KR20150036838A - Markers for detecting adipogenic differentiation of mesenchymal stem cells and its uses - Google Patents

Abstract

Disclosed are a composition for detecting adipogenic differentiation of mesenchymal stem cells which uses fatty acid methyl ester as markers, and a use thereof. According to the present invention, the composition allows to nondestructively identify whether a cultivation of stem cells which is essentially required with amplification and differentiation through an in vitro cultivation is differentiated or not, thereby improving efficiency and convenience of effective differentiation of the stem cells and clinical application thereof.

Description

Markers for differentiation of mesenchymal stem cells into adipocytes and their use {Markers for detecting adipogenic differentiation of mesenchymal stem cells and their uses}

This is a technical field related to stem cell differentiation detection.

Stem cells are the omnipotent cells of the embryo that can differentiate into any tissue. Representative stem cells include embryonic stem cells and adult stem cells using human embryos. These adult stem cells are small numbers of cells in each tissue of the body, and they constitute specific tissues. Typical examples are pluripotent stem cells and mesenchymal stem cells. These adult stem cells provide the cells necessary for the body to remain healthy at all times and, when the damage occurs, the stem cells in the other organs that have migrated to the damaged area are differentiated into damaged tissues and restored.

Mesenchymal stem cells can differentiate into various cells such as adipocytes, osteoblasts, chondrocytes, cardiac cells, muscle cells and neurons. However, since they have limited proliferative power unlike embryonic stem cells, there is no possibility of cancer cell formation, There is no ethical problem. In addition, since mesenchymal stem cells do not induce an immune response during transplantation, they are becoming an effective means of cell therapy.

However, it is pointed out that the disadvantage that the number of stem cells obtained from an individual is low and cultivation is difficult, and that it can be differentiated into specific cells, is pointed out as a limitation of the study, and studies are being conducted to overcome this problem.

U.S. Published Patent Application No. 2011-0129918 relates to a method for culturing mesenchymal stem cells at a low density under hypoxic conditions and a method for culturing mesenchymal stem cells without deteriorating the proliferative capacity, aging and differentiation ability of mesenchymal stem cells .

Korean Patent Laid-Open Publication No. 2009-0052704 discloses an adipocyte differentiation detection marker and a method for regulating differentiation of mesenchymal stem cells using the marker. The expression level of protein tyrosine phosphatase (PTP) selected from the group consisting of PTPRQ (protein tyrosine phosphatase receptor type Q), SSH2 (slingshothomolog 2) and MTMR11 (myotubularin related protein 11) was measured to identify differentiation markers and differentiation In the case of the above-mentioned method.

However, since adult stem cells can not be obtained from an individual, an artificial amplification is necessary in Invitro. Since mesenchymal stem cells have not yet established an efficient mass differentiation technique into specific cells, there is a great limitation in actual clinical and industrial use.

Therefore, it is necessary to develop a technique for culturing cells that have differentiated into specific stages and have homogeneity.

The present invention provides a method for determining whether adult stem cells are differentiated into adipocytes.

In one embodiment, the present invention is directed to a process for the preparation of a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein the compound is selected from the group consisting of Methyl laurate, Methyl myristate, Methyl palmitate, Methyl linoleate, Methyl oleate, elaidate, and methyl stearate. The present invention also provides a composition for detecting the differentiation of mesenchymal stem cells into adipocytes.

The mesenchymal stem cells according to the present invention are not limited to bone marrow, blood, dermis or periosteum, but are not limited thereto.

The detection substance contained in the composition according to the present invention includes a substance used in an antibody analysis, a chemiluminescence assay, or a mass spectrometry method. For example, include receptors, ligands, substrates, antibodies, antibody fragments, antibody mimetics, aptamers, avidity multimers, or peptidomimetics that specifically recognize the lipids herein. However, it is not limited thereto.

In another aspect, the present invention provides a kit for detecting the differentiation of mesenchymal stem cells into adipocytes, comprising the composition according to the present invention.

In another embodiment, the present invention also provides a pharmaceutical composition comprising at least one compound selected from the group consisting of methyl laurate, methyl myristate, methyl palmitate, methyl linolate, methyl oleate, methyl elaidate, methyl stearate and derivatives thereof in mesenchymal stem cells And detecting fatty acid methyl esters of the mesenchymal stem cells.

In the method according to the present invention, the step of extracting the fatty acid methyl ester may be performed by a solid-phase microextraction (SPME) method or a head-SPME (HS-SPME) But is not limited thereto. In one embodiment, the fiber used in the SPME is not limited to polyacrylate (PA), polydimethylsiloxane (PDMS), Carboxen / PDMS, or PDMS / DVB (Divinylbenzene).

The fatty acid methyl ester detection in the method according to the present invention may be performed by one or more of antibody analysis, chemiluminescence assay, or mass spectrometry analysis method, but is not limited thereto.

In another embodiment, the method further comprises the step of determining the cell as an adipocyte when the amount of at least one of the fatty acid methyl ester or derivative thereof is increased in the mesenchymal stem cell compared to the control.

