KR101950072B1 - Methods and compositions for enhancing clinical therapeutic effect of stem cells medicine - Google Patents

Abstract

The present invention relates to a composition for improving migration or differentiation of a stem cell containing CSF-2 (colony stimulating factor-2) protein as an active ingredient, and a method for culturing stem cells having improved migration and differentiation ability using the same. Induced mesenchymal stem cell line to increase osteoblast differentiation into osteoblast when cultured with the addition of the protein to the osteoblast differentiation medium to increase the stemness of CSF- And the expression of fibronectin (FN 1), snail, twist, and vimentin, which are EMT marker genes, was significantly increased, ) Capability is greatly improved and that the cytoskeletal proteins of the stem cells are rearranged into a spindle shape, -2 protein may be useful as a composition for improving the therapeutic effect of a stem cell therapeutic agent.

Description

METHODS AND COMPOSITIONS FOR IMPROVING THERAPEUTIC EFFECTS OF STEM CELL CERTIFICATES [0002]

The present invention relates to a composition for improving migration or differentiation of stem cells containing CSF-2 (colony stimulating factor-2) protein as an active ingredient, and a method for improving the therapeutic effect of a stem cell treatment agent using the same.

Stem cells are cells capable of differentiating the organisms that constitute biological tissues into various cells. These stem cells are collectively referred to as the "undifferentiated cells" before the differentiation that can be obtained from the embryonic, fetal and adult tissues do. Stem cells are differentiated into specific cells by differentiation stimuli (environment), and unlike the differentiated cells in which cell division is stopped, they can self-renew themselves by cell division, ; expansion, and it can be differentiated into other cells by different environments or differentiation stimuli, and is characterized by plasticity in differentiation.

Stem cells can be divided into embryo-derived stem cells derived from the developing embryo and adult stem cells existing in various adult tissues such as bone marrow and cord blood. Embryonic stem cells have a high degree of differentiation and therapeutic efficacy, but their clinical applications are limited due to ethical problems and carcinogenicity of the cells themselves. Although the ability of differentiation is lower than that of embryonic stem cells, adult stem cells, which have no ethical problems and low carcinogenicity, are now attracting much attention as cell therapy agents such as cancer, diabetes, intractable diseases and neurodegenerative diseases, In addition to the development of information science, adult stem cells have been actively studied as a platform for development of related drugs and toxicity tests in the field of regenerative medicine using stem cells. Therefore, in order to regulate various intracellular signaling systems involved in the differentiation of adult stem cells into specialized cells and to obtain stable and mature cells for clinical application, it is necessary to control elaborate and appropriate cell culture conditions.

In the treatment of diseases using adult stem cells, the effects are often very limited. Since adult stem cells are present in an extremely small amount in the body at a ratio of about 0.01 to 0.001% in the body, a process of culturing a large amount in vitro is indispensably required for therapeutic purposes. In the therapeutic agent using adult stem cells, During the culturing process, stem cells such as migration potential of stem cells capable of moving to the wound site, differentiation potential of stem cells capable of differentiating into specific (target) cells, and stem cells, It is known that inherent functions are degraded or lost. In this context, there is an urgent need to develop a novel stem cell in vitro culture method which can suppress the decrease of the therapeutic effect of stem cells during in vitro culture and further improve the therapeutic ability.

To control stem cell proliferation and differentiation, researchers worldwide have developed a variety of chemical methods that inject growth factors, hormones, and cytokines to improve the differentiation and therapeutic efficacy of stem cell therapies. Paul W Burridge et al. Have disclosed a technique for differentiation of stem cells through the regulation of chemical culture conditions (Nature methods, 2014). However, these chemical methods have limitations in the efficiency of obtaining stem cells, .

Accordingly, the present inventors have found that when stem cells are in vitro cultured in culture medium supplemented with growth factor protein CSF-2 (colony stimulating factor-2) while trying to solve the problems of the existing stem cell in vitro culture method The present inventors have accomplished the present invention by confirming that the migration and differentiation ability of stem cells, which play an important role in the therapeutic effect as a stem cell treatment agent, are greatly improved.

It is an object of the present invention to provide a composition for improving the migration and differentiation of stem cells containing CSF-2 (colony stimulating factor-2) protein as an active ingredient.

In order to achieve the above object, the present invention provides a composition for improving migration or differentiation of stem cells containing CSF-2 (colony stimulating factor-2) protein as an active ingredient.

In addition,

1) culturing human stem cells in vitro;

2) culturing the cultured cells of step 1) in a culture medium supplemented with CSF-2 (colony stimulating factor-2) protein to induce differentiation. Thereby providing a culture method.

The present invention relates to a composition for improving migration or differentiation of a stem cell containing CSF-2 (colony stimulating factor-2) protein as an active ingredient, and a method for culturing stem cells having improved migration and differentiation ability using the same. Induced mesenchymal stem cell line to increase osteoblast differentiation into osteoblast when cultured with the addition of the protein to the osteoblast differentiation medium to increase the stemness of CSF- And the expression of fibronectin (FN 1), snail, twist, and vimentin, which are EMT marker genes, was significantly increased, ) Capability is greatly improved and that the cytoskeletal proteins of the stem cells are rearranged into a spindle shape, -2 protein may be useful as a composition for improving the therapeutic effect of a stem cell therapeutic agent.

