Methods for preparing cardiac organoids derived from human pluripotent stem cells and the resulting cardiac organoids derived from human pluripotent stem cells.

Abstract

This invention relates to a method for preparing cardiac organoids derived from human pluripotent stem cells, comprising: Step A: culturing human pluripotent stem cells (hPSCs) to prepare embryonic bodies with a diameter of 100 μm to 130 μm; Step B: culturing the embryonic bodies prepared in Step A to a diameter of 200 μm to 250 μm; Step C: differentiating the embryonic bodies derived from human pluripotent stem cells with a diameter of 200 μm to 250 μm prepared in Step B into constructs containing mesodermal cells and endoderm cells; Step D: differentiating the constructs containing mesodermal cells and endoderm cells differentiated in Step C into constructs containing cardiomyocytes, fibroblasts, and vascular endothelial cells. Self-organized cardiac organoids (cells); and, step E: culturing and maturing the self-organized cardiac organoids differentiated through step D, wherein the cardiac organoids that have completed the maturation process through step E have cardiomyocytes, fibroblasts, and endothelial cells forming a self-organized structure with a cell ratio of 55 to 65:15 to 30:15.

Description

Technical Field

[0001] This invention relates to a method for preparing cardiac organoids derived from human pluripotent stem cells, and the cardiac organoids derived from human pluripotent stem cells prepared therefrom. Background Technology

[0002] Recently, with the help of continuously developing technologies, a differentiation method has been developed to differentiate human pluripotent stem cells (hPSCs) into most of the cells that make up the human body. However, cells differentiated from two-dimensional cultured cells have relatively low maturity and therefore relatively low functionality compared to actual human cells. As a result, there are still obvious limitations in applying them to drug toxicity testing, drug screening, or cell therapy.

[0003] Accordingly, many technical studies on cell differentiation and culture are currently being conducted to prepare cells that have reached the maturity level of real human cells and have full function. In particular, on a global scale, there is ongoing research and development of organoids that simulate the form of cells in real organs, which exist in multiple cell clusters rather than in a single form, and can fully reflect the corresponding functions.

[0004] Organoids that mimic the structure and function of real human organs can be prepared by the continuous cell division and differentiation of organ stem cells, such as those in the intestines and brain, which are considered to have organ-specific stem cells. However, the heart does not have organ-specific stem cells, so this method cannot be used to prepare heart organoids. The only proposed preparation method is to differentiate the various cells that make up the heart separately and then mix them.

[0005] In this regard, the prior art for preparing heart organoids is U.S. Patent Publication No. 2020-0283735, entitled "Organoid and method for producing the same" (hereinafter referred to as "prior art").

[0006] However, previous methods for preparing heart organoids, including existing technologies, only involve mixing the differentiated heart-forming cells or culturing them using an extracellular matrix composed of specific components. This presents a problem: the diverse cells that make up the heart have a characteristic that they begin to interact and develop gradually in the early stages of development, and this characteristic affects the function of the heart. The methods described above would lose this effect on the heart.

[0007] As a result, if the various cells that make up the heart differentiate simultaneously but fail to self-organize through interaction at the beginning of early development, their state will not be sufficient to simulate the structure and function of the real heart. Consequently, when developing new drugs, it will be difficult to use disease modeling for drug screening and cardiotoxicity testing to derive meaningful results. Summary of the Invention

[0008] The problem to be solved

[0009] This invention is proposed to solve the above-mentioned problems. The purpose of this invention is to provide a method for preparing cardiac organoids using human pluripotent stem cells. Specifically, multiple cells that make up the heart differentiate simultaneously and begin to self-organize through interaction in the early stages of development. They are then cultured and differentiated so that the cardiac organoid has a state that can fully simulate the structure and function of a real heart.

[0010] Problem-solving methods

[0011] To achieve the above objectives, the present invention provides a method for preparing cardiac organoids derived from human pluripotent stem cells, characterized by comprising: Step A: culturing human pluripotent stem cells (hPSCs) to prepare embryonic bodies with a diameter of 100 μm to 130 μm; Step B: culturing the embryonic bodies prepared in Step A to a diameter of 200 μm to 250 μm; Step C: differentiating the embryonic bodies derived from human pluripotent stem cells with a diameter of 200 μm to 250 μm prepared in Step B into constructs containing mesodermal cells and endoderm cells; Step D: differentiating the constructs containing mesodermal cells and endoderm cells differentiated in Step C into constructs containing cardiomyocytes, fibroblasts, and vascular endothelial cells. Self-organized cardiac organoids (cells); and, step E: culturing and maturing the self-organized cardiac organoids differentiated through step D, wherein the cardiac organoids that have completed the maturation process through step E have cardiomyocytes, fibroblasts, and endothelial cells forming a self-organized structure with a cell ratio of 55 to 65:15 to 30:15.

[0012] In step A above, the human pluripotent stem cell is at least one of human embryonic stem cells (hESC) or human induced pluripotent stem cells (hiPSC).

[0013] Furthermore, step A above involves seeding human pluripotent stem cells in mTeSR culture medium supplemented with ROCK (Rho-associated kinase) inhibitor (Y-27632) and culturing for 1 day to prepare embryoid bodies with a diameter of 100 μm to 130 μm.

