JP6719114B2 - Kit and method for determining the ability of mesenchymal stem cells or chondrocytes to form bone tissue or cartilage tissue - Google Patents

Description

The present invention relates to a marker showing the ability of somatic stem cells or chondrocytes to differentiate into bone tissue or cartilage tissue and its use. More specifically, a marker showing the differentiation ability of somatic stem cells or chondrocytes into bone tissue or cartilage tissue, a kit for determining differentiation ability, a method for determining differentiation ability, and bone tissue or cartilage of somatic stem cells or chondrocytes The present invention relates to a method for producing a marker showing the ability to differentiate into tissues.

Differentiation of somatic stem cells or chondrocytes into bone tissue or cartilage tissue for application to regenerative medicine is under study. Further, a marker showing that the cells are stem cells, a marker showing that the stem cells have been differentiated into bone tissue, cartilage tissue, etc. have been found. For example, in Patent Document 1, cell detection comprising a combination of a gene marker and a gene, which is used for monitoring a series of steps of culturing bone marrow cells and differentiating into osteoblasts via mesenchymal stem cells. Genetic markers for are described.

Patent No. 5243021

However, a useful marker that serves as an index of the differentiation ability of somatic stem cells or chondrocytes into bone tissue or cartilage tissue is not known. As described later in Examples, the inventors have found that there is a difference in the ability to differentiate into bone tissue or cartilage tissue depending on the state of stem cells. In order to apply stem cells to regenerative medicine, it may be effective to use stem cells having high differentiation potential. Therefore, an object of the present invention is to provide a marker showing the ability of somatic stem cells or chondrocytes to differentiate into bone tissue or cartilage tissue.

The present invention includes the following aspects.
(1) A marker showing the ability to differentiate somatic stem cells or chondrocytes into bone tissue or cartilage tissue, which contains the genes listed in Table 1 below or the proteins encoded by the genes listed in Table 1 below.
(2) A gene selected from the group consisting of the genes shown in Tables 2 and 3 below, or a protein encoded by the gene selected from the group consisting of the genes shown in Tables 2 and 3 below. A marker showing the ability to differentiate somatic stem cells or chondrocytes into bone tissue or cartilage tissue according to (1), further comprising:
(3) Soluble carrier family 35, member F2 (SLC35F2) gene or a protein encoded by SLC35F2 gene, and a Tumor necrosis receptor receptor superfamily, member 11b (TNFN11F gene including combination of TNFRSF11B) gene (TNFNFN11 gene) or a gene encoding TNFRSF11B. A marker showing the ability of the somatic stem cells or chondrocytes described in 2) to differentiate into bone tissue or cartilage tissue.
(4) A primer set for amplifying cDNA of the gene described in Table 1 described in (1), a probe that specifically hybridizes to mRNA of the gene described in Table 1 above, or A kit for determining the ability to differentiate somatic stem cells or chondrocytes into bone tissue or cartilage tissue, which comprises a specific binding substance for the protein encoded by the described gene.
(5) A primer set for amplifying cDNA of a gene selected from the group consisting of the genes listed in Tables 2 and 3 listed in (2), from the genes listed in Tables 2 and 3 above A probe that specifically hybridizes to mRNA of a gene selected from the group consisting of: or a specific binding substance for a protein encoded by the gene selected from the group consisting of the genes listed in Table 2 and Table 3; The kit for determining the ability to differentiate somatic stem cells or chondrocytes into bone tissue or cartilage tissue according to (4), further comprising:
(6) A primer set for amplifying SLC35F2 gene cDNA, a probe that specifically hybridizes to SLC35F2 gene mRNA, or a specific binding substance for a protein encoded by the SLC35F2 gene, and a cDNA of TNFRSF11B gene are amplified. Bone tissue of somatic stem cell or chondrocyte according to (5), which comprises a primer set for Alternatively, a kit for determining the ability to differentiate into cartilage tissue.
(7) A step of quantifying the expression level of a marker gene or a marker protein in somatic stem cells or chondrocytes, wherein the marker gene or the marker protein is the gene described in Table 1 of (1) or the A method for determining the ability of somatic stem cells or chondrocytes to differentiate into bone tissue or cartilage tissue, which comprises proteins encoded by the genes listed in Table 1.
(8) The marker gene or the marker protein is a gene selected from the group consisting of the genes listed in Tables 2 and 3 of (2), or the genes listed in Tables 2 and 3 above. The method for determining the ability of somatic stem cells or chondrocytes to differentiate into bone tissue or cartilage tissue according to (7), further comprising a protein encoded by a gene selected from the group consisting of:
(9) The marker gene or the marker protein is selected from the group consisting of the genes listed in Table 1 or the proteins encoded by the genes listed in Table 1 and the genes listed in Table 2 above. Or a protein encoded by a gene selected from the group consisting of the genes listed in Table 2 above, and the expression level of the marker gene or the marker protein in somatic stem cells or chondrocytes in control cells is The determination method according to (8), wherein when the expression level of the marker gene or the marker protein is higher, the somatic stem cells or the chondrocytes are determined to have a high ability to differentiate into bone tissue or cartilage tissue.
(10) The marker gene or the marker protein is selected from the group consisting of the genes listed in Table 1 or the proteins encoded by the genes listed in Table 1 and the genes listed in Table 3 above. A gene or a protein encoded by a gene selected from the group consisting of the genes listed in Table 3 above, the gene listed in Table 1 above in somatic stem cells or chondrocytes, or the protein listed in Table 1 above The expression level of the protein encoded by the gene is higher than the expression level of the gene described in Table 1 or the protein encoded by the gene described in Table 1 in the control cells, and the somatic stem cell or The expression level of the gene selected from the group consisting of the genes listed in Table 3 above or the protein encoded by the gene selected from the group consisting of the genes listed in Table 3 above in the chondrocytes in the control cells The determination method according to (8), wherein when the expression level of the gene or the protein is lower, the somatic stem cell or the chondrocyte is determined to have a high ability to differentiate into bone tissue or cartilage tissue.
(11) The marker gene or the marker protein comprises a combination of the SLC35F2 gene or the protein encoded by the SLC35F2 gene and the protein encoded by the TNFRSF11B gene or the TNFRSF11B gene, and the SLC35F2 gene in somatic stem cells or chondrocytes or The expression level of the protein encoded by the SLC35F2 gene was higher than the expression level of the SLC35F2 gene or the protein encoded by the SLC35F2 gene in the control cells, and was encoded by the TNFRSF11B gene or the TNFRSF11B gene in somatic stem cells or chondrocytes When the expression level of the protein is lower than the expression level of the TNFRSF11B gene or the protein encoded by the TNFRSF11B gene in the control cells, it is determined that the somatic stem cell or the chondrocyte has a high ability to differentiate into bone tissue or cartilage tissue. Yes, the determination method according to (10).
(12) The marker gene or the marker protein is a combination of the SLC35F2 gene or the protein encoded by the SLC35F2 gene and the protein encoded by the TNFRSF11B gene or the TNFRSF11B gene, and the TNFRSF11B gene in somatic stem cells or chondrocytes Of the expression level of the SLC35F2 gene to the expression level of the SLC35F2 gene is higher than the expression level of the SLC35F2 gene to the expression level of the TNFRSF11B gene in control cells, or is encoded by the TNFRSF11B gene in somatic stem cells or chondrocytes When the ratio of the expression amount of the protein encoded by the SLC35F2 gene to the expression amount of the protein is greater than the ratio of the expression amount of the protein encoded by the SLC35F2 gene to the expression amount of the protein encoded by the TNFRSF11B gene in control cells In addition, the determination method according to (8), wherein the somatic stem cells or the chondrocytes are determined to have a high ability to differentiate into bone tissue or cartilage tissue.
(13) Measurement of expression level of gene or protein in somatic stem cells or chondrocytes having high differentiation ability to bone tissue or cartilage tissue and somatic stem cells or chondrocytes having low differentiation ability to bone tissue or cartilage tissue And a somatic stem cell or chondrocyte having a high differentiation ability to a bone tissue or a cartilage tissue, and a somatic stem cell or chondrocyte having a low differentiation ability to a bone tissue or a cartilage tissue, a gene having a different expression level Or the step of determining that the protein is a marker showing the ability of somatic stem cells or chondrocytes to differentiate into bone tissue or cartilage tissue, the ability to differentiate somatic stem cells or chondrocytes into bone tissue or cartilage tissue A method for producing a marker.

