Myocardial cell population, pharmaceutical composition, method for preparing myocardial cell population, and myocardial cell spheroid

Abstract

The myocardial cell population comprises myocardial cells obtained by differentiation of human pluripotent stem cells, and the myocardial cell population has the following characteristics: (1) cardiac troponin T positive cells account for 90% or more; (2) the spontaneous pulsation is 0 to 60 times per minute; and (3) the myocardial cell population is a cell population within 30 days from the start of differentiation. The invention also relates to a pharmaceutical composition which comprises the myocardial cell population and / or myocardial cell spheres obtained by forming the myocardial cell population into spheres, and a pharmaceutically acceptable carrier. A method of preparing the myocardial cell population comprises: (a) expanding human pluripotent stem cells; (b) culturing the expanded human pluripotent stem cells under conditions to differentiate into cardiomyocytes to produce a cell population comprising 60% or more of cardiomyocytes; and (c) removing human pluripotent stem cells and non-myocardial cells from the cell population.

Description

TECHNICAL FIELD The present application relates to a cardiac myocyte population, a pharmaceutical composition, a method for producing a cardiac myocyte population, and a cardiac myocyte spheroid. BACKGROUND When a large number of cardiac myocytes are lost due to a heart attack, the cardiac muscle tissue is eventually replaced with fibrous tissue that has no contractile function, resulting in heart failure throughout the patient's life. At present, clinical applications of cardiac repair based on human pluripotent stem cell-derived cardiac myocytes are under investigation. It is expected that by transplanting human pluripotent stem cell-derived cardiac myocytes (hereinafter referred to as "hPSC-CM") into the cardiac muscle tissue, regeneration of the cardiac muscle tissue can be achieved. Since hPSC-CM can be theoretically produced in large quantities in vitro, regenerative medicine using hPSC-CM is considered to have great potential.

[0003] To date, numerous researchers including the present inventors have disclosed methods for producing high-purity human pluripotent stem cell-derived cardiac myocytes in large quantities (see Patent Documents 1 to 3). Proof-of-concept experiments involving transplantation of these cardiac myocytes into animal models include, for example, experiments in which a cardiac myocyte population obtained by inducing differentiation of human ES cells using activin A and BMP4 was transplanted into an induced myocardial infarction rat (see Non-Patent Document 1), and experiments in which cardiac myocytes differentiated from human iPS cells were transplanted into an infarcted heart of an immunodeficient mouse (see, for example, Non-Patent Document 2). In these experiments, it was confirmed that the transplanted cardiac myocyte population survived in the recipient heart for a certain period of time. In addition, in order to improve the chimeric rate of the cardiac myocyte population in the recipient cardiac muscle tissue, various strategies were evaluated, including co-transplantation with Matrigel (registered trademark) and promotion of PSC-CM proliferation by genetic modification. These techniques showed effects in improving the chimeric rate, but most of them are currently not suitable for clinical applications.

[0004] In addition, simply chimerizing hPSC-CM into a recipient heart is not sufficient to achieve cardiac regeneration therapy. For cardiac regeneration therapy, electrical integration between the transplanted cardiac myocytes and the recipient cardiac myocytes is essential.

[0005] In experiments in which human ES cell-derived cardiac myocytes were transplanted into a damaged heart of a guinea pig, it was confirmed that electrical integration between the recipient cardiac myocytes and the transplanted cardiac myocytes was achieved in the damaged heart (see, for example, Non-Patent Documents 3 and 4, and Patent Document 4).

[0006] However, the results obtained from these experiments cannot confirm whether cardiac regeneration can be safely implemented and achieved in a clinical-scale cardiac myocyte transplantation, because the experimental animal models used are not physiologically suitable for simulating clinical applications.

[0007] In addition, experiments have been conducted using primate and pig models closer to humans. However, the cardiomyocytes differentiated from human pluripotent stem cells generally exhibit a fetal or neonatal phenotype. Thus, there are significant differences between human pluripotent stem cell-derived cardiomyocytes and human heart cardiomyocytes in terms of electrical properties, particularly maturity. Thus, new problems have been reported, such as limited survival of transplanted pluripotent stem cell-derived cardiomyocytes, and induction of transplant-related arrhythmia in the recipient heart by pluripotent stem cell-derived transplanted cardiomyocytes (see Non-Patent Literature 5, 6, 7, 8). In order to suppress transplant-related arrhythmia, methods for reducing arrhythmia by anti-arrhythmic drugs have been disclosed (see Non-Patent Literature 9), but these methods have problems such as a greater burden on the recipient. In addition, there are reports on modification of cells related to ion channels (see Non-Patent Literature 10). However, the use of genetically edited cells as transplanted cells has risks such as adverse mutations and unpredictable effects due to unintended mutations at unintended sites caused by gene editing, and is not ideal because of the complexity of the manufacturing and quality control processes.

[0008]

Prior Art Documents

SUMMARY

[0009] II. Solution to the problem Therefore, the present application aims to provide a human pluripotent stem cell-derived cardiomyocyte population capable of satisfying the conditions required for clinical applications, a cardiomyocyte spheroid formed from the cardiomyocyte population, a pharmaceutical composition comprising the cardiomyocyte population and / or the cardiomyocyte spheroid as an active ingredient, a method for manufacturing the cardiomyocyte population, and a method for manufacturing the cardiomyocyte spheroid.

[0010] The present application includes the following aspects: [1] A cardiomyocyte population comprising ventricular cardiomyocytes differentiated from human pluripotent stem cells, wherein the cardiomyocyte population has the following characteristics: (1) cardiomyosin T-positive cells account for 90% or more of all viable cells; (2) the spontaneous beating frequency is 0 to 60 beats per minute; and (3) the cardiomyocyte population is a population of cells within 30 days from the start of self-differentiation.

[0011] [2] The cardiomyocyte population according to [1], wherein the spontaneous beating frequency is 0 to 50 beats per minute.

[0012] [3] A pharmaceutical composition comprising at least one of the following groups: the cardiomyocyte population according to [1] or [2], and a cardiomyocyte spheroid obtained by forming the cardiomyocyte population into a spheroid, and a pharmaceutically acceptable carrier.

[0013] [4] The pharmaceutical composition according to [3], wherein the viable cells in the cardiomyocyte population account for 80% or more of all cells.

[0014] [5] The pharmaceutical composition according to [3], wherein the cells constituting the cardiomyocyte spheroid account for 60% or more of all cells in the pharmaceutical composition.

[0015] [6] The pharmaceutical composition according to [3] for use in the treatment of heart failure.

[0016] [7] A method for preparing a population of cardiomyocytes according to [1] or [2], comprising: (a) expanding human pluripotent stem cells in culture; (b) culturing the expanded human pluripotent stem cells under conditions for inducing differentiation into cardiomyocytes to generate a cell population comprising more than 60% cardiomyocytes; and (c) removing human pluripotent stem cells and non-cardiomyocytes from the cell population.

[0017] [8] A spheroid of cardiomyocytes, obtained by forming a spheroid shape from a group of cardiomyocytes as described in [1] or [2].

[0018] [9] The cardiomyocyte spheres according to [8], wherein the diameter of the spheres is 50 to 300 micrometers.

[0019]

[10] A method for preparing cardiomyocyte spheroids, comprising: suspending a group of cardiomyocytes according to [1] or [2] in a culture medium and culturing the suspension in a culture container with micropores at the bottom.

[0020] III. Effects of the Invention According to the present invention, by optimizing the purity, maturity, and spontaneous beating frequency of cardiomyocyte populations, cardiomyocyte populations suitable for safe clinical transplantation can be obtained. These cardiomyocyte populations can achieve electrical integration with recipient cardiomyocytes after transplantation, maintain maturity, and survive long-term. They can effectively regenerate damaged myocardial tissue and exhibit a low incidence of persistent severe ventricular arrhythmias induced in the recipient. Therefore, the cardiomyocyte populations, their derived cardiomyocyte spheroids, and pharmaceutical compositions provided by the present invention are suitable for the treatment of heart diseases such as heart failure. [Attached Image Description] Figure 1A (b) Flow cytometry analysis results of the cardiac troponin T positivity rate of the human iPS cell-derived cardiomyocyte population prepared in Example 1 (1).

[0022] (a) Flow cytometry analysis results showing the positive rate of cardiac troponin T in the pre-purification cell population.

[0023] Figure 1B The action potential pattern obtained by patch-clamp electrophysiological analysis of the human iPS cell-derived cardiomyocyte population prepared in Example 1 (1) is shown.

[0024] Figure 1C The spontaneous beat rate, maximum diastolic potential (MDP), and action potential duration (APD90) at 90% repolarization are shown in the electrophysiological analysis of the human iPS cell-derived cardiomyocyte population prepared in Example 1(1) using the patch-clamp method.

[0025] Figure 1DThe action potential waveforms obtained from the drug responsiveness analysis of the human iPS cell-derived cardiomyocyte population prepared in Example 1(1) under the action of isoproterenol, amiodarone and ivabradine are shown.

[0026] Figure 1E The spontaneous beat frequency per minute of the human iPS cell-derived cardiomyocyte population prepared in Example 1 (1) is shown under the action of isoproterenol, amiodarone and ivabradine.

[0027] Figure 1F The results of immunocytochemical analysis of the human iPS cell-derived cardiomyocyte population prepared in Example 1 (1) are shown.

[0028] Figure 1G The results of spontaneous beating frequency measurements of purified human iPS cell-derived cardiomyocytes (purified hiPSC-CMs) and unpurified cell populations (pre-purified hiPSC-CMs) prepared in Example 1(1) are shown.

[0029] Figure 2A The survival rate of cardiomyocytes contained in the cardiomyocyte spheres prepared in Example 2(1) is shown.

[0030] Figure 2B The results of measuring the sarcomere length of the host cardiomyocytes and the transplanted cardiomyocytes after transplanting the cardiomyocytes prepared in Example 2 (1) into cynomolgus monkeys are shown in Example 3.

[0031] Figure 2C The results of long-term culture and immunohistochemical staining of cardiomyocytes prepared in Example 2(1) are shown in Example 2(2).

[0032] Day 28: 28 days after the start of culture; Day 56: 56 days after the start of culture; Day 84: 84 days after the start of culture; A, D, G: stained with CytoRed and DAPI; B, E, H: stained with cardiac troponin T (cTnT) and DAPI; C, F, I: stained with CytoRed, cTnT, and DAPI.

[0033] Figure 2D The results of bioluminescence signal detection are shown in Example 2(2), after human iPS cell-derived dispersed cardiomyocytes expressing modified luciferase and cardiomyocyte spheroids expressing modified luciferase (hiPSC-CSs) were transplanted into mature male NOG mice.

[0034] Figure 2E ShowFigure 2D A graph showing the bioluminescent signals detected in the sample.

[0035] Figure 2F In Example 3, cardiomyocyte spheroids (equivalent to 2 × 10⁻⁶ cells) prepared in Example 2(1) are shown. 7 After transplanting cardiomyocytes into cynomolgus monkeys, the trough concentration of cyclosporine in their peripheral blood was measured. The shaded area represents the range of trough cyclosporine concentrations required to suppress acute rejection after heart transplantation.

[0036] Figure 2G In Example 3, cardiomyocytes prepared in Example 2(1) (equivalent to 6 × 10⁻⁶ cells) are shown. 7 The results of measuring the trough concentration of cyclosporine in the peripheral blood of cynomolgus monkeys after transplantation of cardiomyocytes (number of cardiomyocytes) were obtained. The shaded area represents the range of trough concentrations of cyclosporine used to suppress acute rejection after heart transplantation.

[0037] Figure 3A The results of left ventricular shortening rate (FS) measured by echocardiography in Example 3 are shown before transplantation of cardiomyocytes or vectors prepared in Example 2(1) in cynomolgus monkeys, 4 weeks post-transplantation, and 12 weeks post-transplantation.

[0038] Figure 3B The Holter electrocardiogram results of cynomolgus monkeys that exhibited transient ventricular tachycardia after transplantation of cardiomyocyte spheroids prepared in Example 2 (1) are shown in Example 3.

[0039] Figure 3C The Holter electrocardiogram results after transplanting cardiomyocyte spheres or carriers prepared in Example 2(1) into cynomolgus monkeys are shown in Example 3.

[0040] Figure 4A The histological examination results of the myocardial cell spheroids prepared in Example 2(1) after transplantation into the cynomolgus monkey in Example 3 are shown.

[0041] Figure 4B The histological examination results of the myocardial cell spheroids prepared in Example 2(1) after transplantation into the cynomolgus monkey in Example 3 are shown.

[0042] Figure 5A The following are cardiac ultrasound results of the cynomolgus monkeys transplanted with cardiomyocyte spheres or carriers prepared in Example 2 (1) before transplantation, 4 weeks after transplantation, and 12 weeks after transplantation. Scale bar is 5 cm.

