Method for purification and production of ventricular cardiomyocytes

Abstract

The present invention relates to a method for the purification and production of ventricular cardiomyocytes. According to the present invention, a high concentration of ventricular cardiomyocytes can be obtained through physical methods using the surface markers of CD81, IL1RL1, and CD59 as identifiers.

Description

Method for purifying and manufacturing ventricular cardiomyocytes

[0001] The present invention relates to a method for purifying and manufacturing ventricular cardiomyocytes, and according to the present invention, a high concentration of ventricular cardiomyocytes can be obtained through a physical method using surface markers of CD81, IL1RL1, and CD59 as markers.

[0002] Myocardial infarction is a disease in which cardiomyocytes die due to a decrease in the supply of oxygen and nutrients to all or part of the heart, and cardiac failure is a disease in which the heart's function declines and it cannot properly supply blood to the body. Both myocardial infarction and heart failure can be caused by the loss of cardiomyocytes (heart muscle cells).

[0003] Myocardial infarction and heart failure are on the rise worldwide, but because damaged or defective hearts have difficulty repairing themselves, chemical treatments for these conditions only delay the progression of the disease.

[0004] Meanwhile, heart transplantation can be a fundamental treatment for restoring heart function, but it can cause problems such as a shortage of organ donors, medical ethics, and physical and financial burden on patients.

[0005] For this reason, cardiomyocytes derived from stem cells, such as induced pluripotent stem cells (iPSCs) or embryonic stem cells (ESCs), are being utilized in regenerative medicine for cardiac disease. Cardiomyocytes differentiated from stem cells can include three subtypes of cardiomyocytes: nodal, atrial, and ventricular, and may additionally include non-cardiomyocytes, such as undifferentiated cells.

[0006] Among these, in the method of isolating cardiomyocytes, cell surface markers that have been discovered so far have been identified and markers specifically expressed in cardiomyocytes have been searched for, and representative examples include CD177z, CD42, CD236a, SIRPA, and CD71. However, the surface markers discovered so far are only used as markers for purifying cardiomyocytes and do not distinguish between the types and functions of atrial and ventricular cells.

[0007] Ventricular cardiomyocytes may be directly involved in the development of cardiac contraction in myocardial infarction. Therefore, isolating pure ventricular cardiomyocytes may be crucial for cell regenerative therapy to restore cardiac function. Furthermore, transplanting differentiated cardiomyocytes containing different cardiomyocyte subtypes into disease models can induce arrhythmias. Therefore, the development of a technology to purify pure ventricular cardiomyocytes is urgently needed.

[0008] Accordingly, the inventors of the present invention have developed a method for purifying and manufacturing ventricular cardiomyocytes by confirming that when only CD81, IL1RL1, and CD59 positive cells are separated by a physical method in order to isolate and purify ventricular cardiomyocytes at a high concentration from a cell population containing cardiomyocytes, only single-cell ventricular cardiomyocytes can be effectively separated and purified.

[0009] The purpose of the present invention is to provide a method for purifying ventricular cardiomyocytes.

[0010] Another object of the present invention is to provide a method for producing ventricular cardiomyocytes.

[0011] The present invention relates to a method for purifying and manufacturing ventricular cardiomyocytes, and according to the present invention, a high concentration of ventricular cardiomyocytes can be obtained through a physical method using surface markers of CD81, IL1RL1, and CD59 as markers.

[0012] Hereinafter, the present invention will be described in more detail.

[0013] One aspect of the present invention relates to a method for purifying ventricular cardiomyocytes, comprising: i) a step of confirming the expression of at least one marker selected from the group consisting of IL1RL1, CD81, and CD59 in cardiomyocytes; and ii) a step of isolating cells positive for the expression of at least one marker selected from the group consisting of IL1RL1, CD81, and CD59 confirmed in step i) at the cell level.

