KR101919662B1 - UPSA-1 as a surface marker of undifferentiated dental pulp stem cells and a monoclonal antibody specific thereto - Google Patents

Abstract

The present invention relates to an undifferntiation-specific pulp stem cell surface antigen-1 (UPSA-1), which is a surface marker of undifferentiated stem cells. Specifically, the present invention relates to an undifferentiated stem cell pulp stem cells), a method for quantifying or separating undifferentiated stem cells using the composition, a method for inducing differentiation of undifferentiated stem cells, and an antibody specific for UPSA-1.
The composition comprising the agent capable of detecting UPSA-1 of the present invention can be used not only to confirm the undifferentiated state of the stem cell, but also to separate the stem cell of the undifferentiated state. In addition, since the differentiation induction method of the present invention increases the bone differentiation and dentin differentiation efficiency of undifferentiated stem cells, it can be usefully used in the field of cell therapy or tissue engineering.

Description

The surface markers of undifferentiated stem cells, UPSA-1 and its specific monoclonal antibody, {UPSA-1 as a surface marker of undifferentiated dental pulp stem cells and a monoclonal antibody specific}

The present invention relates to an undifferntiation-specific pulp stem cell surface antigen-1 (UPSA-1), which is a surface marker of undifferentiated stem cells. Specifically, the present invention relates to an undifferentiated stem cell pulp stem cells), a method for quantifying or separating undifferentiated stem cells using the composition, a method for inducing differentiation of undifferentiated stem cells, and an antibody specific for UPSA-1.

Human dental pulp stem cells (hDPSCs) are pluripotent stem cells necessary for regeneration of dentin and dentin, and can differentiate into various tissues such as bone, adipose tissue, neurons, and dentin. hDPSCs are obtained by primary culturing of dental tissues. The population of embryonic stem cells is a mixture of differentiated cells, precursors of various lines, and undifferentiated stem cells. In addition, hDPSCs exhibit inhomogeneous cellular phenotypes and characteristics depending on culture conditions and number of passages, so surface markers specific for undifferentiated hDPSCs are required to identify and culture a uniform population of undifferentiated hDPSCs.

Specifically, the surface markers described above include STRO-1, which is used to identify subgroups of cells having dentate and osteogenic capacity from pulp cells (Yang, X., et al., MJ Dent Res, 2009 (1999), pp. 1020-5), and other markers expressed in dendritic cells include CD29 and CD44 as well as markers of mesenchymal stem cells such as CD73 and CD105 (Jo, YY, et al. Tissue Eng, 2007. 13 (4): p. 767-73 .; Pivoriuunas, A., et al., Stem Cells Dev, 2010. 19 (7): p. In addition, CD271 is a neurotrophin receptor, a protein growth factor that stimulates neuronal cells to survive. It is known that MSCs including DPSCs inhibit the differentiation into bone, fat, cartilage, and muscular system. (Waddington, RJ, et al., Cells Tissues Organs, 2009. 189 (1-4): p. 268-74). However, the above-mentioned cell surface markers have a low expression level in DPSCs, and the expression of the cell surface markers is decreased during long-term culture. Accordingly, specific and reliable protein markers or carbohydrate epitopes that characterize undifferentiated hDPSCs need to be identified.

On the other hand, stem cells can be developed in the form of cell therapy, new drug development, biomedical engineering, and the like, and thus have possibilities of being used for treatment of a wide range of diseases. Specifically, as a cell therapy agent, stem cells extracted from a patient can be differentiated and proliferated in a specific environment and re-injected into the patient to replace the functions of damaged cells and tissues; As a new drug development tool, it is possible to differentiate stem cells into specific cells and to easily and quickly verify the toxicity of new drug candidates during the clinical trial process during the development of new drugs; As a tool of biomedical engineering, tissues and organs that have been variously differentiated from stem cells in an appropriate manner can treat diseases by regenerating the functions of tissues or organs damaged by disease. Accordingly, a lot of research has been conducted to develop a technique for separating undifferentiated stem cells. Specifically, a method for separating undifferentiated spermatogonial stem cells from pigs has been developed (Korean Patent Publication No. 2013-0088632). However, although undifferentiated hDPSCs may be useful for the prevention or treatment of periodontal disease, there has been no study of techniques for the isolation of undifferentiated hDPSCs.

Under these circumstances, the present inventors have made intensive efforts to identify undifferentiated hDPSCs-specific markers in order to isolate undifferentiated hDPSCs. As a result, UPSA-1 (undifferntiation-specific pulp stem cell surface antigen-1) specifically expressed in undifferentiated hDPSCs , And it was confirmed that it is possible to detect, quantify and isolate undifferentiated hDPSCs through the antigen and to promote its differentiation. Thus, the present invention has been completed.

One object of the present invention is to provide a composition for the detection of undifferentiated dental pulp stem cells, which comprises an agent capable of detecting undifferntiation-specific pulp stem cell surface antigen-1 (UPSA-1) .

Another object of the present invention is to provide a kit for detecting the undifferentiation of stem cells, which comprises the above composition.

It is another object of the present invention to provide a method for detecting UPSA-1 comprising: (a) contacting a cell or cell culture medium with an agent capable of detecting UPSA-1 on Invitro; And (b) confirming whether the agent and the cell or the cell culture fluid are bound to each other, and a method for quantifying undifferentiated stem cells.

Another object of the present invention is to provide a composition for the separation of undifferentiated stem cells, which comprises an agent capable of specifically binding to UPSA-1.

It is another object of the present invention to provide a method for producing a stem cell comprising: (a) subculturing a stem cell in vitro; (b) contacting the cell obtained from step (a) with an antibody specific for UPSA-1; And (c) recovering the complex of said stem cell and said antibody specific for UPSA-1.

Another object of the present invention is to provide a method for inducing differentiation of undifferentiated stem cells, which comprises culturing the undifferentiated stem cell separated by the above separation method in a medium containing a differentiation inducing compound.

Still another object of the present invention is a heavy chain CDR1 as set forth in SEQ ID NO: 1; A heavy chain CDR2 as set forth in SEQ ID NO: 2; And a heavy chain variable region comprising the heavy chain CDR3 as set forth in SEQ ID NO: 3; and a light chain CDR1 as set forth in SEQ ID NO: 5; A light chain CDR2 as set forth in SEQ ID NO: 6; And a light chain variable region comprising the light chain CDR3 as set forth in SEQ ID NO: 7, or a fragment thereof.

In order to achieve the above object, one aspect of the present invention relates to a method for detecting undifferentiation-specific pulp stem cell surface antigen-1 (UPSA-1) in a dental pulp stem cell A composition for detection is provided.

Another aspect provides a kit for detecting the undifferentiation of pulp stem cells, comprising the above composition.

(A) contacting the cell or cell culture with an agent capable of detecting UPSA-1 on Invitro; And (b) confirming whether the agent and the cell or cell culture medium are bound to each other, and a method for quantifying undifferentiated stem cells.

In the present invention, since undifferntiation-specific pulp stem cell surface antigen-1 (UPSA-1) is a cell surface antigen specifically expressed in human dental pulp stem cells (hDPSCs), it can be used as a marker of undifferentiated hDPSCs Respectively. Furthermore, it was confirmed that the isolated monoclonal antibody, UPSA-1, binds to the UPSA-1 antigen specifically expressed on the cell surface of the undifferentiated stem cell, and thus, the UPSA-1 specific binding agent, -1 can be detected by using a preparation capable of detecting undifferentiated stem cells.

