KR101171958B1 - Method for the differentiation of adipose-derived stem cells into adipocytes using heparin-binding domain - Google Patents

Abstract

The present invention relates to a method for differentiation of adipose stem cells into adipocytes using a heparin-binding domain (HBD), and specifically, HBP having adhesion to adipose-derived stem cells (ASCs). Culture vessel is fixed to the surface; And inoculating the adipose stem cells into the culture vessel and culturing the adipose stem cells in a state of being adhered to the surface of the culture vessel. According to the present invention, when HBD having adhesion to adipose stem cells is fixed in a culture vessel, cell adhesion is induced by the interaction of HBD and adipose stem cells to culture the adipose stem cells adhered to the surface of the culture vessel. It can effectively induce mass differentiation into adipocytes. Therefore, the present invention can be very usefully applied to the fields of differentiation and proliferation of adipose stem cells and regenerative medicine such as tissue engineering and cell therapy.

Adipocyte, adipocyte, cell adhesion, heparin-binding domain, cell differentiation

Description

METHODS FOR THE DIFFERENTIATION OF ADIPOSE-DERIVED STEM CELLS INTO ADIPOCYTES USING HEPARIN-BINDING DOMAIN}

The present invention relates to a method for mass differentiation of adipocytes into adipocytes using a heparin-binding domain (HBD) having adhesion to adipocytes.

Recently, with the development of biotechnology, methods for regenerating tissues and organs using stem cells and restoring their functions have been actively attempted. Stem cells are cells that have the ability of self-replicating and differentiate into two or more types of cells. Totipotent stem cells, pluripotent stem cells, and multipotent stem cells cells).

Recently, it has been reported that human adipose tissue contains an undifferentiated cell population that is capable of differentiating into a variety of cells such as liver, bone, cartilage, fat, blood vessels, heart, and nervous system (B). Cousin et al., BBRC 301: 1016, 2003; Miranville et al., Circulation 110: 349, 2004; S. Gronthos et al., J. Cell. Physiol . 189: 54, 2001; MJ Seo et al., BBRC 328: 258, 2005). As the adipose-derived multipotent stem cells, adipose-derived stem cells (ASCs) are easy and safe to acquire in that they can extract a large amount of adipose tissue in comparison with mesenchymal-based stem cells. It is attracting attention as a new source of multipotent stem cells because of its advantages in terms of tissue accessibility, stability, efficacy, and economics due to its advantages without restriction on tissue supply and demand, and in vitro culture.

On the other hand, extracellular matrix (ECM) adhesion of cells is the basis for regulating life phenomena such as development, wound healing, organ formation, and cancer metastasis. Some extracellular substrates, such as fibronectin, collagen, and laminin, are known to have molecules that can promote cell adhesion. The components that make up the cell's in vivo environment can be used to regulate cell proliferation and differentiation in vitro. For this purpose, it is important to make components such as extracellular matrix as materials for cell adhesion or tissue development. , SH and Akaike, T., Tissue Eng . 13: 601-609, 2007).

Along with these studies, studies are being actively conducted to understand biological adhesion to stem cells, tissue engineering and extracellular matrix. In one example, intracellular proliferation of bone marrow-derived mesenchymal stem cells has shown potential for differentiation from collagen type I to adipocytes (Mauney, JR, et al . , J. Biomed . Mater . Res. 79: 464-475, 2006 Mesenchymal stem cells adhere to titanium surfaces, proliferate well and have the potential for bone differentiation (Maeda, M., et al ., J. Biochem . 141: 729-736, 2007). Fibronectin also plays an important role in cell morphology and function through cell-fibronectin interactions (Dalton, BA, et al., J. Cell Sci . 108: 2083-2092, 1995), type III connecting segments and neighboring heparin-binding domains have been shown to promote adhesion of melanoma cells (McCarthy, JB, et al., J. Cell Biol . 102: 179-88, 1986). Heparan sulfate proteoglycan (HSPG) is involved in the regulation of osteoblast differentiation of mesenchymal stem cells in the cell adhesion and bone formation protein signaling pathways through the heparin-binding domain of fibronectin (Drake, SL, et al. J. Biol . Chem .. 268: 15859-15867, 1993; Manton, KJ, et al., Stem Cells 25: 2845-2854, 2007).

However, most stem cell cultures, including adipose stem cells, have been performed using commercially available culture vessels. Since this study does not consider the effect of the substrate to which the cells adhere, it is necessary to establish the optimal conditions for differentiation. impossible. Therefore, in order to utilize adipose stem cells in regenerative medicine fields such as tissue engineering and cell therapy, it is essential to increase the efficiency of differentiation of adipose stem cells into adipocytes.

Accordingly, the present inventors have made intensive research efforts to develop efficient differentiation methods of adipose stem cells. As a result, the present inventors have found the relationship between the substrates adhering to the adipose stem cells in the differentiation of the adipose stem cells into the adipocytes. The differentiation efficiency of the cells into adipocytes was evaluated. As a result, when the adipose stem cells were cultured in a culture vessel having a fixed heparin-binding domain, the adipose stem cells proliferated in a state in which they were effectively adhered to the culture vessel, thereby confirming that differentiation efficiency into adipocytes was increased.

Accordingly, an object of the present invention is to provide a method of using heparin binding domain (HBD) having adhesion to adipose stem cells for efficient differentiation of adipose stem cells into adipocytes.

In order to achieve the above object, the present invention provides a culture vessel in which heparin binding domain (HBD) is fixed to the surface.

In another aspect, the present invention provides a method for differentiating adipose stem cells into adipocytes comprising inoculating the adipose stem cells in the culture vessel and culturing the adipocytes adhered to the surface of the culture vessel.

According to the present invention, when culturing adipose stem cells in a culture vessel in which a heparin binding domain having adhesion to adipose stem cells is fixed to the surface, heparan sulfate proteoglycan (HSPG) and heparin binding present in the cell membranes of adipose stem cells Since the adipose stem cells are adhered to the culture vessel surface by the interaction with the domain, the differentiation of the adipose stem cells into the adipocytes can be effectively induced. Therefore, the present invention can be applied to culture techniques related to the differentiation and proliferation of adipose stem cells to provide a large amount of adipocytes that can be usefully used as functional cells of cell support and complex support for tissue engineering.

The present invention provides a method of using heparin binding domain for efficient differentiation of fat stem cells into fat cells.