[0043] Figure 5B This illustrates Example 3, where cynomolgus monkeys were transplanted with cardiomyocyte spheroids prepared in Example 2(1) (equivalent to 6 × 10⁻⁶ cells).7 The results of left ventricular ejection fraction (LVEF) and left ventricular shortening rate (FS) measurements were obtained from cardiomyocytes or vectors before transplantation, 4 weeks after transplantation, and 12 weeks after transplantation.

[0044] Figure 5C The Holter electrocardiogram results after transplanting cardiomyocyte spheres or carriers prepared in Example 2(1) into cynomolgus monkeys are shown in Example 3.

[0045] Figure 5D The results of left ventricular shortening fraction (FS) measured by echocardiography were shown in Example 3 after cynomolgus monkeys were transplanted with human iPS cell-derived cardiomyocytes or vectors.

[0046] hiPSC-CSs (purified): Cardiac cell spheroids prepared in Example 2(1); hiPSC-CSs (unpurified): Cardiac cell spheroids prepared from unpurified hiPSC-CSs.

[0047] Figure 5E The results of serum cardiac troponin T (cTnT) and brain natriuretic peptide (BNP) measurements are shown in Example 3 after cynomolgus monkeys transplanted cardiomyocyte spheres or carriers prepared in Example 2 (1).

[0048] A, B: Transplantation 2×10 7 C, D: 6 × 10 cardiomyocytes transplanted; 7 Each myocardial cell.

[0049] Figure 5F The duration of arrhythmia in an individual cynomolgus monkey that developed transient arrhythmia after transplantation of cardiomyocytes prepared in Example 2 (1) is shown in Example 3.

[0050] *p<0.05 compared to pre-Tx; #p<0.05 indicates difference between the vector group and the hiPSC-CSs group.

[0051] Figure 6 The histological examination results of the heart (A), lung (B, F), liver (C, G), kidney (D, H) and spleen (E, I) after transplantation of cardiomyocyte spheroids prepared in Example 2(1) in cynomolgus monkeys are shown in Example 3.

[0052] The scale in A–D is 1 cm; the scale in E is 5 mm; and the scale in I is 200 μm.

Detailed Implementation Methods

[0054] The term "comprise" indicates that the target component may also contain other components besides the target component. The term "consist of" indicates that the target component does not contain other components besides the target component. The term "consist essentially of" indicates that other components besides the target component are not present in a manner that performs a particular function (e.g., in a manner that does not completely negate the effects of the invention). In this specification, when "comprise" is used, it also includes the meanings of "consist of" and "consist essentially of".

[0055] The proteins, peptides, polynucleotides (DNA, RNA), carriers, cells, cell populations, and cardiomyocytes described herein can be "isolated." "Isolated" means separated from its natural state or from other components. "Isolated" can also mean that the component is substantially free of other components. "Substantially free of other components" means that the amount of other components contained in the isolated component is negligible. The content of other components in the isolated component can, for example, be no more than 10% by mass, no more than 5% by mass, no more than 4% by mass, no more than 3% by mass, no more than 2% by mass, no more than 1% by mass, no more than 0.5% by mass, or no more than 0.1% by mass. The proteins, peptides, polynucleotides (DNA, RNA), carriers, cells, cell populations, and cardiomyocytes described herein can each be isolated proteins, isolated peptides, isolated polynucleotides (isolated DNA, isolated RNA), isolated carriers, isolated cells, isolated cell populations, and isolated cardiomyocytes, respectively.

[0056] In this specification, the percentage of a specific cell in a cell population refers to the proportion of that specific cell number to the total number of cells in the cell population, calculated by the following formula: Percentage of a specific cell type (%) = Number of specific cells / Total number of cells in the cell population × 100 (cardiomyocyte population) The first embodiment of this disclosure relates to a cardiomyocyte population comprising ventricular cardiomyocytes differentiated from human pluripotent stem cells. This cardiomyocyte population has the following characteristics (1), (2), and (3): (1) Cardiac troponin T positive cells accounted for more than 90% of all surviving cells; (2) The spontaneous pulsation rate is 0–60 beats per minute; (3) The cardiomyocyte population is within 30 days of the start of self-differentiation.

[0057] The myocardial cell population of this embodiment, having the above-mentioned characteristics (1) to (3), can meet the following conditions (i) to (iv) required for clinical application: (i) The cardiomyocyte population is of high purity and can be easily manufactured; (ii) It can be safely transplanted into the human body; (iii) It can achieve electrical synchronization with the cardiomyocytes of the recipient and survive for a long time while maintaining maturity, thereby regenerating the damaged parts of the recipient's heart; (iv) The incidence of severe ventricular arrhythmias that persist for a period of time in the receptor is low.

[0058] The cardiomyocyte population of this embodiment preferably satisfies one or more of the above conditions (i) to (iv), more preferably satisfies two or more, even more preferably satisfies three or more, and especially preferably satisfies all four conditions. Because it satisfies the above conditions (i) to (iv), the cardiomyocyte population of this embodiment can be considered a clinically usable cardiomyocyte population.

[0059] Human pluripotent stem cells In this article, "pluripotent stem cells" refers to cells with self-renewal capacity and the ability to differentiate into multiple lineages. Specific examples include embryonic stem cells (ES cells), induced pluripotent stem cells (iPS cells), embryonic germ cells (EG cells), germ cell stem cells (GS cells), and cells derived from these pluripotent stem cells that possess pluripotency. "Pluripotent stem cells" are not limited to known cells with self-renewal capacity and the ability to differentiate into multiple lineages; they may also include unknown cells with properties equivalent to the aforementioned ES cells or iPS cells.

[0060] Whether a cell is a pluripotent stem cell can be determined based on whether it possesses pluripotent stem cell-specific properties or expresses pluripotent stem cell-specific markers. For example, pluripotent stem cell-specific properties include self-renewal capacity and the ability to differentiate into other cell types that differ from pluripotent stem cell properties. Teratoblastic and chimeric cell formation abilities are also examples of pluripotent stem cell-specific properties.

[0061] Pluripotent stem cell-specific markers (hereinafter referred to as "pluripotent markers") refer to factors specifically expressed in pluripotent stem cells, such as Oct3 / 4, Nanog, Sox2, SSEA-1, SSEA-3, SSEA-4, TRA1-60, TRA1-81, Lin28, Fbx15, SSEA-5, GDF3, KLF4, CLDN6, etc. If the expression of at least one of the above pluripotent markers is observed, the cell can be identified as a pluripotent stem cell. Pluripotent markers can be used alone or in combination of two or more. As an example, cells expressing Oct3 / 4 can be identified as pluripotent stem cells. The expression of pluripotent markers in cells can be confirmed using known methods such as RT-PCR or microarray analysis.

[0062] Human pluripotent stem cells can be human ES cells, human iPS cells, human EG cells, or human GS cells. Human ES cells or human iPS cells are preferred, with human iPS cells being more preferred. "Human iPS cells" refers to pluripotent stem cells artificially induced from non-pluripotent cells (such as adult somatic cells), and their preparation methods are well known in the art. Specifically, they can be prepared by introducing one or more reprogramming factors into somatic cells such as fibroblasts, hematopoietic cells, or epidermal cells. Examples of reprogramming factors include Oct3 / 4, Sox2, c-Myc, l-Myc, Klf4, Nanog, and Lin28. Pluripotency can be confirmed by detecting the expression of the above stem cell-specific genes and / or proteins, and cells can be selected accordingly.

[0063] The Kyoto University iPS Cell Research Foundation (53 Shogoin-Kawahara-cho, Sakyo-ku, Kyoto-shi, Kyoto Prefecture, Japan, Public Interest Incorporated Foundation) provides several human iPS cell lines. These human iPS cell lines can be used as human pluripotent stem cells. Specific examples of the human iPS cell lines provided by the foundation include the Ff-I01, Ff-I14, Ff-I01s01, Ff-I14s03, Ff-I14s04, QHJI-I14s04, Ff-MH09s01, and Ff-MH15s02 lines. The Ff-I14, Ff-I14s04, and QHJI-I14s04 lines are particularly preferred. All of the above iPS cells are HLA haplotype homozygous human iPS cells. For clinical use, clinical-grade cell lines are preferred. Human iPS cells can also be obtained from cell banks such as ATCC, and commercially available human iPS cells can also be used.

[0064] <cardiac cell population> The cardiomyocyte population of this embodiment comprises cardiomyocytes differentiated from human pluripotent stem cells (hPSC-CMs), and particularly includes mature ventricular cardiomyocytes. In this document, cardiomyocytes are defined as cells positive for at least one of sarcomeric α-actinin, cardiac troponin T (cTnT), or troponin I type 1 (TNNI1). Ventricular cardiomyocytes are defined as cells positive for cTnT and / or myosin light chain 2v (MLC2v). Furthermore, cardiomyocytes typically exhibit spontaneous beating activity. Ventricular cardiomyocytes are a class of cardiomyocytes that can constitute ventricular myocardium.

[0065] Furthermore, the term "cardiac progenitor cells" as used in this article refers to precursor cells of cardiomyocytes, which are positive for at least one of Nkx2.5, GATA4, MEF2C, or MES1. The term "non-cardiomyocytes" as used in this article refers to cells that are neither cardiomyocytes nor cardiac progenitor cells, specifically including smooth muscle cells and endothelial cells.

[0066] The cardiomyocyte population in this embodiment meets all of the following conditions: (1) cTnT positive cells accounted for more than 90% of all surviving cells; (2) The spontaneous pulsation rate is 0–60 beats per minute; and (3) The myocardial cell population is within 30 days of the start of self-differentiation.

[0067] In this document, "cell population" refers to a collection of two or more cell types of the same or different kinds. "Cardiomyocyte population" refers to a cell population containing cardiomyocytes, which may consist solely of cardiomyocytes or further include non-cardiomyocytes. In this embodiment, at least a portion of the cardiomyocytes in the cardiomyocyte population are ventricular cardiomyocytes. This cell population may also include cardiomyocytes other than ventricular cardiomyocytes (e.g., cardiac progenitor cells, atrial cardiomyocytes, etc.). The cardiomyocyte population of this invention can be prepared from human pluripotent stem cells according to the following method.

[0068] <(1) cTnT positive cells account for more than 90% of all surviving cells> cTnT is a protein that makes up myofibrils of the cardiac muscle and is a well-known marker of cardiomyocytes. cTnT-positive cells are considered cardiomyocytes. Human cTnT is a protein encoded by the TNN2 gene (Gene ID: 7139).

[0069] In this article, "positive" means that the expression of a target protein or gene can be detected in cells using methods known in the art. Protein detection can be performed using immunological assays using antibodies, such as ELISA, immunofluorescence staining, or flow cytometry. For proteins expressed intracellularly rather than on the cell surface (e.g., transcription factors or their subunits), the target protein can also be detected by co-expressing a reporter protein and detecting that reporter protein. Gene detection can be performed using nucleic acid amplification and detection methods, such as RT-PCR, microarray analysis, or RNAseq.

[0070] In this article, "negative" means that the expression level of the target protein or gene in cells is below the detection limit of any of the methods mentioned above. The detection limit varies depending on the method and can be determined using conventional techniques.

[0071] The proportion of cTnT-positive cells in the cardiomyocyte population is not particularly limited as long as it reaches 90% or more of the surviving cells. Preferably, it is not less than 90%, more preferably not less than 95%, further preferably not less than 96%, even more preferably not less than 97%, still further preferably not less than 98%, and especially preferably not less than 99% or not less than 99.5%. The percentage of cTnT-positive cells, with the total number of surviving cardiomyocytes as the denominator, can be, for example, 90–100%, 95–100%, 96–100%, 97–100%, 98–100%, 99–100%, or 99.5–100%.

[0072] The percentage of cTnT-positive cells among all surviving cells can be calculated by determining the number of cTnT-positive cells using any of the methods described above and then comparing it to the total number of surviving cardiomyocytes determined by a commonly used method (e.g., flow cytometry threshold). Preferably, flow cytometry analysis using an antibody against cTnT is used to confirm the number of cTnT-positive cardiomyocytes. Anti-cTnT antibodies for flow cytometry are commercially available. For example, surviving cells in a cardiomyocyte population can be selected by flow cytometry or similar methods, and the number of cTnT-positive cells among the selected surviving cells can be determined, thereby calculating the ratio of cTnT-positive cells to all surviving cardiomyocytes. For example, in the data obtained from flow cytometry analysis of a cardiomyocyte population, a surviving cell population can be selected by thresholding, and the ratio of cTnT-positive cells can be obtained based on the expression level of cTnT in the selected surviving cell population.

[0073] The selection of viable cells from a myocardial cell population can be carried out using methods such as forward scattering, side scattering, or staining with live dyes (e.g., nucleic acid-binding dyes, protein-binding dyes).

