CN113924359A - Method for promoting stem cells to differentiate into mature myocardial cells through tomato hypokaline treatment - Google Patents

Method for promoting stem cells to differentiate into mature myocardial cells through tomato hypokaline treatment Download PDF

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CN113924359A
CN113924359A CN202080040203.7A CN202080040203A CN113924359A CN 113924359 A CN113924359 A CN 113924359A CN 202080040203 A CN202080040203 A CN 202080040203A CN 113924359 A CN113924359 A CN 113924359A
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cardiomyocytes
stem cells
solanesoximine
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金载皓
金艺瑟
尹停媛
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Hessel Technology Co ltd
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Abstract

The invention relates to a method for promoting differentiation and maturation of stem cells into myocardial cells by treating the stem cells with solanesoximine. The present invention improves the efficiency of cardiomyocyte differentiation by treating stem cells with solanesol during the differentiation of stem cells into cardiomyocytes, and exhibits the following remarkable effects: cardiomyocytes produced by the treatment with solanesoximine are similar in structure and function to mature human cardiomyocytes. Therefore, the present invention is expected to be effectively applied to an evaluation platform for developing a cell therapeutic agent for treating myocardial infarction or a drug for testing cardiotoxicity or treating heart disease.

Description

Method for promoting stem cells to differentiate into mature myocardial cells through tomato hypokaline treatment
Technical Field
The invention relates to a method for promoting differentiation and maturation of stem cells into myocardial cells by treating the stem cells with solanesoximine.
Background
Due to the limitation of the difficulty in obtaining human cardiac myocytes, development of new drugs for cardiovascular diseases and toxicity tests are mainly performed using cells derived from experimental animals such as mice and rats. However, the results of pharmacodynamic and toxicity tests of new drugs in animal cells and animal models may differ from those of human cardiomyocytes due to species-to-species differences. Therefore, various methods for differentiating stem cells including human embryonic stem cells and induced pluripotent stem cells into cardiomyocytes have been studied for the last 10 years. Differentiation of stem cells into functional cardiomyocytes provides a platform for regenerative medicine, developmental research, tissue engineering, disease model establishment, and drug toxicity assessment.
The differentiated cardiomyocytes repeatedly contract and relax at the same time, express the cardiac sarcomere protein, have the action potential of the cardiomyocytes, and express Ca2+Transient increases and decreases in concentration (calcium transients). It is well known that differentiated cardiomyocytes exhibit similar function during development as cardiomyocytes due to contractile and diastolic action and neurohormonal induced signaling.
However, these differentiated cardiomyocytes were still immature, had lower contractile and diastolic function than adult cardiomyocytes, and showed morphologically inappropriate protein expression patterns. The use of differentiated cardiomyocytes for the treatment of diseases or the research and development of new drugs is limited. Therefore, a step of separately maturing the differentiated cardiomyocytes is required. Representative recent studies associated with this report that when cardiomyocytes derived from human pluripotent stem cells are treated with growth hormone T3 (triiodo-L-thyronine) which is most suitable for cardiac leaf formation, both the size of the cardiomyocytes and the length of the sarcomere increase and the expression of cardiac structural proteins increases.
On the other hand, tomato hypo (tomatidine) belongs to the family of steroidal alkaloids, which are produced by chemical degradation of α -tomatidine (α -tomatine) produced from two molecules of glucose (D-glucose). These steroidal alkaloid substances are mainly present in the plant, animal and fungal kingdoms. In recent studies of skeletal muscle atrophy, it was reported that solanesoximine increases mTORC1 signaling to reduce skeletal muscle atrophy and can increase skeletal muscle strength and motor capacity, and can improve longevity and health through selective mitochondrial autophagy (mitophage) through SKN-1/Nrf2 signaling pathway in c. However, there has been no report on the effect of treating stem cells with solanesoximine on improving the structure and function of cardiomyocytes.
Disclosure of Invention
Technical problem
The present inventors attempted to differentiate stem cells into cardiomyocytes having a structure and function close to that of adult cardiomyocytes. The results confirmed that the expression of proteins associated with ion active channels of cardiomyocytes increased after treatment with solanesoximine, thereby effectively promoting the maturation of cardiomyocytes. Based on this, the present inventors have completed the present invention.
Accordingly, it is an object of the present invention to provide a composition for inducing differentiation from stem cells into cardiomyocytes or inducing maturation of differentiated cardiomyocytes, comprising solanesoximine, and a method for inducing differentiation from stem cells into cardiomyocytes or inducing maturation of differentiated cardiomyocytes using the same.
Solution scheme
In order to achieve the above objects, the present invention provides a composition for inducing differentiation from stem cells into cardiomyocytes or inducing maturation of differentiated cardiomyocytes, the composition comprising solanesoximine.
