JP6733889B2 - Cell differentiation potential determination method - Google Patents

Description

The present invention relates to a method for determining the cell differentiation potential of somatic stem cells using a culture supernatant, particularly a method for determining the cell differentiation potential of somatic stem cells into osteoblasts or chondrocytes, and a kit therefor.

Regenerative medicine is the restructuring of tissues by supplementing tissues or organs with impaired functions with pluripotent stem cells such as somatic stem cells or iPS cells collected from themselves or others, or cells differentiated from these cells. It is a medical treatment that regenerates organs. In recent years, great expectations have been placed on this as a medical treatment that can fundamentally repair and regenerate a disease that has only symptomatic treatment such as drug treatment. In particular, since mesenchymal stem cells can be collected from autologous tissues such as bone marrow and fat, clinical application is progressing mainly on autologous transplantation without immune rejection.
However, somatic stem cells such as mesenchymal stem cells are heterogeneous cell populations, and their properties (cell differentiation potential, proliferative capacity, migration capacity) differ greatly depending on the age, tissue, passage number, etc. of the individual from which they are derived. In order to evaluate the efficacy of treatment, a quality verification method for stably obtaining a cell population suitable for transplantation is important. The method for verifying the quality of cells before transplantation is also highly important in evaluating the efficacy and risk of cell transplantation using somatic stem cells.
However, in somatic stem cells, since there is no unique marker for identifying except hematopoietic stem cells, it is possible to verify the quality of cells by confirming the expression of multiple cell surface markers at the time of transplantation. The current situation.
Under such circumstances, there has been a strong demand for the development of a determination method capable of quantitatively evaluating the quality characteristics of somatic stem cells such as mesenchymal stem cells, particularly the cell differentiation potential (differentiation capacity) directly linked to the therapeutic efficacy.

The term “cell differentiation potential” used in the present invention means that when a certain cell or cell population is placed in an appropriate differentiation-inducing state, it can be transformed into another type of cell or cell population such as progenitor cells and tissue cells. It means having the ability. Here, when the “cell differentiation potential is high” of a certain cell or cell population, regardless of the number of types of directionality that changes, even if one type has a high ability to change to another type of cell, It is described as "high in cell differentiation potential". In addition, since it is a concept different from “undifferentiation marker” that simply distinguishes whether a cell is in an undifferentiated state or a differentiated state, it is a cell or cell population that highly expresses the undifferentiated marker. However, there are cases where the “cell differentiation potential” is low.

The present inventors have recently found that α2-6 sialic acid is a cell surface sugar chain marker that serves as an index of cell differentiation potential from the results of cell surface sugar chain analysis in early and late passages of somatic stem cells such as mesenchymal stem cells. As a result, they found out that an assay system using SSA lectin or the like that recognizes α2-6 sialic acid as an epitope was constructed. In addition, a sandwich assay system for observing changes in the amount of α2-6 sialic acid in CD29 and CD49e glycoprotein sugar chains on the surface of stem cells was also constructed as an assay system for evaluating and determining the potential of cell differentiation, particularly to osteoblasts or chondrocytes. (Patent Document 1).
This makes it possible to quantitatively evaluate the cell differentiation potential of somatic stem cell samples such as mesenchymal stem cells, and to obtain a cell population suitable for regenerative treatment using somatic stem cells, particularly bone or cartilage regenerative treatment. We were able to provide a quality verification method for stable acquisition.
However, the verification method not only requires a complicated step of collecting somatic stem cells in order to detect sugar chains or glycoproteins on the surface of somatic stem cells, but is also valuable for originally used for transplantation therapy. There is a problem in that stem cells are used for inspection.

Further, as a concept for evaluating the quality of stem cells similar to the cell differentiation potential, there is a concept of “replicative senescence” that includes division ability, proliferation ability as well as differentiation ability, and PARG1 in mesenchymal stem cells, Measurement of the expression level of each gene of CDKN2B, PTN and MCM3 (patent document 2 and non-patent document 1), and measurement of the degree of methylation of GRM7, CASR, PRAMEF2, SELP, CASP14, KRTAP13-3 gene in CpG-island. (Patent Document 3) proposes a quality evaluation method for determining and evaluating the state of replication senescence of a cell culture containing mesenchymal stem cells.
However, these quality determination methods are also similar in that stem cells themselves need to be used for analysis, and in addition, a recovery step from the culture solution is required, and more complicated measurement and analysis steps of expression levels of multiple genes are required. ing.

From the above, it has been strongly desired to establish a simple and accurate method for quantitatively examining the cell differentiation potential of stem cells using the culture supernatant of somatic stem cells.

WO2016/006712 WO2011/000833 WO2013/017701

AGING,April 2010,Vol.2,No4,p.224-230 Molecular & Cellular Proteomics, 2012, Vol.11, p.1913-1923 Glycobiology,2014,Vol.24,No.5,p.458-468

The methods currently being developed as quality evaluation methods to determine the therapeutic efficacy of somatic stem cells analyze differences in intracellular gene expression, cell surface markers, etc., and therefore all of them need to use the cells themselves for analysis. However, there is a problem that a complicated process of collecting cells is required and that valuable cells originally used for transplantation treatment are used in the test.
The object of the present invention is to solve such problems, a method of non-invasively detecting the cell differentiation potential of somatic stem cells without reducing the number of test cells, quantifying and determining, and a culture supernatant. An object of the present invention is to provide a simple and reliable method for controlling the quality of somatic stem cells using the method.
The present invention is to easily and efficiently determine and evaluate the cell differentiation potential of somatic stem cells by an assay system using the cell culture supernatant of somatic stem cells. It is an object of the present invention to provide a method for determining a cell differentiation potential of somatic stem cells into osteoblasts or chondrocytes by a sandwich assay system using a culture supernatant.

Therefore, the present inventors focused their attention on α2-6 sialic acid (Patent Document 1), which was previously identified as a cell surface sugar chain marker that serves as an index of cell differentiation potential on the surface of somatic stem cells, and showed the somatic character for each passage number. When the amount of α2-6 sialic acid in the complex sugar chain secreted into the culture supernatant of stem cells was measured, no significant change was observed in response to the increase in passage number. Moreover, since the measured values are not stable, it was revealed that α2-6 sialic acid itself does not serve as an index of cell differentiation potential in the culture supernatant.
The present inventors have found that, during subculture of somatic stem cells, α2-6-sialic acid-containing glycoconjugates secreted in the culture supernatant are mixed with α2- in the sugar chain as the cell differentiation potential decreases. We came up with the possibility that there are glycoconjugates with varying amounts of 6 sialic acid, and further conducted extensive research using α2-6 sialic acid-specific lectins. As a result, among the α2-6 sialic acid-containing glycoproteins secreted in the culture fluid of bone marrow-derived mesenchymal stem cells, the amount of α2-6 sialic acid tends to decrease as the number of passages of the stem cells increases. Multiple secretory glycoproteins could be identified. Among these secretory glycoproteins, fibronectin showed almost no change in the amount secreted into the culture supernatant even when the passage number increased, and α2- in the sugar chain contained in the sugar chain increased with the passage number. It has been found that the amount of 6 sialic acid tends to decrease greatly.

Then, various somatic stem cells were subcultured, and the amount of α2-6 sialic acid on fibronectin in the culture supernatant at each passage number was measured and the change in the amount was observed. When the early passage cells and the late passage cells of the stem cells were subjected to the differentiation treatment into osteoblasts and chondrocytes, both the early passage and the late passages of the osteoblasts and chondrocytes were differentiated. There was a clear difference between and.
This indicates that the amount of α2-6 sialic acid in the fibronectin-containing sugar chain in the culture supernatant of somatic stem cells serves as an index of the cell differentiation potential of osteoblasts or chondrocytes, and This means that the cell differentiation potential of somatic stem cells can be determined and evaluated by measuring the amount of α2-6 sialic acid in the fibronectin-containing sugar chain in the supernatant.

In response to the above experimental results, we constructed a sandwich assay system using a lectin that recognizes α2-6 sialic acid and an antibody that recognizes fibronectin, and transformed it into osteoblasts or chondrocytes using the culture supernatant of somatic stem cells. The present invention related to the method for determining the cell differentiation potential of the above has been completed.

