KR100948170B1 - Method for inducing the defferentiation of embryonic stem cells into hemangioblast - Google Patents

Abstract

The present invention provides a composition for inducing embryonic stem cell differentiation comprising MEK / ERK (mitogen-activated protein kinase kinase / extracellular regulated kinase) signaling inhibitor and BMP (bone morphogenetic protein) and mesenchymal from embryonic stem cells using the composition It relates to a method of inducing differentiation into a cell (mesoderm cell). In addition, the mesenchymal cells obtained by the above method can be differentiated into various mesenchymal tissue cells. As a specific example, the present invention provides the vascular endothelial cell growth factor (VEGF) and the basic fibroblast growth factor (BFGF). And inducing differentiation into hemangioblasts by culturing in the presence of Hemangioblasts differentiated as described above can be effectively induced differentiation into vascular endothelial cells, vascular smooth muscle cells and hematopoietic stem cells under various culture conditions.

Embryonic stem cells, mesenchymal cells, hemangioblasts, MEK / ERK signaling inhibitors, BMP

Description

Method for inducing the defferentiation of embryonic stem cells into hemangioblast}

The present invention relates to a composition for inducing stem cell differentiation comprising a MEK / ERK signaling inhibitor and BMP, and a method of inducing differentiation from embryonic stem cells to mesoderm cells using the composition. In addition, the mesenchymal cells obtained by the above method can be differentiated into various mesenchymal tissue cells. As a specific example, the present invention provides the vascular endothelial cell growth factor (VEGF) and the basic fibroblast growth factor (BFGF). And inducing differentiation into hemangioblasts by culturing in the presence of Hemangioblasts differentiated as described above can be effectively induced differentiation into vascular endothelial cells, vascular smooth muscle cells and hematopoietic stem cells under various culture conditions.

Human embryonic stem cells (hESCs) are cells derived from the inner cell mass of human blastocysts (JA Thomson et al, Science 282, 1145-1147, 1998), and autologous growth ( It has the ability to self-renewal and the pluripotency to differentiate into various cell types in the human body (KS O'Shea et al, Anat Rec 257, 32-41, 1999; AM Wobus et al). , Preface, Cells Tissues Organs 165, 129-130, 1999). Due to the characteristics of these human embryonic stem cells, human embryonic stem cells are known to be used for the treatment of diseases caused by deterioration or death of the cell itself, such as diabetes and Parkinson's disease (JH Kim et al. Nature 418, 50-56, 2002; S. Gerecht-Nir et al, Transpl Immunol 12, 203-209, 2004; Y. Hori et al, Proc Natl Acad Sci USA 99, 16105-16110, 2002). To date, several research groups have used human embryonic stem cells to induce differentiation into various cell types. Differentiation of human embryonic stem cells can be largely divided into three methods. First, an embryonic body is formed from human embryonic stem cells (Itskovitz-Eldor J et al. Mol Med 6: 88-95, 2000). Secondly, animal embryos, such as FBS or FCS, are added to human embryonic stem cells growing into a monolayer to differentiate them naturally (Wang et al. Nature biotech 25, 317-318, 2007). ). Finally, human embryonic stem cells are co-cultured with other differentiated cells (Vodyanik, M. A. et al. Blood 105, 617-626, 2005). These three differentiation methods create an environment in which human embryonic stem cells can naturally differentiate, induce the production of various cell types, and take a method of separating and obtaining a cell type desired by an experimenter among the differentiated cells. Therefore, the differentiation efficiency to a specific cell type is low, especially when animal-derived factors or serum are contained, there are great difficulties in studying early human developmental mechanisms through human embryonic stem cells due to various factors in serum.

Embryonic stem cells, on the other hand, can differentiate into ectoderm, mesoderm and endoderm stem cells, among which pluripotent stem cells of mesodermal origin are bone, cartilage tissue, tendons, muscle, fat and blood vessels at the beginning stage. Differentiate into endothelium and the like (Minguell et al., Esp. Biol. Med. 226, 507-520, 2001). Representative cell groups derived from such mesodermal stem cells include so-called hemangioblasts. Pluripotent stem cells of mesodermal origin have self-proliferative properties, and transplanting these cells into various animal model systems is useful for cell therapy because these cells are differentiated at localized sites and differentiated into blood vessels and various blood cells. . In order to obtain sufficient amounts of such stem cells of mesodermal origin to be used for cell therapy, a technique for inducing differentiation from embryonic stem cells to stem cells of mesodermal origin is required. However, the method of effectively inducing differentiation of embryonic stem cells into stem cells of mesodermal origin has not been known so far at home and abroad.

In addition, as a technique for inducing differentiation from embryonic stem cells to hemangioblasts, endothelial cells are cultured by culturing embryonic stem cells in a medium containing VEGF, bFGF, insulin-like growth factor (IGF) and epidermal growth factor (EGF). Method for inducing differentiation of the embryonic stem cells (international patent publication WO 03/040319), embryonic stem cells selected from SCF (stem cell factor), FLT-3 ligand, IL-3, IL-6 and granulocyte colony stimulating factor (G-CSF) Method of producing hematopoietic lineage by culturing in an environment containing hematopoietic growth factor (US Patent Publication No. US 2003/0153082), human embryonic body by complex culture with human placental matrix cells Method of inducing differentiation into hematopoietic stem cells (Domestic Patent Application No. 10-2006-0009934) and method of inducing differentiation of human embryoid bodies into hematopoietic stem cells by complex culture of human embryoid bodies with human bone marrow stromal cells (Domestic Patent Application No. 10-2006) 2004-00975 38). However, there is no known attempt to induce differentiation into hemangioblasts by regulating the signaling system of embryonic stem cells.

