KR101715464B1 - Monoclonal antibody specific binding undifferentiated dental pulp stem cells and uses thereof - Google Patents

Abstract

The present invention relates to a composition comprising a monoclonal antibody capable of specifically binding to undifferentiated dendritic cells, a hybridoma cell line producing the monoclonal antibody, a composition for confirming the differentiation of stem cells, including the monoclonal antibody, A kit for confirming whether the stem cells are differentiated, a method for confirming whether the stem cells are differentiated using the composition or kit, a composition for separating undifferentiated stem cells containing the monoclonal antibody, And a method for separating undifferentiated stem cells using the composition or kit. The use of the monoclonal antibody of the present invention not only confirms the undifferentiated state of the stem cell, but also can separate the stem cell of undifferentiated state, so that it can be widely used for various stem cell studies using the stem cell will be.

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a monoclonal antibody that specifically binds to undifferentiated stem cells,

The present invention relates to a monoclonal antibody that specifically binds to undifferentiated stem cells and a use thereof. More particularly, the present invention relates to a monoclonal antibody capable of specifically binding to undifferentiated dimensional cells, A composition for confirming the differentiation of mesenchymal stem cells including the above monoclonal antibody, a kit for confirming differentiation of stem cells containing the above composition, a kit for confirming the differentiation of stem cells, A composition for separating undifferentiated stem cells comprising said monoclonal antibody, a kit for separating undifferentiated stem cells comprising said composition, and a method for separating undifferentiated stem cells using said composition or kit will be.

Adult stem cells are present in various adult tissues such as skin, nervous tissue, and bone marrow. These adult stem cells are known to be more useful for cell mediated therapy and regenerative medicine applications than embryonic stem cells because they have no ethical problems to overcome and are less likely to produce cancer. In particular, mesenchymal stem cells, a type of adult stem cells, are pluripotent stem cells capable of differentiating into various cells such as osteoblast (bone), chondrocyte (cartilage), and adipocyte (fat) , It is known that it can be easily separated from various tissues such as dental tissues.

The teeth contain a population of mesenchymal stem cells with higher self-proliferating and differentiating capacity than any other tissue. Recently, because of the advantage of easily obtaining mesenchymal cells even in naturally occurring or surgically removed teeth, There is growing interest in the use of adult stem cells as a source. The cell population with the characteristics of the stem cells is divided into two groups according to the dimensions of the tooth or permanent teeth, the periodontal ligament connecting between the root and the bone, the growing root, the crown surrounding the unerupted tooth, the apical papilla, Separated from various parts, tooth stem cells are also separated from the gums. These cells express specific gene markers in vitro and ex vivo and are capable of differentiating into osteoblasts, adipocytes, chondrocytes, etc. of mesenchymal cell lines and differentiating them into cells of other lines. Dimensional stem cells isolated from human dimensions have high proliferative capacity and can differentiate into nerve cells, adipocyte dentin cells, induce bone formation after in vivo transplantation, produce dentin and cells other than teeth Has been reported to have the ability to differentiate into dental caries. Therefore, studies on the use of such dental stem cells have been actively conducted.

For example, Korean Patent Laid-Open Publication No. 2009-0033641 discloses a human dental stem cell having a mineralization and a culturing method thereof, and Korean Patent Laid-Open Publication No. 2009-0033643 discloses a new dental follicular stem stem cell and its culture Korean Patent Laid-Open Publication No. 2008-0089547 discloses a composition for hard tissue formation and dentin or dental tissue regeneration comprising enamel cells, epidermal cells or a culture thereof as an active ingredient. Open Publication No. 2014-0006323 discloses a pharmaceutical composition for preventing or treating osteoporosis, which comprises a stem cell culture liquid as an active ingredient.

However, there is a difficulty in using such a tooth stem cell as a stem cell for research or industrial use because the above-mentioned tooth stem cell is a mixture of undifferentiated stem cell and differentiated or differentiating stem cell. That is, the stem cells contained in various oral tissues such as dentate glands, permanent bodies, dentures, and periodontal tissues exhibit relatively superior differentiation ability, and therefore, the stem cells include stem cells having already undergone differentiation or completed differentiation There is a disadvantage that uniformity and uniformity are low. To overcome these disadvantages, it is necessary to distinguish differentiated tissue cells from undifferentiated stem cells from various oral tissue-derived stem cells, but no means for effectively distinguishing such stem cells has been developed.

Under these circumstances, the present inventors have made extensive efforts to develop a method for distinguishing differentiated tissue cells from undifferentiated stem cells from a variety of oral tissue-derived stem cells. As a result, they have found monoclonal antibodies specific to undifferentiated stem cells The present inventors completed the present invention by confirming that when the above monoclonal antibody is used, the differentiated tissue cells and undifferentiated stem cells can be effectively distinguished.

It is an object of the present invention to provide a monoclonal antibody capable of specifically binding to undifferentiated dimensional cells.

Another object of the present invention is to provide a hybridoma cell line which produces said monoclonal antibody.

It is still another object of the present invention to provide a composition for confirming the differentiation of stem cells comprising the above monoclonal antibody.

It is still another object of the present invention to provide a kit for confirming the differentiation of stem cells, which comprises the composition.

It is still another object of the present invention to provide a method for confirming the differentiation of pulp stem cells using the composition or kit.

It is still another object of the present invention to provide a composition for separating undifferentiated stem cells comprising the above monoclonal antibody.

It is still another object of the present invention to provide a kit for separating undifferentiated stem cells comprising the above composition.

It is still another object of the present invention to provide a method for separating undifferentiated stem cells using the composition or kit.

