WO2024162806A1 - Method for preparing aging-inhibited stem cells - Google Patents
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- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
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Definitions
- the present invention relates to a method for producing anti-aging stem cells, and more specifically, to a method for producing anti-aging stem cells, including a step of treating stem cells with a GLS1 inhibitor, and a composition for anti-aging of stem cells containing a GLS1 inhibitor as an active ingredient.
- Mesenchymal stem cells are stem cells with multipotent properties derived from various adult cells such as bone marrow, umbilical cord blood, placenta (or placental tissue cells), and fat (or adipose tissue cells).
- mesenchymal stem cells derived from bone marrow are being studied in various ways for development as cell therapy due to their multipotent properties that can differentiate into adipose tissue, bone/cartilage tissue, and muscle tissue.
- mesenchymal stem cells isolated from the human body so that they are suitable for treatment.
- many patients who are the subjects of cell therapy are elderly, and mesenchymal stem cells extracted from the tissues of the elderly tend to have low proliferative and differentiation abilities, resulting in low therapeutic efficacy. Therefore, a technology to activate mesenchymal stem cells derived from the elderly, like mesenchymal stem cells derived from young people, is demanded.
- Mesenchymal stem cells similar to other human primary cultured cells, are known to rapidly decrease in cell division by a senescence mechanism independent of telomere shortening when cultured in vitro (Shibata, KR et al., Stem cells , 25;2371-2382, 2007). Although the mechanism of this senescence phenomenon has not yet been clearly identified, it is pointed out that the main cause is the accumulation of environmental stress due to long-term in vitro culture, which causes the expression and accumulation of the Cdk inhibitory protein p16 (INK4a), thereby suppressing the activity of the Cdk protein responsible for cell growth.
- Cdk inhibitory protein p16 INK4a
- the inventors of the present invention have made extensive efforts to develop a method for effectively inhibiting the aging of mesenchymal stem cells, and as a result, have confirmed that when treating aged mesenchymal stem cells with a GLS1 inhibitor, mesenchymal stem cells having activity similar to that of young mesenchymal stem cells can be produced, thereby completing the present invention.
- the purpose of the present invention is to provide a method for producing anti-aging stem cells.
- Another object of the present invention is to provide a method for inhibiting aging of stem cells.
- Another object of the present invention is to provide a composition for inhibiting aging of stem cells.
- the present invention provides a method for producing aging-inhibiting stem cells, which comprises a step of treating stem cells with a GLS1 inhibitor.
- the present invention also provides a method for inhibiting aging of stem cells, comprising a step of treating aging stem cells with a GLS1 inhibitor.
- the present invention also provides a composition for inhibiting aging of stem cells containing a GLS1 inhibitor as an active ingredient.
- Figure 1a shows the results of senescence associated beta-galactosidase (SA- ⁇ -gal) staining of young WJ-MSC (senescence-uninduced MSC; U-MSC) and aged WJ-MSC (replicative senescence MSC; rS-MSC) groups.
- SA- ⁇ -gal senescence associated beta-galactosidase
- Figure 1b shows the results of measuring the cell numbers of young WJ-MSCs (control group) and aged WJ-MSCs for 3 days.
- Figure 1c shows the results of confirming the doubling time of young WJ-MSCs (control group) and aged WJ-MSCs.
- Figure 1d shows the results of immunofluorescence staining (ICC) for lamin B1, a nuclear envelope marker, in young and aged WJ-MSCs.
- Figure 2a shows the mRNA expression levels of GLS1 in young WJ-MSCs and aged WJ-MSCs.
- Figure 2b shows the protein expression levels of GLS1 in young WJ-MSCs and aged WJ-MSCs.
- Figure 2c shows the results of confirming the decrease in mRNA expression of GLS1 after siGLS1 transfection into young and aged WJ-MSCs.
- Figure 2d shows the results of confirming cell viability after transfecting young WJ-MSCs and aged WJ-MSCs with siGLS1.
- Figure 2e shows the results of confirming cell viability after BPTES treatment in young WJ-MSCs and aged WJ-MSCs.
- Figure 3a is a schematic diagram of the BPTES treatment and experimental procedures for aged WJ-MSCs.
- Figure 3b shows the results of measuring glutamine levels in the cell culture medium of aged WJ-MSCs and BPTES-treated aged WJ-MSCs.
- Figure 3c shows the results of measuring intracellular glutamine levels in aged WJ-MSCs and BPTES-treated aged WJ-MSCs.
- Figure 3d shows the results of measuring intracellular glutamate levels in aged WJ-MSCs and BPTES-treated aged WJ-MSCs.
- Figure 3e shows the results of measuring intracellular ⁇ -ketoglutarate levels in aged WJ-MSCs and BPTES-treated aged WJ-MSCs.
- Figure 3f shows the results of measuring the cell number for 3 days after BPTES treatment on aged WJ-MSCs.
- Figure 3g shows the results of confirming the expression levels of senescence-related proteins p16, p21, and GLB1 in young WJ-MSCs, aged WJ-MSCs, and BPTES-treated aged WJ-MSCs.
- Figure 3h shows the results of confirming the mRNA expression levels of p16 and p21, which are aging-related genes, in young WJ-MSCs, aged WJ-MSCs, and BPTES-treated aged WJ-MSCs.
- Figure 3i shows the results of confirming the mRNA expression levels of SASP-related genes IGFBP3, IGFBP5, and IGFBP7 in young WJ-MSCs, aged WJ-MSCs, and aged WJ-MSCs treated with BPTES.
- Figure 4 shows the results of confirming the stem cell capacity of young WJ-MSCs and aged WJ-MSCs, respectively, through FACS when treated with BPTES.
- Figure 5a shows the results of confirming relative cell viability after siGLS1 transfection into aged WJ-MSCs.
- Figure 5b shows the results of confirming the mRNA expression levels of p16 and p21, which are senescence-related genes, after siGLS1 transfection into senescent WJ-MSCs.
- Figure 5c shows the results of confirming the protein expression levels of GLS1 and the senescence-related proteins p16, p21, and GLB1 after siGLS1 transfection into senescent WJ-MSCs.
- Figure 5d shows the results of confirming the mRNA expression levels of SASP-related genes IGFBP3, IGFBP5, and IGFBP7 after transfection of siGLS1 into aged WJ-MSCs.
- Figure 6a shows the results of examining cell viability for 3 days after treating aged WJ-MSCs with CB-838 and C968, respectively.
- Figure 6b shows the results of confirming the decrease in the expression of aging-related genes, p16 and p21, after treating aging WJ-MSCs with CB-838 and C968, respectively.
- Figure 6c shows the results of confirming the decrease in the expression of aging-related proteins p16, p21, and GLB1 after treating aging WJ-MSCs with CB-838 and C968, respectively.
- Figure 6d shows the results of confirming a decrease in the expression of SASP-related genes IGFBP3, IGFBP5, and IGFBP7 after treating aged WJ-MSCs with CB-838 and C968, respectively.
- Figure 7a is a schematic diagram of the experimental design for a strategy to inhibit senescence in mesenchymal stem cells.
- Figure 7b shows the results of SA- ⁇ -gal staining to evaluate the degree of senescence of young WJ-MSCs, senescent WJ-MSCs, and replicative senescence-alleviated MSCs (rSA-MSCs).
- Figure 7c shows the results of measuring the cell area of young WJ-MSCs, aged WJ-MSCs, and senescence-inhibited WJ-MSCs.
- Figure 7d shows the results of confirming the doubling time of young WJ-MSCs, aged WJ-MSCs, and aged-inhibited WJ-MSCs.
- Figure 7e shows the results of SA- ⁇ -gal staining to evaluate the degree of senescence in recovered senescent WJ-MSCs after treatment with GLS1 inhibitors CB-839 and C968.
- Figure 8a shows the results of confirming the protein expression levels of apoptosis markers, cleaved PARP and cleaved caspase 3, in muscle cells after co-culturing apoptosis-induced muscle cells with young WJ-MSCs, aged WJ-MSCs, and aged-inhibited WJ-MSCs.
- Figure 8b shows the results of Annexin V immunofluorescence staining to evaluate the therapeutic efficacy of WJ-MSCs against apoptosis.
- Figure 9a shows the results of a behavioral assessment of grip strength after administering young WJ-MSCs, aged WJ-MSCs, and aged-inhibited WJ-MSCs to DMD disease model mice (MDX).
- Figure 9b shows the results of measuring Creatine kinase (CK) activity in the control group and the experimental groups administered young WJ-MSCs, aged WJ-MSCs, and aged-inhibited WJ-MSCs.
- CK Creatine kinase
- Figure 9c shows the results of confirming the expression levels of annexin V, MHC, and Fibronectin proteins in the control group and the experimental groups administered young WJ-MSCs, aged WJ-MSCs, and aged-inhibited WJ-MSCs.
- Figure 9d shows the results of numerically quantifying the protein expression level for annexin V in Figure 9c.
- Figure 9e shows the results of numerically quantifying the protein expression level for MHC in Figure 9c.
- Figure 9f shows the results of numerically quantifying the protein expression level for fibronectin in Figure 9c.
- Figure 9g shows the results of immunofluorescence staining and sirius red staining for annexin V and MHC in the control group and the experimental groups administered young WJ-MSCs, aged WJ-MSCs, and aged-inhibited WJ-MSCs.
- Figure 9h shows the result of numerically quantifying the immunofluorescence color for MHC in Figure 9g.
- Figure 9i shows the numerical results of immunofluorescence staining for annexin V in Figure 9g.
- Figure 9j shows the numerical results of the sirius red staining results of Figure 9g.
- Figure 10a is a Venn diagram showing the differentially expressed genes (DEGs) with FC > 3 for young WJ-MSCs, aged WJ-MSCs, and BPTES-treated aged WJ-MSCs.
- DEGs differentially expressed genes
- Figure 10b shows the results of a heat map showing DEGs with FC > 3 for young WJ-MSCs, aged WJ-MSCs, and BPTES-treated aged WJ-MSCs.
- Figure 10c shows the results of gene categories related to DEGs with FC > 3 in BPTES-treated aged WJ-MSCs compared to aged WJ-MSCs.
- Figure 10d shows the results of confirming the expression levels of CTSC, E2F7, and SORBS2 mRNA in young WJ-MSCs and aged WJ-MSCs in 3 lots.
- Figure 10e shows the normalized log2 values of mRNA sequence analysis for CTSC, E2F7, and SORBS2 in young WJ-MSCs, aged WJ-MSCs, and BPTES-treated aged WJ-MSCs.
- Figure 10f shows the results of confirming the mRNA expression levels of CTSC, E2F7, and SORBS2 in young WJ-MSCs, aged WJ-MSCs, and BPTES-treated aged WJ-MSCs.
- Figure 10g shows the results of confirming the expression levels of CTSC, E2F7, and SORBS2 mRNA after siGLS1 transfection into aged WJ-MSCs.
- Figure 11a is a heat map showing DEGs with FC > 1.5 and p ⁇ 0.05 in three lots of young WJ-MSCs and aged WJ-MSCs.
- Figure 11b shows the top 10 categories based on p-values through DAVID analysis using the GO database.
- Figure 11c shows the top 10 categories based on p-values through DAVID analysis using the KEGG database.
- Figure 11d shows the analysis results based on gene set enrichment analysis (GSEA) showing that the replicative senescence of WJ-MSCs is associated with “cell cycle DNA replication” and “cell-to-cell signaling by Wnt.”
- GSEA gene set enrichment analysis
- Figure 11e shows the results of confirming the expression levels of p21, a aging-related protein, and ⁇ -catenin, a Wnt signaling pathway-related protein, in three lots of young WJ-MSCs and aged WJ-MSCs.
- Figure 11f shows the results of confirming the protein expression of GLS1 and the protein expression levels of ⁇ -catenin and phosphorylated GSK3 ⁇ (ser9), which are proteins related to the Wnt signaling pathway, in young WJ-MSCs, aged WJ-MSCs, and aged-inhibited WJ-MSCs.
- Figure 11g shows the results of confirming the protein expression levels of GLS1 protein and ⁇ -catenin and phosphorylated GSK3 ⁇ (ser9), proteins related to the Wnt signaling pathway, after transfection of siGLS1 into aged WJ-MSCs.
- Figure 11h shows the results of confirming the protein expression levels of ⁇ -catenin and phosphorylated GSK3 ⁇ (ser9), proteins related to the Wnt signaling pathway, after treatment of aged WJ-MSCs with CB-839 and C968.
- Figure 12a shows the results of the analysis showing that “positive regulation of autophagy” increases due to aging in WJ-MSCs based on gene set enrichment analysis (GSEA).
- GSEA gene set enrichment analysis
- Figure 12b shows the results of confirming the protein expression level of the autophagy-related protein LC3II in young WJ-MSCs, aged WJ-MSCs, and senescence-inhibited WJ-MSCs.
- Figure 12c shows the results of confirming the protein expression levels of GLS1 protein and autophagy-related protein LC3II after transfection of siGLS1 into aged WJ-MSCs.
- Figure 12d shows the results of confirming the protein expression level of LC3II, a protein related to autophagy, after treatment of aged WJ-MSCs with CB-839 and C968.
- ⁇ -galactosidase positive cells increased in aged mesenchymal stem cells compared to young-derived mesenchymal stem cells isolated from umbilical cord blood, and that mRNA expression of GLS1 in aged WJ-MSCs was significantly increased compared to young WJ-MSCs, but that mRNA expression of GLS1 was significantly decreased when treated with a GLS1 inhibitor, and that the doubling time of aged mesenchymal stem cells was also shortened by treatment with a GLS1 inhibitor.
- the present invention relates, in one aspect, to a method for producing anti-aging stem cells, comprising a step of treating stem cells with a GLS1 inhibitor.
- the GLS1 inhibitor may be BPTES (bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl)ethyl sulfide), C968, CB-839 (Telaglenastat), DON (6-Diazo-5-oxo-L-norleucine), etc., and preferably, BPTES, CB-839 or C968 may be used, but is not limited thereto.
- BPTES bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl)ethyl sulfide
- C968, CB-839 Telaglenastat
- DON 6-Diazo-5-oxo-L-norleucine
- senescent stem cells refer to cells that are passage 15 or more and have 25% or more positive cells as a result of beta-galactosidase staining.
- rS-MSC senescent stem cells
- the p16-pRB pathway and the p53-p21 pathway related to cell cycle arrest are activated, and the expression of proteins such as p16, p21, and p53 increases.
- proteins such as p16, p21, and p53 increases.
- abnormal structural changes occur, and the cell size increases and the morphology becomes longer and wider.
- SA senescence-associated beta-galactosidase activity
- SASP senescence associated secretion phenotype
- young stem cells refer to cells that are less than passage 6 and have less than 1% positive cells in the beta-galactosidase staining result. Unlike senescent stem cells, young stem cells have inactivated p16-pRB pathway and p53-p21 pathway related to cell cycle arrest, and thus have low expression of proteins such as p16, p21, and p53. In addition, they have a smaller cell size than senescent cells. Young stem cells do not show the phenomenon of increased beta-galactosidase expression or increased SASP-related protein expression, which are characteristics of aging.
- WJ-MSC refers to mesenchymal stem cells derived from Wharton's jelly of umbilical cord blood.
- the level of aging-related mRNA expression which was increased in aged WJ-MSCs compared to young WJ-MSCs, was significantly reduced when treated with BPTES, a GLS1 inhibitor, and that the expression of SASP-related genes was increased in aged WJ-MSCs compared to young WJ-MSCs and that the mRNA level was recovered when treated with BPTES.
- the doubling time which was 24.97 hours in young WJ-MSCs, increased to 56.67 hours in aged WJ-MSCs. It was confirmed that the doubling time was faster in aged WJ-MSCs recovered after BPTES treatment, at 42.86 hours, compared to aged WJ-MSCs.
- BPTES treatment at 42.86 hours
- the present invention relates to a method for inhibiting aging of stem cells, comprising a step of treating aging WJ-MSCs with a GLS1 inhibitor.
- the GLS1 inhibitor may be BPTES (bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl)ethyl sulfide), C968, CB-839 (Telaglenastat), DON (6-Diazo-5-oxo-L-norleucine), etc., and preferably, BPTES, CB-839 or C968 may be used, but is not limited thereto.
- BPTES bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl)ethyl sulfide
- C968, CB-839 Telaglenastat
- DON 6-Diazo-5-oxo-L-norleucine
- the gene expression pattern of cells in which aging was recovered by BPTES treatment was similar to that of young WJ-MSCs.
- genes whose expression was different during aging were confirmed, and among them, CTSC, E2F7, and SORBS2 are genes known to be associated with aging, and it was confirmed that other aging-related genes, in addition to GLS1, were affected in expression due to the recovery of aging.
- FACS was performed on young WJ-MSCs, aged WJ-MSCs, and aged WJ-MSCs treated with BPTES to determine their stem cell potential.
- the markers of mesenchymal stem cells were identified through the expression of CD44, CD73, CD90, CD105, and CD166, and the markers of hematopoietic stem cells were CD14, CD11b, HLA-DR (MHCII), CD34, CD45, and CD19 (BD Biosciences, USA) to compare their stem cell potential.
- MHCII HLA-DR
- CD34 CD45
- CD19 BD Biosciences, USA
- the present invention relates to an anti-aging composition of stem cells containing a GLS1 inhibitor as an active ingredient.
- muscle cells in which apoptosis was induced were co-cultured with young WJ-MSCs, senescent WJ-MSCs, and senescence-inhibited WJ-MSCs, and it was confirmed that the expression of cleaved PAPR and cleaved capase 3 was significantly reduced in muscle cells co-cultured with young WJ-MSCs and senescence-inhibited WJ-MSCs.
- a mouse animal experiment was conducted to explore the therapeutic efficacy of anti-aging WJ-MSCs on muscular dystrophy (DMD) disease.
- DMD muscular dystrophy
- CK activity decreased in the experimental group administered young WJ-MSCs, and when aged WJ-MSCs were administered, a similar trend was observed as in the control group.
- WJ-MSCs that inhibited aging through BPTES treatment
- CK activity was confirmed to decrease compared to the experimental group administered aged WJ-MSCs.
- WJ-MSCs treated with BPTES to inhibit aging had an enhanced therapeutic effect similar to that of young WJ-MSCs.
- the present invention confirmed that replicative senescence can be suppressed by activating the Wnt signaling pathway through inhibition of GLS1 in aged WJ-MSCs, although the Wnt signaling pathway is inactivated due to aging, and that autophagy is related to replicative senescence of WJ-MSCs and can be regulated by GLS1.
- Example 1 Establishment of a replicative aging model of human mesenchymal stem cells
- Wharton’s Jelly-mesenchymal stem cells were isolated from the umbilical cord and placenta through a collaborative study with the Department of Obstetrics and Gynecology, Samsung Medical Center, in accordance with the standards of the IRB (IRB# 2016-07-102-043) approved by Samsung Medical Center.
- Umbilical cord-derived mesenchymal stem cells were isolated from the umbilical cord according to the method of PARK et al. (Park et al., Arch. Pharm. Res. 39: 1171-1179, 2016).
- the umbilical cord was cut into 3-4 cm lengths, the tissue was finely chopped, and treated with collagenase solution (Gibco, USA) for 60-90 minutes to decompose the extracellular matrix, and then 0.25% trypsin (Gibco, USA) was added to induce further digestion for 30 minutes at 37°C. Afterwards, fetal bovine serum (FBS; Biowest, USA) was added, and the cells were obtained by centrifugation at 1000 g for 10 minutes.
- collagenase solution Gibco, USA
- trypsin Gibco, USA
- FBS fetal bovine serum
- WJ-MSC cells were cultured in a 5% CO2 environment at 37°C using MEM Alpha (Minimum Essential Medium, Invitrogen-Gibco, Rockville, MD) medium containing 10% FBS (fetal bovine serum, Invitrogen-Gibco) and 50 ⁇ g/mL gentamycin (Invitrogen-Gibco) for use in subsequent experiments.
- MEM Alpha Minimum Essential Medium, Invitrogen-Gibco, Rockville, MD
- FBS fetal bovine serum, Invitrogen-Gibco
- gentamycin Invitrogen-Gibco
- the isolated WJ-MSCs were seeded at a density of 3 x 10 3 cells/cm 2 in MEM Alpha (Minimum Essential Medium, Invitrogen-Gibco, Rockville, MD) medium containing 10% FBS (fetal bovine serum, Invitrogen-Gibco) and 50 ⁇ g/mL gentamycin (Invitrogen-Gibco) and serially passaged at 37°C and 5% CO 2 .
- MEM Alpha Minimum Essential Medium, Invitrogen-Gibco, Rockville, MD
- FBS fetal bovine serum
- Invitrogen-Gibco 50 ⁇ g/mL gentamycin
- WJ-MSC refers to human mesenchymal stem cells derived from Wharton's jelly of umbilical cord blood.
- Young WJ-MSC used passage 5 senescence-uninduced MSC (U-MSC)
- senescent WJ-MSC used replicative senescent mesenchymal stem cells (rS-MSC) that were serially subcultured for passage 18 or more of "young WJ-MSC”.
- U-MSC senescence-uninduced MSC
- rS-MSC replicative senescent mesenchymal stem cells
- SA- ⁇ -gal staining was performed on the young and aged WJ-MSC groups. Specifically, young WJ-MSCs and aged WJ-MSCs were cultured in 6-well plates using MEM Alpha (Minimum Essential Medium, Invitrogen-Gibco, Rockville, MD) medium containing 10% FBS until 70-80% confluent, and then washed twice with PBS. Afterwards, they were fixed for 5 minutes at room temperature with 1X fixation solution. After washing twice with PBS, they were incubated for 16 hours at 37°C with SA- ⁇ -gal staining solution (cell signaling) adjusted to pH 6. The staining images were confirmed through an optical microscope, and ⁇ -galactosidase-positive cells were quantitatively analyzed using Image J.
- ⁇ -galactosidase-positive cells in aged WJ-MSCs significantly increased to 38.48% compared to young WJ-MSCs.
- WJ-MSCs To determine the cell proliferation rate of young and aged WJ-MSCs, cell numbers were measured for 3 days. Specifically, WJ-MSCs were seeded at 2 ⁇ 103 cells/well in 6-well plates using MEM Alpha (Minimum Essential Medium, Invitrogen-Gibco, Rockville, MD) medium containing 10% FBS, and the cell number was counted using a hemocytometer at 0, 24, 48, and 72 hours after seeding, and the doubling time was calculated using this.
- MEM Alpha Minimum Essential Medium, Invitrogen-Gibco, Rockville, MD
- young WJ-MSCs showed a significantly faster cell proliferation rate than aged WJ-MSCs.
- the doubling time of young WJ-MSCs was 27.02 hours, whereas that of aged WJ-MSCs was significantly increased to 47.93 hours.
- Immunofluorescence staining was performed on lamin B1, a nuclear protein whose expression is characterized by a decrease with aging in young and aged WJ-MSCs. Specifically, mesenchymal stem cells were fixed with 4% PFA for 5 minutes at room temperature, and then permeabilized with 0.25% Trition X-100 in PBS for 5 minutes at room temperature. Then, the cells were blocked with blocking solution (2% BSA, 0.1% Tween 20 in PBS) for 1 hour at room temperature, and the primary antibody was diluted in the blocking solution and reacted overnight at 4°C. The results were confirmed using a confocal microscope Zeiss LSM700, and fluorescence quantitative analysis was performed using Image J.