In addition, the present invention provides a medium composition for inducing differentiation from stem cells into cardiomyocytes or inducing maturation of differentiated cardiomyocytes, the medium composition comprising the composition.
In addition, the present invention provides a method for inducing differentiation from stem cells into cardiomyocytes or inducing maturation of differentiated cardiomyocytes, the method comprising culturing stem cells or differentiated cardiomyocytes in a medium comprising solanesoximine.
Further, the present invention provides a cell therapeutic agent for preventing or treating heart disease, which comprises the differentiated mature cardiomyocytes.
Effects of the invention
The present invention improves the efficiency of cardiomyocyte differentiation by treating stem cells with solanesol during the differentiation of stem cells into cardiomyocytes, and exhibits the following remarkable effects: cardiomyocytes produced by the treatment with solanesoximine are similar in structure and function to mature human cardiomyocytes. Therefore, the present invention is expected to be effectively applied to an evaluation platform for developing a cell therapeutic agent for treating myocardial infarction or a drug for testing cardiotoxicity or treating heart disease.
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FIG. 1 is a schematic representation of an experimental protocol for the differentiation or maturation of human embryonic stem cells into cardiomyocytes using solanesoximine;
FIG. 2 shows the results obtained by confirming the effect of the concentration of solasonine on the differentiation from human embryonic stem cells into cardiomyocytes (one-way analysis of variance;. p value < 0.05,. p value < 0.01,. p value < 0.005);
figure 3 shows the results obtained when treated with 1 μ M solanesoximine by: (A) analyzing by flow cytometry; and (B) Western blotting to analyze the expression level of each cardiomyocyte marker and confirm the results obtained from the efficiency of the differentiation of human embryonic stem cells into cardiomyocytes based on the treatment with solanesoximine;
figure 4 shows the results obtained when treated with 1 μ M solanesoximine by: (A) an optical microscope; and (B) results obtained by confocal laser microscopy comparing cell sizes (student t test;. p value < 0.01);
fig. 5 shows: (A) changes in expression of genes encoding sarcoskeletal proteins (MYL2(MLC2v), MYL7(MLC2a), MYH7(β myosin heavy chain, β MHC)), MYH6(α myosin heavy chain, α MHC)), TNN I type 1 and TNN I type 3) in cardiomyocytes treated with 1 μ M solanine were confirmed by western blotting by harvesting cardiomyocytes differentiated from human embryonic stem cells on each day of differentiation (14 days, 30 days and 60 days); and (B) the results obtained by quantifying them (unpaired student t test;. p value < 0.05,. p value < 0.01,. p value < 0.005);
fig. 6 shows the results obtained by analyzing the expression level of cTnI shown in mature cardiomyocytes on day 30 after differentiation using flow cytometry;
FIG. 7 shows the expression level of disease-associated genes (NPPA) in cardiomyocytes differentiated by treatment with solanesoximine (unpaired student's t-test;. p-value < 0.005);
fig. 8 shows the results obtained by staining with BIN1 antibody and comparing and confirming the expression of BIN1, which is a component of t tubules (transverse tubules), in cardiomyocytes differentiated by treatment with solanesoximine using confocal laser microscopy;
fig. 9 shows: (A) confirming the expression level of BIN1 and JPH2 constituting t tubules (transverse tubules) in cardiomyocytes differentiated by treatment with solanesol by western blotting; and (B) the results obtained by quantifying them (unpaired student t test;. p value < 0.05,. p value < 0.01,. p value < 0.005);
FIG. 10 shows the results obtained by confirming the arrangement of t tubules (transverse tubules) in the tomato hypoline-treated differentiated cardiomyocytes by Di-8-ANEPPS staining;
FIG. 11 shows the results obtained by quantifying the density of t tubules (transverse tubules) in the cardiac myocytes differentiated by the treatment with solasonine (unpaired student t-test;. p values < 0.05,. p values < 0.01,. p values < 0.005);
FIG. 12 shows the results obtained by performing a multi-electrode array assay to confirm whether the differentiated cardiomyocytes treated with solasonine are mature (student's t-test;. p values < 0.05,. p values < 0.01,. p values < 0.005).
Detailed Description
The present invention provides a composition for inducing differentiation from stem cells into cardiomyocytes and/or inducing maturation of differentiated cardiomyocytes, the composition comprising solanesoximine.
The present invention will be described in detail below.
Terms not otherwise defined herein have meanings commonly used in the art to which the present invention pertains.
