JP4540948B2 - Method for identifying and separating mesenchymal stem cells using differentiation markers - Google Patents

Description

  The present invention relates to detection, identification and separation of mesenchymal stem cells, and in particular, mesenchymal stem cell detection gene markers and / or mesenchymal stem cell detection, the expression of which differs between mesenchymal stem cells and fibroblasts. The present invention relates to a method for identifying and separating mesenchymal stem cells using a protein marker.

  Mesenchymal stem cells exist in mammalian bone marrow and are known as pluripotent stem cells that differentiate into adipocytes, chondrocytes, and bone cells. Mesenchymal stem cells are attracting attention as transplant materials for regenerative medicine of many tissues such as bone, cartilage, tendon, muscle, fat and periodontal tissue because of their pluripotency (gene medicine, Vol. .4, No. 2 (2000) p58-61). Recently, a review about the current state and prospects of mesenchymal stem cell research has been published, and reports on the collection and culture of mesenchymal stem cells have been made (Experimental Medicine, Vol. 19, No. 3 (February) 2001). p350-356). Furthermore, it was reported that mesenchymal stem cells also exist in adipose tissue (Tissue Engineering, PA Zuk et al., Multilineage cells from human adipose tissue: implications for cell-based therapies. 7: 211-228, 2001) .

  In recent years, several patent applications have been published regarding the culture and differentiation of mesenchymal stem cells. For example, JP-A-11-506610 discloses a composition and method for maintaining the survival of human mesenchymal progenitor cells in a serum-free environment, and JP-T-10-512756 discloses a mesenchymal stem cell. To induce differentiation, a method comprising contacting with a bioactive factor such as osteoinductive factor, differentiation-associated factor, cartilage-inducing factor such as prostaglandin, ascorbic acid, collagen extracellular matrix, etc. No. 217576 discloses an invention for a method of culturing pluripotent mesenchymal stem cells in the presence of prolactin or its equivalent and differentiating mesenchymal stem cells into adipocytes.

  The inventor previously proliferated mesenchymal stem cells significantly faster by culturing mesenchymal stem cells in the presence of a basement membrane extracellular matrix or in a medium containing fibroblast growth factor (FGF) or the like. And a culture method for obtaining significantly more mesenchymal stem cells than the conventional culture method was disclosed (Japanese Patent Laid-Open No. 2003-52360). In addition, a method for separating and collecting mesenchymal stem cells from oral tissues has been disclosed in order to collect mesenchymal stem cells that are safe and easy to collect in the mother body (Japanese Patent Laid-Open No. 2003-52365). Publication).

  In recent years, with the advancement of regenerative medicine, mesenchymal stem cells have attracted attention as transplant materials for regenerative medicine of many tissues such as bone, cartilage, tendon, muscle, fat and periodontal tissue due to their pluripotency. Has been. In order to use mesenchymal stem cells for tissue regenerative medicine, it is first necessary to collect these stem cells from living tissue, proliferate them, and further differentiate and proliferate them to prepare the tissue. Become. Although mesenchymal stem cells are present in the bone marrow and periosteum, it is necessary to develop a method for safely and easily collecting mesenchymal stem cells from these tissues for practical use in tissue regenerative medicine. is there. In order to put mesenchymal stem cells into tissue regeneration medicine, it is necessary to develop a technique for securing a sufficient amount of mesenchymal stem cells. For that purpose, it is important to develop a technique for culturing and growing the collected mesenchymal stem cells while maintaining the differentiation ability.

As described above, the present inventor has developed a method for separating and collecting a mesenchymal stem cell that is safe and easy to collect when collecting mesenchymal stem cells. Furthermore, the present inventor maintains the differentiation ability of mesenchymal stem cells by culturing mesenchymal stem cells in the presence of a basement membrane extracellular matrix or in a medium containing fibroblast growth factor (FGF) or the like. Developed a method to make it grow explosively. However, in order to put the cultured mesenchymal stem cells into regenerative medicine, it is necessary to confirm that the cultured cells are mesenchymal stem cells, and a method for detecting and identifying the mesenchymal stem cells It is necessary to develop. Conventionally, in mesenchymal stem cells, a marker gene that characterizes the cells has not been identified. Therefore, identifying a marker gene that characterizes mesenchymal stem cells and developing a method for detecting, separating and identifying mesenchymal stem cells has been solved in order to put mesenchymal stem cells into practical use for regenerative medicine. It is an important issue that must be done.
JP 10-512756 Gazette. Japanese National Patent Publication No. 11-506610. JP 2000-217576 A. JP2003-52360A. JP2003-52365A. Gene Medicine Vol. 4, no. 2 (2000) p58-61. Experimental Medicine Vol. 19, no. 3 (February issue) 2001, p350-356. Tissue Engineering, PA Zuk et al., Multilineage cells from human adipose tissue: implications for cell-based therapies. 7: 211-228, 2001.

