Method for producing in vitro disease simulation model in which adult acute myocardial infarction and heart failure diseases are induced by ischemia-reperfusion injury using human pluripotent stem cell-derived cardiac organoid, and in vitro disease simulation model produced thereby

Abstract

The present invention relates to a method for preparing an in vitro disease simulation model in which adult acute myocardial infarction and heart failure diseases are induced by ischemia-reperfusion injury using human pluripotent stem cell-derived cardiac organoids. The method includes: a step A of culturing human pluripotent stem cells to prepare a human pluripotent stem cell-derived cardiac organoid self-organized to include cardiomyocytes, fibroblasts, and vascular endothelial cells; a step B of culturing the human pluripotent stem cell-derived cardiac organoid prepared through step A in RPMI 1640 medium with cobalt (II) chloride added and glucose excluded to simulate ischemic conditions; a step C of culturing the human pluripotent stem cell-derived cardiac organoid that underwent ischemic condition simulation in step B in RPMI 1640 medium with D-glucose and calcium chloride added to simulate a reperfusion injury condition; and a step D of culturing the human pluripotent stem cell-derived cardiac organoid that underwent ischemia-reperfusion injury simulation in step C in RPMI 1640 medium with TGF-β and B-27 Supplement added to simulate a state of fibrosis resulting from heart failure.

Description

Method for producing an in vitro disease-simulating model in which adult acute myocardial infarction and heart failure are induced by ischemia-reperfusion injury using human pluripotent stem cell-derived cardiac organoids, and an in vitro disease-simulating model produced thereby

[0001] The present invention relates to a method for producing an in vitro disease-simulating model in which adult acute myocardial infarction and heart failure are induced by ischemia-reperfusion injury using human pluripotent stem cell-derived cardiac organoids, and to an in vitro disease-simulating model produced thereby.

[0002] Currently, disease-simulating models using organoids are being used to analyze the causes and mechanisms of diseases by creating mutant cells in which disease-related genes in pluripotent stem cells are deleted using gene scissors using gene editing technology, or by creating congenital defect models using immature organoids at the fetal level. However, because they are disease-simulating models in an immature state at the fetal level, they have limitations in representing diseases that occur in adult humans.

[0003] Diseases occurring in human adults occur through the interaction between various cells and the surrounding microenvironment, but an adult-level disease simulation model using organoids has not yet been developed.

[0004] In addition, there are still limitations in responding to inflammation, infection, etc. because the immune response, which is one of the most important functions of the body to defend itself from external organisms including pathogens, is not displayed at the organoid level.

[0005] To overcome this, the applicant of the present invention has presented a technology related to a method for producing cardiac organoids that are sufficiently capable of simulating the structure and function of an actual heart through Korean Patent Publication No. 10-2022-0153170 entitled “Method for producing cardiac organoids derived from human pluripotent stem cells and human pluripotent stem cell-derived cardiac organoids produced thereby” (hereinafter referred to as “prior art”).

[0006] Furthermore, the present applicant proposes a method for producing an in vitro disease-simulating model that can be utilized for screening new drugs for myocardial infarction and heart failure in adults and for elucidating the cause of the disease mechanism by simulating a fibrotic state that progresses to heart failure after ischemia-reperfusion injury in adults based on prior art.

[0007] In this regard, ischemia-reperfusion injury in human adults refers to a situation where, when blood supply is temporarily interrupted and blood flow is restored to the area where ischemia occurred, cells and tissues that should normally be promoted for recovery instead deteriorate.

[0008] The representative mechanisms are known to be intracellular calcium overload (intracellular calcium overload) and excessive nutrient supply (high glucose) due to reperfusion, which causes calcium ions to move from the endoplasmic reticulum into the cytoplasm.

[0009] This causes the mitochondrial permeability transition pores in the mitochondrial membranes of heart cells to open, allowing excessive water to flow in, ultimately leading to mitochondrial death. If this destruction continues and mitochondria fail to function properly, adenosine triphosphate (ATP, the cellular energy source) is depleted, exacerbating ischemic symptoms and ultimately leading to cell death and tissue damage.

[0010] Additionally, if tissue damage persists, fibrosis progresses due to activation of fibroblasts present in the heart, leading to structural or functional abnormalities that reduce the relaxation-pumping function and lead to heart failure, which is unable to properly supply the blood needed by the body tissues.

[0011] The present invention was created to solve the above problems, and the purpose of the present invention is to provide a method for producing an in vitro disease-simulating model that can be utilized for screening new drugs for myocardial infarction and heart failure in adults and for identifying the cause of the disease mechanism by simulating a fibrotic state that progresses to heart failure after ischemia-reperfusion injury in adults.

[0012] In order to achieve the above object, the present invention provides a method for producing an in vitro disease-simulating model in which adult acute myocardial infarction and heart failure are induced by ischemia-reperfusion injury using human pluripotent stem cell-derived cardiac organoids, comprising: a step A of culturing human pluripotent stem cells (hPSCs) to prepare self-organized human pluripotent stem cell-derived cardiac organoids including cardiomyocytes, fibroblasts, and endothelial cells; a step B of culturing the human pluripotent stem cell-derived cardiac organoids prepared through step A in RPMI 1640 medium supplemented with cobalt(II) chloride (CoCl2) and excluding glucose to simulate an ischemic condition; A step C of culturing human pluripotent stem cell-derived cardiac organoids, which have undergone ischemic condition simulation through the above step B, in RPMI 1640 medium supplemented with D-glucose and calcium chloride (CaCl2) to simulate reperfusion injury; and a step D of culturing human pluripotent stem cell-derived cardiac organoids, which have undergone ischemic-reperfusion injury simulation through the above step C, in RPMI 1640 medium supplemented with TGF-β (Transforming Growth Factor-β) and B-27 Supplement to simulate fibrosis due to heart failure.

[0013] Here, the step A comprises: step A-1 of culturing human pluripotent stem cells, which are at least one of human embryonic stem cells (hESCs) or human induced pluripotent stem cells (hiPSCs), to prepare an embryonic body having a diameter of 100 μm to 130 μm; step A-2 of culturing the embryonic body prepared through step A-1 until it has a diameter of 200 μm to 250 μm; step A-3 of differentiating the human pluripotent stem cell-derived embryonic body having a diameter of 200 μm to 250 μm through step A-2 into a structure including mesoderm cells and endoderm cells; A step A-4 in which a construct including mesodermal cells and endoderm cells differentiated through the above step A-3 is differentiated into a self-organized cardiac organoid including cardiomyocytes, fibroblasts, and endothelial cells; and a step A-5 in which the self-organized cardiac organoid differentiated through the above step A-4 is cultured and matured.