That is, the present invention includes the following inventions.
[1] A method for determining or evaluating cell differentiation potential of somatic stem cells using a culture supernatant, which comprises the amount of α2-6 sialic acid in a secretory core protein-containing sugar chain in the culture supernatant of somatic stem cells A method comprising the step of measuring
The present invention is preferably applied to a method of determining or evaluating the cell differentiation potential of osteoblasts or chondrocytes, among the cell differentiation potentials of somatic stem cells.
[2] The method according to [1] above, wherein the secretory core protein is fibronectin.
[3] The method according to [1] or [2] above, wherein the somatic stem cell is a bone marrow-derived mesenchymal stem cell or a chondrocyte stem cell.
[4] The step of measuring the amount of α2-6 sialic acid in a secretory core protein-containing sugar chain comprises a lectin that specifically recognizes α2-6 sialic acid and an antibody that specifically recognizes the secretory core protein. The method according to any one of [1] to [3] above, wherein the method is carried out using the sandwich assay method of [1].
[5] The method according to [4] above, wherein the lectin that specifically recognizes α2-6 sialic acid is an SNA lectin or an SSA lectin.
[6] The method according to any one of [1] to [5] above, which further comprises the step of measuring the total amount of secretory core protein contained in the culture supernatant of somatic stem cells.
[7] A method for determining or evaluating the cell differentiation potential of somatic stem cells using a culture supernatant, which comprises the following (1) and (2):
(1) The test stem cell culture supernatant sample
A step of overlaying a probe on which either one of (a) or (b) below is immobilized and then allowing the other labeled probe to act, or (a) and (b) below Overlaying the probe onto the immobilized substrate, and then allowing the labeled probe of (b) to act;
(A) a lectin that specifically recognizes α2-6 sialic acid,
(B) an antibody that specifically recognizes a secretory core protein,
(2) A step of measuring the labeled amount of the labeled probe.
[8] The method according to [7] above, further including the following steps (3) and (4):
(3) Using the remaining sample of the test culture supernatant sample used in step (1), the total amount of secretory core protein per unit volume by the antibody that specifically recognizes the labeled secretory core protein Measuring the amount of labeling corresponding to
(4) A step of calculating the ratio of the amount of the secretory core protein containing α2-6 sialic acid to the total amount of the secretory core protein in the culture supernatant, based on the labeling amount measured in the steps (2) and (3).
[9] The method according to [7] or [8] above, wherein the secretory core protein is fibronectin, and the antibody that specifically recognizes the secretory core protein is an anti-fibronectin antibody.
[10] A kit for determining the cell differentiation potential of somatic stem cells for use as a culture supernatant of somatic stem cells, which is a sandwich assay kit containing the probes of (a) and (b) below:
(A) a lectin that specifically recognizes α2-6 sialic acid,
(B) An antibody that specifically recognizes a secretory core protein.
Of the cell differentiation potentials of somatic stem cells, the present invention preferably applies the cell differentiation potential of osteoblasts or chondrocytes to the determination kit.
Further, a standard substance comprising a recombinant secretory core protein labeled as (c) may be combined.
[11] The kit according to [10] above, wherein either one of the probes (a) or (b) is immobilized on a substrate and the other probe is labeled.
[12] The kit according to [11], wherein the lectin that specifically recognizes α2-6 sialic acid is SNA lectin or SSA lectin.
[13] The kit according to any one of claims 10 to 12, wherein the antibody that specifically recognizes the secretory core protein is an anti-fibronectin antibody.
[14] A method for controlling the quality of somatic stem cells using a culture supernatant, comprising:
A method, comprising the step of measuring the amount of α2-6 sialic acid in a secretory core protein-containing sugar chain in the culture supernatant of somatic stem cells.
[15] The method according to [14] above, wherein the secretory core protein is fibronectin.
[16] The step of measuring the amount of α2-6 sialic acid in the secretory core protein-containing sugar chain comprises a lectin that specifically recognizes α2-6 sialic acid and an antibody that specifically recognizes the secretory core protein. The method according to [14] or [15] above, which is carried out using the sandwich assay method according to [1].
[17] A somatic stem cell quality control kit for use as a somatic stem cell culture supernatant, which comprises the following probes (a) and (b):
(A) a lectin that specifically recognizes α2-6 sialic acid,
(B) An antibody that specifically recognizes a secretory core protein.
The present kit may be further combined with a standard substance comprising a recombinant secretory core protein labeled as (c).
[18] The kit according to the above [17], wherein either one of the probes (a) or (b) is immobilized on a substrate and the other probe is labeled.
[19] The kit according to [17] or [18], wherein the lectin that specifically recognizes α2-6 sialic acid is SNA lectin or SSA lectin.
[20] The kit according to any one of [17] to [19] above, wherein the antibody that specifically recognizes the secretory core protein is an anti-fibronectin antibody.

According to the method for determining the cell differentiation potential of osteoblasts or chondrocytes in somatic stem cells according to the present invention, α2-6 sialic acid is recognized using the culture supernatant of the test cells themselves without using them. A sandwich assay system using a lectin and an antibody that recognizes fibronectin can noninvasively evaluate the state of cell differentiation potential of cells. In particular, since it is possible to perform the measurement using the culture solution discarded at the time of subculture as the culture supernatant, there is an advantage that the measurement can be performed easily without reducing the number of valuable cultured cells. Therefore, the present invention can easily and efficiently determine and evaluate the quality of a somatic stem cell sample for transplantation, particularly for regeneration of osteoblasts or chondrocytes.

Identification of α2-6 sialic acid-containing glycoprotein in cartilage stem cell culture supernatant (reactivity between anti-fibronectin antibody and SNA) Preparation of standard curve of anti-fibronectin antibody labeling intensity on SNA-immobilized plate using recombinant fibronectin Verification of lectin plate performance for detection of cell differentiation potential markers in culture supernatant Analysis results of long-term subculture of bone marrow-derived mesenchymal stem cells, cartilage tissue-derived chondrocytes, and skin fibroblasts (analysis by SNA-αFib sandwich assay) Comparison of osteogenic potential of bone marrow-derived mesenchymal stem cells in early and late passages used in FIG. Comparison of chondrogenic potential of cartilage-derived chondrocytes (Yub625) in the early and late passages used in FIG. Comparison of bone differentiation potential of bone marrow-derived mesenchymal stem cells used in FIG. 4 in early and late passages Comparison of long-term subculture of skin fibroblasts used in FIG. 4 and bone and adipose differentiation capacity of early and late passage skin fibroblasts Evaluation of cell differentiation potential in chondrogenic stem cells (Yub625) using lectin plate and antibody plate Changes in the amount of α2-6 sialic acid-containing fibronectin per total fibronectin in the culture supernatant by subculture using polydactyl bone marrow-derived mesenchymal stem cells (Yub622)

1. Regarding α2-6 sialic acid that is a sugar chain epitope to be detected in the present invention, in the present invention, when it is referred to as “α2-6 sialic acid”, N-acetylneuraminic acid is generally a hydroxyl group at the 6-position of galactose. It refers to both “Neu5Acα2-6Gal”, which is an α2-6-linked sugar chain, and “Neu5Gcα2-6Gal”, which is an α2-6-linked sugar chain in which N-glycolyl-neuraminic acid is also α2-6-linked. However, in glycoproteins that have human-derived α2-6 sialic acid at the non-reducing end, α2-6 sialic acid is produced in the body only in the case of "Neu5Acα2-6Gal" in which the amino group at the 5-position of neuraminic acid is acetylated. Since it is not synthesized, the present invention mainly deals with “Neu5Acα2-6Gal”. Also in the following description, α2-6 sialic acid is described as “Neu5Acα2-6Gal”.
Therefore, in the present invention, typically α2-6 sialic acid (Neu5Acα2-6Gal) present at the non-reducing end of a secretory glycoprotein-containing sugar chain such as fibronectin secreted from human somatic stem cells such as human mesenchymal stem cells is typically used. ) Is a sugar chain epitope to be detected for evaluation and determination of the cell differentiation potential of somatic stem cells.
It should be noted that, in mammals other than humans such as pigs and mice, since it also includes the case of "Neu5Gcα2-6Gal" as α2-6 sialic acid, if the target stem cells are derived from mammals other than humans, "Neu5Acα2-6Gal" And “Neu5Gcα2-6Gal” serve as a sugar chain epitope to be detected for determining and evaluating the cell differentiation potential of the present invention.

Sialic acid (N-acetylneuraminic acid) exists at the non-reducing end of sugar chains of secreted glycoproteins such as mucin and serum proteins, and membrane proteins, and gives negative charges to the surface of proteins and cells It has the function of suppressing aggregation of each other, increasing the solubility of proteins, and protecting against attack from proteases. It is also known to function as a ligand for viruses and endogenous receptors. It is also known that it is recognized by Siglec, which is a type of endogenous lectin that is mainly expressed on immune cells, and regulates the function of immune cells.

In addition, it has been known for a long time that α2-6 sialic acid is expressed in large amounts on the surface of undifferentiated cells such as iPS cells (Non-patent Documents 2 and 3), and recently, by the present inventors, on the surface of somatic stem cells. It was also elucidated that the expressed α2-6 sialic acid serves as a cell surface sugar chain marker and is an excellent indicator of the potential of cell differentiation into osteoblasts or chondrocytes (Patent Document 1).
On the other hand, in the case of using the culture supernatant, even if attention is paid to the change in the total amount of α2-6 sialic acid, it does not serve as an index for determining the cell differentiation potential. It was clarified that a glycoprotein as a target can be an excellent indicator of the potential of cell differentiation into osteoblasts or chondrocytes by focusing on the change in the amount of α2-6 sialic acid in the contained sugar chains.
That is, for the first time in the present invention, a specific secretory glycoprotein such as fibronectin functions as an excellent core protein of α2-6 sialic acid, which is a “cell differentiation potential” sugar chain epitope, in the culture supernatant of somatic stem cells. It was shown that That is, by measuring the amount of α2-6 sialic acid present at the non-reducing end of the sugar chain contained in a specific secretory glycoprotein such as fibronectin in the culture supernatant of somatic stem cells, osteoblasts of the subject stem cells can be measured. It has become possible to quantitatively determine the “cell differentiation potential” of cells and chondrocytes.

