WO2011049099A1 - Procédé de production de cellules souches cancéreuses - Google Patents

Procédé de production de cellules souches cancéreuses Download PDF

Info

Publication number
WO2011049099A1
WO2011049099A1 PCT/JP2010/068406 JP2010068406W WO2011049099A1 WO 2011049099 A1 WO2011049099 A1 WO 2011049099A1 JP 2010068406 W JP2010068406 W JP 2010068406W WO 2011049099 A1 WO2011049099 A1 WO 2011049099A1
Authority
WO
WIPO (PCT)
Prior art keywords
gene
cancer
cancer stem
stem cell
stem cells
Prior art date
Application number
PCT/JP2010/068406
Other languages
English (en)
Japanese (ja)
Inventor
正樹 森
健一 永井
慶人 富丸
範克 三吉
宏光 星野
秀始 石井
Original Assignee
国立大学法人大阪大学
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 国立大学法人大阪大学 filed Critical 国立大学法人大阪大学
Publication of WO2011049099A1 publication Critical patent/WO2011049099A1/fr

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0693—Tumour cells; Cancer cells
    • C12N5/0695—Stem cells; Progenitor cells; Precursor cells
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/60—Transcription factors
    • 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
    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/30—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from cancer cells, e.g. reversion of tumour cells

Definitions

  • the present invention relates to a cancer stem cell and a method for producing the same.
  • the present invention also relates to various screening methods using cancer stem cells, methods for evaluating the efficacy of cancer therapeutics, and the like.
  • Cancer cells have the property of being capable of self-proliferation and being able to wet to surrounding tissues or metastasize to distant tissues. However, not all cancer cells forming a cancer tissue have these characteristics, and there are very few cancer cells that develop cancer or advance cancer. It is known to be a cancer stem cell. Cancer stem cells exhibit undifferentiated surface characteristics like normal stem cells, have self-renewal ability and differentiation ability, and have the property of producing all cancer cells in various differentiation stages constituting cancer tissue. That is, cancer stem cells are thought to be responsible for generating the majority of cancer cells by differentiation while maintaining the same cells as themselves by self-replication in cancer tissues.
  • cancer stem cells are useful as a development tool for drug discovery and diagnostics, and the establishment of their preparations is eagerly desired.
  • cancer stem cells are only a few percent or less of the cancer cells that make up cancer tissue, and the purification process is extremely difficult. The current situation is that the product has not been established.
  • An object of the present invention is to provide a technique for artificially producing cancer stem cells. Furthermore, an object of the present invention is to provide various methods for cancer treatment and anticancer drug discovery using artificially produced cancer stem cells.
  • the present inventors did not isolate a cancer stem cell from a cancer tissue, but conducted a diligent study from a new and unprecedented viewpoint of newly producing a cancer stem cell. It should be said that for cancer cells, the group consisting of (1) KLF family gene, (2) OCT family gene, (3) SOX family gene, and (4) NANOG, LIN28, KRAS, BCL2, BMI1, and ERAS At least one undifferentiation-inducing factor gene selected from and / or at least one tumor suppressor gene expression inhibitor selected from the group consisting of CDKN2A, FHIT, TP53, RB1, PTEN, and LZTS1 Thus, the present inventors have found that the cancer cells can be induced into cancer stem cells that have self-replicating ability and have acquired undifferentiation. The present invention has been completed by further studies based on this finding.
  • this invention provides the invention of the aspect hung up below.
  • Item 1. (1) KLF family gene, (2) OCT family gene, (3) SOX family gene, and (4) at least one undifferentiated selected from the group consisting of NANOG, LIN28, KRAS, BCL2, BMI1, and ERAS A cancer comprising a step of introducing into a cancer cell an inducer gene and / or an expression suppressor of at least one tumor suppressor gene selected from the group consisting of CDKN2A, FHIT, TP53, RB1, PTEN, and LZTS1 A method for producing stem cells.
  • Item 2. The production method according to Item 1, wherein the KLF family gene is a KLF4 gene.
  • Item 3. Item 3.
  • Item 1 or 2 wherein the OCT family gene is an OCT3 / 4 gene.
  • Item 4. The production method according to any one of Items 1 to 3, wherein the SOX family gene is a SOX2 gene.
  • Item 5. (1) KLF family gene, (2) OCT family gene, (3) SOX family gene, and (4) at least one undifferentiated selected from the group consisting of NANOG, LIN28, KRAS, BCL2, BMI1, and ERAS It is obtained by introducing into a cancer cell an inducer gene and / or an expression suppressor of at least one tumor suppressor gene selected from the group consisting of CDKN2A, FHIT, TP53, RB1, PTEN, and LZTS1, Cancer stem cells.
  • Item 6. Item 6.
  • Item 7 An antibody that specifically binds to the cancer stem cell of Item 5.
  • Item 7. A screening method for a substance that induces differentiation of cancer stem cells, comprising the following steps: 6. A step of bringing a test substance into contact with the cancer stem cell according to Item 5 and measuring the presence or absence of differentiation of the cancer stem cell, and a step of selecting the test substance obtained by differentiating the cancer stem cell as a substance that induces differentiation of the cancer stem cell.
