JP6692028B2 - Stem cell quality control method - Google Patents

Description

  The present invention relates to a stem cell quality control method using detection of methylated DNA. More specifically, after the differentiated cells are subjected to the reprogramming treatment, the methylation state of DNA extracted from the cells derived from the reprogrammed cells is analyzed, and the reprogramming of the cells at the gene level is performed. Relates to a method of determining the success or failure of.

  Since pluripotent stem cells have the ability to be differentiated into various tissues of the living body (pluripotency), their application to drug discovery screening, disease mechanism research, and regenerative medicine materials has been studied. In particular, induced pluripotent stem cells (hereinafter, also referred to as iPS cells) derived from cells are promising as regenerative medical materials because they have no ethical problems.

  As a method for identifying stem cells, a morphological identification method by microscopic observation, a method of staining a cell surface marker with an antibody, a method of dyeing cells using an enzymatic activity such as alkaline phosphatase activity, and the like are known. There is. Patent Document 1 discloses a method of determining the degree of differentiation of pluripotent stem cells by analyzing the flatness of cultured cells with a microscope.

  When pluripotent stem cells are subcultured, the properties of the cells are gradually changed. Examples of changes in the properties of cells include changes in differentiation tendency with respect to differentiation-inducing operations, changes in cell growth rate, changes in resistance to canceration, and the like. It is known that changes in cell properties associated with these subcultures may occur while morphological changes in cells are not observed. Therefore, conventional stem cell identification methods may not be able to detect changes in cell properties.

  It is said that the above-mentioned changes in cell properties associated with subculture are due to epigenetic changes in genes, particularly changes in the methylation modification state of DNA. A method for evaluating stem cells using the methylation modification state of DNA or its expression profile as an index has been proposed. For example, Patent Document 2 discloses a method for evaluating characteristics such as pluripotency of cells by comparing and analyzing a non-coding RNA profile characteristic of each cell with a reference profile. Patent Document 3 discloses a method of selectively staining only undifferentiated stem cells and evaluating the differentiated state of the stem cells by using a probe that binds to a sugar chain specific to the undifferentiated cells. .. Patent Document 4 discloses that chromosomal DNA extracted from a test stem cell is analyzed for methylation and demethylation patterns by detecting methylated cytosine and hydroxymethylated cytosine and comparing them with a reference stem cell. Disclosed is a method of identifying stem cells, which associates with the stage of stem cell passage or differentiation.

  On the other hand, as an already established method for analyzing the methylation state of DNA, there is a method using a bisulfite (bisulfite, bisulfite, bisulfite) reaction. This method is the most widely used method for the analysis of methylated DNA. When single-stranded DNA is treated with bisulfite, cytosine is converted to uracil through sulfonation, hydrodeamination, and desulfonation. On the other hand, methylated cytosine has a very slow reaction rate of sulfonation that occurs first, and therefore remains methylated cytosine during the reaction time of the actual bisulfite treatment. Therefore, when PCR (Polymerase Chain Reaction) is carried out using DNA treated with bisulfite, methylated cytosine remains as cytosine, but unmethylated cytosine is amplified by replacing uracil with thymine. The methylation state is analyzed by utilizing the difference in the bases of cytosine and thymine generated in the sequence of this PCR amplification product. The method widely used based on this is the method described in Patent Document 5 and Non-Patent Document 1, the methylation-specific PCR method (methylation-specific PCR analysis method), and the bisulfite described in Non-Patent Documents 2 and 3. It is a sequence method.

  The methylation-specific PCR analysis method is carried out by sequentially performing PCR amplification using a methylated sequence-specific primer and a non-methylated sequence-specific primer after a bisulfite treatment of DNA, and agarose gel electrophoresis. This is a method of determining the DNA methylation state of the target region based on the presence or absence. Although it is a methylated DNA analysis method that is still widely used in the point that quantitative analysis of methylated DNA can be performed without a special device, it takes time and effort to use the electrophoresis method for the analysis. There were challenges.

  In the bisulfite sequencing method, after bisulfite treatment of DNA, PCR amplification using a primer common to methylated DNA and unmethylated DNA is performed, and the obtained PCR product is cloned into TOPO-cloning vector (Invitrogen) or the like. The presence or absence of methylation is detected by sequencing. Although it has an advantage that the methylation state of all CpG sequences in the amplified region can be known, it may not be suitable for analysis of a large number of samples because labor is required for analysis. Further, for example, in the case of analyzing the DNA methylation rate of a hemimethylated DNA or a gene having a different DNA methylation state by allele using the bisulfite sequence method, in order to accurately calculate the DNA methylation rate, a large number of The clone needs to be sequenced. That is, when analyzing changes in the methylation state of DNA by using the bisulfite sequence method, a large number of samples must be analyzed, and there was a problem that a very large amount of time and labor were required. ..

  Ion exchange chromatography is widely used in fields such as biochemistry and medicine as a method for easily and accurately separating and analyzing biopolymers such as nucleic acids, proteins, and polysaccharides in a short time. For example, when a PCR amplification product of a nucleic acid is separated using ion exchange chromatography, anion exchange chromatography is generally used that separates by utilizing the negative charge of phosphate contained in a nucleic acid molecule. An anion exchange chromatography column packed with a column packing having a cationic functional group as an ion exchange group has already been marketed and used in various research fields.

  Furthermore, by ion-exchange chromatography using a column packed with a column packing material having both a strong cationic group and a weak cationic group as a cationic functional group, a single base between 20 mer non-methylated synthetic oligonucleotides was detected. It has been reported that the difference can be separated and analyzed, and that the PCR amplification product after bisulfite treatment can be separated and analyzed due to the difference in DNA methylation rate (Patent Documents 6 and 7).

International Publication No. 2014/041935 pamphlet International Publication No. 2012/025709 Pamphlet International Publication No. 2013/128914 Pamphlet International Publication No. 2014/112655 Pamphlet US Pat. No. 5,786,146 International Publication No. 2012/108516 Pamphlet International Publication No. 2014/136930 Pamphlet

Proc. Natl. Acad. Sci. USA, 93, pp. 9821-9826 (1996) Clark, S.M. J. et al. , Nucleic acids Res 22, pp. 2990-2997 (1994) New genetic engineering handbook revised 4th edition (edited by Masami Muramatsu and Masaru Yamamoto), Yodosha, pp. 99-106, 2003

  As described above, as methods for evaluating the quality of iPS cells, miRNA analysis, detection of cell surface markers, image analysis of cell morphology, epigenome analysis and the like are known. However, a method for evaluating epigenetic changes in iPS cells, particularly changes in the methylation state of DNA, rapidly, simply, and with high throughput has not been realized.

  An object of the present invention is to provide a method for analyzing the quality of iPS cells at a gene level quickly, simply and with high throughput.

  The present inventors separated the PCR-amplified product obtained by treating the DNA from the reprogrammed cells with bisulfite and then amplifying by PCR by ion exchange chromatography. It was found that the retention times of the resulting signal peaks differ between reprogrammed iPS cells and non-reprogrammed somatic cells. By the method using the above PCR amplification and ion exchange chromatography, it is possible to detect a change in DNA methylation associated with cell reprogramming.

  Furthermore, the method using the above PCR amplification and ion exchange chromatography determines whether or not cell reprogramming is successful; whether or not the undifferentiated state of pluripotent stem cells (iPS cells, embryonic stem cells, etc.) is maintained; and It is possible to determine whether or not undifferentiated cells are mixed in a somatic cell culture differentiated from pluripotent stem cells.

Therefore, the present invention provides the following:
[1] A quality control method for induced pluripotent stem cells, which comprises:
Treating genomic DNA prepared from reprogrammed cells or cultures thereof with bisulfite;
PCR amplifying the bisulfite treated DNA with a primer set that amplifies the DNA of the CpG region of the demethylated gene;
Subjecting the resulting PCR amplification product to ion exchange chromatography to obtain a detection signal; and
When the retention time of the peak of the detection signal obtained from the reprogrammed cells or its culture is longer than the retention time of the peak of the control detection signal, the reprogrammed cells are artificially engineered. Determining that the cells are pluripotent stem cells,
Here, the detection signal of the control was obtained by treating genomic DNA prepared from uninitialized cells or cultures thereof with bisulfite, and then PCR-amplifying using the primer set. Is a detection signal obtained by subjecting the PCR amplification product to ion exchange chromatography,
Including the method.
[2] A quality control method for induced pluripotent stem cells, which comprises:
Treating genomic DNA prepared from reprogrammed cells or cultures thereof with bisulfite;
PCR-amplifying the bisulfite-treated DNA with a primer set that amplifies the DNA in the CpG region of the methylated gene;
Subjecting the resulting PCR amplification product to ion exchange chromatography to obtain a detection signal; and
When the retention time of the peak of the detection signal obtained from the reprogrammed cells or the culture thereof is shorter than the retention time of the peak of the control detection signal, the reprogrammed cells are artificially engineered. Determining that the cells are pluripotent stem cells,
Here, the detection signal of the control was obtained by treating genomic DNA prepared from uninitialized cells or cultures thereof with bisulfite, and then PCR-amplifying using the primer set. Is a detection signal obtained by subjecting the PCR amplification product to ion exchange chromatography,
Including the method.
[3] A quality control method for induced pluripotent stem cells, which comprises:
(1-1) treating genomic DNA prepared from reprogrammed cells or a culture thereof with bisulfite;
(1-2) treating genomic DNA prepared from uninitialized cells or a culture thereof with bisulfite;
(2-1) PCR-amplifying the bisulfite-treated DNA obtained in step (1-1) by using a primer set that amplifies the DNA in the CpG region of the demethylation gene;
(2-2) PCR amplification of the bisulfite-treated DNA obtained in step (1-1) by using a primer set that amplifies the DNA in the CpG region of the methylation gene;
(2-3) PCR-amplifying the bisulfite-treated DNA obtained in step (1-2) using a primer set that amplifies the DNA in the CpG region of the demethylated gene;
(2-4) PCR amplification of the bisulfite-treated DNA obtained in step (1-2) using a primer set that amplifies the DNA in the CpG region of the methylated gene;
(3-1) subjecting the PCR amplification product obtained in step (2-1) to ion exchange chromatography to obtain a detection signal;
(3-2) subjecting the PCR amplification product obtained in step (2-2) to ion exchange chromatography to obtain a detection signal;
(3-3) subjecting the PCR amplification product obtained in step (2-3) to ion exchange chromatography to obtain a detection signal;
(3-4) subjecting the PCR amplification product obtained in step (2-4) to ion exchange chromatography to obtain a detection signal;
(4) The retention time of the peak of the detection signal obtained in step (3-1) is longer than the retention time of the peak of the detection signal obtained in step (3-3), and -2) when the retention time of the peak of the detection signal obtained in step (3-4) is shorter than the retention time of the peak of the detection signal obtained in step (3-4), Determining that the cells are induced pluripotent stem cells,
Including the method.
[4] A method for controlling the quality of induced pluripotent stem cells, which comprises:
(1-1) treating genomic DNA prepared from reprogrammed cells or a culture thereof with bisulfite;
(1-2) treating genomic DNA prepared from induced pluripotent stem cells or a culture thereof with bisulfite;
(2-1) A primer set for amplifying the DNA of the CpG region of the demethylation gene, and / or the DNA of the CpG region of the methylation gene, which is treated with the bisulfite salt obtained in the step (1-1), PCR amplification using a primer set that amplifies DNA;
(2-2) A primer set for amplifying the DNA of the CpG region of the demethylation gene, and / or the DNA treated with the bisulfite salt obtained in the step (1-2) of the CpG region of the methylation gene PCR amplification using a primer set that amplifies DNA;
(3-1) Step (2-1) subjecting the obtained PCR amplification product to ion exchange chromatography to obtain a detection signal;
(3-2) Step (2-2) subjecting the obtained PCR amplification product to ion exchange chromatography to obtain a detection signal;
(4) The reprogrammed cells when there is no difference in peak retention time between the detection signal obtained in step (3-1) and the detection signal obtained in step (3-2) Is determined to be induced pluripotent stem cells,
Including the method.
[5] The method according to [1], [3] or [4], wherein the demethylated gene is at least one selected from the group consisting of NANOG, Oct3 / 4, RAB25, SALL4 and SLC22A3.
[6] The method according to [2], [3] or [4], wherein the methylated gene is at least one selected from the group consisting of SP100 and UBE1L.
[7] The primer set for amplifying the DNA in the CpG region of the demethylation gene comprises a primer consisting of SEQ ID NOs: 1 and 2, a primer consisting of SEQ ID NOs: 3 and 4, a primer consisting of SEQ ID NOs: 5 and 6, SEQ ID NO: 7 The method according to [1], [3] or [4], which is a set of the primer consisting of SEQ.
[8] A primer set for amplifying the DNA in the CpG region of the methylated gene comprises a primer consisting of SEQ ID NOs: 9 and 10, a primer consisting of SEQ ID NOs: 17 and 10, a primer consisting of SEQ ID NOs: 11 and 12, or SEQ ID NO: 18 The method according to [2], [3] or [4], which is a set of primers consisting of
[9] The method according to any one of [1] to [8], wherein the initialized cells are initialized skin fibroblasts.