[0014] In addition, the above step B is a step of culturing the human pluripotent stem cell-derived cardiac organoid prepared through the above step A in 2 to 4 ml of RPMI 1640 medium with cobalt (II) chloride added at a concentration of 50 μM to 100 μM and glucose excluded, for 5 to 7 hours using a cell incubator with a temperature environment of 35°C to 40°C.

[0015] In addition, the above step C is a C-1 step in which the human pluripotent stem cell-derived cardiac organoids that have gone through the above step B are cultured for 46 to 50 hours in 2 to 4 ml of RPMI 1640 medium supplemented with D-glucose at a concentration of 23 to 25 mM and calcium chloride (CaCl2) at a concentration of 1.8 to 2 mM using a cell incubator with a temperature environment of 35 to 40°C; And step C-2, which includes adding 0.5 ml to 1.5 ml of RPMI 1640 medium containing D-glucose at a concentration of 23 mM to 25 mM and calcium chloride (CaCl2) at a concentration of 1.8 mM to 2 mM to the RPMI 1640 medium used in step C-1, and then culturing the human pluripotent stem cell-derived cardiac organoids that have undergone step C-1 for 22 to 26 hours using a cell incubator with a temperature environment of 35°C to 40°C.

[0016] In addition, the above step D is a step of culturing the human pluripotent stem cell-derived cardiac organoids that have gone through the above step C for 6 to 8 days in RPMI 1640 medium supplemented with TGF-β (Transforming Growth Factor-β) and B-27 Supplement at a concentration of 5 to 10 ng / ml using a cell incubator with a temperature environment of 35 to 40°C.

[0017] Meanwhile, in order to achieve the above purpose, an in vitro disease simulating model according to the present invention is prepared through a method for manufacturing an in vitro disease simulating model in which adult acute myocardial infarction and heart failure are induced by ischemia-reperfusion injury using a human pluripotent stem cell-derived cardiac organoid as described above.

[0018] According to the present invention, the following effects are achieved.

[0019] First, by simulating the fibrotic state that progresses to heart failure after ischemia-reperfusion injury in adults, an in vitro disease-simulating model can be created that can be utilized for screening new drugs for myocardial infarction and heart failure in adults and for elucidating the causes of disease mechanisms.

[0020] Second, it can build a platform that can screen new drugs for myocardial infarction and heart failure and elucidate the causes of disease mechanisms.

[0021] Figure 1 is a flow chart of a method for producing an in vitro disease-simulating model in which adult acute myocardial infarction and heart failure are induced by ischemia-reperfusion injury using a human pluripotent stem cell-derived cardiac organoid of the present invention.

[0022] Figure 2 is a photograph comparing the differences in organoid structure depending on whether the ischemic condition induction step is performed in the method for producing an in vitro disease-simulating model in which adult acute myocardial infarction and heart failure are induced by ischemia-reperfusion injury using human pluripotent stem cell-derived cardiac organoids of the present invention.

[0023] Figure 3 shows the results of comparing the expression levels of hypoxia-inducible factors according to the progress of the ischemic state induction step in the method for producing an in vitro disease-simulating model in which adult acute myocardial infarction and heart failure disease are induced by ischemia-reperfusion injury using human pluripotent stem cell-derived cardiac organoids of the present invention.

[0024] Figure 4 shows the results of comparing the expression levels of apoptosis markers according to the progress and degree of the reperfusion state induction step in the method for producing an in vitro disease-simulating model in which adult acute myocardial infarction and heart failure disease are induced by ischemia-reperfusion injury using human pluripotent stem cell-derived cardiac organoids of the present invention.

[0025] Figure 5 is a graph comparing the extracellular release amounts of acute myocardial infarction markers according to the progress of the reperfusion injury induction step in the method for manufacturing an in vitro disease-simulating model in which adult acute myocardial infarction and heart failure disease are induced by ischemia-reperfusion injury using human pluripotent stem cell-derived cardiac organoids of the present invention.

[0026] Figures 6 and 7 are graphs comparing the expression levels of inflammation-related markers according to the progress of the reperfusion injury induction step in the method for producing an in vitro disease-simulating model in which adult acute myocardial infarction and heart failure disease are induced by ischemia-reperfusion injury using human pluripotent stem cell-derived cardiac organoids of the present invention.

[0027] Figures 8 to 11 are results of comparing the expression levels of proteins involved in inflammatory response and immune system regulation according to the progress of the reperfusion injury induction step in the method for producing an in vitro disease-simulating model in which adult acute myocardial infarction and heart failure disease are induced by ischemia-reperfusion injury using human pluripotent stem cell-derived cardiac organoids of the present invention.

[0028] Figure 12 is a photograph comparing the differences in organoid structure depending on whether the heart failure state induction step is progressed in the method for producing an in vitro disease-simulating model in which adult acute myocardial infarction and heart failure disease are induced by ischemia-reperfusion injury using human pluripotent stem cell-derived cardiac organoids of the present invention.

[0029] Figures 13 to 15 are graphs comparing the expression levels of fibrosis and collagen-related markers according to the progress of the heart failure state induction step in the method for producing an in vitro disease-simulating model in which adult acute myocardial infarction and heart failure disease are induced by ischemia-reperfusion injury using human pluripotent stem cell-derived cardiac organoids of the present invention.

[0030] Figure 16 is a photograph comparing the degree of fibrosis according to the progress of the heart failure state induction step in the method for producing an in vitro disease-simulating model in which adult acute myocardial infarction and heart failure disease are induced by ischemia-reperfusion injury using human pluripotent stem cell-derived cardiac organoids of the present invention.

[0031] A preferred embodiment of the present invention will be described in more detail with reference to the attached drawings, but already well-known technical parts will be omitted or compressed for the sake of brevity.

[0032] <Description of a method for producing an in vitro disease-simulating model of adult acute myocardial infarction and heart failure induced by ischemia-reperfusion injury using human pluripotent stem cell-derived cardiac organoids>

[0033] Hereinafter, a detailed description will be given of the process for producing an in vitro disease-simulating model in which adult acute myocardial infarction and heart failure are induced by ischemia-reperfusion injury using a human pluripotent stem cell-derived cardiac organoid according to the present invention, with reference to FIG. 1.

