WO2020218427A1 - Method for evaluating quality of somatic stem cells - Google Patents

Method for evaluating quality of somatic stem cells Download PDF

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WO2020218427A1
WO2020218427A1 PCT/JP2020/017514 JP2020017514W WO2020218427A1 WO 2020218427 A1 WO2020218427 A1 WO 2020218427A1 JP 2020017514 W JP2020017514 W JP 2020017514W WO 2020218427 A1 WO2020218427 A1 WO 2020218427A1
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stem cells
somatic stem
lectin
sialic acid
cell
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Japanese (ja)
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一隆 的場
泰斗 西野
浩章 舘野
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日産化学株式会社
国立研究開発法人産業技術総合研究所
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • 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
    • 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
    • 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
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals

Definitions

  • the present invention relates to a method for evaluating the quality of somatic stem cells and a method for controlling the quality, including a step of measuring the surface marker of extracellular vesicles in the culture supernatant of somatic stem cells.
  • mesenchymal stem cells MSC: Mesenchymal Stem Cell
  • surface markers such as CD73, CD90, and CD105 are analyzed for quality evaluation.
  • extracellular vesicles secreted extracellularly from cells play an important role in cell-cell interactions, and are rapidly attracting attention.
  • Patent Document 1 identifies cell surface sugar chain markers that are highly correlated with the differentiation potential of somatic stem cells such as mesenchymal stem cells, and differentiates somatic stem cells using the cell surface sugar chain markers. It describes the method for determining and evaluating the potential, and the method for isolating and enriching somatic stem cells with high differentiation potential.
  • This document describes a method for determining the high differentiation potential of somatic stem cells by measuring ⁇ 2-6 sialic acid expressed on the surface of somatic stem cells by using ⁇ 2-6 sialic acid-binding lectin or antibody. Somatic stem cells having a high expression level of ⁇ 2-6 sialic acid are isolated and concentrated on the cell surface from the somatic stem cell-containing sample.
  • Patent Document 2 describes that the cell differentiation potential of somatic stem cells is determined and evaluated by an assay system using a cell culture supernatant of somatic stem cells, and that of somatic stem cells. The quality control method using the culture supernatant is described. In the method described in this document, the expression level of ⁇ 2-6 sialic acid in a specific secretory protein-containing sugar chain such as fibronectin in a culture supernatant sample of a subject stem cell is measured.
  • Non-Patent Document 1 describes Glycome analysis of extracellular vesicles (EV: extracellular vesicles) derived from human iPS cells using a lectin microarray.
  • Patent Document 3 is a type of extracellular vesicle secreted from the mesenchymal stem cell as an index for monitoring the state of the mesenchymal stem cell transformed by introducing an oncogene. Techniques using miRNA in exosomes are disclosed.
  • Patent Document 4 discloses a method for evaluating the aging of the cells using miRNA contained in exosomes in the culture supernatant of cultured cells used for transplantation or the like as an index.
  • An object of the present invention is to find a new method for evaluating the quality of somatic stem cells, which can reduce the number of steps and is easy to pretreat.
  • the gist of the present invention is as follows.
  • a method for evaluating the quality of somatic stem cells which comprises a step of measuring surface markers of extracellular vesicles in a culture supernatant of somatic stem cells.
  • the somatic stem cell is a mesenchymal stem cell.
  • the surface marker is ⁇ 2-6 sialic acid.
  • the surface marker is an MSC marker.
  • the step of measuring ⁇ 2-6 sialic acid is (1) A step of overlaying the culture supernatant of the target somatic stem cell on a substrate on which one of the following probes (a) or (b) is immobilized, and then allowing the labeled other probe to act.
  • A At least one lectin selected from SSA lectins, SNA lectins, and TJAI lectins
  • B At least one protein selected from anti-CD9 antibody, anti-CD63 antibody, anti-CD81 antibody, and TIM4, and
  • the step of measuring ⁇ 2-6 sialic acid is (1) Step of reacting the culture supernatant of the target somatic stem cell with the labeled lectin of (a) below (a) At least one lectin selected from SSA lectin, SNA lectin, and TJAI lectin. And (2) the method according to [3], which comprises a step of measuring the labeled amount. [7] The method according to any one of [1] to [6], which comprises or does not include a step of purifying extracellular vesicles in advance. [8] A method for quality control of somatic stem cells, which comprises a step of measuring a surface marker of extracellular vesicles in a culture supernatant of somatic stem cells. [9] The method according to [8], wherein the surface marker is ⁇ 2-6 sialic acid. [10] The method according to [8], wherein the surface marker is an MSC marker.
  • the present invention provides a method for evaluating the quality of somatic stem cells, which comprises a step of measuring surface markers of extracellular vesicles in a culture supernatant of somatic stem cells, which has not been known conventionally. ..
  • the method according to the present invention is a simpler method than the conventional method. It is also possible to evaluate quality without consuming or stimulating valuable cells.
  • FIG. 1 shows the results of examining the ability of adipose-derived mesenchymal stem cells to differentiate into osteoblasts and adipocytes in Example 1.
  • FIG. 2 shows the results of examining the ability of excess finger-derived cartilage stem cells to differentiate into cartilage in Example 2.
  • FIG. 3 shows the results of examining the ability of fibroblasts to differentiate into osteoblasts and adipocytes in Example 3.
  • FIG. 4 shows human skin fibroblasts (hFibs), human iPS cells (201B7), adipose-derived mesenchymal stem cells (ADSC, Lot # 2117), and surplus finger bone marrow-derived mesenchymal stem cells (Yub621c) in Example 4.
  • FIG. 5 shows human skin fibroblasts (hFibs), human iPS cells (201B7), adipose-derived mesenchymal stem cells (ADSC, Lot # 2117), and surplus finger bone marrow-derived mesenchymal stem cells (Yub621c) in Example 5.
  • hFibs human skin fibroblasts
  • ADSC adipose-derived mesenchymal stem cells
  • Yub621c surplus finger bone marrow-derived mesenchymal stem cells
  • FIG. 6 shows the results of examining the MSC markers of extracellular vesicles and MSC markers on the cell surface of the cell culture supernatant of surplus finger-derived cartilage stem cells (Yub625) in Example 6.
  • FIG. 7 shows the results of examining the reactivity of ⁇ 2-6 sialic acid-binding lectin in the exosomes of the cell culture supernatant of bone marrow-derived mesenchymal stem cells (Yub622) in Example 7.
  • the present invention provides a method for evaluating or controlling the quality of somatic stem cells, which comprises the step of measuring the surface markers of extracellular vesicles in the culture supernatant of somatic stem cells.
  • the term "somatic stem cell” may be mainly a somatic stem cell or a cell in which the somatic stem cell is subcultured.
  • the somatic stem cells include various somatic stem cells such as nerve stem cells, epithelial stem cells, hepatic stem cells, reproductive stem cells, hematopoietic stem cells, mesenchymal stem cells, cartilage stem cells, and skeletal muscle stem cells. Stem cells are preferred.
  • the method of the present invention can be carried out not only in the state of cloned somatic stem cells but also in the state of a culture of living tissue consisting of a heterogeneous cell population from which somatic stem cells have been collected.
  • the term stem cells also includes cultures containing somatic stem cells.
  • the somatic stem cell contained in the sample is a cell isolated from a living body, a primary culture thereof, a subculture, or a cultured cell line that has been established. You may.
  • the culture method may be not only normal plane culture (two-dimensional) but also three-dimensional culture. Normally, when analyzing a spheroid (cell mass) produced by three-dimensional culture, it is necessary to perform flow cytometry on each cell separately, but in the present invention, since the culture supernatant is used, the spheroid is used. It has the advantage of being able to analyze without destroying it.
  • Mesenchymal stem cells can be collected from adipose tissue, umbilical cord blood, umbilical cord, amniotic membrane, placenta, or from bone marrow puncture from jawbone or femoral 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), which is 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 finger excised by surgery in a patient with polyphinopathy
  • cartilage tissue-derived cartilage stem cells (cartilage stem cells derived from polyphinopathy) can be obtained from the cartilage tissue. it can. It can also be obtained from RIKEN BioResource Center, JSRB Cell Bank, etc.
  • chondrocyte maintenance medium MesenPRO RS ⁇ TM> medium (Life Technologies), which is generally used as a mesenchymal stem cell maintenance medium, may be used, but chondrocyte basal medium and chondrocyte proliferation medium (Takara Bio) ) And other maintenance media for chondrocytes can also be used.
  • the present invention not only can the quality of stem cells collected from a tissue having a body be evaluated or controlled, but also the quality of somatic stem cells after expanded culture can be evaluated or controlled.
  • the present invention can also be used for isolating and concentrating cells having excellent quality and high differentiation potential.
  • stem cells of the present invention include non-human mammals such as monkeys, pigs, cows, goats, and sheep. , Mouse, rat-derived stem cells.
