JP5243021B2 - Genetic markers for detecting cells such as mesenchymal stem cells - Google Patents

Description

  The present invention provides a genetic marker for detecting cells at each stage in the process of differentiating bone marrow cells into mesoblasts via mesenchymal stem cells, and detecting the cells at each stage using the genetic markers. It relates to a method of separation. The present invention further relates to a method for monitoring a culture process in which bone marrow cells are differentiated into osteoblasts via mesenchymal stem cells.

  A mesenchymal stem cell is an undifferentiated cell that has multipotency capable of differentiating into various types of cells such as muscle, bone, and fat, and has the ability to self-replicate. A representative mesenchymal stem cell is a cell that is slightly present in bone marrow or umbilical cord blood, but in recent years, a method for separating mesenchymal stem cells from adipose tissue and oral periosteum has been reported. Mesenchymal stem cells are differentiated into various cells by the addition of various hormones and cytokines (small secreted proteins), so they are the most promising cells for transplantation used in regenerative medicine and cell medical technology. In addition to myocardial regeneration, clinical application of immunosuppressive therapy is being started.

  For example, Prochymal is a therapy that suppresses Graft vs Host Disease, a side effect by administering mesenchymal stem cells before hematopoietic stem cell transplantation during leukemia treatment, and has completed Phase 1 clinical trials. The company entered Phase 2 in 2005 and plans to launch in the second half of 2007. Provacel is a therapy that repairs myocardial damage caused by myocardial infarction with mesenchymal stem cells. It entered Phase 1 in the first quarter of 2005, entered Phase 2/3 in the middle of 2006, and is scheduled to launch in 2009. doing. In addition, Chondrogen is a therapy that repairs joint meniscal damage with mesenchymal stem cells and is pending a clinical trial with the FDA.

  It is important to secure a sufficient amount of mesenchymal stem cells for the practical application of mesenchymal stem cells as regenerative medicine. For that purpose, it is slightly contained in bone marrow and adipocytes. It is necessary to efficiently isolate and purify mesenchymal stem cells, and to proliferate mesenchymal stem cells while maintaining pluripotency by culturing them.

  However, it is known that relatively small cells among human bone marrow stromal cells exhibit pluripotency, but in fact, the origin and properties of mesenchymal stem cells are not fully understood. Therefore, in order to efficiently isolate and purify mesenchymal stem cells, a search for a marker that specifically identifies mesenchymal stem cells is being conducted.

  To date, Patent Documents 1 and 2 and Non-Patent Document 1 have been reported as markers for mesenchymal stem cells. However, all these reports are intended only for the separation of mesenchymal stem cells and fibroblasts. Since fibroblasts are mixed when isolating and purifying mesenchymal stem cells from bone marrow, they proliferate in the same way as mesenchymal stem cells during culture, and morphologically indistinguishable from mesenchymal stem cells, Markers that separate these are selected. Marker extraction is determined from the difference in expression profiles between mesenchymal stem cells and fibroblasts.

  In addition, there is also a report on a marker for identifying a precursor mesenchymal stem cell having differentiation ability into a mesenchymal stem cell (Patent Document 3). That is, when embryonic stem cells were cultured in the presence of retinoic acid, progenitor mesenchymal stem cells appeared at an early stage of differentiation, and this cell population expressed Sox1, which is a neuroectodermal marker. In this method, Sox1 is used as a marker to select progenitor mesenchymal stem cells differentiated from embryonic stem cells.

  Among the things that are being studied to improve the yield of mesenchymal stem cells are the development and use of equipment and equipment for separation and purification, the device of the separation source, and the enrichment of mesenchymal stem cells in vivo. There are administration of a compound to a living body and administration of a protein molecule to a living body. In addition, with regard to the proliferation of mesenchymal stem cells by culturing, attempts have been made to solve the problem by devising medium components and additives.

  In addition, when the proliferated mesenchymal stem cells are put to practical use in regenerative medicine, it is important from the aspect of ensuring safety to guarantee the quality of the cells. In the culturing process, cell mutation and canceration, differentiation into unintended cells, contamination by bacteria and viruses, etc. may occur, and it is essential to manage the state of the cells. When applying for biological cells as pharmaceuticals, mycoplasma and virus sterility tests are listed as one of the items in the guidelines by the Ministry of Health, Labor and Welfare, and detection by the PCR method using culture methods and nucleic acid sequences is known as an existing method. It has been. As a method for predicting canceration, there is a method for measuring the expression level of cancer-related genes such as hTERT and c-myc. However, in order to satisfy all of these items with the existing method, it is necessary to perform a wide variety of inspections, which is difficult in practice. In order to commercialize regenerative medicine, it is necessary to construct a simple and inexpensive inspection system for quality control of cells as well as basic techniques such as cell and scaffold preparation techniques.

  Furthermore, as a report investigating changes in the expression profile in the culturing process so far, for example, Patent Document 4 (a method for inducing differentiation into adipocytes, a compound for controlling differentiation into adipocytes and a screening method thereof), Patent Document 2 (Investigation of changes in gene expression profile in the process of differentiation into osteoblasts by inducing BMP2 expression using C9 cells with rhBMP2 gene introduced to express BMP-2 in mouse MSC cell line) Non-patent document 3 (C2C12 of premyoblasts differentiates into osteoblasts depending on BMP-2, but in the process, microarray data were analyzed at 7 locations within 24 hours after BMP-2 treatment. And changes in gene expression during bone differentiation, and there are descriptions that list genes classified by function).

Molecular marker distinguish bone marrow mesenchymal stem cells from fibroblasts, Biochem. Biophys. Res Commun. 332,297-303, 2005 DNA microarray analysis using statistical methods and clustering: three case studies Osnat Ravid-Amir M. Sc. Thesis submitted to the Scientific Council of the weizmann Institute of Science Prof. Eytan Domany (Research conducted under the supervision February (2003) J. Cellular Biochemistry 89: 401-426 2003 JP 2004-290189 A JP 2005-27579 A JP 2005-304443 JP 2000-217576 A

  The present invention relates to a gene marker useful for detecting and / or identifying a cell, particularly a mesenchymal stem cell, at each stage in the process of differentiating bone marrow cells into osteoblasts via mesenchymal stem cells; A probe or primer for detecting a cell; a method for detecting and / or identifying a cell at each stage in a process of differentiating from a bone marrow cell into an osteoblast via a mesenchymal stem cell, in particular, a mesenchymal stem cell, and a bone marrow cell It is an object to be solved to provide a method for monitoring a culture process in which cells are differentiated into osteoblasts from mesenchymal stem cells.

  As a result of diligent investigations to achieve the above-mentioned problems, the inventors of the present invention have changed the culture process from the state of cells into five groups in the culture process for differentiating bone marrow fluid into mesenchymal stem cells and further osteoblasts. Further, the contents were further subdivided, and an expression profile at 8 points in all steps was obtained for 30,000 genes. These were performed on five specimens, and all data were examined carefully to select gene groups that are characteristically expressed in mesenchymal stem cells, gene groups that are characteristically expressed in osteoblasts, and the like. Of these genes, by using genes that are characteristically expressed in each group, not only the cells contained in each group, that is, mesenchymal stem cells and osteoblasts, but also other groups of cells included in the culture process. It has become possible to discriminate and separate from fibroblasts and the like not included in these culture processes. Furthermore, it became possible to monitor the culture process by combining genes that are characteristically expressed in each group. The present invention has been completed based on these findings.

That is, according to the present invention, the following inventions (1) to (13) are provided.
(1) The gene marker for cell detection according to any of (i) to (v) below.
(I) To detect bone marrow cells comprising a gene having the base sequence described in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13 in the sequence listing Genetic markers of
(Ii) Bone marrow cells comprising a gene having the base sequence described in SEQ ID NO: 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 in the sequence listing A marker for detecting a cell after culturing a cell but before only mesenchymal stem cells are selectively cultured;
(Iii) a genetic marker for detecting mesenchymal stem cells, comprising a gene having the base sequence set forth in SEQ ID NO: 20, 21, 22, 23, 24 or 25 in the sequence listing;
(Iv) a gene marker for detecting osteoprogenitor cells, comprising a gene having the nucleotide sequence set forth in SEQ ID NO: 26, 27 or 28 in the sequence listing; and (v) SEQ ID NO: 28, 29, 30 in the sequence listing. , 31, 32, 33, 34, 35 or 36, a gene marker for detecting osteoblasts, comprising a gene having the base sequence of
(Vi) A cell after culturing bone marrow cells and bone marrow cells, which is composed of the gene having the nucleotide sequence set forth in SEQ ID NO: 37 or 7 in the sequence listing and before only mesenchymal stem cells are selectively cultured. Genes that show differential expression levels between:
(Vii) A cell after culturing bone marrow cells consisting of a gene having the nucleotide sequence set forth in SEQ ID NO: 38 or 39 in the sequence listing, and a cell before only mesenchymal stem cells are selectively cultured and mesenchymal system Genes that show differential expression levels among stem cells:
(Viii) A gene showing a difference in expression level between a mesenchymal stem cell and an osteoprogenitor cell comprising a gene having the base sequence set forth in SEQ ID NO: 40 or 21 in the sequence listing:
(Ix) A gene showing a difference in expression level between osteoblasts and osteoprogenitor cells comprising a gene having the base sequence set forth in SEQ ID NO: 28, 41 or 42 in the sequence listing:

(2) A probe for detecting the gene marker for cell detection according to (1), which has a DNA sequence that hybridizes with the gene marker for cell detection according to (1) under stringent conditions.
(3) The probe according to (2), comprising all or part of the antisense strand of the base sequence of the gene marker for cell detection according to (1).