In another embodiment, the present invention also provides a method of producing a stem cell comprising contacting a mesenchymal stem cell with a test material; Quantifying at least one substance selected from the group consisting of methyl laurate, methyl myristate, methyl palmitate, methyl linoleate, methyl oleate, methyl elaidate, and methyl stearate in the mesenchymal stem cells after contact with the test substance; And comparing the amount of the substance with the amount of the at least one substance quantified in the control cells not in contact with the test substance or in the control cells before contact with the test substance, The method comprising the step of selecting a stem cell as a differentiation promoting material for a fat cell, and a method for selecting a differentiation regulator as a fat cell of a mesenchymal stem cell.

The present invention provides a composition for detecting the differentiation of mesenchymal stem cells into adipocytes using a fatty acid methyl ester as a marker is capable of nondestructively identifying whether or not differentiation occurs in the culture of stem cells in which amplification and differentiation are essential through in vitro culture, Efficiency and convenience are improved in efficient differentiation and clinical application of stem cells. In addition, it is possible to identify the differentiation before application of the quality control of the cell as well as the clinical application, thereby increasing the success rate of the clinical application.

FIG. 1A is a schematic diagram of the entire process of extracting a fatty acid methyl ester. FIG.
1B is a schematic diagram of a method of performing HS-SPME using a fiber in a cell culture plate.
Fig. 1C shows the results of differentiation of mesenchymal stem cells into adipocytes and undifferentiated states by oil red O staining.
FIG. 1D shows the results of analysis of the expression level of the marker gene induced by differentiation after differentiation using RT-PCR.
FIG. 2A shows a result of staining a fibroblast-like morphology of a mesenchymal stem cell with a crystal violet upon 2D culture.
FIG. 2B is a flow cytometric analysis result of a marker expressed in mesenchymal stem cells.
FIG. 2c shows the results of differentiation of mesenchymal stem cells into adipocytes, bone cells, and chondrocytes using tissue-specific staining and mRNA expression of each differentiation marker.
3A is a chromatogram obtained by GC-MS analysis of stem cells differentiated into mesenchymal stem cells and adipocytes.
FIG. 3B is a histogram showing quantitative values of fatty acid methyl esters detected in FIG. 3A.
FIG. 4 is a result of quantitative analysis of suitability for extracting fatty acid methyl esters of widely used PA, PDMS, CAR / PDMS and PDMS / DVB among SPME fibers.
FIG. 5 shows the results of analyzing the linearity, the limit of detection, and the limit of quantification of fatty acid methyl esters in order to verify the method of analyzing fatty acid methyl esters.
FIG. 6 is a result of confirming the reproducibility in order to confirm the deterioration of the components and the error of the components that can appear in the analysis of the respective fatty acid methyl esters. Intra-day and inter-day were identified at three different concentrations.
FIG. 7 shows mass spectrometry spectral results of methyl laurate. FIG.
8 is a mass spectrometry spectral result of methyl myristate.
9 is a mass spectrometry spectral result of methyl palmitate.
10 is a mass spectrometry spectral result of methyl linoleate.
11 is a mass spectrometry spectral result of methyl oleate.
12 shows mass spectrometry spectral results of methyl elaidate.
13 is a mass spectrometry spectral result of methyl stearate.

The present invention is based on the detection of volatile metabolic markers which can detect the metabolism of adipocytes in adipose tissue differentiation.

Accordingly, in one aspect, the present invention relates to a composition for detecting the differentiation of mesenchymal stem cells into adipocytes, comprising a detection substance of at least one fatty acid methyl ester selected from the group consisting of the fatty acid methyl esters listed in Table 1 below.

[Table 1]

Methyl laurate, Methyl myristate, Methyl palmitate, Methyl linoleate, Methyl oleate, Methyl oleate, Methyl oleate, And a detection substance of at least one fatty acid methyl ester selected from the group consisting of Methyl elaidate, Methyl stearate and derivatives thereof. The present invention also relates to a composition for detecting the differentiation of mesenchymal stem cells into adipocytes.

Since mesenchymal stem cells can not be obtained from an individual, an artificial amplification is essential. In order to confirm whether the mesenchymal stem cells are differentiated, it is necessary to monitor the activity and the characteristics of the cells with increasing number of cells in the cell culture process, Is very important for the quality control that we want to acquire. In one embodiment according to the present application, mesenchymal stem cells are used for detection of adipocytes.

As used herein, the term "mesenchymal stem cell" is a stem cell which is isolated from bone marrow, blood, dermis or periosteum. It is a pluripotent stem cell capable of differentiating into various cells such as adipocytes, chondrocytes, Cells. The mesenchymal stem cells are derived from an animal, preferably a mammal, more preferably a human.