The present inventors cultured human dermal stem cells (hDPSCs) obtained from a patient's wisdom tooth and compared the surface markers expressed on the surface thereof with the markers of stem cells. As a result, CD44, CD90 and CD146 were detected on the surface of undifferentiated hDPSCs It was confirmed that the level of expression of CD44, CD90, CD146 and STRO-1 abruptly decreased on the surface of the differentiated hDPSCs, while the level of expression of STRO-1 was slightly increased, And expected to exhibit the same characteristics as stem cells.

Thus, the undifferentiated hDPSCs were immunized with a mouse, lymphocyte cells were obtained from the mouse, and a monoclonal antibody capable of binding to the surface antigen of the undifferentiated hDPSCs was excavated therefrom. As a result of investigating the characteristics of the excised monoclonal antibody, the monoclonal antibody exhibited IgG, IgG2b subtype, including kappa light chain, and exhibited a higher level of binding affinity to the surface of undifferentiated hDPSCs than the surface of differentiated hDPSCs Respectively. In addition, the surface antigen of undifferentiated hDPSCs that specifically binds to the monoclonal antibody has a size of about 180 kDa and is N-glycosylated, and it is confirmed that it is associated with the expression of the mesenchymal stem cell marker. In addition, it was confirmed that the undifferentiated hDPSCs and the differentiated hDPSCs can be clearly separated using the above monoclonal antibody.

In order to achieve the above object, the present invention provides monoclonal antibodies capable of specifically binding to undifferentiated sized pleural cells as one embodiment.

As an example, the monoclonal antibody provided in the present invention may comprise a heavy chain CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 1; A heavy chain CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 2; And a heavy chain variable region comprising a heavy chain CDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 3; And light chain CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 4; A light chain CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 5; And a light chain variable region comprising a light chain CDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 6.

As another example, as with conventional monoclonal antibodies, the monoclonal antibodies provided herein also consist of two full-length heavy chains and two full-length light chains.

The heavy chain is not particularly limited, but includes, for example, a heavy chain CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 1; A heavy chain CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 2; And a heavy chain variable region comprising a heavy chain CDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 3, and as another example, a polypeptide encoded by the amino acid sequence set forth in SEQ ID NO: 9 or the nucleotide sequence set forth in SEQ ID NO: 8 or 10 ≪ / RTI >

The light chain is also not particularly limited. For example, a light chain CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 4; A light chain CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 5; And a light chain variable region comprising the light chain CDR3 consisting of the amino acid sequence of SEQ ID NO: 6, and as another example, the amino acid sequence of SEQ ID NO: 11 or 13 or the nucleotide sequence of SEQ ID NO: ≪ / RTI >

As another example, the monoclonal antibodies provided in the present invention can be prepared by methods known in the art. For example, it may be prepared by a chemical peptide synthesis method, or may be prepared by amplifying the gene encoding the monoclonal antibody by polymerase chain reaction (PCR) or synthesizing it by a known method and then cloning it into an expression vector for expression , A hybridoma cell line derived from a lymphocyte obtained by injection of an immunogen of the above monoclonal antibody into an animal, or as another example, a protein derived from undifferentiated stem cells to which said monoclonal antibody is specifically bound Can be prepared using a hybridoma cell line prepared by inoculating an animal with an immunogen and using a lymphocyte obtained from the animal.

The monoclonal antibody may be a complete form having two full-length light chains and two full-length heavy chains as well as a form in which some amino acid sequences constituting them are mutated or functional fragments of antibody molecules.

In proteins and polypeptides, amino acid exchanges that do not globally alter the activity of the molecule are known in the art. The most commonly occurring exchanges involve amino acid residues Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Thy / Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / VaI, Ala / Glu and Asp / Gly. By this exchange, the monoclonal antibody of the sequence mutated from the amino acid sequence of the monoclonal antibody May be included in the scope of the present invention. In addition, a monoclonal antibody having a mutation in which the structural stability against the heat, pH, etc. of the above monoclonal antibody is increased or the activity of the antibody is increased by mutation or modification of the amino acid sequence may also be included in the scope of the present invention.

The functional fragment of the antibody molecule refers to a fragment having at least an antigen-binding function, and examples thereof may be Fab, F (ab ') 2, F (ab') ₂ and Fv.

The monoclonal antibody can bind to the stem cell, which can bind the undifferentiated stem cell with higher affinity and specificity than the differentiated stem cell. Therefore, the monoclonal antibody can be used for confirming the differentiation of the stem cells or for separating the undifferentiated stem cells from the stem cells.

The term " dental pulp stem cells (DPSCs) "of the present invention means stem cells present in the dermal tissue. The stem cells are known to have high proliferative capacity and high cell-forming ability and thus form dense calcified nodules and have the ability to differentiate into dentin cells, adipocytes, chondrocytes, and osteoblasts.

In the present invention, the DPSCs may be interpreted to mean undifferentiated DPSCs, wherein the undifferentiated DPSCs express higher levels of CD44, CD90 and CD146 than the differentiated DPSCs, and the expression level of STRO-1 The monoclonal antibody of the present invention not only confirms the differentiation of DPSCs but also can separate undifferentiated DPSCs from differentiated DPSCs.

In addition, since the monoclonal antibody of the present invention can bind to human periodontal ligament cells (hPDLCs), root fibroblasts (hGFs) and myeloid mesenchymal stem cells (BMMSCs) with high specificity in addition to the DPSCs, May be used to isolate undifferentiated stem cells contained in periodontal ligament cells (hPDLCs) or root fibroblasts (hGFs).

In another aspect, the present invention provides a hybridoma cell line producing the above monoclonal antibody.

The term "hybridoma cell line" of the present invention refers to a cell made by artificially fusing two different kinds of cells, using a substance causing cell fusion such as polyethylene glycol or a virus of any kind, Means a cell or cell line in which cells are fused to integrate different functions of different cells into one cell. In particular, hybrid myeloma cells and B cells, the precursor cells of antibody-producing cells among the lymphocytes in the spleen or lymph nodes, are used to produce monoclonal antibodies, which are widely used in research and clinical applications. In addition, T cells that produce lymphocaine (physiologically active substance) and hybrid cells that are tumor cells thereof have been put to practical use.

