Composition for detecting mesenchymal stem cells in relation to senescence, and use thereof

Abstract

The present invention relates to a composition for detecting senescent tonsil-derived mesenchymal stem cells, and a use thereof. By using a marker according to the present invention, cellular senescence of tonsil-derived mesenchymal stem cells may be accurately detected. In addition, by treating cells or a culture medium with the composition according to the present invention, senescence of tonsil-derived mesenchymal stem cells may be inhibited and the multipotency thereof may be maintained, even throughout a long culture period, and thus tonsil-derived mesenchymal stem cells cultured as such may be usefully employed as a therapeutic application resource or a therapeutic resource in the field of regenerative medicine.

Description

Composition for detecting mesenchymal stem cells associated with aging and use thereof

[0001] The present invention relates to a composition for detecting aged mesenchymal stem cells and its use.

[0002] Mesenchymal stem cells (MSCs) are a promising cell source for therapeutic applications in regenerative medicine. MSCs can be derived from various adult tissues, including bone marrow, adipose tissue, tonsils, and umbilical cord, thereby avoiding ethical concerns associated with embryonic stem cells. To date, a growing number of studies have demonstrated therapeutic effects utilizing the properties of MSCs, including in vitro self-renewal, multilineage differentiation, and immunomodulatory effects that promote in vivo regenerative processes. Furthermore, trophic factors released by MSCs exhibit additional therapeutic effects, such as pro-angiogenic or anti-angiogenic effects. For example, exosomes derived from mouse bone marrow-derived mesenchymal stem cells (BM-MSCs) significantly inhibit tumor progression and angiogenesis by suppressing vascular endothelial growth factor (VEGF) in 4T1 breast cancer cells both in vitro and in vivo. While MSCs offer numerous advantages as therapeutic tools for regenerative medicine, consistent quality control under laboratory culture conditions poses several challenges. Although MSCs can be expanded in vitro with increased passage numbers to obtain sufficient cell numbers, proliferation of MSCs is markedly reduced, resulting in cellular senescence.

[0003] Cellular senescence is a physiological process in which cells lose their replicative capacity. Common hallmarks of cellular senescence include arrest of cell cycle progression, structural changes related to the lysosomal compartment or the extracellular matrix, and metabolic adaptations, including increased ROS or dysfunctional mitochondria. Furthermore, cellular senescence can also lead to changes in the morphological and functional properties of mesenchymal stem cells (MSCs). Specifically, senescence of human BM-MSCs, through extensive in vitro expansion, is known to significantly impair MSC phenotype and differentiation potential. Furthermore, while long-term cultured human umbilical cord-derived MSCs (UC-MSCs) can be expanded for numerous passages, most suffer from genomic alterations, such as chromosomal aberrations or changes in epigenetic properties. While these characteristics of cellular senescence have been previously reported in MSCs of various origins, the detailed molecular mechanisms underlying the senescence process in MSCs remain insufficient due to its complexity and comprehensive nature.

[0004] Despite the ISCT (International Society for Cellular Therapy) definition, the cell population of mesenchymal stem cells (MSCs) is highly heterogeneous due to various factors, including donor, tissue source, and cell culture environment. Many researchers have established approaches using surface phenotypic profiling to minimize the heterogeneity and senescence of MSCs. CD264+ induced cellular senescence during serial passage and reduced proliferation and multilineage differentiation in human BM-MSCs compared to CD264-. In contrast, downregulation of CD146 resulted in lower growth rates, multilineage differentiation, expression of pluripotency-associated transcription factors (NANOG, OCT4, and SOX2), and elevated expression of senescence-associated markers such as p16, p21, and p53. All these reports suggest that the discovery of MSC senescence-associated biomarkers is crucial for achieving improved in vitro biological properties and in vivo therapeutic efficacy.

[0005] Accordingly, the inventors of the present invention have made extensive efforts to develop a composition for detecting aged mesenchymal stem cells and a use thereof, and as a result, have confirmed that tonsil-derived mesenchymal stem cells (TMSCs) are a new source of mesenchymal stem cells with a high differentiation potential into various lineage cells and a relatively higher proliferation rate than mesenchymal stem cells derived from other types of tissues. Based on these advantages, the inventors have explored the cellular mechanisms of mesenchymal stem cells or MSC markers using TMSCs in regenerative medicine, and have confirmed that aged tonsil-derived mesenchymal stem cells can be detected by utilizing podoplanin (PDPN), a small transmembrane glycoprotein expressed on the surface of some MSCs, including TMSCs, thereby completing the present invention.

[0006] One object of the present invention is to provide a composition and / or kit for detecting senescence of tonsil-derived mesenchymal stem cells, comprising an antibody or aptamer that specifically binds to podoplanin protein.

[0007] Another object of the present invention is to provide a method for detecting senescence of tonsil-derived mesenchymal stem cells, comprising the steps of (a) detecting protein expression of podoplanin in tonsil-derived mesenchymal stem cells; and (b) measuring the expression level of the podoplanin protein.

[0008] Another object of the present invention is to provide a method for screening for a senescence inhibitor in tonsil-derived mesenchymal stem cells, comprising the steps of: (a) contacting a sample to be analyzed with a cell in which the protein expression of podoplanin in tonsil-derived mesenchymal stem cells is reduced; (b) measuring the expression level of the podoplanin protein; and (c) determining the sample as a senescence inhibitor in tonsil-derived mesenchymal stem cells when the expression of the podoplanin protein is increased.

[0009] Another object of the present invention is to provide a composition for inhibiting cellular senescence of tonsil-derived mesenchymal stem cells containing podoplanin.

[0010] Another object of the present invention is to provide a method for inhibiting senescence of tonsil-derived mesenchymal stem cells, comprising the step of treating tonsil-derived mesenchymal stem cells with podoplanin and then subculturing them.

[0011] To avoid confusion due to overlapping content, the description of redundant content will be omitted below. In other words, the content of the invention is not limited to the content described below, and the content of the invention should be interpreted based on the overall content of the invention.

[0012] Mesenchymal stem cells, possessing self-renewal and differentiation potential, are being utilized as a promising cell source for developing clinical applications in regenerative medicine. While mesenchymal stem cell-based therapeutic approaches are rapidly expanding for various diseases, concerns about functional decline in mesenchymal stem cells due to aging necessitate securing high-quality efficacy.

