WO2016088931A1 - Method for preparing induced pluripotent stem cells from adipose-derived mesenchymal stem cells and differentiating same into osteoblasts - Google Patents

Abstract

The present invention relates to a culture medium composition, for induced pluripotent stem cell dedifferentiation, comprising an Ecklonia cava extract. In addition, the present invention relates to a method for differentiating induced pluripotent stem cells, which are prepared by means of the culture medium composition, into osteoblasts. By means of the culture medium composition, according to the present invention, induced pluripotent stems cell can be efficiently prepared by using mesenchymal stem cells, and the prepared pluripotent stem cells can be differentiated into osteoblasts. Therefore, the present invention can be utilized for a patient-specific cell therapy product.

Description

Method of producing induced pluripotent stem cells from adipose-derived mesenchymal stem cells to differentiate into osteoblasts

The present invention relates to a method for preparing patient-specific induced pluripotent stem cells using the pluripotent stem cell induction medium composition of human fat-derived mesenchymal stem cells and differentiating them into osteoblasts.

Stem cells collectively refer to the undifferentiated cells in the pre-differentiated stage that can be obtained from each tissue. In the undifferentiated state, it is capable of continuously producing the same cells as itself for a certain period of time, and under proper conditions, it is capable of differentiating into various cells constituting biological tissues.

Stem cells can be largely divided into embryonic stem cells and adult stem cells according to their differentiation capacity and production time. Another classification is according to the differentiation capacity of stem cells, which can be divided into pluripotency, multipotency and unipotency stem cells.

Adult stem cells can be divided into multipotent or unipotent stem cells. Representative adult stem cells include mesenchymal stem cells (MSCs) and hematopoietic stem cells (HSCs). Mesenchymal stem cells differentiate into chondrocytes, osteoblasts, adipocytes, myocytes, and neurons. Hematopoietic stem cells mainly contain blood cells such as red blood cells, white blood cells, and platelets. It is known to differentiate into cells.

Pluripotent stem cells, on the other hand, refer to stem cells that have the versatility to differentiate into all three germ layers that make up a living body and to differentiate into all cells or organ tissues of the human body, and generally embryonic stem cells. This is the case. Human embryonic stem cells have many ethical problems because they are made from embryos that can occur in human life, but they are known to have superior cell proliferation and differentiation capacity as adult stem cells. Adult stem cells can be obtained from bone marrow, blood, brain, skin, etc., so there are few ethical problems, but they have limited differentiation ability compared to embryonic stem cells.

As an alternative to overcome this problem, various methods have been attempted to prepare customized pluripotent stem cells similar to embryonic stem cells by dedifferentiating adult-derived cells. Representative methods include fusion with ES cells, somatic cell nuclear transfer, and reprogramming by gene factor. The cell fusion method has a problem in that the induced cells have two more pairs of genes, and the somatic cell nuclear transfer method requires a large amount of eggs and a very low efficiency. In addition, the specific factor injection method uses a virus containing a carcinogenic gene to induce differentiation by inserting a specific gene, which poses a high risk of cancer, and in view of the possibility of developing a cell therapy due to low efficiency and difficulty in terms of methodology. It is a problem.

In order to obtain pluripotent stem cells successfully and in large quantities, the culture composition at the stage of culturing isolated adipose-derived mononuclear cells is very important. It is a state.

Meanwhile, a composition for treating or preventing atopic diseases using Ecklonia cava (Patent No. 10-2009-0043115), a skin composition such as cosmetics (Patent No. 10-2013-0017159), or a hair dye composition for oxidation dyeing (publishing Patent No. 10-2012-0126148), but is used to dedifferentiate adipose-derived mesenchymal stem cells into induced pluripotency stem cells. There is nothing.

The matters described as the background art are only for the purpose of improving the understanding of the background of the present invention and should not be taken as acknowledging that they correspond to the related art already known to those skilled in the art.

