WO2013186946A1 - Méthode de sélection d'une cellule souche pluripotente - Google Patents

Méthode de sélection d'une cellule souche pluripotente Download PDF

Info

Publication number
WO2013186946A1
WO2013186946A1 PCT/JP2012/078651 JP2012078651W WO2013186946A1 WO 2013186946 A1 WO2013186946 A1 WO 2013186946A1 JP 2012078651 W JP2012078651 W JP 2012078651W WO 2013186946 A1 WO2013186946 A1 WO 2013186946A1
Authority
WO
WIPO (PCT)
Prior art keywords
hexnac
hex
fuc
glcnac
man
Prior art date
Application number
PCT/JP2012/078651
Other languages
English (en)
Japanese (ja)
Inventor
康郎 篠原
潤一 古川
直樹 藤谷
香代 荒木
中村　幸夫
Original Assignee
国立大学法人北海道大学
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 国立大学法人北海道大学 filed Critical 国立大学法人北海道大学
Priority to JP2014521190A priority Critical patent/JP5979452B2/ja
Publication of WO2013186946A1 publication Critical patent/WO2013186946A1/fr

Links

Images

Classifications

    • 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
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56966—Animal cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2400/00—Assays, e.g. immunoassays or enzyme assays, involving carbohydrates
    • G01N2400/10—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • G01N2400/38—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence, e.g. gluco- or galactomannans, e.g. Konjac gum, Locust bean gum, Guar gum