[0013] Accordingly, the present inventors have confirmed through extensive efforts that podoplanin (PDPN) is significantly reduced in aged tonsil-derived mesenchymal stem cells, thereby confirming that podoplanin can be used as a marker for detecting cellular senescence of tonsil-derived mesenchymal stem cells or screening for inhibitors of cellular senescence of tonsil-derived mesenchymal stem cells. In addition, by confirming that the characteristics of tonsil-derived mesenchymal stem cells, such as multilineage differentiation potential and cell cycle progression, were lost in cells in which PDPN expression was suppressed by siPDPN or the like, it was confirmed that podoplanin can inhibit cellular senescence of tonsil-derived mesenchymal stem cells.

[0014] Accordingly, the present invention provides a composition for detecting senescence of tonsil-derived mesenchymal stem cells, a kit for detecting senescence, a method for detecting senescence of tonsil-derived mesenchymal stem cells, a method for screening a senescence inhibitor of tonsil-derived mesenchymal stem cells, a composition for inhibiting senescence of tonsil-derived mesenchymal stem cells, and a method for inhibiting senescence of tonsil-derived mesenchymal stem cells.

[0015] As one embodiment for achieving the above purpose, the present invention provides a composition for detecting senescence of tonsil-derived mesenchymal stem cells, each comprising an antibody or aptamer that specifically binds to podoplanin protein.

[0016] The present inventors have dedicated themselves to discovering a novel marker capable of accurately detecting cellular senescence in tonsil-derived mesenchymal stem cells (MSCs), a useful tool for regenerative medicine cell therapy. As a result, we identified podoplanin (PDPN), a small transmembrane glycoprotein expressed on the surface of some MSCs, and specifically the podoplanin protein, as a marker capable of accurately detecting cellular senescence.

[0017] According to one embodiment of the present invention, down-regulation of PDPN at the protein level, rather than mRNA expression, was observed in a passage-dependent manner, and it was confirmed that expression of PDPN protein was significantly reduced in both the lysate and cell surface of aged tonsil-derived mesenchymal stem cells after 15 or more passages.

[0018] Podoplanin (PDPN) is a mucin-like membrane protein with a mass of 36–43 kDa. The gene encodes a type I, integral membrane, and highly O-glycosylated glycoprotein, which is widely distributed across human tissues. PDPN has been extensively studied in the field of cancer, but its association with cellular senescence is currently unknown.

[0019] Adult-derived mesenchymal stem cells (MSCs) have been extensively investigated in numerous reports because they do not have the ethical concerns or carcinogenicity restrictions associated with embryonic-derived MSCs or induced pluripotent mesenchymal stem cells (iPSCs). The present invention utilizes tonsil-derived MSCs, as the inventors' previous studies have shown that tonsil-derived MSCs (TMSCs) represent a novel source of MSCs with a high differentiation potential into various cell lineages and a relatively higher proliferation rate than MSCs derived from other types of tissues.

[0020] In this specification, the term "cellular senescence" refers to a state in which cell division irreversibly ceases after a certain number of divisions. Cellular senescence differs from apoptosis in that while cell proliferation and division are impaired, cellular metabolism persists for a significant period. Cellular senescence is triggered by a wide variety of factors, including DNA damage, telomere disruption, oncogene activation, and organelle stress.

[0021] The characteristics that appear in the aging of various cells are as follows. The primary characteristics of cellular aging include genetic instability that can be confirmed by the increase and phosphorylation of oncogenes such as p16, p21, and p53, telomere disorders such as telomere shortening, epigenetic changes such as DNA methylation and post-translational modification of histones, and loss of protein production function. In addition, methods for directly confirming cellular aging in cells or tissues include increased single cell size, measurement of SA-β-gal, and decline in mitochondrial function. In the present invention, the characteristics of the term “aged tonsil-derived mesenchymal stem cells” may basically exhibit the characteristics of a decrease in cell proliferation ability, loss of cell division ability, decrease in cell migration, decrease in cell differentiation ability, shortened telomere length, increase in single cell size and SA-β-gal, or all of these.

[0022] In one specific example of the present invention, by confirming an increase in doubling time, cell size, and cell staining for SA-β-gal, a shortening of telomere length, and a decrease in differentiation potential (adipogenesis, chondrogenesis, and osteogenesis) in tonsil-derived mesenchymal stem cells passaged 15 or more times, it was confirmed that tonsil-derived mesenchymal stem cells passaged 15 or more times were senescent cells, and it was demonstrated that the characteristics of tonsil-derived mesenchymal stem cells were affected by in vitro aging.

[0023] The marker of the present invention can be an indicator of cellular senescence and can be used to detect the occurrence and development of cellular senescence.

[0024] The term “detection” herein refers to determining the level of a marker (i.e., podoplanin protein) in a biological sample, such as cells, cell culture or cell lysate.

[0025] As one embodiment for achieving the above purpose, the present invention provides a kit for detecting senescence of tonsil-derived mesenchymal stem cells, comprising the composition for detecting senescence of tonsil-derived mesenchymal stem cells.

[0026] The above kit may be an immunoassay kit. That is, it can detect cellular senescence using an antigen-antibody reaction. In this case, an antibody or aptamer that specifically binds to the cellular senescence marker of the present invention may be used.

[0027] The antibody used in the present invention is a polyclonal or monoclonal antibody, and is preferably a monoclonal antibody. The antibody can be produced by methods commonly practiced in the art, such as the fusion method (Kohler and Milstein, European Journal of Immunology, 6:511-519 (1976)), the recombinant DNA method (U.S. Patent No. 4,816,56), or the phage antibody library method (Clackson et al., Nature, 352:624-628 (1991) and Marks et al., J. Mol. Biol., 222:58, 1-597 (1991)). General procedures for antibody production are described in Harlow, E. and Lane, D., Using Antibodies: A Laboratory Manual, Cold Spring Harbor Press, New York, 1999; Zola, H., Monoclonal Antibodies: A Manual of Techniques, CRC Press, Inc., Boca Raton, Florida, 1984; and Coligan, CURRENT PROTOCOLS IN IMMUNOLOGY, Wiley / Greene, NY, 1991.

[0028] For example, hybridoma cells that produce monoclonal antibodies are produced by fusing immortalized cell lines with antibody-producing lymphocytes. The techniques required for this process are well known and readily available to those skilled in the art. Polyclonal antibodies can be obtained by injecting a protein antigen into a suitable animal, collecting antisera from the animal, and then isolating the antibody from the antisera using known affinity techniques.

