KR101541958B1 - Markers for detecting senescence in adult stem cells and its uses - Google Patents

Abstract

The present invention discloses a stem cell aging discrimination composition comprising a substance for the detection of lysophosphatidylcholine and lysophosphatidylethanolamine which can be used as a cell senescence marker and a use thereof. The composition according to the present invention can improve the success rate of clinical application because it can determine whether or not it is aged before culturing stem cells, which is essential for amplification through incubation with incubation, as well as quality control of cells.

Description

Markers for detecting senescence of adult stem cells and their uses {

It is a technical field related to stem cell culture.

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.

Since mesenchymal stem cells have a limited ability to proliferate unlike embryonic stem cells, there is no possibility of cancer cell formation, and there is no ethical problem because there is no destruction of embryo. As a result, the mesenchymal stem cells have reached a stage where they can be rapidly applied for clinical application.

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.

United States Patent Application Publication No. 2001-0129918 discloses a method for culturing mesenchymal stem cells at a low density under hypoxic conditions and discloses a method for culturing mesenchymal stem cells without lowering the proliferative capacity, aging and differentiation ability of MSCs have.

Korean Patent Laid-Open Publication No. 2013-0057682 discloses a method for inducing cell senescence in cord blood-derived mesenchymal stem cells, a method for inhibiting cell senescence, and a composition for inhibiting cell senescence, and a method for inhibiting senescence using an antioxidant.

However, there is still a problem that it is difficult to maintain and manage cell activity and characteristics due to aging of cells in the process, since artificial amplification is essential in Invitro since there is little amount available from the individual. Therefore, it is necessary to develop a method to observe the regulation of aging and its change during the cultivation of homogeneous cells.

The present invention provides a method for determining whether or not aging occurs during culturing of adult stem cells.

In one embodiment, the subject is a lysophosphatidylcholine (LPC) 16: 0; LPC 18: 0; LPC 18: 1; LPC 20: 4; LPC 22: 5; Lysophosphatidylethanolamine (LPE) 22: 4; LPE 22: 5; And a detection substance of at least one lipid selected from the group consisting of LPE 22: 6 and derivatives thereof.

The composition according to the present invention can be used for adult stem cells, for example, for determining the aging of mesenchymal stem cells.

Detecting materials that may be included in the compositions according to the present invention include, but are not limited to, those used in antibody analysis, chemiluminescence assay, or liquid chromatography mass spectrometry. Detecting materials that can be used in the compositions according to the present invention include receptors, ligands, substrates, antibodies, antibody fragments, antibody mimetics, aptamers, avidity multimers, or peptides that specifically recognize the lipid But are not limited to, peptidomimetics.

In another aspect, the disclosure also provides a kit for detecting adult stem cell senescence comprising a composition according to the present disclosure.

In another embodiment, the present invention provides a method of producing lysophosphatidylcholine (LPC) 16: 0 in mesenchymal stem cells; LPC 18: 0; LPC 18: 1; LPC 20: 4; LPC 22: 5; Lysophosphatidylethanolamine (LPE) 22: 4; LPE 22: 5; And LPE 22: 6 and at least one lipid selected from the group consisting of derivatives thereof. In one embodiment according to the present invention, adult stem cells include, but are not limited to, mesenchymal stem cells, for example. In the method according to the present invention, detection may be performed by one or more of antibody analysis, chemiluminescence assay, or liquid chromatography mass spectrometry, but is not limited thereto. Further comprising the step of determining the cell as an aged cell when the expression level of at least one of the lipids used as a marker of the present invention in the method according to the present invention is increased in the mesenchymal stem cell compared to the control.

In another aspect, the disclosure also provides a method of screening for an agent for inhibiting adult stem cell senescence. In one embodiment according to the present application, the method comprises contacting an adult stem cell with a test substance; After contact with the test substance, lysophosphatidylcholine (LPC) 16: 0 in the adult stem cells; LPC 18: 0; LPC 18: 1; LPC 20: 4; LPC 22: 5; Lysophosphatidylethanolamine (LPE) 22: 4; LPE 22: 5; And LPE 22: 6; And comparing the amount of the substance with the amount of the at least one substance quantified in the control cell not contacted with the test substance or in the control cell before contact with the test substance, And a step of selecting the material for inhibiting stem cell senescence.