[0034] (1) Heart organoid preparation stage<S110, A단계>

[0035] In this step, human pluripotent stem cells (hPSCs) are cultured to prepare self-organized human pluripotent stem cell-derived cardiac organoids containing cardiomyocytes, fibroblasts, and endothelial cells.

[0036] Here, it can be interpreted as referring to human pluripotent stem cells (hPSCs) themselves, but in a more specific sense, it can also be interpreted as cells composed of at least one of human embryonic stem cells (hESCs) or human induced pluripotent stem cells (hiPSCs).

[0037] In addition, this step (S110) corresponds to the process of manufacturing a human pluripotent stem cell-derived cardiac organoid according to the method for manufacturing a human pluripotent stem cell-derived cardiac organoid by the applicant's prior published patent document No. 10-2022-0153170, 'Method for manufacturing a human pluripotent stem cell-derived cardiac organoid and human pluripotent stem cell-derived cardiac organoid manufactured thereby'.

[0038] Specifically, in the embryoid body preparation stage (Stage A-1), human pluripotent stem cells in culture are harvested and then 1.5×10 6 Plating was performed using mTeSR medium (Stem Cell Technologies, Canada) supplemented with ROCK (Rho-associated kinase) inhibitor (Y-27632, Tocris Bioscience, UK) at a concentration of 10 μM per well.

[0039] Here, it is important that no coating is used for cell attachment when plating human pluripotent stem cells on a 6-well plate, and that Matrigel, which is used in general organoid culture, is not used; alternatively, a low attachment plate can be used.

[0040] Additionally, after one day of culture, embryoid bodies consisting of approximately 200 to 500 cells and having a diameter of 100 μm to 130 μm are prepared in each well.

[0041] Therefore, 1.5×10 in each well of a 6-well plate 6 When human pluripotent stem cells are plated, approximately 3,000 to 7,500 embryoid bodies with a diameter of 100 to 130 μm are produced after 1 day of culture.

[0042] Next, the embryoid body culture step (Step A-2) is performed by culturing the embryoid bodies with a diameter of 100 µm to 130 µm prepared through the embryoid body preparation step (S110) in an mTeSR culture medium without (or without) the ROCK (Rho-associated kinase) inhibitor (Y-27632) for 2 to 3 days to prepare human pluripotent stem cell-derived embryoid bodies with a diameter of 200 µm to 250 µm.

[0043] Here, the culture process using mTeSR culture medium without (without) ROCK (Rho-associated kinase) inhibitor (Y-27632) is performed for 2 to 3 days, and it is preferable to replace the culture medium with a new one every day.

[0044] Next, the mesoderm and endoderm cell differentiation stage (Stage A-3) is subdivided and sequentially performed in two stages. First, through the embryoid body culture stage (Stage A-2), human pluripotent stem cell-derived embryoid bodies with a diameter of 200 μm to 250 μm are cultured for two days in RPMI 1640 medium supplemented with B-27 Supplement excluding insulin, CHIR99021 with a concentration of 6 μM to 8 μM, BMP4 with a concentration of 9 ng / ㎖ to 10 ng / ㎖, and Activin A with a concentration of 8 ng / ㎖ to 10 ng / ㎖.

[0045] Afterwards, the embryoid bodies derived from human pluripotent stem cells that have undergone the primary differentiation process of the mesoderm and endoderm cell differentiation stage (Stage A-3) are cultured for another 2 days in RPMI 1640 medium supplemented with B-27 Supplement excluding insulin, XAV939, XAV939 at a concentration of 8 μM to 12 μM, and L-Ascorbic acid at a concentration of 40 μg / mL to 60 μg / mL, followed by a secondary differentiation process to differentiate them into constructs including mesoderm cells and endoderm cells.

[0046] Following this, the cardiac organoid self-organization stage (Stage A-4) is further subdivided into four stages, and first, through the mesoderm and endoderm cell differentiation stage (Stage A-3), a construct including differentiated mesoderm cells and endoderm cells is cultured for two days in RPMI 1640 medium supplemented with B-27 Supplement excluding insulin and L-Ascorbic acid at a concentration of 40 μg / ml to 60 μg / ml, thereby inducing differentiation into cardiomyocytes.

[0047] After that, the construct in which differentiation of cardiomyocytes has been induced through the first organizational process is cultured for 2 days by adding BMP4 at a concentration of 20 ng / ml to 40 ng / ml, VEGF (Vascular endothelial growth factor) at a concentration of 20 ng / ml to 40 ng / ml, and FGF2 (Fibroblast Growth Factor 2) at a concentration of 20 ng / ml to 40 ng / ml to the RPMI 1640 medium used in the first organizational process, and a second organizational process is performed to induce differentiation of fibroblasts and endothelial cells.

[0048] Next, a third organizational process is performed in which the constructs in which differentiation of cardiomyocytes, fibroblasts, and endothelial cells has been induced through a secondary organizational process are cultured for 2 days in RPMI 1640 medium supplemented with B-27 Supplement excluding vitamin A, L-Ascorbic acid at a concentration of 40 μg / ml to 60 μg / ml, BMP4 at a concentration of 20 ng / ml to 40 ng / ml, VEGF (Vascular endothelial growth factor) at a concentration of 20 ng / ml to 40 ng / ml, and FGF2 (Fibroblast Growth Factor 2) at a concentration of 20 ng / ml to 40 ng / ml.

[0049] Finally, the constructs that have completed culture through the third organizational process are cultured for 2 days in RPMI 1640 medium supplemented with B-27 Supplement (excluding Vitamin A) except for L-Ascorbic acid, BMP4 at a concentration of 20 to 40 ng / ml, VEGF (Vascular endothelial growth factor) at a concentration of 20 to 40 ng / ml, and FGF2 (Fibroblast Growth Factor 2) at a concentration of 20 to 40 ng / ml, to achieve the fourth organizational process of self-organization of cardiac organoids through differentiation.

[0050] After completing the cardiac organoid self-organization stage (Stage A-4) through a series of detailed processes, cardiac organoids are formed by self-organizing cardiomyocytes, fibroblasts, and endothelial cells through differentiation.

[0051] Finally, the cardiac organoid maturation stage (Stage A-5) is performed by culturing the cardiac organoids, which have been self-organized to include cardiomyocytes, fibroblasts, and endothelial cells through the cardiac organoid self-organization stage (Stage A-4), in a maturation medium for 10 to 20 days to proceed with maturation.

