EP2627779A1 - Quality determination of stem cells - Google Patents

Quality determination of stem cells

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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
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Prior art keywords
genes
methylation
pluripotent stem
cpg
stem cell
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EP11767256.8A
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German (de)
French (fr)
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Simeon Santourlidis
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Ghanjati Foued
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Ghanjati Foued
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Priority to EP11767256.8A priority Critical patent/EP2627779A1/en
Publication of EP2627779A1 publication Critical patent/EP2627779A1/en
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    • 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

Abstract

The invention relates to a method for determining the quality of a pluripotent stem cell, comprising the following steps: measuring the DNA methylation of at least one CpG in a CpG island in at least two genes of the pluripotent stem cell, comparing it to the DNA methylation of the at least one CpG in a CpG island in the at least two genes of at least one reference cell, wherein the genes are located on different chromosomes and belong to the gene family of olfactory receptor genes.

Description

Qualitätsbestimmunq von Stammzellen  Quality determination of stem cells
Die vorliegende Erfindung betrifft ein Verfahren zur Qualitätsbestimmung von pluripotenten Stammzellen. The present invention relates to a method for quality determination of pluripotent stem cells.
In der regenerativen Medizin werden sehr intensiv vielfältige Ansätze verfolgt, um Stammzellen in zellersatztherapeutischer Anwendung den, unter degenerativer Erkrankung leidenden Patienten, verabreichen zu können. Bereits jetzt erfolgen Therapien in diversen Kliniken unter Verwendung von Stammzellen, die der Knorpelregeneration dienen . In absehbarer Zukunft wird sich die Anwendung von Stammzellen potenzieren. Hierzu wird die Methode der Reprogrammierung von körpereigenen, terminal differenzierten Zellen mittels Induktion von Pluripotenz beitragen. Hierdurch werden sowohl Probleme der Im m unabstoßung als auch ethische Aspekte umgangen. In regenerative medicine, very intensive approaches are pursued in order to be able to administer stem cells in cell replacement therapy application to patients suffering from degenerative disease. Therapies are already taking place in various clinics using stem cells that are used for cartilage regeneration. In the foreseeable future, the use of stem cells will increase. To this end, the method of reprogramming of endogenous, terminally differentiated cells by means of induction of pluripotency will contribute. This avoids both problems of imputation and ethical aspects.
Bei allen existierenden Ansätzen erfordert dies jedoch die Zellkultivierung in- vitro, wobei keine einheitlichen Standards existieren . Für eine Anwendung beim Menschen ist es erforderlich, die Güte- bzw. Qualität der Stammzellen zu bestimmen. However, in all of the existing approaches, this requires cell cultivation in vitro, with no uniform standards. For human use, it is necessary to determine the quality of the stem cells.
Chuanying Pan et al., J. Genet. Genomics 37 (2010) 241-248 untersuchten die Demethylierung der Promotoren von NANOG und OCT4 in induzierten pluripo- tenten Stammzellen von Fibroblasten. Chuanying Pan et al., J. Genet. Genomics 37 (2010) 241-248 investigated the demethylation of the promoters of NANOG and OCT4 in induced pluripotent stem cells of fibroblasts.
Prashant Mali et al., Stern Cells 28 (2010) 713-720 untersuchen die Demethylierung von induzierten pluripotenten Stammzellen mit Hilfe eines HumanMethylation27 BeadChips. Prashant Mali et al., Star Cells 28 (2010) 713-720 investigate the demethylation of induced pluripotent stem cells using a HumanMethylation27 beadchip.
Aufgabe der vorliegenden Erfindung war es, ein einfaches Verfahren zur Güte- und Qualitätsbestimmung bereitzustellen. Object of the present invention was to provide a simple method for quality and quality determination.
Gelöst wi rd die Aufgabe du rch ein Verfahren zur Qualitätsbestimmung von pluripotenten Stammzellen umfassend die Schritte: - Messen der DNA-Methylierung mindestens eines CpG in einer CpG-Insel in mindestens drei Genen der pluripotenten Stammzelle The task is solved by a method for determining the quality of pluripotent stem cells comprising the steps: Measuring the DNA methylation of at least one CpG in a CpG island in at least three genes of the pluripotent stem cell
- Vergleich mit der DNA-Methylierung des mindestens einen CpG in einer CpG-Insel in den mindestens drei Genen mindestens einer Referenzzelle wobei die Gene auf verschiedenen Chromosomen lokalisiert sind und zur Genfamilie der olfaktorischen Rezeptorgene gehören. Comparison with the DNA methylation of the at least one CpG in a CpG island in the at least three genes of at least one reference cell wherein the genes are located on different chromosomes and belong to the gene family of the olfactory receptor genes.
Die Ausprägung eines speziellen Zelltyps z. B. einer Stammzelle erfordert eine zelltypenspezifische Genexpression. Diese setzt eine qualitativ und quantitativ angepasste Genregulation voraus. Einer der wesentlichen Mechanismen zur Genregulation ist die DNA- Methylierung. Schätzungsweise 60% der menschlichen Gene werden in differenzierten Zel len du rch D NA-Methylierung beei nfl usst . I n der D NA kann Cytosi n, welches sich im Kontext eines pali ndrom ischen CpG Dinukleotids befi ndet, ei ne zusätzl iche Methylg ru ppe tragen . Solche entsprechend methylierten Gene werden nicht oder stark vermindert exprimiert. The expression of a special cell type z. B. 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.
Die Methylierung ist in den Genen aber nicht genomweit gleichmäßig verteilt, sondern liegt in sogenannten CpG-Inseln vermehrt vor, die in den 5' Regionen der Gene liegen. However, the methylation is not uniformly distributed in the genes throughout the genome, but is increasingly present in so-called CpG islands, which are located in the 5 'regions of the genes.
Wenn eine CpG-Insel lückenlos methyliert ist, ist eine Transkription des kor- respondierenden Gens nicht möglich . Im Falle einer Unmethylierung hat das Gen transkriptioneile Kompetenz, d.h. es kann transkribiert werden. If 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.
