WO2016006712A1 - Method for determining cell differentiation potential - Google Patents

Method for determining cell differentiation potential Download PDF

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WO2016006712A1
WO2016006712A1 PCT/JP2015/070053 JP2015070053W WO2016006712A1 WO 2016006712 A1 WO2016006712 A1 WO 2016006712A1 JP 2015070053 W JP2015070053 W JP 2015070053W WO 2016006712 A1 WO2016006712 A1 WO 2016006712A1
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sialic acid
stem cells
lectin
probe
differentiation potential
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PCT/JP2015/070053
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French (fr)
Japanese (ja)
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浩章 舘野
弓弦 伊藤
泰子 小沼
淳 平林
英憲 阿久津
雅士 豊田
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国立研究開発法人産業技術総合研究所
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Priority to JP2016532993A priority Critical patent/JP6478418B2/en
Publication of WO2016006712A1 publication Critical patent/WO2016006712A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
    • G01N30/02—Column chromatography
    • G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor

Definitions

  • the present invention relates to a method for discriminating differentiation potential, which is the ability of somatic stem cells to change to another type of cell, and a method for concentrating somatic stem cells having high differentiation potential.
  • somatic stem cells with high differentiation potential can be identified and concentrated efficiently and easily, leading to acceleration of regenerative medicine using somatic stem cells.
  • Somatic stem cells such as mesenchymal stem cells are stem cells that can be collected from autologous tissues such as bone marrow and fat, and autologous transplantation without immune rejection has already been clinically applied.
  • somatic stem cells such as mesenchymal stem cells are heterogeneous cell populations, and their properties (differentiation potential, proliferative ability, migration ability) vary greatly depending on the age, tissue, passage number, etc. of the individual from which they are treated. It is difficult to evaluate the effectiveness of the product and its quality control is extremely difficult.
  • somatic stem cells such as mesenchymal stem cells except for hematopoietic stem cells.
  • cell identification is performed by confirming the expression of a plurality of cell surface markers, but in the first place, the expression of these markers and their therapeutic efficacy have not yet been correlated.
  • Patent Document 2 analysis of antigens specifically expressed on the surface of mesenchymal stem cells showed that the antigen CD146 is a marker showing pluripotency into bone, cartilage and fat in mesenchymal stem cells.
  • Patent Document 3 Non-Patent Documents 2 and 3).
  • the expression level of NG2 on the cell surface was found to be highly correlated with the pluripotency of mesenchymal stem cells, and the differentiation potential of somatic stem cells such as mesenchymal stem cells was determined by measuring the expression levels of antigens CD146 and NG2.
  • a method of determining or evaluating has been proposed (Patent Document 2).
  • sugar chain is located on the outermost side of the group of substances covering the cell surface, and is known to change dramatically in response to changes in cell status such as cell differentiation and malignancy.
  • stem cell markers and cancer markers are sugar chain markers. Therefore, it has been attracting attention that sugar chains on the cell surface of somatic stem cells such as mesenchymal stem cells change according to their differentiation state, and particularly as an indicator of the differentiation state of mesenchymal stem cells that have been put to practical use.
  • sugar chain markers There are many studies on sugar chain markers. For example, as a method of evaluating the differentiation state of stem cells, the cell state and cell differentiation are evaluated from changes in the sugar chain type of the sugar chain group in the quantitative profile obtained for N-linked sugar chains on the surface of differentiated stem cells.
  • Patent Document 4 pluripotent stem cells are strictly analyzed by analyzing the absolute amount of N-linked sugar chains, O-linked sugar chains, GSL, GAG, and FOS on the stem cell surface
  • a comprehensive evaluation method such as an evaluation and selection method (Patent Document 5) has been proposed.
  • a method for evaluating the differentiation state of pluripotent stem cells using the undifferentiated sugar chain marker “Fuc ⁇ 1-2Gal ⁇ 1-3GlcNAc / Fuc ⁇ 1-2Gal ⁇ 1-3GalNAc” has been proposed (Patent Document 6).
  • a sugar chain marker (Patent Document 7) for bone differentiation determination for accurately determining the bone differentiation state is also provided.
  • somatic stem cells such as mesenchymal stem cells composed of heterogeneous cell populations cannot be used for strict quality evaluation or isolation / concentration of populations with high differentiation potential.
  • An object of the present invention is to identify a cell surface sugar chain marker that is an excellent index of differentiation potential that ensures the therapeutic efficacy of somatic stem cells, particularly mesenchymal stem cells, and to make the cell surface sugar chain marker simple and efficient. It is intended to provide a method for determining the differentiation potential of somatic stem cells by detecting and quantifying them. Furthermore, a method for isolating or concentrating somatic stem cells having high differentiation potential from a heterogeneous cell population containing somatic stem cells using the cell surface sugar chain marker and a quality control method for somatic stem cells are provided. It is.
  • the term “differentiation potential” has a potential ability to change into another type of cell such as a progenitor cell or tissue cell when a cell is placed in an appropriate differentiation-inducing state. It means that Here, when a cell has “high differentiation potential”, it does not depend on whether or not there are multiple types of cells that change. If the ability to change to a different type of cell is high, it is expressed as “high differentiation potential”.
  • mesenchymal stem cells can be differentiated into many tissues such as bone, cartilage, and fat, and there are sources that are available in relatively large quantities like adipose tissue. Is the most advanced application to regenerative treatment.
  • mesenchymal stem cells are collected mainly from bone marrow, fetal appendages or adipose tissue, but they are collected as part of a very diverse cell population, so progenitor cells that have already started to differentiate in a specific direction Are also included, and there are large variations from lot to lot.
  • a membrane fraction is prepared from human adipose-derived mesenchymal stem cells, and after fluorescent labeling, it is subjected to a high-density lectin array, and has high proliferation potential as well as high differentiation potential into osteoblasts and adipocytes.
  • a lectin having a markedly different binding property between cells in early passages and cells in late passages in which proliferative ability was reduced and differentiation potential was lost was statistically extracted.
  • ADSC P3 human fat-derived mesenchymal stem cells
  • ADSC P26 human fat-derived mesenchymal stem cells
  • Fibroblast human skin fibroblasts
  • the sugar chain was excised from the protein fractions of these human iPS cells (strain 201B7), ADSC ⁇ P3, ADSC P26, and human skin fibroblasts (Fibroblast) by hydrazine degradation, converted to 2-pyridylamino (PA), then Mono-Q
  • the glycans were fractionated according to the acidity of the glycans by ion exchange chromatography using a column.
  • cartilage tissue-derived chondrocyte (Yub621c strain), which is a type of cartilage stem cell, is subcultured.
  • the differentiation potential of osteoblasts and adipocytes in the early and late passages was analyzed, and the reactivity of the above four lectins in flow cytometry was analyzed.
  • the same tendency as in the case of adipose tissue-derived mesenchymal stem cells was observed in the cartilage stem cells.
  • a polychondrogenic cartilage tissue-derived chondrocyte in order to clearly distinguish a polychondrogenic cartilage tissue-derived chondrocyte from a differentiated chondrocyte, it may be referred to as a polydactyly-derived cartilage stem cell.
  • ⁇ 2-6 sialic acid is an excellent cell surface sugar chain marker that serves as an indicator of the differentiation potential of somatic stem cells. The present invention has been completed by obtaining the above knowledge.
  • the present invention includes the following inventions.
  • Discrimination and evaluation of differentiation potential of somatic stem cells comprising the step of detecting ⁇ 2-6 sialic acid expressed on the surface of somatic stem cells or the step of measuring the amount of ⁇ 2-6 sialic acid how to.
  • ⁇ 2-6 sialic acid to be detected or measured in the present invention is a sugar chain present at the non-reducing end in the N-linked sugar chain of glycoprotein expressed on the surface of somatic stem cells. Therefore, it can be expressed as follows.
  • [1 '] Includes the step of detecting ⁇ 2-6 sialic acid or measuring the amount of ⁇ 2-6 sialic acid present at the non-reducing end of the N-linked sugar chain of glycoprotein expressed on the surface of somatic stem cells A method for discriminating and evaluating the differentiation potential of somatic stem cells.
  • the somatic stem cells are mesenchymal stem cells or cartilage stem cells
  • the differentiation potential of the somatic stem cells is differentiation potential to osteoblasts or chondrocytes.
  • the mesenchymal stem cells are preferably bone marrow-derived mesenchymal stem cells or adipose-derived mesenchymal stem cells
  • the chondrocyte stem cells are preferably polydactyly chondrocyte-derived cartilage stem cells.
  • the step of detecting ⁇ 2-6 sialic acid or measuring the amount of ⁇ 2-6 sialic acid is performed using a probe that recognizes ⁇ 2-6 sialic acid as an epitope.
  • the probe that recognizes ⁇ 2-6 sialic acid as an epitope specifically refers to at least one protein selected from a lectin that recognizes ⁇ 2-6 sialic acid as an epitope and an antibody. Since a fragment in which the antigen recognition site of the antibody is conserved or a derivative of the antibody is also included, it can be expressed as follows.
  • [3 ′] The step of detecting ⁇ 2-6 sialic acid or measuring the amount of ⁇ 2-6 sialic acid is performed using at least one protein selected from a lectin and an antibody that recognize ⁇ 2-6 sialic acid as an epitope.
  • the method according to [1] or [2] above, wherein [3 ′′] The step of detecting or measuring ⁇ 2-6 sialic acid comprises a lectin that recognizes ⁇ 2-6 sialic acid as an epitope, an antibody that recognizes ⁇ 2-6 sialic acid as an epitope, and an antigen recognition site of the antibody.
  • the probe according to [3], wherein the probe that recognizes ⁇ 2-6 sialic acid as an epitope includes at least one lectin selected from TJAI lectin, SSA lectin, SNA lectin, and PSL1a lectin.
  • the above [3 ′] can be cited as follows.
  • the lectin that recognizes ⁇ 2-6 sialic acid as an epitope includes at least one lectin selected from TJAI lectin, SSA lectin, SNA lectin, and PSL1a lectin. ] The method of description.
  • [5] A step of detecting or measuring a glycoprotein expressed on the surface of a somatic stem cell and having ⁇ 2-6 sialic acid as a non-reducing terminal sugar chain using a probe that specifically recognizes the glycoprotein.
  • the glycoprotein is preferably a glycoprotein that is specifically and / or expressed in a large amount in the target somatic stem cells.
  • the probe that specifically recognizes the glycoprotein is at least one antibody selected from an anti-CD29 antibody and an anti-CD49e antibody.
  • the anti-CD29 antibody and the anti-CD49e antibody may be a polyclonal antibody, a monoclonal antibody, an antibody fragment such as a Fab fragment in which the antigen recognition site is conserved, a humanized antibody, a single chain antibody, or the like.
  • a method for discriminating or evaluating the differentiation potential of somatic stem cells into osteoblasts or chondrocytes comprising the following (1) and (2); (1) Overlaying a specimen stem cell-containing sample on a substrate on which either one of the following probes (a) or (b) is immobilized, and then allowing the other labeled probe to act , (A) at least one lectin selected from SSA lectin, SNA lectin, and PSL1a lectin, (B) at least one antibody selected from an anti-CD29 antibody and an anti-CD49e antibody, (2) A step of measuring the labeled amount.
  • the following method is included.
  • [8 ′] A method for discriminating or evaluating the differentiation potential of somatic stem cells into osteoblasts or chondrocytes, comprising the following (1) and (2): (1) Overlaying a specimen-containing stem cell-containing sample on a substrate on which the following (a) is immobilized, and then allowing (b) labeled with a fluorescent dye such as Cy3 to act; (A) at least one lectin selected from SSA lectin, SNA lectin, and PSL1a lectin, (B) at least one antibody selected from an anti-CD29 antibody and an anti-CD49e antibody, (2) A step of measuring the amount of fluorescent label.
  • a fluorescent dye such as Cy3
  • a reagent for determining or evaluating the differentiation potential of somatic stem cells comprising a probe that recognizes ⁇ 2-6 sialic acid as an epitope. Or it can be expressed as follows.
  • a reagent for determining or evaluating the differentiation potential of somatic stem cells comprising at least one protein selected from a lectin that recognizes ⁇ 2-6 sialic acid as an epitope and an antibody.
  • a kit for determining differentiation potential of somatic stem cells comprising a probe that recognizes ⁇ 2-6 sialic acid as an epitope. Or it can be expressed as follows.
  • a probe that recognizes the ⁇ 2-6 sialic acid as an epitope (A) at least one lectin that recognizes ⁇ 2-6 sialic acid as an epitope, and (b) at least one anti- ⁇ 2-6 sialic acid antibody that recognizes ⁇ 2-6 sialic acid as an epitope,
  • a probe that recognizes the ⁇ 2-6 sialic acid as an epitope (A) at least one lectin selected from TJAI lectin, SSA lectin, SNA lectin, and PSL1a lectin, and (b) at least one anti- ⁇ 2-6 sialic acid antibody that recognizes ⁇ 2-6 sialic acid as an epitope ,
  • the somatic stem cell differentiation potential determination kit comprises: (A) a probe that recognizes ⁇ 2-6 sialic acid as an epitope, and (b) a probe that specifically recognizes a glycoprotein expressed on the surface of a somatic stem cell and having ⁇ 2-6 sialic acid as a non-reducing terminal sugar chain ,
  • the kit according to [12] wherein either one of the probes (a) or (b) is immobilized on a substrate and the other probe is labeled. For example, the following cases are included.
  • the somatic stem cell differentiation potential determination kit comprises: (A) At least one lectin selected from TJAI lectin, SSA lectin, SNA lectin, and PSL1a lectin, and (b) expressed on the surface of somatic stem cells, and ⁇ 2-6 sialic acid as a non-reducing terminal sugar chain A probe that specifically recognizes the glycoprotein it has, The kit according to [12], wherein either one of the probes (a) or (b) is immobilized on a substrate and the other probe is labeled.
  • a kit for discriminating or evaluating the differentiation potential of somatic stem cells into osteoblasts or chondrocytes (A) at least one lectin selected from SSA lectin, SNA lectin, and PSL1a lectin, (B) at least one antibody selected from an anti-CD29 antibody and an anti-CD49e antibody, A kit in which either one of the probes (a) or (b) is immobilized on a substrate and the other probe is labeled. For example, the following cases are included.
  • a kit for discriminating or evaluating the differentiation potential of somatic stem cells into osteoblasts or chondrocytes comprising (a) and (b); (A) a substrate (for example, a lectin array) on which at least one lectin selected from SSA lectin, SNA lectin, and PSL1a lectin is immobilized; (B) At least one antibody selected from a labeled anti-CD29 antibody and an anti-CD49e antibody.
  • a kit for discriminating or evaluating the differentiation potential of somatic stem cells into osteoblasts or chondrocytes comprising (a) to (c); (A) a substrate on which at least one lectin selected from SSA lectin, SNA lectin, and PSL1a lectin is immobilized; (B) at least one antibody selected from a labeled anti-CD29 antibody and an anti-CD49e antibody, (C) A device for measuring the amount of label.
  • kits for discriminating or evaluating the differentiation potential of somatic stem cells into osteoblasts or chondrocytes comprising (a) to (d); (A) a streptavidin-coated carrier (for example, streptavidin-coated magnetic beads) on which at least one antibody selected from a biotin-labeled anti-CD29 antibody and an anti-CD49e antibody is immobilized, (B) a substrate on which at least one lectin selected from SSA lectin, SNA lectin, and PSL1a lectin is immobilized; (C) at least one antibody selected from a fluorescently labeled anti-CD29 antibody and an anti-CD49e antibody, (D) An apparatus for measuring the amount of fluorescent label.
  • a streptavidin-coated carrier for example, streptavidin-coated magnetic beads
  • a method for separating or concentrating somatic stem cells having a high differentiation potential from a cell sample containing somatic stem cells A method comprising a step of contacting a cell sample containing somatic stem cells with a probe that recognizes ⁇ 2-6 sialic acid as an epitope. Or it can be expressed as follows.
  • [16 ′] A method for separating or concentrating somatic stem cells having high differentiation potential from a cell sample containing somatic stem cells A method comprising contacting a cell sample containing somatic stem cells with at least one protein selected from a lectin that recognizes ⁇ 2-6 sialic acid as an epitope and an antibody.
  • a method for separating or concentrating somatic stem cells having high differentiation potential from a cell sample containing somatic stem cells (1) Mesenchymal stem cells or cartilage stem cells from a tissue or body fluid selected from a living body, adipose tissue, umbilical cord blood, umbilical cord, amniotic membrane, placenta, cartilage tissue, jaw bone or femur bone marrow, or polydactyly-derived bone marrow tissue Collecting a cell sample containing (2) A method comprising contacting a probe that recognizes ⁇ 2-6 sialic acid as an epitope.
  • [16 ′ ′′] Use of a probe that recognizes ⁇ 2-6 sialic acid as an epitope in a method for separating or enriching somatic stem cells having high differentiation potential from a cell sample containing somatic stem cells, The method is Use comprising the step of contacting a cell sample containing somatic stem cells with at least one protein selected from lectins and antibodies that recognize ⁇ 2-6 sialic acid as an epitope.
  • a probe that recognizes ⁇ 2-6 sialic acid as an epitope in a method for separating or enriching somatic stem cells having high differentiation potential from a cell sample containing somatic stem cells,
  • the method is Mesenchymal stem cell or chondrocyte-containing cell sample selected from adipose tissue, umbilical cord blood, umbilical cord, amniotic membrane, placenta, cartilage tissue, jaw bone or femur bone marrow, or polydactyly-derived bone marrow tissue isolated from a living body
  • the method comprises a step of contacting a probe that recognizes ⁇ 2-6 sialic acid as an epitope.
  • the step of contacting the probe that recognizes ⁇ 2-6 sialic acid as an epitope includes any one of a cell fractionation step by flow cytometry, or a separation step using a carrier on which the probe is immobilized.
  • the step of contacting at least one protein selected from a lectin that recognizes ⁇ 2-6 sialic acid as an epitope and an antibody is a cell fractionation step by flow cytometry, or a carrier on which the protein is immobilized
  • a method for separating or concentrating somatic stem cells having high differentiation potential into osteoblasts or chondrocytes from a cell sample containing somatic stem cells For cell samples containing somatic stem cells (1) A step of contacting a biotin-labeled anti-CD29 antibody and a streptavidin-coated carrier (for example, streptavidin-coated magnetic beads) to which at least one antibody selected from anti-CD49e antibodies is immobilized, (2) contacting at least one lectin selected from SSA lectin, SNA lectin, and PSL1a lectin; Including methods.
  • a biotin-labeled anti-CD29 antibody and a streptavidin-coated carrier for example, streptavidin-coated magnetic beads
  • a method for separating or concentrating mesenchymal stem cells having a high differentiation potential into osteoblasts or chondrocytes from a mesenchymal stem cell or chondrocyte-containing cell sample A method comprising the step of contacting the stem cell-containing sample with a probe that recognizes ⁇ 2-6 sialic acid as an epitope. Or it can be expressed as follows.
  • [21 ′] A method for separating or concentrating mesenchymal stem cells having high differentiation potential into osteoblasts or chondrocytes from a mesenchymal stem cell or chondrocyte-containing cell sample, A method comprising contacting the stem cell-containing sample with at least one protein selected from lectins and antibodies that recognize ⁇ 2-6 sialic acid as an epitope.
  • the mesenchymal stem cell or chondrocyte-containing cell sample is selected from a living body, adipose tissue, umbilical cord blood, umbilical cord, amniotic membrane, placenta, cartilage tissue, jaw bone or femur bone marrow, or polydactyly-derived bone marrow tissue.
  • a kit for isolating or concentrating somatic stem cells with high differentiation potential comprising a carrier on which a probe that recognizes ⁇ 2-6 sialic acid as an epitope is immobilized. Or it can be expressed as follows. [24 ′] Isolation of somatic stem cells having high differentiation potential, comprising a carrier on which at least one protein selected from a lectin that recognizes ⁇ 2-6 sialic acid as an epitope and an antibody is immobilized, or Concentration kit.
  • a kit for isolating or concentrating bone marrow-derived mesenchymal stem cells having high differentiation potential into osteoblasts comprising a carrier on which a probe that recognizes ⁇ 2-6 sialic acid as an epitope is immobilized. Or it can be expressed as follows. [25 ′] derived from bone marrow with high differentiation potential into osteoblasts, comprising a carrier on which at least one protein selected from a lectin that recognizes ⁇ 2-6 sialic acid as an epitope and an antibody is immobilized
  • a kit for isolating or concentrating mesenchymal stem cells comprising a carrier on which at least one protein selected from a lectin that recognizes ⁇ 2-6 sialic acid as an epitope and an antibody is immobilized.
  • a method of preparing a transplant material for osteoblast or chondrocyte proliferation using a mesenchymal stem cell or chondrocyte-containing cell sample comprising the following steps (1) to (5): (1) A step of expanding the explanted mesenchymal stem cell or chondrocyte-containing cell sample ex vivo, (2) contacting the cell sample obtained in step (1) with a carrier immobilized with a probe that recognizes ⁇ 2-6 sialic acid as an epitope; (3) a step of washing the immobilization support obtained in step (2) with a phosphate-containing buffer to remove non-specific binders, (4) a step of washing the immobilized carrier obtained in step (3) with a saccharide-containing buffer solution to release cells bound to the probe from the immobilized carrier; (5) A step of collecting the cells obtained in step (4) and preparing a transplant material.
  • a method for preparing a transplant material for osteoblast or chondrocyte proliferation using a mesenchymal stem cell or chondrocyte-containing cell sample comprising the following steps (1) to (5) ; (1) a step of expanding the explanted mesenchymal stem cell-containing cell or cartilage stem cell sample ex vivo, (2)
  • the cell sample obtained in step (1) is at least one selected from an antibody that recognizes at least one lectin selected from SSA lectin, SNA lectin, and PSL1a lectin or ⁇ 2-6 sialic acid as an epitope.
  • a method for preparing a transplant material for osteoblast or chondrocyte proliferation using a mesenchymal stem cell or chondrocyte-containing cell sample comprising the following steps (1) to (6) Method; (1) a step of expanding the explanted mesenchymal stem cell-containing cell or cartilage stem cell sample ex vivo, (2) A step of concentrating the stem cells using the immune reaction of the cell sample obtained in step (1) with at least one antibody selected from anti-CD29 antibody and anti-CD49e antibody, (3) The cell sample concentrated in step (2) is at least one selected from an antibody that recognizes at least one lectin selected from SSA lectin, SNA lectin, and PSL1a lectin or ⁇ 2-6 sialic acid as an epitope.
  • ⁇ 2-6 sialic acid serves as a sugar chain marker indicating the differentiation potential of somatic stem cells such as mesenchymal stem cells.
  • Lectins or antibodies that specifically bind to ⁇ 2-6 sialic acid can be used as reagents for discriminating the differentiation potential of cells. Since the differentiation potential of somatic stem cells used for transplantation treatment can be evaluated in advance from the reactivity of the ⁇ 2-6 sialic acid-binding lectin of the present invention, application to regenerative medicine using somatic stem cells is expected.
  • mesenchymal stem cells or cartilage stem cells having a certain quality can be isolated from a diverse cell population including differentiation potential at each stage, such as bone marrow-derived mesenchymal stem cell-containing cultured cells, using flow cytometry, magnetic beads, etc. Can be separated and concentrated.
  • changes in the amount of ⁇ 2-6 sialic acid as the terminal sugar chain of CD29 and CD49e glycoproteins, which are surface antigens of somatic stem cells such as mesenchymal stem cells correlate with changes in differentiation potential to osteoblasts or chondrocytes Found it expensive.
  • somatic stem cells into osteoblasts or chondrocytes by using an assay system that combines anti-CD29 antibody and / or anti-CD49e antibody and ⁇ 2-6 sialic acid reactive lectin (SSA, SNA, rPSL1a) Can be accurately determined and evaluated.
  • assay system that combines anti-CD29 antibody and / or anti-CD49e antibody and ⁇ 2-6 sialic acid reactive lectin (SSA, SNA, rPSL1a)can be accurately determined and evaluated.
  • ADSC adipose-derived mesenchymal stem cells
  • (a) shows no differentiation induction
  • (b) and (c) and (d) show cells induced to differentiate into osteoblasts.
  • (b) is a control without sialidase treatment
  • (c) is a case where differentiation induction treatment is performed after sialidase treatment
  • (d) is a case where differentiation induction is performed in the presence of sialidase.
  • ⁇ 2-6 sialic acid is generally a sugar chain in which N-acetylneuraminic acid is ⁇ 2-6 linked to a hydroxyl group at the 6-position of galactose. Similarly, it refers to both “Neu5Ac ⁇ 2-6Gal” and the sugar chain “Neu5Gc ⁇ 2-6Gal” in which N-glycolyl-type neuraminic acid is similarly ⁇ 2-6 linked.
  • ⁇ 2-6 sialic acid is produced in the body only in the case of ⁇ Neu5Ac ⁇ 2-6Gal '' in which the amino group at position 5 of neuraminic acid is acetylated. Since it is not synthesized, the present invention mainly targets only “Neu5Ac ⁇ 2-6Gal”. In the following description, ⁇ 2-6 sialic acid is described as “Neu5Ac ⁇ 2-6Gal”.
  • ⁇ 2-6 sialic acid (Neu5Ac ⁇ 2-6Gal), which is typically present at the non-reducing end of the glycoprotein on the cell surface of human somatic stem cells such as human mesenchymal stem cells, is differentiated from stem cells.
  • a sugar chain marker for potential evaluation and determination that is, an epitope to be detected.
  • the target stem cells are derived from mammals other than humans includes “Neu5Gc ⁇ 2-6Gal” as ⁇ 2-6 sialic acid, so that “Neu5Ac ⁇ 2-6Gal” And “Neu5Gc ⁇ 2-6Gal” is the epitope to be detected for determining and evaluating the differentiation potential of the present invention.
  • Sialic acid (N-acetylneuraminic acid) is an acidic sugar with a carboxyl group that exists at the end of a complex sugar chain on the cell surface.
  • ⁇ 2-6 sialic acid of the type bonded to and ⁇ 2-3 sialic acid of the type bonded to the hydroxyl group at the 3-position As a special glycoprotein or glycolipid, ⁇ 2-8 sialic acid of the type bonded to the hydroxyl group at the 8-position of sialic acid is also known.
  • Sialic acid is present at the non-reducing end of sugar chains of secreted glycoproteins such as mucin and serum proteins, and membrane proteins.
  • Non-Patent Document 4 Non-Patent Document 4
  • Non-patent Document 5 sialic acid residues on the airway epithelial cells are ⁇ 2-6 sialic acid in humans compared to ⁇ 2-3 sialic acid in birds, and both ⁇ 2-6 sialic acid and ⁇ 2-3 sialic acid in pigs. Expression is a rare reason that avian influenza directly infects humans and is usually the reason for infection via swine.
  • somatic stem cells such as mesenchymal stem cells before differentiation induction
  • sugar chain structure change in cell surface complex sugar chains For the first time in the present invention, it has been found that the expression level of non-reducing end ⁇ 2-6 sialic acid-containing sugar chains in complex sugar chains on the surface of somatic stem cells such as mesenchymal stem cells has a correlation with the differentiation potential of the cells.
  • ⁇ 2-6 sialic acid was found to be an excellent indicator of the degree of differentiation potential of somatic stem cells.
  • the differentiation potential of somatic stem cells can be determined by quantitatively measuring the expression level of ⁇ 2-6 sialic acid on the surface of somatic stem cells such as mesenchymal stem cells using an ⁇ 2-6 sialic acid-binding probe. It became possible to do.
  • the expression level of ⁇ 2-6 sialic acid on the surface of bone marrow-derived mesenchymal stem cells is particularly correlated with the differentiation potential of osteoblasts or chondrocytes among the differentiation potentials of various cells. It has been found that ⁇ 2-6 sialic acid can be used as a marker for evaluation and determination of differentiation potential into osteoblasts or chondrocytes.
  • ⁇ 2-6 sialic acid binding probe (2-1) ⁇ 2-6 sialic acid binding probe
  • ⁇ 2-6 sialic acid is specifically detected in order to detect ⁇ 2-6 sialic acid on the surface of somatic stem cells.
  • a probe that recognizes as an epitope is used.
  • Proteins that recognize and bind to a specific sugar chain structure are collectively called “lectins”, and typical ⁇ 2-6 sialic acid-binding probes include various ⁇ 2-6 sialic acid-binding lectins.
  • the present invention is not limited thereto, and an anti- ⁇ 2-6 sialic acid antibody or a derivative thereof that recognizes ⁇ 2-6 sialic acid as a sugar chain antigen (epitope) is also preferably used.
  • ⁇ 2-6 sialic acid can be obtained by immunizing an animal as it is or bound to a carrier protein such as albumin or KLH.
  • a 6-sialic acid antibody (Non-patent Document 5) can also be used.
  • the antibody is not particularly limited as long as it has the ability to specifically recognize and bind to ⁇ 2-6 sialic acid as an epitope.
  • Polyclonal antibody, monoclonal antibody, and Fab fragment in which the antigen recognition site is conserved In addition to antibody fragments such as these, humanized antibodies, single chain antibodies, and the like can also be used.
  • anti- ⁇ 2-6 sialic acid antibody or “antibody recognizing ⁇ 2-6 sialic acid as an epitope” or the like, not only the antibody but also a fragment or derivative in which the antigen recognition site is conserved, etc. Used as an included term.
  • the ⁇ 2-6 sialic acid binding probe of the present invention may be used alone, or a plurality of probes may be used in combination.
  • a plurality of types of ⁇ 2-6 sialic acid-binding lectins or further anti- ⁇ 2-6 sialic acid antibodies can be used in combination.
  • the ⁇ 2-6 sialic acid binding lectin used as the ⁇ 2-6 sialic acid binding probe of the present invention is a non-reducing end of a sugar chain of a glycoprotein on the cell surface. Any lectin may be used as long as it can recognize ⁇ 2-6 sialic acid.
  • TJAI lectin Trichosanthes japonica lectin-I
  • SSA lectin Sudbucus sieboldiana lectin
  • SNA lectin Sambucus nigra lectin (SNA)
  • PSL1a lectin Polyporus squamosus lectin
  • TJAI lectin can be extracted from Kikarasuuri
  • SSA lectin can be extracted from Japanese elderberry
  • SNA lectin can be extracted from elderberry
  • PSL1a lectin can be extracted from Ahihiratake
  • rPSL1a lectin that retains ⁇ 2-6 sialic acid specificity is commercially available from Wako Pure Chemical Industries. Both lectins are known to specifically recognize ⁇ 2-6 sialic acid (Neu5Ac ⁇ 2-6Gal and Neu5Gc ⁇ 2-6Gal) constituting the non-reducing end of glycoconjugates on the cell surface.
  • FIG. 5 shows the results of frontal affinity chromatography (FAC) analysis of the reactivity of each of these four lectins (TJAI, SSA, SNA, rPSL1a) to various complex sugar chains.
  • FAC frontal affinity chromatography
  • the above four lectins are lectins having binding specificity to ⁇ 2-6 sialic acid residues.
  • ⁇ 2-6 sialic acid-specific lectins other than these four types of lectins include lacanka lectins extracted from Arachnaceae plants (Patent Document 8).
  • it can be obtained according to information from the lectin frontier database (LfDB) or the like. It is also possible to screen from natural or artificial protein samples using a sugar chain array containing ⁇ 2-6 sialic acid.
  • ⁇ 2-6 sialic acid-containing glycoprotein ⁇ 2-6 sialic acid on the surface of the somatic stem cell to be detected in the present invention binds to asparagine in the extracellular domain of the glycoprotein present on the surface of the somatic stem cell.
  • N-linked sugar chain N-linked sugar chain
  • ⁇ 2-6 sialic acid may be added to the non-reducing end in the N-type sugar chain, increasing the content of ⁇ 2-6 sialic acid.
  • a body related to the core protein of N-linked glycoprotein ( ⁇ 2-6 sialic acid-containing N-linked glycoprotein) having ⁇ 2-6 sialic acid at the non-reducing end as an index for judging and evaluating the differentiation potential of stem cells The expression level on the surface of sex stem cells can be used.
  • the core protein of the ⁇ 2-6 sialic acid-containing N-linked glycoprotein can be easily determined by those skilled in the art because it can identify the glycoprotein collected using ⁇ 2-6 sialic acid as an index.
  • the membrane fraction or protein fraction of somatic stem cells in the early passage is enriched with ⁇ 2-6 sialic acid-containing glycoproteins using beads immobilized with at least one of TJAI, SSA, SNA, and rPSL1a lectin It can be easily determined by detecting with an antibody against the core protein or by examining the amino acid sequence with a mass spectrometer or the like.
  • Such a core protein of a glycoprotein having ⁇ 2-6 sialic acid at the non-reducing end of the N-linked sugar chain may also be a marker for determining and evaluating the differentiation potential of somatic stem cells. That is, in that case, the differentiation potential of somatic stem cells can be determined and evaluated by measuring the expression level of the core protein at the mRNA level or at the protein level by a known method. Specifically, for example, the core protein mRNA level is measured by quantitative RT-PCR, the measurement is performed by a next-generation sequencer, the measurement is performed by a DNA microarray, and a core protein-specific antibody is prepared or purchased. ELISA, flow cytometry, Western blot, immunostaining, or sandwich assay using antibody overlay lectin microarrays.
  • (2-4) Glycoprotein for determining differentiation potential into osteoblasts or chondrocytes In order to identify a core protein for determining differentiation potential, the present inventors have expressed ⁇ 2- that is highly expressed on the surface of mesenchymal stem cells. 6-sialic acid-containing N-linked glycoprotein antigens were searched and CD29, CD49e and CD13 glycoproteins were selected as candidates. Next, prepare cells with different passage numbers of bone marrow-derived mesenchymal stem cells and cartilage stem cells, and immobilize each hydrophobic fraction (membrane fraction) with anti-CD29 antibody, anti-CD49e antibody and anti-CD13 antibody. Were immunoprecipitated with the prepared beads.
  • CD29, CD49e and CD13 glycoproteins were subjected to gel electrophoresis, and changes in the amount of reaction with rPSL1a lectin according to different passage numbers were observed.
  • rPSL1a lectin changes in the amount of reaction with rPSL1a lectin according to different passage numbers were observed.
  • CD29 and CD49e glycoproteins were used to measure ⁇ 2-6 sialic acid content. The effectiveness as a core protein was judged to be high.
  • the amount of ⁇ 2-6 sialic acid possessed by CD29 and CD49e on the surface of bone marrow-derived mesenchymal stem cells and cartilage stem cells reflects the degree of differentiation potential to osteoblasts or chondrocytes that decreases with successive passages. In other words, it means a decrease with high correlation. Differentiating into osteoblasts or chondrocytes more quantitatively by simultaneously measuring the expression level of CD29 and CD49e on bone marrow-derived mesenchymal stem cells and cartilage stem cells and ⁇ 2-6 sialic acid contained in CD29 and CD49e The potential can be determined. Both CD29 and CD49e belong to the integrin family involved in signaling through cell membranes in addition to cell adhesion. CD29 is the integrin ⁇ 1 chain and CD49e is the integrin ⁇ 5 chain.
  • SSA, SNA and rPSL1a were found to be lectins that more accurately reflect the decreasing rate of the expression level of ⁇ 2-6 sialic acid on CD29 and CD49e. That is, it was found that SSA, SNA and rPSL1a are suitable as lectins used in an assay system using an anti-CD29 antibody or an anti-CD49e antibody.
  • the anti-CD29 antibody and the anti-CD49e antibody are not particularly limited as long as they have the ability to recognize and specifically bind CD29 and CD49e, or antigenic fragments thereof, respectively, as polyclonal antibodies, In addition to monoclonal antibodies and antibody fragments such as Fab fragments in which the antigen recognition site is conserved, humanized antibodies, single chain antibodies, and the like can also be used.
  • Anti-CD29 antibody and anti-CD49e antibody are commercially available from Abcam, R & D, Beckman Coulter, and the like.
  • test stem cells to be examined and their collection sources are mainly somatic stem cells or cells obtained by subculturing the somatic stem cells. It can also be applied to stem cells (ES cells) and stem cells (iPS cells, etc.) that have been dedifferentiated by introducing genes into somatic cells.
  • the somatic stem cells include various somatic stem cells such as neural stem cells, epithelial stem cells, hepatic stem cells, reproductive stem cells, hematopoietic stem cells, mesenchymal stem cells, cartilage stem cells, skeletal muscle stem cells, etc. Stem cells and cartilage stem cells are preferred.
  • the differentiation potential can be determined and evaluated not only in cloned somatic stem cells but also in the state of a culture of a living tissue consisting of a heterogeneous cell population from which somatic stem cells are collected.
  • a culture containing somatic stem cells is also included. That is, when the subject stem cell-containing sample is referred to in the present invention, the somatic stem cell contained in the sample is a cell isolated from a living body, a primary culture or a subculture thereof, or an established cultured cell line. It is.
  • Mesenchymal stem cells can be collected from liposuctioned adipose tissue, umbilical cord blood, umbilical cord, amniotic membrane, placenta, or bone marrow puncture from jawbone or femur-derived bone marrow.
  • mesenchymal stem cells are also commercially available.
  • 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.
  • MesenPRO RS TM medium (Life Technologies) generally used as a mesenchymal stem cell maintenance medium can be appropriately used.
  • bone marrow-derived mesenchymal stem cells can be obtained from the bone marrow tissue of the fingers excised by surgery for patients with polydactyly, and cartilage tissue-derived chondrocytes (polydactyly derived cartilage stem cells) can be obtained from the cartilage tissue. it can. Also available from RIKEN BioResource Center, JSRB Cell Bank, etc.
  • cartilage stem cell maintenance medium As the cartilage stem cell maintenance medium, MesenPRO RS TM ground (Life Technologies), which is generally used as a mesenchymal stem cell maintenance medium, may be used, but chondrocyte basic medium, chondrocyte growth medium (Takara Bio), etc. A maintenance medium for cartilage stem cells can also be used.
  • the present invention can be used not only to determine the differentiation potential of stem cells collected from a body tissue, but also to determine the differentiation potential after expanded culture. It can also be used to isolate and concentrate cells with high differentiation potential.
  • stem cells are considered to be controlled by a common mechanism in mammals, not limited to humans, the stem cells of the present invention include mammals other than humans, such as monkeys, pigs, cows, goats, sheep. It can also be applied when using stem cells derived from mice or rats.
  • the present invention relates to somatic stem cells such as somatic stem cell-containing tissue obtained from a living body.
  • the present invention relates to a method for determining or evaluating the differentiation potential of a somatic stem cell sample by specifically detecting (in vitro) ⁇ 2-6 sialic acid expressed on the cell surface of the somatic stem cell in a sample or somatic stem cell culture . And the method of performing quality control of a somatic stem cell sample according to the determination result is also included.
  • Mass spectrometry, liquid chromatography, and MALDI-TOF MS can be used for detection of ⁇ 2-6 sialic acid on the surface of stem cells and measurement of expression level, but the method using ⁇ 2-6 sialic acid binding probe is the cell surface. It is most suitable for detecting ⁇ 2-6 sialic acid at the non-reducing end of glycoconjugates.
  • ⁇ 2-6 sialic acid binding probe is the cell surface. It is most suitable for detecting ⁇ 2-6 sialic acid at the non-reducing end of glycoconjugates.
  • the ⁇ 2-6 sialic acid-binding lectin can be washed by washing the stem cells with a saccharide-containing buffer such as galactose or lactose. Can be liberated.
  • the amount of label bound to ⁇ 2-6 sialic acid on the stem cell surface can be accurately measured even in somatic stem cell culture media containing other cells. Therefore, it is possible to easily evaluate and determine the differentiation potential of stem cells.
  • labeling probes such as lectins, fluorescent labels such as R-Phycoerythrin (PE) and FITC, enzyme labels such as peroxidase, labels with biotin (+ HRP-labeled avidin), and the like can be used.
  • PE R-Phycoerythrin
  • enzyme labels such as peroxidase
  • the ratio of ⁇ 2-6 sialic acid to ⁇ 2-3 sialic acid is also an indicator of differentiation potential.
  • the differentiation potential of the specimen stem cell sample can also be increased by desorbing ⁇ 2-6 sialic acid and ⁇ 2-3 sialic acid using specific sialidase and ⁇ 2-6 sialic acid specific sialidase and measuring the ratio. Can be determined.
  • CD29 and CD49e in experiments using adipose-derived mesenchymal stem cells and cartilage stem cells, the amount of ⁇ 2-6 sialic acid possessed by CD29 and CD49e on the surface of somatic stem cells decreases with successive passages, that is, CD29 And CD49e was found to be the core protein of ⁇ 2-6 sialic acid in somatic stem cells that correlates with reduced differentiation potential. It was also found that SSA, SNA, and rPSL1a lectin serve as probes that more accurately reflect the rate of decrease in ⁇ 2-6 sialic acid expression on CD29 and CD49e.
  • an anti-CD29 antibody or an anti-CD49e antibody Concentration / isolation method using a carrier immobilized with ⁇ 2-6 sialic acid reactive lectin (SSA, SNA, rPSL1a, etc.) or anti- ⁇ 2-6 sialic acid antibody. Can be applied.
  • a sandwich assay system using an anti-CD29 antibody or an anti-CD49e antibody together with an ⁇ 2-6 sialic acid reactive lectin (SSA, SNA, rPSL1a, etc.) or an anti- ⁇ 2-6 sialic acid antibody for example, a subject stem cell stem cell
  • SSA ⁇ 2-6 sialic acid reactive lectin
  • SNA SNA
  • rPSL1a anti- ⁇ 2-6 sialic acid antibody
  • the sample is applied to a substrate containing SSA, SNA, rPSL1a, etc., and the labeling intensity is measured with a labeled anti-CD29 antibody or anti-CD49e antibody.
  • the differentiation potential into osteoblasts or chondrocytes can be accurately evaluated and determined.
  • the cell population used for such evaluation and determination is preferably a population containing 1 ⁇ 10 4 or more somatic stem cells, and more preferably 1 ⁇ 10 5 .
  • the reference fluorescence intensity is appropriately set according to the type of the subject stem cell and the fluorescent dye used.
  • a protein (glycoprotein) is prepared from a stem cell-containing sample, labeled with a fluorescent dye, and then subjected to a lectin array containing at least one ⁇ 2-6 sialic acid-binding lectin, and is evanescent wave excited fluorescence type The fluorescence intensity is measured with a detection system.
  • a protein is prepared from a stem cell-containing sample, applied to a lectin array containing at least one ⁇ 2-6 sialic acid-binding lectin, and overlaid with a labeled antibody or labeled lectin that recognizes the protein or sugar chain, and evanescent waves The fluorescence intensity is measured with an excitation fluorescence type detection system.
  • E A protein prepared from a stem cell-containing sample, subjected to a plate on which at least one ⁇ 2-6 sialic acid-binding lectin is immobilized, and overlaid with a labeled antibody or labeled lectin that recognizes the protein or sugar chain, Measure absorbance, fluorescence intensity, and luminescence with a reader.
  • the lectin blotting method is applied. After SDS-PAGE, transfer to a membrane such as a nitrocellulose membrane or PVDF membrane, or blot directly on the membrane, and then react with a labeled ⁇ 2-6 sialic acid-binding lectin. For example, when a biotin-labeled lectin is used, color development by an avidin reaction with an HRP-labeled avidin solution is observed after washing with a blocking buffer.
  • the present invention relates to a somatic stem cell sample or somatic stem cell such as a somatic stem cell-containing tissue obtained from a living body. By contacting the culture with a lectin or antibody that specifically recognizes ⁇ 2-6 sialic acid expressed on the cell surface of somatic stem cells, that is, an ⁇ 2-6 sialic acid-binding probe, a body with high differentiation potential
  • the present invention relates to a method for isolating or enriching sex stem cells.
  • the method for isolating and concentrating stem cells with high differentiation potential using an ⁇ 2-6 sialic acid-binding probe is not particularly limited, and general cell separation methods can be applied.
  • cell fractionation by flow cytometry cell fractionation using a carrier for immobilization such as magnetic beads on which lectin or antibody is immobilized, and affinity chromatography such as lectin column chromatography on which lectin or antibody is immobilized
  • a photographic separation method can be used.
  • these methods see, for example, 1996, published by Shujunsha, Glycobiology Experimental Protocol, Cell Engineering Separate Volume, Experimental Protocol Series and 1999, published by Ishiyaku Shuppan Publishing Co., Ltd. It is described in the electrophoresis experiment method.
  • the ⁇ 2-6 sialic acid-binding probe of the present invention When isolating and concentrating stem cells with high differentiation potential using the ⁇ 2-6 sialic acid-binding probe of the present invention, it is a lectin that does not inhibit stem cell growth and differentiation induction even if it remains in the stem cell culture. In addition, it is preferable to use a lectin that has extremely low toxicity to the human body even if it remains in cells for living transplantation, that is, a lectin that has no or negligible cytotoxicity. It is more preferable to employ an isolation / concentration method in which direct labeling with a fluorescent dye or the like is not performed.
  • a stem cell with high differentiation potential can be isolated and concentrated by isolating and concentrating the cells having high reactivity with the ⁇ 2-6 sialic acid-binding probe by flow cytometry. Specifically, for example, after a fluorescently labeled ⁇ 2-6 sialic acid-binding probe is reacted with a sample containing an analyte stem cell for about 1 hour at 4 ° C., unbound cells are washed away with a phosphate buffer or the like. Next, after collecting cells with high fluorescence intensity, the ⁇ 2-6 sialic acid binding probe fluorescently labeled with a buffer containing galactose or lactose is removed.
  • stem cells with high differentiation potential expressing ⁇ 2-6 sialic acid can also be isolated and concentrated by using magnetic beads, affinity columns or the like on which ⁇ 2-6 sialic acid-binding probe is immobilized.
  • an ⁇ 2-6 sialic acid-binding probe is immobilized on a magnetic bead, reacted with an analyte stem cell-containing sample at 4 ° C. for about 1 hour, and then washed with a phosphate buffer. After collecting the magnetic beads using a magnet, the cells are detached from the magnetic beads using a buffer containing galactose or lactose.
  • the somatic stem cells isolated and concentrated in this manner can be promptly subjected to differentiation differentiation into desired cells by being subjected to a known differentiation induction medium.
  • the cells after differentiation induction or the cells after the isolation and concentration can be transplanted as cells for regenerative medicine.
  • what is necessary is just to apply a known method for the preparation method of transplant material.
  • the mesenchymal stem cell-containing cell group collected from the bone marrow can be evaluated or judged as it is, or before transplantation after ex vivo expansion culture, the differentiation potential of the culture to osteoblasts.
  • a part of the culture containing preferably 1 ⁇ 10 4 or more mesenchymal stem cells is collected and ⁇ 2-6 on the surface of mesenchymal stem cells is obtained by the method described in the above (4-2).
  • the amount of sialic acid is measured, and the differentiation potential into osteoblasts is evaluated and judged. For example, when the average fluorescence intensity obtained by flow cytometry is high, it is determined that the cell group has a high differentiation potential into osteoblasts, and whether to perform transplantation is determined according to the determination result.
  • the isolation and concentration method of (5-1) above to a mesenchymal stem cell-containing cell group collected from bone marrow, only stem cells with high differentiation potential into osteoblasts are isolated and concentrated. can do.
  • Differentiation Potential Determination Kit As described in (2-4) above, in the present invention, the amount of ⁇ 2-6 sialic acid possessed by CD29 and CD49e on the surface of mesenchymal stem cells or cartilage stem cells is Or it discovered that it had a high correlation with the grade of the differentiation potential to a chondrocyte etc. Further, among the ⁇ 2-6 sialic acid-reactive lectins, the lectins that can determine changes in the amount of ⁇ 2-6 sialic acid on CD29 and CD49e with higher correlation are SSA, SNA, and rPSL1a. I found out.
  • anti-CD29 antibody and / or anti-CD49e antibody and ⁇ 2-6 sialic acid are used as an assay system for more accurately determining and evaluating the differentiation potential in subject stem cells such as test mesenchymal stem cells or cartilage stem cells.
  • a sandwich assay system for determining differentiation potential was established by combining reactive lectins (SSA, SNA, rPSL1a).
  • anti-CD29 antibody after reacting the fraction enriched with anti-CD29 antibody or anti-CD49e antibody to a substrate on which ⁇ 2-6 sialic acid-reactive lectin (SSA, SNA, rPSL1a) is immobilized, anti-CD29 antibody
  • SSA ⁇ 2-6 sialic acid-reactive lectin
  • rPSL1a ⁇ 2-6 sialic acid-reactive lectin
  • detection and quantification are possible by combining a substrate with an anti-CD29 antibody or anti-CD49e antibody immobilized thereon and a labeled ⁇ 2-6 sialic acid reactive lectin (SSA, SNA, rPSL1a) or antibody.
  • the somatic stem cell differentiation potential determination kit is a kit containing a probe that recognizes at least ⁇ 2-6 sialic acid as an epitope, specifically, (A) a lectin that recognizes ⁇ 2-6 sialic acid as an epitope (TJAI, SSA, SNA, rPSL1a, etc.), and (b) an anti- ⁇ 2-6 sialic acid antibody that recognizes ⁇ 2-6 sialic acid as an epitope, It may be a kit including a combination.
  • (A) a probe that recognizes ⁇ 2-6 sialic acid as an epitope, and (b) a probe that has ⁇ 2-6 sialic acid as a non-reducing terminal sugar chain and recognizes a glycoprotein expressed in a target stem cell It is preferable that one of (a) and (b) is immobilized on a substrate and the other is labeled.
  • somatic stem cells preferably mesenchymal stem cells or cartilage stem cells as target cells
  • kits refers to a case where reagents mainly composed of respective probes are combined.
  • a measuring device may be included.
  • a standard strain of the same somatic stem cells as the target somatic stem cell-containing sample is used.
  • a calibration curve for the degree of differentiation potential according to the expression level of ⁇ 2-6 sialic acid it is possible to quantitatively evaluate the sample containing test stem cells. Specifically, a standard strain is subcultured, and a part of cells of a plurality of subcultures during subculture is collected, and labeling strength is obtained using the sandwich assay kit comprising (a) and (b) above. Measure.
  • the passage cells are induced to differentiate into the differentiated cells of interest, and numerical values indicating the differentiation potential such as differentiated cell marker strength are measured, and a calibration curve is obtained from both values.
  • a calibration curve is obtained from both values.
  • Example 1 Subculture of adipose-derived mesenchymal stem cells
  • Adipose-derived mesenchymal stem cells (ADSC, Life Technologies, Lot #: 2118) were added to MesenPRO RS TM medium (Life Technologies) (FIG. 1).
  • PD indicates the division index.
  • FIG. 2 the differentiation potential of cells in the early passage (P5) and late passage (P28) into osteoblasts and adipocytes was examined (FIG. 2).
  • the number of passages varies depending on the cell type and culture conditions.
  • the period when the cell growth curve rises linearly in the early stage of stem cell culture is called “early passage”, and the period when the cell growth curve continues or becomes gentle or flat becomes “late stage”.
  • a portion of cells from the early passage (P5) and a portion of cells from the late passage (P28) of the adipose-derived mesenchymal stem cells were taken out, and differentiation induction into osteoblasts and adipocytes was performed. Differentiation into osteoblasts was performed using hMSC differentiation BulletKit-osteogenic (Cat #: PT-3002, Lonza), and adipocyte differentiation was performed using hMSC differentiation BulletKit-adipogenic (Cat #: PT-3004, Lonza).
  • Example 2 Extraction of lectins having significantly different binding properties at the early passage and late passage of adipose-derived mesenchymal stem cells.
  • the sugar chain analysis by a high-density lectin array was performed.
  • Membrane fractions were prepared from human adipose-derived mesenchymal stem cells, labeled with fluorescence, subjected to a high-density lectin array, and fluorescence intensity was measured with an evanescent wave excitation fluorescence detection system.
  • FIG. 3 shows the mean value and standard deviation of the fluorescence signal intensity of each lectin for early passage cells (P2-5; black bars) and late passage cells (P25-29; white bars). Moreover, the graph which showed the average value of each lectin in each passage of a human adipose origin mesenchymal stem cell is shown in FIG. It was found that the fluorescence signals of these four lectins gradually decreased with each passage number (FIG. 4).
  • FIG. 5 shows the reactivity of each of these four lectins to various complex sugar chains. Each lectin specifically binds only to sugar chains 501 to 506 having an ⁇ 2-6 sialic acid residue.
  • sialic acid residues it has no binding property to sugar chains 601 and 602 containing only ⁇ 2-3 sialic acid residues, and has ⁇ 2-6 sialic acid residues together with ⁇ 2-3 sialic acid. It can be seen that the sugar chain 506 has binding properties. From this, it can be said that the above four lectins are lectins having binding specificity to ⁇ 2-6 sialic acid residues. On the other hand, there are many ⁇ 2-6 sialic acid residues on the surface of human adipose-derived mesenchymal stem cells in the early passage, where lectins having binding specificity to these ⁇ 2-6 sialic acid residues exhibit remarkable specific binding.
  • ⁇ 2-6 sialic acid residues decreases dramatically as it is present and later in passage.
  • a decrease in proliferation potential as well as a decrease in differentiation potential were observed, so ⁇ 2-6 sialic acid residues on the surface of mesenchymal stem cells were It was suggested that it may be a sugar chain marker indicating the differentiation potential of leaf stem cells.
  • Example 3 Verification of reactivity of ⁇ 2-6 sialic acid-binding lectin to adipose-derived mesenchymal stem cells by flow cytometry The same human adipose-derived mesenchymal stem cells as used in Example 1 were subcultured in the same manner.
  • FIG. 7 shows a graph comparing the average fluorescence intensity obtained by flow cytometry with human iPS cells (201B7 strain) and human skin fibroblasts (Fibroblast, ATCC).
  • the four lectins were highly reactive against ADSC P3, which has differentiation potential, whereas ADSC P26 and human skin fibroblasts (Fibroblast), which have no differentiation potential, were almost reactive. There wasn't.
  • the reactivity of four lectins against pluripotent human iPS cells (201B7 strain, RIKEN BioResource Center), which are considered to have the highest differentiation potential, was 2 to 4 times that of ADSC P3. High reactivity was shown.
  • the above results also suggested that the reactivity of the four lectins TJAI, SSA, SNA, and rPSL1a is highly correlated with the differentiation potential of the cells.
  • Example 4 Sialic Acid Binding Modes of Glycoprotein Sugar Chains Expressed in Various Cells Human adipose-derived mesenchymal stem cells (ADSC P3, early passage (P3) and late passage (P26) used in Example 3 ADSC P26) and gas phase hydrazine degradation method applied to human iPS cells (201B7 strain) and human skin fibroblasts (Fibroblast) to cut out glycoprotein sugar chains and fractionate them according to the acidity of the sugar chains did.
  • ADSC P3, early passage (P3) and late passage (P26) used in Example 3 ADSC P26
  • gas phase hydrazine degradation method applied to human iPS cells 201B7 strain
  • human skin fibroblasts fibroblast
  • the glycan was excised by treatment with hydraclub Y2100 (J-Oil Mills) using anhydrous hydrazine at 100 ° C for 4 hours, and after the reaction, anhydrous hydrazine was dried under reduced pressure.
  • the free sugar chain was N-acetylated, desalted with Dowex 50WX2, and lyophilized.
  • the free sugar chain is converted to 2-pyridylamino (PA) with GlycoTAG (Takara Bio), and the PA-modified sugar chain is subjected to ion exchange chromatography using a Mono-Q column (GE), depending on the acidity of the sugar chain.
  • the sugar chain was fractionated.
  • the fraction with one sialic acid added is designated as A1, and the fraction with two sialic acids added is designated as A4.
  • A1 The fraction with one sialic acid added
  • A2 the fraction with two sialic acids added
  • A4 the fraction with two sialic acids added
  • ⁇ 2-3 sialic acid-specific sialidase derived from Salmonella typhimurimum LT2 (Takara Bio) Perfringens sialidase (Merck) was reacted to calculate the ratio of ⁇ 2-3 sialic acid and ⁇ 2-6 sialic acid.
  • the proportion of ⁇ 2-6 sialic acid (black bar) in the A1 fraction was 83% for human iPS cells, 25% for ADSC P3, and 0% for ADSC P28 and Fibroblast.
  • the sugar chains containing two ⁇ 2-6 sialic acids (black bars) and one ⁇ 2-6 sialic acid (black dots) are 58% and 15% in human iPS cells and 27% in ADSC P3, respectively.
  • the ratio of ⁇ 2-6 sialic acid was highest in human iPS cells, and was hardly confirmed in ADSC P3, ADSC P28 and Fibroblast. From the above results, it was found that the ratio of ⁇ 2-6 sialic acid is a good index for determining the height of cell differentiation potential.
  • Example 5 Proliferation ability, differentiation potential, and reactivity of ⁇ 2-6 sialic acid-binding lectin in subcultured early cells and late cells of polydactyly cartilage tissue- derived chondrocytes (Yub621c strain) Verification was performed using somatic stem cells derived from tissues other than adipose tissue-derived mesenchymal stem cells. Multi-deficient cartilage tissue-derived chondrocytes (Yub621c strain, RIKEN BioResource Center), a type of cartilage stem cell, are subcultured in the same manner as in Example 1 and the early passage (P7) to late passage (P28) The proliferative ability (number of divisions) leading to is plotted.
  • chondrocytes polydactyly derived cartilage stem cells
  • Example 6 Response of ⁇ 2-6 sialic acid-binding lectin by proliferation ability, differentiation potential, and flow cytometry in subcultured early and late cells of mesenchymal stem cells derived from polydactyly marrow (Yub622 strain) sexually polydactyly bone marrow-derived mesenchymal stem cells (Yub622 strain, RIKEN Bioresource Center) were subcultured in the same manner as the first embodiment and the like, and proliferative capacity ranging from early passages (P5) in late passage (P17) When the (number of divisions) was plotted, the proliferation ability decreased at the late passage (P17), as in the case of the adipocyte-derived mesenchymal stem cells analyzed in Example 2 and the like.
  • the reactivity of the four lectins (TJAI, SSA, SNA, rPSL1a) in flow cytometry showed high reactivity in the early passage (P4) and slightly to the late passage (P15) cells. Although a decrease in reactivity was observed, no significant decrease was observed.
  • the differentiation potentials of osteoblasts and adipocytes in each cell were analyzed, both differentiation potentials were high in the early passage and the adipocyte differentiation potential was lost in the late passage (P17) cells. However, the differentiation potential into osteoblasts was maintained (FIG. 10).
  • the reactivity of the four lectins that is, the amount of ⁇ 2-6 sialic acid on the surface of the bone marrow-derived mesenchymal stem cells was not related to the proliferation ability, It was suggested that it is highly related to the differentiation potential into cells.
  • Example 7 Reactivity of ⁇ 2-6 sialic acid-binding lectin by subculture of bone marrow-derived mesenchymal stem cells, differentiation potential, and flow cytometry ⁇ 2-6 sialic acid on the cell surface in bone marrow-derived mesenchymal stem cells
  • bone marrow-derived mesenchymal stem cells of a type different from Example 6 purchased from Lonza, Analysis of differentiation potential into adipocytes and reactivity with four lectins were performed.
  • the degree of decrease in reactivity of the four lectins reflected the degree of decrease in osteoblast differentiation potential as compared with the adipocyte differentiation potential. From the above results, it was verified that the amount of ⁇ 2,6-sialic acid on the cell surface in bone marrow-derived mesenchymal stem cells is highly related to the differentiation potential to osteoblasts.
  • Example 8 The differentiation potential of osteoblasts in the subcultured early and late cells of polydactyly marrow-derived mesenchymal stem cells, and the reactivity of ⁇ 2-6 sialic acid-binding lectin by flow cytometry ( Using the polydactyly marrow-derived mesenchymal stem cells (Yub622 strain, RIKEN BioResource Center) used in Example 6), another differentiation induction experiment into osteoblasts was performed. Subculture was performed in the same manner as in Example 6. In Example 6, although the difference in reactivity between ⁇ 2-6 sialic acid-binding lectin was not clear in the early and late passages, the differentiation potential induced by fat differentiation was increased according to the increase in passage number.
  • the fluorescence intensity of four lectins was measured using flow cytometry for the cells at the early passage (P5) and late passage (P15), and the average value was obtained.
  • the SSA lectin decreased from 3225 in (P5) to 1227 in (P15). This result means that the amount of ⁇ 2-6 sialic acid on the cell surface that reacts with SSA lectin was reduced to about 1/3.
  • the results show that the reactivity with TJA1 lectin, SNA lectin, and rPSL1a lectin is reduced to 1/3 to 1/4.
  • Example 9 Mesenchymal stem cells derived from polydactyly bone marrow Subculture in early and late cells Differentiation potential to osteoblasts and reactivity of ⁇ 2-6 sialic acid-binding lectin by flow cytometry ( An experiment similar to Example 8) was performed using another cell line (Yub10F, RIKEN BioResource Center) of polydactyly marrow-derived mesenchymal stem cells.
  • the cells used for osteoblast differentiation and the cells used for flow cytometry are the same cells, and the correlation between differentiation potential and lectin reactivity ( ⁇ 2-6 sialic acid content) is directly Can be analyzed.
  • the integrated value of the fluorescence intensity of ⁇ 2-6 sialic acid-binding lectin by flow cytometry measured for cells in early passage (P5) and late passage (P15) is In the early passage (P5) cells and late passage (P15) cells, there was a significant decrease of about 1/4 to 1/5.
  • the differentiation induction kit made by Lonza was used to induce differentiation into bone differentiated cells for 2 weeks and stained with alizarin red.
  • the late passage (P15) almost all differentiation into osteoblasts can be observed.
  • a large number of osteoblasts were observed in the early passage (P5) cells, and it was confirmed that the differentiation potential to osteoblasts was lost in the late passage (P15) (Fig. 13).
  • This result also shows a high correlation between the amount of ⁇ 2-6 sialic acid on the surface of polymyelinating bone marrow-derived mesenchymal stem cells and the differentiation potential into osteoblasts.
  • Example 11 Reactivity change of ⁇ 2-6 sialic acid-binding lectin (rPSL1a) in cell extracts of adipose-derived mesenchymal stem cells (ADSC) and polydactyly-derived chondrocyte stem cells (Yub621c) of different passage numbers (11 -1) Change in reactivity of ⁇ 2-6 sialic acid-binding lectin (rPSL1a) in immunoprecipitates with anti-CD29 antibody In this experiment, passage number was used to identify the core protein of ⁇ 2-6 sialic acid on the stem cell surface.
  • ADSC adipose-derived mesenchymal stem cells
  • Yub621c polydactyly-derived chondrocyte stem cells
  • stem cells such as mesenchymal stem cells
  • ADSC adipose-derived mesenchymal stem cells
  • polydactyly-derived cartilage stem cells in three stages: early, middle, and late, and mesenchymal stem cell markers
  • Changes in the expression levels of CD29, CD49e and CD13 glycoprotein antigens, also known as ⁇ , and the amount of ⁇ 2-6 sialic acid which is a non-reducing terminal sugar chain constituting the complex sugar chain are observed (FIGS. 18 to 20).
  • ADSC adipose-derived mesenchymal stem cells
  • Example 1 The same ADSC as the adipose-derived mesenchymal stem cells (ADSC) used in Example 1 was subcultured in the same manner, and the cells from different passage numbers (P5, P19, P28) were cultured in CelLytic TM MEM Protein Extraction Kit. A cell extract was obtained as a hydrophobic fraction. Streptavidin-coated magnetic beads (Dynabeads M-280 Streptavidin, Dynabeads) immobilized with biotin-labeled anti-CD29 antibody (Abcam) were allowed to act on each cell extract and left overnight at 4 ° C. Immunoprecipitation. The resulting immunoprecipitate was solubilized with SDS sample buffer, and then run by SDS-PAGE.
  • ⁇ 2-6 sialic acid-binding lectin decreases with increasing passage number, although there is no change in the reactivity of anti-CD29 antibody due to the difference in passage number in any cell. I understood it. From this, the expression level of CD29 glycoprotein on the cell surface does not differ greatly depending on the passage number, whereas the amount of terminal ⁇ 2-6 sialic acid in the complex sugar chain of CD29 glycoprotein is the passage number. It can be seen that it decreases with increasing. In other words, ⁇ 2-6 sialic acid on CD29 was high in early passage cells with high cell differentiation potential, but ⁇ 2-6 sialic acid on CD29 was low in late passage cells with low cell differentiation potential. .
  • ⁇ 2-6 sialic acid-binding lectin rPSL1a
  • CD13 reacted with ⁇ 2-6 sialic acid-binding lectin (rPSL1a), so it was found that it is one of the core proteins with ⁇ 2-6 sialic acid at the non-reducing end.
  • the amount of ⁇ 2-6 sialic acid was slightly decreased in the late passage of the strain (P28), and no significant difference was observed in the amount of ⁇ 2-6 sialic acid on CD13 depending on the passage number. That is, the amount of ⁇ 2-6 sialic acid on CD13 was not correlated with the differentiation potential.
  • CD29 glycoprotein and CD49e glycoprotein are suitable as ⁇ 2-6 sialic acid core proteins for observing the correlation between changes in ⁇ 2-6 sialic acid content and differentiation potential in somatic stem cells. I understood.
  • Example 12 Reactivity change of ⁇ 2-6 sialic acid-binding lectin (rPSL1a) to immunoprecipitation with anti-CD29 antibody and anti-CD49e antibody from cell extract of iPS cells (201B7 strain ) iPS cells (201B7 strain)
  • the cell extract obtained as a hydrophobic fraction was subjected to immunoprecipitation using an anti-CD29 antibody (R & D) and an anti-CD49e antibody (R & D).
  • a change in reactivity with ⁇ 2-6 sialic acid-binding lectin (rPSL1a) was observed on the precipitate (FIG. 21).
  • the immunoprecipitates of the anti-CD29 antibody and anti-CD49e antibody of iPS cells had a very large amount of ⁇ 2-6 sialic acid and were a core protein of ⁇ 2-6 sialic acid on the stem cell surface. That is, it was strongly suggested that the height of cell differentiation potential can be estimated by measuring the amount of ⁇ 2-6 sialic acid on CD29 and CD49e as a marker.
  • Example 13 Construction of four ⁇ 2-6 sialic acid-binding lectins (SNA, SSA, TJAI, rPSL1a) and anti-CD29 antibody sandwich assay system (13-1) Sandwich assay system polydactyly with CD29 antibody Immunoprecipitation was performed with anti-CD29 antibody from the hydrophobic fraction of cells of various passage numbers (P7, P16, P28) of the derived cartilage stem cells (Yub621c), and each of the immunoprecipitates was bound to 4 types of ⁇ 2-6 sialic acid.
  • a sandwich assay was performed by reacting a sex lectin (SNA, SSA, TJAI, rPSL1a) to an immobilized array and reacting with an anti-CD29 antibody fluorescently labeled with Cy3.
  • SNA, SSA, TJAI, rPSL1a sex lectin
  • rPSL1a anti-CD29 antibody fluorescently labeled with Cy3.
  • SNA, SSA, and rPSL1a lectins other than TJAI lectin all showed a decreasing tendency as the passage number increased.
  • the sandwich assay system combining anti-CD29 antibody and SNA lectin is an ⁇ 2-6 sialic acid for estimating the differentiation potential.
  • somatic stem cells such as mesenchymal stem cells and cartilage stem cells
  • a calibration curve is obtained from numerical values such as the labeling intensity measured in the assay system and the differentiated cell marker intensity specific to the target differentiated cell, so that the adipocytes, osteoblasts, or chondrocytes for the subject stem cells are obtained.
  • the differentiation potential as a transplant material, it is possible to perform quantitative evaluation and determination with high certainty. That is, a sandwich assay system combining an anti-CD29 antibody and SNA lectin, SSA lectin, and rPSL1a lectin can be used for quantitative evaluation and determination of differentiation potential of somatic stem cells into various cells.
  • ⁇ 2-6 on the surface of the subject stem cell can be obtained by combining SNA lectin, SSA lectin, and rPSL1a lectin other than TJAI lectin among ⁇ 2-6 sialic acid-binding lectins. It was found that a sandwich assay system capable of quantifying changes in the expression level of sialic acid could be constructed. That is, a sandwich assay system in which an anti-CD49e antibody is combined with SNA lectin, SSA lectin, and rPSL1a lectin can also be used for quantitative evaluation and determination of differentiation potential of somatic stem cells into various cells.
  • Example 14 Osteoblast differentiation of sialidase-treated polydactyly marrow-derived mesenchymal stem cells (Yub621c) In osteoblast differentiation of polydactyly marrow-derived mesenchymal stem cells (Yub621c), In order to confirm that expression plays an important role, the effect on the osteoblast differentiation by the presence or absence of sialidase treatment was observed before and during differentiation induction.
  • sialidase Arthrobacter ureafaciens sialidase (Roche Life Science Co., Ltd.) that cleaves both ⁇ 2-3 sialic acid and ⁇ 2-6 sialic acid was used.