Definitions

  • the present invention relates to a method for evaluating the differentiation state of cells, specifically, a method for evaluating the differentiation state of pluripotent stem cells.
  • iPS cells pluripotent stem cells
  • Non-patent Document 1 A sorting method using SSEA-1, SSEA-3, SSEA-4, and PECAM-1 as a sorting method for undifferentiated ES cells (Patent Document 1), a sorting method based on detection of a Psbp gene expression product (Patent Document 2), Methods for using antibodies against Alkaline phosphatase, TRA-1-60, TRA-1-81, CD30, Cripto, etc. (Non-patent Document 1) have been proposed as various selection markers.
  • sugar chains having the same epitope structure may be contained in different classes of glycoconjugates.
  • Lewis X antigen (Lex) is often observed on complex N-linked sugar chains, but may also be expressed on glycosphingolipids at the same time. When expressed on glycosphingolipid, this becomes a glycolipid widely known as an undifferentiated marker called SSEA-1 (CD15).
  • Non-patent Documents 4-7 a sugar chain structure that is characteristically expressed in undifferentiated cells is recognized by a lectin and fluorescence is detected.
  • Lectins can often recognize the binding mode of sugar chains, but on the other hand, they only recognize sugar chains of 1 to several residues, and it is unclear which complex carbohydrates on the cell surface are derived, such as cluster effects. Since the amount of binding is affected by this, quantification is difficult and cross-reaction problems remain.
  • the present invention provides a new method for efficiently and strictly evaluating and selecting pluripotent stem cells by eliminating the cross-reactions seen in antigen-antibody reactions.
  • Non-Patent Document 11 N-linked sugar chains, O-linked sugar chains (Non-Patent Document 11), glycosphingolipid (GSL) sugar chains (Non-Patent Document 12), Focusing on glycosaminoglycan (GAG) (Non-patent Document 13), a method for analyzing individual absolute amounts was developed.
  • GSL glycosphingolipid
  • GAG Focusing on glycosaminoglycan
  • N-linked sugar chains and free oligosaccharides (FOS) of cells have been integrated.
  • a method was established for quantitative analysis of all N-linked sugar chains, O-linked sugar chains, GSL, GAG, and FOS of proteins from one cell sample. Then, by conducting comprehensive quantitative and qualitative analysis of these glycoconjugates, we searched for novel glycoconjugates that are specifically expressed in pluripotent stem cells.
  • glycoconjugate sugar chains of individual classes of cells N-linked sugar chains, FOS, GSL, GAG and O-linked types
  • WO2006 / 030584, WO2007 / 108204, and WO2008 / 018170 Comprehensive approach to structural and functional glycomics based on chemoselective glycoblotting and sequential tag conversion "Jun (2008) Anal. Chem. 80, 1094-1101, Glyco blotting method and BEP method, etc.
  • the present invention (1) A method for determining the differentiation state of pluripotent stem cells,
  • Group B selected from the group consisting of (neo) lacto series, globo series and ganglio series
  • Glycosphingolipid group C selected from the group consisting of (Man) n (GlcNAc) type 2 and (Man) n (GlcNAc) type 1, selected from CS ( ⁇ UA ⁇ 1-3GalNAc)
  • Glysaminoglycan E group selected from the group consisting of chondroitin sulfate / dermatan sulfate, heparan sulfate selected from HS ( ⁇ UA ⁇ 1-4GlcNAc), and hyaluronic acid constituent disaccharides selected from HA ( ⁇ UA ⁇ 1-3GlcNAc) : (NeuAc) n (HexNAc) n (Fuc
  • a method comprising the step of comparing with an amount; (2) Each of the groups A to E has the following sugar chains: Group A: Group B Group C Group D Group E
  • the amount of the sugar chain in the cells other than the pluripotent stem cells is decreased compared to the amount of sugar chains in the cells other than the pluripotent stem cells.
  • the test cell is determined to be a pluripotent cell, the method according to any one of (1) to (13); (15) A sugar chain having a plurality of fucose residues is (Hex) 1 (HexNAc) 2 (Fuc) 2+ (Man) 3 (GlcNAc) 2, (Hex) 3 (HexNAc) 3 (Fuc) 2 (NeuAc) 1+ (Man) 3 (GlcNAc) 2, (Hex) 2 (HexNAc) 2 (Fuc) 2 (NeuAc) 1+ (Man) 3 (GlcNAc) 2, (Hex) 3 (HexNAc) 3 (Fuc) 2+ (Man) 3 (GlcNAc) 2, (Hex) 3 (HexNAc) 3 (Fuc) 2+ (Man) 3 (GlcNAc)
  • test cell is determined to be a pluripotent cell when increasing; (17) Of the FOS in the test cell, the amount of (Hex) 1 (HexNAc) 1, (Hex) 1 (HexNAc) 2 is compared with the amount of sugar chains in cells other than the pluripotent stem cells.
  • the test cell is a pluripotent cell when it is decreased or when the relative quantity ratio of FOS and N-linked sugar chain (FOS / N-linked sugar chain) is 2.0% or less
  • the method of the present invention it is possible to accurately obtain the amounts and expression profile information of all major glycoconjugates of various pluripotent stem cells including ES cells and iPS cells. By comparing with a control group, it is possible to accurately evaluate the undifferentiated state of cells and search for cell markers for efficiently selecting pluripotent stem cells.
  • the MALDI spectrum of the sugar chain in various cells is shown.
  • IS indicates an internal standard ((Hex) 2 (HexNAc) 2 (NeuAc) 2+ (Man) 3 (GlcNAc) 1).
  • mass spectra of the N-linked sugar chain of HL60, the N-linked sugar chain of iPS1A, the free sugar chain of HL60, and the free sugar chain of iPS1A are shown.
  • the results of zwitterionic hydrophilic chromatography are shown.
  • IS indicates an internal standard (isomaltose).
  • (+) Shows the HPLC chromatograph of hyaluronidase, chondroitinase ABC, heparinase / heparitinase digested
  • ( ⁇ ) shows the hyaluronidase, chondroitinase ABC, heparinase / heparitinase digested.
  • the quantification result of the sugar chain in various cells is shown.