By the way, in general, somatic stem cells gradually decrease in proliferative capacity as they are passaged, and at the same time, the “cell differentiation potential”, which is the ability to differentiate into various cells, tends to decrease. Therefore, also in the present invention, a cell group with a small number of passages (early passage) is used as a cell group model having a high cell differentiation potential, and a cell group with a large number of passages (late passage) is differentiated into cells with sufficient passages. It is handled as a cell group model with low potential. In the present invention, when somatic stem cells are subcultured, the time when the cell growth curve rises linearly when the proliferative capacity (number of divisions) is plotted for each passage is called the “early passage”. The time when the cell growth curve becomes gentle or flat as the cells are passaged is called "late passage".

2. Regarding α2-6 sialic acid-containing secretory glycoprotein in culture supernatant (2-1) Target α2-6 sialic acid-containing secretory glycoprotein in the present invention In the present invention, the α2-6 sialic acid-containing secretory glycoprotein in the culture supernatant of somatic stem cells is used. The “cell differentiation potential” of the subject stem cell is quantitatively determined by measuring the amount of α2-6 sialic acid present at the non-reducing end of the sugar chain contained in the specific secretory glycoprotein.
There are many kinds of carbohydrates and sugar chain-containing substances in the culture supernatant of somatic stem cells. For example, carbohydrates, glycoproteins, glycolipids, other complex carbohydrates secreted by somatic stem cells and In addition to various sugar chain-containing substances cleaved from membrane proteins and membrane lipids, carbohydrates in medium components are included.
The total amount of α2-6 sialic acid contained in each of these sugar chain-containing substances does not change significantly even after repeated passage of somatic stem cells, but the amount is not always stable at a constant amount.
On the other hand, the α2-6 sialic acid-containing secretory glycoprotein, which is a target of the cell differentiation potential determination index of the present invention, has a constant amount secreted from somatic stem cells as a protein, whereas -6 Only the amount of sialic acid decreases as the cell differentiation potential decreases.
That is, the sugar chain-containing substance targeted in the present invention is always stably secreted not only in somatic stem cells but also in differentiated cells, and the content of complex sugar chains including α2-6 sialic acid is sufficiently high. It is a large secretory glycoprotein. Fibronectin etc. can be illustrated as such a secretory glycoprotein.
This means that the core protein containing the sugar chain epitope α2-6 sialic acid to be detected and measured in the culture supernatant of somatic stem cells is a specific secretory glycoprotein such as fibronectin. The specific secretory glycoprotein may also be referred to as the “secretory core protein” of the present invention.
Hereinafter, fibronectin will be mainly described as a typical “secretory core protein”, but the present invention is not limited to fibronectin.

(2-2) Fibronectin According to the cell differentiation potential determination method of the present invention, “on a sugar chain contained in fibronectin, which is a secretory core protein of the sugar chain epitope to be measured in the culture supernatant of somatic stem cells, is included. The amount of α2-6 sialic acid” sugar chain epitope is detected or measured.
Here, fibronectin (Gene ID: 2335 in NCBI database) that is a typical detection target of this method is mesenchymal stem cells, chondrocyte stem cells (undifferentiated chondrocytes), fibroblasts, epithelial cells, myocytes , Is synthesized in many cells such as neutrophils, macrophages, Schwann cells, and keratinocytes, and is secreted extracellularly. It is known to bind to integrin, which is a receptor protein on the cell membrane, and as a cell adhesion molecule, adhesion to extracellular matrix, formation and retention of connective tissue, wound healing, morphology of tissues and organs during embryogenesis. -It is considered to have many functions that support the normal vital functions of spinal function, such as the formation and maintenance of compartments.
It was demonstrated in the present invention that this fibronectin functions as an excellent secretory core protein of α2-6 sialic acid sugar chain epitope in determining the cell differentiation potential of somatic stem cells.
Fibronectin monomer is a soluble glycoprotein having a molecular weight of 210 to 250 kDa (2,146 to 2,325 amino acid residues), and is known to have seven N-type sugar chains and one O-type sugar chain. "Α2-6 sialic acid" is considered to be bound to the non-reducing end of one or more sugar chains of these seven N-type sugar chains (eg, Gu J. et al., Biol Pharm Bull. 2009 May;32(5):780-5.). Recombinant fibronectin produced from mouse myeloma cells and the like is also commercially available (R&D, etc.), and was also used as a standard substance in the examples of the present invention.

3. Method for Detecting and Measuring "Cell Differentiation Potential" in Culture Supernatant (3-1) "Cell Differentiation Potential Determination Method" of the Present Invention In the cell differentiation potential determination method of the present invention, somatic stem cells secrete into a culture solution. The amount of "α2-6 sialic acid" added to the non-reducing end of the secretory core protein-containing sugar chain such as fibronectin was detected or measured to differentiate somatic stem cells into osteoblasts or chondrocytes. The degree of potential (hereinafter, sometimes simply referred to as “cell differentiation potential”) is determined.
Typically, the amount of α2-6 sialic acid on the fibronectin-containing sugar chain in the culture supernatant of somatic stem cells such as mesenchymal stem cells was determined using anti-fibronectin antibody and α2-6 sialic acid-specific binding lectin. It is a method of measuring using the sandwich assay method described above, and evaluating and determining the degree of “cell differentiation potential” according to the amount, which is simple and preferable. Furthermore, by simultaneously measuring the amount of fibronectin in the remaining culture supernatant and accurately calculating the amount of change in α2-6 sialic acid per unit fibronectin, it is possible to improve the determination accuracy of the “cell differentiation potential”.
As another method, first, a test culture supernatant sample is preliminarily immunoprecipitated with fibronectin in the culture supernatant using beads, a plate, or the like on which an anti-fibronectin antibody is immobilized, and α2-6 on the obtained fibronectin is preliminarily immunoprecipitated. There is a method of detecting sialic acid using an α2-6 sialic acid-specific lectin. This method is complicated in that multiple steps are added, but it is easy to grasp the total amount of fibronectin in the culture supernatant, and it is possible to accurately calculate the amount of change in the amount of α2-6 sialic acid per total fibronectin. There are advantages.

(3-2) Collection Method of Culture Supernatant In the present invention, the culture supernatant of a somatic stem cell culture fluid collected from a living body or subcultured in an undifferentiated state is used as a test sample. A certain amount is collected using a micropipette or the like, and the reactivity with a protein that specifically binds to α2-6 sialic acid on the fibronectin of the present invention is analyzed. The culture medium is generally replaced with fresh culture medium at regular intervals. Therefore, the sugar chain marker is detected after the sugar chain marker is secreted in the culture medium after exchange in a detectable amount. The time required to elute a detectable amount of sugar chain marker in the culture medium after the culture medium exchange may differ depending on the type of cells and culture conditions. Therefore, the time from the culture medium exchange to the collection of the culture supernatant used for the detection of the sugar chain marker can be appropriately set depending on the cell type and the culture conditions, but is, for example, about 18 to 30 hours. Usually, the medium is replaced every 1 to 3 days, so it is preferable to use the culture supernatant that is discarded when the medium is replaced.

(3-3) Lectin-antibody sandwich method In the present invention, in order to easily and reliably detect α2-6 sialic acid in a secretory core protein-containing sugar chain such as fibronectin secreted in a culture solution, α2-6 is used. A lectin-antibody sandwich method using a lectin that specifically recognizes sialic acid and a secretory core protein-specific antibody such as an anti-fibronectin antibody is used. A lectin-antibody sandwich ELISA is preferred.
In the sandwich method, either a secretory core protein-specific antibody such as an anti-fibronectin antibody or an α2-6 sialic acid-specific lectin such as SNA is bound to a solid phase. The case where the lectin is immobilized will be described below.