  The present invention enables rapid, simple, and high-throughput quality evaluation of pluripotent stem cells by measuring the methylation state of DNA extracted from cells. According to the present invention, whether iPS cells have been established by inducing reprogramming of differentiated somatic cells such as skin fibroblasts to iPS cells, pluripotency of established stem cells, or differentiation state of differentiated cells. Etc. can be determined at the gene level. The present invention analyzes the epigenetic state of cells, which cannot be evaluated only by conventional morphological observation and expression analysis of undifferentiated markers by detecting the difference in methylation state of chromosomal DNA.

HPLC chromatogram of PCR amplification products in the NANOG gene region of iPS cells and human skin fibroblasts. HPLC chromatogram of PCR amplification products in the Oct3 / 4 gene region of iPS cells and human skin fibroblasts. HPLC chromatogram of PCR amplification products in the RAB25 gene region of iPS cells and human skin fibroblasts. HPLC chromatogram of PCR amplification products in the SALL4 gene region of iPS cells and human skin fibroblasts. HPLC chromatogram of PCR amplification products in the SP100 gene region of iPS cells and human skin fibroblasts. HPLC chromatogram of PCR amplification products in the UBE1L gene region of iPS cells and human skin fibroblasts. HPLC chromatogram of PCR amplification products in the SLC22A3 gene region of iPS cells and human skin fibroblasts. HPLC chromatograms of PCR amplification products with different primers in the Oct3 / 4 gene region of iPS cells and human skin fibroblasts. The figure which shows the methylation analysis result of the CpG site of NANOG, Oct3 / 4, RAB25, and SALL4 gene region by a bisulfite sequence method. Black circles: methylated CpG sites, open circles: non-methylated CpG sites, the numbers below each population represent the methylation rate (%). The figure which shows the methylation analysis result of CpG site of SP100 and UBE1L gene region by the bisulfite sequence method. Black circles: methylated CpG sites, open circles: non-methylated CpG sites, the numbers below each population represent the methylation rate (%).

  In the present specification, the “stem cell” has the ability to differentiate into a specialized cell other than self (may be referred to as “maturation”) (differentiation capacity) and the same property as self even after cell division. A cell that has the ability to generate cells (self-renewal ability). Stem cells include highly pluripotent stem cells such as embryonic stem cells (ES cells) and less pluripotent stem cells such as adult stem cells. As a method of culturing stem cells, a method of culturing adherent cells in a culture container is generally used, but is not limited to this.

  As used herein, "pluripotent" means the ability to differentiate into any of the three germ layers: endoderm, mesoderm, or ectoderm. As used herein, “pluripotent cell” refers to a cell that is capable of differentiating into cells belonging to any of the three germ layers, including direct progeny of totipotent cells, embryonic stem cells, adult stem cells, and artificial stem cells. An example is pluripotent stem cells.

  In the present specification, the “undifferentiated state” means a state having both the differentiation ability and the self-renewal ability which are characteristics of stem cells. However, the daughter cells are considered to be differentiated when the characteristics of the cells change, such as the difference in the differentiation ability between the parent cells and the daughter cells. The "treatment for maintaining an undifferentiated state" is a treatment for maintaining the differentiation ability and the self-renewal ability without loss, and is not particularly limited, and for example, bFGF (basic Fibroblast) in a stem cell culture medium is used. Processing such as addition of Growth Factor) or LIF (Leukemia Inhibitory Factor).

  In the present specification, the term “induced pluripotent stem cell” is a somatic cell originally, and is capable of differentiating into a cell having all the properties of three germ layers by pluripotency induction treatment (pluripotency). , Means a cell that has also become capable of self-renewal. Induced pluripotent stem cells are generally called iPS cells. Further, “reprogramming treatment” (also synonymous with “pluripotency inducing treatment”) is a treatment for imparting pluripotency and self-renewal ability. For example, four genes of Sox2, Oct3 / 4, Klf4, and Myc The processing such as introduction can be exemplified, but the processing is not limited thereto.

  In the present specification, “differentiation” means that a relatively undifferentiated cell such as a stem cell is changed and specialized, and the “differentiated cell” means the specialized cell. "Differentiation" includes various stages, from totipotent cells to less pluripotent stem cells to terminally differentiated cells. Examples of the "differentiated cell" include somatic cells of a living body, cells obtained by inducing differentiation of stem cells to differentiate in a tissue-specific manner, and the like. “Differentiation induction” refers to specialization of relatively undifferentiated cells such as stem cells. Examples of the method for inducing differentiation include adding growth factors such as BMP (Bone Morphogenic Protein) and Wnt to the cell culture medium, adding differentiation inducing factors such as retinoic acid to the cell culture medium, or introducing a gene such as MyoD. However, the present invention is not limited to these.

  In the present specification, "reprogramming" is used synonymously with induction of pluripotency, and eliminates and reconstitutes epigenetic markers such as DNA methylation so that the differentiation state of a cell is unresolved. It means to be in a differentiated state. For example, an example of cell reprogramming is the induction of pluripotent stem cells from cells that do not have pluripotency. The term “dedifferentiation” used herein means to bring a certain cell into a more immature state. Dedifferentiation may be the return of a cell to the initial or developing state in which it has differentiated, and it can differentiate into cells of other germ layers from cells that cannot generate cells other than the germ layer type to which they belong. It may be a state. The dedifferentiation includes, for example, acquisition of a cell having an ability of differentiating from three germ layers to obtain the ability of differentiating from a germ layer. Dedifferentiation also includes the generation of pluripotent stem cells. Any method may be used as the method for initializing or dedifferentiating (initializing treatment or dedifferentiating treatment), and known methods such as the method described in WO 2011/016588 may be applied. it can.

  As used herein, the term "somatic cell" refers to a cell other than a germ cell, such as an egg or sperm, which directly transfers its DNA to the next generation. Somatic cells include, for example, cells that remain pluripotent such as adult stem cells, and terminally differentiated cells such as skin fibroblasts. Usually, somatic cells have limited pluripotency, or somatic cells have no pluripotency. Somatic cells as used herein may be naturally occurring, genetically modified, or cultures thereof.

  In the present specification, the term “demethylation gene” means that the DNA of the CpG site is methylated in somatic cells before the initialization treatment, whereas the iPS produced by the initialization treatment is prepared. In cells, it means a gene that is not methylated. On the other hand, in the present specification, the term “methylation gene” means that the DNA of the CpG site is not methylated in somatic cells before the initialization treatment, but is prepared by the initialization treatment. It means a gene that is methylated in iPS cells. For example, examples of demethylation genes include NANOG, Oct3 / 4, RAB25, SALL4, and SLC22A3, and examples of methylation genes include GBP3, SP100, LYST, and UBE1L.

  The NANOG gene is a gene encoding a protein (homebox protein NANOG isoform 1) specified by RefSeq ID: NP — 079141. The Oct3 / 4 gene is a protein identified by RefSeq ID: NP_002692 (Octamer-binding transcribing factor 3; Octamer-binding transcription factor 4; POU dominion transcription 1, clath). The RAB25 gene is a gene encoding a protein (ras-related protein Rab-25) specified by RefSeq ID: NP — 065120. The SALL4 gene is a gene encoding a protein (sal-like protein 4) specified by RefSeq ID: NP — 065169. The GBP3 gene is a gene encoding a protein (guanylate-binding protein 3) specified by RefSeq ID: NP — 060754. The SP100 gene is a gene encoding a protein (nuclear autoantigen Sp-100) specified by RefSeq ID: NP_001073860 or NP_003104. The LYST gene is a gene encoding a protein (lysosomal trafficking regulator, CHS1) specified by RefSeq ID: AAI44703. The UBE1L gene is a gene encoding a protein (ubiquitin-activating enzyme E1-like) specified by RefSeq ID: AAG49557. The SLC22A3 gene is a gene encoding a protein (Solute carrier family 22 member 3) specified by RefSeq ID: NP — 068812.

  In the present specification, “DNA methylation” means that in carbon, the carbon at the 5-position of cytosine is methylated. In the present specification, "detecting methylation" of DNA means the presence or absence or the amount of methylated DNA in the DNA to be detected, the ratio of the amount, or the methylation rate of the DNA. Means to do. In the present specification, the "DNA methylation rate" means the proportion of cytosine at the CpG site that is methylated in the DNA to be detected. For example, in the CpG island of the specific DNA to be detected. , The total number of cytosines (methylated cytosine and non-methylated cytosine) to the ratio of the number of methylated cytosine.

  In the present specification, the “CpG site” means a site where a phosphodiester bond (p) is formed between cytosine (C) and guanine (G) in DNA. In the present specification, the term “CpG region (or CpG island)” means that a 2-nucleotide sequence of cytosine (C) -guanine (G) via a phosphodiester bond (p) (ie, CpG site) frequently appears. Area CpG islands often exist in the promoter region upstream of the gene. In the present specification, “the CpG region of a (some) gene” preferably means the CpG region existing in the coding region of the gene or a region close thereto, or the “CpG site of the (some) gene” It preferably means a CpG site existing in the coding region of the gene or a region close thereto, and more preferably a CpG site contained in the CpG region. Further, preferably, the “CpG site or CpG region of (a) gene” means the CpG site or CpG region existing in the promoter of the gene and the peripheral region thereof. As used herein, the term “(a) gene promoter and its peripheral region” refers to a promoter region, an exon region adjacent to the downstream thereof, and an intron region adjacent to the exon region. The CpG site or CpG region of a specific gene can be identified based on the methods such as MassARRAY method and pyrosequencing.