  • the cell name is displayed in the center of the pentagon, and the expression profiles of N-linked sugar chains (N), FOS, GAG, GSL, and O-linked sugar chains (O) are shown as pie charts at the respective vertices. .
  • the size of the circle reflects the amount of sugar chain expressed, and the color (shading) reflects the structure of each sugar chain.
  • 4 is a legend of the graph of FIG. 3.
  • the color (shading) in each pie chart corresponds to the structure of each sugar chain.
  • “N” in the center of the pie chart represents an N-linked sugar chain, and “O” represents an O-linked sugar chain.
  • the result of the correlation matrix between cells is shown.
  • the correlation coefficient (r) between cells was calculated using quantitative data of all sugar chains observed in 18 types of cells.
  • a cell surrounded by a dotted line indicates a combination of cells having a large correlation coefficient. The darker the cell color, the higher the correlation.
  • a cell not surrounded by a dotted line indicates a combination of cells having a small correlation coefficient.
  • Hierarchical cluster analysis is a method of classifying target data groups into mathematically similar data groups, using Euclidean distance as the similarity between individuals, and using group average method as the similarity between clusters. It was. SSEA-3, 4, 5, GloboGH and Tra-1 epitopes, which are widely used as stem cell markers, were grouped together in a branch group surrounded by a dotted line in the phylogenetic tree.
  • Box-and-whisker plots showing a comparison of the expression levels of various N-linked sugar chains (sugar chains having multiple fucose residues) that were significantly different between stem cells (Stem) and non-stem cells (Non-stem) is there.
  • Box-and-whisker plots showing a comparison of the expression levels of various N-linked sugar chains (sugar chains having multiple fucose residues) that were significantly different between stem cells (Stem) and non-stem cells (Non-stem) Yes (continuation of FIG. 7).
  • FIG. Box-and-whisker showing a comparison of the expression levels of various N-linked glycans (bisecto-type, LacdiNAc-type, or multi-branched glycans) that showed a significant difference between stem cells (Stem) and non-stem cells (Non-stem)
  • FIG. Box showing comparison of expression levels of various N-linked glycans (3-branched 3 sialylated glycans and degradable glycans) that were significantly different between stem cells (Stem) and non-stem cells (Non-stem)
  • FIG. 11 is a box-and-whisker diagram showing a comparison of the expression levels of various glycosphingolipids ((neo) lacto series) in which a significant difference was observed between stem cells (Stem) and non-stem cells (Non-stem).
  • FIG. 11 is a box-and-whisker diagram showing a comparison of the expression levels of various glycosphingolipids ((neo) lacto series) in which significant differences were observed between stem cells (Stem) and non-stem cells (Non-stem) (continuation of FIG. 11). .
  • FIG. 17 is a box-and-whisker diagram showing a comparison of expression levels of various glycosaminoglycans in which a significant difference was observed between stem cells (Stem) and non-stem cells (Non-stem) (continuation of FIG. 16).
  • FIG. 4 is a box and whisker plot showing a comparison of expression levels of various O-linked sugar chains in which a significant difference was observed between stem cells (Stem) and non-stem cells (Non-stem).
  • FIG. 4 is a box and whisker plot showing a comparison of expression levels of various free oligosaccharides between normal iPS cells (good) and cells incompletely converted to iPS HiPS-RIKEN-3C (bad). It is a box-and-whisker plot showing a comparison of the expression levels of various O-linked sugar chains between normal iPS cells (good) and cells with incomplete iPS formation HiPS-RIKEN-3C (bad).
  • the “pluripotent stem cell” means a stem cell having the ability to differentiate into all tissue cells constituting an individual, that is, “pluripotency”.
  • pluripotent stem cells include embryonic stem cells (ES cells), tissue stem cells, living stem cells, and induced pluripotent stem cells (iPS cells).
  • ES cells embryonic stem cells
  • tissue stem cells tissue stem cells
  • iPS cells induced pluripotent stem cells
  • the cells are not limited to the above cells as long as they have pluripotency.
  • pluripotent stem cells can be derived from animals, and can be, for example, mammals, fish and the like.
  • mammals include humans, mice, rats, sheep, pigs, monkeys and the like, and examples of fish include medaka and zebrafish, but are not limited to these examples.
  • Cells other than pluripotent stem cells include all except the pluripotent stem cells described above.
  • normal cells derived from tissues, cancer cells, or established normal cells, cancer cells, immortal cells Although a cancerous cell can be mentioned, it is not limited to these examples.
  • pluripotent stem cells and “cells other than pluripotent stem cells” used in the present invention can be obtained commercially or by distribution, and can be easily produced by conventional techniques in the art. can do.
  • the “high mannose type sugar chain” used in the present invention means a sugar chain in which the extension of the core structure ((Man) 3 (GlcNAc) 2) is composed only of mannose residues.
  • the “complex-type sugar chain” used in the present invention is a sugar chain in which the extension of the core structure ((Man) 3 (GlcNAc) 2) is composed of residues such as glucosamine, galactose, sialic acid, and fucose. This means that a single strand, a double strand, a triple strand, or a quadruplex is formed according to the difference in the number of branches.