In the present invention, "tomato hypoline (tomato hypoline)" may be a compound represented by 5 α,20 β (F),22 β 1(F),25 β 0(F),27-azaspirostan-3 β 2-ol, { tomato hypoline, (3 β 4,5 β 3,22 β 6,25R) -isomer }, 77-59-8, Tomatidin, UNII-2B73S48786, (3 β 5,5 β 8,22 β 7,25S) -spirostan-3-ol, EINECS 201-040-3, NSC 27592, NSC 226903, CHEBI: 9629, 5 α -Tomatodan-3 β 9-ol, (22S,25S) -5 α 1-spironolan-3 α 0-ol, 2B73S48786, { spironolan-3-ol, (3 α 2,5 α 3,22 α 5,25S) - }, SMR001233234, 5 α 4-Tomatodan-3 β -ol (8CI), SR-05000002324, (3 β,5 α,25S) -spironolan-3-ol, Prestwick _473, Prestwick3_000573, BSPBIo _000386, MLS002153880, MLS002222215, SCHEMBIML L335093, AC1L23J5, BPS 0025 _000426, CHEMBBL 2165711, DTXSID0037102, HMS 2146D 08, N-N639, ZI MBM 81640, NCZ 438182, BP-588294, BP _000426, CHEMBBL 2165711, DTXSID0037102, HMS 2146D 08, NCSANJO-N639, NCSAC 438182, NCHRONO-3, SAC 10823, SAC-3-O-3-O-H-O-H-O-H-O-H-O-H-O-H-H, c27H45NO2HCl), etc., characterized by having the following structural formula.
Figure BDA0003382090220000031
The solanesoximine is a substance based on steroid alkaloid, and is a compound formed by chemically degrading two molecules of D-glucose, one molecule of D-xylose and one molecule of D-galactose of the tomatidine extracted from tomatoes. Solanesoximine is normally present in immature green tomatoes.
In the present invention, the solanesoximine may be characterized in that it is contained at a concentration of 0.01 μ M to 100 μ M. When treated in a differentiation medium, solanesoximine can be obtained by treatment in methanol (CH)3OH) chloroform (CHCl)3) The compound (1: 1) can be used by dissolving in DMSO ((CH): 1)3)2SO) or methanol. In one embodiment of the invention, methanol (CH) is dissolved at a concentration of 10mM to 50mM3OH) chloroform (CHCl)3) Solanine in a mixed solution of 1:1 was prepared diluted to 1mM in DMSO and used for experiments at a final cell treatment concentration of 1 μ M or 0.1% in cell culture broth.
In the present specification, the term "stem cell" is a cell that forms the basis of a cell or tissue of a subject, and refers to a cell having pluripotency, which is characterized in that it can self-renew through repeated division and can differentiate into cells having a specific function according to circumstances.
In the present invention, stem cells can be classified into pluripotent stem cells, multipotent stem cells and unipotent stem cells according to their differentiation ability. Pluripotent stem cells are pluripotent cells having the potential to differentiate into all cells (totipotency), and include Embryonic Stem Cells (ESC), induced pluripotent stem cells (iPS), and the like. In the present invention, the stem cell may be a pluripotent stem cell, and may preferably be a human pluripotent stem cell, but is not limited thereto. In one embodiment of the present invention, a method of differentiating or maturing human embryonic stem cells from among stem cells into cardiomyocytes is disclosed.
In the present specification, the term "cardiomyocyte" includes, but is not limited to, cardiomyocyte precursor cells having the ability to become functional cardiomyocytes in the future, or fetal cardiomyocytes, cells of all differentiation stages of adult cardiomyocytes, which means cells that can be identified by at least one, preferably multiple, markers or criteria by at least one, preferably multiple, of the methods described below. The expression of various markers specific to cardiomyocytes can be detected by known biochemical or immunochemical methods, and these methods can be used without limitation. In this method, a polyclonal antibody or a monoclonal antibody specific for a marker that binds to a cardiac muscle precursor cell or a cardiac muscle cell can be used. Without limitation, antibodies targeting a single specific marker may be commercially available or prepared by known methods. Examples of cardiac muscle precursor cells or cardiac muscle cell-specific markers may include aSA (α -sarcomere actin), myosin heavy/light chain (MHC/MLC), cTnT (cardiac troponin-T), MLC2a (atrial myosin light chain-2), MLC2v (ventricular myosin light chain-2), MYH7(β myosin heavy chain, β MHC), MYH6(α myosin heavy chain, α MHC), TNN I type 1, TNN I type 3, ANP, GATA4, nkx2.5, and MEF-2c, and the like.
The present invention is technically characterized in that the solanesoximine is used for improving the differentiation rate of human pluripotent stem cells, such as stem cells, particularly human embryonic stem cells or induced pluripotent stem cells, into myocardial cells, promoting the differentiation from the human embryonic stem cells or induced pluripotent stem cells into mature myocardial cells, and increasing the expression of the muscle skeleton system of the differentiated myocardial cells, the structural protein of t tubules (transverse tubules) and genes thereof.