Hierbei sind die methylierbaren CpG Dinukleotide jedoch nicht gleichwertig für die Regulation des Gens, dem sie angehören . Die Methylierung von bestimmten CpGs, die über ihre Assoziation mit Zielsequenzen von Transkriptionsfakto- ren wi rken, üben den größten Ei nfl uss auf die Genexpression aus. Es wird vermutet, dass die methylierten CpGs über ihre sterische Positionierung in die großen Furchen der DNA die Transkriptionsfaktorbindung beeinflussen. So kann eine nicht lückenlos methylierte CpG-Insel, je nachdem welche, CpGs methyliert sind, einen Transkriptionsblock oder eine verminderte Transkription zur Folge haben, es kann aber auch gar keinen reprimierenden Einfluss auf die Transkription geben. Erfindungsgemäß wird die DNA-Methylierung mindestens eines CpG in einer CpG-Insel in mindestens drei Genen einer zu untersuchenden pluripotenten Stammzelle analysiert, d . h . es werden Gene ausgewählt und an denen von diesen Genen bekannten CpG-Inseln wird an mindestens einer Position der CpG-Insel die Methylierung gemessen . Bevorzugt wird eine Position in einer CpG-Insel gemessen, die für die Transkriptionssteuerung möglichst relevant ist. Anschließend erfolgt ein Vergleich dieser Methylierung mit der Methylierung einer Referenzzelle, wobei bei den gleichen Genen die gleichen CpG- Inseln und die gleichen Positionen innerhalb der CpG-Inseln der Referenzzelle verglichen werden. In einer Ausführungsform der Erfindung kann es sich bei der untersuchten pluripotenten Stammzelle um eine induzierte pluripotente Stammzelle handeln, dabei eignet sich dann als Referenzzelle eine bekannte pluripotente Stammzelle. However, 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. According to the invention, 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 . genes are selected and methylation is measured on CpG islands known by these genes at at least one position of the CpG island. Preferably, a position in a CpG island is measured that is as relevant as possible for the transcription control. Subsequently, a comparison of this methylation with the methylation of a reference cell, wherein the same genes the same CpG islands and the same positions within the CpG islands of the reference cell are compared. In one embodiment of the invention, 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.
Weisen die untersuchte Stammzelle und die Referenzzelle ähnliche oder über- ei nsti mmende Methylierungsmuster auf, spricht dies für eine erfolgreiche Reprogrammierung und damit eine hohe Qualität der erhaltenen induzierten, pluripotenten Stammzelle. Zusätzlich oder alternativ kann als Referenzzelle auch die Ausgangszelle eingesetzt werden, beispielsweise eine Fibroblastenzel- le. Aus dem Umfang der Änderung der DNA-Methylierung kann ebenfalls auf den Erfolg der Induktion geschlossen werden, d .h . je ähnlicher die ausdifferenzierte Zelle und die vermeintlich induzierte Stammzelle sind, desto schlechter ist die Qualität der induzierten Stammzelle. If the investigated stem cell and the reference cell have similar or superior methylation patterns, this indicates a successful reprogramming and thus a high quality of the induced, pluripotent stem cell obtained. Additionally or alternatively, 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.
Stammzellen werden häufig längere Zeit in Kultur gehalten. Es gibt Stammzelll inien, tei lweise auch aus embryonalen Stammzellen, die über Jahre in Kultur gehalten werden. Es ist in solchen Fällen nicht auszuschließen, dass die Stammzellen im Rahmen der Kultivierung Schäden erleiden, die über lange Zeit unbemerkt bleiben können. Hierbei kann das erfindungsgemäße Verfahren eingesetzt werden, um im Laufe einer Kultivierung den Methylierungsgrad einer oder mehrerer CpG-Inseln von zwei oder mehr Genen zu beobachten und aus einem Auftreten von Unterschieden auf eine Veränderung der kultivierten Zelle zu schließen. Mit anderen Worten, eine Zelle, die gegenüber ihrer ursprünglich kultivierten Zel le ei n a bweichendes D NA-Methylierungsmuster aufweist, ist qualitativ weniger hochwertig. 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. In this case, 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.
Es wird erfindungsgemäß bevorzugt, dass nicht nur die DNA-Methylierung von drei Genen bestimmt wird, sondern mehr Gene eingesetzt werden, um Unterschiede deutlicher zu machen . Erfindungsgemäß kann es sich dabei um drei oder vier oder fünf oder sieben oder zehn oder mehr Gene handeln. It is preferred according to the invention that not only the DNA methylation of three genes is determined, but more 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.
Um einen repräsentativen Eindruck des Methyloms zu erhalten werden Gene, die auf verschiedenen Chromosomen lokalisiert sind untersucht, weil dies ei- nen verbesserten Überblick über die Situation der Zelle gibt. In order to obtain a representative impression of the methylome, genes that are located on different chromosomes are examined because this gives an improved overview of the situation of the cell.
In vielen Fällen wird es sinnvoll sein, Gene zu untersuchen, die zumindest teilweise einer Genfamilie angehören. In anderen Ausführungsformen kann es auch sinnvoll sein, beispielsweise zehn Gene zu untersuchen, wobei einige Gene zu einer Genfamilie gehören und die anderen Gene zu einer anderen Genfamilie gehören. 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.
Wichtig für das erfindungsgemäße Verfahren ist, dass immer nur der Methylierungsgrad identischer Positionen in identischen CpG-Inseln von identischen Genen miteinander verglichen werden können. It is important for the method according to the invention that only the degree of methylation of identical positions in identical CpG islands of identical genes can always be compared with one another.
Erfindungsgemäß ist es bevorzugt, dass an jeder der gemessenen CpG-Inseln nicht nur eine einzelne Position, sondern mehrere Positionen analysiert werden oder bei Vorhandensein mehrerer CpG-Inseln mehrere Positionen in mehreren CpG-Inseln. Die Genfamilie, die erfindungsgemäß eingesetzt wird, ist die Genfamilie der olfaktorischen Rezeptor-Gene (The h u man olfactory receptor gene family, Malnic B, Godfrey PA, Buck LB. Proc Natl Acad Sei U S A. 2004 Feb 24; 101(8) : 2584-9. Erratum in : Proc Natl Acad Sei U S A. 2004 May 4; 101(18) : 7205 und The mouse olfactory receptor gene family. Godfrey PA, Malnic B, Buck LB. Proc Natl Acad Sei U S A. 2004 Feb 17; 101(7) : 2156-61. Epub 2004 Feb 9). Die olfaktorischen Rezeptor-Gene sind die größte Genfamilie im humanen Genom (ca. 1.000 Gene). Diese Gene sind in embryonalen und in induzierten pluripotenten Stammzellen in ihrem 5' Bereich mit einer CpG- Insel assoziiert und diese ist in beiden Stammzelltypen dicht methyliert. Hingegen sind dieselben CpG-Inseln derselben Gene in Fibroblasten weitestgehend unmethyliert. Da dieser Unterschied in jedem Chromosom mehrfach zu finden ist, stellt er eine ausgeprägte Repräsentanz für die DNA-Methylierung des Genoms ("Methylom") dar. Der besondere Vorteil der Genfamilie der olfak- torischen Rezeptor-Ge n e l i egt i n i h re r g ro ße n A n za h l , d i e ü be r a l l e Chromosonen verteilt sind und der dichten Methylierung bei embryonalen und induzierten pluripotenten Stammzellen. Damit ist es möglich, die Methylierung an einer Vielzahl von differentiell methylierbaren genomischen Stellen zu ermitteln. Bevorzugt werden mindestens 10 Stellen, mehr bevorzugt mindestens 20, mindestens 50, mindestens 100 oder mindestens 1000 Stellen analysiert und für die Bewertung herangezogen. According to the invention, it is preferable that not only a single position, but several positions are analyzed on each of the measured CpG islands, or multiple positions in multiple CpG islands in the presence of multiple CpG islands. 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.
