Osteogenic differentiation ability determination method

Abstract

Provided are: an osteogenic differentiation ability determination method; a method for isolating or enriching somatic stem cells having osteogenic differentiation ability; a kit; a pharmaceutical composition production method; and a culturing condition selection method.　The present invention pertains to: a method comprising detecting α1,2 fucose derived from somatic stem cells in a sample; and a kit comprising a probe that specifically binds to α1,2 fucose.

Description

Method for determining bone differentiation potential

[0001] The present disclosure relates to a marker for determining bone differentiation potential, a method for determining bone differentiation potential, and a method for isolating or concentrating somatic stem cells having bone differentiation potential.

[0002] The development of treatments that use stem cells, including mesenchymal stem cells, to repair and regenerate damaged cells and tissues in patients is progressing.Mesenchymal stem cells have a problem with heterogeneity, which occurs due to various factors such as the tissue of origin, donor, and culture method, and there is a need to develop technologies to detect and separate the functionality of mesenchymal stem cells.

[0003] Hiroaki Tateno et al. α2-6 sialylation is a marker of the differentiation potential of human mesenchymal stem cells, Glycobiology, 2016 Dec;26(12):1328-1337. doi: 10.1093 / glycob / cww039.Mabuchi Y et al. LNGFR(+)THY-1(+)VCAM-1(hi+) cells reveal functionally distinct subpopulations in mesenchymal stem cells, Stem Cell Reports, 2013 Jul 11;1(2):152-65. doi: 10.1016 / j.stemcr.2013.06.001.Eriko Grace Suto et al. Prospectively isolated mesenchymal stem / stromal cells are enriched in the CD73+ population and exhibit efficacy after transplantation, Sci Rep. 2017 Jul 6;7(1):4838. doi: 10.1038 / s41598-017-05099-1.

[0004] The present disclosure aims to provide a marker for determining bone differentiation potential, a method for determining bone differentiation potential, a method for isolating or concentrating somatic stem cells with bone differentiation potential, a kit, a method for producing a pharmaceutical composition, and a method for selecting culture conditions.

[0005] The present disclosure includes the following aspects. [1-1] A method for determining the bone differentiation potential of somatic stem cells, comprising detecting somatic stem cell-derived α1,2 fucose in a sample. [1-2] The method according to [1-1], wherein the somatic stem cells are mesenchymal stem cells. [1-3] The method according to [1-1] or [1-2], further comprising detecting somatic stem cell-derived α2,6 sialic acid in a sample. [1-4] The method according to any one of [1-1] to [1-3], wherein α1,2 fucose is detected using a lectin that specifically binds to α1,2 fucose. [1-5] The method according to [1-3] or [1-4], wherein α2,6 sialic acid is detected using a lectin that specifically binds to α2,6 sialic acid. [2-1] A method for separating or enriching somatic stem cells with bone differentiation potential, comprising: contacting somatic stem cells in a sample with a probe that specifically binds to α1,2 fucose; and separating and removing the somatic stem cells bound to the probe that specifically binds to α1,2 fucose from other cells in the sample. [2-2] The method according to [2-1], wherein the somatic stem cells are mesenchymal stem cells. [2-3] The method according to [2-1] or [2-2], further comprising: contacting somatic stem cells in a sample with a probe that specifically binds to α2,6 sialic acid; and separating and maintaining the somatic stem cells bound to the probe that specifically binds to α2,6 sialic acid from other cells in the sample. [3-1] A kit for determining the bone differentiation potential of somatic stem cells, comprising a probe that specifically binds to α1,2 fucose. [3-2] A kit for separating or enriching somatic stem cells with bone differentiation potential, comprising a probe that specifically binds to α1,2 fucose. [3-3] The kit according to [3-1] or [3-2], wherein the probe that specifically binds to α1,2-fucose is UEA1 lectin. [3-4] The kit according to any one of [3-1] to [3-3], wherein the somatic stem cells are mesenchymal stem cells. [3-5] The kit according to any one of [3-1] to [3-4], further comprising a probe that specifically binds to α2,6-sialic acid.[3-6] The kit according to [3-5], wherein the probe that specifically binds to α2,6 sialic acid is at least one lectin selected from the group consisting of TJAI, SSA, SNA, and PSL1a. [4-1] A method for producing a pharmaceutical composition for treating or preventing a bone disease, comprising the method according to any one of [2-1] to [2-3]. [4-2] A method for selecting culture conditions suitable for culturing somatic stem cells with bone differentiation potential, comprising the method according to any one of [1-1] to [1-5]. [4-3] The method according to [4-2], wherein the somatic stem cells are mesenchymal stem cells.

[0006] According to the present disclosure, it is possible to provide a marker for determining bone differentiation potential, a method for determining bone differentiation potential, a method for isolating or concentrating somatic stem cells having bone differentiation potential, a kit, a method for producing a pharmaceutical composition, and a method for selecting culture conditions.

[0007] 1 shows a microscopic image (A) of Alizarin Red staining of an MSC3 line that had been induced to differentiate into bone, and a graph (B) of the absorbance (Mean ± SD) of the eluate of the staining dye (N = 3). Scale bar: 100 μm. 1 shows a microscopic image (A) of Oil Red staining of an MSC3 line that had been induced to differentiate into adipocytes, and a graph (B) of the absorbance (Mean ± SD) of the eluate of the staining dye (N = 3). Scale bar: 100 μm. 1 shows a list of the lectins used in the DNA-labeled lectins used in the search for glycan markers by scGR-seq in Example 3. 1 shows the results of analyzing an MSC3 line (N = 3149 cells) by scGR-seq, and dimensionally compressing the obtained RNA information and glycan information using the UMAP method, followed by depiction. (A) MSC3 line (N = 3149 cells) was analyzed by scGR-seq, and the resulting RNA and glycan information was subjected to dimensionality reduction using the UMAP method. This is a dot plot of differentially expressed genes (top 10 genes) for each cluster classified by clustering analysis. The color shading indicates the average expression level of the gene, and the size of the circle indicates the percentage of cells expressing each gene. The solid-line box indicates the cluster in which the gene showed the highest expression. (B) This is a dot plot of only lectins (variably reactive lectins) that have high binding affinity and a high percentage of bound cells in the MSC3 line clusters. The color shading indicates the average binding level of the lectin, and the size of the circle indicates the percentage of cells bound by each lectin. The solid-line box indicates the cluster in which the lectin showed the highest binding. This is a graph showing the results of analyzing the content of clusters 0 to 9 in the MSC3 line. The content of cluster 5 in each line is connected by a solid line. This is a graph showing the results of analyzing the binding amount of PE-labeled UEA1 to the MSC3 line by flow cytometry. The light-colored peak represents the negative control PE-BSA, and the dark-colored peak represents PE-UEA1. Mean PE-A represents the mean fluorescence intensity of PE-UEA1, the vertical axis represents the number of cells normalized by the mode, and the horizontal axis represents the amount of PE fluorescence. This graph shows the results of FACS analysis of the gates for positive or negative fractions in each MSC line and the amount of UEA1 binding before and after sorting. The vertical axis represents the number of cells, and the horizontal axis represents the amount of PE fluorescence.UEA1-positive MSC cells and UEA1-negative MSC cells were sorted by FACS and induced to differentiate into bone. (A) Microscopic images of alizarin red staining and (B) a graph of the absorbance (mean ± SD) of the dye eluate (N = 3) are shown. Scale bar: 100 μm. This is a dot plot diagram of lectins that showed varying reactivity between MSC lines in scGR-seq analysis. The shade of color indicates the average binding amount of the lectin, and the size of the circle indicates the percentage of cells bound by each lectin. The solid line frame indicates the cluster with the highest lectin binding. This graph shows the results of flow cytometry analysis of the binding amount of PE-labeled SSA to MSC3 lines. The light-colored peak indicates the negative control PE-BSA, and the solid line peak indicates PE-SSA. Mean PE-A indicates the mean fluorescence intensity of PE-SSA. The vertical axis indicates the number of cells normalized by the mode, and the horizontal axis indicates the fluorescence intensity of PE. Figure 1 shows the results of FACS analysis of the amount of SSA binding before and after sorting, as well as the gates for positive and negative fractions in each MSC line. The vertical axis indicates the number of cells, and the horizontal axis indicates the fluorescence intensity of PE. SSA-positive MSC cells and SSA-negative MSC cells sorted by FACS were subjected to osteogenic differentiation induction. (A) shows a microscopic image stained with Alizarin Red, and (B) shows a graph of the absorbance (Mean ± SD) of the dye eluate (N = 3). Scale bar: 100 μm. Figure 1 shows the results of an analysis of the amount of UEA1 and SSA binding for MSC cells before sorting by FACS, SSA-positive MSC cells sorted by FACS, and UEA1-negative MSC cells. The vertical axis indicates the fluorescence intensity of APC-SSA, and the horizontal axis indicates the fluorescence intensity of PE-UEA1. MSC cells before sorting by FACS, SSA-positive MSC cells sorted by FACS, and UEA1-negative MSC cells were induced to differentiate into bones. (A) Microscopic images of Alizarin Red staining and (B) a graph of absorbance (Mean ± SD) of the eluate (N = 4) are shown. Scale bar: 100 μm. SSA-positive MSC cells and SSA-negative MSC cells sorted by FACS were induced to differentiate into cartilage. (B) Graph of absorbance (Mean ± SD) of the eluate stained with Alcian Blue stain (N = 8).(A) A graph showing the results of flow cytometry demonstrating the binding of PE-UEA1 to MSC cells co-administered with fucose (Con = 0, 10, 100 mM) and UEA1, and to MSC cells previously treated with fucosidase (ExoFuc). The light gray peak represents PE-BSA (negative control), and the dark gray peak represents PE-UEA1. The vertical axis represents the number of cells normalized by the mode, and the horizontal axis represents the fluorescence intensity of PE. (B) A graph showing the difference between the mean fluorescence intensity of PE-UEA1 and the mean fluorescence intensity of PE-BSA in each group. (A) A graph showing the results of flow cytometry analysis of the amount of PE-UEA1 binding to MSCs pretreated with 2-FF (0.1, 0.3, 1 mM) for 3 days. The light gray peak represents PE-BSA (negative control), and the dark gray peak represents PE-UEA1. The vertical axis shows the number of cells normalized by the mode, and the horizontal axis shows the fluorescence intensity of PE. (B) A diagram showing the difference between the mean fluorescence intensity of PE-UEA1 and the mean fluorescence intensity of PE-BSA in each group. Osteogenesis induction was performed in the presence or absence of 2-FF, and microscopic images (A) were stained with Alizarin Red, and a graph (B) of the absorbance of the eluate (Mean ± SD) was shown (N = 6). Scale bar: 100 μm.