Under these backgrounds, the present inventors have made efforts to find ways to induce differentiation of embryonic stem cells into mesodermal stem cells by controlling the signaling system of embryonic stem cells. As a result, MEK / ERK signaling is transmitted to embryonic stem cells. It was found that treatment with inhibitors and BMP can induce differentiation into mesodermal stem cells with high efficiency, and furthermore, the treatment of VEGF and bFGF in differentiated mesenchymal stem cells by the above method is excellent for inducing differentiation into hemangioblasts. The present invention has been completed by finding an effect. This differentiation induction method has the advantage that can effectively produce a variety of cells by controlling the signaling system of human embryonic stem cells without the addition of serum derived from animals.

One object of the present invention is to provide a composition for inducing embryonic stem cell differentiation comprising a MEK / ERK signaling inhibitor and BMP.

Another object of the present invention is to provide a method of inducing differentiation from embryonic stem cells to mesoderm cells using the composition.

Still another object of the present invention is to provide a method of inducing differentiation into hemangioblasts by culturing the mesenchymal cells obtained by the above method in the presence of VEGF and bFGF.

Still another object of the present invention is to provide a method of inducing differentiation into vascular endothelial cells by culturing hemangioblasts obtained by the above method in the presence of VEGF and bFGF.

Still another object of the present invention is to provide a method of inducing differentiation of vascular smooth muscle cells by culturing hemangioblasts obtained by the above method in the presence of PDGF-BB.

Still another object of the present invention is to provide a method of inducing differentiation into hematopoietic stem cells by culturing hemangioblasts obtained by the above method in MethCult GF H4434 (Stem Cell Technologies, Canada).

Accordingly, in one aspect, the present invention relates to a composition for inducing embryonic stem cell differentiation comprising a MEK / ERK signaling inhibitor and BMP.

As used herein, the term "embryonic stem cell" refers to a cell cultured in vitro by extracting an inner cell mass from an blastocyst embryo just before the fertilized egg implants in the mother's womb. It refers to a cell that may be pluripotent or totipotent capable of differentiating into cells, and broadly 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 of the present invention can be derived from a mammal, including human, preferably human embryonic stem cells.

As used herein, the term “differentiation” refers to a phenomenon in which a cell's structure or function is specialized during cell division and proliferation. Pluripotent stem cells can differentiate into lineage-defining progenitor cells (eg, ectoderm cells, mesoderm cells, or endoderm cells, etc.) and then further differentiate into other types of progenitor cells (eg, hemangioblasts, etc.). Then, they may be differentiated into terminal differentiated cells (eg, vascular endothelial cells and vascular smooth muscle cells, etc.) which play a characteristic role in specific tissues (eg, blood vessels, etc.).

As used herein, the term "MEK / ERK signaling inhibitor" refers to MEK1 / 2, which is an upstream molecule of ERK1 / 2 and ERK1 / 2 involved in mitogen-activated protein kinase kinase / extracellular regulated kinase (MEK) signaling. Means the substances that are targeted.

Mitogen-activated protein kinase kinase (MEK) is an enzyme that acts at the end of the MAP kinase signaling system in the cytoplasm and acts as an important mediator of extracellular signals into the nucleus and threonine (Thr) of the myelin basic protein. The residues are phosphorylated in vitro. ERK is a representative MAP kinase present in higher organisms that phosphorylates threonine (Thr) and tyrosine (Tyr) residues by external signals. Since such phosphorylation of threonine and tyrosine residues plays a crucial role in MAP kinase activation, it has been reported that enzymes are not activated when these amino acid residues are substituted with other amino acid residues.

The present invention demonstrates for the first time that the inhibition of signaling by MEK / ERK is effective in regulating stem cell differentiation, and the MEK / ERK signaling inhibitor included in the composition of the present invention preferably means PD98059 or U0126. PD98059 is 2- (2-amino-3-methoxyphenyl) -4H-1-Benzopyran-4-one2- (2'-Amino-3'-methoxy) -flavone2- (2-amino-3-methoxyphenyl) -chromone , U0126 is represented by 1,4-diamino-2,3-dicyano-1,4-bis [2-aminophenylthio] butadiene. However, it will be apparent to those skilled in the art that all MEK / ERK signaling inhibitors in addition to the above compounds fall within the scope of the present invention. ERK1 / 2 is activated by MEK 1/2, and inhibiting the activity of MEK 1/2 immediately inhibits the activity of ERK1 / 2, thus MEK 1/2 is a direct upstream signaling molecule of ERK1 / 2.

    In the present invention, the term "BMP (bone morphogenetic proteins)" is a bone growth factor, known as a substance that promotes bone formation, but in the present invention is used as a substance for controlling the differentiation of embryonic stem cells. BMP of the present invention preferably means BMP-2, BMP-4, or BMP-7.