Specifically, the solanesoximine is characterized in that it can increase the expression level of at least one gene or protein selected from the group consisting of: aSA (α -sarcomeric actin), cTnT (cardiac troponin-T), MLC2a (atrial myosin light chain-2 or MYL7), MLC2v (ventricular myosin light chain-2 or MYL2), cTnI (cardiac troponin 1), MYH7(β myosin heavy chain), MYH6(α myosin heavy chain), JPH2 (catenin-2), and BIN1 (bridging integrin 1).
Furthermore, solanesoximine is characterized in that it reduces the expression level of NPPA (a-type natriuretic peptide precursor) gene or protein in differentiated cardiomyocytes.
Furthermore, solanesoximine is characterized in that it aligns the arrangement of transverse tubules (t tubules) in differentiated cardiomyocytes in one direction.
The expression of cardiomyocyte-specific markers can be confirmed by conventional and commonly used molecular biological methods for amplifying, detecting and analyzing mRNA encoding any marker protein, such as reverse transcriptase-mediated polymerase chain reaction (RT-PCR) or hybridization analysis, but is not limited to a specific method. Nucleic acid sequences encoding cardiomyocyte-specific marker proteins are known, available from public databases such as GenBank, and the marker-specific sequences required for use as primers or probes can be readily determined.
In addition, in order to confirm whether the differentiated cardiomyocytes are mature, immunofluorescent staining may be performed on t tubules (transverse tubules) in the cardiomyocytes, or electrophysiological criteria may be additionally used.
In addition, the present invention provides a medium composition for inducing differentiation from stem cells into cardiomyocytes or inducing maturation of differentiated cardiomyocytes, the medium composition comprising the composition.
In the present invention, the medium composition may further comprise CHIR99021(6- [ [2- [ [4- (2, 4-dichlorophenyl) -5- (5-methyl-1H-imidazol-2-yl) -2-pyrimidinyl ] amino ] ethyl ] amino ] -3-pyridinecarbonitrile) and IWP2(N- (6-methyl-2-benzothiazolyl) -2- [ (3,4,6, 7-tetrahydro-4-oxo-3-phenylthieno [3,2-d ] pyrimidin-2-yl) thio ] -acetamide).
On the other hand, in the present invention, CHIR99021 acts as a Glycogen Synthase Kinase (GSK) inhibitor/Wnt signaling inducer. It will be apparent to those of ordinary skill in the art that, in addition to CHIR99021, all GSK inhibitors involved in GSK signaling including 1-AKP (1-azakenpaullon) may fall within the scope of the present invention without limitation.
In addition, in the present invention, IWP2 is used as a Wnt signaling inhibitor. It will be apparent to those of ordinary skill in the art that all Wnt signaling inhibitors other than IWP2 may fall within the scope of the present invention without limitation.
Various inducers contained in the medium composition according to the present invention may be purchased or prepared from the market, and the effective concentration thereof may be adjusted according to factors known in the art, such as the kind of culture solution, the culture method, and the like. For example, CHIR99021 and IWP2 may be added to the medium at concentrations of 8. mu.M to 16. mu.M and 1. mu.M to 10. mu.M, respectively, and in one embodiment of the present invention, CHIR99021 and IWP2 are added at concentrations of 10. mu.M to 14. mu.M and 5. mu.M, respectively, but are not limited thereto.
In the present invention, the medium composition includes all medium culture liquids generally used for culturing stem cells in the art to which the present invention pertains. The culture solution used for the cultivation generally includes a carbon source, a nitrogen source and trace element components.
In addition, the present invention provides a method for inducing differentiation from stem cells into cardiomyocytes or inducing maturation of differentiated cardiomyocytes, the method comprising culturing stem cells or differentiated cardiomyocytes in a medium comprising solanesoximine.
In the present invention, before culturing the stem cells or differentiated cardiomyocytes in a medium comprising solanesoximine, the method may further comprise the steps of: (a) culturing the stem cells in a medium containing a supplement of B27 supplemented with CHIR99021 from which insulin is removed for 20 to 30 hours to differentiate into cardiomyocytes; (b) culturing the differentiated cardiomyocytes in a medium without CHIR99021 for 1 to 3 days; and (c) culturing the cultured cardiomyocytes in a medium supplemented with IWP2 for 1 to 3 days. Preferably, before culturing the stem cells or differentiated cardiomyocytes in a medium comprising solanesoximine, the method may further comprise the steps of: (a) culturing stem cells in a medium containing supplement of B27 supplemented with CHIR99021 and with insulin removed for 24 hours to differentiate into cardiomyocytes; (b) culturing the differentiated cardiomyocytes in a medium without CHIR99021 for 2 days; and (c) culturing the cultured cardiomyocytes in a medium supplemented with IWP2 for 2 days.