(A1) A gene selected from the group consisting of the genes listed in Tables 2 and 3 of (2), or a gene selected from the group consisting of the genes listed in Tables 2 and 3 above. A marker comprising the encoded protein, which shows the ability of somatic stem cells or chondrocytes to differentiate into bone tissue or cartilage tissue.
(A2) Tumor necrosis factor receptor receptor superfamily, member 11b (TNFRSF11B) gene or a protein encoded by the TNFRSF11B gene, showing the ability of somatic stem cells or chondrocytes to differentiate into bone tissue or cartilage tissue according to (A1). marker.
(A3) A primer set for amplifying a cDNA of a gene selected from the group consisting of the genes listed in Tables 2 and 3 of (2), from the genes listed in Tables 2 and 3 above. A probe that specifically hybridizes to mRNA of a gene selected from the group consisting of: or a specific binding substance for a protein encoded by the gene selected from the group consisting of the genes listed in Table 2 and Table 3; A kit for determining the ability of somatic stem cells or chondrocytes to differentiate into bone tissue or cartilage tissue.
(A4) A primer set for amplifying cDNA of TNFRSF11B gene, a probe that specifically hybridizes to mRNA of TNFRSF11B gene, or a specific binding substance for a protein encoded by the TNFRSF11B gene, described in (A3) A kit for determining the ability to differentiate somatic stem cells or chondrocytes into bone tissue or cartilage tissue.
(A5) A step of quantifying the expression level of the marker gene or marker protein in somatic stem cells or chondrocytes is provided, and the marker gene or the marker protein is described in Table 2 and Table 3 described in (2). Bone tissue or cartilage of somatic stem cells or chondrocytes, which is a gene selected from the group consisting of genes, or a protein encoded by a gene selected from the group consisting of the genes listed in Tables 2 and 3 above A method for determining the ability to differentiate into a tissue.
(A6) The marker gene or the marker protein is encoded by a gene selected from the group consisting of the genes listed in Table 2 above or a gene selected from the group consisting of the genes listed in Table 2 above. A somatic stem cell or chondrocyte which is a protein and the expression level of the marker gene or the marker protein in somatic stem cells or chondrocytes is higher than the expression level of the marker gene or the marker protein in control cells. Is determined to have a high ability to differentiate into bone tissue or cartilage tissue, (A5).
(A7) The marker gene or the marker protein is encoded by a gene selected from the group consisting of the genes listed in Table 3 above or a gene selected from the group consisting of the genes listed in Table 3 above. Protein, the expression level of the gene or protein in somatic stem cells or chondrocytes is lower than the expression level of the gene or protein in control cells, the somatic stem cells or chondrocytes are bone tissue or The determination method according to (A5), which determines that the ability to differentiate into cartilage tissue is high.
(A8) The marker gene or the marker protein is a protein encoded by the TNFRSF11B gene or the TNFRSF11B gene, and the expression level of the protein encoded by the TNFRSF11B gene or the TNFRSF11B gene in somatic stem cells or chondrocytes is in the control cells. The determination according to (A7), wherein when the expression level of the TNFRSF11B gene or the protein encoded by the TNFRSF11B gene is lower, it is determined that the somatic stem cell or the chondrocyte has a high ability to differentiate into bone tissue or cartilage tissue. Method.

According to the present invention, a marker showing the ability of somatic stem cells or chondrocytes to differentiate into bone tissue or cartilage tissue can be provided.

(A) is a growth curve of Yub622 cells measured in Experimental Example 1. (B) is a photograph showing the result of alizarin red staining of Yub622 cells that had been induced to differentiate into bone tissue in Experimental Example 1. (A) is a growth curve of hMSC cells measured in Experimental Example 1. (B) is a photograph showing the result of alizarin red staining of hMSC cells that were induced to differentiate into bone tissue in Experimental Example 1. (A) is a growth curve of BM-AT cells measured in Experimental Example 1. (B) is a photograph showing the results of alizarin red staining of BM-AT cells that had been induced to differentiate into bone tissue in Experimental Example 1. (A) is a growth curve of StemPro human ADSC cells measured in Experimental Example 1. (B) is a photograph showing the results of alizarin red staining of StemPro human ADSC cells that were induced to differentiate into bone tissue in Experimental Example 1. (A) is a hematoxylin-eosin stain (HE) and an alcian blue stain (AB) of a sample obtained by inducing differentiation of Yub621c cells at passage number 7 (P7) and passage number 9 (P9) into cartilage tissue in Experimental Example 2. It is a photograph showing the result of. (B) is a hematoxylin-eosin stain (HE) and alcian blue stain (AB) of a sample obtained by inducing differentiation of Yub625 cells at passage number 5 (P5) and passage number 22 (P22) into cartilage tissue in Experimental Example 2. It is a photograph showing the result of. 7 is a graph showing the results of examining the expression level of the SLC35F2 gene by microarray analysis in Experimental Example 4. 7 is a graph showing the results of examining the expression level of the RBP1 gene by microarray analysis in Experimental Example 4. 9 is a graph showing the results of examining the expression level of AMPH gene by microarray analysis in Experimental Example 4. 7 is a graph showing the results of examining the expression level of the EFNB3 gene in Experimental Example 4 by microarray analysis. 9 is a graph showing the results of examination of the expression level of the NTNG1 gene by microarray analysis in Experimental Example 4. 9 is a graph showing the results of examining the expression level of the NRGN gene by microarray analysis in Experimental Example 4. 9 is a graph showing the results of examination of the expression level of BAALC gene by microarray analysis in Experimental Example 4. 9 is a graph showing the results of examining the expression level of the TMSB15A gene in Experimental Example 4 by microarray analysis. 9 is a graph showing the results of examining the expression level of GALNT14 gene in Experimental Example 4 by microarray analysis. 7 is a graph showing the results of examination of the expression level of SGOL1 gene by microarray analysis in Experimental Example 4. 7 is a graph showing the results of examining the expression level of HIST1H2BF gene by microarray analysis in Experimental Example 4. 9 is a graph showing the results of examination of the expression level of MDFI gene by microarray analysis in Experimental Example 4. 7 is a graph showing the results of examining the expression level of HIST1H4L gene by microarray analysis in Experimental Example 4. 9 is a graph showing the results of examining the expression level of the COLEC12 gene in Experimental Example 4 by microarray analysis. 9 is a graph showing the results of examining the expression level of the GFRA2 gene in Experimental Example 4 by microarray analysis. 9 is a graph showing the results of examining the expression level of KCNJ8 gene by microarray analysis in Experimental Example 4. 9 is a graph showing the results of examining the expression level of the PTGER2 gene in Experimental Example 4 by microarray analysis. 9 is a graph showing the results of examining the expression level of the SLC43A3 gene in Experimental Example 4 by microarray analysis. 9 is a graph showing the results of examining the expression level of the SLC7A2 gene by microarray analysis in Experimental Example 4. 9 is a graph showing the results of examining the expression level of the TNFRSF11B gene in Experimental Example 4 by microarray analysis. 7 is a graph showing the results of examining the expression level of the IGFBP2 gene in Experimental Example 4 by microarray analysis. 7 is a graph showing the results of examining the expression level of the ELN gene by microarray analysis in Experimental Example 4. 9 is a graph showing the results of examining the expression level of the ACAN gene by microarray analysis in Experimental Example 4. 9 is a graph showing the results of examining the expression level of the CRYAB gene in Experimental Example 4 by microarray analysis. 9 is a graph showing the results of examining the expression level of KRT16P2 gene in Experimental Example 4 by microarray analysis. 9 is a graph showing the results of examining the expression level of the KRT16P6 gene in Experimental Example 4 by microarray analysis. 7 is a graph showing the results of examining the expression level of KRT14 gene by microarray analysis in Experimental Example 4. 7 is a graph showing the results of examining the expression level of the SLC35F2 gene in Yub622 cells in Experimental Example 5 by real-time PCR analysis. 7 is a graph showing the results of examining the expression level of the SLC35F2 gene in hMSC cells by real-time PCR analysis in Experimental Example 5. 9 is a graph showing the results of examining the expression level of the SLC35F2 gene in BM-AT cells by real-time PCR analysis in Experimental Example 5. 9 is a graph showing the results of examining the expression level of the SLC35F2 gene in Yub621c cells and Yub625 cells in Experimental Example 5 by real-time PCR analysis. (A) is a growth curve of Yub2505 cells measured in Experimental Example 6. (B) is a photograph showing the results of alizarin red staining of Yub2505 cells that were induced to differentiate into bone tissue in Experimental Example 6. Fig. 6 is a photograph showing the results of Alcian blue staining (AB) of Yub2505 cells that have been induced to differentiate into cartilage tissue. (A) is a growth curve of UDE-BM cells measured in Experimental Example 6. (B) is a photograph showing the results of alizarin red staining of UDE-BM cells that were induced to differentiate into bone tissue in Experimental Example 6. 3 is a photograph showing the results of Alcian blue staining (AB) of UDE-BM cells that have been induced to differentiate into cartilage tissue. 7 is a graph showing the results of examining the expression level of the SLC35F2 gene in Experimental Example 7 by microarray analysis. 9 is a graph showing the results of examination of the expression level of RBP1 gene by microarray analysis in Experimental Example 7. 9 is a graph showing the results of examining the expression level of AMPH gene by microarray analysis in Experimental Example 7. 9 is a graph showing the results of examining the expression level of the EFNB3 gene in Experimental Example 7 by microarray analysis. 9 is a graph showing the results of examination of the expression level of the NTNG1 gene by microarray analysis in Experimental Example 7. 9 is a graph showing the results of examining the expression level of the BAALC gene by microarray analysis in Experimental Example 7. 9 is a graph showing the results of examining the expression level of the TMSB15A gene in Experimental Example 7 by microarray analysis. 7 is a graph showing the results of examining the expression level of GALNT14 gene in Experimental Example 7 by microarray analysis. 9 is a graph showing the results of examination of the expression level of MDFI gene by microarray analysis in Experimental Example 7. 9 is a graph showing the results of examining the expression level of the GFRA2 gene in Experimental Example 7 by microarray analysis. 9 is a graph showing the results of examining the expression level of the PTGER2 gene in Experimental Example 7 by microarray analysis. 7 is a graph showing the results of examination of the expression level of the SLC43A3 gene by microarray analysis in Experimental Example 7. 9 is a graph showing the results of examining the expression level of the TNFRSF11B gene in Experimental Example 7 by microarray analysis. It is a graph which shows the result of having examined the expression level of MDFI gene by the microarray analysis in Experimental example 8. 9 is a graph showing the results of examining the expression level of the IGFBP2 gene in Experimental Example 8 by microarray analysis. 9 is a graph showing the results of examining the expression level of the ELN gene by microarray analysis in Experimental Example 8. 9 is a graph showing the results of examining the expression level of the KRT16P2 gene by microarray analysis in Experimental Example 8. 9 is a graph showing the results of examining the expression level of KRT16P6 gene in Experimental Example 8 by microarray analysis. 9 is a graph showing the results of examining the expression level of KRT14 gene by microarray analysis in Experimental Example 8. 9 is a graph showing the results of examining the expression level of the SLC35F2 gene in StemPro human ADSC cells in Experimental Example 9 by real-time PCR analysis. 9 is a graph showing the results of examining the expression level of the SLC35F2 gene in Yub2505 cells in Experimental Example 9 by real-time PCR analysis. 9 is a graph showing the results of examining the expression level of the SLC35F2 gene in UDE-BM cells in Experimental Example 9 by real-time PCR analysis. 11 is a graph showing the results of examining the expression level of the TNFRSF11B gene in Yub622 cells in Experimental Example 10 by real-time PCR analysis. 11 is a graph showing the results of examining the expression level of the TNFRSF11B gene in hMSC cells by a real-time PCR analysis in Experimental Example 10. 9 is a graph showing the results of examining the expression level of the TNFRSF11B gene in BM-AT cells by real-time PCR analysis in Experimental Example 10. 9 is a graph showing the results of examining the expression level of the TNFRSF11B gene in Yub621c cells and Yub625 cells in Experimental Example 10 by real-time PCR analysis. 9 is a graph showing the results of examining the expression level of the TNFRSF11B gene in StemPro human ADSC cells in Experimental Example 10 by real-time PCR analysis. 9 is a graph showing the results of examining the expression level of the TNFRSF11B gene in Yub2505 cells in Experimental Example 10 by real-time PCR analysis. 9 is a graph showing the results of examining the expression level of the TNFRSF11B gene in UDE-BM cells in Experimental Example 10 by real-time PCR analysis. 20 is a graph showing the results of measuring the ratio of the expression level of the SLC35F2 gene to the expression level of the TNFRSF11B gene in various cells in Experimental Example 11. 20 is a graph showing the results of measuring the ratio of the expression level of the SLC35F2 gene to the expression level of the TNFRSF11B gene in various cells in Experimental Example 11.

