EP2627779A1 - Détermination de la qualité de cellules souches - Google Patents

Détermination de la qualité de cellules souches

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Publication number
EP2627779A1
EP2627779A1 EP11767256.8A EP11767256A EP2627779A1 EP 2627779 A1 EP2627779 A1 EP 2627779A1 EP 11767256 A EP11767256 A EP 11767256A EP 2627779 A1 EP2627779 A1 EP 2627779A1
Authority
EP
European Patent Office
Prior art keywords
genes
methylation
pluripotent stem
cpg
stem cell
Prior art date
Legal status (The legal status 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 status listed.)
Withdrawn
Application number
EP11767256.8A
Other languages
German (de)
English (en)
Inventor
Simeon Santourlidis
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Ghanjati Foued
Original Assignee
Ghanjati Foued
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 Ghanjati Foued filed Critical Ghanjati Foued
Priority to EP11767256.8A priority Critical patent/EP2627779A1/fr
Publication of EP2627779A1 publication Critical patent/EP2627779A1/fr
Withdrawn legal-status Critical Current

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Classifications

    • 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
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • 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
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0696—Artificially induced pluripotent stem cells, e.g. iPS
    • 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
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6881—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for tissue or cell typing, e.g. human leukocyte antigen [HLA] probes
    • 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/154—Methylation markers