FIG. 1 is a graph comparing the expression pattern of CSF-2 (colony stimulating factor-2) gene in mesenchymal stem cell lines derived from fibroblasts and undifferentiated adipose tissues using Real-time PCR It is a degree.
FIG. 2 is a graph showing the result of MTT assay after treating CSF-2 protein in adipose derived mesenchymal stem cell line (recombinant CSF-2; hereinafter referred to as CSF 2).
FIG. 3 is a graph comparing the expression patterns of SOX2 and NANOG, which are closely related to stemness, after the treatment of CSF-2 protein with adipose-derived mesenchymal stem cell line using Real-time PCR Also:
A: Expression pattern of SOX2; And
B: Expression pattern of NANOG.
FIG. 4 is a graph showing the results of induction of osteoblast differentiation after treatment of adipose-derived mesenchymal stem cell line with CSF-2 protein:
A: Photo-induced staining with alizarin red after induction of osteoblast differentiation; And
B: Diagram showing measurement of the degree of differentiation through absorbance (570 nm).
FIG. 5 shows the results of the transwell assay and measurement of MMP-2 / MMP-9 expression pattern (western blotting) after treating CSF-2 protein in adipose-derived mesenchymal stem cell line Fig.
A: Transwell assay results; And
B: Results of western blotting of MMP-2 / MMP-9 expression pattern.
FIG. 6 is a graph showing the result of immunofluorescence staining of phalloidin after treatment of CSF-2 protein in an adipose-derived mesenchymal stem cell line (DAPI: nuclear staining, Merge: photograph binding).
FIG. 7 is a graph showing the results of comparative analysis of EMT (Epithelial-Mesenchymal Transition) marker gene expression patterns of stem cells after treatment with CSF-2 protein in adipose derived mesenchymal stem cell line using Real-time PCR to be:
A: Expression pattern of fibronectin 1 (FN 1);
B: Expression pattern of snail;
C: Expression pattern of twist; And
D: Expression pattern of vimentin.

Hereinafter, the present invention will be described in detail.

The present invention provides a composition for improving migration and differentiation of stem cells containing CSF-2 (colony stimulating factor-2) protein as an active ingredient.

The colony stimulating factor-2 (CSF-2) protein used in the present invention is known as a cytokine involved in the production, differentiation and function of granulocytes and macrophages.

The efficacy of stem cell-based therapies is largely determined by the ability of two stem cells, one is the migration potential to move to the damaged site and the other is the differentiation that can differentiate into target cells It is a differential potential.

The CSF-2 (colony stimulating factor-2) protein is a growth factor protein. Preferably, the CSF-2 protein may be composed of the amino acid sequence of SEQ ID NO: 1, but is not limited thereto. But it is not limited thereto.

In addition, mutants of the above base sequences are also included within the scope of the present invention. Specifically, the base sequence is at least 70%, more preferably at least 80%, even more preferably at least 90%, most preferably at least 95 Or more of the sequence homology. &Quot;% of sequence homology to polynucleotides " is ascertained by comparing the comparison region with two optimally aligned sequences, and a portion of the polynucleotide sequence in the comparison region is the reference sequence for the optimal alignment of the two sequences (I. E., A gap) relative to the < / RTI >

In the present invention, the gene consisting of the nucleotide sequence of SEQ ID NO: 2 may be inserted into a recombinant expression vector. The term " recombinant expression vector " means a bacterial plasmid, a phage, a yeast plasmid, a plant cell virus, a mammalian cell virus, or other vector. In principle, it can be used if any vector can replicate and stabilize within the host. An important characteristic of the expression vector is that it has a replication origin, a promoter, a marker gene and a translation control element. That is, the expression vector may be a genetic construct comprising an essential regulatory element operably linked to the expression of the gene insert capable of expressing the desired protein in a suitable host cell.

The term " recombinant " refers to a cell in which a cell replicates a heterologous nucleic acid, expresses the nucleic acid, or expresses a protein encoded by a peptide, heterologous peptide or heterologous nucleic acid. The recombinant cell can express a gene or a gene fragment that is not found in the natural form of the cell in one of the sense or antisense form. In addition, the recombinant cell can express a gene found in a cell in its natural state, but the gene has been modified and reintroduced intracellularly by an artificial means.

An expression vector comprising the RNA sequence and appropriate transcription / translation control signals can be constructed by methods known to those skilled in the art. Such methods include in vitro recombinant DNA technology, DNA synthesis techniques, and in vivo recombination techniques. The DNA sequence can be effectively linked to appropriate promoters in the expression vector. The expression vector may also include a ribosome binding site and a transcription terminator as a translation initiation site.

The expression vector of the present invention may include a plasmid vector, a cosmid vector, an episome vector, a viral vector, and the like, preferably a viral vector. As the viral vector, vectors derived from retrovirus, lentivirus, adenovirus, adeno-associated virus, herpes simplex virus, Sendai virus, etc. can be used, and a lentiviral vector can be preferably used, Most preferably, a plasmid vector such as pCDH, pECFP, or pLKO can be used.

The composition is characterized in that the expression of SOX2 or NANOG gene is increased to increase the stemness of stem cells.