[0008] An embodiment of the present disclosure will be described in detail below. However, the scope of the present disclosure is not limited to the embodiment described herein, and various modifications can be made without departing from the spirit of the present disclosure. Each aspect disclosed in this specification can be combined with any other feature disclosed in this specification. Furthermore, when multiple upper and lower limits are described for a particular parameter, any of these upper and lower limits can be combined to form a suitable numerical range. Furthermore, the lower and / or upper limits of a numerical range described in this disclosure may be replaced with numerical values ​​within that range and shown in the examples. The expression "X to Y" indicating a numerical range means "X or greater and Y or less." If a specific description described for one embodiment also applies to other embodiments, that description may be omitted in other embodiments. Each configuration and combination thereof in each embodiment is merely an example, and additions, omissions, substitutions, and other modifications of configurations are possible as appropriate within the spirit of the present disclosure. The present disclosure is not limited by the embodiments, but is limited only by the claims. Unless otherwise specified in the embodiments and examples, methods described in standard protocol collections such as J. Sambrook, E. F. Fritsch & T. Maniatis (Eds.), Molecular cloning, a laboratory manual (3rd edition), Cold Spring Harbor Press, Cold Spring Harbor, New York (2001); F. M. Ausubel, R. Brent, R. E. Kingston, D. D. Moore, J. G. Seidman, J. A. Smith, K. Struhl (Eds.), Current Protocols in Molecular Biology, John Wiley & Sons Ltd., or modified or altered methods thereof, are used. Furthermore, when commercially available reagent kits or measuring devices are used, the protocols attached thereto are used unless otherwise specified.

[0009] ==First embodiment (marker for determining bone differentiation potential)== The marker according to this embodiment is a marker for determining the bone differentiation potential of somatic stem cells, which is composed of a sugar chain containing α1,2 fucose derived from somatic stem cells.

[0010] (α1,2 Fucose) α1,2 fucose is a sugar chain in which the hydroxyl group at position 1 of fucose is α-linked to the hydroxyl group at position 2 of galactose. The marker containing α1,2 fucose is not limited as long as it is a sugar chain containing α1,2 fucose, and may be a glycoprotein or a glycolipid. Sugar chains containing α1,2 fucose include H type 1 (Fucα1-2Galβ1-3GlcNAc), H type 2 (Fucα1-2Galβ1-4GlcNAc), H type 3 (Fucα1-2Galβ1-3GalNAcα1-), H type 4 (Fucα1-2Galβ1-3GalNAcβ1-), A type 1 (Fucα1-2(GalNAcα1-3)Galβ1-3GlcNAc), A type 2 (Fucα1-2(GalNAcα1-3)Galβ1-4GlcNAc), A type 3 (Fucα1-2(GalNAcα1-3)Galβ1-3GalNAcα1-), and A type 4 (Fucα1-2(GalNAcα1 -3)Galβ1-3GalNAcβ1-), B type 1 (Fucα1-2(Galα1-3)Galβ1-3GlcNAc), B type 2 (Fucα1-2(Galα1-3)Galβ1-4GlcNAc), B type 3 (Fucα1-2(Galα1-3)Galβ1-3GalNAcα1-), B type 4 (Fucα1-2(Galα1-3)Galβ1-3GalNAcβ1-), LeY (Fucα1-2Galβ1-4(Fucα1-3)GlcNAc), LeB (Fucα1-2Galβ1-3(Fucα1-4)GlcNAc), etc., but any sugar chain containing α1,2 fucose at the non-reducing end of the sugar chain is acceptable. The α1,2-fucose may be L-fucose or D-fucose.

[0011] The marker according to this embodiment is a sugar chain containing α1,2 fucose derived from somatic stem cells whose bone differentiation potential is to be assessed. The sugar chain is expressed on the cell surface of somatic stem cells. The sugar chain may remain attached to the cell surface of somatic stem cells, or the sugar chain released from the cell surface of somatic stem cells and present in the sample may be used as the marker. For example, α1,2 fucose or a marker (sugar chain) containing α1,2 fucose can be released from somatic stem cells using enzymatic cleavage with peptide N-glycosidase or endo-type O-glycanase, or chemical cleavage methods such as hydrazinolysis or alkaline β-elimination. For example, when assessing the bone differentiation potential of homogeneous somatic stem cells, such as cultured cells, the released sugar chain may be used as a marker. The bone differentiation potential of a cell population can be assessed.

[0012] When the sugar chain is attached to the cell surface of the somatic stem cell, it may be bound to, for example, a protein on the cell or a lipid on the cell. When bound to a protein, the amino acid may be serine and / or threonine (i.e., the sugar chain is a serine-threonine-linked sugar chain (O-glycan)), or asparagine (i.e., the sugar chain is an asparagine-linked sugar chain (N-glycan)).

[0013] Somatic stem cells expressing α1,2 fucose on their cell surface are likely to have no or low bone differentiation potential. In one embodiment, somatic stem cells expressing α1,2 fucose on their cell surface have no or low bone differentiation potential. Therefore, a sugar chain containing α1,2 fucose derived from somatic stem cells can be used as a negative marker for determining the bone differentiation potential of somatic stem cells. Furthermore, in a somatic stem cell population containing a higher number or proportion of somatic stem cells expressing sugar chains containing α1,2 fucose on their cell surface compared to a comparison cell population, the somatic stem cell population has lower bone differentiation potential than the comparison cell population. Therefore, a sugar chain containing α1,2 fucose derived from somatic stem cells can be used as a negative marker for determining the bone differentiation potential of a somatic stem cell population.

[0014] (Somatic stem cells) Somatic stem cells include somatic stem cells isolated from biological samples collected from an animal individual and cells obtained by subculture of such somatic stem cells. That is, somatic stem cells may be cells isolated from an animal individual, their primary cultured cells, subcultured cells, or established cultured cell lines. Examples of somatic stem cells include neural stem cells, epithelial stem cells, hepatic stem cells, germline stem cells, hematopoietic stem cells, mesenchymal stem cells, chondrocyte stem cells, and skeletal muscle stem cells. Considering the relationship between α-1,2-fucose and the bone differentiation ability of cells, somatic stem cells are preferably mesenchymal stem cells or chondrocyte stem cells, and more preferably mesenchymal stem cells.

[0015] The animal from which the somatic stem cells are derived may be, for example, a mammal or any other species, such as a bird, reptile, amphibian, or fish, but is preferably a mammal. Stem cells are believed to be regulated in a common way in mammals other than humans. The mammal may be a human or a non-human animal, and the non-human animal species may be, for example, a monkey, dog, cat, horse, cow, pig, sheep, goat, rabbit, guinea pig, hamster, mouse, and / or rat, and is not limited by its use as a livestock animal, pet animal, or laboratory animal.

[0016] Somatic stem cells can be obtained from animal umbilical cord blood, umbilical cord, amniotic membrane, placenta, adipose tissue, etc., or from bone marrow obtained by bone marrow aspiration of the jawbone, femur, ilium, etc. Commercially available mesenchymal stem cells can also be purchased. For example, adipose tissue-derived mesenchymal stem cells can be purchased from Life Technologies, Inc., and bone marrow-derived mesenchymal stem cells can be purchased from Lonza, PromoCell, etc. Furthermore, bone marrow-derived mesenchymal stem cells can be obtained from bone marrow tissue of fingers surgically removed from polydactyly patients, and cartilage tissue-derived chondrocyte stem cells (polydactyly-derived chondrocyte stem cells) can be obtained from cartilage tissue. They can also be obtained from the RIKEN BioResource Center, JSRB Cell Bank, etc.

[0017] The culture conditions for somatic stem cells are not particularly limited and can be appropriately determined by those skilled in the art depending on the type of cell; however, the culture temperature is preferably 36 to 37°C, which is similar to the body temperature of an animal. For example, when the somatic stem cells are mesenchymal stem cells, the medium may be MesenPRO RST M medium (Life Technologies), which is commonly used as a mesenchymal stem cell maintenance medium, or other medium for maintaining chondrocytes, such as chondrocyte basal medium or chondrocyte proliferation medium (Takara Bio).

[0018] (Bone differentiation ability) "Bone differentiation" with respect to the marker according to this embodiment encompasses the phenomenon of bone or cartilage formation, regardless of the mechanism of action. Cells that constitute animal bones include osteoblasts, osteocytes, and osteoclasts. Cells that constitute cartilage include chondrocytes. Since osteoblasts are bone-forming cells differentiated from mesenchymal stem cells, bone differentiation according to this embodiment, in one embodiment, refers to the differentiation of osteoblasts from somatic stem cells. In another embodiment, bone differentiation refers to the differentiation of chondrocytes from somatic stem cells. In one embodiment, bone differentiation refers to the differentiation of osteoblasts from mesenchymal stem cells. In another embodiment, bone differentiation refers to the differentiation of chondrocytes from mesenchymal stem cells.

[0019] "Osteoblast differentiation potential" refers to the potential of somatic stem cells to undergo bone differentiation when maintained under appropriate culture conditions. Therefore, assessment of bone differentiation potential may be a determination of the bone differentiation potential of the subject's somatic stem cells at the time the assessment result is obtained, or a prediction of the future possibility of bone differentiation. In one embodiment, bone differentiation potential refers to the potential of somatic stem cells to differentiate into osteoblasts or chondrocytes. In one embodiment, bone differentiation potential refers to the potential of mesenchymal stem cells to differentiate into osteoblasts or chondrocytes.

[0020] As used herein, "having bone differentiation ability" may mean either having bone differentiation ability or having a higher bone differentiation ability than a comparison subject. "Not having bone differentiation ability (no bone differentiation ability)" may mean either not having bone differentiation ability or having a lower bone differentiation ability than a comparison subject.

[0021] That is, the marker according to this embodiment may be a marker for determining the differentiation potential of somatic stem cells to differentiate into osteoblasts or chondrocytes, or may be a marker for determining the differentiation potential of mesenchymal stem cells or chondrocytes to differentiate into osteoblasts or chondrocytes. In one embodiment, the marker according to this embodiment may be a marker for determining the differentiation potential of mesenchymal stem cells to differentiate into osteoblasts or chondrocytes.

[0022] (Combined Markers) In one embodiment, the marker according to this embodiment may be used in combination with one or more other markers for assessing the bone differentiation potential of somatic stem cells. For example, the marker according to this embodiment, which is composed of a sugar chain containing α-1,2 fucose, can detect somatic stem cells with low bone differentiation potential, i.e., it is a negative marker for assessing the bone differentiation potential of somatic stem cells. Therefore, using a marker capable of detecting somatic stem cells with high bone differentiation potential in combination with this embodiment facilitates the determination of somatic stem cells with high or low bone differentiation potential with greater accuracy / precision. Therefore, it is preferable that the other marker used in combination with the marker according to this embodiment is a marker capable of detecting somatic stem cells with high bone differentiation potential. An example of such a marker is a sugar chain containing α-2,6 sialic acid derived from somatic stem cells. Here, as with the sugar chain containing α-1,2 fucose, the sugar chain containing α-2,6 sialic acid is not limited to a sugar chain containing α-2,6 sialic acid as long as it is a sugar chain containing α-2,6 sialic acid as part of the sugar chain, and may be either a homo- or hetero-sugar chain.