[Marker showing the ability of somatic stem cells or chondrocytes to differentiate into bone tissue or cartilage tissue]
In one embodiment, the present invention may be the SLC35F2 gene described in Table 1 above or a protein encoded by the SLC35F2 gene (hereinafter, referred to as “SLC35F2 protein”, and the same applies to proteins encoded by other genes. A marker showing the ability of somatic stem cells or chondrocytes to differentiate into bone tissue or cartilage tissue.

As will be described later in Examples, the present inventors have found that mesenchymal stem cells that have a high ability to differentiate into bone tissue or cartilage tissue highly express the SLC35F2 gene. In addition, a positive correlation was observed between the ability to differentiate into bone tissue or cartilage tissue and the expression level of SLC35F2 gene. That is, it was recognized that the higher the expression level of the SLC35F2 gene, the higher the ability to differentiate into bone tissue or cartilage tissue.

Therefore, the SLC35F2 gene or SLC35F2 protein can be used as a marker showing the ability of target cells to differentiate into bone tissue or cartilage tissue. Here, the target cells are cells having the ability to differentiate into bone tissue or cartilage tissue, and include somatic stem cells or chondrocytes. That is, somatic stem cells or chondrocytes that have a high expression level of the SLC35F2 gene or SLC35F2 protein have a high ability to differentiate into bone tissue or cartilage tissue.

Here, examples of somatic stem cells include stem cells capable of differentiating into bone tissue or cartilage tissue. More specifically, stem cells derived from mesenchyme such as fat, bone marrow, cord blood, umbilical cord, amniotic membrane, placenta, endometrium, synovium, tendon, bone, cartilage, dental pulp, periodontal ligament (ie, mesenchymal stem cells) And progenitor cells, and stem cells and progenitor cells derived from tissues other than mesenchyme such as vascular endothelium and skin. Further, the chondrocytes may be those recovered from cartilage tissue. Somatic stem cells or chondrocytes may be those obtained by inducing differentiation from ES cells, iPS cells and the like.

As will be described later in Examples, as markers showing the differentiation ability of somatic stem cells or chondrocytes into bone tissue or cartilage tissue, in addition to the SLC35F2 gene or SLC35F2 protein, the markers shown in Table 2 and Table 3 above are listed. A gene selected from the group consisting of the above genes, or a protein encoded by the gene selected from the group consisting of the genes described in Tables 2 and 3 above can be used.

Table 4 below shows the accession numbers of typical transcripts transcribed from the genes specified by the gene symbols shown in Tables 1 to 3 above. The nucleotide sequences identified by the accession numbers shown in Table 4 were published in the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/) in December 2015. Based on information dated 2nd.

The marker of the present embodiment is not limited to the transcript identified by the accession number shown in Table 4 or a translated product thereof, but is a splicing variant or transcription start point transcribed from the gene identified by the gene symbol shown in Table 4. It may be a variant such as a variant or a translated product thereof.

In addition, the base sequence of the marker gene of the present embodiment may include mutations due to the presence of single nucleotide polymorphisms and the like. Therefore, the base sequence of the marker gene of the present embodiment is, for example, 90% or more, for example 95% or more, for example 98% or more, for example 99% or more with the base sequence specified by the accession number shown in Table 4 below. As long as they have sequence identity, they may have a base sequence different from the base sequence specified by the accession number. This is also the case when the marker gene of the present embodiment is a variant such as a splicing variant of a nucleotide sequence specified by an accession number shown in Table 4 below or a variant due to a difference in transcription start point.

The gene selected from the group consisting of the genes listed in Tables 2 and 3 above, or the protein encoded by the gene selected from the group consisting of the genes listed in Tables 2 and 3 above is SLC35F2. It can be used in combination with a gene or SLC35F2 protein, or independently of the SLC35F2 gene or SLC35F2 protein, as a marker showing the ability of somatic stem cells or chondrocytes to differentiate into bone tissue or cartilage tissue.

As a marker showing the ability of somatic stem cells or chondrocytes to differentiate into bone tissue or cartilage tissue, any one of the above genes or proteins may be used alone, or two or more may be used in combination.