The term "undifferntiation-specific pulp stem cell surface antigen-1" (UPSA-1) of the present invention means an antigen expressed on the surface of undifferentiated stem cells.

The term " agent capable of detecting UPSA-1 " means a substance capable of specifically binding to UPSA-1 through high affinity, and specifically refers to an antibody, an aptamer, a protein, Small molecule, nucleic acid, cell, and more specifically may be an antibody specific for UPSA-1, but is not limited thereto.

The agent may be labeled directly or indirectly for detection and isolation of undifferentiated stem cells. Specifically, the label may be a ligand, a bead, a radionuclide, an enzyme, a substrate, a cofactor, an inhibitor, a fluorescer, a chemiluminescent material, a magnetic particle, a hapten and a dye. It does not. Specific examples of the ligand include biotin, avidin and streptavidin, and the enzyme includes luciferase, peroxidase, and beta-galactosidase, and the fluorescent substance includes fluorescein, coumarin, rhodamine, But are not limited to, fumaric acid, fumaric acid, fumaric acid, fumaric acid, fumaric acid, fumaric acid, fumaric acid, fumaric acid, fumaric acid, fumaric acid, Many of the known markers may be used as such detectable markers, and those skilled in the art will be able to select appropriate markers for the purpose of the invention.

The term " antibody " of the present invention means a protein molecule that acts as a receptor for an antigen specifically recognizing an antigen, including immunoglobulin molecules immunologically reactive with specific antigens, and includes polyclonal antibodies, Antibodies, whole antibodies, and antibody fragments. The whole antibody is a structure having two full-length light chains and two full-length heavy chains, and each light chain is linked to a heavy chain by a disulfide bond. The whole antibody includes IgA, IgD, IgE, IgM and IgG, and IgG is a subtype and includes IgG1, IgG2, IgG3 and IgG4.

The antibody fragment refers to a part of the whole antibody, and includes a fragment having an antigen-binding function. Examples include, but are not limited to, Fc fragments, Fab, Fab ', F (ab') _2, and Fv. The Fc fragment refers to the terminal region of an antibody capable of binding to a cell surface receptor such as an Fc receptor and consists of the second or third constant domain of two heavy chains of the antibody. The Fab has one antigen-binding site in a structure having a variable region of a light chain and a heavy chain, a constant region of a light chain, and a first constant region (CH1) of a heavy chain. Fab 'differs from Fab in that it has a hinge region that contains at least one cysteine residue at the C-terminus of the heavy chain CH1 domain. The F (ab ') ₂ antibody is produced when the cysteine residue of the hinge region of the Fab' forms a disulfide bond. Fv (variable fragment) refers to the smallest antibody fragment that has only a heavy chain variable region and a light chain variable region. The double chain Fv (disulfidestabilized variable fragment) is a disulfide bond in which the heavy chain variable region and the light chain variable region are connected. The single chain variable fragment (scFv) is generally linked to the variable region (VH) of the heavy chain through a peptide linker The variable region (VL) of the light chain is linked by covalent bonds. In the present invention, the scFv refers to a minimal antibody fragment having a complete antigen-recognition and antigen-binding site and includes the VH and VL regions of the antibody, wherein the domain is present in a single polypeptide chain. Such an antibody fragment can be obtained using a protein hydrolyzing enzyme (for example, a whole antibody can be restricted to papain and a Fab can be obtained, and when cut with pepsin, an F (ab ') ₂ fragment can be obtained) , Preferably through recombinant DNA technology.

As used herein, the term " an antibody specific for UPSA-1 " refers to an antibody or a fragment thereof recognizing and binding to UPSA-1 as an antigen. In the present specification, the antibody includes 'UPSA-1 monoclonal antibody' Monoclonal antibodies UPSA-1 ', and' mAb UPSA-1 '.

Specifically, the antibody comprises a heavy chain CDR1 as set forth in SEQ ID NO: 1, a heavy chain CDR2 as set forth in SEQ ID NO: 2; And a heavy chain variable region comprising the heavy chain CDR3 as set forth in SEQ ID NO: 3; and a light chain CDR1 as set forth in SEQ ID NO: 5; A light chain CDR2 as set forth in SEQ ID NO: 6; And a light chain variable region comprising the light chain CDR3 as set forth in SEQ ID NO: 7.

The antibody may also be an antibody comprising a light chain comprising the amino acid sequence of SEQ ID NO: 4 and a light chain comprising the amino acid sequence of SEQ ID NO: 8, a heavy chain and sequence encoding by the nucleotide sequence of SEQ ID NO: But is not limited to, an antibody comprising a light chain encoded by the nucleotide sequence of SEQ ID NO: 10.

In a specific example of the present invention, it was confirmed that monoclonal antibodies cultured and purified in hybridoma cells specifically bind to UPSA-1, a cell surface antigen of undifferentiated human dental mesencephalic cells (FIGS. 1A and 1B) , And the gene sequences of the heavy chain and the light chain and the peptide sequence were confirmed by analyzing the gene base sequence of the monoclonal antibody (FIGS. 2 to 4d).

The term " dental pulp stem cell " of the present invention means a stem cell present in the pulp tissue. Specifically, the stem cell may be isolated from a third molar of a human, but is not limited thereto. In this specification, the stem cells can be named as' human dermal stem cells', hDPSCs' or 'DPSCs'.

The term " differentiation " of the present invention refers to a phenomenon in which the structure or function of a cell is specialized while cells are divided and proliferated to grow an entire individual. In other words, it refers to a process in which cells, tissues, etc. of a living organism are changed into appropriate forms and functions to perform their respective roles. For example, the processes in which the pluripotent stem cells such as embryonic stem cells are transformed into ectoderm, mesoderm, and endoderm cells are also differentiated, and narrowly, the processes in which hematopoietic stem cells change into red blood cells, white blood cells, platelets, etc. are also differentiated.

The term " undifferentiated " of the present invention means a state in which the cells are not finally differentiated, and means a state in which the differentiation step is not further advanced or de-differentiated in the initial state.

The term " detection of undifferentiated stem cells " of the present invention means that the agent capable of detecting UPSA-1 of the present invention binds to UPSA-1, an undifferentiation-specific antigen of stem cells, and confirms the undifferentiated state of the stem cells .

The kit for detecting pulmonary fibroblast differentiation of the present invention includes not only a marker gene whose expression is increased or decreased in the undifferentiated state of stem cells, a primer or an antibody capable of selectively recognizing the protein, And tools and reagents commonly used in the art. Such tools and reagents include, but are not limited to, suitable carriers, labeling agents capable of generating a detectable signal, solubilizers, cleaning agents, buffers, stabilizers, and the like. When the labeling substance is an enzyme, it may include a substrate capable of measuring the activity of the enzyme and a reaction terminator. Suitable carriers include, but are not limited to, soluble carriers, e. G., Physiologically acceptable buffers such as PBS, insoluble carriers such as polystyrene, polyethylene, polypropylene, Polyacrylonitrile, fluororesin, crosslinked dextran, polysaccharide, polymer such as magnetic fine particles plated with metal on latex, other paper, glass, metal, agarose, and combinations thereof.

The kit for detecting a pulmonary fibroblast differentiation of the present invention may specifically be an RT-PCR kit, a DNA kit or a protein chip kit. When an antibody against the protein encoded by the gene is provided on the protein chip, formation of an antigen-antibody complex for two or more antibodies can be observed, which is more advantageous in confirming the differentiation of stem cells.