(4) The gene marker for cell detection according to (1), comprising a combination of a sense primer and an antisense primer capable of specifically amplifying the gene marker for cell detection according to (1) by a PCR method Primer for detecting.

(5) A microarray or DNA chip for detecting a marker gene for cell detection obtained by immobilizing at least one of the probes according to (2) or (3) on a support.

(6) Among gene markers having a base sequence described in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13 of the sequence listing in the test cell A method for detecting and / or separating bone marrow cells, comprising detecting the expression level of at least one gene marker and comparing the expression level with the same gene marker expression level in the bone marrow cells.
(7) A gene having the base sequence described in SEQ ID NO: 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or 19 in the sequence table in the test cell The expression level of at least one gene marker among the markers is detected, and the expression level of the same genetic marker in the cells after culturing bone marrow cells and before only mesenchymal stem cells are selectively cultured A method for detecting and / or isolating cells after culturing bone marrow cells, wherein only cells that have been mesenchymal stem cells are selectively cultured, including comparison.

(8) detecting the expression level of at least one of the genetic markers having the base sequence set forth in SEQ ID NO: 20, 21, 22, 23, 24 or 25 in the sequence listing in the test cell; A method for detecting and / or separating mesenchymal stem cells, comprising comparing the expression level of the same gene marker in mesenchymal stem cells.
(9) The expression level of at least one of the gene markers having the nucleotide sequence set forth in SEQ ID NO: 26, 27 or 28 in the sequence listing in the test cell is detected, and the same in the osteoprogenitor cell A method for detecting and / or separating osteoprogenitor cells, comprising comparing the expression level of a gene marker.

(10) detecting the expression level of at least one of the gene markers having the base sequence described in SEQ ID NO: 28, 29, 30, 31, 32, 33, 34, 35 or 36 in the sequence listing; A method for detecting and / or isolating osteoblasts, comprising comparing the expression level of the same gene marker in osteoblasts.
(11) The expression level of the gene marker is detected using the probe according to (2) or (3), the primer according to (4), or the microarray or DNA chip according to (5). ) To (10).

(12) In the step of culturing bone marrow cells and differentiating into target cells, the expression level of any one or more gene markers selected from the genes described in (a) to (i) below is detected, A culture step of differentiating into any one or more cells of (A) to (E) below, comprising comparing the expression level of the same gene marker in any one or more cells of (A) to (E) How to monitor.
(A) the nucleotide sequence of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13 in the sequence listing, which is a gene marker for detecting bone marrow cells (B) a gene marker for detecting a cell after culturing bone marrow cells but before only mesenchymal stem cells are selectively cultured, SEQ ID NO: 5, 6, A gene having the base sequence described in 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 or 19;
(C) a gene having a base sequence set forth in SEQ ID NO: 20, 21, 22, 23, 24 or 25 in the sequence listing, which is a gene marker for detecting mesenchymal stem cells;
(D) a gene having a base sequence set forth in SEQ ID NO: 26, 27 or 28 in the sequence listing, which is a gene marker for detecting osteoprogenitor cells;
(E) a gene having a base sequence set forth in SEQ ID NO: 28, 29, 30, 31, 32, 33, 34, 35 or 36 in the sequence listing, which is a gene marker for detecting osteoblasts;
(F) a gene that shows a difference in expression level between culturing bone marrow cells and culturing cells after culturing bone marrow cells but before only mesenchymal stem cells are selectively cultured. A gene having the nucleotide sequence set forth in SEQ ID NO: 37 or 7 in the sequence listing;
(G) It is a gene showing a difference in expression level between the culture of bone marrow cells and a cell before only mesenchymal stem cells are selectively cultured and the culture of mesenchymal stem cells A gene having the nucleotide sequence set forth in SEQ ID NO: 38 or 39 in the Sequence Listing;
(H) a gene having a base sequence set forth in SEQ ID NO: 40 or 21 in the sequence listing, which is a gene showing a difference in expression level between culture of mesenchymal stem cells and culture of osteoprogenitor cells;
(I) a gene having a base sequence set forth in SEQ ID NO: 28, 41 or 42 in the sequence listing, which is a gene showing a difference in expression level between culture of osteoprogenitor cells and culture of osteoblasts;
(A) Bone marrow cells (B) Cells after culturing bone marrow cells and before only mesenchymal stem cells are selectively cultured (C) Mesenchymal stem cells (D) Osteoprogenitor cells (E) Bone Blast

(13) The expression level of the gene marker is detected using the probe according to (2) or (3), the primer according to (4), or the microarray or DNA chip according to (5). ) Method.

  By using the gene marker of the present invention, it has become possible to detect and separate cells at each stage in the culture process in which bone marrow cells are differentiated into mesoblasts via mesenchymal stem cells. In addition, by using the gene marker of the present invention, cells at each stage in the culture process for differentiation from bone marrow cells to mesenchymal stem cells into osteoblasts can be monitored. That is, when a cell is infected with a virus or the like during culture, it can be distinguished from a cell included in a normal culture process. In addition, if there is a growth delay in the same culturing step, it can be distinguished from the cells in the originally intended step, so quality control in the culturing step becomes possible.

Hereinafter, the present invention will be described in more detail.
The present invention relates to a genetic marker for cell detection or separation or monitoring described in any of (i) to (ix) below.
(I) To detect bone marrow cells comprising a gene having the base sequence described in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or 13 in the sequence listing Genetic markers of
(Ii) Bone marrow cells comprising a gene having the base sequence described in SEQ ID NO: 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 in the sequence listing A marker for detecting a cell after culturing a cell but before only mesenchymal stem cells are selectively cultured;
(Iii) a genetic marker for detecting mesenchymal stem cells, comprising a gene having the base sequence set forth in SEQ ID NO: 20, 21, 22, 23, 24 or 25 in the sequence listing;
(Iv) a gene marker for detecting osteoprogenitor cells, comprising a gene having the nucleotide sequence set forth in SEQ ID NO: 26, 27 or 28 in the sequence listing; and (v) SEQ ID NO: 28, 29, 30 in the sequence listing. , 31, 32, 33, 34, 35 or 36, a gene marker for detecting osteoblasts, comprising a gene having the base sequence of
(Vi) A cell after culturing bone marrow cells and bone marrow cells, which is composed of the gene having the nucleotide sequence set forth in SEQ ID NO: 37 or 7 in the sequence listing and before only mesenchymal stem cells are selectively cultured. Genes that show differential expression levels between:
(Vii) A cell after culturing bone marrow cells consisting of a gene having the nucleotide sequence set forth in SEQ ID NO: 38 or 39 in the sequence listing, and a cell before only mesenchymal stem cells are selectively cultured and mesenchymal system Genes that show differential expression levels among stem cells:
(Viii) A gene showing a difference in expression level between a mesenchymal stem cell and an osteoprogenitor cell comprising a gene having the base sequence set forth in SEQ ID NO: 40 or 21 in the sequence listing:
(Ix) A gene showing a difference in expression level between osteoblasts and osteoprogenitor cells comprising a gene having the base sequence set forth in SEQ ID NO: 28, 41 or 42 in the sequence listing:

  Bone marrow is a soft tissue that fills the bone lumen and is a hematopoietic organ. Bone marrow fluid is present in the bone marrow and can be collected from the bone marrow. In the present specification, “bone marrow cell” refers to a cell present in bone marrow fluid. Bone marrow cells include erythrocytes, granulocytes, megakaryocytes, lymphocytes, adipocytes, and the like, as well as small amounts of mesenchymal stem cells, hematopoietic stem cells, vascular endothelial progenitor cells, and the like. Bone marrow cells can be collected from, for example, human iliac, long bone, or other bone from which bone marrow cells can be collected.

  “Mesenchymal stem cells” are bone marrow-derived stem cells having multipotency into osteoblasts, chondrocytes, adipocytes, myoblasts, fibroblasts, nerve cells, vascular endothelial cells and the like. The “osteoprogenitor cell” is a cell after inducing bone differentiation into a mesenchymal stem cell, which has not yet expressed a marker for mature osteoblasts. An “osteoblast” is a cell that expresses at least one marker of osteoblasts such as ALP activity and osteocalcin.

  In the present invention, bone marrow fluid is collected from the bone marrow and bone marrow cells in the bone marrow fluid are used. As described above, the bone marrow fluid contains various bone marrow cells such as mesenchymal stem cells, hematopoietic stem cells, vascular endothelial progenitor cells in addition to erythrocytes, granulocytes, megakaryocytes, lymphocytes, adipocytes and the like. That is, in the present specification, “bone marrow cell” means a group of cells composed of various cells obtained from bone marrow. By subjecting the bone marrow cells to the culture method described later, only the mesenchymal stem cells contained in the cell group are highly selectively cultured, and a cell group containing almost pure mesenchymal stem cells only is obtained. Can do. Furthermore, these mesenchymal stem cells can be differentiated into osteoblasts and the like by differentiation induction. In the method of the present invention, these steps can be reliably managed.

  Detected by the method described later with a gene marker for detecting mesenchymal stem cells, comprising the gene having the base sequence set forth in SEQ ID NO: 20, 21, 22, 23, 24 or 25 in the above (iii) Sequence Listing In the isolated mesenchymal stem cells, the cell population usually contains 70% or more, preferably 80% or more, more preferably 90% or more of mesenchymal stem cells.