(3-4) α2-6 sialic acid-binding probe of the present invention In the present invention, in order to detect α2-6 sialic acid at the non-reducing end of the secretory core protein-containing sugar chain in the culture supernatant, A probe that specifically recognizes α2-6 sialic acid as an epitope is used. Proteins that recognize and bind to a specific sugar chain structure are collectively called “lectins”, and typical α2-6 sialic acid-binding probes include various α2-6 sialic acid-binding lectins. However, the anti-α2-6 sialic acid antibody or its derivative that recognizes α2-6 sialic acid as a sugar chain antigen (epitope) is not limited thereto. Further, the α2-6 sialic acid-binding probe of the present invention may be used alone, or a plurality of probes may be used in combination. For example, a plurality of types of α2-6 sialic acid-binding lectins, or an anti-α2-6 sialic acid antibody can also be used in combination.
The α2-6 sialic acid-binding lectin used as the α2-6 sialic acid-binding probe of the present invention includes α2-6 sialic acid at the non-reducing end of the secretory core protein-containing sugar chain such as fibronectin in the culture supernatant. Any lectin may be used as long as it can be recognized. For example, SNA lectins (Sambucus nigra lectin (SNA), SSA lectins (Sambucus sieboldiana lectin), and PSL1a lectins (Polyporus squamosus lectin) which are lectins specifically recognizing various α2-6 sialic acids described in Patent Document 1 and the like. , And TJAI lectin (Trichosanthes japonica lectin-I) can be used. Particularly, SNA and SSA lectin are preferable, SNA lectin from Elderberry, SSA lectin from Japanese elderberry, PSL1a lectin from Astragalus edulis, and TJAI lectin from Kiki. It can also be extracted from crows, but SNA is VECTOR Laboratories, SSA and TJAI lectins are marketed by Seikagaku Corp. PSL1a lectin is a recombinant rPSL1a lectin that retains α2-6 sialic acid specificity. Is marketed by Wako Pure Chemical Industries.

(3-5) Preparation of immobilized lectin As a substrate (solid phase) on which a lectin is immobilized, a plate (eg, microwell plate), a microarray substrate (eg, slide glass for microarray), a tube, beads (eg,) , Plastic beads, magnetic beads, chromatography carriers (eg Sepharose (trademark)), membranes (eg nitrocellulose membrane, PVDF membrane), gels (eg polyacrylamide gel) and the like. Among them, plates, beads and membranes are preferably used, and plates are most preferably used because of the ease of handling.
The most preferable method for immobilizing the lectin is immobilization by biotin-avidin bond. In that case, the lectin is biotinylated in advance, and the biotinylated lectin is prepared in a solid-phased form on a substrate (well) coated with streptavidin, whereby the detection sensitivity is improved and the background is greatly reduced.

As another immobilization method, a physical adsorption method or a chemical bonding method can be used. When the physical adsorption method is used, a method of activating the surface by γ-ray treatment, electron beam treatment, UV treatment, plasma treatment, corona treatment, etc. may be mentioned. When the chemical bonding method is used, p-nitrophenyl is used. A substituted or unsubstituted phenol active ester group such as a group; and N-hydroxy such as N-hydroxysuccinimide group, N-hydroxyphthalimide group, N-hydroxy-5-norbornene-2,3-dicarboximide, etc. It is preferable that the substrate surface has an imide active ester group, a carboxyl group, a carbonyl group, an aldehyde group, an epoxy group, an amino group, a thiol group, a maleimide group, or the like.
Both plates are commercially available, and as the "physical adsorption method" plates, "ELISA plate (blockingless type)" manufactured by Sumitomo Bakelite Co., Ltd., "Maxi-Soap plate" manufactured by NUNC, etc. "The active ester plate kit" manufactured by Sumitomo Bakelite Co., Ltd., the "immobilizer amino plate" manufactured by NUNC Co., Ltd., and the "avidin plate" is the "avidin plate (blockingless type)" manufactured by Sumitomo Bakelite Co., Ltd. Etc. can be used.
Alternatively, a commercially available lectin array containing SNA lectin or SSA lectin may be used. For example, a lectin array (Kuno et al., Nature Methods 2, 851-856, 2005) in which 45 types of plant lectins with different specificities are immobilized on the same substrate and LecChip ^™ Ver.1.0 (Glyco Technica) ) Can be used.

(3-6) Addition and Washing of Test Culture Supernatant Sample The test culture supernatant sample is diluted with a buffer solution or added undiluted to the immobilized lectin wells for interaction, and then Contaminants that bind specifically are washed with a lectin array buffer (commercially available).
Then, a buffer containing an antibody specific to the “secretory core protein” of the present invention, such as an indirectly or directly labeled anti-fibronectin antibody, is added and reacted.

(3-7) Preparation of “secretory core protein”-specific antibody of the present invention The “secretory core protein” of the present invention includes α2-6 sialic acid from somatic stem cells such as fibronectin as described above. Since there are secretory glycoproteins, an anti-fibronectin antibody or the like can be used as the “secretory core protein”-specific antibody of the present invention.
For example, in the case of an anti-fibronectin antibody for detecting fibronectin, as long as it has the ability to recognize and specifically bind to fibronectin as an epitope, a polyclonal antibody, a monoclonal antibody, and its antigen recognition site are preserved. In addition to antibody fragments such as Fab fragments, humanized antibodies, single chain antibodies and the like can also be used. As such an antibody, it is convenient to use a commercially available anti-fibronectin antibody (α-Fib: R&D Systems, etc.), but a commercially available recombinant fibronectin (R&D) or a fibronectin protein prepared based on the sequence information of fibronectin. Using, the specific antibody can be appropriately produced by a conventional method.

When a secretory core protein-specific antibody such as an anti-fibronectin antibody is overlaid, it may be labeled with a labeling substance indirectly or directly. Examples of labeling substances include fluorescent substances (eg, FITC, rhodamine, Cy3, Cy5), radioactive substances (eg, ¹³ C, ³ H), enzymes (eg, alkaline phosphatase, peroxidase (horseradish peroxidase, etc.), glucose oxidase. , Β-galactosidase), and the like. Alternatively, the antibody may be labeled with biotin, and (strept)avidin may be labeled with the above-mentioned labeling substance to utilize the binding between biotin and (strept)avidin.
In the case of sandwich ELISA, a general method in which a secondary antibody for detection is detected by using an anti-mouse IgG antibody labeled with horseradish peroxidase (HRP) or the like can also be used.

When an enzyme is used as the labeling substance, detection is performed using an appropriate substrate according to the enzyme used. For example, when using peroxidase as the enzyme, o-phenylenediamine (OPD), tetramethylbenzidine (TMB), etc. are used as the substrate, and when using alkaline phosphatase, p-nitrophenyl phosphate (PNPP), etc. Is used. As the enzyme reaction stopping solution and the substrate solution, conventionally known ones can be appropriately selected and used according to the selected enzyme.

(3-8) Measuring method and device α2-6 sialic acid in sugar chain of secretory core protein such as fibronectin in culture supernatant sample and lectin on substrate form a complex and bind to secretory core protein The α2-6 sialic acid on the secretory core protein in the sample is detected and quantified by measuring the signal generated by the labeled antibody. The signal may be measured using an appropriate measuring device depending on the labeling substance used.

When the sandwich method is used to measure the α2-6 sialic acid-containing sugar chain on the secretory core protein, the measurement method is ELISA, immunochromatography, radioimmunoassay (RIA), fluorescent immunoassay (FIA method), chemiluminescence immunoassay. , An evanescent wave analysis method can be used. These methods are known to those skilled in the art, and any method may be selected. In addition, these methods may be carried out according to a usual procedure, and the actual setting of reaction conditions and the like are within the technical range that can be usually carried out by those skilled in the art.
In general, the binding between sugar chains and lectins is weaker than that between antibodies (generally, the binding constant for antigen-antibody reaction is said to be 10 ⁶ to 10 ⁹ M ⁻¹ , whereas between lectin and sugar chains It is said that the binding constant of is 10 ⁴ to 10 ⁷ M ⁻¹ .) Therefore, the binding signal between the lectin and the target sugar chain is preferably detected using an evanescent wave excitation type fluorescence detection method. The evanescent wave excitation type fluorescence detection method is described in Kuno et al., Nature Methods, 2, 851-856 (2005) and the like. GlycoStation ^™ Reader 1200 (Moritex) or the like can be used for this detection.

(3-9) Standard curve (calibration curve)
For quantitative detection of cell differentiation potential by this method, labeled secretory core protein, for example, labeled recombinant fibronectin was used as a standard substance and overlaid on the substrate on which α2-6 sialic acid-specific lectin was immobilized. The amount of labeled signal can be converted as the amount of α2-6 sialic acid on fibronectin by plotting a calibration curve by blotting the labeled signal amount (main wavelength 450 nm, sub wavelength 620 nm) according to the amount of standard substance. it can. For example, a commercially available recombinant fibronectin derived from recombinant human cells (manufactured by R&D) can be used as a standard substance.

(3-10) Measurement of Labeled Lectin When Using Immobilized Antibody The antibody side can be immobilized on the surface of a plate, a bead, etc. by a general method to overlay the labeled lectin. At that time, as the labeling method, the same labeling method as the labeling of the antibody described in (3-7) with a fluorescent substance, an enzyme, biotin or the like is used, but biotin-labeled lectin and HRP-labeled avidin were used. A detection system utilizing the biotin-avidin reaction is simple and preferable.
For detecting the binding signal between the fluorescently labeled lectin and the target sugar chain, it is preferable to use the above-mentioned evanescent wave excitation type fluorescence detection method.

