Methods for enriching cardiomyocytes

Abstract

A method for enriching atrial myocytes or ventricular myocytes in a cell population containing cardiomyocytes induced by stem cells is provided. The method is a technique for enriching atrial myocytes or ventricular myocytes in a cell population containing cardiomyocytes induced by stem cells, comprising the step of recovering atrial myocytes or ventricular myocytes from the cell population using the expression level of CD151 as an indicator.

Description

Technical Field

[0001] The present invention relates to a method for enriching atrial myocytes or ventricular myocytes in a cell population containing cardiomyocytes induced from stem cells, etc. Background Art

[0002] Attempts have been made to induce cardiomyocytes from stem cells such as induced pluripotent stem cells (iPSCs) and embryonic stem cells (ESCs) and use them in regenerative medicine for heart disease.

[0003] Atrial myocytes and ventricular myocytes have different functions. In the cell population containing cardiomyocytes induced by stem cells, there are various cardiomyocytes including atrial myocytes and ventricular myocytes, and non-cardiomyocytes such as undifferentiated cells may also be contained. Therefore, when a cell population containing cardiomyocytes induced by stem cells is applied to regenerative medicine, it is expected that one of atrial myocytes or ventricular myocytes is enriched in the cell population according to the therapeutic purpose.

[0004] Non-Patent Document 1 describes a technique for isolating ventricular myocytes and atrial myocytes from cardiomyocytes isolated from tissues of mouse embryos and newborn mice using integrin 6 as a marker.

[0005] In addition, Non-Patent Document 2 describes a technique for separating cardiomyocytes induced from iPSCs into ventricular myocytes and atrial myocytes using CD235a and RALDH2 as markers.

[0006] Prior art literature

[0007] Non-patent literature

[0008] Non-patent document 1: "Differential Expression Levels of Integrin α6 Enable theSelective Identification and Isolation of Atrial and VentricularCardiomyocytes", PLoS One, 2015, 10(11):e0143538.

[0009] Non-patent document 2: "Human Pluripotent Stem Cell-Derived Atrial andVentricular Cardiomyocytes Develop from Distinct Mesoderm Populations", CellStem Cell, 2017, 21(2): 179-194. Summary of the invention

[0010] Problems to be solved by the invention

[0011] The main object of the present invention is to provide a technique for enriching atrial myocytes or ventricular myocytes in a cell population containing cardiomyocytes induced from stem cells.

[0012] Means for solving problems

[0013] In order to solve the above-mentioned problems, the present invention provides the following [1] to

[20] .

[0014] [1] A method for enriching atrial myocytes or ventricular myocytes in a cell population containing cardiomyocytes induced from stem cells, the method comprising the following steps:

[0015] Atrial myocytes or ventricular myocytes are collected from the above cell population using the expression level of CD151 as an indicator.

[0016] [2] A method for enriching atrial myocytes according to [1],

[0017] The method comprises the step of recovering CD151 low-expressing cells from the above cell population.

[0018] [3] A method, which is the method of [1] for enriching ventricular myocytes,

[0019] The method comprises the step of recovering CD151 high-expressing cells from the above cell population.

[0020] [4] The method according to any one of [1] to [3], wherein the stem cells are induced pluripotent stem cells.

[0021] [5] A cell population enriched in atrial myocytes or ventricular myocytes, obtained by the method of [1].

[0022] [6] A cell population enriched for atrial myocytes, obtained by the method of [2].

[0023] [7] A cell population enriched for ventricular myocytes, obtained by the method of [3].

[0024] [8] A medicine comprising the cell population described in any one of [5] to [7].

[0025] [9] A preparation method, which is a method for preparing atrial myocytes or ventricular myocytes from stem cells, the method comprising the following steps:

[0026] Step (A), inducing a cell population containing cardiomyocytes from the above stem cells; and

[0027] Step (B), using the expression level of CD151 as an indicator, recovering atrial myocytes or ventricular myocytes from the above cell population.

[0028]

[10] A preparation method, which is the method of [9] for preparing atrial myocytes, comprising the following steps:

[0029] Step (A), inducing a cell population containing cardiomyocytes from the above stem cells under atrial myocyte differentiation conditions; and

[0030] Step (B), recovering CD151 low-expressing cells from the above cell population.

[0031]

[11] A preparation method, which is the method of [9] for preparing ventricular myocytes, comprising the following steps:

[0032] Step (A), inducing a cell population containing cardiomyocytes from the above stem cells under conditions of ventricular myocyte differentiation; and

[0033] Step (B), recovering CD151 high-expressing cells from the above cell population.

[0034]

[12] The method according to any one of [9] to

[11] , wherein the stem cells are induced pluripotent stem cells.

[0035]

[13] Atrial myocytes or ventricular myocytes obtained by the preparation method of [9].

[0036]

[14] Atrial myocytes obtained by the preparation method of

[10] .

[0037]

[15] Ventricular myocytes obtained by the preparation method of

[11] .

[0038]

[16] A medicine comprising atrial myocytes or ventricular myocytes obtained by the production method according to any one of [9] to

[11] .

[0039]

[17] A reagent for enriching atrial myocytes or ventricular myocytes in a cell population containing cardiomyocytes induced from stem cells, the reagent comprising a CD151 detection probe.

[0040]

[18] The reagent of

[17] , wherein the CD151 detection probe is an anti-CD151 antibody.

[0041]

[19] Use of a CD151 detection probe for enriching atrial myocytes or ventricular myocytes in a cell population containing cardiomyocytes induced from stem cells.

[0042]

[20] Use of CD151 as a marker for enriching atrial myocytes or ventricular myocytes in a cell population containing cardiomyocytes induced from stem cells.

[0043] [definition]

[0044] In the present invention, "cell population" refers to a collection of two or more cells of different types. "Cell subpopulation" refers to a collection of the same or different cells constituting a cell population, which is a group of cells having at least one common property.

[0045] The so-called "enrichment" and "enrichment" refer to increasing the amount of a specific constituent in a composition such as a composition of cells. The so-called "enriched", when used to describe a composition of cells, such as a cell group, means that the amount of a specific constituent is increased compared to the proportion of the constituent in the cell group before enrichment. For example, a composition such as a cell group can be enriched according to the target cell type, so that the proportion of the target cell type is increased compared to the proportion of target cells present in the cell group before enrichment. The cell group can be enriched for the target cell type by a cell selection and screening method known in the art. The cell group can also be enriched by a specific selection or screening process described in this specification. In a specific embodiment of the present invention, the enriched cell population is enriched for the target cell population by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 97%, 98% or 99% relative to the cell population before enrichment. In a specific embodiment of the present invention, the enriched cell population contains at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% of the target cell population.

[0046] "CD151" is a 4-transmembrane protein belonging to the 4-transmembrane protein family. CD151 plays an important role in signal transduction related to cell differentiation, proliferation and movement. In addition, CD151 is known to form a complex with integrin and participate in functions such as cell adhesion and fusion.

[0047] In the present invention, CD151 is used as a marker for enriching atrial myocytes or ventricular myocytes in a cell population containing cardiomyocytes induced from stem cells.

[0048] "Marker" refers to "marker protein" or "marker gene", which is a protein or gene specifically expressed on the cell surface, in the cytoplasm and / or in the nucleus of a specific cell type. The marker can be a positive selection marker or a negative selection marker. Preferably, the marker is a cell surface marker, and the cell surface selection marker can be used to concentrate, separate and / or detect viable cells.

