Method for improving myocardial function

Abstract

Provided are: cardiomyocytes with which, by overexpressing NCAM1 in iPS cell-derived cardiomyocytes, iPS cell-derived cardiomyocytes having high adhesion ability can be obtained, and by producing EHT using the same, the organization of the EHT can be further promoted and the function of the EHT can be enhanced; a cellular tissue-like structure; and a method for improving the function of cardiomyocytes. Provided are cardiomyocytes into which an expression vector of an NCAM1 protein has been introduced. Moreover, provided are: a cellular tissue-like structure comprising said cardiomyocytes; and a method for improving the function of cardiomyocytes, the method comprising a step for inducing the expression of an NCAM1 protein in cardiomyocytes.

Description

Method for improving myocardial function

[0001] The present invention relates to cardiomyocytes, cellular tissue-like structures, and methods for improving cardiomyocyte function. This application claims priority to Japanese Patent Application No. 2024-149349, filed August 30, 2024, the contents of which are incorporated herein by reference.

[0002] Engineered heart tissue (EHT) is a technology that mimics cardiac tissue in vitro, and is expected to be applied to cardiotoxicity assessment, drug discovery research by creating cardiac disease models, and regenerative medicine for cardiac disease.

[0003] As a technique for obtaining artificial myocardial tissue, for example, Patent Document 1 discloses a cardiomyocyte maturation promoter containing one or more specific compounds such as 2-methoxy-5-((Z)-2-(3,4,5-trimethoxyphenyl)vinyl)phenol. This technique aims to obtain a useful cardiomyocyte maturation promoter because the compound or a salt thereof has the effect of maturing cardiomyocytes, and also aims to obtain a method for promoting cardiomyocyte maturation that uses the compound to mature immature cardiomyocytes at low cost in a short period of time, compared to methods of culturing immature cardiomyocytes for a long period of time.

[0004] Patent Literature 2 discloses a method for producing atrial myocytes from early stage cardiomyocytes, which includes a step of culturing a cell population containing early stage cardiomyocytes in the presence of a Notch signal inhibitor. This technology aims to produce atrial myocytes from early stage cardiomyocytes with high efficiency by including a step of culturing a cell population containing early stage cardiomyocytes in the presence of a Notch signal inhibitor.

[0005] International Publication No. 2019 / 189554 International Publication No. 2024 / 029617

[0006] Unlike actual cardiac tissue, EHT produced by the above-mentioned conventional techniques has a sparse cell density and the sarcomere sequences contained in the EHT are not uniformly aligned or oriented. Considering these factors, it is difficult to say that the produced EHT is organized, and its function is immature. Therefore, there is a need for a technique that promotes the organization of EHT and matures its function.

[0007] The present invention was made in consideration of the above circumstances, and its purpose is to establish a method for producing iPS cell-derived cardiomyocytes with improved adhesive ability, and to establish a method for using such cells to promote the organization of EHT and further mature its function.

[0008] As a result of intensive research into achieving the above-mentioned object, the present inventors discovered that neural cell adhesion molecule 1 (NCAM1) enhances the adhesive ability of iPS cell-derived cardiomyocytes, and further discovered that using it to produce EHTs enhances sarcomere orientation and strengthens the function of the EHTs, thereby completing the present invention.

[0009] The present invention was completed based on these findings and through further investigation, and broadly encompasses the following inventions. [1] Cardiomyocytes into which an expression vector for NCAM1 protein has been introduced. [2] The cardiomyocytes according to [1], wherein the cardiomyocytes are cells induced to differentiate from pluripotent stem cells. [3] The cardiomyocytes according to [1] or [2], wherein the cardiomyocytes are cardiomyocytes induced to differentiate from iPS cells. [4] The cardiomyocytes according to any of [1] to [3], which are used for cell transplantation therapy. [5] A cellular tissue-like structure comprising the cardiomyocytes according to any of [1] to [4]. [6] The cellular tissue-like structure according to [5], which is a sheet composed of the cardiomyocytes. [7] The cellular tissue-like structure according to [5] or [6], which is a cardiac organoid or artificial myocardial tissue (Engineered Heart Tissue, EHT) comprising the cardiomyocytes. [8] The cellular tissue-like structure according to any one of [5] to [7], wherein one or more of the functions of cell adhesiveness, myocardial pulsation propagation velocity, calcium handling, tissue density, and contractile force are improved. [9] The cellular tissue-like structure according to any one of [5] to [8], wherein the length of sarcomeres and the orientation of sarcomeres in one direction are improved.

[10] A method for improving cardiomyocyte function, comprising the step of inducing expression of NCAM1 protein in cardiomyocytes.

[11] The method for improving cardiomyocyte function according to

[10] , wherein the cardiomyocytes are cells induced to differentiate from pluripotent stem cells.

[12] The method for improving cardiomyocyte function according to

[10] or

[11] , wherein the cardiomyocytes are iPS cell-derived cardiomyocytes.

[13] The method for improving cardiomyocyte function according to any one of claims

[10] to

[12] , further comprising the step of arranging the cardiomyocytes to form a cellular tissue-like structure.

[14] The method for improving cardiomyocyte function according to any one of

[10] to

[13] , wherein the cellular tissue-like structure is a sheet comprising arranged cardiomyocytes.

[15] The method for improving cardiomyocyte function according to any one of

[10] to

[14] , wherein the cellular tissue-like structure is a cardiac organoid or an engineered heart tissue (EHT) containing the cardiomyocytes.

