Purification and preparation method of ventricular myocardial cells

Abstract

The present invention relates to a ventricular myocardial cell purification and preparation method, according to the present invention, surface marker factors of CD81, IL1RL1 and CD59 are used as markers, and high concentration ventricular myocardial cells can be obtained by a physical method.

Description

[Technical field]

[0001] The present invention relates to a method for purifying and preparing ventricular cardiomyocytes. According to the present invention, surface marker factors of CD81, IL1RL1 and CD59 are used as markers, and high-concentration ventricular cardiomyocytes can be obtained by physical methods. [Background technology]

[0002] Myocardial infarction is a disease in which the myocardial cells die due to a decrease in oxygen and nutrients to the whole or part of the heart, and heart failure is a disease in which the heart's function declines and cannot supply blood to the body normally. Myocardial infarction and heart failure can be induced by damage to myocardial cells (heart muscle cells).

[0003] Myocardial infarction and heart failure are on the rise worldwide, but because a damaged or defective heart has difficulty recovering on its own, chemical treatments for these diseases can only delay the progression of the disease.

[0004] On the other hand, heart transplantation can be a fundamental treatment method to restore heart function, but it may cause problems such as insufficient organ donors, medical ethics, and patient physical and economic burdens.

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

[0006] Among them, in the method of isolating cardiomyocytes, markers specifically expressed in cardiomyocytes are explored by identifying cell surface markers discovered so far, 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 cells that are directly related to the occurrence of cardiac contraction in myocardial infarction. That is, in cell regeneration therapy for restoring cardiac function, it may be more important to simply separate ventricular cardiomyocytes. In addition, when differentiated cardiomyocytes with different subtypes of cardiomyocytes are transplanted into disease models, there is a problem that arrhythmia may be induced. Therefore, it is urgent to develop a technology that purifies only simple ventricular cardiomyocytes. [Summary of the invention]

[0008] [Technical Problems to be Solved by the Invention]

[0009] Therefore, in order to isolate and purify ventricular cardiomyocytes at high concentration in a cell population containing cardiomyocytes, the inventors confirmed that single-cell units of ventricular cardiomyocytes could be effectively isolated and purified when only CD81, IL1RL1 and CD59 positive cells were separated by physical methods, thereby developing a method for purifying and preparing ventricular cardiomyocytes.

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

[0011] Another object of the present invention is to provide a method for preparing ventricular cardiomyocytes.

[0012]

Technical solution

[0013] The present invention relates to a method for purifying and preparing ventricular cardiomyocytes. According to the present invention, surface marker factors of CD81, IL1RL1 and CD59 are used as markers, and high-concentration ventricular cardiomyocytes can be obtained by a physical method.

[0014] Hereinafter, the present invention will be described in more detail.

[0015] One aspect of the present invention relates to a method for purifying ventricular cardiomyocytes, comprising: step i), confirming the expression of one or more markers selected from the group consisting of IL1RL1, CD81 and CD59 in cardiomyocytes; and step ii), separating at the cellular level the 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).

[0016] The term "cardiomyocytes" in this specification refers to muscle cells that constitute the heart wall. Cardiomyocytes can be divided into nodular cardiomyocytes, atrial cardiomyocytes and ventricular cardiomyocytes according to the structure of the heart. On the other hand, cardiomyocytes can be damaged or apoptotic when subjected to stress such as myocardial infarction or myocarditis. Damage or apoptosis of cardiomyocytes can induce decreased myocardial function, thereby inducing heart disease. Therefore, cardiomyocytes differentiated from pluripotent stem cells can be used in regenerative cell therapy for cardiac function recovery or heart disease treatment.

[0017] On the other hand, in cell regeneration therapy for myocardial infarction, it may be more important to simply isolate ventricular cardiomyocytes from differentiated cardiomyocytes. This is because the purity of ventricular cardiomyocytes can affect the effect of cell regeneration therapy. For example, when different subtypes of cardiomyocytes are used together during cell regeneration therapy, or when immature cardiomyocytes or general muscle cells are mixed, the prognosis may be worse than when using simple ventricular cardiomyocytes. Therefore, a method for purifying only ventricular cardiomyocytes from cardiomyocytes differentiated from stem cells is important in regenerative cell therapy.