[0023] Somatic stem cells expressing glycans containing α2,6 sialic acid on their cell surface are more likely to have bone differentiation potential than somatic stem cells that do not have glycans containing α2,6 sialic acid. Therefore, glycans containing α2,6 sialic acid derived from somatic stem cells can be used as a positive marker for determining the bone differentiation potential of somatic stem cells.

[0024] The sugar chains containing α2,6 sialic acid may be sugar chains containing α2,6 sialic acid on CD29 and / or CD49e derived from somatic stem cells. The differentiation potential of somatic stem cells is more accurately reflected by the α2,6 sialic acid contained in the sugar chains on CD29 and / or CD49e than by the total α2,6 sialic acid expressed on the cell surface of somatic stem cells. Therefore, by using the marker according to this embodiment (sugar chains containing α1,2 fucose) in combination with sugar chains containing α2,6 sialic acid on CD29 and / or CD49e, it becomes easier to determine somatic stem cells with high or low bone differentiation potential with higher accuracy / precision.

[0025] Here, in the present specification, "α2,6 sialic acid" refers to both "Neu5Acα2-6Gal," a sugar chain in which N-acetylneuraminic acid is α2,6-linked to the hydroxyl group at position 6 of galactose, and "Neu5Gcα2-6Gal," a sugar chain in which N-glycolyl neuraminic acid is similarly α2,6-linked. However, in glycoproteins having human-derived α2,6 sialic acid at the non-reducing end, only "Neu5Acα2-6Gal," in which α2,6 sialic acid is biosynthesized in vivo, is "Neu5Acα2-6Gal," in which the amino group at position 5 of neuraminic acid is acetylated. Therefore, in the present specification, "α2,6 sialic acid" mainly refers only to "Neu5Acα2-6Gal." In mammals other than humans, such as pigs and mice, the α2-6 sialic acid may be "Neu5Gcα2-6Gal." Therefore, when the target stem cells are derived from a mammal other than humans, "Neu5Acα2-6Gal" and "Neu5Gcα2-6Gal" are considered to be "α2,6 sialic acid."

[0026] Another example of a marker to be used in combination with the marker according to this embodiment is the ratio of α2,6 sialic acid to α2,3 sialic acid. In one embodiment, the ratio of α2,6 sialic acid to α2,3 sialic acid may be the ratio of glycans containing α2,3 sialic acid to glycans containing α2,6 sialic acid.

[0027] In one embodiment, the marker of this embodiment comprises a glycan containing α1,2 fucose and a glycan containing α2,6 sialic acid, a glycan containing α2,6 sialic acid on CD29 and / or CD49e, and / or the ratio of α2,6 sialic acid to α2,3 sialic acid.

[0028] In one embodiment, the marker of this embodiment may be a combination of a sugar chain containing α1,2 fucose and one or more other markers for assessing bone differentiation potential. Examples include a combination of a sugar chain containing α1,2 fucose and a sugar chain containing α2,6 sialic acid, a combination of a sugar chain containing α1,2 fucose and a sugar chain containing α2,6 sialic acid on CD29 and / or CD49e, or a combination of a sugar chain containing α1,2 fucose and the ratio of α2,6 sialic acid to α2,3 sialic acid (or the ratio of sugar chains containing α2,6 sialic acid to sugar chains containing α2,3 sialic acid). These markers are derived from somatic stem cells.

[0029] (Uses) The marker according to this embodiment can be used to determine the bone differentiation potential of somatic stem cells isolated from an animal, their primary cultured cells, subcultured cells, or established cultured cell lines. This use will be described in the second embodiment. Furthermore, the marker according to this embodiment can be used to detect, isolate, or enrich somatic stem cells with bone differentiation potential, using the expression of the marker on the cell surface of somatic stem cells as an indicator. This use will be described in the third embodiment. Furthermore, the marker according to this embodiment can be used as a kit containing a probe that specifically binds to the marker. This use will be described in the fourth embodiment. Furthermore, the marker according to this embodiment can be used to isolate or enrich somatic stem cells with bone differentiation potential, using the expression of the marker on the cell surface of somatic stem cells as an indicator, and to manufacture a pharmaceutical for treating bone diseases. This use will be described in the fifth embodiment. Furthermore, the marker according to this embodiment can be used to select culture conditions for culturing somatic stem cells with bone differentiation potential, using the expression of the marker on the cell surface of somatic stem cells as an indicator. This use will be described in the sixth embodiment.

[0030] ==Second embodiment (determination method)== The method according to this embodiment is a method for determining the bone differentiation ability of somatic stem cells, and includes detecting α1,2 fucose derived from somatic stem cells in a sample.

[0031] In this embodiment, "α1,2 fucose," "somatic stem cells," "bone differentiation potential," and "combined marker" are as described in embodiment 1. The method according to this embodiment is a method for determining the bone differentiation potential of somatic stem cells using the marker according to embodiment 1 as an indicator.

[0032] (Detection Method) The detection of "α1,2 fucose" in this embodiment is a method for detecting the marker according to the first embodiment in a sample. That is, the detection of "α1,2 fucose" is not limited to the scope of detecting the presence or absence of α1,2 fucose derived from somatic stem cells in a sample, and the α1,2 fucose portion contained in the sugar chain may be directly detected, or the entire sugar chain marker containing α1,2 fucose, or a portion thereof, may be detected. Hereinafter, these modes of detection will be described using the term "detection of α1,2 fucose."

[0033] The detection in this embodiment may include measuring the amount of α1,2 fucose, where the amount of α1,2 fucose may be an absolute value or a relative value.

[0034] In this embodiment, the sample for detecting α1,2 fucose is not limited to a specific sample containing α1,2 fucose. For example, if the somatic stem cells are cultured cells, the sample may be a culture medium. For example, if the somatic stem cells are cells isolated from a living organism, the sample may be a buffer solution known to those skilled in the art. As long as the sample contains detectable α1,2 fucose derived from somatic stem cells, the sample may contain somatic stem cells or may have been removed from the sample at the time of carrying out the determination method. Release of glycans containing α1,2 fucose from somatic stem cells can be carried out as described in the first embodiment. Considering the ease and / or sensitivity of detecting α1,2 fucose, it is preferable that the sample contains the somatic stem cells to be determined. That is, it is more preferable that glycans containing α1,2 fucose are attached to the cell surface of the somatic stem cells at the time of detection, and that α1,2 fucose is detected.

[0035] Prior to the detection of α1,2 fucose, the sample may be subjected to a pretreatment suitable for detection, or may be subjected to detection without any pretreatment. The pretreatment step for detection can be appropriately selected by those skilled in the art according to the type of sample, the content or content rate of α1,2 fucose in the sample (or the estimated content or estimated content rate), etc., according to a method well known to those skilled in the art. For example, when measuring the amount of α1,2 fucose in two or more samples, it is preferable to make the number of somatic stem cells contained or contained in the two samples the same or similar by dilution, etc. For example, to determine the bone differentiation ability of somatic stem cells in a sample based on the detection of α1,2 fucose, it is preferable to use a dilution of 1 x 10 ４ or more, more preferably 1×10 ５ It is preferable to use a cell population containing at least one somatic stem cell.

[0036] In this embodiment, detection of α1,2 fucose can be performed by methods well known to those skilled in the art and is not limited by the technique, and may be performed by any method, such as a method using a probe that specifically binds to α1,2 fucose (e.g., flow cytometry, microscopic observation, array, blotting, etc.), mass spectrometry, liquid chromatography, MALDI-TOF MS, etc. In consideration of the specificity and / or simplicity of detection of α1,2 fucose, detection of α1,2 fucose is preferably performed by a method that uses a probe that specifically binds to α1,2 fucose. When a probe that specifically binds to α1,2 fucose is used, the α1,2 fucose moiety can be directly detected in a sugar chain containing α1,2 fucose.

[0037] When a probe that specifically binds to α1,2-fucose is used, the probe is not limited by other properties as long as it specifically binds to α1,2-fucose, and may be, for example, one or more lectins or one or more antibodies, or one or more types of lectins may be used in combination with one or more types of antibodies.

[0038] In one embodiment, the method according to this embodiment is a method comprising detecting α1,2 fucose using a lectin that specifically binds to α1,2 fucose.

[0039] In this embodiment, examples of lectins that specifically bind to α1,2-fucose include UEA1 (Ulex europaeus agglutinin-1), rBC2LCN (recombinant N-terminal domain of BC2L-C lectin), rAOL (recombinant Aspergillus oryzae lectin), rAAL (recombinant Aleuria aurantia lectin), and / or TJAII (Trichosanthes japonica II lectin). Two or more of these lectins may be used as a probe for detection. In this case, any lectins may be combined, but it is preferable to use at least UEA1. UEA1 alone may also be used. The use of UEA1 as a probe facilitates specific detection of α1,2-fucose and / or facilitates the determination of the bone differentiation ability of somatic stem cells with high accuracy / precision. Here, the lectin that specifically binds to α1,2 fucose also includes proteins or peptides other than full-length proteins, as long as they maintain their binding ability to the target α1,2 fucose, and may be, for example, a structure in which a portion of the protein is deleted, a structure in which an arbitrary peptide is added to a full-length protein, an extracellular domain, a protein or peptide containing an extracellular domain, a sugar-binding domain, a protein or peptide containing a sugar-binding domain, etc.

[0040] Monoclonal and / or polyclonal antibodies can be prepared by methods well known to those skilled in the art. For example, they can be obtained by administering to an animal a portion or all of a sugar chain having α1,2-fucose bound to a carrier protein as an immunogen. The antibody is not particularly limited as long as it has the ability to recognize and specifically bind to a portion or all of a sugar chain having α1,2-fucose, including the α1,2-fucose portion, as an epitope, and may be a polyclonal antibody, a monoclonal antibody, an antibody fragment such as a Fab fragment in which the antigen recognition site is preserved, a humanized antibody, a single-chain antibody, or the like. If an antibody is commercially available, it may be purchased.

[0041] The probe may be labeled by a well-known technique, and examples thereof include probes labeled with R-Phycoerythrin (PE), fluorescent labels such as FITC, enzyme labels such as peroxidase, biotin (+HRP-labeled avidin), etc. Alternatively, a lectin or antibody against an antibody (primary antibody) that binds to α1,2-fucose may be labeled and used as a labeled secondary antibody.