As will be described later in Examples, the expression levels of the genes described in Table 1 and Table 2 above or the proteins encoded by the genes described in Table 1 and Table 2 above are determined as somatic stem cells or chondrocytes. Tend to have a positive correlation with the ability to differentiate into bone tissue or cartilage tissue. That is, somatic stem cells or chondrocytes in which the expression amount of any of these genes or proteins is high has a high ability to differentiate into bone tissue or cartilage tissue.

On the other hand, the expression levels of the genes listed in Table 3 above or the proteins encoded by the genes listed in Table 3 above are negatively correlated with the ability of somatic stem cells or chondrocytes to differentiate into bone tissue or cartilage tissue. Tends to show. That is, somatic stem cells or chondrocytes in which the expression level of any of these genes or proteins is low has a high ability to differentiate into bone tissue or cartilage tissue.

As will be described later in Examples, it is higher when a combination of SLC35F2 gene or SLC35F2 protein and TNFRSF11B gene or TNFRSF11B protein is used as a marker showing the differentiation ability of somatic stem cells or chondrocytes into bone tissue or cartilage tissue. It tends to be possible to evaluate the ability of somatic stem cells or chondrocytes to differentiate into bone tissue or cartilage tissue with high accuracy.

Therefore, the marker of the present embodiment may be a combination of the SLC35F2 gene or SLC35F2 protein and the TNFRSF11B gene or TNFRSF11B protein.

[Differentiating ability determination kit]
In one embodiment, the present invention provides a primer set for amplifying the SLC35F2 gene cDNA described in Table 1 above, a probe that specifically hybridizes to the SLC35F2 gene mRNA, or a specific binding substance for the SLC35F2 protein, A kit for determining the ability of somatic stem cells or chondrocytes to differentiate into bone tissue or cartilage tissue is provided.

(Primer set)
The primer set is not particularly limited as long as it can amplify the cDNA of the SLC35F2 gene. For example, a set of a sense primer having the base sequence shown in SEQ ID NO: 1 and an antisense primer having the base sequence shown in SEQ ID NO: 2 can be mentioned.

(probe)
The probe is not particularly limited as long as it specifically hybridizes with mRNA of SLC35F2 gene. The probe may be immobilized on a carrier to form a DNA microarray or the like.

(Specific binding substance)
Specific binding substances include, for example, antibodies, antibody fragments, aptamers and the like. The antibody may be prepared, for example, by immunizing an animal such as a mouse with the target protein or its partial peptide as an antigen. Alternatively, it can be prepared by screening an antibody library such as a phage library. Examples of antibody fragments include Fv, Fab and scFv. The above-mentioned antibody or antibody fragment may be polyclonal or monoclonal.

An aptamer is a substance having a specific binding ability to a labeling substance. Examples of aptamers include nucleic acid aptamers and peptide aptamers. Nucleic acid aptamers capable of specifically binding to a target protein can be selected by, for example, the systematic evolution of ligand by exponential enrichment (SELEX) method. Moreover, the peptide aptamer having the specific binding ability to the target protein can be selected by, for example, the Two-hybrid method using yeast.

The specific binding substance is not particularly limited as long as it can specifically bind to the target protein, and may be commercially available. The specific binding substance may be immobilized on a carrier to form a protein chip or the like.

By using the differentiation capacity determination kit of the present embodiment, the expression level of the SLC35F2 gene in the target somatic stem cell or chondrocyte can be qualitatively or quantitatively measured. When the expression level of the SLC35F2 gene in somatic stem cells or chondrocytes is higher than that in control cells, it can be determined that the somatic stem cells or chondrocytes have a high ability to differentiate into bone tissue or cartilage tissue.

Here, as the control cells, for example, mesenchymal stem cells and the like, which have been previously confirmed to have a low ability to differentiate into bone tissue or cartilage tissue, can be used. More specifically, for example, mesenchymal stem cells with a high passage number can be mentioned.

In one embodiment, the kit for determining a differentiation potential is a primer set for amplifying the SLC35F2 gene cDNA described in Table 1 above, a probe that specifically hybridizes to the SLC35F2 gene mRNA, or a specific SLC35F2 protein. A binding substance, a primer set for amplifying a cDNA of a gene selected from the group consisting of the genes listed in Tables 2 and 3 above, and a group consisting of the genes listed in Tables 2 and 3 above A probe that specifically hybridizes to mRNA of a selected gene, or a substance that specifically binds to a protein encoded by a gene selected from the group consisting of the genes shown in Tables 2 and 3 above May be

Alternatively, the kit for determining a differentiation potential of the present embodiment comprises a primer set for amplifying cDNA of a gene selected from the group consisting of the genes shown in Tables 2 and 3 above, Tables 2 and 3 above. A probe that specifically hybridizes to mRNA of a gene selected from the group consisting of the genes described in 1. or a protein encoded by a gene selected from the group consisting of the genes described in Tables 2 and 3 above. May be provided with a specific binding substance for. That is, the kit for determining a differentiation potential of the present embodiment may be for measuring the expression level of a marker gene or marker protein other than SLC35F2.

The kit for determining a differentiation potential of the present embodiment independently measures the expression level of any one of the genes listed in Tables 1 to 3 above or the proteins encoded by the genes listed in Tables 1 to 3 above. One may be used, or two or more types of expression levels may be combined and measured.

As described later in Examples, the expression levels of the genes listed in Tables 1 and 2 above or the proteins encoded by the genes listed in Tables 1 and 2 above are different from those of somatic stem cells or chondrocytes. It tends to show a positive correlation with the ability to differentiate into bone tissue or cartilage tissue. That is, the somatic stem cells or chondrocytes confirmed to have a higher expression level of any of these genes or proteins by the kit for determining a differentiation potential of the present embodiment as compared to control cells are bone tissue or cartilage. It can be determined that the ability to differentiate into tissues is high. As the control cells, the same cells as described above can be used.

On the other hand, the expression levels of the genes listed in Table 3 above or the proteins encoded by the genes listed in Table 3 above are negatively correlated with the ability of somatic stem cells or chondrocytes to differentiate into bone tissue or cartilage tissue. Tend to show. That is, the somatic stem cells or chondrocytes confirmed to have a low expression level of any of these genes or proteins by the kit for determining a differentiation potential of the present embodiment as compared with control cells are bone tissue or cartilage. It can be determined that the ability to differentiate into tissues is high. As the control cells, the same cells as described above can be used.

As will be described later in Examples, it is higher when a combination of SLC35F2 gene or SLC35F2 protein and TNFRSF11B gene or TNFRSF11B protein is used as a marker showing the differentiation ability of somatic stem cells or chondrocytes into bone tissue or cartilage tissue. It tends to be possible to evaluate the ability of somatic stem cells or chondrocytes to differentiate into bone tissue or cartilage tissue with high accuracy.

Therefore, the kit for determining a differentiation potential of the present embodiment comprises a primer set for amplifying the cDNA of the SLC35F2 gene, a probe that specifically hybridizes to the mRNA of the SLC35F2 gene, or a specific binding substance for the SLC35F2 protein, and a TNFRSF11B gene. It may be provided with a primer set for amplifying cDNA, a probe that specifically hybridizes to mRNA of TNFRSF11B gene, or a specific binding substance for TNFRSF11B protein.

[Determination of differentiation ability]
In one embodiment, the present invention comprises the step of quantifying the expression level of the marker gene or marker protein in somatic stem cells or chondrocytes, wherein the marker gene or marker protein is the SLC35F2 gene described in Table 1 above. Also provided is a method for determining the ability of somatic stem cells or chondrocytes to differentiate into bone tissue or cartilage tissue containing the SLC35F2 protein.

The quantification of the expression level of the SLC35F2 gene or SLC35F2 protein can be performed by using, for example, the above-mentioned kit for determining differentiation ability. More specifically, for example, quantitative PCR using a primer set capable of amplifying the SLC35F2 gene cDNA, gene expression analysis using a microarray on which a probe that specifically hybridizes to the SLC35F2 gene mRNA is immobilized. The expression level of the SLC35F2 gene or SLC35F2 protein can be quantified by immunostaining using an antibody against the SLC35F2 protein.

As described above, when the expression level of the SLC35F2 gene in somatic stem cells or chondrocytes is higher than that in control cells, it is determined that the somatic stem cells or chondrocytes have a high ability to differentiate into bone tissue or cartilage tissue. can do. As the control cells, the same cells as described above can be used.

In one embodiment, the method for determining the differentiation potential comprises a step of quantifying the expression level of a marker gene or marker protein in somatic stem cells or chondrocytes, wherein the marker gene or the marker protein is described in Table 1 above. SLC35F2 gene or SLC35F2 protein, and a gene selected from the group consisting of the genes listed in Tables 2 and 3 above or a gene selected from the group consisting of the genes listed in Tables 2 and 3 above. It may be a combination with the encoded protein.