[0029] When the method of the present invention is performed using an antibody or aptamer, the present invention can be used to detect cellular senescence by performing it according to a conventional immunoassay method. Such immunoassay can be performed according to various quantitative or qualitative immunoassay protocols developed in the past. The immunoassay formats include, but are not limited to, radioimmunoassay, radioimmunoprecipitation, immunoprecipitation, immunohistochemical staining, enzyme-linked immunosorbent assay (ELISA), capture-ELISA, inhibition or competition assay, sandwich assay, flow cytometry, immunofluorescence staining, and immunoaffinity purification. The immunoassay or immunostaining method is described in Enzyme Immunoassay, E.T. Maggio, ed., CRC Press, Boca Raton, Florida, 1980; Gaastra, W., Enzymelinked immunosorbent assay (ELISA), in Methods in Molecular Biology, Vol. 1, Walker, JM ed., Humana Press, NJ, 1984; and Ed Harlow and David Lane, Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1999.

[0030] For example, when the method of the present invention is performed according to a radioimmunoassay method, antibodies labeled with radioactive isotopes (e.g., C14, I125, P32 and S35) can be used to detect the marker molecule of the present invention.

[0031] When the method of the present invention is performed by ELISA, a specific embodiment of the present invention comprises the steps of (i) coating an unknown cell culture fluid (or cell lysate) to be analyzed on the surface of a solid substrate; (ii) reacting the cell culture fluid (or cell lysate) with an antibody against a marker as a primary antibody; (iii) reacting the resultant of step (ii) with a secondary antibody conjugated to an enzyme; and (iv) measuring the activity of the enzyme.

[0032] Suitable solid substrates include hydrocarbon polymers (e.g., polystyrene and polypropylene), glass, metal or gel, most preferably a microtiter plate.

[0033] The enzyme bound to the secondary antibody includes, but is not limited to, an enzyme that catalyzes a color reaction, a fluorescent reaction, a luminescent reaction, or an infrared reaction, and includes, for example, alkaline phosphatase, β-galactosidase, horseradish peroxidase, luciferase, and cytochrome P450. When alkaline phosphatase is used as an enzyme binding to the secondary antibody, color reaction substrates such as bromochloroindolyl phosphate (BCIP), nitro blue tetrazolium (NBT), naphthol-AS-B1-phosphate and enhanced chemifluorescence (ECF) are used as substrates, and when horseradish peroxidase is used, chloronaphthol, aminoethylcarbazole, diaminobenzidine, D-luciferin, lucigenin (bis-N-methylacridinium nitrate), resorufin benzyl ether, luminol, Amplex Red reagent (10-acetyl-3,7-dihydroxyphenoxazine), HYR (p-phenylenediamine-HCl and pyrocatechol), TMB (tetramethylbenzidine), Substrates such as ABTS (2,2'-Azine-di[3-ethylbenzthiazoline sulfonate]), o-phenylenediamine (OPD), and naphthol / pyronine, glucose oxidase, and t-NBT (nitroblue tetrazolium) and m-PMS (phenzaine methosulfate) can be used.

[0034] When the method of the present invention is carried out in a capture-ELISA manner, a specific embodiment of the present invention comprises the steps of (i) coating an antibody against a marker of the present invention as a capturing antibody on the surface of a solid substrate; (ii) reacting the capturing antibody with a cell sample; (iii) reacting the resultant of step (ii) with a detecting antibody that is bound to a label that generates a signal and that specifically reacts with the podoplanin protein; and (iv) measuring a signal generated from the label.

[0035] The detection antibody has a label that generates a detectable signal. The label includes, but is not limited to, chemicals (e.g., biotin), enzymes (e.g., alkaline phosphatase, β-galactosidase, horseradish peroxidase, and cytochrome P450), radioactive materials (e.g., C14, I125, P32, and S35), fluorescent materials (e.g., fluorescein), luminescent materials, chemiluminescent materials, and fluorescence resonance energy transfer (FRET). Various labels and labeling methods are described in Ed Harlow and David Lane, Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1999.

[0036] In the above ELISA and capture-ELISA methods, the final enzyme activity measurement or signal measurement can be performed using various methods known in the art. Detection of such signals enables qualitative or quantitative analysis of the marker of the present invention. If biotin is used as a label, the signal can be easily detected with streptavidin, and if luciferase is used, the signal can be easily detected with luciferin.

[0037] According to another variation of the present invention, an aptamer that specifically binds to the marker of the present invention can be used instead of an antibody. An aptamer is an oligonucleotide or peptide molecule, and general information about aptamers is described in detail in Bock LC et al., Nature 355(6360):5646(1992); Hoppe-Seyler F, Butz K, "Peptide aptamers: powerful new tools for molecular medicine", J Mol Med. 78(8):42630(2000); Cohen BA, Colas P, Brent R, "An artificial cell-cycle inhibitor isolated from a combinatorial library", Proc Natl Acad Sci USA. 95(24):142727(1998).

[0038] Cellular senescence can be detected by analyzing the intensity of the final signal from the above-described immunoassay process. That is, if the signal for the marker of the present invention is weaker than that of a normal sample, the sample is judged to be cellular senescence.

[0039] In addition to the components described above, the kit of the present invention may additionally include other components. For example, it may further include a user guide describing optimal reaction procedures (conditions). The guide is a printed document explaining how to use the kit, such as the order of application of the samples and kit contents. The guide includes a pamphlet or leaflet-style guidebook, a label attached to the kit, and a description on the surface of the package containing the kit. The guide also includes information provided through electronic media, such as the Internet.

[0040] The marker of the present invention is a biomolecule that is underexpressed in aged tonsil-derived mesenchymal stem cells. The underexpression of this marker can be measured at the protein level. As used herein, the term "underexpression" refers to a case where the expression level of the target protein in the sample under investigation is lower than that in a normal sample. For example, if the protein expression of PDPN is underexpressed by 90% or less, preferably 85% or less, and more preferably 80% or less, compared to early-passage (young) stem cells, cellular senescence is determined.

[0041] As one embodiment for achieving the above object, the present invention provides a method for detecting cell senescence of tonsil-derived mesenchymal stem cells, comprising: (a) a step of detecting protein expression of podoplanin in tonsil-derived mesenchymal stem cells; and (b) a step of measuring the expression level of the podoplanin protein.

[0042] At this time, the method further includes a step of (c) determining cell senescence when the expression of the podoplanin protein is low compared to that of primary tonsil-derived mesenchymal stem cells.

