KR101170366B1 - Biomarker for the predicting stem cell differentiate to osteoblast - Google Patents

Abstract

The present invention provides a composition for predicting osteogenic potential of stem cells comprising a protein secreted during the differentiation of stem cells into osteoblasts, an agent for measuring the expression level of the protein at the mRNA or protein level, and the composition of the protein. It relates to a method for predicting the osteogenic capacity of stem cells by measuring the amount of expression.

In addition, the present invention is a composition for inducing differentiation of stem cells into osteoblasts (SMOC1) containing SMOC1 (SPARC-related Modular Calcium-binding Protein 1) and by controlling the expression of SMOC1 in stem cells to regulate differentiation into osteoblasts It is about how to.

Stem Cells, Osteoblasts, SMOC1

Description

Biomarker for the predicting stem cell differentiated to osteoblast}

The present invention measures a composition for predicting bone formation ability of stem cells, including a protein secreted during the process of differentiating stem cells into osteoblasts, an agent for measuring the expression level of the protein at the mRNA or protein level and the expression amount of the protein It relates to a method for predicting the bone formation ability of stem cells. The present invention also relates to a composition for inducing differentiation of stem cells comprising SMOC1 into osteoblasts and a method for controlling differentiation into osteoblasts by controlling the expression of SMOC1 in stem cells.

Mesenchymal stem cells (MSCs), also known as pluripotent stromal cells, have spindle-like and fibroblast-like phenotypes and are present in a variety of tissues including bone marrow, muscle, dermis and fat. MSCs can be differentiated into various lineages, including osteoblasts, adipocytes, chondrocytes, and myoblast lineages under appropriate culture conditions. Mechanisms for lineage specific differentiation have been extensively studied. In particular, osteoblast differentiation is regulated by a variety of growth factors, including hedgehog proteins, bone morphogenic proteins (BMP), transforming growth factor-β (TGF-β), parathyroid hormone (PTH) and WNT proteins. The growth factors transduce their signals through a set of transcription factors that regulate the expression of genes that play an important role in osteoblast differentiation and bone matrix formation. In particular, run-related transcription factor 2 (RUNX2) is a transcription factor essential for osteoblast differentiation. Damage to RUX2 causes a rare skeletal disease called cleidocranial dysplasia in humans and complete bone loss in mice. RUNX2 regulates the expression of zinc pinker-containing transcription factor OSX (Osterix), another major factor involved in bone development. Transcription factors including nuclear factor for activated T cells 2 (NFAT2), homeobox proteins such as Msx2, Dlx-3, Dlx-5, and Dlx-6, and activator protein 1 proteins such as Fos, Fra, and ATF4 Play an important role in bone development by interacting directly or indirectly with RUNX2 or OSX. The transcription factor induces the expression of an array of genes encoding components of the bone matrix, including mineralization related proteins such as ECM proteins and alkaline phosphatase (ALP). RUNX2 and OSX regulate the expression of osteoblast-specific genes including COL1 (collagen type 1), ALP, OPN (osteopontin), SPARC (osteonectin, or ON) and osteocalcin (OC). Thus, expression of the target gene can be used as a marker for osteoblast differentiation.

Osteoblast differentiation may be regulated by ECM proteins in addition to growth factor signaling through transcription factors. The most abundant ECM protein in bone is COL1. In MC-4 preosteoblasts growing on COL1 fibrous substrates, RUNX2 is stabilized through acetylation and then induces osteoblast differentiation. Serine phosphorylation residues in the activation domain of RUNX2 are targeted through mitogen activated protein kinase kinase / extracellular signal-regulated kinse (MEK / ERK) signaling pathways by integrin binding. Another ECM protein that can regulate osteoblast function is SPARC. ON deficiency causes osteopenia due to the reduction of osteoblasts and osteoclasts. This means that SPARC regulates the recruitment or proliferation of osteoblast precursors. ECM proteins may also indirectly regulate osteoblast differentiation by interacting with growth factors. ON, OPN, and TSP1 are platelet-derived growth factor (PDGF), TGFβ1, vascular endothelial growth factor (VEGF), fibroblast growth factor-2 (FGF-2), insulin-like growth factor (IGF), and epihelial growth factor (EGF). Can interact with a variety of growth factors that regulate osteoblast differentiation, such as parathyroid hormone (PTH) and IGF-binding protein (IGF-BP). Thus, ECM proteins not only contain organic bone matrices but can also directly or indirectly regulate osteoblast differentiation and proliferation.

The inventors of the present invention have tried to discover factors involved in the differentiation of stem cells into osteoblasts, and identify proteins secreted during the process of differentiating stem cells with osteoblasts into different osteoblasts. High stem cells express SMOC1 highly among the above proteins, and stem cells overexpressing SMOC1 promoted differentiation into osteoblasts, thus completing the present invention.

One object of the present invention is to provide a protein that secretes stem cells in the process of differentiation into osteoblasts.

Another object of the present invention is to provide a composition for predicting osteogenic potential of stem cells comprising an agent for measuring the expression level of the protein at the mRNA or protein level.

Another object of the present invention to provide a method for predicting the osteogenic capacity of stem cells by measuring the amount of expression of the protein.