[0049] The detection of marker protein can be carried out by immunological analysis using specific antibodies of the marker protein, such as ELISA, immunostaining, flow cytometry, etc. As a specific antibody for the marker protein, an antibody that binds to a specific amino acid sequence in the marker protein, or a specific sugar chain bound to the marker protein, etc. can be used. In addition, in the case of a marker protein (such as a transcription factor or its subunit, etc.) that is expressed in the cell and does not appear on the cell surface, a reporter protein can be expressed together with the marker protein, and the marker protein as the object is detected by detecting the reporter protein. This method is preferably used in cases where a suitable cell surface marker cannot be confirmed. The detection of marker genes can be carried out using nucleic acid amplification methods and / or nucleic acid detection methods known in the field, such as RT-PCR, microarrays, biochips, and RNAseq, etc.

[0050] "Expression" is defined as the transcription of a specific nucleotide sequence driven by a promoter and / or the translation of the transcription product.

[0051] "Positive" or "expression" means that a protein or mRNA is expressed in an amount that can be detected by methods known in the art. Detection of proteins can be performed using immunological analysis using antibodies, such as ELISA, immunostaining, and flow cytometry. In addition, in the case of proteins (such as transcription factors or their subunits, etc.) that are expressed in cells and do not appear on the cell surface, a reporter protein can be expressed together with the protein, and the protein as the object can be detected by detecting the reporter protein. Detection of mRNA can be performed using nucleic acid amplification methods and / or nucleic acid detection methods such as RT-PCR, microarrays, biochips, and RNAseq.

[0052] "Negative" or "no expression" means that the expression level of a protein or gene is lower than the detection limit of all or any one of the above-mentioned known methods. The detection limit of protein or gene expression varies according to each method.

[0053] "Pluripotency" refers to the ability to differentiate into tissues and cells with various morphologies and functions, and the ability to differentiate into cells of any lineage of the three germ layers. "Pluripotency" cannot differentiate into a blastoderm, and therefore has no ability to form an individual, which is different from "totipotency" which can differentiate into any tissue of an organism, including the blastoderm.

[0054] "Multipotency" refers to the ability to differentiate into multiple cells of a limited number of lineages. For example, mesenchymal stem cells, hematopoietic stem cells, and neural stem cells are multipotent but not pluripotent.

[0055] "Cultivation" refers to maintaining cells in an in vitro environment to allow them to proliferate (grow) and / or differentiate. "Cultivating" refers to maintaining cells outside of a tissue or in vitro, such as in a cell culture dish, plate, bottle or culture tank (tank), to allow them to proliferate (grow) and / or differentiate.

[0056] "Sustain" means culturing a desired cell population while maintaining its number. The maintenance of the cell number can be achieved by keeping the cells alive without proliferation, or by offsetting the increase in the number of cells due to proliferation with the decrease in the number of cells due to death. For the maintenance of the cell number, it is not necessary to maintain the number of cells to be exactly the same, as long as the number of cells is maintained to be substantially the same according to the purpose of the present invention.

[0057] "Expanding culture (Expand)" refers to cultivating for the purpose of making the desired cell group proliferate and increasing the number of cells. The increase in the number of cells is achieved as long as the increase caused by the proliferation of the cells exceeds the reduction caused by the death, and all cells of the cell group are not required to proliferate. Compared with before starting to expand the culture, the increase in the number of cells can be 1.1 times, 1.2 times, 1.5 times, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 15 times, 20 times, 30 times, 40 times, 50 times, 100 times, 300 times, 500 times, 1000 times, 3000 times, 5000 times, 10000 times, 100000 times or more.

[0058] “comprise(s) or comprising…” means including but not limited to the elements following the phrase. Therefore, it is implied that the elements following the phrase are included, but it is not implied that any other elements are excluded.

[0059] "About" or "approximately" means a value fluctuating by 30%, 25%, 20%, 15%, 10%, 8%, 6%, 5%, 4%, 3%, 2% or 1% increase or decrease, respectively, relative to a reference value. Preferably, the term "about" or "approximately" means a range of 15%, 10%, 5%, or 1% increase or decrease, respectively, relative to a reference value.

[0060] Effects of the Invention

[0061] According to the present invention, there is provided a technique for enriching atrial myocytes or ventricular myocytes in a cell population containing cardiomyocytes induced from stem cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0062] Figure 1 The figure shows typical action potential waveforms exhibited by ventricular myocytes and atrial myocytes.

[0063] Figure 2 This figure shows the results of analyzing the expression of CD151 in a cell population containing cardiomyocytes induced by iPSCs under ventricular myocyte induction conditions by flow cytometry. The horizontal axis represents the fluorescence intensity of EmGFP, and the vertical axis represents the fluorescence intensity of Alexa (registered trademark) 647. In the figure, "Q2" represents a "CD151-high" cell subpopulation, and "Q4" represents a "CD151-low" cell subpopulation.

[0064] Figure 3 This figure shows the results of analyzing the expression of CD151 in a cell population containing cardiomyocytes induced by iPSCs under atrial myocyte induction conditions using a flow cytometer. The horizontal axis represents the fluorescence intensity of EmGFP, and the vertical axis represents the fluorescence intensity of Alexa (registered trademark) 647. In the figure, "Q2" represents a "CD151-high" cell subpopulation, and "Q4" represents a "CD151-low" cell subpopulation.

[0065] Figure 4 The chart shows the expression levels of marker genes (KCNA5, KCNJ3, NPPA, NR2F1, NR2F2, TBX5) of atrial myocytes and marker genes (HEY2, MYL2) of ventricular myocytes in the cell subpopulation "CD151-high" obtained under ventricular myocyte differentiation conditions and "CD151-low" obtained under atrial myocyte differentiation conditions. The expression levels of marker genes of atrial myocytes are expressed as relative values ​​(mean ± standard error) when the expression levels in the cell subpopulation "CD151-high" obtained under atrial myocyte differentiation conditions are set to 1. The expression levels of marker genes of ventricular myocytes are expressed as relative values ​​(mean ± standard error) when the expression levels in the cell subpopulation "CD151-high" obtained under ventricular myocyte differentiation conditions are set to 1.

[0066] Figure 5 The graphs show the responses of the cell subpopulations "CD151-high" obtained under ventricular myocyte differentiation conditions and "CD151-low" obtained under atrial myocyte differentiation conditions to inhibitors and agonists of atrial myocardial specific channels. (A) shows the response to the inhibitor (4-aminopyridine), and (B) shows the response to the agonist (carbachol). DETAILED DESCRIPTION

[0067] Hereinafter, preferred embodiments of the present invention will be described. It should be noted that the embodiments described below are examples of typical embodiments of the present invention, and the scope of the present invention is not to be interpreted as being limited thereto.

[0068] 1. A method for enriching atrial myocytes or ventricular myocytes in a cell population containing cardiomyocytes induced from stem cells,

[0069] The method for enriching atrial myocytes or ventricular myocytes of the present invention comprises the following steps.

[0070] Step (1), using the expression level of CD151 as an indicator, recovering atrial myocytes or ventricular myocytes from a cell population containing cardiomyocytes.

[0071] In the method of the present invention, when atrial myocytes are enriched, step (1) is specifically the following step (1-1).

[0072] Step (1-1), recovering CD151 low-expressing cells from a cell population containing cardiomyocytes.