Definitions

  • the present invention relates to a method for quality determination of pluripotent stem cells.
  • Object of the present invention was to provide a simple method for quality and quality determination.
  • a stem cell requires a cell-type specific gene expression. This requires a qualitatively and quantitatively adjusted gene regulation.
  • One of the key mechanisms for gene regulation is DNA methylation. It is estimated that 60% of human genes will be secreted in differentiated cells by DNA methylation. In the D NA, cytosine, which is in the context of a palpable CpG dinucleotide, can carry an additional methyl group. Such appropriately methylated genes are not expressed or greatly reduced.
  • a CpG island is fully methylated, transcription of the corresponding gene is not possible. In the case of unmethylation, the gene has transcriptional competence, i. it can be transcribed.
  • the methylatable CpG dinucleotides are not equivalent to the regulation of the gene to which they belong.
  • the methylation of certain CpGs that are associated with transcriptional factor targeting sequences exerts the greatest influence on gene expression.
  • the methylated CpGs are thought to influence transcription factor binding through their steric positioning in the major grooves of DNA. So for example, an incompletely methylated CpG island may result in a transcriptional block or decreased transcription, depending on which CpGs are methylated, or it may not have a repressive effect on transcription.
  • the DNA methylation of at least one CpG in a CpG island is analyzed in at least three genes of a pluripotent stem cell to be investigated, i. H .
  • the investigated pluripotent stem cell may be an induced pluripotent stem cell, in which case a known pluripotent stem cell is suitable as the reference cell.
  • the reference cell can also be the reference cell, for example a fibroblast cell. From the extent of the change in DNA methylation can also be concluded on the success of the induction, ie. the more similar the differentiated cell and the supposedly induced stem cell, the worse the quality of the induced stem cell.
  • Stem cells are often kept in culture for a long time. There are also stem cell lines, sometimes also from embryonic stem cells, which are kept in culture for years. It is not excluded in such cases that the In the course of cultivation, stem cells suffer damage that may go unnoticed for a long time.
  • the inventive method can be used to observe the degree of methylation of one or more CpG islands of two or more genes in the course of a cultivation and to conclude from an occurrence of differences on a change in the cultured cell. In other words, a cell that has a different DNA methylation pattern from its originally cultured cell is of lower quality.
  • genes are used to make differences clearer. According to the invention, these may be three or four or five or seven or ten or more genes.
  • genes that are located on different chromosomes are examined because this gives an improved overview of the situation of the cell.
  • genes that at least partially belong to a gene family In many cases, it will be useful to study genes that at least partially belong to a gene family. In other embodiments, it may also be useful to examine, for example, ten genes, where some genes belong to one gene family and the other genes belong to a different gene family.
  • the gene family used according to the invention is the gene family of the olfactory receptor genes (The hu man olfactory receptor gene family, Malnic B, Godfrey PA, Buck LB Proc Natl Acad Be USA 2004 Feb 24, 101 (8): Erratum in: Proc Natl Acad See USA 2004 May 4; 101 (18): 7205 and The mouse olfactory receptor gene family Godfrey PA, Malnic B, Buck LB Proc Natl Acad Sei USA 2004 Feb 17; 101 (7): 2156-61, Epub 2004 Feb 9).
  • the olfactory receptor genes are the largest gene family in the human genome (about 1,000 genes). These genes are associated with a CpG island in their 5 'region in embryonic and induced pluripotent stem cells, and this is densely methylated in both stem cell types. In contrast, the same CpG islands of the same genes are largely unmethylated in fibroblasts. Since this difference can be found multiple times in each chromosome, it represents a pronounced representation for the DNA methylation of the genome ("methylom").
  • the particular advantage of the gene family of olfactory receptor genes is inferior A n za hl distributed throughout the chromosome and dense methylation in embryonic and induced pluripotent stem cells. This makes it possible to determine the methylation at a variety of differentially methylatable genomic sites. Preferably at least 10 digits, more preferably at least 20, at least 50, at least 100 or at least 1000 digits are analyzed and used for the evaluation.
  • n is an integer representing the family, with members having a sequence identity greater than 40%;
  • X is a single letter representing a subfamily, with members having more than 60% sequence identity
  • OR1A1 is the first isoform of subfamily A of the olfactory receptor family 1.
  • receptors of the same subfamily recognize similar molecules. There are two large groups, the fish-like receptor class I with the OR families 51 to 56 and the class II (tetrapod) with the OR families 1 to 13.
  • OR2AT2P OR2AT4; OR2B2; OR2B3; OR2B4P; OR2B6; OR2B7P; OR2B8P;
  • OR2S1P OR2S2; OR2T1; OR2T2; OR2T3; OR2T4; OR2T5; OR2T6; OR2T7;
  • OR4A4P OR4A5; OR4A6P; OR4A7P; OR4A8P; OR4A9P; OR4A10P; OR4A11P;
  • OR4B2P OR4C1P; OR4C2P; OR4C3; OR4C4P; OR4C5; OR4C6; OR4C7P;
  • OR4C9P OR4C10P; OR4C11; OR4C12; OR4C13; OR4C14P; OR4C15;
  • OR4F8P OR4F13P; OR4F14P; OR4F15; OR4F16; OR4F17; OR4F21; OR4F28P;
  • OR4F29 OR4G1P; OR4G2P; OR4G3P; OR4G4P; OR4G6P; OR4G11P; OR4H6P;
  • OR5BT1P OR5C1; OR5D2P; OR5D3P; OR5D13; OR5D14; OR5D15P; OR5D16;
  • OR5G5P OR5H1; OR5H2; OR5H3P; OR5H4P; OR5H5P; OR5H6; OR5H7P;
  • OR7D4 OR7D11P; OR7E1P; OR7E2P; OR7E4P; OR7E5P; OR7E7P; OR7E8P;
  • OR7E10P OR7E11P; OR7E12P; OR7E13P; OR7E14P; OR7E15P; OR7E16P;
  • OR7E18P OR7E19P; OR7E21P; OR7E22P; OR7E23P; OR7E24; OR7E25P; OR7E26P; OR7E28P; OR7E29P; OR7E31P; OR7E33P; OR7E35P; OR7E36P;
  • OR7E85P OR7E86P; OR7E87P; OR7E89P; OR7E90P; OR7E91P; OR7E93P;
  • a method for the determination of DNA methylation a variety of methods are in use.
  • a common method is the methylation-specific PCR.
  • the methylated cytosine is converted into uracil.
  • specific primers it can be examined whether the respective sites to be examined are methylated or not.
  • the measuring method is a real-time PCR, in which labeled markers or labeled probes are used.
  • the methylation-specific PCR reactions used can in principle also be carried out in a PCR with a large number of primers. In order to increase the specificity of the PCR, it is preferred that the respective examinations are carried out separately. In many embodiments, it will be helpful to add additional controls to verify the quality of the measurement.
  • the invention also provides a method for determining the quality of a pluripotent stem cell comprising the steps
  • Figure 3 shows the DNA methylation status in the 5 ' region of Olfactory receptor genes on chromosome 11 in fibroblasts, IPS fibroblasts and ES cells (13).
  • FIG. 4 shows the DNA methylation status in the 5 ' region of olefinic receptor genes on chromosome 19 in fibroblasts, IPS fibroblasts and ES cells.
  • FIG. 5 shows the DNA methylation status in the 5 ' region of olfactory receptor genes on chromosome 17 in fibroblasts, IPS fibroblasts and ES cells (FIG. 13)
  • Figure 6 shows the DNA methylation status in the 5 ' region of chromosome 3 olfactory receptor genes in fibroblasts, IPS fibroblasts and ES cells (13).
  • Genomic DNA was isolated as follows: The genomic DNA was isolated from the cells using the Qiagen DNAeasy blood & tissue DNA isolation kit. 2. Isolation of methylated DNA
  • Genomic DNA was converted by ultrasound sonication into a fragment size of about 300 to 1,000 base pairs.
  • the methylated DNA fragments were precipitated by means of a methyl cytosine specific antibody; a Methylamp Methylated DNA Capture Kit (MeDIP from Diagenode) was used. 3. Analysis of Methylierunq
  • the hybridized arrays were scanned with a microarray scanner (molecular devices) and images were generated using the Axon Genepix software. Nimble-Scan Version 2.5 and Signal-Map Version 1.9 were used for the analysis. 4. Quality determination

