WO2014133090A1 - Cellule souche pluripotente pour traiter une tumeur cérébrale - Google Patents

Cellule souche pluripotente pour traiter une tumeur cérébrale Download PDF

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WO2014133090A1
WO2014133090A1 PCT/JP2014/054910 JP2014054910W WO2014133090A1 WO 2014133090 A1 WO2014133090 A1 WO 2014133090A1 JP 2014054910 W JP2014054910 W JP 2014054910W WO 2014133090 A1 WO2014133090 A1 WO 2014133090A1
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cells
gene
negative
cell
cell preparation
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Japanese (ja)
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宏樹 難波
真理 出澤
正順 吉田
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Tohoku University NUC
Clio Inc
Hamamatsu University School of Medicine NUC
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Tohoku University NUC
Clio Inc
Hamamatsu University School of Medicine NUC
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • 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
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0662—Stem cells
    • C12N5/0668—Mesenchymal stem cells from other natural sources
    • 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
    • C12N2510/00—Genetically modified cells

Definitions

  • the present invention relates to a cell preparation containing pluripotent stem cells into which a suicide gene effective for treatment of brain tumors is introduced.
  • Glioma (glioma), which accounts for about 1 ⁇ 4 of brain tumors, grows infiltrating into the surrounding normal brain tissue, so that if the brain function is to be preserved, it is difficult to remove it by surgery. Furthermore, radiation therapy and anticancer drug treatment are often used after surgery, but the cure rate is low, and the average malignant glioblastoma (glioblastoma) has an average survival time of only about 1 year. Moreover, despite the development of various treatment strategies, this result has hardly changed for the past 30 years and is said to be one of the last malignant tumors left in the 21st century, and the development of new treatment strategies is urgently needed. It is.
  • suicide gene therapy introduced in the 1990s is a therapy that uses genes such as viruses to change harmful drugs in mammals into toxic substances (anticancer drugs) in transgenic cells and kill cancer cells. is there.
  • suicide gene therapy using the herpes simplex virus thymidine kinase gene (HSVtk gene) and the antiviral agent ganciclovir (GCV)
  • HSVtk gene herpes simplex virus thymidine kinase gene
  • GCV antiviral agent ganciclovir
  • Non-Patent Document 2 Attempts have been made to treat brain tumors by incorporating suicide genes into such migratory neural stem cells and expressing cytotoxic substances in the tumor site (Patent Document 1).
  • Non-patent Document 3 neural stem cells are extremely effective as a vector for carrying a suicide gene to a tumor site, but when considering clinical application, it is invasive and extremely difficult to remove neural stem cells from a patient. So far, the present inventors have used a mesenchymal stem cell collected from the bone marrow as a vector carrying a suicide gene to the tumor site as a more general-purpose vector having the same mobility as that of a neural stem cell. A treatment test was attempted (Patent Document 2). However, the mesenchymal stem cells used in this treatment test were heterogeneous and the effects were not constant.
  • SSEA-3 Stage-Specific Embryonic Antigen-3
  • Muse cells Multilineage-differentiating Stress Enduring cells; Muse cells
  • An object of the present invention is to provide a new medical use using pluripotent stem cells (Muse cells) in brain tumor treatment. More specifically, an object of the present invention is to provide a cell preparation for the prevention and / or treatment of brain tumor, comprising Muse cells into which a suicide gene has been introduced and a prodrug corresponding to the suicide gene.
  • Muse cells pluripotent stem cells
  • HSVtk herpes simplex virus thymidine kinase
  • GCV ganciclovir
  • the present invention is as follows.
  • a cell preparation for treating a brain tumor comprising a pluripotent stem cell into which a suicide gene has been introduced, wherein the pluripotent stem cell is derived from a mesenchymal tissue or a cultured mesenchymal cell in a living body.
