WO2016060264A1 - Procédé de contrôle de la qualité de cellules souches - Google Patents

Procédé de contrôle de la qualité de cellules souches Download PDF

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WO2016060264A1
WO2016060264A1 PCT/JP2015/079370 JP2015079370W WO2016060264A1 WO 2016060264 A1 WO2016060264 A1 WO 2016060264A1 JP 2015079370 W JP2015079370 W JP 2015079370W WO 2016060264 A1 WO2016060264 A1 WO 2016060264A1
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dna
detection signal
pcr amplification
cells
cell
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卓也 與谷
博暁 太平
有理子 根本
栄一郎 砂村
健夫 久保田
邦夫 三宅
美沙 武居
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積水メディカル株式会社
国立大学法人山梨大学
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids

Definitions

  • the present invention relates to a stem cell quality control method using detection of methylated DNA. More specifically, after initializing the differentiated cell, the methylation state of DNA extracted from the cell derived from the cell subjected to the initialization process is analyzed, and the cell is initialized at the gene level. It is related with the method of determining the success or failure of.
  • pluripotent stem cells have the ability to differentiate into various tissues in the living body (differentiation versatility), application to drug discovery screening, disease mechanism research, and regenerative medical materials has been studied.
  • iPS cells induced pluripotent stem cells derived from cells are promising as regenerative medical materials because they have no ethical problems.
  • Patent Document 1 discloses a method of discriminating the degree of differentiation of pluripotent stem cells by analyzing the flatness of cultured cells with a microscope.
  • pluripotent stem cells when pluripotent stem cells are subcultured, the cell properties gradually change.
  • changes in cell properties 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 without morphological changes in the cells. Therefore, a conventional stem cell identification method may not be able to detect changes in cell properties.
  • Patent Document 2 discloses a method for evaluating characteristics such as pluripotency of a cell 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 stem cells by using a probe that binds to a sugar chain specific to undifferentiated cells. .
  • Patent Document 4 analyzes methylated and demethylated patterns of chromosomal DNA extracted from test stem cells by detecting methylated cytosine and hydroxymethylated cytosine and comparing them with reference stem cells.
  • a method of stem cell identification is disclosed that associates with the stage of stem cell passage or differentiation.
  • PCR Polymerase Chain Reaction
  • methylated cytosine remains cytosine, but unmethylated cytosine is amplified by replacing uracil with thymine.
  • the methylation state is analyzed using the difference between the bases cytosine and thymine generated in the sequence of the PCR amplification product.
  • the methods widely used based on this principle are the Methylation-Specific PCR method (methylation-specific PCR analysis method) described in Patent Document 5 and Non-Patent Document 1, and the bisulfite described in Non-Patent Documents 2 and 3. It is a sequence method.
  • methylation-specific PCR analysis method After DNA bisulfite treatment, PCR amplification using a methylated sequence-specific primer and an unmethylated sequence-specific primer, and agarose gel electrophoresis were sequentially performed. This is a method for determining the DNA methylation state of a target region based on the presence or absence. Although it is a methylated DNA analysis method that is still widely used today in that it allows quantitative analysis of methylated DNA without a special device, it requires labor and time in terms of using electrophoresis for analysis. There was a problem.
  • DNA is bisulfite treated, PCR amplified using a common primer for methylated DNA and unmethylated DNA, and the resulting PCR product cloned into TOPO-cloning vector (Invitrogen), etc.
  • TOPO-cloning vector Invitrogen
  • Ion exchange chromatography is widely used in fields such as biochemistry and medicine as a method that allows simple and accurate separation and analysis of biopolymers such as nucleic acids, proteins, and polysaccharides in a short time.
  • anion exchange chromatography is generally used in which the negative charges of phosphate contained in the nucleic acid molecules are used for separation.
  • Anion exchange chromatography columns packed with a column filler having a cationic functional group as an ion exchange group are already commercially available and used in various research fields.
  • methods for evaluating the quality of iPS cells include miRNA analysis, cell surface marker detection, cell morphology image analysis, epigenome analysis, and the like.
  • a method for evaluating epigenetic changes in iPS cells, particularly changes in the methylation state of DNA, in a rapid, simple and high throughput manner has not been realized.
  • the object of the present invention is to provide a method for analyzing the quality of iPS cells at a gene level quickly, conveniently and with high throughput.
  • the present inventors treated the initialization-treated cell DNA with bisulfite, and then amplified the PCR amplification product obtained by PCR. As a result, the ion exchange chromatography separated the PCR amplification product. It was found that the retention time of the resulting signal peak was different between reprogrammed iPS cells and non-reprogrammed somatic cells. Changes in DNA methylation accompanying cell reprogramming can be detected by the method using PCR amplification and ion exchange chromatography.
  • the method using the PCR amplification and ion exchange chromatography described above is based on the success or failure of cell reprogramming; 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 culture of somatic cells differentiated from pluripotent stem cells.
  • pluripotent stem cells iPS cells, embryonic stem cells, etc.
  • a method for quality control of induced pluripotent stem cells Treating genomic DNA prepared from reprogrammed cells or culture thereof with bisulfite; PCR amplification of the DNA treated with the bisulfite using 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 If the retention time of the detection signal peak obtained from the initialized cell or its culture is longer than the retention time of the control detection signal peak, the initialized cell is artificially Determining that it is a pluripotent stem cell;
  • the detection signal of the control was obtained by treating a genomic DNA prepared from uninitialized cells or a culture thereof with bisulfite and then PCR-amplifying with the primer set.
  • a method for quality control of induced pluripotent stem cells Treating genomic DNA prepared from reprogrammed cells or culture thereof with bisulfite; PCR amplification of the DNA treated with bisulfite using a primer set that amplifies the DNA of 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 detection signal peak obtained from the initialized cell or its culture is shorter than the retention time of the control detection signal peak, the initialized cell is artificially treated.
  • the detection signal of the control was obtained by treating a genomic DNA prepared from uninitialized cells or a culture thereof with bisulfite and then PCR-amplifying with the primer set.
  • a detection signal obtained by subjecting a PCR amplification product to ion exchange chromatography. Including a method.
  • a method for quality control of induced pluripotent stem cells (1-1) treating genomic DNA prepared from reprogrammed cells or culture thereof with bisulfite; (1-2) treating genomic DNA prepared from uninitialized cells or a culture thereof with bisulfite; (2-1) PCR amplification of the DNA treated with bisulfite obtained in step (1-1) using a primer set that amplifies the DNA of the CpG region of the demethylated gene; (2-2) PCR amplification of the DNA treated with bisulfite obtained in step (1-1) using a primer set for amplifying the DNA of the CpG region of the methylated gene; (2-3) PCR amplification of the DNA treated with bisulfite obtained in step (1-2) using a primer set that amplifies the DNA of the CpG region of the demethylated gene; (2-4) PCR amplification of the DNA treated with bisulfite obtained in step (1-2) using a primer set that amplifies the DNA in the CpG region of the methylated gene; (3
  • a method for quality control of induced pluripotent stem cells (1-1) treating genomic DNA prepared from reprogrammed cells or culture thereof with bisulfite; (1-2) treating genomic DNA prepared from an induced pluripotent stem cell or a culture thereof with bisulfite; (2-1) The primer set for amplifying the DNA of the CpG region of the demethylated gene and / or the CpG region of the methylated gene of the DNA treated with the bisulfite obtained in step (1-1) PCR amplification using a primer set that amplifies DNA; (2-2) The primer set for amplifying the DNA of the CpG region of the demethylated gene and / or the CpG region of the methylated gene of the DNA treated with the bisulfite obtained in step (1-2) PCR amplification using a primer set that amplifies DNA; (3-1) Step (2-1) Obtaining a detection signal by subjecting the obtained PCR amplification product to ion exchange chromatography; (3-2) Step (2-2) Obtaining
  • a primer set for amplifying DNA of the CpG region of the demethylated gene is 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, and SEQ ID NO: 7
  • a primer set for amplifying DNA of the CpG region of the methylated gene is 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 present invention makes it possible to evaluate the quality of pluripotent stem cells quickly, simply, and at high throughput by measuring the methylation state of DNA extracted from cells.