  • a culture supernatant of a somatic stem cell culture solution collected from a living body or commercially available, or a culture solution subcultured in an undifferentiated state is used as a test sample.
  • the culture medium is generally replaced with fresh culture medium at regular intervals.
  • the surface markers of extracellular vesicles in the culture supernatant are measured.
  • the measurement target may be any marker as long as it is a surface marker of extracellular vesicles of somatic stem cells, and examples thereof include ⁇ 2-6 sialic acid or MSC markers.
  • ⁇ 2-6 sialic acid generally refers to "Neu5Ac ⁇ 2-6Gal” and N, which are sugar chains in which N-acetylneuraminic acid is ⁇ 2-6 bonded to the hydroxyl group at the 6-position of galactose.
  • -Glycolyl-type neuramate refers to both sugar chains “Neu5Gc ⁇ 2-6Gal” that are also ⁇ 2-6 bound.
  • the term "MSC marker” may be any cell surface of mesenchymal stem cells or any marker molecule related to MSC expressed on the surface of extracellular vesicles.
  • adhesion molecules CD106, CD166 and CD29 examples thereof include CD105 (SH2), CD73 (SH3 / 4), CD44, CD90 (Thy-1), CD7, CD13 and Stro-1.
  • the negative marker examples include adhesion molecules CD31, CD18, CD56, hematopoietic markers CD45, CD34, CD14, CD11 and co-stimulatory molecules CD80, CD86, and CD40.
  • a surface marker of extracellular vesicles released in a culture medium of somatic stem cells for example, " ⁇ 2-6" added to the non-reducing end of a sugar chain
  • the abundance of "sialic acid” is detected or measured to determine the degree of cell differentiation potential of somatic stem cells into, for example, osteoblasts or chondrocytes.
  • the amount of ⁇ 2-6 sialic acid or MSC marker on the surface of extracellular vesicles in the culture supernatant of somatic stem cells such as mesenchymal stem cells, ⁇ 2-6 sialic acid-specific binding lectin or It is convenient and preferable to evaluate or control the quality of somatic stem cells by measuring using an antibody against the MSC marker and a sandwich assay method using a protein or antibody that can be used for isolation of extracellular vesicles.
  • all extracellular vesicles in the culture supernatant are previously purified using beads, plates, etc. on which proteins or antibodies that can be used for isolation of extracellular vesicles are immobilized, and ⁇ 2-6 sialic acid is used.
  • the amount of MSC marker is detected using an ⁇ 2-6 sialic acid-specific lectin or an antibody against the MSC marker.
  • a probe that specifically recognizes the ⁇ 2-6 sialic acid or MSC marker as an epitope in order to detect the ⁇ 2-6 sialic acid or MSC marker on the surface of extracellular vesicles in the culture supernatant, a probe that specifically recognizes the ⁇ 2-6 sialic acid or MSC marker as an epitope.
  • proteins that exhibit binding activity to sugar chains are collectively called "lectins", and typical ⁇ 2-6 sialic acid-binding probes include, but are limited to, various ⁇ 2-6 sialic acid-binding lectins.
  • ⁇ 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 ⁇ 2-6 sialic acid-binding lectins, or an anti- ⁇ 2-6 sialic acid antibody can also be used in combination.
  • Examples of the ⁇ 2-6 sialic acid-binding lectin used as the ⁇ 2-6 sialic acid-binding probe of the present invention include lectins and antibodies capable of recognizing ⁇ 2-6 sialic acid on the surface of extracellular vesicles in the culture supernatant. Either may be used.
  • SNA lectin Sudbucus nigra lectin
  • SSA lectin Sudbucus sieboldiana lectin
  • PSL1a lectin Polyporus squamosus lectin
  • TJAI lectin Trichosans
  • SNA and SSA lectins are preferred.
  • SNA lectins can be extracted from elder, SSA lectins from Japanese elders, PSL1a lectins from dryad's saddle, and TJAI lectins from Trichosanthes chinensis. It is commercially available.
  • PSL1a lectin a recombinant rPSL1a lectin having ⁇ 2-6 sialic acid specificity is commercially available from Wako Pure Chemical Industries.
  • Examples of the antibody for detecting the MSC marker of the present invention include anti-CD44 antibody, anti-CD73 antibody, anti-CD90 antibody, anti-CD105 antibody, anti-CD146 antibody, anti-CD271 antibody, anti-CD11b antibody, anti-CD19 antibody, anti-CD31 antibody.
  • Examples thereof include anti-CD34 antibody, anti-CD45 antibody, anti-CD144 antibody, and anti-HLA-DR antibody, but various commercially available antibodies may be used.
  • Proteins or antibodies that can be used to isolate extracellular vesicles include, but are not limited to, anti-CD9 antibody, anti-CD63 antibody, anti-CD81 antibody, and TIM4, which may be used alone or in combination. can do.
  • a plate eg, microwell plate
  • a microarray substrate eg, slide glass for microarray
  • a tube e.g., Beads (eg, plastic beads, magnetic beads), chromatographic carriers (eg, Sepharose TM), membranes (eg, nitrocellulose membranes, PVDF membranes), gels (eg, polyacrylamide gels) and the like. .. Among them, plates, beads and membranes are preferably used.
  • a commercially available lectin array may be used.
  • a high-density lectin microarray in which 96 types of lectins having different specificities are immobilized and a lectin array in which 45 types of plant lectins are immobilized on the same substrate (Kuno et al., Nature Methods 2, 851-856). , 2005) and LecChipTM Ver.1.0 (manufactured by Glyco Technica) can be used.
  • test culture supernatant sample is added to the immobilized lectin well without dilution or dilution with a buffer solution and allowed to interact with each other, and then the non-specifically bound impurities are added to the lectin array buffer solution. Clean with (commercially available). Then, it may be measured by flow cytometry.
  • an ⁇ 2-6 sialic acid-binding probe or MSC marker It may be measured by sandwiching with a binding probe. Elisa may be used for the measurement. Further, in addition to the usual ELISA, it is possible to measure with higher sensitivity by using a digital ELISA such as SIMOA (Quantix). When magnetic beads are used, the measurement may be performed by flow cytometry.
  • ⁇ 2-6 sialic acid-binding probe or MSC marker-binding property using a nanopore device such as qNano (IzonScience) or NanoSight (MalvernPanalytical) without isolating or isolating extracellular vesicles from cell culture supernatant.
  • the quality of the original cell or cell mass may be assessed by measuring with or without a probe and classifying and assessing extracellular vesicles with surface markers.
  • iPS cell Human iPS cell (201B7 strain) Human adipose-derived mesenchymal stem cells (ADSC) Human skin fibroblasts (hFibs) Excess finger-derived chondrocytes (Yub625 strain) Bone marrow-derived mesenchymal stem cells (Yub621c strain, Yub622 strain)
  • Example 1 Adipose-derived mesenchymal stem cells (ADSC, Life Technologies, Lot #: 2118) were cultured in MesenPRO RS TM Medium (Life Technologies), which is a common mesenchymal stem cell medium. The differentiation potential of cells in early passage (P5) and late passage (P28) into osteoblasts and adipocytes was investigated.
  • early passage early passage
  • late passage late passage
  • the number of passages differs depending on the cell type and culture conditions.
  • the time when the cell growth curve rises linearly is called “early passage”
  • late passage the time when the cell growth curve becomes loose or flat after continuing passage
  • Example 2 Excess finger-derived chondrocyte stem cells (Yub625 strain, RIKEN BioResource Center), which is a type of chondrocyte, are subcultured in the same manner as in Example 1, and cartilage is subjected to cartilage for early passage (P5) and late passage (P22). Differentiation into cells was induced. Chondrogenic differentiation was performed using the hMSC-Human Mesenchymal Stem Cell Chondrogenic Differentiation Medium BulletKit (Cat #: PT3003, Lonza). Cartilage formation was confirmed by staining with Alcian Blue (Muto Pure Chemicals Co., LTD., Cat #: 20121). The results are shown in FIG. Chondrocytes are stained light blue. Differentiation into chondrocytes was observed in the cells in the early passage (P5), but almost no cartilage differentiation was observed in the cells in the late passage (P22).
  • Example 3 Human skin fibroblasts (ATCC, #: PCS-201-012, Lot: 58605481) were subcultured in MesenPRO RS TM Medium (Life Technologies) in the same manner as in Example 1 and were subcultured in the early stage of passage (P5). Differentiation into osteoblasts and adipocytes was induced and confirmed in the late passage (P20) by the same method as in Example 1. The results are shown in FIG. Since no red-stained cells were confirmed, it was confirmed that there was no potential for differentiation into osteoblasts and adipocytes.