Abstract

The present invention addresses the problem of identifying a cell surface sugar chain marker that has high correlation with the differentiation potential of a somatic stem cell such as a mesenchymal stem cell, and providing a method for determining/evaluating the differentiation potential of a somatic stem cell using the cell surface sugar chain marker and a method for isolating/concentrating a somatic stem cell having high differentiation potential using the cell surface sugar chain marker. The present invention provides: a method for determining the degree of differentiation potential of a somatic stem cell by measuring α2-6 sialic acid expressed on the surface of the somatic stem cell using α2-6 sialic acid-binding lectin1 or an α2-6 sialic acid-binding antibody; and a method for isolating/concentrating a somatic stem cell, in which α2-6 sialic acid is expressed on the surface thereof in a large amount, from a sample containing somatic stem cells using α2-6 sialic acid-binding lectin1 or an α2-6 sialic acid-binding antibody.

Description

細胞分化ポテンシャル判別法Cell differentiation potential discrimination method
　本発明は体性幹細胞の別種の細胞に変化する能力である分化ポテンシャルを判別する方法、及び分化ポテンシャルの高い体性幹細胞を濃縮する方法に関する。当該方法により、分化ポテンシャルの高い体性幹細胞を効率よく簡便に判別し、濃縮することができるため、体性幹細胞を用いた再生医療の加速化につながる。 The present invention relates to a method for discriminating differentiation potential, which is the ability of somatic stem cells to change to another type of cell, and a method for concentrating somatic stem cells having high differentiation potential. By this method, somatic stem cells with high differentiation potential can be identified and concentrated efficiently and easily, leading to acceleration of regenerative medicine using somatic stem cells.
　再生医療とは機能が損なわれた場所に必要な細胞を補う医療のことであり、従来の薬物治療等の対症療法しかない疾病に対しても根本的な修復と再生が可能な医療として大きな期待が寄せられている。間葉系幹細胞などの体性幹細胞は、骨髄や脂肪などの自己組織からでも採取可能な幹細胞であり、免疫拒絶がない自家移植は既に臨床応用が行われている。しかし、間葉系幹細胞等の体性幹細胞はヘテロな細胞集団であり、その性質（分化ポテンシャル、増殖能、遊走能）は由来する個人の年齢、組織、継代数などにより大きく異なることから、治療の有効性を評価することが難しく、その品質管理に関しても困難を極めている。現在、造血幹細胞を除いて間葉系幹細胞等の体性幹細胞を同定するための唯一無二のマーカーは存在しない。そのため、間葉系幹細胞等では複数の細胞表面マーカーの発現を確認することにより細胞同定を行っているが、そもそもこれらマーカーの発現と、その治療有効性を関連づけることは未だできていない。 Regenerative medicine is a medicine that supplements necessary cells in places where functions are impaired, and it is highly expected as a medicine that can fundamentally repair and regenerate even for diseases that only have symptomatic treatment such as conventional drug treatment Has been sent. Somatic stem cells such as mesenchymal stem cells are stem cells that can be collected from autologous tissues such as bone marrow and fat, and autologous transplantation without immune rejection has already been clinically applied. However, somatic stem cells such as mesenchymal stem cells are heterogeneous cell populations, and their properties (differentiation potential, proliferative ability, migration ability) vary greatly depending on the age, tissue, passage number, etc. of the individual from which they are treated. It is difficult to evaluate the effectiveness of the product and its quality control is extremely difficult. Currently, there is no unique marker for identifying somatic stem cells such as mesenchymal stem cells except for hematopoietic stem cells. For this reason, in mesenchymal stem cells and the like, cell identification is performed by confirming the expression of a plurality of cell surface markers, but in the first place, the expression of these markers and their therapeutic efficacy have not yet been correlated.
　このような事情を受け、間葉系幹細胞等の体性幹細胞の品質特性、特に治療有効性に直結する分化ポテンシャルを定量的に評価可能な細胞表面マーカーの開発が近年活発化してきている。
　特に間葉系幹細胞の遺伝子発現量の解析が進み、PARG1、CDKN2B、PTN及びMCM3遺伝子の発現量が、いずれも幹細胞の***能及び分化能にあたる「複製老化（replicative senescence）」の評価の指標となることが示され、これら各遺伝子の発現量の測定により幹細胞を含有する細胞培養物の品質評価が提案された（特許文献１、非特許文献１）。また、間葉系幹細胞表面で特異的に発現する抗原の解析から、抗原CD146が間葉系幹細胞における骨、軟骨、脂肪への多分化能を示すマーカーとなることが示された（特許文献２、非特許文献２、３）。同様に細胞表面でのNG2の発現量も間葉系幹細胞の多分化能との相関が高いことが見いだされ、間葉系幹細胞など体性幹細胞の分化能を抗原CD146およびNG2の発現量測定により判定又は評価する方法が提案されている（特許文献２）。他に、細胞培養物の「複製老化」の指標としてCpG-アイランドでのメチル化に着目し、GRM7,CASR,PRAMEF2,SELP,CASP14,KRTAP13-3遺伝子のCpG-アイランドでのメチル化が「複製老化」と正又は負の相関があることを見いだした例がある（特許文献３）。
　このように、細胞表面の抗原タンパク質マーカーとして、細胞の分化ポテンシャル、分化状態の評価に有効なマーカーが多数同定されているが、特に間葉系幹細胞において、その分化ポテンシャルを簡便かつ正確に評価するための実用性の高いマーカーとするには限界があり、依然として治療に用いる間葉系幹細胞の規格化が困難な状況にあった。 Under such circumstances, development of cell surface markers capable of quantitatively evaluating the quality characteristics of somatic stem cells such as mesenchymal stem cells, particularly the differentiation potential directly linked to therapeutic efficacy, has been activated recently.
In particular, the analysis of gene expression levels of mesenchymal stem cells has progressed, and the expression levels of PARG1, CDKN2B, PTN and MCM3 genes are all indicators of evaluation of “replicative senescence”, which are the ability to divide and differentiate stem cells. It was shown that the quality of cell cultures containing stem cells was evaluated by measuring the expression levels of these genes (Patent Document 1, Non-Patent Document 1). Furthermore, analysis of antigens specifically expressed on the surface of mesenchymal stem cells showed that the antigen CD146 is a marker showing pluripotency into bone, cartilage and fat in mesenchymal stem cells (Patent Document 2). Non-Patent Documents 2 and 3). Similarly, the expression level of NG2 on the cell surface was found to be highly correlated with the pluripotency of mesenchymal stem cells, and the differentiation potential of somatic stem cells such as mesenchymal stem cells was determined by measuring the expression levels of antigens CD146 and NG2. A method of determining or evaluating has been proposed (Patent Document 2). In addition, focusing on methylation at CpG-islands as an indicator of “replication aging” in cell culture, methylation at CpG-islands of GRM7, CASR, PRAMEF2, SELP, CASP14, and KRTAP13-3 genes There is an example where it has been found that there is a positive or negative correlation with “aging” (Patent Document 3).
As described above, many markers effective for evaluating the differentiation potential and differentiation state of a cell have been identified as antigen protein markers on the cell surface. In particular, the differentiation potential of mesenchymal stem cells is easily and accurately evaluated. Therefore, there is a limit to making it a highly practical marker, and it was still difficult to standardize mesenchymal stem cells used for treatment.
　一方、細胞表面を覆う物質群のうちで糖鎖は最も外側に位置しており、細胞分化や悪性化など細胞の状態変化に応じて劇的に変化することが知られており、事実、既知の幹細胞マーカーや癌マーカーの多くが糖鎖マーカーである。したがって、間葉系幹細胞など体性幹細胞の細胞表面の糖鎖がその分化状態に応じて変化することは従来から注目されており、特に実用化の進んだ間葉系幹細胞の分化状態の指標となる糖鎖マーカーの研究が盛んに行われている。例えば、幹細胞の分化状態を評価する方法として、分化誘導した幹細胞表面のN-結合型糖鎖について取得した定量的プロファイル中での糖鎖群の糖鎖タイプの変化から細胞状態、細胞分化を評価する方法（特許文献４）、幹細胞表面のN-結合型糖鎖、O-結合型糖鎖、GSL、GAG、FOSの個々の糖鎖の絶対量を解析することで多能性幹細胞を厳密に評価、選別する方法（特許文献５）などの総合的な評価方法が提案されている。また、未分化糖鎖マーカー「Fucα1-2Galβ1-3GlcNAc/Fucα1-2Galβ1-3GalNAc」を用いて多能性幹細胞の分化状態を評価する方法（特許文献６）も提案されており、間葉系幹細胞の骨分化状態を正確に判定するための骨分化判定用の糖鎖マーカー（特許文献７）も提供されている。しかしながら、これら総合的評価方法や、提案された各糖鎖マーカーは、いずれも対象の幹細胞がどのような分化状態にあるかを判定するためのものであって、分化ポテンシャル（又は複製老化）を評価、判定するためのものではない。そのため、特にヘテロな細胞集団からなる間葉系幹細胞など体性幹細胞において、厳密な品質評価や分化ポテンシャルの高い集団の単離、濃縮に用いることはできない。 On the other hand, the sugar chain is located on the outermost side of the group of substances covering the cell surface, and is known to change dramatically in response to changes in cell status such as cell differentiation and malignancy. Many of the stem cell markers and cancer markers are sugar chain markers. Therefore, it has been attracting attention that sugar chains on the cell surface of somatic stem cells such as mesenchymal stem cells change according to their differentiation state, and particularly as an indicator of the differentiation state of mesenchymal stem cells that have been put to practical use. There are many studies on sugar chain markers. For example, as a method of evaluating the differentiation state of stem cells, the cell state and cell differentiation are evaluated from changes in the sugar chain type of the sugar chain group in the quantitative profile obtained for N-linked sugar chains on the surface of differentiated stem cells. (Patent Document 4), pluripotent stem cells are strictly analyzed by analyzing the absolute amount of N-linked sugar chains, O-linked sugar chains, GSL, GAG, and FOS on the stem cell surface A comprehensive evaluation method such as an evaluation and selection method (Patent Document 5) has been proposed. In addition, a method for evaluating the differentiation state of pluripotent stem cells using the undifferentiated sugar chain marker “Fucα1-2Galβ1-3GlcNAc / Fucα1-2Galβ1-3GalNAc” has been proposed (Patent Document 6). A sugar chain marker (Patent Document 7) for bone differentiation determination for accurately determining the bone differentiation state is also provided. However, these comprehensive evaluation methods and the proposed sugar chain markers are all for determining the differentiation state of the target stem cell, and have a differentiation potential (or replication aging). It is not for evaluation and judgment. Therefore, in particular, somatic stem cells such as mesenchymal stem cells composed of heterogeneous cell populations cannot be used for strict quality evaluation or isolation / concentration of populations with high differentiation potential.
　以上のことから、間葉系幹細胞などの体性幹細胞の分化ポテンシャルを評価、判定でき、かつ分化ポテンシャルの高い集団の単離、濃縮に有用な、細胞表面の糖鎖マーカーの提供が切望されていた。 Based on the above, there is an urgent need to provide cell surface sugar chain markers that can evaluate and determine the differentiation potential of somatic stem cells such as mesenchymal stem cells, and are useful for isolating and enriching populations with high differentiation potential. It was.
ＷＯ２０１１／０００８３３WO2011 / 000833 特表２０１４－５０１１１０号公報Special table 2014-501110 gazette ＷＯ２０１３／０１７７０１WO2013 / 017701 ＷＯ２０１０／０５８６０５WO2010 / 058605 ＷＯ２０１３／１８６９４６WO2013 / 186946 ＷＯ２０１３／０６５３０２WO2013 / 065302 特開２０１２－３７４１６号公報JP 2012-37416 A 特開２０１２－３７４１６号公報JP 2012-37416 A 特許第５６９８５８８号公報Japanese Patent No. 5698588
　本発明の課題は、体性幹細胞、特に間葉系幹細胞の治療有効性を担保する分化ポテンシャルの優れた指標となる細胞表面糖鎖マーカーを同定し、当該細胞表面糖鎖マーカーを簡便且つ効率的に検出し定量化することによる体性幹細胞の分化ポテンシャルの判定方法を提供することである。また、さらに当該細胞表面糖鎖マーカーを利用して体性幹細胞を含む不均一な細胞集団から分化ポテンシャルの高い体性幹細胞を単離または濃縮する方法及び体性幹細胞の品質管理方法を提供することである。
　なお、本発明で「分化ポテンシャル」というとき、ある細胞が適切な分化誘導状態に置かれた場合に、前駆細胞、組織細胞などの別種の細胞に変化することができる潜在的な能力を有していることをいう。ここで、ある細胞の「分化ポテンシャルが高い」というとき、変化する細胞の種類が複数種類であるか否かにはかかわらない。一種類であっても別種類の細胞へと変化する能力が高ければ「分化ポテンシャルが高い」と表現する。従来、間葉系幹細胞ではその***能及び分化能を評価する際の概念として「複製老化（特許文献１、非特許文献１）」という用語があるが、本発明における「分化ポテンシャル」の用語は、専ら分化能に着目した「別種の細胞に変化できる潜在能力」を評価する概念を表し、かつ間葉系幹細胞に留まらず、広く幹細胞全体に適用可能な用語である。 An object of the present invention is to identify a cell surface sugar chain marker that is an excellent index of differentiation potential that ensures the therapeutic efficacy of somatic stem cells, particularly mesenchymal stem cells, and to make the cell surface sugar chain marker simple and efficient. It is intended to provide a method for determining the differentiation potential of somatic stem cells by detecting and quantifying them. Furthermore, a method for isolating or concentrating somatic stem cells having high differentiation potential from a heterogeneous cell population containing somatic stem cells using the cell surface sugar chain marker and a quality control method for somatic stem cells are provided. It is.
In the present invention, the term “differentiation potential” has a potential ability to change into another type of cell such as a progenitor cell or tissue cell when a cell is placed in an appropriate differentiation-inducing state. It means that Here, when a cell has “high differentiation potential”, it does not depend on whether or not there are multiple types of cells that change. If the ability to change to a different type of cell is high, it is expressed as “high differentiation potential”. Conventionally, in mesenchymal stem cells, there is a term “replication aging (Patent Document 1, Non-Patent Document 1)” as a concept when evaluating the division ability and differentiation ability, but the term “differentiation potential” in the present invention is It is a term that expresses the concept of evaluating the “potential of being able to change into a different type of cell” focusing exclusively on differentiation ability, and is applicable not only to mesenchymal stem cells but also to the whole stem cells.
　体性幹細胞のうちでもとりわけ間葉系幹細胞（MSC）は、骨、軟骨、脂肪などの多くの組織に分化可能な上に、脂肪組織のように比較的大量に入手可能なソースがあり、調製が容易であるため、再生治療への応用が最も進んでいる。しかし、間葉系幹細胞は、骨髄、胎児付属物又は脂肪組織から主に採取されるが、非常に雑多な細胞集団の一部として採取されるため、すでに特定方向への分化を開始した前駆細胞も含まれており、ロットごとに大きなばらつきがある。そのため、再生治療に供する前の段階で、その分化ポテンシャルを評価することが安全で効果的な再生医療のために重要であるが、未だ間葉系幹細胞の分化ポテンシャルを簡便にかつ正確に評価する手法は確立されていない。実際の医療の現場では、幹細胞の分化ポテンシャルを評価せずに患者に移植しているのが現状であり、移植効果にばらつきがでる一因となっている。 Among the somatic stem cells, especially mesenchymal stem cells (MSCs) can be differentiated into many tissues such as bone, cartilage, and fat, and there are sources that are available in relatively large quantities like adipose tissue. Is the most advanced application to regenerative treatment. However, mesenchymal stem cells are collected mainly from bone marrow, fetal appendages or adipose tissue, but they are collected as part of a very diverse cell population, so progenitor cells that have already started to differentiate in a specific direction Are also included, and there are large variations from lot to lot. Therefore, it is important for safe and effective regenerative medicine to evaluate its differentiation potential before it is used for regenerative treatment, but it is still easy and accurate to evaluate the differentiation potential of mesenchymal stem cells. The method has not been established. In actual medical practice, transplantation to patients without evaluating the differentiation potential of stem cells is the current situation, which is a cause of variation in transplantation effects.
　間葉系幹細胞を長期培養すると徐々にその増殖能と共に分化ポテンシャルが低下する傾向があることはこの分野の研究者が以前から実感しているが、それを定量的に評価できる指標（マーカー）は存在しなかった。本発明者らは、その点に着目して脂肪由来間葉系幹細胞を長期培養し、継代初期と継代後期の細胞の骨芽細胞と脂肪細胞への分化ポテンシャルを解析した結果、継代初期で観察された骨芽細胞及び脂肪細胞への分化ポテンシャルは、細胞***がほぼ停止した継代後期ではほとんど失われることが分かった。そこで、高密度レクチンアレイを用いて各継代数のヒト脂肪由来間葉系幹細胞（ADSC）の糖鎖解析を行った結果、分化ポテンシャルの低下した継代後期の細胞では、継代初期の細胞と比較して、細胞表面のα2-6シアル酸の発現量が顕著に減少することを見いだした。脂肪組織由来以外に骨髄由来間葉系幹細胞、及び多指症軟骨組織由来軟骨細胞（軟骨幹細胞）においても同様の結果が得られたことから、体性幹細胞における細胞表面のα2-6シアル酸発現量の定量的な解析が、体性幹細胞の分化ポテンシャルの優れた指標となることがわかった。 Researchers in this field have long felt that when mesenchymal stem cells are cultured for a long time, their differentiation potential tends to decrease gradually with their proliferative ability. Did not exist. The present inventors focused on this point and cultured long-term adipose-derived mesenchymal stem cells and analyzed the differentiation potential of early and late passage cells into osteoblasts and adipocytes. It was found that the differentiation potential to osteoblasts and adipocytes observed in the early stage was almost lost at the late passage when cell division was almost stopped. Therefore, as a result of glycan analysis of human adipose-derived mesenchymal stem cells (ADSC) at each passage number using a high-density lectin array, late passage cells with reduced differentiation potential In comparison, it was found that the expression level of α2-6 sialic acid on the cell surface was significantly reduced. In addition to adipose tissue, bone marrow-derived mesenchymal stem cells and polydactyly cartilage tissue-derived chondrocytes (chondral stem cells) showed similar results, so that α2-6 sialic acid expression on the surface of somatic stem cells The quantitative analysis of the amount was found to be an excellent indicator of the differentiation potential of somatic stem cells.
　具体的には、ヒト脂肪由来間葉系幹細胞から膜画分を調製し、蛍光ラベル化後、高密度レクチンアレイに供して、高い増殖能と共に骨芽細胞及び脂肪細胞への分化ポテンシャルも高い継代初期の細胞と、増殖能も低下し、分化ポテンシャルの失われた継代後期の細胞とで顕著に異なる結合性を示すレクチンを統計的に抽出した。その結果、α2-6シアル酸に結合特異性を示す４種のレクチン、Trichosanthes japonica lectin-I (TJAI), Sambucus sieboldiana lectin (SSA), Sambucus nigra lectin (SNA), recombinant Polyporus squamosus lectin (rPSL1a)を選択した。この結果をフローサイトメトリーを用いて検証したところ、いずれのレクチンも、継代初期の細胞に顕著に高い反応性を示すのに対して、継代後期の細胞にはバックグラウンドレベルの反応性しか示さなかった。これら４種のレクチンは、いずれもα2-6シアル酸に結合特異性を示すことから、α2-6シアル酸が間葉系幹細胞の分化ポテンシャルを示すマーカーである可能性が示唆された。 Specifically, a membrane fraction is prepared from human adipose-derived mesenchymal stem cells, and after fluorescent labeling, it is subjected to a high-density lectin array, and has high proliferation potential as well as high differentiation potential into osteoblasts and adipocytes. A lectin having a markedly different binding property between cells in early passages and cells in late passages in which proliferative ability was reduced and differentiation potential was lost was statistically extracted. As a result, four lectins with binding specificity for α2-6 sialic acid, Trichosanthes japonica lectin-I (TJAI), Sambucus sieboldiana lectin (SSA), Sambucus nigra lectin (SNA), recombinant Polyporus squamosr Lin Selected. When this result was verified using flow cytometry, all lectins showed significantly higher reactivity to early passage cells, whereas late-passage cells only had a background level of reactivity. Not shown. These four lectins all showed binding specificity to α2-6 sialic acid, suggesting that α2-6 sialic acid may be a marker indicating the differentiation potential of mesenchymal stem cells.
　次いで、上記４種のレクチンの、継代初期のヒト脂肪由来間葉系幹細胞（ADSC P3）、継代後期のヒト脂肪由来間葉系幹細胞（ADSC P26）、及びヒト皮膚繊維芽細胞（Fibroblast）に対する反応性を比較したところ、これら４種のレクチンはいずれも分化ポテンシャルを有するADSC P3に対して高い反応性が確認されたのに対して、分化ポテンシャルが失われたADSC P26とヒト皮膚繊維芽細胞にはほとんど反応性が見られなかった。分化ポテンシャルが最も高いと考えられるヒトiPS細胞（201B7株）でも確認すると、それぞれADSC P3の２～４倍の反応性が観察された。この結果からも、幹細胞表面のα2-6シアル酸が細胞の分化ポテンシャルと高い相関性を示す可能性が示唆された。 Next, human fat-derived mesenchymal stem cells (ADSC P3) in the early passage, human fat-derived mesenchymal stem cells (ADSC P26) in the late passage, and human skin fibroblasts (Fibroblast). When these four lectins were all highly reactive against ADSC P3, which has differentiation potential, ADSC26P26 and human skin fibroblasts, which lost differentiation potential, were confirmed. The cells showed little reactivity. When confirmed in human iPS cells (201B7 strain), which is considered to have the highest differentiation potential, 2 to 4 times the reactivity of ADSC P3 was observed. This result also suggests that α2-6 sialic acid on the stem cell surface may be highly correlated with the differentiation potential of the cells.
　そこで、これらヒトiPS細胞（201B7株）、ADSC P3、ADSC P26、ヒト皮膚繊維芽細胞（Fibroblast）のタンパク質画分から、糖鎖をヒドラジン分解で切り出し、２－ピリジルアミノ（PA）化後、Mono-Qカラムを用いたイオン交換クロマトグラフィーに供して、糖鎖の酸性度に応じて糖鎖を分画した。シアル酸が１個付加されたA1画分、シアル酸が２個付加されたA4画分に対して、α2-3シアル酸特異的シアリダーゼと、α2-3及びα2-6シアル酸の両方を切断するシアリダーゼを反応させ、A1及びA4分画それぞれでのα2-3シアル酸とα2-6シアル酸の割合を計算した。その結果、いずれの画分においても、α2-6シアル酸の割合は、ヒトiPS細胞で最も高く、次にADSC P3であり、ADSC P26とFibroblastではほとんど確認されないという傾向が見られた。以上の結果から、細胞表面のα2-6シアル酸とα2-3シアル酸との割合も、細胞分化ポテンシャルの高さを判定するための優れた指標となることがわかった。 Therefore, the sugar chain was excised from the protein fractions of these human iPS cells (strain 201B7), ADSC 皮膚 P3, ADSC P26, and human skin fibroblasts (Fibroblast) by hydrazine degradation, converted to 2-pyridylamino (PA), then Mono-Q The glycans were fractionated according to the acidity of the glycans by ion exchange chromatography using a column. Cleavage of α2-3 sialic acid-specific sialidase and both α2-3 and α2-6 sialic acid from A1 fraction with one sialic acid and A4 fraction with two sialic acids The ratio of α2-3 sialic acid and α2-6 sialic acid in each of the A1 and A4 fractions was calculated. As a result, in any fraction, the ratio of α2-6 sialic acid was the highest in human iPS cells, followed by ADSC P3, and there was a tendency that ADSC P26 and Fibroblast were hardly confirmed. From the above results, it was found that the ratio of α2-6 sialic acid and α2-3 sialic acid on the cell surface is also an excellent index for determining the height of cell differentiation potential.
　次いで、脂肪組織由来間葉系幹細胞以外の他の組織由来の体性幹細胞を用いて検証を行うため、軟骨幹細胞の一種である多指症軟骨組織由来軟骨細胞（Yub621c株）を継代培養し、継代初期と継代後期での骨芽細胞と脂肪細胞への分化ポテンシャルを解析し、フローサイトメトリーにおける上記４種のレクチンの反応性を解析した。その結果、軟骨幹細胞においても脂肪組織由来間葉系幹細胞の場合と同様の傾向を示した。なお、以下、多指症軟骨組織由来軟骨細胞を分化した軟骨細胞と明確に区別するため、多指症由来軟骨幹細胞ということもある。 Next, in order to perform verification using somatic stem cells derived from other tissues other than the adipose tissue-derived mesenchymal stem cells, a multi-digitated cartilage tissue-derived chondrocyte (Yub621c strain), which is a type of cartilage stem cell, is subcultured. The differentiation potential of osteoblasts and adipocytes in the early and late passages was analyzed, and the reactivity of the above four lectins in flow cytometry was analyzed. As a result, the same tendency as in the case of adipose tissue-derived mesenchymal stem cells was observed in the cartilage stem cells. In addition, hereinafter, in order to clearly distinguish a polychondrogenic cartilage tissue-derived chondrocyte from a differentiated chondrocyte, it may be referred to as a polydactyly-derived cartilage stem cell.
　これに対し、多指症骨髄由来間葉系幹細胞（Yub622株）の場合、当初の実験系では、フローサイトメトリーにおける上記４種のレクチンの反応性は、継代初期と比較して、継代後期の細胞での反応性に大きな低下は認められなかったにもかかわらず、脂肪細胞への分化ポテンシャルについては、継代初期細胞で活発であった分化が、継代後期ではほぼ起こらなくなり、α2-6シアル酸量との相関はみられず、むしろ継代数に依存して低下すると解された。一方、骨芽細胞への分化については、継代後期になってもその分化ポテンシャルは維持されていた。別の骨髄由来間葉系幹細胞（Lonza社から購入）においても同様の現象がみられた。
　以上の結果から、少なくとも骨髄由来間葉系幹細胞の場合では、４種のレクチンの反応性に示される細胞表面でのα2-6シアル酸の発現量の高さは、骨芽細胞分化ポテンシャルの高さを表す優れた指標となることが示唆された。 On the other hand, in the case of polydactyly derived bone marrow-derived mesenchymal stem cells (Yub622 strain), in the initial experimental system, the reactivity of the above four lectins in flow cytometry was compared to that in the early passage. Despite the fact that there was no significant decrease in reactivity in late cells, the differentiation potential to adipocytes was almost the same as that in early passage cells, but almost no longer occurred in late passage cells. It was understood that there was no correlation with the amount of -6 sialic acid, rather it decreased depending on the passage number. On the other hand, regarding differentiation into osteoblasts, the differentiation potential was maintained even at the later passage. The same phenomenon was observed in other bone marrow-derived mesenchymal stem cells (purchased from Lonza).
From the above results, at least in the case of bone marrow-derived mesenchymal stem cells, the high expression level of α2-6 sialic acid on the cell surface indicated by the reactivity of the four lectins indicates a high osteoblast differentiation potential. It was suggested that this would be an excellent indicator of stagnation.
　すなわち、α2-6シアル酸を認識する４種のレクチンの反応性は、体性幹細胞の増殖能ではなく分化ポテンシャルとの相関を示唆するものであるが、その幹細胞の由来によって、多分化能全ての分化ポテンシャルと関連する場合、又は特定の分化能の分化ポテンシャルにのみ関連する場合があることが示唆された。そして、いずれの場合であっても、α2-6シアル酸は、体性幹細胞の分化ポテンシャルの指標となる優れた細胞表面の糖鎖マーカーであると結論付けた。
　以上の知見を得たことで本発明を完成するに至った。 In other words, the reactivity of the four lectins that recognize α2-6 sialic acid suggests a correlation with the differentiation potential rather than the proliferation ability of somatic stem cells. It was suggested that it may be related to the differentiation potential of, or may be related only to the differentiation potential of a specific differentiation ability. In any case, it was concluded that α2-6 sialic acid is an excellent cell surface sugar chain marker that serves as an indicator of the differentiation potential of somatic stem cells.
The present invention has been completed by obtaining the above knowledge.
　その後、各種多指症軟骨組織由来の軟骨細胞及び間葉系幹細胞を用いて繰り返し実験を行った結果、α2-6シアル酸の発現量の高さと、骨芽細胞若しくは軟骨細胞への分化ポテンシャルの高さとの相関性の高さを実証した。また、特に、骨芽細胞もしくは軟骨細胞への分化ポテンシャルの変化に応じたα2-6シアル酸量の変化の観察に適したコアタンパク質として、幹細胞表面のCD29及びCD49e糖タンパク質を同定し、体性幹細胞の分化ポテンシャル判定用の優れたサンドイッチアッセイ系を構築することができた。 Subsequently, as a result of repeated experiments using chondrocytes derived from various polydactyly cartilage tissues and mesenchymal stem cells, the expression level of α2-6 sialic acid and the differentiation potential into osteoblasts or chondrocytes The high correlation with the height was demonstrated. In particular, we identified CD29 and CD49e glycoproteins on the stem cell surface as core proteins suitable for observing changes in the amount of α2-6 sialic acid in response to changes in differentiation potential into osteoblasts or chondrocytes, An excellent sandwich assay system for determining the differentiation potential of stem cells could be constructed.
　すなわち、本発明は以下の発明を包含する。
〔１〕　体性幹細胞表面に発現するα2-6シアル酸を検出する工程、又はα2-6シアル酸量を測定する工程を包含することを特徴とする、体性幹細胞の分化ポテンシャルを判別、評価する方法。
　ここで、本発明で検出もしくは測定の対象となるα2-6シアル酸は、体性幹細胞表面に発現している糖タンパク質のN-結合型糖鎖中の非還元末端に存在する糖鎖であるため、以下の様にも表現できる。
〔１'〕　体性幹細胞表面に発現している糖タンパク質のN-結合型糖鎖中の非還元末端に存在するα2-6シアル酸を検出又はα2-6シアル酸量を測定する工程を包含することを特徴とする、体性幹細胞の分化ポテンシャルを判別、評価する方法。 That is, the present invention includes the following inventions.
[1] Discrimination and evaluation of differentiation potential of somatic stem cells, comprising the step of detecting α2-6 sialic acid expressed on the surface of somatic stem cells or the step of measuring the amount of α2-6 sialic acid how to.
Here, α2-6 sialic acid to be detected or measured in the present invention is a sugar chain present at the non-reducing end in the N-linked sugar chain of glycoprotein expressed on the surface of somatic stem cells. Therefore, it can be expressed as follows.
[1 '] Includes the step of detecting α2-6 sialic acid or measuring the amount of α2-6 sialic acid present at the non-reducing end of the N-linked sugar chain of glycoprotein expressed on the surface of somatic stem cells A method for discriminating and evaluating the differentiation potential of somatic stem cells.
〔２〕　体性幹細胞が間葉系幹細胞又は軟骨幹細胞であって、体性幹細胞の分化ポテンシャルが、骨芽細胞又は軟骨細胞への分化ポテンシャルである、前記〔１〕に記載の方法。
　ここで、間葉系幹細胞としては、骨髄由来間葉系幹細胞又は脂肪由来間葉系幹細胞が好ましく、軟骨幹細胞は、多指症軟骨細胞由来軟骨幹細胞が好ましい。 [2] The method according to [1] above, wherein the somatic stem cells are mesenchymal stem cells or cartilage stem cells, and the differentiation potential of the somatic stem cells is differentiation potential to osteoblasts or chondrocytes.
Here, the mesenchymal stem cells are preferably bone marrow-derived mesenchymal stem cells or adipose-derived mesenchymal stem cells, and the chondrocyte stem cells are preferably polydactyly chondrocyte-derived cartilage stem cells.
〔３〕　α2-6シアル酸を検出又はα2-6シアル酸量を測定する工程が、α2-6シアル酸をエピトープとして認識するプローブを用いて行うことを特徴とする、前記〔１〕又は〔２〕に記載の方法。
　ここで、α2-6シアル酸をエピトープとして認識するプローブとは、具体的にはα2-6シアル酸をエピトープとして認識するレクチン及び抗体から選択された少なくとも１種のタンパク質を指し、当該抗体としては、当該抗体の抗原認識部位が保存されたフラグメントもしくは当該抗体の誘導体も含まれるため、以下の様にも表現できる。
〔３’〕　α2-6シアル酸を検出又はα2-6シアル酸量を測定する工程が、α2-6シアル酸をエピトープとして認識するレクチン及び抗体から選択された少なくとも１種のタンパク質を用いて行うことを特徴とする、前記〔１〕又は前記〔２〕に記載の方法。
〔３’’〕　α2-6シアル酸を検出又は測定する工程が、α2-6シアル酸をエピトープとして認識するレクチン、並びにα2-6シアル酸をエピトープとして認識する抗体、当該抗体の抗原認識部位が保存された抗体フラグメント及び当該抗体の誘導体から選択された少なくとも１種のタンパク質を用いて行うことを特徴とする、前記〔１〕又は前記〔２〕に記載の方法。 [3] The step of detecting α2-6 sialic acid or measuring the amount of α2-6 sialic acid is performed using a probe that recognizes α2-6 sialic acid as an epitope. 2].
Here, the probe that recognizes α2-6 sialic acid as an epitope specifically refers to at least one protein selected from a lectin that recognizes α2-6 sialic acid as an epitope and an antibody. Since a fragment in which the antigen recognition site of the antibody is conserved or a derivative of the antibody is also included, it can be expressed as follows.
[3 ′] The step of detecting α2-6 sialic acid or measuring the amount of α2-6 sialic acid is performed using at least one protein selected from a lectin and an antibody that recognize α2-6 sialic acid as an epitope. The method according to [1] or [2] above, wherein
[3 ″] The step of detecting or measuring α2-6 sialic acid comprises a lectin that recognizes α2-6 sialic acid as an epitope, an antibody that recognizes α2-6 sialic acid as an epitope, and an antigen recognition site of the antibody. The method according to [1] or [2] above, wherein the method is performed using at least one protein selected from a conserved antibody fragment and a derivative of the antibody.
〔４〕　α2-6シアル酸をエピトープとして認識するプローブが、TJAIレクチン、SSAレクチン、SNAレクチン、及びPSL1aレクチンから選択された少なくとも１種のレクチンを含むことを特徴とする、前記〔３〕に記載の方法。
　または、前記〔３’〕を引用して以下の様にも表現できる。
〔４’〕　α2-6シアル酸をエピトープとして認識するレクチンが、TJAIレクチン、SSAレクチン、SNAレクチン、及びPSL1aレクチンから選択された少なくとも１種のレクチンを含むことを特徴とする、前記〔３’〕に記載の方法。 [4] The probe according to [3], wherein the probe that recognizes α2-6 sialic acid as an epitope includes at least one lectin selected from TJAI lectin, SSA lectin, SNA lectin, and PSL1a lectin. The method described.
Alternatively, the above [3 ′] can be cited as follows.
[4 ′] The lectin that recognizes α2-6 sialic acid as an epitope includes at least one lectin selected from TJAI lectin, SSA lectin, SNA lectin, and PSL1a lectin. ] The method of description.
〔５〕　さらに、体性幹細胞表面で発現し、かつα2-6シアル酸を非還元末端糖鎖として有する糖タンパク質を、当該糖タンパク質を特異的に認識するプローブを用いて検出又は測定する工程を包含する、〔１〕～〔４〕のいずれかに記載の方法。
　ここで、前記糖タンパク質は、対象とする体性幹細胞で特異的に及び／又は大量に発現している糖タンパク質であることが好ましい。 [5] A step of detecting or measuring a glycoprotein expressed on the surface of a somatic stem cell and having α2-6 sialic acid as a non-reducing terminal sugar chain using a probe that specifically recognizes the glycoprotein. The method according to any one of [1] to [4].
Here, the glycoprotein is preferably a glycoprotein that is specifically and / or expressed in a large amount in the target somatic stem cells.
〔６〕　前記糖タンパク質が、CD29又はCD49eである、〔５〕に記載の方法。 [6] The method according to [5], wherein the glycoprotein is CD29 or CD49e.
〔７〕　前記糖タンパク質を特異的に認識するプローブが、抗CD29抗体、及び抗CD49e抗体から選択された少なくとも１種の抗体である前記〔６〕に記載の方法。
　ここで、抗CD29抗体、及び抗CD49e抗体は、ポリクローナル抗体、モノクローナル抗体、及びその抗原認識部位が保存されたFabフラグメントなどの抗体フラグメント、ヒト化抗体、単鎖抗体などであってもよい。 [7] The method according to [6], wherein the probe that specifically recognizes the glycoprotein is at least one antibody selected from an anti-CD29 antibody and an anti-CD49e antibody.
Here, the anti-CD29 antibody and the anti-CD49e antibody may be a polyclonal antibody, a monoclonal antibody, an antibody fragment such as a Fab fragment in which the antigen recognition site is conserved, a humanized antibody, a single chain antibody, or the like.
〔８〕　体性幹細胞の骨芽細胞又は軟骨細胞への分化ポテンシャルを判別又は評価する方法であって、下記の（１）及び（２）を含む方法；
（１）被検体性幹細胞含有試料を、下記の（ａ）又は（ｂ）のいずれか一方のプローブが固定化された基板上にオーバーレイし、次いで、標識化された他方のプローブを作用させる工程、
（ａ）SSAレクチン、SNAレクチン、及びPSL1aレクチンから選択された少なくとも１種のレクチン、
（ｂ）抗CD29抗体、及び抗CD49e抗体から選択された少なくとも１種の抗体、
（２）標識量を測定する工程。
　例えば、以下の方法を含む。
〔８’〕　体性幹細胞の骨芽細胞又は軟骨細胞への分化ポテンシャルを判別又は評価する方法であって、下記の（１）及び（２）を含む方法；
（１）被検体性幹細胞含有試料を、下記の（ａ）を固定化された基板上にオーバーレイし、次いでCy3等の蛍光色素で標識化された（ｂ）を作用させる工程、
（ａ）SSAレクチン、SNAレクチン、及びPSL1aレクチンから選択された少なくとも１種のレクチン、
（ｂ）抗CD29抗体、及び抗CD49e抗体から選択された少なくとも１種の抗体、
（２）蛍光標識量を測定する工程。 [8] A method for discriminating or evaluating the differentiation potential of somatic stem cells into osteoblasts or chondrocytes, comprising the following (1) and (2);
(1) Overlaying a specimen stem cell-containing sample on a substrate on which either one of the following probes (a) or (b) is immobilized, and then allowing the other labeled probe to act ,
(A) at least one lectin selected from SSA lectin, SNA lectin, and PSL1a lectin,
(B) at least one antibody selected from an anti-CD29 antibody and an anti-CD49e antibody,
(2) A step of measuring the labeled amount.
For example, the following method is included.
[8 ′] A method for discriminating or evaluating the differentiation potential of somatic stem cells into osteoblasts or chondrocytes, comprising the following (1) and (2):
(1) Overlaying a specimen-containing stem cell-containing sample on a substrate on which the following (a) is immobilized, and then allowing (b) labeled with a fluorescent dye such as Cy3 to act;
(A) at least one lectin selected from SSA lectin, SNA lectin, and PSL1a lectin,
(B) at least one antibody selected from an anti-CD29 antibody and an anti-CD49e antibody,
(2) A step of measuring the amount of fluorescent label.
〔９〕　あらかじめ、前記試料中の被検体性幹細胞を、前記（ａ）又は（ｂ）のプローブとの相互作用を利用して濃縮する工程を設けることを特徴とする、前記〔８〕に記載の方法。
　例えば、以下の方法を含む。
〔９’〕　あらかじめ、前記試料中の被検体性幹細胞を、標識化した（ａ）のレクチンと反応させフローサイトメトリーにより細胞分画する工程、又は標識化した（ａ）のレクチンを固定化した担体による分離工程を設けることを特徴とする、前記〔８〕に記載の方法。 [9] The method according to [8], wherein a step of concentrating the analyte stem cells in the sample in advance using the interaction with the probe (a) or (b) is provided. the method of.
For example, the following method is included.