  • whether or not iPS cells have been established by induction of iPS cells from differentiated somatic cells such as dermal fibroblasts, the pluripotency of established stem cells, or the differentiation state of differentiation-induced cells. Can be determined at the gene level.
  • the present invention analyzes the epigenetic state of a cell, which cannot be evaluated only by conventional morphological observation or expression analysis of an undifferentiated marker, by detecting a difference in the methylation state of chromosomal DNA.
  • HPLC chromatograms of PCR amplification products in the NANOG gene region of iPS cells and human skin fibroblasts HPLC chromatograms of PCR amplification products in the Oct3 / 4 gene region of iPS cells and human skin fibroblasts.
  • HPLC chromatograms of PCR amplification products in the RAB25 gene region of iPS cells and human skin fibroblasts HPLC chromatograms of PCR amplification products in the SALL4 gene region of iPS cells and human skin fibroblasts.
  • HPLC chromatograms of PCR amplification products in the UBE1L gene region of iPS cells and human skin fibroblasts HPLC chromatograms 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.
  • Black circle methylated CpG site, white circle: unmethylated CpG site, the numerical value below each group represents the methylation rate (%).
  • stem cell has the same property as self even after cell division through the ability to differentiate into cells specialized for other than self (may be referred to as “mature”). It means a cell that has the ability to generate cells (self-replicating ability).
  • Stem cells include highly pluripotent stem cells such as embryonic stem cells (ES cells), less pluripotent stem cells such as adult stem cells, and the like.
  • ES cells embryonic stem cells
  • pluripotent stem cells such as adult stem cells, and the like.
  • an adherent cell culture method in a culture vessel is generally used, but is not limited thereto.
  • pluripotency means the ability to differentiate into one of the three germ layers: endoderm, mesoderm, or ectoderm.
  • pluripotent cell means a cell capable of differentiating into a cell belonging to any of the three germ layers, a direct progeny of an totipotent cell, an embryonic stem cell, an adult stem cell, and an artificial cell A pluripotent stem cell is illustrated.
  • the “undifferentiated state” means a state having both the differentiation ability and the self-replication ability, which are the characteristics of stem cells. However, if the cell properties change, such as a change in differentiation potential between the parent cell and the daughter cell, the daughter cell is differentiated.
  • the “treatment for maintaining the undifferentiated state” is a treatment for maintaining the differentiation ability and the self-replication ability so as not to be lost, and is not particularly limited. For example, bFGF (basic Fibroblast in a stem cell culture medium) Processing such as addition of Growth Factor) or LIF (Leukemia Inhibitory Factor) is included.
  • an “artificial pluripotent stem cell” is an original somatic cell that has the ability to differentiate into cells having all the properties of three germ layers by pluripotency induction treatment (pluripotency). Means cells that have both self-replicating ability. Induced pluripotent stem cells are generally referred to as iPS cells. “Initialization process” (also synonymous with “pluripotency induction process”) is a process for imparting pluripotency and self-replication ability. For example, four genes of Sox2, Oct3 / 4, Klf4, and Myc are assigned. Examples of the processing such as introduction are not limited to these.
  • differentiation means that a relatively undifferentiated cell such as a stem cell changes and specializes, and “differentiated cell” means the specialized cell.
  • “Differentiation” includes various stages from differentiation from totipotent cells to less pluripotent stem cells to differentiation into terminally differentiated cells. Examples of “differentiated cells” include somatic cells of a living body, cells obtained by inducing differentiation of stem cells and differentiated in a tissue-specific manner. “Differentiation induction” refers to the specialization of relatively undifferentiated cells such as stem cells.
  • Methods for inducing differentiation include addition of growth factors such as BMP (Bone Morphogenic Protein) and Wnt into the cell culture medium, addition of differentiation induction factors such as retinoic acid into the cell culture medium, or gene transfer such as MyoD.
  • growth factors such as BMP (Bone Morphogenic Protein) and Wnt
  • differentiation induction factors such as retinoic acid into the cell culture medium
  • gene transfer such as MyoD.
  • the present invention is not limited to these.
  • initialization (reprogramming) is used synonymously with pluripotency induction, erases and reconstitutes epigenetic markers such as DNA methylation, and changes the differentiation state of a certain cell. It means to be differentiated.
  • an example of cell reprogramming is to induce pluripotent stem cells from cells that do not have pluripotency.
  • dedifferentiation means that a certain cell is brought into an immature state. Dedifferentiation may mean returning to the initial or developing state in which a cell has differentiated, and can differentiate from a cell that cannot produce cells other than the germ layer to which it belongs to another germ layer cell. It may be a state.
  • Dedifferentiation includes, for example, that a cell having no ability to differentiate from three germ layers acquires the ability to differentiate from three germ layers. Dedifferentiation also includes the generation of pluripotent stem cells. Any method may be used as a method for initialization or dedifferentiation (initialization treatment or dedifferentiation treatment). For example, a known method such as the method described in International Publication No. 2011-016588 is applied. it can.
  • somatic cell means a cell other than a cell that directly transmits its DNA to the next generation, such as a germ cell, for example, an egg or sperm.
  • Somatic cells include, for example, cells that retain pluripotency such as adult stem cells, and terminally differentiated cells such as dermal fibroblasts. Usually, the pluripotency of somatic cells is limited or somatic cells have no pluripotency at all.
  • somatic cells may be naturally occurring, genetically altered, or cultures thereof.
  • demethylated gene refers to an iPS prepared by performing an initialization process, whereas the DNA of the CpG site is methylated in a somatic cell before the initialization process. It means a gene that is not methylated in cells.
  • a “methylated gene” is prepared by performing an initialization process, whereas the DNA of the CpG site is not methylated in a somatic cell before the initialization process. It means a methylated gene in iPS cells.
  • examples of demethylated genes include NANOG, Oct3 / 4, RAB25, SALL4, and SLC22A3, and examples of methylated 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 transcription factor 3; Octer-binding transcription factor 4; POU dominant, cranc1c5, POU dominant, cranc 1
  • 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 specified by RefSeq ID: NP_001073860 or NP_003104 (nuclear autoantigen Sp-100).
  • 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 that encodes a protein (Solution carrier family 22 member 3) specified by RefSeq ID: NP_068812.
  • DNA methylation means that the carbon at the 5-position of cytosine is methylated in DNA.
  • detecting methylation of DNA means measuring the presence / absence or abundance of methylated DNA in the DNA to be detected, the ratio of the abundance, or the methylation rate of the DNA. It means to do.