[0010] The present invention also includes the following inventions:

[16] A method for screening for myocardium-related drugs, comprising administering a candidate substance to cardiomyocytes according to any one of [1] to [4] or to a cellular tissue-like structure according to any one of [5] to [9], and determining that a substance that has an effect on early stage cardiomyocytes or the cellular tissue-like structure is a myocardium-related drug.

[0011] According to the present invention, by overexpressing NCAM1 in iPS cell-derived cardiomyocytes, it is possible to obtain iPS cell-derived cardiomyocytes with high adhesive ability, and by using these to produce EHT, it is possible to further promote EHT organization and enhance EHT function.

[0012] FIG. 1 is a bar graph showing the results of evaluation by the adhesion ability evaluation assay in Test 4 of this Example. FIG. 2 is a bar graph showing the results measured by the propagation velocity evaluation in Test 5 of this Example. FIG. 3 is a line graph showing the measurement of intracellular calcium levels over time in Test 6 of this Example. FIG. 4 is a bar graph showing the results of sarcomere orientation in Test 8 of this Example. FIG. 5 is a box plot showing the results of sarcomere length in Test 8 of this Example. FIG. 6 is a bar graph showing the results of measuring contractile force in Test 9 of this Example. FIG. 7 is a graph showing the results of measuring the contraction and expansion times of EHT 26 days after preparation with overexpression of NCAM1, before drug addition and after addition of isoproterenol. FIG. 8 is a graph showing the results of measuring the contraction and expansion times of EHT 28 days after preparation with overexpression of NCAM1, before drug addition and after addition of isoproterenol and E-4031.

[0013] Hereinafter, cardiomyocytes, cellular tissue-like structures, methods for improving cardiomyocyte function, and methods for screening myocardium-related drugs according to the present invention will be described with reference to embodiments, although the present invention is not limited to the following embodiments.

[0014] (Cardiomyocytes) The cardiomyocytes of this embodiment are cardiomyocytes into which an expression vector for the NCAM1 protein has been introduced. In other words, the cardiomyocytes of this embodiment can be said to be those that are equipped with, have, or retain an expression vector for the NCAM1 protein.

[0015] An expression vector refers to a molecule containing a gene encoding a specific protein and used to introduce the genetic material into a cell, particularly an expression vector in which the entire vector containing the genetic material is composed of a nucleic acid molecule. The expression vector may further contain a sequence for promoting or identifying the expression of the protein. Cells into which an expression vector has been introduced preferably possess (are equipped with or retain) the gene in an expressible state. Here, "an expression vector being introduced into a cell" refers to the incorporation of the expression vector into the cell, particularly the incorporation of a gene contained in the expression vector into the cell. In the present invention, cells into which an expression vector has been introduced are sometimes referred to as cells transformed by the expression vector or transfected cells. Furthermore, cells into which a viral vector has been introduced as an expression vector are sometimes referred to as transduced cells.

[0016] The expression vector is not particularly limited as long as it does not impair the effects of the cardiomyocytes of this embodiment. For example, an expression vector for the NCAM1 protein described below can be suitably used.

[0017] The most appropriate conditions for the method of introducing the expression vector can be selected by taking into account the characteristics of the expression vector to be introduced and the target cardiomyocytes. The vector to be used can be selected appropriately taking into account the above conditions, but it is particularly preferable to select a viral vector that can be easily introduced into cells under a wide range of conditions, including living cells. For example, an adeno-associated virus (AAV) vector, a lentivirus vector, a retrovirus vector, or a ppiggybag vector can be used. For example, if an adeno-associated virus (AAV) is suitable as an expression vector for the NCAM1 protein described below, it can be introduced by contacting the target cardiomyocytes with the AAV under appropriate conditions. Furthermore, for example, if the protein is to be expressed at the stage of iPS cells, a ppiggybag vector can also be effectively used. Note that an example of a means of increasing NCAM1 expression is a method of increasing NCAM1 expression by adding hormones, compounds, etc. to cardiomyocytes.

[0018] In the cardiomyocytes of this embodiment, an expression vector for the NCAM1 protein has been introduced, and expression of the NCAM1 protein is induced by the gene contained in the vector. In this embodiment, cardiomyocytes are cells that form cardiac muscle tissue, and include not only ventricular myocardial cells and atrial myocardial cells, but also pacemaker cells that control the rhythm of heartbeats. More specifically, cardiomyocytes refer to cells that express at least one marker gene selected from the group consisting of cardiac troponin (cTNT), αMHC (α myosin heavy chain, MYH6), and βMHC (MYH7). The NCAM1 protein is a glycoprotein known to be involved in cell adhesion (human NCAM1, Homo sapiens neural cell adhesion molecule 1: NM_000615.7). The present inventors have discovered that expressing NCAM1 protein in cardiomyocytes can enhance cell adhesion ability, promote EHT organization, and provide mature myocardial tissue with functional maturation. In particular, cardiomyocytes overexpressing NCAM1 protein exhibit improved function compared to cells that do not express or express low levels of NCAM1 protein. Here, cardiomyocytes overexpressing NCAM1 protein refer to cardiomyocytes in which the expression level of NCAM1 protein, either in terms of protein amount or RNA amount, is increased compared to normal cardiomyocytes (WT: wild-type) that have not undergone expression induction or the like. Furthermore, the percentage increase in expression level refers to cardiomyocytes in which the expression level is increased by preferably 50% or more, more preferably 100% or more.

