Method for preparing cardiac organoids derived from human pluripotent stem cells, and efficacy and toxicity evaluation technique

Abstract

The present invention relates to a method for preparing cardiac organoids derived from human pluripotent stem cells, and an efficacy and toxicity evaluation technique. When the cardiomyocytes and cardiac organoids purified by a CD47 surface marker, of the present invention, are used, mature cardiomyocytes and cardiac organoids can be formed, and the cardiomyocytes and cardiac organoids are characterized in that mature cardiomyocytes having uniform contractile ability and a large surface area are formed at a high rate, and thus the present invention can be effectively used in cardiovascular disease modeling including that in toxicity evaluation and drug screening.

Description

Methods for producing cardiac organoids derived from human pluripotent stem cells, and techniques for evaluating their efficacy and toxicity.

[0001] This patent application claims priority to Republic of Korea Patent Application No. 10-2023-0090709, filed with the Korean Intellectual Property Office on July 12, 2023, and Republic of Korea Patent Application No. 10-2023-0130635, filed with the Korean Intellectual Property Office on September 27, 2023, the disclosures of which are incorporated herein by reference.

[0002] The present invention relates to a method for producing cardiac organoids derived from human pluripotent stem cells and a technique for evaluating their efficacy and toxicity.

[0003] Stem cells are undifferentiated cells that can differentiate into cells with specific functions through cell division, or they can self-renew through cell division into undifferentiated and pluripotent cells. Among stem cells, pluripotent stem cells (human pluripotent stem cells) are cells that develop from the initial fertilized egg cell before differentiating into various organs. They have the property of dividing into all cells of a fetus or adult. Embryonic stem cells and induced pluripotent stem cells (iPSCs) fall into this category.

[0004] Recently, the technology of creating organoids, organ-specific cell aggregates, using pluripotent stem cells has been attracting attention. Organoids are organoids created by culturing cells isolated from stem cells or organ-derived cells in 3D and then aggregating and recombining them. Organoids are characterized by their ability to form specific cells from organs such as the heart, stomach, intestines, and liver, reproducing specific organ functions, and spatially organizing them into structures similar to actual organs. Because organoids can mimic complex biological organs more effectively than conventional cultured cells due to their self-organization capabilities, they are expected to serve as valuable 3D models for understanding the development of early-stage organs, tissue interactions, and disease pathogenesis.

[0005] In particular, it is expected that leveraging the similarity of organoids to actual organs will overcome the limitations of existing animal models in clinical trials. Organoid models that recreate actual organs can address the challenges inherent in interspecies differences between humans and animals, address ethical concerns surrounding animal testing, and yield more biocompatible results than studies utilizing monolayer cultures or simply organized cells, thanks to organoids possessing organ-level three-dimensional structures.

[0006] Furthermore, organoids can be used to construct only the necessary organs, offering greater experimental accessibility compared to conventional bio-based research, facilitating the elucidation of molecular mechanisms and the application of cutting-edge research techniques. These characteristics make organoids ideal test subjects for assessing drug efficacy and safety during drug development. They are also a key next-generation biotechnology, applicable not only for toxicity and efficacy assessments in drug development but also for disease research, cancer treatment, and regenerative therapies.

[0007] Organs recreated through organoids include the stomach, intestines, liver, lungs, and brain. However, cardiac organoids fail to fully replicate the electrophysiological characteristics of the heart, exhibiting characteristics of immature cardiomyocytes. Structurally, sarcomere formation and arrangement are incomplete, and they do not resemble the atrium and ventricle of the living heart. Furthermore, long-term culture in a culture medium rich in oxygen and nutrients for vascular network formation is difficult, and ion channel formation is weak and drug responsiveness is incomplete. Furthermore, due to the wide range of beating capacity of cardiomyocytes, cardiac organoids produced using purified cardiomyocytes present difficulties in accurately assessing drug toxicity.

[0008] Since most cardiovascular heart diseases are characterized by the development of diseases after adulthood, the acquisition, culture, and maturation of a sufficient number of cardiomyocytes with a consistent contractile range are necessary to produce cardiac organoids for drug and toxicity evaluation of cardiovascular heart diseases.

[0009] Therefore, there is a need to develop cardiac organoids that can be manufactured by inducing differentiation and isolation of cardiomyocytes with a constant contractile range from pluripotent stem cells with high efficiency and by inducing differentiation of the cardiomyocytes.

[0010] Against this backdrop, the inventors of the present invention discovered that the contraction interval of cardiomyocytes purified with the CD47 surface marker was constant, and purified cardiomyocytes with high yield and high efficiency through the cultivation of the cardiomyocytes, and produced cardiac organoids using the same, thereby completing the present invention.

