KR20090090586A - How to induce differentiation of embryonic stem cells into cardiomyocytes - Google Patents

Abstract

The present invention relates to a method for inducing differentiation of embryonic stem cells or embryonic stem cell-derived embryoid bodies into cardiomyocytes using an inhibitor of HIF-1 (Hypoxia-inducible factor-1) and a composition for inducing such differentiation.

Description

How to induce the differentiation of embryonic stem cells into myocardial cells}

The present invention relates to a method for effectively differentiating embryonic stem cells into cardiomyocytes and to a composition for inducing differentiation by adding and culturing a substance that inhibits HIF-1 activity to the culture medium of embryonic stem cells.

Embryonic stem cells are excellent in their ability to continuously proliferate while maintaining normal karyotype without differentiation in vitro culture conditions, and also to the ability to differentiate into almost all cell types that theoretically constitute living bodies, depending on the culture environment. Due to its characteristics, the importance of research and technology development is emphasized.

Stem cell differentiation technology is evaluated as a core technology in the development of next generation regenerative medicine and medical industry. In particular, tissue-specific differentiated cells derived from embryonic stem cells are emphasized not only as a cell therapeutic agent but also as a biological screening system for drug screening and toxicity testing during drug development. Therefore, the demand for a technique for inducing differentiation into tissue cells that play a specific function using embryonic stem cells with excellent differentiation inducing ability is increasing rapidly. In order to develop a standardized differentiation induction protocol, tissue cell specific differentiation It can be said that it is necessary to secure the promoting material.

In 1981, when Evans and Kaufman succeeded in culturing embryonic stem cells in mouse embryos, mouse embryonic stem cells have been used as the basis for research and development of embryonic stem cell research and differentiation induction technology. Since the successful culture of human embryonic stem cells in the blastocyst masses, the development of technology using human embryonic stem cells, in particular the development of tissue cell specific differentiation induction technology has been actively progressed. Many studies have reported that human embryonic stem cells are retinal cell progenitor cells (Lamba DA et al., 2006), neurons (Li XJ et al., 2006; Zhang SC et al., 2001), hematopoiesis under in vitro culture conditions. Cells (Tian X et al., 2005; Kaufman DS et al., 2005; Kaufman DS et al., 2001), cardiomyocytes (Kehat I et al., 2003; Kehat I et al., 2001) and pancreatic cells ( Assady S et al., 2001), it is reported that the differentiation of human embryonic stem cells into specific cells in vitro culture conditions depending on the additive composition of the culture medium, the culture conditions of the cells, including feeder cells. It is suggested that it is possible, and the therapeutic effect in the disease animal model is suggested.

Cardiac-cell based replacement therapy has been recognized as a fundamental treatment for heart disease. To date, skeletal myoblasts, bone means including embryonic stem cells Nuclear cells (bone marrow mononuclear cells, BMC) and circulating progenitor cells, endothelial progenitor cells, mesenchymal cells, cardiac stem cells, etc. And the therapeutic effect using this has been intensively studied. In particular, since the cardiogenial potential of embryonic stem cells was reported by Doetschman in 1985, many reports have suggested that embryonic stem cells can differentiate into cardiomyocytes and induce differentiation (Kehat I et al, 2004, Denning C et al. , 2006 Xu C et al., 2006; Xu C et al., 2002 Mummery C et al., 2002). Embryonic stem cell utilization has become a new therapeutic paradigm for heart-related diseases.

To date, agents that promote differentiation from embryonic stem cells to cardiomyocytes include retinoic acid (RA) (Wobus AM et al., 1997), dimethyl sulfoxide (DMSO) (Ventura C et al., 2000), Corbic acid (vitamin C) (Takahashi T et al., 2003), cardiogenol C (Wu X et al., 2004), Nogin (Yuasa S et al., 2005), etc. are well known, but clinically excellent function Cardiomyocyte differentiation induction technology using human embryonic stem cells capable of performing is still weak. To overcome this, it is important to understand the factors and mechanisms involved in inducing cardiomyocyte differentiation in human embryonic stem cells, and it is very important to find and control substances that control their activity and use them in differentiation induction technology. .

In early development, normal organ formation and differentiation-inducing processes are performed in the hypoxic state, and understanding the molecular mechanisms regulating the hypoxic state may provide important clues in understanding the differentiation of stem cells that can survive in the hypoxic state. It is believed to be able. In particular, the transcription regulator Hypoxia-inducible factor 1 (HIF-1), which plays a representative role in mediating cellular responses in the hypoxic state of the cell, increases the stability and activity in the hypoxic state and the hypoxia of the target gene. Defects in the Hypoxia response element (HRE) are known to increase or decrease the expression of related genes. HIF-1, which functions in the form of a heterodimer consisting of HIF-1α and HIF-1β, is involved in genes involved in cell differentiation related to angiogenesis (erythropoietin (EPO), VEGF, FLK-1, bFGF, etc.). It is known to directly or indirectly regulate the expression of is an important molecular target in understanding the stem cell differentiation process associated with angiogenesis.

In addition, prior art related to the method of inducing differentiation of cardiomyocytes from stem cells are published in International Publication No. 2005-033298 (METHOD OF INDUCING THE DIFFERENTIATION OF STEM CELLS INTO MYOCARDIAL CELLS), International Publication No. 2003-006950 ( CELLS OF THE CARDIOMYOCYTE LINEAGE PRODUCED FROM HUMAN PLURIPOTENT STEM CELLS and Korean Registered Publication No. 0484550 (Method for Producing Cells for Cell Transplantation), but To date, Differentiation of Stem Cells into Cardiomyocytes by Controlling HIF-1 Activity No technique related to the method has been disclosed.