[0019] Specifically, the functions of mature myocardial tissue include phenomena such as increased cell-to-cell adhesion in structures primarily composed of NCAM1-expressing cardiomyocytes, the formation of dense tissue, improved pulsatile propagation velocity, improved calcium handling, and improved sarcomere orientation.

[0020] Here, the increase in cell-to-cell adhesion ability can be evaluated by an adhesion ability evaluation assay or the like. The adhesion ability evaluation assay can be performed by seeding and culturing target cells on a plate on which other cells have been cultured, treating the target cells with a cell separation solution, and then detecting the signal of the target cells remaining on the plate. Here, myoblasts or the like can be used as the other cells. The cell separation solution can be a known solution containing an enzyme with proteolytic activity. Examples of methods for detecting cell signals include immunostaining the cells and measuring the signal (e.g., fluorescence intensity) obtained.

[0021] The formation of a dense tissue can be evaluated, for example, by measuring the number of cells per unit area in a microscopic image.

[0022] The improvement in the pulsation propagation velocity can be evaluated by observing the cell movement over time, for example, by using a cell motion imaging system.

[0023] The improvement of calcium handling can be evaluated by quantifying the intracellular calcium concentration. Quantification of the calcium concentration is performed by measuring the intracellular calcium ion (Ca ２＋ ) The evaluation can be performed by measurement using a measurement reagent (for example, Fluo4 reagent).

[0024] The improvement in sarcomere orientation can be evaluated by observing the cultured tissue under a microscope and analyzing it using image processing software (for example, ImageJ).

[0025] The improvement in myocardial tissue function described above can be evaluated as an improvement when a significant difference is observed, such as when the results are improved compared to when a similar test is performed using normal cardiomyocytes (WT: wild-type) that have not been subjected to expression induction, etc. In the description of this embodiment, the improvement in myocardial tissue function may be collectively referred to as myocardial function improvement.

[0026] Any known expression vector for the NCAM1 protein can be used as long as it is capable of expressing a protein in cells. For example, a viral vector can be used. In this embodiment, it is preferable to use an adeno-associated virus (AAV) that expresses the human NCAM1 protein.

[0027] In this embodiment, the cardiomyocytes may be cells that form myocardial tissue, or cultured cells for artificially forming myocardial tissue may be used. Among these, the cardiomyocytes are preferably cells induced to differentiate from pluripotent stem cells. Furthermore, it is also preferable that the cardiomyocytes are cardiomyocytes induced to differentiate from iPS cells (iPS-derived cardiomyocytes). By using pluripotent stem cells or iPS-derived cardiomyocytes, artificial cardiomyocytes can be effectively produced, and this technology can promote the organization of artificial cardiomyocytes and mature their functions.

[0028] In this embodiment, the cardiomyocytes are also preferably used for cell transplantation therapy. Cell transplantation therapy refers to applications such as transplanting cells into an organ in the body and using them to treat the organ. The cardiomyocytes for cell transplantation therapy can be used to treat various heart diseases. Here, treatment refers to various treatments, and broadly includes medical procedures such as treatment, prevention, and recurrence prevention of disease. In addition to treating diseases, treatment also includes strengthening cardiac function. The treatment according to this embodiment can be used to treat humans and other animals, particularly mammals.

[0029] (Cellular tissue-like structure) The cellular tissue-like structure of this embodiment contains the cardiomyocytes described above. In view of the inclusion of the cardiomyocytes described above, the cellular tissue-like structure of this embodiment is in a form including cells expressing NCAM1 protein. Expression of NCAM1 protein in cardiomyocytes enhances the cell adhesion ability of cardiomyocytes, effectively achieving myocardial tissue-like organization, and therefore the cellular tissue structure of this embodiment achieves the same effect of improving myocardial function as the cardiomyocytes of this embodiment. The cardiomyocytes contained in the cellular tissue-like structure of this embodiment are excellent in physical properties such as cell adhesion, tissue density, and orientation. Furthermore, they are also excellent in myocardial functions such as myocardial pulsation propagation velocity, calcium handling, and contractile force. Therefore, the structure can be widely and effectively used as an artificial myocardial tissue-like structure.

[0030] The cell tissue-like structure of this embodiment may contain other components as appropriate in addition to the cardiomyocytes described above. For example, it may contain other cells, other chemical components, etc. These cells and chemical components can be selected from known ones that are effective in the process of cell culture or tissue formation, for example.

[0031] The cell tissue-like structure of this embodiment is also preferably a sheet composed of the cardiomyocytes. A sheet mainly refers to a membrane-like or plate-like structure, and its shape and size can be selected appropriately. As described above, the sheet of this embodiment may contain components other than cardiomyocytes.

[0032] The cell tissue-like structure of this embodiment is also preferably a cardiac organoid or an artificial myocardial tissue (Engineered Heart Tissue, EHT) containing the cardiomyocytes. Any known cardiac organoid or EHT form can be used as appropriate. Since the cell tissue-like structure of this embodiment contains the myocardial tissue of this embodiment as described above, the cell tissue-like structure of this embodiment will have many of the mature functions of the cardiomyocytes, making it particularly suitable for use as a cardiac organoid or artificial myocardial tissue.

