Method for purifying and producing ventricular cardiomyocytes

Abstract

The present invention relates to a method for purifying and producing ventricular cardiomyocytes, and according to the present invention, high concentrations of ventricular cardiomyocytes can be obtained by physical methods using surface markers CD81, IL1RL1 and CD59 as markers.

Description

[Technical Field]

[0001] The present invention relates to a method for purifying and producing ventricular cardiomyocytes, and according to the present invention, high concentrations of ventricular cardiomyocytes can be obtained by physical methods using surface markers CD81, IL1RL1 and CD59 as markers. [Background technology]

[0002] Myocardial infarction is a disease in which myocardial cells die due to a decrease in the supply of oxygen and nutrients to all or part of the heart, and heart failure is a disease caused by an insufficient supply of blood to the body due to a decrease in the heart's function.Such myocardial infarction and heart failure can be caused by the loss of myocardial cells (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 diseases only slow the progression of the disease.

[0004] On the other hand, heart transplantation can be a fundamental therapeutic therapy for restoring cardiac function, but it can lead to problems such as a shortage of organ donors, medical ethics, and physical and economic burdens on patients.

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

[0006] Among these, methods for isolating cardiomyocytes have sought to identify markers specifically expressed by cardiomyocytes by identifying previously known cell surface markers, and representative examples have been proposed, such as CD177z, CD42, CD236a, SIRPA, and CD71. However, the surface markers identified to date are only used as markers for purifying cardiomyocytes and cannot distinguish the types and functions of atrial and ventricular cells.

[0007] Ventricular cardiomyocytes may be directly involved in cardiac contraction in myocardial infarction. Therefore, the isolation of pure ventricular cardiomyocytes may be crucial for cell regenerative therapy to restore cardiac function. Furthermore, there is a risk of arrhythmias when differentiated cardiomyocytes with different cardiomyocyte subtypes are transplanted into disease models. Therefore, the development of a technology for purifying pure ventricular cardiomyocytes is highly desirable. Summary of the Invention [Problem to be solved by the invention]

[0008] Therefore, the inventors confirmed that when CD81-, IL1RL1-, and CD59-positive cells are separated by a physical method to separate and purify ventricular cardiomyocytes at a high concentration from a cell group containing cardiomyocytes, it is possible to effectively separate and purify only single-cell ventricular cardiomyocytes, and have developed a method for purifying and producing ventricular cardiomyocytes.

[0009] An object 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. [Means for solving the problem]

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

[0012] The present invention will now be described in more detail.

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

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

[0015] Meanwhile, in cell regeneration therapy for myocardial infarction, it may be more important to isolate pure ventricular cardiomyocytes from differentiated cardiomyocytes, because the purity of ventricular cardiomyocytes can affect the efficacy of cell regeneration therapy. For example, when different subtypes of cardiomyocytes are used in cell regeneration therapy, or when immature cardiomyocytes or general muscle cells are mixed, the prognosis may be worse than when pure ventricular cardiomyocytes are used. Therefore, a method for purifying only ventricular cardiomyocytes from cardiomyocytes differentiated from stem cells is important in 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 cells may be human pluripotent stem cells (hPSCs), such as induced pluripotent stem cells (iPSCs) or embryonic stem cells (ESCs), but are not limited thereto.

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

[0019] In the present invention, the markers in steps i) and ii) may further include CD151, which can further increase the efficiency of ventricular cardiomyocyte purification.

[0020] In the present invention, one or more markers selected from the group consisting of IL1RL1, CD81, CD59 and CD151 are used as markers for purifying ventricular cardiomyocytes in a cardiomyocyte-containing cell population.

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

[0022] In the present invention, the method for purifying ventricular cardiomyocytes may include: i) confirming the expression of one or more markers selected from the group consisting of IL1RL1, CD81, and CD59, and CD151 in cardiomyocytes; and ii) isolating at the cellular level the cells confirmed in step i) that are positive for the expression of one or more markers selected from the group consisting of IL1RL1, CD81, and CD59, and CD151.