[0052] Here, the medium for maturation of cardiac organoids corresponds to RPMI 1640 medium supplemented with B-27 Supplement excluding Vitamin A, VEGF (Vascular endothelial growth factor) at a concentration of 20 ng / ml to 40 ng / ml, FGF2 (Fibroblast Growth Factor 2) at a concentration of 20 ng / ml to 40 ng / ml, and SB431542 as a component for suppressing TGF-β (Transforming Growth Factor-β) signaling at a concentration of 10 ng / ml to 15 ng / ml.

[0053] As a result, human induced pluripotent stem cells (hiPSCs) were derived, and through embryoid bodies (EBs), differentiated into mesoderm and endoderm cells on Day 6, then differentiated into cardiomyocytes, fibroblasts, and endothelial cells on Day 10 into a self-organized cardiac organoid state, and finally, the mature cardiac organoid state on Day 24 could be confirmed.

[0054] Above all, after completing the cardiac organoid maturation stage (S150) through a series of detailed processes, self-organized cardiac organoids are prepared through differentiation with a cell number ratio of cardiomyocytes, fibroblasts, and endothelial cells of 55 to 65:15 to 30:15, which is very close to the cell number ratio of 6:2:1.5 of cardiomyocytes, fibroblasts, and endothelial cells that constitute the actual human heart.

[0055]

[0056] (2) Ischemic state induction stage<S120, B단계>

[0057] In this step, the human pluripotent stem cell-derived cardiac organoids prepared through the previously performed cardiac organoid preparation step (S110) are cultured in RPMI 1640 medium with cobalt(II) chloride (CoCl2) added and glucose excluded to simulate ischemic conditions.

[0058] Specifically, in each well of the plate on which the culture is performed, the human pluripotent stem cell-derived cardiac organoids prepared through the cardiac organoid preparation step (S110) are positioned, and a culture medium corresponding to RPMI 1640 medium with cobalt (II) chloride added at a concentration of 50 μM to 100 μM and glucose excluded is dispensed into each well at 2 to 4 ml (most preferably 3 ml).

[0059] Following this, the plate in which the human pluripotent stem cell-derived cardiac organoids and culture medium have been dispensed into each well is cultured in a cell incubator with a temperature environment of 35°C to 40°C (most preferably 37°C) and a CO2 environment of 5% for 5 to 7 hours (most preferably 6 hours), and the results are as shown in Fig. 2.

[0060] As shown in Fig. 2, compared to the 'Control' that did not go through this step (S120), the appearance of the heart organoids in the 'Ischemia' that went through this step (S120) is different because the ischemic condition is simulated.

[0061] Furthermore, to confirm whether the human pluripotent stem cell-derived cardiac organoids that went through this step (S120) were ready to simulate ischemic conditions by inducing ischemia, the normal group (Control) that did not go through this step (S120) and the experimental group (Ischemia) that went through this step (S120) were divided into the following groups: the expression level of HIF-1α (Hypoxia-inducible factor 1-alpha), a hypoxia-inducible factor, was confirmed through Western blot.

[0062] First, to prepare Western blot samples, collect the cardiac organoids from the normal and experimental groups in a 1.5 ml Eppendorf tube, remove the remaining culture medium, wash once with DPBS, and add cell lysis buffer in an amount 2 to 3 times the sample pellet size after removing DPBS.

[0063] Here, the cell lysis buffer is made by diluting the original solution with distilled water at a volume ratio of 1:10, and then adding protein-decomposing enzyme and phosphatase inhibitor at a volume ratio of 1:100.

[0064] All subsequent steps are carried out on ice to prevent protein denaturation. To effectively dissolve the proteins in the organoids, the cells are sonicated 5 to 10 times and centrifuged at 13,000 rpm for 30 minutes to extract the proteins.

[0065] Additionally, the supernatant containing the protein is transferred to a new 1.5 ml Eppendorf tube, and then quantified using the Bradford assay to ensure that 20 μg of protein is present in 20 μl per sample to perform protein quantification.

[0066] In addition, by treating the sample buffer reagent with a volume ratio of 1:4 per sample and using a heat block to place it at 90℃ for 5 minutes, the tertiary and quaternary structures of the protein are transformed into linear structures. After loading 20㎕ of the prepared sample into a 1.0 mm thick mini protein gel containing 4 to 12% Bis-Tris and adding MES SDS running buffer, it is run under electrophoresis at 200 V for 25 minutes.

[0067] Next, to transfer the proteins on the gel to a nitrocellulose membrane, remove the plastic cover around the mini protein gel, place the gel on the membrane, and perform the transfer process at 15 to 20 V for 7 minutes. To wash the membrane, wash it three times using TBS-T buffer.

[0068] Additionally, to prevent contamination of the non-transferred portion of the membrane with other proteins (non-specific protein binding), 10 ml of TBS-T buffer with 5% skim milk was added to each membrane for 1 hour, and the membrane was washed three times with TBS-T buffer to remove any skim milk remaining on the membrane.

[0069] And in order to confirm the expression of HIF-1α, a hypoxia-induced factor among the proteins transferred to the membrane, the primary antibodies GAPDH and HIF-1α antibodies were each diluted in a volume ratio of 1:1000 in TBS-T buffer containing 5% BSA, dispensed at 10 ml per membrane, and treated on a shaker at 4°C at a speed of 70 rpm for 24 hours. After removing the solution containing the primary antibody, the membrane was washed three times using TBS-T buffer.

[0070] Next, to bind the secondary antibody that specifically binds to the primary antibody, the secondary antibody, Rabbit immunoglobulin G, was diluted in a volume ratio of 1:2000 in TBS-T buffer containing 5% BSA, dispensed at 10 ml per membrane, and reacted for 1 hour on a shaker at room temperature at a speed of 70 rpm. After removing the solution containing the secondary antibody, the membrane was washed three times using TBS-T buffer.

[0071] Finally, to detect the presence of the target protein by adding a substrate for the enzyme, Western peroxide reagent and Western luminol / enhancer reagent were reacted in a 1:1 volume ratio. The membrane was evenly wetted with this solution about 10 to 20 times, and then photographed using a Chemi doc (FUSION SOLO 6S) device. Based on the photographed images, the protein expression level was quantified using the Image J program, and the difference between the normal group and the experimental group was represented in a graph as shown in Figure 3.

