue 240 placed between the tips of the pair of support pillars 220. The artificial cardiac tissue can be manufactured using an artificial cardiac tissue device 200 shown on the left side of Figure 2. Details will be described later.

[0026] In the production method of this embodiment, the immature cardiomyocytes or the epicardial cells may have a mutation in a myocardial disease-related gene. In this case, as will be described later in Examples, the obtained myocardial model can reproduce an in vivo disease phenotype as a myocardial disease model.

[0027] The cardiomyopathy-related gene is not particularly limited, and examples thereof include the dystrophin (DMD) gene. An example of the NCBI accession number for the human DMD gene is Gene ID: 1756. Examples of mutations in the DMD gene include mutations similar to those found in DMD patients.

[0028] In the step of culturing the mature cardiac tissue in the presence of inflammatory cytokines, a cyclic electrical stimulus may be further applied to the mature cardiac tissue. As will be described later in the Examples, applying a cyclic electrical stimulus to the mature cardiac tissue together with the inflammatory cytokine stimulus can more effectively reproduce the in vivo phenotype. Examples of the cyclic electrical stimulus include an electrical stimulus of 1 to 20 V, 1 to 10 ms, and 0.5 to 6 Hz.

[0029] The medium used in the production method of this embodiment may be a basal medium to which various additives have been added.

[0030] As the basal medium, any conventionally known medium can be used without any particular limitation. For example, IMDM medium, Medium 199 medium, Eagle's Minimum Essential Medium (EMEM) medium, αMEM medium, Dulbecco's modified Eagle's Medium (DMEM) medium, Ham's F12 medium, RPMI1640 medium, Fischer's medium, Neurobasal Medium, StemPro-34, StemFit AK02, medium obtained by removing solution C from StemFit AK02 (AJINOMOTO AK02 solution A 400 mL and solution B 100 mL, total 500 mL), Essential 6 medium, mixed media thereof, and the like.

[0031] Examples of additives include serum, albumin, transferrin, Knockout Serum Rep lacement (KSR), N2 supplement, B27 supplement, fatty acids, insulin, collagen precursors, trace elements, 2-mercaptoethanol, 1-thiolglycerol, lipids, amino acids, L-glutamine, L-alanyl-L-glutamine, Glutamax (Thermo Fisher Scientific), non-essential amino acids, vitamins, growth factors, low molecular weight compounds, antibiotics, antioxidants, pyruvic acid, buffers, inorganic salts, and the like.

[0032] [Cardiomyocyte Disease Model] In one embodiment, the present invention provides a cardiomyocyte disease model that includes mature cardiomyocytes having a mutation in a cardiomyocyte disease-associated gene and exhibits a cardiomyocyte disease phenotype. As described below in the Examples, the cardiomyocyte disease model of this embodiment can reproduce a disease phenotype in vivo. As described above, it has traditionally been difficult to produce in vitro human tissue that reproduces a disease phenotype.

[0033] The myocardial disease model of this embodiment may be produced by the above-described production method. That is, the myocardial disease model of this embodiment may be produced by a production method including a step of culturing mature cardiac tissue in the presence of inflammatory cytokines for two days or more to obtain a myocardial disease model, wherein the mature cardiac tissue is obtained by maturing immature cardiac tissue obtained by co-culturing immature cardiomyocytes and epicardial cells, and the immature cardiomyocytes and the epicardial cells have mutations in myocardial disease-related genes.

[0034] Here, the immature cardiomyocytes, epicardial cells, immature cardiac tissue, mature cardiac tissue, inflammatory cytokines, etc. are the same as those described above.

[0035] During the step of culturing the mature cardiac tissue in the presence of inflammatory cytokines, the mature cardiac tissue may further be subjected to a cyclic electrical stimulus, which is similar to that described above.

[0036] The cardiomyopathy-related gene is not particularly limited and may be, for example, the DMD gene, which is the same as that described above.

[0037] In the myocardial disease model of this embodiment, examples of myocardial disease phenotypes include a decrease in contractile force over time, DNA damage, etc. The contractile force of the myocardial disease model can be calculated, for example, by using a myocardial disease model in the form of an artificial cardiac tissue, recording the movement of the support body connected to the artificial cardiac tissue, and analyzing the video, as will be described later in the Examples.

[0038] The method for detecting DNA damage in a myocardial disease model is not particularly limited, and can be measured, for example, by detecting intranuclear granules by immunostaining for phosphorylated histone H2AX (γH2AX).