(3-11) Measurement of total amount of secretory core protein in culture supernatant The total amount of secretory core protein of the present invention such as the amount of fibronectin secreted in the culture solution of somatic stem cells is almost unchanged even after passage. Therefore, it is not essential to measure the amount of secretory core protein in the culture supernatant.
However, as one of the methods for increasing the accuracy of determination of “cell differentiation potential”, when measuring the amount of α2-6 sialic acid at the non-reducing end of the sugar chain contained in the secretory core protein by the sandwich method described above. , The total amount of secretory core protein per unit volume was also measured with a secretory core protein-specific antibody against the remaining culture supernatant, and the amount of change in α2-6 sialic acid per unit secretory core protein was accurately calculated. There is a method of doing.
As a measuring method at that time, a general protein measuring method such as a known Western blotting method, ELISA, and sandwich assay method can be used, and the antibody-antibody sandwich ELISA method is particularly preferable.
When the amount of α2-6 sialic acid on secretory core protein is measured by the lectin-antibody sandwich method with immobilized lectin side, it recognizes an epitope different from the labeled secretory core protein-specific antibody on the detection side If the secretory core protein-specific antibody to be immobilized is immobilized in the same manner as the lectin, overlay of the culture supernatant can be simultaneously performed on both, and the labeled amount by the subsequent binding of the labeled antibody can be measured. From the ratio, the amount of α2-6 sialic acid per unit secretory core protein can be calculated.

4. Target subject stem cells and collection source thereof In the present specification, determination of cell differentiation potential, subject stem cells to be evaluated are mainly mesenchymal stem cells or cartilage stem cells, and cloned somatic stem cells In addition, it also includes a culture of a biological tissue composed of a heterogeneous cell population obtained by collecting somatic stem cells.
Therefore, the term "culture supernatant" of the subject stem cells in the present invention means a subculture of somatic stem cells (medium) or a washing solution thereof, or a culture supernatant from a biological tissue culture solution in which somatic stem cells are collected. is there.
Further, stem cells are not limited to humans and are controlled by a mechanism common to a considerable part in mammals, somatic stem cells of the present invention include mammals other than humans, for example, monkey, pig, cow, goat, It can also be applied when using somatic stem cells derived from sheep, mice and rats.

Mesenchymal stem cells can be collected from lipoaspirated adipose tissue, umbilical cord blood, umbilical cord, amniotic membrane, placenta, or bone marrow aspirate from jaw bone or femur bone marrow. In addition, mesenchymal stem cells are commercially available. For example, adipose tissue-derived mesenchymal stem cells can be purchased from Life Technologies, and bone marrow-derived mesenchymal stem cells can be purchased from Lonza, PromoCell and the like. The culture conditions are not particularly limited, but the culture temperature is preferably 36 to 37°C, which is the same as the body temperature. As the medium, a MesenPRO RS ^™ medium (Life Technologies) generally used as a mesenchymal stem cell maintenance medium can be appropriately used. In addition, bone marrow-derived mesenchymal stem cells can be obtained from the bone marrow tissue of the finger excised by surgery for polydactyly.
Chondrocyte stem cells can be collected from cartilage tissue. For example, cartilage tissue-derived chondrocytes (polydactyly-derived cartilage stem cells) can be obtained from the cartilage tissue of the excised finger of a polydactyly patient. It can also be obtained from RIKEN BioResource Center, JSRB Cell Bank, etc. As the chondrocyte maintenance medium, although MesenPRO RS ^™ medium (Life Technologies) generally used as a mesenchymal stem cell maintenance medium may be used, chondrocyte basic medium, chondrocyte growth medium (Takara Bio Inc.), etc. It is also possible to use a maintenance medium for chondrocyte stem cells.

5. Determination and Evaluation Method of Somatic Stem Cell Sample and Quality Control (5-1) Quality Control Based on Determination and Evaluation of Cell Differentiation Potential of Somatic Stem Cell Sample The present invention relates to somatic stem cell-containing tissues and the like obtained from a living body. In a stem cell sample or somatic stem cell culture, α2-6 sialic acid specifically expressed at the non-reducing end of the secretory core protein-containing sugar chain of the present invention such as fibronectin secreted in the culture solution of somatic stem cells is specifically detected. The present invention relates to a method for determining or evaluating the cell differentiation potential of a somatic stem cell sample. It also includes a method of performing quality control of a somatic stem cell sample according to the determination result.
Detection of α2-6 sialic acid on the secretory core protein of the present invention such as fibronectin in somatic stem cell culture medium and measurement of the expression level include α2-6 sialic acid-binding lectin (SSA, The most suitable method is to apply a sandwich assay method with secretory core protein-specific antibodies such as SNA) and anti-fibronectin antibody. When evaluating and determining the cell differentiation potential of the entire culture lot, and when performing quality control based on that evaluation, the culture supernatant that does not contain cells is used, so when changing the medium during passage. Since the discarding culture solution and the washing solution that are always available can be used, precious somatic stem cells themselves are not consumed.

Using a sandwich assay system that combines secretory core protein-specific antibodies such as anti-fibronectin antibody and α2-6 sialic acid-reactive lectin (SSA, SNA), measure the labeling intensity of secretory core protein-specific antibodies such as anti-fibronectin antibody. By doing so, the cell differentiation potential to osteoblasts or chondrocytes can be accurately evaluated and determined.
In such a sandwich assay for determining a cell differentiation potential, whichever may be immobilized, a substrate (well) on which α2-6 sialic acid-reactive lectin (SSA, SNA) is immobilized by the avidin method or the physical adsorption method On the other hand, it is preferable to supply the culture supernatant of the analyte stem cells and overlay the labeled secretory core protein-specific antibody. In the present invention, the term "labeled secretory core protein-specific antibody" includes the case where the antibody itself is not labeled and is detected by a labeled secondary antibody such as a labeled antiglobulin antibody.

Further, by labeling the α2-6 sialic acid-binding lectin or the secretory core protein-specific antibody, even for the culture supernatant containing contaminants, the α2-6 sialic acid on the secretory core protein such as fibronectin can be labeled. Since the acid expression amount can be accurately measured, the cell differentiation potential of somatic stem cells can be easily evaluated and determined. When overlaying with lectins, labeling of lectins can be performed by using fluorescent labels such as R-Phycoerythrin (PE) and FITC, enzyme labels such as peroxidase, and labeling with biotin (+HRP labeled avidin). Alternatively, a labeled antibody against lectin may be used as a secondary antibody to measure the labeled amount.

6. Kit for Judging Cell Differentiation Potential of Somatic Stem Cell As described in (3-1) above, in the present invention, a secretory core protein such as fibronectin in the culture supernatant secreted from mesenchymal stem cells or chondrocyte stem cells. It was found that the amount of α2-6 sialic acid possessed by S. alba has a high correlation with the degree of cell differentiation potential into osteoblasts or chondrocytes.
As a result, the cell differentiation potential of the test stem cells such as mesenchymal stem cells or chondrocyte stem cells to osteoblasts or chondrocytes is accurately determined from the culture supernatant, and as an assay system for evaluation, a secretory core is used. It was possible to construct a sandwich assay system for determining the cell differentiation potential, which is a combination of a protein-specific antibody and α2-6 sialic acid-reactive lectin.

Specifically, after reacting a test stem cell culture supernatant with a substrate (well) on which α2-6 sialic acid-reactive lectin was immobilized by avidin method or physical adsorption method, secretory core protein-specific A method for overlaying and detecting or quantifying an antibody can be exemplified. On the contrary, detection and quantification can also be performed by a combination of a secretory core protein-specific antibody-immobilized substrate and a labeled α2-6 sialic acid-reactive lectin.
That is, in the present invention, when the kit for cell differentiation potential determination of somatic stem cells, a kit for determining and evaluating the cell differentiation potential to osteoblasts or chondrocytes using somatic stem cell culture supernatant,
(A) a lectin that specifically recognizes α2-6 sialic acid, and (b) a secretory core protein-specific antibody,
It is a kit containing the probe of.
It is preferable that one of the probes (a) and (b) is immobilized on a substrate and the other probe is labeled, and in particular (a) is immobilized by an avidin method or a physical adsorption method. A kit comprising the prepared substrate (well) and (b) for overlay is preferred. As a method for immobilizing a lectin on a plate, the avidin method of binding to avidin is the most excellent, and then the physical adsorption method of physically adsorbing a lectin is excellent. Further, as described above, as the α2-6 sialic acid-reactive lectin, SSA and SNA, especially SNA is a lectin that can determine changes in the amount of α2-6 sialic acid on a secretory core protein with higher correlation, which is preferable. .. Examples of secretory core protein-specific antibodies include anti-fibronectin antibodies.
In addition, when measuring the total amount of secretory core protein in the culture supernatant, after measuring the amount of α2-6 sialic acid on the secretory core protein with a kit combining (a) and (b) above, the remaining culture is measured. It can be measured with the antibody probe of (b) directly or indirectly labeled with the supernatant, but both (a) and (b) should be placed in separate regions on the substrate. For example, after overlaying the test sample, the α2-6 sialic acid on the secretory core protein is subjected to a single label detection step using a labeled antibody of the secretory core protein-specific antibody which is the same as or different from that of (b). The measurement of the amount and the measurement of the total amount of secretory core protein can be performed simultaneously.
Furthermore, a recombinant secretory protein with stable quality such as recombinant fibronectin can be included in the reagents of the kit as a standard substance.
In the present invention, the term "kit" refers to a case where reagents mainly consisting of probes are combined, but it may include a container for the reagent, a package, an instruction manual, and the like, as well as a measuring device and the like.