Lyophosphatidylcholine and lysophosphatidylethanolamine which can be used as cell senescence markers according to the present invention can be used to determine whether or not aging has occurred in the culture of stem cells in which amplification through in vitro culture is essential, Therefore, the success rate of clinical application can be increased.

FIG. 1A is a micrograph showing the cell morphology of hcMSCs isolated from human bone marrow. FIG.
FIG. 1B is a flow cytometric analysis result of observation of markers expressed in stem cells.
Figure 1c is a micrograph (x40 and x100 magnification) observing pluripotency that can differentiate into adipocytes, bone and cartilage of hcMSC.
FIG. 2A shows the results of observation of beta-galactosidase staining for the characteristics of hcMSC in the cell senescence process.
FIG. 2B shows the results of observing the characteristics of hcMSC in cell senescence through immunofluorescence staining for caveolin-1.
FIG. 2C shows the results of reverse transcription-PCR of the expression of the molecular marker of hcMSC in the cell senescence process.
FIG. 2d shows the results of flow cytometry analysis of changes in hcMSC surface marker expression in the cell senescence process.
Figures 3a and 3b are ion chromatograms of hcMSC cells observed with UPLC-Q-TOF-MS.
FIG. 4 shows the result of analyzing the metabolite of hcMSC with PCA.
FIG. 5 is a histogram showing the mean and variance of the metabolomic analysis results according to FIG.
FIG. 6 is a scatter plot showing the mean and variance of the metabolomic analysis results according to FIG.
Figures 7A and 7B are MS / MS spectral results of the LPC 16: 0 standard and the impact energy is 10-50 eV.
Figures 8A and 8B are MS / MS spectral results of the LPC 18: 0 standard and the impact energy is 10-50 eV.
Figures 9a and 9b are MS / MS spectral results of endogenous LPC 16: 0 showing changes in expression levels during the aging process of hcMSC, with collision energy of 10-50 eV.
10a and 10b are MS / MS spectral results of endogenous LPC 18: 0 showing changes in the expression level during the aging process of hcMSC, and the collision energy is 10-50 eV.
FIGS. 11A and 11B are MS / MS spectral results of endogenous LPC 18: 1 showing changes in the expression level during the aging process of hcMSC, and the collision energy is 10-50 eV.
12A and 12B are MS / MS spectral results of endogenous LPC 20: 4 showing changes in the expression level during the aging process of hcMSC, and the collision energy is 10-50 eV.
13A and 13B are MS / MS spectral results of an endogenous LPC 22: 5 showing changes in the expression level during the aging process of hcMSC, and the collision energy is 10-50 eV.
14A and 14B are MS / MS spectral results of the LPE 16: 0 standard material, with an impact energy of 5-25 eV.
Figures 15A and 15B are MS / MS spectral results of the LPE 18: 0 standard and the impact energy is 5-25 eV.
16A and 16B are MS / MS spectral results of endogenous LPE 22: 4 showing changes in the expression level during the aging process of hcMSC and the collision energy is 5-25 eV.
FIGS. 17A and 17B are MS / MS spectral results of endogenous LPE 22: 5 showing changes in the expression level during the aging process of hcMSC, and the collision energy is 5-25 eV.
Figures 18a and 18b are MS / MS spectral results of endogenous LPE 22: 6 showing changes in expression levels during the aging process of hcMSCs and the collision energy is 5-25 eV.
Figure 19 is a graph comparing the expression of selected MSC senescence markers with progression of cell logarithm.

The present invention is based on the discovery that lysophosphatidylcholine (LPC) and lysophosphatidylethanolamine (LPE) can act as markers for the progression of senescence of stem cells in the culture of adult stem cells.

Cellular senescence is a phenotype caused by changes in the cell's replication and overall activity as the number of cells in the cell increases, and is subject to complex regulation throughout DNA, protein, and endogenous metabolism And ultimately stops the proliferation.

