KR101092916B1 - Biomarker for purification or identification of mesenchymal stem cells and methods of purification of mesenchymal stem cells by using it - Google Patents

Abstract

The present invention relates to the identification of mesenchymal stem cells (aka mesenchymal stromal cells) isolation biomarker and method for isolating mesenchymal stem cells using the same, comprising an anti-fibroblast activation protein α ( FAPα ) antibody. It provides a composition for separating or identifying mesenchymal stem cells, characterized in that it provides a mesenchymal stem cell separation apparatus and identification kit comprising an anti-FAPα antibody. The present invention also provides a method for identifying and separating and purifying mesenchymal stem cells. Specifically, contacting the anti-FAPα antibody with a sample cell; Providing a method for identifying mesenchymal stem cells, characterized in that it comprises the step of confirming the antigen-antibody complex formation of the FAPα and the anti-FAPα antibody of the sample cells, the liquid biological medium containing mesenchymal stem cells Contacting with an anti-FAPα antibody to form an antigen-antibody complex; And it provides a method for separating and purifying mesenchymal stem cells comprising the step of recovering the antigen-antibody complex.

Mesenchymal stem cells (MSCs), fibroblast active protein α (FAPα)

Description

Biomarker for purification or identification of mesenchymal stem cells and methods of purification of mesenchymal stem cells by using it}

The present invention is a fibroblast activation protein α ( FAP α ) which is a surface protein marker for identifying and isolating mesenchymal stem cells from human bone marrow, peripheral blood, cord blood and adipose tissue and mesenchymal stem cells using the same. To a separation method.

Mesenchymal stem cells ( MSCs ), also commonly called mesenchymal stromal cells [1], are present in bone marrow ( BM ) but migrate signals to maintain connective tissue homeostasis. In response to release into the circulation. Recently, mesenchymal stem cells have received widespread interest in the field of cellular and immunotherapy because of their versatility, including tissue regeneration, hematopoietic support, and immunomodulation [2-4]. However, rapid development into clinically useful products has been delayed due to the lack of critical markers. Although glycolipid markers such as SSEA-4 [5] and GD2 [6] are known in bone marrow-derived mesenchymal stem cells, no single protein marker has been developed that distinguishes mesenchymal stem cells from other cell types. Did. Therefore, in order to clarify human mesenchymal stem cells, it has conventionally been necessary to analyze a number of surface protein markers. International Association for cell therapy (International Society for Cellular Therapy) are CD14 ^- (or CD11b ^-) CD19 ^- (or ^{CD79α -) CD34 - CD45 - CD73} + CD90 + CD105 + HLA-DR + combination appropriate proposed that 7 of ].

Differential gene expression profiling based on DNA microarray was performed by the present inventors to reveal specific molecular signals of human mesenchymal stem cells [8, 9]. This document reports a number of genes whose expression is preferred in mesenchymal stem cells, unlike peripheral blood-derived mononuclear cells. However, there has been no report that the fibroblast activating protein α is specifically expressed in mesenchymal stem cells so that the mesenchymal stem cells can be isolated and identified.

International patent application PCT / JP2004 / 002457 (International Application Date February 27, 2004) provides a marker for detecting, separating and identifying mesenchymal stem cells, and a method for detecting, separating and identifying mesenchymal stem cells using the marker. do. In addition, the probe for detecting the mesenchymal stem cell marker gene and the mesenchymal stem cell gene marker, a PCR primer for amplifying the gene of the test cell, and the polypeptide expressing the mesenchymal stem cell marker gene A polypeptide marker for detecting mesenchymal stem cells, the antibody specifically binding to the polypeptide marker for detecting the polypeptide marker, a probe for detecting the mesenchymal stem cell marker gene, and an antibody specifically binding to the polypeptide marker Disclosed is a method for identifying and isolating used mesenchymal stem cells. However, there is no mention of FAPα as a marker for isolating and identifying mesenchymal stem cells.

We have known that fibroblast activation protein α ( FAP α ), also called seprase, encodes an essential membrane protein with a large spherical ectodomain [10]. It is known that its activity is inhibited [11-14], and it has good properties as a mesenchymal stem cell-specific marker. Further studies on this have been performed to isolate and identify mesenchymal stem cells from human bone marrow The present invention has been completed confirming that FAPα can be used as a surface protein marker.

