Method for producing cardiac organoids derived from human pluripotent stem cells and cardiac organoids produced thereby

Abstract

The present invention relates to a method for producing cardiac organoids derived from human pluripotent stem cells, comprising the steps of: Step A: culturing human pluripotent stem cells to form embryoid bodies at a concentration of 100-130 μm; Step B: culturing the embryoid bodies obtained in Step A until they reach a diameter of 200-250 μm; Step C: differentiating the 200-250 μm diameter human pluripotent stem cell-derived embryoid bodies obtained in Step B into constructs containing mesodermal and endodermal cells; Step D: differentiating the constructs containing mesodermal and endodermal cells differentiated in Step C into cardiac organoids that have self-organized to contain cardiomyocytes, fibroblasts, and vascular endothelial cells; and Step E: culturing and maturing the cardiac organoids differentiated and self-organized in Step D. The vascular endothelial cells obtained through the maturation process in Step E are characterized by self-organization of cardiomyocytes, fibroblasts, and vascular endothelial cells in a cell number ratio of 55-65:15 to 30:15. [Representative diagram] Figure 1

Description

[Technical field]

[0001] The present invention relates to a method for producing cardiac organoids derived from human pluripotent stem cells and cardiac organoids derived from human pluripotent stem cells produced thereby. [Background technology]

[0002] With the recent continuous development of technology, methods have been developed to differentiate most of the cells that make up the human body from human pluripotent stem cells (hPSCs). However, cells differentiated through 2D culture are significantly less mature and less functional than actual human cells, which means there are clear limitations to their use in drug toxicity evaluation, drug screening, cell therapy, etc.

[0003] Therefore, various cell differentiation and culture related technologies are being researched in order to produce cells that have reached the maturity level of cells that make up an actual human and have sufficient functionality. In particular, research and development is being conducted around the world on organoids that reproduce the morphology of an actual organ in which various cells exist as a group, rather than as a single cell, and that are also fully functional.

[0004] Organoids that can mimic the structure and function of actual human organs can be produced by continuous cell division and differentiation from organ stem cells such as the intestine and brain, which are believed to have organ-specific stem cells. However, in the case of the heart, which does not have organ-specific stem cells, there is no room for applying such methods. Therefore, the only method proposed for producing organoids is to differentiate the various cells that make up the heart separately and then mix them together.

[0005] In this regard, a prior art document corresponding to the conventional technology for producing cardiac organoids is U.S. Patent Publication No. 2020-0283735, entitled "Organoid and method for producing the same" (hereinafter referred to as "Conventional Technology").

[0006] However, existing methods for producing cardiac organoids, including those in the prior art, simply involve mixing differentiated cardiac cells or culturing them in an extracellular matrix consisting of specific components. As a result, they have the limitation of overlooking the fact that the property of the various cells that make up the heart developing while interacting with each other from the early stages of development affects cardiac function.

[0007] As a result, unless the various cells that make up the heart are able to differentiate simultaneously and self-organize through interactions from the early stages of development, the state will be insufficient to reproduce the structure and function of the actual heart.Therefore, when developing new drugs, it is problematic that the state is not sufficient to perform drug screening and cardiac toxicity evaluation through disease modeling and derive significant results. Summary of the Invention [Problem to be solved by the invention]

[0008] The present invention has been made to solve the above-mentioned problems, and its purpose is to provide a method for producing cardiac organoids in which various cells that constitute the heart are simultaneously differentiated from human pluripotent stem cells, self-organize through interactions from the early stages of development, and are cultured and differentiated in a state sufficient to reproduce the structure and function of an actual heart. [Means for solving the problem]

[0009] In order to achieve the above object, the method for producing cardiac organoids derived from human pluripotent stem cells according to the present invention includes the steps of: Step A: culturing human pluripotent stem cells (hPSCs) to form embryonic bodies having a diameter of 100 to 130 μm; Step B: culturing the embryonic bodies obtained in Step A until they reach a diameter of 200 to 250 μm; Step C: differentiating the human pluripotent stem cell-derived embryonic bodies having a diameter of 200 to 250 μm obtained in Step B into constructs containing mesodermal cells and endodermal cells; and Step D: differentiating the constructs containing mesodermal cells and endodermal cells differentiated in Step C into cardiomyocytes, fibroblasts, and vascular endothelial cells. The method includes step D of differentiating cardiac organoids into self-organized cardiac organoids containing cardiac myocytes (cardiomyocytes), fibroblasts, and vascular endothelial cells (vascular endothelial cells) and step E of culturing and maturing the cardiac organoids that have been differentiated and self-organized in step D. The cardiac organoids obtained through the maturation process in step E are characterized in that cardiomyocytes, fibroblasts, and vascular endothelial cells are self-organized in a cell number ratio of 55-65:15-30:15.

[0010] Here, the human pluripotent stem cells in step A are at least one of human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs).

[0011] Furthermore, step A is a step in which human pluripotent stem cells are plated in mTeSR culture medium supplemented with a Rho-associated kinase (ROCK) inhibitor (Y-27632), cultured for one day, and embryoid bodies with a diameter of 100 to 130 μm are formed.

[0012] Furthermore, step B is a step of culturing the embryoid bodies having a diameter of 100 to 130 μm obtained in step A for 2 to 3 days in mTeSR culture medium containing no ROCK inhibitor (Y-27632) to form human pluripotent stem cell-derived embryoid bodies having a diameter of 200 to 250 μm.

