WO2020149391A1 - Method for evaluating differentiation resistance of undifferentiated cells - Google Patents

Method for evaluating differentiation resistance of undifferentiated cells Download PDF

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WO2020149391A1
WO2020149391A1 PCT/JP2020/001417 JP2020001417W WO2020149391A1 WO 2020149391 A1 WO2020149391 A1 WO 2020149391A1 JP 2020001417 W JP2020001417 W JP 2020001417W WO 2020149391 A1 WO2020149391 A1 WO 2020149391A1
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undifferentiated
cell
promoter
gene
mir886
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英樹 谷口
圭輔 関根
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公立大学法人横浜市立大学
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Definitions

  • the present invention relates to a method for evaluating differentiation resistance of undifferentiated cells.
  • Non-patent Documents 1 and 2 Regarding the involvement of differentiation resistance (difficulty in differentiation) of undifferentiated cells, there are reports of DNA methylation and gene expression (Non-patent Documents 1 and 2).
  • Non-Patent Documents 3 and 4 there has been a report on a method for detecting and evaluating residual/contamination of undifferentiated iPS cells in differentiated cells (Non-Patent Documents 3 and 4), but undifferentiated cells remain at the stage of undifferentiated iPS cells. / There is no method to evaluate contamination risk.
  • the reculturing method has the advantage of being highly accurate because it forms colonies from contaminating undifferentiated iPS cells, but it takes more than a week to detect it, so the method using quantitative PCR is simple and easy. ⁇ It is excellent in that it can be implemented quickly. Moreover, it is considered that whether or not the undifferentiated cells remain in the differentiated cells is already defined not only in the differentiation process but also in the stage of the undifferentiated iPS cells. In fact, it has been reported that iPS cell clones are resistant to differentiation, and the methylation and expression genes characteristic of differentiation resistant clones have also been analyzed.
  • undifferentiated cells were targeted, and marker genes were identified for evaluating the risk of undifferentiated iPS cells remaining/contaminating in the differentiated cells when differentiation is induced.
  • the amount of undifferentiated iPS cells remaining in the differentiated cells of the same iPS cell clone with different passage numbers etc. is evaluated, and DNA methylation analysis is performed on iPS cells that have undifferentiated iPS cells remaining and those that do not. For genes with different states, genes that correlate with undifferentiated survival were identified. As a result, it was found that ZNF354C, C12orf56, ZNF578 and MIR886 are expressed in iPS cells in which undifferentiated iPS cells do not remain, and DPP6 is expressed in iPS cells in which undifferentiated iPS cells remain. Furthermore, it was revealed that in other iPS cell clones that were not used in the methylation analysis, the presence or absence of undifferentiated iPS cells in the differentiated cells of these clones was correlated with the expression of the gene identified this time.
  • the gist of the present invention is as follows.
  • a method for evaluating the differentiation resistance of undifferentiated cells which comprises measuring the following (i) and/or (ii).
  • (ii) Methylation status of the promoter of at least one gene selected from the group consisting of ZNF354C, C12orf56, ZNF578, DPP6 and MIR886 At least one gene selected from the group consisting of ZNF354C, C12orf56, ZNF578 and MIR886 Expression level and/or promoter activity is high, the differentiation resistance of undifferentiated cells is evaluated to be low, and the expression level of at least one gene selected from the group consisting of ZNF354C, C12orf56, ZNF578 and MIR886 and/or
  • the undifferentiated cells are embryonic tumor cells (EC cells), embryonic stem cells (ES cells), induced pluripotent stem cells (iPS cells) or embryonic germ cells (EG cells) (1) to ( The method according to any one of 5).
  • the method for measuring the expression level of a gene is qPCR, digital PCR, immunostaining, in situ hybridization, RNA sequence, microarray, NanoString, antibody array, FlowCytometry, mass spectrometry or a combination thereof (7) Method.
  • the method for measuring the methylation status of the gene promoter is to concentrate methylated DNA and then detect the concentrated DNA. Decoding (sequencing) the base sequence after base substitution by bisulfite treatment.
  • Method a method of detecting the nucleotide sequence after hybridization with bisulfite treatment by hybridization, a methylation-specific PCR (MSP) method, a method of detecting with or without cleavage by a methylation-sensitive restriction enzyme, a methylated cytosine
  • MSP methylation-specific PCR
  • the method according to any one of (1), (4) to (6), which is a method of glucosylation and detection with an enzyme sensitive to glucosylated cytosine, or a combination thereof.
  • a method for selecting an undifferentiated cell line having low or high differentiation resistance by measuring the following (i) and/or (ii) in the undifferentiated cell population.
  • the expression level and/or promoter activity of DPP6 is high, it is selected as an undifferentiated cell line having high differentiation resistance, and when the expression level and/or promoter activity of DPP6 is low, the differentiation resistance is low.
  • a promoter of at least one gene selected from the group consisting of ZNF354C, C12orf56, ZNF578 and MIR886 has a low methylation state, it is selected as an undifferentiated cell line having low differentiation resistance, and ZNF354C, C12orf56, ZNF578 is selected.
  • an undifferentiated cell line having high differentiation resistance is selected when the promoter has a high methylation state of at least one gene selected from the group consisting of MIR886 and MIR886.
  • the methylation status of the DPP6 promoter is low, it is selected as an undifferentiated cell line with high differentiation resistance, and when the methylation status of the DPP6 promoter is high, it is selected as an undifferentiated cell line with low differentiation resistance.
  • the undifferentiated cell line is an embryonal tumor cell (EC cell) line, an embryonic stem cell (ES cell) line, an induced pluripotent stem cell (iPS cell) line, or an embryonic germ cell (EG cell) line
  • the method according to any one of (10) to (14).
  • a kit for evaluating the differentiation resistance of undifferentiated cells which comprises a reagent capable of measuring the following (i) and/or (ii).
  • Expression level and/or promoter activity of at least one gene selected from the group consisting of ZNF354C, C12orf56, ZNF578, DPP6 and MIR886 (ii) The methylation state of the promoter of at least one gene selected from the group consisting of ZNF354C, C12orf56, ZNF578, DPP6 and MIR886 (18)
  • the reagent capable of measuring the expression level of the gene is a primer, a probe or an antibody.
  • the kit according to (17). (19) The kit according to (17), wherein the reagent capable of measuring the promoter activity of the gene is a gene sequence in which a reporter protein is linked downstream of the promoter or a vector incorporating this gene sequence.
  • a reagent capable of measuring the methylation state of a gene promoter is bisulfite (bisulfite), a microarray reagent for methylation analysis, a sequencing reagent by the Sanger method, a sequencing reagent for a next-generation sequencer, a 5-mC antibody, 5-hmC antibody, methyladenosine antibody, 5'-methyl-2'-deoxycytidine antibody, HRP-labeled DNA antibody, 5-hmC glucosyltransferase, glucosyl-5hmC sensitive restriction enzyme endonuclease, MBD1 (Methyl-CpG Binding Domain Protein1), The kit according to (17), which is MBD2 (Methyl-CpG Binding Domain Protein2), a specific PCR primer, a specific probe or a DNA purification kit.
  • undifferentiated cells for example, undifferentiated pluripotent stem cells
  • organoids formed from undifferentiated cells eg, liver organ bud (iPS cell liver bud, etc.
  • This specification includes the content described in the Japan patent application, Japanese Patent Application No. 2019-005892, and/or drawing which are the foundations of the priority of this application.
  • the present invention provides a method for evaluating differentiation resistance of undifferentiated cells, which comprises measuring the following (i) and/or (ii).
  • (ii) Methylation status of the promoter of at least one gene selected from the group consisting of ZNF354C, C12orf56, ZNF578, DPP6 and MIR886 There is only one kind of gene for measuring the expression level, promoter activity and methylation status of the promoter. It may be a combination of two or more kinds.
  • the undifferentiated cell to be evaluated may be a cell having pluripotency, and for example, the undifferentiated cell is an embryonal tumor cell (EC cell), an embryonic stem cell (ES cell) or an induced pluripotent stem cell. (IPS cells) and embryonic germ cells (EG cells).
  • the undifferentiated cell may be derived from human or any animal other than human.
  • differentiation resistance refers to difficulty in differentiation, and preferably resistance to hepatocyte differentiation. It is known that the differentiation resistance of undifferentiated cells differs depending on the cell line, but it may change depending on the culture conditions such as the number of passages, the passage method and the cell density at the passage. Alternatively, it may change depending on the culture substrate or medium. Differentiation resistance is determined by re-seeding a certain number of differentiated cells after induction of differentiation, culturing under undifferentiated culture conditions, and proliferating colonies by immunostaining with an undifferentiated marker (SOX2, etc.). It can be evaluated by confirming that there is one and calculating one colony as one undifferentiated iPS cell.
  • SOX2 undifferentiated marker
  • DPP6 When the expression level and/or promoter activity of DPP6 is high, it is evaluated that the differentiation resistance of undifferentiated cells is high, and when the expression level and/or promoter activity of DPP6 is low, the differentiation resistance of undifferentiated cells is low Can be evaluated.
  • the methylation status of the promoter of at least one gene selected from the group consisting of ZNF354C, C12orf56, ZNF578 and MIR886 is low, it is evaluated that the differentiation resistance of undifferentiated cells is low, and it is determined from ZNF354C, C12orf56, ZNF578 and MIR886.
  • the methylation status of the promoter of at least one gene selected from the group is high, it can be evaluated that the undifferentiated cells have high differentiation resistance.
  • the methylation status of the DPP6 promoter When the methylation status of the DPP6 promoter is low, it is evaluated that the differentiation resistance of the undifferentiated cells is high, and when the methylation status of the DPP6 promoter is high, the differentiation resistance of the undifferentiated cells is evaluated as low.
  • “high” or “low” expression level of a gene means that it is higher or lower than a predetermined numerical value capable of discriminating between high differentiation resistance and low differentiation resistance of undifferentiated cells.
  • the numerical value varies depending on the sensitivity and specificity to be sought, and may vary depending on the type of undifferentiated cell (for example, difference between iPS cell and ES cell) and difference in cell clone. The same applies to "high” or “low” promoter activity of a gene and "high” or “low” methylation status.
  • the value of the methylation state of the promoter of ZNF354C was When it is 40% or less or less, it is evaluated that the differentiation resistance of undifferentiated cells is low, and when the methylation state value of the promoter of ZNF354C is 60% or more, the differentiation resistance of undifferentiated cells is high. Can be evaluated.
  • the value of methylation status of the promoter of C12orf56 is 40% or less or less
  • the value of methylation status of the promoter of ZNF578 is 40% or less or less
  • the value of methylation status of the promoter of DPP6 is 60% or less.
  • the MIR886 promoter methylation state value is 40% or less, or less, if the differentiation resistance of undifferentiated cells is evaluated as low, C12orf56 promoter methylation state value of 60% or more ,
  • the value of the methylation status of the promoter of ZNF578 is 60% or more
  • the value of the methylation status of the promoter of DPP6 is 40% or less, or less
  • the value of the methylation status of the promoter of MIR886 is 60% or more. It can be evaluated that the differentiation resistance of the differentiated cells is high.
  • the expression level of a gene can be measured as the amount of mRNA transcribed from the gene or the amount of protein translated from mRNA. Specifically, the gene expression level can be measured by qPCR, digital PCR, immunostaining, in situ hybridization, RNA sequence, microarray, NanoString, antibody array, FlowCytometry, mass spectrometry, a combination thereof and the like.
  • the methods for analyzing the methylation status of the gene promoter are: 1. A method in which methylated DNA is concentrated using an anti-methylated cytosine or adenosine antibody, etc., and the concentrated DNA is detected and quantified by a sequence, microarray, qPCR, etc. after concentration. After the base substitution by bisulfite treatment, a method of detecting the base sequence by decoding (sequencing) or by using hybridization such as microarray or MLPAR (Multiplex Ligation Probe Amplification) method. 3. A method of detecting the presence or absence of amplification by PCR after the base substitution by bisulfite treatment (methylation-specific PCR (MSP) method), 4. 4. 4.
  • a method for detecting the presence or absence of cleavage using a methylation-sensitive restriction enzyme There is a method in which methylated cytosine is glucosylated and detected with an enzyme sensitive to glucosylated cytosine, and any of these methods or a combination thereof can be used.
  • the present invention provides a method for selecting an undifferentiated cell line with low or high differentiation resistance by measuring the following (i) and/or (ii) in the undifferentiated cell population.
  • (ii) Methylation status of the promoter of at least one gene selected from the group consisting of ZNF354C, C12orf56, ZNF578, DPP6 and MIR886 There is only one kind of gene for measuring the expression level, promoter activity and methylation status of the promoter. It may be a combination of two or more kinds.
  • the undifferentiated cell line to be selected may be a pluripotent cell line.
  • the undifferentiated cell line may be an embryonal tumor cell (EC cell) line, an embryonic stem cell (ES cell) line, Induced pluripotent stem cell (iPS cell) line or embryonic germ cell (EG cell) line, and iPS cell line is preferable.
  • the undifferentiated cell line may be derived from human or any animal other than human.
  • the expression level and/or promoter activity of at least one gene selected from the group consisting of ZNF354C, C12orf56, ZNF578 and MIR886 is high, it is selected as an undifferentiated cell line having low differentiation resistance, and ZNF354C, C12orf56, ZNF578 and When the expression level and/or promoter activity of at least one gene selected from the group consisting of MIR886 is low, it can be selected as an undifferentiated cell line having high differentiation resistance.
  • DPP6 When the expression level and/or promoter activity of DPP6 is high, it is selected as an undifferentiated cell line with high differentiation resistance, and when the expression level and/or promoter activity of DPP6 is low, undifferentiated cell line with low differentiation resistance Can be sorted as.
  • the methylation status of the promoter of at least one gene selected from the group consisting of ZNF354C, C12orf56, ZNF578 and MIR886 is low, it is selected as an undifferentiated cell line with low differentiation resistance, and selected from ZNF354C, C12orf56, ZNF578 and MIR886.