  • a screening method for a substance that can suppress the growth of cancer stem cells or a substance that promotes the growth of cancer stem cells comprising the following steps: A step of contacting a test substance with the cancer stem cell according to Item 5 and measuring the degree of proliferation of the cancer stem cell, and a test substance that suppresses the growth of the cancer stem cell, or a test substance that promotes the growth of the cancer stem cell. The process of selecting as a substance which can suppress the proliferation of a cancer stem cell, or a substance which promotes the proliferation of a cancer stem cell.
  • a screening method for a therapeutic agent for cancer comprising the following steps: 6.
  • Item 10 A method for evaluating the efficacy of a therapeutic agent for cancer, comprising the following steps: The step of bringing a cancer therapeutic agent into contact with the cancer stem cell according to Item 5 to determine the degree of inhibition of the growth of the cancer stem cell, and the greater the degree of inhibition of the growth of the cancer stem cell, the higher the efficacy of the therapeutic agent for cancer is determined. Process. Item 11.
  • KLF family gene (2) OCT family gene, (3) SOX family gene, and (4) at least one undifferentiated selected from the group consisting of NANOG, LIN28, KRAS, BCL2, BMI1, and ERAS Cancer stem cell induction characterized by comprising an inducer gene and / or an expression suppressor of at least one tumor suppressor gene selected from the group consisting of CDKN2A, FHIT, TP53, RB1, PTEN, and LZTS1 Agent.
  • cancer stem cells that could not be established conventionally can be artificially produced.
  • the cancer stem cells produced according to the present invention can be used for screening of therapeutic drugs for cancer, evaluation of the efficacy of cancer therapeutic drugs, etc., and can contribute to the improvement of cancer treatment technology.
  • by clarifying the characteristics of cancer stem cells produced by the present invention it is possible to elucidate the mechanisms of cancer development, progression, metastasis, etc., and thus innovative drug discovery and treatment methods that lead to the fundamental treatment of cancer Can contribute to the development of
  • the results of measuring the expression level of the Nanog gene 20 days after transfection of the PLC cells into which the combination of each gene (Example 1-6) was introduced are shown.
  • the vertical axis (NANOG / GAPDH) in the figure indicates the ratio of the NANOG gene expression level to the GAPDH gene expression level.
  • FIG. 2-3 The results of measuring the expression level of Nanog gene 12 days after transfection of HuCCT cells into which a combination of genes (Examples 1, 2 and 7-12) was introduced are shown.
  • the results of measuring the expression level of Nanog gene 12 days after transfection of DLD-1 cells into which a combination of genes (Examples 3 and 7-11) has been introduced are shown.
  • cultivated for 20 days is shown.
  • cancer stem cell means a cell that has self-replicating ability and maintains an undifferentiated state and can produce a cancer cell by differentiation.
  • the method for producing cancer stem cells of the present invention comprises: (1) KLF family gene, (2) OCT family gene, (3) SOX family gene, and (4) NANOG, LIN28, KRAS, BCL2, BMI1, and Expression of at least one undifferentiation inducer gene selected from the group consisting of ERAS and / or at least one tumor suppressor gene selected from the group consisting of CDKN2A, FHIT, TP53, RB1, PTEN, and LZTS1
  • the method includes a step of introducing an inhibitory factor.
  • the manufacturing method of this invention is explained in full detail.
  • the types of cancer cells induced by cancer stem cells are not particularly limited, and cancer cells derived from any cancer can be used.
  • cancer cells used in the present invention include colorectal cancer cells, colon cancer cells, esophageal cancer cells, gastric cancer cells, pancreatic cancer cells, liver cancer cells, bile duct cancer cells and the like.
  • these cancer cells from the viewpoint of efficiently inducing cancer stem cells, colon cancer cells, pancreatic cancer cells, liver cancer cells, cholangiocarcinoma cells, and more preferably colon cancer cells are exemplified.
  • the said cancer cell what was extracted from the cancer patient may be used, and a commercial item may be used.
  • the cancer cells are appropriately selected from those derived from mammals such as humans, mice, rats, hamsters, rabbits, cats, dogs, sheep, pigs, cows, goats, monkeys, etc., depending on the intended use of cancer stem cells.
  • mammals such as humans, mice, rats, hamsters, rabbits, cats, dogs, sheep, pigs, cows, goats, monkeys, etc.
  • those derived from humans are preferable.
  • human-derived somatic cells they may be derived from any of fetuses, infants, children, and adults.
  • KLF family genes as factors for reprogramming cancer cells into cancer stem cells, (1) KLF family genes, (2) OCT family genes, (3) SOX family genes, and (4) specific undifferentiation inducer genes and Use of a specific tumor suppressor gene suppression factor.
  • KLF family genes as factors for reprogramming cancer cells into cancer stem cells.
  • OCT family genes as factors for reprogramming cancer cells into cancer stem cells.
  • SOX family genes as factors for reprogramming cancer cells into cancer stem cells.
  • specific undifferentiation inducer genes Use of a specific tumor suppressor gene suppression factor.
  • KLF family genes include KLF1, KLF2, KLF4, and KLF5. These KLF family genes may be used alone or in combination of two or more. Among these KLF family genes, the KLF4 gene is preferably used from the viewpoint of efficient induction into cancer stem cells.
  • the base sequence of the KLF4 gene is known (NCBI accession Number NM_010637 (human), NM_004235 (Mouse)).
  • nucleotide sequence of the KLF1 gene (NCBI accession Number NM_006563 (human), NM_010635 (Mouse)), the nucleotide sequence of the KLF2 gene (NCBI accession Number NM_016270 (human), NM_008452 (Mouse)), and the nucleotide sequence of the KLF5 gene (NCBI accession Number NM_001730 (human), NM_009769 (Mouse)) is also known.