[0013] Furthermore, step C includes step C-1 of culturing the human pluripotent stem cell-derived embryoid bodies having a diameter of 200 to 250 μm obtained in step B for two days in RPMI1640 medium supplemented with B-27 supplement (minus insulin), CHIR99021, bone morphogenetic protein-4 (BMP4) and activin A, and step C-2 of culturing the human pluripotent stem cell-derived embryoid bodies cultured in step C-1 for two days in RPMI1640 medium supplemented with B-27 supplement (minus insulin), XAV939 and L-ascorbic acid to differentiate them into constructs containing mesodermal cells and endodermal cells.

[0014] Here, step C-1 is a step of culturing the human pluripotent stem cell-derived embryoid bodies having a diameter of 200 to 250 μm obtained in step B for two days in RPMI1640 medium supplemented with B-27 supplement (minus insulin), CHIR99021 at a concentration of 6 to 8 μM as a glycogen synthase kinase-3β (GSK-3β) inhibitor for Wnt signal activation, BMP4 at a concentration of 9 to 10 ng / ml as a component for BMP signal activation, and activin A at a concentration of 8 to 10 ng / ml as a component for transforming growth factor-β (TGF-β) signal activation.

[0015] In addition, step C-2 is a step of culturing the human pluripotent stem cell-derived embryoid bodies cultured in step C-1 for two days in RPMI1640 medium supplemented with B-27 supplement (minus insulin), XAV939 at a concentration of 8 to 12 μM as a component for inhibiting Wnt signaling, and L-ascorbic acid at a concentration of 40 to 60 μg / ml as a component for activating BMP signaling.

[0016] Further, the step D includes a step D-1 of incubating the construct including the mesodermal cells and endodermal cells differentiated in the step C for 2 days in RPMI1640 medium supplemented with B-27 supplement (minus insulin) and L-ascorbic acid at a concentration of 40 to 60 μg / ml to induce differentiation into cardiomyocytes, and a step D-2 of incubating the construct in which differentiation into cardiomyocytes has been induced in the step D-1 in the RPMI1640 medium used in the step D-1, supplemented with BMP4 at a concentration of 20 to 40 ng / ml, vascular endothelial growth factor (VEGF) at a concentration of 20 to 40 ng / ml, and fibroblast growth factor 2 (FGF2) at a concentration of 20 to 40 ng / ml. 2) and incubating for 2 days to induce differentiation of fibroblasts and vascular endothelial cells; step D-3 incubating the construct in which differentiation of cardiomyocytes, fibroblasts and vascular endothelial cells was induced in step D-2 for 2 days in RPMI1640 medium supplemented with B-27 supplement (minus vitamin A), L-ascorbic acid at a concentration of 40 to 60 μg / ml, BMP4 at a concentration of 20 to 40 ng / ml, VEGF at a concentration of 20 to 40 ng / ml and FGF2 at a concentration of 20 to 40 ng / ml for 2 days; and step D-4 incubating the construct cultured in step D-3 for 2 days in RPMI1640 medium supplemented with B-27 supplement (minus vitamin A), BMP4 at a concentration of 20 to 40 ng / ml, VEGF at a concentration of 20 to 40 ng / ml and FGF2 at a concentration of 20 to 40 ng / ml to perform self-organization of cardiac organoids by differentiation.

[0017] Furthermore, step E is a step in which the cardiac organoids that have differentiated in step D-4 and self-organized to contain cardiomyocytes, fibroblasts and vascular endothelial cells are matured by culturing them for 10 to 20 days in RPMI1640 medium supplemented with B-27 supplement (minus vitamin A), VEGF at a concentration of 20 to 40 ng / ml, FGF2 at a concentration of 20 to 40 ng / ml, and SB431542 at a concentration of 10 to 15 ng / ml as a component for inhibiting TGF-β signaling.

[0018] On the other hand, in order to achieve the above-mentioned object, the human pluripotent stem cell-derived cardiac organoid according to the present invention is produced by the above-mentioned method for producing a human pluripotent stem cell-derived cardiac organoid. Effect of the Invention

[0019] The present invention has the following advantages.

[0020] First, it overcomes the limitation of not having cardiac stem cells, and can produce cardiac organoids in which the cardiomyocytes, cardiac fibroblasts, and vascular endothelial cells that make up the heart are simultaneously differentiated from human pluripotent stem cells, self-organize through interactions from the early stages of development, and are in a state sufficient to reproduce the structure and function of an actual heart through culture differentiation.

[0021] Second, cardiac organoids, which are well-equipped to recapitulate cardiac structure and function, can be used for drug screening and cardiac toxicity assessment through disease modeling during drug development, dramatically improving the efficiency of drug development.

[0022] Third, human pluripotent stem cell-derived cardiac organoids can be used as an alternative model to study cardiac-related disease mechanisms, aiding technological advances in the field of disease research. [Brief description of the drawings]