Still another object of the present invention is to provide a composition for inducing differentiation of stem cells into osteoblasts, including SMOC1 (SPARC-related Modular Calcium-binding Protein 1).

Still another object of the present invention is to provide a method of controlling differentiation into osteoblasts by controlling the expression of SMOC1 in stem cells.

In the present invention, the term 'osteoblastic ability of stem cells' means the extent to which stem cells are differentiated into osteoblasts.

In the present invention, the term 'stem cells' can be isolated and purified from bone marrow (bone-marrow, BM), peripheral nerve blood, umbilical cord blood, periosteum, dermis (dermis) and mesodermal origin. Stem cells in the present invention is preferably isolated from the bone marrow.

Bone marrow derived stem cells are separated by a number of different mechanical separation methods, depending on the source of bone marrow, and such separation methods are known in the art. The most critical step in the isolation of stem cells is to use a specially prepared medium. The medium is characterized in that it contains an agent capable of growing stem cells without differentiation and directly attaching only stem cells to the plastic or glass surface of the culture dish. For example, by using a medium that enables selective attachment of stem cells present in the bone marrow in small amounts, it is possible to separate stem cells from other cells (ie, red blood cells and white blood cells, etc.) present in the bone marrow.

The present invention relates to a protein secreted in the process of differentiating stem cells into osteoblasts, preferably in the early stage of differentiation, more preferably 16 hours after the onset of differentiation. The protein is as shown in Table 1.

Among the proteins shown in Table 1 above, proteins that specifically increase and secrete in the process of differentiating stem cells with high osteogenic capacity into osteoblasts are shown in Table 2.

The protein shown in Table 1 is a protein secreted during the process of differentiation of stem cells into osteoblasts, the protein shown in Table 2 is specifically increased during the process of differentiation of stem cells with high osteogenic capacity into osteoblasts to secrete Since the protein, it is possible to predict the bone formation ability of stem cells by measuring the expression level of at least one of these proteins.

In one aspect, the present invention provides for the expression of at least one or more proteins of Table 1, preferably at least one or more proteins of Table 2, more preferably SMOC1 (SPARC-related Modular Calcium-binding Protein 1). The present invention relates to a composition for predicting osteogenic potential of stem cells, including an agent for measuring an amount at an mRNA or protein level.

In one aspect, the invention comprises an agent for measuring at the mRNA level the expression level of at least one or more of the proteins shown in Table 1, preferably at least one or more of the proteins shown in Table 2, more preferably SMOC1. It relates to a composition for predicting bone formation ability of stem cells. The agent for measuring the expression level of the protein at the mRNA level is preferably a primer pair or probe specific for the gene of the protein.

Specifically, the primer is a nucleic acid sequence having a short free 3 'hydroxyl group, which forms a complementary template and base pair and serves as a starting point for template strand copying. Means.

The primers of the present invention may, if necessary, comprise a label that can be detected directly or indirectly by spectroscopic, photochemical, biochemical, immunochemical or chemical means. Examples of the label include enzymes such as horseradish peroxidase, alkaline phosphatase, and the like, radioisotopes such as 33P, chemical groups such as biotin, and fluorescent molecules.

Probe means a nucleic acid fragment such as RNA or DNA that can achieve specific binding with mRNA and can be labeled to confirm the presence or absence of a specific mRNA. The probe may be prepared in the form of an oligonucleotide probe, a single stranded DNA probe, a double stranded DNA probe, or an RNA probe.

A person skilled in the art can probe from a nucleic acid sequence of a known gene to specifically recognize a primer pair or a specific region of the gene in consideration of variables such as the degree of hybridization between sequences within a target gene. You can design

In another aspect, the invention comprises an agent for measuring at the protein level the expression level of at least one or more of the proteins shown in Table 1, preferably at least one or more of the proteins shown in Table 2, more preferably SMOC1. It relates to a composition for predicting bone formation ability of stem cells. The agent to be measured at the protein level is preferably an antibody specific for the protein and includes all polyclonal antibodies, monoclonal antibodies and recombinant antibodies. In addition, the antibodies of the present invention are functional fragments of the antibody molecule in full form with two full length light chains and two full length heavy chains, eg, Fab, F (ab '), F (ab') 2 And Fv.

The composition for predicting bone formation ability of stem cells according to the present invention may be applied to and prepared as a kit for predicting differentiation ability of stem cells into osteoblasts, further including other components or devices widely used in the art.

Specific examples include primer pairs specific for the gene and, in addition, test tubes or other suitable containers, reaction buffers, enzymes such as deoxynucleotides (dNTPs), Taq-polymerases and reverse transcriptases, DNAse inhibitors, RNAse inhibitors, It can be prepared as a kit for RT-PCR, including DEPC-water, sterile water and the like. As another example, the kit may be manufactured as a kit for a DNA chip containing essential elements necessary for carrying out the DNA chip.

As another example, the antibody comprises a specific antibody to a protein of the gene and additionally a reagent capable of detecting the bound antibody, such as labeled secondary antibodies, chromophores, enzymes and substrates thereof. It can be produced as a kit for ELISA. As another example, the kit may be manufactured as a kit for a protein chip containing essential elements necessary for carrying out the protein chip.