Thus, in the present invention, as a HIF-1 inhibitor for inhibiting the activity of the transcriptional regulator hypoxia-inducible factor 1 (HIF-1), which plays a representative role in mediating cellular responses in a hypoxic state, When the synthesized low molecular weight compound was added to the culture medium of embryonic stem cells, it was confirmed that differentiation into the cardiomyocyte lineage was effectively induced, and the present invention was completed by analyzing the characteristics of the differentiated cells.

An object of the present invention comprises an embryonic stem cell or embryonic stem cell-derived embryonic body comprising an inhibitor of HIF-1 (Hypoxia-inducible factor-1), a transcriptional regulator that plays a representative role in mediating cellular responses in a hypoxic state. It is to provide a composition for inducing differentiation into cardiomyocytes.

Still another object of the present invention is to provide a method of inducing differentiation of embryonic stem cells or embryonic stem cell-derived embryoid bodies into cardiomyocytes by culturing in a culture medium comprising the composition.

In one embodiment, the present invention relates to a composition for inducing differentiation of embryonic stem cells or embryonic stem cell-derived embryoid bodies into cardiomyocytes, including a HIF-1 (Hypoxia-inducible factor-1) inhibitor.

As used herein, the term "HIFoxia-inducible factor-1" (HIF-1) is a transcriptional regulator that plays a representative role in mediating the response of cells in a hypoxic state, and increases the stability and activity in the hypoxic state and hypoxia of the target gene. Defects in the Hypoxia response element (HRE) are known to increase or decrease the expression of related genes. In addition, HIF-1 functions in the form of a heterodimer consisting of HIF-1α and HIF-1β. In the hypoxic state, HIF-1α binds to HIF-1β to be activated in the form of a heterodimer and hypoxia responsive. It is known to play a pivotal role in regulating the expression of target genes by binding to the element (HRE) base sequence.

As used herein, the term "HIF-1 inhibitor" refers to a substance that inhibits the activity of HIF-1 and interferes with normal transcriptional regulation, and is preferably an HIF-1α inhibitor. In addition, the HIF-1 inhibitor may be an antagonist of HIF-1α, more preferably a compound represented by the following formula (1) or (2).

[Formula 1]

[Formula 2]

The present inventors first synthesized the compounds represented by Formula 1 and Formula 2 as HIF-1 inhibitors and received a patent in Korea (Korean Patent Registration No. 10-0786336). Methods for the preparation of compounds of Formula 1 and Formula 2 are described in detail in the above registered patents, the entire contents of which are related to the compounds of Formula 1 and Formula 2 are incorporated herein by reference.

On the other hand, the registered patent is not known at all the physiological function of the stem cell differentiation of the compound of Formula 1 and Formula 2. On the other hand, the present invention first demonstrated that the compounds of Formula 1 and Formula 2 have an excellent effect on inducing differentiation of stem cells into cardiomyocytes. The present invention is characterized by culturing stem cells under the stimulation of the compounds of formulas (1) and (2), and the stimulation method is not particularly limited. Preferably, the compound may be added to the medium. In addition, all the methods which show the same effect as the method of adding the said compound to a medium can be used.

In addition, in the composition for inducing differentiation of embryonic stem cells or embryonic stem cell-derived embryoid bodies into cardiomyocytes, the concentration of the compound represented by Formula 1 or Formula 2 used as an HIF-1 inhibitor is 0.1 to 20. It is preferably μM, most preferably 1 μM.

In the present invention, the term "embryonic stem cell" is an culturing in vitro by extracting the inner cell mass from the blastocyst embryo just before the fertilized egg implants in the mother's womb, and can differentiate into cells of all tissues of the individual. It refers to a cell that may be pluripotent or pluripotent, and in the broad sense also includes embryoid bodies derived from embryonic stem cells. Embryos are intermediate structures formed by stem cells during spontaneous differentiation of embryonic stem cells into various tissue forms, and aggregate forms formed during the culture of embryonic stem cells. On the other hand, the embryonic stem cells and embryonic stem cell-derived embryoid body of the present invention is preferably derived from human or mouse.

In the present invention, "cardiomyocytes" includes not only precursors of cardiac cells capable of differentiating into cardiac muscle cells of all types, including ventricular, atrial and nodular cells, but also cells of all cardiomyocyte lineages that can be formed during differentiation. can do.

The composition of the present invention may further comprise a type 1 transforming growth factor beta receptor kinase (TβRI) inhibitor. TβRI is a group of receptors for TGF-β, a multifunctional peptide that functions on cell proliferation, differentiation and various types of cells, also known as activin receptor like kinase 5 (ALK5). Means a substance that interferes with the delivery process. At this time, the TβRI inhibitor included in the composition of the present invention is preferably added at a concentration of 0.1 to 20 μM, more preferably 0.1 to 5 μM concentration.

In addition, the composition of the present invention may further comprise a known substance that promotes the differentiation of embryonic stem cells into cardiomyocytes, for example, retinoic acid, dimethyl sulfoxide, ascorbic acid (vitamin C) and cardio The game is not limited to this.

On the other hand, the composition of the present invention may be preferably provided in the form of a medium composition, wherein the medium composition may include a common base additive, for example, serum, amino acids, antibiotics and differentiation regulators, It is not limited to this.

In a specific embodiment of the present invention, as a HIF-1 inhibitor, by culturing human embryonic stem cells or mouse embryonic stem cells in a culture medium composition containing a compound represented by the formula (1) or (2) at various concentrations to differentiate into a cardiomyocyte lineage After induction, the degree of differentiation into cardiomyocytes was examined. Semi-quantitative RT-PCR and immunofluorescence staining were performed to evaluate the expression level and protein levels of cardiomyocyte-specific genes, compared to the control group without HIF-1 inhibitor. In addition, the expression level of the cardiomyocyte-specific genes and protein expression levels were increased, and the concentration of the HIF-1 inhibitor showed no cytotoxicity at 1 μM and effectively induced differentiation into cardiomyocytes. This means that the composition comprising the HIF-1 inhibitor of the present invention can effectively differentiate embryonic stem cells into the cardiomyocyte lineage.