An object of the present invention is to provide a method for separating and identifying mesenchymal stem cells from human bone marrow, cord blood, peripheral blood and adipose tissue.

It is also an object of the present invention to provide a composition, device or kit that can specifically identify and identify mesenchymal stem cells.

In order to achieve the above object, the present invention provides a composition for separating or identifying mesenchymal stem cells, comprising an anti-fibroblast activation protein α ( FAP α ) antibody. The antibody may be a monoclonal antibody, a polyclonal antibody or a single chain antibody or a recombinant antibody. Ligands, magnetic beads, enzymes, and the like may be bound to the antibody to facilitate the binding of the antibody to FAPα specific for mesenchymal stem cells. The ligand may be, but is not limited to, biotin, avidin or streptoavidin, and the enzyme may be, but is not limited to, luciferase, peroxidase or beta galactosidase. Those skilled in the art can select and use appropriate ligands, beads, and enzymes for the identification of antibodies.

  The present invention provides an apparatus for separating mesenchymal stem cells comprising an anti-FAPα antibody. In order to separate the complex of the anti-FAPα antibody and FAPα it can be separated by using an antibody and a cell separator to which the fluorescent material is bound, and can be separated by using a magnetic device with an antibody connected with magnetic beads, specific ligand binding and It can be separated using a column for separating it.

The present invention provides a mesenchymal stem cell identification kit comprising an anti-FAPα antibody. In order to identify the complex of the anti-FAPα antibody and FAPα can be confirmed by using an antibody and a fluorescence spectrometer combined with a fluorescent material, it can be confirmed by using an antibody and a magnetic device connected with magnetic beads, specific enzyme and substrate reaction You can check through The identification kit may be specifically a protein chip for identifying mesenchymal stem cells including an anti-FAPα antibody.

The present invention also provides a method of contacting an anti-FAPα antibody with sample cells; It provides a method for identifying mesenchymal stem cells comprising the step of confirming the antigen-antibody complex formation of the FAPα and the FAPα antibody of the sample cells. The antigen-antibody complex formation may be determined by using mesenchymal stem cells by radioimmunoassay (RIA), enzyme immunoassay (ELISA), immunofluorescence, chromosomal particle binding, or chemiluminescent binding.

The present invention also comprises the steps of contacting a liquid biological medium containing mesenchymal stem cells with an anti-FAPα antibody to form an antigen-antibody complex; And it provides a method for separating and purifying mesenchymal stem cells comprising the step of recovering the antigen-antibody complex. The antigen-antibody complex recovery step may be recovered using any one ligand selected from the group consisting of biotin, avidin, and streptavidin, or may be recovered using magnetic beads and magnetic devices.

A first aspect of the invention relates to a composition for isolating or identifying mesenchymal stem cells comprising an anti-FAPα antibody.

In the present invention, "fibroblast activation protein α ( FAP α )" is a cell membrane endogenous gelatinase of homodimers belonging to the serine protease family. This protein is thought to be involved in epithelial-mesenchymal interaction or growth of fibroblasts during development, tissue repair, and cancer of epithelial cells. The sequence for these genes is known as NCBI Accession No. NM_004460, and the sequence for this protein is known as NCBI Accession Nos. NP_004451, AAH26250 and the like.

As used herein, an "antibody" includes a molecule having at least one antigen binding site formed by a variable region of the H chain or L chain, or a combination thereof, in addition to the antibody in the form normally present in vivo. For example, a peptide comprising only the variable region of the H chain, a Fab consisting of one set of H chain fragments and an L chain fragment, (Fab ') ₂ consisting of two sets of H chain fragments and an L chain fragment, an H chain fragment and an L chain Single chain antibody “ScFv” and the like, in which the fragments are bound in series on the same peptide, are also included.

The antibody may be a monoclonal antibody, a polyclonal antibody or a single chain antibody or a recombinant antibody. Ligands, magnetic beads, enzymes, and the like may be bound to the antibody to facilitate confirmation of binding of the antibody to FAPα specific to the mesenchymal stem cells. The ligand may be, but is not limited to, biotin, avidin or streptoavidin, and the enzyme may be, but is not limited to, luciferase, peroxidase or beta galactosidase. Those skilled in the art can select and use appropriate ligands, beads, and enzymes for the identification of antibodies.