In another aspect, the present invention predicts the bone formation ability of stem cells by measuring the expression amount of at least one or more proteins of the protein shown in Table 1, preferably at least one or more proteins of the protein shown in Table 2, more preferably SMOC1 It is about how to. Specifically, the expression level of the protein can be detected at the mRNA level or the protein level.

In a specific embodiment, the present invention uses primer pairs or probes specific for at least one or more proteins of the proteins shown in Table 1, preferably at least one or more proteins of the proteins shown in Table 2, more preferably, SMOC1, It relates to a method for predicting the bone formation ability of stem cells, comprising the step of measuring the expression level of the protein at the mRNA level from a biological sample to be predicted osteogenic capacity.

Specific analysis methods for measuring the expression level at the mRNA level include RT-PCR, competitive RT-PCR, real-time RT-PCR, RNase protection assay, Northern blotting, DNA chip, etc., but is not limited thereto.

In another specific embodiment, the present invention uses an antibody specific for at least one or more proteins of Table 1, preferably at least one or more proteins of Table 2, more preferably SMOC1, The present invention relates to a method for predicting bone formation ability of stem cells, including measuring the expression level of the protein at the protein level by contacting a biological sample to be formed to form an antigen-antibody complex.

Specific analytical methods for measuring the expression level at the protein level include Western blot, ELISA, radioimmunoassay, radioimmunoproliferation, oukteroni immunodiffusion, rocket immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation Assays, FACS, protein chips, and the like, but are not limited to such. ELISA is a direct sandwich ELISA using another labeled antibody that recognizes the antigen in a complex of antibody and antigen attached to a solid support, followed by reaction with another antibody that recognizes the antigen in a complex of antibody and antigen attached to a solid support. Various ELISA methods, such as indirect sandwich ELISA using the labeled secondary antibody which recognizes this antibody, are included.

In the present invention, the "biological sample to predict bone formation ability" may be a culture obtained by culturing stem cells in bone formation medium for a predetermined time. The osteogenic medium may be a osteogenic medium known in the art, and in one embodiment, an osteogenic medium including dexamethasone, ascorbic acid, and glycerophosphate may be used (see Example 1-1). ). Incubation time is 12-16 hours.

In addition, the method may include comparing the expression level of the gene in the biological sample by using the expression level of the gene as a control in the stem cells verified to have high or low bone formation ability. This can be compared with absolute or relative differences in the amount of expression between the control and the biological sample.

Through the above-described detection methods, the expression level of at least one or more proteins of Table 1, preferably at least one or more proteins of Table 2, more preferably SMOC1, is quantitatively analyzed at the mRNA or protein level. Through this, it is possible to predict the differentiation capacity of stem cells into osteoblasts.

As another aspect, the present invention relates to a composition for inducing differentiation of stem cells into osteoblasts containing a SMOC1 protein or a gene carrier comprising an SMOC1 gene.

In the present invention, the gene carrier comprising the SMOC1 gene can be prepared using recombinant DNA methods known in the art.

First, SMOC1 gene can be prepared using techniques known in the art. For example, the primer can be prepared by referring to a known SMOC1 gene sequence and then subjected to PCR using genomic DNA as a template. Moreover, it can also synthesize | combine using a chemical synthesis method or DNA automatic synthesis apparatus with reference to a well-known SMOC1 gene sequence.

In the present invention, a gene carrier useful for introducing SMOC1 into cells is a virus (or viral vector). The virus includes adenovirus, adeno-associated virus, retrovirus, lentivirus, herpes simplex virus, alpha virus, etc. It is not limited to this.

Another gene carrier useful for introducing SMOC1 into cells is a nonviral vector. Non-viral vectors refer to all vectors commonly used for intracellular delivery of genes except for the viral vectors described above, and examples include various plasmids and liposomes that can be expressed in eukaryotic cells.

On the other hand, the gene carrier comprising the SMOC1 of the present invention will need to be operably linked to the promoter in order for the SMOC1 to be transcribed in the cell. The promoter includes a binding site for the RNA polymerase and may be located upstream of the coding region. The promoter may be any promoter capable of initiating transcription in eukaryotic cells, but preferred promoters are CMV (Cytomegalovirus), SV40 (Simian virus 40), TK (Thymidine kinase), RSV (Respiratory syncytial virus), CMV5, and the like. Is a promoter derived from.

In addition, the gene carrier comprising the SMOC1 of the present invention may include the origin of replication of eukaryotic cells, initiation codon, termination codon, polyadenylation signal, Kozak (Kozak) for efficient transcription or translation of nucleic acid molecules encoding SMOC1 ), An enhancer, a signal sequence for membrane targeting and secretion, an internal ribosome entry site (IRES), and the like.

As used herein, the term "operably linked" means that the binding between nucleic acid sequences is functionally related. The case where any nucleic acid sequence is operably linked is when any nucleic acid sequence is positioned to be functionally related to another nucleic acid sequence. In the present invention, when any transcriptional regulatory sequence affects the transcription of a nucleic acid molecule encoding SMOC1, the transcriptional regulatory sequence is said to be operably linked to the nucleic acid molecule.

In another aspect, the present invention relates to a method for controlling differentiation into osteoblasts by controlling the expression of SMOC1 in stem cells.