As used herein, the term "marker or marker" refers to a substance capable of distinguishing cells that have been differentiated or undergone differentiation from adipocyte differentiation cells before they are differentiated, Or reduced biologically active forms, or organic biomolecules such as nucleic acids, lipids, glycolipids, glycoproteins, and the like. In one embodiment herein, fatty acid methyl esters whose expression increases with cell differentiation, such as methyl laurate, methyl myristate, methyl palmitate, methyl linoleate, methyl oleate, methyl elaidate, methyl stearate And derivatives thereof.

As used herein, the term " detection or discrimination "includes quantitative and / or qualitative analysis, including detection of presence and absence and determination of concentration, which methods are well known in the art and are well known to those skilled in the art You will be able to choose a method.

The methyl esters or derivatives thereof according to the present invention can be detected through a variety of known methods, and a suitable detection method can be selected by those skilled in the art. See, for example, Lipids, 2005, Volume 40, Issue 4, pp 419-428.

In one embodiment according to the present invention, the methyl ester or derivative thereof may be a receptor, ligand, substrate, antibody, antibody fragment, aptamer, avidity multimer or peptidomimetics that specifically recognize it. . The detecting substance can be used for antibody analysis, chemiluminescence assay, mass spectrometry or the like, and of course, the method can be used for the detection of a marker according to the present invention.

According to one embodiment of the present invention, markers can be detected using mass spectrometry, particularly mass spectrometry suitable for analysis of volatile metabolites. For example, Applied Biochemistry and Biotechnology Jul-Sep 2000, Volume 88, Issue 1-3, pp 33-44.

In one embodiment according to the present invention, HS-SPME-GC-MS (HS-SPME) Gas Chromatography Mass Spectrometry (HSSM) analysis can be used.

In other implementations, multiple reaction monitoring (MRM) techniques using, for example, Triple Quadrupole LC-MS / MS are used. MRM is a method for quantitatively and precisely measuring multiple markers such as biomarkers present in a sample. The first mass filter (Q1) is used to measure a precursor ion or a parent ion among the ion fragments generated in the ionization source Optionally to the collision tube. Then, the precursor ions reaching the impingement tube collide with the inner impact gas and are split to generate product ions or daughter ions and are sent to the second mass filter Q2, where only the characteristic ions are transmitted to the detection unit. It is a selective and sensitive analytical method that can detect only the information of the desired component in this way. See, for example, Gillette et al., 2013, Nature Methods 10: 28-34.

In another embodiment according to the present application, gas or liquid chromatography mass spectrometry is used for detection of the marker. The data of the gas or liquid chromatography mass spectrometry can also be applied to selected ion monitoring (SIM) or MRM (mass spectrometry) analyzes, which analyze based on the exact molecular weight value of the mass spectrometer without markers on the markers and the reproducibility of the retention time Can be used.

In another embodiment, antibody assays can be used. Antibody assays can be performed using materials that specifically recognize the markers herein, such as polyclonal antibodies, monoclonal antibodies, receptors, ligands, antibody fragments, aptamers, avidity multimers, or peptides For example, sandwich immunoassay such as ELISA (Enzyme Linked Immuno Sorbent Assay), RIA (Radio Immuno Assay) or the like can be used for analysis using a mimetic substance. This method can be applied to a first antibody conjugated to a bead, membrane, slide or microtiter plate made of a solid substrate such as glass, plastic (e.g. polystyrene), polysaccharide, nylon or nitrocellulose, A fluorescent substance such as 3H or 125I, a fluorescent substance, a chemiluminescent substance, a hapten, a biotin, a digoxigenin or the like, which can be directly or indirectly detected, Alternatively, the protein can be detected qualitatively or quantitatively by binding to an enzyme conjugated with an enzyme such as luminescent horseradish peroxidase, alkaline phosphatase, or malate dehydrogenase.

In other embodiments, Immunoelectrophoresis such as Ouchterlony plates, Western blot, Crossed IE, Rocket IE, Fused Rocket IE, Affinity IE can be used which can simply detect the marker through antigen-antibody binding.

Reagents or substances used in such methods are well known and include, for example, antigen-antibody reactions, substrates that specifically bind to the markers, peptide aptamers, receptors or ligands that specifically interact with the markers, , Or a mass spectrometer can be used.

Reagents or substances that specifically interact or bind to the markers herein for detection of the markers herein may be used in conjunction with chip-based or nanoparticles.

Markers in accordance with the present disclosure may also be used in one or more combinations, e.g., two, three, four, five, six, seven, or more combinations, A combination of markers satisfying the desired sensitivity and specificity can be selected.

In another aspect, the invention also provides a kit for the differential discrimination or detection comprising a composition according to the present invention. The kit of the present invention can determine the onset or progress of differentiation through quantitative and / or qualitative analysis of the differentiation marker marker according to the present invention.