  • NCBI NCBI accession Number NM_006563 (human), NM_010635 (Mouse)
  • nucleotide sequence of the KLF2 gene NCBI accession Number NM_016270 (human), NM_008452 (Mouse)
  • nucleotide sequence of the KLF5 gene NCBI accession Number NM_001730 (human), NM_009769 (Mouse)
  • OCT family genes include OCT3 / 4, OCT1A, and OCT6. These OCT family genes may be used alone or in combination of two or more. Among these OCT family genes, the OCT3 / 4 gene is preferably used from the viewpoint of efficient induction into the cancer stem cells.
  • the base sequence of OCT3 / 4 is known (NCBI accession Number NM_002701 (human), NM_013633 (Mouse)).
  • the base sequence of the OCT1A gene NCBI accession Number NM_002697 (human), NM_198934 (Mouse)
  • the base sequence of the OCT6 gene NCBI accession Number NM_002699 (human), NM_011141 (Mouse) are also known.
  • SOX family genes include SOX1, SOX2, SOX3, SOX7, SOX15, SOX17, and SOX18. These SOX family genes may be used alone or in combination of two or more. Among these SOX family genes, the SOX2 gene is preferably used from the viewpoint of efficient induction into cancer stem cells.
  • the base sequence of the SOX2 gene is known (NCBI accession Number NM_003106 (human), NM_011443 (Mouse)).
  • nucleotide sequence of SOX1 gene (NCBI accession Number NM_005986 (human), NM_009233 (Mouse)), the nucleotide sequence of SOX3 gene (NCBI accession Number ⁇ NM_005634 (human), NM_009237 (Mouse)), the nucleotide sequence of SOX7 gene (NCBI accession Number NM_031439 (human), NM_011446 (Mouse)), SOX15 gene nucleotide sequence (NCBI accession Number NM_006942 (human), NM_009235 (Mouse)), SOX17 gene nucleotide sequence (NCBI accession Number NM_0022454 (human), NM_011441 (Mouse) ) And the nucleotide sequence of the SOX18 gene (NCBI accession Number NM_018419 (human), NM_009236 (Mouse)) are also known.
  • NANOG gene base sequence NCBI accession Number NM_024865 (human), NM_0280162 (Mouse)
  • LIN28 gene base sequence NCBI accession Number NM_024674 (human) , NM_145833 (Mouse)
  • nucleotide sequence of KRAS gene NCBI accession Number NM_004985, NM_033360 (human), NM_021284 (Mouse)
  • nucleotide sequence of BCL2 gene NCBI accession Number NM_000633, NM_000657 (human), NM_009741, NM_177410 (Mouse)
  • BMI1 gene base sequence NCBI accession Number NM_005180 (human), NM_007552 (Mouse)
  • ERAS gene base sequence NCBI accession Number NM_181532 (human), NM_181548
  • the tumor suppressor gene expression inhibitor is not particularly limited as long as it can suppress the expression of the cancer suppressor gene in cancer cells induced by cancer stem cells.
  • shRNA shRNA, miRNA, and Examples having an RNA interference effect such as siRNA are exemplified.
  • shRNA is preferable because it enables silencing of a target tumor suppressor gene stably in a cancer cell induced by cancer stem cells for a long period of time.
  • the tumor suppressor gene expression inhibitory factor may be directly introduced into the target cancer cells shRNA, miRNA, siRNA and the like, from the viewpoint of effectively suppressing the expression of the target cancer suppressor gene, shRNA, In order for miRNA, siRNA, and the like to be expressed in the target cancer cell, it is desirable to incorporate a DNA fragment encoding them into the vector and introduce it into the target cancer cell.
  • shRNA when shRNA is used as the tumor suppressor gene expression inhibitor, in order to enhance the silencing effect of the target tumor suppressor gene, 2 out of shRNAs that can exert an RNA interference effect on the target cancer suppressor gene. It is desirable to select and use a combination of two or more different base sequences.
  • Suppressors such as shRNA, miRNA, and siRNA are constructed by a known technique based on information on the base sequence of a tumor suppressor gene that is subject to expression suppression.
  • the base sequence of the target cancer suppressor gene is also known as shown below: CDKN2A gene base sequence (NCBI accession Number NM_000077, NM_058195, NM_058197 (human), NM_001040654, NM_009877 (Mouse)), FHIT Base sequence of the gene (NCBI accession Number NM_002012 (human), NM_010210 (Mouse)), base sequence of the TP53 gene (NCBI accession Number NM_000546, NM_001126112,26NM_001126114, NM_001126115, NM_001126116, NM_00112610011 (27), NM_0011260011 (27) )), RB1 gene base sequence (NCBI accession Number NM_000321 (human), NM_009029 (Mous
  • undifferentiation-inducing factor genes and / or tumor suppressor gene expression inhibitory factors may be used singly or in combination of two or more. Specifically, one or more of the genes of the undifferentiation inducing factor may be used, and one or more of the tumor suppressor gene expression suppressing factors may be used. Furthermore, one or more types of undifferentiation-inducing factor genes and one or more types of tumor suppressor gene expression suppressing factors may be used in combination.
  • LIN28 gene, KRAS gene, FHIT gene suppressor, PTEN gene The inhibitory factor of is mentioned.
  • the LIN gene when hepatoma cells are used as cancer cells to be induced into cancer stem cells, the LIN gene is suitable; when bile duct cancer cells are used as cancer cells to be induced into cancer stem cells, the LIN gene, KRAS gene, FHIT gene suppressors and PTEN gene suppressors are preferred; when cholangiocarcinoma cells are used as cancer cells induced in cancer stem cells, FHIT gene suppressors are preferred.