  • hybrid sugar chain used in the present invention means a sugar chain in which a part of a branch is composed only of mannose, such as a high mannose type, and the other branch includes a glucosamine residue or the like, such as a complex type. To do.
  • the major glycoconjugates of the cells detected and used in the present invention include N-linked sugar chains, free oligosaccharides (hereinafter sometimes referred to as “FOS”), glycosphingolipids (hereinafter referred to as “GSL”). And sugar chains, glycosaminoglycans (hereinafter sometimes referred to as “GAG”), and O-linked sugar chains.
  • FOS free oligosaccharides
  • GSL glycosphingolipids
  • GAG glycosphingolipids
  • GAG glycosaminoglycans
  • these complex sugar chains contained in cells are subjected to a treatment for separating and purifying, and a quantitative profile can be individually obtained for the separated and purified sugar chains.
  • Separation and purification of sugar chains can be performed by, for example, glycoblotting methods (for example, the above-mentioned WO2006 / 030684, WO2007 / 108204 and WO2008 / 018170, Comprehensive approach to structural and functional glycomics based on chemoselective glycoblotting and sequential tag conversion Junichi , Yasuro Shinohara, Hiromitsu Kuramoto, Yoshiaki Miura, Hideyuki Shimaoka, Masaki Kurogochi, Mika Nakano, Shin-ichiro Nishimura. (2008) Anal. Chem. 80, 1094-1101) and BEP method (non-patent literature 11) This is preferable from the viewpoint that the desired sugar chain is not lost and can be quickly separated and purified.
  • N-linked glycan fractions Add 2% SDS 100 mM Tris-acetate buffer to various pluripotent stem cell pellets containing ES cells and iPS cells, and recover the cell extract by sonication .
  • the cell extract is reduced with TCEP, treated with DNase, and alkylated with iodoacetamide, then added with ethanol, allowed to stand at ⁇ 30 ° C. for 3 hours, and the pellet is recovered by centrifugation.
  • the collected pellet is digested with trypsin and then cleaved with N-linked sugar chain using peptide N glycanase F.
  • the recovered sugar chain is purified and labeled using a glycoblotting method as described in the above document.
  • the solution obtained in the above steps 1 to 3 is centrifuged at 20000 g for 10 minutes to precipitate the protein fraction, and the obtained supernatant is collected as a total lipid extract containing glycosphingolipid in a new sample tube. .
  • the solvent is removed from the collected solution with a centrifugal evaporator. 5.
  • the obtained cell-derived total lipid fraction was suspended in 50 mM Tris acetate buffer (pH 5.0) containing 40 microliters of 0.2% Triton-X100 (or 0.2% sodium cholate).
  • glycosaminoglycan chains After inactivating pronase (90 ° C., 10 minutes), 20 microliters of 30% aqueous sodium acetate solution is added, and 480 microliters of ice-cold ethanol is added to precipitate glycosaminoglycan chains ( ⁇ 30 ° C., 2 hours). Glycosaminoglycan chain precipitates are collected by centrifugation (5000 rpm, 10 minutes, 4 ° C.), dried, dissolved in 80 microliters of 100 mM ammonium acetate buffer (including 5 mM calcium acetate), and 5 ⁇ m each.
  • the precipitate (protein fraction) and supernatant are separated by centrifugation (20000 ⁇ g, 10 minutes, 4 ° C.), and the precipitate is used for O-linked sugar chain analysis. 5.
  • the precipitate is dissolved in ultrapure water, made into a fraction having a molecular weight of 3000 or more by Amicon, and then concentrated to dryness. 6).
  • the precipitate is redissolved with 10 ⁇ L of ultrapure water, tetraacetylchitotetraose (10 ⁇ M, 10 ⁇ L) is added to the internal standard, and 20 ⁇ L of 0.4 M NaOH and 20 ⁇ L of 0.5 M 3-methyl-1-phenyl are added.
  • the quantitative glycan profile is preferably determined by separating the purified glycan from MALDI-TOF / MS (matrix-assisted laser desorption / ionization-time of-flight / mass spectrometry) or LC-ESI / SSI-TOF / MS ( liquid chromatography-electrospray ionization / sonic spray ionization-time of flight / mass spectrometry).
  • MALDI-TOF / MS matrix-assisted laser desorption / ionization-time of-flight / mass spectrometry
  • LC-ESI / SSI-TOF / MS liquid chromatography-electrospray ionization / sonic spray ionization-time of flight / mass spectrometry.
  • N-linked glycans For example, purified and labeled N-linked glycans, O-linked glycans, FOS, GSL glycans are mixed with 2,5-dihydroxybenzoic acid (10mg / mL 30% acetonitrile) and analyzed by MALDI-TOF MS. Can be used.
  • UltraFlex II TOF / TOF manufactured by Bruker Daltonics is used, and all spectra can be acquired in the reflector mode.
  • Signal detection and quantification can be performed using, for example, the company's FlexAnalysis 3.0. Using a standard sugar chain of known concentration ((Neu5Ac) 2 (Gal) 2 (GlcNAc) 2+ (Man) 3 (GlcNAc) 1) as an internal standard, each signal can be quantified from the area ratio.
  • the purified sugar chain is subjected to quantitative analysis using MALDI-TOF / MS.
  • the sugar chain is quantified by comparing the peak area value of each sugar chain with an internal standard of known concentration.
  • the purified sugar chain is subjected to quantitative analysis using MALDI-TOF / MS.
  • the sugar chain is quantified by comparing the peak area value of each sugar chain with an internal standard of known concentration.
  • the purified sugar chain is subjected to quantitative analysis using MALDI-TOF / MS.
  • the sugar chain is quantified by comparing the peak area value of each sugar chain with an internal standard of known concentration.
  • Glysaminoglycan Analysis of the purified GAG sugar chain is performed by a zwitterionic hydrophilic chromatography method. Add isomaltotriose as an internal standard and perform quantitative analysis by fluorescence detection.