[0023] [Figure 1] 1 is a flowchart of the method for producing cardiac organoids derived from human pluripotent stem cells of the present invention. [Diagram 2] Photographs illustrating the results of observing the changes in the size and number of constituent cells of embryoid bodies due to the embryoid body preparation and culture steps in the method for producing cardiac organoids derived from human pluripotent stem cells of the present invention. [Diagram 3] Photographs illustrating the results of observing the changes in the size and number of constituent cells of embryoid bodies due to the embryoid body preparation and culture steps in the method for producing cardiac organoids derived from human pluripotent stem cells of the present invention. [Figure 4] 1 shows microscopic photographs of the state at each stage during the process of differentiation from human pluripotent stem cells, via embryoid bodies, to cardiac organoids, using the method of the present invention for producing cardiac organoids derived from human pluripotent stem cells. [Diagram 5] 1 is a graph showing the change in diameter size over time during the process of differentiation from human pluripotent stem cells to cardiac organoids via embryoid bodies using the method of the present invention for producing cardiac organoids derived from human pluripotent stem cells. [Figure 6] 1 is a graph showing the change in pulsation ratio by period during the process of differentiation from human pluripotent stem cells to embryoid bodies and then to cardiac organoids using the method of the present invention for producing cardiac organoids derived from human pluripotent stem cells. [Figure 7] 1 shows fluorescent staining photographs of human pluripotent stem cell-derived cardiac organoids produced by the method of the present invention for producing human pluripotent stem cell-derived cardiac organoids. [Figure 8] FIG. 1 is a schematic diagram summarizing the step-by-step culture conditions and periods for each experimental group according to the method for producing cardiac organoids derived from human pluripotent stem cells of the present invention. [Figure 9] 1 is a graph showing the results of flow cytometry for each experimental group of human pluripotent stem cell-derived cardiac organoids produced by the method for producing human pluripotent stem cell-derived cardiac organoids of the present invention. [Figure 10] 1 is a graph showing the results of flow cytometry for each experimental group of human pluripotent stem cell-derived cardiac organoids produced by the method for producing human pluripotent stem cell-derived cardiac organoids of the present invention. [Figure 11]1 is a graph showing the results of gene expression by experimental group for human pluripotent stem cell-derived cardiac organoids produced by the method for producing human pluripotent stem cell-derived cardiac organoids of the present invention. [Figure 12] 1 is a graph showing the results of gene expression by experimental group for human pluripotent stem cell-derived cardiac organoids produced by the method for producing human pluripotent stem cell-derived cardiac organoids of the present invention. [Figure 13] 1 is a graph showing the results of gene expression by experimental group for human pluripotent stem cell-derived cardiac organoids produced by the method for producing human pluripotent stem cell-derived cardiac organoids of the present invention. [Figure 14] 1 shows photographs comparing the results of fluorescent staining of human pluripotent stem cell-derived cardiac organoids by experimental group, the cardiac organoids being produced by the method of the present invention for producing human pluripotent stem cell-derived cardiac organoids. [Figure 15] 1 shows photographs comparing the results of fluorescent staining of human pluripotent stem cell-derived cardiac organoids by experimental group, the cardiac organoids being produced by the method of the present invention for producing human pluripotent stem cell-derived cardiac organoids. [Figure 16] 1 shows photographs and graphs comparing calcium imaging results by experimental group for human pluripotent stem cell-derived cardiac organoids produced by the method for producing human pluripotent stem cell-derived cardiac organoids of the present invention. [Figure 17] 1 shows photographs and graphs comparing calcium imaging results by experimental group for human pluripotent stem cell-derived cardiac organoids produced by the method for producing human pluripotent stem cell-derived cardiac organoids of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0024] Hereinafter, preferred embodiments of the present invention will be described in more detail with reference to the accompanying drawings. However, in order to simplify the description, already known technical parts will be omitted or simplified.

[0025] 1. Description of the method for producing human pluripotent stem cell-derived cardiac organoids Hereinafter, with reference to FIG. 1, the steps of the method for producing cardiac organoids derived from human pluripotent stem cells according to the present invention will be described in detail.

[0026] (1) Embryoid body preparation step<S110、ステップA> In this step (S110), human pluripotent stem cells are cultured to form embryonic bodies having a diameter of 100 to 130 μm.

[0027] Here, the term may refer to human pluripotent stem cells (hPSCs) themselves, but more specifically, it can also be interpreted as cells consisting of at least one of human embryonic stem cells (hESCs) or human induced pluripotent stem cells (hiPSCs).

[0028] Specifically, in the embryoid body preparation step (S110), human pluripotent stem cells in culture are harvested and then cultured in a 6-well plate at 1.5 × 10 6 The cells are plated in mTeSR medium (Stem Cell Technologies, Canada) supplemented with a Rho-associated kinase (ROCK) inhibitor (Y-27632, Tocris Bioscience, UK) at a concentration of 10 μM cells / well.

[0029] When plating human pluripotent stem cells onto 6-well plates, it is important not to coat the plates for cell adhesion, and it is also important not to use Matrigel, which is commonly used in organoid culture; instead, low attachment plates can be used.

[0030] Furthermore, as shown in FIG. 2, after culturing for one day, embryoid bodies consisting of approximately 200 to 500 cells and measuring 100 to 130 μm in diameter were obtained in each well.

[0031] Therefore, 1.5 x 10 cells were placed in each well of a 6-well plate. 6When human pluripotent stem cells are plated, approximately 3,000 to 7,500 embryoid bodies with diameters of 100 to 130 μm are generated after one day of culture.

[0032] (2) Embryoid body culture step<S120、ステップB> In this step (S120), the embryoid bodies formed in the previous embryoid body preparation step (S110) are further cultured until they reach a diameter of 200 to 250 μm.

[0033] Specifically, in the embryoid body culture step (S120), embryoid bodies with a diameter of 100 to 130 μm formed in the embryoid body preparation step (S110) are cultured for 2 to 3 days in mTeSR culture medium to which a ROCK inhibitor (Y-27632) has not been added (additive-free), to obtain human pluripotent stem cell-derived embryoid bodies with a diameter of 200 to 250 μm.