[0014] The term "cardiomyocyte" used herein refers to the muscle cells that form the heart wall. Cardiomyocytes can be divided into nodal, atrial, and ventricular cardiomyocytes, depending on the structure of the heart. Cardiomyocytes can be damaged or destroyed when exposed to stressors such as myocardial infarction or myocarditis. Damage or destruction of cardiomyocytes can lead to a decline in myocardial function, which can lead to heart disease. Therefore, cardiomyocytes differentiated from pluripotent stem cells can be utilized in regenerative cell therapy for restoring cardiac function or treating heart disease.

[0015] Meanwhile, in cell regenerative therapy for myocardial infarction, the isolation of pure ventricular cardiomyocytes among differentiated cardiomyocytes may be more important. This is because the purity of ventricular cardiomyocytes can affect the efficacy of cell regenerative therapy. For example, when different subtypes of cardiomyocytes are used in cell regenerative therapy, or when immature cardiomyocytes or normal myocytes are mixed in, the prognosis may be worse than when pure ventricular cardiomyocytes are used. Therefore, methods for purifying ventricular cardiomyocytes alone among cardiomyocytes differentiated from stem cells are crucial for regenerative cell therapy.

[0016] In the present invention, ventricular cardiomyocytes may be derived from stem cells, but are not limited thereto.

[0017] In the present invention, the stem cell may be a ventricular cardiomyocyte derived from a human pluripotent stem cell (hPSC), and may be, for example, a ventricular cardiomyocyte derived from an induced pluripotent stem cell (iPSC) or an embryonic stem cell (ESC), but is not limited thereto.

[0018] The term "marker" as used herein means a marker protein or marker gene, and means a protein or its gene that is specifically expressed on the cell surface, cytoplasm and / or nucleus of a given cell, for example, on the cell surface.

[0019] In the present invention, the markers of steps i) and ii) may additionally include CD151, thereby further increasing the purification efficiency of ventricular cardiomyocytes.

[0020] In the present invention, at least one marker selected from the group consisting of IL1RL1, CD81, CD59 and CD151 is used as a marker for purifying ventricular cardiomyocytes from a cell population containing cardiomyocytes.

[0021] In the present invention, the cells that do not express the marker, i.e., cells that are negative for the marker, may be undifferentiated stem cells or embryoid bodies, or nodal cardiomyocytes or atrial cardiomyocytes.

[0022] In the present invention, the method for purifying ventricular cardiomyocytes may include: i) a step of confirming the expression of at least one marker selected from the group consisting of IL1RL1, CD81, and CD59, and a CD151 marker in cardiomyocytes; and ii) a step of isolating cells in which the expression of at least one marker selected from the group consisting of IL1RL1, CD81, and CD59, and the CD151 marker, confirmed in step i), is positive at the cell level.

[0023] In the present invention, the method for purifying ventricular cardiomyocytes may additionally include, before performing step i), a step of isolating cells expressing CD71 positively from cardiomyocytes at the cell level to purify only contractile cardiomyocytes.

[0024] In the present invention, in the case of contractile cardiomyocytes, since it is possible to analyze changes in the level of contractile ability according to the drug response, it is desirable to isolate and use only contractile cardiomyocytes in a research process using cardiomyocytes, such as in the cardiotoxicity drug evaluation of a new drug candidate, and since it can also be applied as a cell treatment for heart failure using contractile cardiomyocytes, the process of distinguishing and separating contractile cardiomyocytes and non-contractile cardiomyocytes among differentiated cardiomyocytes can also play an important role.

[0025] In the present invention, CD71 can be used as a surface marker specifically expressed in contractile cardiomyocytes. Therefore, when cardiomyocytes are stained with the CD71 marker, cells that do not express CD71, i.e., CD71-negative cells, may be non-contractile cardiomyocytes.

[0026] Cardiomyocytes that have not undergone the ventricular cardiomyocyte purification method according to the present invention may have a ventricular cardiomyocyte purity of 10 to 70%, 10 to 60%, 10 to 50%, 20 to 70%, 20 to 60%, 20 to 50%, 30 to 70%, 30 to 60%, 30 to 50%, 40 to 70%, 40 to 60%, or 40 to 50%.