- mesenchymal stem cells After culturing mesenchymal stem cells, they were washed with PBS and lysed with RIPA buffer (BIOSESANG, Sungnam, Gyeonggi, Korea) containing protease inhibitor cocktail (Amresco, Solon, OH, USA), centrifuged at 4°C, 15,000g for 30 minutes, and the supernatant was obtained. 10 ⁇ g of protein was electrophoresed to separate by size using SDS-PAGE and transferred to a PVDF (polyvinylidene difluoride) membrane. The membrane was blocked with TBST containing 5% skim milk for 1 hour at room temperature, and the primary antibody was diluted in TBST containing 5% skim milk and reacted overnight at 4°C.
- RIPA buffer BIOOSESANG, Sungnam, Gyeonggi, Korea
- Amresco Amresco, Solon, OH, USA
- the membrane was washed three times for 10 minutes with TBST, and the secondary antibody was diluted in TBST containing 5% skim milk and reacted for 1 hour at room temperature. Afterwards, the membrane was washed three times with TBST for 10 minutes each, treated with ECL solution (Advansta, USA), and the band image was confirmed in a gel imaging system (Amersham Imager 600, GE Healthcare, Buckinghamshire, UK). The protein expression level was measured using Image J and corrected with ⁇ -actin. The primary antibody used was GLS1, ⁇ -actin (Santa Cruz Biotechnology, Dallas, TX, USA).
- siGLS1 was transfected into WJ-MSCs, RNA was extracted using the AccuPrep Universal RNA Extraction Kit, and qRT-PCR was performed using 2X Power SYBR Green Master Mix (AB) (see Table 1).
- mesenchymal stem cells cultured at about 50% confluence in growth medium were transferred to serum-free medium, and two candidate sequences (bioneer) (Table 2) for GLS1 from the siRNA library were transfected at a concentration of 25 nM using lipofectamine RNAiMax (Invitrogen). 72 hours after transfection, qRT-PCR was performed using the same method as in Example 2.
- a CCK8 assay was performed to confirm cell viability. Specifically, mesenchymal stem cells were treated with DMSO (control) or 30 ⁇ M BPTES, a solvent for BPTES, for 72 hours, and the CCK8 assay was performed using the same method as in Example 1.
- Glutaminolysis is a metabolic process in which glutamine is broken down into glutamate and then into TCA cycle metabolites such as ⁇ -ketoglutarate. Since GLS1 is an enzyme that catalyzes the process of converting glutamine to glutamate, when BPTES, which inhibits it, was treated, changes in the products of the glutaminolysis process were confirmed.
- Glutamine assay was performed to determine the level of glutamine in the cell culture medium and cells. Specifically, aged WJ-MSCs were treated with DMSO or BPTES 30 ⁇ M for 24 hours, and then the cell culture medium and cells were harvested. Glutamine in the cell culture medium was measured using a glutamine assay kit (Abnova), and glutamine in the cells was measured using a glutamine assay kit (dojindo).
- a glutamate assay was performed. Specifically, aged WJ-MSCs were treated with 30 ⁇ M DMSO or BPTES for 24 h, and then the cells were harvested. Intracellular glutamate was measured using a glutamate assay kit (dojindo).
- ⁇ -ketoglutarate assay was performed. Specifically, aged WJ-MSCs were treated with 30 ⁇ M DMSO or BPTES for 24 h, and then the cells were harvested. Intracellular ⁇ -ketoglutarate was measured using an ⁇ -ketoglutarate assay kit (dojindo).
- Figure 3g shows the results of Western blot performed on p16 and p21 to evaluate the level of protein expression of aging-related genes. It was confirmed that the level of aging-related protein expression, which was increased in aged WJ-MSCs compared to young WJ-MSCs, was significantly reduced when treated with BPTES.
- qRT-PCR was performed on p16 and p21 using the same method as in Example 2.
- SASP senescence associated secretion phenotype
- the mRNA levels of SASP-related genes were confirmed through qRT-PCR for IGFBP3, IGFBP5, and IGFBP7 using the same method as in Example 2.
- GLS1 plays an important role in regulating replicative senescence of WJ-MSCs.
- markers of mesenchymal stem cells were identified through the expression of CD44, CD73, CD90, CD105, and CD166, and markers of hematopoietic stem cell lineage were used to compare stem cell potential using CD14, CD11b, HLA-DR (MHCII), CD34, CD45, and CD19 (BD Biosciences, USA). At this time, 10,000 events were acquired and analyzed using a BD FACS Verse flow cytometer.
- young WJ-MSCs expressed positive markers CD44, CD73, CD90, CD105, and CD166 at more than 99%, but negative markers CD14, CD11b, HLA-DR (MHCII), CD34, CD45, and CD19 at less than 1.5%.
- MHCII HLA-DR
- CD34 CD45
- CD19 CD19 at less than 1.5%.
- the expression of CD90 and CD166 in aged WJ-MSCs decreased compared to young WJ-MSCs, but increased again to more than 93% when treated with BPTES.
- Example 5 Confirmation of inhibition of aging after GLS1 knockdown in a human mesenchymal stem cell aging model
- siGLS1 was transfected into senescent WJ-MSCs using the same method as in Example 2, and then qRT-PCR was performed.
- qRT-PCR was performed to determine the mRNA levels of SASP-related genes IGFBP3, IGFBP5, and IGFBP7 using the same method as in Example 2.
- IGFBP5 was significantly reduced when treated with CB-839 and C968, and IGFBP3 and IGFBP7 showed a tendency to decrease.
- Example 7 Obtaining anti-senescence cells with enhanced cell proliferation by utilizing selective senescent cell death (senolysis) induced by BPTES in aged WJ-MSCs
- Figure 7 shows the results of evaluating the in vitro therapeutic efficacy of anti-aging and anti-aging cells after recovery of aged WJ-MSCs treated with a GLS1 inhibitor.
- ⁇ -galactosidase positive cells which increased to 26.33% in aged WJ-MSCs, decreased to 5.82% in anti-aging WJ-MSCs, confirming that aging was alleviated.
- Figure 7d shows the results of a CCK8 assay performed using the same method as Example 1 to confirm the doubling time of senescent WJ-MSCs and senescence-inhibited WJ-MSCs.
- Figure 7e shows that after treating aged WJ-MSCs with 1 ⁇ M CB-839 or 10 ⁇ M C968, a GLS1 inhibitor, for 24 hours, ⁇ -gal staining was performed using the same method as in Example 1 to evaluate the degree of senescence.
- Figure 8 shows the results of confirming the anti-apoptotic effect of anti-aging WJ-MSC on muscle cells. Specifically, the anti-apoptotic effect was confirmed by co-culturing WJ-MSC with muscle cells that had undergone apoptosis. After induced apoptosis in muscle cells through serum starvation, co-culturing was performed with young WJ-MSC, aged WJ-MSC, and anti-aging WJ-MSC for 24 hours.
- Figure 8a shows the results of western blot performed in the same manner as Example 1 to confirm the protein expression levels of cleaved PARP and cleaved caspase 3, which are apoptosis markers, in co-cultured apoptotic muscle cells with young WJ-MSCs, senescent WJ-MSCs, and senescence-inhibited WJ-MSCs.
- the primary antibodies used were cleaved PARP, cleaved caspase 3 (Cell signaling), and ⁇ -actin (Santa Cruz Biotechnology).
- cleaved PAPR and cleaved capase 3 increased in the control group, which was muscle cells in which apoptosis was induced by serum-free medium (SF media), compared to normal muscle cells.
- SF media serum-free medium
- the expression of these markers did not differ in the muscle cell group co-cultured with aged WJ-MSCs compared to the control group, but it was confirmed that it significantly decreased in the muscle cells co-cultured with young WJ-MSCs and senescence-inhibited WJ-MSCs.
- Figure 8b shows the results of immunofluorescence staining for Annexin V performed in the same manner as Example 1 to evaluate the therapeutic efficacy of WJ-MSCs on apoptosis.
- the proportion of annexin V-positive cells significantly increased in the control group (myocytes in which apoptosis was induced) compared to the normal muscle cell group, but significantly decreased in all muscle cell groups co-cultured with WJ-MSCs.
- the proportion of annexin V-positive cells significantly decreased in muscle cells co-cultured with young WJ-MSCs and senescence-inhibited WJ-MSCs compared to muscle cells co-cultured with aged WJ-MSCs.
- Example 9 Enhanced efficacy of anti-aging WJ-MSCs on skeletal muscle recovery in mdx mice
- mice To explore the therapeutic efficacy of anti-aging WJ-MSCs on muscular dystrophy (DMD), mouse animal experiments were conducted. Specifically, young WJ-MSCs, aged WJ-MSCs, and anti-aging WJ-MSCs were administered to DMD disease model mice (MDX) through the tail vein, and the mice were observed for one week before being sacrificed and muscle tissues were observed.
- MDX DMD disease model mice
- the grip strength behavioral evaluation of the control group and the experimental group administered 4 x 104 cells of young WJ-MSCs, aged WJ-MSCs, and aged-inhibited WJ-MSCs was confirmed. Specifically, the grip strength of the forepaws and hind paws was measured and analyzed before and after mesenchymal stem cell administration using a grip strength meter (BIO GS3), and the results were corrected by the body weight of the experimental animals.
- BIO GS3 grip strength meter
- the creatine kinase (CK) activity of the control group and the experimental groups administered young WJ-MSCs, aged WJ-MSCs, and aged-inhibited WJ-MSCs was measured. Specifically, blood was collected before and after mesenchymal stem cell administration through orbital blood collection, and the serum was separated to measure creatine kinase (CK) activity using CPK-PIII (Fujifilm).
- CK activity significantly decreased in the young WJ-MSC administration group and the anti-aging WJ-MSC administration group compared to the MDX control group, but there was no statistical difference in the aged WJ-MSC administration group.
- Figures 9c and 9d show the proportion of mature myofibers according to the expression level of MHC proteins in the control group and the experimental group administered with young WJ-MSCs, aged WJ-MSCs, aged-inhibited WJ-MSCs, and 4 x 104 cells.
- Figures 9c and 9e show the results of confirming the difference in inhibitory efficacy on apoptosis of muscle tissue according to the amount of annexinV protein expression in the control group and the experimental group administered young WJ-MSCs, aged WJ-MSCs, aged-inhibited WJ-MSCs, and 4 x 104 cells.
- annexin V expression in the skeletal muscles of the control group significantly increased compared to the normal group (WT).
- the young WJ-MSC and aging-inhibited WJ-MSC administration groups showed a significant decrease in annexin V expression compared to the control group, but no significance was observed in the aging WJ-MSC administration group.
- aging-inhibited WJ-MSCs have a greater effect on inhibiting apoptosis of damaged muscle cells than aging WJ-MSCs, and thus, the mesenchymal stem cells can be used as a therapeutic agent for muscle diseases.
- Figures 9c and 9f show the results of confirming the difference in muscle tissue fibrosis inhibition efficacy according to the amount of fibronectin protein expression in the control group and the experimental group administered young WJ-MSCs, aged WJ-MSCs, aged-inhibited WJ-MSCs, and 4 x 104 cells.
- the young WJ-MSC and anti-aging WJ-MSC administration groups showed a significant decrease in fibronectin expression compared to the control group, but there was no difference in the anti-aging WJ-MSC administration group.
- anti-aging WJ-MSC can effectively suppress fibrosis of damaged muscle cells by reducing the expression of fibronectin.
- Figure 9g shows the results of immunofluorescence staining (IHC) performed on calf muscle tissue of the control group and the experimental group administered 4 x 104 cells of young WJ-MSCs, aged WJ-MSCs, and aged-inhibited WJ-MSCs.
- IHC immunofluorescence staining
- muscle tissues were fixed with 4% PFA at room temperature and then made into paraffin blocks. Paraffin blocks were sectioned to 4 ⁇ m thickness. Tissue slides were blocked with blocking solution (2% BSA, 0.1% Tween20 in PBS) at room temperature for 1 hour, and primary antibodies were diluted in the blocking solution and reacted overnight at 4°C. The results were confirmed using a fluorescence microscope, and the results were subjected to fluorescence quantitative analysis using Image J. MHC (R&D) and AnnexinV (Abcam) were used as primary antibodies.
- blocking solution 2% BSA, 0.1% Tween20 in PBS
- the young WJ-MSC administration group and the aging-inhibited WJ-MSC administration group showed a significant increase in MHC protein expression levels, but there was no difference in the aged WJ-MSC administration group.
- the young WJ-MSC administration group and the aging-inhibited WJ-MSC administration group showed a significant increase in MHC protein expression levels compared to the aged WJ-MSC administration group.
- Figures 9g and 9i show the results of immunofluorescence staining for AnnexinV in the control group and the experimental group administered 4 x 104 cells of young WJ-MSCs, senescent WJ-MSCs, and senescence-inhibited WJ-MSCs.
- Annexin V expression in the skeletal muscles of the control group significantly increased compared to the normal group (WT).
- Annexin V expression in the young WJ-MSC and aging-inhibited WJ-MSC administration groups significantly decreased compared to the control group, but no difference was observed in the aging WJ-MSC administration group.
- Figures 9g and 9j show the results of sirius red staining to confirm the fibrosis inhibition effect of muscle tissue of the control group and the experimental group administered 4 x 104 cells of young WJ-MSCs, aged WJ-MSCs, aged-inhibited WJ-MSCs, and collagen accumulation.
- the calf muscle tissue of the MDX mouse model was washed with PBS and then reacted with picro-sirius red (solution A) at room temperature for 1 hour. Afterwards, it was washed twice with acidified water (solution B), mounted, and images were obtained using Scanscope. The captured images were quantitatively analyzed using Image J software to obtain relative values to the MDX control group.
- the collagen fibrosis accumulation significantly increased in the control group compared to the normal group.
- the WJ-MSC administration group showed a significant decrease in collagen accumulation compared to the control group.
- the young WJ-MSC and aging-suppressed WJ-MSC administration groups showed a significant decrease in collagen fibrosis accumulation compared to the aging WJ-MSC administration group.
- Example 10 Restoration of expression of cathepsin C (CTSC), E2F transcription factor 7 (E2F7), and sorbin SH3 domain containing 2 (SORBS2) through GLS1 inhibition in aged WJ-MSCs
- CSC cathepsin C
- E2F7 E2F transcription factor 7
- SORBS2 sorbin SH3 domain containing 2
- RNA sequencing RNA samples with an RNA Integrity Number (RIN) value of 7.0 or higher and a 28S:18S ratio of 1 or higher were used, and mRNA libraries were constructed using QuantSeq 3' mRNA-Seq Library Prep Kit (eBiogen) according to the manufacturer's instructions. Differentially expressed genes (DEGs) analysis was performed using ExDEGA2.0.
- Figure 10a shows the results of constructing a Venn Diagram of genes with FC differences of 3 or more in young WJ-MSCs and BPTES-treated aged WJ-MSCs compared to aged WJ-MSCs. There were 25 genes that were commonly up-regulated and 58 genes that were commonly down-regulated in young WJ-MSCs and BPTES-treated aged WJ-MSCs compared to aged WJ-MSCs.
- Figure 10b is a heat map showing 83 DEG genes that were commonly changed in young WJ-MSCs, aged WJ-MSCs, and BPTES-treated aged WJ-MSCs.
- Figure 10c shows the result of creating the GO categories of the above genes.
- Up means a gene that is highly expressed in BPTES-treated senescent WJ-MSCs compared to senescent WJ-MSCs
- down means a gene that is lowly expressed in BPTES-treated senescent WJ-MSCs compared to senescent WJ-MSCs.
- genes related to cell cycle and DNA repair were found to be upregulated more, while genes related to aging, extracellular matrix (ECM), secretion, inflammatory response, and immune response were found to be downregulated more.
- ECM extracellular matrix
- CTSC, E2F7, and SORBS2 are genes known to be associated with aging and gene expression, but no study has been conducted on their association with aging in WJ-MSCs. Therefore, we verified whether these genes showed differences in expression according to aging in WJ-MSCs in 3 lots.
- Gene expression of CTSC, E2F7, and SORBS2 in young and aged WJ-MSCs from 3 donors was performed using the same method as in Example 2.
- the mRNA levels of CTSC and E2F7 were significantly decreased, and the mRNA level of SORBS2 was significantly increased in aged WJ-MSCs compared to young WJ-MSCs of WJ-MSC 3 lots.
- Figure 10e is a graph showing the expression of CTSC, E2F7, and SORBS2 confirmed by mRNA-seq as normalized log2.
- CTSC and E2F7 were significantly decreased in aged WJ-MSCs compared to young WJ-MSCs, while SORBS2 was significantly increased.
- aged WJ-MSCs treated with BPTES recovered to the same pattern as young WJ-MSCs.
- qRT-PCR was performed using the same method as in Example 2.
- the mRNA expression of CTSC and E2F7 decreased in the aged WJ-MSC control group compared to young WJ-MSCs, and significantly increased when BPTES was treated in aged WJ-MSCs.
- the mRNA expression of SORBS2 increased in the aged WJ-MSC control group compared to young WJ-MSCs, and significantly decreased when BPTES was treated in aged WJ-MSCs. This result is consistent with the trend of Fig. 10e above.
- the GLS1 gene was knocked down by transfecting siGLS1 into aged WJ-MSCs using the same method as in Example 2, and the gene expression levels for CTSC, E2F7, and SORBS2 were confirmed through qRT-PCR.
- the expression levels of CTSC and E2F7 in the siGLS1 treatment group were statistically significantly increased compared to the siNC treatment control group in aged WJ-MSCs, while the expression level of SORBS2 was significantly decreased.
- the expression of CTSC, E2F7, and SORBS2 genes was affected by the inhibition of GLS1 in aged WJ-MSCs.
- Example 11 Association between inhibition of senescence through GLS1 inhibition and WNT signaling pathway in replicative senescent mesenchymal stem cells
- Example 11 presents the results of mRNA-seq analysis to identify genetic changes due to replicative aging in the transcriptomes of young and aged WJ-MSCs from 3 lots.
- 320 genes were up-regulated and 225 genes were down-regulated in young WJ-MSCs compared to aged WJ-MSCs.
- Figure 11b shows the top 10 categories based on p-value using the GO database
- Figure 11c shows the top 10 categories based on p-value using the KEGG database (p ⁇ 0.05).
- the GO category “Wnt signaling pathway, planar cell polarity pathway” in the biological process was confirmed to be up-regulated in young WJ-MSCs compared to aged WJ-MSCs.
- GSEA Gene set enrichment analysis
- p21 a senescence-related protein, and ⁇ -catenin, a Wnt signaling pathway-related protein, in young and aged WJ-MSCs from lot 3 was confirmed through western blot using the same method as in Example 2.
- Wnt signaling-related proteins in aging-inhibited WJ-MSCs was confirmed via Western blot using the same method as in Example 2.
- the primary antibodies used were GLS1 (abcam), ⁇ -catenin, p-GSK3 ⁇ , GSK3 ⁇ (cell signaling), and ⁇ -tubulin (sigma).
- Wnt signaling-related proteins were examined by Western blot using the same method as in Example 2.
- the primary antibodies used were GLS1 (abcam), ⁇ -catenin, p-GSK3 ⁇ , GSK3 ⁇ (cell signaling), and ⁇ -tubulin (sigma).
- Example 12 Relationship between autophagy and inhibition of aging through GLS1 inhibition in replicative senescent mesenchymal stem cells
- GSEA Gene set enrichment analysis
- the enrichment score in the “positive regulation of autophagy” gene set was higher in aged WJ-MSCs than in young WJ-MSCs.
- LC3II a protein related to autophagy
- LC3II cell signaling
- ⁇ -tubulin a protein related to autophagy
- aging can be suppressed so that aging stem cells maintain high cell proliferation capacity and stem cell properties, thereby improving the therapeutic efficacy of cell therapy using mesenchymal stem cells.
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Abstract
The present invention relates to: a method for preparing aging-inhibited stem cells, comprising a step of treating stem cells with a GLS1 inhibitor; and a composition for aging inhibition or aging alleviation of stem cells, comprising the GLS1 inhibitor as an active ingredient. According to the present invention, aging of aged stem cells can be alleviated such that high cell proliferation capacity and stem cell properties are maintained, and thus the efficacy of cell therapy using mesenchymal stem cells can be improved.
Description
본 발명은 노화 억제 줄기세포의 제조방법에 관한 것으로, 더욱 자세하게는 줄기세포를 GLS1 저해제로 처리하는 단계를 포함하는 노화 억제 줄기세포의 제조방법 및 GLS1 저해제를 유효성분으로 함유하는 줄기세포의 노화억제용 조성물에 관한 것이다.The present invention relates to a method for producing anti-aging stem cells, and more specifically, to a method for producing anti-aging stem cells, including a step of treating stem cells with a GLS1 inhibitor, and a composition for anti-aging of stem cells containing a GLS1 inhibitor as an active ingredient.
중간엽 줄기세포(mesenchymal stem cells)는 골수, 제대혈, 태반(또는 태반 조직세포), 지방(또는 지방조직 세포) 등의 다양한 성체 세포로부터 유래하는 다분화성의 성질을 갖는 줄기세포이다. 예를 들어, 골수(bonemarrow)로부터 유래된 중간엽 줄기세포는 지방조직, 뼈/연골 조직, 근육조직으로 분화될 수 있는 다분화성에 의해 세포치료제로서의 개발을 위해 다양한 연구가 진행되고 있다.Mesenchymal stem cells are stem cells with multipotent properties derived from various adult cells such as bone marrow, umbilical cord blood, placenta (or placental tissue cells), and fat (or adipose tissue cells). For example, mesenchymal stem cells derived from bone marrow are being studied in various ways for development as cell therapy due to their multipotent properties that can differentiate into adipose tissue, bone/cartilage tissue, and muscle tissue.
최근, 중간엽 줄기세포를 이용한 세포치료기술이 각광을 받기 시작하면서, 인체로부터 분리된 중간엽 줄기세포를 치료에 적합하도록 활성화시키는 기술의 개발이 요구되고 있으며, 특히, 세포치료 대상이 되는 환자의 경우, 고령자가 많고, 고령자의 조직으로부터 채취된 중간엽 줄기세포의 경우, 증식능과 분화능이 떨어져 치료효율이 낮은 경향이 있으므로, 고령자 유래 중간엽 줄기세포를 젊은이 유래의 중간엽 줄기세포와 같이 활성화시키는 기술이 요구되고 있다.Recently, as cell therapy technology using mesenchymal stem cells has begun to receive attention, there is a demand for the development of technology to activate mesenchymal stem cells isolated from the human body so that they are suitable for treatment. In particular, many patients who are the subjects of cell therapy are elderly, and mesenchymal stem cells extracted from the tissues of the elderly tend to have low proliferative and differentiation abilities, resulting in low therapeutic efficacy. Therefore, a technology to activate mesenchymal stem cells derived from the elderly, like mesenchymal stem cells derived from young people, is demanded.