The composition increases the expression of MMP-2 (Matrix metallopeptidase-2) or MMP-9 (Matrix metallopeptidase-9) gene and induces epithelial-mesenchymal transition (EMT) markers fibronectin 1, , Twist, or vimentin gene expression in a mammal.

The stem cells may be embryonic stem cells (ESC), adult stem cells, or induced pluripotent stem cells (iPSC), but the present invention is not limited thereto.

In a specific experimental example of the present invention, it was confirmed that CSF-2 protein was markedly expressed in mesenchymal stem cells that had been differentiated from fibroblasts that had already undergone differentiation, so that CSF-2 protein could be a stem cell-specific expression marker (See FIG. 1), CSF-2 protein did not significantly affect the growth of mesenchymal stem cell line when adipose-derived mesenchymal stem cell line, an adult stem cell, was treated with CSF-2 protein (See FIG. 2), the expression of SOX2 and NANOG, which are closely related to the stemness of the adipose-derived mesenchymal stem cell line, was significantly increased when the CSF-2 protein was treated (see FIG. 3 ), And that the ability of the CSF-2 protein to differentiate into an osteoblast from an adipose-derived mesenchymal stem cell line was greatly improved (see FIG. 4).

In addition, the ability of the CSF-2 protein to migrate through the transwell of the adipose-derived mesenchymal stem cell line is greatly enhanced, and MMP-2, an important factor in the regulation of stem cell migration, And MMP-9 (Matrix metallopeptidase-2/9) were significantly increased (see FIG. 5). When CSF-2 protein was treated with adipose-derived mesenchymal stem cell line, cytoskeletal protein was rearranged into a spindle shape with increased mobility (see FIG. 6), and the EMT marker gene, which is well known to be involved in cell migration, The expression of fibronectin (FN 1), snail, twist, and vimentin was markedly increased (see FIG. 7).

Therefore, the CSF-2 protein (colony stimulating factor-2) protein of the present invention increases the migration and differentiation of stem cells. Therefore, when the CSF-2 protein is cultured in vitro for a large number of adult stem cells, Can improve the inherent properties of stem cells such as migration ability and can be usefully used as a composition for improving the therapeutic effect of a stem cell therapeutic agent.

In addition,

1) culturing human stem cells in vitro;

2) culturing the cultured cells of step 1) in a culture medium supplemented with CSF-2 (colony stimulating factor-2) protein to induce differentiation. Thereby providing a culture method.

The stem cell of step 1) is preferably an embryonic stem cell (ESC), an adult stem cell, or an induced pluripotent stem cell (iPSC), but is not limited thereto. Preferably, it is not limited to adult stem cells.

The adult stem cells of the present invention refers to undifferentiated cells having multipotency derived from a mammal including a human, preferably human adult cells. Examples thereof include bone marrow, blood, brain, skin, fat (i.e., Adipose tissue or adipocyte), umbilical cord blood, umbilical cord blood wharton's jelly, nerve, epithelium, or skin, more preferably derived from adipose tissue or bone marrow, Mesenchymal stem cells are most preferred, but are not limited thereto.

The treatment concentration of the CSF-2 (colony stimulating factor-2) protein of the present invention is preferably 10 to 500 ng / ml, but is not limited thereto. When the concentration of the protein is less than 10 ng / ml, there is a problem that the efficiency of stem cell migration or differentiation is lowered. When the concentration of the protein is more than 500 ng / ml, toxicity to stem cells may occur. In this respect, the CSF-2 protein concentration in the method and composition for improving the therapeutic effect of the stem cell therapeutic agent of the present invention may be preferably 50 to 300 ng / ml, more preferably 200 ng / ml But is not limited thereto.

In addition, the treatment of the CSF-2 (colony stimulating factor-2) protein of the present invention is preferably 48 to 72 hours, but is not limited thereto.

The present invention also provides a composition for enhancing the therapeutic effect of a stem cell therapeutic agent by adding CSF-2 (colony stimulating factor-2) protein to a culture solution of stem cells, May contain one or more kinds of effective ingredients.

The composition of the present invention may further comprise a pharmaceutically acceptable additive, wherein pharmaceutically acceptable additives include starch, gelatinized starch, microcrystalline cellulose, lactose, povidone, colloidal silicon dioxide, calcium hydrogen phosphate, lactose Starch glycolate, sodium starch glycolate, carnauba wax, synthetic aluminum silicate, stearic acid, magnesium stearate, aluminum stearate, calcium stearate, calcium stearate, , White sugar, dextrose, sorbitol and talc. The pharmaceutically acceptable additives according to the present invention are preferably included in the composition in an amount of 0.1 to 90 parts by weight, but are not limited thereto.

Hereinafter, the present invention will be described in detail with reference to Examples and Experimental Examples.

However, the following Examples and Experimental Examples are merely illustrative of the present invention, and the present invention is not limited by the following Examples and Experimental Examples.

< Example  1> Local origin Intermediate lobe  Preparation of stem cell lines

Prior to the experiment, adipocyte mesenchymal stem cell lines (MSCs) were prepared as follows.

