Generation of cardiac mesoderm cell-based mature ventricular cardiac organoids

Abstract

The present invention relates to a mature ventricular-type cardiac organoid derived from cardiac mesenchymal cells and a method for producing the same. The cardiac organoid derived from cardiac mesenchymal cells produced by the method of the present invention is a mature ventricular-type cardiac organoid. It has structural maturation with better formation and arrangement of myomas and better formation of T-tubules compared to previously reported human pluripotent stem cell-derived cardiomyocytes, and has more uniform and synchronized characteristics of the cardiomyocytes' beating characteristics. Therefore, it can replace existing animal experiments, thereby resolving ethical issues and having the effect of being used as a three-dimensional cardiac tissue mimic that can reduce differences in responsiveness to drugs between different species.

Description

Technology Field

[0001] The present invention relates to a mature ventricular-type cardiac organoid derived from cardiac mesenchymal cells and a method for manufacturing the same. Background Technology

[0002] Ischemic heart disease has a very high prevalence worldwide and ranks first among all causes of death from a single disease. In Korea as well, the number of patients with ischemic heart disease is rapidly increasing due to socioeconomic development and Westernized lifestyles. Furthermore, for patients with severe end-stage heart failure, there is currently no cure other than heart transplantation or mechanical left ventricular assist devices. However, the disease faces challenges such as a shortage of donor organs, difficulties in securing organs, high mortality rates, and high treatment costs.

[0003] Meanwhile, transplantation methods for reconstructing damaged human tissues include xenograft, allograft, and autograft. Xenograft presents problems such as immunological incompatibility and the transmission of zoonotic pathogens, including retroviruses. Furthermore, allograft faces issues of donor immune rejection and unavailability, while autograft presents challenges such as difficulty in obtaining the appropriate amount of tissue required and an increased risk of trauma to the patient. To address these issues, technologies involving the direct transplantation of artificial substitutes or tissues formed by culturing cells are gaining attention. Following the publication of a study on retinal regeneration using cell sheets fabricated from pure cells in the New England Journal of Medicine in 2004, research on three-dimensional artificial tissues utilizing only cells has been actively conducted in the field of tissue engineering. Biomaterials refer to materials that come into direct contact with biological tissues as a means of treating diseases and regeneration; in particular, they serve as the basic materials for artificial organs and tissues used to replace damaged or dysfunctional human tissues and organs. Artificial tissues include joints, bones, skin, the heart, and blood vessels, and their range of applications is highly diverse. Tissue engineering technology can not only solve the problem of donor shortages but also regenerate all tissues and organs of the human body without inducing immune responses or cancer. To date, biocompatibility has been an indispensable characteristic required for artificial tissues intended for transplantation. In this field of tissue engineering, organoids—also known as artificial organs or mini-organs—have recently been receiving more attention than artificial tissues. Currently, various types of organoid models derived from stem cells of different in vivo organs have been established, and research on organoids is actively underway as a tool for regenerative medicine and cell therapy. Organoids are three-dimensional cellular structures formed through the development and differentiation of stem cells, serving as miniature organ mimics that reproduce the structural and functional characteristics of the corresponding organ.Organoids can mimic the environment of biological tissues, serve as models to replace animal testing, and are utilized in various applied research fields such as new drug development, disease modeling, and regenerative therapy. However, most cardiomyocytes differentiated from human pluripotent stem cells (HPCs) possess characteristics of immature cardiomyocytes, exhibiting traits similar to those of embryonic or fetal cardiomyocytes. Structural features of HPC-derived immature cardiomyocytes include incomplete formation and arrangement of myamina, as well as incomplete T-tubules. Additionally, mitochondria are small and irregularly distributed, and cristae occupy a lower density compared to mature cardiomyocytes. Electrophysiological and pulsatile characteristics are reported to be unsynchronized, and the formation of ion channels and responsiveness to drugs are incomplete. Recently, it has been reported that the degree of maturation of cardiomyocytes derived from HPCs is an important indicator determining drug responsiveness when assessing arrhythmia development and cardiotoxicity. Although methods have been reported to induce the maturation of immature myocardial cells using cardiac organoids, there have been problems with the low number and maturity of heart organoids. The problem to be solved

[0004] The present invention aims to provide a method for producing organoids with a higher number of heartbeats and maturity using cardiac mesenchymal cells during the differentiation induction stage from human pluripotent stem cells, and a composition for inducing cardiac organoid maturation, in order to solve the problem that conventional cardiomyocytes differentiated from human pluripotent stem cells exhibit structural and functional characteristics of embryonic or immature cardiomyocytes, and reported cardiac organoids exhibit low maturation and pulsation characteristics, making them unsuitable for disease modeling and drug toxicity assessment. means of solving the problem

[0005] To solve the above problem, the present invention provides a method for producing a cardiac organoid derived from cardiac mesenchymal cells.

[0006] In addition, the present invention provides a cardiac organoid derived from cardiac mesenchymal cells prepared by the above method.

[0007] In addition, the present invention provides a composition for inducing cardiac organoid maturation. Effects of the invention

[0008] The cardiac organoid derived from cardiac mesenchymal cells produced by the method of the present invention is a mature ventricular-type cardiac organoid. It has structural maturation with better formation and arrangement of myomas and better formation of T-tubules compared to previously reported human pluripotent stem cell-derived cardiomyocytes, and has more uniform and synchronized characteristics of the heartbeats of the cardiomyocytes. Therefore, it can replace conventional animal experiments, thereby resolving ethical issues and having the effect of being used as a three-dimensional cardiac tissue mimic that can reduce differences in responsiveness to drugs between different species. Brief explanation of the drawing

[0009] Figure 1 is a figure showing the differentiation of human pluripotent stem cells into cardiac mesoderm cells (CMCs) and cardiomyocytes (CMs), respectively, and the analysis of their characteristics. Figure 2 is a figure showing the fabrication of an organoid using cardiac mesenchymal cells and cardiomyocytes differentiated from human pluripotent stem cells and the confirmation of its characteristics. Figure 3 is a figure confirming the optimal number of CMCs isolated as single cells during organoid production. Fig. 4 is 2 x 10 6 This is a figure showing the ratio of the size of the organoid formed when cells are inoculated (A) and the size of the pulsating organoid (B). Figure 5 is a figure confirming the differentiation of cardiac organoids derived from cardiac mesenchymal cells and cardiac organoids derived from cardiac muscle cells into cardiac muscle cell types. Figure 6 is a figure confirming the percentage and beating characteristics of cardiac organoids derived from cardiac mesenchymal cells and cardiac organoids. Figure 7 is a figure analyzing the action potentials of cardiac organoids derived from cardiac mesenchymal cells and cardiac organoids derived from myocardial cells. Figure 8 is a diagram analyzing the junctions between cells in a pulsating organoid. Figure 9 is an analysis of the ultrastructure of cardiomyocytes (A and B), sarcomere length and Z-line width (C and D), and T-tubules (Figures E to H) in cardiac mesenchymal cell-derived cardiac organoids and cardiomyocyte-derived organoids: DJ: desmosomal junction; GJ: gap junction; AJ: adherens junction; and Z: Z-line. Figure 10 is a figure confirming the shape and area of ​​a cavity similar to the ventricular cavity, which is a morphological feature observed in the ventricle: H: H-band; LD: lipid droplet; Mt: mitochondria; TT: T-tubule; and Z: Z-line. Figure 11 is a figure showing the metabolic maturity of cardiac organoids derived from cardiac mesenchymal cells and cardiac organoids derived from myocardial cells through the analysis of mitochondrial structure and metabolic markers. Figure 12 is a figure showing the metabolic maturity of cardiac organoids derived from cardiac mesenchymal cells and cardiac organoids derived from myocardial cells through metabolic analysis by oxygen respiration. Figure 13 is a figure confirming the types of cardiac constituent cells and their composition ratios in cardiac mesenchymal cell-derived cardiac organoids and myocardial cell-derived organoids. Figure 14 is a figure showing the signaling mechanisms related to the characteristics and maturation of cardiac organoids identified through next-generation sequencing (RNA-Seq) and the list of regulated genes identified through gene ontology (GO). Figure 15 shows the signaling mechanism affecting the maturation of cardiac organoids derived from cardiac mesenchymal cells, identified through KEGG (Kyoto Encyclopedia of Genes and Genomes) analysis. Figure 16 is a figure showing the experimental results of gene and protein expression of molecules related to extracellular matrix-integrin interaction, focal adhesion, and LEFTY signaling, which were found to have increased expression in cardiac mesenchymal cell-derived cardiac organoids compared to cardiac muscle-derived organoids in next-generation sequencing. Figure 17 is a figure confirming the effect of LEFTY on the maturation mechanism of cardiac organoids derived from cardiac mesenchymal cells. Figure 18 is a figure confirming the characteristics of CMC-CO and CM-CO of induced pluripotent stem cell-derived organoids. Specific details for implementing the invention

[0010] Hereinafter, the present invention will be described in detail with reference to the attached drawings for embodiments of the present invention. However, the following embodiments are presented as examples of the present invention, and if it is determined that a detailed description of a technology or configuration well known to those skilled in the art may unnecessarily obscure the essence of the present invention, such detailed description may be omitted, and the present invention is not limited thereby. The present invention is capable of various modifications and applications within the scope of the claims set forth below and the equivalents interpreted therefrom.