In another aspect, the present invention relates to a method for inducing differentiation of embryonic stem cells or embryonic stem cell-derived embryoid bodies into cardiomyocytes using a composition comprising the inhibitor of HIF-1 (Hypoxia-inducible factor-1). .

In the present invention, a method of inducing differentiation of embryonic stem cells into cardiomyocytes may use an adhesion culture method, an embryonic method, and a known method of inducing differentiation of cardiomyocytes, and preferably, but not limited thereto. no. Adhesion culture method is a method of inducing differentiation by attaching and culturing undifferentiated embryonic stem cells to the surface of the incubator during differentiation induction process, and via embryonic body method, stem cells are cultured by suspension culture for a few days. Refers to a method of inducing differentiation while producing and attaching it to the culture vessel again.

Preferably, the method of inducing stem cell differentiation of the present invention comprises the steps of preparing a stem cell; Obtaining an embryoid body from stem cells; And culturing an embryonic body in a medium comprising a composition for inducing stem cell differentiation comprising an HIF-1 inhibitor to separate the differentiated cells from the embryonic body.

More preferably, the method of inducing stem cell differentiation of the present invention comprises the steps of: 1) co-culturing undifferentiated embryonic stem cells with feeder cells; 2) floating the embryonic stem cells to establish an embryo; 3) attaching and culturing the established embryoid body; And 4) culturing an embryoid body in a medium comprising a composition for inducing stem cell differentiation comprising an HIF-1 inhibitor to differentiate into cardiomyocytes.

First, step 1) is a step of propagating undifferentiated embryonic stem cells by attaching undifferentiated embryonic stem cells onto feeder cells and co-cultured. Embryonic stem cells used in this step may use embryonic stem cells derived from humans or mice, and mouse embryonic fibroblasts that have stopped proliferation by treating mitomycin C as feeder cells. Undifferentiated embryonic stem cells are preferably co-cultured with feeder cells.

Step 2) is a step of floating culture the undifferentiated embryonic stem cells cultured in the above to establish an embryo. Specifically, in the case of undifferentiated human embryonic stem cells, cell embryos are formed in 500-800 μm sizes of human embryonic stem cells in all directions during subculture in order to obtain a uniform size of embryonic bodies after attaching them to the feeder cells and co-culturing them. Human embryonic stem cell-derived embryoid bodies can be established by floating culture. In addition, in the case of undifferentiated mouse embryonic stem cells, the embryonic stem cells were adhered to the feeder cells and co-cultured, and then mouse embryonic stem cells were suspended and cultured through suspension culture at a concentration of 800 cells / 20 μl (culture medium). Cell-derived embryoid bodies can be established. Step 2) is generally carried out for a sufficient time for cell aggregates to form in a suspended culture, preferably 1 to 5 days, more preferably 4 days.

 Step 3) is a step of stabilizing the embryo body by attaching and incubating the embryo embryo body established above. Thereafter, the attached embryonic stem cell-derived embryoid bodies may be treated with differentiation inducing agents to specific cells to induce differentiation of embryonic stem cells into specific cells.

Step 4) is a step of inducing differentiation into cardiomyocytes by treating the attachment-cultured embryonic stem cell-derived embryos with a HIF-1 inhibitor. The HIF-1 inhibitor is not particularly limited as long as it inhibits the activity of HIF-1 and interferes with the normal transcription control process. Preferably, the compound represented by Formula 1 or Formula 2 may be used alone or in combination. At this time, the HIF-1 inhibitor is treated at a concentration in the culture medium of 0.1 to 20 μM, more preferably at a concentration of 1 μM. In addition, when inducing differentiation into cardiomyocytes, in addition to the HIF-1 inhibitor, it may further include a known substance and a general medium additive for promoting the induction of differentiation of embryonic stem cells into cardiomyocytes, and may preferably include a TβRI inhibitor. have. Step 3) is generally carried out for a sufficient time for the differentiated cells to form, preferably 3 to 15 days, more preferably 7 days.

Specific examples of the present invention also induced differentiation of human embryonic stem cells into cardiomyocytes using the culture medium transfection method according to step 4 (FIG. 2A). First, in the first stage, human embryonic stem cells were subjected to 20% knockout serum replacement (Invitrogen, USA), 0.1 mM non-essential amino acid (NEAA; Invitrogen, USA) after 0.1 passage. DMEM / F12 (Invitrogen, USA) supplemented with mM beta-mercaptoethanol, 4 ng / ml recombinant human basic FGF (Invitrogen, USA), and 1X penicillin-streptomycin (Invitrogen, USA) USA) cocultured with MEF (mouse embryonic fibroblast) inhibited proliferation with mitomycin-C in the culture medium, the cells were attached and exchanged with fresh culture medium for 5 additional days. Human embryonic stem cells in colony form were constructed to have a size of 500 to 800 µm in all directions using a glass pipette. In step 2, the human embryonic stem cells clumps were constructed for 4 days in embryonic medium ( Embryos were established by suspension culture in 80% Knockout DMEM, 20% fetal bovine serum, 1 mM L-glutamine, 0.1 mM β-mercaptoethanol and 1 mM NEAA). In step 3, the established embryoid bodies were transferred to a cell culture vessel coated with 0.1% gelatin and cultured for 1 day with the differentiation medium. In step 4, the HIF-1α inhibitor was added to the embryos in the attached culture and cultured for 7 days or more.