In the present invention, the hybridoma cell line can be interpreted as a hybridoma cell line capable of producing a monoclonal antibody capable of specifically binding to the undifferentiated dimensional cells provided in the present invention.

According to one embodiment of the present invention, human dermal stem cells (hDPSCs) obtained from patient's wisdom teeth were cultured and surface markers expressed on the surface thereof were compared with the markers of stem cells. As a result, on the surface of undifferentiated hDPSCs CD44, CD90, and CD146 were expressed at high levels and the level of expression of STRO-1 was slightly increased, while the level of expression of CD44, CD90, CD146 and STRO-1 was drastically reduced at the surface of differentiated hDPSCs. In addition, it was confirmed that CD24 and CD106, surface markers of dental apical papilla stem cells, were not expressed on the surface of undifferentiated or differentiated hDPSCs (Fig. 1). Thus, a mouse is inoculated with undifferentiated hDPSCs to immunize the lymph node, lymph node cells are obtained from the lymph node, and then the obtained lymphocyte and myeloma cells are fused to obtain hybridoma cells capable of producing various monoclonal antibodies Respectively. Among the respective monoclonal antibodies expressed from the hybridoma cells, a monoclonal antibody (α-1G9) which binds to hDPSCs that are undifferentiated more than the differentiated hDPSCs with higher affinity was selected (Example 2-1).

In another aspect, the present invention provides a composition for identifying or separating pulp stem cells comprising the monoclonal antibody.

In the present invention, the above monoclonal antibody can be used in a method for distinguishing or separating undifferentiated DPSCs by specifically binding to surface antigens expressed on the surface of undifferentiated DPSCs. Therefore, for convenience, The material may be used in a combined form. The labeling substance is not particularly limited as long as it can be used for distinguishing or separating undifferentiated DPSCs to which the desired surface antigen is expressed by binding to the monoclonal antibody. For example, ligands, beads, radionuclides, An enzyme, a substrate, a cofactor, an inhibitor, a fluorescer, a chemiluminescent material, a magnetic particle, a hapten, a dye, and the like; as another example, a ligand such as biotin, avidin or streptavidin; Enzymes such as luciferase, peroxidase, and beta-galactosidase; 6-carboxyfluorescein, VIC, JOE, 5- (2'-aminoethyl) aminonaphthalene-1, 2-carboxyfluorescein, 6-carboxyfluorescein, -Sulfonic acid, coumarin and derivatives thereof, fluorescent substances such as cyanine-5, lucifer yellow, Texas red, tetramethylrhodamine, yakima yellow, calf fluoride 610, coumarin, derivatives thereof and the like.

The term "differentiation" of the present invention means a part of the development process in which the structure and function of a cell are specialized while cells are divided and proliferated and the entire individual is grown. Specifically, It can be interpreted as a developmental process that changes to the appropriate form and function to perform the given role. For example, the process may be a process in which the pluripotent stem cells such as embryonic stem cells are transformed into ectodermal, mesodermal, or endodermal cells. In another example, DPSCs may be transformed into bone tissue or dentin tissue.

The term " osteogenic differentiation "as used herein refers to a series of physiological responses that occur in the course of animal development, in which osteoblasts or osteoclasts differentiate to form bone matrix, and the formed bone matrix is calcified to form bone It refers to the developmental process of bone formation during osteogenesis.

In the present invention, the bone differentiation can be interpreted as a development process in which DPSCs are converted into bone tissue by artificially inducing differentiation.

The term "dentinogenic differentiation" of the present invention refers to the developmental process in which extraocular cells appear during development of an animal to form dentin cells.

In the present invention, the dentin differentiation can be interpreted as a development process in which DPSCs are induced to artificially differentiate into dentin tissues.

According to another embodiment of the present invention, the monoclonal antibody (a-1G9) has IgG, IgG2b subtype, and kappa light chain (Table 2) (Fig. 3), showing a higher level of binding affinity on the surface of undifferentiated hDPSCs than on the surface of hDPSCs (Fig. 3), showing affinity for differentiated hDPSCs versus differentiated hDPSCs compared to conventional mesenchymal stem cell markers (CD44 or CD106) (Table 3) showed similar affinity to CD44 for human periodontal ligament cells (hPDLCs), root fibroblasts (hGFs) and myeloid mesenchymal stem cells (BMMSCs) The surface antigen specifically bound to the above monoclonal antibody has a size of about 180 kDa and is N-glycosylated (FIG. 5). The surface antigen bound to the above monoclonal antibody (? -1G9) Associated with the expression of cell markers (Fig. 6). In order to confirm whether the monoclonal antibody can be used for screening the undifferentiated hDPSCs, hDPSCs were distinguished from undifferentiated hDPSCs that bind strongly to the monoclonal antibody and the weakly bound hDPSCs to the monoclonal antibody, As a result of comparing the osteoclast differentiation ability of each hDPSCs, it was confirmed that the undifferentiated hDPSCs strongly binding to the monoclonal antibody exhibited a relatively high level of bone differentiation ability (Fig. 7).

In another aspect, the present invention provides a kit for identifying or separating pulp stem cells comprising the composition.

Since the composition for confirming or separating the undifferentiated DPSCs of the stem cell can specifically detect undifferentiated DPSCs, the kit for identifying or separating the undifferentiated DPSCs containing the same can be used for identifying or separating the undifferentiated DPSCs (RIA) kit, an enzyme immunoassay (ELISA) kit, an immunofluorescence kit, a chemiluminescent substance binding method kit, a protein chip kit, a chromatography kit , A magnetic activated cell sorting (MACS) kit, and the like.