  • the gene of (1) to (3) and the gene of the undifferentiation inducer of (4) above are commonly present in mammals including humans, and those derived from any mammal can be used. It is desirable to select appropriately according to the origin of the cancer cells to be introduced. For example, when human-derived cancer cells are used, the genes (1) to (3) and the undifferentiation inducer gene (4) introduced into the cancer cells are derived from humans. It is desirable. In addition to the wild-type gene, the gene of (1) to (3) and the gene of the undifferentiation inducer of (4) are one or several (for example, 1 to 10) in the amino acid sequence of the gene product.
  • the genes (1) to (3) and the gene for the undifferentiation inducer (4) can be prepared according to conventional methods based on known sequence information.
  • cDNA of a target gene can be prepared by extracting RNA from a mammal-derived cell and cloning according to a conventional method.
  • Introduction of the above genes (1) to (3) and (4) a specific undifferentiation inducer gene and / or a specific tumor suppressor gene expression suppressor into a cancer cell can be performed by a known technique. it can.
  • a method for introducing a gene of the above (1) to (3) and (4) a specific undifferentiation inducer gene and / or a specific tumor suppressor gene expression suppressor into a cancer cell a vector Examples thereof include calcium phosphate method; lipofection method; electroporation method; microinjection method and the like. Among these, the method using a vector is preferable from the viewpoint of introduction efficiency.
  • a vector When using a vector to introduce the above genes (1) to (3), and (4) a specific undifferentiation inducer gene and / or a specific tumor suppressor gene expression suppressor into cancer cells, as vectors, viral vectors, non-viral vectors, artificial viruses and the like can be used, and viral vectors such as adenoviruses and retroviruses are preferred.
  • the above genes (1) to (3) and (4) a specific undifferentiation inducer gene and / or a specific tumor suppressor gene expression suppressor are in separate vectors. It may be integrated, or two or more genes may be integrated in one vector.
  • cancer cells into which the above genes (1) to (3) and (4) specific undifferentiation-inducing factor genes and / or specific tumor suppressor gene expression suppressors have been introduced are By culturing in a viable medium for about 7 to 35 days, the cancer cells are reprogrammed and induced into cancer stem cells that have acquired pluripotency as well as self-renewal ability.
  • the genes of the above (1) to (3), and (4) a gene of a specific undifferentiation inducer and / or a specific tumor suppressor gene are introduced
  • the genes of the above (1) to (3), and (4) a gene of a specific undifferentiation inducer and / or a specific tumor suppressor gene About 7 days after the introduction of the expression inhibitory factor, the cells were cultured in a medium containing 10% by volume of fetal bovine serum (FBS), and then (4-5 ng / ml wt% basic) in a dish coated with matrix protein. It is desirable to culture in a medium containing fibroblast growth factor (bFGF), where the matrix protein is exemplified by matrigel, fibronectin, collagen, laminin, etc. Among these, matrigel is preferred.
  • bFGF fibroblast growth factor
  • a cancer stem cell It is induced into a cancer stem cell from among the cancer cells into which the above genes (1) to (3) and (4) the gene of a specific undifferentiation inducer and / or the expression suppressor of a specific tumor suppressor gene are introduced.
  • the selection of the cells can be performed using as an index whether or not the cells have the ability to proliferate and whether or not they have the characteristics of cancer stem cells. Specifically, the selection of such cancer stem cells is carried out using the cell shape, presence / absence of specific staining for cancer stem cells, presence / absence of expression of a marker gene for cancer stem cells, etc., from among cells having proliferative ability. be able to.
  • a reporter gene construct prepared by previously binding a drug resistance gene to a cancer stem cell marker gene promoter is introduced into a cancer cell, cells that have acquired the characteristics of the cancer stem cell can grow in the presence of the drug. Therefore, cells that have acquired the characteristics of cancer stem cells can be selected using growth in the presence of a drug as an index.
  • cells induced by cancer stem cells proliferate and exhibit a circular shape when cultured in a medium coated with matrix protein in a medium containing basic fibroblast growth factor (bFGF). The above index can be used.
  • the cancer stem cell vesicle expresses a cancer stem cell marker gene (Nanog, BMI1, etc.), the presence or absence of the expression of the marker gene can also be used as the index.
  • CD24 has also been shown to be a surface marker for cancer stem cells (Eyal Sagiv et al., Targeting CD24 for Treatment of Colorectal and Pancreatic Cancer by Monoclonal Antibodies or small interfering RNA, Cancer Research, Vol. 68, pages, 2803-2812, 2008), CD24 expression can also be used as an indicator of cancer stem cell selection.
  • the characteristics of the cancer stem cells can be clarified, and further, a substance capable of inducing differentiation and controlling proliferation can be screened.
  • the test substance (candidate substance to be used for screening) is contacted with the cancer stem cells obtained above, the presence or absence of differentiation of the cancer stem cells is measured, and the test substance that has differentiated the cancer stem cells is selected By doing so, it becomes possible to screen for substances that induce differentiation of cancer stem cells.
  • a test substance or cancer in which the test substance (candidate substance to be used for screening) is brought into contact with the cancer stem cells obtained above, the degree of cancer stem cell proliferation is measured, and cancer stem cell proliferation is suppressed.