[0073] In the cell population containing cardiomyocytes induced by stem cells, the cell subpopulation identified based on high expression of CD151 contains ventricular myocytes at a higher ratio than the cell subpopulation identified based on low expression of CD151. Therefore, by removing the cell subpopulation identified based on high expression of CD151 from the cell population containing cardiomyocytes induced by stem cells, or recovering the cell subpopulation identified based on low expression of CD151 from the cell population containing cardiomyocytes, atrial myocytes can be enriched in the cell population.

[0074] On the other hand, in the method of the present invention, when ventricular myocytes are enriched, step (1) is specifically the following step (1-2).

[0075] Step (1-2), recovering cells with high CD151 expression from the cell population containing cardiomyocytes.

[0076] In the cell population containing cardiomyocytes induced by stem cells, the cell subpopulation identified based on high expression of CD151 contains ventricular myocytes at a higher ratio than the cell subpopulation identified based on low expression of CD151. Therefore, by removing the cell subpopulation identified based on low expression of CD151 from the cell population containing cardiomyocytes induced by stem cells, or recovering the cell subpopulation identified based on high expression of CD151 from the cell population containing cardiomyocytes, ventricular myocytes can be enriched in the cell population.

[0077] [CD151 detection probe]

[0078] The expression level of CD151 in cells can be determined using a CD151 detection probe (hereinafter also simply referred to as a “probe”) that has binding properties to CD151.

[0079] The probe may be, for example, an antibody or a nucleic acid (aptamer, etc.), and is preferably an antibody.

[0080] Preferably, the probe is labeled to be detectable optically, electrically or magnetically. In this case, the cell can be contacted with the probe, and the signal from the label of the probe bound to CD151 can be detected optically, electrically or magnetically, and the expression level of CD151 in the cell can be determined based on the signal intensity.

[0081] In addition, for example, when the probe is an antibody, the expression level of CD151 in cells can also be determined using a labeled secondary antibody that binds to the antibody.

[0082] The present invention also provides a reagent for enriching atrial myocytes or ventricular myocytes in a cell population containing cardiomyocytes induced from stem cells, the reagent comprising the above-mentioned probe.

[0083] For the expression level of CD151 in cells, a certain reference value can be set based on the distribution of expression levels in the cell population, and cells presenting expression levels above the reference value are determined as high expression, and cells presenting expression levels below the reference value are determined as low expression. As the reference value in this case, for example, it can be the maximum value, average value, median value or mode of the expression level of each cell, preferably the maximum value. The reference value can be appropriately set according to the target cell enrichment rate, for example, it can be set to 1% larger or smaller than the maximum value, average value, median value or mode (preferably the maximum value) of the expression level of each cell, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or a larger or smaller value.

[0084] In addition, for the expression level of CD151 in cells, the expression level in stem cells can also be set as a reference value, and cells showing an expression level above the reference value are determined as high expression, and cells showing an expression level below the reference value are determined as low expression. As the reference value in this case, for example, it can be the maximum value, average value, median value or mode of the expression level in stem cells, preferably the maximum value. The reference value can be appropriately set according to the target cell enrichment rate, for example, it can be set to 1% larger or smaller than the maximum value, average value, median value or mode (preferably the maximum value) of the expression level in stem cells, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or a larger or smaller value.

[0085] Alternatively, for the expression level of CD151 in cells, the signal intensity detected in the same manner as the cells contacted with the probe for cells not contacted with the probe (negative control) can also be set as a reference value, and cells showing a signal intensity above the reference value are determined as high expression, and cells showing a signal intensity below the reference value are determined as low expression. The reference value can be appropriately set according to the target cell enrichment rate, for example, it can be set to 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or a larger or smaller value than the maximum value, average value, median value or mode (preferably the maximum value) of the expression level of the negative control.

[0086] The recovery of the target cell subpopulation or the removal of the non-target cell subpopulation can be applied to the conditions known in the past, and can be suitably applied to the cell sorting using, for example, a flow cytometer. As an example, first, an anti-CD151 primary antibody is reacted with the cells. After washing the cells to remove the anti-CD151 primary antibody that is not combined with the cells, a fluorescently labeled secondary antibody is reacted with the cells. After further washing the cells to remove the fluorescently labeled secondary antibody that is not combined with the primary antibody, the fluorescence intensity of the cells is measured by flow cytometry, and the cells presenting the expression level above the above-mentioned reference value are sorted as CD151 high-expressing cell subpopulations (CD151-high), and the cells presenting the expression level below the reference value are sorted as CD151 low-expressing cell subpopulations (CD151-low).

[0087] 2. Cardiac myocyte populations enriched with atrial or ventricular myocytes

[0088] The present invention also provides a cell population enriched in atrial myocytes and a cell population enriched in ventricular myocytes obtained by the above-mentioned enrichment method.

[0089] The proportion of atrial myocytes or ventricular myocytes in the cell population is not particularly limited. The proportion in the recovered target cell subpopulation is, for example, 5%, 10%, 20%, 30%, 40%, 50% higher than the proportion in the initial cell population containing cardiomyocytes induced from stem cells, preferably 60%, 70%, 80%, 90% higher, more preferably 100%, 150%, 200%, 300%, 400% higher, and most preferably 500% higher. The proportion of atrial myocytes or ventricular myocytes in the cell population enriched with atrial myocytes or in the cell population enriched with ventricular myocytes is, for example, 5% or more, 10% or more, 20% or more, 30% or more, 40% or more, 50% or more, preferably 60% or more, 70% or more, 80% or more, 90% or more, and more preferably 99.5% or more, 99.9% or more.

[0090] Whether the cell is an atrial myocyte or a ventricular myocyte can be identified by a conventionally known method, and for example, identification based on the electrophysiological analysis, marker expression analysis, and drug response analysis described below can be employed.

[0091] Identification based on electrophysiological analysis can employ any of the following (1) to (7) based on the patch clamp method, for example. Figure 1 Typical action potential waveforms of ventricular and atrial myocytes are shown.

[0092] (1) The ratio of the action potential duration at 30% repolarization (APD30) to the action potential duration at 90% repolarization (APD90) (APD30 / 90) is 0.3 or more, and the maximum rate of waveform rise (dv / dt max ) is 10 or more action potential waveform is defined as ventricular myocardial cells. On the other hand, cells with APD30 / 90 less than 0.3 and dv / dt max Cells with action potential waveforms of 10 or more are defined as atrial myocytes. In this case, dv / dt max Cells with an action potential waveform of less than 10 and a spontaneous action potential period of more than 1 s are defined as immature cardiomyocytes that are not classified as either ventricular myocytes or atrial myocytes (Cell Stem Cell, 2017, 21, 179-194).

[0093] (2) Cells showing an action potential waveform with a significantly longer action potential duration (APD50) or APD90 value at 50% repolarization are defined as ventricular myocytes, and cells showing an action potential waveform with a significantly shorter APD50 or APD90 value are defined as atrial myocytes (JCI Insight, 2018; 3(12): e99941, Eur. Heart J. 2017, 38, 292-301).

[0094] (3) The action potential duration (APD20) or APD50 or APD90 values ​​at 20% repolarization were significantly longer, and the amplitude of the action potential plateau (APA Plat ) were defined as ventricular myocytes, and cells with significantly larger action potential waveforms were defined as those with significantly shorter APD20, APD50, or APD90 values, and APA Plat Cells with significantly smaller action potential waveforms were defined as atrial myocytes (Stem Cell Reports. 2017 Dec 12; 9(6): 1765-1779).