The kit of the present invention can be used for the various detection methods described above. For example, examples of the kit in the present invention include mass spectrometry, immunochromatography strip kit, ELISA kit, chemiluminescence assay kit, and luminex kit. ELISA kits contain antibodies specific for biomarkers. Antibodies are monoclonal antibodies, polyclonal antibodies or recombinant antibodies with high specificity and affinity for each biomarker and little cross reactivity to other biomarkers. The ELISA kit may also include antibodies specific for the control. Other ELISA kits can be used to detect antibodies that can bind a reagent capable of detecting the bound antibody, such as a labeled secondary antibody, chromophores, an enzyme (e. G., Conjugated to an antibody) Other materials, and the like.

Luminex kit is a high-throughput quantitative assay capable of simultaneously measuring up to 100 kinds of analytes without pretreating a small sample (10-20 μl) of patient sample. It has excellent sensitivity (pg unit ), Which can be quantified in a short time (3-4 hours) and can be used as an alternative to conventional ELISA or ELISPOT. The Luminex Assay is a multiplex fluorescence microplate assay method capable of analyzing more than 100 biological samples simultaneously in each well in a 96-well plate and using two kinds of laser detectors to carry out signal transmission in real time Polystyrene beads of more than 100 different color groups are distinguished and quantified. The 100 beads are configured to be distinguished in the following manner. On one side, red fluorescence bead is divided into more than ten stages, and on the other side, orange fluorescence bead is divided into ten stages, and intensity difference is shown. Between them, red and orange beads Are mixed at different ratios to form a total of 100 color-coded bead sets. In addition, each bead has an antibody attached to the substance to be analyzed, so that a biomarker can be quantified by the immune antibody reaction using the antibody.

Luminex kits capable of carrying out the Luminex assays of the present invention include antibodies specific for biomarkers. Antibodies are monoclonal antibodies, polyclonal antibodies or recombinant antibodies with high specificity and affinity for each biomarker and little cross-reactivity to other markers. The lumenex kit may also include antibodies specific for the control material. Other luminex kits may also include reagents capable of detecting bound antibodies, such as labeled secondary antibodies, chromophores, enzymes (e.g., conjugated to antibodies) and other substrates capable of binding to the substrate or antibody . ≪ / RTI > The antibody may be an antibody conjugated with a micro particle, and the fine particle may be a colored latex or a colloidal gold particle.

The markers according to the present invention can be used for discriminating whether or not the mesenchymal stem cells are differentiated into adipocytes. From this viewpoint, the present invention also relates to a method for the treatment of mesenchymal stem cells by using methyl laurate, methyl myristate, methyl palmitate, The present invention relates to a method for discriminating the differentiation of mesenchymal stem cells into adipocytes, in particular, to an in vitro method, comprising the step of detecting one or more fatty acid methyl esters selected from the group consisting of methyl estradate, methyl elaidate, methyl stearate and derivatives thereof will be. The markers used in the method of the present invention, the detection method and the like can be referred to above.

In the method or composition according to the invention, it may comprise the step of extracting the fatty acid methyl ester prior to the detection of the methyl ester according to the invention, which extraction can be carried out via a destructive or non-destructive method, for example SPME (Solid-phase microextraction) or HS-SPME (Headspace SPME) can be used (see Journal of biochemical and biophysical methods, 2007, Volume 70, pp 181-193). SPME is a method of directly contacting a fiber with a sample and selecting a substance having an affinity with a fiber. HS-SPME is a method of selectively extracting only volatile substances without contact among affinity materials. See, for example, Metabolomics, 2007, Volume 3, Issue 1, pp 13 - 17.

The fibers used in the SPME herein include but are not limited to PA (polyacrylate), PDMS (polydimethylsiloxane), CAR (Carboxen) / PDMS or PDMS / DVB (Divinylbenzene) Divinylbenzene) is used.

The method further comprises the step of determining the cell as an adipocyte when the amount of at least one of the fatty acid methyl ester or the derivative thereof is increased in the mesenchymal stem cell compared with the control. The discrimination of cell differentiation can be performed, for example, as follows. The cutoff of the normal range of the marker in the pre-differentiation control group (the upper limit in the case of an increase or the lower limit in the case of an increase) is determined and compared with the threshold value in cells requiring suspected differentiation or detection of differentiation In the case of variation, that is, increase or decrease, it can be discriminated by differentiation. For example, if the amount of the marker is increased by about 1.5 times or more than the threshold value, it can be judged that the differentiation is started. The stage of differentiation can be divided into before and after differentiation. If the threshold value is increased about 1.5 times or more, it can be judged that the differentiation has proceeded.

Whether or not the increase or decrease is greater than or less than the threshold value and the judgment based on the threshold value may be determined by various factors such as the origin of the cell, the type of the cell, the sex and age of the object from which the cell is derived, Equipment, and the like, and those skilled in the art will be able to determine appropriate values in light of these factors.

The detection method according to the present invention can be used alone or in combination with other known methods capable of detecting differentiation.

In another embodiment, the present invention relates to a method of selecting a substance capable of promoting or inhibiting the differentiation of mesenchymal stem cells, for example. The marker according to the present invention has an increased amount in the mesenchymal stem cells in which the differentiation has been initiated and can be usefully used for screening substances that promote or inhibit the differentiation of mesenchymal stem cells into adipocytes.