In one example, the kit can be an immunofluorescence kit comprising a substrate on which the formulation of the composition is immobilized, and a secondary antibody specific for the DPSCs and conjugated with a fluorescent material.

As another example, the kit may be a chromatography kit for separating undifferentiated DPSCs comprising a bead with a formulation of the composition, a column into which the bead can be filled, and a buffer for development.

As another example, the kit may be a MACS kit comprising biotin, a magnetic bead-bound streptavidin, and a magnetic generator, wherein the formulation of the composition is combined.

According to another aspect of the present invention, there is provided a method for confirming whether a stem cell is differentiated using the composition or kit.

Specifically, the method of the present invention for determining the differentiation of stem cells comprises: (a) adding the monoclonal antibody to the separated DPSCs to obtain a reaction product; And (b) measuring the content of DPSCs bound to the monoclonal antibody.

In the above method, when the monoclonal antibody is bound to the differentiated DPSCs as a control, and the content measured in the step (b) is relatively higher than the content measured in the control, the separated DPSCs Can be judged to be undifferentiated DPSCs.

According to another aspect of the present invention, there is provided a method for separating undifferentiated stem cells using the composition or kit.

Specifically, a method of isolating undifferentiated stem cells of the present invention comprises: (a) adding the monoclonal antibody or composition to isolated DPSCs to obtain a reaction product; And (b) recovering the DPSCs bound to the monoclonal antibody.

In the above method, the DPSCs may be recovered using a method commonly used in the art. For example, flow cytometry, immunoprecipitation, autologous cell separation, chromatography, or the like can be used As another example, flow cytometry using a composition labeled with a fluorescent substance, immunoprecipitation using a composition comprising a preparation combined with gold particles, magnetic separation using a composition combined with a magnetic bead, A chromatography method using a packed column, or the like.

In the present invention, a biotin-conjugated monoclonal antibody is firstly reacted with DPSCs, streptavidin conjugated with magnetic beads (MicroBeads) is added to the reaction product, the magnetic beads are labeled on the DPSCs, ) Were used to separate undifferentiated DPSCs.

The use of the monoclonal antibody of the present invention not only confirms the undifferentiated state of the stem cell, but also can separate the stem cell of undifferentiated state, so that it can be widely used for various stem cell studies using the stem cell will be.

1 is a graph showing the results of analysis of the expression levels of various mesenchymal stem cell markers in undifferentiated or differentiated hDPSCs (human dental pulp stem cells) through flow cytometry analysis, and FIG. 1 B is a graph showing the results of flow cytometry And a graph showing the results of quantitative analysis of the analysis results.
Fig. 2 is a schematic diagram showing the flow of a method for producing a hybridoma cell producing a monoclonal antibody capable of binding to the undifferentiated hDPSCs of the present invention. Fig.
3 is a graph showing the binding affinity of a monoclonal antibody (α-1G9) to cell surface molecules of hDPSCs.
FIG. 4 is a graph showing the results of comparing the average binding affinity of the monoclonal antibody (α-1G9) provided in the present invention for undifferentiated or differentiated hDPSCs.
FIG. 5 is a Western blot analysis photograph showing the molecular weight of cell surface markers of undifferentiated hDPSCs to which the monoclonal antibody (? -1G9) provided in the present invention binds.
FIG. 6 shows the results of two-color flow cytometry analysis using the respective monoclonal antibodies against the monoclonal antibody (? -1G9) and the mesenchymal stem cell markers (CD44, CD73 or CD90) provided by the present invention in which FITC was conjugated to undifferentiated hDPSCs In the graph of FIG.
FIG. 7 is a photograph (left) and a graph (right) showing the results of comparing the bone-dividing capacities of the undifferentiated or differentiated hDPSCs selected using the monoclonal antibody (? -1G9).
FIG. 8 is a graph showing the effect of osteocalcin, austenectin, alkaline phosphatase, DSPP and DMP-1 expressed in undifferentiated hDPSCs (Undiff) and differentiated hDPSCs (Diff) selected using the monoclonal antibody (? -1G9) Lt; RTI ID = 0.0 > expression level. ≪ / RTI >

Hereinafter, the present invention will be described in more detail with reference to examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

Example  1: undifferentiated hDPSCs ( human dental pulp stem cells ) For cell surface marker analysis

We collected normal wisdom teeth discarded from adult patients between 20 and 25 years of age. In order to cultivate human dental cells, the wisdom teeth were ruptured and the dental tissues were separated from the upper and root portions. In order to cultivate periodontal ligament cells and gingival fibroblasts, dentate gyrus and protruding layer of root were collected respectively. The tissue sections were resolved in a solution containing 3 mg / ml collagenase type I and 4 mg / ml dispase at 37 ° C for 1 hour. Cell suspensions were obtained through a 70-μM strainer and inoculated into α-MEM medium containing 20% FBS and antibiotics and incubated at 37 ° C. and 5% CO ₂ . To differentiate into mineralized traits, cells were inoculated into differentiation medium (α-MEM medium containing 10% FBS, 5 mM β-glycerophosphate, 100 nM dexamethasone and 100 μM ascorbic acid) and cultured for 7-14 days to obtain hDPSCs And the undifferentiated state in the cells was confirmed by immunophenotype analysis using a normal mesenchymal stem cell marker.

At this time, the immunophenotyping analysis was performed as follows: Cells were separated using inactivated isolation buffer, and recovered and suspended in PBS containing 5% FBS. Each monoclonal antibody against various mesenchymal stem cell markers (CD44, CD90, CD146, Stro-1, CD24 and CD106) conjugated with PE (phycoerythrin) was treated with 2 x 10 ⁶ cells / ml of cells. As a negative control, anti-mouse IgG or IgM treated was used. The cells were incubated in an ice bath for 30 minutes. Flow cytometry was performed on the cells. The obtained results were quantitatively analyzed using Cell Quest and WinMDI 2.9 program (FIGS. 1A and 1B).