The present invention is characterized in that the embryonic stem cells are cultured under the stimulation of MEK / ERK signaling inhibitor and BMP, the stimulation method is not particularly limited, but preferably the method of adding the substance in the medium. In addition, any method having the same effect as that of adding the substance to the medium can be used.

The compositions of the present invention may comprise only MEK / ERK signaling inhibitors and BMPs as stem cell differentiation inducing agents, or may further include other differentiation inducing agents to cause synergistic effects between these differentiation inducing agents. Differentiation inducing agents that can be additionally used in the compositions of the present invention can be any known as inducing agents of differentiation of stem cells. Preferably, the composition of the present invention is preferably included at a concentration of 20 μM to 50 μM in the medium composition in the case of MEK / ERK signaling inhibitor, and is preferably included at a concentration of 10 μM to 20 μM in the medium composition in the case of BMP. .

In addition, when the composition of the present invention is used in the form of a medium composition, it may include a general base medium additive, for example, serum, amino acids, antibiotics and differentiation regulators are possible, but are not limited thereto.

Embryonic stem cell differentiation inducing composition comprising MEK / ERK signaling inhibitor and BMP of the present invention can effectively induce differentiation of embryonic stem cells into mesodermal series cells including mesenchymal cells. Such mesodermal cells include mesenchymal cells, hemangioblasts, vascular endothelial cells, vascular smooth muscle cells, or hematopoietic stem cells. .

As used herein, the term "mesoderm cell" refers to a pluripotent stem cell of mesodermal origin, which mesenchymal cells are bone, cartilage tissue, tendons, muscle, fat and blood vessels at the developmental stage. Differentiate into endothelium and the like (Minguell et al., Esp. Biol. Med. 226, 507-520, 2001). Representative cell groups derived from such mesodermal stem cells include so-called hemangioblasts. Mesenchymal cells of the present invention have self proliferation and pluripotency.

In order to confirm differentiation from embryonic stem cells to mesenchymal cells, detection of markers specific for mesenchymal cells can be used. Examples of markers specific for mesenchymal cells include Goosecoid, Brachyury, TBX-4, TBX-5 and TBX-6. The method for detecting the expression of various markers specific for such mesenchymal cells is not particularly limited, but amplification, detection, detection of mRNA encoding any marker protein as reverse transcriptase mediated polymerase chain reaction (RT-PCR) or hybridization assay, It can be confirmed by molecular biological methods commonly used in the art for interpretation. Nucleic acid sequences encoding marker proteins specific for mesenchymal cells are already known so that genes are available from public databases such as GenBank and can easily determine the marker specific sequences required for use as primers or probes. . More preferably, expression of the marker can be confirmed at the protein level using immunochemical methods such as immunohistochemical staining or immunoelectrophoresis. In immunochemical methods, marker-specific polyclonal antibodies or monoclonal antibodies that bind to mesenchymal cells can be used, and commercially available antibodies can be used.

In addition, the composition of the present invention may further comprise VEGF and bFGF, VEGF and bFGF can induce differentiation of mesenchymal cells into hemangioblasts (hemangioblast).

As another aspect, the present invention relates to a method for inducing differentiation from embryonic stem cells to mesoderm cells using a composition for inducing embryonic stem cell differentiation comprising a MEK / ERK signaling inhibitor and BMP. .

In addition, the present invention relates to a method of inducing differentiation into hemangioblasts by culturing the mesenchymal cells obtained by the above method in the presence of VEGF and bFGF. The present invention also relates to a method of inducing differentiation into vascular endothelial cells by culturing hemangioblasts obtained by the above method in the presence of VEGF and bFGF. The present invention also relates to a method of inducing differentiation into vascular smooth muscle cells by culturing hemangioblasts obtained by the above method in the presence of PDGF-BB. The present invention also relates to a method of inducing differentiation into hematopoietic stem cells by culturing hemangioblasts obtained by the above method in MethCult GF H4434 medium.

Specifically, embryonic stem cell differentiation induction method of the present invention comprises the steps of 1) preparing undifferentiated embryonic stem cells; 2) culturing the embryonic stem cells in a medium comprising a composition for inducing embryonic stem cell differentiation comprising a MEK / ERK signaling inhibitor and BMP to differentiate into mesenchymal cells; 3) culturing the differentiated mesenchymal cells in the presence of VEGF and bFGF to differentiate into hemangioblasts; And 4) differentiating the differentiated hemangioblasts into vascular endothelial cells, vascular smooth muscle cells, and hematopoietic stem cells.

First, step 1) is to prepare embryonic stem cells in undifferentiated state, it is preferable to co-culture embryonic stem cells with feeder cells. Generally, serum is used to proliferate cells. However, embryonic stem cells can be co-cultured by attaching onto feeder cells instead of serum because they cannot maintain undifferentiated state when cultured with serum. As a mitomycin C-treated mouse-derived fibroblast-derived mouse fibroblasts (MEF (mouse embryonic fibroblast, MEF) or STO (ATCC, USA)) can be used.