The RT-PCR kit may comprise a respective pair of primers specific for the marker gene, and may include other test tubes or appropriate containers, reaction buffers (varying in pH and magnesium concentration), deoxynucleotides (dNTPs), Taq- Enzymes such as polymerase and reverse transcriptase, DNAse, RNAse inhibitor DEPC-water, sterile water, and the like.

The DNA chip kit may include a substrate on which a cDNA or an oligonucleotide corresponding to a gene or a fragment thereof is attached, and a reagent, a preparation, and an enzyme for producing a fluorescent-labeled probe. The substrate may be a control gene Or a cDNA or oligonucleotide corresponding to the fragment thereof.

The protein chip kit may be a kit in which one or more antibodies against a marker are arranged at predetermined positions on a substrate and fixed at high density. In the method using a protein chip, a protein is separated from a sample, and a separated protein is hybridized with a protein chip to form an antigen-antibody complex, and the presence or the expression level of the protein can be confirmed by reading it.

In the present invention, the method for quantifying undifferentiated stem cells includes the steps of contacting the agent capable of detecting the UPSA-1 with a cell or a cell culture medium; And confirming whether or not it is a combination thereof. The cell may be a cell contained in the dermal tissue, and more specifically, it may be, but is not limited to, a mesenchymal stem cell or a dermal cell in the undifferentiated, differentiated, or differentiated state contained in the dermal tissue.

Whether the preparation is bound to a cell or a cell culture medium can be confirmed by immunoassay or flow cytometry, but is not limited thereto.

The term " immunoassay " of the present invention refers to a biochemical test method for measuring the presence or concentration of a polymer in a solution using an antibody or an immunoglobulin, and examples thereof include radioimmunoassay (RIA), enzyme immunoassay ELISA), immunofluorescence or chemiluminescent substance binding method, but the present invention is not limited thereto.

The term " flow cytometry " Refers to a technique that counts the number of cells, classifies species, or detects biomarkers. Specifically, it detects, but is not limited to, cells labeled with fluorescent substances or isotopes.

Using the above method, the number of cells bound to the preparation can be counted, or the concentration can be measured to quantitate the cells. For example, it is possible to quantify undifferentiated stem cells by performing immunostaining with an antibody specific for UPSA-1 (Example 5 and Fig. 5).

Another embodiment provides a composition for the separation of undifferentiated stem cells, which comprises an agent capable of specifically binding to UPSA-1.

Another aspect relates to a method of producing a stem cell comprising: (a) subculturing a stem cell in vitro; (b) contacting the cell obtained from step (a) with an antibody specific for UPSA-1; And (c) recovering a complex of said stem cell and said antibody specific for UPSA-1. The present invention also provides a method for separating undifferentiated stem cells.

The above-described UPSA-1, antibody and pulmonary stem cell differentiation are as described above.

Using the agent specifically binding to UPSA-1 of the present invention, it is possible not only to detect undifferentiated stem cells as described above, but also to isolate a homogeneous population of undifferentiated cells from stem cells have.

The term " agent specifically binding to UPSA-1 " of the present invention means a substance capable of binding to UPSA-1 with high affinity and specificity, and specifically includes an antibody, an aptamer, Small molecule, nucleic acid, cell, and more specifically may be an antibody specific for UPSA-1, but is not limited thereto.

Specific methods for separating only pulmonary stem cells using an antibody specific to UPSA-1 include, for example, a method of labeling with a fluorescent antibody and separating into a cell sorter, a method of separating pulmonary stem cells A method of immobilizing an anti-UPSA-1 antibody having affinity to a specific UPSA-1 and directly or indirectly binding the stem cell, a separation method using an immunoadsorption column, and a separation method using immuno magnetic beads But is not limited thereto.

In addition, the separation method may be performed by a fluorescence activated cell sorter or a magnetic activated cell sorter, but is not limited thereto.

The term 'complex' refers to a conjugate in which the stem cell and the monoclonal antibody UPSA-1 are specifically bound.

In a specific embodiment of the present invention, the monoclonal antibody UPSA-1 bound to the undifferentiated stem cell is labeled with magnetic beads, and then the cell is separated into a self-activating cell sorter, whereby the separated undifferentiated stem cells are differentiated (Fig. 8A), and the expression levels of bone cell and dentin marker proteins were increased (Fig. 8B).

This suggests that the use of an antibody specific for UPSA-1 according to the present invention can clearly distinguish and separate undifferentiated or differentiated hDPSCs.

Another embodiment provides a method for inducing differentiation of undifferentiated stem cells, comprising the step of culturing the undifferentiated stem cell separated by the separation method in a medium containing the differentiation inducing compound.

In one embodiment of the present invention, the undifferentiated stem cells isolated through the present invention have excellent differentiation ability, and thus it has been confirmed that the cells can be differentiated into bone or dentin.

The above-mentioned mesenchymal stem cells and differentiation are as described above.

In the present invention, the differentiation-inducing compound may be at least one selected from the group consisting of ascorbic acid, dexamethasone, and beta-glycerophosphate, although the present invention is not limited thereto. Suitable compounds can be selected for the purposes of the invention.

In the present invention, the differentiation may be bone or dentin differentiation.

The term " osteogenic differentiation " of the present invention refers to a series of physiological responses that occur during the development of an animal, in which osteoblast or osteoclast differentiated to form bone matrix, and the bone matrix formed was calcified to form bone May mean the step of forming bone matrix during the osteogenesis process or may be a process of naturally or artificially inducing differentiation of stem cells of an animal to form bone matrix. In the present invention, the 'bone differentiation' may be used in combination with 'bone cell differentiation'.

The term " dentinogenic differentiation " of the present invention may refer to a step in which extraocular cells appear during the development of an animal to form dentin cells and naturally or artificially induce the differentiation of animal stem cells Which may be a process of forming dentin cells.

In one specific embodiment of the present invention, undifferentiated stem cells isolated using monoclonal antibody UPSA-1 are cultured in a medium containing 100 μM ascorbic acid, 500 nM dexamethasone and 5 mM beta-glycerol As shown in FIG. 8A, the expression level of osteocyte and dentin marker protein was increased when cultured for 14 days in medium supplemented with phosphate (β-glycerophosphate) to induce differentiation (Fig. 8B).

Another embodiment is a heavy chain CDR1 as set forth in SEQ ID NO: 1; a heavy chain CDR2 as set forth in SEQ ID NO: 2; And a heavy chain variable region comprising the heavy chain CDR3 as set forth in SEQ ID NO: 3; and a light chain CDR1 as set forth in SEQ ID NO: 5; A light chain CDR2 as set forth in SEQ ID NO: 6; And a light chain variable region comprising the light chain CDR3 as set forth in SEQ ID NO: 7, or a fragment thereof.

The antibody may also be an antibody comprising a light chain comprising the amino acid sequence of SEQ ID NO: 4 and a light chain comprising the amino acid sequence of SEQ ID NO: 8, a heavy chain and sequence encoding by the nucleotide sequence of SEQ ID NO: But is not limited to, an antibody comprising a light chain encoded by the nucleotide sequence of SEQ ID NO: 10.