Since adult stem cells are small in amount that can be obtained from an individual, an artificial amplification is essential in the In Vitro. In order to proliferate without losing the differentiation ability, the cell activity and the characteristic of the stem cell are monitored It is essential that markers of the present invention can be used to detect adult stem cell senescence. In one embodiment according to the present application, it is used for detecting senescence of mesenchymal stem cells.

As used herein, the term "mesenchymal stem cell" is a stem cell which is isolated from bone marrow, blood, dermis and periosteum. It is an omnipotent stem cell capable of differentiating into various cells such as adipocytes, chondrocytes, Means pluripotent 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 the onset or progression of senescence or senescent cells from cells before senescence has progressed, in which the senescence is increased Or reduced biologically active forms, or organic biomolecules such as nucleic acids, lipids, glycolipids, glycoproteins, and the like. In one embodiment of the present invention, lysophosphatidylcholine (LPC) and lysophosphatidylethanolamine (LPE), whose expression increases with aging of cells, are included.

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 biomarker according to the present invention is LPC of the following formula (1) or LPE of the formula (2) or a derivative thereof.

[Chemical Formula 1]

Wherein R1 and R2 are C16 to C22 saturated or unsaturated fatty acids or H; In one embodiment according to the present application, LPC or a derivative thereof is selected from the group consisting of lysophosphatidylcholine (LPC) 16: 0; LPC 18: 0; LPC 18: 1; LPC 20: 4; And LPC 22: 5.

(2)

Wherein R1 and R2 are C22 saturated or unsaturated fatty acid or H. In one embodiment according to the present application, LPE or a derivative thereof is lysophosphatidylethanolamine (LPE) 22: 4; LPE 22: 5; And LPE 22: 6.

The LPC or LPE or derivative thereof according to the present invention can be detected through various known methods and a suitable detection method can be selected by those skilled in the art. For example, The Analyst 8th June 2001, Volume 126, Issue 7, pp 1042-1050.

Ligands, substrates, antibodies, antibody fragments, aptamers, avidity multimers, or peptidomimetics that specifically recognize LPC or LPE or derivatives thereof, in one embodiment according to the present invention. . The detection substance can be used for antibody analysis, chemiluminescence assay, or liquid chromatography mass spectrometry, etc., 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, the markers can be detected using mass spectrometry. For example, refer to Applied Biochemistry and Biotechnology Jul-Sep 2000, Volume 88, Issue 1-3, pp 33-44 have.

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, And then labeled or labeled with a labeling substance capable of direct or indirect detection, for example, a radioactive substance such as ³ H or ¹²⁵ I, a fluorescent substance, a chemiluminescent substance, a hapten, a biotin, a digoxigenin, Protein can be detected qualitatively or quantitatively through the conjugation of an enzyme conjugated with an enzyme such as horseradish peroxidase, alkaline phosphatase, or malate dehydrogenase capable of coloring or luminescence.

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 by using a mass spectrometer. ≪ RTI ID = 0.0 >

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. Methods of immunoassay or immunostaining are described in Enzyme Immunoassay, E. T. Maggio, ed., CRC Press, Boca Raton, Florida, 1980; Gaastra, W., Enzyme-linked immunosorbent assay (ELISA), in Methods in Molecular Biology, Vol. 1, Walker, J.M. Ed., Humana Press, NJ, latest edition. By analyzing the intensity of the final signal by the above-described immunoassay, that is, performing signal contrast with the sample before aging, it is possible to detect the aging or the stage of aging.

Detection of the markers herein can be performed using mass spectrometry. See, for example, Kim, et al. 2010 J Proteome Res. 9: 689-99; Anderson, L et al 2006 Mol Cell Proteomics 5: 573-88. In one embodiment, for example, multiple reaction monitoring (MRM) techniques using 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 invention, liquid chromatography mass spectrometry is used for detection of the marker. In addition, the data of the liquid chromatography mass spectrometry can be obtained by using Selected Ion Monitoring (SIM) or MRM, which analyzes based on the accurate molecular weight value of the mass spectrometer and the retention time of the retention time of separation of the peptides in liquid chromatography without labeling the protein or peptide Can be used.