[0011] The purpose of the present invention is to provide a method for producing cardiomyocytes purified with a CD47 surface marker and a method for producing cardiac organoids through a culturing process of the cardiomyocytes.

[0012] Another object of the present invention is to provide a human pluripotent stem cell-derived cardiac organoid manufactured by the above manufacturing method.

[0013] Another object of the present invention is to provide a method for evaluating drug toxicity using the cardiac organoid.

[0014] Another object of the present invention is to provide a method for screening candidate drugs for treating cardiac diseases using the cardiac organoid.

[0015] In order to achieve the above purpose, the present invention provides a method for producing a human pluripotent stem cell, comprising the steps of: i) culturing human pluripotent stem cells to differentiate into embryonic stem cells;

[0016] ii) a step of culturing the differentiated embryonic body to differentiate into mesoderm;

[0017] iii) a step of culturing the differentiated mesoderm into cardiomyocytes; and

[0018] iv) Provided is a method for producing cardiomyocytes, including a step of purifying the cultured cardiomyocytes using a CD47 surface marker.

[0019] The present invention also provides a method for producing cardiac organoids derived from human pluripotent stem cells, which comprises a step of culturing cardiomyocytes produced through the above method for producing cardiomyocytes in a medium for differentiation of cardiac organoids.

[0020] The present invention also provides a human pluripotent stem cell-derived cardiac organoid manufactured through the above manufacturing method.

[0021] The present invention also provides a method for evaluating the toxicity of a drug, comprising the steps of treating the cardiac organoid with a drug; and measuring the activity of the organoid.

[0022] The present invention also provides a method for screening a candidate drug for treating a heart disease, comprising: treating a cardiac organoid with a candidate drug for treating a heart disease; and selecting the candidate drug as a therapeutic agent for treating a heart disease if the expression of a biomarker protein for a heart disease or an mRNA encoding the same in a group treated with the candidate drug is increased or decreased compared to before the treatment.

[0023] A method for producing purified cardiomyocytes using the CD47 surface marker of the present invention; a method for producing cardiac organoids through culturing the produced cardiomyocytes; and a method for producing cardiac organoids using the cardiac organoids produced by the method, wherein mature cardiomyocytes and cardiac organoids can be formed, and the cardiomyocytes and cardiac organoids exhibit the characteristics of having a high proportion of mature cardiomyocytes with uniform contractile ability and a large surface area, and thus can be usefully utilized in modeling cardiovascular diseases, including toxicity assessment and drug screening.

[0024] Figure 1 is a drawing that explores cardiomyocyte-specific surface markers among various surface markers available on the market.

[0025] Figure 2 is a diagram showing the differentiation process of cardiomyocytes purified using the iPSC-derived CD47 surface marker of the present invention.

[0026] Figure 3 is a diagram showing the contraction interval of cardiomyocytes purified using the CD47 surface marker.

[0027] Figure 4 is a diagram showing the results of maturation of purified cardiomyocytes using the CD47 surface marker and the results of immunostaining of matured cardiomyocytes.

[0028] Figure 5 is a diagram showing the results of electrophysiological analysis of cardiomyocytes purified with the CD47 surface marker.

[0029] Figure 6 is a diagram showing the results of a drug toxicity analysis using purified cardiomyocytes with a CD47 surface marker.

[0030] Figure 7 is a drawing showing the process of producing cardiac organoids using cardiomyocytes purified with a CD47 surface marker and the produced cardiac organoids.

[0031] i) A step of culturing human pluripotent stem cells to differentiate into embryonic stem cells;

[0032] ii) a step of culturing the differentiated embryonic body to differentiate into mesoderm;

[0033] iii) a step of culturing the differentiated mesoderm into cardiomyocytes; and

[0034] iv) A method for producing cardiomyocytes, comprising a step of purifying the cultured cardiomyocytes using a CD47 surface marker.

[0035] Hereinafter, the present invention will be described in detail.

[0036] Method for producing cardiomyocytes

[0037] In one aspect, the present invention comprises the steps of: i) culturing human pluripotent stem cells to differentiate into embryonic stem cells;

[0038] ii) a step of culturing the differentiated embryonic body to differentiate into mesoderm;

[0039] iii) a step of culturing the differentiated mesoderm into cardiomyocytes; and

[0040] iv) It relates to a method for producing cardiomyocytes, including a step of purifying the cultured cardiomyocytes using a CD47 surface marker.

[0041] The term “human Pluripotent Stem Cell (hPSC)” in this specification may refer to a cell that has the ability to differentiate into all cells that constitute the human body.