  • Isolated SSEA-3-positive cells wherein the cell preparation is used with a prodrug of a drug that kills or inhibits the growth of a brain tumor, the prodrug being a substrate for an enzyme produced by expression of the suicide gene A cell preparation.
  • pluripotent stem cells are CD34 negative, CD117 negative, CD146 negative, CD271 negative, NG2 negative, vWF negative, Sox10 negative, Snai1 negative, Slug negative, Tyrp1 negative, and Dct negative.
  • the prodrug is gancyclovir (GCV), acyclovir, penciclovir, PMEA adefovir or PMPA tenofovir when the suicide gene is an HSVtk gene, and 5-fluorocytosine when the suicide gene is a cytosine deaminase gene, 5-fluorouracil when the suicide gene is a uracil phosphoribosyltransferase gene, 6-thioxanthine or 6-thioguanine when the suicide gene is a gpt gene, and a prodrug when the suicide gene is a nitroreductase (ntr) gene
  • GCV gancyclovir
  • penciclovir penciclovir
  • PMEA adefovir or PMPA tenofovir when the suicide gene is an HSVtk gene
  • 5-fluorocytosine when the suicide gene is a cytosine deaminase gene
  • 5-fluorouracil when the
  • the present invention relates to the expression of a suicide gene by administering a suicide gene-introduced Muse cell and a prodrug for the suicide gene from a vein or the like to a subject suffering from a brain tumor, thereby accumulating the Muse cell in the brain tumor. Based on the activation of the prodrug, tumor cells can be killed or their growth can be suppressed.
  • FIG. 2 shows the results of observing the culture state of glioma cells over time when glioma cells and Muse-TK are co-cultured at different cell ratios.
  • FIG. 3 is a diagram showing the decrease in glioma cells by counting the number of cells based on the results obtained in FIG. The result of having measured the migration ability of the Muse cell with respect to a glioma cell using the Boyden chamber is shown. The result of having verified the bystander effect by Muse-TK in vivo is shown.
  • the present invention relates to a cell preparation for treating a brain tumor, comprising pluripotent stem cells (Muse cells) into which a suicide gene has been introduced.
  • Muse cells pluripotent stem cells
  • the present invention aims at treatment of brain tumors, in particular, treatment of killing glioma cells or inhibiting their growth, using cell preparations containing pluripotent stem cells (Muse cells) into which a suicide gene has been introduced.
  • “brain tumor” refers to a state in which any type of nerve cell proliferates abnormally.
  • Examples of brain tumors include, but are not limited to, glioma, medulloblastoma, neuroblastoma, meningioma, pituitary adenoma, schwannoma, primary central nervous system lymphoma, sarcoma, and spinal cord tumor Can be mentioned.
  • the cell preparation of the present invention can target the above-mentioned various brain tumors, but it is preferable to treat gliomas that account for about 1/4 of the brain tumors as treatment targets.
  • Pluripotent stem cells The pluripotent stem cell used in the cell preparation of the present invention was found by Dezawa, one of the present inventors, in the human body and named “Muse (Multilineage-differentiating Stress Ending) cell”. It is. Muse cells can be obtained from skin tissues such as bone marrow fluid and dermal connective tissue, and are also scattered in connective tissues of each organ. In addition, this cell is a cell having the properties of both pluripotent stem cells and mesenchymal stem cells. For example, the cell surface markers “SSEA-3 (Stage-specific embryonic antigen-3)” and “ Identified as "CD105" double positive.
  • SSEA-3 Serial-specific embryonic antigen-3
  • Muse cells or cell populations containing Muse cells can be separated from living tissues using, for example, these antigen markers as indicators. Details such as a method for separating Muse cells, an identification method, and characteristics are disclosed in International Publication No. WO2011 / 007900. Also, as reported by Wakao et al. (2011, supra), when mesenchymal cells are cultured from bone marrow, skin, etc. and used as the population of Muse cells, all SSEA-3 positive cells are CD105 It is known to be a positive cell.