중간엽 줄기세포는, 다른 인체 초대배양 세포와 유사하게, 체외(in vitro) 배양 시 텔로미어 소실(telomere shortening)과 무관한 노화(senescence) 기전에 의해 세포의 분열이 급격히 감소하는 것으로 알려져 있다(Shibata, K.R. et al., Stem cells, 25;2371-2382, 2007). 이러한 노화 현상은, 그 기전이 아직 명확히 밝혀진 것은 아니나, 장기간의 체외 배양에 의한 환경적 스트레스의 축적으로 Cdk 저해 단백질인 p16(INK4a)가 발현되고 축적되어, 세포의 성장을 담당하는 Cdk 단백질의 활성이 억제되어 현상이 주요 원인으로 지적되고 있다. 중간엽 줄기세포에서는 Bmi-1이라는 종양유전자를 발현시켜 p16의 발현을 저해하였을 때, 세포의 노화가 억제되는 것이 확인되었으며(Zhang, X. et al., Biochemical and biophysical research communications 351;853-859, 2006), 또한 배양 중에 FGF-2를 처리하여 p21(Cip1), p53, 및 p16(INK4a)의 mRNA 발현을 억제되었을 때, G1 시기의 성장 정지된 중간엽 줄기세포의 성장 정지가 억제되는 것이 보고된 바 있다(Ito, T. et al., Biochemical and biophysical research communications, 359;108-114 2007). 또한 대한민국공개특허 제10-2009-0108141호에서는 Wip1 단백질을 코딩하는 유전자를 중간엽 줄기세포에 형질전환시켜, 중간엽 줄기세포의 노화를 억제시키는 방법을 제시하였다.Mesenchymal stem cells, similar to other human primary cultured cells, are known to rapidly decrease in cell division by a senescence mechanism independent of telomere shortening when cultured in vitro (Shibata, KR et al., Stem cells , 25;2371-2382, 2007). Although the mechanism of this senescence phenomenon has not yet been clearly identified, it is pointed out that the main cause is the accumulation of environmental stress due to long-term in vitro culture, which causes the expression and accumulation of the Cdk inhibitory protein p16 (INK4a), thereby suppressing the activity of the Cdk protein responsible for cell growth. In mesenchymal stem cells, it was confirmed that when the expression of p16 was inhibited by expressing the oncogene Bmi-1, cell senescence was suppressed (Zhang, X. et al., Biochemical and biophysical research communications 351;853-859, 2006). In addition, it was reported that when the mRNA expression of p21 (Cip1), p53, and p16 (INK4a) was suppressed by treating FGF-2 during culture, the growth arrest of mesenchymal stem cells that had arrested growth in the G1 phase was suppressed (Ito, T. et al., Biochemical and biophysical research communications , 359;108-114 2007). In addition, Korean Patent Publication No. 10-2009-0108141 proposed a method of suppressing the senescence of mesenchymal stem cells by transforming the gene encoding the Wip1 protein into mesenchymal stem cells.
이에, 본 발명자들은 중간엽 줄기세포의 노화를 효과적으로 억제하는 방법을 개발하고자 예의 노력한 결과, 노화 중간엽 줄기세포에 GLS1 저해제를 처리하는 경우, 젊은 중간엽 줄기세포와 유사한 활성을 가지는 중간엽 줄기세포를 제조할 수 있다는 것을 확인하고, 본 발명을 완성하게 되었다.Accordingly, the inventors of the present invention have made extensive efforts to develop a method for effectively inhibiting the aging of mesenchymal stem cells, and as a result, have confirmed that when treating aged mesenchymal stem cells with a GLS1 inhibitor, mesenchymal stem cells having activity similar to that of young mesenchymal stem cells can be produced, thereby completing the present invention.
발명의 요약Summary of the invention
본 발명의 목적은 노화 억제 줄기세포의 제조방법을 제공하는데 있다. The purpose of the present invention is to provide a method for producing anti-aging stem cells.
본 발명의 다른 목적은 줄기세포의 노화 억제방법을 제공하는데 있다. Another object of the present invention is to provide a method for inhibiting aging of stem cells.
본 발명의 또 다른 목적은 줄기세포의 노화억제용 조성물을 제공하는데 있다.Another object of the present invention is to provide a composition for inhibiting aging of stem cells.
상기 목적을 달성하기 위하여, 본 발명은 줄기세포를 GLS1 저해제로 처리하는 단계를 포함하는 노화 억제 줄기세포의 제조방법을 제공한다.To achieve the above purpose, the present invention provides a method for producing aging-inhibiting stem cells, which comprises a step of treating stem cells with a GLS1 inhibitor.
본 발명은 또한, 노화 줄기세포를 GLS1 저해제로 처리하는 단계를 포함하는 줄기세포의 노화 억제방법을 제공한다.The present invention also provides a method for inhibiting aging of stem cells, comprising a step of treating aging stem cells with a GLS1 inhibitor.
본 발명은 또한, GLS1 저해제를 유효성분으로 함유하는 줄기세포의 노화억제용 조성물을 제공한다.The present invention also provides a composition for inhibiting aging of stem cells containing a GLS1 inhibitor as an active ingredient.
도 1a는 젊은 WJ-MSC(senescence-uninduced MSC; U-MSC) 그룹과 노화 WJ-MSC(replicative senescence MSC; rS-MSC) 그룹의 노화 관련 베타 갈락토시데이즈(senescence associated beta-galactosidase, SA- β-gal) 염색을 수행한 결과를 나타낸 것이다.Figure 1a shows the results of senescence associated beta-galactosidase (SA-β-gal) staining of young WJ-MSC (senescence-uninduced MSC; U-MSC) and aged WJ-MSC (replicative senescence MSC; rS-MSC) groups.
도 1b는 젊은 WJ-MSC(대조군)와 노화 WJ-MSC의 세포 수를 3일 동안 측정한 결과를 나타낸 것이다.Figure 1b shows the results of measuring the cell numbers of young WJ-MSCs (control group) and aged WJ-MSCs for 3 days.
도 1c는 젊은 WJ-MSC(대조군)와 노화 WJ-MSC의 배가시간(doubling time)을 확인한 결과를 나타낸 것이다.Figure 1c shows the results of confirming the doubling time of young WJ-MSCs (control group) and aged WJ-MSCs.
도 1d는 젊은 WJ-MSC와 노화 WJ-MSC에서 핵막(nuclear envelope) 마커인 lamin B1에 대해 면역형광염색(ICC)를 수행한 결과를 나타낸 것이다.Figure 1d shows the results of immunofluorescence staining (ICC) for lamin B1, a nuclear envelope marker, in young and aged WJ-MSCs.
도 2a는 젊은 WJ-MSC, 노화 WJ-MSC의 GLS1의 mRNA 발현양을 나타낸 것이다.Figure 2a shows the mRNA expression levels of GLS1 in young WJ-MSCs and aged WJ-MSCs.
도 2b는 젊은 WJ-MSC와 노화 WJ-MSC의 GLS1의 단백질 발현양을 나타낸 것이다.Figure 2b shows the protein expression levels of GLS1 in young WJ-MSCs and aged WJ-MSCs.
도 2c는 젊은 WJ-MSC와 노화 WJ-MSC에 siGLS1 형질주입 후, GLS1의 mRNA 발현 감소를 확인한 결과를 나타낸 것이다.Figure 2c shows the results of confirming the decrease in mRNA expression of GLS1 after siGLS1 transfection into young and aged WJ-MSCs.
도 2d는 젊은 WJ-MSC와 노화 WJ-MSC에 siGLS1을 형질주입 후, 세포 생존력(cell viability)을 확인한 결과를 나타낸 것이다.Figure 2d shows the results of confirming cell viability after transfecting young WJ-MSCs and aged WJ-MSCs with siGLS1.
도 2e는 젊은 WJ-MSC와 노화 WJ-MSC에 BPTES 처리 후, 세포 생존력(cell viability)을 확인한 결과를 나타낸 것이다. Figure 2e shows the results of confirming cell viability after BPTES treatment in young WJ-MSCs and aged WJ-MSCs.
도 3a는 노화 WJ-MSC에 BPTES 처리 및 실험 절차에 대한 모식도이다.Figure 3a is a schematic diagram of the BPTES treatment and experimental procedures for aged WJ-MSCs.
도 3b는 노화 WJ-MSC와 BPTES 처리한 노화 WJ-MSC의 세포배양액에서 글루타민(glutamine) 수준을 측정한 결과를 나타낸 것이다.Figure 3b shows the results of measuring glutamine levels in the cell culture medium of aged WJ-MSCs and BPTES-treated aged WJ-MSCs.
도 3c는 노화 WJ-MSC와 BPTES 처리한 노화 WJ-MSC에서 세포 내 글루타민 수준을 측정한 결과를 나타낸 것이다.Figure 3c shows the results of measuring intracellular glutamine levels in aged WJ-MSCs and BPTES-treated aged WJ-MSCs.
도 3d는 노화 WJ-MSC와 BPTES 처리한 노화 WJ-MSC에서 세포 내 글루타메이트(glutamate) 수준을 측정한 결과이다.Figure 3d shows the results of measuring intracellular glutamate levels in aged WJ-MSCs and BPTES-treated aged WJ-MSCs.
도 3e는 노화 WJ-MSC와 BPTES 처리한 노화 WJ-MSC에서 세포 내 α-케토글루타레이트(α-ketoglutarate) 수준을 측정한 결과를 나타낸 것이다.Figure 3e shows the results of measuring intracellular α-ketoglutarate levels in aged WJ-MSCs and BPTES-treated aged WJ-MSCs.
도 3f는 노화 WJ-MSC에 BPTES 처리 후 세포 수를 3일 동안 측정한 결과를 나타낸 것이다.Figure 3f shows the results of measuring the cell number for 3 days after BPTES treatment on aged WJ-MSCs.
도 3g는 젊은 WJ-MSC, 노화 WJ-MSC, BPTES 처리한 노화 WJ-MSC에서 노화관련 단백질인 p16, p21 및 GLB1의 발현양을 확인한 결과를 나타낸 것이다. Figure 3g shows the results of confirming the expression levels of senescence-related proteins p16, p21, and GLB1 in young WJ-MSCs, aged WJ-MSCs, and BPTES-treated aged WJ-MSCs.
도 3h는 젊은 WJ-MSC, 노화 WJ-MSC, BPTES 처리한 노화 WJ-MSC에서 노화관련 유전자인 p16 및 p21의 mRNA 발현양을 확인한 결과를 나타낸 것이다.Figure 3h shows the results of confirming the mRNA expression levels of p16 and p21, which are aging-related genes, in young WJ-MSCs, aged WJ-MSCs, and BPTES-treated aged WJ-MSCs.
도 3i는 젊은 WJ-MSC, 노화 WJ-MSC, BPTES 처리한 노화 WJ-MSC의 SASP관련 유전자인 IGFBP3, IGFBP5 및 IGFBP7의 mRNA 발현양을 확인한 결과를 나타낸 것이다.Figure 3i shows the results of confirming the mRNA expression levels of SASP-related genes IGFBP3, IGFBP5, and IGFBP7 in young WJ-MSCs, aged WJ-MSCs, and aged WJ-MSCs treated with BPTES.
도 4는 BPTES 처리시, 젊은 WJ-MSC, 노화 WJ-MSC에 대해 각각 FACS를 통해 줄기세포능을 확인한 결과를 나타낸 것이다. Figure 4 shows the results of confirming the stem cell capacity of young WJ-MSCs and aged WJ-MSCs, respectively, through FACS when treated with BPTES.
도 5a는 노화 WJ-MSC에 siGLS1 형질주입 후, 상대적인 세포 생존력(cell viability)을 확인한 결과이다.Figure 5a shows the results of confirming relative cell viability after siGLS1 transfection into aged WJ-MSCs.
도 5b는 노화 WJ-MSC에 siGLS1 형질주입 후, 노화관련 유전자인 p16 및 p21의 mRNA 발현양을 확인한 결과를 나타낸 것이다.Figure 5b shows the results of confirming the mRNA expression levels of p16 and p21, which are senescence-related genes, after siGLS1 transfection into senescent WJ-MSCs.
도 5c는 노화 WJ-MSC에 siGLS1 형질주입 후, GLS1 단백질 발현양 및 노화관련 단백질인 p16, p21 및 GLB1의 단백질 발현양을 확인한 결과를 나타낸 것이다.Figure 5c shows the results of confirming the protein expression levels of GLS1 and the senescence-related proteins p16, p21, and GLB1 after siGLS1 transfection into senescent WJ-MSCs.
도 5d는 노화 WJ-MSC에 siGLS1를 형질주입 후, SASP관련 유전자인 IGFBP3, IGFBP5 및 IGFBP7의 mRNA 발현양을 확인한 결과를 나타낸 것이다.Figure 5d shows the results of confirming the mRNA expression levels of SASP-related genes IGFBP3, IGFBP5, and IGFBP7 after transfection of siGLS1 into aged WJ-MSCs.
도 6a는 노화 WJ-MSC에 CB-838와 C968을 각각 처리 후, 3일 동안 세포생존력(cell viability)을 확인한 결과를 나타낸 것이다.Figure 6a shows the results of examining cell viability for 3 days after treating aged WJ-MSCs with CB-838 and C968, respectively.
도 6b는 노화 WJ-MSC에 CB-838와 C968을 각각 처리 후, 노화관련 유전자인 p16 및 p21의 발현 감소를 확인한 결과를 나타낸 것이다.Figure 6b shows the results of confirming the decrease in the expression of aging-related genes, p16 and p21, after treating aging WJ-MSCs with CB-838 and C968, respectively.
도 6c는 노화 WJ-MSC에 CB-838와 C968을 각각 처리 후, 노화관련 단백질인 p16, p21 및 GLB1의 발현 감소를 확인한 결과를 나타낸 것이다.Figure 6c shows the results of confirming the decrease in the expression of aging-related proteins p16, p21, and GLB1 after treating aging WJ-MSCs with CB-838 and C968, respectively.
도 6d는 노화 WJ-MSC에 CB-838와 C968을 각각 처리 후, SASP관련 유전자인 IGFBP3, IGFBP5 및 IGFBP7의 발현 감소를 확인한 결과를 나타낸 것이다.Figure 6d shows the results of confirming a decrease in the expression of SASP-related genes IGFBP3, IGFBP5, and IGFBP7 after treating aged WJ-MSCs with CB-838 and C968, respectively.
도 7a는 중간엽 줄기세포의 노화 억제 전략에 대한 실험 설계 모식도이다.Figure 7a is a schematic diagram of the experimental design for a strategy to inhibit senescence in mesenchymal stem cells.
도 7b는 젊은 WJ-MSC, 노화 WJ-MSC, 노화억제 WJ-MSC(replicative senescence-alleviated MSC ; rSA-MSC)의 노화 정도를 평가하기 위해 SA- β-gal 염색을 수행한 결과를 나타낸 것이다.Figure 7b shows the results of SA-β-gal staining to evaluate the degree of senescence of young WJ-MSCs, senescent WJ-MSCs, and replicative senescence-alleviated MSCs (rSA-MSCs).
도 7c는 젊은 WJ-MSC, 노화 WJ-MSC, 노화억제 WJ-MSC의 세포 면적을 측정한 결과를 나타낸 것이다.Figure 7c shows the results of measuring the cell area of young WJ-MSCs, aged WJ-MSCs, and senescence-inhibited WJ-MSCs.
도 7d는 젊은 WJ-MSC, 노화 WJ-MSC, 노화억제 WJ-MSC의 배가시간(doubling time)을 확인한 결과를 나타낸 것이다. Figure 7d shows the results of confirming the doubling time of young WJ-MSCs, aged WJ-MSCs, and aged-inhibited WJ-MSCs.
도 7e는 GLS1 억제제인 CB-839와 C968를 처리한 후, 회복한 노화 WJ-MSC의 노화 정도를 평가하기 위해 SA- β-gal 염색을 수행한 결과를 나타낸 것이다.Figure 7e shows the results of SA-β-gal staining to evaluate the degree of senescence in recovered senescent WJ-MSCs after treatment with GLS1 inhibitors CB-839 and C968.
도 8a는 세포사멸이 유도된 근육세포를 젊은 WJ-MSC, 노화 WJ-MSC, 노화억제 WJ-MSC와 공동배양을 진행한 후, 근육세포에서 세포사멸 마커인 cleaved PARP와 cleaved caspase 3의 단백질 발현 수준을 확인한 결과를 나타낸 것이다.Figure 8a shows the results of confirming the protein expression levels of apoptosis markers, cleaved PARP and cleaved caspase 3, in muscle cells after co-culturing apoptosis-induced muscle cells with young WJ-MSCs, aged WJ-MSCs, and aged-inhibited WJ-MSCs.
도 8b는 세포사멸에 대한 WJ-MSC의 치료효능을 평가하기 위해 Annexin V 면역형광염색을 수행한 결과를 나타낸 것이다.Figure 8b shows the results of Annexin V immunofluorescence staining to evaluate the therapeutic efficacy of WJ-MSCs against apoptosis.
도 9a는 DMD 질병 모델 마우스(MDX)에 젊은 WJ-MSC, 노화 WJ-MSC 및 노화억제 WJ-MSC를 투여 후, 악력(grip strength) 행동평가를 확인한 결과를 나타낸 것이다.Figure 9a shows the results of a behavioral assessment of grip strength after administering young WJ-MSCs, aged WJ-MSCs, and aged-inhibited WJ-MSCs to DMD disease model mice (MDX).
도 9b는 대조군과 젊은 WJ-MSC, 노화 WJ-MSC, 노화억제 WJ-MSC를 투여한 실험군의 Creatine kinase(CK) 활성을 측정한 결과를 나타낸 것이다.Figure 9b shows the results of measuring Creatine kinase (CK) activity in the control group and the experimental groups administered young WJ-MSCs, aged WJ-MSCs, and aged-inhibited WJ-MSCs.
도 9c는 대조군과 젊은 WJ-MSC, 노화 WJ-MSC, 노화억제 WJ-MSC를 투여한 실험군의 annexin V, MHC 및 Fibronectin 단백질 발현양을 확인한 결과를 나타낸 것이다.Figure 9c shows the results of confirming the expression levels of annexin V, MHC, and Fibronectin proteins in the control group and the experimental groups administered young WJ-MSCs, aged WJ-MSCs, and aged-inhibited WJ-MSCs.
도 9d는 도 9c의 annexin V에 대한 단백질 발현양을 수치화한 결과를 나타낸 것이다.Figure 9d shows the results of numerically quantifying the protein expression level for annexin V in Figure 9c.
도 9e는 도 9c의 MHC에 대한 단백질 발현양을 수치화한 결과를 나타낸 것이다.Figure 9e shows the results of numerically quantifying the protein expression level for MHC in Figure 9c.
도 9f는 도 9c의 fibronectin에 대한 단백질 발현양을 수치화한 결과를 나타낸 것이다.Figure 9f shows the results of numerically quantifying the protein expression level for fibronectin in Figure 9c.
도 9g는 대조군과 젊은 WJ-MSC, 노화 WJ-MSC, 노화억제 WJ-MSC를 투여한 실험군의 annexin V 및 MHC에 대한 면역형광염색과 시리우스 레드(sirius red)염색 결과를 나타낸 것이다.Figure 9g shows the results of immunofluorescence staining and sirius red staining for annexin V and MHC in the control group and the experimental groups administered young WJ-MSCs, aged WJ-MSCs, and aged-inhibited WJ-MSCs.
도 9h는 도 9g의 MHC에 대한 면역형광색을 수치화한 결과를 나타낸 것이다.Figure 9h shows the result of numerically quantifying the immunofluorescence color for MHC in Figure 9g.
도 9i는 도 9g의 annexin V에 대한 면역형광염색을 수치화한 결과를 나타낸 것이다.Figure 9i shows the numerical results of immunofluorescence staining for annexin V in Figure 9g.
도 9j는 도 9g의 sirius red 염색 결과를 수치화한 결과를 나타낸 것이다.Figure 9j shows the numerical results of the sirius red staining results of Figure 9g.
도 10a는 젊은 WJ-MSC, 노화 WJ-MSC, BPTES 처리한 노화 WJ-MSC에 대한 FC > 3 이상인 DEG(differentially expressed genes)를 벤 다이어그램으로 나타낸 것이다.Figure 10a is a Venn diagram showing the differentially expressed genes (DEGs) with FC > 3 for young WJ-MSCs, aged WJ-MSCs, and BPTES-treated aged WJ-MSCs.
도 10b는 젊은 WJ-MSC, 노화 WJ-MSC, BPTES 처리한 노화 WJ-MSC에 대한 FC > 3 이상인 DEG를 heat map으로 나타낸 결과이다.Figure 10b shows the results of a heat map showing DEGs with FC > 3 for young WJ-MSCs, aged WJ-MSCs, and BPTES-treated aged WJ-MSCs.
도 10c는 노화 WJ-MSC 대비 BPTES 처리한 노화 WJ-MSC의 FC > 3 이상인 DEG와 관련된 유전자 카테고리를 나타낸 결과이다.Figure 10c shows the results of gene categories related to DEGs with FC > 3 in BPTES-treated aged WJ-MSCs compared to aged WJ-MSCs.
도 10d는 3 lot에서 젊은 WJ-MSC, 노화 WJ-MSC의 CTSC, E2F7 및 SORBS2 mRNA발현양을 확인한 결과를 나타낸 것이다.Figure 10d shows the results of confirming the expression levels of CTSC, E2F7, and SORBS2 mRNA in young WJ-MSCs and aged WJ-MSCs in 3 lots.
도 10e는 젊은 WJ-MSC, 노화 WJ-MSC, BPTES 처리한 노화 WJ-MSC의 CTSC, E2F7 및 SORBS2에 대한 mRNA 서열 분석을 통해 정규화한 log2 값으로 나타낸 것이다.Figure 10e shows the normalized log2 values of mRNA sequence analysis for CTSC, E2F7, and SORBS2 in young WJ-MSCs, aged WJ-MSCs, and BPTES-treated aged WJ-MSCs.
도 10f는 젊은 WJ-MSC, 노화 WJ-MSC, BPTES 처리한 노화 WJ-MSC의 CTSC, E2F7 및 SORBS2 mRNA발현양을 확인한 결과를 나타낸 것이다.Figure 10f shows the results of confirming the mRNA expression levels of CTSC, E2F7, and SORBS2 in young WJ-MSCs, aged WJ-MSCs, and BPTES-treated aged WJ-MSCs.
도 10g는 노화 WJ-MSC에 siGLS1 형질주입 후, CTSC, E2F7, SORBS2 mRNA발현양을 확인한 결과를 나타낸 것이다.Figure 10g shows the results of confirming the expression levels of CTSC, E2F7, and SORBS2 mRNA after siGLS1 transfection into aged WJ-MSCs.
도 11a는 3 lot의 젊은 WJ-MSC, 노화 WJ-MSC에서 FC > 1.5이며 p < 0.05인 DEG를 heat map으로 나타낸 것이다.Figure 11a is a heat map showing DEGs with FC > 1.5 and p < 0.05 in three lots of young WJ-MSCs and aged WJ-MSCs.
도 11b는 GO 데이터베이스를 이용한 DAVID분석을 통해 p-값을 기준으로 상위 10개 카테고리를 나타낸 것이다.Figure 11b shows the top 10 categories based on p-values through DAVID analysis using the GO database.