In addition, when embryonic stem cells are cultured without feeder cells (feeder-free culture), they may be cultured in a matrigel-coated culture dish to which a conditioned medium (CM) by feeder cells is added. As used herein, the term "conditioned medium" refers to a culture solution produced by culturing vegetative MEF or STO in human embryonic stem cell medium to which bFGF is added. Specifically, in the present invention, STO (ATCC, USA) was used as a feeder cell to prepare a conditioned medium, which was 10% fetal bovine serum (Hyclone, USA), 0.1 mM non-essential amino acid, 1X penicillin / Incubated in DMEM (Invitorgen, USA) medium supplemented with streptomycin and 0.5 mM beta-mercaptoethanol, and then inactivated in 10 μg / ml mitomycin-C (Sigma, USA) for 2 hours and 30 minutes. It was.

Step 2) is a step of inducing differentiation into mesenchymal cells by treating the prepared embryonic stem cell-derived embryos with MEK / ERK signaling inhibitor and BMP. The MEK / ERK signaling inhibitor is not particularly limited as long as it inhibits MEK / ERK signaling and interferes with the normal signaling process. Preferably, PD98059 or U0126 may be used. At this time, the MEK / ERK signaling inhibitor is preferably treated at a concentration in the culture medium of 20 to 50 μM. BMP is preferably BMP-2,4 or 7 and treated at a concentration in culture medium of 10-20 μM. It is preferable to treat MEK / ERK signaling inhibitors and BMP simultaneously than to treat each of them alone (FIG. 2A).

In addition, when inducing differentiation into mesenchymal cells, in addition to the MEK / ERK signaling inhibitor and BMP, it may further include a known substance and a general medium additive for promoting the induction of differentiation of embryonic stem cells into mesenchymal cells. Step 2) is generally carried out for a sufficient time for the differentiated cells to form, preferably 3 to 5 days.

Step 3) is a step of culturing the differentiated mesenchymal cells in the presence of VEGF and bFGF to differentiate into hemangioblasts. VEGF and bFGF are preferably included in the medium at a concentration of 50 ng / ml. In addition, the VEGF and bFGF may further include a known substance and a general medium additive which promotes differentiation induction into hemangioblasts. Step 3) is generally carried out for a sufficient time for differentiated cells to form, preferably 3 to 5 days.

Step 4) is a step of differentiating the differentiated hemangioblasts into vascular endothelial cells, vascular smooth muscle cells and hematopoietic stem cells under appropriate culture conditions. In order to differentiate the hemangioblasts into vascular endothelial cells, it is preferable to culture in the presence of VEGF and bFGF. At this time, VEGF and bFGF are preferably cultured for 3 to 5 days at a concentration of 50 ng / ml. In addition, in order to differentiate the hemangioblasts into vascular smooth muscle cells, it is preferable to culture in the presence of platelet-derived growth factor (BB) (PDGF-BB), wherein the PDGF-BB concentration in the medium to 50ng / ㎖ 3 days It is preferable to incubate for 5 days. In addition, in order to differentiate the hemangioblasts into hematopoietic stem cells, it is preferable to culture in MethCult GF H4434 (Stem Cell Technologies, Canada), preferably 15 days.

In a specific embodiment of the present invention, human embryonic stem cells were differentiated according to the above four steps (FIG. 1). First, in step 1, human embryonic stem cells were co-cultured on STO cell line, which is a feeder cell line, and then one colony was cut into 300 to 500 μm diameter using a 10 ml syringe needle for feeder-free culture. It was placed on top and incubated for 2 days in a medium containing 4-8 ng / ml bFGF. In the second step, human embryonic stem cells cultured in a conditioned medium for 2 days were added to the unconditioned medium at concentrations of 20-50 μM and 10-20 ng / ml of MEK1 / 2 inhibitors PD98059 and BMP-4, respectively, and cultured for 3 days. . At this time, "unconditioned medium" refers to embryonic stem cell culture medium to which bFGF is not added. In step 3, VEGF and bFGF were added to an unconditioned medium at a concentration of 50 ng / ml and incubated for 3 days. In step 4, cells expressing CD34, an angioblast marker, were obtained by separating only CD34 positive cells using CD34 microbeads. Obtained CD34 positive cells were cultured for about 5 days by adding VEGF and bFGF to EGM (Endothelial Cell Growth Medium, clonetics, USA) to differentiate into vascular endothelial cells. In addition, in order to differentiate CD34 positive cells into vascular smooth muscle cells, 50 ng / ml PDGF-BB was added to EGM (Endothelial cell Growth Medium, clonetics, USA) and cultured for about 5 days. In addition, to differentiate CD34 positive cells into hematopoietic stem cells, the cells were cultured in MethCult GF H4434 (Stem Cell Technologies, Canada) for about 15 days, and the experimental method was performed according to the manufacturer's protocol.