[9 ′] In advance, the step of subjecting the stem cells in the sample to the labeled lectin (a) and fractionating the cells by flow cytometry, or the labeled lectin (a) was immobilized. The method according to [8] above, wherein a separation step using a carrier is provided.
〔１０〕　前記〔８〕又は〔９〕の方法に、さらに
（３）測定した標識量と、被検体性幹細胞の分化ポテンシャルとを相関させる工程、
を設けることを特徴とする、体性幹細胞の骨芽細胞又は軟骨細胞への分化ポテンシャルを定量的に判別又は評価する方法。 [10] The method according to [8] or [9], further comprising (3) correlating the measured labeling amount with the differentiation potential of the subject stem cell,
A method for quantitatively discriminating or evaluating the differentiation potential of somatic stem cells into osteoblasts or chondrocytes.
〔１１〕　α2-6シアル酸をエピトープとして認識するプローブを含むことを特徴とする、体性幹細胞の分化ポテンシャルの判定又は評価用試薬。
　または、以下の様にも表現できる。
〔１１’〕　α2-6シアル酸をエピトープとして認識するレクチン及び抗体から選択された少なくとも１種のタンパク質を含むことを特徴とする、体性幹細胞の分化ポテンシャルの判定又は評価用試薬。 [11] A reagent for determining or evaluating the differentiation potential of somatic stem cells, comprising a probe that recognizes α2-6 sialic acid as an epitope.
Or it can be expressed as follows.
[11 ′] A reagent for determining or evaluating the differentiation potential of somatic stem cells, comprising at least one protein selected from a lectin that recognizes α2-6 sialic acid as an epitope and an antibody.
〔１２〕　α2-6シアル酸をエピトープとして認識するプローブを含む、体性幹細胞の分化ポテンシャル判定用キット。
　または、以下の様にも表現できる。
〔１２’〕　α2-6シアル酸をエピトープとして認識するレクチン及び抗体から選択された少なくとも１種のタンパク質を含むことを特徴とする、体性幹細胞の分化ポテンシャルの判定用キット。
〔１２’’〕　TJAIレクチン、SSAレクチン、SNAレクチン、及びPSL1aレクチン、並びにα2-6シアル酸をエピトープとして認識する抗α2-6シアル酸抗体の少なくとも１種のタンパク質を含むことを特徴とする、体性幹細胞の分化ポテンシャルの判定用キット。 [12] A kit for determining differentiation potential of somatic stem cells, comprising a probe that recognizes α2-6 sialic acid as an epitope.
Or it can be expressed as follows.
[12 ′] A kit for determining differentiation potential of somatic stem cells, comprising at least one protein selected from a lectin that recognizes α2-6 sialic acid as an epitope and an antibody.
[12 ″] TJAI lectin, SSA lectin, SNA lectin, and PSL1a lectin, and at least one protein of anti-α2-6 sialic acid antibody that recognizes α2-6 sialic acid as an epitope, A kit for determining the differentiation potential of somatic stem cells.
〔１３〕　前記α2-6シアル酸をエピトープとして認識するプローブが、
（ａ）α2-6シアル酸をエピトープとして認識するレクチンの少なくとも１つ、及び
（ｂ）α2-6シアル酸をエピトープとして認識する抗α2-6シアル酸抗体の少なくとも１つ、
との組み合わせを含み（ａ）又は（ｂ）のいずれか一方のプローブが基板に固定化され、他方のプローブが標識化されている、前記〔１２〕に記載のキット。
　例えば、以下の場合を含む。
〔１３’〕　前記α2-6シアル酸をエピトープとして認識するプローブが、
（ａ）TJAIレクチン、SSAレクチン、SNAレクチン、及びPSL1aレクチンから選択された少なくとも１種のレクチン、及び
（ｂ）α2-6シアル酸をエピトープとして認識する抗α2-6シアル酸抗体の少なくとも１つ、
との組み合わせを含み（ａ）又は（ｂ）のいずれか一方のプローブが基板に固定化され、他方のプローブが標識化されている、前記〔１２〕に記載のキット。 [13] A probe that recognizes the α2-6 sialic acid as an epitope,
(A) at least one lectin that recognizes α2-6 sialic acid as an epitope, and (b) at least one anti-α2-6 sialic acid antibody that recognizes α2-6 sialic acid as an epitope,
The kit according to [12] above, wherein either one of the probes (a) or (b) is immobilized on a substrate and the other probe is labeled.
For example, the following cases are included.
[13 ′] A probe that recognizes the α2-6 sialic acid as an epitope,
(A) at least one lectin selected from TJAI lectin, SSA lectin, SNA lectin, and PSL1a lectin, and (b) at least one anti-α2-6 sialic acid antibody that recognizes α2-6 sialic acid as an epitope ,
The kit according to [12] above, wherein either one of the probes (a) or (b) is immobilized on a substrate and the other probe is labeled.
〔１４〕　前記体性幹細胞の分化ポテンシャル判定用キットが、
（ａ）α2-6シアル酸をエピトープとして認識するプローブ、と共に
（ｂ）体性幹細胞表面で発現し、かつα2-6シアル酸を非還元末端糖鎖として有する糖タンパク質を特異的に認識するプローブ、
を含み、（ａ）又は（ｂ）のいずれか一方のプローブが基板に固定化され、他方のプローブが標識化されている、前記〔１２〕に記載のキット。
　例えば、以下の場合を含む。
〔１４’〕　前記体性幹細胞の分化ポテンシャル判定用キットが、
（ａ）TJAIレクチン、SSAレクチン、SNAレクチン、及びPSL1aレクチンから選択された少なくとも１種のレクチン、と共に
（ｂ）体性幹細胞表面で発現し、かつα2-6シアル酸を非還元末端糖鎖として有する糖タンパク質を特異的に認識するプローブ、
を含み、（ａ）又は（ｂ）のいずれか一方のプローブが基板に固定化され、他方のプローブが標識化されている、前記〔１２〕に記載のキット。 [14] The somatic stem cell differentiation potential determination kit comprises:
(A) a probe that recognizes α2-6 sialic acid as an epitope, and (b) a probe that specifically recognizes a glycoprotein expressed on the surface of a somatic stem cell and having α2-6 sialic acid as a non-reducing terminal sugar chain ,
The kit according to [12], wherein either one of the probes (a) or (b) is immobilized on a substrate and the other probe is labeled.
For example, the following cases are included.
[14 ′] The somatic stem cell differentiation potential determination kit comprises:
(A) At least one lectin selected from TJAI lectin, SSA lectin, SNA lectin, and PSL1a lectin, and (b) expressed on the surface of somatic stem cells, and α2-6 sialic acid as a non-reducing terminal sugar chain A probe that specifically recognizes the glycoprotein it has,
The kit according to [12], wherein either one of the probes (a) or (b) is immobilized on a substrate and the other probe is labeled.
〔１５〕　体性幹細胞の骨芽細胞又は軟骨細胞への分化ポテンシャルを判別又は評価するためのキットであって、
（ａ）SSAレクチン、SNAレクチン、及びPSL1aレクチンから選択された少なくとも１種のレクチン、
（ｂ）抗CD29抗体、及び抗CD49e抗体から選択された少なくとも１種の抗体、
の組み合わせを含み（ａ）又は（ｂ）のいずれか一方のプローブが基板に固定化され、他方のプローブが標識化されているキット。
　例えば、以下の場合を含む。
〔１５’〕　体性幹細胞の骨芽細胞又は軟骨細胞への分化ポテンシャルを判別又は評価するためのキットであって、（ａ）及び（ｂ）を含むキット；
（ａ）SSAレクチン、SNAレクチン、及びPSL1aレクチンから選択された少なくとも１種のレクチンが固定化された基板（例えば、レクチンアレイ）、
（ｂ）標識化された抗CD29抗体、及び抗CD49e抗体から選択された少なくとも１種の抗体。
〔１５’’〕　体性幹細胞の骨芽細胞又は軟骨細胞への分化ポテンシャルを判別又は評価するためのキットであって、（ａ）～（ｃ）を含むキット；
（ａ）SSAレクチン、SNAレクチン、及びPSL1aレクチンから選択された少なくとも１種のレクチンが固定化された基板、
（ｂ）標識化された抗CD29抗体、及び抗CD49e抗体から選択された少なくとも１種の抗体、
（ｃ）標識量を測定するための装置。
〔１５’’’〕　体性幹細胞の骨芽細胞又は軟骨細胞への分化ポテンシャルを判別又は評価するためのキットであって、（ａ）～（ｄ）を含むキット；
（ａ）ビオチン標識した抗CD29抗体、及び抗CD49e抗体から選択された少なくとも１種の抗体を固定化したストレプトアビジン被覆担体（例えば、ストレプトアビジン被覆磁気ビーズ）、
（ｂ）SSAレクチン、SNAレクチン、及びPSL1aレクチンから選択された少なくとも１種のレクチンが固定化された基板、
（ｃ）蛍光標識化された抗CD29抗体、及び抗CD49e抗体から選択された少なくとも１種の抗体、
（ｄ）蛍光標識量を測定するための装置。 [15] A kit for discriminating or evaluating the differentiation potential of somatic stem cells into osteoblasts or chondrocytes,
(A) at least one lectin selected from SSA lectin, SNA lectin, and PSL1a lectin,
(B) at least one antibody selected from an anti-CD29 antibody and an anti-CD49e antibody,
A kit in which either one of the probes (a) or (b) is immobilized on a substrate and the other probe is labeled.
For example, the following cases are included.
[15 ′] A kit for discriminating or evaluating the differentiation potential of somatic stem cells into osteoblasts or chondrocytes, comprising (a) and (b);
(A) a substrate (for example, a lectin array) on which at least one lectin selected from SSA lectin, SNA lectin, and PSL1a lectin is immobilized;
(B) At least one antibody selected from a labeled anti-CD29 antibody and an anti-CD49e antibody.
[15 ″] A kit for discriminating or evaluating the differentiation potential of somatic stem cells into osteoblasts or chondrocytes, the kit comprising (a) to (c);
(A) a substrate on which at least one lectin selected from SSA lectin, SNA lectin, and PSL1a lectin is immobilized;
(B) at least one antibody selected from a labeled anti-CD29 antibody and an anti-CD49e antibody,
(C) A device for measuring the amount of label.
[15 ′ ″] a kit for discriminating or evaluating the differentiation potential of somatic stem cells into osteoblasts or chondrocytes, the kit comprising (a) to (d);
(A) a streptavidin-coated carrier (for example, streptavidin-coated magnetic beads) on which at least one antibody selected from a biotin-labeled anti-CD29 antibody and an anti-CD49e antibody is immobilized,
(B) a substrate on which at least one lectin selected from SSA lectin, SNA lectin, and PSL1a lectin is immobilized;
(C) at least one antibody selected from a fluorescently labeled anti-CD29 antibody and an anti-CD49e antibody,
(D) An apparatus for measuring the amount of fluorescent label.
〔１６〕　体性幹細胞を含む細胞試料から分化ポテンシャルが高い体性幹細胞を分離又は濃縮する方法であって、
体性幹細胞を含む細胞試料に対し、α2-6シアル酸をエピトープとして認識するプローブを接触させる工程を含む、方法。
　または、以下の様にも表現できる。
〔１６’〕　体性幹細胞を含む細胞試料から分化ポテンシャルが高い体性幹細胞を分離又は濃縮する方法であって、
体性幹細胞を含む細胞試料に対し、α2-6シアル酸をエピトープとして認識するレクチン及び抗体から選択された少なくとも１種のタンパク質を接触させる工程を含む、方法。
〔１６’’〕　体性幹細胞を含む細胞試料から分化ポテンシャルが高い体性幹細胞を分離又は濃縮する方法であって、
（１）生体から脂肪組織、臍帯血、臍帯、羊膜、胎盤、軟骨組織、顎骨もしくは大腿骨由来骨髄、又は多指症由来骨髄組織から選択される組織又は体液から、間葉系幹細胞又は軟骨幹細胞を含有する細胞試料を採取する工程、
（２）α2-6シアル酸をエピトープとして認識するプローブを接触させる工程
を含む、方法。
〔１６’’’〕　α2-6シアル酸をエピトープとして認識するプローブの
体性幹細胞を含む細胞試料から分化ポテンシャルが高い体性幹細胞を分離又は濃縮する方法における使用であって、
当該方法は、
体性幹細胞を含む細胞試料に対し、α2-6シアル酸をエピトープとして認識するレクチン及び抗体から選択された少なくとも１種のタンパク質を接触させる工程を含む、使用。
〔１６’’’’〕 　α2-6シアル酸をエピトープとして認識するプローブの
体性幹細胞を含む細胞試料から分化ポテンシャルが高い体性幹細胞を分離又は濃縮する方法における使用であって、
当該方法は、
生体から単離された脂肪組織、臍帯血、臍帯、羊膜、胎盤、軟骨組織、顎骨もしくは大腿骨由来骨髄、又は多指症由来骨髄組織からから選択される間葉系幹細胞又は軟骨幹細胞含有細胞試料に対し、α2-6シアル酸をエピトープとして認識するプローブを接触させる工程を含む、使用。 [16] A method for separating or concentrating somatic stem cells having a high differentiation potential from a cell sample containing somatic stem cells,
A method comprising a step of contacting a cell sample containing somatic stem cells with a probe that recognizes α2-6 sialic acid as an epitope.
Or it can be expressed as follows.
[16 ′] A method for separating or concentrating somatic stem cells having high differentiation potential from a cell sample containing somatic stem cells,
A method comprising contacting a cell sample containing somatic stem cells with at least one protein selected from a lectin that recognizes α2-6 sialic acid as an epitope and an antibody.
[16 ″] A method for separating or concentrating somatic stem cells having high differentiation potential from a cell sample containing somatic stem cells,
(1) Mesenchymal stem cells or cartilage stem cells from a tissue or body fluid selected from a living body, adipose tissue, umbilical cord blood, umbilical cord, amniotic membrane, placenta, cartilage tissue, jaw bone or femur bone marrow, or polydactyly-derived bone marrow tissue Collecting a cell sample containing
(2) A method comprising contacting a probe that recognizes α2-6 sialic acid as an epitope.
[16 ′ ″] Use of a probe that recognizes α2-6 sialic acid as an epitope in a method for separating or enriching somatic stem cells having high differentiation potential from a cell sample containing somatic stem cells,
The method is
Use comprising the step of contacting a cell sample containing somatic stem cells with at least one protein selected from lectins and antibodies that recognize α2-6 sialic acid as an epitope.
[16 ″ ″] Use of a probe that recognizes α2-6 sialic acid as an epitope in a method for separating or enriching somatic stem cells having high differentiation potential from a cell sample containing somatic stem cells,
The method is
Mesenchymal stem cell or chondrocyte-containing cell sample selected from adipose tissue, umbilical cord blood, umbilical cord, amniotic membrane, placenta, cartilage tissue, jaw bone or femur bone marrow, or polydactyly-derived bone marrow tissue isolated from a living body On the other hand, the method comprises a step of contacting a probe that recognizes α2-6 sialic acid as an epitope.
〔１７〕　前記α2-6シアル酸をエピトープとして認識するプローブを接触させる工程が、フローサイトメトリーによる細胞分画工程、又は前記プローブを固定化した担体による分離工程のいずれかの方法を含む、前記〔１６〕に記載の方法。
　または、前記〔１６’〕を引用して以下の様にも表現できる。
〔１７’〕　前記α2-6シアル酸をエピトープとして認識するレクチン及び抗体から選択された少なくとも１種のタンパク質を接触させる工程が、フローサイトメトリーによる細胞分画工程、又は前記タンパク質を固定化した担体による分離工程のいずれかの方法を含む、前記〔１６’〕に記載の方法。 [17] The step of contacting the probe that recognizes α2-6 sialic acid as an epitope includes any one of a cell fractionation step by flow cytometry, or a separation step using a carrier on which the probe is immobilized. [16] The method described in [16].
Alternatively, it can be expressed as follows by quoting [16 ′].
[17 ′] The step of contacting at least one protein selected from a lectin that recognizes α2-6 sialic acid as an epitope and an antibody is a cell fractionation step by flow cytometry, or a carrier on which the protein is immobilized The method according to [16 ′] above, comprising any method of the separation step according to.
〔１８〕　前記プローブを固定化した担体が、磁気ビーズ又はアフィニティカラムである、前記〔１７〕に記載の方法。
　または、前記〔１７’〕を引用して以下の様にも表現できる。
〔１８’〕　前記タンパク質を固定化した担体が、磁気ビーズ又はアフィニティカラムである、前記〔１７’〕に記載の方法。 [18] The method according to [17] above, wherein the carrier on which the probe is immobilized is a magnetic bead or an affinity column.
Alternatively, the above [17 ′] can be cited as follows.
[18 ′] The method according to [17 ′] above, wherein the carrier on which the protein is immobilized is a magnetic bead or an affinity column.
〔１９〕　α2-6シアル酸をエピトープとして認識するプローブが、TJAIレクチン、SSAレクチン、SNAレクチン、及びPSL1aレクチンから選択された少なくとも１種のレクチンを含むことを特徴とする、前記〔１６〕～〔１８〕のいずれかに記載の方法。
　または、前記〔１６’〕～〔１８’〕を引用して以下の様にも表現できる。
〔１９’〕　α2-6シアル酸をエピトープとして認識するレクチンが、TJAIレクチン、SSAレクチン、SNAレクチン、及びPSL1aレクチンから選択された少なくとも１種のレクチンを含むことを特徴とする、前記〔１６’〕～〔１８’〕のいずれかに記載の方法。 [19] The above-mentioned [16] to [16], wherein the probe that recognizes α2-6 sialic acid as an epitope includes at least one lectin selected from TJAI lectin, SSA lectin, SNA lectin, and PSL1a lectin. [18] The method according to any one of [18].
Alternatively, the above [16 ′] to [18 ′] can be cited as follows.
[19 ′] The lectin that recognizes α2-6 sialic acid as an epitope includes at least one lectin selected from TJAI lectin, SSA lectin, SNA lectin, and PSL1a lectin. ] To [18 '].
〔２０〕　あらかじめ、前記試料中の体性幹細胞を、体性幹細胞表面で発現し、かつα2-6シアル酸を非還元末端糖鎖として有する糖タンパク質を特異的に認識するプローブとの相互作用を利用して濃縮する工程を設けることを特徴とする、前記〔１６〕～〔１９〕のいずれかに記載の方法。
　例えば、体性幹細胞を含む細胞試料から骨芽細胞又は軟骨細胞への分化ポテンシャルが高い体性幹細胞を分離又は濃縮する方法の場合、以下の方法を含む。
〔２０’〕　体性幹細胞を含む細胞試料から骨芽細胞又は軟骨細胞への分化ポテンシャルが高い体性幹細胞を分離又は濃縮する方法であって、
体性幹細胞を含む細胞試料に対し、
　（１）ビオチン標識した抗CD29抗体、及び抗CD49e抗体から選択された少なくとも１種の抗体を固定化したストレプトアビジン被覆担体（例えば、ストレプトアビジン被覆磁気ビーズ）と接触させる工程、
　（２）SSAレクチン、SNAレクチン、及びPSL1aレクチンから選択された少なくとも１種のレクチンを接触させる工程、
　を含む方法。 [20] Interacting with a probe that specifically recognizes a somatic stem cell in the sample on the surface of the somatic stem cell and specifically recognizes a glycoprotein having α2-6 sialic acid as a non-reducing terminal sugar chain The method according to any one of [16] to [19] above, which comprises a step of using and concentrating.
For example, in the case of a method for separating or concentrating somatic stem cells having high differentiation potential into osteoblasts or chondrocytes from a cell sample containing somatic stem cells, the following methods are included.
[20 ′] A method for separating or concentrating somatic stem cells having high differentiation potential into osteoblasts or chondrocytes from a cell sample containing somatic stem cells,
For cell samples containing somatic stem cells
(1) A step of contacting a biotin-labeled anti-CD29 antibody and a streptavidin-coated carrier (for example, streptavidin-coated magnetic beads) to which at least one antibody selected from anti-CD49e antibodies is immobilized,
(2) contacting at least one lectin selected from SSA lectin, SNA lectin, and PSL1a lectin;
Including methods.
〔２１〕　間葉系幹細胞又は軟骨幹細胞含有細胞試料から、骨芽細胞又は軟骨細胞への分化ポテンシャルが高い間葉系幹細胞を分離又は濃縮する方法であって、
前記幹細胞含有試料に対し、α2-6シアル酸をエピトープとして認識するプローブを接触させる工程を含む、方法。
　または、以下の様にも表現できる。
〔２１’〕　間葉系幹細胞又は軟骨幹細胞含有細胞試料から、骨芽細胞又は軟骨細胞への分化ポテンシャルが高い間葉系幹細胞を分離又は濃縮する方法であって、
前記幹細胞含有試料に対し、α2-6シアル酸をエピトープとして認識するレクチン及び抗体から選択された少なくとも１種のタンパク質を接触させる工程を含む、方法。 [21] A method for separating or concentrating mesenchymal stem cells having a high differentiation potential into osteoblasts or chondrocytes from a mesenchymal stem cell or chondrocyte-containing cell sample,
A method comprising the step of contacting the stem cell-containing sample with a probe that recognizes α2-6 sialic acid as an epitope.
Or it can be expressed as follows.
[21 ′] A method for separating or concentrating mesenchymal stem cells having high differentiation potential into osteoblasts or chondrocytes from a mesenchymal stem cell or chondrocyte-containing cell sample,
A method comprising contacting the stem cell-containing sample with at least one protein selected from lectins and antibodies that recognize α2-6 sialic acid as an epitope.
〔２２〕　あらかじめ、前記試料中の体性幹細胞を、CD29又はCD49e糖タンパク質を特異的に認識するプローブとの相互作用を利用して濃縮する工程を設けることを特徴とする、前記〔２１〕に記載の方法。 [22] The method according to [21], wherein a step of concentrating somatic stem cells in the sample in advance using an interaction with a probe that specifically recognizes CD29 or CD49e glycoprotein is provided. The method described.
〔２３〕　CD29又はCD49e糖タンパク質を認識するプローブとの相互作用が、抗CD29抗体又は抗CD49e抗体との免疫反応である、前記〔２２〕に記載の方法。
　または、以下の方法を含む。
〔２３’’〕　間葉系幹細胞又は軟骨幹細胞含有細胞試料から骨芽細胞又は軟骨細胞への分化ポテンシャルが高い体性幹細胞を分離又は濃縮する方法であって、
体性幹細胞を含む細胞試料に対し、
　（１）ビオチン標識した抗CD29抗体、及び抗CD49e抗体から選択された少なくとも１種の抗体を固定化したストレプトアビジン被覆担体（例えば、ストレプトアビジン被覆磁気ビーズ）と接触させる工程、
　（２）SSAレクチン、SNAレクチン、及びPSL1aレクチンから選択された少なくとも１種のレクチンを接触させる工程、
を含む方法。
　ここで、間葉系幹細胞又は軟骨幹細胞含有細胞試料は、生体から、脂肪組織、臍帯血、臍帯、羊膜、胎盤、軟骨組織、顎骨もしくは大腿骨由来骨髄、又は多指症由来骨髄組織から選択される組織又は体液から採取された細胞試料、もしくはその初代培養物、継代培養物、又は株化された培養間葉系幹細胞株又は培養軟骨幹細胞株を含む。以下も同様である。 [23] The method described in [22] above, wherein the interaction with a probe that recognizes CD29 or CD49e glycoprotein is an immune reaction with an anti-CD29 antibody or an anti-CD49e antibody.
Or the following method is included.
[23 ″] a method for separating or concentrating somatic stem cells having high differentiation potential into osteoblasts or chondrocytes from a mesenchymal stem cell or chondrocyte-containing cell sample,
For cell samples containing somatic stem cells
(1) A step of contacting a biotin-labeled anti-CD29 antibody and a streptavidin-coated carrier (for example, streptavidin-coated magnetic beads) to which at least one antibody selected from anti-CD49e antibodies is immobilized,
(2) contacting at least one lectin selected from SSA lectin, SNA lectin, and PSL1a lectin;
Including methods.
Here, the mesenchymal stem cell or chondrocyte-containing cell sample is selected from a living body, adipose tissue, umbilical cord blood, umbilical cord, amniotic membrane, placenta, cartilage tissue, jaw bone or femur bone marrow, or polydactyly-derived bone marrow tissue. Cell samples collected from tissues or body fluids, or primary cultures, subcultures thereof, or established cultured mesenchymal stem cell lines or cultured cartilage stem cell lines. The same applies to the following.
〔２４〕　α2-6シアル酸をエピトープとして認識するプローブを固定化した担体を含むことを特徴とする、分化ポテンシャルの高い体性幹細胞の単離又は濃縮用キット。
　または、以下の様にも表現できる。
〔２４’〕　α2-6シアル酸をエピトープとして認識するレクチン及び抗体から選択された少なくとも１種のタンパク質を固定化した担体を含むことを特徴とする、分化ポテンシャルの高い体性幹細胞の単離又は濃縮用キット。 [24] A kit for isolating or concentrating somatic stem cells with high differentiation potential, comprising a carrier on which a probe that recognizes α2-6 sialic acid as an epitope is immobilized.
Or it can be expressed as follows.
[24 ′] Isolation of somatic stem cells having high differentiation potential, comprising a carrier on which at least one protein selected from a lectin that recognizes α2-6 sialic acid as an epitope and an antibody is immobilized, or Concentration kit.
〔２５〕　α2-6シアル酸をエピトープとして認識するプローブを固定化した担体を含むことを特徴とする、骨芽細胞への分化ポテンシャルの高い骨髄由来間葉系幹細胞の単離又は濃縮用キット。
　または、以下の様にも表現できる。
〔２５’〕　α2-6シアル酸をエピトープとして認識するレクチン及び抗体から選択された少なくとも１種のタンパク質を固定化した担体を含むことを特徴とする、骨芽細胞への分化ポテンシャルの高い骨髄由来間葉系幹細胞の単離又は濃縮用キット。 [25] A kit for isolating or concentrating bone marrow-derived mesenchymal stem cells having high differentiation potential into osteoblasts, comprising a carrier on which a probe that recognizes α2-6 sialic acid as an epitope is immobilized.
Or it can be expressed as follows.
[25 ′] derived from bone marrow with high differentiation potential into osteoblasts, comprising a carrier on which at least one protein selected from a lectin that recognizes α2-6 sialic acid as an epitope and an antibody is immobilized A kit for isolating or concentrating mesenchymal stem cells.
〔２６〕　前記プローブを固定化した担体が、磁気ビーズ又はアフィニティカラムである、前記〔２４〕又は〔２５〕に記載のキット。
　または、前記〔２４’〕及び〔２５’〕を引用して以下の様にも表現できる。
〔２６'〕　前記タンパク質を固定化した担体が、磁気ビーズ又はアフィニティカラムである、前記〔２４’〕又は〔２５’〕に記載のキット。 [26] The kit according to [24] or [25], wherein the carrier on which the probe is immobilized is a magnetic bead or an affinity column.
Alternatively, the above [24 ′] and [25 ′] can be cited as follows.
[26 ′] The kit according to [24 ′] or [25 ′], wherein the carrier on which the protein is immobilized is a magnetic bead or an affinity column.
〔２７〕　さらに、CD29又はCD49e糖タンパク質を認識するプローブを含む前記〔２５〕又は〔２６〕に記載のキット。 [27] The kit according to [25] or [26], further comprising a probe that recognizes CD29 or CD49e glycoprotein.
〔２８〕　間葉系幹細胞又は軟骨幹細胞含有細胞試料を用いた骨芽細胞又は軟骨細胞増殖のための移植材料の調製方法であって、下記の（１）～（５）の工程を含む方法；
（１）採取した間葉系幹細胞又は軟骨幹細胞含有細胞試料をex vivoで拡大培養する工程、
（２）工程（１）で得られた細胞試料を、α2-6シアル酸をエピトープとして認識するプローブを固定化した担体と接触させる工程、
（３）工程（２）で得られた固定化担体をリン酸含有緩衝液により洗浄し、非特異的な結合物を除去する工程、
（４）工程（３）で得られた前記固定化担体を糖類含有緩衝液により洗浄し、前記プローブと結合している細胞を固定化担体から遊離させる工程、
（５）工程（４）で得られた細胞を集めて移植用材料を調製する工程。
　または、以下の様にも表現できる。
〔２８’〕　間葉系幹細胞又は軟骨幹細胞含有細胞試料を用いた骨芽細胞又は軟骨細胞増殖のための移植材料の調製方法であって、下記の（１）～（５）の工程を含む方法；
（１）採取した間葉系幹細胞含有細胞又は軟骨幹細胞試料をex vivoで拡大培養する工程、
（２）工程（１）で得られた細胞試料を、SSAレクチン、SNAレクチン、及びPSL1aレクチンから選択された少なくとも１種のレクチンまたはα2-6シアル酸をエピトープとして認識する抗体から選択された少なくとも１種のタンパク質を固定化した担体と接触させる工程、
（３）工程（２）で得られた固定化担体をリン酸含有緩衝液により洗浄し、非特異的な結合物を除去する工程、
（４）工程（３）で得られた固定化担体を糖類含有緩衝液により洗浄し、前記タンパク質と結合している細胞を固定化担体から遊離させる工程、
（５）工程（４）で得られた細胞を集めて移植用材料を調製する工程。
〔２８’’〕　間葉系幹細胞又は軟骨幹細胞含有細胞試料を用いた骨芽細胞又は軟骨細胞増殖のための移植材料の調製方法であって、下記の（１）～（６）の工程を含む方法；
（１）採取した間葉系幹細胞含有細胞又は軟骨幹細胞試料をex vivoで拡大培養する工程、
（２）工程（１）で得られた細胞試料を、抗CD29抗体、及び抗CD49e抗体から選択された少なくとも１種の抗体との免疫反応を利用して幹細胞を濃縮する工程、
（３）工程（２）で濃縮された細胞試料を、SSAレクチン、SNAレクチン、及びPSL1aレクチンから選択された少なくとも１種のレクチンまたはα2-6シアル酸をエピトープとして認識する抗体から選択された少なくとも１種のタンパク質を固定化した担体と接触させる工程、
（４）工程（３）で得られた固定化担体をリン酸含有緩衝液により洗浄し、非特異的な結合物を除去する工程、
（５）工程（４）で得られた固定化担体を糖類含有緩衝液により洗浄し、前記タンパク質と結合している細胞を固定化担体から遊離させる工程、
（６）工程（５）で得られた細胞を集めて移植用材料を調製する工程。 [28] A method of preparing a transplant material for osteoblast or chondrocyte proliferation using a mesenchymal stem cell or chondrocyte-containing cell sample, comprising the following steps (1) to (5):
(1) A step of expanding the explanted mesenchymal stem cell or chondrocyte-containing cell sample ex vivo,
(2) contacting the cell sample obtained in step (1) with a carrier immobilized with a probe that recognizes α2-6 sialic acid as an epitope;
(3) a step of washing the immobilization support obtained in step (2) with a phosphate-containing buffer to remove non-specific binders,
(4) a step of washing the immobilized carrier obtained in step (3) with a saccharide-containing buffer solution to release cells bound to the probe from the immobilized carrier;
(5) A step of collecting the cells obtained in step (4) and preparing a transplant material.
Or it can be expressed as follows.
[28 ′] A method for preparing a transplant material for osteoblast or chondrocyte proliferation using a mesenchymal stem cell or chondrocyte-containing cell sample, comprising the following steps (1) to (5) ;
(1) a step of expanding the explanted mesenchymal stem cell-containing cell or cartilage stem cell sample ex vivo,
(2) The cell sample obtained in step (1) is at least one selected from an antibody that recognizes at least one lectin selected from SSA lectin, SNA lectin, and PSL1a lectin or α2-6 sialic acid as an epitope. Contacting one kind of protein with an immobilized carrier;
(3) a step of washing the immobilization support obtained in step (2) with a phosphate-containing buffer to remove non-specific binders,
(4) a step of washing the immobilized carrier obtained in step (3) with a saccharide-containing buffer to release cells bound to the protein from the immobilized carrier;
(5) A step of collecting the cells obtained in step (4) and preparing a transplant material.
[28 ″] A method for preparing a transplant material for osteoblast or chondrocyte proliferation using a mesenchymal stem cell or chondrocyte-containing cell sample, comprising the following steps (1) to (6) Method;
(1) a step of expanding the explanted mesenchymal stem cell-containing cell or cartilage stem cell sample ex vivo,
(2) A step of concentrating the stem cells using the immune reaction of the cell sample obtained in step (1) with at least one antibody selected from anti-CD29 antibody and anti-CD49e antibody,
(3) The cell sample concentrated in step (2) is at least one selected from an antibody that recognizes at least one lectin selected from SSA lectin, SNA lectin, and PSL1a lectin or α2-6 sialic acid as an epitope. Contacting one kind of protein with an immobilized carrier;
(4) a step of washing the immobilization support obtained in step (3) with a phosphate-containing buffer to remove non-specific binding substances,
(5) A step of washing the immobilized carrier obtained in step (4) with a saccharide-containing buffer to release cells bound to the protein from the immobilized carrier;
(6) A step of collecting the cells obtained in step (5) and preparing a transplant material.
　本発明により、α2-6シアル酸は間葉系幹細胞などの体性幹細胞の分化ポテンシャルを示す糖鎖マーカーとなることが見いだされた。α2-6シアル酸に特異的に結合性を示すレクチン又は抗体などは、細胞の分化ポテンシャルを判別する試薬として用いることができる。本発明のα2-6シアル酸結合性レクチンの反応性から、移植治療に用いる体性幹細胞の分化ポテンシャルを事前に評価することができるため、体性幹細胞を用いた再生医療への応用が期待される。また、骨髄由来間葉系幹細胞含有培養細胞などの各段階の分化ポテンシャルを含む雑多な細胞集団から、フローサイトメトリー、磁気ビーズなどを用いて一定の品質を有する間葉系幹細胞又は軟骨幹細胞を単離、濃縮することができる。
　また、間葉系幹細胞など体性幹細胞の表面抗原であるCD29及びCD49e糖タンパク質の末端糖鎖としてのα2-6シアル酸量の変化が骨芽細胞又は軟骨細胞への分化ポテンシャル変化との相関性が高いことを見いだした。抗CD29抗体及び／又は抗CD49e抗体とα2-6シアル酸反応性レクチン（SSA,SNA,rPSL1a）とを組み合わせたアッセイ系を用いることで、体性幹細胞の骨芽細胞又は軟骨細胞への分化ポテンシャルを正確に判定、評価することができる。 According to the present invention, it has been found that α2-6 sialic acid serves as a sugar chain marker indicating the differentiation potential of somatic stem cells such as mesenchymal stem cells. Lectins or antibodies that specifically bind to α2-6 sialic acid can be used as reagents for discriminating the differentiation potential of cells. Since the differentiation potential of somatic stem cells used for transplantation treatment can be evaluated in advance from the reactivity of the α2-6 sialic acid-binding lectin of the present invention, application to regenerative medicine using somatic stem cells is expected. The In addition, mesenchymal stem cells or cartilage stem cells having a certain quality can be isolated from a diverse cell population including differentiation potential at each stage, such as bone marrow-derived mesenchymal stem cell-containing cultured cells, using flow cytometry, magnetic beads, etc. Can be separated and concentrated.
In addition, changes in the amount of α2-6 sialic acid as the terminal sugar chain of CD29 and CD49e glycoproteins, which are surface antigens of somatic stem cells such as mesenchymal stem cells, correlate with changes in differentiation potential to osteoblasts or chondrocytes Found it expensive. Differentiation potential of somatic stem cells into osteoblasts or chondrocytes by using an assay system that combines anti-CD29 antibody and / or anti-CD49e antibody and α2-6 sialic acid reactive lectin (SSA, SNA, rPSL1a) Can be accurately determined and evaluated.
脂肪由来間葉系幹細胞（ADSC）の継代培養による増殖能の変化Changes in proliferative ability of subcultured adipose-derived mesenchymal stem cells (ADSC) 脂肪由来間葉系幹細胞（ADSC）の骨芽細胞および脂肪細胞分化ポテンシャルの解析Analysis of osteoblast and adipocyte differentiation potential of adipose-derived mesenchymal stem cells (ADSC) 脂肪由来間葉系幹細胞（ADSC）の継代初期及び継代後期におけるα2-6シアル酸結合性レクチンとの反応性の変化Changes in reactivity of adipose-derived mesenchymal stem cells (ADSC) with α2-6 sialic acid-binding lectin during early passage and late passage 脂肪由来間葉系幹細胞（ADSC）の培養過程におけるα2-6シアル酸結合性レクチン（TJAI、SSA、SNA、及びrPSL1a）との反応性の変化Changes in reactivity with α2-6 sialic acid-binding lectins (TJAI, SSA, SNA, and rPSL1a) during culture of adipose-derived mesenchymal stem cells (ADSC) TJAI、SSA、SNA、及びrPSL1aレクチンの各種糖鎖への結合性TJAI, SSA, SNA, and rPSL1a lectin binding to various sugar chains レクチンへの結合性を観察した各種糖鎖の糖鎖構造Sugar chain structures of various sugar chains observed for binding to lectins フローサイトメトリーによる脂肪由来間葉系幹細胞（ADSC）へのα2-6シアル酸結合性レクチンの反応性Reactivity of α2-6 sialic acid-binding lectin to adipose-derived mesenchymal stem cells (ADSC) by flow cytometry 継代初期及び継代後期のADSCにおけるフローサイトメトリーによるα2-6シアル酸結合性レクチン（TJAI、SSA、SNA、及びrPSL1a）への反応性の変化、及びiPS細胞、Fibroblast細胞との反応性比較Changes in reactivity to α2-6 sialic acid-binding lectins (TJAI, SSA, SNA, and rPSL1a) by flow cytometry in ADSC at early and late passages, and comparison of reactivity with iPS cells and Fibroblast cells 各種細胞に発現する糖タンパク質シアル酸結合様式：（Ａ）シアル酸が１個付加された画分でのα2-6シアル酸とα2-3シアル酸の発現割合　（Ｂ）シアル酸が２個付加された画分でのα2-6シアル酸とα2-3シアル酸の発現割合Glycoprotein sialic acid binding mode expressed in various cells: (A) Expression ratio of α2-6 sialic acid and α2-3 sialic acid in the fraction with one sialic acid added (B) Two sialic acids added Of α2-6 sialic acid and α2-3 sialic acid in selected fractions 多指症軟骨組織由来軟骨細胞（軟骨幹細胞：Yub621c株）の継代培養：（Ａ）継代培養細胞の増殖性　（Ｂ）フローサイトメトリーによるα2-6シアル酸結合性レクチンの反応性　（Ｃ）分化ポテンシャルSubculture of polydactyly cartilage tissue-derived chondrocytes (chondral stem cells: Yub621c strain): (A) Proliferation of subcultured cells (B) Reactivity of α2-6 sialic acid-binding lectin by flow cytometry (C ) Differentiation potential 多指症骨髄由来間葉系幹細胞(Yub622株)の継代培養：（Ａ）継代培養細胞の増殖性　（Ｂ）フローサイトメトリーによるα2-6シアル酸結合性レクチンの反応性　（Ｃ）分化ポテンシャルSubculture of polydactyly marrow derived mesenchymal stem cells (Yub622 strain): (A) Proliferation of subcultured cells (B) Reactivity of α2-6 sialic acid binding lectin by flow cytometry (C) Differentiation potential 骨髄由来間葉系幹細胞（Lonza株）の継代培養：（Ａ）継代培養細胞の増殖性　（Ｂ）フローサイトメトリーによるα2-6シアル酸結合性レクチンの反応性Subculture of bone marrow-derived mesenchymal stem cells (Lonza strain): (A) Proliferation of subcultured cells (B) Reactivity of α2-6 sialic acid-binding lectin by flow cytometry 多指症骨髄由来間葉系幹細胞(Yub622株)の継代初期及び継代後期細胞：（Ａ）フローサイトメトリーによるα2-6シアル酸結合性レクチンの反応性　（Ｂ）骨芽細胞分化（Lonza、2週間）Early and late passage cells of polydactyloid bone marrow-derived mesenchymal stem cells (Yub622 strain): (A) Reactivity of α2-6 sialic acid-binding lectin by flow cytometry (B) Osteoblast differentiation (Lonza 2 weeks) 多指症骨髄由来間葉系幹細胞(Yub10F株)の継代初期及び継代後期細胞：（Ａ）フローサイトメトリーによるα2-6シアル酸結合性レクチンの反応性　（Ｂ）骨芽細胞分化（Lonza、2週間）Early and late passage cells of polydactyloid bone marrow-derived mesenchymal stem cells (Yub10F strain): (A) Reactivity of α2-6 sialic acid-binding lectin by flow cytometry (B) Osteoblast differentiation (Lonza 2 weeks) 継代初期と後期の多指症由来軟骨幹細胞であるYub621c株、Yub625株から分化誘導した軟骨細胞の軟骨マーカーの発現、図中（＋）は、軟骨分化誘導後の細胞、（－）は分化誘導をしていないことを表す。継代初期（Yub621c P7、Yub625 P5）で軟骨分化率が高かったのに対し、継代後期（Yub625 P22）では軟骨分化率が極めて低いことが示された。Expression of cartilage markers in chondrocytes derived from Yub621c and Yub625 strains, which are polydactyly-derived chondrogenic stem cells in early and late passages, (+) indicates cells after induction of cartilage differentiation, (-) indicates differentiation Indicates that no guidance has been given. It was shown that the cartilage differentiation rate was high in the early passage (Yub621cYP7, Yub625 P5), whereas the cartilage differentiation rate was extremely low in the late passage (Yub625 P22). 継代初期と後期のYub621c株、Yub625株を軟骨分化誘導後のHE染色像とアルシアンブルー染色像HE staining image and Alcian blue staining image after induction of cartilage differentiation in early and late passages of Yub621c and Yub625 strains 多指症由来軟骨幹細胞(Yub621株)の継代初期及び継代後期細胞のフローサイトメトリーによるα2-6シアル酸結合性レクチンの反応性Reactivity of α2-6 sialic acid-binding lectin by flow cytometry of early and late passage cells of polydactyly derived cartilage stem cells (Yub621 strain) 多指症由来軟骨幹細胞(Yub625株)の継代初期及び継代後期細胞のフローサイトメトリーによるα2-6シアル酸結合性レクチンの反応性Reactivity of α2-6 sialic acid-binding lectin by flow cytometry of early and late passage cells of polydactyly derived cartilage stem cells (Yub625 strain) 異なる継代数の脂肪由来間葉系幹細胞（ADSC、Ａ）と多指症由来軟骨幹細胞（Yub621c、Ｂ）の細胞抽出液からCD29抗体で免疫沈降したCD29へのα2-6シアル酸結合性レクチン（rPSL1a）の反応性変化Α2-6 sialic acid binding lectin to CD29 immunoprecipitated with CD29 antibody from cell extracts of adipose-derived mesenchymal stem cells (ADSC, A) and polydactyly-derived chondrocyte stem cells (Yub621c, B) of different passage numbers ( rPSL1a) reactivity change 異なる継代数の脂肪由来間葉系幹細胞（ADSC、Ａ）と多指症骨髄由来間葉系幹細胞（Yub621c、Ｂ）の細胞抽出液からCD49e抗体で免疫沈降したCD49eへのα2-6シアル酸結合性レクチン（rPSL1a）の反応性変化Α2-6 sialic acid binding to CD49e immunoprecipitated with CD49e antibody from cell extracts of adipose-derived mesenchymal stem cells (ADSC, A) and polydactyly bone marrow-derived mesenchymal stem cells (Yub621c, B) of different passage numbers In the reactivity of the bacterial lectin (rPSL1a) 異なる継代数の脂肪由来間葉系幹細胞（ADSC、Ａ）と多指症由来軟骨幹細胞（Yub621c、Ｂ）の細胞抽出液からCD13抗体で免疫沈降したCD13へのα2-6シアル酸結合性レクチン（rPSL1a）の反応性変化Α2-6 sialic acid-binding lectin to CD13 immunoprecipitated with CD13 antibody from cell extracts of adipose-derived mesenchymal stem cells (ADSC, A) and polydactyly-derived chondrocyte stem cells (Yub621c, B) of different passage numbers rPSL1a) reactivity change iPS細胞（201B7株）の細胞抽出液から免疫沈降したCD29（Ａ）とCD49e（B）へのα2-6シアル酸結合性レクチン（rPSL1a）の反応性変化Changes in reactivity of α2-6 sialic acid-binding lectin (rPSL1a) to CD29 (A) and CD49e (B) immunoprecipitated from cell extracts of iPS cells (strain 201B7) ４種のα2-6シアル酸結合性レクチン(SNA、SSA、TJAI、rPSL1a)とCD29抗体のサンドイッチアッセイ系の構築Construction of sandwich assay system of 4 kinds of α2-6 sialic acid binding lectins (SNA, SSA, TJAI, rPSL1a) and CD29 antibody ４種のα2-6シアル酸結合性レクチン(SNA、SSA、TJAI、rPSL1a)とCD49e抗体のサンドイッチアッセイ系の構築Construction of sandwich assay system of 4 kinds of α2-6 sialic acid binding lectins (SNA, SSA, TJAI, rPSL1a) and CD49e antibody シアリダーゼ処理した多指症由来軟骨幹細胞（Yub621c）の骨芽細胞分化。図中、(a)は分化誘導なし、(b)(c)(d)は骨芽細胞への分化誘導をした細胞を示す。(b)はシアリダーゼ処理のないコントロールであり、(c)はシアリダーゼ処理後に分化誘導処理を行った場合、(d)はシアリダーゼ存在下で分化誘導を行った場合である。Osteoblast differentiation of sialidase-treated polydactyly-derived cartilage stem cells (Yub621c). In the figure, (a) shows no differentiation induction, and (b), (c) and (d) show cells induced to differentiate into osteoblasts. (b) is a control without sialidase treatment, (c) is a case where differentiation induction treatment is performed after sialidase treatment, and (d) is a case where differentiation induction is performed in the presence of sialidase.