  • An undifferentiated cell line with high differentiation resistance can be selected when the methylation status of the promoter of at least one gene selected from the group is high.
  • the expression level of the gene, the promoter activity, and the methylation state of the promoter of the gene is “high” or “low” as described above.
  • ZNF354C, C12orf56, ZNF578, DPP6 and MIR886 can be used as a marker gene for detecting undifferentiated cells with low or high differentiation resistance present in the undifferentiated cell population. Therefore, the present invention, in the undifferentiated cell population, in order to detect undifferentiated cells with low or high differentiation resistance, at least one selected from the group consisting of ZNF354C, C12orf56, ZNF578, DPP6 and MIR886. Methods of using genes as markers are provided.
  • the gene used as a marker may be one type or a combination of two or more types.
  • the present invention also provides a kit for evaluating the differentiation resistance of undifferentiated cells, which comprises a reagent capable of measuring the following (i) and/or (ii).
  • a reagent capable of measuring the following (i) and/or (ii).
  • Methylation status of the promoter of at least one gene selected from the group consisting of ZNF354C, C12orf56, ZNF578, DPP6 and MIR886 There is only one kind of gene for measuring the expression level, promoter activity and methylation status of the promoter. It may be a combination of two or more kinds.
  • Examples of the reagent capable of measuring the expression level of a gene include a primer, a probe and an antibody.
  • the set of oligonucleotide primers is a target sequence (usually about 50 to 180 bp) in the nucleotide sequence of a transcript (mRNA) or cDNA of at least one gene selected from the group consisting of ZNF354C, C12orf56, ZNF578, DPP6 and MIR886.
  • mRNA transcript
  • cDNA of at least one gene selected from the group consisting of ZNF354C, C12orf56, ZNF578, DPP6 and MIR886.
  • the target sequence may be designed to have a sequence complementary to both ends of the target sequence.
  • the length of the oligonucleotide primer may be, for example, 15 to 35 nucleotides, preferably 18 to 27 nucleotides.
  • the nucleotide probe may hybridize to a transcript (mRNA) of at least one gene selected from the group consisting of ZNF354C, C12orf56, ZNF578, DPP6 and MIR886 (mRNA) or cDNA under stringent conditions. Alternatively, it may be designed to have a part or all of the nucleotide sequence of the cDNA or a sequence complementary thereto. Stringent conditions can be appropriately determined.
  • the length of the nucleotide probe is usually 1000 nucleotides or less, preferably 100 nucleotides or less, more preferably 50 nucleotides or less, and even more preferably 14 to 30 nucleotides.
  • the nucleotide probe may be single-stranded or double-stranded.
  • the antibody may be either a monoclonal antibody or a polyclonal antibody.
  • the antibody in addition to full-length antibodies, Fab, F(ab)' 2 , ScFv, Diabody, V H , VL , Sc(Fv) 2 , Bispecific sc(Fv) 2 , Minibody, ScFv- It is a concept that includes low molecular weight compounds such as Fc monomer and ScFv-Fc dimer.
  • the probe or antibody may be immobilized on a solid phase (eg, substrate, beads, membrane, etc.).
  • the reagent of the present invention may be labeled.
  • the primer may be labeled with a fluorescent substance or a quenching substance
  • the probe and the antibody may be labeled with a radioisotope, an enzyme, a luminescent substance, a fluorescent substance, biotin or the like.
  • the primary antibody that specifically binds to the target molecule in the present invention, the protein that is the expression product of at least one gene selected from the group consisting of ZNF354C, C12orf56, ZNF578, DPP6 and MIR886)
  • the primary antibody When the target molecule is detected by reacting a secondary antibody that binds to the antibody, the secondary antibody may be labeled (the primary antibody is not labeled).
  • Examples of the reagent capable of measuring the promoter activity of a gene include a gene sequence in which a reporter protein is linked downstream of the promoter or a vector incorporating this gene sequence.
  • reporter proteins include luciferase, fluorescent proteins such as GFP, and proteins expressed on cell membranes such as CD antigen.
  • the vector is preferably a plasmid vector.
  • a reagent that can measure the methylation state of the gene promoter bisulfite (bisulfite), a microarray reagent for methylation analysis such as Illumina Infinity Methylation EPIC BeadChip, a sequencing reagent by the Sanger method, a sequencing reagent for the next-generation sequencer, 5-mC antibody, 5-hmC antibody, methyladenosine antibody, 5'-methyl-2'-deoxycytidine antibody, HRP-labeled DNA antibody, 5-hmCglucose transferase, glucosyl-5hmC sensitive restriction endonuclease (GSRE: MspI, GlaI) , Csp6I, HaeIII, Taq ⁇ I, MboI, McrBC), MBD1 (Methyl-CpGBinding Domain Protein1), MBD2 (Methyl-CpG Binding Domain Protein2), specific PCR primer, specific probe or DNA purification kit. it can.
  • the kit of the present invention further detects with a reagent for detection with a primer (DNA polymerase, buffer, magnesium ion, dNTPs, probe, etc.), a reagent for detection with a probe (buffer, antibody, substrate, etc.), and antibody.
  • a primer DNA polymerase, buffer, magnesium ion, dNTPs, probe, etc.
  • a reagent for detection with a probe buffer, antibody, substrate, etc.
  • antibody buffer, antibody, substrate, etc.
  • Reagents secondary antibodies, substrates, buffers, etc.
  • reagents for measuring gene promoter activity buffers, luminescent substrates, antibodies, etc.
  • instruments reaction vessels, pipettes, etc.
  • kit instructions controls Samples for use, control data for analyzing measurement results, and the like.
  • the marker genes (ZNF354C, C12orf56, ZNF578, DPP6 and MIR886) of the present invention were found as follows. The amount of undifferentiated iPS cells remaining in differentiated cells of the same iPS cell clone with different passage numbers was evaluated, and DNA methylation analysis was performed on iPS cells that remained undifferentiated iPS cells and those that did not, to determine the DNA methylation status. For genes that have a difference in expression, the gene expression at the time of iPS cells before differentiation is examined to identify genes that differ in expression between clones that are likely to remain undifferentiated and clones that are unlikely to remain undifferentiated. The identified gene becomes a candidate for the marker gene.
  • the present invention evaluates the undifferentiated cell residual amount in the differentiated cells of the same undifferentiated cell clone having different culture conditions such as the number of passages, and the DNA in the clone in which the undifferentiated cell remains and the clone in which the undifferentiated cell does not remain.
  • Methylation analysis was performed to examine gene expression in undifferentiated cells before differentiation for genes that differ in DNA methylation status, between clones that tend to remain undifferentiated and clones that remain difficult to remain undifferentiated.
  • the present invention provides a method for searching for a marker gene for detecting undifferentiated cells with low or high differentiation resistance, which are present in an undifferentiated cell population, which comprises identifying genes that differ in expression. If "genes with different DNA methylation status" are common to other clones and have a correlation with the survival of undifferentiated cells, it can be predicted by evaluating methylation status instead of gene expression. Become a marker.
  • the undifferentiated cell may be a cell having pluripotency, and for example, the undifferentiated cell is an embryonal tumor cell (EC cell), an embryonic stem cell (ES cell) or an induced pluripotent stem cell (iPS cell), It is an embryonic germ cell (EG cell).
  • the undifferentiated cell may be derived from human or any animal other than human.
  • Examples of the culture state include culture conditions such as the number of passages, a passage method and cell density at the passage, a substrate for culture and a medium.
  • the undifferentiated cells may be passaged at least once or more.
  • the passage number is preferably 40 to 100, and when the undifferentiated cells have low differentiation resistance.
  • the passage number is preferably 8 to 30. Since iPS cells are considered to grow indefinitely, the passage number can be any number of times.
  • the differentiated cell may be any differentiated cell of endoderm, mesoderm and ectoderm.
  • endoderm differentiated cells include, but are not limited to, hepatic endoderm cells and the like.
  • mesodermal differentiated cells include, but are not limited to, transseptal mesenchymal cells, mesenchymal cells, vascular endothelial cells, and the like.
  • ectodermal differentiated cells include, but are not limited to, neural stem cells, neural crest cells, and neural cells.
  • the amount of undifferentiated cells remaining can be evaluated by the method described in Examples below, but is not limited thereto.
  • Example 1 Identification of markers capable of evaluating undifferentiated survival at the time of iPS cells Purpose Detection and elimination of undifferentiated cell contamination in iPS(ES) cell-derived differentiated cells, which contributes to regenerative medicine applications, is an important issue in ensuring the safety of all iPS(ES) cell-derived cell processed products. To date, rapid evaluation of undifferentiated cell contamination by verification of LIN28A expression in retinal pigment epithelial cells (RPE) has been reported, but we found that LIN28A was unsuitable for multiple cell processing products. ing.
  • RPE retinal pigment epithelial cells
  • genes that differ in the DNA methylation status of iPS cell clones that are prone to undifferentiated residuals and iPS cell clones that are unlikely to undergo undifferentiated residuals are extracted, and genes that are correlated with expression and undifferentiated residuals by quantitative PCR ( MIR886 (VTRNA2-1), DPP6, ZNF578, C12orf56, ZNF354C) were extracted.
  • One undifferentiated colony is a colony formed from one undifferentiated iPS cell contained in the differentiated cell, and the number of undifferentiated iPS cells contained in the differentiated cell is evaluated.
  • Induction of hepatocyte differentiation from human iPS cells was performed as described in Nature. 499(7459):481-4.(2013); Cell Rep. 21(10):2661-2670.(2017).
  • Methodhylation analysis For the methylation analysis, the DNA of cells at each differentiation stage was subjected to bisulfite treatment and then subjected to DNA methylation analysis using Infinia MethylationEPIC BeadChip Kit from Illumina. [Quantitative evaluation of expression] Quantitative PCR was performed as described in Nature. 499(7459):481-4.(2013); Cell Rep. 21(10):2661-2670.(2017).
  • the present invention can be used for quality evaluation of undifferentiated cells such as iPS cells used in regenerative medicine. Specifically, it can be used for detection and evaluation of residual/contamination of undifferentiated cells in the process of establishing iPS cells, the process of manufacturing master cell banks, working cell banks, and the process of manufacturing products such as regenerative medicine.

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Abstract

Provided is a method for predicting, in the stage of undifferentiated cells, the possibility of contamination with undifferentiated cells after the differentiation of the cells. The differentiation resistance of undifferentiated cells is evaluated by measuring (i) and/or (ii). (i) Expression level and/or promoter activity of at least one gene selected from the group consisting of ZNF354C, C12orf56, ZNF578, DPP6 and MIR886. (ii) Methylation state of the promoter of at least one gene selected from the group consisting of ZNF354C, C12orf56, ZNF578, DPP6 and MIR886.

Description

未分化細胞の分化抵抗性評価法Evaluation method for differentiation resistance of undifferentiated cells
　本発明は、未分化細胞の分化抵抗性評価法に関する。 The present invention relates to a method for evaluating differentiation resistance of undifferentiated cells.
　再生医療に用いる未分化多能性幹細胞の分化抵抗性、すなわち分化誘導した細胞集団における未分化細胞の残存/混入のしやすさを評価することは癌化リスクの観点から極めて重要である。 From the perspective of canceration risk, it is extremely important to evaluate the differentiation resistance of undifferentiated pluripotent stem cells used in regenerative medicine, that is, the susceptibility of undifferentiated cells to the remaining/contaminated cells in the differentiation-induced cell population.
　未分化細胞の分化抵抗性(分化しにくさ)の関与についてはDNAメチル化および遺伝子発現の報告がある（非特許文献１、２）。 Regarding the involvement of differentiation resistance (difficulty in differentiation) of undifferentiated cells, there are reports of DNA methylation and gene expression (Non-patent Documents 1 and 2).
　また、これまでに、分化細胞での未分化iPS細胞の残存/混入を検出、評価する手法は報告があるが（非特許文献３、４）、未分化iPS細胞の段階で未分化細胞の残存/混入リスクを評価する手法は存在しない。 In addition, there has been a report on a method for detecting and evaluating residual/contamination of undifferentiated iPS cells in differentiated cells (Non-Patent Documents 3 and 4), but undifferentiated cells remain at the stage of undifferentiated iPS cells. / There is no method to evaluate contamination risk.
　従来技術の中で、再培養法は混入する未分化iPS細胞からコロニーを形成させるため正確性が高いという利点が有る一方、検出までに1週間以上かかることから、定量PCRを用いた方法が簡便・迅速に実施可能な点で優れている。
　また、分化細胞に未分化細胞が残存するか否かは、分化過程だけで無く、未分化iPS細胞の段階ですでに規定されていると考えられる。実際に、iPS細胞のクローンによって分化抵抗性があることが報告されており、分化抵抗性クローンに特徴的なメチル化や発現遺伝子も解析されている。
　一方、本発明者らの研究から、分化能が高いiPS細胞クローンであっても継代を重ねることなどによって、分化能に変化が生じ、未分化iPS細胞が混入しやすくなる場合が有ることが明らかとなってきた。したがって、分化能が良好なクローンであっても、未分化iPS細胞混入リスクを日常的に評価することが重要である。
　しかしながら、これまでに未分化iPS細胞の段階で分化細胞に未分化iPS細胞が混入する可能性を予測する手法は存在しなかった。
　本発明は、未分化細胞の段階で、細胞が分化したときに未分化なままの細胞が残存/混入する可能性を予測する手法を提供することを目的とする。 Among the conventional techniques, the reculturing method has the advantage of being highly accurate because it forms colonies from contaminating undifferentiated iPS cells, but it takes more than a week to detect it, so the method using quantitative PCR is simple and easy.・It is excellent in that it can be implemented quickly.
Moreover, it is considered that whether or not the undifferentiated cells remain in the differentiated cells is already defined not only in the differentiation process but also in the stage of the undifferentiated iPS cells. In fact, it has been reported that iPS cell clones are resistant to differentiation, and the methylation and expression genes characteristic of differentiation resistant clones have also been analyzed.