[0072] As shown in Figure 3, it can be seen that the expression level of HIF-1α (Hypoxia-inducible factor 1-alpha), a hypoxia-inducible factor, increased by approximately 15 times in the experimental group compared to the normal group, and as a result, it can be seen that the experimental group (Ischemia) that went through this step (S120) simulated the ischemic condition of adults.

[0073]

[0074] (3) Reperfusion injury induction stage<S130, C단계>

[0075] In this step, human pluripotent stem cell-derived cardiac organoids, which have undergone an ischemic condition simulation through the previously performed ischemic condition induction step (S120), are cultured in RPMI 1640 medium supplemented with D-glucose and calcium chloride (CaCl2) to simulate reperfusion injury.

[0076] First, in this step (S130), human pluripotent stem cell-derived cardiac organoids, which have undergone ischemic condition simulation through the previously performed ischemic condition induction step (S120), are placed in each well of a plate on which culture is performed, and then 2 to 4 ml (most preferably 3 ml) of a culture solution corresponding to RPMI 1640 medium supplemented with D-glucose at a concentration of 23 to 25 mM and calcium chloride (CaCl2) at a concentration of 1.8 to 2 mM is dispensed into each well.

[0077] Next, a process (Step C-1) is performed in which the plate, in which the human pluripotent stem cell-derived cardiac organoids and culture medium, in which the ischemic condition is simulated, are dispensed into each well, is cultured in a cell incubator with a temperature environment of 35°C to 40°C (most preferably 37°C) and a CO2 environment of 5% for 46 to 50 hours (most preferably 48 hours).

[0078] Next, after 48 hours have passed, 0.5 ml to 1.5 ml (most preferably 1 ml) of RPMI 1640 medium containing D-glucose at a concentration of 23 mM to 25 mM and calcium chloride (CaCl2) at a concentration of 1.8 mM to 2 mM is added to the culture medium corresponding to the RPMI 1640 medium filled in each well of the plate, and then the plate is incubated again in a cell incubator with a temperature environment of 35°C to 40°C (most preferably 37°C) and a CO2 environment of 5% for 22 to 26 hours (most preferably 24 hours) (Step C-2).

[0079] Finally, to confirm whether the human pluripotent stem cell-derived cardiac organoids that went through this step (S130) were ready to simulate the reperfusion injury state by inducing reperfusion injury, the normal group (Control) that did not go through the ischemic condition induction step (S120), the first experimental group (Ischemia) that went through the ischemic condition induction step (S120), the second experimental group (IR 24h) that went through the 24-hour point of this step (S130) (part of step C-1), the third experimental group (IR 48h) that went through the 48-hour point of this step (S130) (step C-1), and the fourth experimental group (IR 72h) that went through the 72-hour point of this step (S130) (step C-2) were divided into groups, and the expression level of cleaved caspase-3, an apoptosis marker, was confirmed through Western blot to confirm cell death.

[0080] Here, caspase, which acts as an important regulatory factor in inducing apoptosis, exists in an inactive state within the cell and is activated (cleaved caspase) by external stimuli.

[0081] First, to prepare Western blot samples, the cardiac organoids from each of the normal group and experimental groups 1 to 4 were collected in a 1.5 ml Eppendorf tube, the remaining culture medium was removed, and the tubes were washed once with DPBS. After removing the DPBS, cell lysis buffer was added in an amount 2 to 3 times the sample pellet size.

[0082] Here, the cell lysis buffer is made by diluting the original solution with distilled water at a volume ratio of 1:10, and then adding protein-decomposing enzyme and phosphatase inhibitor at a volume ratio of 1:100.

[0083] All subsequent steps are carried out on ice to prevent protein denaturation. To effectively dissolve the proteins in the organoids, the cells are sonicated 5 to 10 times and centrifuged at 13,000 rpm for 30 minutes to extract the proteins.

[0084] Additionally, the supernatant containing the protein is transferred to a new 1.5 ml Eppendorf tube, and then quantified using the Bradford assay to ensure that 20 μg of protein is present in 20 μl per sample to perform protein quantification.

[0085] In addition, by treating the sample buffer reagent with a volume ratio of 1:4 per sample and using a heat block to place it at 90℃ for 5 minutes, the tertiary and quaternary structures of the protein are transformed into linear structures. After loading 20㎕ of the prepared sample into a 1.0mm thick mini protein gel containing 4 to 12% Bis-Tris and adding MES SDS running buffer, it is lowered using an electrophoresis device at 200V for 25 minutes.

[0086] Next, to transfer the proteins on the gel to a nitrocellulose membrane, remove the plastic cover around the mini protein gel, place the gel on the membrane, and perform the transfer process at 15 to 20 V for 7 minutes. To wash the membrane, wash it three times using TBS-T buffer.

[0087] Additionally, to prevent contamination of the non-transferred portion of the membrane with other proteins (non-specific protein binding), 10 ml of TBS-T buffer with 5% skim milk was added to each membrane for 1 hour, and the membrane was washed three times with TBS-T buffer to remove any skim milk remaining on the membrane.

[0088] And to confirm the expression of phosphorylated cleaved caspase-3 among the proteins transferred to the membrane, the primary antibody GAPGH was diluted in a volume ratio of 1:1000 in TBS-T buffer containing 5% BSA, and caspase-3 and cleaved caspase-3 were each diluted in a volume ratio of 1:500, dispensed at 10 ml per membrane, and treated on a shaker at 4°C and 70 rpm for 24 hours.

[0089] Also, to wash the membrane after removing the solution containing the primary antibody, wash 3 times using TBS-T buffer. To bind the secondary antibody that specifically binds to the primary antibody, dilute the secondary antibody, Rabbit immunoglobulin G, in a volume ratio of 1:2000 in TBS-T buffer containing 5% BSA, dispense 10㎖ per membrane, and react on a shaker at room temperature at a speed of 70 rpm for 1 hour. After removing the solution containing the secondary antibody, wash the membrane 3 times using TBS-T buffer.

[0090] Next, to detect the presence of the target protein by adding a substrate for the enzyme, 500 ㎕ of Western peroxide reagent and Western luminol / enhancer reagent are reacted at a volume ratio of 1:1.

[0091] Finally, the solution was evenly applied to the membrane about 10 to 20 times, and then images were taken using a Chemi doc (FUSION SOLO 6S) device. Based on the images taken, the amount of protein expression was quantified using the Image J program, and the differences between the normal group and the first to fourth experimental groups were graphed. The results are as shown in Figure 4.