도 11c는 KEGG 데이터베이스를 이용한 DAVID분석을 통해 p-값을 기준으로 상위 10개 카테고리를 나타낸 것이다.Figure 11c shows the top 10 categories based on p-values through DAVID analysis using the KEGG database.
도 11d는 유전자 집합 농축 분석(GSEA)을 기반으로 WJ-MSC의 복제 노화가 “세포 주기 DNA 복제” 및 “Wnt에 의한 세포-세포 신호 전달”에 연관이 있음을 보여주는 분석 결과를 나타낸 것이다.Figure 11d shows the analysis results based on gene set enrichment analysis (GSEA) showing that the replicative senescence of WJ-MSCs is associated with “cell cycle DNA replication” and “cell-to-cell signaling by Wnt.”
도 11e는 3 lot의 젊은 WJ-MSC와 노화 WJ-MSC에서 노화관련 단백질인 p21과 Wnt 신호전달 경로 관련 단백질인 β-catenin의 발현양을 확인한 결과를 나타낸 것이다.Figure 11e shows the results of confirming the expression levels of p21, a aging-related protein, and β-catenin, a Wnt signaling pathway-related protein, in three lots of young WJ-MSCs and aged WJ-MSCs.
도 11f는 젊은 WJ-MSC, 노화 WJ-MSC, 노화억제 WJ-MSC에서 GLS1 단백질 발현 및 Wnt 신호전달 경로 관련 단백질인 β-catenin과 인산화된 GSK3β(ser9)의 단백질 발현양을 확인한 결과를 나타낸 것이다.Figure 11f shows the results of confirming the protein expression of GLS1 and the protein expression levels of β-catenin and phosphorylated GSK3β (ser9), which are proteins related to the Wnt signaling pathway, in young WJ-MSCs, aged WJ-MSCs, and aged-inhibited WJ-MSCs.
도 11g는 노화 WJ-MSC에 siGLS1을 형질주입후, GLS1 단백질 발현 및 Wnt 신호전달 경로 관련 단백질인 β-catenin과 인산화된 GSK3β(ser9)의 단백질 발현양을 확인한 결과를 나타낸 것이다.Figure 11g shows the results of confirming the protein expression levels of GLS1 protein and β-catenin and phosphorylated GSK3β (ser9), proteins related to the Wnt signaling pathway, after transfection of siGLS1 into aged WJ-MSCs.
도 11h는 CB-839와 C968을 노화 WJ-MSC에 처리 후, Wnt 신호전달 경로 관련 단백질인 β-catenin과 인산화된 GSK3β(ser9)의 단백질 발현양을 확인한 결과를 나타낸 것이다.Figure 11h shows the results of confirming the protein expression levels of β-catenin and phosphorylated GSK3β (ser9), proteins related to the Wnt signaling pathway, after treatment of aged WJ-MSCs with CB-839 and C968.
도 12a는 유전자 집합 농축 분석(GSEA)을 기반으로 WJ-MSC의 노화로 인해 “자가포식작용(autophagy)의 양성 조절”이 증가함을 보여주는 분석 결과를 나타낸 것이다.Figure 12a shows the results of the analysis showing that “positive regulation of autophagy” increases due to aging in WJ-MSCs based on gene set enrichment analysis (GSEA).
도 12b는 젊은 WJ-MSC, 노화 WJ-MSC, 노화억제 WJ-MSC에서 자가포식작용 관련 단백질 LC3II의 단백질 발현양을 확인한 결과를 나타낸 것이다.Figure 12b shows the results of confirming the protein expression level of the autophagy-related protein LC3II in young WJ-MSCs, aged WJ-MSCs, and senescence-inhibited WJ-MSCs.
도 12c는 노화 WJ-MSC에 siGLS1을 형질주입후, GLS1 단백질 및 자가포식작용 관련 단백질 LC3II의 단백질 발현양을 확인한 결과를 나타낸 것이다.Figure 12c shows the results of confirming the protein expression levels of GLS1 protein and autophagy-related protein LC3II after transfection of siGLS1 into aged WJ-MSCs.
도 12d는 CB-839와 C968을 노화 WJ-MSC에 처리 후, 자가포식작용 관련 단백질 LC3II의 단백질 발현양을 확인한 결과를 나타낸 것이다.Figure 12d shows the results of confirming the protein expression level of LC3II, a protein related to autophagy, after treatment of aged WJ-MSCs with CB-839 and C968.
발명의 상세한 설명 및 바람직한 구현예Detailed description of the invention and preferred embodiments
다른 식으로 정의되지 않는 한, 본 명세서에서 사용된 모든 기술적 및 과학적 용어들은 본 발명이 속하는 기술 분야에서 숙련된 전문가에 의해서 통상적으로 이해되는 것과 동일한 의미를 갖는다. 일반적으로 본 명세서에서 사용된 명명법은 본 기술 분야에서 잘 알려져 있고 통상적으로 사용되는 것이다.Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein is well known and commonly used in the art.
본 발명에서는 제대혈에서 분리한 젊은 유래 중간엽 줄기세포보다 노화 중간엽 줄기세포에서 β-galactosidase 양성세포가 증가하는 것을 확인하였고, 노화 WJ-MSC에서 GLS1의 mRNA 발현이 젊은 WJ-MSC에 비해 크게 증가되었으나, GLS1 저해제 처리시 GLS1의 mRNA 발현이 현저하게 감소하는 것을 확인하였으며, 노화 중간엽 줄기세포의 배가시간(doubling time) 또한 GLS1 저해제 처리에 의하여 단축되는 것을 확인하였다. In the present invention, it was confirmed that β-galactosidase positive cells increased in aged mesenchymal stem cells compared to young-derived mesenchymal stem cells isolated from umbilical cord blood, and that mRNA expression of GLS1 in aged WJ-MSCs was significantly increased compared to young WJ-MSCs, but that mRNA expression of GLS1 was significantly decreased when treated with a GLS1 inhibitor, and that the doubling time of aged mesenchymal stem cells was also shortened by treatment with a GLS1 inhibitor.
따라서, 본 발명은 일 관점에서, 줄기세포를 GLS1 저해제로 처리하는 단계를 포함하는 노화 억제 줄기세포의 제조방법에 관한 것이다.Accordingly, the present invention relates, in one aspect, to a method for producing anti-aging stem cells, comprising a step of treating stem cells with a GLS1 inhibitor.
본 발명에서 상기 GLS1 저해제는 BPTES(bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl)ethyl sulfide), C968, CB-839(Telaglenastat), DON(6-Diazo-5-oxo-L-norleucine) 등을 사용할 수 있으며, 바람직하게는 BPTES, CB-839 또는 C968를 사용할 수 있으나, 이에 한정되는 것은 아니다.In the present invention, the GLS1 inhibitor may be BPTES (bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl)ethyl sulfide), C968, CB-839 (Telaglenastat), DON (6-Diazo-5-oxo-L-norleucine), etc., and preferably, BPTES, CB-839 or C968 may be used, but is not limited thereto.
본 발명에서 "노화 줄기세포"(rS-MSC)는 passage 15 이상이면서 beta-galactosidase staining 결과 positive cell이 25% 이상인 세포를 의미한다. 줄기세포 노화시, cell cycle arrest 관련 p16-pRB pathway와 p53-p21 pathway가 활성화되어 p16, p21, p53과 같은 단백질 발현이 증가한다. 또한, 비정상적인 구조적 변화가 일어나 cell size가 커지고 morphology가 길고 넓어지는 특징을 가진다. 세포노화시, lysosome의 beta-galactosidase 발현이 증가하여 senescence-associated(SA) beta-galactosidase activity의 활성이 증가한다. 또한 SASP(senescence associated secretion phenotype)관련 단백질의 발현이 증가한다.In the present invention, "senescent stem cells" (rS-MSC) refer to cells that are passage 15 or more and have 25% or more positive cells as a result of beta-galactosidase staining. When stem cells age, the p16-pRB pathway and the p53-p21 pathway related to cell cycle arrest are activated, and the expression of proteins such as p16, p21, and p53 increases. In addition, abnormal structural changes occur, and the cell size increases and the morphology becomes longer and wider. When cells age, the expression of lysosome beta-galactosidase increases, and the activity of senescence-associated (SA) beta-galactosidase activity increases. In addition, the expression of SASP (senescence associated secretion phenotype)-related proteins increases.
본 발명에서 "젊은 줄기세포"(U-MSC)는 passage 6 미만이면서 beta-galactosidase staining 결과 positive cell이 1% 미만인 세포를 의미한다. 노화 줄기세포와 달리 젊은 줄기세포는 cell cycle arrest 관련 p16-pRB pathway와 p53-p21 pathway가 비활성화되어 p16, p21, p53과 같은 단백질 발현이 낮게 나타난다. 또한, 노화세포에 비해 작은 cell size를 가진다. 젊은 줄기세포에서는 노화의 특징인 beta-galactosidase 발현 증가 또는 SASP관련 단백질 발현이 증가하는 현상이 나타나지 않는다.In the present invention, "young stem cells" (U-MSC) refer to cells that are less than passage 6 and have less than 1% positive cells in the beta-galactosidase staining result. Unlike senescent stem cells, young stem cells have inactivated p16-pRB pathway and p53-p21 pathway related to cell cycle arrest, and thus have low expression of proteins such as p16, p21, and p53. In addition, they have a smaller cell size than senescent cells. Young stem cells do not show the phenomenon of increased beta-galactosidase expression or increased SASP-related protein expression, which are characteristics of aging.
본 발명에서 "WJ-MSC"는 제대혈의 와튼젤리(waton's jelly) 유래의 중간엽 줄기세포를 의미한다. In the present invention, "WJ-MSC" refers to mesenchymal stem cells derived from Wharton's jelly of umbilical cord blood.
본 발명의 일 양태에서는 젊은 WJ-MSC보다 노화 WJ-MSC에서 증가하였던 노화관련 mRNA 발현 정도가 GLS1 저해제인 BPTES 처리시 유의하게 감소한 것을 확인할 수 있었으며, SASP관련 유전자의 발현이 젊은 WJ-MSC에 비하여 노화 WJ-MSC에서 증가하였고 BPTES 처리시 mRNA 수준이 회복되는 것을 확인하였다.In one embodiment of the present invention, it was confirmed that the level of aging-related mRNA expression, which was increased in aged WJ-MSCs compared to young WJ-MSCs, was significantly reduced when treated with BPTES, a GLS1 inhibitor, and that the expression of SASP-related genes was increased in aged WJ-MSCs compared to young WJ-MSCs and that the mRNA level was recovered when treated with BPTES.
아울러, 본 발명의 다른 양태에서는 노화 WJ-MSC에서 26.33%로 증가하였던 b-gal 양성 세포가 BPTES 처리시 5.82%로 감소하여 노화억제가 진행되는 것을 확인하였으며, 젊은 WJ-MSC에서는 노화 WJ-MSC에 비해 빠른 세포증식율을 보였다. BPTES 처리 후 회복한 노화 WJ-MSC에서는 노화 WJ-MSC 보다 세포증식율이 유의미하게 증가하는 것을 확인하였다.In addition, in another embodiment of the present invention, it was confirmed that b-gal positive cells, which had increased to 26.33% in aged WJ-MSCs, decreased to 5.82% upon BPTES treatment, indicating that aging was inhibited, and that young WJ-MSCs showed a faster cell proliferation rate than aged WJ-MSCs. It was confirmed that aged WJ-MSCs recovered after BPTES treatment had a significantly higher cell proliferation rate than aged WJ-MSCs.
본 발명의 다른 양태에서는 젊은 WJ-MSC에서는 24.97시간이였던 배가시간이 노화 WJ-MSC에서는 56.67시간으로 증가하였다. BPTES 처리후 회복한 노화 WJ-MSC에서는 42.86시간으로 노화 WJ-MSC에 비해 배가시간이 빨라진 것을 확인할 수 있었다. 이러한 결과를 종합적으로 보았을 때, 노화 WJ-MSC에 GLS1 저해제를 처리하였을 때 노화진행을 현저하게 저해함을 밝혀냈다. 이는 WJ-MSC노화 억제전략의 기초가 될 수 있다.In another embodiment of the present invention, the doubling time, which was 24.97 hours in young WJ-MSCs, increased to 56.67 hours in aged WJ-MSCs. It was confirmed that the doubling time was faster in aged WJ-MSCs recovered after BPTES treatment, at 42.86 hours, compared to aged WJ-MSCs. When these results were comprehensively considered, it was revealed that when a GLS1 inhibitor was treated to aged WJ-MSCs, the aging process was significantly inhibited. This can be the basis for a WJ-MSC aging inhibition strategy.
따라서, 본 발명은 다른 관점에서, 노화 WJ-MSC를 GLS1 저해제로 처리하는 단계를 포함하는 줄기세포의 노화 억제방법에 관한 것이다. Therefore, the present invention, from another perspective, relates to a method for inhibiting aging of stem cells, comprising a step of treating aging WJ-MSCs with a GLS1 inhibitor.
본 발명에서 상기 GLS1 저해제는 BPTES(bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl)ethyl sulfide), C968, CB-839(Telaglenastat), DON(6-Diazo-5-oxo-L-norleucine) 등을 사용할 수 있으며, 바람직하게는 BPTES, CB-839 또는 C968를 사용할 수 있으나, 이에 한정되는 것은 아니다.In the present invention, the GLS1 inhibitor may be BPTES (bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl)ethyl sulfide), C968, CB-839 (Telaglenastat), DON (6-Diazo-5-oxo-L-norleucine), etc., and preferably, BPTES, CB-839 or C968 may be used, but is not limited thereto.
본 발명의 일 양태에서는 BPTES 처리에 의해 노화가 회복된 세포의 유전자 발현 양상도 젊은 WJ-MSC와 유사하게 회복이 되는지 확인하였다. 노화시 GLS1 외에도 발현에 차이가 나는 유전자를 확인하였으며, 그 중 CTSC, E2F7 및 SORBS2는 노화와 연관이 되어 있다고 알려진 유전자들로 노화가 회복됨으로 인해 GLS1외에 다른 노화 관련 유전자들도 발현에 영향을 받는다는 것을 확인할 수 있었다.In one embodiment of the present invention, it was confirmed whether the gene expression pattern of cells in which aging was recovered by BPTES treatment was similar to that of young WJ-MSCs. In addition to GLS1, genes whose expression was different during aging were confirmed, and among them, CTSC, E2F7, and SORBS2 are genes known to be associated with aging, and it was confirmed that other aging-related genes, in addition to GLS1, were affected in expression due to the recovery of aging.
아울러, 젊은 WJ-MSC, 노화 WJ-MSC 및 BPTES 처리한 노화 WJ-MSC에 각각 FACS를 수행하여 줄기세포능을 확인하였다. 중간엽 줄기세포의 마커는 CD44, CD73, CD90, CD105 및 CD166의 발현을 통해 확인하였고, 조혈모세포 계통의 마커는 CD14, CD11b, HLA-DR (MHCII), CD34, CD45 및 CD19 (BD Biosciences, USA)을 사용하여 줄기세포능을 비교하였다. 중간엽 줄기세포에서 노화가 진행되었을 때 줄기세포능(stemness)이 감소하였으나, BPTES 처리시 줄기세포능이 회복되는 것을 확인하였다.In addition, FACS was performed on young WJ-MSCs, aged WJ-MSCs, and aged WJ-MSCs treated with BPTES to determine their stem cell potential. The markers of mesenchymal stem cells were identified through the expression of CD44, CD73, CD90, CD105, and CD166, and the markers of hematopoietic stem cells were CD14, CD11b, HLA-DR (MHCII), CD34, CD45, and CD19 (BD Biosciences, USA) to compare their stem cell potential. When aging progressed in mesenchymal stem cells, the stem cell potential decreased, but it was confirmed that the stem cell potential was restored when BPTES was treated.
또 다른 관점에서, 본 발명은 GLS1 저해제를 유효성분으로 함유하는 줄기세포의 노화억제 조성물에 관한 것이다.From another perspective, the present invention relates to an anti-aging composition of stem cells containing a GLS1 inhibitor as an active ingredient.
본 발명의 일 양태에서는 세포사멸이 유도된 근육세포를 젊은 WJ-MSC, 노화 WJ-MSC, 노화억제 WJ-MSC와 공동배양을 진행하여, cleaved PAPR와 cleaved capase 3의 발현은 젊은 WJ-MSC, 노화억제 WJ-MSC와 공동배양한 근육세포에서는 유의미하게 감소한 것을 확인하였다.In one embodiment of the present invention, muscle cells in which apoptosis was induced were co-cultured with young WJ-MSCs, senescent WJ-MSCs, and senescence-inhibited WJ-MSCs, and it was confirmed that the expression of cleaved PAPR and cleaved capase 3 was significantly reduced in muscle cells co-cultured with young WJ-MSCs and senescence-inhibited WJ-MSCs.
또한, 노화 WJ-MSC를 공동배양한 근육세포에 비해 젊은 WJ-MSC, 노화억제 WJ-MSC와 공동배양을 진행한 근육세포에서 annexin V 양성세포의 비율이 유의하게 감소하는 것을 확인하여, WJ-MSC의 노화로 인해 감소된 항세포사멸 효능은 BPTES 처리를 통한 노화억제 WJ-MSC에서 회복될 수 있음을 확인하였다.In addition, we confirmed that the proportion of annexin V-positive cells was significantly decreased in muscle cells co-cultured with young WJ-MSCs and anti-aging WJ-MSCs compared to muscle cells co-cultured with aged WJ-MSCs, confirming that the anti-apoptotic efficacy of WJ-MSCs reduced due to aging can be recovered in anti-aging WJ-MSCs treated with BPTES.
본 발명의 다른 양태에서는, 노화 억제 WJ-MSC의 근디스트로피(DMD) 질병에 대한 치료효능을 탐색하기 위해 마우스 동물실험을 진행한 결과, 젊은 WJ-MSC 투여군에서는 악력(grip strength)이 대조군(MDX Ctrl)에 비해 증가하였으며, 노화 WJ-MSC를 투여했을 때는 대조군과 비슷한 경향을 보였다. BPTES 처리를 통한 노화억제 WJ-MSC를 투여한 실험군에서는 노화 WJ-MSC 투여한 실험군에 비해 악력(grip strength)가 증가하는 것을 확인하였다.In another aspect of the present invention, a mouse animal experiment was conducted to explore the therapeutic efficacy of anti-aging WJ-MSCs on muscular dystrophy (DMD) disease. As a result, in the young WJ-MSC administration group, grip strength increased compared to the control group (MDX Ctrl), and when aged WJ-MSCs were administered, a similar tendency was observed to the control group. In the experimental group administered anti-aging WJ-MSCs through BPTES treatment, it was confirmed that grip strength increased compared to the experimental group administered aged WJ-MSCs.
MDX대조군에 비해 젊은 WJ-MSC를 투여한 실험군에서 CK activity가 감소하였으며, 노화 WJ-MSC를 투여했을 때는 대조군과 비슷한 경향을 보였다. BPTES 처리를 통한 노화억제 WJ-MSC를 투여한 실험군에서는 노화 WJ-MSC 투여한 실험군에 비해 CK activity가 감소함을 확인하였다.Compared to the MDX control group, CK activity decreased in the experimental group administered young WJ-MSCs, and when aged WJ-MSCs were administered, a similar trend was observed as in the control group. In the experimental group administered WJ-MSCs that inhibited aging through BPTES treatment, CK activity was confirmed to decrease compared to the experimental group administered aged WJ-MSCs.
따라서, 노화가 진행된 WJ-MSC는 MDX 마우스의 질병에 대한 치료 효과가 없지만, BPTES 처리하여 노화를 억제시킨 WJ-MSC는 젊은 WJ-MSC만큼 치료 효능이 증대되는 것을 확인할 수 있었다.Therefore, it was confirmed that while aged WJ-MSCs had no therapeutic effect on diseases in MDX mice, WJ-MSCs treated with BPTES to inhibit aging had an enhanced therapeutic effect similar to that of young WJ-MSCs.
또한, 본 발명에서는 노화가 진행된 WJ-MSC는 노화로 인해 Wnt 신호전달 경로가 비활성되지만, GLS1의 억제를 통해 Wnt 신호전달 경로의 활성화시킴으로써 복제성 노화를 억제할 수 있다는 것을 확인하였으며, 자가포식작용이 WJ-MSC의 복제성 노화와 관련이 있으며, GLS1에 의해 조절될 수 있다는 것을 확인하였다. In addition, the present invention confirmed that replicative senescence can be suppressed by activating the Wnt signaling pathway through inhibition of GLS1 in aged WJ-MSCs, although the Wnt signaling pathway is inactivated due to aging, and that autophagy is related to replicative senescence of WJ-MSCs and can be regulated by GLS1.
이하, 실시예를 통하여 본 발명을 더욱 상세히 설명하고자 한다. 이들 실시예는 오로지 본 발명을 예시하기 위한 것으로서, 본 발명의 범위가 이들 실시예에 의해 제한되는 것으로 해석되지 않는 것은 해당 업계에서 통상의 지식을 가진 자에 있어서 자명할 것이다.Hereinafter, the present invention will be described in more detail through examples. These examples are only intended to illustrate the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as being limited by these examples.
실시예 1. 인체 중간엽 줄기세포의 복제성 노화모델 수립Example 1. Establishment of a replicative aging model of human mesenchymal stem cells
제대 중간엽 줄기세포(Wharton’s Jelly-mesenchymal stem cell, WJ-MSC)는 삼성서울병원에서 승인한 IRB (IRB# 2016-07-102-043)의 기준에 따라 삼성서울병원 산부인과와의 협업 연구를 통하여 탯줄 및 태반을 확보한 후 탯줄 유래 중간엽 줄기세포(WJ-MSC)는 PARK 등의 방법(Park et al., Arch. Pharm. Res. 39: 1171-1179, 2016)에 따라 탯줄로부터 분리하였다. Wharton’s Jelly-mesenchymal stem cells (WJ-MSC) were isolated from the umbilical cord and placenta through a collaborative study with the Department of Obstetrics and Gynecology, Samsung Medical Center, in accordance with the standards of the IRB (IRB# 2016-07-102-043) approved by Samsung Medical Center. Umbilical cord-derived mesenchymal stem cells (WJ-MSC) were isolated from the umbilical cord according to the method of PARK et al. (Park et al., Arch. Pharm. Res. 39: 1171-1179, 2016).