[Preceding technical literature]

Patent Document 1. Korean Patent Publication No. 10-2009-0043115

Patent Document 2. Korea Patent Publication No. 10-2013-0017159

Patent Document 3. Korean Patent Publication No. 10-2012-0126148

The present inventors endeavored to find a method of inducing pluripotent stem cells with high efficiency for the practical use of cell therapy with high safety and production efficiency. As a result, when the Ecklonia cava extract, which is a safe natural extract, was added to the cell culture medium, the present invention was completed by confirming that induced pluripotent stem cells can be prepared at a safe and high efficiency using fat-derived mesenchymal stem cells. .

Accordingly, it is an object of the present invention to provide a medium composition for dedifferentiating adipose derived mesenchymal stem cells into induced pluripotent stem cells comprising Ecklonia cava extract.

Another object of the present invention to provide a method for producing induced pluripotent stem cells comprising the step of dedifferentiating adipose-derived mesenchymal stem cells into induced pluripotent stem cells in a medium containing Ecklonia cava extract. Another object of the present invention to provide an induced pluripotent stem cell prepared by the above method. Another object of the present invention is to provide a method for dedifferentiating mesenchymal stem cells into induced pluripotent stem cells in the medium containing the Ecklonia cava extract and then differentiating them into osteoblasts. Another object of the present invention to provide an osteoblast prepared by the above method. Still another object of the present invention is to provide a composition for treating a patient customized cell comprising induced pluripotent stem cells prepared by the above method by separating stem cells from the fat of the patient.

Other objects and advantages of the present invention will become apparent from the following detailed description, claims and drawings.

According to one aspect of the present invention, the present invention is to differentiate the adipose-derived mesenchymal stem cells containing Ecklonia cava extract into induced pluripotency stem cells It provides a medium composition for.

According to another aspect of the present invention, the present invention provides a method for differentiating osteoblasts from mesenchymal stem cells, comprising the steps of: (a) Ecklonia cava extract Adding to the cell culture medium; (b) dedifferentiating the adipose derived mesenchymal stem cells into induced pluripotency stem cells in the medium; And (c) differentiating the induced pluripotent stem cells into osteoblasts.

The present inventors have tried to find a method of inducing high efficiency pluripotent stem cells for the practical use of developing high-efficiency cell therapies without the risk of cancer cell formation without the ethical problem of destroying embryos and without the use of viruses. As a result, when the Ecklonia cava extract, which is a safe natural extract, was added to the cell culture medium, it was confirmed that the induced pluripotent stem cells could be produced with surprisingly high efficiency.

Ecklonia cava ( Ecklonia cava ), an active ingredient included in the medium composition of the present invention is a perennial seaweed of seaweed kelp kelp, which is inhabited in the southern coast, Jeju island coast and Ulleungdo coastal area, and is mainly fed as abalone and conch. It is also used as a major raw material for making alginic acid, iodine, and potassium, and for food.

Ecklonia cava extract according to the present invention comprises water, (a) anhydrous or hydrous lower alcohol having 1 to 4 carbon atoms (methanol, ethanol, propanol, butanol, normal-propanol, iso-propanol and normal-butanol, etc.), (b) the Mixed solvent of lower alcohol and water, organic solvents such as (c) acetone, (d) ethyl acetate, (e) chloroform, (f) 1,3-butylene glycol, (g) hexane, (h) diethyl ether The extraction may be performed using a solvent, and preferably, extraction may be performed using a mixed solvent of methanol or ethanol and water or each of them. When extracted using a mixed solvent, the content of methanol or ethanol is preferably 50-80 v / v%.

As used herein, the term "embryonic stem cell" refers to a cell having pluripotency as a cell cultured by separating from an inner cell mass of a blastocyst that is early in development after fertilization. As used herein, the term "pluripotent stem cell" refers to a stem having pluripotency capable of differentiating into three germ layers constituting a living body, that is, endoderm, mesoderm, and ectoderm. Refers to a cell.

As used herein, the term "differentiation" refers to a phenomenon in which structures or functions are specialized while cells divide and proliferate and grow, that is, a cell or tissue of an organism has a shape or function to perform a task given to each. It means to change.