[0092] As shown in Figure 4, it can be seen that the expression level of HIF-1α (Hypoxia-inducible factor 1-alpha), a hypoxia-inducible factor, increased by about 15 times in the experimental group compared to the normal group, and the protein expression level of cleaved caspase-3, which corresponds to a state in which caspase, which acts as an important regulatory factor in inducing cell death, is activated by external stimuli, was significantly increased in human pluripotent stem cell-derived cardiac organoids that went through this step (S130) corresponding to experimental groups 2 to 4.

[0093] In addition, to confirm whether the human pluripotent stem cell-derived cardiac organoids that went through this step (S130) were ready to simulate a reperfusion injury state by inducing reperfusion injury, the normal group (Control) that did not go through the ischemic condition induction step (S120) and the test group (IR) that went through this step (S120) were divided into two groups and compared using an ELISA experiment to see whether acute myocardial infarction markers were released outside the cells.

[0094] Here, the samples were cardiac organoid cultures from the normal group (Control) and the test group (IR), and each specific marker was measured using an ELISA kit according to the manufacturer's recommended instructions.

[0095] Additionally, the cell culture solution was concentrated by centrifugation at 3000 rpm for 30 minutes using an Amicon Ultra filter, and the concentrated sample was placed on a plate coated with each antigen and reacted at room temperature for 2 hours and 30 minutes.

[0096] Next, wash three times using the washing solution included in the kit, treat the detection antibody at room temperature for 1 hour, and then wash three times using the washing solution after removing the detection antibody solution.

[0097] Following this, the secondary antibody, HRP, is reacted at room temperature for 45 minutes, then removed, and the TMB solution is treated to undergo a color development process, after which the reaction is stopped using a stop solution.

[0098] Finally, to detect the degree of color development, the absorbance at 450 nm was measured using a microplate reader device, and the value was normalized to show the difference between the normal group (Control) and the test group (IR) in a graph, as shown in Fig. 5.

[0099] As shown in Figure 5, in the test group (IR), all acute myocardial infarction markers (Creatine kinase M; CKM, Cardiac troponin T; cTnI, Myoglobin; MB) were released extracellularly and increased compared to the normal group (Control).

[0100] Furthermore, in order to confirm whether the human pluripotent stem cell-derived cardiac organoids that went through this step (S130) were ready to simulate a reperfusion injury state by inducing reperfusion injury, the normal group (Control) that did not go through the ischemic condition induction step (S120) and the test group (IR) that went through this step (S120) were divided into messenger RNA (mRNA) level inflammation-related markers (Interleukin 1 alpha; IL1α, Interleukin 1 beta; IL1β, Interleukin 6; IL6, Interleukin 8; IL8, Cyclooxygenase; COX) were confirmed through real-time polymerase chain reaction analysis, and the results are as shown in Figures 6 and 7.

[0101] At the same time, the expression levels of NF-ĸB, a protein involved in inflammatory response and immune system regulation, and ERK, JNK, and p38, proteins related to the MAPK signaling pathway, a cellular inflammation signaling system induced by external stimuli, were confirmed through Western blot.

[0102] First, to prepare Western blot samples, collect the cardiac organoids of the normal and test groups in a 1.5 ml Eppendorf tube, remove the remaining culture medium, wash once with DPBS, and add cell lysis buffer in an amount 2 to 3 times the sample pellet size after removing DPBS.

[0103] Here, the cell lysis buffer is made by diluting the original solution with distilled water at a volume ratio of 1:10, and then adding protein-decomposing enzyme and phosphatase inhibitor at a volume ratio of 1:100.

[0104] All subsequent steps are carried out on ice to prevent protein denaturation. To effectively dissolve the proteins in the organoids, the cells are sonicated 5 to 10 times and centrifuged at 13,000 rpm for 30 minutes to extract the proteins.

[0105] Additionally, the supernatant containing the protein is transferred to a new 1.5 ml Eppendorf tube, and then quantified using the Bradford assay to ensure that 20 μg of protein is present in 20 μl per sample to perform protein quantification.

[0106] In addition, by treating the sample buffer reagent with a volume ratio of 1:4 per sample and using a heat block to place it at 90℃ for 5 minutes, the tertiary and quaternary structures of the protein are transformed into linear structures. After loading 20㎕ of the prepared sample into a 1.0mm thick mini protein gel containing 4 to 12% Bis-Tris and adding MES SDS running buffer, it is lowered using an electrophoresis device at 200V for 25 minutes.

[0107] Next, to transfer the proteins on the gel to a nitrocellulose membrane, remove the plastic cover around the mini protein gel, place the gel on the membrane, and perform the transfer process at 15 to 20 V for 7 minutes. To wash the membrane, wash it three times using TBS-T buffer.

[0108] Additionally, to prevent contamination of the non-transferred portion of the membrane with other proteins (non-specific protein binding), 10 ml of TBS-T buffer with 5% skim milk was added to each membrane for 1 hour, and the membrane was washed three times with TBS-T buffer to remove any skim milk remaining on the membrane.

[0109] Then, dilute the primary antibodies GAPGH 1:1000, NF-ĸB 1:500, pNF-ĸB 1:500, p38 1:1000, pp38 1:1000, ERK 1:1000, pERK 1:1000, JNK 1:500, and pJNK 1:500 in TBS-T buffer containing 5% BSA, dispense 10 ml per membrane, and treat at 70 rpm on a shaker at 4°C for 24 hours. After removing the solution containing the primary antibody, wash the membrane three times using TBS-T buffer.

[0110] Next, in order to bind the secondary antibody that specifically binds to the primary antibody, the secondary antibody, Rabbit immunoglobulin G, is diluted 1:2000 in TBS-T buffer containing 5% BSA, 10 ml is dispensed per membrane, and the reaction is performed on a shaker at room temperature at a speed of 70 rpm for 1 hour. After removing the solution containing the secondary antibody, the membrane is washed three times using TBS-T buffer.

[0111] Finally, to detect the presence of the target protein by adding a substrate for the enzyme, Western peroxide reagent and Western luminol / enhancer reagent were reacted at a volume ratio of 1:1, 500 ㎕ each, and the membrane was evenly wetted with this solution about 10 to 20 times, and then photographed using a Chemi doc (FUSION SOLO 6S) device, and the protein expression level was quantified using the Image J program based on the photographed image, and the results of the graph showing the difference between the normal group and the experimental group are as shown in Figures 8 to 11.