  • a test substance that promotes the proliferation of stem cells it becomes possible to screen for a substance that can suppress the proliferation of cancer stem cells or a substance that promotes the proliferation of cancer stem cells.
  • cancer stem cells are responsible for the onset, progression, and metastasis of cancer
  • the cancer stem cells obtained above can also be used for screening for cancer therapeutics and for evaluating the efficacy of cancer therapeutics.
  • a test substance candidate substance to be used for screening
  • the cancer stem cell obtained above is brought into contact with a cancer therapeutic agent, and the degree of inhibition of cancer stem cell growth is measured. It can be evaluated that the lower the degree of inhibition of cancer stem cell growth, the lower the efficacy of the therapeutic agent for cancer.
  • the growth inhibition of cancer stem cells includes not only the case of stopping the growth of cancer stem cells or attenuating the growth ability but also the case of killing cancer stem cells.
  • the present invention provides an antibody that specifically binds to the cancer stem cells obtained above.
  • the antibody may be either a monoclonal antibody or a polyclonal antibody, but is preferably a monoclonal antibody.
  • the antibody can be used for the purpose of detecting cancer stem cells and can also be used as a therapeutic agent for cancer.
  • Cancer stem cell inducer As described above, (1) KLF family gene, (2) OCT family gene, (3) SOX family gene, and (4) NANOG, LIN28, KRAS, BCL2, BMI1, and ERAS At least one selected undifferentiation-inducing factor gene and / or at least one tumor suppressor gene expression inhibitor selected from the group consisting of CDKN2A, FHIT, TP53, RB1, PTEN, and LZTS1, Cancer stem cells can be prepared by introducing into cells. Therefore, the present invention is further selected from the group consisting of (1) KLF family gene, (2) OCT family gene, (3) SOX family gene, and (4) NANOG, LIN28, KRAS, BCL2, BMI1, and ERAS.
  • a cancer comprising at least one gene for an undifferentiated inducer and / or an expression suppressor for at least one tumor suppressor gene selected from the group consisting of CDKN2A, FHIT, TP53, RB1, PTEN, and LZTS1
  • a stem cell inducer comprises a set of various factors used for reprogramming cancer cells to induce cancer stem cells, the gene of (1) to (3) above, and (4) specific It is desirable that the undifferentiation-inducing factor gene and / or the expression suppressor of a specific tumor suppressor gene be contained in a form that can be introduced into cancer cells.
  • Examples include vectors incorporating the genes (1) to (3) above and (4) a specific undifferentiation-inducing factor gene and / or a specific tumor suppressor gene expression suppressor.
  • the above genes (1) to (3), and (4) a specific undifferentiation-inducing factor gene and / or a specific tumor suppressor gene expression suppressor may be incorporated into different vectors.
  • two or more genes may be simultaneously incorporated into one vector.
  • the genes and vectors used for the soft cancer stem cell inducer are as described above.
  • ⁇ Experiment method> 1 ⁇ 10 6 cancer cells were inoculated into 2 ml of 10% FBS-containing DMEM medium (containing 2 ⁇ g / ml puromycin) in a 30 mm dish, and 10 ⁇ l of Lipofectamine 2000 transfection reagent and a retroviral receptor were incorporated into the expression. 3-5 ⁇ g of lentiviral vector was added and incubated at room temperature for 20 minutes. Next, after removing the supernatant and washing the cells with 10% FBS-containing DMEM medium, 2 ml of 10% FBS-containing DMEM medium was added, and 10 ⁇ l of Lipofectamine 2000 transfection reagent and the gene or DNA fragment shown in Table 1 were incorporated.
  • 10% FBS-containing DMEM medium containing 2 ⁇ g / ml puromycin
  • each gene was transfected into cancer cells by adding 5 ⁇ g each of pMXs-based retroviral vectors (4 types in total) and incubating at 37 ° C. for 24 hours.
  • the retrovirus vector used for the transfection uses a vector in which one kind of gene or DNA fragment is incorporated, and according to the combination of genes to be transfected, each cancer cell is combined with a retrovirus vector in which each gene is incorporated. Transfected.
  • Each cancer cell after transfection is cultured in DMEM medium containing 10% FBS for 7 days while changing the medium every other day.
  • DMEM medium containing 10% FBS
  • mTeSRTM1 and matrigel for BD human ES cells (StemCell Technologies Technologies Inc) was cultured while changing the medium every other day.
  • the expression level of Nanog gene was measured by RT-PCR.
  • the expression level of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene was also measured by RT-PCR.
  • the primers used for this measurement are as follows.
  • the Forward Primer and Reverse Primer for the Nanog gene below are mixed with UPL (# 66) tag (Roche) and PCR reaction is performed.
  • the Forward Primer and Reverse Primer for the GAPDH gene below are UPL (# 60) tag (Roche PCR product was mixed.
  • FIGS. 1-12 show the results of measuring the expression level of NANOG gene in PLC cells (20 days after transfection) into which the combination of each gene was introduced (Example 1-6).
  • FIG. 2 shows the combination of each gene (Example As a result of measuring the expression level of NANOG gene in HuCCT-1 cells (12 days after transfection) introduced with 1, 2, and 7-12),
  • FIG. 3 shows combinations of the genes (Examples 1-3 and 7-).
  • transduced 11) (12 days after transfection) is shown.
  • the NANOG gene is specifically expressed in liver cancer cells, and its expression level is an indicator that it has been induced in cancer stem cells.
  • FIG. 4 shows the results of observing the shape of cells when the combinations of the genes of Examples 2, 5 and 6 were transfected into colon cancer cells and cultured for 20 days.
  • DLD-1 cancer cells