Die Nomenklatur der Gene lautet "ORnXm", wobei The nomenclature of genes is "ORnXm", where
"OR" für die Olfactory Receptor Superfamily steht; "OR" stands for the Olfactory Receptor Superfamily;
"n" eine ganze Zahl ist, die die Familie repräsentiert, wobei die Mitglieder eine Sequenzidentität von mehr als 40% aufweisen; "n" is an integer representing the family, with members having a sequence identity greater than 40%;
"X" ein einzelner Buchstabe ist, der eine Unterfamilie repräsentiert, wobei die Mitglieder mehr als 60% Sequenzidentität haben und "X" is a single letter representing a subfamily, with members having more than 60% sequence identity and
"m" eine ganze Zahl ist, die ein individuelles Familienmitglied bezeichnet. So ist beispielsweise OR1A1 die erste Isoform der Subfamilie A der olfaktorischen Rezeptorfamilie 1. "m" is an integer that designates an individual family member. For example, OR1A1 is the first isoform of subfamily A of the olfactory receptor family 1.
Es wird davon ausgegangen, dass Rezeptoren der gleichen Subfamilie ähnliche Moleküle erkennen. Es existieren zwei große Gruppen, die Klasse I (fish-like receptor) mit den OR- Familien 51 bis 56 und die Klasse II (tetrapod) mit den OR-Familien 1 bis 13. It is believed that 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.
Bevorzugt verwendete Rezeptorgene sind folgende: Preferred receptor genes are the following:
OR1A1; OR1A2; OR1AA1P; OR1AB1P; OR1AC1P; OR1B1; OR1C1; OR1D2; OR1A1; OR1A2; OR1AA1P; OR1AB1P; OR1AC1P; OR1B1; OR1C1; OR1D2;
OR1D3P; OR1D4; OR1D5; OR1E1; OR1E2; OR1E3; OR1F1; OR1F2P; OR1F12; OR1G1; OR1H1P; OR1I1; OR1J1; OR1J2; OR1J4; OR1K1; OR1L1; OR1L3;OR1D3P; OR1D4; OR1D5; OR1E1; OR1E2; OR1E3; OR1F1; OR1F2P; OR1F12; OR1G1; OR1H1P; OR1I1; OR1J1; OR1J2; OR1J4; OR1K1; OR1L1; OR1L3;
OR1L4; OR1L6; OR1L8; OR1M1; OR1M4P; OR1N1; OR1N2; OR1P1; OR1Q1;OR1L4; OR1L6; OR1L8; OR1M1; OR1M4P; OR1N1; OR1N2; OR1P1; OR1Q1;
OR1R1P; OR1S1; OR1S2; OR1X1P; OR1X5P; OR2A1; OR2A2; OR2A3P;OR1R1P; OR1S1; OR1S2; OR1X1P; OR1X5P; OR2A1; OR2A2; OR2A3P;
OR2A4; OR2A5; OR2A7; OR2A9P; OR2A12; OR2A13P; OR2A14; OR2A15P;OR2A4; OR2A5; OR2A7; OR2A9P; OR2A12; OR2A13P; OR2A14; OR2A15P;
OR2A20P; OR2A25; OR2A41P; OR2A42; OR2AD1P; OR2AE1; OR2AF1P; OR2AG1; OR2AG2; OR2AH1P; OR2AI1P; OR2AJ1; OR2AK2; OR2AL1P;OR2A20P; OR2A25; OR2A41P; OR2A42; OR2AD1P; OR2AE1; OR2AF1P; OR2AG1; OR2AG2; OR2AH1P; OR2AI1P; OR2AJ1; OR2AK2; OR2AL1P;
OR2AM1P; OR2A01P; OR2AP1; OR2AQ1P; OR2AS1P; OR2AS2P; OR2AT1P;OR2AM1P; OR2A01P; OR2AP1; OR2AQ1P; OR2AS1P; OR2AS2P; OR2AT1P;
OR2AT2P; OR2AT4; OR2B2; OR2B3; OR2B4P; OR2B6; OR2B7P; OR2B8P;OR2AT2P; OR2AT4; OR2B2; OR2B3; OR2B4P; OR2B6; OR2B7P; OR2B8P;
OR2B11; OR2BH1P; OR2C1; OR2C3; OR2D2; OR2D3; OR2E1P; OR2F1;OR2B11; OR2BH1P; OR2C1; OR2C3; OR2D2; OR2D3; OR2E1P; OR2F1;
OR2F2; OR2G1P; OR2G2; OR2G3; OR2G6; OR2H1; OR2H2; OR2H4P; OR2H5P; OR2I1P; OR2J1; OR2J2; OR2J3; OR2J4P; OR2K2; OR2L1P; OR2L2;OR2F2; OR2G1P; OR2G2; OR2G3; OR2G6; OR2H1; OR2H2; OR2H4P; OR2H5P; OR2I1P; OR2J1; OR2J2; OR2J3; OR2J4P; OR2K2; OR2L1P; OR2L2;
OR2L3; OR2L5; OR2L6P; OR2L8; OR2L9P; OR2L13; OR2M1P; OR2M2;OR2L3; OR2L5; OR2L6P; OR2L8; OR2L9P; OR2L13; OR2M1P; OR2M2;
OR2M3; OR2M4; OR2M5; OR2M7; OR2N1P; OR2P1P; OR2Q1P; OR2R1P;OR2M3; OR2M4; OR2M5; OR2M7; OR2N1P; OR2P1P; OR2Q1P; OR2R1P;
OR2S1P; OR2S2; OR2T1; OR2T2; OR2T3; OR2T4; OR2T5; OR2T6; OR2T7;OR2S1P; OR2S2; OR2T1; OR2T2; OR2T3; OR2T4; OR2T5; OR2T6; OR2T7;
OR2T8; OR2T10; OR2T11; OR2T12; OR2T27; OR2T29; OR2T32P; OR2T33; OR2T34; OR2T35; OR2U1P; OR2U2P; OR2V1; OR2V2; OR2W1; OR2W2P;OR2T8; OR2T10; OR2T11; OR2T12; OR2T27; OR2T29; OR2T32P; OR2T33; OR2T34; OR2T35; OR2U1P; OR2U2P; OR2V1; OR2V2; OR2W1; OR2W2P;
OR2W3; OR2W4P; OR2W5; OR2W6P; OR2X1P; OR2Y1; OR2Z1; OR3A1;OR2W3; OR2W4P; OR2W5; OR2W6P; OR2X1P; OR2Y1; OR2Z1; OR3A1;
OR3A2; OR3A3; OR3A4P; OR3B1P; OR3D1P; OR4A1P; OR4A2P; OR4A3P;OR3A2; OR3A3; OR3A4P; OR3B1P; OR3D1P; OR4A1P; OR4A2P; OR4A3P;