구체적으로, 탯줄을 3-4 cm 길이 자른 다음, 조직을 잘게 자르고 세포외기질을 분해하기 위해 콜라게나아제 용액(Gibco, USA)을 60~90분 동안 처리한 다음, 0.25% 트립신(Gibco, USA)을 추가하여 30분 동안, 37℃에서 분해를 더 유도하였다. 이후, 소태아혈청(FBS; Biowest, USA)을 첨가하여, 1000g에서 10분 동안 원심분리하여 세포를 얻고, 10% FBS(fetal bovine serum, Invitrogen-Gibco) 및 50 ㎍/mL 젠타마이신(Invitrogen-Gibco)이 포함되어 있는 MEM Alpha(Minimum Essential Medium, Invitrogen-Gibco, Rockville, MD) 배지를 이용하여 37℃, 5% CO2 환경에서 배양한 WJ-MSC 세포를 이후 실험에 사용하였다. Specifically, the umbilical cord was cut into 3-4 cm lengths, the tissue was finely chopped, and treated with collagenase solution (Gibco, USA) for 60-90 minutes to decompose the extracellular matrix, and then 0.25% trypsin (Gibco, USA) was added to induce further digestion for 30 minutes at 37°C. Afterwards, fetal bovine serum (FBS; Biowest, USA) was added, and the cells were obtained by centrifugation at 1000 g for 10 minutes. WJ-MSC cells were cultured in a 5% CO2 environment at 37°C using MEM Alpha (Minimum Essential Medium, Invitrogen-Gibco, Rockville, MD) medium containing 10% FBS (fetal bovine serum, Invitrogen-Gibco) and 50 ㎍/mL gentamycin (Invitrogen-Gibco) for use in subsequent experiments.
중간엽 줄기세포의 복제성 노화모델을 제작하기 위해 10% FBS(fetal bovine serum, Invitrogen-Gibco) 및 50 ㎍/mL 젠타마이신(Invitrogen-Gibco)이 포함되어 있는 MEM Alpha(Minimum Essential Medium, Invitrogen-Gibco, Rockville, MD) 배지에 상기 분리된 WJ-MSC를 3 x 103 cells/㎝2의 밀도로 분주하여 37℃ 및 5% CO2 조건하에서 연속적으로 계대배양하였다. To create a replicative senescence model of mesenchymal stem cells, the isolated WJ-MSCs were seeded at a density of 3 x 10 3 cells/cm 2 in MEM Alpha (Minimum Essential Medium, Invitrogen-Gibco, Rockville, MD) medium containing 10% FBS (fetal bovine serum, Invitrogen-Gibco) and 50 ㎍/mL gentamycin (Invitrogen-Gibco) and serially passaged at 37°C and 5% CO 2 .
본 발명에서 "WJ-MSC"는 제대혈의 와튼젤리(waton's jelly) 유래의 인체 중간엽 줄기세포를 의미한다. "젊은 WJ-MSC"로는 passage 5인 노화가 유도되지 않은 중간엽 줄기세포 (senescence-uninduced MSC; U-MSC)를 사용하였으며, “노화 WJ-MSC”는 "젊은 WJ-MSC"를 연속적으로 passage 18이상으로 계대배양한 복제성 노화 중간엽 줄기세포 (replicative senescnece MSC; rS-MSC)를 사용하였다. 도면에서는 "젊은 WJ-MSC"를 “U-MSC“로, “노화 WJ-MSC”는 “rS-MSC“로 표기하였다. In the present invention, "WJ-MSC" refers to human mesenchymal stem cells derived from Wharton's jelly of umbilical cord blood. "Young WJ-MSC" used passage 5 senescence-uninduced MSC (U-MSC), and "senescent WJ-MSC" used replicative senescent mesenchymal stem cells (rS-MSC) that were serially subcultured for passage 18 or more of "young WJ-MSC". In the drawings, "young WJ-MSC" is expressed as "U-MSC", and "senescent WJ-MSC" is expressed as "rS-MSC".
노화 정도를 확인하기 위하여, 젊은 WJ-MSC 그룹과 노화 WJ-MSC 그룹의 노화 관련 베타-갈락토시데이즈(senescence associated beta-galactosidase, SA- β-gal) 염색을 수행하였다. 구체적으로 6웰 플레이트에서 젊은 WJ-MSC 및 노화 WJ-MSC를 각각 10% FBS가 첨가되어 있는 MEM Alpha(Minimum Essential Medium, Invitrogen-Gibco, Rockville, MD) 배지를 이용해 70-80% 밀도가 될 때까지 배양한 뒤 PBS를 이용하여 2번 세척하였다. 이후 1X fixation solution으로 5분동안 실온에서 고정시켰다. PBS를 이용하여 2번 씻어준 뒤 pH6으로 맞춘 SA-β-gal 염색 용액(cell signaling)으로 16시간동안 37℃에서 인큐베이션하였다. 광학현미경을 통해 염색 이미지를 확인하고, image J를 이용하여 β-galactosidase 양성세포를 정량분석하였다.To determine the degree of aging, senescence-associated beta-galactosidase (SA- β-gal) staining was performed on the young and aged WJ-MSC groups. Specifically, young WJ-MSCs and aged WJ-MSCs were cultured in 6-well plates using MEM Alpha (Minimum Essential Medium, Invitrogen-Gibco, Rockville, MD) medium containing 10% FBS until 70-80% confluent, and then washed twice with PBS. Afterwards, they were fixed for 5 minutes at room temperature with 1X fixation solution. After washing twice with PBS, they were incubated for 16 hours at 37°C with SA-β-gal staining solution (cell signaling) adjusted to pH 6. The staining images were confirmed through an optical microscope, and β-galactosidase-positive cells were quantitatively analyzed using Image J.
그 결과, 도 1a에 나타난 바와 같이, 노화 WJ-MSC에서 β-galactosidase 양성 세포가 38.48%로 젊은 WJ-MSC에 비해 유의한 차이로 증가하였다.As a result, as shown in Fig. 1a, β-galactosidase-positive cells in aged WJ-MSCs significantly increased to 38.48% compared to young WJ-MSCs.
젊은 WJ-MSC, 노화 WJ-MSC의 세포증식율(proliferation)을 확인하기 위해 3일 동안 세포 수를 측정하였다. 구체적으로 WJ-MSC를 10% FBS가 첨가되어 있는 MEM Alpha(Minimum Essential Medium, Invitrogen-Gibco, Rockville, MD) 배지를 이용해 6-웰 플레이트에 2 Х103 cells/well로 분주하고, 분주 후 0, 24, 48, 72시간에 hemocytometer기를 이용하여 세포 수를 계수하고, 이를 이용하여 배가시간(doubling time)을 계산하였다. To determine the cell proliferation rate of young and aged WJ-MSCs, cell numbers were measured for 3 days. Specifically, WJ-MSCs were seeded at 2 Х103 cells/well in 6-well plates using MEM Alpha (Minimum Essential Medium, Invitrogen-Gibco, Rockville, MD) medium containing 10% FBS, and the cell number was counted using a hemocytometer at 0, 24, 48, and 72 hours after seeding, and the doubling time was calculated using this.
그 결과, 도 1b에 나타난 바와 같이, 젊은 WJ-MSC에서는 노화 WJ-MSC에 비해 유의하게 빠른 세포증식율을 보였다. 또한, 도 1c에 나타난 바와 같이, 젊은 WJ-MSC의 배가시간(doubling time)은 27.02시간인데 반해, 노화 WJ-MSC의 배가시간(doubling time)은 47.93시간으로 유의하게 증가하였다.As a result, as shown in Fig. 1b, young WJ-MSCs showed a significantly faster cell proliferation rate than aged WJ-MSCs. In addition, as shown in Fig. 1c, the doubling time of young WJ-MSCs was 27.02 hours, whereas that of aged WJ-MSCs was significantly increased to 47.93 hours.
젊은 WJ-MSC, 노화 WJ-MSC에서 노화가 진행됨에 따라 발현이 감소되는 특징을 가지는 핵 단백질인 lamin B1에 대해 면역형광염색(ICC)를 수행하였다. 구체적으로 중간엽 줄기세포를 4% PFA로 상온에서 5분동안 고정한 뒤, 0.25% Trition X-100이 들어있는 PBS로 상온에서 5분동안 permeabilization과정을 수행하였다. 그 다음, 상온에서 1시간동안 blocking solution (2% BSA, 0.1% Tween20 in PBS)으로 blocking과정을 하고 blocking solution에 1차 항체를 희석하여 4℃에서 밤새 반응시켰다. Confocal microscope Zeiss LSM700을 이용하여 결과를 확인하였으며, image J를 통해서 형광 정량분석하였다.Immunofluorescence staining (ICC) was performed on lamin B1, a nuclear protein whose expression is characterized by a decrease with aging in young and aged WJ-MSCs. Specifically, mesenchymal stem cells were fixed with 4% PFA for 5 minutes at room temperature, and then permeabilized with 0.25% Trition X-100 in PBS for 5 minutes at room temperature. Then, the cells were blocked with blocking solution (2% BSA, 0.1% Tween 20 in PBS) for 1 hour at room temperature, and the primary antibody was diluted in the blocking solution and reacted overnight at 4℃. The results were confirmed using a confocal microscope Zeiss LSM700, and fluorescence quantitative analysis was performed using Image J.
그 결과, 도 1d에 나타난 바와 같이, 젊은 WJ-MSC에 비해 노화 WJ-MSC에서 노화가 진행됨에 따라 lamin B1의 발현양이 38.37% 약해진 것을 확인할 수 있었다.As a result, as shown in Fig. 1d, it was confirmed that the expression of lamin B1 was weakened by 38.37% as aging progressed in aged WJ-MSCs compared to young WJ-MSCs.
상기 결과를 통해서 중간엽 줄기세포를 이용한 복제성 노화모델을 수립하였다. Through the above results, a replicative aging model using mesenchymal stem cells was established.
실시예 2. 인체 중간엽 줄기세포에서 GLS1 억제로 인한 노화 줄기세포 선택적 사멸 확인Example 2. Confirmation of selective death of senescent stem cells due to GLS1 inhibition in human mesenchymal stem cells
노화 WJ-MSC 및 젊은 WJ-MSC에서의 GLS1의 발현을 확인하였다. 중간엽 줄기세포에 AccuPrep Universal RNA Extraction Kit을 이용하여 중간엽 줄기세포에서 RNA를 추출하였고, 2X Power SYBR Green Master Mix (AB)를 사용하여 qRT-PCR을 수행하였다.The expression of GLS1 in aged WJ-MSCs and young WJ-MSCs was confirmed. RNA was extracted from mesenchymal stem cells using the AccuPrep Universal RNA Extraction Kit, and qRT-PCR was performed using 2X Power SYBR Green Master Mix (AB).
그 결과, 도 2a에 나타난 바와 같이, GLS1의 mRNA 발현이 젊은 WJ-MSC에 비해 노화 WJ-MSC에서 크게 증가된 것을 확인하였다.As a result, as shown in Fig. 2a, it was confirmed that the mRNA expression of GLS1 was significantly increased in aged WJ-MSCs compared to young WJ-MSCs.
아울러, 젊은 WJ-MSC 그룹 및 노화 WJ-MSC 그룹의 GLS1의 단백질 발현을 Western blot으로 확인하였다. In addition, the protein expression of GLS1 in the young WJ-MSC group and the aged WJ-MSC group was confirmed by Western blot.
구체적으로, 중간엽 줄기세포를 배양 후 PBS로 세척한 후, protease inhibitor cocktail (Amresco, Solon, OH, USA)을 넣은 RIPA 버퍼(BIOSESANG, Sungnam, Gyeonggi, Korea)로 용해시킨 뒤, 4℃, 15,000g에서 30분 동안 원심분리하여 상층액을 수득하였다. 10 ㎍의 단백질을 SDS-PAGE를 통해 크기 별로 분리되도록 전기영동한 뒤, PVDF (polyvinylidene difluoride) 멤브레인에 옮겼다. 상기 멤브레인을 5% skim milk가 포함된 TBST로 상온에서 1시간 동안 블로킹을 수행하고, 5% skim milk가 포함된 TBST에 1차 항체를 희석하여 4℃에서 하룻밤 반응시켰다. 이후, 멤브레인을 TBST로 10분간 3회 씻어낸 뒤 다시 5% skim milk가 포함된 TBST에 2차 항체를 희석하여 상온에서 1시간 동안 반응시켰다. 이후, 멤브레인을 TBST로 10분씩 3회 씻어내고 ECL solution(Advansta, USA)을 처리한 뒤 gel imaging system (Amersham Imager 600, GE Healthcare, Buckinghamshire, UK)에서 밴드의 이미지를 확인하였다. 단백질의 발현 정도는 image J를 이용하여 측정하고 β-actin으로 보정하였다. 1차 항체는 GLS1, β-actin (Santa Cruz Biotechnology, Dallas, TX, USA)을 사용하였다.Specifically, after culturing mesenchymal stem cells, they were washed with PBS and lysed with RIPA buffer (BIOSESANG, Sungnam, Gyeonggi, Korea) containing protease inhibitor cocktail (Amresco, Solon, OH, USA), centrifuged at 4°C, 15,000g for 30 minutes, and the supernatant was obtained. 10 μg of protein was electrophoresed to separate by size using SDS-PAGE and transferred to a PVDF (polyvinylidene difluoride) membrane. The membrane was blocked with TBST containing 5% skim milk for 1 hour at room temperature, and the primary antibody was diluted in TBST containing 5% skim milk and reacted overnight at 4°C. Thereafter, the membrane was washed three times for 10 minutes with TBST, and the secondary antibody was diluted in TBST containing 5% skim milk and reacted for 1 hour at room temperature. Afterwards, the membrane was washed three times with TBST for 10 minutes each, treated with ECL solution (Advansta, USA), and the band image was confirmed in a gel imaging system (Amersham Imager 600, GE Healthcare, Buckinghamshire, UK). The protein expression level was measured using Image J and corrected with β-actin. The primary antibody used was GLS1, β-actin (Santa Cruz Biotechnology, Dallas, TX, USA).
그 결과, 도 2b에 나타난 바와 같이, 노화 WJ-MSC에서 GLS1의 단백질 발현이 젊은 WJ-MSC에 비해 증가하는 것을 확인하였다.As a result, as shown in Fig. 2b, it was confirmed that the protein expression of GLS1 increased in aged WJ-MSCs compared to young WJ-MSCs.
노화 WJ-MSC에서 높게 발현되는 GLS1이 노화 WJ-MSC의 생존력에 끼치는 영향을 확인하기 위하여 WJ-MSC에 siGLS1을 형질주입 후 AccuPrep Universal RNA Extraction Kit을 이용하여 RNA를 추출하였고, 2X Power SYBR Green Master Mix (AB)를 사용하여 qRT-PCR을 수행하였다(표 1 참조).To determine the effect of GLS1, which is highly expressed in aged WJ-MSCs, on the viability of aged WJ-MSCs, siGLS1 was transfected into WJ-MSCs, RNA was extracted using the AccuPrep Universal RNA Extraction Kit, and qRT-PCR was performed using 2X Power SYBR Green Master Mix (AB) (see Table 1).
구체적으로, 성장 배지에서 50%정도 배양된 중간엽 줄기세포를 무혈청 배지로 옮긴 뒤 siRNA 라이브러리에서 GLS1에 대한 2가지 후보 서열(bioneer)(표 2)을 lipofectamine RNAiMax(invitrogen)을 이용하여 25nM의 농도로 형질 주입하였다. 형질주입 72시간 후, 실시예 2와 동일한 방법으로 qRT-PCR을 수행하였다. Specifically, mesenchymal stem cells cultured at about 50% confluence in growth medium were transferred to serum-free medium, and two candidate sequences (bioneer) (Table 2) for GLS1 from the siRNA library were transfected at a concentration of 25 nM using lipofectamine RNAiMax (Invitrogen). 72 hours after transfection, qRT-PCR was performed using the same method as in Example 2.
그 결과, 도 2c에서 나타난 바와 같이, 젊은 WJ-MSC와 노화 WJ-MSC에서 75%이상 GLS1의 knock down 됨을 확인할 수 있었다.As a result, as shown in Fig. 2c, it was confirmed that GLS1 was knocked down by more than 75% in young WJ-MSCs and aged WJ-MSCs.
젊은 WJ-MSC와 노화 WJ-MSC에 실시예 1과 동일한 방법으로 siGLS1 형질전환 후, 세포 생존력(cell viabiliity)을 확인하기 위해 CCK8 assay를 수행하였다.After siGLS1 transfection into young and aged WJ-MSCs using the same method as in Example 1, a CCK8 assay was performed to confirm cell viability.
그 결과, 도 2d에서 나타남과 같이 노화 WJ-MSC에서는 GLS1 knock down으로 인해 세포 생존력이 유의하게 억제함을 확인하였다. As a result, as shown in Fig. 2d, it was confirmed that cell viability was significantly inhibited due to GLS1 knock down in aged WJ-MSCs.
젊은 WJ-MSC 또는 노화 WJ-MSC에서 BPTES을 30μM로 처리 후, 세포생존력(cell viabiliity)을 확인하기 위해 CCK8 assay를 수행하였다. 구체적으로 중간엽 줄기세포에 BPTES의 용매인 DMSO(대조군) 또는 BPTES 30μM을 72시간동안 처리 후, 실시예1과 동일한 방법으로 CCK8 assay를 수행하였다.After treating young or aged WJ-MSCs with 30 μM BPTES, a CCK8 assay was performed to confirm cell viability. Specifically, mesenchymal stem cells were treated with DMSO (control) or 30 μM BPTES, a solvent for BPTES, for 72 hours, and the CCK8 assay was performed using the same method as in Example 1.
이를 통해서, 노화 WJ-MSC에서 증가한 GLS1은 노화 WJ-MSC의 세포생존력에 관여하며, GLS1 저해제인 BPTES를 사용하여 노화세포만을 선택적으로 사멸시킬 수 있다는 것을 확인하였다.Through this, we confirmed that increased GLS1 in senescent WJ-MSCs is involved in the cell viability of senescent WJ-MSCs, and that BPTES, a GLS1 inhibitor, can selectively kill only senescent cells.
실시예 3. 인체 중간엽 줄기세포에서 BPTES 처리 후 노화억제 확인Example 3. Confirmation of inhibition of aging after BPTES treatment in human mesenchymal stem cells
글루타민 분해(Glutaminolysis)는 글루타민(glutamine)이 글루타메이트(glutamate)로 분해된 다음 α-케토글루타레이트(α-ketoglutarate) 등의 TCA 회로 대사산물로 분해되는 대사과정이다. GLS1이 글루타민이 글루타메이트로 전환되는 과정을 촉매하는 효소이기 때문에 이를 억제하는 BPTES를 처리하였을 때, 글루타민 분해 과정의 산물 변화를 확인하였다. Glutaminolysis is a metabolic process in which glutamine is broken down into glutamate and then into TCA cycle metabolites such as α-ketoglutarate. Since GLS1 is an enzyme that catalyzes the process of converting glutamine to glutamate, when BPTES, which inhibits it, was treated, changes in the products of the glutaminolysis process were confirmed.
노화 WJ-MSC에 BPTES 30μM 처리 후 GLS1의 활성 억제를 확인하기 위해 글루타민, 글루타메이트, α-케토글루타레이트의 수준을 확인하였다. 세포배양배지 및 세포 내의 글루타민 정도를 확인하기 위해 글루타민 어세이를 수행하였다. 구체적으로 노화 WJ-MSC에 DMSO 또는 BPTES 30μM을 24시간 동안 처리한 후, 세포배양배지와 세포를 수확하였다. 글루타민 어세이 키트(Abnova)를 이용하여 세포배양배지 내의 글루타민을 측정하였고, 세포 내의 글루타민은 글루타민 어세이 키트(dojindo)를 이용하여 측정하였다.To confirm the inhibition of GLS1 activity after BPTES 30 μM treatment in aged WJ-MSCs, the levels of glutamine, glutamate, and α-ketoglutarate were determined. Glutamine assay was performed to determine the level of glutamine in the cell culture medium and cells. Specifically, aged WJ-MSCs were treated with DMSO or BPTES 30 μM for 24 hours, and then the cell culture medium and cells were harvested. Glutamine in the cell culture medium was measured using a glutamine assay kit (Abnova), and glutamine in the cells was measured using a glutamine assay kit (dojindo).
그 결과, 도 3b와 도 3c에 나타난 바와 같이, 노화 WJ-MSC에 BPTES 처리시 세포배양액 내의 글루타민 농도와 세포 내의 글루타민 농도가 대조군에 비해 증가한 것을 확인할 수 있었다.As a result, as shown in Figures 3b and 3c, it was confirmed that the glutamine concentration in the cell culture medium and the glutamine concentration within the cells increased compared to the control group when BPTES was treated in aged WJ-MSCs.
노화 WJ-MSC에서 BPTES처리 후, 세포 내의 글루타메이트 수준을 확인하기 위해 글루타메이트 어세이를 수행하였다. 구체적으로 노화 WJ-MSC에 DMSO 또는 BPTES 30μM을 24시간동안 처리 후, 세포를 수확하였다. 세포 내의 글루타메이트는 글루타메이트 어세이 키트(dojindo)를 이용하여 측정하였다.To determine the intracellular glutamate levels after BPTES treatment in aged WJ-MSCs, a glutamate assay was performed. Specifically, aged WJ-MSCs were treated with 30 μM DMSO or BPTES for 24 h, and then the cells were harvested. Intracellular glutamate was measured using a glutamate assay kit (dojindo).
그 결과, 도 3d에 나타난 바와 같이, 노화 WJ-MSC에 BPTES 처리시 세포내의 글루타메이트 농도가 대조군에 비해 감소한 것을 확인할 수 있었다.As a result, as shown in Fig. 3d, it was confirmed that the intracellular glutamate concentration decreased when BPTES was treated in aged WJ-MSCs compared to the control group.
노화 WJ-MSC에서 BPTES처리 후, 세포 내의 α-케토글루타레이트 수준을 확인하기 위해 α-케토글루타레이트 어세이를 수행하였다. 구체적으로 노화 WJ-MSC에 DMSO 또는 BPTES 30μM을 24시간동안 처리 후, 세포를 수확하였다. 세포 내의 α-케토글루타레이트는 α-케토글루타레이트 어세이 키트(dojindo)를 이용하여 측정하였다.To determine the intracellular α-ketoglutarate level after BPTES treatment in aged WJ-MSCs, an α-ketoglutarate assay was performed. Specifically, aged WJ-MSCs were treated with 30 μM DMSO or BPTES for 24 h, and then the cells were harvested. Intracellular α-ketoglutarate was measured using an α-ketoglutarate assay kit (dojindo).
그 결과, 도 3e에 나타난 바와 같이, 노화 WJ-MSC에 BPTES 처리시 세포내의 α-케토글루타레이트 농도가 대조군에 비해 감소한 것을 확인할 수 있었다.As a result, as shown in Fig. 3e, it was confirmed that the intracellular α-ketoglutarate concentration decreased compared to the control group when BPTES was treated in aged WJ-MSCs.
따라서, 노화 WJ-MSC에 BPTES를 처리하여 GLS1을 억제하면 글루타민이 글루타메이트로 변환되지 않아 글루타민이 축적되고 글루타메이트와 α-케토글루타레이트의 생성이 감소되는 것을 확인함하여, 30μM의 BPTES가 노화 WJ-MSC에서 글루타민 분해를 정상적으로 억제시키는 것을 확인하였다.Therefore, we confirmed that when BPTES was treated in aged WJ-MSCs to inhibit GLS1, glutamine was not converted to glutamate, resulting in glutamine accumulation and a decrease in the production of glutamate and α-ketoglutarate, confirming that 30 μM BPTES normally inhibited glutaminolysis in aged WJ-MSCs.
BPTES 처리후 72시간 동안 노화 WJ-MSC의 세포생존력(cell viability)을 확인하기 위해 실시예 1과 동일한 방법으로 세포 수를 측정하였다. To confirm the cell viability of aged WJ-MSCs for 72 hours after BPTES treatment, the cell number was measured using the same method as in Example 1.
