KR100943985B1 - Compound and its derivatives which have cardiomyocyte differentiating effect from stem cells - Google Patents

Abstract

The present invention relates to a compound and derivatives thereof used to induce differentiation from embryonic stem cells of a mammal into cardiomyocytes or to improve cardiac function, and comprising culturing stem cells in the presence of the compound or derivatives thereof. Method for inducing differentiation from cells to cardiomyocytes, stem cell differentiation induction medium composition comprising the compound or derivatives thereof, cardiomyocytes prepared by the differentiation induction method, the cardiomyocytes as an active ingredient To provide a pharmaceutical composition for treating heart disease, using the cardiomyocytes to screen for candidates for promoting the induction, differentiation, regeneration or survival of cardiomyocytes.

Embryonic Stem Cells, Cardiomyocytes, Differentiation Accelerators, Proliferation Accelerators, Cell Therapies, Cardiac Regenerative Therapies

Description

Compounds and derivatives that promote differentiation of stem cells into cardiomyocytes {Compound and its derivatives which have cardiomyocyte differentiating effect from stem cells}

The present invention relates to a compound and derivatives thereof used to induce differentiation from embryonic stem cells of a mammal into cardiomyocytes or to improve cardiac function, and more specifically, to culture stem cells in the presence of the compound or derivatives thereof. Method for inducing differentiation from stem cells to cardiomyocytes, comprising the composition for inducing stem cell differentiation comprising the compound or a derivative thereof, cardiomyocytes prepared by the differentiation induction method, the cardiomyocytes It relates to a pharmaceutical composition for treating heart disease, comprising as an active ingredient, a method for screening a candidate substance for promoting the induction, differentiation, regeneration or survival of cardiomyocytes using the cardiomyocytes.

Stem cells are capable of differentiating into a variety of cells and have self-proliferation ability. They are embryonic stem cells (ES cells) isolated from early embryos, and embryonic primitive germ cells. Three types of embryonic germ cells (EG cells) isolated from, and multipotent adult progenitor cells (MAPC cells) isolated from adult bone marrow are best known. Stem cells have been the subject of research as cell resources for functional recovery of various organs. The process of inducing differentiation from stem cells to desired cells can vary, and many studies have been conducted to determine the effects of cytokines, bioactive proteins, and simple compounds in order to induce differentiation into specific cells. ought.

On the other hand, cardiomyocytes actively divide and actively divide cells before birth, but they lose their division function immediately after birth and do not have undifferentiated progenitor cells, and thus are not affected by various stresses such as myocardial infarction and myocarditis. Exposed cardiac muscle cells die when exposed and are not replenished. As a result, the remaining cardiomyocytes try to maintain cardiac function due to the target hypertrophy, but when various stresses persist and exceed the allowable range, the cardiac failure occurs due to the decline and death of new cardiomyocytes, leading to deterioration of myocardial function. do.

In the past, as a therapeutic agent for heart failure, cardiac agents such as digitalis or xanthine, which increase the contractile force of the myocardium, have been used. However, when these drugs are administered for a long time, it is known that the condition of the disease is exacerbated by excessive consumption of myocardial energy. have. In addition, recently, treatment with beta-blockers or angiotensin converting enzyme (ACE) inhibitors to reduce excessive heart load caused by hyperactivity of the sympathetic nervous system or the renin-angiotensin system is mainly symptomatic. Is not a way to recover the heart tissue itself. Compared to these methods, heart transplantation is a fundamental treatment for severe heart failure, but there are problems such as lack of organ providers, medical ethics, and physical and economic burdens of patients, making it difficult to use heart transplantation as a general treatment.

Therefore, stem cells have received great attention as a source of cardiomyocytes for cell therapy in studies to improve or restore heart function due to various causes such as myocardial infarction. In particular, embryonic stem cells have been known to induce differentiation into cardiomyocytes by in vitro culture. In particular, animal experiments in which cardiac muscle cells derived from embryonic stem cells were transplanted into adult heart tissues were found to be highly effective, almost identical to those in embryonic myocardium (US Pat. No. 6,015,671; Klug et al. , J. Clin. Invest., 98: 216, 1996). It is also known to induce differentiation from human embryonic stem cells to cardiomyocytes (Kehat et al., J. Clin. Invest., 108: 407, 2001; Chunhui et al., Circ.Res., 91: 508, 2002; International Publication No. 03/06950), according to these studies, cardiomyocytes induced by differentiation from human embryonic stem cells not only have a function of autonomous beating but also myosin heavy / light chain, α-actinin And express myocardial specific proteins such as troponin and atrial natriuretic peptide (ANP) and myocardial specific transcription factors such as GATA-4, Nkx2.5 and MEF-2c. Observation and electrophysiological analysis confirmed that the immature cardiomyocytes of immature traits were maintained, and their use in regenerative medicine is expected.

