WO2017126616A1 - Modèle cellulaire pour des tumeurs de la famille du sarcome d'ewing, et procédé de criblage d'agents antitumoraux utilisant le modèle - Google Patents

Modèle cellulaire pour des tumeurs de la famille du sarcome d'ewing, et procédé de criblage d'agents antitumoraux utilisant le modèle Download PDF

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WO2017126616A1
WO2017126616A1 PCT/JP2017/001778 JP2017001778W WO2017126616A1 WO 2017126616 A1 WO2017126616 A1 WO 2017126616A1 JP 2017001778 W JP2017001778 W JP 2017001778W WO 2017126616 A1 WO2017126616 A1 WO 2017126616A1
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cells
ews
ewing sarcoma
cell
sarcoma family
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泰広 山田
真吾 河村
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国立大学法人京都大学
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • 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
    • C12N5/12—Fused cells, e.g. hybridomas
    • C12N5/16—Animal cells
    • 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
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms

Definitions

  • the present invention relates to a cell and a method for screening a drug using the cell, and specifically relates to a screening method for an Ewing sarcoma family tumor model cell and an antitumor agent using the same.
  • Ewing's sarcoma of family tumors are sarcomas that affect bone and soft tissue from childhood to adolescence.
  • the site of onset varies, and there are many metastatic sites, such as lung, bone marrow, and bone.
  • Chimeric genes have been detected in most Ewing sarcoma family tumor patients, and the chimeric genes are considered to be the causative genes for carcinogenesis. If a genetic test detects chimeric genes such as EWS-FLI, EWS-ERG, EWS-ETV1, and EWS-FEV, it will be a definitive diagnosis of Ewing sarcoma family tumors.
  • Chemotherapy that is highly effective against Ewing sarcoma family tumors includes doxorubicin, cyclophosphamide, vincristine, ifosfamide, etoposide, and actinomycin, and multidrug chemotherapy combining these is performed.
  • progress in chemotherapy has improved treatment outcomes, there is nothing definitive, and multidisciplinary treatment combining chemotherapy, surgery, and radiation therapy is necessary.
  • the prognosis is poor when it occurs in a region where it is difficult to perform surgery and radiation irradiation such as the head and neck, spine, and pelvis.
  • long-term survivors after treatment have increased secondary cancer caused by anticancer drugs and radiation, and long-term follow-up of late complications including secondary cancer is important.
  • Non-Patent Documents 1 and 2 the chimeric gene EWS-FLI1 is introduced into a primary-cultured bone-derived cell or bone marrow-derived mesenchymal progenitor cell using a retroviral vector to produce a tumor, but EWS-FLI1 is made constitutively. It is unclear whether tumor cells have acquired cell proliferation and tumorigenicity depending on EWS-FLI1.
  • an object of the present invention is to establish a model cell of an Ewing sarcoma family tumor that can be used for screening of an effective new anticancer substance against an Ewing sarcoma family tumor and that forms a tumor by directly reacting with a chimeric gene. To do.
  • EWS-FLI1-dependent osteosarcoma model was constructed from mouse bone marrow stromal cells using the doxycycline-inducible EWS-FLI1 system. did. Furthermore, using artificial pluripotent stem (iPS) cell technology, we have succeeded in obtaining EWS-FLI1-dependent osteosarcoma-derived iPS cells having cancer-related gene abnormalities. And it discovered that these cells could be used suitably for the screening of the medicine with respect to Ewing sarcoma family tumor, and came to complete this invention.
  • iPS artificial pluripotent stem
  • Ewing sarcoma family tumor cells derived from bone marrow stromal cells, in which an EWS chimeric gene placed under the control of a promoter containing an inducible transcriptional regulatory sequence is introduced onto the chromosome and grows upon expression of the EWS chimeric protein Ewing sarcoma family tumor cells that are capable and have acquired infinite growth potential (immortalized).
  • EWS chimeric gene placed under the control of a promoter containing an inducible transcriptional regulatory sequence is introduced onto the chromosome and grows upon expression of the EWS chimeric protein Ewing sarcoma family tumor cells that are capable and have acquired infinite growth potential (immortalized).
  • EWS chimeric gene is the EWS-FLI1 gene.
  • Ewing sarcoma family tumor model non-human mammal which is transplanted with the Ewing sarcoma family tumor cell according to any one of [1] to [5] and develops Ewing sarcoma family tumor depending on the expression of EWS chimeric protein .
  • [8] A step of adding a test compound to the Ewing sarcoma family tumor cell according to any one of [1] to [5] cultured under the induction of EWS chimeric gene expression, Examining a tumor phenotype and selecting a test compound as a therapeutic drug candidate compound for an Ewing sarcoma family tumor, using as an index the reduction of the proliferative ability of the Ewing sarcoma family tumor cells or the reduction of the tumor phenotype
  • a screening method for an Ewing sarcoma family tumor therapeutic agent comprising a step.
  • a step of differentiating the induced pluripotent stem cell according to [6] into a bone cell, a step of inducing expression of an EWS chimeric protein, and a test compound on the Ewing sarcoma family tumor cell expressing the obtained EWS chimeric protein The step of adding, the step of determining the proliferative ability or tumor phenotype of the Ewing sarcoma family tumor cells, and reducing the proliferative ability of the Ewing sarcoma family tumor cells or reducing the tumor phenotype,
  • a method for screening an Ewing sarcoma family tumor therapeutic agent comprising a step of selecting a test compound as a therapeutic drug candidate compound for an Ewing sarcoma family tumor.
  • a step of culturing the induced pluripotent stem cell according to [6] in a bone differentiation-inducing medium to which a test compound is added in a state where no EWS chimeric protein is expressed to differentiate into an osteocyte, and a degree of differentiation into an osteocyte A screening method for a bone differentiation promoting substance, comprising a step of determining, and a step of selecting a test compound as a bone differentiation promoting substance using as an index an increase in the degree of differentiation into the bone cells.
  • sarcoma cells capable of controlling the expression of chimeric genes (such as EWS-FLI1) detected in Ewing sarcoma family tumors by stimulation with drugs and the like, and iPS cells derived from sarcoma cells were obtained.
  • This sarcoma cell differs from conventional cell lines in that cell proliferation activity is induced depending on the expression of the chimeric gene.