  • the “DNA methylation rate” means the rate at which cytosine at the CpG site is methylated in the DNA to be detected. For example, in the CpG island of the specific DNA to be detected , Expressed as a ratio of the number of methylated cytosines to the total number of cytosines (methylated cytosine and unmethylated cytosine).
  • CpG site means a site where cytosine (C) and guanine (G) have a phosphodiester bond (p) in DNA.
  • CpG region or CpG island
  • p phosphodiester bond
  • the CpG region of a (some) gene preferably means a CpG region existing in the coding region of the gene or a region close thereto, or “the CpG site of a (some) gene” Preferably, it means a CpG site present in the coding region of the gene or a region close thereto, more preferably a CpG site contained in the CpG region. Also preferably, “the CpG site or CpG region of a gene” means a CpG site or CpG region present in the promoter of the gene and its surrounding region.
  • the “promoter of gene (and) and its peripheral region” means an exon region adjacent to the promoter region and its downstream, and an intron region adjacent to the exon region.
  • a CpG site or CpG region of a specific gene can be identified based on a method such as MassARRAY method or pyrosequencing.
  • whether or not the test cell is a pluripotent stem cell is determined using the demethylated gene of the test cell or DNA methylation in the methylated gene as an index.
  • the demethylation of the demethylated gene DNA and the methylation of the methylated gene DNA both mean that the test cell has been initialized, that is, the test cell is a pluripotent stem cell.
  • DNA methylation is detected 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 using the peak height and retention time of the detection signal in the ion exchange chromatography as an index.
  • the retention time of the detection signal peak is related to the DNA methylation rate; that is, an increase in the retention time represents a decrease in the DNA methylation rate, whereas a decrease in the retention time represents an increase in the DNA methylation rate.
  • the peak derived from DNA having a high methylation rate in the detection signal is high, the presence ratio of methylated DNA is high.
  • the detection signal from the test cell group is compared with the detection signal from the control cell group.
  • the control cell group include negatively differentiated adult tissue cells and uninitialized somatic cells.
  • Positive controls include pluripotent stem cells and established artificial pluripotent stem cells. Etc. If the detection signal from the test cell group has a pattern different from the detection signal from the control cell group, the target DNA of the test cell group is determined to be demethylated or methylated compared to 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, it is determined that the DNA methylation states of the test cell group and the control cell group are equivalent.
  • the present invention provides a method for quality control of induced pluripotent stem cells comprising: Treating genomic DNA prepared from reprogrammed cells or culture thereof with bisulfite; PCR amplification of the DNA treated with the bisulfite using 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 If the retention time of the detection signal peak obtained from the initialized cell or its culture is longer than the retention time of the control detection signal peak, the initialized cell is artificially Determining that it is a pluripotent stem cell;
  • the detection signal of the control was obtained by treating a genomic DNA prepared from uninitialized cells or a culture thereof with bisulfite and then PCR-amplifying with the primer set. This is a detection signal obtained by subjecting the PCR amplification product to ion exchange chromatography.
  • the present invention provides a method for quality control of induced pluripotent stem cells comprising: Treating genomic DNA prepared from reprogrammed cells or culture thereof with bisulfite; PCR amplification of the DNA treated with bisulfite using a primer set that amplifies the DNA of 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 detection signal peak obtained from the initialized cell or its culture is shorter than the retention time of the control detection signal peak, the initialized cell is artificially treated.
  • the detection signal of the control was obtained by treating a genomic DNA prepared from uninitialized cells or a culture thereof with bisulfite and then PCR-amplifying with the primer set. This is a detection signal obtained by subjecting the PCR amplification product to ion exchange chromatography.
  • the present invention provides a method for quality control of induced pluripotent stem cells comprising: (1) treating genomic DNA prepared from reprogrammed cells or culture thereof, and genomic DNA prepared from non-initialized cells or culture thereof with bisulfite; (2) PCR amplification of each DNA treated with the bisulfite obtained in step (1) using a primer set for amplifying 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 initialization-treated cell or culture thereof obtained in step (3) is the detection signal obtained from the cell not subjected to initialization treatment or the culture thereof. When the retention time is longer than the peak retention time, the initialized cell is determined to be an induced pluripotent stem cell.
  • the present invention provides a method for quality control of induced pluripotent stem cells comprising: (1 ′) treatment of genomic DNA prepared from cells that have been reprogrammed or culture thereof, and genomic DNA prepared from cells that have not been reprogrammed or culture thereof, with bisulfite; (2 ′) PCR amplification of each DNA treated with the bisulfite obtained in step (1 ′) using 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 time obtained from the cells that have not been initialized or the culture thereof, in which the retention time of the peak of the detection signal obtained from the cells or cultures that have been initialized in step (3 ′) When the retention time of the signal peak is shorter than that, the initialized cell is determined to be an induced pluripotent stem cell.
  • the present invention provides a method for quality control of induced pluripotent stem cells comprising: (1 ′′ -1) treating genomic DNA prepared from the reprogrammed cells or culture thereof with bisulfite; (1 ''-2) treating genomic DNA prepared from uninitialized cells or culture thereof with bisulfite; (2 ′′ -1) PCR amplification of the DNA treated with bisulfite obtained in step (1 ′′ -1) using a primer set that amplifies the DNA of the CpG region of the demethylated gene ; (2 ′′ -2) PCR amplification of the DNA treated with bisulfite obtained in step (1 ′′ -1) using a primer set that amplifies the DNA of the CpG region of the methylated gene; (2 ''-3) PCR amplification of the DNA treated with bisulfite obtained in step (1 ''-2) using a primer set that amplifies the DNA of the CpG region of the demethylated gene ; (2 ′′ -4) PCR amplification of the DNA
  • the present invention provides a method for quality control of induced pluripotent stem cells comprising: (1 ''') treating genomic DNA prepared from reprogrammed cells or culture thereof, as well as genomic DNA prepared from non-initialized cells or culture thereof, with bisulfite. ; (2 ′′ ′-1) Each DNA treated with the bisulfite obtained in step (1 ′ ′′) was subjected to PCR using a primer set that amplifies the DNA in the CpG region of the demethylated gene.
  • the signal obtained from the cell subjected to the initialization treatment is longer than the retention time of the peak of the signal obtained from the cell or its culture. If the retention time of the peak is shorter than the retention time of the peak of the signal obtained from the uninitialized cell, Determining that the cell that has been initialized is an induced pluripotent stem cell.
  • the present invention provides a method for quality control of induced pluripotent stem cells comprising: (1 ''''-1) treating genomic DNA prepared from the reprogrammed cells or culture thereof with bisulfite; (1 ''''-2) treating genomic DNA prepared from an induced pluripotent stem cell or a culture thereof with bisulfite; (2 ''''-1) Primer set for amplifying the DNA of the CpG region of the demethylated gene from the DNA treated with bisulfite obtained in step (1 ''''-1), and / or Or PCR amplification using a primer set that amplifies the DNA of the CpG region of the methylated gene; (2 ′′ ′′-2) a primer set for amplifying the DNA treated with the bisulfite obtained in step (1 ′′ ′′-2), the CpG region of the demethylated gene, and / or Or PCR amplification using a primer set that amplifies the DNA of the CpG region of the methylated gene; (3 ′′
  • the present invention provides a method for quality control of induced pluripotent stem cells comprising: (1 '''') treating genomic DNA prepared from reprogrammed cells or culture thereof, as well as genomic DNA prepared from induced pluripotent stem cells or culture thereof, with bisulfite.