In a specific example of the present invention, it was confirmed that monoclonal antibodies cultured and purified in hybridoma cells specifically bind to UPSA-1, a cell surface antigen of undifferentiated human dental mesencephalic cells (FIGS. 1A and 1B) , And the gene sequences of the heavy chain and the light chain and the peptide sequence were confirmed by analyzing the gene base sequence of the monoclonal antibody (FIGS. 2 to 4d).

This suggests that the antibody according to the present invention specifically binds to UPSA-1 and thus can be usefully used for detection, quantification and isolation of undifferentiated hDPSCs.

The composition comprising the agent capable of detecting UPSA-1 of the present invention can be used not only to confirm the undifferentiated state of the stem cell, but also to separate the stem cell of the undifferentiated state. In addition, since the differentiation induction method of the present invention increases the bone differentiation and dentin differentiation efficiency of undifferentiated stem cells, it can be usefully used in the field of cell therapy or tissue engineering.

Figure 1A shows the binding affinities of the monoclonal antibody UPSA-1 to the UPSA-1, CD24, CD44, CD73, CD90 and Stro-1 antigens expressed on the surface of human dental pulp stem cells (hDPSCs) Showing flow cytometry results.
FIG. 1B is data obtained by quantifying the binding affinity analyzed in FIG. 1A.
Figure 2 is a Western blot image showing the results of detection of heavy and light chain peptides of the undifferntiation-specific Pulp stem cell Surface Antigen-1 (UPSA-1).
3 is a table showing the results of analysis of the gene of monoclonal antibody UPSA-1. a is a heavy chain variable region, and b is a light chain variable region.
Figure 4a shows the DNA sequence of the heavy chain of monoclonal antibody UPSA-1.
Figure 4b is an image showing the amino acid sequence and variable region of the heavy chain of monoclonal antibody UPSA-1.
Figure 4C shows the DNA sequence of the light chain of the monoclonal antibody UPSA-1.
Figure 4D is an image showing the amino acid sequence and variable region of the light chain of monoclonal antibody UPSA-1.
Figure 5 is an immunofluorescent staining image showing monoclonal antibody UPSA-1 bound or not bound to hDPSCs cell surface.
Figure 6 is a Western blot image showing that the epitope of monoclonal antibody UPSA-1 is a carbohydrate moiety.
Fig. 7a shows the cell surface binding affinity of monoclonal antibody UPSA-1 according to the treatment with tunicamycin, wherein a is a flow cytometric analysis result, and b is data quantifying the binding affinity analyzed in a above.
FIG. 7B is a graph showing the levels of RNA expression of bone and dentin-related markers (osteonectin, Runx2, ColI and ALP, DMP-1, DSPP) in hDPSCs differentiated after treatment with tunicamycin.
Figure 8a shows the results of comparing the ability of differentiation of Ab-negative or differentiated hDPSCs (Ab-positive) using monoclonal antibody UPSA-1 (a-UPSA-1). a is an image showing the degree of staining of alizarin red S, and b is a result of quantifying the degree of staining of a.
FIG. 8B shows the results of comparing the marker expression levels of undifferentiated hDPSCs (-) and differentiated hDPSCs (+) using the monoclonal antibody UPSA-1. Specifically, protein expression levels of bone-related markers (osteonectin, Runx2, ColI) and dentin-related markers (ALP, DMP-1, DSPP) were analyzed.
Fig. 9 is an image showing the schematic (a) of the dimensional tissue and the histological fragment (b) of the potential stem cell Nisshl present in the dimension. Arrows point to the stem cells of the dimension.

Hereinafter, embodiments of the present invention will be described in detail to facilitate understanding of the present invention. However, the embodiments according to the present invention can be modified into various other forms, and the scope of the present invention should not be construed as being limited to the following embodiments. Embodiments of the invention are provided to more fully describe the present invention to those skilled in the art.

Example  1. Cell culture

Example  1-1. Isolation and Culture of Human Dimension Stem Cells

For the first culture of human dental pulp stem cells (hDPSCs), the third molar was extracted from patients 17-28 years old according to the guidelines approved by the clinical examination committee of the Dental Hospital of Dankook University. Dimensional cells were separated by a conventional method.

In order to isolate hDPSCs, the dental tissues were separated by removing the head of the teeth. Then, three dimensional tissue was stained with 3 mg / ml collagenase type I (millipore) and 4 mg / ml Deg.] C and dispase (Sigma) solution at 37 [deg.] C for 1 hour. The isolated single cell suspension was incubated with α-MEM (hyclone) supplemented with 20% fetal bovine serum (FBS) at 37 ° C. and 5% carbon dioxide concentration.

Example  1-2. Differentiation of Human Dimension Stem Cells

The isolated hDPSCs were differentiated by culturing for 14 days in medium supplemented with 100 μM ascorbic acid, 500 nM dexamethasone, and 5 mM β-glycerophosphate.

Example  1-3. Of Human Dimension Stem Cells Deglycosylation

To perform deglycosylation of the isolated hDPSCs, tunicamycin (Sigma) with a final concentration of 1 mg / ml in the culture medium was treated for 24, 48, 72 hours and protein immobilization (New England Biolab, P6039) and reacted at 37 ° C for 4 hours.

Example  2. Antibody purification

In order to purify the undifferntiation-specific Pulp stem cell surface antigen-1 (UPSA-1) antibody, column chromatography was performed on the hybridoma cell culture supernatant.

Specifically, the culture supernatant was transferred to rProtein G Incospharm column, washed with PBS (pH 8.0), and bound IgG was eluted with 0.1 M glycine buffer (pH 3.0). Thereafter, 1.0 M Tris-HCl (pH 9.0) was added at a ratio of 10: 1 to neutralize the eluted fraction. After dialysis, antibody concentration was analyzed at OD ₂₈₀ .

Example  3. Antibody Gene Sequence Analysis

Total RNA was isolated from 1x10 ⁶ UPSA-1 hybridoma cells using easy-spin ^TM (total RNA) extraction kit (intron). DNA was then synthesized using the ONE-STEP RT-PCR Premix kit (intron). (Ning, B., et al., Oncol Lett, 2012.3 (5): p. 1083-1086), and the sequences thereof are listed in Table 1 below.

division primer
name order Mouse heavy chain constant region primers, VH primers SEQ ID NO: 11 IgG 5'-ATA GAC AGA TGG GGG TGT CGT TTT GGC -3 ' SEQ ID NO: 12 5 'MH1 5'-SAR CTN MAG CTG SAG SAG TC -3 ' SEQ ID NO: 13 5 'MH 2 5'-SAR GTN MAG CTG SAG SAG TCW GG -3 ' Mouse kappa chain constant region primers, VL primers SEQ ID NO: 14 5 'Mk 5'- GAY ATT GTG MTS ACM CAR WCT MCA -3 ' SEQ ID NO: 15 Primer 6 5'-GAC ATT GTG CTG ACC CAA TCT CCA GCT TCT -3 ' SEQ ID NO: 16 Primer 7 5'-GAC ATT CAG CTG ACC CAG TCT CCA -3 '

The following conditions were set for the PCR amplification: reverse transcription reaction at 45 ° C for 30 min, cDNA fusion at 94 ° C for 5 min, denaturation at 94 ° C for 1 min (30 cycles), annealing at 45 ° C 1 min at extension step, 1 min at 72 < 0 > C, and 5 min at 72 < 0 > C for extension of the last addition. The PCR products were purified using the Expin ^TM Clean Up kit (GeneALL).

