Method for selecting high-efficiency stem cells

Abstract

The present invention relates to a method for selecting high-efficiency stem cells by confirming the expression of an LY6K gene in stem cells or measuring the content of a protein encoded by the LY6K gene. According to the present invention, it is possible to select high-efficiency stem cells having excellent cell proliferation ability, cell migration ability, adhesion ability, and apoptosis inhibitory ability, and to select stem cells having excellent therapeutic efficiency by eliminating the problem of differences in stem cell efficiency due to donor-to-donor variations in stem cells. Therefore, when stem cell therapeutic agents are mass-produced, production costs can be reduced.

Description

Methods for Selecting High-Efficacy Stem Cells

[0001] The present invention relates to a method for selecting high-potency stem cells, and more particularly, to a method for selecting high-potency stem cells by confirming the expression of the LY6K gene in stem cells or measuring the content of a protein encoded by the LY6K gene.

[0002]

[0003] Various studies are underway to explore the potential use of stem cells for cell therapy by differentiating them into various cell types. Pluripotent embryonic stem cells, which can differentiate into various cell types, have attracted attention as a potential cell therapy. However, ethical issues surrounding their use have hindered their practical application in cell therapy. To circumvent these ethical issues, active research is being conducted using adult stem cells (Trends in Neurosciences 23:450, 2000).

[0004] Among adult stem cells, mesenchymal stem cells (MSCs) are multipotent stem cells derived from various adult sources, including bone marrow, umbilical cord blood, placenta (or placental tissue cells), and adipose tissue (or adipose tissue cells). For example, bone marrow-derived MSCs are being studied extensively for their potential as cell therapy agents due to their multipotent differentiation potential into adipose tissue, bone / cartilage tissue, and muscle tissue.

[0005] Recently, as cell therapy using mesenchymal stem cells has begun to receive attention, there is a need for the development of a technology to activate mesenchymal stem cells isolated from the human body to be suitable for treatment. In addition, there is a need for a technology to resolve the problem of differences in cell therapy efficacy due to differences in stem cell donors during mass production of stem cell therapeutics.

[0006] Accordingly, the inventors of the present invention have conducted extensive research efforts to find a selection marker capable of selecting only high-potency stem cells, and as a result, have selected high-potency mesenchymal stem cells that exhibit a high cell death inhibition effect when co-cultured with a myoblast apoptosis model, and confirmed that the LY6K gene in the selected high-potency mesenchymal stem cells exhibits a specifically higher expression than in low-potency mesenchymal stem cells, thereby completing the present invention.

[0007]

[0008] Summary of the invention

[0009] The purpose of the present invention is to provide a method for selecting high-efficiency stem cells.

[0010] Another object of the present invention is to provide a method for quality control of a stem cell therapeutic agent.

[0011] Another object of the present invention is to provide a method for producing high-efficiency stem cells.

[0012] Another object of the present invention is to provide a composition for producing high-efficiency stem cells.

[0013] Another object of the present invention is to provide a composition for selecting high-efficiency stem cells.

[0014] Another object of the present invention is to provide a high-efficiency stem cell selection kit comprising the above composition.

[0015] In order to achieve the above purpose, the present invention provides a method for confirming the expression of the LY6K gene in stem cells or measuring the content of a protein encoded by the LY6K gene; and

[0016] A method for selecting high-potency stem cells is provided, including a step of selecting high-potency stem cells when the expression of the LY6K gene or the content of the protein encoded by the LY6K gene is higher than that of other stem cells.

[0017] The present invention also provides a method for confirming LY6K gene expression in cultured stem cells or measuring the content of a protein encoded by the LY6K gene; and

[0018] A method for quality control of a stem cell therapeutic agent is provided, including a step of selecting the stem cell as a therapeutic agent when the expression of the LY6K gene or the content of the protein encoded by the LY6K gene is higher than that of other stem cells.

[0019] The present invention also provides a method for producing high-efficiency stem cells, comprising a step of treating stem cells with a protein encoded by the LY6K gene.

[0020] The present invention also provides a composition for producing high-efficiency stem cells containing a protein encoded by the LY6K gene as an effective ingredient.

[0021] The present invention also provides a composition for selecting high-efficiency stem cells, comprising an agent capable of measuring the expression level of mRNA of the LY6K gene or the level of protein encoded by LY6K.

[0022] The present invention also provides a high-efficiency stem cell selection kit comprising the composition.

[0023]

[0024] Figure 1a shows the results of the apoptosis inhibition efficacy of mesenchymal stem cells.

[0025] Figure 1b shows the results of a heatmap showing differentially expressed genes (DEGs) in the high-efficacy group compared to the low-efficacy group.

[0026] Figure 1c is a graph showing the expression level of LY6K, the gene showing the greatest difference in expression between the high-efficacy group and the low-efficacy group, as Normalized Data (log2).

[0027] Figure 1d is a graph comparing the expression levels of the LY6K gene, which was selected as a high-efficacy MSC gene candidate, in the high-efficacy group and the low-efficacy group.

[0028] Figure 2a is a graph comparing the relative mRNA expression of LY6K when a selected siRNA sequence was transfected.

[0029] Figure 2b shows the results of analysis using gene set enrichment analysis (GSEA) showing that the expression of LY6K is associated with “cell cycle” and “DNA replication.”

[0030] Figure 2c shows the genes whose expression changed in siLY6K-treated mesenchymal stem cells compared to siNC-treated mesenchymal stem cells, and is divided into the top 14 categories based on p-values ​​through DAVID analysis using the Gene ontology (GO) database.

[0031] Figure 2d shows the results of confirming the cell proliferation ability and replication time of mesenchymal stem cells after knocking down siLY6K by transfection into mesenchymal stem cells.

[0032] Figure 2e shows the results of confirming the phosphorylation of AKT and ERK, kinases involved in cell proliferation, after knocking down siLY6K by transfecting it into mesenchymal stem cells.

[0033] Figure 3a shows the results of confirming the migratory ability of mesenchymal stem cells after knocking down siLY6K by transfection into mesenchymal stem cells.

[0034] Figure 3b shows the results of confirming the adhesive ability of mesenchymal stem cells after knocking down siLY6K by transfection into mesenchymal stem cells.

[0035] Figure 3c shows the results of confirming the phosphorylation of Smad1 / 5 and Smad2 / 3 in mesenchymal stem cells transfected with siLY6K, when treated with or without BMP-6 and TGF-β3.

[0036] Figure 4a shows the results of confirming stem cell function through FACS after knocking down siLY6K by transfection into mesenchymal stem cells.

[0037] Figure 4b shows the results of confirming differentiation ability after knocking down siLY6K by transfection into mesenchymal stem cells.

[0038] Figure 5a shows the results of confirming the expression of Cleaved PARP and Cleaved Caspase 3 proteins, which are apoptosis markers, in a myoblast apoptosis model co-cultured with 17 lots of mesenchymal stem cells.

[0039] Figure 5b shows the results of classifying 17 lots of mesenchymal stem cells by the gene expression level of LY6K and listing the protein expression levels of Cleaved PARP and Cleaved Caspase 3 in myoblasts in order of the gene expression level of LY6K.

[0040] Figure 5c shows the results of comparing the protein expression levels of Cleaved PARP and Cleaved Caspase 3 in myoblasts by mesenchymal stem cell group of LY6K_L, LY6K_M, and LY6K_H.

[0041] Figure 5d shows the results of analyzing the correlation between the protein expression levels of Cleaved PARP and Cleaved Caspase 3 in myoblasts and the LY6K gene expression level in mesenchymal stem cells.

[0042] Figure 6a shows the results of Western blotting confirming the decrease in anti-apoptotic efficacy against myoblasts due to inhibition of LY6K gene expression in mesenchymal stem cells.

[0043] Figure 6b shows the results of confirming the expression of Annexin V and 7AAD, which are apoptosis-related proteins, in co-cultured myoblasts following inhibition of LY6K gene expression in mesenchymal stem cells, using a flow cytometer.

[0044] Figure 7a shows the results of Western blotting to confirm the expression of MHC, a muscle regeneration marker, following the inhibition of LY6K gene expression in mesenchymal stem cells in damaged myotubes treated with lovastatin.