Alternatively, in the method for determining the differentiation potential of the present embodiment, the marker gene or marker protein for quantifying the expression level is a gene selected from the group consisting of the genes listed in Table 2 and Table 3 above or Table 2 above. It may be a protein encoded by a gene selected from the group consisting of the genes listed in Table 3 above. That is, the marker gene or marker protein whose expression level is quantified may be a marker gene or marker protein other than SLC35F2.

In the method for determining the differentiation potential of the present embodiment, the marker gene or the marker protein for quantifying the expression amount is a gene encoded in the genes listed in Tables 1 to 3 or the genes listed in Tables 1 to 3 above. Any one type may be used alone, or a combination of two or more types may be used.

As described above, the expression levels of the genes described in Tables 1 and 2 above or the proteins encoded by the genes described in Tables 1 and 2 above are determined by the bone tissue of somatic stem cells or chondrocytes or It tends to show a positive correlation with the ability to differentiate into cartilage tissue. That is, in the differentiation potential determination method of the present embodiment, when the expression level of any of these genes or proteins in the target somatic stem cells or chondrocytes is higher than the expression level of the gene or protein in control cells It can be determined that the target somatic stem cell or the chondrocyte has a high ability to differentiate into bone tissue or cartilage tissue. As the control cells, the same cells as described above can be used.

In addition, as described above, the expression level of the gene described in Table 3 above or the protein encoded by the gene described in Table 3 above is determined by the ability of somatic stem cells or chondrocytes to differentiate into bone tissue or cartilage tissue. Tends to have a negative correlation with. That is, in the differentiation potential determination method of the present embodiment, when the expression level of any of these genes or proteins in the target somatic stem cells or chondrocytes is lower than the expression level of the gene or protein in control cells It can be determined that the target somatic stem cell or the chondrocyte has a high ability to differentiate into bone tissue or cartilage tissue. As the control cells, the same cells as described above can be used.

As will be described later in Examples, it is higher when a combination of SLC35F2 gene or SLC35F2 protein and TNFRSF11B gene or TNFRSF11B protein is used as a marker showing the differentiation ability of somatic stem cells or chondrocytes into bone tissue or cartilage tissue. It tends to be possible to evaluate the ability of somatic stem cells or chondrocytes to differentiate into bone tissue or cartilage tissue with high accuracy.

Therefore, the method for determining the differentiation potential of the present embodiment comprises a step of quantifying the expression level of the SLC35F2 gene or SLC35F2 protein and the expression level of the TNFRSF11B gene or TNFRSF11B protein in somatic stem cells or chondrocytes. Good.

The expression level of SLC35F2 gene or SLC35F2 protein in somatic stem cells or chondrocytes is higher than the expression level of SLC35F2 gene or SLC35F2 protein in control cells, and expression of TNFRSF11B gene or TNFRSF11B protein in somatic stem cells or chondrocytes When the amount is lower than the expression level of the TNFRSF11B gene or TNFRSF11B protein in the control cells, it may be determined that the somatic stem cells or the chondrocytes have a high ability to differentiate into bone tissue or cartilage tissue.

As a result, the ability of somatic stem cells or chondrocytes to differentiate into bone tissue or cartilage tissue can be determined with higher accuracy.

Alternatively, as described below in Examples, the method for determining the differentiation potential of the present embodiment is to quantify the expression level of the SLC35F2 gene or SLC35F2 protein and the expression level of the TNFRSF11B gene or TNFRSF11B protein in somatic stem cells or chondrocytes. And a bone tissue of somatic stem cells or chondrocytes based on the ratio of the expression level of the TNFRSF11B gene and the expression level of the SLC35F2 gene, or the ratio of the expression level of the TNFRSF11B protein and the expression level of the SLC35F2 protein. The ability to differentiate into cartilage tissue may be determined.

Then, when the ratio of the expression level of the SLC35F2 gene to the expression level of the TNFRSF11B gene or the ratio of the expression level of the SLC35F2 protein to the expression level of the TNFRSF11B protein is larger than those in control cells, It may be determined that stem cells or chondrocytes have a high ability to differentiate into bone tissue or cartilage tissue. As the control cells, the same cells as described above can be used.

Also by such a method, the differentiation ability of somatic stem cells or chondrocytes into bone tissue or cartilage tissue can be determined with higher accuracy.

When the differentiation potential is determined based on the ratio of the expression level of the SLC35F2 gene or protein to the expression level of the TNFRSF11B gene or protein, more specifically, it is calculated based on the following formula (F2) or the following formula (F3). If the ratio is, for example, 0.5 or more, preferably 1 or more, more preferably 1.5 or more, even more preferably 2 or more, it is determined that the target cell has a high ability to differentiate into bone tissue or cartilage tissue. You can
Ratio of expression amount of SLC35F2 gene to expression amount of TNFRSF11B gene=(expression amount of SLC35F2 gene)÷(expression amount of TNFRSF11B gene)...(F2)
Ratio of SLC35F2 protein expression level to TNFRSF11B protein expression level=(SLC35F2 protein expression level)/(TNFRSF11B protein expression level)...(F3)

Here, as the gene expression level, for example, the copy number of the mRNA transcribed from the TNFRSF11B gene or the mRNA transcribed from the SLC35F2 gene may be measured, and the copy number may be determined as the expression level. Alternatively, for each gene, the relative value of the expression level based on the expression level of the housekeeping gene such as glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was measured, and the measured relative value was used as the expression level. You may judge. As the protein expression level, for example, the number of molecules of TNFRSF11B protein or SLC35F2 protein may be calculated and the number of molecules may be determined as the expression level.

In calculating the ratio of the TNFRSF11B gene or protein to the SLC35F2 gene or protein, any gene may be used as a reference. For example, when the SLC35F2 gene or protein is used as a reference, the ratio of the expression level of the TNFRSF11B gene to the expression level of the SLC35F2 gene or the expression of the protein encoded by the TNFRSF11B gene to the expression level of the protein encoded by the SLC35F2 gene. When the ratio of the amounts is smaller than those of the control cells, it is determined that the somatic stem cells or chondrocytes have a high differentiation ability into bone tissue or cartilage tissue.

[Method for producing a marker showing the ability of somatic stem cells or chondrocytes to differentiate into bone tissue or cartilage tissue]
In one embodiment, the present invention provides a gene or a somatic stem cell or chondrocyte having a high differentiation ability to a bone tissue or a cartilage tissue, and a somatic stem cell or chondrocyte having a low differentiation ability to a bone tissue or a cartilage tissue, Expression in the step of measuring the expression level of the protein, somatic stem cells or chondrocytes with high differentiation ability to bone tissue or cartilage tissue, and somatic stem cells or chondrocytes with low differentiation ability to bone tissue or cartilage tissue Determining that the gene or protein having a different amount is a marker showing the ability of somatic stem cells or chondrocytes to differentiate into bone tissue or cartilage tissue, the bone tissue of somatic stem cells or chondrocytes, or Provided is a method for producing a marker showing the ability to differentiate into cartilage tissue.

As will be described later in Examples, a marker showing the ability of somatic stem cells or chondrocytes to differentiate into bone tissue or cartilage tissue can be obtained by the method of the present embodiment. The “marker manufacturing method” of the present embodiment can be restated as a “marker screening method”, a “marker acquisition method”, a “marker determination method”, or the like.

In the method for producing a marker of the present embodiment, it is efficient to comprehensively measure the expression level of a gene or protein by, for example, DNA microarray or RNA sequencing (RNA-Seq) using a next-generation sequencer.

Further, as the somatic stem cells or chondrocytes having a high differentiation ability into bone tissue or cartilage tissue, for example, somatic stem cells or chondrocytes with a low passage number may be used. Further, as the somatic stem cells or chondrocytes having a low ability to differentiate into bone tissue or cartilage tissue, somatic stem cells or chondrocytes with a high passage number may be used. The high level of differentiation into bone tissue or cartilage tissue is preferably determined by actually inducing differentiation of somatic stem cells or chondrocytes into bone tissue or cartilage tissue and confirming the differentiation potential.

Next, the present invention will be described in more detail with reference to examples, but the present invention is not limited to the following examples.