[0018] In the present invention, the ventricular cardiomyocytes may be derived from stem cells, but are not limited thereto.

[0019] In the present invention, the stem cells may be ventricular cardiomyocytes derived from human pluripotent stem cells (hPSCs), for example, ventricular cardiomyocytes derived from reverse pluripotent stem cells (induced pluripotent stem cells (iPSCs)) or embryonic stem cells (ESCs), but are not limited thereto.

[0020] The term "marker" in the present specification refers to a marker protein or a marker gene, and refers to a protein or a gene thereof that is specifically expressed on the cell surface, cytoplasm and / or nucleus of a given cell, for example, on the cell surface.

[0021] In the present invention, the markers in step i) and step ii) may further include CD151, thereby further improving the purification efficiency of ventricular cardiomyocytes.

[0022] 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 from a cell population containing cardiomyocytes.

[0023] In the present invention, the cells that do not express the marker, that is, the cells that are negative for the marker, may be undifferentiated stem cells or embryoid bodies, or may be nodal cardiomyocytes or atrial cardiomyocytes.

[0024] In the present invention, the method for purifying ventricular cardiomyocytes may include: step i), confirming the expression of one or more markers selected from the group consisting of IL1RL1, CD81 and CD59 and CD151 in cardiomyocytes; and step ii), separating at the cellular level the cells that are confirmed in step i) to be positive for the expression of one or more markers selected from the group consisting of IL1RL1, CD81 and CD59 and CD151.

[0025] In the present invention, the method for purifying ventricular cardiomyocytes may further include the following steps before performing step i): separating cells positive for CD71 expression from cardiomyocytes at the cellular level, and purifying only the beating cardiomyocytes.

[0026] In the present invention, with regard to autonomous cardiomyocytes, since changes in the level of autonomous ability according to drug response can be analyzed, in the process of research using cardiomyocytes (such as cardiotoxic drug evaluation of new drug candidates), it is preferred to isolate and use only autonomous cardiomyocytes. Therefore, this can also be used as a cell therapy for heart failure using autonomous cardiomyocytes. Therefore, the process of distinguishing and separating autonomous cardiomyocytes and non-autonomous cardiomyocytes in differentiated cardiomyocytes may also be important.

[0027] In the present invention, CD71 can be used as a surface marker factor specifically expressed in autonomous cardiomyocytes. Therefore, when cardiomyocytes are stained with the CD71 marker, cells that do not express CD71, that is, CD71-negative cells, can be non-autonomous cardiomyocytes.

[0028] In cardiomyocytes that have not been purified by the ventricular cardiomyocyte purification method of the present invention, the purity of the ventricular cardiomyocytes can be 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%.

[0029] In the cardiomyocytes purified by the method for purifying ventricular cardiomyocytes of the present invention, 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%.

[0030] Another aspect of the present invention relates to a method for preparing ventricular cardiomyocytes, comprising: step i), inducing stem cells to differentiate into cardiomyocytes; step ii), confirming the expression of one or more markers selected from the group consisting of IL1RL1, CD81 and CD59 in the cardiomyocytes; and step iii), performing cell-level separation on cells that are confirmed in step ii) to be positive for the expression of one or more markers selected from the group consisting of IL1RL1, CD81 and CD59.

[0031] In the present invention, the stem cell may be a human pluripotent stem cell (hPSC), for example, an inversely induced pluripotent stem cell (iPSC) or an embryonic stem cell (ESC), but is not limited thereto.

[0032] In the present invention, the markers in step ii) and step iii) may further include CD151, thereby preparing ventricular cardiomyocytes with higher purity.

[0033] 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 from a cell population containing cardiomyocytes.

[0034] In the present invention, the cells that do not express the marker, that is, the cells that are negative for the marker, may be undifferentiated stem cells or embryoid bodies, or may be nodal cardiomyocytes or atrial cardiomyocytes.