[0033] It is also preferable that the cell tissue-like structure of this embodiment has improved functions in one or more of cell adhesiveness, myocardial pulsation propagation velocity, calcium handling, tissue density, and contractile force. As described above, expression of NCAM1 in cardiomyocytes improves one of the effects of improving myocardial function in tissues primarily containing these cardiomyocytes, specifically, the aforementioned properties. The cell tissue-like structure of this embodiment preferably has improved two or more of the aforementioned properties, more preferably three or more, even more preferably four or more, and particularly preferably all of the aforementioned properties.

[0034] The cell tissue-like structure of this embodiment preferably has improved length and unidirectional orientation of the sarcomeres contained therein. Here, sarcomere is an assembly formed by the assembly of various molecules in tissues, etc., and refers to the filamentous structure in muscle tissue, etc. Here, it refers to the filamentous structure of muscle tissue in cardiac muscle. The cell tissue-like structure of this embodiment has improved length and unidirectional orientation of the sarcomeres contained therein, and therefore the contractile force of the sarcomeres also increases.

[0035] (Method for improving cardiomyocyte function) The method for improving cardiomyocyte function of this embodiment includes a step of inducing expression of NCAM1 protein in cardiomyocytes. As described above, by inducing expression of NCAM1 protein in cardiomyocytes, mature function as myocardial tissue can be obtained, and the effect of improving myocardial function can be achieved. As a means for inducing expression of NCAM1 protein in cardiomyocytes, an expression vector for NCAM1 protein may be introduced as described above. Other known means for expressing proteins may also be used.

[0036] The cardiomyocytes used in the method for improving cardiomyocyte function of this embodiment can be selected without limitation on their origin. For example, as described above, cells induced to differentiate from pluripotent stem cells may be used as the cardiomyocytes. Alternatively, iPS cell-derived cardiomyocytes may be used as the cardiomyocytes.

[0037] The method for improving cardiomyocyte function of this embodiment may further include a step of appropriately arranging the cardiomyocytes to form a cellular tissue-like structure. The cellular tissue-like structure may be a sheet formed by arranging cardiomyocytes, as described above. The cellular tissue-like structure may be a cardiac organoid or engineered heart tissue (EHT) containing cardiomyocytes, as described above.

[0038] (Method for Screening Myocardium-Related Drugs) The method for screening myocardium-related drugs of this embodiment involves administering a candidate substance to the cardiomyocytes or the cellular tissue-like structure, and determining that the substance has an effect on the cardiomyocytes or the cellular tissue-like structure as a myocardium-related drug. Here, the myocardium-related drug refers to a drug (component, compound, composition, etc.) that may have an effect on the cardiomyocytes or the cellular tissue-like structure. "Affecting" refers to both a drug that has a positive effect and a drug that has a negative effect. For example, a positive effect refers to a drug that has a therapeutic or preventive effect or improves a condition in treatment, and identifying a drug that has a positive effect allows it to be used in treatment, etc. A negative effect refers to a drug that worsens a condition or inhibits the positive effect, and identifying a drug that has a negative effect allows for the identification of a drug that should not be used in treatment, etc. The candidate substance is a drug to be screened for its effect, and can preferably be appropriately selected from drugs that may have an effect. Specifically, a candidate substance is administered to the cellular tissue-like structure, and if an effect on the morphology, structure, etc. of the cellular tissue-like structure is observed, the substance is determined to be a drug that may have an effect on cardiomyocytes, myocardial tissue, or myocardium. For example, if administration of a candidate substance results in a change in the contraction time or expansion time of the cell tissue-like structure compared to when the substance is not administered, the substance can be determined to be a substance (drug) that affects the contraction or expansion of the myocardium, respectively. Note that the above screening can also be performed after appropriately treating the cardiomyocytes or cell-like structures with a drug that imparts cardiotoxicity (e.g., doxorubicin, etc.).

[0039] (Other Aspects of the Present Embodiment) Furthermore, examples of other aspects of the present embodiment include the following. Another aspect of the present embodiment is an expression vector for NCAM1 protein for use in a method for improving the function of cardiomyocytes. Another aspect of the present embodiment is use of cardiomyocytes for producing a cellular tissue-like structure. Another aspect of the present embodiment is a cell transplantation therapy method using cardiomyocytes. Another aspect of the present embodiment is a method for treating heart disease using the cell transplantation therapy method.

[0040] Although the embodiment of the present invention has been described above, the present invention is not limited to the above embodiment and various modifications can be made.

[0041] Examples are shown below, but the present invention is not limited to these examples.

[0042] [Test 1: Cell Culture] iPS cells were used, 1390D4. iPS cells were cultured using a known method ("A novel efficient feeder-free culture system for the derivation of human induced pluripotent stem cells," Masato Nakagawa, et al., Scientific Reports, 2014, 4:3594) on a culture dish coated with iMatrix-511 (Nippi) using StemFit AK02N (Ajinomoto) medium.

[0043] Human cardiac fibroblasts (HCF) (Promo Cell) were cultured on culture dishes using Fibroblast Growth Medium 3 Kit (Promo Cell). Rat myoblasts (H9C2 cells) were cultured on culture dishes using DMEM (high glucose medium) supplemented with 10% FBS.