[0039] As described later in the Examples, the myocardial disease model may be in the form of a cardiac organoid or an engineered heart tissue (EHT). The cardiac organoid and EHT are similar to those described above.

[0040] [Method for screening for therapeutic agents for myocardial disease] In one embodiment, the present invention provides a method for screening for therapeutic agents for myocardial disease, comprising the steps of culturing the above-described myocardial disease model in the presence of a test substance and evaluating the myocardial disease phenotype of the myocardial disease model, wherein the myocardial disease phenotype approaches a wild-type phenotype in the presence of the test substance compared to the absence of the test substance, indicating that the test substance is a therapeutic agent for myocardial disease.

[0041] According to the screening method of this embodiment, it is possible to obtain results with high clinical applicability, thereby enabling efficient screening of therapeutic drugs for myocardial diseases. Furthermore, since no laboratory animals are used, ethical issues can be alleviated.

[0042] Examples of test substances include natural compound libraries, synthetic compound libraries, existing drug libraries, and metabolite libraries.

[0043] Examples of myocardial disease phenotypes include a decrease in contractile force over time, DNA damage, etc. The methods for measuring these are similar to those described above.

[0044] When the myocardial disease phenotype approaches the wild-type phenotype compared to the absence of the test substance, the test substance can be determined to be a therapeutic agent for myocardial disease. The myocardial disease phenotype approaches the wild-type phenotype means that in the presence of the test substance, the measured values ​​of contractile force, DNA damage, etc. of the myocardial disease model approach the measured values ​​when measured using a wild-type myocardial model compared to the absence of the test substance. Here, the measured values ​​when measured using a wild-type myocardial model may be measured simultaneously with the myocardial disease model, or may be measured in advance. In addition, the measured values ​​when measured using a wild-type myocardial model may be values ​​measured in the presence of the test substance, or values ​​measured in the absence of the test substance.

[0045] The present invention will now be described in more detail with reference to examples, but the present invention is not limited to the following examples.

[0046] Test Example 1: Preparation of a Myocardial Model Figure 1 is a schematic diagram of the experimental process of this test example. iPSCs were used: an iPSC line (CiRA00111, hereinafter sometimes referred to as the "DMD line") established from a patient lacking exon 44 of the dystrophin gene (NCBI Gene ID: 1756); and a control line in which the defect had been repaired by genome editing (hereinafter sometimes referred to as the "mutation-repaired line"). In addition, an iPSC line (1390D4) derived from a healthy individual was used for epicardial cell differentiation. Both the CiRA00111 and 1390D4 lines were established at the Center for iPS Cell Research and Application, Kyoto University. iPSC maintenance culture was performed according to a conventional method (Nakagawa M., et al., A novel efficient feeder-free culture system for the derivation of human induced pluripotent stem cells, Scientific Reports, 4, 3594, 2014).

[0047] iPSCs were differentiated into immature cardiomyocytes according to a previously published method (Miki K., et al., ERR-gamma enhances cardiac maturation with T-tubule formation in human iPSC-derived cardiomyocytes, Nat Commun., 12, 3596, 2001). More specifically, after forming embryoid bodies in aggregation medium, the cells were cultured for two days in mesoderm induction medium containing BMP4, activin A, and bFGF. Subsequently, the cells were cultured for three days in early cardiomyocyte induction medium containing VEGF and a WNT inhibitor. Subsequently, the cells were cultured in cardiomyocyte induction medium containing VEGF, thereby differentiating the iPSCs into immature cardiomyocytes. The cells were then cultured for three days in cardiomyocyte selection medium, followed by four days in cardiomyocyte induction medium. The composition of the cardiomyocyte selection medium was glucose- and glutamine-free DMEM (Thermo Fisher Scientific) containing sodium pyruvate (1 mM, Thermo Fisher Scientific) and L-lactic acid (4 mM, Fujifilm Wako Pure Chemical Industries).

[0048] The differentiation of iPSCs into epicardial cells was carried out by partially modifying a conventionally known method (Non-Patent Document 1). Specifically, iPSCs were suspended in an aggregation medium and 2 × 10 ６ The cells were seeded onto a 6-well ultra-low attachment plate (Corning) at 1.5 mL / well to form embryoid bodies (day 0). The aggregation medium consisted of L-glutamine (2 mM, Thermo Fisher Scientific), transferrin (150 μg / mL, Roche), ascorbic acid (50 μg / mL, Sigma), monothioglycerol (450 mM, Sigma), Y-27632 (10 μM, Fujifilm Wako Pure Chemical Industries), BMP4 (2 ng / mL, R&D Systems), and Matrigel. （Ｒ） The medium was StemPro-34 SFM (Thermo Fisher Scientific) containing Growth Factor Reduced (0.5%, Corning).