  An object of the present invention is a method for effectively identifying and separating mesenchymal stem cells and fibroblasts that are morphologically similar and difficult to distinguish, and in particular, mesenchymal stem cells and fibroblasts. To provide a method for detecting, identifying and separating mesenchymal stem cells using a mesenchymal stem cell detection gene marker and / or a mesenchymal stem cell detection protein marker whose expression is different from that of a cell. .

  The present inventor has intensively studied to solve the above problems, and that mesenchymal stem cells have a higher level of vitamin D response ability than fibroblasts, and target genes for vitamin D receptors Elucidating that vitamin D receptor-related genes such as osteocalcin and osteopontin genes are expressed in mesenchymal stem cells before inducing bone differentiation, and the vitamin D receptor gene or the gene is encoded Or the vitamin D receptor-related gene or the protein encoded by the gene as a differentiation marker between mesenchymal stem cells and fibroblasts, and detecting the expression of the gene and protein in a test cell, It was found that mesenchymal stem cells and fibroblasts can be effectively identified and separated, and the present invention has been completed. In the present invention, a target gene of a vitamin D receptor such as osteocalcin gene and osteopontin gene is added in a mesenchymal stem cell by adding a gene expression activator such as 1,25 hydroxylated vitamin D. The inventors have found that its expression is enhanced, and that it is possible to identify and separate mesenchymal stem cells effectively by detecting the expression of the enhanced gene, and further have made the present invention.

  That is, mesenchymal stem cells are differentiated into bone, cartilage, fat, muscle, tendon, nerve, etc., and are expected to be transplanted cells for repairing damage to these tissues from the viewpoint of use in regenerative medicine. However, since mesenchymal stem cells and fibroblasts cannot be distinguished morphologically, in order to confirm that they are mesenchymal stem cells, in vitro or in vivo There was no reliable way to prove pluripotency. However, in order to put mesenchymal stem cells into practical use for tissue regenerative medicine, it is necessary to predict that the cells are mesenchymal stem cells and that the mesenchymal stem cells maintain pluripotency. There is a need to develop a method for distinguishing from fibroblasts in advance. To date, it has been reported that many cell surface antigens are expressed in mesenchymal stem cells, but these cell surface antigens are specific or selective for mesenchymal stem cells. There is no clear evidence.

  The present inventor has compared the expression levels of many cell surface antigens between human mesenchymal stem cells and human fibroblasts, but no antigens showing clear differences have been found so far. Therefore, the present inventor first conducted an extensive search to find a gene serving as a marker for detecting mesenchymal stem cells, and as a result, 13 genes were specifically expressed in mesenchymal stem cells. A method for discriminating mesenchymal stem cells and fibroblasts was developed using the gene as a marker for detecting mesenchymal stem cells, and a patent application was filed (Japanese Patent Application No. 2003-63077).

  Furthermore, as a result of earnest search for markers for effectively discriminating and separating mesenchymal stem cells and fibroblasts, the present inventors have found that mesenchymal stem cells have a higher level of vitamin D response ability than fibroblasts. And that a vitamin D receptor-related gene such as osteocalcin gene has been expressed in mesenchymal stem cells before inducing bone differentiation, and the vitamin D receptor gene or It has been found that the protein, or the vitamin D receptor-related gene or protein, can be used as a differentiation marker between mesenchymal stem cells and fibroblasts. Furthermore, in the present invention, a target gene of a vitamin D receptor such as osteocalcin gene and osteopontin gene is added to a mesenchymal stem cell by adding a gene expression activator such as active 1,25-hydroxyvitamin D. It was found that the expression was enhanced in the medium. On the other hand, these expressions were hardly detectable in fibroblasts regardless of the addition or non-addition of 1,25-hydroxyvitamin D.