[0044] [Test 2: Cardiomyocyte Differentiation] To differentiate the iPS cells cultured in Test 1 into cardiomyocytes, 2 × 10 iPS cells obtained in Test 1 were plated in a low-adhesion 6-well plate. ６The cells were seeded at 1.5 mL / well and statically cultured at 37°C under 5% oxygen and 5% carbon dioxide conditions (day 0 of differentiation initiation). The culture medium used was StemPro-34 SFM (Thermo Fisher Science) supplemented with 2 mM L-glutamine (Invitrogen), 0.4 mM monothioglycerol (Sigma), 50 μg / mL ascorbic acid (Sigma), 150 μg / mL Transferrin (Wako), 10 μM Rock inhibitor (Y-27632), 2 ng / mL BMP4 (R&D Systems), and 0.5% Matrigel (Corning).

[0045] The next day (day 1 after the start of differentiation), 1.5 mL of StemPro-34 SFM supplemented with 2 mM L-glutamine, 0.4 mM monothioglycerol, 50 μg / mL ascorbic acid, 150 μg / mL Transferrin, 18 ng / mL BMP4, 5 ng / mL bFGF (R&D Systems), and 6 ng / mL Activin A (R&D Systems) was added, and the cells were statically cultured at 37°C under 5% oxygen and 5% carbon dioxide conditions.

[0046] Three days after seeding (day 3 of differentiation), the medium was replaced with 3 mL of StemPro-34 SFM (Thermo Fisher Science) supplemented with 2 mM L-glutamine, 0.4 mM monothioglycerol, 50 μg / mL ascorbic acid, 150 μg / mL Transferrin, 1 μM IWP-3 (Stemgent), 0.6 μM Dorsomorphin (Sigma-Aldrich), 5.4 μM SB431542 (Sigma-Aldrich), and 10 ng / mL VEGF (R&D Systems). The cells were then cultured at 37°C under 5% oxygen and 5% carbon dioxide conditions.

[0047] Six days after seeding (six days after the start of differentiation), the medium was replaced with 3 mL of StemPro-34 SFM (maintenance medium) supplemented with 2 mM L-glutamine, 0.4 mM monothioglycerol, 50 μg / mL ascorbic acid, 150 μg / mL transferrin, and 5 ng / mL VEGF (R&D Systems), and the cells were statically cultured at 37°C and 5% oxygen. Thereafter, the medium was replaced every 2-3 days, and from day 10 onwards, the cells were statically cultured at 37°C and 5% carbon dioxide.

[0048] [Test 3: Overexpression of NCAM1 using AAV] To remove non-cardiomyocytes, embryoid bodies obtained in Test 2 on day 13 or later after differentiation were statically cultured for 3 days at 37°C under 5% carbon dioxide in DMEM (glucose-, glutamic acid-, and phenol red-free) containing 4 mM lactate and 1 mM pyruvate. The medium was then replaced with 3 mL of maintenance medium, and the embryoid bodies were maintained at 37°C under 5% carbon dioxide.

[0049] To obtain singled iPS cell-derived cardiomyocytes, the purified embryoid bodies were incubated for 1 hour at 37°C in IMDM (Thermo Fisher Scientific) supplemented with 10-20 μg / mL Liberase™ (Sigma-Aldrich), and then the medium was replaced with TrypLE Select (Thermo Fisher Scientific) supplemented with 10 μg / mL DNase I. After incubation at 37°C for 15-30 minutes, IMDM was added, and singled iPS cell-derived cardiomyocytes were obtained by pipetting.

[0050] The singled iPS cell-derived cardiomyocytes were suspended with AAV expressing NCAM1 or control AAV at a multiplicity of infection of 200-1000, seeded onto fibronectin-coated plates, and cultured in maintenance medium.

[0051] [Test 4: Assay for evaluating adhesion ability] 1 to 2 × 10 H9C2 cells cultured in Test 1 were ４The cells were seeded at 1 cell / well in a 96-well plate and statically cultured in a DMEM (high glucose) medium supplemented with 10% FBS at 37°C under 5% carbon dioxide conditions until they became 100% confluent.

[0052] Three days after infection with the same AAV as in Experiment 3, the iPS cell-derived cardiomyocytes were incubated at 37°C for 10 to 30 minutes in Accumax and dissociated from the bottom of the plate. The resulting iPS cell-derived cardiomyocytes were collected in a volume of 0.5 x 10 ５ The cells were seeded onto H9C2 cells in a 10% FBS-supplemented DMEM high glucose medium at 37°C and 5% carbon dioxide for 3 hours, then washed three times with PBS to remove unadhered cells, followed by static culture at 37°C and 5% carbon dioxide.

[0053] After 2 days, the cells were washed three times with PBS, and 4% PFA was added and allowed to stand at room temperature for 15 minutes. To quantify the number of cells adhering to H9C2, immunostaining was performed using a mouse anti-human nuclear antibody as the primary antibody and the anti-mouse IgG antibody Alexa488 (Invitrogen) as the secondary antibody. The mean fluorescence intensity of Alexa488 was quantified using a plate reader (PerkinElmer).

[0054] Figure 1 is a bar graph showing the results of the adhesive ability evaluation assay in Test 4 for the control (Ctrl) and NCAM1-overexpressing iPS cell-derived cardiomyocytes obtained in Test 3. The vertical axis shows the relative values, with the fluorescence intensity of the anti-human nuclear antibody (hNuc intensity), which indicates the adhesive ability of the control, set at 1, and the graph shows the mean ± standard deviation. As shown in the figure, the fluorescence intensity of NCAM1-overexpressing iPS cell-derived cardiomyocytes was significantly increased compared to the control. These results demonstrate that NCAM1 overexpression increases the adhesive ability of cardiomyocytes.