The genes having the base sequences described in SEQ ID NOs: 1 to 36 in the sequence listing are the following genes, respectively.
Sequence number 1: Homo sapiens CREBBP / EP300 inhibitor 1 (CRI1), mRNA
Sequence number 2: Homo sapiens peroxiredoxin 1 (PRDX1), transcript variant 1, mRNA
Sequence number 3: Homo sapiens diazepam binding inhibitor (GABA receptor modulator, acyl-Coenzyme A binding protein) (DBI), mRNA
Sequence number 4: Homo sapiens cyclin L1 (CCNL1), mRNA
SEQ ID NO: 5: Homo sapiens annexin A2 (ANXA2), transcript variant 3, mRNA
Sequence number 6: Homo sapiens arginase, liver (ARG1), mRNA
SEQ ID NO: 7: Homo sapiens matrix metallopeptidase 9 (gelatinase B, 92 kDa gelatinase, 92 kDa type IV collagenase) (MMP9), mRNA
Sequence number 8: Homo sapiens peroxiredoxin 4 (PRDX4), mRNA
SEQ ID NO: 9: Homo sapiens ATP synthase, H + transporting, mitochondrial F0 complex, subunit d (ATP5H), nuclear gene encoding mitochondrial protein, transcript variant 1, mRNA
SEQ ID NO: 10: Homo sapiens NADH dehydrogenase (ubiquinone) 1 beta subcomplex, 3,
12kDa (NDUFB3), mRNA
SEQ ID NO: 11: Homo sapiens microsomal glutathione S-transferase 3 (MGST3), mRNA
SEQ ID NO: 12: Homo sapiens pIFI27-like protein mRNA, partial cds
SEQ ID NO: 13: Homo sapiens transgelin (TAGLN), transcript variant 2, mRNA
SEQ ID NO: 14: Homo sapiens CD163 molecule (CD163), transcript variant 1, mRNA
SEQ ID NO: 15: Homo sapiens allograft inflammatory factor 1 (AIF1), transcript variant 2, mRNA
SEQ ID NO: 16: Homo sapiens legumain (LGMN), transcript variant 1, mRNA
SEQ ID NO: 17: Homo sapiens cathepsin C (CTSC), transcript variant 1, mRNA
SEQ ID NO: 18: Homo sapiens CD14 molecule (CD14), transcript variant 1, mRNA
Sequence number 19: Homo sapiens interferon, gamma-inducible protein 30 (IFI30), mRNA
SEQ ID NO: 20: Homo sapiens insulin receptor substrate 2 (IRS2), mRNA
Sequence number 21: Homo sapiens latexin (LXN), mRNA
SEQ ID NO: 22: Homo sapiens TSC22 domain family, member 1 (TSC22D1), transcript variant 2, mRNA
SEQ ID NO: 23: Homo sapiens secreted protein, acidic, cysteine-rich (osteonectin)
(SPARC), mRNA
Sequence number 24: Homo sapiens connective tissue growth factor (CTGF), mRNA
Sequence number 25: Homo sapiens metallothionein 1H (MT1H), mRNA
SEQ ID NO: 26: Homo sapiens solute carrier family 40 (iron-regulated transporter), member 1 (SLC40A1), mRNA SEQ ID NO: 27: Homo sapiens similar to P311 protein (H. sapiens) (LOC157630), mRNA
SEQ ID NO: 28: Homo sapiens collagen, type XI, alpha 1 (COL11A1), transcript variant A, mRNA
SEQ ID NO: 29: Homo sapiens major histocompatibility complex, class II, DR alpha (HLA-DRA), mRNA
Sequence number 30: Homo sapiens fibulin 5 (FBLN5), mRNA
Sequence number 31: Homo sapiens frizzled-related protein (FRZB), mRNA
SEQ ID NO: 32: HLA-DP (DPB1 * 02012) = major histocompatibility complex class II antigen beta chain [human, LCL 45.1, mutant 45.EM19, mRNA Partial Mutant, 171 nt]
SEQ ID NO: 33: Homo sapiens nuclear protein 1 (NUPR1), transcript variant 2, mRNA
SEQ ID NO: 34: Homo sapiens high-mobility group box 2, mRNA (cDNA clone MGC: 2393 IMAGE: 2963045), complete cds
SEQ ID NO: 35: Homo sapiens heterogeneous nuclear ribonucleoprotein A1 (HNRPA1), transcript variant 1, mRNA
SEQ ID NO: 36: Homo sapiens cDNA clone IMAGE: 3680553

The genes having the base sequences described in SEQ ID NOs: 37 to 42 in the sequence listing are the following genes, respectively.
SEQ ID NO: 37: Homo sapiens cathepsin B (CTSB), transcript variant 1, mRNA
SEQ ID NO: 38: Homo sapiens collagen, type I, alpha 2 (COL1A2), mRNA
SEQ ID NO: 39: Homo sapiens interleukin 8 (IL8), mRNA
SEQ ID NO: 40: Homo sapiens pentraxin-related gene, rapidly induced by IL-1 beta (PTX3), mRNA
SEQ ID NO: 41: Homo sapiens apolipoprotein D (APOD), mRNA
SEQ ID NO: 42: Homo sapiens transforming growth factor, beta-induced, 68 kDa (TGFBI), mRNA

  The gene marker of the present invention measures the expression level in cells at each stage of differentiation from bone marrow cells through mesenchymal stem cells to osteoblasts using a known method such as a microarray method, and the like. Cell expression level in a cell at a specific differentiation stage can be distinguished from the expression level in a cell at all other differentiation stages, or It is selected as an index that it can be distinguished from the expression level. Since the gene marker of the present invention is also used for monitoring the culture process of cells at each stage of differentiation from bone marrow cells through mesenchymal cells to osteoblasts, expression in cells that are not normally differentiated What is distinguishable also when compared with the amount is preferable.

  Cells at each stage of differentiation from bone marrow cells used for selecting genetic markers to mesenchymal stem cells via osteoblasts are cultured from bone marrow cells as known as on-spec cells, while inducing differentiation, It can be prepared by observing morphological changes and proliferation under a microscope, or measuring bone matrix in the process of differentiation of mesenchymal stem cells into osteoblasts.

  In addition, cells that are not normally differentiated (hereinafter sometimes referred to as “off-spec cells”) are cells that have been cultured by changing the culture conditions in each culture step, specifically the passage number and culture solution composition. Cells cultured under different conditions, or cells that were not cultured normally, for example, cells differentiated other than bone, such as adipocytes and chondrocytes, cells contaminated with bacteria and viruses, and cancerous cells Different types of cells, such as fibroblasts, can be used.

  In the present invention, bone marrow cells and bone marrow cells are cultured by detecting the expression level of any one or more gene markers among the genes described in (i) to (ix) above in the test cells. And mesenchymal stem cells, mesenchymal stem cells, osteoprogenitor cells, or osteoblasts before being selectively cultured can be detected and / or separated. Furthermore, it is possible to monitor the culture process in which the test cells differentiate from bone marrow cells to mesenchymal stem cells into osteoblasts.

  In the present invention, bone marrow cells, cells after culturing bone marrow cells, and before mesenchymal stem cells are selectively cultured, mesenchymal stem cells, osteoprogenitor cells, or osteoblasts (hereinafter referred to as “ The expression level of the marker gene of the present invention in a test cell may be used for detection and / or separation of a cell from a bone marrow cell to an osteoblast (sometimes abbreviated as “cell from bone marrow cell to osteoblast cell”) As a detection method, a method known to those skilled in the art can be used. For example, Northern blotting can be used to detect the expression level of a gene at the mRNA level. In this case, a probe having a DNA sequence that hybridizes with the DNA sequence of the gene marker of the present invention under stringent conditions can be used. Detection of the expression level of the gene marker in “cells from bone marrow cells to osteoblasts” using the probe can be appropriately performed using a known method. For example, a DNA probe of an appropriate length is prepared from the DNA sequence of the gene marker shown in the sequence table, labeled with a radiolabel or a fluorescent label, etc., and this is hybridized with a test sample, and the amount of the label The method etc. which measure are mentioned. As the DNA probe, a probe comprising all or part of the antisense strand of the base sequence of the gene marker of the present invention shown in the sequence table can be used. The probe can also be used as a microarray or DNA chip by immobilizing at least one probe on a support.

  In the preparation of the DNA probe described above, the conditions for hybridizing with the gene marker under stringent conditions in the base sequence of the present invention include, for example, hybridization at 42 ° C. and 1 × SSC (0.15 M NaCl, 0.015M sodium citrate), 0.1% SDS (Sodium dodecyl sulfate), and a condition of washing at 42 ° C. with a buffer solution, more preferably hybridization at 65 ° C., and 0 The conditions may include washing at 65 ° C. with a buffer containing 1 × SSC and 0.1% SDS.

  The expression level of the marker gene can also be detected using quantitative or semi-quantitative PCR. RT-PCR (reverse transcription PCR) can be used as the PCR. In performing the PCR, a primer comprising a sense primer and an antisense primer for amplifying the gene marker of the present invention can be used. Further, only a primer for one gene marker may be added to the reaction solution for performing PCR, and the PCR may be performed for each gene marker, or primers for a plurality of gene markers may be simultaneously added to the reaction solution for performing PCR, The PCR may be performed on a plurality of gene markers at once.

  Specific examples of the probe and primer for detecting the cell detection marker gene of the present invention include those described in Table 4, but are not limited thereto, and those skilled in the art can Based on the base sequences of the marker genes described in SEQ ID NOs: 1 to 42, the base sequences of probes and primers that can detect the target marker gene can be appropriately set.