  Therefore, in the present invention, the addition of a vitamin D receptor-related gene such as the vitamin D receptor, osteocalcin gene, or a gene such as active 1,25-hydroxyvitamin D Because it was found that by detecting the expression of target genes of vitamin D receptors such as osteocalcin and osteopontin with enhanced expression, it becomes possible to distinguish fibroblasts as a new marker of mesenchymal stem cells By using these new markers, it becomes possible to construct a method for effectively discriminating and separating mesenchymal stem cells from other cell populations such as fibroblasts. The method of the present invention also makes it possible to test the quality of mesenchymal stem cells grown in a test tube. Therefore, the present invention can contribute to the use of mesenchymal stem cells for regenerative medicine.

Specifically, the present invention is a gene expression activator in mesenchymal stem cells and fibroblasts using the osteocalcin gene or osteopontin gene, which is the target gene of the vitamin D receptor gene, as a differentiation marker 1 , 25 by adding hydroxylated vitamin D and / or detecting the expression of the gene activated and / or the protein encoded by the activated gene, the difference in expression between the mesenchymal stem cells and fibroblasts of the gene by detecting, consisting identification method from fibroblasts mesenchymal stem cells, characterized by identifying the mesenchymal stem cells and fibroblasts (claim 1).

Further, the present invention provides a method for distinguishing mesenchymal stem cells from fibroblasts according to claim 1 , wherein the detection of the differentiation marker gene includes the use of quantitative or semi-quantitative PCR (claims). 2) or the method of identifying mesenchymal stem cells from fibroblasts according to claim 2 , wherein the use of quantitative or semi-quantitative PCR is RT-PCR (claim 3) , The mesenchymal stem cell of the mesenchymal stem cell according to claim 1 , wherein the detection of the differentiation marker gene is performed using a gene detection probe comprising a DNA sequence that hybridizes with the base sequence of the gene under stringent conditions. identification method from fibroblasts (claim 4) or, for gene detection probe, the fibroblasts mesenchymal stem cells of claim 4, wherein comprising all or part of the antisense strand of the nucleotide sequence of the gene identifying how Consists (claim 5).

The present invention, detection of protein fractionation marker gene encodes, induced by the protein, mesenchymal stem cells of claim 1, wherein a is performed using an antibody that specifically binds to the protein identification method from fibroblasts (claim 6) and the antibody, the identification method from fibroblasts mesenchymal stem cells according to claim 6, characterized in that it is a monoclonal antibody (claim 7) and, mesenchymal stem cells in a test cell, fractionation marker gene labeled by a fluorescent antibody technique using an antibody that specifically binds to the protein encoded, to separate and identify mesenchymal stem cells said labeled it identification method (claim 8) or from fibroblast mesenchymal stem cells of claim 6 or 7, wherein the genetic osteocalcin is a target gene of the vitamin D receptor gene Or fractionation marker for gene detection probe consisting of osteopontin gene, and either equipped with a 1,25 hydroxide Vitamin D is an expression activator fractionation marker gene, and / or is the target gene of the vitamin D receptor gene antibodies for detection of proteins the gene or osteopontin gene osteocalcin encodes, and identify from fibroblasts of mesenchymal stem cells comprising equipped with 1,25 hydroxide vitamin D is an expression activator fractionation marker gene Kit (claim 9) .

  According to the present invention, a mesenchymal stem cell and a fibroblast are discriminated by detecting a difference in expression between the mesenchymal stem cell and the fibroblast in the vitamin D receptor gene and its related gene as a differentiation marker.・ Establishing a method for separating mesenchymal stem cells and fibroblasts, which were difficult to distinguish in their form, can be easily and effectively separated. This overcomes the problem of differentiation from fibroblasts, which has been an obstacle when using pluripotent mesenchymal stem cells such as bone, cartilage, fat, muscle, tendon, and nerve for regenerative medicine. Therefore, the present invention is expected to greatly contribute to the use of mesenchymal stem cells for regenerative medicine and the like.