[0074] <(2) Spontaneous pulsation rate is 0–60 beats per minute> The spontaneous pulsation rate of the myocardial cell population in this embodiment is 0-60 beats per minute, preferably 0-50 beats per minute, more preferably 0-45 beats per minute, and particularly preferably 0-35 beats per minute. The spontaneous pulsation rate can also be 10-60, 10-50, 10-45, 10-40, or 10-35 beats per minute; or it can be 20-60, 20-50, 20-45, 20-40, or 20-35 beats per minute.

[0075] The spontaneous beat rate of a cardiomyocyte population (i.e., the number of spontaneous beats per unit time) can be determined using methods known in the art. Methods that can be used to determine the spontaneous beat rate are those capable of analyzing cell motion. For example, images or videos of the cardiomyocyte population can be acquired under a microscope and analyzed using motion vectors; or the potential patterns (spontaneous action potentials) that change with cell beating can be recorded. Specific examples include using a patch-clamp amplifier to measure the action potential duration (APD90) and spontaneous action potentials at 90% repolarization, or using multi-electrode systems, calcium imaging, impedance measurements, etc.

[0076] The aforementioned spontaneous beat rate is preferably measured with an appropriate number of cells suitable for the above method; multiple measurements can be performed, and the results can be statistically processed to obtain numerical values. Specifically, a patch-clamp amplifier can be used to record the APD90 and spontaneous action potentials of a cardiomyocyte population. The intracellular fluid used for action potential measurement may contain: potassium gluconate (130 mmol / L), KCl (10 mmol / L), NaCl (5 mmol / L), MgCl2 (1 mmol / L), ethylene glycol bis(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA: 0.1 mmol / L), magnesium-bound adenosine triphosphate (0.1 mmol / L), and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES: 10 mmol / L). Extracellular fluid may contain: NaCl (136.5 mmol / L), KCl (5.4 mmol / L), CaCl2 (1.8 mmol / L), MgCl2 (0.53 mmol / L), HEPES (85.5 mmol / L) and glucose (5.5 mmol / L).

[0077] <(3) Within 30 days of the start of differentiation> In this article, "initiation of differentiation" refers to the point in time when human pluripotent stem cells are placed in a differentiation induction medium (i.e., a medium containing substances that induce differentiation into cardiomyocytes) to begin culturing. "Within 30 days from the initiation of differentiation" refers to the period from the start of the culture until 30 days have elapsed.

[0078] As described herein, the cardiomyocyte population of this embodiment can be obtained through: (A) a differentiation induction process that induces pluripotent stem cells to differentiate into cardiomyocytes, and (B) a process that removes pluripotent stem cells and non-cardiomyocytes. The cardiomyocyte population of this embodiment is obtained by completing process (B) within 30 days from the start of process (A).

[0079] Traditionally, cells cultured for approximately 30 days from the induction of human pluripotent stem cells into cardiomyocytes correspond to the fetal-neonatal stage of human cardiomyocytes, being smaller in size and less mature than adult cardiomyocytes. On the other hand, it is known that extending the culture time to more than 40 days improves the maturity of cardiomyocytes (see, for example, Dhahri W. et al., Circulation, 145:1412–1426 (2022)). However, long-term culture is expensive and impractical, and there are problems such as an increase in non-cardiomyocytes and decreased cell adhesion with prolonged culture, making it unsuitable for clinical application.

[0080] The cardiomyocyte population of this embodiment retains the characteristics of (1) and (2) described above even during the culture period of up to 30 days from the start of differentiation, and contains mature ventricular cardiomyocytes with high purity. Therefore, it is a cardiomyocyte population suitable for clinical application.

[0081] Furthermore, as described later in the method for manufacturing cardiomyocyte populations, the method for obtaining mature and highly pure cardiomyocytes by limiting the culture period from the initiation of cardiomyocyte differentiation to the completion of purification to within 30 days has significant advantages for clinical use.

[0082] If the cardiomyocyte population of this embodiment is cryopreserved after being manufactured through processes (A) and (B) described above, the cryopreservation period is excluded from the calculation of the period "within 30 days from the start of differentiation". That is, the period from the start of cryopreservation of the cardiomyocyte population to the completion of thawing of the cryopreserved cardiomyocyte population is excluded from the period "within 30 days from the start of differentiation".

[0083] <Other characteristics> The aforementioned characteristic (2) of the cardiomyocyte population reflects the maturity of the cardiomyocytes, and the characteristic of "spontaneous beat rate of 0-60 beats per minute" indicates that the cell population contains a large number of ventricular cardiomyocytes. Furthermore, since the presence of pacemaker cells and / or atrial cardiomyocytes in the population increases the beat rate, characteristic (2) also indicates a low content of these types of cells. The cardiomyocyte population of this embodiment can alternatively or additionally be characterized by containing a high proportion of cells positive for the ventricular marker MLC2v. Preferably, when the cardiomyocyte population of this embodiment is transplanted into the recipient myocardial tissue, the proportion of MLC2v positive cells is at least 70%, more preferably at least 80%, and most preferably at least 90%.

[0084] In clinical use, cardiomyocyte populations are typically cryopreserved and thawed before administration. Known cell cryopreservation solutions can be used for this purpose, and such preparations are commercially available. For example, STEM-CELLBANKER® GMP grade (manufactured by Zenogen Pharma Co., Ltd.). Cryopreservation can be performed by suspending the cardiomyocytes in a cryopreservation solution and then freezing them. The cardiomyocyte populations of this embodiment can also exhibit responsiveness to antiarrhythmic drugs. Examples of such drugs include adrenergic receptor agonists (such as isoproterenol), HCN channel blockers (such as ivabradine), and potassium channel blockers (such as amiodarone).

[0085] Because it possesses all the features of (1) to (3), the cardiomyocyte population of this embodiment can be used clinically as transplantable cardiomyocytes. The cardiomyocyte population with these features is a high-purity cardiomyocyte population, especially a population of mature ventricular cardiomyocytes. As shown in the examples described later, these cardiomyocytes can be electrically synchronized with the cardiomyocytes of the recipient, maintain maturity, survive for a long time, and regenerate damaged areas of the recipient's heart.

[0086] Furthermore, as shown in the embodiments described later, the incidence of persistent and severe ventricular arrhythmias is low after transplantation of the cardiomyocytes of this embodiment. Since these cardiomyocytes are derived from human pluripotent stem cells and have high purification levels, they can be safely transplanted into the human body and can be easily and industrially manufactured. Therefore, the cardiomyocytes of this embodiment are highly useful as transplantable cardiomyocytes.

[0087] (Myocardial cell spheroids) The cardiomyocyte population in this embodiment can also be used in the form of cardiomyocyte spheroids (hereinafter also referred to as "cardiomyocyte spheroids"). The "cardiomyocyte spheroids" in the second embodiment of this disclosure refer to the aggregates of the above-mentioned cardiomyocyte populations, which can be formed by the following methods.

[0088] There is no limitation on the diameter of the cardiomyocyte spheroids, as long as cell death does not occur near the center of the spheroid and its maturity is not significantly reduced compared to the surrounding cells. For example, the diameter can be 50 μm to 300 μm. The preferred range is 100 μm to 250 μm, and more preferably 150 μm to 200 μm. The shape of the spheroids is preferably close to spherical, but any shape is acceptable as long as it can maintain the viability of the cardiomyocytes. The "diameter of the cardiomyocyte spheroids" refers to the span through the center point of the aggregate. For elliptical aggregates, the minimum diameter should be within the lower limit mentioned above, and the maximum diameter should be within the upper limit mentioned above. For a population containing multiple cardiomyocyte spheroids, preferably at least 60%, more preferably at least 70%, and most preferably at least 80% of the spheroids are located within the above diameter range, and spheroids of other sizes may also coexist.

[0089] The diameter and distribution of cardiomyocyte spheroids can be determined by known methods, such as image analysis of spheroid photographs or using particle size distribution measurement devices. The spheroids may be aggregates consisting solely of cardiomyocytes, or they may contain cells aggregated around microcarrier-like materials suitable for transplantation (e.g., extracellular matrix particles).

[0090] The cardiomyocyte spheroids of this embodiment can be used as the active ingredient in the pharmaceutical composition described later. In this case, it is preferable that the cells composed of the spheroids account for at least 60%, more preferably at least 70%, and most preferably at least 80% of all cells in the pharmaceutical composition.

[0091] The number of cells within a cardiomyocyte sphere can be determined using any suitable method. For example, a correlation between sphere diameter and cell number can be established first, and then the cell number can be calculated based on the diameter and number of sample spheres. This correlation can be obtained by measuring the diameter of multiple spheres, dissociating them into individual cardiomyocytes, and counting them. The largest diameter can be used for this determination.

[0092] The cardiomyocyte spheroids in this embodiment can be prepared from the cardiomyocyte population of the first embodiment. The spheroids themselves are not required to be within 30 days of the start of differentiation, but the cardiomyocyte population used to prepare the spheroids can be obtained within 30 days of the start of differentiation.

[0093] The inventors have discovered that when cardiomyocytes are prepared in spherical form, cell viability can be maintained at a high level. Therefore, by forming a population of cardiomyocytes into spheroids and then recovering them, a population with a high proportion of surviving cells can be obtained. The cardiomyocyte spheroids (spheroid population) obtained from the cardiomyocyte population of the first embodiment contain highly pure and highly active mature cardiomyocytes; when transplanted to a recipient, they can easily colonize in the recipient's myocardium, and the incidence of persistent severe ventricular arrhythmias is low, thus making them suitable for clinical application.

[0094] (Pharmaceutical composition) The third embodiment of this disclosure is a pharmaceutical composition comprising at least one type of pharmaceutically acceptable carrier selected from the group consisting of the cardiomyocyte population of the first embodiment and the cardiomyocyte spheroids of the second embodiment. The pharmaceutical composition of this embodiment uses at least one of the cardiomyocyte population and the cardiomyocyte spheroids obtained by spheroidizing the cardiomyocyte population as its active ingredient. Preferably, the proportion of viable cells in the pharmaceutical composition of this embodiment is not less than 60%, more preferably not less than 70%.

[0095] Preferably, the proportion of surviving cells is maintained throughout the storage and / or transportation of the pharmaceutical composition, and more preferably until administration.

[0096] The number of surviving cells can be determined using known methods. For example, surviving cells can be distinguished from dead cells by trypan blue staining, and the surviving and dead cells can be counted separately. The surviving cell ratio can then be calculated as the proportion of surviving cells to the total number of cells.

[0097] Furthermore, the cardiomyocyte population contained in the pharmaceutical composition of this embodiment is preferably such that the number of cells constituting cardiomyocytes accounts for not less than 60%, more preferably not less than 70%, and even more preferably not less than 80% of the total number of cells in the pharmaceutical composition. During the storage and / or transportation of the pharmaceutical composition, the proportion of cardiomyocytes constituting cardiomyocyte spheroids is also preferably maintained, and more preferably maintained until administration.

[0098] <Cardiomyocyte Clusters and Cardiomyocyte Spheroids> "Myocardial cell cluster" and "myocardial cell sphere" refer to the myocardial cell cluster of the first embodiment and the myocardial cell sphere of the second embodiment of this disclosure, respectively.

[0099] <Pharmaceutical Acceptable Carrier> "Pharmaceutically acceptable carrier" refers to a carrier that does not inhibit the physiological activity of the active ingredient and does not exhibit substantial toxicity to the target organism. "Does not exhibit substantial toxicity" means that it does not exhibit toxicity to the target organism at the usual dosage. In the pharmaceutical composition of this embodiment, the carrier does not damage the cardiomyocyte population of the first embodiment and does not exhibit substantial toxicity to the target organism. Pharmaceutically acceptable carriers include any known pharmaceutically acceptable ingredient that is generally considered an inert component. For example, they may include solvents, diluents, carriers, excipients, glidants, binders, granulators, dispersants, suspending agents, wetting agents, lubricants, disintegrants, solubilizers, stabilizers, emulsifiers, fillers, etc. The carrier may be used alone or in combination of two or more.

[0100] Pharmaceutically acceptable carriers can be buffer solutions. Examples of buffer solutions include physiological saline, general-purpose buffers (such as phosphate, citric acid, and other organic acids), etc.

[0101] In addition to the components mentioned above, the pharmaceutical composition may further contain other components. These other components are not limited and can be any components used in the pharmaceutical field. For example, pharmaceutical excipients other than those mentioned above may be included. Examples of pharmaceutical excipients include, but are not limited to, preservatives (such as antioxidants), chelating agents, flavoring and odor-correcting agents, sweeteners, thickeners, buffers, coloring agents, etc.