[0043] The term “early tonsil-derived mesenchymal stem cells” as used herein refers to tonsil-derived mesenchymal stem cells isolated from a human body at passage 1 or passages 3 to 7. Alternatively, it refers to tonsil-derived mesenchymal stem cells that do not exhibit characteristics of reduced cell proliferation capacity, reduced cell differentiation capacity, or shortened telomere length.

[0044] As one embodiment for achieving the above object, the present invention provides a method for screening for a senescence inhibitor in tonsil-derived mesenchymal stem cells, comprising the steps of: (a) contacting a sample to be analyzed with a cell in which the protein expression of podoplanin in tonsil-derived mesenchymal stem cells is reduced; (b) measuring the expression level of the podoplanin protein; and (c) determining the sample as a senescence inhibitor in tonsil-derived mesenchymal stem cells when the expression of the podoplanin protein is increased.

[0045] According to the method of the present invention, a sample to be analyzed is first brought into contact with tonsil-derived mesenchymal stem cells in which protein expression of the marker of the present invention is reduced. The term "sample" used in referring to the screening method of the present invention refers to an unknown substance used in screening to determine whether it affects the expression level of the marker of the present invention. The sample includes, but is not limited to, chemicals, nucleotides, antisense RNA, siRNA (small interference RNA), and natural product extracts.

[0046] Next, the expression level of the marker of the present invention is measured in the cells treated with the sample. The expression level can be measured as described above, and if the measurement results show that the protein level of the marker of the present invention is high, the sample can be determined to be a cell senescence inhibitor.

[0047] As one embodiment for achieving the above purpose, the present invention provides a composition for inhibiting cellular senescence of tonsil-derived mesenchymal stem cells containing podoplanin.

[0048] The composition for inhibiting aging of the present invention inhibits aging, which is a phenomenon in which the number and regenerative ability of tonsil-derived mesenchymal stem cells decrease due to the loss of tonsil-derived mesenchymal stem cell characteristics, such as self-renewal and multipotency, after a certain number of divisions.

[0049] In an embodiment according to the present invention, PDPN+ cells showed significantly higher proliferation ability than PDPN- cells at 48 hours, and showed significantly higher scratch recovery ability, confirming that they maintained the characteristics of tonsil-derived mesenchymal stem cells, such as self-proliferation and cell migration.

[0050] Meanwhile, the present invention provides a method for culturing tonsil-derived mesenchymal stem cells so as to secure a sufficient number of separated tonsil-derived mesenchymal stem cells after isolating tonsil-derived mesenchymal stem cells from tissue.

[0051] As one embodiment for achieving the above object, the present invention provides a method for culturing tonsil-derived mesenchymal stem cells, comprising: a step of culturing isolated tonsil-derived mesenchymal stem cells; a step of measuring the expression level of podoplanin in the tonsil-derived mesenchymal stem cells; and a step of taking only tonsil-derived mesenchymal stem cells having a high expression level of podoplanin.

[0052] As another aspect for achieving the above object, the present invention provides a method for inhibiting senescence of tonsil-derived mesenchymal stem cells, comprising the step of treating tonsil-derived mesenchymal stem cells with podoplanin and then subculturing them.

[0053] For example, it is known that human bone marrow-derived mesenchymal stem cells, like general somatic cells, show characteristic changes such as a decrease in proliferation rate and a decrease in differentiation ability as the number of passages increases during in vitro culture. In addition, it has been reported that in the later stages of passage culture, the length of mTRF (mean telomere restriction fragment) decreases, the number of cells stained with SA β-gal (senescence-associated β-galactosidase) increases, and changes in the expression of genes and proteins related to cell growth, proliferation, and the cell cycle occur. In order to minimize such characteristic changes in stem cell passage culture and to obtain only tonsil mesenchymal stem cells with excellent activity, in the present invention, podoplanin can be used as a biomarker for stem cell differentiation, and also directly treated or overexpressed to inhibit the aging of mesenchymal stem cells.

[0054] The present invention can effectively suppress cell senescence of tonsil-derived mesenchymal stem cells by including a step of treating tonsil-derived mesenchymal stem cells with podoplanin and then subculturing them.

[0055] The above subculture is a maintenance culture in which the identity of the tonsil-derived mesenchymal stem cells is maintained while being proliferated. In this case, the tonsil-derived mesenchymal stem cells may preferably be tonsil-derived mesenchymal stem cells. The tonsil-derived mesenchymal stem cells are cells that possess multipotency and can differentiate into cells including adipocytes, osteocytes, chondrocytes, muscle cells, nerve cells, and cardiomyocytes.

[0056] Preferably, the subculture may be performed at least once, twice, three times, four times, five times, ten times or more, but is not limited thereto.

[0057] Preferably, podoplanin can be added to a culture medium for tonsil-derived mesenchymal stem cells. The medium is a mixture containing essential sugars, amino acids, water, and other nutrients required for cell survival. A basic medium, excluding serum, nutrients, and various tonsil-derived mesenchymal stem cell growth factors, can be used. The basic medium can be either synthetically synthesized or a commercially available, chemically defined medium. Commercially prepared media include DMEM (Dulbecco's Modified Eagle's Medium), MEM (Minimal Essential Medium), BME (Basal Medium Eagle), RPMI 1640, DMEM / F-10 (Dulbecco's Modified Eagle's Medium: Nutrient Mixture F-10), DMEM / F-12 (Dulbecco's Modified Eagle's Medium: Nutrient Mixture) F-12), a-MEM (a-Minimal essential Medium), G-MEM (Glasgow's Minimal Essential Medium), and IMDM (Isocove's Modified Dulbecco's Medium).

[0058] The medium used for culturing tonsil-derived mesenchymal stem cells refers to a culture solution that can support the growth and survival of tonsil-derived mesenchymal stem cells under in vitro culture conditions, and includes all commonly used media in the relevant field suitable for culturing tonsil-derived mesenchymal stem cells. In addition, to prevent infection with bacteria, fungi, etc., antibiotics, antifungals, and substances commonly used in the relevant industry to prevent the growth of mycoplasma that causes contamination can be used. Antibiotics commonly used in cell culture, such as penicillin-streptomycin, can all be used, and antifungals such as alporelycin B, and mycoplasma inhibitors commonly used substances such as gentamicin, ciprofloxacin, and azithromycin can be used.