1 is a graph showing the results of analysis of the expression levels of various mesenchymal stem cell markers in undifferentiated or differentiated hDPSCs (human dental pulp stem cells) through flow cytometry analysis, and FIG. 1 B is a graph showing the results of flow cytometry And a graph showing the results of quantitative analysis of the analysis results. As shown in FIG. 1, CD44, CD90, CD146 and STRO-1 are expressed on the surface of differentiated hDPSCs while the level of expression of CD44, CD90 and CD146 is high and the expression level of STRO-1 is slightly increased on the surface of undifferentiated hDPSCs And the level of expression of the protein decreased sharply. In addition, it was confirmed that the surface markers of dental apical papilla stem cells, CD24 and CD106, were not expressed on the surface of undifferentiated or differentiated hDPSCs.

Example  2: hDPSCs Of cell surface molecules Monoclonal antibody  Production and characterization

Example  2-1: hDPSCs Of cell surface molecules Monoclonal antibody  production

In undifferentiated or differentiated hDPSCs at 1 x 10 < ⁶ > cells / 30 [mu] l of concentration in 10 solitary Balb / c mice on the soles of the hind paw and on the soles of the left hind paw, Immunization was induced by scanning for 8 days under one condition (Table 1).

Injection plan of undifferentiated or differentiated hDPSCs Number of injections hDPSCs 1st 2nd 3rd 4th 5th 6th 7th 8th Left foot floor
Right foot Differentiated type
Undifferentiated type injection
- injection
injection injection
injection injection
injection injection
injection injection
injection injection
injection -
injection

After induction of immunization, the lymph nodes were extracted from the left footpad area where the undifferentiated hDPSCs were injected, from which a lymphocyte suspension was obtained. The lymphocytes were fused with FO myeloma cells to prepare hybridomas , And each of the prepared hybridomas was cultured in a 96-well plate containing DMEM medium containing 20% FBS / HAT (FIG. 2).

Fig. 2 is a schematic diagram showing the flow of a method for producing a hybridoma cell producing a monoclonal antibody capable of binding to the undifferentiated hDPSCs of the present invention. Fig.

A total of 245 hybridomas were prepared according to the method described above, and a monoclonal antibody (? -1G9) that binds to hDPSCs with higher affinity than undifferentiated hDPSCs among the monoclonal antibodies produced in the respective hybridomas was selected Respectively.

Example  2-2: Monoclonal antibody Subtype analysis

Subtypes of the mAb selected in Example 2-1 were analyzed.

Specifically. Mouse IgG1, anti-mouse IgG2a, anti-mouse IgG2b, anti-mouse IgG3, anti-mouse IgM, anti-mouse IgA, anti-mouse Igκ and anti-mouse Igλ were coated on a 96 well plate, After blocking with PBS containing 10% FBS, the antigen-antibody reaction was carried out by treating the monoclonal antibody (α-1G9). The optical density of the reaction product was measured at 450 nm to measure the monoclonal antibody -1G9) were analyzed (Table 2).

Analysis of subtype signal of monoclonal antibody (α-1G9) Monoclonal antibody IgG IgM IgA κ λ G1 G2a G2b G3 α-1G9 - - + - - - + -

As shown in Table 2, it was found that the monoclonal antibody was an IgG, an IgG2b subtype, and contained a kappa light chain.

Example  2-3: hDPSCs Of cell surface molecules Monoclonal antibody  Binding affinity analysis

FITC was labeled on the monoclonal antibody (α-1G9) selected in Example 2-1, and then flow cytometry was performed on the undifferentiated or differentiated hDPSCs using the labeled monoclonal antibody, and the undifferentiated or differentiated Binding affinity for the hDPSCs (Fig. 3).

3 is a graph showing the binding affinity of a monoclonal antibody (α-1G9) to cell surface molecules of hDPSCs. As shown in FIG. 3, it was confirmed that the above monoclonal antibody (? -1G9) exhibited a higher level of binding affinity to the surface of undifferentiated hDPSCs than the surface of differentiated hDPSCs.

In order to compare the binding affinity for the undifferentiated or differentiated hDPSCs to those of conventional mesenchymal stem cell markers (CD44 or CD106), five undifferentiated or differentiated hDPSCs (Case 1-5) were individually prepared, The binding affinity of each antibody was measured using each of the prepared hDPSCs, and the average value thereof was calculated (Table 3), and the measured average values were compared (FIG. 4). FIG. 4 is a graph showing the results of comparing the average binding affinity of the monoclonal antibody (α-1G9) provided in the present invention for undifferentiated or differentiated hDPSCs.

The binding affinity of the antibody to the cell surface of hPDSCs Monoclonal antibody CD106 CD44 1G9 Undifferentiated hDPSCs
Case1
Case2
Case3
Case4
Case5 12.16
27.93
22.07
0
27.94 93.76
93.63
94.55
84.14
85.29 89.8
67.86
53.72
80.94
81.81 Average 18.02 90.27 74.83 Differentiated hDPSCs
Case1
Case2
Case3
Case4
Case5 0
0
0
0
24.17 31.32
35.81
46.59
78.05
78.12 12.82
0
22.71
63.68
59.31 Average 4.83 53.98 31.70

As shown in Table 3 and FIG. 4, the difference in binding affinity between the undifferentiated or differentiated hDPSCs measured by treating the conventional mesenchymal stem cell marker CD44 or CD106 with the monoclonal antibody was 36.29 and 13.19, respectively, Since the difference in binding affinity measured using the monoclonal antibody (α-1G9) provided by the present invention is 43.13, the monoclonal antibody (α-1G9) provided by the present invention has a higher binding affinity than the antibody against the conventional mesenchymal stem cell marker And showed improved binding affinity for undifferentiated hDPSCs.