[Experimental Example 1]
(Examination of the relationship between the number of passages of mesenchymal stem cells and the ability to differentiate into bone tissue)
The mesenchymal stem cells were passaged and induced to differentiate into bone tissue over time, and the ability to differentiate into bone tissue at each passage was examined. Examples of the mesenchymal stem cells are Yud622 (RIKEN BioResource Center), which is a polydactyl digital bone marrow-derived mesenchymal stem cell, hMSC (Lonza), which is an adult bone marrow-derived mesenchymal stem cell, and adult bone marrow-derived mesenchymal stem cell. Bone Marrow Derived Mesenchymal Stem Cells; Normal, Human (BM-AT) (ATCC) and adult fat-derived mesenchymal stem cells, StemPro human ADSC (Thermo Fisher Scientific) were used.

At the passage of each cell, the cumulative PDL was calculated by integrating the cell division number (PDL) calculated by the following formula (F1).
PDL=log ₁₀ (the number of cells at the end of culture/the number of cells at the start of culture)×3.33 (F1)

In addition, at the time of each passage, some cells were transferred to a new dish, and a commercially available medium (type “PT-3002”, trade name “hMSC differential Bullet Kit-osteogenic”, Lonza) was used for 2 weeks (Yub622, hMSC, BM-AT) or 4 weeks (StemPro human ADSC) culture to induce differentiation into bone tissue. As a control, cells cultured for the same period without induction of differentiation were prepared.

Subsequently, it was confirmed whether or not the cells were induced to differentiate into bone tissue by staining the cells after induction of differentiation into bone tissue with alizarin red. When the cells have differentiated into bone tissue, they are stained reddish orange.

1 to 4 are graphs and photographs showing the experimental results. FIG. 1( a) is a growth curve of Yub622 cells. Numbers in the graph indicate passage numbers. FIG. 1(b) is a photograph showing the results of alizarin red staining of Yub622 cells that have been induced to differentiate into bone tissue. In the figure, "bone differentiation induction (+)" indicates the result of induction of differentiation into bone tissue, and "bone differentiation induction (-)" indicates the result of control cells. "P6" means that cells that were passaged 6 times were differentiated into bone tissue, "P7" means that cells that were passaged 7 times were differentiated into bone tissue, and so on. ..

2(a) and 2(b) show the results of the same experiment as in FIGS. 1(a) and 1(b) except that hMSCs were used as cells. 3(a) and 3(b) show the results of the same experiment as in FIGS. 1(a) and 1(b) except that BM-AT was used as the cells. 4(a) and 4(b) show the results of the same experiment as in FIGS. 1(a) and 1(b), except that StemPro human ADSC was used as the cells. In the figure, "ADSC" indicates that the results were obtained using StemPro human ADSC cells.

As a result, in any of the mesenchymal stem cells, the smaller the passage number, the higher the differentiation ability to bone tissue, and the tendency to decrease the differentiation ability to bone tissue as the passage number increased was observed.

[Experimental Example 2]
(Examination of the relationship between the number of passages of mesenchymal stem cells and the ability to differentiate into cartilage tissue)
Mesenchymal stem cells were passaged to induce differentiation into cartilage tissue, and the relationship between the number of passages and the ability to differentiate into cartilage tissue was examined. As the mesenchymal stem cells, Yub621c (RIKEN BioResource Center) and Yub625 (RIKEN BioResource Center), which are polydactyl finger bone marrow-derived mesenchymal stem cells, were used.

Yub621c cells at passage numbers 7 and 9 and Yub625 cells at passage numbers 5 and 22 were transferred to a commercially available culture medium kit (type “PT-3003”, trade name “hMSC differential Bullet Kit-chondrogenic”, Lonza) using the TransformingGrowth. Differentiation into cartilage tissue was induced by pellet culture of 2×10 ⁵ cells as one pellet for 28 to 29 days using a medium supplemented with −β3 (TGFβ3) and bone morphogenetic protein 2 (BMP2).

Subsequently, each cell hull after differentiation induction was fixed with formalin to prepare a paraffin section. Then, each prepared section was stained with hematoxylin-eosin and alcian blue. The cartilage tissue is stained with Alcian blue.

5A and 5B are photographs showing the experimental results. FIG. 5(a) is a result of hematoxylin-eosin staining (HE) and alcian blue staining (AB) of a sample in which Yub621c cells at passage number 7 (P7) and passage number 9 (P9) were induced to differentiate into cartilage tissue. Is a photograph showing.

In addition, FIG. 5( b) shows hematoxylin-eosin staining (HE) and alcian blue staining (AB) of a sample obtained by inducing differentiation of Yub625 cells at passage number 5 (P5) and passage number 22 (P22) into cartilage tissue. It is a photograph showing the result of.

As a result, it was confirmed that Yub621c cells at passage numbers 7 and 9 and Yub625 cells at passage number 5 were differentiated into cartilage tissue. On the other hand, it was revealed that cartilage tissue was not formed in the Yub625 cells at passage number 22.

This result indicates that mesenchymal stem cells with a small number of passages have a high ability to differentiate into cartilage tissue, but that the number of passages increases, the ability to differentiate into cartilage tissue decreases.

[Experimental Example 3]
(Microarray analysis 1)
There is a correlation with the ability to differentiate into bone tissue by extracting total RNA from Yub622 cells and hMSC cells that have high ability to differentiate into bone tissue, and Yub622 cells and hMSC cells that have low ability to differentiate into bone tissue. The gene was extracted. As cells having a high ability to differentiate into bone tissue, Yub622 cells with passage numbers of 5 to 9 were used. In addition, as cells having a medium level of differentiation into bone tissue, Yub622 cells having a passage number of 10 and hMSC cells having a passage number of 5 to 8 and 10 were used. In addition, as cells having a low ability to differentiate into bone tissue, Yub622 cells having a passage number of 11 to 16 and hMSC cells having a passage number of 9,11 to 16 were used. As a result, the genes shown in Tables 1 and 2 above were found as genes having a positive correlation with the ability to differentiate into bone tissue.

In addition, the genes listed in Table 3 above were found as genes having a negative correlation with the ability to differentiate into bone tissue.

[Experimental Example 4]
(Microarray analysis 2)
Total RNA was extracted from mesenchymal stem cells at various passage numbers, and the expression level of each gene extracted in Experimental Example 3 was examined by microarray analysis.

The above-mentioned StemPro human ADSC cells, BM-AT cells, Yub622 cells and hMSC cells were used as the mesenchymal stem cells. A commercially available kit (model “311-02501”, trade name “Isogen”, Nippon Gene Co., Ltd.) was used for extraction of total RNA. A commercially available microarray (model “G4851A”, trade name “SurePrint G3 Human GE 8×60K”, Agilent Technologies) was used.

6A to 6S and FIGS. 7A to 7H are graphs showing the results of microarray analysis. In the figure, "ADSC" indicates that the results were obtained using StemPro human ADSC cells. Further, "P4" indicates that it is the analysis result of total RNA extracted from cells at passage number 4, "P5" indicates that it is the analysis result of total RNA extracted from cells at passage number 5, and The same is true.

As a result, it was confirmed that the genes listed in Tables 1 and 2 tended to have high expression levels in cells having a high ability to differentiate into bone tissue.

In addition, it was confirmed that the genes listed in Table 3 above had a low expression level in cells having a high ability to differentiate into bone tissue.

[Experimental Example 5]
(Real-time PCR analysis 1)
Total RNA was extracted from mesenchymal stem cells at various passage numbers, and the expression level of SLC35F2 gene was examined by real-time PCR analysis.

As the mesenchymal stem cells, the above-mentioned Yub622 cells, hMSC cells, BM-AT cells, Yub621c cells and Yub625 cells were used. A commercially available kit (model “311-02501”, trade name “Isogen”, Nippon Gene Co., Ltd.) was used for extraction of total RNA. In addition, the real-time PCR reaction is carried out using a commercially available kit (type “RR096A”, trade name “One Step SYBR PrimeScript PLUS RT-PCR Kit”, Takara Bio Inc.), and real-time PCR device (trade name “StepOnePlus real-time PCR system”, Applied Bio). Systems).

The PCR of the SLC35F2 gene cDNA was performed using a sense primer having the base sequence shown in SEQ ID NO: 1 and an antisense primer having the base sequence shown in SEQ ID NO: 2. As a control, real-time PCR of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was performed, and the expression level of the SLC35F2 gene was corrected based on the measured expression level of the GAPDH gene. PCR of the GAPDH gene cDNA was performed using a sense primer having the base sequence shown in SEQ ID NO:3 and an antisense primer having the base sequence shown in SEQ ID NO:4.