A second aspect of the present invention relates to a mesenchymal stem cell separation apparatus and an identification kit using an anti-FAPα antibody.

 Mesenchymal stem cell separation apparatus comprising an anti-FAPα antibody of the present invention can be separated by using an antibody and cell separator to which the fluorescent material is bound to separate the complex of the anti-FAPα antibody and FAPα. The magnetic beads may be separated using an antibody and a magnetic device to which magnetic beads are connected, and may be separated using a specific ligand bond and a column for separating the same.

Mesenchymal stem cell identification kit, characterized in that it comprises an anti-FAPα antibody of the present invention can be identified using an antibody and a fluorescence spectrometer combined with a fluorescent material to identify the complex of the anti-FAPα antibody and FAPα The magnetic beads may be identified using an antibody and a magnetic device to which magnetic beads are connected, and may be identified through a specific enzyme and substrate reaction. The identification kit may be specifically a protein chip for identifying mesenchymal stem cells including an anti-FAPα antibody.

Examples of the container shape used in the present invention include a column filled with a water insoluble carrier, a flask type filled with a water insoluble carrier, or a flask type case. The FAPα positive cell separation apparatus can be manufactured by combining such a container shape with an insoluble carrier in which antibodies are directly or indirectly immobilized. In addition, these devices can be used as a cell separation system by combining with a pump that flows cell suspension, saline, and the like.

When the antibody is immobilized on a water insoluble carrier, it is also useful to bind the antibody and the water insoluble carrier through a spacer. In addition, if necessary, the water-insoluble carrier and the compound may be bonded to each other through molecules (spacers) of any length. For details regarding the spacer, see, for example, "Affinity Chromatography" K. Kasai et al., Tokyo Kagaku Dojin Publishing (1991) p. 105-108. Examples of the spacers include polymethylene chains and polyethylene glycol chains. It is preferable that it is 500 kPa or less, and, as for the length of a spacer, it is more preferable that it is 200 kPa or less. As a method of bonding the compound to the water-insoluble carrier through a spacer, for example, when agarose is used as the water-insoluble carrier, the hydroxyl group of the agarose and one isocyanate group of the hexamethylene diisocyanate used as the spacer are reacted and bound. And a method of reacting the remaining isocyanate group with the amino group, hydroxyl group or carboxyl group of the antibody and the like and bonding them.

The water-insoluble carrier referred to in the present invention refers to a solid state in an aqueous solution at room temperature, and may be in any shape. Examples of the shape include spherical, cubic, flat, chip, fibrous, flat film, sponge, hollow fiber, and the like. Among them, it is preferable that the fine packing is easy, the antibody can be kept relatively homogeneous on the surface, the effective effective area can be secured relatively high, and the spherical, particulate and fibrous ones are preferable in terms of fluidity of the cell suspension.

The material of the water-insoluble carrier may be an inorganic compound or an organic compound as long as the antibody can be retained on its surface. However, an organic polymer compound is preferable in that the amount of the eluent is small when contacting the cell suspension and the shape control is easier. Examples thereof include polymers and copolymers of vinyl-based compounds or derivatives thereof such as polypropylene, polystyrene, polymethacrylate ester, polyacrylate ester, polyacrylic acid and polyvinyl alcohol; Polyamide compounds such as nylon 6 or nylon 66; Polyester-based compounds such as polyethylene terephthalate; And plant-derived polysaccharide compounds such as cellulose. Moreover, these water-insoluble carriers can also be easily recovered by combining these with magnets.