[0034] Here, the culture process using mTeSR culture medium to which the ROCK inhibitor (Y-27632) has not been added (additive-free) is carried out for 2 to 3 days, and it is preferable to replace the culture medium with a new one every day.

[0035] Therefore, as shown in Figure 3, on the first day of culture in the embryoid body preparation step (S110), embryoid bodies with a diameter of 100 to 130 μm are obtained, as shown in the photograph marked "Day-3." When the embryoid culture step (S120) then changes the culture medium conditions and cultures for an additional 2 to 3 days, the diameter gradually increases to 200 to 250 μm, as shown in the photograph marked "Day 0."

[0036] (3) Mesodermal and endodermal cell differentiation steps<S130、ステップC> In this step (S130), a process is carried out in which the human pluripotent stem cell-derived embryoid bodies having a diameter of 200 to 250 μm obtained in the embryoid body culture step (S120) are differentiated into constructs containing mesodermal cells and endodermal cells.

[0037] The mesodermal and endodermal cell differentiation step (S130) is more specifically subdivided and is performed sequentially in two steps. First, a primary differentiation process (step C-1) is performed in which the human pluripotent stem cell-derived embryoid bodies with a diameter of 200 to 250 μm obtained in the embryo-like culture step (S120) are cultured for two days in RPMI1640 medium supplemented with B-27 supplement (minus insulin), CHIR99021, bone morphogenetic protein 4 (BMP4), and activin A.

[0038] Specifically, the medium in which the human pluripotent stem cell-derived embryoid bodies having a diameter of 200 to 250 μm obtained in the embryoid body culture step (S120) are primarily cultured for 2 days corresponds to RPMI1640 medium supplemented with B-27 supplement (minus insulin; Thermo Fisher Scientific, USA), CHIR99021 at a concentration of 6 to 8 μM, BMP4 at a concentration of 9 to 10 ng / ml, and activin A at a concentration of 8 to 10 ng / ml.

[0039] Here, CHIR99021, which is added to the RPMI1640 medium used in the primary differentiation process of the mesoderm and endoderm cell differentiation step (S130), is a glycogen synthase kinase 3β (GSK-3β) inhibitor for Wnt signal activation, and most preferably has a concentration of 6 μM.

[0040] Furthermore, BMP-4, which is added to the RPMI1640 medium used in the primary differentiation process of the mesoderm and endoderm cell differentiation step (S130), is a component for activating BMP signals, and most preferably has a concentration of 10 ng / ml.

[0041] Next, activin A, which is added to the RPMI1640 medium used in the primary differentiation process of the mesoderm and endoderm cell differentiation step (S130), is a component for transforming growth factor β (TGF-β) signal activation, and most preferably has a concentration of 10 ng / ml.

[0042] Then, the human pluripotent stem cell-derived embryoid bodies cultured during the primary differentiation process of the mesoderm and endoderm cell differentiation step (S130) are cultured for an additional two days in RPMI1640 medium supplemented with B-27 supplement (minus insulin), XAV939, and L-ascorbic acid to perform the secondary differentiation process (step C-2) in which they are differentiated into constructs containing mesoderm and endoderm cells.

[0043] Specifically, the medium in which the human pluripotent stem cell-derived embryoid bodies cultured during the primary differentiation process of the mesoderm and endoderm cell differentiation step (S130) are secondary cultured for 2 days corresponds to RPMI1640 medium supplemented with B-27 supplement (minus insulin; Thermo Fisher Scientific, USA), XAV939 at a concentration of 8 to 12 μM, and L-ascorbic acid at a concentration of 40 to 60 μg / ml.

[0044] Here, XAV939 added to the RPMI1640 medium used in the secondary differentiation process of the mesoderm and endoderm cell differentiation step (S130) is a component for inhibiting Wnt signaling, and most preferably has a concentration of 10 μM.

[0045] In addition, XAV939 added to the RPMI1640 medium used in the secondary differentiation process of the mesoderm and endoderm cell differentiation step (S130) is Inhibitor of Wnt Production-2, 3, 4 (IWP-2, 3, 4) depending on the implementation, and IWP-2, 3, 4 can be substituted at the same concentration.

[0046] Furthermore, L-ascorbic acid added to the RPMI1640 medium used in the secondary differentiation process of the mesoderm and endoderm cell differentiation step (S130) is a component for inhibiting Wnt signaling, and most preferably has a concentration of 50 μg / ml.

[0047] (4) Cardiac organoid self-assembly step<S140、ステップD> In this step (S140), a process is carried out in which the construct containing the mesodermal and endodermal cells differentiated in the mesodermal and endodermal cell differentiation step (S130) is differentiated into a self-organized heart organoid containing cardiomyocytes, fibroblasts and vascular endothelial cells.

[0048] This cardiac organoid self-organization step (S140) is more specifically subdivided and is carried out sequentially over four steps. First, the construct containing mesodermal and endodermal cells differentiated in the mesodermal and endodermal cell differentiation step (S130) is cultured for two days in RPMI1640 medium supplemented with B-27 supplement (minus insulin) and L-ascorbic acid at a concentration of 40 to 60 μg / ml to induce differentiation into cardiomyocytes, thereby carrying out the primary organization process (step D-1).

[0049] Here, it is most preferable that the L-ascorbic acid added to the RPMI1640 medium used in the primary organization process of the cardiac organoid self-organization step (S140) has a concentration of 50 μg / ml.