Whether or not the cells are aged or the aging step can be determined, for example, as follows. The cutoff of the normal range of the marker in the control group before aging is determined (upper limit in case of increase / lower limit in case of increase), and compared with the threshold value in cells which are suspected of aging or in need of detection of aging If it fluctuates, that is, increases or decreases, it can be determined as aging. For example, if the amount of the biomarker is increased by about 1.5 times or more than the threshold value, it can be judged that aging is started. The stage of aging can be divided into before and after aging. If the threshold value is increased or decreased by 1.5 times or more, it can be judged that aging has progressed.

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 aging.

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 present invention also provides a kit for detecting aging comprising the composition according to the present invention. The kit of the present invention can determine the onset or progress of aging through quantitative and / or qualitative analysis of aging markers 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 (R) 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.

In another aspect, the present invention relates to a method for screening a substance capable of inhibiting the senescence of adult stem cells. The marker according to the present invention increases in the amount of adult stem cells whose aging has been initiated, and thus can be useful for screening substances that inhibit the aging of adult stem cells.

The method according to the present invention comprises the steps of contacting an adult stem cell with a test substance; After contact with the test substance, lysophosphatidylcholine (LPC) 16: 0 in the adult stem cells; LPC 18: 0; LPC 18: 1; LPC 20: 4; LPC 22: 5; Lysophosphatidylethanolamine (LPE) 22: 4; LPE 22: 5; And LPE 22: 6; And comparing the amount of the substance with the amount of the at least one substance quantified in the control cell not contacted with the test substance or before the contact with the test substance, And selecting the test substance as a substance for inhibiting adult stem cell senescence.

The detection for adult stem cells and quantification according to the present invention is as mentioned 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 test, substances which, in the presence of the test substance, result in a reduction of the markers according to the present invention, are selected as candidates as compared with the control group which is not in contact with the test substance. Less than about 99%, less than about 95%, about 90%, about 85%, about 80%, about 75%, about 70% less, less than about 65% , About 55% reduction, about 50% reduction, about 45% reduction, about 40% reduction, about 30% reduction, about 20% reduction or less.

As used herein, the term "test substance" means a substance that is expected to inhibit the aging of adult stem cells as described above, and may be a low molecular weight compound, a high molecular weight compound, a mixture of compounds (e.g., a natural extract or a cell 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 application, a test substance may be used which has a therapeutic effect of 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

The experimental method used in this embodiment is as follows.

Isolation and culture 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 experiment was performed according to the approved protocol of the Medical Research Ethics Review Board of Inha University and a written explanation was obtained from each patient based on sufficient explanation. The isolated mesenchymal stem cells were named D103 and D106, respectively, and were cultured in DMEM (Dulbecco's modified essential medium, Gibco BRL, USA) containing 10% heat-inactivated FBS (fetal bovine serum) and 1% antibiotic and antifungal agent In a 5% CO ₂ incubator. After subculturing for 7, 15 and 20 times, they were kept frozen at -80 ° C until use.

Characterization of mesenchymal stem cells

The characteristics of mesenchymal stem cells were confirmed by the expression and differentiation ability of cell surface markers. The surface markers of the cells were labeled with anti-CD14, anti-CD34, anti-CD44, anti-CD73, anti-CD90, anti-CD105, anti-HLA Class I, anti-HLA- Was purchased from BD Biosciences Pharmingen, USA using a flow cytometer (BD Bioscience) using a flow cytometry analyzer in a 6-pass subcultured hsMSC.