Cells induced differentiation from embryonic stem cells to cardiomyocytes through the differentiation induction method of the present invention has the morphological, physiological or immunological characteristics of cardiomyocytes, preferably the gene expression level of cardiomyocyte-specific markers is increased. Can be increased. For cardiomyocytes induced differentiation from human embryonic stem cells, cardiomyocyte specific markers such as MLC-2α, MLC-2v, Cardiac Actin, cTnT, cTnl, α-actinin, MEF-2C, α-MHC and The degree of expression of one or more genes in ANP may be increased. In addition, in the case of cardiomyocytes differentiated from mouse embryonic stem cells, the expression level of one or more genes of Nkx2.5, α-MHC, β-MHC, MLC-2v, GATA4 and α-actinin may be increased. .

In a specific embodiment of the present invention, semi-quantitative RT-PCR was performed to analyze the expression variation of cardiomyocyte-specific genes of cells differentiated into cardiomyocytes through the embryonic method as described above. The HIF-1α inhibitor at a concentration of 1 μM showed no cytotoxicity and increased the expression of cardiomyocyte specific genes. Specifically, after 7 days of incubation with HIF-1α inhibitor, the expression variation of cardiomyocyte-specific genes was analyzed using semi-quantitative RT-PCR. As a result, MLC-2α and MLC-2v were analyzed. , Cardiac Actin, cTnT, cTnl, α-actinin, MEF-2C, α-MHC and ANP expression was increased (Fig. 3), the compound represented by the formula 1 or formula 2 in the compound group is excellent in efficacy Confirmed (not shown).

In addition, in order to further verify the ability of HIF-1α inhibitors to promote differentiation into cardiomyocytes, expression of cardiomyocyte markers was verified at the protein level using immunofluorescence. Cardiomyocyte differentiation markers α-MHC and α-actinin positive staining reactions were confirmed in each of the treated cell groups (Fig. 4). It was excellent and the treatment concentration showed the best effect at 1 μM (not shown in the results). From these results, it was confirmed that the HIF-1α inhibitor of the present invention has the ability to promote differentiation into cardiomyocytes in human embryonic stem cells. In addition, it was examined whether the beating cells characteristic of cells differentiated into functional cardiomyocytes were produced in a cell group induced by differentiation by HIF-1α inhibitors. As a result, the pulsating cell production rate of the cell groups treated with the formulas (1) and (2), respectively, increased about 4 times compared to the control group (Fig. 5).

On the other hand, in a specific embodiment of the present invention it was confirmed that the HIF-1α inhibitor used in the present invention also has cardiomyocyte differentiation induction in mouse embryonic stem cells RW.4 (ATCC SCRC-1018). That is, the expression of Nkx2.5, α-MHC, β-MHC, MLC-2v, GATA4, and α-actinin, which are effective cardiomyocyte markers, was effectively treated with 1 μM HIF-1α inhibitors on embryonic cells for 7 days. This increase was confirmed via semi-quantitative RT-PCR (FIG. 6). Similar to human embryonic stem cells, HIF-1α inhibitor compounds showed the best ability to promote differentiation of the compounds of Formula 1 and Formula 2, and the treatment concentration showed the best effect at 1 μM (not shown). In addition, the ability of HIF-1α inhibitors to promote differentiation into cardiomyocytes was also confirmed by the results of investigating the rate of generation of pulsating colonies. Increased degree (FIG. 7).

Myocardial progenitor cells induced by differentiation from embryonic stem cells produced in the present invention can be used to obtain a large amount of cardiomyocytes as they continue to grow and proliferate, and can be used as cell compositions for cell replacement therapy for treating heart muscle related diseases. .

The composition according to the present invention can effectively induce differentiation from embryonic stem cells to cardiomyocytes with a simple composition containing a HIF-1 inhibitor, and a large amount of cardiomyocytes through a method of inducing differentiation into cardiomyocytes using such a composition. Can be produced by These cardiomyocytes can be used in the development of new drugs, such as cell therapy for the treatment of heart-related diseases.

Hereinafter, the present invention will be described in more detail with reference to Examples and Preparation Examples. These examples and production examples are only for illustrating the present invention more specifically, and the scope of the present invention is not limited by these examples and production examples.

< Production Example  1> HIF -1 Inhibitors-Preparation of Compounds of Formula 1

[Formula 1]

2- [2- (4- Tert Butyl Phenoxy )- Acetylamino ]- Isnicotinamide

4-tert-butylphenoxy acetic acid (60.1 mg, 0.29 mmol), 2-amino isonicotinamide (60.3 mg, 0.44 mmol), DIPEA (0.1 mL, 0.58 mmol) and PyBOP (301.8 mg, 0.58 mmol) were added to DMF 4.0. Put into mL and stirred. Separated with EtoAC and water, the organic layer was washed with aqueous sodium chloride solution, dried over anhydrous MgSO ₄ and concentrated. Purification by Prep-TLC ( n -Hexane: EtoAc: MeOH = 6: 3: 1) gave the desired compound as a white solid (53.6 mg, 56.5% yield).

¹ H-NMR (DMSO-d ₆ , 300 Hz) 10.64 (1H, s, NH), 8.42-8.46 (2H, m, aromatic-H), 8.20 (1H, s, NH ₂ ), 7.68 (1H, s , NH ₂ ), 7.49-7.51 (1H, m, aromatic-H), 7.28-7.32 (2H, m, aromatic-H) ,. 6.86-6.91 (2H, m, aromatic-H), 4.78 (2H, s, CH ₂ ), 1.24 (9H, s, CH ₃ ).