On the other hand, from the studies by the present inventors, even in the case of iPS cell clones with high differentiation potential, due to repeated passages, changes in differentiation potential may occur, and undifferentiated iPS cells may be easily mixed. It has become clear. Therefore, it is important to routinely evaluate the risk of undifferentiated iPS cell contamination even with clones with good differentiation potential.
However, there has been no method for predicting the possibility of undifferentiated iPS cells contaminating differentiated cells at the stage of undifferentiated iPS cells.
It is an object of the present invention to provide a method of predicting the possibility that undifferentiated cells will remain/contaminate when the cells are differentiated at the stage of undifferentiated cells.
　本発明では未分化細胞を対象とし、分化誘導した際に分化細胞に未分化iPS細胞が残存/混入するリスクを評価するためのマーカー遺伝子の同定を行った。 In the present invention, undifferentiated cells were targeted, and marker genes were identified for evaluating the risk of undifferentiated iPS cells remaining/contaminating in the differentiated cells when differentiation is induced.
　継代回数などが異なる同一iPS細胞クローンの分化細胞における未分化iPS細胞残存量を評価し、未分化iPS細胞が残存するiPS細胞と残存しないiPS細胞におけるDNAメチル化解析を実施し、DNAメチル化状態に差が有る遺伝子について、未分化残存と相関する遺伝子を特定した。
　その結果、ZNF354C, C12orf56, ZNF578, MIR886は未分化iPS細胞が残存しないiPS細胞において発現が有り、DPP6は未分化iPS細胞が残存するiPS細胞において発現することを見出した。さらに、メチル化解析に用いなかった別のiPS細胞クローンにおいても、これらクローンの分化細胞における未分化iPS細胞残存の有無と、今回同定した遺伝子の発現に相関が有ることが明らかとなった。 The amount of undifferentiated iPS cells remaining in the differentiated cells of the same iPS cell clone with different passage numbers etc. is evaluated, and DNA methylation analysis is performed on iPS cells that have undifferentiated iPS cells remaining and those that do not. For genes with different states, genes that correlate with undifferentiated survival were identified.
As a result, it was found that ZNF354C, C12orf56, ZNF578 and MIR886 are expressed in iPS cells in which undifferentiated iPS cells do not remain, and DPP6 is expressed in iPS cells in which undifferentiated iPS cells remain. Furthermore, it was revealed that in other iPS cell clones that were not used in the methylation analysis, the presence or absence of undifferentiated iPS cells in the differentiated cells of these clones was correlated with the expression of the gene identified this time.
　これら遺伝子を用いることで、従来評価不可能であった未分化iPS細胞段階で、分化誘導した細胞への未分化iPS細胞の残存/混入の予測が可能となる。この手法は、iPS細胞だけでなく、ES細胞など他の未分化細胞についても応用可能である。 By using these genes, it becomes possible to predict the survival/contamination of undifferentiated iPS cells in the cells that have been induced to differentiate at the stage of undifferentiated iPS cells that could not be evaluated conventionally. This method can be applied not only to iPS cells but also to other undifferentiated cells such as ES cells.
　本発明の要旨は以下の通りである。
（１）下記の(i)及び／又は(ii)を測定することを含む、未分化細胞の分化抵抗性を評価する方法。
(i)ZNF354C、C12orf56、ZNF578、DPP6及びMIR886からなる群より選択される少なくとも１つの遺伝子の発現レベル及び／又はプロモーター活性
(ii) ZNF354C、C12orf56、ZNF578、DPP6及びMIR886からなる群より選択される少なくとも１つの遺伝子のプロモーターのメチル化状態
（２）ZNF354C、C12orf56、ZNF578及びMIR886からなる群より選択される少なくとも１つの遺伝子の発現レベル及び／又はプロモーター活性が高い場合に、未分化細胞の分化抵抗性が低いと評価し、ZNF354C、C12orf56、ZNF578及びMIR886からなる群より選択される少なくとも１つの遺伝子の発現レベル及び／又はプロモーター活性が低い場合に、未分化細胞の分化抵抗性が高いと評価する（１）記載の方法。
（３）DPP6の発現レベル及び／又はプロモーター活性が高い場合に、未分化細胞の分化抵抗性が高いと評価し、DPP6の発現レベル及び／又はプロモーター活性が低い場合に、未分化細胞の分化抵抗性が低いと評価する（１）記載の方法。
（４）ZNF354C、C12orf56、ZNF578及びMIR886からなる群より選択される少なくとも１つの遺伝子のプロモーターのメチル化状態が低い(メチル化状態が高いと発現(=プロモーター活性)が低いため)場合に、未分化細胞の分化抵抗性が低いと評価し、ZNF354C、C12orf56、ZNF578及びMIR886からなる群より選択される少なくとも１つの遺伝子のプロモーターのメチル化状態が高い(メチル化状態が高いと発現が低いため)場合に、未分化細胞の分化抵抗性が高いと評価する（１）記載の方法。
（５）DPP6のプロモーターのメチル化状態が低い場合に、未分化細胞の分化抵抗性が高いと評価し、DPP6のプロモーターのメチル化状態が高い場合に、未分化細胞の分化抵抗性が低いと評価する（１）記載の方法。
（６）未分化細胞が、胚性腫瘍細胞（ＥＣ細胞）、胚性幹細胞（ＥＳ細胞）、人工多能性幹細胞（ｉＰＳ細胞）又は胚性生殖細胞（ＥＧ細胞)である（１）～（５）のいずれかに記載の方法。
（７）遺伝子の発現レベルをmRNAの量又はタンパク質の量として測定する（１）～（３）又は（６）のいずれかに記載の方法。
（８）遺伝子の発現レベルを測定する方法が、qPCR、デジタルPCR、免疫染色、in situ hybridization、RNAシークエンス、マイクロアレイ、NanoString、抗体アレイ、FlowCytometry、質量分析又はそれらの組み合わせである（７）記載の方法。
（９）遺伝子のプロモーターのメチル化状態を測定する方法が、メチル化 DNA を濃縮した後に、濃縮されたDNAを検出する方法、バイサルファイト処理による塩基置換した後の塩基配列を解読(シークエンス)する方法、バイサルファイト処理による塩基置換した後の塩基配列をハイブリダイゼーションにより検出する方法、メチル化特異的PCR(MSP)法、メチル化感受性の制限酵素による切断の有無で検出する方法、メチル化シトシンをグルコシル化し、グルコシル化シトシン感受性の酵素で検出する方法又はそれらの組み合わせである（１）、（４）～（６）のいずれかに記載の方法。
（１０）未分化細胞集団における、下記の(i)及び／又は(ii)を測定することによって、分化抵抗性の低いあるいは高い未分化細胞株を選別する方法。
(i)ZNF354C、C12orf56、ZNF578、DPP6及びMIR886からなる群より選択される少なくとも１つの遺伝子の発現レベル及び／又はプロモーター活性
(ii) ZNF354C、C12orf56、ZNF578、DPP6及びMIR886からなる群より選択される少なくとも１つの遺伝子のプロモーターのメチル化状態
（１１）ZNF354C、C12orf56、ZNF578及びMIR886からなる群より選択される少なくとも１つの遺伝子の発現レベル及び／又はプロモーター活性が高い場合に、分化抵抗性が低い未分化細胞株として選別し、ZNF354C、C12orf56、ZNF578及びMIR886からなる群より選択される少なくとも１つの遺伝子の発現レベル及び／又はプロモーター活性が低い場合に、分化抵抗性が高い未分化細胞株として選別する（１０）記載の方法。
（１２）DPP6の発現レベル及び／又はプロモーター活性が高い場合に、分化抵抗性が高い未分化細胞株として選別し、DPP6の発現レベル及び／又はプロモーター活性が低い場合に、分化抵抗性が低い未分化細胞株として選別する（１０）記載の方法。
（１３）ZNF354C、C12orf56、ZNF578及びMIR886からなる群より選択される少なくとも１つの遺伝子のプロモーターのメチル化状態が低い場合に、分化抵抗性が低い未分化細胞株として選別し、ZNF354C、C12orf56、ZNF578及びMIR886からなる群より選択される少なくとも１つの遺伝子のプロモーターのメチル化状態が高い場合に、分化抵抗性が高い未分化細胞株として選別する（１０）記載の方法。
（１４）DPP6のプロモーターのメチル化状態が低い場合に、分化抵抗性が高い未分化細胞株として選別し、DPP6のプロモーターのメチル化状態が高い場合に、分化抵抗性が低い未分化細胞株として選別する（１０）記載の方法。
（１５）未分化細胞株が、胚性腫瘍細胞（ＥＣ細胞）株、胚性幹細胞（ＥＳ細胞）株、人工多能性幹細胞（ｉＰＳ細胞）株又は胚性生殖細胞（ＥＧ細胞)株である（１０）～（１４）のいずれかに記載の方法。
（１６）未分化細胞集団中に存在する、分化抵抗性の低いあるいは高い未分化細胞を検出するために、ZNF354C、C12orf56、ZNF578、DPP6及びMIR886からなる群より選択される少なくとも１つの遺伝子をマーカーとして使用する方法。
（１７）下記の(i)及び／又は(ii)を測定可能な試薬を含む、未分化細胞の分化抵抗性を評価するためのキット。
(i)ZNF354C、C12orf56、ZNF578、DPP6及びMIR886からなる群より選択される少なくとも１つの遺伝子の発現レベル及び／又はプロモーター活性
(ii) ZNF354C、C12orf56、ZNF578、DPP6及びMIR886からなる群より選択される少なくとも１つの遺伝子のプロモーターのメチル化状態
（１８）遺伝子の発現レベルを測定可能な試薬が、プライマー、プローブ又は抗体である（１７）記載のキット。
（１９）遺伝子のプロモーター活性を測定可能な試薬が、プロモーター下流にレポータータンパク質を連結した遺伝子配列又はこの遺伝子配列を組み込んだベクターである（１７）記載のキット。
（２０）遺伝子のプロモーターのメチル化状態を測定可能な試薬が、バイサルファイト（亜硫酸水素塩）、メチル化解析用マイクロアレイ試薬、Sanger法によるシークエンス試薬、次世代シークエンサー用シークエンス試薬、5-mC抗体、5-hmC抗体、メチルアデノシン抗体、5’-methyl-2’-deoxycytidine抗体、HRP標識DNA抗体、5-hmC グルコシルトランスフェラーゼ、グルコシル-5hmC感受性制限酵素エンドヌクレアーゼ、MBD1 (Methyl-CpG Binding Domain Protein1)、MBD2 (Methyl-CpG Binding Domain Protein2)、特異的PCRプライマー、特異的プローブ又はDNA精製キットである（１７）記載のキット。 The gist of the present invention is as follows.
(1) A method for evaluating the differentiation resistance of undifferentiated cells, which comprises measuring the following (i) and/or (ii).
(i) Expression level and/or promoter activity of at least one gene selected from the group consisting of ZNF354C, C12orf56, ZNF578, DPP6 and MIR886
(ii) Methylation status of the promoter of at least one gene selected from the group consisting of ZNF354C, C12orf56, ZNF578, DPP6 and MIR886 (2) At least one gene selected from the group consisting of ZNF354C, C12orf56, ZNF578 and MIR886 Expression level and/or promoter activity is high, the differentiation resistance of undifferentiated cells is evaluated to be low, and the expression level of at least one gene selected from the group consisting of ZNF354C, C12orf56, ZNF578 and MIR886 and/or The method according to (1), wherein the differentiation resistance of undifferentiated cells is evaluated to be high when the promoter activity is low.
(3) When the expression level of DPP6 and/or the promoter activity is high, it is evaluated that the differentiation resistance of the undifferentiated cell is high, and when the expression level and/or the promoter activity of DPP6 is low, the differentiation resistance of the undifferentiated cell is The method according to (1), which is evaluated as having low property.
(4) If the methylation status of the promoter of at least one gene selected from the group consisting of ZNF354C, C12orf56, ZNF578 and MIR886 is low (because the expression (=promoter activity) is low when the methylation status is high, it is It is evaluated that the differentiation resistance of differentiated cells is low, and the methylation status of the promoter of at least one gene selected from the group consisting of ZNF354C, C12orf56, ZNF578 and MIR886 is high (because the expression is low when the methylation status is high). In some cases, the method according to (1), wherein the undifferentiated cells are evaluated to have high differentiation resistance.
(5) When the methylation status of the DPP6 promoter is low, it is evaluated that the differentiation resistance of the undifferentiated cells is high, and when the methylation status of the DPP6 promoter is high, the differentiation resistance of the undifferentiated cells is low. The method described in (1) for evaluation.
(6) The undifferentiated cells are embryonic tumor cells (EC cells), embryonic stem cells (ES cells), induced pluripotent stem cells (iPS cells) or embryonic germ cells (EG cells) (1) to ( The method according to any one of 5).
(7) The method according to any one of (1) to (3) or (6), wherein the expression level of the gene is measured as the amount of mRNA or the amount of protein.
(8) The method for measuring the expression level of a gene is qPCR, digital PCR, immunostaining, in situ hybridization, RNA sequence, microarray, NanoString, antibody array, FlowCytometry, mass spectrometry or a combination thereof (7) Method.
(9) The method for measuring the methylation status of the gene promoter is to concentrate methylated DNA and then detect the concentrated DNA. Decoding (sequencing) the base sequence after base substitution by bisulfite treatment. Method, a method of detecting the nucleotide sequence after hybridization with bisulfite treatment by hybridization, a methylation-specific PCR (MSP) method, a method of detecting with or without cleavage by a methylation-sensitive restriction enzyme, a methylated cytosine The method according to any one of (1), (4) to (6), which is a method of glucosylation and detection with an enzyme sensitive to glucosylated cytosine, or a combination thereof.