  The present invention uses a vitamin D receptor gene and / or a related gene thereof as a differentiation marker, detects a difference in expression between mesenchymal stem cells and fibroblasts, and detects mesenchymal stem cells and fibroblasts. And mesenchymal stem cells are obtained. The difference in expression of the discrimination marker is detected by detecting the expression of the vitamin D receptor gene and / or its related gene, or detecting the protein encoded by the gene. As a related gene of a vitamin D receptor, the target gene of a vitamin D receptor like the gene of osteocalcin can be mentioned. The vitamin D receptor gene used as a differentiation marker for mesenchymal stem cells and fibroblasts in the present invention and the base sequence and amino acid sequence of the protein encoded by the gene are already known (Proc. Natl. Acad. Sci. USA 85, 10, 3294-3298, 1988), DNA sequence information of the gene can be approached by accession number: NM_000376 in NCBI gene database. Regarding the vitamin D receptor gene, several gene polymorphisms are known (Japanese Patent Laid-Open No. 2001-333798, Japanese Patent Laid-Open No. 2001-29088, and Japanese Translation of PCT International Publication No. 2002-525074).

  Further, in the present invention, the base sequence of osteocalcin gene and the amino acid sequence of osteocalcin, which are target genes of vitamin D receptor used as a differentiation marker for mesenchymal stem cells and fibroblasts, are already known (J. Biol Chem. 255, 8685-8691, 1980), the DNA sequence information of the gene can be approached by accession number: NM_000711 in NCBI gene database. Osteocalcin (BGP) is a protein that occupies about 20% of bone non-collagenous protein, and human osteocalcin has a molecular weight of 5800 and is composed of 49 amino acids.

  Further, in the present invention, the base sequence of osteopontin gene and the amino acid sequence of osteopontin, which are target genes of vitamin D receptor used as a differentiation marker between mesenchymal stem cells and fibroblasts, are already known (Proc. Natl Acad. Sci. USA, 83, 8819-8823, 1986), the DNA sequence information of the gene can be approached by the accession number: NM_000582 in NCBI gene database. Osteopontin (bone sialoprotein-1: BSP-1) is the main phosphoprotein of bone and contains sialic acid. Osteopontin consists of 301 amino acids and has 12 residues of phosphoserine and 1 residue of phosphothreonine.

  In the present invention, the difference in the expression of the differentiation marker gene as described above between mesenchymal stem cells and fibroblasts is detected using a gene detection means known per se to identify mesenchymal stem cells.・ Separate. When detecting the expression of the discrimination marker gene of the present invention as a protein encoded by the gene, an antibody that specifically binds to the protein is prepared using the protein, Expression in the mesenchymal stem cells and fibroblasts is detected by a method known per se, and the mesenchymal stem cells are identified and separated. As an antibody used for detecting the expression of the fractional marker protein, either a monoclonal antibody or a polyclonal antibody can be used.

  In the present invention, expression of a target gene of a vitamin D receptor such as an osteocalcin gene or an osteopontin gene by adding a gene expression activator such as 1,25 hydroxylated vitamin D in mesenchymal stem cells. Has been found to be specifically enhanced. Therefore, in the present invention, by adding a gene expression activator, the expression of a differentiation marker gene such as the osteocalcin gene or the osteopontin gene is enhanced, and the expression of the enhanced gene is detected, thereby It is possible to effectively identify and separate leaf stem cells.

  In the present invention, in order to detect the expression of the marker gene in the present invention, a method used for detecting the expression of a known gene can be used. For example, the Northern blotting method can be used for detecting the expression of the marker gene in the present invention. In addition, in order to detect and identify the expression of the gene marker in the present invention, a probe having a DNA sequence that hybridizes with the DNA sequence of the gene marker in the present invention under stringent conditions can be used. Detection of gene expression in mesenchymal stem cells and fibroblasts using the probe can be appropriately performed using a known method. For example, detection of mesenchymal stem cells by preparing a DNA probe of an appropriate length from a DNA sequence of a known gene marker, appropriately attaching a label such as a fluorescent label, and hybridizing it with a specimen I do. As the DNA probe, a marker gene detection probe consisting of all or part of the antisense strand of the base sequence of a known gene marker can be used. In addition, the probe can be used in the form of a microarray or DNA chip for detecting a marker gene having at least one or more immobilized thereon.