However, embryonic bodies (EBs) formed from embryonic stem cells or germ germ cells by conventional methods are not only cardiomyocytes but also hematopoietic cells, vascular cells, nervous system cells, intestinal cells, bone and cartilage cells. There is a problem that occurs in a mixed form of cells. The efficiency of differentiating cardiomyocytes from stem cells is very low, and the rate of cardiomyocytes in these differentiated cells is only about 5 to 20% of the total. Therefore, increasing the efficiency of differentiation into cardiomyocytes is considered to be a very important electricity in the development of cell therapies for improving cardiac function.

Compounds of the present invention and derivatives thereof are the core precursors of porphyrin synthesis and are used in the synthesis of porphyrins. The compound of formula 1 is Bull. Korean Chem. Soc. (WS Cho et al., 1998, 19, 314), Tetrahedron (C. Lee and J. Lindsey, 1994, 50, 11427) and US Patent Publication No. 2005/038262, which is a cell differentiation. The physiological function of is not known at all. Its derivative, the compound of formula 2, is only known as an intermediate of porphyrin synthesis (J.-W. Ka, Tetrahedron Lett. 2000, 41, 4609).

As a result of efforts to develop cell therapies for improving heart function, the present inventors have found a novel use that the compounds represented by the formula (1) or derivatives thereof represented by the formula (2) have an excellent effect on inducing differentiation from stem cells to cardiomyocytes. The present invention has been completed by discovering.

One object of the present invention is to provide a method of inducing differentiation from stem cells to cardiomyocytes, comprising culturing stem cells in the presence of a compound represented by formula (1) or a derivative thereof represented by formula (2). will be.

Another object of the present invention is to provide a medium composition for inducing differentiation from stem cells to cardiomyocytes, comprising the compound or a derivative thereof.

Still another object of the present invention is to provide cardiomyocytes produced by the differentiation induction method.

Still another object of the present invention is to provide a composition for treating heart disease comprising the cardiomyocytes as an active ingredient.

Still another object of the present invention is to provide a kit for assaying a candidate substance that promotes induction, differentiation, regeneration, or survival of cardiomyocytes, including the cardiomyocytes.

Still another object of the present invention is to provide a method for screening candidate substances for promoting induction, differentiation, regeneration or survival of cardiomyocytes using the cardiomyocytes.

Thus, in one embodiment, the present invention comprises culturing stem cells in the presence of a compound represented by formula (1) or a derivative thereof represented by formula (2), the method of inducing differentiation from stem cells to cardiomyocytes It is about:

In the above formula, R ₁ Is selected from the group consisting of hydrogen, alkyl, alkoxy, hydroxy, halide, nitro, cyano group, alkoxycarbonyl, amino group, acetamido group and substituted or unsubstituted aryl group or heteroaryl group, and R2, R3 and R4 Is the same or different and is selected from the group consisting of hydrogen, alkyl, alkoxy, hydroxy, halide, nitro, cyano group and alkoxycarbonyl group, X is C, N, O or S.

As used herein, the term "stem cell" refers to a cell having the ability to differentiate into various cells and having self-proliferative capacity, embryonic stem cells (ES cells) isolated from early embryos, Three types of embryonic germ cells (EG cells) isolated from embryonic primitive germ cells and multipotent adult progenitor cells (MAPC cells) isolated from adult bone marrow are best known. Specific examples of mouse-derived embryonic stem cells include EB3 cells, E14 cells, D3 cells, CCE cells, R1 cells, 129SV cells and J1 cells. In addition, standard protocols already established for the preparation, passage and preservation of embryonic stem cells, embryonic germ cells and pluripotent adult stem cells (Matsui et al., Cell, 70: 841, 1992; Shamblott et al., Proc Natl.Acad. Sci. USA, 95: 13726, 1998; US Pat. No. 6,090,622; Jiang et al., Nature, 418: 41, 2002; International Publication No. 01/11011) to facilitate these stem cells. Can be used.

In addition, the cells usable in the present invention are not limited to the above three types, and include all pluripotent stem cells derived from mammalian embryos, fetuses, umbilical cord blood, or adult tissues such as adult organs or bone marrow, or blood. And include stem cells having traits similar to embryonic stem cells. In this case, the term "embryonic stem cell-like trait" means that there are surface (antigen) markers specific for embryonic stem cells, express embryonic stem cell-specific genes, or have teratoma-forming functions, Or it can be defined as cell biological properties specific to embryonic stem cells, such as chimeric mouse formation ability.