In the present invention, the method may further include: the differentiated cardiomyocytes were purified by maturation of the differentiated cardiomyocytes by treatment with solanesol, followed by their culture in medium (glucose-, lactate) excluding glucose and supplemented with lactate. The purification is characterized in that the medium does not contain solanesoximine.
In addition, the method may further include: solanesoximine was added to RPMI1640 medium (RBin) containing B27 supplement depleted of insulin and cultured after purification of differentiated cardiomyocytes.
In one embodiment of the present invention, on day 20 after the differentiation of human embryonic stem cells was started according to CHIR99021, human embryonic stem cells were cultured in a medium (glucose-, lactate) excluding glucose and to which lactate was added for 4 days, the cells differentiated into cardiomyocytes were purified, solanesoximine was further added to RPMI1640 medium (RBin) containing B27 additive from which insulin was removed, and the cells were continuously cultured for 6 days to prepare mature differentiated cardiomyocytes.
Further, the present invention provides a cell therapeutic agent for preventing or treating heart disease, which comprises cardiomyocytes differentiated into mature cells by the method according to the present invention.
In the present invention, the term "cell therapeutic agent" refers to cells and tissues prepared by isolation, culture and special manipulation from humans, which are drugs for therapeutic, diagnostic and prophylactic purposes (FDA regulation in the united states), and refers to drugs for therapeutic, diagnostic and prophylactic purposes by a series of actions, such as proliferation in vitro and selection of viable autologous, allogeneic or xenogeneic cells or otherwise altering the biological properties of cells to restore the function of cells or tissues.
The term "prevention" as used herein refers to any effect of inhibiting or delaying the progression of a cardiac disease by administering a cell therapeutic according to the invention.
As used herein, the term "treatment" refers to any effect that ameliorates or beneficially alters a cardiac disease by administration of a cellular therapeutic agent according to the invention.
In the present invention, the heart disease may be at least one selected from the group consisting of, but not limited to: myocardial infarction, angina pectoris, ischemic cardiomyopathy, primary cardiomyopathy, secondary cardiomyopathy, and heart failure.
In the present invention, the cardiomyocytes are characterized in that they mature to a degree very similar in morphology and function to adult cardiomyocytes, as compared to cardiomyocytes differentiated by conventional differentiation methods of solanesoximine treatment.
Hereinafter, the present application will be described in detail by examples. These examples are only for illustrating the present invention in more detail, and it is obvious to those skilled in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. Statistical significance was analyzed using a one-way analysis of variance (unpaired) student t-test, indicating a significance value with a p-value < 0.05, a significance value with a p-value < 0.01, and a significance value with a p-value < 0.005.
Modes for carrying out the invention
Example 1 Caryophylline treatment during differentiation of human embryonic Stem cells into cardiomyocytes
The following experiment was performed to obtain cardiomyocytes differentiated from human embryonic stem cells. Human embryonic stem cells H9(Wi institute) were maintained in mTeSR1 medium (stem cell technologies) for about 3 to 5 days and cultured (5% CO) in matrigel-coated 6-well plates (Nunc, Denmark)2At 37 ℃ C. When the cells were seeded on the plate, they were treated with 5 μ M ROCK inhibitor (Y27632) for 24 hours to increase cell viability.
Thereafter, when the plates were full of human embryonic stem cells, 10 μ M to 14 μ M of GSK inhibitor and Wnt signaling inducer CHIR99021(6- [ [2- [ [4- (2, 4-dichlorophenyl) -5- (5-methyl-1H-imidazol-2-yl) -2-pyrimidinyl ] amino ] ethyl ] amino ] -3-pyridinecarbonitrile) were added, and the cells were continuously cultured in rp1640 mi medium (RBin) containing insulin-removed B27 supplement for 24 hours. The following day, cells were cultured in RBin without GSK inhibitor (CHIR99021) for an additional 48 hours. Then, 5 μ M of IWP2(N- (6-methyl-2-benzothiazolyl) -2- [ (3,4,6, 7-tetrahydro-4-oxo-3-phenylthieno [3,2-d ] pyrimidin-2-yl) thio ] -acetamide) which inhibits the Wnt signaling pathway was added to RBin, and the cells were cultured for 48 hours, followed by addition of 1 μ M of solanesoximine to the culture broth. From day 8 of differentiation by treatment with GSK inhibitors, cells were cultured in RPMI1640 medium containing B27 supplement in the presence of 1 μ M of solanesoximine. On the 20 th day from the start of differentiation, the cells were cultured in a medium (glucose-, lactic acid) containing no glucose and to which 4mM lactate was added for 4 days, and the cells differentiated into cardiomyocytes were purified. At this time, the medium was changed every 48 hours, and the medium contained no solanesoximine. Thereafter, solanesoximine was added again to RPMI1640 medium (RB +) containing B27 supplement containing insulin, and the culture was continued until day 60 after differentiation. The experimental process is schematically shown in FIG. 1.