[0027] Cardiomyocytes obtained through the ventricular cardiomyocyte purification method according to the present invention may have a ventricular cardiomyocyte purity of 70 to 95%, 70 to 92%, 70 to 90%, 70 to 85%, 70 to 80%, 73 to 95%, 73 to 92%, 73 to 90%, 73 to 85%, 73 to 80%, 80 to 95%, 80 to 92%, 80 to 90%, or 80 to 85%.

[0028] Another aspect of the present invention relates to a method for producing ventricular cardiomyocytes, comprising: i) a step of inducing differentiation of stem cells into cardiomyocytes; ii) a step of confirming the expression of at least one marker selected from the group consisting of IL1RL1, CD81, and CD59 in the cardiomyocytes; and iii) a step of isolating cells positive for the expression of at least one marker selected from the group consisting of IL1RL1, CD81, and CD59 confirmed in step ii) at the cell level.

[0029] In the present invention, the stem cell may be a human pluripotent stem cell (hPSC), and may be, for example, an induced pluripotent stem cell (iPSC) or an embryonic stem cell (ESC), but is not limited thereto.

[0030] In the present invention, the markers of steps ii) and iii) may additionally include CD151, thereby enabling the production of ventricular cardiomyocytes of higher purity.

[0031] In the present invention, at least one marker selected from the group consisting of IL1RL1, CD81, CD59 and CD151 is used as a marker for purifying ventricular cardiomyocytes from a cell population containing cardiomyocytes.

[0032] In the present invention, the cells that do not express the marker, i.e., cells that are negative for the marker, may be undifferentiated stem cells or embryoid bodies, or nodal cardiomyocytes or atrial cardiomyocytes.

[0033] In the present invention, the method for producing ventricular cardiomyocytes may include: i) a step of inducing differentiation of stem cells into cardiomyocytes; ii) a step of confirming the expression of at least one marker selected from the group consisting of IL1RL1, CD81, and CD59, and a CD151 marker in the cardiomyocytes; and iii) a step of isolating cells in which the expression of at least one marker selected from the group consisting of IL1RL1, CD81, and CD59, and the CD151 marker, confirmed in step ii), is positive at the cell level.

[0034] In the present invention, the method for producing ventricular cardiomyocytes may include a step of first purifying cardiomyocytes using a metabolic control culture medium between steps i) and ii), thereby removing cells other than cardiomyocytes and further increasing the efficiency of purifying cardiomyocytes.

[0035] In the present invention, the metabolic control culture medium may contain human serum albumin and lactate.

[0036] In the present invention, the metabolism control culture medium may be a culture medium containing human serum albumin and lactate in a culture medium excluding glucose, and by creating an environment in which cells other than cardiomyocytes cannot perform cell activity, the efficiency of purification of cardiomyocytes can be further increased.

[0037] In the present invention, the method for producing ventricular cardiomyocytes may include a step of maturing cardiomyocytes using a cardiomyocyte maturation induction culture medium between steps i) and ii), thereby shortening the maturation period of cardiomyocytes.

[0038] In the present invention, the culture medium for inducing cardiomyocyte maturation may include at least one selected from the group consisting of B27 (B27 supplement), T3 (3,3′,5-Triiodo-L-thyronine), and Dex (Dexamethasone).

[0039] In the present invention, the culture medium for inducing cardiomyocyte maturation may be an RPMI1640 culture medium to which at least one selected from the group consisting of B27 (B27 supplement), T3 (3,3′,5-Triiodo-L-thyronine), and Dex (Dexamethasone) is added.

[0040] In the present invention, the culture medium for inducing cardiomyocyte maturation may contain B27 at a concentration of 0.1 to 5 wt%, 0.1 to 4 wt%, 0.1 to 3 wt%, 0.1 to 2 wt%, 0.5 to 5 wt%, 0.5 to 4 wt%, 0.5 to 3 wt%, 0.5 to 2 wt%, 1 to 5 wt%, 1 to 4 wt%, 1 to 3 wt%, or 1 to 2 wt%.