[0102] The dosage form of the pharmaceutical composition is not particularly limited and can be any dosage form commonly used in pharmaceutics. The pharmaceutical composition of this embodiment is typically a non-oral formulation and can be formulated into an appropriate dosage form according to the following administration methods, such as an injectable formulation or a myocardial patch. The pharmaceutical composition of this embodiment can be stored and transported cryopreserved as needed; when in cryopreservation, it can be thawed before use and further diluted with a sterile carrier corresponding to the target cell type. The pharmaceutical composition of this embodiment can be, for example, sealed in vials for patient use, or sealed in syringes or other devices according to the administration method. Furthermore, after administration, commonly used immunosuppressants can be given to the recipient patient. Immunosuppressants can be appropriately selected according to the patient's condition; examples include agents that act on immunophilins (such as cyclosporine, tacrolimus hydrate, etc.). The pharmaceutical composition of this embodiment can also be combined with these immunosuppressants to form a combination formulation.

[0103] The preferred route of administration for the pharmaceutical composition of this embodiment is non-oral administration. As a non-oral route of administration, any known method of delivering cells to a patient can be used, such as any method that allows for minimally invasive delivery of cells to the patient's heart. Specifically, local administration to the heart (especially the ventricles or myocardial tissue) is preferred, for example, administration via a graft needle into the ventricular myocardial tissue from the epicardial side, or administration via a catheter from the endocardial side or pericardium.

[0104] The pharmaceutical composition can deliver a "therapeutic effective amount" of cardiomyocytes of the first embodiment and / or cardiomyocyte spheroids of the second embodiment. "Therapeutic effective amount" refers to the dosage of a drug that has a therapeutic or preventative effect on the treated disease. For example, the therapeutic effective amount of the cardiomyocytes and / or cardiomyocyte spheroids may be the dosage that produces a cardiac repair effect on the receptor. The therapeutic effective dose can be appropriately determined based on the patient's symptoms, weight, age, sex, etc., as well as the formulation and route of administration of the pharmaceutical composition. For example, the pharmaceutical composition may be administered in a single dose of 10... 5 ~10 10 10 cardiomyocytes, preferably 10 7 ~10 9 Each myocardial cell.

[0105] The pharmaceutical composition can be administered once or multiple times. For example, after administration to a patient, the patient's cardiac function and the colonization of transplanted cardiomyocytes can be monitored, and the drug can be administered again if necessary.

[0106] The pharmaceutical composition of this embodiment is administered to a human. The pharmaceutical composition can be used to treat heart diseases such as heart failure. Heart failure is a disease characterized by impaired cardiac function. Its causes include, for example, myocardial infarction, angina pectoris, hypertension, valvular heart disease, cardiomyopathy, arrhythmia, and congenital diseases. The heart failure to be treated can be caused by any of the above causes. The pharmaceutical composition of this embodiment is suitable for treating heart failure caused by myocardial tissue damage due to the above causes. By administering the pharmaceutical composition of this embodiment (i.e., transplanted cardiomyocytes and / or cardiomyocyte spheroids) to a patient with heart failure, damaged myocardial tissue can be regenerated, thereby improving cardiac function. The pharmaceutical composition of this embodiment specifically has the following functions: (1) the transplanted contractile cardiomyocytes adhere to the damaged myocardium of the recipient and directly enhance the contractility of the damaged area of ​​the recipient's heart; and / or (2) release paracrine factors with pro-angiogenic, cardioprotective, anti-inflammatory, and anti-fibrotic effects.

[0107] (Methods for preparing cardiomyocyte populations) The fourth embodiment of this disclosure provides a method for preparing a population of cardiomyocytes. The method comprises the following steps (a) to (c) in the listed order: (a) Expanding human pluripotent stem cells in culture; (b) Expanded human pluripotent stem cells are cultured under conditions that induce differentiation into cardiomyocytes to obtain a cell population containing a sufficient proportion of cardiomyocytes, specifically, to achieve a cTnT positive cell proportion of over 60%, so as to reach over 90% after process (c); and (c) Remove human pluripotent stem cells and non-myocardial cells from the said cell population.

[0108] <Process (a)> The expansion and culture of human pluripotent stem cells can be carried out using known methods. Examples include Thomson et al., Science (1998) 282(5391):1145–7; Hovatta et al.; Ludwig et al., Nat Methods (2006) 3:637–46; Kennedy et al., Blood (2007) 109:2679–87; Nat Methods (2011) 8:424–9; Wang et al., StemCell Res. (2013) 11(3):1103–16; and the methods described in International Publication 2018 / 181342, etc.

[0109] The culture medium can be one commonly used as a basal medium for mammalian cell culture. Examples of basal media include BME, BGJb, CMRL 1066, Glasgow MEM, Improved MEM Zinc Option, IMDM, Medium 199, EagleMEM, αMEM, DMEM, F-12, DMEM / F12, IMDM / F12, Ham, RPMI 1640, Fischer's, etc., or mixtures thereof.

[0110] Alternatively, commercially available stem cell culture media can be used. Examples include StemFit AS103C (Ajinomoto Co., Ltd.), StemFit® AK series media (such as StemFit AK03N, StemFit AK02N) (Ajinomoto Co., Ltd.), Essential 8 (Life Technologies Corporation), mTESR1 (STEMCELL Technologies Canada Inc.), TESR2 (same as above), RHBS (same as above), TeSR™-E6 (same as above), hESF-GRO (Nipron Co., Ltd.), hESF-DIF (same as above), CSIT-7 (Cell Science Research Institute Co., Ltd.), Essential 6 (Life Technologies Corporation), etc.

[0111] To avoid the contamination of undefined chemical components, the culture medium is preferably a chemically defined medium (CDM), and more preferably a serum-free medium. "Serium-free medium" refers to a medium that does not contain unreconstituted or unpurified serum. Medium containing purified blood-derived components and / or purified animal tissue-derived components (such as growth factors like bFGF) also fall within the definition of serum-free medium. Serum-free medium may contain serum substitutes. Examples of serum substitutes include serum albumin, transferrin, fatty acids, collagen precursors, trace elements, 2-mercaptoethanol, or 3'-thioglycerol. These substitutes can be commercially available products, such as KnockOut™ Serum Replacement, Chemically-defined Lipid Concentrate, GlutaMAX™, B27, and N2 (all products of Life Technologies Corporation), but are not limited to these.

[0112] The culture medium may also selectively contain some or all of the essential amino acids (except L-tryptophan, such as L-leucine, L-lysine, L-phenylalanine, L-isoleucine, L-threonine, L-histidine, L-methionine, L-valine), some or all of the non-essential amino acids (such as L-alanine, L-arginine, L-asparagine, L-aspartic acid, glycine, L-glutamine, L-glutamic acid, L-cysteine, L-serine, L-tyrosine, L-proline), and other amino acids such as L-cysteine.

[0113] The culture medium may also contain additives. Examples include ROCK (Rho-associated protein kinase) inhibitors such as Y-27632, antibiotics such as penicillin-streptomycin, vitamins, L-ascorbic acid, L-ascorbic acid magnesium phosphate, sodium pyruvate, 2-aminoethanol, glucose, sodium bicarbonate, HEPES, insulin, progesterone, sodium selenite, putrescine, etc. The additives are preferably present in concentrations within their known ranges.

[0114] The culture medium may contain fatty acids. Examples include oleic acid, linoleic acid, α-linolenic acid, γ-linolenic acid, palmitic acid, stearic acid, arachidonic acid, eicosapentaenoic acid, docosahexaenoic acid, butyric acid, acetic acid, valeric acid, hexanoic acid, heptanoic acid, octanoic acid, nonanoic acid, decanoic acid, lauric acid, myristic acid, pentadecanoic acid, heptadecanic acid, ceramide, tung oil acid, 8,11-eicosadienoic acid, 5,8,11-eicostrienoic acid, behenic acid, ceramide, nervonic acid, hexacosanoic acid, triacontanic acid, beeswax acid, linolenic acid, etc. Fatty acids can be saturated or unsaturated. The culture medium can also be a basal medium containing L-ascorbic acid, selenium, transferrin, insulin, FGF2, and TGFβ1, supplemented with L-tryptophan or its derivatives.

[0115] The pH of the culture medium is preferably about 6.0 to 8.5, more preferably about 7.0 to 7.5. The culture medium is preferably sterilized by means of membrane filtration or the like.

[0116] Human pluripotent stem cells can be cultured using conventional methods, with or without the use of feeder cells such as fibroblasts.

[0117] The culture vessel used is not limited as long as it is suitable for cell culture. Examples include bottles, tissue culture flasks, dishes, culture dishes, multi-well dishes, microplates, multi-well plates, multi-well culture plates, microslides, chamber slides, tubes, trays, culture bags, roller bottles, etc. The culture vessel can be cell-adhesive or non-adhesive. Adhesive vessels can be coated with a matrix, such as Matrigel (Niwa A, et al. PLoS One. 6(7):e22261, 2011), gelatin, collagen, elastin, fibronectin, hyaluronic acid, chondroitin sulfate, glycosaminoglycans, proteoglycans, or artificial materials that mimic their functions. Adhesive culture is preferably carried out using the above-mentioned coated vessels.

[0118] The cultivation temperature is approximately 30–40℃, preferably approximately 37℃. The CO2 concentration is approximately 1–10%, preferably approximately 2–5%. The oxygen concentration is generally 1–40%, and can be adjusted appropriately according to the cultivation conditions.

[0119] <Process (b)> As long as the cardiomyocyte population of this embodiment can be obtained, any method for inducing differentiation of human pluripotent stem cells into cardiomyocytes can be used. Examples include: (i) activating Wnt / β-catenin signaling in human pluripotent stem cells to obtain a first cell population (i.e., culturing in a first differentiation medium), and (ii) inhibiting Wnt / β-catenin signaling in the first cell population to obtain a second cell population containing cardiomyocyte precursor cells (i.e., culturing in a second differentiation medium). Subsequently, the cardiomyocyte precursor cells are further cultured in media such as RPMI, DMEM, or StemPro-34 for 1–5 days to obtain a cardiomyocyte population containing mature ventricular cardiomyocytes. Through the above process, a cell population in which more than 60% of all surviving cells differentiate into cardiomyocytes can be obtained.

[0120] Wnt / β-catenin signaling can be activated by exposing pluripotent stem cells to Wnt agonists (such as activin A, BMP4, or bFGF). Wnt agonists include GSK-3β inhibitors such as CHIR-99021, TW-119, SB216763, SB415286, and CHIR-98014. Cells can be used at concentrations that yield cardiomyocytes with the desired characteristics. Cells are cultured in primary differentiation medium for 1–6 days. Differentiation into mesodermal cells can be confirmed by detecting the expression of markers such as T, MIXL1, and NODAL.

[0121] The primary differentiation medium was then replaced with a secondary differentiation medium containing Wnt antagonists and VEGF. Wnt antagonists included IWP-2, IWP-3, IWP-4, IWR-1, PNU-74654, XAV939, and KY02111. These were used at concentrations that yielded cardiomyocytes with the desired characteristics. Cells were cultured in the secondary differentiation medium for 1–7 days.

[0122] The total incubation period for steps (i) and (ii) (including the subsequent 1 to 5 days of incubation in RPMI, DMEM or StemPro-34 and the incubation period for process (c)) is preferably completed within 30 days.

[0123] The culture containers and conditions used can be the same as those described in procedure (a). Differentiation is preferably performed using the method described by Tohyama S. et al., StemCell Reports (2017) 9:1406–1414. Suitable containers include four-layer plates (Thermo Fisher Scientific), and suitable culture media include StemFit AS301 or StemFit AS501 (Ajinomoto Co., Ltd.).

[0124] The proportion of cardiomyocytes in the cell population obtained through this process is not limited as long as it is sufficient to achieve a cTnT positive cell proportion of over 90% after process (c). Specifically, when the purification process (c) is completed approximately 4 days later, cardiomyocytes preferably constitute more than 60% at the end of process (b); if the cardiomyocytes are further purified during process (c), a lower proportion can be used. The proportion of cardiomyocytes can be determined by flow cytometry to measure the proportion of cells positive for markers such as cTnT.

[0125] <Process (c)> The method for removing human pluripotent stem cells and non-cardiomyocytes from the cell population after procedure (b) can be a known method. See, for example, International Publication 2018 / 3031074457, Tanosaki S. et al., iScience. 2020;23:101535; Tanosaki S. et al., STAR Protocols. 2022;3:101360; Tohyama S. et al., Cell Metabolism. 2016;23:663–74; Tohyama S. et al., Cell Stem Cell. 2013;12:127–37, etc.

[0126] Specifically, for example, cell death of residual undifferentiated cells and non-myocardial cells can be induced by culturing the cell population obtained in process (b) in a culture medium containing at least one compound selected from the following group: fatty acid synthesis inhibitors, fatty acid utilization inhibitors, and cholesterol synthesis inhibitors.