The method according to the present invention comprises contacting a mesenchymal stem cell with a test substance; Measuring the amount of at least one substance selected from the group consisting of methyl laurate, methyl myristate, methyl palmitate, methyl linolate, methyl oleate, methyl elaidate, methyl stearate and derivatives thereof in the mesenchymal stem cells after contact with the test substance step; And comparing the amount of the substance with the amount of the at least one substance quantified in the control cells not contacted with the test substance or before the contact with the test substance, And selecting each of mesenchymal stem cells as an agent for promoting or inhibiting differentiation into adipocytes.

The detection method for mesenchymal stem cell and marker determination according to the present invention is as described above.

The type of cell used and the amount and type of test substance used in the present method will vary depending on the specific experimental method to be used and the kind of the test substance, and a person skilled in the art will be able to select an appropriate amount. As a result of the experiment, it is possible to select, as candidates, substances that cause an increase (differentiation promoter) or decrease (differentiation inhibitor) of the marker according to the present invention in the presence of the test substance as compared with the control group not in contact with the test substance, (Differentiation-promoting agent) or reduction (differentiation-inhibiting agent) according to the present invention is used as a control group, and cells which are not in contact with the test substance are used as a control group. can do.

As used herein, the term "test substance" means a substance that is expected to inhibit or promote the differentiation of adult stem cells into adipocytes as described above, and may be a low molecular weight compound, a high molecular weight compound, a mixture of compounds Or tissue culture), or biopharmaceuticals (e.g., proteins, antibodies, peptides, DNA, RNA, antisense oligonucleotides, RNAi, aptamers, RNAzymes and DNAzymes) or sugars and lipids. The test substance may be a polypeptide having two or more amino acid residues, such as six, ten, twelve, twenty or fewer or more than twenty such as 50 amino acid residues. The test materials can be obtained from libraries of synthetic or natural compounds, and methods for obtaining libraries of such compounds are known in the art. Synthetic compound libraries are commercially available from Maybridge Chemical Co., Comgenex (USA), Brandon Associates (USA), Microsource (USA) and Sigma-Aldrich (USA) ) And MycoSearch (USA). The test materials can be obtained by various combinatorial library methods known in the art and include, for example, biological libraries, spatially addressable parallel solid phase or solution phase libraries, deconvolution By the desired synthetic library method, "1-bead 1-compound" library method, and by synthetic library methods using affinity chromatography screening. Methods for synthesis of molecular libraries are described in DeWitt et al., Proc. Natl. Acad. Sci. USA 90, 6909, 1993; Erb et al. Proc. Natl. Acad. Sci. USA 91, 11422, 1994; Zuckermann et al., J. Med. Chem. 37, 2678, 1994; Cho et al., Science 261, 1303, 1993; Carell et al., Angew. Chem. Int. Ed. Engl. 33,2059,1994; Carell et al., Angew. Chem. Int. Ed. Engl. 33, 2061; Gallop et al., J. Med. Chem. 37, 1233, 1994, and the like.

In one embodiment according to the present invention, a test substance may be used which has an effect of promoting the differentiation of adipocytes into a low molecular weight. For example, compounds having a weight of about 1000 Da such as 400 Da, 600 Da or 800 Da can be used. Depending on the purpose, such compounds may constitute a part of a library of compounds, and the number of compounds constituting the library may vary from several tens to several millions. Such a library of compounds can be prepared by reacting peptides, peptoids and other cyclic or linear oligomeric compounds, and low molecular weight compounds based on a template, such as benzodiazepines, hydantoins, biaryls, carbocycles, and polycycle compounds such as naphthalene, Carbodihydrate and amino acid derivatives, dihydropyridines, benzhydryls and heterocycles (such as triazine, indole, thiazolidine, etc.), but this is merely exemplary It is not limited thereto.

For example, biologics can also be used for screening. Biologics refers to a cell or a biomolecule, which refers to a substance produced using biomolecules, proteins, nucleic acids, carbohydrates, lipids, or in vivo and in vitro cell systems. Biomolecules may be provided alone or in combination with other biomolecules or cells. Biomolecules include, for example, proteins or biological organic materials found in polynucleotides, peptides, antibodies, or other plasma.

Hereinafter, embodiments are provided to facilitate understanding of the present invention. However, the following examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited to the following examples.

Example

Experimental Method

Isolation and identification of mesenchymal stem cells

Human clonal mesenchymal stem cells were isolated from human bone marrow (BM) and separated by subfractionation culturing method (Song SU, et al . (2008) Stem Cells Dev 17 (3): 451 -461). The experiments were performed according to the approved protocol of the Medical Research Ethics Review Board, and written informed consent was obtained from each patient. The isolated mesenchymal stem cells were cultured in DMEM (Dulbecco's modified essential medium, Gibco BRL, USA) containing 10% heat-inactivated fetal bovine serum and 1% antibiotic and antifungal agent in 5% CO ₂ And cultured in an incubator.