As used herein, the term "cell therapeutic agent" is a cell and tissue manufactured by separating, culturing and special manipulation from humans and used as a medicine for the purpose of treatment, diagnosis and prevention, and to restore the function of the cell or tissue. It refers to a medicament used for the purpose of treatment, diagnosis and prevention through a series of actions such as proliferating, selecting, or otherwise altering the biological characteristics of allogeneic or heterologous cells in vitro. Cell therapy agents are largely classified into somatic cell therapy and stem cell therapy according to the degree of differentiation of cells, and the present invention relates in particular to stem cell therapy.

"Mesenchymal stem cells" of the present invention are cells isolated from embryonic stem cells or adult stem cells derived from mammals, preferably fat-derived mesenchymal stem cells, and more preferably fat-derived mesenchyme of the human body Stem cells. The fat-derived stem cells can be obtained from adipose tissue of the human body. Sampling of mesenchymal stem cells from fat can be performed using a variety of methods. For example, in order to separate mononuclear cells from adipose tissue, adipose tissue is collected from the human body to DPBS (Dulbecco's Phosphate-Buffered Saline). Rinse until no blood comes out, and the washed umbilical cord is cut with a surgical blade and incubated at 37 ° C. to obtain a mononuclear cell-containing solution.

As used herein, the term "medium" refers to cells of cells such as stem cells in vitro , including elements essential for the growth and proliferation of cells such as sugars, amino acids, various nutrients, serum, growth factors, and minerals. Refers to a mixture for incubation or differentiation.

In particular, the medium of the present invention is a medium for culturing mesenchymal stem cells. Herein, the mesenchymal stem cells are cells isolated from embryonic stem cells or adult stem cells derived from mammals, and have the ability to feed indefinitely and various cell types (eg, fat cells, chondrocytes, muscle cells, bones). Cells, etc.). In the present invention, a multipotent mesenchymal stem cell having an immunophenotype that shows a positive response to antibodies against CD73, CD90 and CD105, and a negative response to antibodies to CD34 and CD45 is used.

Various media are commercially available in the art, and may be artificially prepared and used. For example, commercially available media include Dulbecco's Modified Eagle's Medium (DMEM), Minimal Essential Medium (MEM), Basic Medium Eagle (BME), RPMI 1640, F-10, F-12, DMEM F-12, and α-MEM ( α-Minimal Essential Medium, G-MEM (Glasgow's Minimal Essential Medium), IMPM (Iscove's Modified Dulbecco's Medium), AmnioMax, AminoMax II complete Medium (Gibco, Newyork, USA), Chang's Medium MesemCult-XF Medium (STEMCELL Technologies, Vancouver, Canada) and the like, and can be used as a basal medium included in the medium composition of the present invention together with a medium that can be artificially produced.

Serum components (for example, Fetal Bovine Serum (FBS)) and antibiotics (for example, penicillin, streptomycin), which are commonly added, may be added to the basal medium. The concentration of serum component or antibiotic component added to the basal medium may vary within a range capable of achieving the effect of the present invention, preferably 10% FBS, 100 unit / ml penicillin, 50 μg / ml streptomycin, or the like. Can be added.

In addition, the medium of the present invention may further comprise a nutrient mixture (Nutrient Mixture). The nutrition mixture is a mixture containing various amino acids, vitamins, inorganic salts, and the like generally used in cell culture, and may be prepared by mixing the amino acids, vitamins, inorganic salts, and the like, or use a commercially prepared nutrition mixture. Commercially prepared nutrient mixtures include, but are not limited to, M199, MCDB110, MCDB202, MCDB302, and the like.

In addition, the medium of the present invention may further include energy water for induction and stabilization of pluripotent stem cells. The energy water is preferably added at 0.01 to 10 v / v%, more preferably at 0.05 to 0.5 v / v%.

The medium composition of the present invention is a medium specific for pluripotent stem cell induction, and may be achieved by adding Ecklonia cava extract to the basal medium, and may preferably comprise Ecklonia cava extract at a concentration of 10 to 400 μg / ml.

According to another aspect of the invention, the present invention comprises the steps of adding the Ecklonia cava extract to the cell culture medium; And dedifferentiating adipose-derived mesenchymal stem cells into induced pluripotency stem cells in the medium.