  In the present invention, whether or not the test cell is a pluripotent stem cell is determined using the DNA methylation in the demethylation gene or methylation gene of the test cell as an index. Demethylation of the DNA of the demethylation gene and methylation of the DNA of the methylation gene both mean that the test cell has been reprogrammed, that is, the test cell is a pluripotent stem cell.

  In the present invention, detection of DNA methylation is carried out by a method using bisulfite treatment of target DNA, PCR amplification, and ion exchange chromatography analysis. Whether or not the target DNA is methylated can be determined by using the peak height and retention time of the detection signal in the ion exchange chromatography as an index. The retention time of the peak of the detection signal is related to the DNA methylation rate; that is, the extension of the retention time represents the decrease of the DNA methylation rate, and conversely, the reduction of the retention time shows the increase of the DNA methylation rate. Represent On the other hand, a high peak derived from DNA having a high methylation rate in the detection signal indicates that the abundance ratio of methylated DNA is high.

  Preferably, the detection signal from the test cell population is compared to the detection signal from the control cell population. As a control cell group, a negative control includes adult tissue cells that have undergone terminal differentiation, somatic cells that have not been subjected to reprogramming, and a positive control includes pluripotent stem cells and established induced pluripotent stem cells. And so on. If the detection signal from the test cell group has a different pattern from the detection signal from the control cell group, it is determined that the target DNA of the test cell group is demethylated or methylated as compared with the control cell group. .. On the other hand, if the detection signal from the test cell group has the same pattern as the detection signal from the control cell group, the DNA methylation states of the test cell group and the control cell group are determined to be equivalent.

Therefore, in one embodiment, the present invention provides a method for quality control of induced pluripotent stem cells, which comprises:
Treating genomic DNA prepared from reprogrammed cells or cultures thereof with bisulfite;
PCR amplifying the bisulfite treated DNA with a primer set that amplifies the DNA of the CpG region of the demethylated gene;
Subjecting the resulting PCR amplification product to ion exchange chromatography to obtain a detection signal; and
When the retention time of the peak of the detection signal obtained from the reprogrammed cells or its culture is longer than the retention time of the peak of the control detection signal, the reprogrammed cells are artificially engineered. Determining that the cells are pluripotent stem cells,
Here, the detection signal of the control was obtained by treating genomic DNA prepared from uninitialized cells or cultures thereof with bisulfite, and then PCR-amplifying using the primer set. It is a detection signal obtained by subjecting the PCR amplification product to ion exchange chromatography.

In another embodiment, the present invention provides a method for quality control of induced pluripotent stem cells, which comprises:
Treating genomic DNA prepared from reprogrammed cells or cultures thereof with bisulfite;
PCR-amplifying the bisulfite-treated DNA with a primer set that amplifies the DNA in the CpG region of the methylated gene;
Subjecting the resulting PCR amplification product to ion exchange chromatography to obtain a detection signal; and
When the retention time of the peak of the detection signal obtained from the reprogrammed cells or the culture thereof is shorter than the retention time of the peak of the control detection signal, the reprogrammed cells are artificially engineered. Determining that the cells are pluripotent stem cells,
Here, the detection signal of the control was obtained by treating genomic DNA prepared from uninitialized cells or cultures thereof with bisulfite, and then PCR-amplifying using the primer set. It is a detection signal obtained by subjecting the PCR amplification product to ion exchange chromatography.

In a further embodiment, the present invention provides a method for quality control of induced pluripotent stem cells, which comprises:
(1) treating genomic DNA prepared from reprogrammed cells or cultures thereof, and genomic DNA prepared from non-reprogrammed cells or cultures thereof with bisulfite;
(2) PCR amplification of each of the bisulfite-treated DNAs obtained in step (1) with a primer set that amplifies the DNA of the CpG region of the demethylated gene;
(3) subjecting each PCR amplification product obtained in step (2) to ion exchange chromatography to obtain a detection signal;
(4) The retention time of the peak of the detection signal obtained from the reprogrammed cells or the culture thereof obtained in step (3) depends on the detection signal obtained from the non-reprogrammed cells or the culture thereof. To judge that the reprogrammed cells are induced pluripotent stem cells when the retention time of the peak is extended.

In a further embodiment, the present invention provides a method for quality control of induced pluripotent stem cells, which comprises:
(1 ′) treating genomic DNA prepared from reprogrammed cells or cultures thereof and genomic DNA prepared from non-reprogrammed cells or cultures thereof with bisulfite;
(2 ′) PCR amplification of each of the bisulfite-treated DNAs obtained in step (1 ′) with a primer set that amplifies the DNA of the CpG region of the methylated gene;
(3 ') subjecting each PCR amplification product obtained in step (2') to ion exchange chromatography to obtain a detection signal;
(4 ′) Detection obtained from the uninitialized cells or culture thereof in which the peak retention time of the detection signal obtained from the initialized cells or culture thereof obtained in step (3 ′) is To judge that the reprogrammed cells are induced pluripotent stem cells when the retention time of the signal peak is shorter than the retention time.

In yet another embodiment, the present invention provides a method for quality control of induced pluripotent stem cells, which comprises:
Treating the genomic DNA prepared from (1 ''-1) reprogrammed cells or a culture thereof with bisulfite;
(1 ''-2) treatment of genomic DNA prepared from uninitialized cells or cultures thereof with bisulfite;
(2 ″ -1) PCR amplification of the bisulfite-treated DNA obtained in step (1 ″ -1) using a primer set for amplifying the DNA in the CpG region of the demethylation gene ;
(2 ″ -2) PCR amplification of the bisulfite-treated DNA obtained in step (1 ″ -1) using a primer set that amplifies the DNA of the CpG region of the methylation gene;
(2 ″ -3) PCR amplification of the bisulfite-treated DNA obtained in step (1 ″ -2) using a primer set for amplifying the DNA in the CpG region of the demethylation gene ;
(2 ″ -4) PCR-amplifying the bisulfite-treated DNA obtained in step (1 ″ -2) using a primer set that amplifies the DNA in the CpG region of the methylated gene;
(3 ″ -1) subjecting the PCR amplification product obtained in step (2 ″ -1) to ion exchange chromatography to obtain a detection signal;
(3 ″ -2) subjecting the PCR amplification product obtained in step (2 ″ -2) to ion exchange chromatography to obtain a detection signal;
(3 ″ -3) subjecting the PCR amplification product obtained in step (2 ″ -3) to ion exchange chromatography to obtain a detection signal;
(3 ″ -4) subjecting the PCR amplification product obtained in step (2 ″ -4) to ion exchange chromatography to obtain a detection signal;
(4 '') The retention time of the peak of the detection signal obtained in the step (3 ''-1) is longer than the retention time of the peak of the detection signal obtained in the step (3 ''-3). And the retention time of the peak of the detection signal obtained in step (3 ″ -2) is shorter than the retention time of the peak of the detection signal obtained in step (3 ″ -4) First, determining that the reprogrammed cells are induced pluripotent stem cells.

In a further embodiment, the present invention provides a method for quality control of induced pluripotent stem cells, which comprises:
(1 ′ ″) Treatment of genomic DNA prepared from reprogrammed cells or cultures thereof, and genomic DNA prepared from non-reprogrammed cells or cultures thereof with bisulfite ;
(2 ′ ″-1) Each of the DNAs treated with the bisulfite obtained in the step (1 ′ ″) was subjected to PCR using a primer set for amplifying the DNA of the CpG region of the demethylation gene. Amplifying;
(2 ′ ″-2) Each of the bisulfite-treated DNAs obtained in step (1 ′ ″) is PCR-amplified using a primer set that amplifies the DNA in the CpG region of the methylated gene. thing;
(3 ′ ″-1) subjecting each PCR amplification product obtained in step (2 ′ ″-1) to ion exchange chromatography to obtain a detection signal;
(3 ′ ″-2) subjecting each PCR amplification product obtained in step (2 ′ ″-2) to ion exchange chromatography to obtain a detection signal;
(4 ″ ′) In the detection signal obtained in the step (3 ′ ″-1), the retention time of the peak of the signal obtained from the initialized cells or the culture thereof is not initialized. The retention time of the peak of the signal obtained from the cell or its culture was extended, and in the detection signal obtained in the step (3 ′ ″-2), the signal obtained from the reprogrammed cells was If the retention time of the peak is shorter than the retention time of the peak of the signal obtained from the uninitialized cells,
To determine that the reprogrammed cells are induced pluripotent stem cells.

In yet another embodiment, the invention provides a method for quality control of induced pluripotent stem cells, including:
Treating the genomic DNA prepared from (1 ''''-1) reprogrammed cells or cultures thereof with bisulfite;
(1 ''''-2) treatment of genomic DNA prepared from induced pluripotent stem cells or cultures thereof with bisulfite;
(2 ''''-1) A primer set for amplifying the DNA in the CpG region of the demethylation gene, which is obtained by treating the DNA treated with bisulfite obtained in the step (1 ''''-1), and / Or PCR amplification using a primer set that amplifies DNA in the CpG region of the methylated gene;
(2 ''''-2) A primer set for amplifying the DNA of the CpG region of the demethylation gene, which is obtained by treating the DNA obtained by the step (1 ''''-2) with bisulfite, and / Or PCR amplification using a primer set that amplifies DNA in the CpG region of the methylated gene;
(3 ″ ″-1) Step (2 ″ ″-1) subjecting the PCR amplification product obtained to ion exchange chromatography to obtain a detection signal;
(3 ″ ″-2) Step (2 ″ ″-2) subjecting the PCR amplification product obtained to ion exchange chromatography to obtain a detection signal;
(4 '''') Difference in peak retention time between the detection signal obtained in step (3 ''''-1) and the detection signal obtained in step (3 ''''-2) When there is no such thing, it is determined that the reprogrammed cells are induced pluripotent stem cells.

In a further embodiment, the present invention provides a method for quality control of induced pluripotent stem cells, which comprises:
(1 ′ ″ ″) Treatment of genomic DNA prepared from reprogrammed cells or cultures thereof, as well as genomic DNA prepared from induced pluripotent stem cells or cultures thereof with bisulfite ;
(2 ′ ″ ″) A primer set for amplifying the DNA of the CpG region of the demethylated gene, and / or each DNA treated with bisulfite obtained in the step (1 ′ ″ ″) PCR amplification using a primer set that amplifies the DNA in the CpG region of the methylated gene;
(3 ′ ″ ″) subjecting each PCR amplification product obtained in step (2 ′ ″ ″) to ion exchange chromatography to obtain a detection signal;
(4 ″ ″ ″) Detection signal obtained from the reprogrammed cells or culture thereof obtained in step (3 ′ ″ ″) and detection obtained from induced pluripotent stem cells or culture thereof To judge that the reprogrammed cells are induced pluripotent stem cells when there is no difference in the retention time of the peak from the signal.

  In yet another embodiment, a method of using the uninitialized cells as a control and a method of using the induced pluripotent stem cells as a control, the initialized cells are induced pluripotent You may determine whether it is a sex stem cell.