  • step (2 ′′ ′ ′′) a primer set for amplifying the DNA of the CpG region of the demethylated gene from each DNA treated with the bisulfite obtained in step (1 ′ ′′ ′′), and / or PCR amplification using primer sets that amplify DNA of the CpG region of the methylated gene, respectively;
  • step (2 ′ ′′ ′′) Each PCR amplification product obtained in step (2 ′ ′′ ′′) is subjected to ion exchange chromatography to obtain a detection signal;
  • (4 ''''') Detection signal obtained from the initialized cell obtained in step (3''''') or its culture and detection obtained from induced pluripotent stem cell or its culture When there is no difference in peak retention time from the signal, the initialized cell is determined to be an induced pluripotent stem cell.
  • the method of using the uninitialized cell as a control and the method of using the induced pluripotent stem cell as a control, and the initialized cell is an induced pluripotent You may determine whether it is a sex stem cell.
  • the “initialized cell” to be tested in the method of the present invention is preferably a nucleated cell subjected to an initialization process, more preferably a tissue cell subjected to an initialization process, such as an initial cell. Skin fibroblasts that have been subjected to an aging treatment, peripheral blood cells that have been subjected to an initialization treatment, and the like.
  • the initialization process is not particularly limited as long as it can induce cell initialization. For example, a procedure for producing iPS cells (for example, see Japanese Patent No. 5467223). Gene transfer according to (but not limited to).
  • an “uninitialized cell” may be the same type of cell as the above-mentioned initialized cell, but not initialized.
  • an “artificial pluripotent stem cell” as a control in the method of the present invention a cell derived from an established artificial pluripotent stem cell line, an induced pluripotent stem cell that has been confirmed to be initialized, and the like can be used. .
  • the organism serving as a source of “cells” such as stem cells and differentiated cells used in the present invention is not limited to humans, but mammals other than humans, such as monkeys, apes, dogs, mice, rats, pigs, goats, Examples include guinea pigs, birds and fish.
  • the method for preparing genomic DNA from the above “cells” and “cell cultures” is not particularly limited, and known methods can be appropriately selected and used.
  • Known methods for preparing DNA include the phenol chloroform method, or commercially available DNA extraction kits such as DNeasy Blood & Tissue Kit (Qiagen), QIAamp DNA Mini kit (Qiagen), and Clean Columns (NexTec).
  • DNA extraction method using 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.
  • the extracted genomic DNA is treated with bisulfite.
  • a method of the bisulfite treatment of DNA A well-known method can be selected suitably and can be used.
  • Known methods for treating bisulfite include, for example, EZ DNA Methylation-Lighting Kit (manufactured by ZYMO Research), EpiTect Bisulfite Kit (48) (manufactured by Qiagen), and MethylEasy (HumanGenet). And a commercially available kit such as Cells-to-CpG Bisulfite Conversion Kit (Applied Biosystems), CpGenome Turbo Bisulfite Modification Kit (manufactured by MERCK MILLIPORE).
  • the DNA treated with bisulfite is 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.
  • 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 includes SP100 and UBE1L.
  • the group can be mentioned. More preferably, one or more selected from the group consisting of the RAB25 gene, the SALL4 gene, and the SLC22A3 gene having a large difference in retention time of detection signals by chromatography and a good peak shape is selected.
  • the peak shape is good generally means that the peak is approximately symmetric, no peak reading or tailing is observed, the peak has no shoulder, the peak is sharp, and the like.
  • the chain length of the PCR amplification product can be appropriately selected in consideration of factors such as shortening the PCR amplification time, shortening the analysis time in ion exchange chromatography, and maintaining separation performance.
  • the chain length of the PCR amplification product when DNA bisulfite having a large number of CpG sites is used as a template is preferably 1000 bp or less, more preferably 700 bp or less, and even more preferably 500 bp or less.
  • the chain length of the PCR amplification product when DNA bisulfite having a small number of CpG sites is used as a template is preferably 1000 bp, more preferably 700 bp, and even more preferably 500 bp, and nonspecific hybridization in PCR.
  • the lower limit is 30 to 40 bp which is the chain length of the PCR amplification product when using a primer near 15 mer that can avoid the above.
  • the PCR amplification product has a chain length of preferably 100 to 500 bp, and more preferably 250 to 450 bp.
  • the primer it is preferable to design the primer so that the content of the region corresponding to the CpG site in the PCR amplification product is rich.
  • the number of bases derived from cytosine at the CpG site is preferably 2% or more, more preferably 5% or more with respect to the number of bases (chain length) of the PCR amplification product.
  • the PCR amplification product contains at least 3, preferably 3 to 15, bases derived from cytosine at the CpG site where the methylation state changes before and after the initialization process. The change in the DNA methylation rate can be detected by ion exchange chromatography analysis described later.
  • the primer set shown in Table 1 can be mentioned.
  • the primer set used in the method of the present invention to amplify the DNA of the CpG region of the methylated gene the primer sets described in Table 2 can be mentioned.
  • the ion exchange chromatography performed in the present invention is preferably anion exchange chromatography.
  • the column packing material 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 surface of the packing shown in Patent Document 6 is strongly cationic. Base particles having both groups and weak cationic groups are preferred.
  • the strong cationic group means a cationic group that dissociates in a wide range of pH 1 to 14. That is, the strong cationic group can be kept dissociated (cationized) without being affected by the pH of the aqueous solution.
  • the quaternary ammonium group is an example of the strong cationic group.
  • Specific examples include trialkylammonium groups such as a trimethylammonium group, a triethylammonium group, and a dimethylethylammonium group.
  • Examples of the counter ion of the strong cationic group include halide ions such as chloride ions, bromide ions, and iodide ions.
  • the amount of the strong cationic group introduced onto the surface of the substrate particles is not particularly limited, but a preferable lower limit per dry weight of the filler is 1 ⁇ eq / g, and a preferable upper limit is 500 ⁇ eq / g.
  • a preferable lower limit per dry weight of the filler is 1 ⁇ eq / g
  • a preferable upper limit is 500 ⁇ eq / g.
  • the amount of the strong cationic group is less than 1 ⁇ eq / g, the holding power is weak and the separation performance may be deteriorated.
  • the amount of the strong cationic group exceeds 500 ⁇ eq / g, the holding power becomes too strong and the PCR amplification product cannot be easily eluted, and problems such as an excessive analysis time may occur.
  • 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 dissociation state changes. That is, when the pH is higher than 8, the protons of the weak cationic group are dissociated, and the proportion not having a positive charge increases. On the other hand, when the pH is lower than 8, the weak cationic group becomes protonated and the proportion of positive charges increases.
  • 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 desirable.
  • the amount of the weak cationic group introduced onto the surface of the base particle is not particularly limited, but a preferable lower limit per dry weight of the filler is 0.5 ⁇ eq / g, and a preferable upper limit is 500 ⁇ eq / g.
  • a preferable lower limit per dry weight of the filler is 0.5 ⁇ eq / g
  • a preferable upper limit is 500 ⁇ eq / g.
  • the amount of the weak cationic group is less than 0.5 ⁇ eq / g, the separation performance may not be improved because the amount is too small. If the amount of the weak cationic group exceeds 500 ⁇ eq / g, the holding power becomes too strong as in the case of the strong cationic group, so that 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 substrate particle can be measured by quantifying the nitrogen atom contained in the amino group.
