KR20090076483A - Method for the differentiation of mesenchymal stem cells to osteoblasts and medium for the differentiation thereof - Google Patents

Abstract

A method for differentiating a human mesenchymal stem cell which is positive to CD73, CD90, CD105, and CD166 to osteoblast is provided to shorten differentiation time and have stability in use. An osteroblast is differentiated by culturing a human mesenchymal stem cell which is positive to CD73, CD90, CD105, and CD166 in medium with a fucoidan. The concentration of fucoidan is 0.1-5 mg/ml. The medium obtained by adding the fucoidan comprises the fucoidan, fetal bovine serum(FBS), dexamethasone, beta-glycerophospyhate, and low-glucose DMEM (Dulbecco's Modified Eagle Medium ) having ascorbic acid. The mesenchymal stem cell is bone marrow, umbilical cord blood, or adipose-derived mesenchymal stem cell.

Description

Method for differentiation of mesenchymal stem cells to osteoblasts and medium for the differentiation according to differentiation of mesenchymal stem cells into osteoblasts

The present invention relates to a method for differentiating mesenchymal stem cells into osteoblasts and a medium for differentiation used in the differentiation method.

Mesenchymal stem cells are cells that can be separated from bone marrow, umbilical cord blood, adipose tissue, and the like, and refer to primitive cells immediately before they are differentiated into human tissue cells, which play an important role in tissue regeneration. The mesenchymal stem cells are cells capable of differentiation into bone, cartilage, fat, blood vessels, liver, and the like.

Mesenchymal stem cells have the disadvantages of small cell number and difficulty in proliferation, but are free from ethical debate because they are easy to obtain and are extracted from human tissues that have grown, unlike embryonic stem cells. Among the many tissues from which mesenchymal stem cells can be extracted, the most representative ones are bone marrow, umbilical cord blood, fetal tissue, and adipose tissue. Among these, adipose derived mesenchymal stem cells have the same characteristics as bone marrow-derived mesenchymal stem cells and can be obtained from tissue discarded after liposuction and are relatively easy to separate.

At present, there is an active research on a method for using stem cells in tissue engineering. However, inducers for differentiating mesenchymal stem cells can cause problems in safety because they can affect the human body after transplantation.

On the other hand, fucoidan is a kind of water-soluble dietary fiber found in brown algae such as seaweed and kelp used for food, and has been reported to have apoptosis activity of cancer cells. Treatment of fucoidan with cancer cells causes DNA to be cut into small fragments, causing cell death. This process of cell death occurred in cancer cells, but not in the case of treatment with fucoidan on stem cells. There are many studies on the mechanism of the death of cancer cells by fucoidan, but the process of fucoidan affects the growth and differentiation of stem cells has not been revealed at all.

[Reference 1] Ronan O'LEARY, Mark REREK, and Edward John WOOD Fucoidan Modulates the Effect of Transforming Growth Factor (TGF) -b 1 on Fibroblast Proliferation and Wound Repopulation in in Vitro Models of Dermal Wound Repair Biol . Pharm . Bull . 27 (2) 266-270 (2004)

[2] Laura B. Grabel, Charles G. Glabe, Mark S. Singer, Gail R. Martin and Steven D. Rosen A FUCAN SPECIFIC LECTIN ON TERATOCARCINOMA STEM CELLS BBRC . 102 (4) 1165-1171 (1981)

[Reference 3] KIMINORI MATSUBARA, CHANGHU XUE, XUE ZHAO4 MASAHARU MORI, TATSUYA SUGAWARA and TAKASHI HIRATA Effects of middle molecular weight fucoidans on in in vitro and ex vivo angiogenesis of endothelial cells. INTERNATIONAL JOURNAL OF MOLECULAR MEDICINE 15: 695-699 (2005)

[4] Karim Senni, Farida Gueniche, Alexandrine Foucault-Bertau, Sylvie Igondjo-Tchen, Florence Fioretti, Sylvia Colliec-Jouault, Patrick Durand, Jean Guezennec, Gaston Godeau, Didier Letourneur. Fucoidan a sulfated polysaccharide from brown algae is a potent modulator of connective tissue proteolysis. Archives of Biochemistry and Biophysics 445: 5664 (2006)