[0042] The probe may be immobilized on any carrier suitable for the method of this embodiment. The carrier can be appropriately selected from carriers known to those skilled in the art and may be in the form of a microplate, tube, disk-shaped piece, particle (beads), etc. When the carrier is a particle, the carrier may be packed into a purification or separation column. The method for immobilizing the probe on the carrier can be appropriately selected from methods known to those skilled in the art, and may include, for example, affinity binding, chemical binding, or physical adsorption. More specifically, for example, when the probe is a lectin, the Fc region of an antibody may be attached to the lectin, and the lectin may be bound to beads to which protein G has been attached by utilizing the binding force between the Fc region and protein G. Alternatively, biotinylated lectin may be immobilized on streptavidin-immobilized beads. Alternatively, the lectin may be immobilized on beads activated with N-hydroxysuccinimide (NHS) or epoxy groups by amine coupling.

[0043] Detection of α1,2 fucose in a sample using a probe that specifically binds to α1,2 fucose involves, for example, contacting the probe with α1,2 fucose in a sample containing α1,2 fucose. For example, the probe can be introduced into a sample containing α1,2 fucose to bring the probe into contact with α1,2 fucose. By contacting the probe with α1,2 fucose, the probe binds to α1,2 fucose in the sample. Detecting the probe that specifically binds to α1,2 fucose allows detection of α1,2 fucose in the sample. Using a labeled probe or a secondary labeled antibody, flow cytometry, microscopy, array analysis, or blotting can be performed to not only detect the presence or absence of α1,2 fucose, but also to measure its amount. Measuring the amount of α1,2 fucose can determine not only the presence or absence of bone differentiation potential of somatic stem cells, but also the level (absolute or relative) of the bone differentiation potential of somatic stem cells compared to a control and / or the level of bone differentiation potential of somatic stem cells.

[0044] More specific, non-limiting examples of the method include the following (Chapter 45, Antibodies and Lectins in Glycan Analysis, pages 537-550, of "Glycobiology," 2nd Edition, Cold Spring Harbor). (a) A fluorescent dye-labeled α1,2 fucose-binding probe is reacted with a somatic stem cell-containing sample, and after washing, the fluorescence intensity is analyzed using flow cytometry or a fluorescence microscope. (b) A somatic stem cell-containing sample is reacted with an α1,2 fucose-binding probe, and then the amount of labeling of the secondary antibody is measured using a secondary labeled antibody that can detect the probe. (c) Proteins (glycoproteins) are prepared from the somatic stem cell-containing sample, labeled with a fluorescent dye, and then subjected to a lectin array containing at least one α1,2 fucose-binding lectin, and the fluorescence intensity is measured using an evanescent wave excitation fluorescence detection system. (d) Proteins are prepared from the somatic stem cell-containing sample, applied to a lectin array containing at least one α1,2-fucose-binding lectin, and overlaid with a labeled antibody or labeled lectin that recognizes the protein or sugar chain, and the fluorescence intensity is measured using an evanescent wave excitation fluorescence detection system. (e) Proteins are prepared from the somatic stem cell-containing sample, applied to a plate on which at least one α1,2-fucose-binding lectin has been immobilized, and overlaid with a labeled antibody or labeled lectin that recognizes the protein or sugar chain, and the absorbance, fluorescence intensity, and luminescence are measured using a plate reader. (f) After preparing proteins from the somatic stem cell-containing sample, lectin blotting is performed. After SDS-PAGE, the proteins are transferred to a membrane such as a nitrocellulose membrane or a PVDF membrane, or directly blotted onto the membrane, and then reacted with labeled α1,2-fucose-binding lectin. For example, when a biotin-labeled lectin is used, after washing with a blocking buffer, color development due to an avidin reaction with an HRP-labeled avidin solution is observed.

[0045] In one embodiment, the method for assessing bone differentiation potential according to this embodiment may further comprise detecting one or more other markers for assessing the bone differentiation potential of somatic stem cells in a sample. Examples and preferred examples of the one or more other markers for assessing the bone differentiation potential of somatic stem cells are as described in the "Combined Markers" section of the first embodiment. Examples include glycans containing α2,6 sialic acid, glycans containing α2,6 sialic acid on CD29 and / or CD49e, and the ratio of α2,6 sialic acid to α2,3 sialic acid (which may also be the ratio of glycans containing α2,6 sialic acid to glycans containing α2,3 sialic acid). Because the differentiation potential of somatic stem cells is more accurately reflected by the α2,6 sialic acid contained in the glycans on CD29 and / or CD49e than by the total α2,6 sialic acid expressed on the cell surface of somatic stem cells, the one or more other markers for assessing bone differentiation potential are preferably glycans containing α2,6 sialic acid on CD29 and / or CD49e.

[0046] Here, the detection of "one or more other markers" in this embodiment is not limited to the extent that the presence or absence of one or more other markers derived from somatic stem cells in a sample can be detected. Therefore, for example, when the marker is a sugar chain containing α2,6 sialic acid, the α2,6 sialic acid portion contained in the sugar chain may be detected directly, or the entire sugar chain marker containing α2,6 sialic acid, or a portion thereof, may be detected. Hereinafter, these detection modes will be described using the term "detection of α2,6 sialic acid."

[0047] In one embodiment, the method for determining the bone differentiation potential of somatic stem cells preferably further comprises detecting somatic stem cell-derived α2,6 sialic acid in the sample in addition to detecting somatic stem cell-derived α1,2 fucose in the sample. In one embodiment, the somatic stem cell-derived α2,6 sialic acid may be α2,6 sialic acid on CD29 and / or CD49e. In one embodiment, the somatic stem cell-derived α2,6 sialic acid may be α2,6 sialic acid on CD29 and / or CD49e. In one embodiment, the somatic stem cell-derived α2,6 sialic acid may be the ratio of α2,6 sialic acid to α2,3 sialic acid (the ratio of glycans containing α2,6 sialic acid to glycans containing α2,3 sialic acid).

[0048] α1,2-fucose is a negative marker for determining the bone differentiation potential of somatic stem cells, which can detect somatic stem cells with low bone differentiation potential. By using the above-mentioned other markers that can detect somatic stem cells with high bone differentiation potential, it becomes easier to determine somatic stem cells with high or low bone differentiation potential with higher accuracy / precision.

[0049] Similar to the method for detecting α1,2-fucose, one or more other markers for assessing bone differentiation potential may be detected by any method, such as a method using a probe that specifically recognizes the marker (e.g., flow cytometry, microscopic observation, array, blotting, etc.), mass spectrometry, liquid chromatography, MALDI-TOF MS, etc. Considering the specificity and / or simplicity of marker detection, it is preferable to detect the marker by a method using a probe that specifically binds to the marker. When a probe that specifically binds to the marker is used, for example, the α2,6 sialic acid moiety can be directly detected in a sugar chain containing α2,6 sialic acid.

[0050] When a probe that specifically binds to a marker is used, the probe is not limited by other properties as long as it specifically binds to the marker, and may be, for example, one or more lectins or one or more antibodies, or one or more types of lectins may be used in combination with one or more types of antibodies.

[0051] In one embodiment, the method according to this embodiment further comprises detecting α2,6 sialic acid using a lectin that specifically binds to α2,6 sialic acid.

[0052] For example, when the marker is a sugar chain containing α2,6 sialic acid, from the viewpoint of the binding ability of the marker, the probe is preferably at least one lectin selected from TJAI (Trichosanthes japonica lectin-I), SSA (Sambucus sieboldiana lectin), SNA (Sambucus nigra lectin), and PSL1a (recombinant Polyporus squamosus lectin); when the marker is a sugar chain containing α2,6 sialic acid on CD29 and / or CD49e, the probe is more preferably at least one lectin selected from SSA (Sambucus sieboldiana lectin), SNA (Sambucus nigra lectin), and PSL1a (recombinant Polyporus squamosus lectin). When a sugar chain containing α2,6 sialic acid on CD29 and / or CD49e is the marker, the probe may be, for example, an anti-CD29 antibody and / or an anti-CD49e antibody for detecting CD29 and / or CD49e, and the above-mentioned lectin or antibody for detecting α2,6 sialic acid. α2,6 sialic acid on CD29 and / or CD49e can be detected by detecting CD29 and / or CD49e and α2,6 sialic acid, respectively. When the ratio of α2,6 sialic acid to α2,3 sialic acid (which may be the ratio of sugar chains containing α2,6 sialic acid to sugar chains containing α2,3 sialic acid) is the marker, the ratio of α2,6 sialic acid to α2,3 sialic acid can be detected by reacting an α2,3 sialic acid-specific sialidase with a sialidase that cleaves both α2,3 and α2,6 sialic acid.

[0053] One or more other markers for assessing bone differentiation potential can be detected in the same manner as in the above-described method for detecting α1,2-fucose. For example, the detection may involve contacting the other marker with a probe that specifically binds to the other marker in a sample containing the other marker.

[0054] The detection of α1,2-fucose and the detection of one or more other markers may be carried out simultaneously, or one may be carried out first and the other may be carried out later.

[0055] In detecting "one or more other markers," the lectin, like a lectin that specifically binds to α1,2-fucose, also includes proteins or peptides other than full-length proteins, as long as they maintain their binding ability to the target marker. In detecting "one or more other markers," like an antibody that specifically binds to α1,2-fucose, monoclonal and / or polyclonal antibodies can be prepared by methods well known to those skilled in the art, and the form of the antibody is not limited as long as it has the ability to specifically bind to the marker. In detecting "one or more other markers," the probe may be immobilized on any carrier, like a probe that specifically binds to α1,2-fucose. In detecting "one or more other markers," for example, the probe may be brought into contact with the marker, and further, for example, the probe may be brought into contact with the marker by introducing the probe into a sample containing the marker, as described for the probe that specifically binds to α1,2-fucose. In detecting "one or more other markers," specific examples of detection and the use of reference cells, etc. to measure the amount of the marker are the same as described for the probe that specifically binds to α1,2-fucose.

[0056] (Method of Determination) The bone differentiation potential of the somatic stem cells to be determined can be determined based on the detection results of α1,2 fucose described above in the "Detection Method." In one embodiment, the method for determining the bone differentiation potential of somatic stem cells according to this embodiment comprises detecting α1,2 fucose derived from somatic stem cells in a sample, and determining the bone differentiation potential of the somatic stem cells based on the detection results. The method for determining the bone differentiation potential of somatic stem cells according to this embodiment does not include a step in which a doctor or other medical professional determines the detection results of detecting α1,2 fucose derived from somatic stem cells in a sample.