[0112] As shown in Figures 6 and 7, it can be seen that the expression level of inflammation-related markers (Interleukin 1 alpha; IL1α, Interleukin 1 beta; IL1β, Interleukin 6; IL6, Interleukin 8; IL8, Cyclooxygenase; COX) in the test group (IR) was significantly increased compared to the normal group (Control).

[0113] In addition, as shown in Figures 8 to 11, in the test group (IR), compared to the normal group (Control), the expression levels of NF-ĸB, a protein involved in the regulation of the immune system and the immune response, and ERK, JNK, and p38, proteins related to the MAPK signaling pathway, a cellular inflammation signal transmission system caused by external stimuli, were all significantly increased.

[0114] Considering these results comprehensively, it can be seen that the fourth experimental group (IR 72h) that progressed (stage C-2) up to the 72-hour point of this stage (S130) and the test group (IR) that went through this stage (S130) simulated the ischemia-reperfusion injury state of adults.

[0115]

[0116] (4) Heart failure induction stage<S140, D단계>

[0117] In this step, human pluripotent stem cell-derived cardiac organoids, which have undergone the previous reperfusion injury induction step (S130) to simulate ischemia-reperfusion injury, are cultured in RPMI 1640 medium supplemented with TGF-β (Transforming Growth Factor-β) and B-27 Supplement to simulate fibrosis caused by heart failure.

[0118]

[0119] Specifically, in each well of the plate where culture is performed, human pluripotent stem cell-derived cardiac organoids that have undergone the reperfusion injury state induction step (S130) are placed, and a culture medium corresponding to RPMI 1640 medium supplemented with TGF-β (Transforming Growth Factor-β) and B-27 Supplement at a concentration of 5 ng / ml to 10 ng / ml is dispensed into each well.

[0120] Following this, the plate in which the human pluripotent stem cell-derived cardiac organoids and the culture medium simulating the ischemia-reperfusion injury condition were dispensed into each well was cultured in a cell incubator with a temperature environment of 35°C to 40°C (most preferably 37°C) and a CO2 environment of 5% for 6 to 8 days (most preferably 7 days), with the culture medium replaced every 2 days, and the results are as shown in Fig. 12.

[0121] As shown in Fig. 12, compared to the normal group (Control) that did not undergo the ischemic condition induction step (S120), the experimental group (IR-Fibrosis) that underwent this step (S140) showed a different appearance of the cardiac organoids, as the fibrosis state that progresses to heart failure after ischemia-reperfusion injury was simulated.

[0122] More specifically, to confirm whether human pluripotent stem cell-derived cardiac organoids that went through this step (S140) were ready to simulate the fibrotic state that progresses to heart failure after ischemia-reperfusion injury, time-lapse polymerase chain reaction analysis was performed on the experimental group (IR-Fibrosis) that went through this step (S140) compared to the normal group (Control) that did not go through the ischemic state induction step (S120) to confirm fibrosis and collagen-related markers at the messenger RNA (mRNA) level.

[0123] First, to prepare RNA samples, cardiac organoids from the normal group (Control) and experimental group (IR-Fibrosis) were transferred to a 1.5 ml Eppendorf tube, the culture medium was removed, and then washed once with DPBS.

[0124] Following this, after removing DPBS, 1 ml of Trizol is dispensed per sample and the cells are disrupted by sonication about 4 to 5 times using an ultrasonic disruptor. 200 μl of chloroform is added to the sample, mixed for 15 seconds using a shaker, and centrifuged at 15,000 rpm for 30 minutes at 4°C.

[0125] Next, transfer the upper, clear layer to a new 1.5 ml Eppendorf tube, add an equal volume of isopropanol, and mix the sample by inverting. Incubate at room temperature for 15 minutes, then centrifuge at 15,000 rpm for 15 minutes at 4°C.

[0126] Additionally, after removing the supernatant, add 1 ml of 75% ethanol per sample and centrifuge at 7,500 rpm for 5 minutes at 4°C. This process is repeated twice, and after removing the supernatant, the pellet is dissolved in DEPC-treated water and quantified using a microplate reader.

[0127] Following this, the RNA concentration per sample was adjusted to 500 ng, and then complementary DNA (cDNA) was synthesized. Using the synthesized cDNA as a template, primers for fibrosis-related genes ACTA2, POSTN, Vimentin, and MMP2 and primers for collagen-related genes COL1A1, COL1A2, and COL3A1 were added at a concentration of 10 pmol / ㎕, respectively, to perform polymerase chain reaction.

[0128] Here, the mRNA expression values ​​are normalized to the housekeeping gene GAPDH and compared between the normal group (Control) and the experimental group (IR-Fibrosis), and the results are as shown in Figures 13 to 15.

[0129] As shown in Figures 13 and 15, it can be seen that markers related to fibrosis (Actin alpha 2; ACTA2, Periostin; POSTN, Vimentin, Matrix metalloproteinase-2; MMP2) and collagen (Collagen type I alpha 1 chain; COL1A1, Collagen type I alpha 2; COL1A2, Collagen type III alpha 1 chain; COL3A1) were significantly increased in the experimental group (IR-Fibrosis) compared to the normal group (control).

[0130] Furthermore, to confirm whether the human pluripotent stem cell-derived cardiac organoids that went through this step (S140) were ready to simulate the fibrotic state that progresses to heart failure after ischemia-reperfusion injury, the degree of fibrosis was confirmed using Masson's trichrome staining, which distinguishes collagen fibers, in the experimental group (IR-Fibrosis) that went through this step (S140) compared to the normal group (Control) that did not go through the ischemic state induction step (S120).

[0131] First, the cardiac organoids of the normal group (Control) and the experimental group (IR-Fibrosis) were transferred to a 1.5 ml Eppendorf tube, the culture medium was removed, and after removing the culture medium, 1 ml of DPBS was dispensed and washed twice.

[0132] Next, after removing DPBS, add 1 ml of 4% PFA solution and leave at 4°C for about 15 minutes. After removing 4% PFA, eosin staining is performed to identify organoids and paraffin blocks are made.

[0133] Next, the paraffin block produced using a cutter is cut into a certain thickness, a sample is prepared on a slide glass, and the prepared sample is treated twice for 5 minutes each using xylene to remove paraffin.