[0127] As an inhibitor of fatty acid synthesis, a compound that inhibits fatty acid synthesis by targeting at least one factor selected from the group consisting of ATP citrate lyase, fatty acid synthase, acetyl-CoA carboxylase, and malonyl-CoA decarboxylase is preferred; more preferably, a compound that targets at least one of ATP citrate lyase and fatty acid synthase. Examples of inhibitors that inhibit fatty acid synthesis by targeting fatty acid synthase include orlistat, C75, flavonoids, and epigallocatechin gallate (EGCG); among which orlistat and C75 are preferred, and orlistat is more preferred. Examples of inhibitors that target ATP citrate lyase include LY294002 and SB204990. Examples of inhibitors that target acetyl-CoA carboxylase include Soraphen A, TOFA, A769662, metformin, and AICAR; among which TOFA and A769662 are preferred.

[0128] As an inhibitor of fatty acid utilization, compounds that target carnitine palmitoyltransferase 1 to inhibit fatty acid utilization are preferred. Examples of inhibitors that target carnitine palmitoyltransferase 1 to inhibit fatty acid breakdown include etomoxir, perhexiline, and ranolazine; among which etomoxir and perhexiline are preferred.

[0129] As a cholesterol synthesis inhibitor, a compound that inhibits cholesterol synthesis by targeting at least one factor selected from the group consisting of acetyl-CoA acetyltransferase, HMG-CoA synthase, and HMG-CoA reductase is preferred; more preferably, a compound that inhibits cholesterol synthesis by targeting HMG-CoA reductase is preferred. Examples of cholesterol synthesis inhibitors targeting HMG-CoA reductase include pravastatin, simvastatin, fluvastatin, atorvastatin, pitavastatin, rosuvastatin, cerivastatin, lovastatin, and mevastatin; among which pravastatin, simvastatin, fluvastatin, atorvastatin, pitavastatin, and rosuvastatin are preferred, and simvastatin is more preferred.

[0130] Preferably, at least one selected from the group consisting of orlistat, C75, LY294002, SB204990, etomoxir, perhexiline, simvastatin, and their salts is used, with orlistat being more preferred. The concentrations of fatty acid synthesis inhibitors, fatty acid utilization inhibitors, and cholesterol synthesis inhibitors in the culture medium can be appropriately selected according to their types. For example, the concentration of the aforementioned fatty acid synthesis inhibitors or fatty acid utilization inhibitors can be 0.1–500 μM; the concentration of cholesterol synthesis inhibitors can be 0.01–50 μM. For instance, the culture medium used in this invention can be prepared by adding at least one substance from the group consisting of fatty acid synthesis inhibitors, fatty acid utilization inhibitors, and cholesterol synthesis inhibitors, and at least one compound from the group consisting of cholesterol synthesis inhibitors, to the aforementioned basal culture medium. This culture medium may selectively contain at least one compound from the group consisting of glucose, glutamine, and methionine; the inclusion of glucose, glutamine, and / or methionine is expected to promote good cell growth.

[0131] Cell culture in process (c) can be performed under general cell culture conditions. The culture temperature is approximately 30–40°C, preferably approximately 37°C; the CO2 concentration is approximately 1–10%, preferably approximately 2–5%; and the oxygen concentration is typically 1–40%, which can be selected as needed. The culture time is not limited, but is preferably more than 24 hours, more preferably more than 48 hours, specifically 1–5 days. Passaging can be performed if necessary. By culturing the cell population obtained in process (b) in a culture medium containing at least one of the above-mentioned inhibitors, human pluripotent stem cells and non-cardiac cells can be removed from the cell population.

[0132] Furthermore, by adding the following process: culturing cells in a culture medium (based on a commonly used cell culture medium formulation) that does not contain glucose, glutamine, or glutamate but contains lactate, selectively proliferates cardiomyocytes and induces the death of non-cardiomyocytes, thereby further purifying cardiomyocytes. This method is specifically described in international publications WO2007 / 088874 and WO2016 / 010165.

[0133] It should be noted that the characteristic of the cardiomyocyte population in the first embodiment being "within 30 days from the start of differentiation" is taken as the starting point when the differentiation induction medium (a medium containing substances that induce differentiation into cardiomyocytes; for example, the first differentiation medium) is used, and the period from the start of culturing the cells in a medium containing substances that induce differentiation into cardiomyocytes to the end of process (c) is within 30 days.

[0134] <Optional Procedure> Selection process; After the above processes (a) to (c), the manufacturing method of this embodiment may optionally include a selection process for selecting myocardial cell populations having the above characteristics (1), (2), and (3).

[0135] The selection process can be performed with reference to the method described in the "cardiomyocyte population" section above. Specifically, for example, the following steps can be performed: Select a cell population (d1) from the cell population obtained in process (c) whose self-differentiation has started (i.e., process (b)) within 30 days. Subsequently, select a cardiomyocyte population in which cTnT positive cells account for more than 90% of all surviving cells. For example, perform flow cytometry analysis on the cell population obtained in process (c) using an antibody against cTnT, and select a cell population in which the proportion of cTnT positive cells in all surviving cells is not less than 90% (d2). Next, select a cardiomyocyte population from the selected cell populations with a spontaneous beat rate of 0 to 60 beats per minute. For example, record the action potential duration (APD90) and spontaneous action potentials of the cardiomyocyte population at 90% repolarization using a patch-clamp amplifier, and select a cell population in which the spontaneous beat rate is 0 to 60 beats per minute (d3). The order of the above processes (d1) to (d3) is not limited to the aforementioned order and can be performed in any order.

[0136] The process of cryopreservation of cardiomyocytes; The manufacturing method of this embodiment may include a step of cryopreserving cardiomyocytes after processes (a) to (c) and optional selection processes described above. Preferably, the cardiomyocytes obtained by the above method are removed from the culture plate using known methods, washed if necessary, and suspended in a cell cryopreservation solution commonly used for cell cryopreservation. Examples of cell cryopreservation solutions include STEM-CELLBANKER® GMP grade (manufactured by Zenogen Pharma Co., Ltd.).

[0137] The concentration of cardiomyocytes in the cell suspension to be cryopreserved should be such that freeze-thaw cycles do not lead to cell death; specifically, the viability of surviving cells after freeze-thaw should be no less than 50% of the viability of surviving cells before cryopreservation. For example, the concentration of cardiomyocytes in the cell suspension could be 1 × 10⁻⁶. 5 ~1×10 8 Cells / mL, preferably 1×10⁻⁶ 6 ~1×10 7 Cells / mL. Cryopreservation containers can be vials or bags used for cell cryopreservation.

[0138] Cryopreservation and preservation can be carried out using conventional methods. For example, cells can be initially frozen in a freezer at -60°C to -80°C, then transferred to a container filled with liquid nitrogen, or transferred to a cryogenic freezer at -150°C or lower for long-term storage. Alternatively, a programmable cryostat can be used to cryopreserve cells under controlled temperature. For example, cells can be cooled from -40°C to -60°C at a rate of 1–10°C / min, held at -40°C to -60°C for 10–60 minutes, and then cooled to -80°C at a rate of 1–10°C / min and held at -80°C for several days. The thawing process for cryopreserved cardiomyocytes can be carried out using known methods, such as removing the cryopreserved cells from a liquid nitrogen container or cryogenic freezer and thawing them in a 37°C water bath.

[0139] (Methods for preparing cardiomyocyte spheroids) This disclosure provides a fifth embodiment of a method for preparing cardiomyocyte spheroids. The method includes suspending a cardiomyocyte population of the first embodiment in a culture medium and then statically culturing it in a culture vessel with micropores on the bottom. This method can produce cardiomyocyte spheroids of the second embodiment. Specifically, cardiomyocytes of the first embodiment can be suspended in a culture medium and statically cultured in a culture vessel with micropores on the bottom to form cardiomyocyte spheroids. The cardiomyocytes of the first embodiment used herein are not in spherical form. The cardiomyocytes can be those obtained according to the manufacturing method of the fourth embodiment. The culture medium can be the aforementioned basal culture medium, preferably a serum-free culture medium. The culture medium may contain insulin (0.1–10 mg / L), transferrin (0.1–10 μg / L), selenium (0.1–10 μg / L), bFGF (1–100 ng / mL), EGF (1–1000 ng / mL), PDGF (1–1000 ng / mL), and endothelin-1 (ET-1) (1×10⁻⁶ mg / mL). -8 ~1×10 -6 M). A specific example is StemFit AS301 (Ajinomoto Co., Ltd.). As a culture container with micropores on the bottom surface, commercially available culture dishes specifically for pelleting can be used. Micropores refer to openings with diameters in the micrometer range (e.g., 100–1000 μm, preferably 100–800 μm, more preferably 200–500 μm, and especially preferably 300–500 μm). The culture container is preferably one with a low-adhesion culture surface; alternatively, a material that inhibits bioadhesion, such as Prevlex® (Nissan Chemical Industries, Ltd.), can be coated on its surface. Specific examples include six-well plates coated with Prevlex (Elplasia RB 500 400 NA 6, manufactured by Corning). The preparation of cardiomyocyte spheroids can be carried out according to existing techniques (e.g., WO2009 / 017524, Kawaguchi S. et al., JACC Basic Transl Sci. 2021;6:239–254; Tabei R. et al., J Heart Lung Transplant. 2019;38:203–214).

[0140] (Methods for treating heart failure) The sixth embodiment of this disclosure is a method for treating heart failure, comprising administering to a subject requiring treatment for heart failure at least one type selected from the group consisting of cardiomyocyte populations of the first embodiment and cardiomyocyte spheroids of the second embodiment. The administered cardiomyocyte populations and / or cardiomyocyte spheroids may be in the form of the pharmaceutical composition of the third embodiment. The cardiomyocyte populations and / or cardiomyocyte spheroids may be injected directly into myocardial tissue, for example, by injection. The delivery device may be any device capable of injecting cardiomyocyte populations and / or cardiomyocyte spheroids into target tissue, such as the device described in WO2020 / 013125. The needle used is of gauge 24 to 30. Catheters may also be used.

[0141] A therapeutically effective dose of cardiomyocytes and / or cardiomyocyte spheroids can be administered to patients requiring treatment for heart failure. The therapeutically effective dose can be appropriately determined based on the degree of myocardial tissue damage in the patient. For example, a single administration of the cardiomyocyte spheroids and / or cardiomyocyte spheroids may be 10... 5 ~10 10 10 cardiomyocytes, preferably 10 7 ~10 9 Each cardiomyocyte. Here, when administering cardiomyocyte spheroids, the dose is measured in terms of the number of cardiomyocytes in the group of cardiomyocytes forming the cardiomyocyte spheroids.

[0142] The treatment method described in this embodiment is applicable to humans. Heart failure is a disease characterized by impaired cardiac function. Its causes include, for example, myocardial infarction, angina pectoris, hypertension, valvular heart disease, cardiomyopathy, arrhythmia, and congenital abnormalities. The heart failure to be treated can be caused by any of the above causes. The pharmaceutical composition of this embodiment is suitable for treating heart failure caused by myocardial tissue damage due to the above causes. By administering the pharmaceutical composition of this embodiment (transplanted cardiomyocytes and / or cardiomyocyte spheroids) to the subject, damaged myocardial tissue can be regenerated, thereby improving cardiac function. The pharmaceutical composition specifically has the following functions: (1) the transplanted contractile cardiomyocytes adhere to the damaged myocardium of the recipient and directly enhance the contractility of the damaged site of the recipient's heart; and / or (2) the function of releasing paracrine factors with pro-angiogenic, cardioprotective, anti-inflammatory, and anti-fibrotic effects.

[0143] (Other implementation methods) Other embodiments of this disclosure include: using at least one selected from the group consisting of a myocardial cell population of the first embodiment and a myocardial cell sphere of the second embodiment to prepare a pharmaceutical composition for treating heart failure.

[0144] Other embodiments of this disclosure include: at least one selected from the group consisting of a myocardial cell population of the first embodiment and a myocardial cell sphere of the second embodiment for use in treating heart failure.

[0145] Other embodiments of this disclosure include: using at least one selected from the group consisting of a myocardial cell population of the first embodiment and a myocardial cell sphere of the second embodiment in the treatment of heart failure.

Detailed Implementation Methods

[0147] Example 1: Preparation and analysis of cardiomyocyte populations derived from human iPS cells (1) Preparation of purified cardiomyocyte populations derived from human iPSCs Human iPS cells (FfI14s04 strain) (provided by the iPS Cell Research Foundation, Kyoto University) were cultured in a medium containing iMatrix511 (Nippi Inc.) and induced to differentiate into cardiomyocytes according to the differentiation induction protocol. The human iPS cells (FfI14s04 strain) were cultured on culture plates coated with iMatrix 511 (Nippi Inc.) using StemFit AS103C medium (Ajinomoto Co., Ltd.). Differentiation induction into cardiomyocytes was performed on four-layer culture plates (ThermoFisher Scientific Inc.) using StemFit AS301 medium (Ajinomoto Co., Ltd.) and according to the method described in Tohyama S. et al., Stem Cell Reports 2017;9:1406–1414.