OR4A4P; OR4A5; OR4A6P; OR4A7P; OR4A8P; OR4A9P; OR4A10P; OR4A11P;OR4A4P; OR4A5; OR4A6P; OR4A7P; OR4A8P; OR4A9P; OR4A10P; OR4A11P;
OR4A12P; OR4A13P; OR4A14P; OR4A15; OR4A16; OR4A17P; OR4A18P; OR4A19P; OR4A21P; OR4A40P; OR4A41P; OR4A42P; OR4A43P; OR4A44P;OR4A12P; OR4A13P; OR4A14P; OR4A15; OR4A16; OR4A17P; OR4A18P; OR4A19P; OR4A21P; OR4A40P; OR4A41P; OR4A42P; OR4A43P; OR4A44P;
OR4A45P; OR4A46P; OR4A47; OR4A48P; OR4A49P; OR4A50P; OR4B1;OR4A45P; OR4A46P; OR4A47; OR4A48P; OR4A49P; OR4A50P; OR4B1;
OR4B2P; OR4C1P; OR4C2P; OR4C3; OR4C4P; OR4C5; OR4C6; OR4C7P;OR4B2P; OR4C1P; OR4C2P; OR4C3; OR4C4P; OR4C5; OR4C6; OR4C7P;
OR4C9P; OR4C10P; OR4C11; OR4C12; OR4C13; OR4C14P; OR4C15;OR4C9P; OR4C10P; OR4C11; OR4C12; OR4C13; OR4C14P; OR4C15;
OR4C16; OR4C45; OR4C46; OR4C48P; OR4C49P; OR4C50P; OR4D1; OR4D2; OR4D5; OR4D6; OR4D7P; OR4D8P; OR4D9; OR4D10; OR4D11; OR4D12P;OR4C16; OR4C45; OR4C46; OR4C48P; OR4C49P; OR4C50P; OR4D1; OR4D2; OR4D5; OR4D6; OR4D7P; OR4D8P; OR4D9; OR4D10; OR4D11; OR4D12P;
OR4E1; OR4E2; OR4F1P; OR4F2P; OR4F3; OR4F4; OR4F5; OR4F6; OR4F7P;OR4E1; OR4E2; OR4F1P; OR4F2P; OR4F3; OR4F4; OR4F5; OR4F6; OR4F7P;
OR4F8P; OR4F13P; OR4F14P; OR4F15; OR4F16; OR4F17; OR4F21; OR4F28P;OR4F8P; OR4F13P; OR4F14P; OR4F15; OR4F16; OR4F17; OR4F21; OR4F28P;
OR4F29; OR4G1P; OR4G2P; OR4G3P; OR4G4P; OR4G6P; OR4G11P; OR4H6P;OR4F29; OR4G1P; OR4G2P; OR4G3P; OR4G4P; OR4G6P; OR4G11P; OR4H6P;
OR4H12P; OR4K1; OR4K2; OR4K3; OR4K4P; OR4K5; OR4K6P; OR4K7P; OR4K8P; OR4K11P; OR4K12P; OR4K13; OR4K14; OR4K15; OR4K16P; OR4K17; OR4L1; OR4M1; OR4M2; OR4N1P; OR4N2; OR4N3P; OR4N4;OR4H12P; OR4K1; OR4K2; OR4K3; OR4K4P; OR4K5; OR4K6P; OR4K7P; OR4K8P; OR4K11P; OR4K12P; OR4K13; OR4K14; OR4K15; OR4K16P; OR4K17; OR4L1; OR4M1; OR4M2; OR4N1P; OR4N2; OR4N3P; OR4N4;
OR4N5; OR4P1P; OR4P4; OR4Q1P; OR4Q2; OR4Q3; OR4R1P; OR4R2P;OR4N5; OR4P1P; OR4P4; OR4Q1P; OR4Q2; OR4Q3; OR4R1P; OR4R2P;
OR4R3P; OR4S1; OR4S2; OR4T1P; OR4U1P; OR4V1P; OR4W1P; OR4X1;OR4R3P; OR4S1; OR4S2; OR4T1P; OR4U1P; OR4V1P; OR4W1P; OR4X1;
OR4X2; OR4X7P; OR5A1; OR5A2; OR5AC1; OR5AC2; OR5AC4P; OR5AH1P; OR5AK1P; OR5AK2; OR5AK3P; OR5AK4P; OR5AL1; OR5AL2P; OR5AM1P;OR4X2; OR4X7P; OR5A1; OR5A2; OR5AC1; OR5AC2; OR5AC4P; OR5AH1P; OR5AK1P; OR5AK2; OR5AK3P; OR5AK4P; OR5AL1; OR5AL2P; OR5AM1P;
OR5AN1; OR5AN2P; OR5A01P; OR5AP1P; OR5AP2; OR5AQ1P; OR5AR1;OR5AN1; OR5AN2P; OR5A01P; OR5AP1P; OR5AP2; OR5AQ1P; OR5AR1;
OR5AS1; OR5AU1; OR5AW1P; OR5AZ1P; OR5B1P; OR5B2; OR5B3; OR5B10P;OR5AS1; OR5AU1; OR5AW1P; OR5AZ1P; OR5B1P; OR5B2; OR5B3; OR5B10P;
OR5B12; OR5B15P; OR5B17; OR5B19P; OR5B21; OR5BA1P; OR5BB1P;OR5B12; OR5B15P; OR5B17; OR5B19P; OR5B21; OR5BA1P; OR5BB1P;
OR5BC1P; OR5BD1P; OR5BE1P; OR5BH1P; OR5BJ1P; OR5BK1P; OR5BL1P; OR5BM1P; OR5BN1P; OR5BN2P; OR5BP1P; OR5BQ1P; OR5BR1P; OR5BS1P;OR5BC1P; OR5BD1P; OR5BE1P; OR5BH1P; OR5BJ1P; OR5BK1P; OR5BL1P; OR5BM1P; OR5BN1P; OR5BN2P; OR5BP1P; OR5BQ1P; OR5BR1P; OR5BS1P;
OR5BT1P; OR5C1; OR5D2P; OR5D3P; OR5D13; OR5D14; OR5D15P; OR5D16;OR5BT1P; OR5C1; OR5D2P; OR5D3P; OR5D13; OR5D14; OR5D15P; OR5D16;
OR5D17P; OR5D18; OR5E1P; OR5F1; OR5F2P; OR5G1P; OR5G3; OR5G4P;OR5D17P; OR5D18; OR5E1P; OR5F1; OR5F2P; OR5G1P; OR5G3; OR5G4P;
OR5G5P; OR5H1; OR5H2; OR5H3P; OR5H4P; OR5H5P; OR5H6; OR5H7P;OR5G5P; OR5H1; OR5H2; OR5H3P; OR5H4P; OR5H5P; OR5H6; OR5H7P;
OR5H8P; OR5H14; OR5H15; OR5I1; OR5J1P; OR5J2; OR5J7P; OR5K1; OR5K2; OR5K3; OR5K4; OR5L1; OR5L2; OR5M1; OR5M2P; OR5M3; OR5M4P;OR5H8P; OR5H14; OR5H15; OR5I1; OR5J1P; OR5J2; OR5J7P; OR5K1; OR5K2; OR5K3; OR5K4; OR5L1; OR5L2; OR5M1; OR5M2P; OR5M3; OR5M4P;
OR5M5P; OR5M6P; OR5M7P; OR5M8; OR5M9; OR5M10; OR5M11; OR5M12P;OR5M5P; OR5M6P; OR5M7P; OR5M8; OR5M9; OR5M10; OR5M11; OR5M12P;
OR5M13P; OR5M14P; OR5P1P; OR5P2; OR5P3; OR5P4P; OR5R1; OR5S1P;OR5M13P; OR5M14P; OR5P1P; OR5P2; OR5P3; OR5P4P; OR5R1; OR5S1P;
OR5T1; OR5T2; OR5T3; OR5V1; OR5W1P; OR5W2; OR6A2; OR6B1; OR6B2;OR5T1; OR5T2; OR5T3; OR5V1; OR5W1P; OR5W2; OR6A2; OR6B1; OR6B2;