In one aspect, the present invention relates to a method of treating SMOC1 protein on stem cells to differentiate them into osteoblasts. That is, by culturing stem cells in a medium containing the SMOC1 protein can be differentiated into osteoblasts. The medium is preferably a bone formation medium. SMOC1 is preferably treated at an early stage in the process of differentiating stem cells into osteoblasts, that is, within 1 to 15 days after initiation of differentiation induction or induction of differentiation.

In another aspect, the present invention relates to a method of introducing a gene carrier comprising an SMOC1 gene into stem cells to differentiate into osteoblasts. In other words, by introducing a gene carrier containing the SMOC1 gene into stem cells, it can be differentiated into osteoblasts by culturing in bone formation medium.

The method of introducing the gene carrier comprising SMOC1 of the present invention into stem cells can be carried out based on known techniques, for example, calcium phosphate transfection, DEAE-dextran mediated transfection (DEAE-dextran). Mediated transfection, microinjection, electroporation, cationic lipid-mediated transfection, ballistic introduction or infection and the like can be used.

According to the present invention, the protein composition secreted by stem cells showing high bone formation ability can be identified by comparing and analyzing proteins secreted by high and low bone formation ability by stem cell analysis.

The present invention revealed that stem cells with high bone formation ability express high SMOC1. Thus, osteogenic capacity can be predicted by measuring the expression level of SMOC1.

In addition, the present invention revealed that stem cells overexpressing SMOC1 promotes differentiation into osteoblasts. Therefore, differentiation into osteoblasts can be promoted by processing SMOC1 protein or introducing SMOC1 gene into stem cells.

Hereinafter, the examples are only for illustrating the present invention in more detail, and the scope of the present invention is not limited by these examples in accordance with the gist of the present invention, those skilled in the art. Will be self-evident.

Example 1 Materials and Methods

1-1. Cell culture

Human bone marrow was obtained from patients undergoing total hip replacement surgery. Prior consent was obtained from the patient prior to surgery. BMSCs were isolated according to known methods (Park EK et al. 2004, Biomaterials 25 (17) : 3403-11). Bone marrow samples were diluted with phosphate buffered saline (PBS) at pH 7.4 and gradient centrifugation was performed using histopaque (Sigma, St Louis, MO). After centrifugation, MNCs (mononuclear cells) were collected and washed twice with PBS. Recovered MNCs were plated in α-MEM containing 10% fetal bovine serum (FBS), 100 U / ml penicillin and 100 μg / ml streptomycin (all purchased from Gibco BRL, Gaithersburg, MD), 5% Incubated at 37 ° C. for 12 h in humid air of CO ₂ . Non-adherent cells were removed and adherent cells were recovered as BMSCs fractions and expanded for subsequent analysis. Growth medium was exchanged every other day and cells were used up to 5 passages in all examples.

To induce differentiation into osteoblasts, BMSCs were seeded in standard medium at a density of 1.5 × 10 ⁵ cells per 35 mm dish (70-80% confluence). After stabilizing overnight, the culture medium was exchanged with osteogenic medium comprising 10 nM dexamethasone (Sigma, St. Louis, MO), 50 μg / ml ascorbic acid-2-phosphate and 10 mM β-glycerophosphate (Sigma). . Mineralization was measured by staining with alizarin red S, which stains the inorganic substrate.

1-2. Surface marker analysis by flow cytometry

BMSCs were removed with a solution of 0.05% trypsin / 1 mM EDTA and washed with PBS containing 1% BSA (bovine serum albumin). BMSCs (1 × 10 ⁵ cells per tube) were fluorescently conjugated mouse anti-human CD14, CD29, CD34, CD44, CD90, CD106, HLA-ABC (BD Biosciences, San Jose, Calif.), CD105 (R & D Systems) and CD146 (Chemicon, Temecula, CA) antibody was incubated for 1 hour at 4 ℃. Cells were washed twice with cold PBS containing 1% BSA. Anti-CD14, 29, 44, 105 and HLA-ABC antibodies were conjugated with PE (phycoerythrin) and anti-CD34, 90, 106 and 146 were conjugated with fluorescein isothiocyanate (FITC). After antibody binding, cells were analyzed using a FACSAria Sorting Flow Cytometer (Beckton Dickinson).

1-3. Proliferation measurement

BMSCs were plated on 96 spheres at a density of 700 cells / sphere and incubated overnight. The cells were incubated in standard medium for 0, 1, 3, 5 and 7 days. By the indicated time, MTT (3- (4,5-Dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide, a tetrazole) reagent was added to a final concentration of 50 µg / ml and left to incubate at 37 ° C. for 3 hours. . Intracellular formazan was measured after cell lysis. Absorbance at 570 nm was measured using an ELISA plate reader (SHIMADZU, Kyoto, Japan).

1-4. ALP activity measurement

ALP activity was measured according to the manufacturer's instructions using ALP yellow liquid substrate (pNPP) (Sigma). BMSCs were plated on 85-spheres (Corning, Lowell, Mass.) With 85-90% confluence and incubated for 7 days in α-MEM medium containing 10% FBS with or without osteogenic medium. Cells were washed twice with PBS and lysed with 0.1% SDS. The lysates were left incubated at 37 ° C. for 30 minutes with pNPP, after which the reaction was stopped by addition of 2N NaOH. Absorbance was measured at 405 nm.