  • sarcoma cell-derived iPS cells can form Ewing sarcoma family tumors by expressing a chimeric gene after induction of bone differentiation. These are considered cell lines that can directly monitor the effects of the chimeric gene.
  • Cell lines that respond to the Ewing sarcoma family tumor-associated chimeric gene are useful for screening the development of direct molecular target-specific inhibitors against the chimeric gene. Moreover, since ON / OFF of the expression of the chimeric gene can be regulated and the phenotype of the tumor can be reversibly controlled, it can be suitably used for analyzing the mechanism of osteosarcoma generation.
  • EWS-FLI1 expression system using lentivirus Lentiviral vectors are introduced into bone marrow stromal cells collected from Rosa26-M2rtTA mice, and EWS-FLI1-expressing neomycin resistant cells are selected.
  • the microscope picture which shows the growth in the Dox containing medium or Dox free medium of the immortalized cell (EFN # 2) in each cell. EFN # 2 grew rapidly in Dox-containing medium. Dox removal caused delayed growth and morphological changes in EWS-FLI1-expressing cells (4 days after removal).
  • the graph which shows the result of qRT-PCR which shows the mRNA expression of EWS-FLI1 with and without Dox in a Dox treatment sample. Data are presented as mean ⁇ SD.
  • the expression level of Dox-untreated cells was set to 1.
  • the figure (photograph) which shows the result of the western blotting which uses an anti- HA antibody.
  • EWS-FLI1 protein was detected in the presence of Dox.
  • EFN # 2 developed tumors in immunocompromised mice only in the presence of Dox (10 weeks after transplantation).
  • FIG. 6 shows the results of an in vivo tumor formation assay using sarcoma cell line SCOS # 2. Dox treatment was discontinued at 3 weeks and mice were sacrificed at 7 weeks. The micrograph of the obtained iPS cell-like cell. IPS cell-like cells were constructed from sarcoma cells by introducing reprogrammed transcription factors. The graph which shows the result of qRT-PCR which shows the expression level of a pluripotency related gene in a sarcoma origin iPS cell-like cell. It was revealed that the expression level of pluripotency-related genes is equivalent to that of ES cells.
  • FIG. 1 Schematic diagram of in vitro bone differentiation. The figure which shows the result of the qRT-PCR analysis of a bone differentiation related gene. Wild type ES cells (V6.5) or EWS-FLI1-inducible ES cells (Rosa-rtTA / Rosa :: tetO-EWS-FLI1-ires-mCherry) were used as controls in bone differentiation experiments. Regarding the expression of bone differentiation-related genes, sarcoma-derived iPS cells and control ES cells on day 0 and day 17 during bone differentiation were examined. Mean values ⁇ SD of three independent experiments are shown. The average expression level of ES cells on day 17 was set to 1.
  • EWS-FLI1-induced ES cells (Rosa-M2rtTA / Col1a1 :: tetO-EWS-FLI1-ires-mCherry) were used as a control. Shown is the mean ⁇ SD of 6 independent osteogenic regions in 2 independent sarcoma-derived iPS cell teratomas or 9 independent osteogenic regions in 2 independent ES cell teratomas. Mann-Whitney U test was used for statistical analysis. Graph showing proliferation of iPS cells or control ES cells with and without Dox.
  • EWS-FLI1 expression does not promote proliferation of undifferentiated pluripotent stem cells.
  • Osteogenic cells derived from iPS cells with EWS-FLI1 developed tumors in immunocompromised mice only in the presence of Dox (3-7 weeks after treatment).
  • the Ewing sarcoma family tumor cells of the present invention are derived from bone marrow stromal cells, and an EWS chimeric gene placed under the control of a promoter containing an inducible transcriptional regulatory sequence is introduced onto the chromosome, and during expression of the EWS chimeric protein, It is a cell that can grow and has infinite proliferation ability.
  • the Ewing sarcoma family tumor cell of the present invention introduces an EWS chimera gene that is placed under control of a promoter containing an inducible transcription control sequence into bone marrow stromal cells derived from a mammal, It can be obtained by selecting immortalized cells that can grow in a state in which the protein is expressed.
  • Bone marrow stromal cells can be obtained from a variety of sources. Sources of bone marrow stromal cells and methods for obtaining bone marrow stromal cells from those sources and methods for culturing them have been described in the prior art (eg Friedenstein et al., 1987, Cell Tissue Kinetics 20: 263- 272; Castro-Malaspina et al., 1980, Blood 56: 289-301; Mets et al., 1981, Mech. Ageing Develop. 16: 81-89; Piersma et al., 1985, Exp. Hematol. 13: 237 -243; Caplan, 1991, J.Orthoped.Res.
  • Bone marrow stromal cells can be obtained from any bone marrow, including, for example, cells from the bone marrow obtained by aspiration of the iliac crest of a human donor. Methods for obtaining bone marrow from a donor are well known techniques.
  • bone marrow stromal cells can also be obtained commercially, for example, bone marrow stromal cells isolated from humans, mice, rats, rabbits, dogs, goats, sheep, pigs and horses can be obtained from Cognate® Bioservices (Baltimore , MD).
  • EWS chimeric genes examples include EWS-FLI, EWS-ERG, EWS-ETV1, and EWS-FEV as described in Cancer Res. 2000 Mar 15; 60 (6): 1536-40 etc.
  • EWS-FLI1 is preferable.
  • EWS-FLI1 is preferably human-derived EWS-FLI1, for example, a fusion protein of EWS (SEQ ID NO: 2) and FLI1 (SEQ ID NO: 3) encoded by the nucleotide sequence of base numbers 1-1494 of SEQ ID NO: 1. Examples include proteins containing amino acid sequences.
  • an amino acid sequence having 80% or more, preferably 90% or more, more preferably 95% or more identity with the above-mentioned amino acid sequence is included as long as the function of inducing cells into cancer and inducing the Ewing sarcoma family tumor is maintained. It may be a protein.
  • inducible transcription control sequences include sequences that initiate transcription from a promoter in response to stimuli such as drugs. Examples include Tet operator sequences, Cumate operator sequences (System Biosciences: SparQ Cumate Switch Inducible System), ⁇ An operator sequence (Special Table 2006-526991) and the like can be mentioned.
  • the Tet operator sequence is a sequence that binds a reverse tetracycline-regulated transactivator (tetracycline response factor: TRE), and reverse tetracycline expressed from the host cell depending on reverse tetracycline such as doxycycline (Dox) added to the cell.