To this end, the present invention provides a culture vessel in which heparin binding domain (HBD) having adhesion to adipose stem cells is fixed to the surface.

The culture vessel according to the present invention maintains the inherent biological activity of the heparin binding domain (HBD) having an adhesive activity capable of binding to heparan sulfate proteoglycan (HSPG) present in the cell membranes of adipose stem cells. While being fixed to the surface of the culture vessel. Thus, by adhering the adipose stem cells to the surface using the adhesion activity to the adipocytes of the HBD fixed on the surface of the culture vessel it can be effective differentiation of the adipocytes into adipocytes.

Heparin binding domain (HBD) is a polypeptide encoded by the nucleotide sequence corresponding to 5081 to 5893 bp of human fibronectin cDNA (NCBI GenBank Accession No. X02761.1), which binds to HSPG present in the cell membranes of adipose stem cells to form fat. In the culture of stem cells, they exhibit biological functions important for their differentiation and proliferation. Heparin binding domain (HBD) having this feature is preferably fixed to a concentration of 10 to 100 ㎍ / ㎖ on the surface of the culture vessel. If the concentration of HBD fixed on the surface of the culture vessel is less than 10 ㎍ / ㎖ may cause a problem that cell adhesion does not occur, it is preferable to maintain the concentration range.

Immobilization of HBD to the culture vessel surface can be accomplished by any method known in the art that is used to immobilize polypeptides on solid substrate surfaces, traditionally shared by physical adsorption, non-selective chemical reactions. Bonding and the like can be used. An example of such an immobilization method uses biotin-streptavidin / avidin binding by binding biotin to a protein and then applying the protein to a solid surface treated with streptavidin or avidin. To fix the protein by means of; Fixing a protein by integrating an active group (chemical functional group for fixing the protein by chemical bonding) on the substrate using a plasma; Forming a porous sol-gel thin film having a sufficiently increased specific surface area using a sol-gel method on a surface of a solid substrate, and then fixing the protein by physical adsorption to the porous thin film; Immobilizing antithrombogenic proteins on polytetrafluoroethylene (PTFE) surfaces by plasma reaction; A method of immobilizing a protein by binding an enzyme in which two or more consecutive fused cationic amino residues are fused to two enzymes; Fixing the protein to a hydrophobic polymer layer bound to a solid phase support using a substrate; Fixing the protein using a buffer component on the plastic surface; There is known a method of fixing a protein by contacting the protein to a solid surface having a hydrophobic surface in an alcohol solution.

In one embodiment of the invention, a polypeptide linker wherein a amino terminus of a heparin binding domain (HBD) is fused to a carboxyl terminus of the polypeptide linker using a polypeptide linker that is recombinantly capable of mass expression and easy purification. Immobilization is performed in the form of HBD recombinant protein.

The polypeptide used as a linker in the present invention binds to the amino terminus or carboxyl terminus of HBD via its carboxyl or amino terminus and adsorbs to a culture vessel having a hydrophobic surface via a hydrophobic domain present at its amino or carboxyl terminus. As it can be, any one can be used as long as it is capable of mass expression and easy purification recombinantly and does not affect the culture of stem cells. Examples of such polypeptide linkers include maltose-binding protein (MBP), hydrophobin, Fc domain of immunoglobulin, hydrophobic cell penetrating peptides (CPPs), and the like.

In another embodiment of the present invention, immobilization is achieved by covalently binding HBD to chemically active surfaces such as aldehyde, catechol, carboxyl, and amine groups using amino, carboxyl or thiol groups present in the HBD. To perform. For example, if polydopamine is coated on the surface of the culture vessel and reacted with HBD under oxidative conditions, the amine group of HBD may be reacted by Michael addition reaction or Schiff-base reaction. Alternatively, the catechol of thiol and polydopamine may be covalently bound to fix HBD.

In another embodiment of the present invention, immobilization of HBD to the culture vessel surface is carried out using sugar polymers having amphipathicity. For example, PVMA (poly- ( N - p -vinylbenzyl-4- O- aD-glucopyranosyl-)-D-gluconamide) or PVLA (poly- ( N - p- vinylbenzyl-4- O -aD-glucopyranosyl-)- Polyvinlybenzyl-conjugated sugars such as D-galactonamide) expose the hydrophilic sugar to the outside as the hydrophobic polyvinylbenzyl moiety is spontaneously adsorbed onto the hydrophobic surface. When the surface of the polyvinylbenzyl-conjugated sugar polymer is adsorbed with an oxidizing agent such as sodium periodate, the diol portion of the sugar is broken to generate an aldehyde group, and the produced aldehyde group and the amine group of HBD are shifted. HBD can be immobilized covalently by a base reaction.

In a preferred embodiment of the invention, HBD immobilization to the culture vessel surface is carried out using maltose binding protein (MBP) as the polypeptide linker. MBP (NCBI GenBank Accession No. AAB59056) is Escherichia coli ) is a periplasmic protein that is located in the plasma membrane space across the cell membrane and is involved in the transport of sugars such as maltose and maltodextrin into the cell.

MBP is mainly used to produce useful foreign proteins in the form of recombinant proteins, which are made by translating from the gene malE in the cell and inserting the gene of the foreign protein downstream of the cloned malE gene to express it in the cell. Can easily produce large quantities of recombinant protein fusion of dog proteins. In particular, if the protein to be expressed is small in size or foreign protein with less stability in other host cells, it is advantageous to express it in the cell in the form of recombinant protein using MBP as described above. The foreign protein expressed from the gene to which the malE gene is bound can be easily isolated and purified using the property that MBP has a binding affinity for maltose. For example, by reacting amylose-coated resin and cell lysate with maltose-multiplexed form, washing the reacted resin several times to remove other contaminated proteins, and then adding high concentrations of maltose to compete. Only the desired protein can be eluted simply.

In one embodiment of the present invention, the carboxyl terminus of MBP and the amino terminus of HBD are prepared using maltose binding protein (MBP), which has the advantage of being easily expressed and purified with excellent binding ability with maltose as a protein expressed in E. coli. The combined MBP-HBD recombinant protein was prepared and the recombinant protein was immobilized by simple physical adsorption on the surface of the hydrophobic culture vessel using the hydrophobic domain of MBP as a linker. At this time, the carboxyl terminus of MBP is used for binding to HBD for the production of recombinant protein, while the amino terminus including hydrophobic domain is used for spontaneous physical adsorption to hydrophobic surface in a later step.