Example 2 confirmation of differentiation efficiency of differentiated cardiomyocytes
2-1 exploring concentration of solanesoximine to maximize differentiation efficiency
In order to find the optimum concentration of solanesoximine for maximizing the differentiation efficiency of cardiomyocytes differentiated from human embryonic stem cells, in the experimental protocol of example 1, cells were treated with solanesoximine at concentrations of 0.1 μ M, 0.5 μ M, 1 μ M, 2 μ M and 5 μ M, and the expression of cardiac skeletal protein aSA (α -sarcomere actin) in cardiomyocytes differentiated from human embryonic stem cells was analyzed by western blot on day 20 of differentiation. The results are shown in FIG. 2.
As shown in fig. 2, it was confirmed that the differentiation rate of cardiomyocytes was most significantly improved at a concentration of 1 μ M of solanesol.
2-2 analysis of expression patterns of Each cardiomyocyte marker
Next, western blotting and flow cytometry were performed to compare how the expression pattern of each cardiomyocyte marker is displayed differently in one cell. As markers for evaluating the ability to differentiate into cardiomyocytes, sarcomeric protein aSA involved in cardiomyocyte contraction and cTnT (cardiac troponin-T), MLC2a (atrial myosin light chain-2) and MLC2v (ventricular myosin light chain-2) which appear specifically in cardiomyocytes as structural proteins of cardiomyocytes were used, and each marker was measured in DMSO-treated + differentiated cardiomyocytes and solanesoximine (1 μ M) -treated + differentiated cardiomyocytes as control groups on day 20 of differentiation. The results are shown in FIG. 3.
As shown in fig. 3, it was confirmed that the expression ratio of the cardiomyocyte structural protein in the differentiated cardiomyocytes was significantly higher according to the 1 μ M solanesol treatment than the control group. This expression pattern was also confirmed by flow cytometry. The results above confirmed that the expression level of cardiomyocyte-specific protein was significantly increased by the treatment with solanesol, and that human embryonic stem cells were successfully differentiated into cardiomyocytes. In addition, it was confirmed that the concentration of solanesoximine maximizing the differentiation rate was 1. mu.M.
Example 3 study of Effect of tomato Hibiscine on promotion of cardiomyocyte maturation
3-1, confirmation of cardiomyocyte size
Cardiomyocytes differentiated from human embryonic stem cells were treated with lactic acid and placed on a 0.1% gelatin-coated coverslip, fixed with 4% Paraformaldehyde (PFA), washed 3 times with Phosphate Buffer (PBS), and blocked with 5% Bovine serum albumin (Bovine serum album, BSA; sigma Aldrich) for 30 minutes. Next, primary antibodies against aSA, cTnT and MLC2v (abcam) were incubated overnight at 4 ℃ and fluorescently labeled with secondary antibodies to which Alexa- fluro 488 or 647 was attached. Thereafter, for nuclear staining, immobilization was performed using vectashield (vector laboratories) containing DAPI (4', 6-diamidino-2-phenylindole). After fixation, immunofluorescent-stained cell samples were photographed at 600-fold magnification using a laser scanning confocal microscope (Olympus fluoroview FV 1000). The results are shown in FIG. 4.
As shown in fig. 4A, the cell size was significantly larger in the tomato hypoline-treated group compared to the control group, and by this morphological difference, it was confirmed that the differentiated cardiomyocyte maturation was induced by the treatment with tomato hypoline. Furthermore, as shown in fig. 4B, it was observed that the patterns of aSA, cTnT and MLC2v staining in the solanesoximine-treated group also showed a more pronounced trend compared to the control group. From the above results, it was confirmed that maturation of differentiated cardiomyocytes was promoted by treatment with solanesol.
3-2 confirmation of the expression level of the muscle skeleton structural protein in cardiomyocytes
The following experiment was performed to analyze whether there was a difference in the expression level of the proteins constituting the cardiomyocytes according to the duration of the tomato hypoline treatment. First, cardiomyocyte cells differentiated from human embryonic stem cells were collected on each differentiation day (14 days, 30 days, and 60 days), and the expression level of cardiomyocyte marker markers was identified by western blotting using antibodies. In addition, real-time PCR was performed to confirm that the expression of genes encoding sarcoskeletal proteins (MYL2(MLC2v), MYL7(MLC2a), MYH7(β myosin heavy chain, β MHC), MYH6(α myosin heavy chain, α MHC), TNN I type 1 and TNN I type 3) was increased in cardiomyocytes differentiated according to the tomato subalkali treatment. The results are shown in FIG. 5.