  • Example 4 Human skin fibroblasts (hFibs), human iPS cells (201B7), adipose-derived mesenchymal stem cells (ADSC, Lot # 2117), surplus finger bone marrow-derived mesenchymal stem cells (Yub621c), surplus finger-derived cartilage stem cells (Yub625) Extracellular vesicles (EV) were purified from the cell culture supernatant of MagCapture TM Exosome Isolation Kit PS (Fuji Film Wako Pure Drug, 293-77601). An adsorption inhibitor (Exocap Ultracentrifugation Storage Booster, MBL Science, MEK-USB) was added to the obtained EV and stored at -80 ° C.
  • Example 5 Human skin fibroblasts (hFibs), human iPS cells (201B7), adipose-derived mesenchymal stem cells (ADSC, Lot # 2117), surplus finger bone marrow-derived mesenchymal stem cells (Yub621c), surplus finger-derived cartilage stem cells (Yub625) From 40 mL of cell culture supernatant of SNA, SSA, TJAI lectin (final concentration: 10 ⁇ g), EV was captured using PS Capture TM Exosome Flow Cytometry Kit (Wako, # 297-79701) containing Tim4 binding beads and PE-labeled.
  • PS Capture TM Exosome Flow Cytometry Kit (Wako, # 297-79701) containing Tim4 binding beads and PE-labeled.
  • Example 6 EV from 40 mL of cell culture supernatant of surplus finger-derived cartilage stem cells (Yub625) using PS Capture TM Exosome Flow Cytometry Kit (Wako, # 297-79701) containing Tim4 binding beads in the same manner as in Example 5.
  • Various antibodies (all manufactured by BD (Becton, Dickinson and Company), 10 ⁇ g / mL) of MSC markers (CD13, CD29, CD73, CD90, CD105) captured and PE-labeled on 10 ⁇ L of beads were reacted with flow cytos. Analysis was performed with a meter (CytoFLEX, Beckman).
  • the MSC markers on the cell surface were similarly reacted with various antibodies of the PE-labeled MSC markers (all made by BD, 10 ⁇ g / mL), and flowed. It was analyzed with a cytometer (CytoFLEX, Beckman). The results based on the average fluorescence intensity are shown in FIG. Each antibody showed a similar pattern of reactivity in both extracellular vesicles and cells. From this result, it can be seen that the quality of somatic stem cells could be easily evaluated without consuming the cells themselves by measuring the MSC marker using EV.
  • Example 7 Exosomes were purified from the culture supernatant of bone marrow-derived mesenchymal stem cells (Yub622) using MagCapture TM Exosome Isolation Kit PS MagCapture TM Exosome Isolation Kit PS (manufactured by Fuji Film Wako Pure Chemical Industries, Ltd.). The purified exosome was reacted with PE-labeled TJA1 ( ⁇ 2-6 sialic acid-binding lectin) and analyzed by a flow cytometer (CytoFLEX, equipped with a violet laser). As a result, it was found that the amount of exosomes showing strong reactivity with PE-TJA1 was 2.3% in the cells in the early stage of passage, whereas it was 0.99% in the late passage.
  • PE-labeled TJA1 ⁇ 2-6 sialic acid-binding lectin

Abstract

The present invention provides a method for evaluating the quality of somatic stem cells and a method for controlling the quality of the same, comprising a step of measuring a surface marker for extracellular vesicles in the culture supernatant of somatic stem cells.

Description

体性幹細胞の品質を評価する方法How to evaluate the quality of somatic stem cells
　本発明は、体性幹細胞の培養上清中の、細胞外小胞の表面マーカーを測定する工程を含む、体性幹細胞の品質を評価する方法、および品質を管理する方法に関する。 The present invention relates to a method for evaluating the quality of somatic stem cells and a method for controlling the quality, including a step of measuring the surface marker of extracellular vesicles in the culture supernatant of somatic stem cells.
　細胞の品質を評価する方法としては様々な方法がある。顕微鏡を用いた画像解析、フローサイトメーターを用いた免疫表現型解析、細胞内サイトカイン解析、あるいは次世代シーケンサーを用いたゲノム変異解析などである。これらの中でもフローサイトメーターを用いた免疫表現型解析は広く普及した定量的な品質評価法である。
　フローサイトメトリーの際に染色する表面マーカーはタンパク質抗原、糖鎖抗原、脂質分子、接着分子、サイトカインレセプターなど多くの種類があり、それらのうちの複数の表面マーカーによって、細胞を分類解析でき、品質評価、品質管理に利用されている。例えば間葉系幹細胞（ＭＳＣ：Ｍｅｓｅｎｃｈｙｍａｌ　Ｓｔｅｍ　Ｃｅｌｌ）を評価する際には、ＣＤ７３、ＣＤ９０、ＣＤ１０５などの表面マーカーを解析し、品質評価を行っている。
　一方で、近年、細胞から細胞外に分泌される細胞外小胞が、細胞間相互作用において重要な役割を持つことが明らかとなり急速に注目を集めている。 There are various methods for evaluating the quality of cells. Image analysis using a microscope, immunophenotypic analysis using a flow cytometer, intracellular cytokine analysis, or genome mutation analysis using a next-generation sequencer. Among these, immunophenotypic analysis using a flow cytometer is a widely used quantitative quality evaluation method.
There are many types of surface markers to be stained during flow cytometry, such as protein antigens, sugar chain antigens, lipid molecules, adhesion molecules, and cytokine receptors, and multiple surface markers among them can be used to classify and analyze cells for quality. It is used for evaluation and quality control. For example, when evaluating mesenchymal stem cells (MSC: Mesenchymal Stem Cell), surface markers such as CD73, CD90, and CD105 are analyzed for quality evaluation.
On the other hand, in recent years, it has become clear that extracellular vesicles secreted extracellularly from cells play an important role in cell-cell interactions, and are rapidly attracting attention.
　ＷＯ２０１６／００６７１２（特許文献１）は、間葉系幹細胞など体性幹細胞の分化ポテンシャルと相関性の高い細胞表面の糖鎖マーカーを同定し、当該細胞表面糖鎖マーカーを利用した体性幹細胞の分化ポテンシャルの判定、評価方法、分化ポテンシャルの高い体性幹細胞の単離、濃縮方法について記載している。この文献は、α２－６シアル酸結合性レクチン又は抗体を用いることにより、体性幹細胞表面で発現するα２－６シアル酸を測定して体性幹細胞の分化ポテンシャルの高さを判定する方法を記載しており、体性幹細胞含有試料から、細胞表面でα２－６シアル酸の発現量の高い体性幹細胞を単離、濃縮している。 WO2016 / 006712 (Patent Document 1) identifies cell surface sugar chain markers that are highly correlated with the differentiation potential of somatic stem cells such as mesenchymal stem cells, and differentiates somatic stem cells using the cell surface sugar chain markers. It describes the method for determining and evaluating the potential, and the method for isolating and enriching somatic stem cells with high differentiation potential. This document describes a method for determining the high differentiation potential of somatic stem cells by measuring α2-6 sialic acid expressed on the surface of somatic stem cells by using α2-6 sialic acid-binding lectin or antibody. Somatic stem cells having a high expression level of α2-6 sialic acid are isolated and concentrated on the cell surface from the somatic stem cell-containing sample.
　特開２０１７－１８４７１３（特許文献２）は、体性幹細胞の細胞培養上清を用いたアッセイ系によって体性幹細胞の細胞分化ポテンシャルを判定、評価することを記載しており、また体性幹細胞の培養上清を用いた品質管理方法を記載している。この文献に記載される方法では、被検体性幹細胞の培養上清試料中のフィブロネクチンなどの特定の分泌性タンパク質含有糖鎖中のα２－６シアル酸発現量を測定している。 Japanese Patent Application Laid-Open No. 2017-184713 (Patent Document 2) describes that the cell differentiation potential of somatic stem cells is determined and evaluated by an assay system using a cell culture supernatant of somatic stem cells, and that of somatic stem cells. The quality control method using the culture supernatant is described. In the method described in this document, the expression level of α2-6 sialic acid in a specific secretory protein-containing sugar chain such as fibronectin in a culture supernatant sample of a subject stem cell is measured.
　Ｓａｉｔｏ，Ｓ．ｅｔ　ａｌ．（非特許文献１）は、レクチンマイクロアレイを用いて、ヒトｉＰＳ細胞に由来する細胞外小胞（ＥＶ：ｅｘｔｒａｃｅｌｌｕｌａｒ　ｖｅｓｉｃｌｅｓ）のグライコーム分析を記載している。 Saito, S.A. et al. (Non-Patent Document 1) describes Glycome analysis of extracellular vesicles (EV: extracellular vesicles) derived from human iPS cells using a lectin microarray.
　ＷＯ２０１１／０５３２５７（特許文献３）には、癌遺伝子を導入して形質転換させた間葉系幹細胞の状態をモニタリングする指標として、前記間葉系幹細胞から分泌される細胞外小胞の一種であるエクソソーム中のｍｉＲＮＡを用いる技術が開示されている。 WO2011 / 053257 (Patent Document 3) is a type of extracellular vesicle secreted from the mesenchymal stem cell as an index for monitoring the state of the mesenchymal stem cell transformed by introducing an oncogene. Techniques using miRNA in exosomes are disclosed.