  The "initialized cells" to be tested in the method of the present invention are preferably initialized nucleated cells, and more preferably initialized tissue cells such as initial cells. Examples include dermal fibroblasts that have been subjected to chemical treatment, peripheral blood cells that have undergone initialization treatment, and the like. The initialization treatment is not particularly limited as long as it is a treatment capable of inducing cell initialization. For example, a procedure for producing iPS cells (see, for example, Japanese Patent No. 5467223 can be referred to. (Not limited).

  As a "non-initialized cell", which is a control in the method of the present invention, a cell of the same type as the above-mentioned initialized cell, which is not subjected to the initialization treatment, can be used. .. Further, as a control in the method of the present invention, as the "induced pluripotent stem cells", cells derived from the established induced pluripotent stem cell line, induced induced pluripotent stem cells, etc. may be used. ..

  The organism used as a source of "cells" such as stem cells and differentiated cells used in the present invention is not limited to humans, mammals other than humans, for example, monkeys, apes, dogs, mice, rats, pigs, goats, Guinea pigs, as well as birds, fishes and the like can be mentioned.

  The method for preparing genomic DNA from the “cell” and “cell culture” is not particularly limited, and a known method can be appropriately selected and used. As a known method for preparing DNA, a phenol chloroform method, or a commercially available DNA extraction kit, for example, DNeasy Blood & Tissue Kit (manufactured by Qiagen), QIAamp DNA Mini kit (manufactured by Qiagen), or Clean Columns (NexTec) described below is used. (Manufactured by the same company), AquaPure (manufactured by Bio-Rad), ZR Plant / Seed DNA Kit (manufactured by Zymo Research), prepGEM (manufactured by ZyGEM), BuccalQuick (manufactured by TrimGen), and the like.

  In the method of the present invention, the extracted genomic DNA is treated with bisulfite. The method for treating the bisulfite salt of DNA is not particularly limited, and a known method can be appropriately selected and used. Known methods for the treatment with bisulfite include, for example, EZ DNA Methylation-Lighting Kit (manufactured by ZYMO Research Co., Ltd.), EpiTect Bisulfite Kit (48) (manufactured by Qiagen Co., Ltd.), and MethylEasygen (Human Siengen). Commercially available kits such as Cells-to-CpG Bisulfite Conversion Kit (manufactured by Applied Biosystems), CpGenome Turbo Bisulfite Modification Kit (manufactured by MERCK MILIPORE), and the like.

  The bisulfite treated DNA is then amplified by PCR. The PCR amplification method is not particularly limited, and a known method can be appropriately selected and used according to the sequence, length, amount, etc. of the DNA to be amplified.

  In the PCR, the DNA of the CpG region of one or both of the demethylated gene and the methylated gene is amplified. Preferably, the demethylated gene includes one or more genes selected from the group consisting of NANOG, Oct3 / 4, RAB25, SALL4, and SLC22A3, and the methylated gene consists of SP100 and UBE1L. One or more genes selected from the group are included. More preferably, at least one selected from the group consisting of RAB25 gene, SALL4 gene, and SLC22A3 gene, which have a large difference in retention time of detection signals by chromatography and have a good peak shape, is selected. In addition, "the peak has a good shape" generally means that the peak is approximately symmetrical, no reading or tailing of the peak is observed, the peak has no shoulder, and the peak is sharp.

  The chain length of the PCR amplification product can be appropriately selected in consideration of factors such as shortening of PCR amplification time, shortening of analysis time in ion exchange chromatography, and maintenance of separation performance. For example, when a bisulfite salt of DNA having many CpG sites is used as a template, the chain length of the PCR amplification product is preferably 1000 bp or less, more preferably 700 bp or less, and further preferably 500 bp or less. On the other hand, when the bisulfite salt of DNA having a small number of CpG sites is used as a template, the chain length of the PCR amplification product is preferably 1000 bp, more preferably 700 bp, further preferably 500 bp as the upper limit, and nonspecific hybridization in PCR is performed. The lower limit is 30 to 40 bp, which is the chain length of the PCR amplification product when using a primer around 15 mer that can avoid In the demethylated gene or methylated gene region used in the present invention, the chain length of the PCR amplification product is, for example, preferably 100 to 500 bp, more preferably 250 to 450 bp. On the other hand, it is preferable to design the primer so that the content of the region corresponding to the CpG site in the PCR amplification product becomes rich. For example, the number of bases derived from cytosine at the CpG site is preferably 2% or more, and more preferably 5% or more, with respect to the number of bases (chain length) of the PCR amplification product. On the other hand, in the method of the present invention, if the PCR amplification product contains at least 3 bases derived from cytosine at the CpG site whose methylation state changes before and after the initialization treatment, preferably 3 to 15 bases. The change in the DNA methylation rate can be detected by the ion exchange chromatography analysis described later.

  Preferred examples of the primer set for amplifying the DNA of the CpG region of the demethylated gene used in the method of the present invention include the primer set shown in Table 1. Further, as a preferred example of the primer set for amplifying the DNA of the CpG region of the methylated gene used in the method of the present invention, the primer set shown in Table 2 can be mentioned.

  Subsequently, the resulting PCR amplification product is subjected to ion exchange chromatography. The ion exchange chromatography performed in the present invention is preferably anion exchange chromatography. The packing material of the column used in the ion exchange chromatography performed in the present invention is not particularly limited as long as it is a base particle having a strong cationic group on the surface, but the packing material shown in Patent Document 6 has a strong cationic property. Substrate particles having both groups and weakly cationic groups are preferred.

  In the present specification, the strong cationic group means a cationic group that dissociates in a wide pH range of 1 to 14. That is, the strong cationic group can maintain a dissociated (cationized) state without being affected by the pH of the aqueous solution.

  Examples of the strong cationic group include a quaternary ammonium group. Specific examples include a trialkylammonium group such as a trimethylammonium group, a triethylammonium group, and a dimethylethylammonium group. Further, examples of the counter ion of the strong cationic group include halide ions such as chloride ion, bromide ion, and iodide ion.

  The amount of the strong cationic group introduced onto the surface of the base particles is not particularly limited, but the preferable lower limit per dry weight of the filler is 1 μeq / g, and the preferable upper limit is 500 μeq / g. When the amount of the strong cationic group is less than 1 μeq / g, the holding power may be weak and the separation performance may be deteriorated. When the amount of the strong cationic group exceeds 500 μeq / g, the retentivity becomes too strong to allow the PCR amplification product to be easily eluted and the analysis time may become too long.

  In the present specification, the weak cationic group means a cationic group having a pKa of 8 or more. That is, the weak cationic group is affected by the pH of the aqueous solution, and the dissociated state changes. That is, when the pH is higher than 8, the protons of the weak cationic group are dissociated, and the proportion of those having no positive charge increases. On the contrary, when the pH is lower than 8, the weak cationic group is protonated and the proportion of positive charges is increased.

  Examples of the weak cationic group include a tertiary amino group, a secondary amino group and a primary amino group. Of these, a tertiary amino group is preferable.

  The amount of the weak cationic groups introduced onto the surface of the base particles is not particularly limited, but the preferable lower limit per dry weight of the filler is 0.5 μeq / g, and the preferable upper limit is 500 μeq / g. If the amount of the weak cationic group is less than 0.5 μeq / g, the amount may be too small to improve the separation performance. When the amount of the weak cationic group exceeds 500 μeq / g, the retention strength becomes too strong like the strong cationic group, the PCR amplification product cannot be easily eluted, and the analysis time becomes too long. May occur.

  The amount of the strong cationic group or the weak cationic group on the surface of the base particle can be measured by quantifying the nitrogen atom contained in the amino group. The method of quantifying nitrogen includes, for example, the Kjeldahl method. In the case of the fillers described in the examples below, first, the nitrogen contained in the strong cationic group after polymerization is quantified, and then the strong cationic group and the weak cationic group after introducing the weak cationic group. By quantifying the nitrogen contained in, it is possible to calculate the amount of weakly cationic groups introduced later. By quantifying as described above, the amount of the strong cationic group and the amount of the weak cationic group can be adjusted within the above range when the filler is prepared.

  As the base particles, for example, synthetic polymer fine particles obtained by using a polymerizable monomer or the like, silica-based inorganic fine particles, or the like can be used, and a hydrophobic cross-linked polymer made of a synthetic organic polymer. It is preferably a particle.

  The hydrophobic crosslinked polymer is a hydrophobic crosslinked polymer obtained by copolymerizing at least one kind of hydrophobic crosslinkable monomer and at least one kind of monomer having a reactive functional group, and at least one kind of hydrophobic crosslinked polymer. Any of a hydrophobic cross-linked polymer obtained by copolymerizing a hydrophobic cross-linkable monomer, a monomer having at least one reactive functional group and at least one hydrophobic non-crosslinkable monomer It may be.

  The hydrophobic crosslinkable monomer is not particularly limited as long as it has two or more vinyl groups in one molecule of the monomer, and examples thereof include ethylene glycol di (meth) acrylate and polyethylene glycol di (meth). Di (meth) acrylic acid ester such as acrylate, propylene glycol di (meth) acrylate, polypropylene glycol di (meth) acrylate, tri (meth) acrylate such as trimethylolmethane tri (meth) acrylate, tetramethylolmethane tri (meth) acrylate Examples thereof include acrylic acid ester or tetra (meth) acrylic acid ester, and aromatic compounds such as divinylbenzene, divinyltoluene, divinylxylene and divinylnaphthalene. In this specification, the (meth) acrylate means acrylate or methacrylate, and the (meth) acrylic means acrylic or methacrylic.

  Examples of the monomer having a reactive functional group include glycidyl (meth) acrylate and isocyanate ethyl (meth) acrylate.

  The hydrophobic non-crosslinkable monomer is not particularly limited as long as it is a non-crosslinkable polymerizable organic monomer having a hydrophobic property, and examples thereof include methyl (meth) acrylate, ethyl (meth) acrylate, Examples thereof include (meth) acrylic acid esters such as butyl (meth) acrylate and t-butyl (meth) acrylate, and styrene-based monomers such as styrene and methylstyrene.

  When the hydrophobic cross-linked polymer is obtained by copolymerizing the hydrophobic cross-linkable monomer and the monomer having the reactive functional group, the hydrophobic cross-linked polymer in the hydrophobic cross-linked polymer The preferable lower limit of the content ratio of the segment derived from the crosslinkable monomer is 10% by weight, and the more preferable lower limit is 20% by weight.

  The filler for ion exchange chromatography used in the present invention preferably has a polymer layer having the strong cationic group and the weak cationic group on the surface of the base particle. In the polymer having the strong cationic group and the weak cationic group, the strong cationic group and the weak cationic group are preferably derived from independent monomers. Specifically, the packing material for ion exchange chromatography used in the present invention comprises the above hydrophobic crosslinked polymer particles and a hydrophilic polymer having a strong cationic group copolymerized on the surface of the above hydrophobic crosslinked polymer particles. It is preferable that the weakly cationic group is introduced on the surface of the coated polymer particle composed of a coalesced layer.