[0148] Following differentiation induction, residual human iPS cells in the resulting cell population were removed using the fatty acid synthesis inhibitor orlistat, according to the methods described in Tanosaki S. et al., iScience 2020;23:101535 and Tanosaki S. et al., STAR Protoc 2022;3:101360. Subsequently, the cells were cultured using StemFit AS501 (Ajinomoto Co., Ltd.) according to the methods described in Tohyama S. et al., Cell Metab 2016;23:663–74 and Tohyama S. et al., Cell Stem Cell 2013;12:127–37. The purified cardiomyocyte population was cryopreserved in STEM-CELLBANKER® GMP grade (Zenogen Pharma Inc.). Differentiation induction culture lasted 7–15 days; culture to remove residual human iPS cells lasted 5–10 days.

[0149] (2) Flow cytometry and electrophysiological analysis of human iPSC-derived cardiomyocyte populations The human iPS cell-derived cardiomyocyte population prepared and cryopreserved in (1) was thawed according to the methods described in Tohyama S. et al., Cell Metab 2016;23:663–74 and Tohyama S. et al., Cell StemCell 2013;12:127–37. The results are shown in Figure 1A -1F.

[0150] Figure 1A (b) shows the flow cytometry results of the positivity rate of cardiac troponin T (cTnT) in the cardiomyocyte population after thawing. Dead cells were excluded by thresholding, and the cTnT positivity rate in the surviving cells was determined. The cardiomyocyte population prepared in (1) had a cTnT positivity rate of 99.8% after thawing ( Figure 1A (b) It should be noted that the cTnT positivity rate of the pre-purification cardiomyocyte population was 81.0% ( Figure 1A (a)).

[0151] Furthermore, following the method described by Ichimura H. et al., Sci Rep 2020;10:11883, electrophysiological analysis of the thawed cardiomyocyte population was performed using patch-clamp technique. Specifically, the cryopreserved cardiomyocyte population prepared in (1) was thawed, reseeded, and cultured for an additional 4 days. To examine the automatic beat frequency, action potential duration at 90% repolarization (APD90), and spontaneous action potentials, recordings were performed using a patch-clamp amplifier (Axopatch 200B, Molecular Devices Inc.) under ruptured whole-cell patch-clamp conditions.

[0152] The intracellular fluid used for action potential recording contained potassium gluconate (130 mmol / L), KCl (10 mmol / L), NaCl (5 mmol / L), MgCl2 (1 mmol / L), ethylene glycol tetraacetic acid (EGTA: 0.1 mmol / L), magnesium-bound adenosine triphosphate (0.1 mmol / L), and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid: HEPES (10 mmol / L). The extracellular perfusion fluid contained NaCl (136.5 mmol / L), KCl (5.4 mmol / L), CaCl2 (1.8 mmol / L), MgCl2 (0.53 mmol / L), HEPES (85.5 mmol / L), and glucose (5.5 mmol / L).

[0153] The results of a total of 65 analyses (65 cells) are shown in Figure 1B and Figure 1C .like Figure 1BAs shown, the human iPS cell-derived cardiomyocyte population prepared in (1) exhibited a ventricular-type action potential pattern after freeze-thaw. Figure 1C As shown in the left figure (BeatRate), the spontaneous beat rate is 34 ± 13 beats / min. Figure 1C The maximum diastolic potential (MDP) shown in the middle plot (MDP) is –58.4 ± 0.64 mV. Figure 1C The action potential duration (APD90) at 90% repolarization, as shown in the right figure (APD90), is 465 ± 25 ms.

[0154] In contrast, for a cell population that had only undergone cardiomyocyte differentiation without purification (n = 7), the results are shown in... Figure 1G .like Figure 1G As clearly shown, the average spontaneous beat rate of the purified cardiomyocyte population is approximately 38 beats per minute, while that of the unpurified cell population is approximately 60 beats per minute.

[0155] (3) Drug response of human iPS cell-derived cardiomyocyte population After thawing the human iPS cell-derived cardiomyocyte population prepared and frozen in (1), it was cultured for about 1 week in MEMα (Thermo Fisher Scientific Inc., catalog no. 40610) containing 2.5% FBS (Merck Inc.) and sodium pyruvate (Merck Inc., catalog no. S8636). On the day of detection, the cells were placed in HBSS (Thermo Fisher, catalog no. 1402504092) containing 5 mMCal520AA (AAT Bioquest, catalog no. 21131), and pluronic F-127 (AAT Bioquest, catalog no. 20053) and probenecid (AAT Bioquest Inc., catalog no. 20062) were added, and incubated at 37°C for 1 hour.

[0156] The adrenergic receptor agonist isoproterenol (Merck Inc., catalog no. I6504) (0.01 nM, 0.1 nM, 1 nM, 10 nM, 100 nM, or 1 μM) was added to the culture medium, and Ca2+ was measured using FDSS / μCELL (Hamamatsu Photonics Co., Ltd.) 2+Transient. In addition, the HCN channel blocker ivabradine (Merck Inc., catalog no. SML02C81) (10 nM, 30 nM, 100 nM, 300 nM, or 1 μM) was added to the culture medium, and Ca was measured in the same manner. 2+ Transient. Further, the potassium channel blocker amoodarone (Merck Inc., catalogo. A8423) (0.1 μM, 0.3 μM, 1 μM, 3 μM, or 10 μM) was added to the culture medium, and Ca was measured in the same manner. 2+ Transient change.

[0157] The results are shown in Figure 1D and Figure 1E The analysis was performed based on three different batches of cell populations prepared according to the method described in (1). Figure 1D and Figure 1E As clearly shown, the human iPS cell-derived cardiomyocyte population obtained in (1) exhibited concentration-dependent responses to the adrenergic receptor agonist isopreterenol, the HCN channel blocker ivabradine, and the potassium channel blocker amoodarone.

[0158] (4) Immunocytochemical analysis of cardiomyocyte populations derived from human iPS cells The human iPS cell-derived cardiomyocyte population prepared and cryopreserved in (1) was thawed according to the methods described by Tohyama S. et al., CellMetab 2016;23:663–74 and Tohyama S. et al., Cell Stem Cell 2013;12:127–37. Following standard procedures, the thawed cardiomyocyte population was immunolabeled using α-actin antibody, α-vimentin antibody, and DAPI. The results are shown in… Figure 1F (a). In the cardiomyocyte population prepared by (1), most cells were actin-positive and vimentin-negative.

[0159] In addition, the same cell population was immunolabeled using anti-MLC2v antibody, anti-MLC2a antibody, and DAPI, and the results are shown in... Figure 1F (b) and Figure 1F (e). Of the cardiomyocyte population prepared in (1), 85% were mature ventricular cardiomyocytes that were positive only for MLC2v, while 7% were immature cardiomyocytes that were positive only for MLC2a, or cells that were positive for both MLC2v and MLC2a. Figure 1F (b) and Figure 1F (e) Furthermore, analysis of the expression of connexin 43 and N-cadherin showed that both were expressed at high levels.Figure 1F (c) and Figure 1F (d)).

[0160] In summary, the cardiomyocyte population prepared in (1) consists of nearly purified ventricular cardiomyocytes, and its maturity is similar to that of adult cardiomyocytes.

[0161] Example 2: Preparation and analysis of cardiomyocyte spheroids (1) Preparation of cardiomyocyte spheroids from human iPS cell-derived cardiomyocyte population The frozen human iPS cell-derived cardiomyocyte population from Example 1 (1) was suspended in StemFit AS301 medium and cultured according to the methods described in Kawaguchi S. et al., JACC Basic Transl Sci 2021;6:239–254, or Tabei R. et al., JHeart Lung Transplant 2019;38:203–214. The cells were then seeded onto six-well plates coated with Prevelex® (Elplasia RB500 400 NA 6, Corning Inc.) and cultured for 2 days. Subsequently, the medium was replaced at half volume every 2 days for a total of 7 days, and human iPS cell-derived cardiomyocyte spheroids were collected.

[0162] The size of cardiomyocyte spheroids was determined using a particle size distribution analyzer (Beckman Coulter, Inc.). The results showed that the average diameter of the cardiomyocyte spheroids was approximately 150 μm.

[0163] In addition, cardiomyocyte spheroids were recovered immediately after collection and again 4 hours later. These spheroids were treated with a 3:1 mixture of trypsin / EDTA and Accumax at 37°C for 10–15 minutes to dissociate them into single cells, followed by trypan blue staining. The viability of cardiomyocytes in each spheroid is shown in [Figure / Table / Insert Table ... Figure 2A . Figure 2A In this context, "253G4" indicates the result obtained by differentiating, purifying, and cryopreserving the research-grade human iPS cell line 253G4 (provided by Kyoto University) according to Example 1 (1), and then forming cardiomyocyte spheroids according to the method described in this embodiment. Figure 2A As shown, for both the 253G4 and FfI14s04 strains, the cardiomyocyte viability in cardiomyocyte spheroids stained with trypan blue immediately after collection (0 h) and 4 h (4 h) exceeded 90%. The viability of the FfI14s04 strain was 95.8 ± 2.2% (N = 3).

[0164] Furthermore, under a fixed field of view of the recovered spheroid suspension, the number of cardiomyocyte spheroids with a diameter ≥50 μm was counted to calculate the number of cardiomyocytes constituting the spheroids. Simultaneously, under the same field of view, the number of cardiomyocyte spheroids with a diameter ≤50 μm and the number of single cells (non-spheroid cardiomyocytes) were counted to calculate the total cell number. The proportion of cardiomyocytes to the total cell number was calculated according to the following formula, ranging from 74% to 86%.

[0165] Percentage of cardiomyocytes (%) = (Number of cardiomyocytes) / [(Number of cardiomyocytes) + (Number of non-cardiomyocytes)] × 100 (2) Long-term culture of cardiomyocyte spheroids derived from human iPS cells The cardiomyocyte spheroids obtained in (1) were further cultured in the same medium for extended periods. After 28, 56, or 84 days of culture, the cardiomyocyte spheroids were cultured for 1 hour in Cytored solution (molecular weight = 313.31, Fujifilm and Kojun Pharmaceutical Co., Ltd.). Furthermore, the cardiomyocyte spheroids were embedded in Tissue-Tek OCT Compound (SAKURA Inc.), and 10 μm thick frozen sections were prepared using a cryostat (CM3050S, Leica Microsystems Inc.). Immunohistochemical analysis was performed on the sections using α-DAPI antibody, α-human specific cardiac troponin I antibody (cTnI, ab52862; Abcam Inc.) as primary antibodies, and biotin-labeled secondary antibody (Vector Laboratories Inc.). The results are shown in... Figure 2C .like Figure 2C As shown in Figures A, D, and G, it was observed that the external culture medium (including CytoRed solution) could penetrate the central region of the cardiomyocyte spheroids, indicating that the cardiomyocytes inside the spheroids can directly obtain nutrients from the external culture medium. Furthermore, as... Figure 2C As shown in B, E, and H, even after 12 weeks of culture, the cardiomyocyte spheroids still maintain the purity of cardiomyocytes.

[0166] (3) Transplantation of human iPS cell-derived cardiomyocytes into mature male NOG mice Mature male NOG mice (In Vivo Science Co., Ltd.) were anesthetized with a low dose of isoflurane and ventilated using a rodent ventilator. A left thoracotomy was performed at the 4th intercostal space, and spheroids of cardiomyocytes expressing modified luciferase (Akaluc) or 1×10⁻⁶ cells were removed. 6HiPSC-CSs expressing the modified luciferase (Akaluc) were co-injected into the myocardium with 60 μL of PBS. In these transplanted mice, anesthesia was repeated on days 7, 14, and 28 post-transplantation, and Akaluminen-Hydrochloride (Fujifilm Corporation, 20 nmol / g body weight) was injected intraperitoneally. Ten minutes after administration, imaging was performed using an imaging system (NEWTON 7.0 FT500, Vilber Inc.) at a 20 cm × 20 cm field of view and a 1-minute exposure. Image analysis was performed using Kuant software. Results are shown in... Figure 2D and Figure 2E .like Figure 2D and Figure 2E The results clearly show that spherical cardiomyocytes transplanted into animal myocardium exhibit more efficient survival compared to dispersed cardiomyocytes transplanted into animal myocardium.