  • TRE reverse tetracycline response factor
  • Dox doxycycline
  • a regulatory transactivator binds and induces expression of a gene linked downstream thereof.
  • Examples of the reverse tetracycline-regulated transactivator include a fusion protein composed of a mutant Tet repressor protein (tTetR) and a VP16 activation domain (AD) included in Clonetech's Tet-On® System.
  • tTetR Tet repressor protein
  • AD VP16 activation domain
  • An EWS chimeric gene linked to a promoter capable of functioning in mammalian cells is arranged downstream of the inducible transcription control sequence.
  • promoters that can function in mammalian cells include ⁇ -actin promoters, CMV promoters, CAG (CAGGS) promoters, etc., but many types of promoters are known and are not particularly limited thereto.
  • the expression construct for gene transfer includes a tag sequence (for example, Flag tag: SEQ ID NO: 4, HA tag: SEQ ID NO: 5), drug in addition to the above-described inducible transcription control sequence, promoter, and EWS chimeric gene. Selection marker sequences such as resistance genes, ribosome binding sequences, photoprotein coding sequences and the like may be included.
  • a tag sequence for example, Flag tag: SEQ ID NO: 4, HA tag: SEQ ID NO: 5
  • Selection marker sequences such as resistance genes, ribosome binding sequences, photoprotein coding sequences and the like may be included.
  • the EWS chimeric gene and the drug resistance gene under the control of the Tet operon, it becomes possible to induce the EWS chimeric gene and the drug resistance gene simultaneously by administration of doxycycline (Dox).
  • Dox doxycycline
  • the EWS chimeric gene is an oncogene, it contributes to carcinogenesis only in certain cell types and causes toxicity in many cell tumors.
  • Dox + drugs such as neomycin
  • the method of gene transfer onto the chromosome is not particularly limited, and examples thereof include a method using a viral vector, a method using a plasmid vector, a method using an artificial chromosome, and the like.
  • a method using a viral vector is preferable, and a lentiviral vector is used. The method is more preferred.
  • a simian immunodeficiency virus vector As a lentiviral vector, a simian immunodeficiency virus vector, an equine infectious anemia virus (EIAV) vector, a human immunodeficiency virus (HIV, for example, HIV1 or HIV2) vector, a feline immunodeficiency virus (FIV) vector, etc. can be used.
  • EIAV equine infectious anemia virus
  • HAV human immunodeficiency virus
  • HIV human immunodeficiency virus
  • FMV feline immunodeficiency virus
  • a commercially available lentiviral vector can also be used.
  • EWS chimeric gene placed under the control of a promoter containing an inducible transcription control sequence is introduced into a bone marrow stromal cell derived from a mammal and then added to a chromosome, and then a stimulating substance for gene expression induction is added.
  • the EWS chimeric protein of the present invention can be obtained by expressing an EWS chimeric protein, and culturing and growing cells in that state, and selecting immortalized cells.
  • Ewing sarcoma family tumor cells are characterized by chromosomal abnormalities, tumor-specific genes, and expression of cell cycle markers (eg, Ki67) in addition to their proliferative ability.
  • the Ewing sarcoma family tumor cells of the present invention may be established.
  • an Ewing sarcoma family tumor model animal that produces an Ewing sarcoma family tumor depending on the expression of the EWS chimeric protein can be obtained.
  • the transplant site and the amount of cells used for transplantation may be appropriately determined according to the body weight of the animal, the desired phenotype, and the like.
  • iPS cells ⁇ Artificial pluripotent stem (iPS) cells>
  • Ewing sarcoma family tumor cells of the present invention By initializing the Ewing sarcoma family tumor cells of the present invention, it is possible to obtain iPS cells that can differentiate into cells exhibiting the Ewing sarcoma family tumor phenotype depending on the expression of the EWS chimeric protein.
  • iPS cells introduce certain nuclear reprogramming substances into the Ewing sarcoma family tumor cells of the present invention in the form of DNA or protein, or increase the endogenous mRNA and protein expression of the nuclear reprogramming substances by drugs.
  • the nuclear reprogramming substance is not particularly limited as long as it is a gene specifically expressed in ES cells, a gene that plays an important role in maintaining undifferentiation of ES cells, or a gene product thereof.
  • Oct3 / 4 Klf4, Klf1, Klf2, Klf5, Sox2, Sox1, Sox3, Sox15, Sox17, Sox18, c-Myc, L-Myc, N-Myc, TERT, SV40 Large T antigen, HPV16 E6, HPB28 E7 Examples are Lin28b, Nanog, Esrrb, or Esrrg.
  • These reprogramming substances may be used in combination when iPS cells are established.
  • Nucleotide sequences of mouse and human cDNAs of each nuclear reprogramming substance and amino acid sequence information of the protein encoded by the cDNA can be obtained by referring to NCBI accession numbers described in WO 2007/079666.
  • a person skilled in the art can prepare a desired nuclear reprogramming substance by a conventional method based on the cDNA sequence or amino acid sequence information.
  • nuclear reprogramming substances may be introduced into tumor cells to be reprogrammed in the form of proteins, for example, by lipofection, binding to cell membrane permeable peptides, microinjection, or in the form of DNA.
  • it can be introduced into tumor cells by techniques such as vectors such as viruses, plasmids, artificial chromosomes, lipofection, liposomes, and microinjection.
  • viral vectors examples include retroviral vectors and lentiviral vectors (above, Cell, 126, pp.663-676, 2006; Cell, 131, pp.861-872, 2007; Science, 318, pp.1917-1920, 2007 ), Adenovirus vectors (Science, 322, 945-949, 2008), adeno-associated virus vectors, Sendai virus vectors (Proc Jpn Acad Ser B Phys Biol Sci. 85, 348-62, 2009) and the like.
  • artificial chromosome vectors include human artificial chromosomes (HAC), yeast artificial chromosomes (YAC), and bacterial artificial chromosomes (BAC, PAC).
  • a plasmid for mammalian cells can be used (Science, 322: 949-953, 2008).
  • the vector can contain regulatory sequences such as a promoter, an enhancer, a ribosome binding sequence, a terminator, and a polyadenylation site so that a nuclear reprogramming substance can be expressed.