As shown in fig. 5, in the case of expressing cTnI (cardiac troponin 1) which differs depending on the degree of maturation of cardiomyocytes, it was confirmed that the expression increased with the increase of the time of the treatment with solanesoximine as compared with the control group. On the other hand, in the case of the myocardial skeletal-constituting proteins as a, cTnT and MLC2a, which were expressed at the early stage of differentiation of cardiomyocytes, it was confirmed that there was a difference in the expression level after 14 days from the start of differentiation, but there was no difference in the expression level of the remaining differentiated cardiomyocytes after purification with lactic acid (lactate selection). However, as the tomato hypoline treatment time increased, the expression of MYL2, MYL7, MYH7, MYH6, TNN I type 1 and TNN I type 3 increased.
Next, the expression level of cTnI at day 30 after differentiation was analyzed using a flow cytometer, and the results are shown in fig. 6.
As shown in fig. 6, it was confirmed that the expression of cTnI in the tomato-treated group was about 2-fold or more compared to the control group. From the above results, it was confirmed that the maturation of differentiated cardiomyocytes can be effectively induced after the treatment with solanesoximine.
In addition, real-time PCR was performed to confirm whether expression of a gene indicative of pathological disease (NPPA) was shown in differentiated cardiomyocytes treated with solanesoximine. The results are shown in FIG. 7.
As shown in fig. 7, it was confirmed that the expression of cardiac hypertrophy-associated gene NPPA (a type-a natriuretic peptide precursor) was significantly reduced in the tomato hypoline-treated group. From the above results, it was confirmed that solanesoximine does not induce myocardial hypertrophy by inducing the maturation of differentiated cardiomyocytes by inhibiting the expression of NPPA.
3-3 confirmation of the structure of the t tubule in cardiomyocytes
One of the organelles in cardiomyocytes, the t-tubule (transverse tubule), contains membrane microdomains rich in ion channels and signal molecules. Immunofluorescent staining of cells was performed to confirm that solanesoximine induced the formation of t tubules important for the contraction and relaxation of cardiomyocytes. Cells from day 60 of differentiation were plated on 0.1% gelatin-coated coverslips, cultured in medium mixed with RPMI1640 and 20% fetal bovine serum for 2 to 3 days, and then adapted in RPMI1640 medium (RB +) containing B27 supplement as a cardiomyocyte growth medium. Thereafter, cells were fixed with 4% paraformaldehyde, washed 3 times with PBS, and blocked with 5% bovine serum albumin for 30 minutes. To confirm the expression levels of BIN1 as a t-tubule component and a gene involved in calcium channel transport and aSA as a cardiac myocyte marker, BIN1(abcam) primary antibody was incubated at 4 ℃ for 12 hours and fluorescently labeled with a secondary antibody linked to Alexa- fluor 488, 647. Thereafter, for nuclear staining, fixation was performed using Vectashield containing DAPI. After fixation, the t tubule structural protein immunofluorescent stained cell samples were photographed at 600 x magnification using a laser scanning confocal microscope. The results are shown in FIG. 8.
In addition, among the t tubule proteins, in order to confirm the expression amounts of JPH2 (adiponectin-2) and BIN1 (bridging integrin 1) associated with plasma membrane L-type calcium channel and sarcoplasmic reticuline (ryanodine) receptor associated with calcium-induced calcium release, which physically link the t tubule membrane and sarcoplasmic omentum in muscle cells, western blotting was performed using JPH2 antibody and BIN1 antibody. The results are shown in FIG. 9.
As shown in fig. 8 and 9, it was confirmed that the expression of BIN1 and JPH2 in t-tubules, which are essential components of cardiomyocytes, was significantly increased according to the treatment with solasonine.
Next, the arrangement and density were confirmed by staining the differentiated cardiomyocytes with Di-8-ANEPPS (a fluorescent marker based on the movement of ions in the t-vial). Specifically, 2. mu.M Di-8-ANEPPS (thermo) was mixed with RB + as a growth medium for cardiomyocytes and incubated (20 min, 37 ℃). After the incubation was completed, the cardiomyocytes were washed twice with the medium without the reagent and then photographed at 450nm, 510nm and 570nm using a laser scanning confocal microscope. The imaging results were analyzed using the Image J program. The results are shown in fig. 10 and 11.
It is known that cardiomyocytes undergoing the maturation process have uniformly arranged t tubules, and the higher the density of t tubules, the more simultaneously contraction and relaxation of large areas of cardiomyocytes occur. As shown in fig. 10, it was confirmed that the t-tubule arrangement of the solanesoximine-treated group was significantly uniform compared to the control group. Furthermore, as shown in fig. 11, it was confirmed that the density in the area occupied by the t tubules was higher in the cardiomyocytes in the tomato subalkali-treated group compared to the control group.