[0011] Furthermore, the terminology used in this specification is used to appropriately describe preferred embodiments of the present invention, and may vary depending on the intent of the user or operator, or the conventions of the field to which the present invention belongs. Accordingly, the definitions of these terms should be based on the content throughout this specification. Throughout the specification, when a part is described as "comprising" a certain component, unless specifically stated otherwise, this means that it does not exclude other components but may include additional components.

[0012] All technical terms used in this invention, unless otherwise defined, are used in the sense generally understood by those skilled in the art in the relevant field of this invention. Additionally, while preferred methods or samples are described herein, similar or equivalents are also included within the scope of this invention. The contents of all publications cited as references in this specification are incorporated into this invention.

[0014] In one aspect, the present invention relates to a method for producing a cardiac organoid derived from cardiac mesenchymal cells, comprising the steps of: a) differentiating human pluripotent stem cells into cardiac mesenchymal cells; b) separating and dispensing the cardiac mesenchymal cells into single cells; c) culturing in a culture medium containing RPMI1640, B27, and insulin; and d) culturing in a culture medium containing RPMI1640, B27, and vitamin A.

[0015] In one embodiment, human pluripotent stem cells can be differentiated into cardiac mesenchymal cells by a method comprising: i) separating human pluripotent stem cells into single cells; ii) dispensing and culturing in E8 medium containing a Rho kinase inhibitor; iii) culturing in a culture medium containing RPMI1640, B27, and insulin containing a GSK-3 inhibitor; and iv) treating with IWP2 and culturing.

[0016] In one embodiment, step a) can be performed for 4 days.

[0017] In one embodiment, in step b), the cardiac mesenchymal cells are 2 x 10 5 Cells / cm 2 It can be divided at a density of.

[0018] In one embodiment, step c) can be performed for 3 days and cultured until the 7th day of differentiation during the entire period.

[0019] In one embodiment, step c) can be performed for 23 days, and culture can be performed up to day 30 of differentiation during the entire period.

[0020] According to the present invention, the 4 days, the 7th day and the 30th day of differentiation during the entire period may refer to the 4 days from the date of isolating pluripotent stem cells into single cells and dispensing them into E8 medium containing a Rho kinase inhibitor to culture, and the 7th day and the 30th day from the date of dispensing them into E8 medium containing a Rho kinase inhibitor to culture (see FIG. 2A).

[0021] In one embodiment, the method may further include a step of selecting organoids of 70 to 100 μm, which may be performed between steps c) and d) and during step d), and may be performed on day 7 and day 20 of differentiation during the entire period.

[0022] The term "pluripotent stem cell (PSC)" as used in this specification refers to a stem cell capable of induced differentiation into any form of cell that makes up the body, and pluripotent stem cells include embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs).

[0023] Specifically, embryonic stem cells are induced from the inner cell mass of a blastocyst at the pre-implantation stage. The induced cells are maintained in a specific environment and are capable of unlimited culture and pluripotent differentiation. Furthermore, induced pluripotent stem cells may refer to pluripotent differentiated cells created by dedifferentiation from somatic cells, and are formed by making somatic cells very similar to embryonic stem cells through a process called reprogramming, such as cell fusion, nuclear transfer, and the overexpression of pluripotency regulatory factors. Furthermore, pluripotent stem cells are not limited to embryonic stem cells and induced pluripotent stem cells, but may include all cells possessing both pluripotency and self-replicating ability. However, preferably, pluripotent stem cells may be mammalian cells, and more preferably human-derived pluripotent stem cells.

[0024] The "organoid" described in this specification is a "miniature organ-like structure" created using stem cells to perform minimal functions, characterized by its three-dimensional structure that allows for the creation of an environment similar to actual body organs even in a laboratory. In other words, an "organoid" refers to a cell having a three-dimensional structure and signifies a model similar to organs, such as nerves or intestines, manufactured through an artificial culture process rather than being collected or acquired from animals. The origin of the cells constituting it is not limited. The organoid may have an environment that allows it to interact with the surrounding environment during the cell growth process. Unlike 2D culture, 3D cell culture allows cells to grow in all directions in vitro. Accordingly, the 3D organoid in this invention can almost perfectly mimic organs that actually interact within the body, serving as an excellent model for observing the development of treatments for diseases and the like.

[0025] In the present invention, the term "differentiation" refers to the phenomenon in which the structure or function of cells becomes specialized while cells divide and proliferate and the entire organism grows. That is, it refers to the process in which the cells, tissues, etc. of an organism change into a form and function suitable for performing the roles assigned to them. For example, differentiation can include not only the process of pluripotent stem cells changing into ectoderm, mesoderm, and endoderm cells, but also the process in which progenitor cells come to express specific differentiation traits.

[0026] In one aspect, the present invention relates to a cardiac organoid derived from cardiac mesenchymal cells produced by the method of the present invention.

[0027] In one embodiment, the heart organoid may be ventricular-like.

[0028] In one embodiment, the cardiac organoid derived from cardiac mesenchymal cells of the present invention may have increased gene expression of ITGA5, ITGA7, ITGB1, ITGB3, COL4A1, LAMB1, LAMC1, FN1, LAMA2, NODAL, LEFTY1, LEFTY2, or PITX2 compared to a cardiac organoid derived from myocardial cells, increased protein expression of LIMK, SMAD4, COL1A, ITGA5, ITGAV, ITGB1, ITGB3, ITGB4, LEFTY, NODAL, or PITX2, and increased phosphorylation of FAK, ROCK1, ROCK2, MLC2, RAC, LIMK, SMAD2, or SMAD3.

[0029] In one aspect, the present invention relates to a composition for inducing cardiac organoid maturation comprising LEFTY or an expression promoter thereof as an active ingredient.

[0030] In the present invention, the expression promoter of LEFTY may be a substance that promotes the expression of LEFTY or induces overexpression.

[0031] In one aspect, the present invention relates to a composition for inducing cardiac organoid maturation comprising a polynucleotide encoding LEFTY, or a vector containing said polynucleotide, as an active ingredient.

[0032] The LEFTY-PITX2 signaling mechanism described in this invention is involved in left-right asymmetry during mammalian cardiac development, and LEFTY is also known to be important for maintaining the self-renewal of stem cells. Furthermore, during cardiac muscle development, fibronectin, collagen type V, and fibrillin, which are components of the fetal cardiac extracellular matrix, are replaced by collagen type I, which is a component of the adult cardiac extracellular matrix. Collagen type I has been reported to aid in the maturation of mouse and hiPSC-derived cardiomyocytes. Additionally, PITX2 has been reported to regulate ECM degradation in ovaries with hypothyroidism through the regulation of collagen biosynthesis. ECM-activating integrins further induce a downstream signaling cascade by inducing focal adhesion complex formation, actin polymerization, actin myosin stress fiber formation, and FAK phosphorylation. FAK activation also influences the activity of Rho-family GTPases, which can affect the actin cytoskeletal tissue. In addition, it has been reported that integrin β1 and α5 gene expression are induced during the maturation process, and that FAK activity is required for maturation in hPSC-derived cardiomyocytes. It has been reported that ROCK1 mediates the destabilization of the actin cytoskeleton through the regulation of MLC2 phosphorylation, whereas ROCK2 plays a role in stabilizing the actin cytoskeleton via cofilin. ROCK phosphorylates and activates LIM kinase, thereby phosphorylating ADF / Cofilin and inactivating actin depolymerization activity.