  • the quantitative profile of O-linked glycans is preferably determined by separating the purified glycans from MALDI-TOF / MS (matrix-assisted laser desorption / ionization-time of flight / mass spectrometry). ).
  • the sugar chain portion purified by the above method is analyzed by MALDI-TOF (/ TOF) (UltraFlex II, Bruker) using 2,5-dihydrobenzoic acid (10 mg / mL) as a matrix.
  • N-linked sugar chains N-glycan
  • SGL glycosphingolipids
  • FOS free oligosaccharides
  • GAG glycaminoglycans
  • O-linked O-linked
  • sugar chains having a plurality of fucose residues include: (Hex) 1 (HexNAc) 2 (Fuc) 2+ (Man) 3 (GlcNAc) 2, (Hex) 3 (HexNAc) 3 (Fuc) 2 (NeuAc) 1+ (Man) 3 (GlcNAc) 2, (Hex) 2 (HexNAc) 2 (Fuc) 2 (NeuAc) 1+ (Man) 3 (GlcNAc) 2, (Hex) 3 (HexNAc) 3 (Fuc) 2+ (Man) 3 (GlcNAc) 2, (Hex) 3 (HexNAc) 3 (Fuc) 2 (NeuAc) 1 (NeuGc) 1+ (Man) 3 (GlcNAc) 2, (Hex) 3 (HexNAc) 3 (Fuc) 2 (NeuAc) 1 (NeuGc) 1+ (Man) 3 (GlcNAc) 2, (Hex) 1 (HexNAc) 3 (Fuc
  • a bisecting type or LacdiNAc type or multi-branched sugar chain for example, (Hex) 3 (HexNAc) 3+ (Man) 3 (GlcNAc) 2, (HexNAc) 3+ (Man) 3 (GlcNAc) 2, (Hex) 1 (HexNAc) 4 (Fuc) 1+ (Man) 3 (GlcNAc) 2 Is mentioned.
  • degradable sugar chains include: (Man) 2 (GlcNAc) 2, (Man) 3 (GlcNAc) 2 or (Man) 3 (GlcNAc) 2 (Fuc) 1 Is mentioned.
  • 3-sialylated tri-branched sugar chains include: (Hex) 3 (HexNAc) 3 (NeuAc) 3+ (Man) 3 (GlcNAc) 2 Is mentioned.
  • glycosphingolipids examples include: (Hex) 3 (HexNAc) 1 ((n) Lc4), (Hex) 4 (HexNAc) 1 (Gal- (n) Lc4), (Hex) 3 (HexNAc) 1 (Fuc) 1 (SSEA-1 / 5), (Hex) 3 (HexNAc) 1 (Fuc) 2 (diFuc- (n) Lc4), (Hex) 3 (HexNAc) 2 ((n) Lc5), (Hex) 2 (HexNAc) 1 (Lc3), (Hex) 4 (HexNAc) 2 (Fuc) 1 (NeuAc) 2 Is mentioned.
  • glycosphingolipid for example, (Hex) 3 (Gb3), (Hex) 3 (HexNAc) 1 (Gb4), (Hex) 4 (HexNAc) 1 (Gb5, SSEA-3), (Hex) 4 (HexNAc) 1 (Neu5Ac) 1 (SSEA4), (Hex) 4 (HexNAc) 1 (Fuc) 1 (globo H) Is mentioned.
  • Examples of free oligosaccharides include: (Hex) 1 (HexNAc) 1, (Hex) 1 (HexNAc) 2 Is mentioned.
  • Glycosaminoglycan Glycosaminoglycan disaccharides are shown in Table 16.
  • the HS (heparan sulfate) system includes eight types shown in a).
  • the CS (chondroitin sulfate) system there are 8 types shown in b).
  • HA (hyaluronic acid) includes those shown in c). More preferably, as HS, HS-0S (non-sulfated heparan sulfate disaccharide), HS-6S (6-position sulfated heparan sulfate disaccharide), HS-NS (N-sulfated heparan sulfate disaccharide) are CS.
  • CS-0S non-sulfated chondroitin sulfate disaccharide
  • CS-2S (2-position sulfated chondroitin sulfate disaccharide)
  • CS-4S 4-position sulfated chondroitin disaccharide
  • CS-2S4S (2, 4-position) Sulfated chondroitin sulfate disaccharide
  • O-linked sugar chains examples include: (Hex) 3 (HexNAc) 3, (Hex) 2 (HexNAc) 2 (Fuc) 1, (Hex) 2 (HexNAc) 2, (Hex) 1 (HexNAc) 2, (HexNAc) 2 Is mentioned.
  • the present inventors are a group of sugar chains whose expression kinetics are remarkably changed in so-called “deficient iPS” in which iPS conversion having differentiation resistance is incomplete among sugar chains that are expressed in normal iPS cells. I found out. Therefore, another embodiment of the present invention provides a method for detecting defective iPS using these sugar chains, and the use of these sugar chains as detection markers for defective iPS. These sugar chains are also useful as quality control markers that serve as indicators of the pluripotency of the produced iPS cells.
  • N-linked sugar chains for detecting non-pluripotent cells from established iPS cells include: (Hex) 2 (HexNAc) 3 (Fuc) 1 (NeuAc) 2+ (Man) 3 (GlcNAc) 2, (Hex) 1 (HexNAc) 3 (Fuc) 1 (NeuAc) 1+ (Man) 3 (GlcNAc) 2, (Hex) 2 (HexNAc) 2 (Fuc) 2 (NeuAc) 1+ (Man) 3 (GlcNAc) 2, (Hex) 2 (HexNAc) 2 (Fuc) 2+ (Man) 3 (GlcNAc) 2
  • glycosphingolipids include: (Hex) 2 (HexNAc) 1 (Fuc) 1 ("SSEA-5"), (Hex) 3 (HexNAc) 1 (Fuc) 2 ("diFuc- (n) Lc4"), (Hex) 3 ("Gb3"), (Hex) 4 (Hex)
  • Pluripotent stem cells 5 types of iPS cells HiPS-RIKEN-1A, 2A, 12A established from umbilical cord-derived cells HiPS-RIKEN-3A established from decidual tissue HiPS-RIKEN-11A established from tissues around the amniotic membrane 5 types of human ES cells KhES-1, KhES-2, KhES-3, KhES-4, KhES-5
  • Non-pluripotent stem cells control human cells: HL60 (human promyelocytic leukemia-derived cell line) HeLa (human cervical cancer-derived cell line) A549 (human non-small cell lung cancer-derived cell line) KLM-1 (human pancreatic cancer cell-derived cell line) Caco-2 (human colorectal cancer-derived cell line) HepG2 (human liver cancer-derived cell line) NEC8 (human test test).
  • N-linked sugar chains, O-linked sugar chains, FOS, GSL sugar chains, and GAG of each cell were extracted as follows.
  • Sample preparation for analysis of N-linked glycans and FOS 1. 1 ⁇ 10 6 cell pellets in tris acetate buffer containing 2% sodium dodecyl sulfate (100 microliters) and sonicated (5 Second irradiation + 10 seconds interval (4 sets). 2. Add 2 microliters of 0.5 M tris (2-carboxyethyl) phosphine (final concentration about 100 mM) and 200 mM iodoacetamide (final concentration about 20 mM) to reductively alkylate the disulfide bonds of the protein (room temperature 30 minutes). 3.
  • the supernatant was concentrated to dryness and then resuspended in 50 mM Tris acetate buffer containing a surfactant (2% Triton X-100 or sodium cholate) to obtain 25 mU of endoglucoceramidase I from Rhodocossus and II was added to cleave the sugar chain from the ceramide chain (37 ° C., overnight). 3.
  • the precipitate was dissolved in 90 microliters of 50 mM ammonium acetate buffer and the protein was digested with 10 microliters of pronase solution (10 mg / mL) (overnight at 37 ° C.).
  • FIG. 1 shows a representative MALDI-TOF / MS spectrum and liquid chromatography results.