[0057] The determination method may be a method for determining the bone differentiation potential of a single somatic stem cell, or a method for determining the bone differentiation potential of a somatic stem cell population contained in a sample as a whole. The method for determining the bone differentiation potential of a somatic stem cell population contained in a sample as a whole may be a method for determining whether the somatic stem cell population contains cells that may have bone differentiation potential, or a method for determining whether the somatic stem cell population contains more or fewer cells that may have bone differentiation potential than a comparison cell population. If the somatic stem cell population contains more cells that may have bone differentiation potential than the comparison cell population, it can be determined that the somatic stem cell population has higher bone differentiation potential than the comparison cell population, and if it contains fewer cells that may have bone differentiation potential than the comparison cell population, it can be determined that the somatic stem cell population has lower bone differentiation potential than the comparison cell population.

[0058] As described in the first embodiment, sugar chains containing α1,2 fucose can be used as a negative marker for determining the bone differentiation potential of somatic stem cells. Thus, in one embodiment, the detection of α1,2 fucose is the detection of the presence or absence of α1,2 fucose. If α1,2 fucose is detected in a sample, it can be determined that the somatic stem cells contained in or that were contained in the sample do not have bone differentiation potential. In another embodiment, the detection of α1,2 fucose is the detection of the presence or absence of α1,2 fucose. If α1,2 fucose is detected in a sample, it can be determined that the somatic stem cells contained in the sample have lower bone differentiation potential than somatic stem cells contained in or that were contained in a sample in which α1,2 fucose is not detected. On the other hand, in one embodiment, if α1,2 fucose is not detected in the sample to be evaluated, it can be determined that the somatic stem cells contained in or that were contained in the sample have bone differentiation potential. In another embodiment, if α1,2 fucose is not detected in a sample, it can be determined that the somatic stem cells contained in the sample have higher bone differentiation potential than somatic stem cells contained in or that were contained in a sample in which α1,2 fucose is detected.

[0059] In one embodiment, when the amount of α1,2 fucose in a sample is measured by detecting α1,2 fucose, it can be determined that the higher the amount of α1,2 fucose, the more somatic stem cells contained in or that have been contained in the sample do not have the ability to differentiate into bone. That is, it can be determined that the somatic stem cell population contained in the sample as a group has lower bone differentiation ability compared to a comparison cell population. In another embodiment, the lower the amount of α1,2 fucose, the more somatic stem cells contained in or that have been contained in the sample have the ability to differentiate into bone. That is, it can be determined that the somatic stem cell population contained in the sample as a group has higher bone differentiation ability compared to a comparison cell population.

[0060] In one embodiment, for example, before carrying out the above detection method, the amount of α1,2-fucose may be measured in somatic stem cells (reference cells) or a cell population (reference population) whose bone differentiation potential and its level have already been determined, and a reference value may be set. Depending on whether the amount of α1,2-fucose in the cell population to be evaluated is higher or lower than the reference value in the reference cells or reference population, it is possible to determine not only the presence or absence of the cell differentiation potential of the somatic stem cells to be evaluated, but also the level of bone differentiation potential and the like compared to a comparison subject. Furthermore, if the reference cells or reference population have the desired bone differentiation potential, it is possible to determine whether the somatic stem cells or cell population to be evaluated have the desired bone differentiation potential by comparing them with the reference value.

[0061] As described in the "Detection Method" section, the determination method according to this embodiment may be a method based on the detection results of one or more other markers for determining the bone differentiation potential of somatic stem cells in a sample, in addition to α1,2 fucose. The other markers and probes that specifically bind to the other markers are as described in the "Detection Method" section. Determination of the bone differentiation potential of somatic stem cells based on the detection method of other markers can be performed in accordance with the determination method based on the detection results of α1,2 fucose, as explained above. However, when the other marker is a positive marker for determining the bone differentiation potential of somatic stem cells, its detection can determine that somatic stem cells or a population thereof have bone differentiation potential, or that somatic stem cells or a population thereof have high bone differentiation potential.

[0062] In one embodiment, the determination method according to this embodiment comprises detecting α1,2 fucose derived from somatic stem cells in a sample, and detecting α2,6 sialic acid derived from somatic stem cells, α2,6 sialic acid on CD29 and / or CD49e, or the ratio of α2,6 sialic acid to α2,3 sialic acid in the sample. In one embodiment, the determination method according to this embodiment comprises detecting α1,2 fucose derived from somatic stem cells in a sample, and detecting α2,6 sialic acid on CD29 and / or CD49e.

[0063] ==Third embodiment (separation or concentration method)== The method for separating or concentrating somatic stem cells with bone differentiation ability according to this embodiment is a method comprising detecting α1,2 fucose derived from somatic stem cells.

[0064] In this embodiment, "α1,2-fucose," "somatic stem cells," "bone differentiation potential," and "combined marker" are as described in Embodiment 1. The method according to this embodiment is a method for separating or concentrating somatic stem cells with bone differentiation potential using the marker according to Embodiment 1 as an indicator.

[0065] In this embodiment, detection of α1,2 fucose derived from somatic stem cells can be carried out according to the description of the "Detection Method" in the second embodiment. However, since cell separation or enrichment is performed using α1,2 fucose derived from somatic stem cells as an indicator, it is preferable that α1,2 fucose be attached to the surface of the somatic stem cells at the time of detection. In one embodiment, a method for separating or enriching somatic stem cells with bone differentiation potential according to this embodiment includes contacting somatic stem cells in a sample with a probe that specifically binds to α1,2 fucose, and separating and removing somatic stem cells bound to the probe that specifically binds to α1,2 fucose from other cells in the sample. In this method, somatic stem cells that specifically bind to the probe express α1,2 fucose on their cell surface.

[0066] Examples and preferred examples of probes for detecting α1,2-fucose are as described in the "Detection method" of the second embodiment.

[0067] For example, a probe that specifically binds to α1,2 fucose can be used to detect α1,2 fucose by flow cytometry or the like, and somatic stem cells expressing α1,2 fucose on their cell surface can be separated from other cells. Somatic stem cells that express a sugar chain containing α1,2 fucose on their cell surface, which is a negative marker for determining the bone differentiation potential of somatic stem cells, can be separated and removed from a sample containing other cells, thereby isolating and concentrating somatic stem cells with bone differentiation potential or a somatic stem cell population with high bone differentiation potential. More specifically, for example, a fluorescently labeled probe that specifically binds to α1,2 fucose can be reacted with a somatic stem cell-containing sample at 4°C for about 1 hour, after which unbound cells can be washed away with phosphate buffer or the like, and then cells with high fluorescence intensity can be removed.

[0068] In addition, somatic stem cells expressing α1,2 fucose on their cell surface can be detected, separated, and removed by using magnetic beads, affinity columns, or the like, to which a probe that specifically binds to α1,2 fucose is immobilized, and somatic stem cells with high bone differentiation potential can be isolated or concentrated. Specifically, for example, a probe that specifically binds to α1,2 fucose is immobilized on magnetic beads, and the beads are reacted with a test stem cell-containing sample at 4°C for approximately 1 hour, followed by washing with phosphate buffer. The magnetic beads are then collected using a magnet, and somatic stem cells expressing α1,2 fucose on their cell surface can be separated and removed from other cells in the sample, allowing the isolation or concentration of somatic stem cells with bone differentiation potential or a somatic stem cell population with high bone differentiation potential. The isolated or concentrated somatic stem cells with high bone differentiation potential can then be rapidly placed in a differentiation-inducing medium well known to those skilled in the art to induce differentiation into the desired cells.

[0069] In one embodiment, the method for isolating or enriching somatic stem cells capable of bone differentiation according to this embodiment may comprise detecting α1,2 fucose and one or more other markers for determining the bone differentiation potential of somatic stem cells, which are expressed on the cell surface of somatic stem cells. In one embodiment, the method for isolating or enriching somatic stem cells capable of bone differentiation according to this embodiment may comprise contacting somatic stem cells in a sample with a probe that specifically binds to α1,2 fucose, and separating and removing the somatic stem cells bound to the probe from other cells in the sample, and may further comprise contacting the somatic stem cells in the sample with probes that specifically bind to one or more other markers for determining the bone differentiation potential of somatic stem cells, and isolating and maintaining the somatic stem cells that specifically bind to the probes that specifically bind to the one or more other markers.

[0070] Examples and preferred examples of one or more other markers for determining the bone differentiation potential of somatic stem cells are as described in the first embodiment, namely, sugar chains containing α2,6 sialic acid, sugar chains containing α2,6 sialic acid on CD29 and / or CD49e present on the surface of somatic stem cells, and the ratio of α2,6 sialic acid to α2,3 sialic acid (or the ratio of sugar chains containing α2,6 sialic acid to sugar chains containing α2,3 sialic acid).

[0071] α1,2-fucose is a negative marker for determining the bone differentiation potential of somatic stem cells, which can detect somatic stem cells with low bone differentiation potential. By using the above-mentioned other markers that can detect somatic stem cells with high bone differentiation potential, it becomes easier to further concentrate somatic stem cells with high bone differentiation potential.

[0072] In this embodiment, the detection of one or more other markers for determining the bone differentiation potential of somatic stem cells can be carried out according to the description of the "detection method" in the second embodiment.

[0073] That is, in one embodiment, the method for separating or enriching somatic stem cells capable of bone differentiation according to this embodiment may comprise detecting α1,2 fucose expressed on the cell surface of somatic stem cells, and detecting α2,6 sialic acid, α2,6 sialic acid on CD29 and / or CD49e, and / or the ratio of α2,6 sialic acid to α2,3 sialic acid (which may also be the ratio of sugar chains containing α2,6 sialic acid to sugar chains containing α2,3 sialic acid). In one embodiment, from the viewpoint of ease of separating or enriching somatic stem cells, the method for separating or enriching somatic stem cells capable of bone differentiation is preferably a method comprising detecting α1,2 fucose expressed on the cell surface of somatic stem cells, and detecting α2,6 sialic acid on CD29 and / or CD49e.

[0074] In one embodiment, the method for isolating or enriching somatic stem cells with high bone differentiation potential according to this embodiment comprises contacting somatic stem cells in a sample with a probe that specifically binds to α1,2 fucose, and separating and removing the somatic stem cells that specifically bind to the probe from other cells in the sample, and may further comprise contacting the somatic stem cells with a probe that specifically binds to α2,6 sialic acid, a probe that specifically binds to α2,6 sialic acid on CD29 and / or CD49e, and / or a probe that specifically detects the ratio of α2,6 sialic acid to α2,3 sialic acid, and separating and maintaining the somatic stem cells that specifically bind to the probe from other cells in the sample. From the viewpoint of ease of isolating or enriching somatic stem cells, a method using a probe for α2,6 sialic acid on CD29 and / or CD49e is preferred.

[0075] The isolation and removal of somatic stem cells using a probe that specifically binds to α1,2 fucose and the isolation and maintenance (or removal) of somatic stem cells using a probe that specifically binds to one or more other markers for determining bone differentiation ability may be performed simultaneously, or one may be performed first and the other may be performed later.