[0042] The above human pluripotent stem cells may be embryonic stem cells or induced pluripotent stem cells (iPSCs). Specifically, the embryonic stem cells are derived from the inner cell mass of a blastocyst prior to implantation, and the derived cells are capable of unlimited culture and pluripotent differentiation. The induced pluripotent stem cells may refer to pluripotent differentiated cells created through dedifferentiation from somatic cells, and may be formed by making somatic cells into a state similar to embryonic stem cells through a reprogramming process such as cell fusion, nuclear transfer, and overexpression of pluripotent regulatory factors.

[0043] In one embodiment of the present invention, the human pluripotent stem cell may be either an embryonic stem cell or an induced pluripotent stem cell, and for example, the human pluripotent stem cell may be an induced pluripotent stem cell.

[0044] The term "differentiation" in this specification generally refers to a phenomenon in which a relatively simple system is separated into two or more qualitatively different subsystems. Specifically, it can refer to a phenomenon in which structures or functions become specialized while cells divide and proliferate and grow, that is, a phenomenon in which cells, tissues, etc. of a living organism change in form or function in order to perform their respective assigned tasks.

[0045] The term "cardiomyocyte" as used herein may include, without limitation, a cell at any stage of differentiation of a cardiomyocyte precursor cell, a fetal cardiomyocyte, or an adult cardiomyocyte that has the ability to become a functional cardiomyocyte in the future, and may mean a cell that can be identified by multiple markers or criteria.

[0046] In cells having the characteristics of the above-mentioned cardiomyocytes, for example, gene expression of cardiomyocyte-specific transcription factors such as CD47, GATA-4, TEF-1, Tbx-5, MEF2, and MLC-2v, or expression of proteins such as sarcomere, myosin, troponin I, α-actinin, and ANP, which are cardiomyocyte-specific markers, can be confirmed.

[0047] According to a feature of the present invention, the cardiomyocytes express a CD47 surface marker, and by using the CD47 surface marker, the cardiomyocytes can be purified with high purity and high efficiency.

[0048] In one embodiment of the present invention, the step i) may include a step of culturing in a first differentiation medium containing Y27632 and EGF and then treating with EDTA; and a step of culturing in a second differentiation medium containing Pluronic F-127, N2 supplement, and FBS.

[0049] The concentration of Y27632 in the first differentiation medium of step i) above may be 5 to 20 μM / mL, 5 to 15 μM / mL, or 5 to 10 μM / mL, but is not limited thereto.

[0050] The concentration of EGF in the first differentiation medium of the above step i) may be, but is not limited to, 5 to 50 ng / mL, 5 to 45 ng / mL, 5 to 40 ng / mL, 5 to 35 ng / mL, 5 to 30 ng / mL, or 5 to 25 ng / mL.

[0051] The above step i) may be performed for 1 to 5 days, for example, for 1 day, or 2 days, or 3 days, or 4 days.

[0052] The concentration of EDTA in the above step i) may be, but is not limited to, 0.1 to 1.0 mM, 0.1 to 0.8 mM, or 0.1 to 0.6 mM.

[0053] The concentration of Pluronic F-127 in the second differentiation medium of step i) above may be 0.01 to 0.1 g / L, 0.01 to 0.08 g / L, or 0.01 to 0.06 g / L, but is not limited thereto.

[0054] The concentration of the N2 supplement in the second differentiation medium of step i) above may be, but is not limited to, 5 to 20%, 5 to 15%, or 5 to 10%.

[0055] The concentration of FBS in the second differentiation medium of step i) above may be 5 to 20%, 5 to 15%, or 5 to 10%, but is not limited thereto.

[0056] In one embodiment of the present invention, the differentiation medium of step ii) may include Pluronic F-127, BMP4, and FBS.

[0057] The concentration of Pluronic F-127 in the differentiation medium of step ii) above may be 0.01 to 0.1 g / L, 0.01 to 0.08 g / L, or 0.01 to 0.06 g / L, but is not limited thereto.

[0058] The concentration of BMP4 in the differentiation medium of step ii) above may be, but is not limited to, 5 to 50 ng / mL, 5 to 45 ng / mL, 5 to 40 ng / mL, 5 to 35 ng / mL, 5 to 30 ng / mL, or 5 to 25 ng / mL.

[0059] The concentration of FBS in the differentiation medium of step ii) above may be 5 to 20%, 5 to 15%, or 5 to 10%, but is not limited thereto.

[0060] The above step ii) may be performed for 1 to 8 days, for example, for 1 day, or 2 days, or 3 days, or 4 days, or 5 days, or 6 days.

[0061] In one embodiment of the present invention, step iii) may be a step in which cardiomyocytes are differentiated in a differentiation medium containing Pluronic F-127 and FBS.

[0062] The concentration of Pluronic F-127 in the differentiation medium of step iii) above may be 0.01 to 0.1 g / L, 0.01 to 0.08 g / L, or 0.01 to 0.06 g / L, but is not limited thereto.