Characterization of mesenchymal stem cells

The characteristics of mesenchymal stem cells were confirmed by the expression and differentiation ability of cell surface markers. For analysis of cell surface markers, anti-CD14, anti-CD34, anti-CD44, anti-CD73, anti-CD90, anti-CD105, anti-HLA Class I, anti-HLA- All antibodies were purchased from BD Biosciences Pharmingen, USA) and analyzed using a flow cytometer (FACSCalibur flow cytometer, BD Bioscience) in 6-passaged mesenchymal stem cells.

Identification of the ability of mesenchymal stem cells to differentiate

To confirm the differentiation potential of mesenchymal stem cells, it was confirmed to differentiate into three types of cell types: adipocyte, chondrocyte, and osteocyte.

In order to differentiate into fat cells and then dispensed at a density of 6x10 ⁴ cells / well in 4-well plate at ^{10% calf serum, 10 -7 M} dexamethasone (DEX; Sigma, USA), 10mg / mL insulin, 0.5mM 1-methyl- 3-isobutylxanthine (IBMX; Sigma), and 50 mg / mL indomethacine (Sigma) to confirm the differentiation into mast cells after 4-5 days. The cells were fixed with 4% formaldehyde, stained with oil red O for 30 minutes, and stained with hematoxylin for 10 minutes.

To differentiate into chondrocytes, 2.0 × 10 ⁵ mesenchymal stem cells are placed in a 15-mL tube using pellet culture, and the pellet is separated and cultured in a medium. ng / mL transforming growth factor-b1, 10 ng / mL TGF-β3 and 1% insulin-transferrin-seleous acid. After the differentiation, the cell pellet was embedded in OCT compound (Sakura Finetek, USA), frozen, and then 8 mm slice was prepared and stained with toluidine blue.

Cells were seeded in the same manner to differentiate into osteoblasts and cultured in osteocyte differentiation medium (10% FBS, 50 μg / mL ascorbic acid (Sigma), 10 ^-8 M DEX, and α- MEM containing 10 mM β-glycerophosphate ) For 3 weeks while exchanging the medium at 3-day intervals. The cells were then fixed with 4% formaldehyde and stained with alizarin red S for 30 min.

In the case of differentiation into adipocytes, the expression of mRNA expressed upon adipocyte differentiation was re-confirmed using a reverse transcription-polymerase chain reaction (RT-PCR). Total RNA was extracted from the mesenchymal stem cells using the easy-Blue RNA extraction reagent (Intron Biotechnology, Korea) according to the manufacturer's instructions. Then, 1 mg of total RNA was used as a template and PCR was performed after synthesizing cDNA using AccuPower cDNA synthesis kit (Bioneer, Korea) according to the manufacturer's instructions. PCR was performed using the AccuPower cDNA synthesis kit (Bioneer). The primers used in the PCR were as follows. FABP4 is forward (f) 5'-CATCAGTGTGAATGGGGATG-3 ', reverse (r) 5'-GTGGAAGTGACGCCTTTCAT-3', PPARγ2 is (5'-GACCACTCCCACTCCTTTGA-3 ', 5'-CGACATTCAATTGCCATGAG- LPL (5'-TATGAAAAACCAAGTAGCAAGGTTC-3 ', (r) 5'-GTAATCTGACTCTGTCCTTGTGGAT-3', osteocalcin, (f) 5'-TACAGGGCGGCCACAAGTTTT-3 ', (r) 5'- ATGGAGAGCAAAGCCCTGCTC- (f) 5'-GTGCAGAGTCCAGCAAAGGT-3 ', (5'-CTAGCCAACTCGTCACAGTC-3', Col2 is 5'-TTTCCCAGGTCAAGATGGTC-3 ', 5'-TCACCTGGTTTTCCACCTTC- (F) 5'-GCCCAAGAGGTGCCCCTGGAATAC-3 ', (r) 5'-CCTGAGAAAGAGGAGTGGACATAC-3', aggrecan (f) 5'-GCTACACCCTAAAGCCACTGCT- '-AACGGATTTGGTCGTATTGG-3', (r) 5'-TGTGGTCATGAGTCCTTCCA-3 '.

The PCR reaction conditions for each gene were as follows; ^{FABP4 (Ta = 56 o C,} 30 cycles, products = 399bp), PPARg (Ta = 60 o C, 32 cycles, products = 257bp), LPL (Ta = 60 o C, 30 cycles, products = 717bp), Runx2 ( ^{Ta = 58 o C, 38 cycles} , products = 336bp), Osteocalcin (Ta = 56 o C, 35 cycles, products = 175bp), Col2 (Ta = 56 o C, 40 cycles, products = 498bp), Col10A1 (Ta = ^{57 o C, 35 cycles, products} = 703bp), Aggrecan (Ta = 60 o C, 35 cycles, products = 350bp), GAPDH (Ta = 56 o C, 25 cycles, products = 476bp).