그 결과, 도 3f에서 나타남과 같이 BPTES처리시 24시간부터 노화 WJ-MSC의 생존력에 크게 영향을 주는 것을 확인하였다. 따라서 이후 실험에서 BPTES를 24시간 처리 후 노화 억제를 확인하였다.As a result, as shown in Fig. 3f, it was confirmed that BPTES treatment significantly affected the viability of aged WJ-MSCs from 24 hours. Therefore, in the subsequent experiment, inhibition of aging was confirmed after 24 hours of BPTES treatment.
노화 WJ-MSC에 BPTES 30μM로 24시간동안 처리한 후, 실시예 2와 동일한 방법으로 Western blot을 통해 확인하였다. 1차 항체로 P16 및 P21 (cell signaling), GLB1(Abcam), β-actin (Santa Cruz Biotechnology)을 사용하였다.After treating aged WJ-MSCs with 30 μM BPTES for 24 hours, Western blot was performed in the same manner as in Example 2. P16 and P21 (cell signaling), GLB1 (Abcam), and β-actin (Santa Cruz Biotechnology) were used as primary antibodies.
도 3g는 노화관련 유전자의 단백질 발현 정도를 평가하기 위해 p16 및 p21에서 Western blot을 진행한 결과로, 젊은 WJ-MSC보다 노화 WJ-MSC에서 증가하였던 노화관련 단백질 발현 정도가 BPTES 처리시 유의하게 감소한 것을 확인할 수 있었다. Figure 3g shows the results of Western blot performed on p16 and p21 to evaluate the level of protein expression of aging-related genes. It was confirmed that the level of aging-related protein expression, which was increased in aged WJ-MSCs compared to young WJ-MSCs, was significantly reduced when treated with BPTES.
노화관련 유전자의 mRNA level을 평가하기 위해, 실시예 2와 동일한 방법으로 p16, p21에 대해 qRT-PCR을 진행하였다. To evaluate the mRNA levels of aging-related genes, qRT-PCR was performed on p16 and p21 using the same method as in Example 2.
그 결과, 도 3h에 나타난 바와 같이, 젊은 WJ-MSC보다 노화 WJ-MSC에서 증가하였던 노화관련 mRNA 발현 정도가 BPTES 처리시 노화 WJ-MSC에 비해 유의하게 감소한 것을 확인할 수 있었다. As a result, as shown in Fig. 3h, it was confirmed that the level of aging-related mRNA expression, which was increased in aged WJ-MSCs compared to young WJ-MSCs, was significantly reduced compared to aged WJ-MSCs when treated with BPTES.
세포 노화로 인해 증가하는 노화 관련 분비 표현형(Senescence associated secretion phenotype, SASP) 유전자의 발현을 확인하기 위하여 qRT-PCR을 수행하였다.To confirm the expression of senescence associated secretion phenotype (SASP) genes that increase with cellular senescence, qRT-PCR was performed.
SASP 관련 유전자의 mRNA 수준은 실시예 2와 동일한 방법으로 IGFBP3, IGFBP5 및 IGFBP7에 대한 qRT-PCR을 통해 확인하였다. The mRNA levels of SASP-related genes were confirmed through qRT-PCR for IGFBP3, IGFBP5, and IGFBP7 using the same method as in Example 2.
그 결과, 도 3i에 나타난 바와 같이, SASP 관련 유전자의 발현이 젊은 WJ-MSC에 비하여 노화 WJ-MSC에서 증가하였고 BPTES 처리시 노화 WJ-MSC에 비해 유의하게 감소된 것을 확인할 수 있었다.As a result, as shown in Fig. 3i, it was confirmed that the expression of SASP-related genes increased in aged WJ-MSCs compared to young WJ-MSCs and significantly decreased compared to aged WJ-MSCs when treated with BPTES.
상기 결과를 통해서 GLS1이 WJ-MSC의 복제성 노화 조절에 중요한 역할을 한다는 것을 확인하였다.Through the above results, we confirmed that GLS1 plays an important role in regulating replicative senescence of WJ-MSCs.
실시예 4. 인체 중간엽 줄기세포에서 BPTES 처리 후 줄기세포능 확인Example 4. Confirmation of stem cell capacity after BPTES treatment in human mesenchymal stem cells
젊은 WJ-MSC와 노화 WJ-MSC에 DMSO 또는 BPTES 30μM을 24시간동안 처리한 후, FACS를 수행하여 줄기세포능을 확인하였다. After treating young and aged WJ-MSCs with 30 μM DMSO or BPTES for 24 hours, stem cell potential was confirmed by performing FACS.
구체적으로 중간엽 줄기세포의 마커는 CD44, CD73, CD90, CD105 및 CD166의 발현을 통해 확인하였고, 조혈모세포 계통의 마커는 CD14, CD11b, HLA-DR (MHCII), CD34, CD45 및 CD19 (BD Biosciences, USA)을 사용하여 줄기세포능을 비교하였다. 이때 BD FACS Verse flow cytometer를 이용하여 10000 event를 획득하여 분석하였다.Specifically, markers of mesenchymal stem cells were identified through the expression of CD44, CD73, CD90, CD105, and CD166, and markers of hematopoietic stem cell lineage were used to compare stem cell potential using CD14, CD11b, HLA-DR (MHCII), CD34, CD45, and CD19 (BD Biosciences, USA). At this time, 10,000 events were acquired and analyzed using a BD FACS Verse flow cytometer.
그 결과, 도 4에 나타낸 바와 같이, 젊은 WJ-MSC는 positive marker인 CD44, CD73, CD90, CD105 및 CD166은 99% 이상 발현하지만, negative marker인 CD14, CD11b, HLA-DR (MHCII), CD34, CD45 및 CD19는 1.5% 미만으로 거의 발현하지 않았다. 또한, 젊은 WJ-MSC에 BPTES 처리시 양성 마커와 음성 마커의 발현에 차이를 보이지 않았다. 반면, 노화 WJ-MSC는 젊은 WJ-MSC에 비해 CD90과 CD166의 발현이 감소하였으나, BPTES 처리 시 다시 93% 이상으로 증가하였다. As a result, as shown in Fig. 4, young WJ-MSCs expressed positive markers CD44, CD73, CD90, CD105, and CD166 at more than 99%, but negative markers CD14, CD11b, HLA-DR (MHCII), CD34, CD45, and CD19 at less than 1.5%. In addition, when young WJ-MSCs were treated with BPTES, there was no difference in the expression of positive and negative markers. On the other hand, the expression of CD90 and CD166 in aged WJ-MSCs decreased compared to young WJ-MSCs, but increased again to more than 93% when treated with BPTES.
즉, 중간엽 줄기세포에서 노화가 진행되었을 때 줄기세포능(stemness)가 감소하였으나 BPTES 처리시 줄기세포능이 회복되는 것을 확인하였다.That is, it was confirmed that stemness decreased when aging progressed in mesenchymal stem cells, but stemness was restored when treated with BPTES.
실시예 5. 인체 중간엽 줄기세포 노화모델에서 GLS1 넉다운(knock down) 후 노화 억제 확인Example 5. Confirmation of inhibition of aging after GLS1 knockdown in a human mesenchymal stem cell aging model
노화 WJ-MSC에 siGLS1를 형질전환한 후, 세포생존력(cell viability)을 확인하기 위해 실시예 1과 동일한 방법으로 수행하였다. After transfecting aged WJ-MSCs with siGLS1, cell viability was confirmed using the same method as in Example 1.
그 결과, 도 5a에서 나타남과 같이 GLS1 넉다운(knock down) 시 노화 WJ-MSC가 사멸되는 것을 확인하였다. As a result, it was confirmed that aged WJ-MSCs were killed when GLS1 was knocked down, as shown in Fig. 5a.
이러한 GLS1에 의한 노화세포의 사멸로 인해 노화가 억제되는지 확인하기 위해 실시예 2와 동일한 방법으로 노화 WJ-MSC에 siGLS1 형질주입 후, qRT-PCR을 수행하였다.To confirm whether aging is suppressed by the death of senescent cells by GLS1, siGLS1 was transfected into senescent WJ-MSCs using the same method as in Example 2, and then qRT-PCR was performed.
그 결과, 도 5b에서 나타남과 같이, 노화 WJ-MSC에서 80%이상 GLS1의 넉다운되는 것을 확인할 수 있었다. 또한, 노화 관련 유전자 p16이 siGLS1 형질주입 하였을 때 유의하게 감소하는 것을 확인하였다.As a result, as shown in Fig. 5b, it was confirmed that GLS1 was knocked down by more than 80% in aged WJ-MSCs. In addition, it was confirmed that the aging-related gene p16 was significantly reduced when siGLS1 was transfected.
아울러, 노화 WJ-MSC에서 siNC그룹과 siGLS1그룹의 GLS1 발현 및 노화관련 단백질 발현을 실시예 2와 동일한 방법으로 Western blot을 수행하였다. 1차 항체로 P16, P21 (cell signaling), GLS1, GLB1(Abcam) 및 β-actin (Santa Cruz Biotechnology)을 사용하였다.In addition, Western blot was performed on the expression of GLS1 and senescence-related proteins in the siNC group and siGLS1 group in aged WJ-MSCs using the same method as in Example 2. P16, P21 (cell signaling), GLS1, GLB1 (Abcam), and β-actin (Santa Cruz Biotechnology) were used as primary antibodies.
그 결과, 도 5c에 나타난 바와 같이, GLS1의 넉 다운 시 노화관련 유전자의 단백질인 p16, p21 및 GLB1의 발현이 감소하는 것을 확인하였다.As a result, as shown in Fig. 5c, it was confirmed that the expression of p16, p21, and GLB1, which are proteins of aging-related genes, decreased when GLS1 was knocked down.
노화 WJ-MSC에서 GLS1 넉 다운으로 인한 SASP의 감소를 확인하기 위해 SASP 관련 유전자인 IGFBP3, IGFBP5 및 IGFBP7의 mRNA 수준을 실시예 2와 동일한 방법으로 qRT-PCR을 수행하였다. To confirm the decrease in SASP due to GLS1 knockdown in aged WJ-MSCs, qRT-PCR was performed to determine the mRNA levels of SASP-related genes IGFBP3, IGFBP5, and IGFBP7 using the same method as in Example 2.
그 결과, 도 5d에 나타난 바와 같이, SASP 관련 유전자의 발현이 노화 WJ-MSC 대조군에 비하여 siGLS1 형질전환된 세포에서 감소된 것을 확인할 수 있었다.As a result, as shown in Fig. 5d, it was confirmed that the expression of SASP-related genes was reduced in siGLS1-transfected cells compared to the aged WJ-MSC control group.
이를 통해서 노화 WJ-MSC에서 GLS1 유전자가 직접적으로 노화를 억제하는데 관여하는 것을 확인하였다.Through this, we confirmed that the GLS1 gene is directly involved in suppressing aging in aged WJ-MSCs.
실시예 6. 인체 중간엽 줄기세포에서 다른 GLS1 저해제 처리 시 노화 억제 확인Example 6. Confirmation of inhibition of aging by treatment with different GLS1 inhibitors in human mesenchymal stem cells
BPTES 이외의 GLS1 저해제인 CB-839와 C968처리 후, 노화 WJ-MSC에 대한 영향을 추가적으로 확인하였다. We additionally examined the effects of CB-839 and C968, GLS1 inhibitors other than BPTES, on senescent WJ-MSCs.
구체적으로 CB-839(1μM)와 C968(10μM)를 24시간 동안 처리 후, 노화 WJ-MSC의 세포생존력 (cell viability)을 확인하기 위해 CCK8 assay를 실시예 1 과 동일한 방법으로 수행하였다. Specifically, to confirm the cell viability of senescent WJ-MSCs after treatment with CB-839 (1 μM) and C968 (10 μM) for 24 hours, a CCK8 assay was performed using the same method as in Example 1.
그 결과, 도 6a에 나타난 바와 같이, 노화 WJ-MSC에 DMSO를 처리한 대조군에 비해 CB-839(1μM)와 C968(10μM)를 처리하였을 때 세포생존력이 유의하게 감소하는 것을 확인하였다.As a result, as shown in Fig. 6a, it was confirmed that cell viability significantly decreased when CB-839 (1 μM) and C968 (10 μM) were treated compared to the control group treated with DMSO in aged WJ-MSCs.
GLS1 저해제인 CB-839와 C968처리 후, 노화 관련 유전자의 mRNA발현을 확인하기 위해 실시예 2와 동일한 방법으로 qRT-PCR을 수행하였다.To confirm the mRNA expression of aging-related genes after treatment with GLS1 inhibitors CB-839 and C968, qRT-PCR was performed using the same method as in Example 2.
그 결과, 도 6b에서 나타난 바와 같이, 노화 WJ-MSC에 GLS1 저해제인 CB-839와 C968 처리시 p21과 p16의 유전자 발현이 유의하게 감소하는 것을 확인하였다.As a result, as shown in Fig. 6b, it was confirmed that the gene expression of p21 and p16 was significantly reduced when aging WJ-MSCs were treated with GLS1 inhibitors, CB-839 and C968.
또한, 노화 WJ-MSC에서 CB-839(1μM)와 C968(10μM) 처리 후, 노화관련 단백질 발현을 실시예 2와 동일한 방법으로 Western blot을 수행하였다. 1차 항체로 P16, P21 (cell signaling), GLB1(Abcam) 및 GAPDH(Santa Cruz Biotechnology)을 사용하였다.In addition, after treatment with CB-839 (1 μM) and C968 (10 μM) in aged WJ-MSCs, Western blot was performed using the same method as in Example 2 to determine the expression of senescence-related proteins. P16, P21 (cell signaling), GLB1 (Abcam), and GAPDH (Santa Cruz Biotechnology) were used as primary antibodies.
그 결과, 도 6c에서 나타난 바와 같이, 노화 WJ-MSC에 GLS1 저해제인 CB-839와 C968처리시 p21, p16, GLB1의 단백질 발현이 감소되는 것을 확인하였다.As a result, as shown in Fig. 6c, it was confirmed that the protein expression of p21, p16, and GLB1 was reduced when CB-839 and C968, which are GLS1 inhibitors, were treated in aged WJ-MSCs.
노화 WJ-MSC에서 CB-839(1μM)와 C968(10μM) 처리 시, SASP 관련 유전자의 mRNA 수준을 확인하기 위해 실시예 2와 동일한 방법으로 IGFBP3, IGFBP5 및 IGFBP7에 대한 qRT-PCR을 수행하였다. To confirm the mRNA levels of SASP-related genes when CB-839 (1 μM) and C968 (10 μM) were treated in aged WJ-MSCs, qRT-PCR for IGFBP3, IGFBP5, and IGFBP7 was performed using the same method as in Example 2.
그 결과, 도 6d에 나타난 바와 같이, 노화 WJ-MSC에서 DMSO를 처리한 대조군에 비하여 CB-839와 C968처리 시, IGFBP5는 유의하게 감소되었고, IGFBP3과 IGFBP7은 감소하는 경향성을 확인할 수 있었다.As a result, as shown in Fig. 6d, compared to the control group treated with DMSO in aged WJ-MSCs, IGFBP5 was significantly reduced when treated with CB-839 and C968, and IGFBP3 and IGFBP7 showed a tendency to decrease.
이를 통해서, BPTES 이외의 GLS1 저해제인 CB-839와 C968처리를 하였을 때에도 WJ-MSC에서 노화가 억제되는 것을 확인하였다. 이는 BPTES 약물 특이적인 노화억제효과가 아닌 GLS1 억제를 통한 노화 억제임을 나타낸다.Through this, we confirmed that senescence was suppressed in WJ-MSCs even when treated with CB-839 and C968, which are GLS1 inhibitors other than BPTES. This indicates that senescence inhibition is through GLS1 inhibition and not a drug-specific senescence inhibition effect of BPTES.
실시예 7. 노화 WJ-MSC에서 BPTES로 인한 노화세포 선택적 사멸 (senolysis)를 활용하여 세포증식능이 향상된 노화억제세포 획득Example 7. Obtaining anti-senescence cells with enhanced cell proliferation by utilizing selective senescent cell death (senolysis) induced by BPTES in aged WJ-MSCs
도 7은 노화 WJ-MSC에 GLS1 억제제를 처리한 뒤, 회복(recovery)후 노화억제 및 노화 억제세포의 in vitro 치료 효능을 평가한 결과이다.Figure 7 shows the results of evaluating the in vitro therapeutic efficacy of anti-aging and anti-aging cells after recovery of aged WJ-MSCs treated with a GLS1 inhibitor.
구체적으로 노화 WJ-MSC에 BPTES(30μM)를 24시간 동안 처리 후 일반 배지로 바꿔서 회복(recovery) 후 노화가 경감된 세포(replicative senescence-alleviated MSC ; rSA-MSC)를 확인하였고, 이를 “노화억제 WJ-MSC“라 명명하였다. 노화억제 WJ-MSC의 노화 정도를 평가하기 위해 실시예 1과 동일한 방법으로 SA-β-gal 염색을 수행하였다. Specifically, after treating aged WJ-MSCs with BPTES (30 μM) for 24 hours and then replacing them with normal medium, cells with alleviated senescence (replicative senescence-alleviated MSC; rSA-MSC) were identified after recovery, and these were named “senescence-inhibited WJ-MSCs.” To evaluate the degree of senescence of senescence-inhibited WJ-MSCs, SA-β-gal staining was performed using the same method as in Example 1.
그 결과, 도 7b에 나타난 바와 같이, 노화 WJ-MSC에서 26.33%로 증가하였던 β-galactosidase 양성세포는 노화억제 WJ-MSC에서 5.82%로 감소함에 따라 노화가 경감되었음을 확인하였다.As a result, as shown in Fig. 7b, β-galactosidase positive cells, which increased to 26.33% in aged WJ-MSCs, decreased to 5.82% in anti-aging WJ-MSCs, confirming that aging was alleviated.
노화가 진행되면 세포의 면적이 넓어지므로 젊은 WJ-MSC, 노화 WJ-MSC, 노화 억제 WJ-MSC의 세포 면적을 image J 소프트웨어를 이용하여 측정하였다. As aging progresses, the cell area increases, so the cell area of young WJ-MSCs, senescent WJ-MSCs, and senescence-inhibited WJ-MSCs was measured using Image J software.
그 결과, 도 7c에 나타난 바와 같이, 젊은 WJ-MSC보다 노화 WJ-MSC에서 증가하였던 세포 면적이 노화 억제 WJ-MSC에서 유의하게 감소한 것을 확인할 수 있었다. As a result, as shown in Fig. 7c, it was confirmed that the cell area, which had increased in aged WJ-MSCs compared to young WJ-MSCs, significantly decreased in aged-inhibited WJ-MSCs.
도 7d는 노화 WJ-MSC, 노화억제 WJ-MSC의 배가시간(doubling time)을 확인하기 위해 실시예 1과 동일한 방법으로 CCK8 assay를 수행한 결과를 나타낸 것이다. Figure 7d shows the results of a CCK8 assay performed using the same method as Example 1 to confirm the doubling time of senescent WJ-MSCs and senescence-inhibited WJ-MSCs.
그 결과, 도 7d에 나타난 바와 같이, 노화 WJ-MSC의 배가시간은 56.67시간으로 노화로 인해 세포 증식능이 감소되지만, 노화억제 WJ-MSC에서는 42.86시간으로 세포 증식능이 노화 WJ-MSC에 비해 향상되는 것을 확인하였다.As a result, as shown in Fig. 7d, the doubling time of aged WJ-MSCs was 56.67 hours, indicating that cell proliferation ability decreased due to aging, but in senescence-inhibited WJ-MSCs, it was 42.86 hours, confirming that cell proliferation ability was improved compared to aged WJ-MSCs.
따라서 노화 WJ-MSC에서 BPTES로 인한 선택적 사멸(senolysis)을 활용하여 세포증식능이 향상된 노화억제 WJ-MSC를 획득할 수 있었다.Therefore, we were able to obtain anti-senescence WJ-MSCs with enhanced cell proliferation potential by utilizing BPTES-induced selective senolysis in aged WJ-MSCs.
도 7e는 노화 WJ-MSC에 GLS1 저해제인 CB-839 1μM 또는 C968 10μM을 24시간동안 처리 후, 노화 정도를 평가하기 위해 실시예 1과 동일한 방법으로 β-gal 염색을 수행하였다. Figure 7e shows that after treating aged WJ-MSCs with 1 μM CB-839 or 10 μM C968, a GLS1 inhibitor, for 24 hours, β-gal staining was performed using the same method as in Example 1 to evaluate the degree of senescence.
그 결과, GLS1 저해제 CB-839, C968에서도 β-galactosidase 양성세포 비율이 노화 WJ-MSC에서 감소하는 것을 확인하였다.As a result, we confirmed that the proportion of β-galactosidase-positive cells decreased in aged WJ-MSCs even with GLS1 inhibitors CB-839 and C968.
실시예 8. 노화 억제 WJ-MSC의 근육세포에 대한 항세포사멸 효능 확인Example 8. Confirmation of anti-apoptotic effect of WJ-MSC on muscle cells that inhibits aging
도 8은 노화억제 WJ-MSC의 근육세포 대한 항세포사멸 효능을 확인한 결과이다. 구체적으로 항세포사멸 효능은 세포사멸을 유도한 근육세포와 WJ-MSC를 공동배양하여 확인하였다. serum starvation을 통해 근육세포에 세포사멸을 유도한 뒤 24시간동안 젊은 WJ-MSC, 노화 WJ-MSC, 노화억제 WJ-MSC와 각각 공동배양을 진행하였다. Figure 8 shows the results of confirming the anti-apoptotic effect of anti-aging WJ-MSC on muscle cells. Specifically, the anti-apoptotic effect was confirmed by co-culturing WJ-MSC with muscle cells that had undergone apoptosis. After induced apoptosis in muscle cells through serum starvation, co-culturing was performed with young WJ-MSC, aged WJ-MSC, and anti-aging WJ-MSC for 24 hours.
도 8a는 세포사멸이 유도된 근육세포를 젊은 WJ-MSC, 노화 WJ-MSC, 노화억제 WJ-MSC와 공동배양하여 세포의 세포사멸 마커인 cleaved PARP와 cleaved caspase 3의 단백질 발현 수준을 확인하기 위해 실시예 1과 동일한 방법으로 western blot을 수행한 결과를 나타낸 것이다. 1차 항체는 cleaved PARP, cleaved caspase 3(Cell signaling) 및 β-actin(Santa Cruz Biotechnology)을 사용하였다.Figure 8a shows the results of western blot performed in the same manner as Example 1 to confirm the protein expression levels of cleaved PARP and cleaved caspase 3, which are apoptosis markers, in co-cultured apoptotic muscle cells with young WJ-MSCs, senescent WJ-MSCs, and senescence-inhibited WJ-MSCs. The primary antibodies used were cleaved PARP, cleaved caspase 3 (Cell signaling), and β-actin (Santa Cruz Biotechnology).