1-5. Preparation and Trypsin Digestion of Secretory Proteins

BMSCs were inoculated in 100 mm culture dishes with 85-90% confluence and allowed to stabilize overnight. The cells were washed twice with serum-free α-MEM medium and incubated for 16 hours in 7 ml serum-free α-MEM medium with or without osteogenic medium. The conditioned media (CM) was recovered and centrifuged to remove cell debris. Proteins in CM were precipitated for 60 minutes with 10% v / v trichloroacetic acid (Sigma) on ice. The precipitate was recovered by centrifugation at 4 ° C. for 15 minutes at 14,000 rpm and then washed twice with ice-cold acetone. Protein pellets were dried in air and protein concentrations were measured according to BCA protein assay (Thermo Scientific, Rockford, IL). 20 μg of protein was isolated by 10% polyacrylamide gel electrophoresis (SDS-PAGE) and visualized by colloidal staining using 0.25% Pierce Coommassie Brilliant Blue G-250 dissolved in distilled water. The gel was sliced into 15 pieces and each piece was bleached by standing in a solution of 75 mM ammonium bicarbonate / 40% ethanol (1: 1 v / v). After bleaching, the gels were left incubated for 30 minutes at room temperature with a solution of 55 mM iodoacetoamide to alkylate the protein and then the gel pieces were dehydrated in 100% ACN (acetonitrile) and dried. Gel pieces are 20 μg / ml of modified sequencing grade trypsin, Roche Applied Science) was expanded in 10 µl of 25 mM ammonium bicarbonate buffer solution and then incubated overnight at 37 ℃. Trypsinized peptide mixture was eluted from the gel with 0.1% formic acid.

1-6. LC-ESI-MS / MS Analysis

LC-MS / MS analysis was performed as known (Heo SH et al. 2007, Proteomics 7 (23): 4292-302; Ha BG et al. 2008, Proteomics 8 (13): 2625-39). LC-MS / MS analysis using Finnigan's ProteomeX workstation LTQ linear ion trap MS, Thermo Electron, San Jose, CA with NSI source (San Jose, CA) Was performed. 12 μl of peptide mixture was injected and loaded into peptide trap cartridges (Agilent, Palo Alto, Calif.). The peptide was eluted on a 10-cm reversed phase PicoFrit column filled with 5 μm, 300 mm diameter C18 silica gel in a house and then separated by gradient elution on a RP column. As the mobile phase, H ₂ O (A) and ACN (B) were used, respectively, and both solutions contained 0.1% (v / v) formic acid. The flow rate was maintained at 200 nL / min. Gradients started at 2% B, pulled up to 60% B in 50 minutes, 80% B in the next 5 minutes, and 100% A in the last 15 minutes. A data-dependent acquisition mode (m / z 300-1800) was possible and five MS / MS scans were performed with a 30 second dynamic exclusion option operating after each survey MS scan. The spray voltage was 1.9 kV and the temperature of the ion transfer tube was set to 195 ° C. Standardized collision energy was set at 35%.

1-7. Data Analysis

Data analysis was performed as known (Park ES et al. 2009 Proteomics Analysis of Human Dentin Reveals Distinct Protein Expression Profiles. J Proteome Res. 2009 Mar; 8 (3): 1338-46). In the database investigation, serial mass spectra were extracted by SORCERER version 3.4 beta 2. Charge state deconvolution and deisotoping did not proceed. All MS / MS samples were tested with SEQUEST (ThermoFinnigan, San Jose, CA; version v.27, rev. 11), which was set up to examine the ipiHUMAN3.49 database (unknown version, 74017 entries) to estimate non-specific digestive enzymes. The analysis was carried out. SEQUEST was investigated to allow a fragment ion mass error of 1.0 Da and a parent ion error of 1.5 Da. To improve false-positive statistics, a decoy option was selected during the data lookup process in the SORCERER program, which can reduce noise effects and improve the quality of the results. The oxidation of methionine in SEQUEST has been characterized by various formulas.

For protein identification, MS / MS based peptide and protein identification was verified using Scaffold (version Scaffold-01_07_00, Proteome Software Inc., Portland, OR). A probability of at least 95.0% is established, as specified in the Peptide Prophet algorithm (Keller A, et al. 2002 Anal Chem 74 (20) : 5383-92; Nesvizhskii AI et al. 2003 Anal Chem 75 (17) : 4646-58). And peptide identification was allowed when at least two peptides were included. Proteins containing similar peptides and indistinguishable from MS / MS analysis alone were grouped to meet the principles of parsimony.

Gene ontology analysis was performed using DAVID (database for annotation, visualization and integrated discovery, http://david.abcc.ncifcrf.gov/home.jsp). Proteins were grouped by DAVID into genetic symbols according to biological processes.

1-8. Electroporation

BMSCs were electroporated with either SMOC1 shRNA or wild type SMOC1 cDNA using Microporator ^™ (NanoEnTek Inc., Seoul, Korea). Cells (2 × 10 ⁵ ) were transfected with 1 μg shRNA or cDNA using 1000 pulse voltage, 40 pulse width and 1 pulse. SMOC1 shRNA was obtained from SABioscience (Frederick, MD). The sequence of each shRNA is as follows: shRNA-1 (Sh-1), 5'-GCCTCAGGAAGCTGTGTTTGT-3 '(nucleotide no. 324-345) (SEQ ID NO: 1); siRNA-2 (Sh-2), 5'-GGTGATCAAGGACTCCAAACT-3 '(nucleotide no. 618-639) (SEQ ID NO: 2); Negative control shRNA (N / C), 5'-GGAATCTCATTCGATGCATAC-3 '(SEQ ID NO: 3). SMOC1 cDNA was obtained from Thermo Scientific (Huntsville, AL).