  In the preparation of the DNA probe, the conditions for “hybridize with the DNA sequence of the marker gene under stringent conditions” in the base sequence of the present invention include, for example, hybridization at 42 ° C. and 1 × SSC. (0.15M NaCl, 0.015M sodium citrate), washing treatment at 42 ° C. with a buffer containing 0.1% SDS (Sodium dodecyl sulfate), hybridization at 65 ° C., and A washing treatment at 65 ° C. with a buffer containing 0.1 × SSC and 0.1% SDS can be mentioned more preferably. In addition to the above temperature conditions, there are various factors that influence the stringency of hybridization. Those skilled in the art can combine various elements to obtain a string equivalent to the above-described hybridization stringency. It is possible to realize a genie.

  Furthermore, in the present invention, when detecting the protein marker in the present invention, an antibody that is induced by the protein and specifically binds to the protein can be used. Examples of the antibody include a monoclonal antibody and a polyclonal antibody. The antibody can be prepared by a conventional method using the polypeptide marker of the present invention as an antigen. Detection of marker protein expression in a test cell using the antibody of the present invention can be carried out using an immunoassay using a known antibody. Examples of the immunological measurement method include known immunological measurement methods such as RIA method, ELISA method, and fluorescent antibody method.

  In the present invention, the expression of a marker gene and / or marker polypeptide in a test cell is detected using the gene marker and protein marker detection probe and / or detection antibody of the present invention, and discriminated from fibroblasts. Then, mesenchymal stem cells can be isolated and obtained. In detecting a marker gene in a test cell, quantitative or semi-quantitative PCR can be used to amplify the gene of the test cell. RT-PCR (reverse transcription PCR) can be used as the PCR. In carrying out the PCR, a primer comprising a sense primer and an antisense primer for amplifying the marker gene in the present invention is used.

  The mesenchymal stem cells can be labeled by the fluorescent antibody method using the antibody of the present invention, the expression of the mesenchymal stem cell marker polypeptide in the test cell can be detected, and the mesenchymal stem cells can be identified and separated. . In order to label undifferentiated hematopoietic cells by the fluorescent antibody method, an antibody that specifically binds to the protein marker in the present invention is fluorescently labeled, and this is bound to a mesenchymal stem cell expressing an antigen. Either label the leaf stem cells (direct fluorescent antibody method) or bind the unlabeled specific antibody of the present invention to the mesenchymal stem cells expressing the antigen, and then label the secondary antibody ( Anti-immunoglobulin antibody) is bound to label mesenchymal stem cells (indirect fluorescent antibody method), and the labeled mesenchymal stem cells are separated and collected.

  The probe for detecting a marker gene used for detection and identification of the mesenchymal stem cell of the present invention, the microarray or DNA chip for detecting the marker gene to which the probe is immobilized, and the antibody for detecting the mesenchymal stem cell of the present invention are equipped with them. It can be commercialized as a kit for identifying mesenchymal stem cells.

EXAMPLES Hereinafter, although an Example demonstrates this invention more concretely, the technical scope of this invention is not limited to these illustrations.
[Materials and methods]
This example compares the expression levels of human bone marrow mesenchymal cells and human fibroblast bone, cartilage, and adipose-related genes in the growth phase (70-80% confluent) in which no lineage has been determined to differentiate. did.
Human bone marrow mesenchymal stem cells were isolated from the iliac bone or alveolar bone. Or purchased. Human fibloblasts were purchased or cultured separately from gingival tissue.
All of these cells were cultured in a medium containing 1 ng / ml bFGF and 10% fetal calf serum (FCS) at 37 ° C. in the presence of 5% carbon dioxide (Biochemical and Biophysical Research Communication, 288, 413). -419, 2001). In this example, the one that was passaged 3 to 7 times was used. Bone differentiation was performed at 37 ° C. using a bone differentiation-inducing medium (a medium in which 100 nM dexamethasone, 10 mM β-glycerol phosphate, 50 μg / mL ascorbic acid-2-phosphate was added to αMEM containing 10% FBS) that reached confluence. It was induced by culturing for 28 days in the presence of 5% carbon dioxide (Science 284, 2, 1999). Under these conditions, the human bone marrow mesenchymal cell culture system was uniformly and extensively stained with alizarin red, indicating that bone differentiation and calcification had occurred.