< Production Example  2> HIF -1 Inhibitor-Preparation of Compound of Formula 2

[Formula 2]

2- (2,4- Dichloro - Phenoxymethyl )- Benzoxazole -6-carboxylic acid

2- (2,4-dichloro-phenoxymethyl) -benzoxazole-6-carboxylic acid methyl ester (100 mg, 0.29 mmol) was added to 8 ml of dimethyl sulfide and 58 ml of CH ₂ Cl ₂ , followed by aluminum bromide (1.24 g, 4.64 mmol) was added. Stirred at room temperature for 2.5 hours. Water and 10% HCl were added, stirred at room temperature for 1 hour, and then separated into EtoAC and water. The organic layer was washed with aqueous sodium chloride solution, dried over anhydrous MgSO ₄ , concentrated and purified by flash chromatography (CH ₂ Cl ₂ : MeOH = 4: 1) to give the title compound as a white solid (59.15 mg mg, 61.2% yield).

¹ H-NMR (DMSO-d ₆ , 300 Hz) 13.22 (1H, s, COOH), 8.28 (1H, s, aromatic-H), 8.00-8.03 (1H, m, aromatic-H), 7.88 (1H, d, J = 8.4 Hz, aromatic-H), 7.64-7.65 (1H, m, aromatic-H), 7.34-7.43 (2H, m, aromatic-H), 5.66 (2H, s, CH ₂ ).

Example  1: Culture of Human Embryonic Stem Cells

In this example, to maintain the undifferentiated state of human embryonic stem cells, 20% knockout serum replacement (Knockout serum replacement, Invitrogen, USA), 0.1 mM non-essential amino acid (NEAA; Invitrogen, USA), DMEM / F12 with 0.1 mM beta-mercaptoethanol, 4 ng / ml recombinant human basic FGF (Invitrogen, USA) and 1X penicillin-streptomycin (Invitrogen, USA) (Invitrogen, USA) medium was co-cultured with mitomycin-C (Sigma, USA) treated MEF (mouse embryonic fibroblast) feeder cells. MEF feeder cells were treated with DMEM supplemented with 10% fetal bovine serum (Hyclone, USA), 0.1 mM non-essential amino acids, 1X penicillin / streptomycin and 0.5 mM beta-mercaptoethanol one day prior to human embryonic stem cell passage. (Invitorgen, USA) incubated in medium, and then inactivated in 10 ㎍ / ㎖ mitomycin-C (Sigma, USA) for 1 hour 30 minutes 0.1% gelatin at a concentration of 7.5 X 10 ⁴ cells / cm ² ( Sigma, USA) was attached to a culture vessel coated with human embryonic stem cell co-culture. For passage of human embryonic stem cells, cells of colony-like human embryonic stem cells are constructed in 500-800 μm size on all sides using an apparatus made of glass Pasteur pipette, and then placed on the feeder cells at appropriate intervals. . The cell mass adhered to the feeder cells, and the culture medium was exchanged daily from about 2 days thereafter, and subcultured at 5-6 day intervals.

Example  2: from human embryonic stem cells Embryonic  formation

In the passage of human embryonic stem cells, human embryonic stem cells in colony state were cut into 500 to 800 μm size in four directions with the glass pipette described in Example 1 to obtain a uniform embryonic body, and then recovered in a suspended state. 10% Knockout Serum Replacement (Invitrogen, USA), 0.1 mM Non-essential amino acid (NEAA; Invitrogen, USA), 0.1 mM Beta- Suspension culture was carried out in an EB cultrure medium containing mercaptoethanol and 1 × penicillin-streptomycin (Invitrogen, USA).

Example  3: human embryonic stem cells Into cardiomyocytes  differentiation

In order to induce differentiation of human embryonic stem cells obtained in Example 1 into cardiomyocytes, the embryos derived from human embryonic stem cells obtained in Example 2 were cultured for 4 days using embryonic culture medium and then attached for 1 day. Incubated. Thereafter, the cells were cultured for 7 days or more in embryonic culture medium to which HIF-1α inhibitors of different concentrations (0.1 to 50 μM) were added (FIG. 2A).

Example  4: mouse embryonic stem cell culture

In this example, in order to maintain the undifferentiated state of mouse embryonic stem cells, 20% fetal bovine serum, 0.1 mM non-essential amino acid (NEAA; Invitrogen, USA), 1 mM sodium pyruvate (Invitrogen), 0.1 Mitomyma in DMEM (GIBCO-BRL, USA) medium containing mM beta-mercaptoethanol and 1 × penicillin-streptomycin (Invitrogen, USA, 1000 U / ml leukemia inhibitory factors (LIF); Chemicon, USA) Co-cultured with isine-C (Sigma, USA) treated MEF (mouse embryonic fibroblast) feeder cells. MEF feeder cells were treated with DMEM supplemented with 10% fetal bovine serum (Hyclone, USA), 0.1 mM non-essential amino acids, 1X penicillin / streptomycin and 0.5 mM beta-mercaptoethanol one day prior to mouse embryonic stem cell passage. (Invitorgen, USA) incubated in medium, and then inactivated in 10 μg / ml mitomycin-C (Sigma, USA) for 1 hour 30 minutes to 0.1% gelatin (Sigma) at a concentration of 7.5 × 10 ⁴ cells / cm ² . , USA) was used for co-culture with mouse embryonic stem cells after attaching to a culture vessel coated with. Mouse embryonic stem cells were passaged using Trypsin / EDTA solution every two days.

Example  5: mouse embryonic stem cell from Embryonic  formation

10% fetal bovine serum, 0.1 mM non-essential amino acid (NEAA; Invitrogen, USA) by hanging drops method in mouse embryonic stem cells at a concentration of 800 cells / 20 ml (culture medium) during subculture. Embryos were formed by suspension culture in EB cultrure medium containing 0.1 mM beta-mercaptoethanol and 1 × penicillin-streptomycin (Invitrogen, USA).