[0023] In the present invention, the method for purifying ventricular cardiomyocytes may further include, before performing step i), a step of separating cardiomyocytes that are positive for CD71 expression at the cellular level to purify only contractile cardiomyocytes.

[0024] In the present invention, in the case of contractile cardiomyocytes, changes in contractile ability level due to drug response can be analyzed, so it is preferable to isolate and use only contractile cardiomyocytes in research processes using cardiomyocytes, such as cardiotoxicity drug evaluation of new drug candidate substances.As such, the process of separating and isolating contractile cardiomyocytes from non-contractile cardiomyocytes among differentiated cardiomyocytes can also play an important role, as contractile cardiomyocytes can also be used as a cellular therapeutic agent for heart failure.

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

[0026] Cardiomyocytes that have not been subjected to the method for purifying ventricular cardiomyocytes according to the present invention may have a purity of ventricular cardiomyocytes 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] The cardiomyocytes that have undergone the method for purifying ventricular cardiomyocytes according to the present invention may have a purity of ventricular cardiomyocytes 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] Yet another aspect of the present invention relates to a method for producing ventricular cardiomyocytes, comprising: i) inducing differentiation of stem cells into cardiomyocytes; ii) confirming the expression of one or more markers selected from the group consisting of IL1RL1, CD81, and CD59 in the cardiomyocytes; and iii) isolating at the cellular level cells that are positive for the expression of one or more markers selected from the group consisting of IL1RL1, CD81, and CD59 confirmed in step ii).

[0029] In the present invention, stem cells may be human pluripotent stem cells (hPSCs), such as induced pluripotent stem cells (iPSCs) or embryonic stem cells (ESCs), but are not limited thereto.

[0030] In the present invention, the markers in steps ii) and iii) may further include CD151, which allows for the production of ventricular cardiomyocytes with higher purity.

[0031] In the present invention, one or more markers selected from the group consisting of IL1RL1, CD81, CD59 and CD151 are used as markers for purifying ventricular cardiomyocytes in a cardiomyocyte-containing cell population.

[0032] In the present invention, cells that do not express the marker, i.e., cells that are negative for the marker, may be undifferentiated stem cells or embryonic 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 one or more markers selected from the group consisting of IL1RL1, CD81, and CD59, and CD151 in the cardiomyocytes; and iii) a step of isolating at the cellular level the cells confirmed in step ii) that are positive for the expression of one or more markers selected from the group consisting of IL1RL1, CD81, and CD59, and CD151.

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

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

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

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

[0038] In the present invention, the culture medium for inducing cardiomyocyte maturation may contain one or more members 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 cardiomyocyte maturation-inducing culture medium may be an RPMI1640 culture medium supplemented with one or more members selected from the group consisting of B27 (B27 supplement), T3 (3,3',5-Triiodo-L-thyronine), and Dex (Dexamethasone).

[0040] In the present invention, the cardiomyocyte maturation-inducing culture medium 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 cardiomyocyte maturation-inducing culture medium 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 cardiomyocyte maturation-inducing culture medium 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 allowing for the production of highly pure ventricular cardiomyocytes.

[0044] In the present invention, step ii) may be performed after primary purification of the cardiomyocytes induced to differentiate in step i) using a metabolism control culture medium, followed by culturing in a cardiomyocyte maturation induction culture medium 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.

[0045] In the present invention, the method for producing ventricular cardiomyocytes may further include, between steps i) and ii), a step of separating cardiomyocytes that are positive for CD71 expression at the cellular level to purify only contractile cardiomyocytes.

[0046] In the present invention, in the case of contractile cardiomyocytes, changes in contractile ability level due to drug response can be analyzed, so it is preferable to isolate and use only contractile cardiomyocytes in research processes using cardiomyocytes, such as cardiotoxicity drug evaluation of new drug candidate substances.As such, contractile cardiomyocytes can also be used as a cellular therapeutic agent for heart failure, so the process of separating and isolating contractile cardiomyocytes from non-contractile cardiomyocytes among differentiated cardiomyocytes can also play an important role.