[0014] Furthermore, step B above involves placing the embryoid bodies with a diameter of 100 μm to 130 μm obtained through step A into mTeSR culture medium without the addition of ROCK (Rho-associated kinase) inhibitor (Y-27632) and culturing them for 2 to 3 days to prepare embryoid bodies derived from human pluripotent stem cells with a diameter of 200 μm to 250 μm.

[0015] Furthermore, step C above includes: step C-1: placing the human pluripotent stem cell-derived embryoids with a diameter of 200μm to 250μm obtained by step B above in RPMI 1640 medium supplemented with B-27 Supplement (minus Insulin), CHIR99021, BMP4 (Bone morphogenetic protein-4) and Activin A, and culturing for 2 days; and step C-2: placing the human pluripotent stem cell-derived embryoids cultured by step C-1 above in RPMI 1640 medium supplemented with B-27 Supplement (minus Insulin), XAV939, and L-ascorbic acid, and culturing for 2 days to differentiate them into constructs containing mesodermal cells and endoderm cells.

[0016] The C-1 step described above involves placing the human pluripotent stem cell-derived embryoid bodies with a diameter of 200 μm to 250 μm obtained through step B into RPMI 1640 medium and culturing them for 2 days. The RPMI 1640 medium contains the following: B-27 Supplement (minus insulin), CHIR99021 at a concentration of 6 μM to 8 μM as an inhibitor of GSK-3β (Glycogen Synthase Kinase-3β) for activating Wnt signaling, BMP4 at a concentration of 9 ng / ml to 10 ng / ml as a component for activating BMP signaling, and Activin A at a concentration of 8 ng / ml to 10 ng / ml as a component for activating TGF-β (Transforming Growth Factor-β) signaling.

[0017] Furthermore, step C-2 above involves placing the human pluripotent stem cell-derived embryoids cultured in step C-1 into RPMI 1640 medium and culturing for 2 days. The RPMI 1640 medium contains: B-27 Supplement (minus insulin), XAV939 at a concentration of 8 μM to 12 μM as a component for inhibiting Wnt signaling, and L-ascorbic acid at a concentration of 40 ng / ml to 60 ng / ml as a component for activating BMP signaling.

[0018] Furthermore, step D above includes: Step D-1: The construct containing mesodermal and endoderm cells differentiated through step C is placed in RPMI 1640 medium supplemented with B-27 Supplement (minus insulin) and L-ascorbic acid at a concentration of 40 ng / ml to 60 ng / ml, and cultured for 2 days to induce cardiomyocyte differentiation; Step D-2: BMP4 at a concentration of 20 ng / ml to 40 ng / ml, VEGF (Vascular Endothelialgrowth Factor) at a concentration of 20 ng / ml to 40 ng / ml, and FGF2 (Fibroblast Growth Factor) at a concentration of 20 ng / ml to 40 ng / ml are added to the RPMI 1640 medium used in step D-1 above. 2) Subsequently, the cardiomyocyte differentiation-inducing constructs formed through step D-1 were transferred into the solution and cultured for 2 days to induce the differentiation of fibroblasts and vascular endothelial cells; Step D-3: The cardiomyocyte, fibroblast, and vascular endothelial cell differentiation-inducing constructs formed through step D-2 were placed in RPMI supplemented with B-27 Supplement (minus 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. In RPMI 1640 medium, cultured for 2 days; and, D-4 step: The constructs cultured in step D-3 were placed in RPMI 1640 medium supplemented with B-27 Supplement (minus Vitamin A), 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, and cultured for 2 days to form self-organized cardiac organoids through differentiation.

[0019] Furthermore, step E above involves placing the self-organized cardiac organoids differentiated through step D-4 and containing cardiomyocytes, fibroblasts, and vascular endothelial cells in RPMI 1640 medium supplemented with B-27 supplement (minus 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 at a concentration of 10 ng / ml to 15 ng / ml (as a component that inhibits TGF-β (Transforming Growth Factor-β) signaling) for 10 to 20 days to allow them to mature.

[0020] In addition, in order to achieve the above objectives, the human pluripotent stem cell-derived heart organoids according to the present invention are prepared by the human pluripotent stem cell-derived heart organoid preparation method described above.

[0021] The effects of the invention

[0022] The effects of this invention are as follows:

[0023] First, it overcomes the limitation that the heart does not contain stem cells. It can prepare cardiac cardiomyocytes, cardiac fibroblasts and vascular endothelial cells that make up the heart from human pluripotent stem cells. At the same time, they can differentiate and achieve self-organization through interaction in the early stage of development, and be cultured and differentiated, so as to have a state that can fully simulate the structure and function of the real heart.

[0024] Secondly, when developing new drugs, cardiac organoids that can fully simulate the structure and function of the heart can be used in drug screening and cardiotoxicity testing through disease modeling, thereby significantly improving the efficiency of new drug development.

[0025] Third, using human pluripotent stem cell-derived heart organoids as alternative models for studying the mechanisms of heart-related diseases will contribute to the technological development of the disease research field. Attached Figure Description

[0026] Figure 1 This is a sequence diagram of the method for preparing cardiac organoids derived from human pluripotent stem cells according to the present invention;

[0027] Figure 2 and Figure 3The photographs are used to illustrate the observation results of changes in embryoid size and constituent cell number during the embryoid preparation and culture steps in the method for preparing cardiac organoids derived from human pluripotent stem cells according to the present invention.