  In the present invention, the expression level of the marker gene can also be detected at the protein level using an antibody against the protein encoded by any one of SEQ ID NOs: 1 to 42 in the sequence listing. The above antibody may be a monoclonal antibody or a polyclonal antibody. The above-described antibody can be prepared by a conventional method using a protein encoded by any one of the nucleotide sequences of SEQ ID NOS: 1-42 or a partial peptide thereof as an antigen. In order to detect the expression of a gene marker in a test cell using the above-described antibody, for example, a known immunological measurement method such as an RIA method, an ELISA method, or a fluorescent antibody method can be used.

  When the expression level of the gene marker of the present invention in the test cell is detected using the above-mentioned antibody and “cells from bone marrow cells to osteoblasts” such as mesenchymal stem cells are separated by the method described later, it is known. By this method, “cells from bone marrow cells to osteoblasts” such as mesenchymal stem cells can be labeled and separated using the labeling strength or the like as an index. Examples of the labeling method include a fluorescent antibody method. In order to label a protein encoded by a gene marker expressed in “cells from bone marrow cells to osteoblasts” such as mesenchymal stem cells by the fluorescent antibody method, it is specific to the protein encoded by the gene marker of the present invention. The antibody that binds to the protein is fluorescently labeled, and this is bound to “cells from bone marrow cells to osteoblasts” such as mesenchymal stem cells that express the protein encoded by the gene marker, and the cells are labeled (Direct fluorescent antibody method) or a secondary antibody (anti-immunoglobulin antibody) after binding an unlabeled specific antibody to a mesenchymal stem cell expressing a protein encoded by a genetic marker ) To label a protein encoded by a genetic marker in “cells from bone marrow cells to osteoblasts” such as mesenchymal stem cells (indirect fluorescent antibody method), The reduction cells, separating the labeled quantity as an index, may be taken.

  In the present invention, the above-described probe or antibody is used to detect the expression level of the gene marker of the present invention in a test cell, thereby identifying the state of the cell and culturing bone marrow cells and bone marrow cells. It is possible to isolate and obtain a cell, a mesenchymal stem cell, an osteoprogenitor cell, or an osteoblast before only the mesenchymal stem cell is selectively cultured.

  For the specific detection of “cells from bone marrow cells to osteoblasts”, the method of identifying the cells is to detect the expression level of one or more gene markers in the test cells as described above, This is compared with the expression level of the same gene marker in “cells from bone marrow cells to osteoblasts” as an index. The comparison of the expression level of the gene marker may be an absolute value of the expression level of one gene marker or a ratio of the expression levels of two or more gene markers.

  The expression level of the same gene marker in “cells from bone marrow cells to osteoblasts” was confirmed to be a specific cell by the method described later (hereinafter, sometimes referred to as “on-spec cell”) It is preferable to obtain in advance as the expression level of the same gene marker (hereinafter sometimes referred to as “on-spec cell reference value”).

  When obtaining the reference value, the on-spec cell may be a single culture system, but is preferably obtained from a plurality of culture systems and used, and the expression level of each gene marker is statistically processed in the test cell. It is preferable to compare with the expression level of the marker gene. These analyzes can be easily performed using a known method such as statistical analysis software R using the R language or S-PLUS, SPSS, SAS, StatView using the S language.

  The genetic markers to be compared are those that are known to show expression levels different from other differentiation stages in any differentiation stage of cells from bone marrow cells to osteoblasts to be detected and / or separated. Use. Specific examples include those described in (i) to (ix) above. Among these, use one or more, or use any of multivariate analysis in which a plurality of gene markers are combined and analyzed. Can do. In order to determine a certain differentiation stage, when only the differentiation stage is to be measured, those described in the above (i) to (v) are preferable, while the differentiation stage and before the differentiation stage are preferred. In addition, in the case where the subsequent differentiation stage is a measurement target, in addition to those described in (i) to (v) above, the above (vi) to (vi) to show a remarkable expression level difference between two adjacent steps. Those described in (ix) are also preferable.

  When one gene marker is used, specifically, for example, using a plurality of specific on-spec cells, the average expression level ± 2XSD or ± 3XSD of the expression level of the marker gene in the cell is the reference value of the on-spec cell. And the determination method using as an index that the expression level of the marker gene in the test cell falls within this reference value is used.

  In addition, using a plurality of specific on-spec cells, an ROC curve is drawn for the expression level of the marker gene in the cells, and this is used as a reference value to determine the cut-off value of the on-spec cells. It can also be determined as an index whether or not the expression level of the marker gene is included therein. Moreover, cluster analysis etc. can also be used.

  On the other hand, when analyzing two or more gene markers, for example, for the expression level of the marker in a specific on-spec cell, a reference value or a range thereof is determined by the same method as described above, and the expression level of the marker in the test cell is determined. It can be determined whether or not the expression level of the marker falls within this reference range. Specific analysis methods include prediction (multiple regression analysis, quantification type I), discrimination (discriminant analysis, quantification type II), machine learning (nearest neighbor method, decision tree, support vector machine, neural network), phase Similarity analysis using the number of relations or distance function is used.

  As another example, based on the expression level of two or more gene markers, the reference value group of on-spec cells and the expression level value group of the marker in the test substance are compared with the group as a group. Can be determined. Specific analysis methods include cluster analysis, multidimensional scaling, principal component analysis, factor analysis, quantification type III, quantification type IV, self-organizing map, network estimation (boolean network, Bayesian network), etc. Use.

  More specifically, for example, in the determination method using the Spearman correlation coefficient, the correlation coefficient value is 0.6 or more, preferably 0.8 or more, more preferably 0 with respect to the reference value of the on-spec cell. When it is 9 or more, the test cell is determined to be a cell at the specific differentiation stage.

  In the hierarchical cluster analysis, if the expression level value of the test cell is included in the reference value group (cluster) of the on-spec cell, it is determined that the cell is in a specific differentiation stage, and a different cluster is formed. It can be determined that the cells are not in the differentiation stage.

  In the multidimensional scaling method based on the Euclidean distance, when the expression level value of the test cell is plotted in the same region where the reference value of the on-spec cell is plotted, the cell is in a specific differentiation stage. And when plotted in a distant region, it can be determined that the cell is not in the differentiation stage.

  Thus, cells determined to be any cell from bone marrow cells to osteoblasts can be separated by a known method.

  In the present invention, the cell culture process from bone marrow cells to osteoblasts can also be monitored (hereinafter sometimes referred to as “monitoring”) using the gene marker. Monitoring means that cells in each differentiation stage are detected by the method described above, and whether or not the cells in each culture process are on-spec cells and is intended for quality control in the culture process It is.

  Each culture step to be monitored includes (A) culture of bone marrow cells, (B) culture of cells after culturing bone marrow cells and before only mesenchymal stem cells are selectively cultured, and (C) Leaf culture stem cell culture, (D) osteoprogenitor cell culture, and (E) osteoblast culture process.

  Detection of the expression level of the gene marker for monitoring may be performed each time in each culture step, or may be performed across a plurality of steps. Moreover, after all the culture | cultivation processes which are scheduled, you may perform with respect to all or one part of each culture | cultivation process.

  Detection of the expression level of the gene marker in each culture step, comparison with the reference value of the on-spec cell, and determination can be performed by obtaining a part of the cells in the culture step to be measured as test cells and using the above method. .

  A combination of culture steps for monitoring can be determined as appropriate, and one or more of the culture steps (A) to (E) can be monitored very easily depending on the purpose. For example, when culturing mesenchymal stem cells from bone marrow cells (corresponding to steps (A) to (C) above), the above (a), (b), (c), (f) and (g) Monitoring may be performed using one or more genes selected from the genes described in. Preferably, any one or more gene markers selected from the gene markers described in (a), (b), (c) are used, and more preferably, described in (a), (b), (c) One or more of these genetic markers may be selected and monitored for each step.

  In the case of performing differentiation-inducing culture from mesenchymal stem cells to osteoblasts (corresponding to the above steps (C) to (E)), the above (c), (d), (e), (h ) And (i) may be monitored using any one or more genes selected from the gene markers described in (i). Preferably, any one or more gene markers selected from the gene markers described in (c), (d), (e) are used, and more preferably, described in (c), (d), (e) Any one or more of these genetic markers may be selected and used for each step.

  A probe for detecting the gene marker of the present invention, a microarray or DNA chip on which the probe is immobilized, a primer for amplifying the gene marker of the present invention, and an antibody against the protein encoded by the gene marker of the present invention, It can be commercialized as a kit for identifying “cells from bone marrow cells to osteoblasts” such as mesenchymal stem cells equipped.

  EXAMPLES Hereinafter, although an Example demonstrates this invention more concretely, the technical scope of this invention is not limited to these illustrations.

Example 1 [Material Preparation]
(Preparation of culture reagents)
10% fetal bovine serum and antibiotics are added to Dulbecco's Modified Eagle Medium (manufactured by Invitrogen) to the mesenchymal stem cell culture solution from bone marrow fluid (hereinafter referred to as “mesenchymal stem cell culture solution”), and the final concentration is 50 μg. A medium supplemented with / mL vitamin C and a final concentration of 10 ng / mL bFGF was prepared.

  10% fetal bovine serum and antibiotics are added to Dulbecco's Modified Eagle Medium (manufactured by Invitrogen) in the differentiation-inducing medium for osteoblasts (hereinafter referred to as osteoblast-inducing culture medium), and a final concentration of 50 μg / mL vitamin A medium supplemented with C, a final concentration of 100 nM dexamethasone and a final concentration of 10 mM β-glycerophosphate was prepared.