Modification of the water-insoluble carrier and imparting hydrophilicity to the surface are also preferably used in the present invention. Hydrophilicity can be imparted to the surface of the water-insoluble carrier by immobilizing proteins derived from living bodies such as albumin and globulin and introducing a synthetic functional group. Examples of synthetic functional groups that impart hydrophilicity include polyethyleneglycol chains, hydroxyl groups, amide groups, ether groups and ester groups of repeating units 2 to 1500. Provided that the polyethyleneglycol chain and the hydroxyl group may be bonded to each other or may be present separately. Examples of the monomer having a polyethylene glycol chain that imparts hydrophilicity include terminal methoxy methacrylates such as methoxy diethylene glycol methacrylate and methoxy triethylene glycol methacrylate; Terminal methoxy acrylates such as methoxy diethylene glycol acrylate and methoxy triethylene glycol acrylate; And methacrylates and acrylates having terminal hydroxyl groups bonded to hydrogen atoms instead of methyl groups; Or the whole monomer which has a polyethyleneglycol chain of repeating units 2-1500 which has one or more polymerizable functional groups at the terminal is mentioned. In addition, when directly bonded to a carrier, polyethylene glycol derivatives of repeating units 2 to 1500 are also included. Examples of the monomer having other substituents that impart hydrophilicity include vinylpyrrolidone and the like, but are not limited thereto. In addition, one or more polymerizable functional groups may be present. The polymerizable functional group represents a functional group capable of polymerizing alone or by two or more functional groups, and examples thereof include carbon-carbon multiple bonds such as vinyl groups, acetylene groups, and diene groups, and ring structures such as epoxy groups and oxetane groups. Although it is mentioned, it is not limited to this.

Moreover, nonionic functional group may exist with the said functional group, For example, Nonionic functional group, Especially, Amide group, such as a dimethylamide group, diethylamide group, diisopropylamide group for the purpose of improving hydrophilicity; Polyester chains such as aromatic polyester chains such as polyethylene terephthalate chain and polybutylene terephthalate chain, and aliphatic polyester chains; Polyether chains such as methylene glycol chains and propylene glycol chains; Nonionic hydrophilic functional groups, such as a polycarbonate chain, or all nonionic functional groups, such as an alkyl chain, an alkyl fluoride chain, and an allyl chain, for the purpose of hydrophobic provision are included. Although any of the above functional groups may coexist, preferably having a nonionic hydrophilic functional group provides excellent results.

Methods for modifying the water-insoluble carrier and imparting hydrophilicity to the surface thereof include graft methods using covalent bonds, ionic bonds, radiation, or plasma; Physical adsorption; Embedding; Or all known methods such as insolubilization of the precipitate to the substrate surface. Therefore, surface modification is carried out by known methods such as graft polymerization of polymer compounds or monomers thereof, or formation of covalent bonds using radiation or plasma, etc. (Japanese Patent Laid-Open No. 1-249063, Japanese Patent Publication (Hi) 3-502094) is preferably used in the present invention.

As a method of immobilizing the antibody on the surface of the water-insoluble carrier, a method of covalently binding the antibody to the surface of the water-insoluble carrier by chemical means or graft using radiation or electron beam, or by a functional means on the surface of the water-insoluble carrier Through covalent bonding. Of these, the covalent binding method through the functional group is preferable because there is no risk of antibody elution at the time of use. When the water insoluble carrier has a coating layer, the antibody may be insolubilized on the surface of the coating layer.

As an example of the method of obtaining the active group for antibody immobilization on the surface of a water-insoluble carrier or its coating layer, there are cyanide halogenation method, epichlorohydrin method, bisepoxide method, bromoacetylbromide method, halogenated acetamide method and the like. Specifically, an amino group, a carboxyl group, a hydroxyl group, a thiol group, an acid anhydride, a succinylamide group, a substituted halogen group, an aldehyde group, an epoxy group, a trisil group, etc. are mentioned. The bromocyan method, the N-hydrosuccinimide group, and the halogenated acetamide method are especially preferable from the viewpoint of the ease of antibody immobilization.

As the haloacetaminomethylating agent used in the activator, N-hydroxymethylchloroacetamide, N-hydroxymethylfluoroacetamide, N-hydroxymethylbromoacetamide, N-hydroxymethyliodoacetamide, etc. It can be used advantageously. Among them, preferably N-hydroxymethylbromoacetamide, N-hydroxymethyliodoacetamide or the like can be advantageously used in view of economy and stability. The fluoro group or chloro group introduced to the water insoluble carrier can be easily converted to an iodine group or bromine group by introducing an active group and then treating with potassium iodide or potassium bromide solution. The acid catalyst used in the preparation of the water-insoluble carrier incorporating these active groups may be any strong acid, especially protonic acid, but non-limiting examples include sulfonic acid derivatives such as trifluoromethane, methane, benzene and toluene; And Friedel-Crafts catalysts such as sulfuric acid, zinc chloride, aluminum chloride, tin tetrachloride and the like can be advantageously used.