Differentiation was induced in three different cell types to identify differentiation potential. To differentiate into adipocytes, the cells were seeded at a density of ⁶ × 10 ⁴ cells / well in a 4-well plate and cultured in 10% calf serum, 10 ^-7 M dexamethasone (DEX; Sigma, USA), 10 mg / mL insulin, 0.5 mM 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. 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. Pelleted cultures were used to differentiate into cartilage tissue. Insert the 2.0x10 ⁵ hcMSC a 15mL test tube was precipitated cells. The cells were then suspended in 500 μl of serum-free chondrogenic differentiation medium (10 ng / ml TGF-ß1 (R & D Systems, Minneapolis, MN, USA), 10 ng / ml TGF-b3 -selenious acid premix (BD Biosciences) for 3 weeks at 3-day intervals. The cell pellet was embedded in OCT compound (Sakura Finetek, USA), frozen, and then 8 mm slice was prepared and stained with saporin O.

β-Gal staining and immunofluorescence staining analysis

Β-gal staining kit (Cell Biolabs, USA) was used as the manufacturer's method to examine the activity of SA-β-gal (senescence-associated β-galactosidase) Using a microscope, the results of staining were checked to confirm whether or not the cells were aged, and blue cells showed aged cells.

For immunofluorescence staining, hcMSC cells were plated on a slide glass, fixed with 4% paraformaldehyde, and permeabilized with 0.1% triton X-100. Cells were then blocked with 5% normal goat serum for 30 min at room temperature and then incubated overnight at 4 ° C with anti-caveolin-1 antibody (Cell signaling Technology, USA). Cells were washed with TBS containing 0.1% Tween 20 and incubated with FITC-conjugated secondary antibody (Molecular Probes, USA) for 1 hour at room temperature. For F-actin, cells were incubated with AlexaFluor 597-conjugated phalloidin (Molecular Probes) for 30 min at room temperature. The nuclei were stained with 4 ', 6'-diamidino-2-phenylindole (DAPI; Molecular Probes). After washing, the cells were mounted and observed with a confocal microscope (Olympus FluoView FV1000, Tokyo, Japan).

In the case of immunoblot, hcMSC cells in different numbers of passages were diluted in a buffer solution (10 mM Tris-Cl pH 7.5, 150 mM NaCl, 1 mM EDTA (pH 8.0), 1% triton X-100, 0.5% sodium deoxycholate, 0.1 % SDS, 1 mM PMSF, and protease inhibitor cocktail solution), and then transferred to PVDF membrane after electrophoresis (SDS-PAGE). The membranes were blocked with 5% non-fat milk, incubated with each antibody, developed with a chemical luminescence kit, and visualized on a LAS4000 mini (Fuji PhotoFilm, Japan).

Reverse transcription-polymerase chain reaction (RT-PCR)

Total RNA was extracted from hcMSC 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 and conditions used in the PCR are shown in Table 1. The amplification product was visualized as LAS4000 mini after electrophoresis in 1% agarose gel containing SyberSafe.

[Table 1]

Extraction of endogenous metabolites from mesenchymal stem cells

The experiment was performed by Yanes O, et al. (2010) Nat Chem Biol 6 (6): 411-417. To summarize, approximately 600 ml of cold acetone was added to approximately 3 million cultured mesenchymal stem cells. After mixing, the cells were frozen in liquid nitrogen for 1 minute, dissolved at room temperature, and then ultrasonicated for 10 minutes. Then, the solution was stored at -20 ° C for 1 hour, centrifuged, and the supernatant was taken. Add 400 μL of a mixture of methanol / water / formic acid (86.5 / 12.5 / 1.0) to the remaining residue, mix and conduct ultrasonic extraction. After storing at -20 ° C for 1 hour, the supernatant was centrifuged and combined with the supernatant which had already been obtained and dried. The residue was mixed with 100 μL of 95% acetonitrile / water, centrifuged at 13,000 rpm, and the supernatant was used for analysis. The samples were stored at 4 ° C in the autoclave and repeated three times for each sample (n = 3).