  10% fetal bovine serum and antibiotics are added to Dulbecco's Modified Eagle Medium (manufactured by Invitrogen) to the differentiation induction medium for chondrocytes (hereinafter referred to as “cartilage differentiation induction culture medium”), and a final concentration of 50 μg / mL vitamin C, a medium supplemented with dexamethasone at a final concentration of 100 nM, TGFβ3 at a final concentration of 10 ng / mL, and ITS was prepared.

  10% fetal bovine serum and antibiotics were added to Dulbecco's Modified Eagle Medium (Invitrogen), and dexamethasone at a final concentration of 1 μM was added to the adipocyte differentiation induction medium (hereinafter referred to as “adip differentiation induction culture medium”). A medium supplemented with 10 μg / mL insulin, final concentration of 0.2 μM indomethacin and final concentration of 0.5 mM IBMX (3-isobuthyl-1-methyl xanthine: Sigma) was prepared.

  The culture solution of mesenchymal stem cells used for studying the conditions of the culture solution (hereinafter referred to as “mesenchymal stem cell culture solution (for study of conditions)”) is Dulbecco's Modified Eagle Medium Nutrient Mixture F-12 (manufactured by Invitrogen) Was prepared by adding 10% fetal bovine serum and antibiotics.

(Cell culture)
A mesenchymal stem cell culture medium was used for the proliferation culture of human-derived mesenchymal stem cells. An osteoblast differentiation culture medium was used for bone differentiation culture of human-derived mesenchymal stem cells, a cartilage differentiation induction culture medium was used for cartilage differentiation culture, and a fat differentiation induction culture medium was used for fat differentiation culture. For growth culture of human fibroblasts, fibroblast medium kit-2 (2% FBS) (manufactured by Cambrex) or mesenchymal stem cell culture solution was used. These cells were cultured at 37 ° C. and 5% carbon dioxide gas concentration.

(Culture process)
A basic protocol for a series of culturing procedures after separating mesenchymal stem cells from bone marrow fluid, through proliferation culture and differentiation into osteoblasts, chondrocytes, and adipocytes is shown below.

Mesenchymal stem cell proliferation culture from bone marrow fluid: On day 1, bone marrow fluid was suspended in mesenchymal stem cell culture solution and seeded in a flask. On the 4th day, 70% of the culture broth was changed, and thereafter, the whole medium was changed every 3-4 days. When the cells became confluent, the cells were passaged. When the cells became confluent again, the cells were collected and used for differentiation induction.
Osteoblast differentiation induction: The collected cells were suspended and seeded in an osteoblast differentiation medium. The whole medium was changed every 3 to 4 days, and differentiation culture was performed for 14 days.
Induction of chondrocyte differentiation: The collected cells were suspended and seeded in a mesenchymal stem cell culture medium. The day after sowing, the medium was replaced with a cartilage differentiation-inducing culture medium, and the whole medium was replaced every 3 to 4 days, and differentiation culture was performed for 21 days.
Adipocyte differentiation induction: The recovered cells were suspended and seeded in a mesenchymal stem cell culture medium. The day after sowing, the culture medium was replaced with an adipose differentiation-inducing culture medium, and the whole medium was replaced every 3 to 4 days, followed by differentiation culture for 14 days.

(Recovery of cells)
Cells were collected in the following eight culture steps (stage) (FIG. 1).
Stage 1: Purchased bone marrow fluid Stage 2: Early culture cells on the 4th day of culture Stage 3: Passage 1 culture medium phase Stage 4: Passage 2 culture late cells Stage 5: Cells cultured for 1 day after induction of differentiation Stage 6: Early differentiation cells cultured for 4 days after differentiation induction Stage 7: Middle differentiation cells cultured for 7 days after differentiation induction Stage 8: Late differentiation cells cultured for 14 days after differentiation induction

(Definition of on-spec model and off-spec model)
Cells that were normally cultured under the above-mentioned prescribed culture conditions between bone marrow fluid and bone differentiation were defined as an on-spec model. On the other hand, cells cultured under different conditions in this step, specifically cells cultured by changing passage number and culture solution composition, or cells that were not cultured normally, such as fat cells, Off-spec models were chondrocytes, cells differentiated other than bone, cells contaminated with bacteria / viruses, cancerous cells, cells of different lineages, such as fibroblasts.

Example 2 [microarray analysis]
(Microarray experiment method)
After extracting total RNA from cells in stages 1 to 8 of the culture process shown in FIG. 1 using RNeasy Mini Kit (QIAGEN), complementary mRNA is amplified using Amino Allyl Message Amp aRNA Kit (Ambion), 5 μg of the mixture was hybridized with a DNA microarray (AceGene Human oligo chip 30K 1 chip version). The hybridization method followed AceGene ^R- 1 Chip Version-instruction manual. Further, as a reference, a bone marrow RNA mixed solution purchased from Becton Dickinson was used. A mixed solution of bone marrow RNA was used to amplify complementary mRNA in the same manner as described above, and this RNA was used as a reference for all DNA microarray experiments. Scan Array Express (PerkinElmer) was used for scanning of the DNA microarray. ImaGene (manufactured by BioDiscovery) was used for digitizing the acquired scanning image, and GeneSight (manufactured by BioDiscovery) was used for normalization. For statistical processing of microarray data, spreadsheet software Excel (manufactured by Microsoft) and statistical analysis software R (free software) were used.

(Microarray analysis results)
1. Selection of marker gene candidates From the bone marrow fluid to bone differentiation, each of the 5 samples (A, B, C, D, E) using the on-spec model that was successfully cultured as 8-stage cells shown in Fig. 1, Expression profile data was obtained from

  Regarding the expression level of each gene, first, the average value of the above five samples was calculated, and genes having a difference between the maximum and the minimum of 2 times or more were extracted. From the extracted gene group, A specimen out of 5 specimens is used as a reference, and Pearson correlation coefficients of specimen A and the other 4 specimens are expressed as specimens A and B, specimens A and C, for the 8-stage variation pattern. Calculations were made for combinations of Samples A and D and Samples A and E, respectively. The average value of the calculated Pearson correlation coefficient was calculated, and 2080 genes showing 0.4 or more were extracted.

2. Extraction of gene groups characteristic to each culture step For the 2080 gene extracted in step 5, the 8-stage culture process shown in FIG. 1 is performed in five groups (group 1 = stage 1 (bone marrow fluid), group 2 = stage 2, group 3 = stages 3 and 4 (between (Leaf stem cells), group 4 = stage 5 (osteoprogenitor cells), group 5 = stages 6, 7, and 8 (osteoblasts)), multiple comparison test according to the decision tree flow shown in Figure 3 Went.
As a result of the multiple comparison test per group total, 1. From the 2080 genes extracted in (1), 36 genes (P <0.01) (Table 1) that can define each group by specific expression level, and (2) significant expression difference between two adjacent groups Nine genes (overlap with (1) were 3 genes) (Table 2) were narrowed down. The 42 genes excluding duplication from the total 45 genes were hereinafter referred to as “marker candidate genes”.

  Of the 36 genes in (1), 9 genes were duplicated in 13 genes defining group 1 and 15 genes defining group 2. However, even if the genes are the same, the expression level detected in the cells recovered from the bone marrow fluid of group 1 is significantly different from the expression level detected in the cultured early cells of group 2. It turns out that it can be clearly defined. The same was true for genes that overlap between other groups.

Example 3 [Verification of genes capable of defining each group of culture process]
(Preparation of off-spec model)
As an off-spec model, cells differentiated from a sample of mesenchymal stem cells other than bone were prepared. Specifically, cartilage differentiated cells (differentiation cultures 4 days and 14 days) and adipose differentiation cells (differentiation cultures 4 days and 14 days) were prepared. Each cell was cultured under the conditions described in Example 1. In addition, mesenchymal stem cells artificially inoculated with fibroblasts (Cambrex, product code CC-2511) and HSV-I (herpes simplex type I) virus were prepared. Two types of fibroblasts were prepared using fibroblast medium kit-2 (2% FBS) (manufactured by Cambrex) or mesenchymal stem cell culture medium. The mesenchymal stem cells artificially inoculated with HSV-I (herpes simplex type I) virus were inoculated with about 10 ⁷ copies of HSV-I to the stage 4 mesenchymal stem cells shown in FIG. The culture was continuously prepared for 72 hours.

(Microarray analysis results)
Expression profile data was obtained for all cells prepared as an off-spec model according to the method described in Example 2.
Example 2-2. 36 genes (Table 1) predicted to be able to define groups 1-5 of the culture process extracted in step 1 were evaluated by adding off-spec model data to on-spec 5 specimen data. Then, there was a difference in gene expression level in each group, and the 36 genes (Table 1) showed a particularly large difference in expression level. Further, even in off-spec cells, there was a difference in the expression level of the 36 genes, and it was confirmed that these 36 genes can distinguish the on-spec cells of each group from other cells. Representative examples of this result are shown in FIGS. 4 to 8, groups 1 to 5 show the expression levels of the 36 genes in the on-spec cells of each group. Chondrocytes, adipocytes, fibroblasts, and HSV-I inoculated mesenchymal stem cells are off-spec cells. The expression level of a gene that can define each of the above groups in the cell is shown.

  From these results, it was found that these 36 genes (Table 1) can be gene markers that can define each group depending on their expression levels.

Example 4 [Verification of whether or not a culture process can be identified-1]
(Stage discrimination confirmation of 5 on-spec standard specimens by hierarchical clustering)
Hierarchical clustering analysis using the statistical analysis software R (free software) was performed on the expression level of 42 candidate genes for 5 on-spec standards (A, B, C, D, E). In each group, all on-spec reference samples were classified into the same cluster. From this, it was possible to discriminate among the five culture process groups by hierarchical clustering analysis based on the expression level of the marker candidate 42 gene (Group 1: on-spec A, B, C, D, E_stage 1 in FIG. 9). , Group 2: On-Spec A, B, C, D, E_Stage 2, Group 3: On-Spec A, B, C, D, E_ Stage 3 and Stage 4, Group 4: On-Spec A, B, C , D, E_stage 5, group 5: On-spec A, B, C, D, E_ stage 6, stage 7 and stage 8).