1-9. Semi-quantitative RT-PCR

BMSCs were incubated for a designated time with or without osteogenic medium. Total RNA was extracted using Tri reagent (Molecular Research Center Inc., Cincinnati, OH) and cDNA was synthesized from 1 μg of RNA using Superscript II (Invitrogen). In performing semi-quantitative PCR, the following heat cycle parameters were used: after 2 minutes at 95 ° C., 60 seconds at 95 ° C., 60 seconds at the corresponding annealing temperature, 90 seconds at 72 ° C. and then 10 minutes at 72 ° C. During the final extension. Human GAPDH (glyceraldehyde-3-phosphate dehydrogenase) was used as an internal control. All PCR products were electrophoresed on 1% agarose gels and visualized by ethidium bromide staining. Gels were photographed using the ChemiDoc ^™ XRS system (Bio-Rad, Hercules, CA). Specific primers used for PCR are shown in Table 3.

1-10. Western blot analysis

In Western blot analysis, concentrated CM or cell lysates were separated by 7.5 or 10% SDS-PAGE. BMSCs were prepared in RIPA buffer (50 mM Tris-HCl, pH 8.0, 5 mM EDTA, 10% glycerol, 150 mM NaCl, 1% Triton). X-100, 1 mM EGTA, 10 mM NaF and protease inhibitor mixture) were lysed and centrifuged. The gel was transferred to a nitrocellulose membrane in 25 mM Tris-HCl buffer (pH 7.4) containing 20% methanol, 192 mM glycine and 0.01% SDS. The membrane was incubated in 5% fat-free dry milk dissolved in TBS-T (25 mM Tris-HCl, pH 7.4, 150 mM NaCl and 0.2% Tween 20) to block nonspecific binding and anti-CHI3L1 (Novus Biologicals). , Littleton, CO), -CTGF, -IGFPB5 (Santa Cruz Biotechnology, Santa Cruz, CA), -SMOC1 (Abnova, Taipei, Taiwan) or -TGFB1 (abcam, Cambridge, MA) antibody, followed by horseradish peroxy Probe was probed with a secondary antibody conjugated to a multidase. Immunoreactive proteins were visualized using the ECL system (GE Healthcare, Buckinghamshire, UK). Chemiluminescence was detected using an X-ray film.

Example 2: Results

2-1. Osteoblast Differentiation of Donor BMSCs

To investigate the osteoblast differentiation mechanism, the osteoblast differentiation capacity of BMSCs isolated from six donors was compared. When the BMSCs isolated from each donor were stimulated with osteoblastic medium, the differentiation of osteoblast differentiation was confirmed by alizarin red S staining (FIG. 1A). In particular, BMSCs isolated from providers 4 and 6 were mineralized higher than BMSCs isolated from other providers. BMSCs isolated from providers 2 and 3 were found to be less mineralized than other samples. These results indicate that BMSCs isolated from each donor have different osteoblast differentiation capacity. Since osteoblast differentiation is associated with proliferation, we investigated whether BMSCs with high osteoblast differentiation also have high proliferative capacity. As shown in FIG. 1B, all BMSCs showed a similar proliferation pattern except for provider # 2. Therefore, it can be seen that there is no correlation between osteoblast differentiation and proliferative capacity. MSCs express specific cell surface markers, and the expression of these markers is dependent on the stage of differentiation. Flow cytometry was used to investigate the expression of MSC cell surface markers. In each donor BMSCs most of the BMSCs (more than 80%) were positive for MSC surface markers such as CD29, CD44, CD90, CD105 and HLA-ABC and there was no significant difference between the six donor BMSCs (FIG. 1C). The proportion of cells that were CD146 positive varied between BMSCs, but was independent of osteoblast differentiation (FIGS. 1A and 1C). The expression of negative markers such as CD14, CD34, CD106 was very low in each BMSCs population (FIG. 1C). These results suggest that the difference in osteoblast differentiation capacity of BMSCs is not due to differences in proliferative capacity or surface marker expression.

2-2. Secretome analysis of low osteogenic potential (LOP) and high osteogenic potential (HOP) BMSCs (see FIG. 2)

During osteoblast differentiation, BMSCs constantly interact with ECM proteins secreted by BMSCs in an autocrine or paracrine manner. To investigate the effect of BMSC microenvironment on osteoblast differentiation, the secreted protein profiles of HOP and LOP BMSCs selected from six BMSC providers were examined. HOP and LOP BMSCs were selected based on the results of FIG. 1A. The secreted protein was recovered from each sample and analyzed using LC-MS / MS. To rule out false positives, the decoy option was chosen during the SORCERER program's data search process, which improves the quality of the search results by reducing noise effects. As a result, 208 secreted proteins were identified in LOP BMSCs and 202 secreted proteins were identified in HOP BMSCs. Of these proteins, 138 were common in LOP and HOP BMSCs, while 70 and 64 proteins were selectively secreted in LOP and HOP BMSCs, respectively (FIG. 3A). The secreted protein profile of HOP BMSCs was focused on 64 proteins selectively secreted by HOP BMSCs, assuming that it would affect osteoblast differentiation more than that of LOP BMSCs (Table 2). The full list of secreted proteins is shown in Table 1.