According to one embodiment of the present invention, when using the medium composition containing the Ecklonia cava extract of the present invention as the experimental group (medium extract containing Ecklonia cava extract, and energy water in DMEM F-12 medium) and only DMEM F-12 medium as a control group Unlike when used, it was confirmed that pluripotent stem cell colonies were formed on day 8-10 (FIG. 2 and FIG. 3).

According to another aspect of the present invention, the present invention provides an induced pluripotent stem cell prepared by the above method.

Induced pluripotent stem cells of the present invention have the same differentiation capacity as embryonic stem cells, and are almost identical to embryonic stem cells in the shape of cells (FIGS. 2 and 3). According to an embodiment of the present invention, as a result of examining the expression of the genes (Oct4, Sox-2) and protein (SSEA-4) characteristic to the embryonic stem cells embryonic stem cells and pluripotent stem cells induced by the present invention It was confirmed that the same genes and proteins are expressed (Figs. 4 and 5).

According to another embodiment, mesenchymal stem cells (MSC) that have not undergone the induction process have low expression levels of OCT4, SOX2 and Nanog, which are characteristic genes of pluripotent stem cells, whereas pluripotency induced by the method of the present invention In stem cells (Experimental Example 1-1: EtOH EPN, Experimental Example 1-2: Sonic EPN), these characteristic genes were significantly expressed (Figs. 6 and 7). Therefore, the induced pluripotent stem cells of the present invention can effectively differentiate into osteoblasts.

According to one embodiment of the present invention, it was confirmed that cells expected to be pluripotent stem cells can be differentiated into osteoblasts (FIG. 8).

According to another aspect of the present invention, the present invention provides a cell therapy composition comprising the differentiated osteoblasts.

The composition of the present invention can be administered by any route of administration, specifically by intraperitoneal or thoracic cavity administration, by intravenous or arterial vascular administration, or by topical administration by injection.

In the present invention, the composition can be administered in the form of injections, suspensions, emulsifiers and the like based on conventional methods, and if necessary, suspended in an adjuvant such as Freund's complete adjuvant or adjuvant activity such as BCG. It is also possible to administer with the substance having. The composition may be sterile or contain adjuvants such as stabilizers, hydrating or emulsifying accelerators, salts or buffers for controlling osmotic pressure and other therapeutically valuable substances.

The cell therapy composition of the present invention can be applied to arthritis, nervous system diseases, endocrine diseases, liver diseases, and the like, and according to the results of clinical trials for humans in the future, there is also the possibility of treating allogeneic cells for humans.

The features and advantages of the present invention are summarized as follows:

(i) The present invention provides a medium composition for induced pluripotent stem cell reverse differentiation comprising Ecklonia cava extract.

(ii) The present invention also provides a method for producing pluripotent stem cells using the medium composition.

(iii) By using the medium composition according to the present invention, it is possible to efficiently produce induced pluripotent stem cells using adipose-derived mesenchymal stem cells, and the prepared pluripotent stem cells are capable of differentiating into various cells, thereby treating cells. It can be usefully used as.

(iv) The present invention eliminates ethical problems caused by embryo destruction by not using embryonic stem cells by producing pluripotent stem cells having the same differentiation capacity as embryonic stem cells, and thus does not use viruses that can cause cancer. Safe pluripotent stem cells can be produced without risk.

(v) Furthermore, since natural extracts are used, pluripotent stem cells can be produced very easily and significantly higher than conventional methods, and mesenchymal stem cells isolated from adipocytes of patients can be used for the practical application of stem cell therapeutics tailored to patients. Expect to be able to advance. It is believed that the present invention will greatly contribute to the treatment of various intractable diseases such as neurological diseases and immune diseases.

1 is a diagram showing that the pluripotent stem cells that are almost identical to embryonic stem cells are induced when cultured by injecting Ecklonia cava extract medium from adipose-derived mesenchymal stem cells.

Figure 2 shows the pluripotent stem cell colony formation induced by the concentration of Ecklonia cava extract (ethanol extract) by the method of the present invention (Example 1-1).