  The hydrophilic polymer having a strong cationic group is composed of a hydrophilic monomer having a strong cationic group, and is derived from one or more hydrophilic monomers having a strong cationic group. It may contain a segment. That is, as a method for producing the hydrophilic polymer having a strong cationic group, a method in which a hydrophilic monomer having a strong cationic group is polymerized alone, a hydrophilic polymer having two or more strong cationic groups is used. Examples thereof include a method of copolymerizing monomers, a method of copolymerizing a hydrophilic monomer having a strong cationic group and a hydrophilic monomer having no strong cationic group, and the like.

  The hydrophilic monomer having a strong cationic group preferably has a quaternary ammonium group. Specifically, for example, ethyltriethylammonium methacrylate, ethyldimethyldimethylammonium chloride, ethyldimethyldimethylbenzylammonium chloride, ethyldimethylbenzylammonium acrylate, ethyltriethylammonium acrylate, ethyldimethylethylammonium acrylate. Examples thereof include chloride, acrylamidoethyltrimethylammonium chloride, acrylamidoethyltriethylammonium chloride, acrylamidoethyldimethylethylammonium chloride and the like.

  As a method for introducing the weak cationic group to the surface of the coated polymer particles, a known method can be used. Specifically, for example, as a method of introducing a tertiary amino group as the weak cationic group, a hydrophobic crosslinked polymer particle composed of a hydrophobic crosslinked polymer having a segment derived from a monomer having a glycidyl group is used. A method of copolymerizing the hydrophilic monomer having a strong cationic group on the surface and then reacting a reagent having a tertiary amino group with a glycidyl group; hydrophobic having a segment derived from a monomer having an isocyanate group A method of copolymerizing the hydrophilic monomer having a strong cationic group on the surface of a hydrophobic crosslinked polymer particle composed of a crosslinked polymer, and then reacting a reagent having a tertiary amino group with an isocyanate group; Of copolymerizing the above-mentioned hydrophilic monomer having a strong cationic group and a monomer having a tertiary amino group on the surface of the crosslinkable polymer particles; Method of introducing a tertiary amino group onto the surface of a coated polymer particle having a layer of the hydrophilic polymer having a strong cationic group using a silane coupling agent having a cation group; On the surface of the hydrophobic cross-linked polymer particles composed of the hydrophobic cross-linked polymer having a segment derived from, the hydrophilic monomer having the strong cationic group is copolymerized, and then a reagent having a carboxy group and a tertiary amino group. Are condensed with carbodiimide; a hydrophilic monomer having a strong cationic group on the surface of a hydrophobic crosslinked polymer particle comprising a hydrophobic crosslinked polymer having a segment derived from a monomer having an ester bond. After copolymerizing the monomer and hydrolyzing the ester bond part, the carboxy group generated by hydrolysis and a reagent having a tertiary amino group are then capsulated. And a method such as condensation with Bojiimido. Among them, a hydrophilic monomer having a strong cationic group is copolymerized on the surface of a hydrophobic crosslinked polymer particle composed of a hydrophobic crosslinked polymer having a segment derived from a monomer having a glycidyl group, and then , A method of reacting a reagent having a tertiary amino group with a glycidyl group, or the above strong cationic property on the surface of a hydrophobic crosslinked polymer particle composed of a hydrophobic crosslinked polymer having a segment derived from a monomer having an isocyanate group. A method in which a hydrophilic monomer having a group is copolymerized and then a reagent having a tertiary amino group is reacted with an isocyanate group is preferable.

  The reagent having a tertiary amino group that reacts with a reactive functional group such as a glycidyl group or an isocyanate group is not particularly limited as long as it has a tertiary amino group and a functional group capable of reacting with a reactive functional group. Not done. Examples of the functional group capable of reacting with the reactive functional group include a primary amino group and a hydroxyl group. Of these, a group having a primary amino group at the terminal is preferable. Specific reagents having the functional group include N, N-dimethylaminomethylamine, N, N-dimethylaminoethylamine, N, N-dimethylaminopropylamine, N, N-dimethylaminobutylamine, N, N- Diethylaminoethylamine, N, N-diethylaminopropylethylamine, N, N-diethylaminobutylamine, N, N-diethylaminopentylamine, N, N-diethylaminohexylamine, N, N-dipropylaminobutylamine, N, N-dibutylaminopropyl Examples include amines.

  The relative positional relationship between the strong cationic group, preferably a quaternary ammonium salt, and the weak cationic group, preferably a tertiary amino group is such that the strong cationic group is a base material rather than the weak cationic group. It is preferable that the particle is located far from the surface of the particle, that is, outside. For example, it is preferable that the weakly cationic group is within 30Å from the surface of the base material particle, and the strong cationic group is within 300Å from the surface of the base material particle and is outside the weakly cationic group.

  The average particle size of the base particles used in the packing material for ion exchange chromatography used in the present invention is not particularly limited, but a preferred lower limit is 0.1 μm, and a preferred upper limit is 20 μm. If the average particle size is less than 0.1 μm, the pressure inside the column may become too high, resulting in poor separation. If the average particle diameter exceeds 20 μm, the dead volume in the column becomes too large, and separation failure may occur. In addition, in this specification, the said average particle diameter shows a volume average particle diameter, and it can measure it using a particle size distribution measuring device (AccuSizer780 / Particle Sizing Systems company make etc.).

  As the composition of the eluent used in the ion exchange chromatography performed in the present invention, known conditions can be used.

  As the buffer solution used in the above-mentioned eluent, it is preferable to use a buffer solution containing a known salt compound or an organic solvent. Specifically, for example, Tris-hydrochloric acid buffer solution, TE buffer solution containing Tris and EDTA, Tris And a TBA buffer solution composed of boric acid and EDTA.

  The pH of the eluent is not particularly limited, but the preferred lower limit is 5 and the preferred upper limit is 10. It is considered that by setting this range, the weak cationic group also effectively acts as an ion exchange group (anion exchange group). The more preferable lower limit of the pH of the eluent is 6, and the more preferable upper limit thereof is 9.

  Examples of the salt contained in the eluent include salts of halides such as sodium chloride, potassium chloride, sodium bromide, potassium bromide and the like and alkali metals; calcium chloride, calcium bromide, magnesium chloride, magnesium bromide. And the like, and salts of a halide with an alkaline earth metal; inorganic acid salts such as sodium perchlorate, potassium perchlorate, sodium sulfate, potassium sulfate, ammonium sulfate, sodium nitrate and potassium nitrate. Further, organic acid salts such as sodium acetate, potassium acetate, sodium succinate, potassium succinate can also be used. Any of the above salts may be used alone or in combination.

  The salt concentration of the eluent may be appropriately adjusted according to the analysis conditions, but a preferred lower limit is 10 mmol / L, a preferred upper limit is 2000 mmol / L, a more preferred lower limit is 100 mmol / L, and a more preferred upper limit is 1500 mmol / L. It is L.

  Further, the eluent used in the ion exchange chromatography used in the present invention contains antichaotropic ions in order to further enhance the separation performance. The anti-chaotropic ion has a property opposite to that of the karotopic ion and has a function of stabilizing the hydrated structure. Therefore, it has the effect of enhancing the hydrophobic interaction between the packing material and the nucleic acid molecule. The main interaction of the ion exchange chromatography used in the present invention is electrostatic interaction, but in addition to that, the action of hydrophobic interaction is also utilized to enhance the separation performance.

The antichaotropic ions contained in the eluent include phosphate ions (PO ₄ ³⁻ ), sulfate ions (SO ₄ ²⁻ ), ammonium ions (NH ₄ ⁺ ), potassium ions (K ⁺ ), sodium ions (Na ⁺ ) And the like. Among these combinations of ions, sulfate ion and ammonium ion are preferably used. Any of the above antichaotropic ions may be used alone or in combination. It should be noted that a part of the above-mentioned antichaotropic ion contains a salt or a buffer component contained in the eluent. When such a component is used, it has both the property as a salt or a buffering property contained in the eluent and the property as an antichaotropic ion, and is therefore suitable for the present invention.

  The concentration at the time of analysis of the antichaotropic ion in the eluent for ion exchange chromatography used in the present invention may be appropriately adjusted according to the analyte, but it is preferably 2000 mmol / L or less as an antichaotropic salt. Specifically, a method in which the concentration of the antichaotropic salt is gradient eluted in the range of 0 to 2000 mmol / L can be mentioned. Therefore, the concentration of the antichaotropic salt at the start of the analysis need not be 0 mmol / L, and the concentration of the antichaotropic salt at the end of the analysis need not be 2000 mmol / L. The gradient elution method may be a low-pressure gradient method or a high-pressure gradient method, but a method of eluting while precisely adjusting the concentration by the high-pressure gradient method is preferable.

  The antichaotropic ion may be added to only one kind of eluent used for elution, or may be added to plural kinds of eluents. Further, the antichaotropic ion may have both the role of enhancing the hydrophobic interaction between the packing material and the PCR amplification product or the buffering ability, and the role of eluting the PCR amplification product from the column.

  The column temperature when the PCR amplification product is analyzed by the ion exchange chromatography performed in the present invention is preferably 30 ° C or higher, more preferably 40 ° C or higher, and further preferably 45 ° C or higher. When the column temperature of ion exchange chromatography is lower than 30 ° C, the hydrophobic interaction between the packing material and the PCR amplification product is weakened, and it becomes difficult to obtain a desired separation effect. When the column temperature of ion exchange chromatography is lower than 45 ° C., a PCR amplification product of a bisulfite-treated methylated DNA (methylated DNA sample) and a PCR amplification product of a bisulfite-treated unmethylated DNA ( The difference in retention time with the non-methylated DNA sample) is small. Furthermore, when the column temperature is 55 ° C. or higher, preferably 60 ° C. or higher, the difference in retention time between the methylated DNA sample and the non-methylated DNA sample further widens, and the respective peaks become clearer, resulting in higher accuracy. It is possible to detect DNA methylation with good quality.

  Furthermore, when the column temperature of the ion exchange chromatography is increased, the methylated DNA sample and the unmethylated DNA sample are clearly separated, so that both of the methylated DNA and the unmethylated DNA in the sample DNA are present according to the ratio of the two. A difference easily occurs in the peak area or peak height of the retention time. Therefore, if the column temperature is increased, the presence of methylated DNA and unmethylated DNA in the sample DNA is determined based on the area or height of the peak of the retention time between the methylated DNA sample and the unmethylated DNA sample. It becomes easier to measure the amount and the abundance ratio.

  On the other hand, when the column temperature of ion exchange chromatography is 90 ° C. or higher, the double strands of the nucleic acid molecule in the PCR amplification product are dissociated, which is not preferable for analysis. Further, if the column temperature is 100 ° C. or higher, boiling of the eluent may occur, which is not preferable for analysis. Therefore, the column temperature at the time of analyzing the PCR amplification product by the ion exchange chromatography performed in the present invention may be 30 ° C or higher and lower than 90 ° C, preferably 40 ° C or higher and lower than 90 ° C, and more preferably 45. ℃ or more and less than 90 ℃, more preferably 55 ℃ or more and less than 90 ℃, even more preferably 55 ℃ or more and 85 ℃ or less, still more preferably 60 ℃ or more and 85 ℃ or less.