(10) A method for selecting an undifferentiated cell line having low or high differentiation resistance by measuring the following (i) and/or (ii) in the undifferentiated cell population.
(i) Expression level and/or promoter activity of at least one gene selected from the group consisting of ZNF354C, C12orf56, ZNF578, DPP6 and MIR886
(ii) methylation status of the promoter of at least one gene selected from the group consisting of ZNF354C, C12orf56, ZNF578, DPP6 and MIR886 (11) at least one gene selected from the group consisting of ZNF354C, C12orf56, ZNF578 and MIR886 When the expression level and/or the promoter activity is high, it is selected as an undifferentiated cell line with low differentiation resistance, and the expression level of at least one gene selected from the group consisting of ZNF354C, C12orf56, ZNF578 and MIR886 and/or The method according to (10), wherein when the promoter activity is low, it is selected as an undifferentiated cell line having high differentiation resistance.
(12) When the expression level and/or promoter activity of DPP6 is high, it is selected as an undifferentiated cell line having high differentiation resistance, and when the expression level and/or promoter activity of DPP6 is low, the differentiation resistance is low. The method according to (10), wherein the cell line is selected as a differentiated cell line.
(13) When a promoter of at least one gene selected from the group consisting of ZNF354C, C12orf56, ZNF578 and MIR886 has a low methylation state, it is selected as an undifferentiated cell line having low differentiation resistance, and ZNF354C, C12orf56, ZNF578 is selected. The method according to (10), wherein an undifferentiated cell line having high differentiation resistance is selected when the promoter has a high methylation state of at least one gene selected from the group consisting of MIR886 and MIR886.
(14) When the methylation status of the DPP6 promoter is low, it is selected as an undifferentiated cell line with high differentiation resistance, and when the methylation status of the DPP6 promoter is high, it is selected as an undifferentiated cell line with low differentiation resistance. The method described in (10) of selecting.
(15) The undifferentiated cell line is an embryonal tumor cell (EC cell) line, an embryonic stem cell (ES cell) line, an induced pluripotent stem cell (iPS cell) line, or an embryonic germ cell (EG cell) line The method according to any one of (10) to (14).
(16) A marker for at least one gene selected from the group consisting of ZNF354C, C12orf56, ZNF578, DPP6 and MIR886 in order to detect undifferentiated cells with low or high differentiation resistance present in the undifferentiated cell population. As a method to use.
(17) A kit for evaluating the differentiation resistance of undifferentiated cells, which comprises a reagent capable of measuring the following (i) and/or (ii).
(i) Expression level and/or promoter activity of at least one gene selected from the group consisting of ZNF354C, C12orf56, ZNF578, DPP6 and MIR886
(ii) The methylation state of the promoter of at least one gene selected from the group consisting of ZNF354C, C12orf56, ZNF578, DPP6 and MIR886 (18) The reagent capable of measuring the expression level of the gene is a primer, a probe or an antibody. The kit according to (17).
(19) The kit according to (17), wherein the reagent capable of measuring the promoter activity of the gene is a gene sequence in which a reporter protein is linked downstream of the promoter or a vector incorporating this gene sequence.
(20) A reagent capable of measuring the methylation state of a gene promoter is bisulfite (bisulfite), a microarray reagent for methylation analysis, a sequencing reagent by the Sanger method, a sequencing reagent for a next-generation sequencer, a 5-mC antibody, 5-hmC antibody, methyladenosine antibody, 5'-methyl-2'-deoxycytidine antibody, HRP-labeled DNA antibody, 5-hmC glucosyltransferase, glucosyl-5hmC sensitive restriction enzyme endonuclease, MBD1 (Methyl-CpG Binding Domain Protein1), The kit according to (17), which is MBD2 (Methyl-CpG Binding Domain Protein2), a specific PCR primer, a specific probe or a DNA purification kit.
　本発明により、未分化細胞（例えば、未分化多能性幹細胞）の良否の早期判定による製造コストの抑制および未分化細胞から形成されるオルガノイド（例えば、肝臓器官芽(iPS細胞肝芽など)）の安全性の確保が達成される。
　本明細書は、本願の優先権の基礎である日本国特許出願、特願2019‐005892の明細書および／または図面に記載される内容を包含する。 According to the present invention, undifferentiated cells (for example, undifferentiated pluripotent stem cells) early determination of quality of the production cost suppression and organoids formed from undifferentiated cells (eg, liver organ bud (iPS cell liver bud, etc.)) The safety of is secured.
This specification includes the content described in the Japan patent application, Japanese Patent Application No. 2019-005892, and/or drawing which are the foundations of the priority of this application.
各マーカー遺伝子の発現量と、再培養法にて評価した肝内胚葉細胞(HE)での残存未分化iPS細胞数の相関。未分化残存しやすい株では、再培養法で未分化iPS細胞の残存が見られ、未分化残存し難い株では未分化残存しやすい株で、未分化iPS細胞の残存が検出されない。各マーカー遺伝子は未分化残存した時(未分化残存し難い株)と未分化残存しなかった時(未分化残存しやすい株)の間で明確な発現の違いが有る。Correlation between the expression level of each marker gene and the number of undifferentiated iPS cells remaining in hepatic endoderm cells (HE) evaluated by the reculturing method. Undifferentiated iPS cells remain in the reculturing method in undifferentiated cells that remain easily, and undifferentiated iPS cells remain in unredifferentiated cells that do not remain undifferentiated. There is a clear difference in expression between each marker gene when it remains undifferentiated (a strain that is unlikely to remain undifferentiated) and when it is not undifferentiated (a strain that easily remains undifferentiated).
　以下、本発明を詳細に説明する。 The present invention will be described in detail below.
　本発明は、下記の(i)及び／又は(ii)を測定することを含む、未分化細胞の分化抵抗性を評価する方法を提供する。
(i)ZNF354C、C12orf56、ZNF578、DPP6及びMIR886からなる群より選択される少なくとも１つの遺伝子の発現レベル及び／又はプロモーター活性
(ii) ZNF354C、C12orf56、ZNF578、DPP6及びMIR886からなる群より選択される少なくとも１つの遺伝子のプロモーターのメチル化状態
　発現レベル、プロモーター活性及びプロモーターのメチル化状態を測定する遺伝子は、１種類であってもよいし、２種類以上の組み合わせでもよい。 The present invention provides a method for evaluating differentiation resistance of undifferentiated cells, which comprises measuring the following (i) and/or (ii).
(i) Expression level and/or promoter activity of at least one gene selected from the group consisting of ZNF354C, C12orf56, ZNF578, DPP6 and MIR886
(ii) Methylation status of the promoter of at least one gene selected from the group consisting of ZNF354C, C12orf56, ZNF578, DPP6 and MIR886. There is only one kind of gene for measuring the expression level, promoter activity and methylation status of the promoter. It may be a combination of two or more kinds.
　評価の対象とする未分化細胞は、多能性を有する細胞であるとよく、例えば、未分化細胞は、胚性腫瘍細胞（ＥＣ細胞）、胚性幹細胞（ＥＳ細胞）又は人工多能性幹細胞（ｉＰＳ細胞）、胚性生殖細胞(EG細胞)である。未分化細胞は、ヒトあるいはヒト以外のいかなる動物に由来するものであってもよい。 The undifferentiated cell to be evaluated may be a cell having pluripotency, and for example, the undifferentiated cell is an embryonal tumor cell (EC cell), an embryonic stem cell (ES cell) or an induced pluripotent stem cell. (IPS cells) and embryonic germ cells (EG cells). The undifferentiated cell may be derived from human or any animal other than human.
　本明細書において、「分化抵抗性」とは、分化しにくさをいい、肝細胞への分化しにくさであることが好ましい。
　未分化細胞の分化抵抗性は、細胞株によって異なることが知られているが、継代回数、継代方法や継代時の細胞密度などの培養条件によって、変化する可能性がある。あるいは培養用基質や培地によっても変化する可能性がある。
　分化抵抗性は、分化誘導後に一定の細胞数の分化細胞を再播種し、未分化培養条件下で培養し、増殖したコロニーを未分化マーカー(SOX2など)による免疫染色で、未分化iPS細胞であることを確認し、1コロニーを1個の未分化iPS細胞であるとして算出することで評価できる。 In the present specification, “differentiation resistance” refers to difficulty in differentiation, and preferably resistance to hepatocyte differentiation.
It is known that the differentiation resistance of undifferentiated cells differs depending on the cell line, but it may change depending on the culture conditions such as the number of passages, the passage method and the cell density at the passage. Alternatively, it may change depending on the culture substrate or medium.
Differentiation resistance is determined by re-seeding a certain number of differentiated cells after induction of differentiation, culturing under undifferentiated culture conditions, and proliferating colonies by immunostaining with an undifferentiated marker (SOX2, etc.). It can be evaluated by confirming that there is one and calculating one colony as one undifferentiated iPS cell.
　ZNF354C、C12orf56、ZNF578及びMIR886からなる群より選択される少なくとも１つの遺伝子の発現レベル及び／又はプロモーター活性が高い場合に、未分化細胞の分化抵抗性が低いと評価し、ZNF354C、C12orf56、ZNF578及びMIR886からなる群より選択される少なくとも１つの遺伝子の発現レベル及び／又はプロモーター活性が低い場合に、未分化細胞の分化抵抗性が高いと評価することができる。 When the expression level and/or promoter activity of at least one gene selected from the group consisting of ZNF354C, C12orf56, ZNF578 and MIR886 is high, it is evaluated that the differentiation resistance of undifferentiated cells is low, and ZNF354C, C12orf56, ZNF578 and When the expression level and/or promoter activity of at least one gene selected from the group consisting of MIR886 is low, it can be evaluated that the undifferentiated cells have high differentiation resistance.
　DPP6の発現レベル及び／又はプロモーター活性が高い場合に、未分化細胞の分化抵抗性が高いと評価し、DPP6の発現レベル及び／又はプロモーター活性が低い場合に、未分化細胞の分化抵抗性が低いと評価することができる。 When the expression level and/or promoter activity of DPP6 is high, it is evaluated that the differentiation resistance of undifferentiated cells is high, and when the expression level and/or promoter activity of DPP6 is low, the differentiation resistance of undifferentiated cells is low Can be evaluated.
　ZNF354C、C12orf56、ZNF578及びMIR886からなる群より選択される少なくとも１つの遺伝子のプロモーターのメチル化状態が低い場合に、未分化細胞の分化抵抗性が低いと評価し、ZNF354C、C12orf56、ZNF578及びMIR886からなる群より選択される少なくとも１つの遺伝子のプロモーターのメチル化状態が高い場合に、未分化細胞の分化抵抗性が高いと評価することができる。 When the methylation status of the promoter of at least one gene selected from the group consisting of ZNF354C, C12orf56, ZNF578 and MIR886 is low, it is evaluated that the differentiation resistance of undifferentiated cells is low, and it is determined from ZNF354C, C12orf56, ZNF578 and MIR886. When the methylation status of the promoter of at least one gene selected from the group is high, it can be evaluated that the undifferentiated cells have high differentiation resistance.
　DPP6のプロモーターのメチル化状態が低い場合に、未分化細胞の分化抵抗性が高いと評価し、DPP6のプロモーターのメチル化状態が高い場合に、未分化細胞の分化抵抗性が低いと評価することができる。 When the methylation status of the DPP6 promoter is low, it is evaluated that the differentiation resistance of the undifferentiated cells is high, and when the methylation status of the DPP6 promoter is high, the differentiation resistance of the undifferentiated cells is evaluated as low. You can
　本明細書において、遺伝子の発現レベルが「高い」あるいは「低い」とは、未分化細胞の分化抵抗性の高いものと低いものを判別できる所定の数値よりも高いあるいは低いことを意味する。その数値は、求める感度や特異度によって異なるものであり、また、未分化細胞の種類（例えば、iPS細胞かES細胞かといった違い）、細胞のクローンの違いなどによって変化しうる。遺伝子のプロモーター活性が「高い」あるいは「低い」、メチル化状態の「高い」あるいは「低い」も同様である。 In the present specification, “high” or “low” expression level of a gene means that it is higher or lower than a predetermined numerical value capable of discriminating between high differentiation resistance and low differentiation resistance of undifferentiated cells. The numerical value varies depending on the sensitivity and specificity to be sought, and may vary depending on the type of undifferentiated cell (for example, difference between iPS cell and ES cell) and difference in cell clone. The same applies to "high" or "low" promoter activity of a gene and "high" or "low" methylation status.
　後述の実施例での実験によれば、遺伝子の発現レベルをqPCRで測定した場合、ZNF354Cの発現レベルの値が18Sを内部標準としたとき2x10^-7以上である場合に、未分化細胞の分化抵抗性が低いと評価でき、ZNF354Cの発現レベルの値が18Sを内部標準としたとき1x10^-7以下又は未満である場合に、未分化細胞の分化抵抗性が高いと評価できる。同じ測定法によれば、C12orf56の発現レベルの値が18Sを内部標準としたとき2.5x10^-7以上、ZNF578の発現レベルの値が18Sを内部標準としたとき1x10^-7以上、DPP6の発現レベルの値が18Sを内部標準としたとき3x10^-7以下又は未満、MIR886の発現レベルの値が18Sを内部標準としたとき1x10^-6以上である場合に、未分化細胞の分化抵抗性が低いと評価でき、C12orf56の発現レベルの値が18Sを内部標準としたとき2x10^-7以下又は未満、ZNF578の発現レベルの値が18Sを内部標準としたとき5x10^-8以下又は未満、DPP6の発現レベルの値が18Sを内部標準としたとき3x10^-7以上、MIR886の発現レベルの値が18Sを内部標準としたとき1x10^-7以下又は未満である場合に、未分化細胞の分化抵抗性が高いと評価できる。 According to the experiments in Examples described below, when the gene expression level was measured by qPCR, when the expression level value of ZNF354C was 2x10^-7 or more when 18S was used as an internal standard, undifferentiated cells It can be evaluated that differentiation resistance is low, and when the expression level of ZNF354C is 1x10^-7 or less or less when 18S is used as an internal standard, it can be evaluated that differentiation resistance of undifferentiated cells is high. According to the same measurement method, the expression level value of C12orf56 is 2.5x10^-7 or more when 18S is used as an internal standard, and the expression level value of ZNF578 is 1x10^-7 or more when 18S is used as an internal standard. Differentiation resistance of undifferentiated cells when the expression level value is 3x10^-7 or less when 18S is used as an internal standard, and when the expression level value of MIR886 is 1x10^-6 or more when 18S is used as an internal standard. If the expression level of C12orf56 is 18x as an internal standard, it is 2x10^-7 or less or less, and if the expression level of ZNF578 is 18S as an internal standard, 5x10^-8 or less or less. , Undifferentiated cells when the expression level value of DPP6 is 3x10^-7 or more when 18S is used as an internal standard, and when the expression level value of MIR886 is 1x10^-7 or less when 18S is used as an internal standard. Can be evaluated as having high differentiation resistance.