  • promoter used examples include EF1 ⁇ promoter, CAG promoter, SR ⁇ promoter, SV40 promoter, LTR promoter, CMV (cytomegalovirus) promoter, RSV (rous sarcoma virus) promoter, MoMuLV (Moloney murine leukemia virus) LTR, HSV- A TK (herpes simplex virus thymidine kinase) promoter or the like is used.
  • EF1 ⁇ promoter, CAG promoter, MoMuLV LTR, CMV promoter, SR ⁇ promoter and the like can be mentioned.
  • drug resistance genes for example, kanamycin resistance gene, ampicillin resistance gene, puromycin resistance gene, etc.
  • thymidine kinase gene diphtheria toxin gene and other selectable marker sequences
  • green fluorescent protein (GFP) green fluorescent protein
  • GUS ⁇ -glucuronidase
  • reporter gene sequences such as FLAG, and the like.
  • the above vector contains a LoxP sequence before and after the gene or promoter encoding the nuclear reprogramming substance and the gene encoding the nuclear reprogramming substance that binds to it. You may have.
  • transposons include, for example, piggyBac, a transposon derived from lepidopterous insects (Kaji, K. et al., (2009), Nature, 458: 771-775, Woltjen et al., (2009), Nature, 458: 766-770, WO 2010/012077).
  • the vector replicates without chromosomal integration and is present episomally, so that the origin and replication of lymphotrophic herpes virus, BK virus, and bovine papillomavirus
  • sequence which concerns on may be included. Examples include EBNA-1 and oriP or Large T and SV40ori sequences (WO 2009/115295, WO 2009/157201 and WO 2009/149233).
  • an expression vector for polycistronic expression may be used.
  • the gene-encoding sequence may be linked by an IRES or foot-and-mouth disease virus (FMDV) 2A coding region (Science, 322: 949-953, 2008 and WO 2009/092042, WO 2009/152529).
  • FMDV foot-and-mouth disease virus
  • HDAC histone deacetylase
  • VPA valproic acid
  • MC 1293 sodium butyrate
  • M344 small molecule inhibitors
  • siRNA and shRNA against HDAC e.g., HDAC1 siRNA (Smartpool® (Millipore), HuSH 29mer shRNA Nucleic acid expression inhibitors such as Constructs against HDAC1 (OriGene) etc.
  • DNA methyltransferase inhibitors eg 5'-azacytidine
  • G9a Histone methyltransferase inhibitors [eg, small molecule inhibitors such as BIX-01294 (Cell Stem Cell, 2: 525-528 (2008)), siRNA and shRNA against G9a (eg, G9a siRNA (human) (Santa Cruz Biotechnology) Etc.), L-channel] calcium] agonist (eg B ayk8644) (Cell Stem Cell, 3, 568-574 (2008)), p53 inhibitors (eg siRNA and shRNA against p53) (Cell Stem Cell, 3, 475-479 (2008)), Wnt Signaling activator (eg soluble Wnt3a ) (Cell Stem Cell, 3, 132-135 (2008)), growth factors such as LIF or bFGF, ALK5 inhibitors (eg, SB431542) (Nat.
  • small BIX-01294 Cell Stem Cell, 2: 525-528 (2008)
  • siRNA and shRNA against G9a eg, G9a siRNA (human) (Santa Cruz
  • Examples of the drug in the method for increasing the expression of the endogenous protein of the nuclear reprogramming substance by the drug include 6-bromoindirubin-3'-oxime, indirubin-5-nitro-3'-oxime, valproic acid, 2- (3- (6-methylpyridin-2-yl) -lH-pyrazol-4-yl) -1,5-naphthyridine, 1- (4-methylphenyl) -2- (4,5,6,7-tetrahydro-2-imino- 3 (2H) -benzothiazolyl) ethanone HBr (pifithrin-alpha), prostaglandin J2, and prostaglandin E2 are exemplified (WO 2010/068955).
  • Examples of the culture medium for iPS cell induction include (1) DMEM, DMEM / F12 or DME medium containing 10-15% FBS (these media include LIF, penicillin / streptomycin, puromycin, L-glutamine). , (2) ES cell culture medium containing bFGF or SCF, such as mouse ES cell culture medium (for example, TX-WES medium, thrombos. X) or primate ES cell culture medium (for example, primate (human & monkey) ES cell culture medium (Reprocell, Kyoto, Japan), mTeSR-1).
  • mouse ES cell culture medium for example, TX-WES medium, thrombos. X
  • primate ES cell culture medium for example, primate (human & monkey) ES cell culture medium (Reprocell, Kyoto, Japan), mTeSR-1).
  • a tumor cell and a nuclear reprogramming substance are contacted in DMEM or DMEM / F12 medium containing 10% FBS in the presence of 5% CO 2 at 37 ° C.
  • Culture for ⁇ 7 days then re-spread cells onto feeder cells (eg, mitomycin C-treated STO cells, SNL cells, etc.), and bFGF-containing primate ES cells approximately 10 days after contact between tumor cells and nuclear reprogramming substance
  • the cells can be cultured in a culture medium and ES cell-like colonies can be generated about 30 to about 45 days or more after the contact.
  • the cells may be cultured under conditions of an oxygen concentration as low as 5-10%.
  • 10% FBS-containing DMEM medium including LIF, penicillin / streptomycin, puromycin, L-glutamine, etc.
  • feeder cells eg, mitomycin C-treated STO cells, SNL cells, etc.
  • Non-essential amino acids, ⁇ -mercaptoethanol, etc. can be included as appropriate.
  • ES-like colonies can be formed after about 25 to about 30 days or more.
  • the medium is replaced with a fresh medium once a day from the second day after the start of the culture.
  • the number of tumor cells used for nuclear reprogramming is not limited, but ranges from about 5 ⁇ 10 3 to about 5 ⁇ 10 6 cells per 100 cm 2 of culture dish.
  • marker gene-expressing cells When a gene containing a drug resistance gene is used as the marker gene, marker gene-expressing cells can be selected by culturing in a medium (selective medium) containing the corresponding drug.
  • the marker gene when the marker gene is a fluorescent protein gene, the marker gene-expressing cells can be obtained by observing with a fluorescence microscope, by adding a luminescent substrate in the case of a luminescent enzyme gene, Can be detected.