Document 5 Guangxia Gao and Mitchell Goldfarb. Heparin can activate a receptor tyrosine kinase. The EMBO Journal 14 (10): 2183-2190 (1995)

Document 6 ELIZABETH A. SWEENEY AND THALIA PAPAYANNOPOULOU. Increase in Circulating SDF-1 after Treatment with Sulfated Glycans. The Role of SDF-1 in Mobilization. Ann NY Acad Sci . 2001 Jun; 938: 48-52

Document 7 DELPHINE CHABUT, ANNE-MARIE FISCHER, SYLVIA COLLIEC-JOUAULT, INGRID LAURENDEAU, SABINE MATOU, BERNARD LE BONNIEC, and DOMINIQUE HELLEY. Low Molecular Weight Fucoidan and Heparin Enhance the Basic Fibroblast Growth Factor-Induced Tube Formation of Endothelial Cells through Heparan Sulfate-Dependent 6 Overexpression. Mol Pharmacol 64: 696702 (2003)

8 Faouzia Zemani, Danielle Benisvy, Isabelle Galy-Fauroux, Anna Lokajczyk, Sylvia Colliec-Jouault, Georges Uzan, Anne Marie Fischer, Catherine Boisson-Vidal. Low-molecular-weight fucoidan enhances the proangiogenic phenotype of endothelial progenitor cells. Biochemical Pharmacology 70: 1167-1175 (2005)

[9] Johannes Meiler and Martin Schuler. Therapeutic Targeting of Apoptotic Pathways in Cancer. Current Drug Targets , 7: 1361-1369 (2006)

[Patent 10] Japanese Patent Laid-Open No. 04-91027

Document 11 US Patent Publication No. 2006/0051316

The present inventors conducted various searches for biocompatible natural products as various differentiation inducers capable of effectively inducing differentiation of mesenchymal stem cells. As a result, fucoidan, a seaweed polysaccharide that is harmless to the human body, is used as osteoblasts of mesenchymal stem cells. It was found that it effectively promotes the differentiation of.

Accordingly, an object of the present invention is to provide a method for differentiating mesenchymal stem cells into osteoblasts using fucoidan.

In addition, an object of the present invention is to provide a medium for differentiation of mesenchymal stem cells into osteoblasts used in the differentiation method.

According to one aspect of the present invention, in a method for differentiating human mesenchymal stem cells showing osteoblasts (CD73 positive, CD90 positive, CD105 positive, and CD166 positive) into osteoblasts, the fucoidan in the osteoblast differentiation medium Provided is a method for differentiating mesenchymal stem cells into osteoblasts, which comprises culturing the mesenchymal stem cells in a medium obtained by adding a).

The fucoidan may be added at a concentration of 0.1-5 mg / ml. The medium obtained by adding fucoidan to the osteoblast differentiation medium is low-glucose supplemented with fucoidan, fetal bovine serum (FBS), dexamethasone, β-glycerophosphate, and ascorbic acid. glucose) DMEM medium, more preferably low supplemented with 0.1-5 mg / ml fucoidan, 10% fetal calf serum, 0.1 μM dexamethasone, 10 mM β-glycerophosphate, and 0.05 mM ascorbic acid -Glucose DMEM medium.

In addition, the medium obtained by adding fucoidan to the osteoblast differentiation medium may be a medium containing no differentiation-inducing substance, and preferably low-glucose supplemented with fucoidan, fetal calf blood, β-glycerophosphate, and ascorbic acid. DMEM medium, more preferably low glucose DMEM medium supplemented with 0.1-5 mg / ml fucoidan, 10% fetal calf serum, 10 mM β-glycerophosphate, and 0.05 mM ascorbic acid have.

In the differentiation method of the present invention, the mesenchymal stem cells may be mesenchymal stem cells derived from bone marrow, umbilical cord blood, or fat, and preferably may be mesenchymal stem cells derived from fat.