[0028] Figure 4 These are photographs taken at various stages of the process by which human pluripotent stem cells differentiate into heart organoids from embryoid bodies, as observed under a microscope according to the method for preparing heart organoids derived from human pluripotent stem cells according to the present invention.

[0029] Figure 5 The diagram illustrates the changes in diameter of human pluripotent stem cells during different stages of their differentiation into cardiac organoids, in accordance with the method for preparing cardiac organoids derived from human pluripotent stem cells according to the present invention.

[0030] Figure 6 The diagram illustrates the changes in the pulsation ratio during different stages of the process of human pluripotent stem cells differentiating into cardiac organoids from embryoid bodies, in order to prepare cardiac organoids from human pluripotent stem cells according to the present invention.

[0031] Figure 7 A fluorescently stained photograph of a human pluripotent stem cell-derived heart organoid prepared according to the method for preparing human pluripotent stem cell-derived heart organoids of the present invention;

[0032] Figure 8 This is a schematic diagram summarizing the culture conditions and time periods for each step of each experimental group in order to prepare cardiac organoids derived from human pluripotent stem cells according to the present invention.

[0033] Figure 9 and Figure 10 A graph showing the flow cytometry analysis results of various experimental groups of human pluripotent stem cell-derived heart organoids prepared according to the method of preparing human pluripotent stem cell-derived heart organoids according to the present invention.

[0034] Figures 11 to 13 A graph showing the gene expression results of each experimental group of human pluripotent stem cell-derived heart organoids prepared by the method of preparing human pluripotent stem cell-derived heart organoids according to the present invention.

[0035] Figure 14 and Figure 15 Comparative photographs of the fluorescence staining results of different experimental groups of human pluripotent stem cell-derived heart organoids prepared according to the method of preparing human pluripotent stem cell-derived heart organoids according to the present invention.

[0036] Figure 16 and Figure 17Comparative photographs and graphs of calcium imaging results of different experimental groups of human pluripotent stem cell-derived heart organoids prepared according to the method of preparing human pluripotent stem cell-derived heart organoids of the present invention. Detailed Implementation

[0037] The preferred embodiments of the present invention will be described in more detail with reference to the accompanying drawings. For the sake of brevity, well-known technical parts will be omitted or shortened.

[0038] 1. Preparation method of cardiac organoids derived from human pluripotent stem cells

[0039] The following will refer to Figure 1 The preparation process of the method for preparing cardiac organoids derived from human pluripotent stem cells according to the present invention is described in detail.

[0040] (1) Embryoid body preparation step <S110, Step A>

[0041] The process performed in this step (S110) is to culture human pluripotent stem cells (hPSCs) to prepare embryonic bodies with a diameter of 100 μm to 130 μm.

[0042] Here, it refers 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).

[0043] Specifically, in the embryoid preparation step (S110), after harvesting the cultured human pluripotent stem cells, they are seeded in 6-well plates at a rate of 1.5 × 10⁶ cells / well using mTeSR culture medium (Stem Cell Technologies, Canada) supplemented with 10 μM ROCK (Rho-associated kinase) inhibitor (Y-27632, Tocris Bioscience, UK).

[0044] Here, when seeding human pluripotent stem cells into a 6-well plate, no coating for adsorbing cells is applied, and importantly, the substrate gel commonly used in organoid culture is not used; instead, a low-attachment culture dish can be used.

[0045] Also, such as Figure 2 As shown, after one day of culture, embryoid bodies with a diameter of 100μm to 130μm, consisting of about 200 to 500 cells, are formed in each well.

[0046] Therefore, when human pluripotent stem cells are seeded at a unit of 1.5 × 10⁶ cells / well in each well of a 6-well plate, after culturing for 1 day, approximately 3,000 to 7,500 embryoid bodies with a diameter of 100 μm to 130 μm will be generated.

[0047] (2) Embryoid body culture step <S120, Step B>

[0048] The process implemented in step (S120) is to further culture the embryoid obtained by the above embryoid preparation step (S110) until the diameter of the embryoid reaches 200 μm to 250 μm.

[0049] Specifically, the embryoid culture step (S120) is as follows: embryoids with a diameter of 100 μm to 130 μm, obtained by the embryoid preparation step (S110), are placed in mTeSR culture medium without the addition of ROCK (Rho-associated kinase) inhibitor (Y-27632) and cultured for 2 to 3 days to prepare human pluripotent stem cell-derived embryoids with a diameter of 200 μm to 250 μm.

[0050] During the 2-3 day culture period using mTeSR medium without the addition of ROCK (Rho-associated kinase) inhibitor (Y-27632), it is best to change the medium daily.

[0051] Therefore, as Figure 3 As shown, on the first day of culture following the embryoid preparation step (S110), embryoids with a diameter of 100 μm to 130 μm were prepared, as shown in the photograph marked 'Day ? ? 3'. Then, following the embryoid culture step (S120), after changing the conditions of the culture medium, further culture was carried out. After 2 to 3 days of culture, the diameter gradually increased and reached the range of 200 μm to 250 μm, as shown in the photograph marked 'Day 0'.

[0052] (3) Mesoderm and endoderm cell differentiation step <S130, Step C>

[0053] The process performed in this step (S130) is to differentiate the embryoid body with a diameter of 200 μm to 250 μm, which was prepared by the embryoid body culture step (S120), into a construct containing mesodermal cells and endoderm cells.