[0055] [Test 5: Evaluation of propagation velocity] Three days after infection with AAV in Test 4, the iPS cell-derived cardiomyocytes were incubated in Accumax at 37°C for 10 to 30 minutes and dissociated from the bottom of the plate. ５Droplets were seeded onto fibronectin-coated 35 mm glass-bottom dishes (Matsunami Glass Industry) at 5 μL / cell and statically cultured in maintenance medium supplemented with 10 μM Rock inhibitor (Y27632) at 37°C and 5% CO2 for 2 hours, followed by static culture in 2 mL of maintenance medium supplemented with 10 μM Rock inhibitor (Y27632) at 37°C and 5% CO2.

[0056] The next day, the medium was replaced with a maintenance medium, and the cells were statically cultured at 37° C. and 5% carbon dioxide. After 4 to 6 days, images were taken using a cell motion imaging system SI8000 (SONY), and the propagation speed was quantified.

[0057] Figure 2 is a bar graph showing the results of measuring the propagation velocity of control (Ctrl) and NCAM1-overexpressing iPS cell-derived cardiomyocytes obtained in Test 3 by the propagation velocity evaluation in Test 5. The vertical axis represents propagation velocity (µm / sec), and the graph is shown as the mean ± standard deviation. As shown in the figure, the propagation velocity was significantly increased in NCAM1-overexpressing iPS cell-derived cardiomyocytes compared to the control. These results demonstrate that overexpression of NCAM1 increases the intercellular propagation velocity of cardiomyocytes.

[0058] [Test 6: Quantification of Intracellular Calcium Concentration] Three days after infection with AAV in Test 4, iPS cell-derived cardiomyocytes were incubated at 37°C for 10 to 30 minutes in Accumax and dissociated from the plate bottom. Subsequently, the cells were spotted onto a fibronectin-coated 35 mm glass-bottom dish (Matsunami Glass Industry) at 25,000 cells / 5 μL and statically cultured for 2 hours at 37°C and 5% carbon dioxide in maintenance medium supplemented with 10 μM Rock inhibitor (Y27632). Subsequently, the cells were statically cultured at 37°C and 5% carbon dioxide in maintenance medium supplemented with 2 mL of 10 μM Rock inhibitor (Y27632).

[0059] The next day, the medium was replaced with a maintenance medium, and the cells were statically cultured at 37°C and 5% carbon dioxide. After 5 days, the cells were cultured in modified Tyrode's solution [129 mM NaCl, 2.5 mM KCl, 1 mM MgCl ２, 30 mM D-glucose, 2 mM CaCl ２ After washing with 25 mM HEPES, the cells were incubated in modified Tyrode's solution containing 5 μM Fluo4 (DOJIN) for 30 minutes at 37°C and 5% carbon dioxide. The medium was then replaced with 2 mL of modified Tyrode's solution, and the dish was placed in an incubator fitted to a fluorescence microscope. After incubation at 37°C for 30 minutes, the time-dependent changes in Fluo4 fluorescence intensity were recorded under 1.5 Hz electrical stimulation. The resulting changes in fluorescence intensity over time were analyzed using Matlab (Mathworks).

[0060] Figure 3 is a line graph showing the time course of intracellular calcium levels measured in Test 6 for control (Ctrl) and iPS cell-derived cardiomyocytes overexpressing NCAM1 obtained in Test 3. The vertical axis shows the corrected value of Fluo4 fluorescence intensity ((F-F ０ ) / F ０ , F ０ : baseline fluorescence intensity, F: fluorescence intensity at the time of measurement), and the graph shows the average value. As shown in the figure, the cardiomyocytes derived from NCAM1-overexpressing iPS cells showed a significant increase in the fluorescence intensity (F-F) compared to the control. ０ ) / F ０ These results demonstrate that overexpression of NCAM1 increases the intracellular calcium level in cardiomyocytes over time.

[0061] [Test 7: Preparation of artificial cardiac tissue] The iPS cell-derived cardiomyocytes 3 days after infection with AAV in Test 4 were incubated in Accumax at 37°C for 10 to 30 minutes and detached from the bottom of the plate. ５ iPS cell-derived cardiomyocytes from cells and 1 x 10 ５The cells were mixed with HCF and then added to 100-150 μL of 10 μM Rock inhibitor (Y27632), 2.5-5 mg / mL fibrinogen (Sigma-Aldrich), 10% fetal bovine serum, 5-20 μg / mL aprotinin, 0.75-3 units thrombin, and 10% Geltrex™ LDEV-Free reduced growth factor basement membrane matrix (Invitrogen). The cells were suspended in a maintenance medium supplemented with erythrocytes (Bio-Rad Corporation), added to a cardiac tissue device (BS-X9606, manufactured by Sumitomo Bakelite), and subjected to static culture for 1-2 hours at 37°C under 5% carbon dioxide conditions.

[0062] Then, 1 mL of IMDM was added, and the myocardial tissue device with the attached cell suspension mass was removed from the mold plate and statically cultured in a 24-well plate containing maintenance medium supplemented with 10 μM Rock inhibitor (Y27632) at 37°C under 5% carbon dioxide conditions. The medium was replaced with maintenance medium every few days to maintain the culture.