After removing the adipose tissue, the fibrin material and the red blood vessel, which are pink in the washed fat tissue, were removed with sterilized tweezers and surgical scissors. 10 ml collagenase enzyme preparation solution prepared beforehand was added to the fat tissue, and the fat tissue was minced to a size of 1 mm ³ or less with surgical scissors, transferred to sterilized 500 ml Erlenmeyer flask, Approximately 30 ml to 40 ml of a collagenase enzyme cleavage solution was added. Next, the inlet of the Erlenmeyer flask was sealed with a foil, and an enzyme reaction was carried out in a constant temperature water bath set at 37 ° C. and 100 rpm for one day. The adipose tissue was transferred back into a 50 ml centrifuge tube and centrifuged at 2500 rpm for 10 minutes The extracellular matrix of the supernatant and the yellow oil layer were removed using a vacuum pump. Next, the stromal vascular cell fraction (SVF) was suspended in the culture medium DMEM, and the suspended SVF was slowly passed through a 100-μm cell strainer in a new centrifugation tube . The filtered SVF was passed through a 40-μm cell strainer in the same manner. The filtered SVF was added to the culture medium DMEM, and the cells were transferred to collagen I cell ware 100 mm culture dish. Then, the cells were incubated in a carbon dioxide incubator (37 ° C., 5% ₂ ) until the cells were adhered. When the cells were confirmed to be adhered, they were subcultured and used in the experiment.

< Experimental Example  1 > stem cell-specific expression CSF -2 protein

First, the present inventors used real-time PCR for mesenchymal stem cell lines (MSCs) derived from fibroblasts and two undifferentiated adipose tissue differentiated as mesenchymal stem cells, 2 (colony stimulating factor-2) gene, the following experiment was conducted.

The above fibroblasts and mesenchymal stem cell lines (MSCs) were seeded on a 60 mm plate to a cell number of about 2 x 10 ⁵ and cultured for 48 hours. The cells were washed with 10 ml of D-PBS, and 500 μl of Tizol (based on a 6-well plate) was added and reacted for 15 minutes. Cells were collected again by pipetting, transferred to a tube, and 200 μl of chloroform was added thereto, followed by centrifugation at 4 ° C. and 13,000 rpm for 15 minutes. Thereafter, the supernatant was separated, transferred to a new tube, reacted with 500 μl of isopropanol for 15 minutes, and centrifuged again at 4 ° C. and 13,000 rpm for 15 minutes. Next, the supernatant was discarded and only the precipitated pellets were separated, washed with 75% ethanol, and then centrifuged again at 4 ° C and 13,000 rpm for 15 minutes. Next, the ethanol was removed, and the RNA was completely dried. The pellet was dissolved in sterilized water and the RNA was quantified by nano-drop. Random 6mers, dNTP mixture, Rnase inhibitor and Primescript RTase were added to the diluted RNA CDNA was synthesized by mixing Buffer. The synthesized cDNA was diluted 1/10 and RT-qPCR was performed by mixing CSF-2 Primer and SYBER Green mixture shown in Table 1.

name SEQ ID NO: order CSF-2 amino acid sequence SEQ ID NO: 1 MAPARSPSPS TQPWEHVNAI QEARRLLNLS RDTAAEMNET VEVISEMFDL QEPTCLQTRL ELYKQGLRGS LTKLKGPLTM MASHYKQHCP PTPETSCATQ IITFESFKEN LKDFLLVIPF DCWEPVQE CSF-2 base sequence SEQ ID NO: 2 5'atggcgccggcgcgcagcccgagcccgagcacccagccgtgggaacatgtgaacgcgattcaggaagcgcgccgcctgctgaacctgagccgcgataccgcggcggaaatgaacgaaaccgtggaagtgattagcgaaatgtttgatctgcaggaaccgacctgcctgcagacccgcctggaactgtataaacagggcctgcgcggcagcctgaccaaactgaaaggcccgctgaccatgatggcgagccattataaacagcattgcccgccgaccccggaaaccagctgcgcgacccagattattacctttgaaagctttaaagaaaacctgaaagattttctgctggtgattccgtttgattgctgggaaccggtgcaggaa-3 ' CSF-2 forward primer SEQ ID NO: 3 5'-ATGATGGCCAGCCACTACAA-3 ' CSF-2 reverse primer SEQ ID NO: 4 5'-AGCAGTCAAAGGGGATGACA-3 '

As a result, as shown in Fig. 1, CSF-2 protein was more than 100 times more expressed in mesenchymal stem cells undifferentiated than the already differentiated fibroblasts (Fig. 1). This means that the CSF-2 protein may be a stem cell-specific expression marker.

< Experimental Example  2> CSF -2 Confirmation of effect on stem cell growth through protein treatment

Next, in order to examine the effect of CSF-2 protein on the growth of stem cells, the adipose-derived mesenchymal stem cell line was treated with recombinant CSF-2 protein (rCSF2) The MTT assay, an experimental method for measuring survival, was performed as follows.

First, the adipose-derived mesenchymal stem cell line was seeded in a 96-well plate at a cell number of about 2 x 10 ³ , treated with 1% FBS (Fetal bovine serum) in a basic culture medium, / ml &lt; / RTI &gt; recombinant CSF-2 protein for 72 hours. At this time, the medium was changed every day, and the recombinant CSF-2 protein was treated at a constant concentration. Next, MTT solution (MTT (3- [4,5-dimethylthiazol-2-yl] -2,5-diphenyl-tetrazolium bromide agent) was added to 1/20 volume of the culture medium After incubation for 4 hours in a CO2 incubator, the culture medium was removed, and 200 μl of DMSO was added thereto, and the mixture was reacted at room temperature for 3 to 5 minutes in a state of blocking light. Next, using a microplate reader, MTT Was reduced to formazan by mitochondria at an absorbance of 570 nm and the absorbance of the mesenchymal stem cell line treated with the recombinant CSF-2 protein versus the absorbance of the control cells (%) was measured. The effect on cell growth was evaluated.