The method described in the present invention may be any cell that does not have a trait similar to embryonic stem cells or a pluripotent stem cell, but which has a property capable of differentiating into a cell having a trait of cardiomyocyte type in at least in vitro culture. Can be used for Examples of such cells include bone marrow mesenchymal stem cells derived from bone marrow cells (Bruder et al., US Pat. No. 5,736,396; pittenger et al., Science 284: 143, 1999) or CMG cells (Makino et al. , J. Clin. Invest., 103: 697, 1999; International Publication No. 01/048151) or Spoc cells derived from muscle tissue (International Publication No. 03/035382).

As used herein, the term "cardiomyocytes" is used in the same sense as "cardiomyocytes", cardiomyocyte progenitor cells, or fetal cardiomyocytes, having the ability to be functional cardiomyocytes in the future, It includes cells of all differentiation stages of adult cardiomyocytes. That is, the term "induction of differentiation from stem cells to cardiomyocytes" in the present invention refers to intermediate stages before completely differentiation from stem cells to cardiomyocytes as well as when differentiation is induced from stem cells to cardiomyocytes. In addition to the differentiation into cardiomyocyte progenitor cells formed in the present invention, the compounds of the present invention not only effectively achieve full differentiation of stem cells into cardiomyocyte cells but also are very large in differentiation from stem cells to cardiomyocyte progenitor cells. Indicates efficiency.

In order to confirm the differentiation from stem cells to cardiomyocytes, detection of markers specific for cardiomyocyte progenitor cells or cardiomyocytes can be used. Examples of markers specific for cardiomyocyte progenitor cells or cardiomyocytes include myosin heavy chain / light chain, α-actinine, troponin, ANP, GATA-4, Nkx2.5 and MEF-2c. The method for detecting the expression of various markers specific for such cardiomyocytes is not particularly limited, but amplification, detection, detection of mRNA encoding any marker protein by reverse transcriptase mediated polymerase chain reaction (RT-PCR) or hybridization assay It can be confirmed by the molecular biology method commonly used for interpretation. Nucleic acid sequences encoding marker proteins specific for myocardial progenitor cells or cardiomyocytes are already known and are available from public databases such as GenBank and facilitate marker specific sequences necessary for use as primers or probes. Can decide. More preferably, immunochemical methods such as immunohistochemical staining or immunoelectrophoresis can be used. In immunochemical methods, marker specific polyclonal antibodies or monoclonal antibodies that bind to cardiomyocyte progenitor cells or cardiomyocytes can be used, and commercially available antibodies can be used.

In addition, in order to confirm the differentiation from stem cells to cardiomyocytes, physiological criteria may be additionally used. In other words, the stem cell-derived cells can be a useful indicator, such as having an independent rhythm, expressing various ion channels and responding to electrophysiological stimulation.

In the present invention, the compound represented by the formula (1) or its derivatives represented by the formula (2), specifically include the following compounds represented by the formula (3) to (14). In addition, these derivatives include various pharmaceutically acceptable inorganic or organic salts.

The present invention is characterized in that the stem cells are cultured under the stimulation of the compound represented by the formula (1) or its derivatives represented by the formula (2), the stimulation method is not particularly limited, but preferably a method for adding the compound in the medium Can be mentioned. In addition, all the methods which show the same effect as the method of adding the said compound to a medium can be used.

More specifically, the stem cell differentiation induction method of the present invention can be largely carried out in three steps: (a) preparing a stem cell; (b) obtaining an embryoid body from stem cells; And (c) isolating differentiated cells from the embryos by culturing the embryos in a medium comprising a medium composition for inducing stem cell differentiation comprising the compound of the present invention.

The culture dish used in step (b) is preferably a bacterial grade culture dish without a functional group to which cells can be adhered for efficient formation of an embryo. Step (b) is generally carried out for a sufficient time for embryonic bodies to form, preferably 2 to 5 days.

In step (c), the compound of the present invention and another stem cell differentiation inducing agent can be treated together, and there is no time limit for each other in the treatment. That is, the compounds of the present invention can be treated before, simultaneously or after other differentiation inducing agents. Separation of individual cells from embryonic bodies, which are cell aggregates, can be carried out according to conventional methods known in the art. For example, it can be carried out by mechanical and enzymatic methods, and an enzymatic separation method using an enzyme such as trypsin is preferred. Step (c) is generally carried out for a sufficient time for the differentiated cells to form, preferably 3 to 15 days. On the other hand, in step (c), tissue culture dishes, multi-well plates or poly-L-lysine coating-glass coverslips which are commonly used in animal cell culture and capable of attachment of animal cells may be used.