[0049] The next day (Day 1), 1.5 mL of mesoderm induction medium was added to each well, and the cells were cultured for an additional 3 days at 37°C under 5% carbon dioxide and 5% oxygen conditions. The mesoderm induction medium consisted of StemPro-34 SFM containing L-glutamine (2 mM), transferrin (150 μg / mL), ascorbic acid (50 μg / mL), monothioglycerol (450 mM), BMP4 (4 ng / mL), activin A (8 ng / mL, R&D Systems), and bFGF (10 ng / mL, R&D Systems).

[0050] On day 4 of differentiation, the embryoid bodies were switched to monolayer culture. The embryoid bodies were collected by centrifugation and then incubated with TrypLE. ＴＭThe embryoid bodies were treated with Express Enzyme (Thermo Fisher Scientific) at 37°C for 5 minutes and dispersed into single cells by pipetting. The cells were precipitated by centrifugation (1,000 rpm, 3 minutes). The supernatant was removed and the cells were suspended in StemPro-34 SFM containing L-glutamine (2 mM), transferrin (150 μg / mL), ascorbic acid (50 μg / mL), monothioglycerol (450 mM), retinol (2 μM, Sigma), BMP4 (10 ng / mL), SB431542 (6 μM, Fujifilm Wako Pure Chemical Industries), and CHIR99021 (1 μM, Fujifilm Wako Pure Chemical Industries).

[0051] 1 × 10 cells were placed in a 6-well plate (Corning) coated with GLS250 gelatin solution (Fujifilm Wako Pure Chemical Industries, Ltd.) at 37°C for 1 hour. ６ The cells were seeded at 2 cells / well. After 3 days of culture at 37°C and 5% carbon dioxide, the medium was replaced with epicardial cell maintenance medium. The composition of the epicardial cell maintenance medium was StemPro-34 SFM containing 2 mM L-glutamine, 150 μg / mL transferrin, 50 μg / mL ascorbic acid, 450 mM monothioglycerol, and 6 μM SB431542.

[0052] On the 8th day after the start of differentiation induction, the monolayer cells were dispersed and replated. The medium was replaced with IMDM (Thermo Fisher Scientific) containing Liberase (100 μg / mL, Roche) and DNase I (10 μg / mL, Millipore), and the cells were treated at 37°C and 5% carbon dioxide for 1 hour. The monolayer cells that had floated up in a sheet form were collected in a 15 mL tube (Falcon) and treated with TrypLE. ＴＭ After treatment with Express Enzyme at 37°C for 5 minutes, the cells were dispersed into single cells by pipetting. The cells were precipitated by centrifugation (1,000 rpm, 3 minutes), the supernatant was removed, and the cells were suspended in epicardial cell maintenance medium. 1 × 10 cells were placed on a 6-well plate coated with GLS250 gelatin solution at 37°C for 1 hour. ６ The cells were seeded at 2 cells / well and cultured at 37° C. in 5% carbon dioxide. Thereafter, the medium was replaced with epicardial cell maintenance medium every 2 or 3 days, and the cells were cultured until the 21st day.

[0053] Immature cardiac tissue was formed by mixing iPSC-derived immature cardiomyocytes on day 20 of differentiation with iPSC-derived epicardial cells on day 21 of differentiation. To disperse embryoid bodies, the iPSC-derived immature cardiomyocytes were washed once with phosphate-buffered saline (PBS, Fujifilm Wako Pure Chemical Industries, Ltd.), and then treated with IMDM containing Liberase (100 μg / mL) and DNase I (10 μg / mL) at 37°C under 5% carbon dioxide for 1 hour. Subsequently, the solution was diluted with TrypLE containing DNase I (10 μg / mL) for 1 hour. ＴＭ The medium was replaced with Express Enzyme and incubated at 37°C for 10 minutes under 5% carbon dioxide. The embryoid bodies were dispersed into single cells by pipetting and then centrifuged (1,000 rpm, 3 minutes) to precipitate the cells. After removing the supernatant, the cells were suspended in cardiomyocyte induction medium. The cardiomyocyte induction medium consisted of StemPro-34 SFM containing L-glutamine (2 mM), transferrin (150 μg / mL), ascorbic acid (50 μg / mL), and monothioglycerol (450 mM).