The DNA fragment (insert) and the pBluescript KS (+) vector were ligated in a ratio of 3: 1 (v / v) using a T4 ligase (TaKaRa) Lt; / RTI > The product was then transformed into DH5α competent cells and then separated into Ampicillin in Luria-Bertani (LB) containing IPTG and X-gal. The selected colonies were treated with Exprep ^™ Plasmid kit (GeneALL ).

Antibody gene sequences were analyzed using Solgent Co., Ltd and confirmed using the IMGT / V-QUEST program (version 3.2.21).

Example  4. Flow cell  Analysis cytometry )

To confirm the binding characteristics of the antibody and hDPSCs, flow cytometry was performed.

Specifically, each antibody (mAb UPSA-1, Stro-1, PE-conjugated CD24, CD44, CD73 and CD90) contained in PBS solution (containing 1% BSA) was added to 1x10 ⁶ cells in a ratio of 1: 100 Lt; / RTI > for 1 hour. The cells were then washed three times with PBS and reacted for 1 hour on ice with FITC-conjugated anti-mouse IgG (1: 100 dilution). Cells were washed three times and analyzed using a FACS Calibur flow cytometer (BD Biosciences). Binding affinities were quantified using the Cell Quest and WinMDI program.

Example  5. Immunofluorescence  analysis

To immunostain the cell surface, cells were cultured in mAb UPSA-1 in an unfixed state.

On the other hand, to immunostain the cells, the cells were placed on a cover slip and fixed with PBS containing 4% paraformaldehyde / methanol. Then, mAb UPSA-1 was treated with cells overnight at 4 ° C and then cultured for 1 hour with FITC-conjugated anti-mouse IgG (1: 100 dilution) contained in blocking solution (PBS containing 10% FBS) Respectively. Cell nuclei were detected with DAPI (4,6-diamidino-2-phenylindole).

Example  6. Western blot  Analysis and immunoprecipitation

Cells were treated with EZ-Link sulfo-NHS-LC-Biotin (EZ-Link Sulfo-NHS-LC-Biotin, Thermo Scientific) for 30 min at 4 ° C to detect glycosylated surface antigens on the cell surface . Biotin labeled cells were incubated with 1% NP-40 dissolution buffer (20 mM Tris-HCl, pH 8.0, 100 mM NaCl, 2 mM EDTA, PD 8.0, 2 mM EGTA pH 8.0, 1% nonidet P- Enzyme inhibitor) was dissolved and treated. The cell extracts were then cultured in 10 μg of mAb UPSA-1. Thereafter, the sample was incubated with rProtein G agarose (Incospharm) at 4 ° C for 3 hours.

To perform deglycosylation, the sample was reacted with a deglycosylation mix (NEW ENGLAND BioLabs; NEB). SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and then transferred to PVDF membrane (Millipore) and detected with mAb UPSA-1 primary antibody and streptavidin-HRP (GE Healthcare). Signals were visualized using an ECL Western blotting detection kit (GE healthcare).

Example  7. Quantitative real time PCR  ( qRT - PCR )

The mRNA expression levels of the cells treated or not treated with tunicamycin were analyzed using qRT-PCR (quantitative real-time PCR).

Specifically, cDNA was synthesized using ReverTra Ace ^® qPCR RT Kit (Toyobo Corporation) after extracting total RNA using Total RNA Extraction Kit (intron), and iTaq ^™ Universal SYBR ^® Green Supermix (Bio-Rad) system Were used to perform qRT-PCR. The primers used were listed in Table 2 below.

gene division direction The sequence (5 '- > 3') hGAPDH SEQ ID NO: 17 Forward GGAGTCCACTGGCGTCTTCAC SEQ ID NO: 18 Reverse GCTGATGATCTTGAGGCTGTTGTC Osteonectin SEQ ID NO: 19 Forward CTGTTGCCTGTCTCTAAACC SEQ ID NO: 20 Reverse CACCATCATCAA ATTCTCCT Runx2 SEQ ID NO: 21 Forward GTCTCACTGCCTCTCACT SEQ ID NO: 22 Reverse TACACACATCTCCTCCCTTC COLI SEQ ID NO: 23 Forward GGAGGAGAGTCAGGAAGG SEQ ID NO: 24 Reverse TCAGCAACACAGTTACACAA ALP SEQ ID NO: 25 Forward CTTGACCTCCTCGGAAGACACTC SEQ ID NO: 26 Reverse CGCCCACCACCTTGTAGCC DMP1 SEQ ID NO: 27 Forward GACTCTCAAGAAGACAGCAA SEQ ID NO: 28 Reverse GACTCACTCACCACCTCT DSPP SEQ ID NO: 29 Forward CAGTACAGGATGAGTTAAATGCCAGTG SEQ ID NO: 30 Reverse CCATTCCCTTCTCCCTTGTGACC

The following conditions were set for performing qPCR: 1 minute (1 cycle) at 95 ° C, 15 seconds (40 cycles) at 95 ° C, 1 minute (1 cycle) at 60 ° C. Then, a dissociation curve was designed in the range of 65 占 폚 and 95 占 폚. GAPDH was used as an internal control to standardize the diversity of expression levels.

The relative expression of each gene (ΔCq) was calculated using the following equation: ΔCq = 2 ^{( Cq (Min) - Cq (Sample))} .

Cq (Min) represents the lowest average value of the GOI (Gene of Interest), and Cq (Sample) represents the average Cq of the samples.

The normalized expression amount (ΔΔCq) was calculated using the following equation: ΔΔCq = ΔCq _(Sample) / ΔCq _(Ref) .

At this time, the normalized expression amount (DELTA DELTA Cq) means that the relative expression amount of each gene is normalized to the expression amount of GAPDH gene.

Statistical analysis was performed with three numerical values. Student's t test was used and a p- value less than 0.05 was considered significant: T-test; * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001. All results were expressed as mean ( n = 3) with error bars and t test was used with GraphPad Prism 6 program.

Example  8. Undifferentiated or differentiated of hDPSCs  detach

One μg of biotin-conjugated monoclonal antibody (α-UPSA-1) was added to hDPSCs (about 1 × 10 ⁶ cells) of Example 1-1, reacted at 4 ° C. for 1 hour and then washed. Streptavidin magnetic beads (Streptavidin MicroBeads) were then treated and allowed to react for 1 hour at 4 ° C under dark conditions. The magnetic bead-labeled hDPSCs were separated on a MACS column system to obtain Ab-positive hDPSCs (Group 1) or Ab-negative hDPSCs (Group 2).

Example  9. Alizarin Red  S (alizarin red S) staining

70% ethanol was added to the hDPSCs of Example 1-1 at room temperature for 5 minutes to fix and then dried. 2% alizarin red S (pH 4.5) was added to each of the dried cells to perform a staining reaction, followed by washing and comparing the color development level.

In order to quantitatively analyze the color development level, 10% acetic acid was added to the stained cells to extract a dye, neutralized by treatment with 10% ammonium hydroxide, and analyzed at 405 nm.

Example  10. Immunohistochemical staining ( immunohistochemistry )

Dimensional tissues were fixed in 10% formaldehyde for 5 days. Thereafter, the above-mentioned tissue was flaked to a thickness of 5 mu m to prepare a sample. Of these, 6 samples were randomly selected, mounted on slides and reacted with anti-UPSA-1 antibody for 1 hour. This was followed by reaction with a biotinylated secondary antibody and HRP enzyme-avidin conjugate.