According to another aspect of the present invention, there is provided a medium for differentiating mesenchymal stem cells into osteoblasts, which is obtained by adding 0.1-5 mg / ml of fucoidan to osteoblast differentiation medium.

The medium for differentiation of mesenchymal stem cells into osteoblasts may be low glucose DMEM medium supplemented with 0.1-5 mg / ml of fucoidan, fetal bovine serum, dexamethasone, β-glycerophosphate, and ascorbic acid, and more. Preferably it may be low-glucose DMEM medium supplemented with 0.1-5 mg / ml fucoidan, 10% fetal calf serum, 0.1 μM dexamethasone, 10 mM β-glycerophosphate, and 0.05 mM ascorbic acid.

In addition, the medium for differentiating mesenchymal stem cells into osteoblasts may be low glucose DMEM medium supplemented with 0.1-5 mg / ml of fucoidan, fetal bovine serum, β-glycerophosphate, and ascorbic acid, and more. Preferably, low glucose DMEM medium supplemented with 0.1-5 mg / ml fucoidan, 10% fetal calf serum, 10 mM β-glycerophosphate, and 0.05 mM ascorbic acid.

The differentiation method of mesenchymal stem cells into osteoblasts according to the present invention is performed in the presence of fucoidan, a seaweed polysaccharide, which can greatly shorten the differentiation period by 50% or more. In other words, the eruption period, which took about four weeks, can be greatly reduced to two weeks. In addition, since the differentiation method of the present invention can eliminate the use of cytokines, ie, differentiation-inducing substances such as dexamethasone, it is possible to avoid safety problems that may arise when transplanting the obtained osteoblasts into the human body.

The present invention is a method for differentiating human mesenchymal stem cells (CD73 positive, CD90 positive, CD105 positive, and CD166 positive) into osteoblasts, a medium obtained by adding fucoidan to osteoblast differentiation medium. And the mesenchymal stem cells, characterized in that the culturing of the mesenchymal stem cells comprising the method of differentiation.

In the present specification, the "fucoidan" is a kind of water-soluble dietary fiber found in brown seaweed such as seaweed and kelp used for food, and contains about 4-5% of dried brown algae, antithrombogenicity, anticancer, It has been reported to have anti-inflammatory, anti-allergic, anti-dementia, antiviral, anti-diabetic, anti-obesity activity. Fucoidan is a generic term for sulfated fucose-containing polysaccharides. Fucoidan can be obtained from brown algae containing the same according to conventional methods (Japanese Patent Laid-Open No. 04-91027, etc.). Fucoidan can be divided into glucuronic acid-containing fucoidan (U-fucoidan) and glucuronic acid non-containing fucoidan (F-fucoidan), and the term "fucoidan" (fucoidan) "includes both of these. In addition, fucoidan may be present in the form combined with galactose, uronic acid, mannose, xylose, and the like, and the term "fucoidan" includes all of the above forms of fucoidan. The molecular weight of the fucoidan is not particularly limited and may be, for example, about 200,000, but may have various molecular weights by sugar chains and the like.

Differentiation method according to the present invention comprises the step of culturing the mesenchymal stem cells in a medium obtained by adding fucoidan to osteoblast differentiation medium.

The content of fucoidan in the medium may be added at a concentration of 0.1 to 5 mg / ml, preferably 0.1 to 0.5 mg / ml. When the fucoidan is in the form of a dry powder, if necessary, the fucoidan may be added to the medium, followed by filtration with a filter (for example, a 0.45 um filter).

In addition, the osteoblast differentiation medium is a conventional medium used to differentiate mesenchymal stem cells into osteoblasts, for example, fetal bovine serum (FBS), dexamethasone, β-glycerophosphate (β-glycerophosphate). ), And a medium obtained by adding fucoidan to a low-glucose DMEM medium supplemented with ascorbic acid. More preferably, the medium used for the differentiation method according to the present invention is 0.1-5 mg / ml of fucoidan, 10% fetal calf serum, 0.1 μM dexamethasone, 10 mM β-glycerophosphate, and 0.05 mM ascorbate. Acid- supplemented low-glucose DMEM medium.