[0041] In the present invention, the culture medium for inducing cardiomyocyte maturation may contain T3 at a concentration of 5 to 50 ng / ml, 5 to 40 ng / ml, 5 to 30 ng / ml, 5 to 20 ng / ml, 10 to 50 ng / ml, 10 to 40 ng / ml, 10 to 30 ng / ml, or 10 to 20 ng / ml.

[0042] In the present invention, the culture medium for inducing cardiomyocyte maturation may contain Dex at a concentration of 1 to 10 nM, 1 to 9 nM, 1 to 8 nM, 1 to 7 nM, 1 to 6 nM, 1 to 5 nM, 3 to 10 nM, 3 to 9 nM, 3 to 8 nM, 3 to 7 nM, 3 to 6 nM, or 3 to 5 nM.

[0043] In the present invention, step ii) may be performed after culturing the cardiomyocytes induced to differentiate in step i) for 14 to 28 days, 14 to 26 days, 14 to 24 days, 14 to 22 days, 14 to 20 days, 14 to 18 days, or 14 to 16 days, for example, 14 days, thereby obtaining highly pure ventricular cardiomyocytes.

[0044] In the present invention, step ii) may be performed by first purifying the cardiomyocytes induced to differentiate in step i) using a metabolic control culture medium, and then culturing them for 14 to 28 days, 14 to 26 days, 14 to 24 days, 14 to 22 days, 14 to 20 days, 14 to 18 days, or 14 to 16 days, for example, 14 days, using a cardiomyocyte maturation induction culture medium, thereby obtaining highly pure ventricular cardiomyocytes.

[0045] In the present invention, the method for producing ventricular cardiomyocytes may additionally include, between steps i) and ii), a step of isolating cells expressing CD71 positively in cardiomyocytes at the cell level to purify only contractile cardiomyocytes.

[0046] In the present invention, in the case of contractile cardiomyocytes, since it is possible to analyze changes in the level of contractile ability according to the drug response, it is desirable to isolate and use only contractile cardiomyocytes in a research process using cardiomyocytes, such as in the cardiotoxicity drug evaluation of a new drug candidate, and since it can also be applied as a cell treatment for heart failure using contractile cardiomyocytes, the process of distinguishing and separating contractile cardiomyocytes and non-contractile cardiomyocytes among differentiated cardiomyocytes can also play an important role.

[0047] In the present invention, CD71 can be used as a surface marker specifically expressed in contractile cardiomyocytes. Therefore, when cardiomyocytes are stained with the CD71 marker, cells that do not express CD71, i.e., CD71-negative cells, may be non-contractile cardiomyocytes.

[0048] High-purity ventricular cardiomyocytes can be obtained by the method for producing ventricular cardiomyocytes according to the present invention, and specifically, the purity of the ventricular cardiomyocytes can be 70 to 95%, 70 to 92%, 70 to 90%, 70 to 85%, 70 to 80%, 73 to 95%, 73 to 92%, 73 to 90%, 73 to 85%, 73 to 80%, 80 to 95%, 80 to 92%, 80 to 90%, or 80 to 85%.

[0049] The present invention relates to a method for purifying and manufacturing ventricular cardiomyocytes, and according to the present invention, a high concentration of ventricular cardiomyocytes can be obtained through a physical method using surface markers of CD81, IL1RL1, and CD59 as markers.

[0050] Figure 1 is a schematic diagram showing a method for differentiating cardiomyocytes and a method for purifying ventricular cardiomyocytes according to one embodiment of the present invention.

[0051] Figure 2 is a graph showing the results of analyzing the action potential of cardiomyocytes before performing secondary purification using a marker for purifying ventricular cardiomyocytes according to one embodiment of the present invention.

[0052] FIG. 3 is a graph showing the results of RNA-seq analysis performed to secure a list of markers for purifying ventricular cardiomyocytes according to one embodiment of the present invention.