As a result of sequencing, the amino terminal of the heparin binding domain (HBD) is fused to the carboxyl end of the maltose binding protein (MBP) provided as described above, and the MBP-HBD recombinant protein having adhesion to stem cells is SEQ ID NO: It has an amino acid sequence of 2, which is encoded by a polynucleotide having a nucleotide sequence of SEQ ID NO: 1.

Polypeptide linker-HBD recombinant proteins according to the present invention can be prepared using conventional chemical synthesis or genetic recombination techniques, and the like. For example, a recombinant expression vector comprising a polynucleotide encoding a polypeptide linker-HBD recombinant protein according to the present invention is prepared and transformed into a suitable host cell to obtain a transformed bacterium. .

In a preferred embodiment of the present invention, amplified HBD by polymerase chain reaction (PCR) and then cloned the amplified HBD gene into a vector containing the MBP gene to include a recombinant gene fragment linked to the HBD gene at the carboxyl end of MBP To prepare an expression vector. The recombinant expression vector of the present invention thus prepared may be, for example, pMAL-c2X-HBD. The recombinant expression vector pMAL-c2X-HBD is a vector in which the HBD gene amplified by PCR is inserted into an open reading frame (ORF) site of the carboxyl terminus of the MBP gene in the pMAL-c2X vector (New England Biolabs). .

A transformed bacterium is obtained by transforming the host cell with the recombinant expression vector thus prepared. In a preferred embodiment of the present invention, the recombinant expression vector pMAL-c2X-HBD comprising a recombinant gene fragment fused with HBD at the carboxyl terminus of MBP is transformed into E. coli K12 TB1 to MBP-HBD recombinant protein. E. coli transformants expressing are obtained.

The obtained transformed bacteria were cultured under appropriate conditions, such as in the presence of isopropyl-β-D-thiogalactoside (IPTG), a gene expression inducing agent, and then the recombinant protein was recovered from the culture. do. Recovery of the recombinant protein expressed in the transgenic bacteria can be carried out through various separation and purification methods known in the art.

  In a preferred embodiment of the present invention, affinity column chromatography using a material, such as amylose resin, that can specifically bind maltose using the characteristics of MBP in MBP-HBD recombinant protein Recombinant protein is isolated and purified. HBD expressed in the form of recombinant protein with MBP by the above process retains its original biological activity because amino terminus participates in binding to MBP and carboxyl terminus plays an important role in maintaining HBD activity. Can be.

"Maintaining biological activity" in the present invention means maintaining at least 50%, preferably at least 60% and more preferably at least 70% of the original biological activity or function even after HBD is fused to the polypeptide linker. . Even more preferably the fused HBD retains at least 80%, most preferably at least 90% of the original biological activity or function. In the most preferred embodiment of the invention, HBD expressed and purified in the form of recombinant protein with a polypeptide linker retains at least 99% of its original biological activity or function.

For immobilization of the thus prepared MBP-HBD recombinant protein, the recombinant protein is first added to a suitable buffer such as buffered phosphate saline (PBS), Tween20 / PBS, Tris-HCl buffer, bicarbonate buffer, and the like. After dilution at a concentration of ng / ml to 0.5 mg / ml, this dilution is added to a culture vessel with a hydrophobic surface. When the culture vessel is reacted at 4 to 25 ° C. for 1 to 24 hours, the recombinant protein is immobilized on the hydrophobic surface by physical adsorption of the hydrophobic domain and the hydrophobic domain located at the amino terminal of MBP. At this time, the concentration of the recombinant protein immobilized on the hydrophobic surface is preferably 10 to 100 ㎍ / ㎖.

Any culture vessel suitable for the present invention can be used without limitation as long as it has a hydrophobic surface, for example, silanized surface, carbon nanotube (CNT) surface, hydrocarbon coated surface, Having a polymer (eg, polystyrene, polycarbonate, polypropylene, polyethylene, Teflon, polytetrafluoroethylene, polyester-containing biodegradable polymer, etc.) surface, metal (eg, stainless steel, titanium, gold, platinum, etc.) surface Can be.

As described above, the culture vessel according to the present invention having a surface to which the heparin binding domain is immobilized by various immobilization methods has a bioactive surface having excellent adhesion to adipose stem cells, and differentiates the adipose stem cells into adipocytes and It can be usefully used for proliferation studies.

In another aspect, the present invention provides a method for differentiating adipose stem cells into adipocytes comprising inoculating the adipose stem cells in the culture vessel and culturing the adipocytes adhered to the surface of the culture vessel.

When inoculated and cultured with adipose stem cells in a culture vessel having a fixed heparin binding domain (HBD) according to the invention, heparan sulfate sulfate (HSPG) present in the cell membrane of the adipose stem cells is made of fat The culture proceeds with the stem cells adhered to the culture vessel. In this case, the adipose stem cells maintain a rounded cell morphology during the culture period, thereby promoting differentiation into adipocytes.

The culturing of adipose stem cells according to the present invention is inoculated with adipose stem cells at a concentration of 1 × 10 ⁴ to 4 × 10 ⁴ cells / cm 2 in the adipocyte differentiation medium, which is performed at 35 to 38.5 ° C. for 14 to 28 days. It is preferable.

Adipose stem cells that can be used in the present invention are isolated from human adipose tissue, and can be easily obtained from the patient himself or others with the same phenotype. At this time, any adipose tissue obtained by any method used for collecting fat may be used regardless of the position of the body. Typically, subcutaneous adipose tissue, bone marrow adipose tissue, mesenteric adipose tissue, gastrointestinal adipose tissue, retroperitoneal adipose tissue, etc. are possible. Adipose stem cells are derived from adipose tissue by a known method from human adipose tissue, for example, as disclosed in WO2000 / 53795 and WO2005 / 04273. It can be obtained through the process of enzymatic treatment such as collagenase, removal of floating cells such as erythrocytes by centrifugation.