The differentiation method according to the present invention can significantly shorten the differentiation period by 50% or more by using fucoidan as described above, and can also eliminate the use of cytokines, that is, differentiation-inducing substances such as dexamethasone. That is, the medium used in the differentiation method of the present invention does not contain differentiation-inducing substances. For example, it may be dexamethasone-free medium. As an example, the medium used in the differentiation method of the present invention may be low-glucose DMEM medium supplemented with fucoidan, fetal calf blood, β-glycerophosphate, and ascorbic acid, more preferably 0.1-5 mg / ml Low-glucose DMEM medium supplemented with fucoidan, 10% fetal calf serum, 10 mM β-glycerophosphate, and 0.05 mM ascorbic acid.

The differentiation method of the present invention can be applied to all mesenchymal stem cells derived from adult cells. That is, mesenchymal stem cells derived from adult cells all have the characteristics that the immunological phenotype is D73 positive, CD90 positive, CD105 positive, and CD166 positive. Thus, as long as the immunological phenotype has the characteristics of D73 positive, CD90 positive, CD105 positive, and CD166 positive, mesenchymal stem cells from various adult cells, for example from bone marrow, umbilical cord blood, or adipose, preferably The same applies to mesenchymal stem cells derived from fat (fat cells or tissues).

The present invention also includes a medium for differentiation of mesenchymal stem cells into osteoblasts, which is obtained by adding 0.1-5 mg / ml, preferably 0.1-0.5 mg / ml, of fucoidan to osteoblast differentiation medium. .

According to one embodiment of the present invention, the medium for differentiation of mesenchymal stem cells into osteoblasts is 0.1-5 mg / ml of fucoidan, fetal bovine serum (FBS), dexamethasone, β-glycerophosphate ( β-glycerophosphate), and low-glucose DMEM medium supplemented with ascorbic acid, more preferably 0.1-5 mg / ml of fucoidan, 10% fetal bovine serum (FBS) , Low-glucose DMEM medium supplemented with 0.1 μM dexamethasone, 10 mM β-glycerophosphate, and 0.05 mM ascorbic acid.

In addition, the medium for differentiation of the mesenchymal stem cells into osteoblasts may be a medium in which cytokines such as dexamethasone, that is, differentiation-inducing substances are excluded, for example, dexamethasone hand-free (dexamethasone-free) medium Can be. Thus, according to one embodiment of the present invention, the medium for differentiation of mesenchymal stem cells into osteoblasts is low-filled with 0.1-5 mg / ml of fucoidan, fetal bovine serum, β-glycerophosphate, and ascorbic acid. Glucose DMEM medium, more preferably low glucose DMEM medium supplemented with 0.1-5 mg / ml fucoidan, 10% fetal calf serum, 10 mM β-glycerophosphate, and 0.05 mM ascorbic acid Can be.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, the following examples are intended to illustrate the invention and not to limit the invention.

Example 1 Isolation of Mesenchymal Stem Cells from Human Adipose Tissue

The patient received the subcutaneous fat removal procedure, and the adipose tissue was removed from the patient by liposuction method. The collected adipose tissue was washed three times with phosphate buffered saline containing no calcium and potassium. Treated with 0.075% collagenase and left in a 37 ° C. incubator for 1 hour to disrupt adipose tissue and separate cells. DMEM medium containing fetal calf serum was added in the same amount as collagenase and centrifuged at 250 g for 10 minutes. The supernatant was removed and red blood cells were removed by treating the settled precipitate with 0.16 M NH ₄ Cl for 10 minutes. Was separated 10 minutes centrifugation at a rate of 250g in tissue to remove the red blood cells were suspended into the DMEM to the obtained precipitate, divides by 1 X 10 ⁵ on a tissue culture plate with a diameter of 10cm showing the fibroblast form and then cultured for 3 to 4 Cells were selected and passaged according to the condition of the cells (ie, when the cells were filled with 70-80% tissue culture dishes).