[0076] Fourth Embodiment (Kit) A kit according to this embodiment is a kit for determining the bone differentiation potential of somatic stem cells, and includes a probe that specifically binds to α1,2-fucose. Such a kit can be used in the method according to the second embodiment for determining the bone differentiation potential of somatic stem cells.

[0077] Another kit according to this embodiment is a kit for isolating or enriching somatic stem cells capable of bone differentiation, which kit includes a probe that specifically binds to α1,2-fucose. Such a kit can be used to carry out the method for isolating or enriching somatic stem cells capable of bone differentiation according to the third embodiment.

[0078] In this embodiment, the "α1,2 fucose," "somatic stem cells," "bone differentiation potential," and "combined marker" are as described in the first embodiment.

[0079] Examples and preferred examples of probes for detecting α1,2-fucose are as described in the "Detection method" of the second embodiment.

[0080] In addition to the probe, the kit may also include one or more reagents necessary for carrying out the method for determining the osteogenic differentiation potential of somatic stem cells in a sample according to the second embodiment or the method for isolating or concentrating somatic stem cells with high osteogenic differentiation potential according to the third embodiment. The reagents may be, for example, a buffer solution, a culture medium, a washing solution, a reagent for labeling the probe, and / or a labeled secondary antibody. In the kit, the probe may be immobilized on a carrier known to those skilled in the art, such as beads, chips, tubes, plates, or separation columns, depending on the process to be performed. The material of the carrier may also be appropriately selected from substances known to those skilled in the art, and may include polystyrene, polyacrylic acid, polymethacrylic acid, polymethyl methacrylate, polyacrylamide, polyglycidyl methacrylate, polypropylene, polyolefin, polyimide, polyurethane, polyester, polyvinyl chloride, polyethylene, polychlorocarbonate, silicone resin, silicone rubber, agarose, dextran, glass, silicon oxide, diatomaceous earth, porous glass, ground glass, alumina, silica gel, metal oxide, iron, cobalt, nickel, magnetite, chromite, etc. The method for immobilizing a C-type lectin on a carrier can be appropriately selected from methods well known to those skilled in the art. For example, the C-type lectin may be bound by affinity binding, chemical binding, or physical adsorption.

[0081] In one embodiment, the kit for determining the bone differentiation potential of somatic stem cells according to this embodiment and the kit for separating or enriching somatic stem cells with bone differentiation potential according to this embodiment may contain probes for one or more other markers for determining the bone differentiation potential of somatic stem cells. Examples and preferred examples of the one or more other markers for determining the bone differentiation potential of somatic stem cells are as described in the "Combined Markers" section of the first embodiment. Furthermore, examples and preferred examples of probes for one or more other markers for determining the bone differentiation potential of somatic stem cells are as described in the "Detection Method" section of the second embodiment. When a sugar chain containing α2,6 sialic acid on CD29 and / or CD49e is the marker, the probe may be, for example, an anti-CD29 antibody and / or an anti-CD49e antibody for detecting CD29 and / or CD49e, and the above-mentioned lectin or antibody for detecting α2,6 sialic acid.

[0082] The kit according to this embodiment may be a kit for, for example, EIA (Enzyme Immunoassay) including chemiluminescent enzyme immunoassay (CLEIA), fluorescent immunoassay, ELISA (Enzyme-Linked Immunosorbent Assay), RIA (Radioimmunoassay), Western blotting, latex agglutination, immunochromatography, sandwich method, flow cytometry, or the like.

[0083] The kit according to this embodiment optionally contains the probe described above, one or more reagents described above, and may further contain instructions for using the kit.

[0084] The kit for separating or concentrating somatic stem cells with high bone differentiation potential according to this embodiment can also be used to manufacture a pharmaceutical according to the fifth embodiment.

[0085] ==Fifth embodiment (pharmaceutical composition and its production)== The method for producing a pharmaceutical composition for treating or preventing bone diseases according to this embodiment is a method that includes the method for isolating or concentrating somatic stem cells with bone differentiation ability according to the third embodiment.

[0086] In this embodiment, the "α1,2 fucose," "somatic stem cells," "bone differentiation potential," and "combined marker" are as described in the first embodiment.

[0087] In this embodiment, the method for isolating or concentrating somatic stem cells with bone differentiation potential may use somatic stem cells collected from the transplant recipient or from an individual other than the transplant recipient, and may use either somatic stem cells isolated from an individual or somatic stem cells that have been subcultured. In one embodiment, the isolated or concentrated somatic stem cells with bone differentiation potential may be subjected to the method for determining bone differentiation potential of the second embodiment before transplantation to confirm whether the bone differentiation potential of the somatic stem cells is maintained.

[0088] The separation or enrichment method according to the third embodiment allows somatic stem cells with osteogenic differentiation potential to be separated from other cells in a sample. A cell population containing somatic stem cells separated in this manner is enriched in somatic stem cells with osteogenic differentiation potential compared to a cell population containing somatic stem cells that have not been separated in this manner. Therefore, the cell population containing somatic stem cells separated has a higher osteogenic differentiation potential compared to a cell population containing somatic stem cells that have not been separated.

[0089] A cell population with osteogenic differentiation potential can be differentiated into desired cells by differentiation induction. Because the cell population obtained by the method for isolating or concentrating somatic stem cells with osteogenic differentiation potential according to the third embodiment has high osteogenic differentiation potential as described above, the obtained cell population can be suitably used as a pharmaceutical composition for treating diseases requiring bone formation or repair. Examples of diseases requiring bone formation or repair include, but are not limited to, osteogenesis imperfecta, achondroplasia, hypochondroplasia, osteoporosis, and osteoarthritis.

[0090] "Treatment" includes the reduction, alleviation, or relief of disease symptoms, and "prevention" includes protection against future onset of a disease or symptom and inhibition of progression. Desirable therapeutic effects of treatment include alleviation of symptoms, improvement of direct or indirect pathological consequences of a disease, reduction in the rate of progression of worsening symptoms, recovery or alleviation of the disease state, and improvement in prognosis. For example, treatment or prevention of a bone disease includes administering the pharmaceutical composition of this embodiment to a subject with or diagnosed as having a bone disease, for the purpose of achieving desired effects such as promotion of bone formation and reduction in the rate of bone tissue deterioration.

[0091] The prepared cell population of somatic stem cells may be directly transplanted into the subject to be treated using methods well known to those skilled in the art, or the cell population may be induced to differentiate into desired cells or tissues in vivo, and the cells or tissues may then be transplanted into the subject to be treated.

[0092] According to one embodiment, a pharmaceutical composition for treating a bone disease comprises a population of somatic stem cells capable of osteogenic differentiation. The pharmaceutical composition is produced by the method according to this embodiment, and thereby has higher osteogenic differentiation potential than a population of somatic stem cells that have not been separated or enriched. According to another embodiment, a pharmaceutical composition for treating a bone disease comprises cells or tissues induced to differentiate from a population of somatic stem cells capable of osteogenic differentiation.

[0093] A method for treating bone diseases according to one embodiment comprises administering or transplanting a population of somatic stem cells capable of bone differentiation to a subject. The somatic stem cell population capable of bone differentiation is produced by the production method according to this embodiment, and thus has higher bone differentiation potential than a cell population of somatic stem cells that have not been separated or enriched. A method for treating bone diseases according to another embodiment comprises administering or transplanting cells or tissues induced to differentiate from the population of somatic stem cells capable of bone differentiation to a subject.

[0094] One embodiment of the use is the use of a somatic stem cell population with bone differentiation potential in the manufacture of a pharmaceutical composition for the treatment of bone diseases. By being produced by the production method according to this embodiment, the pharmaceutical composition has a higher bone differentiation potential than a cell population of somatic stem cells that has not been separated or enriched. Another embodiment of the use is the use of cells or tissues induced to differentiate from a somatic stem cell population with bone differentiation potential in the manufacture of a pharmaceutical composition for the treatment of bone diseases.

[0095] One embodiment of this embodiment is a somatic stem cell population with bone differentiation potential for use in the treatment of bone diseases. The somatic stem cell population with bone differentiation potential is produced by the production method of this embodiment, and thereby has higher bone differentiation potential than a cell population of somatic stem cells that have not been separated or enriched. Another embodiment is a somatic stem cell population with bone differentiation potential for use in the treatment of bone diseases.

[0096] ==Sixth embodiment (screening method)== The method for selecting culture conditions suitable for culturing somatic stem cells with bone differentiation ability according to this embodiment is a method that includes the method for determining the bone differentiation ability of somatic stem cells according to the second embodiment.

[0097] In this embodiment, the "α1,2 fucose," "somatic stem cells," "bone differentiation potential," and "combined marker" are as described in the first embodiment.

[0098] Somatic stem cells that express α1,2-fucose on their cell surface are unlikely to have bone differentiation potential. Therefore, the bone differentiation potential of somatic stem cells in a culture environment can be determined using the method for determining bone differentiation potential according to the second embodiment, and based on whether the somatic stem cells have bone differentiation potential, it can be determined whether the culture conditions are suitable for the somatic stem cells to maintain or acquire bone differentiation potential. Therefore, the method according to this embodiment allows for the selection of culture conditions suitable for the culture of somatic stem cells with bone differentiation potential. That is, in one embodiment, the method according to this embodiment for selecting culture conditions suitable for the culture of somatic stem cells with bone differentiation potential comprises carrying out the method for determining the bone differentiation potential of somatic stem cells according to the second embodiment, and, if the somatic stem cells have bone differentiation potential, selecting the culture conditions as suitable for the culture of somatic stem cells.

[0099] The present disclosure will be explained in more detail below by showing examples, but interpretation of the present disclosure is not limited to these examples.

[0100] Example 1: Culturing of mesenchymal stem cells Three human adipose-derived mesenchymal stem cell lines (ADSC550, ADSC561, ADSC894) were obtained from LONZA (Table 1). The cells were maintained in MesenPRO RS Medium (Thermo Fisher Scientific Inc.) on plastic dishes. When the cells reached 70-80% confluency, they were detached using TrypLE Express (Thermo Fisher Scientific Inc.) and approximately 1 / 3 to 1 / 4 of the cells were seeded onto a new dish for passage. In the following examples, mesenchymal stem cells (MSCs) at passage numbers 4 to 6 were used.