Extraction and detection of fatty acid methyl esters released from stem cells

Fatty acid methyl ester was analyzed by headspace-solid phase microextraction-gas chromatography-mass spectrometry (HS-SPME-GC-MS). For this purpose, four types of fibers were used: polyacrylate (PA), polydimethylsiloxane (PDMS), carboxen / polydimethylsiloxane (CAR / PDMS) and polydimethylsiloxane / divinylbenzene (PDMS / DVB). The extractability of each fiber was compared using seven standard fatty acid methyl esters. PA and PDMS did not detect any fatty acid methyl esters, and CAR / PDMS did not extract methyl linoleate and Methyl elaidate. Therefore, PDMS / DVB extracted from all 7 fatty acid methyl esters were used for extraction.

For extraction of the fatty acid methyl esters released from the stem cells, the fibers were activated at 250 ° C for 20 minutes. The fiber was then irradiated with ultraviolet light for 15-30 minutes to prevent contamination of the cell culture. The fiber was then placed in a cell culture plate and extracted for about 4 days. The fibers were then injected into GC-MS (agilent 7890A GC system and 5975C inert XL mass spectrometer interfaced GC-MS) using a MultiStructural Sampler (MPS) from GERSTEL. A DB-5 capillary column (length 30 m, ID 0.25 mm, film thickness 0.25 μm) from agilent was used for the efficient separation of fatty acid methyl esters. An inert gas, helium gas, was used as carrier gas. The flow rate was 1 mL / min and the sample was injected into the instrument at 250 ° C injection temperature in splitless mode. The temperature program of the GC-MS was set up so that the fatty acid methyl ester was detected. The temperature program was set at 100 ° C and raised to 215 ° C at a rate of 5 ° C / min, maintained at that temperature for 2 minutes, Speed up to 230 ℃. The separated components enter the MS along with the helium gas. To detect this, the ion source was set at 250 ° C, the transfer line was set at 240 ° C, and the quadrupole was set at 150 ° C. The ionization energy was 70 eV, which is commonly used in electron impact (EI).

Example 1 Isolation and Characterization of Mesenchymal Stem Cells

The morphology of the mesenchymal stem cells isolated from the donor as described in the experimental method was analyzed to reveal the morphology of fibroblasts as shown in Fig. In addition, high expression levels of CD44, CD73, CD90, CD105, HLA Class I, OCT-4 and PODXL, which are surface markers of mesenchymal stem cells, were confirmed, and endothelial or hematopoietic cell markers such as CD14, CD34, CD45, HLA- DR was negative (Fig. 2B).

Example 2. Confirmation of differentiation potential of mesenchymal stem cells

When only the expression of surface markers of mesenchymal stem cells is analyzed, the ability to differentiate these stem cells can not be confirmed. In order to confirm the presence of multiple pluripotency, differentiation into three types of cells (adipocytes, osteocytes, chondrocytes) was confirmed.

As shown in FIG. 1C and FIG. 1D (differentiation into adipocytes) and FIG. 2C, when the differentiated cells were differentiated into mast cells, osteocytes and chondrocytes, (Oil red O), bone cells (alizarin red S), and cartilage cells (toluidine blue). In addition, mRNA expression of differentiation markers of each cell was confirmed.

FABP4, PPARγ2, and LPL were highly induced in differentiation into adipocytes, and runx2 and OCN were highly expressed in bone cell differentiation. Expression of chondrocytes was confirmed by expression of mRNA, and it was confirmed that type II collagen, type X collagen and aggrecan were increased.

These results indicate that isolated cells have the characteristics of mesenchymal stem cells.

Example 3. Extraction of Fatty Acid Methylester Using HS-SPME Fiber and Analysis of Fatty Acid Methylester Using GC-MS

As described in the experimental method, PDMS / DVB most suitable for extraction of fatty acid methyl esters was used for extraction (see FIG. 4). Fatty acid methyl esters were extracted using PDMS / DVB, then the fiber was inserted into GC-MS and the fatty acid methyl ester was desorbed by increasing the injection temperature. Agilent 7890A GC and Agilent 5975C inert XL mass sepctrometer were then analyzed using an instrument interlocked. For the separation of the components, DB-5 capillary column was used as described in the experimental method. The length was 30 m, the particle size was 0.25 mm, and the film thickness was 0.25 μm. The maximum temperature of the column was 350 ° C and the initial temperature was 100 ° C and the final temperature was 325 ° C. However, in the case of fatty acid methyl esters detected, all of them were detected when the temperature was increased up to about 230 ° C., and the final temperature was set to 230 ° C. in order to minimize the time. The temperature was raised at a slow rate of 5 [deg.] C / min to ensure efficient separation of the components. Helium was used as an inert gas to transport these components and the flow rate was 1 mL / min. For the efficient detection of the mass spectrometer, the condition of the mass spectrometer was also optimized. The ion source was set at 250 ° C, the transfer line was set at 240 ° C, and the quadrupole was set at 150 ° C. In order to make an efficient comparison with the database used for identification of fatty acid methyl esters, 70 eV was used as a universal collision energy in electron impact mode.