  The sample injection amount into the ion exchange chromatography column is not particularly limited and may be appropriately adjusted according to the ion exchange capacity of the column and the sample concentration. The flow rate is preferably 0.1 mL / min to 3.0 mL / min, more preferably 0.5 mL / min to 1.5 mL / min. If the flow rate is slow, improvement in separation can be expected, but if it is too slow, analysis may take a long time, and the separation performance may be deteriorated due to broadening of peaks. On the contrary, when the flow velocity is high, there is an advantage in that the analysis time is shortened, but the peak is compressed, which causes the deterioration of the separation performance. Therefore, although it is a parameter that is appropriately adjusted depending on the performance of the column, it is desirable to set it within the range of the flow velocity. The retention time of each sample can be pre-determined by performing preliminary experiments on each sample. As the liquid feeding method, a known liquid feeding method such as a linear gradient elution method or a stepwise elution method can be used, but the linear gradient elution method is preferable as the liquid feeding method in the present invention. The size of the gradient (gradient) may be appropriately adjusted depending on the separation performance of the column and the characteristics of the analyte (here, PCR amplification product) in the range of 0% to 100% for the eluent used for elution.

  In the present invention, the DNA methylation in the genomic DNA prepared from cells is detected by subjecting the PCR-amplified product of the bisulfite-treated DNA to the ion exchange chromatography in the procedure described above.

  When the DNA is treated with bisulfite, unmethylated cytosine in the DNA is converted to uracil, but methylated cytosine remains cytosine. When PCR amplification of bisulfite-treated DNA is performed, uracil derived from unmethylated cytosine is further replaced with thymine, so that there is a difference in the abundance ratio of cytosine and thymine between methylated DNA and unmethylated DNA. Therefore, the DNA in the PCR amplification product has a different sequence depending on the methylation rate of the original DNA. When the PCR amplification product is subjected to ion exchange chromatography, a chromatogram showing different signals depending on the base sequence of the DNA contained in the amplification product is obtained. Therefore, methylation of DNA can be detected based on the detection signal obtained by ion exchange chromatography of sample DNA.

  That is, the more methylated the DNA extracted from cells, the shorter the retention time of the peak of the detection signal obtained from the PCR amplification product, and conversely, if the degree of methylation is low, the retention time of the peak of the detection signal. Becomes longer. In addition, the height of the peak of each detection signal indicates that the abundance ratio of DNA having a methylation rate corresponding to the peak is high.

  For example, in the case of producing iPS cells by subjecting differentiated cells such as skin fibroblasts to reprogramming, when reprogramming is performed normally, methylated cytosine in the CpG region of the demethylated gene region is demethylated. It That is, the methylation rate of the demethylated gene region is lower in iPS cells than in the differentiated cells that have not been subjected to the reprogramming treatment. Therefore, in the chromatographic analysis of the demethylated gene, the peak of the detection signal derived from the differentiated cell is detected at a retention time earlier than the peak of the detection signal derived from the iPS cell.

  On the other hand, when the reprogramming of the differentiated cells is normally performed, unmethylated cytosine in the CpG region of the methylated gene region is methylated. That is, the methylation rate of the methylated gene region is higher in iPS cells than in the differentiated cells that have not been subjected to the reprogramming treatment. Therefore, in the chromatographic analysis of the methylated gene, the peak of the detection signal derived from iPS cells is detected at a retention time earlier than the peak of the detection signal derived from the differentiated cells.

  In the method of the present invention, as a method for determining the presence or absence of a peak of a detection signal by chromatography, existing data processing software, for example, LCsolution (Shimadzu Corporation), GRAMS / AI (Thermo Fisher Scientific), IgorPro (WaveMetrics). The peak detection using a company etc. is mentioned. To exemplify a method for determining the presence or absence of a peak using LCsolution, specifically, first, a section of a retention time at which a peak is desired to be detected is designated. Next, various parameters are set in order to remove unnecessary peaks such as noise. For example, the parameter “WIDTH” is made larger than the half-value width of the unnecessary peak, the parameter “SLOPE” is made larger than the rising slope of the unnecessary peak, and the peak with low resolution is changed vertically by changing the setting of the parameter “DRIFT”. It is possible to select whether to perform division or baseline division. Since different chromatograms can be obtained depending on the analysis conditions, the type of selected genetic marker, the amount of the sample, etc., the parameter values may be set to appropriate values according to the chromatogram.

  Whether or not there is a difference in the retention time of the peaks of the detection signals obtained by chromatography is determined by comparing the retention times of the peak tops of the respective detection signals obtained by the data processing software. When the retention time difference between the two detection signals is preferably 2 seconds or more, more preferably 5 seconds or more, it is judged that the two signals have different retention times. On the other hand, if the difference in retention time between the two detection signals is preferably less than 2 seconds, more preferably less than 1 second, it is judged that there is no difference in the retention time of the two signals.

  The retention time of the peak of the detection signal obtained by chromatography may vary depending on the PCR amplification product, the conditions of chromatography and the like. Therefore, the detection signal by chromatography from the control cells is preferably newly obtained each time the detection signal is obtained from the test cells, but separately obtained under predetermined conditions and stored as control data. You may refer to it as needed.

  In the method of the present invention, for each gene region, the retention time of the peak of the detection signal of cells derived from the test cells that have been subjected to the reprogramming treatment is set to the differentiation time of iPS cells (positive control), human skin fibroblasts, etc. The retention time of the peak of the detection signal of cells (negative control) or both is compared. If the retention time differs between the test cell and the negative control, the test cell has a demethylated DNA in the demethylated gene region or is methylated in the methylated gene region than the differentiated cell. That is, the test cell is a reprogrammed iPS cell. Alternatively, if the retention time is not different between the test cell and the positive control, the test cell is similar to iPS cells in that the DNA of the demethylation gene region is demethylated, or the methylation gene region is demethylated. The DNA is methylated, that is, the test cell is a reprogrammed iPS cell. Therefore, the success or failure of cell reprogramming (iPS cell production) can be determined by the method of the present invention.

  Furthermore, the method of the present invention can be applied not only to the success or failure of cell reprogramming but also to the judgment of whether or not the established iPS cells maintain their pluripotency. For example, using a cell of a subculture of the established iPS cell line as a test subject, a detection signal by ion exchange chromatography is obtained by the same procedure as above, and the retention time of the peak of the detection signal is compared with a control. .. Whether or not the demethylation of the demethylation gene region DNA or the methylation of the methylation gene region DNA is maintained in the test target cell based on the presence or absence of the difference in retention time from the control, that is, the test target cell It is possible to determine whether or not the plant retains pluripotency without differentiation.

  Therefore, the above-mentioned method of the present invention enables quality control of iPS cells.

  Furthermore, the iPS cell quality control method of the present invention can be carried out in combination with a conventional iPS cell identification method. Conventional methods for identifying iPS cells include confirmation by the activity of reporters bound to NANOG, Oct3 / 4, etc. (puromycin resistance, GFP positive, etc.), visual confirmation of formation of ES cell-like colonies, and the like. Accurate methods include alkaline phosphatase staining, expression analysis of various ES cell-specific genes, and confirmation test of teratoma formation by transplanting cells into mice. Preferably, these conventional methods are performed for secondary evaluation after the method of the invention.

In a further embodiment, the method of the present invention has the following applications:
Determining whether or not cell pluripotency is maintained in a subculture of stem cells other than iPS cells (eg ES cells, adult stem cells, etc.);
Detecting contamination of differentiated cells into stem cell subcultures;
Detection of undifferentiated cell contamination in differentiated cell cultures (eg, undifferentiated cell contamination in somatic cell cultures differentiated from pluripotent stem cells).

  In the case of determining pluripotency with respect to stem cells other than iPS cells, in the method of the present invention, a culture of reprogrammed cells to be tested is used as ES cells, adult stem cells (neural stem cells, hematopoietic stem cells, Substitute cultures of stem cells such as leaf-like stem cells), and replace the control with differentiated cells (negative control) or the same type of stem cells that have been confirmed to be pluripotent (positive control), The same steps as described above are performed to determine whether the test subject cells are stem cells. Alternatively, the control cell may be a primary cell that has not been passaged, or a cell of a subculture in which the number of passages is small and the properties of the primary cell have been inherited.

  When detecting the contamination of differentiated cells into a subculture of stem cells, in the above-mentioned method of the present invention, a culture of reprogrammed cells to be tested is used as iPS cells, ES cells, adult stem cells, etc. Perform the same steps as above, substituting a subculture of stem cells, and replacing the control with a differentiated cell (negative control), or a stem cell of the same type with confirmed pluripotency (positive control), etc. , It is determined whether or not the differentiated cells are mixed in the test target cells.

  When detecting undifferentiated cell contamination in a differentiated cell culture, in the method of the present invention, the culture of the reprogrammed cells to be tested is replaced with a differentiated cell culture, and the control is a stem cell. (Negative control), differentiated cells (positive control), etc. are replaced, and the same steps as above are performed to determine whether the test target cells are differentiated cells.

  Conventionally, when identifying iPS cells by morphological observation, in the preparation of iPS cells to be described later, it took time to identify the colonies on the 21st to 30th days of culture. On the other hand, according to the present invention, after the 7th day of culture in which the cells subjected to the initialization treatment are seeded on the feeder cells, the steps similar to the above are performed without waiting for the formation of colonies, and the test target cells are Whether or not it is a stem cell can be determined.

  Further, when the DNA methylation state of iPS cells is analyzed by the bisulfite sequencing method, many steps of PCR-amplifying the target gene region after bisulfite conversion, further cloning, and then sequencing are required. . Furthermore, it is desirable to confirm by electrophoresis whether or not the desired PCR amplification product was obtained before cloning. On the other hand, according to the present invention, since the PCR amplification product is directly subjected to the chromatographic analysis, the step of cloning is unnecessary, and the success or failure of PCR amplification can be confirmed by the peak intensity of the detection signal of chromatography. Therefore, according to the present invention, the DNA methylation state can be analyzed in fewer steps than in the bisulfite sequencing method.

  Further, in the case of accurately analyzing the DNA methylation state of a hemimethylated DNA or a gene having a different DNA methylation state among alleles, it is necessary to analyze many clones by the bisulfite sequencing method. On the other hand, in the chromatographic analysis according to the present invention, since hemimethylation of DNA and the difference in DNA methylation state between alleles are detected as the bimodal peak of the detection signal, the accuracy is improved even when the number of samples is small. The DNA methylation status can be analyzed well.

  Hereinafter, the present invention will be described in detail with reference to Examples, but the present invention is not limited to the following Examples.