그 결과, cleaved PAPR와 cleaved capase 3의 발현은 정상(Normal) 근육세포보다 무혈청 배지(SF media)로 인해 세포사멸이 유도된 근육세포인 대조군에서 증가하였다. 또한, 이러한 마커들의 발현은 대조군과 비교하여 노화 WJ-MSC와 공동배양한 근육세포 그룹에서는 차이가 없었으나, 젊은 WJ-MSC, 노화억제 WJ-MSC와 공동배양한 근육세포에서는 유의미하게 감소한 것을 확인하였다. As a result, the expression of cleaved PAPR and cleaved capase 3 increased in the control group, which was muscle cells in which apoptosis was induced by serum-free medium (SF media), compared to normal muscle cells. In addition, the expression of these markers did not differ in the muscle cell group co-cultured with aged WJ-MSCs compared to the control group, but it was confirmed that it significantly decreased in the muscle cells co-cultured with young WJ-MSCs and senescence-inhibited WJ-MSCs.
도 8b는 세포사멸에 대한 WJ-MSC의 치료효능을 평가하기 위하여, 실시예 1과 동일한 방법으로 Annexin V에 대한 면역형광염색을 수행한 결과를 나타낸 것이다. annexin V 양성세포의 비율은 정상 근육세포 그룹보다 대조군(세포사멸이 유도된 근육세포)에서 유의하게 증가하였으나 WJ-MSC와 공동배양한 근육세포 그룹에서 모두 유의미하게 감소하였다. 또한, 노화 WJ-MSC를 공동배양한 근육세포에 비해 젊은 WJ-MSC, 노화억제 WJ-MSC와 공동배양을 진행한 근육세포에서 annexin V 양성세포의 비율이 유의하게 감소하였다. Figure 8b shows the results of immunofluorescence staining for Annexin V performed in the same manner as Example 1 to evaluate the therapeutic efficacy of WJ-MSCs on apoptosis. The proportion of annexin V-positive cells significantly increased in the control group (myocytes in which apoptosis was induced) compared to the normal muscle cell group, but significantly decreased in all muscle cell groups co-cultured with WJ-MSCs. In addition, the proportion of annexin V-positive cells significantly decreased in muscle cells co-cultured with young WJ-MSCs and senescence-inhibited WJ-MSCs compared to muscle cells co-cultured with aged WJ-MSCs.
이를 통해서, WJ-MSC의 노화로 인해 감소된 항세포사멸 효능은 BPTES 처리를 통한 노화억제 WJ-MSC에서 회복될 수 있음을 확인하였다.Through this, it was confirmed that the anti-apoptotic efficacy of WJ-MSCs decreased due to aging could be restored in aging-inhibited WJ-MSCs through BPTES treatment.
실시예 9. mdx 마우스의 근골격근 회복에 대한 노화 억제 WJ-MSC의 효능 증대Example 9. Enhanced efficacy of anti-aging WJ-MSCs on skeletal muscle recovery in mdx mice
노화 억제 WJ-MSC의 근디스트로피(DMD) 질병에 대한 치료효능을 탐색하기 위해 마우스 동물실험을 진행하였다. 구체적으로 DMD질병모델 마우스(MDX)에 젊은 WJ-MSC, 노화 WJ-MSC 및 노화 억제 WJ-MSC를 각각 마우스의 꼬리 정맥을 통해 투여한 뒤 1주일 동안 관찰하고 희생(sacrifice)하여 근육 조직을 관찰하였다.To explore the therapeutic efficacy of anti-aging WJ-MSCs on muscular dystrophy (DMD), mouse animal experiments were conducted. Specifically, young WJ-MSCs, aged WJ-MSCs, and anti-aging WJ-MSCs were administered to DMD disease model mice (MDX) through the tail vein, and the mice were observed for one week before being sacrificed and muscle tissues were observed.
대조군과 젊은 WJ-MSC, 노화 WJ-MSC, 노화 억제 WJ-MSC, 4 x 104cells을 투여한 실험군의 악력(grip strength) 행동평가를 확인하였다. 구체적으로 앞발과 뒷발의 악력을 grip strength meter (BIO GS3)를 이용하여 중간엽 줄기세포 투여 전/후를 측정하여 분석하였으며, 실험동물 체중으로 결과를 보정하였다.The grip strength behavioral evaluation of the control group and the experimental group administered 4 x 104 cells of young WJ-MSCs, aged WJ-MSCs, and aged-inhibited WJ-MSCs was confirmed. Specifically, the grip strength of the forepaws and hind paws was measured and analyzed before and after mesenchymal stem cell administration using a grip strength meter (BIO GS3), and the results were corrected by the body weight of the experimental animals.
그 결과, 도 9a에 나타난 바와 같이, 젊은 WJ-MSC 투여군과 노화 억제 WJ-MSC 투여군에서는 악력이 대조군(MDX Ctrl)에 비해 유의하게 증가하였으나, 노화 WJ-MSC를 투여했을 때는 대조군과 차이가 없었다. 또한, 젊은 WJ-MSC 투여군과 노화 억제 WJ-MSC 투여군에서는 노화 WJ-MSC 투여한 실험군에 비해 악력이 유의하게 증가하는 것을 확인하였다.As a result, as shown in Fig. 9a, in the young WJ-MSC administration group and the aging-inhibited WJ-MSC administration group, grip strength significantly increased compared to the control group (MDX Ctrl), but there was no difference from the control group when aged WJ-MSCs were administered. In addition, it was confirmed that in the young WJ-MSC administration group and the aging-inhibited WJ-MSC administration group, grip strength significantly increased compared to the experimental group administered aged WJ-MSCs.
대조군과 젊은 WJ-MSC, 노화 WJ-MSC, 노화 억제 WJ-MSC를 투여한 실험군의 creatine kinase (CK) 활성을 측정하였다. 구체적으로, 안와채혈을 통해 중간엽 줄기세포 투여 전/후 혈액을 채취하였고, 혈청을 분리하여 CPK-PIII (Fujifilm)을 이용하여 creatine kinase (CK) 활성을 측정하였다.The creatine kinase (CK) activity of the control group and the experimental groups administered young WJ-MSCs, aged WJ-MSCs, and aged-inhibited WJ-MSCs was measured. Specifically, blood was collected before and after mesenchymal stem cell administration through orbital blood collection, and the serum was separated to measure creatine kinase (CK) activity using CPK-PIII (Fujifilm).
그 결과, 도 9b에 나타난 바와 같이, MDX 대조군에 비해 젊은 WJ-MSC 투여군과 노화 억제 WJ-MSC 투여군에서는 CK 활성이 유의하게 감소하였으나, 노화 WJ-MSC 투여군에서는 통계적 차이는 없었다.As a result, as shown in Fig. 9b, CK activity significantly decreased in the young WJ-MSC administration group and the anti-aging WJ-MSC administration group compared to the MDX control group, but there was no statistical difference in the aged WJ-MSC administration group.
대조군과 젊은 WJ-MSC, 노화 WJ-MSC, 노화 억제 WJ-MSC, 4 x 104cells을 투여한 실험군의 종아리 근육조직에서 MHC, AnnexinV, Fibronectin의 단백질 발현양을 확인하기 위해 실시예 2와 동일한 방법으로 Western blot을 진행하였다. 1차 항체는 AnnexinV(abcam), MHC, fibronectin(cell signaling) 및 GAPDH(santa cruz biotechnology)를 이용하였다.To confirm the protein expression levels of MHC, AnnexinV, and Fibronectin in the calf muscle tissue of the control group and the experimental group administered 4 x 104 cells of young WJ-MSCs, aged WJ-MSCs, and aged-inhibited WJ-MSCs, Western blot was performed using the same method as in Example 2. The primary antibodies used were AnnexinV (abcam), MHC, fibronectin (cell signaling), and GAPDH (santa cruz biotechnology).
도 9c 및 도 9d는 대조군과 젊은 WJ-MSC, 노화 WJ-MSC, 노화 억제 WJ-MSC, 4 x 104cells을 투여한 실험군의 MHC 단백질 발현양에 따른 성숙한 근섬유(mature myofiber) 비중을 확인하였다.Figures 9c and 9d show the proportion of mature myofibers according to the expression level of MHC proteins in the control group and the experimental group administered with young WJ-MSCs, aged WJ-MSCs, aged-inhibited WJ-MSCs, and 4 x 104 cells.
그 결과, 도 9c 및 도 9d에 나타낸 바와 같이, 대조군에 비해 노화 WJ-MSC를 투여하였을 때 MHC 단백질 발현양이 유의하게 감소하였으나, 노화 억제 WJ-MSC를 투여하였을 때는 발현양이 유의하게 증가하는 것을 확인하였다. As a result, as shown in Figures 9c and 9d, it was confirmed that when aged WJ-MSCs were administered compared to the control group, the expression level of MHC protein significantly decreased, but when aged WJ-MSCs were administered, the expression level significantly increased.
도 9c 및 도 9e는 대조군과 젊은 WJ-MSC, 노화 WJ-MSC, 노화 억제 WJ-MSC, 4 x 104cells을 투여한 실험군의 annexinV 단백질 발현양에 따른 근육 조직의 세포사멸에 대한 억제 효능의 차이를 확인한 결과이다.Figures 9c and 9e show the results of confirming the difference in inhibitory efficacy on apoptosis of muscle tissue according to the amount of annexinV protein expression in the control group and the experimental group administered young WJ-MSCs, aged WJ-MSCs, aged-inhibited WJ-MSCs, and 4 x 104 cells.
그 결과, 도 9c 및 도 9e에 나타난 바와 같이, 정상군(WT)에 비해 대조군의 근골격근의 annexin V 발현이 유의하게 증가하였다. 젊은 WJ-MSC와 노화 억제 WJ-MSC 투여군은 대조군에 비해 annexin V 발현이 유의하게 감소하였으나, 노화 WJ-MSC 투여군에서는 유의성은 나타나지 않았다. 즉, 노화 억제 WJ-MSC는 노화 WJ-MSC에 비해 손상된 근육세포의 세포사멸을 억제하는 효능이 더 큰 바, 상기 중간엽 줄기세포는 근육 질환의 치료제로 이용할 수 있다.As a result, as shown in Figs. 9c and 9e, annexin V expression in the skeletal muscles of the control group significantly increased compared to the normal group (WT). The young WJ-MSC and aging-inhibited WJ-MSC administration groups showed a significant decrease in annexin V expression compared to the control group, but no significance was observed in the aging WJ-MSC administration group. In other words, aging-inhibited WJ-MSCs have a greater effect on inhibiting apoptosis of damaged muscle cells than aging WJ-MSCs, and thus, the mesenchymal stem cells can be used as a therapeutic agent for muscle diseases.
도 9c 및 도 9f는 대조군과 젊은 WJ-MSC, 노화 WJ-MSC, 노화 억제 WJ-MSC, 4 x 104cells을 투여한 실험군의 fibronectin 단백질 발현양에 따른 근육 조직의 섬유화 억제 효능의 차이를 확인한 결과이다.Figures 9c and 9f show the results of confirming the difference in muscle tissue fibrosis inhibition efficacy according to the amount of fibronectin protein expression in the control group and the experimental group administered young WJ-MSCs, aged WJ-MSCs, aged-inhibited WJ-MSCs, and 4 x 104 cells.
그 결과, 도 9c, 도 9f에 나타난 바와 같이, 젊은 WJ-MSC와 노화 억제 WJ-MSC 투여군은 대조군에 비해 fibronectin 발현이 유의하게 감소하였으나 노화 WJ-MSC 투여군에서는 차이가 없었다. 즉, 노화 억제 WJ-MSC는 fibronectin의 발현을 감소시킴으로써 손상된 근육세포의 섬유화를 효과적으로 억제할 수 있다.As a result, as shown in Fig. 9c and Fig. 9f, the young WJ-MSC and anti-aging WJ-MSC administration groups showed a significant decrease in fibronectin expression compared to the control group, but there was no difference in the anti-aging WJ-MSC administration group. In other words, anti-aging WJ-MSC can effectively suppress fibrosis of damaged muscle cells by reducing the expression of fibronectin.
도 9g는 대조군과 젊은 WJ-MSC, 노화 WJ-MSC, 노화 억제 WJ-MSC, 4 x 104cells을 투여한 실험군의 종아리 근육조직에서 면역형광염색(IHC)을 수행한 결과이다. Figure 9g shows the results of immunofluorescence staining (IHC) performed on calf muscle tissue of the control group and the experimental group administered 4 x 104 cells of young WJ-MSCs, aged WJ-MSCs, and aged-inhibited WJ-MSCs.
구체적으로 근육조직은 상온에서 4% PFA로 고정한 후, 파라핀 블록으로 제작하였다. 파라핀 블록은 4μm 두께로 절단되었다. 조직슬라이드는 상온에서 1시간 동안 blocking solution (2% BSA, 0.1% Tween20 in PBS)으로 blocking하고 blocking solution에 1차 항체를 희석하여 4℃에서 밤새 반응시켰다. 형광 현미경을 이용하여 결과를 확인하였으며 그 결과는 image J를 통해서 형광정량분석을 진행하였다. 1차 항체로는 MHC(R&D) 및 AnnexinV(Abcam)을 사용하였다.Specifically, muscle tissues were fixed with 4% PFA at room temperature and then made into paraffin blocks. Paraffin blocks were sectioned to 4 μm thickness. Tissue slides were blocked with blocking solution (2% BSA, 0.1% Tween20 in PBS) at room temperature for 1 hour, and primary antibodies were diluted in the blocking solution and reacted overnight at 4°C. The results were confirmed using a fluorescence microscope, and the results were subjected to fluorescence quantitative analysis using Image J. MHC (R&D) and AnnexinV (Abcam) were used as primary antibodies.
그 결과, 도 9g, 도 9h에 나타난 바와 같이, 대조군에 비해 젊은 WJ-MSC 투여군과 노화 억제 WJ-MSC 투여군은 MHC 단백질 발현양이 유의하게 증가하였으나, 노화 WJ-MSC 투여군은 차이가 없었다. 또한, 젊은 WJ-MSC 투여군과 노화 억제 WJ-MSC 투여군에서는 노화 WJ-MSC 투여군에 비해서도 MHC 단백질 발현양이 유의하게 증가하는 것을 확인하였다. As a result, as shown in Fig. 9g and Fig. 9h, compared to the control group, the young WJ-MSC administration group and the aging-inhibited WJ-MSC administration group showed a significant increase in MHC protein expression levels, but there was no difference in the aged WJ-MSC administration group. In addition, it was confirmed that the young WJ-MSC administration group and the aging-inhibited WJ-MSC administration group showed a significant increase in MHC protein expression levels compared to the aged WJ-MSC administration group.
도 9g, 도 9i는 대조군과 젊은 WJ-MSC, 노화 WJ-MSC, 노화 억제 WJ-MSC, 4 x 104cells을 투여한 실험군의 AnnexinV에 대한 면역형광염색을 수행한 결과이다.Figures 9g and 9i show the results of immunofluorescence staining for AnnexinV in the control group and the experimental group administered 4 x 104 cells of young WJ-MSCs, senescent WJ-MSCs, and senescence-inhibited WJ-MSCs.
그 결과, 도 9g 및 도 9i에 나타난 바와 같이, 정상군(WT)에 비해 대조군의 근골격근의 Annexin V 발현이 유의하게 증가하였다. 젊은 WJ-MSC와 노화 억제 WJ-MSC 투여군은 대조군에 비해 Annexin V 발현이 유의하게 감소하였으나, 노화 WJ-MSC 투여군에서는 차이가 나타나지 않았다.As a result, as shown in Fig. 9g and Fig. 9i, Annexin V expression in the skeletal muscles of the control group significantly increased compared to the normal group (WT). Annexin V expression in the young WJ-MSC and aging-inhibited WJ-MSC administration groups significantly decreased compared to the control group, but no difference was observed in the aging WJ-MSC administration group.
도 9g 및 도 9j는 대조군과 젊은 WJ-MSC, 노화 WJ-MSC, 노화 억제 WJ-MSC, 4 x 104cells을 투여한 실험군의 근육조직의 섬유화 억제 효능을 콜라겐 축적 여부를 통해 확인하기 위해 시리우스 레드(sirius red) 염색을 수행한 결과이다. Figures 9g and 9j show the results of sirius red staining to confirm the fibrosis inhibition effect of muscle tissue of the control group and the experimental group administered 4 x 104 cells of young WJ-MSCs, aged WJ-MSCs, aged-inhibited WJ-MSCs, and collagen accumulation.
구체적으로, MDX 마우스 모델의 종아리 근육 조직을 PBS로 세척한 후, picro-sirius red (solution A)로 실온에서 1시간 동안 반응시켰다. 이후 Acidified water (solutaion B)로 2회 세척하고, 마운팅한 후 Scanscope를 이용하여 이미지를 얻었다. 촬영된 이미지는 Image J 소프트웨어를 통해 정량분석을 하여 MDX 대조군에 대한 상대적인 값을 구하였다.Specifically, the calf muscle tissue of the MDX mouse model was washed with PBS and then reacted with picro-sirius red (solution A) at room temperature for 1 hour. Afterwards, it was washed twice with acidified water (solution B), mounted, and images were obtained using Scanscope. The captured images were quantitatively analyzed using Image J software to obtain relative values to the MDX control group.
그 결과, 도 9g 및 도 9j에 나타난 바와 같이, 대조군에서는 정상군에 비해 콜라겐의 섬유화 축적이 유의하게 증가하였다. WJ-MSC 투여군은 대조군에 비해 콜라겐의 축적이 유의하게 감소하였다. 그러나 젊은 WJ-MSC와 노화 억제 WJ-MSC 투여군은 노화 WJ-MSC 투여군에 비해 콜라겐의 섬유화 축적이 유의하게 감소하는 것을 확인할 수 있었다.As a result, as shown in Fig. 9g and Fig. 9j, the collagen fibrosis accumulation significantly increased in the control group compared to the normal group. The WJ-MSC administration group showed a significant decrease in collagen accumulation compared to the control group. However, it was confirmed that the young WJ-MSC and aging-suppressed WJ-MSC administration groups showed a significant decrease in collagen fibrosis accumulation compared to the aging WJ-MSC administration group.
따라서, WJ-MSC는 노화가 진행되면 MDX 마우스의 질병에 대한 치료 효과가 감소하지만, BPTES 처리하여 노화를 경감시킨 노화 억제 WJ-MSC는 젊은 WJ-MSC 만큼 치료 효능이 증대되는 것을 확인할 수 있었다.Therefore, although the therapeutic efficacy of WJ-MSCs for diseases in MDX mice decreases with aging, it was confirmed that the therapeutic efficacy of aging-inhibited WJ-MSCs treated with BPTES to alleviate aging was enhanced to the same extent as that of young WJ-MSCs.
실시예 10. 노화 WJ-MSC에서 GLS1 억제를 통한 cathepsin C(CTSC), E2F transcription factor 7(E2F7), sorbin SH3 domain containing 2(SORBS2)의 발현 회복Example 10. Restoration of expression of cathepsin C (CTSC), E2F transcription factor 7 (E2F7), and sorbin SH3 domain containing 2 (SORBS2) through GLS1 inhibition in aged WJ-MSCs
상기 결과를 통해 GLS1 억제제로 인해 노화 WJ-MSC에서 다양한 노화 마커들의 발현이 감소하였고, 노화가 억제되는 것이 확인되었다. 따라서 노화가 억제된 세포의 유전자 발현 양상도 젊은 WJ-MSC와 유사하게 회복이 되는지 확인하고자 하였다. The above results confirmed that the expression of various senescence markers was reduced and senescence was suppressed in aged WJ-MSCs due to GLS1 inhibitor. Therefore, we wanted to confirm whether the gene expression pattern of cells with suppressed senescence was restored to a similar state as young WJ-MSCs.
구체적으로 젊은 WJ-MSC, 노화 WJ-MSC, BPTES 처리한 노화 WJ-MSC에서 RNA를 분리하였고 mRNA 시퀀싱을 진행하여 발현 차이가 나는 유전자를 확인하였다. RNA 시퀀싱에는 RNA Integrity Number(RIN) 값이 7.0 이상이고 28S:18S 비율이 1 이상인 RNA 검체를 사용했으며 제조업체의 지침에 따라 QuantSeq 3' mRNA-Seq Library Prep Kit(eBiogen)를 사용하여 mRNA 라이브러리 구성을 하였다. DEGs(differentially expressed genes)분석은 ExDEGA2.0을 이용하여 수행하였다.Specifically, RNA was isolated from young WJ-MSCs, aged WJ-MSCs, and BPTES-treated aged WJ-MSCs, and mRNA sequencing was performed to identify genes with differential expression. For RNA sequencing, RNA samples with an RNA Integrity Number (RIN) value of 7.0 or higher and a 28S:18S ratio of 1 or higher were used, and mRNA libraries were constructed using QuantSeq 3' mRNA-Seq Library Prep Kit (eBiogen) according to the manufacturer's instructions. Differentially expressed genes (DEGs) analysis was performed using ExDEGA2.0.
도 10a는 노화 WJ-MSC에 비해 젊은 WJ-MSC과 BPTES를 처리한 노화 WJ-MSC에서 FC가 3 이상 차이 나는 유전자를 Venn Diagram으로 작성한 결과를 나타낸 것이다. 노화 WJ-MSC에 비해 젊은 WJ-MSC와 BPTES 처리한 노화 WJ-MSC에서 공통적으로 상향조절된 유전자는 25개였으며, 공통적으로 하향조절된 유전자는 58개였다. Figure 10a shows the results of constructing a Venn Diagram of genes with FC differences of 3 or more in young WJ-MSCs and BPTES-treated aged WJ-MSCs compared to aged WJ-MSCs. There were 25 genes that were commonly up-regulated and 58 genes that were commonly down-regulated in young WJ-MSCs and BPTES-treated aged WJ-MSCs compared to aged WJ-MSCs.
도 10b는 젊은 WJ-MSC, 노화 WJ-MSC, BPTES 처리한 노화 WJ-MSC에서 공통적으로 변화된 83개의 DEG 유전자를 heat map으로 나타낸 것이다.Figure 10b is a heat map showing 83 DEG genes that were commonly changed in young WJ-MSCs, aged WJ-MSCs, and BPTES-treated aged WJ-MSCs.
도 10c는 위의 유전자들의 GO 카테고리를 작성한 결과이다. Up은 노화 WJ-MSC에 비해 BPTES 처리한 노화 WJ-MSC에서의 높게 발현되는 유전자를, down은 노화 WJ-MSC에 비해 BPTES 처리한 노화 WJ-MSC에서의 낮게 발현되는 유전자를 의미한다. 그 결과, cell cycle과 DNA repair 관련된 유전자는 상향조절되는 수가 더 많은 것으로 나타났으며, 노화(aging), 세포외기질(extracellular matrix, ECM), 분비(secretion), 염증반응(inflammatory response), 면역반응(immune response)과 관련된 유전자는 하향조절되는 수가 더 많은 것으로 나타났다.Figure 10c shows the result of creating the GO categories of the above genes. Up means a gene that is highly expressed in BPTES-treated senescent WJ-MSCs compared to senescent WJ-MSCs, and down means a gene that is lowly expressed in BPTES-treated senescent WJ-MSCs compared to senescent WJ-MSCs. As a result, genes related to cell cycle and DNA repair were found to be upregulated more, while genes related to aging, extracellular matrix (ECM), secretion, inflammatory response, and immune response were found to be downregulated more.