[0033] In one embodiment of the present invention, in the step of differentiating stem cells into cardiomyocytes, a three-dimensional structure was formed to confirm that the degree of maturation into cardiomyocytes increased. In addition, to confirm that LEFTY acted in this process, the degree of maturation was analyzed through the decrease in the function of LEFTY. It was confirmed that differentiation into mature cardiomyocytes and differentiation into ventricular type cardiomyocytes were enhanced in the organoids that were differentiated into cardiac mesenchymal cells and then cultured in three dimensions, compared to the organoid control group that was differentiated into cardiomyocytes under monolayer culture conditions and then cultured in three dimensions.

[0034] In one embodiment of the present invention, it was confirmed that in an organoid derived from cardiac mesenchymal cells, the formation and arrangement of cardiomyocytes are well-arranged compared to an organoid derived from cardiomyocytes, the size of mitochondria is large, and cristae are formed at a high density, and it was confirmed that the heartbeats of the cardiomyocytes are synchronized and exhibit a heartbeat similar to that of ventricular-type cardiomyocytes.

[0035] In one embodiment of the present invention, it was confirmed that the protein expression levels of COL1A, ITGB1, and ITGB3 were significantly reduced through functional degradation caused by treatment with LEFTY siRNA in cardiac mesenchymal cell-derived cardiac organoids, and that the activation of pLIMK, LIMK, ROCK1, and ROCK2 was inhibited during the generation of cardiac mesenchymal cell-derived cardiac organoids through treatment with LEFTY siRNA. In addition, the mRNA and protein expression of MLC2v and CAV3 were significantly reduced by functional degradation caused by LEFTY siRNA during the generation of cardiac mesenchymal cell-derived cardiac organoids. Furthermore, treatment with LEFTY siRNA in cardiac mesenchymal cell-derived cardiac organoids reduced the phosphorylation of FAK, confirming that LEFTY-PITX2 can regulate the extracellular matrix-integrin-focal adhesion signaling pathway during the generation of cardiac mesenchymal cell-derived cardiac organoids. Furthermore, upon investigating the downstream signaling of extracellular matrix-integrin-FAK involved in the maturation of cardiac mesenchymal cell-derived cardiac organoids, LIMK1 and pLIMK1 were increased in cardiac mesenchymal cell-derived cardiac organoids compared to cardiomyocyte-derived organoids. Additionally, ROCK1 and ROCK2 proteins, downstream signals known to be activated by integrins, were significantly increased in cardiac mesenchymal cell-derived cardiac organoids compared to cardiomyocyte-derived organoids. Therefore, it can be inferred that ROCK1, through extracellular matrix-integrin activation, primarily induces the activation of pMLC2, thereby inducing cardiomyocyte maturation.

[0036] In one embodiment of the present invention, it was confirmed that genes encoding extracellular matrix-integrin interactions and focal adhesion signaling factors are upregulated in cardiac mesenchymal cell-derived cardiac organoids, and that the phosphorylation of FAK is increased in cardiac mesenchymal cell-derived cardiac organoids.

[0037] In this invention, it was confirmed that cardiac organoids derived from cardiac mesenchymal cells exhibited improved structural, metabolic, functional, and molecular maturity of cardiomyocytes compared to organoids derived from cardiomyocytes, and that genes related to cardiac maturation and ventricular-type cardiomyocytes were upregulated.

[0038] In one aspect, the present invention relates to a method for evaluating drug toxicity using a cardiac organoid derived from cardiac mesenchymal cells, comprising the steps of: reacting a cardiac organoid derived from cardiac mesenchymal cells of the present invention with a drug; washing the cardiac organoid after the drug reaction is complete; culturing the washed cardiac organoid; capturing images of the reaction, washing, and culturing steps; obtaining the captured images; and analyzing the obtained images.

[0040] The present invention will be explained in more detail through the following examples. However, the following examples are intended only to illustrate the content of the present invention and do not limit the present invention.

[0042] Example 1. Confirmation of characteristics of cardiac mesenchymal cells and cardiomyocytes differentiated from stem cells

[0043] Differentiation from human pluripotent stem cells into cardiac mesoderm cells (CMCs) and cardiomyocytes (CMs), respectively, was induced, and their characteristics were analyzed. Specifically, the human embryonic stem cell line H9 hESC was isolated into single cells using Accutase, and then 2 x 10⁶ cells were placed in Matrigel-coated 6-well culture dishes using E8 medium (STEMCELL Technologies) containing 2 μM Thiazovivin (Rho kinase inhibitor). 6 cells (2 x 10 5 cells / cm 2Cells were seeded at a density of . Subsequently, after culturing for 1 day until the cells filled the 6-wells, 6 μM of the GSK-3 inhibitor CHIR99021 (Sigma-Aldrich) was added to RPMI1640+B27-insulin medium and cultured for 48 hours. On the 2nd day of differentiation, 2 μM IWP2 was added to the medium and cultured for 2 days. On the 4th day of differentiation, the medium was switched to RPMI1640+B27-insulin medium for culture, and on the 7th day of differentiation, the medium was switched to RPMI1640+B27-vitamin A medium while cultured until the 11th day of differentiation (Fig. 1A). To confirm the expression of each cell-specific marker, polymerase chain reaction (PCR) was performed for each differentiation day. Specifically, total RNA was extracted using TRIzol, and the concentration and purity of the RNA were measured using a Nanodrop spectrophotometer. Extracted total RNA was reacted with 20 μL of M-MLV reverse transcriptase at 37°C for 50 minutes to generate cDNA, and polymerase chain reaction was performed using iQ™ SYBR Green supermix. The results were recorded using the MYIQ2 Detection System. Gene expression levels were compared by quantification based on Ct values; GAPDH was used as the reference gene, and nuclear DNA was used for standardization when determining the ratio of mitochondrial DNA. Intron-spanning primers were designed using ProbeFinder software to avoid amplification of genomic DNA. Additionally, fluorescence staining was performed to confirm marker expression. Specifically, cardiac mesenchymal cells (CMC) on days 4 to 5 of differentiation and cardiomyocytes (CM) on day 11 of differentiation were washed twice with PBS and fixed with 4% paraformaldehyde (PFA) for 20 minutes. Fixed cells were permeated with 0.25% Triton X-100 for 30 minutes and blocked with 5% normal goat serum (NGS) for 1 hour at room temperature.Then, the cells were incubated overnight at 4°C with the primary antibodies against each marker: anti-T (1:400), anti-VEGFR2 (1:400), anti-NESTIN (1:400), anti-FOXA2 (1:400), anti-cardiac troponin T (cTnT, 1:400), and anti-Myosin light chain 2a (MLC2a, 1:400). Each cell was washed three times with PBST containing 0.1% Tween 20 in PBS, then reacted with secondary antibodies Alexa Fluor 488 chicken anti-rabbit IgG (1:1000), Alexa Fluor 594 goat anti-mouse IgG (1:1000), and Alexa Fluor 594 goat anti-rabbit IgG (1:1000) for 2 hours at room temperature, and the nuclei were stained with 1 μg / mL DAPI. Fluorescence images were obtained using a fluorescence microscope and a confocal fluorescence microscope.

[0044] As a result, mesoendodermal cell markers MIXL1 and T increased on days 1-2 of differentiation, and cardiac mesodermal cell markers MESP1 and VEGFR2 increased on days 4-5 of differentiation (Fig. 1B). Additionally, fluorescence staining confirmed the expression of the mesoendodermal cell marker T and the cardiac mesodermal cell marker VEGFR2, while the expression of the ectoderm marker NESTIN and the endoderm marker FOXA2 was not observed (Fig. 1C). Furthermore, on day 11 of differentiation, the cardiomyocyte marker cTnT and the atrial type cardiomyocyte marker MLC2a increased (Fig. 1B). Moreover, fluorescence staining of cardiomyocyte marker expression in cells on day 11 of differentiation confirmed the expression of the cardiomyocyte marker cTnT and the atrial type cardiomyocyte marker MLC2a (Fig. 1D).