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

[0048] Highly pure ventricular cardiomyocytes can be obtained by the method for producing ventricular cardiomyocytes according to the present invention. Specifically, the purity of the ventricular cardiomyocytes may 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%. [Effects of the Invention]

[0049] The present invention relates to a method for purifying and producing ventricular cardiomyocytes, and according to the present invention, high concentrations of ventricular cardiomyocytes can be obtained by physical methods using surface markers CD81, IL1RL1 and CD59 as markers. [Brief explanation of the drawings]

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

[0051] [Figure 2] 10 is a graph showing the results of analyzing the action voltage of cardiomyocytes before secondary purification using a marker for ventricular cardiomyocyte purification according to one embodiment of the present invention.

[0052] [Figure 3] 1 is a graph showing the results of RNA-seq analysis performed to obtain a marker list for ventricular cardiomyocyte purification according to one embodiment of the present invention.

[0053] [Figure 4] 10 shows a method for measuring cell images and action voltages after secondary purification using a marker for ventricular cardiomyocyte purification according to one embodiment of the present invention.

[0054] [Figure 5] 10 is a graph showing the results of analyzing the action voltage of cardiomyocytes after secondary purification using a marker for ventricular cardiomyocyte purification according to one embodiment of the present invention.

[0055] [Figure 6] 1 is a graph showing gene expression profiles before and after purification of ventricular cardiomyocytes according to one example of the present invention.

[0056] [Figure 7]This figure shows the results of confirming that CD71 staining can specifically label only contracting cardiomyocytes in differentiated cardiomyocytes (Figure 7a), and the results of flow cytometry analysis of control groups of SIRPA-labeled cells and CD71-labeled cells (Figure 7b). DETAILED DESCRIPTION OF THE INVENTION

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

[0058] The present invention will be described in more detail below with reference to the following examples, but these examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

[0059] Example 1. Selection and efficacy of ventricular cardiomyocyte purification markers

[0060] 1-1. Human induced pluripotent stem cell (hiPSC) generation

[0061] Somatic cells from healthy donors who have completed the donor consent form are used with vector- and virus-free reprogramming kits (stemgent, StemRNA TM Human induced pluripotent stem cell (hiPSC) cell lines were generated using a 3rd Gen Reprogramming Kit according to the manufacturer's protocol.

[0062] 1-2. Induction of differentiation of human induced pluripotent stem cells (hiPSCs) into cardiomyocytes

[0063] Cardiomyocyte differentiation was induced from human induced pluripotent stem cells (hiPSCs) using the following method, according to the schedule in Figure 1. Human induced pluripotent stem cell (hiPSC) cell lines were cultured on Matrigel in iPS-BREW XF medium (StemMACS™, Mitenyi Biotec) for 4 days in an undifferentiated state. For initial differentiation, the cultured stem cells were sequentially treated with the small molecule compounds CHIR99021 and Wnt-C59. The cells were then 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.

[0064] Eight days after differentiation induction, contractile cardiomyocytes were observed and were first purified for three days using a metabolic control medium containing glucose-depleted RPMI 1640 medium, human serum albumin (Sigma, 2%), and lactate (Sigma, 1 mM / ml).

[0065] The cells were then cultured for 14 days in a cardiomyocyte maturation induction medium (RPMI1640 + B27 (B27 supplement) (Gibco, 1%) + T3 (3,3',5-Triiodo-L-thyronine) (Sigma, 10 ng / ml) + dexamethasone (Stemcell Technology, 5 nM).

[0066] Cultured cardiomyocytes were dissociated into single cells using Triple LE-select (Gibco), and then the voltage and current of all three types of cardiomyocytes (ventricular, atrial, and nodal) were recorded using electrophysiological analysis (patch clamp), and the results are shown in Figure 2. As shown in Figure 2, the action voltage of cardiomyocytes after primary purification alone was found to be a mixed distribution of 4% nodal cardiomyocytes, 46% atrial cardiomyocytes, and 50% ventricular cardiomyocytes.