　特開２０１６－１４４４３９（特許文献４）は、移植等に用いる培養細胞の培養上清中のエクソソーム中に含まれるｍｉＲＮＡを指標として、前記細胞の老化を評価する方法を開示している。 Japanese Patent Application Laid-Open No. 2016-144439 (Patent Document 4) discloses a method for evaluating the aging of the cells using miRNA contained in exosomes in the culture supernatant of cultured cells used for transplantation or the like as an index.
ＷＯ２０１６／００６７１２WO2016 / 006712 特開２０１７－１８４７１３JP 2017-184713 ＷＯ２０１１／０５３２５７WO2011 / 053257 特開２０１６－１４４４３９JP 2016-144439
　本発明は、工程数削減が可能で、前処理が簡便な体性幹細胞の品質を評価するための新たな方法を見出すことを課題とする。 An object of the present invention is to find a new method for evaluating the quality of somatic stem cells, which can reduce the number of steps and is easy to pretreat.
　本発明者らは鋭意研究の末、体性幹細胞の培養上清中の、細胞外小胞の表面マーカーを測定することにより、上記課題を解決できることを見出し、本発明を完成させるに至った。 After diligent research, the present inventors have found that the above-mentioned problems can be solved by measuring the surface markers of extracellular vesicles in the culture supernatant of somatic stem cells, and have completed the present invention.
　すなわち、本発明の要旨は、以下である。
［１］　体性幹細胞の培養上清中の、細胞外小胞の表面マーカーを測定する工程を含む、体性幹細胞の品質を評価する方法。
［２］　上記体性幹細胞が、間葉系幹細胞である、［１］に記載の方法。
［３］　表面マーカーが、α２－６シアル酸である、［１］又は［２］に記載の方法。
［４］　表面マーカーが、ＭＳＣマーカーである、［１］又は［２］に記載の方法。
［５］　α２－６シアル酸を測定する工程が、
（１）対象の体性幹細胞の培養上清を、下記（ａ）又は（ｂ）のいずれか一方のプローブが固定された基板上にオーバーレイし、つ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ＴＪＡＩレクチンから選択される少なくとも１種のレクチン
　（ｂ）抗ＣＤ９抗体、抗ＣＤ６３抗体、抗ＣＤ８１抗体、及びＴＩＭ４から選択される少なくとも１種のタンパク質、及び
（２）標識量を測定する工程
を含む、［３］に記載の方法。
［６］　α２－６シアル酸を測定する工程が、
（１）対象の体性幹細胞の培養上清と、標識化された下記（ａ）のレクチンとを作用させる工程
　（ａ）ＳＳＡレクチン、ＳＮＡレクチン、及びＴＪＡＩレクチンから選択される少なくとも１種のレクチン
及び
（２）標識量を測定する工程
を含む、［３］に記載の方法。
［７］　あらかじめ細胞外小胞を精製する工程を含む又は含まない、［１］～［６］のいずれか一項に記載の方法。
［８］　体性幹細胞の培養上清中の、細胞外小胞の表面マーカーを測定する工程を含む、体性幹細胞の品質管理方法。
［９］　表面マーカーが、α２－６シアル酸である、［８］に記載の方法。
［１０］表面マーカーが、ＭＳＣマーカーである、［８］に記載の方法。 That is, the gist of the present invention is as follows.
[1] A method for evaluating the quality of somatic stem cells, which comprises a step of measuring surface markers of extracellular vesicles in a culture supernatant of somatic stem cells.
[2] The method according to [1], wherein the somatic stem cell is a mesenchymal stem cell.
[3] The method according to [1] or [2], wherein the surface marker is α2-6 sialic acid.
[4] The method according to [1] or [2], wherein the surface marker is an MSC marker.
[5] The step of measuring α2-6 sialic acid is
(1) A step of overlaying the culture supernatant of the target somatic stem cell on a substrate on which one of the following probes (a) or (b) is immobilized, and then allowing the labeled other probe to act. (A) At least one lectin selected from SSA lectins, SNA lectins, and TJAI lectins (b) At least one protein selected from anti-CD9 antibody, anti-CD63 antibody, anti-CD81 antibody, and TIM4, and ( 2) The method according to [3], which comprises a step of measuring the labeled amount.
[6] The step of measuring α2-6 sialic acid is
(1) Step of reacting the culture supernatant of the target somatic stem cell with the labeled lectin of (a) below (a) At least one lectin selected from SSA lectin, SNA lectin, and TJAI lectin. And (2) the method according to [3], which comprises a step of measuring the labeled amount.
[7] The method according to any one of [1] to [6], which comprises or does not include a step of purifying extracellular vesicles in advance.
[8] A method for quality control of somatic stem cells, which comprises a step of measuring a surface marker of extracellular vesicles in a culture supernatant of somatic stem cells.
[9] The method according to [8], wherein the surface marker is α2-6 sialic acid.
[10] The method according to [8], wherein the surface marker is an MSC marker.
　本発明により、従来には知られていなかった、体性幹細胞の培養上清中の、細胞外小胞の表面マーカーを測定する工程を含む、体性幹細胞の品質を評価する方法が提供される。本発明による方法は、従来の方法に比べて、より簡便な方法である。また、貴重な細胞を消費または刺激することなく、品質を評価することが可能である。 INDUSTRIAL APPLICABILITY The present invention provides a method for evaluating the quality of somatic stem cells, which comprises a step of measuring surface markers of extracellular vesicles in a culture supernatant of somatic stem cells, which has not been known conventionally. .. The method according to the present invention is a simpler method than the conventional method. It is also possible to evaluate quality without consuming or stimulating valuable cells.
図１は、実施例１において、脂肪由来間葉系幹細胞の骨芽細胞及び脂肪細胞への分化能を調べた結果である。FIG. 1 shows the results of examining the ability of adipose-derived mesenchymal stem cells to differentiate into osteoblasts and adipocytes in Example 1. 図２は、実施例２において、余剰指由来軟骨幹細胞の軟骨への分化能を調べた結果である。FIG. 2 shows the results of examining the ability of excess finger-derived cartilage stem cells to differentiate into cartilage in Example 2. 図３は、実施例３において、線維芽細胞の骨芽細胞及び脂肪細胞への分化能を調べた結果である。FIG. 3 shows the results of examining the ability of fibroblasts to differentiate into osteoblasts and adipocytes in Example 3. 図４は、実施例４において、ヒト皮膚線維芽細胞（hFibs）、ヒトiPS細胞（201B7）、脂肪由来間葉系幹細胞（ADSC、Lot#2117）、余剰指骨髄由来間葉系幹細胞（Yub621c）、余剰指由来軟骨幹細胞（Yub625）の細胞培養上清の細胞外小胞のα２－６シアル酸量を、レクチンＳＮＡにより調べた結果である。FIG. 4 shows human skin fibroblasts (hFibs), human iPS cells (201B7), adipose-derived mesenchymal stem cells (ADSC, Lot # 2117), and surplus finger bone marrow-derived mesenchymal stem cells (Yub621c) in Example 4. , The result of examining the amount of α2-6 sialic acid in the extracellular vesicles of the cell culture supernatant of surplus finger-derived cartilage stem cells (Yub625) by Lectin SNA. 図５は、実施例５において、ヒト皮膚線維芽細胞（hFibs）、ヒトiPS細胞（201B7）、脂肪由来間葉系幹細胞（ADSC、Lot#2117）、余剰指骨髄由来間葉系幹細胞（Yub621c）、余剰指由来軟骨幹細胞（Yub625）の細胞培養上清の細胞外小胞のα２－６シアル酸量を各種レクチンにより調べた結果である。FIG. 5 shows human skin fibroblasts (hFibs), human iPS cells (201B7), adipose-derived mesenchymal stem cells (ADSC, Lot # 2117), and surplus finger bone marrow-derived mesenchymal stem cells (Yub621c) in Example 5. This is the result of examining the amount of α2-6 sialic acid in extracellular vesicles in the cell culture supernatant of surplus finger-derived cartilage stem cells (Yub625) with various lectins. 図６は、実施例６において、余剰指由来軟骨幹細胞（Yub625）の細胞培養上清の細胞外小胞のＭＳＣマーカーと細胞表面のＭＳＣマーカーを調べた結果である。FIG. 6 shows the results of examining the MSC markers of extracellular vesicles and MSC markers on the cell surface of the cell culture supernatant of surplus finger-derived cartilage stem cells (Yub625) in Example 6. 図７は、実施例７において、骨髄由来間葉系幹細胞（Yub622）の細胞培養上清のエクソソームの、α２－６シアル酸結合性レクチンの反応性を調べた結果である。FIG. 7 shows the results of examining the reactivity of α2-6 sialic acid-binding lectin in the exosomes of the cell culture supernatant of bone marrow-derived mesenchymal stem cells (Yub622) in Example 7.