[0167] Example 3: Transplantation of cardiomyocyte spheroids into cynomolgus monkeys (1) Induction of myocardial ischemia-reperfusion injury in cynomolgus monkeys Based on Japanese domestic regulations and guidelines, all experimental procedures were reviewed by the Animal Experimentation Committee of Shinshu University and ultimately approved by the university president (No. 300023), Keio University (No. A2022-180), and Ina Research (No. 18088). Two weeks prior to cardiomyocyte spheroid transplantation, myocardial infarction was induced in a total of 10 cynomolgus monkeys (4 males and 6 females) according to the following method. The surgery was performed according to the methods described in Ichimura H. et al., Sci Rep. 2020;10:11883, or Kobayashi H. et al., Methods Mol Biol. 2021;2320:295–3023.

[0168] Specifically, cynomolgus monkeys were anesthetized via intramuscular injection of ketamine and toluidine, and a 3.5 mm diameter endotracheal tube was inserted and maintained with 2% isoflurane ventilation. Subcutaneous injection of buprenorphine was routinely administered to reduce postoperative pain. Blood pressure, oxygen saturation, and electrocardiogram (ECG) were monitored intraoperatively. Phenylephrine was administered intravenously to maintain appropriate blood pressure. After a median thoracotomy, a 4-0 silk suture was inserted into the myocardium of the mid-segment of the left anterior descending artery (LAD) and then threaded into a section of polyethylene tubing; a silicone tubing was then inserted above the polyethylene tubing and ligated with sutures. Before inducing ischemia, lidocaine 1 mg / kg and heparin 200 U / kg were administered intravenously; the same dose of heparin was administered hourly before reperfusion. After 180 minutes of mid-segment LAD occlusion, cardiac reperfusion was achieved by removing the tubing.

[0169] (2) Preparation and transport of cardiomyocyte spheroids for transplantation Cardiac cell spheroids prepared according to Example 2(1) were collected on the day of transplantation, diluted with physiological saline, placed in Eppendorf tubes, and transported to the transplantation facility at 4°C. Upon arrival at the transplantation facility, transplantation was immediately performed on cynomolgus monkeys according to the following method.

[0170] (3) Transplantation of cardiomyocyte spheroids The four male and six female cynomolgus monkeys that underwent the aforementioned induction of myocardial infarction were divided into two groups based on sex. One group was administered the equivalent of 2 × 10⁻⁶ mg / L of the drug prepared according to the method described in (2) above. 7 One group received cardiomyocyte spheroids, while the other group received a vector and were treated as described below. Euthanasia was performed 12 weeks post-transplantation for histological analysis; cardiac function and arrhythmias were monitored during the observation period. In addition, in a separate experiment, 10 cynomolgus monkeys (4 males and 6 females) induced with myocardial infarction were divided into two groups; one group received the equivalent of 6 × 10⁶ cardiomyocytes. 7 One group of myocardial cells were given myocardial cell spheres (as described in (2) above), and another group was given a carrier.

[0171] The specific transplantation method is as follows: On the 14th day after induced MI, a second median thoracotomy was performed to expose the heart. The myocardial cell spheroids obtained in (2) suspended in physiological saline, or the physiological saline carrier, were injected into the infarct area and marginal area 5–6 times, 100 μL each time, through a 27G injection needle.

[0172] Immunosuppression was achieved via intravenous administration of methylprednisolone and abatacept (Bristol Myers Squibb Co.) and subcutaneous administration of cyclosporine (Novartis International AG). Methylprednisolone was administered at 50 mg / kg / day for 3 consecutive days starting from day 1 post-transplantation, followed by 2 mg / kg / day. Abatacept was administered at 12.5 mg / kg starting from day 1 post-transplantation, and then every 2 weeks. Cyclosporine was administered at a daily dose of 5–10 mg / kg, adjusted according to peripheral blood trough concentrations from day 5 post-transplantation until the end of the experiment. Cyclosporine tracer levels are shown below. Figure 2F (Transplantation is equivalent to 2 × 10) 7 (a number of myocardial cells) and Figure 2G (Transplantation is equivalent to 6 × 10) 7 (1 cardiomyocyte).

[0173] (4) Analytical methods and procedures for animals after myocardial cell spheroid transplantation (Confirmation of the myocardial infarction model) Myocardial infarction is confirmed by transient ST-segment elevation on electrocardiogram and elevated serum cTnT.

[0174] (Echocardiography) Echocardiography was performed using a VIVID 7 system (GE Healthcare Biosciences Inc.) on days 0, 28, and 84 post-transplantation. The heart was observed parasternal short-axis after intramuscular injection of ketamine and methaqualone. Left ventricular end-diastolic diameter (LVEDD) and end-systolic diameter (LVESD) were measured in M-mode, and the fractional shortening (FS) was calculated using the following formula: FS = 100 × [(LVEDD – LVESD) / LVEDD].

[0175] (Computed Tomography) CT scans were performed on day 2 before transplantation, day 28 after transplantation, and day 84 after transplantation. CT imaging was performed according to the method described by Shiba Y. et al., Nature. 2016;538:388–391. Animals were anesthetized, intubated, and mechanically ventilated with 2% isoflurane. Cardiac imaging was performed using a R_mCT AX (Rigaku Corporation, Tokyo, Japan) with contrast agent (Iopamidol, 8 ml / min). Cardiac contraction and ventilation cycles were automatically synchronized. Left ventricular end-diastolic volume (LVEDV) and end-systolic volume (LVESV) were measured using Ziostation2 software (Amin, Tokyo, Japan). Left ventricular ejection fraction (LVEF) was calculated using the following formula: LVEF = 100 × [(LVEDV - LVESV) / LVEDV].

[0176] (Holter electrocardiogram) Holter monitoring was performed on days 4-4 before transplantation and on days 4, 7, 14, 28, 42, 56, 70, and 83 post-transplantation. A 2-lead precordial system was used to connect the Holter recorder. Animals were fitted with vests to protect the equipment, and ECGs were recorded for 24 hours. Ventricular tachycardia was defined as four or more consecutive premature ventricular contractions (PVCs) with a ventricular rate exceeding 150 bpm; sustained ventricular tachycardia was defined as a ventricular tachycardia lasting longer than 30 seconds. All analyses were performed by operators unaware of the grouping information.

[0177] (Hematological testing) Peripheral blood was collected, and plasma was separated to determine brain natriuretic peptide (BNP) levels. Peripheral blood cTnT levels were measured on day -12 (48 hours after MI) and day -10 (96 hours after MI) before transplantation. Whole blood was used to determine cyclosporine trough concentration using electrochemiluminescence immunoassay.

[0178] (Histology) On day 84 post-transplantation, animals receiving CSs were euthanized and subjected to full-body necropsy. Hearts were transversely sectioned into 5 mm thick slices and fixed with 4% paraformaldehyde. All sections were routinely stained with hematoxylin-eosin (HE) and picric acid-sirius red (PSR) to determine the scar area. If the graft was located in the scar area, the scar area was calculated by subtracting the graft area from the total area of ​​fibers stained red by PSR. Immunohistochemical analysis was performed on the sections using a primary antibody against human-specific cTnI (ab52862; Abcam Inc.) and a biotinylated secondary antibody (Vector Laboratories, Inc.). Colorimetric detection was performed using an HRP-labeled streptavidin ABC kit (Vector Laboratories Inc.), followed by a DAB substrate kit (Vector Laboratories Inc.).

[0179] The slides were also subjected to immunohistochemical analysis using the following primary antibodies: myosin light chain 2A (MLK-2A; S 58-205, Becton, Dickinson and Company), myosin light chain 2V primary antibody (MLC-2V; ab92721, Abcam), anti-α-actin primary antibody (ab137346, Abcam), anti-vimentin primary antibody (Merck & Co.), anti-connexin 43 primary antibody (Cx43, ab11370; Abcam Inc.), anti-N-cadherin primary antibody (Thermo Fisher Scientific Inc.), anti-cTnT primary antibody (clone: ​​13-11), anti-CD45 primary antibody (clone: ​​2B11 & PD), anti-Ki67 primary antibody (ab16667, Abcam), anti-pan-cadherin primary antibody (clone: ​​CH-19), and anti-tomato lectin primary antibody (Tomato Lectin, Dylight 594 Labeled Tomato). Lectin (Vector Laboratories, Inc.) and species-specific fluorescent dyes (ThermoFisher Scientific, Inc.).

[0180] Simultaneously, brain, lung, liver, kidney, and spleen tissues were collected and fixed with 4% PFA. Unless obvious abnormalities were found, several sections were randomly selected and immunohistochemically stained with hematoxylin and eosin (HE) and human-specific cTnI antibody to detect ectopic cardiomyocyte transplantation. Stained sections were imaged using a NanoZoomer 2.0-RS microscope (Hamamatsu Photonics Co., Ltd.) or a BZ-X700 microscope (KEYENCE Co., Ltd.).

[0181] (Statistical Analysis) Analysis of variance (ANOVA) was used to evaluate echocardiography, CT scans, BNP levels, graft area, and outcomes, with post-hoc comparisons between time points performed using Tukey's multiple comparisons test. Unpaired t-tests were used for comparisons between groups at different time points. The percentage of cTnT was analyzed using ANOVA with post-hoc comparisons performed using Tukey's multiple comparisons test. All summary data are expressed as mean ± standard error (SEM). All statistical analyses were performed using GraphPad Prism (GraphPad Software Inc.), and a significance threshold of P < 0.05 was set.

[0182] (5) Analysis of animals after myocardial cell spheroid transplantation (Histology) As described in Example 3(3), on day 84 (12 weeks) post-transplantation, the recipient received an equivalent of 2 × 10⁻⁶ cells / mL. 7 Hearts were harvested from animals with cardiomyocyte spheroids and stained with Picrosirius Red. Results showed no significant difference in scar area between the vector transplant animals and the cardiomyocyte spheroid transplant animals (9.12 ± 1.74% vs. 5.24 ± 0.49% of total left ventricular area, p = 0.15). When comparing transplanted 6 × 10⁶ heart cells... 7 One cardiomyocyte and 2 × 10 7 In animals with a number of cardiomyocytes, there was no difference in scar area between the two groups. Figure 4B (a)). The transplanted area is 6 × 10 7 Group 2 × 10 7 The group is 13 times larger ( Figure 4B (b), 4B(c)). The transplanted tissue accounted for 11.0 ± 1.6% of the scar area, a considerable size. Furthermore, the transplanted cardiomyocytes were identified using a human-specific cTnI antibody, and no signs of graft rejection were observed in the hearts of 4 out of 5 animals that received the transplant.

[0183] Furthermore, the cardiomyocytes of the transplanted animals exhibited a myogenic pattern similar to that of the host cardiomyocytes, with cTnT expression being equivalent to sarcomere length. Figure 2B This indicates that, 12 weeks post-transplantation, the maturity of the transplanted myocardial tissue was almost identical to that of the host myocardial cells.

[0184] In Example 3(3), for receiving human iPS cell-derived cardiomyocyte spheroids (equivalent to 6 × 10⁻⁶), 7 In animals that underwent ectopic cardiomyocyte transplantation (with 1000 cardiomyocytes), no signs of ectopic cardiomyocyte transplantation were observed in the lungs, liver, kidneys, or spleen. Figure 6 A–I).

[0185] All transplanted cells were cTnT-positive cardiomyocytes. Most cardiomyocytes were MLC2v-positive mature ventricular subtypes, with only a small number expressing MLC2a (…). Figure 4A (F) Ki67-positive cells were also observed in the transplantation area, but most of them were cTnT-negative non-cardiomyocytes; Ki67-positive cardiomyocytes were extremely rare at 12 weeks post-transplantation. Figure 4A (G)). The transplanted cardiomyocytes showed good vascularization. Figure 4A (J), 4A(M)), and highly expresses connexin 43 and cadherin ( Figure 4A (H), (I), (K), (L)). These results indicate that the transplanted cardiomyocytes survive at a maturity level similar to that of the host cardiomyocytes.