[0054] iPSC-derived epicardial cells were cultured at 37°C under 5% carbon dioxide for 1 hour after replacing the medium with IMDM (Thermo Fisher Scientific) containing Liberase (100 μg / mL, Roche) and DNase I (10 μg / mL, Millipore). The monolayer cells that emerged as a sheet were collected in a 15 mL tube (Falcon) and transfected with TrypLE. ＴＭ After treatment with Express Enzyme for 5 minutes at 37°C, the cells were dispersed into single cells by pipetting. The cells were precipitated by centrifugation (1,000 rpm, 3 minutes), the supernatant was removed, and the cells were suspended in cardiomyocyte induction medium.

[0055] Immature cardiomyocytes 1 x 10 ６ and epicardial cells 0.5 × 10 ６ After mixing, the mixture was centrifuged (1,000 rpm, 3 minutes) to precipitate the particles, the supernatant was removed, and the particles were suspended in 100 μL of fibrinogen gel. The composition of the fibrinogen gel was aprotinin (20 μg / mL, Sigma), fibrinogen (5 mg / mL, Sigma), Matrigel （Ｒ）The cardiomyocyte induction medium contained Growth Factor Reduced (10%) and fetal bovine serum (FBS, 10%, Thermo Fisher Scientific).

[0056] The engineered heart tissue (EHT) was fabricated as follows. The left side of Figure 2 is a cross-sectional view illustrating the structure of an artificial cardiac tissue device 200. The artificial cardiac tissue device 200 had a substrate 210 and a pair of elastically deformable support pillars 220 suspended from the substrate 210. When fabricating the artificial cardiac tissue, the tips of the pair of support pillars 220 of the artificial cardiac tissue device 200 were placed inside a recess 231 of a mold 230 having a recess 231. Next, a fibrinogen gel containing immature cardiomyocytes and epicardial cells was injected into the recess 231 of the mold 230. Next, 2 μL of thrombin (2 U / mL, Sigma) was dropped into the fibrinogen gel to polymerize the fibrinogen, and cardiac tissue 240 was formed between the tips of the pair of support pillars 220. After standing for 30 minutes under conditions of 37°C and 5% carbon dioxide, 500 μL of cardiomyocyte induction medium was added, and the artificial cardiac tissue was removed from the mold 230. The right side of Figure 2 is a schematic diagram of the artificial cardiac tissue removed from the mold 230. The artificial cardiac tissue including the substrate 210, a pair of support pillars 220, and cardiac tissue 240 disposed between the tips of the pair of support pillars 220 was mounted on a 24-well plate (Corning), and the artificial cardiac tissue was cultured for 7 days under conditions of 37°C and 5% carbon dioxide in cardiomyocyte induction medium containing retinol (2 μM). The medium was changed once every 3 days.

[0057] Cardiac organoids were prepared as follows. iPSC-derived cardiomyocytes on day 20 of differentiation and iPSC-derived epicardial cells on day 21 of differentiation were dispersed into single cells, and then cultured in a cardiomyocyte induction medium containing retinol (2 μM) at a cell density of 2.5 × 10 ５ The resulting cell mixture was seeded (3 × 10 cells / mL) in a 96-well transparent round-bottom ultra-low attachment surface microplate (Corning). ４Cells were cultured at 60 μL / well and centrifuged at 700 rpm for 2 minutes to form cell clusters. After 3 days, 60 μL / well of cardiomyocyte induction medium containing 2 μM retinol was added. Thereafter, the medium was replaced every 3 days by removing 50 μL / well of the medium and adding 50 μL / well of fresh cardiomyocyte induction medium containing 2 μM retinol.

[0058] Eight days after the formation of the artificial cardiac tissue or cardiac organoids, maturation was induced. A previously known method (Non-Patent Document 2) was used with some modifications. The culture medium for the artificial cardiac tissue or cardiac organoids was replaced with a metabolic maturation medium, and the tissue was cultured at 37°C and 5% carbon dioxide. The metabolic maturation medium was a low-glucose medium supplemented with dexamethasone (0.25 μM, Sigma), T3 hormone (4 nM, Sigma), and PPARα agonist GW7647 (1 μM). The low-glucose medium was supplemented with palmitic acid (200 μM, Sigma), B-27 ＴＭ The medium was DMEM (Thermo Fisher Scientific) containing supplement, minus insulin (1%, Thermo Fisher Scientific), and 2 g / L glucose. The medium was changed every three days, and the cells were cultured for nine days to complete maturation.