OR6B3; OR6C1; OR6C2; OR6C3; OR6C4; OR6C5P; OR6C6; OR6C7P; OR6C64P; OR6C65; OR6C66P; OR6C68; OR6C69P; OR6C70; OR6C71P;OR6B3; OR6C1; OR6C2; OR6C3; OR6C4; OR6C5P; OR6C6; OR6C7P; OR6C64P; OR6C65; OR6C66P; OR6C68; OR6C69P; OR6C70; OR6C71P;
OR6C72P; OR6C73P; OR6C74; OR6C75; OR6C76; OR6D1P; OR6E1P; OR6F1;OR6C72P; OR6C73P; OR6C74; OR6C75; OR6C76; OR6D1P; OR6E1P; OR6F1;
OR6J1; OR6K1P; OR6K2; OR6K3; OR6K4P; OR6K5P; OR6K6; OR6L1P;OR6J1; OR6K1P; OR6K2; OR6K3; OR6K4P; OR6K5P; OR6K6; OR6L1P;
OR6L2P; OR6M1; OR6M2P; OR6M3P; OR6N1; OR6N2; OR6P1; OR6Q1;OR6L2P; OR6M1; OR6M2P; OR6M3P; OR6N1; OR6N2; OR6P1; OR6Q1;
OR6R1P; OR6R2P; OR6S1; OR6T1; OR6U2P; OR6V1; OR6W1P; OR6X1; OR6Y1; OR7A1P; OR7A2P; OR7A3P; OR7A5; OR7A8P; OR7A10; OR7A11P;OR6R1P; OR6R2P; OR6S1; OR6T1; OR6U2P; OR6V1; OR6W1P; OR6X1; OR6Y1; OR7A1P; OR7A2P; OR7A3P; OR7A5; OR7A8P; OR7A10; OR7A11P;
OR7A15P; OR7A17; OR7A18P; OR7A19P; OR7C1; OR7C2; OR7D1P; OR7D2;OR7A15P; OR7A17; OR7A18P; OR7A19P; OR7C1; OR7C2; OR7D1P; OR7D2;
OR7D4; OR7D11P; OR7E1P; OR7E2P; OR7E4P; OR7E5P; OR7E7P; OR7E8P;OR7D4; OR7D11P; OR7E1P; OR7E2P; OR7E4P; OR7E5P; OR7E7P; OR7E8P;
OR7E10P; OR7E11P; OR7E12P; OR7E13P; OR7E14P; OR7E15P; OR7E16P;OR7E10P; OR7E11P; OR7E12P; OR7E13P; OR7E14P; OR7E15P; OR7E16P;
OR7E18P; OR7E19P; OR7E21P; OR7E22P; OR7E23P; OR7E24; OR7E25P; OR7E26P; OR7E28P; OR7E29P; OR7E31P; OR7E33P; OR7E35P; OR7E36P;OR7E18P; OR7E19P; OR7E21P; OR7E22P; OR7E23P; OR7E24; OR7E25P; OR7E26P; OR7E28P; OR7E29P; OR7E31P; OR7E33P; OR7E35P; OR7E36P;
OR7E37P; OR7E38P; OR7E39P; OR7E41P; OR7E43P; OR7E46P; OR7E47P;OR7E37P; OR7E38P; OR7E39P; OR7E41P; OR7E43P; OR7E46P; OR7E47P;
OR7E53P; OR7E55P; OR7E59P; OR7E62P; OR7E66P; OR7E83P; OR7E84P;OR7E53P; OR7E55P; OR7E59P; OR7E62P; OR7E66P; OR7E83P; OR7E84P;
OR7E85P; OR7E86P; OR7E87P; OR7E89P; OR7E90P; OR7E91P; OR7E93P;OR7E85P; OR7E86P; OR7E87P; OR7E89P; OR7E90P; OR7E91P; OR7E93P;
OR7E94P; OR7E96P; OR7E97P; OR7E99P; OR7E100P; OR7E101P; OR7E102P; OR7E104P; OR7E105P; OR7E106P; OR7E108P; OR7E109P; OR7E110P;OR7E94P; OR7E96P; OR7E97P; OR7E99P; OR7E100P; OR7E101P; OR7E102P; OR7E104P; OR7E105P; OR7E106P; OR7E108P; OR7E109P; OR7E110P;
OR7E111P; OR7E115P; OR7E116P; OR7E117P; OR7E121P; OR7E122P;OR7E111P; OR7E115P; OR7E116P; OR7E117P; OR7E121P; OR7E122P;
OR7E125P; OR7E126P; OR7E128P; OR7E129P; OR7E130P; OR7E136P;OR7E125P; OR7E126P; OR7E128P; OR7E129P; OR7E130P; OR7E136P;
OR7E140P; OR7E145P; OR7E148P; OR7E149P; OR7E154P; OR7E155P;OR7E140P; OR7E145P; OR7E148P; OR7E149P; OR7E154P; OR7E155P;
OR7E156P; OR7E157P; OR7E158P; OR7E159P; OR7E160P; OR7E161P; OR7E162P; OR7G1; OR7G2; OR7G3; OR7G15P; OR7H1P; OR7H2P; OR7K1P;OR7E156P; OR7E157P; OR7E158P; OR7E159P; OR7E160P; OR7E161P; OR7E162P; OR7G1; OR7G2; OR7G3; OR7G15P; OR7H1P; OR7H2P; OR7K1P;
OR7L1P; OR7M1P; OR8A1; OR8A2P; OR8A3P; OR8B1P; OR8B2; OR8B3;OR7L1P; OR7M1P; OR8A1; OR8A2P; OR8A3P; OR8B1P; OR8B2; OR8B3;
OR8B4; OR8B5P; OR8B6P; OR8B7P; OR8B8; OR8B9P; OR8B10P; OR8B12;OR8B4; OR8B5P; OR8B6P; OR8B7P; OR8B8; OR8B9P; OR8B10P; OR8B12;
OR8C1P; OR8D1; OR8D2; OR8D4; OR8F1P; OR8G1; OR8G2; OR8G3P;OR8C1P; OR8D1; OR8D2; OR8D4; OR8F1P; OR8G1; OR8G2; OR8G3P;
OR8G5; OR8G7P; OR8H1; OR8H2; OR8H3; OR8I1P; OR8I2; OR8I4P; OR8J1; OR8J2; OR8J3; OR8K1; OR8K2P; OR8K3; OR8K4P; OR8K5; OR8L1P; OR8Q1P;OR8G5; OR8G7P; OR8H1; OR8H2; OR8H3; OR8I1P; OR8I2; OR8I4P; OR8J1; OR8J2; OR8J3; OR8K1; OR8K2P; OR8K3; OR8K4P; OR8K5; OR8L1P; OR8Q1P;
OR8R1P; OR8S1; OR8S21P; OR8T1P; OR8U1; OR8U8; OR8U9; OR8V1P;OR8R1P; OR8S1; OR8S21P; OR8T1P; OR8U1; OR8U8; OR8U9; OR8V1P;
OR8X1P; OR9A1P; OR9A2; OR9A3P; OR9A4; OR9G1; OR9G2P; OR9G3P;OR8X1P; OR9A1P; OR9A2; OR9A3P; OR9A4; OR9G1; OR9G2P; OR9G3P;