Figure 3 shows the pluripotent stem cell colony formation induced by the concentration of Ecklonia cava extract (water extract) by the method of the present invention (Example 1-2).

Figure 4 confirms that pluripotent stem cells induced by the method of the present invention (Experimental Example 1-1) by using specific protein expression.

5 shows that pluripotent stem cells induced by the method of the present invention (Experimental Example 1-2) are pluripotent stem cells using specific protein expression.

6 is a graph showing the gene expression of pluripotent stem cells induced by the method of the present invention (Experimental Example 1-1) and graphs thereof.

Figure 7 shows the gene expression of pluripotent stem cells induced by the method of the present invention (Experimental Example 1-2) and graphically depicted them.

Figure 8 is the result of differentiation into osteoblasts using the osteoblast differentiation medium with pluripotent stem cells induced by the method of the present invention.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention. .

Example 1: Preparation of Ecklonia cava Extract

Example 1-1 Preparation of Ecklonia cava Extract Using Ethanol Solvent

The herbal samples used in the experiment were purchased from Jeju Island and used in the experiment after accurate evaluation by experts. 100 g of the dried herbal sample was placed in 1 L of 70% ethanol, and ethanol was extracted under reflux for 16 hours and filtered using a filter paper. The filtrate was concentrated in a rotary evaporator and immediately freeze dried.

Example 1-2 Preparation of Ecklonia cava Extract Using Water

The herbal samples used in the experiment were purchased from Jeju Island and used in the experiment after accurate evaluation by experts. 100 g of the dried herbal sample was placed in 1 L of water, and water was extracted using an ultrasonic extractor for 16 hours, and filtered using a filter paper. The filtrate was concentrated in a rotary evaporator and immediately freeze dried.

Example 2: Isolation and Culture of Mesenchymal Stem Cells from Human Adipose Tissue

Example 2-1: Collection of Human Adipose Tissue

Adipose tissue is collected immediately after liposuction. The sample collects aspirated adipose tissue in 500 ml sterile glass bottles before being transferred to the laboratory. The sterile glass bottle is then sealed and transferred to the laboratory. In the laboratory, mesenchymal stem cells are extracted in a flow hood of class 100 under sterile conditions. The sample is first transferred to a sterile stainless steel container. The PBS was washed several times and then the adipose tissue sample was then cut into 2 cm lengths and transferred to a 50 ml tube where additional washing and anti-infection with 70% ethanol and antibiotic mixture (50 IU / ml penicillin, 50 ug / ml) Wash several times with PBS added with streptomycin (purchased from Invitrogen) until the solution is clear.

Example 2-2: Isolation and Culture of Mesenchymal Stem Cells from Human Adipose Tissue

The isolated adipose tissue was washed with PBS, the tissue was chopped, and digested for 1 hour while shaking once every 10 minutes at 37 ° C. using DMEM medium to which collagenase type 1 (1 mg / ml) was added. Next, washed with PBS and centrifuged for 5 minutes at 1000 rpm. The supernatant was aspirated and the pellet remaining on the bottom was washed with PBS and centrifuged at 1000 rpm for 5 minutes. Filtered with a 100 μm mesh size filler to remove debris and washed with PBS.

For the isolation / culture of mesenchymal cells, the explanted tissue was treated with 5 ml of DMEM (Dulbecco's modified eagle medium) F-12 (Gibco), 10% FBS, 100 units with 10% fetal calf serum (FBS, Hyclone). / Ml penicillin, 50 ㎍ / ㎖ streptomycin in a 95% nitrogen, 5% carbon dioxide cell culture incubator at 37 ℃ to maintain the hypoxic state to kill the cells other than stem cells, the purity of mesenchymal stem cells Raised. Medium was changed every 3 or 4 days. Outgrowth of cells was monitored by light microscopy. Elongating cells were trypsinized (0.125% trypsin / 0.05% EDTA) for further expansion and cryopreservation (using DMEM / 10% FBS).