Liquid Chromatography-MS (LC-MS)

Analysis was performed using an UPLC system (Waters) Acquity ™ UPLC column (BEH C18, 1.7 μm, 2.1 mm × 100 mm) and a micrOTOF-QII mass spectrometer (Bruker Daltonics, Germany). The mobile phases were analyzed using water containing 0.1% formic acid and acetonitrile containing 0.1% formic acid at a flow rate of 0.2 mL / min and a column temperature of 40 ° C. In order to calibrate the instrumental deviations during the analysis process, the samples were analyzed in arbitrary order and the reproducibility was confirmed by repeatedly analyzing 18 mixtures of the whole samples as QC (quality control) samples. The analytical range of the mass spectrometer was 50 ~ 1000 m / z, capillary voltage was 4.5 kV, and nebulizer pressure was 1.2 bar. The drying gas flow rate at 200 ℃ was 8.0 L / min. The quadrupole and the collision cell were optimized to set the quadropole ion energy to 15.0 eV and the collision energy to 10.0 eV. A high purity nitrogen gas was used as the collision gas, and lithium formic acid was injected before the injection to ensure accuracy.

The measured results were calibrated using MZmine software. The relative standard deviation (RSD%) was calculated for each variable (Hu C et al. (2011) PLoS One 6 (5): e19423 and Dunn WB, et al. Protoc 6 (7): 1060-1083).

Data processing

The original data were analyzed using MZmine software (Pluskal T, et al. (2010) BMC Bioinformatics 11: 395). During the mass spectrometry, MS spectra were calculated as m / z and intensity pairs (noise level set at 10 ³ , Centroid algorithm). The conditions for constructing the chromatogram are as follows: minimum height; 1.5 x 10 < ³ > and m / z tolerance; 0.05. The peak deconvolution used the "Savitzky-Golay" function (1.5 × 10 ³ , derivative threshold level; 80%) and the peak array was the "RANdom SAmple Consensus (RANSAC) ; 0.05, absolute retention time (RT) tolerance; 0.1 min, RANSAC iterations; 10000). Principal component analysis (PCA) was performed using the calculated peak data and converted to a csv file.

LC-Multiple reaction monitoring (MRM) mass spectrometry for LPC and LPE

Data were collected by MRM using a UPLC system (Waters) and a micrOTOF-QII mass spectrometer (Bruker Daltonics) (positive ion mode). 1-palmitoyl (C16: 0) lysophosphatidylcholine (LPC), 1-stearoyl (C18: 0) LPC, 1-palmitoyl (C16: 0) lysophosphatidylethanolamine (LPE) and 1-stearoyl Alabaster, USA). Lipids were dissolved in chloroform / methanol / water (65: 35: 8). Each reference material was dissolved in methanol (1-> 10) just before use. In MS / MS analysis, the daughter ions of the target molecule were identified using MRM. The precursor ion [M + H] was selected under the following conditions: LPC 16: 0: 496.3397, LPC 18: 0: 524.3710, LPE 16: 0: 454.2928 and LPE 18: 0: 482.3241.

In the case of LPC, the collision energy (10, 20, 30, 40, 50 eV) was gradually applied. On the other hand, for LPE, slightly weaker impact energy (5, 10, 15, 20, 25 eV) was applied. The isolation width was 10.0. All MS and MS / MS base peak chromatogram (BPC), spectra and m / z errors were calibrated to less than 5 ppm and collected using DataAnalysis 4.0 (Bruker Daltonics). Other chromatographic conditions are as described above.

Metabolism analysis

All targets were characterized for lysophosphatidylcholine (LPC) or lysophosphatidylethanolamine (LPE) with neutral loss of m / z 184 and 141 Da. The measured results were converted into metabolites using databases and standards. Among the measured m / z values, only the values showing that the expression changes with aging were selected and the structure was predicted through the MS / MS spectrum. Based on the predicted results, the standard products were purchased and analyzed, and the spectral results were compared to confirm the agreement. I used the LIPID MAPS (http://www.lipidmaps.org) database.

Example 1 Characterization of hcMSC

Analysis of the morphology of hsMSC cells isolated from the two donors as described in the experimental method revealed the morphology of fibroblasts as shown in Fig. In addition, CD14, CD73, CD90, CD105, HLA Class I and PODXL, which are surface markers of MSC, were found to be highly expressed and CD14, CD34, CD45 and HLA-DR, markers of endothelial or hematopoietic cells, 1b). As shown in FIG. 1C, the mast cells (oil red O), bone cells (alizarin red S ) And cartilage cells (ToluDain Blue). These results show that the separated hcMSC has the characteristics of MSC.