[0059] The contractile force of the artificial cardiac tissue was measured before and after maturation. The contractile force of the artificial cardiac tissue was calculated by recording the movement of the support body connected to the artificial cardiac tissue under periodic electrical stimulation conditions of 3 V, 7 ms, and 1.5 Hz and analyzing the video. The periodic electrical stimulation was applied using a C-Pace system (IonOptix). The video was converted into a motion waveform using a Python program, and the movement distance of the support body as the artificial cardiac tissue went from relaxation to contraction was calculated. The contractile force was calculated using the formula shown in Equation (1) below (the formula for a cantilever beam with a tip load).

[0060] [In formula (1), P represents the contractile force, Wmax represents the movement distance of the support tip, b represents the support width, h represents the support thickness, E represents Young's modulus, and l represents the support height.]

[0061] Figure 3 is a graph showing the measurement results of the contractile force of the artificial cardiac tissue. In Figure 3, "ns" indicates no significant difference, "**" indicates a significant difference at P<0.01, and "***" indicates a significant difference at P<0.001.

[0062] Comparing the results before and after maturation, no increase in contractile force was observed in the DMD strain, but a significant increase in contractile force was observed in the mutation-repaired strain. Furthermore, after maturation, the contractile force of the DMD strain was significantly reduced compared to the mutation-repaired strain. This result indicates that maturation of cardiac tissue resulted in a significant decrease in contractile force in the DMD strain.

[0063] [Test Example 2: Change in contractile force of artificial cardiac tissue due to periodic electrical stimulation] Figure 4 is a schematic diagram of the experimental process of this test example. Periodic electrical stimulation (2.5 V, 5 ms, 2 Hz) was applied to the matured artificial cardiac tissue, and the tissue was cultured in a low-glucose medium at 37°C and 5% carbon dioxide for 8 days to evaluate the effect on contractile force. Contractile force was measured every two days using the same method as in Test Example 1.

[0064] Figure 5 is a graph showing the results of measuring the contractile force of the artificial cardiac tissue. In Figure 5, "*" indicates a significant difference at P<0.05, "**" indicates a significant difference at P<0.01, and "***" indicates a significant difference at P<0.001. The results showed that the contractile force of the DMD strain was significantly lower than that of the mutation-repaired strain at every measurement point. This result further supports the idea that maturation of cardiac tissue resulted in a significant decrease in contractile force in the DMD strain.

[0065] Figure 6 is a graph showing the change in contractile force relative to the value before electrical stimulation (day 0). In Figure 6, "*" indicates a significant difference at P<0.05. As a result, the DMD strain showed a tendency for contractile force to decrease over time, but a significant decrease was only observed on day 4. On the other hand, no change in contractile force was observed in the mutation-repaired strain during the observation period.

[0066] Test Example 3: Changes in contractile force of artificial cardiac tissue due to cyclic electrical stimulation and inflammatory cytokine stimulation. Figure 7 is a schematic diagram of the experimental process of this test example. After maturation, artificial cardiac tissue was subjected to cyclic electrical stimulation (2.5 V, 5 ms, 2 Hz) in the presence of inflammatory cytokines, and cultured in low-glucose medium at 37°C and 5% carbon dioxide for 8 days to evaluate the effect on contractile force. Human IL-6 recombinant (1 ng / mL, R&D Systems) and human TNF-α recombinant (2 ng / mL, R&D Systems) were added as inflammatory cytokines. Contractile force was measured every two days using the same method as in Test Example 1.

[0067] Figure 8 is a graph showing the results of measuring the contractile force of the artificial cardiac tissue. In Figure 8, "*" indicates a significant difference at P<0.05, and "**" indicates a significant difference at P<0.01. The results showed that the contractile force of the DMD strain was significantly lower than that of the mutation-repaired strain from day 0 to day 4.

[0068] Figure 9 is a graph showing the change in contractile force relative to the value before electrical stimulation (day 0). In Figure 9, "*" indicates a significant difference at P<0.05, and "**" indicates a significant difference at P<0.01. As a result, the contractile force of the DMD strain decreased over time, with significant decreases observed on days 2, 6, and 8. On the other hand, no significant decrease in contractile force was observed in the mutation-repaired strain except on day 4.