In a preferred embodiment of the present invention, the human adipose tissue obtained incidentally in liposuction is washed with phosphate buffer (PBS), and then the tissue is chopped and 37 ° C. using serum-free medium to which collagenase type 1 is added. Treatment for 1 to 6 hours. Subsequently, after washing with PBS, the supernatant is removed by centrifugation at 1000 rpm for 5 minutes and the pellet remaining on the bottom is separated. The separated pellets were washed with PBS and centrifuged for 5 minutes at 1000 rpm. The supernatant obtained therefrom is filtered to remove suspended cells such as red blood cells and cell debris, and then washed with PBS. After 24 hours of incubation in serum-containing medium, the cells that do not adhere to the bottom of the culture vessel are washed with PBS, and cultured with replacing the serum-containing medium every two days to obtain multipotent adipose stem cells. Adipocytes isolated as described above show excellent proliferation rate up to passage number 16 even in several passages. Therefore, according to the present invention, the multipotent adipose stem cells isolated from human adipose tissue may be used as-differentiated cells for subsequent adipocyte differentiation and proliferation, or cells cultured for more than 10 passages at 60% confluency. Can be used. The use of adipose stem cells, which have been sufficiently propagated by subculture, can induce the differentiation of adipocytes in a large amount within a short time.

In a preferred embodiment of the present invention, 2 × 10 ⁴ cells are stored in each well of a well plate in which MBP-HBD recombinant protein is immobilized after the isolated adipocytes are suspended in serum medium to induce differentiation of adipocytes into adipocytes. Inoculate at a concentration of / ml and incubate for 28 days. In this case, any medium suitable for the culture may be used without limitation as long as it is a medium commonly used for culturing and / or differentiating fat stem cells. For example, 10 μg / ml of Dulbeco's modified eagle medium (DMEM), Ham's F12, etc. Medium added with insulin, 115 μg / ml methyl-isobutylxanthine, 1 μM dexamethasone, 20 μM indomethacin and fetal bovine serum (FBS) can be used.

Cells cultured according to the above method were stained with oil-red O, which is a fat-specific stain, and observed by electron microscopy. As a result, lipid droplets were detected and the fat stem according to the method of the present invention. It was confirmed that the cells were differentiated into adipocytes.

As described above, the method of differentiating adipose stem cells into adipocytes according to the present invention can induce the adhesion activity of the cells using a culture vessel in which heparin binding domains are fixed, thereby allowing the simple and effective differentiation of the adipose stem cells into adipocytes. have. In addition, the differentiation method according to the present invention does not require the use of expensive medium or special growth factors, and can apply adipose stem cells proliferated in several passages to differentiate adipose cells from adipose stem cells in large quantities. That has the advantage.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only intended to further illustrate the present invention and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention.

< Reference Example  1> from human adipose tissue Multiplicity  Isolation of Stem Cells

Subcutaneous adipose tissue from the Catholic University Plastic Surgery Laboratory was washed three times with PBS containing 2% penicillin / streptomycin to remove contaminated blood and chopped with surgical scissors. The adipose tissue was immersed in the prepared tissue solution (serum-free DMEM + 1% BSA (w / v) + 0.3% collagenase type 1), stirred at 37 ° C for 2 hours, and centrifuged at 1000 rpm for 5 minutes. The supernatant and the pellet were separated. The supernatant was discarded and the remaining pellet was collected, washed with PBS, and centrifuged at 1000 rpm for 5 minutes to separate the supernatant. The separated supernatant was filtered through a 100 μm mesh to remove tissue debris and washed with PBS. Cells isolated therefrom were cultured in DMEM / F12 medium (Welgene) containing 10% FBS. After 24 hours of incubation, non-adherent cells were removed by washing with PBS, and the adhered cells were cultured every 2 days with DMEM / F12 medium containing 10% FBS to obtain human subcutaneous adipose tissue-derived stem cells.

1A is a photograph of multipotent stem cells isolated from human subcutaneous adipose tissue according to the above method under a phase contrast microscope (Nikon) at 100 × magnification, and FIG. 1B is expression of cell surface antigens in the multipotent stem cells. The results were analyzed by flow cytometry. At this time, CD29, CD90 and CD105 were used as surface antigens to identify mesenchymal stem cells, and CD34 and HLA-DR were used as surface antigens to observe whether or not other cells were mixed during stem cell isolation and culture. It was. From the above results, it was confirmed that the cells isolated from human subcutaneous fat tissue were adipose stem cells having a phenotype of mesenchymal stem cells.

< Reference Example  2> Extracellular  Analysis of Adhesion Activity of Adipose Stem Cells to Substrates

To analyze the adhesion activity of adipose stem cells to extracellular matrix (ECM) proteins, the cells were placed in a non-tissue culture treated 96-well plate (“PS plate”). As the extracellular protein, collagen type I, collagen type IV, fibronectin (FN) and laminine were added to each 20 µg / ml, and the well plate was stored at 25 ° C for 4 hours, respectively. Extracellular matrix protein was immobilized on the well surface and washed three times with PBS. 100 μg / ml of 1% BSA was added to the well plate and blocked for 1 hour at 25 ° C., and then washed three times with PBS.

The adipose stem cells prepared in Reference Example 1 were suspended in serum-free DMEM-F12 medium at a concentration of 1 × 10 ⁴ cells / ml, and the suspension was inoculated into each well of the well plate and incubated in a 37 ° C. incubator for 30 minutes. Then washed three times with PBS. The cells adhered to the surface of each well were lysed with RIPA buffer (Pierce), and then the adhered cells were quantified by BCA (Bicinchoninic Acid) protein analysis.

As a result, as shown in Figure 2 , the adipose stem cells showed a slight difference in adhesion rate on the surface coated with collagen type I (Col I), type IV (Col IV), fibronectin (Fn) and laminin (Ln) Cell attachment occurred within 30 minutes of inoculation, and it was confirmed that the morphology of the cells changed to a spread state.

On the other hand, to investigate the role of Integrin β1 in the adhesion of ECM components and adipose stem cells, the adipose stem cells prepared in Reference Example 1 in serum-free DMEM-F12 medium at a concentration of 1 × 10 ⁵ cells / ml Suspension was added to the suspension with an integrin β1 blocking antibody (anti-CD29, Mab 2253) at a concentration of 10 or 40 μg / ml. This was inoculated into a well plate in which extracellular matrix protein was fixed on the surface as described above, and reacted at room temperature for 10 minutes, followed by incubation for 30 minutes in a 37 ° C. incubator. The well plates were washed three times with PBS and then the adhered cells were lysed with RIPA buffer and the adhered cells were quantified by BCA protein analysis.