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  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Analytical Chemistry (AREA)
  • Biotechnology (AREA)
  • Genetics & Genomics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biomedical Technology (AREA)
  • Immunology (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Cell Biology (AREA)
  • Physics & Mathematics (AREA)
  • Biophysics (AREA)
  • Molecular Biology (AREA)
  • Transplantation (AREA)
  • Developmental Biology & Embryology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'invention concerne un procédé de détermination de la qualité d'une cellule souche pluripotente comprenant les étapes consistant à : mesurer la méthylation de l'ADN d'au moins un CpG dans un îlot CpG dans au moins deux gènes de la cellule souche pluripotente, comparer avec la méthylation de l'ADN dudit CpG dans un îlot CpG dans au moins deux gènes d'au moins une cellule de référence, les gènes étant localisés sur différents chromosomes et faisant partie de la famille génique des gènes récepteurs olfactifs.
EP11767256.8A 2010-10-11 2011-10-11 Détermination de la qualité de cellules souches Withdrawn EP2627779A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
EP11767256.8A EP2627779A1 (fr) 2010-10-11 2011-10-11 Détermination de la qualité de cellules souches

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
EP10187137 2010-10-11
EP11767256.8A EP2627779A1 (fr) 2010-10-11 2011-10-11 Détermination de la qualité de cellules souches
PCT/EP2011/067697 WO2012049154A1 (fr) 2010-10-11 2011-10-11 Détermination de la qualité de cellules souches

Publications (1)

Publication Number Publication Date
EP2627779A1 true EP2627779A1 (fr) 2013-08-21

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Family Applications (1)

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EP11767256.8A Withdrawn EP2627779A1 (fr) 2010-10-11 2011-10-11 Détermination de la qualité de cellules souches

Country Status (4)

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US (1) US20140004512A1 (fr)
EP (1) EP2627779A1 (fr)
JP (1) JP2014500708A (fr)
WO (1) WO2012049154A1 (fr)

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016060264A1 (fr) * 2014-10-17 2016-04-21 積水メディカル株式会社 Procédé de contrôle de la qualité de cellules souches
JP6886893B2 (ja) * 2017-08-23 2021-06-16 花王株式会社 嗅覚受容体の応答測定方法

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1567644A4 (fr) * 2002-04-16 2006-04-05 Origene Technologies Inc Genes et batteries de genes specifiques de tissus
WO2007026255A2 (fr) * 2005-06-22 2007-03-08 Universitetet I Oslo Cellules dedifferenciees et procedes permettant de realiser et d'utiliser des cellules dedifferenciees
WO2010069008A1 (fr) * 2008-12-19 2010-06-24 Griffith University Cellule à compétence germinale dérivée de tissu adulte
WO2010115052A2 (fr) * 2009-04-03 2010-10-07 The Mclean Hospital Corporation Cellules souches pluripotentes induites

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
None *
See also references of WO2012049154A1 *

Also Published As

Publication number Publication date
US20140004512A1 (en) 2014-01-02
WO2012049154A1 (fr) 2012-04-19
WO2012049154A8 (fr) 2013-04-18
JP2014500708A (ja) 2014-01-16

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