Example 2: Identification of immunological phenotype of mesenchymal stem cells

FACS analysis was performed to confirm the immunological phenotype of the mesenchymal stem cells obtained in Example 1. After subcultured 4 ~ 5 times from the adipose tissue, the obtained cells were treated with trypsin-EDTA, separated and collected from the culture dish, and each 1 × 10 ^{4 was placed in} a 1.5 ml tube, each containing calcium and potassium phosphate buffered saline ( phosphate buffered saline). Antibodies that can be used as specific markers of mesenchymal stem cells were treated in each tube, left for 1 hour, and then transferred to a tube for FACS, and the cell surface phenotype was confirmed by FACS analyzer. As the antibody, CD73 (PE), CD90 (FITC), CD105 (FITC), CD166 (FITC) were used as positive markers, and HLA-DR (FITC), CD34 (PE), CD45 (PE) as negative markers. Was used.

The FACS analysis results are shown in FIG. 1. As can be seen in Figure 1, mesenchymal stem cells isolated from adipose tissue are conventionally the same as bone marrow-derived mesenchymal stem cells, CD73 (PE), CD90 (FITC), CD105 (FITC), and CD166 ( FITC), and negative for HLA-DR (FITC), CD34 (PE), and CD45.

Example 3: Induction of Differentiation of Adipose Tissue-derived Mesenchymal Stem Cells into Osteoblasts

To confirm the differentiation ability of adipose tissue-derived mesenchymal stem cells, osteoblast differentiation induction medium was added to a culture dish filled with 70 to 80% of mesenchymal stem cells that passed through 5 to 6 passages, and the medium was twice a week. Replace and incubate for 4 weeks. The composition of the osteoblast differentiation medium was as follows: 10% fetal bovine serum (Ftal bovine serum, FBS, Sigma), 0.1 μM dexamethasone (Sigma), 10 mM β-glycerophosphate (β- glycerophosphate, Sigma), and low-glucose DMEM medium (Gibco BRL) supplemented with 0.05 mM ascorbic acid (Sigma).

Example 4 Induction of Differentiation of Adipose Tissue-derived Mesenchymal Stem Cells into Osteoblasts by Treatment of Fucoidan

Differentiation of mesenchymal stem cells was induced for 2 weeks in the same manner as in Example 3, except that DMEM medium treated with fucoidan at a concentration of 0.1 mg / ml was used. Each time the medium was changed, the fucoidan was treated at the same concentration of 0.1 mg / ml.

Test Example 1 Microscopic Observation of Differentiated Induced Cells

The results of observing the cells differentiated in Example 3 and 4 under an optical microscope are shown in FIG. 2. Figure 2 is the result measured after induction of differentiation for 2 weeks in Examples 3 and 4. As can be seen in Figure 2, when inducing differentiation by culturing mesenchymal stem cells in differentiation medium containing fucoidan, it can be seen that differentiation into osteoblasts was effectively induced in two weeks. On the contrary, when the differentiation medium containing no fucoidan was used, almost no differentiation was performed for 2 weeks (in the middle of FIG. 2), and when the differentiation was induced for 4 weeks, some degree of differentiation was obtained (data Not listed).

Test Example 2: Calcium Deposition Measurement for Differentiation Confirmation

After 2 weeks of induction of differentiation in Examples 3 and 4, the mesenchymal stem cells were differentiated into osteoblasts after 2 weeks through mineralized matrix staining according to von Kossa's method (Silver 10) using silver-nitrate staining. Whether or not was measured. The result is shown in FIG. 3.

As can be seen in Figure 3, it can be seen that when the treatment of fucoidan induces differentiation, it is effectively promoted differentiation of mesenchymal stem cells into osteoblasts.