　また、後述の実施例での実験によれば、バイサルファイト処理実施後のイルミナ社Infinium MethylationEPIC BEadChip Kitを用いて遺伝子のプロモーターのメチル化状態を測定した場合、ZNF354Cのプロモーターのメチル化状態の値が40%以下又は未満である場合に、未分化細胞の分化抵抗性が低いと評価し、ZNF354Cのプロモーターのメチル化状態の値が60%以上である場合に、未分化細胞の分化抵抗性が高いと評価することができる。同じ測定法によれば、C12orf56のプロモーターのメチル化状態の値が40%以下又は未満、ZNF578のプロモーターのメチル化状態の値が40%以下又は未満、DPP6のプロモーターのメチル化状態の値が60%以上、MIR886のプロモーターのメチル化状態の値が40%以下又は未満、である場合に、未分化細胞の分化抵抗性が低いと評価し、C12orf56のプロモーターのメチル化状態の値が60%以上、ZNF578のプロモーターのメチル化状態の値が60%以上、DPP6のプロモーターのメチル化状態の値が40%以下又は未満、MIR886のプロモーターのメチル化状態の値が60%以上である場合に、未分化細胞の分化抵抗性が高いと評価することができる。 Further, according to the experiment in the example described below, when the methylation state of the promoter of the gene was measured using Illumina Infinity Methylation EPIC BEAD Chip Kit after bisulfite treatment, the value of the methylation state of the promoter of ZNF354C was When it is 40% or less or less, it is evaluated that the differentiation resistance of undifferentiated cells is low, and when the methylation state value of the promoter of ZNF354C is 60% or more, the differentiation resistance of undifferentiated cells is high. Can be evaluated. According to the same measurement method, the value of methylation status of the promoter of C12orf56 is 40% or less or less, the value of methylation status of the promoter of ZNF578 is 40% or less or less, and the value of methylation status of the promoter of DPP6 is 60% or less. % Or more, the MIR886 promoter methylation state value is 40% or less, or less, if the differentiation resistance of undifferentiated cells is evaluated as low, C12orf56 promoter methylation state value of 60% or more , The value of the methylation status of the promoter of ZNF578 is 60% or more, the value of the methylation status of the promoter of DPP6 is 40% or less, or less, and the value of the methylation status of the promoter of MIR886 is 60% or more. It can be evaluated that the differentiation resistance of the differentiated cells is high.
　遺伝子の発現レベルは、遺伝子から転写されたmRNAの量あるいはmRNAから翻訳されたタンパク質の量として測定することができる。具体的には、遺伝子の発現レベルは、qPCR、デジタルPCR、免疫染色、in situ hybridization、RNAシークエンス、マイクロアレイ、NanoString、抗体アレイ、FlowCytometry、質量分析、それらの組み合わせなどで測定することができる。 The expression level of a gene can be measured as the amount of mRNA transcribed from the gene or the amount of protein translated from mRNA. Specifically, the gene expression level can be measured by qPCR, digital PCR, immunostaining, in situ hybridization, RNA sequence, microarray, NanoString, antibody array, FlowCytometry, mass spectrometry, a combination thereof and the like.
　遺伝子のプロモーターのメチル化状態を解析する手法としては、１． 抗メチル化シトシンやアデノシン抗体などを用いてメチル化 DNA を濃縮し、濃縮後にシークエンス、マイクロアレイ、qPCR等によって、濃縮されたDNAを検出、定量する方法、２． バイサルファイト処理による塩基置換した後、塩基配列を解読(シークエンス)あるいはマイクロアレイ、MLPAR（Multiplex Ligation Probe Amplification）法等のハイブリダイズを用いて検出する方法、３．バイサルファイト処理による塩基置換した後、PCRによる増幅の有無で検出する方法（メチル化特異的PCR(MSP)法）、４． メチル化感受性の制限酵素を利用して切断の有無で検出する方法、５．メチル化シトシンをグルコシル化し、グルコシル化シトシン感受性の酵素で検出する方法等があり、これらのいずれかの方法あるいはその組み合わせなどを用いることができる。 The methods for analyzing the methylation status of the gene promoter are: 1. A method in which methylated DNA is concentrated using an anti-methylated cytosine or adenosine antibody, etc., and the concentrated DNA is detected and quantified by a sequence, microarray, qPCR, etc. after concentration. After the base substitution by bisulfite treatment, a method of detecting the base sequence by decoding (sequencing) or by using hybridization such as microarray or MLPAR (Multiplex Ligation Probe Amplification) method. 3. A method of detecting the presence or absence of amplification by PCR after the base substitution by bisulfite treatment (methylation-specific PCR (MSP) method), 4. 4. A method for detecting the presence or absence of cleavage using a methylation-sensitive restriction enzyme. There is a method in which methylated cytosine is glucosylated and detected with an enzyme sensitive to glucosylated cytosine, and any of these methods or a combination thereof can be used.
　未分化細胞集団における、上記の(i)及び／又は(ii)を測定することによって、分化抵抗性の低いあるいは高い未分化細胞株を選別することができる。よって、本発明は、未分化細胞集団における、下記の(i)及び／又は(ii)を測定することによって、分化抵抗性の低いあるいは高い未分化細胞株を選別する方法を提供する。
(i)ZNF354C、C12orf56、ZNF578、DPP6及びMIR886からなる群より選択される少なくとも１つの遺伝子の発現レベル及び／又はプロモーター活性
(ii) ZNF354C、C12orf56、ZNF578、DPP6及びMIR886からなる群より選択される少なくとも１つの遺伝子のプロモーターのメチル化状態
　発現レベル、プロモーター活性及びプロモーターのメチル化状態を測定する遺伝子は、１種類であってもよいし、２種類以上の組み合わせでもよい。 By measuring the above (i) and/or (ii) in the undifferentiated cell population, undifferentiated cell lines with low or high differentiation resistance can be selected. Therefore, the present invention provides a method for selecting an undifferentiated cell line with low or high differentiation resistance by measuring the following (i) and/or (ii) in the undifferentiated cell population.
(i) Expression level and/or promoter activity of at least one gene selected from the group consisting of ZNF354C, C12orf56, ZNF578, DPP6 and MIR886
(ii) Methylation status of the promoter of at least one gene selected from the group consisting of ZNF354C, C12orf56, ZNF578, DPP6 and MIR886. There is only one kind of gene for measuring the expression level, promoter activity and methylation status of the promoter. It may be a combination of two or more kinds.
　選別の対象とする未分化細胞株は、多能性を有する細胞株であるとよく、例えば、未分化細胞株は、胚性腫瘍細胞（ＥＣ細胞）株、胚性幹細胞（ＥＳ細胞）株、人工多能性幹細胞（ｉＰＳ細胞）株又は胚性生殖細胞（ＥＧ細胞)株であり、ｉＰＳ細胞株が好ましい。未分化細胞株は、ヒトあるいはヒト以外のいかなる動物に由来するものであってもよい。 The undifferentiated cell line to be selected may be a pluripotent cell line. For example, the undifferentiated cell line may be an embryonal tumor cell (EC cell) line, an embryonic stem cell (ES cell) line, Induced pluripotent stem cell (iPS cell) line or embryonic germ cell (EG cell) line, and iPS cell line is preferable. The undifferentiated cell line may be derived from human or any animal other than human.
　ZNF354C、C12orf56、ZNF578及びMIR886からなる群より選択される少なくとも１つの遺伝子の発現レベル及び／又はプロモーター活性が高い場合に、分化抵抗性が低い未分化細胞株として選別し、ZNF354C、C12orf56、ZNF578及びMIR886からなる群より選択される少なくとも１つの遺伝子の発現レベル及び／又はプロモーター活性が低い場合に、分化抵抗性が高い未分化細胞株として選別することができる。 When the expression level and/or promoter activity of at least one gene selected from the group consisting of ZNF354C, C12orf56, ZNF578 and MIR886 is high, it is selected as an undifferentiated cell line having low differentiation resistance, and ZNF354C, C12orf56, ZNF578 and When the expression level and/or promoter activity of at least one gene selected from the group consisting of MIR886 is low, it can be selected as an undifferentiated cell line having high differentiation resistance.
　DPP6の発現レベル及び／又はプロモーター活性が高い場合に、分化抵抗性が高い未分化細胞株として選別し、DPP6の発現レベル及び／又はプロモーター活性が低い場合に、分化抵抗性が低い未分化細胞株として選別することができる。 When the expression level and/or promoter activity of DPP6 is high, it is selected as an undifferentiated cell line with high differentiation resistance, and when the expression level and/or promoter activity of DPP6 is low, undifferentiated cell line with low differentiation resistance Can be sorted as.
　ZNF354C、C12orf56、ZNF578及びMIR886からなる群より選択される少なくとも１つの遺伝子のプロモーターのメチル化状態が低い場合に、分化抵抗性が低い未分化細胞株として選別し、ZNF354C、C12orf56、ZNF578及びMIR886からなる群より選択される少なくとも１つの遺伝子のプロモーターのメチル化状態が高い場合に、分化抵抗性が高い未分化細胞株として選別することができる。 When the methylation status of the promoter of at least one gene selected from the group consisting of ZNF354C, C12orf56, ZNF578 and MIR886 is low, it is selected as an undifferentiated cell line with low differentiation resistance, and selected from ZNF354C, C12orf56, ZNF578 and MIR886. An undifferentiated cell line with high differentiation resistance can be selected when the methylation status of the promoter of at least one gene selected from the group is high.
　DPP6のプロモーターのメチル化状態が低い場合に、分化抵抗性が高い未分化細胞株として選別し、DPP6のプロモーターのメチル化状態が高い場合に、分化抵抗性が低い未分化細胞株として選別することができる。 When the methylation status of the DPP6 promoter is low, select as an undifferentiated cell line with high differentiation resistance, and when the methylation status of the DPP6 promoter is high, select as undifferentiated cell line with low differentiation resistance You can
　遺伝子の発現レベル、プロモーター活性及び遺伝子のプロモーターのメチル化状態の「高い」あるいは「低い」については上述した通りである。 “The expression level of the gene, the promoter activity, and the methylation state of the promoter of the gene is “high” or “low” as described above.
　ZNF354C、C12orf56、ZNF578、DPP6及びMIR886は、未分化細胞集団中に存在する、分化抵抗性の低いあるいは高い未分化細胞を検出するためのマーカー遺伝子として使用できる。よって、本発明は、未分化細胞集団中に存在する、分化抵抗性の低いあるいは高い未分化細胞を検出するために、ZNF354C、C12orf56、ZNF578、DPP6及びMIR886からなる群より選択される少なくとも１つの遺伝子をマーカーとして使用する方法を提供する。
　マーカーとして使用する遺伝子は、１種類であってもよいし、２種類以上の組み合わせでもよい。 ZNF354C, C12orf56, ZNF578, DPP6 and MIR886 can be used as a marker gene for detecting undifferentiated cells with low or high differentiation resistance present in the undifferentiated cell population. Therefore, the present invention, in the undifferentiated cell population, in order to detect undifferentiated cells with low or high differentiation resistance, at least one selected from the group consisting of ZNF354C, C12orf56, ZNF578, DPP6 and MIR886. Methods of using genes as markers are provided.
The gene used as a marker may be one type or a combination of two or more types.
　また、本発明は、下記の(i)及び／又は(ii)を測定可能な試薬を含む、未分化細胞の分化抵抗性を評価するためのキットを提供する。
(i)ZNF354C、C12orf56、ZNF578、DPP6及びMIR886からなる群より選択される少なくとも１つの遺伝子の発現レベル及び／又はプロモーター活性
(ii) ZNF354C、C12orf56、ZNF578、DPP6及びMIR886からなる群より選択される少なくとも１つの遺伝子のプロモーターのメチル化状態
　発現レベル、プロモーター活性及びプロモーターのメチル化状態を測定する遺伝子は、１種類であってもよいし、２種類以上の組み合わせでもよい。 The present invention also provides a kit for evaluating the differentiation resistance of undifferentiated cells, which comprises a reagent capable of measuring the following (i) and/or (ii).
(i) Expression level and/or promoter activity of at least one gene selected from the group consisting of ZNF354C, C12orf56, ZNF578, DPP6 and MIR886
(ii) Methylation status of the promoter of at least one gene selected from the group consisting of ZNF354C, C12orf56, ZNF578, DPP6 and MIR886. There is only one kind of gene for measuring the expression level, promoter activity and methylation status of the promoter. It may be a combination of two or more kinds.