Example 5 [Verification of whether culture process is discriminable-2]
(Stage discrimination confirmation with additional samples added)
In order to verify the validity of the 42 genes selected for the purpose of discriminating the culture process, 6 new on-spec models (F, G, H, I, J, The same analysis as in Example 4 was performed using K). The expression profile data of stage 4 and stage 6 shown in Fig. 1 is obtained using a microarray, and together with the data obtained from 5 reference specimens (A, B, C, D, E), the marker candidate 42 genes Hierarchical clustering analysis was performed using the expression level. As a representative example of the results of the hierarchical clustering analysis, the results for two stages (stages 4 and 6) of six on-spec models (F, G, H, I, J, K) are shown in FIG. As shown in FIG. 9, it was confirmed that 6 on-spec models were included in the same cluster as the case of 5 reference samples (A, B, C, D, E) (Group 3: On-spec in FIG. 9). F, G, H, I, J, K_stage 4, on-spec F, G, H, I, J, K_stage 6). From these results, it was shown that by acquiring 42 gene expression profile data, it was possible to monitor that the culture process had taken a normal course.

Example 6 [Verification of whether culture process is discriminable-3]
(Verification using off-spec)
1. Verification using adipose differentiated cells Gene expression data of cells differentiated into fat obtained in Example 3 (differentiated culture 4 days, 14 days) is added to the on-spec model data, and the hierarchical clustering analysis described above Went. As a result, it was confirmed that cells differentiated into fat form different clusters from any culture stage (fat differentiation 4 day and fat differentiation 14 day in FIG. 9). From this, it was found that abnormalities that change normal culture, such as differentiation into other than osteoblasts, can be monitored during the culture process.

2. Verification using fibroblasts The gene expression data of two off-spec (fibroblast) samples obtained in Example 3 were added to the on-spec model data, and the above-described hierarchical clustering analysis was performed. As a result, it was confirmed that two fibroblast specimens formed different clusters from any culture stage of mesenchymal stem cells (fibroblast 1 and fibroblast 1.1 in FIG. 9). This shows that mesenchymal stem cells and fibroblasts that are similar in cell morphology can be distinguished very clearly.

3. Verification using mesenchymal stem cells inoculated with virus The gene expression data of off-spec (mesenchymal stem cells artificially inoculated with HSV-I virus) obtained in Example 3 was added to the on-spec model data. Thus, the hierarchical clustering analysis described above was performed. As a result, it was confirmed that cells artificially inoculated with virus formed clusters different from all culture stages of normally cultured mesenchymal stem cells (72 hr after HSVI inoculation in FIG. 9). This suggests the possibility of monitoring abnormalities that affect normal culture such as viral contamination during the culture process.

Example 7 [Verification by real-time PCR]
(Real-time PCR method)
Total RNA was extracted from the test cells using RNeasy Mini Kit (QIAGEN), and then cDNA was synthesized using 1 μg of total RNA as a template. The synthesis procedure was in accordance with the protocol (First-Strand cDNA Synthesis Using SuperScript II RT) described in the attached material of Super Script II Reverse Transcriptase (Invitrogen). For the primer, Random Primer (6mer) (TaKaRa) was used. After synthesizing cDNA, the synthesized cDNA solution was diluted 5-fold. Real-time PCR was performed using 1 μL of this diluted solution as a template. The primer / probe reagents used are shown in Table 3.

About the measurement, ABI PRISM 7700 Sequence Detector (made by ABI) was used, and it implemented by the probe method or SYBR GreenI method. If the probe method ^{is, TaqMan R Universal PCR Master Mix,} No AmpErase reaction solution ^R UNG (ABI Company), in the case of SYBR Green I method reaction solution SYBR ^R GREEN PCR Master Kit (ABI Company) Was used. The probes beginning with “Hs” shown in Table 3 were manufactured by ABI and indicated product numbers.

(Comparison with microarray data)
The gene expression data of the DNA microarray is quantified as a relative value, but the expression level analysis by real-time PCR is quantified as an absolute value. For 42 genes selected for the purpose of discriminating the culture process, gene expression data was evaluated by real-time PCR. For the five on-spec model standards used in Example 2, the gene expression level of the cells at the 8th stage of the culturing process was measured using real-time PCR. Of the five reference samples, data on the stage 2 of the sample B and stages 1 and 2 of the sample D were insufficient because the total RNA amount was insufficient. After measuring the Ct value of 42 genes, ΔCt value (40-Ct value) was calculated. From the calculated ΔCt value, the expression variation with time in the 8 stages of the culture process is shown in a graph (the graph on the left side of FIGS. 10 to 15), and the expression variation with time of the microarray obtained in Example 2 (of FIGS. 10 to 15). (Right graph).

  As a result, regarding the 32 genes (Table 4) that can define each culture process group and the 9 genes (Table 2) that show a significant difference in expression between the two adjacent groups, the expression variation of 38 genes excluding 3 overlapping genes It was confirmed that the pattern showed a similar type (FIGS. 10 to 15). Furthermore, for the 38 genes that showed similar types, as in the microarray data, the genes that can define group 1 are significantly different (P <0.01) from all other groups, and groups 2 to 5 can be defined. The same was true for genes.

Example 8 [Verification of genes capable of defining each group of culture steps]
(Acquisition of gene expression data of off-spec model by real-time PCR)
Cells obtained by bone differentiation from mesenchymal stem cells prepared in Example 3 (differentiated culture 4 days and 14 days), cartilage differentiated cells (differentiated culture 4 days and 14 days), and fat differentiated cells ( Differentiated cultures (4th and 14th), 2 types of fibroblasts prepared using 2 cultures, and mesenchymal stem cells artificially inoculated with HSV-I (herpes simplex virus) virus Expression profile data of marker candidate genes in the middle were obtained according to the method described in Example 7 using real-time PCR.

  For 32 genes (Table 4) that are predicted to be able to define each group of the culture process shown in Example 7, the data of the on-spec standard 5 samples (A, B, C, D, E) and the data of the off-spec model In addition, in the on-spec cells, there was a difference in the expression level of the gene in each group, and the 32 genes (Table 4) showed a particularly large difference in the expression level. Moreover, even in off-spec cells, there was a difference in the expression level of the 32 genes, and it was confirmed that these 32 genes can distinguish the on-spec cells of each group from other cells. Representative examples of the results are shown in FIGS. 16 to 20, groups 1 to 5 show the expression levels of the 32 genes in the on-spec cells of each group, and chondrocytes, adipocytes, fibroblasts, and HSV-I inoculated mesenchymal stem cells are off-spec cells. The expression level of the gene in the cell is shown.

  From this, it was found that these 32 genes can be gene markers that can define each group depending on their expression levels.

  From gene expression data by real-time PCR of 5 on-spec standards (A, B, C, D, E) and off-spec model, group 3 out of 32 genes that can define each culture process group shown in Example 7 3 genes (IRS2, LXN, and TSC22D1) that can regulate the number of genes and 3 genes (COL11A1, FBLN5, and FRZB) that are significantly different in the two adjacent groups of group 5 are selected, and 2 by multidimensional scaling based on Euclidean distance Dimensional plot analysis was performed. The results are shown in FIGS. 21 and 22, A to E shown in the plots indicate specimens, and the numbers indicate stages (FIGS. 1 and 2). In FIG. 21, the stages 3 and 4 of the reference specimens A, B, C, D, and E of the group 3 are all present in the same region. In FIG. 22, the stages 6, 7, and 8 of the group 5 reference specimens A, B, C, D, and E are all present in the same region. The plots in the group of specimens defined by each marker gene were concentrated in a close range and showed a distribution different from the plots of other groups and off-spec model specimens. For multidimensional scaling, total analysis software R (free software) was used.

Example 9 [Verification-1 of whether or not the culture process can be discriminated]
(Stage discrimination check of five reference samples (A, B, C, D, E) by hierarchical clustering)
Among the 32 genes that can define each culture process group (Table 4) and 9 genes that show a significant difference in expression between the two adjacent groups (Table 2), the expression profile data of marker candidate genes in each cell, Obtained according to the method described in Example 7 using real-time PCR.

  A hierarchical clustering analysis was performed using the statistical analysis software R (free software) for the ΔCt values (40-Ct values) of a total of 38 genes excluding the overlapping 3 genes. Samples were classified into the same cluster. From this, it was possible to discriminate among the five culture steps (Group 1: On-Spec A, B, C, E_Stage 1, Group 2: On-Spec A, C, E_Stage 2, Group 3: On-Spec A, B, C, D, E_Stage 3 and Stage 4, Group 4: On-Spec A, B, C, D, E_Stage 5, Group 5: On-Spec A, B, C, D , E_stage 6 and stage 7 and stage 8).

Example 10 [Verification of whether culture process is discriminable-2]
(Stage discrimination confirmation with additional samples added)
In order to verify the validity of the 38 genes selected for the purpose of discriminating the culture process, 6 new on-spec models (F, G, H, I, J, The gene expression data of all culture stages of K) were obtained according to the method described in Example 7 using real-time PCR.