Test Example 3: Measurement of Osteoblast Marker Gene Expression

Gene expression was confirmed through RT-PCR to confirm the differentiation of adipose tissue-derived mesenchymal stem cells. Collagen type I, osteopontin, and alkaline phosphatase were identified as genes used as markers for differentiation into osteoblasts. PCR conditions such as primers and Tm (Melting temperature) are shown in Table 1 below, and the results are shown in FIG. 4.

gene primer SEQ ID NO: Tm (℃) Col i Forward direction 5'-GGACACAATGGATTGCAAGG-3 ' One 60 Reverse 5'-TAACCACTGCTCCACTCTGG-3 ' 2 OP Forward direction 5'-AGCCAGGACTCCATTGACTCGAAC-3 ' 3 68 Reverse 5'-GTTTCAGCACTCTGGTCATCCAGC-3 ' 4 ALP Forward direction 5'-ACCATTCCCACGTCTTCACATTTG-3 ' 5 57 Reverse 5'-AGACATTCTCTCGTTCACCGCC-3 ' 6

As can be seen in Figure 4, the mesenchymal stem cell control (no control) did not express any genes used as differentiation markers in the control group, the group treated with fucoidan to the mesenchymal stem cells OP and Weak expression of ALP could be observed and it was confirmed that Col I was strongly expressed. In addition, when the differentiation medium and the fucoidan were treated together, there was a strong expression of Col I, OP and ALP compared to the conventional differentiation medium. When the gene expression patterns were compared between the group in which the fucoidan was added to the mesenchymal stem cells and the group treated with the differentiation medium, the expression of Col I was stronger in the group in which the fucoidan was added to the culture medium than in the differentiation medium. Therefore, it can be seen that the differentiation into osteoblasts was promoted by the addition of fucoidan.

Example 5 Induction of Differentiation of Adipose Tissue-derived Mesenchymal Stem Cells into Osteoblasts by Treatment of Fucoidan in Dexamethasone-Free Medium

A medium obtained by treating Fucoidan with osteoblast differentiation induction medium was added to a culture dish filled with 70 to 80% of mesenchymal stem cells passaged 5 to 6 times, and cultured for 2 weeks while changing medium twice a week. The composition of the medium was as follows: 0.1 mg / ml of fucoidan, 10% fetal bovine serum (FBS, Sigma), 10 mM β-glycerophosphate (Sigma), and Low-glucose DMEM medium (Gibco BRL) supplemented with 0.05 mM ascorbic acid (Sigma). Each time the medium was changed, the fucoidan was treated at the same concentration of 0.1 mg / ml.

Test Example 4 Calcium Deposition Measurement and Osteoblast Marker Gene Expression Pattern

After two weeks of induction of differentiation in Example 3 and Example 5, the results of measuring calcium deposition and osteoblast marker gene expression in the same manner as in Test Examples 2 and 4 are as shown in Figures 5 and 6.

As can be seen in Figure 5, induction of differentiation by treating fucoidan can be seen that mesenchymal stem cells are effectively differentiated into osteoblasts despite the addition of differentiation-inducing substances such as dexamethasone. 6, it can be seen that differentiation into osteoblasts is effectively induced by the addition of fucoidan.

1 is an immunological phenotypic analysis of mesenchymal stem cells isolated from adipose tissue.

Figure 2 is a result of measuring the differentiation of mesenchymal stem cells isolated from adipose tissue in fucoidan-treated and untreated conditions for 2 weeks, and then measured with an optical microscope.

Figure 3 is a result of induction of differentiation of mesenchymal stem cells isolated from adipose tissue in fucoidan-treated and untreated conditions for 2 weeks, and then the differentiation into osteoblasts measured by silver-nitrate staining method.

4 is a result of measuring the specific marker gene of osteoblasts by PT-PCR by separating the RNA of mesenchymal stem cells induced differentiation into osteoblasts.

FIG. 5 shows the results of differentiation of mesenchymal stem cells isolated from adipose tissue in dexamethasone-free medium for 2 weeks after treatment with fucoidan and non-treatment, followed by differentiation into osteoblasts by silver-nitrate staining.

6 is a result of measuring specific marker genes of osteoblasts by PT-PCR by separating RNA of mesenchymal stem cells induced by differentiation into osteoblasts when treated with decomethasone-free medium in fucoidan.