Example 4. Identification and Quantification of Fatty Acid Methyl Ester

The chromatogram and mass spectrometry spectra were used to identify fatty acid methyl esters in the GC-MS data. Once all the peaks in the chromatogram were found and compared with the NIST database, a total of 7 fatty acid methyl esters were detected. Methyl laurate, Methyl myristate, Methyl palmitate, Methyl linoleate, Methyl oleate, Methyl elaidate, and Methyl stearate. Retention times were 12.23, 16.57, 20.64, 23.86, 23.99, 24.13 and 24.54 min, respectively. This is because the components having similar characteristics are emitted in order of decreasing molecular weight due to the nature of GC-MS. The results are shown in FIG.

Retention times and mass spectra of the respective fatty acid methyl esters were obtained using standard products for more accurate identification. The results are described in Figures 7-13.

In order to confirm the linearity with increasing the concentration of the standard product, a range of 1 ppm to 250 ppm was prepared and R2, which is an index of linearity, was obtained. The results are shown in FIG. The R2 of all the fatty acid methyl esters showed a good linearity over 0.99, indicating that the method of detecting the fatty acid methyl ester is suitable. Precision and accuracy were also checked to confirm that there was no change in data due to alteration of the sample or changes in the instrument. The results are shown in FIG. These results indicate that the reproducibility results are not due to the component concentration, alteration and instrumental error.

As a result of the analysis as described above, it was confirmed that no fatty acid methyl ester was detected in mesenchymal stem cells before differentiation according to FIG. 3B. However, seven samples of fatty acid methyl esters were detected in samples after the differentiation of mesenchymal stem cells into adipocytes, and quantitatively high values were obtained. This indicates that there is a pattern of a lot of positive and not negative, and it can be used as a marker of good fat differentiation.

While the present invention has been described in connection with what is presently considered to be the preferred embodiments, it is to be understood that the invention is not limited to the disclosed embodiments, but, on the contrary, .

All technical terms used in the present invention are used in the sense that they are generally understood by those of ordinary skill in the relevant field of the present invention unless otherwise defined. The contents of all publications referred to herein are incorporated herein by reference.

Claims (11)

Methyl laurate, Methyl myristate, Methyl palmitate, Methyl linoleate, Methyl oleate, Methyl elaidate, and methyl A composition for detecting the differentiation of mesenchymal stem cells into adipocytes, comprising a detection substance of one or more fatty acid methyl esters selected from the group consisting of methyl stearate.
The composition according to claim 1, wherein the mesenchymal stem cells are derived from bone marrow, blood, dermis or periosteum.
The composition for detecting the differentiation of mesenchymal stem cells into adipocytes according to claim 1, wherein the substance for detection is a substance used in antibody analysis, chemiluminescence assay, or mass spectrometry.
The method of claim 1, wherein the detecting substance is a receptor, ligand, substrate, antibody, antibody fragment, antibody mimetic, aptamer, avidity multimeric, or peptidomimetic that specifically recognizes the lipid A composition for detecting the differentiation of mesenchymal stem cells into adipocytes, comprising peptidomimetics.
A kit for detecting the differentiation of mesenchymal stem cells into adipocytes, comprising the composition according to any one of claims 1 to 4.
A method for detecting at least one fatty acid methyl ester selected from the group consisting of methyl laurate, methyl myristate, methyl palmitate, methyl linolate, methyl oleate, methyl elaidate, methyl stearate and derivatives thereof in mesenchymal stem cells A method for differentiation of mesenchymal stem cells into adipocytes.
7. The method of claim 6, further comprising extracting the fatty acid methyl ester prior to the detecting step, wherein the extraction is performed through a solid-phase microextraction (SPME) or a head-space-SPME (HS-SPME) Way.
The method of claim 7, wherein the fiber used in the SPME is polyacrylate (PA), polydimethylsiloxane (PDMS), Carboxen / PDMS, or PDMS / DVB (Divinylbenzene).
7. The method according to claim 6, wherein the detection is performed by at least one of antibody analysis, chemiluminescence assay, or mass spectrometry.
7. The method of claim 6, further comprising the step of determining the cell as an adipocyte when the amount of at least one of the fatty acid methyl ester or the derivative thereof is increased in the mesenchymal stem cell compared with the control Of differentiation of mesenchymal stem cells into adipocytes.
Contacting mesenchymal stem cells with a test material;
Measuring the amount of at least one substance selected from the group consisting of methyl laurate, methyl myristate, methyl palmitate, methyl linolate, methyl oleate, methyl elaidate, methyl stearate and derivatives thereof in the mesenchymal stem cells after contact with the test substance step; And
Comparing the amount of the substance to the amount of the at least one substance quantified in the control cells not contacted with the test substance or in the control cells before contact with the test substance and when the amount of the substance is increased, Selecting a cell as an adipocyte differentiation promoting substance, and selecting a differentiation regulating substance from a mesenchymal stem cell adipocyte.

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