In addition, as a medium used in the present invention, any one conventionally used for culturing stem cells may be used. For example, α-MEM medium containing serum, a buffer, an antioxidant, and the like may be used.

In order to confirm the effect of inducing stem cell differentiation of the compound of the present invention and its derivatives, twelve compounds (compounds of Formulas 3 to 14) belonging to the compound of the present invention were treated with KH2 cells, which are mouse embryonic stem cell lines, to treat α-myosin. Fluorescence of EGFP by heavy chains was confirmed, and the expression of cardiac muscle cell specific marker genes, Nkx2.5 and GATA4, MLC-2V and α-MHC, was confirmed by electrophoresis (FIGS. 1 and 2). In addition, it was confirmed that cells expressing EGFP (green fluorescence) were cardiomyocytes by immunohistochemical staining (FIG. 4). In addition, it was confirmed that the differentiation of stem cells in a concentration-dependent manner to the compound of the present invention (Fig. 5).

In addition, the embodiment of the present invention through the fluorescent immunostaining showing the rate of production of the heart muscle cells and the expression of cardiomyocyte markers, the compounds and derivatives of the present invention is excellent in not only mouse embryonic stem cells but also human embryonic stem cells It was confirmed that there is an induction effect (Figs. 7 and 8).

As another aspect, the present invention relates to a medium composition for inducing differentiation from stem cells to cardiomyocytes, comprising a compound represented by the formula (1) or a derivative thereof represented by the formula (2).

The composition of the present invention may include a compound represented by formula (1) or a derivative thereof represented by formula (2) alone as a stem cell differentiation inducing agent, or may further include other differentiation inducing agents, thereby synergizing between these differentiation inducing agents. Effect can be generated. Differentiation inducers that can be used in the composition of the present invention can be any known as the differentiation inducer of stem cells, and preferably include retinoic acid and dimethyl sulfoxide (DMSO).

Preferably, the composition of the present invention is included in the medium composition at a concentration of 0.1 μM to 100 μM.

As another aspect, the present invention relates to cardiomyocytes produced by the differentiation induction method.

Cardiomyocytes induced by differentiation from stem cells including embryonic stem cells by the differentiation induction method can be efficiently and in large quantities obtained high-quality cardiomyocytes by continuously recovering, isolating and purifying cells according to known methods. . The cardiomyocytes thus obtained are hereinafter referred to as cardiomyocytes produced by the present invention.

Purification of cardiac muscle cells can be applied to all known cell separation and purification methods, and specific examples thereof include antigen-antibody reactions such as flow cytometry, magnetic beads, or panning methods (Monoclonal Antibodies: principles and practice, Third). Edition (Acad. Press, 1993); Antibody Engineering: A Practical Approach (IRL Pressat Oxford University Press, 1996), cell differentiation by density gradient centrifugation using a carrier such as sucrose or Percoll. have. In addition, as a screening method for cardiomyocytes, artificially modifying genes of pluripotent stem cells, such as embryonic stem cells, which are the underlying cells, and imparting drug resistance or ectopic protein expression ability to recover cells having traits as cardiac muscle cells. How to do this.

As another aspect, the present invention relates to a pharmaceutical composition for treating heart disease comprising the cardiomyocytes as an active ingredient.

Diseases or conditions that can be treated by the pharmaceutical composition of the present invention include all heart diseases caused by damage to cardiomyocytes. Preferably, the heart disease includes myocardial infarction, ischemic heart disease, congestive heart failure, hypertrophic cardiomyopathy, dilated cardiomyopathy, myocarditis and chronic heart failure.

The pharmaceutical composition of the present invention may further comprise a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers are those commonly used in the preparation, such as lactose, dex, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose , Polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, and the like. In addition to the above components, the pharmaceutical composition of the present invention may further include a lubricant, a humectant, a sweetener, a flavoring agent, an emulsifier, a suspending agent and a preservative. Pharmaceutically acceptable carriers and formulations are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).

In addition, if the pharmaceutical composition of the present invention contains the cardiomyocyte cells produced by the present invention in high purity, the cells are suspended in an aqueous carrier such as a medium, and the cells are contained in a support such as a biodegradable substrate. Can be used, or any shape, such as processed into a single or multiple layers of cardiomyocyte cells (Shimizu et al., Circ. Res. 90: e40, 2002). In addition, according to methods which can be easily carried out by those skilled in the art, the present invention may be prepared in a unit dose form or formulated using a pharmaceutically acceptable carrier and / or excipient, or in a multi-dose container. It can be prepared by incorporation. The formulations may then be in the form of solutions, suspensions or emulsions in oils or aqueous media, and may further comprise dispersants or stabilizers.