  • An example of a method for quantifying nitrogen is the Kjeldahl method.
  • the nitrogen contained in the strong cationic group after the polymerization is quantified, and then the strong cationic group and the weak cationic group after the introduction of the weak cationic group.
  • the amount of weak cationic group introduced later can be calculated by quantifying the nitrogen contained in. By quantifying in this manner, the amount of strong cationic group and the amount of weak cationic group can be adjusted within the above range when preparing the filler.
  • the base particle for example, synthetic polymer fine particles obtained using a polymerizable monomer, inorganic fine particles such as silica, etc. can be used.
  • the particles are desirable.
  • the hydrophobic crosslinked polymer is a hydrophobic crosslinked polymer obtained by copolymerizing at least one hydrophobic crosslinkable monomer and a monomer having at least one reactive functional group. Any of the hydrophobic cross-linked polymers obtained by copolymerizing a hydrophobic cross-linkable monomer, a monomer having at least one reactive functional group and at least one hydrophobic non-cross-linkable monomer There may be.
  • the hydrophobic crosslinkable monomer is not particularly limited as long as it has two or more vinyl groups in one monomer molecule.
  • ethylene glycol di (meth) acrylate, polyethylene glycol di (meth) Di (meth) acrylates such as acrylate, propylene glycol di (meth) acrylate, polypropylene glycol di (meth) acrylate, tri (meth) such as trimethylol methane tri (meth) acrylate, tetramethylol methane tri (meth) acrylate
  • acrylic esters, tetra (meth) acrylic esters, and aromatic compounds such as divinylbenzene, divinyltoluene, divinylxylene, and divinylnaphthalene.
  • the above (meth) acrylate means acrylate or methacrylate
  • (meth) acryl means acryl or methacryl.
  • 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 hydrophobic properties.
  • 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 monomers such as styrene and methylstyrene.
  • the hydrophobic cross-linked polymer is obtained by copolymerizing the hydrophobic cross-linkable monomer and the monomer having a reactive functional group, the hydrophobic property 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.
  • the strong cationic group and the weak cationic group are preferably derived from independent monomers.
  • the filler for ion exchange chromatography used in the present invention is a hydrophilic polymer having a strong cationic group copolymerized on the surface of the hydrophobic crosslinked polymer particles and the hydrophobic crosslinked polymer particles. It is preferable that a weak 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 a hydrophilic monomer having one or more strong cationic groups. What is necessary is just to contain a segment. That is, as a method for producing the hydrophilic polymer having a strong cationic group, a method of polymerizing a hydrophilic monomer having a strong cationic group alone, a hydrophilic property having two or more strong cationic groups. 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.
  • the hydrophilic monomer having a strong cationic group is preferably one having a quaternary ammonium group.
  • ethyl triethylammonium chloride ethyl dimethylethylammonium methacrylate, ethyl dimethylbenzylammonium methacrylate, ethyl dimethylbenzylammonium acrylate, ethyl triethylammonium acrylate, ethyl dimethylethylammonium acrylate
  • Examples include chloride, acrylamidoethyltrimethylammonium chloride, acrylamidoethyltriethylammonium chloride, acrylamidoethyldimethylethylammonium chloride, and the like.
  • a method for introducing the weak cationic group into the surface of the coated polymer particle 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 comprising a hydrophobic crosslinked polymer having a segment derived from a monomer having a glycidyl group is used.
  • the carboxy group produced by hydrolysis and the reagent having a tertiary amino group are then combined with the reagent. And a method such as condensation with Bojiimido.
  • the 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 comprising a hydrophobic crosslinked polymer having a segment derived from a monomer having an isocyanate group A method of copolymerizing a hydrophilic monomer having a group and then reacting a reagent having a tertiary amino group with an isocyanate group is preferred.
  • the reagent having a tertiary amino group to be reacted with a reactive functional group such as a glycidyl group or an isocyanate group is not particularly limited as long as the reagent has a tertiary amino group and a functional group capable of reacting with the reactive functional group.
  • a 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 such functional groups 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 An amine etc. are mentioned.
  • the relative position 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 substrate rather than the weak cationic group. It is preferable to be at a position far from the surface of the particle, that is, outside. For example, it is preferable that the weak cationic group is within 30 mm from the surface of the base particle, and the strong cationic group is within 300 mm from the base particle surface, and is outside the weak cationic group.
  • the average particle diameter of the base particles used in the ion exchange chromatography filler used in the present invention is not particularly limited, but a preferred lower limit is 0.1 ⁇ m and a preferred upper limit is 20 ⁇ m. If the average particle size is less than 0.1 ⁇ m, the inside of the column may become too high, resulting in poor separation. When the average particle diameter exceeds 20 ⁇ m, the dead volume in the column becomes too large, which may cause poor separation.
  • the average particle diameter indicates a volume average particle diameter, and can be measured using a particle size distribution measuring apparatus (such as AccuSize 780 / Particle Sizing Systems).
  • composition of the eluent used in the ion exchange chromatography performed in the present invention known conditions can be used.
  • buffers or organic solvents containing known salt compounds it is preferable to use buffers or organic solvents containing known salt compounds. Specifically, for example, Tris-HCl buffer, TE buffer consisting of 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. By setting in this range, it is considered that 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 is 9.
  • Examples of the salt contained in the eluent include salts consisting of halides such as sodium chloride, potassium chloride, sodium bromide, potassium bromide and alkali metals; calcium chloride, calcium bromide, magnesium chloride, magnesium bromide. Salts composed of halides such as alkaline earth metals and the like; inorganic acid salts such as sodium perchlorate, potassium perchlorate, sodium sulfate, potassium sulfate, ammonium sulfate, sodium nitrate, and potassium nitrate can be used. Moreover, 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 the preferred lower limit is 10 mmol / L, the preferred upper limit is 2000 mmol / L, the more preferred lower limit is 100 mmol / L, and the more preferred upper limit is 1500 mmol / L. L.
  • the eluent used in the ion exchange chromatography used in the present invention contains anti-chaotropic ions in order to further improve the separation performance.
  • Anti-chaotropic ions have a property opposite to that of kaorotopic ions and have a function of stabilizing the hydration structure. Therefore, there is an effect of strengthening the hydrophobic interaction between the filler and the nucleic acid molecule.
  • the main interaction of the ion exchange chromatography used in the present invention is electrostatic interaction, but in addition, separation performance is enhanced by utilizing the action of hydrophobic interaction.
  • Anti-chaotropic 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 + ). Among these ion combinations, sulfate ions and ammonium ions are preferably used.
  • the anti-chaotropic ions can be used either alone or in combination.
  • a part of the above-mentioned antichaotropic ion includes a salt or a buffer component contained in the eluent. When such a component is used, it has both a property as a salt or a buffer capacity contained in the eluent and a property as an anti-chaotropic ion, which is preferable for the present invention.
  • the concentration of anti-chaotropic ions in the eluent for ion-exchange chromatography used in the present invention may be appropriately adjusted according to the analysis target, but is preferably 2000 mmol / L or less as the anti-chaotropic salt.
  • a method of performing gradient elution with the concentration of the antichaotropic salt in the range of 0 to 2000 mmol / L 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 performing precise concentration adjustment by the high pressure gradient method is preferred.