[0050] Thereafter, the construct in which differentiation of cardiomyocytes has been induced in the primary organization process (step D-1) is cultured for two days in the RPMI1640 medium used in the primary organization process (step D-1) with the addition of BMP4 at a concentration of 20-40 ng / ml, vascular endothelial growth factor (VEGF) at a concentration of 20-40 ng / ml, and fibroblast growth factor 2 (FGF2) at a concentration of 20-40 ng / ml, thereby carrying out the secondary organization process (step D-2) in which differentiation of fibroblasts and vascular endothelial cells is induced.

[0051] Here, BMP4, which is further added to the RPMI1640 medium used in the secondary organization process of the cardiac organoid self-organization step (S140), is a component for BMP signal activation, and preferably has a concentration of 30 ng / ml.

[0052] In addition, VEGF, which is further added to the RPMI1640 medium used in the secondary organization process of the cardiac organoid self-organization step (S140), is a component involved in vascular differentiation and formation, and preferably has a concentration of 30 ng / ml.

[0053] Furthermore, FGF2, which is further added to the RPMI1640 medium used in the secondary organization process of the cardiac organoid self-organization step (S140), is a component for FGF signal activation, and preferably has a concentration of 30 ng / ml.

[0054] Next, the constructs in which differentiation of cardiomyocytes, fibroblasts and vascular endothelial cells was induced in the secondary organization process (step D-2) are cultured for two days in RPMI1640 medium supplemented with B-27 supplement (minus vitamin A), L-ascorbic acid at a concentration of 40-60 μg / ml, BMP4 at a concentration of 20-40 ng / ml, VEGF at a concentration of 20-40 ng / ml and FGF2 at a concentration of 20-40 ng / ml, in the tertiary organization process (step D-3).

[0055] Here, it is most preferable that the L-ascorbic acid added to the RPMI1640 medium used in the tertiary organization process of the cardiac organoid self-organization step (S140) has a concentration of 50 μg / ml.

[0056] In addition, BMP4 added to the RPMI1640 medium used in the tertiary organization process of the cardiac organoid self-organization step (S140) is a component for BMP signal activation, and preferably has a concentration of 30 ng / ml.

[0057] Furthermore, VEGF, which is further added to the RPMI1640 medium used in the tertiary organization process of the cardiac organoid self-organization step (S140), is a component involved in vascular differentiation and formation, and preferably has a concentration of 30 ng / ml.

[0058] Furthermore, FGF2, which is further added to the RPMI1640 medium used in the tertiary organization process of the cardiac organoid self-organization step (S140), is a component for FGF signal activation, and preferably has a concentration of 30 ng / ml.

[0059] Finally, the constructs cultured in the tertiary organization process (step D-3) are cultured for two days in RPMI1640 medium supplemented with B-27 supplement (minus vitamin A), BMP4 at a concentration of 20-40 ng / ml, VEGF at a concentration of 20-40 ng / ml, and FGF2 at a concentration of 20-40 ng / ml, except for L-ascorbic acid, to perform the quaternary organization process (step D-4) to achieve self-organization of cardiac organoids through differentiation.

[0060] Here, BMP4 added to the RPMI1640 medium used in the quaternary organization process of the cardiac organoid self-organization step (S140) is a component for BMP signal activation, and preferably has a concentration of 30 ng / ml.

[0061] In addition, VEGF, which is further added to the RPMI1640 medium used in the quaternary organization process of the cardiac organoid self-organization step (S140), is a component involved in vascular differentiation and formation, and preferably has a concentration of 30 ng / ml.

[0062] Furthermore, FGF2, which is further added to the RPMI1640 medium used in the quaternary organization process of the cardiac organoid self-organization step (S140), is a component for FGF signal activation, and preferably has a concentration of 30 ng / ml.

[0063] Once the cardiac organoid self-organization step (S140) is completed through this series of detailed processes, cardiac organoids in which cardiomyocytes, fibroblasts, and vascular endothelial cells are self-organized through differentiation are obtained.

[0064] (5) Cardiac organoid maturation step<S150、ステップE> In this step (S150), the cardiac organoids that have differentiated and self-organized in the cardiac organoid self-organization step (S140) are cultured and matured.

[0065] Specifically, in the cardiac organoid maturation step (S150), the cardiac organoids that have self-organized to contain cardiomyocytes, fibroblasts, and vascular endothelial cells in the cardiac organoid self-organization step (S140) are cultured in a maturation medium for 10 to 20 days to mature them.

[0066] Here, the medium for maturation of cardiac organoids corresponds to RPMI1640 medium supplemented with B-27 supplement (minus vitamin A), VEGF at a concentration of 20 to 40 ng / ml, FGF2 at a concentration of 20 to 40 ng / ml, and components for inhibiting TGF-β signaling, and SB431542 at a concentration of 10 to 15 ng / ml.

[0067] In addition, VEGF added to the RPMI1640 medium used in the cardiac organoid maturation step (S150) is a component involved in vascular differentiation and formation, and preferably has a concentration of 30 ng / ml.

[0068] Furthermore, FGF2 added to the RPMI1640 medium used in the cardiac organoid maturation step (S150) is a component for FGF signal activation and preferably has a concentration of 30 ng / ml.

[0069] Furthermore, SB431542 added to the RPMI1640 medium used in the cardiac organoid maturation step (S150) is a component for inhibiting TGF-β signaling, and preferably has a concentration of 10 ng / ml.