8A to 8D are graphs showing the results of real-time PCR analysis. 8A is the analysis result of Yub622 cells, FIG. 8B is the analysis result of hMSC cells, FIG. 8C is the analysis result of BM-AT cells, and FIG. 8D is the analysis result of Yub621c cells and Yub625 cells. In the figure, "P5" indicates that the results of analysis of total RNA extracted from cells at passage number 5 and "P6" indicate that of results of analysis of total RNA extracted from cells at passage 6 , Is the same.

As a result, it was confirmed that the SLC35F2 gene had a high expression level in cells having a high differentiation ability into bone tissue and cartilage tissue, and a low expression level in cells having a low differentiation ability into bone tissue and cartilage tissue.

[Experimental Example 6]
(Examination of the relationship between the passage number of mesenchymal stem cells and the ability to differentiate into bone tissue and cartilage tissue)
The mesenchymal stem cells were passaged and induced to differentiate into bone tissue and cartilage tissue at successive passages, and the ability to differentiate into bone tissue and cartilage tissue at each passage was examined. As the mesenchymal stem cells, polydactyly finger cartilage-derived mesenchymal stem cells Yub2505 (provided by growth bioresource) and UDE-BM (provided by growth bioresource), which are multiarm humeral bone marrow-derived mesenchymal stem cells were used.

At the passage of each cell, the integrated PDL was calculated by integrating the number of cell divisions (Population doubling, PDL) calculated by the above formula (F1).

In addition, at the time of each passage, transfer some cells to a new dish, and culture for 2 weeks using a commercially available medium (type "PT-3002", trade name "hMSC differential Bullet Kit-osteogenic", Lonza). To induce differentiation into bone tissue. As a control, cells cultured for the same period without induction of differentiation were prepared.

Subsequently, it was confirmed by staining the cells after the differentiation induction into the bone tissue with alizarin red whether or not the differentiation into the bone tissue was induced. When the cells have differentiated into bone tissue, they are stained reddish orange.

At the time of each passage, a part of the cells was transferred to a commercially available medium kit (type “PT-3003”, trade name “hMSC differential Bullet Kit-chondrogenic”, Lonza) and Transforming Growth Factor-β3 (TGFbeta3) and bone. Differentiation into cartilage tissue was induced by pellet culture of 2.5×10 ⁵ cells as one pellet for 2 weeks using a medium to which protein 2 (BMP2) was added.

Subsequently, each cell hull after differentiation induction was fixed with formalin to prepare a paraffin section. Then, each prepared section was stained with Alcian blue. The cartilage tissue is stained with Alcian blue.

FIGS. 9A and 9B, FIG. 10, FIGS. 11A and 11B, and FIG. 12 are graphs and photographs showing the experimental results. FIG. 9( a) is a growth curve of Yub2505 cells. Numbers in the graph indicate passage numbers. FIG. 9( b) is a photograph showing the results of alizarin red staining of Yub2505 cells that have been induced to differentiate into bone tissue. In the figure, "bone differentiation induction (+)" indicates the result of induction of differentiation into bone tissue, and "bone differentiation induction (-)" indicates the result of control cells. "P6" means that cells that were passaged 6 times were differentiated into bone tissue, "P7" means that cells that were passaged 7 times were differentiated into bone tissue, and so on. .. FIG. 10 is a photograph showing the results of Alcian blue staining (AB) of Yub2505 cells that had been induced to differentiate into cartilage tissue.

In addition, FIGS. 11A, 11B, and 12 show the results of the same experiment as FIGS. 9A, 9B, and 10 except that UDE-BM was used as the cells.

As a result, in any mesenchymal stem cell, the smaller the passage number, the higher the differentiation ability into bone tissue and cartilage tissue, and the tendency that the differentiation ability into bone tissue and cartilage tissue decreases as the passage number increases. Admitted. This result further supports that the mesenchymal stem cells with a small number of passages have a high ability to differentiate into cartilage tissue, but the increasing the number of passages reduces the ability to differentiate into cartilage tissue.

[Experiment 7]
(Microarray analysis 3)
Total RNA was extracted from mesenchymal stem cells at various passage numbers, and the expression levels of the genes listed in Tables 1 to 3 found in Experimental Example 3 were examined by microarray analysis.

As the mesenchymal stem cells, the above-mentioned Yub2505 cells, UDE-BM cells and Yub622 cells were used. A commercially available kit (model “311-02501”, trade name “Isogen”, Nippon Gene Co., Ltd.) was used for extraction of total RNA. A commercially available microarray (model “G4851A”, trade name “SurePrint G3 Human GE 8×60K”, Agilent Technologies) was used.

13A to 13M are graphs showing the results of microarray analysis. In the figure, “P4” indicates the analysis result of total RNA extracted from cells at passage number 4, “P5” indicates the analysis result of total RNA extracted from cells at passage number 5, , Is the same.

As a result, it was confirmed that the genes described in Tables 1 and 2 tend to have high expression levels in cells having a high differentiation ability into bone tissue and cartilage tissue.

In addition, it was confirmed that the genes listed in Table 3 above had a low expression level in cells having a high ability to differentiate into bone tissue.

[Experimental Example 8]
(Microarray analysis 4)
Total RNA was extracted from mesenchymal stem cells at various passage numbers, and the expression levels of the genes listed in Tables 1 to 3 found in Experimental Example 3 were examined by microarray analysis.

As the mesenchymal stem cells, the above-mentioned StemPro human ADSC cells, BM-AT cells and Yub622 cells were used. A commercially available kit (model “311-02501”, trade name “Isogen”, Nippon Gene Co., Ltd.) was used for extraction of total RNA. A commercially available microarray (model “G4851A”, trade name “SurePrint G3 Human GE 8×60K”, Agilent Technologies) was used.

14A to 14F are graphs showing the results of microarray analysis. In the figure, “P4” indicates the analysis result of total RNA extracted from cells at passage number 4, “P5” indicates the analysis result of total RNA extracted from cells at passage number 5, , Is the same.

As a result, it was confirmed that the genes listed in Tables 1 and 2 tend to be highly expressed in cells having a high ability to differentiate into bone tissue and cartilage tissue.

In addition, it was confirmed that the genes listed in Table 3 above had a low expression level in cells having a high ability to differentiate into bone tissue.

[Experimental Example 9]
(Real-time PCR analysis 2)
Total RNA was extracted from mesenchymal stem cells at various passage numbers, and the expression level of SLC35F2 gene was examined by real-time PCR analysis.

As the mesenchymal stem cells, the above-mentioned StemPro human ADSC cells, Yub2505 cells and UDE-BM cells were used. A commercially available kit (model “311-02501”, trade name “Isogen”, Nippon Gene Co., Ltd.) was used for extraction of total RNA. In addition, the real-time PCR reaction is carried out using a commercially available kit (type “RR096A”, trade name “One Step SYBR PrimeScript PLUS RT-PCR Kit”, Takara Bio Inc.), and real-time PCR device (trade name “StepOnePlus real-time PCR system”, Applied Bio). Systems).

The PCR of the SLC35F2 gene cDNA was performed using a sense primer having the base sequence shown in SEQ ID NO: 1 and an antisense primer having the base sequence shown in SEQ ID NO: 2. As a control, real-time PCR of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was performed, and the expression level of the SLC35F2 gene was corrected based on the measured expression level of the GAPDH gene. PCR of the GAPDH gene cDNA was performed using a sense primer having the base sequence shown in SEQ ID NO:3 and an antisense primer having the base sequence shown in SEQ ID NO:4.

15A to 15C are graphs showing the results of real-time PCR analysis. FIG. 15A is the analysis result of StemPro human ADSC cells, FIG. 15B is the analysis result of Yub2505 cells, and FIG. 15C is the analysis result of UDE-BM cells. In the figure, "P5" indicates that the results of analysis of total RNA extracted from cells at passage number 5 and "P6" indicate that of results of analysis of total RNA extracted from cells at passage 6 , Is the same.

As a result, it was confirmed that the SLC35F2 gene had a high expression level in cells having a high differentiation ability into bone tissue and cartilage tissue, and a low expression level in cells having a low differentiation ability into bone tissue and cartilage tissue.

[Experimental Example 10]
(Real-time PCR analysis 3)
Total RNA was extracted from mesenchymal stem cells at various passage numbers, and the expression level of the TNFRSF11B gene was examined by real-time PCR analysis.