[0035] In the present invention, the method for preparing ventricular cardiomyocytes may include: step i), inducing stem cells to differentiate into cardiomyocytes; step ii), confirming the expression of one or more selected from the group consisting of IL1RL1, CD81 and CD59 and CD151 markers in the cardiomyocytes; and step iii), performing cell-level separation on the cells that are confirmed in step ii) to be positive for the expression of one or more selected from the group consisting of IL1RL1, CD81 and CD59 and CD151 markers.

[0036] In the present invention, the method for preparing ventricular cardiomyocytes may include a step of using a metabolism control culture medium to purify the cardiomyocytes for the first time between step i) and step ii), thereby further improving the purification efficiency of the cardiomyocytes by removing cells other than the cardiomyocytes.

[0037] In the present invention, the metabolic control culture medium may contain human serum albumin and lactate.

[0038] In the present invention, the metabolic control culture medium can be a culture medium that contains human serum albumin and lactate in a culture medium that has been deprived of glucose, and by creating an environment in which cells other than cardiomyocytes cannot carry out cell activities, the purification efficiency of cardiomyocytes can be further improved.

[0039] In the present invention, the method for preparing ventricular cardiomyocytes may further include, between step i) and step ii), a step of maturing the cardiomyocytes using a cardiomyocyte maturation induction culture medium, thereby shortening the maturation period of the cardiomyocytes.

[0040] In the present invention, the cardiomyocyte maturation induction culture medium may contain one or more selected from the group consisting of B27 (B27 supplement), T3 (3,3',5-triiodo-L-thyronine) and Dex (dexamethasone).

[0041] In the present invention, the cardiomyocyte maturation induction culture medium may be prepared by adding one or more selected from the group consisting of B27 (B27 supplement), T3 (3,3',5-triiodo-L-thyronine) and Dex (dexamethasone) to RPMI1640 culture medium.

[0042] In the present invention, the cardiomyocyte maturation induction culture medium can contain B27 at a concentration of 0.1 wt % to 5 wt %, 0.1 wt % to 4 wt %, 0.1 wt % to 3 wt %, 0.1 wt % to 2 wt %, 0.5 wt % to 5 wt %, 0.5 wt % to 4 wt %, 0.5 wt % to 3 wt %, 0.5 wt % to 2 wt %, 1 wt % to 5 wt %, 1 wt % to 4 wt %, 1 wt % to 3 wt % or 1 wt % to 2 wt %.

[0043] In the present invention, the cardiomyocyte maturation induction culture medium may contain T3 at a concentration of 5ng / ml to 50ng / ml, 5ng / ml to 40ng / ml, 5ng / ml to 30ng / ml, 5ng / ml to 20ng / ml, 10ng / ml to 50ng / ml, 10ng / ml to 40ng / ml, 10ng / ml to 30ng / ml or 10ng / ml to 20ng / ml.

[0044] In the present invention, the cardiomyocyte maturation induction culture medium may contain Dex at a concentration of 1nM to 10nM, 1nM to 9nM, 1nM to 8nM, 1nM to 7nM, 1nM to 6nM, 1nM to 5nM, 3nM to 10nM, 3nM to 9nM, 3nM to 8nM, 3nM to 7nM, 3nM to 6nM or 3nM to 5nM.

[0045] In the present invention, step ii) can be performed after culturing the differentiated cardiomyocytes induced 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, it can be performed after culturing for 14 days, thereby obtaining high-purity ventricular cardiomyocytes.

[0046] In the present invention, step ii) can be performed after the cardiomyocytes induced for differentiation in step i) are first purified using a metabolic control culture medium and then cultured with 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, it can be performed after 14 days of culture, thereby obtaining highly pure ventricular cardiomyocytes.

[0047] In the present invention, the method for preparing ventricular cardiomyocytes may further include the following step between step i) and step ii): performing cell-level separation on cells that are positive for CD71 expression among cardiomyocytes, and purifying only autonomous cardiomyocytes.

[0048] In the present invention, with regard to autonomous cardiomyocytes, since changes in the level of autonomous ability according to drug response can be analyzed, in the process of research using cardiomyocytes (such as cardiotoxic drug evaluation of new drug candidates), it is preferred to isolate and use only autonomous cardiomyocytes. Therefore, this can also be used as a cell therapy for heart failure using autonomous cardiomyocytes, so the process of distinguishing and separating autonomous cardiomyocytes and non-autonomous cardiomyocytes in differentiated cardiomyocytes may also be important.