[0070] As a result, as shown in Figure 4, the organoids are derived from human induced pluripotent stem cells, pass through embryoid bodies (EBs), and differentiate into mesodermal and endodermal cells on day 6 (Day 6), then differentiate into self-organized cardiac organoids of cardiomyocytes, fibroblasts, and vascular endothelial cells on day 10 (Day 10), and finally reach a mature cardiac organoid state on day 24 (Day 24).

[0071] In this process of diameter change at each step, as shown in Figure 5, it can be confirmed that from human pluripotent stem cell-derived embryoid bodies with a diameter of 200-250 μm, mature cardiac organoids with a diameter of 300-400 μm can finally be obtained on day 25 (Day 25).

[0072] Furthermore, as shown in Figure 6, in the process of change in the pulsation ratio at each step, from day 0 (Day 0), which corresponds to the state of human pluripotent stem cell-derived embryoid bodies with a diameter of 200 to 250 μm, to the final mature cardiac organoids at days 21 (Day 21) to 30 (Day 30), it was confirmed that more than 96% of individuals showed pulsation.

[0073] In particular, when the cardiac organoid maturation step (S150) is completed through a series of such detailed processes, cardiac organoids are obtained in which cardiomyocytes, fibroblasts and vascular endothelial cells are self-organized through differentiation in a cell number ratio of 55-65:15-30:15, which is very close to the cell number ratio of cardiomyocytes, fibroblasts and vascular endothelial cells that make up an actual human heart, which is 6:2:1.5.

[0074] In connection with this, fluorescent staining was performed on cardiac organoids derived from human pluripotent stem cells, and the expression of a cardiomyocyte marker (cTnT) and a ventricular-type cardiomyocyte marker (MYL2) was confirmed by image analysis, and the results are shown in Figure 7.

[0075] Cardiac organoids, which have similar or identical cell ratios of cardiomyocytes, fibroblasts, and vascular endothelial cells that are similar to those of the actual heart and which have self-organized and matured through simultaneous differentiation and interactions from the early developmental stage, are in a state sufficient to reproduce the structure and function of the actual heart. By using them to perform drug screening and cardiac toxicity evaluation through disease modeling during new drug development, the efficiency of new drug development can be dramatically improved.

[0076] 2. Explanation of the experiment to confirm the functionality of human pluripotent stem cell-derived cardiac organoids obtained by the manufacturing method for human pluripotent stem cell-derived cardiac organoids First, human pluripotent stem cell-derived cardiac organoids produced by the above-mentioned method for producing human pluripotent stem cell-derived cardiac organoids were set as one experimental group, and another experimental group was set up to compare the degree of compatibility with an actual heart from the standpoint of mutual structure and functionality. The results are described below.

[0077] (1) Experimental production of cardiac organoids derived from human pluripotent stem cells As shown in Figure 8, human pluripotent stem cell-derived cardiac organoids differentiated under different culture conditions were set as experimental group 1 and experimental group 2, respectively.

[0078] In the case of experimental group 1 and experimental group 2, the process of forming human pluripotent stem cell-derived embryoid bodies through the embryoid body preparation step (S110) and embryoid body culture step (S120) was commonly performed in the same manner.

[0079] In addition, human pluripotent stem cell-derived embryoid bodies with a diameter of 200 to 250 μm are subjected to primary culture for two days in RPMI 1640 medium supplemented with B-27 supplement (minus insulin), 6 μM CHIR99021, 10 ng / ml BMP4, and 10 ng / ml activin A, and the human pluripotent stem cell-derived embryoid bodies that have undergone primary culture are then cultured for two days in RPMI 1640 medium supplemented with B-27 supplement (minus insulin), 10 μM XAV939, and 50 μg / ml L-ascorbic acid to differentiate into constructs containing mesodermal cells and endodermal cells, and subsequent processes are performed under other culture conditions.

[0080] -Experimental Group 1 In experimental group 1, as shown in Figure 8, constructs containing differentiated mesodermal and endodermal cells were cultured for 2 days in RPMI1640 medium supplemented with B-27 supplement (minus insulin) and L-ascorbic acid at a concentration of 50 μg / ml to induce differentiation of cardiomyocytes, then cultured for 2 days in RPMI1640 medium supplemented with B-27 supplement (minus vitamin A) and L-ascorbic acid at a concentration of 50 μg / ml, and then L-ascorbic acid was removed and cultured for 2 days in RPMI1640 medium supplemented with B-27 supplement (minus vitamin A) to induce self-organization of cardiac organoids.

[0081] Next, the self-organized cardiac organoids corresponding to experimental group 1 were cultured in RPMI1640 medium supplemented with B-27 supplement (minus vitamin A) for 10 to 30 days to undergo the maturation process, completing experimental group 1.

[0082] -Experimental Group 2 In experimental group 2, as shown in FIG. 8, constructs containing differentiated mesodermal and endodermal cells were cultured for 2 days in RPMI 1640 medium supplemented with B-27 supplement (minus insulin) and 50 μg / ml L-ascorbic acid to induce differentiation into cardiomyocytes, and then the same RPMI 1640 medium was added with 30 ng / ml BMP4, 30 ng / ml VEGF, and 30 ng / ml FGF2 to culture for 2 days, and then cultured for 2 days in RPMI 1640 medium supplemented with B-27 supplement (minus vitamin A), 30 μg / ml L-ascorbic acid, 30 ng / ml BMP4, 30 ng / ml VEGF, and 30 ng / ml FGF2. After that, the L-ascorbic acid was removed and the cardiac organoids were allowed to self-organize by culturing for two days in RPMI1640 medium supplemented with B-27 supplement (minus vitamin A), 30 ng / ml BMP4, 30 ng / ml VEGF, and 30 ng / ml FGF2.