Example 2 Confirmation of senescence of mesenchymal stem cells

Two separate ganglion mesenchymal stem cell lines were subcultured to induce senescence. In order to confirm the expression of the metabolites in the process of aging, samples were collected from 7 (early stage, high proliferative capacity), 15 (intermediate stage, low proliferative capacity before aging), and 20 And β-Gal staining was performed to confirm the aging process. As shown in FIG. 2A, in the passage numbers 7 and 15, which were not stained with β-Gal, it was confirmed that the aging did not progress significantly, and the cells of passage number 20 were stained strongly and the aging progressed considerably. Aging was also confirmed by caveolin-1 staining, another marker of aging (see Fig. 2b).

As another evidence of aging, RT-PCR showed that expression of PCNA decreased and expression of p16 ^INK4a and p19 ^ARF increased with increasing number of passages (see FIG. 2C).

Next, the changes in cell markers due to aging of the cells were observed by flow cytometry. As a result, the markers of hematopoietic stem cells (CD13, CD34, and CD45) were not affected by cell senescence (see FIG. On the other hand, MSC markers were affected by aging. The expression of CD73 and CD105 in D106 was not influenced by the passage number of 15 and the mean fluorescence intensity (MFI) of CD73 was significantly CD105 was not detected. In the case of D103, CD73 and CD105 were not detected at passage number 15, whereas expression was restored at 20 times. These results indicate that the expression pattern of the surface marker is somewhat variable during the cell aging process.

Example 3 Analysis of Endogenous Metabolites

In order to investigate the expression of endogenous metabolites associated with aging, metabolites were analyzed as described in the experimental method using cells of passage number 7, 15 and 20. The experiment was repeated three times for each sample and the same experiment was performed twice using two cell lines derived from each donor to correct individual differences derived from the cells. As shown in Fig. 3, although there was a difference between the two samples in the amount, the main peak and the tendency were found to agree with each other. The intensity and standard deviation of the main peak were found to increase compared to the number of passages 20 to 7 and 15.

These results were analyzed by PCA for endogenous metabolites before and after cell senescence. The PCA is an analysis method that finds the variance that maximizes the variance and shortens the data from it and expresses the individual information of the data more easily. The principal component variable is obtained by the original variable and the variation of the original variable is investigated by the principal component.

In the PCA analysis, each sample was labeled with a different color point and plotted on PC1 and PCA major component axes. Some samples formed clusters and showed similar composition of endogenous metabolites. Directional movement was detected as aging progressed from 7 to 15 and 20 in the passage number. Also, as the aging process progresses, the variation may also increase. These results suggest that the deviation is due to the aging process, since the hsMSCs used herein are homogeneous cells.

That is, the data obtained through the UPLC Q-TOF MS was analyzed through statistical analysis after correction based on MZmine. In order to understand the overall tendency of the data, principal component analysis was performed and it was confirmed that the homogeneity of the same cells was altered during the aging of the mesenchymal stem cells. The mesenchymal stem cells cultured through the layer separation culture method are the clone cells originating from a single cell and have an advantage of maintaining the inherent characteristics in the culture process. Nevertheless, the loss of intracellular homogeneity in the number of transgenic samples of 20 indicates that aging is an ongoing process that does not maintain homeostasis of endogenous metabolite expression. On the basis of this, a variance test was conducted to select the metabolites having greatly changed homeostasis as markers.

Example 4 Identification of Endogenous Metabolites of Aging Index of Cells

Identification was carried out centering on the metabolites selected above. In order to obtain the reliability of the results, we performed spectral - based structural analysis only on metabolites measured as reproducible in QC (quality control) samples. Some of the selected metabolites showed peaks with a value of m / z 184 on the spectrum, confirming that they are lipids in the metabolites, especially lysophosphatidylcholine (LPC). Accordingly, the reference material was obtained based on the molecular weight and identification was carried out. In the case of the remaining metabolites, a peak having a characteristic of 'precursor ion-141 Da' was observed on the spectrum, and lysophosphatidylethanolamine (LPE) . At this time, the collision energy of 10, 20, 30, 40, 50 eV and the collision energy of 5, 10, 15, 20, 25 eV were applied to LPC and LPE, respectively. The results are shown in Figures 7 to 18 and Table 2 below.