[0053] Figure 4 illustrates a method of measuring cell images and action potential after performing secondary purification using a marker for purifying ventricular cardiomyocytes according to one embodiment of the present invention.

[0054] Figure 5 is a graph showing the results of analyzing the action potential of cardiomyocytes after performing secondary purification using a marker for purifying ventricular cardiomyocytes according to one embodiment of the present invention.

[0055] Figure 6 is a graph confirming the gene expression pattern before and after purification of ventricular cardiomyocytes according to one embodiment of the present invention.

[0056] Figure 7 shows the results (7a) confirming that only contractile cardiomyocytes can be specifically labeled through CD71 staining in differentiated cardiomyocytes, and the results (7b) of flow cytometry analysis of SIRPA-labeled cells and CD71-labeled cells as a control group.

[0057] The present invention relates to a method for purifying ventricular cardiomyocytes, comprising: i) a step of confirming the expression of at least one marker selected from the group consisting of IL1RL1, CD81, and CD59 in cardiomyocytes; and ii) a step of isolating cells positive for the expression of at least one marker selected from the group consisting of IL1RL1, CD81, and CD59 confirmed in step i) at the cell level.

[0058] Hereinafter, the present invention will be described in more detail with reference to the following examples. However, these examples are only intended to illustrate the present invention, and the scope of the present invention is not limited by these examples.

[0059]

[0060] Example 1. Selection and Efficacy Confirmation of Ventricular Cardiomyocyte Purification Markers

[0061] 1-1. Production of human induced pluripotent stem cells (hiPSCs)

[0062] Human induced pluripotent stem cells (hiPSC) cell lines were created using somatic cells from normal individuals for whom donor consent was completed, using a vector- and virus-free reprogramming kit (stemgent, StemRNA™ 3rd Gen Reprogramming Kit) according to the manufacturer's protocol.

[0063]

[0064] 1-2. Induction of differentiation into cardiomyocytes from human induced pluripotent stem cells (hiPSCs)

[0065] According to the schedule of Fig. 1, cardiomyocyte differentiation was induced from human induced pluripotent stem cells by the following method. Human induced pluripotent stem cells (hiPSC) cell lines were maintained in an undifferentiated state on Matrigel in iPS-BREW XF medium (StemMACS TM, Mitenyi Biotec) for 4 days. For initial differentiation, the cultured stem cells were sequentially treated with small molecule compounds CHIR 99021 and Wnt-C59, and the cells were differentiated and cultured for 4 days in cardiomyocyte differentiation medium (CDM; RPMI1640 (ThermoFisher Scientific) + 500 μg / ml Human serum albumin (Sigma Aldrich) + 213 μg / ml Ascorbic acid (Sigma Aldrich)) to induce differentiation into contracting cardiomyocytes.

[0066] After 8 days of differentiation induction, contractile cardiomyocytes were observed and were first purified for 3 days using a metabolic control medium containing human serum albumin (Huamn Serum Albumn) (Sigma, 2%) and lactate (Sigma, 1 mM / ml) in glucose-free RPMI1640 medium.

[0067] Then, the culture medium was replaced with a cardiomyocyte maturation induction medium (RPMI1640 + B27 (B27 supplement) (Gibco, 1%) + T3 (3,3′,5-Triiodo-L-thyronine) (Sigma, 10ng / ml) + Dexamethasone (Stemcell technology, 5nM)) for 14 days.

[0068] After cultured cardiomyocytes were separated into single cells using TyprLE-select (Gibco), the voltage and current of all three types of cardiomyocytes, including ventricular, atrial, and nodal, were recorded through an electrophysiological analysis method (patch clamp), and the results are shown in Fig. 2. As shown in Fig. 2, it was confirmed that the action potential of cardiomyocytes after performing only the first purification was mixed, with 4% nodal cardiomyocytes, 46% atrial cardiomyocytes, and 50% ventricular cardiomyocytes.