[0095] (4) Cells presenting an action potential waveform with a significantly larger value of the ratio of APD20 to the action potential duration at 80% repolarization (APD80) (APD20 / 80) were defined as ventricular myocytes, and cells presenting an action potential waveform with a significantly smaller value of APD20 / 80 were defined as atrial myocytes (JCI Insight, 2018; 3(12): e99941).

[0096] (5) Cells showing an action potential waveform with a significantly smaller ratio of APD90 to APD50 (APD90 / 50) are defined as ventricular myocytes, and cells showing an action potential waveform with a significantly larger APD90 / 50 value are defined as atrial myocytes (Eur. Heart J., 2017, 38, 292-301, Eur. Heart J., 2011, 32, 952-962).

[0097] (6) Cells showing an action potential waveform with an APD90 / 50 of less than 1.4 are defined as ventricular myocytes, and cells showing an action potential waveform with an APD90 / 50 of more than 1.7 are defined as atrial myocytes. In this case, cells showing an action potential waveform with an APD90 / 50 of 1.4 or more and 1.7 or less are defined as pacemaker cells (Eur. Heart J., 2011, 32, 952-962).

[0098] (7) The plateau period with membrane potential changes within 20 mV is longer than 50 ms, and the dv / dt max Cells with an action potential waveform with an APA greater than 50, an action potential amplitude (APA) greater than 85 mV, and an APD90 / 50 less than 2.3 were defined as ventricular myocytes. Cells with an action potential waveform that differed from the same action potential waveform only in the absence of a plateau were defined as atrial myocytes, and cells with an action potential waveform that differed in the absence of a plateau and an APD90 / 50 greater than 2.3 were defined as pacemaker cells (PNAS, 2017, E8372-E8381).

[0099] For identification based on marker expression analysis, the expression of marker proteins or marker genes is measured, and as long as the cell expresses atrial myocyte markers, preferably without expressing ventricular myocyte markers, the cell can be determined to be an atrial myocyte. Conversely, as long as the cell expresses ventricular myocyte markers, preferably without expressing atrial myocyte markers, the cell can be determined to be a ventricular myocyte. As marker genes for atrial myocytes, KCNA5 (Potassium voltage-gated channel subfamily A member 5), KCNJ3 (Potassium voltage-gated channel subfamily J member 3), NPPA (Natriuretic peptide A), NR2F1 (Nuclear receptor subfamily 2 group F member 1), NR2F2 (Nuclear receptor subfamily 2 group F member 2), and TBX5 (T-Box 5) are known. As marker genes for ventricular myocytes, HEY2 (Hes related family BHLH transcription factor with YRPW motif 2) and MYL2 (Myosin light chain 2) are known.

[0100] It should be noted that the cardiomyocytes including atrial myocytes and ventricular myocytes refer to cells expressing at least one marker selected from the group consisting of at least cardiac troponin (cTNT), αMHC (αmyosin heavy chain, α myosin heavy chain, MYH6) and βMHC (MYH7). For the cTNT gene, in the case of humans, NCBI accession number NM_000364 can be exemplified, and in the case of mice, NM_001130176 can be exemplified. For the αMHC gene, in the case of humans, NCBI accession number NM_002471 can be exemplified, and in the case of mice, NM_001164171 can be exemplified. For the βMHC gene, in the case of humans, NCBI accession number NM_000257 can be exemplified, and in the case of mice, NM_080728 can be exemplified.

[0101] The detection of the marker protein can be performed by immunological analysis using specific antibodies to the marker protein, such as ELISA, immunostaining, flow cytometry, etc. The detection of the marker gene can be performed by nucleic acid amplification methods and / or nucleic acid detection methods known in the art, such as RT-PCR, microarrays, biochips, etc.

[0102] As identification based on drug response analysis, there can be mentioned a method of detecting the response of cells to drugs that activate or inhibit channels specifically expressed in atrial myocytes or ventricular myocytes. For example, carbachol is a drug that activates muscarinic potassium channels (I K channels) specifically expressed in atrial myocytes. K,Ach ) drug, when added to cells, shortening of action potential duration (APD) was observed only in atrial myocytes, but it did not affect the APD of ventricular myocytes. In addition, 4-aminopyridine is a drug that inhibits ultrafast activated delayed rectifier potassium channels (IK ur ) drugs, when added to cells, the prolongation of APD20 was observed only in atrial myocytes, but did not affect the APD of ventricular myocytes (Stem Cell Reports, 2018; 11(6): 1378-1390.). Atrial myocytes or ventricular myocytes can be identified by detecting the responsiveness of cells to these drugs.

[0103] 3. Method for preparing atrial myocytes or ventricular myocytes from stem cells

[0104] The method for preparing atrial myocytes or ventricular myocytes from the stem cells of the present invention comprises the following steps.

[0105] Step (A) is to induce a cell population containing cardiomyocytes from the above stem cells.

[0106] Step (B), using the expression level of CD151 as an indicator, recovering atrial myocytes or ventricular myocytes from the above cell population.

[0107] In the preparation method of the present invention, when preparing atrial myocytes, steps (A) and (B) are specifically the following steps (A-1) and (B-1).

[0108] Step (A-1), inducing a cell population containing cardiomyocytes from the above stem cells under atrial myocyte differentiation conditions.

[0109] Step (B-1), recovering CD151 low-expressing cells from the above cell population.

[0110] On the other hand, in the preparation method of the present invention, when preparing ventricular myocytes, steps (A) and (B) are specifically the following steps (A-2) and (B-2).

[0111] Step (A-2), inducing a cell population containing cardiomyocytes from the above stem cells under conditions of ventricular myocyte differentiation,

[0112] Step (B-2), recovering CD151 high-expressing cells from the above cell population.

[0113] [Induction step]

[0114] In steps (A), (A-1), and (A-2), a cell population including cardiomyocytes is induced from stem cells.

[0115] [Stem Cells]

[0116] Examples of "stem cells" include pluripotent stem cells.

[0117] "Pluripotent stem cells" that can be used in the present invention refer to stem cells with the ability to differentiate into tissues and cells with various forms and functions of an organism, and cells that can differentiate into any one of the three germ layers (endoderm, mesoderm, ectoderm) of the lineage. It is not particularly limited, and examples thereof include embryonic stem cells (ESC), embryonic stem cells derived from cloned embryos obtained by nuclear transplantation, spermatogonial stem cells, embryonic germ cells, induced pluripotent stem cells (sometimes referred to as "iPSC" in this specification), etc. In addition, "pluripotent stem cells" that can be used in the present invention refer to stem cells with the ability to differentiate into cells of a plurality of limited number of lineages. As "pluripotent stem cells" that can be used in the present invention, examples thereof include dental pulp stem cells, stem cells derived from oral mucosa, hair follicle stem cells, fibroblasts derived from culture, adult stem cells of bone marrow stem cells, etc. Preferred pluripotent stem cells are ESC and iPSC.

[0118] As "ESC", if it is mouse ESC, various mouse ESC strains established by inGenious targeting laboratory, RIKEN (Rikagakuin), etc. can be used, and if it is human ESC, various human ESC strains established by the University of Wisconsin, NIH, RIKEN, Kyoto University, National Center for Developmental Medical Research, Cellartis, etc. can be used. For example, as human ESC strains, CHB-1 to CHB-12 strains, RUES1 strains, RUES2 strains, HUES1 to HUES28 strains, etc. sold by ESI Bio, H1 strains, H9 strains, etc. sold by WiCell Research, KhES-1 strains, KhES-2 strains, KhES-3 strains, KhES-4 strains, KhES-5 strains, SSES1 strains, SSES2 strains, SSES3 strains, etc. sold by RIKEN can be used.