Example  6: mouse embryonic stem cell of Into cardiomyocytes  differentiation

In order to induce differentiation of mouse embryonic stem cells obtained in Example 4 into cardiomyocytes, the embryonic stem cells derived from mouse embryos obtained in Example 5 were cultured in embryos for 3 days and then cultured for 1 day. It was. Thereafter, the cells were cultured for 7 days or more in embryonic medium containing different concentrations (0.1 to 50 μM) of HIF-1α inhibitor (FIG. 2B).

Example  7: Heartbeat generation rate investigation

In this example, it was examined whether beating cells, which can be characteristically observed in cells differentiated into functional cardiomyocytes, were produced by differentiation inducers.

7-1. Derived from human embryonic stem cells Embryonic  Survey of pulsating cell production rate

In order to observe the pulsating cell production rate, 20 embryos obtained in Example 2 (human-derived, FIG. 2A stage II) were attached to a 60 mm dish (x 5), and treated with and not treated with differentiation inducers. The number of beating embryos was counted by observing the beats appearing in the microscope daily. This experiment was repeated three times or more.

As a result, the pulsating cell production rate (7 days after the compound treatment) of the groups treated with the formulas (1) and (2) increased about 4 times compared to the untreated control (Fig. 5).

7-2. Derived from mouse embryonic stem cells Embryonic  Survey of pulsating cell production rate

To observe the pulsating cell production rate, 20 embryos obtained in Example 5 (mouse derived, FIG. 2B stage II) were attached to a 35 mm dish (x 5), and treated with and not treated with differentiation inducers. The number of beating embryos was counted by observing the beats appearing in the microscope daily. This experiment was repeated three times or more.

As a result, the pulsating cell production rate (7 days and 14 days after compound treatment) of the cell groups treated with Formulas 1 and 2 respectively increased about 7 times compared to the control group (FIG. 7).

Example  8 : Cardiomyocytes  Differentiation related marker factor expression

8-1. Semi Quantitative  ( semi - quantitative ) RT - PCR

Semi-quantitative RT-PCR was performed on the cells obtained in Example 3 (derived from humans) or 6 (derived from mice) to identify cardiomyocyte differentiation patterns. First, the cells treated with the HIF-1α inhibitor and the untreated cells were recovered at different concentrations by differentiation stages, and then all RNAs were isolated using Trizol reagent (Invitrogen, USA), and the reverse transcriptase (Superscript) was used as a template. CDNA was synthesized using II reverse transcriptase) and oligo (dT) primers. The primers shown in Table 1 and Table 2 were used to confirm the expression of cardiomyocyte specific marker genes.

Polymerase Reagent Primer for Identification of Human Cardiomyocyte Specific Marker Gene Expression Name PCR primer Size  ( bp ) Gene Bank Accession No . ANP Forward GGAACCAGAGGGGAGAGACAGAG (SEQ ID NO: 1) 406 NM_006172 Reverse CGCCCTCAGCTTGCTTTTTAGGAG (SEQ ID NO: 2) α-MHC Forward ATGACCGATGCCCAGATGGCTGA (SEQ ID NO: 3) 424 NM_002471 Reverse TCACTCCTCTTCTTGCCCCGGTA (SEQ ID NO: 4) MLC-2a Forward ACAGAGTTTATTGAGGTGCCCC (SEQ ID NO: 5) 381 NM_021223 Reverse AAGGTGAAGTGTCCCAGAGG (SEQ ID NO: 6) MLC-2v Forward TATTGGAACATGGCCTCTGGAT (SEQ ID NO: 7) 382 BC015821 Reverse GGTGCTGAAGGCTGATTACGTT (SEQ ID NO: 8) cTnT Forward GGCAGCGGAAGAGGATGCTGAA (SEQ ID NO: 9) 152 NM_001001431 Reverse GAGGCACCAAGTTGGGCATGAACGA (SEQ ID NO: 10) cTnl Forward CCTGCGGAGAGTGAGGATCT (SEQ ID NO: 11) 218 NM_000363 Reverse TAGGCAGGAAGGCTCAGCTC (SEQ ID NO: 12) MEF-2C Forward ATGGATGAACGTAACAGACAGGT (SEQ ID NO: 13) 99 S57212 Reverse CGCAATCTCACAGTCACACAG (SEQ ID NO: 14) α-actinin Forward GGCGTGCAGTACAACTACGTG (SEQ ID NO: 15) 580 BC051770 Reverse AGTCAATGAGGTCAGGCCGGT (SEQ ID NO: 16) Cardiac actin Foreard TCTATGAGGGCTACGCTTTG (SEQ ID NO: 17) 630 NM_005159 Reverse CCTGACTGGAAGGTAGATGG (SEQ ID NO: 18) GAPDH Forward GAAGGTGAAGGTCGGAGTC (SEQ ID NO: 19) 226 NM_002046 Reverse GAAGATGGTGATGGGATTTC (SEQ ID NO: 20)

Polymerase Reaction Primer for Identification of Mouse Cardiomyocyte Specific Marker Gene Expression Name PCR primer Size  ( bp ) Gene Bank Accession No . α-MHC Forward GCCCAGTACCTCCGAAAGTC (SEQ ID NO: 21) 110 NM_101856 Reverse GCCTTAACATACTCCTCCTTGTC (SEQ ID NO: 22) β-MHC Forward ACTGTCAACACTAAGAGGGTCA (SEQ ID NO: 23) 144 NM_080728 Reverse TTGGATGATTTGATCTTCCAGGG (SEQ ID NO: 24) GATA4 Forward TCTCACTATGGGCACAGCAG (SEQ ID NO: 25) 135 NM_008092 Reverse GCGARGRCRGAGRGACAGGA (SEQ ID NO: 26) Nkx2.5 Forward CAAGTGCTCTCCTGCTTTCC (SEQ ID NO: 27) 136 NM_008700 Reverse GGCTTTGTCCAGCTCCACT (SEQ ID NO: 28) MLC-2v Forward TGTGGGTCACCTGAGGCTGTGGTTCAG (SEQ ID NO: 29) 189 NM_016754 Reverse GAAGGCTGACTATGTCCGGGAGATGC (SEQ ID NO: 30) α-actinin Forward TGGCACCCAGATCGAGAAC (SEQ ID NO: 31) 121 NM_033268 Reverse GTGGAACCGCATTTTTCCCC (SEQ ID NO: 32) GAPDH Forward GTGTTCCTACCCCCAATGTG (SEQ ID NO: 33) 215 NM_008084 Reverse TGTCATTGAGAGCAATGCCA (SEQ ID NO: 34)