As a result, as shown in Fig. 2, it was confirmed that the CSF-2 protein had no significant effect on the growth of the mesenchymal stem cell line (Fig. 2).

< Experimental Example  3> CSF -2 Stem Cellularity of Stem Cells through Protein Treatment stemness ) Check the improvement effect

The present inventors examined the effect of CSF-2 protein on the stemness of stem cells. After treating CSF-2 protein, markers closely related to stemness (stemness) The expression of SOX2 and NANOG genes was measured by real-time PCR.

First, medium containing 1% FBS (Fetal bovine serum) and 200 ng / ml CSF-2 protein were added daily for 48 hours in a basic culture medium of adipose derived mesenchymal stem cell line. After the culture was completed, the medium was removed and 1 ml of trizol was added thereto. The mixture was shaken at room temperature for 5 minutes and 1.5 ml of trizol-added cell solution was pipetted into a tube. Next, 200 μl of chloroform was added and mixed well. The mixture was allowed to react at room temperature for 5 minutes, and centrifuged at 12,000 rpm for 15 minutes at 4 ° C. Next, the supernatant was transferred to a new tube, and 500 μl of isopropanol was added and the tube was mixed upside down. It was left at room temperature for 15 minutes. Thereafter, the supernatant was discarded by centrifugation at 12,000 rpm at 4 ° C for 15 minutes, 1 ml of ethanol (75%) was added, and the mixture was thoroughly mixed and then centrifuged at 12,000 rpm at 4 ° C for 12 minutes. Next, the supernatant was discarded, the tube was turned upside down so that ethanol was not left, the RNA was dissolved in sterilized water, and RNA concentration was quantified using nanodrop. Then, cDNA was synthesized with 2 μg / 8 μl of the quantified RNA. RT-qPCR was performed using the synthesized cDNA using the SOX2 and NANOG primers shown in Table 2 below, and the expression patterns of the respective genes were compared.

name SEQ ID NO: order SOX2 forward primer SEQ ID NO: 5 5'-AAATGGGAGGGGTGCAAAAGAGGAG-3 ' SOX2 forward primer SEQ ID NO: 6 5'-CAGCTGTCATTTGCTGTGGGTGATG-3 ' NANOG forward primer SEQ ID NO: 7 5'-ACACCATTGCTATTCTTCGGCCAGTTG-3 ' NANOG forward primer SEQ ID NO: 8 5'-CAGCTGTGTGTACTCAATGATAGATTT-3 '

As a result, as shown in Fig. 3, when the CSF-2 protein was treated, expression of SOX2 (A in Fig. 3) and NANOG (B in Fig. 3), which are marker genes closely related to stemness, Of the total number of patients.

< Experimental Example  4> CSF 2 Differentiation of stem cells through treatment of protein Ability increase  Confirm

To examine the effect of CSF-2 protein on the differentiation ability of stem cells when treated with stem cells, the inventors treated CSF-2 protein in adipose-derived mesenchymal stem cell lines and then cultured osteoblast ).

First, the adipose-derived mesenchymal stem cell line was cultured in a 6-well plate. When the cell number reached a confluency of about 70%, DMEM High Glucose with 10 mM β-glycerophosphate, 50 μM ascorbic acid, 100 μM dexamethasone) was treated with 1% FBS and cultured for about 2 weeks while changing the medium every 3 days. At this time, 200 ng / ml CSF-2 protein was treated together with each time the medium was changed. Next, the medium was removed from a 6-well plate in which differentiation-induced cells were grown, and the cells were washed twice with 2 ml of sterilized water. Next, 1 ml of ethanol (70%) was added to fix the cells, and the plate was dried at 4 ° C for 30 minutes to 1 hour. Then, 1 ml of D-PBS was added to the fixed cells, And stained with 1 ml alizarin red solution for 10 minutes to 1 hour. Thereafter, the cells were washed five times with sterilized water or D-PBS, and 2 ml of sterilized water was added to the washed cells, and the degree of differentiation was visually confirmed by confocal microscopy. The degree of differentiation was measured by measuring the absorbance at 570 nm.

As a result, as shown in FIG. 4, when the CSF-2 protein was treated, the ability of the adipose-derived mesenchymal stem cell line to differentiate into osteoblasts was remarkably improved by the alizarin red staining (Fig. 4A), and graphs obtained by measuring the degree of differentiation by absorbance showed that the ability to differentiate into CSF-2 protein when treated with adipose-derived mesenchymal stem cell line CSF-2 protein (Fig. 4B).

< Experimental Example  5> CSF 2 protein, the migration of stem cells Ability increase  Confirm

<5-1> Transwell Assay ( transwell  assay

The present inventors conducted transwell assay experiments after treating CSF-2 protein to measure the effect of CSF-2 protein on stem cell migration ability.