[0059] By utilizing the podoplanin marker according to the present invention, cellular senescence of tonsil-derived mesenchymal stem cells can be accurately detected. Furthermore, by treating cells or culture medium with the composition according to the present invention, senescence of tonsil-derived mesenchymal stem cells can be suppressed and their pluripotency maintained even over long-term culture periods. Therefore, tonsil-derived mesenchymal stem cells cultured in this manner can be usefully utilized as therapeutic resources or therapeutic agents in the field of regenerative medicine.

[0060] Figure 1 shows the results of confirming the characteristics of cell senescence in tonsil-derived mesenchymal stem cells (TMSCs) (A: Measurement of cell growth time according to continuous culture, B: Measurement of cell length and width by passage-dependent method, C: Results of senescence-associated β-galactosidase (SA-β-gal) assay (blue-stained cells are senescent cells (black arrows)), D: Measurement of telomere length). Early represents young TMSCs passaged 3 to 7 times, and Late represents aged TMSCs passaged 15 or more times. Bars represent the mean ± SD, and statistically significant differences are indicated by *p<0.05 and ***p<0.001. Alphabet indicates significant differences between experimental groups (*p<0.05).

[0061] Figure 2 shows the results of confirming the senescence characteristics of early and late tonsil-derived mesenchymal stem cells (TMSCs) proposed by the ISCT minimum criteria (A: Flow cytometry analysis to evaluate cell size and particle size and analyze Forward Scatter (FSC) and Side Scatter (SSC) values ​​to evaluate MSC-specific surface markers such as hematopoietic surface markers (CD14, CD34, and CD45) and primitive cell markers (CD73, CD90, and CD105); B: Gene expression analysis of pluripotency-related transcription factors (NANOG, OCT4, and SOX2); C: Evaluation of multilineage differentiation levels including adipogenesis, chondrogenesis, and osteogenesis). Bars represent mean ± SD, and statistically significant differences are indicated by *p<0.05 and ***p<0.001. ns is not significant.

[0062] Figure 3 shows the results of confirming changes in PDPN expression through replicative senescence (A: quantitative mRNA expression measurement results, B: protein level measurement results of PDPN and GAPDH, C: immunophenotypic analysis results of early and late PDPN). Bars represent mean ± SD, and letters indicate significant differences between experimental groups (*p < 0.05).

[0063] Figure 4 shows the results of identifying MSC characteristics in PDPN+ TMSCs or PDPN- TMSCs sorted by FACS (A: Immunophenotypic analysis of PDPN for PDPN+ cells and PDPN- cells, B: Telomere length determination by T / S ratio measurement, C: Analysis of differences between MSC-specific surface markers, D: Analysis of pluripotency markers, E: Analysis of multilineage differentiation potential of PDPN+ cells and PDPN- cells). Bars represent mean ± SD, and statistically significant differences are indicated by ***p<0.001. ns: not significant.

[0064] Figure 5 shows the results of confirming the effects of PDPN on cell proliferation, cell cycle, and cell motility (A: Cell proliferation analysis results of PDPN+ cells and PDPN- cells, B: Cell cycle analysis results of PDPN+ cells and PDPN- cells, C: Protein level measurement results of G0 / G1 phase-related markers, CDK4 and cyclin D1, of PDPN+ cells and PDPN- cells, D: Cell motility analysis results of PDPN+ cells and PDPN- cells (Scratch recovery rate is calculated as the percentage of the remaining scratch area compared to the initial scratch area)). Bars represent mean ± SD, and statistically significant differences are indicated by *p<0.01 and ***p<0.001.

[0065] Figure 6 shows the results of evaluating the cell cycle and cell migration ability after transfection with PDPN siRNA (A: Quantitative mRNA expression level measurement result, B: Protein level measurement result of PDPN and GAPDH, C: Cell cycle analysis result, D: Protein level measurement result of G0 / G1 phase-related markers CDK4 and cyclin D1, E: Cell migration ability analysis result (Scratch recovery rate is calculated as the percentage of the remaining scratch area compared to the initial scratch area)). Bars represent mean ± SD, and statistically significant differences are indicated by *p<0.01 and ***p<0.001. ns means not significant.

[0066] Hereinafter, preferred examples are presented to aid in understanding the present invention. However, the following examples are provided solely to facilitate a better understanding of the present invention, and the scope of the present invention is not limited by the following examples.

[0067] Materials and Methods

[0068] 1. Isolation and expansion of tonsil-derived mesenchymal stem cells (TMSCs)

[0069] TMSCs were isolated according to a protocol (Cytotherapy. 2012;14(10):1193-202) approved by the Institutional Review Board of Ewha Womans University Mokdong Hospital, Korea (EUMC-2019-08-030). In addition, informed written consent was obtained from the legal guardians of patients under 10 years of age undergoing tonsillectomy. Specifically, tonsil tissues collected from four donors were cut into small pieces using scissors. The cut tissues were enzymatically treated with 210 U / mL collagenase type I (Thermo Fisher Scientific, Waltham, MA, USA) and 10 mg / mL DNase (Sigma-Aldrich, St. Louis, MO, USA) at 37°C for 30 minutes. The tissues were filtered and centrifuged with Ficoll-Paque (GE Healthcare, Piscataway, NJ, USA) to obtain mononuclear cells. Mononuclear cells were plated on culture plates and cultured for 48 hours. Non-attached cells were discarded and adherent cells (TMSCs) were used in the following experiments.

[0070] In the following experiments, TMSCs were cultured and expanded in DMEM (DMEM-LG; Welgene Inc.) containing low glucose (1,000 mg / L) supplemented with 10% fetal bovine serum (FBS; Young In Frontier, Seoul, Korea), 100 U / mL penicillin, 100 U / mL streptomycin, and 0.25 μg / mL amphotericin B in a humidified CO2 incubator at 37°C. For isolation experiments, TMSCs were divided into two groups with different passage ranges. Early passage TMSCs (Early) were between passages 3 and 7, and late passage TMSCs (Late) were passaged 15 or more times.

[0071] 2. Measurement of doubling time and cell size

[0072] Tonsil-derived mesenchymal stem cells (TMSCs) were cultured for 72 or 96 hours and then harvested at 80–90% confluence using a trypsin-EDTA (trypsin-ethylenediaminetetraacetic acid) solution (Thermo Fisher Scientific). Viable cells in the suspension mixed with trypan blue were counted using a hemocytometer. Initial and final cell counts were collected at each passage of TMSCs, and the doubling time was calculated using the following formula: Equation 1.