A third aspect of the present invention relates to a method for identifying and identifying mesenchymal stem cells using an anti-FAPα antibody. More specifically, contacting the anti-FAPα antibody with the sample cell; It provides a method for identifying mesenchymal stem cells comprising the step of confirming the antigen-antibody complex formation of the FAPα and the anti-FAPα antibody of the sample cells.

The antigen-antibody complex formation method is an immunological assay (immunoassay) using the antigen antibody response. Among the immunoassays, the most common measurement method is enzyme immunoassay (ELISA). Examples of the ELISA method include radioimmunoassay (RIA), fluorescent immunoassay (FIA), enzyme immunoassay (EIA, ELISA), color particle binding method, chemiluminescence binding method, and the like. Known.

In another aspect, the present invention comprises the steps of contacting a liquid biological medium containing mesenchymal stem cells with an anti-FAPα antibody to form an antigen-antibody complex; And it provides a method for separating and purifying mesenchymal stem cells comprising the step of recovering the antigen-antibody complex. The antigen-antibody complex recovery step may be recovered using any one ligand selected from the group consisting of biotin, avidin, and streptavidin, or may be recovered using magnetic beads and magnetic devices.

As a method of specifically separating mesenchymal stem cells using an antibody, a method of labeling them with a fluorescent antibody to separate them with a cell sorter or affinity for FAPα specific for mesenchymal stem cells on a water-insoluble carrier Immobilized anti-FAPα antibody and a method of directly or indirectly binding mesenchymal stem cells, separation by an immunosorbent column, and separation using immunomagnetic beads.

Separation of cells labeled with a fluorescent antibody is first incubated the cell mixture with a fluorescently labeled monoclonal antibody that recognizes the membrane antigen expressed on the target cells, and then treated with a laser sorter (cell sorter) on the treated cells. Is a method of separating the cells to which the fluorescent antibody is bound by emitting only the cells to which the antibody is bound.

The method of immobilizing a monoclonal antibody on a water insoluble carrier is a cell separation method in which the antigen-positive cells are directly or indirectly bound to a monoclonal antibody immobilized on a carrier or device.

International Patent Publication No. W0 87-04628 discloses the use of monoclonal antibodies directed against antigens present on the membrane surface of a target cell directly on the surface of the separation device, and the method of first binding the cell mixture to the membrane antigen on the target cell. A method of incubating with a monoclonal antibody and then treating in a cell separation device immobilized with a ligand such as an anti-immunoglobulin antibody that binds to an antibody on the cell surface is described. In these methods separation occurs on a plastic chalet to which the antibody is immobilized, the cell mixture is poured onto a plastic chalet to which the initial antibody is immobilized, and the antibody is incubated to bind the membrane antigen on the target cell. After incubation, the plastic chalet is washed to remove unbound cells and separated.

The immunosorbent column method is to immobilize a ligand such as an antibody against a membrane antigen on a target cell to the bead surface, and charge the column to perform cell separation therefrom.

International Patent Publication No. W0 91-16116 discloses a cell-antibody-biotin bond by incubating by adding a biotin-labeled antibody to a membrane antigen on a target cell in a cell mixture to immobilize avidin on a porous acrylamide gel. A method of binding and separating target cells into a column using strong binding of biotin-avidin is described by passing the prepared beads through a packed column.

In the immunomagnetic bead method, the cell mixture is first incubated with the magnetic beads to which the antibody is bound to label target cells with magnetic beads. After labeling, labeled cells are separated from unlabeled cells using a magnetic device.