[0186] (Echocardiography) In Example 3(3), the acceptance is equivalent to 2 × 10 7 Human iPS cell-derived cardiomyocyte spheroid transplantation animals and vector group animals were compared. Left ventricular short-axis diameter (FS) was measured before transplantation and at 4 and 12 weeks post-transplantation. Results are shown in... Figure 3A .like Figure 3A As clearly shown, animals receiving cardiomyocyte spheroid transplantation exhibited significant improvement in contractile function 4 weeks post-transplantation. Furthermore, animals receiving the equivalent of 6 × 10⁻⁶ cells showed improved contractile function. 7 Animals that underwent cardiomyocyte spheroid transplantation of individual cardiomyocytes were examined by ultrasound before transplantation, at 4 weeks post-transplantation, and at 12 weeks post-transplantation. Their LVEF values ​​are shown below. Figure 5A and Figure 5B Echocardiography showed that animals receiving cardiomyocyte spheroid transplantation had a higher shortening fraction than the vector group, and this advantage persisted until 12 weeks post-transplantation. On the other hand, we also conducted experiments using cardiomyocytes derived from human iPS cells that were differentiated but not purified, and transplanted them into cynomolgus monkeys using the same method as in Example 3(3). Left ventricular short axis diameter (FS) was measured by ultrasound in animals receiving spheroid transplantation from unpurified cardiomyocyte populations and in the vector group before transplantation, at 4 and 12 weeks post-transplantation, and compared with the results of animals receiving cardiomyocyte spheroid transplantation. See [link to relevant documentation]. Figure 5D .like Figure 5D As shown, the FS value of animals transplanted with iPSC-derived cardiomyocyte spheroids prepared from unpurified cardiomyocyte populations was significantly lower than that of the vector group animals. As... Figure 5DAs clearly shown, animals receiving cardiomyocyte spheroids prepared from purified human iPS cell populations (hiPSC-CSCs(purified)) had significantly higher FS values ​​than animals receiving cardiomyocyte spheroids prepared from unpurified cardiomyocyte populations (hiPSC-CSCs(unpurified)). Compared to the unpurified group, the purified group showed significantly improved contractile function 4 weeks post-transplantation.

[0187] (Computed Tomography) In Example 3, human iPS cell-derived cardiomyocyte spheroids (equivalent to 6 × 10⁻⁶) were received. 7 Animals that underwent cardiomyocyte transplantation (with a number of myocardial cells) and the vector group underwent CT scans 1 day before transplantation, 28 days after transplantation, and 84 days after transplantation. The LVEF calculated from the results is shown in [image / data / image / etc.]. Figure 5B (a). At 4 and 12 weeks post-transplantation, the LVEF of animals with cardiomyocyte spheroids prepared from hiPSC-CSCs was significantly higher than that of the vector group.

[0188] (Hematological testing) In Example 3(3), peripheral blood (equivalent to 2 × 10⁻⁶ cells) was collected from animals that received transplanted human iPS cell-derived cardiomyocyte spheroids on days 2 and 4 post-transplantation. 7 cardiomyocytes ( Figure 5E -A and B), 6 × 10 7 cardiomyocytes ( Figure 5E Serum cTnT levels were measured using C and D assays; additionally, peripheral blood was collected on day 14 post-transplantation to measure serum BNP levels. Results are shown in... Figure 5E .like Figure 5E As shown in -A and 5E-C, both the cardiomyocyte spheroid transplanted animals and the vector group animals showed elevated serum cTnT, confirming the ischemic state. Figure 5E -A and C). In addition, serum BNP levels were roughly equivalent between the vector group and the cardiomyocyte spheroid transplantation group.

[0189] (Holter electrocardiogram) Holter electrocardiograms were recorded before transplantation and on days 4, 7, and 14 post-transplantation, as well as every two weeks thereafter, in both the animal and vector groups that received cardiomyocyte spheroids prepared from human iPS cell-differentiated cardiomyocyte populations. No significant ventricular arrhythmias were observed in the vector group. Figure 3C (a) and 3C(c)). Furthermore, among the five animals that received cardiomyocyte spheroids prepared from a population of cardiomyocytes differentiated from human iPS cells, four showed no or very few ventricular arrhythmias during the experiment. Figure 3C(b) and 3C(d)). It is worth noting that one of the animals (ID: C067, see Figure 5D A transient ventricular tachycardia (lasting 9 minutes and 14 seconds in a single day) occurred within 2 weeks post-transplantation. Figure 5D No further ventricular arrhythmias were observed thereafter. Figure 3B , 3C (b), 3C(d)).

[0190] Furthermore, in Example 3, cardiomyocytes prepared from a population differentiated from human iPS cells and equivalent to 6 × 10⁻⁶ were used. 7 Animals that underwent myocardial cell spheroid transplantation of individual myocardial cells were analyzed in the same way as the vector group animals, and the results are shown in... Figure 5C .Depend on Figure 5C It is evident that two-quarters of the transplanted animals (ID: C096 and C100, see Figure 5) exhibited persistent ventricular arrhythmias, but no persistent ventricular tachycardia was observed after day 14. Figure 5C (b) and 5C(d)). The highest heart rate of these ventricular arrhythmias was 176 bpm, and the duration was only 11 minutes and 6 seconds and 16 minutes and 3 seconds, respectively. Figure 5D Compared to previous studies (Shiba Y. et al., Nature. 2016;538:388–391), transient ventricular arrhythmias observed in some transplanted animals in Example 3 were shorter in duration and less frequent. This shorter duration and lower incidence can be explained by differences in the human iPS cell-derived cardiomyocytes transplanted; these differences are thought to stem from factors such as the higher purity and maturity of the cell population prepared in Example 1 and the higher proportion of viable cells in the cardiomyocyte population transplanted to cynomolgus monkeys in Example 3.

[0191] The inventors have developed a method for directly transplanting pluripotent stem cell-derived cardiomyocytes (PSC-CMs) into the myocardium, as illustrated in Example 3. This method carries potential risks associated with needle injection, such as tissue damage, bleeding, thrombosis, and cardiac edema; however, the transplanted PSC-CMs have been shown to contract synchronously with adjacent recipient cardiomyocytes, a phenomenon known as cardiac regeneration therapy.

[0192] The cardiomyocyte spheroids prepared in Example 2 have several advantages and may become an ideal active ingredient for cardiac regeneration therapy. Firstly, the preparation process for cardiomyocyte spheroids is simple, requiring only seeding of cardiomyocyte populations on a specific culture plate and allowing them to naturally form cell clusters. This simplicity is particularly advantageous for preparing large quantities of cardiomyocyte populations for clinical use (such as those required for human cardiac regeneration therapy).

[0193] Secondly, cardiomyocyte spheroids can maintain a survival rate of at least 24 hours in physiological saline at 4°C, allowing the drug composition to be transported from CPC to multiple medical institutions without pre-transplantation cell processing. Thirdly, cardiomyocyte spheroids can be easily expanded in vitro, enabling the mass production of cells for transplantation.

[0194] Fourth, the cardiomyocyte spheroids prepared in Example 2 can be effectively transplanted and colonized in the recipient's myocardial tissue without the need for additives. As described in Example 3, the transplantation is equivalent to 6 × 10 7 Animals with cardiomyocyte spheroids showed significant recovery of contractile function even 12 weeks post-transplantation, and the graft accounted for 11.0 ± 1.6% of the scar area. The post-transplant arrhythmias observed in Example 3 were similar to those in previous studies (Shiba Y. et al., Nature 2016;538:388–391, where cardiomyocyte purity was 63%), however, their duration was significantly shorter and their incidence lower. Figure 5F The low incidence of post-transplant arrhythmias may be due to differences in the origin of the transplanted cardiomyocytes. Previous studies have focused on cardiomyocytes derived from allogeneic monkeys, while Example 3 used allogeneic human iPS cells.

[0195] Nevertheless, other allogeneic transplantation studies (Liu YW et al., Nat Biotechnol. 2018;36:597–605; Chong JJ et al., Nature 2014;510:273–277; Romagnuolo R. et al., Stem Cell Reports 2019;12:967–981; Wahiba D. et al., Circulation 2022;145:1412–1426 [US20200407687]) have reported a high incidence of persistent ventricular arrhythmias after transplantation.

[0196] In other xenotransplantation studies, such as Liu YW et al. (Nat Biotechnol. 2018;36:597–605), the purity of transplanted cardiomyocytes was 86–99%, but the frequency and onset time of persistent ventricular arrhythmias were both high. Similarly, Chong JJ et al. (Nature 2014;510:273–277) used cardiomyocytes with a purity of 73 ± 12%, yet still observed a high frequency and early onset time of persistent ventricular arrhythmias. Furthermore, Romagnuolo R. et al. (Stem Cell Reports 2019;12:967–981) used cardiomyocytes with a purity of 81.9 ± 3.9% and a viable cell percentage of 80.7 ± 2.4%, but the frequency and duration of ventricular arrhythmias remained high. Wahiba D. et al. (Circulation 2022;145:1412–1426) reported that the spontaneous beat rate of the transplanted cardiomyocytes was 45 bpm on day 20 of culture and about 30 bpm on day 40, and ventricular arrhythmias were still observed.

[0197] Based on the above findings, it is demonstrated for the first time that to achieve the low incidence and short duration of ventricular arrhythmias after transplantation as seen in Example 3, at least two conditions must be met: (1) the purity of the cardiomyocytes contained in the transplanted cardiomyocyte spheroids is 99.8%; and (2) the spontaneous beat rate is 34 ± 13 beats / min. Furthermore, the human iPS cell-derived cardiomyocytes prepared in Example 1 underwent a total differentiation and purification process of up to 25 days, unlike methods that obtain mature cardiomyocytes through long-term culture.

[0198] In summary, when clinical-grade iPSC-CM formulations were transplanted into the hearts of infarcted primates, transient, non-fatal arrhythmias occurred, but long-term graft survival and improved contractile function were confirmed. These arrhythmias were considered manageable within acceptable safety limits. The human iPS cell-derived cardiomyocyte population prepared in Example 1 was characterized by over 90% of cells being positive for cardiac troponin T (…). Figure 1A (b) and spontaneous contractions occur at a rate of 0–60 times / min. Figure 1C Furthermore, the cardiomyocyte spheroid system prepared in Example 2 was manufactured using the cardiomyocytes obtained in Example 1 within 30 days of the initiation of self-differentiation (excluding the cryopreservation period). It is expected that these cardiomyocyte populations will regenerate the damaged areas of the recipient's heart by electrically synchronizing with the recipient cardiomyocytes, maintaining their maturity, and surviving long-term. Figure 2B , 3A 4A, 4B, 5A, 5B). Furthermore, in animals that received these cardiomyocyte spheroid transplants, some did not develop ventricular arrhythmias; even when they did, they were significantly less frequent than in previous studies, transient, and clinically manageable.Figure 3C , 5C 5D).

[0199] Based on the above properties, the cardiomyocytes prepared in Example 1 and the cardiomyocyte spheroids manufactured in Example 2 can be used as cell therapy products suitable for clinical application.

[0200] [Industry Applicability] According to the present invention, the following can be provided: — Human pluripotent stem cell-derived cardiomyocyte populations and cardiomyocyte spheroids that may meet the requirements for clinical application; — A pharmaceutical composition with the above-mentioned myocardial cell populations and / or myocardial cell spheroids as active ingredients; — The method for manufacturing the aforementioned cardiomyocyte population; and — The method for manufacturing the cardiomyocyte spheroids.

[0201] The above description illustrates preferred embodiments of the present invention; however, the present invention is not limited thereto.

[0202] Various additions, deletions, substitutions and modifications may be made without departing from the spirit and scope of this invention.

[0203] This invention should not be limited to the foregoing description, but should be construed solely by the scope of the appended claims.

Claims

1. A population of cardiomyocytes comprising ventricular myocytes differentiated from human pluripotent stem cells, wherein, This population of cardiomyocytes has the following characteristics: (1) Cardiac troponin T positive cells accounted for more than 90% of all surviving cells; (2) Spontaneous pulsation ranges from 0 to 60 beats per minute; and, (3) The cardiomyocyte population is a cell population that has been in the population for less than 30 days since the start of differentiation.

2. The cardiomyocyte population according to claim 1, wherein, Spontaneous pulsation ranges from 0 to 50 beats per minute.

3. A pharmaceutical composition comprising: at least one selected from the group consisting of a cardiomyocyte population as described in claim 1 or 2 and cardiomyocyte spheres obtained by forming the cardiomyocyte population into a spherical shape, and a pharmaceutically acceptable carrier.

4. The pharmaceutical composition according to claim 3, wherein, In the aforementioned cardiomyocyte population, surviving cells account for more than 80% of all cells.

5. The pharmaceutical composition according to claim 3, wherein, The cells constituting the myocardial cell spheres account for more than 60% of all cells in the pharmaceutical composition.

6. The pharmaceutical composition according to claim 3, for the treatment of heart failure.

7. A method for preparing the cardiomyocyte population of claim 1 or 2, comprising: (a) Expanding human pluripotent stem cells; (b) Culture the expanded human pluripotent stem cells under conditions that allow them to differentiate into cardiomyocytes to generate a cell population containing more than 60% cardiomyocytes; and, (c) Remove human pluripotent stem cells and non-myocardial cells from the cell population.

8. A cardiomyocyte spheroid obtained by forming a group of cardiomyocytes as described in claim 1 or 2 into a spherical shape.

9. The cardiomyocyte spheroids according to claim 8, wherein, The diameter of the myocardial cell spheres is 50 to 300 micrometers.

10. A method for preparing cardiomyocyte spheroids, comprising: After suspending the cardiomyocyte population as described in claim 1 or 2 in a culture medium, it is then statically cultured in a culture vessel with micropores at the bottom.

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