[0073] [Mathematical Formula 1]

[0074] Doubling time (h) = [{T-T0}(log0) / (logN-logN0)}]

[0075] In separate experiments, TMSCs were seeded onto culture dishes at the indicated passages and visualized in at least five randomly selected fields after 24 h under a light microscope (Olympus, BX51, Japan). The cell length and extension width of each cell were measured using Image J software (National Institutes of Health, Bethesda, MD, USA), respectively.

[0076] 3. Analysis of β-galactosidase associated with aging

[0077] Senescence-associated β-galactosidase (SA-β-gal) activity was detected in two groups of TMSCs with different passage ranges using the SA-β-gal staining kit (Cell Signaling Technology, Boston, MA, USA) according to the manufacturer's instructions. Briefly, TMSCs were fixed with 1x fixative for 15 min at room temperature and then washed twice with Dulbecco's phosphate-buffered saline (DPBS; Welgene Inc.). The fixed cells were incubated with β-gal staining solution (pH 6.0) for 24 h in a CO2-free, 37°C dry incubator. Senescent cells were observed in blue and visualized by light microscopy.

[0078] 4. Reverse transcription polymerase chain reaction (RT-PCR)

[0079] Total RNA of TMSCs was extracted using TRizol® reagent (Thermo Fisher Scientific), and 2 μg of total RNA was used to synthesize cDNA using Superscript reverse transcriptase, dNTPs, and oligo-dT primers according to the manufacturer's instructions. The reaction conditions for PCR amplification were 1 cycle at 94°C for 2 min, followed by 36 cycles of 94°C for 30 s, 56°C for 30 s, 72°C for 1 min, and 72°C for 5 min.

[0080] 5. Measuring telomere length

[0081] Genomic DNA was extracted from TMSCs using the DNeasy Blood & Tissue kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. The extracted genomic DNA was used to determine relative telomere length, a measure of the ratio of telomeres to single-copy genes (T / S ratio). Briefly, genomic DNA (200 ng / μL) was synthesized using Power SYBR™ Green PCR Master Mix (Thermo Fisher Scientific) and a telomere primer pair (telg and telc) or a 36B4 primer pair for single-copy gene amplification. All PCR reactions were performed on a QuantStudio 3 Real-time PCR system (Thermo Fisher Scientific).

[0082] 6. Primer sequence

[0083] The primer sequences for amplification of pluripotency-related transcription factors, PDPN, telomere, and single-copy genes used in this experiment are shown in Table 1 below.

[0084]

[0085]

[0086] 7. Flow cytometry

[0087] The immunophenotype of MSCs was investigated using flow cytometry. TMSCs were collected as single cells and stained with phycoerythrin (PE)-conjugated anti-human antibodies against CD14, CD34, CD73, and PDPN, or fluorescein isothiocyanate (FITC)-conjugated anti-human antibodies against CD45, CD90, and CD105 for 30 min at 4°C. After staining, cells were analyzed using an ACEA NovoCyte flow cytometer (Agilant Technologies Inc., Santa Clara, CA, USA). All antibodies were purchased from BD Biosciences (San Jose, CA, USA).

[0088] 8. Fluorescence-activated cell sorting (FACS)

[0089] MSCs were prepared as single cells and labeled with PE-conjugated anti-human PDPN for 30 min at 4°C. The cells were washed twice with cell sorting buffer containing FBS and then filtered using a 70 μm cell strainer (Corning Inc., Corning, NY, USA). The filtered single cells were sorted into PDPN+ or PDPN− cells using an S3e™ cell sorter (Bio-Rad Laboratories Inc., Hercules, CA, USA). After sorting, the cells were seeded onto culture plates and cultured for further experiments.

[0090] 9. Multilineage differentiation of TMSC

[0091] To investigate the level of multilineage differentiation, TMSCs were cultured for 2 weeks in commercially available adipogenic, chondrogenic, and osteogenic differentiation media (Thermo Fisher Scientific). Each differentiation medium was replaced every 3–4 days. Differentiated TMSCs were fixed with 4% paraformaldehyde for 30 minutes and washed with DPBS. Oil red O, Alcian blue, and Alizarin red S staining were used to verify the adipogenic, chondrogenic, and osteogenic differentiation capacity of the cells, respectively. After 30 minutes of staining, the cells were washed twice with DPBS and observed under a microscope. At least three randomly acquired images were converted to RGB stacks and the differentiation level was quantified using Image J software.

[0092] 10. Western blot analysis

[0093] TMSCs were extracted using a lysis buffer containing 20 mM Tris-HCl (pH 7.5), 1% Triton X-100, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA (ethylene glycol-bis (β-aminoethyl ether)-N, N, N′, N′-tetraacetic acid), and Protease Inhibitor Cocktail™. Equal volumes (20 μg) of protein were separated by SDS-PAGE and transferred to nitrocellulose membranes using a Trans-Blot Turbo system (Bio-Rad Laboratories Inc.). The transferred membranes were fixed with 5% skim milk and incubated with primary antibodies against PDPN, CDK4 (cyclin-dependent kinase 4), and cyclin D1 (1:1000 dilution; Cell signaling Technology, Boston, MA, USA), and GAPDH (glyceraldehyde 3-phosphate dehydrogenase; 1:5000 dilution; AbFrontier, Seoul, Korea), respectively. The membranes were then incubated with the corresponding secondary antibodies. Proteins were developed using a chemiluminescence reagent (Amersham, Buckinghamshire, UK) on a luminescence image analyzer (Cytiva, Marlborough, MA, USA).

[0094] 11. Cell viability / proliferation analysis

[0095] After sorting cells into PDPN+ and PDPN- cells, cell viability / proliferation assays were performed with slight modifications. Briefly, sorted cells were seeded in 24-well plates at a density of 1 × 10 4Cells were seeded per well, and MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) solution (Sigma-Aldrich) was added at the indicated times (0, 24, 48, and 72 h). After incubation for 2 h in a humidified CO2 incubator at 37°C, the dyed purple formazan was dissolved using dimethyl sulfoxide (Sigma-Aldrich), and an equal amount of the dissolved solution was transferred to a 96-well plate. The absorbance was measured at 540 nm using a microplate reader and expressed as a value relative to the value at time 0.

[0096] 12. Cell cycle analysis

[0097] TMSCs(1×10 5 Cells) were prepared as single cells using trypsin-EDTA solution and fixed with 70% ethanol solution for 1 h at 4°C. The fixed cells were washed with cold DPBS containing 2% FBS and stained with RNase A (Sigma-Aldrich) and propidium iodide (PI; BD Biosciences) for 30 min in a humidified CO2 incubator at 37°C in the dark. Analysis of cell cycle distribution was performed using an ACEA NovoCyte flow cytometer.