Landscapes

  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Biomedical Technology (AREA)
  • Wood Science & Technology (AREA)
  • Organic Chemistry (AREA)
  • Chemical & Material Sciences (AREA)
  • Biotechnology (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Genetics & Genomics (AREA)
  • Microbiology (AREA)
  • Cell Biology (AREA)
  • Oncology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Developmental Biology & Embryology (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

La présente invention concerne principalement une technique pour construire artificiellement une cellule souche cancéreuse à partir d'une cellule cancéreuse. Une cellule cancéreuse est dérivée en une cellule souche cancéreuse ayant une capacité d'auto-renouvellement et acquérant une capacité de non-différentiation par l'introduction dans la cellule cancéreuse de (1) un gène de la famille KLF, (2) un gène de la famille OCT, (3) un gène de la famille SOX et (4) un gène d'au moins un type d'un facteur d'induction de non-différentiation choisi dans le groupe constitué par NANOG, LIN28, KRAS, BCL2, BMI1 et ERAS, et/ou un inhibiteur d'expression d'au moins un type d'un gène suppresseur de cancer choisi dans le groupe constitué par CDKN2A, FHIT, TP53, RB1, PTEN et LZTS1.
PCT/JP2010/068406 2009-10-20 2010-10-19 Procédé de production de cellules souches cancéreuses WO2011049099A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2009-241322 2009-10-20
JP2009241322 2009-10-20

Publications (1)

Publication Number Publication Date
WO2011049099A1 true WO2011049099A1 (fr) 2011-04-28

Family

ID=43900322

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2010/068406 WO2011049099A1 (fr) 2009-10-20 2010-10-19 Procédé de production de cellules souches cancéreuses

Country Status (1)

Country Link
WO (1) WO2011049099A1 (fr)