  • the Muse cells when separating Muse cells from living mesenchymal tissue or cultured mesenchymal stem cells, the Muse cells can be purified and used simply with SSEA-3 as an antigen marker.
  • pluripotent stem cells isolated from living mesenchymal tissue or cultured mesenchymal tissue using SSEA-3 as an antigen marker which can be used in cell preparations for treating brain tumors ( Muse cells) or a cell population containing Muse cells may be simply referred to as “SSEA-3 positive cells”.
  • “non-Muse cells” refer to cells other than “SSEA-3-positive cells”, which are cells contained in a mesenchymal tissue or cultured mesenchymal tissue in a living body.
  • Muse cells or cell populations containing Muse cells can be obtained from living tissue (eg, using antibodies against the cell surface marker SSEA-3 alone, or both antibodies against SSEA-3 and CD105, respectively) , Mesenchymal tissue).
  • living tissue eg, using antibodies against the cell surface marker SSEA-3 alone, or both antibodies against SSEA-3 and CD105, respectively
  • Mesenchymal tissue e.g., Mesenchymal tissue.
  • “living body” means a living body of a mammal. In the present invention, the living body does not include embryos whose developmental stage is earlier than the fertilized egg or blastocyst stage, but includes embryos in the developmental stage after the blastocyst stage including the fetus and blastocyst.
  • Mammals include, but are not limited to, primates such as humans and monkeys, rodents such as mice, rats, rabbits, guinea pigs, cats, dogs, sheep, pigs, cows, horses, donkeys, goats, ferrets, etc. It is done.
  • Muse cells used in the cell preparation of the present invention are clearly distinguished from embryonic stem cells (ES cells) and embryonic germ stem cells (EG cells) in that they are derived from living tissues.
  • ES cells embryonic stem cells
  • EG cells embryonic germ stem cells
  • “Mesenchymal tissue” refers to tissues existing in various organs such as bone, synovium, fat, blood, bone marrow, skeletal muscle, dermis, ligament, tendon, dental pulp, and umbilical cord.
  • Muse cells can be obtained from bone marrow or skin.
  • the Muse cell used may be autologous to the recipient who receives the cell transplant, or may be another family.
  • a Muse cell or a cell population containing a Muse cell can be separated from a living tissue using, for example, SSEA-3 positive and SSEA-3 and CD105 double positive as an index.
  • SSEA-3 positive and SSEA-3 and CD105 double positive are known to include various types of stem cells and progenitor cells.
  • Muse cells are not the same as these cells.
  • Such stem cells and progenitor cells include skin-derived progenitor cells (SKP), neural crest stem cells (NCSC), melanoblast (MB), perivascular cells (PC), endothelial progenitor cells (EP), adipose-derived stem cells (ADSC). ).
  • Muse cells can be isolated using “non-expression” of a marker unique to these cells as an index.
  • Muse cells are CD34 (EP and ADSC markers), CD117 (c-kit) (MB markers), CD146 (PC and ADSC markers), CD271 (NGFR) (NCSC markers), NG2 (PC marker), vWF factor (von Willebrand factor) (EP marker), Sox10 (NCSC marker), Snai1 (SKP marker), Slug (SKP marker), Tyrp1 (MB marker), and At least one of 11 markers selected from the group consisting of Dct (MB marker), for example 2, 3, 4, 5, 6, 7, 8, 9, 10 The non-expression of individual or eleven markers can be separated into indicators.
  • non-expression of CD117 and CD146 can be separated as an index
  • non-expression of CD117, CD146, NG2, CD34, vWF and CD271 can be separated as an index
  • the non-expression of 11 markers can be separated as an index.
  • the Muse cell having the above characteristics used in the cell preparation of the present invention is as follows: (I) low or no telomerase activity; (Ii) has the ability to differentiate into cells of any germ layer of the three germ layers; It may have at least one property selected from the group consisting of (iii) showing no neoplastic growth; and (iv) having a self-renewal capability.