[0054] The mesodermal and endoderm cell differentiation step (S130) will be further subdivided into two steps and implemented sequentially. The first differentiation process (C-1 step) is implemented first: human pluripotent stem cell-derived embryoids with a diameter of 200 μm to 250 μm obtained through the embryoid culture step (S120) are placed in RPMI 1640 medium supplemented with B-27 Supplement (minus Insulin), CHIR99021, BMP4 (Bone morphogenetic protein-4) and Activin A and cultured for 2 days.

[0055] Specifically, the first culture of human pluripotent stem cell-derived embryoids with a diameter of 200 μm to 250 μm is carried out through the embryoid culture step (S120), and the culture period is 2 days. The culture medium used at this time is equivalent to RPMI1640 medium supplemented with B-27 Supplement (minus Insulin; Thermo Fisher Scientific, USA), CHIR99021 at a concentration of 6 μM to 8 μM, BMP4 at a concentration of 9 ng / ml to 10 ng / ml, and Activin A at a concentration of 8 ng / ml to 10 ng / ml.

[0056] Here, CHIR99021 added to the RPMI1640 medium used in the first differentiation process of the mesodermal and endoderm cell differentiation step (S130) is a GSK-3β (Glycogen Synthase Kinase-3β) inhibitor used to activate Wnt signaling, and its concentration is preferably 6 μM.

[0057] Furthermore, the BMP-4 (Bone morphogenetic protein-4) added to the RPMI 1640 medium used in the first differentiation process of the mesodermal and endoderm cell differentiation step (S130) is preferably 10 ng / ml as a component for activating BMP signaling.

[0058] Secondly, the Activin A added to the RPMI1640 medium used in the first differentiation process of the mesodermal and endoderm cell differentiation step (S130) is a component used to activate TGF-β (Transforming Growth Factor-β) signal, and its concentration is preferably 10 ng / ml.

[0059] Then, the second differentiation process (C-2 step) is carried out: the embryoids derived from human pluripotent stem cells that have undergone the first differentiation process (S130) and have been cultured are placed in RPMI 1640 medium supplemented with B-27 Supplement (minus Insulin), XAV939, and L-ascorbic acid, and cultured for another 2 days to differentiate them into constructs containing mesodermal cells and endoderm cells.

[0060] Specifically, the second culture of embryoids derived from human pluripotent stem cells, which undergo the first differentiation process of mesodermal and endoderm cell differentiation (S130), lasts for 2 days. The culture medium used at this time is equivalent to RPMI1640 medium supplemented with B-27 Supplement (minus Insulin; Thermo Fisher Scientific, USA), 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.

[0061] Here, the XAV939 added to the RPMI1640 medium used in the second differentiation process of the mesodermal and endoderm cell differentiation step (S130) is, as a component for inhibiting Wnt signaling, and its concentration is preferably 10 μM.

[0062] Furthermore, the XAV939 added to the RPMI1640 medium used in the second differentiation process of the mesodermal and endoderm cell differentiation step (S130) can be replaced by IWP-2, 3, and 4 at an equivalent concentration, depending on the implementation, by the Inhibitor of Wnt Production-2, 3, and 4.

[0063] Furthermore, the L-ascorbic acid added to the RPMI 1640 medium used in the second differentiation process of the mesodermal and endoderm cell differentiation step (S130) is, as a component for inhibiting Wnt signaling, and its concentration is preferably 50 μg / ml.

[0064] (4) Cardiac organoid self-organization step <S140, Step D>

[0065] The process performed in this step (S140) is to differentiate the construct containing mesodermal cells and endoderm cells, which was differentiated through the mesodermal and endoderm cell differentiation step (S130), into a self-organized heart organoid containing cardiomyocytes, fibroblasts, and endothelial cells.

[0066] The cardiac organoid self-organization step (S140) is further subdivided into four steps and implemented sequentially. The first step is the first organization process (D-1 step): the construct containing mesodermal and endoderm cells differentiated through the mesodermal and endoderm cell differentiation step (S130) is placed in RPMI 1640 medium supplemented with B-27 Supplement (minus Insulin) and L-ascorbic acid at a concentration of 40 μg / ml to 60 μg / ml and cultured for 2 days to induce differentiation into cardiomyocytes.

[0067] Here, the optimal concentration of L-ascorbic acid added to the RPMI 1640 medium used in the first tissue process of the cardiac organoid self-organization step (S140) is 50 μg / ml.

[0068] Then, the second tissue process (step D-2) is performed: the differentiation-induced constructs of cardiomyocytes formed through the first tissue process (step D-1) are transferred into a culture medium and cultured for 2 days to induce the differentiation of fibroblasts and vascular endothelial cells. The culture medium is RPMI 1640 medium used in the first tissue process (step D-1) with the addition of 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.

[0069] Here, the BMP-4 (Bone morphogenetic protein-4) added to the RPMI 1640 medium used in the second tissue process of the cardiac organoid self-organization step (S140) is, as a component for activating BMP signaling, and its concentration is preferably 30 ng / ml.

[0070] Furthermore, the VEGF (Vascular endothelial growth factor) added to the RPMI 1640 medium used in the second tissue process of the cardiac organoid self-organization step (S140) is a component used to interfere with the differentiation and formation of blood vessels, and its concentration is preferably 30 ng / ml.