[0045] Figure 7b shows the results of immunocytochemistry staining to confirm the decreased recovery of atrophied myotube cells due to inhibition of LY6K gene expression in mesenchymal stem cells caused by lovastatin treatment.

[0046] Figure 8a shows the results of confirming the change in grip strength of mice according to the amount of LY6K gene expression of mesenchymal stem cells administered to a muscular dystrophy mouse model.

[0047] Figure 8b shows the results of confirming the difference in creatine kinase activity according to the amount of LY6K gene expression in mesenchymal stem cells administered to a muscular dystrophy mouse model.

[0048] Figure 9a shows the results of examining the degree of H&E and Tunel staining in mouse calf muscle tissue according to the level of LY6K gene expression of mesenchymal stem cells administered to a muscular dystrophy mouse model.

[0049] Figure 9b shows the results of confirming the expression level of Annexin V protein, a cell death marker, in mouse calf muscle tissue according to the expression level of LY6K gene in mesenchymal stem cells administered to a muscular dystrophy mouse model.

[0050] Figure 9c shows the results of confirming the regenerative effect of muscle tissue according to the difference in the expression level of the LY6K gene of mesenchymal stem cells administered to a muscular dystrophy mouse model through MHC staining, a muscle marker.

[0051] Figure 9d shows the results of confirming the expression level of MHC protein in mouse calf muscle tissue according to the expression level of LY6K gene in mesenchymal stem cells administered to a muscular dystrophy mouse model.

[0052] Figure 9e shows the results of confirming the efficacy of reducing fibrosis in muscle tissue according to the difference in the amount of LY6K gene expression of mesenchymal stem cells administered to a muscular dystrophy mouse model using Sirius Red staining.

[0053] Figure 9f shows the results of confirming the expression level of fibronectin protein in mouse calf muscle tissue according to the expression level of LY6K gene in mesenchymal stem cells administered to a muscular dystrophy mouse model.

[0054]

[0055] Detailed description of the invention and preferred embodiments

[0056] 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 pertains, unless otherwise defined herein. Generally, the nomenclature used herein is well known and commonly used in the art.

[0057] In the present invention, in order to develop a method capable of selecting only high-potency stem cells, high-potency mesenchymal stem cells that exhibit a high cell death inhibition effect when co-cultured with a myoblast apoptosis model were selected, and it was confirmed that the LY6K gene was expressed at a higher level in the selected high-potency mesenchymal stem cells than in low-potency mesenchymal stem cells. In addition, it was confirmed that cell proliferation and cell migration were reduced in mesenchymal stem cells in which the expression of the LY6K gene was suppressed by siRNA.

[0058] Therefore, the present invention provides, in one aspect, a method for detecting LY6K gene expression in stem cells or measuring the content of a protein encoded by the LY6K gene; and

[0059] The present invention relates to a method for selecting high-potency stem cells, including a step of selecting high-potency stem cells when the expression of the LY6K gene or the content of the protein encoded by the LY6K gene is higher than that of other stem cells.

[0060] In the present invention, “high-efficiency stem cells” refer to stem cells that have very high cell proliferation, cell migration, adhesion, and cell death inhibition abilities, and thus are highly effective as cell therapy agents.

[0061] In one embodiment of the present invention, mRNA Quantseq was performed on high-potency stem cells and low-potency stem cells, and genes with a log2 value of 4.0 or higher and a fold change of 2 times or more were compared and analyzed to find genes that were highly expressed in the high-potency stem cell group compared to the low-potency stem cell group among the genes that had a log2 value of 4.0 or higher and a fold change (FC) of 2 times or more, and 17 genes that were highly expressed in the high-potency stem cell group compared to the low-potency stem cell group were identified, and among them, LY6K (Fold change=6.902), which was most highly expressed in the high-potency stem cell group compared to the low-potency stem cell group, was selected as the final candidate for the high-potency stem cell selection gene.

[0062] In the present invention, the step of selecting high-efficiency stem cells may be characterized by selecting cells in which the mRNA expression level of LY6K has a log2 value of 4.0 or higher and a fold change of 2 times or more.

[0063] In the present invention, the step of selecting high-potency stem cells can select stem cells with high expression of LY6K compared to a control group of cells, and the control group can be low-potency stem cells.

[0064] In one aspect of the present invention, it was confirmed that cell proliferation ability was reduced and doubling time was increased in mesenchymal stem cells in which expression of the LY6K gene was suppressed by siRNA compared to the control group.

[0065] In another aspect of the present invention, it was confirmed through a wound healing assay that the migratory ability of mesenchymal stem cells was reduced in mesenchymal stem cells in which the expression of LY6K was suppressed.

[0066] In another aspect of the present invention, it was confirmed that the adhesion ability of mesenchymal stem cells was reduced in mesenchymal stem cells in which the expression of LY6K was suppressed.

[0067] In another embodiment of the present invention, mesenchymal stem cells and muscle cells in which apoptosis was induced were co-cultured, and the expression of apoptosis-related proteins according to the inhibition of LY6K gene expression was confirmed. As a result, it was confirmed that the expression levels of cleaved PARP and cleaved Caspase 3 were reduced in the experimental group co-cultured with the siNC-treated control group compared to the control group in which apoptosis was induced. However, there was no difference in the expression levels of cleaved PARP and cleaved Caspase 3 in the experimental group co-cultured with the siLY6K-treated group. In other words, it was confirmed that when LY6K expression was inhibited in mesenchymal stem cells, the ability to inhibit apoptosis of muscle cells was significantly reduced.

[0068] In another embodiment of the present invention, it was confirmed that the thickness of myotubes in which damage was induced by treatment with lovastatin was reduced compared to normal myotubes, but the thickness of myotubes co-cultured with mesenchymal stem cells with low LY6K gene expression was restored to a level similar to that before treatment with lovastatin. Therefore, it was confirmed that when LY6K expression is suppressed in mesenchymal stem cells, the muscle cell regeneration efficacy is reduced.

[0069] In another aspect of the present invention, as a result of confirming the change in mouse grip strength according to mesenchymal stem cells administered to a muscular dystrophy mouse model (Mdx), it was confirmed that the grip strength value tended to be measured lower in mice administered mesenchymal stem cells with low LY6K gene expression than in mice administered mesenchymal stem cells with high LY6K gene expression. Therefore, it was confirmed that the recovery of motor ability was reduced in mice administered mesenchymal stem cells with low LY6K gene expression.

[0070] In another aspect of the present invention, when changes in creatine kinase activity were confirmed in mesenchymal stem cells administered to a muscular dystrophy mouse model, it was confirmed that creatine kinase activity was significantly reduced in mice administered mesenchymal stem cells with high LY6K gene expression, but that there was almost no difference in CK activity between mice administered mesenchymal stem cells with low LY6K gene expression and the control group of the muscular dystrophy mouse model. Therefore, it was confirmed that the therapeutic efficacy for damaged muscle cells was reduced in mice administered mesenchymal stem cells with low LY6K gene expression.

[0071] In another aspect of the present invention, it was confirmed that the ratio of TUNEL-positive cells (←) was significantly reduced when mesenchymal stem cells with high LY6K expression were administered in a muscular dystrophy mouse model compared to when mesenchymal stem cells with low LY6K expression were administered. Since TUNEL binds to and stains DNA ends generated by apoptosis, it was confirmed through H&E staining and Western blotting that apoptosis was reduced when mesenchymal stem cells with high LY6K expression were administered, as evidenced by the decrease in the ratio of TUNEL-positive cells (see Fig. 9a).

[0072] In addition, in another aspect, when mesenchymal stem cells with high LY6K gene expression were administered to a muscular dystrophy mouse model, a tendency for the expression of AnnexinⅤ to decrease was confirmed compared to the MDX control group, and the group administered mesenchymal stem cells with high LY6K gene expression was confirmed to have a significant decrease in the expression of AnnexinⅤ compared to the group administered mesenchymal stem cells with low LY6K gene expression, confirming that the expression of LY6K is related to the anti-apoptotic efficacy in the muscular dystrophy mouse model (see Fig. 9b).