Reference Example 1 Preparation of cells [Preparation of human iPS cells]
The human iPS cells were prepared based on the protocol “Human iPS cell establishment method using episomal vector” published by the Institute for iPS Cell Research, Kyoto University.
Three types of plasmids, pCXLE-hOct4-shp53, pCXLE-hSK, and pCXLE-hUL, were used as the plasmids used for the initialization.
Human dermal fibroblasts (cell number: JCRB0541, cell name: TIG-111) established from the skin of a 34-year-old Japanese woman were used in the experiment. The culture medium of the fibroblasts used was a DMEM (Sigma-Aldrich) supplemented with 10% Fetal bovine serum (Sigma-Aldrich) (10% FBS medium). The fibroblasts were cultured and maintained in a 100 mm culture dish at 37 ° C. and 5% CO ₂ . The plasmid was introduced by the following procedure. First, the medium supernatant of fibroblasts that had become confluent in a 100 mm culture dish was removed by suction, and 10 mL of PBS was added to wash the cells. PBS was removed by suction, 1 mL of 0.25% Trypsin / 1 mM EDTA solution (Invitrogen) was added per dish, and the dish was incubated at 37 ° C. for 3 minutes. When the cells floated, 4 mL of 10% FBS medium was added and suspended, and 6 × 10 ⁵ cells were collected in a 15 mL centrifuge tube. PBS was added to bring the total volume to 10 mL, followed by centrifugation at 200 × g for 5 minutes to remove the supernatant. The plasmid solution was prepared using pCXLE-hOct4-shp53, pCXLE-hSK, and pCXLE-hUL (1 μg each, 3 μg in total) using the Neon Transfection kit (Invitrogen). The cells were suspended in the plasmid solution, and the cell suspension was sucked with a Neon 100 μL chip and set in a gene transfer device Neon Transfection System (Invitrogen). Electroporation was performed under the conditions of Voltage: 1650 V, Width: 10 ms, and Pulse Number: 3 to introduce the plasmid into the cells.
After the introduction, the cells were rapidly seeded on a 6-well culture plate (Falcon) in which 3 mL of 10% FBS medium was previously dispensed and incubated under the conditions of 37 ° C. and 5% CO ₂ . On the next day (the second day of culture), the medium supernatant was removed by suction, 2 mL of 10% FBS medium was added, and the medium was replaced. Thereafter, the medium was exchanged once every two days.
Subcultured on feeder cells on day 7 of culture. The medium was removed by suction, and 2 mL of PBS was added to wash the cells. PBS was removed by suction, 0.3 mL per well of 0.25% Trypsin / 1 mM EDTA solution (Invitrogen) was added, and the mixture was incubated at 37 ° C. for 3 minutes. When the cells floated up, 2 mL of 10% FBS medium was added and suspended by pipetting. A 10% FBS medium was added to adjust the concentration to 1 × 10 ⁴ / mL, and 10 mL of the cell suspension was seeded on a 100 mm dish on which feeder cells had been seeded. As the feeder cells, SNL76 / 7 treated with mitomycin C was used.
On the 8th day of culture, the medium was replaced with a medium for human iPS cells. A medium for human iPS cells was prepared by adding 2.5 µL of 1 µg / µL bFGF solution (Reprocell) to 500 mL of Primate ES medium (Reprocell). Thereafter, the medium was exchanged once every two days.

Isolation of iPS cell colonies was performed between 21st and 30th day of culture. After the colonies grew and became visible, the colonies were picked before differentiation started. A colony having a size of about 2 mm or less was targeted for collection. To transfer the collected colonies, 200 μL of human iPS cell culture medium was dispensed per well into a 96-well plate. Using a small volume pipette such as a pipette for 10 μL, the colonies were removed under a stereoscopic microscope. The collected colonies were transferred to a 96-well plate and disrupted by pipetting until the colonies became small clusters (however, before the single cell). SNL feeder cells treated with mitomycin C were prepared in a 24-well plate, and the collapsed colonies were seeded on the SNL feeder cells. 300 μL of human iPS cell culture medium was added to the 24-well plate and cultured at 37 ° C. in a 5% CO ₂ incubator until it became 80% to 90% confluent.

  Passage of iPS cells was performed according to the method of Suemori et al. (Int. J. Biol. 48: 1149-1154, 2004).

[Preparation of human skin fibroblasts]
Human dermal fibroblasts (cell number: JCRB0541, cell name: TIG-111) were used as control cells before initialization. The human skin fibroblasts were purchased from JCRB cell bank, National Institute of Pharmaceutical Sciences. As the culture medium of the fibroblasts, 10% FBS medium was used. The fibroblasts were cultured and maintained in a 100 mm culture dish under the conditions of 37 ° C. and 5% CO ₂ .

Reference Example 2 Selection of iPS cells using phase contrast microscope iPS cells were selected by morphological observation of cells using a phase contrast microscope. The degree of cell differentiation was classified into the following three stages. Colonies with a compact sphere and clear edges are in the most undifferentiated stage, colonies in a stage where the thickness has decreased and the sphere has started to collapse a little are colonies in a moderately differentiated stage, cohesive The colonies whose shape collapsed and became flat were regarded as the most differentiated colonies. The most differentiated colonies were cut out with a pipette tip. The colonies at the most undifferentiated stage were selected as iPS cells and used for analysis. These cells were selected visually.

Reference Example 3 Preparation of anion exchange column In 2000 mL of 3 wt% polyvinyl alcohol (manufactured by Nippon Synthetic Chemical Industry) aqueous solution in a reactor equipped with a stirrer, 200 g of tetraethylene glycol dimethacrylate (manufactured by Shin Nakamura Chemical Co., Ltd.) and triethylene glycol diethylene A mixture of 100 g of methacrylate (manufactured by Shin-Nakamura Chemical Co., Ltd.), 100 g of glycidyl methacrylate (manufactured by Wako Pure Chemical Industries, Ltd.) and 1.0 g of benzoyl peroxide (manufactured by Kishida Chemical Co., Ltd.) was added. It heated with stirring and superposed | polymerized at 80 degreeC under nitrogen atmosphere for 1 hour. Next, as a hydrophilic monomer having a strong cationic group, 100 g of ethyl trimethyl ammonium ammonium chloride (manufactured by Wako Pure Chemical Industries, Ltd.) was dissolved in ion-exchanged water. This was added to the same reactor, and similarly, polymerization was carried out at 80 ° C. for 2 hours under a nitrogen atmosphere while stirring. The resulting polymer composition was washed with water and acetone to obtain coated polymer particles having a hydrophilic polymer layer having a quaternary ammonium group on the surface. The obtained coated polymer particles were measured using a particle size distribution measuring device (manufactured by AccuSizer780 / Particle Sizing Systems), and the average particle size was 10 μm.

  10 g of the obtained coated polymer particles were dispersed in 100 mL of ion-exchanged water to prepare a pre-reaction slurry. Next, 10 mL of N, N-dimethylaminopropylamine (manufactured by Wako Pure Chemical Industries, Ltd.) was added while stirring the slurry, and the mixture was reacted at 70 ° C. for 4 hours. After completion of the reaction, the supernatant was removed using a centrifuge (“Himac CR20G” manufactured by Hitachi Ltd.) and washed with ion-exchanged water. After washing, the supernatant was removed using a centrifuge. This washing with ion-exchanged water was repeated four more times to obtain a packing material for ion-exchange chromatography having a quaternary ammonium group and a tertiary amino group on the surface of the base material particles.

  The above-mentioned packing material for ion exchange chromatography was packed in a stainless steel column (column size: inner diameter 4.6 mm × length 20 mm) of a liquid chromatography system.

Reference Example 4 HPLC Analysis In the following example, the ion exchange chromatography analysis of the PCR amplification product was performed under the following conditions using the anion exchange column prepared in Reference Example 3.
System: LC-20A series (made by Shimadzu Corporation)
Eluent: Eluent A 25 mmol / L Tris-HCl buffer (pH 7.5)
Eluent B 25 mmol / L Tris-HCl buffer (pH 7.5)
+1 mol / L ammonium sulfate Analysis time: 15 minutes Elution method: The mixing ratio of the eluent B was linearly increased under the following gradient conditions.
0 minutes (eluent B 40%) → 10 minutes (eluent B 100%)
Sample: PCR amplification product obtained in Examples 1 to 3 described later Flow rate: 1.0 mL / min
Detection wavelength: 260nm
Sample injection volume: 5 μL
Column temperature: 70 ℃

Example 1 Discrimination between human iPS cells and human skin fibroblasts using determination of DNA methylation state in NANOG gene region by HPLC [Extraction of genomic DNA and bisulfite treatment]
For the promoter region of NANOG, which is one of the demethylation genes, a measurement sample was prepared in order to analyze the DNA methylation of the CpG site.
First, for the CpG island containing the above-mentioned CpG site, a primer was designed using Methyl Primer Express (registered trademark) software (Life Technologies).

  DNA was extracted from the iPS cells (4 strains) and the skin fibroblasts (TIG-111) prepared in Reference Example 1 using DNeasy Blood & Tissue Kit (manufactured by Qiagen) according to the manufacturer's instructions. The extracted DNA was treated with bisulfite using EZ DNA Methylation-Lighting Kit (ZYMO Research).

[PCR]
Using the treated DNA as a template, a 336 bp region containing the promoter region of the NANOG gene was PCR amplified. As the PCR primer, primer set 1 (SEQ ID NOS: 1 and 2) was used. The sequences of each primer are shown in Table 3. PCR was carried out using 25 ng of template DNA, 1 × Ex Taq Buffer (20 mM Mg ² + plus) (TaKaRa BIO), 200 μmol / L dNTP, 1.25 U TaKaRa Ex Taq HS (TaKaRa BIO), and 0.2 μmol / L. The reaction was carried out with 50 μL of the reaction solution containing the forward and reverse primers. The polymerase was activated at 95 ° C for 10 minutes. The DNA was amplified by a Veriti thermal cycler manufactured by Applied Biosystems under the conditions of 94 ° C. for 30 seconds, 55 ° C. for 30 seconds and 72 ° C. for 30 seconds for 38 cycles, and finally, extension at 72 ° C. for 7 minutes. After completion of PCR, 5 μL of the reaction solution was mixed with 1 μL of loading dye solution on a 2% agarose gel to which ethidium bromide had been added in advance, and the mixture was applied and electrophoresed to observe the PCR amplification product to obtain the desired PCR amplification product. I confirmed that.

[DNA methylation analysis of pluripotent stem cells and skin fibroblasts by HPLC method]
The PCR amplification products obtained from the iPS cells and human skin fibroblasts prepared above were subjected to HPLC analysis by the procedure of Reference Example 4, respectively. The obtained HPLC chromatogram is shown in FIG. The chromatogram in FIG. 1 is adjusted so that the peak heights are the same. As shown in FIG. 1, the retention time of the peak of the detection signal from iPS cells was later than the retention time of the peak of the detection signal of human dermal fibroblasts. This indicates that in the PCR-amplified gene region, the CpG site that was methylated in human skin fibroblasts was demethylated in iPS cells. Since this result is consistent with the finding that NANOG is a demethylated gene, it was shown that iPS cells can be identified by measuring the retention time of the peak of the chromatogram according to the method of the present invention. .. In addition, detection signals were obtained at substantially the same retention time between different iPS cell lines, indicating that iPS cells can be distinguished with good reproducibility.

Example 2 Discrimination of Human iPS Cells and Human Skin Fibroblasts Using Determination of DNA Methylation State by HPLC Method From each of the iPS cell line and human skin fibroblasts (TIG-111) prepared in Reference Example 1, Genomic DNA was extracted by the same procedure as in Example 1, treated with bisulfite, and subjected to PCR and HPLC. In PCR, the CpG region of each gene of Oct3 / 4, RAB25, SALL4, SP100, and UBE1L was amplified. PCR sets used were primer sets 2 to 6 (SEQ ID NOS: 3 to 12). The sequence and product size of each primer are shown in Table 4.

  The HPLC chromatograms obtained for each gene region (PCR products by primer sets 2 to 6) are shown in FIGS. Oct3 / 4 (primer set 2) in Fig. 2, RAB25 (primer set 3) in Fig. 3, SALL4 (primer set 4) in Fig. 4, SP100 (primer set 5) in Fig. 5, UBE1L (primer set). 6) is shown in FIG. The chromatograms in each figure are adjusted so that the peak heights are the same.