１．α2-6シアル酸について
　本発明において、「α2-6シアル酸」というときは、一般に、N-アセチルノイラミン酸が、ガラクトースの6位の位置の水酸基とα2-6結合した糖鎖である「Neu5Acα2-6Gal」及びN-グリコリル型ノイラミン酸が同様にα2-6結合した糖鎖「Neu5Gcα2-6Gal」の両方を指す。しかし、ヒト由来のα2-6シアル酸を非還元末端に有する糖タンパク質では、α2-6シアル酸がノイラミン酸の5位のアミノ基がアセチル化された「Neu5Acα2-6Gal」の場合しか体内で生合成されないため、本発明では主として「Neu5Acα2-6Gal」のみを対象とする。以下の説明でもα2-6シアル酸が「Neu5Acα2-6Gal」であるとして説明する。
　したがって、本発明においては、典型的にはヒト間葉系幹細胞などのヒト体性幹細胞の細胞表面の糖タンパク質の非還元末端に存在するα2-6シアル酸（Neu5Acα2-6Gal）を、幹細胞の分化ポテンシャルの評価、判定用の糖鎖マーカー、すなわち検出対象エピトープとする。
　なお、ブタ、マウスなどヒト以外の哺乳動物では、α2-6シアル酸として「Neu5Gcα2-6Gal」である場合も含むので、対象幹細胞がヒト以外の哺乳動物に由来する場合は、「Neu5Acα2-6Gal」及び「Neu5Gcα2-6Gal」が本発明の分化ポテンシャルを判定、評価するための検出対象エピトープとなる。 1. About α2-6 sialic acid In the present invention, “α2-6 sialic acid” is generally a sugar chain in which N-acetylneuraminic acid is α2-6 linked to a hydroxyl group at the 6-position of galactose. Similarly, it refers to both “Neu5Acα2-6Gal” and the sugar chain “Neu5Gcα2-6Gal” in which N-glycolyl-type neuraminic acid is similarly α2-6 linked. However, in glycoproteins with 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 position 5 of neuraminic acid is acetylated. Since it is not synthesized, the present invention mainly targets only “Neu5Acα2-6Gal”. In the following description, α2-6 sialic acid is described as “Neu5Acα2-6Gal”.
Therefore, in the present invention, α2-6 sialic acid (Neu5Acα2-6Gal), which is typically present at the non-reducing end of the glycoprotein on the cell surface of human somatic stem cells such as human mesenchymal stem cells, is differentiated from stem cells. A sugar chain marker for potential evaluation and determination, that is, an epitope to be detected.
In addition, in mammals other than humans such as pigs and mice, the case where the target stem cells are derived from mammals other than humans includes “Neu5Gcα2-6Gal” as α2-6 sialic acid, so that “Neu5Acα2-6Gal” And “Neu5Gcα2-6Gal” is the epitope to be detected for determining and evaluating the differentiation potential of the present invention.
　シアル酸（N-アセチルノイラミン酸）は、細胞表面の複合糖鎖末端に存在するカルボキシル基を持つ酸性糖であって、シアル酸残基ともよばれ、還元末端側の単糖の6位の水酸基に結合したタイプのα2-6シアル酸及び3位の水酸基に結合したタイプのα2-3シアル酸が知られている。またある特殊な糖タンパク質や糖脂質では、シアル酸の８位の水酸基に結合したタイプのα2-8シアル酸も知られている。シアル酸はムチンや血清タンパク質などの分泌糖タンパク質、膜タンパク質の糖鎖の非還元末端に存在し、タンパク質や細胞の表面に負電荷を付与することにより、細胞同士の凝集を抑えたり、タンパク質の溶解性を高めたり、プロテアーゼからの攻撃から防御したりする機能をもつ。またウイルスや内在性リセプターのリガンドとして機能することが知られている。主に免疫細胞上に発現する内在性レクチンの一種であるシグレックに認識されて、免疫細胞の機能を調節していることも知られている。
　とりわけ、α2-6シアル酸は、血管内皮細胞表面に存在する接着分子PECAMの細胞内局在と機能を調節して、細胞死を制御しているという報告（非特許文献４）もあり、α2-6シアル酸を標的とした抗血管新生阻害剤の可能性も検討されている。癌細胞表面のN-結合型糖タンパク質中でのα2-6シアル酸の発現増加が、癌の進行、転移、予後不良と関係があることも報告されている(非特許文献５)。
　また、気道の上皮細胞表面のシアル酸残基がヒトではα2-6シアル酸であるのに対してトリではα2-3シアル酸、ブタではα2-6シアル酸とα2-3シアル酸の両方の発現であることが、トリインフルエンザが直接ヒトに感染することがまれであり、通常ブタを介して感染する理由とされている。 Sialic acid (N-acetylneuraminic acid) is an acidic sugar with a carboxyl group that exists at the end of a complex sugar chain on the cell surface. There are known α2-6 sialic acid of the type bonded to and α2-3 sialic acid of the type bonded to the hydroxyl group at the 3-position. As a special glycoprotein or glycolipid, α2-8 sialic acid of the type bonded to the hydroxyl group at the 8-position of sialic acid is also known. Sialic acid is present at the non-reducing end of sugar chains of secreted glycoproteins such as mucin and serum proteins, and membrane proteins. By adding a negative charge to the surface of proteins and cells, aggregation of cells can be suppressed, It has functions to increase solubility and protect against attacks 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 kind of endogenous lectin expressed mainly on immune cells, and regulates the functions of immune cells.
In particular, there is a report that α2-6 sialic acid regulates cell death by regulating the intracellular localization and function of the adhesion molecule PECAM present on the surface of vascular endothelial cells (Non-Patent Document 4). The possibility of anti-angiogenesis inhibitors targeting -6 sialic acid is also being investigated. It has also been reported that increased expression of α2-6 sialic acid in N-linked glycoproteins on the surface of cancer cells is associated with cancer progression, metastasis, and poor prognosis (Non-patent Document 5).
In addition, sialic acid residues on the airway epithelial cells are α2-6 sialic acid in humans compared to α2-3 sialic acid in birds, and both α2-6 sialic acid and α2-3 sialic acid in pigs. Expression is a rare reason that avian influenza directly infects humans and is usually the reason for infection via swine.
　近年、再生治療への実用化が進んでいる間葉系幹細胞（MSC）から骨芽細胞への分化技術においても、分化誘導後の細胞表面の複合糖鎖の糖鎖構造が質量分析によって解析され、骨芽細胞への分化が十分に進んだ場合に増加する糖鎖構造が複数種類同定されている。その中には、α2-6結合かα2-3結合かは不明であるがシアル酸が非還元末端に結合した糖鎖構造も見いだされ、骨芽細胞を同定するためのマーカーとして用いることが提案されている（特許文献８）。
　しかし、分化誘導前の間葉系幹細胞などの体性幹細胞の品質評価に直接関わる分化ポテンシャルと細胞表面複合糖鎖における糖鎖構造変化との相関について報告された例はない。本発明において初めて、間葉系幹細胞など体性幹細胞表面の複合糖鎖における非還元末端のα2-6シアル酸含有糖鎖の発現量が、当該細胞の分化ポテンシャルと相関性を有することが見いだされ、α2-6シアル酸が体性幹細胞の分化ポテンシャルの程度の優れた指標となることがわかった。すなわち、例えば、間葉系幹細胞などの体性幹細胞表面におけるα2-6シアル酸の発現量を、α2-6シアル酸結合性プローブを用いて定量測定することにより、体性幹細胞の分化ポテンシャルを判別することが可能となった。
　また、間葉系幹細胞のうちでも骨髄由来間葉系幹細胞表面のα2-6シアル酸発現量については、各種細胞への分化ポテンシャルのうちでも特に骨芽細胞又は軟骨細胞への分化ポテンシャルとの相関性が高い傾向が見いだされており、α2-6シアル酸を骨芽細胞又は軟骨細胞への分化ポテンシャルの評価、判定用マーカーとして用いることができる。 In recent years, even in the differentiation technology from mesenchymal stem cells (MSC) to osteoblasts, which has been put to practical use for regenerative treatment, the sugar chain structure of the complex sugar chain on the cell surface after differentiation induction has been analyzed by mass spectrometry. Several types of sugar chain structures have been identified that increase when differentiation into osteoblasts is sufficiently advanced. Among them, it is unknown whether it is α2-6 or α2-3, but a sugar chain structure in which sialic acid is bound to the non-reducing end was also found, and it was proposed to use it as a marker for identifying osteoblasts. (Patent Document 8).
However, there has been no report on the correlation between the differentiation potential directly related to the quality evaluation of somatic stem cells such as mesenchymal stem cells before differentiation induction and the sugar chain structure change in cell surface complex sugar chains. For the first time in the present invention, it has been found that the expression level of non-reducing end α2-6 sialic acid-containing sugar chains in complex sugar chains on the surface of somatic stem cells such as mesenchymal stem cells has a correlation with the differentiation potential of the cells. Thus, α2-6 sialic acid was found to be an excellent indicator of the degree of differentiation potential of somatic stem cells. That is, for example, the differentiation potential of somatic stem cells can be determined by quantitatively measuring the expression level of α2-6 sialic acid on the surface of somatic stem cells such as mesenchymal stem cells using an α2-6 sialic acid-binding probe. It became possible to do.
Among the mesenchymal stem cells, the expression level of α2-6 sialic acid on the surface of bone marrow-derived mesenchymal stem cells is particularly correlated with the differentiation potential of osteoblasts or chondrocytes among the differentiation potentials of various cells. It has been found that α2-6 sialic acid can be used as a marker for evaluation and determination of differentiation potential into osteoblasts or chondrocytes.
２．α2-6シアル酸結合性プローブについて
（２－１）α2-6シアル酸結合性プローブ
　本発明において、体性幹細胞表面のα2-6シアル酸を検出するためには、α2-6シアル酸を特異的にエピトープとして認識するプローブを用いる。特定の糖鎖構造を認識して結合するタンパク質は「レクチン」と総称されており、典型的なα2-6シアル酸結合性プローブとしては、各種のα2-6シアル酸結合性レクチンが挙げられるが、これらに限定されるものではなく、α2-6シアル酸を糖鎖抗原（エピトープ）として認識する抗α2-6シアル酸抗体又はその誘導体も好ましく用いられる。抗α2-6シアル酸抗体を調製するためには、例えば、α2-6シアル酸をそのままもしくはアルブミンやKLHなどのキャリアータンパク質に結合させて動物に免疫することで得られ、また既知の抗α2-6シアル酸抗体（非特許文献５）を用いることもできる。当該抗体は、α2-6シアル酸をエピトープとして認識して特異的に結合する能力を有するものであれば、特に限定はなく、ポリクローナル抗体、モノクローナル抗体、及びその抗原認識部位が保存されたFabフラグメントなどの抗体フラグメントの他、ヒト化抗体、単鎖抗体などを使用することもできる。本発明において、「抗α2-6シアル酸抗体」、又は「α2-6シアル酸をエピトープとして認識する抗体」などというとき、抗体のみならず、その抗原認識部位が保存されたフラグメント又は誘導体などを含めた用語として用いる。
　また、本発明のα2-6シアル酸結合性プローブは単独で用いても良いが、複数のプローブを組み合わせて用いても良い。例えば、複数種のα2-6シアル酸結合性レクチン、またはさらに抗α2-6シアル酸抗体も併用することができる。 2. About α2-6 sialic acid binding probe (2-1) α2-6 sialic acid binding probe In the present invention, α2-6 sialic acid is specifically detected in order to detect α2-6 sialic acid on the surface of somatic stem cells. A probe that recognizes 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 present invention is not limited thereto, and an anti-α2-6 sialic acid antibody or a derivative thereof that recognizes α2-6 sialic acid as a sugar chain antigen (epitope) is also preferably used. In order to prepare an anti-α2-6 sialic acid antibody, for example, α2-6 sialic acid can be obtained by immunizing an animal as it is or bound to a carrier protein such as albumin or KLH. A 6-sialic acid antibody (Non-patent Document 5) can also be used. The antibody is not particularly limited as long as it has the ability to specifically recognize and bind to α2-6 sialic acid as an epitope. Polyclonal antibody, monoclonal antibody, and Fab fragment in which the antigen recognition site is conserved In addition to antibody fragments such as these, humanized antibodies, single chain antibodies, and the like can also be used. In the present invention, when referring to “anti-α2-6 sialic acid antibody” or “antibody recognizing α2-6 sialic acid as an epitope” or the like, not only the antibody but also a fragment or derivative in which the antigen recognition site is conserved, etc. Used as an included term.
In addition, 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 further anti-α2-6 sialic acid antibodies can be used in combination.
（２－２）α2-6シアル酸結合性レクチン
　本発明のα2-6シアル酸結合性プローブとして用いられるα2-6シアル酸結合性レクチンとしては、細胞表面の糖タンパク質の糖鎖の非還元末端のα2-6シアル酸を認識できるレクチンであればいずれのレクチンを用いてもよい。例えば、TJAIレクチン（Trichosanthes japonica lectin-I)、SSAレクチン（Sambucus sieboldiana lectin)、SNAレクチン（Sambucus nigra lectin (SNA)、PSL1aレクチン（Polyporus squamosus lectin)を用いることができる。
　TJAIレクチンはキカラスウリから、SSAレクチンはニホンニワトコから、またSNAレクチンはニワトコからそれぞれ抽出することもできるが、TJAI及びSSAレクチンは生化学工業株式会社、SNAはVECTOR Laboratories社により市販されている。PSL1aレクチンはアミヒラタケから抽出することもできるが、α2-6シアル酸特異性を保持したリコンビナント体のrPSL1aレクチンが和光純薬工業により市販されている。いずれのレクチンも細胞表面の複合糖質の非還元末端を構成するα2-6シアル酸（Neu5Acα2-6Gal及びNeu5Gcα2-6Gal）を特異的に認識することが知られている。 (2-2) α2-6 sialic acid binding lectin The α2-6 sialic acid binding lectin used as the α2-6 sialic acid binding probe of the present invention is a non-reducing end of a sugar chain of a glycoprotein on the cell surface. Any lectin may be used as long as it can recognize α2-6 sialic acid. For example, TJAI lectin (Trichosanthes japonica lectin-I), SSA lectin (Sambucus sieboldiana lectin), SNA lectin (Sambucus nigra lectin (SNA), PSL1a lectin (Polyporus squamosus lectin) can be used.
TJAI lectin can be extracted from Kikarasuuri, SSA lectin can be extracted from Japanese elderberry, and SNA lectin can be extracted from elderberry, but TJAI and SSA lectin are commercially available from Seikagaku Corporation and SNA is sold by VECTOR Laboratories. Although PSL1a lectin can be extracted from Ahihiratake, recombinant rPSL1a lectin that retains α2-6 sialic acid specificity is commercially available from Wako Pure Chemical Industries. Both lectins are known to specifically recognize α2-6 sialic acid (Neu5Acα2-6Gal and Neu5Gcα2-6Gal) constituting the non-reducing end of glycoconjugates on the cell surface.
　これら４種のレクチン（TJAI、SSA、SNA、rPSL1a）それぞれの各種複合糖鎖に対する反応性をフロンタルアフィニティークロマトグラフィー（FAC）で解析した結果を図５に示す。いずれのレクチンもα2-6シアル酸残基を有する糖鎖501～506のみに特異的に結合することが見て取れる。同じシアル酸残基のうちでもα2-3シアル酸残基のみを含む糖鎖601及び602に対しては全く結合性を示さず、α2-3シアル酸と共にα2-6シアル酸残基を有している糖鎖506に対しては結合性を有していることがわかる。このことから、上記４種のレクチンが、α2-6シアル酸残基に結合特異性を有するレクチンであることが確認できる。
　これら４種類のレクチン以外のα2-6シアル酸特異的レクチンとしては、羅漢果科植物から抽出されたラカンカレクチン（特許文献８）等が例示できる。その他、レクチンフロンティアデータベース（LfDB）等からの情報に従って入手可能である。また、α2-6シアル酸を含む糖鎖アレイを用いて、天然もしくは人工のタンパク質試料からスクリーニングすることもできる。 FIG. 5 shows the results of frontal affinity chromatography (FAC) analysis of the reactivity of each of these four lectins (TJAI, SSA, SNA, rPSL1a) to various complex sugar chains. It can be seen that any lectin specifically binds only to sugar chains 501 to 506 having α2-6 sialic acid residues. Among the same sialic acid residues, it has no binding property to sugar chains 601 and 602 containing only α2-3 sialic acid residues, and has α2-6 sialic acid residues together with α2-3 sialic acid. It can be seen that the sugar chain 506 has binding properties. From this, it can be confirmed that the above four lectins are lectins having binding specificity to α2-6 sialic acid residues.
Examples of α2-6 sialic acid-specific lectins other than these four types of lectins include lacanka lectins extracted from Arachnaceae plants (Patent Document 8). In addition, it can be obtained according to information from the lectin frontier database (LfDB) or the like. It is also possible to screen from natural or artificial protein samples using a sugar chain array containing α2-6 sialic acid.
（２－３）α2-6シアル酸含有糖タンパク質
　本発明の検出対象となる体性幹細胞表面のα2-6シアル酸は、体性幹細胞表面に存在する糖タンパク質の細胞外ドメイン中のアスパラギンに結合した複合糖鎖（N-結合型糖鎖）の非還元末端に存在している。
　つまり、継代初期など分化ポテンシャルの高い時期の体性幹細胞表面においては、非還元末端にα2-6シアル酸を有するN-結合型糖タンパク質（α2-6シアル酸含有N-結合型糖タンパク質）が顕著に高発現している可能性が高い。またはN-型糖鎖中の非還元末端にα2-6シアル酸が大量に付加され、α2-6シアル酸含有量が増大している可能性もあるが、前者の場合であれば、体性幹細胞の分化ポテンシャルを判定、評価するための指標として、非還元末端にα2-6シアル酸を有するN-結合型糖タンパク質（α2-6シアル酸含有N-結合型糖タンパク質）のコアタンパク質に関する体性幹細胞表面での発現量を利用することができる。
　α2-6シアル酸含有N-結合型糖タンパク質のコアタンパク質は、α2-6シアル酸を指標に採取した糖タンパク質を同定すればいいので、当業者は容易に決定することができる。例えば継代初期の体性幹細胞の膜画分もしくはタンパク質画分をTJAI、SSA、SNA、rPSL1aレクチンの少なくともいずれか１種を固定化したビーズでα2-6シアル酸を含有する糖タンパク質を濃縮後、そのコアタンパク質に対する抗体などで検出するか、質量分析計等でアミノ酸配列を調べれば容易に決定できる。 (2-3) α2-6 sialic acid-containing glycoprotein α2-6 sialic acid on the surface of the somatic stem cell to be detected in the present invention binds to asparagine in the extracellular domain of the glycoprotein present on the surface of the somatic stem cell. Present at the non-reducing end of the complex sugar chain (N-linked sugar chain).
In other words, N-linked glycoproteins with α2-6 sialic acid at the non-reducing end (α2-6 sialic acid-containing N-linked glycoprotein) on the surface of somatic stem cells with high differentiation potential such as early passage Is highly likely to be highly expressed. Or a large amount of α2-6 sialic acid may be added to the non-reducing end in the N-type sugar chain, increasing the content of α2-6 sialic acid. A body related to the core protein of N-linked glycoprotein (α2-6 sialic acid-containing N-linked glycoprotein) having α2-6 sialic acid at the non-reducing end as an index for judging and evaluating the differentiation potential of stem cells The expression level on the surface of sex stem cells can be used.
The core protein of the α2-6 sialic acid-containing N-linked glycoprotein can be easily determined by those skilled in the art because it can identify the glycoprotein collected using α2-6 sialic acid as an index. For example, the membrane fraction or protein fraction of somatic stem cells in the early passage is enriched with α2-6 sialic acid-containing glycoproteins using beads immobilized with at least one of TJAI, SSA, SNA, and rPSL1a lectin It can be easily determined by detecting with an antibody against the core protein or by examining the amino acid sequence with a mass spectrometer or the like.
　このようなN-結合型糖鎖の非還元末端にα2-6シアル酸を有する糖タンパク質のコアタンパク質もまた、体性幹細胞の分化ポテンシャルを判定、評価用のマーカーとなる可能性がある。すなわち、その場合は当該コアタンパク質の発現量を既知の手法によりmRNAレベルで又はタンパク質レベルで測定することで、体性幹細胞の分化ポテンシャルの判定、評価できることになる。具体的には、例えばコアタンパク質mRNAレベルを定量的RT-PCRで測定する、次世代シーケンサーで測定する、DNAマイクロアレイで測定する、などの方法、コアタンパク質特異的な抗体を作製、もしくは購入して、ELISA法、フローサイトメトリー、ウエスタンブロット、免疫染色、又は抗体オーバーレイ・レクチンマイクロアレイによるサンドイッチアッセイ法が用いられる。 Such a core protein of a glycoprotein having α2-6 sialic acid at the non-reducing end of the N-linked sugar chain may also be a marker for determining and evaluating the differentiation potential of somatic stem cells. That is, in that case, the differentiation potential of somatic stem cells can be determined and evaluated by measuring the expression level of the core protein at the mRNA level or at the protein level by a known method. Specifically, for example, the core protein mRNA level is measured by quantitative RT-PCR, the measurement is performed by a next-generation sequencer, the measurement is performed by a DNA microarray, and a core protein-specific antibody is prepared or purchased. ELISA, flow cytometry, Western blot, immunostaining, or sandwich assay using antibody overlay lectin microarrays.
（２－４）骨芽細胞又は軟骨細胞への分化ポテンシャル判定用糖タンパク質
　本発明者らは、分化ポテンシャル判定用コアタンパク質を特定するために、間葉系幹細胞表面に多く発現しているα2-6シアル酸含有N-結合型糖タンパク質抗原を検索し、その候補としてCD29、CD49e及びCD13糖タンパク質を選定した。
　次いで、骨髄由来間葉系幹細胞及び軟骨幹細胞の継代数の異なる細胞を用意し、それぞれの疎水性画分（膜画分）を、抗CD29抗体、抗CD49e抗体及び抗CD13抗体のそれぞれを固定化したビーズで免疫沈降させた。得られたCD29、CD49e及びCD13糖タンパク質をゲル泳動させて異なる継代数に応じたrPSL1aレクチンとの反応量の変化を観察した。
　その結果、CD29及びCD49e糖タンパク質のα2-6シアル酸含量が、継代数の増加に従って明確に減少する傾向を示したことから、CD29及びCD49e糖タンパク質がα2-6シアル酸含有量測定のためのコアタンパク質として有効性が高いと判定した。すなわち、骨髄由来間葉系幹細胞及び軟骨幹細胞表面のCD29及びCD49eが有しているα2-6シアル酸量が、継代を重ねるに従って低下する骨芽細胞又は軟骨細胞への分化ポテンシャルの程度を反映し、高い相関性をもって低下することを意味する。骨髄由来間葉系幹細胞及び軟骨幹細胞表面のCD29及びCD49e発現量と、CD29及びCD49eが含有するα2-6シアル酸とを同時に測定することで、より定量的に骨芽細胞又は軟骨細胞への分化ポテンシャルの判定が可能となる。
　なお、CD29及びCD49eは、いずれも細胞接着の他、細胞膜を介したシグナリングに関与するインテグリンファミリーに属する。CD29は、インテグリンβ1鎖であり、CD49eは、インテグリンα5鎖である。 (2-4) Glycoprotein for determining differentiation potential into osteoblasts or chondrocytes In order to identify a core protein for determining differentiation potential, the present inventors have expressed α2- that is highly expressed on the surface of mesenchymal stem cells. 6-sialic acid-containing N-linked glycoprotein antigens were searched and CD29, CD49e and CD13 glycoproteins were selected as candidates.
Next, prepare cells with different passage numbers of bone marrow-derived mesenchymal stem cells and cartilage stem cells, and immobilize each hydrophobic fraction (membrane fraction) with anti-CD29 antibody, anti-CD49e antibody and anti-CD13 antibody. Were immunoprecipitated with the prepared beads. The resulting CD29, CD49e and CD13 glycoproteins were subjected to gel electrophoresis, and changes in the amount of reaction with rPSL1a lectin according to different passage numbers were observed.
As a result, since the α2-6 sialic acid content of CD29 and CD49e glycoproteins tended to decrease clearly as the passage number increased, CD29 and CD49e glycoproteins were used to measure α2-6 sialic acid content. The effectiveness as a core protein was judged to be high. That is, the amount of α2-6 sialic acid possessed by CD29 and CD49e on the surface of bone marrow-derived mesenchymal stem cells and cartilage stem cells reflects the degree of differentiation potential to osteoblasts or chondrocytes that decreases with successive passages. In other words, it means a decrease with high correlation. Differentiating into osteoblasts or chondrocytes more quantitatively by simultaneously measuring the expression level of CD29 and CD49e on bone marrow-derived mesenchymal stem cells and cartilage stem cells and α2-6 sialic acid contained in CD29 and CD49e The potential can be determined.
Both CD29 and CD49e belong to the integrin family involved in signaling through cell membranes in addition to cell adhesion. CD29 is the integrin β1 chain and CD49e is the integrin α5 chain.
　さらに、α2-6シアル酸反応性レクチンのうちでもSSA、SNA及びrPSL1aは、CD29及びCD49e上のα2-6シアル酸発現量の減少割合を、より的確に反映するレクチンであることが判明した。つまり、抗CD29抗体又は抗CD49e抗体を用いるアッセイ系で用いるレクチンとしては、SSA、SNA及びrPSL1aが適していることがわかった。
　具体的には、抗CD29抗体又は抗CD49e抗体と共に、α2-6シアル酸反応性レクチン（SSA、SNA、rPSL1aなど）もしくは抗α2-6シアル酸抗体とを用いたサンドイッチアッセイ系により、被検骨髄由来間葉系幹細胞又は被検軟骨幹細胞の、骨芽細胞又は軟骨細胞への分化ポテンシャルを正確に評価、判定することが可能となった。 Furthermore, among the α2-6 sialic acid-responsive lectins, SSA, SNA and rPSL1a were found to be lectins that more accurately reflect the decreasing rate of the expression level of α2-6 sialic acid on CD29 and CD49e. That is, it was found that SSA, SNA and rPSL1a are suitable as lectins used in an assay system using an anti-CD29 antibody or an anti-CD49e antibody.
Specifically, by using a sandwich assay system using an anti-CD29 antibody or an anti-CD49e antibody and an α2-6 sialic acid-reactive lectin (SSA, SNA, rPSL1a, etc.) or an anti-α2-6 sialic acid antibody, It has become possible to accurately evaluate and determine the differentiation potential of derived mesenchymal stem cells or test cartilage stem cells into osteoblasts or chondrocytes.
　ここで、抗CD29抗体及び抗CD49e抗体は、それぞれCD29及びCD49e、またはそれらの抗原性フラグメントをエピトープとして認識して特異的に結合する能力を有するものであれば、特に限定はなく、ポリクローナル抗体、モノクローナル抗体、及びその抗原認識部位が保存されたFabフラグメントなどの抗体フラグメントの他、ヒト化抗体、単鎖抗体などを使用することもできる。抗CD29抗体及び抗CD49e抗体はAbcam社、R&D社、ベックマン・コールター社などから市販されているので簡単に入手できる。 Here, the anti-CD29 antibody and the anti-CD49e antibody are not particularly limited as long as they have the ability to recognize and specifically bind CD29 and CD49e, or antigenic fragments thereof, respectively, as polyclonal antibodies, In addition to monoclonal antibodies and antibody fragments such as Fab fragments in which the antigen recognition site is conserved, humanized antibodies, single chain antibodies, and the like can also be used. Anti-CD29 antibody and anti-CD49e antibody are commercially available from Abcam, R & D, Beckman Coulter, and the like.
３．対象となる被検幹細胞及びその採取源
　本明細書において分化ポテンシャルの判定、評価の対象となる被検幹細胞は、主として体性幹細胞もしくは当該体性幹細胞を継代培養した細胞であるが、胚性幹細胞（ES細胞）、体細胞に遺伝子などを導入して脱分化させた幹細胞（iPS細胞等）にも適用できる。ここで、体性幹細胞としては、神経幹細胞、上皮幹細胞、肝幹細胞、生殖幹細胞、造血幹細胞、間葉系幹細胞、軟骨幹細胞、骨格筋幹細胞等の様々な体性幹細胞が含まれるが、間葉系幹細胞及び軟骨幹細胞が好ましい。また、分化ポテンシャルの判定、評価は、クローン化された体性幹細胞のみならず、体性幹細胞を採取したヘテロな細胞集団からなる生体組織の培養物の状態でも行うことができるので、本発明において体性幹細胞というとき、体性幹細胞を含有する培養物をも包含する。
　すなわち、本発明において被検体性幹細胞含有試料というとき、試料に含まれる体性幹細胞は、生体から単離された細胞、その初代培養物、もしくは継代培養物、又は株化された培養細胞株である。 3. Test stem cells to be examined and their collection sources In the present specification, test stem cells to be evaluated and evaluated for differentiation potential are mainly somatic stem cells or cells obtained by subculturing the somatic stem cells. It can also be applied to stem cells (ES cells) and stem cells (iPS cells, etc.) that have been dedifferentiated by introducing genes into somatic cells. Here, the somatic stem cells include various somatic stem cells such as neural stem cells, epithelial stem cells, hepatic stem cells, reproductive stem cells, hematopoietic stem cells, mesenchymal stem cells, cartilage stem cells, skeletal muscle stem cells, etc. Stem cells and cartilage stem cells are preferred. In addition, the differentiation potential can be determined and evaluated not only in cloned somatic stem cells but also in the state of a culture of a living tissue consisting of a heterogeneous cell population from which somatic stem cells are collected. When referring to somatic stem cells, a culture containing somatic stem cells is also included.
That is, when the subject stem cell-containing sample is referred to in the present invention, the somatic stem cell contained in the sample is a cell isolated from a living body, a primary culture or a subculture thereof, or an established cultured cell line. It is.
　間葉系幹細胞は、脂肪吸引された脂肪組織や臍帯血、臍帯、羊膜、胎盤から、又は顎骨や大腿骨由来骨髄からの骨髄穿刺から採取できる。また、間葉系幹細胞は市販もされており、例えば脂肪組織由来間葉系幹細胞は、Life Technologies社、骨髄由来間葉系幹細胞は、Lonza社、PromoCell社などから購入することもできる。培養条件は、特に限定されないが、培養温度は、体温と同様の36～37℃が好ましい。培地は、間葉系幹細胞維持培地として一般に用いられているMesenPRO RSTM培地(Life Technologies社)などを適宜用いることができる。さらに、多指症患者の手術で切除された指の骨髄組織からは、骨髄由来間葉系幹細胞が取得でき、また軟骨組織からは、軟骨組織由来軟骨細胞（多指症由来軟骨幹細胞）が取得できる。また、理研バイオリソースセンター、JSRB細胞バンクなどから入手できる。軟骨幹細胞維持培地は、間葉系幹細胞維持培地として一般に用いられているMesenPRO RSTM地(Life Technologies社)などを用いても良いが、軟骨細胞基本培地、軟骨細胞増殖培地（タカラバイオ社）などの軟骨幹細胞用の維持培地を用いることもできる。 Mesenchymal stem cells can be collected from liposuctioned adipose tissue, umbilical cord blood, umbilical cord, amniotic membrane, placenta, or bone marrow puncture from jawbone or femur-derived bone marrow. In addition, mesenchymal stem cells are also 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, MesenPRO RS ™ medium (Life Technologies) generally used as a mesenchymal stem cell maintenance medium can be appropriately used. Furthermore, bone marrow-derived mesenchymal stem cells can be obtained from the bone marrow tissue of the fingers excised by surgery for patients with polydactyly, and cartilage tissue-derived chondrocytes (polydactyly derived cartilage stem cells) can be obtained from the cartilage tissue. it can. Also available from RIKEN BioResource Center, JSRB Cell Bank, etc. As the cartilage stem cell maintenance medium, MesenPRO RS TM ground (Life Technologies), which is generally used as a mesenchymal stem cell maintenance medium, may be used, but chondrocyte basic medium, chondrocyte growth medium (Takara Bio), etc. A maintenance medium for cartilage stem cells can also be used.
　本発明は、体のある組織から採取した幹細胞の分化ポテンシャルを判定することができるばかりでなく、拡大培養した後の分化ポテンシャルの判定にも用いることができる。また、分化ポテンシャルの高い細胞を単離、濃縮する際にも用いることができる。
　また、幹細胞はヒトに限らず哺乳動物ではかなりの部分で共通したしくみで制御されていると考えられるので、本発明の幹細胞としてはヒト以外の哺乳動物、例えばサル、ブタ、ウシ、ヤギ、ヒツジ、マウス、ラット由来の幹細胞を用いる場合にも適用できる。 The present invention can be used not only to determine the differentiation potential of stem cells collected from a body tissue, but also to determine the differentiation potential after expanded culture. It can also be used to isolate and concentrate cells with high differentiation potential.
In addition, since stem cells are considered to be controlled by a common mechanism in mammals, not limited to humans, the stem cells of the present invention include mammals other than humans, such as monkeys, pigs, cows, goats, sheep. It can also be applied when using stem cells derived from mice or rats.
４．体性幹細胞試料の判定及び評価方法並びに品質管理
（４－１）体性幹細胞試料の分化ポテンシャルの判定及び評価に基づく品質管理
　本発明は、生体から取得した体性幹細胞含有組織などの体性幹細胞試料または体性幹細胞培養物において、体性幹細胞の細胞表面で発現するα2-6シアル酸を特異的に検出する（in vitro）ことにより、体性幹細胞試料の分化ポテンシャルを判定または評価する方法に関する。そして、その判定結果に従って体性幹細胞試料の品質管理を行う方法も包含する。
　幹細胞表面のα2-6シアル酸の検出及び発現量の測定には質量分析、液体クロマトグラフィー及びMALDI-TOF MSを用いることができるが、α2-6シアル酸結合性プローブを用いる方法が、細胞表面の複合糖質の非還元末端のα2-6シアル酸検出には最も適している。培養物のロット全体の分化ポテンシャルを評価、判定する場合、及びその評価に基づく品質管理を行う場合には、通常は培養物の一部を取り出して行うため、一般にプローブの残存に基づく幹細胞への悪影響は考慮する必要は無い。仮に、分化ポテンシャルを評価、判定後の体性幹細胞をそのまま利用する必要がある場合は、ガラクトースやラクトースなどの糖類含有緩衝液などを用いて幹細胞を洗浄することでα2-6シアル酸結合レクチンを遊離させることができる。 4). Method for determining and evaluating somatic stem cell sample and quality control (4-1) Quality control based on determination and evaluation of differentiation potential of somatic stem cell sample The present invention relates to somatic stem cells such as somatic stem cell-containing tissue obtained from a living body. The present invention relates to a method for determining or evaluating the differentiation potential of a somatic stem cell sample by specifically detecting (in vitro) α2-6 sialic acid expressed on the cell surface of the somatic stem cell in a sample or somatic stem cell culture . And the method of performing quality control of a somatic stem cell sample according to the determination result is also included.
Mass spectrometry, liquid chromatography, and MALDI-TOF MS can be used for detection of α2-6 sialic acid on the surface of stem cells and measurement of expression level, but the method using α2-6 sialic acid binding probe is the cell surface. It is most suitable for detecting α2-6 sialic acid at the non-reducing end of glycoconjugates. When evaluating and judging the differentiation potential of the whole lot of culture, and when performing quality control based on that evaluation, a part of the culture is usually taken out, and in general, stem cells based on the remaining probe are used. There is no need to consider adverse effects. If it is necessary to use the somatic stem cells after evaluating and determining the differentiation potential, the α2-6 sialic acid-binding lectin can be washed by washing the stem cells with a saccharide-containing buffer such as galactose or lactose. Can be liberated.
　また、α2-6シアル酸結合性プローブを標識化することで、他の細胞が混在する体性幹細胞培養液に対しても、幹細胞表面のα2-6シアル酸と結合した標識量を正確に測定することができるため、簡便に幹細胞の分化ポテンシャルの評価、判定を行うことができる。レクチンなどプローブの標識には、R-Phycoerythrin(PE)、FITCなどの蛍光標識、パーオキシダーゼなどの酵素標識、ビオチンによる標識（＋HRP標識アビジン）などを用いることができる。プローブとして用いたレクチンに対する抗体を二次抗体として用いて、幹細胞表面のα2-6シアル酸と結合した標識量を測定し、細胞の分化ポテンシャルの評価、判定を行うことができる。 In addition, by labeling the α2-6 sialic acid-binding probe, the amount of label bound to α2-6 sialic acid on the stem cell surface can be accurately measured even in somatic stem cell culture media containing other cells. Therefore, it is possible to easily evaluate and determine the differentiation potential of stem cells. For labeling probes such as lectins, fluorescent labels such as R-Phycoerythrin (PE) and FITC, enzyme labels such as peroxidase, labels with biotin (+ HRP-labeled avidin), and the like can be used. By using an antibody against lectin used as a probe as a secondary antibody, the amount of label bound to α2-6 sialic acid on the stem cell surface can be measured to evaluate and determine the differentiation potential of the cell.
　さらに、本発明ではα2-6シアル酸のα2-3シアル酸に対する割合も分化ポテンシャルの指標となることが示されていることから、被検体性幹細胞試料に対して、既知のα2-3シアル酸特異的シアリダーゼ及びα2-6シアル酸特異的シアリダーゼなどを用いてα2-6シアル酸及びα2-3シアル酸を脱離させ、その比率を測定することによっても被検体性幹細胞試料の分化ポテンシャルの高さを判定することができる。 Further, in the present invention, it has been shown that the ratio of α2-6 sialic acid to α2-3 sialic acid is also an indicator of differentiation potential. The differentiation potential of the specimen stem cell sample can also be increased by desorbing α2-6 sialic acid and α2-3 sialic acid using specific sialidase and α2-6 sialic acid specific sialidase and measuring the ratio. Can be determined.
　本発明において、脂肪由来間葉系幹細胞及び軟骨幹細胞を用いた実験で、体性幹細胞表面のCD29及びCD49eが有しているα2-6シアル酸量が継代を重ねるに従って低下すること、すなわちCD29及びCD49eが、分化ポテンシャルの低下と相関する体性幹細胞におけるα2-6シアル酸のコアタンパク質であることがわかった。そして、SSA、SNA及びrPSL1aレクチンが、CD29及びCD49e上のα2-6シアル酸発現量の減少割合をより的確に反映するプローブとなることも判明した。
　したがって、間葉系幹細胞、軟骨幹細胞など体性幹細胞を含む細胞集団から、骨芽細胞又は軟骨細胞などへの分化ポテンシャルが高い細胞集団を濃縮もしくは単離しようというときには、抗CD29抗体又は抗CD49e抗体による免疫沈降により濃縮した細胞集団に対して、さらにα2-6シアル酸反応性レクチン（SSA、SNA、rPSL1aなど）もしくは抗α2-6シアル酸抗体を固定化した担体を用いた濃縮、単離方法を適用することができる。 In the present invention, in experiments using adipose-derived mesenchymal stem cells and cartilage stem cells, the amount of α2-6 sialic acid possessed by CD29 and CD49e on the surface of somatic stem cells decreases with successive passages, that is, CD29 And CD49e was found to be the core protein of α2-6 sialic acid in somatic stem cells that correlates with reduced differentiation potential. It was also found that SSA, SNA, and rPSL1a lectin serve as probes that more accurately reflect the rate of decrease in α2-6 sialic acid expression on CD29 and CD49e.
Therefore, when concentrating or isolating a cell population having a high differentiation potential into osteoblasts or chondrocytes from a cell population containing somatic stem cells such as mesenchymal stem cells and cartilage stem cells, an anti-CD29 antibody or an anti-CD49e antibody Concentration / isolation method using a carrier immobilized with α2-6 sialic acid reactive lectin (SSA, SNA, rPSL1a, etc.) or anti-α2-6 sialic acid antibody. Can be applied.
　また、抗CD29抗体又は抗CD49e抗体と共に、α2-6シアル酸反応性レクチン（SSA、SNA、rPSL1aなど）もしくは抗α2-6シアル酸抗体とを用いたサンドイッチアッセイ系、例えば、被検体性幹細胞幹細胞を、培養液のまま、もしくは抗CD29抗体又は抗CD49e抗体で濃縮後、SSA、SNA、rPSL1aなどを含む基板上に供し、標識化した抗CD29抗体又は抗CD49e抗体で標識強度を測定することで、骨芽細胞又は軟骨細胞への分化ポテンシャルを正確に評価、判定することができる。 In addition, a sandwich assay system using an anti-CD29 antibody or an anti-CD49e antibody together with an α2-6 sialic acid reactive lectin (SSA, SNA, rPSL1a, etc.) or an anti-α2-6 sialic acid antibody, for example, a subject stem cell stem cell In a culture medium or after concentration with an anti-CD29 antibody or anti-CD49e antibody, the sample is applied to a substrate containing SSA, SNA, rPSL1a, etc., and the labeling intensity is measured with a labeled anti-CD29 antibody or anti-CD49e antibody. The differentiation potential into osteoblasts or chondrocytes can be accurately evaluated and determined.