For extraction of mesenchymal stem cells, pellets of cells were resuspended and counted in medium DMEM F-12 (Gibco), 10% FBS, 100 unit / ml penicillin, 50 μg / ml streptomycin, and 10 cm tissue culture dishes. Were inoculated at a density of 1 × 10 ⁶ cells / dish. The medium was changed every 3 or 4 days. Cell growth and cloning were monitored by light microscopy. At about 90% confluence, the cells were sub-cultured as described above.

Experimental Example 1 Induction of Pluripotent Stem Cells from Adipose-Derived Mesenchymal Stem Cells

Experimental Example 1-1 Human fat-derived mesenchyme according to Ecklonia cava extract concentration of Example 1-1 Pluripotent stem cell production of stem cells

Experiments to induce pluripotent stem cells from human adipose-derived stem cells according to the concentration of Jeju Ecklonia cava extract. The control group was a medium of MSC. DMEM F-12 (Gibco), 10% FBS, 100 unit / ml penicillin, / Ml streptomycin was used as the primary medium, and the experimental group used the Jeju Ecklonia cava extract prepared in Example 1-1 in medium using human adipose-derived mesenchymal stem cells subjected to subculture 3, Normal, 1 ㎍ / ml Concentrations of 20 μg / ml, 50 μg / ml, 100 μg / ml, 400 μg / ml, 800 μg / ml, 1 mg / ml and energy water (SiO ₂ , Al ₂ O ₃ , TiO ₃ , Fe ₂ O ₃ , CaO, Na ₂ O, K ₂ O, purified de-ionized water containing LiO, Estinicin) 0.1 v / v% was added (FIG. 1). Monocytes isolated from human adipose derived mesenchymal stem cells were inoculated with 1 × 10 ⁴ cells in 6-well plates and cultured at 37 ° C. and 5% CO ₂ .

As a result, in the experimental group, the colony was clearly formed after 10 days when the concentration of the Jeju Ecklonia cava extract was 100 to 400 ㎍ / ㎖ (Fig. 2), the microscope magnification was observed at a 200-fold ratio.

Experimental Example 1-2: Preparation of Pluripotent Stem Cells of Human Adipose-derived Mesenchymal Stem Cells According to Ecklonia cava Extract Concentrations of Example 1-2

Experimental in the same manner as in Experimental Example 1-1, the Jeju Ecklonia cava extract was prepared in Example 1-2 was used. As a result, in the experimental group, the colony was clearly formed after 10 days when the concentration of the Jeju Ecklonia cava extract was 20 to 50 μg / ml (FIG. 3), and the microscope magnification was observed at a 200-fold ratio.

Experimental Example 1-3 Analysis of Immunochemical Staining of Pluripotent Stem Cells Induced by the Method of the Present Invention

Whether the expression of OCT4, SOX2, or the protein SSEA-4 (stage-specific embryonic antigen-4), which is a specific gene of embryonic stem cells, was expressed in the pluripotent stem cells induced by the methods of Experimental Examples 1-1 and 1-2 above Antibodies were used to analyze protein expression using immunochemical staining.

The staining process was first fixed with 4% paraformaldehyde (Paraformaldehyde), and then washed with PBS and blocked (blocking) with 1% BSA solution. After treatment with the primary antibody against OCT4, SOX3, SSEA-4 for 18 hours at 4 ℃, washed with PBS and treated with a secondary antibody with a fluorescent pigment (FITC) for the primary antibody at room temperature The reaction was carried out for 1 hour.

After washing with PBS, the expression was analyzed using a fluorescence microscope, and the results are shown in FIGS. 4 and 5. Figure 4 is a result for pluripotent stem cells induced by Ecklonia cava extract extracted with ethanol, Figure 5 is a result for pluripotent stem cells induced by Ecklonia cava extract extracted with water.

The shape of stem cell colonies can be seen by the bright field results of FIGS. 4 (a) and 5 (b).

In addition, in the staining results for proteins expressed from the pluripotent stem cell specific markers OCT4, SOX2, and SSEA-4 of FIGS. 4 (b) and 5 (b), all positive reactions occurred, indicating that pluripotent stem cells were prepared. I could confirm it.

The presence of stem cells was once again confirmed through staining of the cell nuclei by DAPI of FIGS. 4 (c) and 5 (c).