  • the anti-chaotropic ion may be added to only one type of eluent used for elution, or may be added to a plurality of types of eluent.
  • the anti-chaotropic ion may have both the role of enhancing the hydrophobic interaction between the packing material and the PCR amplification product or the buffering capacity, and the effect of eluting the PCR amplification product from the column.
  • the column temperature when analyzing PCR amplification products by 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.
  • the column temperature of the ion exchange chromatography is less than 30 ° C., the hydrophobic interaction between the packing material and the PCR amplification product becomes weak, and it becomes difficult to obtain a desired separation effect.
  • the column temperature of ion exchange chromatography is less than 45 ° C.
  • the PCR amplification product (methylated DNA sample) of methylated DNA treated with bisulfite and the PCR amplified product of unmethylated DNA treated with bisulfite The difference in retention time from the unmethylated DNA sample is small.
  • 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 is further widened, and each peak becomes clearer. It is possible to detect methylation of DNA with good quality.
  • both of the methylated DNA sample and the unmethylated DNA sample are clearly separated when the column temperature of the ion exchange chromatography is increased, both of the methylated DNA sample and the unmethylated DNA sample are present according to the ratio of the methylated DNA to the unmethylated DNA in the sample DNA. A difference tends to occur in the peak area or peak height of the holding time. Therefore, the higher the column temperature, the presence of each of methylated and unmethylated DNA in the sample DNA based on the area or height of the retention time peak between the methylated and unmethylated DNA samples It becomes easier to measure the quantity and the abundance ratio.
  • the column temperature of ion exchange chromatography is 90 ° C. or higher, the double strands of the nucleic acid molecules in the PCR amplification product are dissociated, which is not preferable for analysis. Furthermore, if the column temperature is 100 ° C. or higher, the eluent may be boiled, which is not preferable for analysis. Therefore, the column temperature when analyzing the PCR amplification product by 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., more preferably 45 ° C. It is more than 55 degreeC and less than 90 degreeC, More preferably, it is 55 degreeC or more and 85 degrees C or less, More preferably, it is 60 degreeC or more and 85 degrees C or less.
  • the amount of sample injected into the ion exchange chromatography column is not particularly limited, and may be appropriately adjusted according to the ion exchange capacity and sample concentration of the column.
  • the flow rate is preferably from 0.1 mL / min to 3.0 mL / min, more preferably from 0.5 mL / min to 1.5 mL / min. If the flow rate is slow, improvement of the separation can be expected. However, if the flow rate is too slow, it may take a long time for the analysis, or the separation performance may be lowered due to broad peaks. Conversely, an increase in the flow rate has an advantage in terms of shortening the analysis time, but the peak is compressed, leading to a decrease in separation performance.
  • the holding time of each sample can be determined in advance by conducting a preliminary experiment on each sample.
  • a liquid feeding method a known liquid feeding method such as a linear gradient elution method or a stepwise elution method can be used, but a linear gradient elution method is preferred as the liquid feeding method in the present invention.
  • the size of the gradient may be appropriately adjusted in accordance with the separation performance of the column and the characteristics of the analyte (here, PCR amplification product) in the range of 0 to 100% of the eluent used for elution.
  • DNA methylation in genomic DNA prepared from cells is detected by subjecting the PCR amplification product of DNA treated with bisulfite in the above-described procedure to ion exchange chromatography.
  • DNA in the PCR amplification product has a different sequence depending on the methylation rate of the original DNA.
  • ion exchange chromatography a chromatogram showing different signals according to the base sequence of the DNA contained in the amplification product is obtained. Therefore, DNA methylation can be detected based on a detection signal obtained by ion exchange chromatography of sample DNA.
  • the more the DNA extracted from the cells is methylated the shorter the retention time of the detection signal peak obtained from the PCR amplification product. Conversely, if the degree of methylation is low, the retention time of the detection signal peak Becomes longer. Further, the peak height of each detection signal indicates that the presence ratio of DNA having a methylation rate corresponding to the peak is high.
  • iPS cells are prepared by subjecting differentiated cells such as skin fibroblasts to initialization
  • differentiated cells such as skin fibroblasts
  • the initialization is performed normally, methylated cytosine in the CpG region of the demethylated gene region is demethylated.
  • 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 initialization process. 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.
  • the 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 initialization treatment. Therefore, in the chromatographic analysis of the methylated gene, the detection signal peak derived from the iPS cell is detected at a retention time earlier than the detection signal peak derived from the differentiated cell.
  • existing data processing software such as LCsolution (Shimadzu Corporation), GRAMS / AI (Thermo Fisher Scientific), IgorPro (WaveMetrics) can be used to determine the presence or absence of a detection signal peak by chromatography.
  • peak detection using To illustrate a method for determining the presence / absence of a peak using LCsolution, specifically, a holding time interval in which a peak is to be detected is first specified. Next, various parameters are set in order to remove unnecessary peaks such as noise.
  • the parameter “WIDTH” is set to be larger than the half width of the unnecessary peak
  • the parameter “SLOPE” is set to be larger than the rising slope of the unnecessary peak
  • the setting of the parameter “DRIFT” is changed so that the low resolution peak is vertically For example, selecting whether to divide or to divide the baseline. Since different chromatograms can be obtained as parameter values depending on the analysis conditions, the type of gene marker selected, the amount of specimen, etc., appropriate values may be set according to the chromatogram.
  • Whether there is a difference in the retention time of the peak of the detection signal obtained by chromatography is determined by comparing the retention time of the peak top of each detection signal obtained by the data processing software. If the difference in retention time between the two detection signals is preferably 2 seconds or more, more preferably 5 seconds or more, it is determined that there is a difference in the retention times of the two signals. On the other hand, when the difference in the retention time between the two detection signals is preferably less than 2 seconds, more preferably less than 1 second, it is determined that there is no difference in the retention times of the two signals.
  • the retention time of the peak of the detection signal obtained by chromatography can vary depending on the PCR amplification product, the chromatography conditions, and the like. Therefore, it is preferable to obtain a detection signal by chromatography from the control cell every time the detection signal is obtained from the test cell, but it is obtained separately under predetermined conditions and stored as control data. You may refer to them as needed.
  • the retention time of the peak of the detection signal of the cell derived from the test cell subjected to the initialization treatment is differentiated from iPS cell (positive control), human skin fibroblast, etc. Compare to the retention time of the peak of the detection signal of the cells (negative control) or both. If the retention time differs between the test cell and the negative control, the test cell is more demethylated in the demethylated gene region than the differentiated cell, or the methylated gene region DNA is methylated. That is, the test cell is a reprogrammed iPS cell.
  • the test cell is the same as the iPS cell, and the DNA of the demethylated gene region is demethylated or the methylated gene region
  • the DNA is methylated, that is, the test cell is an initialized iPS cell. Therefore, the success or failure of cell reprogramming (iPS cell production) can be determined by the method of the present invention.
  • the above-described method of the present invention can be applied not only to the success or failure of cell initialization, but also to the determination of whether or not the established iPS cells maintain their pluripotency.
  • a detection signal by ion exchange chromatography is obtained in the same procedure as described above, and the retention time of the peak of the detection signal is compared with a control .
  • the demethylated gene region DNA demethylation or methylated gene region DNA demethylation in the test subject cell is maintained based on whether there is a difference in retention time from the control, ie, the test subject cell It can be determined whether or not pluripotency is maintained without differentiation.