[0046] Example 2. Preparation of embryonic stem cell-derived organoids

[0047] To compare the characteristics of organoids using cardiac mesenchymal cells and cardiomyocytes differentiated from human pluripotent stem cells (Fig. 2A), cardiac mesenchymal cells on day 4 of differentiation and cardiomyocytes on day 11 of differentiation were isolated as single cells using Accutase and inoculated into Poly-HEMA-coated 6-well culture dishes, respectively, during the differentiation step of human embryonic stem cells (H9 hESCs) into cardiomyocytes. At this time, to determine the optimal number of cells, aggregates were compared by inoculating various cell numbers, and as a result, the most appropriate number of cells was found to be 2 x 10⁶. 6 pieces (2 x 10 5 Cells / cm 2 A density of 2 x 10 per well of a 6-well culture dish 6 It was observed as (Fig. 3), and in subsequent experiments, 2 x 10 5 Cells / cm 2Cells were seeded at a density. Cardiac mesenchymal cells were cultured in RPMI1640+B27-insulin medium until day 7 of differentiation, then switched to RPMI1640+B27-vitamin A medium and cultured until day 30 of differentiation. Cardiac myocardial cells were cultured in RPMI1640+B27-vitamin A medium from day 11 of differentiation to day 30 of differentiation (Fig. 2A). Additionally, single cells were isolated to induce the formation of cardiac organoids (day 4 of differentiation for CMC / day 11 of differentiation for CM), and three days later, cardiac organoids were separated using a 70 to 100 μm cell strainer to select organoids of uniform size. On day 20 of differentiation, cardiac organoids were separated using a 100 μm cell strainer to remove self-aggregated cardiac organoids. Subsequently, cardiac mesenchymal cell-derived cardiac organoids (CMC-CO) and cardiomyocyte-derived organoids (CM-CO) were maintained in RPMI / B27-Vitamin A, with the medium changed every 2 days (Fig. 2A). After 15 days of differentiation, the formation of CMC or CM-derived cell aggregates, i.e., organoids, was confirmed (Fig. 2B), and the size of the formed organoids was determined to be 2 x 10⁶. 5 Cells / cm 2When cells were seeded at a density of [value], a large number of organoids exhibited a size of 70 to 100 μm (Fig. 4A), and organoids of 70 to 100 μm were selected. In addition, to analyze the pulsation of the organoids with uniform size, video was recorded using a microscope (4x, 50 frames per second), and then the light intensity in a selected area was analyzed using NIS for 15 seconds. It was assumed that the pulsation dynamics and regularity of the selected area were observed through the synchronization of the cardiac organoid. As a result, at 15 days of differentiation, the cardiac mesenchymal cell-derived cardiac organoids and myocardial cell-derived organoids accounted for 73.8% and 75.1%, respectively; at 20 days of differentiation, 86.0% and 83.2%, respectively; at 25 days of differentiation, 90.8% and 85.8%, respectively; and at 30 days of differentiation, 92.8% and 86.4%, respectively (Fig. 2C).

[0049] Example 3. Confirmation of myocardial cell type in cardiac organoids

[0050] To confirm the differentiation of the cardiac organoids derived from cardiac mesenchymal cells and cardiac organoids derived from cardiac cells prepared in Example 2 above into cardiac cell types, polymerase chain reaction, fluorescence staining, flow cytometry, and Western blot analysis were performed on day 30 of differentiation of each cardiac organoid using primers for cTnT, a cardiac cell-specific marker; MLC2a, an atrial type cardiac cell marker; MLC2v, a ventricular type cardiac cell marker; and TBX18, a nodal type cardiac cell marker. The polymerase chain reaction was performed in the same manner as in Example 1, and the fluorescence staining method was performed in the same manner as in Example 1 using antibodies against anti-cardiac troponin T (cTnT, 1:400), anti-Myosin light chain 2a (MLC2a, 1:400), anti-Myosin light chain 2v (MLC2v, 1:400), and anti-TBX18 (1:400). Specifically, for flow cytometry analysis, cardiac mesenchymal cells and myocardial cell-derived organoids were each dissociated into single cells using Accutase, fixed with 4% PFA for 20 minutes, and then washed with PBS+2% FBS. The cells were incubated on ice with ice-cold 100% methanol for 20 minutes and reacted with primary antibodies cTnT (1:100), MLC2a (1:100), MLC2v (1:100), and TBX18 (1:100), respectively, at room temperature for 45 minutes. Subsequently, the cells were reacted with secondary antibodies goat anti-mouse IgG1 Alexa 488, goat anti-rabbit IgG Alexa 594, and goat anti-rabbit IgG Alexa 647, respectively, at room temperature for 20 minutes each. The cells were then analyzed using a BD FACSCanto™II flow cytometer, and the data were analyzed using FlowJo software.Additionally, for Western blot analysis, cardiac mesenchymal and cardiomyocyte-derived organoids were washed in PBS and lysed in 1X cell lysis buffer containing 1 mM phenylmethylsulfonyl fluoriderk. Protein concentrations were measured using the Bradford assay, after which 15 μg of protein was mixed with 1X loading stain and boiled for 8 minutes. Subsequently, each protein sample was separated by electrophoresis on a 10% SDS-acrylamide gel and transferred to a polyvinylidene fluoride membrane. The membrane was blocked in TBST containing 5% skim milk powder or BSA for 1 hour at room temperature. The blocked membrane was then incubated overnight at 4°C with primary antibodies anti-cTnT (1:1000), anti-MLC2a (1:1000), anti-MLC2v (1:1000), and anti-TBX18 (1:1000). Subsequently, the membrane was washed three times with TBST and reacted with a horseradish peroxidase-conjugated secondary antibody at room temperature for one hour. Protein bands were visualized using ECL and obtained using X-ray film and a ChemiDoc imaging system.

[0051] As a result of the polymerase chain reaction, it was confirmed that the gene expression of cTnT, MLC2a, and MLC2v was significantly increased in cardiac mesenchymal cell-derived cardiac organoids, while the expression of TBX18 was significantly decreased in cardiac mesenchymal cell-derived cardiac organoids (Fig. 5A). Furthermore, fluorescence staining analysis revealed that, similar to the polymerase chain reaction, the expression of cTnT, MLC2a, and MLC2v was increased in cardiac mesenchymal cell-derived cardiac organoids, whereas the expression of TBX18 was almost non-existent in cardiac mesenchymal cell-derived and cardiomyocyte-derived organoids (Figs. 5B to 5D). In addition, flow cytometry analysis revealed that cTnT-positive cells accounted for 76.6% and 54.5% of cardiac mesenchymal cell-derived and cardiomyocyte-derived organoids, respectively; MLC2v-positive cells accounted for 53.6% and 28.9%, respectively; MLC2a-positive cells accounted for 10.6% and 7.30%, respectively; and TBX18-positive cells accounted for 1.65% and 3.55%, respectively, confirming that cTnT, MLC2a, and MLC2v-positive cells were more prevalent in cardiac mesenchymal cell-derived cardiac organoids (Fig. 5E). Furthermore, Western blot analysis confirmed that not only gene expression but also protein expression of cTnT, MLC2a, and MLC2v was increased in cardiac mesenchymal cell-derived cardiac organoids (Fig. 5F).

[0053] Example 4. Analysis of heartbeats in cardiac organoids

[0054] As in Example 2 above, the percentage of beating organoids and pulsating characteristics of cardiac mesenchymal cell-derived cardiac organoids and cardiac organoids were confirmed using video. Pulsating characteristics were verified by establishing five spots of a fixed size within the beating cardiac organoids in the video, measuring the beats per minute and peak-to-peak intervals within those areas, and plotting the results on a graph. In addition, intracellular Ca2+ For transient analysis, each cardiac organoid was reacted with 4 μg / mL of Fluo-4 AM at 37°C for 45 minutes to label intracellular calcium, and after observing the fluorescence using a confocal fluorescence microscope, the intensity was measured using Image J software and quantified into a graph.

[0055] As a result, there was no significant difference in the percentage of beating cardiac mesenchymal cell-derived cardiac organoids and cardiomyocyte-derived organoids (Figs. 2C and 6A); however, quantitative analysis confirmed that cardiac mesenchymal cell-derived cardiac organoids had a lower beat rate per minute of cardiomyocytes and wider, more uniform beat intervals compared to cardiomyocyte-derived organoids (Figs. 6B and 6C). In addition, it was observed that the intensity of Fluo 4-AM was significantly increased in cardiac mesenchymal cell-derived cardiac organoids compared to cardiomyocyte-derived organoids (Figs. 6D and E).