[0067] 1-3. Selection of markers for ventricular cardiomyocyte purification using RNA-based gene analysis (single cell array)

[0068] The differentiated cardiomyocytes were first purified and cultured in maturation-inducing medium for 10, 55, and 100 days, and then manually isolated in groups of 100 single cells. Each cell was then lysed to obtain cell extracts, which were then subjected to global transcriptome assay. From the results of the global transcriptome assay, hierarchical clustering analysis of the global gene expression profiles was performed using average linkage, and the results are shown in Figure 3a.

[0069] As a result, as can be seen from FIG. 3a, it was confirmed that all of the cells analyzed based on changes in cardiomyocyte-specific genes in cardiomyocytes cultured for 10, 55, and 100 days were cardiomyocytes.

[0070] Among the genes analyzed, cardiomyocyte genes and ventricular cardiomyocyte genes were first separated, and then genes that showed differences in expression levels between 55 and 100 days of culture were analyzed, focusing on genes encoding cell surface markers (CDs).The results are shown in Figure 3b.From the results in Figure 3b, CD59, IL1RL1, CD81, and CD151, which are surface markers specific to ventricular cardiomyocytes, were selected.

[0071] 1-4. Confirmation of efficacy of purified ventricular cardiomyocyte markers

[0072] First, among the genes that showed differential expression levels in differentiation-induced cardiomyocytes based on the results analyzed in the previous example, genes encoding cell surface tagging factors (CD) were analyzed. To efficiently purify ventricular cardiomyocytes, pure cardiomyocytes were first purified using a metabolism control medium.

[0073] We investigated whether CD59, IL1RL1, CD81, and CD151 could be used as markers to specifically select ventricular cardiomyocytes from cardiomyocytes differentiated from hPSCs. After culturing in maturation-inducing medium for 14 days, the cells were stained for CD59, IL1RL1, CD81, and CD151, and then purified using a flow cytometer.

[0074] Purified cardiomyocytes isolated using CD59, IL1RL1, CD81, and CD151 markers were cultured in maturation-inducing medium for 24 hours. After confirming that the purified cells exhibited contraction (Figure 4a), action voltage was recorded using the patch clamp technique (Figure 4b), and the results are shown in Figure 5.

[0075] As a result, as shown in Figure 5, it was confirmed that the action voltage of cardiomyocytes isolated and purified using the marker CD59 was 87% ventricular cardiomyocytes, the action voltage of cardiomyocytes isolated and purified using the marker IL1RL1 was 80% ventricular cardiomyocytes, the action voltage of cardiomyocytes isolated and purified using the marker CD81 was 89% ventricular cardiomyocytes, and the action voltage of cardiomyocytes isolated and purified using the marker CD151 was 92% ventricular cardiomyocytes.

[0076] Furthermore, genetic analysis was performed on cardiomyocytes after only primary purification and cardiomyocytes after secondary purification using the CD59 marker, and the results are shown in Figure 6. As shown in Figure 6, in cardiomyocytes after secondary purification using the CD59 marker (after sorting), compared to cardiomyocytes after only primary purification (before sorting), the expression of the cardiomyocyte-specific gene TNNT2 and the cardiomyocyte-related ion channel genes SCN5A and CACN1C was increased, the expression of the ventricular cardiomyocyte-specific genes MYL2 and MLC2V was increased, and the expression of the atrial cardiomyocyte MLC2a gene was decreased.

[0077] This confirmed that ventricular cardiomyocytes can be specifically purified and separated from cardiomyocytes using the markers CD59, IL1RL1, CD81, and CD151 selected in the present invention.