LPC 18: 0, LPC 18: 1, LPC 20: 4, LPC 22: 5, lysophosphatidylethanolamine (LPE), and lysophosphatidylethanolamine ) 22: 4, LPE 22: 5, and LPE 22: 6, indicating that the expression of this substance is a metabolic breakdown in the course of senescence of mesenchymal stem cells. In the name of each compound, the number in front indicates the number of carbon atoms, the number in the back indicates the number of double bonds, and the number of double bonds in 0 indicates the saturated fatty acid.

Eight metabolites were interpreted as a change in expression due to damage to the cell as the cell aged, a phenomenon that changes in expression when cells are oxidatively stressed. In addition, when the expression of this indicator substance changes, it affects the cell cycle, resulting in a change in phenotype such as differentiation ability. Therefore, in the present invention, the 8 metabolites have been developed as an indicator substance for confirming the senescence of mesenchymal stem cells. Based on this, it is possible to confirm the degree of senescence of the mesenchymal stem cells in the future and to control the culture, Stem cells were made available.

The expression level of these 8 markers was expressed as a percentage at the number of passages 7 and 15 compared with the number of passages 20. The results are shown in Fig. 19, in which all metabolites show a decrease in expression amount as the aging progresses. As a result, the variance increases with aging.

[Table 2]

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 (10)

Lysophosphatidylcholine (LPC) 16: 0; LPC 18: 0; LPC 18: 1; LPC 20: 4; LPC 22: 5; Lysophosphatidylethanolamine (LPE) 22: 4; LPE 22: 5; And lipids of LPE 22: 6, wherein the numbers preceding the lipids in each lipid are carbon numbers and the numbers after the lipids indicate the number of double bonds, and when the double bond number is 0 Wherein said lipid is saturated with said lipid.
delete The composition for detecting mesenchymal stem cell senescence according to claim 1, wherein the substance for detection is a substance used in antibody analysis, chemiluminescence assay, or liquid chromatography 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 mesenchymal stem cell senescence comprising peptidomimetics. A kit for detecting mesenchymal stem cell senescence comprising a composition according to any one of claims 1, 3 or 4.
In mesenchymal stem cells, lysophosphatidylcholine (LPC) 16: 0; LPC 18: 0; LPC 18: 1; LPC 20: 4; LPC 22: 5; Lysophosphatidylethanolamine (LPE) 22: 4; LPE 22: 5; And a lipid of LPE 22: 6, wherein the number preceding the lipid in each lipid is the number of carbon atoms, the number after the lipid indicates the number of double bonds, and the double bond number 0 , The lipid is saturated. ≪ / RTI >
delete 7. The method according to claim 6, wherein the detection is performed by at least one of antibody analysis, chemiluminescence assay, or liquid chromatography mass spectrometry.
7. The method of claim 6, wherein the method further comprises determining the cell as an aged cell if the amount of at least one of the lipids is increased in the mesenchymal stem cell compared to the control. Cell aging discrimination method.
Contacting mesenchymal stem cells with a test material;
After contact with the test material, lysophosphatidylcholine (LPC) 16: 0 in the mesenchymal stem cells; LPC 18: 0; LPC 18: 1; LPC 20: 4; LPC 22: 5; Lysophosphatidylethanolamine (LPE) 22: 4; LPE 22: 5; And LPE 22: 6; And
Comparing the amount of lipid to the amount of the at least one lipid quantified in the control cells not contacted with the test substance or in the control cells before contact with the test substance, And a step of selecting as a substance for inhibiting stem cell senescence,
Wherein the numbers in the respective lipids indicate the number of carbon atoms and the numbers after the double bonds indicate the number of double bonds and when the double bond number is 0, the lipids are saturated.

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