[0049] In the present invention, CD71 can be used as a surface marker factor specifically expressed in autonomous cardiomyocytes. Therefore, when cardiomyocytes are stained with the CD71 marker, cells that do not express CD71, that is, CD71-negative cells, can be non-autonomous cardiomyocytes.

[0050] By the method for preparing ventricular cardiomyocytes of the present invention, high-purity ventricular cardiomyocytes can be obtained. 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%.

[0051]

Beneficial Effects

[0052] The present invention relates to a method for purifying and preparing ventricular cardiomyocytes. According to the present invention, surface marker factors of CD81, IL1RL1 and CD59 are used as markers, and high-concentration ventricular cardiomyocytes can be obtained by physical methods.

Brief Description of the Drawings

[0053] Figure 1 Schematic diagram showing a method for differentiating cardiomyocytes and a method for purifying ventricular cardiomyocytes according to an embodiment of the present invention.

[0054] Figure 2 This is a graph showing the results of analyzing the action potential of cardiomyocytes before the second purification using the marker for purifying ventricular cardiomyocytes according to one embodiment of the present invention.

[0055] Figure 3 This is a graph showing the results of RNA-seq analysis performed to obtain a marker for purifying ventricular cardiomyocytes according to one embodiment of the present invention.

[0056] Figure 4 A method for measuring cell images and action potentials after secondary purification using a marker for purifying ventricular cardiomyocytes according to an embodiment of the present invention is shown.

[0057] Figure 5 This is a graph showing the results of analyzing the action potential of cardiomyocytes after the second purification using the marker for purifying ventricular cardiomyocytes according to one embodiment of the present invention.

[0058] Figure 6 This is a graph confirming the gene expression pattern before and after purification of ventricular cardiomyocytes according to one embodiment of the present invention.

[0059] Figure 7The results show that among differentiated cardiomyocytes, only autonomous cardiomyocytes can be specifically labeled by CD71 staining ( Figure 7 Part A) and flow cytometry analysis of SIRPA-labeled cells and CD71-labeled cells in the control group ( Figure 7 Part B of the [Specific implementation method]

[0060] The present invention relates to a method for purifying ventricular cardiomyocytes, comprising: step i), confirming the expression of one or more markers selected from the group consisting of IL1RL1, CD81 and CD59 in cardiomyocytes; and step ii), separating cells at the cellular level 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). [Specific embodiment]

[0062] Hereinafter, the present invention will be described in more detail by the following examples. However, these examples are only used to exemplify the present invention, and the scope of the present invention is not limited to these examples.

[0063] [Example 1. Selection of ventricular cardiomyocyte purification markers and confirmation of efficacy]

[0064] [1-1. Preparation of human induced pluripotent stem cells (hiPSC)]

[0065] Normal human somatic cells with consent from donors were used and reprogrammed using a vector-free and virus-free reprogramming kit (Stemgent, StemRNA TM The third generation reprogramming kit (3rd Gen Reprogramming Kit) was used to prepare human induced pluripotent stem cells (hiPSC) cell lines according to the manufacturer's protocol.

[0066] [1-2. Inducing human induced pluripotent stem cells (hiPSCs) to differentiate into cardiomyocytes]

[0067] according to Figure 1In the schedule, human induced pluripotent stem cells were induced to differentiate into cardiomyocytes by the following method. Human induced pluripotent stem cells (hiPSC) cell lines were cultured on Matrigel in iPS-BREW XF medium (StemMACS TM , Mitenyi Biotec) in an undifferentiated state for 4 days. The stem cells cultured for initial differentiation were treated with low molecular weight compounds CHIR 99021 and Wnt-C59 in sequence, and the cells were differentiated and cultured for 4 days using 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.

[0068] After 8 days of differentiation induction, contractile cardiomyocytes were observed and the first purification was performed for 3 days in RPMI1640 medium without glucose using a metabolic control medium containing human serum albumin (Huamn Serum Albumn) (Sigma, 2%) and lactate (Sigma, 1 mM / ml).

