WO2016006712A1 - Procédé pour déterminer un potentiel de différenciation cellulaire - Google Patents

Procédé pour déterminer un potentiel de différenciation cellulaire 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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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/ja
Publication of WO2016006712A1 publication Critical patent/WO2016006712A1/fr

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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.

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Abstract

La présente invention aborde le problème de l'identification d'un marqueur à chaîne de sucre de surface cellulaire qui présente une haute corrélation avec le potentiel de différenciation d'une cellule souche somatique, telle qu'une cellule souche mésenchymateuse, et décrit un procédé permettant de déterminer/évaluer le potentiel de différenciation d'une cellule souche somatique à l'aide du marqueur à chaîne de sucre de surface cellulaire et un procédé permettant d'isoler/de concentrer une cellule souche somatique ayant un potentiel élevé de différenciation à l'aide du marqueur à chaîne de sucre de surface cellulaire. La présente invention concerne : un procédé pour déterminer le degré de potentiel de différentiation d'une cellule souche somatique en mesurant l'acide α2-6 sialique exprimé à la surface de la cellule souche somatique en utilisant la lectine 1 de liaison à l'acide α2-6 sialique ou un anticorps de liaison à l'acide α2-6 sialique ; et un procédé pour isoler/concentrer une cellule souche somatique, selon lequel l'acide α2-6 sialique est exprimé sur la surface de celle-ci dans une grande quantité, à partir d'un échantillon contenant des cellules souches somatiques en utilisant la lectine 1 de liaison à l'acide α2-6 sialique ou un anticorps de liaison à l'acide α2-6 sialique.
PCT/JP2015/070053 2014-07-11 2015-07-13 Procédé pour déterminer un potentiel de différenciation cellulaire WO2016006712A1 (fr)

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WO2019045649A1 (fr) * 2017-08-29 2019-03-07 Agency For Science, Technology And Research Méthodes pour enrichir des cellules souches mésenchymateuses
WO2020209316A1 (fr) 2019-04-08 2020-10-15 有限会社ジーエヌコーポレーション Culture de chondrocytes avec capacité de régénération tissulaire élevée
WO2020218427A1 (fr) * 2019-04-25 2020-10-29 日産化学株式会社 Procédé d'évaluation de la qualité de cellules souches somatiques
JP2021501189A (ja) * 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 癌の治療方法
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JP2017184713A (ja) * 2016-03-31 2017-10-12 国立研究開発法人産業技術総合研究所 細胞分化ポテンシャル判定法
JP2019000063A (ja) * 2017-06-19 2019-01-10 東ソー株式会社 未分化細胞の剥離回収方法
WO2019045649A1 (fr) * 2017-08-29 2019-03-07 Agency For Science, Technology And Research Méthodes pour enrichir des cellules souches mésenchymateuses
JP2021501189A (ja) * 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 癌の治療方法
WO2020209316A1 (fr) 2019-04-08 2020-10-15 有限会社ジーエヌコーポレーション Culture de chondrocytes avec capacité de régénération tissulaire élevée
WO2020218427A1 (fr) * 2019-04-25 2020-10-29 日産化学株式会社 Procédé d'évaluation de la qualité de cellules souches somatiques
JP2022551702A (ja) * 2019-10-08 2022-12-13 株式会社ジンケム 幹細胞能を増大させるための組成物及びその用途
JP2022035915A (ja) * 2020-08-21 2022-03-04 遵義医科大学附属医院 抗ヒト間葉系幹細胞老化およびその幹細胞性特徴増強方法
JP7119044B2 (ja) 2020-08-21 2022-08-16 遵義医科大学附属医院 抗ヒト間葉系幹細胞老化およびその幹細胞性特徴増強方法

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