In the present invention, the total amount of secretory core proteins such as fibronectin secreted in the culture medium is hardly changed even if the passage of somatic stem cells is increased, and α2-6 sialic acid expression on secretory core proteins such as fibronectin is not changed. Since it has been found that the amount decreases, it is sufficient to measure the α2-6 sialic acid expression level on the secretory core protein for qualitative determination and evaluation.
At that time, using a standard strain of somatic stem cells that is the same as the target somatic stem cell-containing sample, the expression amount of α2-6 sialic acid on secretory core proteins such as fibronectin secreted in the culture medium in advance. It is also possible to quantitatively evaluate the test stem cell-containing sample by using a calibration curve for the degree of cell differentiation potential according to. Specifically, a standard strain is subcultured, a part of cells at a plurality of subcultures in the subculture is collected, and the labeling intensity is measured using the sandwich assay kit consisting of (a) and (b) above. To measure. At the same time, the passage cell is induced to differentiate into the desired differentiated cell, and the numerical value indicating the cell differentiation potential such as the strength of the differentiated cell marker is measured, and the calibration curve is obtained from the both values. By using the obtained calibration curve, the labeling intensity measured by the assay system for the sample containing the subject stem cells can be converted into a quantitative cell differentiation potential amount to the target differentiated cells, It is possible to accurately evaluate and determine the cell differentiation potential of a sex stem cell-containing sample.

Furthermore, in order to more quantitatively and accurately determine and evaluate the cell differentiation potential, it is preferable to simultaneously measure the total amount of secretory core proteins such as fibronectin secreted in the culture medium.
Specifically, the α2-6 sialic acid expression level on fibronectin in the culture supernatant of the analyte stem cells was measured, and the total amount of secretory core proteins such as fibronectin in the remaining culture supernatant was measured at the same time. From the measured values, "α2-6 sialic acid expression on secretory core protein/amount of secretory core protein", for example, "α2-6 sialic acid expression on fibronectin/fibronectin amount" is calculated.
For the expression level of α2-6 sialic acid on the secretory core protein, the labeling intensity measured using the sandwich assay kit consisting of (a) and (b) above can be used, and the secretory property in the culture supernatant can be determined. The amount of core protein may be measured by any measurement method as long as it can be quantified, and can be easily measured by a usual ELISA method. For example, it can be accurately measured by a sandwich ELISA method comprising (c) an antibody specific to a secretory core protein such as an anti-fibronectin antibody and (d) an anti-globulin antibody.

Hereinafter, the present invention will be described specifically with reference to Examples, but the present invention is not limited thereto.
Other terms and concepts in the present invention are based on the meaning of terms commonly used in the art, and various techniques used for carrying out the present invention are notably defined by the sources. Except for this, those skilled in the art can easily and surely carry out the implementation based on known documents and the like. In addition, various analyzes and the like were carried out by applying the methods described in the analytical instruments or reagents used, the instruction manual of the kit, the catalog, and the like.
The description in the technical documents, patent publications, and patent application specifications cited in this specification shall be referred to as the description of the present invention.

Example 1 Identification of α2-6 Sialic Acid-Containing Glycoprotein in Cartilage Stem Cell Culture Supernatant Cartilage tissue-derived chondrocyte stem cells (Yub625 strain) were used as a general mesenchymal stem cell medium, MesenPRO RS ^™ medium (Life Technologies). 100 μL of culture supernatant for each passage number P3, P6, P9, P12, P15, P18 and each control medium were mixed with anti-fibronectin antibody (R&D)-immobilized beads. And reacted overnight at 4°C. After washing the beads, the bound fraction was eluted by heating with 20 μL of 0.2% SDS at 95° C. for 5 minutes. 15 μL of each binding fraction was mixed with streptavidin-immobilized beads to remove biotinylated anti-fibronectin antibody, and then 2.5 μL of 5×SDS sample buffer was added to 10 μL of the sample, and the mixture was incubated at 95° C. for 5 minutes. After heat denaturation, electrophoresis was performed on an acrylamide gel, and blotting was performed with an anti-fibronectin monoclonal antibody (manufactured by R&D) and HRP-SNA. In addition, as each test culture supernatant sample, a culture supernatant to be discarded when the medium was exchanged every 1 to 2 days was collected and used (the same applies to other examples unless otherwise specified).
As a result, it was observed that the amount of fibronectin showing 250 kDa or more did not change and that the reactivity of SNA with respect to the fibronectin-containing sugar chain decreased (FIG. 1).
That is, it was confirmed that the amount of fibronectin in the culture supernatant did not change, and the amount of α2-6 sialic acid on fibronectin decreased, even though the cell differentiation potential decreased with the increase in the number of subcultures of chondrocyte stem cells. ..
From the above results, it was suggested that the cell differentiation potential of somatic stem cells can be determined by measuring the amount of α2-6 sialic acid in the fibronectin-containing sugar chain in the culture supernatant. This also indicates that the secretory core protein retaining the cell differentiation potential marker α2-6 sialic acid is fibronectin.

Example 2 Preparation of Standard Curve Using Recombinant Fibronectin In this example, labeled recombinant fibronectin was used as a standard substance, and a standard curve for converting the amount of labeled signal as the amount of α2-6 sialic acid on fibronectin. (Calibration curve) was created.
Specifically, different amounts of recombinant fibronectin (manufactured by R&D) were reacted for 1 hour at room temperature on a plate on which 1 μg/well of SNA was immobilized. After washing, an HRP-labeled anti-fibronectin monoclonal antibody (manufactured by R&D) was reacted, and after 1 hour at room temperature, a TMB solution was added to perform a color reaction. After 30 minutes, 1N hydrochloric acid was added to stop the color reaction, and then the absorbance at 450 nm to 620 nm was measured (FIG. 2).

(Example 3) Verification of reactivity of ELISA system for detecting α2-6 sialic acid containing fibronectin in culture supernatant (3-1) Verification of difference in detectability by various immobilized lectins In this example, α2- 6 In order to establish an optimal sandwich ELISA system of a lectin specifically reacting with sialic acid and an anti-fibronectin antibody, it is immobilized on a substrate (microarray plate) as the following (Experimental Example 1) to (Experimental Example 9). The type of lectin and the method of immobilizing the lectin were changed, and a signal value by a lectin-antibody sandwich ELISA system was obtained using an anti-fibronectin antibody (R&D), and the difference in reactivity was verified.
As a method for immobilizing lectin, "Physical adsorption method" plate that physically adsorbs lectin directly on the substrate surface is used as "ELISA plate (blockingless type) (Sumitomo Bakelite Co., Ltd.)""Active Ester Plate Kit (Sumitomo Bakelite Co., Ltd.)" as a "chemical binding plate" for chemically binding, and "Avidin plate (blocking-less type) as an "avidin plate" for binding a biotinylated lectin by coating the substrate surface with avidin ) (Manufactured by Sumitomo Bakelite Co., Ltd.)”.
Then, as the mesenchymal stem cells, polydactyly digitized bone marrow-derived mesenchymal stem cells (Yub622 strain, RIKEN BioResource Center) were subcultured by the same method as in (Example 1) to obtain osteoblast differentiation ability. The above-mentioned ELISA system was applied to the cell culture supernatant in the state of having the above (passage number 3) and the cell culture supernatant in the state of not having the osteoblast differentiation ability (passage number 15), and the absorbance was measured.
In addition, the absorbance of MesenPRO RS ^™ medium (Life Technologies) alone (“medium”), which is an exchange medium, and PBS (“cell-free”) were used as controls.

[Experimental Example 1] When SNA and avidin plate were used:
(1) 50 μL of a solution prepared by diluting SNA (manufactured by Vector) biotin-labeled using Dojindo Biotin labeling kit with PBS (manufactured by Nissui) to a final concentration of 1 μg/mL on an avidin plate (Sumitomo Bakelite). Each was dispensed and left at room temperature for 1 hour.
(2) After discarding the solution, 300 μL of the washing liquid was dispensed and discarded, and the operation of tapping was repeated 3 times for washing.
(3) 50 μL of each culture supernatant diluted 10-fold with PBS was added to the plate, and the plate was allowed to stand at room temperature for 1 hour.
(4) After discarding the solution, 300 μL of the washing solution was dispensed and discarded, and the operation of tapping was repeated 3 times for washing.
(5) HRP-labeled anti-fibronectin antibody diluted with 0.05% Triton-X100/PBS solution to a final concentration of 1 μg/mL (manufactured by R&D, using HRP kit manufactured by Dojindo Co., Ltd.) was added by 50 μL each, and allowed to stand at room temperature for 1 hour. did.
(6) After discarding the solution, 300 μL of the washing solution was dispensed and discarded, and the operation of tapping was repeated 3 times for washing.
(7) 100 μL each of the color-developing reagent prepared by mixing the color-developing solution and the substrate solution at 100:1 was added to each well, and the mixture was allowed to stand at room temperature for 15 minutes.
(8) 100 μL of the stop solution was added to each well, and the absorbance was measured with a microplate reader.