[0063] [Test 8: Immunostaining of Artificial Heart Tissue] After tissue preparation in Test 7, EHTs on day 14 were fixed using 4% PFA and then immersed in PBS. Immunostaining was performed using mouse anti-NCAM1 antibody and rabbit anti-ACTN2 antibody as primary antibodies, and anti-mouse IgG antibody Alexa488 (Invitrogen), anti-rabbit IgG antibody Alexa546 (Invitrogen), and Hoechst 3342 as secondary antibodies. Sarcomere orientation was quantified using ImageJ. Sarcomere length was measured using ImageJ as the distance between the z-zones of sarcomeres stained with anti-ACTN2 antibody.

[0064] 4 is a bar graph showing the results of sarcomere orientation 14 days after production of control (Ctrl) and NCAM1-overexpressing EHTs obtained in Experiment 8. The vertical axis shows the relative value, with the sarcomere orientation of the control set at 1, and the graph shows the mean ± standard deviation.

[0065] Figure 5 is a box plot showing the results of sarcomere length 14 days after generation of control (Ctrl) and NCAM1-overexpressing EHTs obtained in Experiment 8. The vertical axis shows the distance between the z-bands of sarcomeres stained with anti-ACTN2 antibody, and the maximum, minimum, and quartiles are shown on the graph.

[0066] As shown in the figure, in the artificial cardiac tissue using cardiomyocytes derived from NCAM1-overexpressing iPS cells, both sarcomere orientation and length were significantly increased compared to the control. These results demonstrate that overexpression of NCAM1 increases both sarcomere orientation and length in cardiomyocytes.

[0067] [Test 9: Measurement of Contractile Force of Artificial Cardiac Tissue] EHTs on the 14th day after tissue preparation in Test 7 were statically cultured in Gay's Balanced Salt Solution (GBSS) at 37°C and 5% carbon dioxide for 30 minutes, and then videotaped under a microscope the contraction under 1.5 Hz electrical stimulation. The contractile force was calculated by measuring the distance traveled from the relaxation state of the tip of the strut of the myocardial tissue device to the contraction state using the following formula. The diameter of the EHT at the relaxation state was calculated from the obtained video, and the cross-sectional area was calculated assuming the EHT was a perfect circle.

[0068]

[0069] In the above formula, b is width (2 mm), h is thickness (0.5 mm), E is Young's modulus (3000 MPa), W is movement distance (mm), and l is length (11.3 mm).

[0070] 6 is a bar graph showing the results of measurement in Test 9 of the contractile force 14 days after the preparation of control (Ctrl) and NCAM1-overexpressing EHTs obtained in Test 8. The vertical axis represents the contractile force as a function of the cross-sectional area of ​​the EHT (mN / mm ２ ) and the graph shows the mean ± standard deviation.

[0071] As shown in the figure, the contractile force of the artificial cardiac tissue using cardiomyocytes derived from NCAM1-overexpressing iPS cells was significantly increased compared to the control. These results demonstrate that overexpression of NCAM1 increases the contractile force of cardiomyocyte tissue.

[0072] [Test 10: Evaluation of drug response of artificial cardiac tissue overexpressing NCAM1] After preparing artificial cardiac tissue overexpressing NCAM1 using the same method as in Test 7, the tissue was statically cultured for 2 days in a maintenance medium containing 5% FBS (StemPro-34 SFM supplemented with 2 mM L-glutamine, 0.4 mM monothioglycerol, 50 μg / mL ascorbic acid, 150 μg / mL transferrin, and 5 ng / mL VEGF) at 37°C under 5% carbon dioxide conditions. Thereafter, the tissue was statically cultured in the maintenance medium at 37°C under 5% carbon dioxide conditions for 26 days.

[0073] To evaluate the drug response to the contractile function of artificial cardiac tissue overexpressing NCAM1, first, for the purpose of acclimatizing to the reaction solution, the culture medium was replaced with Gay's Balanced Salt Solution (GBSS), and then statically cultured for 30 minutes or more at 37 ° C and 5% carbon dioxide conditions, and then the contraction state under 1.5 Hz electrical stimulation was videotaped under a microscope. Next, to evaluate the contractile function before the drug reaction, the medium was replaced with GBSS containing 0.1% (v / v), and then statically cultured for 30 minutes or more at 37 ° C and 5% carbon dioxide conditions, and then the contraction state under 1.5 Hz electrical stimulation was videotaped under a microscope. Next, the medium was replaced with GBSS containing 1 μM isoproterenol, and then statically cultured for 30 minutes or more at 37 ° C and 5% carbon dioxide conditions, and then the contraction state under 1.5 Hz electrical stimulation was videotaped under a microscope.

[0074] After the measurement, the artificial tissue was subjected to static culture in the maintenance medium at 37 ° C and 5% carbon dioxide for another 2 days. Subsequently, to confirm the reproducibility of drug responsiveness, a similar experiment was conducted to confirm the drug response to contractile function. First, to allow the cells to become accustomed to the reaction solution, the culture medium was replaced with GBSS, and then the cells were statically cultured at 37 ° C and 5% carbon dioxide for 30 minutes or more. Then, the contraction was videotaped under a microscope at 1.5 Hz electrical stimulation. Next, to evaluate the contractile function before drug response, the medium was replaced with GBSS containing 0.1% (v / v) DMSO, and the cells were statically cultured at 37 ° C and 5% carbon dioxide for 30 minutes or more. Then, the contraction was videotaped under a microscope at 1.5 Hz electrical stimulation. Next, the medium was replaced with GBSS containing 1 μM isoproterenol, and the cells were statically cultured at 37 ° C and 5% carbon dioxide for 30 minutes or more. Then, the contraction was videotaped under a microscope at 1.5 Hz electrical stimulation. To further evaluate the reactivity of other compounds, the medium was replaced with GBSS containing 5 μM E-4031, and the cells were statically cultured at 37°C and 5% carbon dioxide for 30 minutes or more. Then, the contraction was observed under a microscope with electrical stimulation at 1.5 Hz. Using the obtained video data, the contraction and expansion times were calculated from the change in the movement distance of the tip of the strut of the myocardial tissue device accompanying the contraction and expansion.