OR9G4; OR9G9; OR9H1P; OR9I1; OR9I2P; OR9I3P; OR9K1P; OR9K2; OR9L1P; OR9M1P; OR9N1P; OR9P1P; OR9Q1; OR9Q2; OR9R1P; OR9S24P OR10A2; OR10A3; OR10A4; OR10A5; OR10A6; OR10A7; OR10AA1P OR10AB1P; OR10AC1P; OR10AD1; OR10AE1P; OR10AE3P; OR10AF1P OR10AG1; OR10AH1P; OR10AK1P; OR10B1P; OR10C1; OR10D1P; OR10D3 OR10D4P; OR10D5P; OR10G1P; OR10G2; OR10G3; OR10G4; OR10G5P OR10G6; OR10G7; OR10G8; OR10G9; OR10H1; OR10H2; OR10H3; OR10H4 OR10H5; OR10J1; OR10J2P; OR10J3; OR10J4; OR10J5; OR10J6P; OR10J7P OR10J8P; OR10J9P; OR10K1; OR10K2; OR10N1P; OR10P1; OR10Q1 OR10Q2P; OR10R1P; OR10R2; OR10R3P; OR10S1; OR10T1P; OR10T2 OR10U1P; OR10V1; OR10V2P; OR10V3P; OR10V7P; OR10W1; OR10X1 OR10Y1P; OR10Z1; OR11A1; OR11G1P; OR11G2; OR11H1; OR11H2 OR11H3P; OR11H4; OR11H5P; OR11H6; OR11H7; OR11H12; OR11H13P OR11I1P; OR11J1P; OR11J2P; OR11J5P; OR11K1P; OR11K2P; OR11L1 OR11M1P; OR11N1P; OR11P1P; OR11Q1P; OR12D1P; OR12D2; OR12D3 OR13A1; OR13C1P; OR13C2; OR13C3; OR13C4; OR13C5; OR13C6P, OR13C7P; OR13C8; OR13C9; OR13D1; OR13D2P; OR13D3P; OR13E1P OR13F1; OR13G1; OR13H1; OR13I1P; OR13J1; OR13K1P; OR13Z1P OR13Z2P; OR13Z3P; OR14A2; OR14A16; OR14C36; OR14I1; OR14J1 OR14K1; OR14L1P; OR51A1P; OR51A2; OR51A3P; OR51A4; OR51A5P OR51A6P; OR51A7; OR51A8P; OR51A9P; OR51A10P; OR51AB1P; OR51B2 OR51B3P; OR51B4; OR51B5; OR51B6; OR51B8P; OR51C1P; OR51C4P OR51D1; OR51E1; OR51E2; OR51F1; OR51F2; OR51F3P; OR51F4P; OR51F5P OR51G1; OR51G2; OR51H1P; OR51H2P; OR51I1; OR51I2; OR51J1, OR51K1P; OR51L1; OR51M1; OR51N1P; OR51P1P; OR51Q1; OR51R1P OR51S1; OR51T1; OR51V1; OR52A1; OR52A4; OR52A5; OR52B1P; OR52B2 OR52B3P; OR52B4; OR52B5P; OR52B6; OR52D1; OR52E1; OR52E2 OR52E3P; OR52E4; OR52E5; OR52E6; OR52E7P; OR52E8; OR52H1 OR52H2P; OR52I1; OR52I2; OR52J1P; OR52J2P; OR52J3; OR52K1; OR52K2 OR52K3P; OR52L1; OR52L2P; OR52M1; OR52M2P; OR52N1; OR52N2 OR52N3P; OR52N4; OR52N5; OR52P1P; OR52P2P; OR52Q1P; OR52R1, OR52S1P; OR52T1P; OR52U1P; OR52V1P; OR52W1; OR52X1P; OR52Y1P OR52Z1; OR55B1P; OR56A1; OR56A3; OR56A4; OR56A5; OR56A7P; OR56B1; OR56B2P; OR56B3P; OR56B4. OR9G4; OR9G9; OR9H1P; OR9I1; OR9I2P; OR9I3P; OR9K1P; OR9K2; OR9L1P; OR9M1P; OR9N1P; OR9P1P; OR9Q1; OR9Q2; OR9R1P; OR9S24P OR10A2; OR10A3; OR10A4; OR10A5; OR10A6; OR10A7; OR10AA1P OR10AB1P; OR10AC1P; OR10AD1; OR10AE1P; OR10AE3P; OR10AF1P OR10AG1; OR10AH1P; OR10AK1P; OR10B1P; OR10C1; OR10D1P; OR10D3 OR10D4P; OR10D5P; OR10G1P; OR10G2; OR10G3; OR10G4; OR10G5P OR10G6; OR10G7; OR10G8; OR10G9; OR10H1; OR10H2; OR10H3; OR10H4 OR10H5; OR10J1; OR10J2P; OR10J3; OR10J4; OR10J5; OR10J6P; OR10J7P OR10J8P; OR10J9P; OR10K1; OR10K2; OR10N1P; OR10P1; OR10Q1 OR10Q2P; OR10R1P; OR10R2; OR10R3P; OR10S1; OR10T1P; OR10T2 OR10U1P; OR10V1; OR10V2P; OR10V3P; OR10V7P; OR10W1; OR10X1 OR10Y1P; OR10Z1; OR11A1; OR11G1P; OR11G2; OR11H1; OR11H2 OR11H3P; OR11H4; OR11H5P; OR11H6; OR11H7; OR11H12; OR11H13P OR11I1P; OR11J1P; OR11J2P; OR11J5P; OR11K1P; OR11K2P; OR11L1 OR11M1P; OR11N1P; OR11P1P; OR11Q1P; OR12D1P; OR12D2; OR12D3 OR13A1; OR13C1P; OR13C2; OR13C3; OR13C4; OR13C5; OR13C6P, OR13C7P; OR13C8; OR13C9; OR13D1; OR13D2P; OR13D3P; OR13E1P OR13F1; OR13G1; OR13H1; OR13I1P; OR13J1; OR13K1P; OR13Z1P OR13Z2P; OR13Z3P; OR14A2; OR14A16; OR14C36; OR14I1; OR14J1 OR14K1; OR14L1P; OR51A1P; OR51A2; OR51A3P; OR51A4; OR51A5P OR51A6P; OR51A7; OR51A8P; OR51A9P; OR51A10P; OR51AB1P; OR51B2 OR51B3P; OR51B4; OR51B5; OR51B6; OR51B8P; OR51C1P; OR51C4P OR51D1; OR51E1; OR51E2; OR51F1; OR51F2; OR51F3P; OR51F4P; OR51F5P OR51G1; OR51G2; OR51H1P; OR51H2P; OR51I1; OR51I2; OR51J1, OR51K1P; OR51L1; OR51M1; OR51N1P; OR51P1P; OR51Q1; OR51R1P OR51S1; OR51T1; OR51V1; OR52A1; OR52A4; OR52A5; OR52B1P; OR52B2 OR52B3P; OR52B4; OR52B5P; OR52B6; OR52D1; OR52E1; OR52E2 OR52E3P; OR52E4; OR52E5; OR52E6; OR52E7P; OR52E8; OR52H1 OR52H2P; OR52I1; OR52I2; OR52J1P; OR52J2P; OR52J3; OR52K1; OR52K2 OR52K3P; OR52L1; OR52L2P; OR52M1; OR52M2P; OR52N1; OR52N2 OR52N3P; OR52N4; OR52N5; OR52P1P; OR52P2P; OR52Q1P; OR52R1 , OR52S1P; OR52T1P; OR52U1P; OR52V1P; OR52W1; OR52X1P; OR52Y1P OR52Z1; OR55B1P; OR56A1; OR56A3; OR56A4; OR56A5; OR56A7P; OR56B1; OR56B2P; OR56B3P; OR56B4.