　本発明は、体性幹細胞の培養上清中の、細胞外小胞の表面マーカーを測定する工程を含む、体性幹細胞の品質を評価又は管理する方法を提供する。 The present invention provides a method for evaluating or controlling the quality of somatic stem cells, which comprises the step of measuring the surface markers of extracellular vesicles in the culture supernatant of somatic stem cells.
　本発明において、「体性幹細胞」というときは、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ればよい。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、間葉系幹細胞が好ましい。また、本発明の方法は、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。 In the present invention, the term "somatic stem cell" may be mainly a somatic stem cell or a cell in which the somatic stem cell is subcultured. Here, the somatic stem cells include various somatic stem cells such as nerve stem cells, epithelial stem cells, hepatic stem cells, reproductive stem cells, hematopoietic stem cells, mesenchymal stem cells, cartilage stem cells, and skeletal muscle stem cells. Stem cells are preferred. In addition, the method of the present invention can be carried out not only in the state of cloned somatic stem cells but also in the state of a culture of living tissue consisting of a heterogeneous cell population from which somatic stem cells have been collected. The term stem cells also includes cultures containing somatic stem cells.
　すなわち、本発明において被検体性幹細胞というとき、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ってもよい。また、培養方法は、通常の平面培養（２次元）のみならず、３次元培養であってもよい。通常、３次元培養で作製したスフェロイド（細胞塊）を分析する際には、１細胞ずつに分けてフローサイトメトリーを行う必要があるが、本発明においては、培養上清を使うので、スフェロイドを破壊せずに分析できる利点がある。 That is, when the subject stem cell 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 thereof, a subculture, or a cultured cell line that has been established. You may. In addition, the culture method may be not only normal plane culture (two-dimensional) but also three-dimensional culture. Normally, when analyzing a spheroid (cell mass) produced by three-dimensional culture, it is necessary to perform flow cytometry on each cell separately, but in the present invention, since the culture supernatant is used, the spheroid is used. It has the advantage of being able to analyze without destroying it.
　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°Ｃ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 RS < TM > 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、軟骨組織由来軟骨幹細胞（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も入手できる。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 RS < TM > 培地(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 adipose tissue, umbilical cord blood, umbilical cord, amniotic membrane, placenta, or from bone marrow puncture from jawbone or femoral 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 <TM> medium (Life Technologies), which is 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 finger excised by surgery in a patient with polyphinopathy, and cartilage tissue-derived cartilage stem cells (cartilage stem cells derived from polyphinopathy) can be obtained from the cartilage tissue. it can. It can also be obtained from RIKEN BioResource Center, JSRB Cell Bank, etc. As the chondrocyte maintenance medium, MesenPRO RS <TM> medium (Life Technologies), which is generally used as a mesenchymal stem cell maintenance medium, may be used, but chondrocyte basal medium and chondrocyte proliferation medium (Takara Bio) ) And other maintenance media for chondrocytes can also be used.
　本発明によれば、体のある組織から採取した幹細胞の品質を評価又は管理することができるばかりでなく、拡大培養した後の体性幹細胞の品質を評価又は管理することもできる。また、本発明は、品質の優れた、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。 According to the present invention, not only can the quality of stem cells collected from a tissue having a body be evaluated or controlled, but also the quality of somatic stem cells after expanded culture can be evaluated or controlled. The present invention can also be used for isolating and concentrating cells having excellent quality and high differentiation potential.
　体性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であってもよい。 Since somatic stem cells are considered to be controlled by a mechanism common not only to humans but also to mammals in a considerable part, the stem cells of the present invention include non-human mammals such as monkeys, pigs, cows, goats, and sheep. , Mouse, rat-derived stem cells.
　本発明では、生体から採取された、もしくは市販の体性幹細胞の培養液、又は未分化状態で継代培養した培養液の培養上清を検査試料として用いる。培養液は一般に一定期間毎に新鮮な培養液と交換する。本発明では、培養上清中の、細胞外小胞の表面マーカーを測定する。 In the present invention, a culture supernatant of a somatic stem cell culture solution collected from a living body or commercially available, or a culture solution subcultured in an undifferentiated state is used as a test sample. The culture medium is generally replaced with fresh culture medium at regular intervals. In the present invention, the surface markers of extracellular vesicles in the culture supernatant are measured.
　本発明では、測定対象は、体性幹細胞の細胞外小胞の表面マーカーであれば、いずれのマーカーであってもよいが、例えば、α２－６シアル酸又はＭＳＣマーカーが挙げられる。 In the present invention, the measurement target may be any marker as long as it is a surface marker of extracellular vesicles of somatic stem cells, and examples thereof include α2-6 sialic acid or MSC markers.
　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。 In the present invention, the term "α2-6 sialic acid" generally refers to "Neu5Acα2-6Gal" and N, which are sugar chains in which N-acetylneuraminic acid is α2-6 bonded to the hydroxyl group at the 6-position of galactose. -Glycolyl-type neuramate refers to both sugar chains "Neu5Gcα2-6Gal" that are also α2-6 bound.
　本発明において、「ＭＳＣマーカー」というときは、間葉系幹細胞の細胞表面或いは細胞外小胞の表面で発現している、ＭＳＣに関連しているマーカー分子であれば、いずれであってもよいが、例えば、接着分子ＣＤ１０６、ＣＤ１６６、ＣＤ２９と同様に、ＣＤ１０５（ＳＨ２）、ＣＤ７３（ＳＨ３／４）、　ＣＤ４４、ＣＤ９０（Ｔｈｙ－１）、ＣＤ７、ＣＤ１３及びＳｔｒｏ－１が挙げられる。また、ネガティブマーカーとしては、接着分子ＣＤ３１、ＣＤ１８、ＣＤ５６、造血性マーカーＣＤ４５、ＣＤ３４、ＣＤ１４、ＣＤ１１や共刺激分子ＣＤ８０、ＣＤ８６、ＣＤ４０が挙げられる。 In the present invention, the term "MSC marker" may be any cell surface of mesenchymal stem cells or any marker molecule related to MSC expressed on the surface of extracellular vesicles. However, as with the adhesion molecules CD106, CD166 and CD29, examples thereof include CD105 (SH2), CD73 (SH3 / 4), CD44, CD90 (Thy-1), CD7, CD13 and Stro-1. Examples of the negative marker include adhesion molecules CD31, CD18, CD56, hematopoietic markers CD45, CD34, CD14, CD11 and co-stimulatory molecules CD80, CD86, and CD40.
　本発明の体性幹細胞の品質を評価又は管理する方法では、体性幹細胞の培養液中に遊離した細胞外小胞の表面マーカー、例えば、糖鎖の非還元末端に付加された「α２－６シアル酸」の存在量を検出又は測定し、体性幹細胞の、例えば、骨芽細胞又は軟骨細胞への細胞分化ポテンシャルの程度を判定する。 In the method for evaluating or controlling the quality of somatic stem cells of the present invention, a surface marker of extracellular vesicles released in a culture medium of somatic stem cells, for example, "α2-6" added to the non-reducing end of a sugar chain The abundance of "sialic acid" is detected or measured to determine the degree of cell differentiation potential of somatic stem cells into, for example, osteoblasts or chondrocytes.
　典型的には、間葉系幹細胞など体性幹細胞の培養上清中の細胞外小胞の表面にあるα２－６シアル酸又はＭＳＣマーカーの量を、α２－６シアル酸特異的結合性レクチン又はＭＳＣマーカーに対する抗体、及び細胞外小胞の単離に使用可能なタンパク質又は抗体を利用したサンドイッチアッセイ法を用いて測定し、体性幹細胞の品質を評価又は管理することが、簡便で好ましい。 Typically, the amount of α2-6 sialic acid or MSC marker on the surface of extracellular vesicles in the culture supernatant of somatic stem cells such as mesenchymal stem cells, α2-6 sialic acid-specific binding lectin or It is convenient and preferable to evaluate or control the quality of somatic stem cells by measuring using an antibody against the MSC marker and a sandwich assay method using a protein or antibody that can be used for isolation of extracellular vesicles.
　一実施態様として、細胞外小胞の単離に使用可能なタンパク質又は抗体を固定化したビーズ、プレートなどを用いて培養上清中の細胞外小胞をあらかじめ全て精製し、α２－６シアル酸又はＭＳＣマーカーの量を、α２－６シアル酸特異的レクチン又はＭＳＣマーカーに対する抗体を用いて検出する。 In one embodiment, all extracellular vesicles in the culture supernatant are previously purified using beads, plates, etc. on which proteins or antibodies that can be used for isolation of extracellular vesicles are immobilized, and α2-6 sialic acid is used. Alternatively, the amount of MSC marker is detected using an α2-6 sialic acid-specific lectin or an antibody against the MSC marker.