As a result, as shown in Fig . 3 , when adipocytes adhere to the surface to which collagen type I (ColI), type IV (ColIV) and laminin (Ln) are fixed, their adhesion is prevented by integrin β1 blocking antibodies. While completely inhibited, it was confirmed that when adhered to fibronectin (Fn) coated surface, their adhesion was hardly inhibited by the integrin β1 blocking antibody. The results indicate that other biomolecules other than integrin β1 are involved in the adhesion activity of the adipose stem cells and fibronectin.

< Reference Example  3> Fibronectin  Analysis of Adhesion Activity of Adipose Stem Cells

In addition to peptides that recognize integrin β1, fibronectin contains heparin binding domains (HBDs) that bind to heparan sulfate proteoglycans (HSPG) on cell membranes, and in neurons or bone cells, HBDs affect cell adhesion and morphological changes. The fact is known. Based on this fact, the following experiment was conducted to investigate whether HBD affects the adhesion activity of adipose stem cells to fibronectin.

First, the adipose stem cells prepared in Reference Example 1 were suspended in serum-free DMEM-F12 medium at a concentration of 1 × 10 ⁵ cells / ml, and in the suspension, Fn-C / H-II (KNNQKSEPLIGRKKT; a peptide that recognizes HSPG); Sigma) was added at a concentration of 3 mg / ml, integrin β1 blocking antibody was added at a concentration of 20 μg / ml, or both. Each mixture was added to a well plate fixed with fibronectin (Fn) as in Reference Example 2, a well plate fixed with BSA (bovine serum albumin) as a control, reacted at room temperature for 10 minutes, and then incubated in a 37 ° C. incubator. Incubate for minutes. The well plates were washed three times with PBS and then the adhered cells were lysed with RIPA buffer and the adhered cells were quantified by BCA protein analysis.

The results are shown in Figure 4 , where a represents the adhesion of fat stem cells to the BSA-fixed surface, b is the adhesion of fat stem cells to the Fn-fixed surface, and c is Fn-C / H. Adipose stem cells adhere to Fn-fixed surfaces in the presence of -II, d denotes adherence of fat stem cells to Fn-fixed surfaces in the presence of Fn-C / H-II and integrin β1 blocking antibodies. . As shown in FIG. 4 , adhesion of fat stem cells to Fn-fixed surfaces was not inhibited by treatment with integrin β1 blocking antibody alone (b) or Fn-C / H-II alone (c), whereas integrin When the β1 blocking antibody and Fn-C / H-II were simultaneously treated (d), the number of adherent adipose stem cells was significantly reduced. Figure 5 is a quantitative analysis of the results of Figure 4 , when the integrin β1 blocking antibody and Fn-C / H-II was treated at the same time confirmed that more than 50% cell adhesion is inhibited. The results indicate that integrin β1 and HSPG of the cell membrane cooperatively induce cell adhesion when adipocytes adhere to fibronectin.

From the results of Reference Examples 2 and 3, it can be seen that the adipose stem cells have a mechanism of inducing cell adhesion by binding to the heparin binding domain in fibronectin via the heparin motif present in the cell membrane.

< Example  1> MBP - HBD  Expression and Purification of Recombinant Proteins

To prepare a recombinant protein fused with a heparin binding domain (HBD) to a maltose binding protein (MBP) as a polypeptide linker, the HBD gene fragment (5081-5893 bp) of human fibronectin cDNA (NCBI GenBank Accession No. X02761.1) The recombinant expression vector pMAL-c2X-HBD inserted in pMAL vector (New England Biolabs) containing MBP gene was produced by requesting to Genotech.

E. coli transformants obtained by transforming the recombinant expression vector pMAL-c2X-HBD into E. coli K12 TB1 were inoculated in LB medium containing 60 μg / ml of ampicillin and then cultured overnight at 37 ° C. 10 ml of the culture was added to 1 L of LB medium and shaken at 37 ° C., and IPTG was added at a final concentration of 3 mM when the absorbance of the culture was about 0.6 at a wavelength of 650 nm. After 2 hours after the addition of IPTG, the culture was stopped, and the culture was centrifuged at 4000 × g for 20 minutes (Combi-514R, Hanil) to collect cell precipitates. Suspension was added to the cell precipitate by adding 50 ml of buffer (20 ml of 1 M Tris-HCl, pH 7.5, 11.7 g of NaCl, and 2 ml of 0.5 M EDTA), and then EDTA (ethylenediaminetetraacetic acid) and PMSF were added to a final concentration of 1 mM. (phenylmethanesulphonyl fluoride) was added. The cell mixture was repeatedly frozen and thawed at −20 ° C. prior to purification to facilitate cell disruption. The cell mixture was then sonicated for about 10 seconds at a 10% power using an ultrasonic grinder (Fisher Scientific Model 500 Sonic Dismembrator) in an ice bath to break up the cells and left in an ice bath for 30 seconds. The above procedure was repeated twice to completely disrupt the cells. The cell homogenate thus obtained was centrifuged at 9000 × g for 1 hour (Combi-514R, Hanil) to obtain a supernatant containing soluble protein, and then buffered (1 M Tris-HCl 20 ml, pH 7.5, NaCl 11.7 g). , 0.5 M EDTA, 2 ml).

On the other hand, in order to separate the MBP-HBD recombinant protein expressed from E. coli transformants, affinity chromatography using amylose resin (New England Biolabs) was prepared. The column was previously equilibrated by washing with about 8 × bed volume of buffer (20 mL of 1 M Tris-HCl, pH 7.5, 11.7 g NaCl, 2 mL of 0.5 M EDTA). The equilibrated amylose resin affinity chromatography was loaded with the supernatant of the soluble protein obtained above at a rate of 1 ml per minute. A buffer of at least 12 × bed volume (20 mL of 1 M Tris-HCl, pH 7.5, 11.7 g NaCl, 2 mL of 0.5 M EDTA) was flowed into the column to remove unadsorbed protein from the resin. Then, 10 mM maltose elution buffer (20 ml of 1 M Tris-HCl, pH 7.5, 11.7 g NaCl, 2 ml of 0.5 M EDTA, 10 mM maltose) was added to elute the proteins adsorbed on the resin at a rate of 1 ml per minute. Was recovered. The protein recovered therefrom was electrophoresed on a 10% polyacrylamide gel to confirm the approximate molecular weight and purity of the purified protein. As a result, the purity of purified protein was over 95% and the approximate molecular weight was about 80,000 Da.