[Experimental Example 2] When SSA and avidin plate were used:
Instead of SNA in Experiment 1 operation (1), SSA (manufactured by JOM) was immobilized on the plate, and the subsequent protocol was the same as in Experiment 1 operation (2) to (8).

[Experimental Example 3] When PSL and avidin plate were used:
PSL (manufactured by Wako Pure Chemical Industries, Ltd.) was immobilized in place of the SNA in Experiment 1 operation (1), and the subsequent protocol was the same as in Experiment 1 operation (2) to (8).

[Experimental Example 4] When SNA and physical adsorption plate were used:
(1) 50 μL of SNA (manufactured by Vector) diluted to a final concentration of 1 μg/mL with PBS (manufactured by Nissui) was dispensed into a maxisorp plate (manufactured by NUNC) and allowed to stand at room temperature for 2 hours.
In the protocol after (2), the same operations as in Experiment 1 operations (2) to (8) were performed.

[Experimental Example 5] When SSA and physical adsorption plate were used:
Experimental Example 4 SSA (manufactured by JOM) was immobilized instead of SNA in the operation (1), and the subsequent protocol was the same as the operations (2) to (8) in the experimental example 1.

[Experimental Example 6] When using PSL and a physical adsorption plate:
Experimental Example 4 PSL (manufactured by Wako Pure Chemical Industries, Ltd.) was immobilized instead of SNA in Operation (1), and the subsequent protocol was the same as in Experimental Example 1 operations (2) to (8).

[Experimental Example 7] When SNA and chemically bonded plate were used:
(1) 50 μL of SNA (manufactured by Vector) diluted with PBS (manufactured by Nissui) to a final concentration of 1 μg/mL was dispensed into immobilizer amino plates (manufactured by NUNC) and allowed to stand at room temperature for 2 hours.
In the protocol after (2), the same operations as in Experiment 1 operations (2) to (8) were performed.

[Experimental Example 8] When SSA and a chemically bonded plate were used:
Experimental Example 7 SSA (manufactured by JOM) was immobilized instead of SNA in Operation (1), and the subsequent protocol was the same as in Experimental Example 1 operations (2) to (8).

[Experimental Example 9] When PSL and chemically bonded plate were used:
Experimental Example 7 PSL (manufactured by Wako Pure Chemical Industries, Ltd.) was immobilized instead of SNA in Operation (1), and the subsequent protocol was the same as in Experimental Example 1 operations (2) to (8).

<Results>
The above results are summarized as shown in (Table 1) and (FIG. 3) below.
That is, as the α2-6 sialic acid-specific lectin, it was confirmed that the reactivity was decreased in response to the decrease in the cell differentiation potential in any of the lectins. I found out. It was also confirmed that the avidin-bound plate had good sensitivity as a method for immobilizing the lectin.
Therefore, as the lectin for the lectin-ELISA system for determining the cell differentiation potential using the somatic stem cell culture supernatant, any lectin that specifically reacts with α2-6 sialic acid can be used. However, it was found that SNA lectin or SSA lectin is preferable, particularly SNA lectin is preferable, and that when a lectin is immobilized, the use of an avidin-bound plate is the most sensitive.

(Example 4) Changes in α2-6 sialic acid expression level on fibronectin in the culture supernatant in long-term subculture of bone marrow-derived mesenchymal stem cells, cartilage tissue-derived chondrocytes, and skin fibroblasts With this example, According to Examples 5 to 7 and Comparative Example 1 to be described later, changes in the expression level of α2-6 sialic acid on fibronectin secreted into the culture medium of subculture of various somatic stem cells, and bone cells of somatic stem cells Or, verify the relationship with the cell differentiation potential to chondrocytes.
The bone marrow-derived mesenchymal stem cells Yub622 strain (RIKEN BioResource Center), Yub10F strain (RIKEN BioResource Center), cartilage tissue-derived chondrocyte stem cells Yub621c strain (RIKEN BioResource Center), Yub625 strain (RIKEN BioResource Center), and human skin fibroblast The cells (hFibs, ATCC) were subcultured for a long period of 90 to 190 days by the same method as in (Example 1). For all cells, the medium was exchanged when the cells reached 80% confluence in a 10-cm culture dish, and the culture supernatant was collected each time the medium was exchanged, and the SNA immobilized in the avidin-coated wells was collected. Reacted with lectin. Then, an HRP-labeled anti-fibronectin antibody (α-Fib, manufactured by R&D) was overlaid. The amount of labeling was measured according to the passage number, and changes in the expression level of α2-6 sialic acid on fibronectin in each culture supernatant were analyzed.
As a result, bone marrow-derived mesenchymal stem cells and cartilage tissue-derived chondrogenic stem cells rapidly decreased the α2-6 sialic acid expression level on fibronectin as the passage number increased, whereas in human skin fibroblasts, There was no reactivity at the passage number (Fig. 4).

This strongly suggests that the expression level of α2-6 sialic acid on fibronectin in the culture supernatant of somatic stem cells is highly related to the potential of somatic stem cells to differentiate into osteoblasts or chondrocytes. ing.
In view of the results of this Example and the following Examples 5 to 7 and Comparative Example 1, the α2-6 sialic acid on fibronectin secreted into the culture medium of somatic stem cells is It was found to be an excellent indicator for determining the cell differentiation potential of chondrocytes.

(Example 5) Subculture of Yud622 strain of polydactyly bone marrow-derived mesenchymal stem cells used in (Example 4) in comparison of osteodifferentiation ability of polydactyly bone marrow-derived mesenchymal stem cells in early and late passages The early cells (P5) and the late cells (P15) and the passage early cells (P9) and the late cells (P14) of the Yub10F strain were respectively transformed into an osteoblast differentiation medium (hMSC differentiation BulletKit-osteogenic (Cat#: PT)). -3002, Lonza)), and osteoblasts were stained with alizarin red S. In the early passage cells, the number of cells differentiated into osteoblasts is large and the cell differentiation potential is high, whereas in the late-stage cells, osteoblast differentiation hardly occurs and it is confirmed that the cell differentiation potential is low. (Fig. 5).

From these results, the expression amount of α2-6 sialic acid on fibronectin in the culture supernatant of the bone marrow-derived mesenchymal stem cells used in Example 4 was determined to be the cell differentiation potential of the bone marrow-derived mesenchymal stem cells into osteoblasts. It was confirmed that it is an index.

(Example 6) Comparison of osteodifferentiation ability of chondrocyte derived from polydactyly at the early and late stages of passage In this example, Yud621c strain of chondrocyte derived from polydactyly used in (Example 4) (RIKEN BioResource Center) and Using the Yub625 strain (RIKEN BioResource Center), the subculture early cells (P8) and late cells (P28) of the Yub621c strain, and the passage early cells (P8) and late cells (P19) of the Yub625 strain, respectively, were performed. Differentiation was induced into osteoblasts in the same manner as in Example 5), and the osteoblasts were stained with alizarin red S. In the early passage cells, the number of cells differentiated into osteoblasts is large and the cell differentiation potential is high, whereas in the late-stage cells, osteoblast differentiation hardly occurs and it is confirmed that the cell differentiation potential is low. It was
From these results, it was confirmed that the expression level of α2-6 sialic acid on fibronectin in the culture supernatant of the chondrocyte stem cells used in Example 4 is an index of the cell differentiation potential of chondrocyte stem cells to osteoblasts. It was done (Fig. 6).

(Example 7) Comparison of chondrogenic potential of chondrocyte-derived chondrocytes (Yub625 strain) in the early and late passages , polydactyly-derived chondrocyte stem cells used in (Example 4), early passage cells of the Yub625 strain (P5) Each of the cells and the late phase cells (P22) was subjected to chondrocyte induction treatment using a chondrocyte differentiation induction kit manufactured by Lonza, and stained with Alcian blue (Muto Chemical Co., Ltd.) at pH 2.5. As a result, the cartilage-derived strain derived from the early-passage strain was clearly strongly stained with cyan blue, indicating that cartilage differentiation was actively occurring.
This is also high in cartilage-inducing ability in early passage cells in which a large amount of α2-6 sialic acid is present on fibronectin in the culture supernatant, and late cells in which α2-6 sialic acid amount is rapidly lost. It was confirmed that it will be almost lost.
That is, it was confirmed that the expression level of α2-6 sialic acid on fibronectin in the culture supernatant of the chondrocyte stem cells used in Example 4 also serves as an index of the cell differentiation potential of chondrocyte stem cells into chondrocytes (Fig. 7).

(Comparative Example 1) Long-term subculture of skin fibroblasts and comparison of bone differentiation ability and fat differentiation ability of early and late passage skin fibroblasts (Example 4) are used for comparison. The human fibroblast cell line hFibs (obtained from ATCC) was also treated for inducing differentiation into osteoblasts in the same manner as in Examples 5 to 6 and stained with alizarin red S that stains osteoblasts. did. As a precaution, differentiation induction into adipocytes was also performed using hMSC differentiation BulletKit-adipogenic (Cat#: PT-3004, Lonza), and the differentiation state was confirmed by staining with oil red O which adipocytes are stained.
As a result, no staining was confirmed (Fig. 8).