[0101] Example 2: Evaluation of mesenchymal stem cell bone differentiation potential. MSCs were induced to differentiate into bone, and osteoblasts were evaluated by alizarin red staining. MSCs were seeded onto collagen-coated 12-well plates (Sumitomo Bakelite) and differentiated using the Human Mesenchymal Stem Cell Osteogenic Differentiation Medium Bullet Kit (LONZA) according to the manufacturer's protocol (N=3). Three weeks after osteoblast induction, cells were washed twice with phosphate buffered saline (PBS) (Fujifilm Wako Pure Chemicals), then fixed with 4% paraformaldehyde in phosphate buffered saline (Fujifilm Wako Pure Chemicals) for 30 minutes at room temperature. After washing with MilliQ water, 1% alizarin red solution (pH 6.3-6.4) (Muto Chemicals) was added and the cells were allowed to stand at room temperature for 20 minutes. After washing with MilliQ water, PBS was added and the plate was left to stand at room temperature for 15 minutes. The PBS was then replaced and images were taken under a microscope (BZ-9000, Keyence). After imaging, the PBS was removed, and calcified nodule dissolving solution (PG Research) was added. The plate was shaken for 10 minutes to elute the dye. The eluate was transferred to a 96-well plate, and the absorbance at 450 / 620 nm was measured using a microplate reader (Spectra Max M3, Molecular Devices). Figure 1 (A) shows a microscopic image of alizarin red staining, and (B) shows the absorbance of the eluate. The black areas in the microscopic image represent cells stained with alizarin red, indicating the formation of bone nodules with calcium deposits due to the action of osteoblasts. The absorbance of the eluate indicates the amount of alizarin red staining of the sample. Bone differentiation potential varied significantly between MSC lines, with the number of cells with bone differentiation potential increasing in the order ADSC561 > ADSC894 > ADSC550, indicating a significantly higher bone differentiation potential. One-way ANOVA analysis of the three groups showed p = 0.0000000249. Post-hoc Holm test analysis showed the difference between ADSC550 and ADSC894 was p = 0.00012, the difference between ADSC550 and ADSC561 was p = 0.000000032, and the difference between ADSC561 and ADSC894 was p = 0.000000086.

[0102] Example 3: Evaluation of adipogenic potential of mesenchymal stem cells. MSCs were induced to differentiate into adipocytes, and adipocytes were evaluated by Oil Red staining. MSCs were seeded onto 12-well plates and differentiated using the Human Mesenchymal Stem Cell Adipogenic Differentiation Bullet Kit (LONZA) according to the manufacturer's protocol (N=3). Three weeks after induction of adipogenic differentiation, the cells were washed twice with phosphate buffered saline (PBS) (Fujifilm Wako Pure Chemicals), then fixed with 10% formalin solution (Fujifilm Wako Pure Chemicals) for 10 minutes at room temperature. The cells were then washed twice with PBS and immersed in 60% isopropanol solution for 1 minute. Oil Red staining solution (pH 6.3-6.4) (Muto Chemicals) was added and allowed to stand at room temperature for 20 minutes. After washing once with 60% isopropanol solution and twice with PBS, PBS was added and the plate was photographed under a microscope (BZ-9000, Keyence). After photographing, the PBS was removed, isopropanol was added, and the plate was shaken for 10 minutes to elute the dye. The eluate was transferred to a 96-well plate, and the absorbance at 450 / 620 nm was measured using a plate reader (Spectra Max M3). Figure 2 (A) shows a microscopic image of oil red staining, and (B) shows the absorbance of the eluate. The black areas in the micrograph indicate adipocytes stained with oil red. The absorbance of the eluate indicates the amount of oil red staining of the sample. Unlike the osteogenic differentiation potential in Example 2, no significant differences were observed between MSC lines in adipogenic differentiation potential.

[0103] [Example 4] Search for glycosylation markers To search for glycosylation markers of functional subpopulations of MSCs, analysis was performed using single-cell glycan and RNA sequencing (scGR-seq). Three MSC lines (ADSC550, ADSC561, ADSC894) were detached using TrypLE Express to prepare single cell suspensions. 1 × 10 cells were used for each sample. ５Cells were collected and washed three times with PBS containing 1% bovine serum albumin (BSA, Sigma), then blocked with Human TruStain FcX (1 / 20, Abcam) on ice for 10 minutes. Subsequently, the cells were incubated on ice for 1 hour in 1% BSA / PBS containing DNA-labeled lectin (1 μg / ml, Figure 3) and TotalSeq™-C0251-C0253 anti-human hash tag antibody (1 μg / ml, Biolegends). After washing three times with 1% BSA / PBS, cell clumps were removed using a Flowmi cell strainer (porosity 40 μm, Bel-Art Products). Equal amounts of cells from each sample were mixed and strained at 1 × 10 ５The cell suspension was treated with Chromium Controller (10X Genomics), and RNA and lectin DNA barcode libraries were generated using ChromiumNext GEM Single Cell 5' Reagent Kits v2 (Dual Index) (10X Genomics) according to the manufacturer's protocol. Library quality was analyzed using a MultiNA (Shimadzu Corporation), and sequencing was performed using a DNBSEQ-G400RS (100 bp, paired-end, 400 million read pairs). The raw count data for lectin barcodes and RNA was subjected to quality control and normalization on the Seurat R package (version 4.0.2). Then, glycan (lectin barcode) and RNA expression data were integrated using the weighted nearest neighbor method, and dimensionality reduction and clustering, differential expression gene analysis, and reactive variable lectin extraction were performed using the Uniform Manifold Approximation and Projection (UMAP) method. Figure 4 shows the results of dimensionality reduction and clustering analysis of the RNA and glycan information of each cell, depicting the cells (UMAP plot) (N = 3149 cells). MSCs were classified into 10 cell clusters, from cluster 0 to 9. Figure 5 shows dot plots of the differentially expressed genes (top 10 genes) and reactive variable lectins for each cluster. Genes characteristic of each cluster were extracted (Figure 5A). Furthermore, clusters 2, 5, 6, 7, and 9 showed distinctive glycan profiles (Figure 5B). Next, the abundance of cluster populations in each MSC line was analyzed to narrow down the clusters with distinctive glycans to those associated with osteogenic differentiation potential (Figure 6). It was found that the abundance of cluster 5 was high in the ADSC550 line, which has low osteogenic differentiation potential.

[0104] [Example 5] Verification of Cluster 5-associated Lectin Reactivity by Flow Cytometry The reactivity of lectins that showed high reactivity in Cluster 5 was analyzed by flow cytometry. Each MSC line (ADSC550, ADSC561, ADSC894) was detached using TrypLE Express to prepare a single cell suspension. 1 × 10 cells were collected for each sample. ５ Cells were sorted, washed with 1% BSA / PBS, and then incubated with phycoerythrin (PE)-labeled lectin (1 μg / ml) on ice for 1 hour. After washing twice with 1% BSA / PBS, analysis was performed using a flow cytometer (CytoFLEX, Beckman Coulter). The phycoerythrin (PE)-labeled lectins used were UEA1, rPhoSL (rPTL), TJAII, and HPA, which were found to be highly reactive with cluster 5 in Example 4. Reactivity was observed with all evaluated lectins in each MSC line (ADSC550, ADSC561, and ADSC894). Consistent with the scGR-seq results, the α1,2-fucose-binding lectin UEA1 (Ulex europaeus agglutinin-1) was found to be the lectin that showed the highest reactivity with the ADSC550 line (Figure 7). It was shown that UEA1-negative and -positive cells were present in both cell lines.

[0105] Example 6 Evaluation of the Osteodifferentiation Potential of UEA1-Positive / Negative Mesenchymal Stem Cells (Sorting of Mesenchymal Stem Cells Based on UEA1 Reactivity) UEA1-positive and -negative cells present in each MSC line (ADSC550, ADSC561, ADSC894) were sorted by fluorescence-activated cell sorting (FACS). As in Example 5, each MSC line was incubated with PE-labeled UEA1 and stained, and positive and negative fractions were sorted using a cell sorter (ARIAIII, BD Bioscience). A portion of the cells was analyzed before and after sorting to confirm UEA1 reactivity. Figure 8 shows the gates for the positive and negative fractions in each MSC line and the results of analyzing the amount of UEA1 binding before and after sorting. It was confirmed that UEA1-negative and -positive cells were sorted in all lines.

[0106] (Evaluation of bone differentiation potential of UEA1-positive / negative cells) The isolated MSC UEA1-positive / negative cells were induced to differentiate into bone, and their bone differentiation potential was evaluated by Alizarin Red staining and absorbance measurement of the dye eluate as in Example 2. Figure 9 shows a microscopic image of Alizarin Red staining (A) and the absorbance of the eluate (B). In the low bone differentiation potential line ADSC550, no significant difference was observed between UEA1-positive cells (Poj) and negative (Neg) cells (p = 0.516942315). However, in the medium bone differentiation potential line ADSC894 and the high bone differentiation potential line ADSC561, it was found that UEA1-positive cells had significantly lower bone differentiation potential than negative cells (p = 0.000389752 (ADSC894), p = 0.008890271 (ADSC561)). These results indicate that UEA1-positive cells are a cluster with low bone differentiation potential, and that the bone differentiation potential of the cell population can be improved by enriching UEA1-negative cells. Statistical analysis was performed using a t-test.

[0107] Example 7 Extraction and flow cytometry analysis of lectins showing varying reactivity between MSC lines To further explore glycomarkers that characterize differences in bone differentiation potential between MSC lines, lectins showing varying reactivity between MSC lines in scGR-seq analysis were extracted. Figure 10 shows a dot plot of the varying lectin reactivity. rLSLN and SSA showed high reactivity with ADSC561, an MSC line with high bone differentiation potential.

[0108] Focusing on lectins that showed high reactivity with ADSC561, an MSC line with high bone differentiation potential, the reactivity of these lectins was analyzed by flow cytometry as in Example 5. Similar to scGR-seq, the α2,6 sialic acid-binding lectin Sambucus sieboldiana agglutinin (SSA) was found to be a lectin that showed high reactivity with ADSC561 ( FIG. 11 ). α2,6 sialic acid has also been identified as a glycan correlated with MSC differentiation potential in lectin array analysis ( Patent Nos. 6478418 and 6733889 ), consistent with the results of this analysis.

[0109] Example 8 Evaluation of the osteogenic differentiation potential of SSA-positive / -negative mesenchymal stem cells (Sorting of mesenchymal stem cells based on SSA reactivity) SSA-positive and -negative cells present in each MSC line (ADSC550, ADSC561, ADSC894) were sorted by fluorescein-activated cell sorting (FACS) in the same manner as in Example 6. Figure 12 shows the results of analyzing the amount of SSA binding before and after sorting for each MSC line. It was confirmed that SSA-negative and -positive cells were successfully sorted for each line.