The protein sample was placed in a dialysis membrane (MWCO12-14,000, Spectrum laboratories, Inc.) and diluted with PBS for 3 days to obtain only the protein without maltose. Then, centrifuged filter (Centrifugal Filter, Amicon Ultra-15 MWCO 5,000, Millipore) was centrifuged at 4000 x g for 45 minutes (Combi-514R, one day) to concentrate the protein to obtain MBP-HBD recombinant protein. .

As a result of sequencing analysis, the MBP-HBD recombinant protein, in which the amino terminal of HBD is fused to the carboxyl terminal of MBP, has an amino acid sequence of SEQ ID NO: 2, which is encoded by a polynucleotide having a nucleotide sequence of SEQ ID NO: 1 do.

< Example  2> HBD  Analysis of Adhesion Activity of Adipose Stem Cells to Immobilized Surface

In order to investigate whether adipose stem cells can directly adhere to the surface where HBD is immobilized, MBP-HBD recombinant protein prepared in Example 1 was added to each well of a 96-well PS plate, respectively, 0, 1, 5, 10, 50 and 100 μg / ml concentrations were added and fixed at room temperature for 4 hours and then washed three times with PBS. 1% BSA was added to the well plate, blocked for 1 hour, washed three times with PBS, and then adhered to serum-free DMEM / F12 medium by adding fat stem cells suspended at a concentration of 1 × 10 ⁵ cells / ml for 1 hour. I was. The well plates were washed three times with PBS and then the adhered cells were lysed with RIPA buffer and the adhered cells were quantified by BCA protein analysis.

As a result, as shown in Figure 6a , the adhesion activity of fat stem cells on the surface of the MBP-HBD recombinant protein fixed at a concentration of 10 ㎍ / ㎖ or more was confirmed. In particular, the adhesion of adipose stem cells observed on the surface of the MBP-HBD recombinant protein fixed at a concentration of 100 μg / ml was almost similar to that of the Tissue Culture Treated Well Plate (“TCP”). , Fibronectin (Fn) corresponded to about 80% of the adhesion rate to the fixed surface.

To investigate the effect of heparin on the adhesion of adipose stem cells to HBD, a 100 μg / ml concentration of MBP-HBD recombinant protein or a 20 μg / ml fibronectin-immobilized surface as a control was applied at a concentration of 1 μg / ml. After 1-hour pre-treatment with heparin, cell adhesion experiments were performed by inoculating fat stem cells in the same manner as above.

As a result, as shown in FIG. 6B , adhesion of fat stem cells was hardly inhibited by heparin on the surface where fibronectin (Fn) was fixed, whereas adhesion of fat stem cells was on the surface where MBP-HBD recombinant protein was fixed. It can be seen that at least 50% is inhibited by heparin. From the above results, it can be seen that when adipose stem cells adhere to HBD-fixed surfaces, heparin is directly involved with HBD, which is responsible for the adhesion activity of HBD to adipose stem cells on the cell membranes of adipose stem cells. This indicates that the existing HSPG is involved.

< Example  3> HBD Changes in Cell Morphology by Adhesion of Human and Adipose Stem Cells

In order to observe the direct morphological changes of adipose stem cells according to cell adhesion with HBD, as shown in Example 2, the suspension of fat stem cells in serum-free DMEM / F12 medium was fixed to TCP and FN (20 μg / ml). The immobilized surface and the MBP-HBD recombinant protein (100 μg / ml) were immunized respectively. After incubating each well plate for 2 hours, the morphology of the cells was observed under a phase contrast microscope.

As a result, as shown in FIG . 7 , a small podia was formed when cultured in TCP (A), and the cells were completely spread when cultured on the surface B fixed with Fn. ) Was observed, but the cells remained round when cultured on the surface (C) where the MBP-HBD recombinant protein was fixed. In general, the morphology and differentiation of cells are closely related to each other. It is known that it is advantageous to maintain a cell spreading shape for cell proliferation, while maintaining a rounded shape for cell differentiation. In particular, it has been reported that the size and shape of cultured cells are important factors in the differentiation of mesenchymal stem cells including adipocytes into adipocytes (R. McBeath et al . , Develop . Cell 6: 483, 2004; W. Luo et al. , Langmuir 24: 12129, 2009).

< Example  Induction of Differentiation of Adipose Stem Cells into Adipocytes by Cell Adhesion

To differentiate adipocytes into adipocytes, adipocytes on a TCP surface and on a 24-well PS plate surface immobilized with MBP-HBD recombinant protein (100 μg / ml) and fibronectin (20 μg / ml), respectively, according to the present invention. Differentiation medium or normal growth medium was added and 2 × 10 ⁴ cells / ml of fat stem cells were inoculated and incubated in a 37 ° C. incubator for 28 days. As the adipocyte differentiation medium, 10 μg / ml insulin, 115 μg / ml methyl-isobutylxanthine, 1 μM dexamethasone, 20 μM indomethacin and fetal bovine serum (FBS) were added to DMEM-F12 medium. DMEM-F12 medium to which FBS was added was used as a growth medium. At this time, the medium was replaced with a new one every three days. After 28 days of culture, the lipid droplets were stained with oil-red O, which is a staining of adipose tissue, to confirm the differentiation of adipose stem cells into adipocytes.

As a result, as shown in FIG . 8 , when the adipose stem cells were cultured in the normal growth medium rather than the adipose cell differentiation medium (A, B, C), no differentiation of adipocytes was observed in all well plates. . On the other hand, when cultured in adipocyte differentiation medium (D, E, F), all well plates showed differentiation into adipocytes above a certain level. In particular, MBP-HBD recombinant protein induced differentiation on a fixed surface. More cells were stained red than when (F) induced differentiation on Fn fixed surface (B) or TCP surface (D). This indicates that the differentiation of adipose stem cells into adipocytes on the surface where the MBP-HBD recombinant protein was immobilized was more effective.

9 is To obtain quantitative information on the differentiation of adipocytes into adipocytes, the area of the stained cells (A), the amount of stained oil-red cucumber (B), and the adipocytes stained with oil-red o were obtained on the whole culture surface. This is the result of analyzing the number (C). From the results of FIG. 9 , it was confirmed that culturing the adipose stem cells on the surface where the MBP-HBD recombinant protein was fixed than on the surface where the Fn was fixed or the TCP surface showed higher differentiation efficiency to the adipocytes. The results indicate that the efficiency of differentiating fat stem cells into adipocytes can be improved by controlling the cell adhesion mechanism using the heparin binding domain.