The sections were analyzed on a Leica DFC280 optical microscope and Leica Q Win Image Analysis System (Leica Micros Imaging Solutions Ltd., Cambridge, UK).

The contents confirmed through the above examples are summarized in the following experimental examples.

Experimental Example 1. Monoclonal antibody specifically binding to UPSA-1 antigen UPSA-1

The present inventors prepared constructs of antibody sets for screening cell surface molecules specifically expressed in undifferentiated human dental mesencemental stem cells (hDPSCs). Among the above antibody sets, monoclonal antibody UPSA-1 Additional studies have been conducted to characterize mAb UPSA-1.

First, the binding ability of mAb UPSA-1 to cell surface markers expressed in hDPSCs (UPSA-1, CD24, CD44, CD73, CD90, Stro-1) was confirmed by flow cytometry analysis according to Example 4. At this time, 2-3 undigested hDPSCs were used as undifferentiated cells according to Experimental Example 1-1, and the differentiated cells were separated according to Experimental Example 1-1, HDPSCs differentiated by the method according to Example 1-2 were used.

As a result, it was confirmed that the binding affinity of mAb UPSA-1 to the differentiated cells was reduced to less than 4.6 times as compared to the undifferentiated cells, as a result of the UPSA-1 antigen (FIGS. 1A and 1B). In addition, the binding affinity for differentiated cells was 3.2, 1.7, or 3.2 times lower than that of undifferentiated cells, respectively (Fig. 1a, c-e and Fig. 1b), also for the CD44, CD90 or CD73 antigen.

The above results indicate that the UPSA-1 antigen is expressed at a higher level on the surface of undifferentiated hDPSCs than the differentiated cells, and the degree of expression is decreased in the differentiated state compared to other antigens. Thus, it is known that the antigen is specific for undifferentiated hDPSCs I could. In addition, mAb UPSA-1 specifically binds to undifferentiated hDPSCs, specifically UPSA-1 expressed in undifferentiated hDPSCs.

Experimental Example  2. Monoclonal antibody UPSA -1 Features

MAb UPSA-1 was isolated from the hybridoma culture medium by the method according to Example 2 and analyzed by SDS-PAGE to detect the heavy and light chain peptides of the antibody (FIG. 2). Specifically, the V-, J-, and D- portions of the heavy chain of the antibody were found to be Musmus IGHV1-9 * 01F, Musmus IGHJ4 * 01F, and Musmus IGHD1-1 * 01F, respectively (FIG. The V- and J- parts of the light chain were confirmed to be Musmus IGκV4-77 * 01P and Musmus IGκJ1 * 01F, respectively, and the variable region of the UPSA-1 light chain was Ig _κ gene belongs to the V4 subgroup (Fig. 3b).

In addition, the nucleotide sequence and amino acid sequence thereof were confirmed by analyzing the gene base sequence of the antibody by the method according to Example 3 (FIGS. 4A to 4D), and the sequence thereof is listed in Table 3 below.

division order mAb UPSA-1 heavy chain amino acid sequence SEQ ID NO: 4 EVQLEESGAELMKPGASVKISCKATGYTFSSYWIEWVKQRPGHGLEWIGEILPGSDNTNYNEKFKGKATFTADTSSNTAYMQLSSLTSEDSAVYYCTRATTVYAMDYWGQGTSVTVSSAKTTPPSVY mAb UPSA-1 light chain amino acid sequence SEQ ID NO: 8 DIQLTQSPAILSASPGEKVTMTCSASSSSSYMHWFQQKPGSSPKLWIYIISNLASGVPARQWQWVWDLLFSHNQQREGARCCHLLLPAVEHFPTGRSVEAPSWKSNGLMLHQLYP mAb UPSA-1 heavy chain DNA sequence SEQ ID NO: 9 GAGGTTCAGCTGGAGGAGTCTGGAGCTGAGCTGATGAAGCCTGGGGCCTCAGTGAAGATATCCTGCAAGGCTACTGGCTACACATTCAGTAGCTACTGGATAGAGTGGGTAAAGCAGAGGCCTGGACATGGCCTTGAGTGGATTGGAGAGATTTTACCTGGAAGTGATAATACTAACTACAATGAGAAGTTCAAGGGCAAGGCCACATTCACTGCAGATACATCCTCCAACACAGCCTACATGCAACTCAGCAGCCTGACATCTGAGGACTCTGCCGTCTATTACTGTACAAGGGCGACTACGGTCTATGCTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCAGCCAAAACGACACCCCCATCTGTCTAT mAb UPSA-1 light chain DNA sequence SEQ ID NO: 10 GACATTCAGCTGACCCAGTCTCCAGCAATCCTGTCTGCATCTCCAGGGGAGAAGGTCACCATGACCTGCAGTGCCAGCTCAAGTGTAAGTTACATGCACTGGTTCCAGCAGAAGCCAGGATCCTCCCCCAAACTCTGGATTTATATCATATCCAACCTGGCTTCTGGAGTCCCTGCTCGCTTCAGTGGCAGTGGGTCTGGGACCTCTTATTCTCTCACAATCAGCAGCGTGAAGGCCGAAGATGCTGCCACTTATTACTGCCAGCAGTGGAGCAGTTCCCCACCGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAACGGGCTGATGCTGCACCAACTGTATCCC

EXPERIMENTAL EXAMPLE 3 Monoclonal Antibody Recognizing Carbohydrate Epitope Expressed on hDPSCs Surface UPSA-1

Since most cell surface antigens are known as glycoproteins or glycolipids, the present inventors have investigated whether the epitope of the UPSA-1 antigen is a carbohydrate residue.

First, immunofluorescence staining was performed with mAb UPSA-1 without the fixing or permeation step according to the method according to Example 5. As a result, relatively strong cell membrane staining and internalization of the antibody were observed in most of the cells (Fig. 5 (a)). However, treatment with tunicamycin, an N-glycosylation inhibitor of a protein that inhibits glycosylation, resulted in a sharp decrease in the mAb UPSA-1 binding signal (FIG. 5 b). In addition, it was confirmed that the mAb UPSA-1 signal on the cell surface disappeared without treatment with tunicamycin (FIG. 5C) when paraformaldehyde and methanol were treated and fixed and permeated. From the above results, it was found that mAb UPSA-1 binds to cell surface antigens.

Then, to confirm whether the antigen molecule is a carbohydrate residue, the UPSA-1 antigen was immunoprecipitated using hDPSCs treated with tunicamycin by the method according to Example 6. As a result, it was confirmed that when the tunicamycin was treated, the amount of two bands larger than 135 kDa decreased and the band of 75 kDa disappeared completely (Fig. 6, lanes 2-3). In addition, when the deglycosylation mixture solution according to the existing literature (Koutsioulis, D., et al., Glycobiology, 2008. 18 (10): p. 799-805) -PAGE (Fig. 6, lanes 4-5).

The above results show that mAb UPSA-1 binds to the carbohydrate epitope on the cell surface of hDPSCs.

Experimental Example  4. of hDPSCs  Distinguish the state of differentiation Monoclonal antibody UPSA -One

Experimental Example  4-1. Tunicamycin  Depending on processing of hDPSCs Ability to distinguish  Confirm

Whether inhibition of glycosylation affects the differentiation efficiency of undifferentiated hDPSCs was investigated.