[0057] Example 5. Analysis of action potentials in cardiac organoids

[0058] To analyze the action potentials of cardiac organoids derived from cardiac mesenchymal cells and organoids derived from cardiomyocytes, patch clamp experiments were performed. Specifically, cardiac organoids derived from cardiac mesenchymal cells and cardiomyocytes, respectively, were inoculated into 35 mm culture dishes coated with Matrigel. Action potentials were measured using the whole cell composition, and the patch clamp experiments were conducted at room temperature using an Axopatch 200B amplifier. The cardiac organoids were placed in a chamber mounted on an inverted microscope and sequentially supplied with a tierod solution containing 143 mM NaCl, 5.4 mM KCl, 5 mM HEPES, 0.33 mM NaH2PO4, 5.5 mM glucose, 1.8 mM CaCl2, and 0.5 mM MgCl2 (pH 7.4). The patch pipette solution contained 140 mM KCl, 5 mM EGTA, 5 mM glucose, 5 mM HEPES, 5 mM Mg-ATP, and 1 mM MgCl2 (pH 7.2). Action potentials were recorded in spontaneously beating cells for 5 minutes using a gapless mode current clamp. Action potential amplitude, maximum upward stroke rate, and repolarization of the action potential duration at 50% and 90% were analyzed for five consecutive action potentials using the software pClamp 10.7. The patch pipette was pulled into a thin capillary using a P-97 Flaming / Brown Micropipette Puller, and the glass microelectrode was pulled and set so that the solution filling the pipette had a tip resistance of 4–6 MΩ. Current clamp recordings were digitally filtered through a 10 kHz, 4-pole Bessel-type low-pass filter and sampled at a rate of 25 kHz. Data collection and analysis were performed using a digitizer (DigiData 1550B) and analysis software pClamp 10.7 (Molecular Devices).

[0059] As a result of examining the ratio of atrial-like cardiomyocyte action potentials to ventricular-like cardiomyocyte action potentials in cardiac mesenchymal and cardiomyocyte-derived organoids, it was found that among cardiac mesenchymal cell-derived cardiac organoids, approximately 33.3% were atrial-like cardiomyocyte action potentials and approximately 66.7% were ventricular-like cardiomyocyte action potentials, while among cardiomyocyte-derived organoids, approximately 66.7% were atrial-like cardiomyocyte action potentials and approximately 33.3% were ventricular-like cardiomyocyte action potentials (Fig. 7B). In addition, resting membrane potentials were examined to confirm the maturity of cardiomyocytes, and it was observed that cardiac mesenchymal cell-derived cardiac organoids had significantly lower resting membrane potentials (Fig. 7C). In addition, although there was no significant difference in contraction frequency between cardiac mesenchymal cell-derived cardiac organoids and cardiomyocyte-derived organoids, it was confirmed that the action potential duration was significantly longer in cardiac mesenchymal cell-derived cardiac organoids compared to cardiomyocyte-derived organoids for both 50% (APD 50) and 90% (APD 90) (Figs. 7D to 7F). Since the prolonged action potential duration is a characteristic of ventricular-type cardiomyocytes, the gene expression of potassium ion channels determining this was examined using polymerase chain reaction, and it was observed that the expression of the relevant gene was significantly increased in cardiac mesenchymal cell-derived cardiac organoids compared to cardiomyocyte-derived organoids (Fig. 7G).

[0061] Example 6. Analysis of intercellular junctions in cardiac organoids

[0062] To analyze intercellular junctions in pulsating organoids, transmission electron microscopy images were used to identify the junctions between cells. Specifically, cardiac mesenchymal cell-derived cardiac organoids and cardiomyocyte-derived organoids were fixed overnight at 4°C using 2% PFA / 2.5% glutaraldehyde, respectively. Subsequently, they were post-fixed with 1% osmium tetroxide, dehydrated, and wrapped in Eponate-12 resin. 1 μm thick sections were obtained using a Reichert-Jung Ultracut E ultramicrotome, stained with toluidine blue, and images were acquired using a Carl Zeiss Axio microscope. Then, 60 nm thick sections per block were selected from Formvar-coated slot grids, stained with uranyl acetate / lead citrate, and the samples were recorded in TEM. TEM images were analyzed using Image J image processing software, and statistics were expressed as means ± standard deviation (SD) using Prism software.

[0063] As a result, desmosomal junctions and adherens junctions belonging to the intercalated disc were observed in both cardiac mesenchymal cell-derived cardiac organoids and cardiac muscle-derived organoids, but gap junctions were observed only in cardiac mesenchymal cell-derived cardiac organoids (Fig. 8A). In addition, to confirm the gene expression and protein expression related to gap junctions, the expression of Cx43, a gap junction marker, and ZO-1, known to support adherens junctions, was confirmed using polymerase chain reaction, Western blot analysis (anti-Cx43 (1:1000) and anti-ZO-1 (1:1000)) and fluorescence staining (anti-Connexin 43 (Cx43, 1:400) and anti-ZO-1 (1:400)). Polymerase chain reaction and Western blot analysis showed that the gene and protein expression of Cx43 and ZO-1 were significantly increased in cardiac mesenchymal cell-derived cardiac organoids compared to myocardial cell-derived organoids (Figs. 8B and C), and fluorescence staining results showed that the expression of Cx43 was uniform at the junctions between cells in cardiac mesenchymal cell-derived cardiac organoids (Fig. 8D).

[0065] Example 7. Analysis of the structural maturity of cardiac organoids

[0066] 7-1. Confirmation of the Microstructure of Cardiac Myocardial Cells

[0067] To analyze the ultrastructure of cardiomyocytes in cardiac organoids derived from cardiac mesenchymal cells and organoids derived from cardiomyocytes, the microstructure of cardiomyocytes was examined using transmission electron microscopy. As a result, in cardiac organoids derived from cardiac mesenchymal cells, well-ordered long myomers and mitochondria aligned along the myomers were observed, and H-bands as well as Z-lines were identified in the myomers (Fig. 9A). In contrast, in organoids derived from cardiomyocytes, irregularly arranged short myomers and unordered mitochondria were found, and only Z-lines were observed in the myomers (Fig. 9B).

[0069] 7-2. Verification of sarcomere length and Z-line width

[0070] Since sarcomere length and Z-line width can serve as indicators of cardiac cell maturity, sarcomere length and Z-line width were quantified and plotted. The results showed that the sarcomere length was approximately 1.8 μm in cardiac mesenchymal cell-derived cardiac organoids and approximately 1.6 μm in cardiac cell-derived organoids (Fig. 9C). It was also found that the Z-line width was wider in cardiac mesenchymal cell-derived organoids compared to cardiac cell-derived organoids (Fig. 9D).

[0072] 7-3. Check T-Tubule

[0073] Since T-tubules found in mature myocardial cells were observed in cardiac organoids derived from cardiac mesenchymal cells (Fig. 9A), the gene expression and protein expression of T-tubule markers CAV3, JPH2, and BIN1 were confirmed through polymerase chain reaction, Western blot analysis, and fluorescence staining.

[0074] As a result, gene expression of CAV3, JPH2, and BIN1 was found to be increased in cardiac organoids derived from cardiac mesenchymal cells compared to organoids derived from myocardial cells (Fig. 9E), and CAV3 and JPH2 were observed uniformly in cardiac organoids derived from cardiac mesenchymal cells (Figs. 9F to 9G). Similarly to these results, protein expression also showed increased expression of CAV3 and JPH2 in cardiac organoids derived from cardiac mesenchymal cells compared to organoids derived from myocardial cells (Fig. 9H).

[0076] 7-4. Comparison of Lumen Areas

[0077] Cavity morphology similar to the ventricular cavity, a morphological feature seen in the ventricle, was observed in cardiac mesenchymal cell-derived cardiac organoids and cardiac muscle cell-derived organoids (Fig. 10A). When the number of organoids with lumens and the area of ​​the lumen in a single organoid were compared and plotted, it was found that the number of organoids with lumens was higher in cardiac mesenchymal cell-derived cardiac organoids compared to cardiac muscle cell-derived organoids, and the area of ​​the lumen in a single organoid was also larger in cardiac mesenchymal cell-derived cardiac organoids compared to cardiac muscle cell-derived organoids (Figs. 10B and 10C).