　被検体性幹細胞を含有している試料中の体性幹細胞標識量を正確に評価、測定するためには、試料中の体性幹細胞の数をほぼ一定数になるように調整する工程を設けることが好ましい。
　このような評価、判定に供する細胞集団は、体性幹細胞が1×104個以上含まれる集団であることが好ましく、1×105個含まれているとさらに好ましい。例えば蛍光標識を用いたフローサイトメトリー解析の場合は、平均蛍光強度が高い場合には、被検体性幹細胞の分化ポテンシャルが高いと判断する。また、平均蛍光強度が低い場合には、分化ポテンシャルが低いと判断する。基準となる蛍光強度は被検体性幹細胞の種類や、使用する蛍光色素によって適宜設定する。 In order to accurately evaluate and measure the amount of somatic stem cells labeled in a sample containing analyte stem cells, a process for adjusting the number of somatic stem cells in the sample to an almost constant number is provided. Is preferred.
The cell population used for such evaluation and determination is preferably a population containing 1 × 10 4 or more somatic stem cells, and more preferably 1 × 10 5 . For example, in the case of flow cytometry analysis using a fluorescent label, if the average fluorescence intensity is high, it is determined that the differentiation potential of the subject stem cell is high. When the average fluorescence intensity is low, it is determined that the differentiation potential is low. The reference fluorescence intensity is appropriately set according to the type of the subject stem cell and the fluorescent dye used.
（４－２）α2-6シアル酸結合性プローブを用いたα2-6シアル酸の検出、測定方法
　α2-6シアル酸結合性プローブを用いて幹細胞表面の糖タンパク質が有するα2-6シアル酸を測定する具体的な方法としては、これに限られるわけではないが、例えば以下の様な方法がある（コールドスプリングハーバー第２版「糖鎖生物学」４５章　グリカン分析における抗体とレクチン　p537-550など）。
　（ａ）幹細胞含有試料に、蛍光色素で標識したα2-6シアル酸結合性プローブを反応させ、洗浄後に、フローサイトメトリーや蛍光顕微鏡で蛍光強度を解析する。
　（ｂ）幹細胞含有試料に、α2-6シアル酸結合性プローブを反応させ、次いで当該プローブを検出可能な第２次標識抗体を用いて第二次抗体の標識量を測定する。
　（ｃ）幹細胞含有試料からタンパク質（含糖タンパク質）を調製し、蛍光色素による標識を行った後、α2-6シアル酸結合性レクチンの少なくとも１つを含むレクチンアレイに供し、エバネッセント波励起蛍光型検出系で蛍光強度を測定する。
　（ｄ）幹細胞含有試料からタンパク質を調製し、α2-6シアル酸結合性レクチンの少なくとも１つを含むレクチンアレイに供し、タンパク質や糖鎖を認識する標識抗体又は標識レクチンをオーバーレイして、エバネッセント波励起蛍光型検出系で蛍光強度を測定する。
　（ｅ）幹細胞含有試料からタンパク質を調製し、α2-6シアル酸結合性レクチンの少なくとも１つを固定化したプレートに供し、タンパク質や糖鎖を認識する標識抗体又は標識レクチンをオーバーレイして、プレートリーダーで吸光度、蛍光強度、発光を測定する。
　（ｆ）幹細胞含有試料からタンパク質を調製後、レクチンブロッティング法を適用する。SDS-PAGE後にニトロセルロース膜、PVDF膜などのメンブレンに転写するか、又は直接メンブレンにブロットした後、標識したα2-6シアル酸結合性レクチンと反応させる。例えば、ビオチン標識レクチンを用いた場合には、ブロッキングバッファーによる洗浄後、HRP標識アビジン溶液によるアビジン反応による発色を観察する。 (4-2) α2-6 sialic acid detection and measurement method using α2-6 sialic acid binding probe α2-6 sialic acid possessed by glycoprotein on stem cell surface using α2-6 sialic acid binding probe Specific methods for measurement include, but are not limited to, for example, the following method (Cold Spring Harbor 2nd edition “Glycobiology”, Chapter 45: Antibodies and lectins in glycan analysis p537-550 Such).
(A) An α2-6 sialic acid-binding probe labeled with a fluorescent dye is reacted with a stem cell-containing sample, and after washing, the fluorescence intensity is analyzed by flow cytometry or a fluorescence microscope.
(B) The α2-6 sialic acid-binding probe is reacted with the stem cell-containing sample, and then the amount of secondary antibody labeled is measured using a secondary labeled antibody capable of detecting the probe.
(C) A protein (glycoprotein) is prepared from a stem cell-containing sample, labeled with a fluorescent dye, and then subjected to a lectin array containing at least one α2-6 sialic acid-binding lectin, and is evanescent wave excited fluorescence type The fluorescence intensity is measured with a detection system.
(D) A protein is prepared from a stem cell-containing sample, applied to a lectin array containing at least one α2-6 sialic acid-binding lectin, and overlaid with a labeled antibody or labeled lectin that recognizes the protein or sugar chain, and evanescent waves The fluorescence intensity is measured with an excitation fluorescence type detection system.
(E) A protein prepared from a stem cell-containing sample, subjected to a plate on which at least one α2-6 sialic acid-binding lectin is immobilized, and overlaid with a labeled antibody or labeled lectin that recognizes the protein or sugar chain, Measure absorbance, fluorescence intensity, and luminescence with a reader.
(F) After preparing the protein from the stem cell-containing sample, the lectin blotting method is applied. After SDS-PAGE, transfer to a membrane such as a nitrocellulose membrane or PVDF membrane, or blot directly on the membrane, and then react with a labeled α2-6 sialic acid-binding lectin. For example, when a biotin-labeled lectin is used, color development by an avidin reaction with an HRP-labeled avidin solution is observed after washing with a blocking buffer.
５．体性幹細胞ソースからの分化ポテンシャルの高い幹細胞の単離、濃縮方法
（５－１）単離、濃縮方法
　本発明は、生体から取得した体性幹細胞含有組織などの体性幹細胞試料または体性幹細胞培養物に対して、体性幹細胞の細胞表面で発現するα2-6シアル酸を特異的に認識するレクチンまたは抗体、すなわちα2-6シアル酸結合性プローブを接触させることにより、分化ポテンシャルの高い体性幹細胞を単離または濃縮する方法に関する。
　α2-6シアル酸結合性プローブによる分化ポテンシャルの高い幹細胞の単離、濃縮方法は、特に限定されるものではなく、一般的な細胞分離方法が適用できる。例えば、フローサイトメトリーによる細胞分画法、又はレクチンもしくは抗体を固定化した磁気ビーズなどの固定化用担体を用いる細胞分画法、及びレクチンもしくは抗体を固定化したレクチンカラムクロマトグラフィーなどのアフィニティクロマトグラフィーによる分離法などを用いることができる。これらの方法の詳細については例えば、１９９６年、秀潤社発行、グライコバイオロジー実験プロトコール、細胞工学別冊、実験プロトコールシリーズや１９９９年、医歯薬出版株式会社発行、日本電気泳動学会編集、最新電気泳動実験法などに記載されている。
　本発明のα2-6シアル酸結合性プローブにより、分化ポテンシャルの高い幹細胞の単離、濃縮する場合には、幹細胞培養物中に残存していても幹細胞の生育及び分化誘導を阻害しないレクチンであり、かつ生体移植用細胞中に残存していても人体への毒性がきわめて低いレクチン、すなわち細胞毒性が無いか無視できる程度のレクチンを用いることが好ましい。また蛍光色素などによる直接的な標識を行わない単離、濃縮法を採用することがより好ましい。 5. Isolation and enrichment method of stem cells with high differentiation potential from somatic stem cell source (5-1) Isolation and enrichment method The present invention relates to a somatic stem cell sample or somatic stem cell such as a somatic stem cell-containing tissue obtained from a living body. By contacting the culture with a lectin or antibody that specifically recognizes α2-6 sialic acid expressed on the cell surface of somatic stem cells, that is, an α2-6 sialic acid-binding probe, a body with high differentiation potential The present invention relates to a method for isolating or enriching sex stem cells.
The method for isolating and concentrating stem cells with high differentiation potential using an α2-6 sialic acid-binding probe is not particularly limited, and general cell separation methods can be applied. For example, cell fractionation by flow cytometry, cell fractionation using a carrier for immobilization such as magnetic beads on which lectin or antibody is immobilized, and affinity chromatography such as lectin column chromatography on which lectin or antibody is immobilized For example, a photographic separation method can be used. For details of these methods, see, for example, 1996, published by Shujunsha, Glycobiology Experimental Protocol, Cell Engineering Separate Volume, Experimental Protocol Series and 1999, published by Ishiyaku Shuppan Publishing Co., Ltd. It is described in the electrophoresis experiment method.
When isolating and concentrating stem cells with high differentiation potential using the α2-6 sialic acid-binding probe of the present invention, it is a lectin that does not inhibit stem cell growth and differentiation induction even if it remains in the stem cell culture. In addition, it is preferable to use a lectin that has extremely low toxicity to the human body even if it remains in cells for living transplantation, that is, a lectin that has no or negligible cytotoxicity. It is more preferable to employ an isolation / concentration method in which direct labeling with a fluorescent dye or the like is not performed.
　α2-6シアル酸結合性プローブに対して反応性の高い細胞をフローサイトメトリーで単離、濃縮することで、分化ポテンシャルの高い幹細胞を単離、濃縮することができる。具体的には、例えば、蛍光標識したα2-6シアル酸結合性プローブを被検体性幹細胞含有試料に４℃、１時間程度反応させた後に、非結合細胞をリン酸緩衝液などで洗い流す。次に蛍光強度の高い細胞を分取後、ガラクトースやラクトースを含む緩衝液で蛍光標識したα2-6シアル酸結合性プローブを除去する。 A stem cell with high differentiation potential can be isolated and concentrated by isolating and concentrating the cells having high reactivity with the α2-6 sialic acid-binding probe by flow cytometry. Specifically, for example, after a fluorescently labeled α2-6 sialic acid-binding probe is reacted with a sample containing an analyte stem cell for about 1 hour at 4 ° C., unbound cells are washed away with a phosphate buffer or the like. Next, after collecting cells with high fluorescence intensity, the α2-6 sialic acid binding probe fluorescently labeled with a buffer containing galactose or lactose is removed.
　また、α2-6シアル酸結合性プローブを固定化した磁気ビーズ、アフィニティカラムなどを用いることによっても、α2-6シアル酸を発現する分化ポテンシャルの高い幹細胞を単離、濃縮することができる。具体的には、例えば、α2-6シアル酸結合性プローブを磁気ビーズに固定化し、被検体性幹細胞含有試料に４℃、１時間程度反応させた後に、リン酸緩衝液で洗浄する。マグネットを用いて磁気ビーズを回収後、ガラクトースやラクトースを含む緩衝液を用いて、細胞を磁気ビーズから剥がす。
　このようにして単離、濃縮された体性幹細胞は速やかに公知の分化誘導培地に供することで所望の細胞への分化誘導を行うことができる。当該分化誘導後の細胞、もしくは上記単離、濃縮後の細胞は、再生医療用の細胞として移植することができる。
　なお、移植材料の調製方法は、既知の手法を適用すればよい。 In addition, stem cells with high differentiation potential expressing α2-6 sialic acid can also be isolated and concentrated by using magnetic beads, affinity columns or the like on which α2-6 sialic acid-binding probe is immobilized. Specifically, for example, an α2-6 sialic acid-binding probe is immobilized on a magnetic bead, reacted with an analyte stem cell-containing sample at 4 ° C. for about 1 hour, and then washed with a phosphate buffer. After collecting the magnetic beads using a magnet, the cells are detached from the magnetic beads using a buffer containing galactose or lactose.
The somatic stem cells isolated and concentrated in this manner can be promptly subjected to differentiation differentiation into desired cells by being subjected to a known differentiation induction medium. The cells after differentiation induction or the cells after the isolation and concentration can be transplanted as cells for regenerative medicine.
In addition, what is necessary is just to apply a known method for the preparation method of transplant material.
（５－２）骨芽細胞移植用骨髄由来間葉系幹細胞の調製
　本発明の糖鎖マーカーである「α2-6シアル酸」を、間葉系幹細胞のうちでも骨髄由来間葉系幹細胞に適用する場合は、特に間葉系幹細胞の骨芽細胞への分化ポテンシャルとの相関が高いと考えられることから、骨芽細胞への分化ポテンシャル評価用マーカーとして用いることができる。 (5-2) Preparation of bone marrow-derived mesenchymal stem cells for osteoblast transplantation “α2-6 sialic acid” which is a sugar chain marker of the present invention is applied to bone marrow-derived mesenchymal stem cells among mesenchymal stem cells. In this case, since it is considered that the correlation with the differentiation potential of mesenchymal stem cells into osteoblasts is particularly high, it can be used as a marker for evaluating differentiation potential into osteoblasts.
　すなわち、骨髄から採取した間葉系幹細胞含有細胞群をそのまま、もしくはex vivoで拡大培養後の移植前に、当該培養物の骨芽細胞への分化ポテンシャルの評価、判定をすることができる。その際、培養物から、間葉系幹細胞が好ましくは1×104個以上含まれるような一部を採取して上記（４－２）で述べた方法により間葉系幹細胞表面のα2-6シアル酸量を測定し、骨芽細胞への分化ポテンシャルの評価、判定を行う。例えば、フローサイトメトリーによって得られた平均蛍光強度が高い場合には、骨芽細胞への分化ポテンシャルが高い細胞群であると判断し、その判定結果に従って、移植を行うか否かを決定する。
　また、骨髄から採取した間葉系幹細胞含有細胞群に対して、上記（５－１）の単離、濃縮法を適用して、骨芽細胞への分化ポテンシャルの高い幹細胞のみを単離、濃縮することができる。 That is, the mesenchymal stem cell-containing cell group collected from the bone marrow can be evaluated or judged as it is, or before transplantation after ex vivo expansion culture, the differentiation potential of the culture to osteoblasts. At that time, a part of the culture containing preferably 1 × 10 4 or more mesenchymal stem cells is collected and α2-6 on the surface of mesenchymal stem cells is obtained by the method described in the above (4-2). The amount of sialic acid is measured, and the differentiation potential into osteoblasts is evaluated and judged. For example, when the average fluorescence intensity obtained by flow cytometry is high, it is determined that the cell group has a high differentiation potential into osteoblasts, and whether to perform transplantation is determined according to the determination result.
In addition, by applying the isolation and concentration method of (5-1) above to a mesenchymal stem cell-containing cell group collected from bone marrow, only stem cells with high differentiation potential into osteoblasts are isolated and concentrated. can do.
６．分化ポテンシャル判定用キット
　上記（２－４）で述べたたように、本発明において、間葉系幹細胞又は軟骨幹細胞表面のCD29及びCD49eが有しているα2-6シアル酸量が、骨芽細胞又は軟骨細胞などへの分化ポテンシャルの程度と高い相関性を有していることを見出した。
　また、本発明者らは、α2-6シアル酸反応性レクチンのうちでも、CD29及びCD49e上のα2-6シアル酸量の変化をより高い相関性をもって判定できるレクチンがSSA,SNA及びrPSL1aであることを見いだした。
　その結果、被検間葉系幹細胞又は軟骨幹細胞など被検体性幹細胞における分化ポテンシャルを、より正確に判定、評価するためのアッセイ系として、抗CD29抗体及び／又は抗CD49e抗体とα2-6シアル酸反応性レクチン（SSA,SNA,rPSL1a）とを組み合わせた分化ポテンシャル判定用サンドイッチアッセイ系を構築することができた。 6). Differentiation Potential Determination Kit As described in (2-4) above, in the present invention, the amount of α2-6 sialic acid possessed by CD29 and CD49e on the surface of mesenchymal stem cells or cartilage stem cells is Or it discovered that it had a high correlation with the grade of the differentiation potential to a chondrocyte etc.
Further, among the α2-6 sialic acid-reactive lectins, the lectins that can determine changes in the amount of α2-6 sialic acid on CD29 and CD49e with higher correlation are SSA, SNA, and rPSL1a. I found out.
As a result, anti-CD29 antibody and / or anti-CD49e antibody and α2-6 sialic acid are used as an assay system for more accurately determining and evaluating the differentiation potential in subject stem cells such as test mesenchymal stem cells or cartilage stem cells. A sandwich assay system for determining differentiation potential was established by combining reactive lectins (SSA, SNA, rPSL1a).
　具体的には、α2-6シアル酸反応性レクチン（SSA,SNA,rPSL1a）を固定化した基板に対して、抗CD29抗体もしくは抗CD49e抗体で濃縮した分画を反応させた後、抗CD29抗体もしくは抗CD49e抗体をオーバーレイして検出又は定量化する方法が例示できる。反対に、抗CD29抗体もしくは抗CD49e抗体を固定化した基板及び標識化したα2-6シアル酸反応性のレクチン（SSA,SNA,rPSL1a）もしくは抗体との組み合わせによっても検出、定量化が可能である。
　すなわち、本発明において、体性幹細胞の分化ポテンシャル判定用キットというとき、少なくともα2-6シアル酸をエピトープとして認識するプローブを含むキットであり、具体的には、
（ａ）α2-6シアル酸をエピトープとして認識するレクチン（TJAI,SSA,SNA,rPSL1aなど）、及び
（ｂ）α2-6シアル酸をエピトープとして認識する抗α2-6シアル酸抗体、
との組み合わせを含むキットであっても良い。 Specifically, after reacting the fraction enriched with anti-CD29 antibody or anti-CD49e antibody to a substrate on which α2-6 sialic acid-reactive lectin (SSA, SNA, rPSL1a) is immobilized, anti-CD29 antibody Alternatively, a method of overlaying an anti-CD49e antibody for detection or quantification can be exemplified. On the other hand, detection and quantification are possible by combining a substrate with an anti-CD29 antibody or anti-CD49e antibody immobilized thereon and a labeled α2-6 sialic acid reactive lectin (SSA, SNA, rPSL1a) or antibody. .
That is, in the present invention, the somatic stem cell differentiation potential determination kit is a kit containing a probe that recognizes at least α2-6 sialic acid as an epitope, specifically,
(A) a lectin that recognizes α2-6 sialic acid as an epitope (TJAI, SSA, SNA, rPSL1a, etc.), and (b) an anti-α2-6 sialic acid antibody that recognizes α2-6 sialic acid as an epitope,
It may be a kit including a combination.
　典型的には、
（ａ）α2-6シアル酸をエピトープとして認識するプローブ、及び
（ｂ）α2-6シアル酸を非還元末端糖鎖として有し、かつ標的幹細胞で発現している糖タンパク質を認識するプローブ、
の組み合わせからなるキットであり、（ａ）（ｂ）の一方が基板上に固定化され、他方が標識化されていることが好ましい。
　特に、体性幹細胞、好ましくは間葉系幹細胞又は軟骨幹細胞を対象細胞とした場合の骨芽細胞又は軟骨細胞などへの分化ポテンシャルを定量的に評価、判定する場合には、一方が基板に固定され、かつ他方が標識化されている、
（ａ）SSA,SNA又はrPSL1aレクチン、
（ｂ）抗CD29抗体又は抗CD49e抗体、
の組み合わせを含むキットが好ましい。
　なお、本発明において、キットというとき、主に各プローブからなる試薬を組み合わせる場合を指すが、試薬用の容器、パッケージ、取扱説明書などの他、測定装置などを含める場合もある。 Typically,
(A) a probe that recognizes α2-6 sialic acid as an epitope, and (b) a probe that has α2-6 sialic acid as a non-reducing terminal sugar chain and recognizes a glycoprotein expressed in a target stem cell,
It is preferable that one of (a) and (b) is immobilized on a substrate and the other is labeled.
In particular, when the differentiation potential to osteoblasts or chondrocytes when using somatic stem cells, preferably mesenchymal stem cells or cartilage stem cells as target cells, is quantitatively evaluated and determined, one is fixed to the substrate. And the other is labeled,
(A) SSA, SNA or rPSL1a lectin,
(B) an anti-CD29 antibody or an anti-CD49e antibody,
A kit comprising a combination of
In the present invention, the term “kit” refers to a case where reagents mainly composed of respective probes are combined. However, in addition to a reagent container, a package, an instruction manual, etc., a measuring device may be included.
　コアタンパク質上のα2-6シアル酸発現量の変化と分化ポテンシャルとの相関を定量的に行うためには、例えば、あらかじめ、対象となる体性幹細胞含有試料と同じ体性幹細胞の標準的な株を用いて、α2-6シアル酸発現量に応じた分化ポテンシャル度についての検量線を用いることで被検幹細胞含有試料の定量的な評価をすることができる。具体的には、標準株を継代培養し、継代培養中の複数の継代数の細胞を１部採取し、上記（ａ）及び（ｂ）からなるサンドイッチアッセイ用キットを用いて、標識強度を測定する。同時に、当該継代細胞を目的とする分化細胞に分化誘導して分化細胞マーカー強度などの分化ポテンシャルを示す数値を測定し、両者の数値から検量線を求める。得られた検量線を用いることで、被検体性幹細胞含有試料に対し当該アッセイ系により測定された標識強度を、目的とする分化細胞への定量的な分化ポテンシャル量に変換できるから、被検体性幹細胞含有試料についての正確な分化ポテンシャルの評価、判定が可能となる。 In order to quantitatively correlate the change in the expression level of α2-6 sialic acid on the core protein and the differentiation potential, for example, in advance, a standard strain of the same somatic stem cells as the target somatic stem cell-containing sample is used. By using a calibration curve for the degree of differentiation potential according to the expression level of α2-6 sialic acid, it is possible to quantitatively evaluate the sample containing test stem cells. Specifically, a standard strain is subcultured, and a part of cells of a plurality of subcultures during subculture is collected, and labeling strength is obtained using the sandwich assay kit comprising (a) and (b) above. Measure. At the same time, the passage cells are induced to differentiate into the differentiated cells of interest, and numerical values indicating the differentiation potential such as differentiated cell marker strength are measured, and a calibration curve is obtained from both values. By using the obtained calibration curve, it is possible to convert the labeling intensity measured by the assay system with respect to the sample containing the analyte stem cells into the quantitative differentiation potential amount to the target differentiated cell. It is possible to accurately evaluate and determine the differentiation potential of the stem cell-containing sample.
　以下に実施例を示し、本発明を具体的に説明するが、本発明はこれらに限定されるものではない。
　本発明におけるその他の用語や概念は、当該分野において慣用的に使用される用語の意味に基づくものであり、本発明を実施するために使用する様々な技術は、特にその出典を明示した技術を除いては、公知の文献等に基づいて当業者であれば容易かつ確実に実施可能である。また、各種の分析などは、使用した分析機器又は試薬、キットの取り扱い説明書、カタログなどに記載の方法を準用して行った。
　なお、本明細書中に引用した技術文献、特許公報及び特許出願明細書中の記載内容は、本発明の記載内容として参照されるものとする。 EXAMPLES The present invention will be specifically described below with reference to examples, but the present invention is not limited to these examples.
Other terms and concepts in the present invention are based on the meanings of terms that are conventionally used in the field, and various techniques used to implement the present invention include those that clearly indicate the source. Except for this, it can be easily and reliably carried out by those skilled in the art based on known documents and the like. In addition, various analyzes were performed by applying the methods described in the analytical instruments or reagents used, kit instruction manuals, catalogs, and the like.
In addition, the description content in the technical literature, the patent gazette, and the patent application specification cited in this specification shall be referred to as the description content of the present invention.
（実施例１）脂肪由来間葉系幹細胞の継代培養
　脂肪由来間葉系幹細胞（ADSC、Life Technologies、Lot#: 2118）を、一般的な間葉系幹細胞培地であるMesenPRO RSTM 培地(Life Technologies)で継代培養した（図１）。図中、PDは***指数を示す。次に継代初期（P5）および継代後期（P28）の細胞の、骨芽細胞、及び脂肪細胞への分化ポテンシャルを調べた（図２）。
　ここで、本発明において「継代初期」又は「継代後期」というとき、それぞれの継代数は、細胞の種類や培養条件によって異なる。幹細胞の培養初期で、細胞増殖カーブが直線的に上昇する時期を「継代初期」、継代を続け、細胞の増殖カーブが緩く、又は平坦となる時期を「継代後期」と呼ぶ。
　脂肪由来間葉系幹細胞の継代初期（P5）の一部の細胞および継代後期（P28）の一部の細胞をそれぞれ取り出し、骨芽細胞及び脂肪細胞への分化誘導を行った。骨芽細胞への分化誘導はhMSC differentiation BulletKit-osteogenic (Cat#: PT-3002, Lonza)、脂肪細胞分化はhMSC differentiation BulletKit-adipogenic（Cat#: PT-3004, Lonza）を用いて行った。骨芽細胞分化はアリザリンレッドS、脂肪細胞分化はオイルレッドOで染色して分化状態を確認した。骨芽細胞と脂肪細胞は赤く染色される。継代初期（P5）の脂肪由来間葉系幹細胞の場合は骨芽細胞及び脂肪細胞への分化が観察されたが、継代後期（P28）の脂肪由来間葉系幹細胞では骨芽細胞及び脂肪細胞はほとんど観察されなかった。すなわち脂肪由来間葉系幹細胞における継代後期の細胞では、骨芽細胞及び脂肪細胞への分化ポテンシャルが失われていることがわかる。 (Example 1) Subculture of adipose-derived mesenchymal stem cells Adipose-derived mesenchymal stem cells (ADSC, Life Technologies, Lot #: 2118) were added to MesenPRO RS ™ medium (Life Technologies) (FIG. 1). In the figure, PD indicates the division index. Next, the differentiation potential of cells in the early passage (P5) and late passage (P28) into osteoblasts and adipocytes was examined (FIG. 2).
Here, in the present invention, when referred to as “early passage” or “late passage”, the number of passages varies depending on the cell type and culture conditions. The period when the cell growth curve rises linearly in the early stage of stem cell culture is called “early passage”, and the period when the cell growth curve continues or becomes gentle or flat becomes “late stage”.
A portion of cells from the early passage (P5) and a portion of cells from the late passage (P28) of the adipose-derived mesenchymal stem cells were taken out, and differentiation induction into osteoblasts and adipocytes was performed. Differentiation into osteoblasts was performed using hMSC differentiation BulletKit-osteogenic (Cat #: PT-3002, Lonza), and adipocyte differentiation was performed using hMSC differentiation BulletKit-adipogenic (Cat #: PT-3004, Lonza). Differentiation state was confirmed by staining with alizarin red S for osteoblast differentiation and oil red O for adipocyte differentiation. Osteoblasts and adipocytes are stained red. In the case of fat-derived mesenchymal stem cells in the early passage (P5), differentiation into osteoblasts and adipocytes was observed, but in the fat-derived mesenchymal stem cells in the late passage (P28), osteoblasts and fat were observed. Little cells were observed. That is, it can be seen that late differentiation cells in adipose-derived mesenchymal stem cells have lost the differentiation potential to osteoblasts and adipocytes.
（実施例２）脂肪由来間葉系幹細胞の継代初期と継代後期で顕著に結合性が異なるレクチンの抽出
　実施例１で用いた各継代数のヒト脂肪由来間葉系幹細胞（ADSC）に対して、高密度レクチンアレイによる糖鎖解析を実施した。ヒト脂肪由来間葉系幹細胞から膜画分を調製し、蛍光ラベル化後、高密度レクチンアレイに供し、エバネッセント波励起蛍光型検出系で蛍光強度を測定した。高い分化ポテンシャル/増殖能を有する継代初期(P2-P6)の細胞では高い蛍光強度を示すのに対して、分化ポテンシャル/増殖能の低下した継代後期(P25-P29)の細胞では、顕著に蛍光強度が低下するレクチンを統計的に抽出した。その結果、４種のレクチン、Trichosanthes japonica lectin-I (TJAI), Sambucus sieboldiana lectin (SSA), Sambucus nigra lectin (SNA), recombinant Polyporus squamosus lectin (rPSL1a)が、p<0.0001で継代前期の細胞に顕著に高い値を示すレクチンとして選択された。図３には、各レクチンの継代前期細胞（P2-5；黒棒）及び継代後期細胞（P25-29；白棒）に対する蛍光シグナル強度の平均値と標準偏差を示す。またヒト脂肪由来間葉系幹細胞の各継代における各レクチンの平均値を示したグラフを図４に示す。これら４種のレクチンの蛍光シグナルは、継代数を重ねる毎に、徐々に低下することが分かった（図４）。これら４種のレクチンそれぞれの各種複合糖鎖に対する反応性を図５に示す。いずれのレクチンもα2-6シアル酸残基を有する糖鎖501～506のみに特異的に結合する。同じシアル酸残基のうちでもα2-3シアル酸残基のみを含む糖鎖601及び602に対しては全く結合性を示さず、α2-3シアル酸と共にα2-6シアル酸残基を有している糖鎖506に対しては結合性を有していることがわかる。このことから、上記４種のレクチンは、α2-6シアル酸残基に結合特異性を有するレクチンである、ということができる。一方、これらα2-6シアル酸残基に結合特異性を有するレクチンが顕著な特異的結合を発揮する継代前期のヒト脂肪由来間葉系幹細胞表面には、α2-6シアル酸残基が多数存在し、継代後期になるに従って、α2-6シアル酸残基の量が激減することが見て取れる。
　そして、上述のように、継代前期から継代後期になるに従って、増殖能の低下と共に分化ポテンシャルの低下も観察されたことから、間葉系幹細胞表面におけるα2-6シアル酸残基は、間葉系幹細胞の分化ポテンシャルを示す糖鎖マーカーである可能性が示唆された。 (Example 2) Extraction of lectins having significantly different binding properties at the early passage and late passage of adipose-derived mesenchymal stem cells. On the other hand, the sugar chain analysis by a high-density lectin array was performed. Membrane fractions were prepared from human adipose-derived mesenchymal stem cells, labeled with fluorescence, subjected to a high-density lectin array, and fluorescence intensity was measured with an evanescent wave excitation fluorescence detection system. Cells in the early passage (P2-P6) with high differentiation potential / proliferation ability show high fluorescence intensity, whereas they are prominent in cells in the late passage (P25-P29) with reduced differentiation potential / proliferation ability The lectins whose fluorescence intensity decreased were statistically extracted. As a result, four kinds of lectins, Trichosanthes japonica lectin-I (TJAI), Sambucus sieboldiana lectin (SSA), Sambucus nigra lectin (SNA), recombinant Polyporus squamosus lectin (rPSL1a), became p <0.0001 cells in the early passage. It was selected as a lectin showing significantly higher values. FIG. 3 shows the mean value and standard deviation of the fluorescence signal intensity of each lectin for early passage cells (P2-5; black bars) and late passage cells (P25-29; white bars). Moreover, the graph which showed the average value of each lectin in each passage of a human adipose origin mesenchymal stem cell is shown in FIG. It was found that the fluorescence signals of these four lectins gradually decreased with each passage number (FIG. 4). FIG. 5 shows the reactivity of each of these four lectins to various complex sugar chains. Each lectin specifically binds only to sugar chains 501 to 506 having an α2-6 sialic acid residue. Among the same sialic acid residues, it has no binding property to sugar chains 601 and 602 containing only α2-3 sialic acid residues, and has α2-6 sialic acid residues together with α2-3 sialic acid. It can be seen that the sugar chain 506 has binding properties. From this, it can be said that the above four lectins are lectins having binding specificity to α2-6 sialic acid residues. On the other hand, there are many α2-6 sialic acid residues on the surface of human adipose-derived mesenchymal stem cells in the early passage, where lectins having binding specificity to these α2-6 sialic acid residues exhibit remarkable specific binding. It can be seen that the amount of α2-6 sialic acid residues decreases dramatically as it is present and later in passage.
As described above, from the early passage to the late passage, a decrease in proliferation potential as well as a decrease in differentiation potential were observed, so α2-6 sialic acid residues on the surface of mesenchymal stem cells were It was suggested that it may be a sugar chain marker indicating the differentiation potential of leaf stem cells.
（実施例３）フローサイトメトリーによる脂肪由来間葉系幹細胞へのα2-6シアル酸結合性レクチンの反応性検証
　実施例１で用いたと同じヒト脂肪由来間葉系幹細胞を同様に継代培養し、その継代初期（P3）及び継代後期（P26）のそれぞれの一部（1×105個）細胞に対して、実施例２で選択された４種類のレクチン（TJAI、SSA、SNA、rPSL1a）をR-Phycoerythrin(PE)で標識した標識レクチンを10 μg/mLで反応させ、1%BSAを含むリン酸緩衝液で洗浄後に、フローサイトメトリー（BD FACSCantoII）で解析した。継代後期(P26、細線)の細胞と比べて、継代初期(P3、太線)の細胞に顕著に高い反応性を示すことが分かった（図６）。継代後期の細胞に対する反応性はほとんどコントロール（グレー）と同程度であった。フローサイトメトリーで得られた平均蛍光強度を、ヒトiPS細胞（201B7株）、ヒト皮膚繊維芽細胞(Fibroblast、ATCC)と比較したグラフを図７に示す。４種のレクチンは、分化ポテンシャルを有するADSC P3に対しては高い反応性が確認されたのに対し、分化ポテンシャルがないADSC P26とヒト皮膚繊維芽細胞（Fibroblast）にはほとんど反応性が見られなかった。また、同時に、分化ポテンシャルが最も高いと考えられる多能性のヒトiPS細胞（201B7株、理化学研究所バイオリソースセンター）に対する４種のレクチンの反応性を測定したところ、ADSC P3の２～４倍もの高い反応性を示した。以上の結果からも、TJAI、SSA、SNA、rPSL1aの４種のレクチンの反応性は、細胞の分化ポテンシャルと高い相関性を示すことが示唆された。 (Example 3) Verification of reactivity of α2-6 sialic acid-binding lectin to adipose-derived mesenchymal stem cells by flow cytometry The same human adipose-derived mesenchymal stem cells as used in Example 1 were subcultured in the same manner. , for the early passages (P3) and the respective part (1 × 10 5) cells late passage (P26), 4 types of lectins selected in example 2 (TJAI, SSA, SNA, The labeled lectin labeled with R-Phycoerythrin (PE) was reacted at 10 μg / mL, washed with a phosphate buffer containing 1% BSA, and analyzed by flow cytometry (BD FACSCantoII). It was found that the cells in the early passage (P3, thick line) showed significantly higher reactivity than the cells in the late passage (P26, thin line) (FIG. 6). The reactivity to late passage cells was almost the same as the control (gray). FIG. 7 shows a graph comparing the average fluorescence intensity obtained by flow cytometry with human iPS cells (201B7 strain) and human skin fibroblasts (Fibroblast, ATCC). The four lectins were highly reactive against ADSC P3, which has differentiation potential, whereas ADSC P26 and human skin fibroblasts (Fibroblast), which have no differentiation potential, were almost reactive. There wasn't. At the same time, the reactivity of four lectins against pluripotent human iPS cells (201B7 strain, RIKEN BioResource Center), which are considered to have the highest differentiation potential, was 2 to 4 times that of ADSC P3. High reactivity was shown. The above results also suggested that the reactivity of the four lectins TJAI, SSA, SNA, and rPSL1a is highly correlated with the differentiation potential of the cells.
（実施例４）各種細胞に発現する糖タンパク質糖鎖のシアル酸結合様式
　実施例３で用いた継代初期（P3）及び継代後期（P26）のヒト脂肪由来間葉系幹細胞（ADSC P3、ADSC P26）と共に、ヒトiPS細胞（201B7株）及びヒト皮膚繊維芽細胞（Fibroblast）に対して気相ヒドラジン分解法を適用して糖タンパク質糖鎖を切り出し、糖鎖の酸性度に応じて分画した。具体的には、上記各細胞を凍結乾燥後、ヒドラクラブY2100（J-オイルミルズ）で、無水ヒドラジンを用いて100℃4時間処理することにより糖鎖を切り出し、反応後、無水ヒドラジンを減圧乾燥して、遊離糖鎖をN-アセチル化し、Dowex 50WX2で脱塩後、凍結乾燥した。次に、遊離糖鎖をGlycoTAG（タカラバイオ）で２－ピリジルアミノ（PA）化し、PA化糖鎖をMono-Qカラム（GE）を用いたイオン交換クロマトグラフィーに供し、糖鎖の酸性度に応じて糖鎖を分画した。シアル酸が１個付加された画分をA1、シアル酸が２個付加された画分をA4とする。これら画分のシアル酸の結合位置を調べるために、Salmonella typhimurimum LT2由来α2-3シアル酸特異的シアリダーゼ（タカラバイオ）と、α2-3シアル酸、及びα2-6シアル酸の両方を切断するClostridium perfringensシアリダーゼ（メルク）を反応させて、α2-3シアル酸とα2-6シアル酸の割合を計算した。その結果、A1画分におけるα2-6シアル酸の割合（黒棒）は、ヒトiPS細胞では83％だったのに対し、ADSC P3では25％、ADSC P28とFibroblastでは0％であった。A4分画における、α2-6シアル酸２個（黒棒）、及びα2-6シアル酸１個（黒点）含む糖鎖は、ヒトiPS細胞ではそれぞれ58％と15％、ADSC P3では27％と11％、ADSC P28では0％と16％、Fibroblastでは5％と5％であった。以上の結果から、α2-6シアル酸の割合は、ヒトiPS細胞で最も高く、次にADSC P3、ADSC P28とFibroblastではほとんど確認されなかった。以上の結果から、α2-6シアル酸の割合は、細胞分化ポテンシャルの高さを判定するためのよい指標となることが分かった。 Example 4 Sialic Acid Binding Modes of Glycoprotein Sugar Chains Expressed in Various Cells Human adipose-derived mesenchymal stem cells (ADSC P3, early passage (P3) and late passage (P26) used in Example 3 ADSC P26) and gas phase hydrazine degradation method applied to human iPS cells (201B7 strain) and human skin fibroblasts (Fibroblast) to cut out glycoprotein sugar chains and fractionate them according to the acidity of the sugar chains did. Specifically, after freeze-drying each of the above cells, the glycan was excised by treatment with hydraclub Y2100 (J-Oil Mills) using anhydrous hydrazine at 100 ° C for 4 hours, and after the reaction, anhydrous hydrazine was dried under reduced pressure. The free sugar chain was N-acetylated, desalted with Dowex 50WX2, and lyophilized. Next, the free sugar chain is converted to 2-pyridylamino (PA) with GlycoTAG (Takara Bio), and the PA-modified sugar chain is subjected to ion exchange chromatography using a Mono-Q column (GE), depending on the acidity of the sugar chain. The sugar chain was fractionated. The fraction with one sialic acid added is designated as A1, and the fraction with two sialic acids added is designated as A4. In order to examine the binding position of sialic acid in these fractions, Clostridium that cleaves both α2-3 sialic acid and α2-6 sialic acid, α2-3 sialic acid-specific sialidase derived from Salmonella typhimurimum LT2 (Takara Bio) Perfringens sialidase (Merck) was reacted to calculate the ratio of α2-3 sialic acid and α2-6 sialic acid. As a result, the proportion of α2-6 sialic acid (black bar) in the A1 fraction was 83% for human iPS cells, 25% for ADSC P3, and 0% for ADSC P28 and Fibroblast. In the A4 fraction, the sugar chains containing two α2-6 sialic acids (black bars) and one α2-6 sialic acid (black dots) are 58% and 15% in human iPS cells and 27% in ADSC P3, respectively. 11%, ADSC P28 0% and 16%, Fibroblast 5% and 5%. From the above results, the ratio of α2-6 sialic acid was highest in human iPS cells, and was hardly confirmed in ADSC P3, ADSC P28 and Fibroblast. From the above results, it was found that the ratio of α2-6 sialic acid is a good index for determining the height of cell differentiation potential.
（実施例５）多指症軟骨組織由来軟骨細胞(Yub621c株)の継代培養初期細胞及び後期細胞における、増殖能、分化ポテンシャル、及びフローサイトメトリーによるα2-6シアル酸結合性レクチンの反応性