The specific parts of the present invention have been described in detail above, and it is apparent to those skilled in the art that such specific descriptions are merely preferred embodiments, and thus the scope of the present invention is not limited thereto. something to do. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.

1A is a photograph (40 ×) of pluripotent stem cells isolated from human subcutaneous adipose tissue observed with a phase contrast microscope,

Figure 1b is the result of analyzing the surface antigen expression pattern of the multipotent stem cells isolated from human subcutaneous adipose tissue by flow cytometry,

2 is a result of examining the adhesion activity of various extracellular matrix proteins to adipose stem cells,

3 is a result of examining the effect of integrin β1 on the adhesion activity of extracellular matrix protein to adipose stem cells,

4 is a result of investigating the effect of Fn-C / H-II peptide on the adhesion activity of fibronectin (Fn) to adipose stem cells,

5 is a quantitative analysis of the results of FIG. 4 ,

Figure 6a is a result of examining the adhesion rate of adipose stem cells according to the fixed concentration of MBP-HBD recombinant protein according to the present invention,

Figure 6b is a result of examining the effect of heparin on the adhesion activity of the MBP-HBD recombinant protein according to the present invention on adipose stem cells,

7 is a phase difference of the morphological changes of cells after culturing the adipocytes on the surface fixed with TCP (A), fibronectin (Fn) (B) and MBP-HBD recombinant protein according to the present invention (C) Is a picture taken under a microscope,

Fig. 8 shows the growth of adipose stem cells on fibronectin (Fn) -fixed surface (A, D), TCP (B, E), and MBP-HBD recombinant protein according to the present invention (C, F). After culturing with (A, B, C) or adipocyte differentiation medium (D, E, F), the result was stained with Oil-Red O,

FIG. 9 analyzes the area (A) of stained cells, the amount of stained oil-red cucumber (B), and the number (C) of fat cells stained with oil-red o in the results of FIG. 8. The result is.