[0083] Next, the self-organized cardiac organoids corresponding to experimental group 2 were cultured for 10 to 20 days in RPMI1640 medium supplemented with B-27 supplement (minus vitamin A), 30 ng / ml VEGF, 30 ng / ml FGF2, and 10 ng / ml SB431542 to undergo a maturation process, completing experimental group 2.

[0084] (2) Comparative experiment of cardiac organoid cell composition by experimental group Using experimental groups 1 and 2 obtained by the above-mentioned method, comparative analysis of the cellular composition of cardiac organoids was performed through various experiments.

[0085] First, flow cytometry was performed using cTnT as a cardiomyocyte marker, CD90 as a fibroblast marker, and VE-cadherin or CD31 as a vascular endothelial cell marker, and the results are shown in FIGS. 9 and 10.

[0086] As shown in Figures 9 and 10, in the case of experimental group 1, the cardiomyocyte marker was analyzed at 92%, the fibroblast marker at 6%, and the vascular endothelial cell marker at 4%, while in the case of experimental group 2, the cardiomyocyte marker was analyzed at 51%, the fibroblast marker at 24%, and the endothelial cell marker at 14%.

[0087] Specifically, when the results of each experiment were converted into percentages, experimental group 1 showed a composition ratio of 90% cardiomyocytes, 6% fibroblasts, and 4% endothelial cells, while experimental group 2 showed a composition ratio of 58% cardiomyocytes, 27% fibroblasts, and 15% endothelial cells.

[0088] Next, we used quantitative real-time PCR to analyze the relative expression of marker genes in cardiac organoids from each experimental group, and used NKX2.5, TNNT2, MYL2, and MYL7 as cardiomyocyte markers.

[0089] As a result, as shown in Figures 11 to 13, it was found that experimental group 2 showed a decrease in expression level compared to experimental group 1, and cardiac fibroblast markers CD90, PDGFR, Vimetin, TCF21, and endoderm markers CD34, PECAM1, Sox17, and FOXA2 showed an increase in expression level in the cardiac organoids of experimental group 2.

[0090] More specifically, immunofluorescence staining tests were performed on the cardiac organoids of experimental groups 1 and 2 obtained by the above-mentioned method. As a result, as shown in Figures 14 and 15, in the cardiac organoids of experimental group 2, the vascular endothelial cell marker (VE-Cadherin) and the fibroblast marker (Vimentin) showed a distribution of increased expression, while the cardiomyocyte marker (cTnT) showed a distribution of decreased expression overall.

[0091] In particular, in the cardiac organoids of experimental group 2, it can be seen that VE-cadherin, a vascular endothelial cell marker, is distributed in a manner that surrounds the outermost part of the cardiac organoids, compared to experimental group 1.

[0092] (3) Comparative experiment of calcium ion channel functionality in cardiac organoids by experimental group Using experimental group 1 and experimental group 2 obtained by the above-mentioned method, the calcium ion channel functionality of cardiac organoids was measured and compared.

[0093] First, to measure calcium concentration in living cells, we used cell-permeable Fluo-4, AM (Thermo Fisher Scientific, Massachusetts, USA) to observe changes in calcium ion concentration in cardiac organoids of each experimental group.

[0094] As a result, as shown in Figures 16 and 17, it was confirmed that calcium ion movement and pulsation were relatively slower in experimental group 2, in which cardiomyocytes, fibroblasts, and vascular endothelial cells were differentiated simultaneously, than in experimental group 1, which consisted of 92% or more cardiomyocytes.

[0095] In summary, cardiac organoids produced as in experimental group 2 overcome the limitation of the absence of cardiac stem cells, and the cardiomyocytes, cardiac fibroblasts, and vascular endothelial cells that make up the heart are simultaneously differentiated from human pluripotent stem cells, self-organize through interactions from the early stages of development, and differentiate in culture, providing a sufficient condition to reproduce the structure and function of an actual heart.

[0096] As a result, cardiac organoids of experimental group 2, which corresponds to a preferred embodiment of human pluripotent stem cell-derived cardiac organoids produced by the above-mentioned method for producing human pluripotent stem cell-derived cardiac organoids, and which are in a state sufficient to reproduce the structure and function of the heart, can be used to perform drug screening and cardiac toxicity evaluation through disease modeling during new drug development, which not only dramatically improves the efficiency of new drug development, but is also suitable for use as an alternative model for research into the mechanisms of heart-related diseases.

[0097] The embodiments disclosed in the present invention are not intended to limit the technical idea of ​​the present invention, but are merely for the purpose of explanation, and such embodiments do not limit the scope of the technical idea of ​​the present invention. The scope of protection of the present invention should be interpreted according to the following claims, and any technical idea within the equivalent range should be interpreted as being included in the scope of the present invention.