　脂肪組織由来間葉系幹細胞以外の他の組織由来の体性幹細胞を用いて検証を行った。軟骨幹細胞の一種である多指症軟骨組織由来軟骨細胞（Yub621c株、理研バイオリソースセンター）を実施例１などと同様の手法で継代培養し、継代初期(P７)から継代後期（P28）に至る増殖能(***回数)をプロットした。継代初期(P７)及び継代後期（P28）細胞に対して骨芽細胞と脂肪細胞への分化誘導を行った結果（図９）、継代初期にはいずれに対しても高い分化ポテンシャルが観察されたが、継代後期になると、脂肪細胞分化は全く観察されず、骨芽細胞分化は、わずかに観察されるもののきわめて低い分化ポテンシャルであった。一方でフローサイトメトリーにおける４種のレクチン（TJAI、SSA、SNA、rPSL1a）の継代初期（P6）から継代後期（P28）での反応性の低下は際だっており、多指症軟骨組織由来軟骨細胞（多指症由来軟骨幹細胞）表面のα2-6シアル酸量の低下は、増殖能とは関連性を示さず、軟骨幹細胞においてもその分化ポテンシャルと高い関連性を示すため、分化ポテンシャルの高さを示す優れた指標となることがわかった。 (Example 5) Proliferation ability, differentiation potential, and reactivity of α2-6 sialic acid-binding lectin in subcultured early cells and late cells of polydactyly cartilage tissue- derived chondrocytes (Yub621c strain) Verification was performed using somatic stem cells derived from tissues other than adipose tissue-derived mesenchymal stem cells. Multi-deficient cartilage tissue-derived chondrocytes (Yub621c strain, RIKEN BioResource Center), a type of cartilage stem cell, are subcultured in the same manner as in Example 1 and the early passage (P7) to late passage (P28) The proliferative ability (number of divisions) leading to is plotted. As a result of induction of differentiation into osteoblasts and adipocytes for early passage (P7) and late passage (P28) cells (FIG. 9), high differentiation potential was found in both early passages. Observed, at the late passage, no adipocyte differentiation was observed, and osteoblast differentiation was a very low differentiation potential, although only slightly observed. On the other hand, the reactivity of the four lectins (TJAI, SSA, SNA, rPSL1a) in flow cytometry was markedly decreased from the early passage (P6) to the later passage (P28), and was derived from polydactyly cartilage tissue. Decrease in the amount of α2-6 sialic acid on the surface of chondrocytes (polydactyly derived cartilage stem cells) is not related to proliferative ability, and is also highly related to its differentiation potential in cartilage stem cells. It turned out to be an excellent indicator of height.
(実施例６) 多指症骨髄由来間葉系幹細胞（Yub622株）の継代培養初期細胞及び後期細胞における、増殖能、分化ポテンシャル、及びフローサイトメトリーによるα2-6シアル酸結合性レクチンの反応性
　多指症骨髄由来間葉系幹細胞（Yub622株、理研バイオリソースセンター）を実施例１などと同様の手法で継代培養し、継代初期(P5) から継代後期（P17）に至る増殖能(***回数)をプロットしたところ、継代後期（P17）になると増殖能が低下する点は実施例２などで解析した脂肪細胞由来間葉系幹細胞の場合と同様であった。一方、フローサイトメトリーにおける４種のレクチン（TJAI、SSA、SNA、rPSL1a）の反応性は、継代初期（P4）では高い反応性を示し、継代後期(P15)の細胞に対しては若干の反応性の低下現象が認められたものの、大きな低下は認められなかった。それぞれの細胞での骨芽細胞と脂肪細胞への分化ポテンシャルを解析したところ、いずれの分化ポテンシャルも継代初期には高く、継代後期(P17)の細胞では脂肪細胞分化ポテンシャルは失われていたが、骨芽細胞への分化ポテンシャルは維持されていた(図１０)。
　以上の結果から、骨髄由来間葉系幹細胞では、４種のレクチンの反応性、すなわち骨髄由来間葉系幹細胞表面のα2-6シアル酸量は、増殖能とは関連性を示さず、骨芽細胞への分化ポテンシャルと高い関連性を示すことが示唆された。 (Example 6) Response of α2-6 sialic acid-binding lectin by proliferation ability, differentiation potential, and flow cytometry in subcultured early and late cells of mesenchymal stem cells derived from polydactyly marrow (Yub622 strain) sexually polydactyly bone marrow-derived mesenchymal stem cells (Yub622 strain, RIKEN Bioresource Center) were subcultured in the same manner as the first embodiment and the like, and proliferative capacity ranging from early passages (P5) in late passage (P17) When the (number of divisions) was plotted, the proliferation ability decreased at the late passage (P17), as in the case of the adipocyte-derived mesenchymal stem cells analyzed in Example 2 and the like. On the other hand, the reactivity of the four lectins (TJAI, SSA, SNA, rPSL1a) in flow cytometry showed high reactivity in the early passage (P4) and slightly to the late passage (P15) cells. Although a decrease in reactivity was observed, no significant decrease was observed. When the differentiation potentials of osteoblasts and adipocytes in each cell were analyzed, both differentiation potentials were high in the early passage and the adipocyte differentiation potential was lost in the late passage (P17) cells. However, the differentiation potential into osteoblasts was maintained (FIG. 10).
From the above results, in the bone marrow-derived mesenchymal stem cells, the reactivity of the four lectins, that is, the amount of α2-6 sialic acid on the surface of the bone marrow-derived mesenchymal stem cells was not related to the proliferation ability, It was suggested that it is highly related to the differentiation potential into cells.
（実施例７）骨髄由来間葉系幹細胞の継代培養、分化ポテンシャル、及びフローサイトメトリーによるα2-6シアル酸結合性レクチンの反応性
　骨髄由来間葉系幹細胞における細胞表面のα2-6シアル酸量と骨芽細胞への分化ポテンシャルとの関連性を検証するために、Lonza社から購入した実施例６とは異なる種類の骨髄由来間葉系幹細胞についても実施例６と同様の骨芽細胞と脂肪細胞への分化ポテンシャル解析及び４種のレクチンとの反応性の解析を行った。フローサイトメトリーによる継代初期及び後期の骨髄由来間葉系幹細胞に対する４種のレクチン（TJAI、SSA、SNA、rPSL1a）の反応性の解析結果では、継代初期(P4)の細胞では反応性が高く、継代後期(P12)の細胞では反応性が若干低下しているが十分に維持されていることが観察された（図１１）。一方、分化ポテンシャルについては、（実施例５）と同様の傾向を示し、継代初期(P5)の細胞では骨芽細胞及び脂肪細胞への分化ポテンシャルが高く、継代後期(P12)の細胞では脂肪細胞分化ポテンシャルがほとんど失われたのに対し、骨芽細胞分化ポテンシャルは若干低下したものの維持されている傾向が観察された（図示せず）。上記４種のレクチンの反応性の低下程度は、脂肪細胞分化ポテンシャルと比較して、より骨芽細胞分化ポテンシャルの低下の程度を反映するものであった。
　以上の結果から、骨髄由来間葉系幹細胞における細胞表面のα2,6シアル酸量は、骨芽細胞への分化ポテンシャルと高い関連性を示すことが検証できた。 (Example 7) Reactivity of α2-6 sialic acid-binding lectin by subculture of bone marrow-derived mesenchymal stem cells, differentiation potential, and flow cytometry α2-6 sialic acid on the cell surface in bone marrow-derived mesenchymal stem cells In order to verify the relationship between the amount and the differentiation potential into osteoblasts, bone marrow-derived mesenchymal stem cells of a type different from Example 6 purchased from Lonza, Analysis of differentiation potential into adipocytes and reactivity with four lectins were performed. Analysis of the reactivity of the four lectins (TJAI, SSA, SNA, rPSL1a) to bone marrow-derived mesenchymal stem cells in early and late passages by flow cytometry showed that the cells in the early passage (P4) were reactive. It was observed that the reactivity of the cells in the late passage (P12) was slightly decreased but sufficiently maintained (FIG. 11). On the other hand, the differentiation potential shows the same tendency as in (Example 5), and the differentiation potential to osteoblasts and adipocytes is high in the early passage cells (P5), and in the late passage cells (P12). While the adipocyte differentiation potential was almost lost, the osteoblast differentiation potential was slightly reduced but maintained (not shown). The degree of decrease in reactivity of the four lectins reflected the degree of decrease in osteoblast differentiation potential as compared with the adipocyte differentiation potential.
From the above results, it was verified that the amount of α2,6-sialic acid on the cell surface in bone marrow-derived mesenchymal stem cells is highly related to the differentiation potential to osteoblasts.
（実施例８）多指症骨髄由来間葉系幹細胞の継代培養初期細胞及び後期細胞における、骨芽細胞への分化ポテンシャル、及びフローサイトメトリーによるα2-6シアル酸結合性レクチンの反応性
　（実施例６）で用いた多指症骨髄由来間葉系幹細胞（Yub622株、理研バイオリソースセンター）を用いて、再度骨芽細胞への分化誘導実験を行った。実施例６と同様の手法で継代培養を行った。実施例６では、継代初期と後期でのα2-6シアル酸結合性レクチンとの反応性の差異が明確でなかったにもかかわらず、脂肪分化誘導性の分化ポテンシャルは、継代数の増加に従って明らかに低下してしまい、α2-6シアル酸結合性レクチンとの反応性との相関は見いだせなかった。むしろ骨髄由来間葉系幹細胞とα2-6シアル酸結合性レクチンとの反応性は、骨芽細胞誘導の分化ポテンシャルとのみ相関する可能性が示唆されたため、今回の追試実験では、新たなYub622株の継代初期(P5)と後期（P15）細胞を用いて骨芽細胞への分化程度と、レクチンとの反応性との間の関連性を観察した。
そして、本実験では、分化ポテンシャルとレクチンの反応性の相関性を直接解析できるように、骨芽細胞分化程度の観察と、フローサイトメトリー用とで同じ細胞を使用した。
　まず、継代初期(P5)の細胞及び継代後期（P15）の細胞について、フローサイトメトリーを用いて４種のレクチン（TJAI、SSA、SNA、rPSL1a）の蛍光強度を測定し、その平均値を求めたところ、SSAレクチンでは(P5)で3225であったものが、（P15）では1227まで減少している。この結果は、SSAレクチンと反応する細胞表面のα2-6シアル酸量が約1/3まで減少したことを意味する。同様に、TJA1レクチン、SNAレクチン、rPSL1aレクチンとの反応性も1/3～1/4まで減少した結果を示している。つまり、多指症骨髄由来間葉系幹細胞（Yub622株）細胞表面のα2-6シアル酸量は、継代初期（P5）には多数存在していたが、継代数を重ね、継代後期(P15)になると、おおよそ初期の1/3～1/4量にまで減少することがわかる。
　次いで、それぞれの細胞をLonza社製の分化誘導キットであるhMSC differentiation BulletKit-osteogenic (Cat#: PT-3002, Lonza)を用いて2週間、骨分化細胞への分化誘導を行った。アリザリンレッドSにより染色したところ、継代後期（P15）では骨芽細胞への分化がほとんど観察できなかったのに対し、継代初期（P5）の細胞は多数の骨芽細胞が観察された。すなわち多指症骨髄由来間葉系幹細胞における継代後期の細胞では、継代初期に高かった骨芽細胞への分化ポテンシャルが失われていることがわかる(図１２)。
　以上の結果から、多指症骨髄由来間葉系幹細胞表面におけるα2-6シアル酸量は、骨芽細胞への分化ポテンシャルと高い相関性を示すことが実証された。 (Example 8) The differentiation potential of osteoblasts in the subcultured early and late cells of polydactyly marrow-derived mesenchymal stem cells, and the reactivity of α2-6 sialic acid-binding lectin by flow cytometry ( Using the polydactyly marrow-derived mesenchymal stem cells (Yub622 strain, RIKEN BioResource Center) used in Example 6), another differentiation induction experiment into osteoblasts was performed. Subculture was performed in the same manner as in Example 6. In Example 6, although the difference in reactivity between α2-6 sialic acid-binding lectin was not clear in the early and late passages, the differentiation potential induced by fat differentiation was increased according to the increase in passage number. It clearly decreased, and a correlation with the reactivity with α2-6 sialic acid-binding lectin could not be found. Rather, it was suggested that the reactivity between bone marrow-derived mesenchymal stem cells and α2-6 sialic acid-binding lectin may only correlate with the differentiation potential induced by osteoblasts. The relationship between the degree of differentiation into osteoblasts and reactivity with lectins was observed using early (P5) and late (P15) cells.
In this experiment, the same cells were used for observation of osteoblast differentiation and for flow cytometry so that the correlation between differentiation potential and lectin reactivity could be directly analyzed.
First, the fluorescence intensity of four lectins (TJAI, SSA, SNA, rPSL1a) was measured using flow cytometry for the cells at the early passage (P5) and late passage (P15), and the average value was obtained. As a result, the SSA lectin decreased from 3225 in (P5) to 1227 in (P15). This result means that the amount of α2-6 sialic acid on the cell surface that reacts with SSA lectin was reduced to about 1/3. Similarly, the results show that the reactivity with TJA1 lectin, SNA lectin, and rPSL1a lectin is reduced to 1/3 to 1/4. In other words, the amount of α2-6 sialic acid on the surface of polydactyly-derived bone marrow-derived mesenchymal stem cells (Yub622 strain) was large in the early passage (P5). When it comes to P15), it can be seen that the amount decreases to the initial 1/3 to 1/4 amount.
Next, each cell was induced to differentiate into bone differentiated cells for 2 weeks using hMSC differentiation BulletKit-osteogenic (Cat #: PT-3002, Lonza), a differentiation induction kit manufactured by Lonza. When stained with alizarin red S, differentiation into osteoblasts was hardly observed in the late passage (P15), whereas many osteoblasts were observed in the early passage (P5) cells. That is, it can be seen that the differentiation potential to osteoblasts, which was high in the early passage, was lost in the cells in the late passage in the polymyelinating bone marrow-derived mesenchymal stem cells (FIG. 12).
From the above results, it was demonstrated that the amount of α2-6 sialic acid on the surface of polymyelinating bone marrow-derived mesenchymal stem cells is highly correlated with the differentiation potential into osteoblasts.
（実施例９）多指症骨髄由来間葉系幹細胞の継代培養初期細胞及び後期細胞における、骨芽細胞への分化ポテンシャル、及びフローサイトメトリーによるα2-6シアル酸結合性レクチンの反応性
　(実施例８)と同様の実験を、多指症骨髄由来間葉系幹細胞の別の細胞株（Yub10F、理研バイオリソースセンター）を用いて行った。本実施例でも骨芽細胞分化に用いた細胞とフローサイトメトリーに用いた細胞とは同じ細胞を使用しており、分化ポテンシャルとレクチン反応性（α2-6シアル酸量）との相関性が直接解析できる。
　（実施例８）の場合と同様に、継代初期(P5)の細胞及び継代後期（P15）の細胞について測定したフローサイトメトリーによるα2-6シアル酸結合性レクチンの蛍光強度の積算値は、継代初期（P5）細胞と継代後期(P15)細胞において約1/4～1/5の大幅な減少を示した。
　次いで、Lonza社製の分化誘導キットにより、2週間骨分化細胞への分化誘導を行い、アリザリンレッドにより染色したところ、同様に、継代後期（P15）では骨芽細胞への分化がほとんど観察できなかったのに対し、継代初期（P5）の細胞は多数の骨芽細胞が観察され、骨芽細胞への分化ポテンシャルが継代後期（P15）では失われていることが確認された(図１３)。
　この結果もまた、多指症骨髄由来間葉系幹細胞表面におけるα2-6シアル酸量と骨芽細胞への分化ポテンシャルとの高い相関性を示している。 (Example 9) Mesenchymal stem cells derived from polydactyly bone marrow Subculture in early and late cells Differentiation potential to osteoblasts and reactivity of α2-6 sialic acid-binding lectin by flow cytometry ( An experiment similar to Example 8) was performed using another cell line (Yub10F, RIKEN BioResource Center) of polydactyly marrow-derived mesenchymal stem cells. In this example, the cells used for osteoblast differentiation and the cells used for flow cytometry are the same cells, and the correlation between differentiation potential and lectin reactivity (α2-6 sialic acid content) is directly Can be analyzed.
As in the case of (Example 8), the integrated value of the fluorescence intensity of α2-6 sialic acid-binding lectin by flow cytometry measured for cells in early passage (P5) and late passage (P15) is In the early passage (P5) cells and late passage (P15) cells, there was a significant decrease of about 1/4 to 1/5.
Next, the differentiation induction kit made by Lonza was used to induce differentiation into bone differentiated cells for 2 weeks and stained with alizarin red. Similarly, in the late passage (P15), almost all differentiation into osteoblasts can be observed. In contrast, a large number of osteoblasts were observed in the early passage (P5) cells, and it was confirmed that the differentiation potential to osteoblasts was lost in the late passage (P15) (Fig. 13).
This result also shows a high correlation between the amount of α2-6 sialic acid on the surface of polymyelinating bone marrow-derived mesenchymal stem cells and the differentiation potential into osteoblasts.
（実施例１０）多指症由来軟骨幹細胞（Yub621c株、Yub625株）の継代培養初期細胞及び後期細胞における、軟骨細胞への分化ポテンシャル、及びα2-6シアル酸結合性レクチンの反応性
（１０－１）Yub621c株及びYub625株の継代培養及び軟骨細胞への分化誘導処理
　（実施例５）で用いたと同じ多指症由来軟骨幹細胞のYub621c株（理研バイオリソースセンター）及び別の多指症由来軟骨幹細胞のYub625株（理研バイオリソースセンター）を（実施例５）と同様に継代培養し、Yub621c株からは継代初期（P7）株及び後期（P22,P27,P28）株を取得し、またYub625株からは継代初期（P5）株及び後期（P18,P22）株を取得し、それぞれの株の１部をLonza社製の軟骨細胞分化誘導キットを用いた軟骨細胞への誘導処理を行った。
　それぞれの株に対して、以下の実験を行った。 (Example 10) Differentiation potential of chondrocytes and reactivity of α2-6 sialic acid-binding lectin in early and late passage cells of polydactyly derived cartilage stem cells (Yub621c strain, Yub625 strain) (10 -1) Yub621c strain (RIKEN BioResource Center) of the same polydactyly-derived chondrocyte stem cell used in subculture of Yub621c strain and Yub625 strain and differentiation induction treatment into chondrocytes (Example 5) and other polydactyly-derived diseases Yub625 strain (RIKEN BioResource Center) of cartilage stem cells is subcultured in the same manner as in (Example 5), and early (P7) and late (P22, P27, P28) strains are obtained from Yub621c strain. Acquired early passage (P5) strain and late (P18, P22) strain from Yub625 strain, and part of each strain was induced to chondrocytes using Lonza chondrocyte differentiation induction kit It was.
The following experiment was conducted for each strain.
（１０－２）Yub621c株（P7）、Yub625株（p5,p22）それぞれから分化誘導した軟骨細胞の軟骨マーカーの発現
　Yub625株由来の継代初期（P5）株及び後期（P22）株、並びにYub621c株の継代初期（P7）株において、軟骨細胞への誘導前と、誘導後の細胞表面の軟骨マーカー（COL2A1）の発現量を、DNAマイクロアレイを用いて測定した。
　その結果、いずれの細胞株でも継代初期細胞は軟骨マーカーの発現量が極めて高く、軟骨分化率が高いことが示されたが、Yub625株由来の継代後期（P22）株では、ほとんど発現せず、軟骨分化率が低いことが確認された（図１４）。すなわち、継代初期細胞で高かった軟骨分化ポテンシャルが継代後期になると失われる、という結果が得られた。 (10-2) Expression of chondrocyte markers of chondrocytes induced to differentiate from Yub621c strain (P7) and Yub625 strain (p5, p22), respectively. In the early passage (P7) strain, the expression level of the cartilage marker (COL2A1) on the cell surface before and after induction into chondrocytes was measured using a DNA microarray.
As a result, it was shown that the early passage cells expressed a very high amount of cartilage marker and the cartilage differentiation rate was high in all cell lines. It was confirmed that the cartilage differentiation rate was low (FIG. 14). That is, the results showed that the cartilage differentiation potential that was high in the early passage cells was lost at the later passage.
（１０－３）Yub621c株（P7,P28）、Yub625株（P5,P22）の軟骨分化誘導後のHE染色像とアルシアンブルー染色像
　（１０－１）で得られた軟骨分化誘導処理後の、Yub625株由来の継代初期（P5）細胞及び後期（P22）細胞、並びにYub621c株の継代初期（P7）細胞及び継代後期（P28）細胞それぞれに対して、HE（ヘマトキシリン・エオジン、武藤化学社製）染色及びpH2.5でのアルシアンブルー（武藤化学社製)染色を行った。その結果、Yub625株、Yub621c株いずれにおいても継代初期株由来の軟骨誘導株のみが、アシアンブルーで強く染色され、軟骨分化が活発に起きていることを示している。この結果も、継代初期細胞で高かった軟骨分化ポテンシャルが継代後期になると失われる、という結果を追認する結果を示している。 (10-3) HEb image after induction of cartilage differentiation and Alcian blue-stained image (10-1) of Yub621c strain (P7, P28) and Yub625 strain (P5, P22) , HE (hematoxylin eosin, Muto) for early (P5) and late (P22) cells derived from Yub625 strain, and early (P7) and late passage (P28) cells of Yub621c strain Chemical Co., Ltd.) and Alcian Blue (manufactured by Muto Chemical Co.) at pH 2.5. As a result, in both the Yub625 strain and the Yub621c strain, only the cartilage induction strain derived from the early passage strain was strongly stained with cyan blue, indicating that cartilage differentiation was actively occurring. This result also confirms the result that the cartilage differentiation potential, which was high in the early passage cells, is lost in the later passage.
（１０－４）Yub621c株（P7,P27）細胞、及びYub625株（P5,P18）細胞のフローサイトメトリーによるα2-6シアル酸結合性レクチンの反応性
　Yub621c株の継代初期(P7)細胞及び継代後期（P27）細胞、並びにYub625株の継代初期(P5)細胞及び継代後期（P18）細胞に対し、（実施例８）の場合と同様に、フローサイトメトリーによるα2-6シアル酸結合性レクチンとの反応性を観察した。その結果、Yub621c株の場合は、継代後期(P27)になると、継代初期（P7）における反応性（すなわち細胞表面のα2-6シアル酸量）が約1/5～1/9にまでに低下することが、またYub625株では、継代後期（P18）では、継代初期（P5）の約1/4～1/6にまで低下することが観察された（図１６、１７）。
　前記（１０－２）及び（１０－３）の結果をあわせると、多指症由来軟骨幹細胞表面におけるα2-6シアル酸量が軟骨細胞への分化ポテンシャルと高い相関性を示していることが実証できた。 (10-4) Reacting α2-6 sialic acid-binding lectin by flow cytometry of Yub621c strain (P7, P27) cells and Yub625 strain (P5, P18) cells Early passage (P7) cells of Yub621c strain and Α2-6 sialic acid by flow cytometry on late passage (P27) cells and early passage (P5) cells and late passage (P18) cells of the Yub625 strain, as in Example 8. The reactivity with the binding lectin was observed. As a result, in the case of the Yub621c strain, the reactivity in the early passage (P7) (ie, the amount of α2-6 sialic acid on the cell surface) reaches about 1/5 to 1/9 at the late passage (P27). In the Yub625 strain, it was observed that it decreased to about 1/4 to 1/6 of the early passage (P5) in the late passage (P18) (FIGS. 16 and 17).
Combining the results of (10-2) and (10-3), it has been demonstrated that the amount of α2-6 sialic acid on the surface of polydactyly-derived chondrocyte stem cells is highly correlated with the differentiation potential into chondrocytes. did it.
（実施例１１）異なる継代数の脂肪由来間葉系幹細胞（ADSC）と多指症由来軟骨幹細胞（Yub621c）の細胞抽出液におけるα2-6シアル酸結合性レクチン（rPSL1a）の反応性変化
（１１－１）抗CD29抗体による免疫沈降物でのα2-6シアル酸結合性レクチン（rPSL1a）の反応性変化
　本実験では、幹細胞表面のα2-6シアル酸のコアタンパク質を特定するために、継代数が初期、中期、後期の3段階に亘る脂肪由来間葉系幹細胞（ADSC）と多指症由来軟骨幹細胞とを用い、間葉系幹細胞など幹細胞表面で高発現しており、間葉系幹細胞マーカーとしても知られるCD29、CD49e及びCD13糖タンパク質抗原発現量と、その複合糖鎖を構成する非還元末端糖鎖であるα2-6シアル酸量の変化を観察する（図１８～２０）。
　実施例１で用いた脂肪由来間葉系幹細胞（ADSC）と同じADSCを同様の手法で継代培養し、異なる継代数の細胞（P5,P19,P28）培養液から、CelLyticＴＭ MEM Protein Extraction Kitを用い疎水性分画として細胞抽出液を取得した。
　各細胞抽出液に対し、ビオチン標識抗CD29抗体（Abcam社製）を固定化したストレプトアビジン被覆磁気ビーズ（Dynabeads M-280 Streptavidin、Dynabeads社製）を作用させ、4℃で一晩放置して、免疫沈降させた。得られた免疫沈降物を、SDSサンプルバッファーで可溶化後、SDS-PAGEで流し、西洋ワサビペルオキシダーゼ（HRP、同人化学社製）で標識した抗CD29抗体との反応性及びHRP標識したα2-6シアル酸結合性レクチン（rPSL1a）との反応性を各継代数ごとに観察した（図１８、Ａ）。
　同様に、実施例１０で用いた多指症由来軟骨幹細胞（Yub621c株）と同じYub621c株についても異なる継代数の細胞（P7,P16,P28）の細胞抽出液を取得し、抗CD29抗体（Abcam社製）による免疫沈降物と、α2-6シアル酸結合性レクチン（rPSL1a）との反応性変化を観察した（図１８、Ｂ）。
　その結果、何れの細胞においても、継代数の違いで抗CD29抗体の反応性に変化は見られないものの、α2-6シアル酸結合性レクチン（rPSL1a）の反応性は継代数の増加とともに低下することがわかった。
　このことから、細胞表面のCD29糖タンパク質の発現量は、継代数ごとにさほど大きな差はみられないのに対し、CD29糖タンパク質の複合糖鎖中の末端α2-6シアル酸量は、継代数の増加とともに減少していくことがわかる。すなわち、細胞分化ポテンシャルが高い継代初期細胞ではCD29上のα2-6シアル酸量は多いものの、細胞分化ポテンシャルが低い継代後期細胞ではCD29上のα2-6シアル酸量は低いことが分かった。 (Example 11) Reactivity change of α2-6 sialic acid-binding lectin (rPSL1a) in cell extracts of adipose-derived mesenchymal stem cells (ADSC) and polydactyly-derived chondrocyte stem cells (Yub621c) of different passage numbers (11 -1) Change in reactivity of α2-6 sialic acid-binding lectin (rPSL1a) in immunoprecipitates with anti-CD29 antibody In this experiment, passage number was used to identify the core protein of α2-6 sialic acid on the stem cell surface. Is highly expressed on the surface of stem cells such as mesenchymal stem cells, using adipose-derived mesenchymal stem cells (ADSC) and polydactyly-derived cartilage stem cells in three stages: early, middle, and late, and mesenchymal stem cell markers Changes in the expression levels of CD29, CD49e and CD13 glycoprotein antigens, also known as γ, and the amount of α2-6 sialic acid which is a non-reducing terminal sugar chain constituting the complex sugar chain are observed (FIGS. 18 to 20).
The same ADSC as the adipose-derived mesenchymal stem cells (ADSC) used in Example 1 was subcultured in the same manner, and the cells from different passage numbers (P5, P19, P28) were cultured in CelLytic ™ MEM Protein Extraction Kit. A cell extract was obtained as a hydrophobic fraction.
Streptavidin-coated magnetic beads (Dynabeads M-280 Streptavidin, Dynabeads) immobilized with biotin-labeled anti-CD29 antibody (Abcam) were allowed to act on each cell extract and left overnight at 4 ° C. Immunoprecipitation. The resulting immunoprecipitate was solubilized with SDS sample buffer, and then run by SDS-PAGE. Reactivity with anti-CD29 antibody labeled with horseradish peroxidase (HRP, manufactured by Dojin Kagaku) and α2-6 labeled with HRP Reactivity with sialic acid-binding lectin (rPSL1a) was observed for each passage number (FIG. 18, A).
Similarly, cell extracts of cells with different passage numbers (P7, P16, P28) were obtained from the same Yub621c strain as the polydactyly derived cartilage stem cells (Yub621c strain) used in Example 10, and anti-CD29 antibody (Abcam The change in reactivity between the immunoprecipitate produced by the company and the α2-6 sialic acid-binding lectin (rPSL1a) was observed (FIG. 18, B).
As a result, the reactivity of α2-6 sialic acid-binding lectin (rPSL1a) decreases with increasing passage number, although there is no change in the reactivity of anti-CD29 antibody due to the difference in passage number in any cell. I understood it.
From this, the expression level of CD29 glycoprotein on the cell surface does not differ greatly depending on the passage number, whereas the amount of terminal α2-6 sialic acid in the complex sugar chain of CD29 glycoprotein is the passage number. It can be seen that it decreases with increasing. In other words, α2-6 sialic acid on CD29 was high in early passage cells with high cell differentiation potential, but α2-6 sialic acid on CD29 was low in late passage cells with low cell differentiation potential. .
（１１－２）CD49e抗体による免疫沈降物でのα2-6シアル酸結合性レクチン（rPSL1a）の反応性変化
　脂肪由来間葉系幹細胞（ADSC）の継代数（P6,P20,P28）細胞、及び多指症由来軟骨幹細胞（Yub621c株）の継代数（P7,P16,P28）細胞からの疎水性分画として得た細胞抽出物に対し、抗CD49e抗体（R&D社製）による免疫沈降を行い、その免疫沈降物と、α2-6シアル酸結合性レクチン（rPSL1a）との反応性変化を観察した（図１９）。
　その結果、CD29の場合と同様に、継代数の違いで抗CD49e抗体の反応性に変化は見られないものの、α2-6シアル酸結合性レクチン（rPSL1a）の反応性は継代数の増加とともに低下することがわかった。すなわち分化ポテンシャルが高い継代初期細胞ではCD49e上のα2-6シアル酸量は多いものの、細胞分化ポテンシャルが低い継代後期細胞ではCD29上のα2-6シアル酸量は低いことが分かった。 (11-2) Reactivity change of α2-6 sialic acid-binding lectin (rPSL1a) in immunoprecipitate by CD49e antibody Passage number (P6, P20, P28) cells of adipose-derived mesenchymal stem cells ( ADSC) , and For the cell extract obtained as the hydrophobic fraction from the passage number (P7, P16, P28) cells of polydactyly derived chondrocyte stem cells (Yub621c strain), immunoprecipitation with anti-CD49e antibody (R & D) was performed, Changes in reactivity between the immunoprecipitate and α2-6 sialic acid-binding lectin (rPSL1a) were observed (FIG. 19).
As a result, the reactivity of α2-6 sialic acid-binding lectin (rPSL1a) decreased with increasing passage number, although there was no change in the reactivity of anti-CD49e antibody due to the difference in passage number, as in CD29. I found out that That is, it was found that α2-6 sialic acid on CD49e was high in early passage cells with high differentiation potential, but α2-6 sialic acid on CD29 was low in late passage cells with low cell differentiation potential.
（１１－３）CD13抗体による免疫沈降物でのα2-6シアル酸結合性レクチン（rPSL1a）の反応性変化
　脂肪由来間葉系幹細胞（ADSC）の継代数（P6,P20,P28）細胞、及び多指症由来軟骨幹細胞（Yub621c株）の継代数（P7,P16,P28）細胞からの細胞抽出液に対し、抗CD13抗体（R&D社製）による免疫沈降を施し、その免疫沈降物と、α2-6シアル酸結合性レクチン（rPSL1a）との反応性変化を観察した（図２０）。
　その結果、CD13はα2-6シアル酸結合性レクチン（rPSL1a）と反応したことから、α2-6シアル酸を非還元末端に有しているコアタンパク質の１つであることは分かったものの、Yub621c株の継代後期（P28）で若干α2-6シアル酸量の低下がみられた程度で、継代数によってCD13上のα2-6シアル酸量に大きな違いは見られなかった。すなわちCD13上のα2-6シアル酸量は分化ポテンシャルとは相関しないことが分かった。 (11-3) Reactivity change of α2-6 sialic acid-binding lectin (rPSL1a ) in CD13 antibody immunoprecipitates Passage number of adipose-derived mesenchymal stem cells ( ADSC) (P6, P20, P28) cells, The cell extract from the passage number (P7, P16, P28) cells of polydactyly derived cartilage stem cells (Yub621c strain) is subjected to immunoprecipitation with anti-CD13 antibody (manufactured by R & D), and the immunoprecipitate and α2 Changes in reactivity with -6 sialic acid-binding lectin (rPSL1a) were observed (FIG. 20).
As a result, CD13 reacted with α2-6 sialic acid-binding lectin (rPSL1a), so it was found that it is one of the core proteins with α2-6 sialic acid at the non-reducing end. The amount of α2-6 sialic acid was slightly decreased in the late passage of the strain (P28), and no significant difference was observed in the amount of α2-6 sialic acid on CD13 depending on the passage number. That is, the amount of α2-6 sialic acid on CD13 was not correlated with the differentiation potential.
　以上の結果から、体性幹細胞におけるα2-6シアル酸量の変化と分化ポテンシャルとの相関を観察するためのα2-6シアル酸コアタンパク質としては、CD29糖タンパク質及びCD49e糖タンパク質が適していることがわかった。 Based on the above results, CD29 glycoprotein and CD49e glycoprotein are suitable as α2-6 sialic acid core proteins for observing the correlation between changes in α2-6 sialic acid content and differentiation potential in somatic stem cells. I understood.
（実施例１２）iPS細胞（201B7株）の細胞抽出液からの抗CD29抗体及び抗CD49e抗体との免疫沈降へのα2-6シアル酸結合性レクチン（rPSL1a）の反応性変化
　iPS細胞（201B7株）の疎水性分画として得た細胞抽出液に対しても、実施例１１と同様に、抗CD29抗体（R&D社製）及び抗CD49e抗体（R&D社製）により免疫沈降を行い、それぞれの免疫沈降物に対して、α2-6シアル酸結合性レクチン（rPSL1a）との反応性変化を観察した（図２１）。
　その結果、iPS細胞の抗CD29抗体及び抗CD49e抗体による免疫沈降物はα2-6シアル酸量がきわめて多く、幹細胞表面におけるα2-6シアル酸のコアタンパク質であることを裏付ける結果であった。すなわち、細胞分化ポテンシャルの高さは、CD29及びCD49e上のα2-6シアル酸量をマーカーとして測定することで推定できることが強く示唆された。 (Example 12) Reactivity change of α2-6 sialic acid-binding lectin (rPSL1a) to immunoprecipitation with anti-CD29 antibody and anti-CD49e antibody from cell extract of iPS cells (201B7 strain ) iPS cells (201B7 strain) In the same manner as in Example 11, the cell extract obtained as a hydrophobic fraction was subjected to immunoprecipitation using an anti-CD29 antibody (R & D) and an anti-CD49e antibody (R & D). A change in reactivity with α2-6 sialic acid-binding lectin (rPSL1a) was observed on the precipitate (FIG. 21).
As a result, it was confirmed that the immunoprecipitates of the anti-CD29 antibody and anti-CD49e antibody of iPS cells had a very large amount of α2-6 sialic acid and were a core protein of α2-6 sialic acid on the stem cell surface. That is, it was strongly suggested that the height of cell differentiation potential can be estimated by measuring the amount of α2-6 sialic acid on CD29 and CD49e as a marker.
（実施例１３）４種のα2-6シアル酸結合性レクチン(SNA、SSA、TJAI、rPSL1a)と抗CD29抗体のサンドイッチアッセイ系の構築
（１３－１）CD29抗体とのサンドイッチアッセイ系
　多指症由来軟骨幹細胞（Yub621c）の各種継代数（P7,P16,P28）の細胞の疎水性分画から抗CD29抗体で免疫沈降を行い、それぞれの免疫沈降物を、４種のα2-6シアル酸結合性レクチン(SNA、SSA、TJAI、rPSL1a)を固定化したアレイに対して反応させて、Cy3で蛍光標識化した抗CD29抗体を反応させるサンドイッチアッセイを行った。
　その結果、α2-6シアル酸結合性レクチンのうち、TJAIレクチン以外のSNA,SSA,rPSL1aレクチンはいずれも継代数の増加に伴い減少傾向を示した。とりわけ、SNAレクチンの場合の継代数の増加に伴う減少傾向がきわめて明瞭であることから、抗CD29抗体とSNAレクチンとを組み合わせたサンドイッチアッセイ系は、分化ポテンシャルを推定するためのα2-6シアル酸発現量測定用アッセイ系として最も有効であるといえる。
　α2-6シアル酸発現量の変化と分化ポテンシャルとの関連性は、用量依存的な相関関係を有する可能性が高いことが期待されるので、あらかじめ間葉系幹細胞、軟骨幹細胞などの体性幹細胞における当該アッセイ系で測定される標識強度と目的分化細胞特異的な分化細胞マーカー強度などの数値とから検量線を求めておくことで、被検体性幹細胞に対する脂肪細胞、骨芽細胞、又は軟骨細胞など移植材料としての分化ポテンシャルに関し、確実性の高い定量的な評価、判定が可能となる。すなわち、抗CD29抗体とSNAレクチン、SSAレクチン、及びrPSL1aレクチンとを組み合わせたサンドイッチアッセイ系は、体性幹細胞の各種細胞への分化ポテンシャルの定量的な評価、判定に用いることができる。 (Example 13) Construction of four α2-6 sialic acid-binding lectins (SNA, SSA, TJAI, rPSL1a) and anti-CD29 antibody sandwich assay system (13-1) Sandwich assay system polydactyly with CD29 antibody Immunoprecipitation was performed with anti-CD29 antibody from the hydrophobic fraction of cells of various passage numbers (P7, P16, P28) of the derived cartilage stem cells (Yub621c), and each of the immunoprecipitates was bound to 4 types of α2-6 sialic acid. A sandwich assay was performed by reacting a sex lectin (SNA, SSA, TJAI, rPSL1a) to an immobilized array and reacting with an anti-CD29 antibody fluorescently labeled with Cy3.
As a result, among the α2-6 sialic acid-binding lectins, SNA, SSA, and rPSL1a lectins other than TJAI lectin all showed a decreasing tendency as the passage number increased. In particular, since the decreasing tendency with increasing passage number in the case of SNA lectin is very clear, the sandwich assay system combining anti-CD29 antibody and SNA lectin is an α2-6 sialic acid for estimating the differentiation potential. It can be said that it is most effective as an assay system for measuring the expression level.
Since it is expected that there is a high possibility of a dose-dependent correlation between the change in the expression level of α2-6 sialic acid and the differentiation potential, somatic stem cells such as mesenchymal stem cells and cartilage stem cells A calibration curve is obtained from numerical values such as the labeling intensity measured in the assay system and the differentiated cell marker intensity specific to the target differentiated cell, so that the adipocytes, osteoblasts, or chondrocytes for the subject stem cells are obtained. With regard to the differentiation potential as a transplant material, it is possible to perform quantitative evaluation and determination with high certainty. That is, a sandwich assay system combining an anti-CD29 antibody and SNA lectin, SSA lectin, and rPSL1a lectin can be used for quantitative evaluation and determination of differentiation potential of somatic stem cells into various cells.
（１３－２）抗CD49e抗体とのサンドイッチアッセイ系
　多指症骨髄由来間葉系幹細胞（Yub621c）の各種継代数（P7,P16,P28）の細胞の疎水性分画から抗CD49e抗体で免疫沈降を行い、それぞれの免疫沈降物を４種のα2-6シアル酸結合性レクチン(SNA、SSA、TJAI、rPSL1a)を固定化したアレイに対して反応させて、Cy3で蛍光標識化した抗CD49e抗体を反応させるサンドイッチアッセイを行った。
　その結果、抗CD49e抗体の場合も、α2-6シアル酸結合性レクチンのうち、TJAIレクチン以外の、SNAレクチン、SSAレクチン、及びrPSL1aレクチンとを組み合わせることで、被検体性幹細胞表面のα2-6シアル酸発現量の変化を定量可能なサンドイッチアッセイ系を構築できることを見いだした。
　すなわち、抗CD49e抗体とSNAレクチン、SSAレクチン、及びrPSL1aレクチンとを組み合わせたサンドイッチアッセイ系もまた、体性幹細胞の各種細胞への分化ポテンシャルの定量的な評価、判定に用いることができる。 (13-2) Sandwich assay with anti-CD49e antibody Immunoprecipitation with anti-CD49e antibody from hydrophobic fractions of various passage numbers (P7, P16, P28) of polydactyly bone marrow-derived mesenchymal stem cells (Yub621c) Each of the immunoprecipitates was reacted with an array immobilized with 4 kinds of α2-6 sialic acid-binding lectins (SNA, SSA, TJAI, rPSL1a), and anti-CD49e antibody fluorescently labeled with Cy3. Sandwich assay was performed.
As a result, in the case of anti-CD49e antibody, α2-6 on the surface of the subject stem cell can be obtained by combining SNA lectin, SSA lectin, and rPSL1a lectin other than TJAI lectin among α2-6 sialic acid-binding lectins. It was found that a sandwich assay system capable of quantifying changes in the expression level of sialic acid could be constructed.
That is, a sandwich assay system in which an anti-CD49e antibody is combined with SNA lectin, SSA lectin, and rPSL1a lectin can also be used for quantitative evaluation and determination of differentiation potential of somatic stem cells into various cells.
（実施例１４）シアリダーゼ処理した多指症骨髄由来間葉系幹細胞（Yub621c）の骨芽細胞分化
　多指症骨髄由来間葉系幹細胞（Yub621c）の骨芽細胞分化において、α2-6シアル酸の発現が重要な役割を担っていることを確認するために、分化誘導前、及び分化誘導中においてシアリダーゼ処理の有無による骨芽細胞分化への影響を観察した。シアリダーゼとしては、α2-3シアル酸、及びα2-6シアル酸の両方を切断するArthrobacter ureafaciensシアリダーゼ（ロシュ・ライフサイエンス社製）を用いた。
　その結果、シアリダーゼ処理を行わない通常の骨芽細胞への分化誘導の場合（コントロール）と比較して、分化誘導前でも分化誘導中でもシアリダーゼ処理を行うと、骨芽細胞分化の効率が下がることが分かった。すなわち、α2-6シアル酸は細胞分化において直接的な機能を担っていることが分かった。 (Example 14) Osteoblast differentiation of sialidase-treated polydactyly marrow-derived mesenchymal stem cells (Yub621c) In osteoblast differentiation of polydactyly marrow-derived mesenchymal stem cells (Yub621c), In order to confirm that expression plays an important role, the effect on the osteoblast differentiation by the presence or absence of sialidase treatment was observed before and during differentiation induction. As the sialidase, Arthrobacter ureafaciens sialidase (Roche Life Science Co., Ltd.) that cleaves both α2-3 sialic acid and α2-6 sialic acid was used.
As a result, compared to the case of differentiation induction into normal osteoblasts without sialidase treatment (control), if sialidase treatment is performed before differentiation induction or during differentiation induction, the efficiency of osteoblast differentiation may be reduced. I understood. That is, it was found that α2-6 sialic acid has a direct function in cell differentiation.