[0110] (Evaluation of bone differentiation potential of SSA-positive / negative cells) Sorted MSC SSA-positive / negative cells were induced to differentiate into bone, and their bone differentiation potential was evaluated by Alizarin Red staining and absorbance measurement of the dye eluate as in Example 2. Figure 13 shows a microscopic image of Alizarin Red staining (A) and the absorbance of the eluate (B). In the low bone differentiation potential line ADSC550, no significant difference was observed between SSA-positive cells (Poj) and SSA-negative cells (Neg) (p=0.26395611). However, in the medium bone differentiation potential line ADSC894 and the high bone differentiation potential line ADSC561, SSA-positive cells were found to have significantly higher bone differentiation potential than SSA-negative cells (p=5.70646E-06 (ADSC894), p=0.001098595 (ADSC561)). These results indicate that SSA-positive cells are a cluster with high bone differentiation potential, and that enrichment of SSA-positive cells can improve the bone differentiation potential of the cell population. Statistical analysis was performed using a t-test.

[0111] Example 9: Evaluation of bone differentiation potential of UEA1-negative / SSA-positive mesenchymal stem cells. By separating UEA1-negative or SSA-positive mesenchymal stem cells, we investigated whether their bone differentiation potential was improved compared to conventionally cultured MSCs. An MSC line (ADSC894 line) was stained with APC-labeled SSA and PE-labeled UEA1, and UEA1-negative cells and SSA-positive cells were separated by FACS in the same manner as in Example 6. Furthermore, the separated cells were induced to differentiate into bone, and their bone differentiation potential was evaluated by Alizarin Red staining in the same manner as in Example 2.

[0112] Figure 14 shows the results of analyzing the amount of UEA1 and SSA binding before and after sorting of MSC lines. It was confirmed that UEA1-negative cells and SSA-positive cells were sorted for both lines. Furthermore, no positive or negative correlation was observed between UEA1 and SSA staining, suggesting that they are independent markers.

[0113] Figure 15 shows the microscopic images of Alizarin Red staining (A) and the absorbance of the eluate (B). Compared with the normal culture group (w / o FACS), the SSA-positive group (p = 0.0127) and the UEA1-negative group (p = 0.0002) each exhibited significantly higher osteogenic differentiation potential. Furthermore, as shown by the microscopic image (A) and the absorbance (B), the UEA1-negative group exhibited higher osteogenic differentiation potential compared with the SSA-positive group (p = 0.0127 (B)). This indicates that sorting using UEA1 or SSA can improve the osteogenic differentiation potential of MSCs. Statistical analysis was performed using one-way ANOVA with a post-hoc Holm test.

[0114] [Example 10] Evaluation of chondrogenic differentiation potential of SSA-positive / negative cells SSA-positive and SSA-negative cells were separated from MSCs (ADSC561 strain) according to the method of Example 8, and chondrogenic differentiation was induced in the SSA-positive and SSA-negative cells. The chondrogenic differentiation potential of each cell type was evaluated by Alcian blue staining, which specifically stains chondrocytes.

[0115] The collected SSA-positive and SSA-negative cells were placed in a 15 ml tapered Falcon tube at 2.5 × 10 ５The cells were seeded individually, centrifuged at 600G for 10 minutes, and pelleted. Differentiation induction was performed using the Human Mesenchymal Stem Cell Chondrogenic Differentiation Medium Bullet Kit (LONZA) according to the manufacturer's recommended protocol. The cells differentiated into cartilage were stained using an Alcian Blue staining kit (Bio Mirai Kobo). Specifically, two weeks after induction of cartilage differentiation, the cell pellet was washed with phosphate buffered saline (PBS) (Fujifilm Wako Pure Chemical Industries) and then fixed for 30-60 minutes with AB fixative (Bio Mirai Kobo). The cells were washed twice with AB wash solution, a pH-adjusted solution was added, and the cells were left to stand at room temperature for 2-3 minutes. After washing with sterile water, AB staining solution was added and the cells were left to stand at room temperature overnight. The cells were placed in a pH-adjusted solution and allowed to stand for 2-3 minutes, then washed twice with sterile water. Sterile water was added and the cells were photographed under a microscope (BZ-9000, Keyence). After photographing, the sterile water was removed, AB elution solution was added, and the cells were allowed to stand overnight to extract the pigment. The elution solution was transferred to a 96-well plate, and the absorbance at 620 nm was measured using a plate reader (Spectra Max M3).

[0116] 16 shows the results of evaluating the chondrogenic differentiation potential of SSA-positive and SSA-negative cells based on the intensity of Alcian blue staining. SSA-positive cells tended to have higher chondrogenic differentiation potential than SSA-negative cells (p=0.052, N=8, t-test). This indicates that SSA-positive cells are a cell population with high chondrogenic differentiation potential, and that the present disclosure makes it possible to separate a cell population with high chondrogenic differentiation potential by enriching SSA-positive cells from MSCs.

[0117] Example 11 Confirmation of the Dependence of UEA1 on Fucosylated Sugar Chains To confirm that UEA1 binds to MSC lines in a manner dependent on fucosylated sugar chains on the cell surface, the effects of co-administration of excess fucose monosaccharide with UEA1 or pretreatment with fucosidase on the reactivity of UEA1 were analyzed. MSC lines (ADSC894) cultured in the same manner as in Example 1 were detached using TrypLE Express to prepare single cell suspensions. 1 x 10 cells per sample were used. ５Cells were collected and washed with 1% BSA / PBS. The fucosidase-pretreated group was treated with 3 units of FucoseExo (Genovis) at 37°C for 1 hour. Then, UEA1 reactivity was analyzed by flow cytometry using PE-labeled UEA1 in the same manner as in Example 5. In the fucose monosaccharide co-administration group, 10 or 100 mM fucose was incubated with PE-labeled UEA1.

[0118] The results are shown in Figure 17. (A) is a graph showing the results of flow cytometry analysis of the amount of PE-labeled UEA1 binding to MSCs co-administered with various concentrations of fucose (Con = 0 mM, 10 mM, 100 mM) and UEA1, or MSCs pre-treated with fucosidase (ExoFuc). The light gray peak represents the negative control PE-BSA, and the dark gray peak represents PE-UEA1. The vertical axis represents the number of cells normalized by the mode, and the horizontal axis represents the fluorescence intensity of PE. (B) is a graph showing the difference between the mean fluorescence intensity of PE-UEA1 and the mean fluorescence intensity of PE-BSA in each group. In the group co-administered with fucose monosaccharide and UEA1, the reactivity of UEA1 decreased depending on the concentration of fucose monosaccharide (Figure 17(A)). Furthermore, a decrease in UEA1 reactivity was observed in the fucosidase pretreatment group. From the above, it was confirmed that UEA1 binds to fucosylated glycans on the cell surface of MSCs.

[0119] Example 12 Control of bone differentiation potential by fucosylation inhibitors To investigate the relationship between fucosylated sugar chains on the cell surface of MSCs and bone differentiation potential, we analyzed the reactivity of UEA1 and the bone differentiation potential of MSCs in cells that had been treated with the fucosylation inhibitor 2-fluorofucose (2-FF) during culture.

[0120] Figure 18(A) is a graph showing the results of flow cytometry analysis of the amount of PE-labeled UEA1 binding to MSCs (ADSC894 strain) after 3 days of culture in medium containing 2-FF (0.1, 0.3, 1 mM), as in Example 5. The light gray peak represents the negative control PE-BSA, and the dark gray peak represents PE-UEA1. The vertical axis represents the number of cells normalized by the mode, and the horizontal axis represents the amount of PE fluorescence. Figure 18(B) is a graph showing the difference between the mean fluorescence intensity of PE-UEA1 and the mean fluorescence intensity of PE-BSA in each group. Figure 19 is a graph showing (A) microscopic images of alizarin red-stained cells after osteogenic differentiation induction in the presence or absence of 2-FF, and (B) the mean absorbance (Mean ± SD) of the eluate (N = 6). The scale bar represents 100 μm.

[0121] The addition of the fucosylation inhibitor 2-FF reduced UEA1 reactivity at all concentrations (0.1 mM, 0.3 mM, 1 mM) (Figure 18). Furthermore, osteogenic differentiation was significantly enhanced in the 2-FF-treated group compared to the untreated group (p=0.0210, N=6, t-test, Figure 19). These findings confirm a negative correlation between fucosylation of MSC cell surface glycans and osteogenic differentiation potential.

[0122] The α1,2-fucose-containing sugar chains of this embodiment can be suitably used in methods for determining bone differentiation potential, methods for isolating or concentrating somatic stem cells with bone differentiation potential, kits, methods for producing pharmaceutical compositions, and methods for selecting culture conditions, and have industrial applicability.

Claims

1. A method for determining the bone differentiation potential of somatic stem cells, comprising detecting α1,2 fucose derived from somatic stem cells in a sample.

2. The method according to claim 1, wherein the somatic stem cells are mesenchymal stem cells.

3. The method of claim 1 or 2, further comprising detecting α2,6 sialic acid derived from somatic stem cells in the sample.

4. The method according to claim 1 or 2, wherein α1,2 fucose is detected using a lectin that specifically binds to α1,2 fucose.

5. The method according to claim 3, wherein α2,6 sialic acid is detected using a lectin that specifically binds to α2,6 sialic acid.

6. A method for isolating or enriching somatic stem cells with bone differentiation potential, comprising: contacting somatic stem cells in a sample with a probe that specifically binds to α1,2 fucose; and separating and removing the somatic stem cells bound to the probe that specifically binds to α1,2 fucose from other cells in the sample.

7. The method according to claim 6, wherein the somatic stem cells are mesenchymal stem cells.

8. The method of claim 6 or 7, further comprising contacting somatic stem cells in the sample with a probe that specifically binds to α2,6 sialic acid, and separating and maintaining the somatic stem cells bound to the probe that specifically binds to α2,6 sialic acid from other cells in the sample.

9. A kit for determining the bone differentiation potential of somatic stem cells, comprising a probe that specifically binds to α1,2 fucose.

10. A kit for isolating or concentrating somatic stem cells having bone differentiation potential, the kit comprising a probe that specifically binds to α1,2 fucose.

11. The kit according to claim 9, wherein the probe that specifically binds to α1,2-fucose is UEA1 lectin (Ulex europaeus agglutinin-1).

12. The kit according to claim 9 or 10, wherein the somatic stem cells are mesenchymal stem cells.

13. The kit according to claim 9 or 10, further comprising a probe that specifically binds to α2,6 sialic acid.

14. The kit described in claim 13, wherein the probe that specifically binds to α2,6 sialic acid is at least one lectin selected from the group consisting of TJAI (Trichosanthes japonica lectin-I), SSA (Sambucus sieboldiana lectin), SNA (Sambucus nigra lectin), and PSL1a (recombinant Polyporus squamosus lectin).

15. A method for producing a pharmaceutical composition for treating or preventing bone diseases, comprising the method according to claim 6 or 7.

16. A method for selecting culture conditions suitable for culturing somatic stem cells capable of bone differentiation, comprising the method of claim 1 or 2.

17. The method of claim 16, wherein the somatic stem cells are mesenchymal stem cells.

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