[0119] “Induced pluripotent stem cells” refer to cells obtained by introducing specific factors (nuclear reprogramming factors) into mammalian somatic cells or undifferentiated stem cells and reprogramming them. Currently, there are many types of "induced pluripotent stem cells". In addition to iPSCs established by Yamanaka et al. by introducing four factors, Oct3 / 4, Sox2, Klf4, and c-Myc, into mouse fibroblasts (Takahashi K, Yamanaka S., Cell, (2006) 126: 663-676), iPSCs derived from human cells established by introducing the same four factors into human fibroblasts (Takahashi K, Yamanaka S. et al., Cell, (2007) 131: 861-872.), Nanog-iPSCs established by introducing the above four factors and screening with Nanog expression as an index (Okita, K., Ichisaka, T., and Yamanaka, S. (2007). Nature 448, 313-317.), iPSCs produced by a method without c-Myc (Nakagawa M, Yamanaka S. et al., Nature Biotechnology, (2008) 26, 101-106), iPSCs established by introducing 6 factors using a virus-free method (Okita K et al., Nat. Methods 2011 May; 8(5): 409-12, Okita K et al., Stem Cells. 31(3): 458-66.), etc. In addition, induced pluripotent stem cells established by introducing four factors, OCT3 / 4, SOX2, NANOG, and LIN28, prepared by Thomson et al. (Yu J., Thomson JA. et al., Science (2007) 318: 1917-1920), induced pluripotent stem cells prepared by Daley et al. (Park IH, Daley GQ. et al., Nature (2007) 451: 141-146), and induced pluripotent stem cells prepared by Sakurada et al. (Japanese Patent Application Laid-Open No. 2008-307007) can also be used.

[0120] In addition, all published papers (e.g., Shi Y., Ding S. et al., Cell Stem Cell, (2008) Vol3, Issue 5, 568-574; Kim JB., Scholer HR. et al., Nature, (2008) 454, 646-650; Huangfu D., Melton, DA. et al., Nature Biotechnology, (2008) 26, No. 7,795-797), or any one of the induced pluripotent stem cells known in the art described in patents (e.g., Japanese Patent Application Publication No. 2008-307007, Japanese Patent Application Publication No. 2008-283972, US2008-2336610, US2009-047263, WO2007-069666, WO2008-118220, WO2008-124133, WO2008-151058, WO2009-006930, WO2009-006997, WO2009-007852).

[0121] As induced pluripotent stem cell strain, various iPSC strains established by NIH, RIKEN, Kyoto University etc. can be utilized.For example, if it is human iPSC strain, then can enumerate RIKEN's HiPS-RIKEN-1A strain, HiPS-RIKEN-2A strain, HiPS-RIKEN-12A strain, Nips-B2 strain etc., 253G1 strain, 201B7 strain, 409B2 strain, 454E2 strain, 606A1 strain, 610B1 strain, 648A1 strain, 1231A3 strain, 1390D4 strain and 1390C1 strain etc., more preferably 1390D4 strain and 1390C1 strain.Or, also can use the cell strain of clinical grade provided by Kyoto University, Cellular Dynamics International etc. and the research purposes and the cell strain of clinical use etc. using these cell strains to make.

[0122] [Conditions for differentiation of atrial myocytes]

[0123] Atrial myocyte differentiation conditions can be applied to previously known conditions (e.g., see EMBO Mol Med (2015) 7: 394-410). For example, embryoid bodies made from iPSCs are cultured for 2 days in a basal medium containing BMP4, activin A, and bFGF, and then cultured for 3 days in a basal medium containing VEGF, Wnt inhibitors, TGF-β inhibitors, and retinoic acid. Further, by culturing in a basal medium containing VEGF, a cell population containing atrial myocytes can be obtained.

[0124] The differentiation induction time is not particularly limited, and is, for example, 7 to 40 days, and may be, for example, 120 days, 90 days, 60 days, 30 days, 28 days, 21 days, 14 days or 7 days depending on the purpose.

[0125] [Ventricular myocyte differentiation conditions]

[0126] The conditions for differentiation of ventricular myocytes can be the conditions known in the past (e.g., see Cell Stem Cell. (2011) 8(2): 228-40). For example, embryoid bodies prepared from iPSCs are cultured for 2 days in a basal medium containing BMP4, activin A, and bFGF, and then cultured for 3 days in a basal medium containing VEGF, a Wnt inhibitor, a BMP4 inhibitor, and a TGF-β inhibitor. Furthermore, by culturing in a basal medium containing VEGF, a cell population containing ventricular myocytes can be obtained.

[0127] The differentiation induction time is not particularly limited, and is, for example, 7 to 40 days, and may be, for example, 120 days, 90 days, 60 days, 30 days, 28 days, 21 days, 14 days or 7 days depending on the purpose.

[0128] As the basal medium, there is no particular limitation, and for example, StemPro-34 SFM (ThermoFisher), STEMdiff APEL2 medium (STEMCELL Technologies, ST-05275), TeSR1 medium, and ChemicallyDefined Medium (CDM) medium can be suitably used. In addition, BME medium, BGJb medium, CMRL 1066 medium, Glasgow MEM medium, Improved MEM (IMEM) medium, Improved MDM (IMDM) medium, Medium 199 medium, Eagle MEM medium, αMEM medium, DMEM medium (high sugar, low sugar), DMEM / F12 medium, Ham medium, RPMI 1640 medium, Fischer's medium, and mixed medium thereof can also be used.

[0129] The CDM medium is not particularly limited, and for example, a medium prepared using Iscove's modified Dulbecco's medium (manufactured by GE Healthcare) can be used.

[0130] The basal medium may be supplemented with substances used in conventional cell culture, such as Ham's F-12 nutrient mixture, albumin such as human serum albumin, polyvinyl alcohol (PVA), deionized BSA, linoleic acid, linolenic acid, cholesterol, insulin, apo-transferrin, selenium, ethanolamine, thioglycerol, protein-free hybridoma mixture II (PFHMII), ascorbic acid, L-alanyl-L-glutamine and / or antibiotics.

[0131] Wnt inhibitors (Wnt signal transduction inhibitors) are substances that inhibit the Wnt-mediated signal transduction pathway, and examples thereof include IWP-2, IWP-3, IWP-4, 2-(4-trifluoromethylphenyl)-7,8-dihydro-5H-thiothiopyrano[4,3-d]pyrimidin-4(3H)-one, IWR1, G-CSF, IGFBP4, Dkk1, Cerberus, anti-Wnt antibodies, Wnt agonists (Wnt receptor inhibitors), soluble Wnt receptor proteins (Frzb-1, etc.), dominant negative mutants, etc. These Wnt inhibitors may be used in combination of two or more.

[0132] BMP4 inhibitors that can be used include Dorsomorphin (6-[4-[2-(1-piperidinyl)ethoxy]phenyl]-3-(4-pyridinyl)pyrazolo[1,5-A]pyrimidine, 6-[4-(2-piperidin-1-ylethoxy)phenyl]-3-pyridin-4-ylpyrazolo[1,5-A]pyrimidine), indirubin-3'-oxime, phenformin hydrochloride, GSK621, WZ4003, HTH-01-015, etc. These BMP4 inhibitors can also be used in combination of two or more.