Claims (28)

  1. 　体性幹細胞の分化ポテンシャルを判別又は評価する方法であって、
    　体性幹細胞表面で発現するα2-6シアル酸を検出する工程、又はα2-6シアル酸量を測定する工程を包含することを特徴とする、方法。     A method for discriminating or evaluating the differentiation potential of somatic stem cells,
    A method comprising the step of detecting α2-6 sialic acid expressed on the surface of a somatic stem cell, or measuring the amount of α2-6 sialic acid.
  2. 　体性幹細胞が間葉系幹細胞又は軟骨幹細胞であって、体性幹細胞の分化ポテンシャルが、骨芽細胞又は軟骨細胞への分化ポテンシャルである、請求項１に記載の方法。 The method according to claim 1, wherein the somatic stem cell is a mesenchymal stem cell or a cartilage stem cell, and the differentiation potential of the somatic stem cell is a differentiation potential into an osteoblast or a chondrocyte.
  3. 　α2-6シアル酸を検出する工程、又はα2-6シアル酸量を測定する工程が、α2-6シアル酸をエピトープとして認識するプローブを用いて行うことを特徴とする、請求項１又は２に記載の方法。 3. The step of detecting α2-6 sialic acid or the step of measuring the amount of α2-6 sialic acid is performed using a probe that recognizes α2-6 sialic acid as an epitope. The method described.
  4. 　α2-6シアル酸をエピトープとして認識するプローブが、TJAIレクチン、SSAレクチン、SNAレクチン、及びPSL1aレクチンから選択された少なくとも１種のレクチンを含むことを特徴とする、請求項３に記載の方法。 The method according to claim 3, wherein the probe that recognizes α2-6 sialic acid as an epitope contains at least one lectin selected from TJAI lectin, SSA lectin, SNA lectin, and PSL1a lectin.
  5. 　さらに、体性幹細胞表面で発現し、かつα2-6シアル酸を非還元末端糖鎖として有する糖タンパク質を、当該糖タンパク質を特異的に認識するプローブを用いて検出又は測定する工程を包含する、請求項１～４のいずれか一項に記載の方法。 And a step of detecting or measuring a glycoprotein expressed on the surface of a somatic stem cell and having α2-6 sialic acid as a non-reducing terminal sugar chain using a probe that specifically recognizes the glycoprotein, The method according to any one of claims 1 to 4.
  6. 　前記糖タンパク質が、CD29又はCD49eである、請求項５に記載の方法。 The method according to claim 5, wherein the glycoprotein is CD29 or CD49e.
  7. 　前記糖タンパク質を特異的に認識するプローブが、抗CD29抗体、及び抗CD49e抗体から選択された少なくとも１種の抗体である、請求項６に記載の方法。 The method according to claim 6, wherein the probe that specifically recognizes the glycoprotein is at least one antibody selected from an anti-CD29 antibody and an anti-CD49e antibody.
  8. 　体性幹細胞の骨芽細胞又は軟骨細胞への分化ポテンシャルを判別又は評価する方法であって、下記の（１）及び（２）を含む方法；
    （１）被検体性幹細胞含有試料を、下記の（ａ）又は（ｂ）のいずれか一方のプローブが固定化された基板上にオーバーレイし、次いで、標識化された他方のプローブを作用させる工程、
    （ａ）SSAレクチン、SNAレクチン、及びPSL1aレクチンから選択された少なくとも１種のレクチン、
    （ｂ）抗CD29抗体、及び抗CD49e抗体から選択された少なくとも１種の抗体、
    （２）標識量を測定する工程。     A method for discriminating or evaluating the differentiation potential of somatic stem cells into osteoblasts or chondrocytes, comprising the following (1) and (2):
    (1) Overlaying a specimen stem cell-containing sample on a substrate on which either one of the following probes (a) or (b) is immobilized, and then allowing the other labeled probe to act ,
    (A) at least one lectin selected from SSA lectin, SNA lectin, and PSL1a lectin,
    (B) at least one antibody selected from an anti-CD29 antibody and an anti-CD49e antibody,
    (2) A step of measuring the labeled amount.
  9. 　あらかじめ、前記試料中の被検体性幹細胞を、前記（ａ）又は（ｂ）のプローブとの相互作用を利用して濃縮する工程を設けることを特徴とする、請求項８に記載の方法。 9. The method according to claim 8, wherein a step of concentrating the analyte stem cells in the sample in advance using the interaction with the probe (a) or (b) is provided.
  10. 　請求項８又は９の方法に、さらに
    （３）測定した標識量と、被検体性幹細胞の分化ポテンシャルとを相関させる工程、
    を設けることを特徴とする、体性幹細胞の骨芽細胞又は軟骨細胞への分化ポテンシャルを定量的に判別又は評価する方法。     The method according to claim 8 or 9, further comprising (3) correlating the measured labeling amount with the differentiation potential of the subject stem cell,
    A method for quantitatively discriminating or evaluating the differentiation potential of somatic stem cells into osteoblasts or chondrocytes.
  11. 　α2-6シアル酸をエピトープとして認識するプローブを含むことを特徴とする、体性幹細胞の分化ポテンシャルの判定又は評価用試薬。 A reagent for determining or evaluating the differentiation potential of somatic stem cells, comprising a probe that recognizes α2-6 sialic acid as an epitope.
  12. 　α2-6シアル酸をエピトープとして認識するプローブを含む、体性幹細胞の分化ポテンシャル判定用キット。 A kit for determining the differentiation potential of somatic stem cells, comprising a probe that recognizes α2-6 sialic acid as an epitope.
  13. 　前記α2-6シアル酸をエピトープとして認識するプローブが、
    （ａ）α2-6シアル酸をエピトープとして認識するレクチンの少なくとも１つ、及び
    （ｂ）α2-6シアル酸をエピトープとして認識する抗α2-6シアル酸抗体の少なくとも１つ、
    との組み合わせを含み（ａ）又は（ｂ）のいずれか一方のプローブが基板に固定化され、他方のプローブが標識化されている、請求項１２に記載のキット。     The probe that recognizes the α2-6 sialic acid as an epitope,
    (A) at least one lectin that recognizes α2-6 sialic acid as an epitope, and (b) at least one anti-α2-6 sialic acid antibody that recognizes α2-6 sialic acid as an epitope,
    The kit according to claim 12, wherein one of the probes (a) and (b) is immobilized on a substrate and the other probe is labeled.
  14. 　前記前記体性幹細胞の分化ポテンシャル判定用キットが、
    （ａ）α2-6シアル酸をエピトープとして認識するプローブ、と共に
    （ｂ）体性幹細胞表面で発現し、かつα2-6シアル酸を非還元末端糖鎖として有する糖タンパク質を特異的に認識するプローブ、
    を含み（ａ）又は（ｂ）のいずれか一方のプローブが基板に固定化され、他方のプローブが標識化されている、請求項１２に記載のキット。     The somatic stem cell differentiation potential determination kit comprises:
    (A) a probe that recognizes α2-6 sialic acid as an epitope, and (b) a probe that specifically recognizes a glycoprotein expressed on the surface of a somatic stem cell and having α2-6 sialic acid as a non-reducing terminal sugar chain ,
    The kit according to claim 12, wherein one of the probes (a) or (b) is immobilized on a substrate and the other probe is labeled.
  15. 　　体性幹細胞の骨芽細胞又は軟骨細胞への分化ポテンシャルを判別又は評価するためのキットであって、
    （ａ）SSAレクチン、SNAレクチン、及びPSL1aレクチンから選択された少なくとも１種のレクチン、
    （ｂ）抗CD29抗体、及び抗CD49e抗体から選択された少なくとも１種の抗体、
    の組み合わせを含み（ａ）又は（ｂ）のいずれか一方のプローブが基板に固定化され、他方のプローブが標識化されているキット。     A kit for discriminating or evaluating the differentiation potential of somatic stem cells into osteoblasts or chondrocytes,
    (A) at least one lectin selected from SSA lectin, SNA lectin, and PSL1a lectin,
    (B) at least one antibody selected from an anti-CD29 antibody and an anti-CD49e antibody,
    A kit in which either one of the probes (a) or (b) is immobilized on a substrate and the other probe is labeled.
  16. 　体性幹細胞を含む細胞試料から分化ポテンシャルが高い体性幹細胞を分離又は濃縮する方法であって、
    体性幹細胞を含む細胞試料に対し、α2-6シアル酸をエピトープとして認識するプローブを接触させる工程を含む、方法。     A method for separating or concentrating somatic stem cells having a high differentiation potential from a cell sample containing somatic stem cells,
    A method comprising a step of contacting a cell sample containing somatic stem cells with a probe that recognizes α2-6 sialic acid as an epitope.
  17. 　前記α2-6シアル酸をエピトープとして認識するプローブを接触させる工程が、フローサイトメトリーによる細胞分画工程、又は前記プローブを固定化した担体による分離工程のいずれかの方法を含む、請求項１６に記載の方法。 The step of contacting the probe that recognizes α2-6 sialic acid as an epitope includes a cell fractionation step by flow cytometry or a separation step using a carrier on which the probe is immobilized. The method described.
  18. 　前記プローブを固定化した担体が、磁気ビーズ又はアフィニティカラムである、請求項１７に記載の方法。 The method according to claim 17, wherein the carrier on which the probe is immobilized is a magnetic bead or an affinity column.
  19. 　α2-6シアル酸をエピトープとして認識するプローブが、TJAIレクチン、SSAレクチン、SNAレクチン、及びPSL1aレクチンから選択された少なくとも１種のレクチンを含むことを特徴とする、請求項１６～１８のいずれかに記載の方法。 The probe for recognizing α2-6 sialic acid as an epitope includes at least one lectin selected from TJAI lectin, SSA lectin, SNA lectin, and PSL1a lectin. The method described in 1.
  20. 　あらかじめ、前記試料中の体性幹細胞を、体性幹細胞表面で発現し、かつα2-6シアル酸を非還元末端糖鎖として有する糖タンパク質を特異的に認識するプローブとの相互作用を利用して濃縮する工程を設けることを特徴とする、請求項１６～１９のいずれかに記載の方法。 In advance, the somatic stem cells in the sample are expressed on the surface of the somatic stem cells, and the interaction with a probe that specifically recognizes a glycoprotein having α2-6 sialic acid as a non-reducing terminal sugar chain is used. The method according to any one of claims 16 to 19, wherein a step of concentrating is provided.
  21. 　間葉系幹細胞又は軟骨幹細胞含有試料から、骨芽細胞又は軟骨細胞への分化ポテンシャルが高い幹細胞を分離又は濃縮する方法であって、
    前記幹細胞含有試料に対し、α2-6シアル酸をエピトープとして認識するプローブを接触させる工程を含む、方法。     A method for separating or concentrating stem cells having a high differentiation potential into osteoblasts or chondrocytes from a mesenchymal stem cell or chondrocyte-containing sample,
    A method comprising the step of contacting the stem cell-containing sample with a probe that recognizes α2-6 sialic acid as an epitope.
  22. 　あらかじめ、前記試料中の体性幹細胞を、CD29又はCD49e糖タンパク質を特異的に認識するプローブとの相互作用を利用して濃縮する工程を設けることを特徴とする、請求項２１に記載の方法。 The method according to claim 21, wherein a step of concentrating somatic stem cells in the sample in advance using an interaction with a probe that specifically recognizes CD29 or CD49e glycoprotein is provided.
  23. 　CD29又はCD49e糖タンパク質を認識するプローブとの相互作用が、抗CD29抗体又は抗CD49e抗体との免疫反応である、請求項２２に記載の方法。 The method according to claim 22, wherein the interaction with a probe recognizing CD29 or CD49e glycoprotein is an immune reaction with an anti-CD29 antibody or an anti-CD49e antibody.
  24. 　α2-6シアル酸をエピトープとして認識するプローブを固定化した担体を含むことを特徴とする、分化ポテンシャルの高い体性幹細胞の単離又は濃縮用キット。 A kit for isolating or concentrating somatic stem cells with high differentiation potential, comprising a carrier on which a probe that recognizes α2-6 sialic acid as an epitope is immobilized.
  25. 　α2-6シアル酸をエピトープとして認識するプローブを固定化した担体を含むことを特徴とする、骨芽細胞又は軟骨細胞への分化ポテンシャルの高い間葉系幹細胞又は軟骨幹細胞の単離又は濃縮用キット。 A kit for isolating or enriching mesenchymal stem cells or chondrocyte stem cells having a high differentiation potential into osteoblasts or chondrocytes, comprising a carrier on which a probe recognizing α2-6 sialic acid as an epitope is immobilized .
  26. 　前記プローブを固定化した担体が、磁気ビーズ又はアフィニティカラムである、請求項２４又は２５に記載のキット。 The kit according to claim 24 or 25, wherein the carrier on which the probe is immobilized is a magnetic bead or an affinity column.
  27. 　さらに、CD29又はCD49e糖タンパク質を認識するプローブを含む請求項２５又は２６に記載のキット。 27. The kit according to claim 25 or 26, further comprising a probe that recognizes CD29 or CD49e glycoprotein.
  28. 　間葉系幹細胞又は軟骨幹細胞含有細胞試料を用いた骨芽細胞又は軟骨細胞増殖のための移植材料の調製方法であって、下記の（１）～（５）の工程を含む方法；
    （１）採取した骨髄由来間葉系幹細胞含有細胞試料をex vivoで拡大培養する工程、
    （２）工程（１）で得られた細胞試料を、α2-6シアル酸をエピトープとして認識するプローブを固定化した担体と接触させる工程、
    （３）工程（２）で得られた固定化担体をリン酸含有緩衝液により洗浄し、非特異的な結合物を除去する工程、
    （４）工程（３）で得られた固定化担体を糖類含有緩衝液により洗浄し、前記プローブと結合している細胞を固定化担体から遊離させる工程、
    （５）工程（４）で得られた細胞を集めて移植用材料を調製する工程。     A method for preparing a transplant material for osteoblast or chondrocyte proliferation using a mesenchymal stem cell or chondrocyte-containing cell sample, comprising the following steps (1) to (5):
    (1) A step of expanding the collected bone marrow-derived mesenchymal stem cell-containing cell sample ex vivo,
    (2) contacting the cell sample obtained in step (1) with a carrier immobilized with a probe that recognizes α2-6 sialic acid as an epitope;
    (3) a step of washing the immobilization support obtained in step (2) with a phosphate-containing buffer to remove non-specific binders,
    (4) a step of washing the immobilized carrier obtained in step (3) with a saccharide-containing buffer, and releasing cells bound to the probe from the immobilized carrier;
    (5) A step of collecting the cells obtained in step (4) and preparing a transplant material.
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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2017184713A (en) * 2016-03-31 2017-10-12 国立研究開発法人産業技術総合研究所 Method for determining cell differentiation potential
JP2019000063A (en) * 2017-06-19 2019-01-10 東ソー株式会社 Method for exfoliating and recovering undifferentiated cells
WO2019045649A1 (en) * 2017-08-29 2019-03-07 Agency For Science, Technology And Research Methods for enriching mesenchymal stem cells
WO2020209316A1 (en) 2019-04-08 2020-10-15 有限会社ジーエヌコーポレーション Chondrocyte culture with high tissue regeneration ability
WO2020218427A1 (en) * 2019-04-25 2020-10-29 日産化学株式会社 Method for evaluating quality of somatic stem cells
JP2021501189A (en) * 2017-10-31 2021-01-14 ナショナル ユニバーシティー オブ アイルランド， ゴールウェイ How to treat cancer
JP2022035915A (en) * 2020-08-21 2022-03-04 遵義医科大学附属医院 Method for aging anti-human mesenchymal stem cells and enhancing their stem cellular characteristics
JP2022551702A (en) * 2019-10-08 2022-12-13 株式会社ジンケム COMPOSITION FOR INCREASING STEM CELL POTENTIAL AND USE THEREOF