As a result of examining the gene expression patterns of human embryonic stem cells by the above method, as shown in FIG. 3, MLC-2α, MLC-2v, Cardiac Actin, cTnT, cTnl, α-actinin, In the case of MEF-2C, α-MHC and ANP, the expression of increased expression in cells treated with HIF-1α inhibitors was observed through semi quantitative RT-PCR.

In mouse embryonic stem cells, gene expression of Nkx2.5, α-MHC, β-MHC, MLC-2v, GATA4 and α-actinin, which are markers of differentiation of cardiomyocytes, was increased in HIF-1α-inhibited cells. Aspects could be observed via semi quantitative RT-PCR (FIG. 6).

8-2. Immunofluorescence Staining  ( Imunofluorescent staining )

In order to confirm the characteristics of the cells obtained in Example 3 at the protein level 1 cm ² Differentiated and propagated cells in cover glass were removed from the culture medium and washed with phosphate buffered saline, and then fixed with a fixed solution (citrate-acetone-formaldehyde). After treatment for 30 minutes in 3% bovine serum albumin / phosphate buffered saline solution and the reaction was added for 12 hours by adding a primary antibody. After washing 15 times in 0.1% Triton x-100 / phosphate buffered saline solution three times for 15 minutes, it is labeled by reacting with Alexa488-conjugated secondary antibody (Molecular probes, USA) for 30 minutes at room temperature. Again washed three times in 0.1% Triton x-100 / phosphate buffered saline solution for 15 minutes and observed with a fluorescent microscope (Olympus, Japan) (Fig. 4). Primary antibodies for confirming the expression of cardiomyocyte differentiation markers were α-MHC (1: 100, Abcam, USA), alpha alpha actinin (1: 250, Sigma, USA).

Human embryonic stem cell-derived embryos were treated with HIF-1α inhibitors and the expression of α-MHC and α-actinin proteins, which are markers for cardiomyocyte differentiation, was verified by immunofluorescence staining. As shown in FIG. 4, HIF- It was confirmed that protein expression including α-MHC and α-actinin was excellent in cells treated with the 1α inhibitor.

The cardiomyocytes produced according to the present invention can contribute to the development of cell therapies for the treatment of heart-related diseases, and the biological efficacy verification system using stem cells required for the development of new drugs, in particular, cardiomyocyte differentiation inducing substances and cardiac function enhancement. It can be used in drug validation system and new drug commercialization to develop materials.

It is also used to identify other new cardiac-related factors by contributing to the signaling process and cardiac surgery and pharmacology research and understanding that controls the differentiation of cardiomyocyte lineages from human embryonic stem cells, or finally differentiated from a clinical perspective. It can be used to secure mature cells and to develop the cause and treatment of heart-related diseases.

1 shows compounds of Formula 1 and Formula 2 which are HIF-1α inhibitors.

FIG. 2 is a simplified diagram of a method for treating the compounds of FIG. 1 on human (A) or mouse (B) embryonic stem cells. FIG.

FIG. 3 shows genes expressing cardiomyocyte-specific genes (MLC-2α, MLC-2v, Cardiac Actin, cTnT, cTnl, when the compounds of FIG. 1 are treated to human embryonic stem cells by the method of FIG. 2A). α-actinin, MEF-2C, α-MHC and ANP) expression is induced electrophoresis confirmed by reverse transcription-polymerase reaction (RT-PCR).

Figure 4 using immunofluorescence staining to increase the expression of protein (α-actinin, α-MHC) that specifically expresses cardiomyocytes when the compounds of Figure 1 is treated to human embryonic stem cells by the method of Figure 2A The cell nuclei were counterstained with DAPI.

FIG. 5 shows the production rate of pulsating human embryonic stem cell-derived embryoid bodies due to differentiation into cardiomyocytes when the compounds of FIG. 1 are treated to human embryonic stem cells by the method of FIG. 2A.

FIG. 6 shows genes expressing cardiomyocyte specific cells (Nkx2.5, α-MHC, β-MHC, MLC-2v, when the compounds of FIG. 1 are treated to mouse embryonic stem cells by the method of FIG. 2B). The expression of GATA4 and α-actinin) was confirmed by reverse transcriptase-polymerase reaction (RT-PCR).

Figure 7 shows the production rate of the pulsating mouse embryonic stem cell-derived embryoid body due to differentiation into cardiomyocytes when the compounds of Figure 1 treated with mouse embryonic stem cells in the modeled method of Figure 2B.