For example, a cell may be prepared by binding a biotin or magnetic beads to an antibody to be immobilized on the cell in advance, and then reacting the biotin or magnetic beads with a water-insoluble carrier including or binding to a substance that is easy to bind, such as avidin or a magnet. The combined water insoluble carrier can be easily recovered. In addition, only target cells can be selectively isolated by reacting the target hematopoietic undifferentiated cells with the fibroblast active protein α antibody and then reacting with the water-insoluble carrier to which the anti-immunoglobulin antibody is bound and washing or recovering the water-insoluble carrier. . If an antibody immobilized on a magnetic bead that is a water-insoluble carrier is used, it can be separated more efficiently by a magnet or the like. Examples of the container shape used in the present invention include a column filled with a water insoluble carrier, a flask type filled with a water insoluble carrier, or a flask type case. The hematopoietic undifferentiated cell separation apparatus can be manufactured by combining such a container shape and the insoluble carrier which directly or indirectly solidified the antibody. In addition, these devices can be used as a cell separation system by combining with a pump that flows cell suspension, saline, and the like.

Hereinafter, the present invention will be described in more detail with reference to Examples.

However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

< Example  1> Bone Marrow-derived Mesenchymal Stem Cells on FAP Confirmation of Selective Expression of α

(1) cell preparation

Cryopreserved human bone marrow and peripheral blood derived monocytes (MNCs) were purchased from Lonza Inc. (Allendale, NJ). Umbilical cord blood and adipose tissue were purchased from a local hospital. Umbilical cord blood was centrifuged at just 200 xg for 10 minutes to give mononuclear cells in the buffy coat layer, and adipose tissue was subjected to collagenase I (Sigma, St.). Louis, MO) was treated for about 1 hour at room temperature, and obtained mononuclear cells of the stromal vascular fraction by centrifugation at 200 xg for 10 minutes and used in this experiment. All human cancer cell lines were obtained from Korean Cell Line Bank, Seoul, Korea.

(2) RT-PCR Analysis

Panels of human peripheral blood (PB) fraction multiple tissue cDNA (MTC) were obtained from Clonetech Laboratories Inc. Mountain View, CA. Total RNA was extracted from all other cell samples using Trizol reagent and reverse transcribed into cDNA. Primers for FAPα amplification were 5'-CAA GTG GCA AGT GGGAGG-3 '(SEQ ID NO: 1) in the forward direction and 5'-GGT TTT CAG ATT CTG ATA-3' (SEQ ID NO: 2) in the reverse direction, and amplified PCR products. Was 1152 bp. Primers for β-actin amplification were 5'-AGA AAA TCT GGC ACC ACA CC-3 '(SEQ ID NO: 3) in the forward direction and 5'-CCA TCT CTT GCT CGA AGT CC-3' (SEQ ID NO: 4) in the reverse direction. The amplified PCR product was 410 bp.

(3) flow cytometry

All assays were performed using mouse monoclonal antibodies against FAPα, CD74, CD90 and CD105 (Santa Cruz Biotechnology Inc., Santa Cruz, Calif.) And Alexa488 conjugated goat anti-mouse IgG, Invitrogen, Carlsbad. , CA) using a Beckman Coulter Epic XL flow cytometer (Miami, FL).

(4) results

Before starting the analysis of FAPα expression in various human cells, the present inventors carefully examined the membrane proteomic results of human mesenchymal stem cells [15]. FAPα was found to be one of the ectodomain containing membrane binding proteins found with high sequence coverage ( FIG. 1 ). This fact has made this type II membrane protein the most important surface protein marker candidate.

First, to find out whether FAPα is a selective marker for bone marrow mesenchymal stem cells, FAPα expression was compared between bone marrow-derived mesenchymal stem cells and monocytes using RT-PCR. Fortunately, FAPα was highly expressed in bone marrow-derived mesenchymal stem cells but not monocytes ( FIG. 2 ). In addition, flow cytometry revealed that FAPα was strongly expressed on the surface of mesenchymal stem cells but insignificant in monocytes ( FIG. 3 ). This suggests that mesenchymal stem cells are the only FAPα expressing cell population in the bone marrow stroma.

Furthermore, RT-PCR analysis was performed to investigate FAPα expression in other human blood cells. Multiple tissue cDNA panel screening revealed that FAPα was not expressed in dormant state but also in active CD8 + (active T lymphocytes), CD4 + (adjuvant T lymphocytes), CD14 + (myeloid cells) or CD19 + (B lymphocytes) blood fractions . 4 ). This indicates that FAPα expressing cells of human blood flow do not belong to these hematopoietic cells.