[0071] Furthermore, the FGF2 (Fibroblast Growth Factor 2) added to the RPMI 1640 medium used in the second tissue process of the cardiac organoid self-organization step (S140) is preferably 30 ng / ml as a component for activating FGF signaling.

[0072] Then, the third tissue process (D-3 step) was carried out: the constructs formed by differentiation induction of cardiomyocytes, fibroblasts and vascular endothelial cells through the second tissue process (D-2 step) were transferred into RPMI 1640 medium supplemented with B-27 Supplement (minus 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, and cultured for 2 days.

[0073] Here, the optimal concentration of L-ascorbic acid added to the RPMI 1640 medium used in the third tissue process of the cardiac organoid self-organization step (S140) is 50 μg / ml.

[0074] Furthermore, the BMP-4 (Bone morphogenetic protein-4) added to the RPMI 1640 medium used in the third tissue process of the heart organoid self-organization step (S140) is preferably 30 ng / ml as a component for activating BMP signaling.

[0075] Furthermore, the VEGF (Vascular endothelial growth factor) added to the RPMI 1640 medium used in the third tissue process of the cardiac organoid self-organization step (S140) is a component used to interfere with the differentiation and formation of blood vessels, and its concentration is preferably 30 ng / ml.

[0076] Furthermore, the FGF2 (Fibroblast Growth Factor 2) added to the RPMI 1640 medium used in the third tissue process of the cardiac organoid self-organization step (S140) is preferably 30 ng / ml as a component for activating FGF signaling.

[0077] Finally, the fourth organization process (D-4 step) is carried out: the constructs cultured through the third organization process (D-3 step) are placed in RPMI 1640 medium containing B-27 Supplement (minus Vitamin A), 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, and cultured for 2 days to form self-organized cardiac organoids through differentiation.

[0078] Here, the BMP-4 (Bone morphogenetic protein-4) added to the RPMI 1640 medium used in the fourth tissue process of the cardiac organoid self-organization step (S140) is, as a component for activating BMP signaling, and its concentration is preferably 30 ng / ml.

[0079] Furthermore, the VEGF (Vascular endothelial growth factor) added to the RPMI 1640 medium used in the fourth tissue process of the cardiac organoid self-organization step (S140) is a component used to interfere with the differentiation and formation of blood vessels, and its concentration is preferably 30 ng / ml.

[0080] Furthermore, the FGF2 (Fibroblast Growth Factor 2) added to the RPMI 1640 medium used in the fourth tissue process of the cardiac organoid self-organization step (S140) is preferably 30 ng / ml as a component for activating FGF signaling.

[0081] After completing the above series of specific processes, once the heart organoid self-organization step (S140) is completed, heart organoids are obtained through differentiation, consisting of cardiomyocytes, fibroblasts, and endothelial cells that can self-organize.

[0082] (5) Cardiac organoid maturation step <S150, Step E>

[0083] The process performed in this step (S150) is to culture and mature the self-organized heart organoids differentiated through the heart organoid self-organization step (S140).

[0084] Specifically, the heart organoid maturation step (S150) is as follows: the self-organized heart organoid containing cardiomyocytes, fibroblasts and endothelial cells obtained through the heart organoid self-organization step (S140) is placed in a maturation culture medium and cultured for 10 to 20 days to allow it to mature.

[0085] Here, the cardiac organoid maturation medium is equivalent to RPMI 1640 medium supplemented with B-27 Supplement (minus 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 at a concentration of 10 ng / ml to 15 ng / ml as a component that inhibits TGF-β (Transforming Growth Factor-β) signaling.

[0086] Furthermore, the VEGF (Vascular endothelial growth factor) added to the RPMI 1640 medium used in the cardiac organoid maturation step (S150) is a component used to interfere with the differentiation and formation of blood vessels, and its concentration is preferably 30 ng / ml.

[0087] Furthermore, the FGF2 (Fibroblast Growth Factor 2) added to the RPMI 1640 medium used in the cardiac organoid maturation step (S150) is preferably 30 ng / ml as a component for activating FGF signaling.

[0088] Furthermore, the SB431542 added to the RPMI 1640 medium used in the cardiac organoid maturation step (S150) is a component for inhibiting TGF-β (Transforming Growth Factor-β) signaling, and its concentration is preferably 10 ng / ml.

[0089] The result is, as Figure 4 As shown, the heart organoids derived from human induced pluripotent stem cells (hiPSCs) and differentiated into mesodermal and endoderm cells on day 6, and then further differentiated into cardiomyocytes, fibroblasts, and endothelial cells on day 10, finally reached their mature state on day 24.

[0090] This process of diameter change in each step, such as Figure 5 As shown, embryoid bodies derived from human pluripotent stem cells with a diameter of 200 μm to 250 μm eventually develop into mature heart organoids with a diameter of 300 μm to 400 μm on day 25.

[0091] Furthermore, regarding the change in the pulsation ratio at each step, such as Figure 6 As shown, from day 0, which is equivalent to an embryoid body derived from human pluripotent stem cells with a diameter of 200 μm to 250 μm, to the mature heart organoids of day 21 to day 30, pulsation was found in more than 96% of the individuals.