First, the adipose-derived mesenchymal stem cell line was cultured for 48 hours with 200 ng / ml CSF-2 protein treated daily. Before carrying out the experiment, 1 ml of trypsin-EDTA was added and the mixture was shaken by hand to spread evenly. The reaction was carried out in a carbon dioxide incubator for 5 minutes. 2 ml of FBS-supplemented culture medium was added to the culture dish, . After incubation, the cells were transferred to a 50-ml centrifuge tube and centrifuged at 2000 rpm for 3 minutes. The supernatant was removed, and 1 ml of the culture medium was added And the mixture was pipetted. Next, 1-ml serum-free medium and 200 ng / ml CSF-2 protein are treated together in a 12-well plate and insert wells are added. The prepared cells were added to each well in an insert well (150 μl), and the cells were shaken to prevent aggregation, followed by culturing for 24 hours. Next, the medium in the insert well was discarded and fixed with paraformaldehyde (PFA; 4%) for 15 minutes. Then, paraformaldehyde (PFA; 4%) in insert wells and plates was discarded, washed twice with 1 ml DPBS, and then hematosillin was added to the plate 700 μl / well was added to each well, and an insert well was reacted. At this time, the reaction was carried out at room temperature for 30 minutes in the state of blocking light. Then, the inside of the insert well was carefully wiped off with a cotton swab, and the number of stained cells was quantitated.

As a result, as shown in Fig. 5, it was confirmed that the migration ability of the adipose-derived mesenchymal stem cell line through the transwell was greatly improved by the CSF-2 protein (Fig. 5, left and right ).

<5-2> MMP -2 and MMP -9 (Matrix 금속opeptidase -2/9) Expression confirmation

In order to further examine the influence of CSF-2 protein on the migration ability of stem cells, the inventors of the present invention examined the effect of CSF-2 protein on MMP -2 and MMP-9 (Matrix metallopeptidase-2/9) were confirmed by western blotting experiments.

First, the basal medium of adipose-derived mesenchymal stem cell line was treated with 1% FBS (Fetal bovine serum) and treated with 200 ng / ml CSF-2 protein and cultured for 72 hours. The CSF-2 protein was treated daily with the medium changed. The pro-prep protein extraction solution was added to the dish on which the cells were cultured. The cells were collected using a scraper, collected in a 1.5 ml tube, and centrifuged at 12,000 rpm at 4 ° C for 15 minutes. After transferring the supernatant to a new tube, proteins were quantified using BCA solution. Next, 10 μg of protein was mixed with distilled water (DW) and 5 × dye, boiled at 99 ° C for 5 minutes, loaded onto a SDS-PAGE gel, loaded with a protein marker and a sample, Followed by electrophoresis for 50-90 minutes to separate the proteins by size. Then, proteins present on the gel were transferred to a nitrocellulose membrane at 100 V for 180 minutes, and then blocked with 5% skim milk. The membrane was reacted with T-PBS containing primary antibody against MMP-2 and MMP-9 (Matrix metallopeptidase-2/9) at 4 ° C for 24 hours. The membrane was washed three times with T-PBS for 10 minutes on the following day, and then the membrane was reacted with T-PBS containing secondary antibody. After 2 hours, T-PBS was washed three times The ECL solution was washed and treated with a membrane to confirm the expression of the protein as a film.

As a result, as shown in Fig. 5, when CSF-2 protein was treated to an adipose-derived mesenchymal stem cell line, MMP-2 and MMP-9 (Matrix metallopeptidase- 2/9) expression was significantly increased, indicating that the migration ability of stem cells was greatly improved (B and L in Fig. 5).

<5-3> Cytoskeleton  Verify rearrangement induction

When stem cells are migrated, rearrangement of cytoskeletal proteins such as actin filaments is very important. The present inventors have found that CSF-2 protein rearrangement of cytoskeletal proteins through immunofluorescence staining experiments of phalloidin with a fluorescent substance that specifically reacts with F-actin, On the other hand.

First, the basal medium of the adipose-derived mesenchymal stem cell line was treated with 1% fetal bovine serum (FBS) and cultured for 72 hours with 200 ng / ml CSF-2 protein treated daily. Cell culture medium was removed and washed with PBS. 2 ml paraformaldehyde (PFA; 4%), a fixation solution, was added and incubated at room temperature for 30 minutes. Then, And washed three times with PBS for 5 minutes each. Next, the phalloidin antibody was treated at a concentration of 1: 1000 in the light-blocked state for 2 hours, and then washed three times with PBS for 5 minutes at room temperature. In order to stain nuclei, DAPI was treated for 1 minute at a concentration of 1: 30,000, washed three times with PBS for 5 minutes at room temperature, mounted on a slide, and analyzed for the expression pattern of phalloidin using a fluorescent microscope Were observed.

As a result, as shown in Fig. 6, when the CSF-2 protein was treated with an adipose-derived mesenchymal stem cell line, the cytoskeletal protein of the stem cell was spindle- shape (Fig. 6).

<5-4> Transitional epithelium (Epithelial- Mesenchymal  Transition; EMT ) Confirmation of increased gene expression

In addition, migration of stem cells may be induced by epithelial-mesenchymal transition (EMT) markers such as fibronectin (FN 1), snail, twist, and vimentin Is also well known. Thus, the present inventors conducted the following experiment to determine the effect of CSF-2 protein on the expression of EMT markers in stem cells.