3-4 confirmation of the electrophysiological Properties of the cardiomyocytes
A multi-electrode array assay was performed to measure conductivity, an electrophysiological property that occurs through contraction and relaxation of cardiomyocytes. Cardiomyocytes at day 60 of differentiation were washed twice with HBSS, then incubated with 0.05% trypsin-EDTA (3 min, 37 ℃) and inactivated with RPMI1640+ 20% FBS (R20) medium, before cell detachment by pipetting. Cells were cultured with R20 on fibronectin-coated multi-electrode array plates (48 h, 37 ℃). After that, the medium was changed to RB +, and then the conductivity was measured. The results are shown in FIG. 12.
As shown in fig. 12, it was confirmed that the cardiomyocytes differentiated by the treatment with solanesoximine strongly received signals inducing contraction by observing an increase in the mean value of the peak amplitudes. Significant increases in Beats Per Minute (BPM) and Field Potential Duration (FPD) were observed, confirming fast intracellular signaling rates, more regular electrophysiological movements, and rapid contraction and relaxation. On the other hand, the beat period average and beat period irregularity were confirmed to be significantly low.
From the above results, it was confirmed that when human pluripotent stem cells are treated with solanesoximine according to the present invention while differentiating into cardiomyocytes, the efficiency of cardiomyocyte differentiation is increased, and a significant effect, i.e., the resulting cardiomyocytes are similar in structure and function to mature human cardiomyocytes, is exhibited. Therefore, when mature cardiomyocytes produced using the present invention are used for new drug development and toxicity assessment, mature human cardiomyocytes that are very similar in morphology and function to adult cardiomyocytes are used, whereby it is expected that the enormous costs required for new drug development may be reduced and the validation period may be shortened, since it can not only address differences due to species differences, but also predict and assess the efficacy and toxicity of new drugs before clinical trials.
While specific details of the invention have been described above, it will be apparent to those skilled in the art that this detailed description is merely a preferred embodiment and that the scope of the invention is not limited thereto. Accordingly, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

Claims (11)

1. A composition for inducing differentiation from stem cells into cardiomyocytes or inducing maturation of differentiated cardiomyocytes, the composition comprising solanesoximine.
2. The composition of claim 1, wherein said solanesoximine is included at a concentration of 0.01 μ Μ to 100 μ Μ.
3. The composition of claim 1, wherein the solanesoximine increases the expression level of at least one gene or protein in differentiated cardiomyocytes selected from the group consisting of: aSA (α -sarcomeric actin), cTnT (cardiac troponin-T), MLC2a (atrial myosin light chain-2 or MYL7), MLC2v (ventricular myosin light chain-2 or MYL2), cTnI (cardiac troponin 1), MYH7(β myosin heavy chain), MYH6(α myosin heavy chain), JPH2 (catenin-2), and BIN1 (bridging integrin 1).
4. The composition of claim 1, wherein the solanesoximine reduces the expression level of an NPPA (a-type natriuretic peptide precursor) gene or protein in differentiated cardiomyocytes.
5. The composition of claim 1, wherein the solanesoximine aligns the arrangement of transverse tubules (t tubules) in differentiated cardiomyocytes in one orientation.
6. A medium composition for inducing differentiation from stem cells into cardiomyocytes or inducing maturation of differentiated cardiomyocytes, the medium composition comprising the composition of any one of claims 1 to 5.
7. The media composition of claim 6, wherein the media composition further comprises CHIR99021(6- [ [2- [ [4- (2, 4-dichlorophenyl) -5- (5-methyl-1H-imidazol-2-yl) -2-pyrimidinyl ] amino ] ethyl ] amino ] -3-pyridinecarbonitrile) and IWP2(N- (6-methyl-2-benzothiazolyl) -2- [ (3,4,6, 7-tetrahydro-4-oxo-3-phenylthieno [3,2-d ] pyrimidin-2-yl) thio ] -acetamide).
8. A method for inducing differentiation from stem cells into cardiomyocytes or inducing maturation of differentiated cardiomyocytes, comprising: culturing the stem cells or differentiated cardiomyocytes in a medium comprising solanesol.
9. The method of claim 8, wherein said solanesoximine is included at a concentration of 0.01 μ Μ to 100 μ Μ.
10. The method of claim 8, wherein said method further comprises the steps of, prior to culturing in a medium comprising solanesoximine:
(a) culturing the stem cells in a medium containing a supplement of B27 supplemented with CHIR99021 from which insulin is removed for 20 to 30 hours to differentiate into cardiomyocytes;
(b) culturing the differentiated cardiomyocytes in a medium without CHIR99021 for 1 to 3 days; and
(c) the cultured cardiomyocytes were cultured in the medium supplemented with IWP2 for 1 to 3 days.
11. A cell therapeutic agent for preventing or treating heart disease, comprising the cardiomyocytes differentiated into mature by the method of claim 8.
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