  • Sample preparation for O-linked glycan analysis The cell pellet consisting of 1 ⁇ 10 6 cells was lysed by sonication (5 seconds irradiation + 10 seconds interval 4 sets) in Tris acetate buffer (100 microliters) containing 2% sodium dodecyl sulfate. 2. 2 microliters of 0.5 M tris (2-carboxyethyl) phosphine (final concentration about 100 mM) and 200 mM iodoacetamide (final concentration about 20 mM) were added to reductively alkylate the disulfide bonds of the protein (room temperature, 30 mM). Min). 3.
  • the precipitate is redissolved with 10 ⁇ L of ultrapure water, tetraacetylchitotetraose (10 ⁇ M, 10 ⁇ L) is added to the internal standard, and 20 ⁇ L of 0.4 M NaOH and 20 ⁇ L of 0.5 M 3-methyl-1-phenyl are added.
  • a methanol solution of ⁇ 5-pyrazolone (PMP) was added, and the mixture was allowed to stand at 85 ° C. for 16 hours. After the reaction, the reaction mixture was neutralized with HCl, and the excess reagent was removed by washing with chloroform, and then the sugar chain portion was purified with graphite carbon and iatrobeads. (According to Non-Patent Document 10).
  • N-linked sugar chain N-glycan
  • O-linked sugar chain O-linked sugar chain
  • FOS free oligosaccharide
  • GSL glycosphingolipid
  • GAG glycaminoglycan
  • Fig. 3 shows an example of the quantitative results for each cell.
  • the apex of the pentagon shows the expression profile of GSL, O-linked glycan, GAG, FOS, and N-linked glycan in the clockwise direction, and the size of the pie chart represents the expression level of the glycan
  • the color reflects the structure.
  • the overall glycome of cells is highly cell specific.
  • FIG. 5 shows the result of the correlation matrix between cells.
  • the pluripotent stem cell group (ES, iPS) and non-pluripotent stem cells were clearly distinguished, and it was shown that the analysis by the comprehensive glycome can be classified according to the properties of the cells.
  • FIG. 6 shows the result of hierarchical cluster analysis.
  • pluripotent stem cell groups ES, iPS
  • non-pluripotent stem cells were clearly distinguished.
  • SSEA which has been proven as an undifferentiated marker, is clustered in the same hierarchy in the same cluster.
  • the sugar chain includes an SSEA-1 / 5 epitope or a sialyl SSEA-1 epitope.
  • Bisect type or LacdiNAc type or hyperbranched sugar chain Hex 3 (HexNAc) 3+ (Man) 3 (GlcNAc) 2, (HexNAc) 3+ (Man) 3 (GlcNAc) 2, (Hex) 1 (HexNAc) 4 (Fuc) 1+ (Man) 3 (GlcNAc) 2 Is significantly higher at p ⁇ 0.01. 3.
  • Degradable sugar chain (Man) 2 (GlcNAc) 2, (Man) 3 (GlcNAc) 2 or (Man) 3 (GlcNAc) 2 (Fuc) 1 Is significantly lower at p ⁇ 0.05.
  • 4.3 Sialylated 3-branched sugar chain (Hex) 3 (HexNAc) 3 (NeuAc) 3+ (Man) 3 (GlcNAc) 2 Is significantly lower at p ⁇ 0.01.
  • FOS recognized the following characteristics. 1. When the amount of (Hex) 1 (HexNAc) 1 was p ⁇ 0.01 and the amount of (Hex) 1 (HexNAc) 2 was p ⁇ 0.05, significant reduction was observed in ES and iPS cells. 2.
  • the relative quantity ratio of FOS and N-linked sugar chains (FOS / N-linked sugar chains) is significantly lower in ES and iPS cells. Since the relative quantity ratio of FOS and N-linked sugar chains (FOS / N-linked sugar chains) is considered to increase when protein synthesis is active in cells, the decline in stem cells is due to stem cell dormancy. It may be possible to reflect the increased state of protein synthesis of the cancer cell used as a state or comparison target.
  • GAG recognized the following characteristics. 1. In iPS cells, a high percentage of sulfate groups in HS was detected. 2. In iPS cells, there was a tendency that CS had few 6-position modifications and many 4-position modifications. 3. In iPS cells, many N-position modifications were detected in HS. 4). Among GAGs in iPS cells, the amount of CS-0S, CS-2S, CS-4S, CS-2S4S, HS-0S is p ⁇ 0.001, and the amount of HS-6S, HS-NS, HA is p ⁇ 0.001. At 0.01, it was increased compared to the amount of sugar chains in non-iPS cells.
  • HiPS-RIKEN-1A, HiPS-RIKEN-2A, HiPS-RIKEN-11A, HiPS-RIKEN-12A, HiPS-RIKEN-3C, HiPS-RIKEN-3C cells are cell lines established simultaneously with HiPS-RIKEN-1A cells from decidual tissue, but do not have the pluripotency characteristic of iPS. "A cell. TRA-1-60 and TRA-1-81, which are undifferentiated markers of human pluripotent stem cells, are not expressed, and the expression level of Nanog is very low.
  • N-linked sugar chain (FIG. 19) Expression of the following sugar chains was markedly increased in cell lines that were “incompletely converted to iPS”. (Hex) 2 (HexNAc) 3 (Fuc) 1 (NeuAc) 2+ (Man) 3 (GlcNAc) 2 (Hex) 1 (HexNAc) 3 (Fuc) 1 (NeuAc) 1+ (Man) 3 (GlcNAc) 2 In addition, the expression of the following sugar chains was remarkably reduced in cell lines in which iPS conversion was incomplete.
  • sugar chains are useful as detection markers for cells with incomplete iPS conversion, and as quality control markers that serve as indicators of the pluripotency of the produced iPS. It was done.
  • the method of the present invention can be used for evaluation and selection of pluripotent stem cells not only for ES cells and iPS cells but also for stem cells in each tissue. It can also be used to evaluate the efficiency of dedifferentiation of general stem cells and to select dedifferentiated cells.
  • Regenerative medicine foundation that evaluates whether or not undifferentiated stem cells are mixed with cells that have been induced to differentiate and cause teratomas if transplanted as they are, and reduce the risk of canceration by removing these pluripotent stem cells It can also be used as a technology.
  • it can be applied to evaluation of materials for regenerative medicine (cell suspension, cell sheet), and there is a possibility that it may be applied to the evaluation of the timing of transplantation and quality control.
  • the method of the present invention can also be applied as a basic technology in disease-specific biomarker search. .