[0063] The concentration of FBS in the differentiation medium of step iii) above may be 5 to 20%, 5 to 15%, or 5 to 10%, but is not limited thereto.

[0064] In one embodiment of the present invention, the differentiated cardiomyocytes may be contractile cardiomyocytes.

[0065] In one embodiment of the present invention, step iv) may be a step of confirming the expression of a CD47 surface marker in cardiomyocytes differentiated in step iii); and a step of isolating and purifying CD47-positive cells in which the expression of the CD47 surface marker is confirmed at the cell level.

[0066] The term "positive" as used herein means that a protein or gene is expressed in a detectable amount by at least any method known in the art. Proteins can be detected by immunological assays using antibodies, such as ELISA, immunostaining, or flow cytometry. For proteins expressed within cells but not on the cell surface (e.g., transcription factors or their subunits), a reporter protein can be co-expressed with the protein, and the target protein can be detected by detecting the reporter protein. Genes can be detected by nucleic acid amplification and / or nucleic acid detection methods, such as RT-PCR, biochips (e.g., microarrays), RNA seq, etc. Expression of a protein or gene can be determined by conventional methods. For example, if flow cytometry is used and the expression level is relatively high compared to the expression level of a control group showing negative protein expression, the protein can be determined to be detectably expressed.

[0067] The term "purification" in this specification means classifying differentiated cardiomyocytes into CD47- or CD47+ (positive) cardiomyocytes and selecting only pure CD47+ (positive) cardiomyocytes among them. In one embodiment of the present invention, "purifying cardiomyocytes with a CD47 surface marker" may mean selecting at least 50%, 55%, 60%, or 65%, preferably at least 70%, of CD47+ (positive) cardiomyocytes among differentiated cardiomyocytes.

[0068] In one embodiment of the present invention, the cardiomyocytes purified with the CD47 surface marker of step iv) may contract at a rate of 60 to 80 times per minute, for example, 65 to 78 times per minute, but are not limited thereto.

[0069] In one embodiment of the present invention, the cardiomyocytes purified with the CD47 surface marker of step iv) may be capable of evaluating the stability against cardiotoxic drugs, but is not limited thereto.

[0070] The above cardiotoxic drugs include, but are not limited to, nifedipine, dofetilide, mexiletine, terfenadine, isoproterenol, doxorubicin, daunorubicin, idarubicin, quinidine, cyclophosphamide, capecitabine, 5-fluorouracil, quinidine, and remdesivir, and safety assessments are available for all drugs to assess cardiotoxicity.

[0071] The term "stability" in this specification means that the cardiotoxicity of a drug can be evaluated by exhibiting a constant field potential, field potential duration, and Na+ peak (amplitude) value within a set error range for a cardiotoxic drug.

[0072] In one embodiment of the present invention, the cardiomyocytes purified with the CD47 surface marker of step iv) may have a mature ventricular type cardiomyocyte distribution of 75% or more, but is not limited thereto.

[0073] In the method for producing cardiomyocytes of the present invention, CD47 can be used as a surface marker specifically expressed in contractile cardiomyocytes. Therefore, when cardiomyocytes are stained with the CD47 marker, cells that do not express CD47, i.e., CD47-negative cells, may be undifferentiated stem cells, embryoid bodies, or non-contractile cardiomyocytes. According to the method for producing cardiomyocytes of the present invention, cases in which differentiation into cardiomyocytes is not induced from stem cells; and / or cases in which stem cells are differentiated into cardiomyocytes but are differentiated into non-contractile cardiomyocytes that do not exhibit contractile characteristics, can be easily excluded.

[0074] Method for producing cardiac organoids

[0075] In another aspect, the present invention relates to a method for producing cardiac organoids, which comprises a step of culturing cardiomyocytes produced by the above method for producing cardiomyocytes in a medium for differentiation of cardiac organoids, to form cardiac organoids derived from human pluripotent stem cells.

[0076] The term "organoid" as used herein may refer to a small cultured organism that possesses a structure, cellular composition, and function similar to a living (human) organ, and may have a three-dimensional structure that can have an environment similar to an actual living organ even in a laboratory. The organoid may have an environment that allows cells to interact with the surrounding environment during the growth process, and may serve as a preclinical and clinical model that can observe the development of disease treatment agents and toxicity assessments by almost perfectly simulating the organs interacting in the body.

[0077] The method for producing cardiac organoids by differentiating the above-mentioned cardiomyocytes into cardiac organoids can be used without limitation as long as it is a method that can be adopted for producing cardiac organoids according to conventional techniques. Specifically, to form cardiac organoids from cardiomyocytes, a cell culture microfluidic chip is used to induce aggregation of vascular endothelial cells for 24 hours, and then the purified cardiomyocytes with the CD47 surface marker are further aggregated for 24 hours to form cardiac organoids, as disclosed in the method disclosed in Korean Patent No. 10-2155868 (Patent Document 1).