  • iPS cells can be confirmed by expression of pluripotency-related genes such as Nanog and Oct3 / 4, demethylation of Nanog promoter and Oct3 / 4 distal enhancer, or teratoma formation.
  • pluripotency-related genes such as Nanog and Oct3 / 4, demethylation of Nanog promoter and Oct3 / 4 distal enhancer, or teratoma formation.
  • iPS cells obtained as described above are cultured and differentiated into bone cells, so that the EWS chimeric protein expression depends on the expression of Ewing sarcoma family tumors. Presenting cells can be obtained.
  • Adhesive culture, suspension culture, or culture using feeder cells may be used.
  • a medium for culturing iPS cells can be prepared using a medium used for culturing animal cells as a basal medium.
  • the basal medium include IMDM medium, Medium ⁇ 199 medium, Eagle's Minimum Essential Medium (EMEM) medium, ⁇ MEM medium, Doulbecco's modified Eagle's Medium (DMEM) medium, Ham's F12 medium, RPMI 1640 medium, and Fischer's medium. Media and the like are included.
  • ⁇ MEM medium is preferable.
  • the medium may contain serum or may be serum-free.
  • albumin transferrin, Knockout Serum Replacement (KSR) (serum substitute for FBS during ES cell culture), fatty acid, insulin, collagen precursor, trace element, 2-mercaptoethanol, 3'-thiol May contain one or more serum replacements such as glycerol, lipids, amino acids, L-glutamine, Glutamax (Invitrogen), non-essential amino acids, vitamins, antibiotics, antioxidants, pyruvate, buffers, inorganic salts , N2 supplement (Invitrogen), B27 supplement (Invitrogen), and one or more cytokines. What is necessary is just to add a component required for the bone differentiation illustrated to triiodothyronine to this. The method of bone differentiation is described, for example, in Nature 467, 285-290. 2010.
  • the culture temperature is not limited to the following, but is about 30 to 40 ° C., preferably about 37 ° C.
  • the culture is performed in an atmosphere of CO 2 -containing air, and the CO 2 concentration is preferably about 2 to 5%. is there.
  • Bone differentiation can be confirmed by expression of bone differentiation markers such as bone differentiation-related genes such as Runx2, Sp7, Pth1r, Col1a1 and Dmp1, and cell morphology.
  • bone differentiation markers such as bone differentiation-related genes such as Runx2, Sp7, Pth1r, Col1a1 and Dmp1, and cell morphology.
  • One embodiment of the screening method of the present invention includes a step of adding a test compound to the Ewing sarcoma family tumor cell of the present invention cultured under the induction of EWS chimeric protein expression, the proliferative ability of the Ewing sarcoma family tumor cell, or Examining a tumor phenotype and selecting a test compound as a therapeutic drug candidate compound for an Ewing sarcoma family tumor, using as an index the reduction of the proliferative ability of the Ewing sarcoma family tumor cells or the reduction of the tumor phenotype Process. If the growth ability or tumor phenotype of the tumor cell line is reduced or decreased as compared with the absence of the compound or the addition of the control compound, the test compound can be selected as a tumor therapeutic drug candidate compound.
  • Other aspects of the screening method of the present invention include the step of differentiating the EPS sarcoma family tumor cell-derived iPS cells of the present invention into bone cells, the step of inducing the expression of EWS chimeric protein, and the expression of the obtained EWS chimeric protein.
  • Adding a test compound to an Ewing sarcoma family tumor cell, determining a growth ability or tumor phenotype of the Ewing sarcoma family tumor cell, and reducing the growth ability of the Ewing sarcoma family tumor cell, or tumor expression Selecting a test compound as a candidate drug for a Ewing sarcoma family tumor using the reduction in type as an index.
  • a compound may be added simultaneously with the induction of EWS chimeric protein expression.
  • the proliferation ability of tumor cells can be examined by a known method.
  • the tumor cell phenotype can be examined by cell morphology, expression of tumor marker genes and cell cycle-related genes such as Ki67, expression of tumor proteins, etc.
  • the expression level of genes and proteins is determined by known methods. can do.
  • screening for therapeutic drugs for Ewing sarcoma family tumors can be performed using a non-human mammal transplanted with the Ewing sarcoma family tumor cells of the present invention.
  • the expression of EWS chimeric protein is induced in a non-human mammal transplanted with the Ewing sarcoma family tumor cell of the present invention, and the obtained EWS chimeric protein is expressed and tested on an Ewing sarcoma family tumor model animal that has produced a tumor.
  • a compound is administered to determine the size or tumor phenotype of the Ewing sarcoma family tumor cell, and to reduce the size of the Ewing sarcoma family tumor or to decrease the tumor phenotype as an index
  • the mode of selecting as a therapeutic drug candidate compound for a family tumor is exemplified.
  • the EPS sarcoma family tumor cell-derived iPS cell of the present invention is cultured in a bone differentiation-inducing medium supplemented with a test compound in a state where no EWS chimeric protein is expressed. And a step of determining the degree of differentiation into bone cells, and a step of selecting a test compound as a bone differentiation promoting substance using as an index the increase in the degree of differentiation into bone cells. Since the EPS sarcoma family tumor cell-derived iPS cells of the present invention have suppressed bone differentiation compared to control cells, screening for a bone differentiation promoting substance can be performed using bone differentiation as an index.
  • the test compound can be selected as a bone differentiation promoting substance.
  • Bone differentiation promoting substances can be therapeutic agents for bone-related diseases such as osteoporosis, and since bone differentiation failure has been shown to be involved in sarcoma formation, bone differentiation promoting substances can also be candidates for sarcoma inhibitory substances.
  • the degree of differentiation into bone cells can be confirmed by the expression of bone differentiation markers such as bone differentiation-related genes such as Runx2, Sp7, Pth1r, Col1a1 and Dmp1, and the morphology of cells.
  • test substance can be used, for example, cell extract, cell culture supernatant, microbial fermentation product, marine organism-derived extract, plant extract, purified protein or crude protein. , Peptides, non-peptide compounds, synthetic low molecular weight compounds, and natural compounds.
  • the test substance may also be (1) a biological library method, (2) a synthetic library method using deconvolution, and (3) a “one-bead one-compound” library method.
  • (4) can be obtained using any of a number of approaches in combinatorial library methods known in the art, including synthetic library methods using affinity chromatography sorting.