Example 3 Discrimination between human iPS cells and human skin fibroblasts using determination of DNA methylation state by HPLC method iPS cell line and human skin fibroblasts (TIG-111) prepared by the same procedure as in Reference Example 1 Genomic DNA was extracted from each of these by the same procedure as in Example 1, treated with bisulfite, and subjected to PCR and HPLC. As the primers for PCR, primer sets 7 to 10 (SEQ ID NOS: 13 to 19) were designed as primers for amplifying the CpG region of each gene of SLC22A3, Oct3 / 4, SP100, and UBE1L. In PCR, the polymerase was activated at 95 ° C for 4 minutes, and the DNA was applied to an Applied Biosystems Veriti thermal cycler for 40 cycles of 95 ° C for 30 seconds, 55 ° C for 30 seconds and 72 ° C for 60 seconds, and finally 72 ° C for 7 minutes. Was amplified under the condition of elongation. The annealing temperature was optimized to a temperature at which the amplification efficiency was high and non-specific amplification did not easily occur after examination. Table 4 shows the sequences and product sizes of the primers used in this analysis.

  The HPLC chromatograms obtained for each gene region of SLC22A3 and Oct3 / 4 (PCR products by primer sets 7 and 8, respectively) are shown in FIGS. The chromatograms in each figure are adjusted so that the peak heights are the same.

For genes classified as demethylated genes, namely Oct3 / 4, RAB25, SALL4, and SLC22A3, as shown in FIGS. 2 to 4, 7 and 8, the retention time of the peak of the detected signal from iPS cells was This was later than the retention time of the detection signal peak of human dermal fibroblasts. This result indicates that demethylation of various demethylated gene regions in iPS cells could be detected by the method of the present invention. On the other hand, in the genes classified as methylated genes, namely SP100 and UBE1L, as shown in FIGS. 5 and 6, the retention time of the peak of the detection signal of iPS cells was shorter than the detection signal of human skin fibroblasts. It was earlier than the peak retention time. This result indicates that the method of the present invention could detect methylation of various methylation gene regions in iPS cells.
From these results, it was shown that the iPS cells can be identified by measuring the retention time of the peak of the chromatogram for the methylated gene or the demethylated gene according to the method of the present invention.

Comparative Example 1 DNA methylation analysis of human iPS cells and human dermal fibroblasts by bisulfite sequencing method

Each PCR amplification product prepared in Example 1 and Example 2 was cloned, and the methylation state of the CpG site in the promoter region of each gene was analyzed using the bisulfite sequencing method.
The PCR amplification product was cloned and transformed using TA Cloning Kit manufactured by Biodynamics, and 20 to 24 colonies were collected. A plasmid was extracted from each colony and sequenced using M13 reverse primer to determine the DNA sequence after bisulfite treatment. In order to confirm the methylation state, analysis was performed using QUMA (http://quma.cdb.riken.jp/).

  The analysis results are shown in FIGS. 9 and 10. The results of determination of the methylation state agreed between the method of the present invention using the HPLC method and the bisulfite sequencing method. However, when analyzing changes in the methylation state of DNA by the bisulfite sequencing method, after PCR amplification, the amplification product is cloned, and a large number of samples obtained by cloning are analyzed one by one by sequencing or the like. Must be done and requires a great deal of time and effort. On the other hand, according to the method of the present invention, since the PCR amplification product is directly subjected to HPLC analysis, the step of cloning is unnecessary and the number of measurement cases can be small. In fact, the analysis by the bisulfite sequencing method takes about several days per gene region, whereas the method of the present invention can obtain the analysis result in about 10 minutes per gene region. Therefore, according to the method of the present invention, iPS cells can be identified quickly, easily, and with high throughput.

Claims (5)

A method for quality control of induced pluripotent stem cells, the method comprising :
Treating genomic DNA prepared from reprogrammed cells or cultures thereof with bisulfite;
PCR amplifying the bisulfite treated DNA with a primer set that amplifies the DNA of the CpG region of the demethylated gene;
Subjecting the resulting PCR amplification product to ion exchange chromatography to obtain a detection signal; and
When the retention time of the peak of the detection signal obtained from the reprogrammed cells or its culture is longer than the retention time of the peak of the control detection signal, the reprogrammed cells are artificially engineered. Determining that the cells are pluripotent stem cells,
Here, the detection signal of the control was obtained by treating genomic DNA prepared from uninitialized cells or cultures thereof with bisulfite, and then PCR-amplifying using the primer set. Is a detection signal obtained by subjecting the PCR amplification product to ion exchange chromatography,
Only including,
The demethylated gene is at least one selected from the group consisting of SLC22A3 and Oct3 / 4,
The primer set for amplifying the DNA of the CpG region of the demethylation gene is as follows:
When the demethylated gene is SLC22A3, a set of a primer consisting of SEQ ID NO: 13 and a primer consisting of SEQ ID NO: 14;
When the demethylation gene is Oct3 / 4, a set of a primer consisting of SEQ ID NO: 15 and a primer consisting of SEQ ID NO: 16,
Is the way. A method for quality control of induced pluripotent stem cells, the method comprising :
Treating genomic DNA prepared from reprogrammed cells or cultures thereof with bisulfite;
PCR-amplifying the bisulfite-treated DNA with a primer set that amplifies the DNA in the CpG region of the methylated gene;
Subjecting the resulting PCR amplification product to ion exchange chromatography to obtain a detection signal; and
When the retention time of the peak of the detection signal obtained from the reprogrammed cells or the culture thereof is shorter than the retention time of the peak of the control detection signal, the reprogrammed cells are artificially engineered. Determining that the cells are pluripotent stem cells,
Here, the detection signal of the control was obtained by treating genomic DNA prepared from uninitialized cells or cultures thereof with bisulfite, and then PCR-amplifying using the primer set. Is a detection signal obtained by subjecting the PCR amplification product to ion exchange chromatography,
Only including,
The methylated gene is at least one selected from the group consisting of SP100 and UBE1L,
The primer set for amplifying the DNA in the CpG region of the methylated gene is as follows:
When the methylated gene is SP100, a set of a primer consisting of SEQ ID NO: 17 and a primer consisting of SEQ ID NO: 10;
When the methylated gene is UBE1L, a set of a primer consisting of SEQ ID NO: 18 and a primer consisting of SEQ ID NO: 19,
Is the way. A method for quality control of induced pluripotent stem cells, the method comprising :
(1-1) treating genomic DNA prepared from reprogrammed cells or a culture thereof with bisulfite;
(1-2) treating genomic DNA prepared from uninitialized cells or a culture thereof with bisulfite;
(2-1) PCR-amplifying the bisulfite-treated DNA obtained in step (1-1) by using a primer set that amplifies the DNA in the CpG region of the demethylation gene;
(2-2) PCR amplification of the bisulfite-treated DNA obtained in step (1-1) by using a primer set that amplifies the DNA in the CpG region of the methylation gene;
(2-3) PCR-amplifying the bisulfite-treated DNA obtained in step (1-2) using a primer set that amplifies the DNA in the CpG region of the demethylated gene;
(2-4) PCR amplification of the bisulfite-treated DNA obtained in step (1-2) using a primer set that amplifies the DNA in the CpG region of the methylated gene;
(3-1) subjecting the PCR amplification product obtained in step (2-1) to ion exchange chromatography to obtain a detection signal;
(3-2) subjecting the PCR amplification product obtained in step (2-2) to ion exchange chromatography to obtain a detection signal;
(3-3) subjecting the PCR amplification product obtained in step (2-3) to ion exchange chromatography to obtain a detection signal;
(3-4) subjecting the PCR amplification product obtained in step (2-4) to ion exchange chromatography to obtain a detection signal;
(4) The retention time of the peak of the detection signal obtained in step (3-1) is longer than the retention time of the peak of the detection signal obtained in step (3-3), and -2) when the retention time of the peak of the detection signal obtained in step (3-4) is shorter than the retention time of the peak of the detection signal obtained in step (3-4), Determining that the cells are induced pluripotent stem cells,
Only including,
The demethylated gene is at least one selected from the group consisting of SLC22A3 and Oct3 / 4,
The primer set for amplifying the DNA of the CpG region of the demethylation gene is as follows:
When the demethylated gene is SLC22A3, a set of a primer consisting of SEQ ID NO: 13 and a primer consisting of SEQ ID NO: 14;
When the demethylation gene is Oct3 / 4, a set of a primer consisting of SEQ ID NO: 15 and a primer consisting of SEQ ID NO: 16,
And
The methylated gene is at least one selected from the group consisting of SP100 and UBE1L,
The primer set for amplifying the DNA in the CpG region of the methylated gene is as follows:
When the methylated gene is SP100, a set of a primer consisting of SEQ ID NO: 17 and a primer consisting of SEQ ID NO: 10;
When the methylated gene is UBE1L, a set of a primer consisting of SEQ ID NO: 18 and a primer consisting of SEQ ID NO: 19,
Is the way. A method for quality control of induced pluripotent stem cells, the method comprising :
(1-1) treating genomic DNA prepared from reprogrammed cells or a culture thereof with bisulfite;
(1-2) treating genomic DNA prepared from induced pluripotent stem cells or a culture thereof with bisulfite;
(2-1) A primer set for amplifying the DNA of the CpG region of the demethylation gene, and / or the DNA of the CpG region of the methylation gene, which is treated with the bisulfite salt obtained in the step (1-1), PCR amplification using a primer set that amplifies DNA;
(2-2) A primer set for amplifying the DNA of the CpG region of the demethylation gene, and / or the DNA treated with the bisulfite salt obtained in the step (1-2) of the CpG region of the methylation gene PCR amplification using a primer set that amplifies DNA;
(3-1) Step (2-1) subjecting the obtained PCR amplification product to ion exchange chromatography to obtain a detection signal;
(3-2) Step (2-2) subjecting the obtained PCR amplification product to ion exchange chromatography to obtain a detection signal;
(4) The reprogrammed cells when there is no difference in peak retention time between the detection signal obtained in step (3-1) and the detection signal obtained in step (3-2) Is determined to be induced pluripotent stem cells,
Only including,
The demethylated gene is at least one selected from the group consisting of SLC22A3 and Oct3 / 4,
The primer set for amplifying the DNA of the CpG region of the demethylation gene is as follows:
When the demethylated gene is SLC22A3, a set of a primer consisting of SEQ ID NO: 13 and a primer consisting of SEQ ID NO: 14;
When the demethylation gene is Oct3 / 4, a set of a primer consisting of SEQ ID NO: 15 and a primer consisting of SEQ ID NO: 16,
And
The methylated gene is at least one selected from the group consisting of SP100 and UBE1L,
The primer set for amplifying the DNA in the CpG region of the methylated gene is as follows:
When the methylated gene is SP100, a set of a primer consisting of SEQ ID NO: 17 and a primer consisting of SEQ ID NO: 10;
When the methylated gene is UBE1L, a set of a primer consisting of SEQ ID NO: 18 and a primer consisting of SEQ ID NO: 19,
Is the way. It said initialization treated cells are initialized treated skin fibroblasts, any one method according to claim 1-4.