[0092] Most importantly, after the heart organoid maturation step (S150) is completed through a series of specific processes, it differentiates to form a self-organized heart organoid with a cell ratio of cardiomyocytes, fibroblasts, and endothelial cells of 55 to 65: 15 to 30: 15. In contrast, the cell ratio of cardiomyocytes, fibroblasts, and endothelial cells in an actual human heart is 6: 2: 1.5. Therefore, in terms of this cell ratio, the self-organized heart organoid is very close to that of an actual human heart.

[0093] To this end, fluorescent staining was performed on cardiac organoids derived from human pluripotent stem cells, and image analysis was conducted to examine the expression of cardiomyocyte markers (cTnT) and ventricular cardiomyocyte markers (MYL2). The results are as follows: Figure 7 As shown.

[0094] This heart organoid has a similar or identical cell ratio to the cardiomyocytes, fibroblasts, and endothelial cells that make up the actual human heart. Furthermore, these cells differentiate simultaneously and begin self-organizing and maturing through interactions from an early stage of development. Therefore, this heart organoid possesses a state that sufficiently mimics the structure and function of a real heart. Thus, in drug development, using this heart organoid in drug screening and cardiotoxicity testing through disease modeling can significantly improve the efficiency of new drug development.

[0095] 2. Functional confirmation experiment description of cardiac organoids derived from human pluripotent stem cells prepared by the preparation method of cardiac organoids derived from human pluripotent stem cells (1) Preparation of experimental groups of cardiac organoids derived from human pluripotent stem cells

[0096] First, human pluripotent stem cell-derived heart organoids prepared using the aforementioned method were designated as one experimental group. Then, another experimental group was set up as a control to compare the structural and functional suitability of the control heart to the actual heart. The results are as follows:

[0097] Figure 8

[0098] like Figure 8 As shown, human pluripotent stem cell-derived cardiac organoids differentiated under different culture conditions were designated as experimental group 1 and experimental group 2, respectively.

[0099] For both experimental groups 1 and 2, the process of preparing human pluripotent stem cell-derived embryoids through embryoid preparation step (S110) and embryoid culture step (S120) is carried out uniformly and identically.

[0100] Furthermore, embryoid bodies derived from human pluripotent stem cells with a diameter of 200 μm to 250 μm were placed in RPMI 1640 medium supplemented with B-27 Supplement (minus Insulin), CHIR99021 at a concentration of 6 μM, BMP4 at a concentration of 10 ng / ml, and Activin A at a concentration of 10 ng / ml for the first culture period of 2 days. Then, the embryoid bodies derived from human pluripotent stem cells after the first culture were placed in RPMI 1640 medium supplemented with B-27 Supplement (minus Insulin), XAV939 at a concentration of 10 μM, and L-ascorbic acid at a concentration of 50 μg / ml for 2 days to differentiate them into constructs containing mesodermal cells and endoderm cells. This differentiation process was carried out identically, while subsequent processes were carried out under different culture conditions.

[0101] -Experimental Group 1

[0102] Experimental group 1 is, for example Figure 8 As shown, constructs containing differentiated mesodermal and endoderm cells were placed in RPMI 1640 medium supplemented with B-27 Supplement (minus Insulin) and L-ascorbic acid at a concentration of 50 μg / ml and cultured for 2 days to induce differentiation into cardiomyocytes. Then, they were placed in RPMI 1640 medium supplemented with B-27 Supplement (minus Vitamin A) and L-ascorbic acid at a concentration of 50 μg / ml and cultured for 2 days. Finally, they were placed in RPMI 1640 medium supplemented with B-27 Supplement (minus Vitamin A) without L-ascorbic acid and cultured for 2 days to form self-organized cardiac organoids.

[0103] Next, the self-organized heart organoids, equivalent to experimental group 1, were placed in RPMI 1640 medium supplemented with B-27 supplement (minus Vitamin A) and cultured for 10 to 30 days to allow them to undergo a maturation process, thus completing experimental group 1.

[0104] -Experimental Group 2

[0105] Experimental group 2, such as (2) Comparison experiment of cell composition of cardiac organoids in each experimental groupAs shown, constructs containing differentiated mesodermal and endoderm cells were placed in RPMI 1640 medium supplemented with B-27 Supplement (minus insulin) and 50 μg / ml L-ascorbic acid for 2 days to induce differentiation into cardiomyocytes. Then, they were placed in RPMI 1640 medium supplemented with B-27 Supplement (minus vitamin A) and 50 μg / ml L-ascorbic acid for 2 days. Next, RPMI 1640 medium supplemented with 30 ng / ml BMP4, 30 ng / ml VEGF (Vascular endothelial growth factor), and 30 ng / ml FGF2 (Fibroblast Growth Factor 2) was added, and the medium was cultured for 2 days. Finally, the medium was placed in RPMI 1640 medium supplemented with B-27 Supplement (minus vitamin A) and 50 μg / ml L-ascorbic acid for 2 days. A) The heart organoids were cultured for 2 days in RPMI 1640 medium containing 30 μg / ml L-ascorbic acid, 30 ng / ml BMP4, 30 ng / ml VEGF (Vascular endothelial growth factor), and 30 ng / ml FGF2 (Fibroblast Growth Factor 2). Then, they were cultured for 2 days in RPMI 1640 medium containing B-27 Supplement (minus Vitamin A), 30 ng / ml BMP4, 30 ng / ml VEGF (Vascular endothelial growth factor), and 30 ng / ml FGF2 (Fibroblast Growth Factor 2) without L-ascorbic acid.