<110> Korea Research Institute of Bioscience and Biotechnology <120> Method for inducing the differentiation of embryonic stem cells          into myocardial cells <130> PA070828 <160> 34 <170> KopatentIn 1.71 <210> 1 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> forward primer for identifying ANP gene <400> 1 ggaaccagag gggagagaca gag 23 <210> 2 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for identifying ANP gene <400> 2 cgccctcagc ttgcttttta ggag 24 <210> 3 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> forward primer for identifying alpha-MHC gene <400> 3 atgaccgatg cccagatggc tga 23 <210> 4 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for identifying alpha-MHC gene <400> 4 tcactcctct tcttgccccg gta 23 <210> 5 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> forward primer for identifying MLC-2a gene <400> 5 acagagttta ttgaggtgcc cc 22 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for identifying MLC-2a gene <400> 6 aaggtgaagt gtcccagagg 20 <210> 7 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> forward primer for identifying MLC-2v gene <400> 7 tattggaaca tggcctctgg at 22 <210> 8 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for identifying MLC-2v gene <400> 8 ggtgctgaag gctgattacg tt 22 <210> 9 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> forward primer for identifying cTnT gene <400> 9 ggcagcggaa gaggatgctg aa 22 <210> 10 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for identifying cTnT gene <400> 10 gaggcaccaa gttgggcatg aacga 25 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> forward primer for identifying cTnl gene <400> 11 cctgcggaga gtgaggatct 20 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for identifying cTnl gene <400> 12 taggcaggaa ggctcagctc 20 <210> 13 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> forward primer for identifying MEF-2C gene <400> 13 atggatgaac gtaacagaca ggt 23 <210> 14 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for identifying MEF-2C gene <400> 14 cgcaatctca cagtcacaca g 21 <210> 15 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> forward primer for identifying alpha-actinin gene <400> 15 ggcgtgcagt acaactacgt g 21 <210> 16 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for identifying alpha-actinin gene <400> 16 agtcaatgag gtcaggccgg t 21 <210> 17 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> forward primer for identifying cardiac actin gene <400> 17 tctatgaggg ctacgctttg 20 <210> 18 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for identifying cardiac actin gene <400> 18 cctgactgga aggtagatgg 20 <210> 19 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> forward primer for identifying GAPDH gene <400> 19 gaaggtgaag gtcggagtc 19 <210> 20 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for identifying GAPDH gene <400> 20 gaagatggtg atgggatttc 20 <210> 21 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> forward primer for identifying alpha-MHC gene <400> 21 gcccagtacc tccgaaagtc 20 <210> 22 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for identifying alpha-MHC gene <400> 22 gccttaacat actcctcctt gtc 23 <210> 23 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> forward primer for identifying beta-MHC gene <400> 23 actgtcaaca ctaagagggt ca 22 <210> 24 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for identifying beta-MHC gene <400> 24 ttggatgatt tgatcttcca ggg 23 <210> 25 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> forward primer for identifying GATA4 gene <400> 25 tctcactatg ggcacagcag 20 <210> 26 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for identifying GATA4 gene <400> 26 gcgargrcrg agrgacagga 20 <210> 27 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> forward primer for identifying Nkx2.5 gene <400> 27 caagtgctct cctgctttcc 20 <210> 28 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for identifying Nkx2.5 gene <400> 28 ggctttgtcc agctccact 19 <210> 29 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> forward primer for identifying MLC-2v gene <400> 29 tgtgggtcac ctgaggctgt ggttcag 27 <210> 30 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for identifying MLC-2v gene <400> 30 gaaggctgac tatgtccggg agatgc 26 <210> 31 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> forward primer for identifying alpah-actinin gene <400> 31 tggcacccag atcgagaac 19 <210> 32 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for identifying alpah-actinin gene <400> 32 gtggaaccgc atttttcccc 20 <210> 33 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> forward primer for identifying GAPDH gene <400> 33 gtgttcctac ccccaatgtg 20 <210> 34 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for identifying GAPDH gene <400> 34 tgtcattgag agcaatgcca 20  

Claims (13)

A method of inducing differentiation of embryonic stem cells or embryonic stem cell-derived embryoid bodies into cardiomyocytes by culturing in a culture medium comprising an inhibitor of HIF-1 (Hypoxia-inducible factor-1). The method of claim 1, wherein the inhibitor of HIF-1 (Hypoxia-inducible factor-1) is a compound represented by Formula 1 or Formula 2 below: [Formula 1]

[Formula 2]

The method of claim 2, wherein the concentration of the compound of Formula 1 or Formula 2 in the culture medium is 0.1 to 20μM. The method of claim 1, wherein the embryonic stem cells and embryonic stem cell derived embryoid bodies are derived from humans or mice. The method according to claim 4, wherein the embryonic stem cells derived from human embryonic stem cells have been established by floating culture of human embryonic stem cells constructed to a size of 500 to 800 µm in all directions when passaged. The cardiomyocytes differentiated from human embryonic stem cells are selected from the group consisting of MLC-2α, MLC-2v, Cardiac Actin, cTnT, cTnl, α-actinin, MEF-2C, α-MHC and ANP. At least one gene expression level is increased. The method according to claim 4, wherein the cardiomyocytes differentiated from mouse embryonic stem cells have an increased expression level of at least one gene selected from the group consisting of Nkx2.5, α-MHC, β-MHC, MLC-2v, GATA4 and α-actinin. Method characterized in that. The method of claim 1, wherein the culture medium further comprises a transforming growth factor-β Type I receptor kinase (TβRI) inhibitor. The method of claim 1, wherein the method of inducing differentiation into cardiomyocytes comprises: 1) co-culturing undifferentiated embryonic stem cells with feeder cells; 2) floating the embryonic stem cells to establish an embryo; 3) attaching and culturing the established embryoid body; And 4) culturing an embryoid body in a medium comprising a composition for inducing stem cell differentiation comprising an HIF-1 inhibitor to differentiate into cardiomyocytes. A composition for inducing differentiation of embryonic stem cells or embryonic stem cell-derived embryoid bodies into cardiomyocytes comprising an inhibitor of HIF-1 (Hypoxia-inducible factor-1). The composition of claim 10, wherein the HIF-1 inhibitor comprises a compound represented by Formula 1 or Formula 2. The composition of claim 11, wherein the concentration of the compound represented by Formula 1 or Formula 2 is 0.1 to 20 μM. The composition of claim 10, further comprising a transforming growth factor-β Type I receptor kinase (TβRI) inhibitor.

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