  • the Muse cell used in the cell preparation of the present invention has all the above properties.
  • telomerase activity is low or absent means that, for example, when telomerase activity is detected using TRAPEZE XL telomerase detection kit (Millipore), it is low or cannot be detected.
  • “Low” telomerase activity means, for example, telomerase having a telomerase activity comparable to that of somatic human fibroblasts, or 1/5 or less, preferably 1/10 or less compared to Hela cells. It means having activity.
  • the Muse cell has the ability to differentiate into three germ layers (endoderm, mesodermal, and ectoderm) in vitro and in vivo, for example, induction culture in vitro Can be differentiated into hepatocytes, nerve cells, skeletal muscle cells, smooth muscle cells, bone cells, fat cells and the like. In addition, when transplanted to the testis in vivo, it may show the ability to differentiate into three germ layers.
  • Muse cells have the property of proliferating at a growth rate of about 1.3 days in suspension culture, but stop growing in about 10 days. Further, when transplanted to the testis, Muse cells have cancer for at least half a year. It has the property of not becoming Moreover, about said (iv), a Muse cell has self-renewal (self-replication) ability.
  • self-renewal refers to culturing cells contained in an embryoid body-like cell mass obtained by suspension culture of one Muse cell, and forming an embryoid body-like cell mass again. .
  • the self-renewal may be repeated once or multiple times.
  • Suicide gene means a gene that can kill itself when expressed in a cell, and typically includes a metabolic toxicity gene.
  • suicide genes include, but are not limited to, herpes simplex virus thymidine kinase (HSVtk) gene (Proc. Natl. Acad. Sci, USA, 78, 1441-1445 (1981)), cytosine deaminase gene (EG11326 codA 355395..356678, E.
  • the cell preparation of the present invention can be used together with a prodrug to treat a brain tumor.
  • the “prodrug” used together with the cell preparation of the present invention is a prodrug of a drug that kills the target tumor cell or inhibits its growth, and itself exhibits such cytotoxicity. It means a drug that you do not have.
  • This prodrug is converted into a drug having pharmacological activity (cytotoxicity) by an enzyme generated by expression of a suicide gene.
  • the prodrug include ganciclovir (GCV), acyclovir, pencyclovir, PMEA adefovir, PMPA tenofovir, etc., preferably GCV, acyclovir, and pencyclovir when the suicide gene is herpes simplex virus thymidine kinase (HSVtk) gene. it can.
  • guanine in the purine form of nucleic acid, and when used for DNA synthesis, DNA synthesis stops there and exerts an antiviral effect.
  • the suicide gene is a cytosine deaminase gene
  • 5-fluorocytosine can be used as the prodrug (Human Gene Therapy, 7, 713-720 (1996)).
  • the suicide gene is a uracil phosphoribosyltransferase gene
  • 5-fluorouracil can be used as a prodrug (Int. J. Oncol, 18, 117-120 (2001)).
  • the suicide gene is a guanine phosphoribosyltransferase gene
  • 6-thioxanthine or 6-thioguanine can be used as the prodrug (Human Gene Therapy, 8, 2043-2055 (1997)).
  • CB1954 can be used as a prodrug (Cancer Gene Therapy, 7, 721-731 (2000)).
  • the reaction mechanism of the enzyme and prodrug produced by the expression of the suicide gene will be described below using, for example, the HSVtk (herpes simplex virus thymidine kinase) gene and GCV (ganciclovir).
  • HSVtk herpes simplex virus thymidine kinase
  • GCV ganciclovir
  • the HSVtk gene is introduced into cancer cells and expressed, and GCV, which is an antiviral agent, is administered as a prodrug.
  • Cancer cells into which the HSVtk gene has been introduced phosphorylate GCV with HSVtk to form a monophosphorylated GCV (GCV-1P).