In addition, the pharmaceutical composition of the present invention can be administered by oral, rectal or intravenous, intramuscular, subcutaneous injection, etc., topical infusion is the most preferred method of administration. More specifically, the pharmaceutical composition of the present invention is injected into the heart directly using a syringe after opening the chest, a method of surgically dissecting a portion of the heart, or implanted by a catheterized transvascular method (Murry et al., Cold Spring Harb. Symp. Quant. Biol., 67: 519, 2002; Menasche, Ann. Thorac. Surg., 75: S20, 2003; Dowell et al., Cardiovasc. Res., 58 : 336, 2003), but is not limited to this. Transplantation of cardiomyocytes recovered from fetal heart into the heart of a cardiac injury animal has been reported to have a fairly good therapeutic effect (Menasche, Ann. Thorac. Surg. 75: S20, 2003; Reffelmann et. al., Heart Fail. Rev. 8: 201, 2003). Cardiomyocytes derived from embryonic stem cells exhibit traits that are quite similar to cardiomyocytes derived from fetal heart (Maltsev et al., Mech. Dev. 44: 41, 1993; Circ. Res. 75: 233, 1994). In addition, it was also confirmed that in the animal experiments in which the cardiac muscle cells derived from embryonic stem cells were transplanted into the adult heart, they showed very high engraftment almost identically to the case in which fetal myocardium was transplanted (Klug et al., J. Clin. Invest. 98: 216, 1996). Therefore, in the above-described cardiac diseases due to the decline and loss of the cardiomyocytes, the cardiomyocytes produced by the method described in the present invention can be supplemented to the diseased heart tissue to promote the improvement of cardiac function.

In another aspect, the present invention provides a kit for assaying a candidate material for promoting the induction, differentiation induction, regeneration or survival of cardiomyocytes, comprising the cardiomyocytes, and a method for screening such candidates. It is about.

The cardiomyocytes produced by the present invention are useful for pharmacological evaluation and activity evaluation of various physiologically active substances (for example, drugs) and novel gene products of unknown function. For example, a substance or drug involved in the control of differentiation from pluripotent stem cells such as embryonic stem cells into cardiomyocytes, or a substance or drug related to the regulation of the function of cardiomyocytes, or cardiomyocytes is toxic or impaired. It can be used for the screening of a substance or a medicament. In particular, since there are few screening methods using human cardiomyocytes at present, the cardiomyocytes produced by the present invention are useful cellular materials for carrying out such screening methods. In another embodiment, an assay kit comprising cardiomyocytes prepared by the present invention is useful for such screening.

The test substance provided for screening may be of any kind that can be added to the culture system, for example, low molecular weight compounds, high molecular weight compounds, organic compounds, inorganic compounds, proteins, peptides, genes, viruses, cells, cell cultures And microbial cultures. Methods of efficiently introducing genes into culture systems include adding them to culture systems using viral vectors such as RNA tumor viruses and adenoviruses, or sealing them to artificial structures such as liposomes and adding them to culture systems. .

More specifically, in the present invention, a method for screening a candidate substance that inhibits or induces differentiation from stem cells to cardiomyocytes comprises (i) a medium comprising a medium composition for inducing stem cell differentiation comprising the compound of the present invention. Culturing the stem cells; (Ii) treating the medium under test with the medium; And (iii) confirming the differentiation of stem cells from cardiac muscle cells before and after treatment with the test substance.

The efficiency of inducing cardiac muscle differentiation of a substance under test is characterized by the expression of various markers specific for cardiac muscle cells at 5 to 15 days, preferably 7 to 12 days after the start of culture of stem cells in culture using the method described in the present invention. Can be determined by detection by biochemical or immunochemical methods. The biochemical or immunochemical method is not particularly limited, but preferably, immunochemical methods such as immunohistochemical staining or immunoelectrophoresis can be used.

In addition, in the present invention, a method for screening a candidate substance for inhibiting or improving the cardiac function of cardiomyocytes comprises the steps of: (i) obtaining cardiomyocytes by the method of claim 1; (Ii) treating the cardiac muscle cells with candidate substances; And (iii) measuring cardiac function of cardiomyocytes before and after treatment with the candidate.