[0079] Example 8. Analysis of metabolic maturity of cardiac organoids

[0080] Since it has been reported that as mitochondria mature, the density of mitochondrial cristae increases and the width of the mitochondria expands, resulting in a complex structure, mitochondrial maturation was compared by graphing the mitochondria of cardiac mesenchymal cell-derived and cardiomyocyte-derived organoids using transmission electron microscopy (Fig. 11A) to analyze the metabolic maturity of these organoids. Furthermore, as mitochondria are involved in cardiac metabolism, the gene and protein expression of SIRT1, PGC1α, TFAM, and CPT1β—markers related to cardiac metabolism—were confirmed through polymerase chain reaction (PCR) and Western blot analysis. In addition, to compare the metabolic maturity of the cardiac organoids, metabolism induced by aerobic respiration was confirmed through the analysis of oxygen consumption rates using the Mitochondrial Stress Test Complete Assay Kit. Specifically, cardiac organoids derived from cardiac mesenchymal cells and myocardial cell-derived organoids were each dispensed into 96-well plates, and a medium containing 1 μM of oligomycin, 2.5 μM of FCCP (carbonyl cyanide-4-phenylhydrazone), and 1 μM of antimycin A was added to each well, along with an extracellular O2 probe. Subsequently, the cardiac organoids were wrapped in preheated HS mineral oil, and fluorescence was measured by confirming excitation and emission at wavelengths of 340 nm and 655 nm using a SpectraMax®i3x microplate reader.

[0081] As a result, the density of mitochondrial cristae structures was found to be more densely formed in cardiac mesenchymal cell-derived organoids compared to cardiac myocardial cell-derived organoids (Fig. 11B), and the width of mitochondria was found to be wider in cardiac mesenchymal cell-derived organoids compared to cardiac myocardial cell-derived organoids (Fig. 11C). In addition, gene expression of cardiac metabolism-related markers SIRT1, PGC1α, TFAM, and CPT1β was significantly increased in cardiac mesenchymal cell-derived organoids compared to cardiac myocardial cell-derived organoids (Fig. 11D), and protein expression of PGC1α, TFAM, and CPT1β was found to be increased in cardiac mesenchymal cell-derived organoids compared to cardiac myocardial cell-derived organoids (Fig. 11E). Furthermore, as a result of examining metabolism by aerobic respiration, it was confirmed that the maximum respiration rate was significantly increased in cardiac mesenchymal cell-derived organoids compared to cardiac myocardial cell-derived organoids (Fig. 12).

[0083] Example 9. Confirmation of cellular composition of cardiac organoids

[0084] 9-1. Identification of Cell Components

[0085] Since the heart contains not only myocardial cells but also vascular cells, smooth muscle cells, and fibroblasts, the cardiac constituent cells of cardiac mesenchymal cell-derived cardiac organoids and myocardial cell-derived organoids were analyzed for vascular cell marker CD31, smooth muscle cell marker α-SMA, and fibroblast marker FSP1 using polymerase chain reaction and fluorescence staining.

[0086] As a result, fluorescence staining using primary antibodies against anti-CD31 (1:400) and anti-SMA (α-smooth muscle actin) (1:400) showed no significant difference in α-SMA expression between cardiac mesenchymal cell-derived cardiac organoids and cardiac muscle-derived organoids, but CD31 was found to be expressed in cardiac mesenchymal cell-derived cardiac organoids (Fig. 13A). In addition, polymerase chain reaction results showed no significant difference between the two groups for α-SMA and FSP1, but gene expression of CD31 was found to be significantly increased in cardiac mesenchymal cell-derived cardiac organoids (Fig. 13B). Accordingly, to identify the vascular cell subtypes, polymerase chain reaction was performed using primers for arterial vascular cell markers CXCR4 and EFNB2, venous vascular cell marker NRF2, and lymphatic vascular cell marker PROX1. As a result, it was confirmed that gene expression of all of the above vascular cell markers was significantly increased in cardiac organoids derived from cardiac mesenchymal cells compared to organoids derived from myocardial cells (Figs. 13C to 13E).

[0088] 9-2. Verification of Cell Composition Ratio

[0089] The ratio of cardiac constituent cells in cardiac mesenchymal cell-derived cardiac organoids and myocardial cell-derived organoids was determined by flow cytometry using primary antibodies of α-SMA (1:100), CD31 (1:100), and FSP1 (1:100). The results showed that in the cardiac mesenchymal cell-derived cardiac organoids and myocardial cell-derived organoids, CD31-positive cells accounted for 4.19% and 0.54%, respectively, α-SMA-positive cells accounted for 8.01% and 7.44%, and FSP1-positive cells accounted for 10.6% and 9.37%, respectively (Fig. 13F).

[0091] Example 10. Identification of cardiac organoid characteristics and signaling mechanisms related to maturation

[0092] 10-1. Next-generation sequencing

[0093] To investigate the characteristics and signaling mechanisms associated with the maturation of cardiac organoids, next-generation sequencing (RNA-Seq) was performed using cardiac mesenchymal cell-derived and cardiomyocyte-derived organoids at differentiation days 15 and 20 (Fig. 14A). Specifically, total RNA from the cardiac organoids was extracted using Trizol, total RNA concentration was measured using NanoDrop, and RNA purity was measured using an Agilent 2100 bioanalyzer with an RNA 6000 Nano Chip. A QuantSeq Library Prep kit was used to prepare a library for next-generation sequencing, and high-throughput sequencing was performed using NextSeq 500. Whole transcripts were compared, and differentially expressed genes (DEGs) showing more than twofold changes in expression in cardiac mesenchymal cell-derived organoids compared to cardiomyocyte-derived organoids were identified. DEGs were analyzed using ExDEGA software, and gene analysis was performed using DAVID (http: / / david.abcc.ncifcrf.gov / ), Medline databases (http: / / www.ncbi.nlm.nih.gov / ), Gene Ontology (https: / / www.ebi.ac.uk / QuickGO / ), Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway (http: / / www.genome.jp / kegg / tool / map_pathway2.html), STRING (http: / / www.string-db.org / ), and R studio (https: / / rstudio.com / ). Venn diagram analysis was performed using a total of 25,737 genes, and at day 15 of differentiation only, the number of genes increased in cardiac mesenchymal cell-derived cardiac organoids compared to myocardial cell-derived organoids was 62, and the number of genes decreased was 26.Furthermore, on day 20 of differentiation alone, cardiac organoids derived from cardiac mesenchymal cells showed an increase of 745 genes and a decrease of 251 genes compared to organoids derived from myocardial cells. Additionally, on days 15 and 20, 117 genes were found to be increased and 10 genes to be decreased in common (Fig. 14B). Moreover, heatmap analysis was performed to investigate gene expression by organ, and it was confirmed that heart-related genes were increased in both cardiac mesenchymal cell-derived and myocardial cell-derived organoids on days 15 and 20 (Fig. 14B). Furthermore, a scatter plot analysis of the genes increased in cardiac mesenchymal cell-derived organoids revealed increased expression of heart-related genes, and it was confirmed that more regulated genes appeared on day 20 than on day 15 (Fig. 14C).

[0095] 10-2. Gene Ontology Analysis

[0096] As a result of investigating the list of regulated genes through gene ontology (GO), the list of genes increased in cardiac mesenchymal cell-derived cardiac organoids compared to myocardial cell-derived organoids included muscle contraction, muscle cell development, regulation of blood circulation, sarcomere organization, regulation of cation transmembrane transport, ventricular cardiac muscle tissue development, regulation of release of sequestered Ca2+ into cytosol by sarcoplasmic reticulum (SR), generation of precursor metabolites and energy, SA node cell to atrial cardiac muscle cell communication, and gap junctions (Fig. 14D).On the other hand, the list of genes reduced in cardiac organoids derived from cardiac mesenchymal cells compared to organoids derived from myocardial cells was Nervous system development, Head development, Cell cycle, Cell proliferation, Brain development, Cell division, Lung development, Programmed cell death, Renal system development, and Kidney development (Fig. 14E).