[0133] TGF-β inhibitors include SB431542 (4-[4-(1,3-benzodioxol-5-yl)-5-(2-pyridinyl)-1H-imidazol-2-yl]-benzamide, 4-[4-(1,3-benzodioxol-5-yl)-5-(2-pyridinyl)-1H-imidazol-2-yl]-benzamide), A83-01 (4-[4-(1,3-benzodioxol-5-yl)-5-(2-pyridinyl)-1H-imidazol-2-yl]-benzamide), (2-pyridyl)-1H-imidazol-2-yl]-benzamide, 4-[4-(1,3-benzodioxol-5-yl)-5-(2-pyridinyl)-1H-imidazol-2-yl]-benzamide), LDN193189 (4-[6-[4-(1-piperazinyl)phenyl]pyrazolo[1,5-a]pyrimidin-3-yl)quinoline, 4-[6-[4-(1-Piperazinyl)phenyl]p yrazolo[1,5-a]pyrimidin-3-yl]-quinoline), GW788388 (4-[4-[3-(2-pyridinyl)-1H-pyrazol-4-yl]2-pyridinyl]-N-(tetrahydro-2H-pyran-4-yl)benzamide, 4-[4-[3-(2-Pyridinyl)-1H-pyrazol-4-yl]-2-pyridinyl]-N-(tetrahydro-2H-pyr an-4-yl)-benzamide), SM16 (4-[4-(1,3-Benzodioxol-5-yl)-5-(6-methyl-2-pyridinyl)-1H-imidazol-2-yl]-bicyclo[2.2.2]octane-1-carboxamide, 4-[4-(1,3-Benzodioxol-5-yl)-5-(6-methyl-2-pyridinyl)-1H-imidazol-2-yl]-bicyclo[2.2.2]octane-1-carboxamide), IN-1130 (3-[[5-(6-methyl-2-pyridinyl)-4-(6-quinoxalinyl)-1H-imidazol-2-yl]methyl]-benzamide, 3-[[5-(6-Methyl-2-pyridinyl)-4-(6-quinoxalinyl)-1H-imidazol-2-yl]methyl]-benzamide), GW6604 (2-phenyl-4-[3-(pyridin-2-yl)-1H-pyrazol-4-yl]pyridine, 2- Phenyl-4-[3-(pyridin-2-yl)-1H-pyrazol-4-yl]pyridine) and SB505124 (2-[4-(1,3-Benzodioxol-5-yl)-2-(1,1-dimethylethyl)-1H-imidazol-5-yl]-6-methyl-pyridine, 2-[4-(1,3-Benzodioxol-5-yl)-2-(1,1-dimethylethyl)-1H-imidazol-5-yl]-6-methyl-pyridine). These TGF-β inhibitors can also be used in combination of two or more. .

[0134] [Separation step]

[0135] In steps (B), (B-1), and (B-2), atrial myocytes or ventricular myocytes are recovered from the obtained cell population using the expression level of CD151 as an index.

[0136] This step is the same as the above-mentioned method for enriching atrial myocytes or ventricular myocytes.

[0137] 4. Atrial and ventricular myocytes

[0138] The present invention also provides a cell population enriched with atrial myocytes and ventricular myocytes obtained by the above preparation method.

[0139] [medicine]

[0140] The cell population, atrial myocytes and ventricular myocytes of the present invention can be applied to cell medicines for treating heart diseases containing the cell population, and methods of treating heart diseases by administering the cell medicines. In addition, the present invention also provides uses of the cell population, atrial myocytes and ventricular myocytes for producing the cell medicines, and the cell population, atrial myocytes and ventricular myocytes for treating heart diseases.

[0141] Cell medicine can be used in regenerative medicine for heart diseases such as myocardial infarction, heart failure, ischemic heart disease, cardiomyopathy, myocarditis, hypertrophic cardiomyopathy, dilated cardiomyopathy, etc.

[0142] The cells contained in the cell medicine can be, for example, cells obtained by peeling off and recovering cells in culture, or cells frozen in a cryopreservation solution. When using cells obtained by expanding the culture and freezing the same batch of cells in small portions, it is preferred in terms of being able to stably obtain the same effect and excellent operability.

[0143] Cell medicine can be a suspension formed by suspending cells in a suitable solvent, a cell aggregate, and any form of a cell sheet such as a monolayer or more than two layers. The solvent can use water, physiological saline or various buffers, cell preservation fluid. In addition, cell aggregates and cell sheets can be formed only by cells, or can be formed by suitable biocompatible materials and cells. As biocompatible materials, collagen, polyglycolic acid (PGA), polylactic acid, alginate, polyethylene oxide, copolymers of polylactic acid-polyglycolic acid, proteoglycans, glycosaminoglycans, human dermis or a combination of these materials can be cited. Biocompatible materials can be porous bodies such as films such as sheets, sponges, or mesh bodies such as fabrics, cloths and nonwovens.

[0144] Cell medicines may contain other components such as pharmaceutically acceptable carriers and additives according to conventional methods depending on the purpose and form. Examples of carriers and additives include isotonic agents, thickeners, sugars, sugar alcohols, preservatives (preservatives), bactericides or antibacterial agents, pH adjusters, stabilizers, chelating agents, oily bases, gel bases, surfactants, suspending agents, fluidizing agents, dispersants, buffers, antioxidants, and the like.

[0145] Using cell medicine, a method for treating the above-mentioned diseases is provided, which comprises administering a therapeutically effective amount of the cell medicine to a patient.

[0146] The so-called therapeutically effective amount refers to the amount of cells that can obtain the therapeutic effect for the above-mentioned disease when the cells are administered to the patient, compared with the control to which the cells are not administered. As a specific therapeutically effective amount, it can be appropriately selected according to the administration form, administration method, purpose of use, and the age, weight, symptoms, etc. of the patient. The effective amount for a human (e.g., adult) per treatment is, for example, 200,000 to 1,000,000 cells / kg body weight.

[0147] Examples of the administration method of the cell medicine include intraperitoneal injection, subcutaneous injection, lymph node injection, intravenous injection, thoracic injection, and local direct injection by laparotomy.

[0148] [Freezing stock solution]

[0149] The present invention also provides a cryopreserved solution containing the cell population and / or atrial myocytes and / or ventricular myocytes obtained by this step.

[0150] The cryostock solution can be prepared by separating the obtained cell group and / or atrial myocytes and / or ventricular myocytes from the culture medium by centrifugation, suspending them in a cryopreservation solution and freezing them. For the cryopreservation solution, it is sufficient to use the reagent used in the cryopreservation of cells in the past. For example, commercially available Cryostem Freezing Medium (trade name) and CELLBANKER (registered trademark) etc.

[0151] The frozen stock solution can be used, for example, to prepare a tissue model (artificial heart) having atrial myocytes and ventricular myocytes as constituent elements.

[0152] Example

[0153] [Test Example 1: Induction of cardiomyocyte population from iPSCs]

[0154] A cell population containing cardiomyocytes is induced from iPSCs under atrial myocyte differentiation conditions or ventricular myocyte differentiation conditions.

[0155] As iPSC, a double knock-in human iPS cell line (1390D4 line) in which EmGFP was inserted into the TNNI1 gene locus and mCherry was inserted into the TNNI3 gene locus as reporter proteins was used.

[0156] iPSCs were maintained and cultured according to a conventionally known method (“An Efficient Nonviral Method to Generate Integration-Free Human-Induced Pluripotent Stem Cells from Cord Blood and Peripheral Blood Cells”, Stem Cells, 2012).