First, the basal medium of adipose-derived mesenchymal stem cell line was treated with 1% FBS (fetal bovine serum) and cultured for 48 hours with 200 ng / ml CSF-2 protein treated daily. When the culture was completed, remove the medium, add 1 ml of trizol, stir at room temperature for 5 minutes, collect the cells again by pipetting, put in a 1.5 ml tube, add 200 μl of chloroform, After 5 minutes at room temperature, the reaction was centrifuged at 12,000 rpm for 15 minutes at 4 ° C. Next, the supernatant is transferred to a new tube, and 500 μl of isopropanol is added. The tube is inverted upside down and reacted at room temperature for 15 minutes. Next, the supernatant was discarded at 12,000 rpm at 4 ° C for 15 minutes, 1 ml of 75% ethanol was added, and the mixture was mixed well. The mixture was centrifuged at 12,000 rpm and 4 ° C for 12 minutes. The supernatant was discarded, the tube was turned upside down to leave no ethanol, the RNA was dissolved in sterilized water, and the RNA concentration was quantified using nanodrop. The synthesized cDNA was used to synthesize fibronectin (FN 1), snail, twist, and vimentin described in Table 3 below. The cDNA was synthesized using 2 μg / 8 μl of the quantified RNA, After RT-qPCR was performed using primers, the expression patterns of the genes were compared.

name SEQ ID NO: order FN 1 forward primer SEQ ID NO: 9 5'-GAGAATGGACCTGCAAGCCCA-3 ' FN 1 reverse primer SEQ ID NO: 10 5'-GGTGCAAGTGATGCGTCCGC-3 ' snail forward primer SEQ ID NO: 11 5'-CCTCCCTGTCAGATGAGGAC-3 ' snail reverse primer SEQ ID NO: 12 5'-CCAGGCTGAGGTATTCCTTG-3 ' twist forward primer SEQ ID NO: 13 5'-GGAGTCCGCAGTCTTACGAG-3 ' twist reverse primer SEQ ID NO: 14 5'-TCTGGAGGACCTGGTAGAGG-3 ' vimentin forward primer SEQ ID NO: 15 5'-ACCCGCACCAACGAGAAGGT-3 ' vimentin reverse primer SEQ ID NO: 16 5'-ATTCTGCTGCTCCAGGAAGCG-3 '

As a result, as shown in FIG. 7, when the CSF-2 protein was treated to an adipose-derived mesenchymal stem cell line, fibronectin (FN 1), an EMT marker gene known to be involved in cell migration, (Figs. 7A, 7B, 7C and 7D) that the expression of snails, twist, and vimentin was significantly increased.