  • the quality control of iPS cells can be performed by the method of the present invention.
  • the quality control method for iPS cells of the present invention can be implemented in combination with a conventional iPS cell identification method.
  • Conventional identification methods of iPS cells include confirmation by reporter activity (puromycin resistance, GFP positive, etc.) bound to NANOG, Oct3 / 4, etc., visual confirmation of formation of ES cell-like colonies, etc.
  • 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.
  • these conventional methods are performed for secondary evaluation after the method of the present invention.
  • the method of the invention can be applied to: Determining whether cell pluripotency is maintained in a subculture of stem cells other than iPS cells (eg, ES cells, adult stem cells, etc.); Detection of contamination of differentiated cells into subcultures of stem cells; Detection of contamination of undifferentiated cells into differentiated cell culture (for example, contamination of undifferentiated cells into a culture of somatic cells induced to differentiate from pluripotent stem cells).
  • stem cells other than iPS cells eg, ES cells, adult stem cells, etc.
  • Detection of contamination of differentiated cells into subcultures of stem cells Detection of contamination of undifferentiated cells into differentiated cell culture (for example, contamination of undifferentiated cells into a culture of somatic cells induced to differentiate from pluripotent stem cells).
  • cultures of cells subjected to reprogramming treatment to be tested are ES cells, adult stem cells (neural stem cells, hematopoietic stem cells, Replaced with a subculture of stem cells such as leaf stem cells) and the control with differentiated cells (negative control), those with the same type of stem cells that have been confirmed pluripotent (positive control),
  • the same process as described above is performed to determine whether or not the test target cell is a stem cell.
  • the control cell may be a primary cultured cell that has not been passaged, or a subcultured cell that has been inherited in a low number of passages and the properties of the primary cultured cell are inherited.
  • a culture of cells subjected to reprogramming treatment such as iPS cells, ES cells, and adult stem cells
  • iPS cells iPS cells
  • ES cells ES cells
  • adult stem cells a culture of cells subjected to reprogramming treatment
  • the culture of the cells subjected to the initialization treatment to be tested is replaced with a differentiated cell culture, and the control is a stem cell.
  • the control is a stem cell.
  • differentiated cells positive control
  • etc. are replaced with the same steps as described above to determine whether or not the test target cells are differentiated cells.
  • the DNA methylation state of iPS cells when analyzing the DNA methylation state of iPS cells by the bisulfite sequencing method, many steps of PCR amplification of the target gene region after bisulfite conversion, further cloning, and subsequent sequencing are required. . Furthermore, it is desirable to confirm by electrophoresis whether or not the desired PCR amplification product has been obtained before cloning.
  • the PCR amplification product is directly subjected to chromatographic analysis, so that a cloning step is unnecessary, and the success or failure of PCR amplification can be confirmed by the peak intensity of the detection signal of the chromatography. Therefore, according to the present invention, the DNA methylation state can be analyzed with fewer steps than the bisulfite sequencing method.
  • DNA methylation status of hemimethylated DNA or genes having different DNA methyl status among alleles it is necessary to analyze many clones by the bisulfite sequencing method.
  • chromatographic analysis according to the present invention DNA hemimethylation and differences in DNA methyl status between alleles are detected as bimodal peaks in the detection signal. The DNA methylation state can be analyzed well.
  • the fibroblast culture medium 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 culture supernatant of confluent fibroblasts in a 100 mm culture dish was removed by aspiration, and 10 mL of PBS was added to wash the cells. PBS was removed by aspiration, 1 mL of 0.25% Trypsin / 1 mM EDTA solution (Invitrogen) was added per dish, and the dish was incubated at 37 ° C.
  • Plasmid solutions were prepared using 1 ⁇ g each of pCXLE-hOct4-shp53, pCXLE-hSK, and pCXLE-hUL (total 3 ⁇ g) using a Neon Transfection kit (Invitrogen).
  • the medium was changed once every two days.
  • the cells were passaged onto feeder cells.
  • the medium was removed by suction, and the cells were washed by adding 2 mL of PBS.
  • PBS was removed by suction, 0.3 mL of 0.25% Trypsin / 1 mM EDTA solution (Invitrogen) was added per well, and the mixture was incubated at 37 ° C. for 3 minutes.
  • 2 mL of 10% FBS medium was added and suspended by pipetting. 10% FBS medium was added and adjusted to 1 ⁇ 10 4 / mL, and 10 mL of the cell suspension was seeded on a 100 mm dish in which feeder cells were seeded in advance.
  • the medium for human iPS cells was prepared by adding 2.5 ⁇ L of 1 ⁇ g / ⁇ L bFGF solution (Reprocell) to 500 mL of Primate ES medium (Reprocell). Thereafter, the medium was changed once every two days.
  • iPS cell colonies were isolated between the 21st and 30th days of culture. After colonies grew and became visible, colonies were collected before differentiation began. A colony having a size of about 2 mm or less was collected. In order to transfer the collected colonies, 200 ⁇ L of human iPS cell culture medium was dispensed into a 96-well plate per well. Using a small-volume pipette such as a 10 ⁇ L pipette, the colonies were removed under a stereomicroscope. The collected colonies were transferred to a 96-well plate, and collapsed by pipetting until the colonies became a small lump (however, in a state before the single cell).
  • SNL feeder cells treated with mitomycin C were prepared in a 24-well plate, and colonies broken up to the small clusters were seeded thereon. 300 ⁇ L 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.
  • Human dermal fibroblasts (cell number: JCRB0541, cell name: TIG-111) were used as control cells before initialization.
  • the human dermal fibroblasts were purchased from JCRB Cell Bank, National Institute of Pharmaceutical Sciences.
  • the fibroblast culture solution used was a 10% FBS medium.
  • the fibroblasts were cultured and maintained in a 100 mm culture dish at 37 ° C. and 5% CO 2 .
  • Reference Example 2 Selection of iPS Cells Using Phase-Contrast Microscope iPS cells were selected by observing cell morphology using a phase-contrast microscope. The degree of cell differentiation was classified into the following three stages. A compact sphere with a clear edge is the most undifferentiated colony, a colony at a stage where the sphere has been reduced in thickness and the sphere has begun to collapse a little, and a colony that is a moderate differentiation stage. The colony whose shape collapsed and became flat was defined as the most differentiated colony. The most differentiated colonies were cut out with a pipette tip. 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 To 2000 mL of 3 wt% polyvinyl alcohol (manufactured by Nippon Synthetic Chemical) in a reactor equipped with a stirrer, 200 g of tetraethylene glycol dimethacrylate (manufactured by Shin-Nakamura Chemical Co., Ltd.), triethylene glycol di A mixture of 100 g of methacrylate (made by Shin-Nakamura Chemical Co., Ltd.), 100 g of glycidyl methacrylate (made by Wako Pure Chemical Industries, Ltd.) and 1.0 g of benzoyl peroxide (made by Kishida Chemical Co., Ltd.) was added.
  • methacrylate made by Shin-Nakamura Chemical Co., Ltd.
  • glycidyl methacrylate made by Wako Pure Chemical Industries, Ltd.
  • benzoyl peroxide made by Kishida Chemical Co., Ltd.
  • the mixture was heated with stirring and polymerized at 80 ° C. for 1 hour in a nitrogen atmosphere.