[0098] Example 11. Analysis of signaling mechanisms specific to the maturation of cardiac mesenchymal cell-derived cardiac organoids

[0099] To analyze the signaling mechanisms affecting the maturation of cardiac organoids derived from cardiac mesenchymal cells based on next-generation sequencing analysis, increased signaling mechanisms were identified through KEGG (Kyoto Encyclopedia of Genes and Genomes) analysis. As a result, cardiac muscle contraction, the calcium ion signaling pathway, focal adhesion, regulation of actin cytoskeleton, adherens junction, TGFβ signaling pathway, tight junction, and extracellular matrix (ECM)-receptor interaction were found to be increased in cardiac organoids derived from cardiac mesenchymal cells compared to organoids derived from cardiomyocytes (Fig. 15A). Accordingly, through heatmap analysis, the expression of genes belonging to each heart-related gene (ventricular CM, atrial CM, nodal CM, cardiac structural maturation, gap junction, cardiac metabolic maturation, cardiac contraction, and calcium ion channel activity) and signaling mechanism (LEFTY signaling, focal adhesion, and extracellular matrix-integrin interaction) was compared.

[0100] As a result, compared to cardiac myocardial cell-derived organoids, cardiac mesenchymal cell-derived cardiac organoids showed increased expression of most genes on day 20 of differentiation compared to day 15, and when comparing heart-related genes with human adult cardiomyocytes, the cardiac mesenchymal cell-derived cardiac organoids at day 20 were found to be the most similar to human adult cardiomyocytes (Figs. 15B and C). In addition, network analysis of the protein-protein interactions of these genes revealed that proteins related to extracellular matrix-integrin interactions, focal adhesion, LEFTY signaling, cardiac maturation, cardiac contraction, and cardiac metabolism all interact with each other (Fig. 15D).

[0102] Example 12. Confirmation of the effect of LEFTY on the maturity of cardiac mesenchymal cell-derived cardiac organoids

[0103] 12-1. Comparison of Gene and Protein Expression

[0104] In Example 11 above, since the expression of genes related to extracellular matrix-integrin interaction, focal adhesion, and LEFTY signaling was increased in cardiac organoids derived from cardiac mesenchymal cells compared to organoids derived from myocardial cells, polymerase chain reaction and Western blot analysis were performed to experimentally verify the validity.

[0105] As a result, gene expression of the extracellular matrix molecules FN1 and LAMA2 was significantly increased in cardiac mesenchymal cell-derived cardiac organoids on days 15 and 20 of differentiation compared to cardiac myocardial cell-derived organoids. Gene expression of COL4A1, LAMB1, and LAMC1 did not show a significant difference between cardiac mesenchymal cell-derived and cardiac myocardial cell-derived organoids on days 15 of differentiation, but it was significantly increased in cardiac mesenchymal cell-derived cardiac organoids on days 20 of differentiation compared to cardiac myocardial cell-derived organoids (Fig. 16A). The integrin ITGA1 was found to show no significant difference between cardiac mesenchymal cell-derived and cardiac myocardial cell-derived organoids on days 15 and 20 of differentiation. There were no significant differences in ITGA5, ITGA7, ITGB1, and ITGB3 between cardiac mesenchymal and cardiomyocyte-derived organoids at day 15 of differentiation, but they were significantly increased in cardiac mesenchymal cell-derived organoids compared to cardiomyocyte-derived organoids at day 20 of differentiation (Fig. 16A). Protein expression of COL1A, ITGA5, ITGAV, ITGB1, and ITGB3 was increased in cardiac mesenchymal cell-derived organoids compared to cardiomyocyte-derived organoids, and was found to be more increased at day 20 than at day 15 of differentiation (Fig. 16B). Protein expression of ITGB4 was increased in cardiac mesenchymal cell-derived organoids compared to cardiomyocyte-derived organoids, and there was no significant difference between day 15 and day 20 of differentiation (Fig. 16B). FN1 was increased in cardiac mesenchymal cell-derived organoids compared to cardiac myocardial cell-derived organoids, but was found to decrease on day 20 compared to day 15 of differentiation (Fig. 16B). ITGAV and ITGB5 were found to be of no significant difference between cardiac mesenchymal cell and cardiac myocardial cell-derived organoids on days 15 and 20 of differentiation (Fig. 16B).

[0107] 12-2. Comparison of Protein Phosphorylation

[0108] Western blot analysis confirmed the expression and phosphorylation of proteins associated with focal adhesion, known as a downstream signaling mechanism of the extracellular matrix-integrin. The results showed that FAK phosphorylation was increased in cardiac mesenchymal cell-derived cardiac organoids compared to cardiomyocyte-derived organoids on days 15 and 20 of differentiation, and that the expression, phosphorylation of LIMK, phosphorylation of RAC, and phosphorylation of ROCK1, ROCK2, and MLC2 were increased in cardiac mesenchymal cell-derived cardiac organoids compared to cardiomyocyte-derived organoids on days 15 and 20 of differentiation (Fig. 16C). On the other hand, it was confirmed that there was no significant difference in Cofilin phosphorylation between cardiac mesenchymal cell-derived cardiac organoids and cardiomyocyte-derived organoids on days 15 and 20 of differentiation (Fig. 16C).

[0110] 12-3. Comparison of Gene Expression and Protein Expression Related to the LEFTY Signaling Mechanism

[0111] Western blot analysis of gene and protein expression related to the LEFTY signaling mechanism revealed that the gene expression of LEFTY1, LEFTY2, and PITX2 was significantly increased in cardiac mesenchymal cell-derived cardiac organoids compared to cardiomyocyte-derived organoids, and this increase was further observed on day 20 compared to day 15 of differentiation (Fig. 16A). Regarding the gene expression of NODAL, there was no significant difference between cardiac mesenchymal cell-derived and cardiomyocyte-derived organoids on day 15 of differentiation, but on day 20 of differentiation, NODAL expression was found to be significantly increased in cardiac mesenchymal cell-derived cardiac organoids compared to cardiomyocyte-derived organoids (Fig. 16A). Similarly, protein expression of LEFTY, NODAL, and PITX2 increased in cardiac mesenchymal cell-derived cardiac organoids (Fig. 16D). In addition, since LEFTY is a superfamily of TGF, the expression and phosphorylation of SMAD were examined, and it was confirmed that SMAD1 / 5 expression was not observed, while the phosphorylation of SMAD2 and SMAD3 and the expression of SMAD4 were increased in cardiac mesenchymal cell-derived cardiac organoids on day 20 of differentiation compared to cardiac muscle-derived organoids (Fig. 16D).

[0113] Example 13. Confirmation of the effect of LEFTY on the maturation mechanism of cardiac mesenchymal cell-derived cardiac organoids

[0114] 13-1. Confirmation of LEFTY-PITX2 Signaling Mechanism

[0115] In Example 12 above, it was confirmed that the expression of LEFTY was significantly increased in cardiac organoids derived from cardiac mesenchymal cells on days 15 and 20 of differentiation. Therefore, to investigate the effect of LEFTY on maturity, knockdown was induced using LEFTY siRNA. Specifically, negative control siRNA (nc siRNA) (sequences are sense 5'-UUCUCCGAACGUGUCACG-3' and antisense 5'-ACGUGACACGUUCGGAGA-3') and LEFTY siRNA (sense 5'-CGUCCAUCACCCAUCCUAA-3' and antisense 5'-UUAGGAUGGGUGAUGGACG-3') were transfected into cardiac organoids derived from cardiac mesenchymal cells on days 15 of differentiation using Lipofectamine RNAiMax. The gene expression and protein expression levels of LEFTY1, LEFTY2, NODAL, and PITX2 in transfected cardiac mesenchymal cell-derived cardiac organoids were confirmed by polymerase chain reaction and Western blot on day 30 of differentiation.

[0116] As a result, it was found that the gene expression of LEFTY1 and LEFTY2 was reduced in the LEFTY siRNA-treated group, and the protein expression of LEFTY was also reduced, and it was confirmed that the gene expression and protein expression of NODAL and PITX2 were reduced (Figs. 17A and 17B).

[0118] 13-2. Confirmation of Extracellular Matrix-Integrin-pFAK-pLIMK-ROCK Signaling Mechanism

[0119] Since PITX2 is reported to regulate collagen synthesis, we examined gene and protein expression related to extracellular matrix-integrins. We found that the gene expression of ITGA7 was reduced in the LEFTY siRNA-treated group, while there was no significant difference in the gene expression of COL4A1 (Fig. 17C). Additionally, protein expressions of COL1A, ITGB1, and ITGB3 were reduced in the LEFTY siRNA-treated group, while FN1 and ITGA5 showed no significant difference (Fig. 17D). Furthermore, examining protein expression related to focal adhesion, we found that the phosphorylation of FAK and LIMK1 was reduced in the LEFTY siRNA-treated group, and protein expressions of LIMK1, ROCK1, and ROCK2 were also reduced in the LEFTY siRNA-treated group (Fig. 17E).