Differentiation-induced cells from embryonic stem cells by the differentiation induction method of the present invention has a morphological, physiological or immunological characteristics, preferably the degree of gene expression of the differentiation-induced cell-specific markers may be increased. . For mesenchymal cells induced differentiation from human embryonic stem cells, among mesenchymal cell specific markers such as Goosecoid, Brachyury, TBX-4, TBX-5 and TBX-6 One or more gene expression levels may be characterized as increased. In addition, hemangioblasts differentiated from mesenchymal cells have CD34 positive characteristics. In addition, vascular endothelial cells induced differentiation from hemangioblasts, von Willerbrand factor (vWF), Ephrin receptor B4 (EphB4), Vascular Endothelial-cadherin (VE-cadherin), CD105 (endoglin) And CD31 (PECAM-1) may be characterized by an increased degree of gene expression. In the case of vascular smooth muscle cells induced differentiation from hemangioblasts, vascular smooth muscle cell specific marker genes SM22α, SM-MHC (smooth muscle-myosin heavy chain), PDGF-B receptor, α-SMA (α-smooth muscle actin) ) And the degree of expression of one or more genes of calponin may be increased.

In a specific embodiment of the present invention, in order to confirm the differentiation-inducing effect of the composition of the present invention and the method of inducing differentiation using the same, mesenchymal cell specificity by treating human embryonic stem cells with MEK / ERK signaling inhibitor and BMP of the present invention Expression of the marker genes Goosecoid, Brachyyu, TBX-4, TBX-5 and TBX-6 were identified by RT-PCR and immunofluorescence staining (FIG. 2). Treatment of mesenchymal cells with VEGF and bFGF confirmed the expression of CD34, an angioblast-specific marker, by RT-PCR (FIG. 3). In addition, RT-PCR expression of markers specific to each cell to confirm whether the differentiation from the differentiated hemangioblasts to vascular endothelial cells (Fig. 4), vascular smooth muscle cells (Fig. 5) and hematopoietic stem cells (Fig. 6) As a result, it was confirmed that the composition of the present invention has an excellent differentiation inducing effect.

As described above, MEK / ERK signaling inhibitors and BMPs can be used to modulate the signaling system of human embryonic stem cells to induce differentiation into mesenchymal cells, and VEGF and bFGF in the differentiation-induced mesenchymal cells. Treatment of the cells can effectively induce hemangioblasts capable of differentiating embryonic stem cells into vascular endothelial cells, vascular smooth muscle cells, and hematopoietic stem cells without serum. This differentiation method can effectively induce differentiation of embryonic stem cells into desired cells and can provide an important mechanism for studying early human development.

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. Culture of Human Embryonic Stem Cells

In the present invention, the human embryonic stem cell culture medium is 20% knockout serum replacement (Knockout serum replacement, Invitrogen, USA), 0.1 mM non-essential amino acid (NEAA; Invitrogen, USA), 0.1 mM DMEM / F12 (Invitrogen, USA) containing beta-mercaptoethanol, 4 ng / ml recombinant human basic FGF (Invitrogen, USA) and 1X penicillin-streptomycin (Invitrogen, USA) A medium was used and used by filtration with a 0.22 mm filter.

After coculture of human embryonic stem cells on feeder cell line, STO cell line, one colony was cut to 300-500 μm in diameter using 10 ml syringe needle for feeder-free culture, and placed on Matrigel, 4 Incubated for 2 days in conditioned medium to which bFGF was added to ˜8 ng / ml.

Example  2. Human Embryonic Stem Cells Mesenchyme  Induce differentiation into cells

2-1. RT - PCR Confirmation by

Human embryonic stem cells cultured in Example 1 were added to MEK1 / 2 inhibitor PD98059 and BMP-4 in condition medium at concentrations of 20-50 μM and 10-20 ng / ml, respectively, and then cultured for 3 days. In order to confirm that the differentiation into mesenchymal cells, the expression of mesenchymal-specific marker genes was examined by RT-PCR. 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 control, a sample treated with only 5 days was used as a control medium, and as an experimental group, PD98059 and BMP-4 were added alone to an unconditioned medium at concentrations of 20 to 50 μM and 10 to 20 ng / ml, respectively, and then put into a cell culture medium. The progress was observed for 3 and 5 days. In addition, the samples treated with PD98059 and BMP-4 simultaneously observed the progress for 3 and 5 days.

As a result, as shown in FIG. 2 (A), mesenchymal cell specific markers BRACHYURY, GOOSECOID, TBX-4, TBX-5, and TBX-6 were expressed only in the samples treated with PD98059 and BMP-4 simultaneously. Expression of mesenchymal cell specific markers did not occur in samples treated with PD98059 or BMP-4 alone.

2-2. Immunofluorescence staining  Confirmed by

In order to confirm the differentiation into mesenchymal cells at the protein level, the embryonic stem cell-specific markers and the mesenchymal cell-specific markers in samples treated with PD98059 and BMP-4 for 5 days in conditioned medium Expression was examined by immunofluorescence staining. In order to stain the mesenchymal-derived human embryonic stem cells with BRACHYURY and GATA-2 markers specific to mesenchymal cells, first, the samples treated with PD98059 and BMP-4 for 3 days were treated with 4% paraformaldehyde for 20 minutes at room temperature. After fixation, wash three times with PBST solution (0.1% Tween-20 in PBS) for 5 minutes. In order to allow the antibody to permeabilize the cells to the nucleus, a permeate solution (addition of 0.1% Triton X-100 to PBS) was placed in a culture dish and left at room temperature for 15 minutes. After 15 minutes, the permeate solution was removed, 4% FBS (Fetal Bovine Serum) was added thereto, and blocking was performed at room temperature for 1 hour. Then, the Goat anti-human OCT4 and Mouse anti-human SSEA-4 antibodies were diluted 1: 300 in the blocking solution, and the Goat anti-human BRACHYURY and Goat anti-human GATA-2 antibodies were diluted 1: 100 and cultured. After putting in a plate, it was placed at 4 ℃ for one day. The next day, secondary antibodies to the antibodies of these markers (Alexa 488) to visually confirm the expression of the above markers (OCT4, SSEA-4, BRACHYURY, GATA-2) using a fluorescent microscope. And Donkey anti-goat IgG conjugated with Alexa 594 and Goat anti-mouse IgG conjugated with Alexa 488) were allowed to stand at room temperature for 1 hour. After 1 hour, and washed 5 times with PBST solution for 10 minutes, and confirmed the expression of markers through a fluorescent microscope.