As the mesenchymal stem cells, the above-mentioned Yub622 cells, hMSC cells, BM-AT cells, Yub621c cells, Yub625 cells, StemPro human ADSC cells, Yub2505 cells, and UDE-BM cells were used. A commercially available kit (model “311-02501”, trade name “Isogen”, Nippon Gene Co., Ltd.) was used for extraction of total RNA. In addition, the real-time PCR reaction is carried out using a commercially available kit (type “RR096A”, trade name “One Step SYBR PrimeScript PLUS RT-PCR Kit”, Takara Bio Inc.), and real-time PCR device (trade name “StepOnePlus real-time PCR system”, Applied Bio). Systems).

The PCR of the cDNA of the TNFRSF11B gene was performed using a sense primer having the base sequence shown in SEQ ID NO:5 and an antisense primer having the base sequence shown in SEQ ID NO:6. As a control, real-time PCR of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene was performed, and the expression level of the TNFRSF11B gene was corrected based on the measured expression level of the GAPDH gene. PCR of the GAPDH gene cDNA was performed using a sense primer having the base sequence shown in SEQ ID NO:3 and an antisense primer having the base sequence shown in SEQ ID NO:4.

16A to 16G are graphs showing the results of real-time PCR analysis. 16A is a result of analysis of Yub622 cells, FIG. 16B is a result of analysis of hMSC cells, FIG. 16C is a result of analysis of BM-AT cells, and FIG. 16D is a result of analysis of Yub621c cells and Yub625 cells. 16E is the analysis result of StemPro human ADSC cells, FIG. 16F is the analysis result of Yub2505 cells, and FIG. 16G is the analysis result of UDE-BM cells. In the figure, "P5" indicates that the results of analysis of total RNA extracted from cells at passage number 5 and "P6" indicate that of results of analysis of total RNA extracted from cells at passage 6 , Is the same.

As a result, it was confirmed that the TNFRSF11B gene had a low expression level in cells having a high differentiation ability into bone tissue and cartilage tissue, and a high expression level in cells having a low differentiation ability into bone tissue and cartilage tissue.

[Experimental Example 11]
(Study of ratio of expression levels of TNFRSF11B gene and SLC35F2 gene)
The ratio of the expression level of the TNFRSF11B gene and the expression level of the SLC35F2 gene in each cell analyzed by real-time PCR in Experimental Examples 5, 9, and 10 was calculated, and the correlation with the ability to differentiate into bone tissue or cartilage tissue was examined.

Specifically, the ratio of the expression level of the SLC35F2 gene to the expression level of the TNFRSF11B gene in each cell was calculated based on the following formula (F2).
Ratio of expression amount of SLC35F2 gene to expression amount of TNFRSF11B gene=(expression amount of SLC35F2 gene)÷(expression amount of TNFRSF11B gene)...(F2)

17A and 17B are graphs showing the ratio of the expression level of the SLC35F2 gene to the expression level of the TNFRSF11B gene in each cell. 17A and 17B also show the ability of each cell to differentiate into bone tissue or cartilage tissue. In the figure, "++", "+", "+/-", and "-" indicate high differentiation ability into bone tissue or cartilage tissue. "++" indicates that the differentiation potential was highest, and "-" indicates that the differentiation potential was not observed. In addition, "ND" indicates that the differentiation ability was not tested.

As a result, it was revealed that cells having a higher ratio of the expression level of the SLC35F2 gene to the expression level of the TNFRSF11B gene tend to have a higher ability to differentiate into bone tissue or cartilage tissue.

According to the present invention, a marker showing the ability of somatic stem cells or chondrocytes to differentiate into bone tissue or cartilage tissue can be provided.

Claims (9)

A primer set for amplifying cDNA of the genes listed in Table 1 below ,
A probe that specifically hybridizes to mRNA of the genes listed in Table 1 below , or
Specific binding agent to the protein encoded by the gene in the following Table 1, comprises, between the mesenchymal stem cells or chondrocytes, kit determination ability formation of bone tissue or cartilage tissue.
A primer set for amplifying cDNA of a gene selected from the group consisting of the genes shown in Tables 2 and 3 below ,
A probe that specifically hybridizes to mRNA of a gene selected from the group consisting of the genes shown in Tables 2 and 3 below , or
Table 2 and the specific binding substance to the protein encoded by the gene selected from the group consisting of the genes listed in Table 3, further comprising a mesenchymal stem cell or a chondrocyte during claim 1, A kit for determining the ability to form bone tissue or cartilage tissue.
Primer set for amplifying cDNA of SLC35F2 gene, probe specifically hybridizing to mRNA of SLC35F2 gene, or specific binding substance for protein encoded by SLC35F2 gene, and primer for amplifying cDNA of TNFRSF11B gene Set, a probe that specifically hybridizes to mRNA of TNFRSF11B gene, or a specific binding substance for a protein encoded by TNFRSF11B gene,
The comprising of mesenchymal stem cells or chondrocytes during claim 2, kit determination ability to form bone tissue or cartilage tissue. A step of quantifying the expression level of a marker gene or a marker protein in somatic stem cells or chondrocytes, wherein the marker gene or the marker protein is the gene described in Table 1 or the table 1 according to claim 1. the described gene containing encoded protein, between mesenchymal stem cells or chondrocytes, determination method of forming ability of bone tissue or cartilage tissue. The marker gene or the marker protein is a gene selected from the group consisting of the genes listed in Tables 2 and 3 of claim 2, or the group consisting of the genes listed in Tables 2 and 3 above. further comprising the mesenchymal stem cells or chondrocytes during claim 4, the determination method of the ability to form bone tissue or cartilage tissue encoded protein more selected genes. The marker gene or the marker protein is a gene selected from the group consisting of the genes listed in Table 1 or the proteins encoded by the genes listed in Table 1, and the genes listed in Table 2; or A protein encoded by a gene selected from the group consisting of the genes listed in Table 2 above,
When the expression level of the marker gene or the marker protein in mesenchymal stem cells or chondrocytes is higher than the expression level of the marker gene or the marker protein in control cells, the mesenchymal stem cells or the chondrocytes are bone. The method according to claim 5 , wherein the ability to form tissue or cartilage tissue is determined to be high. The marker gene or the marker protein is a gene selected from the group consisting of the genes listed in Table 1 or the proteins encoded by the genes listed in Table 1, and the genes listed in Table 3; or A gene encoded by a gene selected from the group consisting of the genes listed in Table 3 above, the gene listed in Table 1 above in mesenchymal stem cells or chondrocytes, or the gene listed in Table 1 above The expression level of the protein encoded by the above is higher than the expression levels of the genes listed in Table 1 or the proteins encoded by the genes listed in Table 1 in control cells, and mesenchymal stem cells or cartilage The expression level of the gene encoded by the gene selected from the group consisting of the genes listed in Table 3 above or the protein encoded by the gene selected from the group consisting of the genes listed in Table 3 above in the cells The determination method according to claim 5 , wherein the mesenchymal stem cells or the chondrocytes are determined to have high bone tissue or cartilage tissue formation ability when the expression level of the gene or the protein is lower. The marker gene or the marker protein comprises a combination of the SLC35F2 gene or the protein encoded by the SLC35F2 gene, and the protein encoded by the TNFRSF11B gene or the TNFRSF11B gene,
The expression amount of the SLC35F2 gene or the protein encoded by the SLC35F2 gene in mesenchymal stem cells or chondrocytes is higher than the expression amount of the protein encoded by the SLC35F2 gene or SLC35F2 gene in control cells, and mesenchymal stem cells or When the expression level of the TNFRSF11B gene or the protein encoded by the TNFRSF11B gene in chondrocytes is lower than the expression level of the protein encoded by the TNFRSF11B gene or TNFRSF11B gene in the control cells, the mesenchymal stem cell or the chondrocyte is The determination method according to claim 7, wherein it is determined that the ability to form bone tissue or cartilage tissue is high. The marker gene or the marker protein is a combination of the SLC35F2 gene or the protein encoded by the SLC35F2 gene, and the protein encoded by the TNFRSF11B gene or the TNFRSF11B gene,
The ratio of the expression level of the SLC35F2 gene to the expression level of the TNFRSF11B gene in mesenchymal stem cells or chondrocytes is higher than the ratio of the expression level of the SLC35F2 gene to the expression level of the TNFRSF11B gene in control cells, or
The ratio of the expression amount of the protein encoded by the SLC35F2 gene to the expression amount of the protein encoded by the TNFRSF11B gene in mesenchymal stem cells or chondrocytes is SLC35F2 relative to the expression amount of the protein encoded by the TNFRSF11B gene in control cells. The determination method according to claim 5 , wherein the mesenchymal stem cells or the chondrocytes are determined to have high bone tissue- or cartilage tissue- forming ability when the expression ratio of the protein encoded by the gene is higher than the ratio.