  • GCV-1P is then phosphorylated to triphosphate by its own thymidine kinase, and in order to inhibit DNA polymerase, cells that undergo DNA replication fall into apoptosis and die. Furthermore, GCV-1P is also taken into neighboring cells through a gap junction, and neighboring cells without gene transfer are also killed due to inhibition of DNA synthesis (bystander effect).
  • a Muse cell into which a suicide gene has been introduced is used.
  • a method of introducing the suicide gene into Muse cells a method of introducing a vector incorporating the gene is common.
  • Gene transfer by use of a vector includes, but is not limited to, viral or non-viral gene transfer (eg, plasmid transfer, phage integrase, transposon, adenovirus, adeno-associated virus, and lentivirus).
  • HSVtk herpes simplex virus thymidine kinase
  • PA317 mouse-derived HSVtk-retrovirus producing cell
  • cytosine deaminase gene it is incorporated into an adenovirus vector with cytomegalovirus early gene enhancer / promoter added to the cytosine deaminase gene cDNA of E. coli (Human Gene Therapy, 6, 1055-1063 (1995)). Can be introduced.
  • the gene When using a uracil phosphoribosyltransferase (UPRT) gene, the gene can be introduced using a retroviral vector LXSN incorporating the UPRT gene of E. coli.
  • the gene When the guanine phosphoribosyltransferase (gpt) gene is used, the gene can be introduced by infecting the cells with the virus using the supernatant of the E. coli gpt-retrovirus producing cell (GP / E86 gpt) culture.
  • a plasmid in which the cytomegalovirus early gene enhancer / promoter is added to the Escherichia coli ntr gene can be prepared, and the gene can be introduced into the cell by electroporation.
  • the cells may be appropriately proliferated.
  • the cell preparation of the present invention can be obtained by suspending Muse cells after introduction of a suicide gene in physiological saline or an appropriate buffer (for example, phosphate buffered saline).
  • physiological saline or an appropriate buffer for example, phosphate buffered saline.
  • the cell preparation may be cultured before being administered to the treatment subject and grown until a predetermined cell concentration is obtained.
  • Muse cells do not become tumors. Therefore, even if cells collected from living tissue remain undifferentiated, there is a possibility of canceration. Low and safe.
  • the culture of the collected Muse cells is not particularly limited, but can be performed in a normal growth medium (for example, ⁇ -minimal essential medium ( ⁇ -MEM) containing 10% calf serum). More specifically, referring to the above International Publication No. WO2011 / 007900, in the culture and proliferation of Muse cells, a medium, additives (for example, antibiotics, serum) and the like are appropriately selected, and Muse cells at a predetermined concentration are selected. A solution containing can be prepared.
  • a normal growth medium for example, ⁇ -minimal essential medium ( ⁇ -MEM) containing 10% calf serum.
  • DMSO dimethyl sulfoxide
  • serum albumin is used to protect the cells
  • antibiotics are used to prevent bacterial contamination and growth. You may make it contain in a cell formulation.
  • other pharmaceutically acceptable ingredients for example, carriers, excipients, disintegrants, buffers, emulsifiers, suspending agents, soothing agents, stabilizers, preservatives, preservatives, physiological saline, etc.
  • Cells or components other than Muse cells contained in mesenchymal stem cells may be contained in the cell preparation.
  • One skilled in the art can add these factors and agents to the cell preparation at appropriate concentrations.
  • the number of Muse cells into which the suicide gene contained in the cell preparation prepared above is introduced depends on the sex of the subject so that a desired effect (eg, disappearance of tumor, reduction of tumor size) can be obtained in the treatment of brain tumors. In consideration of the age, weight, state of the affected area, state of cells to be used, etc., it can be appropriately adjusted.
  • the cell preparation of the present invention can be used a plurality of times (for example, 2 to 10 times) at appropriate intervals (for example, twice a day, once a day, once a week) until a desired therapeutic effect is obtained. 2 times, once a week, once every two weeks).