  • Biological library methods using affinity chromatography sorting are limited to peptide libraries, but the other four approaches can be applied to small molecule compound libraries of peptides, non-peptide oligomers, or compounds (Lam (1997) Anticancer Drug Des . 12: 145-67).
  • Examples of methods for the synthesis of molecular libraries can be found in the art (DeWitt et al. (1993) Proc. Natl. Acad. Sci. USA 90: 6909-13; Erb et al. (1994) Proc. Natl Acad. Sci. USA 91: 11422-6; Zuckermann et al. (1994) J. Med. Chem. 37: 2678-85; Cho et al.
  • the obtained vector was introduced into KH2 ES cells (Genesis 44, 23-28. 2006) having the Rosa26-M2rtTA allele by electroporation.
  • ES cells were cultured in an ES medium containing 15 ⁇ g / ml blasticidin S (Bsd, Funakoshi Co., Ltd.). Bsd resistant colonies were collected and expanded. It was confirmed by Southern blotting that the ES clone was correctly targeted.
  • lentiviral vector Construction of lentiviral vector, lentiviral infection and cell culture
  • pEN-TmiRC3 and pSLIK-Neo lentiviral vector plasmids obtained from Addgene were modified.
  • pEN-TmiRC3 was digested with SpeI and XhoI, and EWS-FLI1-FLAG-HA was ligated downstream of the tetOP-mCMV promoter.
  • the ires-NeoR cassette was ligated to the 3 ′ side of the HA tag, and then the UbiC-rtTA3-ires-NeoR sequence was excised from pSLIK-Neo.
  • the pSLIK-TetO-EWS-FLI1-ires-Neo vector is obtained. It was.
  • Bone marrow stromal cells were obtained from 3-6 week old Rosa26-M2rtTA mice (Genesis 44, 23-28. 2006) as reported in Nat Protoc 4, 102-106. 2009. Non-adherent cells (hematopoietic cells) were removed by exchanging the medium 3 to 4 days after collection of bone marrow cells, and the adherent cells were infected with lentivirus. The cells were then placed in DMEM (Nacalai Tesque) containing 10% FBS (Gibco), penicillin, streptomycin, 200 ⁇ g / ml G418 (Nacalai Tesque) and 2 ⁇ g / ml Dox (Sigma) for 2 months. Cultured and EWS-FLI1-dependent immortalized cells were selected. The same medium was used to maintain the osteosarcoma cell lines, SCOS # 2 and SCOS # 12.
  • Rosa-M2rtTA / Rosa tetO-EWS-FLI1 mice and immunocompromised mice inoculated with sarcoma cells were treated with 2 mg / ml Dox-containing water and 10 mg / ml sucrose. Because of early lethality, Rosa-M2rtTA / Col1a1 :: tetO-EWS-FLI1 mice were treated with lower concentrations of Dox (100 ⁇ g / ml to 2 mg / ml).
  • iPS cells Induction and maintenance of iPS cells Induction of iPS cells by utilizing retroviral vectors (pMX-hOCT3 / 4, pMX-hSOX2, pMX-hKLF4 and pMX-h-cMYC; Addgene) containing reprogramming factors went.
  • Reprogramming factor-induced sarcoma cells are cultured in ES cell medium supplemented with human recombinant LIF (Wako Pure Chemical Industries, Ltd.), 2-mercaptoethanol (Invitrogen) and 50 ⁇ g / ml L-ascorbic acid (Sigma).
  • LIF human recombinant LIF
  • 2-mercaptoethanol Invitrogen
  • 50 ⁇ g / ml L-ascorbic acid Sigma.
  • the constructed iPS cells were maintained in ES cell medium supplemented with LIF, 1 ⁇ M PD0325901 (Stemgent) and 3 ⁇ M CHIR99021 (Stemgent
  • RT-PCR and quantitative real-time RT-PCR RNA was extracted using RNeasy plus mini kit (QIAGEN). Up to 1 ⁇ g of RNA was used for reverse transcription to cDNA.
  • RT-PCR and quantitative real-time PCR were performed using Go-Taq Green Master Mix and Go-Taq qPCR Master Mix (Promega), respectively. Transcript levels were normalized by ⁇ -actin.
  • the cultured cells were washed with immunocytochemistry PBS and fixed with 2% paraformaldehyde for 10 minutes at room temperature.
  • the antibodies used were anti-p53 (Abcam; dilution 1: 200) and anti-p21 (HUGO291) (Abcam; dilution 1: 500).
  • Xenograft assay A total of 3 ⁇ 10 6 EWS-FLI1-dependent immortalized cells, EWS-FLI1-dependent in NOD / ShiJic-scid Jcl mice or BALB / cSLC-nu / nu mice purchased from CLEA Japan and Japan SLC, respectively Sarcoma cells, ES cells or iPS cells were transplanted. NOD / ShiJic-scid Jcl mice were inoculated with EWS-FLI1-dependent immortalized cells and the mice were sacrificed 10 weeks after transplantation. The subcutaneous tissue of BALB / cSLC-nu / nu mice was inoculated with EWS-FLI1-dependent osteosarcoma cells.
  • ES cells / iPS cells were transplanted into BALB / cSLC-nu / nu mice, and teratomas were obtained 3 to 4 weeks later.
  • Cultured cells were washed with ALP stained PBS, fixed, and stained with ALP staining kit (Sigma) according to manufacturer's protocol.
  • Senescence-related ⁇ -gal stained PBS were washed with PBS, fixed, and stained with an aging ⁇ -galactosidase staining kit (# 9860S, Cell Signaling) according to the manufacturer's protocol.
  • the cultured cells were washed with alizarin red-stained PBS and fixed with 4% paraformaldehyde for 5 minutes at room temperature. Fixed cells were washed several times with deionized water and stained for 5 minutes in alizarin red staining solution (alizarin red (Sigma, A5533) 2%, adjusted to pH 4.2 with NH 4 OH). Similarly, deparaffinized sections were stained in alizarin red staining solution for 5 minutes.
  • lentiviral integration site was examined by a slightly modified method described in Stem Cells 27, 300-306. Genomic DNA extracted from SCOS # 2 was digested into 500-800 bp fragments using an ultrasonicator (Covaris E210). The linker cassettes obtained by annealing LC1 and LC2 were ligated to their digested genomic DNA fragments. Then, the first PCR was performed using the primer set of AP1_F and pSLIK1_R, and the nested PCR was performed using the primer set of AP2_F and pSLIK2_R.