[Comparison of marker gene expression levels in human undifferentiated bone marrow mesenchymal cells and human fibroblasts during the growth phase]
After extracting total RNA from each cell using Trizol Reagent (Invitrogen), each microRNA was used as a template with 1 μg as a template using SuperScrit first-strand system or RT-PCR (Invitrogen) at 42 ° C. for 50 minutes. The reverse transcription reaction was performed to synthesize cDNA. Each gene marker was amplified using Advantage 2 PCR enzyme system (Clontech) using the synthesized cDNA as a template. 30 cycles of denaturation at 94 ° C. for 30 minutes and annealing at 68 ° C. for 1 minute were performed. Table 1 summarizes the primer sequences of each gene used for amplification. The amplified genes were separated by electrophoresis using 1% agarose gel and stained by ethidium bromide staining.

Osteocalcin (abbreviated as OC in the figure), osteopontin (OP), bone sialoprotein (OP) in human undifferentiated bone marrow mesenchymal cells and human fibroblasts in proliferative phase (70-80% confluent) BSP), alkaline phosphatase (ALP), type II collagen (Coll 2), aggrecan (AGG), Sox 9, PPAR-gamma 2 (PPAR2) Comparison of mRNA expression levels (FIG. 1).
Undifferentiated bone marrow mesenchymal cells cultured in the growth medium in the growth phase (70-80% confluent), as expected from previous studies, have not added to the bone induction medium because growth has not stopped. It was not differentiated because it was not. In other words, it was not stained with alizarin red staining and was not calcified. However, the expression of osteocalcin mRNA, the most representative bone marker gene, was observed in all human undifferentiated bone marrow mesenchymal cells isolated from 7 individuals (although expression was strong depending on the individual). On the other hand, none of the human fibroblasts isolated from the four individuals expressed osteocalcin mRNA (FIG. 1).

In addition to osteocalcin, osteopontin, bone sialoprotein, and alkaline phosphatase are known to increase in osteoblasts. However, there are individual differences in the expression of these markers, and there are some that are not expressed by each individual. In human fibroblasts isolated from 4 individuals, expression was not observed except for extremely low levels of alkaline phosphatase mRNA detected in cells derived from 2 individuals (FIG. 1).
Regarding the expression of cartilage markers type II collagen and aggrecan, low levels of aggrecan mRNA were detected in most undifferentiated bone marrow mesenchymal cells examined, but one of the most important cartilage marker type II collagen mRNA was detected. Could not (Fig. 1). On the other hand, these cartilage markers (except that very low levels of aggrecan mRNA were detected in cells from two individuals) were not expressed in all human fibroblasts examined (FIG. 1).

The adipocyte marker PPAR-gamma 2 (PPAR2) mRNA was expressed in all bone marrow mesenchymal cells and human fibroblasts (FIG. 1). Even cells could not be detected.
These results indicate that although bone marrow mesenchymal cells are undifferentiated, only a part of each differentiated lineage gene group is already expressed instead of the whole gene group.

[Comparison of expression level of bone marrow mesenchymal cells before and after induction of bone differentiation]
In bone marrow mesenchymal cells derived from three individuals, the expression levels of bone markers after the growth phase and bone differentiation induction were compared. The expression level of osteocalcin was almost the same before and after differentiation, while the expression of other bone markers was all increased after induction of bone differentiation, but it was remarkably low in the proliferative phase or could not be detected (FIG. 2). It was suggested that bone marrow mesenchymal stem cells are proliferating and do not become osteoblasts due to partial expression of bone markers.

[Comparison of action of vitamin D in human undifferentiated, bone marrow mesenchymal cells and human fibroblasts]
When 10 ^-8 M 1,25 hydroxylated vitamin D ₃ (1,25- (OH) ₂ vitamin D3) is added in the growth phase of bone marrow mesenchymal cells derived from 3 individuals, osteocalcin and osteopontin are within 48 hours. Expression was remarkably enhanced (FIG. 3). On the other hand, bone sialoprotein, alkaline phosphatase is the 1,25 hydroxide vitamin D ₃ did not respond. In human fibroblasts, it did not respond to any of the genes 1,25 hydroxide vitamin D _3.
Table 2 shows the results of examining bone markers, cartilage markers, fat markers, and vitamin D responses in human undifferentiated bone marrow mesenchymal cells (MSC) and human fibroblasts using many individual-derived cells. .