First, flow cytometry analysis was performed according to the method of Example 4, and it was confirmed that the affinity of cell surface binding of mAb UPSA-1 was reduced by treatment with tunicamycin (FIG. 7A).

Thereafter, hDPSCs were first treated with tunicamycin for 1-2 days, then dentin / osteoclast differentiation was induced by the method according to Example 1-2, and the expression of dentin and bone cell markers Respectively.

As a result, in the case of hDPSCs treated with tunicamycin, the amount of expression of dentin and bone cell markers was significantly reduced as compared with hDPSCs not treated with tunicamycin (Fig. 7B). Specifically, the levels of RNA expression of bone cell markers such as osteonectin, Runx2, ColI (collagen type 1) and alkaline phosphatase (ALP) were 66%, 15%, 80% Respectively. In addition, RNA expression levels of dentin markers such as DMP-1 (dentin matrix protein-1) and DSPP (dentin sialophosphoprotein) decreased sharply to 96% and 64.3%, respectively.

The above results indicate that glycosylation of undifferentiated hDPSCs is important for stem cell maintenance and that glycosylation is required for the binding of mAb UPSA-1, so that mAb UPSA-1 is an undifferentiated state of hDPSCs And it can be used to confirm it.

Experimental Example 4-2. Identification of the ability of hDPSCs to differentiate according to the presence of monoclonal antibody UPSA-1

Since the undifferentiated hDPSCs can be differentiated according to the differentiation process, the ability to differentiate the undifferentiated or differentiated hDPSCs was compared using the above-mentioned monoclonal antibody UPSA-1 (? -PSAA-1).

First, hDPSCs were isolated by the method according to Example 8. Subsequently, the alizarin red S staining reaction was performed after differentiating hDPSCs (positive reaction to antibody) or differentiated hDPSCs (negative reaction to antibody) by the method according to Example 1-2. At this time, the separated cells were directly cultured without any separate procedure for removing magnetic beads and used in the experiment.

As a result, the Ab-positive hDPSCs of Group 1 selected using the monoclonal antibody of the present invention showed excellent bone differentiation ability, while the Ab-negative hDPSCs of Group 2 selected using the monoclonal antibody showed very low levels (Fig. 8A). As a result, hDPSCs positive for Ab were undifferentiated cells and hDPSCs negative for Ab were differentiated cells.

Furthermore, the undifferentiated hDPSCs selected using the monoclonal antibody of the present invention (α-UPSA-1) or the hDPSCs differentiated by the method according to Example 1-2 were examined for osteocalcin (bone and dentin marker protein Osteocalcin), austenectin, alkaline phosphatase, DSPP, and DMP-1 were compared by the method according to Example 7. As a result, it was confirmed that the expression levels of the bone and dentin marker proteins were significantly increased in the differentiated hDPSCs as compared to undifferentiated hDPSCs (Fig. 8B).

From the above results, it was found that hDPSCs showing positive reaction to the monoclonal antibody UPSA-1 of the present invention are undifferentiated cells having excellent differentiation ability, hDPSCs showing negative responses are cells in differentiated state, It was found that undifferentiated or differentiated hDPSCs can be clearly distinguished and separated.

Stunning  5. hDPSCs  Detect Monoclonal antibody UPSA -One

In order to confirm whether the monoclonal antibody UPSA-1 can directly detect hDPSCs, the antibody was reacted with the dental tissues according to the method according to Example 10, and histochemical staining was performed.

As a result, it was confirmed that the antibody binds to hDPSCs and is stained brown. Thus, stem cells of the dimension can exist in undifferentiated cells adjacent to the odontoblastic layer, or exist in the vicinities of blood vessels adjacent to the blood vessels (Fig. 9).

From the above results, it can be seen that the undifferentiated or differentiated hDPSCs present in the tissue can be clearly identified by using the monoclonal antibody UPSA-1 of the present invention.

Thus, the present inventors confirmed that UPSA-1 is a marker of undifferentiated hDPSCs differentiated from hDPSCs differentiated. In the present invention, it is possible to provide a method for detecting, quantifying, isolating and differentiating hDPSCs.

From the above description, it will be understood by those skilled in the art that the present invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. In this regard, it should be understood that the above-described embodiments are to be considered in all respects as illustrative and not restrictive. The scope of the present invention should be construed as being included in the scope of the present invention without departing from the scope of the present invention as defined by the appended claims.