Cardiac muscle cells, which are indicative of testing a substance under test, may include cardiac muscle cell viability. Specifically, inoculation of the cardiomyocytes prepared by the method described in the culture plate with an appropriate cell density and incubation in a serum-free medium may induce apoptosis. The test substance may be added to the medium to determine the survival rate or mortality rate of cardiomyocytes. Survival rate or mortality rate of cardiac muscle cells can be measured by visual observation using the intake of pigments such as trifan blue as an indicator, and the method using the activity (reducing activity) of dehydrogenated oxygen as an indicator, or specific to apoptotic cells. It can be measured by a method indicating the expression of caspase activity or expression of Annexin V. Kits using this mechanism are commercially available from many brands, such as Sigma, Clonetech, and Promega, and can be easily used.

Since the substance or drug obtained by the screening method has a differentiation-inducing action or a function control action of cardiomyocytes, for example, myocardial infarction, ischemic heart disease, congestive heart failure, hypertrophic cardiomyopathy, enlarged cardiomyopathy, myocarditis and chronic It can be used as a preventive or therapeutic agent for heart diseases such as heart failure.

The compounds and derivatives of the present invention can be used to prevent or recover the progression of cardiac depression due to various causes, and induce differentiation from stem cells to cardiomyocytes in use or research as a cell therapy. It can be usefully used for the purpose.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the following examples are merely illustrative of the present invention and the present invention is not limited thereto.

Example  1. From Mouse Embryonic Stem Cells by Sample Compound Into cardiomyocytes  Confirmation of differentiation induction effect

1-1. Immunofluorescence  Confirmed by

In the specific experiment of the present invention, the mouse embryonic stem cell line KH2 was used. KH2 cells are mouse embryonic stem cells that express enhanced green fluorescence protein (EGFP) enhanced by the promoter of α-myosin heavy chain (α-MHC) gene, a marker specifically expressed in cardiomyocytes. It is a fully engineered embryonic stem cell that does not express EGFP in the undifferentiated state, but expresses EGFP only when differentiated and differentiated into cardiomyocytes.

Embryoid bodies (EBs) were made using KH2 cells as follows. First, embryonic bodies were made by cell suspension to contain 800 cells in 25 μl of differentiation medium, and made 25 μl of droplets on the lid of the culture dish and incubated for 3 days in a suspended state. Three embryos made were placed in each well of a 96 well plate with 225 μl of differentiation medium. Each compound to be tested was dissolved in DMSO (dimethyl sulfoxide) to 10 mM to make a stock (stock) solution. The stock solution was diluted with differentiation medium to a concentration 10 times the concentration to be tested. The 10-fold liquid thus prepared was put in 25 µl of each well of the 96-well plate prepared above and mixed well. The culture solution was changed while culturing for 12 days. The culture solution was changed every two days for the first four days, and thereafter, every day. The medium exchange method is to remove 200 μl of medium from each well, add 180 μl of differentiation medium, and add 20 μl of 10-fold concentration of compound medium prepared within 3 days in the same manner as above. It was set as the method of mixing. On the last day of incubation all medium was removed from each well of a 96 well plate and washed three times with 200 μl of PBS. 50 μl of 0.05% trypsin solution was added to each well and incubated in a 36 ° C. incubator for 3 minutes, and then separated into individual cells using a micropipette. 200% of 1% formalin-PBS solution was separated into each well of the isolated cells and allowed to stand at room temperature for 10 minutes. The isolated cells were analyzed for EGFP fluorescence using FACS caliber.

As a result, as shown in Table 1 and Figure 1, all 12 test substances showed a significant myocardial differentiation promoting effect.

Myocardial Differentiation Promoting Effect of 12 Compounds  No  Id Purity (%) 1st experiment result (5μM) Second Experiment Result (Experiment Concentration, μM) 3rd experiment result 1.0 μM 10.0 μM One 30986 100 37.4 (10) 1.7 15.9 2 30980 90 31.8 41.4 (10) 21.4 24.1 3 30981 100 80 35.5 (1) 16.1 19.6 4 30983 90 57 45.5 (10) 15.6 30.4 5 30984 80 95.4 40.5 (1) 22.0 50.3 6 30985 100 80.6 28.8 (1) 15.3 23.4 7 30987 100 79.1 42.3 (10) 5.1 17.1 8 31002 95 97 43.0 (1) 20.0 51.0 9 117928 85 14.9 41.7 (10) 20.9 27.7 10 117933 79.1 38.3 (1) 12.6 21.8 11 117947 92.3 36.5 (1) 3.2 12.7 12 117970 95 85.2 37.4 (1) 6.5 24.3

1-2. On electrophoresis  Confirmed by

In the process of inducing differentiation from KH2 cells, which are embryonic stem cells, to cardiomyocytes, we analyzed the expression of genes related to cardiomyocyte differentiation when the compound of Formula 3 (30986) was added. In gene expression analysis, total RNA was isolated from cells, and cDNA was synthesized using reverse transcriptase and analyzed by PCR (polymerase-chain reaction) using primers specific to each gene.