　本発明において、培養上清中の細胞外小胞の表面にある、α２－６シアル酸又はＭＳＣマーカーを検出するためには、α２－６シアル酸又はＭＳＣマーカー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。糖鎖に結合活性を示すタンパク質は「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を用いてもよい。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。 In the present invention, in order to detect the α2-6 sialic acid or MSC marker on the surface of extracellular vesicles in the culture supernatant, a probe that specifically recognizes the α2-6 sialic acid or MSC marker as an epitope. Is used. Proteins that exhibit binding activity to sugar chains are collectively called "lectins", and typical α2-6 sialic acid-binding probes include, but are limited to, various α2-6 sialic acid-binding lectins. You may use an anti-α2-6 sialic acid antibody or a derivative thereof that recognizes α2-6 sialic acid as a sugar chain antigen (emetic). Further, the α2-6 sialic acid-binding probe of the present invention may be used alone, or a plurality of probes may be used in combination. For example, a plurality of α2-6 sialic acid-binding lectins, or an anti-α2-6 sialic acid antibody can also be used in combination.
　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、培養上清中の細胞外小胞の表面にあるα２－６シアル酸を認識できるレクチンや抗体などいずれを用いてもよい。例えば、ＳＮＡレクチン（Ｓａｍｂｕｃｕｓ　ｎｉｇｒａ　ｌｅｃｔｉｎ）、ＳＳＡレクチン（Ｓａｍｂｕｃｕｓ　ｓｉｅｂｏｌｄｉａｎａ　ｌｅｃｔｉｎ）、ＰＳＬ１ａレクチン（Ｐｏｌｙｐｏｒｕｓ　ｓｑｕａｍｏｓｕｓ　ｌｅｃｔｉｎ）、及びＴＪＡＩレクチン（Ｔｒｉｃｈｏｓａｎｔｈｅｓ　ｊａｐｏｎｉｃａ　ｌｅｃｔｉｎ－Ｉ）を用いることができる。特に、ＳＮＡ及びＳＳＡレクチンが好ましい。ＳＮＡレクチンはニワトコから、ＳＳＡレクチンはニホンニワトコから、ＰＳＬ１ａレクチンはアミヒラタケから、またＴＪＡＩレクチンはキカラスウリから抽出することもできるが、ＳＮＡはＶＥＣＴＯＲ　Ｌａｂｏｒａｔｏｒｉｅｓ社、ＳＳＡ及びＴＪＡＩレクチンは生化学工業株式会社により市販されている。ＰＳＬ１ａレクチンは、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。 Examples of the α2-6 sialic acid-binding lectin used as the α2-6 sialic acid-binding probe of the present invention include lectins and antibodies capable of recognizing α2-6 sialic acid on the surface of extracellular vesicles in the culture supernatant. Either may be used. For example, SNA lectin (Sambucus nigra lectin), SSA lectin (Sambucus sieboldiana lectin), PSL1a lectin (Polyporus squamosus lectin), and TJAI lectin (Trichosans) that can use TJAI lectin. In particular, SNA and SSA lectins are preferred. SNA lectins can be extracted from elder, SSA lectins from Japanese elders, PSL1a lectins from dryad's saddle, and TJAI lectins from Trichosanthes chinensis. It is commercially available. As the PSL1a lectin, a recombinant rPSL1a lectin having α2-6 sialic acid specificity is commercially available from Wako Pure Chemical Industries.
　本発明のＭＳＣマーカーを検出するための抗体としては、抗ＣＤ４４抗体、抗ＣＤ７３抗体、抗ＣＤ９０抗体、抗ＣＤ１０５抗体、抗ＣＤ１４６抗体、抗ＣＤ２７１抗体、抗ＣＤ１１ｂ抗体、抗ＣＤ１９抗体、抗ＣＤ３１抗体、抗ＣＤ３４抗体、抗ＣＤ４５抗体、抗ＣＤ１４４抗体、及び抗ＨＬＡ－ＤＲ抗体が挙げられるが、市販の各種抗体を使用してもよい。 Examples of the antibody for detecting the MSC marker of the present invention include anti-CD44 antibody, anti-CD73 antibody, anti-CD90 antibody, anti-CD105 antibody, anti-CD146 antibody, anti-CD271 antibody, anti-CD11b antibody, anti-CD19 antibody, anti-CD31 antibody. Examples thereof include anti-CD34 antibody, anti-CD45 antibody, anti-CD144 antibody, and anti-HLA-DR antibody, but various commercially available antibodies may be used.
　細胞外小胞の単離に使用可能なタンパク質又は抗体としては、特に限定はないが、抗ＣＤ９抗体、抗ＣＤ６３抗体、抗ＣＤ８１抗体、及びＴＩＭ４が挙げられ、これらを単独で、又は組み合わせて使用することができる。 Proteins or antibodies that can be used to isolate extracellular vesicles include, but are not limited to, anti-CD9 antibody, anti-CD63 antibody, anti-CD81 antibody, and TIM4, which may be used alone or in combination. can do.
　レクチン又は細胞外小胞の単離に使用可能なタンパク質又は抗体を固相化する基板（固相）としては、プレート（例、マイクロウェルプレート）、マイクロアレイ基板（例、マイクロアレイ用スライドガラス）、チューブ、ビーズ（例、プラスチックビーズ、磁気ビーズ）、クロマトグラフィー用担体（例、Sepharose（商標））、メンブレン（例、ニトロセルロースメンブレン、PVDF膜）、ゲル（例、ポリアクリルアミドゲル）などが例示される。その中でもプレート、ビーズおよびメンブレンが好ましく用いられる。 As the substrate (solid phase) for immobilizing a protein or antibody that can be used for isolating lectin or extracellular vesicles, a plate (eg, microwell plate), a microarray substrate (eg, slide glass for microarray), a tube , Beads (eg, plastic beads, magnetic beads), chromatographic carriers (eg, Sepharose ™), membranes (eg, nitrocellulose membranes, PVDF membranes), gels (eg, polyacrylamide gels) and the like. .. Among them, plates, beads and membranes are preferably used.
　市販のレクチンアレイを用いてもよい。例えば、特異性の異なる９６種類のレクチンが固定化された高密度レクチンマイクロアレイ、４５種の植物レクチンが同一基板上に固定化されているレクチンアレイ（Kuno et al., Nature Methods 2, 851-856, 2005）やLecChipTM Ver.1.0（グライコテクニカ社製）を用いることができる。 A commercially available lectin array may be used. For example, a high-density lectin microarray in which 96 types of lectins having different specificities are immobilized, and a lectin array in which 45 types of plant lectins are immobilized on the same substrate (Kuno et al., Nature Methods 2, 851-856). , 2005) and LecChipTM Ver.1.0 (manufactured by Glyco Technica) can be used.
　被検培養上清試料は、緩衝液で希釈しまたは希釈せずに固相化レクチンウェル内に添加して相互作用させた後、非特異的結合をしている夾雑物をレクチンアレイ用緩衝液（市販されている）で洗浄する。次いで、フローサイトメトリーで測定してもよい。 The test culture supernatant sample is added to the immobilized lectin well without dilution or dilution with a buffer solution and allowed to interact with each other, and then the non-specifically bound impurities are added to the lectin array buffer solution. Clean with (commercially available). Then, it may be measured by flow cytometry.
　抗ＣＤ９抗体、抗ＣＤ６３抗体、抗ＣＤ８１抗体、又はＴＩＭ４などをマイクロプレートまたは磁気ビーズに固定化し、細胞外小胞を含む細胞培養上清を反応後、α２－６シアル酸結合性プローブまたはＭＳＣマーカー結合性プローブでサンドイッチすることにより測定してもよい。測定はＥＬＩＳＡを用いてもよい。さらに通常のＥＬＩＳＡの他にもＳＩＭＯＡ（Ｑｕａｎｔｅｒｉｘ社）のようなデジタルＥＬＩＳＡを用いることでより高感度に測定することもできる。また磁気ビーズを用いた場合はフローサイトメトリーで測定してもよい。 After immobilizing anti-CD9 antibody, anti-CD63 antibody, anti-CD81 antibody, TIM4, etc. on a microplate or magnetic beads and reacting the cell culture supernatant containing extracellular vesicles, an α2-6 sialic acid-binding probe or MSC marker It may be measured by sandwiching with a binding probe. Elisa may be used for the measurement. Further, in addition to the usual ELISA, it is possible to measure with higher sensitivity by using a digital ELISA such as SIMOA (Quantix). When magnetic beads are used, the measurement may be performed by flow cytometry.