  • the therapeutically effective dose is preferably, for example, 1 ⁇ 10 3 cells to 1 ⁇ 10 6 cells per individual and 1 to 10 doses.
  • the total dose in one individual includes, but is not limited to, 1 ⁇ 10 3 cells to 1 ⁇ 10 7 , 1 ⁇ 10 4 cells to 5 ⁇ 10 6 cells, and the like.
  • treatment of brain tumors with the cell preparation of the present invention is based on the expression of a suicide gene introduced into Muse cells, and the subsequent activation of the prodrug by the enzyme that is the gene product. Therefore, in the use of the cell preparation of the present invention, the administration site and administration method (local tumor administration, intracarotid artery administration, intravenous administration) of the cell preparation are not limited.
  • the prodrug used together with the cell preparation of the present invention may be intravenous administration as described above, or may be intraperitoneal administration.
  • a prodrug can be administered intravenously or intraperitoneally to obtain the desired therapeutic effect.
  • the administration time of the prodrug is not limited, but may be any time after administration of the cell preparation of the present invention, at the same time as administration, and before administration as long as it has the above effect.
  • a pharmaceutical composition comprising a Muse cell into which a suicide gene has been introduced, a prodrug, and optionally a carrier or excipient.
  • Example 1 Preparation of Muse cells into which herpes simplex virus thymidine kinase (HSVtk) gene was introduced
  • HSVtk herpes simplex virus thymidine kinase
  • HSVtk gene introduction HSVtk retrovirus-producing cells PA317, mouse fibroblasts, Genetic Therapy Inc.
  • Example 2 Detection of Muse cells around glioblastoma (glioblastoma) It was examined whether or not Muse cells were present in human glioblastoma collected from human subjects. A tissue section of the collected glioblastoma was prepared and immunostained using an antibody against the cell surface antigen (SSEA-3 ) of Muse cells according to a conventional method. As shown in FIG. 1, it was found that Muse cells exist around glioblastoma.
  • SSEA-3 cell surface antigen
  • Example 3 Confirmation of bystander effect in human glioma cells by Muse-TK cells to confirm the bystander effect by Muse-TK prepared in Example 1, co-culture of Muse-TK cells and glioma cells in vitro was examined.
  • 96-well human glioma cells A172 1.5 ⁇ 10 4 cells / well, Muse-TK cells (1/1), 1/4, 1/8, 1/16, 1/32 cells The cells were mixed as a number, cultured in the presence of 2 ⁇ g / ml ganciclovir (GCV) for a predetermined number of days, observed with a phase contrast microscope, and imaged.
  • GCV 2 ⁇ g / ml ganciclovir
  • FIG. 3 shows the results of actually counting glioma cells in culture on the 5th and 9th days after GCV addition based on the results of FIG. As can be seen from this result, cell proliferation of glioma cells was reduced to a cell ratio of 1: 1 to 1: 8. Thus, a bystander effect on glioma cells was observed in Muse-TK cells.
  • Example 4 Confirmation of migration ability to glioma cells by Muse-TK cells
  • Muse cells have the property of accumulating around tumors.
  • Muse cell migration was quantitatively measured using the Boyden chamber method (Boyden, S., J. Exp. Med., Vol. 115, p. 453-466 (1962)).
  • the Boyden chamber used was a QCM Chemotaxis Cell Migration Assay Kit (QCM 24 Well Colorimetric Cell Migration Assay) commercially available from Millipore.
  • the Boyden chamber includes an insert having a filter having uniform fine pores of 8 ⁇ m at the bottom inside the chamber.
  • the culture solution containing Muse cells or non-Muse cells was added to the upper part of the filter of the insert, the culture supernatant of glioma cells was added to the lower part of the insert, and after culturing for 18 hours, the number of cells that passed through the micropores of the filter was counted. .
  • the result is shown in FIG. "Muse A172 "and” Muse " In the “YKG-1” system, Muse cells passed through the micropores and moved significantly to the lower part of the insert.