[0073] In another aspect of the present invention, when mesenchymal stem cells with high LY6K gene expression were administered to a muscular dystrophy mouse model, it was confirmed that the expression level of MHC was significantly increased compared to the MDX control group or the group administered mesenchymal stem cells with low LY6K gene expression. In addition, the group administered mesenchymal stem cells with high LY6K gene expression showed a significant increase in the expression level of MHC compared to the group administered mesenchymal stem cells with low LY6K gene expression. Therefore, it was confirmed that the expression level of LY6K was associated with muscle regeneration in a muscular dystrophy mouse model (see FIGS. 9c and 9d).

[0074] In another aspect of the present invention, it was confirmed that when mesenchymal stem cells were administered to a muscular dystrophy mouse model, collagen accumulation due to fibrosis was significantly reduced compared to the MDX control group. In addition, it was confirmed that the group administered mesenchymal stem cells with high LY6K gene expression showed a significant decrease in collagen accumulation and fibronectin expression compared to the group administered mesenchymal stem cells with low LY6K gene expression, and it was confirmed that LY6K expression was associated with a decrease in muscle fibrosis (see FIGS. 9e and 9f).

[0075] Therefore, from another perspective, the present invention provides a method for confirming LY6K gene expression in cultured stem cells or measuring the content of a protein encoded by the LY6K gene; and

[0076] The present invention relates to a method for quality control of a stem cell therapeutic agent, including a step of selecting the stem cell therapeutic agent when the expression of the LY6K gene or the content of the protein encoded by the LY6K gene is higher than that of other stem cells.

[0077] The selection of the above high-potency stem cells is performed by measuring the expression of LY6K at the mRNA level of a cultured stem cell lot, and selecting stem cells with relatively high LY6K gene expression as high-potency stem cells.

[0078] In another aspect, the present invention relates to a method for producing high-potency stem cells, which comprises a step of treating stem cells with a protein encoded by the LY6K gene.

[0079] From another perspective, the present invention relates to a composition for producing high-efficiency stem cells containing a protein encoded by the LY6K gene as an active ingredient.

[0080] In another aspect, the present invention relates to a composition for selecting high-efficiency stem cells, comprising an agent capable of measuring the expression level of mRNA of the LY6K gene or the level of protein encoded by LY6K.

[0081] In the present invention, the agent capable of measuring the expression level of the mRNA may be characterized by being selected from the group consisting of a primer that specifically binds to and amplifies the mRNA, a probe that specifically binds to the mRNA, and an antisense nucleotide.

[0082] In the present invention, the agent capable of measuring the protein level may be characterized by being selected from the group consisting of antibodies, oligopeptides, ligands, PNA (peptide nucleic acid), and aptamers that specifically bind to the protein.

[0083] In another aspect, the present invention relates to a high-efficiency stem cell selection kit comprising the above composition.

[0084] In the present invention, the kit may be characterized as being an RT-PCR kit, a DNA chip kit, an ELISA kit, a protein chip kit, a rapid kit, or an MRM (Multiple reaction monitoring) kit.

[0085] The kit of the present invention comprises one or more kinds of different component compositions, solutions or devices suitable for an analysis method, and may be an RT-PCR kit, a DNA chip kit or a protein chip kit. In addition to each primer pair specific for a marker gene, the RT-PCR kit may include a test tube or other suitable container, a reaction buffer, deoxynucleotides (dNTPs), enzymes such as Taq polymerase and reverse transcriptase, DNase, RNase inhibitor, DEPC water, sterile water, etc. In addition, it may include a primer pair specific for a gene used as a quantitative control. The DNA chip kit includes a substrate to which a cDNA corresponding to a gene or a fragment thereof is attached as a probe, and the substrate may include a cDNA corresponding to a quantitative structural gene or a fragment thereof.

[0086] In addition, the kit according to the present invention may be a diagnostic kit including a preparation for measuring the protein level, wherein the preparation capable of measuring the protein level is preferably an antibody specific for the protein. Accordingly, the kit including the preparation capable of measuring the protein level may be, for example, a marker detection kit including the essential elements necessary for performing an ELISA, and such a kit may also include reagents capable of detecting antibodies that have formed an "antigen-antibody complex", such as a labeled secondary antibody, chromophores, enzymes (e.g., conjugated to antibodies), and substrates thereof. In addition, an antibody specific for a quantitative control protein may be included.

[0087] Additionally, the amount of antigen-antibody complex formed can be quantitatively measured through the size of the signal of the detection label. Such detection labels can be selected from the group consisting of, but not necessarily limited to, enzymes, fluorescent substances, ligands, luminescent substances, microparticles, redox molecules, and radioisotopes.

[0088] Analytical methods for measuring protein levels include, but are not limited to, Western blot, ELISA, radioimmunoassay, radioimmunodiffusion, aukteroni immunodiffusion, rocket immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation assay, FACS, protein chip, etc., and can be performed using methods known to those skilled in the art.

[0089]

[0090] [Example]

[0091] Hereinafter, the present invention will be described in more detail through examples. These examples are intended solely 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 limited by these examples.

[0092]

[0093] Example 1. Culturing of mesenchymal stem cells

[0094] Mesenchymal stem cells were obtained from umbilical cords and placentas after obtaining prior consent through a collaborative study with the Department of Obstetrics and Gynecology of Samsung Medical Center under the approval of the Institutional Review Board (IRB) of Samsung Medical Center (IRB No. 2016-07-102-043). Mesenchymal stem cells were isolated using a previously known method. Cells were cultured in MEM-alpha (Minimum Essential Medium, Gibco) medium containing 10% FBS (fetal bovine serum, Gibco) and 50 μg / mL gentamicin (Gibco) at 3 × 10 3 The cells were seeded at 100 cells / cm2 and cultured in a 37°C, 5% CO2 incubator.

[0095]

[0096] Example 2. Comparative analysis of gene expression between high-potency and low-potency mesenchymal stem cells.

[0097] To confirm the apoptosis inhibitory effect of mesenchymal stem cells on the myoblast (C2C12, ATCC) apoptosis model, six lots of mesenchymal stem cells were co-cultured with apoptotic myoblasts, and the expression of apoptosis markers cleaved PARP and cleaved Caspase 3 was confirmed.

[0098] Mouse myoblasts C2C12 were cultured in 100 mm culture dishes at 7 Х 10 cm in DMEM (Dulbecco's Modified Eagle's Medium, Biowest) supplemented with 10% FBS and 1 U / ml penicillin / streptomycin (Gibco). 5After culturing for 24 hours, the cells were seeded at a density of 10 cells / dish and starved with FBS for another 24 hours to induce apoptosis. At this time, myoblasts were co-cultured with mesenchymal stem cells using inserts. Western blotting was performed using cleaved poly ADP-ribose polymerase (cleaved PARP) (Cell Signaling Technology), cleaved caspase 3 (Cell Signaling Technology), and beta-actin (Santa Cruz Biotechnology) as primary antibodies, and the degree of apoptosis was compared. Based on these results, the mesenchymal stem cells were classified into high-potency mesenchymal stem cells and low-potency mesenchymal stem cells according to the apoptosis inhibition efficacy.

[0099] Figure 1a shows the results demonstrating the apoptosis inhibition efficacy of mesenchymal stem cells. Compared to the low-efficacy group, the expression of apoptosis markers cleaved PARP and cleaved caspase 3 decreased in a mouse myoblast (C2C12) cell death model co-cultured with the high-efficacy group.

[0100] RNA was extracted from high-efficacy mesenchymal stem cells and low-efficacy mesenchymal stem cells, and mRNA Quantseq was performed to compare and analyze genes that were highly expressed in the high-efficacy group compared to the low-efficacy group among genes with a p-value of 0.05 or less, a log2 value of 4.0 or more, and a fold change of more than 2-fold.

[0101] Figure 1b shows a heatmap of differentially expressed genes (DEGs) in the high-efficacy group compared to the low-efficacy group. Yellow indicates high expression, and blue indicates low expression.

[0102] As shown in Figure 1b, 21 genes were identified with a log2 value of 4.0 or higher and a fold change (FC) difference of more than 2-fold, and among them, 17 genes were identified as being highly expressed in the high-efficacy group compared to the low-efficacy group.

