WO2020149391A1 - Procédé d'évaluation de la résistance à la différenciation de cellules indifférenciées - Google Patents
Procédé d'évaluation de la résistance à la différenciation de cellules indifférenciées Download PDFInfo
- Publication number
- 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
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- undifferentiated
- cell
- promoter
- gene
- mir886
- Prior art date
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6813—Hybridisation assays
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6813—Hybridisation assays
- C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
- C12Q1/6837—Enzymatic or biochemical coupling of nucleic acids to a solid phase using probe arrays or probe chips
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6813—Hybridisation assays
- C12Q1/6841—In situ hybridisation
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6869—Methods for sequencing
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6897—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids involving reporter genes operably linked to promoters
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/10—Cells modified by introduction of foreign genetic material
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.
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Immunology (AREA)
- Analytical Chemistry (AREA)
- Molecular Biology (AREA)
- Physics & Mathematics (AREA)
- Genetics & Genomics (AREA)
- Biotechnology (AREA)
- General Health & Medical Sciences (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Biophysics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biomedical Technology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Urology & Nephrology (AREA)
- Hematology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Cell Biology (AREA)
- Pathology (AREA)
- General Physics & Mathematics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
L'invention concerne un procédé pour prédire, à l'étape de cellules indifférenciées, la possibilité de contamination par des cellules indifférenciées après la différenciation des cellules. La résistance à la différenciation de cellules indifférenciées est évaluée par la mesure de (i) et/ou (ii). (i) Le niveau d'expression et/ou l'activité promotrice d'au moins un gène choisi dans le groupe constitué par ZNF354C, C12orf56, ZNF578, DPP6 et MIR886. (Ii) L'état de méthylation du promoteur d'au moins un gène choisi dans le groupe constitué par ZNF354C, C12orf56, ZNF578, DPP6 et MIR886.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP2020566493A JP7370602B2 (ja) | 2019-01-17 | 2020-01-17 | 未分化細胞の分化抵抗性評価法 |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP2019-005892 | 2019-01-17 | ||
JP2019005892 | 2019-01-17 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2020149391A1 true WO2020149391A1 (fr) | 2020-07-23 |
Family
ID=71614449
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/JP2020/001417 WO2020149391A1 (fr) | 2019-01-17 | 2020-01-17 | Procédé d'évaluation de la résistance à la différenciation de cellules indifférenciées |
Country Status (2)
Country | Link |
---|---|
JP (1) | JP7370602B2 (fr) |
WO (1) | WO2020149391A1 (fr) |
Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2014523735A (ja) * | 2011-07-25 | 2014-09-18 | 国立大学法人京都大学 | 人工多能性幹細胞の選別方法 |
JP2017184623A (ja) * | 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 | 未分化細胞間における分化傾向の評価方法、分化傾向の評価マーカーとしてのSALL3 mRNA、及び、未分化細胞の分化能力の制御方法 |
WO2019078342A1 (fr) * | 2017-10-20 | 2019-04-25 | 国立大学法人大阪大学 | Procédé de sélection d'une cellule souche pluripotente présentant une directivité de différenciation en cardiomyocyte |
WO2019240247A1 (fr) * | 2018-06-15 | 2019-12-19 | 公立大学法人横浜市立大学 | Procédé de détection de cellules indifférenciées |
-
2020
- 2020-01-17 WO PCT/JP2020/001417 patent/WO2020149391A1/fr active Application Filing
- 2020-01-17 JP JP2020566493A patent/JP7370602B2/ja active Active
Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2014523735A (ja) * | 2011-07-25 | 2014-09-18 | 国立大学法人京都大学 | 人工多能性幹細胞の選別方法 |
JP2017184623A (ja) * | 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 | 未分化細胞間における分化傾向の評価方法、分化傾向の評価マーカーとしてのSALL3 mRNA、及び、未分化細胞の分化能力の制御方法 |
WO2019078342A1 (fr) * | 2017-10-20 | 2019-04-25 | 国立大学法人大阪大学 | Procédé de sélection d'une cellule souche pluripotente présentant une directivité de différenciation en cardiomyocyte |
WO2019240247A1 (fr) * | 2018-06-15 | 2019-12-19 | 公立大学法人横浜市立大学 | Procédé de détection de cellules indifférenciées |
Non-Patent Citations (2)
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 * |
Also Published As
Publication number | Publication date |
---|---|
JPWO2020149391A1 (ja) | 2021-12-09 |
JP7370602B2 (ja) | 2023-10-30 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US20210371914A1 (en) | Methods and compositions for identifying or quantifying targets in a biological sample | |
JP2023040233A (ja) | 分子の多重検出方法 | |
US7049103B2 (en) | Method of evaluating drug efficacy and toxicity | |
EP2660331B1 (fr) | Procédé pour l'analyse de génome de cellule unique et trousse correspondante | |
US7906288B2 (en) | Compare-MS: method rapid, sensitive and accurate detection of DNA methylation | |
JP7344565B2 (ja) | 未分化細胞検出法 | |
JP7041934B2 (ja) | 未分化細胞検出法 | |
Van Doorn et al. | Quantitative multiplex detection of plant pathogens using a novel ligation probe-based system coupled with universal, high-throughput real-time PCR on OpenArrays™ | |
WO2021178199A1 (fr) | Procédés et compositions pour détecter des cibles | |
JP2005521409A (ja) | 単一プライマー等温核酸増幅−増強型被分析物検出および定量 | |
AU2006243757A1 (en) | Compositions and methods for the analysis of degraded nucleic acids | |
EP4060039A1 (fr) | Procédé de détection sensible pour des gènes marqueurs indifférenciés | |
WO2020149391A1 (fr) | Procédé d'évaluation de la résistance à la différenciation de cellules indifférenciées | |
KR20160056841A (ko) | Flt3 유전자 변이 정량분석방법 및 분석 키트 | |
Kelkar et al. | A novel method to assess the full genome methylation profile using monoclonal antibody combined with the high throughput based microarray approach | |
KR101683086B1 (ko) | 유전자의 발현량 및 메틸화 프로필을 활용한 돼지의 산자수 예측방법 | |
van Buggenum et al. | Immuno-Detection by sequencing (ID-seq) enables large-scale high-dimensional phenotyping in cells | |
GB2589869A (en) | Method for whole genome sequencing of picogram quantities of DNA | |
US20100167291A1 (en) | Assessing expression of endogenous and exogenous genes | |
Moison et al. | DNA methylation analysis of ChIP products at single nucleotide resolution by Pyrosequencing® | |
JP2018526009A (ja) | ヌクレオチド1個の分解能を有する多価プローブ | |
Morowitz | Modulation of Epigenetic Mark 5-hydroxymethylcytosine in Intestinal Organoid Cultures | |
Powell et al. | Tissue source attribution using the PyroMark® Q48 Autoprep System: Sperm identification in forensic casework | |
Policicchio | Examining epigenetic variation in the brain in mental illness | |
Galán et al. | Monitoring stemness in long-term hESC cultures by real-time PCR |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 20741298 Country of ref document: EP Kind code of ref document: A1 |
|
ENP | Entry into the national phase |
Ref document number: 2020566493 Country of ref document: JP Kind code of ref document: A |
|
NENP | Non-entry into the national phase |
Ref country code: DE |
|
122 | Ep: pct application non-entry in european phase |
Ref document number: 20741298 Country of ref document: EP Kind code of ref document: A1 |