[0078] In one embodiment of the present invention, the medium for differentiation of cardiac organoids may be a cell culture support, and the manufacturing method may use a three-dimensional cell culture microfluidic chip including an inoculation step of sequentially seeding two or more types of cells onto the cell culture support.

[0079] In the method of using the above microfluidic chip for cell culture, the inoculation step may specifically include the following steps (a) and (b).

[0080] (a) aggregation step of seeding and culturing vascular endothelial cells; and

[0081] (b) Co-culturing step of inoculating and culturing the above cardiomyocytes.

[0082] Cardiac organoids manufactured by the manufacturing method

[0083] In another aspect, the present invention relates to a human pluripotent stem cell-derived cardiac organoid produced by the above production method.

[0084] In one embodiment of the present invention, in evaluating the expression level and functionality of cardiac cell-specific genes, the cardiac organoid formed from cardiomyocytes expressing the CD47 surface marker can form cardiomyocytes with a more consistent contraction interval than models used in conventional drug efficacy and toxicity evaluations, and thus can be more similar in form and function to living organs.

[0085] The above cardiac organoid maintains a 1-second pulse interval similar to that of an adult heart, and when vascular endothelial cells are present inside the organoid, the pulse is maintained for more than 2 months compared to when only cardiomyocytes are aggregated, which allows for long-term analysis of drug responses to various drugs.

[0086] The above cardiac organoids may have the potential to be used for drug toxicity assessment in response to drugs and for screening candidate drugs for the treatment of cardiac diseases.

[0087] The above heart disease may be at least one selected from the group consisting of coronary artery disease, hypertension, angina pectoris, myocardial infarction, heart valve disease, heart failure, cardiac hypertrophy, arrhythmia, pericarditis, and endocarditis, but is not limited thereto.

[0088] Methods for evaluating drug toxicity

[0089] In another aspect, the present invention relates to a method for evaluating the toxicity of a drug, comprising the steps of treating a drug to the cardiac organoid; and measuring the activity of the organoid.

[0090] The term "drug" in this specification may refer to an individual nucleic acid, protein, other extract or natural product that is presumed or randomly selected to have the potential to prevent or treat heart disease according to a conventional selection method; and a drug that is presumed or confirmed to have toxic side effects that is currently on the market.

[0091] The above heart disease may be at least one selected from the group consisting of coronary artery disease, hypertension, angina pectoris, myocardial infarction, heart valve disease, heart failure, cardiac hypertrophy, arrhythmia, pericarditis, and endocarditis, but is not limited thereto.

[0092] Method for screening drug candidates for the treatment of heart disease

[0093] In another aspect, the present invention relates to a method for screening a candidate drug for treating a heart disease, comprising the steps of treating a cardiac organoid with a candidate drug for treating a heart disease; and selecting the candidate drug as a therapeutic agent for a heart disease if the expression of a biomarker protein for a heart disease or an mRNA encoding the same in a group treated with the candidate drug is increased or decreased compared to before the treatment.

[0094] The term "candidate drug" as used herein may encompass any substance used to modify or examine a physiological system or disease state for the benefit of an organism. For example, it may be a drug candidate, a test compound, or a test composition.

[0095] The above drug candidate may be at least one selected from the group consisting of cardiotonic agents, hyperlipidemic agents, diuretics, vitamin agents, vaccines, steroid agents, antibiotics, anticancer agents, antihistamines, chemotherapeutic agents, antihypertensive agents, and hormonal agents, but is not limited thereto.

[0096] The test compound or the test composition may be at least one selected from the group consisting of a low molecular weight compound, an antibody, an antisense nucleotide, a short interfering RNA (siRNA), a short hairpin RNA, hexane, a protein, a peptide, other extracts, and a natural extract, but is not limited thereto.

[0097] The above heart disease may be at least one selected from the group consisting of coronary artery disease, hypertension, angina pectoris, myocardial infarction, heart valve disease, heart failure, cardiac hypertrophy, arrhythmia, pericarditis, and endocarditis, but is not limited thereto.

[0098]

[0099] Hereinafter, the present invention will be described in more detail through examples. These examples are intended solely to illustrate the present invention, and the scope of the present invention is not limited by these examples.

[0100] Example 1. Exploration of cardiomyocyte-specific surface markers

[0101] After inducing differentiation of iPSCs into cardiomyocytes, each cell was lysed to obtain cell extracts. A global transcriptome assay was performed to determine the expression levels of mRNA contained in the extracts. From the results of the global transcriptome analysis, the mean and variance of genes were used to derive the cardiac and cardiac-related gene clusters of the global surface marker (CD) gene expression profile.