[0103] Figure 1c is a graph showing the expression level of LY6K, the gene showing the greatest difference in expression between the high-efficacy group and the low-efficacy group, as Normalized Data (log2).

[0104] As shown in Fig. 1c, LY6K (Fold change = 6.902), which was most highly expressed in the high-efficacy group compared to the low-efficacy group, was selected as the final candidate for the high-efficacy selection gene.

[0105] To confirm the expression of the high-efficacy selection gene candidates selected in Fig. 1c in mesenchymal stem cells, qRT-PCR was performed on three lots of high-efficacy group mesenchymal stem cells and three lots of low-efficacy group mesenchymal stem cells to confirm the expression of LY6K.

[0106] RNA was extracted from high-potency group mesenchymal stem cells and low-potency group mesenchymal stem cells using AccuPrep Universal RNA Extraction Kit (bioneer), and qRT-PCR was performed using 2X Power SYBR Green Master Mix (Applied Biosystems) and primers (see Table 1).

[0107]

[0108] Figure 1d is a graph comparing the expression levels of the LY6K gene, which was selected as a high-efficacy MSC gene candidate, in the high-efficacy group and the low-efficacy group.

[0109] As shown in Fig. 1d, LY6K, which showed a statistically significant difference in the mRNA levels of three lots of high-potency mesenchymal stem cells and low-potency mesenchymal stem cells, was selected as the final candidate for the high-potency MSC gene.

[0110]

[0111] Example 3. Correlation between inhibition of LY6K expression in mesenchymal stem cells by LY6K knockdown and cell proliferation and replication time

[0112] 3-1. Selection of LY6K siRNA sequence candidates and verification of knockdown efficacy through mRNA expression level confirmation.

[0113] In Example 2, the optimal sequence was selected through knockdown efficacy verification using an siRNA library for LY6K, which was selected as a high-efficacy MSC gene. Mesenchymal stem cells cultured in cell culture medium were transferred to serum-free medium, and then three candidate sequences for LY6K from the siRNA library (bioneer, see Table 2) were transfected at a concentration of 75 nM using Lipofectamine RNAiMax (Invitrogen). After 48 hours, RNA was extracted from the mesenchymal stem cells using the AccuPrep Universal RNA Extraction Kit (bioneer), and qRT-PCR was performed using 2X Power SYBR Green Master Mix (Applied Biosystems) and primers (see Table 1). Afterwards, the sequence showing the greatest knockdown effect was selected as the optimal sequence for inhibiting LY6K expression by comparing relative mRNA expression.

[0114]

[0115] As a result, in the case of siLY6K, it was confirmed that the sequence of candidate 2 showed the greatest knock-down effect. Therefore, after transfecting mesenchymal stem cells using the candidate 2 sequence of siLY6K, the relative mRNA expression rate by the selected sequence was confirmed.

[0116] Figure 2a is a graph comparing the relative mRNA expression of LY6K when a selected siRNA sequence was transfected.

[0117] As a result, as shown in Fig. 2a, mRNA expression was significantly reduced in the siLY6K treatment group compared to the siNC treatment group. Therefore, the selected siRNA sequence has sequence specificity for the LY6K gene.

[0118]

[0119] 3-2. Confirmation of changes in gene expression in mesenchymal stem cells due to LY6K knockdown

[0120] RNA was extracted from mesenchymal stem cells transfected with the siRNA sequence selected in Example 3-1 to reduce LY6K gene expression, and from control mesenchymal stem cells transfected with a negative control siRNA (siNC), and mRNA Quantseq was performed. Gene set enrichment analysis (GSEA) based on the KEGG pathway was performed to identify a significant gene set related to the expression level of LY6K, and gene ontology related to the expression level of LY6K was confirmed through DAVID analysis.

[0121] Figure 2b is an analysis result showing that the expression of LY6K is associated with “cell cycle” and “DNA replication” through gene set enrichment analysis (GSEA).

[0122] Figure 2c shows the results of DAVID analysis using the Gene ontology (GO) database, showing the top 14 categories based on p-values ​​for genes whose expression changed in mesenchymal stem cells treated with siLY6K compared to mesenchymal stem cells treated with siNC.

[0123] As a result, as shown in Fig. 2b, it was confirmed that the enrichment score was low in the gene set related to the cell cycle and DNA replication pathway in mesenchymal stem cells treated with siLY6K.

[0124] Furthermore, as shown in Figure 2c, when confirmed through Gene Ontology, it was confirmed that genes related to cell division, cell cycle, and DNA replication were significantly reduced in mesenchymal stem cells treated with siLY6K. The results of gene set enrichment analysis (GSEA) and DAVID analysis were consistent, confirming that the expression level of LY6K affects the proliferation of mesenchymal stem cells.

[0125]

[0126] 3-3. Confirmation of the correlation between LY6K expression inhibition and cell proliferation and replication time.

[0127] The correlation between the inhibition of LY6K expression and the cell proliferation and replication time of mesenchymal stem cells was confirmed.

[0128] Mesenchymal stem cells transfected with siNC and siLY6K selected in Example 3-1 were seeded in 6-well plates at 3 × 10 using MEM Alpha (Minimum Essential Medium, Invitrogen-Gibco) medium containing 10% FBS. 4 The cells were dispensed per well, and the cells were harvested at 24-hour intervals for 5 days after dispensing, and the cell count was confirmed.

[0129] Figure 2d shows the results of confirming the cell proliferation ability and replication time of mesenchymal stem cells after knocking down siLY6K by transfection into mesenchymal stem cells.

[0130] As a result, as shown in Fig. 2d, cell proliferation capacity was reduced in the siLY6K treatment group compared to the siNC treatment control group. The replication time of the siNC treatment control group was approximately 38.9 hours, and the replication time of the siLY6K treatment group was approximately 66.9 hours, confirming an increase in replication time. This confirmed that the proliferation capacity of mesenchymal stem cells decreased as the expression of the LY6K gene decreased.

[0131]

[0132] 3-4. Confirmation of the correlation between LY6K expression inhibition and kinase phosphorylation.

[0133] The correlation between the suppression of LY6K expression and the phosphorylation of AKT and ERK was confirmed through Western blotting.

[0134] Specifically, siNC and siLY6K were transfected into mesenchymal stem cells and cultured for 48 hours. After 48 hours, mesenchymal stem cells were pelleted. After lysing with RIPA buffer (BIOSESANG) containing protease inhibitor cocktail (Amresco) and EDTA, the cells were centrifuged at 4°C and 15,000 rpm for 30 minutes to obtain the supernatant. 10 μg of protein was electrophoresed through SDS-PAGE and transferred to PVDF (polyvinylidene difluoride) and NC (nitrocellulose) membranes. Blocking was performed for 1 hour at room temperature with TBST containing 5% skim milk, and primary antibodies were diluted in TBST containing 5% BSA and reacted overnight at 4°C. Afterwards, the membrane was washed three times with TBST for 10 minutes each, and the secondary antibody was diluted in TBST containing 5% BSA 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 (BIO-RAD, USA), and the band image was confirmed using a gel imaging system (Amersham Imager 600). The protein expression level was measured using Image J and corrected with β-actin. AKT, p-AKT (Cell Signaling Technology), ERK, p-ERK (R&D Systems), LY6K (Abcam), and β-actin (Santa Cruz Biotechnology) were used as primary antibodies.

[0135] Figure 2e shows the results of confirming the phosphorylation of AKT and ERK, kinases involved in cell proliferation, after knocking down siLY6K by transfecting it into mesenchymal stem cells.

[0136] As a result, as shown in Fig. 2e, it was confirmed that the phosphorylation of AKT was reduced by the expression of LY6K, while the phosphorylation of ERK was not affected. In other words, it was confirmed that LY6K is involved in the phosphorylation of AKT and affects cell proliferation.

[0137]

[0138] Example 4. Correlation between inhibition of LY6K expression in mesenchymal stem cells by LY6K knockdown and cell migration and cell adhesion.