Example  2-4: Binding Affinity Analysis by Target Organization

Since periodontal ligament and gingival tissues can be used as a source of pluripotent adult stem cells as well as dimensional cells, the potential of antigen recognized by these antibodies as appropriate markers of adult stem cells and progenitor cells Binding affinity of the monoclonal antibody (α-1G9) was analyzed in human periodontal ligament cells (hPDLCs), root fibroblasts (hGFs) and myeloid mesenchymal stem cells (BMMSCs) ). At this time, +++ is 60% or more, ++ is 30-60%, + is 30% or less, - indicates no reactivity.

Binding affinity for monoclonal antibody (α-1G9) Monoclonal antibody hGF hPDLSC hBMMSC CD44
Stro-1
1G9 +++
+++
+++ +++
+
+++ ++
++
++

As shown in Table 4, it was confirmed that CD44 binds strongly to most cell surfaces and Stro-1 strongly binds to the cell surface of hGFs but slightly binds to the surface of hPDLSCs and BMMSCs. In contrast, the monoclonal antibody (? -1G9) provided by the present invention has a comparatively high level of binding affinity similar to that of CD44.

Example  2-5: Analysis of molecular weight of antigen

Tinicamycin, an inhibitor of N-glycosylation, was not treated or treated with undifferentiated hDPSCs and cells were cultured. To bind biotin to the cell surface of the cultured hDPSCs, the cells were washed with cold PBS (pH 7.4), and 1.0 mg of Sulfo-NHS-LC was added to PBS containing 1 x 10 ⁷ cells / -Biotin was added. The biotin-bound cells were disrupted to obtain a cell lysate. Immunoprecipitation analysis was performed by adding the monoclonal antibody (? -1G9) of the present invention to the cell lysate, and a precipitate was obtained from the cell lysate. The precipitate was subjected to western blot analysis using streptavidin-conjugated HRP (FIG. 5).

FIG. 5 is a Western blot analysis photograph showing the molecular weight of cell surface markers of undifferentiated hDPSCs to which the monoclonal antibody (? -1G9) provided in the present invention binds. As shown in FIG. 5, the cell surface marker of the undifferentiated hDPSCs to which the monoclonal antibody (? -1G9) provided in the present invention was bound under the condition of untakamycin treatment showed a molecular weight of about 180 kDa, In the treated condition, it was confirmed that a band having a molecular size of about 50 kDa to 60 kDa newly appeared instead of the previously identified band having a molecular weight of about 180 kDa. Therefore, it was found that the cell surface marker of the undifferentiated hDPSCs to which the monoclonal antibody (α-1G9) provided in the present invention binds has a size of about 180 kDa and is N-glycosylated.

Example  2-6: Intermediate lobe  Stem Cells With a marker  correlation

In order to confirm whether the expression of the surface molecule recognized by the monoclonal antibody (? -1G9) provided in the present invention is associated with other mesenchymal stem cell markers, in the present invention in which FITC is combined with undifferentiated hDPSCs (Fig. 6) using monoclonal antibodies against monoclonal antibody (? -1G9) and mesenchymal stem cell markers (CD44, CD73 or CD90). CD44 mouse antibody, anti-CD90 mouse antibody and anti-CD73 mouse antibody were labeled with biotin, and streptavidin-conjugated PE (phycoerythrin) conjugated with anti- Respectively.

FIG. 6 shows the results of two-color flow cytometry analysis using monoclonal antibodies against the monoclonal antibody (? -1G9) and mesenchymal stem cell markers (CD44, CD73 or CD90) provided by the present invention in which FITC was conjugated to undifferentiated hDPSCs And FIG. As shown in FIG. 6, 79.42%, 58.64% and 75.94% of the 1G9 positive hDPSCs were co-expressed on the cell surface with CD44, CD90 and CD73, respectively.

Therefore, the surface antigen associated with the monoclonal antibody (? -1G9) provided by the present invention was analyzed to be associated with the expression of the mesenchymal stem cell marker.

Example  3: Monoclonal  The antibody (? -1 G9 ) hDPSCs of Osteogenic  prediction

Since the undifferentiated hDPSCs can be differentiated according to the subsequent differentiation process, the bone differentiation ability of the hDPSCs selected using the above monoclonal antibody (? -1G9) was compared.

First, 1 μg of biotin-conjugated monoclonal antibody (α-1G9) was added to hDPSCs (about 1 × 10 ⁶ cells), reacted at 4 ° C. for 1 hour, washed, treated with Streptavidin MicroBeads, And reacted at 4 캜 for 1 hour. Undifferentiated hDPSCs (Ab-positive) or differentiated hDPSCs (Ab-negative) were obtained by separating undifferentiated hDPSCs labeled with magnetic beads (MicroBeads) through a MACS column system.

Next, 70% ethanol was added to the obtained undifferentiated hDPSCs (Ab-positive) or differentiated hDPSCs (Ab-negative) for 5 minutes at room temperature to fix and then dried. Each of the dried cells was stained with 2% alizarin red S (pH 4.5), washed, and compared with the color development level. In order to quantitatively analyze the color development level, 10% acetic acid was added to the stained cells to extract the dye, neutralized by treatment with 10% ammonium hydroxide, and analyzed at 405 nm (FIG. 7).

FIG. 7 is a photograph (left) and a graph (right) showing the results of comparing the bone-dividing capacities of the undifferentiated or differentiated hDPSCs selected using the monoclonal antibody (? -1G9). As shown in FIG. 7, the undifferentiated hDPSCs (Ab-positive) selected using the monoclonal antibody (α-1G9) provided in the present invention exhibited excellent bone differentiation ability, whereas the differentiated hDPSCs Ab-negative) showed a very low level of bone differentiation potential.