As a result, as shown in Figure 2, Nkx2.5 and GATA4 known to be involved in early cardiomyocyte differentiation was found to maintain a high level of expression in cells treated with the compound of formula 3 compared to the control. On the other hand, it was confirmed that MLC-2V and α-MHC, which constitute the cardiomyocyte cells used as markers of cardiomyocytes, exhibited higher levels of expression than the control group as differentiation progressed. Therefore, it can be seen that if the sample compound of the present invention is continuously maintained in the process of determining differentiation from embryonic stem cells to cardiomyocytes, differentiation into cardiomyocytes is promoted.

Example  2. From Mouse Embryonic Stem Cells Depending on Timing of Sample Compound Treatment Into cardiomyocytes  Differentiation induction

In order to determine the experimental results according to the treatment time of the sample compound, the compound of Formula 3 (30986) was treated by 10.0 μM each for each period from the day of induction to 15 days. As a result, as shown in Figure 3, the similar treatment in the experimental group treated with the compound from 3 to 15 days, the experimental group treated with the compound from 5 to 15 days and the experimental group treated with the compound from 8 to 15 days Induced grades were obtained. These results indicate that the compound of the present invention mainly acts in the late stage of differentiation induction, and indicates the possibility of promoting the proliferation of differentiated cells.

Example  3. In mouse embryonic stem cells treated with sample compounds EGFP  Confirm that the expressing cells are cardiomyocytes

Cardiomyocyte markers α-actinine, α-myosin heavy chain (α-MHC) and troponin in KH2 cells obtained by treating 10 uM of the compound of Formula 10 (31002) in differentiation medium from days 3 to 15 of differentiation induction Immunohistochemical staining (red color) for T was performed.

As a result, as shown in Figure 4, it was confirmed that all the cells showing green fluorescence (EGFP positive cells) express the cardiomyocyte markers. Therefore, it was confirmed that the cells expressing EGFP in mouse embryonic stem cells were cardiomyocytes.

Example  4. From Mouse Embryonic Stem Cells According to Concentration of Sample Compound Into cardiomyocytes  Differentiation Induction Effect

To determine whether the concentration-dependent effect of promoting differentiation from embryonic stem cells represented by the sample compound to cardiomyocytes was tested using the compound of Formula 3 (30986). That is, in the process of inducing differentiation from KH2 cells, which are embryonic stem cells, to cardiomyocytes (for 3 to 15 days), the compounds of Formula 3 were respectively added at concentrations of 0 uM, 0.2 uM, 1.0 uM, 5.0 uM and 25.0 uM. Differentiation was induced, and cardiomyocytes differentiated on day 15 were confirmed by EGFP expression.

As a result, as shown in FIG. 5, it was confirmed that differentiation into cardiomyocytes was promoted in a concentration-dependent manner.

Example  5. Representation of Sample Compounds (31002 and 30986) in Human Embryonic Stem Cells Heart muscle cells  Differentiation promoting effect

While differentiating human embryonic stem cells into cardiomyocytes, the effects of promoting the differentiation of cardiomyocytes of the compound of Formula 3 (30986) and the compound of Formula 10 (31002) were analyzed. Embryos were made from human embryonic stem cells, attached to the culture dish, and sample compounds 31002 or 30986 were added to the medium to 10 uM and cultured for 2 days. After incubation, the number of embryoid bodies in which pulsating cells are characteristic of cardiomyocytes was calculated. This experimental procedure is shown in FIG. 6.

As a result, as shown in FIG. 7, in the embryos treated with the compound of Formula 3 (30986) or the compound of Formula 10 (31002), the ratio of the embryonic bodies showing the pulsatile phase was significantly higher than that of the embryos without the sample. Could be increased.

In addition, the expression of marker genes of cardiomyocytes was analyzed by immunocytofluorescence staining to determine whether cells having the characteristics of cardiomyocytes were present in the pulsatum after treatment with the sample compound. ANP and troponin T, which are marker genes of cardiomyocytes, were fixed in cells treated with the compound of Formula 3 (30986) and the compound of Formula 10 (31002) for 2 days as described above, in the pulsating and non-pulsatile cell masses. Expression was analyzed.