[0139] 4-1. Confirmation of the correlation between LY6K expression inhibition and cell migration ability

[0140] To identify homing-related characteristics that affect the therapeutic efficacy of mesenchymal stem cells, a wound healing assay was performed to confirm the correlation between the inhibition of LY6K expression and the cell migration ability of mesenchymal stem cells.

[0141] Mesenchymal stem cells transfected with siNC and siLY6K selected in Example 3-1 were seeded in a 96-well plate at 1.5 x 10 using MEM Alpha (Minimum Essential Medium, Invitrogen-Gibco) medium containing 10% FBS. 4 Cells were seeded at 1 cell / well and cultured for 48 hours. Afterwards, the cells were rinsed twice with MEM Alpha medium without FBS to inhibit cell proliferation, and 10 μg / ml mitomycin C (Sigma-Aldrich) was added to MEM Alpha medium and cultured for 2 hours. Afterwards, the cells were scratched, rinsed twice with culture medium, and images were taken using Incucyte (Sartorius) at 3-hour intervals from 0 to 24 hours. The captured images were expressed as the percentage of wound confluence using the Incucyte analysis tool to show cell migration ability.

[0142] Figure 3a shows the results of confirming the migratory ability of mesenchymal stem cells after knocking down siLY6K by transfection into mesenchymal stem cells.

[0143] As a result, as shown in Fig. 3a, the wound confluence percentage of the siNC-treated control group at the endpoint was approximately 85.3%, and that of the siLY6K-treated group was approximately 73.2%. In other words, it was confirmed that the migratory ability of mesenchymal stem cells decreased as the expression of the LY6K gene decreased.

[0144]

[0145] 4-2. Confirmation of the correlation between LY6K expression inhibition and cell adhesion.

[0146] To identify homing-related characteristics that affect the therapeutic efficacy of mesenchymal stem cells, a cell adhesion assay was performed to confirm the correlation between the inhibition of LY6K expression and the cell adhesion ability of mesenchymal stem cells.

[0147] Mesenchymal stem cells transfected with siNC and siLY6K selected in Example 3-1 were stained with calcein AM using Vybrant Cell Adhesion Assay Kit (Invitrogen), and then seeded in 96-well plates at 1 х 10 using MEM Alpha (Minimum Essential Medium, Invitrogen-Gibco) medium without FBS. 5 Cells were dispensed per well and cultured for 2 hours. Afterwards, the medium was removed to remove non-attached cells, and fluorescence was measured at a wavelength of 494 nm. The fluorescence value of attached cells was divided by the fluorescence value of all cells to express the cell adhesion as a percentage.

[0148] Figure 3b shows the results of confirming the adhesive ability of mesenchymal stem cells after knocking down siLY6K by transfection into mesenchymal stem cells.

[0149] As a result, as shown in Fig. 3b, in the siNC-treated control group, approximately 98.2% of cells were attached after 2 hours of incubation, whereas in the siLY6K-treated group, only approximately 61.6% of cells were attached. In other words, it was confirmed that the adhesive capacity of mesenchymal stem cells decreased as the expression of the LY6K gene decreased.

[0150]

[0151] 4-3. Confirmation of the correlation between LY6K expression and the Smad pathway

[0152] To confirm the genetic changes and signaling pathway changes caused by LY6K knockdown, phosphorylation of Smad1 / 5 and Smad2 / 3 was confirmed through Western blotting (Fig. 3c).

[0153] Mesenchymal stem cells transfected with siNC and siLY6K selected in Example 3-1 were treated with BMP-6 at a concentration of 100 ng / mL for 1 hour, and with TGF-β3 at a concentration of 10 ng / mL for 0.5 hour, after which the cells were harvested. Western blotting was performed in the same manner as in Example 3-2 using Smad1, Phospho-Smad1 / 5, Smad1 / 3, Phospho-Smad2 / 3 (Cell Signaling Technology), LY6K (Abcam), and Beta-actin (Santa Cruz Biotechnology) as primary antibodies.

[0154] Figure 3c shows the results of confirming the phosphorylation of Smad1 / 5 and Smad2 / 3 in mesenchymal stem cells transfected with siLY6K, when treated with or without BMP-6 and TGF-β3.

[0155] As a result, as shown in Fig. 3c, when BMP-6 was treated in mesenchymal stem cells in which LY6K was knocked down, the phosphorylation of Smad1 / 5 was most significantly reduced, confirming that LY6K knockdown affects the Smad1 / 5 signaling pathway. Since Smad1 / 5 is known to be involved in cell migration, it was confirmed that LY6K affects cell migration by being involved in the phosphorylation of Smad1 / 5.

[0156]

[0157] Example 5. Inhibition of LY6K expression and confirmation of stem cell function and differentiation potential.

[0158] 5-1. Inhibition of LY6K expression and confirmation of stem cell function

[0159] Using the sequence selected in Example 3-1, siLY6K was transfected into mesenchymal stem cells to knock them down, and then stem cell function was confirmed using FACS.

[0160] Mesenchymal stem cell markers were identified through the expression of CD44, CD73, CD90, CD105, and CD166, and hematopoietic stem cell lineage markers were CD11b, CD14, CD19, CD34, CD45, and HLA-DR (MHCII) (BD Pharmigen) to compare stem cell capacity with the siNC-treated control group. At this time, 10,000 events were acquired and analyzed using a BD FACS Verse flow cytometer.

[0161] Figure 4a shows the results of confirming stem cell function through FACS after knocking down siLY6K by transfection into mesenchymal stem cells.

[0162] As a result, as shown in Fig. 4a, positive markers CD44, CD73, CD90, CD105, and CD166 were expressed at over 95%, but negative markers CD11b, CD14, CD19, CD34, CD45, and HLA-DR (MHCII) were barely expressed at less than 0.5%. In other words, it was confirmed that even if LY6K was knocked down by siRNA in mesenchymal stem cells, it did not affect stem cell function.

[0163]

[0164] 5-2. Inhibition of LY6K expression and confirmation of differentiation potential

[0165] To confirm the correlation between the inhibition of LY6K expression and the differentiation capacity of mesenchymal stem cells, the degree of differentiation was confirmed by differentiating them into adipocytes, osteocytes, and chondrocytes.

[0166] Adipocyte differentiation was performed by seeding 1X10 mesenchymal stem cells transfected with siLY6K using the sequence selected in Example 3-1 in a 6-well plate using MEM Alpha (Minimum Essential Medium, Invitrogen-Gibco) medium containing 10% FBS. 5 Cells were seeded at 1 / well and cultured until 100% confluent. Afterwards, the cells were replaced with adipocyte differentiation medium using the Stempro Adipocyte Differentiation Kit (Gibco). The differentiation medium was replaced every 3-4 days, and differentiation was performed for 21 days. After differentiation was complete, the cells were fixed at room temperature using 4% PFA and stained using Oil Red O solution (Sigma).

[0167] Osteocyte differentiation was performed by seeding 5X10 mesenchymal stem cells transfected with siLY6K in 6-well plates using MEM Alpha (Minimum Essential Medium, Invitrogen-Gibco) medium containing 10% FBS.4 The cells were seeded at 1 / well and cultured for 24 hours. Afterwards, the osteocyte differentiation medium was replaced using the Stempro Osteocyte Differentiation Kit (Gibco). The differentiation medium was replaced every 3-4 days, and differentiation was performed for 23 days. After differentiation, the cells were fixed at room temperature using 4% PFA and stained using the Alizarin Red S Staining Quantification Assay Kit (Sciencell).

[0168] Chondrocyte differentiation was performed by seeding 2X10 mesenchymal stem cells transfected with siLY6K in 6-well plates using MEM Alpha (Minimum Essential Medium, Invitrogen-Gibco) medium containing 10% FBS. 5 Cells were seeded at 1 / well and cultured for 24 hours. Afterwards, the cells were replaced with chondrocyte differentiation medium using the Stempro Chondrocyte Differentiation Kit (Gibco). The differentiation medium was replaced every 3-4 days, and differentiation was performed for 30 days. After complete differentiation, the cells were fixed at room temperature using 4% PFA and stained with Alcian Blue (IHCworld).

[0169] Figure 4b shows the results of confirming differentiation ability after knocking down siLY6K by transfection into mesenchymal stem cells.