　細胞培養上清から細胞外小胞を単離しまたは単離せずに、ｑＮａｎｏ（Izon Science）などのナノポアデバイスまたはNanoSight（Malvern Panalytical）を用いて、α２－６シアル酸結合性プローブまたはＭＳＣマーカー結合性プローブを添加しまたは添加せずに測定し、細胞外小胞を表面マーカーで分類し、評価することで、もとの細胞または細胞塊の品質を評価してもよい。 Α2-6 sialic acid-binding probe or MSC marker-binding property using a nanopore device such as qNano (IzonScience) or NanoSight (MalvernPanalytical) without isolating or isolating extracellular vesicles from cell culture supernatant. The quality of the original cell or cell mass may be assessed by measuring with or without a probe and classifying and assessing extracellular vesicles with surface markers.
　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。 Examples are shown below, and the present invention will be specifically described, but the present invention is not limited thereto.
細胞
ヒトiPS細胞（201B7株）
ヒト脂肪由来間葉系幹細胞（ADSC）
ヒト皮膚繊維芽細胞（hFibs）
余剰指由来軟骨細胞（Yub625株）
骨髄由来間葉系幹細胞（Yub621c株,Yub622株） Cell Human iPS cell (201B7 strain)
Human adipose-derived mesenchymal stem cells (ADSC)
Human skin fibroblasts (hFibs)
Excess finger-derived chondrocytes (Yub625 strain)
Bone marrow-derived mesenchymal stem cells (Yub621c strain, Yub622 strain)
実施例１
　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 Medium(Life Technologies)で培養した。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。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、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。結果を、図１に示す。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
Adipose-derived mesenchymal stem cells (ADSC, Life Technologies, Lot #: 2118) were cultured in MesenPRO RS TM Medium (Life Technologies), which is a common mesenchymal stem cell medium. The differentiation potential of cells in early passage (P5) and late passage (P28) into osteoblasts and adipocytes was investigated. Here, when the term "early passage" or "late passage" is used in the present invention, the number of passages differs depending on the cell type and culture conditions. In the early stage of stem cell culture, the time when the cell growth curve rises linearly is called "early passage", and the time when the cell growth curve becomes loose or flat after continuing passage is called "late passage".
Some cells in the early stage of passage (P5) and some cells in the late passage (P28) of adipose-derived mesenchymal stem cells were taken out, and differentiation into osteoblasts and adipocytes was induced. Induction of 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). Osteoblast differentiation was stained with alizarin red, and adipocyte differentiation was stained with oil red O to confirm the state of differentiation. The results are shown in FIG. Osteoblasts and adipocytes are stained red. Differentiation into osteoblasts and adipocytes was observed in the early passage (P5) fat-derived mesenchymal stem cells, whereas in the late passage (P28) adipocyte-derived mesenchymal stem cells, osteoblasts and fats were observed. Almost no cells were observed. That is, it can be seen that in the late passage cells of the adipose-derived mesenchymal stem cells, the differentiation potential into osteoblasts and adipocytes is lost.
実施例２
　軟骨幹細胞の一種である余剰指由来軟骨幹細胞（Yub625株、理研バイオリソースセンター）を実施例１と同様の手法で継代培養し、継代初期(P5)と継代後期（P22）に対して軟骨細胞への分化誘導を行った。軟骨分化はhMSC-Human Mesenchymal Stem Cell Chondrogenic Differentiation Medium BulletKit（Cat#: PT3003, Lonza）を用いて行った。軟骨形成はアルシアンブルー(Muto Pure Chemicals Co., LTD., Cat#: 20121)で染色して確認した。結果を、図２に示す。軟骨細胞は水色に染色される。継代初期（P5）の細胞は軟骨細胞への分化が観察されたが、継代後期（P22）の細胞は軟骨分化はほとんど観察されなかった。 Example 2
Excess finger-derived chondrocyte stem cells (Yub625 strain, RIKEN BioResource Center), which is a type of chondrocyte, are subcultured in the same manner as in Example 1, and cartilage is subjected to cartilage for early passage (P5) and late passage (P22). Differentiation into cells was induced. Chondrogenic differentiation was performed using the hMSC-Human Mesenchymal Stem Cell Chondrogenic Differentiation Medium BulletKit (Cat #: PT3003, Lonza). Cartilage formation was confirmed by staining with Alcian Blue (Muto Pure Chemicals Co., LTD., Cat #: 20121). The results are shown in FIG. Chondrocytes are stained light blue. Differentiation into chondrocytes was observed in the cells in the early passage (P5), but almost no cartilage differentiation was observed in the cells in the late passage (P22).
実施例３
　ヒト皮膚線維芽細胞(ATCC, #: PCS-201-012, Lot: 58605481)はMesenPRO RSTM Medium(Life Technologies)で実施例１と同様の手法で継代培養し、継代初期（P5）と継代後期（P20）に対して実施例１と同様の手法で骨芽細胞及び脂肪細胞への分化誘導を行い、確認した。結果を、図３に示す。赤く染まった細胞が全く確認されなかったことから、骨芽細胞及び脂肪細胞への分化ポテンシャルがないことを確認した。 Example 3
Human skin fibroblasts (ATCC, #: PCS-201-012, Lot: 58605481) were subcultured in MesenPRO RS TM Medium (Life Technologies) in the same manner as in Example 1 and were subcultured in the early stage of passage (P5). Differentiation into osteoblasts and adipocytes was induced and confirmed in the late passage (P20) by the same method as in Example 1. The results are shown in FIG. Since no red-stained cells were confirmed, it was confirmed that there was no potential for differentiation into osteoblasts and adipocytes.
実施例４
　ヒト皮膚線維芽細胞（hFibs）、ヒトiPS細胞（201B7）、脂肪由来間葉系幹細胞（ADSC、Lot#2117）、余剰指骨髄由来間葉系幹細胞（Yub621c）、余剰指由来軟骨幹細胞（Yub625）の細胞培養上清からMagCaptureTM Exosome Isolation Kit PS (富士フィルム和光純薬、293-77601)で細胞外小胞（EV）を精製した。得られたEVに吸着防止剤（Exocap Ultracentrifugation Storage Booster、 MBLサイエンス、MEK-USB）を添加して―80℃で保存した。4 ugタンパク量のEVをCy3 NHS Ester Mono-reactive（GE）で蛍光標識し、Sephadex G-25を充てんした脱塩カラムで過剰のCy3を除去後、Probing solution（25 mM Tris-HCl (pH 7.5)、140 mM NaCl,、2.7 mM KCl,、1 mM CaCl2,、1 mM MnCl2及び1% Triton X-100）で0.5 ug/mLに希釈して96種類のレクチンが固定化された高密度レクチンマイクロアレイと反応させ、エバネッセント波励起蛍光型スキャナー（Bio-REX Scan 300、レグザム)で測定した。図４に、SNAレクチンのデータを示す。 Example 4
Human skin fibroblasts (hFibs), human iPS cells (201B7), adipose-derived mesenchymal stem cells (ADSC, Lot # 2117), surplus finger bone marrow-derived mesenchymal stem cells (Yub621c), surplus finger-derived cartilage stem cells (Yub625) Extracellular vesicles (EV) were purified from the cell culture supernatant of MagCapture TM Exosome Isolation Kit PS (Fuji Film Wako Pure Drug, 293-77601). An adsorption inhibitor (Exocap Ultracentrifugation Storage Booster, MBL Science, MEK-USB) was added to the obtained EV and stored at -80 ° C. EV with 4 ug protein amount was fluorescently labeled with Cy3 NHS Ester Mono-reactive (GE), excess Cy3 was removed with a desalting column filled with Sephadex G-25, and then Probing solution (25 mM Tris-HCl (pH 7.5)). ), 140 mM NaCl ,, 2.7 mM KCl ,, 1 mM CaCl 2 , 1 mM MnCl 2 and 1% Triton X-100) diluted to 0.5 ug / mL to a high density with 96 types of lectin immobilized. It was reacted with a lectin microarray and measured with an evanescent wave-pumped fluorescence scanner (Bio-REX Scan 300, dilution). FIG. 4 shows the data of SNA lectin.