As a result, as shown in Fig. 8, it was confirmed that both ANP and troponin T are clearly expressed in the pulmonary cell mass. Therefore, it was confirmed once again that the compound of Formula 3 (30986) and the compound of Formula 10 (31002) promote the differentiation of cardiomyocytes.

Figure 1 shows the results of confirming the effect of differentiation from KH2 cells, which are mouse embryonic stem cell lines to cardiomyocytes, by 12 compounds belonging to the compound of the present invention by immunofluorescence.

Figure 2 is a compound of formula 3 (30986), in order to determine the effect of differentiation from KH2 cells, which are mouse embryonic stem cell lines to cardiomyocytes, cardiomyocyte specific expression genes Nkx2.5 and GATA4, MLC- The result of confirming the expression of 2V and α-MHC by electrophoresis is shown.

Figure 3 shows the results confirming the differentiation-inducing effect from the mouse embryonic stem cell line KH2 cells to cardiomyocytes according to the treatment time to the compound of formula (30986).

Figure 4 shows the simultaneous expression of cardiomyocyte specific markers (α-actinin, α-myosin heavy chain and troponin T) in EGFP expressing cells (green fluorescence) in mouse embryonic stem cells treated with the compound of Formula 10 (31002). Immunohistochemical staining showing (red fluorescence) is shown.

5 is an experimental result showing that the effect of inducing differentiation from mouse embryonic stem cells to cardiomyocytes is concentration-dependent on the sample compound.

Figure 6 shows the myocardial differentiation induction process through embryonic body (EB) formation in human embryonic stem cells.

FIG. 7 shows the ratio of cardiac muscle cells that pulsate in human embryonic stem cells treated with the compound of Formula 3 (30986) or the compound of Formula 10 (31002).

FIG. 8 shows that in human embryonic stem cells treated with the compound of Formula 3 (30986) or the compound of Formula 10 (31002), ANP and troponin T, which are cardiomyocyte-specific marker genes, are expressed in pulsating heart muscle cells. Immune cell fluorescence staining results are shown.

Has been shown to promote the effect of compounds in the differentiation into cardiomyocytes

Claims (12)

A method of inducing differentiation from stem cells to cardiomyocytes, comprising culturing stem cells in the presence of a compound represented by Formula 1 or a derivative thereof represented by Formula 2:

In the above formula, R ₁ Is selected from the group consisting of hydrogen, alkyl, alkoxy, hydroxy, halide, nitro, cyano group, alkoxycarbonyl, amino group, acetamido group and substituted or unsubstituted aryl group or heteroaryl group, R2, R3 and R4 Is the same or different and is selected from the group consisting of hydrogen, alkyl, alkoxy, hydroxy, halide, nitro, cyano group and alkoxycarbonyl group, X is C, N, O or S. The method of claim 1, wherein the compound or a derivative thereof is one of the following compounds represented by Formula 3 to Formula 14.

The method of claim 1, wherein the stem cells are mammalian cells having the ability to differentiate into cardiomyocytes. The method of claim 1, wherein the stem cells are embryonic stem cells. A composition for inducing differentiation from stem cells to cardiomyocytes, comprising a compound represented by Formula 1 or a derivative thereof represented by Formula 2:

In the above formula, R ₁ Is selected from the group consisting of hydrogen, alkyl, alkoxy, hydroxy, halide, nitro, cyano group, alkoxycarbonyl, amino group, acetamido group and substituted or unsubstituted aryl group or heteroaryl group, R2, R3 and R4 Are the same or different and are selected from the group consisting of hydrogen, alkyl, alkoxy, hydroxy, halide, nitro, cyano group and alkoxycarbonyl group, X is C, N, O or S. The composition of claim 5, wherein the compound or derivative is one of the following compounds represented by Formula 3 to Formula 14.

6. The composition of claim 5, wherein said stem cells are mammalian cells having the ability to differentiate into cardiomyocytes. The composition of claim 5, wherein the stem cells are embryonic stem cells. delete delete (Iii) culturing the stem cells in a medium comprising the composition of claim 5; (Ii) treating the medium with a candidate substance; And (iii) identifying whether to differentiate from stem cells to cardiomyocytes before and after treating the candidates, and to screen for candidate substances that inhibit or induce differentiation from stem cells to cardiomyocytes. Way. (Iii) obtaining cardiomyocytes by the method of claim 1; (Ii) treating the cardiac muscle cells with candidate substances; And (iii) measuring cardiac function of cardiomyocytes before and after treatment with the candidate material, wherein the candidate material inhibits or enhances cardiac function of cardiomyocytes.

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