[0157] iPSCs were seeded in 6-well plates with low adhesion (2 × 10 6 The cells were cultured at 37°C and 5% oxygen to form embryoid bodies (day 0). The culture medium used was supplemented with 1% L-glutamine, 150 μg / mL transferrin, 50 μg / mL ascorbic acid, and 4×10 -4 M, Rock inhibitor (Y-27632) 10 μM, BMP4 2 ng / mL and Matrigel 0.5% in StemPro-34 SFM (ThermoFisher).

[0158] [Conditions for differentiation of atrial myocytes]

[0159] The next day (day 1), 1.5 ml / well of atrial myocyte induction medium 1 was added to each well inoculated with cells, and cultured for another 2 days at 37°C and 5% oxygen. Atrial myocyte induction medium 1 was supplemented with 1% L-glutamine, 150 μg / mL transferrin, 50 μg / mL ascorbic acid, 4×10 -4 M, StemPro-34 SFM containing 4 ng / ml BMP4, 8 ng / ml Activin A, and 10 ng / ml bFGF.

[0160] Next (day 3), the culture medium was replaced with atrial myocyte induction medium 2, and the cells were cultured at 37°C and 5% oxygen for 3 days. Atrial myocyte induction medium 2 was supplemented with 1% L-glutamine, 150 μg / mL transferrin, 50 μg / mL ascorbic acid, 4×10 -4 M, VEGF 10 ng / mL, Wnt inhibitor (IWP-3) 1 μM, TGF-β inhibitor (SB431542) 5.4 μM and retinoic acid 1 μM in StemPro-34 SFM.

[0161] [Ventricular myocyte differentiation conditions]

[0162] The next day (day 1), 1.5 ml / well of ventricular myocyte induction medium 1 was added to each well inoculated with cells, and cultured for another 2 days at 37°C and 5% oxygen. Ventricular myocyte induction medium 1 was supplemented with 1% L-glutamine, 150 μg / mL transferrin, 50 μg / mL ascorbic acid, 4×10 -4 M, StemPro-34 SFM containing 18 ng / ml BMP4, 12 ng / ml Activin A, and 10 ng / ml bFGF.

[0163] Next (day 3), the culture medium was replaced with ventricular myocyte induction medium 2, and the cells were cultured at 37°C and 5% oxygen for 3 days. Ventricular myocyte induction medium 2 was supplemented with 1% L-glutamine, 150 μg / mL transferrin, 50 μg / mL ascorbic acid, and 4×10 -4 M, VEGF 10 ng / mL, Wnt inhibitor (IWP-3) 1 μM, BMP4 inhibitor (Dorsomorphin) 0.6 μM and TGF-β inhibitor (SB431542) 5.4 μM in StemPro-34 SFM.

[0164] [Common Operations]

[0165] Under the conditions of ventricular myocyte differentiation or atrial myocyte differentiation, the medium was replaced with cardiomyocyte induction medium 3 on day 6, and cultured at 37°C and 5% oxygen for 14 days (day 20) to obtain a cell population containing ventricular myocytes or atrial myocytes. Cardiomyocyte induction medium 3 was supplemented with 1% L-glutamine, 150 μg / mL transferrin, 50 μg / mL ascorbic acid, 4×10 -4 M and VEGF 5ng / mL StemPro-34 SFM. The medium was changed on days 8, 10, 13, 15, and 17. After the medium was changed on day 10, the cells were cultured under normoxic conditions.

[0166] [Test Example 2: Isolation of cardiomyocyte population based on CD151 expression level]

[0167] The cell group containing ventricular myocytes or atrial myocytes on the 20th day of culture was dispersed into single cells, and the number of cells was counted.

[0168] Anti-CD151 antibody (BD) was added to the cell suspension, and the suspension was left to stand at 4° C. for 30 minutes. A sample without antibody addition was prepared as a negative control.

[0169] After washing, Alexa (registered trademark) 647-labeled secondary antibody was added to the cell suspension, and the suspension was left to stand at 4°C for 30 minutes.

[0170] The expression of CD151 in EmGFP-positive cells was analyzed using a flow cytometer (BD FACSAria Fusion cell sorter). Each cell subpopulation was sorted, and the cell subpopulation showing a fluorescence intensity lower than the maximum value of the fluorescence intensity of the negative control cells was designated as "CD151-low", and the cell subpopulation showing a fluorescence intensity higher than the negative control was designated as "CD151-high". Figure 2 , Figure 3 Flow cytometry graphs are shown, and Table 1 shows the proportions of cell subpopulations.

[0171] [Table 1]

[0172] CD151-High CD151-Low Conditions for ventricular myocyte differentiation 55.4％ 44.5％ Atrial myocyte differentiation conditions 24.7％ 75.0％

[0173] [Test Example 3: Determination of the ratio of atrial myocytes and ventricular myocytes in the cell subpopulations "CD151-high" and "CD151-low"]

[0174] The ratio of atrial myocytes and ventricular myocytes was determined using an electrophysiological method for the "CD151-high" and "CD151-low" cell subpopulations obtained under ventricular myocyte differentiation conditions and atrial myocyte differentiation conditions, respectively.

[0175] Each cell subset was cultured on a coverslip coated with fibronectin. -4 M, VEGF 5ng / mL StemPro-34 SFM, cultured at 37°C, normoxia, and the medium was changed every 3 days. Cells on day 13 to day 16 after culture were used for electrophysiological analysis.

[0176] The whole-cell patch clamp method was used for analysis using an Axopatch 200B amplifier (Molecular Devices) and pCLAMP software. The electrodes were made using glass capillaries (WPI) in a micropipette puller and filled with intracellular solution (130mM KOH, 130mM L-aspartic acid, 20mM KCl, 5mM NaCl, 10mM HEPES, 5mM Mg-ATP, 10mM EGTA, 1mM MgCl2, pH 7.2). The experiment was performed at 35-37°C while refluxing Grignard balanced salt solution (Sigma) as the extracellular solution. The spontaneous action potentials of each cell were recorded for 1 minute in current clamp mode. APD30, APD90, and the maximum speed of waveform rise (dv / dt max ). It will show APD30 / 90≥0.3 and dv / dt max Cells with waveforms ≥10 were defined as ventricular myocytes, which would present APD30 / 90 < 0.3 and dv / dt max Cells with waveforms ≥10 are defined as atrial myocytes. max Cells with waveforms <10 were classified as other cells.

[0177] The results are shown in Table 2.

[0178] [Table 2]

[0179]

[0180] The cell subpopulation "CD151-high" obtained under the ventricular myocyte differentiation conditions contains ventricular myocytes at a high ratio (94.1%). In addition, the cell subpopulation "CD151-low" obtained under the ventricular myocyte differentiation conditions contains mostly ventricular myocytes (71.4%), but also contains a certain number (23.8%) of atrial myocytes. Therefore, it can be seen that a cell population enriched with ventricular myocytes can be obtained by removing the cell subpopulation "CD151-low" (containing 71.4% ventricular myocytes and 23.8% atrial myocytes) from the total cell population obtained under the ventricular myocyte differentiation conditions, or by recovering the cell subpopulation "CD151-high".

[0181] On the other hand, the cell subpopulation "CD151-low" obtained under atrial myocyte differentiation conditions contains atrial myocytes at a high ratio (37.5%). In addition, the cell subpopulation "CD151-high" obtained under atrial myocyte differentiation conditions does not contain atrial myocytes, but contains a certain number (18.8%) of ventricular myocytes. Therefore, it can be seen that a cell population enriched with atrial myocytes can be obtained by removing the cell subpopulation "CD151-high" (containing 18.8% of ventricular myocytes) from the total cell population obtained under atrial myocyte differentiation conditions, or recovering the cell subpopulation "CD151-low".