[0098] 13. In vitro cell migration assay

[0099] TMSCs were seeded at 3 × 10 in 6-well plates. 5Cells were seeded at a concentration of 10 cells / well. After 24 h, cells at 80–90% confluence were treated with 10 μg / mL mitomycin C for 2 h in a humidified CO2 incubator at 37°C. Cells were gently washed with DPBS and cultured with culture medium. A linear scratch was created by scratching with a 200 μL pipette tip. The extent of scratch recovery was imaged using a microscope at each time point (0 and 24 h). The percentage of residual scratch area was measured relative to the initial scratch area.

[0100] 14. Transfection

[0101] TMSCs at 80% confluency were transfected with 100 nM of control small interfering RNA (siRNA) (siCON; Santa Cruz Biotechnology, Santa Cruz, CA, USA) and PDPN siRNA (Bioneer Inc., Oakland, CA, USA) using Lipofectamine 3000 (Thermo Fisher Scientific) according to the manufacturer's instructions. After transfection, cells were incubated for 24 h and then used for further experiments.

[0102] 15. Statistical Analysis

[0103] All results are expressed as mean ± standard deviation (SD). Statistical significance was determined using a one-way analysis of variance (ANOVA). A p-value less than 0.05 was considered statistically significant. All experimental trials were repeated at least three times to verify the reproducibility of the results.

[0104] Example 1. Evaluation of the characteristics and aging-related changes of tonsil-derived mesenchymal stem cells.

[0105] 1. Verification of replicative senescence in TMSCs

[0106] We aimed to evaluate the cell growth rate and morphological changes of TMSCs according to passage number during cell culture. To this end, TMSCs obtained from four different donors were serially cultured, and the doubling time from passage 4 to passage 16 was calculated. As shown in Figure 1A, the average doubling time was 37.42 ± 5.30 h at passage 14, but it significantly increased to 48.41 ± 5.70 h at passages 15 and above.

[0107] Meanwhile, morphological changes were observed by measuring the length and width of cells. As shown in Fig. 1B, the cell length of TMSCs did not differ up to passage 15, with 318.67 ± 75.30 ㎛ at passage 5, 338.46 ± 57.13 ㎛ at passage 10, and 307.28 ± 62.57 ㎛ at passage 15. However, the cell width increased with passage, with 22.61 ± 6.73 ㎛ at passage 5, 25.52 ± 9.50 ㎛ at passage 10, and 36.21 ± 13.50 ㎛ at passage 15, being significantly wider at passage 15 than at passage 5.

[0108] Meanwhile, to confirm the passage-dependent aging of TMSCs based on the above results, TMSCs were divided into two groups: TMSCs cultured within passages 3 to 7 as early passage cells (Early or E; young cells) and TMSCs cultured at passages 15 or more as late passage cells (Late or L; aged cells).

[0109] As shown in Figure 1C, SA-β-gal staining showed that aged cells (Late) exhibited a significantly higher degree of cellular senescence than young cells (Early), and as shown in Figure 1D, the T / S ratio, which suggests shortening of telomere length associated with replicative senescence, was significantly reduced in aged cells.

[0110] These results suggest that TMSCs of passage 15 or more are aged cells.

[0111] 2. Verification of replicative aging-related changes in MSC characteristics

[0112] We investigated whether MSC characteristics such as immunological surface phenotype, pluripotency markers, and multilineage differentiation capacity were altered through replicative senescence.

[0113] As shown in Figure 2A, forward scatter (FSC) and side scatter (SSC) values ​​for assessing cell size and particle size increased in aged cells compared to young cells. However, there was no difference between the two groups in the negative expression of hematopoietic surface markers (CD14, CD34, and CD45) and the positive expression of primitive cell markers (CD73, CD90, and CD105), which are immunophenotypic markers suggested by the ISCT minimum criteria.

[0114] MSCs express NANOG, OCT4, and SOX2, transcription factors associated with pluripotency that maintain the pluripotency of embryonic tonsil-derived mesenchymal stem cells (ESCs). As shown in Figure 2B, RT-PCR revealed that gene expression of OCT4 and SOX2, but not NANOG, was significantly reduced in aged cells undergoing replicative senescence.

[0115] Meanwhile, the effects of replicative aging on multilineage differentiation potential (adipogenesis, chondrogenesis, and osteogenesis) were investigated. To assess differentiation potential, young and aged cells were assessed for differentiation into mesodermal cells such as adipocytes, chondrocytes, or osteocytes.

[0116] As shown in Figure 2C, the levels of Oil red O, Alcian blue, and Alizarin red S staining were confirmed to be reduced in aged cells compared to young cells. These results indicate that mesoderm differentiation potential is significantly reduced in aged cells.

[0117] Example 2. Evaluation of the relationship between aging of tonsil-derived mesenchymal stem cells and podoplanin (PDPN).

[0118] 1. Evaluation of changes in PDPN expression in TMSCs

[0119] Gene and protein expression of PDPN was confirmed during continuous culture of TMSCs.

[0120] As shown in Figures 3A and 3B, the mRNA level of PDPN remained unchanged up to passage 15 (Figure 3A), whereas the protein level of PDPN decreased in a passage-dependent manner (Figure 3B). In particular, a significant decrease in the PDPN protein level was observed at passage 15 compared to passage 5.

[0121] In addition, as shown in Figure 3C, flow cytometry analysis was used to confirm that the PDPN+ group, one of the transmembrane glycoproteins, was reduced to 86.11% in aged cells (94.43% in the young cell PDPN+ group).

[0122] These results indicate that expression of PDPN in the intracellular and membrane proteins decreases with replicative senescence in TMSCs.

[0123] 2. Characteristics of PDPN+ classified TMSCs

[0124] The impact of sorting PDPN+ TMSCs on MSC characteristics was investigated. To isolate PDPN+ or PDPN- TMSCs, single cells stained with or without fluorescent PDPN were sorted using the FACS procedure, and identified as 93.39% and 2.80%, respectively, as shown in Figure 4A.

[0125] As shown in Figure 4B, no significant change in telomere length was observed between PDPN+ and PDPN- cells.