　遺伝子の発現レベルを測定可能な試薬としては、プライマー、プローブ及び抗体などを挙げることができ、例えば、ZNF354C、C12orf56、ZNF578、DPP6及びMIR886からなる群より選択される少なくとも１つの遺伝子の転写産物（mRNA）又はcDNAを特異的に増幅できるオリゴヌクレオチドプライマーのセット、ZNF354C、C12orf56、ZNF578、DPP6及びMIR886からなる群より選択される少なくとも１つの遺伝子の転写産物（mRNA）又はcDNAに特異的にハイブリダイズするヌクレオチドプローブ、ZNF354C、C12orf56、ZNF578、DPP6及びMIR886からなる群より選択される少なくとも１つの遺伝子の転写産物（mRNA）から翻訳されたタンパク（翻訳産物）に特異的に結合する抗体である。オリゴヌクレオチドプライマーのセットは、ZNF354C、C12orf56、ZNF578、DPP6及びMIR886からなる群より選択される少なくとも１つの遺伝子の転写産物（mRNA）又はcDNAのヌクレオチド配列中の標的配列（通常、５０～１８０ｂｐ程度）を増幅できるものであるとよく、標的配列の両末端と相補的な配列を有するように設計されるとよい。オリゴヌクレオチドプライマーの長さは、例えば、１５～３５ヌクレオチドであるとよく、１８～２７ヌクレオチドが好ましい。ヌクレオチドプローブは、ZNF354C、C12orf56、ZNF578、DPP6及びMIR886からなる群より選択される少なくとも１つの遺伝子の転写産物（mRNA）又はcDNAにストリンジェントな条件下でハイブリダイズするものであるとよく、前記mRNA又はcDNAのヌクレオチド配列の一部又は全部又はそれに相補的な配列を有するように設計されるとよい。ストリンジェントな条件は適宜決定することができる。ヌクレオチドプローブの長さは、通常１０００ヌクレオチド以下、好ましくは、１００ヌクレオチド以下、より好ましくは５０ヌクレオチド以下、さらにより好ましくは、１４～３０ヌクレオチドである。ヌクレオチドプローブは、一本鎖であっても、二本鎖であってもよい。抗体は、モノクローナル抗体又はポリクローナル抗体のいずれであってもよい。本明細書において、抗体とは、全長抗体の他、Fab、F(ab)’2、ScFv、Diabody、VH、VL、Sc(Fv)2、Bispecific sc(Fv)2、Minibody、ScFv-Fc monomer、ScFv-Fc dimerなどの低分子化されたものも含む概念である。プローブや抗体は、固相（例えば、基板、ビーズ、膜など）上に固定されていてもよい。 Examples of the reagent capable of measuring the expression level of a gene include a primer, a probe and an antibody. For example, a transcription product of at least one gene selected from the group consisting of ZNF354C, C12orf56, ZNF578, DPP6 and MIR886 ( mRNA) or a set of oligonucleotide primers capable of specifically amplifying cDNA, specifically hybridizing to a transcription product (mRNA) or cDNA of at least one gene selected from the group consisting of ZNF354C, C12orf56, ZNF578, DPP6 and MIR886. Is an antibody that specifically binds to a protein (translation product) translated from a transcription product (mRNA) of at least one gene selected from the group consisting of ZNF354C, C12orf56, ZNF578, DPP6 and MIR886. The set of oligonucleotide primers is a target sequence (usually about 50 to 180 bp) in the nucleotide sequence of a transcript (mRNA) or cDNA of at least one gene selected from the group consisting of ZNF354C, C12orf56, ZNF578, DPP6 and MIR886. Of the target sequence and may be designed to have a sequence complementary to both ends of the target sequence. The length of the oligonucleotide primer may be, for example, 15 to 35 nucleotides, preferably 18 to 27 nucleotides. The nucleotide probe may hybridize to a transcript (mRNA) of at least one gene selected from the group consisting of ZNF354C, C12orf56, ZNF578, DPP6 and MIR886 (mRNA) or cDNA under stringent conditions. Alternatively, it may be designed to have a part or all of the nucleotide sequence of the cDNA or a sequence complementary thereto. Stringent conditions can be appropriately determined. The length of the nucleotide probe is usually 1000 nucleotides or less, preferably 100 nucleotides or less, more preferably 50 nucleotides or less, and even more preferably 14 to 30 nucleotides. The nucleotide probe may be single-stranded or double-stranded. The antibody may be either a monoclonal antibody or a polyclonal antibody. In the present specification, the antibody, in addition to full-length antibodies, Fab, F(ab)' 2 , ScFv, Diabody, V H , VL , Sc(Fv) 2 , Bispecific sc(Fv) 2 , Minibody, ScFv- It is a concept that includes low molecular weight compounds such as Fc monomer and ScFv-Fc dimer. The probe or antibody may be immobilized on a solid phase (eg, substrate, beads, membrane, etc.).
　本発明の試薬は標識されてもよい。例えば、プライマーは、蛍光物質や消光物質などにより標識されていてもよく、プローブ及び抗体は、放射性同位元素、酵素、発光物質、蛍光物質、ビオチンなどで標識されてもよい。また、ターゲット分子（本発明では、ZNF354C、C12orf56、ZNF578、DPP6及びMIR886からなる群より選択される少なくとも１つの遺伝子の発現産物であるタンパク質）に特異的に結合する一次抗体の反応後、この一次抗体に結合する二次抗体を反応させて、ターゲット分子の検出を行う場合には、二次抗体を標識するとよい（一次抗体は標識しない）。 The reagent of the present invention may be labeled. For example, the primer may be labeled with a fluorescent substance or a quenching substance, and the probe and the antibody may be labeled with a radioisotope, an enzyme, a luminescent substance, a fluorescent substance, biotin or the like. In addition, after the reaction with the primary antibody that specifically binds to the target molecule (in the present invention, the protein that is the expression product of at least one gene selected from the group consisting of ZNF354C, C12orf56, ZNF578, DPP6 and MIR886), the primary antibody When the target molecule is detected by reacting a secondary antibody that binds to the antibody, the secondary antibody may be labeled (the primary antibody is not labeled).
　遺伝子のプロモーター活性を測定可能な試薬としては、プロモーター下流にレポータータンパク質を連結した遺伝子配列又はこの遺伝子配列を組み込んだベクターなどを挙げることができる。レポータータンパク質としては、ルシフェラーゼ、GFPなどの蛍光タンパク質、CD抗原などの細胞膜に発現するタンパク質などを例示することができる。ベクターはプラスミドベクターが好ましい。 Examples of the reagent capable of measuring the promoter activity of a gene include a gene sequence in which a reporter protein is linked downstream of the promoter or a vector incorporating this gene sequence. Examples of reporter proteins include luciferase, fluorescent proteins such as GFP, and proteins expressed on cell membranes such as CD antigen. The vector is preferably a plasmid vector.
　遺伝子のプロモーターのメチル化状態を測定可能な試薬としては、バイサルファイト（亜硫酸水素塩）、Illumina 社 Infinium MethylationEPIC BeadChip等のメチル化解析用マイクロアレイ試薬、Sanger法によるシークエンス試薬、次世代シークエンサー用シークエンス試薬、5-mC抗体、5-hmC抗体、メチルアデノシン抗体、5’-methyl-2’-deoxycytidine抗体、HRP標識DNA抗体、5-hmC グルコシルトランスフェラーゼ、グルコシル-5hmC感受性制限酵素エンドヌクレアーゼ（GSRE: MspI、GlaI、Csp6I、HaeIII、TaqαI、MboI、McrBC)、MBD1 (Methyl-CpG Binding Domain Protein1)、MBD2 (Methyl-CpG Binding Domain Protein2)、特異的PCRプライマー、特異的プローブ又はDNA精製キットなどを例示することができる。 As a reagent that can measure the methylation state of the gene promoter, bisulfite (bisulfite), a microarray reagent for methylation analysis such as Illumina Infinity Methylation EPIC BeadChip, a sequencing reagent by the Sanger method, a sequencing reagent for the next-generation sequencer, 5-mC antibody, 5-hmC antibody, methyladenosine antibody, 5'-methyl-2'-deoxycytidine antibody, HRP-labeled DNA antibody, 5-hmCglucose transferase, glucosyl-5hmC sensitive restriction endonuclease (GSRE: MspI, GlaI) , Csp6I, HaeIII, TaqαI, MboI, McrBC), MBD1 (Methyl-CpGBinding Domain Protein1), MBD2 (Methyl-CpG Binding Domain Protein2), specific PCR primer, specific probe or DNA purification kit. it can.
　本発明のキットは、さらに、プライマーで検出するための試薬（ＤＮＡポリメラーゼ、バッファー、マグネシウムイオン、ｄＮＴＰｓ、プローブなど）、プローブで検出するための試薬（バッファー、抗体、基質など）、抗体で検出するための試薬（二次抗体、基質、バッファーなど）、遺伝子のプロモーター活性を測定するための試薬（バッファー、発光基質、抗体など）、器具（反応容器、ピペットなど）、キットの使用説明書、対照用の試料、測定結果を解析するための対照データなどを含んでもよい。 The kit of the present invention further detects with a reagent for detection with a primer (DNA polymerase, buffer, magnesium ion, dNTPs, probe, etc.), a reagent for detection with a probe (buffer, antibody, substrate, etc.), and antibody. Reagents (secondary antibodies, substrates, buffers, etc.), reagents for measuring gene promoter activity (buffers, luminescent substrates, antibodies, etc.), instruments (reaction vessels, pipettes, etc.), kit instructions, controls Samples for use, control data for analyzing measurement results, and the like may be included.
　本発明のマーカー遺伝子（ZNF354C、C12orf56、ZNF578、DPP6及びMIR886）は以下のようにして見出された。継代回数が異なる同一iPS細胞クローンの分化細胞における未分化iPS細胞残存量を評価し、未分化iPS細胞が残存するiPS細胞と残存しないiPS細胞におけるDNAメチル化解析を実施し、DNAメチル化状態に差が有る遺伝子について、分化させる前のiPS細胞時点での遺伝子発現を調べ、未分化残存し易いクローンと未分化残存し難いクローンの間で発現に相違のある遺伝子を同定する。同定された遺伝子がマーカー遺伝子の候補となる。よって、本発明は、継代回数などの培養状態が異なる同一未分化細胞クローンの分化細胞における未分化細胞残存量を評価し、未分化細胞が残存するクローンと未分化細胞が残存しないクローンにおけるDNAメチル化解析を実施し、DNAメチル化状態に差が有る遺伝子について、分化させる前の未分化細胞で遺伝子発現を調べ、未分化のまま残存し易いクローンと未分化のまま残存し難いクローンの間で発現に相違のある遺伝子を同定することを含む、未分化細胞集団中に存在する、分化抵抗性の低いあるいは高い未分化細胞を検出するためのマーカー遺伝子を探索する方法を提供する。"DNAメチル化状態に差が有る遺伝子"が他のクローンなどでも共通して、未分化細胞の残存と相関が有れば、遺伝子発現ではなくメチル化状態を評価することによって予測することが出来るマーカーとなる。 The marker genes (ZNF354C, C12orf56, ZNF578, DPP6 and MIR886) of the present invention were found as follows. The amount of undifferentiated iPS cells remaining in differentiated cells of the same iPS cell clone with different passage numbers was evaluated, and DNA methylation analysis was performed on iPS cells that remained undifferentiated iPS cells and those that did not, to determine the DNA methylation status. For genes that have a difference in expression, the gene expression at the time of iPS cells before differentiation is examined to identify genes that differ in expression between clones that are likely to remain undifferentiated and clones that are unlikely to remain undifferentiated. The identified gene becomes a candidate for the marker gene. Therefore, the present invention evaluates the undifferentiated cell residual amount in the differentiated cells of the same undifferentiated cell clone having different culture conditions such as the number of passages, and the DNA in the clone in which the undifferentiated cell remains and the clone in which the undifferentiated cell does not remain. Methylation analysis was performed to examine gene expression in undifferentiated cells before differentiation for genes that differ in DNA methylation status, between clones that tend to remain undifferentiated and clones that remain difficult to remain undifferentiated. The present invention provides a method for searching for a marker gene for detecting undifferentiated cells with low or high differentiation resistance, which are present in an undifferentiated cell population, which comprises identifying genes that differ in expression. If "genes with different DNA methylation status" are common to other clones and have a correlation with the survival of undifferentiated cells, it can be predicted by evaluating methylation status instead of gene expression. Become a marker.
　未分化細胞は、多能性を有する細胞であるとよく、例えば、未分化細胞は、胚性腫瘍細胞（ＥＣ細胞）、胚性幹細胞（ＥＳ細胞）又は人工多能性幹細胞（ｉＰＳ細胞）、胚性生殖細胞(EG細胞)である。未分化細胞は、ヒトあるいはヒト以外のいかなる動物に由来するものであってもよい。 The undifferentiated cell may be a cell having pluripotency, and for example, the undifferentiated cell is an embryonal tumor cell (EC cell), an embryonic stem cell (ES cell) or an induced pluripotent stem cell (iPS cell), It is an embryonic germ cell (EG cell). The undifferentiated cell may be derived from human or any animal other than human.
　培養状態としては、継代回数、継代方法や継代時の細胞密度などの培養条件、培養用基質や培地などを例示することができる。
　未分化細胞の継代回数は、少なくとも１回以上であればよく、未分化細胞の分化抵抗性が高い場合の継代数は、４０～１００が好ましく、未分化細胞の分化抵抗性が低い場合の継代数は８～３０が好ましい。iPS細胞は無限に増殖すると考えられているので継代数は何回でも無限でも可能である。 Examples of the culture state include culture conditions such as the number of passages, a passage method and cell density at the passage, a substrate for culture and a medium.
The undifferentiated cells may be passaged at least once or more. When the undifferentiated cells have high differentiation resistance, the passage number is preferably 40 to 100, and when the undifferentiated cells have low differentiation resistance. The passage number is preferably 8 to 30. Since iPS cells are considered to grow indefinitely, the passage number can be any number of times.