<110> Gachon University of Industry-Academic cooperation Foundation          Gil Medical Center          Gwangju Institute of Science and Technology          Jung Won University of Industry-Academic cooperation Foundation <120> Methods and compositions for enhancing clinical therapeutic          effect of stem cells medicine <130> 2016P-08-012 <160> 16 <170> KoPatentin 3.0 <210> 1 <211> 128 <212> PRT <213> Artificial Sequence <220> <223> CSF-2 <400> 1 Met Ala Pro Ala Arg Ser Pro Ser Ser Ser Thr Gln Pro Trp Glu His   1 5 10 15 Val Asn Ale Ile Gln Glu Ala Arg Arg Leu Leu Asn Leu Ser Arg Asp              20 25 30 Thr Ala Glu Met Asn Glu Thr Val Glu Val Ile Ser Glu Met Phe          35 40 45 Asp Leu Gln Glu Pro Thr Cys Leu Gln Thr Arg Leu Glu Leu Tyr Lys      50 55 60 Gln Gly Leu Arg Gly Ser Leu Thr Lys Leu Lys Gly Pro Leu Thr Met  65 70 75 80 Met Ala Ser His Tyr Lys Gln His Cys Pro Thr Pro Glu Thr Ser                  85 90 95 Cys Ala Thr Gln Ile Ile Thr Phe Glu Ser Phe Lys Glu Asn Leu Lys             100 105 110 Asp Phe Leu Leu Val Ile Pro Phe Asp Cys Trp Glu Pro Val Gln Glu         115 120 125 <210> 2 <211> 388 <212> PRT <213> Artificial Sequence <220> <223> CSF-2 <400> 2 Ala Ala Ala Thr Gly Gly Cys Gly Cys Cys Gly Gly Cys Gly Cys Gly   1 5 10 15 Cys Ala Gly Cys Cys Cys Gly Ala Gly Cys Cys Cys Gly Ala Gly Cys              20 25 30 Ala Cys Cys Cys Ala Gly Cys Cys Gly Thr Gly Gly Gly Ala Ala Cys          35 40 45 Ala Thr Gly Thr Gly Ala Ala Cys Gly Cys Gly Ala Thr Thr Cys Ala      50 55 60 Gly Gly Ala Ala Gly Cys Gly Cys Gly Cys Cys Gly Cys Cys Thr Gly  65 70 75 80 Cys Thr Gly Ala Ala Cys Cys Thr Gly Ala Gly Cys Cys Gly Cys Gly                  85 90 95 Ala Thr Ala Cys Cys Gly Cys Gly Gly Cys Gly Aly Ala Ala Thr             100 105 110 Gly Ala Ala Cys Gly Aly Ala Ala Cys Cys Gly Aly Ala         115 120 125 Gly Thr Gly Ala Thr Thr Aly Aly Aly Thr Gly Thr     130 135 140 Thr Thr Gly Ala Thr Cys Thr Gly Cys Ala Gly Gly Ala Ala Cys Cys 145 150 155 160 Gly Ala Cys Cys Thr Gly Cys Cys Thr Gly Cys Ala Gly Ala Cys Cys                 165 170 175 Cys Gly Cys Cys Thr Gly Gly Ala Ala Cys Thr Gly Thr Ala Thr Ala             180 185 190 Ala Ala Cys Ala Gly Gly Gys Cys Cys Thr Gly Cys Gly Cys Gly Gly         195 200 205 Cys Ala Gly Cys Cys Thr Gly Ala Cys Cys Ala Ala Cys Thr Gly     210 215 220 Ala Ala Gly Gly Cys Cys Cys Gly Cys Thr Gly Ala Cys Cys Ala 225 230 235 240 Thr Gly Ala Thr Gly Gly Cys Gly Ala Gly Cys Cys Ala Thr Thr Ala                 245 250 255 Thr Ala Ala Cys Ala Gly Cys Ala Thr Thr Gly Cys Cys Cys Gly             260 265 270 Cys Cys Gly Ala Cys Cys Cys Cys Cys Cys Ala         275 280 285 Gly Cys Thr Gly Cys Gly Cys Gly Ala Cys Cys Cys Ala Gly Ala Thr     290 295 300 Thr Ala Thr Thr Ala Cys Cys Thr Thr Thr Gly Ala Ala Ala Gly Cys 305 310 315 320 Thr Thr Thr Ala Ala Gly Ala Ala Ala Ala Cys Cys Thr Gly Ala                 325 330 335 Ala Ala Gly Ala Thr Thr Thr Thr Cys Thr Gly Cys Thr Gly Gly Thr             340 345 350 Gly Ala Thr Thr Cys Cys Gly Thr Thr Thr Gly Ala Thr Thr Gly Cys         355 360 365 Thr Gly Gly Gly Aly Aly Cys Aly Gly Gly Aly Gly Gly     370 375 380 Ala Ala Ala Ala 385 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CSF-2 primer F <400> 3 atgatggcca gccactacaa 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CSF-2 primer R <400> 4 agcagtcaaa ggggatgaca 20 <210> 5 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> SOX2 primer F <400> 5 aaatgggagg ggtgcaaaag aggag 25 <210> 6 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> SOX2 primer R <400> 6 cagctgtcat ttgctgtggg tgatg 25 <210> 7 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> NANOG primer F <400> 7 acaccattgc tattcttcgg ccagttg 27 <210> 8 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> NANOG primer R <400> 8 cagctgtgtg tactcaatga tagattt 27 <210> 9 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> FN 1 primer F <400> 9 gagaatggac ctgcaagccc a 21 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> FN 1 primer R <400> 10 ggtgcaagtg atgcgtccgc 20 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> snail primer F <400> 11 cctccctgtc agatgaggac 20 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> snail primer R <400> 12 ccaggctgag gtattccttg 20 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Twist primer F <400> 13 ggagtccgca gtcttacgag 20 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> twist primer R <400> 14 tctggaggac ctggtagagg 20 <210> 15 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> vimentin primer F <400> 15 acccgcacca acgagaaggt 20 <210> 16 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> vimentin primer R <400> 16 attctgctgc tccaggaagc g 21

Claims (11)

A composition for promoting migration of adipose derived mesenchymal stem cells comprising as an active ingredient CSF-2 (colony stimulating factor-2) protein or a polynucleotide encoding the same.
2. The composition for promoting migration of adipose derived mesenchymal stem cells according to claim 1, wherein the CSF-2 (colony stimulating factor-2) protein is composed of the amino acid sequence shown in SEQ ID NO: 1.
2. The composition for promoting migration of adipose derived mesenchymal stem cells according to claim 1, wherein the CSF-2 (colony stimulating factor-2) polynucleotide comprises the nucleotide sequence of SEQ ID NO: 2.
The composition for promoting migration of adipose derived mesenchymal stem cells according to claim 1, wherein the composition enhances stem cell stem cells.
The composition for promoting migration of adipose derived mesenchymal stem cells according to claim 1, wherein the composition increases expression of SOX2 or NANOG gene.
The composition for promoting migration of adipose derived mesenchymal stem cells according to claim 1, wherein the composition increases the expression of MMP-2 (Matrix metallopeptidase-2) or MMP-9 (Matrix metallopeptidase-9) gene.
2. The method of claim 1, wherein the composition is selected from the group consisting of fibroblasts, fibroblasts, fibroblasts, fibroblasts, fibroblasts, fibroblasts, fibroblasts, / RTI &gt;
delete 1) culturing adipose-derived adipose-derived mesenchymal stem cells in vitro;
2) further culturing the cultured cells of step 1) in a culture medium supplemented with CSF-2 (colony stimulating factor-2) protein;
Derived mesenchymal stem cells. &Lt; RTI ID = 0.0 &gt; 21. &lt; / RTI &gt;
delete [10] The method according to claim 9, wherein the concentration of CSF-2 (colony stimulating factor-2) protein in step 1) is 10 to 500 ng / ml.

Images