[0069]

[0070] 1-3. Selection of ventricular cardiomyocyte purification markers through RNA unit gene analysis (single cell array)

[0071] Differentiated cardiomyocytes were first purified and cultured in a maturation-inducing medium for 10, 55, and 100 days, respectively. These cells were then manually isolated into 100-cell units. Each cell was then lysed to obtain cell extracts, which were then subjected to global transcriptome analysis. From the results of the global transcriptome analysis, hierarchical clustering of the entire gene expression profile was performed using average linkage, and the results are shown in Figure 3a.

[0072] As a result, as can be seen in Fig. 3a, it was confirmed that all cells analyzed through changes in cardiomyocyte-specific genes in cardiomyocytes cultured for 10, 55, and 100 days were cardiomyocytes.

[0073] Among the analyzed genes, cardiomyocyte genes and ventricular cardiomyocyte genes were first isolated, and then, among the genes showing differences in expression levels between 55 and 100 days of culture, the genes encoding cell surface markers (CD) of the increased genes were analyzed, and the results are shown in Fig. 3b. Through the results of Fig. 3b, CD59, IL1RL1, CD81, and CD151, which are surface markers specific for ventricular cardiomyocytes, were selected.

[0074]

[0075] 1-4. Confirmation of the efficacy of purified ventricular cardiomyocyte markers

[0076] First, based on the analysis results from the above examples, we compared and analyzed genes encoding cell surface markers (CDs) among those that exhibited differences in expression levels in differentiated cardiomyocytes. To efficiently purify ventricular cardiomyocytes, pure cardiomyocytes were initially purified using a metabolic control culture medium.

[0077] We sought to determine whether CD59, IL1RL1, CD81, and CD151 could be used as markers to specifically select ventricular cardiomyocytes among cardiomyocytes differentiated from hPSCs. After culturing for 14 days in a maturation-inducing medium, CD59, IL1RL1, CD81, and CD151 were stained and then purified secondarily using flow cytometry.

[0078] Purified cardiomyocytes isolated with CD59, IL1RL1, CD81, and CD151 markers were cultured in a maturation-inducing medium for 24 hours, and as shown in Fig. 4a, it was confirmed that the cells purified with the markers contracted. Then, as shown in Fig. 4b, the action potential was recorded using the patch clamp technique, and the results are shown in Fig. 5.

[0079] As a result, as shown in Fig. 5, it was confirmed that the action voltage of cardiomyocytes isolated and purified with the marker CD59 was distributed in 87% of ventricular cardiomyocytes, the action voltage of cardiomyocytes isolated and purified with the marker IL1RL1 was distributed in 80% of ventricular cardiomyocytes, the action voltage of cardiomyocytes isolated and purified with the marker CD81 was distributed in 89% of ventricular cardiomyocytes, and the action voltage of cardiomyocytes isolated and purified with the marker CD151 was distributed in 92% of ventricular cardiomyocytes.

[0080] In addition, genetic analysis was additionally performed on cardiomyocytes after performing only the first purification and cardiomyocytes after performing the second purification using the CD59 marker, and the results are shown in Fig. 6. As shown in Fig. 6, in the case of cardiomyocytes after performing the second purification using the CD59 marker (After sorting), the expression of the cardiomyocyte-specific gene TNNT2 and the ion channel genes SCN5A and CACN1C involved in cardiomyocytes increased compared to the cardiomyocytes after performing only the first purification (Before sorting), and the expression of the ventricular cardiomyocyte-specific genes MYL2 and MLC2V increased, and the expression of the atrial cardiomyocyte MLC2a gene decreased.

[0081] Accordingly, it was confirmed that ventricular cardiomyocytes within cardiomyocytes can be specifically purified and separated through the markers CD59, IL1RL1, CD81, and CD151 selected in the present invention.

[0082]

[0083] Example 2. Confirmation of the efficacy of CD71, a purified marker for contractile cardiomyocytes.