[0069] Next, the culture medium was replaced with 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) and cultured for 14 days.

[0070] After the cultured cardiomyocytes were separated into single cells using TyprLE-select (Gibco), the voltage and current of all three types of cardiomyocytes (ventricle, atrium, and node, etc.) were recorded by electrophysiological analysis (patch clamp), and the results are shown in Figure 2 In. Figure 2As shown, it was confirmed that the action potential of the cardiomyocytes after the first purification alone was mixed in a distribution of 4% in nodal cardiomyocytes, 46% in atrial cardiomyocytes, and 50% in ventricular cardiomyocytes.

[0071] [1-3. Selection of markers for purification of ventricular cardiomyocytes by RNA unit gene analysis (single cell array)]

[0072] The differentiation-induced cardiomyocytes were purified for the first time and cultured with maturation-inducing culture medium for 10 days, 55 days, and 100 days, and were manually separated into 100 single-cell units. Next, in order to lyse each cell to obtain a cell extract, a global transcriptome assay was performed. Based on the global transcriptome analysis results, the hierarchical clustering analysis results of the overall gene expression profile were derived using average linkage, and the results are shown in Figure 3 in Part A of the .

[0073] Results, such as Figure 3 As shown in Part A of FIG. 1 , it was confirmed that all cells analyzed by changes in cardiomyocyte-specific genes in cardiomyocytes cultured for 10 days, 55 days, and 100 days were cardiomyocytes.

[0074] Among the genes analyzed, the genes encoding cell surface marker factors (CD) whose expression levels were different between 55 and 100 days of culture were analyzed after the first separation of cardiomyocyte genes and ventricular cardiomyocyte genes. The results are shown in Figure 3 In Part B of Figure 3 According to the results of part B, specific surface markers CD59, IL1RL1, CD81 and CD151 were selected in ventricular cardiomyocytes.

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

[0076] First, based on the results of the analysis in the examples, genes encoding cell surface marker factors (CD) were compared and analyzed among genes showing differences in expression levels in differentiation-induced cardiomyocytes. In order to effectively purify ventricular cardiomyocytes, pure cardiomyocytes were first purified using a metabolic control culture medium.

[0077] We confirmed whether CD59, IL1RL1, CD81, and CD151 can be used as markers to specifically select only ventricular cardiomyocyte markers from cardiomyocytes differentiated from hPSCs. Therefore, after 14 days of culture in a maturation induction medium, CD59, IL1RL1, CD81, and CD151 were stained and then purified a second time using a flow cytometer.

[0078] Cardiomyocytes isolated and purified using CD59, IL1RL1, CD81, and CD151 markers were cultured in maturation induction medium for 24 hours. Figure 4 As shown in Part A of FIG. 1 , the cells purified with the markers are confirmed to be autonomous, and then Figure 4 As shown in Part B, action potentials were recorded by patch clamp technique and the results are shown in Figure 5 middle.

[0079] like Figure 5 As shown, the results indicated that: the action potential of the cardiomyocytes separated and purified using the marker CD59 represented 87% of the ventricular cardiomyocytes; the action potential of the cardiomyocytes separated and purified using the marker IL1RL1 represented 80% of the ventricular cardiomyocytes; the action potential of the cardiomyocytes separated and purified using the marker CD81 represented 89% of the ventricular cardiomyocytes; and the action potential of the cardiomyocytes separated and purified using the marker CD151 represented 92% of the ventricular cardiomyocytes.

[0080] In addition, additional gene analysis was performed on cardiomyocytes after only the first purification and cardiomyocytes after the second purification using the CD59 marker, and the results are shown in Figure 6 In. Figure 6 As shown, for cardiomyocytes after the second purification using the CD59 marker (after sorting), compared with cardiomyocytes after only the first purification (before sorting), it was confirmed that the expression of the cardiomyocyte-specific gene TNNT2 and the ion channel genes SCN5A and CACN1C involved in cardiomyocytes increased, the expression of the ventricular cardiomyocyte-specific genes MYL2 and MLC2V increased, and the expression of the atrial cardiomyocyte MLC2a gene decreased.