  • 100 g of ethyl trimethyl ammonium chloride (manufactured by Wako Pure Chemical Industries, Ltd.) as a hydrophilic monomer having a strong cationic group was dissolved in ion-exchanged water. This was added to the same reactor and polymerized in the same manner at 80 ° C. for 2 hours under stirring in a nitrogen atmosphere.
  • the obtained polymerization 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 analyzer (Accumizer 780 / Particle Sizing Systems), and the average particle size was 10 ⁇ m.
  • the above-mentioned packing material for ion exchange chromatography was packed into a stainless steel column (column size: inner diameter 4.6 mm ⁇ length 20 mm) of a liquid chromatography system.
  • Example 1 Discrimination between human iPS cells and human skin fibroblasts using determination of DNA methylation status in the NANOG gene region by HPLC [Extraction of genomic DNA and bisulfite treatment]
  • NANOG which is one of the demethylated genes
  • a measurement sample was prepared in order to analyze DNA methylation at the CpG site.
  • primers were designed for CpG islands containing the above-mentioned CpG sites using Methyl Primer Express (registered trademark) software (Life Technologies).
  • DNA was extracted from iPS cells (4 strains) and 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 (manufactured by 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.
  • Primer set 1 (SEQ ID NOs: 1 and 2) was used as a PCR primer.
  • the sequence of each primer is shown in Table 3.
  • PCR was performed using template DNA 25 ng, 1 ⁇ Ex Taq Buffer (20 mM Mg 2 + plus) (TaKaRa BIO), 200 ⁇ mol / L dNTP, 1.25 U TaKaRa Ex Taq HS (TaKaRa BIO), and 0.2 ⁇ mol / L.
  • the reaction was performed in 50 ⁇ L of a reaction solution containing forward and reverse primers.
  • the polymerase was activated at 95 ° C.
  • the DNA was amplified by an Applied Biosystems Verti thermal cycler under conditions of 38 cycles of 94 ° C. for 30 seconds, 55 ° C. for 30 seconds and 72 ° C. for 30 seconds, and finally extension at 72 ° C. for 7 minutes.
  • mix 1 ⁇ L of loading dye solution with 5 ⁇ L of the reaction solution in 2% agarose gel to which ethidium bromide has been added in advance apply and perform electrophoresis, and observe the PCR amplification product to obtain the desired PCR amplification product. I confirmed that.
  • iPS cells can be identified by measuring the retention time of the chromatogram peak according to the method of the present invention.
  • detection signals were obtained with substantially the same retention time between different iPS cell lines, indicating that iPS cells can be identified with good reproducibility.
  • Example 2 Discrimination of human iPS cells and human skin fibroblasts using determination of DNA methylation status 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 in 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. As PCR primers, primer sets 2 to 6 (SEQ ID NOs: 3 to 12) were used. Table 4 shows the sequence and product size of each primer.
  • FIGS. Oct3 / 4 The HPLC chromatograms obtained for each gene region (PCR product from primer sets 2 to 6) are shown in FIGS. Oct3 / 4 (primer set 2) is shown 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 chromatogram of each figure is adjusted so that the peak height may become the same.
  • Example 3 Discrimination of 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 From each of these, genomic DNA was extracted in the same procedure as in Example 1, treated with bisulfite, and subjected to PCR and HPLC.
  • 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.
  • the polymerase is activated at 95 ° C. for 4 minutes, and the DNA is subjected to 40 cycles of 95 ° C.
  • iPS cells can be identified by measuring the retention time of chromatogram peaks for methylated or demethylated genes according to the method of the present invention.
  • 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.
  • PCR amplification products were cloned and transformed using a TA cloning kit manufactured by Biodynamics, and 20 to 24 colonies were recovered. Plasmids were extracted from each colony, sequenced using M13 reverse primer, and the DNA sequence after bisulfite treatment was determined. In order to confirm the methylation state, analysis was performed using QUAMA (http://quma.cdb.riken.jp/).
  • the analysis results are shown in FIGS.
  • the determination results of the methylation state were consistent between the method of the present invention using the HPLC method and the bisulfite sequencing method.
  • PCR amplification is followed by cloning of amplification products, and analysis of a large number of samples obtained by cloning one by one. Has to do and requires a great deal of time and effort.
  • the PCR amplification product is directly subjected to HPLC analysis, so that a cloning step is unnecessary and the number of measurement examples is small.
  • the bisulfite sequencing method takes about several days to analyze 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.

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Abstract

　L'invention concerne un procédé de contrôle de la qualité de cellules souches pluripotentes induites. Dans ce procédé, l'ADN de cellules initialisées est soumis à un traitement au bisulfite, une amplification par PCR, et une chromatographie par échange d'ions, et les cellules initialisées sont déterminées comme étant des cellules souches pluripotentes induites lorsque le temps de rétention du pic de signal de détection chromatographique est allongé en comparaison à celui de cellules non initialisées.
PCT/JP2015/079370 2014-10-17 2015-10-16 Procédé de contrôle de la qualité de cellules souches WO2016060264A1 (fr)

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2020072734A (ja) * 2014-10-17 2020-05-14 積水メディカル株式会社 幹細胞の品質管理方法

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009057831A1 (fr) * 2007-10-31 2009-05-07 Kyoto University Procédé de re-programmation nucléaire
US20120157339A1 (en) * 2009-06-29 2012-06-21 Guoping Fan Molecular Markers and Assay Methods for Characterizing Cells
US20130295569A1 (en) * 2010-10-28 2013-11-07 Salk Institute For Biological Studies Epigenomic induced pluripotent stem cell signatures
JP2014500708A (ja) * 2010-10-11 2014-01-16 ヴァーネット ピーター 幹細胞の品質判定
WO2014136930A1 (fr) * 2013-03-07 2014-09-12 積水メディカル株式会社 Procédé de détection d'adn méthylé

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP6692028B2 (ja) * 2014-10-17 2020-05-13 積水メディカル株式会社 幹細胞の品質管理方法

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009057831A1 (fr) * 2007-10-31 2009-05-07 Kyoto University Procédé de re-programmation nucléaire
US20120157339A1 (en) * 2009-06-29 2012-06-21 Guoping Fan Molecular Markers and Assay Methods for Characterizing Cells
JP2014500708A (ja) * 2010-10-11 2014-01-16 ヴァーネット ピーター 幹細胞の品質判定
US20130295569A1 (en) * 2010-10-28 2013-11-07 Salk Institute For Biological Studies Epigenomic induced pluripotent stem cell signatures
WO2014136930A1 (fr) * 2013-03-07 2014-09-12 積水メディカル株式会社 Procédé de détection d'adn méthylé

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
NISHINO, K. ET AL.: "DNA Methylation Dynamics in Human Induced Pluripotent Stem Cells over Time", PLOS GENETICS, vol. 7, no. 5, 26 May 2011 (2011-05-26), pages e1002085, ISSN: 1553-7404 *
PAN, C. ET AL.: "SNL fibroblast feeder layers support derivation and maintenance of human induced pluripotent stem cells", JOURNAL OF GENETICS AND GENOMICS, vol. 37, 2010, pages 241 - 248, XP002621302, ISSN: 1673-8527, DOI: doi:10.1016/s1673-8527(09)60042-4 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2020072734A (ja) * 2014-10-17 2020-05-14 積水メディカル株式会社 幹細胞の品質管理方法

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