[FACS analysis using anti-osteocalcin antibody]
First, a primary antibody that recognizes a surface antigen and 5 × 10 ⁵ cells are mixed and incubated at room temperature for 30 minutes. When the primary antibody was recognized by fluorescence, it was measured with a flow cytometer after washing. When the primary antibody was not fluorescently labeled, after washing, the fluorescently labeled secondary antibody having binding activity to the primary antibody and the cells reacted with the primary antibody were mixed and incubated again at room temperature for 30 minutes. After washing, the cells stained with the primary antibody and the secondary antibody were measured with a flow cytometer.
4 and 5 show the results of FACS analysis using an antibody against osteocalcin. With the addition of vitamin D, human undifferentiated bone marrow mesenchymal cells in the proliferating phase of 80% or more were expressed, whereas in fibroblasts, cells producing osteocalcin could not be detected even when vitamin D was added. .

In the Example of this invention, it is a figure which shows the comparison of the expression level of various gene markers in a mesenchymal stem cell (MSC) and a fibroblast. In the Example of this invention, it is a figure which shows the change of the gene marker expression of the mesenchymal stem cell before and behind bone differentiation induction. In the Example of this invention, it is a figure which shows the responsiveness of the mesenchymal stem cell (MSC) with respect to vitamin D, and a fibroblast. In the Example of this invention, it is a figure which shows the detection of the human intracellular (hMSC, hFibroblast) osteocalcin (Osteocalcin) by FACS. In the Example of this invention, it is a figure which shows the comparison of the osteocalcin (Osteocalcin) positive rate of a human MSC and a human fibroblast.

Claims (9)

Using the osteocalcin gene or osteopontin gene, which is the target gene of the vitamin D receptor gene, as a differentiation marker , add 1,25 hydroxylated vitamin D, a gene expression activator, in mesenchymal stem cells and fibroblasts By detecting the expression of the activated gene and / or the protein encoded by the activated gene, the difference in expression between the mesenchymal stem cell and the fibroblast is detected, and the mesenchymal stem cell is detected. A method for discriminating mesenchymal stem cells from fibroblasts, characterized in that they are discriminated from fibroblasts. The method for distinguishing mesenchymal stem cells from fibroblasts according to claim 1 , wherein the detection of the differentiation marker gene comprises the use of quantitative or semi-quantitative PCR. Quantitative or use of semi-quantitative PCR is the identification method from fibroblasts mesenchymal stem cells of claim 2, characterized in that the RT-PCR method. The mesenchymal stem cell of the mesenchymal stem cell according to claim 1 , wherein the detection of the differentiation marker gene is performed using a gene detection probe comprising a DNA sequence that hybridizes with the base sequence of the gene under stringent conditions. Identification method from fibroblasts. The method for discriminating mesenchymal stem cells from fibroblasts according to claim 4 , wherein the gene detection probe comprises all or part of the antisense strand of the base sequence of the gene. The detection of protein fractionation marker gene encodes, induced by the protein, from fibroblasts mesenchymal stem cells of claim 1, wherein a is performed using an antibody that specifically binds to the protein Identification method . The method for discriminating mesenchymal stem cells from fibroblasts according to claim 6 , wherein the antibody is a monoclonal antibody. The mesenchymal stem cells in the test cells are labeled by a fluorescent antibody method using an antibody that specifically binds to the protein encoded by the differentiation marker gene, and the labeled mesenchymal stem cells are separated and identified. 8. A method for identifying mesenchymal stem cells from fibroblasts according to claim 6 or 7 . Equipped with a detection marker gene consisting of osteocalcin gene or osteopontin gene, which is a target gene of the vitamin D receptor gene, and 1,25 hydroxylated vitamin D which is an expression activator of the differentiation marker gene, and / Or equipped with an antibody for protein detection encoded by the osteocalcin gene or osteopontin gene, which is the target gene of the vitamin D receptor gene , and 1,25 hydroxylated vitamin D , which is a substance that activates the expression of the differentiation marker gene A kit for distinguishing mesenchymal stem cells from fibroblasts.

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