　分化細胞は、内胚葉、中胚葉及び外胚葉のいずれの分化細胞であってもよい。内胚葉の分化細胞としては、肝内胚葉細胞などを例示することができるが、これに限定されるわけではない。中胚葉の分化細胞としては、横中隔間充織細胞、間葉系細胞、血管内皮細胞などを例示することができるが、これらに限定されるわけではない。外胚葉の分化細胞としては、神経幹細胞、神経堤細胞、神経細胞などを例示することができるが、これに限定されるわけではない。 The differentiated cell may be any differentiated cell of endoderm, mesoderm and ectoderm. Examples of endoderm differentiated cells include, but are not limited to, hepatic endoderm cells and the like. Examples of mesodermal differentiated cells include, but are not limited to, transseptal mesenchymal cells, mesenchymal cells, vascular endothelial cells, and the like. Examples of ectodermal differentiated cells include, but are not limited to, neural stem cells, neural crest cells, and neural cells.
　未分化細胞残存量の評価は後述の実施例に記載の方法で行うことができるが、これに限定されるわけではない
。 The amount of undifferentiated cells remaining can be evaluated by the method described in Examples below, but is not limited thereto.
　DNAメチル化解析、遺伝子発現の測定法は上述の通りである。 ・ DNA methylation analysis and gene expression measurement methods are as described above.
　以下、実施例により本発明を具体的に説明する。本発明の範囲は以下の実施例に限定されるものではない。
〔実施例１〕iPS細胞時点で未分化残存の評価が可能なマーカーの同定
目的
　再生医療応用に資するiPS(ES)細胞由来分化細胞における未分化細胞の混入の検出および排除は、すべてのiPS(ES)細胞由来細胞加工製品の安全性の確保における重要な課題である。これまでに網膜色素上皮細胞(RPE)におけるLIN28Aの発現検証による迅速な未分化細胞の混入評価が報告されているが、我々は複数の細胞加工製品においてLIN28Aでの評価が不適であることを見出している。また、製造の最終段階における品質評価では、その時点で規格外となった場合に、時間および経済的に多大な損失が生じるという問題があり、製造の遅れは時に患者の予後をも左右することも考えられる。
　iPS(ES)細胞由来細胞加工製品の安全性の確保のため、最終製品の迅速・超高感度な評価手法の開発が必要である。さらに時間的、経済的観点から製造のより早い段階すなわち、未分化iPS細胞の段階で未分化細胞が最終製品に残存する可能性を予測することが望まれる。
　そこで本発明では未分化iPS細胞の段階で最終製品に残存する可能性を予測することが可能な評価手法を開発した。
　これまでにiPS細胞から分化誘導した三胚葉いずれの分化細胞においても利用可能な、汎用性の高い未分化マーカーを同定している（「未分化細胞検出法」(特願2018-115025))。この研究の中で継代回数が長いiPS細胞において未分化残存が起こりやすいクローンを同定している。さらに、この未分化残存の起こりやすさはジェネティックな変異（遺伝子変異）ではなく、エピジェティックな修飾であることを強く示唆するデータを得た。そこで、未分化残存が起こりやすいiPS細胞クローンと未分化残存が起こりにくいiPS細胞クローンのDNAメチル化状態に違いがある遺伝子を抽出し、さらに定量PCRにより発現と未分化残存に相関がある遺伝子(MIR886(VTRNA2-1)、DPP6、ZNF578、C12orf56、ZNF354C)を抽出した。 Hereinafter, the present invention will be specifically described with reference to examples. The scope of the invention is not limited to the examples below.
[Example 1] Identification of markers capable of evaluating undifferentiated survival at the time of iPS cells
Purpose Detection and elimination of undifferentiated cell contamination in iPS(ES) cell-derived differentiated cells, which contributes to regenerative medicine applications, is an important issue in ensuring the safety of all iPS(ES) cell-derived cell processed products. To date, rapid evaluation of undifferentiated cell contamination by verification of LIN28A expression in retinal pigment epithelial cells (RPE) has been reported, but we found that LIN28A was unsuitable for multiple cell processing products. ing. In addition, quality evaluation at the final stage of manufacturing has a problem that if it is out of specification at that time, a large amount of time and money will be lost, and the delay in manufacturing sometimes affects the prognosis of patients. Can also be considered.
In order to secure the safety of cell processing products derived from iPS(ES) cells, it is necessary to develop a rapid and ultrasensitive evaluation method for final products. Furthermore, it is desirable to predict the possibility that undifferentiated cells will remain in the final product at an earlier stage of production, that is, at the stage of undifferentiated iPS cells, from the viewpoint of time and economy.
Therefore, the present invention has developed an evaluation method capable of predicting the possibility of remaining in the final product at the stage of undifferentiated iPS cells.
So far, a highly versatile undifferentiated marker that can be used in any of the three germ layer differentiated cells derived from iPS cells has been identified (“Undifferentiated cell detection method” (Japanese Patent Application No. 2018-115025)). In this study, we identified clones that tend to remain undifferentiated in iPS cells with long passage times. Furthermore, we obtained data that strongly suggest that the likelihood of undifferentiated survival is not genetic mutation (gene mutation) but epigenetic modification. Therefore, genes that differ in the DNA methylation status of iPS cell clones that are prone to undifferentiated residuals and iPS cell clones that are unlikely to undergo undifferentiated residuals are extracted, and genes that are correlated with expression and undifferentiated residuals by quantitative PCR ( MIR886 (VTRNA2-1), DPP6, ZNF578, C12orf56, ZNF354C) were extracted.
方法
[分化抵抗性評価のための未分化残存試験]
　ヒトiPS細胞から肝細胞を分化誘導し、iPS細胞肝芽の作製に最適なタイミングで胚体内胚葉（DE)の2-6日後(HEとする)にPLoS One. 2014 Oct 27;9(10):e110496に記載の方法を一部改変し、ディッシュコーティング剤としてLaminin511E8(iMatrix,ニッピ)、培養培地としてStemFitを用いて1週間培養し、出現したコロニーをSOX2, TRA1-60などの未分化iPS細胞マーカーで免疫染色することにより、出現したコロニーが未分化iPS細胞コロニーであることを確認する。未分化コロニー1つは分化細胞に含まれる未分化iPS細胞1細胞より形成されたコロニーとして、分化細胞に含まれる未分化iPS細胞の細胞数を評価する。
　ヒトiPS細胞から肝細胞の分化誘導はNature. 499(7459):481-4.(2013); Cell Rep. 21(10):2661-2670.(2017)、に記載の通りに行った。
[メチル化解析]
　メチル化解析は各分化段階の細胞のDNA について、バイサルファイト処理実施後、イルミナ社Infinium MethylationEPIC BeadChip Kit を用いて、DNA メチル化解析を行った。
[発現定量評価]
　定量PCRはNature. 499(7459):481-4.(2013); Cell Rep. 21(10):2661-2670.(2017)、に記載の通りに行った。 Method
[Undifferentiated residual test for evaluation of differentiation resistance]
PLoS One. 2014 Oct 27;9(10) 2-6 days after definitive endoderm (DE) (HE) at the optimal timing for inducing hepatocyte differentiation from human iPS cells and producing iPS cell liver buds : partly modified the method described in e110496, Laminin511E8 (iMatrix, Nippi) as a dish coating agent, cultured for 1 week using StemFit as the culture medium, the undifferentiated iPS cells such as SOX2, TRA1-60 emerged colonies It is confirmed by immunostaining with a marker that the emerged colonies are undifferentiated iPS cell colonies. One undifferentiated colony is a colony formed from one undifferentiated iPS cell contained in the differentiated cell, and the number of undifferentiated iPS cells contained in the differentiated cell is evaluated.
Induction of hepatocyte differentiation from human iPS cells was performed as described in Nature. 499(7459):481-4.(2013); Cell Rep. 21(10):2661-2670.(2017).
[Methylation analysis]
For the methylation analysis, the DNA of cells at each differentiation stage was subjected to bisulfite treatment and then subjected to DNA methylation analysis using Infinia MethylationEPIC BeadChip Kit from Illumina.
[Quantitative evaluation of expression]
Quantitative PCR was performed as described in Nature. 499(7459):481-4.(2013); Cell Rep. 21(10):2661-2670.(2017).
結果
　各マーカー遺伝子の発現量と、再培養法にて評価した肝内胚葉細胞(HE)での残存未分化iPS細胞数の相関を図１に示す。未分化残存しやすい株では、再培養法で未分化iPS細胞の残存が見られ、未分化残存し難い株では、未分化iPS細胞の残存が検出されない。各マーカー遺伝子は未分化残存した時(未分化残存し難い株)と未分化残存しなかった時(未分化残存しやすい株)の間で明確な発現の違いが有る。すなわち、ZNF354C、C12orf56、ZNF578及びMIR886では、未分化残存が無い場合に、未分化残存が有る場合に比べ、遺伝子発現が高く、一方DPP6では、未分化残存が無い場合に、未分化残存が有る場合に比べ、遺伝子発現が低い。 Results The correlation between the expression level of each marker gene and the number of undifferentiated iPS cells remaining in hepatic endoderm cells (HE) evaluated by the reculturing method is shown in FIG. Undifferentiated iPS cells remain in the reculture method in undifferentiated strains, and undifferentiated iPS cells do not remain in undifferentiated strains. There is a clear difference in expression between each marker gene when it remains undifferentiated (a strain that is unlikely to remain undifferentiated) and when it is not undifferentiated (a strain that easily remains undifferentiated). That is, in ZNF354C, C12orf56, ZNF578 and MIR886, when there is no undifferentiated residual, gene expression is higher than when there is undifferentiated residual, while in DPP6, when there is no undifferentiated residual, there is undifferentiated residual. Gene expression is lower than in the case.
まとめ
・iPS細胞はクローンの特性によるもの以外に、培養状態(継代回数など)によって、分化細胞に未分化iPS細胞が残存する場合が有る。
・継代回数の増加によって生じる未分化iPS細胞が残存しやすいiPS細胞はエピジェネティックな変化によって生じる。
・メチル化変異によって遺伝子発現が変化し、未分化iPS細胞の残存しやすさと相関する遺伝子を同定した。この遺伝子は、iPS細胞の段階で分化細胞における未分化iPS細胞の残存のしやすさを類推するマーカーとなる。
　本明細書で引用した全ての刊行物、特許および特許出願をそのまま参考として本明細書にとり入れるものとする。 Summary・In addition to the clone characteristics of iPS cells, there are cases where undifferentiated iPS cells remain in the differentiated cells depending on the culture conditions (number of passages, etc.).
-IPS cells, which tend to remain undifferentiated iPS cells caused by an increase in the number of passages, are caused by epigenetic changes.
・We identified a gene whose gene expression was changed by methylation mutation and correlated with the susceptibility of undifferentiated iPS cells to survival. This gene serves as a marker for analogizing the susceptibility of undifferentiated iPS cells to remaining in differentiated cells at the iPS cell stage.
All publications, patents, and patent applications cited in this specification are incorporated herein by reference as they are.
　本発明は、再生医療に用いるiPS細胞などの未分化細胞の品質評価に利用できる。具体的には、iPS細胞の樹立工程と、マスターセルバンク、ワーキングセルバンクの製造工程、再生医療等製品の製造工程などにおける、未分化細胞の残存/混入の検出や評価に利用することができる。 The present invention can be used for quality evaluation of undifferentiated cells such as iPS cells used in regenerative medicine. Specifically, it can be used for detection and evaluation of residual/contamination of undifferentiated cells in the process of establishing iPS cells, the process of manufacturing master cell banks, working cell banks, and the process of manufacturing products such as regenerative medicine.

Claims (20)

  1. 下記の(i)及び／又は(ii)を測定することを含む、未分化細胞の分化抵抗性を評価する方法。
    (i)ZNF354C、C12orf56、ZNF578、DPP6及びMIR886からなる群より選択される少なくとも１つの遺伝子の発現レベル及び／又はプロモーター活性
    (ii) ZNF354C、C12orf56、ZNF578、DPP6及びMIR886からなる群より選択される少なくとも１つの遺伝子のプロモーターのメチル化状態     A method for evaluating the differentiation resistance of undifferentiated cells, which comprises measuring the following (i) and/or (ii).
    (i) Expression level and/or promoter activity of at least one gene selected from the group consisting of ZNF354C, C12orf56, ZNF578, DPP6 and MIR886
    (ii) Methylation status of the promoter of at least one gene selected from the group consisting of ZNF354C, C12orf56, ZNF578, DPP6 and MIR886
  2. ZNF354C、C12orf56、ZNF578及びMIR886からなる群より選択される少なくとも１つの遺伝子の発現レベル及び／又はプロモーター活性が高い場合に、未分化細胞の分化抵抗性が低いと評価し、ZNF354C、C12orf56、ZNF578及びMIR886からなる群より選択される少なくとも１つの遺伝子の発現レベル及び／又はプロモーター活性が低い場合に、未分化細胞の分化抵抗性が高いと評価する請求項１記載の方法。 When the expression level and/or promoter activity of at least one gene selected from the group consisting of ZNF354C, C12orf56, ZNF578 and MIR886 is high, it is evaluated that the differentiation resistance of undifferentiated cells is low, and ZNF354C, C12orf56, ZNF578 and The method according to claim 1, wherein the undifferentiated cells are evaluated to have high differentiation resistance when the expression level and/or promoter activity of at least one gene selected from the group consisting of MIR886 is low.
  3. DPP6の発現レベル及び／又はプロモーター活性が高い場合に、未分化細胞の分化抵抗性が高いと評価し、DPP6の発現レベル及び／又はプロモーター活性が低い場合に、未分化細胞の分化抵抗性が低いと評価する請求項１記載の方法。 When the expression level and/or promoter activity of DPP6 is high, it is evaluated that the differentiation resistance of undifferentiated cells is high, and when the expression level and/or promoter activity of DPP6 is low, the differentiation resistance of undifferentiated cells is low The method according to claim 1, which is evaluated as follows.