As a result, as shown in Figure 2 (B), the expression of embryonic stem cell-specific markers OCT3 / 4 and SSEA-4 decreased with treatment of PD98059 and BMP-4, BRACHYURY mesenchymal cell-specific markers And expression of GATA-2.

Example  3. Differentiation-induced from human embryonic stem cells Mesenchyme  Cellular Into hemangioblasts  Differentiation induction

The mesenchymal cells obtained in Example 2 were incubated for 3 days with VEGF and bFGF at a concentration of 50 ng / ml, respectively, in an unconditioned medium, followed by endothelial cell specific markers (Tie-2, CD31, CD105 and KDR) and embryos. The expression of stem cell specific markers (NANOG and OCT4) and hemangioblast specific markers (CD34) was confirmed by RT-PCR. 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 of treatment of VEGF and bFGF simultaneously for 3 days and 5 days, as shown in FIG. 3, expression of embryonic stem cell specific markers OCT4 and NANOG disappeared and endothelial cell specific markers (Tie-2, CD31, CD105). And KDR) and hemangioblast specific markers increased. The HUVEC used in this experiment is a positive control group and shows the expression level of endothelial cell markers and hemangioblast specific markers in VEGF and bFGF treated samples.

Example  4. derived from human embryonic stem cells Hemangioblast  Isolation and Confirmation of Differentiation into Vascular Endothelial Cells

4-1. RT - PCR Confirmation by

Cells expressing CD34, an angioblast marker, were obtained by separating only CD34 positive cells using CD34 microbeads. The obtained CD34 positive cells were treated with VEGF and bFGF at concentrations of 50 ng / ml in Endothelial Cell Growth Medium (Clontics, USA) in order to differentiate into vascular endothelial cells, and then cultured for about 5 days, and then vascular endothelial cells. The expression of specific markers was examined through RT-PCR. 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 FIG. 4 (a), when CD34-positive cells were cultured in EGM to which VEGF and bFGF were added, vWF (von Willerbrand factor), EphB4 (Ephrin receptor B4), and VE, which are vascular endothelial cell specific markers, were observed. Expression of -cadherin (Vascular Endothelial-cadherin), CD105 (endoglin) and CD31 (PECAM-1) was expressed at levels similar to the positive control HUVEC.

4-2. Immunofluorescence staining  Confirmed by

In order to confirm the differentiation into vascular endothelial cells at the protein level, the expression of vascular endothelial cell-specific markers was examined by immunofluorescence staining. To stain human embryonic stem cells differentiated into vascular endothelial cells, PECAM-1, vWF, and VE-cadherin, the samples were first fixed with 4% paraformaldehyde at room temperature for 20 minutes, followed by PBST solution. (0.1% Tween-20 in PBS) was washed three times for 5 minutes. In order to allow the antibody to permeabilize the cells to the nucleus, a permeate solution (addition of 0.1% Triton X-100 to PBS) was placed in a culture dish and left at room temperature for 15 minutes. After 15 minutes, the permeate solution was removed, 4% NGS (Normal Goat Serum) was added thereto, and blocking was performed at room temperature for 1 hour. Then, the antimicrobial mouse anti-human PECAM-1, rabbit anti-human vWF, and mouse anti-human VE-cadherin antibody were diluted 1: 100 in a blocking solution and placed in a culture dish, and then placed at 4 ° C. for one day. The next day, to visually confirm the expression of the above markers (PECAM-1, vWF, VE-cadherin) using a fluorescent microscope, secondary antibodies to the antibodies of these markers (Alexa 488 and Alexa) 594 conjugated Goat anti-mouse IgG and Alexa 488 conjugated Goat anti-Rabbit poly) were attached and allowed to stand at room temperature for 1 hour. After 1 hour, after washing for 5 minutes with PBST solution for 5 minutes, the expression of the markers was confirmed by fluorescence microscope.

As a result, as shown in Figure 4 (b)-(d), the expression of vascular endothelial cell-specific markers vWF, VE-cadherin and CD31 (PECAM-1) was confirmed.

4-3. AcLDL  Confirm Absorption, Form and Code Structure Formation

In addition, mature endothelial cells have the property of absorbing LDL (Low Density Lipoprotein). To determine whether these characteristics are also in vascular endothelial cells differentiated from human embryonic stem cells, AcLDL (acetylated LDL) is about 4 hours. After the addition in the culture medium, it was confirmed that AcLDL is absorbed into vascular endothelial cells induced by differentiation from human embryonic stem cells through a fluorescence microscope (Fig. 4 (e)).