  • the PCR product was cloned into the pCR4-TOPO vector (Invitrogen) by the TA cloning method, and the DNA sequence of the inserted fragment was analyzed by 3500 ⁇ L Genetic Analyzer (Applied Biosystems) using the seq_LTR_R primer. The resulting sequence was examined by BLAST.
  • Genomic DNA was extracted using the PureLink® Genomic DNA Mini Kit (Invitrogen).
  • Array comparative genomic hybridization analysis was performed using SurePrint G3 mouse genomic CGH microarray kit (Agilent), and analysis was performed using Agilent Genomic Workbench 7.0.
  • Bisulfite genome sequencing Bisulfite treatment was performed using EZ DNA Methylation-Gold Kit TM (ZYMO RESEARCH) according to the manufacturer's protocol. The PCR primers used are indicated in the additional information.
  • the amplification product was cloned into pCR4-TOPO vector (Invitrogen) and transformed into DH5 ⁇ . Colonies were randomly selected and sequenced using M13 forward and reverse primers for each gene.
  • EWS-FLI1-inducible ES cells and mice First, an attempt was made to construct an EWS-FLI1-inducible mouse model by the locus targeting method. Use KH2 mice with tet-inducible cassette and Rosa26 targeting vector (Cell 156, 663-677. 2014; J Clin Invest 123, 600-610. 2013; Genesis 44, 23-28. 2006) Two types of ES cells containing the Dox-inducible EWS-FLI1 allele were constructed.
  • the reverse tetracycline-regulated transactivator (rtTA) is expressed from the Rosa26 locus, while the Tet operator-EWS-FLI1-ires-mCherry construct is integrated into the 3′UTR of the Col1a1 locus.
  • rtTA reverse tetracycline-regulated transactivator
  • Col1a1 the Tet operator-EWS-FLI1-ires-mCherry construct is integrated into the 3′UTR of the Col1a1 locus.
  • Rosa-M2rtTA / Col1a1 :: tetO-EWS-FLI1 the other is incorporated into intron 1 of the Rosa26 locus
  • Both ES cells expressed mCherry fluorescence when treated with Dox in vitro.
  • the inducible EWS-FLI1 expression in ES cells was also confirmed by qRT-PCR and Western blotting.
  • mice blastocyst injection of the ES cells was performed to obtain chimeric mice.
  • EWS-FLI1 was expressed in a wide variety of organs and tissues of the mouse, including bone marrow and bone cortex where Ewing sarcoma often occurs.
  • Some mice (Rosa-M2rtTA / Col1a1 :: tetO-EWS-FLI1) died shortly after EWS-FLI1 induction, which was accompanied by atypical changes in intestinal cells due to abnormal differentiation (14 mice) 8 of them).
  • EWS-FLI1-dependent immortalized cells by Dox-inducible EWS-FLI1 lentiviral system Our results suggested that induction of EWS-FLI1 in mature mice is not sufficient for sarcoma development. Therefore, a lentiviral EWS-FLI1 expression vector having a Dox-inducible expression system was prepared.
  • the TetO-EWS-FLI1-ires-Neo cassette (FIG. 1) was transduced with lentivirus into bone marrow stromal cells of Rosa26-M2rtTA (3-4 weeks old). Transduced bone marrow cells were cultured with Dox and G418.
  • EWS-FLI1-dependent immortalized cells that have formed osteosarcoma in vivo have tumorigenic potential in vivo
  • EFN # 2 and EFN # 12 The mice were transplanted subcutaneously. Mice transplanted 10 weeks after inoculation developed tumors from both cell lines when administered Dox (16/16 for EFN # 2, 2/4 for EFN # 12; FIGS. 5 and 6
  • Dox 16/16 for EFN # 2, 2/4 for EFN # 12; FIGS. 5 and 6
  • no tumor formation was observed in mice (0/16 for EFN # 2, 0/4 for EFN # 12; FIGS. 5 and 6
  • Histological analysis revealed that these tumors consisted of small round blue cells similar to Ewing sarcoma. Many tumor cells showed osteoid formation and were considered small cell osteosarcoma.
  • immunohistochemistry showed that tumor cells expressed EWS-FLI1 and were positive for Ki67, a marker for proliferating cells (data not shown).
  • EWS-FLI1-dependent osteosarcoma cell line In order to investigate the characteristics of EWS-FLI1-induced osteosarcoma in more detail, we investigated the subcutaneous bones of immunocompromised mice inoculated with EFN # 2 and EFN # 12 cells.
  • EWS-FLI1-dependent osteosarcoma cell lines were constructed from sarcomas (SCOS # 2 and SCOS # 12, respectively).
  • the constructed osteosarcoma cell lines expressed EWS-FLI1 in a Dox concentration-dependent manner and actively proliferated in the presence of Dox (Fig. 7).
  • SCOS # 2 and SCOS # 12 changed their morphology after Dox removal and stopped cell growth.
  • EWS-FLI1 whose osteosarcoma cell bone differentiation was promoted by loss of EWS-FLI1 expression
  • SCOS # 2 and SCOS # 12 to identify EWS-FLI1-expressing sarcoma cells.
  • Gene expression profiles were compared among EWS-FLI1 non-expressing sarcoma cells.
  • extracellular matrix-related genes and extracellular region-related genes, often including osteogenesis-related genes and chondrogenesis-related genes compared to Dox-treated EWS-FLI1-expressing sarcoma cells by GO enrichment analysis in both cell lines was significantly enriched in Dox-untreated sarcoma cells (72 hours) (FIGS. 8, 9).
  • SCOS # 2 and SCOS # 12 formed the aforementioned small cell osteosarcoma in immunocompromised mice receiving Dox (FIG. 13). These sarcoma cells had high cell proliferating activity according to Ki67 immunohistochemistry (FIG. 13). Consistent with the in vitro findings that growth of both SCOS # 2 and SCOS # 12 is dependent on EWS-FLI1 expression, the subcutaneous tumors stopped or retarded growth after removal of Dox in vivo (FIGS. 13 and 14). Histological analysis revealed that the tumor from which Dox was removed was composed of osteoid tissue, mature bone tissue and a small number of blue cells (FIG. 13). These results indicated that loss of EWS-FLI1 promotes bone differentiation of osteosarcoma cells in vivo. Based on the above, our results revealed the role of EWS-FLI1 expression in the inhibition of terminal differentiation of osteosarcoma cells.