  As a result of hierarchical clustering analysis by adding six on-spec model samples (F, G, H, I, J, K), each culture stage of the on-spec model six samples has five reference samples (A, B , C, D, E) are confirmed to be included in the same cluster (group 1: on-spec G, H, J, K_stage 1, group 2: on-spec H, J, K in FIG. 23). _Stage 2, group 3: On-spec F, G, H, I, J, K _Stage 3 and stage 4, group 4: On-spec F, G, H, I, J, K _Stage 5, group 5: On-spec F, G, H, I, J, K _ stage 6 and stage 7 and stage 8). From this result, it was shown that by monitoring the gene expression data of 38 genes, it was possible to monitor the normal progress of the culture process.

Example 11 [Verification of whether culture process is discriminable-3]
(Verification using off-spec)
1. Verification using chondrogenic differentiated cells and adipose differentiated cells Cells differentiated into cartilage obtained in Example 8 (differentiated culture 4 days, 14 days)) and fat differentiated cells (differentiated culture 4 days, 14 days) Hierarchical clustering analysis was performed on the gene expression data of Japan and the on-spec model gene expression data in the same manner as in Example 10. As a result, it was confirmed that cartilage differentiated cells and adipose differentiated cells form clusters different from any culture stage (cartilage differentiation 4 day, cartilage differentiation 14 day, adipose differentiation 4 day and adipose differentiation 14 day in FIG. 23). This indicates that abnormalities that change normal culture, such as differentiation into other than osteoblasts, can be monitored during the culture process.

2. Verification using fibroblasts Hierarchical clustering analysis was performed in the same manner as in Example 10 for the gene expression data of the two fibroblasts obtained in Example 8 and the gene expression data of the on-spec model. As a result, it was confirmed that the two fibroblast specimens formed different clusters from any culture stage of mesenchymal stem cells (fibroblast 1 and fibroblast 2 in FIG. 23). This shows that mesenchymal stem cells and fibroblasts that are similar in cell morphology can be distinguished very clearly.

3. Verification using mesenchymal stem cells inoculated with the virus Gene expression data of mesenchymal stem cells artificially inoculated with the HSV-I (herpes simplex virus type I) virus obtained in Example 8 were expressed in the on-spec model. Hierarchical clustering analysis was performed on the data in the same manner as in Example 10. As a result, it was confirmed that the cells artificially inoculated with the virus formed clusters different from all the culture stages of normally cultured mesenchymal stem cells (72 hr after HSVI inoculation in FIG. 23). From this, it was found that abnormalities that change normal culture such as virus contamination during the culture process can be monitored.

4). Verification of the effect of culture conditions on gene expression profile From the mesenchymal stem cells in stage 4 shown in Fig. 1, as mesenchymal stem cells A with 4 passages added in normal culture medium as off-spec cells After the fourth passage, the mesenchymal stem cell B changed to the mesenchymal stem cell culture medium (for condition study) described in Example 1 was prepared, and gene expression data was obtained using real-time PCR. I got it.

  Hierarchical clustering analysis was performed on the gene expression data of 11 on-spec models (A to K) and the gene expression data of the mesenchymal stem cells A and B obtained above in the same manner as in Example 10. As a result, it was found that these mesenchymal stem cells A and B form a cluster different from any onspec cell (FIG. 24, mesenchymal stem cells A and B, group 1, group 2, group 3, group 4, group 5).

  In addition, using the 38 genes selected in Example 10, mesenchymal stem cells (group 3) and osteoblasts (group 5) of 11 on-spec models, and mesenchyme cultured as modified as described above Stem cells A and B were plotted in a two-dimensional diagram by multidimensional scaling based on Euclidean distance. The result is shown in FIG. A to K shown in the plots in the figure indicate specimens, and the numbers indicate stages (FIGS. 1 and 2). The mesenchymal stem cell population (group 3) is separated from the on-spec model in the order of mesenchymal stem cell A and mesenchymal stem cell B, and is also separated from the osteoblast (group 5). Both were remarkably open (Group 3 in FIG. 25: Stages 3 and 4 of reference specimens A, B, C, D, E, Stages 3 and 4 of additional specimens F, G, H, I, J, K, Group 5: Stages 6 and 7 and 8 of reference specimens A, B, C, D and E, stages 6 and 7 and 8 of additional specimens F, G, H, I, J and K, mesenchymal stem cell A, Leaf stem cells B). From this, it was shown that the expression profile changes depending on the passage number of cells and the composition of the culture solution, and it was confirmed that exposure to different culture conditions can be monitored based on such changes in expression profile. .

Example 12 [Examination of culture stage and cell quality judgment method]
(Example 1: Judgment using correlation coefficient)
A representative 23 genes (Table 5) were selected from 32 genes (Table 4) capable of defining each group of culture steps shown in Example 7. For cells belonging to group 1 of 5 on-spec model standards (A, B, C, D, E), the average expression level for each gene was calculated, and the expression pattern of the 23 genes was determined as the reference value for group 1. Stipulated. Such calculation was similarly performed for groups 2 to 5, and the expression pattern of 23 genes serving as a reference for each group was calculated (FIG. 26. Group 1: Onspec A, B, C, E_stage 1 Reference values for pair 1; group 2: on-spec A, C, E_stage 2 vs. reference value for group 2; group 3: on-spec A, B, C, D, E_ stage 3 and 4 vs. group 3 Reference values, group 4: On-spec A, B, C, D, E_stage 5 vs. group 4 reference values, group 5: On-spec A, B, C, D, E_stages 6 and 7, and 8 pairs Reference value of 5).

  Next, for two additional on-spec samples (G, K) and off-spec models used for verification, the Spearman phase relationship between the 23 gene expression patterns of each cell and the five reference expression patterns from Group 1 to Group 5 Numbers were calculated. As a result, the cells in group 1 of the two on-spec additional samples had the highest correlation coefficient with respect to the reference value of group 1 compared to the reference value for the other groups, and the value was 0.9 or more ( 26. On-spec G, K_stage 1 vs. group 1 reference value in FIG. Similar results were obtained for groups 2 to 5 (group 2: on-spec K_stage 2 vs. group 2 reference values in FIG. 26, group 3: on-spec G, K_stage 3 and 4 pairs in FIG. 26). 3 reference values, group 4: reference values for on-spec G, K_stage 5 vs. group 4 in FIG. 26, group 5: reference values for on-spec G, K_stages 6 and 7 and 8 vs. group 5 in FIG. ).

  On the other hand, the off-spec model mesenchymal stem cells B, adipose differentiated cells, 2 specimens of fibroblasts, and HSV-I inoculated cells had a correlation coefficient of less than 0.9 with respect to the reference value of any group. (FIG. 26). However, the mesenchymal stem cell A was 0.9 or more with respect to the reference value of the mesenchymal stem cell. For chondrocytes, the correlation coefficient was 0.9 or more with respect to the reference value of osteoblasts (FIG. 26).

(Example 2: Judgment using Euclidean distance function)
A representative 23 genes (Table 5) were selected from 32 genes (Table 4) capable of defining each group of culture steps shown in Example 7. For cells belonging to group 1 of 5 on-spec standards (A, B, C, D, E), the average expression level was calculated for each gene, and the 23 parameters were defined as the central coordinates of group 1. Such calculation was similarly performed for the groups 2 to 5, and the center coordinates of each group were calculated.

Next, for the two on-spec standards (G, K) used for verification and the above-mentioned off-spec model, the expression level of 23 genes in each cell is the coordinates of each cell, and each center from group 1 to group 5 The Euclidean distance from the coordinates was calculated. As a result, the cells of group 1 tended to gather at a close distance from the central coordinates of group 1. A similar trend was also shown for groups 2-5. As an example, distances from the central coordinates of group 3 (mesenchymal stem cells) and group 5 (osteoblasts) to each cell are shown in FIG. 27 and FIG. As shown in FIG. 27, the Euclidean distance from the center coordinates of the stages 3 and 4 of the seven on-spec reference samples (A, B, C, D, E, G, K,) in the group 3 is between 3.4 and 7.2. The Euclidean distance from the center coordinates of other groups or off-spec models was far apart at 8.1-31.6. Further, as shown in FIG. 28, the Euclidean distance from the central coordinates of the groups 3 of the stages 6 and 7 and 8 of the on-spec reference 7 samples (A, B, C, D, E, G, K,) in the group 5 Gathered close to between 2.9 and 8.0, and the Euclidean distance from the center coordinates of other groups or off-spec models was 9.7 to 39.3 far away.
As shown in Examples 1 and 2, it was shown that culture process stage discrimination and quality control using a statistical analysis technique are possible.

Example 13 [Verification by multiplex PCR detection system]
(multiplex PCR method)
Total RNA was extracted from the test cells using RNeasy Mini Kit (QIAGEN), and then cDNA was synthesized using 1 μg of total RNA as a template. The synthesis procedure was in accordance with the protocol (First-Strand cDNA Synthesis Using SuperScript II RT) described in the attached material of Super Script II Reverse Transcriptase (Invitrogen). For the primer, Random Primer (6mer) (TaKaRa) was used. After synthesizing cDNA, the synthesized cDNA solution was diluted 5-fold. Multiplex PCR was performed using 5 μL of this diluted solution as a template. In addition, representative 23 genes capable of defining each group of the culture process were divided into two sets of 12 genes and 11 genes (Table 6), and PCR reaction was performed using the primers shown in the table (Table 7). Two sets of PCR reaction solutions are Distilled Water, Deionized, Sterile (Wako Pure Chemical Industries, Ltd.), dNTP Mixture (2.5 mM each: TaKaRa), 10 × PCR Buffer (Conteins 15 mM MgCl ₂ : Roche) MgCl ₂ Solution (25 mM: Roche), AmpliTaq Gold ^™ (Roche), template DNA, and primers were mixed and prepared as shown in Table 8. PCR conditions are shown in Table 9. PCR products obtained by the two sets of reactions were electrophoresed using DNA 1000 Kit (Agilent) and detected using 2100 bioanalyzer (Agilent). The electrophoresis results were analyzed with Agilent 2100 Bio Sizing software (Agilent), and the molar concentration (nmol / L) of each gene product was calculated.