<110> Korea Institute of Science and Technology <120> METHOD FOR THE DIFFERENTIATION OF ADIPOSE-DERIVED STEM CELLS INTO          ADIPOCYTES USING HEPARIN-BINDING DOMAIN <130> KC090188 <160> 2 <170> Kopatentin 1.71 <210> 1 <211> 1986 <212> DNA <213> Artificial Sequence <220> <223> nucleotide sequence encoding MBP-HBD recombinant protein <400> 1 atgaaaatcg aagaaggtaa actggtaatc tggattaacg gcgataaagg ctataacggt 60 ctcgctgaag tcggtaagaa attcgagaaa gataccggaa ttaaagtcac cgttgagcat 120 ccggataaac tggaagagaa attcccacag gttgcggcaa ctggcgatgg ccctgacatt 180 atcttctggg cacacgaccg ctttggtggc tacgctcaat ctggcctgtt ggctgaaatc 240 accccggaca aagcgttcca ggacaagctg tatccgttta cctgggatgc cgtacgttac 300 aacggcaagc tgattgctta cccgatcgct gttgaagcgt tatcgctgat ttataacaaa 360 gatctgctgc cgaacccgcc aaaaacctgg gaagagatcc cggcgctgga taaagaactg 420 aaagcgaaag gtaagagcgc gctgatgttc aacctgcaag aaccgtactt cacctggccg 480 ctgattgctg ctgacggggg ttatgcgttc aagtatgaaa acggcaagta cgacattaaa 540 gacgtgggcg tggataacgc tggcgcgaaa gcgggtctga ccttcctggt tgacctgatt 600 aaaaacaaac acatgaatgc agacaccgat tactccatcg cagaagctgc ctttaataaa 660 ggcgaaacag cgatgaccat caacggcccg tgggcatggt ccaacatcga caccagcaaa 720 gtgaattatg gtgtaacggt actgccgacc ttcaagggtc aaccatccaa accgttcgtt 780 ggcgtgctga gcgcaggtat taacgccgcc agtccgaaca aagagctggc aaaagagttc 840 ctcgaaaact atctgctgac tgatgaaggt ctggaagcgg ttaataaaga caaaccgctg 900 ggtgccgtag cgctgaagtc ttacgaggaa gagttggcga aagatccacg tattgccgcc 960 actatggaaa acgcccagaa aggtgaaatc atgccgaaca tcccgcagat gtccgctttc 1020 tggtatgccg tgcgtactgc ggtgatcaac gccgccagcg gtcgtcagac tgtcgatgaa 1080 gccctgaaag acgcgcagac taattcgagc tcgaacaaca acaacaataa caataacaac 1140 aacctcggga tcgagggaag gatttcagaa ttcgctattc ctgcaccaac tgacctgaag 1200 ttcactcagg tcacacccac aagcctgagc gcccagtgga caccacccaa tgttcagctc 1260 actggatatc gagtgcgggt gacccccaag gagaagaccg gaccaatgaa agaaatcaac 1320 cttgctcctg acagctcatc cgtggttgta tcaggactta tggtggccac caaatatgaa 1380 gtgagtgtct atgctcttaa ggacactttg acaagcagac cagctcaggg tgttgtcacc 1440 actctggaga atgtcagccc accaagaagg gctcgtgtga cagatgctac tgagaccacc 1500 atcaccatta gctggagaac caagactgag acgatcactg gcttccaagt tgatgccgtt 1560 ccagccaatg gccagactcc aatccagaga accatcaagc cagatgtcag aagctacacc 1620 atcacaggtt tacaaccagg cactgactac aagatctacc tgtacacctt gaatgacaat 1680 gctcggagct cccctgtggt catcgacgcc tccactgcca ttgatgcacc atccaacctg 1740 cgtttcctgg ccaccacacc caattccttg ctggtatcat ggcagccgcc acgtgccagg 1800 attaccggct acatcatcaa gtatgagaag cctgggtctc ctcccagaga agtggtccct 1860 cggccccgcc ctggtgtcac agaggctact attactggcc tggaaccggg aaccgaatat 1920 acaatttatg tcattgccct gaagaataat cagaagagcg agcccctgat tggaaggaaa 1980 aagaca 1986 <210> 2 <211> 660 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence of MBP-HBD recombinant protein <400> 2 Met Lys Ile Glu Glu Gly Lys Leu Val Ile Trp Ile Asn Gly Asp Lys   1 5 10 15 Gly Tyr Asn Gly Leu Ala Glu Val Gly Lys Lys Phe Glu Lys Asp Thr              20 25 30 Gly Ile Lys Val Thr Val Glu His Pro Asp Lys Leu Glu Glu Lys Phe          35 40 45 Pro Gln Val Ala Ala Thr Gly Asp Gly Pro Asp Ile Ile Phe Trp Ala      50 55 60 His Asp Arg Phe Gly Gly Tyr Ala Gln Ser Gly Leu Leu Ala Glu Ile  65 70 75 80 Thr Pro Asp Lys Ala Phe Gln Asp Lys Leu Tyr Pro Phe Thr Trp Asp                  85 90 95 Ala Val Arg Tyr Asn Gly Lys Leu Ile Ala Tyr Pro Ile Ala Val Glu             100 105 110 Ala Leu Ser Leu Ile Tyr Asn Lys Asp Leu Leu Pro Asn Pro Pro Lys         115 120 125 Thr Trp Glu Glu Ile Pro Ala Leu Asp Lys Glu Leu Lys Ala Lys Gly     130 135 140 Lys Ser Ala Leu Met Phe Asn Leu Gln Glu Pro Tyr Phe Thr Trp Pro 145 150 155 160 Leu Ile Ala Ala Asp Gly Gly Tyr Ala Phe Lys Tyr Glu Asn Gly Lys                 165 170 175 Tyr Asp Ile Lys Asp Val Gly Val Asp Asn Ala Gly Ala Lys Ala Gly             180 185 190 Leu Thr Phe Leu Val Asp Leu Ile Lys Asn Lys His Met Asn Ala Asp         195 200 205 Thr Asp Tyr Ser Ile Ala Glu Ala Ala Phe Asn Lys Gly Glu Thr Ala     210 215 220 Met Thr Ile Asn Gly Pro Trp Ala Trp Ser Asn Ile Asp Thr Ser Lys 225 230 235 240 Val Asn Tyr Gly Val Thr Val Leu Pro Thr Phe Lys Gly Gln Pro Ser                 245 250 255 Lys Pro Phe Val Gly Val Leu Ser Ala Gly Ile Asn Ala Ala Ser Pro             260 265 270 Asn Lys Glu Leu Ala Lys Glu Phe Leu Glu Asn Tyr Leu Leu Thr Asp         275 280 285 Glu Gly Leu Glu Ala Val Asn Lys Asp Lys Pro Leu Gly Ala Val Ala     290 295 300 Leu Lys Ser Tyr Glu Glu Glu Leu Ala Lys Asp Pro Arg Ile Ala Ala 305 310 315 320 Thr Met Glu Asn Ala Gln Lys Gly Glu Ile Met Pro Asn Ile Pro Gln                 325 330 335 Met Ser Ala Phe Trp Tyr Ala Val Arg Thr Ala Val Ile Asn Ala Ala             340 345 350 Ser Gly Arg Gln Thr Val Asp Glu Ala Leu Lys Asp Ala Gln Thr Asn         355 360 365 Ser Ser Ser Asn Asn Asn Asn Asn Asn Asn Asn Asn Asn Leu Gly Ile     370 375 380 Glu Gly Arg Ile Ser Ala Ile Pro Ala Pro Thr Asp Leu Lys Phe Thr 385 390 395 400 Gln Val Thr Pro Thr Ser Leu Ser Ala Gln Trp Thr Pro Pro Asn Val                 405 410 415 Gln Leu Thr Gly Tyr Arg Val Arg Val Thr Pro Lys Glu Lys Thr Gly             420 425 430 Pro Met Lys Glu Ile Asn Leu Ala Pro Asp Ser Ser Ser Val Val Val         435 440 445 Ser Gly Leu Met Val Ala Thr Lys Tyr Glu Val Ser Val Tyr Ala Leu     450 455 460 Lys Asp Thr Leu Thr Ser Arg Pro Ala Gln Gly Val Val Thr Thr Leu 465 470 475 480 Glu Asn Val Ser Pro Pro Arg Arg Ala Arg Val Thr Asp Ala Thr Glu                 485 490 495 Thr Thr Ile Thr Ile Ser Trp Arg Thr Lys Thr Glu Thr Ile Thr Gly             500 505 510 Phe Gln Val Asp Ala Val Pro Ala Asn Gly Gln Thr Pro Ile Gln Arg         515 520 525 Thr Ile Lys Pro Asp Val Arg Ser Tyr Thr Ile Thr Gly Leu Gln Pro     530 535 540 Gly Thr Asp Tyr Lys Ile Tyr Leu Tyr Thr Leu Asn Asp Asn Ala Arg 545 550 555 560 Ser Ser Pro Val Val Ile Asp Ala Ser Thr Ala Ile Asp Ala Pro Ser                 565 570 575 Asn Leu Arg Phe Leu Ala Thr Thr Pro Asn Ser Leu Leu Val Ser Trp             580 585 590 Gln Pro Pro Arg Ala Arg Ile Thr Gly Tyr Ile Ile Lys Tyr Glu Lys         595 600 605 Pro Gly Ser Pro Pro Arg Glu Val Val Pro Arg Pro Arg Pro Gly Val     610 615 620 Thr Glu Ala Thr Ile Thr Gly Leu Glu Pro Gly Thr Glu Tyr Thr Ile 625 630 635 640 Tyr Val Ile Ala Leu Lys Asn Asn Gln Lys Ser Glu Pro Leu Ile Gly                 645 650 655 Arg Lys Lys Thr             660  

Claims (19)

delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete delete Adipocytes are used in culture vessels in which heparin binding domains (HBDs) are fixed to the surface by a maltose binding protein (MBP) linker at a concentration of 10 to 100 µg / ml in the form of recombinant proteins by the maltose binding protein (MBP) linker. Add differentiation medium, Inoculating the medium with a fat stem cells at a concentration of 1 × 10 ⁴ to 4 × 10 ⁴ cells / ㎠, Heparan sulfate sulfate (HSPG) present in the adipose stem cells are cultured in a state of cell adhesion to the heparin binding domain (HBD) in a state in which the fat stem cells were cultured at 35 to 38.5 ℃ for 14 to 28 days to adipocytes Differentiation method of adipose stem cells into adipocytes, characterized in that differentiation. delete 19. The method of claim 18, A method for differentiating adipocytes into adipocytes, characterized in that the recombinant protein has an amino acid sequence as set forth in SEQ ID NO: 2.

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