[0182] [Test Example 4: Analysis of the expression of atrial myocyte markers and ventricular myocyte markers in the cell subpopulations “CD151-high” and “CD151-low”]

[0183] For the “CD151-high” cell subpopulations obtained under the ventricular myocyte differentiation conditions and the “CD151-low” cell subpopulations obtained under the atrial myocyte differentiation conditions in Experimental Example 2, the expression of atrial myocyte markers and ventricular myocyte markers was analyzed.

[0184] According to conventional methods, total RNA was extracted from each cell subpopulation, and the expression of marker genes of atrial myocytes (KCNA5, KCNJ3, NPPA, NR2F1, NR2F2, TBX5) and ventricular myocytes (HEY2, MYL2) was analyzed. The results are shown in Figure 4 The expression level of the marker gene of atrial myocytes is the expression level of each marker in the cells obtained under the atrial myocyte differentiation conditions, and is expressed as a relative value (mean ± standard error) obtained by setting the expression level in the cell subpopulation "CD151-high" obtained under the atrial myocyte differentiation conditions to 1. The expression level of the marker gene of ventricular myocytes is the expression level of each marker in the cells obtained under the ventricular myocyte differentiation conditions, and is expressed as a relative value (mean ± standard error) obtained by setting the expression level in the cell subpopulation "CD151-high" obtained under the ventricular myocyte differentiation conditions to 1.

[0185] In the cell subpopulation "CD151-low" obtained under the atrial myocyte differentiation conditions, the expression of marker genes of atrial myocytes was confirmed to be higher than that of the cell subpopulation "CD151-high". Therefore, it can be seen that a cell population enriched with atrial myocytes can be obtained by removing the cell subpopulation "CD151-high" from the total cell population obtained under the atrial myocyte differentiation conditions, or recovering the cell subpopulation "CD151-low".

[0186] In the cell subpopulation "CD151-high" obtained under the ventricular myocyte differentiation conditions, the expression of marker genes of ventricular myocytes was confirmed to be higher than that of the cell subpopulation "CD151-low". Therefore, it can be seen that by removing the cell subpopulation "CD151-low" from the total cell population obtained under the ventricular myocyte differentiation conditions, or recovering the cell subpopulation "CD151-high", a cell population enriched with ventricular myocytes can be obtained.

[0187] In Experimental Example 1-4, human iPS cell line 1390D4 was used as a double knock-in cell line in which EmGFP was inserted into the TNNI1 gene locus and mCherry was inserted into the TNNI3 gene locus as reporter proteins. Similar results were obtained in experiments using human iPS cell line 1390C1.

[0188] [Test Example 5: Identification of subtype cells of "CD151-high" and "CD151-low" cell subpopulations using atrial myocardium-specific channel inhibitors and agonists]

[0189] The responses of the "CD151-high" cell subpopulations obtained under ventricular myocyte differentiation conditions and the "CD151-low" cell subpopulations obtained under atrial myocyte differentiation conditions in Experimental Example 2 to 4-aminopyridine, an atrial-specific channel inhibitor, and carbachol, an agonist, were analyzed.

[0190] Each cell subset was cultured at 5×10 4 Cells / 5 μL were inoculated in a glass-bottomed dish coated with fibronectin for culture. -4 M, VEGF 5ng / mL StemPro-34 SFM, cultured at 37°C, normoxia, and the medium was changed every 3 days. Cells on the 6th to 10th day after culture were used for membrane potential analysis.

[0191] After removing the culture medium, a solution containing 0.2 μL of voltage-sensitive dye (FluoVolt, Thermofisher scientific, F10488) added to every 200 μL of Grignard balanced salt solution (Sigma) was added to the glass part of the culture dish inoculated with cells, and incubated at 37°C and normoxic conditions for 15 minutes. The solution containing the voltage-sensitive dye was removed, 1 mL of Grignard balanced salt solution was added to the culture dish, and incubated in a culture device (37°C, normoxic) on the microscope stage for 1 hour. Cells derived from "CD151-high" were analyzed at 1 Hz pacing, and cells derived from "CD151-low" were analyzed at 3 Hz pacing.

[0192] The analysis was performed by using AquaCosmos2.6 (Hamamatsu Photonics) to measure the fluorescence response every 5.9 milliseconds for 20 seconds under an excitation light of 490 nm. The measurement range (ROI) was set to 512×64 pixels.

[0193] In order to observe the effect of adding 4-aminopyridine on action potential, the action potential waveform before adding the drug was first obtained. Then, 4-aminopyridine (final concentration 50 μM, sigma) was added to the culture dish, and after standing for 10 minutes, the action potential waveform in the same ROI was obtained.

[0194] In order to observe the effect of adding carbachol on action potential, the action potential waveform before adding the drug was first obtained. Then, carbachol (final concentration 10 μM, sigma) was added to the culture dish, and after standing for 1 minute, the action potential waveform in the same ROI was obtained.

[0195] The obtained action potential waveforms were averaged for 6 to 10 consecutive waveforms. The waveforms before and after the addition of 4-aminopyridine and carbachol are shown in Figure 5 .

[0196] It was confirmed that the action potential duration (APD) was prolonged in the "CD151-low" cell subpopulation obtained under atrial myocardial differentiation conditions by adding 4-aminopyridine. On the other hand, no prolongation of APD was observed in the "CD151-high" cell subpopulation obtained under ventricular myocardial differentiation conditions (see Figure 5 (A)).

[0197] In addition, it was confirmed that the APD was shortened in the "CD151-low" cell subpopulation obtained under atrial myocardial differentiation conditions by adding carbachol. On the other hand, no change in APD was observed in the "CD151-high" cell subpopulation obtained under ventricular myocardial differentiation conditions (see Figure 5 (B)).

[0198] Based on the above drug responses, it was confirmed that the "CD151-low" cell subpopulation obtained under atrial myocardial differentiation conditions was atrial myocytes, and the "CD151-high" cell subpopulation obtained under ventricular myocardial differentiation conditions was ventricular myocytes.

Claims

1. A method for enriching atrial myocytes or ventricular myocytes in a cell population containing cardiomyocytes induced from stem cells, the method comprising the following steps: Using the expression level of CD151 as an index, a cell subpopulation whose CD151 expression level is higher than that of negative control cells is defined as "CD151-high", and a cell subpopulation whose CD151 expression level is lower than that of negative control cells is defined as "CD151-low", and atrial myocytes or ventricular myocytes are collected from the obtained "CD151-high" or "CD151-low" cell subpopulations. in, The negative control cells are the cell population to which no anti-CD151 antibody is added.

2. The method of claim 1, wherein: The stem cells are induced pluripotent stem cells.

3. A preparation method, which is a method for preparing atrial myocytes or ventricular myocytes from stem cells, the method comprising the following steps: Step (A), inducing a cell population containing cardiomyocytes from the stem cells; and Step (B), using the expression level of CD151 as an indicator, defining a cell subpopulation whose CD151 expression level is higher than that of negative control cells as "CD151-high", defining a cell subpopulation whose CD151 expression level is lower than that of negative control cells as "CD151-low", and recovering atrial myocytes or ventricular myocytes from the obtained "CD151-high" or "CD151-low" cell subpopulations, in, The negative control cells are the cell population to which no anti-CD151 antibody is added.

4. Application of CD151 detection probe for enriching atrial myocytes or ventricular myocytes in a cell population containing cardiomyocytes induced from stem cells.

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