[0102] As a result of conducting a marker analysis, it was confirmed that the CD47 surface marker was specifically expressed on most cardiomyocytes. (Figure 1) Accordingly, the following examples were conducted to prepare cardiomyocytes purified with the CD47 surface marker.

[0103] Example 2. Induction of differentiation of cardiomyocytes from stem cells

[0104] 2-1. Formation of the reproductive body

[0105] First, to form human embryoid bodies (hEBs) for cardiomyocyte differentiation, undifferentiated iPSCs (Wicell, KDCA; KNIH) were cultured in medium containing 10 μM / mL Y27632 (Tocris) and 10 ng / mL EGF (R&D systems). After 1 day, 0.5 mM EDTA (Gibco) was treated, and then hEBs were formed by culturing in a medium containing 10% N2 supplement (Gibco) and 8% FBS (Hyclone) on 0.05 g / L Pluronic F-127 (Sigma)-coated culture dishes.

[0106] 2-2. Induction of mesoderm and cardiomyocyte differentiation

[0107] After confirming the formation of embryoid bodies, on the 4th day, the 0.05 g / L Pluronic F-127-coated culture dish was replaced, and the cells were cultured in culture medium containing 12 ng / mL BMP4 (R&D systems) and 8% FBS (Hyclone) to induce mesoderm. Then, to strengthen cardiomyocytes, on the 6th day, the Pluronic F-127-coated culture dish was replaced, and the cells were cultured in culture medium containing only FBS. The same operation was performed on the 8th, 10th, and 12th days to obtain differentiated cardiomyocytes.

[0108] 2-3. Purification of contractile cardiomyocytes

[0109] The obtained cardiomyocytes began to beat from the 10th day, and after confirming that beat occurred, the cardiomyocytes were dissociated with TryPLE (Gibco) and washed with PBS. The dissociated cells were suspended in PBS containing 5% fetal bovine serum (FBS, Hyclone), and the cardiomyocytes were separated using the cardiomyocyte-specific surface marker CD47 identified in Example 1 using an SH800S Cell Sorter flow cytometer and Cell sorter software Ver 2.1.2 (Sony Biotechnology). Flow cytometry analysis revealed that approximately 70% or more of the cells were CD47+ cells.

[0110]

[0111] Example 3. Analysis of purified cardiomyocytes using CD47 surface marker.

[0112] 3-1. Analysis of response axis spacing and shape

[0113] To analyze the contraction interval of purified cardiomyocytes using the CD47 surface marker, the contraction interval of cardiomyocytes observed for 1 minute using a real-time microscope was quantified, and the results are shown in Table 1 below.

[0114] ClassificationInterval (sac)Contracting No. (1 min)Culture PurificationCD47 PurificationCulture PurificationCD47 PurificationNo.10.680.868869No.20.760.887868No.31.020.905866No.40.880.826873No.50.800.787576Range (Interval)0.68 ~ 1.02 (0.34)0.78 ~ 0.90 (0.12)58 ~ 88(30)66 ~ 76(10)

[0115] As can be seen in Table 1 and FIG. 3 of the present invention, CD47 purified cardiomyocytes showed 66 to 76 contractions per minute and a contraction interval of 10, which was close to the heart rate of a typical adult of about 60 to 80 beats per minute, whereas the cardiomyocytes purified in a general culture medium showed 58 to 88 contractions and a contraction interval of 30, which was different from the heart rate of an adult. Therefore, it was confirmed that the cardiomyocytes purified with the CD47 surface marker had a constant contraction interval, similar to an actual heart, compared to the cardiomyocytes purified in a general cardiomyocyte culture medium.

[0116] In addition, as shown in Fig. 4 of the present invention, when CD47 purified cardiomyocytes were cultured for 2 weeks with 10% N2 supplement (Gibco) and 3% FBS (Hyclone), it was confirmed that in the case of CD47 purified cardiomyocytes, more than 90% of the cells were observed as mature cardiomyocytes after only 2 weeks of culture.

[0117] 3-2. Electrophysiological analysis

[0118] Electrophysiological analysis was performed on the mature cardiomyocytes analyzed in Example 3-1 above.

[0119] Specifically, cardiomyocytes sorted by the CD47 surface marker were analyzed in an external solution containing 130 mM NaCl, 5 mM KCl, 1 mM MgCl2, 1.8 mM CaCl2, 10 mM HEPES, and 12.5 mM glucose, pH 7.4 mOsm, at physiological temperature (37°C). To create cell patch clamp conditions, a microglass tube that adheres to the cell membrane was filled with an internal pipette solution consisting of 120 mM K-aspartate, 20 mM KCl, 10 mM HEPES, 5 mM EGTA, 1 mM MgCl2, 1.5 mM Mg-ATP, pH 7.3. Then, the voltage and current of cardiomyocytes were recorded using an Axopatch 200B amplifier, Digidata 1440A, and pClamp software 10.1 (Axon Instruments, USA), and -80 The current was measured in the sequence of 4-second depolarization to +40 mV and 2-second repolarization to -40 mV from the base membrane potential of mV, and the results are shown in Table 2 and Fig. 5.