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012165287A1 (fr) * 2011-05-27 2012-12-06 公立大学法人横浜市立大学 Procédé de production pour une cellule souche cancéreuse artificielle et procédé de différenciation induite associé
WO2013008803A1 (fr) * 2011-07-11 2013-01-17 国立大学法人 熊本大学 Procédé de production de cellules pluripotentes utilisant des bactéries capables de fermentation
US20130145486A1 (en) * 2010-05-31 2013-06-06 Tsuyoshi Akagi Method for producing tumor cell
WO2014038655A1 (fr) * 2012-09-07 2014-03-13 国立大学法人京都大学 Procédé de production de cellules souches somatiques dérivées de l'épithélium intestinal
CN104080907A (zh) * 2011-11-30 2014-10-01 日本国立癌症研究中心 诱导恶性干细胞
US8852941B2 (en) 2010-02-18 2014-10-07 Osaka University Method for producing induced pluripotent stem cells
WO2015199088A1 (fr) * 2014-06-23 2015-12-30 国立大学法人京都大学 Cellules souches cancéreuses induites
JP5920725B2 (ja) * 2010-05-25 2016-05-18 国立研究開発法人国立がん研究センター 生体外で自己複製可能な誘導前がん幹細胞又は誘導悪性幹細胞、これらの製造方法、及び、これらの細胞の応用

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009057831A1 (fr) * 2007-10-31 2009-05-07 Kyoto University Procédé de re-programmation nucléaire

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009057831A1 (fr) * 2007-10-31 2009-05-07 Kyoto University Procédé de re-programmation nucléaire

Non-Patent Citations (14)

* Cited by examiner, † Cited by third party
Title
HONG,H. ET AL.: "Suppression of induced pluripotent stem cell generation by the p53-p21 pathway", NATURE, vol. 460, no. 7259, 27 August 2009 (2009-08-27), LOND, pages 1132 - 1135, XP008129610, DOI: doi:10.1038/nature08235 *
HOSHINO,H. ET AL.: "Hypoxia promotes the generation of the induced pluripotent cancer cells", PROCEEDINGS OF THE JAPANESE CANCER ASSOCIATION, vol. 69, 23 August 2010 (2010-08-23), pages 213 - 214 *
KAWAMURA,T. ET AL.: "Linking the p53 tumour suppressor pathway to somatic cell reprogramming", NATURE, vol. 460, no. 7259, 27 August 2009 (2009-08-27), LOND, pages 1140 - 1144 *
LI,H. ET AL.: "The Ink4/Arf locus is a barrier for iPS cell reprogramming", NATURE, vol. 460, no. 7259, 27 August 2009 (2009-08-27), LOND, pages 1136 - 1139, XP002632651, DOI: doi:10.1038/nature08290 *
LIN,S. ET AL.: "Mir-302 reprograms human skin cancer cells into a pluripotent ES-cell-like state", RNA, vol. 14, no. 10, 2008, pages 2115 - 2124, XP009108022, DOI: doi:10.1261/rna.1162708 *
MARION,R.M. ET AL.: "A p53-mediated DNA damage response limits reprogramming to ensure iPS cell genomic integrity", NATURE, vol. 460, no. 7259, 27 August 2009 (2009-08-27), LOND, pages 1149 - 1153, XP008129617, DOI: doi:10.1038/nature08287 *
MIYOSHI,N. ET AL.: "Defined factors induce reprogramming of gastrointestinal cancer cells", PROC NATL ACAD SCI USA, vol. 107, no. 1, 5 January 2010 (2010-01-05), pages 40 - 45, XP055083357, DOI: doi:10.1073/pnas.0912407107 *
MIYOSHI,N. ET AL.: "Reprogramming in gastrointestinal cancer cells with induction of defined factors", PROCEEDINGS OF THE JAPANESE CANCER ASSOCIATION, vol. 69TH, 23 August 2010 (2010-08-23), pages 232 *
NAGAI,K. ET AL.: "Long-term culture after induction of ES-like genes elicits an aggressive phenotype in mutated cholangiocarcinoma cells", PROCEEDINGS OF THE JAPANESE CANCER ASSOCIATION, vol. 69TH, 23 August 2010 (2010-08-23), pages 214 *
NAGAI,K. ET AL.: "Long-term culture following ES-like gene-induced reprogramming elicits an aggressive phenotype in mutated cholangiocellular carcinoma cells", BIOCHEM BIOPHYS RES COMMUN, vol. 395, no. 2, 30 April 2010 (2010-04-30), pages 258 - 263, XP027035407, DOI: doi:10.1016/j.bbrc.2010.03.176 *
NORIKATSU MIYOSHI ET AL.: "Daichogan no Bunshi Seibutsugaku 'Reprogramming of colorectal cancer cells can be induced?'", FRONTIERS IN COLORECTAL CANCER, vol. 3, no. 1, 31 March 2010 (2010-03-31), pages 69 - 73 *
TAKESHI TANABE ET AL.: "Lin28 no iPS Saibo Yudo ni Okeru Koka", JOURNAL OF JAPANESE BIOCHEMICAL SOCIETY, 2008, pages 1T25 - 11 *
UTIKAL,J. ET AL.: "Immortalization eliminates a roadblock during cellular reprogramming into iPS cells", NATURE, vol. 460, no. 7259, 27 August 2009 (2009-08-27), LOND, pages 1145 - 1148, XP055029395, DOI: doi:10.1038/nature08285 *
UTIKAL,J. ET AL.: "Sox2 is dispensable for the reprogramming of melanocytes and melanoma cells into induced pluripotent stem cells", J CELL SCI, vol. 122, no. 19, 1 October 2009 (2009-10-01), pages 3502 - 3510, XP008142364, DOI: doi:10.1242/jcs.054783 *

Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8852941B2 (en) 2010-02-18 2014-10-07 Osaka University Method for producing induced pluripotent stem cells
JP5920725B2 (ja) * 2010-05-25 2016-05-18 国立研究開発法人国立がん研究センター 生体外で自己複製可能な誘導前がん幹細胞又は誘導悪性幹細胞、これらの製造方法、及び、これらの細胞の応用
US20130145486A1 (en) * 2010-05-31 2013-06-06 Tsuyoshi Akagi Method for producing tumor cell
WO2012165287A1 (fr) * 2011-05-27 2012-12-06 公立大学法人横浜市立大学 Procédé de production pour une cellule souche cancéreuse artificielle et procédé de différenciation induite associé
JPWO2012165287A1 (ja) * 2011-05-27 2015-02-23 公立大学法人横浜市立大学 人工癌幹細胞の作製方法及びその分化誘導方法
US10731134B2 (en) 2011-05-27 2020-08-04 Public University Corporation Yokohama City University Production method for artificial cancer stem cell and induced differentiation method therefor
US9587224B2 (en) 2011-07-11 2017-03-07 National University Corporation Kumamoto University Method for producing pluripotent cell using bacterium having fermentation ability
WO2013008803A1 (fr) * 2011-07-11 2013-01-17 国立大学法人 熊本大学 Procédé de production de cellules pluripotentes utilisant des bactéries capables de fermentation
CN104080907A (zh) * 2011-11-30 2014-10-01 日本国立癌症研究中心 诱导恶性干细胞
EP2749642A4 (fr) * 2011-11-30 2015-03-04 Nat Cancer Ct Cellules souches malignes induites
JPWO2013081188A1 (ja) * 2011-11-30 2015-04-27 独立行政法人国立がん研究センター 誘導悪性幹細胞
WO2014038655A1 (fr) * 2012-09-07 2014-03-13 国立大学法人京都大学 Procédé de production de cellules souches somatiques dérivées de l'épithélium intestinal
WO2015199088A1 (fr) * 2014-06-23 2015-12-30 国立大学法人京都大学 Cellules souches cancéreuses induites

Similar Documents

Publication Publication Date Title
WO2011049099A1 (fr) Procédé de production de cellules souches cancéreuses
JP6934501B2 (ja) 体細胞の再プログラミング
KR101774206B1 (ko) 유도된 다능성 줄기 세포의 효율적인 확립 방법
CN101835890B (zh) 有效建立诱导的多能干细胞的方法
KR102066761B1 (ko) 효율적 인공 다능성 간세포 수립 방법
US8962331B2 (en) Method of making induced pluripotent stem cell from adipose stem cells using minicircle DNA vectors
Toh et al. RNAi reveals phase-specific global regulators of human somatic cell reprogramming
Zhang et al. Telomere-dependent and telomere-independent roles of RAP1 in regulating human stem cell homeostasis
JP5840119B2 (ja) 誘導多能性幹細胞の製造方法
Pina et al. MLLT3 regulates early human erythroid and megakaryocytic cell fate
KR20220165807A (ko) 히스톤 h3-리신 트리메틸화를 제거함으로써 체세포 핵 이식(scnt) 효율을 증가시키는 방법 및 조성물
EP2599859A1 (fr) Cellules haploïdes
JP2021519587A (ja) マイクロｒｎａ発現構築物及びその使用
CN105051188A (zh) 新颖方法
EP2616540A1 (fr) Procédé d'établissement efficace de cellules souches pluripotentes induites
CN109517826B (zh) 一种修饰的Bach1基因及其应用
WO2021172542A1 (fr) Procédé de production de cardiomyocytes matures
EP3041928B1 (fr) Compositions comprenant un inhibiteur de mitofusine pour favoriser la reprogrammation cellulaire et une utilisation correspondante
US20090311789A1 (en) Method for preparing differentiated avian cells and genes involved in maintaining pluripotency
WO2017126616A1 (fr) Modèle cellulaire pour des tumeurs de la famille du sarcome d'ewing, et procédé de criblage d'agents antitumoraux utilisant le modèle
JP7406257B2 (ja) 人工マイクロｒｎａ前駆体およびそれを含む改良されたマイクロｒｎａ発現ベクター
US20140134737A1 (en) Alternative Splicing Modulators and Splice Variants and Their Use in the Control and Detection of Pluripotency and Differentiation
CN114438085A (zh) Mettl3的反义寡核苷酸及其在前列腺癌中的应用
JP2011087475A (ja) 未分化細胞の識別方法
JP2022515211A (ja) 合成マイクロｒｎａミミック

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: 10824948

Country of ref document: EP

Kind code of ref document: A1

122 Ep: pct application non-entry in european phase

Ref document number: 10824948

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: JP