実施例５
　ヒト皮膚線維芽細胞（hFibs）、ヒトiPS細胞（201B7）、脂肪由来間葉系幹細胞（ADSC、Lot#2117）、余剰指骨髄由来間葉系幹細胞（Yub621c）、余剰指由来軟骨幹細胞（Yub625）の細胞培養上清40mLから、Ｔｉｍ４結合ビーズを含むPS CaptureTM Exosome Flow Cytometry Kit (Wako, #297-79701)を用いてEVを捕捉し、PE標識したSNA、SSA、TJAIレクチン（最終濃度：10μg/mL）を反応させ、フローサイトメーター（CytoFLEX, Beckman）で解析した。平均蛍光強度による結果を、図５に示す。いずれのレクチンも多能性をもつヒトiPS細胞（201B7）に最も高い反応を示し、分化能を持たないヒト皮膚線維芽細胞（hFibs）には低い反応性を示した。また継代初期と継代後期の細胞では、いずれの細胞でも分化能を消失した継代後期の細胞と比べて分化能がある継代初期の細胞に対して高い反応性を示した。この結果から、SNA、SSA、TJAIレクチンのEVへの反応性から、細胞の分化ポテンシャルを簡便に評価できたことがわかる。 Example 5
Human skin fibroblasts (hFibs), human iPS cells (201B7), adipose-derived mesenchymal stem cells (ADSC, Lot # 2117), surplus finger bone marrow-derived mesenchymal stem cells (Yub621c), surplus finger-derived cartilage stem cells (Yub625) From 40 mL of cell culture supernatant of SNA, SSA, TJAI lectin (final concentration: 10 μg), EV was captured using PS Capture TM Exosome Flow Cytometry Kit (Wako, # 297-79701) containing Tim4 binding beads and PE-labeled. / mL) was reacted and analyzed with a flow cytometer (CytoFLEX, Beckman). The results based on the average fluorescence intensity are shown in FIG. Both lectins showed the highest reactivity to pluripotent human iPS cells (201B7) and low reactivity to non-differentiating human skin fibroblasts (hFibs). In addition, the cells in the early passage and the late passage showed higher reactivity to the cells in the early passage, which have the ability to differentiate, than the cells in the late passage, which lost the differentiation ability in both cells. From this result, it can be seen that the cell differentiation potential could be easily evaluated from the reactivity of SNA, SSA, and TJAI lectins to EV.
実施例６
　実施例５と同様の手法で、余剰指由来軟骨幹細胞（Yub625）の細胞培養上清40mLから、Ｔｉｍ４結合ビーズを含むPS CaptureTM Exosome Flow Cytometry Kit (Wako, #297-79701)を用いてEVを捕捉し、ビーズ10μLに対してPE標識したＭＳＣマーカー（ＣＤ１３、ＣＤ２９、ＣＤ７３、ＣＤ９０、ＣＤ１０５）の各種抗体（いずれもBD（Becton, Dickinson and Company ）製、10μg/mL）を反応させ、フローサイトメーター（CytoFLEX, Beckman）で解析した。また、余剰指由来軟骨幹細胞（Yub625）の細胞自体を用いて、細胞表面のＭＳＣマーカーについても同様にPE標識したＭＳＣマーカーの各種抗体（いずれもBD製、10 μg/mL）を反応させ、フローサイトメーター（CytoFLEX, Beckman）で解析した。平均蛍光強度による結果を、図６に示す。細胞外小胞と細胞のいずれにおいても、各抗体で同様のパターンの反応性を示した。この結果から、ＥＶを用いてＭＳＣマーカーを測定することにより、細胞自体を消費することなく、体性幹細胞の品質を簡便に評価できたことがわかる。 Example 6
EV from 40 mL of cell culture supernatant of surplus finger-derived cartilage stem cells (Yub625) using PS Capture TM Exosome Flow Cytometry Kit (Wako, # 297-79701) containing Tim4 binding beads in the same manner as in Example 5. Various antibodies (all manufactured by BD (Becton, Dickinson and Company), 10 μg / mL) of MSC markers (CD13, CD29, CD73, CD90, CD105) captured and PE-labeled on 10 μL of beads were reacted with flow cytos. Analysis was performed with a meter (CytoFLEX, Beckman). In addition, using the cells of the surplus finger-derived cartilage stem cells (Yub625) themselves, the MSC markers on the cell surface were similarly reacted with various antibodies of the PE-labeled MSC markers (all made by BD, 10 μg / mL), and flowed. It was analyzed with a cytometer (CytoFLEX, Beckman). The results based on the average fluorescence intensity are shown in FIG. Each antibody showed a similar pattern of reactivity in both extracellular vesicles and cells. From this result, it can be seen that the quality of somatic stem cells could be easily evaluated without consuming the cells themselves by measuring the MSC marker using EV.
実施例７
　ＭａｇＣａｐｔｕｒｅ（商標）エクソソームアイソレーションキットＰＳ　ＭａｇＣａｐｔｕｒｅ（商標）Ｅｘｏｓｏｍｅ　Ｉｓｏｌａｔｉｏｎ　Ｋｉｔ　ＰＳ（富士フィルム和光純薬製）を用いて、骨髄由来間葉系幹細胞（Yub622）の培養上清からエクソソームを精製した。精製されたエクソソームにＰＥ標識ＴＪＡ１（α２－６シアル酸結合性レクチン）を反応させて、フローサイトメーター（ＣｙｔoＦＬＥＸ、バイオレットレーザー搭載）により解析した。その結果、ＰＥ－ＴＪＡ１と強い反応性を示すエクソソームが、継代初期の細胞では２．３％であったのに対して、継代後期では０．９９％であることがわかった。 Example 7
Exosomes were purified from the culture supernatant of bone marrow-derived mesenchymal stem cells (Yub622) using MagCapture ™ Exosome Isolation Kit PS MagCapture ™ Exosome Isolation Kit PS (manufactured by Fuji Film Wako Pure Chemical Industries, Ltd.). The purified exosome was reacted with PE-labeled TJA1 (α2-6 sialic acid-binding lectin) and analyzed by a flow cytometer (CytoFLEX, equipped with a violet laser). As a result, it was found that the amount of exosomes showing strong reactivity with PE-TJA1 was 2.3% in the cells in the early stage of passage, whereas it was 0.99% in the late passage.

Claims (10)

  1. 　体性幹細胞の培養上清中の、細胞外小胞の表面マーカーを測定する工程を含む、体性幹細胞の品質を評価する方法。 A method for evaluating the quality of somatic stem cells, which comprises the step of measuring the surface markers of extracellular vesicles in the culture supernatant of somatic stem cells.
  2. 　上記体性幹細胞が、間葉系幹細胞である、請求項１に記載の方法。 The method according to claim 1, wherein the somatic stem cells are mesenchymal stem cells.
  3. 　表面マーカーが、α２－６シアル酸である、請求項１又は２に記載の方法。 The method according to claim 1 or 2, wherein the surface marker is α2-6 sialic acid.
  4. 　表面マーカーが、ＭＳＣマーカーである、請求項１又は２に記載の方法。 The method according to claim 1 or 2, wherein the surface marker is an MSC marker.
  5. 　α２－６シアル酸を測定する工程が、
    （１）対象の体性幹細胞の培養上清を、下記（ａ）又は（ｂ）のいずれか一方のプローブが固定された基板上にオーバーレイし、つ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ＴＪＡＩレクチンから選択される少なくとも１種のレクチン
    　（ｂ）抗ＣＤ９抗体、抗ＣＤ６３抗体、抗ＣＤ８１抗体、及びＴＩＭ４から選択される少なくとも１種のタンパク質、及び
    （２）標識量を測定する工程
    を含む、請求項３に記載の方法。     The process of measuring α2-6 sialic acid is
    (1) A step of overlaying the culture supernatant of the target somatic stem cell on a substrate on which one of the following probes (a) or (b) is immobilized, and then allowing the labeled other probe to act. (A) At least one lectin selected from SSA lectins, SNA lectins, and TJAI lectins (b) At least one protein selected from anti-CD9 antibody, anti-CD63 antibody, anti-CD81 antibody, and TIM4, and ( 2) The method according to claim 3, which comprises a step of measuring the labeled amount.
  6. 　α２－６シアル酸を測定する工程が、
    （１）対象の体性幹細胞の培養上清と、標識化された下記（ａ）のレクチンとを作用させる工程
    　（ａ）ＳＳＡレクチン、ＳＮＡレクチン、及びＴＪＡＩレクチンから選択される少なくとも１種のレクチン
    及び
    （２）標識量を測定する工程
    を含む、請求項３に記載の方法。     The process of measuring α2-6 sialic acid is
    (1) Step of reacting the culture supernatant of the target somatic stem cell with the labeled lectin of (a) below (a) At least one lectin selected from SSA lectin, SNA lectin, and TJAI lectin. The method according to claim 3, further comprising (2) a step of measuring the labeled amount.
  7. 　あらかじめ細胞外小胞を精製する工程を含む又は含まない、請求項１～６のいずれか一項に記載の方法。 The method according to any one of claims 1 to 6, which includes or does not include a step of purifying extracellular vesicles in advance.
  8. 　体性幹細胞の培養上清中の、細胞外小胞の表面マーカーを測定する工程を含む、体性幹細胞の品質管理方法。 A quality control method for somatic stem cells, which comprises the step of measuring the surface markers of extracellular vesicles in the culture supernatant of somatic stem cells.
  9. 　表面マーカーが、α２－６シアル酸である、請求項８に記載の方法。 The method according to claim 8, wherein the surface marker is α2-6 sialic acid.
  10. 　表面マーカーが、ＭＳＣマーカーである、請求項８に記載の方法。 The method according to claim 8, wherein the surface marker is an MSC marker.
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