[0170] As a result, as shown in Fig. 4b, it was confirmed that differentiation into Adipocyte, Osteocyte, and Chondrocyte progressed in the control group treated with siNC and the siLY6K treated group.

[0171] Therefore, when the results of Figure 4 are summarized, it was found that even if the expression of LY6K is suppressed in mesenchymal stem cells, there is no problem with the multipotency of mesenchymal stem cells.

[0172]

[0173] Example 6. Confirmation of the correlation between the apoptosis inhibition effect of mesenchymal stem cells and LY6K gene expression.

[0174] 6-1. Confirmation of the correlation between apoptosis inhibition and LY6K gene expression in mesenchymal stem cells of various lots.

[0175] To confirm the correlation between the apoptosis inhibition effect of mesenchymal stem cells and LY6K gene expression, 17 lots of mesenchymal stem cells were co-cultured with apoptosis-induced muscle cells, and the expression of apoptosis markers Cleaved PARP and Cleaved Caspase 3 was confirmed.

[0176] Mouse myoblasts C2C12 were cultured in 100 mm culture dishes at 7 Х 10 cm in DMEM (Dulbecco's Modified Eagle's Medium, Biowest) supplemented with 10% FBS and 1 U / ml penicillin / streptomycin (Gibco). 5 Cells were seeded at a density of 10 cells / dish and cultured for 24 hours, and then starved with FBS for another 24 hours to induce apoptosis. At this time, myoblasts were co-cultured with mesenchymal stem cells using inserts. Western blotting was performed using Cleaved Poly ADP-Ribose Polymerase (cleaved PARP) (Cell Signaling Technology), Cleaved Caspase 3 (Cell Signaling Technology), and Beta-actin (Santa Cruz Biotechnology) as primary antibodies, and the degree of apoptosis was compared.

[0177] Figure 5a shows the results of confirming the expression of Cleaved PARP and Cleaved Caspase 3 proteins, which are apoptosis markers, in a myoblast apoptosis model co-cultured with 17 lots of mesenchymal stem cells.

[0178] Figure 5b shows the results of listing the protein expression levels of Cleaved PARP and Cleaved Caspase 3 in myoblasts in the order of the LY6K gene expression levels by classifying 17 lots of mesenchymal stem cells by the gene expression level of LY6K. The LY6K gene expression levels confirmed in the 17 lots of mesenchymal stem cells were classified into the LY6K_L group, 6 lots expressing with a Fold change value of 2 or less based on the LY6K expression level of MSC_A, 5 lots expressing with a Fold change value exceeding 2 and 3.5 or less, LY6K_M group, and 6 lots expressing with a Fold change value exceeding 3.5, LY6K_H group, and the expression of Cleaved PARP, Cleaved Caspase 3, and LY6K genes, which are apoptosis markers, were shown according to these criteria.

[0179] Figure 5c shows the results of comparing the protein expression levels of Cleaved PARP and Cleaved Caspase 3 in myoblasts by mesenchymal stem cell group: LY6K_L, LY6K_M, and LY6K_H.

[0180] Figure 5d shows the results of analyzing the correlation between the protein expression levels of Cleaved PARP and Cleaved Caspase 3 in myoblasts and the LY6K gene expression level in mesenchymal stem cells.

[0181] As a result, the protein expression of Cleaved PARP and Cleaved Caspase 3, which are apoptosis markers, was confirmed to be low in the mouse myoblast model co-cultured with mesenchymal stem cells with high LY6K mRNA expression. When confirmed through Pearson correlation analysis, Cleaved PARP showed a correlation coefficient of r=-0.700**, and Cleaved Caspase 3 showed a correlation coefficient of r=-0.745**, confirming a significant strong negative correlation.

[0182] Therefore, it was confirmed that the higher the expression of LY6K in mesenchymal stem cells, the greater the effect of inhibiting apoptosis of muscle cells.

[0183]

[0184] Example 7. Confirmation of the correlation between the inhibition of LY6K gene expression in mesenchymal stem cells in vitro and the antiapoptotic effect.

[0185] 7-1. Confirmation of expression of apoptosis-related proteins following inhibition of LY6K gene expression in mesenchymal stem cells.

[0186] To determine whether the LY6K gene affects the apoptosis inhibition effect, mesenchymal stem cells, siNC, and mesenchymal stem cells treated with siLY6K selected in Example 3-1 were co-cultured with apoptosis-induced muscle cells, and then the protein expression of Cleaved PARP and Cleaved Caspase 3, which are apoptosis markers, was confirmed.

[0187] Mouse myoblasts C2C12 were cultured in 100 mm culture dishes at 7 Х 10 cm in DMEM (Dulbecco's Modified Eagle's Medium, Biowest) supplemented with 10% FBS and 1 U / ml penicillin / streptomycin (Gibco). 5 Cells were seeded at a density of 10 cells / dish and cultured for 24 hours, and then starved with FBS for another 24 hours to induce apoptosis. At this time, myoblasts were co-cultured with mesenchymal stem cells using inserts. Western blotting was performed using Cleaved Poly ADP-Ribose Polymerase (cleaved PARP) (Cell Signaling Technology), Cleaved Caspase 3 (Cell Signaling Technology), and Beta-actin (Santa Cruz Biotechnology) as primary antibodies, and the degree of apoptosis was compared.

[0188] Figure 6a shows the results of Western blotting confirming the decrease in anti-apoptotic efficacy against myoblasts due to inhibition of LY6K gene expression in mesenchymal stem cells.

[0189] As a result, as shown in Fig. 6a, it was confirmed that the expression levels of Cleaved PARP and Cleaved Caspase 3 decreased in the experimental group co-cultured with the siNC-treated control group compared to the apoptosis-induced control group. However, there was no difference in the expression levels of Cleaved PARP and Cleaved Caspase 3 in the experimental group co-cultured with the siLY6K-treated group. Specifically, in the experimental group co-cultured with the siNC-treated control group, the expression level of Cleaved PARP decreased by 0.67-fold, and the expression level of Cleaved Caspase 3 decreased by 0.68-fold. In the experimental group co-cultured with the siLY6K-treated group, the expression level of Cleaved PARP decreased by 0.98-fold, and the expression level of Cleaved Caspase 3 decreased by 0.93-fold.

[0190] That is, it was confirmed that when LY6K expression was suppressed in mesenchymal stem cells, the ability to suppress apoptosis of muscle cells was significantly reduced.

[0191] Figure 6b shows the results of confirming the expression of Annexin V and 7AAD, which are apoptosis-related proteins, in co-cultured myoblasts following inhibition of LY6K gene expression in mesenchymal stem cells, using a flow cytometer.

[0192] As a result, as shown in Fig. 6b, compared to the control group in which apoptosis was induced, the percentage of live cells increased, while the percentages of dead cells and apoptotic cells decreased in the experimental group co-cultured with siNC-treated mesenchymal stem cells. However, in the experimental group co-cultured with siLY6K-treated cells, compared to the control group in which apoptosis was induced, the percentage of live cells did not change, and while the percentage of dead cells decreased, the percentage of apoptotic cells increased.

[0193] That is, it was confirmed that when LY6K expression was suppressed in mesenchymal stem cells, the ability to suppress apoptosis of muscle cells was reduced.

[0194]

[0195] Example 8. Confirmation of the correlation between LY6K gene expression level and muscle regeneration efficacy in mesenchymal stem cells in vitro.

[0196] 8-1. Confirmation of the expression of muscle regeneration-related proteins following inhibition of LY6K gene expression in mesenchymal stem cells.

[0197] To determine whether knockdown of the LY6K gene affects muscle regeneration, myotube cells induced with damage were co-cultured with siNC and mesenchymal stem cells treated with siLY6K selected in Example 3-1, and then the expression of myosin heavy chain (MHC), a muscle cell marker, was confirmed.