(Experimental Example 8) Evaluation of cell differentiation potential using lectin plate and antibody plate (8-1) Evaluation of cell differentiation potential in cartilage tissue-derived cartilage stem cells (Yub625 strain) In this example, the Yub625 strain of cartilage tissue-derived cartilage stem cells was used. Subculture was performed using (RIKEN BioResource Center) in the same manner as in (Example 1). 100 μL each of the culture supernatant for discarding and each control medium for each medium exchange at passages P3 to P15 were applied to the SNA lectin plate prepared by the procedure of (8-2) below, and sandwich ELISA with an anti-fibronectin antibody was performed. The system measured the amount of α2-6 sialic acid on fibronectin at each passage number. At the same time, the total amount of fibronectin for each passage number was measured using the antibody plate prepared by the procedure of (8-3) below, and the amount of α2-6 sialic acid-containing fibronectin per total passage amount of fibronectin was calculated. The results for typical passage number 3 (p3) and passage number 15 (p15) among them are shown in the following (Table 2).

(8-2) Preparation of lectin plate and lectin-antibody ELISA
An SNA lectin plate was prepared by the following procedure, and a POD-labeled anti-human fibronectin antibody (using a kit manufactured by Takara Bio Inc.) was overlaid, and then the POD label was detected.
(1) A solution of biotin-labeled SNA (manufactured by Vector) diluted with PBS (manufactured by Nissui) to a final concentration of 1 μg/mL was dispensed in 50 μL avidin plates (manufactured by Sumitomo Bakelite Co., Ltd.) at room temperature for 2 hours. I let it stand.
(2) After discarding the solution, 300 μL of the washing liquid was dispensed and discarded, and the operation of tapping was repeated 3 times for washing.
(3) 500 ng/mL of human fibronectin standard antigen (manufactured by PromoCell) diluted with PBS was used as the top concentration, and 50 μL/well was dispensed into the rows A to G of the plate in a 2-fold dilution series. 50 μL of PBS was added to the H row.
(4) 50 μL of each culture supernatant diluted 10-fold with PBS was added to the plate and left standing at 37° C. for 1 hour.
(5) After discarding the solution, 300 μL of the washing solution was dispensed and discarded, and the operation of tapping was repeated 3 times for washing.
(6) POD-labeled anti-human fibronectin antibody (using a kit manufactured by Takara Bio Inc.) was added in an amount of 50 μL each, and the mixture was allowed to stand at 37° C. for 1 hour.
(7) After discarding the solution, 300 μL of the washing solution was dispensed and discarded, and the operation of tapping was repeated 3 times for washing.
(8) 100 μL each of the color-developing reagent prepared by mixing the color-developing solution and the substrate solution at 100:1 was added to all the wells and left standing at room temperature for 15 minutes.
(9) 100 μL of the stop solution was added to each well, and the absorbance was measured with a microplate reader.
The resulting amount of α2-6 sialic acid on fibronectin for each passage number is shown in FIG. 9A.

(8-2) Antibody-antibody ELISA using antibody plate (Takara Bio Inc.)
Using an antibody plate (manufactured by Takara Bio Inc.), the same operations as (3) to (9) in the above (8-2) were performed.
The total amount of fibronectin obtained for each passage number is shown in FIG. 9B.

<Results>
The above results are summarized below (Table 2).
That is, the total amount of fibronectin (secretory core protein) secreted in the culture medium is secreted in a certain amount regardless of the passage number, but the expression amount of α2-6 sialic acid on fibronectin decreases with the passage number. I understood. From the two results, the ratio of α2-6 sialic acid on fibronectin per total fibronectin could be calculated.

(Example 9) Change in percentage of α2-6 sialylated fibronectin in culture supernatant in subculture of polydactyly bone marrow-derived mesenchymal stem cells (Yub622 strain) In this example, polydactyly bone marrow-derived mesenchyme was used. The subculture of the Yub622 strain of lineage stem cells was carried out in the same manner as in (Example 1). 100 μL each of the culture supernatant for discarding and each control medium for each medium exchange at passage numbers P3 to P15 were taken, and SNA lectin-POD labeled anti-human fibronectin antibody (Takara Bio Inc.) was prepared by the same procedure as in (Example 8). By measuring the amount of α2-6 sialic acid on fibronectin for each passage number, by antibody plate and POD-labeled anti-human fibronectin antibody (Takara Bio Inc. kit), measure the total fibronectin amount for each passage number, together, The ratio of α2-6 sialic acid on fibronectin per total fibronectin for each passage was calculated (Fig. 10).
As a result, it can be seen that the proportion of α2-6 sialic acid content on fibronectin in the culture supernatant decreases as the passage number increases.

According to the present invention, it is possible to evaluate and determine the cell differentiation potential of an analyte stem cell sample into osteoblasts or chondrocytes non-invasively, without reducing the analyte stem cells, simply and efficiently. It is a very useful technique for quality control of somatic stem cell samples, especially for quality control of transplant samples for regeneration of osteoblasts or chondrocytes.

Claims (15)

A method for determining or evaluating the cell differentiation potential of mesenchymal stem cells or chondrocyte stem cells using a culture supernatant,
A method comprising the step of measuring the amount of α2-6 sialic acid in a fibronectin- containing sugar chain in the culture supernatant of somatic stem cells. The method according to claim 1 , wherein the mesenchymal stem cells are bone marrow-derived mesenchymal stem cells. The step of measuring the amount of α2-6 sialic acid in the fibronectin- containing sugar chain should be performed using a sandwich assay method using a lectin that specifically recognizes α2-6 sialic acid and an antibody that specifically recognizes fibronectin. The method according to claim 1 or 2 , characterized by: The method according to claim 3 , wherein the lectin that specifically recognizes α2-6 sialic acid is an SNA lectin or an SSA lectin. Further comprising the step of measuring the total amount of fibronectin contained in the culture supernatant of the mesenchymal stem cells or chondrocytes stem cells, the method according to any one of claims 1-4. A method for determining or evaluating the cell differentiation potential of mesenchymal stem cells or chondrocyte stem cells using a culture supernatant, which comprises the following (1) and (2):
(1) A culture supernatant sample of mesenchymal stem cells or test chondrocyte stem cells ,
A step of overlaying a probe on which either one of (a) or (b) below is immobilized and then allowing the other labeled probe to act, or (a) and (b) below Overlaying the probe onto the immobilized substrate, and then allowing the labeled probe of (b) to act;
(A) a lectin that specifically recognizes α2-6 sialic acid,
(B) an antibody that specifically recognizes fibronectin ,
(2) A step of measuring the labeled amount of the labeled probe. The method according to claim 7, further comprising the following steps (3) and (4):
(3) using the residual sample of the test culture supernatant samples used in step (1), the antibody specifically recognizing the labeled fibronectin, a label amount corresponding to the total amount of fibronectin per unit volume Measuring step,
(4) A step of calculating the ratio of the amount of α2-6 sialic acid-containing fibronectin to the total amount of fibronectin in the culture supernatant, based on the labeling amount measured in steps (2) and (3). A kit for determining a cell differentiation potential of mesenchymal stem cells or chondrocyte stem cells for use as a culture supernatant of mesenchymal stem cells or chondrocyte stem cells , which is for a sandwich assay containing the probes of the following (a) and (b): kit;
(A) a lectin that specifically recognizes α2-6 sialic acid,
(B) An antibody that specifically recognizes fibronectin . The kit according to claim 8 , wherein one of the probes (a) and (b) is immobilized on a substrate and the other probe is labeled. The kit according to claim 8 or 9 , wherein the lectin that specifically recognizes α2-6 sialic acid is an SNA lectin or an SSA lectin. A method for quality control of mesenchymal stem cells or chondrocyte stem cells using a culture supernatant,
A method comprising the step of measuring the amount of α2-6 sialic acid in a fibronectin- containing sugar chain in the culture supernatant of mesenchymal stem cells or cartilage stem cells . The step of measuring the amount of α2-6 sialic acid in the fibronectin- containing sugar chain should be performed using a sandwich assay method using a lectin that specifically recognizes α2-6 sialic acid and an antibody that specifically recognizes fibronectin. The method according to claim 11 , characterized by: A mesenchymal stem cell or a quality control kit for chondrocyte stem cells for use in the culture supernatant of the mesenchymal stem cells or chondrocytes stem cells, kits for quality control, including a probe of the following (a) and (b);
(A) a lectin that specifically recognizes α2-6 sialic acid,
(B) An antibody that specifically recognizes fibronectin . The kit according to claim 13 , wherein one of the probes (a) and (b) is immobilized on a substrate and the other probe is labeled. The kit according to claim 13 or 14 , wherein the lectin that specifically recognizes α2-6 sialic acid is an SNA lectin or an SSA lectin.