Next, whether FAPα expression occurs specifically in bone marrow-derived mesenchymal stem cells or in other groups of mesenchymal stem cells derived from umbilical cord blood ( UCB ) or adipose tissue ( AT ) saw. As a result, all three mesenchymal stem cell groups expressed high levels of FAPα ( FIG. 5 ). Of course, it was not expressed in the control monocyte group ( Fig. 2, Fig. 4 and Fig. 5 ). These results indicate that the expression of FAPα is not only derived from bone marrow but also mesenchymal stem cells of other cell origins such as peripheral blood ( PB ), umbilical cord blood ( UCB ) and adipose tissue ( AT ). I found out that it can be distinguished by.

Interestingly, various human cancer cell lines such as Hek293, THP-1, Jurkat, SH-SY5Y and CaCo-2 also did not express FAPα ( FIG. 6 ). FAPα was weakly expressed in HeLa cells, but previous reports have shown that FAPα is often upregulated in HeLa cell derived epithelial cancers [12-14]. All of these results suggest that expression of FAPα is strongly inhibited in most other human cells, if not all but mesenchymal stem cells.

< Example  2> monoclonal anti- FAP using α antibody Mesenchymal stem cells  Selective separation

The present inventors have found that FAPα expressing cells from mononuclear cells extracted from cord blood and adipose tissue as well as bone marrow and peripheral blood-derived mononuclear cells cryopreserved using magnetic bead immunoselection with monoclonal anti-FAPα antibodies. Can be isolated, and cell surface antigen analysis demonstrated that the isolated cells are pure mesenchymal stem cells.

(1) Immunoselection

Approximately 25 μl (containing 10 million beads) of anti-FAPα dynabeid plate mouse lgG (Dynabeads Pan mouse IgG, Dynal Biotech, Oslo, Norway) 0.5 μg of mouse monoclonal antibody (Santa Cruz Biotechnology Inc., Santa Cruz, CA) was mixed and reacted at room temperature for 30 minutes, and then placed in 2 × 10 ⁷ mononuclear cells extracted from bone marrow, peripheral blood, cord blood and adipocytes, respectively. The reaction was carried out at 4 ° C. for 20 minutes. FAPα expressing cells attached to the beads were collected using an external magnet, washed twice with saline, and placed in a culture flask containing a basic cell culture to observe cell proliferation for 3 weeks.

(2) results

Immun selection from bone marrow, peripheral blood, umbilical cord blood and adipocytes using monoclonal anti-FAPα antibodies revealed that all unselected cells (FAPα negative cells) were morphologically heterogeneous but selected cells (FAPα). Positive cells) proliferated rapidly while maintaining a similar shape to mesenchymal stem cells, forming cell colonies consisting of thousands of cells by 2-3 weeks ( FIG. 7 ). Moreover, FAPα positive cells responded positively to CD73, CD90 and CD105, which are characteristic surface markers of mesenchymal stem cells ( FIG. 8 ). These experimental results demonstrate that the cells positively selected by the anti-FAPα antibody are mesenchymal stem cells.

 FAPα protein according to the present invention is very useful as a surface protein marker for separating and identifying mesenchymal stem cells from human bone marrow, peripheral blood, cord blood and adipocytes. Magnetic bead immunoselection with monoclonal anti-FAPα antibodies can be used to rapidly isolate mesenchymal stem cells with FAPα. Human mesenchymal stem cells can differentiate into mesodermal related tissue cells such as bone, cartilage, muscle and adipocytes, as well as their ability to support hematopoietic action by hematopoietic stem cells or to regulate immune function. It is recognized as one of these most extensive cells. Stem cells have fewer ethical and legal problems than other stem cells, have excellent self-proliferation ability, can be frozen for a long time without affecting stem cell characteristics, and have differentiation ability close to pluripotency. It has a high cellular stability after genetic engineering, and has been in the spotlight as a promising cell therapy. Therefore, FAPa, a specific marker of mesenchymal stem cells, will be used to develop mesenchymal stem cell-based cell therapy in the future by easily identifying and separating mesenchymal stem cells from human bone marrow, peripheral blood, cord blood and adipocytes. In addition to its high industrial applicability, it is expected to be used in basic cellular and molecular biology studies of mesenchymal stem cells, which can help identify key cellular signaling pathways involved in autologous proliferation and differentiation studies. .