[0198] Mouse myoblasts C2C12 were cultured in 6-well plates at 1 × 10 in DMEM (Dulbecco's Modified Eagle's Medium, Biowest) supplemented with 10% FBS and 1 U / ml penicillin / streptomycin (Gibco). 5After seeding at a density of 10 cells / well and culturing for 96 hours, the medium was replaced with DMEM (Dulbecco's Modified Eagle's Medium, Biowest) supplemented with 5% horse serum and 1 U / ml penicillin / streptomycin (Gibco) to induce differentiation into myotubes. Differentiated myotubes were treated with 10 μM lovastatin for 24 hours to induce myotube cell damage, and then co-cultured with mesenchymal stem cells using inserts. Western blotting was performed using Myosin Heavy Chain (R&D Systems) and Beta-actin (Santa Cruz Biotechnology) as primary antibodies, and the expression levels of muscle cell markers were compared.

[0199] Figure 7a shows the results of Western blotting to confirm the expression of MHC, a muscle regeneration marker, following the inhibition of LY6K gene expression in mesenchymal stem cells in damaged myotubes treated with lovastatin.

[0200] As a result, as shown in Fig. 7a, in myotubes damaged by lovastatin treatment, MHC expression decreased compared to normal myotubes, and in myotubes co-cultured with mesenchymal stem cells, MHC expression increased. In particular, the group co-cultured with siNC-treated mesenchymal stem cells showed an increase in MHC expression compared to the group co-cultured with siLY6K treatment, confirming that when LY6K expression is inhibited in mesenchymal stem cells, muscle regeneration efficacy is reduced.

[0201] Figure 7b shows the results of immunocytochemistry staining to confirm the decreased recovery of atrophied myotube cells due to inhibition of LY6K gene expression in mesenchymal stem cells caused by lovastatin treatment.

[0202] As a result, as shown in Fig. 7b, the thickness of myotubes treated with lovastatin to induce damage was reduced compared to normal myotubes, but the thickness of myotubes co-cultured with mesenchymal stem cells treated with siNC was confirmed to recover to a level similar to that before lovastatin treatment. In contrast, the thickness of myotubes co-cultured with mesenchymal stem cells treated with siLY6K was confirmed to not show statistical significance compared to the thickness of myotubes treated with lovastatin to induce damage.

[0203] That is, it was confirmed that when LY6K expression was suppressed in mesenchymal stem cells, muscle cell regeneration efficacy was reduced.

[0204]

[0205] Example 9. Confirmation of the correlation between LY6K gene expression level of mesenchymal stem cells in vivo and therapeutic efficacy of muscle tissue.

[0206] 9-1. Confirmation of the effect of LY6K gene expression on the recovery of motor ability in mdx mice through mouse phenotypic behavioral evaluation.

[0207] The mice used for the mouse phenotypic behavioral evaluation were C57BL / 10 strain, and mice of the same age were randomly assigned to each administration group.

[0208] Two phenotypic behavioral assessments were conducted, and a total of three phenotypic behavioral assessments were conducted per session and analyzed as the average value. Specifically, mesenchymal stem cells were administered through the tail vein of a muscular dystrophy mouse model (MDX), and grip strength was measured before administration and 1 week after administration. For each mouse, grip strength was measured in the forelimbs and hindlimbs (forelimb + hindlimb), and the measured grip strength value was divided by the body weight of the mouse to correct it, and the grip strength value after administration was compared with the grip strength value before administration.

[0209] Figure 8a shows the results of confirming changes in mouse grip strength according to the amount of LY6K gene expression of mesenchymal stem cells administered to a muscular dystrophy mouse model.

[0210] As a result of examining changes in mouse grip strength according to mesenchymal stem cells administered to a muscular dystrophy mouse model, as shown in Fig. 8a, in mice administered mesenchymal stem cells with high LY6K gene expression, the grip strength value after administration was confirmed to increase approximately 1.5 times compared to before administration. However, in mice administered mesenchymal stem cells with low LY6K gene expression, the grip strength value after administration was confirmed to show almost no change compared to before administration.

[0211] Therefore, it was confirmed that in a muscular dystrophy mouse model administered with mesenchymal stem cells with high LY6K gene expression, recovery of motor ability increased compared to a mouse model administered with mesenchymal stem cells with low LY6K gene expression.

[0212]

[0213] 9-2. Confirmation of muscle recovery effect in mdx mice according to LY6K gene expression level of mesenchymal stem cells using CK activity assay

[0214] The activity of creatine kinase, which increases when muscles are damaged, was measured to determine the extent to which damaged muscle cells recovered according to the level of LY6K gene expression in the administered mesenchymal stem cells.

[0215] Mesenchymal stem cells were administered via the tail vein to a muscular dystrophy mouse model (MDX) (Central Laboratory Animal), and mouse serum was obtained by orbital blood sampling before and one week after administration. Creatine kinase activity in the serum was measured using Dri-chem slide CPK-PⅢ (FUJI), and the creatine kinase activity values ​​after administration were compared with those before administration.

[0216] Figure 8b shows the results of confirming the difference in creatine kinase activity according to the amount of LY6K gene expression in mesenchymal stem cells administered to a muscular dystrophy mouse model.

[0217] As a result of confirming the change in creatine kinase activity according to the LY6K gene expression level of mesenchymal stem cells administered to a muscular dystrophy mouse model, as shown in Fig. 8b, in mice administered mesenchymal stem cells with high LY6K gene expression, CK activity was significantly reduced after administration, but in mice administered mesenchymal stem cells with low LY6K gene expression, there was almost no difference in CK activity compared to the muscular dystrophy mouse model (MDX) control group. Therefore, it was confirmed that the therapeutic efficacy for damaged muscle cells was reduced in mice administered mesenchymal stem cells with low LY6K gene expression.

[0218]

[0219] Example 10. Correlation between LY6K gene expression level of mesenchymal stem cells identified in mouse muscle tissue and mouse musculoskeletal treatment efficacy.

[0220] 10-1. Confirmation of anti-apoptotic efficacy according to LY6K gene expression level of mesenchymal stem cells through confirmation of expression level of cell death markers.

[0221] To confirm the effect of inhibiting apoptosis according to the expression level of the LY6K gene, the degree of apoptosis in muscle cells was confirmed through immunohistochemistry (IHC) and Western blotting.

[0222] To determine the degree of apoptosis using immunohistochemistry, mesenchymal stem cells were administered via the tail vein of a muscular dystrophy mouse model (MDX). After one week, the mice were sacrificed, and the calf muscles were isolated to obtain tissues. The obtained mouse calf muscle tissues were fixed in 4% PFA and embedded in paraffin to create blocks. They were then serially sectioned to create slides. After removing the paraffin, the slides were tunnel-stained using the ApopTag Peroxidase In Situ Apoptosis Detection Kit (EMD Milipore). Subsequently, hematoxylin & eosin (H&E) staining was performed using Mayer's Hematoxylin (Dako Omnis), and images obtained using an Aperio Slide Scanner 2 (Leica) were quantified using ImageJ.

[0223] Figure 9a shows the results of examining the degree of H&E and TUNEL staining in mouse calf muscle tissue according to the level of LY6K gene expression of mesenchymal stem cells administered to a muscular dystrophy mouse model.

[0224] As a result, as shown in Fig. 9a, the proportion of TUNEL-positive cells significantly increased in the muscular dystrophy mouse model compared to normal mice. In the H&E results, tissues where apoptosis occurred along with inflammation were marked with *.

[0225] We confirmed that the proportion of TUNEL-positive cells (←) was significantly reduced when mesenchymal stem cells with high LY6K expression were administered compared to when mesenchymal stem cells with low LY6K expression were administered. Since TUNEL binds to and stains DNA ends generated by apoptosis, we confirmed that apoptosis was reduced when mesenchymal stem cells with high LY6K expression were administered, as evidenced by the decrease in the proportion of TUNEL-positive cells.

[0226] To determine the degree of cell death using Western blotting, mouse calf muscle tissue was obtained after administering mesenchymal stem cells in the same manner as in Example 10-1. The obtained mouse calf muscle tissue was rapidly frozen and then pulverized. The pulverized tissue was lysed in RIPA buffer (BIOSESANG) containing urea, protease inhibitor cocktail (Amresco), and EDTA, and centrifuged at 8°C and 14,000 rpm for 30 minutes to obtain proteins. Western blotting was performed in the same manner as in Example 3-2, and AnnexinⅤ (Abcam) and Beta-actin (Santa Cruz Biotechnology) were used as primary antibodies.