[0126] Meanwhile, as shown in Figures 4C and 4D, we found that the surface phenotype of tonsil-derived mesenchymal stem cells (Figure 4C) and the pluripotency-related transcription factors NANOG, OCT4, and SOX2 (Figure 4D) were detected at similar expression levels.

[0127] However, as shown in Fig. 4E, the tri-lineage differentiation potential of PDPN+ TMSCs was significantly higher than that of PDPN- TMSCs, as evidenced by increased accumulation of lipid droplets (adipogenesis) stained with Oil Red O solution, glycosaminoglycans (chondrogenesis) stained with Alcian Blue solution, and calcium deposition (osteogenesis) stained with Alizarin Red S solution in PDPN+ TMSCs.

[0128] 3. Evaluation of the effects of PDPN on cell proliferation, cell cycle progression, and cell motility.

[0129] We identified an association between PDPN and aging-related pre-existing mechanisms, such as cell proliferation, cell cycle progression, or cell motility.

[0130] As shown in Figure 5A, PDPN+ cells showed significantly higher proliferation capacity than PDPN- cells at 48 hours.

[0131] Furthermore, to investigate cell cycle arrest, another indicator of proliferative capacity, the signaling pathway of PDPN was investigated using flow cytometry and the expression of cell cycle-related proteins. As shown in Figure 5B, PDPN- TMSCs were confirmed to induce cell cycle arrest in the G0 / G1 phase compared to PDPN+ TMSCs.

[0132] As shown in Figure 5C, further validation revealed that the protein level of cyclin D1, a representative regulatory protein of the G0 / G1 phase, was significantly reduced in PDPN-TMSCs, but the protein level of CDK 4, another marker associated with the G0 / G1 phase, was not.

[0133] Meanwhile, in the scratch recovery assay to evaluate cell migration as shown in Fig. 5D, the scratch recovery levels were 37.28 ± 2.66% and 62.63 ± 12.16% in PDPN- cells and PDPN+ cells, respectively, demonstrating that PDPN+ cells have significantly higher scratch recovery ability than PDPN- cells.

[0134] Meanwhile, the role of PDPN in cell proliferation and cell migration was confirmed by suppressing PDPN expression using siRNA.

[0135] As shown in Figures 6A and 6B, ectopic transfection with PDPN siRNA successfully reduced the expression of both mRNA (Figure 6A) and protein (Figure 6B) of PDPN.

[0136] As shown in Figures 6C and 6D, as expected, siPDPN completely inhibited cell cycle progression in the G0 / G1 phase (Figure 6C), including suppression of Cyclin D1 protein levels (Figure 6D).

[0137] Additionally, as shown in Figure 6E, the degree of scratch recovery was higher in siCON than in siPDPN.

[0138] In summary, the above findings suggest that loss of PDPN leads to downregulation of cell cycle progression as well as proliferation and migration capacity of TMSCs.

Claims

1. A composition for detecting senescence of tonsil-derived mesenchymal stem cells, comprising each antibody or aptamer that specifically binds to podoplanin protein.

2. A composition for detecting cell senescence of tonsil-derived mesenchymal stem cells, wherein the cell senescence in paragraph 1 is characterized by at least one characteristic selected from the group consisting of a decrease in cell proliferation ability, loss of cell division ability, decrease in cell migration, decrease in cell differentiation ability, shortened telomere length, increase in single cell size, and increase in SA-β-gal.

3. A composition for detecting cell senescence of tonsil-derived mesenchymal stem cells, wherein the detection is performed by checking the level of podoplanin protein in cells, cell culture medium, or cell lysate in the first paragraph.

4. A kit for detecting senescence of tonsil-derived mesenchymal stem cells comprising a composition selected from any one of claims 1 to 3.

5. In the fourth paragraph, the kit is a kit for immune assay.

6. In the fifth paragraph, the immunoassay is any one selected from the group consisting of radioimmunoassay, radioimmunoprecipitation, immunoprecipitation, immunohistochemical staining, ELISA (enzyme-linked immunosorbent assay), capture-ELISA, inhibition or competition assay, sandwich assay, flow cytometry, immunofluorescence staining, and immunoaffinity purification. 7.(a) a step of detecting protein expression of podoplanin in tonsil-derived mesenchymal stem cells; and (b) A method for detecting senescence of tonsil-derived mesenchymal stem cells, comprising the step of measuring the expression level of the above-mentioned podoplanin protein.

8. A method for detecting cellular senescence of tonsil-derived mesenchymal stem cells, further comprising the step of determining cellular senescence when the expression of the (c) podoplanin protein is underexpressed compared to primary tonsil-derived mesenchymal stem cells in paragraph 7.

9. A method for detecting senescence of tonsil-derived mesenchymal stem cells, wherein the measurement of protein expression level in paragraph 7 is performed by immunoassay.

10. A method for detecting cellular senescence of tonsil-derived mesenchymal stem cells, wherein the immunoassay is any one selected from the group consisting of radioimmunoassay, radioimmunoprecipitation, immunoprecipitation, immunohistochemical staining, ELISA (enzyme-linked immunosorbent assay), capture-ELISA, inhibition or competition assay, sandwich assay, flow cytometry, immunofluorescence staining, and immunoaffinity purification. 11.(a) a step of contacting a sample to be analyzed with cells in which protein expression of podoplanin is reduced in tonsil-derived mesenchymal stem cells; and (b) a step of measuring the expression level of the above grape planin protein; (c) a step of determining the sample as a senescence inhibitor of tonsil-derived mesenchymal stem cells when the expression of the podoplanin protein is increased; a method for screening for a senescence inhibitor of tonsil-derived mesenchymal stem cells.

12. A method for screening for a senescence inhibitor of tonsil-derived mesenchymal stem cells, wherein the step of measuring the expression level of the protein in claim 11 is performed by immunoassay.

13. A method for screening for a senescence inhibitor of tonsil-derived mesenchymal stem cells, wherein the immunoassay is any one selected from the group consisting of radioimmunoassay, radioimmunoprecipitation, immunoprecipitation, immunohistochemical staining, enzyme-linked immunosorbent assay (ELISA), capture-ELISA, inhibition or competition assay, sandwich assay, flow cytometry, immunofluorescence staining, and immunoaffinity purification.

14. A composition for inhibiting cell senescence of tonsil-derived mesenchymal stem cells containing podoplanin.

15. A method for inhibiting senescence of tonsil-derived mesenchymal stem cells, comprising the step of treating tonsil-derived mesenchymal stem cells with podoplanin and then subculturing them.

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