[0121] 13-3. Confirmation of the effect of LEFTY on the maturation of cardiac mesenchymal cell-derived cardiac organoids

[0122] To determine the effects of LEFTY on myocardial cells and maturation, gene and protein expression of the myocardial cell marker cTnT, the atrial-type myocardial cell marker MLC2a, the ventricular-type myocardial cell marker MLC2v, and the T-tubule marker CAV3 were examined. As a result, there was no significant difference in the case of cTnT and MLC2a, but the expression of MLC2v and CAV3 was found to be decreased in the LEFTY siRNA-treated group (Figs. 17F and 17G).

[0124] Example 14. Production of induced pluripotent stem cell-derived organoids

[0125] To determine whether CMC-CO derived from human induced pluripotent stem cells (hiPSCs) is more mature and generates ventricular type CM compared to CM-CO, similar to what was observed in CMC-CO derived from human embryonic stem cells (hESCs) in Example 2 above, 3 x 10 of the TMOi001-A episomal hiPSC cell line were used. 5 cells / cm2 CMC CO and CM-CO derived from hiPSC were produced using the same method, except for dispensing at a density of , and their characteristics were verified as in the above examples.

[0126] qRT-PCR results showed that the expression of total CM marker (cTnT), arterial CM marker (MLC2a), and ventricular CM marker (MLC2v) was significantly increased in CMC-COs compared to CM-COs, whereas no significant difference was found between the two organoids for the nodular CM marker (TBX18) (Fig. 18A). Furthermore, Western blot analysis of the protein levels of these markers revealed that the expression levels of cTnT and MLC2v proteins were significantly higher in CMC-COs compared to CM-COs (Fig. 18B). Additionally, immunohistochemistry revealed that MLC2a in CMC-COs and CM-COs + CMs and MLC2v + As a result of checking the ratio of CMs, MLC2a + CMs and MLC2v + The proportion of CMs was detected to be abundant in CMC-COs compared to CM-COs (Fig. 18C). In addition, the expression of key indicators of functional and metabolic maturation of CMs was confirmed by qRT-PCR and Western blot analysis, and K +The mRNA expression of channel genes (KCNA4, KCNH2, and KCNJ2) was found to be significantly increased in CMC-COs compared to CM-COs (Fig. 18D), and the mRNA expression of mature cardiac markers (cTnI), t-tubule markers (CAV3, JPH2, and BIN1), and cardiac metabolic markers (PGC1α, TFAM, and CPT1β) was found to be significantly increased in CMC-COs compared to CM-COs (Figs. 18E and F). Furthermore, the protein levels of cTnI, CAV3, and JPH2 were also significantly increased in CMC-COs compared to CM-COs (Fig. 18G). In addition, the mRNA expression levels of Cx43 (gap junction marker) and ZO-1 (adhesion junction marker) were significantly increased in CMC-COs compared to CM-COs (Fig. 18H), and their protein expression was also significantly increased in CMC-COs compared to CM-COs. This was confirmed on day 30 of differentiation (Fig. 18I).

[0128] Through this, mature ventricular-type cardiac organoids were generated using cardiac mesenchymal cells possessing self-organizing capabilities in both hESCs and hiPSCs, and it was confirmed that cardiac mesenchymal cell-derived cardiac organoids exhibited enhanced structural, metabolic, functional, and molecular maturity compared to cardiomyocyte-derived organoids, and that genes related to cardiac maturation and ventricular-type cardiomyocytes were upregulated. Specifically, cardiac mesenchymal cell-derived cardiac organoids displayed 1. more organized myomime structures, 2. more organized mitochondria, 3. well-ordered T-tubules, and 4. more organized and evenly distributed internuclei; and 5. increased gene expression for ventricular-type cardiomyocyte markers, 6. cardiac metabolic markers, 7. T-tubulation markers, 8. potassium ion channel markers, 9. cell junction markers, and 10. vascular cell markers; It exhibited functional maturation such as 11. faster motion vector velocity, 12. reduced heart rate per minute, 13. increased peak-to-peak duration, 14. prolonged action potential duration, and 15. a high proportion of organoids with action potentials similar to ventricular-type cardiomyocytes.

[0129] Here, it was revealed that the LEFTY-PITX2 signaling pathway plays an important role in myocardial maturation and ventricular-type myocardial cells during the generation of cardiac mesenchymal cell-derived cardiac organoids, and it was confirmed that the high self-organization ability of cardiac mesenchymal cells is important for the generation of mature and ventricular cardiac organoids. Therefore, the mature cardiac mesenchymal cell-derived cardiac organoids of the present invention can be usefully utilized for drug screening and disease modeling in the field of cardiology.

Claims

Claim 1 a) a step of differentiating human embryonic stem cells into cardiac mesenchymal cells; b) separating the differentiated cardiac mesenchymal cells into single cells, 2 x 10 5 Cells / cm 2 A method for producing a cardiac organoid derived from cardiac mesenchymal cells, comprising: a step of dispensing at a density of; c) a step of culturing the single-cell separated cardiac mesenchymal cells by replacing the medium with a medium containing RPMI1640 and B27 and not containing insulin; and d) a step of culturing the single-cell separated cardiac mesenchymal cells by replacing the medium with a medium containing RPMI1640 and B27 and not containing vitamin A. Claim 2 A method for producing a cardiac organoid derived from cardiac mesenchymal cells, wherein the human embryonic stem cells differentiate into cardiac mesenchymal cells by a method comprising: i) separating the human embryonic stem cells into single cells; ii) dispensing and culturing in E8 medium containing a Rho kinase inhibitor; iii) adding a GSK-3 inhibitor to a medium containing RPMI1640 and B27 and not containing insulin and culturing; and iv) treating with IWP2 and culturing. Claim 3 A method for preparing a cardiac organoid derived from cardiac mesenchymal cells, wherein, in claim 1, step a) is performed for 4 days. Claim 4 delete Claim 5 A method for preparing a cardiac organoid derived from cardiac mesenchymal cells, wherein, in claim 1, step c) is performed for 3 days. Claim 6 A method for preparing a cardiac organoid derived from cardiac mesenchymal cells, wherein, in claim 1, step c) is performed for 23 days. Claim 7 A method for producing a cardiac organoid derived from cardiac mesenchymal cells according to claim 1, further comprising the step of selecting organoids of 70 to 100 μm. Claim 8 A method for producing a cardiac organoid derived from cardiac mesenchymal cells, wherein, in claim 7, organoid screening is performed between steps c) and d). Claim 9 A method for producing a cardiac organoid derived from cardiac mesenchymal cells, wherein, in claim 7, organoid screening is performed during step d). Claim 10 A cardiac organoid derived from cardiac mesenchymal cells prepared by the method of claim 1. Claim 11 In item 10, a ventricular-like cardiac organoid derived from cardiac mesenchymal cells. Claim 12 In claim 10, a cardiac organoid derived from cardiac mesenchymal cells having increased gene expression of ITGA5, ITGA7, ITGB1, ITGB3, COL4A1, LAMB1, LAMC1, FN1, LAMA2, NODAL, LEFTY1, LEFTY2, or PITX2 compared to a cardiac organoid derived from myocardial cells. Claim 13 In claim 10, cardiac mesenchymal cell-derived cardiac organoids having increased protein expression of LIMK, SMAD4, COL1A, ITGA5, ITGAV, ITGB1, ITGB3, ITGB4, LEFTY, NODAL, or PITX2 compared to cardiac muscle-derived organoids. Claim 14 In claim 10, a cardiac mesenchymal cell-derived cardiac organoid having increased phosphorylation of FAK, ROCK1, ROCK2, MLC2, RAC, LIMK, SMAD2, or SMAD3 compared to a myocardial cell-derived organoid. Claim 15 A composition for inducing cardiac organoid maturation according to claim 1, comprising LEFTY as an active ingredient. Claim 16 A composition for inducing cardiac organoid maturation according to claim 1, comprising a polynucleotide encoding LEFTY, or a vector containing said polynucleotide as an active ingredient.

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