GLS1 외에도 노화로 인한 발현 차이를 보이는 유전자를 확인하였다. 그 중 CTSC, E2F7 및 SORBS2는 노화와 유전자 발현이 연관되어 있음이 알려진 유전자들이지만, WJ-MSC에서 노화와의 연관성에 관한 연구가 진행된 바 없었다. 따라서 3 lot에서 이 유전자들이 WJ-MSC의 노화에 따라 발현의 차이를 보이는지 검증하였다. 3명의 공여자의 젊은 WJ-MSC, 노화 WJ-MSC에서 CTSC, E2F7, SORBS2에 대한 유전자발현을 실시예 2와 동일한 방법으로 qRT-PCR을 수행하였다.In addition to GLS1, genes showing differences in expression due to aging were identified. Among them, CTSC, E2F7, and SORBS2 are genes known to be associated with aging and gene expression, but no study has been conducted on their association with aging in WJ-MSCs. Therefore, we verified whether these genes showed differences in expression according to aging in WJ-MSCs in 3 lots. Gene expression of CTSC, E2F7, and SORBS2 in young and aged WJ-MSCs from 3 donors was performed using the same method as in Example 2.
그 결과, 도 10d에 나타난 바와 같이, WJ-MSC 3 lot의 젊은 WJ-MSC에 비해 노화 WJ-MSC에서 CTSC 및 E2F7의 mRNA 수준은 유의하게 감소하였고, SORBS2의 mRNA 수준은 유의하게 증가하였다.As a result, as shown in Fig. 10d, the mRNA levels of CTSC and E2F7 were significantly decreased, and the mRNA level of SORBS2 was significantly increased in aged WJ-MSCs compared to young WJ-MSCs of WJ-MSC 3 lots.
도 10e는 mRNA-seq에서 확인된 CTSC, E2F7, SORBS2의 발현을 normalized log2로 나타낸 그래프이다. 젊은 WJ-MSC에 비해 노화 WJ-MSC에서 CTSC 및 E2F7의 경우 유의하게 감소하였고, SORBS2에서는 유의하게 증가하였다. 또한, BPTES 처리한 노화 WJ-MSC에서 젊은 WJ-MSC와 같은 패턴으로 회복되는 것을 확인하였다.Figure 10e is a graph showing the expression of CTSC, E2F7, and SORBS2 confirmed by mRNA-seq as normalized log2. CTSC and E2F7 were significantly decreased in aged WJ-MSCs compared to young WJ-MSCs, while SORBS2 was significantly increased. In addition, it was confirmed that aged WJ-MSCs treated with BPTES recovered to the same pattern as young WJ-MSCs.
젊은 WJ-MSC, 노화 WJ-MSC, BPTES 처리한 노화 WJ-MSC에서 CTSC, E2F7 및 SORBS2에 대한 mRNA 수준을 평가하기 위해 실시예 2와 동일한 방법으로 qRT-PCR를 수행하였다.To evaluate the mRNA levels for CTSC, E2F7, and SORBS2 in young WJ-MSCs, aged WJ-MSCs, and BPTES-treated aged WJ-MSCs, qRT-PCR was performed using the same method as in Example 2.
그 결과, 도 10f에 나타난 바와 같이, CTSC와 E2F7의 mRNA발현은 젊은 WJ-MSC에 비해 노화 WJ-MSC 대조군에서 감소하였으며 노화 WJ-MSC에 BPTES 처리시 유의하게 증가하였다. 또한 SORBS2의 mRNA발현은 젊은 WJ-MSC에 비해 노화 WJ-MSC 대조군에서 증가하였으며 노화 WJ-MSC에 BPTES처리시 유의하게 감소하였다. 이는 앞선 도 10e의 경향성과 일치하는 결과이다.As a result, as shown in Fig. 10f, the mRNA expression of CTSC and E2F7 decreased in the aged WJ-MSC control group compared to young WJ-MSCs, and significantly increased when BPTES was treated in aged WJ-MSCs. In addition, the mRNA expression of SORBS2 increased in the aged WJ-MSC control group compared to young WJ-MSCs, and significantly decreased when BPTES was treated in aged WJ-MSCs. This result is consistent with the trend of Fig. 10e above.
실시예 2와 동일한 방법으로 노화 WJ-MSC에 siGLS1을 형질주입하여 GLS1 유전자를 넉다운시킨 후, CTSC, E2F7 및 SORBS2에 대한 유전자 발현양을 qRT-PCR을 통해 확인하였다. The GLS1 gene was knocked down by transfecting siGLS1 into aged WJ-MSCs using the same method as in Example 2, and the gene expression levels for CTSC, E2F7, and SORBS2 were confirmed through qRT-PCR.
그 결과, 도 10g에 나타난 바와 같이, 노화 WJ-MSC에서 siNC 처리 대조군에 비해 siGLS1 처리군에서 CTSC와 E2F7의 발현양은 통계적으로 유의하게 증가하였으며 SORBS2의 발현양은 유의하게 감소하였다. 즉, 노화 WJ-MSC에서 CTSC, E2F7 및 SORBS2 유전자는 GLS1의 억제를 통해 발현이 영향을 받는 것을 확인할 수 있었다.As a result, as shown in Fig. 10g, the expression levels of CTSC and E2F7 in the siGLS1 treatment group were statistically significantly increased compared to the siNC treatment control group in aged WJ-MSCs, while the expression level of SORBS2 was significantly decreased. In other words, it was confirmed that the expression of CTSC, E2F7, and SORBS2 genes was affected by the inhibition of GLS1 in aged WJ-MSCs.
실시예 11. 복제성 노화 중간엽 줄기세포에서 GLS1 억제를 통한 노화 억제와 WNT 신호전달경로과의 연관성Example 11. Association between inhibition of senescence through GLS1 inhibition and WNT signaling pathway in replicative senescent mesenchymal stem cells
실시예 11는 3 lot에서 젊은 WJ-MSC와 노화 WJ-MSC 전사체에서 복제성 노화로 인한 유전적 변화를 확인하기 위해 mRNA-seq을 분석한 결과이다.Example 11 presents the results of mRNA-seq analysis to identify genetic changes due to replicative aging in the transcriptomes of young and aged WJ-MSCs from 3 lots.
3 lot의 젊은 WJ-MSC와 노화 WJ-MSC에서 FC > 1.5, p < 0.05인 DEG를 heatmap으로 나타내었다.DEGs with FC > 1.5, p < 0.05 in 3 lots of young WJ-MSCs and aged WJ-MSCs were displayed as a heatmap.
그 결과, 도 11a에 나타난 바와 같이, 노화 WJ-MSC에 비해 젊은 WJ-MSC에서 320개의 유전자가 상향조절 되었으며 225개의 유전자가 하향 조절되었다.As a result, as shown in Fig. 11a, 320 genes were up-regulated and 225 genes were down-regulated in young WJ-MSCs compared to aged WJ-MSCs.
DEG의 기능적 분석을 위해서 DAVID 분석을 진행하였다. 도 11b는 GO 데이터베이스를, 도 11c는 KEGG 데이터베이스를 이용하여 p-값을 기준으로 상위 10개 카테고리를 나타내었다(p < 0.05).For functional analysis of DEG, DAVID analysis was performed. Figure 11b shows the top 10 categories based on p-value using the GO database, and Figure 11c shows the top 10 categories based on p-value using the KEGG database (p < 0.05).
그 결과, 도 11b에서 나타난 바와 같이, 생물학적 과정에서 GO 카테고리 "Wnt 신호 전달 경로, 평면 세포 극성 경로"는 노화 WJ-MSC에 비해 젊은 WJ-MSC에서 상향조절되는 것을 확인하였다.As a result, as shown in Fig. 11b, the GO category “Wnt signaling pathway, planar cell polarity pathway” in the biological process was confirmed to be up-regulated in young WJ-MSCs compared to aged WJ-MSCs.
KEGG 경로를 통한 DEG분석은 도 11c에서 나타난 바와 같이, “Wnt 신호전달 경로”가 WJ-MSC의 노화와 관련된 것으로 나타났다.DEG analysis through KEGG pathway showed that the “Wnt signaling pathway” was related to the aging of WJ-MSCs, as shown in Fig. 11c.
11,313개의 유전자에 대해 유전자집합농축 분석(GSEA)를 수행하여 복제성 노화와 관련된 중요한 유전자 집합(gene set)를 확인하였다.Gene set enrichment analysis (GSEA) was performed on 11,313 genes to identify important gene sets associated with replicative aging.
그 결과 도 11d에 나타난 바와 같이, 노화 WJ-MSC에 비해 젊은 WJ-MSC에서 “세포 주기 DNA 복제” 및 “Wnt에 의한 세포-세포 신호 전달” 유전자 집합에서 농축 점수(enrichment score)가 높게 나타났다.As a result, as shown in Fig. 11d, the enrichment scores for the “cell cycle DNA replication” and “cell-to-cell signaling by Wnt” gene sets were higher in young WJ-MSCs compared to aged WJ-MSCs.
따라서, 공통적으로 확인된 Wnt 신호 전달 경로는 WJ-MSCs의 복제성 노화와 관련이 있음을 시사한다.Therefore, our results suggest that the commonly identified Wnt signaling pathway is involved in replicative senescence of WJ-MSCs.
3 lot에서 젊은 WJ-MSC와 노화 WJ-MSC에서 노화관련 단백질인 p21과 Wnt 신호전달 경로 관련 단백질인 β-catenin의 발현을 실시예 2와 동일한 방법으로 western blot을 통해 확인하였다. 1차 항체는 p21(abcam), β-catenin(cell signaling) 및 GAPDH(santa cruz biotechnology)을 이용하였다.The expression of p21, a senescence-related protein, and β-catenin, a Wnt signaling pathway-related protein, in young and aged WJ-MSCs from lot 3 was confirmed through western blot using the same method as in Example 2. The primary antibodies used were p21 (abcam), β-catenin (cell signaling), and GAPDH (santa cruz biotechnology).
그 결과, 도 11e에 나타난 바와 같이, 젊은 WJ-MSC에 비해 노화 WJ-MSC에서 p21의 발현이 유의하게 높게 발현하였으며 WJ-MSC에서 노화가 진행되었을 때, β-catenin의 발현이 감소하였다. 즉, 이를 종합하여 보았을 때 WJ-MSC의 복제성 노화와 Wnt 신호전달 경로는 연관되어 있는 것을 확인할 수 있었다.As a result, as shown in Fig. 11e, the expression of p21 was significantly higher in aged WJ-MSCs than in young WJ-MSCs, and the expression of β-catenin decreased as aging progressed in WJ-MSCs. In other words, when looking at this comprehensively, it was confirmed that the replicative senescence of WJ-MSCs and the Wnt signaling pathway are related.
Wnt 신호전달 활성화와 노화 억제와의 관련성을 확인하기 위해 노화억제 WJ-MSC에서 Wnt 신호전달 관련 단백질의 발현을 실시예 2와 동일한 방법으로 Western blot을 통해 확인하였다. 1차 항체는 GLS1(abcam), β-catenin, p-GSK3β, GSK3β(cell signaling) 및 α-tubulin(sigma)을 이용하였다.To confirm the relationship between Wnt signaling activation and aging inhibition, the expression of Wnt signaling-related proteins in aging-inhibited WJ-MSCs was confirmed via Western blot using the same method as in Example 2. The primary antibodies used were GLS1 (abcam), β-catenin, p-GSK3β, GSK3β (cell signaling), and α-tubulin (sigma).
그 결과, 도 11f에서 나타난 바와 같이, β-catenin과 인산화된 GSK3β(ser9)의 발현은 노화가 진행되었을 때 감소하였고, 노화억제 WJ-MSC에서 GLS1 발현이 감소함에 따라 β-catenin과 인산화된 GSK3β(ser9)의 발현이 다시 회복되었다.As a result, as shown in Fig. 11f, the expression of β-catenin and phosphorylated GSK3β (ser9) decreased as aging progressed, and the expression of β-catenin and phosphorylated GSK3β (ser9) was restored as GLS1 expression decreased in senescence-inhibited WJ-MSCs.
노화 WJ-MSC에 siGLS1을 형질주입하여 GLS1 유전자를 넉다운시킨 후, Wnt 신호전달 관련 단백질을 실시예 2와 동일한 방법으로 Western blot을 통해 조사하였다. 1차 항체는 GLS1(abcam), β-catenin, p-GSK3β, GSK3β(cell signaling) 및 α-tubulin(sigma)을 이용하였다.After knocking down the GLS1 gene by transfecting siGLS1 into aged WJ-MSCs, Wnt signaling-related proteins were examined by Western blot using the same method as in Example 2. The primary antibodies used were GLS1 (abcam), β-catenin, p-GSK3β, GSK3β (cell signaling), and α-tubulin (sigma).
그 결과, 도 11g에서 나타난 바와 같이, 노화 WJ-MSC에서 대조군(siNC)에 비해 siGLS1에서 β-catenin과 인산화된 GSK3β(ser9)의 발현은 증가하는 것을 확인할 수 있었다.As a result, as shown in Fig. 11g, it was confirmed that the expression of β-catenin and phosphorylated GSK3β (ser9) increased in siGLS1 compared to the control group (siNC) in aged WJ-MSCs.
또한, BPTES 이외에 다른 GLS1 저해제인 CB-839와 C968을 노화 WJ-MSC에 처리하였을 때, Wnt 신호전달 관련 단백질의 발현을 상기 실시예2와 동일한 방법으로 Western blot을 통해 조사하였다. 1차 항체는 GLS1(abcam), β-catenin, p-GSK3β, GSK3β(cell signaling), α-tubulin(sigma)을 이용하였다. In addition, when CB-839 and C968, other GLS1 inhibitors other than BPTES, were treated to aged WJ-MSCs, the expression of Wnt signaling-related proteins was examined via Western blot in the same manner as in Example 2. The primary antibodies used were GLS1 (abcam), β-catenin, p-GSK3β, GSK3β (cell signaling), and α-tubulin (sigma).
그 결과, 도 11h에 나타난 바와 같이, 노화 WJ-MSC에서 대조군에 비해 GLS1 저해제인 CB-839와 C968을 처리하였을 때, β-catenin과 인산화된 GSK3β(ser9)의 단백질 발현은 유의하게 증가하는 것을 확인할 수 있었다. As a result, as shown in Fig. 11h, it was confirmed that when aging WJ-MSCs were treated with GLS1 inhibitors, CB-839 and C968, compared to the control group, the protein expression of β-catenin and phosphorylated GSK3β (ser9) significantly increased.
이러한 결과는 WJ-MSC는 노화로 인해 Wnt 신호전달 경로가 비활성되지만, GLS1의 억제는 Wnt 신호전달 경로의 활성화시킴으로써 복제성 노화를 억제함을 시사한다.These results suggest that WJ-MSCs undergo aging and the Wnt signaling pathway is inactivated, but inhibition of GLS1 suppresses replicative senescence by activating the Wnt signaling pathway.
실시예 12. 복제성 노화 중간엽 줄기세포의 GLS1 억제를 통한 노화 억제와 자가포식작용의 관련성Example 12. Relationship between autophagy and inhibition of aging through GLS1 inhibition in replicative senescent mesenchymal stem cells
유전자집합농축 분석(GSEA)을 통해 젊은 WJ-MSC에 비해 노화 WJ-MSC에서 농축된 유전자 집합(gene set)을 확인하였다.Gene set enrichment analysis (GSEA) was used to identify enriched gene sets in aged WJ-MSCs compared to young WJ-MSCs.
그 결과, 도 12a에 나타난 바와 같이, 젊은 WJ-MSC에 비해 노화 WJ-MSC에서 "자가포식의 양성 조절" 유전자 집합에서 농축 점수(enrichment score)가 높게 나타났다.As a result, as shown in Fig. 12a, the enrichment score in the “positive regulation of autophagy” gene set was higher in aged WJ-MSCs than in young WJ-MSCs.
자가포식작용과 노화와의 관련성을 확인하기 위해 노화억제 WJ-MSC에서 자가포식작용 관련 단백질 LC3II의 발현을 실시예 2와 동일한 방법으로 Western blot을 통해 조사하였다. 1차 항체는 LC3II(cell signaling) 및 α-tubulin(sigma)을 이용하였다.To confirm the relationship between autophagy and aging, the expression of autophagy-related protein LC3II in senescence-inhibited WJ-MSCs was examined using Western blot in the same manner as in Example 2. LC3II (cell signaling) and α-tubulin (sigma) were used as primary antibodies.
그 결과, 도 12b에서 나타난 바와 같이, LC3II의 발현은 젊은 WJ-MSC에 비해 노화가 진행되었을 때 증가되었고 노화억제 WJ-MSC에서 LC3II의 발현이 다시 회복되었다.As a result, as shown in Fig. 12b, the expression of LC3II increased as aging progressed compared to young WJ-MSCs, and the expression of LC3II was restored in senescence-inhibited WJ-MSCs.
노화 WJ-MSC에 siGLS1을 형질주입하여 GLS1 유전자를 넉다운시킨 후, 자가포식작용 관련 단백질인 LC3II의 발현을 실시예 2와 동일한 방법으로 Western blot을 통해 조사하였다. 1차 항체는 LC3II(cell signaling) 및 α-tubulin(sigma)을 이용하였다.After knocking down the GLS1 gene by transfecting siGLS1 into aged WJ-MSCs, the expression of LC3II, a protein related to autophagy, was examined using Western blot in the same manner as in Example 2. LC3II (cell signaling) and α-tubulin (sigma) were used as primary antibodies.
그 결과, 도 12c에서 나타난 바와 같이, 노화 WJ-MSC에서 대조군(siNC)에 비해 GLS1 유전자가 넉다운된 노화세포에서 LC3II의 단백질 발현은 감소하는 것을 확인할 수 있었다.As a result, as shown in Fig. 12c, it was confirmed that the protein expression of LC3II was decreased in senescent cells in which the GLS1 gene was knocked down compared to the control group (siNC) in senescent WJ-MSCs.
또한, GLS1 저해제인 CB-839와 C968을 노화 WJ-MSC에 처리하였을 때, 자가포식작용 관련 단백질의 발현을 실시예 2와 동일한 방법으로 Western blot을 통해 조사하였다. 1차 항체는 LC3II(cell signaling) 및 α-tubulin(sigma)을 이용하였다. In addition, when CB-839 and C968, which are GLS1 inhibitors, were treated to aged WJ-MSCs, the expression of proteins related to autophagy was examined via Western blot in the same manner as in Example 2. LC3II (cell signaling) and α-tubulin (sigma) were used as primary antibodies.
그 결과, 도 12d에 나타난 바와 같이, 노화 WJ-MSC에서 대조군에 비해 CB-839와 C968을 처리하였을 때, LC3II 단백질 발현은 유의하게 감소하였다. 이러한 결과는 자가포식작용이 WJ-MSC의 복제성 노화와 관련이 있으며, GLS1에 의해 조절될 수 있다는 것을 시사한다.As a result, as shown in Fig. 12d, when CB-839 and C968 were treated in aged WJ-MSCs compared to the control group, LC3II protein expression was significantly reduced. These results suggest that autophagy is related to replicative senescence of WJ-MSCs and can be regulated by GLS1.
본 발명에 따르면, 노화 줄기세포를 높은 세포증식능과 줄기세포성을 유지하도록 노화를 억제시킬 수 있어, 중간엽 줄기세포를 이용한 세포치료제의 치료 효능을 향상시킬 수 있다. According to the present invention, aging can be suppressed so that aging stem cells maintain high cell proliferation capacity and stem cell properties, thereby improving the therapeutic efficacy of cell therapy using mesenchymal stem cells.
이상으로 본 발명 내용의 특정한 부분을 상세히 기술하였는 바, 당업계의 통상의 지식을 가진 자에게 있어서 이러한 구체적 기술은 단지 바람직한 실시태양일 뿐이며, 이에 의해 본 발명의 범위가 제한되는 것이 아닌 점은 명백할 것이다. 따라서, 본 발명의 실질적인 범위는 첨부된 청구항들과 그것들의 등가물에 의하여 정의된다고 할 것이다.While the specific parts of the present invention have been described in detail above, it will be apparent to those skilled in the art that such specific descriptions are merely preferred embodiments and that the scope of the present invention is not limited thereby. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.
전자파일 첨부하였음.Electronic file attached.
Claims (6)
- 줄기세포를 GLS1 저해제로 처리하는 단계를 포함하는 노화 억제 줄기세포의 제조방법.A method for producing anti-aging stem cells, comprising the step of treating stem cells with a GLS1 inhibitor.
- 제1항에 있어서, 상기 GLS1 저해제는 BPTES, CB-839 및 C968으로 구성된 군에서 선택되는 것을 특징으로 하는 방법.A method according to claim 1, characterized in that the GLS1 inhibitor is selected from the group consisting of BPTES, CB-839, and C968.
- 노화 줄기세포를 GLS1 저해제로 처리하는 단계를 포함하는 줄기세포의 노화 회복방법.A method for restoring aging of stem cells, comprising the step of treating aging stem cells with a GLS1 inhibitor.
- 제3항에 있어서, 상기 GLS1 저해제는 BPTES, CB-839 및 C968으로 구성된 군에서 선택되는 것을 특징으로 하는 방법.A method according to claim 3, characterized in that the GLS1 inhibitor is selected from the group consisting of BPTES, CB-839 and C968.
- GLS1 저해제를 유효성분으로 함유하는 줄기세포의 노화억제용 조성물.A composition for inhibiting aging of stem cells containing a GLS1 inhibitor as an active ingredient.
- 제5항에 있어서, 상기 GLS1 저해제는 BPTES, CB-839 및 C968으로 구성된 군에서 선택되는 것을 특징으로 하는 조성물.A composition according to claim 5, wherein the GLS1 inhibitor is selected from the group consisting of BPTES, CB-839, and C968.
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| KR20090108141A (en) * | 2008-04-11 | 2009-10-15 | 차의과학대학교 산학협력단 | Method for inhibiting premature senescence of mesenchymal stem cells |
| KR20200036964A (en) * | 2018-09-28 | 2020-04-08 | 충남대학교산학협력단 | Composition for Preventing Aging of Mesenchymal Stem Cells and Method For Culturing Mesenchymal Stem Cells Using the Same |
| CN113951414A (en) * | 2021-10-22 | 2022-01-21 | 上海舒泽生物科技研究所 | Yeast fermented solid beverage containing GLS1 inhibitor and preparation method thereof |
| JP2022112464A (en) * | 2021-01-21 | 2022-08-02 | 株式会社ユーグレナ | Gls1 gene-inhibiting cosmetic material compositions, gls1 gene inhibitors, cosmetic material compositions for senescent cell removal and senescent cell removing agents |
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| KR20200036964A (en) * | 2018-09-28 | 2020-04-08 | 충남대학교산학협력단 | Composition for Preventing Aging of Mesenchymal Stem Cells and Method For Culturing Mesenchymal Stem Cells Using the Same |
| JP2022112464A (en) * | 2021-01-21 | 2022-08-02 | 株式会社ユーグレナ | Gls1 gene-inhibiting cosmetic material compositions, gls1 gene inhibitors, cosmetic material compositions for senescent cell removal and senescent cell removing agents |
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