In addition, it was confirmed through phase contrast microscopy that differentiation-induced vascular endothelial cells from human embryonic stem cells were similar to the pebble-shaped forms of mature vascular endothelial cells (FIG. 4 (f)).

Finally, vascular endothelial cells, when placed on a material called Matrigel, have the property of forming wire-like structures. In order to confirm that vascular endothelial cells induced by differentiation from human embryonic stem cells maintain the same characteristics, cord-shaped cells were cultured for 24 hours with differentiation-induced vascular endothelial cells placed on human embryonic stem cells on Matrigel. It was confirmed to form the structure of (Fig. 4 (g)).

Example  5. Derived from human embryonic stem cells Hemangioblast Into vascular smooth muscle cells  Differentiation confirmation

5-1. RT - PCR Confirmation by

In order to differentiate CD34 positive cells into vascular smooth muscle cells, 50 ng / ml of PDGF-BB was added to Endothelial Cell Growth Medium (Clontics, USA) and cultured for 5 days, followed by expression of vascular smooth muscle cell markers. It was checked through RT-PCR. 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 5 (a), CD34 positive cells are SM22α, SM-MHC (smooth muscle-myosin heavy chain), which is a marker gene specific for vascular smooth muscle cells in EGM-2 culture medium added with PDGF-BB, PDGF-B receptor, α-smooth muscle actin (α-SMA) and calponin (calponin) were expressed.

5-2. Immunofluorescence staining  Confirmed by

In order to confirm the differentiation into vascular smooth muscle cells at the protein level, the expression of vascular smooth muscle cell-specific markers was examined by immunofluorescence staining. To stain human embryonic stem cells differentiated with vascular smooth muscle cells, α-smooth muscle actin (α-SMA) and calponin, which are specific markers of vascular smooth muscle cells, first, the samples were treated with 4% paraformaldehyde for 20 minutes at room temperature. After fixing at, it was washed three times with PBST solution (0.1% Tween-20 added to PBS) for 5 minutes. In order to allow the antibody to permeabilize the cells to the nucleus, a permeate solution (addition of 0.1% Triton X-100 to PBS) was placed in a culture dish and left at room temperature for 15 minutes. After 15 minutes, the permeate solution was removed, 4% NGS (Normal Goat Serum) was added thereto, and blocking was performed at room temperature for 1 hour. Then, the mouse anti-human α-SMA and rabbit anti-human calponin antibody were diluted 1: 100 in a blocking solution and placed in a culture dish, and then placed at 4 ° C. for 1 day. The next day, in order to visually confirm the expression of the above markers (α-SMA, calponin) using a fluorescence microscope, Goat anti bound to antibodies of these markers (Alexa 488 bound) Goat anti-Rabbit poly) conjugated with -mouse IgG and Alexa 594 was attached and allowed to stand at room temperature for 1 hour. After 1 hour, after washing for 5 minutes with PBST solution for 10 minutes, the expression of the markers was confirmed by fluorescence microscope.

As a result, as shown in Figure 5 (b), it can be seen that the vascular smooth muscle cell-specific markers α-SMA and calponin (calponin) is also expressed at the protein level.

Example  6. Derived from human embryonic stem cells Hemangioblast To hematopoietic stem cells  Differentiation confirmation

To differentiate CD34 positive cells into hematopoietic stem cells, the cells were cultured in MethCult GF H4434 (Stem Cell Technologies, Canada) for about 15 days, and the test method was performed according to the manufacturer's protocol. To determine whether CD34 positive cells differentiate into hematopoietic stem cells, a CFU (colony forming unit) assay was performed. In order to perform the CFU assay, CD34 positive cells were first washed through IMDM (Iscove's MDM) medium, and the cells were serially diluted (500, 1000, 5000, 5x10 ⁴ , 1x10 ⁵ , etc.). It was added to a special medium synthesized by StemCell Technology's own semi-solid methylcellulose polymer. The components added in the medium are as follows.

~ Iscove's MDM

~ 1% Methylcellulose

~ 30% Fetal Bovine serum

~ 1% Bovine Serum Albumin

~ 10 ^-4 M 2-Merchaptoethanol

~ 2 mM L-glutamine

~ 50ng / ml Stem Cell Factor

~ 10ng / ml GM-CSF

~ 10ng / ml IL-3

~ 3U / ml Erythropoietin

After adding CD34 positive cells to the above medium, the cells were cultured at 37 ° C. and 5% CO ₂ conditions for 14 to 20 days to confirm the shape of the cells in the culture dish and the number of colonies. Characterized.

As a result, as shown in Figure 6, it was confirmed that blood cells, such as macrophage (macrophage), erythroid (erythroid) and granulocyte (granulocyte) is generated from the CD34 positive cells. Through this, it can be seen that the CD34 positive cells induced by the present inventors are differentiated into hematopoietic stem cells.

1 is a diagram illustrating a method of inducing differentiation of human embryonic stem cells into hematopoietic stem cells capable of differentiating into hematopoietic stem cells, vascular endothelial cells, and vascular smooth muscle cells.