Claims

1. Step A is to culture human pluripotent stem cells (hPSCs) to form embryonic bodies having a diameter of 100 to 130 μm; Step B: culturing the embryoid bodies obtained in step A until they reach a diameter of 200 to 250 μm; A step C of differentiating the human pluripotent stem cell-derived embryoid bodies having a diameter of 200 to 250 μm obtained in the step B into constructs containing mesodermal cells and endodermal cells; Step D of differentiating the construct including the mesodermal cells and endodermal cells differentiated in step C into a cardiac organoid self-organized to include cardiomyocytes, fibroblasts, and endothelial cells; Step E of culturing and maturing the cardiac organoids differentiated and self-organized in step D; The cardiac organoid obtained through the maturation process of step E is self-organized with cardiomyocytes, fibroblasts and vascular endothelial cells in a cell number ratio of 55 to 65: 15 to 30: 15, Step C comprises: Step C-1: culturing the human pluripotent stem cell-derived embryoid bodies having a diameter of 200 to 250 μm obtained in step B for 2 days in RPMI1640 medium supplemented with insulin-free B-27 supplement, CHIR99021, bone morphogenetic protein-4 (BMP4) and Activin A; and step C-2 of culturing the human pluripotent stem cell-derived embryoid bodies cultured in step C-1 for 2 days in RPMI 1640 medium supplemented with insulin-free B-27 supplement, XAV939, and L-ascorbic acid to differentiate the human pluripotent stem cell-derived embryoid bodies into constructs containing mesodermal cells and endoderm cells. Step D comprises: Step D-1 of inducing differentiation of cardiomyocytes by culturing the construct containing the mesodermal cells and endodermal cells differentiated in step C for 2 days in RPMI 1640 medium supplemented with insulin-free B-27 supplement and L-ascorbic acid at a concentration of 40 to 60 μg / ml; Step D-2 in which the construct in which differentiation into cardiomyocytes has been induced in the step D-1 is cultured for 2 days in the RPMI 1640 medium used in the step D-1, with BMP4 at a concentration of 20 to 40 ng / ml, vascular endothelial growth factor (VEGF) at a concentration of 20 to 40 ng / ml, and fibroblast growth factor 2 (FGF2) at a concentration of 20 to 40 ng / ml, to induce differentiation into fibroblasts and vascular endothelial cells; Step D-3: incubating the construct in which differentiation of cardiomyocytes, fibroblasts and vascular endothelial cells has been induced in step D-2 for 2 days in RPMI 1640 medium supplemented with B-27 supplement (minus vitamin A), L-ascorbic acid at a concentration of 40 to 60 μg / ml, BMP4 at a concentration of 20 to 40 ng / ml, VEGF at a concentration of 20 to 40 ng / ml and FGF2 at a concentration of 20 to 40 ng / ml; Step D-4 of culturing the construct cultured in step D-3 for 2 days in RPMI 1640 medium supplemented with B-27 supplement (minus vitamin A), BMP4 at a concentration of 20 to 40 ng / ml, VEGF at a concentration of 20 to 40 ng / ml, and FGF2 at a concentration of 20 to 40 ng / ml to perform self-organization of cardiac organoids by differentiation; The step E is a step of culturing and maturing the cardiac organoids differentiated in the step D-4 and self-organized to include cardiomyocytes, fibroblasts and vascular endothelial cells for 10 to 20 days in RPMI1640 medium supplemented with B-27 supplement (minus vitamin A), VEGF at a concentration of 20 to 40 ng / ml, FGF2 at a concentration of 20 to 40 ng / ml, and SB431542 at a concentration of 10 to 15 ng / ml as a component for TGF-β signal inhibition. A method for producing cardiac organoids derived from human pluripotent stem cells.

2. The method for producing a cardiac organoid derived from human pluripotent stem cells according to claim 1, wherein the human pluripotent stem cells in step A are at least one of human embryonic stem cells (hESC) and human induced pluripotent stem cells (hiPSC).

3. The method for producing cardiac organoids derived from human pluripotent stem cells according to claim 2, characterized in that step A is a step of plating human pluripotent stem cells in mTeSR (registered trademark) culture medium supplemented with a Rho-associated kinase (ROCK) inhibitor (Y-27632), culturing the cells for one day, and forming embryoid bodies having a diameter of 100 to 130 μm.

4. The method for producing cardiac organoids derived from human pluripotent stem cells according to claim 3, characterized in that step B is a step of culturing the embryoid bodies having a diameter of 100 to 130 μm obtained in step A for 2 to 3 days in mTeSR culture medium to which a ROCK inhibitor (Y-27632) has not been added, to form embryoid bodies derived from human pluripotent stem cells having a diameter of 200 to 250 μm.

5. The step C-1 is a step of culturing the human pluripotent stem cell-derived embryoid body having a diameter of 200 to 250 μm obtained in the step B for 2 days in RPMI1640 medium supplemented with insulin-free B-27 supplement, glycogen synthase kinase 3β (GSK-3β: Glycogen Synthase Kinase-3β) inhibitor for Wnt signal activation at a concentration of 6 to 8 μM CHIR99021, BMP4 at a concentration of 9 to 10 ng / ml as a component for BMP signal activation, and activin A at a concentration of 8 to 10 ng / ml as a component for transforming growth factor β (TGF-β: Transforming Growth Factor-β) signal activation. The method for producing human pluripotent stem cell-derived cardiac organoids according to claim 1.

6. The method for producing human pluripotent stem cell-derived cardiac organoids according to claim 5, characterized in that step C-2 is a step of culturing the human pluripotent stem cell-derived embryoid bodies cultured in step C-1 for 2 days in RPMI1640 medium supplemented with insulin-free B-27 supplement, XAV939 at a concentration of 8 to 12 μM as a component for inhibiting Wnt signaling, and L-ascorbic acid at a concentration of 40 to 60 μg / ml as a component for activating BMP signaling.

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