Using DAVID (http://david.abcc.ncifcrf.gov/home.jsp), the ontology of 64 secreted proteins was analyzed according to biological processes. As shown in FIG. 3B, proteins selectively secreted by HOP BMSCs were broadly classified into cellular and extracellular components. Cell components include metabolic processes (77.27%), gene expression (44.45%), biosynthesis (38.64%), organelle organization (18.18%), cytoskeletal organization (11.64%), cell migration (11.36%) and others (6.82%) Protein involved in the Extracellular components include stimulus response (50%), development and morphogenesis (31.25%), cell adhesion (31.25%), receptor mediated signal transduction (31.25%), regulation of biological quality (31.25%) ), Proteins involved in homeostatic processes (18.75%), cell growth (18.75%), cell migration (18.75%), and bone remodeling (12.5%). The results of this example could be verified by identifying proteins already known as bone formation related proteins such as TGFB1, CTGF, POSTN, IGFBP5, VTN and STC-1 in the secreto of HOP BMSCs. However, several proteins that were not previously known to be involved in osteoblast function, including SMOC-1, EFEMP1, MFAP2, PXDN and DCD, could also be identified.

2-3. Protein expression analysis in a subset of BMSC secretes

To verify the secreto data, expression of a subset of extracellular constituent proteins in LOP and HOP BMSCs was examined by RT-PCR. LOP and HOP BMSCs were cultured in complete medium containing 10% FBS and then stimulated with osteogenic medium for 10 hours. Based on the number of peptide hits that could reflect differential protein expression, eight ECM proteins were selected for further analysis. The expression of SMOC1, CHI3L1, TGFB1, CTGF and EFEMP1 in HOP BMSCs was substantially increased by treatment with osteogenic medium (FIG. 4A). Relatively, the expression of THBS2, TIMP3 and POSTN increased to a lesser extent in HOP BMSCs. The secretion of these 8 proteins was confirmed again by Western blot analysis. As shown in FIG. 4B, the secretion amounts of the eight proteins were higher in HOP BMSCs than in LOP BMSCs, although there were some differences between the proteins regardless of the corresponding mRNA expression amount. (FIG. 4B). These results indicate that protein secretion is regulated at varying levels in BMSCs.

To investigate whether the expression of these 8 proteins is regulated during osteoblast differentiation, gene expression was analyzed after incubating HOP BMSCs with or without osteogenic medium for different periods of time (FIG. 5A). . Surprisingly, the expression of CHI3L1, the highest protein expressed in HOP BMSCs, also increased in control conditions. Similar to the previous results, expression of CTGF, TGFB1 and TSP2 was significantly increased during osteogenic differentiation (FIG. 5). The most significant change in expression during osteogenic differentiation was in SMOC1, which strongly suggests that regulation of SMOC1 expression is associated with osteoblast differentiation. To analyze the secretion of SMOC1 during osteogenic differentiation, HOP BMSCs were incubated for 1, 7 and 14 days, and cells were exposed for an additional 16 hours in serum-free medium containing osteogenic medium at each time point. The lysate was prepared and analyzed by Western blot using anti-SMOC1 antibody. As shown in FIG. 5B, SMOC1 secretion was detected on day 1 in BMSCs treated with control and osteogenic medium. However, in CM derived from BMSCs treated with osteogenic medium, the amount of SMOC1 at day 1 was higher than the control BMSC CM. This means that SMOC1 is secreted in higher amounts in response to osteogenic medium. SMOC1 secretion is somewhat lower but remained for 7 days. On day 7, SMOC1 was still detectable, but more than half of the synthesized SMOC1 was present in the pellet fraction. This suggests that the protein remains in intracellular compartments such as endoplasmic reticulum. These results suggest that SMOC1 functions in the early stages of osteoblast differentiation.

2-4. Analysis of SMOC1 Function in Osteoblast Differentiation of BMSCs

To investigate the function of SMOC1 in osteoblast differentiation of BMSCs, overexpression of shRNA and wild type SMOC1 cDNA was used to produce SMOC1 loss of function and function acquisition variants. To produce SMOC1 knockdown cells, four shRNAs were each transfected with HOP BMSCs using Microporator ^™ . Two shRNAs, namely Sh-1 and Sh-2, significantly reduced the amount of SMOC1 mRNA by 70% and 50% or more of the amount induced by the negative control (N / C) shRNA, respectively ( 6A). SMOC1 knockdown has had no significant impact on the expression of SPARC (FIG. 6A). Analysis of SMOC1 secretion revealed that knockdown of SMOC1 effectively lowered the amount of SMOC1 protein in CM. These results demonstrate that Sh-1 and Sh-2 are effective not only in secreting SMOC1 but also in reducing the amount of endogenous mRNA. To analyze the knockdown effect of SMOC1 on osteoblast differentiation of BMSCs, cells were transfected with either Sh-1 or Sh-2 and then cultured in osteogenic medium for 10 days. Sh-1 and Sh-2 substantially reduced ALP activity, an early marker of osteogenic differentiation (FIG. 6B) and inhibited mineral deposition by BMSCs (FIG. 6C). In addition, the inhibitory effect of shRNA on the expression of osteoblast differentiation markers was investigated. As shown in FIG. 6D, expression of ALP, COL1, ON, OC and BSP was inhibited by SMOC1 knockdown. These results indicated that inhibition of endogenous SMOC1 expression by shRNA inhibited osteoblast differentiation of BMSCs.