[0084] The efficacy of the marker CD71 for specifically selecting only contractile cells among cardiomyocytes differentiated from hPSCs was confirmed (Patent Document KR 10-1916902 B1). Cardiomyocytes differentiated from hPSCs were treated with an anti-CD71 antibody, stained, and then observed under a microscope after developing the anti-CD71 antibody. Furthermore, to determine whether CD71 can be used as a significant surface marker, cardiomyocytes were labeled with SIRPA or CD71, which are known to be useful as cardiomyocyte identification markers, and the degree of fluorescence was analyzed using a flow cytometer (FACS).

[0085] As a result, as shown in Fig. 7, it was confirmed that CD71 can be used as a specific surface marker for contractile cardiomyocytes (Fig. 7a), and the flow cytometry analysis results confirmed that it showed a pattern similar to the SIRPA surface marker, which is a cardiomyocyte surface factor (Fig. 7b).

[0086] In the case of SIRPA, it is known as a specific factor for cardiomyocytes without distinguishing between contractile or non-contractile contractile cells, and it was confirmed that only contractile cardiomyocytes could be specifically selected using the marker CD71.

[0087] The present invention relates to a method for purifying and manufacturing ventricular cardiomyocytes, and according to the present invention, a high concentration of ventricular cardiomyocytes can be obtained through a physical method using surface markers of CD81, IL1RL1, and CD59 as markers.

Claims

1. i) a step of confirming the expression of one or more markers selected from the group consisting of IL1RL1, CD81 and CD59 in cardiomyocytes; and ii) A method for purifying ventricular cardiomyocytes, comprising: a step of separating cells at the cell level that are positive for expression of at least one marker selected from the group consisting of IL1RL1, CD81, and CD59 confirmed in step i); 2. A method for purifying ventricular cardiomyocytes, wherein the cardiomyocytes in paragraph 1 are cardiomyocytes derived from stem cells.

3. A method for purifying ventricular cardiomyocytes, characterized in that the stem cells in the second paragraph are human pluripotent stem cells (hPSCs).

4. A method for purifying ventricular cardiomyocytes in the first paragraph, wherein the markers of steps i) and ii) additionally include CD151.

5. A method for purifying ventricular cardiomyocytes, wherein, in the first paragraph, before performing step i), a step is additionally included for purifying only contractile cardiomyocytes by separating cells expressing CD71 positively at the cell level. 6.i) A step of inducing differentiation from stem cells into cardiomyocytes; ii) a step of confirming the expression of one or more markers selected from the group consisting of IL1RL1, CD81 and CD59 in cardiomyocytes; and iii) A method for producing ventricular cardiomyocytes, comprising: a step of isolating cells at the cell level that are positive for the expression of at least one marker selected from the group consisting of IL1RL1, CD81, and CD59 confirmed in step ii); 7. A method for producing ventricular cardiomyocytes, characterized in that the stem cells in paragraph 6 are human pluripotent stem cells (hPSCs).

8. A method for producing ventricular cardiomyocytes in the sixth paragraph, wherein the markers of steps ii) and iii) additionally include CD151.

9. A method for producing ventricular cardiomyocytes, wherein, in the 6th paragraph, between steps i) and ii), a step of first purifying cardiomyocytes using a metabolic control culture medium is additionally included.

10. A method for producing ventricular cardiomyocytes in claim 9, wherein the metabolic control culture medium contains human serum albumin and lactate.

11. A method for producing ventricular cardiomyocytes, wherein, between steps i) and ii), a step of maturing cardiomyocytes using a cardiomyocyte maturation induction culture medium is additionally included.

12. A method for producing ventricular cardiomyocytes in claim 11, wherein the cardiomyocyte maturation induction culture medium comprises at least one selected from the group consisting of B27 (B27 supplement), T3 (3,3′,5-Triiodo-L-thyronine), and Dex (Dexamethasone).

13. A method for producing ventricular cardiomyocytes, wherein step ii) is performed after culturing the cardiomyocytes induced to differentiate in step i) for 14 to 28 days.

14. A method for purifying ventricular cardiomyocytes, wherein, between steps i) and ii), a step is additionally included to separate cells expressing CD71 positively in cardiomyocytes at the cell level and purify only contractile cardiomyocytes.

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