Meanwhile, the undifferentiated hDPSCs (Undiff) and the differentiated hDPSCs (Diff) selected using the monoclonal antibody (? -1G9) provided in the present invention were examined for osteocalcin, osteonectin ), Alkaline phosphatase, dendin sialophosphoprotein (DSPP), and DMP-1 (Dentin matrix acidic phosphoprotein 1) (Fig. 8).

FIG. 8 is a graph showing the effect of osteocalcin, austenectin, alkaline phosphatase, DSPP and DMP-1 expressed in undifferentiated hDPSCs (Undiff) and differentiated hDPSCs (Diff) selected using the monoclonal antibody (? -1G9) Lt; RTI ID = 0.0 > expression level. ≪ / RTI > As shown in FIG. 8, osteocalcin, osteonectin, alkaline phosphatase, DSPP, and DMP (known as bone and dentin marker proteins) in differentiated hDPSCs rather than undifferentiated hDPSCs selected using the monoclonal antibody (? -1G9) -1 expression level was significantly increased. Thus, it was found that the undifferentiated hDPSCs selected using the above monoclonal antibody (? -1G9) are highly scalable stem cells that are likely to be differentiated into bone or dentin.

Therefore, it was found that the undifferentiated or differentiated hDPSCs can be clearly distinguished by using the monoclonal antibody (? -1G9) provided in the present invention.

<110> Dankook University Cheonan Campus Industry Academic Cooperation Foundation <120> Monoclonal antibody specific binding undifferentiated dental pulp          stem cells and uses thereof <130> KPA141121-KR <160> 14 <170> Kopatentin 2.0 <210> 1 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> heavy chain CDR1 <400> 1 Gly Phe Ser Leu Thr Gly Tyr Gly Val Asn   1 5 10 <210> 2 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> heavy chain CDR2 <400> 2 Met Ile Trp Ser Asp Gly Asn Thr Asp Tyr Asn Ser Ala Leu Lys Ser   1 5 10 15 <210> 3 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> heavy chain CDR3 <400> 3 Asp Gly Gly Asp Tyr Pro Trp Phe Ala Tyr   1 5 10 <210> 4 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> light chain CDR1 <400> 4 Ser Ala Ser Gln Gly Thr Asn Asn Tyr Leu Asn   1 5 10 <210> 5 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> light chain CDR2 <400> 5 Tyr Thr Ser Ser Leu His Ser   1 5 <210> 6 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> light chain CDR3 <400> 6 Gln Gln Tyr Ser Lys Leu Pro Trp Thr   1 5 <210> 7 <211> 116 <212> PRT <213> Artificial Sequence <220> <223> heavy chain 1 <400> 7 Asp Ile Val Met Thr Gln Ser Ser Ser Ser Ser Ser Ser Glu   1 5 10 15 Asp Arg Val Thr Ile Ser Cys Ser Ala Ser Gln Gly Thr Asn Asn Tyr              20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile          35 40 45 Tyr Tyr Thr Ser Ser Leu His Ser Gly Val Ser Ser Arg Phe Ser Gly      50 55 60 Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Pro  65 70 75 80 Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Lys Leu Pro Trp                  85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Ala Asp Ala Ala             100 105 110 Pro Thr Val Ser         115 <210> 8 <211> 393 <212> DNA <213> Artificial Sequence <220> <223> heavy chain 1 <400> 8 gaggttcagc tgcaggagtc aggacctggc ctggtggcgc cctcacagag cctgtccatc 60 acatgcaccg tctcagggtt ctcattaacc ggctatggtg taaactgggt tcgccagcct 120 ccaggagagg gtctggagtg gctgggaatg atatggagtg atggaaacac agactataat 180 tctgctctca aatccagact gagcatcagc aaggacaact ccaagagcca agttttctta 240 aaaatgaaca gtctgcaaac tgatgacaca gccaggtatt attgtgccag agatgggggt 300 gactacccct ggtttgctta ctggggccaa gggactctgg tcactgtctc tgcagccaaa 360 acaacacccc catcagtcta tccactggcc cct 393 <210> 9 <211> 131 <212> PRT <213> Artificial Sequence <220> <223> heavy chain 6 <400> 9 Glu Val Lys Leu Gln Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln   1 5 10 15 Ser Leu Ser Ile Thr Cys Thr Val Ser Gly Phe Ser Leu Thr Gly Tyr              20 25 30 Gly Val Asn Trp Val Arg Gln Pro Pro Gly Glu Gly Leu Glu Trp Leu          35 40 45 Gly Met Ile Trp Ser Asp Gly Asn Thr Asp Tyr Asn Ser Ala Leu Lys      50 55 60 Ser Arg Leu Ser Ile Ser Lys Asp Asn Ser Ser Ser Ser Gln Val Phe Leu  65 70 75 80 Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Arg Tyr Tyr Cys Ala                  85 90 95 Arg Asp Gly Gly Asp Tyr Pro Trp Phe Ala Tyr Trp Gly Gln Gly Thr             100 105 110 Leu Val Thr Val Ser Ala Ala Lys Thr Thr Pro Pro Ser Val Tyr Pro         115 120 125 Leu Ala Pro     130 <210> 10 <211> 393 <212> DNA <213> Artificial Sequence <220> <223> heavy chain 6 <400> 10 gaggtacagc tgcaggagtc tggacctggc ctggtggcgc cctcacagag cctgtccatc 60 acatgcaccg tctcagggtt ctcattaacc ggctatggtg taaactgggt tcgccagcct 120 ccaggagagg gtctggagtg gctgggaatg atatggagtg atggaaacac agactataat 180 tctgctctca aatccagact gagcatcagc aaggacaact ccaagagcca agttttctta 240 aaaatgaaca gtctgcaaac tgatgacaca gccaggtatt attgtgccag agatgggggt 300 gactacccct ggtttgctta ctggggccaa gggactctgg tcactgtctc tgcagccaaa 360 acaacacccc catcagtcta tccactggcc cct 393 <210> 11 <211> 116 <212> PRT <213> Artificial Sequence <220> <223> light chain 1 <400> 11 Asp Ile Val Met Thr Gln Ser Ser Ser Ser Ser Ser Ser Glu   1 5 10 15 Asp Arg Val Thr Ile Ser Cys Ser Ala Ser Gln Gly Thr Asn Asn Tyr              20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile          35 40 45 Tyr Tyr Thr Ser Ser Leu His Ser Gly Val Ser Ser Arg Phe Ser Gly      50 55 60 Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Pro  65 70 75 80 Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Lys Leu Pro Trp                  85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Ala Asp Ala Ala             100 105 110 Pro Thr Val Ser         115 <210> 12 <211> 348 <212> DNA <213> Artificial Sequence <220> <223> light chain 1 <400> 12 gacattcagc tgacccagtc tccatcctcc ctgtctgcct ctctgggaga cagagtcacc 60 atcagttgca gtgcaagtca gggcactaat aattatttaa actggtatca gcagaaacca 120 gatggaactg ttaaactcct gatctattac acatcaagtt tacattcagg agtcccatca 180 aggttcagtg gcagtgggtc tgggacagat tattctctca ccatcagcaa cctggaacct 240 gaagatattg ccacttacta ttgtcagcag tatagtaagc ttccgtggac gttcggtgga 300 ggcaccaagt tggagatcaa acgggctgat gctgcaccaa ctgtatcc 348 <210> 13 <211> 116 <212> PRT <213> Artificial Sequence <220> <223> light chain 6 <400> 13 Asp Ile Gln Leu Thr Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser G   1 5 10 15 Asp Arg Val Thr Ile Ser Cys Ser Ala Ser Gln Gly Thr Asn Asn Tyr              20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile          35 40 45 Tyr Tyr Thr Ser Ser Leu His Ser Gly Val Ser Ser Arg Phe Ser Gly      50 55 60 Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Pro  65 70 75 80 Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Lys Leu Pro Trp                  85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Ala Asp Ala Ala             100 105 110 Pro Thr Val Ser         115 <210> 14 <211> 348 <212> DNA <213> Artificial Sequence <220> <223> light chain 6 <400> 14 gatattgtga tgacacagtc tccatcctcc ctgtctgcct ctctgggaga cagagtcacc 60 atcagttgca gtgcaagtca gggcactaat aattatttaa actggtatca gcagaaacca 120 gatggaactg ttaaactcct gatctattac acatcaagtt tacattcagg agtcccatca 180 aggttcagtg gcagtgggtc tgggacagat tattctctca ccatcagcaa cctggaacct 240 gaagatattg ccacttacta ttgtcagcag tatagtaagc ttccgtggac gttcggtgga 300 ggcaccaagt tggagatcaa acgggctgat gctgcaccaa ctgtatcc 348