  4. ZNF354C、C12orf56、ZNF578及びMIR886からなる群より選択される少なくとも１つの遺伝子のプロモーターのメチル化状態が低い場合に、未分化細胞の分化抵抗性が低いと評価し、ZNF354C、C12orf56、ZNF578及びMIR886からなる群より選択される少なくとも１つの遺伝子のプロモーターのメチル化状態が高い場合に、未分化細胞の分化抵抗性が高いと評価する請求項１記載の方法。 When the methylation status of the promoter of at least one gene selected from the group consisting of ZNF354C, C12orf56, ZNF578 and MIR886 is low, it is evaluated that the differentiation resistance of undifferentiated cells is low, and it is determined from ZNF354C, C12orf56, ZNF578 and MIR886. The method according to claim 1, wherein the differentiation resistance of undifferentiated cells is evaluated to be high when the methylation status of the promoter of at least one gene selected from the group consisting of is high.
  5. DPP6のプロモーターのメチル化状態が低い場合に、未分化細胞の分化抵抗性が高いと評価し、DPP6のプロモーターのメチル化状態が高い場合に、未分化細胞の分化抵抗性が低いと評価する請求項１記載の方法。 When the methylation status of the DPP6 promoter is low, it is evaluated that the differentiation resistance of the undifferentiated cells is high, and when the methylation status of the DPP6 promoter is high, the differentiation resistance of the undifferentiated cells is evaluated as low. The method according to item 1.
  6. 未分化細胞が、胚性腫瘍細胞（ＥＣ細胞）、胚性幹細胞（ＥＳ細胞）、人工多能性幹細胞（ｉＰＳ細胞）又は胚性生殖細胞（ＥＧ細胞)である請求項１～５のいずれかに記載の方法。 The undifferentiated cell is an embryonal tumor cell (EC cell), an embryonic stem cell (ES cell), an induced pluripotent stem cell (iPS cell) or an embryonic germ cell (EG cell), according to any one of claims 1 to 5. The method described in.
  7. 遺伝子の発現レベルをmRNAの量又はタンパク質の量として測定する請求項１～３又は６のいずれかに記載の方法。 7. The method according to claim 1, wherein the expression level of the gene is measured as the amount of mRNA or the amount of protein.
  8. 遺伝子の発現レベルを測定する方法が、qPCR、デジタルPCR、免疫染色、in situ hybridization、RNAシークエンス、マイクロアレイ、NanoString、抗体アレイ、FlowCytometry、質量分析又はそれらの組み合わせである請求項７記載の方法。 The method according to claim 7, wherein the method for measuring the gene expression level is qPCR, digital PCR, immunostaining, in situ hybridization, RNA sequence, microarray, NanoString, antibody array, FlowCytometry, mass spectrometry, or a combination thereof.
  9. 遺伝子のプロモーターのメチル化状態を測定する方法が、メチル化 DNA を濃縮した後に、濃縮されたDNAを検出する方法、バイサルファイト処理による塩基置換した後の塩基配列を解読(シークエンス)する方法、バイサルファイト処理による塩基置換した後の塩基配列をハイブリダイゼーションにより検出する方法、メチル化特異的PCR(MSP)法、メチル化感受性の制限酵素による切断の有無で検出する方法、メチル化シトシンをグルコシル化し、グルコシル化シトシン感受性の酵素で検出する方法又はそれらの組み合わせである請求項１、４～６のいずれかに記載の方法。 The method to measure the methylation status of the gene promoter is to concentrate the methylated DNA and then detect the concentrated DNA, to decode the nucleotide sequence after the base substitution by bisulfite treatment (sequence), A method for detecting the nucleotide sequence after the base substitution by phytotreatment by hybridization, a methylation-specific PCR (MSP) method, a method for detecting with or without cleavage by a methylation-sensitive restriction enzyme, glucosylating methylated cytosine, 7. The method according to claim 1, which is a method of detecting with a glucosylated cytosine-sensitive enzyme or a combination thereof.
  10. 未分化細胞集団における、下記の(i)及び／又は(ii)を測定することによって、分化抵抗性の低いあるいは高い未分化細胞株を選別する方法。
    (i)ZNF354C、C12orf56、ZNF578、DPP6及びMIR886からなる群より選択される少なくとも１つの遺伝子の発現レベル及び／又はプロモーター活性
    (ii) ZNF354C、C12orf56、ZNF578、DPP6及びMIR886からなる群より選択される少なくとも１つの遺伝子のプロモーターのメチル化状態     A method for selecting an undifferentiated cell line having low or high differentiation resistance by measuring the following (i) and/or (ii) in an undifferentiated cell population.
    (i) Expression level and/or promoter activity of at least one gene selected from the group consisting of ZNF354C, C12orf56, ZNF578, DPP6 and MIR886
    (ii) Methylation status of the promoter of at least one gene selected from the group consisting of ZNF354C, C12orf56, ZNF578, DPP6 and MIR886
  11. ZNF354C、C12orf56、ZNF578及びMIR886からなる群より選択される少なくとも１つの遺伝子の発現レベル及び／又はプロモーター活性が高い場合に、分化抵抗性が低い未分化細胞株として選別し、ZNF354C、C12orf56、ZNF578及びMIR886からなる群より選択される少なくとも１つの遺伝子の発現レベル及び／又はプロモーター活性が低い場合に、分化抵抗性が高い未分化細胞株として選別する請求項１０記載の方法。 When the expression level and/or promoter activity of at least one gene selected from the group consisting of ZNF354C, C12orf56, ZNF578 and MIR886 is high, it is selected as an undifferentiated cell line having low differentiation resistance, and ZNF354C, C12orf56, ZNF578 and The method according to claim 10, wherein when the expression level and/or promoter activity of at least one gene selected from the group consisting of MIR886 is low, it is selected as an undifferentiated cell line having high differentiation resistance.
  12. DPP6の発現レベル及び／又はプロモーター活性が高い場合に、分化抵抗性が高い未分化細胞株として選別し、DPP6の発現レベル及び／又はプロモーター活性が低い場合に、分化抵抗性が低い未分化細胞株として選別する請求項１０記載の方法。 When the expression level and/or promoter activity of DPP6 is high, it is selected as an undifferentiated cell line with high differentiation resistance, and when the expression level and/or promoter activity of DPP6 is low, undifferentiated cell line with low differentiation resistance The method according to claim 10, wherein the selection is performed as
  13. ZNF354C、C12orf56、ZNF578及びMIR886からなる群より選択される少なくとも１つの遺伝子のプロモーターのメチル化状態が低い場合に、分化抵抗性が低い未分化細胞株として選別し、ZNF354C、C12orf56、ZNF578及びMIR886からなる群より選択される少なくとも１つの遺伝子のプロモーターのメチル化状態が高い場合に、分化抵抗性が高い未分化細胞株として選別する請求項１０記載の方法。 When the methylation status of the promoter of at least one gene selected from the group consisting of ZNF354C, C12orf56, ZNF578 and MIR886 is low, it is selected as an undifferentiated cell line with low differentiation resistance, and selected from ZNF354C, C12orf56, ZNF578 and MIR886. The method according to claim 10, wherein when the promoter of at least one gene selected from the group has a high methylation state, it is selected as an undifferentiated cell line having high differentiation resistance.
  14. DPP6のプロモーターのメチル化状態が低い場合に、分化抵抗性が高い未分化細胞株として選別し、DPP6のプロモーターのメチル化状態が高い場合に、分化抵抗性が低い未分化細胞株として選別する請求項１０記載の方法。 When the methylation status of the DPP6 promoter is low, it is selected as an undifferentiated cell line with high differentiation resistance, and when the methylation status of the DPP6 promoter is high, it is selected as an undifferentiated cell line with low differentiation resistance. Item 10. The method according to Item 10.
  15. 未分化細胞株が、胚性腫瘍細胞（ＥＣ細胞）株、胚性幹細胞（ＥＳ細胞）株、人工多能性幹細胞（ｉＰＳ細胞）株又は胚性生殖細胞（ＥＧ細胞)株である請求項１０～１４のいずれかに記載の方法。 The undifferentiated cell line is an embryonal tumor cell (EC cell) line, an embryonic stem cell (ES cell) line, an induced pluripotent stem cell (iPS cell) line, or an embryonic germ cell (EG cell) line. 15. The method according to any one of 14 to 14.
  16. 未分化細胞集団中に存在する、分化抵抗性の低いあるいは高い未分化細胞を検出するために、ZNF354C、C12orf56、ZNF578、DPP6及びMIR886からなる群より選択される少なくとも１つの遺伝子をマーカーとして使用する方法。 Use of at least one gene selected from the group consisting of ZNF354C, C12orf56, ZNF578, DPP6 and MIR886 as a marker for detecting undifferentiated cells with low or high differentiation resistance present in an undifferentiated cell population Method.
  17. 下記の(i)及び／又は(ii)を測定可能な試薬を含む、未分化細胞の分化抵抗性を評価するためのキット。
    (i)ZNF354C、C12orf56、ZNF578、DPP6及びMIR886からなる群より選択される少なくとも１つの遺伝子の発現レベル及び／又はプロモーター活性
    (ii) ZNF354C、C12orf56、ZNF578、DPP6及びMIR886からなる群より選択される少なくとも１つの遺伝子のプロモーターのメチル化状態     A kit for evaluating the differentiation resistance of undifferentiated cells, which comprises a reagent capable of measuring the following (i) and/or (ii).
    (i) Expression level and/or promoter activity of at least one gene selected from the group consisting of ZNF354C, C12orf56, ZNF578, DPP6 and MIR886
    (ii) Methylation status of the promoter of at least one gene selected from the group consisting of ZNF354C, C12orf56, ZNF578, DPP6 and MIR886
  18. 遺伝子の発現レベルを測定可能な試薬が、プライマー、プローブ又は抗体である請求項１７記載のキット。 The kit according to claim 17, wherein the reagent capable of measuring the expression level of the gene is a primer, a probe or an antibody.
  19. 遺伝子のプロモーター活性を測定可能な試薬が、プロモーター下流にレポータータンパク質を連結した遺伝子配列又はこの遺伝子配列を組み込んだベクターである請求項１７記載のキット。 The kit according to claim 17, wherein the reagent capable of measuring the promoter activity of the gene is a gene sequence in which a reporter protein is linked downstream of the promoter or a vector incorporating this gene sequence.
  20. 遺伝子のプロモーターのメチル化状態を測定可能な試薬が、バイサルファイト（亜硫酸水素塩）、メチル化解析用マイクロアレイ試薬、Sanger法によるシークエンス試薬、次世代シークエンサー用シークエンス試薬、5-mC抗体、5-hmC抗体、メチルアデノシン抗体、5’-methyl-2’-deoxycytidine抗体、HRP標識DNA抗体、5-hmC グルコシルトランスフェラーゼ、グルコシル-5hmC感受性制限酵素エンドヌクレアーゼ、MBD1 (Methyl-CpG Binding Domain Protein1)、MBD2 (Methyl-CpG Binding Domain Protein2)、特異的PCRプライマー、特異的プローブ又はDNA精製キットである請求項１７記載のキット。 Reagents that can measure the methylation status of gene promoters include bisulfite (bisulfite), microarray reagents for methylation analysis, sequencing reagents by the Sanger method, sequencing reagents for next-generation sequencers, 5-mC antibody, 5-hmC Antibody, methyladenosine antibody, 5'-methyl-2'-deoxycytidine antibody, HRP-labeled DNA antibody, 5-hmCglucosyltransferase, glucosyl-5hmC sensitive restriction enzyme endonuclease, MBD1 (Methyl-CpG Binding Domain Protein1), MBD2 (Methyl) -CpG Binding Domain Protein 2), specific PCR primer, specific probe or DNA purification kit.
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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2014523735A (en) * 2011-07-25 2014-09-18 国立大学法人京都大学 Method for selecting induced pluripotent stem cells
JP2017184623A (en) * 2016-04-01 2017-10-12 公益財団法人ヒューマンサイエンス振興財団 EVALUATION METHOD OF DIFFERENTIATION TENDENCY AMONG UNDIFFERENTIATED CELLS, SALL3 mRNA AS EVALUATION MARKER FOR DIFFERENTIATION TENDENCY, AND METHOD OF CONTROLLING DIFFERENTIATION ABILITY OF UNDIFFERENTIATED CELLS
WO2019078342A1 (en) * 2017-10-20 2019-04-25 国立大学法人大阪大学 Method for selecting pluripotent stem cell having directivity of differentiation to cardiomyocyte
WO2019240247A1 (en) * 2018-06-15 2019-12-19 公立大学法人横浜市立大学 Undifferentiated cell detection method

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2014523735A (en) * 2011-07-25 2014-09-18 国立大学法人京都大学 Method for selecting induced pluripotent stem cells
JP2017184623A (en) * 2016-04-01 2017-10-12 公益財団法人ヒューマンサイエンス振興財団 EVALUATION METHOD OF DIFFERENTIATION TENDENCY AMONG UNDIFFERENTIATED CELLS, SALL3 mRNA AS EVALUATION MARKER FOR DIFFERENTIATION TENDENCY, AND METHOD OF CONTROLLING DIFFERENTIATION ABILITY OF UNDIFFERENTIATED CELLS
WO2019078342A1 (en) * 2017-10-20 2019-04-25 国立大学法人大阪大学 Method for selecting pluripotent stem cell having directivity of differentiation to cardiomyocyte
WO2019240247A1 (en) * 2018-06-15 2019-12-19 公立大学法人横浜市立大学 Undifferentiated cell detection method

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
KOYANAGI-AOI, M. ET AL.: "Differentiation-defective phenotypes revealed by large-scale analyses of human pluripotent stem cells", PNAS, vol. 110, no. 51, 17 December 2013 (2013-12-17), pages 20569 - 20574, XP055173687, DOI: 10.1073/pnas.1319061110 *
SATO, YOJI ET AL.: "Development of cell characteristic analysis method using differentiation propensity as an index, Health and labor sciences research grants regulatory science research project for pharmaceuticals, medical devices, etc. regulatory science research for development of cell and tissue processing products", 2013 SUMMARY/SHARED RESEARCH REPORT, RESEARCH ABSTRACT, vol. 25, 16 June 2014 (2014-06-16), pages 117 - 143 *

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