Als Verfahren zur Bestimmung der DNA-Methylierung sind verschiedenste Verfahren gebräuchlich. Ein gebräuchliches Verfahren ist die methylierungs- spezifische PCR. Hierbei wird durch Einsatz von Bisulfit das methylierte Cytosin in Uracil umgewandelt. Durch die Verwendung spezifischer Primer kann untersucht werden, ob die jeweils zu untersuchenden Stellen methyliert sind oder nicht. Das Messverfahren ist dabei eine Real Time-PCR, bei der markierte Marker oder markierte Sonden eingesetzt werden.  As a method for the determination of DNA methylation a variety of methods are in use. A common method is the methylation-specific PCR. By using bisulfite, the methylated cytosine is converted into uracil. By using 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.
Ein alternatives Verfahren zur Bestimmung ist das sogenannte Nimble-Gene der Firma Roche. Bei diesem Verfahren werden DNA-Fragmente mit Hilfe von 5'-Methylcytidinspezifischen Antikörpern ausgefällt, isoliert und nach Amplifi- kation auf einen Array detektiert. An alternative method of determination is the so-called Nimble gene from Roche. In this method, DNA fragments with the help of 5'-methylcytidine-specific antibodies precipitated, isolated and detected after amplification on an array.
Auch andere Verfahren beispielsweise durch radioaktive Markierung, durch Southern Blot o.ä. sind möglich. Bei der Analyse mehrerer CpG-Inseln oder mehrerer Positionen in einer CpG- Insel oder mehrerer Gene können die dafür verwendeten methylierungsspezi- fischen PCR-Reaktionen grundsätzlich auch in einer PCR mit einer Vielzahl von Primern durchgeführt werden. Um die Spezifität der PCR zu erhöhen, ist es bevorzugt, dass die jeweiligen Untersuchungen getrennt durchgeführt werden. In vielen Ausführungsformen wird es hilfreich sein, zusätzlich Kontrollen einzufügen, um die Qualität der Messung zu überprüfen. Other methods, for example by radioactive labeling, by Southern Blot or the like. are possible. In the analysis of several CpG islands or multiple positions in one CpG island or multiple genes, 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.
Da sich zwischen den Messungen Unterschiede ergeben können, wird es ebenfalls häufig sinnvoll sein, die Referenzzelle und die Stammzelle zeitgleich i n zwei Reaktionen zu prüfen. In vielen Fällen wird es aber auch genügen, für die Methylierung der Stammzellen auf frühere Messungen und entsprechend gespeicherte Daten zurückzugreifen. Since differences may arise between the measurements, it will also often be useful to test the reference cell and the stem cell simultaneously in two reactions. In many cases, however, it will also be sufficient to resort to previous measurements and correspondingly stored data for the methylation of the stem cells.
Gegenstand der Erfindung ist auch ein Verfahren zur Qualitätsbestimmung einer pluripotenten Stammzelle umfassend die Schritte The invention also provides a method for determining the quality of a pluripotent stem cell comprising the steps
- Messen der DNA-Methylierung mindestens eines CpG in einer CpG-Insel in mindestens zwei Genen der pluripotenten Stammzelle  Measuring the DNA methylation of at least one CpG in a CpG island in at least two genes of the pluripotent stem cell
- Vergleich mit der DNA-Methylierung des mindestens einen CpG in einer CpG-Insel in den mindestens zwei Genen mindestens einer Referenzzelle.  Comparison with the DNA methylation of the at least one CpG in a CpG island in the at least two genes of at least one reference cell.
Figur 1 zeigt den DNA Methylierungsstatus im 5 ' Bereich von je drei Olfaktorische Rezeptorgenen auf drei verschiedenen Chromosomen in Fibroblasten, IPSFibroblasten und ES Zellen (13). Figur 2 zeigt den DNA Methylierungsstatus im 5' Bereich von Olfaktorische Rezeptorgenen auf Chromosom 1 in Fibroblasten, IPSFibroblasten und ES Zellen (13) FIG. 1 shows the DNA methylation status in the 5 ' range of three olfactory receptor genes on three different chromosomes in fibroblasts, IPS fibroblasts and ES cells (13). Figure 2 shows DNA methylation status in the 5 ' region of chromosome 1 olfactory receptor genes in fibroblasts, IPS fibroblasts and ES cells (13).
Figur 3 zeigt den DNA Methylierungsstatus im 5' Bereich von Olfaktorische Rezeptorgenen auf Chromosom 11 in Fibroblasten, IPSFibroblasten und ES Zellen (13) 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).
Figur 4 zeigt den DNA Methylierungsstatus im 5' Bereich von Olfaktorische Rezeptorgenen auf Chromosom 19 in Fibroblasten, IPSFibroblasten und ES Zellen (13) Figur 5 zeigt den DNA Methylierungsstatus im 5' Bereich von Olfaktorische Rezeptorgenen auf Chromosom 17 in Fibroblasten, IPSFibroblasten und ES Zellen (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. (13) 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)
Figur 6 zeigt den DNA Methylierungsstatus im 5' Bereich von Olfaktorische Rezeptorgenen auf Chromosom 3 in Fibroblasten, IPSFibroblasten und ES Zellen (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).