[0078] Example 2. Confirmation of the efficacy of CD71, a marker for the purification of contracting cardiomyocytes

[0079] The efficacy of CD71 as a marker for specifically selecting only contracting cells among cardiomyocytes differentiated from hPSCs was confirmed (Patent Document KR10-1916902 B1). Cardiomyocytes differentiated from hPSCs were stained with anti-CD71 antibody, and then the cells were stained with the anti-CD71 antibody and observed under a microscope. To confirm whether CD71 can be used as a significant surface marker, cardiomyocytes were labeled with SIRPA or CD71, both of which are known to be useful as cardiomyocyte differentiation markers, and the degree of fluorescence was analyzed using a flow cytometer (FACS).

[0080] As a result, as shown in Figure 7, we confirmed that CD71 can be used as a specific surface marker for contracting cardiomyocytes (Figure 7a), and flow cytometry analysis confirmed that it showed a pattern similar to that of the SIRPA surface marker, a cardiomyocyte surface factor (Figure 7b).

[0081] In the case of SIRPA, it is known to be a specific factor for cardiomyocytes, regardless of whether they are contracting or non-contracting, but we confirmed that the marker CD71 can be used to specifically select only contracting cardiomyocytes. [Industrial Applicability]

[0082] The present invention relates to a method for purifying and producing ventricular cardiomyocytes, and according to the present invention, high concentrations of ventricular cardiomyocytes can be obtained by physical methods using surface markers CD81, IL1RL1 and CD59 as markers.

Claims

1. i) determining the expression of one or more markers selected from the group consisting of IL1RL1, CD81, and CD59 in cardiomyocytes; ii) isolating at the cellular level cells that are positive for the expression of one or more markers selected from the group consisting of IL1RL1, CD81, and CD59 confirmed in step i).

2. The method for purifying ventricular cardiomyocytes according to claim 1 , wherein the cardiomyocytes are cardiomyocytes induced from stem cells.

3. The method for purifying ventricular cardiomyocytes according to claim 2, wherein the stem cells are human pluripotent stem cells (hPSCs).

4. The method for purifying ventricular cardiomyocytes according to claim 1, wherein the markers in steps i) and ii) further include CD151.

5. The method for purifying ventricular cardiomyocytes according to claim 1, further comprising the step of separating cells that are positive for CD71 expression in cardiomyocytes at the cellular level to purify only contractile cardiomyocytes before performing step i).

6. i) inducing differentiation of stem cells into cardiomyocytes; ii) determining the expression of one or more markers selected from the group consisting of IL1RL1, CD81, and CD59 in the cardiomyocytes; and iii) isolating at the cellular level the cells confirmed in step ii) that are positive for the expression of one or more markers selected from the group consisting of IL1RL1, CD81, and CD59.

7. The method for producing ventricular cardiomyocytes according to claim 6, wherein the stem cells are human pluripotent stem cells (hPSCs).

8. The method for producing ventricular cardiomyocytes according to claim 6, wherein the markers in steps ii) and iii) further include CD151.

9. The method for producing ventricular cardiomyocytes according to claim 6, further comprising a step of primary purification of cardiomyocytes using a metabolism control medium between steps i) and ii).

10. 10. The method for producing ventricular cardiomyocytes according to claim 9, wherein the metabolism control culture medium contains human serum albumin and lactate.

11. The method for producing ventricular cardiomyocytes according to claim 6 , further comprising a step of maturing the cardiomyocytes using a cardiomyocyte maturation-inducing culture medium between steps i) and ii).

12. The method for producing ventricular cardiomyocytes according to claim 11, wherein the cardiomyocyte maturation-inducing culture medium contains one or more compounds selected from the group consisting of B27 (B27 supplement), T3 (3,3',5-Triiodo-L-thyronine), and Dex (Dexamethasone).

13. The method for producing ventricular cardiomyocytes according to claim 6, wherein step ii) is performed after culturing the cardiomyocytes induced to differentiate in step i) for 14 to 28 days.

14. The method for producing ventricular cardiomyocytes according to claim 6, further comprising a step between steps i) and ii) of separating cardiomyocytes that are positive for CD71 expression at the cellular level to purify only contractile cardiomyocytes.

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