  Gene expression data of group 1 (bone marrow fluid), group 3 (mesenchymal stem cells), and group 5 (osteoblasts) for one onspec model (J) that has been successfully cultured from bone marrow fluid to bone differentiation Acquired using a multiplex PCR detection system. Using the obtained molar concentration values of 23 genes, each cell was plotted in a two-dimensional diagram by a multidimensional scaling method based on the Euclidean distance. The result is shown on the left of FIG. In the figure, alphabets indicate specimens and numbers indicate stages. Also, the result of two-dimensional plotting by the multidimensional scaling method based on the Euclidean distance in the same sample using the ΔCt value of real-time PCR (40-Ct) is shown on the right side of FIG. In the figure, alphabets indicate specimens and numbers indicate stages. As a result of comparing the plots of multiplex PCR and real-time PCR, it was confirmed that the patterns were similar and the areas of Group 1, Group 3 and Group 5 were clearly separated. From the above results, it was shown that culture stage discrimination and quality control using a multiplex PCR detection system are possible.

  Table 10 and Table 11 show the gene expression characteristics of the marker genes selected by these studies. 32 genes (Table 4) that can define each group in the culture process from bone marrow fluid to bone differentiation by specific expression level (Table 4), 9 genes (Table 2) showing a significant difference in expression between two adjacent groups Of these, a total of 38 genes excluding 3 overlapping genes are genes that can serve as genetic markers in each group and can be used in combination, but genes that show unique characteristics in each group can be used alone as marker genes It has been shown. Moreover, it was confirmed that the culture stage and the state of the cells can be discriminated by evaluating the expression level of these marker genes quantitatively or by quantifying the data with a semi-quantitative detection system by a statistical analysis method. From this, it is considered that cell normalization and quality control inspection based on gene expression level are possible.

FIG. 1 shows a culture process (stage). FIG. 2 shows a grouping of culture steps (stages). FIG. 3 shows a decision tree. FIG. 4 shows a comparison of the expression level (log ₂ ratio) of human matrix metallopeptidase 9 for group 1 and other cells. FIG. 5 shows a comparison of human interferon-γ-induced protein 30 expression level (log ₂ ratio) for group 2 and other cells. FIG. 6 shows a comparison of human latexin expression levels (log ₂ ratio) for group 3 and other cells. FIG. 7 shows a comparison of the expression level (log ₂ ratio) of human P311 protein-like protein for group 4 and other cells. FIG. 8 shows a comparison of the expression level (log ₂ ratio) of human Frizzled-like protein for group 5 and other cells. FIG. 9 shows inoculation with on-spec reference specimens (A, B, C, D, E), on-spec model specimens (F, G, H, I, J, K), adipose differentiated cells, fibroblasts, and viruses. The hierarchical clustering analysis result by the expression level (log ₂ ratio) of 42 genes in a mesenchymal stem cell is shown. FIG. 10 shows a comparison of real-time PCR data and microarray data of marker candidate genes (7 genes). FIG. 11 shows a comparison of real-time PCR data and microarray data of marker candidate genes (7 genes). FIG. 12 shows a comparison between real-time PCR data and microarray data of marker candidate genes (7 genes). FIG. 13 shows a comparison between real-time PCR data and microarray data of marker candidate genes (7 genes). FIG. 14 shows a comparison between real-time PCR data and microarray data of marker candidate genes (7 genes). FIG. 15 shows a comparison of real-time PCR data and microarray data of marker candidate genes (3 genes). FIG. 16 shows a comparison of expression levels (40-Ct values) for group 1 and other cells. FIG. 17 shows a comparison of expression levels (40-Ct values) for group 2 and other cells. FIG. 18 shows a comparison of expression levels (40-Ct values) for group 3 and other cells. FIG. 19 shows a comparison of expression levels (40-Ct values) for group 4 and other cells. FIG. 20 shows a comparison of expression levels (40-Ct values) for group 5 and other cells. FIG. 21 shows the expression levels of three genes that can define group 3 in on-spec reference samples (A, B, C, D, E), adipose differentiated cells, fibroblasts, and mesenchymal stem cells inoculated with virus (40- The results of two-dimensional plot analysis using multidimensional scaling when Ct values are used are shown. FIG. 22 shows the expression levels of 3 genes that can define group 5 in on-spec reference specimens (A, B, C, D, E), adipose differentiated cells, fibroblasts, and mesenchymal stem cells inoculated with virus (40- The results of two-dimensional plot analysis using multidimensional scaling when Ct values are used are shown. FIG. 23 shows on-spec reference samples (A, B, C, D, E), on-spec verification samples (F, G, H, I, J, K), adipose differentiated cells, cartilage differentiated cells, fibroblasts, The hierarchical clustering analysis result by the expression level (40-Ct value) of 38 genes in the mesenchymal stem cell inoculated with the virus is shown. FIG. 24 shows on-spec specimens (A, B, C, D, E, F, G, H, I, J, K) and 38 gene expression levels (40-Ct values) in mesenchymal stem cells A, B The hierarchical clustering analysis result by is shown. FIG. 25 shows expression levels of 38 genes (40-Ct value) in on-spec samples (A, B, C, D, E, F, G, H, I, J, K) and mesenchymal stem cells A, B The result of 2D plot analysis by multidimensional scaling method is shown. FIG. 26 shows the phases calculated using patterns of expression levels (40-Ct values) of 23 genes in on-spec samples (A, B, C, D, E, F, G, K) and off-spec model samples. Indicates the number of relationships. FIG. 27 shows group 3 calculated using expression levels (40-Ct values) of 23 genes in on-spec samples (A, B, C, D, E, F, G, K) and off-spec model samples. The Euclidean distance from the center coordinates of (mesenchymal stem cells) to each cell is shown. FIG. 28 shows group 5 calculated using expression levels of 23 genes (40-Ct values) in on-spec samples (A, B, C, D, E, F, G, K) and off-spec model samples. The Euclidean distance from the center coordinate of (osteoblast) to each cell is shown. FIG. 29 shows multi-dimensions using expression levels (40-Ct values) of 23 genes in group 1 (bone marrow cells), group 3 (mesenchymal stem cells) and group 5 (osteoblasts) in on-spec specimen J. A comparison between multiplex PCR and real-time PCR is shown for the two-dimensional plot analysis results by the scaling method.

Claims (5)

The following nine gene markers and genes of (i) to (ix) are used for monitoring a series of processes for culturing bone marrow cells and differentiating them into mesoblasts via mesenchymal stem cells. A genetic marker for cell detection consisting of a combination.
(I) a genetic marker for detecting bone marrow cells, comprising a gene having the base sequence set forth in SEQ ID NO: 1 in the sequence listing;
(Ii) Bone marrow cells comprising a gene having the base sequence described in SEQ ID NO: 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 in the sequence listing A marker for detecting a cell after culturing a cell but before only mesenchymal stem cells are selectively cultured;
(Iii) a genetic marker for detecting mesenchymal stem cells, comprising a gene having the base sequence set forth in SEQ ID NO: 20, 21, 22, 23, 24 or 25 in the sequence listing;
(Iv) a gene marker for detecting osteoprogenitor cells, comprising a gene having the nucleotide sequence set forth in SEQ ID NO: 26, 27 or 28 in the sequence listing; and (v) SEQ ID NO: 28, 29, 30 in the sequence listing. , 31, 32, 33, 34, 35 or 36, a gene marker for detecting osteoblasts, comprising a gene having the base sequence of
(Vi) A cell after culturing bone marrow cells and bone marrow cells, which is composed of the gene having the nucleotide sequence set forth in SEQ ID NO: 37 or 7 in the sequence listing and before only mesenchymal stem cells are selectively cultured. Genes that show differential expression levels between:
(Vii) A cell after culturing bone marrow cells consisting of a gene having the nucleotide sequence set forth in SEQ ID NO: 38 or 39 in the sequence listing, and a cell before only mesenchymal stem cells are selectively cultured and mesenchymal system Genes that show differential expression levels among stem cells:
(Viii) A gene showing a difference in expression level between a mesenchymal stem cell and an osteoprogenitor cell comprising a gene having the base sequence set forth in SEQ ID NO: 40 or 21 in the sequence listing:
(Ix) A gene showing a difference in expression level between osteoblasts and osteoprogenitor cells comprising a gene having the base sequence set forth in SEQ ID NO: 28, 41 or 42 in the sequence listing: From the genetic marker and gene according to claim 1 (i) to (ix), hybridization at 65 ° C. and washing at 65 ° C. with a buffer containing 0.1 × SSC, 0.1% SDS The probe for detecting a gene marker for cell detection according to claim 1, comprising a combination of probes having DNA sequences that hybridize under stringent conditions. Probe, comprising all of the antisense strand of the nucleotide sequence of the gene markers and genes according to claim 1 from (i) (ix), probe of claim 2. Of claims 1 to (i) genetic markers and genes according to (ix) a combination of primers consisting of a sense primer and an antisense primer that can specifically amplify by PCR, according to claim 1 Primer for detecting genetic markers for cell detection. A microarray or DNA chip for detecting a marker gene for cell detection obtained by immobilizing the probe according to claim 2 or 3 on a support.

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