[0134] Additionally, to hydrate the sample, it is treated with 100%, 95%, and 70% ethanol for 5 minutes each, and after washing with double-distilled water, it is treated with Weigert's iron hematoxylin for 10 minutes each to stain the cell nuclei.

[0135] Additionally, after washing with running water for 10 minutes, the cells were treated with Biebrich scarlet-acid fuchsin solution for 15 minutes for cytoplasm and muscle fiber staining, and after removing the solution, they were treated with phosphomolybdic-phosphotungstic acid solution for 15 minutes, and then treated with aniline blue for 10 minutes without washing.

[0136] Finally, images were obtained by observing the blue-stained collagen fibers of the samples from the normal group (Control) and the experimental group (IR-Fibrosis) using an optical microscope, and the results are as shown in Fig. 16.

[0137] As shown in Figure 16, it can be seen that the inwardly activated agar fibers are observed in the experimental group (IR-Fibrosis) compared to the normal group (control).

[0138] Considering these results comprehensively, the test group (IR-Fibrosis) that went through this step (S140) simulates the fibrotic state that progresses to heart failure after ischemia-reperfusion injury in adults, and can be utilized as an in vitro disease-simulating model for screening new drugs for myocardial infarction and heart failure in adults and for elucidating the cause of the disease mechanism.

[0139] The embodiments disclosed in the present invention are intended to illustrate, not limit, the technical concepts of the present invention. These embodiments do not limit the scope of the technical concepts of the present invention. The scope of protection should be interpreted according to the following claims, and all technical concepts within the scope equivalent thereto should be construed as being included within the scope of the present invention.

Claims

1. Step A: culturing human pluripotent stem cells (hPSCs) to prepare self-organized human pluripotent stem cell-derived cardiac organoids containing cardiomyocytes, fibroblasts, and endothelial cells; The human pluripotent stem cell-derived cardiac organoids prepared through the above step A were treated with cobalt(II) chloride (CoCl) to simulate ischemic conditions. 2 Stage B: culturing in RPMI 1640 medium supplemented with cobalt(II) chloride and excluding glucose; Human pluripotent stem cell-derived cardiac organoids that simulated ischemic conditions through the above step B were treated with D-glucose and calcium chloride (CaCl) to simulate reperfusion injury conditions. 2 C stage, cultured in RPMI 1640 medium supplemented with calcium chloride; and A method for simulating a fibrotic condition due to heart failure, characterized by comprising a step D in which human pluripotent stem cell-derived cardiac organoids, which have undergone the above step C to simulate ischemia-reperfusion injury, are cultured in RPMI 1640 medium supplemented with TGF-β (Transforming Growth Factor-β) and B-27 Supplement. A method for producing an in vitro disease-mimetic model of adult acute myocardial infarction and heart failure induced by ischemia-reperfusion injury using human pluripotent stem cell-derived cardiac organoids.

2. In paragraph 1, The above step A is, Step A-1 of culturing human pluripotent stem cells, which are at least one of human embryonic stem cells (hESC) or human induced pluripotent stem cells (hiPSC), to prepare embryonic bodies having a diameter of 100 μm to 130 μm; Step A-2, in which the embryoid body prepared through the above step A-1 is cultured until it has a diameter of 200 ㎛ to 250 ㎛; Step A-3, in which human pluripotent stem cell-derived embryoid bodies having a diameter of 200 μm to 250 μm are differentiated into a structure including mesoderm cells and endoderm cells through the above step A-2; Step A-4, in which the construct including the differentiated mesodermal cells and endoderm cells through the above step A-3 is differentiated into a self-organized cardiac organoid including cardiomyocytes, fibroblasts and endothelial cells; and A step A-5 is characterized by culturing and maturing a self-organized cardiac organoid differentiated through the above step A-4. A method for producing an in vitro disease-mimetic model of adult acute myocardial infarction and heart failure induced by ischemia-reperfusion injury using human pluripotent stem cell-derived cardiac organoids.

3. In paragraph 2, The above step B is characterized by being a step of culturing the human pluripotent stem cell-derived cardiac organoid prepared through the above step A in 2 to 4 ml of RPMI 1640 medium containing cobalt (II) chloride at a concentration of 50 μM to 100 μM and excluding glucose, for 5 to 7 hours using a cell incubator with a temperature environment of 35°C to 40°C. A method for producing an in vitro disease-mimetic model of adult acute myocardial infarction and heart failure induced by ischemia-reperfusion injury using human pluripotent stem cell-derived cardiac organoids.

4. In paragraph 2, The above step C is, The human pluripotent stem cell-derived cardiac organoids that passed through the above step B were cultured in a solution containing D-glucose at a concentration of 23 mM to 25 mM and calcium chloride (CaCl) at a concentration of 1.8 mM to 2 mM. 2 C-1 stage, culturing for 46 to 50 hours in a cell incubator with a temperature environment of 35 to 40°C in 2 to 4 ml of RPMI 1640 medium supplemented with calcium chloride; and In the RPMI 1640 medium used in the above step C-1, D-glucose (D-glucose) at a concentration of 23 mM to 25 mM and calcium chloride (CaCl) at a concentration of 1.8 mM to 2 mM were added. 2 , Calcium chloride) is added to 0.5 ml to 1.5 ml of RPMI 1640 medium, and then the human pluripotent stem cell-derived cardiac organoids that have undergone the C-1 step are cultured for 22 to 26 hours using a cell incubator with a temperature environment of 35°C to 40°C. A method for producing an in vitro disease-mimetic model of adult acute myocardial infarction and heart failure induced by ischemia-reperfusion injury using human pluripotent stem cell-derived cardiac organoids.

5. In paragraph 2, The above step D is characterized in that it is a step of culturing the human pluripotent stem cell-derived cardiac organoid that has gone through the above step C in RPMI 1640 medium supplemented with TGF-β (Transforming Growth Factor-β) and B-27 Supplement at a concentration of 5 ng / ㎖ to 10 ng / ㎖ using a cell incubator with a temperature environment of 35℃ to 40℃ for 6 to 8 days. A method for producing an in vitro disease-mimetic model of adult acute myocardial infarction and heart failure induced by ischemia-reperfusion injury using human pluripotent stem cell-derived cardiac organoids.

6. An in vitro disease-mimicking model produced by a method for producing an in vitro disease-mimicking model in which adult acute myocardial infarction and heart failure are induced by ischemia-reperfusion injury using a human pluripotent stem cell-derived cardiac organoid according to any one of claims 1 to 5.

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