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1 shows a tandem mass spectrometry coverage map of fibroblast activation protein α ( FAP α ) sequence. Dark italic sequences indicate the positions of the 12 sequences determined by tandem mass spectrometry (˜24% coverage). The squared sequence at the N-terminus represents a single α-helical transmembrane domain of FAPα.

Figure 2 shows the results of RT-PCR analysis showing that FAPα expression is preferred in bone marrow-derived mesenchymal stem cells compared to monocytes. BM-MSC; Bone marrow (BM) derived mesenchymal stromal cells (MSCs), BM-MNC; Bone marrow (BM) derived mononuclear cell (MNC)

Figure 3 shows the results of flow cytometry showing that FAPα is expressed exclusively in mesenchymal stem cells (grey profile) and not expressed in monocytes (white profile).

4 shows the results of multiple tissue cDNA panel screening showing no FAPα expression in human peripheral blood fractions.

5 shows the results of RT-PCR analysis showing that FAPα is strongly expressed in UCB-MSC, BM-MSC, and AT-MSC, and that FAPα is not expressed in UCB-MNC and AT cells. BM-MSCs: bone marrow derived mesenchymal stem cells; UCB-MSCs: mesenchymal stem cells derived from umbilical cord blood (UCB); AT-MSCs: mesenchymal stem cells derived from adipose tissue (AT); UCB-MNC: umbilical cord blood derived monocytes; AT: adipose tissue

6 shows the results of RT-PCR analysis showing that FAPα is not expressed in human cancer cell lines.

FIG. 7 shows the results of FAPα expressing cells isolated by bone marrow (BM), peripheral blood (PB), umbilical cord blood (UCB) and adipose tissue (AT) by immunoselection to form high-purity cell colonies after 2-3 weeks. (40 × magnification).

8 shows antigenic characteristics of the cell surface for FAPα-positive cells (grey profile) and FAPα-negative cells (white profile).

<110> Genexel-Sein Inc. <120> Biomarker for purification or identification of mesenchymal stem          cells and methods of purification of mesemchymal stem cells by          using it <150> US60969959 <151> 2007-09-05 <150> US60969960 <151> 2007-09-05 <160> 4 <170> KopatentIn 1.71 <210> 1 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 1 caagtggcaa gtgggagg 18 <210> 2 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 2 ggttttcaga ttctgata 18 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 3 agaaaatctg gcaccacacc 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 4 ccatctcttg ctcgaagtcc 20  

Claims (24)

A composition for separating or identifying mesenchymal stem cells from adipocytes, comprising an anti-fibroblast activation protein α (FAPα) antibody to which beads for separation are connected. delete delete delete delete delete A composition for isolating or identifying mesenchymal stem cells from adipocytes, characterized in that it comprises an anti-fibroblast activating protein α antibody linked to a separation ligand. delete delete Composition for separating or identifying mesenchymal stem cells from adipocytes, characterized in that any one of the enzymes selected from the group consisting of luciferase, peroxidase and beta-galactosidase is an anti-fibroblast activating protein α antibody. . delete delete delete delete delete delete delete Contacting the anti-fibroblast active protein α antibody with a sample cell; Separating mesenchymal stem cells from adipocytes comprising the step of confirming the antigen-antibody complex formation of the fibroblast active protein α and the anti-fibroblast active protein α antibody of the sample cell. 19. The method of claim 18, wherein the antigen-antibody complex formation method is radioimmunoassay (RIA), enzyme immunoassay (ELISA), immunofluorescence, chromosome binding method or chemiluminescent material binding method, characterized in that Mesenchymal stem cell separation method. delete delete delete Contacting the anti-fibroblast active protein α antibody with a sample cell; The method of identifying mesenchymal stem cells from adipocytes, characterized in that it comprises the step of confirming the antigen-antibody complex formation of the fibroblast active protein α and the anti-fibroblast active protein α antibody of the sample cell. 24. The method of claim 23, wherein the antigen-antibody complex formation is confirmed by radioimmunoassay (RIA), enzyme immunoassay (ELISA), immunofluorescence, chromosomal binding or chemiluminescent binding method. Mesenchymal stem cell identification method.

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