[0081] Thus, it was confirmed that ventricular cardiomyocytes among cardiomyocytes can be specifically purified and isolated using the markers CD59, IL1RL1, CD81, and CD151 selected in the present invention.

[0082] [Example 2. Confirmation of the efficacy of CD71, a marker for the purification of autonomous cardiomyocytes]

[0083] The efficacy of CD71, a marker for the specific selection of autonomic cells in cardiomyocytes differentiated from hPSC, was confirmed (patent document KR10-1916902B1). Cardiomyocytes differentiated from hPSC were treated with anti-CD71 antibodies and stained, and then the anti-CD71 antibodies were developed and observed under a microscope. In addition, in order to confirm whether CD71 can be used as a significant surface marker, cardiomyocytes were marked with SIRPA or CD71, which are known to be used as cardiomyocyte identification markers, and the degree of fluorescence color development was analyzed in a flow cytometer (FACS).

[0084] like Figure 7 As shown, the results showed that CD71 can be used as a specific surface marker for autonomous cardiomyocytes ( Figure 7 The results of flow cytometry analysis showed that CD71 exhibited a similar pattern to the surface marker of cardiomyocyte surface factor SIRPA ( Figure 7 Part B of the

[0085] SIRPA is known to be a factor specific to cardiomyocytes that does not distinguish whether they are autonomous or non-autonomous, and it was confirmed that only autonomous cardiomyocytes can be specifically selected by using the marker CD71.

[0086] [Industrial Applicability]

[0087] The present invention relates to a method for purifying and preparing ventricular cardiomyocytes. According to the present invention, surface marker factors of CD81, IL1RL1 and CD59 are used as markers, and high-concentration ventricular cardiomyocytes can be obtained by physical methods.

Claims

1. A method for purifying ventricular cardiomyocytes, wherein include: Step (i), confirming the expression of one or more markers selected from the group consisting of IL1RL1, CD81 and CD59 in cardiomyocytes; as well as Step (ii) isolating cells at the cellular level 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 derived from stem cells. The method for purifying ventricular cardiomyocytes according to claim 2 , wherein the stem cells are human pluripotent stem cells. 4 . The method for purifying ventricular cardiomyocytes according to claim 1 , wherein the markers in step (i) and step (ii) further comprise CD151.

5. The method for purifying ventricular cardiomyocytes according to claim 1, wherein before performing step (i), The following steps are involved: Among cardiomyocytes, cells positive for CD71 expression are isolated at the cellular level, thereby purifying only autonomous cardiomyocytes.

6. A method for preparing ventricular cardiomyocytes, wherein include: Step (i), inducing stem cells to differentiate into cardiomyocytes; Step (ii), confirming the expression of one or more markers selected from the group consisting of IL1RL1, CD81 and CD59 in cardiomyocytes; as well as Step (iii) isolating cells at the cellular level 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).

7. The method for preparing ventricular cardiomyocytes according to claim 6, wherein the stem cells are human pluripotent stem cells. 8 . The method for preparing ventricular cardiomyocytes according to claim 6 , wherein the markers in step (ii) and step (iii) further comprise CD151.

9. The method for preparing ventricular cardiomyocytes according to claim 6, wherein between step (i) and step (ii), The following steps are involved: Metabolic control culture medium was used for the first purification of cardiomyocytes. 10 . The method for preparing ventricular cardiomyocytes according to claim 9 , wherein the metabolic control culture medium comprises human serum albumin and lactic acid.

11. The method for preparing ventricular cardiomyocytes according to claim 6, wherein between step (i) and step (ii), The following steps are involved: Cardiomyocyte maturation induction medium was used to mature the cardiomyocytes. 12 . The method for preparing ventricular cardiomyocytes according to claim 11 , wherein the cardiomyocyte maturation induction culture medium comprises at least one selected from the group consisting of B27, T3 and Dex.

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

14. The method for preparing ventricular cardiomyocytes according to claim 6, wherein between step (i) and step (ii), The following steps are involved: Cells positive for CD71 expression among cardiomyocytes are fractionated at the cell level to purify only autonomous cardiomyocytes.

Images