<110> College of Medicine Pochon CHA University Industry-Academic Cooperation Foundation <120> Method for the differentiation of mesenchymal stem cells to          osteoblasts and medium for the differentiation <130> PN0150 <160> 6 <170> KopatentIn 1.71 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 1 ggacacaatg gattgcaagg 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 2 taaccactgc tccactctgg 20 <210> 3 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 agccaggact ccattgactc gaac 24 <210> 4 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 4 gtttcagcac tctggtcatc cagc 24 <210> 5 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 5 accattccca cgtcttcaca tttg 24 <210> 6 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 6 agacattctc tcgttcaccg cc 22  

Claims (14)

In the method for differentiating human mesenchymal stem cells showing CD73 positive, CD90 positive, CD105 positive, and CD166 positive into osteoblasts, the medium in the medium obtained by adding fucoidan to osteoblast differentiation medium. A method of differentiating mesenchymal stem cells into osteoblasts, comprising culturing the mesenchymal stem cells. 2. The method of claim 1, wherein said fucoidan is added at a concentration of 0.1-5 mg / ml. According to claim 1, wherein the medium obtained by adding the fucoidan to the osteoblast differentiation medium is a low-fucoidan, fetal bovine serum (FBS), dexamethasone, β-glycerophosphate, and ascorbic acid supplemented Differentiation method characterized in that (low-glucose) DMEM medium. The medium obtained by adding fucoidan to the osteoblast differentiation medium is 0.1-5 mg / ml of fucoidan, 10% fetal calf serum, 0.1 μM dexamethasone, 10 mM β-glycerophosphate, and 0.05. Differentiation method characterized in that the low-glucose DMEM medium supplemented with mM ascorbic acid. The differentiation method according to claim 1, wherein the medium obtained by adding fucoidan to the osteoblast differentiation medium does not contain differentiation-inducing substances. The method of claim 5, wherein the medium obtained by adding fucoidan to the osteoblast differentiation medium is low glucose DMEM medium supplemented with fucoidan, fetal calf blood, β-glycerophosphate, and ascorbic acid. The medium obtained by adding fucoidan to the osteoblast differentiation medium is supplemented with 0.1-5 mg / ml of fucoidan, 10% fetal calf serum, 10 mM β-glycerophosphate, and 0.05 mM ascorbic acid. Differentiation method characterized in that the low-glucose DMEM medium. The method of any one of claims 1 to 7, wherein the mesenchymal stem cells are mesenchymal stem cells derived from bone marrow, umbilical cord blood, or fat. 9. The method of claim 8, wherein said mesenchymal stem cells are mesenchymal stem cells derived from fat. A medium for differentiation of mesenchymal stem cells into osteoblasts obtained by adding 0.1-5 mg / ml of fucoidan to osteoblast differentiation medium. The low-glucose DMEM of claim 10, wherein the medium for differentiating mesenchymal stem cells into osteoblasts is 0.1-5 mg / ml of fucoidan, fetal bovine serum, dexamethasone, β-glycerophosphate, and ascorbic acid. A medium for differentiation of mesenchymal stem cells into osteoblasts, characterized in that the medium. The medium for differentiating mesenchymal stem cells into osteoblasts according to claim 11, wherein the medium for differentiation into osteoblasts is 0.1-5 mg / ml of fucoidan, 10% fetal calf serum, 0.1 μM dexamethasone, 10 mM β-glycerophosphate, and A medium for differentiation of mesenchymal stem cells into osteoblasts, characterized in that it is a low-glucose DMEM medium supplemented with 0.05 mM of ascorbic acid. The medium for differentiating mesenchymal stem cells into osteoblasts is low glucose DMEM medium supplemented with 0.1-5 mg / ml of fucoidan, fetal bovine serum, β-glycerophosphate, and ascorbic acid. A medium for differentiation of mesenchymal stem cells into osteoblasts. The medium for differentiating mesenchymal stem cells into osteoblasts is 0.1-5 mg / ml of fucoidan, 10% fetal bovine serum, 10 mM β-glycerophosphate, and 0.05 mM ascorbic acid. A medium for differentiation of mesenchymal stem cells into osteoblasts characterized in that it is supplemented low-glucose DMEM medium.

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