[0120] Amplitude (mV)APD 80(ms)Vmax(mV / ms)MDP(mV)Beat period (ms)Atriallike hiPSC-CMs85.21± 7.25131.66± 18.4128.95± 6.20-58.11± 3.46234.08± 65.82Ventricular likehiPSC-CMs109.13± 9.43220.43± 15.6043.27± 9.32-69.10± 5.61648.97± 81.20

[0121] As shown in Table 2 and FIG. 5 of the present invention, the cardiomyocytes purified with the CD47 surface marker exhibited electrical signals of all three types of cardiomyocytes, and it was observed that ventricular-like cardiomyocytes were predominant at a high rate of more than 75% among the three types of cell groups, confirming that the cardiomyocytes purified with the CD47 surface marker had the function of mature ventricular cardiomyocytes.

[0122] Example 4. Toxicity analysis using purified cardiomyocytes with CD47 surface marker

[0123] Using CD47 purified cardiomyocytes, the cardiotoxicity of drugs known to be cardiotoxic was analyzed using remdesivir (MedChem Express) and quinidine (Sigma-Aldrich).

[0124] Field potential (FP), which is measured as spontaneous action potential of CD47+ cardiomyocytes, was recorded through a multi-electrode array (Multi-electrode Array, Maestro Pro, Axion BioSystem; MEA). FP signals were analyzed using the Cardiac Analysis tools 2.2.7 program (Axion Biosystems), and the results are presented in Tables 3 and 4 below and FIG. 6 of the present invention. The percentage changes in the analysis values, such as FP duration (FPD), modified FPD (FPDc; Fredericia's formula), beat period, and Na+ peak amplitude, according to treatment with each concentration of remdesivir (3 μM, 10 μM, 30 μM) and each concentration of quinidine (0.95 μM, 3 μM, 9.5 μM) are presented.

[0125] Amplitude (% change) Remdesivir culture fluid purified CD47 purified 3 μM 8 ± 29 ± 1.5 10 μM 17 ± 3.5 12 ± 1.5 30 μM 21 ± 320 ± 1.5

[0126] Amplitude (% change)Quinidine culture solution purifiedCD47 purified0.95 μM-5 ± 1-8 ± 1.53 μM-10 ± 3.5-12 ± 1.59.5 μM- 15 ± 5.5-22 ± 1.5

[0127] In the case of remdesivir, the risk of cardiotoxicity was detected within the margin of error at a concentration of 10 μM, but no cardiotoxicity was detected within the margin of error in CD47 purified cardiomyocytes. In the case of quinidine, the risk of cardiotoxicity was detected at a concentration of 9.5 μM in CD47 cardiomyocytes, but in the case of the general culture medium purification, the risk of cardiotoxicity was detected within the margin of error, so there may be limitations in securing reliability and stability in the toxicity results using the general culture medium purified cardiomyocytes. However, in the case of CD47 cardiomyocytes, cardiotoxicity was detected within a stable range, confirming the efficacy of the toxicity evaluation of CD47 cardiomyocytes.

[0128] Example 5. Production of cardiac organoids using purified cardiomyocytes with CD47 surface marker.

[0129] To produce cardiac organoids using purified cardiomyocytes labeled with the CD47 surface marker, a 3D cell culture microfluidic chip was used.

[0130] First, vascular endothelial cells were seeded onto a microfluidic chip for cell culture and aggregated, and then, on the second day, CD47+ cardiomyocytes purified using a CD47 surface marker were co-cultured (Fig. 7).

[0131] Specifically, endothelial cells were treated with 0.25% trypsin, incubated at 37°C for 2 minutes, centrifuged at 1,000 rpm for 5 minutes, and resuspended in medium to a final concentration of 5 x 105 cells / mL. Then, 2 days later, 200 mL of CD47+ cardiomyocytes induced to differentiate in Example 2 were seeded, cultured at 37°C for one day, and then further seeded and co-cultured on a culture support containing the endothelial cells. The cell culture medium was injected through the inlet of the microfluidic chip at a rate of 0.8 μL / min, and 500-1,000 μL of fresh culture medium was added to the culture storage container placed at the inlet at a 12-hour cycle.

[0132] After the above co-culture, when aggregation is induced for 24 hours, a cardiac organoid is formed in which vascular cells exist inside and cardiomyocytes exist outside, and its morphology is shown in Fig. 7 of the present invention.