  • the culture supernatant of glioma cells was used for non-Muse cells, and when DMEM was used instead of this culture supernatant, the ability to migrate Muse cells was not exhibited.
  • Example 5 Verification of tumor formation inhibitory effect in vivo
  • the bystander effect by Muse-TK prepared in Example 1 was confirmed in vivo.
  • U87-luc2 cells glioma cell line
  • Muse-TK cells and luciferase gene were introduced were co-transplanted into the right brain of nude mice (male 8 weeks old). More specifically, the numbers of Muse-TK cells and U87-luc2 cells to be transplanted were 2.5 ⁇ 10 4 and 10 ⁇ 10 4 (1: 4), respectively.
  • ganciclovir (GCV) Wired
  • Muse cells into which the suicide gene has been introduced accumulate around the brain tumor, and further, the cells constituting the brain tumor are killed by administration of a prodrug corresponding to the suicide gene. Can be applied to the treatment of brain tumors.

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Abstract

L'objet de la présente invention est de fournir une nouvelle utilisation médicale utilisant une cellule souche pluripotente (cellule Muse). L'invention concerne une préparation de cellules destinée à traiter une tumeur cérébrale, ladite préparation de cellules comprenant des cellules souches pluripotentes positives au SSEA-3 qui sont séparées d'un tissu mésenchymateux dans un organisme ou des cellules mésenchymateuses en culture. La préparation de cellules selon la présente invention se base sur un mécanisme dans lequel des cellules Muse portant un gène suicide qui a été transféré en leur sein sont administrées par voie intraveineuse à un sujet souffrant d'une tumeur cérébrale, et un promédicament correspondant au gène suicide est ensuite administré, de sorte que les cellules Muse sont amenées à s'accumuler de manière sélective autour de la tumeur cérébrale et ainsi, les cellules constituant la tumeur cérébrale sont exterminées ou la croissance des cellules est diminuée par le biais de l'activation du promédicament associée à l'expression du gène suicide.
PCT/JP2014/054910 2013-02-28 2014-02-27 Cellule souche pluripotente pour traiter une tumeur cérébrale Ceased WO2014133090A1 (fr)

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CN107073041A (zh) * 2014-09-05 2017-08-18 国立大学法人东京大学 糖尿病性皮肤溃疡治疗的多功能性干细胞
EP3459553A4 (fr) * 2016-05-16 2020-01-15 National University Corporation Nagoya University Atténuation et traitement de lésions cérébrales périnatales avec des cellules souches pluripotentes
CN110869034A (zh) * 2017-06-20 2020-03-06 国立大学法人名古屋大学 利用多能干细胞进行的伴随胎儿生长迟缓的脑损伤的改善和治疗
EP3622961A4 (fr) * 2017-05-09 2021-01-13 Keio University Formulation à base de cellules pour le traitement d'une tumeur cérébrale

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107073041A (zh) * 2014-09-05 2017-08-18 国立大学法人东京大学 糖尿病性皮肤溃疡治疗的多功能性干细胞
EP3189844A4 (fr) * 2014-09-05 2018-05-02 The University of Tokyo Cellules souches pluripotentes pour le traitement d'un ulcère cutané diabétique
US11000552B2 (en) 2014-09-05 2021-05-11 The University Of Tokyo Pluripotent stem cell for treating diabetic skin ulcer
EP3459553A4 (fr) * 2016-05-16 2020-01-15 National University Corporation Nagoya University Atténuation et traitement de lésions cérébrales périnatales avec des cellules souches pluripotentes
EP3622961A4 (fr) * 2017-05-09 2021-01-13 Keio University Formulation à base de cellules pour le traitement d'une tumeur cérébrale
CN110869034A (zh) * 2017-06-20 2020-03-06 国立大学法人名古屋大学 利用多能干细胞进行的伴随胎儿生长迟缓的脑损伤的改善和治疗

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