[0106] Next, the self-organized heart organoids, equivalent to experimental group 2, were placed in RPMI1640 medium supplemented with B-27 Supplement (minus Vitamin A), VEGF (Vascular endothelial growth factor) at a concentration of 30 ng / ml, FGF2 (Fibroblast Growth Factor 2) at a concentration of 30 ng / ml, and SB431542 at a concentration of 10 ng / ml, and cultured for 10 to 30 days to allow them to undergo a maturation process, thus completing experimental group 2.

[0107] Figure 9

[0108] Using experimental groups 1 and 2 prepared by the methods described above, a comparative analysis of the cellular composition of cardiac organoids was conducted through a variety of experiments.

[0109] First, flow cytometry analysis was performed using cTnT as a marker for cardiomyocytes, CD90 as a marker for fibroblasts, and VE-Cadherin or CD31 as markers for vascular endothelial cells. The results are as follows: Figure 10 and Figure 9 As shown.

[0110] Regarding experimental group 1, such as Figure 10 and Figures 11 to 13 As shown, the percentage of cardiomyocyte markers was 92%, fibroblast markers was 6%, and vascular endothelial cell markers was 4%; for experimental group 2, the percentages were 51%, 24%, and 14%.

[0111] More specifically, when the results of each experiment are converted into percentages, the composition ratio presented by experimental group 1 is 90% cardiomyocytes, 6% fibroblasts, and 4% endothelial cells, while the composition ratio presented by experimental group 2 is 58% cardiomyocytes, 27% fibroblasts, and 15% endothelial cells.

[0112] Secondly, the relative expression of marker genes in cardiac organoids of each experimental group was analyzed using quantitative real-time PCR. NKX2.5, TNNT2, MYL2, and MYL7 were used as markers for cardiomyocytes.

[0113] The result, such as Figure 14As shown, compared with experimental group 1, the expression levels in experimental group 2 were reduced, while the expression of CD90, PDGFR, Vimentin, and TCF21, which are markers of cardiac fibroblasts, and CD34, PECAM1, Sox17, and FOXA2, which are markers of endoderms, were increased in cardiac organoids of experimental group 2.

[0114] More specifically, immunofluorescence staining experiments were performed on the cardiac organoids of experimental groups 1 and 2 prepared by the method described above, and the results are as follows: Figure 15 and (3) Comparison experiment of calcium channel function of cardiac organoids in each experimental group As shown, the distribution of vascular endothelial cell markers (VE-Cadherin) and fibroblast markers (Vimentin) increased in the cardiac organoids of experimental group 2, while the expression of cardiomyocyte marker (cTnT) decreased overall.

[0115] In particular, compared to experimental group 1, the cardiac organoids in experimental group 2 showed that VE-Cadherin, a marker of vascular endothelial cells, was distributed in the outermost morphology surrounding the cardiac organoids.

[0116] Figure 16

[0117] Using experimental groups 1 and 2 prepared by the method described above, the calcium ion channel function of cardiac organoids was measured and compared.

[0118] First, in order to determine the calcium concentration inside living cells, the changes in calcium ion concentration in cardiac organoids of various experimental groups were observed using the cell-permeable Fluo-4 and AM (ThermoFisher Scientific, MA, USA).

[0119] The result, such as Figure 17 and ​ As shown, compared to experimental group 1, which consisted of more than 92% cardiomyocytes, the movement and pulsation of calcium ions were slower in experimental group 2, where cardiomyocytes, fibroblasts, and vascular endothelial cells were differentiated simultaneously.

[0120] In short, the heart organoids prepared by experimental group 2 overcome the limitation that the heart does not contain stem cells. They can be prepared from human pluripotent stem cells to simultaneously differentiate cardiomyocytes, cardiac fibroblasts, and vascular endothelial cells that make up the heart. At the same time, they can achieve self-organization through interaction in the early stage of development and be cultured and differentiated, thus possessing a state that is sufficient to fully simulate the structure and function of the real heart.

[0121] Therefore, the human pluripotent stem cell-derived heart organoids prepared by the above-described method for preparing human pluripotent stem cell-derived heart organoids, as experimental group 2, which is equivalent to an appropriate embodiment, can not only utilize heart organoids with a state that sufficiently simulates the structure and function of a real heart in drug screening and cardiotoxicity testing through disease modeling in new drug development, thereby significantly improving the efficiency of new drug development, but are also suitable as alternative models for studying the mechanisms of heart-related diseases.

[0122] The embodiments described herein are for illustrative purposes only and are not intended to limit the scope of the inventive concept. The scope of the inventive concept is not limited to these embodiments. It should be understood that the scope of protection should be interpreted according to the scope of the appended claims, and all technical concepts within the equivalent scope should fall within the scope of the claims of this invention.