[0133] The formed cardiac organoids are populated with purified cardiomyocytes with a CD47 surface marker, and maintain a regular beat of about 1 second, similar to an adult heart. It was confirmed that the beat was maintained for more than 2 months due to the presence of vascular endothelial cells inside, so cardiac organoids containing purified cardiomyocytes with a CD47 surface marker can be used for long-term analysis in various drug screenings and cardiotoxicity evaluations.

[0134] While the present invention has been described in detail through representative examples above, those skilled in the art will understand that various modifications to the above-described embodiments are possible without departing from the scope of the present invention. Therefore, the scope of the present invention should not be limited to the described embodiments, but should be defined not only by the claims described below but also by all changes or modifications derived from the claims and equivalent concepts.

[0135] The purpose of the present invention is to provide a method for producing cardiomyocytes purified with a CD47 surface marker and a method for producing cardiac organoids through a culturing process of the cardiomyocytes.

[0136] Another object of the present invention is to provide a human pluripotent stem cell-derived cardiac organoid manufactured by the above manufacturing method.

[0137] Another object of the present invention is to provide a method for evaluating drug toxicity using the cardiac organoid.

[0138] Another object of the present invention is to provide a method for screening candidate drugs for treating cardiac diseases using the cardiac organoid.

Claims

1. i) A step of culturing human pluripotent stem cells to differentiate into embryonic stem cells; ii) a step of culturing the differentiated embryonic body to differentiate into mesoderm; iii) a step of culturing the differentiated mesoderm into cardiomyocytes; and iv) A method for producing cardiomyocytes, comprising a step of purifying the cultured cardiomyocytes using a CD47 surface marker.

2. In paragraph 1, A method for producing cardiomyocytes, wherein the human pluripotent stem cells are either embryonic stem cells or induced pluripotent stem cells.

3. In paragraph 1, A method for producing cardiomyocytes, comprising: a step of culturing in a first differentiation medium containing Y27632 and EGF, followed by treatment with EDTA; and a step of culturing in a second differentiation medium containing Pluronic F-127, N2 supplement, and FBS.

4. In paragraph 1, A method for producing cardiomyocytes, wherein the differentiation medium of step ii) above contains Pluronic F-127, BMP4, and FBS.

5. In paragraph 1, The above step iii) is a method for producing cardiomyocytes, wherein the step is a step in which mesoderm is cultured in a differentiation medium containing Pluronic F-127 and FBS to differentiate cardiomyocytes.

6. In paragraph 5, A method for producing cardiomyocytes, wherein the differentiated cardiomyocytes are contractile cardiomyocytes.

7. In paragraph 1, A method for producing cardiomyocytes, wherein step iv) is a step of confirming the expression of a CD47 surface marker in cardiomyocytes differentiated in step iii); and a step of isolating and purifying CD47-positive cells in which the expression of the CD47 surface marker is confirmed at the cell level.

8. In paragraph 1, A method for producing cardiomyocytes, wherein the cardiomyocytes purified with the CD47 surface marker of step iv) above perform contraction at a rate of 60 to 80 times per minute.

9. In paragraph 1, A method for producing cardiomyocytes, wherein the cardiomyocytes purified with the CD47 surface marker of step iv) above can be used to evaluate the stability against cardiotoxic drugs.

10. In paragraph 1, A method for producing cardiomyocytes, wherein the cardiomyocytes purified with the CD47 surface marker of step iv) above have a distribution of mature ventricular type cardiomyocytes of 75% or more.

11. A method for producing cardiac organoids, comprising a step of culturing cardiomyocytes produced by the method of claim 1 in a medium for differentiation of cardiac organoids, to form cardiac organoids derived from human pluripotent stem cells.

12. A cardiac organoid derived from human pluripotent stem cells manufactured by the manufacturing method of Article 11.

13. In paragraph 12, The cardiac organoid is a cardiac organoid having the ability to respond to drugs and to be used for evaluating drug toxicity and screening for candidate drugs for treating cardiac diseases.

14. In Article 13, A cardiac organoid, wherein the above cardiac disease is at least one selected from the group consisting of coronary artery disease, angina pectoris, myocardial infarction, valvular heart disease, heart failure, cardiac hypertrophy, arrhythmia, pericarditis, and endocarditis.

15. A method for evaluating the toxicity of a drug, comprising the step of treating an organoid according to Article 12 with a drug; and the step of measuring the activity of the organoid.

16. A method for screening a candidate drug for treating a heart disease, comprising: treating an organoid according to claim 12 with a candidate drug for treating a heart disease; and selecting the candidate drug as a therapeutic agent for a heart disease when the expression of a biomarker protein for a heart disease or an mRNA encoding the same in the group treated with the candidate drug is increased or decreased compared to before the treatment.

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