[0227] Figure 9b shows the results of confirming the expression level of Annexin V protein, a cell death marker, in mouse calf muscle tissue according to the expression level of LY6K gene in mesenchymal stem cells administered to a muscular dystrophy mouse model.

[0228] As a result, as shown in Fig. 9b, when mesenchymal stem cells with high LY6K gene expression were administered to the muscular dystrophy mouse model (MDX), the expression level of AnnexinⅤ tended to decrease compared to the MDX control group. In addition, the group administered mesenchymal stem cells with high LY6K gene expression showed a significant decrease in the expression level of AnnexinⅤ compared to the group administered mesenchymal stem cells with low LY6K gene expression. Therefore, it was confirmed that the expression level of LY6K is related to the anti-apoptotic efficacy in the muscular dystrophy mouse model.

[0229]

[0230] 10-2. Confirmation of muscle regeneration efficacy according to the expression level of the LY6K gene in mesenchymal stem cells by confirming the expression level of muscle regeneration markers.

[0231] To confirm the muscle regeneration effect according to the expression level of the LY6K gene, the degree of muscle cell regeneration was confirmed through immunohistochemistry (IHC) and Western blotting.

[0232] Calf muscle tissue slides obtained in the same manner as in Example 10-1 were stained with Myosin Heavy Chain (MHC), a muscle marker. After removing paraffin from the slides, antigens were resurrected using EnVision Target Retrieval Solution (Dako Omnis). After reacting with the primary antibody MHC (R&D Systems) for 24 hours, the slides were treated with EnVision System-HRP Labeled polymer (Dako Omnis). After treatment with the Liquid DAB+ Substrate chromegen system (Dako Omnis), images were obtained using an Aperio Slide Scanner 2 (Leica), and quantified using ImageJ.

[0233] Figure 9c shows the results of confirming the regenerative effect of muscle tissue according to the difference in the expression level of the LY6K gene of mesenchymal stem cells administered to a muscular dystrophy mouse model through MHC staining, a muscle marker, within the tissue.

[0234] As a result, as shown in Fig. 9c, when mesenchymal stem cells with high LY6K gene expression were administered to a muscular dystrophy mouse model, it was confirmed that the degree of MHC staining significantly increased compared to the MDX control group or the group administered mesenchymal stem cells with low LY6K gene expression.

[0235] To confirm the degree of cell death using Western blotting, Western blotting was performed using the same method as in Example 10-1, and Myosin Heavy Chain (R&D Systems) and Beta-actin (Santa Cruz Biotechnology) were used as primary antibodies.

[0236] Figure 9d shows the results of confirming the expression level of MHC protein in mouse calf muscle tissue according to the expression level of LY6K gene in mesenchymal stem cells administered to a muscular dystrophy mouse model.

[0237] As a result, as shown in Fig. 9d, when mesenchymal stem cells with high LY6K gene expression were administered to a muscular dystrophy mouse model, the expression level of MHC tended to increase compared to the MDX control group. In addition, the group administered mesenchymal stem cells with high LY6K gene expression showed a significant increase in the expression level of MHC compared to the group administered mesenchymal stem cells with low LY6K gene expression. Therefore, it was confirmed that the expression level of LY6K is related to muscle regeneration in a muscular dystrophy mouse model.

[0238]

[0239] 10-3. Confirmation of muscle fibrosis reduction efficacy according to LY6K gene expression level of mesenchymal stem cells through confirmation of fibrosis marker expression level.

[0240] To confirm the muscle fibrosis reduction effect according to the expression level of the LY6K gene, the fibrosis reduction effect in muscle tissue was confirmed through immunohistochemistry (IHC) and Western blotting.

[0241] After removing paraffin from calf muscle tissue slides obtained in the same manner as in Example 10-1, Sirius Red staining was performed using Picro-Sirius Red Solution (abcam). Images obtained using Aperio Slide Scanner 2 (Leica) were quantified using ImageJ.

[0242] Figure 9e shows the results of confirming the efficacy of reducing fibrosis in muscle tissue according to the difference in the amount of LY6K gene expression of mesenchymal stem cells administered to a muscular dystrophy mouse model using Sirius Red staining.

[0243] As a result, as shown in Fig. 9e, when mesenchymal stem cells were administered to a muscular dystrophy mouse model, it was confirmed that collagen accumulation due to fibrosis was significantly reduced compared to the MDX control group. In addition, the group administered mesenchymal stem cells with high LY6K gene expression showed a significant decrease in collagen accumulation compared to the group administered mesenchymal stem cells with low LY6K gene expression.

[0244] To confirm the degree of fibrosis using Western blotting, Western blotting was performed using the same method as in Example 10-1, and Fibronectin (Abcam) and Beta-actin (Santa Cruz Biotechnology) were used as primary antibodies.

[0245] Figure 9f shows the results of confirming the expression level of fibronectin protein in mouse calf muscle tissue according to the expression level of LY6K gene in mesenchymal stem cells administered to a muscular dystrophy mouse model.

[0246] As a result, as shown in Fig. 9f, when mesenchymal stem cells with high LY6K gene expression were administered to a muscular dystrophy mouse model, the expression level of Fibronectin was significantly reduced compared to the MDX control group. In addition, the group administered mesenchymal stem cells with high LY6K gene expression showed a significant decrease in the expression level of Fibronectin compared to the group administered mesenchymal stem cells with low LY6K gene expression.

[0247] Therefore, we confirmed that LY6K expression level was associated with a decrease in muscle fibrosis in a mouse model of muscular dystrophy.

[0248]

[0249] By selecting high-efficacy stem cells according to the present invention, high-efficacy stem cells with excellent cell proliferation ability, cell migration ability, and apoptosis inhibition ability can be selected, and the problem of differences in stem cell efficacy due to differences between stem cell donors can be eliminated, thereby selecting stem cells with excellent therapeutic efficacy, thereby lowering the production cost when mass-producing stem cell therapeutic agents.

[0250]

[0251] While specific aspects of the present invention have been described in detail above, it will be apparent to those skilled in the art that these specific descriptions merely represent preferred embodiments and are not intended to limit the scope of the present invention. Therefore, the substantial scope of the present invention is defined by the appended claims and their equivalents.

[0252]

[0253] Electronic file attached.

Claims

1. A step of confirming the expression of the LY6K gene in stem cells or measuring the content of the protein encoded by the LY6K gene; and A method for selecting high-potency stem cells, comprising: a step of selecting high-potency stem cells when the expression of the LY6K gene or the content of the protein encoded by the LY6K gene is higher than that of other stem cells.

2. A step of confirming the expression of the LY6K gene in cultured stem cells or measuring the content of the protein encoded by the LY6K gene; and A method for quality control of a stem cell therapeutic agent, comprising a step of selecting the stem cell as a therapeutic agent when the expression of the LY6K gene or the content of the protein encoded by the LY6K gene is higher than that of other stem cells.

3. A method for producing high-efficiency stem cells, comprising a step of treating stem cells with a protein encoded by the LY6K gene.

4. A composition for producing high-efficiency stem cells containing a protein encoded by the LY6K gene as an active ingredient.

5. A composition for high-efficiency stem cell selection comprising a preparation capable of measuring the expression level of mRNA of the LY6K gene or the level of protein encoded by LY6K.

6. A composition for selecting high-efficiency stem cells, characterized in that in paragraph 5, the agent capable of measuring the expression level of the mRNA is selected from the group consisting of a primer that specifically binds to and amplifies the mRNA, a probe that specifically binds to the mRNA, and an antisense nucleotide.

7. A composition for selecting high-efficiency stem cells, characterized in that in paragraph 5, the agent capable of measuring the protein level is selected from the group consisting of antibodies, oligopeptides, ligands, PNA (peptide nucleic acid), and aptamers that specifically bind to the protein.

8. A high-efficiency stem cell selection kit comprising a composition according to any one of claims 5 to 7.

9. A high-efficiency stem cell selection kit according to claim 8, characterized in that the kit is an RT-PCR kit, a DNA chip kit, an ELISA kit, a protein chip kit, a rapid kit, or an MRM (Multiple reaction monitoring) kit.

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