Die Erfindung wird durch die nachfolgenden Beispiele näher erläutert: 1. Isolierung qenomischer DNA The invention is further illustrated by the following examples: 1. Isolation of qenomic DNA
Genomische DNA wurde wie folgt isoliert: Die genomische DNA wurde mittels des Qiagen DNAeasy blood&tissue DNA isolations kit aus den Zellen isoliert. 2. Isolierung von methylierter DNA  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
genomischer DNA wurde durch Ultraschallsonifizierung in eine Fragmentgröße von etwa 300 bis 1.000 Basenpaare überführt. Die methylierten DNA-Fragmente wurden mittels eines methylcytosinspezifischen Antikörpers präzipitiert; verwendet wurde ein Methylamp Methylated DNA Capture Kit (MeDIP der Firma Diagenode). 3. Analyse der Methylierunq 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
Die Präzipitate wurden auf einem Nimble-Gene 385K Ref. Seq. Promoter Array HG18 hybridisiert (Firma Roche). Auf diesem Array befinden sich, kovalent gebunden, die Promoterregion aller bekannten CpG dinukleotidreichen Gene in Form von 50mer Oligonukleotidproben.  The precipitates were assayed on a Nimble Gene 385K Ref. Seq. Promoter Array HG18 hybridized (Roche). Covalently bound to this array are the promoter region of all known CpG dinucleotide-rich genes in the form of 50-mer oligonucleotide probes.
Die hybridisierten Arrays wurden mit einem Microarray-Scanner (molecular devices) gescannt und Bilder mit der Axon Genepix-Software generiert. Zur Analyse wurden Nimble-Scan Version 2.5 und Signal-Map Version 1.9 eingesetzt. 4. Oualitätsbestimmunq 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
Eingesetzt wurden (a) Zellen aus Fibroblasten, (b) pluripotenten Stammzellen, die aus Fibroblasten durch retroviralen Transfer der vier Transkriptionsfaktoren, Oct3/4, Sox2, c-Myc, and Klf4 induziert wurden (analog : Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Takahashi K, Yamanaka S. Cell. 2006 Aug 25; 126(4) : 663-76. Epub 2006 Aug 10 sowie (c) eine bekannte embryonale Zelllinie 1-3. Analysiert wurde die CpG-Methylierung auf olfaktorischen Rezeptorgenen verschiedener Chromosomen. Die Ergebnisse der Analysen sind in den Figuren 1 bis 6 dargestellt.  (A) cells from fibroblasts, (b) pluripotent stem cells induced from fibroblasts by retroviral transfer of the four transcription factors, Oct3 / 4, Sox2, c-Myc, and Klf4 (analogue: Induction of pluripotent stem cells from mouse embryonic Takahashi K, Yamanaka S. Cell, 2006 Aug 25; 126 (4): 663-76, Epub 2006 Aug 10, and (c) A Known Embryonic Cell Line 1-3. Methylation on olfactory receptor genes of different chromosomes The results of the analyzes are shown in FIGS.

Claims

Patentansprüche claims
Verfahren zur Qualitätsbestimmung einer pluripotenten Stammzelle umfassend die Schritte Method for determining the quality of a pluripotent stem cell comprising the steps
- Messen der DNA-Methylierung mindestens eines CpG in einer CpG-Insel in mindestens drei Genen der pluripotenten Stammzelle  Measuring the DNA methylation of at least one CpG in a CpG island in at least three genes of the pluripotent stem cell
- Vergleich mit der DNA-Methylierung des mindestens einen CpG in einer CpG-Insel in den mindestens drei Genen mindestens einer Referenzzelle wobei die Gene auf verschiedenen Chromosomen lokalisiert sind und zur Genfamilie der olfaktorischen Rezeptorgene gehören.  Comparison with the DNA methylation of the at least one CpG in a CpG island in the at least three genes of at least one reference cell wherein the genes are located on different chromosomes and belong to the gene family of the olfactory receptor genes.
Verfahren nach Anspruch 1, wobei die pluripotente Stammzelle eine induzierte pluripotente Stammzelle ist. The method of claim 1, wherein the pluripotent stem cell is an induced pluripotent stem cell.
Verfahren nach Anspruch 1 oder 2, wobei die Referenzzelle eine pluripotente Stammzelle ist. The method of claim 1 or 2, wherein the reference cell is a pluripotent stem cell.
Verfahren nach mindestens einem der Ansprüche 1 bis 3, wobei die Referenzzelle eine differenzierte Zelle ist. The method of any one of claims 1 to 3, wherein the reference cell is a differentiated cell.
Verfahren nach mindestens einem der Ansprüche 1 bis 4, wobei mindestens zwei Referenzzellen eingesetzt werden. Method according to at least one of claims 1 to 4, wherein at least two reference cells are used.
Verfahren nach mindestens einem der Ansprüche 1 bis 5, wobei die Methy- lierung von jeweils mindestens einer CpG-Insel in mindestens vier oder mindestens fünf Genen der pluripotenten Stammzelle gemessen und jeweils mit der Methylierung in Genen der Referenzzellen verglichen wird. The method according to at least one of claims 1 to 5, wherein the methylation of at least one CpG island in at least four or at least five genes of the pluripotent stem cell is measured and in each case compared with the methylation in genes of the reference cells.
Verfahren nach einem der Ansprüche 1 bis 6, wobei die olfaktorischen Rezeptorgene humane Mitglieder der Klasse I oder II sind. A method according to any one of claims 1 to 6, wherein the olfactory receptor genes are human members of class I or II.
Verfahren nach mindestens einem der Ansprüche 1 bis 7, wobei mehrere CpG Inseln in jedem Gen vermessen werden. The method of any one of claims 1 to 7, wherein multiple CpG islands in each gene are measured.
9. Verfahren nach mindestens einem der Ansprüche 1 bis 8, wobei die Methy- lierung von jeweils mindestens einer CpG-Insel in mindestens sieben oder mindestens zehn Genen der pluripotenten Stammzelle gemessen und mit jeweils mit der Methylierung in Genen der Referenzzellen verglichen wird. 10. Verfahren nach mindestens einem der Ansprüche 1 bis 9, wobei die olfaktorischen Rezeptorgene ausgewählt wurden aus OR6K6, OR6N 1, OR6N2, OR51A7, OR51G2, OR51G1, OR1M 1, OR7G2, OR7G1. 9. The method according to at least one of claims 1 to 8, wherein the methylation of each at least one CpG island in at least seven or at least ten genes of the pluripotent stem cell is measured and compared with each with the methylation in genes of the reference cells. 10. The method according to at least one of claims 1 to 9, wherein the olfactory receptor genes were selected from OR6K6, OR6N 1, OR6N2, OR51A7, OR51G2, OR51G1, OR1M 1, OR7G2, OR7G1.
11. Verfahren nach einem der Ansprüche 1 bis 10, wobei zur Messung der DNA-Methylierung eine methylierungsspezifische PCR eingesetzt wird. 12. Verfahren nach Anspruch 11, wobei methylierte Cytosine mit Bisulfit in Uracil umgewandelt werden. 11. The method according to any one of claims 1 to 10, wherein a methylation-specific PCR is used to measure the DNA methylation. 12. The method of claim 11, wherein methylated cytosines are converted with bisulfite into uracil.
13. Verfahren nach einem der Ansprüche 11 oder 12, wobei Real-Time PCR mit markierten Markern oder markierten Sonden eingesetzt wird. 13. The method according to any one of claims 11 or 12, wherein real-time PCR is used with labeled markers or labeled probes.
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