The effect of SMOC1 overexpression on osteoblast differentiation was investigated. RT-PCR and Western blot analysis revealed that SMOC1 is overexpressed in BMSCs electroporated with wild type SMOC1 cDNA. Expression and secretion of SMOC1 were significantly increased after electroporation (FIG. 7A). SMOC1 was expressed in significantly high amounts for 7 days, after which the expression slightly decreased until 14 days after electroporation (FIG. 7B). At day 7, ALP expression was increased in cells overexpressing SMOC1 (left panel in FIG. 7), and at day 14 the expression of several osteoblast differentiation markers (ALP, COL1I, OPN, ON, BSP and OC) were empty. ) Was higher in cells overexpressing SMOC1 than cells expressing the vector. These results strongly suggest that overexpression of SMOC1 induces osteoblast differentiation of BMSCs.

1 shows the differentiation capacity (A), proliferative capacity (B) and surface markers (C) of BMSCs into osteoblasts isolated from six donors.

FIG. 2 briefly illustrates the procedure used for secretome profiling of BMSCs with low or high osteogenic capacity.

FIG. 3 shows a band diagram (A) of proteins secreted from BMSCs having low osteogenicity or high bone formation and proteins secreted from BMSCs having high osteogenicity according to biological processes (B).

Figure 4 shows the results of verifying the protein secreted from BMSCs having low or high osteogenic capacity by RT-PCR (A) and Western blot (B).

5 is a result of analyzing the secreted protein by RT-PCR (B) and the SMOC1 protein by Western blot after incubating the BMSCs having high osteogenic capacity for a predetermined time with or without osteogenic medium (B). It is shown.

Figure 6 shows the effect of SMOC1 knockdown on osteoblast differentiation, (A) the amount of SMOC1 and SPARC expression, (B) the activity of ALP, (C) the degree of mineral deposition, (D) Expression amounts of osteoblast markers are shown respectively.

Figure 7 shows the effect of SMOC1 overexpression on osteoblast differentiation, (A) the amount of SMOC1 expression in cells transformed with wild type SMOC1, (B) 7 in cells transformed with wild type SMOC1 or empty vector Expression levels of SMOC1 and osteoblast markers on day 1 (left) and day 14 (right) are shown, respectively.

<110> Kyungpook National University Industry-Academic Cooperation Foundation <120> Secretory proteins for which stem cell differentiate to          osteoblast <160> 21 <170> Kopatentin 1.71 <210> 1 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> shRNA <400> 1 gcctcaggaa gctgtgtttg t 21 <210> 2 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> shRNA <400> 2 ggtgatcaag gactccaaac t 21 <210> 3 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> shRNA <400> 3 ggaatctcat tcgatgcata c 21 <210> 4 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 4 aacaccatag gtagaatgta aagc 24 <210> 5 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 5 ctgatcagct atatagagtc actc 24 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 6 caccactgac atggttcagg 20 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 7 cgaggcgtcc tacttctttg 20 <210> 8 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 8 tgatgtgacg ctctacggc 19 <210> 9 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 9 aatggcggta ctgacttgat g 21 <210> 10 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 10 ctactacgcc aaggaggtca c 21 <210> 11 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 11 agcagccggt tgctgaggta t 21 <210> 12 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 12 ctgtgtcaac actcagcctg gc 22 <210> 13 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 13 tccttctcat cggtcacacc g 21 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 14 tgccatcaga catcttccag 20 <210> 15 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 15 tgcctgtggt tgactcttag aa 22 <210> 16 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 16 cacaacctgg agactggac 19 <210> 17 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 17 gtgtctgctg gatagaggag 20 <210> 18 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 18 catcaagcag atgaagatgt acc 23 <210> 19 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 19 ggtagtagca ggacttgatc ttgc 24 <210> 20 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 20 ccactggcgt cttcaccac 19 <210> 21 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 21 cctgcttcac caccttcttg 20  

Claims (12)

Biomarker composition for predicting differentiation of stem cells into osteoblasts comprising SMOC1 (SPARC-related Modular Calcium-binding Protein 1). delete delete A composition for predicting bone formation ability of stem cells comprising an agent for measuring the expression level of SMOC1 at the mRNA or protein level. 5. The method of claim 4, The agent measuring at the mRNA level is a composition characterized in that the primer pair or probe specific for the gene of the protein. 5. The method of claim 4, The agent measuring at the protein level is a composition characterized in that the antibody specific for the protein. Method for predicting bone formation ability of stem cells by measuring the expression level of SMOC1 at the mRNA or protein level. 8. The method of claim 7, The amount of expression of the protein is predicted, characterized in that measured in culture obtained by culturing stem cells in bone formation medium. delete delete delete delete

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