Experimental Example 1-4: Pluripotent stem cell gene analysis comparison

After observing the pluripotent stem cells prepared in Experimental Examples 1-1 and 1-2 under a microscope, only colonies were removed using a 200 μl pipette, and total RNA was isolated using TRIzol reagent (manufactured by Invitrogen). CDNA was synthesized by reverse transcriptase-polymerase chain reaction (RT-PCR), and then PCR was performed using primers specific for OCT4, Sox-2, Nanog and the control glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene. . Nanog, OCT4 and Sox-2 are characteristic genes seen in embryonic stem cells. PCR products were analyzed by agarose gel electrophoresis, and the results of confirming the expression of these genes are shown in FIGS. 6 and 7.

As a result, according to FIGS. 6 and 7, the mesenchymal stem cells (control group, MSC) that did not undergo the induction process had low expression levels of OCT4, SOX2 and Nanog, which are characteristic genes of pluripotent stem cells, while Pluripotent stem cells (pluripotent stem cells prepared by Experimental Example 1-1 (denoted by EtOH EPN) and Experimental Example 1-2 (denoted by Sonic EPN)) induced by the expression of these characteristic genes were significantly higher. The expression levels of the stem cell genes OCT4, SOX2 and Nanog can be clearly seen through the graphs of FIGS. 6 and 7.

Experimental Example 2: Differentiation into Osteoblasts

Induce pluripotent stem cells from adipose derived mesenchymal stem cells by culturing in a culture medium of 95%, 37 ° C, and 5% CO ₂ using medium containing Ecklonia cava extract and energy water to induce differentiation into osteoblasts. Then, osteoblast differentiation solution was incubated for 2 weeks in DMEM F-12, 1uM dexamethasone, 10mM, β-glycerol phosphate, 0.2mM ascorbic acid, and 1uM BMP (bone morphogenic protein). As a result of confirming the differentiation into osteoblasts through Von kossa histochemical staining, it was confirmed that the cells that were expected to be pluripotent stem cells could be differentiated into osteoblasts by showing positive reactions to Von kossa (FIGS. 8A and B). .

Having described the specific part of the present invention in detail, it is apparent to those skilled in the art that the specific technology is merely a preferred embodiment, and the scope of the present invention is not limited thereto. Thus, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

Claims (9)

1. A method for differentiating osteoblasts from mesenchymal stem cells, comprising the following steps:

   (a) dedifferentiating adipose-derived mesenchymal stem cells obtained from human fat in a medium comprising Ecklonia cava extract into induced pluripotency stem cells; And

   (b) differentiating the induced pluripotent stem cells into osteoblasts.
2. According to claim 1, wherein the medium of step (a) Dulbecco's Modified Eagle's Medium (DMEM), Minimal Essential Medium (MEM), Basic Medium Eagle (BME), RPMI 1640, F-10, F-12, DMEM-F12 , α-MEM (α-Minimal Essential Medium), G-MEM (Glasgow's Minimal Essential Medium), IMDM (Iscove's Modified Dulbecco's Medium), MacCoy's 5A medium, AmnioMax, AminoMaxII complete Medium or Chang's Medium MesemCult-XF Medium How to.
3. According to claim 1, wherein the Ecklonia cava extract is characterized in that it comprises a concentration of 10 to 400 ㎍ / ㎖ in the medium.
4. The method of claim 1, wherein the Ecklonia cava extract of step (a) is extracted with an ethanol solvent, characterized in that it comprises a concentration of 100 to 400 ㎍ / ㎖ in the medium.
5. The method according to claim 1, wherein the Ecklonia cava extract of step (a) is extracted with water and is contained at a concentration of 20 to 50 μg / ml in the medium.
6. The method of claim 1, wherein the medium of step (a) further comprises an energy water of 0.01 to 10 v / v%.
7. The method of claim 1, wherein step (b) is performed using a medium containing dexamethasone, β-glycerol phosphate, ascorbic acid, and bone morphogenic protein (BMP). Characterized in that.
8. Osteoblasts differentiated by the method of any one of claims 1 to 7.
9. Cell therapy composition comprising osteoblasts of claim 8.

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