[0075] 7 is a graph showing the results of measuring the contraction and diastole times of EHTs 26 days after preparation with overexpression of NCAM1 before drug addition and after the addition of isoproterenol. In (a), the vertical axis represents contraction time, and in (b), the vertical axis represents diastole time. As shown in the figure, EHTs 26 days after preparation with overexpression of NCAM1 showed a shortened contraction and diastole time after the addition of isoproterenol compared to before the addition of the drug.

[0076] 8 is a graph showing the results of measuring the contraction and diastole times of EHTs 28 days after production that overexpressed NCAM1 before drug addition and after the addition of isoproterenol and E-4031. In (a), the vertical axis represents contraction time, and in (b), the vertical axis represents diastole time. In both (a) and (b), the horizontal axis represents the order of addition: before addition, after addition of isoproterenol, and after addition of E-4031, in the order of evaluation. As shown in the figure, in EHTs 28 days after production that overexpressed NCAM1, after the addition of isoproterenol, both the contraction and diastole times were shortened compared to before drug addition, and were further shortened compared to the results 26 days after production. Furthermore, after detecting the EHT response to isoproterenol addition, EHT with E-4031 added showed no significant change in contraction time, but prolonged diastolic time compared to EHT without isoproterenol addition. These results suggest that the artificial cardiac tissue overexpressing NCAM1 exhibited shortened contraction and diastolic time in response to isoproterenol. This indicates rapid contraction and diastolic time due to the action of the drug. Furthermore, prolonged diastolic time was observed in response to E-4031. This indicates that the drug action prolongs the QT interval in vivo. These findings demonstrate high responsiveness of the contractile function to drugs. Isoproterenol and E-4301 are both substances known to have myocardial effects, and these effects could be reproduced using EHT containing myocardium overexpressing NCAM1 used in this test example. This demonstrates that the EHT of this test example can be used in screening methods for substances that affect myocardium.

[0077] Although several embodiments of the present invention have been described above, these embodiments are presented as examples and are not intended to limit the scope of the invention. These embodiments can be implemented in various other forms, and various omissions, substitutions, and modifications can be made without departing from the spirit of the invention. These embodiments and their modifications are included within the scope and spirit of the invention, as well as within the scope of the invention and its equivalents as defined in the claims.

[0078] According to the present invention, it is possible to obtain iPS cell-derived cardiomyocytes with improved adhesion ability. Furthermore, by using these cells, it is possible to enhance the organization of artificial tissues such as EHT and organoids. The present invention can contribute to the development of physiological, pharmacological, and toxicological analysis and screening technologies for the purposes of regenerative medicine, the construction of cardiac disease models, and the development of therapeutic drugs for these diseases.

Claims

1. Cardiomyocytes into which an expression vector for NCAM1 protein has been introduced.

2. The cardiomyocytes according to claim 1, wherein the cardiomyocytes are cells induced to differentiate from pluripotent stem cells.

3. The cardiomyocytes according to claim 1 or 2, wherein the cardiomyocytes are cardiomyocytes induced to differentiate from iPS cells.

4. Cardiomyocytes according to claim 1 or 2, for use in cell transplantation therapy.

5. A cellular tissue-like structure comprising the cardiomyocytes according to claim 1 or 2.

6. The cellular tissue-like structure according to claim 5, which is a sheet composed of the cardiomyocytes.

7. The cell tissue-like structure according to claim 5, which is a cardiac organoid or engineered heart tissue (EHT) comprising the cardiomyocytes.

8. The cell tissue-like structure according to claim 5, which has improved functions in one or more of cell adhesiveness, propagation velocity of cardiac muscle beating, calcium handling, tissue density, and contractile force.

9. A cell tissue-like structure according to claim 5, in which the length of the sarcomeres and the orientation of the sarcomeres in one direction are improved.

10. A method for improving the function of cardiomyocytes, comprising the step of inducing expression of NCAM1 protein in cardiomyocytes.

11. The method for improving cardiomyocyte function according to claim 10, wherein the cardiomyocytes are cells induced to differentiate from pluripotent stem cells.

12. The method for improving cardiomyocyte function according to claim 10 or 11, wherein the cardiomyocytes are iPS cell-derived cardiomyocytes.

13. The method for improving cardiomyocyte function according to claim 10 or 11, further comprising the step of arranging the cardiomyocytes to form a cellular tissue-like structure.

14. The method for improving cardiomyocyte function according to claim 13, wherein the cellular tissue-like structure is a sheet in which the cardiomyocytes are arranged.

15. The method for improving cardiomyocyte function according to claim 13, wherein the cellular tissue-like structure is a cardiac organoid or engineered heart tissue (EHT) containing the cardiomyocytes.

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