Abstract

Cette invention concerne une nouvelle méthode pour évaluer/sélectionner une cellule souche pluripotente à une efficacité élevée et une précision élevée tout en éliminant l'apparition d'une réaction croisée comme observé dans une réaction antigène-anticorps. Dans la présente invention, une chaîne sucre se liant à N, une chaîne sucre se liant à O, une GSL, GAG, et FOS à la surface d'une cellule sont séparées et purifiées à partir d'un échantillon d'une seule cellule par un procédé de glyco-empreinte ou une méthode BET, et l'analyse quantitative/qualitative complète de ces chaînes sucre est mise en œuvre par MALDI-TOF/MS. Ainsi, des marqueurs de chaînes sucre spécifiques d'une cellule souche pluripotente peuvent être déterminés, et les marqueurs de chaînes sucre peuvent être utilisés pour identifier une cellule pluripotente.
PCT/JP2012/078651 2012-06-11 2012-11-05 Méthode de sélection d'une cellule souche pluripotente WO2013186946A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
JP2014521190A JP5979452B2 (ja) 2012-06-11 2012-11-05 多能性幹細胞の選別方法

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2012-132201 2012-06-11
JP2012132201 2012-06-11

Publications (1)

Publication Number Publication Date
WO2013186946A1 true WO2013186946A1 (fr) 2013-12-19

Family

ID=49757803

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2012/078651 WO2013186946A1 (fr) 2012-06-11 2012-11-05 Méthode de sélection d'une cellule souche pluripotente

Country Status (2)

Country Link
JP (1) JP5979452B2 (fr)
WO (1) WO2013186946A1 (fr)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016114341A1 (fr) * 2015-01-14 2016-07-21 国立大学法人京都大学 Composé pour l'identification de cellules pluripotentes
WO2018144864A1 (fr) * 2017-02-03 2018-08-09 The Mclean Hospital Corporation Méthodes de manipulation de destinée cellulaire
JP2018201477A (ja) * 2017-06-09 2018-12-27 国立大学法人北海道大学 ライソゾーム病に対する治療効果を判定するためのマーカー及び方法並びにライソゾーム病治療薬のスクリーニング方法

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008093828A1 (fr) * 2007-02-02 2008-08-07 National Institute Of Advanced Industrial Science And Technology Procede et element pour l'analyse d'une molecule sur la surface d'une cellule vivante
WO2010058605A1 (fr) * 2008-11-21 2010-05-27 国立大学法人 北海道大学 Procédé pour évaluer l'état de cellules
JP2012037416A (ja) * 2010-08-09 2012-02-23 Sumitomo Bakelite Co Ltd 間葉系幹細胞の骨分化判定用マーカーおよび間葉系幹細胞の骨分化判定方法

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU2002364143B2 (en) * 2001-12-07 2008-02-21 Asterias Biotherapeutics, Inc. Islet cells from primate pluripotent stem cells
CA2676323A1 (fr) * 2007-01-18 2008-07-24 Suomen Punainen Risti, Veripalvelu Nouveaux procedes et reactifs pour la production de cellules
MX355340B (es) * 2010-08-31 2018-04-16 Janssen Biotech Inc Diferenciación de células madre embrionarias humanas.