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2009543089A (en) * 2006-07-11 2009-12-03 プロコグニア（イスラエル）エルティーディー Methods and tests for analysis of glycosylation patterns related to cellular status (analysis)
WO2010131641A1 (en) * 2009-05-11 2010-11-18 独立行政法人国立成育医療研究センター Method for determination of condition of cell
JP2011004607A (en) * 2009-06-23 2011-01-13 Nihon Univ New method for maintaining stem cell in undifferentiated state
WO2012039430A1 (en) * 2010-09-21 2012-03-29 株式会社Ｇｐバイオサイエンス Method for isolating cancer stem cells
JP2012526549A (en) * 2009-05-13 2012-11-01 メディポスト カンパニー リミテッド TSP-1, TSP-2, IL-17BR and HB-EGF associated with cellular activity of stem cells and uses thereof
WO2013164970A1 (en) * 2012-05-01 2013-11-07 富士フイルム株式会社 Method for incubating pluripotent stem cells and polypeptide to be used therefor
JP2013545439A (en) * 2010-09-17 2013-12-26 プレジデント・アンド・フェロウズ・オブ・ハーバード・カレッジ Functional genomics assay to characterize the usefulness and safety of pluripotent stem cells

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2009543089A (en) * 2006-07-11 2009-12-03 プロコグニア（イスラエル）エルティーディー Methods and tests for analysis of glycosylation patterns related to cellular status (analysis)
WO2010131641A1 (en) * 2009-05-11 2010-11-18 独立行政法人国立成育医療研究センター Method for determination of condition of cell
JP2012526549A (en) * 2009-05-13 2012-11-01 メディポスト カンパニー リミテッド TSP-1, TSP-2, IL-17BR and HB-EGF associated with cellular activity of stem cells and uses thereof
JP2011004607A (en) * 2009-06-23 2011-01-13 Nihon Univ New method for maintaining stem cell in undifferentiated state
JP2013545439A (en) * 2010-09-17 2013-12-26 プレジデント・アンド・フェロウズ・オブ・ハーバード・カレッジ Functional genomics assay to characterize the usefulness and safety of pluripotent stem cells
WO2012039430A1 (en) * 2010-09-21 2012-03-29 株式会社Ｇｐバイオサイエンス Method for isolating cancer stem cells
WO2013164970A1 (en) * 2012-05-01 2013-11-07 富士フイルム株式会社 Method for incubating pluripotent stem cells and polypeptide to be used therefor

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
ALISSON-SILVA FREDERICO ET AL.: "Evidences for the involvement of cell surface glycans in stem cell pluripotency and differentiation", GLYCOBIOLOGY, vol. 24, no. 5, February 2014 (2014-02-01), pages 458 - 468, XP055129372, DOI: doi:10.1093/glycob/cwu012 *
HASEHIRA KAYO ET AL.: "Structural and Quantitative Evidence for Dynamic Glycome Shift on Production of Induced Pluripotent Stem Cells", MOLECULAR & CELLULAR PROTEOMICS, vol. 11, 2012, pages 1913 - 1923 *
NASIRIKENARI MEHRAB ET AL.: "Remodeling of Marrow Hematopoietic Stem and Progenitor Cells by Non-self ST6Gal-1 Sialyltransferase", THE JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 289, no. 10, March 2014 (2014-03-01), pages 7178 - 7189 *
SWINDALL AMANDA F. ET AL.: "ST6Gal-I Protein Expression Is Upregulated in Human Epithelial Tumors and Correlates with Stem Cell Markers in Normal Tissues and Colon Cancer Cell Lines", CANCER RESEARCH, vol. 73, no. 7, 2013, pages 2368 - 2378 *

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2017184713A (en) * 2016-03-31 2017-10-12 国立研究開発法人産業技術総合研究所 Method for determining cell differentiation potential
JP2019000063A (en) * 2017-06-19 2019-01-10 東ソー株式会社 Method for exfoliating and recovering undifferentiated cells
WO2019045649A1 (en) * 2017-08-29 2019-03-07 Agency For Science, Technology And Research Methods for enriching mesenchymal stem cells
JP2021501189A (en) * 2017-10-31 2021-01-14 ナショナル ユニバーシティー オブ アイルランド， ゴールウェイ How to treat cancer
WO2020209316A1 (en) 2019-04-08 2020-10-15 有限会社ジーエヌコーポレーション Chondrocyte culture with high tissue regeneration ability
WO2020218427A1 (en) * 2019-04-25 2020-10-29 日産化学株式会社 Method for evaluating quality of somatic stem cells
JP2022551702A (en) * 2019-10-08 2022-12-13 株式会社ジンケム COMPOSITION FOR INCREASING STEM CELL POTENTIAL AND USE THEREOF
JP2022035915A (en) * 2020-08-21 2022-03-04 遵義医科大学附属医院 Method for aging anti-human mesenchymal stem cells and enhancing their stem cellular characteristics
JP7119044B2 (en) 2020-08-21 2022-08-16 遵義医科大学附属医院 Anti-human mesenchymal stem cell senescence and method for enhancing its stemness characteristics

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