Figure 2 shows the results of confirming the expression of mesenchymal-specific marker genes by RT-PCR (a) and immunofluorescence staining (b) to confirm whether human embryonic stem cells have been differentiated into mesenchymal cells.

Figure 3 shows endothelial cell specific markers (Tie-2, CD31, CD105 and KDR) and embryonic stem cell specific markers (NANOG and OCT4) to determine whether mesenchymal cells differentiated from human embryonic stem cells are induced differentiation into hemangioblasts. ), And whether hemangioblast specific marker (CD34) is expressed through RT-PCR.

Figure 4 shows whether the expression of vascular endothelial cell-specific markers vWF, EphB4, VE-cadherin, CD105 and CD31 to determine whether hemangioblasts derived from human embryonic stem cells induced vascular endothelial cells RT-PCR (a) And immunofluorescence staining (bd). In addition, AcLDL was confirmed to be absorbed into vascular endothelial cells induced by differentiation from human embryonic stem cells through fluorescence microscopy (e). It was confirmed through a microscope (f), and cultured for 24 hours with differentiation-induced vascular endothelial cells derived from human embryonic stem cells on Matrigel, and confirmed that they formed cord-shaped structures (g). Indicates.

5 is a result of confirming the expression of vascular smooth muscle cell markers by RT-PCR (a) and immunofluorescence staining (b) in order to determine whether hemangioblasts derived from human embryonic stem cells induced differentiation into vascular smooth muscle cells.

6 is a result of performing a colony forming unit (CFU) assay to determine whether hemangioblasts derived from human embryonic stem cells are differentiated into hematopoietic stem cells, macrophage, erythroid, granulocyte, etc. This is a result of confirming that the blood cells are produced.

Claims (20)

Composition for inducing embryonic stem cell differentiation comprising MEK / ERK (mitogen-activated protein kinase kinase / extracellular regulated kinase) signaling inhibitor and BMP (bone morphogenetic protein). The composition of claim 1, wherein the embryonic stem cells are human embryonic stem cells. The composition of claim 1, wherein the BMP is BMP2, BMP4 or BMP7. The composition of claim 1, wherein the MEK / ERK signaling inhibitor is PD98059 or U0126. The composition of claim 1, wherein the concentration of MEK / ERK signaling inhibitor is 20-50 μM. The composition of claim 1 wherein the concentration of BMP is 10-20 μM. The composition of claim 1, further comprising vascular endothelial cell growth factor (VEGF) and basic fibroblast growth factor (bFGF). A method of inducing differentiation of embryonic stem cells into mesodermal lineage cells comprising culturing embryonic stem cells in the presence of a MEK / ERK signaling inhibitor and BMP. The method of claim 8, wherein the mesoderm-based cells are meoderm cells, hemangioblasts, vascular endothelial cells, vascular smooth muscle cells or hematopoietic cells stem cell). The method of claim 8, wherein the BMP is BMP2, BMP4 or BMP7. The method of claim 8, wherein the MEK / ERK signaling inhibitor is PD98059 or U0126. The method of claim 8, wherein the concentration of MEK / ERK signaling inhibitor is 20-50 μM. The method of claim 8, wherein the concentration of BMP is 10-20 μM. The method of claim 8, wherein the culture is incubated for 3 days to 5 days. Iii) culturing embryonic stem cells in the presence of MEK / ERK signaling inhibitor and BMP to induce differentiation into mesenchymal cells; And ii) culturing the mesenchymal cells in the presence of VEGF and bFGF to induce differentiation into hemangioblasts, inducing differentiation of embryonic stem cells into hemangioblasts.  The method of claim 15, wherein the concentrations of VEGF and bFGF are each 50 ng / ml. The method according to claim 15, wherein the culture is carried out in the culture medium of step ii) for 3 to 5 days. Iii) culturing embryonic stem cells in the presence of MEK / ERK signaling inhibitor and BMP to induce differentiation into mesenchymal cells; Ii) culturing the mesenchymal cells in the presence of VEGF and bFGF to induce differentiation into hemangioblasts; And iii) culturing the hemangioblasts in the presence of VEGF and bFGF to induce differentiation into vascular endothelial cells. Iii) culturing embryonic stem cells in the presence of MEK / ERK signaling inhibitor and BMP to induce differentiation into mesenchymal cells; Ii) culturing the mesenchymal cells in the presence of VEGF and bFGF to induce differentiation into hemangioblasts; And iii) culturing the hemangioblasts in the presence of platelet-derived growth factor-BB (PDGF-BB) to induce differentiation into vascular smooth muscle cells, the embryonic stem cells into vascular smooth muscle cells. How to induce differentiation. Iii) culturing embryonic stem cells in the presence of MEK / ERK signaling inhibitor and BMP to induce differentiation into mesenchymal cells; Ii) culturing the mesenchymal cells in the presence of VEGF and bFGF to induce differentiation into hemangioblasts; And iii) inducing the differentiation of embryonic stem cells into hematopoietic stem cells, comprising incubating the hemangioblasts in MethCult GF H4434 (Stem Cell Technologies, Canada) to induce differentiation into hematopoietic stem cells. .

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