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008093828A1 (fr) * 2007-02-02 2008-08-07 National Institute Of Advanced Industrial Science And Technology Procede et element pour l'analyse d'une molecule sur la surface d'une cellule vivante
WO2010058605A1 (fr) * 2008-11-21 2010-05-27 国立大学法人 北海道大学 Procédé pour évaluer l'état de cellules
JP2012037416A (ja) * 2010-08-09 2012-02-23 Sumitomo Bakelite Co Ltd 間葉系幹細胞の骨分化判定用マーカーおよび間葉系幹細胞の骨分化判定方法

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
NORITAKA HASHII ET AL.: "Glycomics in Quality Control of Tissue-engineered Medical Products", JOURNAL OF THE PHARMACEUTICAL SOCIETY OF JAPAN, vol. 132, no. 4, April 2012 (2012-04-01), pages 489 - 497, XP055104616 *
TERUHIDE YAMAGUCHI: "Saisei Iryo Jitsuyoka ni Muketa Saibo Soshiki Kako Iyakuhin no Anzensei Hinshitsu To no Kakuho ni Kansuru Kiban Gijutsu Kaihatsu Kenkyu", SAISEI IRYO JITSUYOKA NI MUKETA SAIBO SOSHIKI KAKO IYAKUHIN NO ANZENSEI HINSHITSU TO NO KAKUHO NI KANSURU KIBAN GIJUTSU KAIHATSU KENKYU HEISEI 22 NENDO SOKATSU BUNTAN KENKYU HOKOKUSHO, 2011, pages 1 - 93 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016114341A1 (fr) * 2015-01-14 2016-07-21 国立大学法人京都大学 Composé pour l'identification de cellules pluripotentes
US10689624B2 (en) 2015-01-14 2020-06-23 Kyoto University Compound for identifying pluripotent cells
WO2018144864A1 (fr) * 2017-02-03 2018-08-09 The Mclean Hospital Corporation Méthodes de manipulation de destinée cellulaire
JP2018201477A (ja) * 2017-06-09 2018-12-27 国立大学法人北海道大学 ライソゾーム病に対する治療効果を判定するためのマーカー及び方法並びにライソゾーム病治療薬のスクリーニング方法

Also Published As

Publication number Publication date
JPWO2013186946A1 (ja) 2016-02-01
JP5979452B2 (ja) 2016-08-24

Similar Documents

Publication Publication Date Title
US8691774B2 (en) Carbohydrate profile compositions from human cells and methods for analysis and modification thereof
EP1913385B1 (fr) Compositions de carbohydrate tissulaire et analyse de celles-ci
US20170002095A1 (en) Cancer specific glycans and use thereof
Satomaa et al. The N-glycome of human embryonic stem cells
JP5116837B2 (ja) グリカンを評価するためのｍｓ法
US20090317834A1 (en) Novel cellular glycan compositions
US20100145032A1 (en) Novel carbohydrate profile compositions from human cells and methods for analysis and modification thereof
CA2712562A1 (fr) Nouveaux agents de liaison cellulaire specifiques
US20090162938A1 (en) Novel Carbohydrate Profile Compositions From Human Cells and Methods for Analysis and Modification Thereof
WO2007054622A1 (fr) Nouvelles compositions a profils glucidiques tirees de cellules humaines et procedes d'analyse et de modification correspondants
Furukawa et al. Glycomics of human embryonic stem cells and human induced pluripotent stem cells
Plaas et al. Keratan sulfate disaccharide composition determined by FACE analysis of keratanase II and endo-β-galactosidase digestion products
JP5979452B2 (ja) 多能性幹細胞の選別方法
Furukawa et al. Recent advances in cellular glycomic analyses
Laremore et al. Electrospray ionization Fourier transform mass spectrometric analysis of intact bikunin glycosaminoglycan from normal human plasma
Yu Methods and Tools to Analyze Glycosylation at a Systems Level
Class et al. Patent application title: CANCER SPECIFIC GLYCANS AND USE THEREOF Inventors: Tero Satomaa (Helsinki, FI) Jari Natunen (Vantaa, FI) Annamari Heiskanen (Helsinki, FI) Anne Olonen (Lahti, FI) Juhani Saarinen (Helsinki, FI) Noora Salovuori (Helsinki, FI) Jari Helin (Vantaa, FI) Assignees: GLYKOS FINLAND OY
WO2013154751A1 (fr) Procédés d'analyse des monosaccharides dérivés des glycanes
WO2019045649A1 (fr) Méthodes pour enrichir des cellules souches mésenchymateuses
CA2669475A1 (fr) Compositions de tissus a base de carbohydrates et leur analyse

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 12878797

Country of ref document: EP

Kind code of ref document: A1

DPE2 Request for preliminary examination filed before expiration of 19th month from priority date (pct application filed from 20040101)
ENP Entry into the national phase

Ref document number: 2014521190

Country of ref document: JP

Kind code of ref document: A

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 12878797

Country of ref document: EP

Kind code of ref document: A1