  • iPS cells from EWS-FLI1-induced osteosarcoma cells Considering that additional genetic abnormalities may be required for EWS-FLI1-induced sarcoma development, multi-potency from EWS-FLI1-induced sarcoma cells Construction of sex stem cells should provide a unique tool to study the effects of genetic abnormalities other than EWS-FLI1 expression on sarcoma development.
  • OCT3 / 4 OCT3 / 4
  • SOX2, KLF4, and cMYC were introduced into the sarcoma cells to obtain iPS cell-like colonies in the absence of EWS-FLI1 expression (colony formation efficiency).
  • iPS cell-like cells expressed pluripotency-related genes such as Nanog and Oct3 / 4 at the same level as ES cells (FIG. 16).
  • the overall gene expression pattern of iPS cell-like cells was similar to that of normal ES cells and control iPS cells.
  • the sarcoma-derived iPS cell-like cells showed demethylation of both Nanog promoter and Oct3 / 4 distal enhancer (FIG. 17). This indicates that these cells have acquired pluripotency via epigenetic reconstitution. Silencing of the exogenous 4 factor expression occurring in the late stage of cell reprogramming was observed in several iPS cell-like clones. This suggests that these cells have been fully reprogrammed.
  • array CGH array comparative genomic hybridization
  • sarcoma-derived iPS cell-like cells have several identical chromosomal abnormalities, confirming that these iPS cell-like clones were obtained from the parent sarcoma cells. These sarcoma-derived iPS cell-like cells have lost the ability to become mature chimeric mice upon blastocyst injection, presumably due to the widespread genetic abnormalities observed in CGH analysis. However, sarcoma-derived iPS cell-like cells formed teratomas composed of cells that differentiated into three different germ layers when inoculated into subcutaneous tissues of immunocompromised mice (FIG. 18). This indicates that these cells are pluripotent. These results indicate that iPS cells were successfully produced from EWS-FLI1-induced osteosarcoma cells.
  • EWS-FLI1-dependent osteosarcoma may arise from osteogenic cells due to increased bone differentiation of sarcoma cells when EWS-FLI1, which shows abnormal bone differentiation independent of EWS-FLI1 expression, disappears It was done. Therefore, in the absence of EWS-FLI1 expression, an attempt was made in vitro to induce osteogenic cells, which are the assumed sarcoma of the sarcoma, from pluripotent stem cells (FIG. 19) (Method of Nature 467, 285-290. 2010). reference). In control ES cells, bone differentiation stimulation induced bone differentiation-related genes such as Runx2, Sp7, Pth1r, Col1a1 and Dmp1 (day 17) (FIG. 20).
  • Bone differentiation stimulation also induced the expression of Runx2, which is an important transcription factor for bone differentiation, in sarcoma-derived iPS cells, but the induction of bone morphogenetic genes downstream of Runx2 decreased even without EWS-FLI1 expression ( Day 17) ( Figure 20).
  • Runx2 is an important transcription factor for bone differentiation
  • Figure 20 the induction of bone morphogenetic genes downstream of Runx2 decreased even without EWS-FLI1 expression
  • Both sarcoma-derived iPS cells and control ES cells formed teratomas, and these teratomas contained osteogenic regions in the absence of EWS-FLI1 expression (FIG. 22).
  • the Ki67 positive rate of sarcoma iPS cell-derived osteogenic cells was significantly higher than that of control ES cell-derived osteogenic cells (P ⁇ 0.01) (FIG. 23).
  • sarcoma-derived iPS cells show abnormal bone differentiation independent of EWS-FLI1 expression, which is also inhibited by differentiation other than EWS-FLI1 fusion and maintains proliferative progenitor cell state It suggests.
  • EWS-FLI1 expression induced rapid sarcoma development from sarcoma-iPS cell-derived osteogenic cells. Analysis of the cooperative action between EWS-FLI1 expression and differentiation abnormalities related to genetic abnormalities on sarcoma development. Tried. In both sarcoma-derived iPS cells and control ES cells (Rosa-M2rtTA / Rosa :: tetO-EWS-FLI1), EWS-FLI1 expression has no promoting effect on cell proliferation under undifferentiated culture conditions (FIG. 24). Next, bone differentiation of sarcoma-derived iPS cells and control ES cells was induced in vitro, followed by EWS-FLI1 expression (FIG. 25).
  • osteogenic precursor cells derived from sarcoma-derived iPS cells and control ES cells were treated with Dox (FIG. 25).
  • sarcoma-derived osteogenic cells showed significant cell proliferation in vitro in response to Dox on day 31 (FIGS. 26 and 27).
  • Histological analysis revealed that these xenograft tumors were sarcomas composed of small round blue cells (FIG. 29).
  • Osteogenic cells derived from control ES cells did not show clear EWS-FLI1-dependent proliferation in vivo (data not shown). This confirms that sarcoma development requires additional abnormalities.

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Abstract

L'invention concerne le développement d'un modèle cellulaire pour des tumeurs de la famille du sarcome d'Ewing qui réagit directement avec des gènes chimères pour former des tumeurs et qui peut être utilisé pour cribler un nouvel agent anticancérogène efficace pour des tumeurs de la famille du sarcome d'Ewing. L'invention concerne une cellule ESFT (Ewing's sarcoma family of tumors, ou tumeurs de la famille du sarcome d'Ewing) dérivée de cellules stromales de moelle osseuse, dans laquelle un gène chimère EWS contrôlé par un promoteur comprenant une séquence régulatrice de transcription inductible est introduit dans un chromosome, et la cellule ESFT est viable et a une capacité de prolifération illimitée pendant l'expression de la protéine chimère EWS.
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JP2020167956A (ja) * 2019-04-03 2020-10-15 学校法人東京医科大学 成熟組織の製造方法、および臓器の製造方法

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WO2019124348A1 (fr) * 2017-12-19 2019-06-27 国立大学法人京都大学 Nouvelle méthode d'induction de différenciation ostéogénique
JPWO2019124348A1 (ja) * 2017-12-19 2020-12-03 国立大学法人京都大学 新規骨分化誘導方法
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