Claims (15)

A heavy chain CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 1; A heavy chain CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 2; And a heavy chain variable region comprising a heavy chain CDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 3; And light chain CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 4; A light chain CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 5; And a light chain variable region comprising a light chain CDR3 consisting of the amino acid sequence set forth in SEQ ID NO: 6.
The method according to claim 1,
A heavy chain composed of the amino acid sequence shown in SEQ ID NO: 9, and a light chain composed of the amino acid sequence shown in SEQ ID NO: 11 or 13. [
The method according to claim 1,
A light chain consisting of a heavy chain composed of a polypeptide encoded by the nucleic acid sequence set forth in SEQ ID NO: 8 or 10 and a light chain comprised of a polypeptide encoded by the nucleic acid sequence set forth in SEQ ID NO: 12 or SEQ ID NO: 14.
The method according to claim 1,
A monoclonal antibody that specifically binds to undifferentiated stem cells.
A hybridoma cell line producing the monoclonal antibody of any one of claims 1 to 4.
A composition for confirming the differentiation of stem cells, which comprises the monoclonal antibody of any one of claims 1 to 4.
A kit for confirming whether a stem cell differentiated according to claim 6 is contained.
8. The method of claim 7,
The kit may be used in a kit such as a radioimmunoassay (RIA) kit, an enzyme immunoassay (ELISA) kit, an immunofluorescence kit, a chemiluminescent substance binding kit, a protein chip kit, a chromatography kit, or a magnetic activated cell sorting Kit.
(a) adding the monoclonal antibody of any one of claims 1 to 4 to isolated stem cells to obtain a reaction product; And
(b) measuring the content of the stem cell bound to the monoclonal antibody.
10. The method of claim 9,
When the above-mentioned monoclonal antibody is bound to the differentiated stem cells and the content measured in the step (b) is relatively higher than the content measured in the control group, the separated stem cells Wherein the cell is determined to be an undifferentiated stem cell.
5. A composition for the detachment of undifferentiated stem cells comprising the monoclonal antibody of any one of claims 1 to 4.
A kit for separating undifferentiated stem cells comprising the composition of claim 11.
13. The method of claim 12,
The kit may be used in a kit such as a radioimmunoassay (RIA) kit, an enzyme immunoassay (ELISA) kit, an immunofluorescence kit, a chemiluminescent substance binding kit, a protein chip kit, a chromatography kit, or a magnetic activated cell sorting Kit.
(a) adding the monoclonal antibody of any one of claims 1 to 4 to isolated stem cells to obtain a reaction product; And
(b) recovering the stem cells bound to the monoclonal antibody.
15. The method of claim 14,
Wherein the step (b) is carried out by flow cytometry, immunoprecipitation, autologous cell separation or chromatography.

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