<110> Dankook University Cheonan Campus Industry Academic Cooperation Foundation <120> UPSA-1 as a surface marker of undifferentiated dental pulp stem          cells and a monoclonal antibody specific <130> KPA160693-KR <160> 30 <170> KoPatentin 3.0 <210> 1 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> CDR1 of mAb UPSA-1 heavy chain <400> 1 Lys Ala Thr Gly Tyr Thr Phe Ser Ser Tyr Trp Ile Glu   1 5 10 <210> 2 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> CDR2 of mAb UPSA-1 heavy chain <400> 2 Glu Ile Leu Pro Gly Ser Asp Asn Thr Asn Tyr Asn Glu Lys Phe Lys   1 5 10 15 Gly     <210> 3 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> CDR3 of mAb UPSA-1 heavy chain <400> 3 Ala Thr Thr Val Tyr Ala Met Asp Tyr   1 5 <210> 4 <211> 127 <212> PRT <213> Artificial Sequence <220> <223> mAb UPSA-1 heavy chain <400> 4 Glu Val Gln Leu Glu Glu Ser Gly Ala Glu Leu Met Lys Pro Gly Ala   1 5 10 15 Ser Val Lys Ile Ser Cys Lys Ala Thr Gly Tyr Thr Phe Ser Ser Tyr              20 25 30 Trp Ile Glu Trp Val Lys Gln Arg Pro Gly His Gly Leu Glu Trp Ile          35 40 45 Gly Glu Ile Leu Pro Gly Ser Asp Asn Thr Asn Tyr Asn Glu Lys Phe      50 55 60 Lys Gly Lys Ala Thr Phe Thr Ala Asp Thr Ser Ser Asn Thr Ala Tyr  65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys                  85 90 95 Thr Arg Ala Thr Thr Val Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr             100 105 110 Ser Val Thr Val Ser Ser Ala Lys Thr Thr Pro Pro Ser Val Tyr         115 120 125 <210> 5 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> CDR1 of mAb UPSA-1 light chain <400> 5 Ser Ala Ser Ser Ser Val Ser Tyr Met His   1 5 10 <210> 6 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> CDR2 of mAb UPSA-1 light chain <400> 6 Ile Ile Ser Asn   1 5 <210> 7 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> CDR3 of mAb UPSA-1 light chain <400> 7 His Leu Leu Leu Pro Ala Val Glu His   1 5 <210> 8 <211> 115 <212> PRT <213> Artificial Sequence <220> <223> mAb UPSA-1 light chain <400> 8 Asp Ile Gln Leu Thr Gln Ser Pro Ala Ile Leu Ser Ala Ser Pro Gly   1 5 10 15 Glu Lys Val Thr Met Thr Cys Ser Ala Ser Ser Ser Val Ser Tyr Met              20 25 30 His Trp Phe Gln Gln Lys Pro Gly Ser Ser Pro Lys Leu Trp Ile Tyr          35 40 45 Ile Ile Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Gln Trp Gln Trp      50 55 60 Val Trp Asp Leu Leu Phe Ser His Asn Gln Gln Arg Glu Gly Ala Arg  65 70 75 80 Cys Cys His Leu Leu Leu Pro Ala Val Glu His Phe Pro Thr Gly Arg                  85 90 95 Ser Val Glu Ala Pro Ser Trp Lys Ser Asn Gly Leu Met Leu His Gln             100 105 110 Leu Tyr Pro         115 <210> 9 <211> 381 <212> DNA <213> Artificial Sequence <220> <223> mAb UPSA-1 heavy chain <400> 9 gaggttcagc tggaggagtc tggagctgag ctgatgaagc ctggggcctc agtgaagata 60 tcctgcaagg ctactggcta cacattcagt agctactgga tagagtgggt aaagcagagg 120 cctggacatg gccttgagtg gattggagag attttacctg gaagtgataa tactaactac 180 aatgagaagt tcaagggcaa ggccacattc actgcagata catcctccaa cacagcctac 240 atgcaactca gcagcctgac atctgaggac tctgccgtct attactgtac aagggcgact 300 acggtctatg ctatggacta ctggggtcaa ggaacctcag tcaccgtctc ctcagccaaa 360 acgacacccc catctgtcta t 381 <210> 10 <211> 347 <212> DNA <213> Artificial Sequence <220> <223> mAb UPSA-1 light chain <400> 10 gacattcagc tgacccagtc tccagcaatc ctgtctgcat ctccagggga gaaggtcacc 60 atgacctgca gtgccagctc aagtgtaagt tacatgcact ggttccagca gaagccagga 120 tcctccccca aactctggat ttatatcata tccaacctgg cttctggagt ccctgctcgc 180 ttcagtggca gtgggtctgg gacctcttat tctctcacaa tcagcagcgt gaaggccgaa 240 gatgctgcca cttattactg ccagcagtgg agcagttccc caccggacgt tcggtggagg 300 caccaagctg gaaatcaaac gggctgatgc tgcaccaact gtatccc 347 <210> 11 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> IgG <400> 11 atagacagat gggggtgtcg ttttggc 27 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> 5 'MH1 <400> 12 sarctnmagc tgsagsagtc 20 <210> 13 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> 5'-MH2 <400> 13 sargtnmagc tgsagsagtc wgg 23 <210> 14 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> 5 'Mk <400> 14 gayattgtgm tsacmcarwc tmca 24 <210> 15 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Primer 6 <400> 15 gacattgtgc tgacccaatc tccagcttct 30 <210> 16 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Primer 7 <400> 16 gacattcagc tgacccagtc tcca 24 <210> 17 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> hGAPDH F-primer <400> 17 ggagtccact ggcgtcttca c 21 <210> 18 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> hGAPDH R-primer <400> 18 gctgatgatc ttgaggctgt tgtc 24 <210> 19 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Osteonectin F-primer <400> 19 ctgttgcctg tctctaaacc 20 <210> 20 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Osteonectin R-primer <400> 20 caccatcatc aaattctcct 20 <210> 21 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> Runx2 F-primer <400> 21 gtctcactgc ctctcact 18 <210> 22 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Runx2 R-primer <400> 22 tacacacatc tcctcccttc 20 <210> 23 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> COLI F-primer <400> 23 ggaggagagt caggaagg 18 <210> 24 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> COLI R-primer <400> 24 tcagcaacac agttacacaa 20 <210> 25 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> ALP F-primer <400> 25 cttgacctcc tcggaagaca ctc 23 <210> 26 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> ALP R-primer <400> 26 cgcccaccac cttgtagcc 19 <210> 27 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> DMP1 F-primer <400> 27 gactctcaag aagacagcaa 20 <210> 28 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> DMP1 R-primer <400> 28 gactcactca ccacctct 18 <210> 29 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> DSPP F-primer <400> 29 cagtacagga tgagttaaat gccagtg 27 <210> 30 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> DSPP R-primer <400> 30 ccattccctt ctcccttgtg acc 23

Claims (19)

A composition for detection of undifferentiated dental pulp stem cells comprising an agent capable of detecting undifferentiation-specific pulp stem cell surface antigen-1 (UPSA-1)
Said agent comprising a heavy chain CDR1 as set forth in SEQ ID NO: 1; A heavy chain CDR2 as set forth in SEQ ID NO: 2; And a heavy chain variable region comprising the heavy chain CDR3 as set forth in SEQ ID NO: 3; and a light chain CDR1 as set forth in SEQ ID NO: 5; A light chain CDR2 as set forth in SEQ ID NO: 6; And a light chain variable region comprising the light chain CDR3 as set forth in SEQ ID NO: 7.
delete delete The composition of claim 1, wherein said antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 4 and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 8.
The composition of claim 1, wherein the antibody comprises a heavy chain encoded by the nucleotide sequence of SEQ ID NO: 9 and a light chain encoded by the nucleotide sequence of SEQ ID NO: 10.
A kit for detecting the differentiation of pulp stem cells, comprising the composition of any one of claims 1, 4 and 5.
(a) contacting an agent capable of detecting undifferentiation-specific pulp stem cell surface antigen-1 (UPSA-1) on a cell or cell culture medium on Invitro; And (b) confirming whether the agent and the cell or cell culture medium are bound to each other, the method comprising:
Said agent comprising a heavy chain CDR1 as set forth in SEQ ID NO: 1; A heavy chain CDR2 as set forth in SEQ ID NO: 2; And a heavy chain variable region comprising the heavy chain CDR3 as set forth in SEQ ID NO: 3; and a light chain CDR1 as set forth in SEQ ID NO: 5; A light chain CDR2 as set forth in SEQ ID NO: 6; And a light chain variable region comprising the light chain CDR3 as set forth in SEQ ID NO: 7.
8. The method of claim 7, wherein the cell is a cell contained in a dermal tissue.
8. The method according to claim 7, wherein step b) uses immunoassay or flow cytometry.
delete 1. A composition for separating undifferentiated stem cells comprising an agent capable of specifically binding to undifferentiation-specific Pulp stem cell surface antigen-1 (UPSA-1)
Said agent comprising a heavy chain CDR1 as set forth in SEQ ID NO: 1; A heavy chain CDR2 as set forth in SEQ ID NO: 2; And a heavy chain variable region comprising the heavy chain CDR3 as set forth in SEQ ID NO: 3; and a light chain CDR1 as set forth in SEQ ID NO: 5; A light chain CDR2 as set forth in SEQ ID NO: 6; And a light chain variable region comprising the light chain CDR3 as set forth in SEQ ID NO: 7.
delete delete delete delete delete Heavy chain CDR1 as set forth in SEQ ID NO: 1 heavy chain CDR2 as set forth in SEQ ID NO: 2; And a heavy chain variable region comprising the heavy chain CDR3 as set forth in SEQ ID NO: 3; and a light chain CDR1 as set forth in SEQ ID NO: 5; A light chain CDR2 as set forth in SEQ ID NO: 6; (Undifferentiation-specific Pulp stem cell Surface Antigen-1) comprising a light chain variable region comprising the light chain CDR3 as set forth in SEQ ID NO: 7, or a fragment thereof.
The antibody or fragment thereof according to claim 17, wherein the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 4 and a light chain comprising the amino acid sequence of SEQ ID NO: 8.
The antibody or fragment thereof according to claim 17, wherein the antibody comprises a heavy chain encoded by the nucleotide sequence of SEQ ID NO: 9 and a light chain encoded by the nucleotide sequence of SEQ ID NO: 10.

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