Claims

1. A method for preparing cardiac organoids derived from human pluripotent stem cells, characterized in that, include: Step A: Culture human pluripotent stem cells (hPSCs) to prepare embryonic bodies with a diameter of 100μm to 130μm; Step B: Cultivate the embryoids prepared in Step A until their diameter reaches 200 μm to 250 μm; Step C: Differentiate the embryoid body derived from human pluripotent stem cells with a diameter of 200 μm to 250 μm obtained in Step B above into a construct containing mesodermal cells and endoderm cells. Step D: Differentiate the construct containing mesodermal and endoderm cells differentiated in Step C into a self-organized heart organoid containing cardiomyocytes, fibroblasts, and endothelial cells; and Step E: Culture and mature the self-organized cardiac organoids differentiated in step D. In this process, the cardiomyocytes, fibroblasts, and endothelial cells of the heart organoids that have completed the maturation process through the above-mentioned step E form self-organizations at a cell ratio of 55 to 65:15 to 30:

15. Step D above includes: Step D-1: The construct containing mesodermal cells and endoderm cells, differentiated through step C, was placed in RPMI 1640 medium supplemented with B-27 Supplement (minus insulin) and L-ascorbic acid at a concentration of 40 ng / ml to 60 ng / ml and cultured for 2 days to induce differentiation of cardiomyocytes. Step D-2: Add 20 ng / ml to 40 ng / ml BMP4, 20 ng / ml to 40 ng / ml VEGF (Vascular endothelial growth factor), and 20 ng / ml to 40 ng / ml FGF2 (Fibroblast Growth Factor 2) to the RPMI 1640 medium used in Step D-1 above. Then, transfer the cardiomyocyte differentiation-inducing construct formed in Step D-1 into the medium and culture for 2 days to induce the differentiation of fibroblasts and vascular endothelial cells. Step D-3: The differentiation-induced constructs of cardiomyocytes, fibroblasts, and vascular endothelial cells formed through step D-2 were placed in RPMI 1640 medium supplemented with B-27 supplement (minus 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, and cultured for 2 days; Step D-4: The constructs cultured in step D-3 were placed in RPMI 1640 medium supplemented with B-27 supplement, 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, and cultured for 2 days to allow for differentiation and the formation of self-organized cardiac organoids. Step E above is as follows: The self-organized heart organoids differentiated through step D-4 and containing cardiomyocytes, fibroblasts, and vascular endothelial cells are placed in RPMI 1640 medium supplemented with B-27 supplement, 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 at a concentration of 10 ng / ml to 15 ng / ml as a component that inhibits TGF-β (Transforming Growth Factor-β) signaling, and cultured for 10 to 20 days to allow them to mature.

2. The method for preparing cardiac organoids derived from human pluripotent stem cells according to claim 1, characterized in that, The human pluripotent stem cells in step A above are at least one of human embryonic stem cells (hESC) or human induced pluripotent stem cells (hiPSC).

3. The method for preparing cardiac organoids derived from human pluripotent stem cells according to claim 2, characterized in that, Step A above is as follows: human pluripotent stem cells are seeded in mTeSR culture medium containing the ROCK inhibitor Y-27632 and cultured for 1 day to prepare embryoid bodies with a diameter of 100μm to 130μm.

4. The method for preparing cardiac organoids derived from human pluripotent stem cells according to claim 3, characterized in that, Step B above is as follows: Place the embryoid bodies with a diameter of 100 μm to 130 μm obtained by step A above in mTeSR culture medium without the addition of ROCK inhibitor Y-27632, and culture for 2 to 3 days to prepare embryoid bodies derived from human pluripotent stem cells with a diameter of 200 μm to 250 μm.

5. The method for preparing cardiac organoids derived from human pluripotent stem cells according to claim 1, characterized in that, Step C above includes: Step C-1: The human pluripotent stem cell-derived embryoid bodies with a diameter of 200 μm to 250 μm prepared in step B above were placed in RPMI 1640 medium supplemented with B-27 Supplement, CHIR99021, BMP4 (Bone morphogenetic protein-4), and Activin A, and cultured for 2 days; Step C-2: The human pluripotent stem cell-derived embryoids cultured in step C-1 are placed in RPMI 1640 medium supplemented with B-27 Supplement, XAV939, and L-Ascorbic acid and cultured for 2 days to differentiate into constructs containing mesodermal and endoderm cells.

6. The method for preparing cardiac organoids derived from human pluripotent stem cells according to claim 5, characterized in that, The above-mentioned C-1 step is as follows: the embryoid body with a diameter of 200 μm to 250 μm obtained by step B above is placed in RPMI 1640 medium and cultured for 2 days. The RPMI 1640 medium contains: B-27 Supplement, CHIR99021 at a concentration of 6 μM to 8 μM as an inhibitor of GSK-3β (Glycogen Synthase Kinase-3β) for activating Wnt signaling, BMP4 at a concentration of 9 ng / ml to 10 ng / ml as a component for activating BMP signaling, and Activin A at a concentration of 8 ng / ml to 10 ng / ml as a component for activating TGF-β (Transforming Growth Factor-β) signaling.

7. The method for preparing cardiac organoids derived from human pluripotent stem cells according to claim 6, characterized in that, The above-mentioned C-2 step is as follows: place the human pluripotent stem cell-derived embryoid bodies cultured in the above-mentioned C-1 step in RPMI 1640 medium and culture for 2 days, wherein the RPMI 1640 medium contains: B-27 supplement, XAV939 at a concentration of 8 μM to 12 μM as a component for inhibiting Wnt signaling, and L-ascorbic acid at a concentration of 40 ng / ml to 60 ng / ml as a component for activating BMP signaling.

8. A cardiac organoid derived from human pluripotent stem cells, characterized in that, Prepared using the method for preparing cardiac organoids derived from human pluripotent stem cells according to any one of claims 1-7.

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