[0069] These results indicate that by simultaneously applying periodic electrical stimulation and exposure to inflammatory cytokines to mature artificial cardiac tissue, it was possible to reproduce the pathological condition of cardiomyopathy, which is characterized by a progressive decline in contractile force.

[0070] Test Example 4: Changes in the phenotype of mature cardiac organoids due to stimulation with inflammatory cytokines. After maturation, cardiac organoids were stimulated with inflammatory cytokines for one week, and cardiomyocytes within the cardiac organoids were isolated using a cell sorter and subjected to gene expression analysis by RNA sequencing. Human IL-6 recombinant (50 ng / mL, R&D Systems), human TNF-α recombinant (50 ng / mL, R&D Systems), and human IFN-γ recombinant (50 ng / mL, R&D Systems) were added as inflammatory cytokines.

[0071] Table 1 shows the gene ontology (GO) terms of the gene group that was highly expressed in cardiomyocytes derived from DMD strains compared to cardiomyocytes derived from mutation repair strains under conditions without inflammatory cytokines.

[0072]

[0073] Table 2 shows the Gene Ontology (GO) terms of the gene group that was highly expressed in cardiomyocytes derived from the DMD strain compared to cardiomyocytes derived from the mutation repair strain in the presence of inflammatory cytokines.

[0074]

[0075] These results showed that in cardiac organoids, inflammation-related gene expression was increased in DMD strain-derived cardiomyocytes, and that in the presence of inflammatory cytokines, cell death-related gene expression was further increased.

[0076] According to the present invention, a technique for producing a myocardial model capable of reproducing an in vivo phenotype can be provided.

[0077] 200...artificial cardiac tissue device, 210...substrate, 220...support body, 231...recess, 230...mold, 240...cardiac tissue.

Claims

1. A method for producing a myocardial model, comprising the step of culturing mature cardiac tissue in the presence of inflammatory cytokines for two days or more to obtain a myocardial model, wherein the mature cardiac tissue is obtained by maturing immature cardiac tissue obtained by co-culturing immature cardiomyocytes and epicardial cells.

2. The method of claim 1, wherein the inflammatory cytokines include interleukin (IL)-6, tumor necrosis factor (TNF)-α, or interferon (IFN)-γ.

3. The manufacturing method according to claim 1 or 2, wherein the step further comprises applying a periodic electrical stimulus to the mature cardiac tissue.

4. The manufacturing method described in claim 1 or 2, wherein the immature cardiomyocytes and the epicardial cells are cells induced to differentiate from pluripotent stem cells.

5. The production method according to claim 1 or 2, wherein the immature cardiomyocytes or the epicardial cells have a mutation in a myocardial disease-related gene.

6. The method according to claim 5, wherein the cardiomyopathy-related gene is the dystrophin (DMD) gene.

7. The manufacturing method according to claim 1 or 2, wherein the myocardial model is a cardiac organoid or an engineered heart tissue (EHT).

8. A myocardial disease model comprising mature cardiomyocytes with a mutation in a cardiomyopathy-associated gene and exhibiting a myocardial disease phenotype.

9. The myocardial disease model according to claim 8, comprising a step of culturing mature cardiac tissue in the presence of inflammatory cytokines for two days or more to obtain a myocardial disease model, wherein the mature cardiac tissue is obtained by maturing immature cardiac tissue obtained by co-culturing immature cardiac cells and epicardial cells, and wherein the immature cardiac cells and the epicardial cells have mutations in myocardial disease-related genes.

10. The myocardial disease model according to claim 8 or 9, wherein the myocardial disease-related gene is the DMD gene.

11. The myocardial disease model according to claim 8 or 9, wherein the myocardial disease phenotype comprises a decrease in contractile force or DNA damage over time.

12. The myocardial disease model according to claim 8 or 9, which is a cardiac organoid or EHT.

13. A screening method for a therapeutic agent for myocardial disease, comprising the steps of: culturing the myocardial disease model according to claim 8 or 9 in the presence of a test substance; and evaluating the myocardial disease phenotype of the myocardial disease model, wherein the myocardial disease phenotype approaching the wild-type phenotype in the presence of the test substance compared to the absence of the test substance indicates that the test substance is a therapeutic agent for myocardial disease.

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